Usage of segmented filamentous bacteria as a prophylactic to improve gut immune maturation in chickens
Date
2025-08
Authors
Meinen-Jochum, Jared Michael
Major Professor
Advisor
Mellata, Melha
Looft, Torey
Zhang, Qijing
Rowling, Matthew
Roth, James A.
Committee Member
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Abstract
Poultry plays a vital role in human nutrition, with global chicken meat production exceeding 100 million tons annually and the U.S. alone producing over 521 million eggs. However, chickens also serve as major reservoirs for Enterobacteriaceae pathogens like Salmonella and Escherichia (E.) coli, which can cause disease in both poultry and humans through contaminated food products. Additionally, these bacteria contribute to the spread of antimicrobial resistance (AMR) via interbacterial transfer of large virulence plasmids. Given the global rise of multidrug-resistant (MDR) bacteria—projected to cost over a trillion dollars in healthcare by 2050—mitigating AMR in poultry is crucial for both animal and human health. While individual prophylactic strategies, such as probiotics and live Salmonella vaccines, have shown promise in reducing Enterobacteriaceae, their combined effects remain uncertain. Furthermore, despite their benefits to poultry productivity, many commercial probiotics fail to induce strong antimicrobial and must be fed continuously. Therefore, novel prophylactic approaches that can stimulate host intestinal immunity are essential to controlling Enterobacteriaceae and curbing AMR spread.
In commercial poultry farms, chicks hatch away from their progenitors from which they acquire key host-specific microbiota, like segmented filamentous bacteria (SFB) involved in gut maturation in early life. SFB, specifically Candidatus arthromitus, are Gram-positive, spore-forming bacteria closely related to Clostridia species. When received at early life, SFB intimately bind to the epithelial layer of the distal small intestine. Rather than causing pathology, the introduction and binding of SFB to the epithelium promotes immune maturation indicated by T-cell differentiation and increased sIgA production. However, most of these studies have been completed in mice, and the impact of SFB in the chicken intestine remains greatly unstudied. Recently, our lab was the first to demonstrate that introducing SFB spores to chickens at hatch increased immunometabolic differentiation compared to chickens that did not receive SFB.
In Chapter Two, we reviewed current strategies for prophylactic and probiotic applications in modern poultry production environments. We reported the demonstrated benefits, limitations, and knowledge gaps presented by commercially available products. The products were generally presented as live bacterial vaccines or direct-fed probiotics. In Chapter Three, we investigated the propensity of ceca content from layer chickens at early, peak, and late lay stages in two environmental conditions (conventionally caged and cage-free) to harbor bacteria that are emergent AMR threats. The objectives of this study were to (i) detect bacteria resistant to colistin, carbapenems, and βlactams in commercial poultry farms, (ii) characterize phylogenetic and virulence markers of E. coli isolates to potentiate virulence risk, and (iii) assess potential transfer of AMR from these isolates to other bacteria via conjugation. Ceca contents from laying hens from the conventional cage (CC) and cage-free (CF) farms at three maturity stages were randomly sampled and screened for extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, carbapenem-resistant Acinetobacter (CRA), and colistin-resistant E. coli (CRE). We found a widespread abundance of ColRE in both CC and CF hens across all three maturity stages. Extraintestinal pathogenic Escherichia coli plasmidic virulence markers iss and iutA, were widely associated with AMR E. coli isolates. ESBL-producing Enterobacteriaceae were uniquely detected in the early lay period of both CC and CF, while multidrug-resistant (MDR) Acinetobacter were found in peak and late lay periods of both CC and CF. CRA was detected in CF hens only. blaCMY was detected in ESBL-producing E. coli in CC and CF and MDR Acinetobacter spp. in CC. Finally, the blaCMY was shown to be transferrable via an IncK/B plasmid in CC. The presence of MDR to the last-resort antibiotics that are transferable between bacteria in food-producing animals is alarming and warrants studies to develop strategies for their mitigation in the environment.
