In this study, the differentiation and characteristics of circulating leukocytes of the studied species was clarified and described in details, each complemented with micrographs.
Most morphological and ultrastructural findings in this study agree with previous studies in similar species, while some discoveries were made. Specifically, heterophil granules were more rounded and less elongated in sea turtles, contrary to earlier descriptions. Heterophil granules in sea turtles were unevenly distributed, and distinct types of granules see on ultra-thin sections can lead to false identification of the granulocytes. Two types of eosinophils were only found in green sea turtles, presumably due to the rareness of small eosinophils. Whether the presence of one type of eosinophils represents immune activation or immaturity remains unknown. Crystalloid granulations were not seen in eosinophils of sea turtles in this study, and previous statement was questionable.
Basophils were rare in sea turtles, but was well documented in this study in the hope to clarify on its morphology for veterinarians. The granulocytes ultrastructure of yellow pond turtles and Chinese bow turtles were similar to those of yellow-bellied sliders and yellow-headed temple turtles.
However, the polygonal crystalloid structures seen in the basophil granules of Chinese box turtles have not been seen in other species of freshwater turtles.
Wright Giemsa stain, Diff-Quik stain and Liu’s stain had similar staining properties and were all suitable for leukocyte differentiation. However, Wright Giemsa satin and Liu’s stain were preferred over Diff-Quik stain for reliable interpretation of heterophil toxic change.
Toxic change morphology in sea turtles and toxic change ultrastructure in reptile species were described for the first time in this study. The characteristics of these cellular changes were demonstrated with both morphologic and ultrastructural descriptions. It is known among
veterinarians working with sea turtles that toxic change was rare in these species, but this study provided evidence on how the presence toxic change was not a sensitive or reliable indicator for a sea turtle’s clinical inflammatory state or prognosis/ treatment outcome.
This study provides fundamental hematologic information and subsequently more sophisticated medical diagnosis and treatment for these endangered species, as well as insights to further investigations on clinical pathology and immunity of these species.
References
Arizza, V., Russo, D., Marrone, F., Sacco, F., & Arculeo, M. (2014). Morphological characterization of the blood cells in the endangered Sicilian endemic pond turtle, Emys trinacris (Testudines: Emydidae). Italian Journal of Zoology, 81(3), 344-353.
Azevedo, A., & Lunardi, L. O. (2003). Cytochemical characterization of eosinophilic leukocytes circulating in the blood of the turtle (Chrysemys dorbignih). Acta histochemica, 105(1), 99.
Bradley, T. A., Norton, T. M., & Latimer, K. S. (1998). Hemogram values, morphological characteristics of blood cells and morphometric study of loggerhead sea turtles, Caretta caretta, in the first year of life. Bulletin of the Association of Reptilian and Amphibian Veterinarians, 8(3), 8-16.
Cambell, T. (2006). Clinical pathology of reptiles. Reptile medicine and surgery, 2nd ed. St. Louis (MO): Saunders, 453-470.
Campbell, T. (2015). Peripheral blood of birds. Exotic Animal Hematology and Cytology. 4th ed.
Ames, Iowa, USA: Wiley-Blackwell, 37-66.
Campbell, T. W. (2012). Hematology of reptiles. Veterinary hematology and clinical chemistry.
Philadelphia: Lippincott Williams & Wilkins, 277-297.
Campbell, T. W. (2015). Peripheral Blood of Reptiles Exotic Animal Hematology and Cytology. 4th ed. Ames, Iowa, USA: Wiley-Blackwell (pp. 67-88).
Cannon, M. S. (1992). The morphology and cytochemistry of the blood leukocytes of Kemp's ridley sea turtle (Lepidochelys kempi). Canadian Journal of Zoology, 70(7), 1336-1340.
Casal, A., Freire, F., Bautista‐Harris, G., Arencibia, A., & Orós, J. (2007). Ultrastructural characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta). Anatomia,
histologia, embryologia, 36(5), 332-335.
Casal, A., & Orós, J. (2007). Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta). Research in veterinary science, 82(2), 158-165.
Chansue, N., Sailasuta, A., Tangtrongpiros, J., Wangnaitham, S., & Assawawongkasem, N. (2011).
Hematology and clinical chemistry of adult yellow‐headed temple turtles (Hieremys annandalii) in Thailand. Veterinary clinical pathology, 40(2), 174-184.
