A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis

Abstract

Background Haemophilus parasuis is the etiological agent of Glässer’s disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and improved PCR designed to detect a different domain of the vtaA genes, the leader sequence (LS) as a diagnostic tool to predict virulence.MethodsA collection of 360 H. parasuis strains was tested by PCR with LS specific primers. Results of the PCR were compared with the clinical origin of the strains and, for a subset of strains, with their phagocytosis and serum resistance using a Chi-square test.ResultsLS-PCR was specific to H. parasuis, and allowed the differential detection of the leader sequences found in clinical and non-clinical isolates. Significant correlation was observed between the results of the LS-PCR and the clinical origin (organ of isolation) of the strains, as well as with their phagocytosis and serum susceptibility, indicating that this PCR is a good predictor of the virulence of the strains. In addition, this new PCR showed a full correlation with the previously validated PCR based on the translocator domain. LS-PCR could be performed in a wide range of annealing temperatures without losing specificity.ConclusionThis newly described PCR based on the leader sequence of the vtaA genes, LS-PCR, is a robust test for the prediction of the virulence potential of H. parasuis strains.

Virulence‐associated gene profiling, DNA fingerprinting and multilocus sequence typing of Haemophilus parasuis isolates in Australia

“…However, this new PCR still needs further study because only a limited number of non‐pathogenic strains were examined in detail. As well, the non‐pathogenic reference strains for serovars 7 and 11 were apparently virulent in this assay, with one of them being sensitive and one resistant, respectively, in a phagocytosis assay …”

“…A UK study that involved whole‐genome sequences of 212 isolates revealed that the group 1 vtaA translocator sequence was found in all isolates; however, there was a difference in the leader sequence of the pathogenic and non‐pathogenic strains . Recently, a PCR was developed for the recognition of potentially virulent strains using a translocator sequence of vtaA . Essentially, those authors argued that VtaA from group 1 does play a role in virulence but that the conditions of a PCR to detect the gene were vital to specificity.…”

“…Essentially, those authors argued that VtaA from group 1 does play a role in virulence but that the conditions of a PCR to detect the gene were vital to specificity. Hence, the need for a more robust PCR . The study suggested that previously positive results for non‐pathogenic strains were related to the conditions of the PCR not being exactly followed, as some non‐pathogenic strains have a very large trimeric autotransporter whose translocator sequence is characteristic of group 1 vtaA .…”

“…Hence, the need for a more robust PCR . The study suggested that previously positive results for non‐pathogenic strains were related to the conditions of the PCR not being exactly followed, as some non‐pathogenic strains have a very large trimeric autotransporter whose translocator sequence is characteristic of group 1 vtaA . However, this new PCR still needs further study because only a limited number of non‐pathogenic strains were examined in detail.…”

“Pathotyping” Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence

“…However, serovar is regarded as a poor proxy for virulence, with the exception of serovars 4 and 5 (12)(13)(14). The vtaA multiplex PCR (mPCR) (15)(16)(17) is the only method that has shown an association between the test result for a given isolate and its virulence, but its wide-scale use has not been reported.…”