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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #430610

Research Project: Detection and Control of Foodborne Parasites for Food Safety

Location: Animal Parasitic Diseases Laboratory

2019 Annual Report

Objective 1: Refine current immunological assays to investigate rates of human exposure to oocysts of Toxoplasma gondii. Subobjective 1.A (Hill): Refine and validate the TgERP ELISA (Toxoplasma gondii Embryogenesis Related Protein) and a Luminex bead-based immunoassay for use in human and veterinary models. Subobjective 1.B (Hill): Evaluate other candidate antigens to enhance the ability to detect exposure to oocysts in individuals with either low (recent infection) or high avidity (chronic infection) antibodies. Subobjective 1.C (Hill): Using sera collected from Americans via NHANES, determine what proportion of those infected with Toxoplasma harbor antibodies to oocysts. Objective 2: Identify mitigation strategies that reduce Toxoplasma oocysts contamination on fruits and leafy greens. Subobjective 2A (Hill): Evaluate the effectiveness of bioassay, tissue culture, and PCR using apoptosis-specific targets for determination of viability of Toxoplasma oocysts after treatment with cold plasma, monochromatic blue light, pulsed light, laser enhanced acoustic waves, gaseous chlorine dioxide, and ozone to inactivate T. gondii oocysts from the surface of fruits, vegetables, and low moisture foods (LMF). Objective 3: Elucidate the molecular epidemiology and molecular genetics of environmental Toxoplasma oocyst contamination and define virulence and persistence of particular genotypes in food animals. Subobjective 3.A (Dubey): Evaluate whether there are genetically distinct subsets of T. gondii in swine and deer. Subobjective 3.B (Dubey, Rosenthal): Evaluate whether the T. gondii oocysts that account for most infections are derived from local, distinct, and genetically homozygous populations. Objective 4: Determine and validate methods for improved inactivation and surveillance of meat-borne exposure to Toxoplasma gondii and Trichinella sprialis. Subobjective 4.A (Hill, Dubey): Develop a model for pork dry curing processes, taking into account five common measurements monitored during curing – salt/brine concentration, water activity (aw), pH, temperature, and time, for inactivation of Trichinella spiralis, Toxoplasma gondii, and Salmonella. The study will be performed in two phases – an initial multi-factorial modeling phase using ARS’s Pathogen Modeling Program and low, internal, and high endpoints for common curing treatments, and a final validation phase. Subobjective 4.B (Hill): Support the technical aspects of the new National Surveillance Program for Trichinella by 1) assisting in the development a sampling framework; 2) development of a high throughput serological assay for Trichinella and Toxoplasma capable of providing the means to document prevalence to less than 1 infection per million pigs; 3) by evaluating more selective diagnostic antigens to improve sensitivity and specificity; and 4) by assisting in the investigation of any positive findings (tracebacks, genotyping).

Our project combines translational and applied research to improve monitoring and surveillance for zoonotic parasites, and develops models for their control. Fundamental research proposes to refine new immunological assays to detect human exposure to the oocyst stage of Toxoplasma, and to develop in vitro assays for Toxoplasma oocyst viability after curative treatment of fruits and vegetables. Applied research will develop methods to monitor and inactivate pathogens associated with pork products. Our overall goal is to mitigate the impact of these potentially harmful parasites, thereby protecting consumers and maintaining the vitality of the U.S. pork industry.

Progress Report
Progress was made on all project objectives, although certain objectives were cut short by the government shutdown, the retirement of the Project Lead, and a program redirection. Bead-based immunoassay was refined for detection of antibodies to Trichinella for use in human and veterinary models (Subobjective 1A) and international partners were identified to evaluate the ability to differentiate individuals with recent exposures to toxoplasmosis from those with chronic infection (Subobjective 1B). ARS developed an assay appropriate for addressing this question and has offered it to research partners who have documented a large-scale soil-borne outbreak including many individuals presumed to share a common, acute exposure. Program redirection will curtail ARS engagement in further developing the application of this assay. SY retirement and program redirection will preclude fulfillment of Subobjective 1C, seeking broader diagnosis among Americans to oocysts of T. gondii. Biosafety and Animal Welfare considerations induced refinement of Subobjective 2A, evaluating various means to inactivate oocysts of T. gondii from the surface of fruits, vegetables, and low moisture foods. Instead, non-zoonotic models of parasitic protozoa are being developed. In particular, Eimeria species are being developed as a model to understand ways to protect produce from contamination with the agent of cyclosporosis. Objective 3 was substantially met by a series of field studies of parasite occurrence and diversity, including major national surveys of parasites in deer and feral swine, as well as targeted studies of other animals such as bobcat, wild turkey, and livestock species. Importantly, clinically-significant infections of Americans were diagnosed with unexpected and disproportionate frequency to derive from unusual parasite genotypes. A series of studies in dry cured sausage and ham were completed providing generally-applicable models for eliminating the risk of viable foodborne parasites in uncooked pork (subobjective 4A). The goals of Subobjective 4B were substantially enlarged by means of a national serosurvey of US slaughterhouses for antibodies to foodborne parasites (in its final phase) and a major APHIS-supported effort to determine rates of trichinellosis in the domestic swine herd as judged by artificial digestion.

