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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

2013 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:
Tested 450 feral swine paired serum and tissue samples for isolation and genotyping of Trichinella and Toxoplasma. For pigs that are raised outdoors, Trichinella may still pose a risk from commingling with infected hosts such as feral swine. For acceptance of pork produced in the U.S. Trichinae Certification Program, recent legislation in the European Union endorses surveillance of wildlife indicator populations; this project provides data to establish feral swine as an indicator population for Trichinella in support of export marketing efforts to answer this requirement from our trading partners (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 Animal and Plant Health Inspection Service (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 (Obj.1.2). Standardized antibody titers in egg yolk preparations for testing HEY therapy in pigs. Determined Toxoplasma-specific isotype concentrations for HEY preparations and prepared dosing regimen for oocyst infected pigs (Obj. 2). Infected pigs with T. spiralis for analysis of effect of length of treatment with a fixed dose of mebendazole on encysted ML. Mebendazole treatment of pigs with 250mg/kg over 3 days or 5 days reduced numbers of recovered ML and rendered ML non-infective to mice. Mebendazole treatment of pigs with 250mg/kg in a single dose was not effective in reducing ML numbers recovered from pigs or in impacting ML infectivity to mice. These data provide a means to evaluate the efficacy of anthelmintic treatment on the viability of Trichinella ML in pig tissues and provide a framework for elimination of Trichinella risk in pigs raised in uncontrolled management systems (Obj. 3.1). Initiated NAHMS swine serological survey for Trichinella and Toxoplasma in collaboration with APHIS-CEAH. This survey will determine the seroprevalence of Trichinella and Toxoplasma in the national swine herd, and provide data on farm management practices that impact pathogen prevalence (Obj. 2). Initiated 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 MSAs nationwide. These data will be used to discern consumer risk from meat products from animals raised in uncontrolled management systems (Obj. 3.2).

4. Accomplishments
1. International Ring Trial to test Trichinella E/S production parameters. For acceptance of pork produced in the U.S. Trichinae Certification Program, recent legislation in the European Union endorses surveillance of wildlife indicator populations as a measure of risk to swine raised in controlled and uncontrolled management systems. Presently accepted testing protocols demand time consuming and costly visual inspection of collected tissues in order to verify that no larval worms are present. The costs of such testing are onerous, and low sensitivity of visual inspection calls into question the public health benefits of the test. Working with a consortium of European Laboratories, scientists at the Agricultural Research Service engaged in collaborative research to identify standardized conditions for producing immunological reagents which offer greater sensitivity at lower cost. These data were used by the World Organization for Animal Health (the OIE, the international body that advises governments on such matters) in developing their new rules on acceptable surveillance methods for wildlife populations and for swine raised in controlled and uncontrolled management systems. These efforts support on-farm certification and export marketing efforts needed by USDA regulatory agencies, and may open new export markets for U.S. producers.

2. Toxoplasma induced abortion in sheep. Sheep are commonly infected with the protozoan parasite, Toxoplasma gondii. Infection may cause early embryonic death and resorption, fetal death and mummification, abortion, stillbirth, and neonatal death. Though sheep frequently acquire T. gondii infection after birth, congenital transmission of T. gondii may be more common than previously believed. Scientists at the Agricultural Research Service investigated a Toxoplasma induced abortion storm which occurred in a flock of purebred Suffolk ewes. Only 14 healthy lambs were born, and 38 abortuses, mummies and weak or stillborn lambs were delivered. Another 15 fetuses identified by ultrasound were presumably resorbed or were aborted undetected. The following year, 2 of 26 ewes delivered T. gondii infected lambs. In subsequent lambing seasons, the ewes lambed normally. These results support the hypothesis that most sheep that have aborted due to T. gondii develop protection against future toxoplasmosis induced abortion, but the protection is not absolute. These data are important to producers who may make culling decisions based on previous history of Toxoplasma-induced abortion; previously exposed ewes are better protected than naïve ewes, making future abortion events less likely.

3. Virulence differences between Toxoplasma and Hammondia. Toxoplasma and Hammondia are very closely related parasites, the former is pathogenic whereas the later is nonpathogenic. To better understand the genetic bases for the differences observed in virulence between Hammondia and Toxoplasma, we sequenced the genome of a H. hammondi isolate (HhCatGer041), and found ~95% similarity between blocks of genes in the same relative positions in the genome (synteny) between the 2 parasites. Using the genome of H. hammondi, the genetic sequence and functionality of two major virulence genes, ROP5 and ROP18, was determined. The H. hammondi ROP5 and ROP18 were both functional, with similar virulence and expression characteristics to the T. gondii ROP5 and ROP18 genes. These data suggest that the virulence differences between Toxoplasma and Hammondia are not solely due to the functionality of these key virulence factors, and other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may also play an important role in the observed disparate pathogenicities of these species. These data are important to researchers and clinicians interested in development of treatments to attenuate virulence during Toxoplasma infection in humans.

