Protected biofactors and antioxidants reduce the negative consequences of virus and cold challenge by modulating immunometabolism via changes in the interleukin-6 receptor signaling cascade in the liver.

Protected biofactors and antioxidants (PBA), and protected biofactors and antioxidants with protected organic acids and essential oils (PBA+POAEO) have been shown to have benefits in stressed or challenged birds. Here, we describe the immunometabolic changes observed in the liver of Ross 308 broilers during feed supplementation and brief physiological stress. These studied additives contain protected essential oils, organic acids, and vitamins which may have protective effects on the liver. Thus, we aimed to determine the signaling changes induced by these supplements and the resultant immunometabolic effects in the liver. All birds received a 2X dose of live bronchitis vaccine at d 0 and a 48-h cold challenge by reducing the temperature from 30 to 32°C, to 20 to 23°C on d 3 to 5. Control birds were fed a standard diet without supplementation. Liver samples were collected to evaluate the effects of these treatments on cytokine gene expression and protein phosphorylation via kinome peptide array. ANOVA was used for statistical analysis of the gene expression data (significance at a p-value of 0.05), and PIIKA2 was used for statistical evaluation and comparative analysis of the kinome peptide array data. At d 15, the kinome peptide array analysis and gene expression data showed stimulation of the interleukin 6 receptor (IL-6R) signal transduction for host protection via heightened immune response while inducing immune modulation and reducing inflammation in both supplement treated groups. Significant changes were observed via IL-6R signaling in the metabolic profiles of both groups compared to control and no significant differences when compared to each other. In the liver, these 2 feed additives induced immunometabolic changes predominantly via the IL-6 receptor family signaling cascade. Differences between the 2 treated groups were predominantly in the metabolic pathways, centered around the mTOR pathway and the proteins AMPK, mTOR and S6K, with a more anabolic phenotype following the addition of essential oils.

Distillers dried grains with soluble and enzyme inclusion in the diet effects broilers performance, intestinal health, and microbiota composition.

This study tested the effect of distillers dried grains with soluble (DDGS) inclusion in a broiler diet, with or without supplementation of exogenous enzymes, on the microbiota composition, intestinal health, diet digestibility and performance. A total of 288 one-day-old chickens was assigned to 6 treatments (8 replicate of 6 birds each) according to a completely randomized design with a 3 × 2 factorial scheme with 3 DDGS levels (0, 7 and 14%) and 2 inclusions of exogenous enzymes (with or without a multicarbohydrase complex + phytase [MCPC]). The results exhibited that DDGS inclusion up to 14% did not impair broilers performance up to 28 d, however, DDGS-fed animals exhibited significant improvement with the MCPC supplementation. No effects of the enzymes in the ileal digestibility were found at 21 d. DDGS inclusion in the diet affected dry matter and gross energy digestibility. Broilers fed diets with MCPC were found to have less intestinal histological alteration thus better gut health. No effect of DDGS, enzyme or interaction of those were observed for intestinal permeability and in the serum inflammatory biomarker (calprotectin) at 7 and 28 d. The increase of DDGS percentage in the diet reduced the diversity of the ileal microbiota but increased the cecal microbiota diversity. The inclusion of DDGS showed positive effects on microbiota composition due to a reduction of Proteobacteria phylum in the ileum at 28d and a reduction in the presence of Enterococcaceae family in the ileum at 14 and 28d. The inclusion of MCPC complex might promote beneficial changes in the ileal and cecal microbiota due reduce of Proteobacteria, Bacillaceae and Enterobacteriaceae. The supplementation of xylanase, ß-glucanase, arabinofuranosidase and phytase to a DDGS diet improves performance and intestinal health allowing the use of these subproduct in the poultry nutrition.

Novel model for chronic intestinal inflammation in chickens: (2) Immunologic mechanism behind the inflammatory response.