In Chapter Four, we aimed to further the investigation of the prophylactic potential of SFB in chickens. One-day-old specific pathogen-free layers were orally treated with either PBS (phosphate-buffered saline; CON) or SFB-based treatment (SFB). At four days post-inoculation (DPI), both CON and SFB groups were orally challenged with Salmonella Typhimurium. Total Enterobacteriaceae and Salmonella were examined by plating and enumeration in feces. The presence and levels of SFB were determined from ilea scrapings via microscopy and qPCR, respectively. Relative gene expression of host-derived antimicrobial peptides and cytokines in the distal ileum was determined by RT-qPCR. A significant decrease in total Enterobacteriaceae was observed in the feces of the SFB group. At necropsy, the level of SFB was significantly higher in the SFB group than in the CON group, while a significant decrease in total Enterobacteriaceae and Salmonella was observed in the ceca of the SFB group. RT-qPCR revealed increased expression of β-defensin 14, and cytokines IL-10 and IFNγ. The introduction of SFB at hatch as a prophylactic treatment may benefit commercial partners as well as consumers by reducing the incidence of Enterobacteriaceae in food animals. Reduction of these bacteria in animals would, in turn, increase animal health, productivity, and food safety for consumers.
Through years of selective breeding, the divergence of chickens raised for meat production (broilers) and egg-laying (layers) has created the need to study prophylactics and probiotics in each model due to their different responses to treatments. In Chapter Five, we investigated whether SFB collected from layer chickens were able to colonize their genetically distinct relative broiler counterparts and impart the same immunomodulation benefits. This study investigated whether providing chicken SFB to newly hatched broilers would increase their gut maturation and resistance to bacteria relevant to broiler and human health. One-day-old Ross308 broilers were orally treated with either PBS (CON) or layer-derived SFB (D-SFB). Feces were collected to detect and enumerate SFB and Enterobacteriaceae. On days 8, 15, 22, and 29, birds were euthanized, and the intestinal samples were collected to detect and enumerate SFB through quantitative PCR (qPCR) and microscopy and expression of genes associated with gut immune function through reverse transcription-qPCR. This study showed that, despite their host specificity, layer SFB can colonize their genetically distinct relative broilers. Ileal SFB colonization was accelerated by a week with the SFB treatment and covered the proximal, medial, and distal sections of the ileum. Colonization of the ileum by SFB in early life highly activated gene expression of intestinal barrier proteins and cytokines, e.g., IL-10 and IFNγ but not IL-17. SFB treatment reduced the level of Enterobacteriaceae in the gut and provided superior resistance to intestinal and extraintestinal pathogens as tested in vitro. Overall, early gut colonization of SFB is imperative for the maturation of the gut immune system and the establishment of a homeostatic gut environment.
Interestingly, we demonstrated that SFB collected from layer chickens were able to colonize broiler chickens, increase immune maturation, and provide protection against intestinal and extraintestinal pathogens. Owing to the host-specificity of SFB, we hypothesized that SFB from avian species would be divergent from SFB collected from mammalian hosts. In Chapter Six, using a combination of Nanopore and Hi-C sequencing, we were able to fully construct and annotate the genome of the chicken SFB collected from Lohman Select Layer chickens. Metabolic network analysis revealed that the chicken SFB genome contained complete pathways for the pentose phosphate and glycolysis processes. However, the electron transport chain and many other pathways necessary for replication were absent. Comparing the biosynthetic, utilization, and transporter pathways between chicken and murine SFB showed that SFB from different hosts have different capabilities and requirements, highlighting the divergent evolution of SFB. Furthermore, we identified two forms of the flagellin subunit (fliC) in the chicken SFB genome. A large fliC that was similar to that found in murine hosts and a small, novel fliC.Analysis of the small fliC interaction with host Toll-like receptor five represents unique host-microbe interactions that may play a role in host-specificity and immune maturation.
The studies described in this dissertation identified the need to explore SFB as a probiotic organism that must be introduced to the layer and broiler chickens at day-of-hatch to promote immune system maturation, reduce Enterobacteriaceae in the gut and promote intestinal and extraintestinal protection. Continued studies are needed to utilize metabolomic analysis to eventually culture chicken SFB in vitro. Continued studies are necessary to elucidate the exact mechanism by which the binding of SFB promotes immune system maturation identified herein. Additionally, further in vivo studies are needed to optimize the dosage quantity and timing of inoculation of SFB.
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