Location: Animal Parasitic Diseases Laboratory
2024 Annual Report
Objectives
Objective 1: Describe natural sporulation in coccidian oocysts, including temporal changes in the expression of genes over the course of sporulation and during the degradation of ‘old oocysts’ and morphological changes over the course of sporulation.
Sub-objective 1.A. Characterize changes in gene expression as oocysts of E. acervulina mature to their sporulated and infectious state using RNA-Seq to elucidate maturation and identify biomarkers for assaying oocyst viability and infectivity.
Sub-objective 1.B: Determine the markers of oocyst senescence by tracking the waning of gene expression and/or the advent of apoptotic signals in E. acervulina.
Sub-objective 1.C: Determine whether developmental gene expression patterns established for E. acervulina hold for other Eimeria species infecting poultry.
Sub-objective 1.D: Characterize morphological changes of E. acervulina oocysts during sporulation and senescence.
Objective 2: Develop assays for coccidian oocyst viability; and using the information from Objective 1, establish gene products strongly up-regulated in infectious, sporulated oocysts of Eimeria. Using comparative genomics, determine homologues in the Cyclospora genome.
Sub-objective 2.A: Establish quantitative assays targeting gene products established (above) to undergo the strongest and most consistent up-regulation in mature oocysts of E. acervulina.
Sub-objective 2.B: Determine the extent to which these genes’ expression levels predict the infectiousness of oocyst cohorts, using cell culture assays and/or in vivo challenge experiments.
Sub-objective 2.C. Identify homologues, in the genome of Cyclospora cayetanensis, of genes consistently and significantly up-regulated in various species of Eimeria infecting poultry.
Sub-objective 2.D: Develop quantitative assays designed to measure expression levels of genes deemed most likely over-expressed in mature, infectious oocysts of C. cayetanensis.
Objective 3: Evaluate the efficacy of interventions for produce safety using the surrogate. Evaluate known interventions on viability, and on the biomarkers to validate assays for coccidian viability.
Sub-objective 3.A: Determine the efficacy of various washing procedures on limiting contamination of produce with oocysts of E. acervulina.
Sub-objective 3.B. Improved detection of E. acervulina and C. cayetanensis DNA in contaminating matrices using genome probe capture arrays.
Objective 4: Continue to advance the molecular epidemiology of other foodborne zoonotic parasites in livestock and wildlife in the U.S. such as Trichinella in feral swine, pastured swine, and wild carnivores, and Sarcocystis zoonotic species in cattle.
Sub-objective 4.A: Determine the efficacy of whole genome and reduced representational sequencing methods to individuate outbreak lineages of Trichinella spiralis.
Sub-objective 4.B: Document prevalence of Trichinella in defined compartments of food production and in wild game populations, and the prevalence of anti-Trichinella antibodies in those animals.
Sub-objective 4.C: Survey U.S. beef for the presence of Sarcocystis species, including the zoonotic species.
Approach
Foodborne parasites exact a serious toll on public health, undermine public confidence in the safety of food, interfere with agricultural marketing and trade, and impose liabilities and exact costs on farmers and food producers. Adopting a “One Health” approach that recognizes commonalities in protecting human, veterinary, and environmental health, we will pursue scientific goals capable of ameliorating burdens imposed by longstanding and emerging problems imposed by parasites contaminating meats and fresh produce. We will first establish a safe and tractable model for Cyclospora cayetanensis, the agent of human cyclosporiasis, using a closely related poultry parasite, Eimeria acervulina. We will use this model to evaluate practical ways to minimize people’s exposure to infection with coccidian oocysts, and will endeavor to supply our regulatory partners with molecular assays to assess parasite viability and infectivity. We will also advance the molecular epidemiology of other foodborne zoonotic parasites in livestock and wildlife in the U.S. such as Trichinella spp. and Sarcocystis zoonotic species. Studies will determine the efficacy of sequencing methods to individuate outbreak lineages of Trichinella spiralis and document prevalence of Trichinella in compartments of food production and in wild game populations. Further studies will analyze the prevalence of Trichinella spp. antibodies in those animals and characterize the presence of Sarcocystis species (including an important zoonotic species) in the U.S. beef supply. Taken together, these studies will address important research gaps and provide powerful tools to producers and food safety regulators for monitoring and ameliorating food safety risks imposed by parasitic infection.
Progress Report
Progress was made towards all project Objectives.