Intestinal inflammation in poultry is a complex response that involves immune and intestinal cells which is still not fully understood. Thus, to better understand the mechanisms that drive the chronic intestinal inflammation in fowl we conducted an experiment applying a previously established nutritional model of low-grade chronic intestinal inflammation to evaluate cytokine and chemokine profiles in the chicken intestine. For this, we placed 90 one-day chickens into two treatments: (1) a control group (CNT) fed a corn-soybean diet, and (2) a group fed a diet high in non-starch polysaccharides (NSP). At days 14, 22, 28 and 36 of age, 6 birds from each treatment were euthanized, jejunal and ileal samples were collected for histological examination and cytokine measurements. The cytokines interferon-alpha (IFN-α), IFN-γ, interleukin-16 (IL-16), IL-10, IL-21, IL-6, macrophage-colony stimulating factor (M-CSF), chemokine C-C motif ligand 20 (CCL20), CCL4, CCL5 and vascular endothelial growth factor (VEGF) were quantified in the intestinal tissue. Histologically, both jejunum and ileum of broilers fed NSP diet showed marked infiltration of mononuclear immune cells into the villi. Further, these birds exhibited a significant (P < 0.05) increase in CCL20 concentration in the jejunum at 14d, but a dramatic reduction of M-CSF at 14 and 21d. Later at 28d and 36d, birds fed the NSP diet exhibited increased IL-16 concentration in the jejunum. Since M-CSF is a monocyte stimulatory cytokine and CCL20 a chemokine of T-cells, the reduced M-CSF and increased production of CCL20 may indicate the involvement of the adaptive immune response, specifically driven by T-cells, occurring around the third week of age in the NSP model. Lastly, as a result of the mononuclear cell infiltration and activation of T-cells, IL-16, a pro-inflammatory T-cell cytokine, increased. Therefore, the current work indicates the importance of adaptive immune cells, especially T-cells, in the chronic intestinal inflammation in broiler chicken.

Protected biofactors and antioxidants reduce the negative consequences of virus and cold challenge while enhancing performance by modulating immunometabolism through cytoskeletal and immune signaling in the jejunum.

The aim of this study was to evaluate the effectiveness and mechanism of action of 2 feed additives in reducing the impacts of virus and temperature stressors. We determined the effects of protected biofactors and antioxidants (P(BF+AOx)), and protected biofactors and antioxidants with protected organic acids and essential oils (P(BF+AOx)+P(OA+EO)) on the immune and metabolic health of Ross 308 broiler chickens. These biofactors and antioxidants were derived from vitamins, and Aspergillus niger, Aspergillus oryzae and Bacillus subtilis fermentation extracts. All Ross 308 chickens were exposed to a double-dose of live bronchitis vaccine at d 0 and environmentally challenged by reducing the temperature from 32°C to 20°C at d 3 for 48 h. Control birds were fed without feed additives in the diet. Performance data and jejunum samples were collected to evaluate the effects of these treatments on growth, cytokine expression, and protein phosphorylation via kinome peptide array. ANOVA was used for statistical analysis of the performance and gene expression data (p-value of 0.05), and PIIKA2 was used for statistical evaluation and comparison of the kinome peptide array data. The P(BF plus;AOx) and P(BF+AOx)+P(OA+EO) treatments significantly increased bird weight gain and decreased feed conversion. The kinome peptide array data analysis showed increased activity of cytoskeletal, cell growth and proliferation proteins, and metabolic signaling in the jejunum of P(BF+AOx)+P(OA+EO) treated chickens. There was a significant decrease in IL-6 gene expression in the jejunum of P(BF+AOx)+P(OA+EO) samples compared to control at d 15. P(BF+AOx)+P(OA+EO) treatments in the jejunum showed strong immunomodulatory effects, perhaps to control inflammation. P(BF+AOx)+P(OA+EO) improves gut health via growth and metabolic signaling in the jejunum while inducing stronger immunomodulation.

A protected complex of biofactors and antioxidants improved growth performance and modulated the immunometabolic phenotype of broiler chickens undergoing early life stress.