Chung, C.-s., Cheng, C.-h., Chin, S.-c., Lee, A.-h., & Chi, C.-h. (2009). Morphologic and cytochemical characteristics of Asian yellow pond turtle (Ocadia sinensis) blood cells and their hematologic and plasma biochemical reference values. Journal of Zoo and Wildlife Medicine, 40(1), 76-85.
Claver, J. A., & Quaglia, A. I. (2009). Comparative morphology, development, and function of blood cells in nonmammalian vertebrates. Journal of exotic pet medicine, 18(2), 87-97.
Di Santi, A., Basile, F., Ferretti, L., Bentivegna, F., Glomski, C., & Pica, A. (2013). Morphology, cytochemistry and immunocytochemistry of the circulating granulocytes of the loggerhead sea turtle Caretta caretta. Comparative Clinical Pathology, 22(3), 481-490.
Fontes Pinto, F., Lopes, C., Malhão, F., & Marcos, R. (2018). Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat. Veterinary clinical pathology.
Hernández, J., Castro, P., Saavedra, P., Ramírez, P., & Orós, J. (2017). Morphologic and Cytochemical Characteristics of the Blood Cells of the Yellow‐bellied Slider (trachemys scripta scripta). Anatomia, histologia, embryologia, 46(5), 446-455.
Hernández, J. D., Orós, J., Artiles, M., Castro, P., & Blanco, A. (2016). Ultrastructural characteristics of blood cells in the Yellow‐Bellied Slider Turtle (Trachemys scripta scripta).
Veterinary clinical pathology, 45(1), 106-109.
Innis, C. J., Tlusty, M., & Wunn, D. (2007). Hematologic and plasma biochemical analysis of juvenile head-started northern red-bellied cooters (Pseudemys rubriventris). Journal of Zoo and Wildlife Medicine, 425-432.
IUCN. (2018). The IUCN Red List of Threatened Species. Version 2018-2.
. Retrieved 13 November 2018 http://www.iucnredlist.org
Javanbakht, H., Vaissi, S., & Parto, P. (2013). The morphological characterization of the blood cells in the three species of turtle and tortoise in Iran. Research in Zoology, 3(1), 38-44.
Latimer, K. S., Tang, K.-N., Goodwin, M. A., Steffens, W., & Brown, J. (1988). Leukocyte changes associated with acute inflammation in chickens. Avian Diseases, 760-772.
Maxwell, M., & Robertson, G. (1998). The avian heterophil leucocyte: a review. World's Poultry Science Journal, 54(2), 155-178.
McCall, C. E., Katayama, I., Cotran, R. S., & Finland, M. (1969). Lysosomal and ultrastructural changes in human" toxic" neutrophils during bacterial infection. Journal of Experimental Medicine, 129(2), 267-293.
Mead, K. F., & Borysenko, M. (1984). Surface immunoglobulin on granular and agranular leukocytes in the thymus and spleen of the snapping turtle, Chelydraserpentina.
Developmental & Comparative Immunology, 8(1), 109-120.
Metin, K., Türkozan, O., Kargin, F., Basumoglu, Y., Taskavak, E., & Koca, S. (2006). Blood cell morphology and plasma biochemistry of the captive European pond turtle Emys orbicularis.
Acta Veterinaria Brno, 75(1), 49-55.
Montali, R. (1988). Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). Journal of comparative pathology, 99(1), 1-26.
Nardini, G., Leopardi, S., & Bielli, M. (2013). Clinical hematology in reptilian species. Veterinary
Clinics: Exotic Animal Practice, 16(1), 1-30.
Oliveira-Júnior, A., Tavares-Dias, M., & Marcon, J. (2009). Biochemical and hematological reference ranges for Amazon freshwater turtle, Podocnemis expansa (Reptilia:
Pelomedusidae), with morphologic assessment of blood cells. Research in veterinary science, 86(1), 146-151.
Oliveira, A. T., Cruz, W. R., Pantoja-Lima, J., Araújo, S. B., Araújo, M. L., Marcon, J. L., &
Tavares-Dias, M. (2011). Morphological and cytochemical characterization of thrombocytes and leukocytes in hatchlings of three species of Amazonian freshwater turtles. Veterinarski arhiv, 81(5), 657-670.
