Serum Fluorescein Isothiocyanate Dextran Research Articles

Dietary Zn proteinate with moderate chelation strength alleviates heat stress-induced intestinal barrier function damage by promoting expression of tight junction proteins via the A20/NF-κB p65/MMP-2 pathway in the jejunum of broilers

BackgroundThe aim of this study was to determine whether and how Zn proteinate with moderate chelation strength (Zn-Prot M) can alleviate heat stress (HS)-induced intestinal barrier function damage of broilers. A completely randomized design was used for comparatively testing the effects of Zn proteinate on HS and non-HS broilers. Under high temperature (HT), a 1 (Control, HT-CON) + 2 (Zn source) × 2 (added Zn level) factorial arrangement of treatments was used. The 2 added Zn sources were Zn-Prot M and Zn sulfate (ZnS), and the 2 added Zn levels were 30 and 60 mg/kg. Under normal temperature (NT), a CON group (NT-CON) and pair-fed group (NT-PF) were included.ResultsThe results showed that HS significantly reduced mRNA and protein expression levels of claudin-1, occludin, junctional adhesion molecule-A (JAMA), zonula occludens-1 (ZO-1) and zinc finger protein A20 (A20) in the jejunum, and HS also remarkably increased serum fluorescein isothiocyanate dextran (FITC-D), endotoxin and interleukin (IL)-1β contents, serum diamine oxidase (DAO) and matrix metalloproteinase (MMP)-2 activities, nuclear factor kappa-B (NF-κB) p65 mRNA expression level, and protein expression levels of NF-κB p65 and MMP-2 in the jejunum. However, dietary supplementation with Zn, especially organic Zn as Zn-Prot M at 60 mg/kg, significantly decreased serum FITC-D, endotoxin and IL-1β contents, serum DAO and MMP-2 activities, NF-κB p65 mRNA expression level, and protein expression levels of NF-κB p65 and MMP-2 in the jejunum of HS broilers, and notably promoted mRNA and protein expression levels of claudin-1, ZO-1 and A20.ConclusionsOur results suggest that dietary Zn, especially 60 mg Zn/kg as Zn-Prot M, can alleviate HS-induced intestinal barrier function damage by promoting the expression of TJ proteins possibly via induction of A20-mediated suppression of the NF-κB p65/MMP-2 pathway in the jejunum of HS broilers.

Impacts of Excreta Exposure and Age on Ileal Microbial Communities, Intestinal Permeability, and Corticosterone in Hens Housed in Enriched Colonies and Cage-Free Housing Systems

To tease apart differences between conventional cage (CC) and cage-free (CF) housing systems, this study focuses on the effects of excreta exposure and age by comparing microbial communities, intestinal permeability, and corticosterone in hens in enriched colonies (EC) and CF housing systems during early- and late-lay. Hens were randomly selected from two rooms of CF (n = 20) and EC (n = 20) at 35 and 76 weeks of age. One hour following an oral gavage of fluorescein isothiocyanate dextran (FITC-D), hens were euthanized, and ileal contents and blood were collected. Serum FITC-D using a fluorescent spectrophotometer and corticosterone using a commercial competitive ELISA kit were analyzed. Following DNA isolation from the ileum contents, the V4 region of the 16S rRNA gene was sequenced. Sequence data were filtered in Mothur v1.43.0, followed by de novo operational taxonomic unit (OTU) clustering and classifying with the SILVA SSU v138 reference database. Serum FITC-D was altered by housing type, age of hens, and the interaction between housing type and age of hens (p < 0.001), with 76-week-old hens housed in EC having the highest FITC-D. Corticosterone increased with age (p = 0.023). Microbial community diversity measurements favored hens housed in the CF housing system as ileal contents tended to have increased species evenness (p = 0.008) and greater alpha diversity (p = 0.006). The majority of the over-representation of OTUs were associated with peak lay.

Investigation of Potential Gut Health Biomarkers in Broiler Chicks Challenged by Campylobacter jejuni and Submitted to a Continuous Water Disinfection Program.

