Multicenter evaluation of the Vetscan Imagyst system using Ocus 40 and EasyScan One scanners to detect gastrointestinal parasites in feces of dogs and cats.

The Vetscan Imagyst system (Zoetis) is a novel, artificial intelligence-driven detection tool that can assist veterinarians in the identification of enteric parasites in dogs and cats. This system consists of a sample preparation device, an automated digital microscope scanner, and a deep-learning algorithm. The EasyScan One scanner (Motic) has had good diagnostic performance compared with manual examinations by experts; however, there are drawbacks when used in veterinary practices in which space for equipment is often limited. To improve the usability of this system, we evaluated an additional scanner, the Ocus 40 (Grundium). Our objectives were to 1) qualitatively evaluate the performance of the Vetscan Imagyst system with the Ocus 40 scanner for identifying Ancylostoma, Toxocara, and Trichuris eggs, Cystoisospora oocysts, and Giardia cysts in canine and feline fecal samples, and 2) expand the assessment of the performance of the Vetscan Imagyst system paired with either the Ocus 40 or EasyScan One scanner to include a larger dataset of 2,191 fecal samples obtained from 4 geographic regions of the United States. When tested with 852 canine and feline fecal samples collected from different geographic regions, the performance of the Vetscan Imagyst system combined with the Ocus 40 scanner was correlated closely with manual evaluations by experts. Sensitivities were 80.0‒97.0% and specificities were 93.7‒100.0% across the targeted parasites. When tested with 1,339 fecal samples, the Vetscan Imagyst system paired with the EasyScan One scanner successfully identified the targeted parasite stages; sensitivities were 73.6‒96.4% and specificities were 79.7‒100.0%.

Diagnosis of canine intestinal parasites: Improved detection of Dipylidium caninum infection through coproantigen testing.

Intestinal parasites, including cestodes like Dipylidium caninum, are common in dogs in the United States of America (USA), but fecal flotation consistently, and, at times, dramatically, fails to identify many of these infections. To determine the extent to which including coproantigen testing for D. caninum would improve the identification of dogs infected with this cestode, we evaluated fecal samples from 877 dogs (589 pet and 288 from municipal shelters) from six USA states using zinc sulfate (specific gravity 1.24) fecal flotation with centrifugation along with coproantigen detection for Giardia sp., hookworms, ascarids, and Trichuris vulpis. For D. caninum, PCR of perianal swabs was included. Intestinal parasite infections were identified, using centrifugal fecal flotation or coproantigen, in 265 dogs (13.2 % pet, 64.9 % shelter). Dipylidium caninum infection was detected in 5.6 % of dogs with the combination of coproantigen and centrifugal fecal flotation, and 7.3 % of dogs when perianal swab results were included; prevalence varied by diagnostic method, population, and geographic region. In pet dogs, D. caninum infection was identified by fecal flotation (0), coproantigen (2.2 %), or perianal swabs (1.2 %). The same methods revealed infection in 0.3 %, 12.5 %, and 11.1 % of shelter dogs, respectively. Frequent use of praziquantel in shelter dogs (116/288; 40.3 %) may have reduced prevalence. Positive and negative agreement of D. caninum coproantigen with perianal swab PCR in pet dogs was 85.7 % and 98.8 %, respectively. Multiple logistic regression analysis accounting for region, population, and age found D. caninum infection to be more common in shelter dogs relative to pet (adjusted OR 4.91 [2.48, 10.24]) and in the Southcentral and Southeast regions relative to North (adjusted OR 9.59 [1.92, 174.13] and 17.69 [3.67, 318.09] respectively). Coproantigen testing also enhanced the detection of other intestinal parasites over fecal flotation alone, including Giardia sp. (14.7 % vs 3.3 %), hookworms (13.8 % vs 8.4 %), ascarids (2.9 % vs 2.2 %), and T. vulpis (2.9 % vs 1.4 %). Together, these data indicate that the coproantigen assay employed increases detection of D. caninum infections several fold, supporting the use of this test in clinical practice, and add to a growing body of research documenting enhanced diagnosis through implementation of multiple laboratory-based methods.

