Development and validation of a TaqMan® probe- based real-time PCR assay for detection of Ehrlichia canis

Abstract

Ehrlichiosis is a potentially fatal zoonotic tick-borne disease, caused by a pleomorphic Gram-negative bacterium. It occurs worldwide and affects humans, domestic and wild animals. Dogs infected with Ehrlichia canis develop canine monocytic ehrlichiosis (CME), a significant infectious disease of canines. TaqMan® based real-time PCR assays to detect Ehrlichia spp. affecting dogs were developed and a real-time PCR assay specific for E. canis validated. The efficiency of the assay was 93% and the 95% limit of detection was 33 E. canis plasmid copies/µl of blood (95% confidence interval: 23 - 58). The assay was specific for E. canis when tested against other haemoparasites. Consistent repeatability was observed, with an inter-run standard deviation (SD) range between 0.33 and 1.29 and an intra-run SD range between 0.04 and 1.14. Field samples were tested in parallel by both the E. canis real-time PCR assay and a reverse line blot hybridization assay. The results were in agreement for the two assays, with an exception of two out of 121 samples. Bayesian latent class analysis was used to calculate a diagnostic sensitivity of the E. canis real-time PCR assay of 90% and a specificity of 92%. This assay is a sensitive and reliable molecular detection method for E. canis and will be a useful tool for early diagnosis and timely treatment for this haemoparasite.