Pesquisa de Leishmania spp. em primatas de cativeiro de cinco regiões brasileiras por diferentes técnicas de diagnóstico
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Data
2020-10-16
Autores
Guiraldi, Lívia Maísa [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Leishmanioses são zoonoses ocasionadas por protozoários da Família Trypanosomatidae e do gênero Leishmania, tendo como vetor diferentes espécies de flebotomíneos pertencentes ao gênero Lutzomyia nas Américas e Phlebotomus no Velho Mundo. Além dos humanos, animais domésticos e silvestres de vida livre e de cativeiro, podem naturalmente se infectar pelo protozoário, podendo atuar como reservatórios, incluindo os primatas não humanos (PNH). Neste trabalho o objetivo foi a pesquisa de Leishmania spp. em 189 PNH cativos, provenientes de quatro zoológicos e um centro de conservação das cinco regiões brasileiras, por meio do diagnóstico direto em esfregaço por extensão sanguínea; Reação de Imunofluorescência Indireta (RIFI), para pesquisa de anticorpos contra o parasito; Reação em Cadeia da Polimerase convencional (cPCR) para amplificação do DNA do protozoário nas amostras de sangue e células conjuntivais; caracterização molecular com o uso de alvos gênicos direcionado ao citocromo de Leishmania spp. com posterior sequenciamento genético para identificação da espécie envolvida na infecção e a discriminação de espécies de Leishmania por análises de dissociação de DNA em alta resolução (HRM). Foram observadas formas amastigotas por diagnóstico direto de quatro primatas. Oito PNH foram reagentes à técnica sorológica de RIFI. A detecção molecular nas amostras de sangue mostrou positividade em 22 PNH e a detecção nas amostras de suabes conjuntivais também foi possível em 22 PNH (sendo cinco deles positivos tanto no sangue como no suabe), em todos os locais amostrados, exceto da região Sul. Para pesquisa de família Trypanosomatidae, recomenda-se o uso de primers direcionados a região V7 e V8 do rRNA que amplificou o DNA em 16 PNH. O DNA de L. infantum foi amplificado em seis animais, bem como a amplificação do DNA de L. braziliensis em nove PNH. Para caracterização molecular, recomenda-se o uso de primers direcionados às sequências do gene de citocromo da subunidade II da oxidase (CoxII) de Leishmania spp., permitindo a identificção de L. infantum em 13 delas. Este é o primeiro trabalho que obteve êxito na utilização da técnica de HRM na discriminação de espécies de Leishmania em amostras de PNH, revelando 11 animais com L. infantum, e cinco positivos para L. braziliensis. Considerando os resultados de todas as técnicas propostas (parasitológico; sorológico e molecular (cPCR de sangue e células conjuntivais), as análises pontuais dos casos positivos revelaram que na região Norte do país foi a que apresentou maior porcentagem de PNH positivos para Leishmania spp. (60%), seguida das regiões Sudeste (35,41%), Centro-Oeste (30,5%) e Nordeste (29,62%). A região Sul não apresentou nenhum animal positivo. Os resultados desse estudo evidenciam a infecção natural por parasitos Leishmania spp. em animais silvestres e apesar do papel dos PNH na epidemiologia das leishmanioses deva ser melhor esclarecido, os resultados sugerem a participação dos PNH na manutenção do ciclo de transmissão em áreas onde a doença é endêmica. Estes animais devem ser levados em consideração para o estabelecimento de medidas de vigilância e controle da doença no contexto One Health visando a participação dos animais (domésticos e silvestres), do homem e do meio ambiente para o entendimento dos processos de saúde e doença contribuindo para o bem-estar comum.
