Commensal Escherichia coli Strains of Bovine Origin Competitively Mitigated Escherichia coli O157:H7 in a Gnotobiotic Murine Intestinal Colonization Model with or without Physiological Stress.

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Currently, there are no effective methods of eliminating this important zoonotic pathogen from cattle, and colonization resistance in relation to EHEC O157:H7 in cattle is poorly understood. We developed a gnotobiotic EHEC O157:H7 murine model to examine aspects of the cattle pathogen-microbiota interaction, and to investigate competitive suppression of EHEC O157:H7 by 18 phylogenetically distinct commensal E. coli strains of bovine origin. As stress has been suggested to influence enteric colonization by EHEC O157:H7 in cattle, corticosterone administration (±) to incite a physiological stress response was included as an experimental variable. Colonization of the intestinal tract (IT) of mice by the bovine EHEC O157:H7 strain, FRIK-2001, mimicked characteristics of bovine IT colonization. In this regard, FRIK-2001 successfully colonized the IT and temporally incited minimal impacts on the host relative to other EHEC O157:H7 strains, including on the renal metabolome. The presence of the commensal E. coli strains decreased EHEC O157:H7 densities in the cecum, proximal colon, and distal colon. Moreover, histopathologic changes and inflammation markers were reduced in the distal colon of mice inoculated with commensal E. coli strains (both propagated separately and communally). Although stress induction affected the behavior of mice, it did not influence EHEC O157:H7 densities or disease. These findings support the use of a gnotobiotic murine model of enteric bovine EHEC O157:H7 colonization to better understand pathogen-host-microbiota interactions toward the development of effective on-farm mitigations for EHEC O157:H7 in cattle, including the identification of bacteria capable of competitively colonizing the IT.

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.

Feather pulp: a novel substrate useful for proton nuclear magnetic resonance spectroscopy metabolomics and biomarker discovery.

Noninvasive biomarkers of stress that are predictive of poultry health are needed. Feather pulp is highly vascularized and represents a potential source of biomarkers that has not been extensively explored. We investigated the feasibility and use of feather pulp for novel biomarker discovery using 1H-Nuclear Magnetic Resonance Spectroscopy (NMR)-based metabolomics. To this end, high quality NMR metabolomic spectra were obtained from chicken feather pulp extracted using either ultrafiltration (UF) or Bligh-Dyer methanol-chloroform (BD) methods. In total, 121 and 160 metabolites were identified using the UF and BD extraction methods, respectively, with 71 of these common to both methods. The metabolome of feather pulp differed in broiler breeders that were 1-, 23-, and 45-wk-of-age. Moreover, feather pulp was more difficult to obtain from older birds, indicating that age must be considered when targeting feather pulp as a source of biomarkers. The metabolomic profile of feather pulp obtained from 12-day-old broilers administered corticosterone differed from control birds, indicating that the metabolome of feather pulp was sensitive to induced physiological stress. A comparative examination of feather pulp and serum in broilers revealed that the feather pulp metabolome differed from that of serum but provided more information. The study findings show that metabolite biomarkers in chicken feather pulp 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.

Fecal 1H-NMR Metabolomics: A Comparison of Sample Preparation Methods for NMR and Novel in Silico Baseline Correction.

Analysis of enteric microbiota function indirectly through the fecal metabolome has the potential to be an informative diagnostic tool. However, metabolomic analysis of feces is hampered by high concentrations of macromolecules such as proteins, fats, and fiber in samples. Three methods-ultrafiltration (UF), Bligh-Dyer (BD), and no extraction (samples added directly to buffer, vortexed, and centrifuged)-were tested on multiple rat (n = 10) and chicken (n = 8) fecal samples to ascertain whether the methods worked equally well across species and individuals. An in silico baseline correction method was evaluated to determine if an algorithm could produce spectra similar to those obtained via UF. For both rat and chicken feces, UF removed all macromolecules and produced no baseline distortion among samples. By contrast, the BD and no extraction methods did not remove all the macromolecules and produced baseline distortions. The application of in silico baseline correction produced spectra comparable to UF spectra. In the case of no extraction, more intense peaks were produced. This suggests that baseline correction may be a cost-effective method for metabolomic analyses of fecal samples and an alternative to UF. UF was the most versatile and efficient extraction method; however, BD and no extraction followed by baseline correction can produce comparable results.

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.

The absence of murine cathelicidin-related antimicrobial peptide impacts host responses enhancing Salmonella enterica serovar Typhimurium infection.

BACKGROUND: Cathelicidins are a class of antimicrobial peptide, and the murine cathelicidin-related antimicrobial peptide (mCRAMP) has been demonstrated in vitro to impair Salmonella enterica serovar Typhimurium proliferation. However, the impact of mCRAMP on host responses and the microbiota following S. Typhimurium infection has not been determined. In this study mCRAMP-/- and mCRAMP+/+ mice (± streptomycin) were orally inoculated with S. enterica serovar Typhimurium DT104 (SA +), and impacts on the host and enteric bacterial communities were temporally evaluated. RESULTS: Higher densities of the pathogen were observed in cecal digesta and associated with mucosa in SA+/mCRAMP-/- mice that were pretreated (ST+) and not pretreated (ST-) with streptomycin at 24 h post-inoculation (hpi). Both SA+/ST+/mCRAMP-/- and SA+/ST-/mCRAMP-/- mice were more susceptible to infection exhibiting greater histopathologic changes (e.g. epithelial injury, leukocyte infiltration, goblet cell loss) at 48 hpi. Correspondingly, immune responses in SA+/ST+/mCRAMP-/- and SA+/ST-/mCRAMP-/- mice were affected (e.g. Ifnγ, Kc, Inos, Il1ß, RegIIIγ). Systemic dissemination of the pathogen was characterized by metabolomics, and the liver metabolome was affected to a greater degree in SA+/ST+/mCRAMP-/- and SA+/ST-/mCRAMP-/- mice (e.g. taurine, cadaverine). Treatment-specific changes to the structure of the enteric microbiota were associated with infection and mCRAMP deficiency, with a higher abundance of Enterobacteriaceae and Veillonellaceae observed in infected null mice. The microbiota of mice that were administered the antibiotic and infected with Salmonella was dominated by Proteobacteria. CONCLUSION: The study findings showed that the absence of mCRAMP modulated both host responses and the enteric microbiota enhancing local and systemic infection by Salmonella Typhimurium.

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.

Systematic individualized narrative language intervention on the personal narratives of children with autism.

PURPOSE: The purpose of this study was to investigate the effect of an individualized, systematic language intervention on the personal narratives of children with autism. METHOD: A single-subject, multiple-baseline design across participants and behaviors was used to examine the effect of the intervention on language features of personal narratives. Three 6- to 8-year-old boys with autism participated in 12 individual intervention sessions that targeted 2-3 story grammar elements (e.g., problem, plan) and 3-4 linguistic complexity elements (e.g., causal subordination, adverbs) selected from each participant's baseline performance. Intervention involved repeated retellings of customized model narratives and the generation of personal narratives with a systematic reduction of visual and verbal scaffolding. Independent personal narratives generated at the end of each baseline, intervention, and maintenance session were analyzed for presence and sophistication of targeted features. RESULTS: Graphical and statistical results showed immediate improvement in targeted language features as a function of intervention. There was mixed evidence of maintenance 2 and 7 weeks after intervention. CONCLUSION: Children with autism can benefit from an individualized, systematic intervention targeting specific narrative language features. Greater intensity of intervention may be needed to gain enduring effects for some language features.