The exploration of novel biomarkers to assess poultry health is of paramount importance, not only to enhance our understanding of the pathogenicity of zoonotic agents but also to evaluate the efficacy of novel treatments as alternatives to antibiotics. The present study aimed to investigate potential gut health biomarkers in broiler chicks challenged by Campylobacter jejuni and subjected to a continuous water disinfection program. A total of 144 one-day-old hatched broiler chicks were randomly allocated to four treatment groups with four replicates each, according to the following experimental design: Group A received untreated drinking water; Group B received drinking water treated with 0.01-0.05% v/v Cid 2000™ (hydrogen peroxide, acetic acid and paracetic acid); Group C was challenged by C. jejuni and received untreated drinking water; and Group D was challenged by C. jejuni and received drinking water treated with 0.01-0.05% v/v Cid 2000™. The use of Cid 2000™ started on day 1 and was applied in intervals until the end of the experiment at 36 days, while the C. jejuni challenge was applied on day 18. Potential biomarkers were investigated in serum, feces, intestinal tissue, intestinal content, and liver samples of broilers. Statistical analysis revealed significant increases (p < 0.001) in serum cortisol levels in C. jejuni-challenged broilers. Serum fluorescein isothiocyanate dextran (FITC-d) increased significantly (p = 0.004) in broilers challenged by C. jejuni and treated with drinking water disinfectant, while fecal ovotransferrin concentration also increased significantly (p < 0.001) in broilers that received the drinking water disinfectant alone. The gene expression levels of occludin (p = 0.003) and mucin-2 (p < 0.001) were significantly upregulated in broilers challenged by C. jejuni, while mucin-2 significantly increased in birds that were challenged and received the drinking water disinfectant (p < 0.001). TLR-4 expression levels were significantly (p = 0.013) decreased in both groups that received the drinking water disinfectant, compared to the negative control group. Finally, the C. jejuni challenge significantly increased (p = 0.032) the crypt depth and decreased (p = 0.021) the villus height-to-crypt-depth ratio in the ileum of birds, while the tested disinfectant product increased (p = 0.033) the villus height in the jejunum of birds. Furthermore, the counts of C. jejuni in the ceca of birds (p = 0.01), as well as its translocation rate to the liver of broilers (p = 0.001), were significantly reduced by the addition of the water disinfectant. This research contributes to novel insights into the intricate interplay of water disinfection and/or C. jejuni challenge with potential intestinal biomarkers. In addition, it emphasizes the need for continued research to unveil the underlying mechanisms, expands our understanding of broiler responses to these challenges and identifies breakpoints for further investigations.

25 Measuring Intestinal Permeability When Urine is not an Option

Abstract The need to understand intestinal health is of increasing importance as the livestock industry seeks to maintain performance efficiency while eliminating prophylactic antibiotic usage. Intestinal permeability is a biomarker of intestinal health as it measures the movement of an indigestible marker across the intestinal barrier with excretion in the urine as the primary biological specimen collected for measurement. The adoption of these methods into poultry has been slow due to the lack of urine excretion. Therefore, the objective of this study is to compare differences in intestinal permeability assays in serum of poultry to determine if these assays could be a reliable method to detect changes in intestinal permeability in poultry under a known stressor. To compare the two commonly used methods [fluorescein isothiocyanate-dextran (FITC-D) method and lactulose/mannitol (LMS) dual sugar method], 2 independent trials were conducted with 40 pens split across 2 fed status (fed or fasted for 12 hours) and 4 sugar treatments (no sugar, FITC-D, LMS, FITC-D+LMS). All pens were sampled at 3 timepoints (14, 28, and 42 days of age) with 5 birds sampled per timepoint. Serum FITC-D was determined through fluorescent spectrophotometry, while mannitol and lactulose concentrations were determined using ion chromatography. Data were analyzed using PROC Glimmix. Fed status, sugar treatment, age, and all 2-and 3-way interactions were included as fixed effects. Trial was included in the model as a random effect with pen included as a repeated effect. Serum lactulose and FITC-D increased in fasted compared with fed birds (P &amp;lt; 0.006). Surprisingly, mannitol, which was expected to be similar across fed and fasted, was increased in fed compared with fasted birds (P &amp;lt; 0.001). The increased concentrations of mannitol in fed birds and measurable concentrations of mannitol in birds not given mannitol suggest another molecule eluted at the same rate as mannitol. Therefore, we propose under these conditions that mannitol, and its ratio with lactulose, are not appropriate methods for measuring intestinal permeability in poultry. Lactulose and FITC-D were increased in serum when the birds were provided those sugars and serum concentration of both sugars decreased at 28 days compared with 14 and 42 days (P &amp;lt; 0.003). FITC-D had a significant 2-way interaction for the sugar by fed status (P &amp;lt; 0.05) whereas other 2-and 3-way interactions were similar across treatments (P &amp;gt; 0.202). Serum lactulose concentrations were affected by all 2-and 3-way interactions with elevated serum lactulose concentrations in broilers provided lactulose and fasted (P &amp;lt; 0.001). Birds that did not receive lactulose had very low to undetectable concentrations of lactulose. This reduced background noise for the detection of serum lactulose and the ability statistically to detect small changes in intestinal permeability make lactulose a potential serum biomarker for intestinal permeability in poultry.

