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

Location: Animal Parasitic Diseases Laboratory

2020 Annual Report

Objectives
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).

Approach
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
This year we delineated new priorities for the project while publishing a series of seminal studies on work conducted in prior years. The new project goals center on how coccidian oocysts develop, how their maturity and infectiousness can be assayed in vitro, and how best to wash them from fresh produce. To accomplish this, we developed a model for Cyclospora cayetanensis (a major human foodborne parasite) using surrogate parasites of chickens (Eimeria). The project plan for this work will soon be reviewed. Meanwhile, we developed promising preliminary RNAseq data tracking consistent patterns of gene expression as oocysts of Eimeria mature to their infectious state. We re-examined histological specimens of a heavy human infection, discovering never-before-seen stages of development. We advanced a series of studies on the epidemiology of other coccidian parasites in food animals, including species of Sarcocystis and Trichinella. Also, we discovered a new, freeze-tolerant species of Trichinella. In addition, we inspected hundreds of thousands of pork samples to advance an Animal and Plant Health Inspection Service (APHIS) funded initiative to determine whether U.S. Pork define a production compartment of negligible risk for Trichinella.

Accomplishments
1. A new reference for coccidian parasites. Coccidian parasites cause enteric disease in people, wildlife, and livestock. ARS researchers in Beltsville, Maryland, authored a textbook covering every aspect of parasite biology, pathology, diagnosis, and control. This text bolsters control efforts by synthesizing current information, rich with illustrations, tables, and references. An expansive chapter summarizes the epidemiology of Cyclospora cayetanensis, a parasite newly prioritized by USDA because of its growing threat to food safety.

2. A human parasite rediscovered. Produce contaminated with the parasite Cyclospora cayetanensis causes human enteric disease. Outbreaks of this parasite impair health and create liability for growers. ARS researchers in Beltsville, Maryland, collaborated with the Food and Drug Administration (FDA) and academic partners to re-examine how disease progresses. By carefully inspecting an unusually heavy infection, they identified new stages of parasite development. This breakthrough aids diagnosis and suggests new approaches to treating disease.

3. Deer: an underappreciated source for zoonotic parasites. Pork, lamb, and venison are infection sources for zoonotic parasites. ARS researchers in Beltsville, Maryland, published the results of years-long surveys for parasites in these meats. By genotyping the isolates, the team discovered how often such parasites move between wildlife and livestock. Deer are frequently infected. Some deer harbor strains responsible for especially severe human disease. These data reshape food safety understanding and help epidemiologists and clinicians address ongoing public health concerns.

