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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #421090


Location: Animal Parasitic Diseases Laboratory

2015 Annual Report

1a. Objectives (from AD-416):
Objective 1: Evaluate the impact of changing management and production practices (e.g. free range, confined, organic) on the incidence of Toxoplasmosis and Trichinella in swine, as it relates to foodborne risk. Objective 2: Assess the effectiveness of on-farm interventions (such as passive immunization therapy as a feed supplement) to prevent enteric Toxoplasma infection in swine, consequent foodborne risk, and potential interaction with Salmonella foodborne infections in swine. C.1., PS 1B, and PS 1.D, Section 4.1. Objective 3: Evaluate the impact of anthelminthic and antiprotozoal treatments on parasitic foodborne infections in swine and the potential foodborne risk. Evaluate the impact of anthelminthic and antiprotozoal treatments on the interaction between foodborne pathogen and foodborne parasitic infections, specficially coinfections of Toxoplasma, Trichinella with Salmonaella and Campylobacter in swine, and changes following treatments. C.1., P.S., 1.A, and P.S., 1.D.

1b. Approach (from AD-416):
Toxoplasma gondii infects 11-20% of the U.S. population, causing birth defects in exposed pregnant women, devastating disease in immunocompromised individuals, and illness and loss of vision in otherwise healthy adults. Consumption of infected pork may be a significant source of infection for consumers in the U.S. Meat derived from pasture-raised pigs is of particular concern, since prevalence in these pigs may exceed 50%. Reducing the risk of foodborne human infection from meat requires adherence to livestock production practices that prevent exposure of animals to the parasite at the farm level, and the development of new treatments which can be used in pasture-raised pigs to prevent infection. Trichinella spiralis is a serious zoonotic pathogen with an unusually broad host and geographic range. Trichinella species infecting game animals pose a risk to humans consuming these meat products as well as a risk to domestic pigs that feed on their carcasses. Understanding the risk to pigs that have access to infected wildlife is an important component of on-farm certification efforts. In addition, the safety of meat from pasture-raised swine needs to be assessed in light of increasing consumer demand for organically-raised meat products. We will evaluate the impact of different management and production practices (e.g., free range, confinement, organically-raised pigs) on the incidence of Toxoplasma and Trichinella in swine, as it relates to foodborne risk, and assess the effectiveness of on-farm interventions (such as passive immunization therapy as a feed supplement) to prevent enteric Toxoplasma infection in swine, consequent foodborne risk, and potential interaction with Salmonella foodborne infections in swine.

3. Progress Report:
In collaboration with scientists at the USDA-ARS Eastern Regional Research Center (ERRC), we completed the 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, water activity, pH, temperature, and time. Currently, models for inactivation of Trichinella and Toxoplasma are not available in the ARS-Pathogen Modeling Program. Consequently, prediction of inactivation of Trichinella and Toxoplasma in pork meat cannot be accomplished with existing models in the PMP, and therefore, such models need to be generated and validated de novo. (Objective 1.2). 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 (Objective 1.2). Continued National Retail Meat Survey for Toxoplasma in organic pork and American lamb. The survey will identify the risk of Toxoplasma infection to consumers from these meat products using samples collected from 25 Metropolitan Statistical Areas (MSAs) nationwide. These data will be used to discern consumer risk from meat products from animals raised in uncontrolled management systems (Objective 3.2).

4. Accomplishments
1. Prevalence and risk factors of Toxoplasma gondii infection in meat animals and meat products destined for human consumption. Toxoplasma gondii is a protozoan parasite that is responsible for approximately 24% of all estimated deaths attributed to foodborne pathogens in the United States. Human infection results from accidental ingestion of oocysts, from the environment, in water or on insufficiently washed produce or through consumption of raw or undercooked meat products that contain T. gondii tissue cysts. Agricultural Research Service (ARS) scientists in Beltsville, Maryland, working with researchers at the Center for Disease Control (CDC), reviewed studies of T. gondii infection in meat because substantial proportions of human T. gondii infection are acquired through consumption of raw or undercooked meat. Prevalence of T. gondii is higher in conventionally reared pigs, sheep and poultry as compared to cattle. Prevalence of T. gondii is greater in meat products from organic compared to conventionally reared meat animals because of outdoor access that poses substantially greater opportunities for exposure to infected rodents, wildlife, and oocyst-contaminated feed, water, or environmental surfaces. Risk factors related to T. gondii exposure for livestock include farm type, feed source, presence of cats, methods of rodent and bird control, carcasses handling and water quality. This review serves as a useful resource and information repository for informing quantitative risk assessment studies for T. gondii infection in humans through meat consumption.

