Molecular analysis of the caecal and tracheal microbiome of heat-stressed broilers supplemented with prebiotic and probiotic.

The gastrointestinal tract commensal microbiome is important for host nutrition, health and immunity. Little information is available regarding the role of these commensals at other mucosal surfaces in poultry. Tracheal mucosal surfaces offer sites for first-line health and immunity promotion in broilers, especially under stress-related conditions. The present study is aimed at elucidating the effects of feed supplementations with mannanoligosaccharides (MOS) prebiotic and a probiotic mixture (PM) on the caecal and tracheal microbiome of broilers kept under chronic heat stress (HS; 35 ± 2°C). Day-old chickens were randomly divided into five treatment groups: thermoneutral control (TN-CONT), HS-CONT, HS-MOS, HS-PM and HS synbiotic (fed MOS and PM). Caecal digesta and tracheal swabs were collected at day 42 and subjected to DNA extraction, followed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. The PCR-DGGE dendrograms revealed significant (49.5% similarity coefficients) differences between caecal and tracheal microbiome. Tracheal microbiome pyrosequencing revealed 9 phyla, 17 classes, 34 orders, 68 families and 125 genera, while 11 phyla, 19 classes, 34 orders, 85 families and 165 genera were identified in caeca. An unweighted UniFrac distance metric revealed a distinct clustering pattern (analysis of similarities, P = 0.007) between caecal and tracheal microbiome. Lactobacillus was the most abundant genus in trachea and caeca and was more abundant in caeca and trachea of HS groups compared with the TN-CONT group. Distinct bacterial clades occupied the caecal and tracheal microbiomes, although some bacterial groups overlapped, demonstrating a core microbiome dominated by Lactobacillus. No positive effects of supplementations were observed on abundance of probiotic bacteria.

Evaluation of feed grade sodium bisulfate impact on gastrointestinal tract microbiota ecology in broilers via a pyrosequencing platform.

The gastrointestinal microbial community in broiler chickens consists of many different species of bacteria, and the overall microbiota can vary from bird to bird. To control pathogenic bacteria in broilers and improve gut health, numerous potential dietary amendments have been used. In this study, we used a pyrosequencing platform to evaluate the effect of sodium bisulfate on microbiota of the crop, cecum, and ileum of broiler chickens grown over several weeks. The diversity information in each digestive organ sample exhibited considerable variation and was clearly separable, suggesting distinct bacterial populations. Although no apparent microbial clustering occurred between the control and the dietary treatments, we did observe shifts in overall microbiota populations in the crop, ileum, and ceca as well as changes in specific microorganisms such as Bacteroides, Clostridium, and Lactobacillus species that were identified as birds became older.

Commercial vaccine provides cross-protection by reducing colonization of Salmonella enterica serovars Infantis and Hadar in turkeys.

Human foodborne outbreaks with antibiotic-resistant Salmonella enterica associated with contaminated poultry products have recently involved serogroup C serovars Infantis and Hadar. The current study evaluated a commercially available Salmonella vaccine for cross-protection against Infantis and Hadar serovars in turkeys. The live, attenuated S. Typhimurium (serogroup B) vaccine significantly reduced colonization of intestinal tissues (cecum, cecal tonsils, and cloaca) by serovars Infantis (C1) and Hadar (C2) and significantly limited systemic dissemination to the spleen. S. Infantis, but not S. Hadar, disseminated to bone marrow in non-vaccinated turkeys, but vaccination prevented S. Infantis dissemination to the bone marrow. The S. Infantis challenge strain contained the pESI megaplasmid, and virulence mechanism(s) residing on this plasmid may support dissemination and/or colonization of systemic niches such as myeloid tissue. Collectively, the data indicate that vaccinating turkeys with the serogroup B S. Typhimurium vaccine limited intestinal colonization and systemic dissemination by serogroup C serovars Infantis and Hadar.

Impact of a Blend of Microencapsulated Organic Acids and Botanicals on the Microbiome of Commercial Broiler Breeders under Clinical Necrotic Enteritis.

