7 Comunicación Corta

P Peruvian journal
J
P of parasitology
Volumen 20- Número 1 - Año 2012
ISSN 2311-4533 (Electronic version)
Comunicación Corta / Short Communication
Biliary hyperplasia in alpacas (Lama pacos) experimentally infected
with Fasciolahepatica metacercariae
Hiperplasia biliar en alpacas (Lama pacos) infectadas experimentalmente
con metacercarias de Fasciola hepatica
1, 2*
2
3
3
Vicente Maco , Vicente P. Maco , Olga Timoteo , Jose R. Espinoza
1.Institute of Tropical Medicine Alexander von Humboldt, Cayetano Heredia University (UPCH), Lima, Peru.
2.Laboratory of Anatomic Pathology, Guadalupe Clinic, Lima, Peru.
3.Institute Molecular Biotechnology Unit, Laboratories for Research and Development,Schoolof Science & Philosophy, UPCH, Lima,Peru.
Abstract
Resumen
Fasciola hepatica infection is one of the major causes of
economic loss in livestock, causing a significant
morbidity among South American camelids such as
alpacas (Lama pacos). In order to better understand the
host-parasite interaction and histopathological insult in
the liver, 6 Huacaya alpacas were infected with a single
dose of 200 viable F. hepatica metacercariae. Adult worms
and eggs were found in tissue preparations of livers of
infected animals after 6 month of experimental
infection. Hepatic lesions as wall thickening of bile ducts,
disorganization of the liver parenchyma, stellate scar with
fibrosis and destruction of bile ducts and liver
parenchyma were observed in all infected animals. The
livers of infected animals showed regions with chronic
inflammation, granuloma containing parasite eggs,
necrosis and cirrhosis. Biliary hyperplasia with multiple
foci of proliferative bile ducts formed by several layers of
transformed epithelial cells was observed adjacent to a
region with extensive liver damage. Chronic F. hepatica
infection in alpacas causes an extensive liver damage and
leads to bridging fibrosis and, characteristically,
adenomatous biliary hyperplasia, suggesting that these
camelids are highly susceptible to this trematode.
Lainfección por Fasciola hepatica representa una de las
mayores causas de perdidas económicas en el ganado a nivel
mundial, causando morbilidad importante entre algunos
camélidos sudamericanos como las alpacas (Lama pacos).
Con el objetivo de entender major la interacción hospederoparásito y el daño histopatológico que causa el parásito en el
hígado, 6 alpacas tipo Huacaya fueron infectadas con una
dosis única de 200 metacercarias de F. hepatica. Se visualizó
parásitos adultos y huevos en los tejidos del hígado de los
animales infectados luego de 6 meses de la infection
experimental. En todos los animales, se observó
engrosamiento de las paredes de los conductos biliares,
parénquima hepático desorganizado, cicatrizaciones
estelares con fibrosis y destrucción de los conductos biliares
y del parénquima hepático. Los hígados de los animales
infectados también mostraron inflamacion crónica,
granulomas conteniendo huevos parasitarios, necrosis y
cirrosis. Se observó hiperplasia biliar con múltiples focos de
conductos biliares proliferativos formados por múltiples
capas de células epiteliales transformadas, adyacentes a la
región con daño hepático extenso. La infeccióncrónica por
F. hepatica en alpacas causa un daño hepático extensor y lleva
a fibrosis portal extensa y,característicamente, a laformación
de adenomas de hiperplasia biliar, lo cual sugiere que estos
camélidos son altamente susceptibles a la infección por este
distoma.
Key words: Fasciola hepatica/ anatomy & histology
|Fasciola hepática /metabolism|Camelids, New World|
Immunohistochemistry|Models, Biological|(Source:
DECS BIREME)
Palabras clave: Fasciola hepatica /anatomía &
histopatología| Fasciola hepatica /metabolismo |Camélidos
Americanos| Inmunohistoquímica| Modelos Biológicos|
(Fuente: DECS BIREME)
Citation: Maco V, Maco VP, Timoteo O, Espinoza JR Biliary hyperplasia in alpacas (Lama pacos) experimentally infected with Fasciola hepatica metacercariae.
