Detection and Control of Foodborne Parasites for Food Safety

Goals / 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).

Project Methods
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 10/01/20 to 09/30/21

Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. We made extensive progress completing important work related to the food safety dangers posed by zoonotic parasites. ARS redirected all its research focused on toxoplasmosis in April 2019, necessitating cessation of activities on many objectives established in May 2016. Some studies relating to experimental and fieldwork concluded before the redirection were published in FY21. These include a series of review articles prepared to enrich a forthcoming revision of Dr. Dubey⿿s seminal textbook on this parasite and a serological survey of the U.S. swine herd; nonetheless, our team⿿s primary focus was on developing and pursuing new food safety priorities. These new priorities shine a focus on poultry coccidiosis as a model to make needed headway in managing the foodborne risk posed by Cyclospora cayetanensis, an enteric parasite imposing increasing burdens on human health and increasingly responsible for multi-state outbreaks attributed to fresh produce. In the last year, our team engaged in significant outreach efforts to new stakeholders (the Center for Produce Safety, United Fresh Incorporated, and individual growers that have experienced product recalls) and partnered with other ARS researchers in the food safety program as well as academic leaders in the field (including the President of the International Association for Food Protection) to define and articulate new program priorities; we wrote a new project organized around goals thereby identified, received a ⿿minor revision⿿ review, and implemented the new project in May 2021. Even prior to the new plan⿿s implementation, the Office National Programs (ONP) endorsed our decision to build stakeholder engagement and seek extramural support for new objectives related to parasitic threats to produce safety; our pre-proposal (to the Center for Produce Safety) delineated sand/zero valent iron filters as a means to remove coccidian oocysts from irrigation waters. We proposed using poultry coccidia (with which our group has ample experience) as a surrogate for Cyclospora, the agent of human disease. The pre-proposal was well received; we were then invited to submit a full proposal, which also appears to have found favor (no formal announcement has yet been made). If filters succeed in removing or harming such parasites, we will deliver a practical pre- harvest control for growers, alleviating concern over a growing food safety threat. We have already made significant progress toward new objectives defined for our successor project (8042-32000-113). In particular, we concluded: ⿢ A study of changing gene expression as coccidian oocysts mature (sporulate), identifying biomarkers for parasite viability. Such assays may provide practical means to assess risk and may suggest new avenues for intervention and hygiene. ⿢ Two studies describing the first-ever means to trace outbreaks and identify sources of contamination with Trichinella spiralis (mentoring a visitor from the National Veterinary Research Institute of Poland, where endemic parasite transmission on low-resource farms continues to threaten human health and impose costs on pork production). Finally, we solidified our team⿿s future by successfully recruiting an outstanding new scientist to fill the vacancy created by Dr. Dolores Hill⿿s retirement. Dr. Asis Khan comes to us from the National Institute of Allergy and Infectious Disease, having previously completed postdoctoral training in one of the Nation⿿s foremost departments of Medical Microbiology, focusing on parasite genetics, genomics, molecular epidemiology, and virulence. He brings extramural support (from the National Institute of Health (NIH), via a collaboration with the University of Georgia) to make new headway on Cryptosporidium epidemiology and prevention. Record of Any Impact of Maximized Teleworking Requirement: The team maintained a commendable record of productivity in spite of the challenges of maximized telework. ACCOMPLISHMENTS 01 A game of cat and mouse. To save human lives, researchers often study animals. The USDA seeks every opportunity to minimize that need. Therefore, USDA researchers and their partners sought to discover a way to grow a cat parasite in mice. This parasite causes toxoplasmosis, sickening pregnant women and their babies. Astonishingly, mice bred and raised to mimic cat digestion supported parasite growth. To hasten the discovery of drugs and vaccines, researchers have sought to build on this landmark achievement. 02 How to prevent foodborne trichinellosis. Many Americans used to become ill by eating pork infected with Trichinella parasites. No longer. Successful measures championed by the USDA reassure consumers, limit producer liability, and promote trade. ARS researchers contributed to new international guidelines for safeguarding meat. ARS partnered with three other USDA agencies (the Agricultural Marketing Service, the Animal and Plant Health Inspection Service, and the Food Safety Inspection Service). 03 A foodborne parasite in meat survives prolonged refrigeration. Refrigeration preserves and protects meat. But USDA researchers suspected that refrigeration does not kill Toxoplasma gondii. The parasite survived in vacuum-packed meat refrigerated for two weeks. Because this parasite causes birth defects and other illnesses, meat should be thoroughly cooked. 04 Dry salt curing kills parasites in meat. Salt curing produces pepperoni and ready-to-eat hams. USDA researchers determined that this procedure kills the parasite causing toxoplasmosis. Salt curing therefore, reduces the risk of birth defects and other illnesses. The findings reassured consumers and helped food producers and regulators manage risk in a cost-effective manner. 05 Dangerous parasites in deer. Many Americans hunt deer and eat venison. Researchers in Beltsville, Maryland, discovered in deer unusual strains of the foodborne parasite causing toxoplasmosis. An outbreak of the severe disease occurred in a group that consumed undercooked venison. These studies remind us that proper cooking ensures venison⿿s safety. 06 Evolution of SARS-CoV-2. COVID-19 upended life and caused great harm. An international team of infectious disease scholars sought ARS expertise to understand how the virus was spreading and changing. These insights helped epidemiologists understand and respond to the emerging threat. 07 A parasite of livestock and dogs has taken the world by storm. Neosporosis is the most important cause of abortion in cattle. USDA researchers and their partners determined that one parasite lineage has expanded globally. This finding gives hope that the same management tools may succeed wherever the parasite causes harm. 08 Against parasites, the best offense is a good defense. Toxoplasma infects many kinds of birds and mammals. It infects millions of people, some of whom suffer birth defects, mental retardation, and blindness. To understand why, USDA researchers worked with scientists at the University of Pittsburgh to compare harmful and harmless parasites. They discovered differences in how these parasites avoid host defenses. These insights suggested new ways to prevent disease. 09 A comprehensive view of an emerging threat to produce safety. Cyclosporiasis is a poorly understood foodborne illness. USDA and FDA scientists reviewed this parasite⿿s spread in the USA and outlined research needs that ARS is now pursuing. Success will reduce harm to consumers, growers and grocers. 10 Better detection for food and water-borne parasites. It is difficult to detect parasites that contaminate food and water. USDA researchers and their partners devised a rapid, low-cost and sensitive method to detect three such parasites. These findings aid public health workers, biologists, and parasitologists seeking to reduce risk. 11 Blue mussels scan for parasites in waterways. Ingesting food and water contaminated with parasites causes illness. Detecting scarce parasites is difficult. To overcome this, USDA scientists used blue mussels. These filter feeders concentrate on such parasites. These findings provided a new means to monitor water quality and safeguard health.

