Topics in Medicine and Surgery Comparative Morphology, Development, and Function of Blood Cells in Nonmammalian Vertebrates Juan A. Claver, DVM, PhD, and Agustin I. E. Quaglia Abstract Much of our knowledge regarding vertebrate blood and blood cells is based on mammalian references. The critical study of nonmammalian vertebrate blood is relatively new, and comparatively few investigations have been published that focus on these animals’ ontogeny and structure-function relationships of blood cells. Nonmammalian vertebrates comprise birds, reptiles, amphibians, and fishes, all of which have a wide range of forms and adaptations. For the clinician, a lack of basic knowledge with these species makes the evaluation of a hemogram more challenging than in mammals. This is a concise review of our current knowledge of comparative morphology that describes routine staining procedures and the development and function of blood cells in nonmammalian vertebrates. Copyright 2009 Elsevier Inc. All rights reserved. Key words: amphibian; bird; fish; hematology; reptile M any general considerations can be made for the blood of nonmammalian vertebrates (NMV). As in mammals, NMV blood cells include erythrocytes, leukocytes, and hemostatic cells. With very few exceptions, red blood cells in NMV are nucleated and oval in shape.1 A cytoskeleton is responsible for their conversion from a sphere to an ellipse, and imparts reversibility of traumatic deformation. All NMV have nucleated thrombocytes instead of platelets, and heterophils with acidophilic granules are, in most cases, the counterpart of mammalian neutrophils. Birds Birds include more than 9000 species of homeothermic vertebrates. Most of the scientific information regarding avian hematology is the result of domestic fowl investigations. The morphology of avian blood cells are comparatively more homogeneous between orders than in other NMV. Hematopoiesis Current scientific knowledge regarding early hematopoiesis in vertebrates came from experiments first performed on the avian embryo. Primitive hematopoiesis in the chicken embryo begins in the blood From the Histology and Embryology Department, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina, and Fundación de Historia Natural Félix de Azara, Buenos Aires, Argentina. Address correspondence to: Juan A. Claver, DVM, PhD, Histology and Embryology Department, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina. E-mail: jclaver@fvet.uba.ar. © 2009 Elsevier Inc. All rights reserved. 1557-5063/09/1802-$30.00 doi:10.1053/j.jepm.2009.04.006 Journal of Exotic Pet Medicine, Vol 18, No 2 (April), 2009: pp 87–97 87 88 Claver and Quaglia Figure 1. Avian blood cells (A, B, C). Erythrocyte (a), polychromatic erythrocyte (b), thrombocytes (c), heterophil (d), eosinophil (e), basophil (f), small lymphocyte (g), large lymphocyte (h), and monocyte (i). islands of the yolk sac during the second day of incubation. At that time, only nucleated erythrocytes and thrombocytes are produced.2,3 Hemoglobin can be detected as early as 24 hours after fertilization (2-4 somite stage). The yolk sac in avian embryos will remain hematopoietic for most of the embryonic development, until bone marrow hematopoiesis is established.3,4 Definitive hematopoiesis begins in the dorsal mesenchyma (aortic and para-aortic foci) from days 5 to 8 of incubation. New stem cells from these areas colonize the spleen, which is hematopoietic from days 9 to 18, and finally the bone marrow from day 12 of incubation into adult life. The fetal liver is not a major hematopoietic organ in birds. Birds were an early model for immunology studies, leading to the identification of the B- and T-lymphocyte lineages. Maturation of T-lymphocytes occurs in the thymus. Birds have a specific organ, the bursa of Fabricius, which is the site of differentiation for Blymphocytes.2 Erythrocytes Avian erythrocytes are oval in shape and nucleated (Fig 1A, a). The nucleus of the avian erythrocyte is also oval in shape, and it becomes more condensed with age. The cytoplasm generally stains uniformly orange-pink, except for a thin, pale perinuclear 89 Morphology of Blood Cells in Nonmammalian Vertebrates band. Hemoglobin is found in both the cytoplasm and nucleus. Erythroplastids (anucleated erythrocytes) and immature erythrocytes are occasionally present in circulating blood. Immature forms of the avian erythrocytes (Fig 1A, b) have an irregular cytoplasmic polychromasia and a more rounded, pale nucleus.5-7 The major function of erythrocytes is oxygen transport, but recently it has been reported that bird erythrocytes, as nonimmune cells, are able to participate in some immune responses that contribute to host defense.8 Leukocytes In the majority of birds, heterophils are the most common white blood cell in circulation. Heterophils have