2009 Annual Report
1a.Objectives (from AD-416)
To develop improved serological detection assays for Salmonella and Toxoplasma in humans, pigs, and chickens. Currently available serological tests for Salmonella in swine contain antigens from serovars that do not predominate in U.S. Midwestern swine. As a result, serological tests perform poorly using sera from these animals. We will use serovar specific antigen isolates from Salmonella to develop a serological test which is more sensitive and specific for detection of Salmonella serovars which predominate in Midwestern swine. Until recently, differentiation of foodborne versus oocyst transmission of oxoplasma to humans was impossible. Identification of stage specific antigens from Toxoplasma oocysts have made the development of a validated serological assay possible. We will use these antigens to develop diagnostic methods to determine the most common transmission route of Toxoplasma in humans, and validate a serological test for detection of Toxoplasma infection in chickens.
1b.Approach (from AD-416)
Though previous studies have identified characteristics of high risk management systems, medium and low risk systems have not been completely characterized. We will identify those management systems and strategies that reduce or eliminate Toxoplasma from swine herds on the farm, and develop a comprehensive risk model for the swine industry. Though pork has been identified as a potential source of human infection for Toxoplasma, recent studies suggest that chicken may also be a risk for human transmission. We will analyze Toxoplasma prevalence in chickens raised in different management systems and develop practical interventions for reducing risk of exposure.
Until recently, differentiation of foodborne versus oocyst transmission of Toxoplasma to humans was impossible. Identification of stage specific antigens from oocysts have made the development of a validated serological assay possible. We will use these antigens to develop diagnostic methods to determine the most common transmission route of Toxoplasma in humans.
The U.S. export market for pork and horsemeat is dependent upon industry compliance with the testing requirements of importing countries. The need for international validation, standardization, and other quality control requirements for digestion-based Trichinella testing is critical as countries compete for export markets. We will collaborate with trading partners and international food safety organizations to harmonize testing procedures for Trichinella in meat products destined for export.
Conducted genotyping on 168 T.gondii isolates (designatedTgPgUs15-182) from various sources using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genotyping data from an additional 14 isolates collected from T.gondii-infected pigs in Maryland were included for analysis. Clonal Type II and III strains predominated in domestic pigs used for human consumption, as well as diverse, previously unrecognized T. gondii lineages.
Examined the prevalence of T. gondii in 152 free-range chickens (Gallus domesticus) from 22 municipalities in 7 northeastern states (Pernambuco, Rio Grande do Norte, Maranhão, Bahia, Ceará, Sergipe, and Alagoas) of Brazil. Results indicate widespread contamination of the rural environment in Brazil with T. gondii oocysts.
Completed isolation of T. gondii from the Bottlenose dolphins (Tursiops truncatus). Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. During 2005, 2006, and 2007 serum or blood clot, and tissues (brain, heart, skeletal muscle) of 52 T. truncatus stranded on the costs of South Carolina were tested for T. gondii; 53% were positive. Three T. gondii isolates (designated TgDoUs103) were recovered and genotyped using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c2208, c29-2, L358, PK1 and Apico). Two genotypes were revealed and belong to the clonal Type II lineage. One isolate has a unique genotype. This is the first report of isolation of viable T. gondii from T. truncatus.
Conducted serological surveillance of wild boar for Trichinella infection in the U.S. For acceptance of pork produced in the U.S. Trichinae Certification Program, recent legislation in the European Union endorses surveillance of wildlife indicator populations. A serological study was undertaken in wild boar collected during annual national surveys conducted by APHIS, NWDP. Results of this study indicate that Trichinella infection does exist in wild boar in the U.S. and these infections are clustered in specific geographic regions. Raising domestic pigs outdoors in these areas could pose a risk for exposure to infection through reservoir or intermediate hosts. The study demonstrated that wild boar should be useful as an indicator species to determine the prevalence of Trichinella spiralis in wildlife populations in the U.S.