Leishmaniasis are zoonoses caused by protozoa of the Family Trypanosomatidae and of the genus Leishmania, having different vector species of sandflies belonging to the genus Lutzomyia in the Americas and Phlebotomus in the Old World. Besides humans, domestic and free ranging or captive wild mammals can naturally become infected by Leishmania spp., and can act as reservoirs, including non-human primates (NHP). The work aimed the research of Leishmania spp. in 189 captive PNH, from four zoos and one conservation center from five Brazilian regions, by direct microscopy research in blood smears; Immunofluorescent Antibody Test (IFAT), to search for antibodies against the parasite; Conventional Polymerase Chain Reaction (cPCR) for the amplification of protozoan DNA from blood samples and conjunctival cells; molecular characterization with the use of gene targets directed to the cytochrome of Leishmania spp. with subsequent genetic sequencing to identify the species involved in the infection and the identification of Leishmania species by High resolution melt (HRM) analyzes. Through the first technique, it was observed amastigotes forms in four primates. Eight PNH were reagents by the IFAT serological technique. Molecular detection in blood samples was positive in 22 NHP and detection in conjunctival swab samples was also possible in 22 NHP (five of which were positive both in blood and swab), in all sampled locations, except in the southern region. For Trypanosomatidae family research, it is recommended to use primers directed to the V7 and V8 regions of the rRNA that amplified the DNA in 16 NHP. The DNA of L. infantum was amplified in six animals, as well as the amplification of the DNA of nine primates for L. braziliensis. For molecular characterization, it is recommended the use of primers directed to sequences of the oxidase subunit II (coxII) cytochrome gene of Leishmania spp., allowing the identification of L. infantum in 13 of them. This is the first research that has been successful in using the HRM technique to discrimination Leishmania species in NHP samples, revealing in 11 animals with L. infantum and five animals positive for L. braziliensis. Considering the results of all the proposed techniques (parasitological; serological and molecular – cPCR and conjuntival cells), the punctual analyzes of positive cases revealed that the Northern region of the country was the one with the highest percentage of NHP positive for Leishmania spp. (60%), followed by the Southeast (35.41%), Midwest (30.5%) and Northeast (29.62%) regions. The South region did not present any positive animals. The results of this study evidences the natural infection by Leishmania spp. parasites in wild animals e and despite the role of NHP in the epidemiology of leishmaniasis should be better clarified, the results obtained suggests the participation of NHP in maintaining the transmission cycle in areas where the disease is endemic. These animals must be taken into consideration for the establishment of disease surveillance and control measures in the context of One Health to complete the participation of animals (domestic and wild), man and the environment for the understanding of health and disease processes, contributing for the common welfare.
Leishmaniasis are zoonoses caused by protozoa of the Family Trypanosomatidae and of the genus Leishmania, having different vector species of sandflies belonging to the genus Lutzomyia in the Americas and Phlebotomus in the Old World. Besides humans, domestic and free ranging or captive wild mammals can naturally become infected by Leishmania spp., and can act as reservoirs, including non-human primates (NHP). The work aimed the research of Leishmania spp. in 189 captive PNH, from four zoos and one conservation center from five Brazilian regions, by direct microscopy research in blood smears; Immunofluorescent Antibody Test (IFAT), to search for antibodies against the parasite; Conventional Polymerase Chain Reaction (cPCR) for the amplification of protozoan DNA from blood samples and conjunctival cells; molecular characterization with the use of gene targets directed to the cytochrome of Leishmania spp. with subsequent genetic sequencing to identify the species involved in the infection and the identification of Leishmania species by High resolution melt (HRM) analyzes. Through the first technique, it was observed amastigotes forms in four primates. Eight PNH were reagents by the IFAT serological technique. Molecular detection in blood samples was positive in 22 NHP and detection in conjunctival swab samples was also possible in 22 NHP (five of which were positive both in blood and swab), in all sampled locations, except in the southern region. For Trypanosomatidae family research, it is recommended to use primers directed to the V7 and V8 regions of the rRNA that amplified the DNA in 16 NHP. The DNA of L. infantum was amplified in six animals, as well as the amplification of the DNA of nine primates for L. braziliensis. For molecular characterization, it is recommended the use of primers directed to sequences of the oxidase subunit II (coxII) cytochrome gene of Leishmania spp., allowing the identification of L. infantum in 13 of them. This is the first research that has been successful in using the HRM technique to discrimination Leishmania species in NHP samples, revealing in 11 animals with L. infantum and five animals positive for L. braziliensis. Considering the results of all the proposed techniques (parasitological; serological and molecular – cPCR and conjuntival cells), the punctual analyzes of positive cases revealed that the Northern region of the country was the one with the highest percentage of NHP positive for Leishmania spp. (60%), followed by the Southeast (35.41%), Midwest (30.5%) and Northeast (29.62%) regions. The South region did not present any positive animals. The results of this study evidences the natural infection by Leishmania spp. parasites in wild animals e and despite the role of NHP in the epidemiology of leishmaniasis should be better clarified, the results obtained suggests the participation of NHP in maintaining the transmission cycle in areas where the disease is endemic. These animals must be taken into consideration for the establishment of disease surveillance and control measures in the context of One Health to complete the participation of animals (domestic and wild), man and the environment for the understanding of health and disease processes, contributing for the common welfare.
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Palavras-chave
Diagnóstico, Leishmaniose, Primata não-humano, Saúde única, Zoológico, Diagnosis, Leishmaniasis, Non-human primates, One health