Prevalence of protozoan parasites in small and medium mammals in Texas, USA.

Wildlife interaction with humans increases the risk of potentially infected ticks seeking an opportunistic blood meal and consequently leading to zoonotic transmission. In the United States, human babesiosis is a tick-borne zoonosis most commonly caused by the intraerythrocytic protozoan parasite, Babesia microti. The presence of Babesia microti and other species of Babesia within Texas has not been well characterized, and the molecular prevalence of these pathogens within wildlife species is largely unknown. Small (e.g. rodents) and medium sized mammalian species (e.g. racoons) represent potential reservoirs for specific species of Babesia, though this relationship has not been thoroughly evaluated within Texas. This study aimed to characterize the molecular prevalence of Babesia species within small and medium sized mammals at two sites in East Texas with an emphasis on detection of pathogen presence in these two contrasting wild mammal groups at these sites. To that end, a total of 480 wild mammals representing eight genera were trapped, sampled, and screened for Babesia species using the TickPath layerplex qPCR assay. Two sites were selected for animal collection, including The Big Thicket National Preserve and Gus Engeling Wildlife Management Area. Molecular analysis revealed the prevalence of various Babesia and Hepathozoon species at 0.09% each, and Sarcocystis at 0.06% . Continued molecular prevalence surveys of tick-borne pathogens in Texas wild mammals will be needed to provide novel information as to which species of Babesia are most prevalent and identify specific wildlife species as pathogen reservoirs.

Ehrlichia canis in dogs of Mexico: Prevalence, incidence, co-infection and factors associated.

Rickettsial infections in dogs of Mexico were investigated. A total of 246 dogs were blood sampled and initially screened to detect Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum and Rickettsia rickettsii by a quantitative real-time PCR (qPCR) assay. Sixty-five dogs were monitored and sampled twice 7-8 months apart. Using the qPCR, 72 positive dogs to E. canis were detected (prevalence of 29.26%). These dogs were also tested by nested PCR to detect the same pathogens. None of the studied dogs were positive to E. chaffeensis, E. ewingii, R. rickettsii nor A. phagocytophilum by both PCR assays. The cumulative incidence of E. canis infection was 38.46%. Sequencing analysis of the nested PCR products revealed 100% and 98.1% identity of E. canis and R. parkeri, respectively. We found a dog co-infected with E. canis and R. parkeri.

TickPath Layerplex: adaptation of a real-time PCR methodology for the simultaneous detection and molecular surveillance of tick-borne pathogens.

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.

Molecular prevalence and ecoregion distribution of select tick-borne pathogens in Texas dogs.

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.

Molecular identification of Ehrlichia ewingii in a polyarthritic Texas dog.

An 8-year-old, neutered male, Golden Retriever presented for bilateral carpal joint effusion. A complete blood count revealed mild leukopenia and marked thrombocytopenia. Samples were sent to the Texas A&M Veterinary Medical Diagnostic Laboratory for blood smear review and serologic testing for tick-borne diseases. Numerous morulae were observed within neutrophils, and antibodies against Ehrlichia canis were detected at a 1:512 dilution via the indirect fluorescent antibody (IFA) test. As neutrophilic morulae are morphologically indistinguishable between Ehrlichia ewingii and Anaplasma phagocytophilum, and genus-wide cross-reactivity is possible with serologic testing, additional molecular testing was performed. Quantitative real-time polymerase chain reaction (qPCR) followed by conventional PCR and Sanger sequencing were performed on serum identified with E ewingii as the sole disease-causing agent. Three months after diagnosis and treatment, no morulae were found, molecular testing for E ewingii detected no DNA, and convalescent IFA testing demonstrated a continued detection of antibodies for E canis at a 1:512 dilution. To the authors' knowledge, this is the first reported case of E ewingii confirmed with molecular diagnostics in a Texas dog. The zoonotic transmission potential of E ewingii should be noted as Texas supports competent tick vectors, and dogs represent effective sentinels for human ehrlichiosis. This report also highlights the utility of molecular diagnostics when serologic and microscopic evaluations are not sufficient in providing the species-level identity of a causative agent.

Ticks infesting dogs in rural communities of Yucatan, Mexico and molecular diagnosis of rickettsial infection.

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.

Novel real-time PCR assays for genomic group identification of tick-borne relapsing fever species Borrelia hermsii.

Borrelia hermsii is a non-Lyme borreliosis pathogen that is responsible for causing tick-borne relapsing fever in humans and animals in the western United States. B. hermsii has been described to encompass two divergent genomic groups, GGI and GGII, which have been suggested to maintain a unique geographical distribution and potential range of pathogenicity. Though the genomic groups have been extensively documented in the literature, a real-time PCR tool for identifying these genomic groups is lacking. This study describes the development and validation of two flaB-based quantitative real-time PCR assays for differentiating between the two genomic groups of B. hermsii while also maintaining specificity against other closely related Borrelia species. The diagnostic specificity of the assays were evaluated using a large panel of various Borrelia species, including a collection of 22 B. hermsii culture isolates purified from various hosts. The high sensitivity and specificity of the assays provide a useful tool for supporting future studies aimed at evaluating the geographical distribution as well as potential intraspecies pathogenicity within arthropod vectors and mammalian hosts.

Molecular detection of rickettsial tick-borne agents in white-tailed deer (Odocoileus virginianus yucatanensis), mazama deer (Mazama temama), and the ticks they host in Yucatan, Mexico.

Twenty-five white-tailed deer (Odocoileus virginianus yucatanensis) (WTD), 4 mazama deer (Mazama temama) (MD), and the ticks they host in Yucatan, Mexico were sampled to run a molecular survey for tick-borne rickettsial agents. The prevalence of rickettsial agents was 20% in WTD (5/25) and 50% in MD (2/4). When sequencing the nested PCR products, E. chaffeensis, A. phagocytophilum and A. odocoilei, were identified as single infection or coinfecting cervids. None of the cervid samples were positive for E. ewingii, E. canis, nor Rickettsia spp. Overall, 355 individual ticks were collected. Species identified based on adult stages infesting cervids included Amblyomma mixtum, A. parvum, A. cf. oblongoguttatum, Ixodes affinis, Rhipicephalus microplus, R. sanguineus sensu lato, and Haemaphysalis juxtakochi. Rhipicephalus microplus was the tick species most commonly found infesting cervids with a frequency of 28.4%, and intensity of 25.2 ticks per animal. A pool of Amblyomma cf. oblongoguttatum adults and one of Amblyomma spp. nymphs were positive for E. canis and E. chaffeensis, respectively. None of the studied tick pools were positive for E. ewingii, A. phagocytophilum, nor R. rickettsii. To the best of our knowledge, this study is the first to report the prevalence of rickettsial agents in WTD and MD in Mexico. Our molecular study is the first to report the detection of E. chaffeensis, A. phagocytophilum, and A. odocoilei in MD in Mexico. The molecular detection of E. chaffeensis, A. phagocytophilum, and A. odocoilei in deer, and E. chaffeensis in Amblyomma spp. nymphs reported here raises the concern for the risk of human exposure to tick-borne rickettsial pathogens. Our findings highlight the need to apply the "One Health" approach to study ticks and tick-borne diseases. This science-based information could be used by state public-health programs to assess the risk for exposure to tick-borne Anaplasmataceae in Yucatan, Mexico.

Development and application of a canine endogenous internal positive control for use in real-time PCR assays.

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.