Previously, the supplementation of a microencapsulated blend of organic acids and botanicals improved the health and performance of broiler breeders under non-challenged conditions. This study aimed to determine if the microencapsulated blend impacted dysbiosis and necrotic enteritis (NE) in broiler breeders. Day-of-hatch chicks were assigned to non-challenge and challenge groups, provided a basal diet supplemented with 0 or 500 g/MT of the blend, and subjected to a laboratory model for NE. On d 20-21, jejunum/ileum content were collected for microbiome sequencing (n = 10; V4 region of 16S rRNA gene). The experiment was repeated (n = 3), and data were analyzed in QIIME2 and R. Alpha and beta diversity, core microbiome, and compositional differences were determined (significance at p ≤ 0.05; Q ≤ 0.05). There was no difference between richness and evenness of those fed diets containing 0 and 500 g/MT microencapsulated blend, but differences were seen between the non-challenged and challenged groups. Beta diversity of the 0 and 500 g/MT non-challenged groups differed, but no differences existed between the NE-challenged groups. The core microbiome of those fed 500 g/MT similarly consisted of Lactobacillus and Clostridiaceae. Furthermore, challenged birds fed diets containing 500 g/MT had a higher abundance of significantly different phyla, namely, Actinobacteriota, Bacteroidota, and Verrucomicrobiota, than the 0 g/MT challenged group. Dietary supplementation of a microencapsulated blend shifted the microbiome by supporting beneficial and core taxa.

Characterization of Salmonella enterica isolates from turkeys in commercial processing plants for resistance to antibiotics, disinfectants, and a growth promoter.

Salmonella enterica isolates from turkeys in two commercial processing plants (1 and 2) were characterized for susceptibility to antibiotics, disinfectants, and the organoarsenical growth promoter, 4-hydroxy-3-nitrophenylarsonic acid (3-NHPAA, roxarsone), and it's metabolites, NaAsO(2) (As(III)) and Na(2)HAsO(4) • 7H(2)O (As(V)). The 130 Salmonella serovars tested demonstrated a low incidence of resistance to the antibiotics gentamicin (GEN), kanamycin (KAN), sulfamethoxazole (SMX), streptomycin (STR), and tetracycline (TET). Isolates resistant to antibiotics were most often multidrug resistant. Serovars Hadar and Typhimurium were resistant to KAN, STR, and TET and GEN, SMX, and STR, respectively. All isolated Salmonella serovars were resistant to the disinfectant chlorhexidine with minimum inhibitory concentrations (MICs; 1-8 µg/mL), and they were susceptible to triclosan and benzalkonium chloride. The didecyldimethylammonium chloride component was the most active ammonium chloride tested. No cross-resistance was observed between antibiotics and disinfectants. The MICs for 3-NHPAA (4096 µg/mL) were consistent between processing Plant 1 and Plant 2, but MICs for the 3-NHPAA metabolites (As(III) and As(V)) were higher in Plant 1 than in Plant 2. In Plant 1, 76% of the isolates had MICs >256 µg/mL for As(III) and 92% of the isolates had MICs >1024 µg/mL for As(V). In Plant 2, all of the isolates had MICs ≤256 µg/mL for As(III) and 90% of the isolates had MICs ≤1024 µg/mL for As(V). Only 4 Salmonella serovars were isolated from Plant 1, but 10 serovars were isolated from Plant 2. S. enterica serovar Derby from Plant 1 was highly resistant to As(III) and As(V) with MICs >1024 and >8192 µg/mL, respectively, suggesting previous exposure to high arsenic metabolite concentrations. These levels may have been high enough to kill other Salmonella serovars, thus possibly explaining the lack of serovar diversity observed in Plant 1. The application of a growth promoter may affect the serovar diversity in treated birds.

Controlling the Colonization of Clostridium perfringens in Broiler Chickens by an Electron-Beam-Killed Vaccine.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Feeding of yeast cell wall extracts during a necrotic enteritis challenge enhances cell growth, survival and immune signaling in the jejunum of broiler chickens.

