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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #421090


Location: Animal Parasitic Diseases Laboratory

2014 Annual Report

1a. Objectives (from AD-416):
Objective 1: Evaluate the impact of changing management and production practices (e.g. free range, confined, organic) on the incidence of Toxoplasmosis and Trichinella in swine, as it relates to foodborne risk. Objective 2: Assess the effectiveness of on-farm interventions (such as passive immunization therapy as a feed supplement) to prevent enteric Toxoplasma infection in swine, consequent foodborne risk, and potential interaction with Salmonella foodborne infections in swine. C.1., PS 1B, and PS 1.D, Section 4.1. Objective 3: Evaluate the impact of anthelminthic and antiprotozoal treatments on parasitic foodborne infections in swine and the potential foodborne risk. Evaluate the impact of anthelminthic and antiprotozoal treatments on the interaction between foodborne pathogen and foodborne parasitic infections, specficially coinfections of Toxoplasma, Trichinella with Salmonaella and Campylobacter in swine, and changes following treatments. C.1., P.S., 1.A, and P.S., 1.D.

1b. Approach (from AD-416):
Toxoplasma gondii infects 11-20% of the U.S. population, causing birth defects in exposed pregnant women, devastating disease in immunocompromised individuals, and illness and loss of vision in otherwise healthy adults. Consumption of infected pork may be a significant source of infection for consumers in the U.S. Meat derived from pasture-raised pigs is of particular concern, since prevalence in these pigs may exceed 50%. Reducing the risk of foodborne human infection from meat requires adherence to livestock production practices that prevent exposure of animals to the parasite at the farm level, and the development of new treatments which can be used in pasture-raised pigs to prevent infection. Trichinella spiralis is a serious zoonotic pathogen with an unusually broad host and geographic range. Trichinella species infecting game animals pose a risk to humans consuming these meat products as well as a risk to domestic pigs that feed on their carcasses. Understanding the risk to pigs that have access to infected wildlife is an important component of on-farm certification efforts. In addition, the safety of meat from pasture-raised swine needs to be assessed in light of increasing consumer demand for organically-raised meat products. We will evaluate the impact of different management and production practices (e.g., free range, confinement, organically-raised pigs) on the incidence of Toxoplasma and Trichinella in swine, as it relates to foodborne risk, and assess the effectiveness of on-farm interventions (such as passive immunization therapy as a feed supplement) to prevent enteric Toxoplasma infection in swine, consequent foodborne risk, and potential interaction with Salmonella foodborne infections in swine.

