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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

2017 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
In collaboration with scientists at the USDA-ARS Eastern Regional Research Center (ERRC), we completed the validation of endpoint defining curing processes necessary for developing a multi-factorial model for pork curing for inactivation of Trichinella and Toxoplasma. The endpoint processes established the survival limits of the 2 parasites with respect to salt/brine concentration, aw (water activity), pH, temperature, and time. Prediction of inactivation of Trichinella and Toxoplasma in pork meat can be accomplished within the defined endpoints which circumscribe most commonly used parameters for curing of fresh pork (Sub-objective 4a). 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 (Sub-objective 4b). Completed the National Retail Meat Survey for Toxoplasma in organic pork and American lamb. The survey identified 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 (Sub-objective 4b).

1. Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms. Toxoplasmosis continues to be a major public health concern in the U.S. The consumption of undercooked meat is suspected as an important mode of this parasite’s transmission, and free range chickens have been found to be infected. In order to determine whether chickens available for purchase pose a significant risk, a large sample of chicken hearts were obtained from local markets and tested for T. gondii infection. Viable T. gondii was not isolated from any hearts by bioassays in mice and cats. These results indicate that unlike free range back yard chickens, chickens from grocery stores do not pose an appreciable risk for consumers. These reassuring data will be helpful to producers, retailers, food safety authorities, and epidemiologists.

2. Curing parameters to inactivate Trichinella spiralis in ready-to-eat meats. Ready-to-eat meats are protected from spoilage and foodborne pathogens through curing processes that use salt, seasonings, and other additives. Past regulations required costly testing of each pork curing process to demonstrate its ability to kill larvae of the foodborne parasite Trichinella spiralis and it would be preferable to predict how well a proposed new method would eliminate risk, especially given growing consumer interest in low-salt meat products. ARS scientists in Beltsville, Maryland, found that salt concentrations above 1.3%, in combination with fermentation to pH 5.2 or below, kills greater than 96% of Trichinella larvae in stuffed sausages within 24-28 hrs and inactivates them all within 10 days. These curing parameters reliably predict inactivation of Trichinella spiralis, eliminating the need for individual product testing. These results will protect public health and will be of great value to food producers seeking protocols and formulas that safeguard consumer health.

Review Publications
Tidy, A., Dubey, J.P., Cardosa, L., Lopes, A., Fangueiro, S. 2017. Seroepidemiology and risk assessment of Toxoplasma gondii infection in captive wild birds and mammals in two zoos in the North of Portugal. Veterinary Parasitology. 235:47-52.
Dubey, J.P., Hemphill, A., Calero-Bernal, R., Schares, G. 2017. Neosporosis of animals. Neosporosis in Animals. CRC Press: Boca Raton, Florida. 530 p.
Gondim, M., Da C.L. Acosta, I., Soares, H.S., Gennari, S., Dubey, J.P., Rossi, J.L. 2017. Occurrence of Toxoplasma gondii antibodies in Lowland Tapirs (Tapirus terrestris) maintained ex-situ in Brazil and Paraguay. Ciencia Rural. 47:03 e20160712.
Calero-Bernal, R., Maur00, N., Hui, S., Kuiken, T., Van De Bildt, M., De Jong, A., Osterhaus, L., Sim, L., Gendron-Fitzpatrick, A., Carmena, D., Cerqueira-Cezar, C., Rosenthal, B.M., Dubey, J.P. 2017. Fatal Sarcocystis canis-like hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong. Veterinary Parasitology. 235:64-68.
Dubey, J.P., Naji, N., Mowery, J.D., Verma, S., Calero-Bernal, R. 2017. Identification of macroscopic sarcocysts of Sarcocystis cameli from camels (Camelus dromedarius) in Iraq. Journal of Parasitic Diseases. 103(2):168-169.
Lindsay, D., Verma, S., Scott, D., Dubey, J.P., Dohlen, A.R. 2016. Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp. ex Accipiter cooperii from a naturally infected Cooper's hawk (Accipiter cooperii). Parasitology International. 66:106-111.
Saraf, P., Shwab, E.K., Dubey, J.P., Su, C. 2017. On the determination of Toxoplasma gondii virulence in mice. Experimental Parasitology. 174:25-30.
Ying, Y., Verma, S.K., Kwok, O.C., Alibana, F., Mcleod, R., Su, C., Dubey, J.P., Pradhan, A.K. 2017. Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms. Parasitology Research. 116:1591-1595.
Bertranpetit, E., Jombart, T., Paradis, E., Pena, H., Dubey, J.P., Su, C., Mercier, A., Devillard, S., Aizenberg, D. 2017. Phylogeography of Toxoplasma gondii points to a South American origin. Infection, Genetics and Evolution. 48:150-155.