We evaluated the supplementation of a protected complex of biofactors and antioxidants [P(BF+AOx)] on growth performance, antioxidant activity, expression of immune-related genes, and immunometabolic phenotype of broilers submitted to early life stressors. The treatments were a nutritionally complete basal diet supplemented or not with P(BF+AOx) (Jefo Nutrition Inc., Saint-Hyacinthe, QC, Canada) from 1 to 14 d of age. 720 one-day old male Ross 308 chickens were placed into pens of 30 birds (12 replicates/treatment). Birds were double-vaccinated against infectious bronchitis (IB; MILDVAC-Ma5T) at the hatchery and submitted, on d 3, to an acute reduction on environmental temperature (from 32° C to 20°C) for 48 h. Feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR) were calculated weekly. On d 7 and 15, samples were collected for expression of immune-related genes and kinome array analysis, and serum to evaluate the antioxidant status. Data were analyzed by ANOVA using SAS (SAS 9.4). From d 1 to 21 and d 1 to 28, the dietary supplementation of P(BF+AOx) significantly increased BWG (P < 0.05) by 3.6 and 3.8%, respectively, and improved FCR (P < 0.05) by 1.2 and 1.8%, respectively. From d 1 to 35, dietary supplementation enhanced BWG (P = 0.03) by 4%. Serum glutathione reductase activity on d 15 was higher in birds fed diets supplemented with P(BF+AOx) compared to the control diet-fed birds (P = 0.04). Dietary supplementation reduced the expression of IL-1ß (P = 0.03) in the lungs on d 7. On d 15, dietary supplementation increased the expression of IL-6 (P = 0.02) and IL-10 (P = 0.03) in the liver. It was observed that, via decreased phosphorylation, catalase was activated in the jejunum and liver, and the phosphorylation of immunoregulatory or proinflammatory proteins was decreased. Other important cellular signaling pathways were also changed in the liver and jejunum due to the supplementation. The supplementation of P(BF+AOx) improves growth performance by promoting a general anti-inflammatory and antioxidant response in chickens undergoing early life stress.

Influential factors on the composition of the conventionally raised broiler gastrointestinal microbiomes.

The microbiome has entered the vernacular of the consumer as well as broiler production and is, therefore, becoming increasingly important to poultry producers to understand. The microbiome is, by definition, compositional and relates to how the microbiological organisms within the gut inhabit that ecological niche. The gut is diverse, flexible, and data acquired requires a greater understanding of the host-microbiome axes, as well as advanced bioinformatics and ecology. There are numerous microbial populations that define the gut microbiome; however, there are even more effects that can influence its composition. As management practices vary between producers, documenting these influences is an essential component of beginning to understand the microbiome. This review targets broiler production and concatenates the currently understood compositional ecology of the broiler gastrointestinal tract microbiome as well as its influences.

Poultry processing and the application of microbiome mapping.

Chicken is globally one of the most popular food animals. However, it is also one of the major reservoirs for foodborne pathogens, annually resulting in continued morbidity and mortality incidences worldwide. In an effort to reduce the threat of foodborne disease, the poultry industry has implemented a multifaceted antimicrobial program that incorporates not only chemical compounds, but also extensive amounts of water application and pathogen monitoring. Unfortunately, the pathogen detection methods currently used by the poultry industry lack speed, relying on microbiological plate methods and molecular detection systems that take time and lack precision. In many cases, the time to data acquisition can take 12 to 24 h. This is problematic if shorter-term answers are required which is becoming more likely as the public demand for chicken meat is only increasing, leading to new pressures to increase line speed. Therefore, new innovations in detection methods must occur to mitigate the risk of foodborne pathogens that could result from faster slaughter and processing speeds. Future technology will have 2 tracks: rapid methods that are meant to detect pathogens and indicator organisms within a few hours, and long-term methods that use microbiome mapping to evaluate sanitation and antimicrobial efficacy. Together, these methods will provide rapid, comprehensive data capable of being applied in both risk-assessment algorithms and used by management to safeguard the public.