colorless cytoplasm and typical eosinophilic, rod-shaped granules, together with some other rounded granules (Fig 1A, d). Specific granules are elliptical, although in some species they may be oval or rounded and have a distinct central body that appears refractile.5 Heterophilic response to infection is similar to that of the mammalian neutrophil, migrating to the sites of inflammation and killing pathogens.9 Heterophils are highly phagocytic and are capable of a broad spectrum of antimicrobial activity. These leukocytes tend to accumulate in inflamed tissue, causing tissue damage and forming heterophilic foci that are morphologically similar to inflammatory lesions in reptiles.10,11 The avian heterophils contain many lysosomal and nonlysosomal enzymes.12 Unlike mammalian neutrophils, avian heterophils are devoid of myeloperoxidase and depend primarily on nonoxidative mechanisms, lysozyme, and acid phosphatase for antimicrobial activity.13 The B defenses have a broad spectrum of activity against the binding abilities of infectious agents. Spherical granules contain acid hydrolases, whereas a third, smaller, round vacuole granule has been identified by electron microscopy.12 Heterophils may show toxic changes in severe systemic illness, including increased cytoplasmic basophilia, degranulation, and abnormal granulation.5 Eosinophils are similar in size to heterophils but contain round, eosinophilic granules and a pale blue cytoplasm (Fig 1A, e).5 In some bird groups, the granules may actually be rod shaped (e.g., Anatidae, Tinamidae, Falconidae; Fig 1D), whereas in others they may have a bluish coloration (e.g., in psittacine birds; Fig 1B and C).14 Crystalline cores have been described in specific granules of eosinophils in some birds such as geese and ducks, but these structures are not commonly found in other species.15 Cytochemically, avian heterophils lack myeloperoxidase, whereas eosinophils are peroxidase positive.13 Addi- tionally, certain hydrolytic enzymes (e.g., acid phosphatase and arylsulphatase) have been detected in avian eosinophilic granules, supporting the theory that these structures are lysosomal in nature. The function of the avian eosinophil is poorly understood. Eosinophilia can be observed after foreign antigen administration and possibly in association with alimentary tract parasitism, but the parasitic response by this cell type in birds has not been scientifically confirmed.11,16 Basophils are recognized by a rounded nucleus and characteristic violet to reddish purple granules, which are much smaller than those of the eosinophil (Fig 1A, f). Little is known about basophil function in birds. Basophils appear to play an important role in early inflammation and immediate hypersensitivity reactions, but differ from those in mammals by not contributing to delayed hypersensitivity.17 Severe stress has also been proposed as an underlying cause for an increased basophilic response in birds.18 Lymphocytes may be the major circulating leukocyte in the blood of some species. Avian lymphocytes are small to large, rounded to irregular cells with a round nucleus and scant to abundant basophilic cytoplasm (Fig 1A, g and f). Small lymphocytes predominate in most birds, with irregular projections or blebs frequently observed on this cell type.7 Birds were used as a pioneer model to study immunology, which resulted in the identification of the B- and T-lymphocyte lineages. Avian lymphocytes appear to function in the same manner as mammalian lymphocytes. The B-cells (bursa dependent) have immunoglobulin receptors for antigens, while T-cells (thymus dependent) are involved in cell-mediated immunity.2,7 Monocytes are the largest cells in bird blood, and they resemble their mammalian counterparts. The typical monocyte is round, with a kidney-shaped nucleus. The cytoplasm is generally deep blue or grayish blue, often presenting a pink- or purple-staining granular area near the nucleus (Fig 1A, i). Monocytes have more cytoplasm and a paler nucleus than large lymphocytes.7 As in mammals, avian monocytes are part of the monocyte-macrophage system and constitute the replacement pool for tissue macrophages.16 Thrombocytes In all NMV, thrombocytes are the functional equivalent of mammalian platelets. In birds, thrombocytes are round to oval cells, smaller than erythrocytes, and contain an oval to rounded nucleus. The cytoplasm is light blue or colorless, often vacuolated with a few acidophilic granulations (Fig 1A, c).7 Thrombocytes are often confused with small lymphocytes and have a ten- 90 dency to clump in smears, particularly when processed without the use of an anticoagulant. When clumping occurs, they may show degranulation of specific granules, cellular degeneration, and nuclear pyknosis.16 As with mammalian platelets, bird thrombocytes aggregate in