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.

A nationwide serological survey for Dirofilaria immitis in companion cats in the United States of America: 3.5% antibody and 0.3% antigen positivity.

BACKGROUND: Feline heartworm disease (HWD) is a complex and often misdiagnosed disease in cats, caused by the filarial nematode Dirofilaria immitis. Despite its significant impact, studies reporting the prevalence of D. immitis in apparently healthy pet cats in the USA are lacking. METHODS: To investigate feline heartworm seroprevalence in apparently healthy pet cats in the USA, serum samples (n = 2165) collected from cats across 47 states and Washington District of Columbia were analyzed for D. immitis antibody (Heska Corp.) and antigen (DiroCHEK®; Zoetis Inc.) with and without acid treatment of the samples. RESULTS: Antibodies to D. immitis antibodies were identified in 3.5% (76/2165) of cats from 26 states, with a significantly higher prevalence in cats from the westernmost US states (West region; 5.4%, 23/429) compared to those from the South (3.8%, 32/847), Midwest (2.7%, 9/338) and Northeast regions (2.2%, 12/551) (P < 0.04). Antigen from D. immitis was detected in 0.3% (6/2165) of cats, which was significantly lower than the antibody detection (P < 10-4), and no samples were positive for both antibody and antigen. CONCLUSIONS: This is the largest antibody-based, nationwide serosurvey of feline heartworm in an apparently healthy cat population, and the results suggest that cats in the USA have a high risk of exposure to D. immitis-infected mosquitoes. The high nationwide prevalence (3.5%) indicates that the true prevalence of cats infected with D. immitis in the USA may be significantly underestimated. Our findings emphasize the need for increased awareness and routine testing of cats for heartworm infection, especially in non-endemic areas of the USA. Clinicians should consider appropriate use of broad-spectrum veterinary-approved parasiticides and lifestyle management in feline patients to reduce the risk of infection. Future studies should focus on evaluating the D. immitis infection status in healthy cats and developing better diagnostic assays to detect this complex infection.

Effect of age on wingbeat frequency of Aedes aegypti and potential application for age estimation of mosquitoes.

To combat mosquito-borne diseases, a variety of vector control tools have been implemented. Estimating age structure in populations of vector species is important for understanding transmission potential. Age-grading techniques have been used as critical methods for evaluating the efficacy of vector control tools. However, methods like mark-release-recapture and ovarian dissection are laborious and require a high level of training. For decades, scientists have discussed the wide array of acoustic signatures of different mosquito species. These distinguishable wingbeat signatures with spatiotemporal classification allow mosquitoes of the same species to locate one another for mating. In recent years, the use of sensitive acoustic devices like mobile phones have proved effective. Wingbeat signatures can be used to identify mosquito species without the challenge of intensive field collections and morphological and molecular identifications. In this study, laboratory Aedes aegypti (L.) female and male wingbeats were recorded using mobile phones to determine whether sex and age differences with chronological time, and across different physiological stages, can be detected. Our results indicate significantly different wingbeat signatures between male and female Ae. aegypti, and a change of wingbeat frequencies with age and reproduction stage in females.

Nationwide molecular survey of Dirofilaria immitis and Dirofilaria repens in companion dogs and cats, United States of America.

BACKGROUND: Heartworms, Dirofilaria immitis, are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens, occurs in the USA. METHODS: To provide this large-scale data, we examined whole blood samples (n = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results. RESULTS: Dirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens. The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis-positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) (P < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) (P < 0.0001). CONCLUSIONS: To our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA.

Detection of zoonotic vector-borne pathogens in domestic dogs in Giza, Egypt.

