Novel primers for the detection and differentiation of S. zooepidemicus and S. equi by PCR

Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) is a commensal opportunistic bacterium associated with outbreaks of equine respiratory disease alongside a diverse range of diseases in various species. The closely related Streptococcus equi subspecies equi (S. equi) is the causative agent of 'strangles', the most frequently diagnosed contagious equine disease. Despite differing clinical signs, the two subspecies share approximately 97 % DNA homology and respectively present serious equine health and welfare concerns. Currently there are few PCR assays targeting unique regions of the S.zooepidemicus genome that facilitate testing independently from S.equi. This study aimed to design a commercially applicable qPCR assay that can differentiate between the two bacterial subspecies. By utilising the species-specific sodA, seeH and lacD genes unique to S. zooepidemicus and S. equi respectively, a rapid, multiplex qPCR was developed. 300 clinical samples positive for S. equi (n = 100), S. zooepidemicus (n = 100) and tracheal wash samples negative for both S. equi and S. zooepidemicus (n = 100) were screened using the validated assay and compared with current diagnostic techniques. The assay was able to differentiate between S. zooepidemicus and S. equi demonstrating assay specificity of 98.0 %. The lowest limit of detection was determined to be between 10 and 100 copies per μL for each target ensuring the identification of low-level infections that risk being missed via culture. The assay provides a rapid, reliable and economical solution to the identification and differentiation of both S. zooepidemicus and S. equi which could offer a more sensitive and time efficient alternative to current techniques.