Changes in immune and metabolic gut response in broilers fed ß-mannanase in ß-mannan-containing diets.

ß-galactomannans found in soy-based broiler feed are known to cause physiological effects that are hypothesized to be related to gut inflammation. Previous studies have shown that the incorporation of ß-mannanase in the diet or as a supplement results in improvements to certain performance parameters related to gut health and feed conversion. Using kinome analysis, we characterized the mechanism of ß-galactomannan activity and supplementation with ß-mannanase on the gut of commercial broilers to understand the mode of action. Two doses of ß-mannanase (200 and 400 g/ton of feed) with and without inclusion of additional ß-galactomannan (3,000 ppm) were tested at 3 time points (d 14, d 28, and d 42 post hatch). Broilers were fed starter (d 0 to 14), grower (d 15 to 28), and finisher diets (d 29 to 42). Jejuna were collected from birds from each treatment condition and time point. Cluster analysis of the kinome data showed that birds clustered first by age, then predominantly by whether ß-mannanase had been included in the diet. Biological pathway analysis showed that the inclusion of additional ß-galactomannan into the diet resulted in increased signaling related to immune response, relative to our normal control diet (with reduced soybean meal). The addition of ß-mannanase to the enhanced ß-galactomannan diet eliminated the majority of this immune-related signaling, indicating that the feed-induced immune response within the jejuna had been eliminated by the addition of ß-mannanase. We also saw changes in specific metabolic and gut function pathways in birds fed ß-mannanase. These observed changes in ß-mannanase-fed birds are likely the mechanism for the enhanced performance and feed conversion observed in birds given ß-mannanase in their diets.

Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni.

Campylobacter spp. are the second leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of nearly $2B USD per year. Most human illness associated with campylobacteriosis is due to infection by C. jejuni and chickens are recognized as a reservoir that could lead to foodborne illness in humans resulting from handling or consuming raw or undercooked chicken. We recently developed a novel breeding strategy based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators including IL-6, CXCLi2, and CCLi2, hereafter referred to as the high and low lines, respectively. We have shown the high line chickens are more resistant to the foodborne and poultry pathogens Salmonella enterica serovar Enteritidis, Eimeria tenella, and Clostridium perfringens-induced necrotic enteritis compared to the low line. The objective of this study was to determine whether the same trend of enhanced resistance in the high line birds was observed for C. jejuni. Birds were challenged at 2 d of age by oral gavage (0.5 mL) with 5 × 106 colony forming units (cfu) of C. jejuni/mL, necropsied 4 d post challenge, and cecal content collected to determine if there was a difference in C. jejuni resistance between the high and low line chickens. There were fewer (P = 0.01) chickens from the high line (28/40 = 71.8%) that were colonized by C. jejuni compared to the low line (37/39 = 94.9%). The amount of C. jejuni recovered from the ceca of infected birds was quantified; however, no differences were observed (P = 0.10). Since the high line birds were also more resistant to C. jejuni, it provides additional validation of selection based on pro-inflammatory mediators producing a line of chickens with increased natural resistance against diverse foodborne and poultry pathogens. The poultry industry is moving towards reduced therapeutics and, as such, our breeding strategy would be a viable method to incorporate into traditional poultry breeding programs.

Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis.

