Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays

Abstract

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

Molecular Characterization and Phylogenetic Analysis of Outer membrane protein P2 (OmpP2) of Glaesserella (Haemophilus) parasuis Isolates in Central State of Peninsular Malaysia

“…The detection of virulent vtaA in Malaysia isolates belonging to OmpP2 genetic type I, and nonvirulent vtaA in isolates belonging to OmpP2 genetic type II, seems to be parallel. However, mapping information from previous literature [ 41 ] showed that strains from genetic type II, such as strain H465 and SW124 of Germany, also possess virulent vtaA , even though all strains in the genetic type I were found to possess virulent vtaA . This may indicate that the two genes are not directly related, but when combined, both may yield more severe clinical infections; this is worth being studied further.…”

Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae

“… a Serovars determined by multiplex PCR (Howell et al, 2015 ) and high‐resolution melting PCR assay (Scherrer et al, 2022 ). b Virulence determined by leader sequence PCR (Galofré‐Milà et al, 2017 ).…”

“… a Serovars determined by multiplex PCR (Howell et al, 2015 ) and high‐resolution melting PCR assay (Scherrer et al, 2022 ). …”

“…All directly extracted DNA samples were tested with the novel multiplex qPCR and compared to the results of culturing. Additionally, an HRM assay for serotyping G. parasuis ‐positive samples (Scherrer et al, 2022 ) and a qPCR detecting M. hyorhinis and M. hyosynoviae (Gomes Neto et al, 2015 ) have been performed (Table A1 ).…”

“…The LS‐PCR was confirmed to be useful for virulence prediction also for global G. parasuis isolates (Macedo et al, 2021 ; Schuwerk et al, 2020 ). Alternatively, a high‐resolution melting assay was developed recently, aiming at serotyping G. parasuis and at the same time targeting vtaA for the prediction of potentially virulent strains (Scherrer et al, 2022 ).…”