Ehrlichia Spp. Of Dogs: Risk of Infection, Persistence of Rickettsemia, and Impact on Reinfection

The research presented in this dissertation was conducted to further understanding of Ehrlichia spp. infections in dogs in the U.S. and Haiti. In chapter 3, to determine the risk of infection posed to dogs exposed to ticks in a natural setting, dogs were walked through tick habitat in northcentral Oklahoma and monitored clinically, serologically, and molecularly for four months for evidence of tick-borne infection. All 10 dogs were shown by serologic and molecular methods to be infected with E. ewingii, and 7 were co-infected with E. chaffeensis; however, no dog exhibited clinical signs of ehrlichiosis. Four of these Ehrlichia spp. infected dogs were monitored for two years (Chapter 4) following tick-exposure to evaluate long-term Ehrlichia spp. infection. In this study, three of four dogs were infected with E. ewingii for at least 460 days while infection with E. chaffeensis was only detectable through day 55. These data demonstrate that dogs may serve as a reservoir host for maintaining E. ewingii. The third study (Chapter 5) was a pilot trial designed to evaluate the impact that previous infection with an Ehrlichia spp. has on reinfection through tick feeding and intravenous sub-inoculation. The Ehrlichia spp. infected dogs from Chapter 4 were exposed to ticks by walks and monitored for four months for evidence of reinfection with an Ehrlichia spp. as previously described in Chapter 3. No reinfections by tick feeding were detected; however, three dogs sub-inoculated with E. ewingii and monitored for 46 additional days demonstrated molecular evidence of reinfection with E. ewingii. Lastly, Chapter 6 determined the prevalence of Ehrlichia spp. infections in 210 dogs from Haiti. Ticks infesting dogs were collected for identification and blood samples were evaluated by serologic and molecular methods for evidence of Ehrlichia spp. infections. Rhipicephalus sanguineus was the only tick collected from dogs. Ehrlichia canis was the only Ehrlichia spp. identified in these dogs; antibodies were present in 69 dogs and DNA was detected in 15 of those dogs. In summary, the risk of infection with an Ehrlichia spp. is high and infections may persist for years and alter future Ehrlichia spp. infections.Veterinary Biomedical Science

Prevalence of Babesia spp., Ehrlichia spp., and tick infestations in Oklahoma black bears (Ursus americanus)

American black bears (Ursus americanus) are commonly infested with ticks throughout their range, but there are few surveys for tick-borne disease agents in bears. To characterize tick infestations and determine the prevalence of current infection with Babesia spp. and past or current infection with Ehrlichia spp. in newly re-established populations of black bears in east central and southeastern Oklahoma, US, we identified adult (n=1,048) and immature (n=107) ticks recovered from bears (n=62). We evaluated serum and whole blood samples from a subset (n=49) for antibodies reactive to, and characteristic DNA fragments of, Ehrlichia spp., as well as characteristic DNA fragments of Babesia spp. Amblyomma americanum, the most common tick identified, was found on a majority (56/62; 90%) of bears and accounted for 697/1,048 (66.5%) of all ticks recovered. Other ticks included Dermacentor variabilis (338/1,048; 32.3%) from 36 bears, Amblyomma maculatum (9/1,048; 0.9%) from three bears, and Ixodes scapularis (4/1,048; 0.4%) from three bears. Antibodies reactive to Ehrlichia spp. were detected in every bear tested (49/49; 100%); maximum inverse titers to Ehrlichia chaffeensis ranged from 64-4,096 (geometric mean titer 1,525). However, PCR failed to identify active infection with E. chaffeensis, Ehrlichia ewingii, or an Ehrlichia ruminantium-like agent. Infection with Babesia spp. was detected by PCR in 3/49 (6%) bears. Together these data confirm that tick infestations and infection with tick-borne disease agents are common in bears in the southern US. The significance of these infestations and infections to the health of bears, if any, and the identity of the Ehrlichia spp. responsible for the antibody reactivity seen, warrant further evaluation.Peer reviewedEntomology and Plant PathologyNatural Resource Ecology and ManagementVeterinary Pathobiolog

Comparative evaluation of commercially available point-of-care heartworm antigen tests using well-characterized canine plasma samples

