Progress 07/01/22 to 02/29/24

Outputs
Target Audience: Nothing Reported 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? Nothing Reported 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 objectives of our Phase I grant were focused on demonstrating whether our product, Prauzni (referred to as FPZ-100 in our Phase I proposal), could be developed into a feed additive for poultry to improve feed efficiency in broiler chickens and reduce pathogen load. The main task of our Phase I proposal was a 750 bird floor pen trial performed at Southern Poultry Research Group, Athens GA to test the effects of Prauzni on both feed conversion and Salmonella reduction. The trial consisted of a control group, and two groups treated with 250 ppm Prauzni in feed for 7 days or 15 days starting at one day of age. Each group consisted of 10 pens with 25 birds in each pen. Feed conversion rate (FCR) and weight gain were measured at days 15, day 28, and day 42 of the trial. As shown in Figure 1, groups treated with Prauzni for either 7 or 15 days showed statistically significantly improved FCR and weight gain compared to untreated birds at day 28 and 42 of the trial (p < 0.05). At 42 days of age, we found that chickens receiving Prauzni for only the first 7 days of life showed a 3.4% FCR improvement compared to the untreated control group. This result was 4.9 times greater than the success criteria target stated in our Phase I Objective. In the same trial, half of the birds in each pen were challenged with Salmonella enteritidis (3.0 x 107 CFU/chick) at day 1 to test the potential of Prauzni to reduce Salmonella load. At 42 days, cecal contents from both challenged and non-challenged birds were collected and cultured. Fifteen days of Prauzni treatment resulted in numerically lower most probable number (MPN) of Salmonella in culture positive cecal samples, especially in the indirect (horizontal) exposed birds, as shown in Figure 2 and Table 2. This trend of lower Salmonella levels in horizontally exposed chickens is encouraging, as in a natural setting this would be more aligned with the primary route of exposure for most birds. These data suggest that in a commercial setting with few Salmonella positive chickens at placement that initiate horizontal spread, Prauzni has the potential to reduce the Salmonella load of contact-infected birds. FCR improvement was obtained with both 7- and 15-day treatments, but we did not see the same numerical reduction with the 7 day treatment. This finding suggests that Prauzni product development could be divided into two product lines; one that is a 7-day treatment for healthy flocks for only FCR improvement, and another that is a 15-day treatment product for both FCR improvement and Salmonella reduction.

Publications

Progress 07/01/22 to 06/30/23

Outputs
Target Audience: Nothing Reported 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? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to continue in vitro assays to determine the mechanisms by which FPZ improves feed efficiency in poultry and further investigate FPZ's antimicrobial action.

Impacts
What was accomplished under these goals? The objectives of our Phase I grant were focused on demonstrating whether our product, Prauzni (referred to as FPZ-100 in our Phase I proposal), could be developed into a feed additive for poultry to improve feed efficiency in broiler chickens and reduce pathogen load. The main task of the first objective of our Phase I proposal was a 750 bird floor pen trial performed at Southern Poultry Research Group, Athens GA to test the effects of Prauzni on both feed conversion and Salmonella reduction. The trial consisted of a control group, and two groups treated with 250 ppm Prauzni in feed for 7 days or 15 days starting at one day of age. Each group consisted of 10 pens with 25 birds in each pen. Feed conversion rate (FCR) and weight gain were measured at days 15, day 28, and day 42 of the trial. As shown in Figure 1, groups treated with Prauzni for either 7 or 15 days showed statistically significantly improved FCR and weight gain compared to untreated birds at day 28 and 42 of the trial (p < 0.05). At 42 days of age, we found that chickens receiving Prauzni for only the first 7 days of life showed a 3.4% FCR improvement compared to the untreated control group. This result was 4.9 times greater than the success criteria target stated in our Phase I Objective. In the same trial, half of the birds in each pen were challenged with Salmonella enteritidis (3.0 x 107 CFU/chick) at day 1 to test the potential of Prauzni to reduce Salmonella load. At 42 days, cecal contents from both challenged and non-challenged birds were collected and cultured. Fifteen days of Prauzni treatment resulted in numerically lower most probable number (MPN) of Salmonella in culture positive cecal samples, especially in the indirect (horizontal) exposed birds. This trend of lower Salmonella levels in horizontally exposed chickens is encouraging, as in a natural setting this would be more aligned with the primary route of exposure for most birds. These data suggest that in a commercial setting with few Salmonella positive chickens at placement that initiate horizontal spread, Prauzni has the potential to reduce the Salmonella load of contact-infected birds. FCR improvement was obtained with both 7- and 15-day treatments, but we did not see the same numerical reduction with the 7 day treatment. This finding suggests that Prauzni product development could be divided into two product lines; one that is a 7-day treatment for healthy flocks for only FCR improvement, and another that is a 15-day treatment product for both FCR improvement and Salmonella reduction. The effect of Prauzni on systemic inflammation was tested by administering Prauzni to LPS-stimulated chicken HD11 macrophage cells in collaboration withDr. Glenn Zhang at Oklahoma State University. Cells were incubated overnight and treated in duplicate with 1 or 5 mg/mL Prauzni for 2 h, followed by stimulation with 10 ng/mL LPS for 3 h. Cells were lysed for RNA isolation and real-time PCR was carried out. mRNA levels of proinflammatory cytokines IL-1β, and IL-8 as well as an anti-inflammatory cytokine, IL-10 were measured by quantitative real-time RT-PCR assays. Relative fold changes of gene expression levels were calculated using the 2-ΔΔCt method normalized against a reference gene, GAPDH. As shown in Figure 4, expression levels of all cytokines (both pro and anti-inflammatory) were increased upon Prauzni treatment. This suggests that the impact of Prauzni on systemic inflammation may be complex. Prauzni Production Process Validation. In anticipation of manufacturing at full commercial capacity, and to provide material for larger trials outlined in Phase II of this grant proposal, Bactana has developed reliable systems to produce up to 6 kg of Prauzni per month (to be scaled to 12 kg in Phase II), in our existing 980 square foot lab space. As part of our 2022 manufacturing scale-up plan, we targeted development of a fermentation protocol that would allow us to reliably ferment our proprietary strains in 90 L fermentation tanks. As pharmaceutical grade equipment can be cost prohibitive for production of products targeting the low margin poultry sector, we developed reliable and cost-effective production methods for low cost anaerobic fermentation and formulation procedures. Based on our success with this initiative, we are now able to produce fermentation batches in these 90 L fermenters with identical growth yields and purity to our lab scale fermentation runs. Importantly, the fermentation equipment we purchased is scalable to 1060 L with the same configuration and protocols as were optimized with our 90 L systems and in accordance with our Standard Operating procedures. This protocol is directly translatable as we build out our production upon completion of the objectives of this proposal. We have also investigated the powder characteristics of Prauzni as consistent mixing with feed will be important for this product. We found that after moderate mixing, Prauzni could be reduced to micron sized powder, (as shown in Figure 5a-b) which is necessary for consistent mixing at commercial feed mills where tonnage scales of feed are used.

Publications