Ruminants Anatomy, Behavior and Diseases

ANIMAL SCIENCE, ISSUES AND PROFESSIONS
RUMINANTS
ANATOMY, BEHAVIOR AND DISEASES
No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or
by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no
expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of information
contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in
rendering legal, medical or any other professional services.
ANIMAL SCIENCE, ISSUES AND PROFESSIONS
Additional books in this series can be found on Nova’s website
under the Series tab.
Additional e-books in this series can be found on Nova’s website
under the e-book tab.
ANIMAL SCIENCE, ISSUES AND PROFESSIONS
RUMINANTS
ANATOMY, BEHAVIOR AND DISEASES
RICARDO EVANDRO MENDES
EDITOR
New York
Copyright © 2012 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical
photocopying, recording or otherwise without the written permission of the Publisher.
For permission to use material from this book please contact us:
Telephone 631-231-7269; Fax 631-231-8175
Web Site: http://www.novapublishers.com
NOTICE TO THE READER
The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or
implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of
information contained in this book. The Publisher shall not be liable for any special,
consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or
reliance upon, this material. Any parts of this book based on government reports are so indicated
and copyright is claimed for those parts to the extent applicable to compilations of such works.
Independent verification should be sought for any data, advice or recommendations contained in
this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage
to persons or property arising from any methods, products, instructions, ideas or otherwise
contained in this publication.
This publication is designed to provide accurate and authoritative information with regard to the
subject matter covered herein. It is sold with the clear understanding that the Publisher is not
engaged in rendering legal or any other professional services. If legal or any other expert
assistance is required, the services of a competent person should be sought. FROM A
DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE
AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data
Ruminants : anatomy, behavior, and diseases / [edited by] Ricardo Evandro Mendes.
p. cm.
Includes bibliographical references and index.
ISBN: (eBook)
1. Ruminants. I. Mendes, Ricardo Evandro.
QL737.U5R875 2011
599.63--dc23
2012010365
Published by Nova Science Publishers, Inc.  New York
Contents
Preface
vii
Chapter I
Neglected Tropical Diseases of Ruminants
Carlos Gutierrez
Chapter II
Liver and Stomach Flukes of Wild and Domestic Ruminants:
Molecular Approach in Their Discrimination
Eva Bazsalovicsová, Ivica Králová-Hromadová,
Katarína Oberhauserová and Marta Špakulová
Chapter III
Fasciolosis
Leandro Buffoni Perazzo
Chapter IV
Evaluating the Whole Production Chain:
A Need for Organic Production Research
Marta López-Alonso, Marta Miranda
and Isabel Blanco-Penedo
Chapter V
Exanthematic Bovine Vaccinia Virus Outbreaks in
Dairy Cattle in Brazil: An Overview of Viral Isolates
and Risk Factors
Jônatas Santos Abrahão, Giliane de Souza Trindade
and Erna Geessien Kroon
1
19
39
55
71
Chapter VI
Behavior Feeding of Ruminants
Christiano Santos Rocha Pitta,
Paulo Fernando Adami
and Laércio Ricardo Sartor
81
Chapter VII
Freemartinism in Cattle
99
Alexandra Esteves, Renée Båge
and Rita Payan-Carreira
Chapter VIII
Fat Depots: A Morphological and
Anatomical Adaptation to Tropical Climates
André Martinho de Almeida
121
vi
Chapter IX
Chapter X
Contents
Bovine Viral Diarrhea Virus: Biology,
Clinical Forms and Pathogenesis
María A. Risalde, Pedro J. Sánchez-Cordón,
Miriam Pedrera, Verónica Molina,
Fernando Romero-Palomo
and José C. Gómez-Villamandos
Adult Mucosa Stomach of Domestic Ruminants: Structural,
Histochemical and Immunocytochemical Study
Gaetano Scala and Lucianna Maruccio
127
149
Chapter XI
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis):
Morphostructural, Microvascular and Immunocytochemical Study 181
Gaetano Scala and Lucianna Maruccio
Chapter XII
Mandible Shape of Ruminants:
Between Phylogeny and Feeding Habits
Mennecart Bastien, Becker Damien
and Berger Jean-Pierre
Chapter XIII
Index
Creating the Tiniest Bison: A System Dynamics Model
of Ecological Evolution
Elin Whitney-Smith
205
227
249
Preface
This book includes thoroughly revised and reviewed information with content of the most
important diseases throughout the world. It presents up-to-date information on diseases
effecting ruminants as Fasciolosis, Freemartism and BVD, as well as, present important
information about behavior and evolutionary and morphological anatomy. Included also are
observations collected during experiments using immunohistochemistry, flow cytometry and
scanning and transmission electron microscopy. This book was written by some of the best
researchers in each area, from universities located in Spain, Portugal, US, Switzerland, Brazil,
Italy and Slovakia. Completely revised chapters reflect the information from current
literature.
Chapter I - Tropical diseases are those that occur principally in the tropics and by
extension in subtropics. Some of them are considered neglected because these diseases persist
exclusively in the poorest and the most marginalized areas of the world, where the interest of
both companies and researchers is limited. There is not an official list of neglected tropical
diseases affecting ruminants, but malignant catarrhal fever, foot and mouth disease,
heartwater, bovine babesiosis, theileriosis, tsetse transmitted and non tsetse transmitted
trypanosomosis, contagious caprine pleuropneumonia or peste des petit ruminant could be
clear examples nowadays. Some initiatives, however, have been successful in terms of control
and eradication of these diseases. Global Rinderpest Eradication Programme under the
auspices of The Food and Agriculture Organization has achieved its eradication in 2011.
Unfortunately many other tropical diseases have not received the same attention. The present
chapter reviews the current status of the most important neglected tropical diseases of
ruminants, including the possible control programs for the different affected areas.
Chapter II - Parasitic diseases represent the most prevalent and important problems of
grazing ruminants that can significantly influence their daily activity and health. The most
ubiquitous are ectoparasites such as biting and sucking lice, bot flies or sarcoptic mange mites
manifested by pruritus, rubbing, scratching and dermal irritation, as well as ticks which are
possible transmitters of tick-borne diseases. Endoparasites similarly pose a potential threat
and constraint of livestock health status. Out of protozoan parasites, coccidiosis may cause
severe profuse watery diarrhoea resulting to dehydration, depression and weakness especially
in young animals. Domestic and free-living ruminants are also frequently infected by various
endohelminths of the classes Nematoda (phyllum Nemathelminthes), Cestoda and Trematoda
(Platyhelminthes) invading organs of the respiratory apparatus, as well as the digestive
system. Out of the trematodes, digenetic liver flukes Fascioloides magna, Fasciola hepatica,
viii
Ricardo Evandro Mendes
Dicrocoelium dendriticum and the stomach fluke Paramphistomum cervi often induce similar
clinical signs as weight loss, diarrhea, anemia, edema and reduced milk production, that
causes difficulties in differential diagnostics. Most of the infections are exactly identified
post-mortem by distinct species-specific morphological characters of adults of flukes. “Giant
liver fluke” F. magna differs mainly in the distinctly large body up to 80 mm long and
rounded anterior body part. Smaller but rather similar “common liver fluke” F. hepatica is up
to 35 mm long possessing anterior cone and ’shoulders’. Adult worms of “lancet liver fluke”
D. dendriticum are rather tiny, measuring up to 10 mm and the body is strongly tapered at
both ends. Mature “stomach flukes” P. cervi have thick robust and cone-shaped body up to
6.5 mm long with conspicuous posterior sucker. Contrary to this, in vivo diagnosis based on
the coprological examination faces serious problems. Eggs, especially those of F. hepatica, F.
magna and P. cervi, are considered to be hardly distinguishable. They are of very similar size,
shape and internal structure and their unequivocal species differentiation is disputable. To
resolve the issue of the accurate in vivo discrimination of liver and stomach flukes in
ruminants, a molecular approach applying PCR-based techniques has recently been designed.
The ribosomal internal transcribed spacer (ITS2, rDNA) provides sufficient species-specific
characters that can be utilized in molecular diagnostics of coprological samples in
parasitological laboratories and veterinary clinics. The chapter summarizes data on taxonomy,
morphology, distribution, clinical signs and PCR-based species differentiation of mentioned
liver and stomach flukes of wild and domestic ruminants.
Chapter III - Fasciolosis is an important parasite disease of livestock that affect the liver.
The life cycle of the parasite is highly complex and requires the presence of snails of the
subfamily Lymnaeinae in the natural environment, which act as intermediate hosts. After
infection, immature worms migrate through the liver parenchyma causing a traumatic
hepatitis.
Then, adult flukes enter the bile ducts and gallbladder where they reach sexual maturity.
Diagnosis is usually achieved by detecting egg in feces or specific antibodies. Control is
mainly based on the use of anthelmintics.
Chapter IV - Organic farming promotes the multiple aims of providing good quality
feedstuffs, appropriate livestock husbandry systems and correct management practices to
promote animal health and welfare. However, benefits of organic systems are primarily
related to environmentally-friendly production and to animal welfare, whereas issues
pertaining to animal health and product quality are more influenced by the specific farm
management than by the production method. Thus, there is a general agreement in the
scientific community for the need to evaluate the whole production chain in a holistic
production such as organic farming. In order to comply with principles of organic production
and consumers’ expectations, a research strategy based on biological indicators, farm
inspections, questionnaires, secondary data and different levels of assessment is needed. This
chapter reviews what has been scientifically proven in linking animal health, husbandry
practices and resultant food product quality. This document addresses critical obstacles not
strong enough to escape from this need-driven research, which at present are still limited, but
where extrapolations from conventional system research are inacceptable. Finally, we also
present the benefits of this integral research for a better knowledge of the current status of
organic production and the potential to improve the organic product quality.
Chapter V - Exanthematous viral diseases are major causes of morbidity and economic
loss associated with dairy cattle. Among the emerging viral exanthematous diseases, we
Preface
ix
highlight bovine vaccinia (BV), of which the Vaccinia virus (VACV) is the etiological agent.
BV outbreaks have been occurring over the past 11 years in Brazilian rural areas and are
associated with ulcerative lesions on the udders and teats of dairy cattle, causing considerable
loss in milk production. BV is a zoonotic disease, and the milker can be infected by direct
contact with an infected animal. Several VACV were isolated in Brazil during BV outbreaks
in recent years, and molecular and biological analyses showed that these viruses can be
segregated into two major groups, based on genetic markers and virulence. Here, we offer a
brief overview of VACV taxonomical location in the Poxviridae family, with an emphasis on
the VACV isolates obtained during BV outbreaks in Brazil, including their geographic
locations, biological characteristics and molecular properties. Risk factors of the disease are
also discussed based on epidemiological information and new findings related to possible
hosts and reservoirs of VAC in Brazil.
Chapter VI - The purpose of this chapter was to discuss how to optimize the grazing
process, relating to the animal decisions taken at different scales, influenced by the pasture
sward and animal supplementation and to highlight the common feed factors that influence
feeding behavior and intake, although their expression and consequences depend on the
environment. Landscape can be considered the largest scales, where biotic and abiotic factors
(topography, water availability, shelters, etc.) are added together influencing the process of
grazing, especially in the continuous stocking system. Another important discussion on these
scales is the number and size of each individual meal, time spent eating and intervals between
meals whereas a meal is defined by a long string of grazing. Animal behavior at a feeding
patch is an important indicator of feeding conditions, by their direct relation with quantitative
attributes, qualitative and pasture sward. Forage intake in each bite and the number of bites
during the grazing period results in total intake of forage. Thus, the maximization of
consumption is directly related to animal production and is dependent on maximizing every
bit over the pasture. Usually the adopted management affects the pasture sward and this along
with the animal supplementation significantly influences consumption and ingestive behavior
of animals. Thus, the process of grazing must be managed considering the relationship
between the components of the system, considering that the plants need its leaves to intercept
solar energy and perform photosynthesis, absorb water and nutrients provided by the soil and
the animal need these leaves to ingest nutrients and due to it, is constantly influencing the
plants growth by the grazing action. Plant-animal relationships interfere on the animal
ingestive behavior and result in different pasture sward what in turn alter the forage search
and apprehension, defoliation patterns of leaves and tillers during the lowering of the pasture.
Pasture sward influences rate of intake and dietary choices. On heterogeneous resources,
animals graze selectively and choose a diet of higher quality than that on offer.
Chapter VII - Freemartinism is one of the most commonly found intersex conditions in
cattle, although it may also occur in small ruminants. The freemartin phenotype appears in a
dizygotic twin pregnancy where one twin is a male and the other is a female. Due to
precocious anastomoses between the placental vascular systems of the two fetuses,
masculinising molecules reach the female twin and disrupt the normal sexual differentiation,
whilst in the male the effects of this association are usually minimal. In cattle, this condition
is observed in 90 to 97% of twin pregnancies.
A freemartin is, by definition, a genetically female fetus masculinised in the presence of a
male co-twin, giving rise to a sterile heifer. Genital tract defects with varying severity can be
observed in freemartin animals, which often present suppression and disorganization of the
x
Ricardo Evandro Mendes
ovary, originating a rudimentary or a testis-like gonad depleted of germ cells. The uterine
horns may be hypoplastic or instead may be reduced to a cord-like structure suspended in the
broad ligament. Anatomic continuity between the uterus and the vagina is frequently absent,
and the existence of rudimentary vesicular glands is typical. The external genitalia commonly
presents enlarged clitoris, small vulva and a prominent, male-like tuft of hair. As a rule,
heifers born twin to a bull have to be considered sterile and should be identified as early as
possible to cull them from replacement stock. Despite its limitations, freemartinism is
currently diagnosed by physical examination, as karyotyping or blood typing is often
considered an unnecessary expense.
In cattle, twinning trend has a genetic background that has been associated to hormonal
regulation in favor of double ovulations. However, the genetic determinant on the basis of
twinning seems to have small importance when compared to environmental or managementassociated factors, particularly in dairy cows. In fact, in dairy animals, in particular in high
milk producing cows, it has long been proven that the increase of twin calvings occurs due to
the hormonal and metabolic disturbances in the energy balance early in the post-partum
period. With increased incidence of twin births in cow it is reasonably expectable a small
increase in the occurrence of freemartins at the farm levels. In this paper it is the intent to
describe the gross and histopathological findings of freemartinism in cattle, using data
gathered from a study at an abattoir (17 cases) and from 3 cases diagnosed in living animals,
supported by a review of the pathophysiology of the process, and to discuss the available
methods for identification of freemartin animals at farm level.
Chapter VIII - Fat depots are appendages that are frequently found in ruminants in
tropical regions. In this article, we will address the issue through two characteristic study
cases, the hump of Bos indicus (Indian cattle) and the sheep’s fat tail, aiming to provide the
reader with an overview of the characteristics that make these animals particularly adapted to
this type of environments.
Chapter IX - Bovine viral diarrhea virus (BVDV) is an endemic ruminant pestivirus in
populations worldwide. Based on antigenic and genetic differences, BVDV isolates can be
classified into two genotypes, BVDV-1 and BVDV-2, which are divided in non-cytopathic
and cytopathic biotypes, depending on their effect on cell cultures. This virus presents a
complex biology which gives rise to a great variety of clinical manifestations, including
subclinical infections of immunocompetent animals, appearance of persistently infected
animals from congenital infections and development of a severe fatal process denominated
mucosal disease. During BVDV pathogenesis, the virus shows a special tropism for cells of
the immune system as monocytes/macrophages, dendritic cells and lymphocytes, inducing
cell death as an extreme event of the infection, or more subtle effects on cytokines and costimulatory molecules, affecting both innate and adaptive immune responses. BVDV can
cause disease on its own, but which is perhaps more important, it also induces a state of
immunosuppression that predisposes calves to the appearance of secondary infections. In
general, the immunosuppressive properties of BVDV produce a reduction in local defense
mechanisms, thereby predisposing calves to other respiratory pathogens, being considered as
the main predisposing factor for the occurrence of Bovine Respiratory Disease Complex.
The study of immune evasion strategies used by BVDV should lead to a better
understanding of the immune system and the interaction of this virus with other pathogens in
the host. This will help to treat virus-induced pathology, identify new mechanisms for
immune modulation, design vaccines and establish control strategies.
Preface
xi
Chapter X - The main objective of the present chapter was to better understand the role of
the domestic ruminants stomach in rumination. The fine structure of the mucosa was studied
in detail to clarify the function of the aliment absorption mechanism. The morpho-structural,
histochemical and immunocytochemical characteristics of the stomach in domestic ruminant
viz cattle, buffalo and sheep was investigated by means of light microscopy (LM),
transmission electron microscopy (TEM), scanning electron microscopy (SEM) and
immunogold-labeling SEM analysis. The stomach specimens of both sexes were processed in
different ways, depending on the type of the technique to be used. Rumen - the mucosa was
covered with a squamous, horny, stratified epithelium. Many intercellular spaces were
observed in the spinous and basal layers. These spaces presented many intercellular
digitations. On the macerated specimens, many folds, which intersected each other, were
observed in the lamina propria, thus giving rise to a mosaic-like structure. Reticulum – the
mucosa epithelium was morphologically similar to the mucosa rumen. The lamina propria
presents well-developed muscularis mucosae. Reticular groove – many glands were observed
in the lamina propria. A large amount of striated muscularis tissue was observed in the tunica
muscularis; this tissue extended up to the middle part of the reticular groove. Omasum- the
tunica mucosa was morphologically different from that of the rumen and reticulum. The
intercellular spaces in the spinous and basal layers were larger in the omasum than in the
rumen and reticulum. Abomasum- the tunica mucosa had many glands and was covered with
a simple columnar epithelium. The lamina propria contained the muscularis mucosae and
glandular adenomeres in the epithelium underlying. By means of the histochemical method,
an intense NADPHd staining in the granulous, spinous and basal layers was observed. By
means of immunogold-labeling, the immunoreactivity to NOS I was found in all specimens of
stomach mucosa of all species studied. Moreover, the immunoreactivity to CD133 was
observed in positive cells of the micro-papillae in the bottom of the reticulum small polygonal
cells and on the papillae of the rumen wall. In conclusion, this data reflects a possible role of
nitric oxide in the delaying the onset of cellular apoptosis in the forestomach domestic
ruminants, by playing a role in the production of cell energy. Moreover, the presence of the
stem cells refers to the renewing of the reticulm micro-papillae and to reconstitute the
damages of the rumen papillae during the normal functional activity.
Chapter XI - The ruminant stomach mucosa shows several morphostructural
characteristics in relation with rumination. It is important and of interest to learn how the
mediterranean buffalo gains a higher body weight and a higher milk production from the
possible relation available for animal production. In this chapter, the morphostructural,
microvascular and immunocytochemical characteristics of the buffalo mucosa stomach during
prenatal period is studied. The fetus specimens of different lengths of both sexes were
processed for light microscopy (LM), scanning electron microscopy (SEM) and immunogoldlabeling SEM analysis. The stomach mucosa in the buffalo fetuses of the crown-rump length
(CRL) 5 cm at LM shows the epithelium with similar characteristics in all areas of the
stomach. The epithelium is composed of several layers of ovoid cells, more dense in the basal
surface. The sub-epithelial tissue, not still very differentiated, is rich with blood vessels. At
the SEM, the apical surface of the epithelial cells have short microvilli. In the buffalo fetuses
of the CRL 7 cm at the LM, the mucosa of abomasum shows the thickening of the elongated
cells. At the SEM, the internal surface of the reticulum shows the reticular groove that begins
to form. Obvious changes are observed in the mucosa stomach buffalo fetuses of CRL 13 cm.
These changes regard the lamina propria organization. In fact, the lamina propria is different
xii
Ricardo Evandro Mendes
in all of the stomach compartments. In the rumen, the connective lamina propria origins of
finger-like protrusions that invade the mucosa epithelium. In the reticulum, the connective
lamina propria describes the polygonal areas. In the omasum, the connective lamina propria
forms the typical papillae of the mucosal laminae.
In theabomasum the connective lamina propria forms the typical mucosa plicae. Then,
the mucosa stomach shows a typical aspect similar to the final organization. From the fetuses
of CRL 13 cm to birth, the blood vessels show the typical organization present in the stomach
compartments. This data confirms the view that the development of sub-epithelial blood
capillaries must take place prior to the development of the mucosa epithelium. In the fetuses
of the CRL 38 cm, in which the morphostructural organization is already well defined, the
epithelium shows an evident immunoreactivity to marker for pluripotent stem cells. In
conclusion the epithelium mucosa of the fetuses buffalo stomach present an intense activity. It
seems that the organization of the lamina propria precedes that of the epithelium mucosa.
Chapter XII - Previous authors have demonstrated that the mandible morphology of
ruminants is strongly related to phylogeny and diet. We performed a geometric morphometric
analysis defined by the landmark configurations of ruminant mandible specimens, using a
Principal Component Analysis (PCA) of the variability in shape. The analysis is based on 21
fossils and 65 extant ruminant species from 65 genera (18 fossil and 47 extant) covering the
diversity of the group. As a weak intraspecific variability is assumed, one mandible specimen
per species has been used in the analysis. The PCA (PC1: 36.0% and PC2 15.3%) reveals
progressive trends in the mandible shape of extant and fossil taxa related to phylogeny and
feeding habits. Within extant ruminants, Tragulina can easily be distinguished from the
Pecora along the PC1 axis. Tragulina possess elongated premolars, a small diastema, an
enlarged angular process, a weak incisura vasorum, a short coronoid process inclined
backwards, a stout condylar process and a subvertical ramus. Bovidae have mainly negative
PC2 values and can broadly be separated from the Cervidae that mostly plot in the positive
side of the axis. However, selective browser Bovidae plot in an overlapped area with
Cervidae in the positive side of the PC2 axis. Bovidae often bear an enlarged corpus
mandibulae, a lengthened molar row, and shortened premolar row and diastema. The ramus is
more backward inclined giving a more efficient lever arm to the masseter muscles, with a less
individualized angular process. Within Bovidae, this mandible shape change is observed
along the PC2 axis. The enlargement of the corpus mandibulae and the elongation of the
molar row are related to the increase of the hypsodonty and the grazing habit. Within
Cervidae, few similar distinctions between the mandible shape and the diet can be observed.
Giraffidae belong to a homogeneous group characterized by an extreme elongation of the
diastema forming a slender corpus mandibulae. Selective browser Bovidae, Moschidae and
Antilocapridae plot within the Cervidae/Bovidae overlapped area.
Regarding the fossil specimens, three different categories are observed. The true
Cervidae, which bear antlers, are within the extant Cervidae. Hoplitomeryx plot close to
extant Antilocapridae within the Cervidae/Bovidae overlapped area. The other Miocene
species, related to either extinct families or ancestral representatives of extant families, plot
between the primitive Gelocus villebramarensis and extant Pecora. The mandible shape of
primitive Tragulina does not permit the distinction of different feeding habits. However,
primitive Tragulina form a separate group that could correspond to the ruminant primitive
state mandible shape.
Preface
xiii
Chapter XIII - The anatomy and behavior of N. American bison were profoundly
modified by the extinction event at the end of the Pleistocene. Plains Bison (Bison bison)
became obligate grazers, and, like musk ox, elk, and other large ruminants, became much
smaller. During the same time period, mastodon, mammoth, horse, ground sloth, and other
non-ruminant herbivores went extinct.
The vegetation of the North American continent shifted from a mixed mosaic to
homogenous stripes: mixed parklands and woodlands throughout the continent were
transformed into the great and treeless prairie, and the mixed parkland/woodlands on the
coasts became closed-canopy forests. The key to understanding the changes in bison anatomy
and behavior, the non-ruminant extinctions, and the vegetative shift is uncovering the
ecological dynamics of the end of the Pleistocene.
Two major theories have been proposed to explain the extinction event:
Overkill – humans hunted animals to extinction and
Climate Change – the end of the Ice Age created ecological stress which caused the
extinctions and the changes in the size of bison.
Both have problems.
A new theory is called for. Second Order Predation hypothesizes that humans reduced
carnivore populations below the level that they controlled herbivore populations, resulting in
an herbivore boom then bust, destabilizing the ecosystem. During the boom phase, herbivores
denuded the environment of vegetation, creating a highly competitive environment – a
bottleneck – where animals that could extract the maximum nutrition from their food –
ruminants –, and reproduce using the minimum amount of food – smaller animals – were
evolutionarily favored, while large non-ruminants went extinct. When a new stability was
reached in the Holocene, huge herds of bison (B. bison bison) dominated and maintained the
grasslands in the center of the country, and trees re-invaded from mountain refugia,where
woodland bison (B. bison athabascae) continued to live in small groups and eat a mixture of
grass and browse. This bottleneck in the environment explains why horses, non-ruminants
that had gone extinct during the transition, could survive in the new stabile ecosystem. Some
scientists postulate that the extinctions can be explained through a combination of the
Overkill and Climate Change hypotheses.
The Pleistocene Extinction Model (PEM) was designed to test all these hypotheses using
the same assumptions. The PEM simulations give surprising results: i) If the model is run
with sufficient Climate Change to produce extinctions, Overkill reduces the impact of
Climate Change, avoiding extinction. ii) Under the same conditions, Second Order Predation
exacerbates the impact of Climate Change, leading to quicker and more extreme extinction.
iii) Second Order Predation alone also causes extinction. iv) Simulations that produce
extinction create a vegetative shift. This new understanding sheds light on the behavioral and
anatomical shifts of bison.
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter I
Neglected Tropical Diseases of
Ruminants
Carlos Gutierrez1
University of Las Palmas de Gran Canaria, Canary Islands, Spain
Abstract
Tropical diseases are those that occur principally in the tropics and by extension in
subtropics. Some of them are considered neglected because these diseases persist
exclusively in the poorest and the most marginalized areas of the world, where the
interest of both companies and researchers is limited. There is not an official list of
neglected tropical diseases affecting ruminants, but malignant catarrhal fever, foot and
mouth disease, heartwater, bovine babesiosis, theileriosis, tsetse transmitted and non
tsetse transmitted trypanosomosis, contagious caprine pleuropneumonia or peste des petit
ruminant could be clear examples nowadays. Some initiatives, however, have been
successful in terms of control and eradication of these diseases. Global Rinderpest
Eradication Programme under the auspices of The Food and Agriculture Organization has
achieved its eradication in 2011. Unfortunately many other tropical diseases have not
received the same attention. The present chapter reviews the current status of the most
important neglected tropical diseases of ruminants, including the possible control
programs for the different affected areas.
Introduction
Neglected tropical diseases are a group of infections that are especially endemic in
certain areas of Africa, Asia, and Latin America. Some of them are considered neglected
because these diseases persist exclusively in the poorest and the most marginalized areas of
these continents, where the interest of both companies and researchers is limited. There is not
an official list of neglected tropical diseases affecting ruminants, but malignant catarrhal
1
E-mail address: cgutierrez@dpat.ulpgc.es.
2
Carlos Gutierrez
fever, foot and mouth disease, heartwater, bovine babesiosis, theileriosis, tsetse transmitted
and non tsetse transmitted trypanosomosis, contagious caprine pleuropneumonia or peste des
petit ruminant could be clear examples currently. Some attempts have been made in terms of
control and eradication of these diseases; such is the case of rinderspest, a disease considered
eradicated in 2011 thanks to the efforts of the Food and Agriculture Organization (FAO).
Unfortunately many other tropical diseases have not received the same attention.
The present chapter supposes a review of the current status of the most important
neglected tropical diseases of ruminants, focusing on clinical aspects, distribution and
possible control programs carried out.
Global Rinderpest Eradication Program, FAO.
An Example to Follow
Rinderpest has devastated many rural areas in the world affecting livestock and wildlife,
impoverishing to rural livelihoods and threading food security. Food and Agriculture
Organization (FAO), since its founding has assisted affected countries in Africa, Asia and
Europe to control rinderpest in their regions. The Global Rinderpest Eradication Programme
(GREP) was created in 1994 in an attempt to consolidate gains in rinderpest control and to
move towards outright eradication of the disease.
In close collaboration with the World Organization for Animal Health (OIE), GREP has
operated as an international coordinator promoting the global eradication of rinderpest
providing several technical supports. From the beginning, GREP was focused to establish the
epidemiology and the geographical distribution of the disease. In a second stage, actions were
promoted to contain the disease within infected eco-systems and to eliminate reservoirs of
infection through epidemiological and intelligence-based control programmes. Finally, when
evidence pointed to the virus had likely been eliminated, GREP established surveillance
systems to verify the absence of the disease.
On June 28, 2011, during the 37th FAO Conference, the member countries of this UN
organization adopted a Resolution declaring global freedom from rinderpest, being the first
animal disease to be eradicated thanks to coordinate human efforts. This fact would suppose
the second eradication of an infectious disease; the smallpox affecting humans was previously
eradicated in 1978 1.
Malignant Catarrhal Fever
Background
Malignant catarrhal fever (MCF) is caused by infection with either alcelaphine
herpesvirus-1 (AIHV-1) or ovine herpesvirus-2 (OvHV-2). It is generally a fatal disease of
cattle and many other species of Artiodactyla 2. Disease produced by AIHV-1 is restricted
to those African areas where wildebeest is present and to zoological collections elsewhere,
and has been referred to as wildebeest-associated MCF. The OvHV-2 form of the disease has
Neglected Tropical Diseases of Ruminants
3
worldwide distribution, is related to ovine husbandry and has been described as sheepassociated (SA) MCF 3.
Clinical Features
Some clinical pictures in cattle affected by MCF include peracute, head and eye,
alimentary, neurological and cutaneous 3. The head and eye form is the most common form
of MCF in cattle. Usual signs include fever, inappetence, ocular and nasal discharge, lesions
of the buccal cavity and muzzle, diarrhea and depression 4. The clinical signs can vary
depending on the infected animal species: most infected deer die within 48 h from the onset
of clinical signs, infected bison generally die within 3 days 5, cattle may survive for a week
or more 4.
Transmission
Both AlHV-1 and OvHV-2 appear to be transmitted by contact or aerosol, mainly from
wildebeest calves (AlHV-1) and lambs (OvHV-2) under 1-year old [6, 7, 8]. The horizontal
route is the common route to transmit the causal viruses between susceptible hosts, although
vertical transmission has also been demonstrated [9, 10]. Tears and nasal secretions are the
main source of free virus in wildebeest [6], while OvHV-2 viral DNA has also been detected
in sheep in samples from respiratory, alimentary and urogenital tracts [11].
Diagnosis
For diagnosis of MCF it is necessary a combination of clinical signs, histopathology and
detection of virus-specific antibodies in blood samples or DNA in blood or tissue samples.
Developments in molecular virology have improved the diagnosis of MCF in susceptible
animals. Sequencing of the genome of AlHV-1 (and genome fragments from other
cherpesviruses) has allowed the development of both generic and specific reagents for
amplification of diagnostic fragments of both AlHV-1 and OvHV-2 genomes by PCR [12, 13,
14, 15].
Treatment and Vaccines
No effective treatment or vaccine against MCF has been developed.
Foot and Mouth Disease
Background
Foot and mouth disease (FMD) is caused by a virus of the genus Aphthovirus, family
Picornaviridae. There are 7 serotypes of FMD virus (FMDV): O, A, C, SAT 1, SAT 2, SAT
4
Carlos Gutierrez
3, and Asia 1, which infect cloven-hoofed animals. Infection with any one serotype does not
confer immunity against another [16].
Of the domesticated species, cattle, pigs, sheep, goats and water buffalo (Bubalus
bubalis) are susceptible to FMD [17]. Many species of cloven-hoofed wildlife may become
infected, and the virus has also been recovered from other animal species. Amongst the
camelidae species, new world camelids and Bactrian camels have been susceptible to the
infection [18]. In Africa, SAT serotypes of FMD viruses are commonly maintained by
African buffalo (Syncerus caffer). There is periodic spillover of infection into livestock or
sympatric cloven-hoofed wildlife. Cattle are usually the main reservoir for FMD viruses
elsewhere, although in some instances the viruses seem to be specifically adapted to pigs [16].
The occurrence of subclinical forms of the disease makes the control of FMD very difficult
[19]. This disease is most important in high-production modern livestock breeds, in particular
dairy cattle and pigs. FMD results in serious economic losses in both, modern and subsistence
farming conditions [20].
Clinical Features
Clinical signs in susceptible animals would be the appearance of vesicles on the feet, in
and around the oral cavity, and on the mammary glands of females. The vesicles rupture and
then heal whilst coronary band lesions may give rise to growth arrest lines that grow down the
side of the hoof.
The change of the involved structures would indicate the age of lesions and,
consequently, the time since infection occurred [16]. Mastitis is a common sequel of FMD in
dairy cattle. The severity of the clinical picture varies with host species and the immunity
status, the strain of virus, the age and breed of animal and the exposure dose. The signs can
range from a mild or inapparent infection to severe forms. Death may result in some cases
[16].
Transmission
FMD viruses can be found in all the secretions and excretions of acutely infected
animals, including expired air. Transmission is generally reached by direct contact or, less
frequently by indirect contact with the excretions and secretions of acutely infected animals
or uncooked meat products. When the animal recovers from the acute stage of FMD, the virus
disappears except from the oropharynx of some ruminants, which persists in low levels.
Animals presenting the virus in the oropharynx for more than 28 days after infection are
considered as carriers [16].
Usually the cattle are carrier for no more than 6 months; however, in some of them the
virus could persist for more than 3 years. In African buffalo, individual animals have been
shown to harbor the virus for at least 5 years, but it is probably not a lifelong phenomenon.
Pigs do not become carriers. Small ruminants do not usually carry FMD viruses for more than
a few months [16].
Neglected Tropical Diseases of Ruminants
5
Diagnosis
To give a diagnosis in suspected animals, the FMD virus should be detected in samples of
vesicular fluid, epithelial tissue, oesophageal–pharyngeal (OP) sample, blood or milk [16]. A
myocarditis would be observed in post-mortem examination (the so-called “tiger heart”) in a
relative proportion of severe cases. However, and due to biosafety and biosecurity reasons,
diagnosis and serotype identification of the FMD virus must be performed in laboratories for
Group 4 pathogens, which are available as OIE reference laboratories for those countries
where these specialized laboratories are not established. Serological diagnoses by ELISA
minimize the risk of life virus management and there are many commercially available test
kits that detect virus antigens or antibodies [16].
Current Geographical Distribution of FMD
An eighth pool of infection, in Western Europe, was present until the 1980s, but has been
eradicated through a combination of preventive vaccination and zoo-sanitary measures [21].
The current worldwide distribution of FMDV affects developing countries where lack of
resources and infrastructure have not made its eradication possible [19]. During 2010, the
disease has been declared to OIE by the following countries: Afghanistan, Bahrain,
Bangladesh, Benin, Bhutan, Botswana, Burkina Faso, Cambodia, Central African Republic,
Chad, China, Chinese Taipei, Comoros, Democratic Republic of the Congo, Cote D’Ivoide,
Ecuador, Ethiopia, Ghana, India, Iran, Iraq, Japan, Kazakhstan, Kenya, Republic of Korea,
Laos, Lebanon, Libya, Malawi, Malaysia, Mali, Mongolia, Mozambique, Myanmar, Namibia,
Nepal, Niger, Nigeria, Oman, Palestinian Autonomous Territories, Qatar, Russia, Rwanda,
Saudi Arabia, Senegal, Somalia, South Africa, Sri Lanka, Sudan, Tanzania, Thailand, Togo,
Turkey, Uganda, United Arab Emirates, Vietnam, Yemen, Zambia and Zimbabwe [22].
Control Programs
The European Commission for the Control of Foot-and-Mouth Disease (EuFMD), a FAO
Commission, was established in 1954 to coordinate foot-and-mouth disease control within
Europe at a time when the disease supposed a severe threat to the continent. The Secretariat is
located at FAO headquarters in Rome and is currently a regional organism focused to
supporting member countries to prevent FMD, although it also facilitates information to
improve preventive measures and the exchange of knowledge and expertise between
countries.
The Southeast Asia Foot and Mouth Disease Campaign (SEAFMD) coordinates the
control programs about Foot and Mouth Disease carried out by eight countries in the region.
These countries are Cambodia, Indonesia, Lao PDR, Malaysia, Myanmar, the Philippines,
Thailand and Vietnam. The campaign is coordinated through an OIE Regional Coordination
Unit placed in Bangkok under the auspicious of the Australian Government's overseas aid
program, administered by AusAID.
In the Americas, PANAFTOSA was founded in 1951 as one of the specialized centers of
the Pan American Health Organization (PAHO/WHO). Located in the Brazilian state of Rio
6
Carlos Gutierrez
de Janeiro, the center is focused to improve the control and eradication of animal diseases,
FMD being one of the most important among them.
Economic Impact
The estimates economic impact to agriculture and industries as consequence of the
outbreak of FMD in the United Kingdom in 2001 was from £5.8 to £6.3 billion [23]. The
Republic of Korea suffered 147 outbreaks of FMD in cattle and swine in six provinces and
four major cities between 29 November 2010 and 15 February 2011. About 3.37 million
cattle, pigs, goats and deer were culled and buried at a cost of almost US$2 billion in direct
expenses and compensation to the farmers. Indirect losses have also been important affecting
in particular the cattle and swine industries [24].
Heartwater
Background
Heartwater (cowdriosis) is a rickettsial disease of domestic and wild ruminants caused by
Ehrlichia ruminantium (formerly Cowdria ruminantium) and transmitted by Amblyomma
ticks [25, 26, 27]. Ehrlichia ruminantium belongs to order Rickettsiales and family
Anaplasmataceae. Ruminants suppose the primary target of the pathogen; however there are
some reports about E. ruminantium infection in canines [28] and, it has recently also been
associated with rapid fatal encephalitis in humans [29]. In these cases, E. ruminantium
infection was evidenced on molecular detection. Isolation and characterization of the
infectious agent is necessary before E. ruminantium can be considered an emerging pathogen
in species other than ruminants and especially in humans [30].
Clinical Signs
The incubation period is ranged 2–3 weeks, and the first clinical sign is usually an acute
fever, with a sudden increase which may exceed 41°C within 1–2 days after the onset of
fever. Fever remains high for 4–5 weeks with little fluctuations and falls shortly before death
[30]. Fever is followed by inappetence, sometimes listlessness, diarrhea particularly in cattle
[31], and dyspnea indicative of lung edema. Neurological symptoms develop progressively.
The animal is restless, walks in circles, makes sucking movements and stands rigidly with
muscular tremors. Cattle may show head press or present anxious or aggressive behavior.
Finally, the animal falls down, presenting pedaling, chewing movements, opisthotonus and
nystagmus. The animal normally dies during or following such an attack. Subacute and
peracute heartwater, with less pronounced signs and sudden death respectively, can also
occur, depending on the breed of ruminant and the strain of E. ruminantium implied [30].
Neglected Tropical Diseases of Ruminants
7
Diagnosis
At post-mortem examination, the most common macroscopic findings are
hydropericardium, petechiae on the epicardium and endocardium, hydrothorax, pulmonary
edema, intestinal congestion, edema of the mediastinal and bronchial lymph nodes,
congestion of the brain and moderate splenomegaly [30, 31]. The presence of Amblyomma sp.
vectors, nervous signs, as well as transudates in the pericardium and thorax on post-mortem
examination could constitute a tentative diagnosis of heartwater. Definitive diagnosis is based
on the observation of colonies of E. ruminantium in capillary endothelial cells of the brain
[30]. DNA and PCR techniques are available to detect E. ruminantium in blood samples of
infected animals. Serological tests include indirect fluorescent antibody (IFA) test, enzymelinked immunosorbent assays (ELISAs) and Western blot, although cross-reactions with
Ehrlichia spp. occur in all these available tests [30].
Treatment and Control
There are no commercial vaccines currently. The immunization method ‘infection and
treatment’ employed in southern Africa is a method that uses infected blood followed by
treatment with tetracycline [31]. New immunizing methods based on attenuated or inactivated
pathogens are being evaluated [30]. Oxitetraciclyne and doxycycline seem to be the election
antibiotics for treatment. Control of tick population is a useful preventive tool in some cases.
Geographical Distribution
Countries where heartwater has been declared to OIE in 2010 have been: Botswana,
Burkina Faso, Comoros, Republic of the Congo, Ethiopia, Ghana, Guadeloupe (France),
Kenya, Madagascar, Mozambique, Namibia, Reunion (France), Somalia, South Africa,
Sudan, Swaziland, Tanzania, Togo, Vietnam, Zambia, Zimbabwe [22]
Economical Impact
The estimated total annual national losses amount to Z$61.3 million (US$ 5.6 million) in
Zimbabwe [32].
Bovine Babesiosis
Background
Bovine babesiosis is due to the infection by protozoan parasites of the genus Babesia,
order Piroplasmida, phylum Apicomplexa. Babesia bovis and Babesia bigemina are widely
distributed around the world and cause an important disease in Africa, Asia, Australia, and
8
Carlos Gutierrez
Latin America [33]. Babesia divergens, on the other hand, is economically relevant in some
European countries.
Transmission
Babesia spp. are transmitted by tick species [34]. Babesia bigemina and B. bovis, which
are widely present in the tropics and subtropics, are mainly transmitted by Rhipicephalus
(Boophilus) microplus. Babesia divergens, however, is transmitted by Ixodes ricinus. Other
important vectors would be Haemaphysalis sp. and other species of Rhipicephalus. Babesia
bovis is generally more pathogenic than B. bigemina or B. divergens, but B. bigemina has the
widest distribution [33].
Clinical Signs
Cattle infected by Babesia bovis show high fever, anorexia, ataxia, general circulatory
shock, and nervous impairments in some cases. Anemia and hemoglobinuria usually appear in
the course of the disease. In acute cases, the grade of parasitemia is low (less than 1%). In B.
bigemina infections, the usual clinical signs are fever, anemia and hemoglobinuria. In acute
cases, parasitemia is often high (> 10%, even 30% in some instances) [33]. The clinical
picture and the parasitemia grade of B. divergens are similar to B. bigemina infections [35].
A life-long immunity against re-infection with the same Babesia species is developed by
infected cattle. Certain degree of cross-protection in B. bigemina-immune animals against
subsequent B. bovis infections has also been evidenced [33]. Calves born from dams
previously infected by any species of Babesia rarely show any clinical signs of disease [35,
34].
Diagnosis
For the identification of the agent, samples of capillary blood are recommended, which
should be fixed with methanol, stained with 10% Giemsa for 15–30 minutes, and examined at
×800–1000 magnification under oil immersion. Available molecular methods (PCR) are
useful to detect and differentiate Babesia spp in cattle. By serology, indirect fluorescent
antibody (IFA) and ELISA are widely used, although the latter is the recommended due to its
better efficiency and objectivity in interpretation of results [33].
Vaccination
A single vaccination usually provides lifelong immunity, developing a protective
immunity in 3–4 weeks [33].
Neglected Tropical Diseases of Ruminants
9
Economical Impact
The overall impact of ticks and tick-borne diseases has been evaluated by McLeod and
Kristjanson (1999) [34] developing a spreadsheet model (Tick Cost). In the case of Australia,
the losses and control of babesiosis and anaplasmosis in cattle industry suppose US$16.9m
per annum with ‘tick worry’ adding US$6.4m to annual losses.
The model also estimated losses about 5.1, 5.4, 6.8, 21.6, 19.4, 57.2, 3.1 and 0.6 million
US dollars annually for Kenya, Zimbabwe, Tanzania, South Africa, China, India, Indonesia
and Philippines, respectively [34].
Bovine Theileriosis
Background
Theileriae are obligated intracellular protozoan parasites that infect both domestic and
wild bovines, although some species also infect sheep and goats. Theileria spp. have complex
life cycles in both vertebrate and invertebrate hosts and are transmitted by ixodid ticks. The
most pathogenic and economically relevant species affecting cattle are T. parva (causing East
Coast Fever) and T. annulata, but other four species can also infect cattle [36].
Clinical Outcome
The severity of East Coast Fever may vary depending on several factors: virulence of the
parasite strain, sporozoite infection rates in ticks and genetic background of the hosts.
Infected cattle develop fever, enlarged lymph nodes, increased respiratory rate, dyspnea and
diarrhea in some cases.
Indigenous cattle in endemic areas usually exhibit mild disease or subclinical infection,
while introduced exotic or indigenous cattle usually show severe disease. Animals infected
with T. parva show enlarged lymph nodes, fever, a gradually increasing respiratory rate,
dyspnea and occasional diarrhea [36].
Diagnosis
Diagnosis is mainly based on clinical signs, epidemiological data as well as the presence
of vectors. The identification of parasites is made on Giemsa-stained blood and lymph node
smears.
The presence of multinucleate intra-cytoplasmic and free schizonts, in lymph node biopsy
smears, is a characteristic diagnostic feature of acute infections with T. parva and T. annulata
[36].
Concerning serological tests, IFA (indirect fluorescent antibody) is the most widely used
test to detect the presence of Theileria spp. The advantage of the IFA is its good sensitivity,
specificity, and that is easy to perform.
10
Carlos Gutierrez
However, there are cross-reactivity problems among some Theileria species, and for this
reason in areas where several species inhabit IFA has serious limitations for large-scale
surveys. Nowadays, ELISA has widely replaced IFA tests for diagnosis of T. parva, and T.
mutans, given its higher sensitivity and specificity.
Molecular diagnostic tests, in particular PCR and reverse line blot hybridization, suppose
new tools for characterizing parasite polymorphisms, defining population genetics and
generating epidemiological data [36].
Geographical Distribution
Theileria parva is present in 13 countries in sub-Saharan Africa causing East Coast fever,
Corridor disease and January disease, while Theileria annulata is found in the Mediterranean
coast of North Africa, southern Europe, the near and Middle East, Central Asia, India and
China. Overlapping does not occur between endemic areas of T. parva and T. annulata. Other
Theileria spp. such as T. taurotragi and T. mutans cause no disease or mild disease, and T.
velifera is considered as non-pathogenic.
These three protozoa are mainly present in Africa, their distribution is overlapping
complicating the epidemiology of bovine theileriosis [36]. The protozoa group composed by
T. sergenti/T. buffeli/T. orientalis is now believed to consist of two species – T. sergenti,
located in the Far East, and T. buffeli/T. orientalis (referred to as T. buffeli) with a wide
distribution [37].
Control Measures
The chemical control of ticks by means of acaricides is the most practical and widely
used method for the control of theileriosis. However, this chemical control could not be fully
effective because of possible acaricide resistance, the cost of acaricides, poor management of
tick control, and illegal cattle movement between regions and countries.
Vaccination with attenuated schizont-infected cell lines has commonly been used against
T. annulata; for T. parva contro; however, infection with tick-derived sporozoites and
treatment with tetracycline is being used in many African countries [36]. Available
chemotherapeutic agents such as parvaquone, buparvaquone and halofuginone are used
against T. annulata and T. parva infections.
Treatments with these agents do not imply the complete eradication of the theileriosis,
which would suppose to develop the state of carrier in the treated individuals [36].
Economic Impact
The total regional loss due to theileriosis in 1989 was estimated to be US$ 168 million in
11 affected countries in Africa, which includes as estimated mortality of 1.1 million cattle
[38].
Neglected Tropical Diseases of Ruminants
11
Trypanosomosis
Background
Trypanosomoses are protozoan diseases, affecting both human and animals, mainly found
in tropical Africa, Latin America and Asia. Depending on the infection route, species of
trypanosomes are divided into two sections [39]: Stercoraria (subgenera Schizotrypanum,
Megatrypanum and Herpetosoma), in which trypanosomes are typically produced in the
hindgut and are then passed on by contaminative transmission from the posterior end of the
digestive tract, and Salivaria (subgenera Duttonella, Nannomonas and Trypanozoon), in
which transmission occurs by the anterior station and it is inoculative. Within section
Stercoraria, trypanosomes of medical and veterinary importance are T. theileri, T. lewisi and
T. cruzi; while within section Salivaria are included T. vivax, T. congolense, including at least
3 types (type Forest, Savannah, Kilifi, Tsavo) or species (T. godfreyi), T. simiae (including
Tsavo-type), T. brucei brucei, T. b. gambiense, T. b.rhodesiense, T. equiperdum, T. evansi
and possibly other species such as T. suis.
Main Characteristics of the most Pathogenic Animal Trypanosomes in
Ruminants
Trypanosoma cruzi infection (Chagas disease) is usually referred to human infections;
however, this protozoon presents a wide host range. Many species of mammals, including
bats, have been reported to be infected with T. cruzi. Chagas disease affects exclusively Latin
American continent where most of the countries have reported this disease. Trypanosoma
cruzi is not a livestock trypanosome as such, but it is sometimes found in domestic ruminants
and horses [40]. Transmission of T. cruzi is essentially cyclical through bugs that belong to
the Reduviidae family: triatomines, or reduviid bugs, of the genera Rhodnius,Panstrongylus
and Triatoma. The metacyclic trypomastigote infective form (metatrypanosome) is present in
the excreta of the bugs that contaminate bite wounds or the mucous membranes, particularly
the eye [41]. For diagnosis, in acute phase (when parasitemia is normally high) conventional
parasitological tests including stained smear examination and micro-hematocrit centrifugation
techniques are usually applied. In chronic phases, indirect immunofluorescence assay (IFA),
indirect hemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA)
have been used.
Trypanosoma vivax, T. congolense, and T. brucei brucei constitute a tsetse-transmitted
group that causes a complex disease known as “nagana”. It is extended over 10 million km2
and affects 37 countries, mostly in Africa. Nagana is a very important disease in cattle, in
which it produces enormous economic losses. Tsetse-transmitted trypanosomes can also
infect humans, in particular T. brucei gambiense or T. brucei rhodesiense, the causal agents of
sleeping sickness. Clinical signs of nagana would include intermittent fever, anemia, edema,
decreased fertility and abortions, loss of milk and meat production and work capacities and
emaciation. Anemia is a typical finding in infected animals and is commonly followed by
weight loss, decreased productivity and mortality [42]. Clinical signs, however, can vary with
the type of cattle; thus the so-called trypano-tolerant Bos taurus exhibit milder signs than the
trypano-sensible zebu cattle [43]. This is also true in sheep and goats to a lesser extent; the
12
Carlos Gutierrez
southern Djallonke sheep and the West African Dwarf (WAD) goat being trypano-tolerant
compared to the large size sheep and goat originating from the northern Sahelian area [44].
There is no clinical sign or post-mortem lesion that can be considered as pathognomonic of
nagana. For that reason, diagnosis must be based on the direct detection of trypanosomes by
microscopic visualization or by means of polymerase chain reaction (PCR) for which positive
results mean active infection, or can be diagnosed indirectly by serological techniques [42].
Trypanosoma evansi, which causes a disease known as surra, is widely distributed and
affects domestic livestock and wildlife in Africa, Asia and Latin America [45]. Recently
severe outbreaks of surra in dromedaries have been described in mainland Europe [46]. The
presence of the parasite causes an important economic impact on the endemic areas. The
transmission of T. evansi is mechanical by biting flies particularly belonging to genus
Tabanus, Stomoxys and Liperosia. Vampire bats are biological vectors in South America,
and, they are considered as host, reservoir and vector of the parasite [47]. The disease is
clinically manifested by pyrexia, progressive anemia, weight loss and lassitude. Edema, in
particular affecting the lower parts of the body, is a common finding. In advanced cases,
parasites invade the central nervous system (CNS), which can lead to nervous signs
(progressive paralysis of the hind quarters and exceptionally paraplegia), especially in horses,
but also in other host species before complete recumbence and death. Abortions and
immunodeficiency associated to T. evansi infection have also been reported in bovines [48].
The clinical signs of T. evansi infection are indicative but are not pathognomonic for
diagnosis; thus, laboratory methods are required. Identification of the agent is needed, which
can be reached by direct, but also by indirect parasitological methods since T. evansi can
grow in laboratory rodents. Within serological tests, enzyme-linked immunosorbent assay
(ELISA), card agglutination tests (CATT/T. evansi), indirect immunofluorescent antibody test
(IFAT) and immune trypanolysis tests are recommended [48].
Control Programs
Control programs against trypanosomosis include both, the use of chemical agents as
therapeutic and/or preventive and, at the same time, a control plan to eradicate or minimize
the vector. Currently there are four trypanocides available for livestock in the market; three of
them (diminazene, isometamidium and homidium) are used against tsetse transmitted
trypanosomosis, while the fourth one (melarsomine) is specifically used for T. evansi. To
control vectors, organochlorides such as DDT, dieldrin or γ-BHC (lindane; gamma isomer of
benzene hexachloride) have been applied to African tsetse areas; these molecules were really
persistent and killed many generations of flies [49]. However, for many decades the wildlife
on the continent was seriously affected due to the indiscriminate use of residual doses of
broad-spectrum insecticides. In Latin America, some attempts have been made to control the
transmission of trypanosomes by vampire bats using net screens, but the results were
insufficient for large scale application. Attempts to control horseflies with insecticide spray
on cattle were somehow successful, but their use could only be restricted to isolated farms,
which limited the extension of the method [47].
Neglected Tropical Diseases of Ruminants
13
Economical Impact
In Africa, tsetse-transmitted trypanosomosis is one of the most important constraints to
agricultural production in the tropical and subtropical areas of the continent. It has been
reported that in an area of 9 million km2 about 50 million people and 46 million cattle are
exposed to the African tsetse transmitted trypanosomosis [50]. In these areas, the disease
decreases the milk and meat productions about 50%. Latin America, on the other hand, is
home to approximately a quarter of the world bovine population, i.e., 280 million heads, most
of which are exposed to T. cruzi, T. vivax and T. evansi infections [47] and even to T. vivax as
recently observed in Brazil [51]. Some studies carried out in Bolivian lowlands and Brazilian
Pantanal have shown that 11 million cattle, with an approximate value of US$3 billion, are at
risk to contract T. vivax infection; thus the economic impact can be more than US$160
million [52]. For cattle ranchers in the Pantanal region, Trypanosoma evansi can produce
economic losses of about US$2.4 million, and about 6,462 per year [53]. Concerning the
Asian continent, official information about Trypanosoma evansi in the Philippines revealed
estimated losses of more than US$1.1 million in nine years caused only by mortality.
However, many cases frequently go unreported and it has been estimated that the true losses
due to the parasite may be higher and are about US$7.9 million for nine years [54]. In
Indonesia, it has been reported that the annual losses from mortality and morbidity associated
with T. evansi were US$28 million [55].
Contagious Caprine Pleuropneumonia
Background
Contagious caprine pleuropneumonia (CCPP) is a severe goat disease affecting many
countries in Africa and Asia where the total goat population is over 500 million [56].
Classical, acute CCPP is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp)
[57], originally known as the F38 biotype.
This organism was first isolated and shown to cause CCPP in Kenya [57]; in 2010
countries declaring to OIE the disease were Afghanistan, Angola, Bahrain, Chad, China,
Ethiopia, India, Iran, Kenya, Kuwait, Niger, Oman, Sierra Leone, Somalia, Tajikistan,
Tanzania, Yemen.
However, the exact distribution of the disease is unknown and may be much more
widespread, because CCPP is often confused with other respiratory diseases and also due to
the isolation of the causative organism is difficult [58].
The disease was also described in mainland Europe in 2004, particularly in Turkey
causing severe outbreaks with losses of up to 25% of kids and adults in some herds [59].
The role of sheep in CCPP is unclear, but it has been considered as a reservoir for the
disease. The detection of antibodies from clinically affected sheep in mixed herds with
infected goats [60] or even the isolation of Mccp from healthy sheep [61] would support this
assumption.
14
Carlos Gutierrez
Diagnosis
Diagnosis requires culture of the causative organism from lung tissue samples and/or
pleural fluid taken at post-mortem. After cloning and purification, isolates can be identified
by several biochemical, immunological and molecular tests, although it is very difficult.
Polymerase chain reaction (PCR) has recently shown to be specific and sensitive, and can be
applied directly to clinical material, such as pleural fluid and lung [58]. Concerning
serological diagnosis, the complement fixation test (CFT) remains as the most recommended
serological test for CCPP, although other tests are being successfully evaluated, such as latex
agglutination test, a pen side test, indirect hemagglutination and ELISA. The latter does not
detect all reactors, but its specificity and suitability for large-scale testing make it an
appropriate test for epidemiological investigations [58].
Vaccine against CCPP caused by Mccp is available commercially.
Peste De Petit Ruminants
Background
Peste des petits ruminants (PPR) is an acute disease of sheep and goats caused by a virus
belonging to genus Morbillivirus, family Paramyxoviridae [62]. The disease is characterized
by fever, pneumonia, oculonasal discharges, stomatitis and diarrhea. PPR exhibits similar
signs to contagious caprine pleuropneumonia or pasteurellosis, and they must be
differentiated. In fact, pasteurellosis can be a secondary infection of PPR, given the
immunodepression induced by the causal agent of PPR, the PPR virus [63]. The disease is
transmitted mainly by aerosols between animals living in close contact [64].
Clinical Signs
PPR affects particularly goats and sheep, being more severe in goats. Cattle can also be
affected but only in a subclinical form. In severe conditions cattle can show clinical evidence
of PPR virus infection, a clinical picture that bears a resemblance to rinderpest infection [63].
The incubation period is about 4–6 days; the onset of the disease is acute showing fever (up to
41°C) for 3–5 days, depression, anorexia and dry muzzle. The oculonasal discharges is serous
in the beginning and become progressively mucopurulent, persisting for around 2 weeks. The
gums show hyperemia and progressive erosive lesions with sialorrea. The animal develops
pneumonia with coughing, pleural rales and abdominal breathing. In a later stage, a watery
blood-stained diarrhea occurs. The morbidity and mortality are very high, except in milder
outbreaks [63].
Diagnosis
For the identification of the agent, samples should be taken in the acute phase of the
disease when clinical signs are evident. The samples would be taken from conjunctival
Neglected Tropical Diseases of Ruminants
15
secretions, nasal discharges, buccal and rectal material and blood as well. Rapid diagnosis is
performed by means of ELISA, counter immunoelectrophoresis and agar gel
immunodiffusion and PCR. Serological tests that are routinely used are the virus
neutralisation and the competitive ELISA [63].
Vaccines based on live attenuated PPR virus are commercially available.
Geographical Distribution
In 2010 the disease has been reported to OIE by Afghanistan, Bangladesh, Benin,
Bhutan, Burkina Faso, Central African Republic, Chad, China, Comoros, Democratic
Republic of the Congo, Republic of the Congo, Cote D’Ivoire, Ethiopia, Gabon, Ghana,
Guinea, Guinea Bissau, India, Iran, Iraq, Kenya, Kuwait, Maldives, Mauritania, Nepal, Niger,
Nigeria, Oman, Palestinian Autonomous Territories, Saudi Arabia, Senegal, Sierra Leone,
Somalia, Sudan, Tanzania, Togo, Turkey, Uganda, United Arab Emirates and Yemen [22].
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Food and Agricultural Organization of the United Nations (FAO), 2011,
http://www.fao.org/ag/againfo/programmes/en/grep/home.html
W.H. Reid and D. Buxton, Proceedings of the Royal Society of Edinburgh B,
Biological Sciences 82, 261–273 (1984)
World Organisation for Animal Health (OIE), Malignant catarrhal fever. In: Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals, Paris, http://www.oie.int/ (2008)
G.C. Russell, J.P. Stewart and D.M. Haig, Vet. J. 179, 324 (2009).
D. O’Toole, H. Li, C. Sourk, D.L. Montgomery and T.B. Crawford, J. Vet. Diagn.
Invest. 14, 183 (2002).
E. Z. Mushi, F.R. Rurangirwa and L. Karstad, L., Vet. Microbiol. 6, 281 (1981).
S.I.F. Baxter, A. Wiyono, I. Pow and H.W. Reid. Arch. Virol. 142, 823 (1997).
H. Li, G. Snowder, D. O’Toole and T.B. Crawford, J. Clin. Microbiol. 36, 223 (1998).
Plowright, W., Res. Vet. Sci. 6, 56 (1965).
P.B. Rossiter, J. Comp. Pathol. 91, 303 (1981).
D. Hüssy, F. Janett, S. Albini, N. Staüber, R. Thun and M. Ackermann, J. Clin.
Microbiol. 40, 4700 (2002).
A. Bridgen and H.W. Reid, Res. Vet. Sci. 50, 38 (1991).
J. Katz, B. Seal and J. Ridpath, J. Vet. Diagn. Invest. 3, 193 (1991).
S.I.F. Baxter, I. Pow, A. Bridgen, and H.W. Reid, Arch. Virol. 132, 145 (1993).
E.J. Flach, H. Reid, I. Pow, and A. Klemt, A., Res. Vet. Sci. 73, 93 (2002).
World Organisation for Animal Health (OIE), Foot and mouth disease. In: Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals, 2009, Paris, www.oie.int
Food and Agricultural Organization of the United Nations (FAO). Emerging Diseases
of Livestock. Vol. 1. W.A. Geering, The Diseases and their Diagnosis, ed. FAO, Rome,
Italy, pp 43–51 (1984).
16
Carlos Gutierrez
[18] M. Larska, U. Wernery, J. Kinne, R. Schuster, G. Alexandersen and S. Alexandersen,
Epidemiol Infect. 137, 549 (2009).
[19] D.J. Paton, K.J. Sumption and B. Charleston, Philos. Trans. R. Soc. Lond. B. Biol. Sci.
364, 2657 (2009).
[20] M.M. Rweyemamu and Y. Leforban, Adv. Virus Res. 53, 111 (1999).
[21] J.F. Valarcher, Y. Leforban, M. Rweyemamu, P.L. Roeder, G. Gerbier, D.K. Mackay,
K.J. Sumption, D. Paton and N. Knowles, Transbound. Emerg. Dis. 55, 14 (2008).
[22] WAHID, Animal Health Information, OIE, 2011, http://web.oie.int/wahis/public.php?
page=disease_status_detail
[23] D. Thompson, P. Muriel, D. Russell, P. Osborne, A. Bromley, M. Rowland, S. CreighTyte and C. Brown. Rev. Sci. Tech. Off. int. Epiz. 21, 675 (2002).
[24] Food and Agricultural Organization of the United Nations (FAO), 2011.
http://www.fao.org/ag/againfo/home/en/news_archive/2011_economic-impactFMD.html
[25] C.P. Bekker, S. de Vos., A. Taoufik, O.A. Sparagano and F. Jongejan (2002). Vet.
Microbiol. 89, 223 (2002).
[26] J.S. Dumler., A.F. Barbet, C.P. Bekker, G.A. Dasch, G.H. Palmer, S.C. Ray, Y.
Rikihisa and F.R. Rurangirwa (2001). Int. J. Syst. Evol. Microbiol. 51, 2145 (2001).
[27] T. F. Peter, M.J. Burridge and S.M. Mahan, Trends Parasitol. 18, 214 (2002).
[28] M.T.E.P. Allsopp and B.A. Allsopp, J. Clin. Microbiol. 39, 4204 (2001).
[29] M. Louw, M.T.E.P. Allsopp and E.C. Meyer, S. Afr. Med. J. 95, 948 (2005).
[30] World Organisation for Animal Health (OIE), Heartwater. In: Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals, 2008, Paris, www.oie.int
[31] J. D. Bezuidenhout, L. Prozesky, J.L. Du Plessis and S.R. van Amstel, Heartwater. In:
J.A.W. Coetzer, G.R. Thomson, R.C. Tustin and N.P.J. Kriek, Infectious Diseases of
Livestock, with special reference to Southern Africa, Oxford University Press, Oxford,
1994, Vol. 1, pp. 351–370.
[32] A.W. Mukhebi, T. Chamboko, C.J. O'Callaghan, T.F. Peter, R.L. Kruska, G.F. Medley,
S.M. Mahan and B.D. Perry, Prev Vet Med. 39, 173 (1999).
[33] World Organisation for Animal Health (OIE), Bovine babesiosis. In: Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals, 2010, Paris, www.oie.int
[34] R. Bock, L. Jackson, A. de Vos and W. Jorgensen, Parasitology 129, S247 (2004).
[35] A. Zintl, G. Mulcahy, H.E. Skerrett, S.M. Taylor amd J.S. Gray, Clin. Microbiol. Rev.
16, 622 (2003).
[36] World Organisation for Animal Health (OIE), Theileriosis. In: Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals, 2008, Paris, www.oie.int
[37] K. Fujisaki, S. Kawazu and T. Kamio, Parasitol. Today 10, 31 (1994).
[38] A.W. Mukhebi, B.D. Perry and R. Kruska, Prev. Vet. Med. 12, 73 (1992).
[39] C.A. Hoare, The trypanosomes of mammals. A zoological monograph. Oxford:
Blackwell Scientific Publications, 1972.
[40] M. Desquesnes and M.L. Dia, Vet. Parasitol. 119, 9 (2004).
[41] M. Desquesnes and C. Gutierrez, Animal trypanosomosis: An important constraint for
livestock in tropical and sub-tropical regions. In: M.T. Javed, Livestock Rearing,
Farming and Practices, Nova Science Publishers, New York, 2011, in press.
Neglected Tropical Diseases of Ruminants
17
[42] World Organisation for Animal Health (OIE), Trypanosomosis (Tsetse-transmitted). In:
Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2008, Paris,
www.oie.int
[43] J. Naessens, Int. J. Parasitol. 36, 521 (2006).
[44] S. Geerts, S. Osaer, B. Goossens and D. Faye, Trends Parasitol. 25, 132 (2009).
[45] A.G. Luckins and R.H. Dwinger, Non-tsetse-transmitted Animal Trypanosomiasis. In:
I. Maudlin, P.H. Holmes and M.A. Miles, The Trypanosomiases, Cabi Publishing,
Trowbidge, 269-281 (2004).
[46] C. Gutierrez, M. Desquesnes, L. Touratier and P. Büscher, Vet. Parasitol. 174, 26
(2010).
[47] M. Desquesnes. Livestock Trypanosomoses and their Vectors in Latin America. Paris,
OIE (World Organisation for Animal Health), 2004, www.oie.int
[48] World Organisation for Animal Health (OIE), Trypanosoma evansi infection (Surra).
In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2010b, Paris,
www.oie.int
[49] I.F. Grant, Trends Parasitol. 17, 10 (2001).
[50] A.P.M. Shaw, Economics of African trypanosomiasis. In: I. Maudlin, P.H. Holmes and
M.A. Miles, The Trypanosomiases, Cabi Publishing, Trowbidge, 269-281 (2004).
[51] A.S. da Silva, H.A. Garcia Perez, M.M. Costa, R.T. Franca, D. de Gasperi, R.A.
Zanette, J.A. Amado, S.T. Lopes, M.M. Teixeira and S.G. Monteiro, Parasitol. Res.
108, 23 (2011).
[52] A.M. Dávila and R.A. Silva, Ann. N. Y. Acad. Sci. 916, 199 (2000).
[53] A. Seidl, A.S. Moraes, R.A.M.S. Silva, Prev. Vet. Med. 33, 219 (1998).
[54] M.F. Manuel, J. Protozool. Res. 8, 131 (1998).
[55] S.A. Reid, Trends Parasitol. 18, 219 (2002).
[56] R.M. Acharya, Goat production. In: Recent advances in goat production. Fifth
International Conference on Goats, New Delhi, India, March 1992, 49–94.
[57] R.H. Leach, H. Erno and K.J. MacOwan, Int. J. Syst. Bacteriol. 43, 603 (1993).
[58] World Organisation for Animal Health (OIE), Contagious Caprine Pleuropneumonia.
In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2008, Paris,
www.oie.int
[59] U. Ozdemir, S. Ozdemir, J. March, C. Churchwood and R.A.J. Nicholas, Vet. Rec. 156
(2005).
[60] A.C. Kibor and P.G. Waiyaki, Anim. Health Prod. Afr. 34, 157 (1986).
[61] J.K Litamoi, S.W Wanyangu and P.K. Simam, Trop. Anim. Health Prod. 22, 260
(1990).
[62] E.P.J. Gibbs, W.P. Taylor, M.J.P. Lawman and J. Bryant, Intervirology 2, 268 (1979).
[63] World Organisation for Animal Health (OIE), Peste des Petits Ruminants. In: Manual
of Diagnostic Tests and Vaccines for Terrestrial Animals, 2008, Paris, www.oie.int
[64] P.C. Lefevre and A. Diallo, Rev. sci. tech. Off. int. Epiz. 9, 951 (1990).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter II
Liver and Stomach Flukes of Wild
and Domestic Ruminants:
Molecular Approach in
Their Discrimination
Eva Bazsalovicsová1, Ivica Králová-Hromadová1,
Katarína Oberhauserová2 and Marta Špakulová1
1
Institute of Parasitology, Slovak Academy of Sciences,
Košice, Slovak Republic
2
The University of Veterinary Medicine and Pharmacy in Košice,
Košice, Slovak Republic
Abstract
Parasitic diseases represent the most prevalent and important problems of grazing
ruminants that can significantly influence their daily activity and health. The most
ubiquitous are ectoparasites such as biting and sucking lice, bot flies or sarcoptic mange
mites manifested by pruritus, rubbing, scratching and dermal irritation, as well as ticks
which are possible transmitters of tick-borne diseases. Endoparasites similarly pose a
potential threat and constraint of livestock health status. Out of protozoan parasites,
coccidiosis may cause severe profuse watery diarrhoea resulting to dehydration,
depression and weakness especially in young animals. Domestic and free-living
ruminants are also frequently infected by various endohelminths of the classes Nematoda
(phyllum Nemathelminthes), Cestoda and Trematoda (Platyhelminthes) invading organs
of the respiratory apparatus, as well as the digestive system. Out of the trematodes,
digenetic liver flukes Fascioloides magna, Fasciola hepatica, Dicrocoelium dendriticum
and the stomach fluke Paramphistomum cervi often induce similar clinical signs as
weight loss, diarrhea, anemia, edema and reduced milk production, that causes
difficulties in differential diagnostics. Most of the infections are exactly identified postmortem by distinct species-specific morphological characters of adults of flukes. “Giant
liver fluke” F. magna differs mainly in the distinctly large body up to 80 mm long and
rounded anterior body part. Smaller but rather similar “common liver fluke” F. hepatica
20
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
is up to 35 mm long possessing anterior cone and ’shoulders’. Adult worms of “lancet
liver fluke” D. dendriticum are rather tiny, measuring up to 10 mm and the body is
strongly tapered at both ends. Mature “stomach flukes” P. cervi have thick robust and
cone-shaped body up to 6.5 mm long with conspicuous posterior sucker. Contrary to this,
in vivo diagnosis based on the coprological examination faces serious problems. Eggs,
especially those of F. hepatica, F. magna and P. cervi, are considered to be hardly
distinguishable. They are of very similar size, shape and internal structure and their
unequivocal species differentiation is disputable. To resolve the issue of the accurate in
vivo discrimination of liver and stomach flukes in ruminants, a molecular approach
applying PCR-based techniques has recently been designed. The ribosomal internal
transcribed spacer (ITS2, rDNA) provides sufficient species-specific characters that can
be utilized in molecular diagnostics of coprological samples in parasitological
laboratories and veterinary clinics. The chapter summarizes data on taxonomy,
morphology, distribution, clinical signs and PCR-based species differentiation of
mentioned liver and stomach flukes of wild and domestic ruminants.
Introduction
Wild and domestic ruminants could in particular be infected with various pathogenic
agents including parasitic species that have the potential to be harmful. Parasites and parasitic
diseases are of great importance in veterinary medicine due to their influence on animal
health and productivity, often causing serious economic losses. Therefore, the parasites
should be monitored and studied at all scientific levels thus enabling us to have a better
understanding of them. In the grazing ruminants, ectoparasite infestations appear to be the
most widespread and common problem. Out of them, sucking lice (order Anoplura), chewing
lice (Mallophaga), biting flies (Diptera), itch mites or sarcoptes mites as well as ticks
(Acarina) are the most important groups of ectoparasites. In general, they are responsible for
restlessness caused by marked irritation, intense itching, rubbing, scratching; they could also
harm the health of their hosts by blood sucking and may transmit (especially ticks) a wide
range of pathogenous bacterial agents e.g. Borrelia spp., Anaplasma spp. and protistan
Babesia spp. [1]. Heavy mortalities caused by the ectoparasites are rather uncommon;
however, a variety of associated clinical signs may result in considerable production losses in
affected animals. Endoparasites represent another group of pathogens in grazing ruminants.
Infections by protozoan parasites, especially Eimeria spp. (Coccidia) may cause such clinical
symptoms as fever, diarrhea, abdominal tenderness, nervous distress, dehydration and
anorexia, primarily in young animals [2]. Serious health problems in ruminants are also
caused by endohelminths of phylla Nemathelminthes (class Nematoda), and Platyhelminthes
(Cestoda and Trematoda) which affect the respiratory as well as digestive systems. Nematode
lungworms in ruminants (e.g. genera Dictyocaulus, Protostrongylus, and Muellerius) can
induce occasional coughing to severe respiratory distress depending on the number of
infected larvae [3]. Species of several tapeworm genera as Taenia and Echinococcus use
domestic and wild ruminants as intermediate hosts, and their larval stages occupy skeletal
muscles or soft tissues (liver and brain more commonly) [4]. Trematode parasitism is
considered to lower significantly ruminant productivity all around the world [3]. Parasites of
the class Trematoda are divided into two subclasses Aspidogastrea and Digenea, and the latter
comprises more than 18 thousands of nominal species being the largest group of metazoan
Liver and Stomach Flukes of Wild and Domestic Ruminants
21
endoparasites of animals [5]. The digenean trematodes, which utilize freshwater snails as
intermediate hosts, are responsible for more or less serious infections of ruminants. Amongst
them, the most common flukes of wild and domestic ruminants are members of the families
Fasciolidae, Dicrocoeliidae and Paramphistomidae, especially common liver fluke Fasciola
hepatica, giant liver fluke Fascioloides magna, lancet liver fluke Dicrocoelium dendriticum,
and the stomach fluke Paramphistomum cervi.
Relevance of these flukes to veterinary medicine is beyond any doubt because the
infections could represent serious health problems for wild and domestic ruminants; the
massive number of flukes may cause acute, occasionally lethal infections.
The flukes are distributed all around the world in large spectrum of grazing ruminants,
but also in other animal hosts and man - in case of F. hepatica and D. dendriticum.
Morphological characters of the fluke species are specific what results in their reliable
differentiation after post mortem examination of the animal hosts (see further). However,
routine examination in parasitological laboratories and veterinary clinics essentially requires
in vivo diagnostics.
Most of these trematode infections are associated with alike clinical signs such as weight
loss, diarrhea and anemia of the definitive hosts, or any marked clinical signs lack; therefore,
a proper assessment of diagnosis is not possible. A standard procedure of in vivo diagnosis is
the coprological examination which relies on the morphology of the eggs. Although this
method is simple to perform and rather routine in parasitological laboratories, it faces a
serious obstacle particularly in case of the discrimination of F. hepatica, F. magna and P.
cervi eggs that are very similar in size, shape and internal structure, causing major doubts in
the unequivocal species differentiation.
In cases of the problematic in vivo discrimination based on morphological characters, a
molecular approach applying PCR-based techniques is very helpful and permits the
determination of fixed genetic interspecific differences.
For the determination of species-specific molecular markers of parasites, several aspects
such as host range, geographic distribution, morphological variability, intra- and interspecific
variation, species limits and definitions of higher taxa have to be considered.
Detailed studies at the DNA level provide exact data that can eliminate taxonomic
problems; however, an appropriate selection of genes or regions which uncover substantial
intra- and interspecific variability plays a key role. Ribosomal DNA clusters, especially the
internal transcribed spacers (ITS1 and ITS2), are predominant and frequently used species
markers [6]. They allow to use the molecular data for the exact molecular discrimination of
parasitic organisms. In general, an intraspecific variation in ITS is low, whereas substantial
differences between well recognized species mostly exists [7].
The main aim of the chapter is to summarize data on the selected liver flukes F. hepatica,
F. magna, D. dendriticum and the stomach fluke P. cervi, common parasites of free living and
domestic ruminants. In particular, the chapter provides data on their systematic classification,
morphology of adult individuals and eggs, hosts spectrum, localization, distribution and
clinical signs. Besides, recently described species-specific ribosomal internal transcribed
spacer 2 (ITS2, rDNA) markers, enabling the reliable molecular discrimination of these
trematodes, are provided.
22
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
Systematic Classification
According to the currently accepted system [8, 9], the classification of F. hepatica, F.
magna, D. dendriticum and P. cervi is as follows:
Out of the discussed flukes, Fascioloides represents a single species genus involving
solely F. magna [9]. The genera Fasciola, Dicrocoelium and Paramphistomum have more or
less rich species spectrum; however, the taxonomy of the taxa is still rather problematic
[10,11,12, 13].
Fasciola Hepatica
Morphology of Adults and Eggs
Adult worms of F. hepatica (Figure 1A) measure in average 30-35 mm in length × 8-15
mm in width. The body is covered by the tegument with small spines; it is dorso-ventrally
flattened, usually foliate but may be oval in outline. The anterior extremity forms a cephalic
cone which is demarcated by typical ’shoulders’ from the remainder of the body. The flukes
belong to the distomic morphological type possessing the oral sucker situated at the anterior
cone and the ventral sucker at the base of cephalic cone.
Figure 1. Adult individuals of Fasciola hepatica (A), Fascioloides magna (B), Dicrocoelium
dendriticum (C) and Paramphistomum cervi (D) fixed in ethanol. Scale bar = 10 mm.
Liver and Stomach Flukes of Wild and Domestic Ruminants
23
Figure 2. Eggs of Fasciola hepatica (A), Fascioloides magna (B), Dicrocoelium dendriticum (C) and
Paramphistomum cervi (D). Scale bar = 100 µm.
The pharynx is well developed, the esophagus is usually very short, and cecum
terminates blindly near the posterior extremity. The male genital system is composed of two
branched testes located in tandem in the middle body region and the cirrus-sac located
anterodorsally to the ventral sucker apparatus. The female genital system is formed by a
branched submedian ovary which lies on the right side, and by the rather short, rosette-like
uterus. The vitteline follicles are situated along left and right body margins, and they lie both
dorsally and ventrally to the intestine [9,14]. Mature flukes of F. hepatica produce
operculated eggs (Figure 2A), their mean size are 135-142 μm in length and 75-82 μm in
width [15]. They are yellowish brown in color, oval in shape and the operculum may be in
some cases indistinct. The eggs consist of fertilized ovum surrounded by large number of
yolk granules [16].
Host Spectrum and Localization of the Parasite
Fasciola hepatica has a very large spectrum of definitive hosts but the most important
and common ruminants infected are cattle (Bos taurus), sheep (Ovis aries) and goat (Capra
hircus). Many other herbivores, like horses (Equus caballus), pigs (Sus scrofa), donkeys
(Eguus africanus), as well as wild ruminants, e.g. red deer (Cervus elaphus elaphus), roe deer
(Capreolus capreolus), fallow deer (Dama dama), and white-tailed deer (Odocoileus
virginianus) are often infected, but in global scale the parasite is of less economic importance
in these hosts [17]. The fluke has adapted also to other autochthonous mammal species such
as camelids in Africa and marsupials in Australia [18]; nutria (Myocastor coypus) and black
rat (Rattus rattus) were described as reservoir hosts [19,20]. Adult individuals of F. hepatica
were also found in cottontail rabbit (Sylvilagus floridanus) [21].
Man also serves as a suitable host of F. hepatica, and infections are not rare in areas
where infected ruminants are present [22]. Epidemic human infections are associated with
24
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
consumption of the watercress on which infective metacercariae are attached, or by drinking
water containing free-floating metacercariae [23]. Major health problems are encountered in
South America (Bolivia, Peru, Chile, Ecuador), the Caribbean area (Cuba), in Western
Europe (Portugal, France and Spain), in the Caspian area (Iran and neighboring countries),
and northern Africa (Egypt) [24].
To complete the life cycle of F. hepatica, the parasite requires freshwater snails of the
family Lymnaeidae as an intermediate host. In the snails, the parasite reproduces asexually
and snails finally release cercariae which encyst on the aquatic or wetland vegetation and
form metacercariae. These infective agents are ingested by a suitable definitive host [15].
Fasciola hepatica is the parasite chiefly confined to the liver, adult individuals occur in bile
ducts of infected animals.
Distribution
Fasciola hepatica is believed to be of an European origin and it has become
cosmopolitan due to its high adaptability to different lymnaeidae snail hosts, a broad spectrum
of definitive hosts, and due to introduction of the susceptible infected intermediate (water
snails) or definitive hosts into new areas [25, 26].
In European countries, most of the F. hepatica infections have been reported in domestic
ruminants, particularly in cattle and sheep. An increasing incidence of fascioliasis in cattle
was described in the Czech Republic during the period of 1996 and 1999 [27]. In Poland,
significant economic loss was caused by F. hepatica infections in cattle [28], as well as in
cattle and sheep in central and eastern regions of the country [29]. Significant prevalence of
bovine fascioliasis was detected in many parts of Switzerland within the last two decades
[30,31,32]. Fascioliasis has also been monitored in Mediterranean countries. Various
prevalence of infection in cattle was detected in Spain and France [33,34], and cattle and
sheep from Italian husbandry were also found to be infected [35, 36]. Bovine fascioliasis was
further documented in Portugal [37], Denmark [38], United Kingdom [39] and Ireland [40].
Various species of cervids and bovids were infected in Slovakia [41], Czech Republic [42],
Austria [43], Poland [44], Italy [45], Portugal [46], Spain [47], Belarus [48], Slovenia [49]
and Lithuania [50]. Infections in wild ruminants resulted mainly from overlapping habitat
with domestic ruminants.
In Asia, fascioliasis was reported in wild ruminants in India and Pakistan [51,52,53], and
in sheep in Saudi Arabia [54]. Among African countries, fascioliasis was determined in wild
ruminants in Nigeria [55], in cattle and goats from Morocco [56], and in sheep from Egypt
[57]. Fascioliasis is the endemic disease of ruminants also in the North America, e.g. in
Canada [58], US states such as Montana, California, Texas, Florida [17], and in Mexico [59].
Particular reports exist from the South America, e.g. in cattle from Brazil [60] and in
domestic ruminants in New Zealand [61].
Clinical Signs
Severity of the fascioliasis depends on the level of infection caused particularly by the
number of ingested metacercariae, the nutritional plane of the animals and also individual
Liver and Stomach Flukes of Wild and Domestic Ruminants
25
resistance of the animals [62]. Clinical signs are generally not specific, the disease has often
subclinical course. In domestic ruminants as obligatory hosts, there is a difference between
the disease manifestations.
