Use of quantitative real-time PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles.

Abstract: In recent years, molecular approaches have been able to characterise the viability of equine upper respiratory tract pathogens using absolute molecular quantitation as well as detection of transcripts for virulence genes. Objective: The objective of this study was to investigate molecular surrogates for S. equi subspecies equi (S. equi) viability in biological samples from horses with strangles. Methods: Retrospective cross-sectional study. Methods: S. equi culture-positive and culture-negative upper airway secretions were assessed by qPCR at the genomic (gDNA) and complimentary DNA (cDNA) level for various target genes (SeM, SEQ2190, eqbE and szpSe). Absolute quantitation was performed using standard curves, and the results were expressed as number of S. equi target genes per μl of gDNA or cDNA. Additionally, the presence or absence of S. equi gene expression for the various target genes was assessed and compared with the culture results. Results: While all 21 culture-positive samples tested S. equiqPCR positive, up to 43.7 and 18.9% of 64 culture-negative samples tested qPCR positive at the gDNA and cDNA level, respectively. Significant differences in absolute quantitation for S. equi at the gDNA level were found between culture-positive and culture-negative samples. When absolute quantitation of S. equi target genes at the gDNA level was assessed with the presence or absence of transcripts, there was a significantly higher S. equi target gene number in samples with expression of transcripts compared with samples with no expression of transcripts. Conclusions: The lack of standardisation of samples collected in the field and the delay from sample collection to samples processing may have negatively affected the cultivability of S. equi and mRNA quality. Conclusions: Molecular viability for S. equi can be investigated by determining absolute quantitation and/or by detecting mRNA for specific target genes. However, veterinarians have to be cautioned that any qPCR-positive result for S. equi needs to be taken seriously and trigger biosecurity protocols aimed at reducing spread.