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. 2025 Jun 27:12:1605316.
doi: 10.3389/fvets.2025.1605316. eCollection 2025.

Novel multiplex PCR approach for the detection of Erysipelothrix rhusiopathiae, Streptococcus suis, and Staphylococcus hyicus in swine

A Arun Prince Milton #  1 , Sabia Khan  1 , Kandhan Srinivas #  1 , Torik Basar  1 , Zakir Hussain  1 , G Bhuvana Priya  2 , M Saminathan  3 , L H Puii  1 , Sandeep Ghatak  1 , Samir Das  1
Affiliations

Novel multiplex PCR approach for the detection of Erysipelothrix rhusiopathiae, Streptococcus suis, and Staphylococcus hyicus in swine

A Arun Prince Milton et al. Front Vet Sci. .

Abstract

Erysipelothrix rhusiopathiae, Streptococcus suis, and Staphylococcus hyicus are neglected, emerging and zoonotic pathogens in pigs that share common clinical signs and lesions, particularly septicaemia and skin lesions, highlighting the need for differential diagnosis. Currently, no single diagnostic assay is available to test samples for confirmation of the presence of all three pathogens. To address this, a novel multiplex PCR-based detection assay was optimized and evaluated for the simultaneous detection of E. rhusiopathiae, S. suis, and S. hyicus targeting the, 23S rRNA, recN, and sodA genes, respectively. The optimized multiplex PCR demonstrated 100% analytical specificity, with a detection limit of 104 copies/μL for E. rhusiopathiae and S. hyicus, and 106 copies/μL for S. suis. The assay was further validated by comparison with previously established uniplex PCR assays and tested on field-collected pig blood samples to assess real-world applicability. The developed assay is rapid, specific and cost-effective and can serve as a handy diagnostic tool for testing suspect samples for these neglected bacterial pathogens of pigs.

Keywords: Erysipelothrix rhusiopathiae; Staphylococcus hyicus; Streptococcus suis; multiplex PCR; pigs.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Gradient PCR analysis. (A) E. rhusiopathiae 399 bp; (B) S. suis 336 bp; (C) S. hyicus 205 bp (M: 100 bp DNA Marker (Thermo Fisher Scientific); NTC, No Template Control).
Figure 2
Figure 2
Multiplex PCR assay revealing simultaneous amplification of E. rhusiopathiae 399 bp; B. S. suis 336 bp; C. S. hyicus 205 bp (M: 100 bp DNA Marker (Thermo Fisher Scientific); NTC: No Template Control).
Figure 3
Figure 3
Analytical specificity analysis of multiplex PCR showing amplification only in E. rhusiopathiae, S. suis and S. hyicus DNA (20 ng) (M: 100 bp DNA Marker (Thermo Fisher Scientific); Lane 1: Erysipelothrix rhusiopathiae ATCC 35456, Lane 2: Streptococcus suis ATCC BAA-853, Lane 3: Staphylococcus hyicus VTCC BAA-60, Lane 4: Staphylococcus aureus ATCC 25923, Lane 5: Staphylococcus xylosus ATCC 29971, Lane 6: Staphylococcus sciuri ATCC 29061, Lane 7: Streptococcus equisimilis ATCC 12388, Lane 8: Streptococcus agalactiae ATCC 13813, Lane 9: Streptococcus bovis ATCC 33317, Lane 10: Campylobacter coli ATCC 33559, Lane 11: Brucella abortus VTCC BAA 465, Lane 12: Clostridium perfringens ATCC 13124, Lane 13: Escherichia coli ATCC 25922, Lane 14: Salmonella Choleraesuis ATCC 10708, and Lane 15: Yersinia enterocolitica ATCC 23715).
Figure 4
Figure 4
Analytical sensitivity. (A) Multiplex PCR; (B) Uniplex PCR of E. rhusiopathiae; (C) Uniplex PCR of S. suis; (D) Uniplex PCR of S. hyicus (M: 100 bp DNA Marker (Thermo Fisher Scientific); Lane 1: 109 copies/μL; Lane 2: 109 copies/μL; Lane 3: 109 copies/μL; Lane 4: 109 copies/μL; Lane 5: 109 copies/μL; Lane 6: 109 copies/μL; Lane 7: 109 copies/μL; NTC: No Template Control).
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References

    1. Van der Waal K, Deen J. Global trends in infectious diseases of swine. Proc Natl Acad Sci U S A. (2018) 115:11495–500. doi: 10.1073/pnas.1806068115, PMID: - DOI - PMC - PubMed
    1. Albernaz-Goncalves R, Antillon GO, Hotzel MJ. Linking animal welfare and antibiotic use in pig farming – a review. Animals. (2022) 12:216. doi: 10.3390/ani12020216, PMID: - DOI - PMC - PubMed
    1. Giles TA, Belkhiri A, Barrow PA, Foster N. Molecular approaches to the diagnosis and monitoring of production diseases in pigs. Res Vet Sci. (2017) 114:266–72. doi: 10.1016/j.rvsc.2017.05.016, PMID: - DOI - PMC - PubMed
    1. Rajkhowa S, Sonowal J, Borthakur U, Pegu SR, Deb R, Das PJ, et al. Meta-analysis of the prevalence of porcine zoonotic bacterial pathogens in India: a 13-year (2010-2023) study. Pathogens. (2023) 12:1266. doi: 10.3390/pathogens12101266, PMID: - DOI - PMC - PubMed
    1. Haldar A, Das D, Saha B, Pal P, Das S, Das A, et al. Smallholder pig farming for rural livelihoods and food security in north East India. J Anim Res. (2017) 7:471–81. doi: 10.5958/2277-940X.2017.00070.5 - DOI

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