| Summary: | Infections with tick-borne pathogens belonging to <i>Anaplasma/Ehrlichia</i> in various vertebrate hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis of single and multi-infections with these pathogens, causing diseases in companion/agricultural animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely used to detect early-phase infections, since these have high sensitivity and specificity. We report the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six <i>Anaplasma/Ehrlichia</i> species pathogens in canine, bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% (7/137). <i>Anaplasma marginale</i> infection in cattle had the highest detection rate (34.4%), followed by canines positive for <i>Anaplasma platys</i> (16.4%) and <i>Ehrlichia canis</i> (13.9%). The assay aided in documenting the first molecular evidence for <i>A. marginale</i> in cattle and small ruminants and <i>Ehrlichia chaffeensis</i> and <i>Ehrlichia ewingii</i> in dogs in the Caribbean island of Grenada.
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