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%.

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.

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.

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.

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.

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.

Genetic diversity of Hepatozoon spp. in coyotes from the south-central United States.

To better define the strains and species of Hepatozoon that infect coyotes in the south-central United States, whole blood and muscle samples were collected from 44 coyotes from 6 locations in Oklahoma and Texas. Samples were evaluated by a nested polymerase chain reaction (PCR) using primers amplifying a variable region of the apicomplexan 18S rRNA gene as well as histopathology (muscle only) for presence of tissue cysts. Hepatozoon spp. infections were identified in 79.5% (35/44) of coyotes tested including 27 of 44 (61.4%) whole blood samples and 17 of 44 (38.6%) muscle samples tested by PCR and 23 of 44 (52.3%) muscle samples evaluated by histological examination. Analysis revealed 19 distinct sequences comprising 3 major clusters of Hepatozoon spp., i.e., 1 most closely related to Hepatozoon americanum, another most closely related to Hepatozoon canis , and the third an intermediate between the 2 groups. The diversity of Hepatozoon spp. in wild canids appears greater than previously recognized and warrants further investigation.

Prevalence of antibodies to spotted fever group Rickettsia spp. and Ehrlichia spp. in coyotes (Canis latrans) in Oklahoma and Texas, USA.

Coyotes (Canis latrans) are commonly infested with ticks, including Amblyomma americanum, the predominant vector of Ehrlichia chaffeensis and Ehrlichia ewingii; Dermacentor variabilis, an important vector of Rickettsia rickettsii; and Amblyomma maculatum, a major vector of Rickettsia parkeri, a spotted fever group (SFG) Rickettsia. To determine the degree to which coyotes are infected with or exposed to tick-borne bacterial disease agents, serum samples collected from coyotes in Oklahoma and Texas were tested for antibodies reactive to R. rickettsii, Ehrlichia canis, E. chaffeensis, E. ewingii, Borrelia burgdorferi, and Anaplasma phagocytophilum by indirect fluorescent antibody (IFA) testing or enzyme-linked immunosorbent assay (ELISA). Of the coyotes tested, 60% (46/77) and 64% (47/74) had antibodies reactive to R. rickettsii and E. chaffeensis, respectively, on IFA. Additionally, 5% (4/77) had antibodies reactive to E. canis, but not B. burgdorferi or A. phagocytophilum, on SNAP(®) 4Dx(®) ELISA; subsequent serologic analysis by plate ELISA using species-specific peptides revealed antibodies to E. ewingii, E. canis, and E. chaffeensis in 46% (23/50), 18% (9/50), and 4% (2/50) of serum samples, respectively. Taken together, these data indicate that coyotes in this region are commonly exposed to SFG Rickettsia and E. ewingii and that further consideration of coyotes as a component of the maintenance cycle for these pathogens may be warranted.