4. Check sample program for Trichinella. For pork to be exported to the European Union, a major trading partner, each swine carcass must be individually tested for the presence of Trichinella spiralis larvae by visual inspection of the meat in order to verify that no larval worms are present. Testing is performed at the packing plant by analysts trained and certified by ARS scientists in the Agricultural Marketing Service Analyst Training and Check Sample Program. In order to maintain integrity of the inspection program, test performance by each analyst is checked 4 times per year by ARS scientists using experimentally infected pork samples (proficiency samples). ARS scientists conducted a standardization trial for proficiency sample preparation for Trichinella infected pork. Optimal worm burden, mixing time, and test sensitivity at 3 infection levels was defined. Determined that using an optimal larval density of 5 larvae per gram with a 2 hour mixing time, test sensitivity was 100% when performed according to International Commission on Trichinellosis recommendations. Proficiency sample preparation for the AMS program was modified to reflect these findings. These efforts support on-farm certification and export marketing efforts as requested by USDA regulatory agencies through determination of risk to domestic pigs from Trichinella.

Review Publications
Schares, G., Langenmayer, M., Scharr, J., Minke, L., Maksimov, P., Maksimov, A., Schares, S., Barwald, A., Basso, W., Dubey, J.P., Conrath, F., Gollnick, N. 2013. Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis. International Journal for Parasitology. 43:143-154.
El-Behairy, A., Choudhary, S., Ferreira, L., Kwok, O., Hilali, M., Su, C., Dubey, J.P. 2013. Genetic characterization of viable Toxoplasma gondii isolates from stray dogs from Giza, Egypt. Veterinary Parasitology. 193:25-29.
Galizi, R., Spano, F., Giubilei, M., Capuccini, B., Magini, A., Urbanelli, L., Ogawa, T., Dubey, J.P., Spaccapelo, R., Emiliani, C., Cristina, M. 2013. Evidence of tRNA cleavage in apicomplexan parasites: half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei. Molecular and Biochemical Parasitology. 188:99-108.
Beltrame, M., Pena, H., Ton, N., Lino, A., Gennari, S., Dubey, J.P., Pereira, F. 2012. Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espírito Santo state, southeastern Brazil. Veterinary Parasitology. 188:255-230.
Buskhin, G., Dubey, J.P., Miska, K.B., Bullitt, E., Costello, C.E., Robbins, P.W., Samuelson, J. 2012. Beta-1,3-glucan1,3-glucan, a druggable target, forms a trabecular scaffold in the oocyst walls of Toxoplasma and Eimeria. mBio. 3(5):e00258-12.
Dubey, J.P., Lago, E., Gennari, S., Su, C., Jones, J. 2012. Toxoplasmosis in humans and animals in Brazil: High prevalence, high burden of disease, and epidemiology. Parasitology. 139:1375-1424.
Gutierrez-Esposito, D., Ortega-Mora, L., Gajadhar, A., Garcia-Lunar, P., Dubey, J.P., Alvarez-Garcia, G. 2012. Serological evidence of besnoitiosis in Canadian wild ruminants and strong cross-reaction between Besnoitia besnoiti and B. tarandi species. Veterinary Parasitology. 190:19-28.
Alvardo-Esquivel, C., Rodriguez-Pena, S., Villena, I., Dubey, J.P. 2012. Seroprevalence of Toxoplasma Gondii infection in domestic horses in Durango State, Mexico. Journal of Parasitology. 98:944-945.
Bossart, G., Mignucci-Giannoni, A., Rivera-Guzman, A., Jimenez-Marrero, N., Camus, A., Bonde, R., Dubey, J.P., Reif, J. 2012. Disseminated toxoplasmosis in Antillean manatees (Trichechus manatus manatus) from Puerto Rico. Diseases of Aquatic Organisms. 101:139-144.
Fomovska, A., Wood, R., Mui, E., Welsh, W., Dubey, J.P., Ferreira, L.R., Hickman, M., Lee, P., Leed, S., Auschwitz, J., Sommerville, C., Woods, S., Roberts, C., Mcleod, R. 2012. Salicylanilide inhibitors of Toxoplasma gondii. Journal of Medicinal Chemistry. 55:8375-8391.
Alvarado-Esquivel, C., Estrada-Malacon, M.A., Reyes-Hernandez, S.O., Perez-Ramirez, J.A., Trujillo-Lopez, J.L., Villena, I., Dubey, J.P. 2012. High prevalance of Toxoplasma Gondii antibodies in domestic pigs in Oaxaca State, Mexico. Veterinary Parasitology. 98:1248-1250.