a site of vascular injury and form a hemostatic plug. Additionally, thrombocytes have phagocytic abilities and probably have some function in nonspecific immunity.19,20 Thrombocyte abnormalities are rarely diagnosed and thrombocyte dysfunction syndromes are not described in birds. Reptiles Reptiles comprise nearly 8000 species of poikilothermic vertebrates. Reptiles represent a diverse group of animals (e.g., lizards, snakes, turtles, crocodiles), with some of them used as pets and others as an economic resource. The American anole, Anolis carolinensis, is the “laboratory mouse” of the reptile world in terms of research investigation. The basic hematology of reptiles is very similar to that of birds, although reptiles are a more heterogeneous class so it may be more difficult to draw inferences between species.21 Hematopoiesis Few studies have been made in primitive hematopoiesis in reptiles. The yolk sac blood islands seem to be the unique primary erythropoietic site during embryonic life in reptiles, with bone marrow not becoming active until birth.22 As in birds, postnatal erythropoiesis and granulopoiesis of reptiles occurs mainly in the bone marrow, although the liver and spleen have some hematopoietic function in the early stages of development. Lymphocytes most likely of bone marrow origin will colonize and differentiate into T-cells in the thymus, which regress with age. A true bursa of Fabricius has not been found in reptiles, and the site where B-lymphocytes develop is currently unknown. The location of lymphopoiesis includes the bone marrow, spleen, and other locations within the body. Mucosal-associated lymphoid tissue is also present in the digestive and respiratory tracts.23 Erythrocytes Erythrocytes of reptiles are similar in function and appearance to those of birds but can vary in size, being relatively smaller in Sauria and larger in some Chelonia, Crocodilia, and Rhynchocephalia (Fig 2A, a and b). The overall number of circulating erythrocytes is lower in reptiles than in mammals or birds. Claver and Quaglia Lizards generally have a greater erythrocyte population than do snakes. There appears to be an inverse correlation between erythrocyte number and cell size.24,25 The nucleus of the reptile erythrocyte is more rounded, particularly in turtles, and often has irregular margins. Immature erythroid forms have basophilic cytoplasm, similar to those in avian species (Fig 2A, c). As reptiles recover from brumation, there may be a marked regenerative response with basophilic erythrocytes.26 The life of a reptile erythrocyte has been reported to be 600 to 800 days, with the extreme longevity being attributed to the low metabolic rate of these animals.27 Leukocytes Classification schemes for leukocytes in reptiles are inconsistent because variable criteria have been used to categorize these cells. Reptile leukocytes have been described according to their appearance rather than function. As in birds, reptiles have heterophils instead of neutrophils. Heterophils may vary greatly between groups, genera, and species. The reptile heterophil has characteristic needle-like acidophilic cytoplasmic granules (Fig 2A, g and B), with crocodilian species having larger granules, although fewer in number, than lizards and snakes (Fig 2A, f).28 The heterophil nucleus has a round shape, and it may have two lobes in some lizards.26 Functionally, the reptilian heterophil is similar to the avian heterophil, with their primary function being phagocytosis.23 Azurophils are leukocytes similar to monocytes but contain prominent azurophilic granules (Fig 2A, i). Azurophils have been described in Squamata (snakes and lizards) and Crocodilia, but are seen rarely in Chelonia.11,29 Some authors consider azurophils as a monocyte variant.30 Eosinophils, Basophils, Lymphocytes, Monocytes. Eosinophils are observed in Crocodilia and Chelonia, but their existence in Squamata is controversial. Even inside a genus of snakes, eosinophils are described in some species and not in others.31,32 Eosinophils have a round nucleus and acidophilic-staining granules, although they stain blue in green iguanas (Fig 2A, h). Cytochemically, heterophils of both avian and reptilian species studied lack myeloperoxidase, whereas eosinophils were peroxidase positive.23 Basophils found in reptile species resemble those of birds, both in appearance and function (Fig 2a, I). Chelonians tend to have higher numbers of circulating basophils than other groups of reptiles; in some cases up to 50% to 60% of their differential may be attrib- 91 Morphology of Blood Cells in Nonmammalian Vertebrates Figure 2. A, Blood cells of reptiles. Erythrocytes of crocodile (a) and iguana (b), polychromatic erythrocyte of turtle (c), normal (d) and reactive (e) thrombocytes, heterophils of crocodile (f) and turtle (g), eosinophil of turtle (h), azurophil (i), monocyte (j), small lymphocyte (k), and basophil (l). B, Heterophil. Trachemys scripta dorbigny. uted to basophils. It is not known why chelonians have such high levels of circulating basophils. For most reptiles, lymphocytes are the most prevalent circulating cell. They are similar in appearance to the avian and mammalian lymphocytes (Fig 2A, k). Band T-lymphocytes have been characterized in reptiles and are functionally similar to those found in birds.23 Lymphocytes show seasonal variations in reptiles, with lower numbers reported during the winter months.33 Monocytes (Fig 2A, j) and thrombocytes (Fig 2A, d and e) are similar in structure and function to those described in the previous avian section.23 Amphibians Very little has been published regarding amphibian (class Amphibia) hematology. This class comprises near 6000 species of poikilothermic vertebrates. Frogs and other amphibians have been widely maintained in captivity as pets and as animal models for physiologic and embryologic studies. Xenopus laevis, the African clawed frog, is the most popular amphibian species used in research. Amphibians are also marketed as culinary delicacies and increasingly studied as bio-indicators of environmental health. The examination of blood samples can be useful in 92 evaluating the status of diseased animals, although the similarity in function of amphibian blood cell types and those of other species is largely unknown. Hematopoiesis The development of hematopoiesis in amphibians has some similarity to that of birds and mammals. The ventral blood islands (e.g., analogous to the yolk sac blood islands of higher vertebrates) and the dorsal lateral plate region are the sites of primitive and definitive hematopoiesis in the amphibian embryo. Hematopoietic stem cells of the dorsal lateral plate give rise to the definitive lineages, which ultimately colonize the fetal liver and thymus.34 The kidneys are the main sites of blood formation in the larval stages of amphibian species. The thymus is the major site of T-cell maturation, but the site of B-cell lymphopoiesis is less clear and amphibians appear to use different sites for this specific cell production depending on the species in question.35 Erythrocyte production, along with thrombocytopoiesis, in adult urodelans is conducted intravascularly in the spleen. In anurans, the spleen is usually the major site of erythrocyte production, although the liver also serves as a secondary locus for this activity. Bone marrow hematopoiesis makes its phylogenetic debut in amphibians and typically occurs after metamorphosis or hibernation. The bone marrow is hematopoietically active in some salamanders and Rana species, but not in Xenopus.36,37 Seasonal changes can cause a shift in the sites (e.g., spleen and bone marrow) of hematopoiesis in some amphibians.38 Erythrocytes Amphibians have nucleated, oval, flattened, biconvex erythrocytes (Fig 3A, a and b). The amphibians have the largest erythrocytes of the animal kingdom, with Amphiuma tridactilum being recorded as having the largest erythrocytes (70 ⫻ 40 m). Anuran erythrocytes are generally much smaller (22 ⫻ 15 m) than those reported for the urodelans. Conversely, the erythrocyte count and hemoglobin concentration of these species are low.38 Although amphibians have nucleated erythrocytes, a percentage of anucleated erythrocytes (erythroplastids) may be observed in salamanders (family Plethodontidae), with Batrachoseps attenuatus possessing the record of nearly 95%.39 Maturation of the erythrocytes in the circulation is common (e.g., Urodeles).38,40 As in reptiles, the amphibian erythrocyte lifespan is longer than that recorded for mammals and birds (e.g., about 700-1400 days in Bufo marinus).27 During brumation, there is an increase in the lifespan of the peripheral Claver and Quaglia blood cells as a consequence of a reduced metabolic activity and a slowing of hematopoiesis.41 Leukocytes, Eosinophils, Basophils, Lymphocytes, Monocytes, Thrombocytes Amphibian leukocytes are larger (30-32 m) than those described for other NMV. Although the primary acute inflammatory cell of the amphibian is the heterophil, these animals also have a small number of cells that have the staining characteristics of neutrophils (Fig 3A, d and e).42 Little is known about the characteristics of these “neutrophils.” Heterophils have acidophilic rod-shaped granulations and a segmented nucleus (Fig 3B). During the hibernation phase of frogs, an increase in the nuclear segmentation of neutrophils has been observed, probably due to cell aging. Amphibian heterophils do phagocytize bacteria.42 Amphibian eosinophils are similar to their heterophils, although the cytoplasmic granules are larger, round to oval, and the nucleus is less segmented (Fig 3A, f).42 