A real-time PCR assay for the quantification ofin tracheal wash samples from Thoroughbred horses.

Abstract

is the predominant equinesp. 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 ofgiven the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of, targeting the hypothetical protein FJM08_00025. We confirmed its high species-specificity forand a limit of detection of 2.9 copies/reaction. We quantifiedin 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 ofinfection. Quantitative evaluation ofover time using our qPCR assay may allow a more accurate assessment ofinfection in coughing horses compared to culture methods.