Orós, J., Casal, A., & Arencibia, A. (2010). Microscopic studies on characterization of blood cells of endangered sea turtles. Microscopy: Science, technology, applications and education, 1(4), 75-84.
Pitol, D. L., Issa, J. P. M., Caetano, F. H., & Lunardi, L. O. (2007). Morphological characterization of the leukocytes in circulating blood of the turtle (Phrynops hilarii). International Journal of Morphology, 677-682.
Salakij, C., Salakij, J., Prihirunkit, K., Narkkong, N. A., Sanyathitiseree, P., & Kranjanapitukkul, K.
(2014). Quantitative and qualitative morphologic, cytochemical, and ultrastructural characteristics of blood cells in captive Asian water monitors. Veterinary clinical pathology, 43(4), 538-546.
Schofield, K., Stone, P., Beddall, A., & Stuart, J. (1983). Quantitative cytochemistry of the toxic granulation blood neutrophil. British journal of haematology, 53(1), 15-22.
Shini, S., Kaiser, P., Shini, A., & Bryden, W. L. (2008). Differential alterations in ultrastructural morphology of chicken heterophils and lymphocytes induced by corticosterone and lipopolysaccharide. Veterinary immunology and immunopathology, 122(1-2), 83-93.
Stacy, B., & Pessier, A. (2007). Host response to infectious agents and identification of pathogens in tissue section. Infectious diseases and pathology of reptiles: color atlas and text. Boca Raton: CRC Press, Taylor & Francis Group, 200, 257-298.
Stacy, N., Fredholm, D., Rodriguez, C., Castro, L., & Harvey, J. (2017). Whip-like heterophil projections in consecutive blood films from an injured gopher tortoise (Gopherus polyphemus) with systemic inflammation. Veterinary Quarterly, 37(1), 162-165.
Stacy, N. I., Alleman, A. R., & Sayler, K. A. (2011). Diagnostic hematology of reptiles. Clinics in laboratory medicine, 31(1), 87-108.
Stacy, N. I., & Raskin, R. E. (2015). Reptilian eosinophils: beauty and diversity by light microscopy.
Veterinary clinical pathology, 44(2), 177-178.
Stacy Nicole I., B. S. (2014). Clinical Pathology of Sea Turtles. Retrieved 2018/7/3, from WIDECAST Technical Report No. 16.
Strik, N., Alleman, A., & Harr, K. (2007). Circulating inflammatory cells. Infectious diseases and pathology of reptiles: color atlas and text, 167-218.
Sykes, J. M., & Klaphake, E. (2015). Reptile hematology. Clinics in laboratory medicine, 35(3), 661-680.
Taylor, K., & Kaplan, H. M. (1961). Light microscopy of the blood cells of pseudemyd turtles.
Herpetologica, 17(3), 186-192.
Tristan, T. (2008). Introduction to Hematology and Biochemistry in Sea Turtles. Paper presented at the ACVP/ASVCP Concurrent Annual Meetings, San Antonio, Texas, USA.
Velásquez, J. C., Cartagena, H. N., Bolaño, C. R., Otero, G. Á ., Pacheco, J. C., & Arias, J. L. (2014).
Caracterización hematológica de hicoteas (Trachemys callirostris Gray, 1856) en Córdoba, Colombia. Revista de Medicina Veterinaria(28), 43-55.
Wilkinson, R., McArthur, S., & Meyer, J. (2004). Clinical pathology. Medicine and surgery of
tortoises and turtles, 141-186.
Wood, F. E., & Ebanks, G. K. (1984). Blood cytology and hematology of the green sea turtle, Chelonia mydas. Herpetologica, 331-336.
Work, T. M., Raskin, R. E., Balazs, G. H., & Whittaker, S. (1998). Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles. American journal of veterinary research, 59, 1252-1257.
Zago, C. E. S., Da Silva, T. L., Da Silva, M. I. A., Venancio, L. P. R., Mendonça, P. P., Junior, L. R.
F., . . . de Azeredo-Oliveira, M. T. V. (2010). Morphological, morphometrical and ultrastructural characterization of Phrynops geoffroanus’(Testudines: Chelidae) blood cells, in different environments. Micron, 41(8), 1005-1010.
Zhang, F.-Y., Li, P.-P., Gu, H.-X., & Ye, M.-B. (2011). Hematology, morphology, and ultrastructure of blood cells of juvenile olive ridley sea turtles (Lepidochelys olivacea).
