Strangles: The Molecular Identification and Epidemiology of Streptococcus equi subsp. equi

Abstract

A conventional PCR diagnostic test was established to confirm the microbiological isolation of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles. This test was based on the amplification of the seeI gene, which is species-specific for S. equi. Further, a multiplex PCR was developed using species-specific primers; to identify the presence of S. equi and two other streptococci know complicate the diagnosis of strangles in horses, Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis). A total of 18 clinical isolates of S. equi plus the Pinnacle IN vaccine isolate, two isolates of S. zooepidemicus and four isolates of S. equisimilis were obtained via culture and used in the development of the multiplex diagnostic tool. Two multiplex tests were trialed; a conventional multiplex PCR and a real-time multiplex PCR.

Both the conventional and real-time multiplex PCR’s were able to distinguish between the streptococci and accurately identified all isolates. However, further testing on 26 field specimens revealed that the real-time multiplex PCR had lower specificity, sensitivity and diagnostic accuracy as compared to the conventional multiplex PCR. This was theorised to be the result of the PEG/KOH solution used in the DNA extraction, possibly interfering with the intercalating dye in the real-time reaction. Based on these preliminary results, the conventional multiplex PCR diagnostic test developed here is recommended for further trials to determine its robustness.

The 19 S. equi isolates obtained, including the vaccine, were further subjected to epidemiological studies. These included sequencing of the variable N-terminal region of the antiphagocytic M-protein SeM to determine SeM allele subtypes and a Sau-PCR amplification method, which previously has not been trialled on S. equi isolates. Sau-PCR involves digestion of genomic DNA and subsequent amplification. Two novel strains of S. equi were found within NZ based on the variable region of the seM gene, SeM alleles 99 and 100. SeM allele 100 had a higher pervalance over allele 99 as it was isolated in 6 out of 9 outbreaks and was found to occur on both the North and South Islands of New Zealand. SeM allele 99 was only found to occur on the North Island. Further to this study, the Pinnacle IN vaccine strain, SeM 2 was isolated from lymph node abcesses of two horses. It was unclear as to whether this ‘vaccine breakdown’ was just a severe adverse reaction to the vaccine or if the vaccine reverted to a more virulent type. The Sau-PCR was able to differentiate between the field isolates of S. equi and the vaccine strain but was unable to further differentiate between the field isolates and was therefore determined not as valuable for S. equi epidemiological studies.