Evaluation of a newly developed rapid ELISA to detect anti-

Canine monocytic ehrlichiosis (CME) is a globally prevalent disease with zoonotic potential primarily caused by the obligate intracellular bacterium Ehrlichia canis, transmitted by the ticks Rhipicephalus spp. to vertebrate hosts. In dogs, CME affects immune system cells, leading to subclinical infection or acute and chronic disease forms that impact multiple organs and potentially result in death if diagnosis is delayed. Diagnosis of canine ehrlichiosis is complex, typically involving cytology, polymerase chain reaction), and serological assays such as the indirect immunofluorescence antibody test (IFAT), considered the gold standard, and enzyme-linked immunosorbent assay (ELISA). Herein, we introduce EhrlichiaCHECK Ab ELISA, a novel rapid indirect ELISA for detecting anti-E. canis antibodies in canine serum or plasma samples. The assay’s performance was validated using 112 canine samples (61 negative, 51 positive). Compared to IFAT, the ELISA exhibited high sensitivity (96.1%, 95% confidence interval [CI]: 85.4%–99.3%) and specificity (95.1%, 95% CI: 85.4%–98.7%). Furthermore, in comparison to the widely used commercial INgezim Ehrlichia ELISA (Gold Standard Diagnostics), EhrlichiaCHECK Ab ELISA demonstrated 98.0% agreement and enhanced specificity attributed to a more specific antigen, corroborated by bioinformatics analysis. The assay also demonstrated accuracy, with low percentage values of intra- and inter-assay coefficients of variation. In conclusion, our data suggest that the newly developed assay is a valuable tool for diagnosing E. canis infections in dogs.