REMOTE BURN INJURY IN AGED MICE INDUCES COLONIC LYMPHOID AGGREGATE EXPANSION AND DYSBIOSIS OF THE FECAL MICROBIOME WHICH CORRELATES WITH NEUROINFLAMMATION.

The Earth's population is aging, and by 2050, one of six people will be 65 years or older. Therefore, proper treatment of injuries that disproportionately impact people of advanced age will be more important. Clinical studies reveal people 65 years or older account for 16.5% of all burn injuries and experience higher morbidity, including neurocognitive decline, and mortality that we and others believe are mediated, in part, by heightened intestinal permeability. Herein, we used our clinically relevant model of scald burn injury in young and aged mice to determine whether age and burn injury cooperate to induce heightened colonic damage, alterations to the fecal microbiome, and whether resultant changes in the microbiome correlate with neuroinflammation. We found that aged, burn-injured mice have an increase in colonic lymphoid aggregates, inflammation, and proinflammatory chemokine expression when compared with young groups and sham-injured aged mice. We then performed fecal microbiota sequencing and found a striking reduction in gut protective bacterial taxa, including Akkermansia , in the aged burn group compared with all other groups. This reduction correlated with an increase in serum fluorescein isothiocyanate-Dextran administered by gavage, indicating heightened intestinal permeability. Furthermore, loss of Akkermansia was highly correlated with increased messenger RNA expression of neuroinflammatory markers in the brain, including chemokine ligand 2, TNF-α, CXC motif ligand 1, and S100 calcium-binding protein A8. Finally, we discovered that postburn alterations in the microbiome correlated with measures of strength in all treatment groups, and those that performed better on the rotarod and hanging wire tests had higher abundance of Akkermansia than those that performed worse. Taken together, these findings indicate that loss of protective bacteria after burn injury in aged mice contributes to alterations in the colon, gut leakiness, neuroinflammation, and strength. Therefore, supplementation of protective bacteria, such as Akkermansia , after burn injury in aged patients may have therapeutic benefit.

Comparison of Intestinal Permeability Methods in Broilers over a 6-Week Growth Period

The adoption of methods detecting intestinal permeability in poultry has been slow due to the lack of urine availability in avian species. The objective of this study was to examine intestinal permeability assays in broilers using serum. Fluorescein isothiocyanate-dextran (FITC-D) and lactulose/mannitol/sucralose (LMS), indigestible sugars, were used to detect intestinal permeability across two fed states (fed or fasted) and four sugar treatments (Control, FITC-D, LMS, or FITC-D+LMS). Broilers housed in pens were assigned one of eight treatments and sampled on 14, 28, and 42 days of age. Data were analyzed using PROC Glimmix for fed state, sugar treatment, age, and all interactions. Serum lactulose and FITC-D increased in fasted compared to fed birds (p &lt; 0.006), whereas mannitol increased in fed compared to fasted birds (p &lt; 0.001). Serum lactulose and FITC-D decreased on day 28 compared to other timepoints (p &lt; 0.003). Serum FITC-D only had a significant sugar by fed state interaction (p &lt; 0.05) with elevated concentrations in fasted and fed birds that received FITC-D. Serum lactulose was significant for all interactions with elevated concentrations in broilers provided lactulose and fasted (p &lt; 0.001). The ability to detect a three-way interaction with serum lactulose suggests an increased sensitivity; however, additional studies are needed.

Sanguinarine-based isoquinoline alkaloids modulated the gut-brain axis and enhanced growth performance and gut integrity in natural heat stress broiler chickens