Planned and conducted research on determination of freezing parameters for destruction of all North American genotypes of Trichinella larvae in pork. Results indicate that freezing of pork containing sylvatic genotypes of Trichinella at temperatures currently proscribed in CFR 318.10 for control of T. spiralis in swine are sufficient to destroy freeze resistant muscle larvae in pork.
Conducted quarterly training program for packer analysts for approved direct detection methods for T. spiralis in pork and horsemeat. Conducted quarterly testing of analysts and evaluated test results in consultation with the Animal and Plant Health Inspection Service (APHIS) and the Agricultural Marketing Service (AMS) to maintain integrity of the analyst training program.
Determination that oocysts from cats are likely the predominant source of T. gondii infections in pregnant women in the U.S. Undetected contamination of food and water by T. gondii oocysts frequently causes infection of humans in North America with T. gondii. We developed an assay that differentiates between oocyst induced and meat-borne Toxoplasma infection in humans. Detection of serum antibody to an 18.3 kDa sporozoite protein was a sensitive and specific method to identify primary infection by sporozoites within the past 6-8 months in persons infected post-natally. This antibody persisted in sera for at least 8 months. Sera from 72 uninfected persons and 59 chronically infected persons were unreactive. Sera from 6 persons in a laboratory epidemic, 17 persons in a riding stable epidemic, 10 persons in a family epidemic, 4 persons part of an epidemic in Victoria Canada, and 76 mothers of congenitally infected children in the National Collaborative Congenital Toxoplasmosis Study (NCCCTS) were tested for antibody. All 6 of the laboratory personnel (100%), 6/10 family members (60%), 15/17 riding stable visitors (88%), and 4/4 Canadian residents (100%), and 59 (78%) of 76 mothers of congenitally infected infants in the NCCCTS had sera that contained antibody to the 18.3 kDa sporozoite protein. Thus, it is likely that these 4 North American outbreaks were due to infection with oocysts. To prevent the suffering and the adverse economic consequences associated with congenital toxoplasmosis in North America, education programs describing hygienic measures and implementation of systematic serologic testing of pregnant women and newborns followed by treatment will prevent most infections. The remaining foodborne infections can be prevented by implementation of farm-level biosecurity measures to prevent animal infections, and education on proper handling and preparation of meats.
Isolate designation and characterization of Toxoplasma gondii isolates from pigs in the United States. Pigs are considered the most important meat source of Toxoplasma gondii for humans in the U.S. In the present study, 168 T. gondii isolates (designatedTgPgUs15-182) from various sources were genotyped using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genotyping data from an additional 14 isolates collected from T. gondii-infected pigs in Maryland were included for analysis. Nine genotypes (#1-9) were recognized from the 182 T. gondii isolates. Most isolates (56%, 102) were clonal Type II (genotypes #1 and #2) and 27% (49) were clonal Type III (genotype #3) strains. Genotype #4 had Type II alleles with the exception of Type I alleles at loci Apico and L358. Eight isolates (genotype #5) from Iowa had a combination of alleles I, II, and III at different loci. The remaining 6 isolates were divided into genotypes # 6-9 and had a combination of different alleles. Eight of the 9 genotypes were previously reported in different animal species and geographical regions. In conclusion, along with the predominance of clonal Type II and III strains, a few diverse, previously unrecognized T. gondii lineages were found circulating in domestic pigs used for human consumption. This work relates to NP 108 Action Plan Component 1a, Pathogens, Toxins, and Chemical Contaminants Preharvest, ii, Epidemiology, to determine the origin and routes of transmission of epizootic pathogens.
