Alexa C. Rosypal

Survey of Antibodies to Trypanosoma cruzi and Leishmania spp. in Gray and Red Fox Populations From North Carolina and Virginia

Abstract American trypanosomiasis and leishmaniasis are caused by related hemoflagellate parasites, Trypanosoma cruzi and Leishmania spp., which share several common host species. Both zoonotic protozoans are endemic in the United States. Canines, including domestic and wild canids, are reservoir hosts for human infections with T. cruzi and Leishmania spp. The present study examined the seroprevalence of T. cruzi and Leishmania spp. in wild canids from North Carolina and Virginia. Wild canine species tested in this work included 49 gray foxes (Urocyon cinereoargenteus) and 5 red foxes (Vulpes vulpes). Overall, sera samples from 54 foxes (North Carolina  =  43; Virginia  =  11) were tested by immunochromatographic strip assays (ICT). Antibodies to T. cruzi were found in 4 (9%) gray foxes from North Carolina and 2 (18%) gray foxes from Virginia. Antibodies to Leishmania spp. were detected in 1 (2%) gray fox from North Carolina. Our results indicate that wild canids are exposed more frequently to T. cruzi in North Carolina than Leishmania spp. and only T. cruzi in Virginia.

Surveillance for Antibodies to Leishmania spp. in Dogs from Sri Lanka

Abstract The global distribution of leishmaniasis is rapidly expanding into new geographic regions. Dogs are the primary reservoir hosts for human visceral leishmaniasis caused by infection with Leishmania infantum. Natural infections with other Leishmania spp. can occur in dogs, but their role as reservoir hosts for other species of Leishmania is uncertain. Leishmania donovani is traditionally considered a visceralizing anthroponotic species; however, cutaneous leishmaniasis caused by L. donovani has been reported in Sri Lanka. In the present study, sera from 114 dogs in Sri Lanka were examined for antibodies to visceralizing Leishmania spp. Sera were tested by the canine immunochromatographic strip assays based on recombinant K39 antigen. Anti-Leishmania spp. antibodies were detectable in 1 of 114 (0.9%) dogs from Sri Lanka. Nonetheless, serological evidence suggests that leishmaniasis may be an emerging zoonosis in Sri Lanka.

Evaluation of a novel dried blood spot collection device (HemaSpot™) to test blood samples collected from dogs for antibodies to Leishmania infantum.

Collection of blood samples from veterinary and wildlife patients is often challenging because the samples have to be collected on farm or in the wild under various environmental conditions. This poses many technical problems associated with venipuncture materials, their safe use and disposal, transportation and processing of collected samples. Dried blood spot (DBS) sample collection techniques offer a simple and practical alternative to traditional blood collection methods to obtain blood samples from animals for parasite antibody evaluation. The DBS collection devices are compact, simple to use, and are particularly useful for large number of samples. Additionally, DBS samples take up less space and they are easier to transport than traditional venipuncture-collected blood samples. Visceral leishmaniasis (VL) is a potentially fatal parasitic disease of dogs and humans and it is frequently diagnosed by antibody tests. Immunochromatographic tests (ICT) for antibodies to Leishmania infantum are commercially available for dogs and they produce qualitative results in minutes. Measurement of canine antibodies to L. infantum with the ICT using traditional venipuncture has been validated previously, but the use of DBS samples has not been evaluated using this method. The purpose of the present study was to determine the ability of DBS samples to detect antibodies to L. infantum in dogs using a commercial ICT assay. One hundred plasma samples from dogs experimentally infected with the LIVT-1 strain of L. infantum were collected by venipuncture and frozen. Individual samples were thawed, and then 80 μl plasma (2 drops) was aliquotted onto the 8-spoked disk pad on individual DBS sample collection devices (HemaSpot™, Spot-On Sciences, Austin, TX), dried, and stored in the dark at room temperature. After one month and six months, respectively, 2 spokes of the 8 spokes of the disk pad of each DBS sample were removed and eluted in 200 μl PBS. The eluate was used to test for antibodies in the ICT and compared to ICT results using thawed plasma (same initial source). Sensitivity and specificity of the ICT using DBS were determined by using ICT results from traditional blood collection samples for comparison. After 1 month, DBS samples showed 100% sensitivity and specificity when compared to ICT results on thawed plasma samples collected by traditional venipuncture. After six months storage at room temperature, DBS samples demonstrated 79% sensitivity and 100% specificity compared to traditional blood collection. Results from this study indicate that dried blood spot collection may be a useful tool for screening dogs for antibodies to L. infantum with the ICT assay.