Impacts
(N/A)

Publications

Thomas, N., White, L.C., Saliki, J., Schuler, K., Lynch, D., Nielsen, O., Dubey, J.P., Knowles, S. 2020. Canine distemper virus in the Washington State sea otter population. Journal of Wildlife Diseases. 56(4):873-883. https://doi.org/10.7589/JWD-D-19-00008.
Tartarelli, I., Tinari, A., Possenti, A., Cherchi, S., Falchi, M., Dubey, J.P., Spano, F. 2020. During host cell traversal and cell-to-cell passage Toxoplasma gondii sporozoites inhabit the parasotophorous vacuole and posteriorly release dense granule proteins-associated membranous trails. International Journal for Parasitology. 50(13):1099-1115. https://doi.org/ 10.1016/j.ijpara.2020.06.012.
Jimenez-Martin, D., Garcia-Bocanegra, I., Almeria, S., Castro-Scholten, S., Dubey, J.P., Amaro-Lopez, M., Cano-Terriza, D. 2020. Epidemiological surveillance of Toxoplasma gondii in small ruminants in southern Spain. Preventive Veterinary Medicine. 183:105137. https://doi.org/10.1016/j. prevetmed.2020.105137.
Almeria, S., Dubey, J.P. 2020. Foodborne transmission of Toxoplasma gondii infection in the last decade: An overview. Research in Veterinary Science. 135:371-385. https://doi.org/10.1016/j.rvsc.2020.10.019.
Dubey, J.P., Murata, F.H., Cerqueira-Cezar, C.K., Kwok, O.C. 2020. Importance of toxoplasmosis in goats: the last decade. Research in Veterinary Science. 132:292-307. https://doi.org/10.1016/j.rvsc.2020.06. 014.
Dubey, J.P., Murata, F.H., Cerqueria-Cezar, C.K., Kwok, O.C., Su, C. 2020. Economic and public health importance of toxoplasmosis in sheep: the last decade. Veterinary Parasitology. 286:109195. https://doi.org/10.1016/j. vetpar.2020.109195.
Rosenthal, B.M. 2021. Zoonotic Sarcocystis. Research in Veterinary Science. 136:151-157. https://doi.org/10.1016/j.rvsc.2021.02.008.
Dubey, J.P., Murata, F.H., Cerqueira-Cezar, C.K., Kwok, O.C., Yang, Y. 2020. Public health significance of Toxoplasma gondii infections in cattle: 2009-2020. Journal of Parasitology. 106(6):772-788. https://doi. org/10.1645/20-82.
Dubey, J.P., Murata, F.H., Cerqueira-Cézar, C., Kwok, O.C. 2020. Public health importance of Toxoplasma gondii infections in horses and donkeys: the last decade. Research in Veterinary Science. 132(492-499). https://doi. org/10.1016/j.rvsc.2020.07.005.
Dubey, J.P., Cerqueira-Cezar, C., Murata, F.H., Kwok, O.C., Hill, D., Yang, Y., Su, C. 2020. All about Toxoplasma gondii infections in pigs: 2009- 2020. Veterinary Parasitology. 288:109185. https://doi.org/10.1016/j. vetpar.2020.109185.
Almeria, S., Cano-Terriza, D., Prieto, P., Dubey, J.P., Jimenez-Martin, D., Castro-Scholten, S., Paniagua, J., Garcia-Bocanegra, I. 2021. Seroprevalence and risk factors of Toxoplasma gondii infection in wild ungulates that cohabit in a natural park with human-animal interaction in the Mediterranean ecosystem. Zoonoses and Public Health. 68(3):263-270. https://doi.org/10.1111/zph.12821.
Dubey, J.P., Murata, F.H., Cerquiera-Cezar, C.K., Kwok, O.C., Yang, Y., Su, C. 2020. Toxoplasma gondii infections in dogs: 2009-2020. Veterinary Parasitology. 287:109223. https://doi.org/10.1016/j.vetpar.2020.109223.
Dubey, J.P., Murata, F.H., Cerqueira-Cezar, C.K., Kwok, O.C., Su, C. 2021. Epidemiological significance of toxoplasma gondii infections in rodents: 2009-2020. Journal of Parasitology. 107(2):182-204. https://doi.org/10. 1645/20-121.
Dubey, J.P., Murata, F., Cerqueira-Cezar, C., Kwok, O.C., Su, C. 2021. Recent evidence for epidemiologic signifcance of Toxoplasma gondii infections in turkeys, ducks, ratites, and other wild birds: 2009-2020. Parasitology. 148(1):1-30. https://doi.org/10.1017/S0031182020001961.
Dubey, J.P., Murata, F.H., Cerqueira-Cezar, C.K., Kwok, O.C. 2020. Recent epidemiologic and clinical importance of Toxoplasma gondii infections in marine mammals: 2009-2020. Veterinary Parasitology. 288:109296. https:// doi.org/10.1016/j.vetpar.2020.109296.
Kimble, K.M., Gomez, G., Szule, J., Dubey, J.P., Buchanan, B., Porter, B.F. 2020. Systemic toxoplasmosis in a horse. Journal of Comparative Pathology. 182:27-31. https://doi.org/10.1016/j.jcpa.2020.11.004.
Dubey, J.P., Murata, F.H., Cerqueira-Cezar, C.K., Kwok, O.C. 2020. Recent epidemiologic and clinical Toxoplasma gondii infections in wild canids and other carnivores: The past decade. Veterinary Parasitology. 290:109337. https://doi.org/10.1016/j.vetpar.2020.109337.
Barroso, P., Garcia-Bocanegra, I., Acevedo, P., Palencia, P., Carro, F., Jiminez-Ruiz, S., Almeria, S., Dubey, J.P., Cano-Terriza, D., Vicente, J. 2020. Long-term determinants of the seroprevalence of Toxoplasma gondii in a mixed ungulate community. Animals. 10(12):2349. https://doi.org/10.3390/ ani10122349.
Schares, G., Globokar Vrhovec, M., Tuschy, M., Joeres, M., Barwald, A., Koudela, B., Dubey, J.P., Maksimov, P., Conraths, F.J. 2021. A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii. Parasites & Vectors. 14(78). https://doi.org/10.1186/s13071-020-04571-8.
