Evaluation of a nested PCR to identify a repeat motif p97 in order to detect Mycoplasma hyopneumoniae genetic diversity

Mycoplasma hyopneumoniae is the primary agent involved in porcine enzootic pneumonia (EP). Different genotypes of M. hyopneumoniae have been described using Multiple Locus Variable-number Tandem Repeat Analysis (MLVA). In our experience it is difficult to perform a MLVA from nasal swab samples due to the sensitivity of some PCRs. In this regard, we have reported the increasing of the sensitivity of p146 locus (Tamiozzo 2011, 2013) developing a nested PCR. Since a recent study (Dos Santos 2015) showed a large number of M. hyopneumoniae types combining p146 and p97 loci and the possibility to type M. hyopneumoniae from minimally invasive samples, such as nasal swabs, without killing animals or performing invasive sampling, the objective of this study was to evaluate a nested PCR for M. hyopneumoniae typing.

A total of 56 M. hyopneumoniae PCR positive (16SrRNA) DNA samples (from nasal swabs, tracheo-bronchial lavages and Mycoplasma hyopneumoniae bacterins) were analyzed, amplifying p97 loci, by 3 PCRs: 1) PCR reported by de Castro (2006), 2) PCR reported by Vranckx (2011) and 3) a nested PCR format using for the first round of amplification, primers and conditions reported by de Castro et al., 2006 and for the second round, primers and conditions reported by Vranckx et al., 2011. Proportions of positives from each single PCR format were compared vs nested PCR format (chi ² test, Epidat).

Fifteen out of 56 (26.8%) samples were positives to the PCR reported by de Castro et al. (2006) whereas 18/56 (32.1%) samples were positives to the PCR reported by Vranckx 2011. Using the nested PCR format, there was 50% of positives. There was statistic significant difference (p=0.019) between the PCR described by de Castro vs the nested PCR format.

The use of the nested format allowed detecting a greater number of positive samples surely due to the increase of the sensitivity. While Vranckx et al. (2011) only worked with bronchoalveolar lavages and tracheal swabs, de Castro et al. (2006) suggested that the PCR assays could have sufficient sensitivity for M. hyopneumoniae typing from clinical samples, we think, a characterization by MLVA is not always possible using conventional PCR (Tamiozzo 2013, 2015), even when working with tracheal and/or bronquial specimens. Kuhnert et al. (2011) noticed that successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in lung samples. We are aware that study of other genomic regions could have been useful to type M. hyopneumoniae, but others specific nPCRs have yet to be developed.

Disclosure of Interest: None Declared.