Persistent hot and humid tropical climate induces heat stress (HS), negatively impacting gut integrity with inflammation and tight junction degradation in broilers. As integrating operations of the digestive tract and hypothalamus in avian are regulated by feed intake, the effect of HS may be eased by feed supplementation. Herein, the effect of isoquinoline alkaloids (IQ) supplementation from Macleaya cordata on integrating gut-brain axis and gut integrity was assessed. 720 male broilers (Ross 308) were treated with IQ at null (control), 60 (IQ60), and 100 mg/kg (IQ100). They were reared in open production house between April and May in Southern Thailand with 33.0–38.0°C (average 35.0°C), 75–80% relative humidity (RH), and 160–200 heat stress index (HI). From day 1 to 42, IQ supplementation positively affected growth performance where body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly higher in birds fed IQs than in the control group (P<0.05), and feed conversion ratio (FCR) was significantly lower in the IQ group (P<0.05). IQ improved gut integrity by reducing inflammation with significant down-regulation of nitric oxide synthase (iNOS), cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), and nuclear factor κB (NF-κB) in the ileum (P<0.05). It strengthened tight junction by upregulates occludin (OCLD), claudin-1 (CLDN1), tight junction proteins-1 (TJP-1) and mucin-2 (MUC-2) in the jejunum (P<0.05). As a result, serum fluorescein isothiocyanate-dextran (FITC-d) and corticosterone levels were significantly lower (P<0.05). In terms of the gut-brain axis, IQ supplementation reduces ghrelin and cholecystokinin (CCK) mRNA expression in the jejunum and hypothalamus (P<0.05), which downregulated the expression of pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotropin-releasing hormone (CRH), and melanocortin four receptor (MC4R). It was the first evidence that IQ influences on modulating the gut-brain axis to suppress anorexigenic regulation in broilers with HS exposure.

Dexmedetomidine alleviates intestinal barrier dysfunction and inflammatory response in mice via suppressing TLR4/MyD88/NF-κB signaling in an experimental model of ulcerative colitis.

Ulcerative colitis (UC) is a nonspecific intestinal inflammatory disease. Dexmedetomidine (DEX) is a selective alpha 2-adrenergic receptor agonist commonly used for analgesia and sedation in intensive care units. Herein, the role and mechanism of DEX in dextran sulfate sodium (DSS)-induced colitis was explored. A murine model of DSS-induced colitis was established by adding 3.5% (w/v) DSS in drinking water to C57BL/6J female mice. The severity of colitis was measured by the disease activity index (DAI) score, colon length and body weight of mice. The serum concentration and mRNA levels of inflammatory cytokines in colon tissues were assessed by ELISA and RT-qPCR, respectively. Protein levels of apoptotic markers, tight junction proteins and genes involved in the TLR4/MyD88/NF-κB signaling were quantified utilizing Western blotting. The pathological changes of colon tissues were evaluated by hematoxylin-eosin (HE) staining and histological score. Intestinal permeability in vivo was assessed by fluorescein isothiocyanate (FITC)-dextran (FITC-D) administration. TUNEL assay was used to determine cell apoptosis in the intestinal epithelium. DSS administration resulted in weight loss, shortening of the colon, increased DAI score, histological abnormalities, and increased serum FITC-D levels in mice, all of which were reversed by DEX injection. Moreover, DEX attenuated DSS-triggered inflammatory response, intestinal barrier injury and intestinal epithelial cell apoptosis. Mechanically, DEX inactivated the TLR4/MyD88/NF-κB signaling in the colon tissues. DEX exerts beneficial effects against the intestinal barrier dysfunction, inflammatory response, and apoptosis of intestinal epithelial cells via inactivation of the TLR4/MyD88/NF-κB signaling in mice with DSS-induced colitis.

Research Note: Virulence gene downregulation and reduced intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate in broilers by a natural antimicrobial (NeutraPath™)

The reduction in antibiotic growth promoter use in poultry, due to antibiotic resistance concerns, has created a need for natural solutions that control enteric pathogens like Salmonella. One of these natural feed additives, a select blend of essential oils, fatty acids, and an enterosorbent mineral (NeutraPath), was assessed for its effects on the intestinal colonization of Salmonella enterica serovar Typhimurium PHL2020 isolate (ST-PHL2020) in broiler chickens and ST-PHL2020 virulence gene expression. An in vitro digestion model simulating the pH and enzymatic conditions of 3 gastrointestinal compartments (crop, proventriculus, and intestine) was first used to evaluate the antibacterial effects of NeutraPath on ST-PHL2020. For the in vivo study, day-old male broilers (n = 90) were randomly allocated to 1 of 3 groups: control or NeutraPath supplemented at 0.25 or 0.5%. The dose rates were chosen to enable observable statistical effects during high Salmonella challenge. All groups were challenged with ST-PHL2020 (106 cfu/bird) via oral gavage on day 9. Bacterial load and prevalence of ST-PHL2020 were examined in ceca-cecal tonsils, and intestinal permeability was assessed via serum recovery of fluorescein isothiocyanate dextran (FITC-d) 24 h postchallenge. NeutraPath inhibited (P < 0.05) ST-PHL2020 growth in the in vitro digestion model compared to the control at all concentrations and in all compartments other than NeutraPath 0.25% in the crop. In vivo, NeutraPath 0.25 and 0.5% reduced (P < 0.05) the total cfu recovered and total prevalence of ST-PHL2020 in the ceca. The serum FITC-d levels were also reduced (P < 0.05) by NeutraPath. Further, NeutraPath's effects on ST-PHL2020’s Salmonella pathogenicity island-1 virulence network development were explored via treating ST-PHL2020 at subinhibitory concentration (1 mg/mL) of NeutraPath in vitro. Compared to the control, NeutraPath downregulated ST-PHL2020 hilA and invF mRNA expression, which further blocked expression of key downstream effectors involved in ST-PHL2020 invasion. Collectively, NeutraPath has the potential to reduce ST-PHL2020 intestinal colonization in broilers and preserve intestinal barrier integrity.