Fabian, B.T., Lepenies, B., Schares, G., Dubey, J.P., Spano, F., Seeber, F. 2021. Expanding the known repertoire of C-type lectin receptors binding to Toxoplasma 2 gondii oocysts using a modified high-resolution immunofluorescence assay. mSphere. 6(2):e01341-20. https://doi.org/10.1128/ mSphere.01341-20.
Almeria, S., Murata, F., Cerqueira-Cezar, C., Kwok, O.C., Shipley, A., Dubey, J.P. 2021. Epidemiological and public health significance of Toxoplasma gondii infection in wild rabbits and hares: 2009-2020. Microorganisms. 9(597). https://doi.org/10.3390/microorganisms9030597.
Noeckler, K., Pozio, E., Van Der Giessen, J., Hill, D.E., Gamble, H. 2019. International commission on Trichinellosis: recommendations on post- harvest control of Trichinella in food animals. Food and Waterborne Parasitology. 14: e00041. https://doi.org/10.1016/j.fawpar.2019.e00041
Rousseau, A., Villena, I., Dumetre, A., Escotte-Binet, S., Favennec, L., Dubey, J.P., Aubert, D., La Carbona, S. 2019. Evaluation of propidium monoazide-based qPCR to detect viable oocysts of Toxoplasma gondii. Parasitology Research. 118:999-1010. https://doi.org/10.1007/s00436-019- 06220-1
Di Genova, B., Wison, S., Dubey, J.P., Knoll, L. 2019. Intestinal delta-6- desaturase activity determines host range for Toxoplasma sexual reproduction. PLoS Biology. 17(8):e3000364. https://doi.org/10.1371/ journal.pbio.3000364.
Sharma, R., Thompson, P., Elkin, B., Mulders, R., Branigan, M., Pongracz, J., Wagner, B., Scandrett, B., Hoberg, E., Rosenthal, B.M., Jenkins, E. 2019. Trichinella pseudospiralis in a wolverine (Gulo gulo) from the Canadian North. Journal of Parasitology. 9:274-280. https://doi.org/10. 1016/j.ijppaw.2019.06.005.
Robertson, L.J., Clark, C., Debenham, J.J., Dubey, J.P., Kvac, M., Li, J., Ponce-Gordo, F., Ryan, U., Schares, G., Su, C., Tsaousis, A.D. 2019. Are molecular tools clarifying or confusing our understanding of the public health threat from zoonotic enteric protozoa in wildlife?. International Journal for Parasitology. 323-341. https://doi.org/10.1016/j.ijppaw.2019. 01.010.
Alves, B., Oliveira, S., Soares, H., Solange, M., Conte-Junior, C., Dubey, J.P., Pena, H.S. 2019. The impact of vaccum packed dry-ageing pork on the viability of Toxoplasma gondii tissue cysts. Food Microbiology. 86(2020) :103331. https://doi.org/10.1016/j.fm.2019.103331.
Sharma, R., Thompson, P., Hoberg, E., Scandrett, B., Konesci, K., Harms, N. , Kukka, P.M., Jung, T.S., Elkin, B., Mulders, R., Larter, N., Branigan, M. , Pongracz, J., Wagner, B., Rosenthal, B.M., Jenkins, E. 2020. Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo). International Journal for Parasitology. 50(4):277-287. https://doi.org/10. 1016/j.ijpara.2020.01.003.
Dubey, J.P., Almeria, S. 2019. Cystoisospora belli infections in humans - the past 100 years. Parasitology. 146(12):1490-1527. https://doi.org/10. 1017/S0031182019000957.
Wong, Z., Sokol, S.L., Dubey, J.P., Boyle, J.P., Olias, P. 2020. Head-to- head comparisons of Toxoplasma gondii and its near relative Hammondia hammondi reveal dramatic differences in the host response and effectors with species-specific functions. PLoS Pathogens. 16(6): e1008528. https:// doi.org/10.1371/journal.ppat.1008528.
Consalter, A., Frazão-Teixeira, E., Dubey, J.P., Zanella, E.L., Da Silva, A.F., De Souza, G.N., Ferreira, A.M. 2019. Epidemiological investigation of Toxoplasma gondii infections in commercial sheep flock in an endemic area for ocular toxoplasmosis in Southern Brazil. Acta Parasitologica. 64:514-519. https://doi.org/10.2478/s11686-019-00081-5.
Abbas, I., Villena, I., Dubey, J.P. 2019. A review on toxoplasmosis in humans and animals from Egypt. Parasitology. 7(9):317. https://doi.org/10. 3390/microorganisms7090317.
Rodrigues, F.T., Moreira, F.A., Coutinho, T., Dubey, J.P., Cardoso, L., Lopes, A. 2019. Antibodies to Toxoplasma gondii in slaughtered free-range and broiler chickens. Veterinary Parasitology. 271(2019):51-53. https:// doi.org/10.1016/j.vetpar.2019.06.007.
Dubey, J.P., Koloren, Z. 2019. A review of toxoplasmosis in humans and animals in Turkey. Parasitology International. 1-60. https://doi.org/10. 1017/S0031182019001318.
Khan, A., Fujita, A., Randle, N., Regidor-Cerrillo, J., Shaik, J., Shen, K. , Oler, A., Quinones, M., Latham, S., Akanmori, B., Cleaveland, S., Ryan, U., Slapeta, J., Schares, G., Ortega-Mora, L., Dubey, J.P., Wastling, J., Grigg, M. 2019. Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum. Nature Communications. 116(45):22764- 22773. https://doi.org/10.1073/pnas.1913531116.
Almeria, S., Cinar, H.N., Dubey, J.P. 2019. Cyclospora cayetanensis and cyclosporiasis: An update. Microorganisms. 7(9):317. https://doi.org/10. 3390/microorganisms7090317.
Ahlers, A., Wolf, T., Windels, S., Olson, B., Matykiewicz, B., Dubey, J.P. 2020. Survey of toxoplasma gondii exposure in muskrats in a relatively pristine ecosystem. Journal of Parasitology. 106(3):346-349.
Fredericks, J.N., Hawkins Cooper, D.S., Hill, D.E., Luchansky, J.B., Porto Fett, A.C., Shoyer, B.A., Fournet, V.M., Urban Jr, J.F., Dubey, J.P. 2020. Inactivation of Toxoplasma gondii bradyzoites after salt exposure during preparation of dry cured hams. Journal of Food Protection. 83(6):1038-1042. https://doi.org/10.4315/0362-028X.JFP-19-461.
