Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Comparative Analysis of the Temporal Impacts of Corticosterone and Simulated Production Stressors on the Metabolome of Broiler Chickens.

The impact of physiological stress on the metabolome of breast muscle, liver, kidney, and hippocampus was investigated in Ross 308 broiler chicks. Simulated on-farm stressors were compared to a corticosterone model of physiological stress. The three different stressors investigated were: (i) corticosterone at a dose of 15 mg/kg of feed; (ii) heat treatment of 36 °C and 40% RH for 8 h per day; and (iii) isolation for 1 h per day. Liver, kidney, breast muscle, and hippocampus samples were taken after 2, 4, 6, and 8 days of stress treatment, and subjected to untargeted 1H-nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis to provide insights on how stress can modulate metabolite profiles and biomarker discovery. Many of the metabolites that were significantly altered in tissues were amino acids, with glycine and alanine showing promise as candidate biomarkers of stress. Corticosterone was shown to significantly alter alanine, aspartate, and glutamate metabolism in the liver, breast, and hippocampus, while isolation altered the same pathways, but only in the kidneys and hippocampus. Isolation also significantly altered the glycine, serine, and threonine metabolism pathway in the liver and breast, while the same pathway was significantly altered by heat in the liver, kidneys, and hippocampus. The study's findings support corticosterone as a model of stress. Moreover, a number of potential metabolite biomarkers were identified in chicken tissues, which may allow producers to effectively monitor stress and to objectively develop and evaluate on-farm mitigations, including practices that reduce stress and enhance bird health.

Microbiota Transplantation in Day-Old Broiler Chickens Ameliorates Necrotic Enteritis via Modulation of the Intestinal Microbiota and Host Immune Responses.

A microbiota transplant (MT) originating from mature adult chicken ceca and propagated in bioreactors was administered to day-old broiler chicks to ascertain the degree to which, and how, the MT affects Clostridium perfringens (Cp)-incited necrotic enteritis (NE). Using a stress predisposition model of NE, birds administered the MT and challenged with Cp showed fewer necrotic lesions, and exhibited a substantially higher α- and ß-diversity of bacteria in their jejunum and ceca. Birds challenged with Cp and not administered the MT showed decreased Lactobacillus and increased Clostridium sensu strico 1 in the jejunum. In ceca, Megamonas, a genus containing butyrate-producing bacteria, was only present in birds administered the MT, and densities of this genus were increased in birds challenged with Cp. Metabolite profiles in cecal digesta were altered in birds administered the MT and challenged with the pathogen; 59 metabolites were differentially abundant following MT treatment, and the relative levels of short chain fatty acids, butyrate, valerate, and propionate, were decreased in birds with NE. Birds administered the MT and challenged with Cp showed evidence of enhanced restoration of intestinal barrier functions, including elevated mRNA of MUC2B, MUC13, and TJP1. Likewise, birds administered the MT exhibited higher mRNA of IL2, IL17A, and IL22 at 2-days post-inoculation with Cp, indicating that these birds were better immunologically equipped to respond to pathogen challenge. Collectively, study findings demonstrated that administering a MT containing a diverse mixture of microorganisms to day-old birds ameliorated NE in broilers by increasing bacterial diversity and promoting positive immune responses.

A stress-induced model of acute necrotic enteritis in broiler chickens using dietary corticosterone administration.

Mounting evidence indicates that stress can predispose chickens to disease. The objective of the current study was to develop a method that utilized physiological stress to predispose Ross 308 broiler chickens to acute necrotic enteritis (NE). Stress was mediated through the administration of the stress hormone, corticosterone. At 11 d posthatch (p.h.), corticosterone (20 mg kg-1) administration commenced. At 12 and 13 d p.h., birds were orally inoculated with a virulent strain of Clostridium perfringens, and at 14 d p.h., birds were euthanized. Birds administered corticosterone exhibited decreased weight gain, and birds co-challenged with C. perfringens and corticosterone were affected to a higher degree. Necrotic lesions were present in birds inoculated with C. perfringens (33%), but a substantially higher prevalence of birds treated with C. perfringens and corticosterone in combination exhibited lesions (100%). Clostridium perfringens densities were correlated with necrotic lesion and histopathologic scores. Both C. perfringens and corticosterone challenge altered mRNA immune responses in the small intestine. In this regard, birds infected with the pathogen showed higher relative mRNA concentrations of toll-like receptor 2A (TLR2A), transforming growth factor beta 2 (TGFß2), and inducible nitric oxide synthase (INOS). Birds co-challenged with C. perfringens and corticosterone showed hindered TLR2A mRNA expression. A reduction in TLR2A responses mediated by corticosterone administration suggests that the glucocorticoid suppresses immune stimulation in jejunal mucosa, which may be the underlying cause for the increased prevalence and intensity of disease observed in corticosterone treated birds. Overall, the corticosterone stress model resulted in levels of NE comparable to other models of NE that currently exist without the use of a co-infection agent. This model may facilitate the exploration of mechanisms of stress-induced NE, and the development of effective alternatives to antibiotics.