Chelonian Conservation and Biology, 10(2), 250-256.
Appendix
Appendix 1: Heterophils of loggerhead sea turtles
A: Wright-Giemsa stain (Stacy Nicole I., 2014); B: Diff-Quik, scale bar= 3.6μm (Casal & Orós, 2007); C: May–Grünwald–Giemsa stain, scale bar=4μm (Di Santi et al., 2013)
Appendix 2: Eosinophils of loggerhead sea turtles
A: Diff-Quik, scale bar= 3.3μm (Casal & Orós, 2007); B: Wright-Giemsa stain (N. I. Stacy &
Raskin, 2015); C: Wright-Giemsa stain (Stacy Nicole I., 2014); D: May–Grünwald–Giemsa stain, scale bar=4μm (Di Santi et al., 2013)
A B C
A B C D
Appendix 3. Lymphocytes of loggerhead sea turtles
A: Wright-Giemsa stain, comparing to a thrombocyte (lower left); B: Wright-Giemsa stain, a reactive lymphocyte (Stacy Nicole I., 2014)
Appendix 4. Monocytes of loggerhead sea turtles
A: Diff-Quik stain, scale bar= 3.2μm (Casal & Orós, 2007); B: Wright-Giensa stain, reactive monocytes (Stacy Nicole I., 2014)
Appendix 5. Heterophils of green sea turtles
A: Leokostat stain (Work et al., 1998); B: modified May-Grünwald-Giemsa stain (Wood & Ebanks, 1984); C: Wright-Giemsa stain (Stacy Nicole I., 2014)
A B
A B
A B C
Appendix 6. Eosinophils of green sea turtles
A: Leokostat stain (Work et al., 1998); B: modified May-Grünwald-Giemsa stain (Wood & Ebanks, 1984); C: Wright-Giemsa stain (Stacy Nicole I., 2014); D: Wright-Giemsa stain (N. I. Stacy &
Raskin, 2015); E: a small eosinophil, Leokostat stain (Work et al., 1998)
Appendix 7. Basophils of green sea turtles
A: Leokostat stain (Work et al., 1998); B: modified May-Grünwald-Giemsa stain (Wood & Ebanks, 1984)
A B C
D E
A B
Appendix 8. Lymphocytes of green sea turtles
A: Leokostat stain (Work et al., 1998); B: modified May-Grünwald-Giemsa stain (Wood & Ebanks, 1984); C: Wright-Giemsa stain (Stacy Nicole I., 2014)
Appendix 9. Monocytes of green sea turtles
A: Leokostat stain (Work et al., 1998); B: Wright-Giemsa stain, a monocyte exhibiting a phagocytized erythrocyte and hemosiderin (Stacy Nicole I., 2014)
Appendix 10. Heterophils of olive ridley sea turtles
A: Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011); B: Wright-Giemsa stain (Stacy Nicole I., 2014)
A B C
A B
A B
Appendix 11. Eosinophils of olive ridley sea turtles
A: a large eosinophil, Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011); B: a large eosinophil, Wright-Giemsa stain (Stacy Nicole I., 2014); C: a small eosinophil, Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011)
Appendix 12. A basophil of a juvenile olive ridley sea turtle
Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011)
Appendix 13. Lymphocytes of juvenile olive ridley sea turtles
A: a small lymphocyte, Wright-Giemsa stain, scale bar=4μm; B: a medium-sized lymphocyte, Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011)
A B C
A B
Appendix 14. Monocytes of juvenile olive ridley sea turtles
Wright-Giemsa stain, scale bar=4μm (Zhang et al., 2011)
Appendix 15. Leukocytes of Kemp's ridley sea turtles
A: a heterophil, Wright-Giemsa stain (Cannon, 1992); B: a heterophil, Wright-Giemsa stain; C: 2 eosinophils, Wright-Giemsa stain (Stacy Nicole I., 2014) D: a lymphocyte, Wright-Giemsa stain; E:
a basophil, Wright-Giemsa stain; F: a monocyte, Wright-Giemsa stain (Stacy Nicole I., 2014)
A B C
D E F
Appendix 16. Heterophil ultrastructure in sea turtles
A: a heterophil of a juvenile loggerhead sea turtle, stained with uranyl acetate (1% methanol) and Stato´s lead solution, scale bar=1.6μm (Orós et al., 2010); B: a heterophil of a green sea turtle, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, scale bar =2μm (Work et al., 1998); C: a heterophil of a juvenile olive ridley sea turtle, stained with uranyl acetate and lead citrate, scale bar =2μm (Zhang et al., 2011); D: a heterophil of a loggerhead sea turtle, stained with uranyl acetate and lead citrate, scale bar =2μm (Di Santi et al., 2013)