The acute stage is characterized by fever, abdominal pain in right quadrant, icterus, pallor
of membranes, weakness and loss of condition. Acute fascioliasis may cause sudden death of
ruminants [62]. In subacute form, the death comes a bit later as a consequence of protracted
anemia, hemorrhages and weight loss. Chronic fascioliasis is accompanied by reduced weight
gain, pale mucosae caused by anemia, unthriftiness, submandibular edema, and reduced milk
secretion [63]. Long-term heavy infestations in sheep are fatal, while the chronic disease in
cattle often manifests as equivocal reduction in milk production or no clinical signs are
present [29]. Infections of cervids are generally not associated with apparent clinical signs.
Depending on the intensity of infection, animal grow, body weight, conception ability and
antler growing are affected [64].
The acute phase of fascioliasis in game is characterized by total weakness, lassitude,
innapetence, anemia, late hair changing, and decrease of antler quality, and sometimes dead.
In the chronic form, diarrhea, weight loss, total weakness and submandibular edemas are
observed; cervids may die due to overall exhaust [64]. In camelids, low infection of F.
hepatica is presented with no clinical evidence, whereas heavily parasitized animals mostly
show anorexia and lethargy [65].
Fascioloides Magna
Morphology of Adults and Eggs
Mature flukes of F. magna (Fig.1B) measure 40-80 mm in length and 20-35 mm in
width, their body are oval, dorso-ventrally flattened, reddish-brown in color, and covered by
tegument with spines. Anterior part of the body is rounded. Fascioloides magna belongs to
the distomic morphological type of flukes; it possesses two suckers – the oral sucker is
localized terminally, and the ventral sucker is situated closely behind it [14,66].
Gastrointestinal system starts with mouth situated in the center of the oral sucker, followed by
small pharynx, esophagus and two long cecum branched into many diverticula. The genital
pore is situated in front of the ventral sucker and is common for both male and female genital
systems.
The male genital system is composed of two deeply lobed testes which are situated side
by side in the middle third of the body, followed by efferent ducts, ductus deferens, vesicular
glands (interna and externa), ejaculatory duct and cirrus-sac not extending posteriorly beyond
ventral sucker [9]. The branched ovary lies on the right side in front of the testes. The uterus
is short forming several folds in the anterior part of body. Profusely developed vitelline
follicles are situated along left and right body margins and are present only on ventral side of
flattened body [14]. Eggs of F. magna (Figure 2B) are symmetric, elliptical or oval, and
operculated at one pole. Proportions of eggs range from 100-175 μm in length and 80-117 μm
in width, their color is grey-yellowish to brownish, immature eggs being yellow. The contents
of the egg consist of germ cells and vitelline cells which are visible through the translucent
cover [14].
26
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
Host Spectrum and Localization of the Parasite
Many free living and domestic ruminants are susceptible to F. magna; however, the
primary host is very probably white-tailed deer, in which the fluke evolved and parasitized for
the longest time [15]. Other obligate North American hosts are wapiti deer (Cervus elaphus
canadensis), caribou (Rangifer tarandus), black-tailed deer (Odocoileus hemionus
columbianus) and mule deer (Odocoileus hemionus hemionus). Red deer and fallow deer
represent obligate hosts of F. magna in Europe. These hosts significantly contribute to
maintaining F. magna populations. Another type of F. magna hosts are aberrant or dead end
hosts including moose (Alces alces), bison (Bison bison), and variety of domesticated or wild
herbivores such as cattle, horse, pig, sheep, goat, llama (Lama glama) and sika deer (Sika
nippon).
The life cycle of F. magna is relatively complex and very similar to the development of
the related fluke F. hepatica. The parasite also utilizes aquatic snails (Lymnaeidae) as
intermediate hosts. Ruminants are infected mostly at pasture by ingestion of infective
metacercariae. Definitive, aberrant or dead end hosts can be infected in enzootic areas, flukes
may successfully reach the liver; however, rarely mature. In liver, the parasites are
surrounded by dense fibrous tissue that may occlude the bile collection system and prevent
eggs from escaping into intestine [15].
Fascioloides magna has in obligate hosts strong predilection to the liver parenchyma,
flukes are localized in thin-walled fibrous pseudocysts where they became mature and
produce eggs which are passed into intestine via the bile system [15]. The very specific
localization differentiates F. magna from other liver flukes, F. hepatica and D. dendriticum.
Distribution
Fascioloides magna is of the North American origin and up to now it parasitizes there in
a variety of wild and domestic ruminants primarily in five major enzootic areas: (1) the Great
Lakes region, (2) Gulf Coast, Lower Mississippi Valley, and southern Atlantic seaboard, (3)
Northern Pacific Coast, (4) Rocky Mountain Trench, and (5) Northern Quebec and Labrador
[15].
The earliest North American record reported flukes in a deer liver collected in 1875 [67].
Actual presence of the giant liver flukes in different North American localities varies from
locally abundant to locally absent [15]. In the first enzootic area, natural infections with F.
magna were described in the State of Minnesota in white-tailed deer, moose, llama and horse
[68,69,70]. In the second enzootic area, F. magna infections were reported e.g. in the states of
Florida and Texas in white-tailed deer, cattle, goat and pig. In the U.S. states of Washington
and Oregon and in the Canadian province British Columbia (northern Pacific Coast), natural
infections were detected in wapiti, black-tailed deer and cattle. Infected wapiti deer, mule
deer, white-tailed deer and cattle were also found in the U.S. state of Montana and Canadian
province Alberta, representing the Rocky Mountain Trench. In the northern Quebec and
Labrador, the last enzootic area, infections in cervids, especially moose and wapiti were also
documented [15]. Fascioloides magna was introduced to Europe from North America along
with its definitive hosts several times [71]. For the first time, the parasite was introduced to
the Regional Park La Mandria near Turin in Italy from Wyoming and this introduction is well
Liver and Stomach Flukes of Wild and Domestic Ruminants
27
documented. In 1865, Savoy King Vittorio Emanuele II commanded to purchase 60 wapiti
from which 47 overlived the trans-ocean ship transport to La Mandria hunting preserve [72].
An undesirable side effect of this deer import was the introduction of giant liver fluke, F.
magna. Since then, its permanent occurrence in this enclosure has been documented with
periodically fluctuating prevalence [73]. Except for Italy, two additional permanent foci of F.
magna occurrence have been established in Europe. The first one encompasses the southwestern and central parts of the Czech Republic. There, the fluke infection was firstly found
in a fallow deer in 1910 [74]. Since then, infections have been recorded in red and roe deer
[14, 75], and sporadic records of natural infections of domestic ruminants, such as sheep and
cattle, were also documented [76]. New foci of F. magna infection were discovered in the
south-western part of the Czech Republic near Austrian border [77]. The floodplain forests
alongside the middle course of the River Danube represent the last European focus of F.
magna. The recent history of formation of the Danube focus showed its potential to spread
quickly into new territories. The first record of F. magna was dated in 1988 [78] situating at
the Slovak side of the Danube near Gabčíkovo. At present, the territory involves Slovakia
[79], Hungary [80], Austria [81] and Croatia [82,83]. The recent molecular population study
proved that the introduction of F. magna to Europe was a result of several transmission
events; mitochondrial haplotypes found in Italy were distinct from those determined in the
remaining European localities. For the Czech populations of the parasite, a south-eastern USA
origin of giant liver fluke was revealed. Identical haplotypes, common for parasites from the
Czech Republic and from expanding focus of the Danube floodplain forests, implies
introduction of F. magna to the Danube region from the already established Czech focus [71].
Sporadic occurrence of the parasite was previously published from Poland, Spain and
Germany [for review see 66]; however, no additional records have been published from these
areas. Similarly, sporadic records on F. magna occurrence were published from South Africa,
Australia and Cuba [for review see 15].
Clinical Signs
Various ruminant species may considerably differ in tolerance against the F. magna
infection. Generally, most of the infections are subclinical; however a course of the disease
depends also on the number of flukes. In obligate hosts, lethargy, depression, weight loss,
innapetence, decrease of antlers and reproduction quality can occur [15]. Game is weaker;
however, the appetite is not changed and submandibular, thoracic and abdominal edemas
appear. Infections of roe deer with three or more flukes may be lethal, while red deer infected
with 10 or sometimes more worms does not show clinical manifestation [64]. It has to be
stressed that although F. magna often do not cause a fatal outcome in red deer, the infection
may lead to a significant loss of body condition and deterioration in value of the trophy [82].
Nervous disorders, as apathy and paralysis can also be observed [84]. Sometimes, severe
infections may be a reason of death [15]. Infections of dead-end and aberrant hosts are not
associated with specific symptoms, but loss of condition and anemia could appear.
In these type of hosts, excessive wandering of juvenile flukes into various abdominal and
pleural organs is characteristic and it may cause the death of the animals [15]. In cattle,
"bottle-jaw" (a swelling in the intermandibular space), anemia and jaundice associated with
unthriftiness, anorexia and weight loss were described [76,85]. Unexpected death without any
28
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
previous specific signs may also be caused by excessive migration of juvenile flukes through
the organism of sheep and goat [15].
Dicrocoelium Dendriticum
Morphology of Adults and Eggs
Adult worms of D. dendriticum (Figure 1C) are 8-10 mm long and 2-3 mm wide and
strongly tapered at both ends. The body is semitransparent, lancet shaped or elongate, and
covered by unspined tegument [86]. The flukes are of distomic morphological type, two
suckers are present in anterior third of body, the oral sucker is usually larger than the ventral
one. Gastrointestinal tract continues by small pharynx, relatively long esophagus and two
cecum that do not reach posterior extremity. The male genital system is composed of two
diagonal and sometimes lobed testes situated immediately posterior to the ventral sucker;
elongate cirrus-sac is well developed. Genital pore is median, just posterior to intestinal
bifurcation. Ovary is smaller than testes and it is situated in anterior half of body close to
testes. Entire hind-body is occupied by uterus. Vitellarium is formed by small follicles
organized in two lateral bands and present exclusively in middle third of body [87]. Due to
the semitransparency of the body, black uterus and white vitellaria are visible by naked eye
[86].
The asymmetrically oval eggs of D. dendriticum (Figure 2C) are rather small 35-45µm in
length and 22-30 µm in width and filled with homogenous dark brown content [86]. Typical,
sometimes indistinct operculum is present at one pole [15]. Embryonated eggs contain a
mature larval stage, the miracidium [86].
Host Spectrum and Localization of the Parasite
Many domestic and wild ruminants, especially sheep, goats, cattle, roe deer, red deer,
moose, sika deer and chamois (Rupicapra rupicapra) are suitable hosts of D. dendriticum.
Low host specificity is exhibited by rabbits, pigs and horses in which the flukes occasionally
parasitize [15]; human infections caused by ingestion of ants containing metacercariae are
also sporadically reported from various parts of the world e.g. Iran [88], Turkey [89] and Italy
[90]. One-off detection of the parasite eggs in human stool can occur after a consumption of
the raw or undercooked animal liver containing mature flukes with eggs, which just pass
through the digestive tract unchanged [91,92].
For the development of the parasite, two intermediate hosts are necessary. The first one is
a terrestrial snail (e.g. genera Helicella and Zebrina), in which cercariae are produced via
asexual development. They are finally excreted by snail in mucous balls which are swallowed
by the second intermediate host, ant (Formica spp.).
The nervous system of infected ants is affected what makes them to remain on plant tops
due to cephalic spasm. The infected hosts can thus be swallowed by grazing ruminants more
easily than the parasite-free ants [93]. The flukes parasitize in the liver, primarily in bile ducts
and gall bladder of infected definitive hosts [94].
Liver and Stomach Flukes of Wild and Domestic Ruminants
29
Distribution
Dicrocoelium dendriticum has a wide distribution in Europe, Asia, and North Africa, but
it also occurs in some focal points in North America. The parasite is apparently of Eurasian
origin, and was introduced to eastern North America [15]. Its occurrence is related to dry and
calcareous or alkaline soils which are suitable biotopes for their intermediate snail hosts [94].
In Europe, a presence of the lancet fluke was reported in several provinces of Spain in
sheep [95] and in grazing beef cattle [96,97]. Cattle and sheep were found to be infected also
in Switzerland [30] and in the region of southern Apennines in Italy [36]. Out of other
European countries, fluctuating prevalence of dicrocoeliosis was detected primarily in
domestic ruminants kept in pastures especially in southern part of Czech Republic, in the
region of central Slovakia, in Germany and in the southern part of France [for review see 93].
Concerning wild ruminants, isolated findings were documented in fallow deer and mufflon
from Hungary [98,99]. Infections of chamois, roe deer and mufflons were detected in
Slovakia and the Czech Republic [100,101]. In Asia, small ruminants, especially sheep and
goats slaughtered in local abattoirs in India, were found to be infected [102]. Various level of
prevalence of D. dendriticum infection in slaughtered cattle, sheep and goats was detected in
northern and south-western Iran [103,104]. Records from the North American continent
include infections of wapiti, mule deer, and white-tailed deer from the Cypress Hills region in
south-eastern Alberta [105]. Recently, the fluke has been confirmed in cattle and cervids from
the same locality [106].
Clinical Signs
It is difficult to identify dicrocoeliosis because of common subclinical or chronic course
of the infection, therefore the disease often remains clinically undetected or undiagnosed [30].
In addition, D. dendriticum infections are frequently concurrent with those of F. hepatica
[15]. Clinical symptoms are usually not manifested although such clinical signs as loss of
condition, anemia, icterus, edemas and emaciation may occur [94]. The health significance is
also due to digestive disorders derived from the hepatobiliary alterations such as decreased
animal weight, growth delay and reduced milk production [11]. Acute fatal infections can
also occur, particularly in sheep [15], which are more susceptible to D. dendriticum than
goats [86]. Intensities of infection in cervids and other wild hosts appear to remain low,
clinical signs are very limited, incidental, or infections take place without any visible
symptoms [15].
Paramphistomum Cervi
Morphology of Adults and Eggs
The body size of adult P. cervi flukes (Figure 1D) reaches up from 4-6 mm in length and
2-2.8 mm in width, worms are pear-shaped, slightly concave ventrally and convex dorsally
[107]. Flukes possess terminal oral opening and medium-sized ventral sucker situated
ventrally near the posterior body end, and so it belongs to amphistomic morphological type.
30
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
Gastrointestinal tract is composed of pharynx, which is without pharyngeal sac and
esophageal bulb. Cecum is straight or sinuous with variable posterior extent.
The male genital system is represented by two lobed testes in tandem or diagonal
position, male ducts and genital pore are situated at a level of intestinal bifurcation. Cirrus sac
is absent. Ovary is post-testicular, rounded or slightly lobed. Uterus of mature flukes is
localized dorsally to testes. Vitelline follicles are present in lateral fields [108]. Mean size of
P. cervi eggs (Figure 2D) is 125-186 μm in length and 62-114 μm in width; they are of oval
up to elliptical shape with operculum at one pole, grey-yellowish to brownish in color [109].
Host Spectrum and Localization of the Parasite
Paramphistomum cervi is the parasite of wild and domestic ruminants which particularly
infects cattle, sheep, goat, red deer and water buffaloes (Bubalus bubalis) [110]. The life
cycle of this parasite is similar to that of F. hepatica and F. magna; the parasite requires
freshwater snails as intermediate host however, they mainly belong to the family Planorbidae
or less often to Lymnaeidae.
Infected snails release cercariae which encyst on subaquatic plants forming
metacercariae. Ruminants can acquire infection by ingestion of metacercariae on pasture
[111]. Adult flukes of P. cervi are the main parasites of fore stomach; they are particularly
localized in the rumen and reticulum of ruminants [112].
Distribution
Paramphistomosis has global distribution with the highest prevalence documented in
subtropical regions, particularly in Asia, Africa and Australia [113,114], and also in Eastern
Europe.
In Europe, P. cervi has been reported in wild and domestic ruminants from Russia,
Bulgaria, Poland, France, Italy, and in Slovakia cervids from different location were also
found to be infected [114,115]. The flukes were also ascertained in the rumen of moose, red
deer and roe deer hunted in Aukštaitija National Park in northeastern Lithuania [50].
In Asia, infections of P. cervi were detected in cattle from Thailand [116], in Black
Bengal goats from Bangladesh [117], in sheep from different localities in China [118], and in
sheep, goats, cattle and buffalo from Pakistan [119].
Paramphistomosis was also found in cattle and sheep in Van Province in Turkey [120]
and in cattle from south-western Saudi Arabia [121]. Records on bovine paramhistomosis
were also described in African countries. The disease is endemic in Tanzania and other East
African countries [122], however recently, P. cervi flukes have been found in sheep
slaughtered in Egypt [123] and in cattle from Ethiopia [124].
High prevalence of P. cervi infection was observed even in North American continent,
where livestock from south-eastern Mexico were found to be infected [125]. In Canadian
province Ontario, P. cervi was found in moose from an agricultural area [126].
Liver and Stomach Flukes of Wild and Domestic Ruminants
31
Clinical Signs
Infections caused by P. cervi were neglected for a long time but they have recently been
recognized as an important cause of productivity loss [110]. Paramphistomosis can cause
significant gastrointestinal disease, drop in production or even death [127]; however, clinical
diagnosis remains difficult [128]. The harm caused by this infection primarily affects
production of animals, since the parasite provokes a lower nutritious conversion followed by
the reduced weight and the decrease in milk production [125]. The disease was also found to
be associated with diarrhea, anemia, weakness and rough hair coat. Effects of
paramphistomes are often underestimated although they should be considered, because of a
risk of the acute infection of immature worms which is characteristic by very difficult
identification [129]. During the acute infections, massive amount of immature flukes migrate
through the intestinal tract causing the acute gastroenteritis with high morbidity and mortality
rates particularly in young animals [111].
Molecular Approach in Discrimination of the Fluke
Species
Adult flukes of each of the four species can be well recognized by the distinct speciesspecific morphological characters such as size and shape of body and inner organs (Fig.1AD). Therefore, the exact post mortem determination is available by isolation of adult
individuals at the necropsy. From a veterinary point of view, in vivo diagnosis of parasitic
infections has a key role. However, above mentioned clinical signs caused by these
trematodes are often mild, very similar, nonspecific or even none, and so they do not provide
sufficient information for the differential diagnosis and the parasitic infections remain often
undiagnosed. Coprological examination is the most frequently used in vivo diagnostic
technique, during which the eggs of the flukes are detected and differentiated according to the
distinct species-specific morphological characters. However, eggs of F. hepatica, F. magna
and P. cervi are of very similar size, shape and internal structure (Figure 2A, B, D).
Therefore, their reliable differentiation based on morphological markers is very problematic
and reliable molecular markers have to be designed. For the design of species-specific
markers in F. hepatica, F. magna, D. dendriticum and P. cervi, ribosomal ITS2 spacer was
selected [130,131].
Figure 3. Complete ITS2 sequence structure of Fasciola hepatica (364 bp) with marked polymorphic
sites. Notes: The sequence is a consensus sequence made by the sequence alignment of distant global
populations of F. hepatica (for details see Králová-Hromadová et al., 2008; Bazsalovicsová et al.,
2010). Nucleotides in bold, polymorphic sites. Nucleotides in boxes, F. hepatica-specific region
applied for the design of F. hepatica-specific primers. Arrows, orientation of primers.
32
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
Figure 4. Complete ITS2 sequence structure of Fascioloides magna (363 bp). Notes: The sequence is
a consensus sequence made by the sequence alignment of North American and European populations of
F. magna (for details see Králová-Hromadová et al., 2008; Bazsalovicsová et al., 2010). Nucleotides in
bold, polymorphic sites. Nucleotides in boxes, F. magna-specific regions applied for the design of F.
magna-specific primers. Arrows, orientation of primers.
Figure 5. Complete ITS2 sequence structure of Dicrocoelium dendriticum (241 bp) with marked
polymorphic sites. Notes: The sequence is a consensus sequence made by the sequence alignment of
Asian and European populations of D. dendriticum (for details see Bazsalovicsová et al., 2010).
Nucleotides in bold, polymorphic sites. Nucleotides in boxes, D. dendriticum-specific regions applied
for the design of D. dendriticum-specific primers. Arrows, orientation of primers.
Figure 6. Complete ITS2 sequence structure of Paramphistomum cervi (286 bp). Notes: The ITS2
sequence determined for Slovak population of the parasite (for details see Bazsalovicsová et al., 2010).
Nucleotides in bold, polymorphic sites. Nucleotides in boxes, P. cervi-specific regions applied for the
design of P. cervi-specific primers. Arrows, orientation of primers.
The ITS2 sequence structure of all studied flukes was analyzed and compared between
different hosts and localities in order to determine a level of intraspecific variation. As a result
of this comparison, no intraspecific variation was detected in F. magna and P. cervi (Figs. 4,
6) while few polymorphic sites were found in F. hepatica and D. dendriticum (Figs. 3, 5).
According to this information, and based on the consensus sequence alignment, regions
displaying no intraspecific variation but specific for particular fluke species were used for the
design of species-specific primers [for details see 130, 131].
Liver and Stomach Flukes of Wild and Domestic Ruminants
33
Figure 7. PCR amplification of genomic DNA of adult individuals of Fasciola hepatica (FH),
Fascioloides magna (FM), Dicrocoelium dendriticum (DD) and Paramphistomum cervi (PC) using
species-specific ITS2 primers.
Each couple of primers designed (Figs. 3-6) annealed only in the DNA of a single species
but did not amplify DNA of other three remaining fluke taxa (Figure 7). The results were
successfully tested on genomic DNA isolated both from fluke tissues and eggs of studied
fluke species [131,132] and have recently been applied for molecular screening of the
infection of snail intermediate hosts [133].
Conclusion
The health of wild and domestic ruminants is the major aspect for their successful
growth, high productivity, reproduction capacity as well as for their welfare. The bacterial,
viral and last but not least parasitic infections represent a serious danger affecting their health.
Therefore, the great effort in veterinary medicine is directed to the effective control of these
infections. For that purpose, the first necessary step is an exact identification of pathogenic
agents, including the parasite species.
It is evident that in vivo identification of the parasite is often not accurate due to many
reasons. In such cases, the proper use of molecular methods undoubtedly provides a good
alternative for the taxonomically precise recognition of the infection. Apart from veterinary
clinics and standard routine in parasitological laboratories, the exact species determination is
an inevitable and crucial step in basic research.
Acknowledgment
This work was supported by the Slovak Research and Development Agency under the
contract of APVV-51-062205. We would like to thank Dr. Marianna Reblánová (Institute of
Parasitology SAS, Košice, Slovakia) for technical assistance with figure graphics.
34
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
H. Mehlhorn, K.A. Al-Rasheid, F. Abdel-Ghaffar, S. Klimpel, and H. Pohle, Parasitol
Res. 107, 425-431 (2010).
D. W. Duzynski and S. J. Upton, Enteric protozoans: Cyclospora, Eimeria, Isospora and
cryptosporidium spp. In: Parasitic diseases of wild mammals (eds W. M. Samuel, M. J.
Pybus, A. A. Kocan) Iowa, USA: Iowa State University Press (2001) 416-442.
J. Vercruysse and E. Claerebout, Vet. Parasitol. 98, 195-214 (2001).
A. Jones and M. J. Pybus, Taeniasis and Echinococcosis. In: Parasitic diseases of wild
mammals (eds W. M. Samuel, M. J. Pybus and A. A. Kocan) Iowa, USA: Iowa State
University Press. (2001) 150-192.
P. D.Olson and V. V. Tkach, Adv. Parasit. 60, 165-243 (2005).
D. M. Hillis and M. T. Dixon, Q Rev. Biol. 66, 411-45 (1991).
L. van Herwerden, R. B. Gasser and D. Blair, Int. J. Parasitol. 30, 157-169 (2000).
P. D. Olson, T. H Cribb, V. V. Tkach, R. A. Bray and D. T. J. Littlewood, Int. J.
Parasitol. 22, 733-755 (2003).
A. Jones, Family Fasciolidae Railliet, 1895. In: Keys to the Trematoda (eds A. Jones, R.
A. Bray, D. I. Gibson). London, United Kingdom: CAB International and Natural
History Museum. (2005a) 79-85.
S. L. Eduardo, Syst. Parasitol. 4, 189-238 (1982).
M. Y. Manga-González, C. González-Lanza, E. Cabanas and R. Campo, Parasitology,
123, 91-114 (2001).
S. Mas-Coma, M. A. Valero and M. D. Bargues, Adv. Parasit. 69, 41-146 (2009).
W. M. Lotfy, S. V. Brant, K. I. Ashmawy, R. Devkota, G. M. Mkoji and E. S. Loker,
Vet. Parasitol. 174, 234-240 (2011).
B. Erhardová-Kotrlá, The occurrence of Fascioloides magna (Bassi, 1875) in
Czechoslovakia. Prague, Czech Republic: Academia (1971).
M. J. Pybus, Liver flukes. In: Parasitic diseases of wild mammals. (eds W. M. Samuel,
M. J. Pybus, A. A. Kocan). Iowa, USA: Iowa State University Press (2001) 121-149.
S. J. Andrews, Life cycle of Fasciola hepatica. In: Fasciolosis. (ed J. P. Dalton).
Cambridge, United Kingdom: CABI Publishing. (1999) 1-29.
P. Torgerson and J. Claxton, Epidemiology and control. In: Fasciolosis. (ed J. P.
Dalton). Cambridge, United Kingdom: CABI Publishing. (1999) 113-139.
S. Mas-Coma, M. D. Bargues, M. A. Valero and M. V. Fuentes, Adaptation capacities
of Fasciola hepatica and their relationships with human fascioliasis: from below sea
level up to the very high altitide. In: Taxonomy, Ecology and Evolution of Metazoan
Parasites (eds C. Combes, J. Jourdane). Perpignan, France: Perpignan University Press
(2003) 81-123.
A. Ménard, A. Agoulon, M. L’Hostis, D. Rondelaud, S. Collard and A. Chauvin, Vet.
Res. 32, 499-508 (2001).
E. Magnanou, R. Fons, C. Feliu and S. Morand, Parasitol. Res. 99, 97-101 (2006).
D. Rondelaud, P. Vignoles, M. Abrous and G. Dreyffuss, Parasitol. Res. 87, 475-478
(2001).
S. Mas-Coma, M. D. Bargues and J. G. Esteban, Human Fasciolosis. In: Fasciolosis.
(ed J. P. Dalton). Cambridge, United Kingdom: CABI Publishing. (1999) 411-429.
Liver and Stomach Flukes of Wild and Domestic Ruminants
35
[23] S. Mas-Coma, M. D. Bargues and M. A. Valero, Plant-borne trematode zoonoses:
fascioliasis and fasciolopsiasis. In: World class parasites, vol. 11. Food-borne parasitic
zoonoses: fish and plant-borne parasites (eds K. D. Murrell, B. Fried). New York:
Springer. (2007) 293-334.
[24] S. Mas-Coma, M. D. Bargues and M. A. Valero, Int. J. Parasitol. 35, 1255-1278
(2005).
[25] S. Mas-Coma, Southeast Asian J. Trop. Med. Publ. Health. 35, 1-11 (2004).
[26] W. M. Lotfy, S. V. Brant, R. J.DeJong, T. H. Le, A. Demiaszkiewicz, R. P. Rajapakse,
V. B. Perera, J. R. Laursen and E. S. Loker, Am. J. Trop. Med .Hyg. 79, 248-255
(2008).
[27] K. Zmuda and K. Chroust, Veterinářství, 4, 181-183 (2001).
[28] A. Deryło, J. Kozłovska-Loj, P. Szilman, N. Najda, A. Seniuk and K. Wasilewski,
Wiadomości parazytologiczne, 47, 775-778 (2001).
[29] M. Kozak-Cieszczyk, Wiadomości parazytologiczne, 52, 137-139 (2006).
[30] D. Ducommun and K. Pfister, Parasitol. Res. 77, 364-366 (1991).
[31] G. Schweizer, G. F. Plebani and U. Braun, Schweizer Archiv fur Tierheilkunde, 145,
177-179 (2003).
[32] C. Rapsch, G. Schweizer, F. Grimm, L. Kohler, C. Bauer, P. Deplazes, U. Braun and P.
R. Torgerson,. Int. J. Parasitol. 36, 1153-1158 (2006).
[33] C. González-Lanza, Y. Manga-González, P. Del-Pozo-Carnero and R. HidalgoArguello, Vet. Parasitol. 34, 35-43 (1989).
[34] C. Mage, H. Bourgne, J. M. Toullieu, D. and G. Dreyfuss, Vet. Res. 33, 439-47 (2002).
[35] T. Balbo, P. Lanfranchi and M. G. Gallo, Parassitologia, 20, 23-28 (1978).
[36] G. Cringoli, L. Rinaldi, V. Veneziano, G. Capelli and J. B. Malone, Vet. Parasitol. 108,
137-143 (2002).
[37] M. A. Conceiçāo, R. M. Durâo, I. M. Costa, A. Castro, A. C. Louza and J. C. Costa,
Vet. Parasitol. 123, 93-103 (2004).
[38] S. A Henriksen, and C. Pilegaard-Andersen, Nord. Vet. Med. 31, 6-13 (1979).
[39] G. C. Pritchard, A. B. Forbes, D. J. Williams, M. R. Salimi-Bejestani and R. G. Daniel,
Vet. Rec. 157, 578-82 (2005).
[40] T. M. Murphy, K. N. Fahy, A. McAuliffe, A. B. Forbes, T. A. Clegg and D. J. O’Brien,
Prev. Vet. Med. 76, 1-10 (2006).
[41] J. Farkaš, S. Findo, M. Stanovský and T. Pataky, Folia Venatoria, 23, 61-68 (1993).
[42] A. Kotrlý and B. Kotrlá, Angewandte Parasitol. 21, 70-78 (1980).
[43] S. Rehbein and M. Visser. Wiener Klinische Wochenschrift, 119, 96-101 (2007).
[44] H. Lis, Zycie Weterynaryjne, 75, 203-205 (2000).
[45] G. Lazzeri, R. Martino, F. di Mancianti, D. Lorenzi, A. Poli, Large Animals Review, 8,
57-61 (2002).
[46] M. J. Maia, Rev. Port. Cienc. Vet. 96, 81-84 (2001).
[47] S. Alasaad, C. Q. Huang, Q. Y. Li, J. E. Granados, C. García-Romero, J. M. Pérez and
X. Q. Zhu, Parasitol. Res. 101, 1245-1250 (2007).
[48] V. V. Shimalov and V. T. Shimalov, Parasitol. Res. 89, 75-76 (2003).
[49] G. Vengušt and A. Bidovec, Helminthologia, 40, 161-164 (2003).
[50] R. Aukstikalniene, E. Bukelskis and E. Kasetaite, Baltic Forestry, 13, 96-102 (2007).
[51] C. S. Hayat, A. Maqbool, B. Hayat, N. Badar and M. Lateef, Pakistan J. Zool. 30, 269270 (1998).
36
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
[52] P. S. Banerjee, R. Garg, C. L. Yadav and H. Ram, Indian J. Anim. Sci. 75, 206-208
(2005).
[53] K. P. Singh, D. A. Chandra, L. M. S. Nayal and R. S. Chauhan, Indian J. Anim. Sci. 77,
1258-1260 (2007).
[54] N. Y. Abou-Zinadah and M. A. Fouad, J. Egyp. Soc. Parasitol. 35, 711-716 (2005).
[55] A. N. Okaeme, Int. J. Zoonoses, 12, 152-155 (1985).
[56] A. Moukrim and D. Rondelaud, Revue de Médécine Véterinaire, 142, 839-843 (1991).
[57] A. M. El-Shazly, A. A. Abdel-Magied, H. A. El-Nahas, M. S. El-Metwaly, T. A.
Morsy, E. M. El Sharkawy and A. T. Morsy, J. Egypt. Soc. Parasitol. 35, 243-52
(2005).
[58] J. C. Haigh, C. Mackintosh and F. Griffin. OIE Revue Scientifique et Technique, 21,
219-248 (2002).
[59] J. A. Munguía-Xóchihua, F. Ibarra-Velarde, A. Ducoing-Watty, N. MontenegroCristino and H. Quiroz-Romero, Parasitol. Res. 101, 127-130 (2007).
[60] L. H. Dutra, M. B. Molento, C. R. C. Naumann, A. W. Biondo, F. S. Fortes, D. Savio
and J. B. Malone, Vet. Par. 169, 76-81 (2010).
[61] W. A. Charleston and P. B. McKenna, New Zeal. Vet. J. 50, 41-47 (2002).
[62] C. A. Behm and N. C. Sangster, Pathology, pathophysiology and clinical aspects. In:
Fasciolosis. (ed J. P. Dalton). Cambridge, United Kingdom: CABI Publishing (1999)
185-217.
[63] H. Mehlhorn, Encyclopedic Reference of Parasitology: Biology, structure, function,
Berlin, Germany, Springer-Verlag (2001).
[64] J. Ciberej, P. Lazar, J. Halász and M. Kačur, Chov a choroby zveri, Magnus, Košice,
Slovakia (1992).
[65] A. N. Hamir and B. B. Smith, Vet. Pathol. 39, 592-594 (2002).
[66] M. Špakulová, D. Rajský, J.Sokol and M. Vodňanský, Giant liver fluke (Fascioloides
magna), an important liver parasite of ruminants. Parpress, Bratislava, Slovakia (2003).
[67] M. C. Hall, (1912). Our present knowledge of the distribution and importance of some
parasitic diseases of sheep and cattle in the United States. In Circular (193), Bureau of
Animal Industry, USA: United States Department of Agriculture.
[68] R. Fenstermacher, O. W. Olsen and B. S. Pomeroy, Cornell. Vet. 33, 323-332 (1943).
[69] G. A. Conboy, T. D. O΄ Brien and D. L. Stevens, J. Parasitol. 74, 345-346 (1988).
[70] S. L. McClanahan, B. E.Stromberg, D. W Hayden, G. A. Averbeck and J. H. Wilson, J.
Vet. Diagn. Investig. 17, 382-385 (2005).
[71] I. Králová-Hromadová, E. Bazsalovicsová, J. Štefka, M. Špakulová, S. Vávrová, T.
Szemes, V. Tkach, A. Trudgett and M. Pybus, Int. J. Parasito. 41, 373-383 (2011).
[72] C. Apostolo, The naturalistic aspects: flora, fauna and the environment. In: La Mandria
Storia e natura del Parco (eds M. Lupo, M. Paglieri, C. Apostolo, E. Vaccarino, M.
Debernardi). Torino, Italy: Edizioni Eda (1996) 67-98.
[73] T. Balbo, L. Rossi and P. G. Meneguez, Parassitologia, 31, 137-144 (1989).
[74] K. Ullrich, Prager. Arch. Tiermed. 10, 19-43 (1930).
[75] K. Chroust and E. Chroustová, Veterinářství, 54, 296-304 (2004).
[76] E. Chroustová, J. Hůlka and J. Jaroš, Vet. Med. (Praha). 25, 557-563 (1980).
[77] A. Novobilský, E. Horáčková, L. Hirtová, D. Modrý, B. Koudela, Parasitol. Res. 100,
549-553 (2007).
[78] D. Rajský, A. Patus and K. Bukovjan, Slov vet čas. 19, 29-30 (1994).
Liver and Stomach Flukes of Wild and Domestic Ruminants
37
[79] D. Rajský, J. Čorba, M. Várady, M. Špakulová and R. Cabadaj, Helminthologia, 39, 6770 (2002).
[80] G. Majoros and V. Sztojkov, Parasit. Hung. 27, 27-38 (1994).
[81] R. Winkelmayer and H. Prosl, Österreichisches Weidwerk, 3, 42-44 (2001).
[82] A. Marinculić, N. Džakula, Z. Janicky, S. Lućinger, T. Živićniak, Vet Archiv. 72, 319325 (2002).
[83] R. Rajković-Janje, S. Bosnić, D. Rimac and T. Gojmerac, Eur. J. Wild Res. 54, 525-528
(2008).
[84] B. Erhardová-Kotrlá and K. Blažek, Acta. Vet. Brno. 39, 287-29 (1970).
[85] A. A. Kingscote, Can. J .Comp. Med. 14, 203-208 (1950).
[86] D. Otranto and D. Traversa, Vet. Par. 107, 317-335 (2002).
[87] T. Pojmańska, Family Dicrocoeliidae Looss, 1899. In: Keys to the Trematoda (eds R.
A. Bray, D. I. Gibson, A. Jones). London, United Kingdom: CAB International and
Natural History Museum. (2008) 233-260.
[88] M. Mahmoodi, A. R. Ramazani, S. Izadi and J. Najafian, Acta. Med. Iran. 48, 198-199
(2010).
[89] Z. T. Cengiz, H. Yilmaz, A. C. Dulger and M.Cicek, Ann. Saudi. Med. 30, 159-161
(2010).
[90] B. Magi, E. Frati, L. Bernini, A. Sansoni and G. Zenelli, Infez. Med., 2, 115-116 (2009).
[91] H. Aspöck, H. Auer and O. Picher, Helminthologia, 36, 139-145 (1999).
[92] F. Schweiger and M. Kuhn, Can. J. Gastroenterol. 22, 571-573 (2008).
[93] Ľ. Ducháček and J. Lamka, Acta. Vet. Brno., 72, 613-626 (2003).
[94] D. Otranto and D. Traversa, Trends Parasitol. 19, 12-15 (2003).
[95] M. Y. Manga-González, C. González-Lanza and P. Del-Pozo, Ann. Parasitol. Hum.
Comp. 66, 57-61 (1991).
[96] C. González-Lanza, M. Y. Manga-González and P. Del-Pozo-Carnero, Parasitol. Res.
79, 488-491 (1993).
[97] [P. Díaz, A. Paz-Silva, R. Sánchez-Andrade, J. L. Suárez, J. Pedreira, M. Arias, P.
Díez-Banos and P. Morrondo, Vet. Par. 149, 285-289 (2007).
[98] T. Andras, Magyar Állatorvosok Lapja, 50, 166-168 (1995).
[99] A. Takacs, Magyar Állatorvosok Lapja, 122, 618-620 (2000).
[100] J. Ciberej, V. Letková and M. Kačur, Slov vet čas. 22, 301-302 (1997).
[101] K. Chroust, Parazitární choroby spárkaté zvěře. Újezd u Brna, Czech Republic: RNDr.
Ivan Straka. (2001).
[102] K. P. Jithendran and T. K. Bhat, Vet. Par. 61, 265-271 (1996).
[103] N. A. Ahmadi and M. Meshkehkar, Journal of Paramedical Sciences, 1, 26-31 (2010).
[104] R. Ahmadi, E. M. Sikeroj and M. Maleki, J. Anim. Vet. Adv. 9, 2723-2724 (2010).
[105] M. J. Pybus, J Wildlife Dis. 26, 453-459. (1990).
[106] C. P. Goater and D. D. Colwell, J Parasitol. 93, 491-494 (2007).
[107] B. Kotrlá and K. Chroust, Acta Vet Brno. 47, 97-101 (1978).
[108] A. Jones, Family Paramphistomidae Fischoeder, 1901. In: Keys to the Trematoda (eds
A. Jones, R. A. Bray, D. I. Gibson). London, United Kingdom: CAB International and
Natural History Museum. (2005b) 229-246.
[109] A. Burgu, Ankara Üniversitesi Veteriner Fakültesi Derg. 28, 50-71 (1981).
[110] P. Anuracpreeda, C. Wanichanon and P. Sobhon, Exp. Parasitol. 118, 203-207 (2008).
[111] R. E. F. Sanabria and J. R. Romero, Helminthologia, 45, 64-68 (2008).
38
Eva Bazsalovicsová, Ivica Králová-Hromadová, Katarína Oberhauserová et al.
[112] B. Panyarachun, P. Sobhon, Y. Tinikul, Ch. Chotwiwatthanakun, V. Anupunpisit and P.
Anuracpreeda, Exp. Parasitol. 125, 95-99 (2010).
[113] J. C. Boray, Aust. Vet. J. 35, 282-287 (1959).
[114] I. G. Horak, Adv. Parasit. 9, 33-72 (1971).
[115] J. Farkaš, Folia Venatoria, 20, 41-52 (1990).
[116] P. Prasitirat, S. Nithiuthai, K. Ruengsuk, P. Kitwan, C. Bunmatid, S. Roopan and H.
Itagaki, Helminthologia, 34, 155-157 (1997).
[117] M. Z. Uddin, T. Farjana, N. Begum and M. M. H. Mondal, Bangladesh J. Vet. Me. 4,
103-106 (2006).
[118] C. R. Wang, Q. J. Hiu, X. Q. Zhu, X. H. Han, H. B. Ni, J. P. Zhao, Q. M. Zhou, H. W.
Zhang and Z. R. Lun, Vet. Par. 140, 378-382 (2006).
[119] M. A. Raza, S. Murtaza, H. A. Bachaya and A. Hussain, Pakistan Vet J. 29, 214-215
(2009).
[120] N. Ozdal, A. Gul, F. Ilhan and S. Deger, Helminthologia, 47, 20-24 (2010).
[121] A. K. Nasher, Vet. Par. 37, 297-300 (1990).
[122] J. D. Keyyu, A. A. Kassuku, L. P. Msalilwa, J. Monrad and N. C. Kyvsgaard, Vet. Res.
Commun. 30, 45-55 (2006).
[123] K. Sultan, A. Y. Desoukey, M. A. Elsiefy and N. M. Elbahy, Global Veterinaria, 5, 8487 (2010).
[124] A. Fromsa, B. Meharenet and B. Mekibib, J. Anim. Vet. Adv. 10, 1592-1597 (2011).
[125] L. J. Rangel-Ruiz, S. T. Albores-Brahms and J. Gamboa-Aguilar, Vet. Par. 116, 217222 (2003).
[126] J. Hoeve, D. G. Joachim, E. M. Addison, J. Wildlife Dis. 24, 371-374 (1988).
[127] P. Dorchies, C. Lacroux, H. Navetat, C. Rizet, E. Gueneau, B. Bisson and H. Ferte,.
Dossiers Techniques Veterinaires, 13, 91-93 (2002).
[128] E. Rieu, A. Recca, J. J. Benet, M. Saana, P. Dorchies and J. Guillot, Vet. Par. 146, 249253 (2007).
[129] P. Dorchies Flukes: Old parasites but new emergence. In Manuscript of 24th World
Buiatrics Congress. Nice, France: Ecole Nationale Vétérinaire, Toulouse, France
(2006).
[130] I. Králová-Hromadová, M. Špakulová, E. Horáčková, L. Turčeková, A. Novobilský, R.
Beck, B. Koudela, A. Marinculić, D. Rajský and M. Pybus, J. Parasitol. 94, 58-67
(2008).
[131] E. Bazsalovicsová, I. Králová-Hromadová, M. Špakulová, M. Reblánová and K.
Oberhauserová, Helminthologia, 47, 76-82 (2010).
[132] K. Oberhauserová, E. Bazsalovicsová, I. Králová-Hromadová, P. Major and M.
Reblánová, Helminthologia, 47, 147-151. (2010).
[133] A. Novobilský, “Molecular screening of the infection of snail intermediate hosts”
Private communication. anovobilsky@yahoo.com.
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter III
Fasciolosis
Leandro Buffoni Perazzo
Parasitology Unit, Animal Health Department,
Faculty of Veterinary Medicine, University of Cordoba,
Cordoba, Spain
Abstract
Fasciolosis is an important parasite disease of livestock that affect the liver. The life
cycle of the parasite is highly complex and requires the presence of snails of the
subfamily Lymnaeinae in the natural environment, which act as intermediate hosts. After
infection, immature worms migrate through the liver parenchyma causing a traumatic
hepatitis.
Then, adult flukes enter the bile ducts and gallbladder where they reach sexual
maturity. Diagnosis is usually achieved by detecting egg in feces or specific antibodies.
Control is mainly based on the use of anthelmintics.
Introduction
Fasciolosis is a major worldwide disease of livestock and responsible for large economic
losses to the agricultural sector estimated at US$ 3 billion per year. Two species are the main
agents that cause the disease: Fasciola hepatica and Fasciola gigantica. Adult worms are
localized in the bile ducts and gallbladder and produce a severe traumatic hepatitis which may
lead to loss of the hepatic function.
The life cycle of the parasite is complex and snails of the subfamily Lymnaeinae act as
intermediate hosts. Since the majority of the literature and studies are regarded to Fasciola
hepatica and both flukes harbor many features in common, description of the disease and
other aspects is firstly made upon Fasciola hepatica.
40
Leandro Buffoni Perazzo
Fasciola Hepatica (Linnaeus, 1758)
Fasciolosis by Fasciola hepatica, also known as distomatosis or liver rot, is caused by
flat worms which affect the liver of ruminants and other domestic animals. It may also occur
in wildlife animals and humans. The parasite is cosmopolitan in distribution and requires the
presence of a suitable intermediate host (freshwater snails) to complete its life cycle.
Classification
Phylum Platyhelminthes, Class Digenea (Van Beneden, 1858), Superorder
Anepitheliocystidia (La Rue, 1957), Order Echinostomatida, Family Fasciolidae (Railliet,
1895), Genus Fasciola (Linnaeus, 1758), Specie hepatica.
Description
The adult fluke is leaf-shaped, dorsoventrally flattened and greyish-brown in color
(Figure 1). It is broader anteriorly with a cone-shaped projection and a pair of “shoulders” and
it is around 3-3.5 cm in length and 1-1.5 cm in width. The tegument is covered with
projecting spines that vary in size and distribution. The oral sucker is usually smaller than the
ventral sucker and it is followed by a well-developed pharynx, a short esophagus and an
intestinal cecum with numerous branches and extended to the posterior extremity. The
trematode is hermaphrodite, the male organs consist of two branched testes situated near the
center of the body. Two sperm ducts come out from each testis and lead to the seminal vesicle
that passes through the cirrus pouch, which is a saccular structure containing the seminal
vesicle, a prostate gland, ejaculate duct, and a cirrus canal or cirrus (when averted from the
cirrus pouch).
Figure 1. Mature Fasciola hepatica.
41
Fasciolosis
(A)
(B)
Figure 2. (A) Non-embryonated egg, (B) Embryonated egg containing a fully-developed miracidium.
The female organs consist of one branched ovary located slightly anterior to the testes
and to the right of the middle, followed by an oviduct that runs toward the ootype and joins a
common vitelline duct. Vitteline cells gather to form vitelline glands resembling follicles or a
bunch of grapes. There is a fine duct coming out from each bunch, which unites into larger
ducts to form two transverse vitelline ducts that finally become a vitelline reservoir. The
ootype is a tubular pouchlike structure, and the uterus is tubular, coiled or rippled, and leads
to a female genital pore. Mehlis gland is round or elliptical in shape, lies in the rear of the
uterus and consists of many unicellular glands [48]. The eggs are yellowish-brown with
granular content and non-embryonated when laid, thin-shelled, oval in shape with an
operculum at one pole and 130-150 µm in length and 70-90 µm in width (Figure 2).
Life Cycle
Mature flukes deposit eggs that are passed out in the feces and develop under favorable
climatic conditions to form a first larval stage, the miracidium. The rate of development
depends on many factors such as temperature, moisture and oxygen tension. At an optimal
temperature of 22-26 ºC the development of the egg takes 10 days but at 10 ºC or 30 ºC, it
requires 6 months or it is completed in 8 days, respectively. The maintenance of moisture is
essential for embryonation whereas the mortality rate is rapidly increased under dry
conditions. The miracidium is broad anteriorly, 130 µm in length and 28 µm in width, it is
covered with a ciliated tegument and has a pair of eye spots (Figure 2B) and a conical papilla
at the anterior end of the body used to penetrate into the intermediate host. When the egg
hatches, the larva is released and starts to actively swim in search of a suitable intermediate
host, a snail of the genus Galba (syn. Lymnaea). Those that fail to find a snail, generally die
within 24 hours. Once the miracidia bores into the snail, it loses its ciliated covering and
transform into an elliptical sacular sporocyst containing germinal cells. By growth and
repeated divisions, germinal cells form germinal balls from which a next larval stage is
developed, the redia. Each sporocyst gives rise to 5-8 rediae. The redia is cylindrical in shape,
has mouth, pharynx and intestine and, like the sporocyst, contains germinal cells which may
produce a generation of daughter redia or cercaria.
42
Leandro Buffoni Perazzo
(A)
(B)
Figure 3. (A) Metacercaria of Fasciola hepatica, (B) Galba truncatula.
Life cycle of Fasciola hepatica.
As redia develops and grows it bursts the sporocyst and is thus released. The cercaria is
tadpole like with an oral and ventral sucker and a discoidal body of 250-350 µm long and a
tail of twice that length. After full development, the cercaria leaves the snail, swim in the
water and attaches to grass losing its tail, and encyst to transform into the infective larval
stage, the metacercaria (Figure 3A). The longevity of the metacercarial cysts depends on
Fasciolosis
43
several factors such as moisture and temperature and may remain viable for long periods
when protected from heat and dryness. The metacercaria is finally ingested by a suitable
definitive host, excyst in the small intestine and liberate the newly excysted juvenile fluke
which rapidly penetrate the intestinal wall and passes into the abdominal cavity. The
immature fluke reaches the liver within 4-6 days by wandering or in response to a stimulus
[45], bores into the liver and migrates through the parenchyma for 5-6 weeks. Then, it enters
the bile ducts and eventually the gallbladder where it becomes sexually mature. The prepatent
period is 10-12 weeks and the entire life cycle takes between 14-23 weeks.
Hosts
Snails of the genus Galba are involved in the life cycle of Fasciola hepatica. Galba
truncatula (Figure 3B) is the most common intermediate host worldwide, though several
species of mollusks have been reported: Galba bulimoides (North America), Galba columella
(Central and North America, Australia, New Zealand), Galba viatrix and Galba cubensis
(South America), Galba tomentosa (Australia and New Zealand). Definitive hosts include
cattle, sheep, goats, buffalo, deer, pigs, horse and other mammals. Humans can also be
affected particularly in endemic areas.
The Disease
The development of fasciolosis depends mainly on two factors, the fluke burden and the
definitive host. Cattle are more resistant to Fasciola hepatica infection than sheep and goats
which are more susceptible hosts and develop lower levels of resistance [20,32]. The chronic
form is the most important in cattle though acute and subacute forms may occur especially in
calves when the infectious dose is high.
Pathogenesis and Pathology
After infection and excystation of the metacercaria in the intestine, immature flukes reach
and penetrate the liver within the first 4-6 days. The disease basically develops in two
different stages: a first stage when the juvenile flukes migrate through the liver parenchyma,
which occur for 5-6 weeks, and a second stage or “biliary” stage when the trematodes enter
the bile ducts.
During the migratory phase flukes burrow into the liver tissue producing “migratory
tracts” and causing severe damage to hepatocytes and sinusoids, which results in destruction
of the normal hepatic architecture, phlebitis, thrombosis, coagulative necrosis and
hemorrhage. The prehensile oral sucker and the prickly tegument enhance erosion of the
parenchyma and although immature flukes may accidentally ingest blood, they essentially
feed on tissue [14,15]. Simultaneously, infiltration of inflammatory cells, mainly eosinophils
and mononuclear cells, occupy the area around the migratory tracts and the portal traids.
44
Leandro Buffoni Perazzo
Between 3-5 weeks after infection lymphocytes and macrophages surround and occupy
the affected tissue. Occasionally, multinucleated giant cells may form an outer layer in order
to phagocytate detritus and necrotic tissue. By the sixth week the deposit of collagen begins
within the tracts and surrounding areas and by the twelfth and sixteenth week cicatrization of
tracts is completed [19]. After the migratory stage at 7-8 weeks post-infection, flukes
penetrate the bile ducts causing hyperplastic cholangitis and chollangiectasis. Spines of the
tegument may produce cell desquamation and ulceration with formation of fibrous tissue
around the ducts. Hepatic fibrosis is the main feature of chronic fasciolosis. As a result of the
restoration and reorganization of the liver, fibrotic zones are irregular in shape and usually
occupy the portal triads, the tracts and the surrounding areas and replace the damaged and
necrotic tissue, which causes disorganization of the parenchymal structure. There is evidence
that suggest that biliary hyperplasia and fibrosis are mainly due to excretion of proline, an
essential amino acid for the synthesis of collagen, by flukes [52]. Calcification of the bile duct
wall and enlargement of the gallbladder is often seen in cattle. Thus, calcium deposits
frequently cause ducts to protrude from the surface, giving them the appearance of a stem of a
clay pipe, hence the commonly known term – “pipe-stem liver”. Although the majority of
parasites reach the liver, in cattle flukes can often migrate and penetrate other organs,
particularly lungs. When this occurs, a calcified cyst containing a dead worm surrounded by
necrotic and fibrous tissue may be observed.
Clinical Sings
Acute fasciolosis is less common and may occur in sheep and calves suffering heavy
infections. It usually occurs in areas with template climate during autumn and early winter,
and presents with sudden deaths and high mortality rates. By contrast, clinical manifestation
of the chronic form differs between animals and includes weight loss, anorexia, lethargy and
weakness.
Anemia is one of the most common symptoms found during infection with Fasciola
hepatica, particularly as a result of the hemorrhage and traumatic hepatitis. The blood loss
caused by one fluke was estimated in 0.2-0.5 ml/day [16,17]. Hypoproteinemia with
hypoalbuminemia is also frequently observed and some studies indicate that the decrease in
the plasmatic level of albumin might induce other sources of protein metabolism, such as the
production of milk and wool, to redirect amino acids toward synthesis of albumin, which
would explain the rapid loss in productivity [13]. When hypoproteinemia is intense and occur
for a long period, submandibular edema may appear.
The plasmatic levels of hepatic enzymes are increased during fasciolosis, though it varies
according to the phase of infection: glutamate dehydrogenase (GLDH), a mitochondrial
enzyme located in hepatocytes (and in many other tissues such as intestine, kidney or
muscles) increases its plasmatic level during the migratory stage, reaching the maximum level
around 8 weeks post-infection, whereas γ-glutamil transferase (GGT), present in the
epithelium of the bile ducts, produces a peak during the early stage of the biliary phase,
between 10-15 weeks post-infection [3,8,34,41].
Fasciolosis
45
Epidemiology
The chronic form of fasciolosis usually takes place during winter and early spring. In
summer, however, disease outbreaks may occur. This is derived from larval stages that have
overwintered within the snails, which makes possible for metacercaria to be present on grass
in spring.
The natural environment plays a key role in maintaining the disease and many
environmental conditions such as temperature or moisture are essential, not only for Galba
spp. snails to survive but also for the larval stages of the parasite to develop within the
mollusk. The optimal temperature for Galba truncatula is 18-27 ºC, but a minimum of 10 ºC
is required to develop. The influence of temperature on egg development is also crucial:
below 10 ºC and above 30 ºC development stops. High humidity, wet pastures and grazing
lands with poor drainage are also suitable conditions for the intermediate host, which usually
inhabits mud instead of flooded lands. However, when high rainfall occurs, clumps of grass,
rushes or reeds are appropriate habitats for the snails.
Diagnosis
Traditionally, diagnosis is based on the coprological analyses of fecal samples and it is
carried out by using concentration methods such as the flotation technique that employs a
high density solution (usually zinc sulphate) or the sedimentation technique which allows
concentrating the eggs in the sediment. However, this methodology is insufficient during the
prepatent period or when animals harbor low fluke burden.
Immunological tests to detect specific antibodies are available, the indirect
haemagglutination test is a reliable method that has been widely used [6,25,30], but in the
past two decades the enzyme-linked immunosorbent assay (ELISA) has become the most
frequently used technique. Originally, the assay was successfully adapted for serological
diagnosis [9,24,22].
A comparative study between the indirect haemagglutination test and the ELISA with
somatic and excretory-secretory antigens was realized, and it was observed that when using
ELISA antibodies were detectable from second week after infection and it was reported a
specificity of 98%, whereas the haemagglutination test showed a specificity of 86% and
detection of antibodies was achieved from third week after challenge [10]. Many variants of
the enzymatic-based assay have been assessed and are used for antibody detection in serum
and milk samples in ruminants [5,31,33,42].
Another feasible alternative for diagnosis is detection of antigens. During infection,
antigens from the parasite are released and may be used for immunodiagnosis as well as for
monitoring chemotherapy. It has been demonstrated that circulating antigens in serum of
infected animals can be detected from two weeks after infection [39]. The use of an antigen
competition enzymatic-based assay in cattle infected with Fasciola hepatica was described,
with antigen detection in serum as early as 6 days after infection [29]. Studies on detection of
antigens have continued and highly specific and sensitive methods for coproantigens
detection in feces are currently available for early diagnosis [35,50].
46
Leandro Buffoni Perazzo
Treatment
A wide variety of drugs are used for chemotherapy. It is of great importance to employ
those with flukicide efficacy against both mature and immature flukes, mainly in acute
fasciolosis. Overall, triclabendazole (TCBZ) is widespread and frequently the drug of choice,
as it is efficacious against adults and juvenile stages [7,44]. The mechanism of action of
TCBZ consists in affecting the tegument of the parasite by disrupting the secretory activity of
tegumental cells and the spermatogenesis [46,47]. In cattle and sheep, it is orally administered
at a dose rate of 12 mg/kg and 10 mg/kg, respectively. However, the withdrawal period in
beef cattle is up to 14-28 days and it must not be employed during lactation.
Albendazole is effective against adult worms and it is mainly used in chronic fasciolosis.
It is given orally at a dose rate of 7.5-10 mg/kg, and it has a withdrawal period of 10-14 days
in beef cattle. Other benzimidazole-derived includes netobimin (pobenzimidazole) and
luxabendazole. The dose rate for netobimin is 20 mg/kg but its administration should be
avoided in lactating cows and during the first trimester of gestation. Nitroxynil, bithionol and
niclofolan are drugs that belong to the phenols spectrum. It has been demonstrated that they
show activity only against adult flukes by affecting the tegument and reproductive system and
producing spastic paralysis of the parasites [17,21,23,26]. Nitroxynil is given subcutaneously
at a dose rate of 10 mg/kg. Clorsulon acts against adult flukes, being more effective in cattle
than in sheep. Other drugs that exhibit high efficacy against Fasciola hepatica include
rafoxanide, closantel and oxyclozanide. Diamphenetide is a phenoyalkane-derived with
fasciolocide activity against adults and newly excysted juvenile flukes from first day after
excystation, though its efficacy decreases as flukes develop and reach maturity [4,28,40].
Control
Due to the complexity of the epidemiological conditions, the life cycle of the parasite and
other features, eradication is difficult to accomplish. Control, however, is achievable by
taking into account some key aspects:
I. Management measures
II. Use of anthelmintics
Management measures are aimed to avoid contact between definitive hosts and infective
pastures and, to reduce or eliminate the intermediate host from the natural environment. One
option for grazing animals are feeding or drinking troughs. Improving drainage would help to
avoid puddles, thus limiting snail habitats. Another alternative is the use of molluscicides to
control snail population, which have shown to be significantly effective [11,49], but it appears
to be not widely accepted due to the risk to contaminate the natural environment and the high
and rapid reproductive capacity of snails to repopulate.
Depending on the farming system, the use of flocks of ducks to reduce snail population
has been also recommended. Combination of two or more methods might be more effective in
reducing the infection rate, but in endemic areas or where high prevalence rates are reported,
management measures are insufficient and the use of drugs to control the disease is essential.
Fasciolosis
47
The use of anthelmintics is the most effective method to controlling the disease. For rural
farmers in developing countries, pharmacological control is often prohibitive but it frequently
implies a positive cost-benefit ratio. As transmission highly depends on local epidemiological
factors, strategic guidelines may vary and should be considered according to the livestock
production and the prevalence rate.
In endemic areas and where infection of definitive hosts occurs throughout the entire year
without regard to seasons, 3-4 treatments are recommended. In countries with warm climate,
where outbreaks take place during summer and spring, animals should be dosed in autumn
and early winter. On the contrary, areas with an average rainfall and cool temperatures,
dosing from early winter to spring and repeating in late summer to winter, would prevent
disease in autumn and winter. Resistance to anthelmintics has been recorded worldwide and
has increased in the last years [2,36,38]. Although it is not regarded as a major problem, some
strategies are suggested to prevent the development of resistance such as the alternation of
drugs. Another option is combination of two or more drugs to enhance synergism, which is
considered to delay resistance.
Immunoprophylaxis
To date, immunological control through the use of vaccines is not a feasible option and
no vaccines are yet available. Nevertheless, many vaccination trials have been carried out
with promising results: in cattle, reductions from 69% to 72.4% of the fluke burden have been
recorded [12,37], whereas in sheep a 78% reduction was also reported [43]. In some
experiments, vaccination not only induced a reduction of fluke burden but also a reduction of
the egg fecundity and of the hepatic damage [1,12,37].
Fasciola Gigantica (Cobbold, 1885)
Fasciola gigantica is the causative agent of the tropical fasciolosis, one of the most
common helminthiasis in Africa and Asia, and also present in the southern Europe, the Pacific
Islands, the southern USA, the Middle East and many other areas with tropical and
subtropical climate. In some countries Fasciola gigantica overlaps with Fasciola hepatica,
despite the fact that epidemiological conditions differ for their intermediate hosts. As
Fasciola hepatica, parasites affect ruminants, herbivores and humans.
Description
Fasciola gigantica is also leaf-shaped and resembles Fasciola hepatica, but adult flukes
are larger and may reach 7.5 cm in length. The conical papilla is shorter, the shoulders are not
well-defined and the eggs are larger, measuring 170-190 µm in length and 90-100 µm in
width.
The tegument is slightly thicker and covered with microvilli and flattened spines. The
reproductive system is similar to Fasciola hepatica, although some slight differences were
48
Leandro Buffoni Perazzo
reported by Terasaki et al. [48]: the gutters are prickly, with spines growing orderly side by
side along the gutters, and the surface of the cirrus is covered by ring-like shallow gutters.
Life Cycle
The life cycle is similar to Fasciola hepatica but it requires more time, hence the
prepatent period is usually longer, being extended up to 16 weeks. Temperature is considered
to be a crucial factor affecting the development of the larval stages within the snail.
Essentially, at 26 ºC the development is completed in around 75 days, but when temperature
drops during winter or cold spells, development decreases until it stops. Under unfavorable
situation of low temperature and high altitude, periods up to 197 days from the moment snails
got infected until cercaria were shed, were reported [18].
Once the cercaria is released from the snail, it encysts to form the metacercaria which
attach to the pasture. Some of the metacercaria do not attach to the grass and become
“floating cysts”. In 1994, Vareille et al. [51] showed that between 18% and 38% of the
metacercaria were floating cysts and that the size of the snails was related to the number of
metacercaria becoming floating cysts.
Hosts
Similarly to Fasciola hepatica, several species of the subfamily Lymnaeinae act as
intermediate hosts, being Radix auricularia the most common in Africa. Other vectors are
Radix natalensis (Africa), Radix rufescens (India, Bangladesh, Pakistan, Malaysia), Radix
acuminata (Indian subcontinent), Radix rubiginosa (South-East Asia), Radix ollula (Hawaii,
Japan), Radix viridis (Japan, China). Definitive hosts include cattle, sheep, goats and buffalo.
Infection of local fauna may sometimes gain importance (camel, deer, and donkeys). Humans
are also susceptible.
The Disease
Fasciolosis due to Fasciola gigantica infection is regarded as one of the most important
parasite disease of ruminants in Asia and Africa and, like in Fasciola hepatica, the course of
the disease is often the chronic form.
Pathogenesis
It is basically the same as that of Fasciola hepatica. In cattle, during the migration
through the liver parenchyma, juvenile worms are often encapsulated in a fibrous cyst, which
results in a lower pathogenicity. Consequently, fewer flukes reach the bile ducts and
gallbladder. Calcified cysts containing flukes are also frequently found.
Fasciolosis
49
Epidemiology
The disease mainly occur in animals grazing flooded areas, as snails are essentially
tropical aquatic living and prefer highly irrigated grounds as habitats such as swamplands,
rice fields, periphery of stagnant water bodies or water channels.
Indeed, in many countries of Asia, irrigated rice fields are considered to be one of the
most major sources of infection for Fasciola gigantica. After encysting, metacercaria usually
attach to the herbage but there is a number of metacercaria that may float freely in water and
move with the flow, thus reaching areas free of snails and widening the source of infection.
There are several reports about prevalence of Fasciola gigantica worldwide, and in some
areas of India, Philippines, Pakistan, Vietnam, Thailand, Iran or Egypt, infection rates up to
80-100 % have been recorded.
Control
The control of the disease follows a similar pattern as in Fasciola hepatica, being the use
of anthelmintics the most effective methodology. However, the majority of studies have been
focused on the effectiveness of the drugs rather than on the frequency and strategy of
application of the drugs.
Moreover, in contrast to Fasciola hepatica, pharmacological control is not broadly used
in tropical fasciolosis and other strategies such as the use of molluscicides, competitor snails,
and ducks as predators or feeding management, gain importance. When a high prevalence is
reported particularly in endemic areas, combination of management measures and
anthelmintics is recommended to reduce the infection rate.
The use of molluscicides is recommended for dams or small areas with stagnant water,
where the source of infection is not widely extended, as for bigger areas such as rice fields,
grazing management or other low-cost means might be more appropriated. Biological control
by the use of ducks that eat snails has been considered. Little information is available on this
issue but to obtain a significant reduction of the infection rate, ducks should populate the
habitat before snails shed the cercaria.
Diagnosis and Treatment
The diagnosis of tropical fasciolosis is similar to that of Fasciola hepatica. It is based on
clinical signs and detection of operculate eggs in feces.
The development of highly sensitive and specific methods for immunodiagnosis has also
been applied to Fasciola gigantica and ELISA and immunofluorescence tests have been used
for detection of specific antibodies and circulating antigens in sera. The anthelmintics used in
the treatment of Fasciola hepatica are also effective against Fasciola gigantica (table 1).
Triclabendazole and clorsulon are the only drugs that have shown to be efficacious against
both mature and immature stages of the parasite.
50
Leandro Buffoni Perazzo
Table 1. Chemotherapy against Fasciola gigantica
Drug
Triclabendazole
Clorsulon
Nitroxynil
Albendazole
Closantel
Rafoxanide
Oxyclozanide
Hexachlorophene
Niclofolan
Bithionol SO4
Host
Cattle
Buffalo
Cattle
Cattle - Buffalo - Sheep
Cattle - Buffalo
Goat
Goat
Sheep
Cattle - Buffalo
Sheep - Goat
Cattle - Buffalo
Sheep - Goat
Buffalo
Sheep
Sheep
Dose - Route of administration
12 mg/kg -intraruminal24 mg/kg -orally2 mg/kg -orally10 mg/kg -subcutaneous injection15 mg/kg -orally7.5 mg/kg -orally20 mg/kg -orally5-7.5 mg/kg -orally10 mg/kg -orally15 mg/kg -orally25 mg/kg -orally30-50 mg/kg -orally0.8 mg/kg -subcutaneous injection2 mg/kg -intramuscular injection80 mg/kg -orally-
Acknowledgment
The author is grateful to Dr. Santiago Hernandez Rodriguez, Dr. Alvaro Martinez
Moreno and Dr. Isabel Acosta Garcia (University of Cordoba - Spain) for their assistance and
for generously providing the images.
References
[1]
[2]
[3]
[4]
[5]
[6]
M.S. Almeida, H. Torloni, P. Lee-Ho, M.M. Vilar, N. Thaumaturgo, A.J.G. Simpson,
M. Tendler, Vaccination against Fasciola hepatica infection using a Schistosoma
mansoni defined recombinant antigen, Smm14, Parasite Immunol., 25, 135-137,
(2003).
M.A. Alvarez-Sanchez, R.C. Mainar-Jaime, J. Perez-Garcia, F.A. Rojo-Vazquez,
Resistance of Fasciola hepatica to triclabendazole and albendazole in sheep in Spain,
Vet. Rec., 159, 424-425, (2006).
P.H. Anderson, J.G. Matthews, S. Berrett, P.J. Brush, D.S.P. Patterson, Changes in
plasma enzyme activity and other blood components in response to acute and chronic
liver damage in cattle, Res. Vet. Sci., 31, 1-4, (1981).
J.M. Annen, J.C. Boray, J. Eckert, Testing of new fasciolicides. II. Efficacy comparison
between rafoxanide and diamphenetide in subacute and chronic fascioliasis of the
sheep, Sweiz. Arch. Tierheilkd., 115,527-538, (1973).
S. Bennema, J. Vercruysse, E. Claerebout, T. Schnieder, C. Strube, E. Ducheyne, G.
Hendrickx, J. Charlier, The use of bulk-tank milk ELISAs to assess the spatial
distribution of Fasciola hepatica, Ostertagia ostertagi and Dictyocaulus viviparus in
dairy cattle in Flanders (Belgium), Vet. Parasitol., 165, 51-57, (2009).
J. Biguet, G. Rose, A. Capron, Diagnosis of distomatosis due to Fasciola hepatica by
the haemagglutination reaction. Comparison with the results of the immuneelectro
Fasciolosis
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
51
phoresis and the hemolysis reaction, Bull. Soc. Pathol. Exot. Filiales, 58, 866-878,
(1965).
J,C. Boray, P.D. Crowfoot, M.B. Strong, J.R. Allison, M. Schellenbaum, M. Von
Orelli, G. Sarasin, Treatment of immature and mature Fasciola hepatica infections in
sheep with triclabendazole, Vet. Rec., 113, 315-317, (1983).
L. Buffoni, R. Zafra, A. Perez-Ecija, F.J. Martinez-Moreno, E. Martinez-Galisteo, T.
Moreno, J. Perez, A. Martinez-Moreno, Immune response of goats immunised with
glutathione S-transferase and experimentally challenged with Fasciola hepatica,
Parasitol. Int., 59, 147-153, (2010).
D.J. Burden, N.C. Hammet, Microplate enzyme linked immunosorbent assay for
antibody to Fasciola hepatica in cattle, Vet. Rec., 103, 158, (1978).
J.B. Cornelissen, W.A. De Leeuw, P.J. Van Der Heijden, Comparison of an indirect
haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in
experimentally and naturally infected sheep, Vet. Q., 14, 152-156, (1992).
N.O. Crossland, The effect of the molluscicide N-tritylmorpholine on transmission of
Fasciola hepatica, Vet. Rec., 98, 45-48, (1976).
J.P. Dalton, S. McGonigle, T.P. Rolph, S.J. Andrews, Induction of protective immunity
in cattle against infection with Fasciola hepatica by vaccination with cathepsin L
proteinases and with hemoglobin, Infect. Immun., 64, 5066-5074, (1996).
J.D. Dargie, C.I. Berry, The hypoalbuminemia of ovine fascioliasis: the influence of
protein intake of the albumin metabolism of infected and of pair-fed control sheep, Int.
J. Parasitol, 9, 17-25, (1979).
B. Dawes, The migration of juvenile forms of Fasciola hepatica L. through the wall of
the intestines in the mouse, with some observations on food and feeding, Parasitol., 53,
109-122, (1963).
B. Dawes, Some observations of Fasciola hepatica L. during feeding operations in the
hepatic parenchyma of the mouse, with notes on the nature of liver damage in this host,
Parasitol., 53, 135-143 (1963).
B. Dawes, D.L. Hughes, Fascioliasis: the invasive stages of Fasciola hepatica in
mammalian hosts, Adv. Parasitol, 2, 97-169, (1964).
B. Dawes, Experimental fascioliasis: some effects on Fasciola hepatica of treatment of
rat hosts with bithionol ('Actamer'), Helminthol., 7, 297-307, (1966).
J.A. Dinnik, N.N. Dinnik, Effect of the seasonal variations of temperatures on the
development of Fasciola gigantica in the snail host in the Kenya highlands, Bull. Ep.
Dis. Afr., 11, 197-207, (1963).
C. Dow, J.G. Ross, J.R. Todd, The histophatology of Fasciola hepatica infections in
sheep, Parasitol., 58, 120-135, (1968).
T.G. Doy, D.L. Hughes, Fasciola hepatica: site of resistance to reinfection in cattle,
Exp. Parasitol., 57, 274-278 (1984).
Fairweather, The quest for an understanding of fasciolicidal action: holy grail or
poisoned chalice? In: J.C. Boray, (ed), Immunology, pathobiology and control of
fasciolosis, MSD AGVET, Rahway, New Jersey, 99-130, (1997).
C.J. Farrel, D.T. Shen, R.B. Wescott, B.Z. Lang, An enzyme-linked immunosorbent
assay for diagnosis of Fasciola hepatica infection in cattle, Am. J. Vet. Res., 42, 237240, (1981).
52
Leandro Buffoni Perazzo
[23] L.M. Gusel'nikova, Effect of some fasciolicide preparations on the tegument on
Fasciola hepatica implanted under the skin of the back of white rats, Med. Parazitol.
Mosk., 43, 214-217, (1974).
[24] G.V. Hillyer, N. Santiago De Weil, Use of immunologic techniques to detect
chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme linked
immunosorbent assay in infected rats and rabbits, J. Parasitol., 65, 680-684, (1979).
[25] G.V. Hillyer, D. Allain, Use of immunologic techniques to detect chemotherapeutic
success in infections with Fasciola hepatica. III. Comparison of counterelectrophoresis
and indirect haemagglutination in infected rabbits, J. Parasitol., 65, 960-963, (1979).
[26] S.D. Holmes, I. Fairweather, Fasciola hepatica: motility response to metabolic
inhibitors in vitro, Exp. Parasitol., 59, 275-289, (1985).
[27] F.W. Jennings, The anaemias of parasitic infections, In: Soulsby, E.J.L. (ed),
Pathophysiology of Parasitic Infection, Academic Press, New York, 41-67, (1976).
[28] S.B. Kendall, J.W. Parfitt, The effect of diamphenetide on Fasciola hepatica at different
stages of development, Res. Vet. Sci., 15, 37-40, (1973).
[29] Th. Leclipteux, P.R. Torgerson, M.L. Doherty, D. McCole, M. Protz, F. Farnir, B.
Losson, Use of excretory-secretory antigens in a competition test to follow the kinetics
of infection by Fasciola hepatica in cattle, Vet. Parasitol., 77, 103-114, (1998).
[30] D. Levieux, A. Levieux, C. Mage, A. Venien, Early immunodiagnosis of bovine
fascioliasis using the specific antigen f2 in a passive haemagglutination test, Vet.
Parasitol., 44, 77-86, (1992).
[31] Martinez, M.S. Martinez-Cruz, F.J. Martinez, P.N. Gutierrez, S. Hernandez, Detection
of antibodies of Fasciola hepatica excretory-secretory antigens in experimentally
infected goats by enzyme immunosorbent assay, Vet. Parasitol., 62, 247-252, (1996).
[32] Martinez-Moreno., F.J. Martinez-Moreno, I. Acosta, P.N. Gutierrez., C. Becerra., S.
Hernandez, Humoral and celular immune responses to experimental Fasciola hepatica
infections in goats, Parasitol. Res., 83, 680-686 (1997).
[33] M. McCann, M. Baylis, D.J. Williams., Seroprevalence and spatial distribution of
Fasciola hepatica-infected dairy herds in England and Wales, Vet. Rec., 166, 612-617,
(2010).
[34] R.E. Mendes, R. Zafra, R.A. Perez-Ecija, L. Buffoni, A. Martinez-Moreno, M. Tendler,
J. Perez, Evaluation of local immune response to Fasciola hepatica experimental
infection in the liver and hepatic lymph nodes of goats immunized with Sm14 vaccine
antigen, Mem. Inst. Oswaldo Cruz, 105, 698-705, (2010).
[35] M. Mezo, M. Gonzalez-Warleta, C. Carro, F.M. Ubeira, An ultrasensitive capture
ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new
monoclonal antibody (MM3), J. Parasitol., 90, 845-852, (2004).
[36] L. Moll, C.P. Gaasenbeek, P. Vellema, F.H. Borgsteede, Resistance of Fasciola
hepatica against triclabendazole in cattle and sheep in the Netherlands, Vet. Parasitol.,
91, 153-158, (2000).
[37] C.A. Morrison, T. Colin, J.L. Sexton, F. Bowen, J. Wicker, T. Friedel, T.W. Spithill,
Protection of cattle against Fasciola hepatica infection by vaccination with glutathione
S-transferase, Vaccine, 14, 1603-1612, (1996).
[38] F. Olaechea, V. Lovera, M. Larroza, F. Raffo, R. Cabrera, Resistance of Fasciola
hepatica against triclabendazole in cattle in Patagonia (Argentina), Vet. Parasitol., 178,
364-366, (2011).
Fasciolosis
53
[39] J. Rodriguez-Perez, G.V. Hillyer, Detection of excretory-secretory circulating antigens
in sheep infected with Fasciola hepatica and with Schistosoma mansoni and F. hepatica,
Vet. Parasitol, 56, 57-66, (1995).
[40] D. ap. T. Rowlands, Diamphenetide: a drug offering a fresh approach to the treatment
of liver fluke disease in sheep, Pest. Sci., 4, 883-889, (1973).
[41] D. ap. T. Rowlands, R.B. Clampitt, Plasma enzyme levels in ruminants infected with
Fasciola hepatica, Vet. Parasitol., 5, 155-175, (1979).
[42] M.R. Salimi-Bejestani, R. Daniel, P. Cripps, S. Felstead, D.J. Williams, Evaluation of
an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica
in milk, Vet. Parasitol, 149, 290-293, (2007).
[43] J.L. Sexton, A.R. Milner, M. Panaccio, J. Waddington, G. Wijffels, D. Chandler, C.
Thompson, L. Wilson, T.W. Spithill, G.F. Mitchell, N.J. Campbel, Glutathione Stransferase. Novel vaccine against Fasciola hepatica infection in sheep, J. Immunol,
145, 3905-3910, (1990).
[44] M.G. Smeal, C.A. Hall, The activity of triclabendazole against immature and adult
Fasciola hepatica infections in sheep, Aust. Vet. J., 60, 329-331, (1983).
[45] M.V.K. Sukhdeo and D.F Mettrick, Parasite behaviour: understanding platyhelminth
responses, Adv. Parasitol., 26, 73-144 (1987).
[46] A.W. Stitt, I. Fairweather, Spermatogenesis in Fasciola hepatica: an ultrastructural
comparison of the effects of the anthelmintic , triclabendazole ('Fasinex') and the
microtubule inhibitor, tubulozole, Invert. Rep. Dev., 22, 139-150, (1992).
[47] A.W. Stitt, I. Fairweather, The effect of the sulphoxide metabolite of triclabendazole
('Fasinex') on the tegument of mature and immature stages of the liver fluke, Fasciola
hepatica, Parasitol., 108, 555-567, (1994).
[48] K. Terasaki, T. Itagaki, T. Shibahara, Y. Noda, N. Moriyama-Gonda, Comparative
study of the reproductive organs of Fasciola groups by optical microscope, J. Vet. Med.
Sci., 63, 735-742, (2001).
[49] G.M. Urquhart, J. Armour, J. Doyle, F.W. Jennings, Studies of ovine fasciolosis. III.
The comparative use of molluscicide and anthelmintic in the control of the disease, Vet.
Rec., 86, 338-345, (1970).
[50] M.A. Valero, F.M. Ubeira, M. Khoubbane, P. Artigas, L. Muiño, M. Mezo, I. PerezCrespo, M.V. Periago, S. Mas-Coma, MM3-ELISA evaluation of coproantigen release
and serum antibody production in sheep experimentally infected with Fasciola hepatica
and Fasciola gigantica, Vet. Parasitol., 159, 77-81, (2009).
[51] M.C. Vareille, G. Dreyfuss, D. Rondelaud, Fasciola gigantica Cobbold and Fasciola
hepatica Linne: the numerical variations of floating cysts in relation to the snail species
and its size at miracidial exposure, Bull. Soc. Franc. Parasitol., 12, 161-166, (1994).
[52] M.L. Wolf-Spengler, H. Isseroff, Fascioliasis: bile duct collagen induced by proline
from the worm, J. Parasitol, 69, 290-294, (1983).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter IV
Evaluating the Whole Production
Chain: A Need for Organic
Production Research
Marta López-Alonso1, Marta Miranda2 and Isabel Blanco-Penedo3
1
Universidade de Santiago de Compostela,
Departamento de Patoloxía Animal,
Facultade de Veterinaria, Lugo, Spain
2
Universidade de Santiago de Compostela,
Departamento de Ciencias Clínicas Veterinarias,
Facultade de Veterinaria, Lugo, Spain
3
Swedish University of Agricultural Sciences,
Department of Clinical Sciences,
Faculty of Veterinary Medicine and Animal Science,
Uppsala, Sweden
Abstract
Organic farming promotes the multiple aims of providing good quality feedstuffs,
appropriate livestock husbandry systems and correct management practices to promote
animal health and welfare. However, benefits of organic systems are primarily related to
environmentally-friendly production and to animal welfare, whereas issues pertaining to
animal health and product quality are more influenced by the specific farm management
than by the production method. Thus, there is a general agreement in the scientific
community for the need to evaluate the whole production chain in a holistic production
such as organic farming. In order to comply with principles of organic production and
consumers’ expectations, a research strategy based on biological indicators, farm
inspections, questionnaires, secondary data and different levels of assessment is needed.
This chapter reviews what has been scientifically proven in linking animal health,
husbandry practices and resultant food product quality.
This document addresses critical obstacles not strong enough to escape from this
need-driven research, which at present are still limited, but where extrapolations from
56
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
conventional system research are inacceptable. Finally, we also present the benefits of
this integral research for a better knowledge of the current status of organic production
and the potential to improve the organic product quality.
Keywords: Organic production, whole production chain, animal health, food quality
Introduction
Livestock production forms an integral part of agricultural holdings practicing organic
farming. The aims of organic farming are to produce milk and meat in a sustainable system
based on good animal health and welfare. Compared to conventional production, organic
production is characterized by a higher roughage/concentrate ratio, a fertilizer- and pesticidefree production of crops and grass, a prolonged grazing season, an anti-parasite strategy
without drugs and emphasis on regular exercise. In organic animal production the principle of
prevention rather than treatment should be emphasized [1]. To fulfill this idea there are two
major approaches.
The first one is to improve immunity and resistance to disease through appropriate
nutrition and breeding and reduction of stress by allowing the animals to express a natural
behavior. The second approach is reducing disease challenge by limiting mixing of animals
from different herds, reduced stocking density, employing good hygiene and grazing
management to prevent parasitic infections [2]. Another aim in organic farming is to reduce
the use of conventional, synthetic veterinary medicinal products both as preventive measures
and in therapy of diseased animals. To deal with this, current EU legislation requires that
individual animals that are treated more than three times in any given year with conventional
medicine lose their organic status. The use of conventional medicine is further discouraged by
the preferred use of alternative medicines, such as phytotherapy and homeopathy, and by the
requirement to use prolonged withholding times for products from animals medicated with
licensed veterinary medicines [1].