3. Progress Report:
A cross-sectional serological survey was completed to estimate the seroprevalence of Trichinella spp. and Toxoplasma gondii, and risk factors associated with infection in feral pigs in the U.S. Serum samples were tested from 3247 feral pigs from 32 states. The overall seroprevalence of antibodies to Trichinella spp. and T. gondii, indicating infection, was 3.0% and 17.7%, respectively. A small proportion of feral pigs (0.7 %) was seropositive for both parasites. Toxoplasma seropositive feral pigs were widespread across the South and Midwest, and more restricted in the arid West. Trichinella infection was significantly higher in the South than in the Midwest, and higher in the Midwest than in the West region (p<0.05). Species distribution modeling indicated that the most probable distribution areas for both parasites are similar, concentrated mostly in the South and the Midwest regions of the U.S. For acceptance of pork produced in the U.S. Trichinae Certification Program, recent legislation in the European Union endorses surveillance of wildlife indicator populations; this project provides data to establish feral pigs as an indicator population for Trichinella in support of export marketing efforts to answer this requirement from our trading partners (Obj. 1.2). Conducted a survey in collaboration with Animal and Plant Health Inspection Service (APHIS)-Wildlife Services, revealing that 28.4% of 984 sampled feral pigs were seropositive for Toxoplasma and 2.9% were seropositive for Trichinella. Of 330 tongues collected from these sampled animals, 1.81% were tissue positive for Trichinella spiralis muscle larvae; no other species or genotypes were found. These data demonstrate that feral pigs serve as a reservoir of infection for Trichinella spiralis and Toxoplasma gondii, both for sylvatic carnivores and domestic pigs. The potential exists for introduction of these pathogens into domestic herds of non-biosecure domestic pigs as a result of increasing overlap of the range of feral pigs with non-biosecure domestic pigs production facilities in the U.S. (Obj. 1.2). Conducted quarterly training program for packer analysts for approved direct detection methods for T. spiralis in pork and horsemeat. Conducted testing of analysts and evaluated test results in consultation with the APHIS and the Agricultural Marketing Service (AMS) to maintain integrity of the analyst training program. These efforts support export marketing efforts as requested by USDA regulatory agencies (Obj.1.2). The minimal effective dose of mebendazole on encysted muscle larvae (ML) of Trichinella spiralis was estimated. Mebendazole treatment of pigs with 5, 50, or 100 mg/kg every other day for 3 days was not effective in reducing numbers of recovered larvae or inactivating ML, while 50 mg/kg for 5 days was effective, as was 83 mg/kg for 3 days. These data provide a means to evaluate the efficacy of anthelmintic treatment on the viability of Trichinella ML in pig tissues and provide a framework for elimination of Trichinella risk in pigs raised in uncontrolled management systems (Obj. 3.1). Completed the National Animal Health Monitoring System 2012 swine serological survey for Trichinella and Toxoplasma, testing 5688 samples in collaboration with APHIS-Center for Epidemiology and Animal Health. Seroprevalence for Toxoplasma was found to be 3.79%. A single Trichinella positive sample was found from pig with outdoor access on a poorly managed farm; this is the first Trichinella positive pig found during the NAHMS survey since 1995. This survey determined the seroprevalence of Trichinella and Toxoplasma in the national swine herd, and provide data on farm management practices that impact pathogen prevalence. These data serve to demonstrate the extremely low prevalence of Trichinella in the national swine herd, assuring trading partners of the safety of U.S. pork (Obj. 2). Continued National Retail Meat Survey for Toxoplasma in organic pork and American lamb. The survey will identify the risk of Toxoplasma infection to consumers from these meat products using samples collected from 25 MSAs nationwide. These data will be used to discern consumer risk from meat products from animals raised in uncontrolled management systems (Obj. 3.2).

4. Accomplishments
1. Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice. Why some people become sick with toxoplasmosis whereas others remain asymptomatic is not fully known. The parasite genetic characteristics are considered one determinant factor. There are many genetic types of Toxoplasma, divided into different haplogroups. Recent studies indicated the predominance of T. gondii haplogroup 12 in wildlife in USA. But still little is known of the genetic diversity of this parasite circulating in wildlife. In the present study, we tested coyotes (Canis latrans), red foxes (Vulpes vulpes), white-tailed deer (Odocoileus virginianus), and geese (Branta canadensis) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 160 of 367 animals, including 92 (34.5%) of 266 coyotes, 49 (62.0%) of 79 white-tailed deer, 17 (85.0%) of 20 red fox, and two of two Canada geese tested by the modified agglutination test (cut off titer 1:25). Tissues from 105 seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 29 animals, including 10 of 53 coyotes, 11 of 16 foxes, seven of 49 deer, and one of one goose. Three isolates were acute virulent to mice, and two others expressed intermediate virulence. To determine the extent of genetic variation of these as well as a few recently reported virulent isolates from wildlife in North America, intron sequences were generated. Analysis of intron sequences and PCR-RFLP genotyping results indicated that the #216 isolates are likely derived from recombination of the clonal type I and III lineages. To determine if T. gondii virulence can be predicted by typing, we genotyped a collection of strains. An association of genotypes of ROP5 and ROP18 with mouse-virulence was thereby demonstrated, however, additional genes may also contribute to virulence in distinct T. gondii genotypes, and virulent forms of Toxoplasma circulate in wildlife.