The public health implications of zoonotic vector-borne pathogens are numerous because domestic animals, such as dogs, live in close proximity to humans. Blood was collected from 116 domestic dogs in Cairo, Egypt from three different settings at the human-animal interface. The three settings the dogs came from were: privately owned animals seeking care at the Cairo University Faculty of Veterinary Medicine Clinic, non-laboratory reared research dogs maintained at the Cairo University Faculty of Veterinary Medicine, and an urban private animal rescue in Shabramont, Giza, Egypt. Enrolled animals were visually inspected for presence of flea or tick ectoparasites, Rhipicephalus sanguineus sensu lato ticks were recovered from 56 enrolled animals and a flea identified as Ctenocephalides felis was recovered from one animal. To test for past and/or current infection with vector-borne pathogens, conventional PCR and IDEXX SNAP® 4Dx® Plus were performed on whole blood. Pathogen targets included: Anaplasma spp., Ehrlichia spp., Babesia spp., Borrelia spp., Bartonella spp., Dirofilaria spp., and Rickettsia spp. Among dogs sampled across all locations, one dog was positive for Babesia sp. infection and one dog was positive for Anaplasma sp. infection as detected by PCR and confirmed by Sanger sequencing. Three additional dogs were positive for infection but had incomplete sequences obtained: two for Ehrlichia sp. and one for Borrelia sp. The SNAP® test results for all sampled dogs included: eight dogs positive for Anaplasma spp., 14 dogs positive for Ehrlichia spp., and five additional dogs positive for both Anaplasma spp. and Ehrlichia spp. SNAP® test results by sampling location showed that 66% of the dogs at the animal rescue were positive for Anaplasma spp. and/or Ehrlichia spp., 17% of the privately owned dogs at the Faculty of Veterinary medicine were positive for Anaplasma spp. and/or Ehrlichia spp., and none of the research dogs were positive for any of the targets on the SNAP® test. This high proportion of seropositivity in the animals sampled indicates a vector population which is not well controlled and a need for continued owner education and promotion of consistent use of preventive medications and the risk for zoonotic transmission.

Serologic evidence of selected vector-borne pathogens in non-owned dogs in the southeast US.

Vector-borne pathogens (VBP) associated with ectoparasitism are of concern for animal health, and there are many gaps in surveillance and reporting data. The purpose of this study was to test for four VBPs in a subset of non-owned dogs from county humane societies in Alabama and Georgia that were admitted to the Auburn University College of Veterinary Medicine Hoerlein Spay/Neuter Program for health exams and routine procedures, including bloodwork and testing with the 4Dx® SNAP® Plus (IDEXX Laboratories, Inc., Westbrook, Maine). Visualized ectoparasites were noted and preserved for identification and analysis. From May-October 2019, residual blood (n = 114) was used for preparing blood smears and DNA extraction and PCR. Out of 114 samples, 35.1% (40/114) were seropositive for one or more VBP: Dirofilaria immitis antigen (20.2%; 23/114) and Ehrlichia spp. antibodies (20.2%; 23/114); six VBD-positive dogs (15%) tested positive for both. No dogs had detectable antibodies to Borrelia burgdorferi or Anaplasma spp. (0%; 0/114). Microfilariae of D. immitis were present in 7 blood smears, all from dogs that were D. immitis antigen positive. Morulae or DNA of Ehrlichia or Anaplasma spp. were not identified in any sample. Fleas were documented in 20.4% (23/113) of dogs, 9.7% (11/113) were infested with ticks, predominantly Amblyomma americanum, and co-infestations were noted in 2.7% (3/113). Our data indicate that there is substantial VBP risk in Alabama and Georgia, and that the reservoir potential of domestic animals, especially non-owned animals, along with potential wildlife reservoirs warrants further investigation.

Retrospective study of canine endoparasites diagnosed by fecal flotation methods analyzed across veterinary parasitology diagnostic laboratories, United States, 2018.