We developed a novel selection method based on an inherently high and low phenotype of pro-inflammatory mediators and produced "high" and "low" line chickens. We have shown high line birds are more resistant to Salmonella enterica serovar Enteritidis and Eimeria tenella compared to the low line. Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illness, and is also an economically important poultry pathogen and known etiologic agent of necrotic enteritis (NE). The objective of this study was to determine if high line birds were also more resistant to NE than low line birds using an established model. Birds were reared in floor pens and challenges were conducted twice (high line = 25/trial, 50 birds total; low line = 26/trial, 52 birds total). Day-old chicks were provided a 55% wheat-corn-based un-medicated starter diet. A bursal disease vaccine was administered at 10× the recommended dose via the ocular route at 14-d-of-age. Birds were challenged daily for 3 d beginning at 16-d-of-age by oral gavage (3 mL) with 10(7) colony forming units (cfu) of C. perfringens/mL then necropsied at 21-d-of-age. All birds had sections of the intestine examined and scored for lesions while the first 10 necropsied also had gut content collected for C. perfringens enumeration. Chickens from the high line were more resistant to C. perfringens-induced NE pathology compared to the low line, as indicated by reduced lesion scores. Ninety percent of the high line birds had lesions of zero or one compared to 67% of the low line birds. Wilcoxon rank sum test showed significantly higher lesion scores in the low line birds compared to the high line (P < 0.0001). There were no differences in the C. perfringens recovered (P = 0.83). These data provide additional validation and support selection based on elevated levels of pro-inflammatory mediators produces chickens with increased resistance against foodborne and poultry pathogens.

Outcomes of emergent embolisation of ruptured hepatocellular carcinoma in a western population.

AIM: To evaluate the authors' experience with interventional radiological management of tumour rupture in hepatocellular carcinoma (HCC) in a Western population. MATERIALS AND METHODS: A retrospective review was performed of all consecutive patients treated at a single institution with transcatheter embolisation for ruptured HCC between 2000 and 2013. Patient age, sex, aetiology of liver disease, degree of underlying liver dysfunction, and clinical presentation were assessed. Embolisation was performed in a selective fashion when possible. Success, complications, and survival were assessed. RESULTS: Twenty-three patients were treated with embolisation for ruptured HCC. Of these patients, nine, nine, and five patients were Child-Pugh Class A, B, and C respectively. Embolisation was successful in 22 patients; one patient remained haemodynamically unstable and transfusion dependent despite embolisation. No major complications occurred. Median survival time was 260 days and the 30 day and 1 year survival rates were 83% and 45%, respectively. Child-Pugh class B or C (p = 0.04), Model for End-Stage Liver Disease score greater than 10 (p = 0.04), lobar embolisation (p = 0.04), and presence of portal vein thrombosis (p = 0.01) were significantly associated with worse prognosis. CONCLUSION: Transcatheter embolisation is effective at controlling haemorrhage in patients with ruptured HCC. Although major procedural complications are low, embolisation should proceed with an understanding of poor prognosis in patients with decompensated liver disease. Superselective embolisation is associated with improved prognosis and should be performed when feasible.

Selection for pro-inflammatory mediators produces chickens more resistant to Eimeria tenella.

We recently developed a novel selection method based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators, including interleukin (IL)-6, CXCLi2, and CCLi2. The resultant high line of chickens is more resistant to Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) compared to the low line. In the current study, we sought to determine if the high line birds were also more resistant to the protozoan parasite Eimeria tenella. In three separate experiments, 14-day-old chickens from the high and low lines were challenged orally with 10×10(3) to 45×10(3) E. tenella oocysts. Birds were sacrificed 6 d postchallenge and the caeca was removed and scored for lesions and body weight gain compared to mock-infected controls. The high line birds were more resistant to intestinal pathology as demonstrated by lower lesion scores (P≤0.04) compared to the low line. There were no differences in body weight gain between the lines. The results from this study showed that in addition to enhanced resistance against Salmonella Enteritidis, high line chickens are also more resistant to the pathology associated with coccidial infections compared to the low line birds. Taken together with our initial study utilizing the high and low lines, selection based on increased pro-inflammatory mediator expression produces chickens that are more resistant to both foodborne and poultry pathogens, including cecal pathology associated with costly coccidial infections.

Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments.