2. Check sample program for Trichinella. For pork to be exported to the European Union, a major trading partner, each swine carcass must be individually tested for the presence of Trichinella spiralis larvae by visual inspection of the meat in order to verify that no larval worms are present. Testing is performed at the packing plant by analysts trained and certified by ARS scientists in the Agricultural Marketing Service Analyst Training and Check Sample Program. In order to maintain integrity of the inspection program, test performance by each analyst is checked 4 times per year by ARS scientists using experimentally infected pork samples (proficiency samples). ARS scientists conducted a standardization trial for proficiency sample preparation for Trichinella infected pork. Optimal worm burden, mixing time, and test sensitivity at 3 infection levels were defined. ARS scientists determined, using an optimal larval density of 5 larvae per gram with a 2 hour mixing time, that test sensitivity was 100% when performed according to recommendations of the International Commission on Trichinellosis. Proficiency sample preparation for the AMS program was modified to reflect these findings. These efforts support on-farm certification and export marketing efforts, as requested by USDA regulatory agencies, through determination of risk to domestic pigs from Trichinella.

3. Isolation and characterization of new genetic types of Toxoplasma gondii and prevalence of Trichinella murrelli from black bear (Ursus americanus). Black bears (Ursus americanus) are hosts for two important zoonotic parasites, Toxoplasma gondii and Trichinella spp., and bears are hunted for human consumption in the USA. Little is known of the genetic diversity of T. gondii circulating in wildlife. In the present study, antibodies to T. gondii were found in juice from tongues of 17 (25.7%) of 66 wild black bear from Maryland during the hunting season of 2010 and 2011. Antibodies to T. gondii were assessed by the modified agglutination test. Tongues of 17 seropositive bears were bioassayed in mice and viable T. gondii was isolated from three samples. These three T. gondii isolates (TgBbMd1-3) were further propagated in cell culture and DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers. Results revealed three genotypes, one of which was highly virulent for outbred mice; all infected mice died of acute toxoplasmosis. Thus, several strains of T. gondii, virulent to mice, are circulating in wildlife in the USA. These 66 tongues, in addition to tongues collected during hunts in previous years, were further investigated for the presence of muscle larvae of Trichinella spp. Two bears were infected with Trichinella murrelli.

4. Assessment of Toxoplasma gondii in goat’s meat, milk, and cheese role in transmission of toxoplasmosis to humans. The consumption of unpasteurized goat cheese and milk has been suggested as a risk factor for toxoplasmosis in humans. ARS researchers in Beltsville, MD studied the survival of Toxoplasma gondii in milk and cheese. Eight goats were inoculated orally with 300-10000 oocysts of T. gondii strain TgGoatUS26. Milk samples were collected daily up to 30 days post infection (p.i.) and bioassayed in mice and cats. By mouse bioassay, T. gondii was detected in milk from all eight goats. The T. gondii excretion in milk was intermittent. Results indicate that T. gondii can be excreted in goat milk, and can survive in fresh cheese made by cold-enzyme treatment. To prevent transmission to humans or animals, milk should not be consumed raw. Raw fresh goat cheese made by cold-enzyme treatment of unpasteurized milk should also be not consumed.

5. Periodic national commodity studies to investigate current issues and examine general health and management practices used on farms. Toxoplasmosis is the cause of economic losses to sheep producers across the U.S. and is also a threat to human health. Here, a survey was conducted on over 4,000 market age lambs (3-12 months of age) from 359 operations in the 22 largest sheep-producing States. Sera were tested for antibodies to T. gondii using the modified agglutination test (MAT). A titer of 25 or above was observed in almost 12% of animals, and at least one positive sample was found in half of the pastured flocks. Lambs in the west had the greatest seroprevalence (17.9%) followed by the east (10.6%) and central U.S. (6.1%). The seroprevalence was 13.1% in small and medium operations with 20-499 ewes and 6.4% in operations with 500 or more ewes. By flock type, seroprevalence was 13.8% in operations that managed their sheep on pasture compared with 4.4% in open range flocks. Of the 359 operations, 45.4% (163) had at least one seropositive sample. By flock size, operation-level prevalence was greatest among operations with 100-499 ewes (48.6%), followed by operations with 20-99 ewes (45.6%) and 500 or more ewes (35.3%). Operation-level prevalence was greatest among operations in the west (58.8%) followed by the east (42.6%) and central U.S. (31.7%). Toxoplasma titers increase with age, which may partially account for this difference along with the size, type and location of the flocks of origin. Increased producer education is needed to reduce the prevalence of T. gondii in sheep operations nationwide.