The EU Regulation [1] was introduced to harmonize the rules of organic livestock
production across member states and to set minimum standards. It follows a system oriented
approach to obtain a good status of animal health and food safety at farm level through
various mainly indirect provisions, such as feeding, husbandry and housing as well as disease
prevention and treatment. Within the EU network ‘Sustaining animal health and food safety
in organic farming’ (SAFO) [3], the work package on standard development has considered
how and to what extent the EU Regulation contributes to high animal health status and food
safety in organic livestock production [4].
Literature reviews reveal that the implementation of organic standards have failed to
clearly improve status of animal health and welfare on many farms in comparison to
conventional production [51]. There is a huge variability with respect to this issue in organic
farms which indicate profound discrepancies between claim and reality of organic livestock
farming.
As a consequence, organic farmers and retailers can no longer stick to the claim that
organic products of animal origin are of higher value with respect to the issue of animal
health and welfare. Reasons for the limited effects of the organic standards are multi-factorial
and assumed to be farm specific in the first place. In order to preserve the credibility of
Evaluating the Whole Production Chain
57
organic agriculture and the confidence of the consumers in organic products there is a need
for more transparency and for a change in the paradigm from a standard-oriented to an
output-oriented approach. Credible information about the specific level of production and
process qualities from each farm has to be provided.
Impacts on Animal Health and Food Quality
Standardization
There is general agreement among the various stakeholders that animal health plays a
dominant role in organic livestock production, but opinions as to what constitutes an
acceptable health status are likely to differ between consumers, farmers and also between
veterinarians. There is no clear science based definition of the term ‘animal health’ (ranging
from the absence of disease to broad definition of health as a state of unrestricted physical,
physiological and psychological well-being) or clear criteria from which the state of animal
health and food quality could be adequately assessed. The improvement of animal living
conditions in organic farms is expected to improve livestock welfare and health status and
subsequently lead to improved product quality.
Based on the literature, we can conclude that at present the health status of farm animals
in both organic and conventional livestock production is similar [6, 7]. Levels of diseases are
high, regardless of the production method. Differences between farms within each group are
greater than differences between the organic and the conventional production method. The
greatest source of variation in relation to disease occurrence is farm management. A high
prevalence of diseases within a farm system is primarily related to the absence of effective
monitoring and feedback mechanisms. Limitations in the availability of labor and capital as
well as structural problems often impede efforts to improve the animal health situation at the
farm level [4]. Organic farming can offer clear advantages for animal health and welfare by
setting limits to the intensification process of animal production, in particular by limiting
growth rates. According to the organic livestock production principles, the emphasis of
disease control is on health promotion based on a broad, holistic approach. One of the aims is
to create a herd/flock and a husbandry system that minimize health and welfare problems by
optimizing production levels and by using suitable breeds and animals for the farm in
question [8]. Organic farms generally use locally produced or on-farm produced roughage as
the main source of animal feed, so, organic animals are highly-dependent on the local
environment. Provision of feed and feeding and husbandry that promotes positive health is
seen to contribute to this aim as well. Feeding is an important tool as many dietary
components are readily transferred from the feed to the fat tissues. Thus, the fatty acid [9] and
vitamin composition (e.g. vitamin E) in the diet [10] has a direct influence on the quality of
the milk and meat. A literature review shows that a large number of traits (including terminal
sire genotype, sex, slaughter weight, feeding and housing regime and slaughter-house) have
been found to have a more or less clear effect on carcass and meat quality [11].
The impact of the EU Regulation on animal health and food quality on organic farms is
therefore difficult to either describe or assess. Data such as mortality or morbidity rates, the
frequency of use of veterinary medicines, or the veterinary costs incurred would be helpful
but do not cover all aspects of animal health and are only available in some regions or farm
58
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
types [7]. They also give no indication of whether such incidences of disease occur ‘because
of’, or ‘in spite of’, adherence to the EU Regulation. The development of disease is often a
consequence of a range of sub-optimal conditions involving various factors. It is difficult to
assess whether any or which requirements in the organic production standards directly
influence animal health because farm animals are complex organisms which react
individually to their environment and any organic farm is a complex system which is
characterized not only by the production method but also by a large number of farm and
management specific aspects [4].
Needs and Priorities to Address Consumers
Concerns
Pressure from society and from within the farming community itself has resulted in a
movement towards a system of animal food production that incorporates a better treatment
and welfare of the animals, takes care of our resources and environment and at the same time
results in products that taste better or more natural and do not contain unwanted chemical
residues. Organic farming addresses all these aspects.
Consumers express concern about possible hormone, antibiotic, pesticide and chemical
residues in animal products and assume that organic products are superior to those produced
conventionally in being lower in residues and higher in nutrient content. To protect public
health, there are continuous efforts by the Member States of the European Union to reduce the
presence of undesirable residues of pharmacologically active substances in food of animal
origin. Improved analytical methods allow for the detection of chemicals in food and feed at
low and very low concentrations, thus resulting in an increased need for assessing the health
significance of detected trace substances. Regulation EC 2377/904 [12] establishes maximum
limits for residues of veterinary medicinal products in food-producing animals and animal
products. Council Directive 96/23/EC [13] lays down measures to monitor certain substances
and residues thereof, mainly veterinary medicinal products, in animals and animal products.
Additionally, Commission Decision 97/747/EC [14] establishes levels and frequencies of
sampling for certain animal products.
The holistic approach of organic farming is also in line with the growing wish for
improved traceability by the consumers. Traceability is defined as “the ability to trace and
follow a food, feed, food producing animal or ingredients, through all stages of production
and distribution” [15]. Some consumers tend to be wary of intensive production systems that
have been associated with food crises or the use of cloned or gene-modified animals [11].
Following the instability in the beef market caused by the bovine spongiform encephalopathy
(BSE) crisis, the European Union promoted the improvement in the transparency of the
conditions for the production and marketing of the products concerned, particularly with
regards to traceability, which exerted a positive influence on consumption of beef. In order to
maintain and strengthen the confidence of consumers in beef and to avoid misleading them, it
is necessary to develop the framework in which the information is made available to
consumers by sufficient and clear labeling of the product. Organic farming practices represent
an alternative to the progressive intensification that has occurred in conventional animal
production [16].
Evaluating the Whole Production Chain
59
Organically-fed consumers look for healthy and tasty food. The aspect of food
composition of greatest interest to the consumer is possibly the type of fat present,
particularly in foods of animal origin. Most research on this topic related to organic products
of animal origin is on the polyunsaturated fatty acid (PUFA) content of milk. Health benefits
of milk have been associated with the n-3 series of PUFA and conjugated linoleic acid (CLA)
contents, including the prevention of carcinogenesis, a reduced incidence of heart disease and
benefits to the immune system. Organic production, with a roughage-based diet, is known to
result in a higher content of PUFAs in milk. The explanation for this is that grasses and pulses
contain significant levels of PUFAs. Results of different studies are not consistent in relation
to the fatty acids profiles [17, 18] and the explanation for the disparity in the findings is
probably related to the amount and type of forage used to feed the cows. High-yielding cows
need to be fed a higher proportion of concentrate to forage in the ration than low-yielding
cows, which affects the fatty acids composition of the milk fat; both grasses and pulses
contain significant levels of PUFAs. However, harvesting these crops as hay or silage rather
than feeding them fresh results in a reduction in the CLA content. Weller and Bowling [19]
showed that changing the feed from conserved forages to fresh herbage increased the CLA
concentration in the milk. The CLA content of milk is also influenced by the breed of cow,
with milk from Jersey cows containing a lower concentration than milk from either Friesian
or Holstein cows. Seasonal variation in fatty acids composition of milk is regarded as being
due to forage growth pattern, succulent growth in the spring and fall being associated with
high intakes of forage and an increased content of PUFA. These influences, combined with a
change in rumen fermentation patterns in high-yielding cows as well as a genetic difference
compared with lower yielding cows, are likely to result in changes to the fatty acid profile of
the milk. All the husbandry and management factors mentioned above are much more
variable in the organic systems compared to the conventional, which could also contribute to
the greater variability on fat composition found in organic milk.
Another important issue is whether taste and consumer acceptability differ for organic
milk. Unfortunately the topic has not been well researched, possibly in part because milk is a
more uniform product than other products such as beef. Milk (conventional and organic) has
to be marketed to the public largely through milk boards which ensure that it is processed
correctly to guarantee its safety for the human consumer. The process may also remove some
of the fat, since there is a strong consumer demand for low-fat foods. One factor complicating
the taste issue is that, as described above, organic milk is often processed using UHT which
gives it a slight nutty taste that some consumers like and others dislike. As a result, the results
of taste tests are not clear-cut. In addition, organic feed is more likely to affect the sensory
properties of milk than conventional feed, due particularly to the presence of certain pulses.
For instance, Bertilsson and Murphy [20] compared the effects of feeding red clover, white
clover, alfalfa and grass silages to dairy cows and found that red clover in particular had a
negative effect on the organoleptic quality of milk; according to the authors the taste of this
milk deviated more frequently from what was expressed as ‘good quality milk’. One study
[21], in which UHT treatment did not complicate the design or findings, reported a lower
taste panel preference for organic milk than for conventional milk. The milk tested was whole
(full-fat) milk from organic, pasture-based, and conventional dairy farms. Organic milk was
the least favoured among the samples, whereas conventional milk and milk from pasture-fed
cows were rated similarly.
60
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
In relation to meat, consumers expect substantially higher quality in meat produced in
organic and pasture-based systems, which is perceived as better not only in terms of how it
was produced, but also in terms of “healthiness” and eating quality. Nutritional quality is
becoming as important to the consumer of organic beef as safety concerns. A factor related to
this is an altered profile of the fat, as indicated previously, which is regarded as being more
favorable in terms of human health. A main difference between the organic and conventional
beef production systems is that the organic animals are fed mainly or exclusively on grass and
other forages, whereas in conventional production cereal grains are also included in the feed.
Consequently organically fed animals generally grow more slowly and take longer to reach
market weight [22] and carcasses show relatively poor muscular development and a reduced
fat content [23]. In a review of organic beef production in Denmark, Nielsen and Thamsborg
[24] found that grazing and increased animal activity, which are inherent components of an
organic beef production system, may affect the eating quality due to darker meat color, risk of
off-flavor, yellow fat, and a higher content of PUFA, including CLA; they concluded,
however, that the overall effect on sensory attributes may be of minor importance. A main
effect of forage feeding on beef composition is to alter the fatty acid composition of the
carcass fat, influencing both the nutritive value of beef and also the organoleptic properties, in
particular flavor. Farm processes and resultant food product quality are also linked through
the health of the animal and its disease status. Thus, to improve meat quality, it is necessary to
examine the whole production chain from breeding to meat processing. So, slaughterhouse
records can be used to evaluate the impact that farming systems have on final beef
performance [25]. In a study developed in NW Spain [26] cattle from organic farms had
lower rates of the predominant diseases that usually occur on beef-cattle farms, and had fewer
condemnations at slaughter. However, this better health status was not reflected by carcass
performance, which seems to be determined more by breed and dietary component.
Furthermore, the suspected higher prevalence of parasitosis in the organic herd compared
with cattle from intensive and conventional systems may decrease beef performance.
Evaluation of condemnations at slaughter could be a useful tool as feedback mechanism to
establish appropriate control measures on farms in order to reduce the high prevalence of subclinical disorders that are mainly related to parasitosis.
Tools for Assessing the Whole Production Chain in
Organic Systems
The benefits of organic systems are primarily related to environmentally-friendly
production and to animal welfare, whereas issues pertaining to animal health and product
quality are more influenced by the specific farm management than by the production method
[27, 28]. For instance, overcrowding and mistakes in feeding regimes, particularly in the case
of intensive practices can lead animals into imbalances or damage on their metabolic
homeostasis, and consequently can result in sub-clinical and clinical disease states. Although
such problems rarely occur in extensive beef production systems [6], animal health issues
remain one of the main difficulties encountered by organic breeders of ruminants [29].
In this scenario, the strategic research priorities should be based on the principles of
organic agriculture, scientific innovation and best integration of indigenous knowledge of
Evaluating the Whole Production Chain
61
farmers. The priorities focus particularly on the conflicts between economy, ecology and
social cohesion/harmony inherent in most concepts for sustainable agriculture and food
production, and they propose research activities and insightful learning concepts far beyond
the niche that organic farming currently still represents [30].
Assessing the whole production chain, with a holistic approach, necessitates a systematic
work. Creating an effective farm animal health plan is vital in maintaining the overall health
and well-being of farm animals. Experience shows that a good animal health plan should
identify the availability of resources and structural problems that influence animal health
situation at the farm, identify management strategies and set targets for reducing the incidence
of disease, and engaging the farmer, veterinarian and advisors in a constructive way. Outside
of the farm, to guarantee a good information flow it’s essential to obtain new knowledge to be
incorporated into the standards and regulations of organic farming. Furthermore, feedback
loops along the food chain, from consumers to producers, will contribute to make the system
more sustainable.
Animal Health and Welfare Plan
When establishing a welfare and health plan we need to be aware that the stock-keeper
has the most significant influence on the welfare and health of the herd. The stock-keeper
should draw up a written health and welfare plan with the herd's veterinary surgeon and,
where necessary, other technical advisors, which should be reviewed and updated each year.
This plan should set out health and husbandry activities that cover the whole year’s cycle of
production, and include strategies to prevent, treat or limit existing disease problems. The
plan should include records to enable us to monitor and assess the health and welfare of the
herd.
The health and welfare of animals depends on them being regularly inspected. All stockkeepers should be familiar with the normal behavior of cattle and should watch for any signs
of distress or disease which include: listlessness; separation from the group; unusual
behavior; loss of body condition; loss of appetite; a sudden fall in milk yield; constipation;
scouring (diarrhea); any discharge from the nostrils or eyes; producing more saliva than
usual; persistent coughing; rapid or irregular breathing; abnormal resting behavior; swollen
joints; lameness and mastitis. It is very important to be able to anticipate problems or
recognize them in their earliest stages. In many cases, you should be able to identify the cause
and put matters right immediately. You should always consider the possibility that cattle may
be affected by a notifiable disease. If the cause is not obvious, or if your immediate action is
not effective, a veterinary surgeon or other expert should be called in immediately - failure to
do so may cause unnecessary suffering
The written health and welfare plan should also, as a minimum, look at: biosecurity
arrangements on-farm and in transport; purchased stock procedures; any specific disease
programs, such as leptospirosis, Johnes disease, salmonella, BVDV and tuberculosis;
vaccination policy and timing; isolation procedures; external and internal parasite control;
lungworm control; lameness monitoring and foot care; routine procedures, such as ear
tagging; mastitis control. The health and welfare plan should make sure that animals get any
necessary medical treatment at the correct time and in the correct dose.
62
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
In geographical areas with known mineral deficiencies and imbalances - and where
vitamin or mineral deficiencies are likely – the need to supplement the animal´s diet should
be considered. Supplementary magnesium should be provided during periods when there is a
recognized risk of deficiency, for example, in early spring or at weaning in sucker herds. This
aspect should be covered in the health and welfare plan.
Equally, too much of a particular vitamin or mineral may cause problems. For example,
too much copper can lead to copper poisoning in ruminants, which makes it necessary to
carefully analyze the amount of copper in the existing diet [16], prior to the administration of
copper orally or by injection.
Body-condition scoring can contribute greatly to good husbandry and help to avoid costly
welfare problems. Condition scoring is an easy technique which allows for a quick
assessment of the body reserves (i.e. fat) of individual animals. The technique will be of
benefit when used as a routine management tool to check that cattle are in the target condition
for each stage of the production cycle, particularly at drying off or weaning; calving; early
lactation in general; and peak yield in particular.
A good biosecurity, which means reducing the risk of disease occurring or spreading to
other animals, can be obtained through good management and husbandry, good hygiene,
reducing stress of the herd and effective disease control systems such as vaccination and
worming programs. A record of mortality that occurs in the herd must be kept, as well as any
medicinal treatment given to the animals. These records need to be kept for at least three
years and should include: the name and address of the medicine supplier, the date the animals
were treated, which animal or group of animals were treated, and the quantity of medicine
used. Although it's not a legal requirement, it is useful to keep a record of specific cases of and treatment given for - certain disorders, for example: mastitis, lameness and milk fever.
Farm Inspections
To ensure that a farm meets the standards of organic agriculture, regular on-farm
inspections are conducted. Farm inspections generally consist of a physical tour, examination
of records, and an oral interview. Inspectors are veterinarians or animal health technicians
trained to evaluate the health of animals and to detect noncompliance in areas such as
structures, housekeeping, and recordkeeping.
During inspections, livestock records are checked for: disease control and tracing, farm
movements, transport, breeding, births, deaths and animal passports, use of medicines,
retention and correct use of animal movement licenses.
Farm inspections pursue to ensure the highest standards of disease control and animal
welfare by: inspecting livestock for signs of disease, ensuring that any sick or injured animals
are cared for promptly, checking that livestock have access to adequate feed and water, and
inspecting buildings to ensure that ventilation, lighting and space allowance meet required
standards, advising on codes of good practice for the welfare of livestock and bio-security.
Identification for traceability, which is vital for notifiable disease control, is specifically
/detailed checked.
Evaluating the Whole Production Chain
63
Questionnaires
Questionnaires are indirect methods frequently used to collect data in epidemiological
studies. Performing questionnaires reflecting what farmers can recall or percept tend to be of
more subjective nature than the more objective registry and cow-side methods. Obtaining
information in questionnaires can be difficult as farmers perceive relevant information as
being sensitive to divulge; consequently, underestimation of the problem (for example:
disease prevalence) may occur due to farmer reticence about admitting the behaviors and
outcomes that are considered professionally transgressive. Like any diagnostic test,
questionnaires have intrinsic precision and accuracy. For questionnaires, the term
repeatability (the ability of a question to give consistent results on more than one occasion) is
often used in place of precision, while validity (the degree to which an answer represents the
true state of nature) is used in place of accuracy [31]. The quality of the procedures used to
develop, administer and validate questionnaires can have a tremendous impact on the quality
of the data collected. Even well designed pre-tested questions can have limited repeatability
or be poorly representative of the process under investigation. Over the years, many concerns
have been raised and recommendations made to ensure rigorous development and validation
of questionnaires [32, 33]. Still, more often than not efforts are allocated to the development
of sophisticated statistical models while no formal evaluation of the quality of the
questionnaire-derived data used in these models is realized. Data of unknown quality can only
yield results of unknown quality [32, 34].
Feedback Loops
All actors in the food chain can potentially play an important role in making the food
system more sustainable. However, relating concerns about the quality of food and
environmental risks to consumer choices or food production methods has become
exceedingly difficult. Signals of unhealthy local ecosystems or production systems are in
danger of being filtered out or masked as a result of the globalization of the food market.
Information on environmental impacts caused by different components of the food production
chain is unlikely to reach consumers, nor is feedback from consumers to producers. This is
because the two have become separated both in time and space, a process enabled through for
example new agricultural and transport technologies and intense trade flows of food products
between distant regions. In other words, feedback loops are loose and as environmental
problems broaden in scale there is a need to establish or strengthen institutions for managing
feedback information between the various parts of the system [35].
It is argued that a more locally based food system with tighter links between producers
and consumers can improve feedback management and thus make the system more
sustainable. Living closer to or having closer contact with food production makes it possible
for the consumer to influence and have better control over how the food they eat is produced.
Where feedback loops are loose and distances are large, feedback needs to be directed
through institutions on an overarching level regarding both the state of the environment,
producer and consumer interests.
Labeling of food items according to environmental or social standards that are controlled
by independent bodies and trusted by consumers is one possible way to deal with the fact that
64
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
there are large distances in the modern food system. When consumers and producers cannot
have direct contact with each other, labels act as information and trust carriers, and normally
also give primary producers better prices.
In order to properly understand and effectively act directly upon feedback from the food
system, organizations and institutions in management positions need to be at the same
temporal and spatial scale as the feedback signals. In the current food system this would
imply a downscaling of structures in order to manage feedback from local ecosystems and
societies, as well as the development of institutions linking together the local and regional
with the global for feedback management [36].
Compendium of Database
A compendium of animal health and welfare information relevant to organic livestock
production has been produced by the Organic Livestock Research Group (OLRG), Veterinary
Epidemiology and Economics Research Unit (VEERU), Department of Agriculture, the
University of Reading. The compendium is supported by full scientific abstracts taken from
the CAB International information database. The objective of the project was to create a
database and archive of information on animal health, welfare and husbandry relevant to
livestock production under organic standards and to assess the database and its relevance to
organic livestock production. The compendia will serve as a resource material for advisors,
inspectors and veterinarians who work with organic or converting farmers. It is also
envisaged that the compendia could be used as a training tool for advisors and veterinarians
learning about the issues related to general and specific animal health and welfare aspects of
organic livestock production; in addition, the material will provide a useful resource material
for the sector bodies and policymakers in the development of organic livestock production
standards and regulations [37].
Critical Obstacles to Enhance Animal Health on
Organic Farms
Contributions to the SAFO workshops revealed a range of constraints to enhancing
animal health and food safety on organic farms. They can be divided into internal (farm level)
and external constraints. A comprehensive overview is presented in the Proceedings of the
Second SAFO Workshop [38]. The most relevant constraints at the farm level are:
management skills of the farmer, availability of capital, labor, nutrients and genotypes
adapted to the specific farm conditions. The nature of these constraints varies considerably;
both between individual farms and between regions and countries. There appears to be a lack
of training and education of farmers in disease prevention and animal health promotion on
organic farms [28] and a lack of analysis of economic implications of disease levels under the
conditions of organic management. External constraints can be categorized broadly into
market factors and institutional support. The market factors encompass those that enhance the
effectiveness of marketing, that provide clear incentives to improve animal health and food
safety, and secure an adequate price that covers the cost of production and yields some profit.
Evaluating the Whole Production Chain
65
Currently, the marketing of organic animal products is difficult in some regions because of
imbalances between supply and demand, a lack of availability of certified abattoirs and
dairies, and a poor flow of information between producers and processors. Prices for organic
products face the same pressure of the market as conventional products and leave little room
for investments in animal health. Lack of knowledge also appears to be an external constraint.
Veterinarians, consultants and inspectors often lack basic knowledge of organic farming
principles and practices and do not have sufficient training in preventive health management
and alternative treatment options. It seems that the standards associated with organic farming
do not per se ensure either high levels of animal health and welfare or safe livestock food
products [28, 39]. Furthermore, there is lack of a uniform standard because countries differ in
characteristics such as climate, availability of resources (feedstuffs, litter, outdoor areas), herd
structures, economic conditions, and disease prevalence, cultural differences in the perception
of problems and expertise to deal with them [28, 40]. Thus, because there are different ways
of adhering to organic principles and standards, developments in organic farming practice
have to be set in a national, regional and local context [28]. Because of the huge variation in
the prevalence of diseases between organic farms, general claims that organic animal
products derive from healthy animals or from animals that are healthier than those in
conventional production cannot be justified, but the organic standards set a framework under
which improvements could be achieved. The modern use of "quality" raises problems: how to
measure and evaluate quality, what is measured and which scale has to be used. It is
commonly acknowledged that meat quality is a difficult characteristic to assess as many
different aspects, both objective and subjective, make up the overall trait. Numerous
literatures are available describing single criteria of meat quality and those describing the
production process. Without a fairly precise idea of what is measured and how the
measurement is performed, a term such as "quality assurance" makes no sense at all [41]. The
current charging system for veterinary support may also not be conducive to the provision of
health promotion management [6]. It would therefore appear likely that animal health and
food safety on organic farms could be enhanced if improved advisory support from veterinary
practitioners were available. In addition, the introduction of an improved certified and quality
management procedure in combination with improved traceability might further improve
animal health and food safety. As the food production system has transformed from one based
locally/regionally to a global system, it has gained in economic efficiency, but it has also
produced many negative social and environmental consequences. Current market trends tend
to impede the reception, understanding and communication of feedback in the food system
[36]. Intensification, specialization, distancing, concentration and homogenization are trends
identified as major constraints for tightened feedback loops. These trends can mask or make it
possible to disregard feedback signals from unhealthy ecosystems and weaken
communication in the food chain. Intensification is associated with social costs as people are
replaced by machinery and ecological costs such as impoverished soils, nutrient and toxin
polluted waters and loss of biodiversity; intensification has increased the dependence on
external resources and hence the motive for sustainable management of local ecosystems is
weaker as resources can be found elsewhere. Farming has become increasingly specialized
and is producing food as a commodity, cultivated or reared in massive monocultures [42]; in
addition, a low level of biodiversity can limit options for adaptations and alternative
solutions. The large distance, both geographically and institutionally, impedes the flow of
information in the food system and blocks ecological feedback along the whole chain from
66
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
extraction to consumer decisions [43]. Globalization of the food system has resulted in
increasingly homogeneous production methods and consumption patterns, since similar
products are marketed and produced worldwide [44], which is in great contrast with organic
products.
Potential for Integral Research of Organic
Farming
The global food system is dynamic, constantly evolving and within the pockets of change
that are created through the dynamic process lie opportunities for innovations or alternative
solutions. As previously aforementioned, tightening feedback loops is one of several strategic
measures that have to be taken to change the current food system in a sustainable direction.
Two strategies for improvement have been proposed: increase the reliance on local
ecosystems, learning from small-scale resource management based on local ecological
knowledge and experience [45, 46] and developing institutions that can deal with spatial,
temporal and cultural distances.
Shifting from external dependence to a more locally -based food supply could have
several positive implications for the sustainability of the food system, locally as well as
globally. Increasing self-reliance may reduce negative effects on the environment, increase
job opportunities related to food production in rural areas and reduce uncertainties about food
production for local consumers [47]. Increased local self-sufficiency can also mean a decrease
in vulnerability of fluctuations in the world economy [48]. The face-to-face ties between
producers and consumers can not only increase communication and learning, but also
revitalize local food production and preserve small-scale family farms [44]. Furthermore,
increased reliance on local resources in farming can potentially create a more knowledgeintensive agriculture on the farm level and provide a possibility for the farmer to see,
understand and react to local agroecosystem feedback [49]. A sustainable food system is only
possible in an overall sustainable society, but with increased local dependence management
errors having a smaller range,these are felt more quickly and should be more reversible [50].
When feedback loops are loose and less direct, there is a need to develop or strengthen
institutions that can handle large geographical and temporal distances. Institutions tuned to
handling feedback in the global food system are needed and they can be directed towards, and
operate on, different levels. For example, on the consumer level tools for enhancing feedback
from production to consumption are the introduction of environmental labels and
environmental management schemes. Environmental labels can serve as a signal of
production practices and the environmental impact of product [51]. Producing food in
accordance with environmental management or quality schemes is a way for the producer to
communicate a quality or environmental commitment [52]. Primary producer organizations,
consumer organizations, retailers and farmer associations are essential to develop initiatives
to communicate improvements in quality and environmental performance. For example, the
organic farming associations, along with control organizations, university departments,
research institutes, etc., are members of the International Federation of Organic Agriculture
Movements (IFOAM) [53], all feedback from members and new knowledge is incorporated
into the standards and regulations of organic farming.
Evaluating the Whole Production Chain
67
Challenges related to the diverse conditions of different regions of Europe needs to be
identified and discussed in relation to the issue of harmonizing organic standards. Farm
practices and research in organic livestock production are not as well developed as in crop
production. As previously indicated, climate, soil, cropping systems, stocking density, market
development and traditions and vary greatly across Europe, creating very different conditions
for livestock production on organic farms; also experiences, expectations and the perception
of problems and challenges vary greatly [4].
All of this ensures that differences between organic farms, not only in terms of husbandry
and management, but overall on the quality of their products, are greater than those when
comparing conventional systems. It is well assumed that farming intensification drastically
reduces diversity at different scales of animal production, this homogenization process
leading to environmental degradation and unsustainability [54]. Taking advantage of diversity
is a new challenge to improve sustainability.The standardization of rules should focus on
areas that show a high level of differences that are important to consumers, that could distort
trade, and/or that could potentially conflict with the core organic values. The overall aims of
organic agriculture should always be considered when harmonizing the rules. Many recorded
differences relate to fertilization and animal feeding which should be considered for
standardization. Harmonization of provisions related to the use of inputs, such as fertilizers,
manure and feed, should follow the overall aim of limiting intensification of organic
production by the reliance on external (conventional) inputs and of reducing environmental
impact.
Also conversion is an area for potential harmonization, both concerning the periods
required for conversion of land and animals and the requirement of whole farm conversion.
Harmonization should also aim to introduce common rules in areas not covered by the EU
regulation but by many other standards, such as specialist plant production systems,
environmental protection and rules for processing. Environmental protection is not considered
by the European Regulation, but is an area of high policy relevance and importance to
European stakeholders as indicated by the high number of differences. Harmonization of the
rules at the EU level should be supported by better communication, more transparency and by
research into areas where limited experience with the implementation of the regulations and
standards exists.
Conclusion
The data presented in this chapter reaffirms the necessity of a systematic control in
organic systems. Studies of the whole production chain address this research strategy, and
provide an accurate reflection of the status of the organic system as a whole. The basis for this
study is to encompass information from the different integral components of any organic
production system and from the time periods along the production cycle.
This type of studycan contribute to a better control of not only thespecific farm, but
itcould also harmonize the needs of food safety, growing awareness of consumers and the aim
of a high level of animal health and welfare.
68
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
EC Council Regulation No 1804/1999 of 19 July 1999, Official Journal of the
European Communities, 1999, L 222, p. 0001–0028.
S. Padel, O. Schmid, and V. Lund, Organic livestock standards. In: Animal Health and
Welfare in Organic Agriculture (eds M. Vaarst, S. Roderick, V. Lund, and W.
Lockeretz). Wallingford. CABI, (2004) 57-72.
SAFO network (Sustaining Animal health and Food safety in Organic farming),
http://safonetwork.org/.
Sundrum, S. Padel, G. Arsenos, A. Kuzniar, B. I. F. Henriksen, M. Walkenhorst, and
M. Vaarst, Current and proposed EU legislation on organic livestock production, with a
focus on animal health, welfare and food safety: a review. In: Future perspectives for
animal health on organic farms: Main findings, conclusions and recommendations from
the SAFO Network (eds C. Rymer, M. Vaarst, and S. Padel). Proceedings of the 5th
SAFO Workshop, Odense, Denmark. University of Reading, UK, 2006.
Sundrum, “Organic livestock production – trapped between aroused consumer
expectations and limited resources”, 16th IFOAM Organic World Congress, Modena,
Italy. 2008. At http://orgprints.org/11813.
M. Hovi, A. Sundrum, and S. M. Thamsborg, Livest. Prod. Sci. 80, 41 (2003).
Sundrum, T. Benninger, and U. RichterPreprint, 2004 at http://orgprints.org/5232/.
IFOAM. IFOAM Norms. II. IFOAM Basic Standards for organic production and
processing. International Federation of Organic Movements, Tholey-Theley, Germany
(2002).
J. D. Wood, and M. Enser, Br. J. Nutr. 78, Suppl. 1, S49 (1997).
D. J. Buckley, P. A. Morrissey, and J. I. Gray, J. An. Sci. 73, 3122 (1995).
QLIF (Quality Low Input Food), 2006 at http://www.qlif.org/research/sub4/
pub/441KUD4a.pdf .
EC Council Regulation No 2377/90 of 26 June 1990, Official Journal of the European
Communities No L 224, 18. 8. 90, p. 224/1-8.
EC Council Directive No 96/23 EC of 29 April 1996, Official Journal L 125 ,
23.05.1996 p. 0003 – 0009.
EC Commission Decision No 97/747/EC of 27 October 1997, Official Journal L 303,
06.11.1997, p. 0012 – 0015.
EC Regulation No 1760/2000 of the European Parliament and of the Council of 17 July
2000, Official Journal of the European Communities L 204/1, 11.08.2000, p. 1-10.
Blanco-Penedo, R. F. Shore, M. Miranda, J. L. Benedito, and M. López-Alonso, Livest.
Sci. 123, 198 (2009).
K. A. Ellis, G. Innocent, D. Grove-White, P. Cripps, W. G. McLean, C. V. Howard, and
M. Mihm, J. Dairy Sci. 89, 1938 (2006).
G. Butler, S. Stergiadis , C. Seal, M. Eyre, and C. Leifert, J. Dairy Sci. 94, 24 (2011).
R. F. Weller, and P. J. Bowling, The yield and quality of plant species grown in mixed
organic swards. In: Proceeding of Organic Meat and Milk from Ruminants (ed Z.
Kyriazakis). Athens, EAAP Publication (2002) 177–180.
J. Bertilsson, and M. Murphy, Grass Forage Sci. 58, 309 (2003).
Evaluating the Whole Production Chain
69
[21] L. P. Valverde, Comparison of sensory characteristics, and instrumental 69lavour
compounds analysis of milk produced by three production methods. Unpublished MS
thesis, Faculty of the Graduate School, University of Missouri-Columbia, USA (2007).
[22] R. Blair, Nutrition and feeding of organic cattle. CAB International (2011).
[23] Russo, and G. Preziuso, Stocarstvo. 59, 23 (2005).
[24] K. Nielsen, and S. M. Thamsborg, Livest. Prod. Sci. 94, 41 (2005).
[25] M. Vaarst, and M. Hovi, “Organic livestock production and food quality: a review of
current status and future challenges”, Proceedings of the 2nd SAFO Workshop,
Witzenhausen, Germany, 2004, pp. 7-15.
[26] Blanco-Penedo, M. López-Alonso, R. F. Shore, M. Miranda, C. Castillo, J. Hernández,
and J. L. Benedito, Agronomy Research 7, 585 (2009).
[27] Sundrum, Organic livestock production. In: Livestock farming and the environment (eds
Hartung, Jörg and Wathes, M. Christopher), Proceedings of Workshop 4 on Sustainable
Animal Production. (2001) 37-38, at: http://orgprints.org/924/.
[28] M. Vaarst, T. W. Bennedsgaard, I. Klaas, T. B. Nissen, S. M. Thamsborg, and S.
Ostergaard, J. Dairy Sci. 89,1842 (2006).
[29] J. Cabaret, Livest. Sci. 80, 99 (2003).
[30] U. Niggli, A. Slabe, O. Schmid, N. Halberg, and M. Schlüter, “Food, Fairness and
Ecology: An organic research agenda for a sustainable future”, 16th IFOAM Organic
World Congress, Modena, Italy, 2008.
[31] S. Dufour, H. W. Barkema, L. DesCôteaux, T. J. DeVries, I. R. Dohoo, K. Reyher, J. P.
Roy, and D. T. Scholl, Prev. Vet. Med. 95, 74 (2010).
[32] Y. H. Schukken, D. Van De Geer, F. J. Grommers, and A. Brand, Prev. Vet. Med. 7,
31–38 (1989).
[33] T. Scholl, P. Dobbelaar, P. And A. Brand, J. Dairy Sci. 75, 615 (1992).
[34] L. Gordis, Am. J. Epidemiol. 109, 21 (1979).
[35] S. Levin, Fragile Dominion: Complexity and the Commons. Perseus Books, Reading,
MA (1999).
[36] Sundkvist, R. Milestad, and A. Jansson, A. On the importance of tightening feedback
loops for sustainable development of food systems. Food Policy 30, 224 (2005).
[37] S. Roderick, and M. Hovi, M, “Organic Livestock: Animal Health, Welfare and
Husbandry Assessment of existing knowledge and production of an advisory resource
compendium (OF0162)”. University of Reading, Veterinary Epidemiology and
Economics Research Unit (VEERU). 2001.
[38] M. Hovi, A. Sundrum, and S. Padel, “Organic livestock farming: potential and
limitations of husbandry practice to secure animal health and welfare and food quality”,
Proceedings of the 2nd SAFO Workshop, Witzenhausen, Germany. University of
Reading, UK. 2004.
[39] N. Fall, U. Emanuelson, K. Martinsson, and S. Jonsson, Prev. Vet. Med. 83, 186 (2008).
[40] V. Lund, and B. Algers, Livest. Sci. 80, 55 (2003).
[41] H. J. Andersen, N. Oksbjerg, J. F. Young, and M. Therkildsen, Meat Sci. 70, 543
(2005).
[42] J. Pretty, Agri-Culture: Reconnecting People, Land and Nature, Earthscan Publications
Ltd, London, Sterling, VA (2002).
[43] T. Princen, Ecol. Econ. 20, 235 (1997).
[44] S. U. O’ Hara, and S. Stagl, Popul. Environ. 22, 533 (2001).
70
Marta López-Alonso, Marta Miranda and Isabel Blanco-Penedo
[45] S. Holling, and S. Sanderson, Dynamics of (dis)harmony in ecological and social
systems. In: Rights to Nature – Ecological, Economic, Cultural, and Political
Principles of Institutions for the Environment (eds S. Hanna, C. Folke, and K. G.
Mäler). Island Press, Washington, DC and Colevo, CA. (1996).
[46] F. Berkes, and C. Folke, Linking social and ecological systems for resilience and
sustainability. In: Linking Social and Ecological Systems – Management Practices and
Social Mechanisms for Building Resilience (eds F. Berkes, C. Folke). University Press,
Cambridge (1998).
[47] G. Feenstra, Agric. Human Values 19, 99 (2002).
[48] H. E. Daly, and J. B. Jr. Cobb, For the Common Good: Redirecting the Economy
toward Community, the Environment, and a Sustainable Future. Beacon Press, Boston
(1989).
[49] N. G. Röling, and J. Jiggins, The ecological knowledge system. In: Facilitating
Sustainable Agriculture. Participatory Learning and Adaptive Management in Times of
Environmental Uncertainty (eds N. G. Röling, and M. A. E. Wagemakers). Cambridge
University Press, Cambridge. 1998.
[50] Borgström Hansson, and M. Wackernagel, Ecol. Econ. 29, 203 (1999).
[51] K. Giannakas, Canadian J. Agric. Econ. 50, 35 (2002).
[52] T. Varzakas, and D. Jukes, Food Policy 22, 501 (1997).
[53] IFOAM. Basic standards for organic production and processing. International
Federation of Organic Agriculture Movements, Tholey-Theley, Germany (2000).
[54] M. Tichit, L. Puillet, R. Sabatier, and F. Teillard, Liv. Sci. 139, 161 (2011).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter V
Exanthematic Bovine Vaccinia Virus
Outbreaks in Dairy Cattle in Brazil:
An Overview of Viral Isolates
and Risk Factors
Jônatas Santos Abrahão, Giliane de Souza Trindade
and Erna Geessien Kroon
Laboratório de Vírus, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais (UFMG),
Brazil
Abstract
Exanthematous viral diseases are major causes of morbidity and economic loss
associated with dairy cattle. Among the emerging viral exanthematous diseases, we
highlight bovine vaccinia (BV), of which the Vaccinia virus (VACV) is the etiological
agent.
BV outbreaks have been occurring over the past 11 years in Brazilian rural areas and
are associated with ulcerative lesions on the udders and teats of dairy cattle, causing
considerable loss in milk production.
BV is a zoonotic disease, and the milker can be infected by direct contact with an
infected animal. Several VACV were isolated in Brazil during BV outbreaks in recent
years, and molecular and biological analyses showed that these viruses can be segregated
into two major groups, based on genetic markers and virulence. Here, we offer a brief
overview of VACV taxonomical location in the Poxviridae family, with an emphasis on
the VACV isolates obtained during BV outbreaks in Brazil, including their geographic
locations, biological characteristics and molecular properties. Risk factors of the disease
are also discussed based on epidemiological information and new findings related to
possible hosts and reservoirs of VAC in Brazil.
72
Jônatas Santos Abrahão, Giliane de Souza Trindade and Erna Geessien Kroon
1. Poxviridae Family
The Poxviridae family is comprised of large and complex DNA viruses with a
cytoplasmic replication cycle. The family is divided into two subfamilies, Chordopoxvirinae
and Entomopoxvirinae, which infect vertebrate and invertebrate hosts, respectively [1]. The
host range of Entomopoxvirinae includes the Insecta species, such as Diptera, Coleoptera,
Orthoptera and Lepidoptera [2,3]. The Chordopoxvirinae are divided into eight genera, of
which four (Orthopoxvirus, Parapoxvirus, Molluscipoxvirus and Yatapoxvirus) are
etiological agents of human diseases [4-7].
Several veterinary diseases are caused by chordopoxviruses, which are associated with
economic losses. Viruses of the Avipoxvirus genus affect a large spectrum of hosts, including
chickens and turkeys, and cause vesicles on the face and feet [8,9]. Members of the
Leporipoxvirus genus cause systemic infections in domestic rabbits with high lethality rates
[10]. Viruses of the Parapoxvirus genus infect caprine, ovine and bovine species [11-13], and
the Orthopoxvirus genus viruses are associated with losses in herds of dairy cattle, buffalo
and camels [14-16]. The chordopoxviruses host range is notably large, and several new viral
species are not classified in a genus. Among the unclassified poxviruses are California
harbor seal poxvirus, Squirrelpox virus, Dolphin poxvirus, Grey kangaroo poxvirus, Red
kangaroo poxvirus, Nile crocodile poxvirus and Spectacled caiman poxvirus [1].
2. The Orthopoxvirus (OPV)
The OPV genus includes nine antigenically related species [1,18], with a wide
geographical distribution and a large host range spectrum [19,20]. Four OPV are able of
infecting humans: Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV)
and Vaccinia virus (VACV) [1,20]. These three species as well as other OPV species such as
Ectromelia virus (ECMV), Camelpox virus, Racconpox virus, Volepox virus and Taterapox
virus are also capable of infecting other animals. Two other species, Skunkpox virus and
Uasin Gishu disease virus, which are etiological agents of disease in opossums and horses,
respectively [1], have not been characterized in detail.
3. Vaccinia Virus
Vaccinia virus, the prototype of OPV, was used in the Smallpox Eradication Campaign
promoted by the World Health Organization (WHO). VACV was used in the smallpox
eradication campaign because it confers high immunological protection, causes localized
lesions, and is less virulent than VARV [21]. The VACV origins remain unknown, although
some theories attempt to explain this: i) VACV is a hybrid of CPXV and VARV; ii) VACV is
derived directly and exclusively from CPXV or iii) VARV; and iv) the natural reservoir of
VACV is currently extinct. Despite these numerous hypotheses, there is no agreement on this
issue to date [14,22,23].
Despite the suspension of the smallpox eradication campaign during the 1980s, the
United States Army Forces continued a program of vaccination, aiming to protect their
Exanthematic Bovine Vaccinia Virus Outbreaks in Dairy Cattle in Brazil
73
soldiers against a hypothetical smallpox bioterrorism attack. Although the soldiers are
immunized, they can still spread infectious viral particles from vaccination scabs. Therefore,
some cases of intra-familial VACV transmission have been described in recent years [24,25].
In March 2007, there was a report from the United States of a severe case of eczema
vaccinatum that caused generalized lesions in a child who was a relative of a vaccinated army
soldier [24].
After smallpox eradication, vaccination was suspended, and interest in VACV was
directed mainly at immunological and virus evasion studies [25,26]. In recent years, VACV
has been considered as a recombinant vector for vaccines [27-29]. However, recent
exanthematic outbreaks affecting dairy cattle and milkers have been reported in Brazil, and
several studies have shown that VACV is the etiological agent of these outbreaks
[12,13,22,30-34].
4. Vaccinia Virus in Brazil
Several studies from Brazilian research institutes have shown that VACV strains are
actively circulating in Brazil [12,13,22,30-34]. During the 1960s, the isolation of the first
VACV strains was performed during a governmental program to investigate unknown viral
diseases reported in Brazilian rural areas [35]. The viruses SPAn232 (SAV) and Cotia were
isolated and re-isolated from sentinel mice in the Cotia forest in São Paulo State. The VACV
strain BeAn58058 (BAV) was isolated from blood samples of a wild rodent of the Oryzomys
genus that was captured in the Utinga forest in Pará State, Amazonia. Recent phylogenetic
analysis showed that all of the above mentioned viruses were VACV [35-38]. In 1993, an
exanthematic outbreak was reported in an animal facility at the Universidade Federal de
Minas Gerais (UFMG), affecting Swiss mice that had been obtained from the Universidade de
Campinas, São Paulo State. Biological tests revealed the presence of poxviruses particles in
the exanthematic lesions of the mice. This virus was named Belo Horizonte (VBH) [40].
Despite the clinical signs suggestive of an ECMV infection, molecular studies showed that
VBH was in fact an additional VACV strain circulating in Brazil [30]. However, despite these
sporadic VACV isolations from rodents, most VACV isolated and characterized since 1999
have come from bovine vaccinia (BV) outbreaks in Brazil [12,13,22,30-34]. The lesions
observed in bovines are typical of poxvirus, which begin with a macula, evolving sequentially
into a papule, a vesicle, a pustule, an ulcer and then a scab. These lesions can favor secondary
infections, such as bacterial mastitis, causing losses in milk production [40]. The calves
usually get infected when suckling sick cows and therefore present lesions on the nose, mouth
and tongue. The occupational infection of milkers is also common, causing lesions on the
fingers, hands, or arms as well as causing myalgia, fever and lymphadenopathy [12,13,22,3034]. In 1999 in Rio de Janeiro State, the VACV Cantagalo (CTGV) was isolated. Initial
molecular analysis suggested that CTGV was derived from VACV-IOC, a vaccine used
during the WHO campaign [22]. In the same year, the VACV-Araçatuba virus (ARAV) was
isolated from an exanthematic outbreak affecting humans and bovines in São Paulo State. The
molecular characterization of ARAV showed high identity with CTGV [41]. Between 2001
and 2003, the Adolfo Lutz Institute investigated vesicle contents collected during outbreaks
occurring in Minas Gerais, Goiás and São Paulo States. Clinical specimens were diagnosed
74
Jônatas Santos Abrahão, Giliane de Souza Trindade and Erna Geessien Kroon
positive for poxviruses. Further molecular characterization grouped these samples
phylogenetically with other Brazilian VACV strains [41]. Other viral strains were isolated in
recent outbreaks, such as the Muriaé virus (MURV) isolated in 2000 from bovine lesions.
Also, in 2003, one more VACV was isolated, in PassaTempo County, Minas Gerais State,
named Passatempo virus (PSTV). Molecular analysis showed that PSTV had the deletion of
18 nucleotides in the A56R gene that is also found in ARAV and CTGV, indicating that those
viruses probably share common origins (Figures 1 and 2) [31]. The isolation of GuaraniP1
virus (GP1V) and GuaraniP2 virus (GP2V) from different rural areas during a single outbreak
in 2001 raised an interesting question regarding VACV origins [13]. The genetic
heterogeneity among GP1V and GP2V suggested that Brazilian VACV probably has more
than one origin, including origins from a non-vaccine strain, as proposed by DAMASO
(2000) [22]. TRINDADE (2007) [13] and DRUMOND (2008) [43], by analyzing conserved
and variable regions of the VACV genome, demonstrated that Brazilian VACV isolated up
until the 2001 outbreak could be grouped into two distinct phylogenetic groups. GP1V and
GP2V each show more similarity to other VACV than they show similarity to each other.
Figure 1. Consensus bootstrap phylogenetic tree based on the nucleotide sequence of OPV ha gene. The
tree was constructed based on ha sequences using the neighbor-joining method, 1,000-bootstrap
replicates, Tamura-3-parameter model (MEGA3.1.). Values >50% are shown. Nucleotide sequences
were obtained from GenBank. The black dot indicates VACV obtained during BV outbreaks in Brazil.
Exanthematic Bovine Vaccinia Virus Outbreaks in Dairy Cattle in Brazil
75
Figure 2. Nucleotide sequences Brazilian VACV samples and comparison to the homologous gene
sequences from several OPV. Alignment of OPV ha nucleotide sequences showing the 18 nt deletion
signature region. The asterisk region indicates this signature, present in several VACV strains isolated
during BV outbreaks. The alignments were performed using the ClustalW method, implemented by the
software MEGA version 3.1.
This genetic variability is reflected directly in certain phenotypic characteristics, such the
dimensions of their viral plaques in BSC-40 cells and their virulence in intranasally-infected
Balb/c mice [44]. More recently, several exanthematic outbreaks caused by VACV affecting
both bovines and humans were described in Espírito Santo State [45], Pernambuco [46],
Rondônia (S. de Saúde de Rôndonia, 2009), Maranhão [47], Tocantins [34], Rio de Janeiro
[48] and São Paulo [33]. In 2010, BRUM and colleagues described a severe exanthematic
outbreak caused by an OPV, which affected horses at a horse-breeding farm in Pelotas City,
Rio Grande do Sul State [49]. Further molecular studies showed that the etiological agent of
the lesions was a VACV, the first isolated from a horse in Brazil [50]. Similar to the Guarani
outbreak, two viral populations were identified in the equine VACV outbreak. However, the
viruses were isolated from the same exanthematic lesion. Therefore, considering all recent
VACV reporting, it is possible to affirm that the virus is widespread in Brazil, affecting all
geographical regions. In 2010, MOTA described a high prevalence (approximately 24%) of
human seropositivity in a rural community in Acre State, Brazilian Amazon [51]. Because no
BV outbreak had been reported to date in that area, the study demonstrated that viral
circulation is not always associated with milk collection procedures.
5. Bovine Vaccinia Risk Factors
The occurrence of BV outbreaks in the last 12 years in Brazil has improved our
knowledge of the disease and its dynamics [12,13,22,30-34]. Although the characterization of
76
Jônatas Santos Abrahão, Giliane de Souza Trindade and Erna Geessien Kroon
the viral strains was an initial priority, epidemiological and ecological studies are the current
focus today, aimed at explaining how BV outbreaks start and spread among rural areas [12].
In this context, a relevant question remains unanswered as to whether BV outbreaks were
under-reported before 1999 or whether the large number of outbreaks simply started at that
time. It is important to mention that during the WHO smallpox vaccination campaign,
exanthematic infections affecting dairy cattle were relatively common, as they were
associated with the spread of VACV from vaccine scabs of recently vaccinated milkers
(reviewed by [13]). However, at the end of the 1970s, smallpox vaccination ceased in Brazil,
and concomitantly, the bovine exanthematic outbreaks associated with recently vaccinated
milkers were no longer reported until 1999 [13]. Because BV resembles other bovine
vesicular diseases, such as foot and mouth disease, bovine herpes and pseudocowpox, the
absence of VACV notifications during the intervening 20 years could be a consequence of
clinical misdiagnosis [40]. However, because VACV strains were originally detected in
Brazilian forests starting in 1960 [36], it is possible that the recent cases of BV are a
consequence of a reemergence of VACV from the wild, probably related to the intensification
of anthropogenic activities in Brazil [52].
In most of cases, the spread of BV during an outbreak is related to an infected milker
with hand lesions that spreads the virus to the herd during the milking process [48]. During
the acute phase of the disease, milkers can present with severe myalgia, high fever, pain, and
lymphadenopathy, which frequently cause them to decrease their occupational activities [48].
However, during the prodromal and convalescence phases, viral particles could in theory also
be spread from the lesions, allowing the direct infection of the herd and the indirect infection
of other milkers [40,48]. In addition, some areas are known to have stringent work regimens
that do not allow sick milkers to take time off from the job. In these specific cases, viral
spread can be rampant among the cattle, affecting almost 100% of the animals in some areas
[40]. The “milker” factor can worsen the BV problem if the same milker keeps occupational
contact with animals from distinct areas, which is very common in Brazilian rural areas.
Another important BV risk factor is the negotiation of infected cattle during the outbreaks
[40,48]. After the occurrence of BV in all Brazilian regions, the veterinary surveillance
institutions have prevented the commercialization of cattle in BV-affected areas. However, on
several occasions, the outbreak notification occurred a few weeks after the outbreak really
began, which allowed the commercialization of infected cattle without inspection. The
introduction of infected cattle into a new area has a great impact on BV spread. Cattle should
be milked every day to avoid udder infections, especially mastitis; therefore, a newly acquired
cow, even when sick, will be promptly milked, leading to direct human infection and to
indirect infection of other dairy cows [40,48]. One way to avoid VACV spread in a bovine
herd is the disinfection of teats and milking devices after each handled animal during the
milking process. However, although disinfection has been shown to be feasible, some farmers
do not apply it correctly, making mistakes in disinfectant dilutions and in using solutions
contaminated with a high content of organic matter. The method of “milking line”, where the
sick cattle are the last to be handled, is another alternative to avoid BV spread, but sometimes,
the farmers are unable to identify an initial VACV lesion.
Despite most of the epidemiological retrospective studies pointing to infected milkers and
cattle commercialization as the initial causes of BV outbreaks, the origins of the outbreaks are
unknown in some cases [12,13,22,30-34]. Some VACV outbreaks are temporally and
spatially distant from previously reported BV outbreak areas [12]. Therefore, the focal origin
Exanthematic Bovine Vaccinia Virus Outbreaks in Dairy Cattle in Brazil
77
of outbreaks is sometimes unknown. Moreover, BV usually occurs during the dry season
when some Brazilian biomes have a scarcity of food, leading some wild species to search for
food in farm storehouses and corrals. Rats, mice, opossums, foxes, wild dogs and small cats
are frequently observed around farming areas [12]. In theory, some of these species,
especially rodents, could be VACV reservoirs. A recent study by our group showed that
peridomestic rodents, such as the Mus musculus and the Rattus rattus species, could be
carriers of VACV between forests and farm facilities. In this study, a new VACV, named
Mariana virus, was concomitantly isolated from a peridomestic mouse, a milker and an
infected cow, indicating that rodents around the farms could represent a hypothetical risk
factor [12]. Moreover, the detection of VACV in rodents and monkeys in Brazilian forests
indicate that VACV may have circulation in the wild [36,52]. The impact of anthropogenic
activities on VACV emergence from the wild is matter of investigation.
Conclusion
In conclusion, BV notifications have increased in the recent years in Brazil. The impact
of the disease in rural areas is considerable, affecting both large and small properties [40].
The co-circulation of distinct VACV and the difficulties of implementing BV control methods
worsen this scenario. Moreover, the complexity of Brazilian ecosystems increases the number
of possible viral hosts and reservoirs and makes the proposition of solid theories about the
VACV life cycle difficult. However, despite these challenges, the discussed BV risk factors
have been consistently identified in the last few years and have been corroborated with each
new study. Together, rapid diagnostic methods, the education of milkers and farmers
regarding BV, the effective inspection of new BV cases and studies on VACV hosts and
reservoirs/host range should be applied as measures of disease control. The development of
vaccines against BV should not be neglected.
References
[1]
[2]
[3]
[4]
[5]
International Committee on Taxonomy of Viruses (Ictv). The big picture Book of
Viruses: Poxviridae (http://www.ncbi.nih.gov/ICTVdb/Ictv/index.htm), 2011.
Damon IK. Poxviruses. In: Knipe DM , Howley PM, editors, Fields virology.
Philadelphia: Lippincott Williams and Wilkins; 2007. p. 2947–75. Becker et al., 2008).
Saral Y, Kalkan A, et al. Detection of Molluscum contagiosum virus (MCV) subtype I
as a single dominant virus subtype in Molluscum lesions from a Turkish population.
Arch. Med. Res. 2006. v.37, n.3, p.388-91.
De Souza Trindade, G., B. P. Drumond, et al. Zoonotic vaccinia virus infection in
Brazil: clinical description and implications for health professionals. J. Clin. Microbiol.
2007. v.23, n.8, p.1370-2.
Lederman ER, Reynolds MG, et al. Prevalence of antibodies against orthopoxviruses
among residents of Likouala region, Republic of Congo: evidence for monkeypox virus
exposure. Am. J. Trop. Med. Hyg. 2007. v.77, n.6, p.1150-6.
78
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
Jônatas Santos Abrahão, Giliane de Souza Trindade and Erna Geessien Kroon
Nazarian SH, Barrett JW, et al. Comparative genetic analysis of genomic DNA
sequences of two human isolates of Tanapox virus. Virus Res. 2007. v.129, n.1, p.1125.
Rapin N, Kesmir C, et al. Modelling the human immune system by combining
bioinformatics and systems biology approaches. J. Biol. Phys. 2006. v.32, n.3-4, p.33553.
Tadese T, Potter AE, et al. Concurrent infection in chickens with fowlpox virus and
infectious laryngotracheitis virus as detected by immunohistochemistry and a multiplex
polymerase chain reaction technique. Avian Dis. 2007. v.3, n.54, p.719-24.
Stanfonrd MM, Werden SJ, et al. Myxoma virus in the European rabbit: interactions
between the virus and its susceptible host. Vet. 2007. v.38, n.2, p.299-318.
Mondal B, Bera AK, et al. Detection of Orf virus from an outbreak in goats and its
genetic relation with other parapoxviruses. Veterinary Research Community. 2006.
v.30, n.5, p.531-9.
Hosamani M, Yadav S, et al. Isolation and characterization of an Indian ORF virus
from goats. Zoonoses Public Health. 2007. v.54, n.5, p.204-8.
Abrahão JS, Guedes MI, et al. One more piece in the VACV ecological puzzle: could
peridomestic rodents be the link between wildlife and bovine vaccinia outbreaks in
Brazil? PLoS One. 2009 v.19, n.10, p.7428.
Trindade GS, Emerson GL, et al. Brazilian vaccinia viruses and their origins. Emerg.
Infect. Dis. 2007 v.13, n.7, p.965-72.
Singh RK, Hosamani M, et al. An outbreak of buffalopox in buffalo (Bubalus bubalis)
dairy herds in Aurangabad, India. Rev. Sci. Tech. 2006. v.25, n.3, p.981-7.
Al-Zi'abi O, Nishikawa H, et al. The first outbreak of camelpox in Syria. J. Vet. Med.
Sci., v.69, n.5, May, p.541-3. 2007. v.69, n.5, p.541-3.
Singh RK, Hosamani M, et al. Buffalopox: an emerging and re-emerging zoonosis.
Anim. Health Res. Rev., 2007. v.8, n.1,p.105-14.
Breman, J. G. and Henderson, D.A. Diagnosis and management of smallpox. N. Engl. J.
Med. 2002. V.346, n.17, p.1300-8.
Loparev VN, Massung RF, et al. Detection and differentiation of old world
orthopoxviruses: restriction fragment length polymorphism of the crmB gene region. J.
Clin. Microbiol. 2001. v.39, n.1, p.94-100.
Mcfadden, G. Poxvirus Tropism. Nature Reviews.2005. v.3, n.3, p.201-13.
Esposito JJ and Fenner F. Poxvirus in fields Virology. Fields, B.N., Knipe, D.M.,
Howley, P.M., Griffin, D.E. 4(2) Lippincott-Raven Publishers, Philadelphia, PA –
USA. 2001.
Fenner, F., Wittek, R. et al. The Ortopoxviruses. 1ª ed. Academic Press. San Diego,
Califórnia. 1989.
Damaso CR, Esposito JJ, et al. An emergent poxvirus from humans and cattle in Rio de
Janeiro State: Cantagalo vírus may derive from brasilian smallpox vaccine. Virology.
2000. v.277, n.2, p.439-49.
Egan C, Kelly CD, et al. Laboratory-confirmed transmission of vaccinia virus infection
through sexual contact with a military vaccinee. J. Clin. Microbiol. 2004. v.42, n.11,
p.5409-11.
Vora S, Damon I, et al. Severe eczema vaccinatum in a household contact of a smallpox
vaccine. J. Clin. Infect. Dis. 2008. v.15, n.54, p.1555-61.
Exanthematic Bovine Vaccinia Virus Outbreaks in Dairy Cattle in Brazil
79
[25] Yao Y, Li P, et al. Vaccinia virus infection induces dendritic cell maturation but inhibits
antigen presentation by MHC class II. Cell Immunol. 2007. v.8, n.12, p.1495-6.
[26] Domingo-Gil E, Perez-Jimenez E, et al. Expression of the E3L gene of vaccinia virus in
transgenic mice decreases host resistance to vaccinia virus and Leishmania major
infections. J. Virol. 2008. v.82, n.1, p.254-67.
[27] Aravindaram K, Yang NS. Gene gun delivery systems for cancer vaccine approaches.
Methods Mol. Biol. 2009. v.542, n.8, p.167-78.
[28] Chian YC, Hwang C J . Induction of tumor antigen-specific immunity in vivo by a
novel vaccinia vector encoding safety-modified simian virus 40 T antigen. Natl. Cancer
Inst. 1999. v.20, n.91, p.169-75.
[29] Kreijtz JH, Suezer Y, et al. MVA-based H5N1 vaccine affords cross-clade protection in
mice against influenza A/H5N1 viruses at low doses and after single immunization.
PLoS One. 2009. v.12, n.4, p.7790-98.
[30] Trindade GS, Da Fonseca FG, et al. Belo Horizonte virus: a vaccinialike virus lacking
the A-type inclusion body gene isolated from infected mice. J. Gen. Virol. 2004. v.434,
n.54, p.56-59.
[31] Leite JA, Drumond BP, et al. Passatempo virus: A novel VACV isolated during a
zoonotic outbreak in Brazil. Emerging Infections Disease. 2005. v.11, n.12, p.1935-8.
[32] Trindade GS, Lobato ZI, et al. Short report: Isolation of two vaccinia virus strains from
a single bovine vaccinia outbreak in rural area from Brazil: Implications on the
emergence of zoonotic orthopoxviruses. Am. J. Trop. Med. Hyg. 2006. v.75, n.3, p.48690.
[33] Megid J, Appolinário CM, et al. Vaccinia virus in humans and cattle in southwest
region of Sao Paulo state, Brazil. Am. J. Trop. Med. Hyg. 2008. v.79, n.5, p.646.
[34] Medaglia ML, Pessoa LC, et al. Spread of cantagalo virus to northern Brazil. Emerg.
Infect. Dis. 2009. v.15, n.7, p.1142-3.
[35] Fonseca FG, Trindade GS, et al. Characterization of a vaccinia-like virus isolated in a
Brazilin forest. J. Gen. Virol. 2002. v.83, n.7, p.223-228.
[36] Lopes S, Lacerda JP, et al. Cotia virus: a new agent isolated from sentinel mice in São
Paulo, Brazil. The Am. J. Trop. Med. Hyg. 1965. v.210, n.1, p.67-72.
[37] Fonseca FG, Lanna MCS, et al. Morphological and molecular characterization of the
poxvirus BeAn 58058. Archives of Virology 1998. v.143, n.89, p.1171-1186.
[38] Marques JT, Trindade GD, et al. The genome of cowpox virus contains a gene related
to those encoding the epidermal growth factor, transforming growth factor alpha and
vaccinia growth factor. Virus Genes. 2001. v.18, n.2, p.151-60.
[39] Diniz S, Trindade G, et al. Surto de varíola murina em camundongos suíços em
biotério– relato de caso. Arq. Bras. Med. Vet. Zootec. 1998. v.8, n.5, p.152-156.
[40] Lobato ZIP, Trindade GS, et al. Surto de vaccínia bovina causada pelo vírus Vaccinia
na região da Zona da Mata Mineira. Arquivo Brasileiro de Medicina Veterinária e
Zootecnia. 2005. v.32, n.54, p.45-52.
[41] Trindade GS, Da Fonseca FG, et al. Araçatuba virus, a vaccinialike virus associated
whit infections in humans and cattle. Emerg. Infect. Dis. 2003. v.9, n.2, p.155-60.
[42] Nagasse-Sugahara TK, Kisielius JJ. Human vaccinia-like vírus outbreaks in São Paulo
and Goiás states, Brazil: virus detection, isolation and identification. Revista do
Instituto de Medicina Tropical de São Paulo 2004. v.46, n.6, p.315-22.
80
Jônatas Santos Abrahão, Giliane de Souza Trindade and Erna Geessien Kroon
[43] Drumond BP, Leite JA, et al. Brazilian Vaccinia virus strains are genetically divergent
and differ from the Lister vaccine strain. Microbes Infect. 2008. v.10, n.2, p.185-97.
[44] Ferreira JM, Drumond BP, et al. Virulence in murine model shows the existence of two
distinct populations of Brazilian Vaccinia virus strains. PLoS One. 2008. v.26, n.3,
p.3043-50.
[45] Donatele DM, Travassos CEPF, et al. Epidemiologic study of bovine poxviral disease
in the southern region of Espírito Santo state, Brazil. Em: XIV Encontro Nacional de
Virologia, 2003, Florianópolis. Vírus Revews and Research – Journal of the Brazilian
Society for Virology. 2007. v.2, n.5, p.45-9.
[46] Damaso CR, Reis AS, et al. A PCR-based assay for detection of emerging vaccinia-like
viruses isolated in Brazil. Diagn. Microbiol. Infect. Dis. 2007 v.57, n.1, p.39-46.
[47] Assis FL, Abrahão JS, et al. Detection and characterization of a Vaccinia virus strain in
Maranhão State. Brazilian Virology Meeting, 2009.
[48] Silva-Fernandes AT, Travassos CE, et al. Natural human infections with Vaccinia virus
during bovine vaccinia outbreaks. J. Clin. Virol. 2009. v.44, n.4, p.308-13.
[49] Brum MC, Anjos BL, et al. An outbreak of orthopoxvirus-associated disease in horses
in southern Brazil. J. Vet. Diagn. Invest. 2010. v.22, n.1, p.143-7.
[50] Campos RK. Diversidade dos Vaccinia virus brasileiros: co-infecção de amostras de
Vaccinia virus com variações fenotípicas durante um surto exantemático em cavalos
crioulos no Brasil. Trabalho de Monografia. 2010. PUC Minas.
[51] Mota BE, Trindade GS, et al. Seroprevalence of orthopoxvirus in an Amazonian rural
village, Acre, Brazil. Arch. Virol. 2010 v.155, n.7, p.1139-44.
[52] Abrahão JS, Silva-Fernandes AT, et al. Vaccinia virus infection in monkeys, Brazilian
Amazon. Emerg. Infect. Dis. 2010 v.16 n.6, p.976-9.
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter VI
Behavior Feeding of Ruminants
Christiano Santos Rocha Pitta, Paulo Fernando Adami
and Laércio Ricardo Sartor
Agronomy Departament, Federal Institute of Paraná – IFPR,
Campus Palmas, Paraná, Brazil
Abstract
The purpose of this chapter was to discuss how to optimize the grazing process,
relating to the animal decisions taken at different scales, influenced by the pasture sward
and animal supplementation and to highlight the common feed factors that influence
feeding behavior and intake, although their expression and consequences depend on the
environment. Landscape can be considered the largest scales, where biotic and abiotic
factors (topography, water availability, shelters, etc.) are added together influencing the
process of grazing, especially in the continuous stocking system. Another important
discussion on these scales is the number and size of each individual meal, time spent
eating and intervals between meals whereas a meal is defined by a long string of grazing.
Animal behavior at a feeding patch is an important indicator of feeding conditions, by
their direct relation with quantitative attributes, qualitative and pasture sward. Forage
intake in each bite and the number of bites during the grazing period results in total intake
of forage. Thus, the maximization of consumption is directly related to animal production
and is dependent on maximizing every bit over the pasture. Usually the adopted
management affects the pasture sward and this along with the animal supplementation
significantly influences consumption and ingestive behavior of animals. Thus, the process
of grazing must be managed considering the relationship between the components of the
system, considering that the plants need its leaves to intercept solar energy and perform
photosynthesis, absorb water and nutrients provided by the soil and the animal need these
leaves to ingest nutrients and due to it, is constantly influencing the plants growth by the
grazing action. Plant-animal relationships interfere on the animal ingestive behavior and
result in different pasture sward what in turn alter the forage search and apprehension,
defoliation patterns of leaves and tillers during the lowering of the pasture. Pasture sward

E-mail: christiano.pitta@ifpr.edu.br.
82
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
influences rate of intake and dietary choices. On heterogeneous resources, animals graze
selectively and choose a diet of higher quality than that on offer.
Introduction
Pasture growth determines the maximum number of animals that can be stocked per
hectare of pasture. However, daily intake of nutrients ultimately determines the productivity
of animals in any livestock operation. Likewise, forage intake is the overall factor that
governs animal productivity on pasture. Moreover, improving animal productivity by
maximizing forage intake is a function of the quantity and quality of the herbage consumed
by the animal.
Feed consumption (intake) depends on the following factors: grazing time (how long
does the animal spend grazing), bite rate (how rapidly does the animal take bites) and on bite
size (how much forage does the animal consume per bite). Considering these factors, daily
intake is a result of grazing time versus bite rate versus bite size. Ruminants, like other
species, seek to adjust food intake to their nutritional needs, especially energy [1]. In specific
situations, feeding behavior of ruminants is characterized by long periods of grazing, from 4
to 12 hours a day, however, for cattle kept in feedlots, these periods range from one hour to
high-energy foods up to six hours or more, to sources with low energy. Even high-quality
pasture herbage may limit the quantity of forage consumed when the supply is inadequate.
For this reason, quantity of available forage more often limits animal production on pasture
than forage quality.
Understanding the behavior of feeding livestock faced with high plant diversity is a
powerful tool available to managers to modify plant-herbivore interactions to achieve targeted
levels in grazing management [2]. A very important aspect for a better use of pastures refers
to the knowledge of the preferred grazing period by the animals [3]. Moreover, understanding
the feeding behavior of livestock faced with high plant diversity is a powerful tool available
to managers to modify plant–herbivore interactions to achieve targets in management grazing
levels [2]. It should be taken into account that besides the pasture, there is a greater
complexity of factors related to management that interfere the feeding behavior [2, 4]. Some
of these management factors can directly affect the feeding behavior, e.g. feed additives [5],
stocking density [6, 7, 8], grazing route [9], pasture height [10] and even the animal’s earlier
experiences [11, 12, 13]. Some other management factors affect the feeding behavior
indirectly by having animals cope with changes in resource availability as a result of changes
in the grazing season [14, 15], the spatial distribution of vegetation [16, 17, 18] or the relative
abundance of species, e.g. with ryegrass and clover associations [19, 20]. The aim of this
chapter was to discuss how to optimize the grazing process once one of the goals of grazing
management is to maximize intake.
Animal Behavior on Pasture
The act of grazing involves a very complex series of decisions that depend on an
elaborate array of mental, motor and digestive abilities. Any animal at grazing, need to take a
Behavior Feeding of Ruminants
83
series of decisions about how to behave in the search, ingestion and digestion of food. Studies
show that animals can recognize the food energy value of a pasture and furthermore, can
account the energy cost to get them, which allows the herbivores to graze, in general, a diet
superior to that present in the average environment [21]. Grazing can be defined as the
process by which animals eat plants for energy and nutrients, using its senses, head and legs
to allocate potential bites and its oral apparatus to bring fodder to the mouth, comprises it
between the teeth, cut with movements of the head - featuring a bite - and then chew it,
forming the bolus, for finally swallowing it [22]. In contrast, this activity includes short
periods in which the animal is not effectively grazing, but involved in activities directly
associated with this process, i.e., selecting its diet and moving to new feeding sites. Temporal
organization of feeding behavior involves grazing shifts and may have several meals along
the day, which are interrupted by intervals of varying length for other activities, among which
stand out rumination, resting, hiking, surveillance, social activities, etc. [23]. A meal is
defined by a long string of grazing [24]. When the meal stops, begin an interval between
meals in which the animal intend to perform other activities besides graze. All these activities
are essential to the existence of animals and none is unnecessary, although each has
different ranges of flexibility, demonstrating a form of competition between them [25]
being the time spent in each of these activities dependent basically on the pasture sward
[26], environmental conditions and nutritional requirements of animals. The number of
meals seems to be an indicator of the quality of the pasture landscape environment [24]. The
animal responds directly to the pasture sward, getting a high rate of intake when the forage
mass is suitable, quickly filling the rumen. Furthermore, in these situations, a high level of
selectivity is allowed and the animals gather a diet of high quality quickly [25]. Multiplying
the number of daily meals by their respective durations makes daily grazing time. Pasture
apprehension and ease of removal or plant resistance at grazing are aspects involved in
acquiring and handling the forage before swallowing and impose limits on the animal
consumption [21]. These pasture characteristics affects the speed of nutrients acquisition by
grazing animals, characteristic directly related to other behavior variable, such as the rate or
frequency of bites carried by animals at grazing (number of bites) that is intimately concerned
with the mass of the bite and reflect on the daily grazing time [24]. Comparing the diurnal
feeding behavior of cattle under continuously and rotated stocking rate, differences between
grazing methods to the grazing period were observed [27].
At fall, grazing period was of 59.8 and 60.7% for males and females, respectively at the
rotational grazing method and 44.4 and 44.1% for males and females, respectively to the
rotational stocking rate assigning lower values for the continuous stocking rate grazing
method.
The temporary higher stocking rate at the rotated grazing method resulted in higher
competitiveness in search of fodder and consequently higher grazing period compared to the
continuous stocking rate grazing method. There was no effect of sex within the grazing
methods.
Average time spent on rumination and on idle activities was higher at the rotational
grazing method in relation to the continuous stocking rate grazing method. The authors
explained that the grazing animals spent longer time in idleness, because they spent less time
with their daytime grazing.
84
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
Forage Intake
Daily intake of forage is the result of the product between the time spent by the animal at
grazing activity and forage intake rate during grazing (Figure 1), which by the way, is the
product of the number of bites per unit of time (rate of bites) and the amount of forage grazed
by each bite (bite size) [28].
The animals at the field may employ different strategies to increase consumption during
grazing, whether by varying the size of the bite, increasing the frequency of bites or by
increasing the grazing time [29]. The animals try to be efficient, since they seek potential
bites while chewing the grass grazed at the previous bite [30]. At pastures whose nutritional
value and availability are not limiting, it is often assumed that the search time for potential bites
is negligible, because the animal chews the grass as it moves from one patch to another one
[31]. Patch or feeding station is defined as a hypothetical semi-circle available in front of the
animal and available to its reach without moving his front foot [32]. In a situation with high
forage mass allowance, a greater time is spent at the feeding patch until the point of
abandonment is reached, represented by the point at which the cost-effective to exploit it
becomes less interesting [25]. When the consumption per bite is reduced, there is a
corresponding decrease in the intake rate, unless there is a compensatory increase in the bites
rate [28].
The consumption per bite is influenced by the tensile strength of material, so that the
mass of the bite can be limited by the maximum force that the animal is capable of exercising
in the apprehension of a bite. There are some characteristics associated with the plant related
to the ease of harvesting forage for animals: the height of the sward, forage mass present per
unit volume, low fibrousness of leaf blades, the spatial distribution of plant tissue, the
presence of barriers to defoliation such as sheaths and stems and its dry matter content [33].
In general, forage mass increase as the pasture height increase and at higher heights, the lower
the number of movements for apprehension and higher the chewing movements [10, 34]. In
contrast, the shorter the plant height the less effective is the ability of the animals to brought
fodder to the mouth [10, 35]. Thus, forage daily intake will also be affected if any reduction
in the rate of intake could not be offset by an increase in daily grazing time [28].
Figure 1. Components of ingestive behavior (Adapted from Hodgson, 1990).
Behavior Feeding of Ruminants
85
Differences in composition between leaves and stems reflect in a higher energy required
to graze stem compared the energy required to graze leaves [36], moreover, leaves have a
lower resistance to break by chewing and shorter retention in the rumen [37]. When referring
about the same tissue, the resistance appears to be greater on the basis than at the apex [38].
Dense pastures with a high proportion of leaves are better consumed by ruminants and
determine more efficiently grazing process and higher animal production [39]. On the other
hand, a pasture with high content of stems and dead material seems to difficult grazing and
limiting the size of the bite [40]. Selectivity is one of the most important aspects to be
observed in pasture management. Depending on the degree of selectivity allowed to the
animal, it will eat a fodder of varying quality what can result in low or high productive
potential. Sheep tend to be more selective than cattle in most circumstances, while young
animals are more selective than adult animals, however, models of diet selection may be more
unstable in young animals than in adults [28]. The diet selection involves the selection of a
local grazing followed by the selection of the bite [37]. The grazing site selection is
influenced by plant species, stage of maturity and deposition of feces and urine, as well as
factors related to variations in micro-topography, shelter, shade and alignment of fences.
Thus, the bite selection is influenced by the preference of the animal to the plant and its
relative accessibility and abundance.
Grazing preference to certain local is mainly a reflection of the animal response to
chemical and physical characteristics of the leaves and stems of plant species and how they
affect the senses of sight, range, taste and smell [28]. According to this author, preference and
selection are both relative terms and the intensity of selection for a particular component in
the pasture depends on one or other component that is present and on the contrast that exists
between them. Pasture sward characteristics determine the degree of selective grazing
performed by the animals as well as the efficiency by which the forage is grazed, determining
the total amount of nutrient intake [39, 42]. Moreover, grazing time increases as the pasture
sward height decrease, ranging from 459 to 380 minutes, respectively, to the heights of 10
and 40 cm [43].
The bite depth seems to have no limitation imposed by the characteristics of the animal's
mouth, being determinate by the pasture characteristic [22]. Animals tend to concentrate their
grazing activity in the canopy layers containing mainly leaf material, and the increase in
depth with increased grazing sward height is parallel to an increase in the depth of the layer of
leaf blades in the pasture [28]. According to the same author, under normal circumstances, the
proportion of total leaf grazed by one defoliation seems to not exceed 25% on average, but in
extreme cases, the combination of a high frequency and high severity of defoliation can result
in a rate removing leaves equivalent to 10% per day, a rate that cannot be sustained for long
periods without affecting the pasture growth.
Thus, the grazing animals bite size (area and depth) are important for both the animal and
the plant. For the animal, the size of the bite along with the volume density of the grazed
stratum defines the size of the bite, which is the most influential variable on animal
consumption [44]. In the case of plant communities, the depth and area of the layer of forage
removed is defined by the plants, thus defining the intensity and spatial pattern of defoliation
[45]. The youngest leaves are more likely to defoliation due to the way in which the animals
graze and the position that it occupy in the tiller [46].
86
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
Daily Activities of Animals
Daily activities of the animals in the pasture include alternating periods of grazing, rest
and rumination. Considering an access period on pasture of 570 minutes a day, equivalent to
9.5 h daily, lambs spent 75.4% of the total time grazing, 13.9% rumination and 10.7% of the
time on idle [47]. However, as previously mentioned, the duration and distribution of these
activities can be influenced by the characteristics of pasture, management, weather conditions
and activity of the animals tested. Grazing time is greatly influenced by time of sunrise and
sunset, but the intensity may vary within and between the main periods of grazing [48].
Time spent on grazing activity is around six to eight hours a day. Grazing time longer
than 8-9 hours per day is an indicative that the pasture conditions are limiting the forage
intake [49]. Thus, under these conditions, interferences may occur over the rumination
activity and other behavioral requirements. Daily grazing time is a direct function of the
quality and availability of forage and thermal balance [50]. Moreover, it is possible to observe
from three to five peaks of grazing throughout the day occurring especially early in the
morning and late afternoon [22]. The grazing activity occurs mainly during the day in
temperate climates, although, short periods of grazing at night are not uncommon [28].
Some authors have reported daily grazing time between 8-9 hours for cattle on winter
pastures [51, 52]. Others, evaluating lactating sheep behavior grazing ryegrass reported daily
grazing time of 9.6 h and 10.9 h respectively to the vegetative and pre-flowering phase of
ryegrass [53]. Also evaluating ryegrass managed at different heights (5 to 20 cm) sheep daily
grazing time ranged from 485 up to 609 minutes/day [54], while [47] observed diurnal
grazing time of 7.2 h, which corresponds to 432 minutes, also evaluating ryegrass managed at
different heights (5 to 20 cm) with sheep. Lambs grazing pearl millet showed daily grazing
time values from 582 to 500 minutes/day from the low to the higher height measured in the
pasture [55]. Regarding to the time spent on rumination, the same author reported that its time
increased as the pasture height management increase, with values from 96 up to 162 minutes
a day, within the limits explored. The time spent resting and on social activities, however,
was not affected by pasture height management. Animal behavior and grazing time on oat
(Avena strigosa Schreb.) plus ryegrass increased as the amount of ryegrass leaves and mass of
green forage decreased during the pasture cycle [56]. Moreover, animal steps between grazing
patches (available hypothetical semi-circle in front of the animal without him move his front
legs) increased as the forage offer increased and the number of stations visited decreased [57].
As the supply of forage green blades decreased, the length of time spent on each patch also
decreased which is also in accordance with the results reported by other authors [10].
After a period of grazing, the animal starts the process of rumination in which fodder is
regurgitated, chewing and again swallowed [21], in order to reduce the size of fodder particles
and allow highest attack of microbial enzymes and consequently increase the passage of
fodder through the reticulo-omasal orifice [21].There are along the day from 15 to 20
rumination periods of widely varying length, dependent on the quality and quantity of forage
consumed, with a peak at the end of the day which gradually decrease until it begins a new
cycle of grazing at dawn [21].This activity takes between 6 and 8 hours daily, and meantime,
15 to 20 thousands chewing movements are performed [28], mainly during the night [52].
Moreover, rumination time is influenced by the nature of diet and seems to be proportional to
the cell wall content of forage [1].
Behavior Feeding of Ruminants
87
Time spent on rumination in cattle is highly correlated (0.96) with neutral detergent fiber
(NDF) intake [58]. In an experiment with cows with three levels of NDF in the diets (26, 30
and 34%) a quadratic response to the time spent on rumination and chewing with maximum
estimated values of 344 and 558; 403 and 651, and 414 days and 674 min/day respectively to
each of the NDF levels and to the rumination and total chewing activity were reported [59].
Young animals are not true ruminants and grazing activity develops more slowly in calves
that receive large amounts of milk and increases rapidly to adult levels after the weaned
process (Figure 2), being this model similar with lambs [28]. The same author reported that
the rumination activity reflects the amount of forage consumed, which shows a similar model
of development. It is also influenced by the digestibility of the diet, being the time spent on
ruminating per kilogram of feed consumed declines as the digestibility of forage increase. In
addition to ensuring their nutritional needs, animals also need to rest which is usually done by
the act of lying down, stand upright and even walk. Animals most often lie down to rest,
varying with the standing up position due to factors associated with the animal (species, age,
physiological state) and the environment (time of day, season and climate) [60].
At winter, the animals rest more than the summer. Rest places and time spent on these
locals depend on the physical environment and on the comfort provided to the animal, with
different locations for the day and night. During the day, the animals rest at their own local
grazing. When the wind is over 38 km/h and the temperature is lower than 15 °C, the animals
seek a refuge in a less exposed area. In the case of sun and high temperature the animals seek
shade. At night, the preference is for higher ground, hot and bare soil and the number of sites
chosen for night rest is limited [61]. The act of walking is associated with the looking for
water, feed and social facilitation. The distance traveled during the day varies greatly
according to each individual and subject to availability of pasture.
Figure 2. Development of grazing and rumination activity of young calves (Adapted from Hodgson,
1990).
