Central Taurine Attenuates Hyperthermia and Isolation Stress Behaviors Augmented by Corticotropin-Releasing Factor with Modifying Brain Amino Acid Metabolism in Neonatal Chicks

The objective of this study was to determine the effects of centrally administered taurine on rectal temperature, behavioral responses and brain amino acid metabolism under isolation stress and the presence of co-injected corticotropin-releasing factor (CRF). Neonatal chicks were centrally injected with saline, 2.1 pmol of CRF, 2.5 μmol of taurine or both taurine and CRF. The results showed that CRF-induced hyperthermia was attenuated by co-injection with taurine. Taurine, alone or with CRF, significantly decreased the number of distress vocalizations and the time spent in active wakefulness, as well as increased the time spent in the sleeping posture, compared with the saline- and CRF-injected chicks. An amino acid chromatographic analysis revealed that diencephalic leucine, isoleucine, tyrosine, glutamate, asparagine, alanine, β-alanine, cystathionine and 3-methylhistidine were decreased in response to taurine alone or in combination with CRF. Central taurine, alone and when co-administered with CRF, decreased isoleucine, phenylalanine, tyrosine and cysteine, but increased glycine concentrations in the brainstem, compared with saline and CRF groups. The results collectively indicate that central taurine attenuated CRF-induced hyperthermia and stress behaviors in neonatal chicks, and the mechanism likely involves the repartitioning of amino acids to different metabolic pathways. In particular, brain leucine, isoleucine, cysteine, glutamate and glycine may be mobilized to cope with acute stressors.

Developmental Change of Yolk Microbiota and Its Role on Early Colonization of Intestinal Microbiota in Chicken Embryo

Although the fertilized eggs were found to contain microbes in early studies, the detailed composition of yolk microbiota and its influence on embryo intestinal microbiota have not been satisfactorily examined yet. In this study, the yolk microbiota was explored by using 16s rRNA sequencing at different developmental stages of the broiler embryo. The results showed that the relative abundance of yolk microbiota was barely changed during embryogenesis. According to the KEGG analysis, the yolk microbiota were functionally related to amino acid, carbohydrate, and lipid metabolisms during chicken embryogenesis. The yolk microbiota influences the embryonic intestinal microbiota through increasing the colonization of Proteobacteria, Firmicutes, and Bacteroidetes in the intestine, particularly. The intestinal microbes of neonatal chicks showed higher proportions of Faecalibacterium, Blautia, Coprococcus, Dorea, and Roseburia compared to the embryonic intestinal microbiota. Our findings might give a better understanding of the composition and developmental change of yolk microbiota and its roles in shaping the intestinal microbiota.

Evaluation of Euthanasia Methods on Behavioral and Physiological Responses of Newly Hatched Male Layer Chicks

Newly hatched male layer chicks are considered as “by-products” in the egg industry and must be humanely euthanized at the hatchery. Instantaneous mechanical destruction (maceration) is the predominant euthanasia method applied in poultry hatcheries and is approved by the American Veterinary Medical Association (AVMA). However, maceration is not perceived by the public to be a humane means of euthanasia. The effects of alternative euthanasia methods, including carbon dioxide (CO2) or nitrogen (N2) inhalation, and a commercial negative pressure stunning system on behavioral and physiological responses of day-of-hatch male layer chicks, were evaluated in a field trial. Chick behaviors, including ataxia, loss of posture, convulsions, cessation of vocalization, and cessation of movement, were monitored. Serum hormones were assessed at the end of each of the alternative euthanasia treatments, including a control group allowed to breathe normal atmospheric air. The N2 method induced unconsciousness and death later than the CO2 and negative pressure methods, and increased serum corticosterone concentrations of neonatal chicks. Carbon dioxide inhalation increased serotonin concentrations as compared to controls, as well as the N2 and the negative pressure methods. The behavioral and physical responses observed in this study suggest that both CO2 inhalation and negative pressure stunning can be employed to humanely euthanize neonatal male layer chicks.

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host’s metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.

