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

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

Location: Animal Parasitic Diseases Laboratory

2018 Annual Report

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

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

Progress Report
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.

1. 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.

2. 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.

3. 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).

4. 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.

5. 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.

Review 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.