The frequency in with animals search for water to drink depends on temperature,
condition of forage and water distribution. When ruminants graze abundant green fodder,
88
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
water consumption reduces. Usually, animals drink after grazing and the higher consumption
occurs from midday up to 6 PM [60]. Although, such behavior is greatly influenced by the
season of the year. Increased distance between the area of grazing and water supply may
decrease linearly or exponentially forage intake to an extent that will also depend on plant
type, topography and age of the animal.
Size and Rate of Animal Bite
Grazing process can be summarized in three stages, not necessarily excluding: time spent
on searching the bite, time spent on the bite action and time spent on bite manipulation [62].
The bite rate measurement estimate how easy forage apprehension occurs, which, along with
the time devoted by the animal on the grazing process as well as the depth and mass of bites,
integrated plant-animal relationship responsible for certain amount of forage consumed [51].
The bite is the fundamental unit of consumption, and its performance is defined as a
series of movements of the head and mouth parts that precedes and includes cutting the grass
and the approach to the mouth [63]. Considering that the forage consumption constitutes a
summation of each action to capture forage by the animal bite, the maximization of
consumption, which is directly related to the magnitude of animal production, goes through
the maximization of each bite deferred in the pasture.
Forage apprehension through the bite is a process that often can reach 35 thousands daily
actions, where animals often graze at a rate of one bite every 1 or 2 seconds [64]. Besides the
number of bites, the size of it, according to [65], is the most important variable in determining
the consumption of grazing animals and this variable is much influenced by the pasture
structure, letting a secondary role to the bite rate and grazing time. Furthermore, when forage
is in short supply bite size is small so animals try to compensate by increasing both their
biting rate and grazing time. When this happens, usually intake is diminished. Bite weight
depends on the amount and distribution of herbage offered within the pasture. The bite size is
determined as by the bite volume versus density of the selected herbage. The bite volume is
the product of bite area and bite depth. Both bite depth and consequently bite volume increase
as sward height increases. If the herbage is very dense, livestock will take smaller bites. Bite
size is directly affect by the sward height, blade width [66], density of forage in the grazing
horizon [67], botanical composition [68], availability and accessibility of leaves [28] and the
developmental stage of the pastures [39]. There was a significant positive correlation between
the bite weight and the pasture height when considering at the evaluations the taller grasses
(above 37 cm), with no significant differences to the grasses with heights ranging from 5.7 to
22.1 cm [69]. Other author assessing the range of herbage mass between 1136 and 1739 kg
ha-1 DM-1, corresponding to heights from 12 up to 19.4 cm over the bite weight, reported no
differences to this variable with a mean value of 0.090 g DM, equivalent to 2.23 mg DM kg-1
PV [47]. Steers behavior on annual ryegrass (Lolium multiflorum Lam) and oats (Avena
strigosa Schreb.) management at four heights (10, 20, 30 and 40 cm), showed that the
stocking rate increased as the pasture height decreased and resulted in lower herbage mass
[10]. Moreover, animals increased the rate of bites, bites per feed station, the total number of
bites, the number of feed station visited and reduced the time spent in the feeding station as
the pasture height management decreased from 40 to 10 cm [10]. These changes on animal
Behavior Feeding of Ruminants
89
behavior resulted in high energy dispended on the forage intake, longer distances traveled by
the animal during the day and higher number of impacts from the animal paw over the soil
what may result in soil degradation, environmental problems and low profit to the farmers.
Assessing the bite weight of sheep grazing ryegrass [53] reported values of 0.098, 0.079 and
0.045 g bite-1 for the vegetative, pre-flowering and flowering stages of the ryegrass,
respectively. In tropical pastures, the density of leaves and the leaf:stem ratio of the grazed
stratum has a greater influence on the size of the bite in relation to the height of the pasture
[70]. The rate of bites, in general, is negatively related to the size of bite. As the animal graze
and dispend more bites on the pasture, he will also spend more time to chew on this bite and,
similarly, the animal that consumes a greater amount of forage during the day, need more
time to ruminate it [71].
The frequency of bites is related to how ease animal apprehends (grasps) the herbage and
takes it into its mouth [72], which is affect by the type and characteristics of the pasture. The
number of bites increases as the supply of green leaves increase [30]. The leaf percentage
decrease as the advance of the grazing period, requiring more time for selection and
manipulation of pasture [65], thus reducing the animal rate of bites [30]. Variations in the rate
of bites in response to different grazing conditions are a result of how the animals distribute
jaw movements to gather, grasp and chew the forage [22]. The height of the pasture, among
other structural parameters that comprise it, is the one that most influences the animal
decision about where to dispend a bite [73]. Bites per minute on pearl millet ranged from 36.9
to 23.1 bites minutes-1 as the pasture height increased [55]. The variation in the rate of bites
according to the pasture structure was demonstrated by steers grazing oats and ryegrass with
different masses of leaf blades [51], and with sheep [53] grazing ryegrass at different
phenological stages. In general, pasture managed at low height difficult animal bite due to the
difficulty in cutting the grass, while pasture managed at taller height, cattle use their tongues
to encircle the herbage and draw it into the mouth, resulting in higher number of movements
for the pasture manipulation, especially for sheep, what may result in higher time spent on the
manipulation of the collected material [22].
A reduction of one bite per minute at each increment of 111 kg ha-1 DM-1 in the herbage
mass was reported [47], and the variation in the animal bite rate values was attributed to the
higher time spent on pasture selection at the pasture managed with high mass of fodder.
According to [74], one of the strategies used by the animal when there is an intake reduction
per bite, due to the unfavorable conditions of the pasture (reduction in height and proportion
of leaves and increased the proportion of stems and dead material), is to increase the bite rate
(bites per minute) or, similarly, positive changes in sward structure can result in increased
intake per bite and decreased the rate of bites once higher bite weights result in longer time
per bite [28].
Effect of Supplementation on the Ingestive
Behavior
Intake is also regulated by the rate at which the forage is digested and passes through the
rumen, which is affected by the diet quality. Low-quality forages, which are slowly digested
due to high fiber content, are retained in the rumen for longer periods. When ingesting low
90
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
quality forage, the animal feels full quickly and ceases to graze. In contrast, highly digestible,
immature forages are slightly laxative, which means it pass quickly through the rumen
enabling the animal to graze and ingest more. Furthermore, when animals are supplemented,
new variables interfere with nutrient intake and are associated with substitution ratios of
forage to supplement and/or addition in the total consumption of dry matter, which change
according to the characteristics of the forage and supplement [28, 75, 76], which usually
changes the feeding behavior (grazing time, ruminating and resting) of grazing animals [77,
78, 79]. Energy supplementation to animal raised on pasture can improve the performance of
grazing ruminants once in temperate species, usually the energy is the main limiting factor for
the performance of these animals. The supply of concentrate to animals in pastures may alter
the feeding behavior, with regard to time of grazing, ruminating and resting, bite rate and size
and due to interactions between plant, animal and supplement.
Supplemented animals travel farther distances per day, choosing grazing stations, than
non-supplemented animals, indicating greater selectivity when compared with animals
exclusively on pasture [80]. Preference for ryegrass leaf blades increases with the level of
supplementation [81]. Supplement levels interfere with the NDF content of forage consumed
[82], showing a higher selectivity of the supplemented animals in relation to nonsupplemented animals. Some research shows that the supplementation with concentrate may
reduce the animals grazing time [83]. If the grazing time decrease, the demand for energy
spend with grazing activities also decrease. Furthermore, the change on the grazing time
associated with supplementation depends on the timing of supplementation [80]. Assessing
the effect of energy supplementation over the grazing time of animals was noted a large
concentration of animals grazing from midday up to 6 PM and to the animals receiving a
supplement, to a lesser extent, there was a concentration of animals grazing on time before
the supply of the supplement, held daily at 2 PM [52]. Other studies have shown a slight
variation in grazing time in response to supplementation [37, 84].A linear decrease in
rumination as the concentrate level increased was reported [85], being this fact attributed to
the decrease of cell wall constituents with the increase of starch content in the diet [86].
Working with beef heifers undergoing supplementation strategies on oats and ryegrass
pastures, was observed that grazing time was greater when the animals started to receive
supplement at 0.6% of BW and this fact occurred due to the higher offer of leaves in the
pasture, especially oats [87]. Regarding for the idle time, daytime observations of animal
behavior observed that non-supplemented animals spend shorter time on rest, while animals
that received supplementation at 1.5% of BW stayed longer periods on rest [83].
Another important factor is the water supply. Keeping an adequate water supply within a
relatively short distance ensures herbage intake is not limited due to water shortage and
walking time. As long as cattle have to walk less than 250 m to water, grazing patterns and
subsequent defecation patterns will be uniform throughout the pasture. Forage located beyond
this distance from a water source will be under-utilized. It is important to note that if the cattle
establish a particular grazing pattern during early grazing cycles, the effect becomes much
greater later in the season. Forage rejected early at far distances from water becomes more
mature and is even more likely to be rejected later in the season [88]. Moreover, encouraging
uniform grazing over the entire paddock by good water supply distribution helps to evenly
distribute dung and urine, and reduce pasture losses due to avoidance. This is best
accomplished by keeping stocking rates high and residency periods short. For high-producing
dairy cattle, the residency period should be no more than a day [88].
Behavior Feeding of Ruminants
91
Conclusion
Animal decisions about the grazing process are constantly influenced by biotic and
abiotic factors such as topography, availability of water and shelter. Meals are divided into
shifts, and animals may have several meals during the day that are interrupted by intervals of
varying length for other activities such as hiking, resting and ruminating. The animal
responds directly to the structure of the pasture, which can be managed aiming to allow a high
rate of intake resulting in high production levels. To ensure maximum intake, livestock must
be provided with herbage of sufficient volume (height and density). This holds as long as
growth stage is not so advanced that quality rather than supply limits intake. Canopy height
and density affect bite size by the grazing animal. Among the plants components, ruminants
select young, green leaf tissue and avoid stems and dead material. In a scenario of high forage
supply, where a high leaf:stem ratio predominates, the animal tends to increase the size and
depth of bite, resulting in greater time spent in the same feeding station, however, when the
animal moves to another feeding station, he travels a higher distance and gives a higher
number of steps between stations due to the greater weight of the last bite, so, the animal
moves meanwhile its mouth is filled to the next station. In the other hand, in a scenario of low
forage supply, the animal bite is smaller what results in a shorter time spend in each feeding
station, consequently, a higher number of station are visited and the animal travel farther until
its satiety which generates increased energy expenditure during the search process. Plantanimal relationships interfere on the animal ingestive behavior and result in different pasture
sward what in turn alter the forage search and apprehension, defoliation patterns of leaves and
tillers during the lowering of the pasture. These effects are more pronounced in tropical
pastures that have a particularly pronounced hardness and density of stems what difficult
animal grazing process, especially the older ones. From the animal point of view, it is aimed
to offer fodder of high nutritional value and at good quantity, and from the point of view of
the plant, it is aimed to promote lower frequencies and intensities of defoliation once plants
leaves allow a structure with greater proportion of leaves in both pre-and post-grazing,
ensuring the maintenance of production and productivity of pastures. The great importance in
understanding these relationships is the establishment of management strategies aimed at
creating environments that promote adequate forage intake and rapid and vigorous regrowth
of the plant.
References
[1]
[2]
[3]
P. J. Van Soest, Nutritional ecology of the ruminant. 2 ed. Ithaca: Cornell University
Press. 1994, 476 p.
K. L. Launchbaugh and Targeted Grazing: A Natural Approach to Vegetation
Management and Landscape Enhancement. Cottrell Printing, Centennial, 2006.
L. H. E. Farinatti, M. G. Rocha, C. H. E. C. Poli, C. C. Pires, L. Potter and J. H. S.
Silva, Desempenho de ovinos recebendo suplementos ou mantidos exclusivamente em
pastagem de azevém (Lolium multiflorum Lam.). Revista Brasilerira de Zootecnia,
v.35, n.2, p.527-534, 2006.
92
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
E. Baraza, J. Villalba and F. Provenza, Nutritional context influences preferences of
lambs for foods with plant secondary metabolites. Appl. Anim. Behav. Sci. 92, 293–305,
2005.
J. Rogosic, R. Estell, S. Ivankovic, J. Kezic, and J. Razov, Potential mechanisms to
increase shrub intake and performance of small ruminants in Mediterranean shrubby
ecosystems. Small Rumin. Res., v.74, p.1–15, 2008.
F. Provenza, J. Bowns, P. Urness, J. Malechek and J. Butcher, Biological manipulation
of blackbrush by goat browsing. J. Range Manage, v.36, p.513–518, 1983.
M. Mellado, R. Valdez, L. Lara and R. Lopez, Stocking rate effects on goats: a research
observation. J. Range Manage, v.56, p.167–173, 2003.
R. Shaw, J. Villalba, and F. Provenza, Resource availability and quality influence
patterns of diet mixing by sheep. J. Chem. Ecol., v.32, p.1267–1278, 2006.
M. Meuret, Organizing a grazing route to motivate intake on coarse resources. Ann.
Zootech, v.45, p.87–88, 1996.
C. Baggio, P. C. F. Carvalho, J. L. S. Silva, I. Anghinoni, M. T. Lopes and J. Thurow,
Padrões de deslocamento e captura de forragem por novilhos em pastagem de azevémanual e aveia-preta manejada sob diferentes alturas em sistema de integração lavourapecuária. Brazilian Journal of Animal Science, v. 38, p. 215-222, 2009.
F. Provenza, Postingestive feedback as an elementary determinant of food preference
and intake in ruminants. J. Range Manage, v.48, p.2–17, 1995.
F. Provenza, J. Villalba, L. Dziba, S. Atwood and R. Banner, Linking herbivore
experience, varied diets, and plant biochemical diversity. Small Rumin. Res., v.49,
p.257–274, 2003.
J. Villalba, F. Provenza and G. Han, Experience influences diet mixing by herbivores:
implications for plant biochemical diversity. Oikos, v.107, p.100–109, 2004.
J. Valderra´bano and L. Torrano, The potential for using goats to control Genista
scorpius shrubs in European black pine stands. For. Ecol. Manage, v.126, p.377–383,
2000.
B. Dumont, P. Renaud, N. Morellet, C. Mallet, F. Anglard and H. Verheyden-Tixier,
Seasonal variations of red deer selectivity on a mixed forest edge. Anim. Res. 54, 369–
381, 2005.
A.J. Hester and G. J. Baillie, Spatial and temporal patterns of heather use by sheep and
red deer within natural heather/grass mosaics. J. Appl. Ecol. 35, 772–784, 1998.
P. J. Holst, C. J. Allan, M. H. Campbell and A. R. Gilmour, Grazing of pasture weeds
by goats and sheep. 2. Scotch broom (Cytisus scoparius). Aust. J. Exp. Agric. 44, 553–
557, 2004.
S. P. Oom, J. A. Beecham, C. J. Legg and A. J. Hester, Foraging in a complex
environment: from foraging strategies to emergent spatial properties. Ecol. Complex. 1,
299–327, 2004.
J. Parsons, J. A. Newman, P. D. Penning, A. Harvey and R. J. Orr, Diet preference of
sheep: effects of recent diet, physiological state and species abundance. J. Anim. Ecol.
v.63, p.465–478, 1994.
P. Penning, J. A. Newman, A. J. Parsons, A. Harvey, R. J. Orr, Diet preferences of adult
sheep and goats grazing ryegrass and white clover. Small Rumin. Res. 24, 175–184,
1997.
Behavior Feeding of Ruminants
93
[21] F. Fraser and D. M. Broom, Alimentação. In: Farm Animal Behavior and Welfare. 3.
ed. NY: CAB International, p. 79-98, 1998.
[22] G. Cosgrove, Animal grazing behaviour and forage intake. In: INTERNATIONAL
SYMPOSIUM OF ANIMAL PRODUCTION UNDER GRAZING, 1997, Viçosa. Anais...
Viçosa: UFV, 1997. p. 59-80.
[23] E. Mayes and P. Duncan, Temporal patterns of feeding behaviour in free - ranging
horses. Behaviour, London, v.96, p. 105-129, 1986.
[24] P. C. F. Carvalho, T. C. M. Genro, E. N. Gonçalves, et al. A estrutura do pasto como
conceito de manejo: reflexos sobre o consumo e a produtividade. In: SIMPÓSIO DE
VOLUMOSOS NA PRODUÇÃO DE RUMINANTES 2, 2005, Jaboticabal. Anais...
Jaboticabal: Unesp, 2005. p.107-124.
[25] P. C. F. Carvalho and A. Moraes, Comportamento ingestivo de Ruminantes: bases para
o manejo sustentável do pasto. In: MANEJO SUSTENTÁVEL EM PASTAGEM, 1.,
2005, Maringá. Anais... Maringá, 2005.
[26] J. M. Thurow, C. Nabinger, Z. M. S. Castilhos, P. C. F. Carvalho, C. M. O. Medeiros,
and M. D. Machado, Estrutura da vegetação e comportamento ingestivo de novilhos em
pastagem natural do Rio Grande do Sul. Brazilian Journal of Animal Science, v. 38, p.
818-826, 2009.
[27] L. C. V. Ítavo, S. R. M. B. O. Souza, J. Rímoli, C. C. B. F Ítavo and A. M. Dias,
Comportamento ingestivo diurno de bovinos em pastejo contínuo e rotacionado. Arch.
Zootec., 57: 43-52, 2008.
[28] J. Hodgson, Grazing Management: Science into Practice. New York: John Wiley and
Sons, 1990. 203p. Longman Handbooks in Agriculture.
[29] J. A. Newman, A. J. Parsons and P. D. Penning, A note on the behavioral strategies
used by grazing animals to alter their intake rates. Grass and Forage Science, v.49,
p.502-505, 1994.
[30] S. Prache, Intake rate, intake per bite of lactating ewes on vegetative e
reproductive swards. Applied Animal Behaviour Science, Champaigne, v.52, p. 53-64,
1997.
[31] E. A. Laca and M. W. Demment, Modelling intake of a grazing ruminant in a
heterogeneous environment. In: INTERNATIONAL SYMPOSIUM ON VEGETATION:
HERBIVORE RELATIONSHIPS, 1992, New York. Proceedings… New York, 1992. p.
57-70.
[32] G. B. Ruyle and D. D. Dwyer, Feeding stations of sheep as an indicator of diminished
forage supply. Journal of Animal Science, Amsterdan, v.61, p.335-353, 1985.
[33] S. Prache and J. Peyraud, Préhensiblite de I´ herbe pâturée chez lês bovins et lês ovins.
INRA Productions Animales, [Paris], v.10, p.377-390, 1997.
[34] P. Penning, A. J. Parsons, R. J. Orr, Intake and behaviour responses by sheep to
changes in sward characteristics under rotational grazing. Grass and Forage Science,
v.49, p.476- 486, 1994.
[35] E. A. Laca, M. W. Demment, R. A. Distel, R.A. et al. A conceptual model to
explain variation in ingestive behavior within a feeding patch. In: INTERNATIONAL
GRASSLAND CONGRESS, 17., 1993, Palmerston North: New Zealand.
Proceedings… Palmerston North: New Zealand, 1993. p. 710-712.
[36] R. Hendricksen and D. J. Minson, The intake and grazing behaviour of cattle a crop of
Lablab purpureus cv. Rongai. Journal of Agricultural Science, v.95, p.547-554, 1980.
94
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
[37] D. L. Minson, Forage in ruminant nutrition. San Diego: Academic Press, 1990. 483p.
[38] R. Greenberg, A. Mehling, M. Lee and J. H. Bock, Tensile behavior of grass. Journal of
Materials Science, v. 24, p. 2549-2554, 1989.
[39] T. H. Stobbs, The effect of plant structure on the intake of tropical pastures. I. Variation
in the bites size of the grazing cattle. Australian Journal of Agricultural Research, v.24,
n.6, p.809-819, 1973.
[40] G. T. Barthram, Sward structure and the depth of grazed horizon. Grass and forage
Science, Oxford, v.36, p.130-131, 1981.
[41] D. P. Poppi, T. P. Hughesc and P. J. L’huillier, Intake of pasture by grazing ruminants.
In: NICOL, A.M. (Ed.). Livestock feeding on pasture. Halminton: New Zealand Society
of Animal Production, v.10, p. 55-64, 1987.
[42] L. Palhano, P. C. F. Carvalho, J. R. Dittrich, A. Moraes, S. C. Silva and A. L. G.
Monteiro, Padrões de deslocamento e procura por forragem de novilhas leiteiras em
pastagem de capim-mombaça. Brazilian Journal of Animal Science, v.35, p.2253-2259,
2006.
[43] Baggio, P. C. F. Carvalho, J. L. S. Silva, L. M. Rocha, C. Bremm, D. T. Santos and A.
L. G. Monteiro, Padrões de uso do tempo por novilhos em pastagem consorciada de
azevém anual e aveia-preta. Brazilian Journal of Animal Science, v.37, n.11, p.19121918, 2008.
[44] S. W. Coleman, Plant-animal interface. Journal of Production Agriculture, v. 5, p.7-13,
1992.
[45] G. R. Edwards, A. J. Parsons, P. D. Penning and J. A. Newman, Relationship between
vegetation state and bite dimensions of sheep grazing contrasting plant species and its
implications for intake rate and diet selection. Grass and Forage Science, v. 50, p. 378388, 1995.
[46] L. S. Pontes, P. C. F. Carvalho, C. Nabinger and A. B. Soareset, Fluxo de biomassa em
pastagem de azevém anual (Lolium multiflorum Lam.) manejada em diferentes alturas.
Brazilian Journal of Animal Science, v.33, n.3, p.529-537, 2004.
[47] J. Roman, Relação Planta-Animal em Diferentes Intensidades de Pastejo com Ovinos
em Azevém Anual (Lolium Multiflorum Lam.). Santa Maria: Universidade Federal de
Santa Maria, 2006, 79p. Dissertação (Mestrado em Zootecnia – Produção Animal) Universidade Federal de Santa Maria, 2006.
[48] J. Hodgson, Ingestive behavior. In: J. D. LEAVER (Ed.) Herbage Intake Handbook.
British Grassland Society, Hurley. 1982. p.113.
[49] J. Hodgson, D. A. Clark and R. J. Mitchell, Foraging behavior in grazing animals and
its impact on plant communities. In: FAHEY, G.C. National conference on forage
quality, evaluation and utilization. Nebraska: University of Nebrasca, 1994. p.796-827.
[50] K. L. Launchbaugh, and L. D. Howery, Understanding landscape use patterns of
livestock as a consequence of foraging behavior. Rangeland Ecol. Manag., 58: 99-108,
2005.
[51] N. B. Trevisan, F. L. F. Quadros, A. C. F. Silva, D. G. Bandinelli, C. E. N. Martins, L.
F. C. Simões, A. R. Maixner and D. R. F. Pires, Comportamento ingestivo de novilhos
de corte em pastagem de aveia preta e azevém com níveis distintos de folhas verdes.
Ciência Rural, v.3 4, n. 5, p. 1543-1548, 2004.
[52] C. Bremm, M. G. Rocha, J. Restle, A. Pilau, D. B. Montagner, F. K. Freitas, S. Macari,
D. A. G. Elejalde, D. Roso, J. Roman, P. Guterres, V. G. Costa and F. P. Neves, Efeito
Behavior Feeding of Ruminants
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
95
de Níveis de Suplementação sobre o Comportamento Ingestivo de Bezerras em
Pastagem de Aveia (Avena strigosa Schreb.) e Azevém (Lolium multiflorum Lam.).
Brazilian Journal of Animal Science, v.34, n.2, p.387-397, 2005.
C. E. S. Pedroso, R. B. Medeiros, M. A. Silva, J. B. J. Jornada, J. C. Saibro and J. R. F.
Teixeira, Comportamento de Ovinos em Gestação e Lactação sob Pastejo em Diferentes
Estágios Fenológicosde Azevém Anual. Brazilian Journal of Animal Science, v.33, n.5,
2004.
E. O. Silveira, Comportamento ingestivo e produção de cordeiros em pastagem de
azevém anual (Lolium multiflorum Lam.) manejada em diferentes alturas. Porto
Alegre: Universidade Federal do Rio Grande do Sul, 2001. 154p. Dissertação
(Mestrado em Zootecnia) – Universidade Federal do Rio Grande do Sul, 2001.
C. R. C. Castro, Relações planta-animal em pastagem de milheto (Pennisetum
clandestinum (L.) Leeke) manejadas em diferentes alturas com ovinos. Porto Alegre:
Universidade Federal do Rio Grande do Sul, 2002. 200p. Dissertação (Mestrado em
Zootecnia) - Universidade Federal do Rio Grande do Sul, 2002.
C. Bremm, M. G. Rocha, F. K. Freitas, S. Macari, D. G. Elejalde and V. G. Costa,
Comportamento ingestivo de bezerras de corte e características da pastagem de 'Avena
strigosa Schreb.' e 'Lolium multiflorum Lam.'. In: II Grassland Ecophysiology and
Grazing Ecology, 2004, Curitiba. Proceedings of II Grassland Ecophysiology and
Grazing Ecology, 2004.
S. Prache and C. Roguet, Influence de la structure du couvert sur le comportement
d´ingestion. Clermont-Ferrand: Institut National de la Recherche Agronomique, 1996.
p.22-24. (Rapport d‘Activité 1992-1995).
J. G. Welch, A. P. Hooper, Ingestion of feed and water. In: CHURCH, D.C. (Ed.). The
ruminant animal: digestive physiology and nutrition. Englewood Cliffs:Reston. p.108116, 1988.
J. L. Albright, Feeding behavior of dairy cattle. J. Dairy Sci., 76(2), 485-498, 1993.
G. W. Arnold and M. L. Dudzinski, Ethology of free-ranging domestic animals.
Amsterdam: Elsevier scientific, p 1-125, 1978.
L. Pérez, Comportamiento alimentario y actividades de cabras em pastoreo sobre
campo natural. Inia On line. 1998, http://www.capraispana.com.es.
P. C. F. Carvalho, C. H. E. C. Poli, C. Nabinger, A. Moraes, Comportamento ingestivo
de bovinos em pastejo e sua relação com a estrutura da pastagem. In: FERRAZ, J.B.S.
(Ed). SIMPÓSIO PECUÁRIA 2000 – PERSPECTIVAS PARA O III MILÊNIO, 1, 2000,
Pirassununga. Anais... Pirassununga: USP/FAZEA, 2000.
E. D. Ungar, Ingestive behaviour. In: HODGSON, J.; ILLIUS, A.W. The ecology and
management of grazing systems. Oxon: CABI, 1996. p. 185-218.
P. C. F. Carvalho, H. M. N. Ribeiro Filho, C. H. E. C. Poli et al. Importância da
estrutura da pastagem na ingestão e seleção de dietas pelo animal em pastejo. In:
REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 38, 2001,
Piracicaba. Anais... Piracicaba, 2001. p.853-871.
E. Chacon and T. H. Stobbs, Influence of progressive defoliation of a grass sward on
the eating behavior of cattle. Australian Journal of Agriculture Research, v.7, n.3,
p.709-727, 1976.
96
Christiano Santos Rocha Pitta, Paulo Fernando Adami and Laércio Ricardo Sartor
[66] E. R. Flores, E. A. Laca, T. C. Griggs and M. W. Demment, Sward height and vertical
morphological differentiation determine cattle bite dimensions. Agronomy Journal,
v.85, n.3, p.527, 1993.
[67] E. A. Laca, T. C. Griggs, R. A. Distel, R.A. et al. Factors and mechanisms that
determine bite weight and grazing behavior of cattle within a feeding station. In: 2ND
GRAZING LIVESTOCK NUTRITION CONFERENCE, 1991. Proceedings...
Steamboat Springs, Colorado. p.174.
[68] G. W. Arnold and M. L. Dudzinsky, Studies on the diet of grazing animals: III. The
effect of pasture species and pasture structure on the herbage intake of sheep.
Australian Journal of Agriculture Research, v.18, p.657-666, 1967.
[69] J. Burlison, J. Hodgson and A. W. Illius, Sward canopy structure and the bite
dimensions and bite weight of grazing sheep. Grass and Forage Science, v.46, p.29-38,
1991.
[70] T. D. A. Forbes, Researching the plant-animal interface: The investigation of ingestive
behaviour of cows and sheep. Journal of Animal Science, v.66, p.2369-2379, 1988.
[71] P. Penning, Some effects of sward conditions on grazing behavior and intake by sheep.
In: Gudmundssun, O. (Ed.), Grazing Research at Northern Latitudes. p.219–226, 1986.
[72] T. H. Stobbs, Rate ob biting by Jersey cows as influenced by the yeld maturity of
pasture swards. Tropical Grassland, Brisbane, v.8, p.81-86, 1974.
[73] P. Penning, A. J. Parsons, R. J. Orr, T. T. Treacher, Intake and behaviour responses by
sheep to changes in sward characteristics under continuous stocking. Grass and Forage
Science, v.46, p.15-28, 1991.
[74] F. C. A. Rego, J. C. Damasceno, N. M. Fukumoto, C. Côrtes, L. Hoeshi, E. N. Martins,
and U. Cecato, Comportamento ingestivo de novilhos mestiços em pastagens tropicais
manejadas em diferentes alturas. Brazilian Journal of Animal Science, v.35, p.16111620, 2006.
[75] D. H. Rearte and G. A. Pieroni, Supplementation of temperate pastures. In:
INTERNATIONAL GRASSLAND CONGRESS, 19., 2001, São Pedro. Proceedings…
São Pedro: SBZ, 2001. p.679-689.
[76] F. Bargo, L. D. Muller, E. S. Kolver and J. E. Delahoy, Invited review: Production and
digestion of supplemented dairy cows on pasture. Journal of Dairy Science, v.86, p.1–
42, 2003.
[77] L. J. Krysl and B. W. Hess, Influence of supplementation on behavior of grazing cattle.
Journal of Animal Science, v.71, p.2546-2555, 1993.
[78] F. Bargo, L. D. Muller, J. E. Delahoy and T. W. Cassidy, Milk response to concentrate
supplementation of high producing dairy cows grazing at two pasture allowances.
Journal of Dairy Science, v. 85, p.1777–1792, 2002.
[79] M. J. Gibb, C. A. Huckle and R. Nuthall, Effects of level of concentrate
supplementation on grazing behaviour and performance by lactating dairy cows grazing
continuously stocked grass swards. Journal of Animal Science, v.74, p.319–335, 2002.
[80] D. C. Adams, Effect of time of supplementation on performance, forage intake and
grazing behavior of yearling beef grazing Russian roildrygrass in the fall. Journal of
Animal Science, v.61, n.4, p.1037-1042, 1985.
[81] Frizzo, M. G. Rocha, J. Restle, M. R. Freitas, G. Biscaíno and A. Pilau, Produção de
forragem e retorno econômico da pastagem de aveia e azevém sob pastejo com bezerras
Behavior Feeding of Ruminants
[82]
[83]
[84]
[85]
[86]
[87]
[88]
97
de corte submetidas a níveis de suplementação energética. Brazilian Journal of Animal
Science, v.32, n.3, p.632-642, 2003.
D. A. G. Elejalde, M. G. Rocha, C. C. Pires, D. B. Montagner, L. Potter, C. Bremm, R.
A. O. Neto and M. G. Kloss, Parâmetros de qualidade da pastagem de azevém “Lolium
multiflorum Lam.” aparentemente consumida por ovelhas de descarte. In: REUNIÓN
DE GRUPO TÉCNICO EN FORRAJERAS DEL CONO SUR, 2004, Salto – Uruguai.
N. M. Patiño Pardo, V. Fischer, M. Balbinotti, C. B. Moreno, E. X. Ferreira, R. I.
Vinhais and P. L. Monks, Comportamento ingestivo diurno de novilhos em pastejo
submetidos a níveis crescentes de suplementação energética. Brazilian Journal of
Animal Science, v.32, n.6, p.1408-1418, 2003.
T. DelCurto, R. C. Cochran, D. L. Harmon, A. A. Beharka, K. A. Jacques, G. Towne,
and E. S. Vanzant Supplementation of dormant tallgrass-prairie forage. I. Influence of
varying supplemental protein and(or) energy levels on forage utilization characteristics
of beef steers in confinement. Journal of Animal Science, v.68, p.515-526, 1990.
P. J. Bürger, J. C. Pereira, A. C. Queiroz, J. F. C. Silva, S. C. V. Filho, P. R. Cecon and
A. D. P. Casali, Comportamento ingestivo em bezerros holandeses alimentados com
dietas contendo diferentes níveis de concentrado. Brazilian Journal of Animal Science,
v.29, n.1, p.236-242, 2000.
J. P. Dulphy, B. Remond and M. Theriez, Ingestive behavior and related activities in
ruminants. In: RUCKEBUSCH, Y.; THIVEND, P. (Ed.) Digestive physiology and
metabolism in ruminants. Lancaster: MTP.1980. p.103-122.
C. Bremm, M. G. Rocha, F. K. Freitas, S. Macari, D. A. G. Elejalde and D. Roso,
Comportamento ingestivo de novilhas de corte submetidas a estratégias de
suplementação em pastagens de aveia e azevém. Brazilian Journal of Animal Science,
v.37, n.7, p.1161-1167, 2008.
T. Barenbrug, Herbage Intake - Forage Factors, 2010 at http://www.farmwest.com/
index.cfm?method=library.showPageandlibrarypageid=101.
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter VII
Freemartinism in Cattle
Alexandra Esteves1, Renée Båge2 and Rita Payan-Carreira1*
1
CECAV [Veterinary and Animal Research Centre], Univ.,
Trás-os-Montes and Alto Douro, Vila Real, Portugal
2
Div. of Reproduction, Dept. of Clinical Sciences,
Faculty of Veterinary Medicine and Animal Science,
Swedish Univ. of Agricultural Sciences, Uppsala, Sweden
Abstract
Freemartinism is one of the most commonly found intersex conditions in cattle,
although it may also occur in small ruminants. The freemartin phenotype appears in a
dizygotic twin pregnancy where one twin is a male and the other is a female. Due to
precocious anastomoses between the placental vascular systems of the two fetuses,
masculinising molecules reach the female twin and disrupt the normal sexual
differentiation, whilst in the male the effects of this association are usually minimal. In
cattle, this condition is observed in 90 to 97% of twin pregnancies.
A freemartin is, by definition, a genetically female fetus masculinised in the presence
of a male co-twin, giving rise to a sterile heifer. Genital tract defects with varying
severity can be observed in freemartin animals, which often present suppression and
disorganization of the ovary, originating a rudimentary or a testis-like gonad depleted of
germ cells. The uterine horns may be hypoplastic or instead may be reduced to a cordlike structure suspended in the broad ligament. Anatomic continuity between the uterus
and the vagina is frequently absent, and the existence of rudimentary vesicular glands is
typical. The external genitalia commonly presents enlarged clitoris, small vulva and a
prominent, male-like tuft of hair. As a rule, heifers born twin to a bull have to be
considered sterile and should be identified as early as possible to cull them from
replacement stock. Despite its limitations, freemartinism is currently diagnosed by
physical examination, as karyotyping or blood typing is often considered an unnecessary
expense.
In cattle, twinning trend has a genetic background that has been associated to
hormonal regulation in favor of double ovulations. However, the genetic determinant on
*
E-mail address: rtpayan@gmail.com.
100
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
the basis of twinning seems to have small importance when compared to environmental
or management-associated factors, particularly in dairy cows. In fact, in dairy animals, in
particular in high milk producing cows, it has long been proven that the increase of twin
calvings occurs due to the hormonal and metabolic disturbances in the energy balance
early in the post-partum period. With increased incidence of twin births in cow it is
reasonably expectable a small increase in the occurrence of freemartins at the farm levels.
In this paper it is the intent to describe the gross and histopathological findings of
freemartinism in cattle, using data gathered from a study at an abattoir (17 cases) and
from 3 cases diagnosed in living animals, supported by a review of the pathophysiology
of the process, and to discuss the available methods for identification of freemartin
animals at farm level.
Introduction
Freemartinism is a particular form of intersexuality in cattle, and represents one of the
most common forms found. This pathology can also be observed in buffalo and small
ruminants [9,16,34,35], although in sheep and goats other forms of intersexuality seems to be
more frequent than the freemartin syndrome [5].
A bovine freemartin is usually defined as a sterile female calf, born co-twin with a male
fetus that shows underdeveloped or misdeveloped genital tract as a result of early
development of vascular anastomoses between fetuses of different gender (Figure 1). As
consequence of placental anastomoses between the heterozygotic twins, blood chimaerism
occurs (60, XX/XY) and passage of male gonad determinants or hormones (such as AntiMüllerian hormone and androgens) are responsible for disrupted differentiation of the female
embryonic gonads and disturbed genital tract development [34,44]. Comparing to the
dramatic changes observed in genital differentiation in the freemartin heifer, the male co-twin
only evidence minimal gross defects, thought a decrease in male fertility have been reported
[11,34].
The external genitalia of the freemartin females is usually feminine in appearance [34,
44], though some minor differences may be perceived at closer examination, and the animal is
commonly raised as female. However, the internal genitalia is masculinized in some extent,
impairing reproduction and fertility. Absence of anatomical continuity between the vagina
and the uterus, hypoplastic or absent uterus, and hypoplastic or streak gonads, co-existing
with vesicular glands, are common findings in freemartin heifers [44].
As a rule, heifers born twin to a bull have to be considered sterile and should be identified
as early as possible to cull them from replacement stock. However, a small number of females
born from twinning pregnancies are not freemartin, although blood chimaerism may be
detected [60]. Furthermore, some freemartin animals have been identified that were born as
singleton due to the death in uterus of its co-twin [34].
This further reinforces the need for an adequate diagnostic approach for the identification
of a freemartin animal, whatever the aptitude (beef vs. dairy) or purpose. It should be
emphasized the importance for discarding the freemartin syndrome during a reproductive
evaluation whenever a heifer is intended to be maintained in the farm for replacement. Its
inability to reproduce demands its early detection, in order to put these animals on the market,
independently of its genetic value.
Freemartinism in Cattle
101
Figure 1. Schematic representation of chorion-allantois anastomoses between two cattle fetus of
different gender, according to the literature.
The freemartin condition may be diagnosed on the basis of physical examination of
suspected animals, as well as by serologic or cytogenetic testing; the later being more
expensive and not always well accepted by the farmer, which often considers it an
unnecessary expense.
In a recent work on the occurrence of congenital abnormalities at an abattoir in the North
of Portugal, and for a 3-month period, a larger number of freemartin situations were detected
in dairy than in beef heifers (68.42% and 31.58%, respectively). In the same study,
freemartinism was found to present an incidence of 1.81% in dairy breeds, in comparison
with only 0.52% in beef breeds. In this chapter we ought to describe the histopathological
findings of 20 situations of freemartinism in cattle, diagnosed either in vivo or at slaughter in
animals older than 6 months of age, supported by a review of the pathophysiology of the
process.
Incidence of the Freemartin Syndrome
Prevalence of the freemartin syndrome in cattle population is directly dependent of the
prevalence of twinning within the population. Although congenital, freemartinism is not a
heritable defect, and consequently does not directly respond to negative selection. However,
twining seems to have some genetic background, and it has been found to present different
incidence among breeds. Additionally, twinning incidence may vary with men-imposed
artificial selection, either by culling or by intentionally use cows with higher twinning rates
[15], or even as consequence of multiple nonsexed embryo transfer, where the deposition of
two or more embryos is currently performed [34]. Multiple pregnancies are strongly affected
by age and parity, but only slightly influenced by season. In table 1, available values for
twinning rates in some cattle breeds are given.
102
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
Table 1. Reported incidence of twin pregnancies in diverse cattle breeds
Breed
Jersey
Holstein
Holstein-Friesian
Guernsey
Simmental
Swedish Frisian
Swedish Red
Brown Swiss
Hereford
German Fleckvieh
Angus
Brahman
Santa Gertrudis
*
cited by Korkman, 1948.
Twinning rate (%)
1.3
1.83
3.4
4.75
2.91
2.33
4.6
1.95
2.57
1.47
8.9
4.08
0.4
3.16
1.1
0.2
0.4
Reference
Rutledge, 1975
Cady and Van Vleck, 1978
Rutledge, 1975
Cady and Van Vleck, 1978
Silva del Rio et al., 2006
Cady and Van Vleck, 1978
Weber, 1944*
Johansson et al., 1974
Gregory, 1990
Johansson et al., 1974
Rutledge, 1975
Cady and Van Vleck, 1978
Rutledge, 1975
Silva del Rio et al., 2006
Rutledge, 1975
Rutledge, 1975
Rutledge, 1975
Twinning in cattle is closely associated with double ovulation rates; therefore a majority
of twins (93-95% of twinning cases) results from the fertilization of two oocytes in the same
cycle [23,47,60]. From those twin pregnancies, about 92% will result in the freemartin
condition [29]. Beside a genetic component, the occurrence of twinning can be favored by the
profound metabolic changes in the early postpartum cows, which are particularly important in
high yielding dairy cows and which is traduced in the occurrence of 25% of twin pregnancies
(against the 4-5% incidence for low and regular milk yielding in Holstein-Frisian) [27,28,59].
Twinning is an undesirable reproductive outcome in dairy cattle systems, even if they have
the potential to improve the efficiency of beef production. It increases energetic demands of
the mother, while decreasing its potential fertility early in the post-partum period [23].
Multiple pregnancies in cattle have some other disadvantages besides the prolonged
postpartum resumption of the ovarian cyclic activity, including an increase on the number of
abortion, the stillbirth and the premature births, and the predisposition to dystocia [6,48].
Hence, it is possible, although rare, the birth of a freemartin female resulting from in uterus
death of her male co-twins after the establishment of fetal vascular anastomoses [20,34,40].
In such situations, single-birth is not always a warranty for the inexistence of
freemartinism [20,34,40], and this condition should be suspected whenever primary anestrus,
irregular cyclicity, genital malformations or perineal abnormalities are clinically detected.
These animals are usually subfertile heifers with an apparent normal genital tract upon
transrectal palpation. With increased incidence of twin births in cow it is reasonable to expect
a small increase in the occurrence of freemartins at the farm levels, and the need for an early
diagnosis and early identification of freemartin calves increases. In a recent survey on the
occurrence of congenital abnormalities at an abattoir in the North of Portugal, and for a 3
Freemartinism in Cattle
103
months period, a larger number of freemartin syndrome conditions (n=17, for a total of 1867
cows) have been detected in dairy than in beef heifers (68.42% and 31.58%, respectively). In
the same study, freemartinism was found to present an incidence of 1.81% in dairy breeds
(mainly Holstein-Friesian), in comparison with only 0.52% in beef breeds.
Pathophysiology of the Freemartin Syndrome
As said, for freemartinism to occur 3 main events have to co-exist: i) a dizygotic twin
pregnancy, ii) with fetuses of different sex and iii) the occurrence of placental anastomosis.
Further, placental anastomoses have to occur early in pregnancy, as it has to be established
before gonadal differentiation, between 2 weeks and 30 days of pregnancy. Vascular
anastomoses occurring later than the female gonadal differentiation or its absence, like it is
most frequently observed in small ruminants, have been observed in only less than 10% of
twin, heterozygotic pregnancies in cattle [60].
Sex differentiation is a sequential, finely orchestrated process that is highly dependent of
the differentiated gonad to fulfill the development of the remainder reproductive structures
(Figure 2). In male calf, gonadal differentiation is coordinated, as in other mammals, with the
entrance of primordial germ cells in the undifferentiated embryonic gonad, by the time that
the cascade of events of gonadal organogenesis is triggered and SRY gene transcription
commence (Figure 2). Consequently, Sertoli cells differentiate and surround primordial germ
cells originating the sex-cords or seminiferous-like testicular cords [53], activating the
recruitment of mesonephric cells (a male-specific event) that will participate in local
vascularization, and the proliferation of interstitial cells with differentiation of Leydig cells,
which soon start producing androgens [2,45]. In the bovine male embryo this occurs by day
38-42 [55]. In the female embryo, gonadal differentiation is slightly postponed [19,40];
ovarian organogenesis occurs later than in the male embryo (Figure 1), apparently without
internal organization until day 70. Pre-meiosis begins by day 75, along with cortical cord
formation (or Pflüger´s cords), and oocyte inclusion within primordial follicles observed
between pregnancy days 130 and 170 [30]. In male embryo, the Anti-Müllerian hormone
produced by the Sertoli cells induces the seminiferous-like cord formation, the regression of
the Müllerian derivates, while androgens promote development of the Wolffian derivates and
the external genitalia masculinisation [19,56]. In female embryo, in the absence of
masculinising substances, regression of Wolffian derivates occur (Figure 2), while
differentiation and development of Müllerian derivates and external genitalia takes place
under the dependence of estrogens and specific signaling proteins [1].
What Happens Then during Gonadal, Internal and External Genitalia
Differentiation in the Freemartin Syndrome?
By the first week following testis differentiation in male embryos, the presumptive
freemartins do not differ significantly from normal females [19]. But by the following 2
weeks, a reduction in gonadal size and disruption of the presumptive ovarian cortex are the
major findings in freemartin females (Figure 2), along with the development of secondary sex
104
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
cords. Yet any morphological masculinisation (i.e. differentiation of seminiferous tubules or
of Leydig cells) is also arrested [19]. The work of Vigier and colleagues [53] showed that
AMH acts on the embryonic gonads by disrupting its ability for early production of sex
steroids; inhibiting the aromatase activity, at the time of sexual differentiation the synthesis of
estradiol observed in normal embryos is switched in the freemartin embryo into the synthesis
of androgens, due to defective aromatization [10]. Consequent to early ovary exposition to
AMH, germ cells loss is observed along with the differentiation of epithelial cells that
resemble Sertoli cells. Those cells aggregate into cord-like structures [55,56], depleted or
containing fewer germ cells [52,55]. Seminiferous cords differentiate around day 80 in the
freemartin gonad, a later event when compared to the male embryo, originating the nodular
areas of testis-like organization that have been described in freemartin gonads. It is also by
this time that seminiferous cords scattered in the freemartin gonads start producing its own
AMH, although the amount of AMH produced is negligible in comparison to the amount
released by the testis of the male twin [10,56]. Nevertheless, presence of circulating AMH at
similar concentrations in the serum of twins of both sexes, at the moment of Müllerian duct
regression, has been demonstrated by Vigier et al. [54], and precedes the gonadal production
of this protein by the freemartin gonad.
By day 55 to 80, in freemartin pregnancies, Müllerian duct regression starts at the same
time in the male and female embryo [19,56]; it begins adjacent to the mesonephros and the
genital folds, and are responsible for a more or less pronounced absence of the Müllerian
ducts derivates, which is always more pronounced on its cranial region. In addition,
circulating androgens allow later incomplete development of the Wolffian ducts derivates in
the female freemartin embryo (Figure 2), like the seminal vesicles that are commonly in the
female freemartin [19,56].
Figure 2. Graphic representation of the major events on the gonadal and genital tract embryonic
development, according to the literature.
However, marked masculinisation of the external genitalia seldom occurs, although
differences in the ano-genital distance are usually found in comparison to that of the normal
Freemartinism in Cattle
105
female. This could be explained by a limited capacity of androgen production by the
freemartin gonad (as most of male embryo androgens are not apparently transferred for the
female embryo), and occurs long after similar events in the male embryo [19]. The severity of
gonadal dysgenesis might be dependent on the extension of the period of AMH exposition
and the age of the embryos at the moment of exposition, which is influenced by the moment
of occurrence of the vascular interchanges. It has been demonstrated that large amounts of
AMH are produced in the male gonad between 50 and 65 days, gradually decreasing in later
stages of pregnancy [52,56]. This may also determine the extent and the severity of
disturbance of the internal and external genitalia development [53,60]. Furthermore, Jost et al
[19] report the existence of a slight lateral asymmetry in freemartins, with the left side
achieving smaller sized structures than the right.
Sporadically, freemartin chimaeric heifers without evidences of genital malformations are
found, and such situations are explained by the absence of vascular anastomoses between the
two placentas [12,49]. Occurrence of fertile female heifers co-twin to a male has been
reported to be less than 10% [60].
Gross Morphological Features in Freemartin
Heifers
There is considerable variation in the genital abnormalities described in freemartin
animals. Nevertheless, association between the severity of malformations (phenotype) and the
percentage of XX/XY cells were not found [35]. The degree of masculinisation of the
genitalia may be important when diagnosing the abnormal animal by means of the physical
evaluation. However, marked masculinisation of the external genitalia is seldom observed.
Also, discrete masculinisation of the external genitalia may difficult initial detection of
freemartin heifers, in particular if arising from singleton calving.
If, most frequently, the external genitalia of the freemartin heifer is feminine in
appearance [29], an enlarged clitoris is commonly found, co-existing with a small vulva and a
prominent tuft of hair (Figure 3A and 3C) [22,31]. It is frequently reported the emission of
urine in upwards spurts (Figure 1B). The small-sized vulva shortens the distance between the
anus and the ventral vulvar commissure, and may also give a different angulation to the
vulvar area (Figure 3D). The internal genital tract of freemartins can be more or less
masculinised. In the 20 situations analysed, the majority of animals presented underdeveloped
uterus (n=14), with uterine horns not longer than the length of the segment of the uterine body
and cervix.
However in a few cases the uterus was rudimentary (n=2) or was reduced to a thin tubular
or cord-like Y-arranged structure (n=4), miming the normal uterine disposition [22,29]. By
transrectal palpation, the later may be apperceived as a rudimentary uterus with the two
uterine horns being longer and thinner than the usual. The presence of vesicular glands was a
constant (Figure 4A), but the prostate is seldom found [58]. In highly masculinised animals, it
is possible to observe some degree of virilization of the phenotype, with the freemartin
displaying a male-like head or body morphology. It is generally accepted that for most
freemartin animals the vagina is shorter than in normally developed females, and frequently is
non-patent or blind ending [34, 44], independently of the development of the uterus.
106
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
Figure 3. The external genitalia of freemartin heifers. A) In freemartin heifers the vulva is smaller and
present a tuft of hair; B) also in these animals, emission of upwards spurts of urine are frequently
reported; C) An enlarged clitoris is a common finding. D) A fish-hook vulva confers a different
angulation of the external genitalia onto the perineum (left), compared to the linear trajectory of this
area in normal females (right), as depicted by the blue lines on the images.
Occasionally, fluid accumulation in the uterus, with subsequent distension of the organ
(Figure 4B), may be found. The cervix is seldom present [29,44] or is underdeveloped, hence
being reduced to a narrowed canal with thick walls (Figure 4C). The cervix with its normal
relief was not detected in none of the cases we had, albeit a cervix was found in one of the
freemartins in the study of Wilkes et al. [58]. Hypoplastic to rudimentary vesicular glands are
commonly found attached to the lower fused segments of the paramesonephric ducts (Figure
4A), and may be considered as key-diagnostic element [44]. They were found in all our cases,
but Khan and Foley [22] only found vesicular glands in about 73% of the analysed genital
tracts. Vesicular glands are frequently palpated as a smooth bilateral mass lateral to the caudal
tubular genitalia. It is also frequent to find one of them more developed than the other, hence
being more easily palpated. In one animal (of Swedish Red breed, SRB) from our survey it
was found duplication of these structures, showing two pairs of vesicular glands, which was
sporadically reported in freemartin heifers [31]. Commonly, on transversal cuts, the ampulla
can be found medial to the seminal vesicles, even if not detected during transrectal palpation
(Figure 4D). In our survey, the prostate was never found. Rudimentary ampulla may also be
Freemartinism in Cattle
107
found within the uterine serosa, on histology slides. If present, the uterus may be hypoplastic
or even rudimentary (Figure 4E). A clear definition of the uterine body and cervical area is
seldom perceptive, and intercornual ligaments are frequently poorly developed or missing.
Lack of communication between the vagina and the uterus may lead to accumulation of the
uterine fluids upstream to the obstruction (Figure 4B).
Figure 4. Gross morphology of the genital tract of freemartin heifers. A) The vesicular glands are
usually present and therefore are considered key-diagnostic. B) Non-patency of the cervical rudiment or
failure of connection between the vagina and uterus lead to uterine fluid accumulation and to enlarged
uterus. C) Often the cervix fails to develop its normal morphology and may remain as a narrowed
structure of thick walls. D) Transversal cuts performed at the vestibule may show the vagina (V) in
ventral position, the vesicular glands (VG) lateral and upwards, and occasionally, in medial position,
rudimentary ampulla (arrows). E) A very rudimentary uterus may be found, co-existing with streak
gonads. F) In one freemartin, a cyst formed a barrier between the uterus and the vagina, leading to
mucometra. G) The uterine horns might resemble finger-ended, due to cranial regression of the
embryonic Müllerian ducts. H) Remnants of the Wolffian ducts (arrows) may occasionally be found
parallel to the caudal female genitalia as tubular, non-contiguous ovoid structures. I) Rudimentary
tubular or cord-like structures may be found surrounded by a dense ligament, in the normal uterus
position. J) Some cord-like structures, when present, do not show anatomical continuity and often fail
to present lumen.
108
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
Figure 5. Morphological appearance of freemartin gonads. A) Frequently, the freemartin female
presents rudimentary gonads and rudimentary epididymis (arrow). It is also often found the presence of
cystic structures at the surface of the gonad, due to lack of connection between the rete and epididymal
structures. B) Smaller gonads are firm, compact, with occasional yellowish nodules. C) Larger gonads
may mimic the ovarian morphology of a cyclic female. D) On longitudinal cuts (same gonad as in C)
some cystic structures similar to follicles may be observed (asterisks), co-existing with a large cavitary
structure; in this case, the cysts corresponded to extended cystic rete tubules conferring a spongiform
appearance to the gonadal parenchyma. E) On ultrasounds the gonads may show areas with regular
hypoechoic mass, somehow similar to that observed in CL, and if cysts exists, some small anechoic
structures that could deceive onto small developing follicles. F) Sporadically, a round rudimentary
gonad (Gon) was found along with a structure similar to a rudimentary epididymis (arrow), which did
not assume it anatomical relation with the testicle.
In one of the situations, diagnosed in a SRB freemartin heifer with a small-sized uterus, a
thin wall (mimicking a vesicle or cyst) was the sole obstruction found, but was at the origin of
fluid accumulation in the uterus (Figure 4F). Due to non-patency of the genitalia uterine
fluids tend to accumulate, distending the uterine walls (Figure 4B). In some animals, the
cranial apexes of the uterine horns are missing, and uterine horns appear as finger-ended
(Figure 4G) [29]. Most frequently, oviducts are not found on gross inspection. Remnants of
mesonephric ducts may be present (Figure 4A and 4H), parallel to the Müllerian derivates.
Though some parts of the mesonephric duct persist in the freemartin animal, and may be
sporadically found in animals presenting a clearly developed uterus, the epididymis and the
spermatic cord are usually absent or poorly developed (Figure 5A and 5 F) unless the genital
tract is markedly masculinised [31], therefore co-existing with cord-like structures within a
ligamentous structure similar to the broad ligament. Although, in some cases these structures
Freemartinism in Cattle
109
are limited to several tubular segments without anatomical continuity (Figure 5I and J), which
not always showed lumen.
Gonads may appear as thickened structure near the apex of the uterine horn (Figure 5A
and 5B) or as a round structure that may present some cyst-like areas that may delude as
ovarian follicles (Figure 5C and 5D). During transrectal palpation, larger gonads may be
mistaken for cyclic ovaries, in particular if the owner has reported erratic or discrete oestrus
behaviour. In contrast, whenever small, rudimentary (streak) gonads exist, they are usually
difficult to palpate and even be missing during transrectal examination or during genital
excision at the abattoir. Larger gonads may present on the surface cut a spongiform, yellowish
to orange parenchyma. Whatever the size of the gonads may be, it is also possible to find
yellow firm areas or cystic follicular-like structures at the surface of the freemartin gonads,
that may induce misjudging of existing ovarian activity (Figure 5D). On ultrasonography
(Figure E), cystic areas may appear as anechoic round structures similar to follicles while the
cord-like areas appear as a slight hypoechoic areas; firm, yellowish parenchyma may give a
homogeneous hypoechoic image, resembling that of the corpus luteum (CL), although not
always as clearly delimited as in the cyclic cow (Figure 5E).
Rarely, in highly masculinised animals, the gonads may be found in the inguinal area, but
not in the scrotum, which does not develop in freemartin animals [29,31]. Peretti et al. [35]
describe 3 cases where inguinal testis were found in the inguinal fat, in freemartin Simmental
animals, two of them also presenting subinguinal vestigial prepuce. In our survey, all the
gonads were found in the abdomen in the normal position for the ovary.
Of the heterosexual twins, all males are sexually competent, albeit with somewhat
reduced fertility compared with that of the singletons [11], whereas 5–10% of the females,
which are not freemartins, are fertile [60].
Though a male born co-twin to a freemartin calf rarely display gross morphological
malformations [25,44], reports of associated male infertility and poor libido exists [11]. Still,
the origin for infertility remains controversial. Some of the reports describe the presence of
focal areas of testicular degeneration [11] and of testicular hypoplasia [34], which could
account to infertility. In addition, the existence of spermatogonial XX/XY chimerism [37]
allied to chromosomal fragility, as demonstrated by Peretti et al. [35], or to increased
degenerative changes could also contribute to loss of fertility in males born co-twin to a
female. XX germ cells do not survive in male gonads [57]. In some situations, infertility has
also been associated to changes in the quality of sperm (motility, concentration, morphology
and viability) co-existing with of acrosome defects [37].
Histopathological Findings in Freemartin Heifers
Evaluation of tissue sections from different segments of the freemartin genital usually
reveal normal to hypoplastic morphology, with exception for the gonads, which showed the
most pronounced changes. Freemartin females most commonly show gonadal dysgenesis
albeit various degrees of modification may be observed. Nevertheless, an apparently normal
ovary with a functional corpus luteum [58] as well as pregnancy and parturition [49] have
been reported in freemartin heifers.
110
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
When a major ovarian morphology is retained, most often the rete ovarii is well
developed whilst the other ovarian structures may be scarce or absent. In contrasts, when
pronounced masculinisation of the gonads occurs, proliferation of seminiferous-like tubules
within a compact interstitial parenchyma resembles a hypoplastic testis [44]. In such cases,
there is also development of the mesonephric ducts, and affected animals show rudimentary
epididymes, deferens ducts and pampiniform plexes [38]. If the percentage of the XX/XY
chimaerism does not correlate to the reproductive phenotype [29,35], the degree of
malformation of the gonads may influence the development of the remainder of the genitalia
considering their androgenic capacity.
According to Willier [57] gonadal dysgenesis could be grouped according to three main
patterns of disturbed differentiation: a more undifferentiated gonad, a gonad where ovarian
morphology prevail and a more masculinized gonad. The prominence of the rete structure is a
common finding in the freemartin gonad (Figure 6A), and usually assumes an eccentric
position in the gonad [31]. Different architecture may be present in the gonads of the same
freemartin animal, with one gonad being less differentiated than the other [29]. In cases where
ovarian morphology dominates, a prominent rete ovarii is frequently found (Figure 6A); the
cortex is poorly developed, with scant follicular structures scattered within a fibrotic
parenchyma (Figure 6B), although the presence of germ cells is rare. Disorganized, poorly
differentiated rete, centrally located within the gonad, may also be observed in less
differentiated gonads (Figure 6 C). Sporadically, masses of tissue resembling corpora lutea
(Figure 6D) may be found; these masses might have their origin from luteinization of atretic
follicles or from the formation of interstitial gland-like masses [22,29,31]; however, most
frequently those masses are not clearly delimited, excluding the first hypothesis. In freemartin
gonads, ovarian-like structures may co-exist with areas of less differentiated structures,
resembling sex-cords, or with testicular tissue, presenting hypoplastic seminiferous tubules
mainly composed by Sertoli cells (Figure 6E). In cases of more pronounced gonadal
masculinization, cord-like structures resembling seminiferous tubules, containing Sertoli cells
predominate [31, 38, 44], accompanied by proliferation of the interstitial tissue, with Leydig
cell hyperplasia. Occasionally only testicular tissue is found, along with rete testis and
epididymis. Even then, germ cells are seldom found [31,44], and when present, they are of
spermatogonial type. Dystrophic calcification or concretions have been reported within the
seminiferous tubules [31,51]. In some situations cystic dilatation of the rete tubules or of the
efferent ducts are observed (Figure 6F to 6H).
The development of the tubular genitalia, either of mesonephric or paramesonephric
origin, varies considerably, but is frequently hypoplastic, or may even be reduced to a thin
tissue band of tubular appearance [51]. The lumen of epididymal tubes and deferens ducts
contains no cells (Figure 6I), as no spermatogenesis exists in freemartin heifers, even if
seminiferous tubules are found in the gonads [31].
Occasionally, failure of the normal differentiation is also observed, with mesonephric
duct remnants appearing as a rope-like structure that may be devoid of lumen (Figure 6J and
6K). If the uterus is present, normal organogenesis may be disturbed, originating
disorganization of the myometrial layers, reduction of the glandular elements and reduced
thickness of the endometrium (Figure 6L and 6M) [22]; the absence of uterine lumen has
been seldom described [18]. Nevertheless, in some situations normal histological architecture
is retained. Vesicular glands, although often present, are usually hypoplastic (Figure 6N),
consisting of relatively few alveoli surrounded by an extensive connective stroma [22].
Freemartinism in Cattle
111
Figure 6. Histological images of diverse segments of different freemartin genital tracts. A) In most
gonads, prominence of the rete structure was found [Scale bar: 300 m]. B) In less developed gonads,
scant follicle-like structures were found scattered within a fibrotic parenchyma [Scale bar: 100 m]. C)
In streak gonads, the most prominent structures correspond to disorganized poorly differentiated rete
[Scale bar: 300 m]. D) Sporadically, masses of luteinized-like cells may be found, resembling a nondelimited corpus luteum [Scale bar: 100 m]. E) Frequently, freemartin gonads show areas composed
of seminiferous-like cords [Scale bar: 100 m]. F) When the rete patency is compromised, cystic
dilatation of rete tubules may be found [Scale bar: 300 m]. G) Multiple cystic rete structures
containing fluid [Scale bar: 300 m]. H) A cystic structure in the pole of the gonad may resemble a
small follicle [Scale bar: 300 m]. I) In freemartins, rudimentary epididymal ducts are devoid of
spermatozoa [Scale bar: 300 m]. J) Mesonephric remnants of disturbed morphology were observed
[Scale bar: 300 m]. K) Some of those remnants were devoid of lumen [Scale bar: 300 m], L)
Hypoplastic uterus showing a decrease in the epithelial elements and absence of caruncular areas [Scale
bar: 300 m]. M) Hypoplastic uterus without surface folds and irregular disposition of the myometrial
layers [Scale bar: 300 m]. N) Rudimentary vesicular glands (right structure) lateral to the undeveloped
ampulla (left) [Scale bar: 300 m]. O) Coiled, penis-like structure with rudimentary cavernous spaces
within connective tissue, surrounded by an albuginea; the central urethra, usually found in the male
penis, is absent [Scale bar: 300 m].
112
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
The glandular elements of the vesicular glands are lined by low cuboid epithelium. In
some animals, the clitoris is greatly enlarged and rather resembles a penis-like structure that
may direct urine spurts upward during urination. One of the freemartins in our survey showed
a enlarged clitoris, similar to a small, highly tortuous penis that extended into the vagina.
Longitudinal histological cuts of this structure presented several almost transversal sections
(compatible with a convoluted trajectory for this structure) composed of scant erectile
elements scattered in a fibrous stroma (Figure 6O).
Diagnosis of the Freemartin Syndrome
If freemartinism is associated with female sterility, diagnosis for freemartin females urges
to be detected as early as possible, particularly in dairy farms. This would allow distinguish
the females bearing freemartin symptoms and those that are potentially fertile from those born
co-twins with a male, the later accounting up to 8%. This would avoid culling animals of high
genetic value that may be used in reproduction, together with identification of animals that
should not be maintained in the farm, unless for beef production, where its sexual competence
is unimportant.
At present, freemartinism may be diagnosed by physical examination, karyotyping, blood
typing, polymerase chain reaction (PCR) or fluorescence identification of Y-chromosome
directed probes (FISH). But each of these methods has limitations and costs, those being
higher when laboratory exams are requested. Still, laboratorial methods also require a correct
planning for the collection and sending of the sample, and obviously additional time for cell
culture, extraction and evaluation of the results. Nevertheless, none of these methods is ideal
in terms of speed, sensitivity, or specificity [50].
All animals born from a heterozygotic pregnancy should be suspected freemartins until
proven otherwise. This procedure is particular important when the owner plan to use the
animal for reproduction, and the freemartins have to be ruled off the replacement stock.
Furthermore, to improve a systematic identification and culling program for freemartinism, it
is very important that the farm records are actual and precise. Also attention should be paid to
singleton females (or those born co-twin with fetal monsters) that present abnormal position
or appearance of the vagina/perineum area, primary anestrus or irregular estrous cycles. The
first approach should be a thorough physical examination of the suspected animal, which may
allow identifying about 80% of the freemartin animals [60]. If inconclusive or when a
confirmation is desired, the adequate laboratory methods may be used.
Identification of freemartin animals by physical examination is based on the fact that,
despite the variations observed within this kind of animals, usually two main clinical signs are
most frequently presented in freemartins: a vagina shorter than the normal, and the existence
of seminal vesicles in a phenotypic female [18,26,29,31,44,60]. A positive diagnostic is far
most easy in highly masculinized animals, but evidences for abnormal development of the
internal and external genitalia should be carefully searched for either by inspection,
transrectal palpation and ultrasonography. The clinical approach is simple, quick and allows
to reduce cost from laboratorial tests, as a large number of animals may be identified through
a careful clinical evaluation.
Freemartinism in Cattle
113
When approaching a suspected freemartin animal, close inspection of the vulva-vagina
segment may reveal the presence of longer vulvar hair, abnormal angulations of the vulva,
resembling a fish-hook shaped vulva, and clitoromegalia. The enlarged clitoris commonly
seems to prolong into the vaginal cavity. Then, the evaluation of the vagina may give some
indications about its size and the existence of a blind-ending structure, as rarely in freemartins
the cervical structure is palpated or visualized at the cranial end of the vagina. A small blindending vagina where the cervical relieve is not possible to palpate or to visualize by
vaginoscopy is a strong indicator for freemartinism. The length of the vagina may be
evaluated in suspected animals from one month of age upwards, and the differences in length
are usually more pronounced in older animals. Measurements can be performed with the well
lubricated round end of a blood collection tube, in younger animals, a commercially available
curved plastic probe (Figure 7), or a blunt-ended probe [21,26,60]. According to our
experience, a plastic sleeve intended for an AI pistolette works quite well: it is very easy to
insert in a small heifer without using too much pressure. The probe must be inserted in the
vagina at an angle of 45º, before be angled downwards to avoid the hymen constriction in
smaller heifers [26]. Particular attention should be paid to obtain the measure of the distance
to the front end of the vagina, but not to the hymen in normal young animals. Freemartin
Holstein calves of up to one month-old present a vagina 5-8cm long, while in normal females
it is 13-15cm long; in mature freemartins, the vaginal length is only 8-10cm, whilst in normal
cows the vagina is about 30cm long [26,29,58]. Peretti et al. [35] refer to vaginal length
varying from 1.5 to 9cm in sixteen freemartin heifers of 10 to 23 years old.
Figure 7. A) Freemartin Probe, also known as Ru-an or as Jor-vet probe [see at www.valeyvet.com or
www.enasco.com]. B) Graphic representation of the differences found when using a freemartin probe
on a normal heifer (Left), where the probe is introduced longer on the vestibule than in the freemartin
heifer (Right), where the probe does no enter more than its first part.
114
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
However, occasionally freemartins heifers present almost normal vaginal length, hence
escaping from this first trial; in such situations, being important to cull potentially sterile
animals, the cytogenetic or serologic tests should be use. In animals older than 6 months, the
existence of vesicular glands from middle to caudal aspect of the tubular genital tract can be
perceived during transrectal palpation near the bladder neck. Usually, the gland in one side is
easier to palpate than the other, due to the fact that small lateral asymmetric development of
the genital structures are common [19]. The small vesicular glands are usually palpated as a
tubular, cigar-shaped, smooth structure; although they present in bulls a lobular surface, this
is seldom apparent during palpation.
The blood grouping is one serologic test was never regularly used for discarding
freemartinism. Although the probability of two dizygotic twins having the same blood group
is quite low, in fact the test for the tolerance onto the co-twin cells could be used for
freemartin diagnosis. If there have been vascular anastomoses between the two placentas,
each fetus will have two populations of erythrocytes and by consequent two sets of red blood
cells antigens [26]. In such tests, blood group markers are used to evaluate for the presence of
hemolysis. Partial hemolysis (when its absence or complete hemolysis is normally expected)
indicates the existence of tolerance, thus of blood chimaerism, and is interpreted as sign for
freemartinism [7, 21, 29]. Blood heparinized samples should preferably be sent for a
reference lab. Nevertheless, this test presents too many inconclusive results to assure the
necessary accuracy. Also, blood grouping test can only be used in animals older than one
month, when surface antigen in blood red cells are matured [26].
On the other hand, the cytogenetic tests are probably the most accurate laboratorial
methods for freemartin testing, and yet its efficiency fairly reaches 90% [7,34]; they are
particular indicated for animals clinically normal. They can be used in all animals, even in
newborns, as once established blood chimaerism persists during the entire life of the animal,
whether it shows a female or a male phenotype. The classical, standard karyotype assesses the
existence of blood chimaerism in leukocyte cultures, hence supporting evidences for the
existence of vascular anastomoses during pregnancy [11,21,34]. However, this method has a
several handicaps: the distribution of the percentage of male cells is quite variable, and
animals with a very low male percentage may fail to be detected unless a large number of
mitosis is analyzed (up to the double than for the normal karyotype) [29]. Further, bacterial
contamination of the sample [21] or ongoing infections in the animal compromise the
lymphocytes cultures and may impair conclusive results. In addition, temperature during
transportation and storage as well as shipping period are determinant for the success of the
technique. The development of new techniques, such as the Polymerase Chain Reaction
(PCR), and the identification of genes in the X- or Y-chromosomes related to the sexual
differentiation that can be demonstrated by in situ hybridization using a fluorescent dye
(FISH), improving the efficiency of freemartin detection up to 98% [7,50]. PCR technique
allows of male-specific DNA amplification [13], thus presenting several advantages in
comparison to karyotyping, as it allows the use of small amounts of sample, is relatively fast
and precise, is a more sensitive assay than the karyotype [13,14,32,50], and it also allow to
use samples other than blood, though the precision of the test on those samples may be lower.
In PCR, a known Y chromosome-specific DNA sequence is used to target XY cells; the
absence of Y-chromosome sequences excludes freemartinism.
Some FISH techniques use the entire blood lymphocytes with or without previous culture
[33,50]. Specific markers or probes similar to those used in PCR technique can be used to
Freemartinism in Cattle
115
detect the Y-chromosome. By using a Y chromosome-specific probe, fluorescence is search
in positive cells within a slide [49]. Those authors report equivalent ratios of the XY to XX
cells or the chimaeric freemartins using his FISH test (using an Y chromosome-specific probe
containing the BC1.2 DNA sequence) as those obtained by karyotyping in the same animals.
However, for maximizing the efficiency of the technique, a large number of cells should be
analyzed, as for the karyotype.
For cytogenetic analysis, blood samples must be aseptically collected into samples to
vacuum heparin (about 5-10 mL; for chromosomal tests) and EDTA probes (5-10 mL; for
DNA tests), and maintained chilled until reaching the lab. Plan the request for cytogenetic
analysis in advance. Talk to the lab, as if cultures are needed it is probable that the blood
ought to arrive at the lab on specific days of the week. Organize the shipment to target those
days, and to achieve adequate periods of transportation. By doing that you minimize any
constrain associated with sample manipulation and assure that your sample arrived at the lab
in good conditions for the analysis. Fundamental information should always accompanies the
samples, like the motive for the request of the analysis, as it may indicate the need for higher
number of observations to be performed, particular on karyotyping. One should be aware that
cytogenetic test and karyotyping only confirm the existence of blood or tissue chimaerism;
hence results should not be used isolated for achieving a freemartin diagnosis, but rather
ought to be cross with the results of the clinical examination. Various alternative diagnostic
methods based on endocrine determinations or hormonal challenge tests have been proposed
besides the cytogenetic tests, based on the observations that freemartins animals present
different gonadal morphology and structures. Differences in sex steroids and gonadotrophins
profiles between freemartin and normal heifers have been studied, as an attempt to establish
gonadal functionality as a predictor of forthcoming fertility. However, these kind of test
present low specificity, and are highly variable with age and the degree of the freemartin
masculinisation.
Plasmatic testosterone levels remained undetectable in freemartins [36,38,41], whilst
those of androstenedione are increased in freemartin animals aged of 24 months but not in
normal or freemartins animals of 10-12 months old [41]. Rota et al [38] report low
progesterone values in freemartins and normal calves until puberty, a time when normal
animals start cycling, but not the freemartin heifers. However, occasional surges on
progesterone have been observed by Saba and colleagues [41]. No differences have been
described in progesterone levels between freemartins and controls [36,41], but blood values
of progesterone surely depend on the presence and on the amount of luteal tissue on the
gonads [34]. Estradiol 17 was found to be lower in freemartin heifers than in the controls,
albeit without statistical significance [36, 46]. Overall steroid production by freemartin
gonads does not clearly differ from the observed in normal animals, in particular in young
animals below 12 months old. This could be detected through challenging tests. Freemartin
heifers fail to respond to hCG administration with an increased in estradiol or progesterone,
as do normal animals [4,43].
Randel et al. [36] report higher plasma LH levels in freemartin heifers when compared to
controls, comparable to those expected following ovariectomy. Saba et al. [42] tested LH
response of freemartin and normal, non-cyclic heifers to exogenous estradiol stimulation, and
observed a consistent LH treatment-induced surge in control heifers at 9-18 hours post
administration; in contrast, in the freemartin group, an absent or diminished response to
stimulation was observed. Later on, Cunningham et al. [8] demonstrated that some of those
116
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
freemartin heifers achieve equivalent LH-induced surges, only delayed in time for 48-72h.
AMH plasmatic levels seems to be slightly increased in freemartin newborn and in animals up
to 5 months of age, in comparison to normal females [38]. Also AMH levels tend to be higher
in freemartins with marked masculinisation of the gonad, thus serving as indicator of the
presence of seminiferous cord-like structures. AMH may be measured by an ELISA
technique in serum or plasma samples. Ideally, blood samples should be centrifuged short
after collection, and sent to the laboratory within short time. Avoid freezing/thawing cycles,
as it may be deleterious for the molecule integrity. Although available, hormone-based
methods are not regularly used for freemartin identification, as their reliability is lower than
most cytogenetic analysis, and their cost is sufficiently lower to become an advantage. Best
results are obtained in older animals [43], where ovarian activity was suspected to exist. The
exception is the AMH determination that should be performed in animals younger than 5
months old. The serological tests only give information on the functional capacity of sex
steroid production by the gonad, and indirectly may inform on the possibility of ovarian
dysgenesis. Nevertheless, information on nutritional deficiencies, thyroid function and other
diseases that may be associated with anestrus should be taken in consideration when
discarding a freemartin situation.
Conclusion
Although not a heritable effect, freemartinism is a congenital syndrome that in
association with twinning may prevail or even increase in cattle farms. Thus, the frequency of
freemartinism in cattle population is directly associated with the prevalence of twinning births
in the population. Most frequently freemartin sterile heifers born co-twin to male calves.
Sterility is related to malformations arising in consequence to precocious anastomoses
between the placental vessels of embryos of distinct gender, which predispose the female
embryo to abnormal differentiation of the genital tract due to the crossing of masculinizing
substances. Nevertheless, in a few situations (less than 10%) vascular anastomoses occurs
latter than the threshold period, and though presenting blood chimaerism, the female calf is
quite normally developed. Further, when in uterus death of a male embryo occurs, singleton
freemartin female may result, despite few cases have been reported.
Consequently, one should be aware of the persistence of freemartinism in cattle farms, of
the possibility for different malformations of the genital tracts, and on the methods that can be
used for early detection of the syndrome in animals not intended for beef production.
Maintenance of freemartin females, potentially infertile, in the replacement stock has negative
results in the farm economy. Ideally, these animals should be early detected and channeled
into the market stock.
The freemartin syndrome is not necessarily a limiting factor in cattle production. It is
important to strengthen that all heifers wished for replacement purposes should be submitted
to a complete reproductive examination to exclude freemartinism and ascertain its fertility
potential. At the moment, it does not exist a simple, 100% trustful test. A first approach
should include the physical and genital tract evaluation. This first trial will allow identifying
the animals with clinical signs. All animals suspected of freemartinism (born for twin
Freemartinism in Cattle
117
pregnancies and showing normal or almost normal genital tracts) with almost normal
genitalia, and all doubtful cases should be submitted to laboratorial studies.
The genetic tests will allow confirming the existence of chimaerism or the existence of
cells carrying the Y-chromosome, but they do not identify potentially fertile females born cotwin with a male calf. Those may be identified through the use of serological tests for the
ovarian function. On respect to the reproductive function of the male calves born co-twin to
freemartins, conflictions reports exist and several questions remain to be answered,
demanding for additional studies that include a larger number of animals. Freemartin animals
may be culled from the reproduction stock, and they can be used for beef production, as the
freemartin carcass has similar characteristics to normal animals.
Acknowledgment
This work was supported by the project from CECAV/UTAD with the reference PEstOE/AGR/UI0772/2011, by the Portuguese Science and Technology Foundation.
References
[1]
Basrur, P.K. (2006). Disrupted sex differentiation and feminization of man and
domestic animals. Environ. Res., 100,18-38.
[2] Brennan, J. and Capel, B. (2004). One tissue, two fates: molecular genetic events that
underlie testis versus ovary development. Nat. Rev. Genet.,5 (7), 509-21.
[3] Cady, R.A. and Van Vleck, L.D. (1978). Factors affecting twinning and effects of
twinning in holstein dairy cattle. J. Anim. Sci., 49, 950-956.
[4] Cavalieri, J. and Farin, P.W. (1998). Birth of a Holstein freemartin calf co-twinned to a
schistosomus reflexus fetus. Theriogenology, 52, 815-826.
[5] Cinzia, S., Iannuzzi, L., Fogu, G., Bonelli, P., Bogliolo, L., Ledda, S., Zedda, M.T. and
Pau, S. (2006). Clinical and cytogenetic studies in intersex ewes. Caryologia, 59 (1),
67-74.
[6] Ḉ obanoglu, O. (2010). Twinning in the cattle: Desirable or undesirable? J. Biol.
Environ. Sci., 4 (10), 1-8.
[7] Corredor Camargo, E.S. and Páez Barón, E.M. (2009). Freemartinismo ó quimerismo
XX/XY em bovino: revision. R. Inv. Agrar. Amb., 0 (1), 7-12.
[8] Cunningham, N.F., Saba, N. and Boarer, C.D. (1977). The acute effect of oestradiol17β and synthetic LH-RH on plasma LH levels in freemartin heifers. J. Reprod. Fert.,
51, 29-33.
[9] Di Meo, G.P. Perucatti A., Di Palo, Iannuzzi, A., Ciotola, F., Peretti, V., Neglia, G.,
Campanile, G., Zicarelli L. and Iannuzzi, L. (2008). Sex chromosome abnormalities and
sterility in river buffalo. Cytogenet. Genome Res., 120 (1-2), 127-31.
[10] Dominguez, M.M., Liptrap, R.M., Croy, B.A. and Basrur, P.K. (1990). Hormonal
correlation of ovarian alterations in bovine freemartin fetuses. Anim. Reprod. Res., 22,
181-201.
118
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
[11] Dunn, H.O., McEntee, K., Hall, C.E., Johnson, R.H. Jr, Stone, W.H. (1979).
Cytogenetic and reproductive studies of bulls born co-twin with freemartins. J. Reprod.
Fertil., 57 (1), 21-30.
[12] Eldridge, F.E. and Blazakb, W.F. (1977). Chromosomal analysis of fertile female
heterosexual twins in cattle. J. Dairy Sci. 60 (3), 458-63.
[13] Ennis, S., Vaughan, L. and Gallagher, T.F. (1999). The diagnosis of freemartinism in
castle using sex-specific DNA sequences. Res. Vet. Sci., 67, 111-112.
[14] Fujishiro, A., Kawakaru, K., Miyake, Y-I. and Kaneda, Y. (1994). A fast convenient
diagnosis of the bovine freemartin syndrome using polymerase chain reaction.
Theriogenology, 43, 883-891.
[15] Gregory, K.E., Bennett, G.L., Van Vleck, L.D., Echternkamp, S.E. and Cundiff, L.V.
(1997). Genetic an environmental parameters for ovulation rate, twinning rate, and
weight traits in a cattle population selected for twinning. J. Anim. Sci., 75, 1213-1222.
[16] Iannuzzi, L., Di Meo, G.P., Perucatti, A., Ciotola, F., Incarnato, D., Di Palo, R., Peretti,
V., Campanile, G. and Zicarelli, L. (2005). Freemartinism in river buffalo: clinical and
cytogenetic observations. Cytogenet. and Genome Res., 108, 355-358.
[17] Johansson, I., Lindhé, B. and Pirchner, F. (1074). Causes of variation in the frequency
of monozygous and dizygous twinning in various breeds of cattle. Hereditas, 78, 201234.
[18] Jones, T.C., Hunt, R.D. and King, N.W. (1997). Genital System - Ambiguous sexual
differentiation. 6th edition, Veterinary Pathology, Lippincott: Williams and Wilkies,,
1149-1153.
[19] Jost, A., Vigier, B. and Prepin, J. (1972). Freemartins in cattle: the first steps of sexual
organogenesis. J. Reprod. Fertil. 29 (3), 349-79.
[20] Kadokawa, H., Minezawa, M., Yamamoto, Y., Takahashi, M., Shimada, K., Takahashi,
H. and Kariya, T. (1995). Freemartinism among singleton bovine females born from
multiple embrio transfer. Theriogenology, 44, 295-306.
[21] Kästli, F. and Hall, J.G. (1978). Cattle twins and freemartin diagnosis. Vet. Rec., 102,
80-83.
[22] Khan, M.Z. and Foley, G.L. (1994). Retrospective studies on the measurements,
karyotyping and pathology of reproductive organs of bovine freemartins. J. Comp.
Path., 110, 25-36.
[23] Komisarek, J. and Dorynek, Z. (2002). Genetic aspects of twinning in cattle. J. Appl.
Genet., 43 (1), 55-68.
[24] Korkman, N. (1948). Genetic variation in the frequency of multiple births in cattle.
Hereditas, 34, 23–34.
[25] Kovacs, A., Stukovszkly, Y., Gippert, E., Csontos, G. and Nagy, Y. (1977). Single-born
XX/XY chimeric bulls whit normal phenotype. Ann. Génét. Sél Anim. 9, 533 [abstract].
