1a. Objectives (from AD-416)
Improve food safety by reducing contamination of fresh produce by improving detection and determining the sources and reducing transmission of protozoan parasites infectious for humans. Objective 1: Improve detection methods for Cryptosporidium, Giardia, and other zoonotic parasites. a) Develop new immunofluorescence and aptamer technologies applicable to detection of zoonotic parasites on fresh fruits and vegetables and in irrigation water and produce washwater. b) Determine effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce. c) Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple infectious organisms thereby reducing the need for multiple testing. Objective 2: Develop prevention and treatment strategies against zoonotic cryptosporidiosis and giardiasis. a) Test anti-viral drugs against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication. b)Test probiotics in mouse models for prevention of cryptosporidiosis. Objective 3: Elucidate the epidemiology of zoonotic Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genotypes using molecular tools. a) Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans. b) Identify emerging zoonotic genotypes and subgenotypes of Cryptosporidium and Giardia with potential to be foodborne pathogens from samples of irrigation water and from manure spread on crop fields.
1b. Approach (from AD-416)
To improve detection methods for zoonotic parasites, studies will develop new immunofluorescence and aptamer technologies applicable to detection of the zoonotic parasites Cryptosporidium oocysts and Giardia cysts on fresh fruits and vegetables and in irrigation water and produce washwater by tagging pathogen-specific antibodies/aptamers with fluorophores with minimal interference from autofluorescing vegetal tissue and other waterborne particles to increase sensitivity, and thereby detection of the parasite eluted from or located on fresh produce. Determine the most effective washing agents and methods for recovering contaminating parasites from the surface of fresh produce including tetrasodium pyrophosphate, 1M Hepes at pH 5.5, 1 M sodium bicarbonate at pH 6.0, 1 M glycine at pH5.5, and 1% lauryl sulfate, and then combine those most effective reagents and test their efficacy. Develop a protocol using amplified fragment length polymorphism analysis (AFLP) to simultaneously detect multiple contamination with Cryptosporidium, G. duodenalis, Blastocystis, and E. bieneusi. Test antiviral drug inhibitors of RNA-dependent RNA polymerase (RdRp) against virus found inside Cryptosporidium parvum stages to inhibit virus and parasite replication using in vitro cell culture methodology. Test probiotics Lactobacillus casei (L. casei) and Bifidobacterium lactis (B. lactis) in mouse models for prevention of cryptosporidiosis and giardiasis. Determine subtypes of Blastocystis and Microsporidia found in food animals that match pathogenic subtypes in humans using DNA samples now held frozen in our inventory from dairy and beef cattle (pre- and post-weaned calves, heifers, and adults) from more than 20 states of the U.S., from sheep (lambs and ewes), as well as from pigs, horses, dogs, cats and alpacas.
3. Progress Report
Contacted potential collaborators at Cornell University and the University of Maryland who have access to orchards where pond water is used for spraying fruit. They agreed to contact farmers who would allow us to collect and test water samples for the presence of Cryptosporidium, Giardia, and Microsporidia. Antiviral drugs are being tested in vitro against the virus found inside Cryptosporidium parvum stages to determine if they can inhibit virus and parasite replication. A DNA based study was initiated to determine the presence of Microsporidia in feces from beef cattle on cow-calf operations on 49 farms in 20 states. Another DNA based study in progress is determining the subtypes of Blastocystis in 30 dairy cattle followed from birth the 2 years of age. ARS collaborator in Nebraska provided samples of runoff from beef cattle feedlots that were filtered through a vegetative treatment system; Cryptosporidium and microsporidia were found.