Progress 08/01/19 to 08/31/20

Outputs
Target Audience:The target audience reached by our efforts during the last 12 months includes: - scientists in animal health companies, like Elanco, Zoetis, Merck and Ceva, - scientists at contract research organizations, like Southern Poultry Research Group, and Colorado Quality Research, - scientists in academic institutions, like the University of Minnesota, and South Dakota University - scientists in government, like the USDA - scientists in poultry production companies, like Tyson and Pilgrim's Pride. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results of this research are proprietary. They are disseminated to interested parties only after the signing of confidentiality agreements. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of the Phase I project (with an end date of August 31, 2020) has been the development of an antimicrobial probiotic and its testing in animal experiments against C. perf. We generated a library of dozens of antimicrobial peptides (AMPs), including enterocins, AMPs known to target Gram-positive bacteria. We screened this library against C. perf. and identified Enterocin A, Enterocin B, and Hiracin JM79 as potent anti-clostridia peptides. We constructed distinct DNA sequences with antimicrobial peptides (enterocin A, enterocin B, hiracin JM79), five DNA promoters (anaerobic, high/medium/low constitutive, native) and four variants of the Microcin V secretion system. We then constructed distinct DNA sequences with polycistronic constructs containing all three peptides. We have found that commercially available probiotics, such as E. coli Nissle 1917, do not colonize well the GI tract of poultry. We decided to use E. coli isolated from healthy chickens. We developed a library of 70 E. coli isolates from ceca of healthy chickens, and of 40 E. coli isolates from the small intestines of healthy chickens. We screened for antibiotic resistance, testing the susceptibility to rifampicin, nalidixic acid, chloramphenicol, ampicillin, kanamycin, and spectinomycin. We also screened for top virulence markers (cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ, ireA, papC), and for enterotoxigenic serotypes (e.g. O157:H7). We selected several strains that are non-virulent, non-pathogenic and have no resistance to tested antibiotics. We built over 500 distinct systems by combining DNA constructs and E. coli isolates. We had agreed with Elanco to test in animals the top five systems in terms of performance in the following antimicrobial assays: The antimicrobial activity of individual recombinant colonies was first evaluated by the stab-on-agar test. The size of the halo gives an indication of activity strength. Survival in chicken stomach contents and in low pH environments. We tested probiotic survival by inoculating stomach contents and minimal media+HCl with 10^7 colony forming units (CFU)/mL of each probiotic. We incubated anaerobically for 45 minutes (approximate residence time in chicken stomach). We then counted the viable CFUs of each probiotic. Survival in bile contents. We emptied bile contents from the duodenum of several chicken intestines and inoculated with 10^7 CFU/mL of each probiotics. We incubated anaerobically for 4 hours and determined the CFUs/mL before and after incubation. Growth of modified probiotics in small intestinal mucus. We expected E. coli to replicate primarily in the mucus layer. Ability to utilize these nutrients is crucial to colonization. We sought to compare growth rates of modified probiotics in small intestinal mucus contents. We isolated mucus from the small intestine then filter-sterilized and diluted 1:10 in minimal medium salts. We devised a scoring system combining the results of activity and stability assays. Using the scoring system we selected the five best performing candidates. Animal experiments The two primary objectives of the animal experiment were: To evaluate the efficacy of five engineered probiotic E.coli. on the control of gross lesion scores and mortality due to necrotic enteritis, when medicated through water from 0-21days, in broilers raised in battery cages for 22days. To select the best probiotic E. coli among five different strains based on the efficacy on controlling gross lesions and mortality caused by necrotic enteritis. This study utilized 540 single-sex commercial broilers in an established NE challenge model. Each cage was the experimental unit. The study began on Study Day 0 (arrival) and birds were enrolled to the study on study day 13. There were nine (n=9) treatment groups (two negative controls, one positive control, five test article groups with challenge and one test article group without challenge). Each treatment group had 6 replicates. Each replicate was represented by 10 birds. The treatment groups were randomly allocated in four different racks. There were 60 birds per treatment group. Treatment groups were represented as T1, T2, T3, T4, T5, T6, T7, T8 and T9. Except T1 and T3 treatment groups, all other treatment groups were medicated with their respective test article from very first (Day 0) drinking water. The medication was provided three times daily. The medicated water was kept until the next medication time period. The same procedure was continued on a daily basis until the last day of the study (Day 21). Except T1 and T2 groups, all other treatment groups were administered with mixture of E. maxima (10,000 sporulated oocyst/mL/bird) + E. acervulina on day 14 through oral gavage. Except T1 and T2 group, all other treatment groups were administered with a C. perf. strain at the rate of 2 x 108 CFU/mL/bird on day 17 & 18 through oral gavage. All the treatment groups were subjected to lesion scoring on day 22. During the period of days 17-22, all mortalities were necropsied. If a dead bird showed any sign of necrotic lesions, then the score was considered as 4 and documented in the lesion scoring sheet. Samples were collected for enumeration of probiotic E. coli from jejunal content, jejunal scraping and cecal content on day 7, 14 and 22. Samples were collected for the enumeration of C. perf. from jejunal contents on Day 22. Results. T6 & T9 construct systems were shown to be effective in controlling mortality and lesion scores in broilers when challenged with C. perf. The mortality rate in the untreated group, T3, was 34% and the lesion score was an average of 1.6. T6 (GPEC2019002) and T9 (GPEC2019005) showed decreased mortality of 14.8% & 11.3% respectively (P<0.05). Similarly, lesion scores were 0.65 & 0.64 respectively (P<0.05). T7 & T8 did not show statistically significant improvements in terms of either mortality or lesion scores. In a separate experiment, we also established the safety profile of various dosing regimens in birds. In a pen-floor experiment involving 1,300 chickens, we demonstrated that there is no measurable morbidity or mortality in birds administered the modified probiotics. We observed a statistically significant improvement in the feed conversion ratio. Birds treated with modified probiotics had an FCR of 1.61, compared to an FCR of 1.72 for untreated birds. We plan to repeat this experiment after a number of improvements on GP837 (the probiotic in T9) described later herein. The design of the experiment is described in detail in the proposed research section. Interactions with the FDA CVM In 2019, we initiated discussions with the FDA and USDA and established that as genetically modified bacteria, our probiotics are considered a live biotherapeutic product and will require FDA approval in the U.S. In November 2019, we presented our technology to the Center for Veterinary Medicine at the FDA. They advised us to open an investigational new animal drug (INAD) file and pursue the path for registration. With the promise and innovative nature of our technology, the FDA has granted us a sponsor fee waiver under Section 740(d)(1)(A) of the FD&C Act. To our knowledge, this waiver by the FDA CVM is a first for a probiotic-based technology and drastically lowers the barrier to product commercialization. There are numerous documents with clear guidance to industry that detail the requirements for assessing and demonstrating effectiveness, target animal safety, human consumer safety, environmental impact and chemistry, manufacturing and controls for live therapeutics. We are using these guidance documents to design experiments for FDA approval.

Publications