A real-time PCR assay for the quantification of Mycoplasma equirhinis in tracheal wash samples from Thoroughbred horses

Abstract

Mycoplasma equirhinis is the predominant equine Mycoplasma sp. isolated from clinically normal horses and is suspected to be associated with inflammatory airway disease in which cough is the primary sign. Quantitative evaluation of bacterial counts is useful in assessing the association between the bacteria in samples and observed clinical signs, but this evaluation has been difficult with conventional culture methods of M. equirhinis given the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of M. equirhinis, targeting the hypothetical protein FJM08_00025. We confirmed its high species-specificity for M. equirhinis and a limit of detection of 2.9 copies/reaction. We quantified M. equirhinis in tracheal wash samples from 20 clinically normal horses and 22 coughing horses. The copy numbers detected by qPCR in 18 of the 22 samples from clinically affected horses were within the range detected in the 20 clinically normal horses (0-84 copies/reaction). The remaining 4 samples had considerably higher copy numbers (734-1,620,000 copies/reaction), suggesting the likely involvement of M. equirhinis infection. Quantitative evaluation of M. equirhinis over time using our qPCR assay may allow a more accurate assessment of M. equirhinis infection in coughing horses compared to culture methods.

Keywords: Mycoplasma equirhinis; horses; quantitative PCR assay.

Figure 1.

Boxplot and scatterplot comparison of copy numbers of Mycoplasma equirhinis detected in tracheal washes from clinically normal and affected horses. Box plots show the interquartile range (IQR; i.e., the bottom of the box indicates the 25th percentile, a black line inside the box indicates the median, and the top of the box indicates the 75th percentile). The whisker extending above the box indicates a range of the 3rd quartile + 1.5 × IQR. Each scatterplot represents a single sample. Samples that fall within the range of clinically normal horses (≤ 84 copies/reaction) are shown as open circles, and those above are shown as black triangles.