Effects of a direct fed microbial (DFM) on broiler chickens exposed to acute and chronic cyclic heat stress in two consecutive experiments

Two consecutive 35 d experiments were conducted to investigate the effects of a multistrain DFM fed continuously to broiler chickens exposed to HS from 28 to 35 d on broiler performance, body composition, ileal digestibility, and intestinal permeability using serum Fluorescein Isothiocyanate Dextran (FITC-d) concentration. The treatments were arranged as a 2 × 2 factorial with temperature: Elevated (HS: 33 ± 2°C for 6 h and 27.7°C for the remaining 18 h from 28 to 35 days of age) and Thermoneutral (TN: 22 to 24°C over the entire 24-h day from 28 to 35 days of age) and diet: corn-soybean meal based with and without DFM (3-strain Bacillus; Enviva PRO) fed over the entire 35-d period as the two factors. Experimental diets were formulated to meet all nutrient recommendations based on breed standards using a starter (0–10 d), grower (10–21 d), and finisher (21–35 d) period. For each of the 2 experiments, 648 Ross 708 broiler chicks were allotted among the treatments with 9 replicate pens of 18 broilers. Data were analyzed as a 2 × 2 factorial within each experiment in JMP 14. In both experiments, cloacal temperatures were increased (P ≤ 0.05) in the broilers subjected to the HS treatment at both 28 d (acute) and 35 d (chronic). Supplementing birds with DFM reduced cloacal temperatures in the Experiment 1 at 28 d, but not at the other time periods. The HS treatment reduced body weight gain and lean tissue accretion from 0 to 35 d in both experiments (P ≤ 0.05). In Experiment 2, when the litter was reused BWG was increased by 36 g/bird with supplementation of DFM (P ≤ 0.05). Ileal digestibility at 28 d (2 h post HS) was improved with DFM supplementation in both experiments (P ≤ 0.05). Serum FITC-d increased with HS at both 28 and 35 d. Serum FITC-d was generally decreased with DFM at 28 d but the response was inconsistent at 35 d. Overall, the results suggest that HS reduced broiler performance and DFM treatment improved intestinal permeability and nutrient digestibility responses to HS in both experiments but did not improve performance until built up litter was used in Experiment 2.

Supplementation of reduced protein diets with

Context Necrotic enteritis (NE) has been considered a major threat to broiler gut health and growth performance. Aims This study aimed at investigating the effects of l-arginine (Arg) or l-citrulline (Cit) supplementation on intestinal morphology, short-chain fatty acid (SCFA), microbiota count, gut permeability, and pH in broilers fed reduced-protein diets during subclinical NE challenge. Methods Ross 308 cockerels (n = 720) were randomly assigned to six experimental treatments with eight replicates of 15 birds per pen. The treatments were standard protein without NE challenge (SP−), or with NE challenge (SP+); reduced protein (two percentage points lower crude protein) without NE challenge (RP−), or with NE challenge (RP+); RP+ plus added Arg (103% of Ross 308 requirement, RPA+) and RPC+ where supplemental Arg in RPA+ was replaced with Cit. A 2 × 2 factorial arrangement was employed for the first four treatments. Factors were NE (− or +) and protein concentration (SP or RP). Treatments SP+, RP+, RPA+, and RPC+ were analysed by one-way ANOVA. Key results Necrotic enteritis × protein interactions were detected for serum fluorescein isothiocyanate dextran (FITC-d) level, C. perfringens (P &lt; 0.05) count in the caeca (P &lt; 0.01), and acetic acid (P &lt; 0.01) and total SCFA concentrations in the ileum on Day 16 (P &lt; 0.001). Feeding the RP diet reduced serum FITC-d concentration, number of C. perfringens in the caeca, and increased acetic acid and total SCFA concentrations in the ileum compared with the SP group only in birds challenged with NE. Birds in the RPC+ treatment had greater jejunal villus height (P &lt; 0.001), and lower caecal C. perfringens and Enterobacteriaceae count than did those in the SP+ treatment (P ≤ 0.001). Conclusions The results indicated a benefit to gut health of broilers during NE challenge when replacing crystalline Arg with Cit in RP diets. Implications In part, replacement of Arg by Cit in the RP diets is of great potential to increase gut health, reduce growth loss, thus, minimising negative effects of NE in broilers.