Corticosterone-Mediated Physiological Stress Alters Liver, Kidney, and Breast Muscle Metabolomic Profiles in Chickens.

The impact of physiological stress on the metabolomes of liver, kidney, and breast muscle was investigated in chickens. To incite a stress response, birds were continuously administered corticosterone (CORT) in their drinking water at three doses (0, 10, and 30 mg L-1), and they were sampled 1, 5, and 12 days after the start of the CORT administration. To solubilize CORT, it was first dissolved in ethanol and then added to water. The administration of ethanol alone significantly altered branched chain amino acid metabolism in both the liver and the kidney, and amino acid and nitrogen metabolism in breast muscle. CORT significantly altered sugar and amino acid metabolism in all three tissues, but to a much greater degree than ethanol alone. In this regard, CORT administration significantly altered 11, 46, and 14 unique metabolites in liver, kidney, and breast muscle, respectively. Many of the metabolites that were affected by CORT administration, such as mannose and glucose, were previously linked to increases in glycosylation and gluconeogenesis in chickens under conditions of production stress. Moreover, several of these metabolites, such as dimethylglycine, galactose, and carnosine were also previously linked to reduced quality meat. In summary, the administration of CORT in chickens significantly modulated host metabolism. Moreover, results indicated that energy potentials are diverted from muscle anabolism to muscle catabolism and gluconeogenesis during periods of stress.

Physiological Stress Mediated by Corticosterone Administration Alters Intestinal Bacterial Communities and Increases the Relative Abundance of Clostridium perfringens in the Small Intestine of Chickens.

A model of physiological stress mediated by the administration of corticosterone (CORT) was used to investigate the impact of stress on the intestinal microbiota of chickens. Birds were administered CORT in their drinking water at 0, 10 (low dose CORT; LDC), and 30 (high dose CORT; HDC) mg/L. Digesta from the small intestine and ceca were examined after 1, 5, and 12 days post-initiation of CORT administration by 16S rRNA gene sequencing. A decrease in phylogenetic diversity and altered composition of bacteria were observed for HDC in the small intestine. Analysis by ANOVA-Like Differential Expression 2 (ALDEx2) showed that densities of Clostridium sensu stricto 1 bacteria were increased in the small intestine for LDC and HDC. Quantitative PCR confirmed that CORT administration increased densities of Clostridium perfringens in the small intestine, but only HDC was associated with increased densities of the bacterium in ceca. Predictive functional analysis by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) showed pathways of carbohydrate metabolism to be enriched with CORT, and amino acid synthesis to be enriched in control birds in the small intestine. In conclusion, physiological stress mediated by CORT modulated bacterial communities in the small intestine and increased densities of C. perfringens. This implicates stress as an important mediator of this important enteric pathogen in poultry.

Host responses to Clostridium perfringens challenge in a chicken model of chronic stress.

BACKGROUND: This study utilized a chicken model of chronic physiological stress mediated by corticosterone (CORT) administration to ascertain how various host metrics are altered upon challenge with Clostridium perfringens. Necrotic enteritis (NE) is a disease of the small intestine of chickens incited by C. perfringens, which can result in elevated morbidity and mortality. The objective of the current study was to investigate how physiological stress alters host responses and predisposes birds to subclinical NE. RESULTS: Birds administered CORT exhibited higher densities of C. perfringens in their intestine, and this corresponded to altered production of intestinal mucus. Characterization of mucus showed that C. perfringens treatment altered the relative abundance of five glycans. Birds inoculated with C. perfringens did not exhibit evidence of acute morbidity. However, histopathologic changes were observed in the small intestine of infected birds. Birds administered CORT showed altered gene expression of tight junction proteins (i.e. CLDN3 and CLDN5) and toll-like receptors (i.e. TLR2 and TLR15) in the small intestine. Moreover, birds administered CORT exhibited increased expression of IL2 and G-CSF in the spleen, and IL1ß, IL2, IL18, IFNγ, and IL6 in the thymus. Body weight gain was impaired only in birds that were administered CORT and challenged with C. perfringens. CONCLUSION: CORT administration modulated a number of host functions, which corresponded to increased densities of C. perfringens in the small intestine and weight gain impairment in chickens. Importantly, results implicate physiological stress as an important predisposing factor to NE, which emphasizes the importance of managing stress to optimize chicken health.

Application of culturomics to characterize diverse anaerobic bacteria from the gastrointestinal tract of broiler chickens in relation to environmental reservoirs.

