Reserpine improves Enterobacteriaceae resistance in chicken intestine via neuro-immunometabolic signaling and MEK1/2 activation.

Salmonella enterica persist in the chicken gut by suppressing inflammatory responses via expansion of intestinal regulatory T cells (Tregs). In humans, T cell activation is controlled by neurochemical signaling in Tregs; however, whether similar neuroimmunological signaling occurs in chickens is currently unknown. In this study, we explore the role of the neuroimmunological axis in intestinal Salmonella resistance using the drug reserpine, which disrupts intracellular storage of catecholamines like norepinephrine. Following reserpine treatment, norepinephrine release was increased in both ceca explant media and Tregs. Similarly, Salmonella killing was greater in reserpine-treated explants, and oral reserpine treatment reduced the level of intestinal Salmonella Typhimurium and other Enterobacteriaceae in vivo. These antimicrobial responses were linked to an increase in antimicrobial peptide and IL-2 gene expression as well as a decrease in CTLA-4 gene expression. Globally, reserpine treatment led to phosphorylative changes in epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and the mitogen-associated protein kinase 2(MEK2). Exogenous norepinephrine treatment alone increased Salmonella resistance, and reserpine-induced antimicrobial responses were blocked using beta-adrenergic receptor inhibitors, suggesting norepinephrine signaling is crucial in this mechanism. Furthermore, EGF treatment reversed reserpine-induced antimicrobial responses, whereas mTOR inhibition increased antimicrobial activities, confirming the roles of metabolic signaling in these responses. Finally, MEK1/2 inhibition suppressed reserpine, norepinephrine, and mTOR-induced antimicrobial responses. Overall, this study demonstrates a central role for MEK1/2 activity in reserpine induced neuro-immunometabolic signaling and subsequent antimicrobial responses in the chicken intestine, providing a means of reducing bacterial colonization in chickens to improve food safety.

Bacteria Broadly-Resistant to Last Resort Antibiotics Detected in Commercial Chicken Farms.

Resistance to last resort antibiotics in bacteria is an emerging threat to human and animal health. It is important to identify the source of these antimicrobial resistant (AMR) bacteria that are resistant to clinically important antibiotics and evaluate their potential transfer among bacteria. The objectives of this study were to (i) detect bacteria resistant to colistin, carbapenems, and ß-lactams 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 via conjugation. Ceca contents from laying hens from conventional cage (CC) and cage-free (CF) farms at three maturity stages were randomly sampled and screened for extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae, carbapenem-resistant Acinetobacter (CRA), and colistin resistant Escherichia coli (CRE) using CHROMagar™ selective media. We found a wide-spread abundance of CRE in both CC and CF hens across all three maturity stages. Extraintestinal pathogenic Escherichia coli phylogenetic groups B2 and D, as well as 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.

Live Bacterial Prophylactics in Modern Poultry.

Commercial poultry farms frequently use live bacterial prophylactics like vaccines and probiotics to prevent bacterial infections. Due to the emergence of antibiotic-resistant bacteria in poultry animals, a closer examination into the health benefits and limitations of commercial, live prophylactics as an alternative to antibiotics is urgently needed. In this review, we summarize the peer-reviewed literature of several commercial live bacterial vaccines and probiotics. Per our estimation, there is a paucity of peer-reviewed published research regarding these products, making repeatability, product-comparison, and understanding biological mechanisms difficult. Furthermore, we briefly-outline significant issues such as probiotic-label accuracy, lack of commercially available live bacterial vaccines for major poultry-related bacteria such as Campylobacter and Clostridium perfringens, as well research gaps (i.e., probiotic-mediated vaccine adjuvancy, gut-brain-microbiota axis). Increased emphasis on these areas would open several avenues for research, ranging from improving protection against bacterial pathogens to using these prophylactics to modulate animal behavior.

Oral Treatment With Ileal Spores Triggers Immunometabolic Shifts in Chicken Gut.

