- Email: [email protected]
Gross and microscopical characterization of the thymus and spleen of Piaractus mesopotamicus (Pacu)
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
305
Detection of antibodies against Ehrlichia canis and Babesia canis in Brazilian wild captive felids
7. Comparative immunology; immunoecology
Marcos R. André 1,∗ , Rosangela Z. Machado 1 , Silmara M. Allegretti 2 , Cristina H. Adania 3 , Paulo A.N. Felippe 2 , Ketty F. Silva 1 , Andréa C.H. Nakaghi 1
Similarities and differences of microscopic structure of spleen in beluga Huso huso with terrestrial animals
1
Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil 2 Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil 3 Centro Brasileiro de Conservac¸ão de Felídeos Neotropicais, Jundiaí, SP, Brazil Keywords: Wild felids; Ehrlichia canis; Babesia canis; Antibodies E-mail address: [email protected] (M.R. André).
Species: Feline Ehrlichiosis is an emergent zoonosis caused by an obligate intracellular bacteria belonging to Anaplasma, Ehrlichia and Neorickettsia genera, present in the environment through complex interactions among invertebrates and vertebrates hosts. Babesiosis is caused by infection with intraerythrocytic parasites of the genus Babesia. Both parasites are transmitted by ticks. Brazilian dogs are commonly infected by E. canis and B. canis, whose vector is the dog brown tick, the Rhipicephalus sanguineus. Little is known about the epidemiology of ehrlichiosis and babesiosis in wild felids, which can be seen as good sentinels for these diseases, since they are hosts for both hemoparasites and vectors. The present work aimed to detect antibodies against E. canis and B. canis vogeli in 72 Brazilian wild captive felids by IFA (indirect immunofluorescent assay), trying to verify if these animals are exposed to these parasites. Blood samples were collected from Puma concolor (cougar), Leopardus pardalis (ocelot), Puma yagouaroundi (aguarondi), Leopardus wiedii (margay), Leopardus tigrinus (little spotted cat), Oncifelis colocolo (pampas cat) and Panthera onca (jaguar) maintained in captivity in Brasilia, Ribeirão Preto, Pedreira, Campinas Zoos and in Associac¸ão Mata Ciliar de Jundiaí (Brazilian Center of Neotropic Felids Conservation). Forty-four (70.96%) of 62 animals were seroreagents for B. canis and 24 % for E. canis. Antibodies titers found ranged from 1:20 (cut-off) to 1:2560 for E. canis and from 1:40 (cut-off) to 1:1280 for B. canis. Fourteen felids were seropositives for both antigens, while seventeen animals were seronegatives for both agents. To authors’s knowledge, this is the first report of seroreactivity of Brazilian wild felids for both E. canis and B. canis vogeli, showing that these animals are exposed to vector and agents of ehrlichiosis and babesiosis. doi:10.1016/j.vetimm.2008.10.202
Mohammadt Sheibani Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Keywords: Spleen; Beluga; Terrestrial animals; Histology E-mail address: [email protected]. Species: Fish One of the largest and the most important sturgeons in the Caspian Sea from the aspect of caviar production is beluga. For this investigation a total number of three belugas fished from southern Caspian Sea, were subjected to study. By an incision on the abdominal wall the spleen was removed and after fixation in 10% buffered formalin, routine histological processes were done, paraffin blocks were sectioned at 6 m and stained with hematoxylin and eosin and studied under light microscope. The results showed that the spleen, unlike to those of mammals, was surrounded with a capsule composed of three layers including an epithelium of cuboidal to columnar cells with some small round secretory cells with eosinophilic granules. Under which a waving layer of elastic fibers and then a condensed connective tissue were located. Under the capsule a dense lymphatic tissue containing some melanomacrophages was observed. The parenchyma of the spleen was composed of white and red pulps as in other vertebrates. White pulps consisted of a lymphatic nodule containing a small branch of splenic artery, as the central artery, like that of mammals, but also a venous branch, unlike mammals that differ from this aspect. The lymphatic nodule is followed by a lymphatic cord consisting of an aggregation of lymphocytes around an arteriole, as periarterial lymphatic sheath. The red pulp contained too many sinusoids filled with