Analise em multilocus de repeticoes em tandem de numero variavel (MLVA) de linhagens vacinais B19 e RB51 de Brucella abortus

Abstract

O atual estudo objetivou analisar a variabilidade genetica de linhagens vacinais de Brucella, avaliando ainda os efeitos de repiques sucessivos sobre as mesmas. Trata-se de doenca zoonotica de alcance mundial e que causa prejuizos economicos na producao animal e danos a saude publica. Visando controlar a doenca, foi instituido o Programa Nacional de Controle e Erradicacao da Brucelose e Tuberculose Animal em 2001, que inclui a vacinacao de femeas. Quanto a avaliacao genetica de linhagens vacinais, a tecnica de analise em multilocus das repeticoes em tandem de numero variavel - MLVA permite identificar a variabilidade genetica microbiana de forma acurada, identificando-se genogrupos de isolados. O atual estudo analisou 63 amostras de linhagens vacinais B19 (n=53) e RB51 (n=10), provenientes de sete laboratorios brasileiros. Tambem foi avaliado o efeito de sucessivos repiques sobre a o genoma microbiano. Desta forma, foram identificados quatro genogrupos distintos. No genogrupo I foram agrupadas as 10 amostras de B19 do laboratorio G e seus 10 repiques. Quarenta e tres amostras de B19 dos laboratorios A, B, D, E, F, H, bem como os 10 repiques das amostras E e da cepa de referencia S (USDA) foram agrupadas no genogrupo II. O genogrupo III foi composto pelas amostras vacinais de RB51 do laboratorio Y e pelos 10 repiques da linhagem Y1. Finalmente, o genogrupo IV incluiu oito amostras vacinais de RB51 e os 10 repiques das amostras X1 e da cepa de referencia Schurig. As linhagens vacinais B19 e RB51 derivaram de ramificacoes geneticas distintas que formaram, cada qual, dois genogrupos, com divergencia apenas nos loci Bruce07 e Bruce43. Concluindo, a tecnica de MLVA mostrou adequada capacidade discriminatoria, podendo ser utilizada para analises de qualidade na producao de vacinas comerciais em nosso Pais. Foi observado, ainda, que 10 passagens sucessivas nao afetam os loci avaliados. ______________________________________________________________________________ ABSTRACT

The present study was aimed to analyze the genetic variability among Brucella abortus lineages present in commercial vaccines from Brazilian laboratories. The brucellosis is a worldwide distributed zoonotic disease. The disease may affect the livestock production as well as the human health. To control the spread of Brucellosis in Brazil, our country established the National Program for Controlling and Eradication of Animal Brucellosis and Tuberculosis – PNCEBT which included the vaccination of female bovines. The multiple locus variable number of tandem repeat analysis (MLVA) is a rapid and accurate method used to access the genetic variability among microbial isolates and to discriminate groups of genetically-related isolates. We used a previously described 16 loci MLVA method to analyze a sample composed by B19 vaccines (n=53) and RB51 vaccines (n=10) from seven Brazilian laboratories. The genetic variability after 10 successive cultures was also accessed. We identified four distinct genogroups of isolates. The genogroup I contained ten B19 samples from the laboratory G and their respective successive cultures. Forty-three B19 samples of the laboratories A, B, D, E, F, and H as well as the successive cultures of the reference S (USDA) strain and the commercial strain E, were all clustered into the genogroup II. The genogroup III included the RB51 laboratory Y’s samples and the Y1’s successive cultures. Finally, eight RB51 samples, the successive X1’s cultures, and the reference Schurig strain, were clustered into the genogroup IV. Our results revealed that the B19 and RB51 lineages derived from two distinct genetic branches that diverged in the locus Bruce07 and Bruce43. In conclusion, the MLVA showed accurate discriminatory results and would be used in future evaluations of the commercial vaccines of Brucellosis. The results of our study also showed that ten successive cultures were not able to generate genetic variability into the loci studied.