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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

2016 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
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.

1. 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.

Service on the National Committee on Neglected Infections of Poverty in the United States, Centers for Disease Control. Member, Working Group on Data collection, surveillance, epidemiology, and mapping, and Working Group, Outreach: Mobilizing Communities/Messaging