Preventive Antibiotic Treatment Increases Susceptibility of Neonatal chicks to Salmonella Infection via Disrupting Gut Microbiota and Linoleic Acid Metabolis

Background: Antibiotics are widely employed in animal husbandry to prevent and treat diseases. Increasing evidence suggests they may alter the animals’ natural microbiota and increase their susceptibility to pathogen. However, the mechanisms linking the gut microbiota and pathogen colonization in poultry have not yet been full elucidated. Herein, we used metagenomic and metabonomic approaches to investigate the effects of florfenicol (FFC) pre-treatment on Salmonella enterica serovar Enteritidis (S. Enteritidis) colonization in the intestines of neonatal chicks in terms of host response, microbiota composition and metabolism. Results: We determined that FFC pre-treatment significantly alters the cecal microbiota and metabolome, and also increases the intestinal permeability and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to S. Enteritidis infection in chicks with FFC pre-treatment relative to without pre-treatment chicks. Prior to Salmonella infection, FFC pre-treatment significantly reduced the abundance of Lactobacillus, and significantly affected linoleic acid metabolism, including significantly reducing the levels of conjugated linoleic acid (CLA), and significantly elevating the abundance of 12,13-EpOME and 12,13-diHOME in cecum. After infection with S. Enteritidis, the abundance of Proteobacteria were significantly increased and host inflammatory responses and intestinal permeability were significantly aggravated relative to without FFC pre-treatment chicks, suggestive of a profound exacerbating of the host response influenced by infection in the context of FFC pre-treatment. The linoleic acid metabolism was still significantly different pathway after Salmonella infection, and we screened CLA and 12,13-diHOME as the target metabolites using a multi-omics technique. Supplementation with CLA maintained intestinal integrity, reduced intestinal inflammation, and accelerated Salmonella clearance from the gut and remission of enteropathy. Whereas, treatment with 12,13-diHOME promoted intestinal inflammation and disrupted the intestinal barrier function to sustain Salmonella infection. Therefore, florfenicol reduces production of CLA by inhibiting Lactobacillus growth, increases 12,13-diHOME level of intestine, thereby reducing colonization resistance of neonatal chicks to Salmonella infection.Conclusion: This study reveals the potential health impact of antibiotics on gut microbiota and linoleic acid metabolism and contributing factors influencing Salmonella colonization in neonatal chicks, and provides mechanistic understanding into the role of the antibiotics promote the colonization of pathogens.

Preventive Antibiotic Treatment Increases Neonatal Chicks Susceptibility to Salmonella Infection Via Disrupting Gut Microbiota and Linoleic Acid Metabolism

Background: Antimicrobial agents have been widely used in animal farms to prevent and treat animal diseases. However, antimicrobial agents may change the bacterial community and increase susceptibility to the pathogenic bacteria infection. Here, we used metagenomic and metabonomic approach to investigate the effects of florfenicol (FFC) pre-treatment on colonization of Salmonella enterica serovar Enteritidis (S. Enteritidis) in intestines of neonatal chicks through analysis of host responses, microbiota and metabolic changes. Results: We observed that FFC pre-treatment significantly increases the level of S. Enteritidis in the cecal contents, spleen and liver and also induces changes to the cecal microbiota and metabolism. Prior to S. Enteritidis infection, FFC significantly reduced the content of Lactobacillus, and significantly affected the linoleic acid metabolism pathway, including significantly reducing the levels of conjugated linoleic acid (CLA), and significantly increasing the abundance of 12,13-EpOME and 12,13-diHOME in cecum. After infection with S. Enteritidis, the abundance of Proteobacteria were significantly increased and the Salmonella-induced intestinal inflammatory responses and intestinal barrier damage were exacerbated. Supplementation with CLA could maintain intestinal integrity, reduce intestinal inflammation, and directly inhibit Salmonella growth to effectively reduce the Salmonella colonization, whereas the 12,13-diHOME through promoting intestinal inflammation and destroying the intestinal barrier function to support the Salmonella infection. Conclusions: Overall, FFC can decrease levels of Lactobacillus and CLA, and elevate cecal 12,13-diHOME concentrations in neonatal chicks and thereby increases susceptibility to Salmonella infection. This study revealed a potential health impact of antibiotics and disturbed gut microbiota and linoleic acid metabolism might be an intestinal health-impairing attribute and may contribute to Salmonella colonization.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 μg of CpG-ODN or saline (n=~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n=3‐4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 μg and 50 μg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1×104 or 1×105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 μg and 50 μg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.