Salmonella and Campylobacter are leading bacterial agents both nationally and globally, making them high public health concerns. Both are significant important zoonotic pathogens commonly found in livestock. Poultry and poultry products are the most common sources of human infections. Infected poultry show little to no clinical signs, and risk the chance of entering the food system, potentially contaminating consumers. Direct links between the high load of bacteria in the chicken intestinal tract and the high contamination of poultry carcasses support the need for pre-harvest control. Previously, antibiotics were used to control bacterial infections and growth; but increasing occurrence of antibiotic resistant bacteria has caused laws and practices to shift. Targeting the control of foodborne pathogens in the pre-harvest stage can improve animal welfare and public health. Alternative treatment methods are needed to combat Salmonella and Campylobacter in production animals, improve antibiotic stewardship, and subsequently strengthen the economy.
We discovered the antimicrobial efficacy of Lactobacillus acidophilus (LA), Lactobacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) in vitro. More importantly, we showed LGG significantly reduced Salmonella in the chicken cecum by 1.9 logs (P<0.001) at 10 days post infection. LGG was also able to inhibit the growth/ presence of other food safety significant Salmonella serovars in an agar well diffusion assay. Characterization of antibacterial activity of LGG revealed that although organic acids are present, the most crucial part of inhibition are the heat and protease stable peptides that were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
After which, we tested the efficacy of Lactobacillus rhamnosus GG (LGG) derived small peptides (P1-NPSRQERR, P2- PDENK, and P4-MLNERVK) against Salmonella Typhimurium (ST) in chickens and examined their antibacterial effects against Salmonella Enteritidis (SE) and other Salmonella serovars in vitro. The antagonistic effects of peptides were heat and protease resistant and antibacterial activity is likely due to their effect on Salmonella membranes. Although, P1, P2, and P4 inhibited ST, SE, and other Salmonella serovars in vitro at 18mM, 15mM, and 18mM respectively, they did not affect the growth of Gram-positive commensal bacteria. P1and P2 at 50mg/kg successfully reduced the colonization of ST by 2.2 logs and 1.8 logs, respectively (P< 0.05), in chickens 7 days post infection. P1, P2, or P4 had no adverse effects on chicken cecum microbial communities, but birds not treated and challenged with Salmonella showed differences in microbial richness and evenness compared to the unchallenged birds. Confocal and transmission electron microscopy revealed that peptides attacked the membrane of Salmonella causing its flaccidity, sloughing, or disruption.
Lastly, we analyzed the ability of Salmonella Typhimurium derived recombinant attenuated Salmonella vaccines (RASV) to reduce the colonization of C. jejuni in chickens. One-day-old Salmonellaand Campylobacter free chickens were orally immunized with RASV constructs (2x108 CFU/ bird), and a booster was given 10 days later. Birds were challenged with a cocktail of 5 C. jejuni strains on day 18. Colonization load was determined by enumerating C. jejuni in the cecum of the birds. Four trials were conducted analyzing the RASVs effect on Campylobacter load in the cecum. Our data suggested that vaccine efficacy was dependent on amount of challenge dose and antigens present in vaccine. RASV90+201 and RASV131 showed consistent reduction across trials 2,3, and 4. The best reduction in Campylobacter load occurred in trial 3 with 103 Campylobacter challenge dose. At 17 days post infection, pG8R-86 (cjaA), pG8R-90+201 (FlaA), and pG8R-131 (FlaA) low challenge dose groups yielded significant reductions in Campylobacter load (approximately 3.7, 2.4, and 1.7 logs, respectively, compared to vector control). Additionally, in multiple trials RASV-90 showed a significant induction of IgY antibody titers compared to the untreated and unchallenged vector control group.
Overall, these studies revealed that antibiotic independent novel therapeutics and vaccines are promising alternatives to control Salmonella and Campylobacter in chickens.