The animal gut is a major site affecting productivity via its role in mediating functions like food conversion and pathogen colonization. Live microorganisms like probiotics are widely used to improve poultry productivity. However, given that chicks receive their microbiota from the environment at-hatch, a bacterial treatment that can stimulate gut immune maturation in early life can benefit animal health. Thus, our lab has begun investigating alternative means to improve poultry health via single inoculation with microbial spores. In this study, we orally-inoculated day-old chicks with ileal scrapings (ISs) enriched for spores via chloroform treatment (SPORE) or non-treated (CON). At 3, 7, and 14 days post-inoculation (dpi), gut permeability was measured via FITC-dextran assay in serum. Additionally, small intestinal scrapings (SISs) were tested for in vitro Salmonella killing and total IgA. Lastly, distal ileum was either fixed or flash-frozen for microscopy or kinome peptide array, respectively. Using bacterial 16S rRNA gene sequencing, SPORE and CON inocula were highly-similar in bacterial composition. However, spores were detected in SPORE but not in CON inoculum. Segmented filamentous bacteria (SFB) filaments were observed in the distal ileum in SPORE birds as early as 3 dpi and all birds at 7 and 14 dpi. Additionally, SFB were detected via PCR in the ceca, colonizing all SPORE birds at 3 dpi. At 3 dpi, SPORE birds exhibited lower gut permeability vs. CON. In SPORE birds, SISs induced greater Salmonella growth in vitro at 3 dpi yet significantly-reduced Salmonella load at 7 and 14 dpi compared to CON in an IgA-independent manner. SPORE distal ileal tissue exhibited unique upregulation of several immunometabolic processes vs. CON birds, including innate (Toll-like receptor, JAK-STAT) and adaptive (T/B cell receptor, TH17 differentiation) immune pathways, PI3K/Akt signaling, mTOR signaling, and insulin-related pathways. Collectively, these data suggest oral inoculation with ileal spores generally-improved gut health. Importance: We report that ileal, spore-forming commensal microbes have potent effects on ileum immunometabolism. Additionally, we identify a functional ileal phenotype in spore-treated chickens, which matched several of the observed immunometabolic changes and was associated with SFB colonization in the ileum.

Microbiome and biological blood marker changes in hens at different laying stages in conventional and cage free housings.

With the majority of conventional cage (CC) laying facilities transitioning into cage-free (CF) systems in the near future, it is important to characterize biological markers of health in layers housed in commercial housings for sustainable production. The objectives of this study were to compare i) blood markers, that is heterophil:lymphocyte (H:L) ratios and susceptibility to avian pathogenic Escherichia coli (APEC) and ii) lung and ceca microbiome between hens at different maturity stages in commercial CC and CF farms. Laying hens at 3 maturity stages were randomly sampled (N = 20 per maturity and per farm). Blood was tested for H:L ratios and APEC killing ability using microscopy and in vitro assay, respectively. Microbiomes were assessed using 16S rRNA sequencing and QIIME2 analysis. Data show H:L ratios did not differ between maturities in both farms. Avian pathogenic Escherichia coli killing was only different in CC hens, where χ7122 level was higher (P < 0.05) in peak compared with early lay. In both farms, microbiome diversity was consistently different (P < 0.05) in both ceca and lung of early lay compared with peak and late lay. In the ceca and lung, relative abundances of the 3 predominant phyla (Bacteroidetes, Firmicutes, and Proteobacteria) did not significantly change with maturity in both farms. Potential pathogens Campylobacter and Staphylococcus reached greater (P < 0.05) abundances in CC lungs in early lay and in CF lungs in late lay, respectively. Overall, this study showed no differences in the stress marker H:L but identified some differences in resistance to APEC and microbiome composition across maturity stages in CC and CF. The lung and gut microbiomes were highly similar, with both serving as potential reservoirs for Campylobacter and Staphylococcus. Future studies on controllable environments for CF and CC are needed to develop adequate strategies for each housing and maturity stage to reduce pathogens and optimize disease-resistance.