red blood cells surrounded by some trabeculae and diffused lymphatic tissues throughout the red pulp of spleen. doi:10.1016/j.vetimm.2008.10.203 Gross and microscopical characterization of the thymus and spleen of Piaractus mesopotamicus (Pacu) G.M. Costa ∗ , C.E. Barroso, A.S. Oliveira, C. Biasi, R.S. Iunes, A.R. Lima, J.M. Monteiro, R.V. Bosch, P. Favaron, M.A. Ivo, J.R. Kfoury Jr. Medical Surgical Department of Veterinary Medicine and Zootenic College of the University of São Paulo, Brazil Keywords: Piaractus mesopotamicus; Anatomy; Histology; Spleen and thymus Species: FishPiaractus mesopotamicus (Pacu) is a fish from the Characidae family, intensively bred in Brazil because of its rusticity, easy raising and adaptation, besides its excellent flavor. In order to produce a healthy fish, information on its immunological system including the histology of the lymphoid organs is needed. Therefore, this
306
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
project performed a gross and histological analysis of the thymus and spleen of five young (about 25 cm in length) and five adult pacus (about 38 cm in length). The thymus appeared as a paired organ, placed on the caudal portion of the head, dorsally to the gills in the opercular cavity and involved by a membranous capsule. The thymus was more evident in fish older than 5 months; it presented a gelatinous consistency varying from vivid to dark red color. Histologically, it showed medullar and cortex regions consisted by conjunctive tissue and colagenous fibers and the presence of thymocytes and lymphocytes. The spleen was located caudally to the liver, ventrally to the swim bladder, dorsally to the medium caudal portion of the biliary vesicle. It was rounded shaped, dorsoventrally flattened and it presented an irregular surface in young and lobed in adult fish. It was delimited by a membrane and, histologically, it was composed by a white pulp, rich in melanomacrophages and extensive red pulp areas. doi:10.1016/j.vetimm.2008.10.204 Immunolocalization of heat shock protein (HSP) 70 in the liver of diploid and triploid rainbow trout R.S. Iunes ∗ , J.M. Monteiro, G.M. Costa, A.R. Lima, R.V. Bosch, J.R. Kfoury Jr. Department Medicine and Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil Keywords: Trout; HSP; Stress; Liver Species: Fish Heat shock proteins (HSPs) belong to a highly conserved family of cellular proteins present in all organisms. HSPs are expressed in different tissues under normal conditions; however it increases substantially when stress is present. Fish show two types of stress response: one is physiological and the other cellular. HSPs play a role at the latter, where a fish may present physiological response to a stressor agent without showing any change of the cellular HSPs profile. Among the HSPs, HSP 70 is the most studied in fish, and we attempted to compare its expression in the liver of diploid and triploid rainbow trout (Oncorhynchus mykiss). Liver from eight diploid and 10 triploid trouts were fixed in 10% formalin and processed for immunohistochemistry, using a monoclonal antibody specific to HSP 70 (Stressgen® mouse anti-HSP 70 monoclonal SPA-810). The immunohistochemistry results showed a cytoplasmatic expression of HSP 70 in hepatocytes and in the epithelial cells of the biliary duct. Differences on the intensity of staining were observed in both ploidy, where triploid fish presented a more evident variation, from intense to negative staining. Negative controls showed no staining in both diploid and triploid fish. Our results suggested triploid rainbow trout show differences on HSP 70 expression characteristics from its diploid counterpart. doi:10.1016/j.vetimm.2008.10.205
Cellular function evaluation of sanguineous leukocytes in Phrynops geoffroanus using flow cytometric methodology A. Genoy-Puerto 1,∗ , S. Rossi 1 , B.O. Ferronato 2 , V.M. SáRocha 3 , C.S. Lisboa 4 , L.C. Sá-Rocha 3 , E.R. Matushima 3 1
