Detecção e caracterização molecular de agentes Anaplasmatceae em Xenarthra no Brazil
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2020-07-08
Autores
Calchi, Ana Cláudia [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os agentes Anaplasmataceae são α-proteobactérias Gram-negativos intracelulares obrigatórias que são transmitidos principalmente por vetores artrópodes. Embora os mamíferos da Superordem Xenarthra (preguiças, tamanduás e tatus) tenham sido implicados como reservatórios de vários agentes zoonóticos, apenas poucos estudos procuraram detectar agentes Anaplasmataceae neste grupo de mamíferos. Este estudo teve por objetivo investigar a ocorrência e diversidade genética de Anaplasma spp. e Ehrlichia spp. em amostras de sangue e baço de Xenarthra de vida livre de quatro estados no Brasil (São Paulo, Mato Grosso do Sul, Rondônia, e Pará). Foram efetuados ensaios de triagem por meio de nPCR e cPCR para detectar os genes rrs e dsb de Anaplasma spp. e Ehrlichia spp., respectivamente. Os ensaios foram positivos em 27,57% (91/330) para Anaplasma spp. e 24,54% (81/330) para Ehrlichia spp. Das 91 amostras positivas para Anaplasma spp., 56,04% foram positivas num ensaio de PCR convencional visando a região intergênica 23S-5S. Análises filogenéticas e de distância baseadas no gene rrs alocaram as sequências de Anaplasma de preguiças (Bradypus variegatus, Bradypus sp., Choloepus spp. e C. didactylus) capturadas nos estados de Rondônia e Pará em um único clado, que estava estreitamente relacionado com o clado de A. marginale, A. ovis, e A. capra. As sequências detectadas nos tamanduás-mirins (Tamandua tetradactyla) de São Paulo foram alocadas em um clado filogeneticamente relacionado com as sequências de Anaplasma spp. detectadas em Nasua nasua, Leopardus pardalis, e Cerdocyon thous no Brasil. Estas sequências foram posicionadas perto das sequências de A. odocoilei. A análise do genótipo corroborou as descobertas anteriores e demonstrou a circulação de dois genótipos distintos de Anaplasma em animais do norte e sudeste do Brasil. O primeiro genótipo era novo. O segundo foi previamente detectado em N. nasua, no estado de Mato Grosso do Sul. As análises da região intergênica também demonstraram dois genótipos distintos de Anaplasma. As sequências detectadas em Xenarthra dos estados do Pará e Rondônia estavam relacionadas com as de A. marginale, A. ovis, e A. capra. Enquanto que as sequências de Anaplasma spp. detectadas em Xenarthra de São Paulo foram alocadas em um clado irmão ao de A. phagocytophilum. As análises baseadas no gene dsb agruparam as sequências de Ehrlichia spp. de São Paulo, Mato Grosso do Sul, e Pará com sequências de E. canis e as de Rondônia e Pará com E. minasensis. Os dados indicam a ocorrência de E. canis e E. minasensis e duas possíveis novas espécies de Anaplasma spp. em mamíferos de vida livre da Superordem Xenarthra no Brasil.
Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.
Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil.
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Diagnóstico molecular, Variação genética, Filogenia, Bacteriologia veterinária, Xenarthra