Protection against avian pathogenic Escherichia coli and Salmonella Kentucky exhibited in chickens given both probiotics and live Salmonella vaccine.

Commercial poultry farms are increasingly threatened by bacterial infections from avian pathogenic Escherichia coli (APEC) and broad-host Salmonella serovars. Recombinant attenuated Salmonella vaccines (RASV) elicit cross-reactive immune responses against APEC in chickens; however, assessment of broad protection is lacking. Probiotics boost chicken immunity and improve vaccination responses. The objective of this study was to determine whether the RASV, the probiotics, or their combination had protection against APEC and Salmonella. White Leghorn chicks were randomly placed into 4 groups: no treatment (CON), probiotics (PRO), RASV (VAX), or both prophylactics (P + V). Chicks in the PRO and P + V groups were fed probiotics daily, beginning at the age of 1-day-old. Chicks in the P + V and VAX groups were orally inoculated with RASV at the age of 4 D and boosted 2 wks later. Total and antigen-specific IgY responses to Salmonella (lipolysaccharide [LPS]) and E. coli (IroN and IutA) were measured in serum samples via ELISA. Bactericidal potential of both serum and blood against 42 APEC isolates comprising 25 serotypes was assessed in vitro. In vivo protection against APEC was evaluated by air sac challenge with APEC χ7122 (O78:K80), gross pathological lesions were scored, and bacterial loads were enumerated. In a second similar study, birds were orally challenged with S. Kentucky (CVM29188), and feces were enumerated for Salmonella at multiple time points. Vaccination elicited significant LPS-specific antibodies regardless of probiotics (P < 0.0001). Chicks in the P + V group demonstrated increased blood and serum bactericidal abilities against multiple APEC strains in vitro compared with the CON group. Following χ7122 challenge, P+V birds had less APEC in their blood (P < 0.001) and lower signs of airsacculitis (P < 0.01) and pericarditis/perihepatitis (P < 0.05) than CON birds. Finally, only P + V birds were negative for fecal Salmonella at all time points. This study shows this combination treatment may be a feasible method to reduce infection by APEC and Salmonella in chickens.

Mouse Genetic Background Affects Transfer of an Antibiotic Resistance Plasmid in the Gastrointestinal Tract.

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.

Evaluation of Live Bacterial Prophylactics to Decrease IncF Plasmid Transfer and Association With Intestinal Small RNAs.

Chicken intestinal Escherichia coli are a reservoir for virulence and antimicrobial resistance (AMR) genes that are often carried on incompatibility group F (IncF) plasmids. The rapid transfer of these plasmids between bacteria in the gut contributes to the emergence of new multidrug-resistant and virulent bacteria that threaten animal agriculture and human health. Thus, the aim of the present study was to determine whether live bacterial prophylactics could affect the distribution of large virulence plasmids and AMR in the intestinal tract and the potential role of smRNA in this process. In this study, we tested ∼100 randomly selected E. coli from pullet feces (n = 3 per group) given no treatment (CON), probiotics (PRO), a live Salmonella vaccine (VAX), or both (P + V). E. coli isolates were evaluated via plasmid profiles and several phenotypic (siderophore production and AMR), and genotypic (PCR for virulence genes and plasmid typing) screens. P + V isolates exhibited markedly attenuated siderophore production, lack of AMR and virulence genes, which are all related to the loss of IncF and ColV plasmids (P < 0.0001). To identify a causal mechanism, we evaluated smRNA levels in the ceca mucus and found a positive association between smRNA concentrations and plasmid content, with both being significantly reduced in P + V birds compared to other groups (P < 0.01). To test this positive association between IncF plasmid transfer and host smRNA concentration, we evenly pooled smRNA per group and treated E. coli mating pairs with serial concentrations of smRNA in vitro. Higher smRNA concentrations resulted in greater rates of IncF plasmid transfer between E. coli donors (APEC O2 or VAX isolate IA-EC-001) and recipient (HS-4) (all groups; P < 0.05). Finally, RNAHybrid predictive analyses detected several chicken miRNAs that hybridize with pilus assembly and plasmid transfer genes on the IncF plasmid pAPEC-O2-R. Overall, we demonstrated P + V treatment reduced smRNA levels in the chicken ceca, which was associated with a reduction in potentially virulent E. coli. Furthermore, we propose a novel mechanism in which intestinal smRNAs signal plasmid exchange between E. coli. Investigations to understand the changes in bacterial gene expression as well as smRNAs responsible for this phenomenon are currently underway.

