Rickettsial infection in dog-associated ticks in three rural communities of Yucatan, Mexico was investigated using qPCR and nested PCR assays. A total of 319 dogs were studied and ticks samples were collected. A total of 170 dogs were infested with ticks (frequency of 53.4%). Overall, 1,380 ticks representing seven species were collected: Amblyomma mixtum, A. ovale, A. parvum, A. cf. oblongoguttatum, Ixodes affinis, Rhipicephalus microplus, and R. sanguineus sensu lato. The most abundant species was R. sanguineus s.l. with a mean intensity of 7.4 ticks/host. Dogs in the communities of Chan San Antonio and Yaxcheku were 2.84 and 2.41 times more likely to be infected with R. sanguineus compared with Sucopo (p < 0.05). Adult pools of A. mixtum, A. parvum, I. affinis, R. microplus, and A. c.f. oblongoguttatum were negative to E. chaffeensis, E. ewingii, A. phagocytophilum, and R. rickettsii. However, pools of R. sanguineus s.l. adults and A. ovale adults, as well as nymphs of Amblyomma spp. were positive to E. canis. Sequencing analysis of the nested PCR products amplifying the 16S rRNA gene fragment of E. canis confirmed the results and revealed 100% identity with sequences of E. canis. This is the first report worldwide of E. canis infection in A. ovale by PCR. This finding does not necessarily indicate that A. ovale is a competent vector of E. canis because pathogen transmission of this specific tick to a naïve dog remains to be documented. This study documented that different tick species parasitize dogs in Yucatan, Mexico, where R. sanguineus s.l., A. ovale, and nymphs of Amblyomma spp. were shown to be infected with E. canis. These findings highlight the need for control strategies against tick infestations in dogs to prevent the risk of tick-borne disease transmission among companion animal and probably human populations.

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Summary Tick‐borne diseases (TBD), caused by borrelial, rickettsial and babesial pathogens, are common across the United States and can cause severe clinical disease in susceptible hosts, such as domestic dogs. However, there are limited TBD molecular epidemiological reports for dogs in Texas, and none for the non‐Lyme borrelial pathogen responsible for causing tick‐borne relapsing fever (TBRF). Therefore, data to support the prevalence of TBRF in the canine population is inadequate. This study aimed to characterize the molecular prevalence of 11 causative agents responsible for three TBD groups within domestic dogs with an emphasis on pathogen distribution within Texas ecoregions. A total representative number of 1,171 whole‐blood samples were collected opportunistically from two Texas veterinary diagnostic laboratories. A layerplex real‐time PCR assay was utilized to screen the dog samples for all 11 pathogens simultaneously. The overall molecular infection prevalence of disease was 0.68% borrelial, 1.8% rickettsial and 0.43% babesial pathogens, for a TBD total of 2.73% across Texas. Higher molecular prevalence was observed when analysed by ecoregion distinction, including 5.78% rickettsial infections by Ehrlichia canis and Anaplasma platys in the Rolling Plains ecoregion, and an average of 1.1% Borrelia turicatae and 1.0% Babesia gibsoni across detected ecoregions. To our knowledge, our findings indicate the first molecular detection of A. platys in Texas, and the first report of coinfections with E. canis and A. platys in dogs of Texas. The zoonotic concerns associated with TBDs, in conjunction with dogs’ implication as an effective sentinel for human disease, highlight the importance of characterizing and monitoring regions associated with active infections in dogs. Surveillance data obtained from this study may aid public health agencies in updating maps depicting high‐risk areas of disease and developing preventative measures for the affected areas.

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

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Tick-borne diseases (TBD) are common across the United States and can result in critical and chronic diseases in a variety of veterinary patients. Moreover, borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, and babesiosis are zoonotic and have been cited as the most common TBDs. Molecular diagnostic methodologies utilized for screening domestic dogs for these causative agents include real-time PCR (qPCR) assays in both singleplex and multiplex formats. However, current limitations of qPCR instruments restrict the number of fluorogenic labels that can be differentiated by the instrument for a given reaction. This study describes the development of the TickPath Layerplex, a diagnostic assay based on qPCR methodology that was adapted for the simultaneous detection and characterization of 11 pathogens responsible for causing 5 common TBDs in domestic dogs. The analytical and diagnostic performance of the layerplex assay was evaluated and shown to be compatible with common instruments utilized in molecular diagnostic laboratories. Test results revealed no inhibition or reduction in sensitivity during validation of the layerplex assay, and the limit of detection was determined to be near 16 genome copy equivalents per microliter. Overall, the high sensitivity, specificity, and screening capability of the assay demonstrate its utility for broadly screening dogs for common TBDs.