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

Abstract

Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the "current standard" of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.

Molecular serotyping ofHaemophilus parasuisisolated from diseased pigs and the relationship between serovars and pathological patterns in Taiwan

“…Previous studies established the serovar and pathotype of H. parasuis are based on differences at the genome level ( Brockmeier et al, 2014 ; Howell et al, 2013 , 2017 ). A multiplex PCR (mPCR) based on a polysaccharide biosynthesis locus analysis was employed to molecularly serotype H. parasuis serovars ( Howell et al, 2015 ).…”

Association betweeniscR-based phylogeny, serovars and potential virulence markers ofHaemophilus parasuis

“…To further confirm the population structure and its relevance with clinical phenotype, 12 PVMs from five pieces of literatures were selected. Primers of one of the selected PVMs ( hsdS ) were newly designed in this study (Table 1), and primers of other selected PVMs are the same with that in the literatures (Sack & Baltes, 2009; Wang et al, 2011; Yu et al, 2014; Howell et al, 2017; Galofre-Mila et al, 2017). The presence or absence of the selected PVMs in all the isolates were determined by PCR.…”

“…An increasing number of potential virulence markers (PVMs) have been identified for H. parasuis , of which some were already used for development of molecular methods for virulence prediction (Sack & Baltes, 2009; Zhou et al, 2010; Wang et al, 2011; Yu et al, 2014; Galofre-Mila et al, 2017; Howell et al, 2017). As clinical data on these PVMs are still limited, its clinical relevance needs to be further confirmed.…”

Peer Review #2 of "Association between iscR-based phylogeny, serovars and potential virulence markers of Haemophilus parasuis (v0.1)"

“…To further confirm the population structure and its relevance with clinical phenotype, twelve PVMs from 5 pieces of literatures were selected. Primers of one of the selected PVMs (hsdS) were newly designed in this study (Table 1), and primers of other selected PVMs are the same with that in the literatures (Sack and Baltes, 2009;Wang et al, 2011;Yu et al, 2014;Howell et al, 2017;Galofre-Mila et al, 2017). The presence or absence of the selected PVMs in all the isolates were determined by PCR.…”

“…An increasing number of potential virulence markers (PVMs) have been identified for H.parasuis, of which some were already used for development of molecular methods for virulence prediction (Sack and Baltes, 2009;Zhou et al, 2010;Wang et al, 2011;Yu et al, 2014;Galofre-Mila et al, 2017;Howell et al, 2017). As clinical data on these PVMs are still limited, its clinical relevance needs to be further confirmed.…”