Histochemically, eosinophils are peroxidase negative and heterophils are peroxidase positive.37 Little is known of eosinophil function in amphibians, although some reaction to trematode infestations has been documented.43 Basophils are morphologically similar to those of other NMV and may be in low or high numbers depending on the species (Fig 3A, g). Heparin-like substances have been described in the basophils of some amphibian species, and they appear to have a similar function to basophils found in other NMV.43 Azurophils have been described in some amphibian species and are believed to be a form of monocyte or neutrophil (Fig 3A, j).42,43 Lymphocytes are generally the smallest leukocyte in amphibians, being half the size of granulocytes. These cells are round to ovoid in shape and contain a small amount of cytoplasm (Fig 3A, h).42 Amphibian lymphocytes seem to act in specific immunity, although urodelans do appear to lack some of the more evolved aspects of lymphocyte-mediated specific immunity present in anurans, birds, and mammals.44 Amphibian monocytes can vary in size, with some being smaller and others being larger than the granulocytes (Fig 3A, i). Monocytes generally have a higher cytoplasmic to nuclear ratio than lymphocytes and a rounded, kidney-shaped or horseshoeshaped nucleus. It can be difficult to differentiate monocytes from large lymphocytes in some cases.42 As with other vertebrates, monocytes serve as phagocytic and antigen-presenting cells.37 Thrombocytes of amphibians are similar in structure and function to those of birds and reptiles (Fig 3A, c).42 93 Morphology of Blood Cells in Nonmammalian Vertebrates Figure 3. A, Blood cells of amphibians. Erythrocyte (a), polychromatic erythrocyte (b), thrombocytes (c), neutrophil (d), heterophil (e), eosinophil (f), basophil (g), small lymphocyte (h), monocyte (i), and azurophil (j). B, Heterophils. Buffo arenarum (Anura). Courtesy of Mariana Cabagna. Fishes Hematopoiesis Fishes are the most numerous (27,000 species) and diverse of all vertebrate groups, making generalizations regarding hematology difficult, if not impossible. The lampreys and the hagfish include about 100 species of jawless fishes. There are also 600 species of chondrichthyes and more than 26,000 bony fishes (osteichthyes). All fish lack bone marrow and lymph nodes.45 Elasmobranchs possess a thymus and spleen, but in the absence of bone marrow and lymph nodes, these fish have evolved unique lymphomyeloid tissues, namely epigonal and Leydig’s organs, associated with the esophagus and gonads, respectively. The epigonal and Leydig’s organs are thought to be vital in generating the immune response in chondrichthyan fishes.45 94 Bony fishes (teleosts) form embryonic erythroid cells in a distinct dorsal-lateral compartment of the embryo known as the intermediate cell mass of Oellacher and are, therefore, an exception to the general vertebrate rule of embryonic hematopoiesis on the yolk sac. Both primitive and definitive cells arise from these cellular precursors.4 In many of the bony fishes, the renal interstitium is the primary site of definitive hematopoiesis, with a significant number of species possessing a discrete anterior (pronephros) kidney that is entirely devoted to hematopoiesis. In the kidney interstitium, all lines of hematopoietic differentiation are observed, including erythropoiesis, granulopoiesis, and lymphopoiesis.46 In some species of bony fishes, other organs (e.g., spleen, heart, meninges, spiral valve) may have lymphomyeloid activity.47 Erythrocytes Almost all fish have nucleated erythrocytes (Fig 4, a). Remarkable exceptions are Maurolicus müelleri, a teleost with small anucleated erythrocytes, and the Antarctic ice fishes (family Channichthyidae), which do not have erythrocytes or hemoglobin in Claver and Quaglia the blood. Erythrocytes in fishes are generally oval in shape with an oval to rounded nucleus.48 The elongated fish erythrocytes appear to represent more mature cells.49 Erythrocytes are rounded in some Agnathae (family Petromyzontidae)50 and tend to be more rounded in elasmobranchs than in teleosts. Fish blood has characteristically low concentrations of erythrocytes (1 to 5 ⫻ 106/mm3) and hemoglobin. A continued increase in size and hemoglobin content occurs in circulating erythrocytes during their lifespan.51 Sedentary fishes have lower erythrocyte levels than those in more active fishes. Recently, it has been observed that, as in birds, fish erythrocytes can act as immune cells, binding and engulfing Candida albicans.52 Leukocytes In fishes, the heterophil has been variably called heterophil or neutrophil depending on the size of cytoplasmic granules. In some instances, fish heterophils are referred to as acidophils.45 To avoid confusion, certain authors propose to use the term “neutrophil” only for mammals.26 Unlike avian and reptilian heterophils, fish heterophils contain large Figure 4. Blood cells of fishes. Erythrocyte (a), normal (b) and activated (c) thrombocytes, neutrophil of trout (d), heterophil of shark (e), eosinophil (f), monocyte (g), basophil (h), and lymphocyte (i). 