Cold stress initiates catecholaminergic and serotonergic responses in the chicken gut that are associated with functional shifts in the microbiome.

Climate change is one of the most significant challenges facing the sustainability of global poultry production. Stress resulting from extreme temperature swings, including cold snaps, is a major concern for food production birds. Despite being well-documented in mammals, the effect of environmental stress on enteric neurophysiology and concomitant impact on host-microbiome interactions remains poorly understood in birds. As early life stressors may imprint long-term adaptive changes in the host, the present study sought to determine whether cold temperature stress, a prominent form of early life stress in chickens, elicits changes in enteric stress-related neurochemical concentrations that coincide with compositional and functional changes in the microbiome that persist into the later life of the bird. Chicks were, or were not, subjected to cold ambient temperature stress during the first week post-hatch and then remained at normal temperature for the remainder of the study. 16S rRNA gene and shallow shotgun metagenomic analyses demonstrated taxonomic and functional divergence between the cecal microbiomes of control and cold stressed chickens that persisted for weeks following cessation of the stressor. Enteric concentrations of serotonin, norepinephrine, and other monoamine neurochemicals were elevated (P < 0.05) in both cecal tissue and luminal content of cold stressed chickens. Significant (P < 0.05) associations were identified between cecal neurochemical concentrations and microbial taxa, suggesting host enteric neurochemical responses to environmental stress may shape the cecal microbiome. These findings demonstrate for the first time that early life exposure to environmental temperature stress can change the developmental trajectory of both the chicken cecal microbiome and host neuroendocrine enteric physiology. As many neurochemicals serve as interkingdom signaling molecules, the relationships identified here could be exploited to control the impact of climate change-driven stress on avian enteric host-microbe interactions.

Catecholamine concentrations in duck eggs are impacted by hen exposure to heat stress.

Rapid "fight-or-flight" responses to stress are largely orchestrated by the catecholamines. Moreover, catecholamines and catecholamine precursors are widely recognized to act as interkingdom signaling molecules among host and microbiota, as well as to serve as chemotactic signals for bacterial foodborne pathogens. While albumen and yolk concentrations of glucocorticoids have received extensive attention as non-invasive indicators of hen response to stress, little is known regarding the impact of the hen's stress response on in ovo catecholamine and catecholamine precursor concentrations. The aim of the present study was to determine norepinephrine and L-dopa concentrations in albumen and yolk of eggs laid by hens maintained under normal or heat stress conditions. Norepinephrine and L-dopa concentrations were also measured in oviductal tissue. Breeder ducks (∼35 weeks/age) were kept under normal (22°C) conditions or subjected to cyclical heat stress (35°C day/29.5°C night) for 3 weeks. Eggs (n = 12 per timepoint/group) were collected on a weekly basis. Hens were sacrificed at baseline or after 3 weeks of heat stress for oviductal tissue collection. Albumen, yolk, and oviduct concentrations of norepinephrine and L-dopa were determined using ultra high-performance liquid chromatography with electrochemical detection. Norepinephrine and L-dopa were detected in oviductal tissue as well as egg albumen and yolk. Norepinephrine concentrations were elevated (p < 0.05) in the yolk of eggs laid by the heat stress group compared to those of the control group. Norepinephrine concentrations in albumen were elevated (p < 0.05) in the heat stress group compared to control group at week 2. L-dopa concentrations were not significantly affected (p > 0.05) by heat stress in albumen, yolk, or oviductal tissue. Together, the present study provides the first evidence of the stress neurohormone, norepinephrine, in duck eggs and identifies that hen exposure to heat stress can affect in ovo norepinephrine concentrations. These data highlight the potential utility of in ovo catecholamine concentrations as non-invasive measures of the hen's response to stress, as well as warrants future research into whether hen deposition of stress-related neurochemicals into the egg could serve as a chemotactic signal in the vertical transmission of foodborne pathogens.

Influence of a sodium-saccharin sweetener on the rumen content and rumen epithelium microbiota in dairy cattle during heat stress.