1. Guidelines to safeguard ready-to-eat meats from parasites compromising food safety. To date, assuring the safety of ready-to-eat meats such as dry-cured sausage required individual assessment of each curing process and assumed, without evidence, that methods effective at killing one parasite species also suffice for others. Here, USDA scientists at Beltsville, Maryland, worked with partners at the National Academy of Sciences and in Canada to discern how little salt serve to inactivate various parasite in sausages, and how quickly that desired result can be achieved. The results show that most current industry practices far exceed what is required, and permit producers to safely predict the adequacy of new formulations without the need for exhaustive and expensive testing. These data will also help producers safely meet the needs of Americans on low-salt diets.

2. Tracking a parasite’s dissemination in swine. Improved biosecurity has reduced the prevalence of zoonotic parasites in swine, but reliable risk assessment requires an understanding of how rapidly parasites disseminate through the body and how many parasites establish themselves in meat tissues. Therefore, USDA researchers at Beltsville, Maryland, working with partners at the University of Maryland, tracked the development of infections, learning that parasites encysted within a week. Even small small helpings of meat were found to harbor infectious tissue cysts, arguing that precautions should be used while handing pork, that all pork should be cooked thoroughly before human consumption, and that uncooked pork should never be fed to cats, who naturally amplify such infections. These results will be of interest to veterinarians, public health workers, and parasitologists, and will serve as the basis for refining quantitative assessments of food safety risk.

3. Unusual foodborne parasite strains are especially likely to sicken Americans. Two decades of research has suggested that American livestock typically harbor only a handful of frequently encountered lineages of a parasite important to food safety and women's reproductive health. Other parasite lineages, by contrast, have been discovered in wildlife, raising concerns as to their proclivity to cause human disease. Therefore, USDA scientists at Beltsville, Maryland, worked with partners at Stanford University, the University of Tennessee, the Palo Alto Medical Foundation, and with partners in France and Romania characterize the parasites responsible for severe human infections. They discovered that nearly half of the severe human cases came from unusual parasite strains. This finding puts the medical community on notice that this disease often derives from poorly-characterized, sometimes especially virulent forms, and focuses attention on environmental and food sources, other than pork, which may be responsible for many such infections.

4. Agricultural impact on the parasite diversity and virulence. Most emerging infectious diseases come from animals, but our human activities can strongly influence where they occur, how they prosper, and how much harm they can cause. USDA scientists at Beltsville, Maryland, worked with partners at the University of Tennessee and in Germany, China, France, and South Africa to determine how agriculture has influenced the epidemiology and the virulence an important parasitic threat to food safety and public health. Linking landscape ecology to parasite virulence, their novel framework contributes fundamental insights on the ecology and evolution of infectious disease, and suggest control strategies that would benefit veterinary and human health.

