Diversidade genética de Anaplasma marginale em bovinos de corte no Pantanal brasileiro
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Data
2018-11-08
Autores
Ramos, Inalda Angélica de Souza
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Universidade Estadual Paulista (Unesp)
Resumo
Poucos são os estudos acerca da diversidade genética de Anaplasma marginale nos rebanhos bovinos brasileiros, principalmente no que diz respeito a bovinos de corte. O presente estudo teve como objetivo avaliar a diversidade genética de A. marginale, com base nos genes msp1α e msp4, em Bos taurus indicus amostrados no Pantanal brasileiro. Alíquotas de sangue com e sem EDTA foram colhidas de 400 bovinos (200 vacas e 200 bezerros) em cinco propriedades de cria e recria extensiva. Enquanto as amostras de soro foram submetidas ao Ensaio Imunoenzimático Indireto (iELISA) para detecção de anticorpos IgG anti-A. marginale, amostras de sangue total foram submetidas à avaliação do hematócrito e confecção de esfregaços sanguíneos corados pelo Giemsa, PCR em tempo real quantitativa (qPCR) baseada no gene msp1β, semi-nested (sn)PCR baseada no gene msp1α e PCR convencional (cPCR) baseada no gene msp4 para A. marginale. Os produtos amplificados nos ensaios de snPCR e cPCR foram purificados e sequenciados pelo Método de Sanger. Sequências do gene msp1α foram submetidas à análise de diversidade pelo software RepeatAnalyser. Sequências do gene msp4 foram submetidas à análise de distância Network pelo software Splitstree e de genótipos pelo Software DnaSP5 e PopART. Cinco animais (duas vacas e três bezerros) apresentaram corpúsculos de A. marginale em esfregaços sanguíneos. As frequências de animais positivos pelo iELISA, qPCR e snPCR foram de 72,25% (289/400), 56,75% (227/400) e 22,9% (52/227), respectivamente. Os testes de iELISA e qPCR mostraram fraca concordância kappa. Vacas (154/200) mostraram-se estatisticamente mais soropositivas que bezerros (135/200) (P<0,05). Na qPCR, o número de bezerros (138/200) e valor médio de quantificação (1,3 x 106 cópias msp1α A. marginale/µL) mostraram-se maiores quando comparados àqueles encontrados para as vacas (89/200; 3,9 x 104) (P<0,05). A rickettsemia estimada pela qPCR não mostrou correlação com o hematócrito dos animais. A análise de microssatélites das 26 sequências msp1α obtidas revelou a presença dos genótipos E (76,9%), C (15,38%) e B (7,69%). O software RepeatAnalyzer mostrou a ocorrência de 14 estirpes circulantes na região sob estudo, com oito nunca antes descritas na literatura (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-β-β-β100; EV7-11-10-15; τ-11-11-27-18; τ-11-10-15; τ-27-13-18). Alta diversidade genética de A. marginale foi encontrada em bovinos de corte no Pantanal Brasileiro. Os índices de diversidade genética revelaram alta diversidade entre o grupo de genótipos/estirpes analisados, bem como na região de colheita, indicando a circulação de novos genótipos/estirpes no país, porém com baixa taxa de dispersão. A cPCR para o gene msp4 de A. marginale revelou 6,16% (14/227; 4 vacas e 10 bezerros) de positividade, Dois genótipos foram identificados entre as 14 sequências do presente estudo. Dezesseis genótipos foram obtidos entre as sequências estudadas em conjunto com as obtidas no GenBank, mostrando que os genótipos msp4 de A. marginale circulantes em bovinos de corte no Pantanal brasileiro são filogeograficamente relacionados àqueles circulantes em países das América do Sul e Central, Europa e Ásia
There are few studies about the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially with regard to beef cattle. The present study aimed to evaluate the genetic diversity of A. marginale based on the msp1α and msp4 genes in Bos taurus indicus, sampled in the Brazilian Pantanal. Blood aliquots with and without EDTA were collected from 400 cattle (200 cows and 200 calves) in five extensive breeding and rearing properties. While the serum samples were submitted to the Indirect Immunoenzyme Assay (iELISA) for the detection of anti-A. marginale IgG antibodies, whole blood samples were submitted to the evaluation of hematocrit and Giemsa-stained blood smears, quantitative real-time PCR (qPCR) based on the msp1β gene, semi-nested (sn) PCR based on the msp1α gene and conventional PCR (cPCR) based on the msp4 gene for A. marginale. The amplified products obtained in the snPCR and cPCR assays were purified and sequenced by the Sanger Method. Sequences of the msp1α gene were submitted to diversity analysis by the RepeatAnalyser software. msp4 gene sequences were submitted to Network distance analysis using the Splitstree software and genotypes analysis by the DnaSP5 and PopART software. Five animals (two cows and three calves) presented A. marginale corpuscles in blood smears. The frequencies of iELISA positive animals, qPCR and snPCR were 72.25% (289/400), 56.75% (227/400) and 22.9% (52/227), respectively. The