The effect of a saccharin-based artificial sweetener was tested on animal performance measures and on the microbial communities associated with the rumen content and with the rumen epithelium during heat stress. Ten cannulated Holstein-Friesian milking dairy cattle were supplemented with 2 g of saccharin-based sweetener per day, top-dressed into individual feeders for a 7-day adaptation period followed by a 14-day heat stress period. A control group of ten additional cows subjected to the same environmental conditions but not supplemented with sweetener were included for comparison. 16S rRNA gene amplicon sequencing was performed on rumen content and rumen epithelium samples from all animals, and comparisons of rumen content microbiota and rumen epithelial microbiota were made between supplemented and control populations. Supplementation of the saccharin-based sweetener did not affect the rumen content microbiota, but differences in the rumen epithelial microbiota beta-diversity (PERMANOVA, P = 0.003, R2 = 0.12) and alpha-diversity (Chao species richness, P = 0.06 and Shannon diversity, P = 0.034) were detected between the supplemented and control experimental groups. Despite the changes detected in the microbial community, animal performance metrics including feed intake, milk yield, and short-chain fatty acid (acetic, propionic, and butyric acid) concentrations were not different between experimental groups. Thus, under the conditions applied, supplementation with a saccharin-based sweetener does not appear to affect animal performance under heat stress. Additionally, we detected differences in the rumen epithelial microbiota due to heat stress when comparing initial, prestressed microbial communities to the communities after heat stress. Importantly, the changes occurring in the rumen epithelial microbiota may have implications on barrier integrity, oxygen scavenging, and urease activity. This research adds insight into the impact of saccharin-based sweeteners on the rumen microbiota and the responsivity of the rumen epithelial microbiota to different stimuli, providing novel hypotheses for future research.

Mitigating the effects of heat stress is becoming more and more important with global increases in temperatures. Heat stress negatively affects livestock health and performance. One way to mitigate the effects of heat stress on livestock is to increase feed intake during stress conditions by enhancing palatability of the feed by adding artificial sweeteners. In this study, we investigated whether supplementation of the diet with a saccharin-based sweetener affected dairy cattle performance and the rumen microbial communities during heat stress. We show that supplementation with a saccharin-based artificial sweetener did not affect the performance of the dairy cattle during heat stress. However, the sweetener resulted in changes in the rumen microbial communities, particularly of the microbial communities attached to the rumen wall. These changes in the rumen wall microbial communities could potentially have implications for the host animal, for example in the integrity of the rumen wall barrier function. Future research will be needed to better understand the role of artificial sweeteners in potentially mitigating stress conditions for livestock and to understand their potential effects on microbial communities.

Distinct Cecal and Fecal Microbiome Responses to Stress Are Accompanied by Sex- and Diet-Dependent Changes in Behavior and Gut Serotonin.

Although diet- and stress-induced perturbations in the microbiome (biotic and abiotic factors) associate with changes in host behavior via the microbiota-gut-brain axis, few mechanisms have been identified. The identification of causative pathways by which the microbiome influences host behavior therefore would benefit from the application of evidence-based conceptual frameworks. One such causal framework is microbial endocrinology which is the study of neuroendocrine axes as avenues of bi-directional neurochemical-based host-microbe crosstalk. As such, we investigated the relationship between diet- and stress-induced alterations in behavior, regional gut serotonergic response, and concomitant changes in the cecal and fecal bacterial populations of male and female mice. Our results demonstrate that sex is a dominant factor in determining compositional changes in the gut microbiome in response to stress and diet modifications. Intestinal serotonergic responses to stress were observed in both sexes but dietary modifications uniquely affected region-specific changes in males and females. Likewise, behavioral alterations diverged between male and female mice. Together, these results demonstrate distinct sex-dependent relationships between cecal and fecal bacterial taxa and behavioral- and serotonergic-responses to stress and diet. The present study demonstrates the importance of including both male and female sexes in the examination of the microbiota-gut-brain axis. As different microbial taxa were identified to associate with the behavioral and gut serotonergic responses of male and female mice, certain bacterial species may hold sex-dependent functional relevance for the host. Future investigations seeking to develop microbiome-based strategies to afford host stress resilience should include sex-based differences in the microbiome.

A neurochemical biogeography of the broiler chicken intestinal tract.