Necrotic enteritis (NE) is one of the most common and costly diseases in the modern broiler industry, having an estimated economic impact of $6 billion dollars annually. Increasing incidents of NE have resulted from restrictions on the use of antibiotic feed additives throughout the broiler industry. As such, finding effective antibiotic alternatives has become a priority. In this study, an experimental model of NE was used, comprising a commercial infectious bursal disease virus vaccine and Clostridium perfringens (C. perfringens) inoculation. Yeast cells wall (YCW) components, ß-glucan (BG), and mannoproteins (MPTs) were evaluated for their effects on disease development. Chicken-specific immunometabolic kinome peptide arrays were used to measure differential phosphorylation between control (uninfected), challenged (infected), and challenged and treated birds in duodenal, jejunal, and ileal tissues. Treatment groups included crude YCW preparation, BG, MPT, or BG+MPT as feed additives. Data analysis revealed kinome profiles cluster predominantly by tissue, with duodenum showing the greatest relative signaling and jejunum showing the greatest response to treatment. BG, MPT, and BG+MPT cluster together, separate from controls and challenge birds in each tissue. Changes in signaling resulting from the treatments were observed in cell growth and survival responses as well as immune responses. None of the treatments of disease challenge returned the profiles to control-like. This is attributable to immune modulation and metabolic effects of the treatments generating distinct profiles from control. Importantly, all the treatments are distinct from the challenge group despite being challenged themselves. Only BG+MPT treatment had a significant effect on bird weight gain compared with the NE challenge group, and this treatment had the greatest impact on gut tissue signaling in all segments. The signaling changes elicited by BG+MPT during an NE challenge were increased cell growth and survival signaling, reducing cell death, apoptosis and innate inflammatory responses, and generating compensatory signaling to reduce disease severity.

Influence of different yeast cell wall preparations and their components on performance and immune and metabolic pathways in Clostridium perfringens-challenged broiler chicks.

A study was conducted to evaluate the influence of the purification of yeast cell wall (YCW) preparations on broiler performance and immunogenic and metabolic pathways under microbial challenge. A total of 240 (day old) chicks were distributed among two battery brooder units (48 pens; 5 birds/pen; 8 replicates/treatment). A basal starter diet was divided into 5 batches to create 6 dietary treatments; non-challenge (NCh-C) and challenge (Ch-C) controls, semi-purified YCW containing cytosol contents (SPYCW; 250 mg/kg), purified YCW (PYCW; 250 mg/kg), 50% purified beta-glucan (BG; 130 mg/kg), and 99.9% purified mannan-oligosaccharide (MOS; 53 mg/kg). All birds were immunocompromised with infectious bursal disease vaccine (10× the recommended dose) on day 10 and then all birds except NCh-C birds were challenged with Clostridium perfringens (Cp) (107 cfu/mL) via oral gavage on days 16 and 17. On day 21, tissue samples were collected from the jejunum and duodenum for analysis with chicken-specific, peptide arrays to study the influence of YCW supplementation on immune and metabolic kinase pathways. On day 16, SPYCW had significantly lower body weight (BW) and weight gain (WG) than other treatments except BG (P < 0.05). The productivity index (PI) was lower in SPYCW and BG than in NCh-C, Ch-C, and PYCW. On day 21, after the Cp challenge, NCh-C was higher than Ch-C, SPYCW, and BG in BW, WG, and PI (P = 0.03). The PI of PYCW was similar to NCh-C. The addition of purified YCW to the starter broiler diets influenced the immune and metabolic pathways in the gut. A total of 459 and 367 peptides in the duodenum and jejunum, respectively, were changed due to the Cp challenge. The YCW treatments had different degrees of influence on these peptides for both the duodenum and jejunum. These results suggest that relative purification of YCW and specific fractions of the YCW can influence broiler performance differently during microbial challenges and can alleviate the impact of these stressors.

Total radioactive residues and residues of [36Cl]chlorate in market size broilers.

