Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine

Abstract

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*10 4 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*10 6 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.Keywords: M. hyopneumoniae. Enzootic pneumonia. Healthy. Quantification. VNTR. ResumoAs diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia têm sido estudadas. Porém, estudos comparativos relativos às estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e análise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Múltiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificação de múltiplo VNTR (VRANCKX et al., 2011). A caracterização molecular das estirpes foi realizada mediante a análise do número de cópias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*10 4 cópias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*10 6 cópias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de cópias de VNTR demonstrou uma elevada variabilidade genética das estirpes d...

Occurrence of Mycoplasma hyopneumoniae in slaughter pigs from Southern Brazil

“…On the other hand, some positive samples in nested-PCR did not present a lung lesion score. This result might be because of the natural process of cicatrization, whereby the microorganism remains in the respiratory tract, turning the animal into an asymptomatic pathogen carrier (Pulgarón et al, 2015;Siqueira et al, 2017). Moreover, the detection of M. hyopneumoniae in lungs without macroscopic pneumonia-like lesions may also be related to the infectious process.…”

Evaluation of two DNA extraction techniques in the detection of Mycoplasma hyopneumoniae by PCR in lungs samples of pigs with and without pneumonia

“…Similar results were obtained by Teixeira et al (2003) when they reported that the macroscopic lesions in the lungs were not sensitive to detect PES, since some samples that presented no macroscopic injuries to PES obtained positive results in PCR, as well as the lung samples with lesions were negative in PCR. Some samples were positive for PCR and presented no pulmonary lesions possibly due to the natural healing process, by which the microorganism remains in the respiratory tract, transforming the animal into an asymptomatic pathogen carrier (PULGARÓN et al, 2015;SIQUEIRA et al, 2017). Moreover, the detection of M. hyopneumoniae in the lungs without macroscopic lesions of the type pneumonia may also be related to the infectious process, and in these cases, the use of PCR capable of detecting small amounts of microorganisms per sample , which would be considered false negatives by less sensitive methods.…”