Catheterization of Intestinal Loops in Ruminants

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the outside world. As such, the intestine plays a pivotal role in immunosurveillance and protection from enteric pathogens. Investigating intestinal physiology and immunology commonly employs 'intestinal loops' as an experimental model. The majority of these loop models are non-recovery surgical procedures that study short-term (<24 hr) changes in the intestine (1-3). We previously created a recovery intestinal loop model to specifically measure long-term (<6 mo) immunological changes in the intestine of sheep following exposure to vaccines, adjuvants, and viruses (4). This procedure localized treatments to a specific 'loop', allowing us to sample this area of the intestine. A significant drawback of this method is the single window of opportunity to administer treatments (i.e. at the time of surgery). Furthermore, samples of both the intestinal mucosa and luminal contents can only be taken at the termination of the project. Other salient limitations of the above model are that the surgical manipulation and requisite post-operative measures (e.g. administration of antibiotics and analgesics) can directly affect the treatment itself and/or alter immune function, thereby confounding results. Therefore, we modified our intestinal loop model by inserting long-term catheters into the loops. Sheep recover fully from the procedure, and are unaffected by the exteriorized catheters. Notably, the establishment of catheters in loops allows us to introduce multiple treatments over an extended interval, following recovery from surgery and clearance of drugs administered during surgery and the post-operative period

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

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 β-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

<i>Campylobacter jejuni</i> Colonization Is Associated with a Dysbiosis in the Cecal Microbiota of Mice in the Absence of Prominent Inflammation

<div><p>Background</p><p><i>Campylobacter jejuni</i> causes enterocolitis in humans, but does not incite disease in asymptomatic carrier animals. To survive in the intestine, <i>C. jejuni</i> must successfully compete with the microbiota and overcome the host immune defense. <i>Campylobacter jejuni</i> colonization success varies considerably amongst individual mice, and we examined the degree to which the intestinal microbiota was affected in mice (i.e. a model carrier animal) colonized by <i>C. jejuni</i> at high relative to low densities.</p> <p>Methods</p><p>Mice were inoculated with <i>C. jejuni</i> or buffer, and pathogen shedding and intestinal colonization were measured. Histopathologic scoring and quantification of mRNA expression for α-defensins, toll-like receptors, and cytokine genes were conducted. Mucosa-associated bacterial communities were characterized by two approaches: multiplexed barcoded pyrosequencing and terminal restriction fragment length polymorphism analysis.</p> <p>Results</p><p>Two <i>C. jejuni</i> treatments were established based on the degree of cecal and colonic colonization; <i>C. jejuni</i> Group A animals were colonized at high cell densities, and <i>C. jejuni</i> Group B animals were colonized at lower cell densities. Histological examination of cecal and colonic tissues indicated that <i>C. jejuni</i> did not incite visible pathologic changes. Although there was no significant difference among treatments in expression of mRNA for α-defensins, toll-like receptors, or cytokine genes, a trend for increased expression of toll-like receptors and cytokine genes was observed for <i>C. jejuni</i> Group A. The results of the two methods to characterize bacterial communities indicated that the composition of the cecal microbiota of <i>C. jejuni</i> Group A mice differed significantly from <i>C. jejuni</i> Group B and Control mice. This difference was due to a reduction in load, diversity and richness of bacteria associated with the cecal mucosa of <i>C. jejuni</i> Group A mice.</p> <p>Conclusions</p><p>High density colonization by <i>C. jejuni</i> is associated with a dysbiosis in the cecal microbiota independent of prominent inflammation.</p> </div

Richness and diversity of bacterial communities.

<p>(A) Richness; (B) Chao1 diversity; (C) Shannon diversity; and (D) phylogenetic diversity. Treatments are: (A) <i>C</i>. <i>jejuni</i> Group A (8.8 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); (B) <i>C</i>. <i>jejuni</i> Group B (6.4 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); and (C) control (not inoculated with <i>C</i>. <i>jejuni</i>). Vertical lines associated with markers are standard error of the means (n=6).</p

Messenger RNA expression of toll-like receptors.

<p>Relative mRNA expression of toll-like receptors (TLR) in cecal tissue where: (A) TLR2; (B) TLR4; (C) TLR5; and (D) TLR9. Treatments are: (A) <i>C</i>. <i>jejuni</i> Group A (8.8 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); (B) <i>C</i>. <i>jejuni</i> Group B (6.4 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); and (C) control (not inoculated with <i>C</i>. <i>jejuni</i>). Vertical lines associated with histogram bars are standard error of the means (n=6).</p

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 IL1β, 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.Arts and Sciences, Irving K. Barber School of (Okanagan)Non UBCBiology, Department of (Okanagan)Chemistry, Department of (Okanagan)ReviewedFacult

Prevalence of <i>Campylobacter jejuni</i> sequences.

<p>Prevalence of sequences (%) identified as <i>C</i>. <i>jejuni</i> by treatment. Treatments are: (A) <i>C</i>. <i>jejuni</i> Group A (8.8 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); (B) <i>C</i>. <i>jejuni</i> Group B (6.4 log₁₀ copy number of <i>C</i>. <i>jejuni</i> g^-1 of cecal tissue); and (C) control (not inoculated with <i>C</i>. <i>jejuni</i>). Vertical lines associated with histogram bars are standard error of the means (n=6). Number associated with histogram bars are the mean number of sequences (± standard error of the means) that were identified as <i>C</i>. <i>jejuni</i> by treatment.</p