Dawson, A.C., Ashander, L.M., Appukuttan, B., Woodman, R.J., Dubey, J.P., Whiley, H., Smith, J.R. 2020. Lamb as a potential source of Toxoplasma gondii infection for Australians. Australian and New Zealand Journal of Public Health. 44(1):49-52. https://doi.org/10.1111/1753-6405.12955.
Neves, M., Lopes, A., Martins, C., Fino, R., Paixao, C., Damil, L., Lima, C., Alho, A., Schallig, H., Dubey, J.P., Cardoso, L. 2020. Survey of Dirofilaria immitis antigen and antibodies to Leishmania infantum and Toxoplasma gondii in cats from Madeira Island, Portugal. Parasites & Vectors. 13:117(2020). https://doi.org/10.1186/s13071-020-3988-4.
Pena, H.S., Ferrari, V.S., Aires, L.E., Soares, H., Oliveira, S., Alves, B. , Gennari, S., Ajzenberg, D., Dubey, J.P., De Mattos, L., De Mattos, C.A., Castiglioni, L. 2020. First isolation and genotyping of Toxoplasma gondii in a free-living giant anteater (Myrmecophaga tridactyla). ACTA TROPICA. https://doi.org/10.1016/j.actatropica.2020.105335.
Dubey, J.P., Cerqueira-Cézar, C.K., Murata, A.A., Verma, S.K., Kwok, O.C., Pedersen, K., Rosenthal, B.M., Su, C. 2020. White-tailed deer (Odocoileus virginianus) are a major reservoir of a diversity of Toxoplasma gondii strains in the USA and pose a risk to consumers of venison. Parasitology. 147(7):1-19. https://doi.org/10.1017/S0031182020000451.
Cano-Terriza, D., Almeria, S., Caballero-Gomez, J., Jimenez-Martin, D., Castro-Scholten, S., Dubey, J.P., Garcia-Bocanegra, I. 2020. Exposure to Toxoplasma gondii in captive zoo animals in Spain. Preventive Veterinary Medicine. 176:104930. https://doi.org/10.1016/j.prevetmed.2020.104930.
El-Alfy, E., Abbas, I., Al-Kappany, Y., Al-Araby, M., Abu-Elwafa, S., Dubey, J.P. 2020. Prevalence of Eimeria species in sheep (Ovis aries) from Dakahlia governorate, Egypt. Journal of Parasitic Diseases. 44:559-573. https://doi.org/10.1007/s12639-020-01229-1.
Neves, M., Lopes, A., Martins, C., Fino, R., Paixao, C., Damil, L., Lima, C., Alho, A., Schallig, H.D., Dubey, J.P., Cardoso, L. 2020. Survey of Dirofilaria immitis antigen and antibodies to Leishmania infantum and Toxoplasma gondii in cats from Madeira Island, Portugal. Parasites & Vectors. 13, 117. https://doi.org/10.1186/s13071-020-3988-4.
Dubey, J.P., Cerqueira-Cezar, C.K., Murata, F.H., Kwok, O.C., Yang, Y., Su, C. 2020. All about toxoplasmosis in cats: the last decade. Preventive Veterinary Medicine. 0304-4017. https://doi.org/10.1016/j.vetpar.2020. 109145.
Sironni, M., Hasnain, S.E., Rosenthal, B.M., Phan, T., Luciani, F., Shaw, M., Sallum, M., Mirhashemi, M.E., Gonzalez-Candelas, F. 2020. SARS-CoV-2 and COVID-19: A genetic, epidemiological, and evolutionary perspective. Infection, Genetics and Evolution. 84. https://doi.org/10.1016/j.meegid. 2020.104384.
Dubey, J.P., Pena, H.F., Cerqueira-Cezar, C., Murata, F.H., Kwok, O.C., Yang, Y., Gennari, S.M., Su, C. 2020. Epidemiologic significance of Toxoplasma gondii infections in chickens (Gallus domesticus): the past decade. Parasitology. 147(12):1263-1289. https://doi.org/10.1017/ S0031182020001134.
Geba, E., Rousseau, A., Le Guernic, A., Escotte-Binet, S., Favennec, L., La Carbona, S., Gargala, G., Dubey, J.P., Villena, I., Betoulle, S., Aubert, D., Bigot-Clivot, A. 2020. Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha. Journal of Applied Microbiology. https://doi.org/10.1111/jam. 14802.
Wadhawan, A., Hill, D.E., Dagdag, A., Mohyuddin, H., Donnelly, P., Jones, J.L., Postolache, T.T. 2018. No evidence for air-borne transmission of Toxoplasma gondii in a very high prevalence area in Lancaster County. Pteridines. 29:172-178. https://doi.org/10.1515/pteridines-2018-0015.
Djurkovic-Djakovic, O., Dupouy-Camet, J., Van Der Giessen, J., Dubey, J.P. 2019. Toxoplasmosis: Overview from a One-Health prospective. Food and Waterborne Parasitology. 15:e00054. https://doi.org/10.1016/j.fawpar.2019. e00054.
Castro, P.E., Dubey, J.P. 2019. Toxoplasma gondii - The Facts. The Veterinary Nurse. 10(4). https://doi.org/10.12968/vetn.2019.10.4.182.
Hollis-Etter, K.M., Anchor, C.L., Chelsvig, J.E., Dubey, J.P., Warner, R.E. 2019. Suburban white-Tailed deer seropositive for Toxoplasma gondii from Chicago, Illinois. Parasitology Research. 118:2271⿿2276. https://doi.org/ 10.1007/s00436-019-06347-1.
Rani, S., Cerqueira-Cezar, C., Murata, F., Kwok, O.C., Dubey, J.P., Pradhan, A. 2020. Distribution of toxoplasma gondii tissue cysts in shoulder muscles of naturally infected goats and lambs. Journal of Food Protection. 83(8):1396-1401. https://doi.org/10.4315/JFP-20-024.
Fredericks, J.N., Hill, D.E., Hawkins Cooper, D.S., Fournet, V.M., Calero- Landa, J., Adams, B.T., Johnson, A.N., Barrow, M., Aquino, J.F., Mahmoud, T., Murphy, V., Barlow, A., Patel, P., George, M., Chehab, N.L., Kramer, M. H., Bauer, N. 2021. Seroprevalence of Toxoplasma gondii in market hogs collected from United States slaugherhouses. Journal of Parasitology. 107(3):404-410. https://doi.org/10.1645/20-142.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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.