The study of neurochemical-based interkingdom signaling and its impact on host-microbe interaction is called microbial endocrinology. Neurochemicals play a recognized role in determining bacterial colonization and interaction with the gut epithelium. While much attention has been devoted to the determination of neurochemical concentrations in the mammalian gut to better understand tissue and region-specific microbial endocrinology-based mechanisms of host-microbe interaction, little is known regarding the biogeography of neurochemicals in the avian gut. Greater resolution of avian gut neurochemical concentrations is needed especially as recent microbial endocrinology-based investigations into bacterial foodborne pathogen colonization of the chicken gut have demonstrated neurochemicals to affect Campylobacter jejuni and Salmonella spp. in vivo and in vitro. The aim of the present study was to determine the concentrations of stress-related neurochemicals in the tissue and luminal content of the duodenum, jejunum, ileum, cecum, and colon of the broiler intestinal tract, and to investigate if this biogeography changes with age of the bird. While all neurochemicals measured were detected in the intestinal tract, many displayed differences in regional concentrations. Whereas the catecholamine norepinephrine was detected in each region of the intestinal tract, epinephrine was present only in the cecum and colon. Likewise, dopamine, and its metabolite 3,4-dihydroxyphenylacetic acid were found in the greatest quantities in the cecum and colon. Serotonin and histamine were identified in each gut region. Region-specific age-related changes were observed (P < 0.05) for serotonin, its metabolite 5-hydroxyindole acetic acid as well as for histamine. Several neurochemicals, including norepinephrine, were found in the contents of each gut region. Epinephrine was not detected in the gut content of any region. Salsolinol, a microbial-produced neuroactive compound was detected in the gut content but not in tissue. Together, our data establish a neurochemical biogeography of the broiler chicken intestinal tract. By providing researchers with a region-by-region map of in vivo gut neurochemical concentrations of a modern broiler chicken breed, this neurochemical map is expected to inform future investigations that seek to utilize avian enteric neurochemistry.

Voluntary binge-patterned alcohol drinking and sex-specific influences on monoamine-related neurochemical signatures in the mouse gut and brain.

BACKGROUND: Altered monoamine (i.e., serotonin, dopamine, and norepinephrine) activity following episodes of alcohol abuse plays key roles not only in the motivation to ingest ethanol, but also physiological dysfunction related to its misuse. Although monoamine activity is essential for physiological processes that require coordinated communication across the gut-brain axis (GBA), relatively little is known about how alcohol misuse may affect monoamine levels across the GBA. Therefore, we evaluated monoamine activity across the mouse gut and brain following episodes of binge-patterned ethanol drinking. METHODS: Monoamine and select metabolite neurochemical concentrations were analyzed by ultra-high-performance liquid chromatography in gut and brain regions of female and male C57BL/6J mice following "Drinking in the Dark" (DID), a binge-patterned ethanol ingestion paradigm. RESULTS: First, we found that alcohol access had an overall small effect on gut monoamine-related neurochemical concentrations, primarily influencing dopamine activity. Second, neurochemical patterns between the small intestine and the striatum were correlated, adding to recent evidence of modulatory activity between these areas. Third, although alcohol access robustly influenced activity in brain areas in the mesolimbic dopamine system, binge exposure also influenced monoaminergic activity in the hypothalamic region. Finally, sex differences were observed in the concentrations of neurochemicals within the gut, which was particularly pronounced in the small intestine. CONCLUSION: Together, these data provide insights into the influence of alcohol abuse and biological sex on monoamine-related neurochemical changes across the GBA, which could have important implications for GBA function and dysfunction.

Serotonin modulates Campylobacter jejuni physiology and invitro interaction with the gut epithelium.

Microbial endocrinology, which is the study of neurochemical-based host-microbe interaction, has demonstrated that neurochemicals affect bacterial pathogenicity. A variety of neurochemicals, including norepinephrine, were shown to enhance intestinal epithelial colonization by Campylobacter jejuni. Yet, little is known whether serotonin, an abundant neurochemical produced in the gut, affects the physiology of C. jejuni and its interaction with the host gut epithelium. Considering the avian gut produces serotonin and serves as a major reservoir of C. jejuni, we sought to investigate whether serotonin can affect C. jejuni physiology and gut epithelial colonization in vitro. We first determined the biogeographical distribution of serotonin concentrations in the serosa, mucosa, as well as the luminal contents of the broiler chicken ileum, cecum, and colon. Serotonin concentrations were greater (P < 0.05) in the mucosa and serosa compared to the luminal content in each gut region examined. Among the ileum, colon, and cecum, the colon was found to contain the greatest concentrations of serotonin. We then investigated whether serotonin may effect changes in C. jejuni growth and motility in vitro. The C. jejuni used in this study was previously isolated from the broiler chicken ceca. Serotonin at concentrations of 1mM or below did not elicit changes in growth (P > 0.05) or motility (P > 0.05) of C. jejuni. Next, we utilized liquid chromatography tandem mass spectrometry to investigate whether serotonin affected the proteome of C. jejuni. Serotonin caused (P < 0.05) the downregulation of a protein (CJJ81176_1037) previously identified to be essential in C. jejuni colonization. Based on our findings, we evaluated whether serotonin would cause a functional change in C. jejuni adhesion and invasion of the HT29MTX-E12 colonic epithelial cell line. Serotonin was found to cause a reduction in adhesion (P < 0.05) but not invasion (P > 0.05). Together, we have identified a potential role for serotonin in modulating C. jejuni colonization in the gut in vitro. Further studies are required to understand the practical implications of these findings for the control of C. jejuni enteric colonization in vivo.