BACKGROUND: Companion animal endoparasites play a substantial role in both veterinary medicine and public health. Updated epidemiological studies are necessary to identify trends in occurrence and distribution of these parasites, and their associated risk factors. This study aimed to assess the occurrence of canine endoparasites  retrospectively, using fecal flotation  test data available through participating academic veterinary parasitology diagnostic laboratories across the United States of America (USA). METHODS: Canine fecal flotation records from ten veterinary diagnostic laboratories located in nine states in the USA acquired from January 1, 2018, to December 31, 2018, were included. RESULTS: A total of 4692 fecal flotation test results were obtained, with a majority comprised of client-owned dogs (3262; 69.52%), followed by research dogs (375; 8.00%), and shelter dogs (122; 2.60%). Samples from 976 (20.80%) dogs were positive for at least one parasite, and co-infections of two or more parasites were found in 3.82% (179/4692) of the samples. The five most commonly detected parasites were: Giardia sp., (8.33%; 391/4692), Ancylostomatidae (5.63%; 264/4692), Cystoisospora spp. (4.35%; 204/4692), Toxocara canis (2.49%;117/4692), and Trichuris vulpis (2.43%; 114/4692). Various other internal parasites, including gastrointestinal and respiratory nematodes, cestodes, trematodes, and protozoans were detected in less than 1% of samples. CONCLUSIONS: These data illustrate the importance of parasite prevention, routine fecal screening, and treatment of pet dogs. Additionally, pet owners should be educated about general parasite prevalence, prevention, and anthelmintic treatment regimens to reduce the risks of environmental contamination and zoonotic transmission.

An investigation of Dirofilaria immitis infection and its effects on mosquito wingbeat frequencies.

Each mosquito species has a different wingbeat frequency by which they attract mates. With just a brief recording (<1/10th of a second) these acoustic signatures can be analyzed to quickly determine if mosquitoes belong to a species that is known to transmit different pathogens. A recent study has shown that mobile phones are capable of capturing acoustic data from mosquito wingbeats. We examined wingbeat signatures and flight duration patterns of D. immitis infected and non-infected Aedes aegypti to determine if mobile phone recordings of wingbeat frequencies can be used to distinguish infected mosquitoes from non-infected ones. Female mosquitoes were recorded prior to and at various time points after feeding on infected or non-infected dog blood by placing individual mosquitoes into a collection vial and recording for 60 s using the Voice Memo app for iPhone 7 plus and 8. To uniformly analyze audio data, recordings were processed using a previously described automated algorithm in Python 3.0 to determine wingbeat frequency. A total of 1669 recordings were gathered, and mosquitoes were dissected to confirm the presence and number of D. immitis larvae. Our findings indicate that there was a significant effect on wingbeat frequency with an increasing number of L3 larvae. Specifically, as the number of L3, infective stage larvae increases, a decrease in wingbeat frequency is seen. However, there was no significant effect of increasing number of L1 or L2 larvae causing increasing wingbeat frequencies. The detection of a significant difference in wingbeat frequencies between mosquitoes harboring infective stage D. immitis larvae is unique and suggests the possibility of using wingbeat recordings as a tool for vector species and pathogen surveillance and monitoring.

Evaluation of oral fluralaner (Bravecto®) for efficacy against nymphs of Amblyomma americanum and Rhipicephalus sanguineus (sensu lato).