[26] Long, S.E. (1990). Development and diagnosis of freemartinism in cattle. In Practice
12, 208-210.
[27] Lopez, H., Caraviello, D.Z., Satter, L.D., Fricke, P.M. and Wiltbank, M.C. (2005).
Relationship between level of milk production and multiple ovulations in lactating dairy
cows. J. Dairy Sci. 88, 2783-2793.
[28] Lopez, H., Satter, L.D. and Wiltbank, M.C. (2004). Relationship between level of milk
production and estrous behavior of lactating dairy cows. Anim. Reprod. Sci. 81, 209223.
Freemartinism in Cattle
119
[29] Marcum, J.B. (1974). The freemartin syndrome. Anim. Breeding Abstracts, 42, 227241.
[30] Matschke, G.H., and Erickson, B.H. (1969). Development and radioresponse of the
prenatal bovine testis. Biol. Reprod., 1 (2), 207-14.
[31] McEntee, K. (1990) Intersexes. In: McEntee, K Edition, Reproductive Pathology of
Domestic Animals, San Diego: Academic Press; 8-30.
[32] McNiel, E.A., Madrill, N.J., Treeful, A.E., Buoen, L.C., Weber, A,F. (2006). J. Vet.
Diagn. Invest., 18, 469-472.
[33] Meinecke, B., Kuiper, H., Drögemülle, C., Leeb, T. and Meinecke-Tillmann, S. (2003).
A mola hydatidosa coexistent with a foetus in a bovine freemartin pregnancy. Placenta,
24(1), 107-12.
[34] Padula, A.M. (2005). The freemartinism syndrome: an update. Anim. Reprod. Sci., 87,
93-109.
[35] Peretti, V., Ciotola, F., Albarella, S., Paciello, O., Dario, C. and Barbieri, V. (2008).
XX/XY Chimerism in cattle: clinical and cytogenetic studies. Sex. Dev., 2, 24-30.
[36] Randel, R.D., Brown, B.L., Erb, R.E., Niswender, G.D. and Callahan, C.J. (1971).
Reproductive steroids in the bovine. II. Comparison of freemartins to fertile heifers. J.
Anim. Sci., 32, 318-326.
[37] Redjuch, B., Slota, E., Gustavsson, I. (2000). 60,XY/60,XX Chimerism in the germ cell
line of mature bulls born in heteroxexual twinning. Theriogenology, 54, 621-627.
[38] Rota, A., Ballarian C., Vigier, B., Cozzi, B.and Rey, R. (2002). Age dependent changes
in plasma anti-Müllerian hormone concentration in the bovine male, female, and
freemartin from birth to puberty: relationship between testosterone production and the
influence on sex differentiation. Gen. Comp. Endocrinol., 129, 39-44.
[39] Rutledge, J.J. (1975). Twinning in cattle. J. Anim. Sci., 40, 803-815.
[40] Ruvinsky, A. and Spicer, L.J. (1999) Developmental genetics: Sex determination and
differentiation. In: Fries, R and Ruvinsky, A. (eds). The Genetics of Cattle.
Wallingford: CABI Publishing and CAB International, 456-461.
[41] Saba, N., Cunningham, N.F. and Millar, P.G. (1975). Plasma progesterone,
androstenedione and testosterone concentration in freemartin heifers. J. Reprod. Fert.,
45, 37-45.
[42] Saba, N., Symons, A.M., Cunningham, N.F. and Boarer, C.D. (1976). The acute effect
of oestrogen injection on plasma LH in freemartin heifers. J. Reprod. Fert., 48, 317321.
[43] Satoh, S., Hirata, T-I., Miyake, Y-I., Kaneda, Y. (1997). The possibility of early
estimation for fertility in bovine heteroxexual twin females. J. Vet. Med. Sci., 59 (3),
221-222.
[44] Schlafer, D.H. and Miller, R.B. (2007). Abnormalities of sexual development - Intersex
conditions. In: 5th Ed Maxie, M.G. (Ed.), Jubb, Kennedy, and Palmer Pathology of
Domestic Animals, Volume 3, chapter 4 (Female genital System) Saunders, St. Louis,
433-440.
[45] Schmahl, J. and Capel, B. (2003). Cell proliferation is necessary for the determination
of male fate in the gonad. Dev. Biol., 258 (2), 264-76.
[46] Shore, L. and Shemesh, M. (1981). Altered steroidogenesis by the fetal bovine
freemartin ovary. J. Reprod. Fert., 63, 309-314.
120
Alexandra Esteves, Renée Båge and Rita Payan-Carreira
[47] Silva del Rio, N., Kirkpatrick, B.W. and Fricke, P.M. (2006). Observed frequency of
monozygotic twinning in Holstein dairy cattle. Theriogenology, 66, 1292-1299.
[48] Silva del Rio, N., Stewart, S., Rapnicki, P., Chang, Y.M. and Fricke, P.M. (2007). An
observational analysis of twin births, calf sex ratio, and calf mortality in Holstein dairy
cattle. J. Dairy Sci., 90, 1255-1264.
[49] Smith, G.S., Van Camp, D. and Basrur, P.K. (1977). A fertile female co-twin to a male
calf. Can. Vet. J., 18 (10), 287-289.
[50] Sohn, S.H., Cho, E.J., Son, W.J. and Lee, C.Y. (2007). Diagnosis of bovine freemartin
by fluorescence in situ hybridization on interphase nuclei using a bovine Y
chromosome-specific DNA probe. Theriogenology, 68, 1003-1011.
[51] Valdovinos, M.A.A., Villagómez, D.A.F. and Benitez, S.L.S. (2000). Estudio
citogenético y anatomopatológico del síndrome freemartin en bovinos (Bos Taurus).
Vet. Méx., 31 (4), 315- 322.
[52] Vigier, B. Watrin, F., Magre, S., Tran, D. and Josso, N. (1987). Purified bovine AMH
induces a characteristic freemartin effect in fetal rat prospective ovaries exposed to it in
vitro. Development, 100 (1), 43-55.
[53] Vigier, B., Forest, M.G., Eychenne, B., Bézard, J., Garrigou, O., Robel, P. and Josso, N.
(1989). Anti-Müllerian hormone produces endocrine sex reversal of fetal ovaries. Proc.
Natl. Acad. Sci. U S A., 86 (10), 3684-8.
[54] Vigier, B., Picard, J.Y., Bézard, J. and Josso, N. (1981). Anti-Miillerian hormone: a
local or long distance morphogenetic factor? Hum. Genet., 58, 85-90.
[55] Vigier, B., Prépin, J. and Jost, A. (1976). Chronologie du développement de l'appareil
génital du foetus de veau. Archs. Anal. Microsc. Morph. Exp., 65, 77-102.
[56] Vigier, B., Tran, D., Legeai, L., Bézard, J. and Josso, N. (1984). Origin of antiMüllerian hormone in bovine freemartin fetuses. J. Rep. Fert., 70, 473-479.
[57] Willier, B. H. (1921) Structure and homologies of free-martin gonads. J. Exp. Zool., 33,
63.
[58] Wilkes, P.R., Wijeratne, W.V., and Munro, I.B. (1981). Reproductive anatomy and
cytogenetics of freemartin heifers. Vet. Rec.,108 (16), 349-53.
[59] Wiltbank, M.C., Fricke, P.M., Sangsritavong, S., Sartori, R. and Ginther, O.J. (2000).
Mechanisms that prevent and produce double ovulations in dairy cattle. J. Dairy Sci.
83, 2998-3007.
[60] Zhang, T., Buoen, L.C., Seguin, B.E., Ruth, G.R. and Webber, A.F. (1994). Diagnosis
of freemartinism in cattle: the need for clinic and cytogenic evaluation. J. Americ. Vet.
Med. Assoc., 204 (10), 1672-1675.
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter VIII
Fat Depots: A Morphological and
Anatomical Adaptation to
Tropical Climates
André Martinho de Almeida
IICT - Instituto de Investigação Científica e Tropical,
Centro de Veterinária e Zootecnia, Faculdade de Medicina Veterinária, Lisboa, Portugal
Abstract
Fat depots are appendages that are frequently found in ruminants in tropical regions.
In this article, we will address the issue through two characteristic study cases, the hump
of Bos indicus (Indian cattle) and the sheep’s fat tail, aiming to provide the reader with an
overview of the characteristics that make these animals particularly adapted to this type
of environments.
Keywords: Fat depot; Adaptation, Zebu, Fat-tailed sheep
Introduction
Tropical climates pose severe constraints to animal production, particularly at the level of
high ambient temperatures and humidity, seasonal weight loss or the existence of tropical
diseases and parasitism. In order to live and produce under such extreme environments, farm
animals have had to adapt to such conditions. One of the most known and perceptible
anatomical adaptation to tropical conditions in domestic livestock is the uneven distribution
of fat in the body, particularly the existence of a fat depot. Such adipose tissues are essential
for maintaining body temperature and act as body reserves that may be depleted in times of
food scarcity. The most well-known of such morphological adipose tissues is the cervo-thorax

E-mail: aalmeida@fmv.utl.pt.
122
André Martinho de Almeida
hump of Zebu (Bos indicus) cattle, native to the Indian sub-continent and widespread
throughout tropical Africa and South America where it plays an important role in the beef
industry. Nevertheless, other examples are available, such as the fat tail in sheep (Ovis aries)
or the hump in the dromedary (Camelus dromedarius). In this article, we will review the first
two study cases in on the interesting morphological adaptation and how it allows animals to
live and produce effectively in such harsh environments.
Bos indicus: A Cattle Species Adapted to Heat
Stress?
Domestic cattle include two distinct species, the European (Bos taurus) and Zebu (Bos
indicus), shown in figure 1. The first originate from the auroch, a wild bovine existing in
Europe and Asia that become extinct in the 19th century. European cattle are presently
distributed across the world. Oppositely, Bos indicus evolved in the Indian peninsula but later
spread to Africa and Americas, the Pacific and Australia.
Both species have a common ancestor that at some point diverged. Both species are
however relatively similar and numerous composite breeds of the two species exist. Zebu
cattle are particularly adapted to the tropics, with special reference to hot ambient
temperatures.
Numerous studies on the comparison of Bos indicus vs. Bos taurus breeds when
subjected to high ambient temperatures are available, particularly from the United States,
Brazil and Australia, either under field conditions or controlled environmental chambers and
including other components like gene expression, reproductive characteristics or carcass traits
[1,2,3].
When subjected to high ambient temperatures, in order to avoid increasing their body
temperature itself, cattlemust match the heat produced by the organism to the heat dissipated.
It is hence imperative to minimize heat gain and maximize heat loss. Bosindicus rely on a
combination of strategies to achieve such goal:
1)
2)
3)
4)
5)
6)
surface area per unit of body weight;
temperature gradient between animal and air;
conduction of heat from the body core to the skin;
solar radiation reflection;
metabolic rate and feed intake and
cellular mechanisms.Such combination of strategies is achieved by a number of
physiological characteristics [1].
Metabolic rate may be defined as the energy produced by unit of surface area. Bos taurus
tend to have higher metabolic rates than Bos indicus, as the latter are less heavy and have
smaller internal organs, as well as larger body surface area. Zebu cattle also have a higher
sweating ability, due to a higher density of sweat glands in comparison to European cattle
breeds. With such increase, zebus are able to dissipate heat more efficiently [1]. Another
remarkable adaptation is the higher resistance to outer influx of heat flow from the milieu.
This is accomplished chiefly the coat cover of Bos indicus that is lighter, sleeker and shinier
Fat Depots
123
contrary to the darker, denser and typically wooly coating of European cattle, particularly in
the cold season. Appendages are also asignificant and identifiable feature of zebu cattle. The
main function of appendages is to increase the surface area of the skin therefore specifically
contributing to the metabolic rate lowering and the increased number of sweat glands [1].
Figure 1. Zebu (Bos indicus) cattle with the characteristic hum and skin appendages, rendering this
group breeds particularly adapted to hot environments.
A very visible characteristic of all Bosindicuscattle breeds is the cervo-thoracic hump. In
zebu, fat deposition is made esswentially at the level of the hump. Localized fat depots allow
the decrease of the body fat throughout the body, which results in the increase in heat
dissipation. Additionally, the hump is in fact an additional appendage that contributes to the
increase of surface area of the body, as previously mentioned [1].
Bos indicus have as well an alleged higher tolerance to ticks and tick borne diseases,
although they are not tolerant to trypanossomiasis (sleeping sickness), a disease that prevents
their use in most of the disease range and particularly West Africa. Additionally, Bos indicus
have other inconvenient: they are regarded by breeders as animals with poorer productive
performance, low meat tenderness, and diminished milk production with a significant
lactation persistency, short estrus and long pre-pubertal periods and finally are animals of
very difficult temperament rendering difficult herd management. Nevertheless, zebus are a
very fascinating subject concerning adaptation to high temperatures. This makes them the
cattle type of the tropics and sub-tropics.
Fat Tailed Sheep – The Desert Ovine?
Arid, steppe like climates, include large areas of the world. In fact, significant areas in all
continents have this type of climate, characterized by rain seasonality and the existence of
periods of prolonged droughts [4]. Some of these areas have however a large human
population that depends on domestic ruminants, particularly sheep and goat, for their
livelihood. In these often termed pastoral, extensive production systems, sheep production
124
André Martinho de Almeida
relies heavily on indigenous breeds with an anatomical distinctiveness: the fat tail or fat rump
(see figure 2). These breeds are an essential ovine genetic resource in the Middle East and
North Africa, Central Asia, Eastern and Southern Africa and have recently been imported to
Brazil and Australia, among other countries [4,5]. Regarding international distribution, only
two fat tailed breeds may be classified as international: the Awassi from Israel and distributed
across the world, particularly in the Mediterranean basin [6], as a dairy sheep; and the
Karakul (Astrakhan) breed form Kazakhstan that gained importance in the pelt production
industry in the 1960’s and 1970’s, particularly in Southern Africa [5].
Fat tailed sheep are characterized by a deposition of fat at the level of the hind quarters.
The shape and size varies considerably with breed, population, gender and particularly
nutritional status. Some of the fat tailed sheep breeds have also fat depots in other parts of the
body, such as the rump or, less frequently, the back of the neck [7]. Fat tailed are usually
shedding hair sheep with a ticker coat on cooler months of extremely diverse color. Fat tailed
sheep are tall and slender and have long legs [5].
Fat tailed sheep are chiefly adapted to arid climates with lengthy and continual dry
seasons. Consequently, the fat tail is able to be mobilized in case of prolonged periods of food
scarcity and decrease its size during the seasonal weight loss periods. On the contrary, during
the rainy season when pastures are available, the size of the fat tail will increase.
Additionally, the uneven distribution of fat in the body of fat tailed sheep has a similar
thermoregulatory effect of the Bos indicus hump previously mentioned, serving as an
appendage that therefore favors heat dissipation. The tall and slender type of bodies of fat
tailed sheep is also an important adaptation to the heat stress characteristic of dry climates.
This trait, as well as the long legs aremainlyappropriate to walk long distances in search of
pastures and water and are consistent with the nomadic and transhumant of herders of semiarid regions. Additionally, fat tailed sheep have an apparent tolerance to diseases and
parasites and have strong gregarious and defense instincts that enable them to defend the
flock from predators. The combination of such characteristics them the ideal sheep breed
group for extensive production systems in arid regions, contributing to food security
supplying milk, meat and fat, but also hides, hair, dung for fertilizing or fire and above with
an important social role as wealth and status indicators and cash reserve.
Figure 2. A van Rooy ram with the characteristic fat tail, an adaptation to nutritional stress imposed by
the arid environments of Southern Africa.
Fat Depots
125
Conclusion
Fat depots such as the hump in zebu cattle or the fat tail in sheep are traits that indicate an
adaptation to environmental conditions of the tropics, including hot temperatures and seasonal
weight loss caused by a scarcity of pastures during the dry season. The presence of fat depots
is therefore an extremely important and desirable trait in the context of animal production in
the tropics, allowing an efficient approach to animal production. Nevertheless, these breeds
are frequently not considered as optimal for animal production due to their modest
productivity. This is a highly controversial argument, given the endurance ability of these
animals, and the fact that they are able to live and produce under conditions in which most
breeds would not even survive. It is therefore highly desirable that the productive and
adaptive physiology traits of these animals to be the objective of further research.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
P.J. Hansen, Animal Reproduction Science 82–83, 349 (2004).
R.J. Collier et al., J. Dairy Sci. 91, 445 (2008).
R.J. Collier et al., J. Anim. Sci. 84, E1 (2006).
A.M. Almeida, Tropical Animal Health and Production 43, 1233 (2011).
A.M. Almeida, Tropical Animal Health and Production 43, 1427 (2011).
E. Gootwine,Tropical Animal Health and Production 43, 1289 (2011).
A.F. Pourlis,Tropical Animal Health and Production 43, 1267 (2011).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter IX
Bovine Viral Diarrhea Virus: Biology,
Clinical Forms and Pathogenesis
María A. Risalde*, Pedro J. Sánchez-Cordón,
Miriam Pedrera, Verónica Molina, Fernando Romero-Palomo
and José C. Gómez-Villamandos
Department of Comparative Pathology, Veterinary Faculty,
University of Córdoba-Agrifood Campus of International Excellence (ceiA3),
Edificio Sanidad Animal, Córdoba, Spain
Abstract
Bovine viral diarrhea virus (BVDV) is an endemic ruminant pestivirus in populations
worldwide. Based on antigenic and genetic differences, BVDV isolates can be classified
into two genotypes, BVDV-1 and BVDV-2, which are divided in non-cytopathic and
cytopathic biotypes, depending on their effect on cell cultures. This virus presents a
complex biology which gives rise to a great variety of clinical manifestations, including
subclinical infections of immunocompetent animals, appearance of persistently infected
animals from congenital infections and development of a severe fatal process
denominated mucosal disease. During BVDV pathogenesis, the virus shows a special
tropism for cells of the immune system as monocytes/macrophages, dendritic cells and
lymphocytes, inducing cell death as an extreme event of the infection, or more subtle
effects on cytokines and co-stimulatory molecules, affecting both innate and adaptive
immune responses. BVDV can cause disease on its own, but which is perhaps more
important, it also induces a state of immunosuppression that predisposes calves to the
appearance of secondary infections. In general, the immunosuppressive properties of
BVDV produce a reduction in local defense mechanisms, thereby predisposing calves to
other respiratory pathogens, being considered as the main predisposing factor for the
occurrence of Bovine Respiratory Disease Complex.
The study of immune evasion strategies used by BVDV should lead to a better
understanding of the immune system and the interaction of this virus with other
*
E-mail address: v12rimom@uco.es.
128
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
pathogens in the host. This will help to treat virus-induced pathology, identify new
mechanisms for immune modulation, design vaccines and establish control strategies.
Introduction
Bovine viral diarrhea virus (BVDV) is an important pathogen of cattle, generating
considerable economic losses for the livestock industry. This pestivirus is the causative agent
of the bovine viral diarrhea (BVD), disease described for the first time by Olafson et al. in
EEUU (1946) as an enteric bovine disease with high morbidity and low mortality that seemed
to have viral etiology [1].
Years later, another process with a sporadic presentation, extremely high mortality and
produced by the same agent was reported in cattle, being named as mucosal disease (MD)
[2,3]. Nowadays, BVD is known as a contagious disease that induces severe reproductive,
respiratory and gastrointestinal pathologies mainly in bovine, although the BVDV can also
infect ovine, caprine, wild ruminants and porcine [4,5].
BVD is widely distributed and presents high prevalence worldwide, seeing increases in
the number of BVDV-seropositive cases with the presence of immunotolerant animals in the
herds. Nevertheless, the prevalence of BVDV infection is variable depending on diverse
circumstances, such as density and size of the herds, housing system and management of the
animals [6].
Since 1946, BVDV has shown to be responsible for a multifactorial disease with
probably the most complicated pathogenesis of the bovine malignances. During the past 50
years, BVDV research has made many advances leading to the development of diagnostic
tests, the production of vaccines and the design of successful control strategies for this virus.
Likewise, the discovery of persistent infection was a key factor to understand BVDV
maintenance in the host and to develop rational eradication strategies [7].
1. Taxonomy, Morphology and Structure of BVDV
BVDV is a pestivirus classified within the Flaviviridae family that displays high
homology with other pestiviruses, such as classical swine fever virus (CSFV) and border
disease virus of sheep [8,9]. Flaviviruses are a closely related group of small enveloped
viruses with a single-stranded, positive sense RNA genome of approximately 12.5 kb in
length [10,11]. This RNA strand comprises an open reading frame (ORF) flanked on the 3'
and 5' ends by untranslated regions (UTR), showing the 5' UTR extreme as the most
conserved region in pestiviruses [10].
The ORF 5' end encodes the structural proteins of the virus (C, Erns, E1, E2), while the
non-structural proteins (Npro, NS23) are encoded at the 3' end [12]. The lipid envelope
derives from the membrane of infected cells with a ranging from 40 to 60 nm in diameter and
is found surrounding an icosahedral nucleocapsid of 25-37 nm [13,14].
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
129
2. Genotypes and Biotypes of BVDV
RNA viruses are characterized by their plasticity and ability to generate a selection of
variants with different antigenic properties [15, 16], which helps BVDV to evade recognition
by neutralizing antibodies and escape the host immune response [17]. BVDV has been
classified into two different genotypes or species, BVDV-1 and BVDV-2, based on genetic
differences [18,19,20]. BVDV-1 includes the most common isolates on herds. This genotype
causes processes with unapparent symptoms, characterized by a slight increase in body
temperature and the presence of moderate lesions restricted to the digestive tract and organs
of the lymphoid system, causing also abortions in pregnant cows and other reproductive
disorders [18,21].
BVDV-2 isolates are associated with acute severe processes, presenting an intense
lymphopenia, thrombocytopenia and body temperatures exceeding 40.6ºC [22,23,24],
sometimes characterized by producing an acute bleeding disease called hemorrhagic
syndrome [15,25]. Independent of the genotype to which it belongs, BVDV is divided in two
biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP), depending on their lytic
activity on cultured epithelial cells [18,19,20].
CP biotypes provoke a cytopathic effect which leads to cytoplasmic vacuolization and
cell death, while NCP biotypes replicate in these cells without causing morphological changes
[8, 14]. The biochemical hallmark of CP strains is the production of high levels of NS3 as a
free protein after the early phase of infection. However, NCP BVDV variants produce small
amounts of NS3 and largely NS23 in the early phase of infection [26,27]. In this regard, there
are evidences that NCP biotype of BVDV may originate CP strains, either by proteolytic
cleavage of the NS23 protein [27,28], gene duplication of the altered NS3 protein [29],
genetic deletion of the NS2 protein [30] or mutation [31]. Cytopathology in vitro does not
correlate with virulence in vivo [32]. Indeed, the most severe clinical form of acute BVDV
infection and the establishment of persistent infections are associated with NCP virus
[18,24,33,34], which is the most common biotype in nature [8,14].
3. Clinical Forms
As it has been reported, the biology of BVDV is very complex, depending on different
factors such as genotype and biotype that causes the infection, the immune status and age of
animals, as well as the gestational period of the cows. This leads to a broad spectrum of
clinical manifestations and lesions that can be classified according to the type of BVDV
infection in: acute infection, congenital infection and MD (Figure 1).
3.1. Acute Infection
The infection of immunocompetent animals with BVDV can originate some clinical
variants: subclinical infection, acute BVDV infection, severe acute BVDV infection and
hemorrhagic BVDV infection [35].
130
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
BVDV
NCP
Acute infection
Congenital infection
Persistently infected (PI)
NCP
Low virulence
- Subclinical
- BVD
- Acute servere
BVD
- Hemorraghic
syndrome
High virulence
< 60
50-120
100-150
> 150
Days of
gestation
- Fetal death
- PI animals
- Malformations
Mutation
of NCP
Infection with
homologous CP
NCP
NCP
CP
CP
+
- Seropositive
healthy calves
Mucosal disease
Figure 1. Schematic summary of different clinical manifestations described in cattle following BVDV
infections.
A significant percentage (70-90%) of BVDV infections results in subclinical infections
[8,36], showing in the animals only a slight increase in body temperature, decreased white
blood cell count and immunosuppression [8,36,37,38,39]. This immunosuppression favors the
emergence of opportunistic infectious agents [40], highlighting those that cause bovine
respiratory disease [41,42]. In these processes, clinical signs will depend on the nature of
secondary infection, so that they are almost never recognized as processes induced by BVDV
[8,36,43]. Acute infection with characteristic clinical symptoms is described as BVD,
observing only moderate clinical manifestations as pyrexia, anorexia, lethargy, salivation,
oculo-nasal discharge, cough and mild diarrhea [44]. Occasionally, erosions and ulcerations
of the oral and gastrointestinal mucosa may be noticed [37,45,46,47]. This form of disease is
usually produced by BVDV-1 strains and some low virulence BVDV-2 strains, and although
it is possible to obtain isolates from both strains, NCP biotypes are more frequent. These
processes that have a high morbidity and very low or no mortality, affect more frequently to
calves with 6-24 months old [37,48].
There is an acute severe form of BVD characterized by high fever (39.7 to 41ºC),
agalactia, watery diarrhea and respiratory disorders. These animals show a minimum
reduction of 50% in the circulating lymphocytes and a marked thrombocytopenia together
with pneumonic lesions, ulcerations in the oral mucosa and depletion of lymphoid organs
[22,36,49,50]. This clinical form is caused by NCP strains of BVDV-2 with high virulence,
presenting elevated morbidity and mortality within 48 hours after the onset and affecting
animals of all ages [23,25,37,50,51,52].
Hemorrhagic syndrome is a grave clinical form evolved from the acute severe form,
where the animals show pyrexia, bloody diarrhea, conjunctiva and mucous congestion,
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
131
petechial hemorrhages and ecchymosis in mucous membranes [15,25,53]. In addition, this
syndrome is characterized by a marked thrombocytopenia, leukopenia and neutropenia [8,54].
Among the most characteristic, lesions highlight an important depletion of lymphoid organs,
increased apoptosis of lymphocytes, vacuolization of epithelial cells and vasculitis in various
organs [24,25,55]. This clinical form is caused by NCP BVDV-2 highly virulent strains and
has a mortality rate near to 25% [8,18,21].
3.2. Congenital Infection
BVDV can also produce reproductive disorders, as it can be removed in the semen of
infected animals, giving rise to venereal infections [56,57] that cause a decline in male
fertility and reduced conception rates [58]. In the case of females, all reproductive organs are
permissive to BVDV, with the ovary being the most affected organ, altering its function and
preventing normal follicular dynamics which produces a temporal infertility [59,60].
Adult immunocompetent pregnant cows can clear the virus and become immune, but
during this period, a transplacental infection involving any of both genotypes may occur,
although in turn, only the NCP biotype causes the fetal infection [61,62]. Fetuses infected
with NCP BVDV present differences in the severity of lesions depending on the stage of
gestation at which the infection takes place [63]. Thus, when the infection occurs before 60
days of gestation, it may lead to fetal death with mummifications or abortions from 10 days to
3 months after the virus entrance [53,64].
The fetus infected between 50-120 days of gestation, which is prior to the development of
fetal lymphoid tissues and to a functional acquired immune response, is unable to recognize
the virus as foreign, which results in the acquisition of immunotolerance to the infecting
BVDV strain and persistent infection [63,65]. After birth, these persistently infected (PI)
animals appear clinically normal but they are viremic and shed virus in all excretions and
secretions continuously, becoming the main reservoir of virus within the herd [8,66,67,68]
and being at risk of suffering MD. Some PI calves have reduced fertility and show
immunosuppression that predispose them to secondary infections [68,69] resulting in the
death of calves during the first year of life [70,71]. Between 100-150 days of gestation,
coinciding with the onset of fetal immunocompetence and organogenesis, congenital
malformations appear and abortions are less frequent. Thus, the most characteristic lesions are
thymic hypoplasia and pulmonary necrosis, alopecia, hypotrichosis, arthrogryposis, growth
retardation and other skeletal and eye abnormalities [72,73,74], presenting also severe
malformations of the nervous system, such as microcephaly, hydrocephaly and cerebellar
hypoplasia [73]. When infection occurs in late stages of the gestation, the immune system is
sufficiently developed and may start an immune response against the infecting virus.
Therefore, these calves are generally born immunocompetent and without problems, although
seropositves to BVDV [63].
3.3. Mucosal Disease (MD)
MD is an sporadic and fatal disease produced by BVDV that only affects PI animals
when a mutation exists of NCP strains into CP or a super-infection shortly after birth by a CP
132
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
biotype antigenically homologous to the NCP biotype that caused the immunotolerance
[12,68,75,76]. Animals that suffer from this disease present bloody diarrhea, mucocutaneous
erosions and death within 2-3 weeks from the onset of clinical signs. Microscopically, calves
have fibrinous enteritis, erosions, ulcerations and hemorrhages in the mucous membranes of
the oral cavity, esophagus, pre-stomachs, abomasum and intestine. Moreover, these animals
show depletion of lymphoid tissues, especially those associated with mucous membranes,
which induces a state of immunosuppression [8,77,78].
4. Pathogenesis
After any infection, there is a complex interaction between the etiologic agent and the
infected host defined as “pathogenesis”. This process evolves from the entry of the pathogen
in the animal to the development of disease and immune response in the host. The pathogenic
mechanisms of BVDV have not yet been clarified, existing discrepancies about whether the
direct action of the virus may or may not be responsible for lesions that appeared in different
locations.
4.1. Distribution and Target Cells Of BVDV
4.1.1. Acute Infections
Regardless of the virulence of the strain, the main route of postnatal infection with
BVDV is the oronasal, being the nasal mucosa and tonsils the primary organs of virus
replication. The virus spreads from the nasal cavity to regional lymph nodes through
lymphatic and blood vessels and then to systemic distribution in a free form or associated
with lymphocytes and monocyte-macrophages (m-MΦs) [41,79,80].
BVDV displays a special tropism for the mucosa-associated lymphoid tissue (tonsils and
intestine) and lymphoid organs as lymph nodes, thymus and spleen. In these locations, the
presence of BVDV antigen is associated with a marked lymphoid depletion. Moreover, the
antigen-presenting cells (APCs) such as dendritic cells (DCs) and m-MΦs, and the
lymphocytes (T and B) appear as the main virus target cells [24,39,79,81,82,83,84,85] (Figure
2). In studies performed after inoculation with strains of low virulence, viral antigen was only
detectable in lymphoid tissues, not observing the presence of virus in the bone marrow during
the infection [24,37,82,83] (Table 1).
However, in experimental infections of colostrum-deprived calves with BVDV strains of
low virulence, the viral antigen can be also detected in the intestinal mucosa, liver, upper and
lower respiratory tract [37,39,82,83,86, 87]. In inoculations with highly virulent strains, it was
observed that the quantity and spread of viral antigen in tissues exceeded that produced by
low virulence strains.
The presence of antigen in processes caused by these strains is not only restricted to the
follicles of lymphoid tissues, but it also extends to other organs as skin, digestive and
respiratory tracts, endocrine tissues, bone marrow and interstitium or vascular walls
[23,24,25,79,55,66) (Table 1).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
133
Figure 2. Histologic section of ileum from a calf inoculated with BVDV displaying a severe depletion
of lymphocytes in the Peyer’s patches (A) associated with the presence of BVDV antigen (B).
Multifocal depletion of lymphocytes in the thymic cortex linked to BVDV distribution (C). The widest
BVDV distribution in the lymph nodes is seen in medullary sinuses (D). Details of the main BVDVtarget cells MΦs, stellate-like cells and some lymphocytes in lymphoid organs (upper right boxes B,
C and D).
Table 1. Distribution of BVDV strains in acute infections
Organs
Lymphoid tissues
Tonsils
Thymus
Spleen
Lymph nodes
Peyer's patches
Bone marrow
Digestive tract
Oral mucosa
Esophagus
Intestines
Respiratory tract
Nasal mucosa
Lung
Heart
Skin
Liver
Pancreas
Kidney
Adrenal
Thyroid
Pituitary
Ovary
Testis
Nervous system
Low virulence
Highly virulence
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
134
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
Furthermore, other cells undergoing BVDV infection to a lesser extent are endothelial
cells, neutrophils, epithelial cells, keratinocytes, megakaryocytes and platelets [23,24,45,88].
Thus, the infection of epithelium in the upper digestive tract may cause erosive to ulcerative
lesions [25,45,55,66]. Moreover, the infection of bone marrow is related with the
development of a marked thrombocytopenia characteristic of the infection with these strains
[23,24,25,46,50,55].
4.1.2. Congenital Infections
Both BVDV biotypes infect the ovaries in experimental inoculations leading to a
decreased conception rate [60,89,90]. In the first stage of gestation, BVDV infects the
placenta and numerous tissues of the fetuses, causing severe damage that is considered as an
important initiator for abortion [91,92].
Nevertheless, if the fetus is not immediately expulsed, it can give rise to mummifications
[93]. PI animals present a wide distribution of BVDV in all their tissues, having more tropism
for epithelial, lymphoid and central nervous system cells, although there are no morphological
lesions associated with the presence of the virus [83,94,95,96]. Teratogenic effects of BVDV
appear when the immune competence begins to develop, the ability to mount an inflammatory
response sets in, and organogenesis is not completed. Such alterations affect mainly to brain
and eyes because the organogenesis of these tissues occurs in the final stages of gestation
[93,97].
4.1.3. Mucosal Disease
Like in PI animals, the NCP BVDV strains can be present in numerous organs and tissues
of calves with MD, but are not associated with tissue lesions. When intranasal infection with
CP BVDV occurs, primary replication of the virus is observed in the epithelium of the tonsil.
Following BVDV spread to the regional lymph nodes, it can be detected in Peyer’s patches,
lymphoid follicles of mucosa-associated to intestinal and respiratory tracts, and to a lesser
extent, in peripheral lymph nodes, thymus and spleen. Therefore, lesions occurring in MD are
associated with the presence of CP BVDV antigen [93,98,99].
4.2. Immune Response and Immunosuppression
The vertebrate immune system possesses sophisticated mechanisms to counter the
multitude of pathogens that establish an infection and cause disease. However, at the same
time, several pathogens evolve continuously developing complicated strategies to suppress or
evade the host immune mechanisms. BVDV is not an exception to this phenomenon, being
able to produce disease on its own and, which is perhaps more important, inducing a state of
immunosuppression that predisposes calves to infections by other micro-organisms. As it has
been seen before, BVDV has a special tropism for cells of the immune system, inducing cell
death as an extreme event of the infection, or more subtle effects on cytokines and costimulatory molecules produced by immune or non-immune cells that could affect to both
innate and adaptive immune response (Table 2).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
135
Table 2. Different mechanisms utilized by BVDV to induce a state of
immunosuppression
BVDV immune evasion strategies
Innate
immunity
Adaptive
immunity
Apoptosis
References
Decreased phagocytic activity
Decreased chemotactic activity
Decreased O2 –synthesis
Increased NO synthesis
Stimulation of PGE2 synthesis
Impaired TNFα response
Inhibition of IL-1 synthesis
Low type I IFN response to NCP strains
Delayed increase of positive APPs
[103]
[103]
[40, 104, 105]
[40, 104, 106]
[107, 108]
[85, 87, 105, 109, 110, 111]
[87, 105, 109, 111, 119]
[71, 121, 122, 123]
[44, 87, 120]
Decreased expression of MHC I in MΦs by NCP
Decreased expression of MHC II in MΦs
Reduced proliferation of CD4+ T cells
Impaired IFNγ response
Delayed neutralizing antibodies response
[50, 122, 133]
[50, 133]
[122]
[145, 146]
[37, 44, 50, 146]
Lymphopenia
Lymphoid depletion in lymphoid tissues
Direct infection of lymphocytes
Release of apoptotic factors by infected MΦs
Activation of extrinsic pathway by NCP
Activation of intrinsic pathway by CP strains
[24, 25, 50, 55, 82, 83, 152]
[23, 39, 45, 55, 83, 85, 111]
[23, 25, 37, 45, 82]
[111, 133, 157, 158, 163]
[157, 158, 170]
[173, 176, 178]
4.2.1. Innate Immune Response
Phagocytosis is a critical innate defense mechanism which implies the intracellular
killing of pathogens and the secretion of pro-inflammatory cytokines. This internalization and
killing of pathogens is an essential pre-requisite for microbial antigen presentation and
induction of a specific adaptive immune response against pathogens [100].
Specialized APCs, such as MΦs and DCs, are considered key components of an innate
immune system, developing the pathogens several strategies to combat these phagocytes
[101]. Particularly, MΦs are of great importance in the defense of the body against viral
infections, so that the valuation of their functions permits to determine the ability of the
individual resistance or susceptibility to infection [102]. Infection of MΦs with BVDV
produce a decrease in chemotactic and phagocytic capacity by altering cellular metabolism
[103], an impaired microbicidal activity by decreasing superoxide anion (O2-) production and
increasing nitric oxide (NO) synthesis in response to lipopolysaccharide [40,104,105,106],
and a stimulation of prostaglandin E2 (PGE2) synthesis [107,108].
Furthermore, various authors have demonstrated that BVDV also induces an impaired
production of cytokines by MΦs both in vitro and in vivo, which could affect the generation
of a subsequent immune response [85,87,105,109,110,111].
Pro-inflammatory cytokines as interleukin (IL)-1 and tumor necrosis factor-α (TNFα) are
of great importance in the innate immune response, being secreted by MΦs in the
inflammatory response or due to the existence of tissue damage [112]. These cytokines,
together with type I IFN, mediate in the acute phase response, an important defense
136
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
mechanism that is activated before the specific immunity against disturbances in homeostasis
by infections, tissue injury or immune disorders [113,114]. This non-specific mechanism
includes, among others, changes in the synthesis of certain plasma proteins denominated
acute phase proteins (APPs) that can be considered as “positive’’ or ‘‘negative’’, depending
on the increase or decrease of their concentration in serum. Haptoglobin (Hp) and serum
amyloid A (SAA), the main positive APPs in cattle, are used as indicators of disease severity
[115,116].
About pro-inflammatory cytokines, in vitro BVDV infection of bovine MΦs does not
provide consistent results; whereas some authors demonstrate a decrease in the production of
TNF with both CP and NCP BVDV strains [105,110], others maintain that CP strains
induce the production of TNF, which contributes to apoptosis of these cells [109]. In this
regard, acute experimental infection with a NCP strain also provided variable results
depending on viral target organs [37,85,87]. The pro-inflammatory capacity of TNFα is
limited by the lack of IL-1α, since these two cytokines act synergistically [117,118]. Thus,
BVDV-associated inhibition of IL-1 has been reported both in vitro and in vivo, regardless of
the biotype of the strain used [85,87,105,109,110,111,119]. On the other hand, the secretion
of these pro-inflammatory cytokines in acute BVDV processes with NCP strains induced a
progressive and late increase of disease indicators as Hp, SAA and fibrinogen in serum
[44,87,120]. Other cytokines as interferons (IFNs) were discovered due to their ability to
protect cells from viral infection, reducing virus replication and dissemination. In acute
processes, infection with CP BVDV strains produces a rapid and powerful local early
response with the release of type I IFN (α/β) by m-MΦs or DCs, activating to the effector
cells of innate immune response and limiting virus replication at the mucosal level [71]. By
contrast, NCP BVDV strains do not stimulate an early local cytokine response and the virus is
conveyed to local lymph nodes where it interacts with DCs that produce large amounts of
IFN, increasing their activation and limiting viral replication. This generates an effective
primary immune response [121,122] which, however, does not prevent the spread of the virus
[71,123], indicating the presence of other factors involved in the immunosuppression induced
by BVDV [44,121]. The infection with both NCP and CP BVDV early in gestation gives rise
to an elevated type I IFN production only in fetuses infected with CP BVDV. Thus, the
evasion of innate immunity by inhibition of type I IFN following NCP BVDV infection
favors the establishment of an immunotolerance state that allows virus persistence in the fetus
[121,124]. Since a functional fetal adaptive immune response does not occur at this time,
virus replication will not be limited and this will spread throughout the body, not developing
in PI calves an acquired immune response to BVDV attributed to tolerance of CD4+ cells
[125,126,127,128].
4.2.2. Adaptive Immune Response
Adaptive immunity is an antigen-specific response with immunologic memory regulated
by T and B lymphocytes, and the soluble factors produced by them –cytokines and antibodies,
respectively. The immune response, whether it is cell or humoral mediated, begins with
antigen recognition, processing and presentation [129,130].
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
137
4.2.2.1. Cell-Mediated Immune Response
This type of adaptive immune response, represented by T cells, recognizes peptide
epitopes presented by the major histocompatibility complex (MHC) molecules on self-cells
such as virus-infected cells. T cells are functionally divided into T-helper (Th) cells that act as
inducers of the immune response through the release of different cytokines and T-cytotoxic
(Tc) cells for exerting a predominantly cytotoxic function, both generally expressing specific
cell surface molecules CD4 and CD8, respectively.
Antigen recognition and its presentation by MHC molecules class I to CD8+
lymphocytes and class II to CD4+ lymphocytes are crucial for their proliferation and the
successful induction of an immune response to any pathogen [131,132]. Many viruses, as
BVDV, have developed strategies for interfering in these processes, compromising the
capacity of infected APCs and affecting the proliferative response of lymphocytes, as means
of immunosuppression. DCs are more resistant than monocytes to in vitro infection with NCP
strains; while DCs are not affected in their ability of viral antigen presentation to T cells,
monocytes infected with NCP strains show an altered presentation function that reduce the
proliferation of CD4+ T cells [122]. Some authors report a decrease in the expression of
MHC class II by monocytes infected with NCP [50,133] and CP strains [133]. In addition, a
decrease of MHC class I in monocytes infected by NCP and an increase in the case of CP has
been observed [50,122,133]. Accordingly, T cell proliferative responses appear later in NCP
BVDV infections than in CP [71,134].
In PI animals, infected monocytes do not seem to have affected their ability to present
antigens, being able to stimulate responses of CD4+ and CD8+ lymphocytes [50,135].
Th1/Th2 paradigm, postulated by Mosmann et al. (1986) from studies on cytokines produced
by T lymphocytes in a murine model, is less well defined in ruminants [136,137]. Th1
cytokines (IFNγ and IL-2) support Ms activation, generation of cytotoxic T cells, induction
of apoptosis and production of opsonizing antibodies, thereby enhancing resistance against
viral infections [112,138]. These chemical mediators are produced by NK cells, lymphocytes
CD8+ and CD4+ Th1 cells in response to IL-12 [130,139]. Th2 cytokines (IL-4, IL-5, IL-10
and IL-13) stimulate the production of IgE by B cells that cause mast cell degranulation and
activation of eosinophils, contributing to immune reactions in allergy and parasitic infections
[140]. IL-4 promotes the development of helper and cytotoxic T cells and the differentiation
of immunoglobulins-producing plasma cells from B cells [130]. This cytokine is produced in
response to antigen activation by CD4+ Th2 cells and some CD8+, NK1+ and γδT cells
[141]. IL-10 is a regulatory cytokine with anti-inflammatory effects produced by all subtypes
of Th cells in humans and cattle, inhibiting the activities initiated by proinflammatory
cytokines [112,142]. Studies on the type of immune response induced by BVDV do not
provide consistent results. While some authors have demonstrate the establishment of a Th1
response [121,143], others maintain that occur a Th2 type immune response associated with a
state of immunosuppression which might interfere with protective Th1 responses against
other pathogens [144]. Thus, experimental acute infections with NCP strains show an
impaired IFNγ response against Mycobacterium bovis and bovine herpesvirus-1 (BHV-1) that
would cause the inhibition of cellular immunity, diminishing host’s ability to contain these
pathogens at the site of entry [145,146].
138
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
4.2.2.2. Humoral Immune Response
B lymphocytes are genetically programmed to recognize a particular antigen, multiply
and differentiate giving rise to plasma cells that produce large amounts of immunoglobulins.
Humoral immunity may be caused by passive immunity for ingesting colostrum antibodies or
by an active immune response after exposure to antigens [130].
High levels of maternal antibodies can block B cell-mediated response to vaccination
with BVDV [147]. However, the vaccination appears to be effective, protecting the animal
against acute infections through a T cell-mediated and memory B cells response [148,149].
Neutralizing antibodies at the portal of entry are perhaps the most effective component of
anti-viral immunity, since they can neutralize the virus and prevent their entry into the host.
However, once the virus enters into the cell, cell-mediated immune response is critical for the
defense against most viral infections. So, the disappearance of BVDV in acute infection
cannot be attributed to the presence of specific antibodies, which have a moderate and
delayed response, not being detected until 2-4 weeks post-infection [37,44,50,146].
4.2.3. Apoptosis
Apoptosis, or programmed cell death, can prevent the replication and spread of the viral
infection, so many viruses have developed strategies to prevent this phenomenon. However,
apoptosis might also facilitate virus dissemination and several viruses have developed
potential mechanisms to activate the apoptotic pathway [150,151]. During the BVD, there is a
decrease in the number of lymphocytes of 50% in infections with strains of low virulence and
90% with high virulence strains [24,25,50,55,82,83,152]. Among T lymphocytes
subpopulations, the number of CD8+ is more reduced than the CD4+, with little affectation of
the γδT circulating cells [41,50,55]. This lymphopenia correlates with infection and lesions in
lymphoid tissues, since severe lymphoid depletion due to apoptosis is prominent in lymphoid
follicles of Peyer’s patches and, to a lesser extent, in thymic cortex [23,25,39,45,55,83,
85,111]. It has not yet been clarified whether these lesions are induced directly by the virus or
the immune response also contributes to its development. In this regard, different studies have
attributed lymphoid depletion to the direct action of BVDV on lymphocytes [23,25,37,45,82].
However, other studies report that this apoptosis occurs subsequent to the elimination of viral
antigen [45,82,83,153,154], suggesting that the cell death process would be mediated by
CD4+ and CD8+ T lymphocytes [82,155,156].
The existence of an indirect mechanism by which infected m-MΦs could play an
important role in apoptosis of T lymphocytes through the release of pro-apoptotic cytokines
[111,157,158], being the principal cause of lymphocyte apoptosis caused by other
pestiviruses, such as CSFV has also been indicated [159,160,161,162]. Thus, apoptosis of
MΦs and epithelial cells induced by the release of type I IFN from MΦs infected with CP
BVDV strains would contribute to the severe lesions observed in MD [47,106,163]. Other
studies in vitro suggest the possibility that only the highly virulent NCP strains could induce
the production by MΦs of these pro-apoptotic factors [133], a fact that rules out the direct
implication of virus replication in the lesions affecting lymphoid tissues [23,24,25] There are
two major regulatory pathways of apoptosis: the extrinsic pathway, which can be induced by
members of the TNF family of cytokine receptors, associated with the cleavage and activation
of caspase-8 [158,164]; and the intrinsic or mitochondria-dependent pathway, governed by
Bcl-2 family proteins [165,166], which promotes the cleavage and activation of the caspase-9
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
139
[167,168]. Both pathways induce apoptosis via activation of effector caspases such as
caspase-3, which, once activated, leads to irreversible cell death [111,169].
The activation of initiator caspase-8 (extrinsic pathway) seems to play a major role in
lymphocyte apoptosis within Peyer’s patches during infection with NCP BVDV strains
[157,158,170]. It has also been reported that the inactivation of caspase-9 accompanied by a
moderate expression of the anti-apoptotic Bcl-2 protein dictates the resistance of MΦs to
some apoptotic stimuli and favors the replication of BVDV within them [170,171,172]. NCP
BVDV replication undermines the biosynthetic functions of MΦs and impairs their
immunological role as effector cells against virus infection [111]. Furthermore, other studies
in vitro show that NCP strains are able to induce an apoptosis-inhibiting effect at the
mitochondrial level in cell culture [173]. This mechanism may be linked to the induction of
bcl-2 overexpression and the lack of increase in effector caspase-3, which would presumably
favor the establishment of persistent infections [174]. CP strains of BVDV induce apoptosis
in cell cultures [47,175], although this phenomenon is not necessary for their replication
[150]. Studies realized on the mechanisms involved in the phenomenon of apoptosis indicate
the activation of the intrinsic pathway [173,176], not ruling out the extrinsic [177], and
pointing to the great presence of virus in cells infected with CP strains as the key factor for
the induction of the intrinsic pathway [178].
4.3. Role of BVDV in the Bovine Respiratory Disease Complex (BRDC)
BRDC is characterized by a primary active viral infection with bovine respiratory viruses
as BVDV, BHV-1, bovine respiratory syncytial virus (BRSV) and parainfluenza-3 (PI-3) that
favors secondary bacterial infections. Bacterial pathogens implicated in this cattle pneumonia
include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni,
Arcanobacterium pyogenes and Mycoplasma bovis [179,180,181,182]. Several immune
evasion strategies have been developed by these respiratory pathogens that help themselves
and also other agents to establish the infection, resulting in an exacerbation of the disease.
BVDV is considered as the main predisposing factor for the occurrence of this process
through an alteration of the immune response, thus favoring the colonization by other
pathogens [181,183] (Figure 3). In this regard, there is evidence that combined infections with
BVDV have a potentiating effect on several pathogens, increasing in a more severe form of
the respiratory disease compared to calves infected only with rota- and coronavirus, [80,184],
BHV-1 [146,185,186], BRSV [41] and Mannheimia haemolytica [120,187].
In these processes, the alteration of pulmonary MΦs activity by respiratory viruses favors
calves susceptibility against secondary infections, since these cells play an important role in
nonspecific primary defense of the lung [102,188], through their phagocytic, microbicidal and
secretory functions [189]. Thus, it has been reported that BVDV infection of pulmonary
alveolar MΦs can lead to a decrease in the expression of Fc receptor and complement C3
required for their phagocytic activity, decreasing its antimicrobial activity and releasing
chemotactic factors [105,107,122,127,190]. The challenge for the animal in the BRDC is to
initiate an innate immune response in order to defeat the virulence mechanisms utilized by the
pathogens without eliciting extensive inflammation that can compromise lung function
[180,191]. Indeed, inflammatory cytokines seem to play a central role in the lung injury
produced during BRDC since high levels of these mediators have been found in the airways
140
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
of cattle infected with viruses or other respiratory pathogens [192,193,194,195]. BVDVinfected calves displayed a great secretion of TNFα and reduced production of IL-10
following BHV-1 infection, leading to an exacerbation of the inflammatory response that
contributes to leukocyte recruitment and activation to the site of infection, favoring viral
spread by increasing the number of target cells [146]. The study of immune evasion strategies
of BVDV should lead to a better understanding of the immune system and the interaction of
this virus with other respiratory pathogens in the host. This will help to treat virus-induced
pathology, identify new mechanisms for immune modulation, design vaccines and establish
control strategies.
Bovine herpesvirus 1
Bovine syncytial virus
Parainfluenza-3
Mannheimia haemolytica
BVDV
Pasteurella multocida
IMMUNOSUPPRESSION
Haemophilus somni
Arcanobacterium pyogenes
Mycoplasma bovis
Figure 3. Schematic depicting the outcome of acute BVDV infection, where the animal is predispose to
suffer from Bovine Respiratory Disease Complex (BRDC) upon infection with secondary viral and
bacterial pathogens.
References
[1]
[2]
[3]
[4]
[5]
P. Olafson, A. D. MacCallum and A. Fox, Cornell Vet. 36, 205 (1946).
F. K. Ramsey and W. H. Chivers, North Am. Vet. 34, 629 (1953).
R. G. Thomson and M. Savan, Can. J. Comp. Med. Vet. Sci. 27, 207 (1963).
R. Tremblay, Vet. Med. 91, 858–866 (1996).
T. R. Ames, Bovine Viral Diarrhea Virus: Diagnosis, Management and Control, Eds.
Goyal and Ridpath, Iowa, USA (2005).
[6] I. Greiser-Wilke, B. Grummer and V. Moenning, Biologicals 31, 113 (2003).
[7] D. Deregt, Bovine Viral Diarrhea Virus: Diagnosis, Management and Control, Eds.
Goyal and Ridpath, Iowa, USA (2005).
[8] S. R. Bolin and D. L. Grooms, Vet. Clin. Food. Anim. 20, 51 (2004).
[9] C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger and L. A. Ball, Virus
Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses, Eds.
Fauquet, Mayo, Maniloff, Desselberger and Ball, San Diego, USA (2005).
[10] J. F. Ridpath and S. R. Bolin, Virus Res. 50, 237 (1997).
[11] B. Grummer, M. Beer, E. Liebler-Tenorio and I. Greiser-Wilke, J. Gen. Virol. 82, 2597
(2001).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
141
[12] H. J. Thiel, P. G. W. Plagemann and V. Moennig, Field’s Virology, Eds. Fields, Knipe
and Hewley, Philadelphia, USA (1996).
[13] B. Lindenbach and C. Rice, Field’s Virology, Eds. Knipe, Howley, Griffin, et al.,
Philadelphia, USA (2001).
[14] J. F. Ridpath, Bovine Viral Diarrhea Virus: Diagnosis, Management and Control, Eds.
Goyal and Ridpath, Iowa, USA (2005).
[15] W. V. Corapi, R. D. Elliott, T. W. French, D. G. Arthur, D. M. Bezek and E. J. Dubovi,
J. Am. Vet. Med. Assoc. 196, 590 (1990).
[16] J. F. Ridpath, “Sequence diversity and genotyping”, International Symposium of
Bovine Viral Diarrhoea Virus: a 50 years Review, Cornell University, USA, pp. 39
(1996).
[17] R. O. Donis, Vet. Clin. North Am. Food Anim. Pract. 11, 393 (1995).
[18] J. F. Ridpath, S. R. Bolin and E. J. Dubovi, Virology 205, 66 (1994).
[19] F. X. Heinz, M. S. Collett, R. H. Purcell, E. A. Gould, C. R. Howard, M. Houghton, R.
J. M. Moorman, C. M. Rice and H. J. Thiel, Virus Taxonomy, Eds. Van Regenmortel,
Fauquet, Bishop, et al., New York, USA (2000).
[20] R. W. Fulton, J. F. Ridpath, A. W. Confer, J. T. Saliki, L. J. Burge and M. E, Payton,
Biologicals 31, 89 (2003).
[21] C. Pellerin, J. Vandenhurk, J. Lecomte and P. Tijssen, Virology 203, 260 (1994).
[22] S. Carman, T. Van Dreumel, J. Ridpath, M. Hazlett, D. Alves, E. Dubovi, R. Tremblay,
S. Bolin, A. Godkin and N. Anderson, J. Vet. Diagn. Invest. 10, 27 (1998).
[23] E. M. Liebler-Tenorio, J. F. Ridpath and J. D. Neill, Am. J. Vet. Res. 63, 1575 (2002).
[24] E. M. Liebler-Tenorio, J. F. Ridpath and J. D. Neill, Biologicals 31, 119 (2003).
[25] B. Stoffregen, S. R. Bolin, J. F. Ridpath, J. Pohlenz, Vet. Microbiol. 77, 157 (2000).
[26] R. O. Donis and E. J. Dubovi, J. Gen. Virol. 68, 1607 (1987).
[27] T. Lackner, A. Müller, A. Pankraz, P. Becher, H. J. Thiel, A. E. Gorbalenya and N.
Tautz, J. Virol. 78, 10765 (2004).
[28] T. Lackner, A. Müller, M. König, H. J. Thiel, and N. Tautz, J. Virol. 79, 9746 (2005).
[29] G. Meyers, N. Tautz, R. Stark, J. Brownlie, E. J. Dubovi, M. S. Collett and H. J. Thiel,
Virology 191, 368 (1992).
[30] N. Tautz, H. J. Thiel, E. J. Dubovi and G. Meyers, J. Virol. 68, 3289 (1994).
[31] B. M. Kümmerer, N. Tautz, P. Becher, H. Thiel and G. Meyers, Vet. Microbiol. 77, 117
(2000).
[32] D. M. Bezek, Y. T. Gröhn and E. J. Dubovi, Am. J. Vet. Res. 55, 1115 (1994).
[33] J. F. Evermann and J. F. Ridpath, Vet. Microbiol. 89, 129 (2002).
[34] R. W. Fulton, J. F. Ridpath, J. T. Saliki, R. E. Briggs, A. W. Confer, L. J. Burge, C. W.
Purdy, R. W. Loan, G. C. Duff and M. E. Payton, Can. J. Vet. Res. 66, 181 (2002).
[35] D. Grooms, J. C. Baker and T. R. Ames, Large Animal Internal Medicine, Ed. Smith,
St. Louis, MO, USA (2002).
[36] E. J. Rickey, “Bovine viral diarrhoea (BVD) in Beef Cattle”, Fact Sheet VM-56.
Department of Large Animal Clinical Sciences, College of Veterinary Medicine,
University of Florida, USA, pp. 1-4 (1996).
[37] C. L. Wilhelmsen, S. R. Bolin, J. F. Ridpath, N. F. Cheville and J. P. Kluge, Vet.
Pathol. 27, 235 (1990).
[38] K. V. Brock, Vet. Clin. North Am. Food Anim. Pract. 11, 549 (1995).
142
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
[39] M. Pedrera, P. J. Sánchez-Cordón, J. L. Romero-Trevejo, M. A. Risalde, I. GreiserWilke and J. C. Gómez-Villamandos, J. Comp. Path. 141, 52 (2009).
[40] L. N. D. Potgieter, Vet. Clin. North Am. Food Anim. Pract. 11, 501 (1995).
[41] B. W. Brodersen and C. L. Kelling, Am. J. Vet. Res. 59, 1423 (1998).
[42] C. Hamers, B. Couvreur, P. Dehan, C. Letellier, P. Lewalle, P. P. Pastoret and P.
Kerkhofs, Vet. J. 160, 250 (2000).
[43] J. Brownlie, Rev. Sci. Tech. Off. Int. Epiz. 9, 43 (1990).
[44] D. Müller-Doblies, A. Arquint, P. Schaller, P. M. Heegaard, M. Hilbe, S. Albini, C.
Abril, K. Tobler, F. Ehrensperger, E. Peterhans, M. Ackermann and A. Metzler, Clin.
Diagn. Lab. Immunol. 11, 302 (2004).
[45] D. J. Marshall, R. A. Moxley and C. L. Kelling, Vet. Pathol. 33, 311 (1996).
[46] M. Spagnuolo-Weaver, G. M. Allan, S. Kennedy, J. C. Foster and B. M. Adair, J. Vet.
Diagn. Invest. 9, 287 (1997).
[47] M. Lambot, E. Hanon, C. Lecomte, C. Hamers, J. J. Letesson and P. P. Pastoret, J. Gen.
Virol. 79, 1745 (1998).
[48] B. R. Cherry, M. J. Reeves and G. Smith, Prev. Vet. Med. 33, 91 (1998).
[49] G. P. David, T. R. Crawshaw, R. F. Gunning, R. C. Hibberd, G. M. Lloyd and P. R.
Marsh, Vet. Rec. 134, 468 (1994).
[50] D. Archambault, C. Beliveau, Y. Couture and S. Carman, Vet. Res. 31, 215 (2000).
[51] J. F. Ridpath and S. R. Bolin, Mol. Cell Probes 12, 101 (1998).
[52] L. Jones and L. Weber, J. Vet. Diagn. Invest. 13, 50 (2001).
[53] J. F. Evermann and G. M. Barrington, Bovine Viral Diarrhea Virus: Diagnosis,
Management and Control, Eds. Goyal and Ridpath, Iowa, USA (2005).
[54] W. C. Rebhun, T. W. French, J. A. Perdrizet, E. J. Dubovi, S. G. Dill and L. F. Karcher,
J. Vet. Intern. Med. 3, 42 (1989).
[55] J. A. Ellis, K. West, V. Cortese, S. L. Myers, S. Carman, K. M. Martin and D. M.
Haines, Can. J. Vet. Res. 62, 161 (1998).
[56] D. H. Schlafer, J. H. Gillespie, R. H. Foote, S. Quick, N. N. Pennow, E. P. Dougherty,
E. I. Schiff, S. E. Allen, P. A. Powers, C. E. Hall and H. Voss, Dtsch. Tierarztl.
Wochenschr. 97, 68 (1990).
[57] P. D. Kirkland, S. G. Richards, J. T. Rothwell and D. F. Stanley, Vet. Rec. 128, 587
(1991).
[58] D. J. Paton, S. Brockman and L. Wood, Br. Vet. J. 146, 171 (1990).
[59] M. D. Fray, D. J. Paton and S. Alenius, Anim. Reprod. Sci. 60-61, 615 (2000).
[60] M. R. McGowan, M. Kafi, P. D. Kirkland, R. Kelly, H. Bielefeldt-Ohmann, M. D.
Occhio and D. Jillella, Theriogenology 59, 1051 (2003).
[61] T. E. Wittum, D. M. Grotelueschen, K. V. Brock, W. G. Kvasnicka, J. G. Floyd, C. L.
Kelling and K. G. Odde, Perv. Vet. Med. 49, 83 (2001).
[62] M. J. Harding, X. Cao, H. Shams, A. F. Johnson, V. B. Vassilev, L. H. Gil, D. W.
Wheeler, D. Haines, G. J. Sibert, L. D. Nelson, M. Campos and R. O. Donis, Am. J. Vet.
Res. 63, 1455 (2002).
[63] S. M. Goyal, Bovine Viral Diarrhea Virus: Diagnosis, Management and Control, Eds.
Goyal and Ridpath, Iowa, USA (2005).
[64] J. T. Done, S. Terlecki, C. Richardson, J. W. Harkness, J. J. Sands, D. S. Patterson, D.
Sweasey, I. G. Shaw, C. E. Winkler and S. J. Duffell, Vet. Rec. 106, 473 (1980).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
143
[65] M. Stokstad and T. Loken, J. Vet. Med. B. Infect. Dis. Vet. Public Health. 49, 494
(2002).
[66] A. C. Odeon, C. L. Kelling, D. J. Marshall, E. S. Estela, E. J. Dubovi and R. O. Donis,
J. Vet. Diagn. Invest. 11, 221 (1999).
[67] K. V. Brock, Biologicals 31, 133 (2003).
[68] A. W. Confer, R. W. Fulton, D. L. Step, B. J. Johnson and J. F. Ridpath, Vet. Pathol.
42, 192 (2005).
[69] C. Muñoz–Zanzi, S. Hietala, M. Thurmond and W. Jonson, Am. J. Vet. Res. 64, 568
(2003).
[70] K. V. Brock, D. R. Lapin and D. R. Skrade, Theriogenology. 47, 837 (1997).
[71] L. S. Brackenbury, B. V. Carr and B. Charleston, Vet. Microbiol. 96, 337 (2003).
[72] J. C. Baker, Vet. Clin. North Am. Food Anim. Pract. 11, 425 (1995).
[73] H. Bielefeldt-Ohmann, Vet. Clin. North Am. Food Anim. Pract. 11, 447 (1995).
[74] R. F. Kahrs, Viral Diseases of Cattle, Ed. Kahrs, Ames, IA, USA (2001).
[75] H. Sentsui, T. Nishimori, R. Kirisawa and A. Morooka, Arch. Virol. 146, 993 (2001).
[76] N. P. Smirnova, H. Bielefeldt-Ohmann, H. Van Campen, K. J. Austin, H. Han, D. L.
Montgomery, M. L. Shoemaker, A. L. van Olphen, and T. R. Hansen, Virus Res. 132,
49 (2008).
[77] E. M. Liebler, J. Waschbusch, J. F. Pohlenz, V. Moennig and B. Liess, Arch. Virol. 3,
109 (1991).
[78] C. L. Wilhelmsen, S. R. Bolin, J. F. Ridpath, N. F. Cheville and J. P. Kluge, Am. J. Vet.
Res. 52, 269 (1991).
[79] C. Bruschke, K. Weerdmeester, J. Van Oirschot and P. Van Rijn, Vet. Microbiol. 64, 23
(1998).
[80] C. L. Kelling, D. J. Steffen, C. L. Topliff, K. M. Eskridge, R. O. Donis and D. S.
Higuchi, Am. J. Vet. Res. 63, 1379 (2002).
[81] U. Teichmann, E. M. Liebler-Tenorio and J. F. Pohlenz, Am. J. Vet. Res. 61, 174
(2000).
[82] E. M. Liebler-Tenorio, J. F. Ridpath and J. D. Neill, J. Vet. Diagn. Invest. 15, 221
(2003).
[83] E. M. Liebler-Tenorio, J. F. Ridpath and J. D. Neill, J. Vet. Diagn. Invest. 16, 388
(2004).
[84] C. L. Kelling, B. D. Hunsaker, D. J. Steffen, C. L. Topliff and K. M. Eskridge, Am. J.
Vet. Res. 68, 788 (2007).
[85] A. I. Raya, J. C. Gómez-Villamandos, P. J. Sánchez-Cordón and M. J. Bautista, Vet.
Pathol. doi number: 10.1177/0300985811414031 (2011).
[86] A. Da Silva, P. J. Sánchez-Cordón, M. Pedrera, J. L. Romero-Trevejo, M. J. Bautista
and J. C. Gómez-Villamandos, “Cytokines expression by pulmonary macrophages
populations during bovine viral diarrhea”, Proceedings of the 19th Meeting of the
Spanish Society of Veterinary Pathology, Murcia, Spain, p. 86 (2007).
[87] M. A. Risalde, J. C. Gómez-Villamandos, M. Pedrera, V. Molina, J. J. Cerón, S.
Martinez-Subiela and P. J. Sánchez-Cordón, Vet. J. doi number:
10.1016/i.tvil.2011.03.003 (2011).
[88] E. M. Liebler, C. Küsters and J. F. Pohlenz, Vet. Immunol. Immunopathol. 48, 233
(1995).
[89] A. Bielanski, T. Sapp and C. Lutze-Wallace, Theriogenology. 49, 1231 (1998).
144
[90]
[91]
[92]
[93]
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
D. L. Grooms, K. V. Brock and L. A. Ward, J. Vet. Diagn. Invest. 10, 125 (1998).
T. V. Baszler, J. F. Evermann and P. S. Kaylor, Vet. Pathol. 32, 609 (1995).
B. Fredriksen, C. Press, T. Loken and S. A. Odegaard, Vet. Microbiol. 64, 109 (1999).
E. M. Liebler-Tenorio, Bovine Viral Diarrhea Virus: Diagnosis, Management and
Control, Eds. Goyal and Ridpath, Iowa, USA (2005).
[94] B. Fredriksen, C. Press, T. Sandvik, S. A. Odegaard and T. Loken, Vet. Pathol. 36, 267
(1999).
[95] B. L. Njaa, E. G. Clark, E. Janzen, J. A. Ellis and D. M. Haines, J. Vet. Diagn. Invest.
12, 393 (2000).
[96] T. Shin and H. Acland, J. Vet. Sci. 2, 81 (2001).
[97] B. Liess, S. Orban, H. R. Frey and G. Trautwein, Dtsch. Tierarztl. Wochenschr. 94, 585
(1987).
[98] E. M. Liebler, J. Waschbüsch, J. F. Pohlenz, V. Moennig and B. Liess, Arch. Virol.
Suppl. 3, 109 (1991).
[99] E. M. Liebler-Tenorio and J. F. Pohlenz, J. Comp. Pathol. 117, 339 (1997).
[100] P. Henneke and D. T. Golenbock, J. Exp. Med. 199, 1 (2004).
[101] B. K.Coombes, Y. Valdez and B. B. Finlay, Curr. Biol. 14, 856 (2004).
[102] D. L. Laskin, B. Weinberger and J. D. Laskin, J. Leukoc. Biol. 70, 163 (2001).
[103] A. T. Ketelsen, D. W. Johnson and C. C. Muscoplat, Infect. Immun. 25, 565 (1979).
[104] H. Adler, B. Frech, P. Meier, T. W. Jungi and E. Peterhans, Biochem. Biophys. Res.
Commun. 202, 1562 (1994).
[105] H. Adler, T. W. Jungi, H. Pfister, M. Strasser, M. Sileghem and E. Peterhans, J. Virol.
70, 2650 (1996).
[106] B. Adler, H. Adler, H. Pfister, T. W. Jungi and E. Peterhans, J. Virol. 71, 3255 (1997).
[107] M. D. Welsh, B. M. Adair and J. C. Foster, Vet. Immunol. Immunopathol. 46, 195
(1995).
[108] K. Van Reeth and B. Adair, Pathol. Biol. 45, 184 (1997).
[109] D. Yamane, M. Nagai, Y. Ogawa, Y. Tohya and H. Akashi, Microbes Infect. 7, 1482
(2005).
[110] S. R. Lee, G. T. Pharr, B. L. Boyd and L. M. Pinchuk, Comp. Immunol. Microbiol.
Infect. Dis. 31, 403 (2008).
[111] M. Pedrera, J. C. Gómez-Villamandos, J. L. Romero-Trevejo, M. A. Risalde, V. Molina
and P. J. Sánchez-Cordón, J. Gen. Virol. 90, 2650 (2009).
[112] C. A. Biron and G. C. Sen, Field’s Virology, Eds. Knipe, Howley, Griffin, Martin,
Roizman and Straus, Philadelphia, USA, (2001).
[113] P. C. Heinrich, J. V. Castell and T. Andus, Interleukin-6 and the acute phase response,
Biochemical Journal. 265, 621 (1990).
[114] C. A. Dinarello, Am. J. Clin. Nut. 83, 447 (2006).
[115] P. D. Eckersall, Rev. Med. Vet. 151, 577 (2000).
[116] H.H. Petersen, J. P. Nielsen and P. M. Heegaard, Vet. Res. 35, 163 (2004).
[117] J. Le and J. Vilcek, Lab. Invest. 56, 234 (1987).
[118] K. Van Reeth, G. Labarque, H. Nauwynck and M. Pensaert, Res. Vet. Sci. 67, 47
(1999).
[119] J. Jensen and R. D. Schultz, Vet. Immunol. Immunopathol. 29, 251 (1991).
[120] C. Gånheim, C. Hultén, U. Carlsson, H. Kindahl, R. Niskanen and K. P. Waller, J. Vet.
Med. B Infect. Dis. Vet. Public Health. 50, 183 (2003).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
145
[121] B. Charleston B, L. S. Brackenbury, B. V. Carr, M. D. Fray, J. C. Hope, C. J. Howard
and W. I. Morrison, J. Virol. 76, 923 (2002).
[122] E. J. Glew, B. V. Carr, L. S. Brackenbury, J. C. Hope, B. Charleston, C. J. Howard, J.
Gen. Virol. 84, 1771 (2003).
[123] K. Palucka and J. Banchereau, Curr. Opin. Immunol. 14, 420 (2002).
[124] B. Charleston, M. D. Fray, S. Baigent, B. V. Carr and W. I. Morrison, J. Gen. Virol. 82,
1893 (2001).
[125] T. Collen, A. J. Douglas, D. J. Paton, G. Zhang and W. I. Morrison, Virology 276, 70
(2000).
[126] M. Schweizer and E. Peterhans, J. Virol. 75, 4692 (2001).
[127] E. Peterhans, T. W. Jungi and M. Schweizer, Biologicals 31, 107 (2003).
[128] N. P. Smirnova, H. Bielefeldt-Ohmann, H. Van Campen, K. J. Austin, H. Han, D. L.
Montgomery, M. L. Shoemaker, A. L. van Olphen and T. R. Hansen, Virus Res. 132, 49
(2008).
[129] R. A. Goldsby, T. K. Kindt, B. A. Osborne and J. Kuby, Immunology, Ed. Freeman and
Company, New York, USA, (2003).
[130] I. R. Tizard, Veterinary Immunology: An Introduction, Ed. Tizard, Philadelphia, USA,
(2008).
[131] J. W. Yewdell and A. B. Hill, Nat. Immunol. 3, 1019 (2002).
[132] E. W. Hewitt, Immunology. 110, 163 (2003).
[133] C. C. L. Chase, G. Elmowalid, A. A. A. Yousif, Vet. Clin. Food Anim. 20, 95 (2004).
[134] T. Collen and W. I. Morrrison, Virus Res. 67, 67 (2000).
[135] E. J. Glew and C. J. Howard, J. Gen. Virol. 82, 1677 (2001).
[136] T. R. Mosmann, H. Cherwinski, M. W. Bond, M. A. Giedlin and R. L. Coffman, J.
Immunol. 136, 2348 (1986).
[137] D. M. Estes and W. C. Brown, Vet. Immunol. Immunopathol. 90, 1 (2002).
[138] C. E. Samuel, J. Biol. Chem. 281, 8305 (2006).
[139] C. A. Hunter, Nat. Rev. Immunol. 5, 521 (2005).
[140] K. M. Murphy and S. L. Reiner, Nat. Rev. Immunol. 2, 933 (2002).
[141] E. Marcenaro, M. Della Chiesa, F. Bellora, S. Parolini, R. Millo, L. Moretta and A.
Moretta, J. Immunol. 174, 3992 (2005).
[142] S. Pestka, C. D. Krause, D. Sarkar, M. R. Walter, Y. Shi and P. B. Fisher, Annu. Rev.
Immunol. 22, 929 (2004).
[143] C. J. Howard, M. C. Clarke, P. Sopp and J. Brownlie, Vet. Immunol. Immunopathol. 32,
303 (1992).
[144] S. G. Rhodes, J. M. Cocksedseg, R. A. Collins and W. I. Morrison, J. Gen. Virol. 80,
1673 (1999).
[145] B. Charleston, J. C. Hope, B. V. Carr and C. J. Howard, Vet. Rec. 149, 481 (2001).
[146] M. A. Risalde, V. Molina, P. J. Sánchez-Cordón, M. Pedrera, R. Panadero, F. RomeroPalomo and J. C. Gómez-Villamandos, Vet. Immunol. Immunopathol. doi number:
10.1016/j.vetimm.2011.07.022 (2011).
[147] Ellis JA, West K, Cortese V, C. Konoby and D. Weigel, J. Am. Vet. Med. Assoc. 219,
3351 (2001).
[148] J. J. Endsley, J. A. Roth, J. F. Ridpath and J. Neill, Biologicals 31, 123 (2003).
[149] J. F. Ridpath, J. D. Neill, J. Endsley and J. A. Roth, Am. J. Vet. Res. 64, 65 (2003).
[150] M. Schweizer and E. Peterhans, J. Gen. Virol. 80, 1147 (1999).
146
María A. Risalde, Pedro J. Sánchez-Cordón, Miriam Pedrera et al.
[151] H. Everett and G. McFadden, Trends Microbiol. 7, 160 (1999).
[152] J. F. Ridpath, J. D. Neill, M. Frey and J. G. Landgraf, Vet. Microbiol. 77, 145 (2000).
[153] S. R. Bolin and J. F. Ridpath, Am. J. Vet. Res. 53, 2157 (1992).
[154] C. J. Bruschke, A. Haghparast, A. Hoek, V. P. Rutten, G. H. Wentink, P. A. van Rijn
and J. T. van Oirschot, Vet. Immunol. Immunopathol. 62, 37 (1998).
[155] S. Hahn, R. Gehri and P. Erb, Immunol. Rev. 146, 57 (1995).
[156] J. A. Ellis and C. Yong, Can. Vet. J. 38, 45 (1997).
[157] L. Zheng, G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch and M. J. Lenardo, Nature.
377, 348 (1995).
[158] H. R. Stennicke, J. M. Jürgensmeier, H. Shin, Q. Deveraux, B. B. Wolf, X. Yang, Q.
Zhou, H. M. Ellerby, L. M. Ellerby, D. Bredesen, D. R. Green, J. C. Reed, C. J.
Froelich and G. S. Salvesen, J. Biol. Chem. 273, 27084 (1998).
[159] J. C. Gómez-Villamandos, E. Ruiz-Villamor, M. J. Bautista, C. P. Sánchez, P. J.
Sánchez-Cordón, F. J. Salguero and A. Jover, J. Comp. Pathol. 125, 98 (2001).
[160] P. J. Sánchez-Cordón, S. Romanini, F. J. Salguero, E. Ruiz-Villamor, M. J. Bautista and
J. C. Gómez-Villamandos, J. Comp. Pathol. 127, 239 (2002).
[161] P. J. Sánchez-Cordón, S. Romanini, F. J. Salguero, E. Ruiz-Villamor, L. Carrasco and J.
C. Gómez-Villamandos, Vet. Pathol. 40, 254 (2003).
[162] P. J. Sánchez-Cordón, A. Núñez, F. J. Salguero, L. Carrasco and J. C. GómezVillamandos, J. Comp. Pathol. 132, 249 (2005).
[163] L. Perler, M. Schweizer, T. W. Jungi and E. Peterhans, J. Gen. Virol. 81, 881 (2000).
[164] D. M. Rasper, J. P. Vaillancourt, S. Hadano, V. M. Houtzager, I. Seiden, S. L. Keen, P.
Tawa, S. Xanthoudakis, J. Nasir, D. Martindale, B. F. Koop, E. P. Peterson, N. A.
Thornberry, J. Huang, D. P. MacPherson, S. C. Black, F. Hornung, M. J. Lenardo, M.
R. Hayden, S. Roy, and D. W. Nicholson, Cell Death Differ. 5, 271 (1998).
[165] S. Cory, D. C. Huang and J. M. Adams, Oncogene. 22, 8590 (2003).
[166] L. C. Miller and J. M. Fox, Vet. Immunol. Immunopathol. 102, 131 (2004).
[167] P. Li, D. Nijhawan, I. Budihardjo, S. M. Srinivasula, M. Ahmad, E. S. Alnemri, X.
Wang, Cell. 91, 479 (1997).
[168] E. A. Slee, M. T. Harte, R. M. Kluck, B. B. Wolf, C. A. Casiano, D. D. Newmeyer, H.
G. Wang, J. C. Reed, D. W. Nicholson, E. S. Alnemri, D. R. Green and S. J. Martin, J.
Cell Biol. 144, 281 (1999).
[169] B. Huppertz, H. G. Frank, J. C. Kingdom, F. Reister and P. Kaufmann, Histochem. Cell
Biol. 110, 495 (1998).
[170] M. Pedrera, J. C. Gómez-Villamandos, M. A. Risalde, V. Molina and P. J. SánchezCordón, J. Comp. Pathol. doi number: 10.1016/j.jcpa.2011.03.015 (2011).
[171] B Levine, Q. Huang, J. T. Isaacs, J. C. Reed, D. E. Griffin and J. M. Hardwick, Nature.
361, 739 (1993).
[172] J. C. Reed, Am. J. Pathol. 157, 1415 (2000).
[173] B. Grummer, S. Bendfeldt, B. Wagner and I. Greiser-Wilke, Virus Res. 90, 143 (2002).
[174] S. Bendfeldt, B. Grummer and I. Greiser-Wilke, Vet. Microbiol. 96, 313 (2003).
[175] J. F. Ridpath, S. Bendfeldt, J. D. Neill and E. M. Liebler-Tenorio, Virus Res. 118, 62
(2006).
[176] B. Grummer, V. Moenning and I. Greiser-Wilke, Dtsch. Tierarztl. Wochenschr. 105, 29
(1998).
[177] M. C. St-Louis, B. Massie and D. Archambault, Vet. Res. 36, 213 (2005).
Bovine Viral Diarrhea Virus: Biology, Clinical Forms and Pathogenesis
147
[178] V. B. Vassilev and R. O. Donis, Virus Res. 69, 95 (2000).
[179] F. M. Shahriar, E. G. Clark, E. Janzen, K. West and G. Wobeser, Can. Vet. J. 43, 863
(2002).
[180] P. D. Hodgson, P. Aich, A. Manuja, K. Hokamp, F. Roche, F. Brinkman, A. Potter, L.
A. Babiuk and P. J. Griebel, Comp. Funct. Genom. 6, 244 (2005).
[181] S. Srikumaran, C. Kelling and A. Ambagala, Anim. Health Res. Rev. 8, 215 (2008).
[182] R. W. Fulton, K. S. Blood, R. J. Panciera, M. E. Payton, J. F. Ridpath, A. W. Confer, J.
T. Saliki, L. T. Burge, R. D. Welsh, B. J. Johnson and A. Reck, J. Vet. Diagn. Invest.
21, 464 (2009).
[183] L. N. Potgieter, Vet. Clin. North. Am. Food Anim. Pract. 13, 471 (1997).
[184] R. Niskanen, A. Lindberg and M. Tråvén, Vet. J. 163, 251 (2002).
[185] L. N. Potgieter, M. D. McCracken, F. M. Hopkins and R. D. Walker, Am. J. Vet. Res.
Apr. 45, 687 (1984).
[186] G. Castrucci, M. Ferrari, V. Traldi and E. Tartaglione, Comp. Immunol. Microbiol.
Infect. Dis. 15, 261 (1992).
[187] L. N. Potgieter, M. D. McCracken, F. M. Hopkins and J. S. Guy, Am. J. Vet. Res. 46,
151 (1985).
[188] P. Zhang, W. R. Summer, G. J. Bagby and S. Nelson, Immunol. Rev. 173, 39 (2000).
[189] L. P. Nicod, Respiration. 66, 2 (1999).
[190] I. Liu, H. D. Lehmkuhl and M. L. Kaeberle, Can. J. Vet. Res. 63, 41 (1999).
[191] C. J. Czuprynski, Anim. Health Res. Rev. 10, 141 (2009).
[192] C. M. Røntved, K. Tjørnehøj, B. Viuff, L. E. Larsen, D. L. Godson, L. Rønsholt and S.
Alexandersen, Vet. Immunol. Immunopathol. 76, 199 (2000).
[193] C. Malazdrewich, T. R. Ames, M. S. Abrahamsen and S. K. Maheswaran, Vet. Pathol.
38, 297 (2001).
[194] C. Avraamides, M. E. Bromberg, J. P. Gaughan, S. M. Thomas, A. Y. Tsygankov and
T. S. Panetti, Am. J. Physiol. Heart Circ. Physiol. 293, 193 (2007).
[195] J. J. Rivera-Rivas, D. Kisiela and C. J. Czuprynski, Vet. Immunol. Immunopathol. 131,
167 (2009).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter X
Adult Mucosa Stomach of Domestic
Ruminants: Structural, Histochemical
and Immunocytochemical Study
Gaetano Scala and Lucianna Maruccio
Department of Biological Structures, Functions and Technologies,
University of Napoli Federico II, Faculty of Veterinary Medicine,
Via Veterinaria,1, Napoli, Italy
Abstract
The main objective of the present chapter was to better understand the role of the
domestic ruminants stomach in rumination. The fine structure of the mucosa was studied
in detail to clarify the function of the aliment absorption mechanism. The morphostructural, histochemical and immunocytochemical characteristics of the stomach in
domestic ruminant viz cattle, buffalo and sheep was investigated by means of light
microscopy (LM), transmission electron microscopy (TEM), scanning electron
microscopy (SEM) and immunogold-labeling SEM analysis. The stomach specimens of
both sexes were processed in different ways, depending on the type of the technique to be
used. Rumen - the mucosa was covered with a squamous, horny, stratified epithelium.