In support of Objective 1, we extended our prior work in two important directions. We published a study comparing gene expression in three species of Eimeria and began validating these predictions in Cyclospora with very promising results. This clarifies what master regulators of physiology and development are conserved among such species. The conserved and strongly expressed genes appear ripe for use as viability markers to better support industry and regulatory decisions and actions intended to mitigate this threat to produce safety. Our completed evaluations of gene expression in aging and dying oocysts were published, building a foundation for viability determination.
Our published work on the benefits of studying surrogates attracted additional attention of stakeholder groups, winning additional grant support (2 new proposals to the Center for Produce Safety) and partnerships to develop aptamer-based detection assays for Cyclospora. Our collaborations with the University of Tennessee produced publications describing Ultraviolet (UV) and Ozone as plausible, enabling the training of a successful Artificial Intelligence-based image processing system for oocyst maturation, and establishing as efficacious disinfection systems for Cyclospora based on UV irradiation and ozonation.
In support of Objective 2, we advanced progress towards viability assays in Eimeria and Cyclospora. Core housekeeping genes provide the most suitable viability markers in Cyclospora, where regulators and industry are plagued with diagnostic assays lacking information about viability (and therefore, risk). We evaluated several of these using quantitative PCR assays.
We completed successfully completed a project, supported by the Center for Produce Safety, defining good performance of filters comprised of sand and zero valent iron to reduce water contamination with oocysts of coccidian parasites. The final report was well received, a manuscript is in review, and industry partners are interested in exploring commercialization of key findings.
A second project funded by the Center for Produce Safety (Viability Assays for Cyclospora) took an unexpected and very promising turn when we refocused our efforts due to the serendipitous discovery of physical changes accompanying parasite senescence. These differences are readily recognized by an artificial intelligence (AI) model, enabling rapid, automated enumeration of live and dead oocysts. Moreover, the fluorescence properties served as a successful basis to physically sort parasites into live and dead categories.
We did not embark on some of the anticipated washing experiments outlined for Objective 3, but we made progress on produce sanitation that we had planned due to excellent collaborations. In particular, our partners at the University of Tennessee (via a Material Transfer Research Agreement) developed a high-throughput image analyzer that they succeeded in using to establish, as efficacious, three wavelengths of Ultraviolet (UV) light, as well as an ozonation procedure, to arrest development of Eimeria oocysts. Ozonation proved efficacious, in a dose-dependent manner, to arrest the development of parasites. This work was favorably reviewed and published.
Supporting Objective 4A, ARS scientists in Beltsville, Maryland, previously published a paper demonstrating Rad-Seq as a powerful, rapid tool to individuate outbreak lineages of Trichinella spiralis. Prior work seeking to trace outbreaks of this foodborne parasite faced steep hurdles owing to the inbred nature of this parasite in Europe and the Americas. The best prior attempts employed a “DNA fingerprinting” method based on characterizing variation in a dozen or more genes harboring repetitive sequences of varying length. Our faster approach simultaneously sequences thousands of genomic fragments from parasites and uses a workflow requiring just a few days.
In support of Objective 4B, we completed a landmark, nationwide survey of pork produced under the Pork Quality Assurance Plus standard and found no instances of infection in over 3.2 million samples tested. This landmark achievement (manuscript in review) will have major benefits for producers, for the Agricultural Marketing Service, for food safety authorities, and for trade negotiators, because it supports with data the benefits of biosecure housing and proper feeding of swine, reducing the public health threat posed by Trichinella in conventionally raised swine as negligible.
In support of Objective 4C, we completed a study of Sarcocystis in beef, finding near universal infection with an enzootic species (acquired from dogs and wild canids) and advanced the first evaluation (using needed metagenomic tools) of the possibility of zoonotic species, as has been reported from Europe and elsewhere.
Accomplishments
1. Parasite viability genes discovered. Managing the harms posed by enteric parasites benefits both animal husbandry and food safety. For example, parasites in the Coccidia family, which includes Eimeria, harms poultry health and production, while Cyclospora Cayetanensis compromises produce safety. ARS researchers in Beltsville, Maryland, examined commonalities in the repertoire of genes expressed by such parasites, focusing on how these parasites develop to become infectious. They discovered broadly shared aspects of parasite development and noted a few genes whose distinct expression profiles may accelerate or delay development, and which may account for differences in virulence. These insights support development of tests to determine where and when viable parasites pose actual risk, tools repeatedly requested by growers and regulators who otherwise are left to manage risk based on equivocal evidence.