Experimental and epidemiological evidence has indicated the respiratory route to be a potential portal of entry for salmonellas in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage, intratracheal or intravenous challenge in chickens. Seven-day-old chicks were challenged with either 10(4) or 10(6) colony-forming units of S. Senftenberg per chick by oral gavage, intratracheal or intravenous challenge, respectively, in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from caecal contents, caecal tissue-caecal tonsils and liver and spleen. In both trials, intratracheal delivery of S. Senftenberg was the only route that allowed colonization of the caeca of chickens when compared with oral gavage or intravenous challenge in a dose response fashion (P < 0.05). Liver and spleen samples yielded no S. Seftenberg after the lower dose challenge by the oral or intratracheal route and only low levels following the high-dose administration by these routes, whereas intravenous challenge resulted in recovery of the organisms after both doses. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize caeca to the same extent as compared with intratracheal challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce the transmission in poultry.

Loxoribine pretreatment reduces Salmonella Enteritidis organ invasion in 1-day-old chickens.

Young poultry exhibit a transient colonization by some food-borne pathogens, including Salmonella, during the first week of life that stems from immature innate and acquired defense mechanisms. Consequently, modulation of the hosts' natural immune response is emerging as an important area of interest for food animal producers, including the poultry industry. Toll-like receptor (TLR) agonists have been shown to boost the innate immune response in young chickens and increase their resistance to colonization by Salmonella enterica serovar Enteritidis. The objective of the present study was to determine if pretreatment with loxoribine, a TLR7 agonist and immune modulator, protects young chicks from Salmonella Enteritidis organ invasion. Loxoribine (0-100 µg) was administered intra-abdominally to 1-d-old broiler chicks, and 4 h later, the birds were challenged orally with Salmonella Enteritidis. Twenty-four hours postchallenge, birds were euthanized and the liver and spleen aseptically removed and cultured for Salmonella Enteritidis. This was carried out on 3 separate occasions using 26 to 50 chicks per dose per experiment. Pretreatment of chicks with loxoribine (6.25-25 µg) significantly (P ≤ 0.05) reduced liver and spleen organ invasion by Salmonella Enteritidis. Higher doses (50-100 µg) of loxoribine had no effect. The results obtained in this study indicate that there is a potential application for using loxoribine to increase protection of young chicks when they are most susceptible to infections with Salmonella.

Effects of avian triggering receptor expressed on myeloid cells (TREM-A1) activation on heterophil functional activities.

A class of innate receptors called the triggering receptors expressed on myeloid cells (TREM) has been discovered and shown to be involved in innate inflammatory responses. The TREM family has been found in the chicken genome and consists of one activating gene (TREM-A1) and two inhibitory genes (TREM-B1 and TREM-B2). However, to date, there have been no reports on the effects of activating the TREM molecules on the functional activity of the primary avian polymorphonuclear cell, the heterophil. To characterize the activation of avian heterophils, we evaluated the effect of receptor ligation on heterophil effector functions. A specific agonistic antibody (Ab) was generated against the peptide sequence of chicken TREM-A1 38-51aa (YNPRQQRWREKSWC). To study TREM-A1 mediated activation, purified peripheral blood heterophils were incubated with various concentrations of the anti-TREM-A1 Ab or control Ab against an irrelevant antigen. Activation via TREM-A1 induces a significant increase in phagocytosis of Salmonella enteritidis, a rapid degranulation, and a dramatic up-regulation in gene expression of the pro-inflammatory cytokine, IL-6, and the inflammatory chemokine, CXCLi2. However, we found no direct TREM-A1 stimulation of the heterophil oxidative burst. Like mammalian TREM, avian TREM-A1 ligation synergizes with the activation of Toll-like receptor-4 (TLR4) ligand, LPS. In addition, the synergistic activity of LPS and TREM-A1 resulted in a significantly (p⩽0.05) increased production of an oxidative burst. Taken together, these results suggest, unlike in mammalian neutrophils, TREM-A1 engagement activates a differential functional activation of avian heterophils, but like mammalian neutrophils, acts in synergy with TLR agonists. These results provide evidence of the function of TREM-A1 in heterophil biology and avian innate immunity.