Development and validation of an Ehrlichia canis real-time PCR assay

Ehrlichiosis is caused by a pleomorphic gram-negative bacteria and is an important zoonotic tick-borne disease, with a potential to be fatal. This bacterium occurs worldwide and species affected by it include humans, domestic and wild animals. Canine monocytic ehrlichiosis develops over a period of about 8-20 days and may progress from an acute phase to subclinical and chronic disease stages. Although direct detection of the bacterial antigen by ELISA has been used successfully to diagnose the disease, a challenge remains in dogs where co-infection of infectious agents is common due to pathogens being transmitted by the same vectors. Cross-reactivity of the serology assays makes it difficult to make species-specific diagnoses. A more sensitive and reliable molecular technique that can detect and identify pathogens at species level is needed to enhance disease diagnosis. In this study, we developed a real-time PCR assay that employs group-specific primers and an Ehrlichia canis TaqMan® minor groove binder probe. The group-specific primers targeted the conserved region of the 16S rRNA gene and the forward primer included redundant base pairs to accommodate other species in this genus. The primer and probe concentrations were optimised to 200 nM and 250 nM respectively. The efficiency of the assay was 93%. The assay was E. canis specific when tested against other canine and livestock pathogens, as no cross-reactivity was observed. The 95% limit of detection was 33 E. canis plasmid copies/µl of blood (95% confidence interval: 23 - 58). Consistent repeatability was observed, where the inter-run standard deviation (SD) ranged between 0.33 - 1.29 and the intra-run SD 0.04 - 1.14. The results for Reverse Line Blot (RLB) hybridization assay and the TaqMan® MGB real-time PCR assay results were compared and found to be in agreement with an exception of three samples out of 121. Diagnostic validation was performed on field samples, the sensitivity of the TaqMan® MGB real-time PCR assay was 90% and the specificity was 92%. This assay will be a useful tool for the early diagnosis of E. canis and this will aid in timely treatment.