Use of a real-time polymerase chain reaction-based fluorogenic 5′ nuclease assay to evaluate insect vectors of Corynebacterium pseudotuberculosis infections in horses

Objective-To develop and use a sensitive molecular assay for detecting The phospholipase D (PLD) exotoxin gene of Corynebacterium pseudo tuberculosis in an attempt to identify insect vectors that may be important in transmission of clinical disease in horses. Sample Population-2,621 flies of various species. Procedure-A real-time polymerase chain reaction (PCR)-based fluorogenic 5' nuclease (TacqMan) system (ie, TaqMan PCR assay) was developed for the detection of the PLD gene in insects. Flies were collected monthly (May to November 2002) from 5 farms in northern California where C pseudo tuberculosis infection in horses is endemic. Three of the 5 farms (which housed a total of 358 horses) had diseased horses during the study period. A total of 2,621 flies of various species were tested for the PLD gene of C pseudotuberculosis. Results-Evidence of bacterial DNA for the PLD gene was detected in skin biopsy specimens from clinically affected horses and from 3 fly species collected from farms where affected horses were housed. Farms with a high incidence of diseased horses had a high proportion of insects carrying the organism. High percentages of flies with positive results for the PLD gene were observed in October when most clinically affected horses were observed. Conclusions and Clinical Relevance-Our results are consistent with the hypothesis that C pseudotuberculosis may be vectored to horses by flies. Three potential vectors were identified, including Haematobia irritans, Stomoxys calcitrans, and Musca domestica. The organism can be identified in up to 20% of house flies (Musca domestica) in the vicinity of diseased horses.