Effect of blending encapsulated essential oils and organic acids as an antibiotic growth promoter alternative on growth performance and intestinal health in broilers with necrotic enteritis

The effects of a blend of encapsulated organic acids with essential oils (EOA) as an alternative to antimicrobial growth promoter (AGP) on growth performance and gut health of Eimeria spp./Clostridium perfringens (C. perfringens) in chickens infected with necrotic enteritis (NE) broilers was investigated. A total of 432 male Arbor Acres broilers (1-day-old) were randomly distributed into 6 treatment groups, namely noninfected negative control (A); NE-infected positive control (D); NE-infected broiler chickens fed a basal diet supplemented with 250 mg/kg bacitracin methylene disalicylate (BMD) plus 90 mg/kg monensin; and NE-infected broiler chicken fed 200; 500; and 800 mg/kg EOA (E, F, G, and H group). Feeding EOA at 200 and 500 mg/kg considerably improved the feed conversion ratio, reduced gut lesions, serum fluorescein isothiocyanate dextran level, and C. perfringens load in the caecum and liver of the NE-infected broiler chickens. This feed was similar to AGP. Furthermore, the increased villous height-to-crypt depth ratio and goblet cells counts, upregulated claudin-1, glucagon-like peptide-2 (GLP-2), insulin-like growth factor-2 (IGF-2) mRNA gene expression, downregulated occludin, zonula occludens-1 (ZO-1), toll-like receptor (TLR-4), interleukin (IL-1β), interferon γ (IFN-γ), TNF receptor-associated factor 6 (TRAF-6), tumor necrosis factor superfamily member 15 (TNFSF15), and Toll-interacting protein (Tollip) genes expression in the jejunum were observed in the NE-infected broiler chickens that received EOA at 200 and 500 mg/kg compared with those of the single NE-challenged groups without EOA supplementations (P < 0.05). The 16S analysis revealed that EOA supplemented with 200 or 500 mg/kg enriched relative abundance of Lactobacillus, unclassified_Lachnospiraceae, and Enterococcus, and carbohydrate metabolic pathways but suppressed unclassified_Erysipelotrichacease and organismal systems involved in the immune system (P < 0.05). Feeding EOA could alleviate NE-induced gut impairment and growth depression and modulate cecal microbiota composition, which has potential as antimicrobial alternatives.

Mitigation of AFB1-Related Toxic Damage to the Intestinal Epithelium in Broiler Chickens Consumed a Yeast Cell Wall Fraction.

In vivo experiments were conducted to evaluate the effectiveness of a yeast cell wall fraction (YCW) to reduce the negative impact of aflatoxin B1 (AFB1) to the intestinal epithelium in broiler chickens. Zeta potential (ζ-potential), point of zero charge (pHpzc), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) techniques were used to characterize the YCW. Two hundred one-day-old male Ross 308 broiler chickens were randomly allocated into four treatments: (1) control, chickens fed an AFB1-free diet; (2) AF, chickens feed an AFB1-contaminated diet (500 ng AFB1/g); (3) YCW, chickens fed an AFB1-free diet + 0.05% YCW; and (4) AF + YCW, chickens fed an AFB1-contaminated diet (500 ng AFB1/g) + 0.05% YCW. At the end of the 21-day feeding period, fluorescein isothiocyanate dextran (FITC-d) was administered to chicks by oral gavage to evaluate gastrointestinal leakage. Blood and duodenum samples were collected to assess serum biochemistry and histomorphology, respectively. Compared to the control group, chicks of the AF group significantly diminished weight gain (WG) and average daily feed intake (ADFI), and increased feed conversion ratio (FCR), mortality rate (MR), and intestinal lesion scores (p < 0.05). Alterations in some serum biochemical parameters, and damage to the intestinal integrity were also evident in the AF-intoxicated birds. YCW supplementation improved WG and FCR and increased villus height, villus area, crypt depth, and the number of goblet cells in villi. The effects of YCW on growth performance were not significant in chicks of the AF + YCW group; however, the treatment decreased MR and significantly ameliorated some biochemical and histomorphological alterations. The beneficial effect of YCW was more evident in promoting gut health since chickens of the AF + YCW group presented a significant reduction in serum FITC-d concentration. This positive effect was mainly related to the changes in negative charges of YCW due to changes in pH, the net negative surface charge above the pHpzc, the higher quantities of negative charged functional groups on the YCW surface, and its ability to form large aggregates. From these results, it can be concluded that YCW at low supplementation level can partially protect broilers' intestinal health from chronic exposure to AFB1.