95 Morphology of Blood Cells in Nonmammalian Vertebrates amounts of myeloperoxidase and their macrophages produce nitric oxide and reactive oxygen.45,53,54 Their morphology is variable, and the nucleus may range in shape between kidney-like to having two or three lobes. The cytoplasm contains very thin, pale eosinophilic granules. Because of the diverse shapes of fish heterophils, a classification scheme has been developed that is based on the size and number of nuclear lobes and shape and staining characteristics of the granules (Fig 4, d and e).46 The presence of peroxidase in fish leukocytes has been associated with bactericidal activity and functions as a defensive immune mechanism.55 Eosinophils (Fig 4, f) are described for some fish but are poorly understood. Granules in fish eosinophils may have bar-shaped crystalloids similar to those described in human eosinophils. There is a lack of knowledge regarding the full function of fish eosinophils, but they seem to function in a similar manner to mammalian mucosal mast cells.56 The eosinophils are found frequently in the digestive tract and gills and have been associated with antigenic stimulation and parasitic infestations.57,58 Basophils are the most variable cell type in fish (Fig 4, h). They are apparently absent in certain species, such as zebra fish (Danio rerio)59 and sea bass (Dicentrarchus labrax),60 but present in others, like the sea bream (Sparus aurata)61 and carp (Cyprinus carpio).62 When present, basophils occur in very low numbers. Fish that do not have basophils also appear to lack mast cells, immunoglobulin E, and serotonin.63 Lymphocytes are the most common circulating leukocyte found in fish. They are generally classified as small (e.g., the predominant form; Fig 4, i) or large.45 Plasma cells are occasionally found in the blood of fishes. T-, B- and NK-lymphocytes are present in bony and cartilaginous fishes, but not in jawless fishes or invertebrates.64 It is generally believed that jawless fishes only have innate immunity.65 However, lymphocytes of lampreys have been shown to transcribe novel variable lymphocyte receptors, consisting of leucine-rich repeats, that can generate diversity through a somatic rearrangement process.66 In contrast to higher vertebrates, most fish hatch at the embryonic stage of life, with the innate immune system serving as their primary defense mechanism. The acquired immune system (B- and T-cells) arises later in life, generally after food consumption.67 Monocytes are large leukocytes with an abundant blue-gray cytoplasm that lacks granules and are occasionally vacuolated (Fig 4, g).45 As in mammals, monocytes migrate to the tissues and became mac- rophages.68 Aggregates of melanin containing macrophages normally occur within the liver, spleen, and kidney of fish and are also found in the liver of reptiles and amphibians.69 In fish, melanomacrophage centers have been shown to trap and retain antigens and immune complexes, thus functioning as a primitive analog to the lymphoid germinal center.70 Thrombocytes Fish thrombocytes are variable in shape and may be rounded to oval or spindle shaped. Activation is frequent in blood smears obtained from the caudal vein, and some of the changes noted in the cells on a smear may be caused by the activation of the clotting cascade (Fig 4, b and c). The nucleus is usually oval, but may be segmented.28 Ultrastructurally, the fish thrombocyte is similar to that found in birds and reptiles. As with other vertebrates, fish thrombocytes are involved in blood clotting, phagocytosis, and other possible immunologic functions.71,72 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Rowley AF, Ratclifffe NA: Vertebrate Blood Cells. Cambridge University Press, 1988 Dieterlen-Lievre F: Birds, in Rowley AF, Ratcliffe NA (eds): Vertebrate Blood Cells. Cambridge, Cambridge University Press, pp 257-336, 1988 Baumann R, Dragon S: Erythropoiesis and red cell function in vertebrate embryos. Eur J Clin Invest 35(Suppl 3):2-12, 2005 Zon, LI: Developmental biology of hematopoiesis. Blood 86:2876-2891, 1995 Campbell TW: Avian Hematology and Cytology. Ames, IA, Iowa State University Press, 1988 Campbell TW: Hematology of common non-domestic animals, in Thrall MA (ed): Veterinary Hematology and Clinical Biochemistry. Philadelphia, Lippincott Williams & Wilkins, pp 225-276, 2004 Campbell TW, Dein FJ: Avian hematology: The basics. Vet Clin North Am Small Anim Pract 14:223-248, 1984 Passantino L, Massaro MA, Jirillo F, et al: Antigenically activated avian