2. Isolation and genetic characterization of Toxoplasma gondii from black bears (Ursus americanus), bobcats (Felis rufus), and feral cats (Felis catus) from Pennsylvania. Toxoplasma gondii infects virtually all warm-blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite 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, Animal Parasitic Disease Laboratory (APDL) tested black bear (Ursus americanus), bobcat (Felis rufus), and feral cat (Felis catus) from the state of Pennsylvania 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 indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risks to infect humans through consumption of game meat.

3. Comparative sequence analysis of Toxoplasma gondii reveals local genomic admixture drives concerted expansion and diversification of secreted pathogenesis determinants. Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To understand this diversity, Animal Parasitic Disease Laboratory (APDL) compared the genomes sequences of 62 globally diverse T. gondii isolates to several closely related apicomplexan parasites. APDL's finding revealed that the tandem amplification outcome and allelic diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. APDL further showed that the unusual population structure of T. gondii is characterized by co-ancestry of large chromosomal haploblocks, suggesting that conserved inheritance of tandemly clustered determinants drives evolution of transmission, host range, and pathogenecity of apicomplexans.

4. Geographic separation of domestic and wild strains of Toxoplasma gondii in French Guiana correlates with a monomorphic version of Chromosome1a. Toxoplasma gondii is a widespread parasite of animals that is easily transmitted to humans. 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. Were Animal Parasitic Disease Laboratory (APDL) characterized the genetic makeup of strains from French Guiana and confirmed that while genetically homogeneous strains exist in anthropized environments, highly divergent and pathogenic isolates are found in jungle environments. The geographic separation of strain types 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. Strains harboring the monomorphic chromosome showed greater potential for transmission in domestic cats, which may contribute to their prevalence in anthropized environments. APDL 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 strain types to expand in the environment as a consequence of enhanced transmission may also lead to the spread of virulence determinants.

5. Toxoplasma gondii host-modulating proteins GRA15 and ROP16 are functionally conserved in Hammondia hammondi, but these species have distinct transcriptomes. The mechanisms underlying the phenotypic differences between the human pathogen Toxoplasma gondii and its nearest extant relative, Hammondia hammondi are unknown, but they are likely to be due to both gene content and gene expression differences. To address this further we tested whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16) were functionally conserved in H. hammondi, and performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. Animal Parasitic Disease Laboratory (APDL) found that both GRA15 and ROP16 from H. hammondi (HhGRA15; HhROP16) modulate the host NF-B and STAT6 pathways, respectively, similar to their T. gondii orthologs. APDL also identified a 16 bp sequence that is deleted in the putative promoter of HhROP16 as a potential core promoter for TgROP16. In contrast to this functional conservation APDL found the transcriptomes of H. hammondi and T. gondii to be 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 T. gondii are upregulated during the tachyzoite to bradyzoite transition 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.

