Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.

A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.