Role of Immunohistochemistry in the Diagnosis of Leptospirosis in Neotropical Primates
Keywords:diagnosis, infectious disease, leptospirosis, primate, pathology, zoonosis.
Background: Leptospirosis is considered the most widespread zoonosis worldwide, occurring more frequently in tropical and developing regions. The aim of the present study was to detect the presence of Leptospira spp. in different primate tissues, using immunohistochemical (IHC) assays, taking advantage of the considerable number of necropsies compatible with a diagnosis of leptospirosis in neotropical primates at the Animal Pathology Laboratory (APL) of the University of Passo Fundo (UPF) in the northern region of Rio Grande do Sul.
Materials, Methods & Results: Paraffin-embedded primate tissue samples were selected from necropsy examinations and subjected to IHC. The streptavidin-biotin-peroxidase method was used with diaminobenzidine chromogen (DAB) to verify immunostaining. Of the101 primates tested for Leptospira spp., 51.48% were positive; taining was distributed between lung (76.92%), liver (44.23%), and kidney (32.69%) tissue. Analysis of the combined anatomopathological verification data of the studied organs revealed a high frequency of lesions commonly observed in the tissues of animals exposed to the pathogen. For complementary diagnosis, an anti-Leptospira spp. antibody test was performed in primates at the UPF-Zoo, from which a population of the necropsied animals originated. The microscopic agglutination test (MAT) was utilized, which demonstrated 90.47% positivity in 21 individuals; sejroe and panama were the most frequent serovars.
Discussion: Different intensities of tissue immunostaining were observed. Areas of fragmented or diffuse staining were considered to indicate equal positivity to that indicated by areas of staining with preserved morphology. Of 52 Leptospirapositive primates, most presented some degree of staining in lung samples, which shows a high level of involvement for this organ in primate leptospirosis. Conventional pathological diagnostic methods do not allow fort issue antigen recognition, thus making the IHC technique important to facilitate conclusive antigen sample verification. In the liver, leptospires were detected mostly between the sinusoids, hepatocytes, and Kupffer cells. In kidney tissues, staining indicated small agglomerates in the tubular lumen, interstitium, and glomeruli. All these forms of presentation have been previously reported. Considering that we detected the highest number of positive samples in lung tissue, followed by those from liver and kidney tissue, we argue that the IHC technique, when applied to samples of these three tissues, decreases the chance of false negatives. Anatomopathological studies of primate leptospirosis are scarce. In dogs, renal lesions are characterized by the necrosis and degeneration of tubular epithelium, cellular debris, and hyaline cylinders. In the liver, hepatocyte cord dissociation and biliary pigment accumulation within the canaliculi and hepatocellular necrosis are observed. These findings are similar to those from our study. In the lung, diffuse alveolar lesions are reported, with hemorrhage and edema, in addition to capillaritis. The high frequency of Leptospira-positive animals determined by serological examination was consistent with the IHC findings, thus confirming the pathogen’s high prevalence in neotropical primate populations in the studied region. Serological surveys on primate populations have already been carried out and have revealed frequency and serovar variations between regions. Immunohistochemical examination allows the detection of leptospires in various tissues and should be used based on the characteristics of the investigated case.
Adin C.A. & Cowgill L.D. 2000. Treatment and outcome of dogs with leptospirosis: 36 casos (1990-1998). Journal of the American Veterinary Medical Association. 216(3): 371-375.
Azizi S., Kheirandish R. & Rahimi E. 2014. Comparison of polymerase chain reaction and Warthin-Starry techniques to detect Leptospira spp. in kidneys of slaughtered cattle. Onderstepoort Journal of Veterinary Research. 81(1): Art. #821, 6 pages. [Fonte: http://dx.doi.org/10.4102/ojvr.v81i1.821].
Baulu J., Everard C.O.R. &Everard J.D. 1987. Leptospires in vervet monkeys (Cercopithecus aethiops sabaeus) on Barbados. Journal of Wildlife Disease. 23(1): 60-66.
Bharti A.R., Nallyb J.E., Ricaldic J.N., Matthiasa M.A., Diazd M.M., Lovettb M.A., Levette P.N., Gilmanf R.H., Willigd M.R., Gotuzzoc E. & Vinetza J.M. 2003. Leptospirosis: a zoonotic disease of global importance. The Lancet Infectious Diseases. 12(3): 757-771.
Brito T., Menezes, L.F., Lima D.M.C., Lourenço S., Silva A.M.G. & Alves V.A.F. 2006. Immunohistochemical and in situ hybridization of the liver and kidney in human leptospirosis. Virchows Archives. 448(5): 576-583.