Palic, J., Parker, V.J., Fales-Williams, A.J., Jarvinen, J.A., Dubey, J.P. 2012. What is your diagnosis? Duodenal brush preparation from a dog. Veterinary Clinical Pathology. 41:431-432.
Dubey, J.P., Darrington, C., Tiao, N., Ferreira, L., Choudhary, S., Molla, B., Saville, W., Tilahun, G., Kwok, O.C., Gebreyes, W. 2013. Isolation of Viable Toxoplasma gondii from Tissues and Feces of Cats from Addis Ababa, Ethiopia. Journal of Parasitology. 99:56-58.
Dubey, J.P., Prowell, M. 2013. Ante-mortem diagnosis, diarrhea, oocyst shedding, treatment, isolation and genetic typing of Toxoplasma gondii associated with clinical Toxoplasmosis in a naturally-infected cat. Journal of Parasitology. 99:158-160.
Rosypal, A.C., Bowman, S.S., Epps, S.A., El Behairy, A.M., Hillali, M., Dubey, J.P. 2013. Serological survey of dogs from Egypt for antibodies to Leishmania spp.. Journal of Parasitology. 99:170-171.
Alvarado-Esquivel, C., Estrada-Malacon, M., Reyes-Hernandez, S., Perez-Ramirez, J., Trujillo-Lopez, J., Villena, I., Dubey, J.P. 2013. Seroprevalence of Toxoplasma Gondii Infection in Domestic Sheep in Oaxaca State, Mexico. Journal of Parasitology. 99:151-152.
Tiao, N., Darrington, C.L., Molla, B., Saville, W.A., Tilahun, G., Kwok, O.C., Gebreyes, W.A., Lappin, M.R., Jones, J.L., Dubey, J.P. 2013. High prevalence of Toxoplasma gondii infection in Ethiopian cats in Addis Ababa, coinfection, and a review of toxoplasmosis in humans and other animals in Ethiopia. Epidemiology and Infection. 141:1029-1033.
Brenner, E.C., Chomel, B.B., Singhasivanon, O., Namekata, D.Y., Kasten, R.W., Kass, P.H., Cortes-Vecino, J., Gennari, S.M., Rajapakse, R., Huong, L., Dubey, J.P. 2013. Bartonella infection of stray dogs from the Tropics: Brazil, Colombia, Sri Lanka and Vietnam. Epidemiology and Infection. 141:54-61.
Walzer, K.A., Dam, R.A., Herrmann, D.C., Schares, G., Dubey, J.P., Boyle, J.P. 2013. Functional conservation of Toxoplasma gondii virulence genes in its avirulent relative, Hammondia hammondi. Proceedings of the National Academy of Sciences. 110:7446-7451.
Dubey, J.P., Choudhary, S., Kwok, O.C., Ferreira, L., Oliveira, S., Verma, S., Marks, D., Perdersen, K., Mickley, R., Randall, A., Arsone, D., Su, C. 2013. Isolation and genetic characterization of Toxoplasma gondii from mute swan (Cygnus olor) from the USA. Veterinary Parasitology. 195:42-46.
Possenti, A., Fratini, F., Pizzi, E., Fantozzi, L., Pozio, E., Dubey, J.P., Ponzi, M., Spano, F. 2013. Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle. Biomed Central (BMC) Genomics. 14:183.
Youn, Z., Luo, S., Dubey, J.P., Zhou, D., Zhu, Y., He, Y., He, X., Zhang, X., Zhu, X. 2013. Serological Evidence of Toxoplasma gondii infection in five species of bats in China. Vector-Borne and Zoonotic Diseases. 13:422-424.
Guy, E., Hill, D.E., Dubey, J.P. 2012. Toxoplasma gondii. In: Robertson, L.J., Smith, H.V., editors. Food Borne Protozoan Parasites. Hauppauge, NY: Nova Publishers. p. 167-188.
Jones, J.L., Dubey, J.P. 2012. Foodborne Toxoplasmosis. Clinical Infectious Diseases. 55(6):845-851.
Edwards, J.F., Dubey, J.P. 2013. Toxoplasma gondii abortion storm in sheep on a Texas farm and isolation of mouse virulent atypical genotype T. gondii from an aborted lamb from a chronically infected ewe. Veterinary Parasitology. 192:129-136.
Dubey, J.P., Simon, A., Poulin, B., Rousseau, A.N., Ogden, N.H. 2013. Toxoplasmosis in Canadian lynx (Lynx canadensis) in Québec, Canada. Journal of Wildlife Diseases. 49(1):39-48.
Costa, G., Marvulo, M., Silva, J., Santana, S., Magalhaes, F., Lima Filho, C., Ribeiro, V., Alves, L., Mota, R., Dubey, J.P., Silva, J. 2012. Seroprevalence of Toxoplasma gondii in domestic and wild animals from the Fernando De Noronha, Brazil. Journal of Parasitology. 98:679-680.
Sukhumavasi, W., Bellosa, M.L., Lucio-Forster, A., Liotta, J., Lee, A., Pornmingmas, P., Chungpivat, S., Mohammed, H.O., Lorentzen, L., Dubey, J.P., Bowman, D.D. 2012. Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand. Veterinary Parasitology. 188:25-30.
Hill, D.E., Dubey, J.P. 2013. Toxoplasma gondii prevalence in farm animals in the U.S. International Journal for Parasitology. 43:107-113.