BACKGROUND: Amblyomma americanum and Rhipicephalus sanguineus (sensu lato) nymphs commonly feed on and transmit pathogens to dogs (Canis familiaris). Control of immature and adult tick life stages is necessary to fully protect animals. We evaluated efficacy of oral fluralaner (Bravecto®) against induced infestations with A. americanum and R. sanguineus (s.l.) nymphs on dogs in two experiments. METHODS: In each experiment, 10 dogs were administered oral fluralaner chewable tablets one time on Day 0 at a targeted minimum dose of 25 mg/kg body weight and 10 dogs remained non-treated controls. Dogs were infested with two groups of 50 A. americanum nymphs and two groups of 50 R. sanguineus (s.l.) nymphs on Days -1, 6, 28, 56 and 84. At 48 h and 72 h post-infestation, nymphs were collected from dogs, assessed as live or dead, and enumerated into categories defining attachment and engorgement status. Fluralaner efficacy was determined in separate analyses against all live nymphs and against live-fed nymphs, i.e. live nymphs that were attached to dogs at the time of collection and/or were engorged. Fluralaner was considered effective when mean numbers of live ticks were reduced in fluralaner-treated dogs by ≥ 90%. RESULTS: Fluralaner efficacy against all live and live-fed A. americanum nymphs in the first experiment was > 94% on all collection days. Efficacy against all live R. sanguineus (s.l.) nymphs in the first experiment was > 96% on all collection days  excluding the 48 h counts for infestations on Days 28 (83.7%), 56 (82.9%) and 84 (86.7%); efficacy against live-fed R. sanguineus (s.l.) nymphs was > 95% on all 48 h/72 h count days. Fluralaner efficacy against all live A. americanum nymphs in the second experiment was > 93% on all collection days for 8 weeks excluding the 48 h count for infestation on Day 56 (87.8%); efficacy against live-fed A. americanum nymphs was > 91% on all count days for 8 weeks. Efficacy against all live R. sanguineus (s.l.) nymphs in the  second experiment was > 91% on all 72 h collection days  except for infestations on Days 28 (76.8%) and 56 (86.3%); efficacy against live-fed R. sanguineus (s.l.) nymphs was 100% on all 72 h count days. CONCLUSIONS: A single administration of oral fluralaner to dogs is effective against A. americanum and R. sanguineus (s.l.) nymphs for up to 12 weeks.

DIVERSE BARTONELLA SPP. DETECTED IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) AND ASSOCIATED KEDS (LIPOPTENA MAZAMAE) IN THE SOUTHEASTERN USA.

There are many known species of Bartonella, Gram-negative bacteria that can cause febrile illness and fatality in humans and animals. These pathogens are often transmitted through hematophagous arthropod vectors such as fleas and lice. Despite increasing awareness about Bartonella spp. and their zoonotic potential, as well as existing literature on Bartonella spp. in cervids, little is known about the diversity of Bartonella spp. in white-tailed deer (Odocoileus virginianus) and their associated keds in the southeastern US. We examined the prevalence and diversity of Bartonella spp. in an enclosed herd of white-tailed deer and their ectoparasites, deer keds (Lipoptena mazamae), in Alabama. The overall prevalence of Bartonella infection in this population of deer was 16% (10/63) and 24% (23/96) in keds associated with deer that we sampled. Three species of Bartonella were identified in both deer and their keds: Bartonella bovis, Bartonella schoenbuchensis, and Bartonella sp. 1. Additionally, Bartonella melophagi was detected in white-tailed deer but not in the sampled keds. The detection of four Bartonella species in one population of white-tailed deer, three of which have known zoonotic potential, highlights the importance of Bartonella diversity within host species.

Coproantigen Detection Augments Diagnosis of Common Nematode Infections in Dogs.