Developed model to predict geographic ranges for North American sylvatic Trichinella species. The geographic distributions of the North American species of encapsulating Trichinella (T. nativa and its variant T6, T. murrelli, and T. spiralis) are poorly characterized. These species are potentially zoonotic, therefore biogeographic information is critical to monitoring their status and any distribution changes due to climatic and man-made environmental impacts. The Maxent program was used to model predicted ranges for these sylvatic Trichinella species using available location records with confirmed species identifications collected over 55 yr throughout North America. Trichinella nativa’s predicted range is primarily north of the 48°-52° latitudes, overlapping the Tundra, sub-Arctic, and Warm Continental eco-regions. Its sympatric genotypic variant, T6, has a predicted range covering primarily the sub-Arctic, and mountainous Temperate Steppe eco-regions, the latter extending below 48°N latitude. In the east, T6’s range includes the Warm Continental and the mountainous Hot Continental eco-regions; the T6 range is also predicted to extend to the Sierra Madre Mountains of Mexico. Trichinella murrelli’s most probable range is centered in the Midwest within the Hot Continental and Prairie eco-regions, with an extension southward to the Subtropical and Tropical/Subtropical Steppe and Desert ecoregions. In the west, it exists in a restricted range characterized as mountainous Mediterranean. The most probable distribution of sylvatic T. spiralis is along the humid east North American coast (Hot Continental south to Subtropical), and along the coast of northwest North America (Marine) to Alaska (Subarctic and Tundra). It's most southerly range extends into central Mexico (Tropical/Subtropical Desert). The difference in relative freeze resistance between T. nativa/T6 and T. murrelli undoubtedly accounts for much of this geographic separation.
Evaluation of Fermentation/Drying and High Pressure Processing on Viability of Trichinella spiralis Larvae in Raw Pork and in Genoa Salami. Relatively little information is known about the effectiveness of high pressure processing (HPP) when used in combination with fermentation and drying to inactivate Trichinella spiralis larvae in ready-to-eat pork products. In this study we evaluated the effectiveness of HPP and fermentation/drying to inactivate T. spiralis larvae in both infected pig muscle and in Genoa salami produced with trichinae infected pork. In part A of the study, in each of two trials, 10 g portions (2 replicates per treatment) of fresh pig masseter muscle (ca. 3.6 log larvae/g) were pressurized at either 70,000 or 87,000 psi (483 or 600 MPa, respectively) for 0.5 to 5.0 min. In part B, Genoa salami prepared with trichinae infected pork (ca. 2.3 log larvae/g of salami batter) was fermented at 20°C for 6 hours and at 27°C for 26 hours and then dried at 20°C for 40 hours and at 17°C for/to: A) 25 days (65 mm casing), B) a target aw of 0.920 (65 mm casing), C) 35 days (105 mm casing), or D) a target aw of 0.940 (105 mm casing). After drying, in each of three trials, for each treatment, salami were post-processed pressurized at 483 MPa or 600 MPa for 0 to 12 min. T. spiralis was inactivated in masseter muscle and Genoa salami by all treatments of fermentation/drying and/or HPP as confirmed by both microscopy and mouse bioassays. Thus, HPP of pork muscle, as well as fermentation/drying or HPP of Genoa salami, are effective for inactivating T. spiralis larvae. These findings will allow producers of RTE pork products to expand the range of processing methods to control for this pathogen in RTE pork products.
Toxoplasma gondii isolates from free-range chickens from the northeast region of Brazil. The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 152 free-range chickens (Gallus domesticus) from 22 municipalities in 7 northeastern states (Pernambuco, Rio Grande do Norte, Maranhão, Bahia, Ceará, Sergipe, and Alagoas) of Brazil was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT); 81 (53.3 %) chickens had titers of 1:5 in 26, 1:10 in 9, 1:20 in 4, 1: 40 in 1, 1: 80 in 6, 1: 160 in 6, 1:320 in 13, 1: 640 in 6, 1:1,280 in 3, 1:2,560 in 6, and 1:5,120 or higher in 1. Hearts and brains of 81 seropositive chickens were bioassayed individually in mice. Toxoplasma gondii was isolated from 23 chickens with MAT titers of 1:5 or higher; the isolates were designated TgCKBr165-187. Five isolates killed all infected mice. Results indicate widespread contamination of rural environment in Brazil with T. gondii oocysts. This work relates to NP 108 Action Plan Component 1a, Pathogens, Toxins, and Chemical Contaminants Preharvest, ii, Epidemiology, to determine the origin and routes of transmission of epizootic pathogens.