Survival of a feline isolate of Tritrichomonas foetus in water, cat urine, cat food and cat litter.

Feline intestinal trichomoniasis caused by Tritrichomonas foetus is associated with large bowel diarrhea in cats from many parts of the world. It has long been recognized as an economically important sexually transmitted disease that causes early abortion in cattle. Isolates of T. foetus from cattle are infectious for the large intestine of cats and isolates of T. foetus from cats are infectious for the reproductive system of cattle. The parasite is maintained by fecal-oral transmission in cats. The present study was conducted to examine the survival of a feline isolate of T. foetus, AUTf-12, under various conditions that are relevant to fecal-oral transmission in cats. Trophozoites were grown in TYM medium and then exposed to water, cat urine, dry cat food, canned cat food, clumping cat litter, or filter paper for various lengths of time and then re-cultured in TYM medium. Trophozoites survived exposure to distilled or tap water for 30 but not 60 min, while they survived for at least 180 min in urine. Trophozoites survived for 30 min on dry cat food but survived for 120-180 min in canned cat food. No survival of trophozoites was observed on cat litter but trophozoites survived for 15 min when placed on filter paper. Our results indicate that T. foetus can survive and be potentially infectious in water, urine, dry cat food and canned cat food.

Toxoplasma gondii and Trypanosoma cruzi Antibodies in Dogs from Virginia

Toxoplasma gondii and Trypanosoma cruzi are zoonotic protozoan parasites that cause disseminated infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and Tr. cruzi in a population of dogs from Virginia. Serum samples were tested from 90 domestic dogs collected from animal shelters in Virginia. Using an indirect immunofluorescent antibody test, sera were examined at a 1 : 50 dilution and antibodies to T. gondii were found in 19 dogs (21%). Antibodies to Tr. cruzi were determined by qualitative immunochromatographic dipstick assay. One (1%) of the 90 dogs had Tr. cruzi antibodies and it was also seropositive for T. gondii. Our findings indicate that dogs are frequently exposed to T. gondii in Virginia, but that antibodies to Tr. cruzi are rare in the same geographical region.

Seroprevalence of Canine Leishmaniasis and American Trypanosomiasis in Dogs from Grenada, West Indies

Abstract Canine leishmaniasis and American trypanosomiasis (AT) are caused by related hemoflagellated parasites, Leishmania spp. and Trypanosoma cruzi, which share several common host species. Dogs are reservoirs for human infections by both pathogens. We determined the prevalence of antibodies to Leishmania spp. and T. cruzi in dogs from Grenada, West Indies. We examined 70 dog sera using the qualitative immunochromatographic dipstick tests (ICTs) based on recombinant antigens specific for visceral leishmaniasis and AT. Antibodies to visceral Leishmania were not detected in Grenadian dogs by ICT. Using the canine dipsticks for AT, antibodies to T. cruzi were determined in 3 (4.3%) of the 70 dogs. Results from this study indicate that dogs in Grenada are exposed in low levels to T. cruzi, but not to visceral Leishmania spp. at all.

Isolation, mouse pathogenicity, and genotyping of Trypanosoma cruzi from an English Cocker Spaniel from Virginia, USA

Trypanosoma cruzi was demonstrated in blood smears and heart tissue from a 5-year old, female, English Cocker Spaniel that had never been outside of the state of Virginia, USA. Plasma from the dog was positive in a commercially available immunochromatographic dipstick assay for T. cruzi and negative in an immunochromatographic dipstick assay for visceral Leishmania spp. The plasma from the dog had an indirect immunofluorescent antibody titer of 1:800 against epimastigotes of T. cruzi while the titer was 1:50 against promastigotes of L. infantum. The parasite was isolated from the blood in vitro from the dog (TcVT-1 isolate) and used to experimentally infect female C3H and ICR mice. The parasite was nonpathogenic for experimentally inoculated mice. DNA was isolated from parasites grown in vitro and used to determine that the genotype of T. cruzi present in the dog was genotype TcIV. This genotype is common in raccoons, Procyon lotor, in North America and suggests that raccoons may serve as reservoirs for canine infection.

High pressure processing treatment prevents embryonation of eggs of Trichuris vulpis and Ascaris suum and induces delay in development of eggs.