2. A drug to inactivate Trichinella in meat. Modern production practices safeguard swine from Trichinella infection, but pastured herds still pose risk to consumers. ARS researchers in Beltsville, Maryland, evaluated four drugs and determined that one, mebendazole, renders encysted larvae non-infective. This finding provides a new tool for producers and regulators to manage food safety risk from this once burdensome parasite.
3. Ozone can stop parasites in their tracks. Coccidian parasites pose food safety risks and compromise the health of livestock and are far less susceptible to sanitizing agents than common bacterial and viral pathogens. ARS researchers in Beltsville, Maryland, partnered with the researchers at the University of Tennessee in Knoxville, Tennessee, to study the promise of ozonated water against such parasites. Using a common chicken parasite as a model, they documented dose-dependent declines injury, approaching 90% success, especially when the ozone was stabilized by addition of citric acid. These studies showed a promising new approach to managing a harmful produce pathogen, have engaged the interest of produce growers and packers, grocers, food safety professionals, and parasitologists, and have spurred new collaborative research with food safety technology companies interested in commercializing the approach.
4. A persistent, widespread parasite. Parasites in the genus Sarcocystis impair livestock wildlife health and can threaten food safety. Such parasites, which cycle between carnivorous hosts and the herbivores upon which they prey, should undergo continuous change because they reproduce via sexual recombination. ARS researchers in Beltsville, Maryland, discovered that the same genotype responsible for a disease outbreak among racoons in the Midwest caused an outbreak, a decade later, in Pacific marine mammals. Fortunately, this particular parasite (Sarcocystis neurona) has not yet imperiled human health. But knowing that this genotype persists and spreads, it is as a concern for veterinary health and underscores that parasites like it can remain genetically stable.
5. A widespread cattle parasite, redescribed. Of the more than 200 known species of Sarcocystis, one occurs in (and sometimes harms) almost all cattle. Its proper identification rests on subtle distinctions from related cattle parasites that can cause human disease via consumption of undercooked beef. ARS researchers in Beltsville, Maryland, reevaluated life cycle of Sarcocystis cruzi, identified additional morphological details of the developmental stages, redescribed the parasite, provided the first molecular characterization from experimentally infected cattle, and deposited life cycle stages in the Smithsonian Museum for future reference. This work will serve as the cornerstone for future diagnoses and studies, benefitting biologists, veterinarians, parasitologists, and beef and dairy farmers.
Review Publications
Gupta, A., Duncan, M., Araujo, L., Kwok, O.C., Rosenthal, B.M., Khan, A., Dubey, J.P. 2023. The same genotype of Sarcocystis neurona responsible for mass mortality in marine mammals induced a clinical outbreak in raccoons (Procyon lotor) 10 years later. International Journal for Parasitology. 53(14):777-785. https://doi.org/10.1016/j.ijpara.2023.08.001.
Dubey, J.P., Gupta, A., De Araujo, L., Kwok, O.C., Khan, A., Rosenthal, B.M. 2023. Sarcocystis cruzi (Hasselmann, 1923) Wenyon, 1926: redescription, molecular characterization, and deposition of life cycle stages specimens in the Smithsonian Museum. Parasitology. 150(13):1192-1206. https://doi.org/10.1017/S003118202300094X.
Tucker, M.S., Obrien, C.N., Johnson, A.N., Dubey, J.P., Rosenthal, B.M., Jenkins, M.C. 2023. Rna-seq of phenotypically distinct Eimeria maxima strains reveals coordinated and contrasting maturation and shared sporogonic biomarkers with Eimeria acervulina. Pathogens. 13(1). Article e13010066. https://doi.org/10.3390/pathogens13010002.
Fredericks, J., Hill, D.E., Zarlenga, D.S., Fournet, V.M., Hawkins-Cooper, D.S., Urban, J.F., Kramer, M.H. 2024. Inactivation of encysted muscle larvae of Trichinella spiralis in pigs using Mebendazole. Veterinary Parasitology. 327: Article e110140. https://doi.org/10.1016/j.vetpar.2024.110140.
Baumann, A.A., Myers, A.K., Khajeh-Kazerooni, N., Rosenthal, B.M., Jenkins, M.C., Obrien, C.N., Fuller, L., Zhong, Q., Morgan, M., Lenaghan, S.C. 2024. Aqueous ozone exposure inhibits sporulation in the Cyclospora cayetanensis surrogate Eimeria acervulina. Journal of Food Protection. 87(5): Article e10026. https://doi.org/10.1016/j.jfp.2024.100260.