Cox, J., Slabach, B., Hast, J., Kwok, O.C., Dubey, J.P. 2017. High seroprevalence of toxoplasma gondii in Elk (Cervus canadensis) of the Central Appalachians, USA. Parasitology Research. 116:1079-1083.
Harito, J., Tysner, K., Campbell, A., Dubey, J.P., Robertson, L. 2017. Lectin-Magnetic Separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR. Water Research. 114:228-236.
Oliveria, S., Aizawa, J., Soares, H., Chiebo, D., De Castro, B., Hora, A., Lopes, M., Schares, G., Jenkins, M.C., Kwok, O.C., Dubey, J.P., Gennari, S., Pena, H. 2018. Experimental neosporosis in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates of Neospora caninum reveals resistance to infection. International Journal for Parasitology. 48: 117–123.
Lopes, A., Oliveira, A., Granada, S., Rodrigues, F., Papadopulos, E., Schallig, H., Dubey, J.P., Cardosa, L. 2017. Antibodies to Toxoplasma gondii and Leishmania spp. in domestic cats from Luanda, Angola. Veterinary Parasitology. 239:15-18.
Dubey, J.P., Calero-Bernal, R., Rosenthal, B.M., Speer, C., Fayer, R. 2015. Sarcocystosis of animals and humans. Boca Raton: CRC Press. 2:1-481 p.
Dubey, J.P., Van Wilpe, E., Hilali, M. 2015. Ultrastructure of Sarcocystis bertrami sarcocysts from naturally infected donkey (Equus asinus) from Egypt. Parasitology. 143(01):18-23.
Trupkiewicz, J.G., Calero-Bernal, R., Verma, S., Mowery, J.D., Davison, S., Habecker, P., Georoff, T.A., Ialeggio, D.M., Dubey, J.P. 2015. Acute, fatal Sarcocystis calchasi-associated hepatitis in Roller pigeons (Columbia livia f. dom.) at Philadelphia Zoo. Veterinary Parasitology. 216:52-58.
Dubey, J.P., More, G., Van Wilpe, E., Calero-Bernal, R., Verma, S.K., Schares, G. 2015. Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from cattle (Bos taurus) and its differentiation from Sarcocystis hominis. Journal of Eukaryotic Microbiology. 63:62-68.
Verma, S., Carstensen, M., Calero-Bernal, R., Moore, S., Jiang, T., Su, C., Dubey, J.P. 2015. Seroprevalence, isolation, first genetic characterization of Toxoplasma gondii, and possible congenital transmission in wild moose from Minnesota, USA. Parasitology Research. 115:687-690.
Dubey, J.P., Kwok, O.C., Gardner, I. 2015. Bayesian estimation of sensitivity and specificity of the modified agglutination test and bioassay for detection of Toxoplasma gondii in free-range chickens. Parasitology. 143:314-319.
Andrade, L., Lugarini, C., Oliveira, R.A., Silva, L.T., Marvulo, M.V., Garcia, J.E., Dubey, J.P., Silva, J.C. 2016. Occurrence of antibodies to Toxoplasma gondii in wild birds from three Federal Conservation Units of Paraíba and Bahia, Brazil. Brazilian Journal of Veterinary Research. 36(2):103-107.
Shwab, E.K., Jiang, T., Pena, H.F., Gennari, S.M., Dubey, J.P., Su, C. 2015. The ROP18 and ROP5 allele types are highly predictive of mouse-virulence across globally distributed strains of Toxoplasma gondii. International Journal for Parasitology. 46:141-146.
Vieira, F.P., Alves, M.G., Martins, L.M., Rangel, A.L., Dubey, J.P., Hill, D.E., Bahia-Oliveira, L.M. 2015. Waterborne toxoplasmosis investigated and analyzed under hydrogeological assessment: new data and perspectives for further research. Memorias Do Instituto Oswaldo Cruz. 113(2):398-403.
Harito, J.B., Campbell, A.T., Prestrud, K.W., Dubey, J.P., Robertson, L.J. 2016. Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts. Experimental Parasitology. 165:88-94.
Guo, M., Mishra, A., Buchanan, R., Dubey, J.P., Hill, D.E., Gamble, R., Jones, J., Du, X., Pradhan, A. 2015. Development of the dose-response relationship for human toxoplasma gondii infection associated with meat consumption. Journal of Risk Assessment. 36(5):926-38.
Cerqueria-Cezar, C., Pedersen, K., Calero-Bernal, R., Kwok, O.C., Villena, I., Dubey, J.P. 2016. Seroprevalence of Neospora caninum in feral swine (Sus scrofa) in the United States. Veterinary Parasitology. 226:35-37.
Verma, S., Lindsay, D., Rosenthal, B.M., Dubey, J.P. 2016. Ancient, globally distributed lineage of Sarcocystis from sporocysts of the Eastern rat snake (Pantherophis alleghaniensis) and its relation to neurological sequalae in intermediate hosts. Parasitology Research. 115:2697-2704.
