Progress report for GNC18-259
Avian pathogenic E. coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), is one of the most common bacterial pathogens of poultry and can be potentially transmitted to humans through consumption of contaminated poultry products. It causes multiple extra-intestinal diseases in poultry, collectively called as avian colibacillosis, which results in high morbidity and mortality (up to 20%), meat and eggs production losses, and increased condemnation of carcasses at slaughter (up to 45%), thus poses significant threat to global poultry production and food security as well as sustainable animal agriculture worldwide. Currently, APEC infections in poultry are controlled by antibiotics medication and vaccination; however, the emergence of multi-drug resistant APEC strains worldwide limits the antibiotics use and vaccines are unable to provide sufficient protection against diverse APEC serotypes. Further, Food and Drug Administration (FDA) in the United States has recommended the voluntarily phasing out the production uses of medically important antibiotics in food-producing animals, with the goal of reducing the selection pressure and the emergence of resistant bacteria (Guidance for Industry, #209,
#213). However, limiting on-farm use of antibiotics could significantly increase animal morbidity and mortality and compromise production efficiency and food security. Therefore, it is critical to replace antibiotics with alternatives that effectively mitigate pathogens on farm for sustainable and safe food production. Towards this end, by conducting multiple in vitro assays, we have identified two probiotics in our lab with strong antimicrobial activity against APEC. We hypothesized that probiotics can effectively prevent APEC infections in poultry, thus provide alternate strategy to control APEC and reduce the reliance on antibiotics. Furthermore, we have prepared probiotics microcapsules using chitosan-alginate microencapsulation technology; encapsulation prolongs the action of probiotics, thereby facilitate efficient delivery of the probiotics to chickens. Here, we propose to test the efficacy of identified probiotics to prevent APEC infections in poultry both in controlled laboratory condition as well as in field conditions. This study will help to develop as well as farmers adoption of non-antibiotic control method for effective management of APEC infections in poultry farms. Through effective control of infections, animal health can be promoted and losses can be minimized ensuring food security. Moreover, public health can be safeguarded by producing safe and wholesome food.
This study will address the issue of antibiotic resistance and focused on developing antibiotic-independent control method.
Learning outcomes: we expect that farmers will increase their knowledge about poultry diseases, antibiotics, antibiotic resistance, and potential non-antibiotic alternatives. The awareness level on prudent use of antibiotics, food security, and food safety is expected to increase. The positive attitude of farmers to enhance the food animal production along with production of safe and wholesome food is expected to nurture. In addition, the husbandry skills of farmers to rear healthy food animals are expected to enhance.
Action outcomes: we expect that farmers will adopt probiotics based control method to control APEC and other poultry associated diseases on their farms. Also, the farmers dependency on antibiotics to control poultry diseases is expected to shift towards possible non-antibiotic alternatives.
We tested the efficacy of two probiotics [Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12)], their microcapsules, and their combination (1:1) in reducing the colonization of APEC in specific pathogen free (SPF) layer chicks (Leghorns). A total of 8 groups (10 chicks/group) were allocated; G-1: APEC infected and LGG gavaged, G-2: APEC infected and Bb12 gavaged, G-3: APEC infected and LGG and Bb12 gavaged, G-4: APEC infected and LGG microcapsules gavaged, G-5: APEC infected and Bb12 microcapsules gavaged, G-6: APEC infected and LGG and Bb12 microcapsules gavaged, G-7: APEC infected and non-gavaged, and G-8: non-infected and non-gavaged. From day 1, probiotics, their microcapsules, and their combination were orally gavaged to chicks (108 CFU/chick) once daily for 12 days (until day 13). On day 6, chicks were infected orally with rifampicin resistant APEC O78 (7.5×107 CFU/chick). Before the infection, rectal swabs were collected randomly from each group to confirm the absence of APEC. The clinical signs of chicks were recorded until 7 days post-infection. On day 14, chicks were necropsied and cecum, liver, and heart were collected for quantitation of APEC load. Spleen and cecal tonsils were collected for analyzing the impact of probiotic treatment on the immune responses of chickens. Cecum is collected to analyze the impact of probiotic treatment on the gut microbiota of chickens. Blood serum is collected to analyze the impact of probiotic treatment on the metabolites of chickens.
The LGG gavaged chicks had 1.6 log less APEC load in chicks cecum compared to non-gavaged chicks; whereas, only 0.6 log less APEC load was observed with Bb12 gavaged chicks. Consistently, LGG microcapsules gavaged chicks had 1.1 log less APEC load in chicks cecum compared to non-gavaged chicks; whereas, only 0.4 log less APEC load was observed with Bb12 microcapsules gavaged chicks. Surprisingly, gavage of LGG and Bb12 and their microcapsules combination reduced only 0.4 and 0.2 log APEC load, respectively, in chicks cecum. Further, APEC was detected in liver of two of the non-gavaged chicks (G-7); whereas, no APEC was detected in liver of LGG gavaged chicks.
Consistent with the better anti-APEC effect of LGG, LGG gavage also increased the body weight of chicks (121±10 grams) compared to non-infected and non-gavaged chicks (109±11 grams; G-8) and infected and non-gavaged chicks (111±10 grams; G-7). The body weights of chicks gavaged with LGG microcapsules, Bb12, Bb12 microcapsules, LGG and Bb12, LGG and Bb12 microcapsules were 113±5, 115±7, 105±31, 111±7, 112±10, respectively.
Overall, LGG and LGG microcapsules gavage reduced the APEC colonization/load in the cecum of chicks and also increased the body weight of chicks
Educational & Outreach Activities
- Deliver brief talk (~10 mins) on the probiotic project at the 2019 TriState Poultry Vet meeting held at the Knights of Columbus Hall in Fort Recovery, Ohio on May 17, 2019. Cooper farms veterinarians, USDA poultry personnel, poultry researchers, and poultry disease diagnosticians are the participants.
- Deliver presentation (~20 mins) on the probiotic project at the 2019 TriState Poultry Vet meeting held at the Knights of Columbus Hall in Fort Recovery, Ohio on Oct 17, 2019. Cooper farms veterinarians, Zoetis personnel, poultry extension personnel, poultry researchers, and poultry disease diagnosticians are the participants. The discussion was also held with Cooper farms veterinarians in regards to accomplishing field trial and workshop.
- Deliver presentation (~30 mins) on the probiotic project at the BioHio Research Park, OARDC, Ohio on Oct 22, 2019. Personnel from the technology commercialization office (TCO), OSU are the participants. The discussion was held with TCO in regard to filing invention disclosure and potential patenting the findings.