Review Publications
Murata, F., Cerqueira-Cezar, C., Kwok, O.C., Tiwari, K., Su, C., Sharma, R., Dubey, J.P. 2018. Role of rats (Rattus norvegicus) in the epidemiology of Toxoplasma gondii infection in Grenada, West Indies. Journal of Parasitology. 104(5):571-573.
Dubey, J.P., Schster, R. 2018. A review of coccidiosis in Old World camels. Veterinary Parasitology. 262:75-83.
Dubey, J.P. 2018. A review of Cystoisospora felis and C. rivolta-induced coccidiosis in cats. Veterinary Parasitology. 263:34-48.
Rosypal, A., Scott, D., Dubey, J.P., Lindsay, D. 2019. Prevalence of sarcocysts in the muscles of raptors from a rehabilitation center in North Carolina. Journal of Parasitology. 105:11-16.
Dubey, J.P., Cerqueira-Cezar, C., Murata, F., Mowery, J.D., Scott, D., Rosypal, A., Lindsay, D. 2019. Confirmation of Sarcocystis jamaicensis sarcocysts in IFN-gamma gene knock out mice orally inoculated with sporocysts from red-tailed hawk (Buteo jamaicensis). Journal of Parasitology. 105(1):143-145.
Dubey, J.P., Lindsay, D. 2019. re-evaluation of asynchronous asexual development of cystoisospora canis in intestines of dogs. Journal of Parasitology. 105:25-28.
Freppel, W., Ferguson, D., Shapiro, K., Dubey, J.P., Puech, P., Dumetre, A. 2018. Structure, composition, and roles of the Toxoplasma gondii oocyst and sporocyst walls. Parasitology Research. 5(2019)100016.
Dubey, J.P. 2019. Re-evaluation of merogony of a Cystoisospora ohioensis-like coccidian and its distinction from gametogony in the intestine of a naturally infected dog. Parasitology. 146:740-745.
Verma, S., Lynch, D., Knowles, S., Cezar, C., Kwok, O.C., Jiang, T., Su, C., Dubey, J.P. 2018. An update on Toxoplasma gondii infections in northern sea otters (Enhydra lutris kenyoni) from Washington State, USA. Veterinary Parasitology. 258:133-137.
Pomares, C., Devillard, S., Holmes, T.H., Olariu, T.R., Press, C.J., Ramirez, R., Talucod, J., Estran, R., Su, C., Dubey, J.P., Aizenberg, D., Montoya, J.G. 2018. Genetic characterization of Toxoplama gondii DNA samples isolated from humans living in North America: An unexpected high prevalence of atypical genotypes. Journal of Infectious Diseases. 218(11):1783-1791.
Dubey, J.P. 2018. Gametogony of Eimeria macusaniensis Guerro, Hernandez, Bazalar and Tabacchi, 1971 in Llama (Lama glama). Parasitology. 145:1540-1547.
Taetzsch, S., Gruszynski, K., Bertke, A., Dubey, J.P., Monti, K., Zajac, A., Lindsay, D. 2018. Prevalence of zoonotic parasites in feral cats of central Virginia, United States. Zoonoses and Public Health. 65:728–735.
Hill, D.E., Luchansky, J.B., Porto Fett, A.C., Gamble, H., Urban Jr, J.F., Fournet, V.M., Hawkins Cooper, D.S., Gajadhar, A., Holley, R., Juneja, V.K., Dubey, J.P. 2018. Rapid inactivation of Toxoplasma gondii bradyzoites in dry cured sausage. Food and Waterborne Parasitology.
Barros, M., Cabezon, O., Dubey, J.P., Almeria, S., Ribas, M., Escobar, L., Ramos, B., Medina-Vogel, G. 2018. Toxoplasma gondii infection in wild mustelids and domestic cats across an urban-rural gradient from Southern Chile. PLoS Pathogens.
Cezar-Cerqueira, C., Thompson, P., Murata, F., Mowery, J.D., Brown, J., Banfield, J., Rosenthal, B.M., Dubey, J.P. 2018. Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA. Parasitology Research. 117:3245-3255.
Horta, M., Guimaraes, M., Arraes-Santos, A., Araujo, A., Dubey, J.P., Labruna, M., Gennari, S., Pena, H. 2018. Detection of anti-Toxoplasma gondii antibodies in small wild mammals from preserved and non-preserved areas in the Caatinga biome, a semi-arid region of Northeast Brazil. Veterinary Parasitology. 14:75-78.