Cezar, C., Thompson, P., Verma, S., Mowery, J., Caler-Bernal, R., Sinnett, D., Van Hemert, C., Rosenthal, B.M., Dubey, J.P. 2017. Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus)from Alaska, USA. Veterinary Parasitology. 116:1871-1878.
Gennari, S., Niemeyer, C., Soares, H., Musso, C., Siqueira, G., Catao-Dias, J., Dias, R., Dubey, J.P. 2016. Seroprevalence of Toxoplasma gondii in seabirds from Abrolhos Archipelago, Brazil. Veterinary Parasitology. 226:50-52.
Mcphillie, M., Zhou, Y., Dubey, J.P., Lorenzi, H., Capper, M., Lukens, A.K., Hickman, M., Muench, S., Verma, S., Weber, C., Wheeler, K., Gordon, J., Sanders, J., Moulton, H., Wang, K., Kim, T., He, Y., Santos, T., Woods, S., Lee, P., Donkin, D., Kim, E., Fraczek, L., Lykins, J., Esaa, F., Alibana-Clouser, F., Dovgin, S., Weiss, L., Brasseur, G., Wirth, D., Kent, M., Hood, L., Meunieur, B., Roberts, C., Hasnain, S., Antonyuk, S.V., Fishwick, C., Mcleod, R. 2016. New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections. Nature Scientific Reports. 6:29179.
Almeria, S., Serrano-Perez, B., Darwich, L., Domingo, M., Mur-Novales, R., Regidor-Cerillo, J., Cabezon, O., Perez-Maillo, M., Lopez-Helguera, I., Fernandez-Alguilar, X., Puig Ribas, M., Ortgea-Mora, L., Garcia-Ispierto, I., Dubey, J.P., Lopez Gatius, F. 2016. Foetal death in naive heifers inoculated with Neospora caninum isolate Nc-Spain7 at 110 days of pregnancy. Experimental Parasitology. 168:62-69.
Love, D., Kwok, O.C., Verma, S.K., Dubey, J.P., Bellah, J. 2016. Seroprevalence and isolation of viable Toxoplasma gondii from raptors in the southeastern USA. Journal of Wildlife Diseases. 52:653-656. doi:10.7589/2015-10-269.
Attademo, F.L., Ribeiro, V.O., Soares, H.S., Luna, F.O., Sousa, G.P., Freire, A.C., Gennari, S.M., Alves, L.C., Marvulo, M.V., Dubey, J.P., Silva, J.C. 2016. Seroprevalence of Toxoplasma gondii in captive antillean manatee (Trichechus manatus manatus) in Brazil. Journal of Zoo and Wildlife Medicine. 47:423-426.
Calero-Bernal, R., Perez-Martin, J.E., Reina, D., Serrano, F.J., Frontera, E., Fuentes, I., Dubey, J.P. 2015. Detection of zoonotic protozoa Toxoplasma gondii and Sarcocystis suihominis in wild boars from Spain. Zoonoses and Public Health. 63:346-350. doi:10.1111/zph.12243.
Adomako-Ankomah, Y., English, E.D., Danielson, J.J., Pernas, L.F., Parker, M.L., Boulanger, M.J., Dubey, J.P., Boyle, J.P. 2016. Host mitochondrial association evolved in the human parasite Toxoplasma gondii via neofunctionalization of a gene duplicate. Genetics. 203:283-298.
Verma, S., Calero-Bernal, R., Carstensen, M., Humpal, C., Dubey, J.P. 2016. Antibody detection and molecular characterization of Toxoplasma gondii from bobcats (Lynx rufus),domestic cats (Felis catus),and wildlife from Minnesota, USA. Journal of Eukaryotic Microbiology. 63:567-571.
Cano-Terriza, D., Puig-Ribas, M., Jimenez-Ruiz, S., Cabezon, O., Almeria, S., Galan-Relano, A., Dubey, J.P., Garcia-Bocanegra, I. 2016. Risk factors of Toxoplasma gondii infection in hunting, pet and watchdogs from Southern Spain and Northern Africa. Parasitology International. 65:363-366.
Bissati, K., Chentoufi, A., Krishank, P., Zhou, X.N., Woods, S., Dubey, J.P., Vang, L., Lykins, J., Broderick, K., Mui, E., Suzuki, Y., Bi, S., Cardona, N. 2016. Adjuvanted multi-epitope vaccines protect HLA-A*1101 transgenic mice against Toxoplasma gondii. Journal of Clinical Immunology Insights (JCI Insights). 1(15):e85955.