Oral Treatments With Probiotics and Live Salmonella Vaccine Induce Unique Changes in Gut Neurochemicals and Microbiome in Chickens.

Cross-talk between the gut microbiota and neurochemicals affects health and well-being of animals. However, little is known about this interaction in chickens despite their importance in food production. Probiotics and live Salmonella vaccines are microbial products commonly given orally to layer pullets to improve health and ensure food safety. This study's objective was to determine how these oral treatments, individually or in combination, would impact the gut environment of chickens. White Leghorn chicks were either non-treated (CON) or orally given probiotics (PRO), a recombinant attenuated Salmonella vaccine (RASV; VAX), or both (P+V). Birds were fed with probiotics daily beginning at 1-day-old and orally immunized with RASV at 4-days-old and boosted 2 weeks post-primary vaccination. At 5 weeks, ceca content, ceca tissues, and small intestinal scrapings (SISs) were collected from ten birds/group post-euthanasia for analyses. Catecholamine, but not serotonergic, metabolism was affected by treatments. Dopamine metabolism, indicated by L-DOPA and DOPAC levels, were increased in P+V birds versus CON and PRO birds. Based on 16S sequencing, beta diversity was more similar among vaccinated birds versus birds given probiotics, suggesting live Salmonella vaccination has a major selective pressure on microbial diversity. Abundances of Akkermansia muciniphila and Enterobacteriaceae positively correlated with levels of tyrosine and norepinephrine, respectively. Both enumeration and 16S sequencing, determined that PRO exhibited the greatest levels of Enterobacteriaceae in the ceca and feces, which was associated with greater IgA production against E. coli virulence factors as tested by ELISA. In summary, we demonstrate that using probiotics alone versus in combination with a live vaccine has major implications in catecholamine production and the microbiota of layer pullets. Additionally, unique correlations between changes in some neurochemicals and specific bacteria have been shown.

Pathogenic and non-pathogenic Escherichia coli colonization and host inflammatory response in a defined microbiota mouse model.

Most Escherichia coli strains in the human intestine are harmless. However, enterohemorrhagic Ecoli (EHEC) is a foodborne pathogen that causes intestinal disease in humans. Conventionally reared (CONV) mice are inconsistent models for human infections with EHEC because they are often resistant to Ecoli colonization, in part due to their gastrointestinal (GI) microbiota. Although antibiotic manipulation of the mouse microbiota has been a common means to overcome colonization resistance, these models have limitations. Currently, there are no licensed treatments for clinical EHEC infections and, thus, new tools to study EHEC colonization need to be developed. Here, we used a defined microbiota mouse model, consisting of the altered Schaedler flora (ASF), to characterize intestinal colonization and compare host responses following colonization with EHEC strain 278F2 or non-pathogenic Ecoli strain MG1655. Significantly higher (P<0.05) levels of both strains were found in feces and cecal and colonic contents of C3H/HeN ASF compared to C3H/HeN CONV mice. GI inflammation was significantly elevated (P<0.05) in the cecum of EHEC 278F2-colonized compared to E. coli MG1655-colonized C3H/HeN ASF mice. In addition, EHEC 278F2 differentially modulated inflammatory-associated genes in colonic tissue of C3H/HeN ASF mice compared to E. coli MG1655-colonized mice. This approach allowed for prolonged colonization of the murine GI tract by pathogenic and non-pathogenic Ecoli strains, and for evaluation of host inflammatory processes. Overall, this system can be used as a powerful tool for future studies to assess therapeutics, microbe-microbe interactions, and strategies for preventing EHEC infections.