Keats, S., Saraf, P., Zhu, X., Zhou, D., Mcferrin, B., Ajzenberg, D., Schares, G., Hammond-Aryee, K., Higgins, S., Gerhold, R., Rosenthal, B.M., Zhao, X., Dubey, J.P., Su, C. 2018. Human impact on the diversity and virulence of the ubiquitous zoonotic parasite 2 Toxoplasma gondii. Proceedings of the National Academy of Sciences.
Ribas, M.P., Almeria, S., Fernandez-Aguilar, X., De Pedro, G., Lizarraga, P., Alarcia-Alejos, O., Molina-Lopez, R., Obon, E., Gholipour, H., Temino, C., Dubey, J.P., Cabezon, O. 2018. Tracking Toxoplasma gondii in freshwater ecosystems: interaction with the invasive American mink (Neovison vison) in Spain. Parasitology Research.
Dubey, J.P., Schuster, R., Kinne, J. 2018. Gametogony of Eimeria cameli in small intestine of one-humped camel (Camelus dromedarius). Parasitology Research. 117:3633-3638.
Dubey, J.P. 2018. Reevaluation of endogenous development of Eimeria bareillyi Gill, Chhabra and Lall, 1963 in water buffalo (Bubalus bubalis). Parasitology.
Verma, S.K., Trupkiewicz, J.G., Georoff, T., Dubey, J.P. 2018. Molecularly confirmed acute, fatal Sarcocystis falcatula infection in the rainbow lorikeets (Trichoglossus moluccanus) at the Philadelphia Zoo, USA. Veterinary Parasitology.
Dubey, J.P., Evason, K., Walther, Z. 2019. Endogenous development of Cystoisospora belli in intestinal and biliary epithelium of humans. Parasitology. 146:865-872.
Dubey, J.P., Lindsay, D. 2019. Coccidiosis in dogs—100 years of progress . Veterinary Parasitology. 266:34-55.
Ballash, G.A., Jenkins, M.C., Kwok, O.C., Dubey, J.P., Shoben, A.B., Robison, T.L., Kraft, T., Shaffer, E.E., Dennis, P.M. 2019. The effect of urbanization on Neospora caninum seroprevalence in white-tailed deer (Odocoileus virginianus) . EcoHealth. 16:109.
Dubey, J.P., Lindsay, D. 2019. Gametogony of Cystoisospora canis and its distinction from meronts in the intestines of dogs. Journal of Parasitology. 105(2)345-350.
Cano-Terriza, D., Almeria, S., Caballero-Gomez, J., Diaz-Cao, J.M., Ruiz-Jiminez, S., Dubey, J.P., García-Bocanegra, I. 2019. Serological survey of Toxoplasma gondii in captive nonhuman primates in zoos in Spain. Comparative Immunology Microbiology and Infectious Diseases. 65:54-57.
Rani, S., Cerqueira-Cézar, C.K., Murata, F.H., Sadler, M., Kwok, O.C., Pradhan, K., Urban Jr, J.F., Hill, D.E., Dubey, J.P. 2019. Toxoplasma gondii tissue cyst formation and quantitative density of tissue cysts in shoulders of pigs 7 and 14 days after feeding infected mice tissues. Veterinary Parasitology. 269:13-15.
Abbas, I., El-Alfy, E., Al-Araby, M., Al-Kappany, Y., El-Seadawy, R., Dubey, J.P. 2019. Prevalence of Eimeria species in camels (Camelus dromedarius) from Egypt and diagnosis of Eimeria cameli oocysts. Journal of Parasitology. 105(3):395-400.
Cerqueira-Cezar, C., Da Silva, A., Murata, F., Sadler, M., Kwok, O.C., Brown, J., Casalena, M., Blake, M., Su, C., Dubey, J.P. 2019. Isolation and genetic characterization of Toxoplasma gondii from tissues of wild turkeys (Meleagris gallopavo) in Pennsylvania. Journal of Parasitology. 105(3):391-394.
Cruz, I., Vinhas, A., Dubey, J.P., Coutinho, T., Cardosa, L., Cotovio, M., Lopes, A. 2019. First report of antibodies to Neospora spp. in horses from Portugal. Brazilian Journal of Veterinary Parasitology. vol 28.
Rodrigues, F., Pereira, C., Dubey, J.P., Novoa, M., Quaresma, M., Schallig, H., Cardoso, L., Lopes, A. 2019. Seroprevalence of Toxoplasma gondii and Leishmania spp. in domestic donkeys from Portugal. Brazilian Journal of Veterinary Parasitology. 28(1).
Anderson, J.A., Alves, D.A., Cerqueira-Cezar, C.K., Da Silva, A.F., Murata, F.H., Norris, J.K., Howe, D.K., Dubey, J.P. 2019. Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus. Veterinary Parasitology. 270:20-24.
Hill D.E., Dubey J.P. (2018) Toxoplasma gondii. In: Ortega Y., Sterling C. (eds) Foodborne Parasites. Food Microbiology and Food Safety. Springer, Cham.