Investigation of a 24-Hour Culture Step to Determine the Viability of Streptococcus equi Subspecies equi Via Quantitative Polymerase Chain Reaction in Nasal Secretions From Horses With Suspected Strangles.

The research study investigates the usage of the quantitative Polymerase chain reaction (PCR) technique, combined with a 24-hour culture step, in evaluating the viability of Streptococcus equi bacteria in horses. The technique attempts to improve the accuracy of identifying strains of this bacteria that are alive through counting of particular gene materials (mRNA transcripts and number of SeM target genes) before and after being cultured.

Objective and Methodology

• The study aimed to investigate a technique to more accurately determine the presence of viable (alive and capable of causing disease) Streptococcus equi subspecies equi bacteria in nasal secretions from horses.
• The traditional PCR detection methods are known to overestimate the prevalence of samples containing live organisms since they only identify the presence of the bacteria DNA without distinguishing between live and dead bacteria.
• This study sought to see if the viability of the bacteria could be determined by quantifying its genome and detecting specific mRNA transcripts for the SeM gene in both pre- and post-cultured samples.
• Nasal secretions were collected from 42 horses suspected to have strangles, a highly contagious equine disease caused by the S. equi bacteria. The secretions were analyzed through culturing and through qPCR, both before and 24 hours after the culture process.

Findings

• The researchers successfully determined the viability of S. equi in 28 out of 42 samples.
• For the 28 positive samples, viable S. equi was identified through detection via culture (11 samples), presence of mRNA transcripts for the SeM gene (25 samples), and/or an increase in the absolute number of SeM target genes between pre- and post-existent samples (17 samples).
• The overall agreement between culture alone (traditional method of detecting bacteria) and the three factors used to determine viability (culture, mRNA, and gene count) stood at 59%.
• The overall agreement for detecting mRNA transcripts and increase in absolute target genes was significantly higher, at 88% and 74% respectively.
• The combination of mRNA transcripts count and the increase in absolute target genes was able to determine the viability status in all 42 samples.

Conclusion

• Applying molecular strategies such as mRNA transcription detection and absolute target gene counting to samples undergoing a 24-hour culture step can successfully determine the viability of S. equi, especially in instances where the culture alone does not yield positive results.
• The new method adds an extra layer of precision that can improve the estimation of live S. equi, consequently enhancing diagnostic accuracy and disease control strategies.