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:
Under Objective 1a, significant progress was made in detecting Blastocystis, a widespread parasite of humans and animals. In collaboration with a small biotech company, a new diagnostic reagent was modified and tested that quickly and accurately identified this organism in livestock feces, demonstrating the practicality of its application for epidemiological studies planned in Objective 3, and for diagnosis in human and animal medicine. Under Objective 2a, progress was made by demonstrating in cell cultures that anti-viral drugs associated with reduction of cryptosporidiosis in AIDS patients do not affect the RNA virus harbored within stages of Cryptosporidium. Therefore, medication with these drugs for treatment of non HIV patients with cryptosporidiosis and for treatment of animals would impose potential health risk and cost without evidence of effective treatment results. Under Objective 3a, significant progress was made by demonstrating that asymptomatic dairy cattle can serve as carriers and sources of multiple concurrent infections with four species of protozoa of public health importance. This objective relates to the safety of farm workers and farm visitors exposed to cattle as well as contamination of milk and meat products. A manuscript describing these infections and the public health implications was published. Further progress was made in Objective 3a to determine the prevalence of Blastocystis in food animals by developing and submitting plans for collaboration with epidemiologists at the National Animal Health Monitoring Service of APHIS to collect specimens from 7500 market pigs in 13 states beginning later in FY2012.
1. Identified giardins in the attachment organelle of Giardia and developed a fluorescence microscopy based diagnostic test for Giardia duodenalis in environmental and biological specimens. ARS scientists in Beltsville, MD identified the location of two unique proteins in the intestinal stage of Giardia parasites in the ventral disc, the organelle used for attaching the parasite to the intestine of an infected host. Because these proteins participate in controlling disc shape, essential for the parasite to attach 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. Antibodies produced against these proteins labeled with a fluorescent dye were found useful for microscopic diagnosis of infection of intestinal tissue and diagnosis of the parasite in feces of infected animals and humans, as well as for the presence of the parasite in the environment (water, soil, plant contamination). A patent application is in the process of being filed with the U.S. Patent Office.
Santin, M., Dargatz, D., Fayer, R. 2012. Prevalence of Giardia duodenalis assemblages in weaned cattle on cow-calf operations in the United States. Veterinary Parasitology. 183:231-236.