Whole genome sequencing of a recombinant type II/III Toxoplasma gondii strain from Uganda reveals chromosome sorting and local allelic variants. Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS associated toxoplasmosis. We here present extensive sequence analysis of eight isolates from Uganda, including the whole genome sequencing of a type II/III recombinant isolate, TgCkUg2. 454 sequencing gave 84% coverage across the >61Mb genome and over 70,000 SNPs were mapped against reference strains. TgCkUg2 was shown to contain entire chromosomes of either type II or type III origin demonstrating chromosome sorting rather than intrachromosomal recombination. 1252 novel polymorphisms were mapped and clusters of new SNPs within coding sequence implied positive selection on a number of genes, including surface antigens and rhoptry proteins. Further sequencing of the remaining isolates, six type II and one type III strain, confirmed the presence of novel SNPs, suggesting these are local allelic variants within Ugandan type II strains. In mice, the type III isolate had parasite burdens at least 30-fold higher than type II isolates, while the recombinant strain had an intermediate burden. The quantity of high confidence SNP data generated in this study and the availability of the putative parental strains to this natural recombinant provide an excellent basis for future studies of the genetic divergence and of genotype phenotype relationships.
Isolation of Toxplasma gondii from Bottlenose dolphins (TursiopsTruncatus). Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In previous serological surveys, > 90% of bottlenose dolphins (Tursiops truncatus) from the coasts of Florida, South Carolina, and California had antibodies to T. gondii by the modified agglutination test (MAT). In the present study, attempts were made to isolate T. gondii from dead T. truncatus. During 2005, 2006, and 2007, serum or blood clot, and tissues (brain, heart, skeletal muscle) of 52 T. truncatus stranded on the coasts of South Carolina were tested for T. gondii. Antibodies to T. gondii (MAT 1:25 or higher) were found in 26 (53%) of 49 dolphins; serum was not available from 3 animals. Tissues (heart, muscle, and sometimes brain) of 32 dolphins (26 seropositive, 3 seronegative, and 3 without accompanying sera) were bioassayed for T. gondii in mice, cats, or both. Tissues of the recipient mice were examined for T. gondii stages. Feces of recipient cats were examined for shedding of T. gondii oocysts, but none excreted oocysts. Toxoplasma gondii was isolated from hearts of the 3 dolphins (2 with MAT titers of 1:200, and 1 without accompanied serum) by bioassay in mice. Genotyping of these 3 T. gondii isolates (designated TgDoUs1-3) using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed 2 genotypes. Two of the 3 isolates have type II alleles at all loci and belong to the clonal Type II lineage. One isolate has a unique genotype. This is the first report of isolation of viable T. gondii from T. truncatus.
Cessation of Trichinella spiralis transmission among scavenging mammals after the removal of infected pigs from a poorly managed farm: Implications for Trichinae transmission in the U.S. Pigs infected with the zoonotic parasite Trichinella spiralis were detected on a farm in Maryland during an animal welfare investigation. Sera and/or tissues were collected from 49 pigs and 3 pig carcasses. The tissues were tested for the presence of T. spiralis muscle larvae by tissue digestion, and the sera were tested for the presence of anti-Trichinella antibodies by ELISA. Seventeen of 50 (34%) pigs were infected with T. spiralis based on tissue digestion. Of these 17 pigs, sera were collected from 16; 9 were serologically positive, 3 sera had OD values that were very close to the positive cut off (0.30), but were still negative, and 4 were negative. All pigs which tested negative by tissue digestion for muscle larvae were also ELISA negative. The farm was subsequently depopulated of pigs. Six months later, testing of trapped scavenging mammals in the farm environment demonstrated that 41% were infected with T. spiralis. After 12 months, 10% of trapped animals were T. spiralis positive, and after 18 months, T. spiralis could not be detected in the scavenging mammal population surrounding the farm. Results of the study suggest that T. spiralis, typically transmitted in the peridomestic rat-pig-human cycle in the U.S., was not maintained in scavenging mammals in the absence of infected pigs. Consequently, our data support the hypothesis that in the absence of significant source of T. spiralis-infected swine (wild or domestic), the risk of infection to wildlife hosts and the development of an independent sylvatic transmission cycle of T. spiralis is minimal. There is little risk of infection to pigs with T. spiralis from wildlife in the U.S. in the absence of a pig reservoir of infection.
