Enteric Diseases of Swine and Cattle: Prevention, Control and Food Safety

NON-TECHNICAL SUMMARY: This project will define mechanisms of pathogen-host-environmental interactions in enteric and food borne diseases, develop and improve diagnostics, treatment, and preventative measures for enteric and food borne diseases and provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians and diagnostic laboratories. <P>

APPROACH: Through its characterization of a porcine rotavirus receptor, Illinois has synthesized a relatively inexpensive neoglycolipid carbomimetic analogue of the active epitope of the intestinal receptor for group A rotavirus. A preliminary trial indicated that the receptor analogue protects pigs from challenge by the OSU strain of rotavirus. IL and OH will collaborate to test the therapeutic efficacy of this neoglycolipid receptor analogue in protecting against various strains of porcine rotavirus in studies conducted at IL. IL will continue its work defining the mechanism of Cryptosporidium parvum sporozoite interaction with host cells using two approaches. One is to identify naturally occurring host enterocye surface molecule(s) that mediate sporozoite recognition and invasion and the other focuses on differential sporozoite gene expression that occurs as a direct consequence of host cell adhesion. IL is also in continuing epidemiologic and overland transport studies with C. parvum in collaboration with the Agricultural Engineering Department at the University of Illinois. IL, in collaboration with OH, will determine the in vivo delivery parameters necessary for optimal therapeutic efficacy of the new synthetic neoglycolipid carbomimetic receptor developed by IL. This will include: dosage, time, frequency of administration, delivery vehicle, intestinal survival, transport, and absorption kinetics, as well as activity against different field strains of porcine rotavirus. These studies will define the dosage parameters necessary for achieving maximum benefit of receptor therapy as a treatment for porcine rotavirus disease. IL will investigate the use of bisphosponates as a therapeutic agent for treatment of cryptosporidiosis using an intestinal xenograft animal mode. Il has identified a colostrum-derived lipid that can block binding of C. parvum, and they will test the ability of this lipid to prevent colonization by C. parvum in intestinal xenografts and newborn calves. In addition to its annual technical report, the NC-1007 committee will assemble a lay document describing major advances in knowledge and technology through research by participating states, with livestock producers, veterinary professionals, and consumers as the target audience. The document will be formatted for dissemination in printed form or over the World Wide Web. Representatives from each participating station will be responsible for submitting pertinent information to the NC-1007 chair, who will appoint an editing committee to assemble information of the highest perceived public interest from individual stations for inclusion in the document. After completion, the document will be emailed to individual station representatives who will disseminate it and pertinent in-state research findings through their respective state's Cooperative Extension Service Agriculture Extension and other producer/consumer education entities for their information and use.

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PROGRESS: 2002/10 TO 2007/09<BR>
Based on our studies showing gangliosides, particularly N-glycolylGM3, are required by sialic acid-dependent strains of porcine rotavirus for enterocyte infectivity, we are actively pursuing synthesis of multivalent, neoglycoconjugate carbomimetics using the native sialyllactose oligo-saccharide moieties of porcine intestinal GM3. We recently synthesized a sialyllactosyl-phosphatidylethanolamine (SLPE) neoglycolipid which displays a potent ability to inhibit both virus binding and infectivity in vitro. We are currently conducting experiments evaluating the in vivo efficacy of this new carbomimetic. In field trials this inhibitor blocked infection, virus shedding and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. We are currently investigating, in collaborative studies with Dr. Sharon Donovan, Department of Food Science and Human Nutrition, the synergistic effect of SLPE and specific porcine milk oligosaccharides as well as soybean derived flavonoids, which we have found to exhibit anti-rotavirus activity that is acting distinct from virus attachment and entry. Previously we described an in vitro cell suspension assay that measures adhesion of sporozoites to host cells (MDBK and Caco-2 cells). This assay involves the incubation of individualized sporozoites and host cells in suspension with end-over-end rotation at 37C. Binding of the sporozoites to host cells is readily observed and quantified by phase contrast microscopy. This assay was used to screen a variety of glycoconjugates for their ability to inhibit parasite binding to host cells. Of the glycoconjugates tested, mucins markedly inhibited binding of sporozoites to host cells. Plasma membrane vesicles (PMV) and fractioned cell membranes, generated from MDBK cells, significantly inhibited binding as well. We have purified and partially characterized this membrane inhibitory activity isolated from MDBK cells, plasma membrane vesicles and bovine intestinal mucosa. Our results indicate natural host intestinal lipid(s) or lipid-like component(s) play an important role in the early stages of host cell invasion. Additionally, we have finished experiments aimed at measuring the rate of sporozooite motility and host cell invasion and correlated these kinetics with the results of experiments aimed at identifying specific sporozoite genes expressed as a consequence of the early stages of host cell invasion or exposure to the host cell membrane component mentioned above. The results of these experiments demonstrate there is no detectable differential gene expression during the very rapid time frame (<5 minutes) of sporozoite host cell invasion. Finally, we have determined the cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil tha occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil.
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IMPACT: 2002/10 TO 2007/09<BR>
Group A rotaviruses are among the most important agents associated with severe diarrhea in the young of both animals and people. Receptor therapeutic approaches aimed at blocking virus attachment, combined with nutritional (isoflavone dietary supplements) therapy capable of blocking virus replication, is likely to be a more field applicable, producer acceptable, and deliverable strategy to combat rotavirus diarrheal disease of agricultural animals than traditional vaccine approaches. Due to the zoonotic nature of Cryptosporidium parvum, the economic loss to the cattle and dairy industries caused by this parasite is compounded by the risk of environmental contamination and human infection. The oocysts are environmentally hearty and exceptionally resistant to chemical eradication. The discovery of a host-derived intestinal lipid which blocks sporozoite invasion may provide novel options for treating or preventing cryptosporidiosis. Our results with vegetative filter strips (VFS) suggest properly constructed VFS are a best management practice for diary farms to reduce or eliminate overland transport and runoff of oocysts in to adjacent waterways.