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United States Department of Agriculture

Agricultural Research Service

Research Project: Integrated Approach to the Detection and Control of Foodborne Parasites and the Impact on Food Safety

Location: Animal Parasitic Diseases

2012 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:
For Objective 1, to determine the effect of primary exposure of pigs to sylvatic species of Trichinella on a challenge infection with T. spiralis muscle larvae (ML), pig muscle samples were enzyme digested to isolate ML 45 days after the challenge infection for determination of worm burden and for analysis of Trichinella genotype composition. Cytokine gene expression by leukocytes from pig whole blood, jejunum, and mesenteric lymph node was analysed after the primary infection and after the challenge. These data are needed to determine the risk to domestic swine from sylvatic species of Trichinella circulating in the environment and consequent risk to consumers. Also for Objective 1, >2500 feral swine serum samples were tested for prevalence estimates of Trichinella and Toxoplasma. In collaboration with Animal and Plant Health Inspection Service (APHIS), over 200 tissue samples were obtained from feral swine for isolation and genotyping of Trichinella ML. Recent legislation in the European Union endorses surveillance of wildlife indicator populations for Trichinella; this project provides data to establish feral swine as an indicator population in support of the U.S. Trichinae Certification Program. A quarterly training and testing program was conducted for packer analysts for approved direct detection methods for T. spiralis in pork. Evaluated test results in consultation with the Agricultural Marketing Service to maintain integrity of the program. These efforts support on-farm certification and export marketing efforts as requested by USDA regulatory agencies. For Objective 2, Toxoplasma infections in mice and cats were initiated to generate infective stages for determination of target level of effective dose of hyperimmune egg yolk (HEY) preparation for prevention of enteric Toxoplasma infection in pigs. A 'proof of principal' study in rats demonstrated that HEY immunotherapy substantially reduces intestinal invasion by T. gondii sporozoites. These data are needed to standardize antibody titers in egg yolk preparations for testing HEY therapy in pigs. For Objective 3, pigs were infected with T. spiralis ML for analysis of effect of larval age on mebendazole activity against encysted ML. Mebendazole treatment of pigs 45 days after infection rendered ML non-viable. Mature nurse cells, older larvae, circulating larvae, and younger nurse cells may be impacted differently by anthelmintic exposure, therefore analysis of the effect of mebendazole on shorter and longer term infections (< and > than 45 days) are being conducted. These data provide a means to evaluate the efficacy of anthelmintic treatment on the viability of Trichinella ML in pig tissues. For Also for Objective 3, pig antibody isotype responses to infection with Toxoplasma, Trichinella, Trichuris and Salmonella were characterized. These data will be used to discern changes during experimentally induced co-infections with helminth and protozoan parasites to evaluate the impact of anthelmintic and antiprotozoal treatments on secondary bacterial infection and pathogen expansion in the swine gut.


4.Accomplishments
1. Demonstrated a protective effect in domestic pigs against infectious Trichinella genotypes by prior exposure to sylvatic genotypes of Trichinella that are carried by feral pigs. To determine if a primary infection with North American sylvatic genotypes of Trichinella (to which domestic pigs are resistant) could protect pigs against a challenge infection with T. spiralis (to which domestic pigs are susceptible), pigs were infected with T. nativa, T. pseudospiralis, and T. murrelli, and then challenged with T. spiralis. Pigs that were exposed to encapsulated genotypes (T. nativa, and T. murrelli) were protected against a challenge infection with T. spiralis, while an un-encapsulated genotype (T. pseudospiralis) provided little protection. Antibody isotype and cytokine gene expression analysis of blood and intestinal tissues from protected (T. nativa, and T. murrelli) versus unprotected animals (T. pseudospiralis) determined that anti-inflammatory, or TH-2 immune, effectors were heightened in early infection in the protected pigs. These data demonstrate that pigs can be rendered resistant to T. spiralis infection by immunization.

2. The U.S. State Department funded Biosecurity Engagement Program on Trichinella spiralis was expanded in the Republic of the Philippines. In collaboration with the Bureau of Animal Industry, Philippine Animal Health Center (PAHC), Republic of the Philippines, a validated Trichinella testing facility was established at PAHC, Quezon City, Luzon and a nationwide prevalence study for Trichinella spiralis in market hogs was conducted. The sample storage capacity of PAHC was expanded for establishment of a swine serum bank for prevalence studies on other swine pathogens of interest. This project provides for improved disease surveillance capacity for a trading partner which in turn reduces risks to U.S. consumers from food-borne pathogens and to U.S. agriculture from importation of foreign animal diseases.