A
B C D
Appendix 17. Eosinophil ultrastructure in sea turtles
A: an eosinophil of a juvenile loggerhead sea turtle, stained with uranyl acetate (1% methanol) and Stato´s lead solution, scale bar=1.3μm (Orós et al., 2010); B: a large eosinophil of a green sea turtle, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, scale bar =2μm (Work et al., 1998); C: a large eosinophil of a juvenile olive ridley sea turtle, stained with uranyl acetate and lead citrate, scale bar =2μm (Zhang et al., 2011); D: a small eosinophil of a green sea turtle with
crystalline structures seen in granules, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate; E: a small eosinophil of a green sea turtle with partially vacuolated granules, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, scale bar =2μm (Work et al., 1998)
A B C
D E
Appendix 18. Lymphocyte ultrastructure in sea turtles
A: a lymphocyte of a juvenile loggerhead sea turtle, comparing to a thrombocyte on the right (arrowhead), stained with uranyl acetate (1% methanol) and Stato´s lead solution, scale bar=1.8μm (Orós et al., 2010); B: a lymphocyte of a green sea turtle, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate (Work et al., 1998); C: a lymphocyte of a juvenile olive ridley sea turtle, stained with uranyl acetate and lead citrate, scale bar =1μm (Zhang et al., 2011)
Appendix 19. Monocyte ultrastructure in sea turtles
A: a monocyte of a juvenile loggerhead sea turtle, stained with uranyl acetate (1% methanol) and Stato´s lead solution, scale bar=1.7μm (Orós et al., 2010); B: a monocyte of a green sea turtle, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, scale bar=2μm (Work et al., 1998); C: a monocyte of a juvenile olive ridley sea turtle, stained with uranyl acetate and lead citrate, scale bar =2μm (Zhang et al., 2011)
A
B
C
A
B
C
Appendix 20. Heterophils of freshwater turtles
A: the D'Orbigny's sliders, Giemsa stain, scale bar=10.0μm (Azevedo & Lunardi, 2003); B: the yellow-bellied sliders, Diff-Quick stain (J. Hernández et al., 2017); C: the common tortoises
(
Testudo graeca) or the Caspian turtles or the European pond turtles, Giemsa stain (Javanbakht et al., 2013); D: the Sicilian pond turtle, May-Grünwald Giemsa stain (Arizza et al., 2014); E: the Chinese stripe-necked turtle, Wright–Giemsa stain, scale bar=10μm (Chung et al., 2009); F: the Hilaire’s side-necked turtle, Leishmann stain (Pitol et al., 2007); G: the European pond turtles, Wright’s stain (Metin et al., 2006); H: the Colombian slider, Wright’s stain (Velásquez et al., 2014);I: the yellow-spotted river turtles, May-Grünwald Giemsa stain (Oliveira et al., 2011); J: the Arrau turtles, May-Grünwald Giemsa stain (Oliveira-Júnior et al., 2009); K: the yellow-headed temple turtles, Wright–Giemsa stain (Chansue et al., 2011); L: yellow-headed temple turtles, Wright’s stain, scale bar=10.0μm (Chansue et al., 2011); M: the Geoffroy’s side-necked turtle, Panoptic stain (Zago et al., 2010)
A B C D E
F G H I J
K L M
Appendix 21. Eosinophils of freshwater turtles
A: the D'Orbigny's sliders, Giemsa stain, scale bar=10.0μm (Azevedo & Lunardi, 2003); B: the yellow-bellied sliders, Diff-Quick stain (J. Hernández et al., 2017); C: the Sicilian pond turtle, May-Grünwald Giemsa stain (Arizza et al., 2014); D: the Chinese stripe-necked turtle,
Wright–Giemsa stain, scale bar=10μm (Chung et al., 2009); E: the Hilaire’s side-necked turtle, Leishmann stain (Pitol et al., 2007); F: the European pond turtles, Wright’s stain (Metin et al., 2006);