Impacts
(N/A)

Publications

Su, C., Dubey, J.P. 2019. Molecular methods, Toxoplasama. Book Chapter. 2071:49-80.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Lindsay, D.S., Dubey, J.P. 2020. Neosporosis, Toxoplasmosis, and Sarcocystosis in Ruminants: An Update. Veterinary Clinics of North America. 36:(205-222).
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.
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.
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.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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 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. Accomplishments 01 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. 02 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. 03 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. 04 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.

Impacts
(N/A)

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.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 (Subobjective 4b). Initiated studies to document inactivation of Toxoplasma gondii in dry cured hams. At the request of APHIS, established a year-long study in collaboration with ARS ERRC laboratory to determine if guidelines for production of dry cured hams in 9 CFR 319.10 for inactivation of Trichinella spiralis also results in inactivation of Toxoplasma gondii. Critical developmental differences were discovered between two closely related parasites, Hammondia hammondi and Toxoplasma gondii. Many genes are common to both organisms, but Toxoplasma gondii is decidedly more pathogenic. Differences were seen in their growth states, the most dramatic of which was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging levels of gene expression after stress exposure. ARS scientists in Beltsville, Maryland also found that H. hammondi could be cultivated in cell culture for up to eight days after a key developmental milestone (excystation) had been reached. Overall the data show that H. hammondi have a distinct, stringently regulated life stage that can be distinguished from those in T. gondii. Those differences may help explain the characteristics that predispose T. gondii to widespread transmission. Accomplishments 01 Rapid inactivation of Toxoplasma gondii during formulation of dry cured ready-to-eat pork sausage. Food producers require generally-applicable rules for ensuring their meat products are safe for consumption, prompting ARS researchers in Beltsville, Maryland, and Wyndmoor, Pennsylvania, to identify the critical point during preparation of dry cured sausage that inactivates Toxoplasma gondii, an important foodborne parasite. Survival of T. gondii at each stage of preparation was assessed, and it was determined that the parasite cannot survive exposure salt concentrations much lower than those typically used for preparation of dry cured sausage. These data suggest that the use of dry curing components, specifically NaCl, are effective in controlling T. gondii potentially transmitted through �ready to eat� meats, rendering these meats safe from risk with respect to T. gondii transmission to human consumers. 02 Detecting Toxoplasma gondii oocyst in ready-to-eat salad. Toxoplasma gondii is an important foodborne parasitic infection as oocysts of this parasite, excreted by cats, are environmentally hardy and highly infectious to people if ingested while drinking contaminated water or eating contaminated food. The detection of oocysts in vegetables is difficult when they are scarce. The increasing consumption of pre- washed ready-to-eat salads may pose a growing risk for consumers. To trace better safeguard ready-to-eat vegetables, ARS researchers developed and validated a sensitive and robust method capable of detecting as few as 25 parasites in 50g in ready-to-eat baby lettuce. The procedure was also adapted for a faster visualization of positive results using a lateral flow dipstick chromatographic detection method. The tool should improve detection and aid efforts to prevent future infections. 03 Protecting blueberries from parasitic infection. The Food and Drug Administration has recently identified fresh fruits and vegetables as commodities with a high potential risk of contamination with Toxoplasma (T.) gondii, a common foodborne parasite. The increasing consumption of fresh produce has resulted in more outbreaks of foodborne illnesses linked to parasites. Current washing steps in produce processing may not effectively eliminate T. gondii from at-risk produce. ARS scientists in Beltsville, Maryland and Albany, California, evaluated low-dose irradiation as a means to inactivate T. gondii oocysts on blueberries. They found conditions that dramatically reduced viable parasite contamination to well-below detection limits without compromising the quality of the berries (as measured by compression firmness, anthocyanins, or color). 04 Partition of Toxoplasma gondii genotypes among areas of human settlement in North America. Toxoplasma gondii is among the most consequential food-borne parasites and while the parasite occurs in a wide range of wild and domesticated animals, domestic farms may constitute a specific and important transmission location. To better understand landscape effects pertinent to this parasite�s transmission, ARS scientists in Beltsville, Maryland, compared the genetic variation in isolates from farm-bound animals, free-roaming animals (with wider home range on or near farms) and wildlife; in addition, parasite genotype distribution in different animal species was analyzed. The project found no absolute limitation of any of the five major genotypes to any one habitat, but genetic diversity was greater in free-roaming than in farm-bound animals. The genotype composition of parasites in wildlife differed from those in farm-bound and free-roaming animals and parasite genotypes differed among host species. These findings reframe the understanding of the exchange of parasites into and out of livestock, and will help farmers, veterinarians, and epidemiologists manage the risk to food safety imposed by various routes of parasite transmission. 05 Environmental and behavioral changes influence exposure of an Arctic apex predator to pathogens and contaminants. Recent decline of sea ice habitat has coincided with increased use of land by polar bears from the southern Beaufort Sea (SB), which may alter the risks of exposure to pathogens and contaminants. ARS scientists in Beltsville, Maryland, assayed blood samples from SB polar bears to assess prior exposure to the pathogens Brucella spp., Toxoplasma gondii, Coxiella burnetii, Francisella tularensis, and Neospora caninum, estimate concentrations of persistent organic pollutants (POPs), and evaluate risk factors associated with exposure to pathogens and POPs. Results indicated that prevalence of Brucella spp. and T. gondii antibodies in serum likely increased through time, and provided the first evidence of exposure of polar bears to C. burnetii, N. caninum, and F. tularensis. Additionally, the odds of exposure to T. gondii were greater for bears that used land than for bears that remained on the sea ice during summer and fall. Infection with T. gondii causes the disease Toxoplasmosis and, in bears this is important from public health and epidemiological viewpoints. In contrast, the mean concentrations of the POP chlordane were lower for land-based bears. In summary, changes in polar bear behavior brought about by climate-induced modifications to the Arctic marine ecosystem may increase exposure risk to certain pathogens and alter contaminant exposure pathways.

