Detecção, quantificação e variabilidade genética de Mycoplasma hyopneumoniae a partir de suínos pneumônicos e aparentemente saudáveis

Molecular differences among Mycoplasma hyopneumoniae strains present in pneumonic lungs of swine have been largely studied. However, no comparative studies concerning the strains present in apparently healthy pigs have been carried out. This study aimed to detect, quantify and perform molecular analysis of M. hyopneumoniae strains in pig lungs with and without pneumonic lesions. The detection of M. hyopneumoniae was performed using multiplex PCR (YAMAGUTI, 2008), real-time PCR (STRAIT et al., 2008) and multiple VNTR amplification (VRANCKX et al., 2011). Molecular characterization of the strains was achieved by analysis of the VNTR copy number in P97R1, P146R3, H2R1 and H4. M. hyopneumoniae was detected in samples from healthy and pneumonic pigs and the amount of M. hyopneumoniae positive samples detected varied with the type of assay. The greater number of positive samples was identified by the multiple VNTR amplification combined with capillary electrophoresis. Using real-time PCR, 4.9*104 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*106 M. hyopneumoniae genome copies/mL was detected in pneumonic lungs. The analysis of VNTR copy number demonstrated a high genetic variability of the M. hyopneumoniae strains present in apparently healthy and pneumonic lungs. Strains having 3 VNTR copy number in P97R1, were detected only in pneumonic lungs and strains having 40 and 43 VNTR copy number in P146R3 were detected only in apparently healthy lungs. Despite the genetic variability of M. hyopneumoniae, predominant strains in the swine farms could be identified... As diferencas moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmoes de suinos com pneumonia tem sido estudadas. Porem, estudos comparativos relativos as estirpes presentes nos suinos aparentemente saudaveis nao foram levados a cabo. O objetivo do estudo foi a deteccao, quantificacao e analise molecular de M. hyopneumoniae nos pulmoes suinos com e sem lesoes pneumonicas. Para a deteccao de M. hyopneumoniae usaramse o PCR Multiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificacao de multiplo VNTR (VRANCKX et al., 2011). A caracterizacao molecular das estirpes foi realizada mediante a analise do numero de copias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suinos saudaveis e pneumonicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior numero de amostras positivas foi identificado pela amplificacao de multiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*104 copias de genoma/mL de M. hyopneumoniae foram detectadas em pulmoes aparentemente saudaveis. Uma quantidade media de 3.9*106 copias de genoma/mL de M. hyopneumoniae foi detectada em pulmoes pneumonicos. A analise do numero de copias de VNTR demonstrou uma elevada variabilidade...