Characterization of Spleen Transcriptome and Immunity Against Avian Colibacillosis After Immunization With Recombinant Attenuated Salmonella Vaccine Strains.

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in poultry. Vaccines targeting APEC in chickens have been partially successful, but many lack heterologous protection. Recombinant attenuated Salmonella vaccine (RASV) strains can induce broad immunity against Salmonella and be modified to deliver E. coli antigens. Along with vaccine characteristics, understanding the host response is crucial for developing improved vaccines. The objectives of this study were to evaluate host responses to vaccination with an RASV producing E. coli common pilus (ECP) and assess protection against APEC infection in chickens. Four-day-old White Leghorn chickens were unvaccinated or orally vaccinated and boosted 2 weeks later with RASV χ8025(pYA3337), RASV χ8025(pYA4428) carrying ecp operon genes, or a combination of χ8025(pYA3337) and χ8025(pYA4428) (Combo). To assess host responses, serum IgY and intestinal IgA antibody titers were measured, and spleen samples (n = 4/group) were collected from unvaccinated and Combo vaccinated 4-week-old chickens for RNA-seq. Vaccine protection potential against Salmonella and APEC was evaluated in vitro using bacterial inhibition assays. Five-week-old chickens were challenged via air sac with either an APEC O2 or O78 strain. E. coli was enumerated from internal organs, and gross colibacillosis lesions were scored at necropsy. RASV immunized chickens elicited anti-E. coli antibodies. The spleen transcriptome revealed that 93% (89/96) of differentially expressed genes (DEG) were more highly expressed in Combo vaccinated compared to unvaccinated chickens, with signal as the most significantly impacted category. RNA-seq analysis also revealed altered cellular and metabolic processes, response to stimulus after vaccination, and immune system processes. Six DEG including genes linked to transcription regulation, actin cytoskeleton, and signaling were highly positively correlated with antibody levels. Samples from RASV immunized chickens showed protection potential against Salmonella strains using in vitro assays, but a variable response was found for APEC strains. After APEC challenges, significant differences were not detected for bacterial loads or gross lesions scores, but χ8025(pYA3337) immunized and χ8025(pYA4428) immunized chickens had significantly fewer number of APEC-O2-positive samples than unvaccinated chickens. This study shows that RASVs can prime the immune system for APEC infection, and is a first step toward developing improved therapeutics for APEC infections in chickens.

Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.

Detection, Prevalence, and Pathogenicity of Non-O157 Shiga Toxin-Producing Escherichia coli from Cattle Hides and Carcasses.

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli (STEC) and harbor these bacteria in the intestinal tract. The prevalence, concentration, and STEC serogroup isolated in cattle varies between individuals. Hide removal at slaughter serves as a major point of carcass contamination and ultimately beef products. Certain STEC serogroups, such as O26, O45, O103, O111, O121, O145, and O157, containing the intestinal adherence factor intimin, pose a large economic burden to food producers because of testing and recalls. Human infection with STEC can cause illnesses ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and is commonly acquired through ingestion of contaminated foods, often beef products. Previously, most studies focused on O157 STEC, but there is growing recognition of the importance of non-O157 STEC serogroups. This review summarizes detection methods, prevalence, and methods for prediction of pathogenicity of non-O157 STEC from cattle hides and carcasses. A synthesis of procedures is outlined for general non-O157 STEC and targeted detection of specific STEC serogroups. Standardization of sample collection and processing procedures would allow for more robust comparisons among studies. Presence of non-O157 STEC isolated from cattle hides and carcasses and specific factors, such as point of sample collection and season, are summarized. Also, factors that might influence STEC survival on these surfaces, such as the microbial population on hides and microbial adherence genes, are raised as topics for future investigation. Finally, this review gives an overview on studies that have used genetic and cell-based methods to identify specific phenotypes of non-O157 STEC strains isolated from cattle to assess their risk to human health.