Study of the population structure of Haemophilus parasuis by multilocus sequence typing.

Haemophilus parasuis is the aetiological agent of Glässer's disease in swine. In addition, this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they had resolution or implementation problems. To overcome these limitations, a multilocus sequence typing (MLST) system, using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB, was developed. Eleven reference strains and 120 field strains were included in this study. The number of alleles per locus ranged from 14 to 41, 6pgd being the locus with the highest diversity. The high genetic heterogeneity of this bacterium was confirmed with MLST, since the strains were divided into 109 sequence types, and only 13 small clonal complexes were detected by the Burst algorithm. Further analysis by unweighted-pair group method with arithmetic mean (UPGMA) identified six clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation (putative non-virulent), while another cluster showed a significant association with isolation from clinical lesions (putative virulent). The remaining clusters did not show a statistical association with the clinical background of the isolates. Finally, although recombination among H. parasuis strains was detected, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. Interestingly, one branch included almost all isolates of the putative virulent UPGMA cluster.