G: the Colombian slider, Wright’s stain (Velásquez et al., 2014); H: the hatchlings of the Arrau turtles, May-Grünwald Giemsa stain (Oliveira et al., 2011); I: the Arrau turtles, May-Grünwald Giemsa stain (Oliveira-Júnior et al., 2009); J: the yellow-headed temple turtles, Wright–Giemsa stain (Chansue et al., 2011); K: the Geoffroy’s side-necked turtle, Panoptic stain (Zago et al., 2010);
L: Northern red bellied turtle (Pseudemys rubriventris), Wright–Giemsa stain (N. I. Stacy & Raskin, 2015); M: the common tortoises or the Caspian turtles or the European pond turtles, Giemsa stain (Javanbakht et al., 2013)
A B C D E
F G H I J
K L M
Appendix 22. Basophils of freshwater turtles
A: the common tortoises or the Caspian turtles or the European pond turtles, Giemsa stain (Javanbakht et al., 2013); B: the Sicilian pond turtle, May-Grünwald Giemsa stain (Arizza et al., 2014); C: the Chinese stripe-necked turtle, Wright–Giemsa stain, scale bar=10μm (Chung et al., 2009); D: the Hilaire’s side-necked turtle, Leishmann stain (Pitol et al., 2007); E: the European pond turtles, Wright’s stain (Metin et al., 2006); F: the Colombian slider, Wright’s stain (Velásquez et al., 2014); G: the hatchlings of the Arrau turtles, May-Grünwald Giemsa stain, scale bar=10.0μm (Oliveira et al., 2011); H: the Arrau turtles, May-Grünwald Giemsa stain, scale bar=11μm
(Oliveira-Júnior et al., 2009); I: the yellow-headed temple turtles, Wright stain (Chansue et al., 2011); J: the yellow-headed temple turtles, Wright–Giemsa stain (Chansue et al., 2011); K: the Geoffroy’s side-necked turtle, Panoptic stain, scale bar=10μm (Zago et al., 2010)
A B C D
E
F G H I
J K
K
Appendix 23. Lymphocytes of freshwater turtles
A: the yellow-bellied sliders, Diff-Quick stain (J. Hernández et al., 2017); B: the common tortoises or the Caspian turtles or the European pond turtles, Giemsa stain (Javanbakht et al., 2013); C: the Sicilian pond turtle, May-Grünwald Giemsa stain (Arizza et al., 2014); D: the Chinese stripe-necked turtle, Wright–Giemsa stain, scale bar=10μm (Chung et al., 2009); E: the Hilaire’s side-necked turtle, Leishmann stain (Pitol et al., 2007); F: the European pond turtles, Wright’s stain (Metin et al., 2006); G: the Colombian slider, Wright’s stain (Velásquez et al., 2014); H: the hatchlings of the six-tubercled Amazon River turtles, May-Grünwald Giemsa stain (Oliveira et al., 2011); I: the Arrau turtles, May-Grünwald Giemsa stain (Oliveira-Júnior et al., 2009); J: the yellow-headed temple turtles, Wright–Giemsa stain (Chansue et al., 2011); K: the yellow-headed temple turtles, Wright–Giemsa stain (Chansue et al., 2011); L: the Geoffroy’s side-necked turtle, Panoptic stain (Zago et al., 2010)
A B C D E
F G H I J
K L
Appendix 24. Monocytes of freshwater turtles
A: the yellow-bellied sliders, Diff-Quick stain (J. Hernández et al., 2017); B: the common tortoises or the Caspian turtles or the European pond turtles, Giemsa stain (Javanbakht et al., 2013); C: the Sicilian pond turtle, May-Grünwald Giemsa stain (Arizza et al., 2014); D: the Chinese stripe-necked turtle, Wright–Giemsa stain, scale bar=10μm (Chung et al., 2009); E: the Hilaire’s side-necked turtle, Leishmann stain (Pitol et al., 2007); F: the European pond turtles, Wright’s stain (Metin et al., 2006); G: the Colombian slider, Wright’s stain (Velásquez et al., 2014); H: the Geoffroy’s
side-necked turtle, Panoptic stain (Zago et al., 2010)
A B C D
E F G H
Appendix 25. Heterophil ultrastructure in freshwater turtles