Microscopic methods which employ active or passive flotation have been used to detect parasite diagnostic stages in the feces of companion animals for many years. More recently, coproantigen ELISAs for the detection of excretory/secretory products from intestinal nematodes have been introduced. These assays can identify the presence of parasites when eggs are not recovered by flotation (e.g. prepatent infection or intermittent egg shedding). The study was designed to assess the added benefit of these coproantigen tests in canine fecal diagnostics. The work was performed at 3 separate sites where canine fecal samples were each independently evaluated by both centrifugal flotation with an expert examiner (CFE) and passive flotation with a less experienced examiner. All samples were also tested using coproantigen ELISA to detect ascarid, hookworm, or whipworm antigen (IDEXX Laboratories, Inc, Westbrook, Maine). A total of 1202 samples were collected; 626 were from shelter dogs and 576 were from pet dogs. CFE recovered ascarid eggs in 58 samples, hookworm eggs in 229 samples, and whipworm eggs in 95 samples. Of the positive samples identified by CFE, the PFE and ELISA identified 40 and 51 ascarid samples, 188 and 203 hookworm samples, and 65 and 67 whipworm positive samples, respectively. The coproantigen ELISA identified 8 ascarid, 82 hookworm, and 22 whipworm positive samples that were not detected by CFE. The combined results of passive flotation and the coproantigen ELISA improved the percent agreement with centrifugal flotation, suggesting that greater sensitivity of detection may be achieved through the use of complementary diagnostic methods. However, errors of misidentification and poor recovery apparently introduced by less experienced examiners using an inferior flotation method remained. A diagnostic approach that combines coproantigen assays with centrifugal flotation and examination by an expert allows detection of more ascarid, hookworm, and whipworm infections.

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

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.

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.

Prevalence of vector-borne pathogens in dogs from Haiti.

Canine vector-borne pathogens are common on some Caribbean islands, but survey data in Haiti are lacking. To determine the prevalence of selected vector-borne pathogens in dogs from Haiti, we tested blood samples collected from 210 owned dogs, 28 (13.3%) of which were infested with Rhipicephalus sanguineus ticks at the time of blood collection. No other tick species were identified on these dogs. A commercially available ELISA identified antibodies to Ehrlichia spp. in 69 (32.9%), antibodies to Anaplasma spp. in 37 (17.6%), and antigen of Dirofilaria immitis in 55 (26.2%); antibodies to Borrelia burgdorferi were not detected in any sample. Molecular assays of whole blood from 207 of the dogs confirmed infection with Ehrlichia canis (15; 7.2%), Anaplasma platys (13; 6.3%), D. immitis (46; 22.2%), Wolbachia spp. (45; 21.7%), Babesia vogeli (16; 7.7%), and Hepatozoon canis (40; 19.3%), but Anaplasma phagocytophilum, Babesia canis, Babesia rossi, Babesia gibsoni, Ehrlichia chaffeensis, Ehrlichia ewingii, or Hepatozoon americanum were not detected. Co-infection with two or more vector-borne pathogens was detected by serology in 42 (20.0%) dogs and by molecular assays in 22 (10.6%) dogs; one dog was co-infected with B. vogeli and E. canis as detected by PCR with D. immitis detected by serology (antigen). Overall, evidence of past or current infection with at least one vector-borne pathogen was identified in 142/210 (67.6%) dogs in this study, underscoring the common nature of these pathogens, some of which are zoonotic, in Haiti.

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

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.

Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure.

Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer=128-4096, GMTMAX=548.7) and E. chaffeensis (10/10, maximum inverse titer=1024-32,768, GMTMAX=4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens.

Pre-treatment with heat facilitates detection of antigen of Dirofilaria immitis in canine samples.

Diagnosis of Dirofilaria immitis infection in dogs is largely dependent on detection of antigen in canine serum, plasma, or whole blood, but antigen may be bound in immune complexes and thus not detected. To develop a model for antigen blocking, we mixed serum from a microfilaremic, antigen-positive dog with that of a hypergammaglobulinemic dog not currently infected with D. immitis and converted the positive sample to antigen-negative; detection of antigen was restored when the mixed sample was heat-treated, presumably due to disruption of antigen/antibody complexes. A blood sample was also evaluated from a dog that was microfilaremic and for which microfilariae were identified as D. immitis by morphologic examination. Antigen of D. immitis was not detected in this sample prior to heating but the sample was strongly positive after heat treatment of whole blood. Taken together, our results indicate that blood samples from some dogs may contain factors that inhibit detection of antigen of D. immitis, and that heat treatment of these samples prior to testing could improve the sensitivity of these assays in some patients.