3. Toxoplasma isolated from organically-raised pigs. Prevalence of T. gondii in organically-raised pigs in two locations (Farm 1 and Farm.
2)in Michigan was investigated. Serum and tissue samples from 33 pigs on the farm were available for T. gondii evaluation at slaughter. Serological testing was performed using both enzyme-linked immunosorbant assay (ELISA) and the modified agglutination test (MAT). Antibodies to T. gondii were detected by both ELISA and MAT in 30 of 33 animals. Hearts of all 33 pigs were bioassayed for T. gondii; T. gondii was isolated from 17 pigs including one from a seronegative (both ELISA and MAT) pig. Genetic typing of 16 of the 17 T. gondii isolates revealed clonal Type II from Farm 1 and clonal Type III on Farm 2. These results revealed very high prevalence of T. gondii in organic pigs for the first time in the U.S., indicating potentially increased health risk of consuming meat products from organically-raised swine.


Review Publications
Maksimov, P., Buschtons, S., Herrmann, D.C., Conraths, F.J., Gorlich, K., Tenter, A.M., Dubey, J.P., Nagel-Kohl, U., Thoms, B., Botcher, L., Kuhne, M., Schares, G. 2011. Serological survey and risk factors for Toxoplasma gondii in domestic ducks and geese in Lower Saxony, Germany. Veterinary Parasitology. 182:140-149.

Dubey, J.P., Lappin, M.R. 2012. Toxoplasmosis and Neosporosis. In: Infectious Diseases of Dogs and Cats. Philadelphia: Elsevier. p. 806-827.

Boyer, K., Hill, D.E., Mui, E., Wroblewski, K., Karrison, T., Dubey, J.P., Sautter, M., Noble, G., Withers, S., Swisher, C., Heydemann, P., Hosten, T., Babiarz, J., Lee, D., Mcleod, R. 2011. Unrecognized ingestion of Toxoplasma gondii oocysts causes congenital toxoplasmosis and epidemics in North America. Clinical Infectious Diseases. 53:1081-1089.

Dubey, J.P., Ferreira, L., Martins, J., Mc Cleod, R. 2012. Oral oocyst-induced mouse model of toxoplasmosis: Effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality. Parasitology. 193:1-13.

Dubey, J.P., Greene, C. 2012. Enteric coccidiosis. In: Infectious Diseases of Dogs and Cats. Philadelphia: Elsevier. p. 828-839.

Chikweto, A., Kumthekar, S., Tiwari, K., Nyack, B., Deokar, M.S., Stratton, G., Macpherson, C.N., Dubey, J.P. 2011. Seroprevalence of Toxoplasma gondii in pigs, sheep, goats, and cattle from Grenada and Carriacou, West Indies. Journal of Parasitology. 97:950-951.

Dubey, J.P., Thomas, N.J. 2011. Sarcocystis neurona retinochoroiditis in a sea otter (Enhydra lutris kenyoni). Veterinary Parasitology. 183:156-159.

Lopes, A.P., Dubey, J.P., Moutinho, O., Gargate, M., Vilares, A., Rodrigues, M., Cardoso, L. 2011. Seroepidemiology of Toxoplasma gondii infection in women from the North of Portugal in their childbearing years. Epidemiology and Infection. DOI: 10.1017/S0950268811001658.1-6.

Alvarado-Esquivel, C., Garcia-Machado, C., Vitela-Corrales, J., Villena, I., Dubey, J.P. 2011. Seroprevalence of Toxoplasma gondii infection in domestic goats in Durango State, Mexico. Veterinary Parasitology. 183:43-46.

Santos, P.S., Albuqurque, G.R., Da Silva, V.M., Martin, A.R., Souza, S.L., Ragozo, A.M., Nascimento, C.C., Gennari, S.M., Dubey, J.P. 2011. Seroprevalence of Toxoplasma gondii in free-living Amazon river dolphins (Inia geoffrensis) from central Amazon, Brazil. Veterinary Parasitology. 183:171-173.

Lindsay, D.S., Dubey, J.P. 2011. Toxoplasma gondii: The changing paradigm of congenital toxoplasmosis. Parasitology. 138:1829-1831.

Garcia-Bocanegra, I., Dubey, J.P., Martinez, F., Vargas, A., Cabezon, O., Zorrilla, I., Arenas, A., Almeria, S. 2012. Prevalence of Toxoplasma gondii and Neosport caninum antibodies in Spanish ibex (Capra pryenaica hispanica). The Veterinary Journal. 191:257-260.

Santos, P.S., Albuquerque, G.R., Da Silva, V.M., Martin, A.R., Marvulo, M.R., Souza, S.L., Souza, S.L., Ragozo, A.M., Nascimento, C.C., Gennari, S.M., Dubey, J.P., Silva, J.C. 2011. Seroprevalence of Toxoplasma gondii in free-living amazon river dolphins (Inia geoffrensis) from central Amazon, Brazil. Veterinary Parasitology. 183:171-173.