A: 2 heterophils of the yellow-bellied sliders, stained withuranyl acetate and lead citrate, scale bar=1.3μm (J. D. Hernández et al., 2016); B: a heterophil of a D'Orbigny's slider, stained with an alcoholic uranyl solution and lead citrate, scale bar=1.0μm (Azevedo & Lunardi, 2003); C: a heterophil of a Hilaire’s side-necked turtles (Pitol et al., 2007); D a heterophil of a yellow-headed temple turtle, stained with uranyl acetate, scale bar=2μm (Chansue et al., 2011); E: a heterophil of a Geoffroy’s side-necked turtle, stained with uranyl acetate and lead citrate (Zago et al., 2010)
A B
C D
F
E
Appendix 26. Eosinophil ultrastructure in freshwater turtles
A: an eosinophil of a yellow-bellied slider, stained withuranyl acetate and lead citrate, scale
bar=1.3μm (J. D. Hernández et al., 2016); B: cytoplasmic granules of an eosinophil of a D'Orbigny's slider, stained with an alcoholic uranyl solution and lead citrate, scale bar=1.0μm (Azevedo &
Lunardi, 2003); C: an eosinophil of a Hilaire’s side-necked turtles (Pitol et al., 2007); D: 2
eosinophils of a yellow-headed temple turtle, stained with uranyl acetate, scale bar=2μm (Chansue et al., 2011); E: cytoplasmic granules of an eosinophil from a yellow-headed temple turtle, stained with uranyl acetate, scale bar=0.5μm (Chansue et al., 2011); F: cytoplasmic granules of an
eosinophil of a Geoffroy’s side-necked turtle, stained with uranyl acetate and lead citrate (Zago et al., 2010)
A B C
D E F
Appendix 27. Basophil ultrastructure in freshwater turtles
A: a basophil of a yellow-bellied slider, stained withuranyl acetate and lead citrate, scale
bar=1.3μm (J. D. Hernández et al., 2016); B: a yellow-bellied slider’s basophil cytoplasmic granules that were rich in microtubules, stained with uranyl acetate and lead citrate, scale bar=0.5μm (J. D.
Hernández et al., 2016); C: a basophil of a Hilaire’s side-necked turtles (Pitol et al., 2007); D: a basophil of a yellow-headed temple turtle, cytoplasmic vacuoles were filled with electron-dense granules, stained with uranyl acetate, scale bar=2μm (Chansue et al., 2011); E: fine lamellar and electron-dense cytoplasmic granules of a basophil from a yellow-headed temple turtle, stained with uranyl acetate, scale bar=2μm (Chansue et al., 2011); F: a basophil of a Geoffroy’s side-necked turtle, stained with uranyl acetate and lead citrate (Zago et al., 2010)
A B C
D E F
Appendix 28. Lymphocyte ultrastructure in freshwater turtles
A: a lymphocyte of a yellow-bellied slider, stained with uranyl acetate and lead citrate, scale bar=1μm (J. D. Hernández et al., 2016); B: a lymphocyte of a Hilaire’s side-necked turtles (Pitol et al., 2007); C: a lymphocyte of a yellow-headed temple turtle, stained with uranyl acetate, scale bar=1μm (Chansue et al., 2011); D: a lymphocyte of a Geoffroy’s side-necked turtle, stained with uranyl acetate and lead citrate (Zago et al., 2010)
A B C
D
Appendix 29. Monocyte ultrastructure in freshwater turtles
A: a monocyte of a yellow-bellied slider, stained withuranyl acetate and lead citrate, scale
bar=1.6μm (J. D. Hernández et al., 2016); B: a monocyte of a Hilaire’s side-necked turtles (Pitol et al., 2007); C: a monocyte of a Geoffroy’s side-necked turtle, stained with uranyl acetate and lead citrate (Zago et al., 2010)
Appendix 30. Avian heterophil toxic change grading system (T. Campbell, 2015)
Toxicity Alternation in cellular morphology 1+ Increase in cytoplasmic basophilia
2+ Deeper cytoplasmic basophilia, partial degranulation
3+ Dark cytoplasmic basophilia, moderate degranulation, abnormal granules, cytoplasmic vacuolization
4+ Deep cytoplasmic basophilia, moderate to marked degranulation, presence of abnormal granules, cytoplasmic vacuolization, karyorrhexis or karyolysis
A B C
