2010 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.
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.
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.
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.
In cooperation with APHIS, feces from cattle on cow-calf operations 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. DNA from that study was purified and stored for continuing studies on additional parasites of public health concern and a request to extend that study to test for the presence of Giardia has been submitted to APHIS. Genetic primers were designed and produced to test for the presence of Blastocystis, a pathogenic microorganism of humans and animals; DNA extracted from feces of dairy cattle, pigs, and humans was tested using these primers and a manuscript describing these primers and their widespread usefulness is in preparation. Using laser confocal microscopy Cryptosporidium oocysts have been found on the surface of apples where they could not be washed off. This could potentially be a food safety issue for fresh fruit or unpasteurized apple juice or cider. A manuscript has submitted and accepted by a peer reviewed journal. Antibodies produced against alpha-, beta-, and delta-giardin from Giardia duodenalis trophozoites blocked attachment of the parasite to surfaces indicating the potential usefulness of antibodies in preventing or treating Giardia infections in humans and animals. Drugs obtained from the National Institutes of Health and used for treatment of RNA virus diseases in humans were not affective against the double stranded RNA virus found within Cryptosporidium and therefore not effective in controlling development of this parasite.
Identified a new species of Cryptosporidium infectious for humans. A new species of Cryptosporidium, infectious for a wide range of animals as well as humans, was discovered and named Cryptosporidium ubiquitum by ARS scientists. This species was found to infect cattle, goats, sheep, numerous wildlife, and rodents as well as humans. Recognition of this organism is important because of its many potential sources of infection and its role as a human pathogen worldwide.
Cryptosporidium remains attached to fruit after washing. Cryptosporidium is a widespread human and animal parasite found in contaminated water and on fresh fruits and vegetables possibly contaminated from irrigation water containing animal feces. In the present study, Cryptosporidium oocysts derived from cattle feces were found to attach firmly to the surface of apples, to resist removal by washing even with strong detergent, and to remain infectious after weeks of storage. These findings relate to published reports of outbreaks in 3 states from contaminated apple cider, suggesting that once contaminated, normal washing may not be effective and fruit can remain infectious for weeks.
Santin, M., Zarlenga, D.S. 2009. A multiplex PCR to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle. Veterinary Parasitology. 166(2009):32-37.
Santin, M., Cortes Vecino, J.A., Fayer, R. 2010. A zoonotic genotype of Enterocytozoon bieneusi in horses. Journal of Parasitology. 96(1):157-161.
Wyatt, C.R., Riggs, M.W., Fayer, R. 2010. Cryptosporidiosis in Neonatal Calves. Veterinary Clinics of North America. 26(1):89-103.
Fayer, R., Santin, M., Dargatz, D. 2010. Species of Cryptosporidium Detected in Weaned Beef Calves on Cow-Calf Operations in the United States. Veterinary Parasitology. 170(3-4):187-192.
Macarisin, D., Bauchan, G.R., Fayer, R. 2010. Spinacia oleracea L. leaf stomata harboring Cryptosporidium parvum oocysts: A potential threat for food safety. Applied and Environmental Microbiology. 76:555-559.
Miska, K.B., Jenkins, M.C., Trout, J.M., Santin, M., Fayer, R. 2009. Detection and comparison of giardiavirus (GLV) from different assemblages of giardiia duodenalis. Journal of Parasitology. 95(5):1197-2000.
Fayer, R., Santin, M. 2009. Cryptosporidium xiaoi n. sp. (Apicomplexa: Cryptosporidiidae) in sheep (Ovis aries). Veterinary Parasitology. 164:192-200.
Fayer, R., Santin, M., Macarisin, D. 2010. Cryptosporidium ubiquitum n.sp. in animals and humans. Veterinary Parasitology. 172(1-2):23-32.