Hill, N.J., Dubey, J.P., Vogelnest, L., Power, M.L., Deane, E.M. 2008. Do free-ranging Common brushtail possums (Trichosurus vulpecula) play a role in the transmission of Toxoplasma gondii within a zoo environment? Veterinary Parasitology. 152:202-209.
Dubey, J.P., Pas, A. 2008. Toxoplasma gondii infection in blanford's fox (vulpes cana). Veterinary Parasitology. 153:147-151.
Dubey, J.P., Jardine, J.E. 2008. Severe intestinal coccidiosis in a newborn lion (Panthera leo). Acta Protozoologica. 47:63-68.
Reichard, M.V., Torretti, L., Garvon, J.M., Dubey, J.P. 2008. Prevalence of antibodies to toxoplasma gondii in wolverines from Nunavut, Canada. Journal of Parasitology. 94:764-765.
Velmurugan, G., Dubey, J.P., Su, C. 2008. Genotyping studies of toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe. Veterinary Parasitology. 155:314-318.
Dubey, J.P., Fair, P.A., Sundar, N., Velmurugan, G., Kwok, O.C., Mcfee, W.E., Majumdar, D., Su, C. 2008. Isolation of toxoplasma gondii from bottlenose dolphins (tursiops truncatus). Journal of Parasitology. 94:821-823.
Dubey, J.P., Crutchley, C. 2008. Toxoplasmosis in wallabies (macropus rufogriseus, macropus eugenii ): blindness, treatment with atovaquone, and isolation of toxoplasma gondii. Journal of Parasitology. 94:929-933.
Pas, A., Dubey, J.P. 2008. Toxoplasmosis in sand fox (vulpus rueppellii). Journal of Parasitology. 94:976-977.
Dubey, J.P., Jones, J.L. 2008. Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology. 38:1257-1278.
Dubey, J.P., Mansfield, K., Hall, B., Kwok, O.C., Thulliez, P. 2008. Seroprevalence of neospora caninum and toxoplasma gondii in black-tailed deer (odocoileus hemionus columbianus) and mule deer (odocoileus hemionus hemionus). Veterinary Parasitology. 156:310-313.
Dubey, J.P., Velmurugan, G., Chockalingam, A., Pena, H., Oliveira, L., Leifer, C., Gennari, S., Oliveira, L., Su, C. 2008. Genetic diversity of Toxoplasma gondii isolates from chickens from Brazil. Veterinary Parasitology. 157:299-305.
Pas, A., Dubey, J.P. 2008. Seroprevalence of antibodies to Toxoplasma gondii in Gordon's wild cat (Felis silvestris gordoni) in the Middle East. Journal of Parasitology. 94:1169.
Yai, L., Ragozo, A.M., Aguiar, D.M., Damaceno, J.T., Oliveira, L.N., Dubey, J.P., Gennari, S.M. 2008. Occurrence of Neospora caninum antibodies in capybaras (Hydrochaeris hydrochaeris) from Sao Paulo State, Brazil. Journal of Parasitology. 94:766.
Dubey, J.P. 2008. The history of Toxoplasma gondii-the first 100 years. Journal of Eukaryotic Microbiology. 55(6):467-475.
Ragozo, A., L.E.O, Y., Oliveira, L., Dias, R., Dubey, J.P., Gennari, S. 2008. Seroprevalence and isolation of Toxoplasma gondii from sheep from Sao Paulo State, Brazil. Journal of Parasitology. 94:1259-1263.
Hartley, W.J., Booth, R., Slocombe, R.F., Dubey, J.P. 2008. Lethal toxoplasmosis in an aviary of kakarikis (Cyanoramphus spp.) in Australia. Journal of Parasitology. 94:1424-1425.