Japanese quail (Coturnix japonica) as a novel model to study the relationship between the avian microbiome and microbial endocrinology-based host-microbe interactions.

BACKGROUND: Microbial endocrinology, which is the study of neuroendocrine-based interkingdom signaling, provides a causal mechanistic framework for understanding the bi-directional crosstalk between the host and microbiome, especially as regards the effect of stress on health and disease. The importance of the cecal microbiome in avian health is well-recognized, yet little is understood regarding the mechanisms underpinning the avian host-microbiome relationship. Neuroendocrine plasticity of avian tissues that are focal points of host-microbiome interaction, such as the gut and lung, has likewise received limited attention. Avian in vivo models that enable the study of the neuroendocrine dynamic between host and microbiome are needed. As such, we utilized Japanese quail (Coturnix japonica) that diverge in corticosterone response to stress to examine the relationship between stress-related neurochemical concentrations at sites of host-microbe interaction, such as the gut, and the cecal microbiome. RESULTS: Our results demonstrate that birds which contrast in corticosterone response to stress show profound separation in cecal microbial community structure as well as exhibit differences in tissue neurochemical concentrations and structural morphologies of the gut. Changes in neurochemicals known to be affected by the microbiome were also identified in tissues outside of the gut, suggesting a potential relationship in birds between the cecal microbiome and overall avian physiology. CONCLUSIONS: The present study provides the first evidence that the structure of the avian cecal microbial community is shaped by selection pressure on the bird for neuroendocrine response to stress. Identification of unique region-dependent neurochemical changes in the intestinal tract following stress highlights environmental stressors as potential drivers of microbial endocrinology-based mechanisms of avian host-microbiome dialogue. Together, these results demonstrate that tissue neurochemical concentrations in the avian gut may be related to the cecal microbiome and reveal the Japanese quail as a novel avian model in which to further examine the mechanisms underpinning these relationships. Video abstract.

Fluoxetine-induced alteration of murine gut microbial community structure: evidence for a microbial endocrinology-based mechanism of action responsible for fluoxetine-induced side effects.

BACKGROUND: Depression and major depressive disorder affect 25% of the population. First line treatment utilizing selective serotonin reuptake inhibitors (SSRIs) have met with limited success due to well-recognized negative side effects which include weight gain or loss. This inability to control unwanted side effects often result in patients stopping their antidepressant medications. The mechanisms underlying the failure of SSRIs are incompletely understood. METHODS: Male CF-1 mice (5 weeks of age, N = 10 per group) were per orally administered fluoxetine (20 mg per kg body weight) or diluent daily for 29 days. During this time fecal specimens were collected at three defined time points (0, 15 and 29 days). At the conclusion of the 29-day dosing regimen, animals were subjected to two behavioral assessments. For bacterial identification of the microbiota, 16S rRNA gene sequencing was performed on 60 fecal specimens (three specimens per mouse time course, N = 20 mice) using Illumina MiSeq. Analysis of community sequence data was done using mothur and LEfSe bioinformatic software packages. RESULTS: Daily per oral administration of fluoxetine for 29 days to male mice resulted in a significant, time dependent, alteration in microbial communities accompanying changes in body weight. The calculated species richness and diversity indicators of the murine fecal microbial communities were inconsistent and not significantly different between the groups. Among the phylotypes decreased in abundance due to fluoxetine administration were Lactobacillus johnsonii and Bacteroidales S24-7 which belong to phyla associated with regulation of body mass. The observed changes in body weight due to fluoxetine administration mimicked the dramatic shifts in weight gain/loss that has been observed in humans. Further, at the conclusion of the 29-day dosing regimen fluoxetine-dosed animals evidenced a mild anxiogenic-like behavior. DISCUSSION: We report that the most widely used antidepressant, fluoxetine, which is an SSRI-type drug, results in the selective depletion of gut microbiota, specifically the Lactobacilli which are involved in the regulation of body weight. Concomitantly, fluoxetine administration increases the abundance of phylotypes related to dysbiosis. Since Lactobacilli have been previously shown to possess a known biogenic amine transporter that regulates the uptake of fluoxetine, it is proposed that a microbial endocrinology-based mechanistic pathway is responsible for the ability of SSRIs to selectively negatively impact beneficial microbiota. The results of this study therefore suggest that the negative clinical side effects due to fluoxetine administration may be due to alterations in gut microbiota. Further, the data also suggests that supplementation of bacterial genera directly affected by fluoxetine administration may prove useful in ameliorating some of the well-known side effects of chronic fluoxetine administration such as weight alterations.