Appendix 31. Heterophil toxic change and/or left-shifting in sea turtles
A: a left-shifted heterophil (upper left) next to a mature heterophil (lower right) in a green sea turtle, Wright-Giemsa stain; B: a mildly toxic and left-shifted heterophil in a green sea turtle,
Wright-Giemsa stain; C: a moderately toxic and left-shifted heterophil in a green sea turtles, Wright-Giemsa stain; D: a moderately toxic and left-shifted heterophil in a green sea turtles,
Wright-Giemsa stain; E: a mildly toxic heterophil in a loggerhead sea turtle, Wright-Giemsa stain; F:
a left-shifted heterophil in a Kemp’s ridley sea turtle, Wright-Giemsa stain (Stacy Nicole I., 2014)
A B C
D
E F
E F
Appendix 32. Heterophil toxic change and/or left-shifting in various reptile species
1-9: Testudines; 10-15: Iguanidae & Agamidae; 16-17:Acrodonta; 18-21: Crocodilia; each from mild to severe on toxicity
1: a non-toxic, immature heterophil with band-shaped nucleus and a few primary, purple-staining
granules in a Gopher tortoise (Gopherus polyphemus). Wright-Giemsa stain. (Strik et al., 2007) 2: a 1 + toxic heterophil in a three-toed box turtle (Terrapene carolina), Wright–Giemsa stain (T. W.
Campbell, 2015)
3: a heterophil with degranulation in a Hermann's tortoise, Hemacolor® rapid stain (Nardini et al., 2013)
4: a 2+ toxic heterophil in a Chinese pond turtle (Chinemys reevesii), Wright–Giemsa stain (T. W.
Campbell, 2015)
5: a 3+ toxic heterophil with partial nuclear lobation in a three-toed box turtle (a species that does not lobe granulocyte nuclei), Wright–Giemsa stain (T. W. Campbell, 2015)
6: a heterophil with abnormal cytoplasmic granules in a Hermann's tortoise, Hemacolor® rapid stain (Nardini et al., 2013)
7: a severely toxic heterophil with cytoplasmic basophilia, degranulation, vacuolation, abnormal granulation, and pleomorphic nuclei in a common tortoise, Wright-Giemsa stain (Strik et al., 2007) 8: a severely toxic heterophil with bilobed nucleus in a common tortoise, Wright-Giemsa stain (Strik et al., 2007)
9: a severely toxic, immature heterophil in a common tortoise, Wright-Giemsa stain (Strik et al., 2007)
10: a heterophil with increased cytoplasmic basophilia in a green iguana, Diff-Quick stain (Nardini et al., 2013)
11: a moderately toxic heterophil with degranulation and cytoplasmic basophilia in a Chinese water dragon (Physignathus cocincinus), Wright-Giemsa stain (Strik et al., 2007)
12: a heterophil with vacuolation in a green iguana, Hemacolor® rapid stain (Nardini et al., 2013) 13: a 2+ toxic heterophil in a green iguana, Wright–Giemsa stain (T. W. Campbell, 2015)
14: a toxic heterophil with abnormal cytoplasmic granules in a green iguana, Diff-Quick stain
(Nardini et al., 2013)
15: a 3+ toxic heterophil in green iguana, Wright–Giemsa stain (T. W. Campbell, 2015) 16: a toxic heterophil with excessive nuclear lobation in a veiled chameleon (Chamaeleo calyptratus), Hemacolor® rapid stain (Nardini et al., 2013)
17: a severely toxic, left-shifted heterophil with cytoplasmic basophilia, degranulation, abnormal granulation, and excessive nuclear lobation in a Fischer’s chameleon (Chameleo fischeri), Wright-Giemsa stain (Strik et al., 2007)
18: a toxic heterophil in an American crocodile (Crocodylus acutus), Wright-Giemsa stain (Strik et al., 2007)
19: a severely toxic, immature heterophil with degranulation, abnormal granulation, cytoplasmic basophilia, and cytoplasmic vacuolation in a Spectacled caiman (Caiman crocodilus),
Wright-Giemsa stain (Strik et al., 2007)
20: a severely toxic heterophil with cytoplasmic basophilia, vacuolation, degranulation, and
20: a severely toxic heterophil with cytoplasmic basophilia, vacuolation, degranulation, and