erythrocytes release cytokine-like factors: a conserved phylogenetic function discovered in fish. Immunopharmacol Immunotoxicol 29: 141-152, 2007 Vegad JL, Katyar AK: The acute inflammatory response in the chicken. Vet Bull 65:309-409, 1995 Harmon BG: Avian heterophils in inflammation and disease resistance. Poult Sci 77:972-977, 1998 Montali RJ: Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). J Comp Pathol 99:1-20, 1988 Maxwell MH, Robertson GW: The avian heterophil leukocyte: a review. World Poult Sci J 54:155-178, 1998 96 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Claver and Quaglia Andreasen CB, Latimer KS: Cytochemical characteristics of chicken heterophils and eosinophils. Vet Clin Pathol 19:51-54, 1990 Campbell TW: Normal hematology of psittacine birds, in Feldman BF, Zinkl JG, Jain NC (eds): Schalm’s Veterinary Hematology. Philadelphia, Lippincott Williams & Wilkins, pp 1155-1160, 2000 Maxwell MH, Siller WG: The ultrastructural characteristics of the eosinophil granules in six species of domestic birds. J Anat 112:289-303, 1972 Latimer KS, Bienzle D: Determination and interpretation of the avian leukogram, in Feldman BF, Zinkl JG, Jain NC (eds): Schalm’s Veterinary Hematology. Philadelphia, Lippincott Williams & Wilkins, pp 417432, 2000 Maxwell MH, Robertson GW: The avian basophilic leukocyte: a review. World Poult Sci J 51:307-325, 1995 Maxwell MH: Avian blood leukocyte response to stress. World Poult Sci J 49:34-43, 1993 Edmonds RH: Electron microscope studies on the hemostatic process in bird embryos I: the initial plug. J Ultrastruct Res 24:295-310, 1968 Bounous DI, Stedman NL: Normal avian hematology. Chicken and turkey, in Feldman BF, Zinkl JG, Jain NC (eds): Schalm’s Veterinary Hematology. Philadelphia, Lippincott Williams & Wilkins, pp 1145-1154, 2000 Martínez-Silvestre I, Marco M, Rodriguez-Dominguez S, et al: Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro. Res Vet Sci 78:127-134, 2005 Vasse J, Beaupain D: Erythropoiesis and haemoglobin ontogeny in the turtle Emys orbicularis L. J Embryol Exp Morphol 62:129-138, 1981 Sypeck J, Borysenko M: Reptiles, in Rowley AF, Ratcliffe NA (eds): Vertebrate Blood Cells. Cambridge, Cambridge University Press, pp 211-256, 1988 Pienaar UDV: Hematology of some south African reptiles. Johannesburg, Witwatersrand University Press, 1962 Hartman FA, Lessler MA: Erythrocyte measurements in fishes, amphibians and reptiles. Biol Bull 126:8388, 1964 Canfield PJ: Comparative cell morphology in the peripheral blood films from exotic and native animals. Aust Vet J 76:793-800, 1998 Altland PD, Brace KC: Red cell life span in the turtle and toad. Am J Physiol 203:1188-1190, 1962 Frye FL: Biomedical and surgical aspects of captive reptilian husbandry (ed 2). Miami, Krieger Publishing, 1991 Dotson TK, Ramsay EC, Bounous DI: A color atlas of blood cells of the Yellow Rat Snake. Compend Contin Educ Pract Vet 17:1013-1016, 1995 Rosskopf WR, Jr: Disorders of reptilian leukocytes and erythrocytes, in Fudge AM (ed): Laboratory Medicine Avian and Exotic Pets. Philadelphia, W.B. Saunders, pp 19-27, 2000 Alleman AR, Jacopson ER, Raskins ER: Morphologic, cytochemical staining and ultrastructural characteristics of blood cells from eastern diamondback rattlesnake (Crotalus adamanteus). Am J Vet Res 60: 507-514, 1992 Troiano JC, Vidal JC, Gould J, et al: Haematologi- 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. cal reference intervals of the South American rattlesnake (Crotalus durissus terrificus, Laurenti, 1768) in captivity. Comp Haematol Int 1:109-112, 1997 Duguy R: Numbers of blood cells and their variation, in Gans C, Pough FH (eds): Biology of Reptilia (vol 3). New York, Academic Press, pp 93-109, 1970 Huber TL, Zon LI: Transcriptional regulation of blood formation during Xenopus development. Semin Immunol 10:103-109, 1998 Hansen JD, Zapata AG: Lymphocyte development in fish and amphibians. Immunol Rev 166:199-220, 1998 Curtis SK, Cowden RR, Nagel JW: Ultrastructure of the bone marrow of the salamander Plethodon glutinosus (Caudata: Plethodontidae). J Morphol 159:151184, 1979 Turner RJ: Amphibians, in Rowley AF, Ratcliffe NA (eds): Vertebrate Blood Cells. Cambridge, Cambridge University, pp 129-209, 1988 Glomski CA, Tamburlin J, Hard R, et al: The phylogenetic odyssey of the erythrocyte. IV. The amphibians. Histol Histopathol 12:147-70, 1997 Villolobos MP, Leon SK, Kezer J: Enucleated erythrocytes in plethodontid salamanders. Herpetologica 44:243-250, 1988 Duellman WE, Trueb L: Biology of Amphibians. McGraw-Hill Book Company, San Francisco, 1986 Nano R, Griffini P, Barni S: Morphohistochemical changes of the blood cells in the hibernating frog (Rana esculenta L.). Comp Haematol Int 1:220-223, 1991 Wright KM: Amphibian hematology, in Wright KM, Whitaker BR (eds): Amphibian Medicine and Captive