Cole Junior J.R., Sulzer C.R. & Pursell A.R. 1973. Improved microtechnique for agglutination test. Applied Microbiology and Biotechnology. 25(6): 970-980.
Corrêa S.H.R., Vasconcellos S.A., Morais Z., Teixeira A.A., Dias R.A., Guimarães M.A.B.V.G., Ferreira F. & Ferreira Neto J.S. 2004. Epidemiologia da leptospirose em animais silvestres na Fundação Parque Zoológico de São Paulo. Brazilian Journal of Veterinary Research and Animal Science. 41(3): 189-193.
Costa S.M. 2010. Frequência de anticorpos contra Leptospira spp. e Trypanosoma cruzy em primatas neotropicais mantidos em cativeiro. 94f. Belém, PA. Dissertação (Mestrado) - Programa de Pós-Graduação em Ciência Animal. Universidade Federal do Pará, Núcleo de Estudos em Ciência Animal, Empresa Brasileira de Pesquisa Agropecuária, Universidade Federal Rural da Amazônia.
Cullen J.M. 2007. Liver, Biliary System and Exocrine Pancreas. In: McGavin M.D. & Zachary J.F. (Eds). Pathology Basis of Veterinary Disease. 4th edn. St. Louis: Mosby Elsevier, pp.393-462.
Ellis T.M, Robertson G.M, Hustas L. & Kirbi M. 1983. Detection of leptospires in tissue using immunoperoxidase staining procedure. Australian Veterinary Journal. 60(12): 364-367.
Ferreira D.R.A., Laroque P.O., Wagner P.G.C., Higino S.S.S., Azevedo S.S., Rego E.W. & Mota R.A. 2011. Ocorrência de anticorpos e fatores de risco associados à infecção por Leptospira spp. em Cebus spp. mantidos em cativeiro no Nordeste do Brasil. Pesquisa Veterinária Brasileira. 31(11): 1019-1023.
Garcia-Vázquez E., Herrero J.A., Hernández A. & Gómez J. 2010. Leptospirosis. Medicine. 10(57): 3896-3902.
Girio R.J.S., Pereira F.L.G., Marchiori Filho M., Mathias L.M., Herreira R.C.P., Alessi A.C. & Girio T.M.S. 2004. Pesquisa de anticorpos contra Leptospira spp. em animais silvestres e em estado feral da região de Nhecolândia, Mato Grosso do Sul, Brasil: utilização da técnica de imuno-histoquímica para detecção do agente. Ciência Rural. 34(1): 165-169.
Haanwinckel M.C.S., Megid J. & Souza L.C. 2004. Avaliação da prova de Imunoperoxidase como recurso diagnóstico na leptospirose animal. Arquivos do Instituto Biológico. 71(3): 293-301.
Hill M.K. & Sanders C.V. 1997. Leptospiral pneumonia. Seminars in Respiratory Infections. 12(1): 44-49.
Pimentel J.S., Gennari S.M., Dubey J.P., Marvulo M.F.V., Vasconcellos S.A., Morais Z.M., Silva J.C.R. & Evêncio Neto J. 2009. Inquérito sorológico para toxoplasmose e leptospirose em mamíferos selvagens neotropicais do Zoológico de Aracaju, Sergipe. Pesquisa Veterinária Brasileira. 29(12): 1009-1014.
Scanziani E, Luini M., Fabbi M. & Pizzocaro P. 1991. Comparison between specific immunoperoxidase staining and bacteriological culture in the diagnosis of renal leptospirosis of pigs. Research in Veterinary Science. 50(2): 229-232.
Silva J.J., Dalston M.O., Carvalho J.E., Setúbal S., Oliveira J.M. & Pereira M.M. 2002. Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Revista da Sociedade Brasileira de Medicina Tropical. 35(4): 395-399.
Tochetto C., Flores M.M., Kommers G.D., Barros C.S.L. & Fighera R.A. 2012. Aspectos anatomopatológicos da leptospirose em cães: 53 casos (1965-2011). Pesquisa Veterinária Brasileira. 32(5): 430-443.
Vinetz J.M., Glass G.E., Flexner C.E., Mueller P. & Kaslow DC. 1996. Sporadic urban leptospirosis. Annals of Internal Medicine. 125(10): 794-798.
Ward J.M. & Rehm S. 1990. Applications of immunohistochemistry in rodent tumor pathology. Experimental Pathology. 40(4): 301-312.
Zachary J.F. 2013. Mecanismos das infecções bacterianas. In: Zachary J.F. & McGavin M.D. (Eds). Bases da Patologia em Veterinária. 5.ed. Rio de Janeiro: Elsevier, pp.147-187.
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