Location: Animal Parasitic Diseases Laboratory
Project Number: 8042-31000-107-10-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Feb 1, 2019
End Date: Jan 31, 2021
Gastrointestinal (GI) tract nematodes or helminths are major infectious parasites of humans and farm animals that cause many adverse outcomes, including malnutrition, stunting, lethargy, immune deficiencies, poor condition and health, and lower productivity. Resistance to current drugs is either widespread or growing and new therapies are urgently needed. This project will produce a radically new cure for these parasites—an engineered food grade/probiotic bacterium capable of curing these parasites in humans and farm animals. This project proposes a radical new approach for deworming—engineering a food grade/probiotic bacterium to express a vertebrate-safe protein, Cry5B, known to target nematodes. Cry5B is a crystal (Cry) protein produced by the bacterium Bacillus thuringiensis. Cry proteins are generally regarded as safe. The related bacterium Bacillus subtilis is widely used in probiotics for human and farm animal health. B. subtilis engineered to express Cry5B results in a strain capable of curing zoonotic hookworm infections in hamsters when given orally. Cry5B has also demonstrated activity against parasites of pigs (in vivo), sheep (in vitro), and horses (in vitro). The aims of this project are (1) broadly test potential spectrum of activity of Cry5B against agriculturally important parasites; (2) optimize Cry5B sequence for efficacy against key parasites of horses (cyathostomins) and small ruminants (Haemonchus contortus); (3) test the hypothesis that Cry5B and nicotinic acetylcholine receptor (nAChR) agonist anthelmintics have ideal, resistance-busting characteristics; (4) build a commercially viable food grade/probiotic bacterium capable of high expression of Cry5B anthelmintic; (5) develop novel formulations of Cry5B to increase its efficacy in the monogastric (e.g., horse) and ruminant (e.g., lambs, goats) GI tracts; (6) test optimized Cry5B and optimized Cry5B formulations for efficacy in vivo against agriculturally important parasites in horses and in small ruminants, namely sheep and goats; and (7) follow any potential changes in the microbiome of treated animals and follow the fate of Cry5B-expressing bacteria after treatment. At the conclusion of this study, our goal is to have optimized a single, engineered bacterial anthelmintic therapy with broad activity against GI tract nematodes of farm animals and, down the road, humans, that is commercially viable. For this research, a unique and expert team of researchers has been assembled with complementary skills in nematicidal Cry proteins, Bacilli, and formulation (University of Massachusetts Medical School), and GI tract nematodes of farm animals (University of Kentucky, Virginia Polytechnical Institute, USDA) in order to ensure its success.
1. Cry5B optimization studies: Optimize Cry5B sequence to target the parasites of interest in this grant, namely cyathostomin parasites of horses (monogastric farm animal) and H. contortus parasites of sheep/goats (small ruminant farm animals). Study Cry5B combinatorial properties with pyrantel and levamisole against wildtype and anthelmintic resistant parasites. Study the spectrum of action of Cry5B against other GI nematodes. 2. Building a commercially-viable food grade/probiotic Cry5B anthelmintic strain: Optimize expression of Cry5B in B. subtilis and integrate into B. subtilis natto without use of antibiotic selection and with an auxotrophic strategy approved by regulatory agencies. Optimize biomass production in fermenters. 3. Formulation studies and in vivo testing in large animals: Formulate food grade/probiotic Cry5B strain to protect Cry5B from premature digestion (stomach of horses; rumen, reticulum, omasum of ruminants) and release Cry5B at parasites (intestine of horses; abomasum of ruminants). Test unformulated and formulated Cry5B in vivo (horses infected with cyathostomins, P. equorum; lambs/goats infected with H. contortus and other strongyles). Follow the fate of bacterial spores and Cry5B responses in large animals, as well as impact of B. subtilis Cry5B strain on microbiome of goats.