Academic Program of Experimental and Compared Pathology, FMVZ/USP, Brazil 2 Academic Program in Applied Ecology, ESALQ/USP, Brazil 3 Departament of Pathology, FMVZ, USP, Brazil 4 Sector of Reptiles of the Foundation Zoological Park of São Paulo, Brazil Keywords: Cellular function; Evaluation; Leukocytes; Flow cytometric; Phrynops geoffroanus E-mail address: [email protected] (A. Genoy-Puerto). Specie: Reptile (turtle) Although Phrynops geoffroanus displays ample geographical distribution in South America, few studies had been developed in relation to the natural history of this freshwater turtle. This species frequently appears in contaminated water sources, but the effect of adverse situations (anthrophic effects) on the animals ‘cellular immune activity is unknown. In this work a method employing flow cytometry was developed in order to evaluate the cellular functions of phagocytosis and spontaneous and induced oxidative burst in leukocytes from blood samples of Phrynops geoffroanus found in contaminated environments of the Piracicaba river and Piracicamirim brook, in São Paulo State. Blood samples were obtained from 8 animals from Piracicaba river, 9 from Piracicamirim brook, and 7 from Zoological Park Foundation of São Paulo; the samples from zoological park were used as control group. The samples had been kept in culture in RPMI medium under refrigeration and directed to the Neuro-immunology Laboratory of Department of Pathology in FMVZ/USP to be processed. The fact that the red blood cells are nucleated in these animals, made necessary the use of Ficoll-PaqueTM PLUS at different densities (1085, 1070 and 1055 g/mL), in order to pre-separate these cells. Then the sample was centrifuged to obtain cells of interest, at 18 ◦ C, 450 × g, acceleration 9 and brake 0, resulting in a white ring of leukocytes located between the superior layers of the Ficoll. The samples were submitted to two washes with RPMI to remove the excesses of Ficoll. After the washes, the samples were suspended again in 500 L RPMI for making a viability counting with Trypan Blue; samples with inferior percentages to 94 % were not used in the stimulates: Zymosan A (Sacaromyces cerevisiae) the Bio Particles® , Alexa Fluor® 594 conjugate to induce phagocytosis and 12-myristate 13-acetic acid (PMA) and Sacaromyces cerevisiae (Zymosan) in the induction of oxidative burst. The animal’s leukocytes of the zoo presented greater phagocytosis intensity induced by Zymosan A, in relation with to the cells of the animals of the Piracicaba river. The intensity of oxidative burst induced by PMA was smaller in the animals of the Piracicamirim brook when compared with turtles of Piracicaba river and the zoo. Financial support: CNPq, CAPES and FAPESP. doi:10.1016/j.vetimm.2008.10.206
305
Detection of antibodies against Ehrlichia canis and Babesia canis in Brazilian wild captive felids
7. Comparative immunology; immunoecology
Marcos R. André 1,∗ , Rosangela Z. Machado 1 , Silmara M. Allegretti 2 , Cristina H. Adania 3 , Paulo A.N. Felippe 2 , Ketty F. Silva 1 , Andréa C.H. Nakaghi 1
Similarities and differences of microscopic structure of spleen in beluga Huso huso with terrestrial animals
1
Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil 2 Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil 3 Centro Brasileiro de Conservac¸ão de Felídeos Neotropicais, Jundiaí, SP, Brazil Keywords: Wild felids; Ehrlichia canis; Babesia canis; Antibodies E-mail address: [email protected] (M.R. André).
Species: Feline Ehrlichiosis is an emergent zoonosis caused by an obligate intracellular bacteria belonging to Anaplasma, Ehrlichia and Neorickettsia genera, present in the environment through complex interactions among invertebrates and vertebrates hosts. Babesiosis is caused by infection with intraerythrocytic parasites of the genus Babesia. Both parasites are transmitted by ticks. Brazilian dogs are commonly infected by E. canis and B. canis, whose vector is the dog brown tick, the Rhipicephalus sanguineus. Little is known about the epidemiology of ehrlichiosis and babesiosis in wild felids, which can be seen as good sentinels for these diseases, since they are hosts for both hemoparasites and vectors. The present work aimed to detect antibodies against E. canis and B. canis vogeli in 72 Brazilian wild captive felids by IFA (indirect immunofluorescent assay), trying to verify if these animals are exposed to these parasites. Blood samples were collected from Puma concolor (cougar), Leopardus pardalis (ocelot), Puma yagouaroundi (aguarondi), Leopardus wiedii (margay), Leopardus tigrinus (little spotted cat), Oncifelis colocolo (pampas cat) and Panthera onca (jaguar) maintained in captivity in Brasilia, Ribeirão Preto, Pedreira, Campinas Zoos and in Associac¸ão Mata Ciliar de Jundiaí (Brazilian Center of Neotropic Felids Conservation). Forty-four (70.96%) of 62 animals were seroreagents for B. canis and 24 % for E. canis. Antibodies titers found ranged from 1:20 (cut-off) to 1:2560 for E. canis and from 1:40 (cut-off) to 1:1280 for B. canis. Fourteen felids were seropositives for both antigens, while seventeen animals were seronegatives for both agents. To authors’s knowledge, this is the first report of seroreactivity of Brazilian wild felids for both E. canis and B. canis vogeli, showing that these animals are exposed to vector and agents of ehrlichiosis and babesiosis. doi:10.1016/j.vetimm.2008.10.202
