Location: Animal Parasitic Diseases Laboratory
Project Number: 8042-31000-107-004-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2014
End Date: Aug 31, 2019
Objective:
There is growing concern from livestock producers and federal agencies that regulate and research animal health and food safety, with the development of anthelmintic resistance in ruminants (including recently domesticated wild ruminants) and anticipated resistance in pigs. Testing of a new class of anthelmintics represented by the Bacillus thuringiensis crystal protein (Cry5B) against larval and adult stages of economically important parasitic nematodes of livestock is not easily accomplished without the partnership developed in this plan. Although the pre-clinical and clinical deliverables from the plan are targeted to humans, the work is basic in nature and applicable to livestock infected with nematode parasites. ARS will provide parasitic nematodes to the Cooperator (University of Massachusetts Medical School) for lethality testing in vitro of Cry5B and related compounds delivered in lysates of strains of Bacillus that are Generally Regarded as Safe (GRAS) and other delivery vehicles. This is a novel and potentially breakthrough technology because it delivers a class of proteins that have been in the human food chain for decades along with a delivery system that is safe and efficient. The in vitro testing of probiotic lysate-derived Cry5B against parasitic animal nematodes can predict efficacy of this preparation in vivo as well as expand information on the mechanism of action of the product. These studies will precede the delivery of the active lysates, and appropriate control lysates that lack expression of Cry5B, to pigs as a proof of principal for efficacy under conditions of experimental exposure to specific parasitic nematode species in confined facilities. Other conditions of delivery of Cry5B and related products will also be evaluated in pigs infected with Ascaris suum and Trichuris suis, as determined by the results of in vitro studies, as well as preliminary analysis of safety of the optimized product delivery by histological and molecular analysis of the clinical status of the treated pigs.
Approach:
1. Propagate, isolate, and deliver larval and adults stages of parasitic animal nematodes. ARS will provide stages of Ascaris suum and Trichuris suis to the Cooperator for testing of Cry5B delivered in lysates of strains of Bacillus that are GRAS approved. We have successfully isolated these stages and shipped them by overnight carrier for testing in the UCSD laboratory in the past, but can conduct in vitro studies in the Beltsville laboratory as an alternative strategy.
2. Conduct natural and experimental infections of pigs with parasitic nematodes and evaluate probiotic lysates-derived Cry5B and other related products. Recently weaned pigs produced at the ARS Beltsville facility will be inoculated with infective eggs of A. suum and T. suis and (depending on the results of in vitro screens) and treated with active and control lysates to evaluate in vivo efficacy against the larval and adult stages of these parasites by assessing worm burden in the intestines. Other potential studies in backup of those with rodents will evaluate the immune neutralization of Cry5B after multiple treatments and for detection of enhanced acquired immunity to parasite nematodes by the release of cryptic antigens following the intoxication of resident parasitic infection with Cry5B in situ. Tissues and bio-fluids will also be collected as specimens for histological evaluation and clinical evaluation of biomarkers evaluation to determine any differences from the norm. Delivery of the Cry5B with mineral supplements that neutralize stomach pH will also be tested for increased efficacy against gastrointestinal parasitic worms.
3. Archive tissues and bio-fluids of pigs for safety evaluation. ARS will also freeze pig intestines, liver, muscle, kidney, brain, whole blood, and lymphoid tissues for RNA extraction and potential transcriptomic evaluation, fix similar tissues for histological examination, and store plasma, urine, and fecal contents for metabolomic and microbiome analysis. These materials represent samples for a safety evaluation of the treatment with probiotic lysate-derived Cry5B in a second species considered similar to humans.