Abstract Background Dirofilaria immitis is a worldwide parasite that is endemic in many parts of the United States. There are many commercial assays available for the detection of D. immitis antigen, one of which was modified and has reentered the market. Our objective was to compare the recently reintroduced Witness® Heartworm (HW) Antigen test Kit (Zoetis, Florham Park, NJ) and the SNAP® Heartworm RT (IDEXX Laboratories, Inc., Westbrook, ME) to the well-based ELISA DiroChek® Heartworm Antigen Test Kit (Zoetis, Florham Park, NJ). Methods Canine plasma samples were either received at the Auburn Diagnostic Parasitology Laboratory from veterinarians submitting samples for additional heartworm testing (n = 100) from 2008 to 2016 or purchased from purpose-bred beagles (n = 50, presumed negative) in 2016. Samples were categorized as “positive,” “borderline” or “negative” using our established spectrophotometric cutoff value with the DiroChek® assay when a sample was initially received and processed. Three commercially available heartworm antigen tests (DiroChek®, Witness® HW, and SNAP® RT) were utilized for simultaneous testing of the 150 samples in random order as per their package insert with the addition of spectrophotometric optical density (OD) readings of the DiroChek® assay. Any samples yielding discordant test results between assays were further evaluated by heat treatment of plasma and retesting. Chi-square tests for the equality of proportions were utilized for statistical analyses. Results Concordant results occurred in 140/150 (93.3%) samples. Discrepant results occurred in 10/150 samples tested (6.6%): 9/10 occurring in the borderline heartworm (HW) category and 1/10 occurring in the negative HW category. The sensitivity and specificity of each test compared to the DiroChek® read by spectrophotometer was similar to what has been reported previously (Witness®: sensitivity 97.0% [94.1–99.4%], specificity 96.4% [95.5–100.0%]; SNAP® RT: sensitivity 90.9% [78.0–100.0%], specificity 98.8% [96.0–100.0%]). There were significant differences detected when comparing the sensitivities of the SNAP® RT and the Witness® HW to the DiroChek® among the 150 total samples (p = 0.003) and the 50 “borderline” samples (p = 0.001). Conclusions In this study, the sensitivity of the Witness® HW was higher than the sensitivity of the SNAP® RT when compared with the DiroChek® test results prior to heat treatment of samples

Haplotypic analysis of cox1 from Toxocara canis demonstrates five distinct clades that are not geographically defined

Background Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. Methods/principle findings Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. Conclusions The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis.This article is published as Martin, Katy A., Jeba RJ Jesudoss Chelladurai, Abrha Bsrat, Cassan Pulaski, Alice CY Lee, Lindsay A. Starkey, and Matthew T. Brewer. "Haplotypic analysis of cox1 from Toxocara canis demonstrates five distinct clades that are not geographically defined." PLOS Neglected Tropical Diseases 17, no. 10 (2023): e0011665. doi: https://doi.org/10.1371/journal.pntd.0011665. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Moxidectin steady state prior to inoculation protects cats from subsequent, repeated infection with Dirofilaria immitis

Abstract Background Infection of cats with Dirofilaria immitis causes seroconversion on antibody tests and pulmonary pathology, often without subsequent development of adult heartworms. Consistent administration of topical 10% imidacloprid-1% moxidectin has been shown to result in sustained plasma levels of moxidectin in cats after three to five treatments, a pharmacokinetic behavior known as “steady state”. Methods To evaluate the ability of moxidectin at “steady state” to protect cats from subsequent infection with D. immitis, cats (n = 10) were treated with the labeled dose of topical 10% imidacloprid-1% moxidectin for four monthly treatments. Each cat was inoculated with 25 third-stage larvae of D. immitis 7, 14, 21, and 28 days after the last treatment; non-treated cats (n = 9) were inoculated on the same days, serving as infection controls. Blood samples were collected from each cat from 1 month prior to treatment until 7 months after the final inoculation and tested for antibody to, and antigen and microfilaria of, D. immitis. Results Measurement of serum levels of moxidectin confirmed steady state in treated cats. Cats treated with topical 10% imidacloprid-1% moxidectin prior to trickle inoculation of D. immitis L3 larvae throughout the 28 day post-treatment period remained negative on antibody and antigen tests throughout the study and did not develop gross or histologic lesions characteristic of heartworm infection. A majority of non-treated cats tested antibody positive by 3–4 months post infection (6/9) and, after heat treatment, tested antigen positive by 6–7 months post-infection (5/9). Histologic lesions characteristic of D. immitis infection, including intimal and medial thickening of the pulmonary artery, were present in every cat with D. immitis antibodies (6/6), although adult D. immitis were confirmed in only 5/6 antibody-positive cats at necropsy. Microfilariae were not detected at any time. Conclusions Taken together, these data indicate that prior treatment with 10% imidacloprid-1% moxidectin protected cats from subsequent infection with D. immitis for 28 days, preventing both formation of a detectable antibody response and development of pulmonary lesions by either immature stages of D. immitis or young adult heartworms