Date of Award
Doctor of Philosophy
Theses & dissertations (Interdisciplinary)
Poultry are a critical source of human nutrition, with over 100 million tons of chicken meat being produced globally per year and over 521 million eggs produced in the United States alone. However, poultry like chickens serve as major reservoirs for bacterial Enterobacteriaceae pathogens like Salmonella and Escherichia coli, which can cause disease in poultry and/or humans via contamination of poultry food products. Furthermore, these Enterobacteriaceae are major reservoirs for antimicrobial resistance (AMR), which is primarily spread through inter-bacterial exchange of large virulence plasmids. Thus, being able to reduce Enterobacteriaceae in poultry and mitigate AMR spread would simultaneously improve poultry and human health. Prophylactic strategies including probiotics and live Salmonella vaccines have individually demonstrated efficacy in reducing Enterobacteriaceae in poultry. However, whether the combination of these live prophylactics produces even greater success (or failure) has yet to be determined. Furthermore, although many commercial probiotics boast a wide number of benefits to poultry productivity (improved feed conversion, pathogen competitive exclusion, inflammation reduction, etc), they induce poor host antimicrobial responses against enteric pathogens. Furthermore, some bacteria like Salmonella actively promote immunotolerance in the chicken gut, which prevents antibacterial host responses and subsequently results in fecal Salmonella shedding and contamination of poultry products. Thus, novel prophylactics which can stimulate host intestinal responses and overcome these immunotolerant mechanisms to clear intestinal Enterobacteriaceae like Salmonella are needed for poultry. Overall, the objective of these studies was to determine whether prophylactics (i.e., probiotics, live Salmonella vaccine, ileal spores, or reserpine) could mitigate Enterobacteriaceae activities in poultry.In the first study of this thesis, we explored whether the combinatorial use of a commercial probiotic mix (Gro-2-Max®) and live Salmonella vaccine (Typhimurium strain) in white leghorn chickens would have superior effects against Enterobacteriaceae intestinal colonization. Birds were received at day-of-hatch and continuously fed probiotics in feed (PRO), orally vaccinated with a live Salmonella vaccine (VAX), given both prophylactics (P+V), or untreated (CON). Using 16S rRNA sequencing and fecal enumeration on MacConkey agar, Enterobacteriaceae ceca abundances and fecal shedding were greatest in PRO birds but was significantly reduced upon Salmonella vaccination, regardless of probiotic supplementation. These changes were positively associated with shifts in fermentative bacterial abundances, supporting the “restaurant hypothesis” in which Enterobacteriaceae rely on fermentative microbes for mono- and disaccharide nutrients. Given the relationship between gut bacteria and neurochemical pro-duction, we hypothesized these live prophylactics induced changes in intestinal neurochemistry. Measuring neurochemical levels via UHPLC-ED, prophylactic treatments induced unique changes in catecholamine metabolism in the chicken intestine. Furthermore, these changes were positively correlated with intestinal bacteria like Enterobacteriaceae and Akkermansia muciniphila. Overall, these live prophylactics induce changes in both neurochemical metabolism and microbial abundances in the chicken intestine, which likely contributed to the loss of Enterobacteriaceae upon Salmonella vaccination. In the second study to determine if these live prophylactics affected E. coli virulence potential from the intestine, a reservoir for pathogens like avian pathogenic E. coli (APEC), ~100 E. coli colonies from each group were isolated and individually screened them for siderophore production, antibiotic resistance, and genotype via PCR. Uniquely, P+V isolates produced significantly less siderophores, were more susceptible to the antibiotics tetracycline and streptomycin, and lacked several APEC virulence factors (iutA¸ iss, hylfA) compared to isolates from all other treatment groups. This loss of virulence potential in P+V E. coli was associated with the absence of IncF and ColV plasmids as well as a loss of total average plasmids per isolate, suggesting that intestinal plasmid transfer may have been reduced in P+V birds. En route to identifying a factor responsible for this observation, ceca mucus total small RNA (smRNA) levels were lowest in P+V birds versus all other groups. Hypothesizing that smRNA concentration was positively associated with IncF plasmid transfer, greater smRNA levels increased IncF plasmid-mediated AMR transfer in vitro, regardless of treatment group. Furthermore, using