Review Publications
Dubey, J.P., Lane, E., Vanwilpe, E., Suleman, E., Reininghaus, B., Verma, S., Rosenthal, B.M. 2014. Sarcocystis cafferi, n. sp. (Protozoa: Apicomplexa) from the African buffalo (Syncerus caffer). Journal of Parasitology. 100(6):817-827.
Dubey, J.P., Verma, S., Ferreira, L., Cassinelli, A., Yuging, Y., Kwok, O.C., Tuo, W., Chiesa, O., Jones, J. 2014. Detection and survival of Toxoplasma gondii in milk and cheese from experimentally infected goats. Journal of Food Protection. 77:1747–1753.
Walzer, K., Dam, R., Srinivasan, A., Borges, A., Hermann, D., Schares, G., Dubey, J.P., Boyle, J. 2014. Hammondia hammondi harbors functional orthologs of the host-modulating effectors GRA15 and ROP16 but is distinguished from toxoplasma gondii by a unique transcriptional profile. Eukaryotic Cell. 13(12):1507-1518.
Dubey, J.P., Hilali, M., Van Wilpeerna, Verma, S., Calero-Bernal, R., Adel-Wahab, A. 2015. Redescription of Sarcocystis fusiformis sarcocysts from the water buffalo (Bubalus bubalis). Veterinary Parasitology. 142(2):385-394.
Ness, S., Schares, G., Peters-Kennedy, J., Mittel, L., Dubey, J.P., Bowman, D., Mohammed, H., Drivers, T. 2014. Serological diagnosis of Besnoitia bennetti infection in donkeys. Journal of Veterinary Diagnostic Investigation. 26(6):778-782.
Dubey, J.P. 2014. Life cycle of Cystoisospora felis (Coccidia: Apicomplexa) in cats and mice. Journal of Eukaryotic Microbiology. 61:637-643.
Verma, S., Chunlei, S., Dubey, J.P. 2015. Toxoplasma gondii isolates from mouflon sheep (Ovis ammon) from Hawaii, USA. Journal of Eukaryotic Microbiology. 62(1):141-143. doi: 10.1111/jeu.12151.
Lopes, A., Dubey, J.P., Darde, M., Cardosa, L. 2014. Epidemiological review of Toxoplasma gondii infection in humans and animals in Portugal. Parasitology. 141:1699-1708.
Calero-Bernal, R., Verma, S., Oliveira, S., Yang, Y., Rosenthal, B.M., Dubey, J.P. 2015. In the United States, negligible rates of zoonotic sarcocystosis occur in feral swine that, by contrast, frequently harbor infections with Sarcocystis meischeriana, a related parasite contracted from dogs. Parasitology. 142:549-556.
Alvarado-Esquivel, C., Romero-Salas, D., Garcia-Vazquez, Z., Cruz-Romero, A., Peniche-Cardena, A., Ibarra-Priego, N., Aguilar-Dominguez, M., Perez-De-Leon, A., Dubey, J.P. 2014. Seroprevalence and correlates of Toxoplasma gondii infection in water buffaloes (Bubalus bubalis) in Veracruz state, Mexico. BioMed Central (BMC) Veterinary Research. 10:232e.
Cabral, A., D'Auria, S., Camargo, M., Rosa, A., Sodre, M., Galvao-Dias, M., Jordao, L., Dubey, J.P., Gennari, S., Pena, H. 2014. Seroepidemiology of Toxoplasma gondii infection in bats from São Paulo city, Brazil. Veterinary Parasitology. 206:3-4.
Lopes, A., Granada, S., Oliveira, A., Brancal, H., Dubey, J.P., Cardosa, L., Vilhena, H. 2014. Toxoplasmosis in dogs: First report of Toxoplasma gondii infection in any animal species in Angola. Pathogens and Global Health. DOI: 10.1179/2047773214Y.0000000160.
Rigoulet, J., Hennache, A., Lagourette, P., George, C., Longeart, L., Le Net, J., Dubey, J.P. 2014. Toxoplasmosis in a bar-shouldered dove (Geopelia humeralis) from the zoo of Clères, France. Parasite. 21:62.
