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A new multiplex real‐time TaqMan® PCR for quantification of Mycoplasma hyopneumoniae, M. hyorhinis and M. flocculare: exploratory epidemiological investigations to research mycoplasmal association in enzootic pneumonia‐like lesions in slaughtered pigs

Fourour, S., Fablet, C., Tocqueville, V., Dorenlor, V., Eono, F., Eveno, E., Kempf, I., Marois‐Créhan, C.
Journal of applied microbiology 2018 v.125 no.2 pp. 345-355
Mycoplasma hyopneumoniae, correlation, enzootic diseases, epidemiological studies, etiology, genes, lungs, pneumonia, quantitative polymerase chain reaction, swine
AIMS: A new multiplex qPCR, targeting Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. flocculare, was developed and the relationship between detection of those mycoplasma species and the extent of gross pneumonia‐like lesions in slaughtered pigs lungs were investigated. METHODS AND RESULTS: The multiplex qPCR method targets the p102, p37 and fruA genes and has detection limits of 14, 146 and 16 genome equivalents μl⁻¹ for M. hyopneumoniae, M. hyorhinis and M. flocculare, respectively. In all, 671 lungs were collected and analysed, among them 666 were scored for macroscopic pneumonia and categorized according to the extent of the lesions (no or minor lesions, moderate lesions and extensive lesions). According to results of multiplex qPCR, 59·5% were positive for M. hyopneumoniae, 3·4% for M. hyorhinis and 34·7% for M. flocculare, with on average, 3·1 × 10⁷, 9·7 × 10⁶ and 5·7 × 10⁶ genome equivalents of mycoplasma ml⁻¹, respectively. More results showed that no or minor lesions were associated with multiplex qPCR‐negative results or qPCR‐positive results for M. flocculare. Moderate to extensive lesions were positively correlated with qPCR‐positive results for M. hyopneumoniae. Extensive lesions were associated with qPCR‐positive results for at least two mycoplasma species (M. hyopneumoniae and M. hyorhinis). CONCLUSION: The findings also indicated that M. hyopneumoniae and M. hyorhinis significantly increased the odds for a lung to have macroscopic pneumonia. No relationship was found between the extent of lesions and the mycoplasma genome load. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex qPCR appears to be specific, sufficiently sensitive and repeatable. The validation of this method with field samples guarantees its use for field epidemiological investigations, particularly to gain more insight into the aetiology of the porcine respiratory disease complex.