6. Toxoplasma gondii oocysts in aquatic environments. Indigenous California blue mussels (Mytilus spp.) were evaluated for their use as biosentinels to monitor the occurrence of Toxoplasma gondii and Cryptosporidium spp. in marine waters. A new method was refined to extract oocyst DNA from mussel hemolymph, gills, and digestive gland tissues, followed by PCR-based detection of these pathogens. This method was able to consistently detect as few as 10 oocysts. A survey of indigenous mussels from Point Lobos and Morro Bay, California, areas known to have high T. gondii seroprevalence in sea otters, were indeed positive for T. gondii (54 % and 33 %, respectively). Mussels from Point Lobos, CA were also contaminated (31 %) with Cryptosporidium oocysts. Phylogenetic analysis using the 18S rRNA gene revealed two distinct Cryptosporidium parvum-like subtypes. These results demonstrate a novel method to isolate parasite DNA from mussel tissue, and demonstrated the feasibility of using indigenous Mytilus spp. as biosentinels for monitoring microbial water quality. More importantly, using this method, a high prevalence of Cryptosporidium spp. and T. gondii oocyst DNA was detected in mussels collected from the California coastline, suggesting widespread environmental contamination.

Review Publications
Dubey, J.P., Choudhary, S., Ferreira, L., Kwok, O.C., Butler, E., Carstensen, M., Yu, L., Su, C. 2013. Isolation and genetic characterization of Toxoplasma gondii from the gray wolf Canis lupus. Veterinary Parasitology. 197:685-690.
Dubey, J.P., Rosenthal, B.M. 2013. Letter to the editor: Identification of Sarcocystis capracanis in cerebrospinal fluid from sheep with neurological disease. Veterinary Parasitology. 197:407-408.
Dubey, J.P., Alvarado-Esquivel, C., Herrera-Valenzuela, V., Gayosso-Dominguez, E., Oliveira, S., Verma, S., Choudhary, S., Kwok, O.C., Su, C. 2013. A new atypical genotype mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught puma (Felis concolor) from Durango, Mexico. Veterinary Parasitology. 197:674-677.
Dubey, J.P., Randall, A., Choudhary, S., Ferreira, L., Verma, S., Oliveira, S., Kwok, O.C., Su, C. 2013. Occurrence, isolation, and genetic characterization of Toxoplasma gondii from white tailed deer (Odocoileus virginianus) in New Jersey. Journal of Parasitology. 99:763-769.
White, L., Schuler, K., Thomas, N., Webb, J., Saliki, J., Ip, H., Dubey, J.P., Frame, E. 2013. Pathogen exposure and blood chemistry in the Washington population of northern sea otters (Enhydra lutris kenyoni). Journal of Wildlife Diseases. 49:887-899.
Choudhary, S., Zieger, U., Sharma, R., Chikweto, A., Tiwari, K., Ferreira, L., Oliveira, S., Barkley, L., Verma, S., Kwok, O.C., Su, C., Dubey, J.P. 2013. Isolation and genetic characterization of Toxoplasma gondii in the mongoose (Herpestes auropunctatus) in Grenada, West Indies. Journal of Zoo and Wildlife Medicine. 44:127-1130.
Bushkin, G., Motari, E., Carpienteri, A., Dubey, J.P., Costello, C., Robbins, P., Samuelson, J. 2013. Acid-fast lipids are important structural components of oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. mBio. DOI: 10.1128/mBio.003787.