Many intercellular spaces were observed in the spinous and basal layers. These spaces
presented many intercellular digitations. On the macerated specimens, many folds, which
intersected each other, were observed in the lamina propria, thus giving rise to a mosaiclike structure. Reticulum – the mucosa epithelium was morphologically similar to the
mucosa rumen. The lamina propria presents well-developed muscularis mucosae.
Reticular groove – many glands were observed in the lamina propria. A large amount of
striated muscularis tissue was observed in the tunica muscularis; this tissue extended up
to the middle part of the reticular groove. Omasum- the tunica mucosa was
morphologically different from that of the rumen and reticulum. The intercellular spaces
in the spinous and basal layers were larger in the omasum than in the rumen and
reticulum. Abomasum- the tunica mucosa had many glands and was covered with a

Email: gaescala@unina.it.
150
Gaetano Scala and Lucianna Maruccio
simple columnar epithelium. The lamina propria contained the muscularis mucosae and
glandular adenomeres in the epithelium underlying. By means of the histochemical
method, an intense NADPHd staining in the granulous, spinous and basal layers was
observed. By means of immunogold-labeling, the immunoreactivity to NOS I was found
in all specimens of stomach mucosa of all species studied. Moreover, the
immunoreactivity to CD133 was observed in positive cells of the micro-papillae in the
bottom of the reticulum small polygonal cells and on the papillae of the rumen wall. In
conclusion, this data reflects a possible role of nitric oxide in the delaying the onset of
cellular apoptosis in the forestomach domestic ruminants, by playing a role in the
production of cell energy. Moreover, the presence of the stem cells refers to the renewing
of the reticulm micro-papillae and to reconstitute the damages of the rumen papillae
during the normal functional activity.
1. Introduction
The domestic ruminant stomach is divided into four compartments: rumen (Rumen),
reticulum (Reticulum), omasum (Omasum) and abomasum (Abomasum). The rumen, the
reticulum and the omasum form the forestomach (Proventriculus), which has a squamous
horn stratified epithelium of the mucosa.
The abomasum has the morpho-structural features similar to the stomach of the
monogastric species. The ruminant domestic forestomach plays an important role in the
digestion of the vegetable foods. Moreover, the physical and biochemical processes related to
rumination take place in the forestomach. The symbiotic bacteria found in the forestomach
carry out an intense fermentative activity and digest cellulose. Furthermore, the forestomach
is involved in other rumination mechanisms and in eructation related muscular activity.
Monogastric herbivores are also able to digest ingesta by means of the fermentative activity
of symbiotic bacteria.
The fermentative activity is less effective in monogastric herbivores than in domestic
ruminants that are able to digest bacteria in the abomasum. Many researchers [1,2,3,4,5] have
investigated the morphology, structure and innervation of the stomach of domestic ruminants.
More recently, the intrinsic innervation in the forestomach of the ovine rumen has been
investigated by means of immunohistochemical methods [6,7,8,9]. The aim of this paper is to
describe the morpho-functional role of the mucosa in each of the three forestomach
compartments in three domestic ruminant species (cattle, buffalo and sheep) using structural,
histochemical and immunocytochemical methods by means of transmission electron
microscopy (TEM), light microscopy (LM) and scanning electron microscopy (SEM). The
histochemical and immunocytochemical methods employed in this paper were chosen to
demonstrate the presence of nitric oxide (NO), which is an important signaling molecule in
the body of mammals that serves to facilitate the communication between neighboring cells
[10].
While the immunocytochemical method was chosen to demonstrate the eventually
presence of the stem cells in the stomach of these three domestic ruminant species,
employing: CD133 or Prominin 1, that is mainly marker to pluri-or-multipotent stem cells
[11].
Adult Mucosa Stomach of Domestic Ruminants
151
2. Materials and Methods
The stomachs of twelve adult cattle (Bos taurus), twenty-four adult water buffalo
(Bubalus bubalis) and six adult sheep (Ovis aries) of both sexes were collected immediately
after slaughter from a government slaughter house in Campania, Italy. Specimens were
collected from the atrium, dorsal and ventral sacs, and pillars of the rumen, the greater
curvature of reticulum and the laminae of the omasum and of the abomasum. The specimens
were processed in different ways, depending on the type of technique to be used.
2.1. Light Microscopy (LM)
The specimens were cut into numerous pieces (0.5 cm long) which were then fixed in
paraformaldehyde and picric acid (PAF) for 12-18 h, rinsed in a glucosate phosphate buffer
for 24-36 h, dehydrated, and pre-embedded twice in 12 h in a solution of 2-hydroxyethil
metacrylate (85 ml), 2-butoxyethanol (15 ml), and benzoyl peroxide (1g). Specimens were
embedded using a mixture of the pre-embedding solution and N, N-dimethylaniline (ratio
30:1).
After polymerization, all specimens were sectioned using a microtome (HistorangeLKB), stained with toluidine blue, and examined under a light microscope (Leitz Orthoplan,
Wetzlar, Germany).
2.2. Transmission Electron Microscopy (TEM)
The specimens were dissected into a small pieces fixed in cacodylate buffered
glutaraldehyde, post-fixed with 2% OsO4 for 2 h, dehydrated and embedded in an EM bed of
812 resin (Electron Microscopy Supplies, Fort Washington, PA, USA). All embedded
specimens were sliced into thin sections using an ultramicrotome (Ultratome IV-LKB,
Bromma, Sweden), stained with uranyl acetate and lead citrate (Ultrastain-LKB), examined
under a transmission electron microscope (Philips EM 201; Eindhoven, The Netherlands) at
40 kV and were photographed.
2.3. Scanning Electron Microscopy (SEM) - Intact Tissue Technique
The stomach specimens were each perfused through the intra-thoracic segment of aorta
discendens (Aorta toracica) with phosphate buffer 0.1 M, pH 7.3, to wash the blood vessels,
and then fixed with Karnovsky’s solution (4% paraformaldehyde, 2.5% glutaraldehyde).
After 12 h, each specimen was cut into numerous pieces (0.5 cm long), which were immersed
in a glucose phosphate buffer for 24-48 h, and dehydrated in ethyl alcohol and critical point
dryer (CPD 030, BALZERS, Liechtenstein).
The piece specimens were mounted on stubs (12.5 mm diameter), examined under SEM
(LEO 435 VP) at 25 kV, and photographed.
152
Gaetano Scala and Lucianna Maruccio
2.4. Scanning Electron Microscopy (SEM) – Maceration Technique
Some specimens, after fixation with Karnovsky’s, were partially macerated with a 15%
KOH solution from 30 min. After maceration, the specimens were immerged in a tannic acid
solution (5%), rinsed with tap water and then treated following the same steps of the others.
2.5. Histochemistry
The specimens were washed in 0.1 M PBS, transferred into a graded series of saccarose
(10%, 20%, 30%), immersed in Tissue Teck OCT compound (Sigma Chemical Co., St.
Louis, MO, USA), frozen in liquid nitrogen and sectioned by a cryostat. To evaluate the
NADPHd activity, sections were incubated with 0.25 mg/ml nitro blue tetrazolium, 1mg/ml
NADPH and 0.5 % Triton X-100 in 0.1 M Tris-HCl buffer, pH 7.3, at 37°C for 10-15 min in
a dark box or at room temperature for 30 min. The reaction was stopped by sample immersion
in 0.1 Tris-HCl buffer. Finally, sections were mounted on cover slips, examined under a light
microscope (Orthoplan; Leitz GmbH, Wetzlar, Germany) and photographed. Control sections
included incubation in media in which substrate was omitted, and pre-incubation with the
sulphydryl inhibitor, 5.5’-dithio-bis-2-nitrobenzoic acid. Neither of these controls produced
staining.
2.6. Immunogold-Labeling SEM Analysis
For the immunogold-labeling SEM analysis, the specimens were immediately immersed
in PBS for 1 h. Then they were incubated for 2 h in a solution containing normal goat serum
(X 0907; Dako Italia s.p.a., Milano, Italy) diluted 1:10 in PBS, and next with following
antibodies: a) rabbit primary polyclonal antibody raised against a peptide mapping the 724739 amino acid sequence of human brain NOS I (AB1632, Chemicon, Temecula, CA, USA),
diluted 1:1500 in PBS; b) rabbit primary polyclonal antibody raised against a peptide
mapping the 508-792 amino acid sequence of human CD133 (sc-30220 Santa Cruz, USA),
diluted 1:500 in PBS; overnight at 4°C. After washing in PBS, the specimens were incubated
with gold-conjugated goat anti-rabbit IgG (E.M.GAR10; Agar Scientific Limited, Stansted,
UK) diluted 1:200 in PBS for 1 h at room temperature. The secondary antibody was
conjugated with gold particles of different size, namely 5 and 15 nm. After washing in PBS,
specimens were fixed by 2.5% glutaraldehyde in 0.1 m cacodylate buffer containing CaCl2,
pH 7.2, for 30 min. After the fixation step and washings with distilled water, specimens were
subjected to silver enhancement (British BioCell International Cardiff, Wales, UK). The
enhancement process enables the use of antibodies conjugated to smaller (5-15 nm) gold
particles, preserving the advantage of faster penetration and higher labeling efficiency [12].
Next specimens were dehydrated through an ethanol series and dried to the critical point. The
specimens, mounted on stubs, were examined under a LEO 435 VP scanning electron
microscope at variable pressure (80-120 Pa) in the backscattered electron mode, which allows
detecting gold particles associated with cells even if they are located intracellular [13]. The
specimens had not been coated by gold, so that only conjugated gold deriving from
immunocytochemical reaction was observed by SEM and were photographed.
Adult Mucosa Stomach of Domestic Ruminants
153
2.7. Western Blotting
The specimens of the stomach removed from all species studied were homogenized with
an Ultraturrax l-407 at 4°C with 5 ml/1.5g tissue of buffer containing 50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10 mm NaF, 0.5% deoxycholic
acid, 0.1% sodium dodecyl sulphate (SDS), 15 Nonited P-40, 1mM phenylmethylsulfonyl
fluoridre, 1.1 U/ml aprotinin, 10mg/ml leupeptin, and 1 mM Na3VO4. Homogenates were
centrifuged at 15,000- g for 20 min at 4°C. Supernatants were divided into small aliquots and
stored at -80°C until used. The amount of total proteins in each sample was determined by the
Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples containing
equal amount of proteins were boiled for 5 min in SDS sample buffer (50 mM Tris-HCl, pH
6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue and 5% b-mercaptoethanol) and run on
a 10% SDS/polycrylamide gel. After electrophoresis, the proteins were transferred to
nitrocellulose using a Mini trans-blot apparatus (Bio-Rad Laboratories) according to the
manufacturer’s instructions. Membranes were blocked for 1 h at room temperature with TBST buffer (150 mM NaCl, 20 mM Tris HCl, pH 7.4, 0.1% Tween 20) containing 5% milk. The
blots were incubated overnight with following antibodies: a) rabbit polyclonal antibody raised
against a peptide mapping the 724-739 amino acid sequence of human brain nitric oxide
synthase I (NOS I) (AB1632; Chemicon International Inc.), diluted 1:1,000 in TBS-T
containing 2.5% milk, b) rabbit polyclonal antibody raised against a peptide mapping the 508792 amino acid sequence of human CD133 (Santa Cruz) diluted 1:1,000 in TBS-T containing
2.5% milk. After incubation, the membranes were washed three times with TBS-T and
incubated for 1 h with horseradish peroxidase-conjugated anti- rabbit IgG (Sigma Chemical)
diluted 1:5,000 in TBT-T containing 2.5% milk. The proteins were visualized by enhanced
chemilumenescence (ECL; Amersham, Little Chalfont, UK). The same blots were stripped
and reprobed using anti-tubulin monoclonal antibody (MAB 1637; Chemicon International
Inc.) to confirm equal loading of proteins in all lanes. All nomenclature in this work was
adopted from the Nomina Anatomica Veterinaria, Nomina Histologica and Nomina
Embryologica Veterinaria (World Association of Veterinary Anatomists, 2005).
3. Results
3.1. Gross Anatomy of the Domestic Ruminant Stomach
The domestic ruminant stomach occupies more than half of the abdomen cavity:
1. The rumen is located on the left side of the median sagittal plane;
2. The reticulum is cranially positioned;
3. The omasum and the abomasum are located on the right side.
3.1.1. Rumen Gross Anatomy
The rumen forms a large sac that extends ventro-caudally on the left side of the middle
sagittal plane from the diaphragm to the pelvic cavity. The left surface of the rumen (Facies
parietalis) is convex and is related to the left abdominal wall, the diaphragm and liver (Figure
154
Gaetano Scala and Lucianna Maruccio
1). The right surface of the rumen (Facies visceralis) is related to the omasum, abomasum,
liver, pancreas and gut (Figure 2). The dorsal curvature of the rumen is related to the sublumbar region and the ventral curvature lies on the abdomen floor. The extremities of the
rumen are cranial and caudal. The right surface has one right longitudinal groove (Sulcus
longitudinalis dexter) and the left surface has one left groove (Sulcus longitudinalis sinister).
Figure 1. The schematic representation of the domestic ruminant stomach. Left surface. R=Esophagus;
Re=Reticulum; Ru=Rumen; S=Reticular groove.
Figure 2. The schematic representation of the domestic ruminant stomach. Right surface.
Ab=Abomasum; O=Omasum; P=Pylorus; R=Esophagus; Ru=Rumen.
These grooves divide the rumen into dorsal (Saccus dorsalis) and ventral (Saccus
ventralis) sacs. Each longitudinal groove has an accessory groove (Sulcus accessorius dexter
and sinister). These two accessory grooves are dorso-caudally directed, but only the right one
reconnects with its longitudinal groove, thus forming the rumen island (Insula ruminis). A
transverse caudal groove (Sulcus caudalis) divides the caudal extremity of the rumen into
dorsal (Saccus caecus caudodorsalis) and ventral (Saccus caecus caudoventralis) blind sacs.
It is connected on both ends to the longitudinal grooves. The blind sacs are separated from the
dorsal and ventral sacs by dorsal (Sulcus coronarius dorsalis) and ventral (Sulcus coronarius
Adult Mucosa Stomach of Domestic Ruminants
155
ventralis) coronary grooves. A cranial groove (Sulcus cranialis) divides the ventral sac and
the ruminal atrium (Atrium ruminis) at the cranial extremity of the rumen. The atrium, in turn,
lies between the reticulum and ventral sac; it is separated from the reticulum by the reticular
groove (Sulcus reticuli). The cranial end of the dorsal sac is contiguous with the ruminal
atrium. The cranial end of the ventral sac is called the ruminal recess (Recessus ruminis).
3.1.2. Reticulum Gross Anatomy
The reticulum is pyriform and slightly flattened cranio-caudally. It is related to the
ruminal atrium and its cranial surface lies on the diaphragm concavity. The reticulum has a
greater curvature (Curvatura major) and a lesser curvature (Curvatura minor): the form is
ventral and lies on the sternal region, and the latter is dorsal. The diaphragmatic surface of the
reticulum is related to the liver; the visceral surface is related to the rumen; and the ventral
part (Fundus reticuli) is related to the abomasum.
3.2.3. Gastric Groove Gross Anatomy
In both monogastrics and ruminants, there is a continuous pathway (Sulcus ventriculi)
from the cardiac portion (Pars cardiaca) to the pylorus.
In monogastrics, the gastric groove is located on the inner side of the lesser curvature and
is shorter than in ruminants. In ruminants, it passes through the reticulo-omasal and omasoabomasal orifices, thus forming the central axis of the stomach. The gastric groove of
ruminants is divided into three parts: the first part, called the reticular groove, runs from the
cardiac portion to the reticulo-omasal orifice and opens to the reticular and ruminal cavities;
the second part (Sulcus omasi) runs from reticulo-omasal to omaso-abomasal orifice and
opens to the omasal cavity; and the third part (Sulcus abomasi) runs along the lesser curvature
of the abomasum (Figure 3).
Figure 3. The schematic representation of the gastric groove in the domestic ruminant stomach.
Ab=Abomasum; O=Omasum; P=Pylorus; R=Esophagus; Re=Reticulum; Ru=Rumen.
156
Gaetano Scala and Lucianna Maruccio
3.2.4. Omasum Gross Anatomy
The omasum is located on the right side of the diaphragm concavity; it is slightly
flattened laterally and is quasi-ellipsoid in shape. The parietal surface (Facies parietalis) of
the omasum is related to the liver, diaphragm and a small tract of the abdominal wall. The
visceral surface (Facies visceralis) is related to the ventral sac of the rumen and the intestine.
The greater curvature (Curvature omasi) is dorsally positioned and related to the liver and
diaphragm. The base (Basis omasi) is cranio-ventrally positioned and related to the reticulum
and abomasum.
3.2.5. Abomasum Gross Anatomy
The abomasum is the glandular section of the ruminant stomach. It lies on the abdomen
floor immediately to the right of the rumen ventral sac; it is elongated and sacciform, and has
a thin distal extremity. The greater curvature (Curvatura major) is ventrally positioned and
connects to the greater omentum; the lesser curvature (Curvatura minor) is dorsally
positioned and connects to the lesser omentum. The parietal surface is related to the abdomen
wall and the visceral surface is related to the rumen and omasum. The abomasum is divided
into three parts: the fundus (Fundus abomasi), the body (Corpus abomasi) and the pylorus
(Pars pylorica). The pylorus is thinner than fundus; it curves dorsally and connects to the
duodenum.
3.2. Structure of the Domestic Ruminant Stomach
The examination of the specimens of the ruminant stomach revealed many particular
structural and ultra-structural features, which varied according to the stomach regions.
3.2.1. Rumen
3.2.1.1. Rumen Structure
The ruminal pillars (Pilae ruminis) were located on the inner surface of the rumen and
corresponded to the external grooves. The pillars were very thick; they were formed by the
depressions of the ruminal surface corresponding to thickness areas behind the external
grooves of the ruminal muscular tissue.
The domestic ruminant rumen had right and left longitudinal pillars, right and left
accessory pillars, cranial and caudal pillars and dorsal and ventral coronary pillars. All of
these pillars divided the ruminal cavity into several sacs. The ruminal mucosa was browncolored and covered with many papillae (Papillae ruminis). These papillae varied in size: the
largest papillae were observed in the atrium, and in the ventral and blind sacs (Figure 4); the
smallest papillae were located in the dorsal sac (Figure 5). There were no papillae on the
mucosa of the pillars. The mucosa of the pillars appeared smooth to the unaided eye, but, in
fact, it was covered with many small grooves, which intersected each other. Since the papillae
vary in size, shape and density, it was difficult to classify them either cylindrical, or roundshaped. All of the morphological types of papillae were to be found throughout the ruminal
mucosa, but the rounded ones were the most widespread. In young animals, the papillae were
smaller than in adults.
Adult Mucosa Stomach of Domestic Ruminants
157
Figure 4. Buffalo rumen. Papillae of the ventral sac.
Figure 5. Buffalo rumen. Papillae of the dorsal sac.
3.2.1.2. Rumen Ultrastructure
The ruminal mucosa was covered with a squamous, horny, stratified epithelium
(Epithelium stratificatum squamosum cornificatum). The LM showed that the epithelium was
tall and had several flat superficial layers. The cells of the basal and spinous layers were
larger than those of the superficial layers; but the cells of the superficial layers had closer
intercellular relationships. Along the border between the epithelium and the lamina propria
(Lamina propria mucosae), there were many alternating epithelial ridges and papilliform
connective protuberances. In the ruminal papillae, the papilliform connective protuberances
penetrated deeply into the epithelium and formed the connective cores of the papillae. These
cores contained blood vessels and nerves, and supported the papillae (Figure 6).
The SEM and TEM showed that the epithelium was composed of several layers of cells.
The SEM showed the elongated ruminal papillae (Figure 7), which presented on the apical
surface many micro-organisms (Figures 8, 9). At TEM, the horny layer had 2-6 rows of
squamous cells (Stratum corneum). These cells had thin intercellular spaces and no nuclei.
The intercellular spaces contain micro-organisms and short micro-ridge-like cellular
protrusions (Figure 10). The granulous layer (Stratum granulosum) was composed of 3-4
rows of elongated, densely-packed cells. The cells of spinous layer (Stratum spinosum) were
arranged in several rows, and each cell was polygonal and had many cytoplasmic digitations.
158
Gaetano Scala and Lucianna Maruccio
These digitations were separated by intercellular spaces containing a dark, granulous matter,
and many desmosomes (Figure 11).
Figure 6. LM papilla of the buffalo rumen. Bc=Blood capillary; E= Mucosa epithelium; Lp= Lamina
propria of the mucosa.
Figure 7. SEM buffalo rumen. Each ruminal papilla has a pedicle-like base.
Adult Mucosa Stomach of Domestic Ruminants
159
Figure 8. SEM inner surface of the buffalo rumen. Note the large number of the ruminal microorganisms.
Figure 9. SEM inner surface of the buffalo rumen. Enlargement of the ruminal micro-organisms.
Figure 10. TEM buffalo rumen. Cells of the epithelial superficial layer. M=Micro-organism; R=Short
microridge-like cellular protrusion.
160
Gaetano Scala and Lucianna Maruccio
Figure 11. TEM buffalo rumen. Cells of the epithelial spinous layer. D=Desmosome.
Figure 12. TEM buffalo rumen. Cells of the epithelial basal layer. Mt=Mithocondria.
Figure 13. TEM buffalo rumen. The mithocondria, which contains intra-mitochondrial matrix granules.
Gr=Intra-mitochondrial matrix granules.
Adult Mucosa Stomach of Domestic Ruminants
161
Figure 14. TEM buffalo rumen. The intercellular spaces. Cd=Cytoplasmic digitations; Hd=Hemidesmosome.
Figure 15. SEM buffalo rumen. The partial macerated papillae. Mo=Mosaic-like structures of the
lamina propria connective.
Figure 16. SEM buffalo rumen. The partial macerated inter-papillar space. Pc=Papillae of the lamina
propria connective.
162
Gaetano Scala and Lucianna Maruccio
Figure 17. SEM cattle rumen. The partial macerated mucosa of the dorsal ruminal sac. Pc=Papillae of
the lamina propria connective.
Figure 18. SEM buffalo rumen. Enlargement of the connective papillae of the partial macerated ruminal
papilla. Pc=Papillae of the lamina propria connective.
Moreover, these cells had many mitochondria in the cytoplasm over their nuclei. The
matrix of many mitochondria contained highly electron-dense intra-mitochondrial matrix
granules (Figures 12, 13). The basal layer (Stratum basale) was composed of a single row of
cells. These cells were tall, had many basal cytoplasmic digitations and were joined the
lamina propria by many hemi-desmosomes (Figure 14). The cytoplasmic digitations varied in
length and formed canaliculi of size different. Some of these digitations were connected to
neighboring cells by desmosomes.
Adult Mucosa Stomach of Domestic Ruminants
163
Figure 19. SEM buffalo rumen. Lamina propria structure.
In the papillae, the lamina propria was composed of dense connective tissue containing
collagen and elastic micro-fibrils and cells. In the inter-papillar spaces, the lamina propria
was also composed of dense connective tissue, and was divided into superficial and deep
layers. A small number of smooth muscular cells were observed in the deep layer, but they
did not form true muscularis mucosae (Lamina muscularis mucosae). On the macerated SEM
specimens, many folds were observed in the lamina propria. These folds intersected each
other and formed a mosaic-like structure (Figure 15). In some areas of the macerated ruminal
mucosa, the connective papillae of the lamina propria similar to flowers were present (Figures
16, 17, 18). The lamina propria was composed of fine collagenous and elastic fibrils that were
loosely packed (Figure 19).
The sub-mucosa (Tela submucosa) was thick and composed of loose connective tissue
containing collagen and elastic micro-fibrils, cells and many vessels and nerves. The
muscularis mucosae was not found, thus making it difficult to distinguish the border between
the tunica sub-mucosa and the lamina propria. A large amount of sub-mucosal connective
tissue penetrated into the tunica muscularis.
The tunica muscularis (Tunica muscularis) was composed of several layers. Large
amounts of connective tissue were observed between the fibro-muscular bundles of the inner
and outer layers. This connective tissue separated the inner layer from the outer one and
contained many blood vessels and nerves. The tunica serosa (Tunica serosa) composed of a
thin layer of connective tissue and was covered with a mesothelium. The serous connective
tissue adhered to the underlying tunica muscularis by means of the sub-serosa. This adherence
did not occur in the grooves of the external surface because these grooves were filled with
loose connective tissue containing blood vessels and nerves, hence causing the tunica serosa
to bridge them. There was no tunica serosa in the areas where the dorsal sac was in contact
with the abdominal wall and spleen. The tunica serosa contained many blood vessels and
nerves.
164
Gaetano Scala and Lucianna Maruccio
3.2.2. Reticulum
3.2.2.1. Reticulum structure
The reticular cavity is connected to the rumen by means of a large rumino-reticular
orifice (Ostium rumino-reticulare). The reticular mucosa was covered with a squamous,
horny stratified epithelium and had many folds (Cristae reticuli). These folds varied in height
and intersected each other, thus forming different polygonal cells (Cellulae reticuli). The
larger of these cells were subdivided by smaller folds and formed smaller cells. These smaller
cells, in turn, were also subdivided in smaller folds, finally resulting overall in 4 size orders of
folds and cells (Figure 20). The largest and deepest cells were found on the fundus and
greater curvature. The floors and the surfaces of the cells, and the free borders of the folds
were studded with either pointed or rounded small, horny papillae. The surfaces of the larger
folds were covered with small protuberances, which were arranged in parallel micro-grooves.
These rows and micro-grooves were perpendicular to the free border of the folds. The folds
became smaller and gradually disappeared as they approached the reticular groove.
3.2.2.2. Reticulum Ultrastructure
The tunica mucosa of the reticulum was covered with a squamous, horny, stratified
epithelium (Epithelium stratificatum squamosum cornificatum). This epithelium was
morphologically similar to the one observed in the rumen. The cells of the horny and
granulous layers were flat.
The cells of the horny layer formed squamae, and those of the basal and spinous layers
were connected to each other by cytoplasmic digitations. The TEM specimens showed these
digitations forming various sized intercellular spaces; the SEM specimens showed the tunica
mucosa studded with pointed micro-papillae. These micro-papillae were on the average 100
m long, and 15-20 m wide at the base (Figure 21). The macerated SEM specimens showed
the micro-papillae composed of a connective core covered with epithelium (Figures 22, 23).
Figure 20. Buffalo reticulum. Inner surface.
Adult Mucosa Stomach of Domestic Ruminants
Figure 21. SEM buffalo reticulum. Reticular cellulae.
Figure 22. SEM buffalo reticulum. Partial macerated reticular cellulae.
Figure 23. SEM buffalo reticulum. Section of the partial macerated reticular cellulae.
165
166
Gaetano Scala and Lucianna Maruccio
The lamina propria was composed of dense connective tissue, which was very thick
within the reticular wall and the distal part of the reticular folds. The lamina propria of the
upper and middle parts of the reticular folds formed the fibro-muscular bundles of the
muscularis mucosae. The muscularis mucosae of the large and medium folds had two layers
of fibro-muscular bundles. These two layers lay side by side; they ran both longitudinally
along the wall of each fold and concentrically around each cell. There was no muscularis
mucosae at the base of the folds, and hence no connection to the tunica muscularis of the
reticular wall. A dense blood capillary network was found throughout the entire lamina
propria, and an even more dense network was located in the sub-endothelial area. The submucosa was composed of loose connective tissue containing collagen and elastic microfibrils, and cells. It was located within the wall of the reticulum and the distal part of the
larger folds. The sub-mucosa extended all around the fibro-muscular bundles of the inner
muscular layer and contained many vessels and nerves. The tunica muscularis was composed
of two or three layers (longitudinal, circular and oblique) depending on their location. A large
amount of connective tissue containing blood vessels and nerves was found between the
muscular layers. The tunica serosa covered the entire free surface of the reticulum. It was
composed of loose connective tissue containing blood vessels and nerves.
3.2.3. Reticular Groove
3.2.3.1. Reticular Groove Structure
The reticular groove (the first part of the gastric groove) began at the cardiac portion,
passed ventrally along the right wall of the ruminal atrium and reticulum and ended at the
reticulo-omasal orifice.
Figure 24. Buffalo forestomach. Reticular groove.
Adult Mucosa Stomach of Domestic Ruminants
167
Along this route, the reticular groove rotated once around its axis. The groove was 10 to
20 cm long, and had right and left lips (Labium dextrum, sinistrum) and a floor (Fundus sulci
reticuli). These lips were very thick and were formed by the distal projection of the cardiac
portion (Figure 24). They delimited the floor and were covered with a brown, wrinkled,
tunica mucosa.
They also had small papillae on the ruminal side, which were similar to those located
near the ruminal pillars. On the reticular side, the lips had small and medium sized folds. The
tunica mucosa on the floor and inner surface of the lips was pale and smooth, and marked by
longitudinal folds, thus resembling the tunica mucosa of the esophagus. The tunica mucosa of
the floor had pointed horny papillae (Papillae unguicoliformes) on its ventral part near the
reticulo-omasal orifice.
3.2.3.2. Reticular Groove Ultrastructure
The tunica mucosa of the lips was histologically and ultra-structurally similar to that of
the rumen and reticulum. The tunica mucosa of the floor and inner surface of the lips had
morphological characteristics, which differed from those of the esophagus and proventricular
cavities. The lamina propria, composed of dense connective tissue, was thick within the floor
and inner sides of the lips and contained a thin and incomplete muscularis mucosae. Since the
sub-mucosa was very thin, or even lacking in some areas of the lips, the lamina propria lays
directly on the tunica muscularis. Many mucous glands were observed in the lamina propria
of the floor and the inner sides of the lips. These glands were tubulo-acinar along the entire
length of the reticular groove and were numerous in the proximal part. The sub-mucosa was
composed of loose connective tissue containing a dense blood vascular network and a welldeveloped plexus of nerves. These nerves were primarily found in the lips. The sub-mucosa
was either very thin or absent in the lips, and very thick in the floor; where it formed many
mucosal folds. The tunica muscularis was composed of longitudinal and circular layers, and
was extensively connected to the tunica muscularis of the reticulum and the rumen. There was
a large amount of connective tissue between the longitudinal and circular layers. The
muscular tissue of the lips was very thick. It was longitudinally directed and ran from the
cardiac portion to the reticulo-omasal orifice. Striated fibro-muscular bundles ran from the
esophagus and cardiac portion to the middle part of the reticular groove, some of these
bundles extended to the neighboring ruminal and reticular areas. These bundles were
disorderly arranged: some were fan-shaped spreading out over the outer layer of the tunica
muscularis; others penetrated into the smooth muscularis tissue of the tunica muscularis.
3.2.4. Omasum
3.2.4.1. Omasum Structure
The omasum mucosa was covered with a squamous horny stratified epithelium
(Epithelium stratificatum squamosum cornificatum) and had many folds (Laminae omasi) of
differing lengths. These folds were tightly packed; they arose from the omasum greater
curvature and the lateral walls, and went towards, but did not reach, the omasum base.
168
Gaetano Scala and Lucianna Maruccio
Figure 25. Buffalo omasum. Op=Papillae of omasal folds.
Each fold had two faces, a border joining the omasum wall, and a free border. The free
border was thick, slightly concave and shorter in length than the joined border. Many horny
papillae were observed on the faces (Figure 25); they were very tall near the reticulo-omasal
orifice. The folds were divided according to their height as follows: high, or first order;
medium, or second order; short or third order; and very short, or fourth order. They were
arranged in regular series or cycles of the following sequence of folds: first order, fourth
order, third order, fourth order, second order, fourth order, third order, fourth order, and first
order (1st, 4th, 3th, 4th, 2nd, 4th, 3th, 4th, 1st).
Deep narrow spaces filled with minced food were observed between the folds. These
spaces together with their corresponding folds occupied almost the entire omasal cavity. The
omasal canal (Canalis omasi), which was located at the omasal base and opened towards the
omasal cavity, ran from the reticulo-omasal to the omaso-abomasal orifice. The omasal canal
was, limited by two lips and had many high pointed papillae near the reticulo-omasal orifice.
Two mucosal folds (Vela abomasica) were observed at the omaso-abomasal orifice. These
two folds delimited the omaso-abomasal orifice and marked the passage between the omasal
and the abomasal mucosae.
3.2.4.2. Omasum Ultrastructure
The horny and granulous layers of the omasum epithelium formed a dense superficial
zone. A deep zone composed of spinous and basal layers contained many intercellular spaces.
These spaces extended up to the lamina propria (Figure 26).
On the SEM specimens, many papillae differing in size and morphology were observed
on the mucosa of the omasal canal and folds (Figures 27, 28). They were either: a) pointed
with a wide base on the omasal canal; b) short and rounded on the faces of the folds, and c)
high and pointed near the joined border of the folds and the reticulo-omasal orifice. On the
partially macerated SEM specimens, the lamina propria, after having the epithelium removed,
Adult Mucosa Stomach of Domestic Ruminants
169
had a wrinkled appearance due to the presence of many pointed protuberances. These
protuberances formed the connective cores of the mucosal papillae. Each core, in turn, had
many small protuberances on its lateral surfaces, which pointed towards the apex. These
small protuberances made impressions on the papillary epithelial sheaths (Figure 29). The
horny and granulous layers of the omasum epithelium were morphologically similar to those
of the rumen and reticular epithelium. On the TEM specimens, the horny layer was composed
of several rows of squamous cells and had small, thin intercellular spaces. The granulous
layer was composed of 3-4 rows of cells joined together by many desmosomes. The spinous
layer was formed of several rows of polyhedral cells having many cytoplasmic digitations
(Figure 30).
Between these digitations, there were large spaces, which contained a weak electrondense material. The basal layer was composed of a single row of tall cells having many
cytoplasmic digitations. Large intercellular spaces extending up to the basement membrane
were found between these digitations. The basal cells were joined to the underlying lamina
propria by many hemi-desmosomes. Many long cytoplasmic digitations joined to the
digitations of their neighboring cells were found in the larger intercellular spaces. Many large
blood capillaries having a fenestrated endothelium were observed in the lamina propria
underneath the basal layer of the epithelium.
On the vascular corrosion casts of the omasum mucosa, a dense blood vascular network
forming several V-shaped capillary loops was observed. These capillary loops extended
underneath the epithelium, thus increasing the exchange surface area between the blood and
the surrounding tissues. The muscular tissue of the folds was composed of fibro-muscular
bundles differing in origin and distribution. Some of these bundles arose from the tunica
muscularis of the omasum, and were arranged parallel to the fold axes and extended up to the
fold free border.
Figure 26. LM mucosa of the buffalo omasum. E=Mucosa epithelium; Lp= Lamina propria of the
mucosa.
170
Gaetano Scala and Lucianna Maruccio
Figure 27. SEM buffalo omasum. Op=Papillae of the omasal fold.
Figure 28. SEM buffalo omasum. Enlargement of the omasal papillae.
Figure 29. SEM buffalo omasum. Op=Papillae of the partial macerated fold.
Adult Mucosa Stomach of Domestic Ruminants
171
Figure 30. TEM buffalo omasum structure. B=Basal layer of the mucosa epithelium; Bc=Blood
capillary of the lamina propria.
Other bundles arose from the muscularis mucosae, and were arranged parallel to the
length of the folds and external to the bundles of the tunica muscularis. Even though the
fibro-muscular bundles of the folds differed in origin and distribution, they were connected to
each other at the free border. In addition, the fibro-muscular bundles of both muscular
systems were irregularly interspersed among each other in the medium tract and near the free
border of the folds. In the smallest folds, the only fibro-muscular bundles observed were those
originating from the muscularis mucosae. Many blood vessels giving rise to a dense subepithelial vascular network were found within the muscular tissue of the folds.
The tunica sub-mucosa was composed of loose connective tissue containing many blood
vessels and nerves, and it sent septa to the underlying tunica muscularis. The tunica
muscularis was composed of outer longitudinal and inner circular layers. It was very thick at
the omasal base, and thicker than the tunica muscularis of the reticular wall. Since the
patterns of distribution of the fibro-muscular bundles were very irregular in some zones of the
omasal wall, it was difficult to individuate between the two muscular layers. The inner
circular layer sent fibro-muscular bundles to the folds. Longitudinal fibro-muscular bundles,
which were the distal projections of those coming from the lips of the reticular groove, were
observed in the lips of the omasal groove. The tunica serosa was composed of loose
connective tissue containing blood vessels, nerves and adipose tissue.
172
Gaetano Scala and Lucianna Maruccio
3.2.5. Abomasum
3.2.5.1. Abomasum Structure
The tunica mucosa of the abomasum had many glands and was covered with a simple
columnar epithelium (Epithelium simplex columnare). The border between the omasal and
abomasal epithelium was clearly distinguishable on the mucosal folds. The epithelium
penetrated into the lamina propria and formed the abomasal glands (Figure 31). Various
morphological types of glands were observed in different regions: a) cardiac glands
(Glandulae cardiacae) in the cardiac region; b) proper gastric glands (Glandulae gastricae
propriae) in the fundus; c) pyloric glands (Glandulae pyloricae) in the pylorus. The cardiac
glands were located in a pale narrow area of the abomasal wall close to the omaso-abomasal
orifice. The proper gastric glands were located in a large gray-reddish area, which primarily
extended throughout the fundus and the corpus. Many standing spiral folds (Plicae spirales
abomasi) that greatly extended the area of the inner surface of the abomasal mucosa were
observed in the region of the proper gastric glands.
Figure 31. LM mucosa of the buffalo abomasum. Pc=Parietal cell.
Adult Mucosa Stomach of Domestic Ruminants
173
The folds began to appear at the omaso-abomasal orifice; they were very high along the
greater curvature, and decreased in size as they approached the faces and the pylorus. They
were no folds along the smaller curvature. The region of the pyloric glands occupied the
entire pylorus and a small area of the corpus. The mucosa of this region was yellowish in
color and had small transitional folds. A 3-4 cm high protuberance (Torus pyloricus) was
observed on the pyloric canal at the end of the smaller curvature. It was hemispherical in
shape and composed of muscular and fatty tissues.
3.2.5.2. Abomasum Ultrastructure
The abomasal mucosa was covered with a simple columnar epithelium containing many
mucous cells. Many small protruding areas (Areae gastricae), which were bounded by a
network of grooves, were found on the abomasal mucosa. Microscopic gastric pits (Foveolae
gastricae), into which abomasal glands opened, were observed on the gastric areas. In the
lamina propria, the glands had a lobular arrangement. This lobular arrangement was due to
the morphological organization of the lamina propria and the muscularis mucosae, which
were composed of a deep layer and an upper layer. The deep layer, which lay on the submucosa, was continuous and larger than the upper layer. The upper layer was transversal and
formed the inter-glandular stroma. It gave rise to septa of various thickness that were directed
upward and perpendicular to the mucosal surface. The larger septa bounded the grooves of
the mucosa and delimited groups of cup-shaped glands located deep within the lamina
propria. Together with the grooves, they formed the gastric glandular lobuli. The smaller
septa partially bounded each gland.
The cardiac glands, which tended to form clusters, were simple-tubular, and in a few
cases, they were forked. Each gland had a narrow neck, a body and a bottom. The necks
opened into a pit and were covered with mucous cells. The bodies were the large parts of the
perspective fundic glands. The bodies and the bottoms were composed of the principal
(Exocrinocyti principales) and parietal (Exocrinocyti parietales) cells. The principal cells,
known to secrete pepsinogen, were polygonal. The parietal cells, know to secrete the
precursors of hydrochloric acid, were globular and located at the periphery of the tubule.
There were fewer parietal cells in the body than in the neck. The pyloric glands were mucous
and ramified.
In addition to the glands, the lamina propria was also composed of the following: dense
connective tissue containing a large blood vascular network, nerves and lymphatics, and
muscularis mucosae. There were many lymphatics located near the omaso-abomasal ostium.
The SEM specimens showed that the openings of the abomasal glands uniformly perforated
the inner surface of the abomasum. On the partially macerated specimens, the connective
framework of the lamina propria delimited the glandular ducts and formed a base for the
secretive cells. The vascular corrosions casts showed that many blood capillaries were located
in the lamina propria surrounding the glandular adenomeres. These blood capillaries ended
underneath the mucosal surface and formed loops around the gland outlets.
The sub-mucosa of the abomasum was thicker than the sub-mucosa of the forestomach,
thus causing the tunica mucosa to form many spiral folds. It was composed of loose
connective tissue that penetrated between the underlying bundles of the tunica muscularis,
and it contributed in forming the pyloric torus. The sub-mucosa had many blood vessels and a
large nerve plexus containing neurons and free sensitive nerve endings. The tunica muscularis
was composed of inner circular and outer longitudinal layers. The inner circular layer was
174
Gaetano Scala and Lucianna Maruccio
thicker than the outer longitudinal layer. The tunica muscularis increased in thickness up to
the pylorus where it formed the pyloric sphincter and the pyloric torus. The connective tissue
dividing the muscular layers contained blood vascular and nerve networks.
The tunica serosa was morphologically similar to the tunica serosa of the forestomach.
There was no tunica serosa in the areas where the abomasum was related to the reticulum and
rumen.
3.3. Histochemistry (LM)
The forestomach mucosa was composed of cells having an intense NADPHd staining in
both the intermediate and deep layers of the epithelium. The staining was located in the
cytoplasm over the nucleus. No staining was highlighted in the cells of superficial layers.
Figure 32. LM NADPHd buffalo mucosa. Gc=Cells of the granulosum layer; Lp= Lamina propria of
the mucosa; Sc=Cells of the spinous layer.
Figure 33. LM NADPHd cattle mucosa. Gc=Cells of the granulosum layer; Lp= Lamina propria of the
mucosa; Sc=Cells of the spinous layer.
Adult Mucosa Stomach of Domestic Ruminants
175
Dense NADPHd staining was found in the endothelium of the epithelial blood capillaries
in the lamina propria. In all three species examined, NADPHd showed a similar staining
pattern (Figures 32, 33).
3.4. Immunogold-Labeling SEM Analysis
The results obtained from the stomach mucosa specimens of all three species examined
were very similar for two antibodies used. Intense NOS I immunoreactivity (IR) was detected
in the forestomach mucosa specimens studied by immunogold-labeling SEM analysis. The IR
was composed of dense gold particles located in the cytoplasm of the cells present in the deep
and intermediate layers. The lamina propria of the mucosa showed IR characterized by
widespread gold particles. A similar IR was found in all three animal species studied (Figures
34, 35, 36, 37, 38).
Figure 34. SEM immunogold of the cattle ruminal mucosa. Intense NOS I immunoreactivity detected in
the cells of the deep and intermediate layers of the epithelium. E=Mucosa epithelium; Lp=Lamina
propria of the mucosa.
Figure 35. SEM immunogold of the cattle reticular mucosa. Intense NOS I immunoreactivity detected
in the cells of the deep and intermediate layers of the epithelium. E=Mucosa epithelium; Lp=Lamina
propria of the mucosa.
176
Gaetano Scala and Lucianna Maruccio
Figure 36. SEM immunogold of the buffalo ruminal mucosa. Intense NOS I immunoreactivity detected
in the cells of the deep and intermediate layers of the epithelium. E=Mucosa epithelium; Lp=Lamina
propria of the mucosa.
Figure 37. SEM immunogold of the buffalo reticular mucosa. Intense NOS I immunoreactivity detected
in the cells of the deep and intermediate layers of the epithelium. E=Mucosa epithelium; Lp=Lamina
propria of the mucosa.
Figure 38. SEM immunogold of the sheep ruminal mucosa. Intense NOS I immunoreactivity detected
in the cells of the deep and intermediate layers of the epithelium. E=Mucosa epithelium; Lp=Lamina
propria of the mucosa.
On the ruminal papillae, the immunopositivity to CD133 was often located in the
discontinuity of the covering mucosa epithelium (Figure 39). Moreover, the immunopositive
cells were observed in the micro-papillae located on the bottom of the reticulum small
Adult Mucosa Stomach of Domestic Ruminants
177
polygonal cells (Figure 40). These positive, long and grouped cells, together in small clusters,
showed an intense immunoreactivity. Intense immunopositivity were observed in the parietal
cells of abomasum glands (Figures 41, 42). No immunoreactivity was observed in the
samples treated with PBS instead of the two primary antibodies (negative control).
Figure 39. SEM immunogold of the buffalo mucosa. Intense CD133 immunoreactivity detected in the
stem cells of the ruminal mucosa epithelium. S=Stem cells.
Figure 40. SEM immunogold of the buffalo mucosa. Intense CD133 immunoreactivity detected in the
stem cells of the reticular mucosa epithelium. S=Stem cells.
178
Gaetano Scala and Lucianna Maruccio
Figure 41. SEM immunogold of the buffalo mucosa. Intense CD133 immunoreactivity detected in the
stem cells of the abomasal mucosa epithelium. S=Parietal stem cells.
Figure 42. SEM immunogold of the buffalo mucosa. Intense CD133 immunoreactivity detected in the
stem cells of the abomasal mucosa epithelium. S=Parietal stem cell.
3.5. Western Blotting
Expression of NOS I and CD133 were found in the forestomach mucosa of all three
domestic ruminants using specific rabbit polyclonal antibodies raised against human brain
NOS I and CD133. The presence of all of the two proteins were detected in the mouse brain
homogenate, which was used as positive control. Finally, the blot was stripped and reprobed
Adult Mucosa Stomach of Domestic Ruminants
179
with a mouse monoclonal anti-tubulin antibody confirming equal loading of proteins in all
lanes.
Conclusion
The conclusions of the present study of the domestic ruminant stomach suggest that:
a) The forestomach mucosa is involved in substance absorption occurring in the
forestomach during rumination;
b) The abomasum mucosa is similar to the gastric mucosa of monogastric species.
Indeed, the morphologies of the rumen papillae, the reticular cells, and the omasum
laminae are related to the absorption of fluids and substances such as the free fatty acids
formed by bacterial fermentative activity. In addition, the role of NO in the forestomach
mucosa of the domestic ruminant is that NO serves to delay the onset of cellular apoptosis of
the mucosa by playing a role in the production of cell energy. NO has important
characteristics that make this delay possible. When the epithelium cells of the forestomach
mucosa migrate from the basal layer to the apical surface, they encounter an O2 deficit. Given
that NO can, under normoxia conditions (O2 concentration <30 lm), bind to the same
mitochondrial cytochrome c oxidase site of the basal mucosa cells to which O2 binds [14,15]
and that NO can play a role in the production of cell energy similar to that of O2, it can be
deduced that NO can make up for the apical surface O2 deficit. In this O2 deficit region, NO
produces the energy that O2 no longer supplies by oxidizing the butyric acid that is normally
present in the forestomach of ruminants [16,17,18,19]. The extent of butyric acid conversion
to ketone bodies in the epithelium of ruminal mucosa is evidently dependent on absorption
rates, which, in turn, are influenced by the ruminal butyric acid concentration and the pH in
the rumen [20]. In addition, because the O2 present in this deficit region is no longer needed
for energy production, it is utilized for other cellular vital functions. These two consequences
of the O2 deficit, namely, the substitution of NO for O2 and the use of O2 for other cellular
vital functions, serve to prolong the life of the cells in the mucosa of forestomach, i.e., to
delay the onset of cellular apoptosis. It should be noted that other studies have demonstrated
that NO plays an important role in delaying the onset of cellular apoptosis by maintaining
mitochondrial integrity, and hence in protecting cells from mitochondrial death [20,21,22,23].
The results regarding the presence of the stem cells in adult buffalo [11] but also in bovine
and adult sheep, refers to renewing of the micro-papilllae located on the bottom of the
reticular small cells or reconstitutes the damages on the rumen papillae during normal
functional activity.
References
[1]
[2]
[3]
E.D. Warner. Am. J. Anat. 102, 33 (1958).
B. G. Panchamukhi, and H. C. Srivastava. Anat. Histol. Embryol. 8, 97 (1979).
A.H.K Osman and R. Berg. Anat. Anz. 149, 232 (1981).
180
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
Gaetano Scala and Lucianna Maruccio
H. Amasaki and M. Daigo. Anat. Histol. Embryol. 17, 1 (1998).
H. Pfannkuche, C. Schellhorn, M. Schermann, J. R. Aschenbach, and G. Gabel. J. Anat.
203, 277 (2003).
H. Pfannkuche, C. Schellhorn, M. Schermann, and G. Gabel. Anat. Rec. A Discov. Mol.
Cell Evol. Biol. 278, 528 (2004).
J. Münnich, G. Gabel, and H. Pfannkuche. J. Anat. 213, 442 (2008).
H. Pfannkuche and G. Gabel. J. Anim. Physiol. Anim. Nutr. 93, 277 (2009).
N. Toda and T. Okamura. Pharmacol. Rev. 55, 271 (2003).
G. R. H. Owen, D. O. Meredith, I. Gwynn, and R.G. Richards. J. Microsc. 207, 27
(2002).
G. Scala, M. Corona, and L. Maruccio. Anat. Histol. Embryol. 40, 47 (2011).
R. G. Richards, M. Stiffanic, G. R. H. Owen, M. Riehle, J. A. P. Gwynn, and A. S. G.
Curtis. Cell Biol. Int. 2, 1237 (2001).
P. R. Castello, P. S. David, T. McClure, Z. Crook, and R. O. Pyton. Cell Metab. 3, 277
(2006).
P. R. Castello, D. K. Woo, K. Ball, J. Wojcik, L. Liu, and R. O. Pyton. Proc. Natl Acad.
Sci. USA 105, 8203 (2008).
C. E. Stevens and Stettler B. K. Am. J. Physiol. 210, 365 (1966).
R. A. Leng and C. E. West. Res. Vet. Sci. 10, 57 (1969).
E. N. Bergman and J. E. Wolff. Am. J. Physiol. 221, 586 (1971).
E. Weigand, J. W. Young, and A. D. Mc Gilliard. J. Dairy Sci. 55, 589 (1972).
J. B. Mannick, X. Q. Miao, and J. S. Stambler. J. Biol. Chem. 272, 24125 (1997).
A. Lòpez-Farré, J. Rodrìguez-Feo, L. Sànchez de Miguel, L. Rico, and S. Casado. Int.
J. Biochem. Cell Biol. 30, 1095 (1998).
P. Ghafourifar, U. Schenk, S. D. Klein, and C. Richter. J. Biol. Chem. 274, 31185
(1999).
T. Inoue, Y. Suzuki, T. Yoshimaru, and C. Ra. J. Leukoc. Bio. 83, 1218 (2008).
G. Scala, M. Corona and L. Maruccio. Anat. Histol. Embryol. 39, 322 (2010).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter XI
Fetal Mucosa Stomach of
the Buffalo (Bubalus Bubalis):
Morphostructural, Microvascular
and Immunocytochemical Study
Gaetano Scala and Lucianna Maruccio
Department of Biological Structures, Functions and Technologies,
University of Napoli Federico II, Faculty of Veterinary Medicine,
Via Veterinaria, 1, Napoli, Italy
Abstract
The ruminant stomach mucosa shows several morphostructural characteristics in
relation with rumination. It is important and of interest to learn how the mediterranean
buffalo gains a higher body weight and a higher milk production from the possible
relation available for animal production. In this chapter, the morphostructural,
microvascular and immunocytochemical characteristics of the buffalo mucosa stomach
during prenatal period is studied. The fetus specimens of different lengths of both sexes
were processed for light microscopy (LM), scanning electron microscopy (SEM) and
immunogold-labeling SEM analysis. The stomach mucosa in the buffalo fetuses of the
crown-rump length (CRL) 5 cm at LM shows the epithelium with similar characteristics
in all areas of the stomach. The epithelium is composed of several layers of ovoid cells,
more dense in the basal surface. The sub-epithelial tissue, not still very differentiated, is
rich with blood vessels. At the SEM, the apical surface of the epithelial cells have short
microvilli. In the buffalo fetuses of the CRL 7 cm at the LM, the mucosa of abomasum
shows the thickening of the elongated cells. At the SEM, the internal surface of the
reticulum shows the reticular groove that begins to form. Obvious changes are observed
in the mucosa stomach buffalo fetuses of CRL 13 cm. These changes regard the lamina
propria organization. In fact, the lamina propria is different in all of the stomach
compartments. In the rumen, the connective lamina propria origins of finger-like

Email: gaescala@unina.it.
182
Gaetano Scala and Lucianna Maruccio
protrusions that invade the mucosa epithelium. In the reticulum, the connective lamina
propria describes the polygonal areas. In the omasum, the connective lamina propria
forms the typical papillae of the mucosal laminae.
In theabomasum the connective lamina propria forms the typical mucosa plicae.
Then, the mucosa stomach shows a typical aspect similar to the final organization. From
the fetuses of CRL 13 cm to birth, the blood vessels show the typical organization present
in the stomach compartments. This data confirms the view that the development of subepithelial blood capillaries must take place prior to the development of the mucosa
epithelium. In the fetuses of the CRL 38 cm, in which the morphostructural organization
is already well defined, the epithelium shows an evident immunoreactivity to marker for
pluripotent stem cells. In conclusion the epithelium mucosa of the fetuses buffalo
stomach present an intense activity. It seems that the organization of the lamina propria
precedes that of the epithelium mucosa.
1. Introduction
The mucosa of the ruminant stomach shows more different morphostructural
characteristics although originating from the by identical ontogenetic type of mucosa. These
different characteristics were brought about functional differentiation of the compartments of
the ruminant stomach in the rumination. It is important and of interest to learn how the
mediterranean buffalo gains a higher body weight and a higher milk production from the
possible relation available for animal production. The mucosa histogenesis of the
compartments of the buffalo stomach from primordial stage to birth was studied by
histological techniques [1,2,3].
However, these studies are insufficient to explain the structural modifications of stomach
mucosa during organogenesis of the buffalo stomach. The knowledge of these structural
modifications is indispensable to evaluate the role of the four compartments of the buffalo
stomach during the rumination.
Some authors [4,5,6,7,8,9,10] have studied the histogenesis of the mucosa of the rumen,
of the reticular groove, of the reticulum, of the omasum and the abomasum using histological
and histochemical techniques. However, the employment of the modern technologies can
supply a contribution to histogenesis of stomach mucosa of the mediterranean buffalo.
Then, in this paper, the organization of the mucosa of each compartment of the buffalo
stomach during the prenatal life was studied in detail in order to clarify the role of the gastric
mucosa in the digestive and nutrient absorption processes in the postnatal life. The
investigation of these processes is necessary for a correct interpretation of the
morphostructural phenomena, particularly pathological phenomena that appear during buffalo
productive activity.
2. Material and Methods
The mediterranean buffalo (Bubalus bubalis)fetuses from the initial prenatal stages until
birth were studied. The fetuses of both sexes were sampled immediately after slaughter from
a government slaughterhouse in Campania, Italy. The specimens were processed in different
ways, depending on the type of techniques to be used.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
183
2.1. Light Microscopy (LM)
Twelve fetuses were cut into numerous pieces (0.5 cm long) which were then fixed in
paraformaldehyde and picric acid (PAF) for 12-18 h, rinsed in a glucose phosphate buffer for
24-36 h, dehydrated, and pre-embedded twice in 12 h in a solution of 2-hydroxyethil
metacrylate (85 ml), 2-butoxyethanol (15 ml), and benzoyl peroxide (1 g). Specimens were
embedded using a mixture of the pre-embedding solution and N, N-dimethylaniline (ratio
30:1).
After polymerization, all specimens were sectioned using a microtome (HistorangeLKB), stained with toluidine blue, and examined under a light microscope (Leitz Orthoplan,
Wetzlar, Germany) to study blood vessel topography.
2.2. Scanning Electron Microscopy (SEM) - Intact Tissue Technique
Twelve fetuses were each perfused through the umbilical vein (Vena ombelicale) with
phosphate buffer 0.1 M, pH 7.3, to wash the blood vessels, and then fixed with Karnovsky’s
solution (4% paraformaldehyde, 2.5% glutaraldehyde). After 12 h, each fetus was cut into
numerous pieces (0.5 cm long), which were immersed in a glucose phosphate buffer for 24-48
h, and dehydrated in ethyl alcohol and critical point dryer (CPD 030, BALZERS,
Liechtenstein). The piece specimens were mounted on stubs (12.5 mm diameter), examined
under SEM (LEO 435 VP) at 25 kV, and photographed.
2.3. Scanning Electron Microscopy (SEM) - Maceration Technique
Specimens of six fetuses, after fixation with Karnovsky’s, were partially macerated with
a 15% KOH solution from 30 min. After maceration, the specimens were immerged in a
tannic acid solution (5%), rinsed with tap water and then treated following the same steps of
the others.
2.4. Scanning Electron Microscopy (SEM) - Vascular Corrosion Cast
Technique
Six fetuses were each perfused through the umbilical vein (Vena ombelicale) with a
physiological solution to wash the blood vessels. They were then injected with a lowviscosity, colored with methylmethacrylate mixture to obtain a cast, and were corroded by
immersion in KOH solution (30%) for 1-2 weeks; the solution was changed every 4-5 days.
Upon complete corrosion, the casts were rinsed with tap water, rinsed with bi-distilled water
and dried in desiccators. Then, the casts were photographed using a digital macrophotographic camera (EOS 7D, Canon, Tokyo, Japan). Each fetus cast was then cut into
numerous 1 cm3 samples that were mounted on stubs (25 mm diameter) and coated with gold
using a sputter coater (SC500, BIORAD, Hemel Hempstead, UK). All gold-coated samples
were examined and photographed under a scanning electron microscope (LEO 435 VP,
Cambridge, UK) at 10 kV.
184
Gaetano Scala and Lucianna Maruccio
2.5. Immunogold-Labeling SEM Analysis
For the immunogold-labeling SEM analysis, the specimens of six fetuses were
immediately immersed in PBS for 1 h. Samples were incubated for 2 h in a solution
containing normal goat serum (X 0907; Dako Italia s.p.a., Milano, Italy) diluted 1:10 in PBS,
and next with a rabbit primary polyclonal antibody raised against a peptide mapping the 508792 amino acid sequence of human CD133 (Santa Cruz, sc-30220 Santa Cruz, USA), diluted
1:500 in PBS, overnight at 4°C. After washing in PBS, the samples were incubated with goldconjugated goat anti-rabbit IgG (E.M.GAR10; Agar Scientific Limited, Stansted, UK) diluted
1:200 in PBS for 1 h at room temperature. The secondary antibody was conjugated with gold
particles of different size, namely 5 and 15nm. After washing in PBS, samples were fixed by
2.5% glutaraldehyde in 0.1 m cacodylate buffer containing CaCl2, pH 7.2, for 30 min. After
the fixation step and washings with distilled water, samples were subjected to silver
enhancement (British BioCell International Cardiff, Wales, UK). The enhancement process
enables the use of antibodies conjugated to smaller (5-15 nm) gold particles, preserving the
advantage of faster penetration and higher labeling efficiency [11]. Next samples were
dehydrated through an ethanol series and dried to the critical point. The specimens, mounted
on stubs, were examined under a LEO 435 VP scanning electron microscope at variable
pressure (80-120 Pa) in the backscattered electron mode, which allows detecting gold
particles associated with cells even if they are located intracellularly [12]. The samples were
not coated by gold, so that only conjugated gold deriving from immunocytochemical reaction
was observed by SEM and were photographed.
2.6. Western Blotting
The specimens of crown-rump length (CRL) 5 cm and 7 cm fetuses and the stomach
removed from all fetuses studied were homogenized an Ultraturrax l-407 at 4°C with 5ml/1.5
g tissue of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
ethylenediaminetetraacetic acid, 10 mm NaF, 0.5% deoxycholic acid, 0.1% sodium dodecyl
sulphate (SDS), 15 Nonited P-40, 1 mM phenylmethylsulfonyl fluoridre, 1.1 U/ml aprotinin,
10 mg/ml leupeptin, and 1 mM Na3VO4. Homogenates were centrifuged at 15,000- g for 20
min at 4°C. Supernatants were divided into small aliquots and stored at -80°C until used. The
amount of total proteins in each sample was determined by the Bio-Rad DC protein assay
(Bio-Rad Laboratories, Hercules, CA, USA). Samples containing equal amount of proteins
were boiled for 5 min in SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 0.1% bromophenol blue and 5% b-mercaptoethanol) and run on a 10%
SDS/polycrylamide gel.
After electrophoresis, the proteins were transferred to nitrocellulose using a Mini transblot apparatus (Bio-Rad Laboratories) according to the manufacturer’s instructions.
Membranes were blocked for 1 h at room temperature with TBS-T buffer (150 mM NaCl, 20
mM Tris HCl, pH 7.4, 0.1% Tween 20) containing 5% milk. The blots were incubated
overnight with a rabbit polyclonal antibody raised against a peptide mapping the 508-792
amino acid sequence of human CD133 (Santa Cruz) diluted 1:1 000 in TBS-T containing
2.5% milk. After incubation, the membranes were washed three times with TBS-T and
incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chemical)
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
185
diluted 1:5 000 in TBT-T containing 2.5% milk. The proteins were visualized by enhanced
chemilumenescence (ECL; Amersham, Little Chalfont, UK).
The same blots were stripped and re-probed using anti-tubulin monoclonal antibody
(MAB 1637; Chemicon International Inc.) to confirm equal loading of proteins in all lanes.
All nomenclature in this work was adopted from the Nomina Anatomica Veterinaria, Nomina
Histologica and Nomina Embryologica Veterinaria (World Association of Veterinary
Anatomists, 2005).
3. Results
At the beginning of the development, the ruminant and mono-gastric species have only
one primordial stomach. During the early stages of ruminant embryonic life, the primordial
stomach is a simple fusiform dilatation of the anterior part of the primitive gut canal. It rotates
around its longitudinal axis, has a convex dorsal greater curvature, a concave dorsal greater
curvature and a concave ventral lesser curvature. The dorsal and ventral mesogastrii are
connected to the primordial stomach; they will become the greater and lesser omentii,
respectively. Then, a little groove begins to appear on the greater curvature and delimits the
rumen-reticulum complex. The omasum (Omasum) then develops from the lesser curvature,
and a little later, the abomasum (Abomasum) develops from the distal part.
Figure 1. Development pattern of the buffalo stomach. a, Stomach embryo CRL 1 cm; b, Stomach
embryo CRL 2 cm; c, Stomach embryo CRL 3 cm; d, Stomach fetus CRL 3-5 cm. A=Abomasum;
O=Omasum; Re=Reticulum; Ru=Rumen.
After another few weeks, the four cavities of the ruminant stomach are well
distinguished: the rumen (Rumen) and the reticulum (Reticulum) are, respectively, on the
dorsal and ventral left side; the omasum and the abomasum are on the right side.
186
Gaetano Scala and Lucianna Maruccio
Subsequently, the rumen increases in size and moves the reticulum anterior-ventrally to a
central position. The omasum and the abomasum are located on the right side. The short tract
of the distal part of the abomasum is, however, also located on the left side (Figure 1). The
mucosa of the four compartments of the buffalo stomach had different structural
modifications that change during prenatal life. Because there is no table of conversion
between the crown-rump lengths (CRL) of buffalo fetuses and their age in literature, it will
estimate only the crown-rump length of the buffalo fetuses.
3.1. Buffalo Fetuses CRL 5 cm
3.1.1. LM
The forestomach (Proventriculus) mucosa shows in this stage the epithelium with similar
characteristics. The epithelium is composed of several layers of ovoid cells with little
intercellular spaces.
The cytoplasm of epithelial cells appears vacuolated and the nucleus, spheroidal,
occupies most of these cells. The mucosa lamina propria appears predominantly cellular
(Figure 2). The abomasum mucosa epithelium is constituted of columnar cells, arranged in
few layers. These columnar cells are disposed perpendicularly to the lamina propria. The
mucosa lamina propria is formed of homogeneous mesenchyme comprising cells of different
type and size (Figure 3).
Figure 2. LM mucosa of the buffalo forestomach of fetus CRL 5 cm. Ca=Epithelial cells of the apical
zone; Cb=Epithelial cells of the basal zone; L= Lamina propria of the mucosa.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
187
Figure 3. LM mucosa of the buffalo abomasum of fetus CRL 5 cm. B=Blood capillary; C=Epithelial
cells; L= Lamina propria of the mucosa.
3.1.2. SEM
The mucosa of the forestomach shows several typical characteristics:
1. The apical surface of mucosa epithelium is covered by short and long microvilli. The
microvilli have a uniform distribution on the cellular surface. The apical surface of
mucosa epithelium is irregular for the different thickness of the apical cells nuclei
(Figure 4).
2. The most of the epithelium cells present spheroidal and largest (12-15 m) nuclei.
The nuclei of the epithelial cells have an evident ovoid nucleolus (Figure 5).
3. The thick (8-10 m) basement membrane forms a support for the epithelium and
connects to the underlying connective tissue of the lamina propria (Figure 6).
4. The lamina propria has a rich blood microvasculature that forms a thick capillary
network (Figure 7).
Figure 4. SEM apical surface of the ruminal epithelium of the buffalo fetus CRL 5 cm. The numerous
microvilli are evident on the apical surface of the ruminal epithelium. Ml=Long microvilli; Ms=Short
microvilli.
188
Gaetano Scala and Lucianna Maruccio
Figure 5. SEM epithelial cells of the ruminal mucosa of the buffalo fetus CRL 5 cm. N=Nucleus; Nc=
Nucleolus.
Figure 6. SEM transversal section of the ruminal mucosa of the buffalo fetus CRL 5 cm. BM=
Basement membrane; Ca=Epithelial cells of the apical zone; L= Lamina propria of the mucosa;
N=Nuclei of the epithelial cells.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
189
Figure 7. SEM lamina propria blood capillaries of the buffalo fetus CRL 5 cm. B=Blood network
capillaries.
3.2. Buffalo Fetuses CRL 7 cm
3.2.1. LM
The stomach mucosa shows only a biggest stratigraphy of the mucosa epithelium. Rumen
– The mucosa epithelium is stratified and it is constituted at the beginning of two-four layers,
later they become 10-12. The apical layer is formed of the vacuolated cells. The deep layer is
formed of densely small cells packed, adherent by the basement membrane to the mucosa
lamina propria. The mucosa lamina propria appears predominantly cellular and it is united
with the underlying submucosa. Reticular groove (Sulcus reticuli) – The mucosa epithelium is
made up of two areas: a deep and a lip. The mucosa epithelium of the deep consists of a
single layer of cuboid or low columnar cells, resting on a basement membrane. The mucosa
epithelium of the lips is formed of several layers of polygonal cells. Omasum – The omasal
mucosa shows laminae of four orders. The laminae of the first order are larger than the
laminae of the second order. The laminae of the second order are shorter than the laminae of
the first order. The laminae of the third order are shorter than the laminae of the second order.
Finally, those of the fourth order are still smaller than the laminae of the third order. The
laminae of the first and second orders show the conical papillary bodies originating as
mesenchyme evaginations into the overlying mucosa epithelium. These papillae never project
into the lumen of the omasum. The laminae of the third order have few smaller papillary
bodies, while those of the fourth order are without. The tip of the first order laminae is
somewhat wide and flat. On the contrary, the tip of the other laminae is rounded. Moreover,
190
Gaetano Scala and Lucianna Maruccio
all of the laminae have a wide base that decreases gradually in breadth. Abomasum – The
abomasal mucosa epithelium shows thin and low folds that are longitudinally orientated.
These folds are, in the initial portion of the abomasum, high while they gradually decrease in
its middle portion. Finally, these folds disappear in the abomasum terminal portion. However,
these folds increase in height, thickness and number with the increasing age.
3.2.2. SEM
The internal surface of the reticulum shows a transversal sulcus. Then, the reticular
groove begins to form. Moreover, the deep and the lips of the reticular groove are evident
(Figure 8).
Figure 8. SEM reticular groove of the buffalo fetus CRL 7 cm. D=Deep of the reticular groove; L=Lip
of the reticular groove.
3.3. Buffalo Fetuses CRL 13-18 cm
3.3.1. LM
Obvious changes are observed in the stomach mucosa regard both the epithelium and the
lamina propria.
Rumen - In the basal area of the mucosa epithelium appear aggregations of circumscribed
cells, forming the primordia of the ruminal papillae. The connective of the mucosa lamina
propria evaginates in the sites of the basal mucosa epithelium to form the cores of the
primordia ruminal papillae.
Reticular groove – The apical surface of the mucosa epithelium is still smooth. Reticulum
– The primordia of the reticular crests are wedge-shaped and consist of a core of cellular
elements of the connective lamina propria that is invaginated into mucosa epithelium. These
crests had pointed apices directed towards the luminal surface of the mucosa epithelium, but
not reaching it in this stage. The basal zone of the mucosa epithelium on the side of the crests
is constituted of a single layer of the cuboid cells (Figure 9). Distally, the mucosa epithelium
is followed by the mucosa lamina propria. The mucosa lamina propria is predominantly
cellular and blends with the underlying submucosa without any line of demarcation.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
191
Figure 9. LM reticular mucosa of the buffalo fetus CRL 13 cm. B=Blood capillary; Ca=Epithelial cells
of the apical zone; Cb=Epithelial cells of the basal zone; E=Evagination of the lamina propria; L=
Lamina propria of the mucosa.
Figure 10. LM omasal mucosa of the buffalo fetus CRL 13 cm. Ca=Epithelial cells of the apical zone;
Cb=Epithelial cells of the basal zone; M=Smooth muscle.
Omasum – The large (1), medium (2), small (3), and smallest (4) omasal laminae are
arranged in the following manner: 1, 4, 3, 4, 2, 4, 3, 4, 1. The middle portion of the cores of
the large, medium and small omasal papillae is occupied by smooth muscle cells with some
elements of the submucosa extending from the inner circular layer of the muscular coat
(Figure 10). Abomasum – The apical surface of the mucosa epithelium is studded with a few,
shallow and V-shaped invaginations that represent the gastric pits. Afterwards, these pits are
enormously increased in number and in depth to involve the whole mucosa epithelium
surface.
192
Gaetano Scala and Lucianna Maruccio
3.3.2. SEM
Rumen – The lamina propria forms the finger-like protrusions (connective papillae) that
invade the mucosa epithelium (Figure 11).
Reticular groove – It is evident, and forms a profound groove bounded of two lips. The
apical surface is smooth.
Reticulum – The apical surface is subdivided into conical formations with a smooth apex
(Figure 12). The conical formations are subdivided by a deep groove that, deeply, is direct
toward the connective reticular crests (Figure 13).
Figure 11. SEM buffalo lamina propria of the fetus CRL 13 cm. The finger-like protrusions (connective
papillae), that invade the mucosa epithelium.
Figure 12. SEM reticular mucosa of the buffalo fetus of CRL 15 cm. The conical formations of the
apical surface.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
193
Figure 13. SEM reticular mucosa of the buffalo fetus of CRL 15 cm. The connective reticular crests of
the lamina propria.
Figure 14. SEM reticular mucosa of the buffalo fetus of CRL 16 cm. The conical formations subdivided
in two parts.
At the beginning, the conical formations are subdivided in two parts (Figure 14).
Successively they are subdivided in four parts (Figure 15), so as to form a cross.
Omasum – The omasal mucosa shows laminae of four orders (first order, second order,
third order and fourth order). The laminal surface is smooth.
Abomasum – The abomasal mucosa is raised in folds longitudinally oriented. The apical
surface of the folds is subdivided on the elongated areas separated by the deep groove.
194
Gaetano Scala and Lucianna Maruccio
Figure 15. SEM reticular mucosa of the buffalo fetus of CRL 17 cm. The conical formations subdivided
in four parts so as to form a cross.
3.4. Buffalo Fetuses CRL 27-38 cm
3.4.1. LM
Rumen – The basal surface of the mucosa epithelium shows circumscribed aggregations
of cells forming the primordia of the ruminal papillae. These aggregations appear firstly in the
basal area of the rumen arium (Atrium ruminis). In some areas of the ruminal mucosa, the
connective lamina propria forms already finger-like formations. Moreover, in the ruminal
mucosa, the lamina propria forms the short and long connective papillae that present a rich
blood microvasculature.
Figure 16. LM of the reticular mucosa in the buffalo fetus of the CRL 30 cm. The connective crests of
the lamina propria. C=Epithelial cells; Cr=Connective crest.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
195
Reticular groove – The epithelium surface appears undulating and forms humps on the
tips of the growing papillary bodies in the reticular groove floor while the epithelium surface
remained smooth in the lips. Reticulum - The luminal surface showed some shallow
invaginations on the tip of the developing reticular crests (Figure 16).
Then, some the cells of the superficial area near the luminal surface begin to decrease in
size. Moreover, each cell has a pyknotic deep nucleus that is also basophilic. This nucleus is
dipped in a translucent eosinophilic material. The total thickness of the mucosa epithelium
increased considerably and the reticular crests begin to differentiate into short and long.
Nevertheless, the crests never projected into the lumen.
Omasum – The mucosa lamina propria evaginates into the lumen of the omasum in few
regions in the interlaminar spaces representing a fifth order lamina. The muscularis mucosae,
begins evident in the cores of the first, the second and third order laminae.
Abomasum – The mucosa epithelium is formed of the glands that then increase greatly in
number and size. These glands take mainly a tubular form with a narrow lumen and a slightly
dilated base. Moreover, these glands are formed of a single layer of low columnar or cubical
vacuolated cells. At the base of these cells, their nuclei are located.
3.4.2. SEM
Rumen – The lamina propria forms finger-like connective papillae that have different
heights, but do not arrive at the surface of the mucosa epithelium. By partial maceration with
KOH, the lamina propria resulted separated from the mucosa epithelium. The inner surface of
mucosa epithelium shows the pits that contain the connective papillae of the lamina propria
(Figure 17). The bottom of these pits is perforated (Figure 18).
The vascular casts of the connective papillae finger-like form the conical vascular
formations that have on the apex the capillary bulges (Figure 19). Reticulum – The lamina
propria forms connective polygonal formations. These polygonal formations are different in
size and in shape.
Figure 17. SEM ruminal mucosa with KOH partial maceration in the buffalo fetus of the CRL 38 cm.
C=Epithelium; F=Finger-like of the connective lamina propria.
196
Gaetano Scala and Lucianna Maruccio
Figure 18. SEM ruminal mucosa with KOH partial maceration in the buffalo fetus of the CRL 38 cm.
Pits of the inner surface of the mucosa epithelium. Perforations of the pits. H=Holes of the bottom pits.
Figure 19. SEM ruminal mucosa of the buffalo fetus of the CRL 38 cm. The vascular casts of the
connective papillae finger-like.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
197
Figure 20. SEM inner surface of the reticulum epithelium of the buffalo fetus of the CRL 38 cm.
Figure 21. SEM lamina propria of the fetus of the CRL 38 cm.
Generally, these formations are hexagonal. By partial maceration with KOH, the lamina
propria is separated from the mucosa epithelium. The inner surface of mucosa epithelium
shows the drills that are the negative image of the lamina propria polygonal formations
(Figures 20, 21).
3.5. Buffalo Fetuses from CRL 40 cm to Birth
Rumen – From fetuses of CRL 40 cm, the inner ruminal surface starts the production of
the epithelial projections that are more visible in the fetuses of CRL 50 cm. Usually one
198
Gaetano Scala and Lucianna Maruccio
papilla begins to form by one connective papillary core. At the birth, the ruminal papillae are
conical.
Reticulum – The histological organization of the reticular mucosa is almost complete.
The formation of the reticular cellules of the first order, the second order and third order is
evident (Figure 22).
Figure 22. LM of the reticular mucosa of the buffalo fetus of the CRL 38 cm.
Figure 23. SEM microvascular casts of the reticular mucosa of the buffalo fetus of the CRL 38 cm.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
199
Figure 24. SEM microvascular casts of the omasal mucosa of the buffalo fetus of the CRL 38 cm.
Figure 25. SEM microvascular casts of the omasal papilla of the buffalo fetus of the CRL 38 cm. Cast
of the omasal papilla in developing.
The mucosa epithelium shows in the basal zone the presence of the elongated cells,
longitudinally arranged, with a dark nucleus. At SEM, the vascular casts of the reticular
mucosa show an arrangement of the blood capillaries that reflects the typical
morphostructural organization (Figure 23). The short vascular formations are located along
the border and on the wall of the reticular crests. Omasum – The papillae are evident along
the surface of the omasal laminae. At SEM, the vascular casts of the omasal laminae appear
like a capillary network located on both sides of the lamina. The capillary network raises the
vascular agglomerated at regular intervals, about 200 µm (Figure 24).
200
Gaetano Scala and Lucianna Maruccio
Figure 26. SEM microvascular casts of the omasal papilla of the buffalo fetus of the CRL 38 cm. Cast
of the omasal papilla in advanced development.
Figure 27. SEM abomasal internal surface of the buffalo fetus of the CRL 38 cm.
Figure 28. SEM microvascular casts of the abomasal mucosa of the buffalo fetus of the CRL 30 cm.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
201
Figure 29. SEM microvascular casts of the abomasal mucosa of the buffalo fetus of the CRL 38 cm.
Figure 30. SEM microvascular casts of the abomasal mucosa of the buffalo fetus of the CRL 38 cm.
Cross-section.
The vascular agglomerates are formed from casts of omasal papillae in different period of
development (Figures 25, 26). Abomasum – At SEM, the inner surface seems a like stamped
carpet (Figure 27). The SEM specimens of the vascular cast of the abomasal mucosa show the
opening of the abomasal glands that uniformly perforate the inner surface of the abomasum
(Figure 28). In advanced development, the capillary networks that circumscribe the gland
openings are elongated and irregular (Figure 29). The capillary network originates from the
blood arterioles, located on the lamina propria (Figure 30The presence of the angiogenesis
phenomena in the capillaries is evident (Figure 31).
202
Gaetano Scala and Lucianna Maruccio
Figure 31. SEM microvascular casts of the abomasal mucosa of the buffalo fetus of the CRL 38 cm. An
angiogenesis case.
3.6. Immunogold-Labeling SEM Analysis
Intense CD133 immunoreactivity was detected in the stomach mucosa of the fetuses of
CRL 60 cm of the mediterranean buffalo with the immunogold-labeling SEM analysis.
The mucosa epithelium shows the presence of the immunoreactive cells on the ruminal
papillae surface (Figure 32), and on the apical surface of the reticular papillae (Figure 33).
Figure 32. SEM immunogold ruminal papilla of the buffalo fetus CRL 60 cm. Anti-CD133
immunoreactivity of the ruminal mucosa epithelium.
Fetal Mucosa Stomach of the Buffalo (Bubalus Bubalis)
203
Figure 33. SEM immunogold reticular papillae of the buffalo fetus CRL 60 cm. Anti-CD133
immunoreactivity of the reticular mucosa epithelium.
Conclusion
The mucosa stomach morphogenesis of the mediterranean buffalo shows several
morphostructural modifications that involve both the epithelium and the lamina propria. The
lamina propria seems to have a greatest role in the mucosa histogenesis. In fact, the lamina
propria observed in early stages of the mucosa stomach organogenesis appears as an abundant
structure of the connective cells and blood capillaries. These blood capillaries are like an
engine that drives the structural modifications of the mucosa epithelium. The structural
modifications observed at SEM increase the knowledge on the mucosa stomach
morphogenesis of the mediterranean buffalo. Morphogenetic and histogenetic observations of
the bovine stomach were carried out in a series of embryos ranging in crown-rump length
from 9.5 mm to 77 mm. A brief comparative survey on mammalian stomach morphology and
histology is made with the purpose of emphasizing the dearth of evidence to support the idea
of an esophageal origin for any stomach component [13]. In addition, the development and
the organogenesis of the stomach mucosa of the domestic ruminants were investigated as
subdivided in four single compartments [14,15,16]. Amasaki and Daigo have studied the
mucosa surface of the fetal bovine rumen with ruminal papillae standing at an interval of 400
m on an average with a slight increase in distance from each other during gestation [17,18].
Therefore, they have hypothesized that the successive formation of new ruminal papillae was
preceded by the formation of sub-epithelial capillary projections in the lamina propria.
Several authors have studied the stomach of the red deer during prenatal development
[19,20,21,22]. Then, the present paper on the fetal mucosa stomach of the mediterranean
buffalo shows an important involvement of the lamina propria in the structure of the mucosa
during prenatal life. Moreover, the presence of blood capillaries in the lamina propria make to
work them like an engine that differentiate the mucosa in four compartments of the buffalo
stomach. This morpho-functional action is observed particularly in the histogenesis of the
reticulum mucosa. The reticular mucosa shows an intense rearrangement due to a complex
structural organization. In fact, the subdivision of the reticular mucosa in the cells forms a
204
Gaetano Scala and Lucianna Maruccio
perfect model of the biological engineering. In conclusion, the fetal mucosa of the buffalo
stomach display the several arrangements during prenatal life that clarify the complexity of
the ruminant mucosa stomach, addressed primarily to cellulose digestion.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
O.P.S. Sengar and S.N. Singh. Agra Univ. J. Res., Agra India 18, 17 (1969).
B.G. Panchamukhi, D.R. Mudholkar, and H. Srivastava. Indian J. Anim. Sci. New Delhi
45, 638 (1977).
B.G. Panchamukhi, D.R. Mudholkar, and H. Srivastava. Indian. J. Anim. Sci. 47, 463
(1977).
A.H.K. Osman and R. Berg. AnatAnz. Jena 149, 232 (1981a).
A.H.K. Osman and R. Berg. Anat. Anz. Jena 150, 516 (1981).
A.H.K. Osman and R. Berg. Anat. Anz, Jena 151, 375 (1982).
A.H.K. Osman and R. Berg. Anat. Anz. Jena 151, 467 (1982).
A.H.K. Osman and R. Berg. Anat. Anz. Jena 155, 217 (1984).
B.G. Panchamukhi and H.C. Srivastava. Anat. Histol. Embryol. 8, 97 (1979).
B.G. Panchamukhi and H.C. Srivastava. Am. J. Res. 43, 346 (1982).
G.R.H. Owen, D.O. Meredith, I. Gwynn, and R.G. Richards. J.Microsc. 207, 27 (2002).
R.G. Richards, M. Stiffanic, G.R.H. Owen, M. Riehle, J.A.P. Gwynn, and A.S.G.
Curtis. Cell Biol. Int. 2, 1237 (2001).
E.D. Warner. Am. J. Anat. 102, 33 (1958).
J.M. Vivo, A. Robina, S. Regodon, M.T. Guillen, A. Franco, A.L. Mayoral. Histol.