Peru j parasitol. 2012;20(1): e25-e29
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Comunicación Corta
Maco et al. Biliary hyperplasia in alpacas infected with Fasciola hepatica
Peruvian journal of parasitology
Volumen 20- Número 1 - Año 2012
ISSN 2311-4533 (Electronic version)
F
asciola hepatica (F. hepatica) infection is
one of the major causes of economic loss
in livestock worldwide, with a great impact
in the economy of endemic areas in Peru.2A wide
range of domestic species is susceptible to the
liver fluke infection, as alpacas (Lama pacos), in
which the infection causes anemia, eosinophilia,
weight loss, emaciation and mortality.3 Despite
the importance of this infection by its negative
impact in fiber productivity and quality, little is
known of the liver pathology caused by F.
hepatica in South American camelids, and of the
parasite factors that produce the pathogenesis of
the disease.
aged 18 to 24 months) from the department of
Junin (Central Highlands of Peru) were infected
with a single dose of 200 metarcariae of F.
hepatica administered orally, after a 30-days
adaption period. The metacercariae were
obtained from lymnaeid snails infected with
laboratory-raised F. hepatica miracidia. Prior to
inoculation, fluke eggs were not detected in stool
samples by the Rapid Sedimentation Technique
(RST) by Lumbreras,8 and enzyme-linked
immunosorbent assays (ELISA) using Fas1, Fas2
and excretory/secretory products were negative.5
All of the infected alpacas and 1 uninfected
control underwent necropsy 6 months after the
first single dose.The study protocol was approved
by the Institutional Ethics Committee for
Animals. Tissue samples from livers were fixed in
formaldehyde, included in paraffin and
processed by hematoxylin-eosin (H&E) and
Masson's trichrome (MT) stains, the latter used
to differentiate between collagen fibers and
smooth muscle from tumors. Von Kossa, a stain
intended for use in the evaluation of calcium
deposits, was not performed since standard
H&E is considered highly sensitive for
mineralization in anatomopathology. The
following variables were recorded: portal
fibrosis, bile duct hyperplasia, cirrhosis, presence
of eggs, granulomas, and the severity of lesions
were graded as mild (fibrosis: enlarged fibrotic
portal tract; hyperplasia: 25%-50% epithelial
height), moderate (fibrosis: periportal fibrosis or
portal to portal fibrosis; hyperplasia: 50%-75%
epithelial height), and severe (fibrosis: bridging
fibrosis with architectural distorsion;
hyperplasia >75% epithelial height).
A differential susceptibility of the host to the
fluke is a feature of the disease, being cattle able
to mount a strong immunological response that
immunoprotects to subsequent infections, and
sheep develop little or absent immunoprotective
response to F. hepatica infection .4
It is not known whether alpacas become resistant
to F. hepatica after a primary infection. However,
field studies suggest that animals
acquiredmultiple infections in endemic areas
and they are re-infected after treatment with
5
flukicide. Alpacas produce antibody response to
liver fluke infection, which peaks 8 weeks after
primary infection, but this response seems
unable to confer immunoprotection to reinfection and does not prevent the liver damage
observed in infected animals.6
U n t i l n o w, n o d e s c r i p t i o n o f t h e
histopathological insults caused by the fluke
infection was reported in alpacas, as it is well
described in sheep, cows and goats. 7In alpacas,
the liver penetration appears to start 4 weeks
after infection suggested by the elevation of
hepatic enzymes observed in infected animals
and bile ducts resident mature flukes shed eggs 8
weeks after the infection. 6
F. hepatica infection of alpacas was confirmed by
the visualization of eggs (Fig. 1) and adult worms
(not shown) in the liver tissue sections. Gross
anatomy of the livers of infected animals showed
hepatomegaly, fibrosis and calcified bile ducts.
Matureflukes were recovered from the livers
atnecropsy. The intensity of infection was
recorded according to the parasite load. The
parasite load averaged 41± 4 (18%-25%) from an
infective dose of 200 metacercaria e per animal.
