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Molecular typing of Mycoplasma hyopneumoniae field strains using p146 gene sequencing  

Qiumei You, Jason Eidt, Hugh Y. Cai

Mycoplasma hyopneumoniae is the causative agent of zoonotic pneumonia world-wide. Genetic strain typing of the pathogen can be useful for disease diagnosis and management. Current M. hyopneumoniae molecular typing methods include multilocus variable number tandem repeat analysis (MLVA) assay and multilocus sequence typing (MLST). Although these molecular methods can generate useful information, they are complicated to run, somewhat difficult to interpret, and not affordable for routine testing. The serine repeat encoding region of the p146 gene was found to be highly pleomorphic and was used for genetic typing of M. hyopneumoniae isolates by PCR-sequencing without cultivation. This method appears to be easy and less expensive to use. However, it is not known if it can be applied to Ontario M. hyopneumoniae isolates. With support from the OMAFRA-AHL Disease Surveillance Program, we validated the p146 gene-sequencing typing method with Ontario, and a few out-of-province, field M. hyopneumoniae isolates.

The p146 PCR-sequencing had specificity and sensitivity similar to our current qPCR and can be used to perform PCR sequence typing directly from field samples without culture. About 190-210 nucleotide sequences were determined for each field strain. Using the standard of 4 nucleotide substitutions (~ 2% nucleotide substitution) as a new strain type, the 113 field strains identified at the AHL from 2009 to 2017 can be grouped into 11 p164 sequence types; most differed from strains described in Switzerland and France.

Most of the strains identified at the AHL in 2017 belong to a large cluster, indicating that the field strains might have been under selective pressure to become more homogeneous (Fig. 1).

Some strains from the same geographic location (based on postal code) and from different years had the same sequence types, indicating that the same strain types can circulate persistently in the field (Fig. 1). In contrast, different subtypes were also identified from similar geographic locations, indicating that a strain of the same subtype can spread widely (Fig. 1). Strains from lung tissue or oral fluid did not form distinct clusters, indicating no difference between these sample sources.

Our study indicated that the p146 PCR-sequencing is a useful typing method for Ontario M. hyopneumoniae strains, although its value in epidemiologic analysis needs further evaluation.

The AHL is offering this typing service for half-price ($40/sample) until May 1, 2018. AHL

Reference   Mayor D, et al. Diversity of Mycoplasma hyopneumoniae in pig farms revealed by direct molecular typing of clinical material. Vet Res 2007;38:391-398.

Figure 1. Phylogenetic tree based on the serine repeat encoding region of the p146 gene of 113 M. hyopneumoniae field strains identified at the AHL. Strain names—first part is the year of isolation and case number; second part is postal code of the location origin of the samples; third part is the tissue origin (empty for lung tissue).