High hydrostatic pressure processing (HPP) is an effective non-thermal treatment used to inactivate pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms and protozoans but has had limited study on metazoans. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and preventing embryonation of Ascaris suum eggs. We conducted experiments using eggs of the canine whipworm Trichuris vulpis collected from naturally infected dogs and A. suum eggs from naturally infected pigs. We observed a delay in development of eggs of T. vulpis in a preliminary experiment and conducted 2 experiments to test the hypothesis that appropriate HPP levels can induce a delay in embryonation of nematode eggs. In experiment 1, nonembryonated T. vulpis eggs in tap water were packaged in sealable bags and exposed to 138-600 megapascals (MPa; 1 MPa=10 atm=147 psi) for 60s in a commercial HPP unit. In a second experiment, nonembryonated eggs of A. suum were exposed to 138-600 MPa and treated for 60s in the same commercial HPP unit. Embyronation of T. vulpis eggs was delayed by 4 and 5 days for eggs treated with 207 and 241 MPa but eventually eggs developed and the numbers of embryonated eggs was similar to controls on day 55. Embryonation of T. vulpis eggs treated with 345 or 350 MPa was delayed by 9 days and never reached more than 5% of eggs embryonated. On day 55 post treatment, 95% of control nontreated T. vulpis eggs were embryonated, 100-65% of eggs treated with 138-276 MPa were embryonated, a maximum of 5% of eggs treated with 345-350 MPa were embryonated, and 0% of eggs treated with ≥ 400 MPa were embryonated. T. vulpis eggs treated with ≥ 400 MPa did not undergo cell division. Embryrnation of A. suum eggs was delayed by 4, 10, and 16 days for eggs treated with 207, 241, and 250MPa, respectively, compared to nontreated control eggs. A. suum eggs treated with 207 MPa eventually embryonated to similar % embryonation values as controls and 138 MPa treated eggs but eggs treated with 241 or 250 MPa were always <5% embryonated. A. suum eggs treated with ≥ 300 MPa did not undergo cell division. On the final day of examination at day 56 after treatment, the % of embryonated eggs was 92% nontreated controls, 94% treated with 138 MPa, 84% treated with 207 MPa, 2% treated with 241 or 250 MPa, and 0% treated with 276, 200, 345, 400, or 414 MPa, respectively.

Survey of dogs from Vietnam for antibodies to visceralizing Leishmania spp.

Abstract Cases of visceral leishmaniasis, one of the most neglected tropical diseases, are increasing globally. Dogs are considered an important reservoir host for visceral leishmaniasis in people. The first cases of human visceral leishmaniasis in Vietnam have recently been reported. Blood samples were collected from 41 dogs in rural Vietnam. Sera were examined for antibodies to visceralizing Leishmania spp. by canine immunochromatographic strip assays based on recombinant K39 antigen. Antibodies to Leishmania spp. were not detected in any of the dogs tested. Results from this study suggest that rural dogs are not likely to be involved in the emergence of human visceral leishmaniasis in Vietnam.

Prevalence of IgG Antibodies to Toxoplasma gondii in Veterinary and Undergraduate Students at Virginia Tech, Blacksburg, Virginia.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects humans and other warm‐blooded vertebrates. Felids are the only definitive host for T. gondii, and they excrete oocysts in their faeces. The national prevalence in humans is declining in the United States. This zoonotic organism is of particular interest due to its importance in pregnant women, in individuals with altered immune systems, and in reactivated ocular infections. Exposure to the parasite in humans is usually associated with consumption of raw or undercooked meat or by accidental ingestion of oocysts. It was hypothesized that veterinary students would have a greater chance at exposure to the parasite than an average population of undergraduate students due to increased contact with cats who are infected. A commercially available ELISA was used to examine serum samples from 336 students (252 veterinary students and 84 undergraduate students) at Virginia Polytechnic Institute and State University and the Virginia‐Maryland Regional College of Veterinary Medicine for serum IgG antibodies to T. gondii antigen. The prevalence of T. gondii in these subjects was 5.6% in veterinary school students (n = 252) and 2.4% in undergraduates (n = 84). There was no significant difference (P > 0.05) in the prevalence of T. gondii antibodies in veterinary versus undergraduate students. The overall prevalence of 4.8% in all students in this study reflects the continuing decline of antibodies to T. gondii in humans in the United States.