In order to gain insight into the
histopathological changes of the fluke in the
liver and biliary track of this South American
camelids, 6 Huacaya alpacas (4 males, 2 females,
Peruv. j. parasitol 2012; 20 (1)
Acceso gratuito en línea a texto completo.
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Comunicación Corta
Maco et al. Biliary hyperplasia in alpacas infected with Fasciola hepatica
Peruvian journal of parasitology
Volumen 20- Número 1 - Año 2012
ISSN 2311-4533 (Electronic version)
E
E
(b)
(a)
Figure 1. (a) Multiple characteristics Fasciola hepatica eggs shells (E) in bile ducts (H&E 40X). (b)
Magnification of parasitic elements in situ (H&E 200X). Scale bars: 100nm.
H & Esections of infected liver tissue showed
extensive tracks of fibrosis (Fig. 2b) as a response
to the liver damage caused by the fluke.The
infection produced an extensive liver damage
that ranges from inflammatory response to
cirrhosis. The liver of infected animals showed
patches of acute and chronic inflammation to
the liver fluke and to the eggs. Granuloma
containing parasite ova was a common finding in
H&E sections of the liver with chronic infection
(Fig. 3a). Inflammatory response recruited
lymphocytes, macrophages and plasma cells. We
did not detected eosinophils in the
inflammatory foci, in spite that peripheral blood
eosinophilia in blood was observed in infected
alpacas. A peripheral ring of fibroblasts with
multinucleated giant cells, lymphocytes and
plasma cells surrounded granuloma containing
F.hepatica ova (Fig. 3b). Multinucleated cell and
lymphocytes were abundant in mineralized
granuloma and in areas with cirrhosis. Mild to
severe grades of bile duct hyperplasia with biliary
adenoma formation (Fig. 4).
C
FB
N
FB
(b)
(a)
Figure 2. (a) Necrosis (N) and calcification (C) in the liver of an alpaca experimentally infected with
Fasciola hepatica metacercariae. Portal triad shows sclerosed bile duct and calcification (H&E 40X).
(b) Different degrees of fibrosis, with nodular to bridging fibrosis (FB), and cirrhosis (MT 40X). Scale
bars: 100nm
Peruv. j. parasitol 2012; 20 (1)
Acceso gratuito en línea a texto completo.
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Comunicación Corta
Maco et al. Biliary hyperplasia in alpacas infected with Fasciola hepatica
Peruvian journal of parasitology
Volumen 20- Número 1 - Año 2012
ISSN 2311-4533 (Electronic version)
The presence of multiple non-encapsulated
biliary adenomes in infected animals is a feature
of this infection not described in other species.7
The neoplastic tissue consists of biliary epithelial
proliferation with no evidence of nuclear or
cellular atypia or the presence of mitotic figures.
Multiple foci of proliferative bile ducts formed by
several layers of transformed epithelial cells were
observed djacent to a region with extensive liver
damage (Figs. 4a and 4b). A severe form of
neoplasia that produced the atrophy of the liver
right lobe was caused by the hyperplasia of biliary
epithelia was previously described in F. hepatica
infected alpaca. 9A similar finding, hyperplasia of
the gall bladder was described in a man with a
chronic infection.10The development of biliary
hyperplasia associated to F. hepatica infection is
probably not an uncommon event andit is
suggestive of the high susceptibility of the alpacas
and other hoststo the liver fluke infection.
G
E
(a)
I
(b)
Figure 3. (a) Portal triads and many hystiocytes forming epithelioid granulomas (G) (H&E 200X). (b)
Portal leukocytic infiltration (I) and dilatation of bile ducts with presence of an egg (E) in the lumen
(H&E 200 X).Scale bars: 100nm. (b) Different degrees of fibrosis, with nodular to bridging fibrosis
(FB), and cirrhosis (MT 40X). Scale bars: 100nm
In conclusion, alpacas are a highly susceptible
species to F. hepatica assuggested by the extensive
liver damage with a distortion of the
liverarchitecture that even leads to tissue
transformation with characteristics of biliary
adenomes in experimentally infected animals.