predictive hybridization analyses, multiple chicken microRNAs (miRNAs), a subset of smRNAs, were aligned with plasmidic genes associated with pilus assembly and plasmid transfer, suggesting that certain smRNAs may drive plasmid transfer. These data identify the combination of probiotics and live Salmonella vaccine as a unique in vivo strategy to mitigate intestinal IncF plasmid transfer and spread of AMR and virulence genes via a smRNA-dependent mechanism. Although Enterobacteriaceae are mainly associated with the intestinal tract, certain members like APEC can bypass host barriers and enter the bloodstream, resulting in avian colibacillosis and high mortality. In the third study, to determine if these live prophylactics affect APEC resistance, blood from P+V birds exhibited greater bactericidal responses against APEC versus CON. However, these responses were independent of IgY antibody response, suggesting that killing from innate immune cells were stimulated upon P+V treatment. Using an airsac-challenge model for APEC in vivo infection, P+V treatment reduced colibacillosis lesions as well as APEC enumeration in the blood compared to CON birds. Overall, in addition to intestinal Enterobacteriaceae resistance, P+V treatment induces extraintestinal protection against APEC infection, meaning that these orally-delivered prophylactics can confer benefits outside of the chicken intestine. Given that probiotics poorly induce poor immunological responses in the chicken intestine, in the fourth study day-old white leghorn chicks were inoculated with a single dose of ileal spores (SPORE) and compared them to untreated controls (CON). Using scanning electron microscopy (SEM) SPORE treatment hastened the rate and consistency of SFB attachment in the ileum, with birds as young as 4 days old exhibiting SFB colonization. Using FITC-dextran to measure gut permeability, SPORE birds exhibited reduced gut leakiness versus CON. To determine immunometabolic shifts in the ileum, the chicken kinome peptide array was used to measure global changes in phosphorylation networks. Generally, SPORE treatment induced drastic shifts in host immunometabolism. Furthermore, these changes were associated with Salmonella resistance, as TH17 cell differentiation was positively associated with in vitro killing of several Salmonella serovars. Overall, we find that a single dose of an ileal spore inoculum was sufficient to induce drastic changes in intestinal immunometabolism and may be an effective strategy to increase Salmonella resistance. In the final study, to investigate the role of the neuroimmunological axis in Salmonella resistance, we developed an ex vivo ceca explant model to use reserpine, which induces intra-cellular catecholamine release, as a novel prophylactic. As expected, reserpine treatment in-duces norepinephrine release from chicken ceca explants and regulatory T cells (Tregs). Furthermore, media supernatants from explant cultures treated with reserpine induced increased antimicrobial responses as well as Salmonella killing compared to untreated explants. Using an in vivo challenge model, oral reserpine treatment increased total Enterobacteriaceae and Salmonella Typhimurium resistance in young birds. Using the immunometabolic kinome peptide array, changes in T cell receptor, epidermal growth factor (EGF), and mTOR signaling were all induced by reserpine treatment. More specifically, EGF receptor (EGFR), the mTOR protein, and the MAPKK MEK2 were all differentially phosphorylated. Using various ligands and inhibitors to determine the role of these pathways, norepinephrine stimulated Salmonella killing in a dose-dependent mechanism. Furthermore, inhibition of beta-adrenergic receptors as well as recombinant EGF suppressed reserpine-induced Salmonella resistance. Furthermore, explant treatment with rapamycin, an mTOR inhibitor, increased Salmonella killing similar to reserpine treatment. Lastly, we found that MEK1/2 signaling was central to antimicrobial responses induced by these neuro-immunometabolic pathways. Overall, this demonstrates a fundamental role for the neuroimmunological axis in Salmonella killing and suggests that reserpine could be a novel prophylactic to improve Salmonella resistance in young birds. The studies described in this dissertation identify multiple prophylactics which improve Enterobacteriaceae resistance in poultry. Future studies are needed to determine whether these strategies can be used in combination as well as changes in specific factors alone (ex: shifts in the gut microbiota and neurochemicals) are sufficient to induce anti-Enterobacteriaceae responses.
Redweik, Graham, "Using prophylactics to improve resistance against Enterobacteriaceae in chickens" (2021). Graduate Theses and Dissertations. 18597.