Impacts
(N/A)

Publications

Verma, S.K., Rosypal, A., Mowery, J.D., Scott, D., Cerqueira-Cezar, C., Dubey, J.P., Lindsay, D., Rosenthal, B.M. 2017. Sarcocystis strixi, n. sp. from barred owls (Strix varia) definitive hosts and gamma interferon gene knockout mice as experimental intermediate host. Journal of Parasitology. 103:768-777.
Verma, S., Lindsay, D., Mowery, J.D., Rosenthal, B.M., Dubey, J.P. 2017. Sarcocystis pantherophis, n. sp. from eastern rat snakes (Pantherophis alleghaniensis) definitive hosts and interferongamma gene knockout mice as experimental intermediate hosts. Journal of Parasitology. 103:547-554.
Dubey, J.P. 2017. Schizogony and gametogony of the vaccine, oocyst- deficient, strain T-263 of Toxoplasma gondii. Veterinary Parasitology. 245:160-162.
Atwood, T., Duncan, C., Patyk, K., Nol, P., Rhyan, J., Mccollum, M., Mckenney, M., Ramey, A., Cezar, C., Kwok, O.C., Dubey, J.P., Hennager, S. 2017. Environmental and behavioral changes may influence the exposure of an Arctic apex predator to pathogens and contaminants. Nature Scientific Reports. 7:13193.
La Rosa, G., Bernal, R., Perez Martin, J., Tonanzi, D., Serrano Aguilera, F., Rosenthal, B.M., Pozio, E. 2018. Rare but evolutionarily consequential outcrossing in a highly inbred zoonotic parasite. International Journal for Parasitology.
Dubey, J.P., Trupkiewicz, J.G., Verma, S.K., Mowery, J.D., Adedoyin, G., Georoff, T., Grigg, M. 2017. Atypical fatal sarcocystosis associated with Sarcocystis neurona in a white-nosed coati (Nasua narica molaris). Veterinary Parasitology. 247:80-84.
Verma, S., Cerqueira-Cezar, C., Murata, F., Lovallo, M., Dubey, J.P., Rosenthal, B.M. 2017. Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi. Veterinary Parasitology. 248:84-89.
Saville, W., Dubey, J.P., Marsh, A., Reed, S., Keene, R., Howe, D., Morrow, J., Workman, J. 2017. Testing the Sarcocystis neurona vaccine using an equine protozoal myeloencephalitis challenge model. Veterinary Parasitology. 247:37-41.
Jiang, T., Shwab, K., Martin, R., Gerhold, R., Rosenthal, B.M., Dubey, J.P. , Su, C. 2018. A partition of Toxoplasma gondii genotypes across spatial gradients and among host species, and decreased parasite diversity towards areas of human settlement in North America. International Journal for Parasitology. 48(8):611-619.
Carstensen, M., Guidice, J.H., Hildebrand, E.C., Dubey, J.P., Erb, J., Stark, D., Hart, J., Barber-Meyer, S., Mech, D.L., Wendels, S.K., Edwards, A.J. 2017. Serological survey of diseases of free-ranging gray wolves (Canis lupus) in Minnesota. Journal of Wildlife Diseases. 53:459-471.
Dubey, J.P. 2018. A review of coccidiosis in South American camelids. Parasitology Research. 117:1999�2013.
Gennari, S., Raso, T., Guida, F., Pena, H., Soares, H., Dubey, J.P. 2017. Occurrence of antibodies to Toxoplasma gondii in the scavenging black vultures (Coragyps atratus) from Brazil. Brazilian Journal of Veterinary Research. 54:197-199.
Alvarado-Esquivel, C., Howe, D., Yeargan, M., Alvarado-Esquivel, D., Zamarripa-Barboza, J., Dubey, J.P. 2017. Seroepidemiology of Sarcocystis neurona and Neospora hughesi infections in domestic donkeys (Equus asinus) in Durango, Mexico. Parasite. 24:27e.
Verma, S., Rosypal Von Dohlen, A., Mowery, J.D., Scott, D., Rosenthal, B.M. , Dubey, J.P., Lindsay, D. 2017. Sarcocystis jamaicensis, n. sp. from red- tailed hawks (Buteo jamaicensis) definitive host and IFN-Gamma gene knockout mice as experimental intermediate host. Journal of Parasitology. 103:555-564.
Saville, W., Reed, S., Dubey, J.P., Granstrom, D., Morley, P., Hinchcliff, K., Kohn, C., Wittum, T., Workman, J. 2017. Inter-observer variation in the diagnosis of neurologic abnormalities in the horse. Journal of Veterinary Internal Medicine. 1:1871�1876.
Dubey, J.P. 2018. A review of coccidiosis in water buffaloes (Bubalus bubalis). Veterinary Parasitology. 256: 50-57.
Dubey, J.P., Bauer, C. 2018. A review of Eimeria infections in horses and other equids. Veterinary Parasitology. 251:1-2.
Sokol, S., Primack, A., Nair, S., Wong, Z., Temboo, M., Dubey, J.P., Verma, S., Boyle, J. 2018. Stress sensitivity in Toxoplasma gondii is linked to its uniquely flexible life cycle. eLife. e36491.
Hill, D.E., Luchansky, J.B., Porto Fett, A.C., Gamble, H., Juneja, V.K., Fournet, V.M., Hawkins Cooper, D.S., Holley, R., Gajadhar, A., Dubey, J.P. 2017. Curing conditions to inactivate Trichinella spiralis muscle larvae in ready-to-eat pork sausage. Food and Waterborne Parasitology. 6:1-8.
Cerqueira-Cezar, C., Calero-Bernal, R., Dubey, J.P., Gennari, S. 2017. All about neosporosis in Brazil. Brazilian Journal of Veterinary Parasitology. 26:253-279.
Dubey, J.P., Jenkins, M.C. 2017. Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus). Parasitology.
Murata, F., Cerqueira-Cezar, C., Thompson, P., Tewari, K., Mowery, J.D., Verma, S., Rosenthal, B.M., Sharma, R., Dubey, J.P. 2018. Sarcocystis cymruensis: Discovery in Western hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: Redescription, molecular characterization, transmission to IFN-gamma gene knockout mice via sporocysts. Parasitology Research. 117:1195�1204.
Lalle, M., Possenti, A., Dubey, J.P., Pozio, E. 2018. Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready to eat salad. Food Microbiology. 70:137- 142.
Lacombe, A.C., Breard, A., Hwang, C., Hill, D.E., Fan, X., Huang, L., Yoo, B.K., Niemira, B.A., Gurtler, J., Wu, V.C. 2016. Inactivation of Toxoplasma gondii on blueberries using low dose irradiation without affecting quality. Journal of Food Protection. 73:981-985.
Zeng, W., Sun, L., Xiang, Z., Li, N., Zhang, J., He, Y., Li, Q., Yang, F., Hu, J., Song, J., Morris, J., Rosenthal, B.M., Yang, Z. 2018. Morphological and molecular characteristics of Sarcocystis bertrami from horses and donkeys in China. Veterinary Parasitology. 252:89-94.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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). Accomplishments 01 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. 02 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.

Impacts
(N/A)