Hoon-Hanks, L., Regan, D., Dubey, J.P., Porter, M., Duncan, C. 2013. Hepatic neosporosis in a dog treated for pemphigus foliaceus. Journal of Veterinary Diagnostic Investigation. 25:807-810.
Alvarado-Equivel, C., Silva-Aguilar, D., Villena, I., Dubey, J.P. 2013. Seroprevalence and correlates of Toxoplasma gondii infection in domestic sheep in Michoacán State, Mexico. Preventive Veterinary Medicine. 112:433-437.
Dubey, J.P., Wilpe, V., Blignaut, D., Williams, J. 2013. Development of early tissue cysts and associated pathology of Besnoitia Besnoiti in a naturally infected bull (Bos Taurus) from South Africa. Journal of Parasitology. 99:459-466.
Alvarado-Esquive, C., Silva-Aguilar, D., Villena, I., Dubey, J.P. 2013. Seroprevalence of Toxoplasma gondii infection in dairy goats in Michoacan, Mexico. Journal of Parasitology. 99:540-542.
Lopes, A., Sousa, S., Dubey, J.P., Ribeiro, A., Silvestre, R., Cotovio, M., Schallig, H., Cardosa, L., Cordeiro-Da-Silva, A. 2013. Prevalence of antibodies to Leishmania infantum and Toxoplasma gondii in horses from the north of Portugal. Parasites & Vectors. 6:178.
Gayosso-Dominguez, C., Gayosso-Dominguez, E., Villena, I., Dubey, J.P. 2013. Seroprevalence of Toxoplasma gondii infection in captive mammals in three zoos in Mexico City, Mexico. Journal of Zoo and Wildlife Medicine. 44:803-806.
Dumètre, A., Dubey, J.P., Ferguson, D., Bongrand, P., Azas, N., Puech, P. 2013. Mechanics of the Toxoplasma gondii oocyst wall. Proceedings of the National Academy of Sciences. 110:11535-11540.
Alvarado-Esquivel, C., Alvarado-Esquivel, D., Villena, I., Dubey, J.P. 2013. Seroprevalence of toxoplasma gondii infection in domestic rabbits in Durango State, Mexico. Preventive Veterinary Medicine. 111:325-328.
Dubey, J.P., Hill, D.E., Zarlenga, D.S., Choudhary, S., Ferreira, L., Oliveira, S., Verna, S., Kwok, O.C., Driscoll, C., Spiker, H., Su, C. 2013. Isolation and characterization of new genetic types of toxoplasma gondii and prevalence of trichinella murrelli from black bear (Ursus americanus). Veterinary Parasitology. 196:24-30.
Dubey, J.P., Choudhary, S., Tilahun, G., Tiao, N., Gebreyes, W., Su, C. 2013. Genetic diversity of Toxoplama gondii isolates from Ethiopian feral cats. Veterinary Parasitology. 196:206-208.
Siqueira, D.B., Alessio, F.M., Mauffrey, J.F., Marvulo, M.F., Ribeiro, V.O., Pena, H.J., Gennari, S.M., Faustino, M.A., Alves, L.C., Gennari, S.M., Dubey, J.P., Silva, J.C. 2013. Seroprevalence of Toxoplasma gondii in wild marsupials and rodents from the Atlantic forest of Pernambuco State, Northeastern region, Brazil. Journal of Parasitology. 99:1140-1143.
Al-Kappany, Y., Abu-Elwafa, S., Hilali, M., Rosenthal, B.M., Dubey, J.P. 2013. Experimental transmission of Sarcocystis muris (Apicomplexa: Sarcocystidae) from the feces of a naturally infected feral cat (Felis catus) to immunocompetent and immunocompromised mice. Journal of Parasitology. 99(6):997-1001.
Yeargan, M., Alvarado-Esquivel, C., Dubey, J.P., Howe, D. 2013. Prevalence of antibodies to Sarcocystis neurona and Neospora hughesi in horses from Mexico. Parasite. 20:29.