Mohammadt Sheibani Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Keywords: Spleen; Beluga; Terrestrial animals; Histology E-mail address: [email protected]. Species: Fish One of the largest and the most important sturgeons in the Caspian Sea from the aspect of caviar production is beluga. For this investigation a total number of three belugas fished from southern Caspian Sea, were subjected to study. By an incision on the abdominal wall the spleen was removed and after fixation in 10% buffered formalin, routine histological processes were done, paraffin blocks were sectioned at 6 m and stained with hematoxylin and eosin and studied under light microscope. The results showed that the spleen, unlike to those of mammals, was surrounded with a capsule composed of three layers including an epithelium of cuboidal to columnar cells with some small round secretory cells with eosinophilic granules. Under which a waving layer of elastic fibers and then a condensed connective tissue were located. Under the capsule a dense lymphatic tissue containing some melanomacrophages was observed. The parenchyma of the spleen was composed of white and red pulps as in other vertebrates. White pulps consisted of a lymphatic nodule containing a small branch of splenic artery, as the central artery, like that of mammals, but also a venous branch, unlike mammals that differ from this aspect. The lymphatic nodule is followed by a lymphatic cord consisting of an aggregation of lymphocytes around an arteriole, as periarterial lymphatic sheath. The red pulp contained too many sinusoids filled with red blood cells surrounded by some trabeculae and diffused lymphatic tissues throughout the red pulp of spleen. doi:10.1016/j.vetimm.2008.10.203 Gross and microscopical characterization of the thymus and spleen of Piaractus mesopotamicus (Pacu) G.M. Costa ∗ , C.E. Barroso, A.S. Oliveira, C. Biasi, R.S. Iunes, A.R. Lima, J.M. Monteiro, R.V. Bosch, P. Favaron, M.A. Ivo, J.R. Kfoury Jr. Medical Surgical Department of Veterinary Medicine and Zootenic College of the University of São Paulo, Brazil Keywords: Piaractus mesopotamicus; Anatomy; Histology; Spleen and thymus Species: FishPiaractus mesopotamicus (Pacu) is a fish from the Characidae family, intensively bred in Brazil because of its rusticity, easy raising and adaptation, besides its excellent flavor. In order to produce a healthy fish, information on its immunological system including the histology of the lymphoid organs is needed. Therefore, this
306
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
project performed a gross and histological analysis of the thymus and spleen of five young (about 25 cm in length) and five adult pacus (about 38 cm in length). The thymus appeared as a paired organ, placed on the caudal portion of the head, dorsally to the gills in the opercular cavity and involved by a membranous capsule. The thymus was more evident in fish older than 5 months; it presented a gelatinous consistency varying from vivid to dark red color. Histologically, it showed medullar and cortex regions consisted by conjunctive tissue and colagenous fibers and the presence of thymocytes and lymphocytes. The spleen was located caudally to the liver, ventrally to the swim bladder, dorsally to the medium caudal portion of the biliary vesicle. It was rounded shaped, dorsoventrally flattened and it presented an irregular surface in young and lobed in adult fish. It was delimited by a membrane and, histologically, it was composed by a white pulp, rich in melanomacrophages and extensive red pulp areas. doi:10.1016/j.vetimm.2008.10.204 Immunolocalization of heat shock protein (HSP) 70 in the liver of diploid and triploid rainbow trout R.S. Iunes ∗ , J.M. Monteiro, G.M. Costa, A.R. Lima, R.V. Bosch, J.R. Kfoury Jr. Department Medicine and Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil Keywords: Trout; HSP; Stress; Liver