Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies

Abstract

Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia 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 Anaplasma/Ehrlichia 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). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada.

Current and Future Molecular Diagnostics of Tick-Borne Diseases in Cattle

“…In many endemic areas, nested PCR’s have been used for the diagnosis of carrier cases of anaplasmosis [ 48 , 49 ]. A multiplex PCR followed by the magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichhia species has been recently used in cattle [ 50 ].…”

“…pathogens in canine, bovine, caprine and ovine blood samples. The assay uses a16S rRNA gene-based real time quantitative PCR assay combined with Luminex xMAP hybridization technology [ 50 ]. Another PCR-based multiplex assay using heat shock protein ( gro EL), the citrate synthase gene ( glt A) and the 18s rDNA gene for Anaplasma , Babesia and Theileria spp.…”

Ticks and Tick-Borne Diseases in Central America and the Caribbean: A One Health Perspective

“…Co-infections due to TBPs are well-characterized in the Caribbean region, notably in dogs. For example, a recent study in Grenada analyzed 455 blood samples from dogs (n = 358), sheep and goats (n = 65), and cattle (n = 32) using a new multiplex real-time PCR [154]. Although co-infections were not detected in ruminants, 1.96% (7/358) dog samples were co-infected with two pathogens of the family Anaplasmataceae (i.e., E. canis and Ehrlichia chaffeensis in two dogs, E. canis and A. platys in four dogs, and A. platys and Ehrlichia ewingii in one dog).…”

“…To overcome the aforementioned drawbacks, a variety of molecular methods have been employed in the CAC region for the surveillance of TBPs. These include conventional PCR (cPCR), nested PCR (nPCR), reverse line blot (RLB), quantitative fluorescence resonance energy transfer (FRET) PCR, multiplex TaqMan ® real-time PCR, and multiplex PCR coupled with oligonucleotide probe based multi-analyte profiling bead (xMAP) [29,62,154,165]. Although molecular assays employed for the detection of TBPs have yielded higher sensitivities and specificities than serological detection methods, the main disadvantage of these approaches is that a limited number of pathogens can be tested simultaneously.…”

Completing the Puzzle: A Cluster of Hunting Dogs with Tick-Borne Illness from a Fishing Community in Tobago, West Indies

“…Tick infestations and TBDs of the canine population have been reported throughout the Caribbean [ 6 , 22 , 33 , 34 , 35 , 36 , 37 ]. However, very little or no reported data is available for some islands, including Tobago, the smaller of the two islands that comprise the Republic of Trinidad and Tobago.…”