Yang, Y., Zhang, Q., Kong, Y., Ying, Y., Kwok, O.C., Llang, H., Dubey, J.P. 2014. Low prevalence of Neospora caninum and Toxoplasma gondii antibodies in dogs in Jilin, Henan and Anhui Provinces of the People’s Republic of China. BioMed Central (BMC) Veterinary Research. 10:295.
Dubey, J.P., Howe, D., Furr, M., Saville, W., Marsh, A., Reed, S., Grigg, M. 2015. An update on Sarcocystis neurona infections in animals and Equine Protozoal Myeloencephalitis (EPM). Veterinary Parasitology. 209:1-42.
Alvarado-Esquivel, C., Alvarado-Esquivel, D., Dubey, J.P. 2015. Prevalence of Toxoplasma gondii antibodies in domestic donkeys (Equus asinus) in Durango, Mexico slaughtered for human consumption. Veterinary Research. 11:6.
Alvarado-Esquivel, C., Vazquez-Morales, R., Colado-Romero, E., Guzman-Sanchez, R., Liesenfeld, O., Dubey, J.P. 2015. Prevalence of infection with Toxoplasma gondii in landrace and mixed breed pigs slaughtered in Baja California Sur state, Mexico. European Journal of Immunology. 5(2015):112-115.
Reichel, M., Moore, D., Hemphill, A., Ortega-Mora, L., Dubey, J.P., Ellis, J. 2015. A live vaccine against Neospora caninum abortions in cattle. Vaccine. 33(2015):1299–1301.
Dubey, J.P., Ionela, H., Olariu, R., Darabus, G., Jones, J. 2014. Epidemiological review of Toxoplasmosis in humans and animals in Romania. Parasitology. 141:311-315.
Dubey, J.P., Ness, S.L., Kwok, O.C., Choudhary, S., Mittel, L., Divers, T. 2014. Toxoplasma gondii seroprevalence in donkeys (Equus asinus) from the U.S.A. and isolation of T. gondii from cats on donkey farms. Veterinary Parasitology. 199:18-23.
Shwab, K., Zhu, X., Majumdar, D., Pena, H., Gennari, S., Dubey, J.P. 2014. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology. 141:453-461.
Dubey, J.P., Johnson, J., Hanson, M., Pierce, V. 2014. Diagnosis of toxoplasmosis-associated abortion in an alpaca (Vicugna pacos) fetus. Veterinary Parasitology. 45:461-464.
Dubey, J.P., Whitesell, L.E., Culp, W.E., Daye, S. 2014. Diagnosis and treatment of Neospora caninum-associated dermatitis in a red fox (Vulpes vulpes) with concurrent Toxoplasma gondii infection. Journal of Zoo and Wildlife Medicine. 45:454-457.
Lindsay, D.S., Dubey, J.P. 2013. Toxoplasmosis in wild and domestic animals. In: Weiss, L., Kim, K., editors. New York, NY: Elsevier. p. 193-215.
Dubey, J.P., Jones, J. 2014. Comments on "Detection of Toxoplasma gondii in raw caprine, ovine, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran". Foodborne Pathogens and Disease. 11(6):500-501.
Dubey, J.P., Newell, T., Verma, S., Edward, S. 2014. Toxoplasma gondii infection in llama (Llama glama): acute visceral disseminated lesions, diagnosis, and development of tissue cysts. Journal of Parasitology. 100:288-295.
Behinke, M., Zhang, T., Dubey, J.P., Sibley, L.D. 2014. Toxoplasma gondii merozoite global gene expression analysis with comparison to the life cycle discloses a unique upregulated expression state during enteric development. Genomics. 15:350.
El Bissati, K., Zhou, Y., Dasgupta, D., Cobb, D., Dubey, J.P., Burkhard, P., Lanar, D., Mcleod, R. 2014. Effectiveness of a novel immunogenic nanoparticle platform for Toxoplasma peptide vaccine in HLA transgenic mice. Vaccine. 32:3243-3248.
Lindsay, D., Houk, A., Mitchel, S., Dubey, J.P. 2014. Developmental biology of Cystoisospora (Apicomplexa: Sarcocystidae) monozoic tissue cysts. Journal of Parasitology. 100:392-398.