Species: Fish Heat shock proteins (HSPs) belong to a highly conserved family of cellular proteins present in all organisms. HSPs are expressed in different tissues under normal conditions; however it increases substantially when stress is present. Fish show two types of stress response: one is physiological and the other cellular. HSPs play a role at the latter, where a fish may present physiological response to a stressor agent without showing any change of the cellular HSPs profile. Among the HSPs, HSP 70 is the most studied in fish, and we attempted to compare its expression in the liver of diploid and triploid rainbow trout (Oncorhynchus mykiss). Liver from eight diploid and 10 triploid trouts were fixed in 10% formalin and processed for immunohistochemistry, using a monoclonal antibody specific to HSP 70 (Stressgen® mouse anti-HSP 70 monoclonal SPA-810). The immunohistochemistry results showed a cytoplasmatic expression of HSP 70 in hepatocytes and in the epithelial cells of the biliary duct. Differences on the intensity of staining were observed in both ploidy, where triploid fish presented a more evident variation, from intense to negative staining. Negative controls showed no staining in both diploid and triploid fish. Our results suggested triploid rainbow trout show differences on HSP 70 expression characteristics from its diploid counterpart. doi:10.1016/j.vetimm.2008.10.205
Cellular function evaluation of sanguineous leukocytes in Phrynops geoffroanus using flow cytometric methodology A. Genoy-Puerto 1,∗ , S. Rossi 1 , B.O. Ferronato 2 , V.M. SáRocha 3 , C.S. Lisboa 4 , L.C. Sá-Rocha 3 , E.R. Matushima 3 1
Academic Program of Experimental and Compared Pathology, FMVZ/USP, Brazil 2 Academic Program in Applied Ecology, ESALQ/USP, Brazil 3 Departament of Pathology, FMVZ, USP, Brazil 4 Sector of Reptiles of the Foundation Zoological Park of São Paulo, Brazil Keywords: Cellular function; Evaluation; Leukocytes; Flow cytometric; Phrynops geoffroanus E-mail address: [email protected] (A. Genoy-Puerto). Specie: Reptile (turtle) Although Phrynops geoffroanus displays ample geographical distribution in South America, few studies had been developed in relation to the natural history of this freshwater turtle. This species frequently appears in contaminated water sources, but the effect of adverse situations (anthrophic effects) on the animals ‘cellular immune activity is unknown. In this work a method employing flow cytometry was developed in order to evaluate the cellular functions of phagocytosis and spontaneous and induced oxidative burst in leukocytes from blood samples of Phrynops geoffroanus found in contaminated environments of the Piracicaba river and Piracicamirim brook, in São Paulo State. Blood samples were obtained from 8 animals from Piracicaba river, 9 from Piracicamirim brook, and 7 from Zoological Park Foundation of São Paulo; the samples from zoological park were used as control group. The samples had been kept in culture in RPMI medium under refrigeration and directed to the Neuro-immunology Laboratory of Department of Pathology in FMVZ/USP to be processed. The fact that the red blood cells are nucleated in these animals, made necessary the use of Ficoll-PaqueTM PLUS at different densities (1085, 1070 and 1055 g/mL), in order to pre-separate these cells. Then the sample was centrifuged to obtain cells of interest, at 18 ◦ C, 450 × g, acceleration 9 and brake 0, resulting in a white ring of leukocytes located between the superior layers of the Ficoll. The samples were submitted to two washes with RPMI to remove the excesses of Ficoll. After the washes, the samples were suspended again in 500 L RPMI for making a viability counting with Trypan Blue; samples with inferior percentages to 94 % were not used in the stimulates: Zymosan A (Sacaromyces cerevisiae) the Bio Particles® , Alexa Fluor® 594 conjugate to induce phagocytosis and 12-myristate 13-acetic acid (PMA) and Sacaromyces cerevisiae (Zymosan) in the induction of oxidative burst. The animal’s leukocytes of the zoo presented greater phagocytosis intensity induced by Zymosan A, in relation with to the cells of the animals of the Piracicaba river. The intensity of oxidative burst induced by PMA was smaller in the animals of the Piracicamirim brook when compared with turtles of Piracicaba river and the zoo. Financial support: CNPq, CAPES and FAPESP. doi:10.1016/j.vetimm.2008.10.206