Histopathol. 5, 461 (1990).
J.M. Vivo and A. Robina. Anat. Histol. Embryol. 19, 208 (1990).
J.M. Vivo and A. Robina. Anat. Histol. Embryol. 20, 10 (1991).
H. Amasaki and M. Daigo. Anat. Anz. 164, 139 (1987).
H. Amasaki and M. Daigo. Anat. Histol. Embryol. 17, 1 (1988).
A. J. Franco, A.J. Masot, M.C. Aguado, L. Gomez, and E. Redondo. J. Anat. 204, 501
(2004).
A.J. Franco, E. Redondo, and A.J. Masot. J. Anat. 205, 277 (2004).
E. Redondo, A.J. Franco, and A.J. Masot. J. Anat. 206, 543 (2005).
A.J. Masot, A.J. Franco, and E. Redondo. J. Anat. 211, 379 (2007).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter XII
Mandible Shape of Ruminants:
Between Phylogeny and Feeding Habits
*
Mennecart Bastien,1, Becker Damien2
and Berger Jean-Pierre1
1
2
Department of Geosciences – Earth Sciences, Fribourg, Switzerland
Section d’archéologie et paléontologie, République et Canton du Jura,
Office de la culture, Switzerland
Abstract
Previous authors have demonstrated that the mandible morphology of ruminants is
strongly related to phylogeny and diet. We performed a geometric morphometric analysis
defined by the landmark configurations of ruminant mandible specimens, using a
Principal Component Analysis (PCA) of the variability in shape. The analysis is based on
21 fossils and 65 extant ruminant species from 65 genera (18 fossil and 47 extant)
covering the diversity of the group. As a weak intraspecific variability is assumed, one
mandible specimen per species has been used in the analysis. The PCA (PC1: 36.0% and
PC2 15.3%) reveals progressive trends in the mandible shape of extant and fossil taxa
related to phylogeny and feeding habits. Within extant ruminants, Tragulina can easily be
distinguished from the Pecora along the PC1 axis. Tragulina possess elongated
premolars, a small diastema, an enlarged angular process, a weak incisura vasorum, a
short coronoid process inclined backwards, a stout condylar process and a subvertical
ramus. Bovidae have mainly negative PC2 values and can broadly be separated from the
Cervidae that mostly plot in the positive side of the axis. However, selective browser
Bovidae plot in an overlapped area with Cervidae in the positive side of the PC2 axis.
Bovidae often bear an enlarged corpus mandibulae, a lengthened molar row, and
shortened premolar row and diastema. The ramus is more backward inclined giving a
more efficient lever arm to the masseter muscles, with a less individualized angular
process. Within Bovidae, this mandible shape change is observed along the PC2 axis. The
enlargement of the corpus mandibulae and the elongation of the molar row are related to
*
E-mail: bastien.mennecart@unifr.ch.
206
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
the increase of the hypsodonty and the grazing habit. Within Cervidae, few similar
distinctions between the mandible shape and the diet can be observed. Giraffidae belong
to a homogeneous group characterized by an extreme elongation of the diastema forming
a slender corpus mandibulae. Selective browser Bovidae, Moschidae and Antilocapridae
plot within the Cervidae/Bovidae overlapped area.
Regarding the fossil specimens, three different categories are observed. The true
Cervidae, which bear antlers, are within the extant Cervidae. Hoplitomeryx plot close to
extant Antilocapridae within the Cervidae/Bovidae overlapped area. The other Miocene
species, related to either extinct families or ancestral representatives of extant families,
plot between the primitive Gelocus villebramarensis and extant Pecora. The mandible
shape of primitive Tragulina does not permit the distinction of different feeding habits.
However, primitive Tragulina form a separate group that could correspond to the
ruminant primitive state mandible shape.
Introduction
The ruminants are one of the most diverse large mammal groups in the world with about
210 species in approximately 70 genera and 6 families (see Table 1). They occupy numerous
ecological niches covering many different types of environment such as deserts, grasslands,
savannas, forested areas, and mountains. Their feeding habits vary from selective browsing to
pure grazing, classically distinguished on the base of their body mass [10,11]. However, some
large forms can feed on leaves while some smaller ruminants can ingest grass (Giraffa
camelopardis and Ourebia ourebi, respectively; see Table 2).
Table 1. Comparison between the exhaustive number of extant ruminants (data from
literature) and the sampling number used in the PCA. Bovidae, [1, 2, 3]; Cervidae, [1, 2,
4]; Giraffidae, [5, 6]; Moschidae, [2, 7]; Antilocapridae, [8]; Tragulidae, [9]. Nb:
number; PCA: this chapter; Sb: selective browser; Fl: folivore; Mx: mixed feeder; Gr,
grazer
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
207
Numerous ecomorphological studies on postcranial bones have recently demonstrated
that the ruminants, especially Bovidae, that cover the largest range of ecological variability
among the ungulates, are good ecological proxies [89,90,91,92,93].
The mandible is considered to be the biological structure that permits mastication [94].
Many authors argued that the mandible’s morphology is directly related to feeding habits
[40,95,96,97,98,99,100,101,102]. Additionally, some research has suggested a phylogenetic
link in the mandible shape evolution [40,96,98,102].
Based on a Principal Component Analysis of ruminant mandible shapes (Relative Warp
Analysis), this chapter basically follows the study of Mennecart et al. [40].
By analysing a larger sample of 86 fossil and extant ruminant species and a reassessment
of 18 anatomical landmarks, this study intends to give a more robust geometric morphometric
analysis of the relationship between mandible morphology, phylogeny and feeding habits.
Table 2. Referred specimens of 21 fossil and 65 extant ruminant mandibles used for
PCA. The feeding categories and the body mass are provided according to literature,
excepted for the species with the mention “this chapter” (Amphitragulus elegans,
Germanomeryx fahlbuschi, Hispanomeryx duriensis, Archaeomeryx optatus, Floridameryx
floridanus), where the body mass is calculated using the methods of Legendre [16],
Martinez and Sudre [42], and Scott [88]. (f), fossil; (e), extant; (Sb), selective browser;
(Fl), folivore; (Mx), mixed feeder; (Gr), grazer. Confident feeding categories of Gelocus
villebramarensis, Germanomeryx fahlbuschi, Hispanomeryx duriensis, Hoplitomeryx
matthei, Archaeomeryx optatus, Bachitherium lavocati, Dorcatherium naui, Floridameryx
floridanus, and Leptomeryx evansi are unknown in the literature. However, based on
their bunoselenodont or well-selenodont molars, we consider that these taxa were likely
to have been either selective browsers (Sb?) or folivores (Fl?) or mixed feeders (Mx?).
Composite specimens: Amphimoschus pontiliviensis (PIMUZ A/V351, PIMUZ A/V358,
PIMUZ A/V360, SMNS 47441); Bachitherium lavocati (USTL PDS1330, two unrefered
specimens from Gaimersheim stored in the BSP); Floridameryx floridanus (UF 13832,
UF 19395); Hispanomeryx matthei (MNCN-33301, MPZ 2008/177, TOR3B-28)
Ruminantia species
Family
Diet
Body mass
(kg)
References
Cervidae
Bovidae
Bovidae?
Housing
institution
Pecora
[12]
[14]
Composite
Alces alces (e)
Ammotragus lervia (e)
Amphimoschus
pontiliviensis (f)
Amphitragulus
elegans (f)
Antidorcas
marsupialis (e)
Antilocapra
americana (e)
Bison antiquus (f)
Bison bison (e)
Blastocerus dichotomus
(e)
Bos grunniens (e)
Bos taurus (e)
Fl
Gr
Fl
500-600
63-145
40
[12, 13]
[14]
[15, 16]
Moschidae?
NMB MA7926
Sb
12
Bovidae
[18]
Mx
30-40
[17, this
chapter]
[18]
Antilocapridae
[19]
Mx
45-70
[19]
Bovidae
Bovidae
Cervidae
ANSP 12976
[20]
[22]
Gr
Gr
Mx
500-700
300-900
80-125
[20, 21]
[20]
[22]
Bovidae
Bovidae
[23]
–
Gr
Mx
350-800
300
[23]
[13, comm.
pers. Vernon]
208
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
Table 2. (Continued)
Ruminantia species
Family
Boselaphus
tragocamelus (e)
Bubalus
mindorensis (e)
Budorcas
taxicolor (e)
Capra
cylindricornis (e)
Capra ibex (e)
Capra sibirica (e)
Capreolus
capreolus (e)
Capreolus
pygargus (e)
Capricornis
crispus (e)
Cephalophus maxwellii
(e)
Cephalophus natalensis
(e)
Cephalophus silvicultor
(e)
Cephalophus
zebra (e)
Cervus nippon (e)
Connochaetes
gnou (e)
Dama dama (e)
Dicrocerus
elegans (f)
Dremotherium feignouxi
(f)
Dremotherium
guthi (f)
Eotragus
sansaniensis (f)
Gazella gazella (e)
Gazella dorcas (e)
Gazella
soemmeringi (e)
Gazella
subgutturosa (e)
Gazella thomsoni (e)
Gelocus
villebramarensis (f)
Germanomeryx
fahlbuschi (f)
Giraffa camelopardalis
(e)
Hispanomeryx
duriensis (f)
Hoplitomeryx
matthei (f)
Hydropotes
inermis (e)
Madoqua
guentheri (e)
Diet
Bovidae
Housing
institution
[24]
References
Mx
Body mass
(kg)
170-270
Bovidae
[25]
Mx
180-300
[25]
Bovidae
[26]
Mx
250-350
[26]
Bovidae
[27]
Mx
55-140
[27]
Bovidae
Bovidae
Gr
Gr
67-117
130
[28]
[29]
Fl
25
[30, 31]
Cervidae
[28]
[29]
MHNF 90171979
[32]
Fl
32-49
[32]
Bovidae
[33]
Fl
30-45
[33]
Bovidae
[34]
Sb
8-10
[34]
Bovidae
NMB 3572
Sb
12-14
[35]
Bovidae
NMB 9279
Sb
43-80
[35]
Bovidae
NMB 2684
Sb
15-20
[35]
Cervidae
Bovidae
[36]
[37]
Mx
Gr
26-33
110-160
[36]
[37]
Cervidae
[38]
Gr
40-70
[38]
Cervidae
[39]
Fl
50
[15, 16, 39]
Moschidae?
NMB MA7925
Fl
15
[7, 40]
Moschidae?
[41]
Mx
11-16
[41, 42, 43]
Bovidae
[44]
Fl
27
[9, 16, 44]
Bovidae
Bovidae
NMB 7035
NMB 11031
Mx
Mx
15-20
15-23
[35]
[13, 35]
Bovidae
NMB 8369
Mx
35-46
[35]
Bovidae
NMB 2497
Mx
20-43
[45]
Cervidae
[24]
Bovidae
NMB 7571
Mx
17-30
[13, 35]
Gelocidae
[46]
Sb?
14
[40, 46]
Palaeomerycidae
Giraffidae
Composite
Fl?
>100
[48]
Fl
1000-1150
[15, 47, this
chapter]
[48, 49]
Moschidae
Composite
Fl?
5-6
Hoplitomerycidae
[52]
Sb?
15-20
[50, this
chapter]
[51, 52]
Cervidae
NMB
Fl
8-12
[13, 53]
Bovidae
[54]
Sb
3-5
[54]
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
Ruminantia species
Family
Housing
institution
Diet
Body mass
(kg)
References
Madoqua kirkii (e)
Madoqua saltiana (e)
Mazama
nemorivaga (e)
Moschus
moschiferus (e)
Muntiacus muntjak (e)
Nemorheadus
goral (e)
Neotragus
moschatus (e)
Odocoileus
hemionus (e)
Odocoileus virginanus
(e)
Okapia johnstoni (e)
Oreamno
americanus (e)
Oreotragus oreotragus
(e)
Ourebia ourebi (e)
Ovis ammon (e)
Ovis canadensis (e)
Ovis dalli (e)
Ozotoceros bezoarticus
(e)
Pantholops
hodgsonii (e)
Procapra
gutturosa (e)
Procervulus
dichotomus (f)
Przewalskium
albirostre (e)
Pseudois nayaur (e)
Pudu puda (e)
Raphicerus
melanotis (e)
Saiga tatarica (e)
Sylvacapra
grimmia (e)
Taurotragus oryx (e)
Tetracerus
quadricornis (e)
Tragelaphus eurycerus
(e)
Bovidae
Bovidae
[55]
NMB 8064
Sb
Fl
4-5
3-4
[55]
[35]
Cervidae
[56]
Sb
8-30
[56, 57]
Moschidae
NMB 5110
Fl
4-12
[7, 30]
Cervidae
Bovidae
NMB 3744
[58]
Fl
Mx
7-12
22-35
[30]
[58]
Bovidae
NMB 2122
Fl
4-9
[35]
Cervidae
[59]
Fl
70-150
[59]
Cervidae
[60]
Fl
45-65
[13, 60]
Giraffidae
Bovidae
[61]
[62]
Fl
Gr
200-300
45-135
[61]
[62]
Bovidae
NMB 8360
Mx
5-16
[13, 35]
Bovidae
Bovidae
Bovidae
Bovidae
Cervidae
NMB 8773
[63]
[64]
[65]
[66]
Gr
Gr
Gr
Mx
Mx
8-21
110-182
70-95
45-85
30-40
[35]
[63]
[64]
[65]
[66]
Bovidae
[67]
Mx
26-39
[67]
Bovidae
[68]
Gr
20-40
[68]
Cervidae
[69]
Mx
40
[15, 16, 69]
Cervidae
[70]
Gr
180-230
[70]
Bovidae
Cervidae
[71]
NMB 2209
Gr
Sb
35-75
10
[71]
[57]
Bovidae
NMB 4228
Mx
9-11
[13, 35]
Bovidae
NMB 1908
Gr
35
[30, 72]
Bovidae
NMB 8400
Fl
11-26
[13, 35]
Bovidae
Bovidae
[73]
[74]
Mx
Sb
340-600
17-22
[73]
[74]
Bovidae
[75]
Fl
150-220
[75]
[76]
Sb?
<5
[76,
this chapter]
NMB Qu.B.63
Sb
7-8
[77, 78]
Coll. Loggia
Fl?
36
[79]
Composite
Mx?
[39]
Sb?
Tragulina
Archaeomeryx optatus
(f)
Bachitherium cf.
curtum (f)
Bachitherium
insigne (f)
Bachitherium
lavocati (f)
Dorcatherium naui (f)
Archaeomerycidae
Bachitheriidae
Bachitheriidae
Bachitheriidae
Tragulidae
7-8
24
[42,
this chapter]
[9, 16, 80]
209
210
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
Table 2. (Continued)
Ruminantia species
Family
Housing
institution
Diet
Body mass
(kg)
References
Floridameryx
floridanus (f)
Hyemoschus aquaticus
(e)
Hypertragulus
hesperius (f)
Gelocidae?
Composite
Sb?
6-10
[81,
this chapter]
Tragulidae
NMB 8699
Sb
12
[13, 30]
Hypertragulidae
Leptomerycidae
[76]
Mx
<5
[76, 82]
[83]
Sb?
3
[83, 84]
Tragulidae
NMB 2588
Sb
5
[9, 13]
Hypertragulidae
[83]
Sb
3
[83, 85]
Tragulidae
NMB 1891
Sb
1-4
[13, 30]
Tragulidae
Tragulidae
NMB 3002
NMB 10007
Sb
Sb
4
2
[86]
[86]
Leptomeryx evansi (f)
Moschiola
meminna (e)
Nanotragulus
loomsi (f)
Tragulus
javanicus (e)
Tragulus kanchil (e)
Tragulus napu (e)
Material and Methods
Sampling
This analysis is based on mandible specimens of 21 fossil and 65 extant ruminant species
from 65 genera (18 fossil and 47 extant), stored pro part in the Bayerische Staatssammlung
für Paläontologie (Germany), the Staatliches Museum für Naturkunde Stuttgart (Germany),
the Université des Sciences et Techniques du Languedoc (France), the Paläontologisches
Institut und Museum der Universität Zürich (Switzerland), the Musée d’histoire naturelle de
Fribourg (Switzerland), and the Naturhistorisches Museum Basel (Switzerland), and
extracted pro parte from literature (see Table 2).
The juvenile specimens and the specimens from captive individuals have been excluded
from the Principal Component Analysis, because they do not always bear all adult characteristics
and sometimes display abnormal shape developments, respectively. On the other hand, a low
intraspecific variability of the mandible morphology being assumed within wild adult
ruminants (personal observation), the sampling included one adult gender-unspecific
specimen per species (priority left mandibles).
The referred specimens of extant ruminants cover all actual ruminant families and
represent two-thirds of the actual generic diversity and one-third of the actual specific
diversity (see Table 1). The sampling strategy aims to optimally cover the size range and the
feeding categories within all infraorders of extant ruminants (Tragulina and Pecora) (Figure 1
and 2). The feeding categories are mainly based on those of Janis [13]: Sb, selective browser
(fruits and dicotyledonous herbage foliage selector); Fl, folivore (at least 90% of
dicotyledonous herbage); Mx, mixed feeder (intermediate feeder with variable diets of
dicotyledonous and monocotyledonous plants); Gr, grazer (at least 90% of monocotyledonous
grass material).
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
211
Regarding the fossil sampling, we focused on primitive Tragulina and Pecora from the
Middle Eocene to the Oligocene of Eurasia and North America, and we completed the
sampling pool with mandibles of derived taxa from the Neogene of the same area. Due to
badly preserved samples, the analysis of Floridameryx floridanus, Bachitherium lavocati,
Amphimoschus pontiliviensis, and Hispanomeryx matthei has been based on composite
specimens.
The feeding habit attributions of the fossil and extant referred species have been
extrapolated from literature (Table 2).
Principal Component Analysis (PCA)
The mandible specimens were photographed in lateral view with a horizontal orientation
of the tooth row, using a camera FinePix S6500fc. The software TpsDig version 2.16 [104]
was used to digitize 18 anatomical landmarks (representing anatomically, geometrically, and
linked homologous points) on each picture, representing the complete mandible shape
(including the ramus and the angular process).
The selected anatomical landmarks, illustrated in Figure 1, are parameters that were
usually included in previous studies on mandible morphology [40,96,97,102,105,106]. In the
species without a first lower premolar, the corresponding landmark (anterior part of p1) is
superimposed to the next landmark (anterior part of premolar row).
The geometric morphometric analysis follows basically the method exposed by several
authors [40,107,108,109]. Conventional morphometric methods use linear distance
measurements, which are strongly correlated to size.
Figure 1. Location of anatomical landmarks used for the PCA on a ruminant mandible (Leptomeryx
evansi; AMNH 11870). Scale bar: 2 cm. 1, posterior part of c; 2, anterior part of p1; 3, anterior part of
premolars row; 4, anterior part of molars row; 5, posterior part of molars row; 6, projection of 10 on the
anterior part of the ramus; 7, maximum of convexity of the coronoid process; 8, maximum of concavity
of the coronoid process; 9, projection of 11 on the posterior part of the coronoid process; 10,
mandibular incisure; 11, condylar process; 12, maximum of concavity of the ramus; 13, largest part of
the angular process; 14, lower part of the angular process; 15, incisura vasorum; 16, projection of 5 on
the lower part of the corpus mandibulae; 17, projection of 4 on the lower part of the corpus
mandibulae; 18, projection of 3 on the lower part of the corpus mandibulae.
212
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
To eliminate the non-shape variation (size) on the landmark configurations, a General
Procrustes Analysis was performed [110]. The coordinates of the mandible landmarks were
processed by the least-square method that transforms the landmark configurations,
superimposing it (translating, scaling and rotating) on a mean shape (consensus) and resulting
in the minimal possible sum of the squares of the distances between the corresponding
homologous points [110,111].
Figure 2. 1) Diagram of body mass distribution of the various species used in the PCA, separated by
function of their family. 2) Diagram of body mass distribution of the various species used in the PCA,
separated by function of their feeding habit. The masses are provided in Table 1. The body mass
categories are very similar to those of Jarman [103] and Gagnon and Chew [35].
Thin-Plate Spline function (TPS) was applied to map the landmark configurations
represented as deformation grids, where one mandible is deformed or “warped” into another.
Shape differences can then be described in terms of deformation-grid differences depicting
the objects [110]. The shape data describing these deformations (partial warps) can be used as
shape variables for statistical comparisons of the variation in mandible shape. Principal
Component Analysis was applied to the partial warp scores resulting in the Relative Warp
Analysis. In order to achieve equal scaling of each regional shape variation, the distortion
parameter of Relative warps (Principal Component axes) was set at  = 0. This procedure is
the most suitable for exploratory and taxonomic studies [112]. The superimposing and
Relative Warp Analysis were performed using the software TpsRelw version 1.46 [113].
Softwares of the “TPS” series used in this chapter are freeware (http://life.bio.sunysb.
edu/morph).
Abbreviations
PCA, Relative Warp Analysis; PC, Principal Component axes; c, canine; p1, lower first
premolar.
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
213
ANSP, Academy of Natural Sciences Philadelphia (United States); BSP, Bayerische
Staatssammlung für Paläontologie (München, Germany); MHNF, Musée d’histoire naturelle
de Fribourg (Switzerland); MNCNB, Museo Nacional de Ciencias Naturales (Madrid,
Spain); NMB, Naturhistorisches Museum Basel (Switzerland); MPZ, Museo Paleontologico
de la Universidad de de Zaragoza (Spain); PIMUZ, Paläontologisches Institut und Museum
der Universität Zürich (Switzerland); SMNS, Staatliches Museum für Naturkunde Stuttgart
(Germany); TOR3B, Toril-3A; UF, Florida Museum of Natural History, University of Florida
(Gainesville, United State of America); USTL, Université des Sciences et Techniques du
Languedoc (Montpellier, France).
Results
The 18 anatomic landmarks generated 32 axes (PC) for each mandible specimen. The
results of the PC’s, using shape components, permitted the distinction of different groups of
ruminants based on the total difference of the mandible shape. PC1 explained 36.0% and PC2
15.3% of total shape variance (Figure 3).
This means that 51.3% of the total shape variance can be explained without the use of
other PC’s. On PC1, elongation of the diastema is positively associated with the thinning-up
of the corpus mandibulae on its posterior part and of the ramus, and the disappearance of p1
(see shape deformation grids in Figure 3.1). On PC2, the diastema and premolar row
elongation, the shallowing-up corpus mandibulae, the forward projection of the ramus, the
salience of the angular process, and the development of the incisura vasorum occur in a
positive variance (see shape deformation grids in Figure 3.1).
Both PC1 and PC2 are informative from both a phylogenetic (Figure 3.2) and ecologic
(Figure 3.3) perspective. These two PC’s permit the net discrimination of the extant
Ruminantia infraorders, Tragulina and Pecora, by a vertical separation line. The character
combination of extant Tragulina (Tragulidae) is a weak incisura vasorum, a sub-vertical
ramus, a short coronoid process inclined backwards, a weakly developed angular process, a
stout condylar process, a rather short diastema (due to the presence of a p1), and a lengthened
premolar row. They plot in a small area from -0.042 to -0.023 on PC1 and from 0.004 to
0.013 on PC2, and are exclusively selective browsers (Figure 3, Table 2). On the other hand,
the mandible shape of extant Pecora (Bovidae, Cervidae, Giraffidae, Moschidae, and
Antilocapridae) is characterized by a strong incisura vasorum, a slender ramus inclined
backwards, a developed coronoid process, a slender condylar process and corpus mandibulae,
a long diastema, and shortened premolar row.
The p1 can be either separated from the other premolars or integrated to the premolar
row, or absent (see shape deformation grids in Figure 3.1). The extant Pecora mainly plot as
positive PC1 values, and negative and positive PC2 values. Bovidae exclusively plot in the
negative-values domain of PC2, whereas Cervidae plot preferentially in the positive-values
domain (only Pudu puda plots in the negative-values domain of both PC1 and PC2, but very
close to 0). An overlapped area between Cervidae and Bovidae occurred in the lowest
positive-values domain of PC2 (from 0 to 0.036). Moschidae and Antilocapridae plot in this
overlapped area. Giraffidae plot in the highest positive-values domain of PC2 (> 0.065), and
are clearly discriminated from other Pecora (Figure 3.2).
FOSSIL
PECORA
FOSSIL
TRAGULINA
214
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
C
1)
B
1. TRAGULINA, Archaeomeryx optatus
2. TRAGULINA, Hypertragulus hesperius
3. TRAGULINA, Bachitherium insigne
4. TRAGULINA, Bachitherium curtum
5. TRAGULINA, Leptomeryx evansi
6. TRAGULINA, Nanotragulus loomsi
7. TRAGULINA, Floridameryx flo ridanus
8. TRAGULINA, Bachitherium lavocati
9. TRAGULINA, Dorcatherium naui
10. PECORA, Gelocus villebramarens is
11. PECORA, Amphimoschus pontiliviensis
12. PECORA, Amphitragulus elegans
13. PECORA, Dremotherium guthi
14. PECORA, Germanomeryx fahlbuschi
15. PECORA, Eotragus sansaniensis
16. PECORA, Hispanomeryx duriensis
17. PECORA, Dremotherium feignouxi
18. PECORA, Procervulus dichotomus
19. PECORA, Dicroceros elegans
20. PECORA, Hoplitomeryx matthei
21. PECORA, Bison antiquus
A
D
a.
2)
0.07
Cervidae
0.06
Moschidae
0.05
Bovidae
17
Fossils
11
0.02
10
0.01
%
.3
5
:1
2
w
R
0.00
Extant
Tragulidae
18
13
15
Overlapped area:
Cervidae/Bovidae
A
a
Primitive
Tragulina
-0.02
1
2
-0.04
-0.05
9
3
4
7
5
21
6
-0.06
Extant Bovidae
-0.07
D
-0.10 -0.09 -0.08 -0.07 -0.06 -0.05 -0.04 -0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
Rw1: 36.0%
3)
C
0.08
Sb
Fl
Mx
Gr
0.07
Tragulidae
0.06
Cervidae
0.05
Moschidae
Bovidae
0.04
Giraffidae
0.03
Antilocapridae
0.02
Fossils
0.01
%
.3
5
1
:
2
w
R
0.00
-0.01
B
4
7
5
20
14
8
9
3
2
19
16
B
-0.01
-0.03
12
14
8
1
0.03
Antilocapridae
18
16
13
10
Extant
Cervidae
0.04
Giraffidae
12
15
PECORA, Pudu puda
C
0.08
Tragulidae
17
11
A
a
-0.02
-0.03
-0.04
-0.05
-0.06
-0.07
D
-0.10 -0.09 -0.08 -0.07 -0.06 -0.05 -0.04 -0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
Rw1: 36.0%
Figure 3. (opposite page). PCA for distortion parameter  = 0 of ruminant mandibles obtained from 21 fossil species
(Archaeomeryx optatus: Shara Murun, Late Eocene; Hypertragulus hesperius: John Day, Late Oligocene; Bachitherium
curtum: Quercy, Oligocene; Bachitherium cf. insigne: Cereste, Early Oligocene; Bachitherium lavocati: Pech Desse and
Gaimersheim1, Late Oligocene; Leptomeryx evansi: Cheyenne River, Early Oligocene; Nanotragulus loomsi: Muddy
Creek, Late Oligocene; Dorcatherium naui: Griesbeckerzell, Middle Miocene; Gelocus villebramarensis: Villebramar,
Early Oligocene; Amphimoschus pontiliviensis: Kapfnach and Languenau1, Early and Middle Miocene; Amphitragulus
elegans: Montaigu le Blin, Early Miocene; Dremotherium guthi: La Milloque, Late Oligocene; Germanomeryx falhbuschi:
Sandelzhausen, Middle Miocene; Eotragus sansaniensis: Sansan, Middle Miocene; Hispanomeryx duriensis: Toril-3 and
Manchones-1, Middle Miocene; Dremotherium feignouxi Montaigu le Blin, Early Miocene; Procervulus dichotomus:
Rauscheröd, Early Miocene; Dicrocerus elegans: Sansan, Middle Miocene; Bison antiquus: Holocene) and 65 extant
species (see Table 1 and 2). 1) Shape deformation grids representing the mean shape (consensus) and the maximum values
of PC1 and PC2. 2) Scatter plots of PC1 versus PC2 with taxonomic characterization. The A-B axis indicates the shape
variation of the mandible “warped” from the mean shape (consensus) into the maximum positive (A) and negative (B)
deviations in the axis of PC1; the C-D axis the shape variation of the mandible “warped” from the mean shape (consensus)
into the maximum positive (C) and negative (D) deviations in the axis of PC2. 3) Same scatter plots as in 2) with diet
characterization.
6
19
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
215
Furthermore, within extant Pecora, the feeding habits are strongly related to variations in
the mandible shape (Figure 3.3).
Grazer Bovidae plot exclusively in the negative-values domain, PC2 and selective
browser Bovidae mainly plot in the positive-values domain PC2. In positive-values domain
PC1 values, a trend from grazers to mixed feeders, folivore and selective browser Bovidae is
discriminated by a progressive increase of PC2 values (Figure 3.3 and Figure 4.1 to 4.4).
There is a progressive transition from a slender corpus mandibulae with an elongated
diastema and premolar row, a marked incisura vasorum and angular process, and subvertical
slender ramus in the selective browser Bovidae to a stocky corpus mandibulae with a short
diastema and premolar row, a few developed incisura vasorum and angular process, and
backward enlarged ramus in the grazer Bovidae. Within extant Cervidae, the selective
browsers and folivores can be differentiated from mixed feeders and grazers (Figure 3.3).
Plotted in the highest positive-values domain of PC1 and the lowest positive-values domain
of PC2, the mixed feeder and grazer Cervidae is characterized by a less marked angular
process, a shorter premolar row, an elongated diastema, and a slightly backward inclined
ramus (Figure 3.3 and Figure 4.12 to 4.15). Moschidae and Antilocapridae, represented by
only one species each (Moschus moschiferus and Antilocapra americana), plot in the same
values domain as folivore Cervidae and mixed feeder Cervidae. Only one feeding habit,
folivore, characterizes the Giraffidae. Regarding the fossil taxa, the separation line of fossil
Tragulina/Pecora is clear but differs from extant Tragulina/Pecora by an anticlockwise
rotation of ca. 45˚. The primitive Tragulina form a distinct group plotted exclusively in the
quadrant defined by negative values of both PC1 and PC2. The primitive ruminant
Archaeomeryx optatus has the most negative value of PC1 (-0.097; Figure 3). The three
species of Bachitheriidae (Bachitherium cf. curtum, B. insigne, B. lavocati) are concentrated
in a very low variation of PC1 values (from -0.068 to -0.082; Figure 3). The Miocene
Tragulidae Dorcatherium naui plots between the primitive Tragulina and the extant
Tragulidae.
Figure 4. Compilation of shape deformation grids indicating the feeding habit trend within Bovidae (14) and Cervidae (5-8). 1, Cephalophus natalensis; 2, Capricornis crispus; 3, Gazella subgutturosa; 4,
Saiga tatarica; 5, Pudu puda; 6, Muntiacus muntjack; 7, Cervus Nippon; 8, Przewalskium albirostre.
The supposed Gelocidae Floridameryx floridanus is very close to the primitive Tragulina
Leptomeryx evansi and Nanotragulus loomsi (Figure 3). Gelocus villebramarensis has the
most negative value of PC1 (-0.066; Figure 3) within Pecora and is somewhat isolated, but
with a positive value of PC2 (0.008; Figure 3) that differs from the negative-values domain of
primitive Tragulina. Most of the Miocene Pecora species plot between Gelocus
216
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
villebramarensis and extant Cervidae area. Even Eotragus sansaniensis, the oldest known
true Bovidae [44,114], plots in this transitional zone. However, the Early/Middle Neogene
Ruminantia Dicroceros elegans and Procervulus dichotomus plot within the values domain of
extant Cervidae. Only the Pliocene Hoplitomerycidae Hoplitomeryx matthei plots in a totally
singular location, close to extant Antilocapridae. Contrary to extant taxa, the distinction of
feeding habits among the fossil specimens is unclear, except for the grazer Bison antiquus,
that is a subactual Bovidae plotted logically within extant grazer Bovidae.
Discussion
Phylogeny
As suggested by Mennecart et al. [40], the results of the PCA confirm that the mandible
shape of ruminants can be used to distinguish the main taxonomic groups, especially at the
infraorder level (Tragulina versus Pecora). However, mandible shapes of primitive Tragulina
and Pecora differ from those of their extant relatives (Figure 3.2). Primitive Tragulina form a
homogenous group plotted in the quadrant defined by both negative PC1 and PC2 values.
They are characterized by a p1 separated from other premolars, a short diastema, a thick
corpus mandibulae, a developed angular process, and a wide vertical ramus. The mandible of
the primitive Tragulina is close to the basic shape observed in ruminant evolution.
Archaeomeryx optatus, often considered as the most primitive grade of assigned ruminants
[76,85,115,116,117], has the lowest PC1 value. This ruminant possesses the characteristic
“fused” cubonavicular bone, but upper incisors are persistent, the median metapods are
unfused, and the lateral ones are not reduced [115]. Bachitherium species are concentrated in
a small PC1 range values. Regarding PC2 values, Bachitherium lavocati is separated from the
very close B. curtum and B. insigne. This medium size Bachitheriidae is the later known
Bachitherium (MP28). It has an elongated diastema, as of the larger B. insigne (Sudre 1986).
Because Brachitherium curtum and B. insigne have a similar biostratigraphic range and have
been discovered in some similar localities, Wehrli [119] suggested a synonymy of these two
species and a marked sexual dimorphism. However, the diastema length and the proportions
of the postcranial remains indicate obvious different ecological niches. Bachitherium curtum
lived in wooded humid area whereas B. insigne lived in light forest habitats [78]. According
to Sudre [120], the most suitable hypothesis is to maintain two distinctive species, despite the
similarity of the mandible shapes.
The mandible shape of Dorcatherium naui discriminates it from primitive Tragulina, but
it also differs from extant Tragulidae by having a p1, a shorter diastema, and a stockier
corpus mandibulae. Extant Tragulidae form a homogenous group composed only of selective
browsers, but it is impossible to determine if the consistency of the PC values are controlled
by a strong taxonomic or feeding affinity (Figure 3). As a result, a general trend within
Tragulina is observed from primitive Ruminantia (Archaeomeryx optatus), primitive
Tragulina (Hypertragulus hesperius), to primitive Tragulidae (Dorcatherium naui), and to
extant Tragulidae (Tragulus napu) (Figure 5.1 to 5.4). The mandible evolves to a slender
shape, a lengthened diastema, a loss of p1, a salient mandible process, and a backward
inclined ramus.
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
217
The Late Miocene Pseudoceratinae Floridameryx floridanus of North America plots
within the primitive Tragulina in the PCA (Figure 3.2). Webb [81] considered this taxon as a
primitive Ruminantia by having an upright lower canine and no appendages, belonging to
Gelocidae on the basis of basicranial features and premolar structures. However, Métais and
Vislobokova [85] argued that Pseudoceratinae, including Pseudoceras, are probably not
Gelocidae, because the related postcranial and molar features are closer to those of Moschus
than Gelocus. Pseudoceratinae have a combination of primitive and derived features that
should be assigned to another family. Moreover, Gelocidae seem to be restricted to the Early
Oligocene of Europe [121]. Hooker and Weidmann [122] argued that the unique and lost
specimen of Gelocus? minor does not come from Mormont (Late Eocene), but from another
unknown locality, dating from after the Grande-Coupure. “Gelocus” whitworthi from the
African Miocene differs from European Gelocus species by having a well-developed
metastylid, an elongated and forked postencristid, and a marked Palaeomeryx fold on the
lower molars [17,123,124, personal observation]. Early Miocene Gelocus gajensis from Asia
clearly displays Lophiomerycidae characteristics such as a rounded metaconid, open trigonid
and talonid, and a crested third basin on lower molars [125, personal observation].
Gelocus villebramarensis has the most negative PC1 value of the Pecora (Figure 3.2) and
is considered as Protopecora or the most primitive Pecora [85,126,127]. It is defined by
having a short diastema and elongated premolars with a relatively large corpus mandibulae in
comparison with extant Pecora, and a p1 either isolated or adjacent to premolars (Figure 5.5).
Tooth p1 is adjacent to premolars in Amphitragulus elegans and Dremotherium guthi, and is
absent in other Pecora. According to the evolutionary trend (Figure 5), the loss of p1 is
associated to a lengthening of the diastema. Based on the PCA (Figure 3.2), Gelocus
villebramarensis seems closer to “tragulid-like” taxa than extant Pecora. However, it clearly
differs from primitive Tragulina by having a more elongated diastema and a more slender
mandible shape.
Figure 5. Compilation of shape deformation grids indicating the evolutionary trends in the mandible
shape from the primitive grade Archaeomeryx optatus (2-4 for Tragulina and 5-9 for Pecora). 1,
Archaeomeryx optatus; 2, Hypertragulus hesperius; 3, Dorcatherium naui; 4, Tragulus napu; 5,
Gelocus villebramarensis; 6, Procervulus dichotomus; 7, Alces alces; 8, Eotragus sansaniensis; 9,
Gazella gazella.
218
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
Regarding the genus Dremotherium (Late Chattian to Aquitanian), the premolars become
shorter and the corpus mandibulae more slender. The two species, D. guthi and D. feignouxi,
have a similar PC1 value and the difference on the PC2 is probably related to their distinctive
feeding habits (Figure 3). The Miocene referred specimens form a homogeneous group
(Figure 3). Miocene Procervulus dichotomus and Dicrocerus elegans cannot be distinguished
from the extant Cervidae. An evolutionary trend can be observed in the shape variation of the
cervid mandibles from the primitive Ruminantia (Archaeomeryx optatus), to the primitive
Pecora (Gelocus villebramarensis), the primitive Cervidae (Procervulus dichotomus) and the
extant Cervidae (Alces alces) (see Figure 5.1, 5.5 to 5.7). The mandible becomes very slender
with a more marked incisura vasorum and an elongated diastema. The premolar row is
slightly reduced and the molar row slightly elongated. The mandible process is smaller, but
more salient, and the ramus is more backward inclined.
Other fossil Pecora, condensed between the primitive Gelocus villebramarensis and
extant Cervidae, are rather removed from extant relatives, except for the subactual Bison
antiquus, which has a logical position within grazer Bovidae, and the insular Pliocene
Hoplitomeryx matthei, which is isolated. The most suitable explanation is that the mandible
shapes of the Early Miocene ruminants are still primitive and quite analogous. Furthermore,
Palaeomerycidae are extinct and the extant Moschidae diversity is not representative of the
past Moschidae.
In the literature, no consensus about the family attribution of Amphitragulus and
Dremotherium has been suggested. These true Eupecora, which appeared during the Late
Oligocene [41,79], have been considered as Palaeomerycidae [17,128], Cervidae [129,130],
and most often as Moschidae [7,41,80,131]. The attribution to Moschidae is mostly based
only on the presence of an elongated saber-like upper canine and the absence of appendages.
But these features are observed in all European ruminants until the Burdigalian.
Amphitragulus and Dremotherium probably do not belong to extant family, but to a
transitional one, which has been replaced consecutively due to the migration of true Cervidae
(Ligeromeryx praestans and Procervulus praelucidus) related to the Brachyodus event during
the Burdigalian [69,132,133].
Many authors argued that Hoplitomeryx and Amphimoschus belong to the family
Hoplitomerycidae on the basis of a large, bicuspid third lobe on m3 in which the median
valley between both cusps is open posteriorly, which appears to be a diagnostic character of
Pecora. Both genera share a large, inflated and smooth surfaced auditory bulla, lack a
Palaeomeryx fold, and have fairly high crowned molars [17,52,134]. However, in the PCA,
these two species do not plot together, Amphimoschus artenensis plotting close to
Amphitragulus elegans and Hoplitomeryx matthei within the extant Cervidae area (Figure 3).
After Gentry [135] and Gentry et al. [80], Amphimoschus seems to be related to Bovidae. On
the other hand, Hoplitomeryx is an insular mammal having singular characteristics, such as
five horns. The resemblance between these two genera is probably due to convergences.
Eotragus sansaniensis is the oldest true Bovidae known [80,195]. Solounias et al. [195]
consider it as an ancestor of the extant Boselaphini (Tetracerus quadricornis and Boselaphus
tragocamelus). These three species have quite similar PC2 values with a rather large range of
PC1 values that confirm the primitive stage of Eotragus sansaniensis. An evolutionary trend
can be observed in the shape variation of the bovid mandibles from the primitive Ruminantia
(Archaeomeryx optatus), to the primitive Pecora (Gelocus villebramarensis), the primitive
Bovidae (Eotragus sansaniensis) and the extant Bovidae (Gazella gazella) (see Figure 5.1,
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
219
5.5, 5.8 and 5.9). The mandible becomes larger with a weakly marked incisura vasorum and
an elongated diastema. The premolar row is reduced and the molar row slightly elongated.
The mandible process is smaller, but more salient, and the ramus is more backward inclined.
Phylogenetic relationships between Giraffidae and primitive Palaeomerycidae are still
under discussion. The most recent analysis suggests that they are a sister group [6]. Extant
Giraffidae plot totally in isolation due to the extreme lengthening of the diastema and the high
gracility of the mandible shape. The PCA does not identify a phylogenetic or feeding control
of the singular and highly homogeneous mandible shape, because extant Giraffidae are
exclusively folivore. After the dental micro-wear analysis of Franz-Odendaal and Solounias
[138], the giraffid Sivatherium hendeyi from Early Pliocene of South Africa was identified as
a mixed feeder. Regrettably, neither direct observation nor literature allowed us to consider
this taxon in our analysis. However, an illustration of Sivatherium hendeyi from Alleyne
Nicholson [138, figure 245] shows a very slender mandible with an elongated diastema very
similar to those of Giraffa camelopardalis and Okapia johnstoni.
Cervidae and Bovidae are highly diversified mammal families, and the specimens of
these families are the most abundant in our PCA (Table 1). The related species analysed
present a very large range of body mass and feeding habits (Figure 2., Table 1). Cervidae
mainly plot in positive-values domain of PC2 and Bovidae in the negative-values domain.
The cervids generally have a more slender mandible with an elongated diastema, a
proportionally larger premolar row, a marked incisura vasorum, and a smaller but more
salient mandible process (Figure 5). However, a mixed area between these two families is
observed. This area groups the most massive Cervidae and the most slender Bovidae.
The extant Moschidae Moschus moschiferus and the past Hispanomeryx duriensis have
similar PC2 values. But, as for Eotragus sansaniensis, the respective PC1 values permits us to
make the distinction between fossil and extant species.
Feeding Habits
The PCA of this chapter reveals the general trends in the mandible shape variation as
related to feeding habits (selective browser, folivore, mixed feeder and grazer) especially of
extant ruminants, but less obviously in fossil ruminants. The families represented by a unique
representative or with an uncertain feeding habit do not discriminate consistent feeding trends
(Antilocapridae, Archaeomerycidae, Gelocidae, Hoplitomerycidae, Hypertragulidae,
Leptomerycidae, Moschidae, Palaeomerycidae). Within primitive ruminants, the feeding habit
related to mandible shape is not apparent. Particularly, the mandible shape of primitive
Tragulina does not permit us to differentiate different feeding behaviours (Figure 3.3). For
example, according to a dental micro-wear study of Blondel [77], Bachitherium curtum was a
selective browser feeding on leaves and fruits, and Bachitherium insigne, which was a larger
form, could have fed on leaves. However, no obvious distinction in mandible shape is
observed (Figure 3.3). Bachitherium lavocati show a very faint difference in mandible shape
compared with other Bachitherium species (especially the ratio between the molars row
length and the diastema length). This taxon, based on postcranial remains analysis, lived in
more open area [78] and could have included more grass in its diet, such as the
contemporaneous Bedenomeryx lamilloquensis and Dremotherium guthi [43].
220
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
Within extant Pecora, an unambiguous trend of the feeding habits, from selective browser
to grazer, is observed in Bovidae (Figure 3.3 and Figure 4.1 to 4.4), which is assumed to be a
good proxy for ecomorphological analyses [90,91,92,93]. The shortening of the diastema and
of the premolar row (compared to the molar row), associated with a smooth incisura vasorum
and a faint angular process, leads to a thickening of the corpus mandibulae that correlates
with the high molar crown, thus with an increase of the hypsodonty index (high crown/molar
length ratio) [97]. The strongly hypsodont dentition permits feeding on grass containing
abrasive phytoliths, typical of grazer habit [97]. Moreover, the backward tilting of the ramus,
also observed in grazer Bovidae, permits them to have a better lever arm of the mastication
muscles located on the ramus (masseter, temporalis). These muscles strongly contribute to
mastication [139]. Consequently, the association of a high hypsondonty index, a stock
mandible and an efficient lever arm characterize the tough food eater Bovidae. These features
are notably well-developed in the subactual grazer Bison antiquus. Extant Cervidae do not
show such a clear feeding trend as within the Bovidae, but we can differentiate between the
group formed by the selective browsers and the folivores, and the group formed by the mixed
feeders and the grazers. Even if it is not so marked within the Bovidae, Cervidae with the
stockiest mandibles feed on more fibrous vegetables, such as grass. The difference of
consistency of results between these two families is probably due to the fact that few
Cervidae have been analyzed in comparison to Bovidae (Table 1), and that Bovidae possess a
larger biodiversity range than Cervidae. In the PCA, the two analyzed Giraffidae are folivores
and are clearly discriminated. The extinct genus Sivatherium (not analyzed in this chapter)
shows a mandible quite similar to extant Giraffidae by having an extremely elongated
diastema and a rather slender shape. However, this genus has been considered as a mixed
feeder by Franz-Odendaal and Solounias [137] and in the illustration of Alleyne Nicholson
[138, Figure 245], the premolar row is reduced, the corpus mandibulae is distally massive,
and the ramus is inclined backwards. These characteristics, similar to those observed within
the mixed feeder Bovidae, suggest that Giraffidae also show an analogous trend of the
mandible shape variation related to feeding habits.
Regarding the fossil taxa, the correlation of the mandible shapes with the feeding habits
is much less apparent, probably due mainly to the feeding specializations being less
developed. For example, the referred Dremotherium species have quite a similar mandible
shape, except a slightly more slender corpus mandibulae in D. feignouxi (Figure 3). However,
according to postcranial and dental micro-wear studies [78,43], Dremotherium guthi probably
lived in open forests and fed on grass, whereas the postcranial remains of the later and larger
D. feignouxi reveal an animal living in open areas [140]. Indeed, the latter has an elongated
cervical [17,130] as in extant folivore antelopes Litocranius walleri and Ammodorcas clarkei
[17,35,141]. We suggest a similar diet for D. feignouxi.
Conclusion
The ruminant mandible shape is a useful proxy to differentiate the main ruminant
taxonomic groups through time. Primitive Tragulina possess a stockier mandible than
primitive Pecora, and it is also the case for the extant representatives. In both cases, the more
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
221
negative PC1 values correspond to more primitive mandible shapes and the evolutionary
trend led to a more slender form with a more elongated diastema and a lost p1.
First assigned to Gelocidae, Floridameryx floridanus seems to belong to Tragulina.
However, without taking into account other morphological and morphometrical
characteristics, the PCA does not supply sufficient information to discuss family level
taxonomy. Within extant Pecora, Giraffidae differ by having an extremely elongated
diastema. Cervidae are generally more slender than Bovidae, but an overlapped area between
these two families groups the most massive Cervidae and the most slender Bovidae together.
Regarding the feeding habits, the well-diversified extant Bovidae show that an
unambiguous trend from selective browser to grazer relates to the variation in mandible
shape. The terminal state of this trend shows the combination of a high hypsondonty index, an
elongated molar row compared to the premolar row, an enlarged corpus mandibulae, a
stockier mandible, and a more efficient lever arm of the mastication muscles (masseter and
temporalis) that permits feeding on tough food such as grass. This trend is also roughly
observed within non-Bovidae Pecora, but to a lesser extent. On the other hand, confident
feeding category discrimination for fossil taxa cannot be clearly identified. Only the cervid
Dicroceros elegans and the subactual bovid Bison antiquus show mandible shapes in
coherence with the feeding habits observed within extant representatives.
Acknowledgements
This project was financially supported by the Swiss National Science Foundation
(projects 200021-115995 and 200021-126420) and the paléojura project of the Canton Jura
(Switzerland). The authors are grateful to Gertrüd Rössner (Bayerische Staatssammlung für
Paläontologie, Germany), Reinhard Ziegler (Staatliches Museum für Naturkunde Stuttgart,
Germany), Heinz Fürrer (Paläontologisches Institut und Museum der Universität Zürich,
Switzerland), Loïc Costeur and Burkart Engesser (Naturhistorisches Museum Basel,
Switzerland), André Fasel (Musée d’histoire naturelle de Fribourg, Switzerland), Suzanne
Jiquel (Université des Sciences et Techniques du Languedoc, France), and Anne-Sophie
Vernon (private collection, Ouchamps, France) for providing access to their collections.
The authors are indebted to Florent Hiard, Soffana Madani, and Laureline Scherler for
their helpful discussions. Special thanks go to Sarah Rollo who kindly improved the English
and to Soledad De Esteban Trivigno who had the kindness to discuss and improve the
geometric morphometrics part of this chapter.
References
[1]
[2]
R. Nowak and J. L. Paradiso, Order Artiodactyla. (1983). R. Nowak and J. L. Paradiso
“Walker's Mammals of the World, Vol. 2 (4th edition)”. The Johns Hopkins University
Press (Baltimore and London). 1173-1306.
E. S. Vrba and G. B. Schaler, Introduction. (2000). E. S. Vrba and G. B. Schaller,
“Antelopes, deer, and relatives: fossil record, behavioral ecology, systematics and
conservation”. Yale University Press (New Haven and London). 1-8.
222
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
N. Solounias, Family Bovidae. (2007). D. R. Prothero and S. E. Foss, “The evolution of
Artiodactyls”. The Johns Hopkins University Press (Baltimore). 278-291.
C. P. Groves, Family Cervidae. (2007). D. R. Prothero and S. E. Foss, “The evolution of
Artiodactyls”. The Johns Hopkins University Press (Baltimore). 249-256.
D. M. Brown, R. A. Brenneman, K.-P. Koepfli, J.P. Pollinger, B. Mila, N. J.
Georgiadis, E. E. Jr Louis, G. F. Grether, D. K. Jacobs, and R. K. Wayne, BMC
Biology, 5(57), 1-13. (2007).
N. Solounias, Family Giraffidae. (2007). D. R. Prothero and S. E. Foss, “The evolution
of Artiodactyls”. The Johns Hopkins University Press (Baltimore). 257-277.
D. R. Prothero, Family Moschidae. (2007). D. R. Prothero and S. E. Foss, “The
evolution of Artiodactyls”. The Johns Hopkins University Press (Baltimore). 221-226.
E. Byrd Davis, Family Antilocapridae. (2007). Prothero, D.R. and Foss, S.E. “The
evolution of Artiodactyls” The Johns Hopkins University Press (Baltimore). 227-240.
G. E. Rössner, Family Tragulidae. (2007). D. R. Prothero and S. C. Foss “The evolution
of artiodactyls”. The Johns Hopkins University Press (Baltimore). 213-220.
M. W. Demment and P. J. Soest van, The American Naturalist, 125, 641 (1985).
R.D. Bodmer, Oikos, 57, 319 (1990).
W. Franzmann, Mammalian Species, 154, 1 (1981).
M. Janis, Mémoires du Museum National d'Histoire Naturelle, Paris (série C), 53, 367
(1986).
G. G. Gray and C. D. Simpson, Mammalian Species, 144, 1 (1980).
T. M. Kaiser and G. E. Rössner, Palaeoecology, 252, 424 (2007).
L. Costeur and S. Legendre, Palaios, 23, 280 (2008).
C. M. Janis and K. M. Scott, American Museum Novitates, 2893, 1 (1987).
J.W. III Cain, P.R. Krausman, and H.L. Germaine, Mammalian Species, 753, 1 (2004).
B. W. O’Gara, Mammalian Species, 90, 1 (1978).
M. Meagher, Mammalian Species, 266, 1 (1986).
F. Rivals, N. Solounias and M. C. Mihlbachler, Quaternary Research, 68, 338 (2007).
L. Pinder and A. P. Grosse, Mammalian Species, 380, 1 (1991).
D. M. Leslie and G. B. Schaller, Mammalian Species, 836, 1 (2009).
D. M. Leslie, Mammalian Species, 813, 1 (2008).
C. C. Custodio, M. V. Lepitens, and R. Heaney, Mammalian Species, 520, 1 (1996).
J. F. Neas and R. S. Offmann, Mammalian Species, 277, 1 (1987).
P. J. Weinberg, Mammalian Species, 695, 1 (2002).
F. Parrini, J. W. III Cain, and P. R. Krausmann, Mammalian Species, 830, 1 (2009).
K. Fedosenko and D. A. Blank, Mammalian Species, 675, 1 (2001).
J. Gordon and A. W. Illius, Functional Ecology, 2, 15 (1988).
M. Fortelius and N. Solounias, American Museum Novitates, 3301, 1 (2000).
A. Danilkin, Mammalian Species, 512, 1 (1995).
N. Jass and J. I. Mead, Mammalian Species, 740, 1 (2004).
K. Ralls, Mammalian Species, 31, 1 (1973).
M. Gagnon and A. E. Chew, Journal of Mammalogy, 81, 490 (2000).
G. A. Feldhamer, Mammalian Species, 128, 1 (1980).
W. von Richter, Mammalian Species, 50, 1 (1974).
G. A. Feldhamer and K. C. Farris-Renner, Mammalian Species, 317, 1 (1988).
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
223
[39] E. P. J. Heizmann, F. Duranthon, and P. Tassy, Stuttgarter Beiträge zur Naturkunde,
Serie C, 39, 1 (1996).
[40] Mennecart, D. Becker, and J.-P. Berger, Swiss Journal of Geosciences. DOI
10.1007/s00015-010-0034-0 (in press).
[41] Y. Jehenne, Münchner Geowissenschaftliche Abhandlungen, 10, 131 (1987).
[42] J.-N. Martinez and J. Sudre, Lethaia, 28, 197 (1995).
[43] Novello, C. Blondel, and M. Brunet, Comptes Rendus Palevol, 9, 471 (2010).
[44] N. Solounias and S. M. C. Moellenken, Journal of Vertebrate Paleontology, 12(2), 250
(1992).
[45] S. C. Kingswood and D. A. Blank, Mammalian Species, 518, 1 (1996).
[46] M. Brunet and Y. Jehenne, Bulletin de la Société Géologique de France, 7, 1659
(1976).
[47] G. E. Rössner, Paläontologische Zeitschrift, 84(1), 123 (2010).
[48] I. Dagg, Mammalian Species, 5, 1 (1971).
[49] M. Janis, Correlation of cranial and dental variables with body size in ungulates and
macropoids. (1990). Damuth, J. and MacFadden, B.J. “Body Size of Mammalian
Paleobiology: Estimating and Biological Implications”. Cambridge University Press
(Cambridge). 255-299.
[50] M. Sanchez, M. Soledad Domingo and J. Morales, Palaeontology, 53(5), 1023 (2010).
[51] J. Leinders, Scripta Geologica, 70, 1 (1983).
[52] A. E. Van der Geer, The effect of insularity on the Eastern Mediterranean early cervoid
Hoplitomeryx: the study of the forelimb. Quaternary International, 182(1), 145 (2008).
[53] J. Kim, N. S. Lee, and S. D. Lee, Conservation Genetics, 12, 851 (2011).
[54] S. C. Kingswood and A. T. Kamumoto, Mammalian Species, 539, 1 (1996).
[55] S. C. Kingswood and A. T. Kamumoto, Mammalian Species, 569, 1 (1997).
[56] W. D. Matthew, Bulletin of the American Museum of Natural History, 23, 535 (1908).
[57] J. F. Eisenberg, The contemporary Cervidae of Central and South America. (2000). E.
S. Vrba and G. B. Schaller, “Antelopes, deer, and relatives: fossil record, behavioral
ecology, systematics and conservation”. Yale University Press (New Haven and
London). 189-202.
[58] J. I. Mead, Mammalian Species, 335, 1 (1989).
[59] E. Anderson and O. C. Wallmo, Mammalian Species, 219, 1-9. (1984).
[60] W. P. Smith, Mammalian Species, 388, 1 (1991).
[61] R. E. Bodmer and G. B. Rabb, Mammalian Species, 422, 1 (1992).
[62] B. Rideout and R. S. Hoffmann, Mammalian Species, 63, 1 (1975).
[63] K. Fedosenko and D. A. Blank, Mammalian Species, 773, 1 (2005).
[64] D. M. Shacleton, Mammalian Species, 230, 1 (1985).
[65] T. Bowyer and D. M. Leslie, Mammalian Species, 393, 1 (1992).
[66] J. E. Jackson, Mammalian Species, 295, 1 (1987).
[67] D. M. Leslie and G. B. Schaller, Mammalian Species, 817, 1 (2008).
[68] V. E. Sokolov and A. A. Luschekina, Mammalian Species, 571, 1 (1997).
[69] G. E. Rössner, Münchner Geowissenchaftliche Abhandlungen, 29, 1 (1995).
[70] D. M. Leslie, Mammalian Species, 849(42), 7 (2010).
[71] X. Wang and R. S. Hoffmann, Mammalian Species, 278, 1 (1987).
[72] V. E. Sokolov, Mammalian Species, 38, 1 (1974).
[73] L. A. Pappas, Mammalian Species, 689, 1 (2002).
224
[74]
[75]
[76]
[77]
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
D. M. Leslie and K. Scharma, Mammalian Species, 843, 1 (2009).
K. Ralls, Mammalian Species, 111, 1 (1978).
E.H. Colbert, American Museum Novitates, 1135, 1 (1941).
C. Blondel, Les ongulés à la limite Eocène/Oligocène et au cours de l’Oligocène en
Europe occidentale: analyses faunistiques, morpho-anatomiques et biogéochimiques
(d13C, d18O). Implications sur la reconstitution des paléoenvironnements. (1996).
Unpublished PhD Thesis: University of Montpellier II, France.
[78] C. Blondel, Comptes Rendus de l’Académie des Sciences de Paris, 326, 527 (1998).
[79] C. Blondel, Geobios, 30, 573 (1997).
[80] W. Gentry, G. E. Rössner, and E. P. J. Heizmann, Suborder Ruminantia. (1999). G. E.
RÖSSNER AND K. HEISSIG, “THE MIOCENE LAND MAMMALS OF EUROPE”. VERLAG DR.
FRIEDRICH PFEIL (MÜNCHEN). 225-258.
[81] D. S. Webb, Bulletin of Florida Museum of Natural History, 48(2), 17 (2008).
[82] W. P. Wall and C. Collins, National Park Service Paleontological Research, 3, 13
(1998).
[83] Frick, American Museum of Natural History Bulletin, 59, 1 (1937).
[84] Zanazzi and M. J. Kohn, Palaeogeography, Palaeoclimatology, Palaeoecology, 257, 22
(2008).
[85] G. Métais, and I. Vislobokova, Basal ruminants. (2007). Prothero, D.R. and S.C. Foss,
“The evolution of artiodactyls”. The Johns Hopkins University Press (Baltimore). 189212.
[86] E. Meijaard and D. Sheil, Ecological Research, 23, 21 (2007).
[87] S. Legendre, Münchner Geowissenschaftliche Abhandlungen, 16, 1 (1989).
[88] K. M. Scott, Postcranial dimensions of ungulates as predictors of body mass. (1990). J.
Damuth and B. J. MacFadden, “Body Size of Mammalian Paleobiology: Estimating and
Biological Implications”. Cambridge University Press (Cambridge). 301-335.
[89] M. Köhler, Münchner Geowissenschaftliche Abhandlungen, 25, 1 (1993).
[90] DeGusta and E. S. Vrba, Journal of Archaeological Science, 30, 1009 (2003).
[91] DeGusta and E. S. Vrba, Journal of Archeological Science, 32, 1099 (2005a).
[92] DeGusta and E. S. Vrba, Journal of Archeological Science, 32, 1115 (2005b).
[93] K. Kovarovic and P. Andrews, Journal of Human Evolution, 52, 663 (2007).
[94] R. Barone, Anatomie comparée des mammifères domestiques Tome 1 Ostéologie (4e
edition). (1999). Vigot (Paris).
[95] N. Solounias, S. M. C. Moelleken and J. M. Plavcan, Journal of Vertebrate
Paleontology, 15(4), 795 (1995).
[96] J. Perez-Barberia and I. J. Gordon, Oecologia, 118, 157 (1999).
[97] J. MacFadden, Annual Review of Ecology and Systematics, 31, 33 (2000).
[98] F. J. Perez-Barberia and I. J. Gordon, Proceedings of the Royal Society B, 268, 1021
(2001).
[99] M. Mendoza, C. M. Janis, and P. Palmqvist, Journal of zoology, 258, 223 (2002).
[100] A. Y. Pavlinov, Geometric morphometrics of the upper antemolar row configuration in
the brown-toothed shrews of the genus Sorex (Mammalia). (2004). A. M. T. Elewa,
“Morphometris Applications in Biology and Paleontology”. Springer (Berlin and
Heidelberg). 223-230.
[101] M. Mendoza and P. Palmqvist, Journal of zoology, 274, 134 (2007).
Mandible Shape of Ruminants: Between Phylogeny and Feeding Habits
225
[102] P. Raia, F. Carotenuto, C. Meloro, P. Piras and D. Pushkina, Evolution, 64(5), 1489
(2010).
[103] P. J. Jarman, Behaviour, 48(3/4), 215 (1973).
[104] F.J. Rohlf, tpsDig 2.16. Stony Brook. Department of Ecology and Evolution, State
Univ. of New York, NY. (2010).
[105] R. M. Joeckel, Paleobiology, 16, 459 (1990).
[106] L. M. Spencer, Journal of Mammalogy, 76, 448 (1995).
[107] F.J. Rohlf, Rotational fit (Procustes) methods. (1990). F.J. Rohlf and F.L. Bookstein,
“Proceedings of the Michigan Morphometrics Workshop”. University of Michigan
Museum of Zoology. 227-236.
[108] F.J. Rohlf and D. Slice, Systematic Biology, 39, 40 (1990).
[109] R. B. Querino, R. C. B. de Moraes and R. Zucchi, Neotropical Entomology, 31, 217
(2002).
[110] D. C. Adams, F. J. Rohlf, and D. E. Slice, Italian Journal of Zoology, 71, 5 (2004).
[111] L. R. Monteiro and S. F. dos Reis, Pincipíos de morfometria geométrica. Holos Editora
(Ribeirão Preto). (1999).
[112] F. J. Rohlf, Relative warps analysis and an example of its application to mosquito
wings. L. F. Marcus, E. Bello and A. Garcia-Valdecasas, “Contributions to
morphometrics”. (1993). Museu Nacional de Ciencias Naturales (Madrid). 131-159.
[113] F.J. Rohlf, tpsRelw v. 1.46. Stony Brook. Department of Ecology and Evolution, State
Univ. of NewYork, NY. (2008).
[114] N. Solounias and S. M. C. Moellenken, Journal of Vertebrate Paleontology, 12(1), 113
(1992).
[115] W. D. Matthew and W. Granger, American Museum Novitates, 196, 1 (1925).
[116] G. G. Simpson, Bulletin of the American Museum of Natural History, 85, 1 (1945).
[117] A. W. Gentry and J. J. Hooker, The phylogeny of the Artiodactyla. M. J. Benton, “The
Phylogeny and Classification of the Tetrapods, volume 2: Mammals”. (1988).
Systematics Association (Special Volume 35B), Clarendon Press (Oxford). 235-272.
[118] J. Sudre, Comptes Rendus de l’Académie des Sciences de Paris, 303, 749 (1986).
[119] H. Wehrli, Notizblatt für Erdkunde und verwandter Wissenschaften, 5(14), 101 (1931).
[120] J. Sudre, Palaeontographica (A), 236, 205 (1995).
[121] J.-W. Guo, T. Qi, and H.-J. Sheng, Vertebrata PalAsiatica, 37(1), 18 (1999).
[122] J. J. Hooker and M. Weidmann, Mémoires suisses de Paléontologie, 120, 1 (2000).
[123] W. R. Hamilton, Bulletin of the British Museum (Natural History) Geology, 21, 73
(1973).
[124] S. Cote, Pecora Incertae sedis. (2010). L. Werdelin and W. J. Sanders, “Cenozoic
Mammals of Africa”. University of California Press (Berkeley). 731-739.
[125] J. C. Barry, S. Cote, L. MacLatchy, E. H. Lindsay, R. Kityo and, A. Rahim Rajppar,
Palaeontologica Electronica, 8, 1 (2005).
[126] D. S. Webb and B. E. Taylor, Bulletin of the American Museum of Natural History,
167, 117 (1980).
[127] C. M. Janis, Journal of Vertebrate Paleontology, 7, 200 (1987).
[128] D. R. Prothero and M. R. Liter, Family Palaeomerycidae. (2007). D. R. Prothero and S.
E. Foss, “The evolution of Artiodactyls”. The Johns Hopkins University Press
(Baltimore). 241-248.
226
Mennecart Bastien, Becker Damien and Berger Jean-Pierre
[129] C. Deperet and H. Douxami, Mémoires de la Société Paléontologique Suisse, 29(1), 1
(1902).
[130] J. Viret, Annales de l’Université de Lyon, Nouvelle Série 47, 1 (1929).
[131] R.L. Caroll, Vertebrate paleontology and evolution. (1988). W.H. Freeman and
Company (New York).
[132] J. Van der Made, Intercontinental relationship Europe Africa and the Indian
Subcontinent. (1999). G. E. RÖSSNER AND K. HEISSIG, “THE MIOCENE LAND MAMMALS
OF EUROPE”. VERLAG DR. FRIEDRICH PFEIL (MÜNCHEN). 457-472.
[133] L. Ginsburg and F. Chevrier, Annales de Paléontologie, 89(4), 253 (2003).
[134] A. E. Van der Geer, The postcranial of the deer Hoplitomeryx (Pliocene; Italy):
another example of adaptive radiation on Eastern Mediterranean islands (2005).J. A.
Alcover and P. Bover, “Proceedings of the International Symposium: Insular
Vertebrate Evolution: the Palaeontological approach”. Monografies de la Societat
d’Història Natural de les Balears, 12. 325-336.
[135] W. Gentry, Historical Biology, 7, 115 (1994).
[136] N. Solounias, J. C. Barry, R. E. Bernor, E. H. Lindsay, and M. Raza, Journal of
Vertebrate Paleontology, 15(4), 806 (1995).
[137] T. A. Franz-Odendaal and N. Solounias, Geodiversitas, 26(4), 675 (2004).
[138] H. Alleyne Nicholson, The ancient life history of the Earth a comprehensive outline of
the principles and leading facts of palæontological science. (1876). D. Appleton and
company (New York).
[139] R. Barone, Anatomie comparée des mammifères domestiques Tome 2 Arthrologie et
myologie (4e edition). (2000). Vigot (Paris).
[140] D. Becker, P.-O. Antoine, B. Engesser, F. Hiard, B. Hostettler, U. Menkveld-Gfeller, B.
Mennecart, L. Scherler, and J.-P. Berger, Annales de Paléontologie, DOI
10.1016/j.annpal.2011.03.001 (2011).
[141] W. Leuthold, Journal of Animal Ecology, 47(2), 561 (1978).
In: Ruminants: Anatomy, Behavior and Diseases
Editor: Ricardo Evandro Mendes
ISBN: 978-1-62081-064-4
© 2012 Nova Science Publishers, Inc.
Chapter XIII
Creating the Tiniest Bison:
A System Dynamics Model of
Ecological Evolution
Elin Whitney-Smith*
Netalyst, Inc., US
Abstract
The anatomy and behavior of N. American bison were profoundly modified by the
extinction event at the end of the Pleistocene. Plains Bison (Bison bison) became obligate
grazers, and, like musk ox, elk, and other large ruminants, became much smaller. During
the same time period, mastodon, mammoth, horse, ground sloth, and other non-ruminant
herbivores went extinct.
The vegetation of the North American continent shifted from a mixed mosaic to
homogenous stripes: mixed parklands and woodlands throughout the continent were
transformed into the great and treeless prairie, and the mixed parkland/woodlands on the
coasts became closed-canopy forests. The key to understanding the changes in bison
anatomy and behavior, the non-ruminant extinctions, and the vegetative shift is
uncovering the ecological dynamics of the end of the Pleistocene.
Two major theories have been proposed to explain the extinction event:
Overkill – humans hunted animals to extinction and
Climate Change – the end of the Ice Age created ecological stress which caused the
extinctions and the changes in the size of bison.
Both have problems.
A new theory is called for. Second Order Predation hypothesizes that humans
reduced carnivore populations below the level that they controlled herbivore populations,
resulting in an herbivore boom then bust, destabilizing the ecosystem. During the boom
phase, herbivores denuded the environment of vegetation, creating a highly competitive
environment – a bottleneck – where animals that could extract the maximum nutrition
from their food – ruminants –, and reproduce using the minimum amount of food –
smaller animals – were evolutionarily favored, while large non-ruminants went extinct.
*
E-mail: elin@quaternary.net.
228
Elin Whitney-Smith
When a new stability was reached in the Holocene, huge herds of bison (B. bison bison)
dominated and maintained the grasslands in the center of the country, and trees reinvaded from mountain refugia,where woodland bison (B. bison athabascae) continued to
live in small groups and eat a mixture of grass and browse. This bottleneck in the
environment explains why horses, non-ruminants that had gone extinct during the
transition, could survive in the new stabile ecosystem. Some scientists postulate that the
extinctions can be explained through a combination of the Overkill and Climate Change
hypotheses.
The Pleistocene Extinction Model (PEM) was designed to test all these hypotheses
using the same assumptions. The PEM simulations give surprising results: i) If the model
is run with sufficient Climate Change to produce extinctions, Overkill reduces the impact
of Climate Change, avoiding extinction. ii) Under the same conditions, Second Order
Predation exacerbates the impact of Climate Change, leading to quicker and more
extreme extinction. iii) Second Order Predation alone also causes extinction. iv)
Simulations that produce extinction create a vegetative shift. This new understanding
sheds light on the behavioral and anatomical shifts of bison.
Introduction
Back when mammoths, mastodonts, and saber tooth cats walked the earth; before the
extinctions at the end of the Pleistocene, North American Bison (Bison bison) were almost as
large as elephants – true megaherbivores – they ate browse as well as grass and lived in
family groups. Their anatomy and behavior were profoundly modified by the extinction
event. Bison, like musk ox, elk, and other large ruminants, became much smaller, and bison
became obligate grazers. They were lucky to be ruminants, since ruminants fared better than
other ungulates [1,2].
During the Ice Age, a diverse complement of animals, dominated by mammoth, bison,
horse, and their predators, lived in mixed parklands and woodlands throughout the continent.
Even in Alaska, there were warm well drained soils that supported low grassy vegetation and
scattered tree cover. Guthrie [3] has called this ecosystem the Mammoth Steppe biome.
After the extinction event, the mixed parklands/woodlands throughout the continent were
transformed, in the center of the continent, into the great and treeless prairie, and, on the
coasts, into closed-canopy forests. Alaska became treeless tundra and boreal forest growing
on poorly-drained, cold soils – permafrost [4,5,6,7,8,9,10,11,12,13,14,15,16].
The great Mammoth Steppe Biome was no more. Many species perished, but bison
survived in two altered forms, plains bison (B. bison bison) and woodland bison (B. bison
athabascae). Understanding modern bison and ruminant survival lies in understanding the
ecological dynamics of the extinction event.
The Theoretical Marketplace
Scientists have proposed various hypotheses to account for the extinctions. Though each
is based on sound observations and good reasoning, together they are like an Escher drawing
where each individual piece is correct and detailed but the overall picture doesn't hang
together well.
Creating the Tiniest Bison
229
Overkill
The Overkill hypothesis, proposed by Paul Martin, [17] holds that H. sapiens entered the
New World and swept through the continent killing herbivores until they were extinct. The
theory is supported by the observation that extinctions throughout the world follow the
migration of H. sapiens and that the extinctions are the greatest where H. sapiens arrived
most recently, reflecting increasing hunting competence. In addition, on Wrangel Island in
Siberia, standard mammoth survived well past the end of the Pleistocene and dwarf mammoth
survived into the historic period – 3,500 BP [18,19]. Since these mammoth were not hunted,
their continued survival supports Overkill. Mossiman and Martin [20] and Whittington and
Dyke [21] created computer models that suggest that it is possible for H. sapiens to over-hunt
herbivores if they kill more herbivores each year than are replaced through natural population
increase. They concentrate on the relationship between H. sapiens and herbivores. Alroy [22]
concentrates on the same relationship, though his model is considerably more complex. All of
these models are simulating a particular hypothesis, not comparing hypotheses. The Overkill
hypothesis would be modeled as figure 1A. The problems with the Overkill hypothesis are:



In general, it is difficult for predators to over-hunt their prey since it is their food
supply and it does not recognize that herbivores are controlled both by the plants
available and the carnivores that prey upon them [23,24].
Animals not hunted by humans, such as the giant ground sloth, also went extinct.
It now appears that the animals did not decline all at once and some survived in
hidden pockets of the world [25].
Clues: Extinctions are the greatest where H. sapiens arrived most recently. Survival, on
Wragel Island, of mammoths past the end of the Pleistocene and dwarf mammoth into the
historic period.
Climate Change
Vastly simplified, the Climate Change hypothesis suggests that as the Ice Age waned,
climate changed so drastically that animals were not able to adapt. There have been a number
of variations on this theme [26,27,28,29,30,31].
One of the major pieces of evidence is based on Hansen's [32,33] study of Ground Sloth
dung from Rampart cave. It shows that over time the sloth ate less and less of its preferred
food and more and more of the food that other herbivores couldn't digest. Clearly, the North
American climate changed at the end of the Pleistocene: At the beginning of the Holocene
(the age following the Pleistocene), the mixed parklands and woodlands throughout the
continent were transformed into the great (and treeless) prairie, and the mixed
parkland/woodland on the coasts has become closed-canopy forest, what Guthrie [3] [34] has
called a shift from plaid to striped. There was an increase in continentality (hotter summers
and colder winters);
There was less rainfall, and it was more variable;
Of course, weather shifts may affect animals directly, e.g. offspring being born into
snowstorms instead of a warm spring. However, the major impact is mediated through
230
Elin Whitney-Smith
vegetation: animals were unable to get enough plants or enough of the right kind of plants at
the right time to eat. Thus they are unable to reproduce, survive pregnancy, or nurse, or the
young are unable to find sufficient food to eat [27,35,36]. The Climate Change hypothesis can
be modeled as shown in figure 1B. There is an exogenous reduction in the capacity of the
land to produce plants, which reduces plant biomass and thus reduces herbivore population.
These hypotheses have been challenged by the following observations. First today's mean
annual temperature is no warmer than that of previous interglacials that are not associated
with extinctions [37,38,39,40,41]. Second that increased continentality resulted in an
increased prevalence of grasses. McDonald [42,43] suggests the horses, which became extinct
on this continent, actually should have prospered during the shift from mixed woodlandparkland to prairie, because their primary food source, grass, was increasing rather than
decreasing, [44] and horses successfully live and reproduce in those same places today.
Further, the increase in continentality was not greater than the continentality of Siberia during
the Pleistocene, where these same animals prospered. Indeed, climate change is associated
with extinctions only in the Americas, and not in Africa, Asia, Europe, or Australia. Finally,
Mammoths, sloths, mastodons, and other animals that went extinct had survived similarly
warm periods during previous inter-glacials, and New World horses, which went extinct at
the end of the Ice Age, are thriving in that same climate today.
Clue: Shift from plaid to striped environments, the lack of preferred food for the ground
sloth, and horses living and reproducing on those same foods today.
Combination of Overkill and Climate Change
Some scientists find both the Overkill and the Climate Change hypotheses unsatisfactory;
they believe that the extinctions are due to some combination of Overkill and Climate
Change. Gary Haynes [45,46] supports a combination hypothesis. His observations suggest
that mammoths were suffering from environmental stress. He compares their condition to that
of African elephants populations that recover from environmental die offs but then are less
likely to survive modern hunting. He reasons that people opportunistically hunted mammoths
as they became more and more stressed and hence more vulnerable to predation. He observes
that on kill sites the long, marrow containing, bones are not broken indicating that the animals
had little fat on them and that most carcasses do not show signs of full utilization by either
people or carnivores indicating environmental stress. Unfortunately, Haynes’ theory of
proboscidean extinction does not account for the extinction of American camels or horses.
Instead, he links their extinctions to increased snow cover. This is a less parsimonious
account of the Pleistocene extinctions. Haynes’ observation that proboscidean populations
increase after camel and horse populations decline is intriguing. R. Dale Guthrie [47] among
others [27,48,49,31,50,51,37,52,53,54, 55,56,57,58,59,60] has also noted patterns of nonsynchronous patterns of species change over the time period – bison (Bison priscus, evolved
to Bison bison), wapiti (Cervus canadensis) and, moose (Alces alces) began to increase in
numbers before the regional extinction of horse (Equus ferus) and mammoth (Mammuthus
primigenius). Though the combination hypothesis seems to be the most intuitive explanation,
there aren’t proposals of how that combination would have worked. The basic concept is
shown in figure 1C.
231
Creating the Tiniest Bison
Clues: 1.Proboscideans suffered from environmental stress, 2. Decline of species was not
synchronous. 2. Ruminant populations increase before the extinctions of horse and mammoth.
a)
b)
c)
Figure 1. Theories of Pleistocene Extinctions A – Overkill H. sapiens kills herbivores, B – Climate
change, exogenous reduced carrying capacity reduces plants, which in turn reduces Herbivores, C –
Combination Hypothesis both arrows into herbivores reduce their population size. Key Plus (+) at the
end of an arrow indicates co-variance (As plants decrease, herbivores decrease). Minus (–) at the end of
an arrow indicates varying inversely (As H. sapiens increase herbivores decrease).
Problems with Climate Change, Overkill, and Combination Hypotheses
The hypotheses do not attempt to explain the pattern of extinctions – the loss of more
browsers and mixed-feeders than grazers, the favoring of ruminant over non-ruminant
grazers, and the general dwarfing of many species including bison, beavers, elk and moose.
None of these hypotheses – Climate Change, Overkill, and a Combination of Climate
Change and Overkill, recognize of the role of carnivores in maintaining the balance on an
ecosystem [61]. In addition, none of the hypotheses recognize that in a stable ecosystem all of
these relationships are balanced relationships. There are feedbacks between the population of
herbivores and plants, between the populations of herbivores and carnivores and between the
populations of H. sapiens and herbivores. Each of the three trophic levels depends upon the
upper level for food and the lower level for controlling population.
A New Hypothesis Is Needed
I have proposed a new theory of extinction – Second Order Predation (2OP) [62] [63] –
and created a modeling environment – the Pleistocene Extinction Model, (PEM) – to test it.
Comparing 2OP to Overkill [64,65,66,20] PEM showed that, using the same structure,
assumptions, and values 2OP produces extinction and Overkill does not.
232
Elin Whitney-Smith
Figure 2. Pleistocene Extinction Model (PEM) The top three trophic levels are divided: Continental
Carrying Capacity is divided into: Carrying capacity for trees and Carrying capacity for grass Plants are
divided into: Large and Small trees, High and Low quality grass Herbivores are divided into: Browsers,
Mixed-feeders, Ruminant grazers and Non-ruminant grazers Key: B in a spiral means that the loop is
balanced. R in a spiral means that the loop is reinforcing. The minus sign on the bottom arrow signifies
that the relationship varies inversely – as H. sapiens increases Carnivores decrease. The dashed line
arrow at the bottom signifies that there are two theories tested with this model – Overkill(OK) without
H. sapiens reducing carnivore populations (without the link) and –Second Order Predation (2OP) H.
sapiens reducing carnivore populations (with the link).
This chapter will review and expand that work to include an examination of Combination
Hypotheses: Overkill plus Climate Change and 2OP plus Climate Change. A first step in
validating a proposed ecosystem model is demonstrating that it can attain a reasonable
equilibrium. Any balanced ecosystem model used to represent a combination hypothesis
needs relationships representing the fact that population levels of a time period determine the
population of the time period following – plants beget plants, herbivores beget herbivores,
carnivores beget carnivores and humans beget humans as shown in figure 2.
The Second Order Predation hypothesis posits that H. sapiens entered the New World,
and not only killed herbivores for food but reduced carnivore populations to the extent that
carnivores were unable to control herbivore populations. The computer model does not
address either why or how humans reduced carnivore populations. Possible mechanisms
include:


Desire for fur
A strategy to reduce competition
Creating the Tiniest Bison


233
A response to carnivore predation on humans, or
The result of an introduced carnivore disease.
We know that when wolves enter a new territory they kill existing predators – fox, coyote
[67,68,69,61]. Humans have the added advantage of planning, policy, rational thought, and
communication. We also know from work done in the Serengeti [70] that when herbivore
populations are reduced through the introduction of a new predator, existing predators will
turn to killing H. sapiens , giving H. sapiens a reason for killing carnivores. Olga Soffer [71]
has documented carnivore killing in Siberia during the Pleistocene/ Holocene transition and
suggests it was for fur. It’s crucial to note that, if such second order predation took place,
regardless of the cause or mechanism, it would – unlike standard predation – proceed without
first order controls, since H. sapiens does not use carnivores for food. Thus, a reduction in
carnivore populations would allow the reinforcing loop – more herbivores today lead to even
more herbivores in the future - to dominate the system, and thus an herbivore population
boom. This boom would kill off most of the vegetation – bust (Figure 3).
Figure 3. H. sapiens reduces carnivore populations. The self-reinforcing loop, Herbivores produce
Herbivores, dominates the system. The heavy arrow from Herbivores to Plants indicates heavy usage of
plants. Then as plants are reduced the plant increase is reduced. The balancing loop between plants and
Herbivores means that herbivores begin to starve as plants disappear. The balancing loops between
Carnivores and Herbivores and between H. sapiens and Herbivores are ineffective and almost
disappear. Key: B in a spiral means that the loop is balanced. R in a spiral means that the loop is
reinforcing. The minus sign on the bottom arrow signifies that the relationship varies inversely – as H.
sapiens increases Carnivores decrease. The dashed line arrow at the bottom signifies that there are two
theories tested with this model – Overkill(OK) without H. sapiens reducing carnivore populations
(without the link) and Second Order Predation (2OP) H. sapiens reducing carnivore populations (with
the link).
234
Elin Whitney-Smith
Pleistocene Extinction Model (PEM):
Second Order Predation vs. Overkill
The initial version of PEM shows how second order predation would lead to extinction.
