Thirty-eight M. hyorhinis isolates obtained in 2010-2011 from disease pigs, in addition to one reference strain, were utilized in this study. These isolates originated from 11 states, 18 systems, three pig stages and different lesion sites within the pigs. Isolates were cultured in Modified Hayflick’s media for 7-14 days followed by DNA extraction. The genome sequences of four M. hyorhinis isolates were utilized to identify potential target genes. The classical MLST scheme was modified to target not only housekeeping genes (slowly accumulate nucleotide changes) but also hyper-variable genes (accumulate changes over a short period of time). This modified schemed would potentially allow us to increase the discriminatory power, making this assay highly useful for outbreak investigations. After selection of the target genes, primers were designed and PCR amplification was carried out. PCR products were purified and sequenced. The data obtained was checked for quality and trimmed to the appropriate length. Sequences from all isolates within each gene were aligned and dendrograms were constructed from individual genes, as well as, the concatenated sequence of all genes. Within each locus (gene) distinct alleles where assigned arbitrary allele numbers. For each isolate, the sequence type (ST) was defined by the combination of the allele number at each of the loci.
More than 25 genes where evaluated as potential gene targets with varying degrees of success. The final MLST protocol included the following genes: ung, pdhB, mtlD, p3, p95. Variation within each gene range from 0.5-20%. The number of alleles per gene varied from 3-11, giving rise to 27 sequence types (STs) within the 39 isolates. The dendrogram constructed based upon concatenated gene sequences revealed genetic variation among the examined isolates, with the greatest similarities belonging from isolates of the same owner/system. Two major lineages were observed; A and B, where lineage B had the majority of the isolates examined and all of the ones from MN. The location of isolation within the pig did not correlate with the dendrogram, however, in lineage A the majority of isolates where cultured from pleura. Isolates belonging to the same system clustered together, and in some instances isolates from different geographic location, but same system where identical. In contrast, three isolates from different systems, states and lesion type had a 100% sequence similarity. Both of these cases suggest a common source of pigs.
Albert Rovira, DVM, MS, PhD
Veterinary Diagnostic Laboratory
University of Minnesota
Tel: 612 625 7702