A Microencapsulated Mixture of Eugenol and Garlic Tincture Supplementation Mitigates the Effect of Necrotic Enteritis on Intestinal Integrity and Increases Goblet Cells in Broilers.

This study was conducted to examine the effects of a plant extract mixture, a microencapsulated product composed of eugenol and garlic tincture (PE), on intestinal health in broilers under necrotic enteritis (NE) challenge. A total of 960 d-old mixed-sex Cobb 500 chicks were randomly distributed to 48-floor pens housing 20 birds per pen. Six treatments were applied: UC, unchallenged control; CC, challenged control; PE, challenged group plus PE; AM, challenged group plus antimicrobial (AM); FAP, challenged group plus a full dose of AM with PE; HAP, challenged group plus a half dose of AM with PE in starter, grower and finisher phases. Birds in the challenged groups were inoculated with Eimeria spp. on d 9 and Clostridium perfringens on d 14. On d 16, the CC group had increased serum fluorescein isothiocyanate dextran (FITC-d), reduced villus surface area, goblet cell number, upregulated CLDN1, JAM2 genes and reduced microbial diversity compared to the UC group (p < 0.05). Birds fed PE had reduced FITC-d, increased goblet cell number and Bifidobacterium compared to the CC group (p < 0.05). Birds fed PE had reduced CLDN5 expression in male birds, and Bacteroides spp. in female birds than CC group (p < 0.05). These findings suggest that PE supplementation mitigates the effect of NE by improving the intestinal health of birds.

Enteric permeability and inflammation associated with day of hatch Enterobacteriaceae inoculation

Early exposure to Enterobacteriaceae may result in inappropriate microbial colonization of the gastrointestinal (GI) tract, induce mild GI inflammation, alter immune system development, and predispose poultry to opportunistic infection. Four experiments were conducted to test Enterobacteriaceae isolates Escherichia coli LG strain (LG), E. coli Huff strain (Huff), Salmonella Enteritidis LB (SE) and Salmonella Typhimurium (ST) on ability to induce GI inflammation. All 4 experiments included a noninoculated control, and day of hatch (DOH) oral inoculation of LG, Huff, SE and ST in experiment 1, LG and SE in experiment 2, and LG, Huff, SE, and ST in experiment 3. Experiment 4 included LG, Huff, a noninoculated control (NIC), and Clostridium perfringens only (NCP) wherein birds received oral C. perfringens challenge on d15-16 to induce necrotic enteritis. Body weight was measured, yolk sacs and spleens were collected, and blood was obtained for serum fluorescein isothiocyanate dextran (FITC-d) recovery and alpha-1-acid glycoprotein (A1GP) concentrations. Samples were taken weekly through 2 wk of age in experiments 1 and 2, or 4 wk of age in experiments 3 and 4. Increased FITC-d recovery was observed for LG and SE on d13 in experiment 2 (P < 0.05), and C. perfringens only birds on d27 in experiment 4 (P < 0.05) as compared to noninoculated controls. Each experiment resulted in notable differences in A1GP serum concentrations over time, with fluctuations in A1GP patterns through d14 based on DOH inoculation (P < 0.05). Over time, A1GP was increased for DOH inoculated birds from d 22 to 29, the fourth wk of life, and d 2-29, the entire experiment, vs. noninoculated controls in experiment 3 (P < 0.05). Similarly, NCP and LGCP showed increased A1GP from d 20 to 27 and d 6 to 27, vs. NIC in experiment 4 (P < 0.05). In experiment 4, C. perfringens challenge resulted in earlier A1GP response in DOH inoculated birds, d 17-20, as compared to NCP birds, d 20-27 (P < 0.05). These results suggest early Enterobacteriaceae exposure may influence early inflammatory state in the GI tract and may also alter patterns of inflammation and responsiveness to pathogens.

