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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.

SUBMITTER: Scherrer S

PROVIDER: S-EPMC9323117 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three-reaction high-resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High-resolution melting assay 1 is described in a previously published report. High-resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High-resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three-reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.

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

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