2011 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 of 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.
Molecular methods were used to determine the prevalence of Giardia genotypes infectious for humans and those that infect only cattle in feces collected from cattle on cow-calf operations in 20 states. 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 Microsporidia was submitted to APHIS. Genetic primers were designed and used successfully 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 found positive, resulting in the discovery of new genotypes.
Giardia, a widespread and highly prevalent parasite of humans and animals, has unique proteins called giardins. Antibodies produced against beta- and delta-giardins were identified by confocal laser scanning microscopy in the ventral disc, an organelle essential for parasite attachment to host cells. This finding provides a basis for developing methods of prevention and treatment of Giardia infections by utilizing anti-giardin antibodies, immunizations, or drugs to block attachment of the parasite to cell surfaces.
In summary, over the past 5 years of this project 4 new species of Cryptosporidium were discovered and named including a widespread zoonotic species found in food animals; Cryptosporidium oocysts were found to strongly attach to fresh leafy greens and apples and resist removal by normal washing procedures; 6 new genotypes of the zoonotic microsporidian parasite E. bieneusi were discovered and gene sequences deposited in GenBank; developed an international consensus paper on taxonomy of zoonotic microsporidia; antibodies to Giardia proteins associated with attachment to host cells blocked experimental attachment; the human pathogen Blastocystis was found in cattle in the U.S.; in cooperation with APHIS zoonotic parasites were identified in cow-calf operations in 20 states; the unit appeared in “The Crypto Parasite” on Animal Planet TV program “Monsters Inside Me”.
Identified giardins in the attachment organelle of Giardia. The location of beta and delta giardin in the ventral disc of Giardia trophozoites was identified using confocal laser scanning microscopy. The two giardin proteins co-localized in the disc but delta giardin was found closest to the ventral surface of the disc, that portion in most direct contact with host cells. Because giardins and other structural proteins are thought to participate in controlling disc shape to enable adherence or attachment to host cells, this finding provides a basis for developing methods of prevention and treatment of Giardia infections by utilizing anti-giardin antibodies, immunizations, or drugs to block attachment of the parasite to cell surfaces.
Developed monoclonal antibodies to Giardia duodenalis for diagnostic purposes. The genes for two giardin proteins associated with trophozoite and cyst stages of Giardia were cloned and expressed. The recombinant proteins from these genes were used to generate monoclonal antibodies that recognize both trophozoites and cysts by immunofluoresence assay. A patent disclosure describing these antigens and use of monoclonal antibodies to giardin proteins was approved by the ARS patent committee and is in the process of being filed with the US Patent Office.
Identified Blastocystis in humans and food animals. The same substypes of Blastocystis spp. were found in feces from several animal species and humans, suggesting a previously unrecognized zoonotic potential of this parasite. A PCR and sequencing protocol was developed with increased sensitive compared with published primers; these were used to detect and subtype Blastocystis spp. specimens from naturally infected humans, primates, cattle, pigs, and chickens. Based on these findings, this method can be used to elucidate the complexity of this enigmatic parasite, its role in human and animal disease, and its zoonotic potential.
Macarisin, D., Bauchan, G.R., Santin, M., Fayer, R. 2010. Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples. Journal of Food Protection. 73(10):1824:1829.
Da Silva Fiuza, V., Juliboni Cosendey, R., Frazao-Teixeira, E., Santin, M., Fayer, R., Rodrigues De Olivira. 2011. Molecular characterization of cryptosporidium in brazilian sheep. Veterinary Parasitology. 175:360-362.
Dixon, B., Fayer, R., Santin, M., Hill, D.E., Dubey, J.P. 2011. Protozoan parisites: Cryptosporidium, Giardia, Cyclospora, and Toxoplasma. In: Hoorfar, J., editor. Foodborne Protozoans. Rapid Detection, Characterization, and Enumeration of Food-Borne Pathogens. Washington, D.C.: American Society for Microbiology. p. 349-370.
Santin, M., Fayer, R. 2010. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Research in Veterinary Science. 90(1):363-371.
Santin, M., Gomez-Munoz, M., Solano Aguilar, G., Fayer, R. 2011. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitology Research. 109(1):205-212.
Jenkins, M.C., Obrien, C.N., Miska, K.B., Schwartz, R., Karns, J.S., Santin, M., Fayer, R. 2011. Gene expression during excystation of Cryptosporidium parvum oocysts. Parasitology Research. 109(2):509-513.