Review Publications
Su, C., Dubey, J.P. 2019. Molecular methods, Toxoplasama. Book Chapter. 2071:49-80. https://doi.org/10.1007/978-1-4939-9857-9_3.
Sokol S.L., Wong Z.S., Boyle J.P., Dubey J.P. 2019. Generation of Toxoplasma gondii and Hammondia hammondi oocysts and purification of their sporozoites for downstream manipulation. In: Tonkin C., editor. Toxoplasma gondii: Methods in Molecular Biology. Humana, New York, NY: vol 2071.
Escotte-Binet, S., Malik Da Silva, A., Cances, B., Aubert, D., Dubey, J.P., La Carbona, S., Villena, I., Poulle, M. 2019. A rapid and sensitive method for Toxoplasma gondii oocyst detection in soil. Veterinary Parasitology. 274(2019):108904. https://doi.org/10.1016/j.vetpar.2019.07.012.
Dubey, J.P., Hill, D., Fournet, V.M., Hawkins Cooper, D.S., Cerqueira-Cezar, C.K., Murata, F.H., Verma, S.K., Kwok, O.C., Rani, S., Fredericks, J.N., Adams, B., Jones, J., Weigand, R., Ying, Y., Guo, M., Su, C., Pradhan, A.K. 2019. Low prevalence of viable Toxoplasma gondii in fresh, unfrozen, American pasture-raised pork and lamb from retail meat stores in the United States. Food Control. 109:106961. https://doi.org/10.1016/j.foodcont.2019.106961.
El-Alfy, E., Abbas, I., Al-Kappany, Y., Al-Araby, M., Abu-Elwafa, S., Dubey, J.P. 2019. Prevalence of Eimeria species in water buffaloes (Bubalus bubalis) from Egypt and first report of Eimeria bareillyi oocysts. Journal of Parasitology. 105:748-754.
Crouch, E.E., Mittel, L.D., Southard, T.L., Cerqueira-Cezar, C.K., Murata, F.H., Kwok, O.C., Su, C., Dubey, J.P. 2019. Littermate cats rescued from a shelter succumbed to acute, primary toxoplasmosis associated with TOXO DB genotype #4, generally circulating in wildlife. Parasitology International. 72(2019):101942. https://doi.org/10.1016/j.parint.2019.101942.
Rousseau, A., Escotte-Binet, S., La Carbona, S., Dumètre, A., Chagneau, S., Favennec, L., Kubina, S., Dubey, J.P., Majou, D., Bigot-Clivot, B., Villena, I., Aubert, D. 2019. Assessing Toxoplasma gondii oocyst infectivity using a sporocyst-based cell-culture assay combined with qPCR for environmental application. Applied and Environmental Microbiology. 85(20):e01189-19. https://doi.org/10.1128/AEM.01189-19.
Dubey, J.P., Cerqueira-Cézar, C.K., Murata, F.H., Verma, S.K., Kwok, O.C., Pedersen, K., Rosenthal, B.M., Su, C. 2019. Genotyping Toxoplasma gondii from the first national survey of feral swine revealed nearly 40% seroprevalence in the adult animals, and the presence of highly virulent parasite genotypes. Parasitology. 1-8. https://doi.org/10.1017/S0031182019001586.
Koloren, Z., Cerqueira-Cezar, C., Murata, F., Kwok, O.C., Banfield, J., Brown, J., Su, C., Dubey, J.P. 2019. High seroprevalence but low rate of isolation for Toxoplasma Gondii from wild elk (cervus canadensis) in Pennsylvania. Journal of Parasitology. 105(6):890–892. https://doi.org/10.1645/19-110.
Zhao, L., Pi, L., Qin, Y., Lu, Y., Zeng, W., Xiang, Z., Wei, X., Chen, X., Li, C., Zhang, Y., Wang, S., Si, Y., Yang, G., Huang, Y., Rosenthal, B.M., Yang, Z. 2019. Malaria parasites imported to China from Central and Western Africa bear high frequencies of mutations conferring resistance to sulfadoxine-pyramethamine, constraining treatment options for source and recipient populations. International Journal for Parasitology: Drug and Drug Resistance. 12:1-6. https://doi.org/10.1016/j.ijpddr.2019.11.002.
Pereira, D.C., Dubey, J.P., Da Mata, A., Neto, H., Cardoso, L., Lopes, A. 2020. Epidemiology of Toxoplasma gondii infection in domestic cattle, sheep, goats and pigs from São Tomé and Príncipe. Brazilian Journal of Veterinary Parasitology. 29(1):eo14819. https://doi.org/10.1590/S1984-29612019101.
Geba, E., Aubert, D., Durand, L., Escotte, S., La Carbona, S., Cazeaux, C., Bonnard, I., Bastien, F., Palos Ladeiro, M., Dubey, J.P., Villena, I., Geffard, A., Bigot-Clivot, A. 2019. Use of bivalve Dreissena polymorpha as biomonitoring tool to reflect the protozoan load in freshwater bodies. Water Research. 170:115297. https://doi.org/10.1016/j.watres.2019.115297.
Sooryanarain, H., C. Lynn, H., Hill, D., Fredericks, J.N., Rosenthal, B.M., Stephen, W., Tanja, O., Xiang-Jing, M. 2020. Hepatitis E virus infection in market weight pigs from slaughterhouses, United States, 2017-2019. Emerging Infectious Diseases. 26(2):354-357.
Dubey, J.P., Lindsay, D.S., Jenkins, M.C., Bauer, C. 2019. Biology of Intestinal Coccidia. In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 1-36. https://doi.org/10.1201/9780429294105.
Almeria, S., Cinar, H.N., Dubey, J.P. 2019. Coccidiosis in Humans. In: Dubey, J.P. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Group. p. 268-312.
Christian, B., Dubey, J.P. 2019. Coccidiosis in horses and other equids. In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 231-243.https://doi.org/10.1201/9780429294105.
Dubey, J.P. 2019. Coccidiosis in Water Buffaloes (Bubalus bubalis). In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. 91-97. https://doi.org/10.1201/9780429294105.
Dubey, J.P. 2019. Coccidiosis in South American Camelids. In: Dubey, J.P. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 153-158. https://doi.org/10.1201/9780429294105.
Dubey, J.P. 2019. Coccidiosis in Cats (felis catus). In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 255-265. https://doi.org/10.1201/9780429294105.
Dubey, J.P., Lindsay, D.S. 2019. Coccidiosis in Dogs (canis familiaris). In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 245-254. https://doi.org/10.1201/9780429294105
Dubey, J.P., Schuster, R.K. 2019. Coccidiosis in Old World Camels. In: Dubey, J.P., editor. Coccidiosis in Livestock, Poultry, Companion Animals, and Humans. 1st Edition. Boca Raton, Florida: CRC Press. p. 147-152. https://doi.org/10.1201/9780429294105.
Lindsay, D.S., Dubey, J.P. 2020. Neosporosis, Toxoplasmosis, and Sarcocystosis in Ruminants: An Update. Veterinary Clinics of North America. 36:(205-222). https://doi.org/10.1016/j.cvfa.2019.11.004.
Durand, L., La Carbona, S., Geffard, A., Possenti, A., Dubey, J.P., Lalle, M. 2019. Comparative evaluation of Loop-mediated isothermal amplification (LAMP) vs qPCR to detect T. gondii oocysts in mussels. Experimental Parasitology. 208:107809. https://doi.org/10.1016/j.exppara.2019.107809.
Schares, G., Dubey, J.P., Rosenthal, B.M., Tuschy, M., Barwald, A., Conraths, F.J. 2020. Immunomagnetic separation of Toxoplasma gondii tissue cysts and sporocysts using a single monoclonal antibody. International Journal of Parasitology. 11:114-119. https://doi.org/10.1016/j.ijppaw.2020.01.011.
Dubey, J.P., Almeria, S., Mowery, J.D., Fortes, J. 2020. Endogenous developmental cycle of the human coccidian, Cyclospora cayetanensis. Parasitology. 106(2):295-307. https://doi.org/10.1645/20-21.