Husbandry. Malabar, FL, Krieger, pp 129-146, 2001 Mitchell JB: The effect of host age on Rana temporaria and Gorgoderina vitelliloba interactions. J Parasitol 12: 601-604, 1982 Terio KA: Comparative Inflammatory Responses of Non-Mammalian Vertebrates, in 55th Annual Meeting of the American College of Veterinary Pathologists (ACVP) & 39th Annual Meeting of the American Society of Clinical Pathology (ASVCP), Orlando, FL, USA, 2004 Campbell TW, Murru BS: An introduction to fish hematology. Comp Cont Ed Vet Sci 12:525-533, 1990 Temmink JH, Bayne CJ: Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio, L.) Dev Comp Immunol 11:125-137, 1987 Peterman AE, Petrie-Hanson L: Ontogeny of American paddle fish lymphoid tissues. J Fish Biol 69(Suppl A):72-88, 2006 Stoskopf MK: Fish Medicine. Philadelphia, Saunders, 1993 Passantino L, Altamura M, Cianciotta A, et al: Maturation of fish erythrocytes coincides with changes in their morphology, enhanced ability to interact with Candida albicans and release of cytokine-like factors active upon autologous macrophages. Immunopharmacol Immunotoxicol 26:573-585, 2004 Potter IC, Percy LR, Barber DL, et al: The morphology, development and physiology of blood cells, in Hardisty MW, Potter IC (eds): The Biology of Lam- 97 Morphology of Blood Cells in Nonmammalian Vertebrates 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. preys (vol 4A). London, Academic Press, pp. 233292, 1982 Speckner W, Schindler JF, Albers C: Age dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio L.) J Exp Biol 141:133-149, 1989 Passantino L, Altamura M, Cianciotta A, et al: Fish immunology. I. Binding and engulfment of Candida albicans by erythrocytes of rainbow trout (Salmo gairdneri Richardson). Immunopharmacol Immunotoxicol 24:665-678, 2002 Lamas J, Ellis AE: Atlantic salmon (Salmo salar) neutrophilic responses to Aeromonas salmonicida. Fish Shellfish Immunol 4:210-294, 1994 Campos-Perez JJ, Allis AE, Secombes CJ: Toxicity of nitric oxide and peroxynitrite to bacterial pathogens of fish. Dis Aquat Organ 43:109-115, 2000 Tavares-Dias M: A morphological and cytochemical study of erythrocytes, thrombocytes and leukocytes in four freshwater teleosts. J Fish Biol 68:1822-1833, 2006 Ellis AE: The immunology of teleosts, in Roberts RJ (ed): Fish Pathology (ed 3). London, Saunders, 2001 Powell MD, Wright GM, Burka JF: Eosinophilic granule cells in the gills of rainbow trout Oncorhynchus mykiss: evidence of migration? J Fish Biol 37:495-497, 1990 Barnett RR, Akindele TC, Orte L, et al: Eosinophilic granulocytes in the epidermis of Oreochromis mossambicus gill filaments studied in situ. J Fish Biol 49:148156, 1996 Crowhurst MO, Layton JE, Lieschke GJ: Developmental biology of zebra fish myeloid cells. Int J Dev Biol 46:483-492, 2002 Esteban MA, Muñoz J, Meseguer J: Blood cells of sea bass (Dicentrarchus labrax L.). Flow cytometric and microscopic studies. Anat Rec 258:80-89, 2001 López-Ruiz A, Esteban MA, Meseguer J: Blood cells of the gilthead seabream (Sparus aurata L.): light and electron microscopic studies. Anat Rec 234:161-171, 1992 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. Tripathi NK, Latimer KS, Burnley V: Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure. Vet Clin Pathol 33:74-83, 2004 Ellis AE: The function of teleost fish lymphocytes in relation to inflammation. Int J Tissue React 8:263270, 1986 Anderson MK, Sun X, Miracle AL, et al: Evolution of hematopoiesis: three members of the PU. 1 transcription factor family in a cartilaginous fish, Raja eglanteria. Proc Natl Acad Sci U S A 98:553-558, 2001 Shintani S, Terzic J, Sato A, et al: Do lampreys have lymphocytes? The Spi evidence. Proc Natl Acad Sci U S A 97:7417-7422, 2000 Pancer Z, Amemiya CT, Ehrhardt GR, et al: Somatic diversification of variable lymphocyte receptors in the Agnathan sea lamprey. Nature 430:174-180, 2004 Rombout J, Huttenhuis HBT, Picchietti S, et al: Phylogeny and ontogeny of fish leucocytes. Fish Shellfish Immunol 19:441-455, 2005 Hrubec TC, Smith AS: Hematology of fish, in Feldman BF, Zinkl JG, Jain NC (eds): Schalm’s Veterinary Hematology. Philadelphia, Lippincott Williams & Wilkins, pp 1120-1125, 2000 Agius C, Roberts RJ: Melano-macrophage centres and their role in fish pathology. J Fish Dis 26:499-509, 2003 Press CMcL, Evensen Ø: The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 9:309-318, 1999 Köllner B, Fischer U, Rombout JHWM, et al: Potential involvement of rainbow trout thrombocytes in immune functions: a study using a panel of monoclonal antibodies and RT-PCR. Dev Comp Immunol 28:1049-1062, 2004 Passantino L, Cianciotta A, Patruno R, et al: Do fish thrombocytes play an immunological role? Their cytoenzymatic profiles and function during an accidental piscine candidiasis in aquarium. Immunopharmacol Immunotoxicol 27:345-356, 2005