Dubey, J.P. 2013. The history and life cycle of Toxoplasma gondii. In: Weiss, L.M. and Kim, K., editors. Toxoplasma gondii, The model apicomplexan: Perspective and methods. 2nd edition. Waltham, MA: Elsevier. p. 1-14.
Aston, E., Mayer, P., Bowman, D., Mohammed, H., Liotta, J., Kwok, O.C., Dubey, J.P. 2014. Use of filter papers to determine seroprevalence of Toxoplasma gondii among hunted ungulates. International Journal for Parasitology: Parasites and Wildlife. 3:15-19.
Dubey, J.P., Why, K., Verma, S., Choudhary, S., Kwok, O.C., Khan, A., Behinke, M., Sibley, L., Ferreira, L., Weaver, M., Stewart, R., Su, C. 2014. Genotyping Toxoplasma gondii from wildlife in Pennsylvania and identification of natural recombinants virulent to mice. Veterinary Parasitology. 200(2014):74-84.
Vitaliano, S., Soares, H., Pena, H., Dubey, J.P., Gennari, S. 2014. Serological evidence of Toxoplasma gondii infection in wild birds and mammals from Southeast region of Brazil. Journal of Zoo and Wildlife Medicine. 45(1):197-199.
Dubey, J.P., Jenkins, M.C., Kwok, O.C.H., Ferreira, L.R., Choudhary, S., Verma, S.K., Villena, I., Butler, E., Carstensen, M., 2013. Congenital transmission of Neospora caninum in white tailed deer (Odocoileus virginianus). Vet Parasitol. 196:519-522.
Dubey, J.P., Dennis, P., Verma, S., Choudhary, S., Ferreira, L., Oliviera, S., Kwok, O.C., Butler, E., Carstensen, M., Su, C. 2014. Epidemiology of toxoplasmosis in white tailed deer (Odocoileus virginianus): occurrence, congenital transmission, correlates of infection, isolation, and genetic characterization of Toxoplasma gondii. Veterinary Parasitology. 202:27-275.
Manciant, F., Nardoni, S., Papini, R., Mugnaini, L., Martini, M., Altomonte, I., Salari, F., D'Ascenzi, C., Dubey, J.P. 2014. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus). Parasites & Vectors. 7:165.
Dubey, J.P., Black, S., Verma, S., Calero-Bernal, R., Morris, E., Hanson, M., Cooley, J. 0204. Sarcocystis neurona schizonts-associated encephalitis,chorioretinitis, and myositis in a two-month-old dog simulating toxoplasmosis, and presence of mature sarcocysts in muscles. Veterinary Parasitology. 202:194-2000.
Alvarado-Esquivel, C., Romero-Salas, D., Garci-Vazquez, Z., Crivelli-Diaz, M., Barrientos-Morales, M., Lopez-De-Buen, L., Dubey, J.P. 2014. Seroprevalence and correlates of Toxoplasma gondii infection in domestic pigs in Veracruz State, Mexico. Tropical Animal Health and Production. 46:705-709.
Dubey, J.P., Jenkins, M.C., Ferreira, L., Choudhary, S., Verma, S., Kwok, O.C., Fetterer, R.H., Butler, E., Carstensen, M. 2014. Isolation of viable neospora caninum from brains of wild gray wolves (canis lupus). Veterinary Parasitology. 201:150-153.
Bartova, E., Machacova, T., Di Loria, A., Sedlak, K., Mariani, U., Fusco, G., Fulgione, D., Veneziano, V., Dubey, J.P. 2014. Toxoplasma gondii seroprevalence in donkeys (Equus asinus) from Italy. Journal of Veterinary Medical Science. 76(2):265-267.
Solange, G., Ogrzewalska, M., Soares, H., Saraiva, D., Pinter, A., Labruna, M., Dubey, J.P. 2014. Occurrence of Toxoplasma gondii antibodies in birds from the Atlantic Forest, state of São Paulo, Brazil. Veterinary Parasitology. 200:193-197.