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:
New polyclonal and monoclonal antibodies were tested that identified Blastocystis in tissues and feces by immunohistochemistry and fluorescence microscopy as part of the goal associated with objective 1. These antibodies were extremely helpful in clearly identifying the parasite in the intestinal tract of pigs and elucidating its role in invasion of host tissue. In addition, a new approach to detect foodborne parasites using transparent adhesive tape to capture organisms combined with molecular and microscopy detection tools, was developed for use on fresh produce and food preparation surfaces. As part of objective 2, progress was made to test anti-viral drugs against Cryptovirus by developing a reproducible cell culture assay for C. parvum with conditions that allowed parasite and Cryptovirus replication. A reproducible in vitro assay that could measure C. parvum development from 24–72 hr using quantitative and semi-quantitative PCR was develped. An increase in Cryptovirus over time in the cell culture media was observed, indicating reproduction of virus in combination with parasite development, suggesting a possible target for parasite control. With confidence in this test system, we will now go back and re-test viral inhibitory drugs for inhibition of parasite development. As part of objective 3, progress was made to elucidate the epidemiology of zoonotic parasites by determining the prevalence and molecularly characterizing Blastocystis subtypes in wildlife, cattle, pigs, and humans. Using DNA primers developed in our laboratory, new variations of the ST 5 subtype of Blastocystis were identified; this may be helpful in epidemiologic traceback studies. Further progress was made to determine the prevalence of Cryptosporidium, Giardia, Microsporidium, and 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 1500 preweaned dairy calves in 13 states beginning in FY2014. Additionally, in collaboration with scientist from Argentina, the first identification and molecular characterization of Enterocytozoon bieneusi in cattle in South America was carried out with the identification of a novel genotype.
1. Giardia duodenalis detected in horses. Giardiasis, caused by G. duodenalis, is a gastrointestinal disease with worldwide distribution. In the United States, it is the most common intestinal parasitic disease causing diarrhea in humans. Giardia duodenalis is not only a widespread parasite in humans but also infects animals that can spread the disease to humans. Because of a lack of data regarding the potential role of horses in the transmission of giardiasis to humans, ARS scientists in Beltsville, MD in collaboration with scientists in Colombia conducted a molecular study of G. duodenalis from horses to determine if horse manure used for crop production posed a public health risk. Using a multilocus molecular approach to characterize isolates from 195 horses, G. duodenalis Assemblages A and B (both infectious for humans) were detected in horses. These findings indicate the potential risk of these companion animals as reservoir of G. duodenalis with the potential to cause disease in humans through direct contact or by contamination of food and water supplies.
2. New method to detect Cryptosporidium. Cryptosporidium is a prevalent and widespread protozoan parasite that causes severe diarrheal disease in humans and livestock. Infection with this parasite is common in children and can be life threatening in immunocompromised persons. Fresh produce contaminated with Cryptosporidium oocysts has been the source of sporadic cases and outbreaks of foodborne infection. Although Cryptosporidium has been detected on the surface of various fresh fruits and vegetables worldwide, detection has been problematic because the parasite adheres tenaciously to surfaces and cannot be grown like bacteria into easily detectable populations on Petri dishes or in tubes. Many current detection methods lack sensitivity and specificity to detect this parasite and others require highly trained personnel and equipment found only in specialized laboratories. ARS scientists in Beltsville, MD developed an adhesive tape recovery method, combined with molecular and microscopic tools, for detection of Cryptosporidium oocysts on fresh produce and on food preparation surfaces. This method is rapid, inexpensive, and can be applied to many products, surfaces, and locations.
Fayer, R., Santin, M., Macarisin, D., Bauchan, G.R. 2013. Adhesive-tape recovery combined with molecular and microscopic testing for the detection of Cryptosporidium oocysts on experimentally contaminated fresh produce. Parasitology Research. 112(4):1567-1574.