Diversidade genética de Anaplasma marginale em bovinos amostrados em Itú, estado de São Paulo
Carregando...
Arquivos
Data
2020-04-08
Autores
Garcia, Amanda Barbosa [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Anaplasma marginale é uma bactéria, Gram negativa, intracelular obrigatória parasita de eritrócitos e o principal agente da Anaplasmose bovina. Esta doença causa anemia severa, provocando a redução do ganho de peso e da produção de leite e gerando grandes perdas econômicas na pecuária mundial. A diversidade genética desta bactéria vem sendo caracterizada com base na sequência das proteínas de superfície (MSPs), principalmente, na proteína MSP1α, sendo possível identificar as diferentes estirpes geográficas de acordo com as diferenças nas sequências de aminoácidos. O presente estudo teve como objetivo investigar a diversidade genética de A. marginale em bovinos de corte da raça Angus naturalmente infectados durante um surto da enfermidade. Vinte bovinos, com idades entre 6 e 9 meses, oriundos de uma fazenda no município de Itú, estado de São Paulo foram acompanhados por quatro meses e quatro colheitas de sangue foram realizadas. Amostras de soro foram submetidas à Ensaio Imunoenzimático Indireto (iELISA) para detecção de anticorpos IgG anti-A. marginale. As oitenta amostras de sangue total obtidas, foram submetidas à extração de DNA, PCR em tempo real quantitativa (qPCR) para o gene msp1β, semi-nested PCR (snPCR) para o gene msp1α, clonagem do fragmento alvo e sequenciamento pelo método de Sanger. As sequências obtidas foram submetidas à analises de diversidade genética pelo software RepeatAnalyzer. O ensaio Imunoenzimático Indireto (iELISA), utilizando Ag total, revelou baixa soroprevalência (22,5%) nos animais amostrados apesar de 100% de positividade na qPCR, com quantificação entre 103 e 107 número de cópias de DNA/μL, mostrando uma possível imunossupressão causada pela presença do patógeno. Na snPCR, baseada no gene msp1α, 57,5% (46/80) das amostras mostraram-se positivas. A análise de microssatélites de 36 sequências obtidas mostrou a presença do genótipo H (58,3%), F (25%), E (19,4%), C (2,7%) e G (2,7%). Através do programa Repeat Analyzer foram identificadas várias estirpes na regiao estudada, algumas delas nunca antes descritas na literatura, como 13 27 13 27 13 F; 16 F F; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91 entre outras. Foi encontrada alta diversidade genética da bactéria A. marginale circulando nos animais estudados, identificando mais de uma estirpe circulante em um mesmo animal.
Anaplasma marginale is a Gram-negative, obligatory intracellular erythrocyte parasite and the main agent of bovine Anaplasmosis. This disease causes severe anemia, causing a reduction in weight gain and milk production and generating major economic losses in livestock worldwide. The genetic diversity of this bacterium has been characterized based on the sequence of surface proteins (MSPs), mainly in the protein MSP1α, making it possible to identify the different geographical strains according to the differences in the amino acid sequences. The present study aimed to investigate the genetic diversity of A. marginale in Angus beef cattle naturally infected during an outbreak of the disease. Twenty cattle, aged 6 to 9 months, from a farm in the city of Itú, state of São Paulo, were followed for four months and four blood samples were taken. Serum samples were subjected to the Indirect Immunoenzymatic Assay (iELISA) to detect antibodies IgG anti-A. marginale. The eighty whole blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing by the Sanger method. The obtained sequences were subjected to genetic diversity analysis using the RepeatAnalyzer software. The Indirect Immunoenzymatic assay (iELISA) revealed low seroprevalence in the sampled animals despite 100% positivity in the qPCR, with quantification between 103 and 107 number of DNA copies/μL, showing a possible immunosuppression caused by the presence of the pathogen. In snPCR, based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis of 36 sequences obtained showed the presence of genotype H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). Through the RepeatAnalyzer software, several strains were identified in the studied region, some of them never before described in the literature, such as 13 27 13 27 13 F; 16 F F; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91 among others. High genetic diversity of A. marginale bacteria was found circulating in the animals studied, identifying more than one circulating strain in the same animal.
Anaplasma marginale is a Gram-negative, obligatory intracellular erythrocyte parasite and the main agent of bovine Anaplasmosis. This disease causes severe anemia, causing a reduction in weight gain and milk production and generating major economic losses in livestock worldwide. The genetic diversity of this bacterium has been characterized based on the sequence of surface proteins (MSPs), mainly in the protein MSP1α, making it possible to identify the different geographical strains according to the differences in the amino acid sequences. The present study aimed to investigate the genetic diversity of A. marginale in Angus beef cattle naturally infected during an outbreak of the disease. Twenty cattle, aged 6 to 9 months, from a farm in the city of Itú, state of São Paulo, were followed for four months and four blood samples were taken. Serum samples were subjected to the Indirect Immunoenzymatic Assay (iELISA) to detect antibodies IgG anti-A. marginale. The eighty whole blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing by the Sanger method. The obtained sequences were subjected to genetic diversity analysis using the RepeatAnalyzer software. The Indirect Immunoenzymatic assay (iELISA) revealed low seroprevalence in the sampled animals despite 100% positivity in the qPCR, with quantification between 103 and 107 number of DNA copies/μL, showing a possible immunosuppression caused by the presence of the pathogen. In snPCR, based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis of 36 sequences obtained showed the presence of genotype H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). Through the RepeatAnalyzer software, several strains were identified in the studied region, some of them never before described in the literature, such as 13 27 13 27 13 F; 16 F F; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91 among others. High genetic diversity of A. marginale bacteria was found circulating in the animals studied, identifying more than one circulating strain in the same animal.
Descrição
Palavras-chave
Anaplasmose bovina, Diversidade genética, Genótipo, Bovine anaplasmosis, Genetic diversity, Microbiologia