Alvarado-Esquivel, C., Garcia-Machado, C., Alvarado-Esquivel, D., Gonzalez-Salazar, A.M., Briones-Fraire, C., Vitela-Corrales, J., Villena, I., Dubey, J.P. 2011. Seroprevalence of Toxoplasma gondii infection in domestic pigs in Durango State, Mexico. Journal of Parasitology. 97:616-619.

Dubey, J.P., Ferreira, L., Martins, J., Jones, J.L. 2011. Sporulation and survival of Toxoplasma gondii oocysts in different types of commercial cat litters. Journal of Parasitology. 97:751-754.

Charles, R.A., Kjos, S., Ellis, A.E., Dubey, J.P., Shock, B.C., Yabsley, M.J. 2012. Parasites and vector-borne pathogens of southern plains woodrats (Neotoma micropus) from southern Texas. Parasitology Research. 110:1855-1862.

Dubey, J.P., Passos, L.M., Chelaiah, R., Ferreira, L., Gennari, S.M., Su, C. 2011. Isolation of viable Toxoplasma gondii from guinea fowl (Numida meleagris) and rabbits from Brazil. Journal of Parasitology. 97:842-845.

Cabezon, O., Garcia-Bocanegra, I., Molina-Lopez, R., Marco, I., Blanco, J.M., Hofle, U., Margalida, A., Bach-Raich, E., Darwich, L., Echeverria, I., Obon, E., Lavin, T., Dubey, J.P., Almeria, S. 2011. Seropostivity and risk factors associated with Toxoplasma gondii infection in wild birds from Spain. PLoS One. 6(12). DOI:6.E29549/Journal.Pone.

Alvarado-Esquivel, C., Sanchez-Okrucky, R., Dubey, J.P. 2012. Seroprevalence of Toxoplasma gondii infection in marine mammals in Mexico. Veterinary Parasitology. 184:321-324.

Lindsay, D., Dubey, J.P., Santin, M., Fayer, R. 2012. Coccidia and Other Protozoa. In: Zimmerman, J., Karriker, L., editors. Diseases of swine. 10th edition. San Francisco, CA: Wiley and Sons, Inc. p. 895-907.

Rajendran, C., Su, C., Dubey, J.P. 2012. Molecular genotyping of Toxoplasma gondii from Central and South America revealed highly diverse populations and suggested possible different origins of the three archetypal lineages. Infection, Genetics and Evolution. 12:356-368.

Su, C., Khan, A., Zhou, P., Majumdara, D., Ajzenberg, D., Darde, M., Zhu, X., Ajioka, J.W., Rosenthal, B.M., Dubey, J.P., Sibley, L.D. 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proceedings of the National Academy of Sciences. 5844-5849.

Alvarado-Esquivel, C., Gonzalez-Salazar, A.M., Alvarado-Esquivel, D., Ontiveros-Vazquez, F., Vitela-Corrales, J., Villena, I., Dubey, J.P. 2012. Seroprevalence of Toxoplasma gondii infection in chickens in Durango State, Mexico. Journal of Parasitology. 98:431-432.

Alvarado-Esquivel, C., Garcia-Machado, C., Alvarado-Esquivel, D., Vitela-Corrales, J., Villena, I., Dubey, J.P. 2012. Seroprevalence of Toxoplasma gondii infection in domestic sheep in Durango State, Mexico. Journal of Parasitology. 98:271-273.

Ness, S.L., Peters-Kennedy, J., Schares, G., Dubey, J.P., Mittel, L.D., Mohamme, H.O., Bowman, D.D., Fellipe, J.B., Wade, S.E., Shultz, N. 2012. Investigation of an outbreak of Besnoitiosis in donkeys in the northeastern United States. Journal of the American Veterinary Medical Association. 240:1329-1337.

Dubey, J.P., Yabsley, M. 2010. Besnoitia neotomofelis n. sp. (Protozoa: Apicomplexa) from the southern plains woodrat (Neotoma micropus). Parasitology. 137:1731-1747.

Rojas-Rivera, A., Estrada-Martinez, S., Sifuentes-Alvarez, A., Dubey, J.P. 2010. Seroepidemiology of Toxoplasma gondii infection in a mennonite community in Durango State, Mexico. Journal of Parasitology. 96:941-945.

Behnke, M., Kahn, A., Wootton, J., Dubey, J.P., Tang, K., Sibley, L.D. 2011. Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proceedings of the National Academy of Sciences. 108:9631-9636.

See, M., Staggs, S., Dubey, J.P., Villegas, E. 2012. Evaluation of four RNA extraction methods used for gene expression analysis of Cryptosporidium parvum and Toxoplasma gondii oocysts. Journal of Microbiological Methods. 89:189-192.

Last Modified: 10/20/2014
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