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United States Department of Agriculture

Agricultural Research Service

Research Project: PROTOZOAN PARASITES AFFECTING FOOD ANIMALS, FOOD SAFETY, AND PUBLIC HEALTH
2009 Annual Report


1a.Objectives (from AD-416)
The objectives are to improve food safety and reduce contamination of drinking water by improving detection, determining sources, and reducing transmission of protozoan parasites infecting humans. Objective 1 a) Improve speed and accuracy of methods to detect Cryptosporidium, Giardia, and Microsporidia in selected environmental specimens and in specimens from food animals, other farm animals, wildlife, and transport hosts that might harbor multiple species or genotypes. b) Develop monoclonal antibodies specifically to identify zoonotic species of Cryptosporidium.

Objective 2 a) Determine the prevalence Blastocystis spp. in 1000 pre- and post-weaned dairy cattle from farms in eastern states utilizing DNA from our immediate past project; determine the prevalence of Microsporidia, Blastocystis, Giardia, and Cryptosporidium in 150 sheep and 500 pigs from birth to market age from multiple farms and states, and from 1000 feedlot beef cattle in Nebraska. b) Determine the presence of these same organisms in environmental specimens provided by NOAA collaborators from waters impacted by agricultural runoff. c) Assess the potential infectivity, duration of infection, and numbers of parasites excreted throughout a period of infection, by experimentally infecting parasite-free cattle, sheep, pigs, chickens, turkeys, and laboratory rodents with any unique genetic isolates found in the field studies described above.

Objective 3 a) Test for protective immunity of HBC fed to neonatal calves experimentally challenged with C. parvum oocysts by observing the severity and duration of infection. b) Conduct biochemical and molecular studies that might serve as a basis for future treatment strategies to interfere with transmission of parasites. c) Test anti-viral drugs associated with reduction of cryptosporidiosis in AIDS patients and in vitro will be tested for efficacy against zoonotic Giardia and Cryptosporidium, both of which have been shown to contain RNA viruses.


1b.Approach (from AD-416)
Studies will identify Giardia, Cryptosporidium, and Microsporidia of livestock and wildlife by developing multiplex PCR techniques and examining new gene sequences to provide improved characterization of these organisms. Viruses have been found within Giardia and Cryptosporidium, and studies will determine if differences in the quality or quantity of such viruses using newly developed reagents can facilitate detection and differentiate pathogenic and non-pathogenic strains.

The prevalence of Giardia, Cryptosporidium, Microsporidia, and Blastocystis in sheep and pigs, and feedlot cattle will be determined. The prevalence of Blastocystis also will be determined in dairy cattle. Unique genotypes of these pathogens from field isolates will be tested in transmission studies to determine their potential to infect other animal hosts. The presence of zoonotic protozoan pathogens in environmental specimens in areas impacted by runoff from agricultural animals will be assessed.

Studies will identify methods to provide protective immunity against Cryptosporidium. Cows will be immunized with recombinant proteins and immune stimulators to produce colostrum with high levels of anti-Cryptosporidium antibody for passive immunization of calves. Biochemical and molecular techniques will be used to study encystation/excystation in Giardia and Cryptosporidium to identify proteins that can be targeted to disrupt transmission. Anti-viral and anti-protozoal drugs will be tested against Cryptosporidium and Giardia using cell culture and animal infectivity models.


3.Progress Report
A multiplex PCR test was developed to detect the major species of Cryptosporidium that infect humans and cattle and differentiate these from minor or noninfectious species. A manuscript has been submitted for publication. In cooperation with APHIS, feces from feedlot cattle in 20 states were examined by molecular methods to determine the prevalence of Cryptosporidium infectious for humans as well as species and genotypes that infect only cattle. A manuscript is in preparation. DNA extracted from feces of dairy cattle from birth to 2 years of age was examined for the presence of Blastocystis, a pathogenic microorganism of humans and animals. Multiple new genotypes of Blastocystis have been discovered based on cloning of specimens. Using laser confocal microscopy Cryptosporidium oocysts have been found inside stomata (openings) in spinach leaves where they could not be washed off. This could potentially be a food safety issue for fresh cut leafy vegetables. A manuscript has been written. Found that antibodies to a recombinant delta-giardin Giardia lamblia trophozoite protein localized the antigen to the ventral disk and could block attachment of the parasite to surfaces. Developed a real-time RT-PCR technique to study the development of Cryptosporidium parvum and its viral symbiont CPV in cell culture, and the effects of irradiation on parasite and virus development. Characterized the expression of mRNA in excysting Cryptosporidium parvum oocysts over time by using subtractive cDNA hybridization techniques. SYs and technicians meet weekly on Monday morning to discuss findings from the previous week and to make plans for the coming week.


4.Accomplishments
1. Cryptosporidium oocysts have been found inside plant stomata. Cryptosporidium is a human and animal parasite found in contaminated water. Fluorescently labeled Cryptosporidium oocysts were found using laser confocal microscopy near and inside plant stomata, the microscopic breathing holes of plants. Washing of contaminated spinach leaves did not remove oocysts protected within the stomata suggesting a potential food safety issue for fresh cut leafy vegetables.


6.Technology Transfer

Number of the New/Active MTAs (providing only)4
Number of Invention Disclosures Submitted1

Review Publications
Dixon, B.R., Parrington, L.J., Parenteau, M., Leclair, D., Santin, M., Fayer, R. 2008. Giardia duodenalis and Cryptosporidium spp. in the intestinal contents of ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) in Nunavik, Quebec, Canada. Journal of Parasitology. 94(5):1161-1163.

Santin, M., Trout, J.M., Fayer, R. 2009. A longitudinal study of Giardia duodenalis genotypes in dairy cows from birth to two years of age. Veterinary Parasitology. 162(1-2):40-45.

Barigye, R., Khaitsa, M.K., Schamber, E., Dyer, N.W., Newell, T.K., Trout, J.M., Santin, M., Fayer, R. 2008. Molecular and Immunohistochemical Detection of Assemblage E Giardia duodenalis in Scouring North Dakota Calves. Veterinary Parasitology. 157(34):196-202.

Santin, M., Fayer, R. 2009. Enterocytozoon bieneusi genotypes nomenclature based on the ITS sequence– a consensus. Journal of Eukaryotic Microbiology. 56(1):34-38.

Fayer, R., Santin, M., Trout, J.M. 2008. Cryptosporidium ryanae n.sp. (Apicomplexa:Cryptosporidiidae)in cattle (Bos Taurus). Veterinary Parasitology. 156:191-198.

Yang, W., Lindquist, H., Cama, V., Schaefer, Iii, F.W., Villegas, E., Fayer, R., Lewis, J., Xiao, L. 2009. Detection of Toxoplasma gondii oocysts in water sample concentrates by real-time PCR. Applied and Environmental Microbiology. 75(11):3477-83.

Last Modified: 10/1/2014
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