However, to account for the pattern of extinction across the various types of herbivores, it is
necessary to expand and disaggregate this into a more elaborate model.
The expansion reflects that there is an upper limit to the ability of the land to produce
plants. The disaggregation splits plants into trees and grass and Herbivores into Browsers,
Mixed-feeders, Ruminant Grazers, and Non-ruminant Grazers. The two top-level predators
remain the same Carnivores and H. sapiens.
The model runs in two modes. The first represents (and tests) the Overkill hypothesis: H.
sapiens hunts Herbivores but doesn't reduce carnivore populations. The second, the Second
Order Predation hypothesis: H. sapiens does reduce carnivore populations. Values used were
based on Mossiman and Martin [72] and Whittington and Dyke [21]. Carnivore values were
based on the needs of modern carnivores – 20 lbs of food per pound of carnivore per year
[68,73]. H. sapiens hunting values were based on food need of 10 lbs of meat per pound of H.
sapiens per year or half the food necessary to support an obligate carnivore.
This is probably an overly high value for human meat consumption, since modern
hunter/gatherers generally have a diet of only 20% meat. This overestimate favors Overkill
rather than Second Order Predation. Otherwise, Carnivores and H. sapiens are modeled
similarly. Both Carnivores and H. sapiens hunt herbivores based on herbivore density. The
graphs below (Figure 4AandB) contrast the Overkill hypothesis (A) with the Second Order
Predation Hypothesis (B).
All simulations begin at equilibrium before either overkill or second order predation
begins, and all variables have been normalized for comparability. In the Overkill scenario
(Figure 4A), as H. sapiens increase, there is a slight increase in trees, a decrease in carnivores
and herbivores, but no extinction.
In the Second Order Predation scenario (Figure 4B), as H. sapiens increase and reduce
carnivore populations as well as herbivores, the decrease in carnivore populations leads to
1.
2.
3.
4.
An initial increase in all herbivore populations and a decrease in plants
A major crash of all populations (first extinction -10735).
A period of equilibrium.
An increase in plants followed by another crash in all other populations (second
extinction -9750).
5. A final equilibrium with plants very high and all other populations reduced
significantly.
Behind the scenes, in Figure 4CandD there is more detail. In the herbivore graph (Figure
4C), the initial equilibrium is followed by:
1. An increase in Browsers, Ruminant and Non-ruminant Grazers and a decline in
Mixed-feeders
2. The extinction of Mixed-feeders followed by the extinction of Browsers
3. A new seemingly steady state for Ruminant and Non-ruminant Grazers, lower than
their earlier equilibrium levels.
Creating the Tiniest Bison
235
4. A decline in Ruminant Grazers and the extinction of Non-ruminant Grazers
5. A new, much lower true equilibrium for Ruminants.
The plant graph (Figure 4 D) shows
1. A decline in tree stocks -- almost to zero -- and an increase in grass stocks
2. Grass stocks decline slightly then recover and then maintain a steady, high level
3. Initially Tree stocks decrease almost to zero and then increase as they begin to
repopulate, at which point grass levels decline.
4. Finally a new plant equilibrium is established with many more trees and much less
grass
a)
b)
Figure 4. Overkill vs. 2OP A – Overkill B – 2OP (H. sapiens kills 0.25lbs of carnivore per year) using
the same assumptions C – 2OP Herbivores D – Plants For all graphs the X axis spans 1.5K years and
curves are normalized at their equilibrium level.
236
Elin Whitney-Smith
Results: Second Order Predation leads to herbivore extinction. Overkill does not.
If we break down the results into phases we can understand what is happening. In the first
phase, between 11117 and 10735 BP, as carnivore stocks are reduced, carnivore consumption
of herbivores no longer keeps up with herbivore reproduction. This allows the reinforcing
loop of herbivore reproduction to dominate the system. This accounts for the increase in
Browsers, and both Grazer stocks. Once carnivores no longer control herbivores, Browser
recruitment becomes greater than the recruitment for trees. This has two impacts:
1. Browsers drive Mixed-feeders to extinction (Browsers only eat Trees. Grazers only
eat Grass. Mixed-feeders need both Trees and Grass and yet are less efficient at
obtaining either. Under equilibrium conditions they buffer the system from shocks
since they are able to substitute grass for trees in small amounts. Under more extreme
conditions they are not able to get enough trees and decline.)
2. Trees recruit more slowly than uncontrolled Browsers resulting in Browsers
overshooting the limit of tree stocks so they follow Mixed-feeders to extinction.
In second phase (between 10735 and 9780 BP), following the extinction of Mixedfeeders and Browsers:
1. Grass, with its high recruitment rate is able to takes over all the available area Trees
formerly occupied, but because grass does not create as much standing crop, the
overall level of plants remains lower than at the simulation start.
2. Non-Ruminant Grazers grow more quickly than Ruminant Grazers because their
recruitment rate is slightly higher.
3. Ruminant and Non-Ruminant Grazers recruit until they reach the new grass limit and
establish an equilibrium, with higher population levels then they had at the beginning
of the simulation (as modeled Ruminants are more efficient at processing grass but
Non-ruminants have a faster reaction time to changes in resources).
4. In the absence of browsers trees begin to re-establish themselves retaking land
previously occupied by grass
5. As the grass stock decreases, Ruminant Grazers, with their more efficient biology,
are able to withstand the reduction in grass better than Non-Ruminant Grazers.
In the final phase (after 9780 BP), after the extinction of Non-ruminant Grazers, the
entire system finds a new equilibrium, with these components:
1. Trees reach their limit, and grass is greatly reduced
2. Ruminant Grazers maintain a stable population, consistent with lower grass limit and
their remaining predators – human and non-human.
3. H. sapiens and Carnivores maintain stable populations, at lower levels than before
second order predation began.
In recent years climate change theorists have highlighted the shift in continental
vegetation patterns from plaid to stripe. They note, during the Pleistocene Holocene
transition, mixed parklands and woodlands throughout the continent transformed into the
great (and treeless) prairie, and the mixed parkland/woodland on the coasts became closed-
Creating the Tiniest Bison
237
canopy forest. In the Arctic, the amount of land covered by permafrost increased significantly
[3]. The interplay of the various plant and herbivore stocks over the three phases of the
Second Order Predation boom bust scenario provides an explanation for these observations.
As H. sapiens reduced Carnivore stocks, herbivore stocks boomed, overgrazing and overbrowsing. Proboscideans (Mammoth and Mastodon) would have knocked over large trees to
get at the tender shoots. (This behavior has been observed in African elephants when they
cannot get enough food because of excess population for the area [74].) Once Browsers and
Mixed Feeders became extinct, trees repopulated from the refugia until they reached the
plains.
On the border between the encroaching trees and the prairie, Ruminant Grazers - new
smaller obligate grazing bison (B. bison bison) - maintain the grassland by eating new shoots
and trampling the ground. We know from modern day evidence that trucks break up
permafrost [75].
These new, truck-produced communities, with warm well drained soils, are made up of
flora and fauna which are normally disjunct [76]. This suggests that the loss of Proboscideans
in the Arctic allowed permafrost to claim more and more area, creating the cold, poorly
drained soils of the Holocene so different from the well-drained Mammoth Steppe of the
Pleistocene.
The pattern of herbivore boom -> plant bust -> herbivore bust also accounts for the size
bias and the ruminant bias observed in the Pleistocene extinctions. In particular, vegetation
scarcity selects for animals that can get the most nutrition from the food available – ruminants
– and those, within a species, that reproduce on scarce resources – smaller animals. Second
Order Predation thus accounts for both the extinctions of the Pleistocene and the shift in
vegetation patterns noted by supporters of the Climate Change hypothesis. No second theory
is needed.
Expand PEM to Include Exogenous Climate
Change
The challenge for this chapter is to test the intuitive notion that Overkill, which is
expected to depress herbivore stocks, and Climate Change, which also has a negative effect
on herbivores, when combined must have an even more negative effect, making the
combination hypothesis a more compelling explanation of the extinctions. To do so, I propose
a simplified version of Climate Change and then combine it in PEM, first with Overkill and
then with Second Order Predation.
The proximate cause of extinction for most of the Climate Change scenarios is related to
changes in the floral environment, i.e., a reduction in plant stocks. Therefore, I use a
reduction in the ability of the land to produce plants as a proxy for Climate Change. Climate
change is modeled as a smooth exogenous 500-year decrease in the capacity of the land to
produce plants. For instance, in a 10% climate change scenario, a land area that, at t=0, was
able to produce 100 plant units annually would, at t=500, only be able to produce 90 plant
units annually. (This model of Climate Change does not take into account the addition of
more land as the ice sheets retreated.)
238
Elin Whitney-Smith
Make It Extreme 13% (vs. 5%)
I wanted Climate Change to have at least as severe an impact as the leading contender –
Second Order Predation.
In addition, to make the Overkill plus Climate Change scenario as strong a contender as
possible, I sought a climate change value large enough to produce extinctions. I found this to
be 13%, a value larger than anyone would think actually occurred [77,78]. I also tested lesser
values of Climate Change, all the way down to 5% to ensure that there were no further
counterintuitive effects or non-linearities. In every case, a smaller value of Climate Change
had less impact on herbivore populations – no extinctions.
Results of Extreme Climate Change by Itself
Figure 5A shows the result of the 13% Climate Change simulation. It is broadly similar to
the graph of Second Order Predation (Figure 1B), with one notable and another minor
exception. Importantly, during this extreme climate change, Ruminant and Non-Ruminant
Grazers do not boom before extinctions occur. (In contrast, in the 2OP scenario, both these
species groups boom first before going extinction.)
Also, in the climate change scenario, the Non-Ruminant extinction comes more quickly
after the Mixed-Feeder and Browser extinctions. (H. sapiens is not included in this
simulation, and thus remains at zero (0)) The behind-the-scenes details are similar to Second
Order Predation (Figure 5B compared with Figure 4C). Again, Mixed-feeders go extinct first,
followed by Browsers. First both Grazer stocks increase, then Non-ruminants go extinct. Only
Ruminants survive.
The Plant graph too is similar to its Second Order Predation counterpart (Figure 5C
compared to Figure 4D): a decrease in plants followed by extinction of Browsers and MixedFeeders, then a decrease in carnivores, and an increase in plants.
As in the Second Order Predation scenario, as trees begin to repopulate, Non-Ruminant
and Ruminant Grazers are in competition with one another for the remaining grass. Ruminant
Grazers win this competition, and the ecosystem reaches a new stability.
Overkill Combined with Extreme Climate Change
Now we add Overkill to this simulation —H. sapiens migrating into the continent killing
herbivores combined with a 13% Exogenous Decline in Carrying Capacity (Figure 6A) –
There is an initial decline in all sectors, but then all sectors recover.
The recovery is not as good as in Overkill alone (Figure 4A). However, a comparison
with Climate Change alone shows that Overkill counteracts the decline in Carrying Capacity
(Figure 6A-C). Behind the scenes, Figures 6BandC show that the increase in H. sapiens
moderates the decline of herbivore stocks, so plant as well as animal stocks regain
equilibrium without extinctions. Overkill reduces herbivore stocks, albeit slightly, and
Extreme Climate Change reduces herbivore stocks, much more significantly.
Creating the Tiniest Bison
239
Figure 5. 13% Exogenous Decline: Climate Change A – Summary, B – Herbivores, C – Plants. For all
graphs, the X axis spans 1.5K years, and curves are normalized at their equilibrium level.
Therefore, we might expect both factors, in combination, to have an even greater impact
on the herbivore stock. Counter-intuitively, the combination has a smaller impact than
Climate Change alone. We can make sense of this by considering the extreme climate change
alone and then in combination with overkill, all from the point of view of plants. Herbivores
are an ongoing constraint on plant stocks. Climate Change (as a 13% reduction in Carrying
Capacity) further reduces the plant stock [In turn, herbivore populations suffer because there
are is less to eat]. When Overkill is added, it reduces herbivore populations, increasing plant
stocks slightly. Briefly: Climate Change is bad for plants, but Overkill is good for plants,
mitigating the impact of Climate Change. Thus plant stocks have time to recover and
equilibrate to the new circumstances. From the plant’s perspective, my enemy’s enemy is my
friend.
240
Elin Whitney-Smith
Figure 6. 13% Exogenous Decline combined with Overkill A – Summary Figure, B – Herbivores C –
Plants. For all graphs, the X axis spans 1.5K years, and curves are normalized at their equilibrium level.
Second Order Predation Combined with Extreme Climate Change
Figure 7A-C shows the results of combining Climate Change with Second Order
Predation. Unlike the Climate Change/Overkill combination, Climate Change/Second Order
Predation produces extinctions, and does so more quickly than Second Order Predation alone
(Figure 4B-C). Behind the scenes, in the CC/2OP scenario, the initial boom is limited to
Browsers. Mixed-Feeders still go extinct first followed by Browsers and as trees reinvade (in
the absence of browsers). As before, Non-ruminant Grazers are not able to successfully
compete with Ruminant Grazers.
Adding 2OP (H. sapiens hunting herbivores and reducing carnivore populations) to
Extreme Climate Change, extinctions happen more quickly than with either Second Order
Predation or Extreme Climate Change alone. Since Second Order Predation allows herbivore
stocks to boom, plant stocks are stressed, and under Extreme Climate Change (as a 13%
reduction in carrying capacity) plant stocks are stressed as well. The relationship between the
impact of Extreme Climate Change and Second Order Predation is additive.
Creating the Tiniest Bison
241
Figure 7. 13% Exogenous Decline combined with Second Order Predation A – Summary Graph, B –
Herbivores C – Plants For all graphs time is 1.5K years and curves are normalized at their equilibrium
level.
Discussion of Second Order Predation Combined with Extreme Climate
Change
Let us revisit the clues and see how Second Order Predation combined with Extreme
Climate Change fits them.
Extinctions followed the introduction of humans becoming more severe as they invaded
continent after continent (Africa least severe – New World most severe).
Second Order Predation, like Overkill, is anthropogenic.
Shift from plaid to striped vegetative environments. Proboscideans were suffering from
environmental stress.
As vegetation became scarce, Proboscideans knocked over the trees in the mixed
parkland/woodland in order to get at the leaves at the tops. Grass, with its short recruitment
time took over. All remaining trees were at the tops of mountains, places inaccessible to
Proboscideans. Once the herbivore boom was over, and herbivore populations stabilized, trees
reinvaded from the mountaintops. Prairie was maintained in the center of the continent by
new smaller obligate grazing bison (B. bison bison).
Survival, on Wrangel Island, of mammoths past the end of the Pleistocene and dwarf
mammoth into the historic period.
242
Elin Whitney-Smith
In the absence of humans, significant predators (human or non-human), or the
competition of large ruminant grazers like bison, the mammoths survived the climate change.
They became dwarfed as the lack of resources selected for smaller animals, which could
survive and reproduce using less food. This dwarfing over time is typical for island fauna.
[79] Species declined non-synchronously (bison up, mammoths down, then vice versa).
In the graphs presented above we see that initially Browser populations increase the most
as Mixed-Feeders crash, and then Grazer populations increase more slowly, supporting the
complex patterns observed.
Horses are able to survive and reproduce on Holocene vegetation.
In both 2OP alone and Climate Change combined with 2OP, there is a bottleneck when
food for herbivores becomes very scarce. This favors smaller animals and animals that can
extract the maximum nourishment from each blade of grass, i.e. ruminants. Horses, nonruminants, are at a selective disadvantage during the bottleneck but once the ecosystem
stabilized horses can once again survive.
Even animals not hunted by humans went extinct. AND Ground sloth could not find its
preferred food.
A scarcity bottleneck affects animals not hunted by man as well as those hunted by man,
and such scarcity would drive animals to eat less favored food when their favored food was
unavailable.
Conclusion
It is highly unlikely that the Pleistocene extinctions were due to a combination of
Overkill and Climate Change. First, Overkill reduces the impact of Climate Change on
herbivore populations – it is a mitigating, not exacerbating influence. Second, therefore, for
Climate Change mitigated by Overkill to be the cause of these extinctions, Climate Change
would have been far more severe than any researcher has proposed and then any evidence in
the climate and fossil record supports, yet we know that the climate change at the PleistoceneHolocene transition was no more severe than earlier climate change events, events that did
not lead to mass extinctions.
Second Order Predation, in contrast, is a much better candidate for a Climate Change
combination hypothesis. As demonstrated, it exacerbates the impact of climate change on
vegetation and herbivores. Subjected to this combination, herbivores would have been
significantly stressed accounting for Haynes’ observation that long bones were not broken
cited above.
This combination hypothesis also explains the sequence of extinctions: first mixed
feeders, then non-ruminant grazer, and finally large ruminant grazers.
This combination hypothesis would also explain why camels (mixed feeder) and horses
(non-ruminant grazer) went extinct before Proboscideans. Mixed feeders go extinct in the
modeling environments first because they are in competition with both grazers and browsers.
If the climate stress was drought then proboscideans could have dominated access to water
excluding horses.
Creating the Tiniest Bison
243
The Implication for Modern Ecology
This work underlines the importance of threshold and combination effects that interact in
counterintuitive ways. Though this is a model of an archaic ecosystem, the same principles
apply in the present day. Something that seems like a good policy, killing off the competition
– carnivores – may have disastrous long-range results and, as in modern ecosystems, the most
likely path of anthropogenic extinction is through habitat destruction.
Implications for System Dynamics and Science
Explanatory factors, hypotheses and policies may have counterintuitive results. System
dynamics models can help scientists to:
1.
2.
3.
4.
Test theories that are not testable in the field,
Make counterintuitive results and theories more understandable,
Test a variety of hypotheses against each other using the same assumptions, and
Test a variety of hypotheses in combination.
Implications for Archaeologists
The obvious issue for archaeologists is to find direct evidence of H. sapiens killing
carnivores and evidence of a reduction in the ability of the land to produce plants at the same
level as during the Pleistocene – e.g. drought or overall reduction in the quality of the soil.
The Second Order Predation with Exogenous Climate Decline results may also explain
global variations in extinction severity during the Pleistocene. H. sapiens appeared first in the
Old World, so the impact of Second Order Predation would not have been exacerbated by
Climate Change, which began much later. In contrast, in the New World, the appearance of
H. sapiens coincided with Climate Change, exacerbating the impact, thus leading to more
severe extinctions, as observed.
Implications for Bison Research
The model, and the assumption of uniformitarianism, provides a way to use modern day
bison research in explaining the Pleistocene extinctions. Environmental exhaustion as the
proximate cause for extinctions suggests a period of extreme scarcity of vegetation. During
that period, ruminants, which extract the maximum nutrition from their food, would be
selectively favored. Environmental exhaustion also accounts for the shift from plaids – patchy
mixed woodland – to stripes – closed canopy forest near the mountain refugia and unbroken
grassland in the centre of the country. The shift, in turn, suggests why two ecophenotypes or
two sub-species of bison emerged – B. bison bison, grazers of the prairie and B. bison
athabascae, mixed feeders of the woodland. Environmental exhaustion also accounts for the
bias in favor of small size observed in many species including bison and the extinction of
244
Elin Whitney-Smith
animals which were not hunted by H. sapiens (e.g. ground sloths) or should not have suffered
from climate change (e.g. horses).
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
R. D. Guthie, “Mosaics, Allelochemics, and Nutrients: An Ecological Theory of Late
Pleistocene Megafaunal Extinctions”, In Quaternary Extinctions: A Prehistoric
Revolution, P. S. Martin and R. G. Klein (Eds) Univ. Arizona Press, Tucson, AZ 259298 (1984).
E. Andersen, “Who’s Who in the Pleistocene: A Mammalian Bestiary”, In Quaternary
extinctions: A prehistoric revolution, P. S. Martin and R. G. Klein, (Eds) Univ. Arizona
Press, Tucson, AZ 40-89 (1984).
R. D. Guthie, Am. Mdl. Nat. 79:346-363 (1968)
G. W. Calef, “Population growth in an introduced herd of wood bison (Bison bison
athabascae)” In: Northern Ecology and Resource Management: Memorial Essays
honoring Don Gill R. Olson, R., Hastings, and F. Geddes, (Eds) Univ. Alberta Press,
Edmonton, Canada. 183-201 (1984).
C. C. Felerov, “On the origin of the mammalian fauna of Canada”, The Bering Land
Bridge D. M. Hopkins, (Ed) Stanford Univ. Press, CA 271-280 (1967).
B. Frenzel, Science 161:637-649 (1968).
W. A. Fuller and L. A. Bayrock, “Late Pleistocene mammals from central Alberta,
Canada”, Vertebrate Paleontology in Alberta, R. E. Folinsbee and D. M. Ross, (Eds)
Univ. Alberta Press, Edmonton, Can. 53-63 (1965).
R. S. Hoffmann and R. D. Taber, “Origin and history of Holarctic tundra ecosystems,
with special reference to their vertebrate faunas”, Arctic and alpine environments H. E.
Wright, Jr. and W. H. Osburn, (Eds) Indiana Univ. Press, Bloomington, ID. 143-170
(1967).
M. L. Hopkins, R. Bonnichsen, and D. Fortsch, Tebiwa 12:1-8 (1969).
B. Kurten, The ice age. G. P. Putnam's sons, NY (1972).
V. C. Matthews, Quaestiones Entomologicae 4:202-24 (1968).
C. A. Repenning, “Palearctic-Nearctic mammalian dispersal in the late Cenozoic”, The
Bering Land Bridge D. M. Hopkins, (Ed) Stanford Univ. Press, CA 288-311 (1967).
J. C. Ritchie and F. K. Hare, Quat. Res. 1:331-342 (1971).
C. L. Sainsbury, “Quaternary geology of western Seward Peninsula, Alaska”, The
Bering Land Bridge D. M. Hopkins, (Ed) Stanford Univ. Press, CA 121-143 (1967).
C. J. Sorenson, Ann. Ass. Am. Geog. 67:214-222 (1977).
B. A. Yurtsev, “Phytogeography of northeastern Asia and the problem of
Transberingian floristic interrelations”, Floristics and paleofloristics of Asia and
eastern North America. A. Graham, (Ed) Elsevier, NY 19-54 (1972).
P. S. Martin, Science 179: 969-974 (1973).
S. L. Vartanyan, Kh. A. Arslanov, T. V. Tertychnaya and S. B. Chernov, Radiocarbon
Volume 37:1 1-6 (1995).
Creating the Tiniest Bison
245
[19] R. D. E. MacPhee, A. N. Tikhonov, D. Mol, C. de Marliave, H. van der Plicht, A. D.
Greenwood, C. Flemming, L. Agenbroad, Russian Federation, Journal of
Archaeological Science 29: 1017–1042 (2002).
[20] J. E. Mosimann, and P. S. Martin, American Scientist 63:304-313 (1975).
[21] S. L. Whittington, and B. Dyke, “Simulating overkill: experiment with the Mossiman
and Martin model”, In Quaternary extinctions: A prehistoric revolution, P. S. Martin
and R. G. Klein, (Eds) Univ. Arizona Press, Tucson, AZ 451-466 (1984).
[22] J. Alroy, Science 292:1893-1896 (2001).
[23] R. M. May, Stability and complexity in model ecosystems. Princeton Univ. Press.
Princeton, NJ (1973).
[24] N. Leader-Williams, J. Wildl. Management, 44, 640-57 (1980)
[25] A. J. Stuart, P. A. Kosintsev, T. F. G. Higham and A. M. Lister, Nature 431: 684-689
(2004).
[26] D. I. Axelrod, Quaternary extinctions of large mammals. Univ of CA Pub in Geological
Sciences 74:1-42 (1967).
[27] B. H. Slaughter, “Animal ranges as a clue to late-Pleistocene extinction”, In Pleistocene
extinctions: The search for a cause, P. S. Martin and H. E. Wright. (Eds) New Haven:
Yale Univ. Press 155-167 (1967).
[28] R. A. Kilti, “Seasonality, gestation time, and large mammal extinctions”, In Quaternary
extinctions: A prehistoric revolution, P. S. Martin and R. G. Klein, (Eds) Univ. Arizona
Press, Tucson, AZ 299-315 (1984).
[29] P. P Hoppe, “Rumen fermentation in African ruminants”, Proceedings of the 13th
Annual Congress of Game Biologists, Atlanta (1978).
[30] J. E. Guilday, “Pleistocene extinction and environmental change: case study of the
Appalachians”, In Quaternary extinctions: A prehistoric revolution, P. S. Martin and R.
G. Klein, (Eds) Univ. Arizona Press, Tucson, AZ 250-259 (1984).
[31] R. W. Graham, and E. L. Lundelius, “Coevolutionary disequilibrium and Pleistocene
extinctions”, In Quaternary extinctions: A prehistoric revolution, P. S. Martin and R. G.
Klein, (Eds) Univ. Arizona Press, Tucson, AZ 223-250 (1984).
[32] R. M. Hansen, Paleobiology 4:302-319 (1978).
[33] A. M. Phillips, “Shasta ground sloth extinction: Fossil packrat midden evidence from
the Western Grand Canyon”, In Quaternary extinctions: A prehistoric revolution, P. S.
Martin and R. G. Klein, (Eds) Univ. Arizona Press, Tucson, AZ 148-159 (1984).
[34] R. D. Guthrie, “Mosaics, Allelochemics, and Nutrients: An Ecological Theory of Late
Pleistocene Megafaunal Extinctions”, In Quaternary Extinctions: A prehistoric
revolution, P. S. Martin and R. G. Klein, (Eds) Univ. Arizona Press, Tucson, AZ 223250 (1984).
[35] D. I. Axelrod. Geol. Sci., 74:1-42 (1967).
[36] R. A. Kilte, “Seasonality, gestation time, and large mammal extinctions”, In
Quaternary extinctions: A prehistoric revolution, Martin, P. S. and Klein, R. G. (Eds)
Univ. Arizona Press, Tucson, AZ 299-311 (1989).
[37] M. B. Davis, “Pleistocene biogeography of temperate deciduous forests”. In:
Geoscience and man: ecology of the Pleistocene. vol:13. School of Geoscience,
Louisiana State Univ., Baton Rouge, LA (1976).
246
Elin Whitney-Smith
[38] E. Andersen, “A survey of the late Pleistocene and Holocene mammal fauna of
Wyoming” Applied geology and archaeology: The Holocene History of Wyoming. M.
Wilson, (Ed) Geol. survey Wyoming Dept. Invest. , no. 10. (1973).
[39] H. J. B. Birks and H. H. Birks, Quaternary paleoecology. Univ. Park Press. Baltimore,
MD (1980).
[40] H. H. Birks Modern macrofossil assemblages in lake sediments in Minnesota. In
Quaternary plant ecology: the 14 symposium of the British Ecological society,
University of Cambridge, March 1972. Birks, H. J. B. and West, R. G. (Eds) Blackwell
scientific pubs. , Oxford, UK 28- 30 (1973).
[41] R. S. Bradely Quaternary Paleoclimatology: Methods of Paleoclimatic Reconstruction.
Allen and Unwin, Winchester, MA (1985).
[42] J. McDonald, North American Bison: Their classification and evolution, Univ. Calif.
Press, Berkeley, CA (1981).
[43] J. McDonald, “The reordered North American selection regime and late Quaternary
megafaunal extinctions”, In Quaternary extinctions: A prehistoric revolution, P. S.
Martin and R. G. Klein, (Eds) Univ. Arizona Press, Tucson, AZ 404-440 (1984).
[44] H. J. B. Birks, and H. H. Birks, Quaternary Paleoecology, Univ. Park Press, Baltimore,
MD (1980).
[45] G. Haynes, Mammoths, mastodonts, and elephants: Biology, behavior, and the fossil
record. Cambridge Univ. Press, Cambridge, UK (1995).
[46] G. Haynes, American megafaunal extinctions at the end of the Pleistocene, Springer,
NY (2008).
[47] R. D. Guthrie, Nature 331:207-209 (2006).
[48] C. A. Ashworth Quat. Res 13:200-212. (1980).
[49] J. V. Matthews. Geol. Soc. Am. Bull. 85:1353-1384 (1979).
[50] J. C. Bernabo and T. Webb III, Quat. Res. 8:64-96 (1977).
[51] G. R. Coope, “Quaternary coleoptera as aids in the interpretation of environmental
history”, British Quaternary Studies, F. W. Shotton (Ed) Clarendon, Oxford, UK 55-68
(1977).
[52] P. A. Delcourt and H. R. Delcourt, Long-term forest dynamics of the temperate zone: a
case study of late Quaternary forests in Eastern North America. Springer-Verlag, NY
(1987).
[53] R. S. Hoffmann and J. K. Jones Jr. , “Influence of late-glacial and post glacial events on
the distribution of recent mammals on the northern Great Plains”, Pleistocene and
recent environments of the central Great Plains, W. Dort, Jr. and J. K. Jones, Jr., (Eds)
Dept. geol. Univ. KS Spec. Pub. 3. Univ. Press, Lawrence, KS 355-394 (1970).
[54] C. W. Hibbard, “Pleistocene mammalian local faunas from the Great Plains and Central
Lowland provinces of the United States”, Pleistocene and recent environments of the
central Great Plains, W. Dort, Jr. and J. K. Jones, Jr., (Eds) Dept. geol. Univ. KS Spec.
Pub. 3. Univ. Press, Lawrence, KS 295-433 (1970).
[55] D. M. Hopkins, “The Cenozoic history of Beringia - a synthesis”, The Bering Land
Bridge D. M. Hopkins, (Ed) Stanford Univ. Press, CA 451-484 (1967).
[56] J. J. Lowe and M. J. C. Walker Reconstructing Quaternary Environments Longman,
London and NY (1984).
[57] J. V. Matthews. Arctic Alpine Res 7:249-59. (1975).
Creating the Tiniest Bison
247
[58] A. V. Morgan and Anne Morgan, “Paleoentomological methods of reconstructing
paleoclimate with reference to interglacial and interstadial insect faunas of southern
Ontario”, Quaternary paleoclimate, W. C. Mahaney (Ed) Univ. E. Anglia, Norwich,
UK 173- 92 (1981).
[59] R. M. Peck, “Pollen in a small river basin: Wilton Creek, Ontario”, In Quaternary plant
ecology: the 14 symposium of the British Ecological society, University of Cambridge,
28- 30 March 1972. Birks, H. J. B. and West, R. G. (Eds) Blackwell scientific pubs. ,
Oxford, UK 61-77 (1973).
[60] S. T. A. Pickett, and P. S. White, The Ecology of Natural Disturbance and Patch
Dynamics. Academic Press, NY (1985).
[61] J. C. Halfpenny, Yellowstone wolves: In the wild, Riverhead Publishing, Helena, MT
67-76 (2003).
[62] E. Whitney-Smith, “Late Pleistocene extinctions through second-order predation”, In
Settlement of the American Continents: A Multidisciplinary Approach to Human
Biogeography, C. M. Barton, G. A. Clark, D. R. Yesner (Eds) University of Arizona
Press, Tucson, AZ 177-188 (2004)).
[63] E. Whitney-Smith, The Second-Order Predation Hypothesis of Pleistocene Extinctions:
A System Dynamics Model, VDM Verlag, Germany (2009).
[64] P. S. Martin, “Catastrophic extinction and Late Pleistocene blitzkrieg: two radiocarbon
tests”, In Extinctions, M. H. Nitecki (Ed) Univ. Chicago Press, Chicago, IL 153-189
(1984).
[65] P. S. Martin, “Prehistoric overkill: A global model”, In Quaternary extinctions: A
prehistoric revolution, P. S. Martin and R. G. Klein, (Eds) Univ. Arizona Press,
Tucson, AZ 354-404 (1984).
[66] J. Alroy, Science 292:1893-1896 (2001).
[67] R. O. Peterson, Wolf ecology and prey relationships on Isle Royale. National Park
Service Scientific Monog. vol. 11, Washington, DC. (1977).
[68] L. N. Carbyn, S. H. Fritts, D. R. Seip, Ecology and conservation of wolves in a
changing world Canadian Circumpolar Institute, Univ. of Alberta, Edmonton, Can.
(1995).
[69] L. N. Carbyn, S. M. Oosenbrug, D. W. Anions, Wolves, bison and the dynamics related
to the Peace-Athabasca delta in Canada's Wood Buffalo National Park. Circumpolar
Research Series, No. 4. Canadian Circumpolar Institute, Univ. of Alberta, Edmonton,
Can. (1993).
[70] A. R. E. Sinclair, “Dynamics of the Serengetti ecosystem: pattern and process”, In
Serengetti. Dynamics of an Ecosystem, A. R. E. Sinclair and M. Norton-Griffiths, (Eds)
Univ. Chicago Press, Chicago, IL 1-31 (1979).
[71] O. Soffer, The Upper Paleolithic of the Central Russian Plain Academic Press,
Orlando, FL (1985).
[72] J. E. Mosimann, and P. S. Martin, American Scientist 63:304-313 (1975).
[73] Cat House, Private e-mail communication from http://www. cathouse-fcc. org (1996).
[74] L. D. Wing, and I. O. Buss, Elephants and Forests. Wildl. Mong. vol. 19 (1970).
[75] G. P. Kershaw, “Tundra plant communities of the Mackenzie mountains, Northwest
Territories; floristic characteristics of long term surface disturbances”, In: Northern
Ecology and Resource Management: Memorial Essays honoring Don Gill, Olson, R. ,
248
[76]
[77]
[78]
[79]
Elin Whitney-Smith
Hastings, R. , and Geddes, F. (Eds) Univ. Alberta Press, Edmonton, Canada. 239-311
(1984).
P. J Webber, P. C. Miller, F. S. Chapin III, and B. H. MacCown, “The vegetation:
pattern and succession”, In: An Arctic ecosystem: the coastal tundra at Barrow Alaska.
US/IBP Synthesis series no. 12, J. Brown, P. C. Miller, L. L. Tieszen, and F. L. Bunnell
(Eds) Dowden Hutchinson and Ross, Stroudsburg PA. 186-219 (1980).
J. Hansen, M. Sato, R Ruedy, K. Lo, D. W. Lea, and M. Medina-Elizade, “Global
temperature change”, Proc. Nat. Acad. Sci. 103 14288-14293 (2006).
W. S. Broecker, Milankovitch and Climate, Reidel, Dordrecht, Germany 687-698
(1984).
R. H. MacArthur, E. O. Wilson, The Theory of Island Biogeography, Princeton
University Press, Princeton, NJ (1967).
Index
A
access, 62, 86, 221
accessibility, 85, 88
accounting, 112
acid, 57, 59, 60, 151, 152, 153, 173, 179, 183, 184
acrosome, 109
acute infection, 9, 31, 129, 133, 137, 138
adaptability, 24
adaptation(s), 65, 121, 122, 123, 124, 125
adaptive radiation, 226
adipose, 121, 171
adipose tissue, 121, 171
adults, viii, 13, 19, 46, 85, 156
aerosols, 14
Afghanistan, 5, 13, 15
Africa, 1, 2, 4, 7, 10, 11, 12, 13, 16, 23, 24, 30, 47,
48, 122, 124, 225, 226, 230
agar, 15
age, 4, 87, 88, 101, 105, 113, 115, 116, 129, 186,
190, 229
agglutination, 12, 14
agglutination test, 12, 14
aggressive behavior, 6
agricultural sector, 39
agriculture, 6, 57, 60, 62, 66, 67
airways, 139
Alaska, 228
albumin, 44, 51
alfalfa, 59
allantois, 101
allergy, 137
alopecia, 131
alternative medicine, 56
alveoli, 110
amino, 44, 152, 153, 184
amino acid(s), 44, 152, 153, 184
ampulla, 106, 107, 111
anastomosis, 103
anatomy, vii, xiii, 120, 227, 228
androgen(s), 100, 103, 104, 105
anemia, viii, 8, 11, 12, 19, 21, 25, 27, 29, 31
angiogenesis, 201, 202
Angola, 13
angulation, 105, 106
animal behavior, 89, 90
animal disease(s), 2, 6
animal welfare, viii, 55, 60, 62
anorexia, 8, 14, 20, 25, 27, 44, 130
anthelmintics, viii, 39, 46, 47, 49
antibiotic, 58
antibody, 7, 8, 9, 12, 45, 51, 53, 152, 153, 179, 184
antigen, 45, 50, 52, 79, 114, 132, 133, 134, 135, 136,
137, 138
antigen-presenting cell(s) (APCs), 132
anus, 105
aorta, 151
apathy, 27
APCs, 135, 137
apex, 85, 109, 169, 192, 195
apoptosis, xi, 131, 136, 137, 138, 139, 150, 179
appetite, 27
aptitude, 100
Argentina, 52
arterioles, 201
arthrogryposis, 131
Asia, 1, 2, 4, 7, 11, 12, 13, 24, 29, 30, 47, 48, 49,
122, 217, 230
assessment, viii, 21, 55, 62
asymmetry, 105
ataxia, 8
atrium, 151, 155, 156, 166
attribution, 218
Austria, 24, 27
avoidance, 90
awareness, 67
250
Index
B
babesiosis, vii, 1, 2, 7, 9, 16
bacteria, 150
bacterial infection, 139
bacterial pathogens, 140
Bahrain, 5, 13
Bangladesh, 5, 15, 30, 38, 48
barriers, 84
basal layer, xi, 149, 160, 162, 168, 169, 179
base, 22, 156, 158, 164, 166, 167, 168, 171, 173,
190, 195, 206
basement membrane, 169, 187, 189
basic research, 33
beef, 29, 46, 58, 59, 60, 90, 96, 97, 100, 101, 102,
103, 112, 116, 117, 122
behaviors, 63
Belarus, 24
Belgium, 50
benefits, viii, 55, 56, 59, 60
benzene, 12
benzoyl peroxide, 151, 183
BHC, 12
Bhutan, 5, 15
bicuspid, 218
bile, viii, 24, 26, 28, 39, 43, 44, 48, 53
bile duct, viii, 24, 28, 39, 43, 44, 48, 53
biochemical processes, 150
biodiversity, 65, 220
bioinformatics, 78
biomass, 230
biopsy, 9
biosafety, 5
biotic, ix, 81, 91
births, x, 62, 100, 102, 116, 118, 120
bison, xiii, 3, 26, 207, 227, 228, 230, 231
bleeding, 129
blends, 190
blood, x, xi, xii, 3, 5, 7, 8, 9, 14, 15, 20, 43, 44, 50,
73, 99, 100, 112, 113, 114, 115, 116, 132, 151,
157, 163, 166, 167, 169, 171, 173, 175, 181, 182,
183, 187, 189, 194, 199, 201, 203
blood group, 114
blood vessels, xi, xii, 132, 151, 157, 163, 166, 171,
173, 181, 182, 183
body fat, 123
body size, 29, 223
body weight, xi, 25, 122, 181, 182
Bolivia, 24
bone, 132, 134, 216
bone marrow, 132, 134
bones, 207, 230
boreal forest, 228
Botswana, 5, 7
bovine babesiosis, vii, 1, 2
bovine vaccinia (BV), ix, 71, 73
brain, 7, 20, 134, 178
Brazil, v, vii, ix, 13, 24, 71, 73, 74, 75, 77, 78, 79,
80, 81, 122, 124
breathing, 14, 61
breeding, 56, 60, 62, 75
Brno, 37
browser, xii, 205, 206, 207, 210, 215, 219, 220, 221
browsing, 92, 206
buffalo, xi, xii, 4, 30, 43, 48, 72, 78, 100, 117, 118,
149, 150, 151, 158, 159, 160, 161, 162, 163, 165,
169, 170, 171, 172, 174, 176, 177, 178, 179, 181,
182, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203
Bulgaria, 30
Burkina Faso, 5, 7, 15
Butcher, 92
C
calcification, 110
calcium, 44
Cambodia, 5
cancer, 79
capillary, 7, 8, 158, 166, 169, 171, 187, 191, 195,
199, 201, 203
carcinogenesis, 59
Caribbean, 24
carnivores, 229, 230, 231
cash, 124
caspases, 139
category d, 221
CD8+, 137, 138
cecum, 23, 25, 28, 40
cell culture, x, 112, 127, 139
cell death, x, 127, 129, 134, 138, 139
cell line(s), 10, 119
cell surface, 137
cellular immunity, 137
cellulose, 150, 204
Central African Republic, 5, 15
Central Asia, 10, 124
central nervous system (CNS), 12, 134
cercaria, 41, 42, 48, 49
cervix, 105, 106, 107
Chad, 5, 13, 15
Chagas disease, 11
challenges, 67, 69, 77
chemical(s), 10, 12, 58, 85, 137
chemotherapeutic agent, 10
chemotherapy, 45, 46
251
Index
Chile, 24
China, 5, 9, 10, 13, 15, 30, 48
cholangitis, 44
chorion, 101
chromosome, 112, 114, 115, 117
circulation, 75, 77
cities, 6
City, 75
classes, vii, 19
classification, 21, 22
cleavage, 129, 138
climate, 44, 47, 65, 67, 87, 123, 229, 230
climate change, 229, 230
climates, 86, 121, 123, 124
clinical diagnosis, 31
clinical disorders, 60
clinical examination, 115
clinical symptoms, 20, 130
cloning, 14
clusters, 21, 173, 177
coherence, 221
collaboration, 2
collagen, 44, 53, 163, 166
colonization, 139
color, 23, 25, 30, 40, 60, 124, 173
colostrum, 132, 138
commercial, 7
commissure, 105
commodity, 65
common findings, 100
common rule, 67
common symptoms, 44
communication, 38, 65, 66, 67, 107, 150
community(s), viii, 55, 58, 75, 85, 94
compensation, 6
competition, 45, 52, 83
competitiveness, 83
complement, 14, 139, 228
complexity, 46, 77, 82, 204
composition, 57, 59, 60, 85, 88
compounds, 69
computer, 229
conception, 25, 131, 134
conceptual model, 93
conduction, 122
conference, 94
configuration, 224
confinement, 97
conflict, 67
congenital malformations, 131
Congo, 15, 77
Congress, 38, 68, 69
conjunctiva, 130
connective tissue, 111, 163, 166, 167, 171, 173, 187
consensus, 31, 32, 74, 212, 214, 218
conservation, 221, 223
constipation, 61
constituents, 90
consumer choice, 63
consumers, viii, 55, 57, 58, 59, 60, 61, 63, 66, 67
consumption, ix, 24, 28, 58, 66, 81, 82, 83, 84, 85,
88, 90
consumption patterns, 66
contamination, 114
control measures, 60
controversial, 109, 125
copper, 62
coronavirus, 139
corpus luteum, 109, 111
correlation, 117, 220
corrosion, 169, 183
cortex, 103, 110, 133, 138
cost, 6, 10, 47, 49, 64, 83, 84, 112, 116
cough, 130
coughing, 14, 20, 61
covering, xii, 41, 176, 205, 206
crises, 58
Croatia, 27
crocodile, 72
crop(s), 56, 59, 67, 93
crop production, 67
crown, xi, 181, 184, 186, 203, 220
crown-rump length (CRL), xi, 181, 184
cryptosporidium, 34
Cuba, 24, 27
cultural differences, 65
culture, 14, 114, 205
cycles, 90, 112, 116, 168
cycling, 115
cyst, 44, 48, 107, 108, 109
cytochrome, 179
cytogenetics, 120
cytokines, x, 127, 134, 135, 136, 137, 138, 139
cytometry, vii
cytoplasm, 162, 174, 175, 186
Czech Republic, 24, 27, 29, 34, 37
D
dairies, 65
damages, xi, 150, 179
danger, 33, 63
database, 64
DDT, 12
deaths, 44, 62
defecation, 90
252
defects, ix, 99, 100, 109
defense mechanisms, x, 127
deficiency(s), 62
deficit, 179
deformation, 212, 213, 214, 215, 217
degradation, 89
dehydration, vii, 19, 20
DEL, 97
dendritic cell, x, 79, 127, 132
Denmark, 24, 60, 68
Department of Agriculture, 36, 64
deposition, 85, 101, 123, 124
deposits, 44
depression, vii, 3, 14, 19, 27
depth, 85, 88, 91, 94, 191
desmosome, 161
destruction, 43
detectable, 45, 132
detection, 3, 6, 12, 13, 28, 45, 49, 52, 53, 58, 77, 79,
80, 100, 105, 114, 116
developing countries, 5, 47
diaphragm, 153, 155, 156
diarrhea, viii, x, 3, 6, 9, 14, 19, 20, 21, 25, 31, 61,
127, 128, 130, 132, 143
diet, ix, xii, 57, 59, 62, 82, 83, 85, 86, 87, 89, 90, 92,
94, 96, 205, 214, 219, 220
differential diagnosis, 31
digestibility, 87
digestion, 83, 96, 150, 204
direct action, 132, 138
direct observation, 219
discharges, 14, 15
discontinuity, 176
discrimination, viii, 20, 21, 213
diseases, vii, viii, 1, 2, 11, 13, 19, 34, 57, 60, 65, 71,
72, 76, 116, 121, 123, 124
disinfection, 76
disposition, 105, 111
distilled water, 152, 183, 184
distress, 20, 61
distribution, viii, 2, 3, 5, 8, 10, 13, 20, 21, 29, 30, 36,
40, 50, 52, 58, 72, 82, 84, 86, 87, 88, 90, 114,
121, 124, 132, 133, 134, 169, 171, 187, 212
diversity, xii, 67, 82, 92, 141, 205, 210, 218
dizygotic, ix, 99, 103, 114
dizygotic twins, 114
DNA, 3, 7, 21, 33, 72, 78, 114, 115, 118, 120
dogs, 77
DOI, 223, 226
dosing, 47
drainage, 45, 46
drawing, 228
drinking water, 24
Index
drugs, 46, 47, 49, 56
dry matter, 84, 90
drying, 62
duodenum, 156
dyspnea, 6, 9
E
East Asia, 48
Eastern Europe, 30
ecchymosis, 131
ecological systems, 70
ecology, 61, 91, 95, 221, 223
economic efficiency, 65
economic losses, 4, 11, 13, 20, 39, 72, 128
ecosystem, xiii, 227, 228, 231
Ecuador, 5, 24
eczema, 73, 78
edema, viii, 6, 7, 11, 19, 25, 44
editors, 77
education, 64, 77
egg, viii, 25, 39, 41, 45, 47
Egypt, 24, 30, 36, 49
election, 7
electron, 151, 152, 162, 169, 183, 184
electrophoresis, 153, 184
elephants, 228, 230
ELISA, 5, 8, 10, 11, 12, 14, 15, 45, 49, 51, 52, 53,
116
elk, xiii, 227, 228, 231
elongation, xii, 205, 213
emission, 105, 106
employment, 182
encephalitis, 6
encephalopathy, 58
encoding, 79
encyst, 24, 30, 42
endemic ruminant pestivirus, x, 127
endocardium, 7
endocrine, 115, 120, 132
endothelial cells, 7, 134
endothelium, 169, 175
endurance, 125
energy, ix, x, xi, 81, 82, 83, 85, 89, 90, 91, 97, 100,
122, 150, 179
energy expenditure, 91
engineering, 204
England, 52
enlargement, xii, 44, 205
enteritis, 132
environment, viii, ix, xiii, 36, 39, 45, 46, 57, 58, 63,
66, 69, 81, 83, 87, 92, 93, 206, 227, 231
environmental conditions, 45, 83, 125
Index
environmental degradation, 67
environmental impact, 63, 66, 67
environmental management, 66
environmental protection, 67
environmental stress, 230, 231
environments, x, 91, 121, 123, 124, 230
enzyme(s), 7, 11, 12, 44, 45, 50, 51, 52, 53, 86
enzyme-linked immunosorbent assay, 7, 11, 12, 45,
51, 53
eosinophils, 43, 137
epicardium, 7
epidemiological investigations, 14
epidemiology, 2, 10
epididymis, 108, 110
epithelial cells, xi, 104, 129, 131, 134, 138, 181, 186,
187, 188
epithelium, xi, xii, 44, 112, 134, 149, 150, 157, 158,
164, 167, 168, 169, 171, 172, 173, 174, 175, 176,
177, 178, 179, 181, 182, 186, 187, 189, 190, 191,
192, 194, 195, 196, 197, 199, 202, 203
epitopes, 137
erosion, 43
erythrocytes, 114
esophagus, 23, 25, 28, 40, 132, 167
ethanol, 22, 152, 184
ethyl alcohol, 151, 183
etiology, 128
EU, 56, 57, 67, 68
Eurasia, 211
Europe, 2, 5, 10, 12, 13, 26, 29, 30, 47, 67, 122, 217,
224, 226, 230
European Commission, 5
European Parliament, 68
European Union, 58
evidence, 2, 14, 25, 44, 77, 100, 139, 203, 229
evolution, 207, 216, 222, 224, 225, 226
excision, 109
excretion, 44
exercise, 56
expertise, 5, 65
exposure, 4, 53, 77, 138
external constraints, 64
extinction, xiii, 227, 228, 230, 231
extraction, 66, 112
F
families, xii, 21, 206, 210, 219, 220, 221
farmers, 6, 47, 56, 57, 61, 63, 64, 76, 77, 89
farms, 12, 56, 57, 59, 60, 64, 66, 67, 68, 77, 112, 116
fat, x, 57, 59, 60, 62, 109, 121, 123, 124, 125, 230
fatty acids, 59, 179
fauna, 36, 48
253
feces, viii, 39, 41, 45, 49, 85
feed additives, 82
feedstuffs, viii, 55, 65
fermentation, 59
fertility, 11, 100, 102, 109, 115, 116, 119, 131
fertilization, 67, 102
fertilizers, 67
fetus, ix, xi, 99, 100, 101, 114, 117, 131, 134, 136,
181, 183, 185, 186, 187, 188, 189, 190, 191, 192,
193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
203
fever, vii, 1, 2, 3, 6, 8, 9, 10, 11, 14, 15, 20, 25, 62,
73, 76, 128, 130
fiber, 87, 89
fiber content, 89
fibrinogen, 136
fibrosis, 44
fibrous tissue, 26, 44
fish, 35, 106, 113
fixation, 14, 152, 183, 184
flavor, 60
flexibility, 83
flora, 36
flotation, 45
flowers, 163
fluctuations, 6, 66
fluid, 5, 14, 106, 107, 108, 111
fluorescence, 112, 115, 120
follicle(s), 23, 25, 28, 30, 41, 103, 108, 109, 110,
111, 132, 134, 138
food, viii, xiii, 2, 51, 55, 56, 57, 58, 59, 60, 61, 63,
64, 66, 67, 68, 69, 77, 82, 83, 92, 121, 124, 168,
220, 221, 227, 229, 230, 231
food chain, 61, 63, 65
food intake, 82
food production, 58, 61, 63, 65, 66
food products, 63, 65
food safety, 56, 64, 67, 68
food security, 2, 124
foot and mouth disease, vii, 1, 2, 76
force, 84
forest habitats, 216
formation, 27, 44, 103, 110, 198, 203
fossils, xii, 205
fragility, 109
fragments, 3
France, 7, 24, 29, 30, 34, 38, 210, 213, 221, 223, 224
freedom, 2
freemartin, ix, x, 99, 100, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120
freezing, 116
freshwater, 21, 24, 30, 40
254
Index
fruits, 210, 219
H
G
Gabon, 15
gallbladder, viii, 39, 43, 44, 48
gastric mucosa, 179, 182
gastroenteritis, 31
gel, 15, 153, 184
gene expression, 122
genes, 21, 114
genetic background, x, 9, 99, 101
genetic marker, ix, 71
genetics, 10, 119
genome, 3, 74, 79, 128
genotype, 57, 129
genotyping, 141
genus, 3, 7, 12, 14, 22, 41, 43, 72, 73, 218, 220, 224
germ cells, x, 25, 99, 103, 104, 109, 110
Germany, 27, 29, 36, 68, 69, 70, 151, 152, 183, 210,
213, 221
gestation, 46, 131, 134, 136, 203
gland, 41, 110, 114, 173, 201
global scale, 23
globalization, 63
glucose, 151, 183
glutamate, 44
glutathione, 51, 52
glycerol, 153, 184
gonadal dysgenesis, 105, 109, 110
gonads, 100, 104, 107, 108, 109, 110, 111, 115, 120
granules, 23, 160, 162
grass(s), xiii, 42, 45, 48, 56, 59, 60, 84, 88, 89, 92,
94, 95, 96, 206, 210, 219, 220, 221, 228, 230
grasslands, xiii, 206, 228
grazers, xiii, 215, 220, 227, 228, 231
grazing, vii, ix, xii, 19, 20, 21, 28, 29, 45, 46, 49, 56,
60, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 206
grids, 212, 213, 214, 215, 217
grouping, 114
growth, ix, 4, 29, 33, 41, 57, 59, 79, 81, 82, 85, 91,
131
growth arrest, 4
growth factor, 79
growth rate, 57
guidelines, 47
Guinea, 15
Gulf Coast, 26
habitat(s), 24, 45, 46, 49
hair, x, 25, 31, 99, 105, 106, 113, 124
haplotypes, 27
hardness, 91
harmonization, 67
harmony, 61, 70
harvesting, 59, 84
Hawaii, 48
health, vii, viii, 19, 20, 21, 24, 29, 33, 55, 56, 57, 58,
60, 61, 62, 64, 67, 68, 69, 77
health problems, 20, 21, 24
health promotion, 57, 64
health status, vii, 19, 56, 57, 60
heart disease, 59
heartwater, vii, 1, 2, 6, 7
heat loss, 122
height, 82, 84, 85, 86, 88, 89, 91, 96, 164, 168, 190
hematocrit, 11
hemoglobin, 51
hemorrhage, 43, 44
hepatitis, viii, 39, 44
hepatocytes, 43, 44
hermaphrodite, 40
herpes, 76
heterogeneity, 74
highlands, 51
histogenesis, 182, 203
histology, 107, 203
history, 27, 226
Holocene, xiii, 214, 228, 229
homeostasis, 60, 136
hormone(s), 100, 103, 119, 120
horses, xiii, 11, 12, 23, 28, 72, 75, 80, 93, 228, 230
host, x, 4, 11, 12, 21, 23, 24, 26, 28, 30, 40, 41, 43,
45, 46, 51, 72, 77, 78, 79, 128, 129, 132, 134,
137, 138, 140
housing, 56, 57, 128
human, 2, 11, 23, 28, 34, 59, 60, 72, 75, 76, 78, 80,
123, 152, 153, 178, 184
human brain, 152, 153, 178
human health, 60
humidity, 45, 121
Hungary, 27, 29
Hunter, 145
hunting, 27, 229, 230
husbandry, viii, 3, 24, 55, 56, 57, 59, 61, 62, 64, 67,
69
hybrid, 72
hybridization, 10
hydrocephaly, 131
hygiene, 56, 62
255
Index
hyperemia, 14
hyperplasia, 44, 110
hypoplasia, 109, 131
hypothesis, 110, 216, 229, 230
hypotrichosis, 131
I
icterus, 25, 29
ideal, 112, 124
identification, x, 5, 8, 9, 14, 31, 33, 79, 100, 102,
112, 114, 116
identity, 73
IFN, 135, 136, 138
IFNγ, 135, 137
IL-13, 137
ileum, 133
image(s), 50, 106, 109, 111, 197
imbalances, 60, 62, 65
immersion, 8, 152, 183
immune disorders, 136
immune modulation, x, 128, 140
immune reaction, 137
immune response, x, 52, 127, 129, 131, 132, 134,
135, 136, 137, 138, 139
immune system, x, 59, 78, 127, 131, 134, 135, 140
immunity, 4, 8, 51, 56, 79, 135, 136, 138
immunization, 7, 79
immunodeficiency, 12
immunoelectrophoresis, 15, 51
immunofluorescence, 11, 49
immunoglobulins, 137, 138
immunogold-labeling SEM analysis, xi, 149, 152,
175, 181, 184, 202
immunohistochemistry, vii, 78
immunoreactivity, xi, xii, 150, 175, 176, 177, 178,
182, 202, 203
immunosuppression, x, 127, 130, 131, 132, 134, 135,
136, 137
impairments, 8
improvements, 65, 66
in situ hybridization, 114, 120
in vitro, 52, 120, 129, 135, 136, 137, 138, 139
in vivo, viii, 20, 21, 31, 33, 79, 101, 129, 135, 136
incidence, x, 24, 59, 61, 100, 101, 102
incisors, 216
incubation period, 6, 14
India, 5, 9, 10, 13, 15, 17, 24, 29, 48, 49, 78, 204
indigenous knowledge, 60
individuals, 10, 21, 22, 23, 24, 31, 33, 210
Indonesia, 5, 9, 13
induction, 135, 137, 139
industry(s), 6, 9, 122, 124, 128
infection, viii, x, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
17, 24, 25, 27, 29, 30, 31, 33, 38, 39, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 73, 76, 78, 127,
128, 129, 130, 131, 132, 134, 135, 136, 137, 138,
139, 140
infectious agents, 130
infertility, 109, 131
infestations, 20, 25
inflammation, 139
inflammatory cells, 43
influenza, 79
infrastructure, 5
ingest, ix, 43, 81, 90, 93, 94, 95, 96, 97, 206
ingestion, 26, 28, 30, 83, 95
ingredients, 58
inguinal, 109
inhibition, 136, 137
inhibitor, 53, 152
injury, 136, 139
innate immunity, 136
inoculation, 132
insecticide, 12
inspections, viii, 55, 62
inspectors, 64, 65
institutions, 63, 64, 66, 76
integration, 60
integrity, 116, 179
intelligence, 2
intercellular relations, 157
interface, 94, 96
interferons, 136
internalization, 135
interphase, 120
intersex, ix, 99, 117
intestinal tract, 31
intestine, 23, 26, 41, 43, 44, 132, 156
intramuscular injection, 50
investments, 65
Iowa, 34, 140, 141, 142, 144
Iran, 5, 13, 15, 24, 28, 29, 37, 49
Iraq, 5, 15
Ireland, 24
islands, 226
isolation, 13, 31, 61, 73, 79, 219
Israel, 124
issues, viii, 55, 60, 64
Italy, vii, 15, 24, 26, 28, 29, 30, 36, 68, 69, 149, 151,
152, 181, 182, 184, 226
J
Japan, 5, 48, 183
jaundice, 27
256
Index
joints, 61
K
karyotype, 114, 115
karyotyping, x, 99, 112, 114, 115, 118
Kazakhstan, 5, 124
Kenya, 5, 7, 9, 13, 15, 51
keratinocytes, 134
kidney, 44
kill, 229, 230
kinetics, 52
KOH, 152, 183, 195, 196, 197
Korea, 5, 6
Kuwait, 13, 15
localization, 21, 26
longevity, 42
loss of appetite, 61
lumen, 107, 109, 110, 111, 189, 195
lung function, 139
lying, 87
lymph, 7, 9, 52, 73, 76, 132, 133, 134, 136
lymph node, 7, 9, 52, 132, 133, 134, 136
lymphadenopathy, 73, 76
lymphocytes, x, 44, 114, 127, 130, 131, 132, 133,
135, 136, 137, 138
lymphoid, 129, 130, 131, 132, 133, 134, 135, 138
lymphoid organs, 130, 131, 132, 133
lymphoid tissue, 131, 132, 135, 138
M
L
labeling, xi, 58, 149, 152, 175, 181, 184, 202
lactation, 46, 62, 123
landscape, 83, 94
Laos, 5
larva, 41
larvae, 20
larval stages, 20, 45, 48
Latin America, 1, 8, 11, 12, 13, 17
lead, x, 12, 27, 39, 40, 57, 60, 62, 107, 127, 131,
139, 151
learning, 61, 64, 66
Lebanon, 5
legislation, 56, 68
legs, 83, 86, 124
Lepidoptera, 72
lesions, ix, 3, 4, 14, 71, 72, 73, 75, 76, 77, 129, 130,
131, 132, 134, 138
lethargy, 25, 27, 44, 130
leukopenia, 131
libido, 109
lice, vii, 19, 20
life cycle, viii, 9, 24, 26, 30, 39, 40, 43, 46, 48, 77
ligament, x, 99, 107, 108
light, xi, xiii, 149, 150, 151, 152, 181, 183, 216, 228
light microscopy (LM), xi, 149, 150, 181
linoleic acid, 59
Lithuania, 24, 30
liver, vii, viii, 19, 20, 21, 24, 26, 28, 36, 39, 40, 43,
44, 48, 50, 51, 52, 53, 132, 153, 155, 156
liver damage, 50, 51
livestock, vii, viii, 2, 4, 11, 12, 16, 19, 30, 39, 47, 55,
56, 57, 62, 64, 65, 67, 68, 69, 82, 88, 91, 94, 121,
128
living conditions, 57
machinery, 65
Mackintosh, 36
macrophages, x, 44, 127, 132, 143
magnesium, 62
magnitude, 88
major histocompatibility complex, 137
majority, 39, 44, 49, 102, 105
Malaysia, 5, 48
malignant catarrhal fever, vii, 1, 2
mammal(s), 11, 16, 23, 34, 43, 103, 150, 206, 218,
219, 224, 226
man, 21, 117
management, viii, ix, x, 5, 10, 46, 49, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 66, 67, 78, 81, 82, 85, 86,
88, 91, 95, 100, 123, 128
mandible, xii, 205, 206, 207, 210, 211, 212, 213,
214, 215, 216, 217, 218, 219, 220, 221
manipulation, 88, 89, 92, 115
manure, 67
mapping, 152, 153, 184
marketing, 58, 64
marrow, 133, 230
mass, 83, 84, 86, 88, 89, 106, 108, 206, 207, 208,
209, 210, 212, 219, 224
masseter, xii, 205, 220, 221
mastitis, 61, 62, 73, 76
matrix, 160, 162
matter, 77, 158
Mauritania, 15
MB, 213
measurement(s), 65, 88, 118, 211
meat, 4, 11, 13, 56, 57, 60, 65, 123, 124
media, 152
median, 28, 153, 216, 218
medical, 11, 61
medicine, 20, 21, 33, 56, 62
257
Index
Mediterranean, 10, 24, 92, 124, 223, 226
Mediterranean countries, 24
meiosis, 103
membranes, 25, 132, 153, 184
memory, 136, 138
memory B cells, 138
mesenchyme, 186, 189
mesothelium, 163
Metabolic, 122
metabolic changes, 102
metabolic disturbances, x, 100
metabolism, 44, 51, 97, 135
metabolites, 92
methanol, 8
methodology, 45, 49
Mexico, 24, 30
MHC, 79, 135, 137
mice, 73, 75, 77, 79
microcephaly, 131
microscope, 53, 151, 152, 183, 184
microscopy, xi, 149, 150, 181
microtome, 151, 183
Middle East, 10, 47, 124
migration, 28, 48, 51, 218, 229
military, 78
Miocene, xii, 206, 214, 215, 217, 218, 224, 226
miracidium, 28, 41
Missouri, 69
mitochondria, 138, 162
mitosis, 114
mixing, 56, 92
models, 63, 85, 229
modifications, 182, 203
moisture, 41, 43, 45
molecules, ix, x, 12, 99, 127, 134, 137
mollusks, 43
Mongolia, 5
monoclonal antibody, 52, 153, 185
Montana, 24, 26
Montenegro, 36
morbidity, viii, 13, 14, 31, 57, 71, 128, 130
Morocco, 24
morphogenesis, 203
morphological variability, 21
morphology, viii, xii, 20, 21, 105, 107, 108, 109,
110, 111, 115, 150, 168, 203, 205, 207, 210, 211
morphometric, xii, 205, 207, 211
mortality, 10, 11, 13, 14, 31, 41, 44, 57, 62, 120,
128, 130, 131
mortality rate, 31, 41, 44, 131
mosaic, xi, xiii, 149, 163, 227
Mozambique, 5, 7
mucosa, xi, xii, 130, 132, 133, 134, 149, 150, 156,
157, 158, 162, 163, 164, 166, 167, 168, 169, 171,
172, 173, 174, 175, 176, 177, 178, 179, 181, 182,
186, 187, 188, 189, 190, 191, 192, 193, 194, 195,
196, 197, 198, 199, 200, 201, 202, 203
mucous membrane(s), 11, 131, 132
multipotent, 150
muscles, xii, 44, 205, 220, 221
muscular tissue, 156, 167, 169, 171
mutation, 129, 131
myalgia, 73, 76
Myanmar, 5
myocarditis, 5
N
NaCl, 153, 184
Namibia, 5, 7
National Park Service, 224
NCP, 129, 130, 131, 134, 135, 136, 137, 138, 139
necrosis, 43, 131
negative effects, 66
negotiation, 76
Nepal, 5, 15
nerve, 173
nervous system, 12, 28, 131
Netherlands, 52, 151
neurons, 173
neutral, 87
neutropenia, 131
neutrophils, 134
New Zealand, 24, 43, 93, 94
Nigeria, 5, 15, 24
Nile, 72
nitric oxide, xi, 135, 150, 153
nitric oxide synthase, 153
nitrogen, 152
NK cells, 137
nodes, 133, 134
nodules, 108
non-structural protein, 128
North Africa, 10, 29, 124
North America, xiii, 24, 26, 29, 30, 32, 43, 211, 217,
227, 228, 229
nuclei, 120, 157, 162, 187, 195
nucleolus, 187
nucleotide sequence, 74, 75
nucleotides, 74
nucleus, 174, 186, 195, 199
nutrient(s), ix, 58, 64, 65, 81, 82, 83, 85, 90, 182
nutrition, xiii, 56, 94, 95, 227
nutritional deficiencies, 116
nutritional status, 124
258
Index
nystagmus, 6
O
objectivity, 8
obstacles, viii, 55
obstruction, 107, 108
oesophageal, 5
OIE, 2, 5, 7, 13, 15, 16, 17, 36
oil, 8
omentum, 156
oocyte, 103
operations, 51
opisthotonus, 6
opportunities, 66
oral cavity, 4, 132
organic matter, 76
organism, 5, 13, 14, 28, 122, 159
organ(s), vii, 19, 27, 31, 40, 41, 44, 106, 122, 129,
131, 132, 134
oryx, 209
ostium, 173
ovariectomy, 115
ovaries, 109, 120, 134
overseas aid, 5
oviduct, 41
ovulation, 102, 118
ovum, 23
ox, xiii, 227, 228
oxygen, 41
P
Pacific, 26, 47, 122
pain, 25, 76
Pakistan, 24, 30, 35, 38, 48, 49
paleontology, 226
pallor, 25
palpation, 102, 105, 106, 109, 112, 114
pancreas, 154
parallel, 85, 107, 108, 164, 169, 171
paralysis, 12, 27, 46
parasite(s), vii, viii, 7, 9, 10, 12, 19, 20, 21, 23, 24,
26, 27, 28, 29, 30, 31, 35, 38, 44, 46, 47, 124
parasitic diseases, 20, 36
parasitic infection, 31, 33, 52, 56, 137
parenchyma, viii, 26, 39, 43, 48, 51, 108, 109, 110,
111
parity, 101
pasture, ix, 26, 30, 48, 59, 60, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 94, 96
pastures, 29, 45, 46, 82, 84, 85, 86, 88, 90, 91, 94,
96, 124, 125
pathogenesis, x, 127, 128, 132
pathogens, x, 5, 7, 20, 127, 128, 134, 135, 137, 139
pathology, x, 100, 118, 128, 140
pathophysiology, x, 36, 100, 101
pathways, 138
PCA, xii, 205, 206, 207, 211, 212, 214, 216, 217,
218, 219, 220, 221
PCR, viii, 3, 7, 8, 10, 12, 14, 15, 20, 21, 33, 80, 112,
114
PCR-based techniques, viii, 20, 21
penis, 111, 112
peptide, 137, 152, 153, 184
pericardium, 7
perineum, 106, 112
permafrost, 228
permission, iv
permit, xii, 206, 213, 219
Peru, 24
pesticide, 56, 58
petechiae, 7
pH, 151, 152, 153, 179, 183, 184
pharynx, 23, 25, 28, 30, 40, 41
phenotype, ix, 99, 105, 110, 114, 118
Philadelphia, 77, 78, 141, 144, 145, 213
Philippines, 5, 9, 13, 49
phlebitis, 43
phosphate, 151, 183
photosynthesis, ix, 81
phylogenetic tree, 74
phylum, 7
physical characteristics, 85
physical environment, 87
physiology, 95, 97, 125
phytotherapy, 56
pigs, 4, 6, 23, 28, 43
placenta, 134
plant type, 88
plants, ix, 30, 81, 83, 85, 91, 210, 229, 230, 231
plasma cells, 137, 138
plasma proteins, 136
plasticity, 129
platelets, 134
Platyhelminthes, vii, 19, 20, 40
playing, xi, 150, 179
plexus, 167, 173
Pliocene, 216, 218, 219, 226
PM, 77, 88, 90
pneumonia, 14, 139
Poland, 24, 27, 30
policy, 61, 67
policymakers, 64
Index
polymerase, 12, 78, 112, 118
polymerase chain reaction, 12, 78, 112, 118
polymerization, 151, 183
polymorphisms, 10
polyunsaturated fat, 59
population, 7, 10, 13, 27, 32, 46, 77, 101, 116, 118,
123, 124, 229, 230, 231
population size, 231
Portugal, vii, 24, 99, 101, 102, 121
positive correlation, 88
predation, 230
predators, 49, 124, 228, 229
pregnancy, ix, 99, 103, 105, 109, 112, 114, 119, 230
premolars, xii, 205, 211, 213, 216, 217, 218
preparation, iv
prepuce, 109
prevention, 56, 59, 64
Principal Component Analysis (PCA), xii, 205, 211
principles, viii, 55, 57, 60, 65, 226
probability, 114
probe, 113, 115, 120
producers, 61, 63, 64, 65, 66
professionals, 77
profit, 64, 89
progesterone, 115, 119
pro-inflammatory, 135, 136
project, 64, 117, 189, 221
proliferation, 103, 110, 119, 135, 137
proline, 44, 53
proposition, 77
prostate gland, 40
protection, 8, 67, 72, 79
proteins, 103, 136, 138, 153, 178, 184, 185
prototype, 72
pruritus, vii, 19
psychological well-being, 57
puberty, 115, 119
public health, 58
pulmonary edema, 7
purification, 14
pylorus, 155, 156, 172, 173, 174
Q
quality assurance, 65
questionnaire, 63
R
radiation, 122
rainfall, 45, 47, 229
rales, 14
259
reactions, 7
reactivity, 10
reading, 128
reagents, 3
reality, 56
reasoning, 228
recall, 63
reception, 65
receptors, 138
recognition, 33, 129, 136, 137
recommendations, 63, 68
red blood cells, 114
regression, 103, 104, 107
regrowth, 91
regulations, 61, 64, 66, 67
relatives, 216, 218, 221, 223
relevance, 64, 67
reliability, 116
relief, 106
replication, 72, 134, 136, 138, 139
reproduction, 27, 33, 100, 112, 117
reproductive organs, 53, 118, 131
Republic of the Congo, 5, 7, 15
requirements, 58, 83, 86
researchers, vii, 1, 150
reserves, 62, 121
residues, 58
resilience, 70
resistance, 10, 25, 43, 47, 51, 56, 79, 83, 85, 122,
135, 137, 139
resource availability, 82
resource management, 66
resources, ix, 5, 58, 61, 65, 66, 68, 82, 92
respiratory disorders, 130
respiratory rate, 9
respiratory syncytial virus, 139
response, 43, 50, 51, 52, 85, 87, 89, 90, 96, 115, 131,
134, 135, 136, 137, 138, 140, 144
restoration, 44
restriction fragment length polymorphis, 78
retardation, 131
reticulum, xi, 30, 149, 150, 151, 153, 155, 156, 164,
165, 166, 167, 174, 176, 181, 182, 185, 190, 197,
203
RH, 117
rice field, 49
rights, iv
risk(s), 5, 13, 31, 46, 60, 62, 63, 76, 77, 131
risk factors, 77
RNA, 128, 129
rodents, 12, 73, 77, 78
room temperature, 152, 153, 184
rowing, 67, 228
260
Index
Royal Society, 15, 224
rules, 56, 67, 138
ruminant stomach mucosa, xi, 181
rural areas, ix, 2, 66, 71, 73, 76, 77
Russia, 5, 30
Rwanda, 5
S
safety, 56, 59, 60, 64, 68, 79
saliva, 61
salmonella, 61
SAS, 33
Saudi Arabia, 5, 15, 24, 30
scaling, 212
scanning electron microscopy (SEM), xi, 149, 150,
181
scarcity, 77, 121, 124, 125
scatter, 214
scatter plot, 214
science, 57, 226
scrotum, 109
sea level, 34
seasonality, 123
secondary data, viii, 55
secrete, 173
secretion, 25, 135, 136, 140
security, 62
sediment, 45
sedimentation, 45
selectivity, 83, 85, 90, 92
self-sufficiency, 66
semen, 131
seminal vesicle, 40, 104, 106, 112
seminiferous tubules, 104, 110
senses, 83, 85
sensitivity, 9, 10, 112
serologic test, 114
serology, 8
Sertoli cells, 103, 104, 110
serum, 45, 53, 104, 116, 136, 152, 184
services, iv
sex, 57, 83, 103, 110, 115, 116, 117, 118, 119, 120
sex ratio, 120
sex reversal, 120
sex steroid, 104, 115, 116
sexual contact, 78
sexual development, 119
sexual dimorphism, 216
shade, 85, 87
shape, viii, xii, 20, 21, 23, 30, 31, 41, 44, 124, 156,
173, 195, 205, 206, 207, 210, 211, 212, 213, 214,
215, 216, 217, 218, 219, 220, 221
sheep, x, xi, 3, 4, 9, 11, 13, 14, 23, 24, 25, 26, 27, 28,
29, 30, 36, 43, 44, 46, 47, 48, 50, 51, 52, 53, 86,
89, 92, 93, 94, 96, 100, 121, 122, 123, 124, 125,
128, 149, 150, 151, 176, 179
shelter, 85, 91
shock, 8
short supply, 88
shortage, 90
showing, 14, 75, 90, 106, 111, 117, 128, 130
shrubs, 92
Siberia, 229, 230
Sierra Leone, 13, 15
signals, 64, 65
signs, viii, 3, 4, 6, 7, 8, 9, 11, 12, 14, 19, 20, 21, 25,
28, 29, 31, 49, 61, 62, 73, 112, 116, 130, 132, 230
silver, 152, 184
simulations, xiii, 228
sinuses, 133
skeletal muscle, 20
skin, 52, 122, 123, 132
sleeping sickness, 11, 123
Slovakia, vii, 24, 27, 29, 30, 33, 36
small intestine, 43
smallpox, 2, 72, 73, 76, 78
smooth muscle, 191
smooth muscle cells, 191
social activities, 83, 86
social costs, 65
society, 58, 66
sodium, 153, 184
software, 75, 211, 212
solution, 45, 151, 152, 183, 184
Somalia, 5, 7, 13, 15
South Africa, 5, 7, 9, 27, 219
South America, 12, 24, 43, 122, 223
Southeast Asia, 5, 35
SP, 213
Spain, vii, 1, 24, 27, 29, 39, 50, 55, 60, 127, 143, 213
spastic, 46
specialization, 65
sperm, 40, 109
spermatogenesis, 46, 110
sphincter, 174
spleen, 132, 134, 163
splenomegaly, 7
squamous cell, 157, 169
Sri Lanka, 5
stability, xiii, 228
stakeholders, 57, 67
standardization, 67
starch, 90
state(s), x, xii, 5, 10, 24, 26, 56, 57, 60, 63, 79, 80,
87, 92, 94, 127, 132, 134, 135, 136, 137, 206, 221
Index
stem cells, xi, xii, 150, 177, 178, 179, 182
sterile, ix, 99, 100, 114, 116
steroids, 119
stillbirth, 102
stimulation, 115, 135
stimulus, 43
stock, 61, 116, 220
stomach, viii, xi, xii, 19, 21, 30, 149, 150, 151, 153,
154, 155, 156, 175, 179, 181, 182, 184, 185, 189,
190, 202, 203
stomatitis, 14
storage, 114
stress, xiii, 56, 62, 124, 227, 230
stroma, 110, 112, 173
structural modifications, 182, 186, 203
structural protein, 128
structure, viii, x, xi, 20, 21, 31, 32, 36, 40, 41, 44, 88,
89, 91, 94, 95, 96, 99, 105, 107, 108, 109, 110,
111, 112, 113, 114, 149, 150, 163, 164, 171, 203,
207, 231
subacute, 25, 43, 50
subcutaneous injection, 50
submucosa, 163, 189, 190, 191
sub-Saharan Africa, 10
subsistence, 4
subsistence farming, 4
substitution, 90, 179
substrate, 152
Sudan, 5, 7, 15
supplementation, ix, 81, 90, 96
supplier, 62
suppression, ix, 99
surface area, 122, 123, 169
surveillance, 2, 76, 83
survival, 228, 229
susceptibility, 135, 139
sustainability, 66, 67, 70
sustainable development, 69
sweat, 122
Sweden, 55, 99, 151
swelling, 27
Switzerland, vii, 24, 29, 205, 210, 213, 221
symptoms, 6, 27, 29, 112, 129
syndrome, 100, 101, 103, 116, 118, 119, 129, 130
synthesis, 44, 104, 135, 136
Syria, 78
T
T cell(s), 135, 137, 138
T lymphocytes, 137, 138
Tajikistan, 13
Tanzania, 5, 7, 9, 13, 15, 30
261
tapeworm, 20
target, 6, 62, 114, 115, 132, 133, 136, 140
target organs, 136
taxa, xii, 21, 22, 33, 205, 207, 211, 215, 216, 217,
220, 221
taxonomy, viii, 20, 22, 221
technical assistance, 33
technical support, 2
techniques, viii, 7, 11, 12, 20, 21, 52, 114, 182
technologies, 63, 182
teeth, 83
TEM, xi, 149, 150, 157, 159, 160, 161, 164, 169, 171
temperament, 123
temperature, 41, 43, 45, 48, 87, 114, 121, 122, 129,
130, 230
tempo, 94
tensile strength, 84
tension, 41
territory, 27
testicle, 108
testing, 14, 101, 114
testis, x, 40, 99, 103, 109, 110, 117, 119
testosterone, 115, 119
Th cells, 137
Thailand, 5, 30, 49
theileriosis, vii, 1, 2, 10
therapy, 56
thinning, 213
thorax, 7, 121
thrombocytopenia, 129, 130, 131, 134
thrombosis, 43
thymus, 132, 134
thyroid, 116
tick-borne disease(s), vii, 9, 19
tillers, ix, 81, 91
time periods, 67
tissue, xi, 3, 5, 14, 43, 44, 84, 85, 91, 109, 110, 115,
117, 134, 135, 149, 153, 163, 166, 167, 171, 174,
181, 184
TNF, 136, 138
Togo, 5, 7, 15
tonsils, 132
tooth, 211, 228
torus, 173
toxin, 65
trade, 63, 67
traditions, 67
training, 64
traits, 57, 118, 122, 125
trajectory, 106, 112
transcription, 103
transforming growth factor, 79
262
Index
transmission, vii, xi, 11, 12, 27, 47, 51, 73, 78, 149,
150, 151
transmission electron microscopy (TEM), vii, xi,
149, 150, 151
transparency, 57, 58, 67
transport, 27, 61, 62, 63
transportation, 114, 115
treatment, 3, 7, 10, 49, 51, 53, 56, 58, 59, 61, 62, 65,
115
trial, 114, 116
tropism, x, 127, 132, 134
trypanosomiasis, 17
tuberculosis, 61
tumor, 79, 135
tumor necrosis factor, 135
tundra, 228
Turkey, 5, 13, 15, 28, 30
twinning, x, 99, 100, 101, 102, 116, 117, 118, 119,
120
twins, 100, 102, 104, 109, 112, 118
U
UK, 68, 69, 152, 153, 183, 184, 185
ulcer, 73
ultrasonography, 109, 112
UN, 2
uniform, 59, 65, 90, 187
United, 5, 6, 15, 16, 24, 34, 36, 37, 72, 122, 213
United Kingdom, 6, 24, 34, 36, 37
United Nations, 15, 16
United States, 36, 72, 122, 213
universities, vii
untranslated regions, 128
urethra, 111
urine, 85, 90, 105, 106, 112
USA, 27, 34, 36, 47, 69, 78, 140, 141, 142, 143, 144,
145, 151, 152, 153, 180, 184
uterus, x, 23, 25, 28, 41, 99, 100, 102, 105, 106, 107,
108, 110, 111, 116
V
vaccine, 3, 52, 53, 73, 76, 78, 79, 80
Vaccinia virus (VACV), ix, 71, 72
vacuum, 115
vagina, x, 99, 100, 105, 107, 112, 113
validation, 63
valuation, 135
variables, 90, 212, 223
variations, 51, 53, 85, 92, 112, 215, 229
vascular system, ix, 99
vascular wall, 132
vascularization, 103
vasculitis, 131
vector, 12, 73, 79
vegetables, 220
vegetation, xiii, 24, 82, 94, 227, 228, 230
vein, 183
ventilation, 62
vertical transmission, 3
vesicle, 40, 73, 108
vessels, 116, 163, 166, 171
Vietnam, 5, 7, 49
viral diseases, viii, 71, 73
viral infection, 135, 136, 137, 138, 139
virology, 3, 77
virus infection, 14, 77, 78, 79, 80, 139
virus replication, 132, 136, 138
viruses, ix, 3, 4, 71, 72, 73, 75, 78, 79, 80, 128, 129,
137, 138, 139
viscosity, 183
visualization, 12
vitamin E, 57
vulnerability, 66
vulva, x, 99, 105, 106, 113
W
Wales, 52, 152, 184
walking, 87, 90
Washington, 26, 70, 151
water, ix, 4, 24, 30, 42, 49, 62, 81, 87, 90, 91, 95,
124, 151, 152, 183
weakness, vii, 19, 25, 31, 44
wealth, 124
wear, 219, 220
web, 16
weight gain, 25
weight loss, viii, 11, 12, 19, 21, 25, 27, 44, 121, 124,
125
welfare, viii, 33, 55, 56, 57, 58, 61, 62, 64, 65, 67,
68, 69
well-being, 61
West Africa, 12, 123
Western blot, 7
Western Europe, 5, 24
white blood cell count, 130
WHO, 5, 73, 76
wildlife, 2, 4, 12, 40, 78
withdrawal, 46
woodland, xiii, 228, 229, 230
wool, 44
World Health Organization (WHO), 72
worldwide, x, 3, 5, 39, 43, 47, 49, 66, 127, 128
263
Index
worms, viii, 20, 22, 27, 28, 29, 31, 39, 40, 46, 48
worry, 9
yield, 61, 62, 63, 68
yolk, 23
Y
Y chromosome, 114, 115, 120
Yale University, 221, 223
Yemen, 5, 13, 15
Z
Zimbabwe, 5, 7, 9
zinc, 45
zoology, 224