iELISA and qPCR tests showed weak kappa concordance. Cows (154/200) were statistically more seropositive than calves (135/200) (P <0.05). In the qPCR, the number of calves (138/200) and mean value of quantification (1.3 x 106 copies msp1α A. marginale/μL) were higher when compared to those found for cows (89/200; 3.9 x 104) (P <0.05). The rickettsemia estimated by qPCR showed no correlation with the hematocrit of the animals. Microsatellite analysis of the obtained 26 msp1α sequences revealed the presence of E (76.9%), C (15.38%) and B (7.69%) genotypes. RepeatAnalyzer software showed the occurrence of 14 circulating strains in the studied region, with eight never previously described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-β-β -β-100; EV7-11-10-15; τ11-11-27-18; τ-11-10-15; τ-27-13-18). High genetic diversity of A. marginale was found in beef cattle in the Brazilian Pantanal. The genetic diversity indexes showed high diversity among the group of genotypes / strains analyzed, as well as in the studied region, indicating the circulation of new genotypes / strains in the country, but with low dispersion rate. The cPCR based on the msp4 gene of A. marginale showed 6.16% (14/227, 4 cows and 10 calves) of positivity. Two genotypes were identified among the 14 sequences of the present study. Sixteen genotypes were obtained among the sequences found in the presente study in conjunction with those obtained in GenBank, showing that A. marginale msp4 genotypes circulating in beef cattle in the Brazilian Pantanal are phylogeographically related to those circulating in countries of South and Central América, Europe and Ásia.
There are few studies about the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially with regard to beef cattle. The present study aimed to evaluate the genetic diversity of A. marginale based on the msp1α and msp4 genes in Bos taurus indicus, sampled in the Brazilian Pantanal. Blood aliquots with and without EDTA were collected from 400 cattle (200 cows and 200 calves) in five extensive breeding and rearing properties. While the serum samples were submitted to the Indirect Immunoenzyme Assay (iELISA) for the detection of anti-A. marginale IgG antibodies, whole blood samples were submitted to the evaluation of hematocrit and Giemsa-stained blood smears, quantitative real-time PCR (qPCR) based on the msp1β gene, semi-nested (sn) PCR based on the msp1α gene and conventional PCR (cPCR) based on the msp4 gene for A. marginale. The amplified products obtained in the snPCR and cPCR assays were purified and sequenced by the Sanger Method. Sequences of the msp1α gene were submitted to diversity analysis by the RepeatAnalyser software. msp4 gene sequences were submitted to Network distance analysis using the Splitstree software and genotypes analysis by the DnaSP5 and PopART software. Five animals (two cows and three calves) presented A. marginale corpuscles in blood smears. The frequencies of iELISA positive animals, qPCR and snPCR were 72.25% (289/400), 56.75% (227/400) and 22.9% (52/227), respectively. The iELISA and qPCR tests showed weak kappa concordance. Cows (154/200) were statistically more seropositive than calves (135/200) (P <0.05). In the qPCR, the number of calves (138/200) and mean value of quantification (1.3 x 106 copies msp1α A. marginale/μL) were higher when compared to those found for cows (89/200; 3.9 x 104) (P <0.05). The rickettsemia estimated by qPCR showed no correlation with the hematocrit of the animals. Microsatellite analysis of the obtained 26 msp1α sequences revealed the presence of E (76.9%), C (15.38%) and B (7.69%) genotypes. RepeatAnalyzer software showed the occurrence of 14 circulating strains in the studied region, with eight never previously described in the literature (τ-10-13-13-18; τ-27-18; EV8-EV8-17; α-β-β -β-100; EV7-11-10-15; τ11-11-27-18; τ-11-10-15; τ-27-13-18). High genetic diversity of A. marginale was found in beef cattle in the Brazilian Pantanal. The genetic diversity indexes showed high diversity among the group of genotypes / strains analyzed, as well as in the studied region, indicating the circulation of new genotypes / strains in the country, but with low dispersion rate. The cPCR based on the msp4 gene of A. marginale showed 6.16% (14/227, 4 cows and 10 calves) of positivity. Two genotypes were identified among the 14 sequences of the present study. Sixteen genotypes were obtained among the sequences found in the presente study in conjunction with those obtained in GenBank, showing that A. marginale msp4 genotypes circulating in beef cattle in the Brazilian Pantanal are phylogeographically related to those circulating in countries of South and Central América, Europe and Ásia.
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Palavras-chave
Anaplasmose bovina, Bos taurus indicus, msp1α, Tandem repeats, msp4, Filogeografia, Brasil