The oral administration of chlorate salts reduces the numbers of Gram-negative pathogens in gastrointestinal tracts of live food animals. Although the efficacy of chlorate salts has been demonstrated repeatedly, the technology cannot be introduced into commercial settings without first demonstrating that chlorate residues, and metabolites of chlorate remaining in edible tissues, represent a negligible risk to consumers. Typically, a first step in this risk assessment is to quantify the parent compound and to identify metabolites remaining in edible tissues of animals treated with the experimental compound. The objectives of this study were to determine the pathway(s) of chlorate metabolism in market broilers and to determine the magnitude of chlorate residues remaining in edible tissues. To this end, 12 broilers (6 weeks; 2.70+/-0.34 kg) were randomly assigned to three treatments of 7.4, 15.0, and 22.5 mM sodium [36Cl]chlorate dissolved in drinking water (n=4 broilers per treatment). Exposure to chlorate, dissolved in drinking water, occurred at 0 and 24 h (250 mL per exposure), feed was withdrawn at hour 38, water was removed at hour 48, and birds were slaughtered at hour 54 (16 h after feed removal and 8 h after water removal). The radioactivity was rapidly eliminated in excreta with 69-78% of the total administered radioactivity being excreted by slaughter. Total radioactive residues were proportional to dose in all edible tissues with chloride ion comprising greater than 98.5% of the radioactive residue for the tissue (9.4-97.8 ppm chlorate equivalents). Chlorate residues were typically greatest in the skin (0.33-0.82 ppm), gizzard (0.1-0.137 ppm), and dark muscle (0.05-0.14 ppm). Adipose, liver, and white muscle tissue contained chlorate concentrations from 0.03 to 0.13 ppm. In contrast, chlorate concentrations in excreta eliminated during the 6 h period prior to slaughter ranged from 53 to 71 ppm. Collectively, these data indicate that broilers rapidly convert chlorate residues to an innocuous metabolite, chloride ion, and that chlorate residues in excreta remain fairly high during the time around slaughter. Because the target tissue of chlorate is the lower gastrointestinal tract, the relatively high distribution of parent chlorate to inedible gastrointestinal tissues and low distribution to edible tissues is favorable for the biological activity and for food safety considerations. These data, when used in conjunction with a toxicological assessment of chlorate, can be used to determine a likely risk/benefit ratio for chlorate.

Reduction of Salmonella Typhimurium in experimentally challenged broilers by nitrate adaptation and chlorate supplementation in drinking water.

The effects of two feed supplements on Salmonella Typhimurium in the ceca of market-age broilers were determined. Broilers orally challenged 6 days before slaughter with a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were divided into one of four groups (20 birds each). The first group (the control group) received no treatment, the second group received sodium nitrate (SN) treatment (574 mg of NaNO3 per kg of feed), the third group received experimental chlorate product (ECP) treatment (15 mM NaClO3 equivalents), and the fourth group received ECP treatment in combination with SN treatment. The SN treatment was administered via feed for 5 days immediately before slaughter, and ECP was provided via ad libitum access to drinking water for the last 2 days before slaughter. Cecal contents were subjected to bacterial analysis. Significant (P < 0.05) Salmonella Typhimurium reductions (ca. 2 log units) relative to levels for untreated control broilers were observed for broilers receiving ECP in combination with SN. The ECP-only treatment resulted in significant (P < 0.05) reductions (ca. 0.8 log) of Salmonella Typhimurium in trial 2. We hypothesize that increasing Salmonella Typhimurium nitrate reductase activity resulted in increased enzymatic reduction of chlorate to chlorite, with a concomitant decrease in cecal Salmonella Typhimurium levels. On the basis of these results, preadaptation with SN followed by ECP supplementation immediately preharvest could be a potential strategy for the reduction of Salmonella Typhimurium in broilers.

Hemolytic and antimicrobial activities differ among saponin-rich extracts from guar, quillaja, yucca, and soybean.

Hemolytic and antibacterial activities of eight serial concentrations ranged from 5-666 microg/mL of saponin-rich extracts from guar meal (GM), quillaja, yucca, and soybean were tested in 96-well plates and read by enzyme-linked immunosorbent assay plate-well as 650 nm. Hemolytic assay used a 1% suspension of chicken red blood cells with water and phosphate buffered saline as positive and negative controls, respectively. Antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were evaluated using ampicillin and bacteria without saponin-rich extract as positive and negative controls, respectively. The 100% MeOH GM and commercial quillaja saponin-rich extracts were significantly the highest in both hemolytic and antibacterial activities against all bacteria at the same concentration tested. Soybean saponin-rich extract had no antibacterial activity against any of the bacteria at the concentrations tested while yucca saponin-rich extract had no antibacterial activity against the gram-negative bacteria at the concentrations tested. GM and quillaja saponin-rich extracts were hemolytic, while yucca and soybean saponin-rich extracts were not hemolytic at the concentrations tested. No saponin-rich extract source had antibacterial activity against S. typhimurium or E. coli at the concentrations tested. Both GM and quillaja saponin-rich extracts exhibited antibacterial activity against S. aureus. Saponin-rich extracts from different plant sources have different hemolytic and antibacterial activities.