Characterization of the microbiota of chickens is of current interest. The goals of the current study were to apply anaerobic isolation methods to comprehensively isolate and identify bacteria from the gastrointestinal tract of chickens and their environment. Bacterial communities within the drinking water were dominated by Escherichia, whereas communities in litter were more representative of the cecum. The crop and small intestine (jejunum and ileum) were dominated by Lactobacillus and Enterococcus spp., and the cecum was dominated by Proteus spp. The collection of bacteria isolated was dominated by Enterococcus spp., Escherichia/Shigella spp., Lactobacillus spp., and Proteus spp.; however, many rare taxa were observed. These included members of the Clostridiales and Clostridium spp., which were commonly isolated from the ileum and cecum. Bacteria isolated by enrichment and direct plating differed. The selective de Man-Rogosa-Sharpe agar was commonly associated with the isolation of Lactobacillus spp. and yielded the lowest diversity of all methods utilized. Increased diversity and frequency of Clostridium spp. was observed in enrichments of blood and mucus or by plating on Columbia agar supplemented with 10% blood and gentamicin. The bacteria isolated from this study provide source material for genomic and functional studies in chicken hosts.

Corticosterone-mediated physiological stress modulates hepatic lipid metabolism, metabolite profiles, and systemic responses in chickens.

The impact of physiological stress on lipid metabolism, the metabolome, and systemic responses was examined in chickens. To incite a stress response, birds were continuously administered corticosterone (CORT) in their drinking water at three doses (0 mg/L, 10 mg/L, and 30 mg/L), and they were sampled 1, 5, and 12 days after commencement of CORT administration. Corticosterone administration to birds differentially regulated lipogenesis genes (i.e. FAS, ACC, ME, and SREBF1), and histopathological examination indicated lipid deposition in hepatocytes. In addition, CORT affected water-soluble metabolite profiles in the liver, as well as in kidney tissue and breast muscle; thirteen unique metabolites were distinguished in CORT-treated birds and this was consistent with the dysregulation of lipid metabolism due to physiological stress. Acute phase responses (APRs) were also altered by CORT, and in particular, expression of SAA1 was decreased and expression of CP was increased. Furthermore, CORT administration caused lymphoid depletion in the bursa of Fabricius and elevated IL6 and TGFß2 mRNA expression after 5 and 12 days of CORT administration. Collectively, incitement of physiological stress via administration of CORT in chickens modulated host metabolism and systemic responses, which indicated that energy potentials are diverted from muscle anabolism during periods of stress.

Structural analysis of broiler chicken small intestinal mucin O-glycan modification by Clostridium perfringens.

Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome.

Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni.

Enrofloxacin is registered for therapeutic use in beef cattle to treat bovine respiratory disease in Canada. A murine model was used to experimentally examine the impact of therapeutic administration of enrofloxacin on fluoroquinolone resistance development in Campylobacter jejuni. Administration of enrofloxacin to mice via subcutaneous injection or per os routes resulted in equivalent levels of bioactive enrofloxacin within the intestine, but bioactivity was short-lived (<48 h after cessation). Enrofloxacin administration did not affect densities of total bacteria, Firmicutes, or Bacteroidetes in digesta and had modest impacts on densities of Enterobacteriaceae. All mice inoculated with C. jejuni NCTC 11168 became persistently colonized by the bacterium. Enrofloxacin reduced C. jejuni cell densities within the cecal and colonic digesta for all treatments, and densities shed in feces as a function of antibiotic duration. None of the C. jejuni isolates recovered from mice after administration of enrofloxacin (n = 260) developed resistance to ciprofloxacin regardless of method or duration of administration. Furthermore, only modest shifts in the minimum inhibitory concentration of the isolates by treatment were noted. The study findings indicate that the risk posed by short-term subcutaneous administration of enrofloxacin for the development of fluoroquinolone resistance in mammals is low.

Antimicrobial growth promoters modulate host responses in mice with a defined intestinal microbiota.

Antibiotics can promote growth in livestock (antimicrobial growth promoters, AGPs), however lack of knowledge regarding mechanisms has hampered the development of effective non-antibiotic alternatives. Antibiotics affect eukaryotic cells at therapeutic concentrations, yet effects of AGPs on host physiology are relatively understudied, partially due to the complexity of host-microorganism interactions within the gastrointestinal tract. To determine the direct effects of AGPs on the host, we generated Altered Schaedler Flora (ASF) mice, and administered chlortetracycline (CTC) and tylosin phosphate (TYL) in feed. Mice were challenged with Citrobacter rodentium to determine how AGPs alter host responses to physiological stress. Although CTC and TYL had inconsistent effects on the ASF taxa, AGPs protected mice from weight loss following C. rodentium inoculation. Mice treated with either CTC or TYL had lower expression of ßd1 and Il17a in the intestine and had a robust induction of Il17a and Il10. Furthermore, AGP administration resulted in a lower hepatic expression of acute phase proteins (Saa1, Hp, and Cp) in liver tissue, and ameliorated C. rodentium-induced reductions in the expression of genes involved in lipogenesis (Hmgcl and Fabp1). Collectively, this indicates that AGPs directly affect host physiology, and highlights important considerations in the development of non-antibiotic alternatives.