Almeria, S., Serrano-Perez, B., Darwich, L., Araujo, R., Lopez-Gatius, F., Dubey, J.P., Gasbarre, L. 2014. Maternal and fetal immune response patterns in heifers experimentally infected with Neospora caninum in the second trimester of pregnancy - A descriptive study. Veterinary Parasitology. 204:146-152.
Dubey, J.P., Fayer, R., Rosenthal, B.M., Calero-Bernal, R., Uggla, A. 2014. Identity of Sarcocystis species of the water buffalo (Bubalus bubalis) and cattle (Bos taurus) and the suppression of Sarcocystis sinensis as a nomen nudum. Veterinary Parasitology. 205:1-6.
Alvarado-Esquivel, C., Romero-Salas, D., Garcia-Vazquez, Z., Cruz-Romero, A., Ibarra-Priego, N., Ahuja-Aguirre, C., Agular-Dominguez, M., Perez De Leon, A.A., Dubey, J.P. 2014. High prevalence of Toxoplasma gondii antibodies in dogs in Veracruz, Mexico. Veterinary Research. 10:191.
Agerholm, J., Dubey, J.P. 2014. Sarcocystosis in a stillborn lamb. Reproduction of Domestic Animals. DOI: 10.1111/rda.12398.
Canon-Franco, W., Lopez-Orozco, N., Gomez-Marin, J., Dubey, J.P. 2014. An overview of seventy years of research (1944–2014) on toxoplasmosis in Colombia, South America. Parasites & Vectors. 7:427.
Dubey, J.P., Cassey, S., Zajac, A., Wildeus, S., Lindsay, D., Verma, S., Oliveira, S., Kwok, O.C., Su, C. 2014. Isolation and genetic characterization of Toxoplasma gondii from alpaca (Vicugna pacos) and sheep (Ovis aries) grazed on pasture in Virginia. Tropical Animal Health and Production. 46:1503-1507.
Cosendey-Kezen Leite, R., Rodrigues De Oliveir, F., Frazao-Teixeira, E., Dubey, J.P., De Souza, G., Lilenbaum, W. 2014. Occurrence and risk factors associated to Toxoplasma gondii infection in sheep from Rio de Janeiro, Brazil. Tropical Animal Health and Production. 46:1463-1466.
Khan, A., Ajzenberg, D., Mercier, A., Demar, M., Simon, S., Darde, M., Rosenthal, B.M., Verma, S., Dubey, J.P., Sibley, L. 2014. Geographic separation of domestic and wild strains of T. gondii in French Guiana correlates with a monomorphic version of chromosome 1a and enhanced transmission in the domestic cat. PLOS Neglected Tropical Diseases. DOI: 10.1371/journal.pntd.0003182.
Grigg, M., Dubey, J.P., Nussenblatt, R. 2015. Ocular Toxoplasmosis: Lessons from Brazil. American Journal of Ophthalmology. 159:999-1001.
Fayer, R., Esposito, D., Dubey, J.P. 2015. Sarcocystis in Humans. Clinical Microbiological Reviews. 28(2):295-311.
Dubey, J.P., Verma, S., Calero-Bernal, R., Cassinelli, A., Kwok, O.C., Van Why, K., Su, C., Humphreys, J. 2014. Isolation and genetic characterization of Toxoplasma gondii from black bears (Ursus americanus), bobcats (Felis rufus), and feral cats (Felis catus) from Pennsylvania. Journal of Eukaryotic Microbiology. DOI: 10.1111/jeu.12196.
Dubey, J.P., Ferguson, D. 2014. Life cycle of Hammondia hammondi (Apicomplexa: Sarcocystidae) in cats. Journal of Eukaryotic Microbiology. 62:346-352.
Verma, S., Ajzenberg, D., Rivera-Sanchez, A., Su, C., Dubey, J.P. 2015. Genetic characterization of Toxoplasma gondii isolates from Portugal, Austria, and Israel reveals higher genetic variability within the type II lineage. Parasitology. 142:948-957.
Ferreira, D., Ribeiro, V., Larque, P., Wagner, P., Pinheiro, J., Silva, J., Dubey, J.P., Rego, E., Mota, R. 2015. Risk factors associated with Toxoplasma gondii infection in captive Sapajus spp. American Journal of Primatology. 77:558–562.