Dubey, J.P., Wouda, W., Muskens, J. 2008. Fatal intestinal coccidiosis in a three week old buffalo calf (Bubalus bubalis). Journal of Parasitology. 94:1289-1294.
Prevalence of antibodies against Toxoplasma gondii in polar bears (Ursus maritimus) from Svalbard and East Greenland. Journal of Parasitology. 95:89-94.
Oliveira, L., Costa Junior, L., Melo, C., Ramos Silva, J., Bevilaqua, C., Azevedo, S., Muradian, V., Araujo, D., Dubey, J.P., Gennari, S. 2009. Toxoplasma gondii isolates from free-range chickens from the northeast region of Brazil. Journal of Parasitology. 95:235-237.
Velmurugan, G., Dubey, J.P., Su, C. 2009. Isolate designation and characterization of Toxoplasma gondii isolates from pigs in the United States. Journal of Parasitology. 95:95-99.
Alvarado-Esquivel, C., Cruz-Magallanes, H.M., Esquivel-Cruz, R., Estrada-Martinez, N., Rivas-Gonzalez, N., Liesenfeld, Q., Martinez-Garcia, S.A., Ramirez, E., Torres-Castorena, A., Castaneda, A., Dubey, J.P. 2008. Seroepidemiology of toxoplasma gondii infection in human adults. From three rural communities in Derango State, Mexico. Journal of Parasitology. 94:811-816.
Dubey, J.P., Velmurugan, G.V., Alvarado-Esquivel, C., Alvarado-Esquivel, D., Rodriquez-Pena, S., Martinez-Garcia, S., Gonzalez-Herrara, A., Ferreira, L.R., Kwok, O.C., Su, C. 2009. Isolation of Toxoplasma gondii from animals in Durango, Mexico. Journal of Parasitology. 95:319-322.
Ragozo, A.M., Yai, L.E., Oliveira, L.N., Dias, R.A., Goncalves, H.C., Azevedo, S.S., Dubey, J.P., Gennari, S.M. 2009. Isolation of Toxoplasma gondii from goats from Brazil. Journal of Parasitology. 95:323-326.
Dubey, J.P., Su, C. 2009. Population biology of Toxoplasma gondii: What's out and where did they come from? Memorias Do Instituto Oswaldo Cruz. 104:190-195.
Dubey, J.P., Moura, L., Sundar, N., Velmurugan, G.V., Kwok, O.C., Kelly, P., Krecek, R.C., Majumdar, D., Su, C. 2009. Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats (felis domesticus) from St. Kitts, West Indies. Parasitology. 136:589-594.
Dubey, J.P. 2009. History of the discovery of the life cycle of Toxoplasma gondii. International Journal for Parasitology. 39:877-882.
Weiss, L.M., Dubey, J.P. 2009. Toxoplasmosis: a history of clinical observations. International Journal for Parasitology. 39:895-901.
Sharma, S., Bal, M.S., Meenakshi, K.K., Sandhu, K.S., Dubey, J.P. 2008. Seroprevalence of neospora caninum antibodies in dogs in india. Journal of Parasitology. 94:303-304.
Pozio, E., Hoberg, E.P., Larosa, G., Zarlenga, D.S. 2009. Molecular Taxonomy, Phylogeny and Biogeography of nematodes belonging to the Trichinella genus. Infection, Genetics and Evolution. 9(4):606-616.
Zarlenga, D.S., Rosenthal, B.M., Hoberg, E.P., Mitreva, M. 2009. Integrating genomics and phylogenetics in understanding the history of trichinella species. Veterinary Parasitology. 159(3-4):210-3.
Hill, D.E., Samuel, M.D., Nolden, C.A., Sundar, N., Zarlenga, D.S., Dubey, J.P. 2008. Trichinella murrelli in scavenging mammals from south-central Wisconsin, USA. Journal of Wildlife Diseases. 44(3):629-635.
Masuoka, P.M., Burke, R., Colaccico, M., Razuri, H., Hill, D.E., Murrell, K. 2009. Predicted geographic ranges for North American sylvatic Trichinella. Journal of Parasitology. 95(4):829-837.