Bigot-Clivot, A., Palos, M.L., Lepoutre, A., Bastein, F., Bonnard, I., Dubey, J.P., Villena, I., Aubert, D., Geffard, O., Geffard, A. 2016. Bioaccumulation of Toxoplasma and Cryptosporidium by the crustacean Gammarus fossarum: involvement in biomonitoring survey and trophic transfer. Water Research. 133:188-194.
Dubey, J.P., Brown, J., Ternent, M., Verma, S., Hill, D.E., Cezar, C., Kwok, O.C., Calero-Bernal, R., Humphreys, J. 2016. Seroepidemiologic study on the prevalence of Toxoplasma gondii and Trichinella spp. infections in black bears (Ursus americanus) in Pennsylvania, USA. Veterinary Parasitology. 229:76-80.
Ojo, K., Dangoudoubiyam, S., Verma, S.K., Scheele, S., Derocher, A.D., Yeargan, M., Choi, R., Smith, T.R., Rivas, K.L., Hulverson, M.A., Barrett, L.K., Fan, E., Maly, D., Parsons, M., Dubey, J.P., Howe, D., Van Voorhis, W.C. 2016. Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy. International Journal for Parasitology. 46:871-880.
Verma, S., Lindsay, D., Grigg, M., Dubey, J.P. 2017. Isolation, Culture and Cryopreservation of Sarcocystis species. Current Protocols in Microbiology. doi: 10.1002/cpmc.32.
Verma, S., Sweeney, A., Lovallo, M., Calero-Bernal, R., Kwok, O.C., Su, C., Grigg, M., Dubey, J.P. 2017. Seroprevalence, isolation, and co-infection of multiple Toxoplasma gondii strains in individual bobcats (Lynx rufus) from Mississippi, USA. International Journal for Parasitology. 47:297-303.
Guo, M., Lambertini, E., Buchanan, R.L., Dubey, J.P., Hill, D.E., Gamble, H., Jones, J.L., Pradhan, A.K. 2016. Quantifying the risk of human Toxoplasma gondii infection due to consumption of fresh pork in the United States. Food Control. 73:1210-1222.
Chikweto, A., Sharma, R., Tiwari, K., Verma, S.K., Calero-Bernal, R., Jiang, T., Su, C., Kwok, O.C., Dubey, J.P. 2017. Isolation, tissue distribution, and molecular characterization of Toxoplasma gondii from chickens in Granada, West Indies. Journal of Parasitology. 103(1):52-55.
Wang, Z., Verma, S., Dubey, J.P., Sibley, L. 2017. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat. PLoS Pathogens. 13(3):e1006272.
Cleveland, C., Denicola, A., Dubey, J.P., Hill, D.E., Bergjaus, R.D., Yabsley, M. 2017. Survey for selected pathogens in wild pigs (Sus scrofa) from Guam, Marianna Islands, USA. Veterinary Microbiology. 205:22-25.
Jenkins, M.C., Dubey, J.P., Miska, K.B., Fetterer, R.H. 2017. Differences in fecundity of Eimeria maxima strains exhibiting different levels of pathogenicity in its avian host. Veterinary Parasitology. 236:1-6.
Silva, J., Ferreira, F., Dias, R., Ajezenberg, D., Marvulo, M., Magalhaes, F., Filho, C., Oliveira, S., Soares, H., Feitosa, T., Aizawa, J., Alves, L., Mota, R., Dubey, J.P., Gennari, S., Pena, H. 2017. Cat-rodent Toxoplasma gondii Type II-variant circulation and limited genetic diversity on the Island of Fernando de Noronha, Brazil. Parasites & Vectors. doi: 10.1186/s13071-017-2150-4.
Freppel, W., Puech, P., Ferguson, D.J., Azas, N., Dubey, J.P., Aurelien, D. 2016. Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalization. Nature Scientific Reports. 6:e33654.
Calero-Bernal, R., Cerqueira-Cezar, C., Verma, S., Mowery, J.D., Carmena, D., Beckman, K., Dubey, J.P. 2016. Sarcocystis arctica (Apicomplexa: Sarcocystidae): ultrastructural description and its new host record, the Alaskan wolf (Canis lupus. Parasitology Research. 115:2893-2897.
Behinke, M., Dubey, J.P., Sibley, L.D. 2016. Genetic approaches to defining pathogenesis of Toxoplasma gondii. Annual Review of Microbiology. 70:63-81.
Hill, D.E., Dubey, J.P. 2016. Toxoplasma gondii as a parasite in food: analysis and control. American Phytopathological Society Press. 2:59-80.
Guo, M., Mishra, A., Buchanan, R.L., Dubey, J.P., Hill, D.E., Gamble, H., Jones, J.L., Pradhan, A.K. 2016. Quantifying the risk of human Toxoplasma gondii infection due to consumption of domestically-produced lamb in the United States. International Association for Food Protection Proceedings. 79:1181-1187.