Publications

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.
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. 2017. 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. 47:279-303.
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 Para�ba 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. doi: 10.1007/s00436-017-5462-6.
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.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. We administered the scientific components of the Agricultural Marketing Service (AMS) Analyst Training and Check Sample Program because fresh pork originating in the U.S. cannot be exported to the European Union (EU) , Russia, Singapore, and other trading partners unless exporting pork packers participate in the Program. Pork exports from the U.S. are required to undergo individual carcass testing by direct visualization of Trichinella muscle larvae (ML); testing is performed at the packing plant. Correct performance of direct visualization testing requires strict adherence to testing protocols. Inspectors from swine slaughter plants across the U.S. were trained and certified to perform the artificial digestion method approved for direct visualization of Trichinella larvae in swine tissues. We provided quarterly quality assurance (proficiency) samples to all currently certified analysts to check accuracy of test performance to maintain the integrity of the Program. The AMS Program preserves an international market for U.S. exporters exceeding 5 billion dollars annually. These efforts support export marketing efforts as requested by USDA regulatory agencies, maintaining and opening new export markets for U.S. producers. The National Retail Meat Survey for Toxoplasma in organic pork and American lamb was completed. The survey identified the risk of Toxoplasma infection to consumers from these meat products using samples collected from 25 Metropolitan Statistical Areas MSAs nationwide. These data were used to develop a systematic meta-analysis of T. gondii prevalence and a quantitative risk assessment for Toxoplasma in organic pork and American lamb in the United States to discern consumer risk from meat products from animals raised in uncontrolled management systems. We defined curing process endpoints necessary for developing a multi- factorial model for pork curing for inactivation of Trichinella and Toxoplasma, in collaboration with scientists at the USDA-ARS Eastern Regional Research Center (ERRC). Models for inactivation of Trichinella and Toxoplasma were not available in the ARS-Pathogen Modeling Program (PMP). Prediction of inactivation of Trichinella and Toxoplasma in pork meat could not be accomplished with the existing bacterial inactivation models in the PMP, and therefore, such models needed to be generated and validated de novo. The endpoint processes established the survival limits of the 2 parasites with respect to salt/brine concentration, water activity, pH, temperature, and time. The publically available models allow producers of ready-to-eat meat products to determine if their specific product formulations will inactivate Toxoplasma and Trichinella without having to complete process validation for each formulation, as would be required by Hazard Analysis and Critical Control Point (HACCP) regulations. Toxoplasma gondii from black bears (Ursus americanus), bobcats (Felis rufus), and feral cats (Felis catus) from Pennsylvania were isolated and genetically characterized. Recently, attention has focused on the genetic diversity of Toxoplasma to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, black bear (Ursus americanus), bobcat (Felis rufus), and feral cat (Felis catus) from the state of Pennsylvania were tested for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. Three genotypes were revealed, adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Three isolates were virulent in mice, causing 100% mortality. Results indicate that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risks to humans through consumption of game meat. Research examined whether or not two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16) were functionally conserved in Hammondia hammondi. To do this, we performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. Toxoplasma gondii host-modulating proteins GRA15 and ROP16 are functionally conserved in H. hammondi, but these species have distinct transcriptomes. The mechanisms underlying the phenotypic differences between T. gondii and its nearest extant relative, H. hammondi are unknown, but they are likely to be due to both gene content and gene expression. In this work, we determined that both GRA15 (HhGRA15) and ROP16 (HhROP16) from H. hammondi modulate the host NF-B and STAT6 pathways, respectively, similar to their T. gondii orthologs. A16 bp sequence that is deleted in the putative promoter of HhROP16 was also identified as a potential core promoter for TgROP16. In contrast to this functional conservation, we showed that the transcriptomes of H. hammondi and T. gondii are distinct. Twelve percent of the genes queried were at least 