(b)
(a)
Figure 4. (a) Prominent adenomatous biliary hyperplasia in alpacas (H&E 40X) with magnification b)
(H&E 200X). Scale bars: 500nm (4a) and 100nm (4b). (H&E 200 X).Scale bars: 100nm. (b) Different
degrees of fibrosis, with nodular to bridging fibrosis (FB), and cirrhosis (MT 40X). Scale bars: 100nm
Peruv. j. parasitol 2012; 20 (1)
Acceso gratuito en línea a texto completo.
28
Comunicación Corta
Maco et al. Biliary hyperplasia in alpacas infected with Fasciola hepatica
Peruvian journal of parasitology
Volumen 20- Número 1 - Año 2012
ISSN 2311-4533 (Electronic version)
Author’s contribution: VM, OT, and JRE
Acknowlegdments. This work was partially
designed the study. VM and VPM performed all
histopathological stains and analyzed the
paraffin-embedded liver tissues.
funded by a grant B/2856-1 from the
International Foundation for Science an
INCAGRO CONTRACT 007-2003 to JRE. OT
received support from the Consortium of
Francophones Universities of Belgium (CIUF)
and RED SAREC. E. Chavarry for providing the
Fas2 antiserum.
Conflict of Interest: The authors declare
none.
References
1. Spithill TW, Smooker PM, Sexton JL, Bozas E,
Morrison CA, Creaney J, Parsons JC. Development
of Vaccines Against Fasciola hepatica. In: Dalton
JP, editor. Fasciolosis. Cambridge, UK: CABI
Publishing; 1999. p. 377-401.
6.Timoteo O, Maco VJr., Maco V, Neyra V, Yi PJ, Leguia
G, Espinoza JR. Characterization of the humoral
immune response in alpacas (Lama pacos)
experimentally infected with Fasciola hepatica against
cysteine proteinases Fas1 and Fas2 and
h i st o p a t h o l o g i c a l f i n d i n g s . Vet I m m u n o l
Immunopathol. 2005; 106: 77-86.
2. Espinoza JR, Terashima A, Herrera-Velit P, Marcos
LA. [Human and animal fascioliasis in Peru: impact
in the economy of endemic zones]. Rev Peru Med Exp
Salud Publica. 2010; 27: 604-612.
7.Behm CA, Sangster NC. Pathology, Pathophysiology
and Clinical Aspects. In: Dalton JP, editor.
Fasciolosis. Cambridge, UK: CABI Publishing; 1999.
p. 377-401.
3. Leguia G, Casas E. Distomatosis hepatica. In: Diaz
FS, editor. Parasitic diseases and parasitological
atlas of South American camelids. First ed. Lima,
Peru: Editorial de Mar; 1999. p. 40-63.
8.Lumbreras H, Cantella R, Burga R, 1962. Acerca de
un procedimiento de sedimentación rápida para
investigar huevos de Fasciola hepatica en las heces, su
evaluación y uso en el campo. Rev Méd Peruana. 1962;
31: 167-74.
4.Mulcahy G, Joyce P, Dalton JP. Immunology of
Fasciola hepatica Infection. In: Dalton JP, editor.
Fasciolosis. Cambridge, UK: CABI Publishing;
1999. p. 377-401.
9.Hamir AN, Smith BB. Severe biliary hyperplasia
associated with liver fluke infection in an adult
alpaca. Vet Pathol. 2002; 39: 592-594.
5.Neyra V, Chavarry E, Espinoza JR. Cysteine
proteinases Fas1 and Fas2 are diagnostic markers
for Fasciola hepatica infection in alpacas (Lama
pacos). Vet Parasitol. 2002; 105: 21-32.
10.Osman M, Lausten SB, El-Sefi T, Boghdadi I,
Rashed MY, Jensen SL. Biliary parasites. Dig Surg.
1998; 15: 287-296.
Correspondence: Vicente Maco. Current address: Honorio Delgado 430, Urb. Ingenieria, San Martin de Porres, Lima, Peru. Tel.: +1 202 247
0505. E-mail address: vicente_maco@hotmail.com
Peruv. j. parasitol 2012; 20 (1)
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