4-fold different between the two species, and some of these were uniquely-expressed in H. hammondi. Moreover, consistent with the rapid conversion of H. hammondi to bradyzoite (e.g., cyst) stages during in vitro growth, a subset of the transcripts that were of higher abundance in H. hammondi as compared to T. gondii are upregulated during the tachyzoite to bradyzoite transition than in T. gondii, suggesting that H. hammondi sporozoites may be more �cyst-like� in their expression profile than T. gondii. These data provide support for the hypothesis that gene deployment may play a more significant role in determining the phenotypic differences between these species than gene content. Geographic separation of domestic and wild strains of Toxoplasma gondii in French Guiana was shown to correlate with a monomorphic version of Chromosome1a. Previous studies have shown that human infections in jungle areas of French Guiana are often quite severe, unlike most human infections that are characterized by mild symptoms in healthy adults. Our work characterized the genetic makeup of strains from French Guiana and confirmed that while genetically homogeneous strains exist in human- adapted environments, highly divergent and pathogenic isolates are found in jungle environments. The geographic separation of strains is also mirrored in conserved genomic regions, including a monomorphic version of chromosome 1a, which has previously been associated with the spread of different lineages around the world. Parasite strains harboring the monomorphic chromosome showed greater potential for transmission in domestic cats, which may contribute to their prevalence in human-adapted environments. Findings also revealed large differences in acute virulence of French Guiana isolates in the laboratory mouse, and these differ from known genetic mechanism that have been defined previously. Hence, the ability of some strains to expand in the environment as a consequence of enhanced transmission may also lead to the spread of virulence determinants. Comparative sequence analysis of T. gondii isolates revealed that local genomic admixture drives concerted expansion and diversification of secreted determinants of pathogenesis. Toxoplasma differs substantially in its broad host distribution, from closely related parasites that typically have narrow, specialized host ranges. To understand this diversity, comparisons of the genome sequences of 62 globally diverse T. gondii isolates to several closely related apicomplexan parasites was completed. Results showed that the tandem amplification outcome and allelic diversification of secretory determinants are the primary features that distinguish the closely related genomes of these biologically diverse parasites. Results also showed that the unusual population structure of T. gondii is characterized by co-ancestry of large sections of the chromosome, suggesting that conserved inheritance of tandemly clustered genetic characters drives evolution of transmission, host range, and pathogenicity of apicomplexans. Finally, we conducted a survey in collaboration with 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 animal tongues collected, 1.81% were tissue positive for T. spiralis ML; no other species or genotypes were found. These data revealed that feral pigs serve as a reservoir for T. spiralis and T. gondii, both for sylvatic carnivores and domestic pigs, and demonstrated the potential for introducing these pathogens into domestic herds of non- biosecure swine production facilities in the U.S. as a result of increasing overlap of in their geographical ranges. Accomplishments 01 Industry practices reduce toxoplasmosis in U.S pork. ARS researchers in Beltsville, Maryland conducted a national serological survey to determine the seroprevalence of T.gondii in the national swine herd. The survey was conducted using a commercially available, USDA-validated ELISA assay and sera collected at slaughter to detect Toxoplasma positive samples. Established seroprevalence of Toxoplasma as measured in a statistically valid sampling of market weight pigs and sows at slaughter, covered 95% of slaughter production in the U.S., and provided the most current national dataset for Toxoplasma seroprevalence in market pigs destined for the fresh meat case and in sows destined for processed meat products. The survey also provided evidence of the impact of industry-led changes in swine management on the reduction of this zoonotic pathogen in the U.S. commercial pork supply. The data will be used to support science-based decisions on the most effective methods to continue decreasing seroprevalence of Toxoplasma in market pigs and sows; the primary livestock commodity infected with Toxoplasma. These data demonstrate the extent to which the level of Toxoplasma has been reduced in commercial pork as compared to previous surveys, and demonstrates the reduction of risk from Toxoplasma transmission from pork to humans in the U.S., which benefits public health and assists food producers and regulators as they seek to improve food safety.

Impacts
(N/A)

Publications