Evaluation of curcumin and copper acetate against Salmonella Typhimurium infection, intestinal permeability, and cecal microbiota composition in broiler chickens

BackgroundInterest in the use of natural feed additives as an alternative to antimicrobials in the poultry industry has increased in recent years because of the risk of bacterial resistance. One of the most studied groups are polyphenolic compounds, given their advantages over other types of additives and their easy potentiation of effects when complexes are formed with metal ions. Therefore, the objective of the present study was to evaluate the impact of dietary supplementation of copper acetate (CA), curcumin (CR), and their combination (CA-CR) against Salmonella Typhimurium colonization, intestinal permeability, and cecal microbiota composition in broiler chickens through a laboratory Salmonella infection model. S. Typhimurium recovery was determined on day 10 post-challenge by isolating Salmonella in homogenates of the right cecal tonsil (12 chickens per group) on Xylose Lysine Tergitol-4 (XLT-4) with novobiocin and nalidixic acid. Intestinal integrity was indirectly determined by the fluorometric measurement of fluorescein isothiocyanate dextran (FITC-d) in serum samples from blood obtained on d 10 post-S. Typhimurium challenge. Finally, microbiota analysis was performed using the content of the left caecal tonsil of 5 chickens per group by sequencing V4 region of 16S rRNA gene.ResultsThe results showed that in two independent studies, all experimental treatments were able to significantly reduce the S. Typhimurium colonization in cecal tonsils (CT, P < 0.0001) compared to the positive control (PC) group. However, only CA-CR was the most effective treatment in reducing S. Typhimurium counts in both independent studies. Furthermore, the serum fluorescein isothiocyanate dextran (FITC-d) concentration in chickens treated with CR was significantly lower when compared to PC (P = 0.0084), which is related to a decrease in intestinal permeability and therefore intestinal integrity. The effect of dietary treatments in reducing Salmonella was further supported by the analysis of 16S rRNA gene sequences using Linear discriminant analysis effect size (LEfSe) since Salmonella was significantly enriched in PC group (LDA score > 2.0 and P < 0.05) compared to other groups. In addition, Coprobacillus, Eubacterium, and Clostridium were significantly higher in the PC group compared to other treatment groups. On the contrary, Fecalibacterium and Enterococcus in CR, unknown genus of Erysipelotrichaceae at CA-CR, and unknown genus of Lachnospiraceae at CA were significantly more abundant respectively.ConclusionsCR treatment was the most effective treatment to reduce S. Typhimurium intestinal colonization and maintain better intestinal homeostasis which might be achieved through modulation of cecal microbiota.

Effects of plant-derived isoquinoline alkaloids on growth performance and intestinal function of broiler chickens under heat stress

Broiler chickens reared under heat stress (HS) conditions have decreased growth performance and show metabolic and immunologic alterations. This study aimed to evaluate the effect of supplementation with a standardized blend of plant-derived isoquinoline alkaloids (IQ) on the growth performance, protein catabolism, intestinal barrier function, and inflammatory status of HS-treated chickens. Three hundred sixty 0-day-old Ross 308 male broiler chickens were randomly distributed into 2 treatment groups: control diet (no additives) or diet supplemented with 100 ppm IQ. At day 14, the chicks in each diet group were further divided into 2 groups, each of which was reared under thermoneutral (TN) (22.4°C) or constant HS (33.0°C) conditions until day 42. Each group consisted of 6 replicates with 15 birds per replicate, and chickens were provided ad libitum access to water and feed. During days 15–21, the body weight gain (BWG) and feed intake (FI) were significantly lower in the HS treatment group than in the TN group, and feed conversion ratio was higher (P < 0.05); these factors were not alleviated by IQ supplementation. During days 22–42, the final BW, BWG, and FI of the HS birds were better among those administered IQ than those that were not (P < 0.05). HS treatment increased plasma lipid peroxide, corticosterone, and uric acid concentrations as well as serum fluorescein isothiocyanate–dextran, a marker of intestinal barrier function, and decreased plasma total protein content (P < 0.05). These changes were not observed in the IQ group, suggesting that IQ supplementation improved oxidative damage, protein catabolism, and intestinal barrier function of chickens under HS. Isoquinoline alkaloid supplementation inhibited the expression of intestinal inflammatory factors, IL-6, tumor necrosis factor–like factor 1A, and inducible nitric oxide synthase under HS treatment (P < 0.05). These results suggest that IQ supplementation can improve the growth performance of broiler chickens under HS conditions, which may be associated with amelioration of oxidative damage, protein catabolism, intestinal barrier function, and inflammation.

Comparison of intestinal permeability, morphology, and ileal microbial communities of commercial hens housed in conventional cages and cage-free housing systems

The gastrointestinal health of poultry can be impacted by a variety of factors including their environment. As egg production moves from conventional cage housing (CC) toward cage-free housing (CF), it is important to understand this impact on intestinal health. This study was conducted to determine if housing type impacted intestinal permeability, morphology, and microbial communities in commercial hens across housing systems. Hens were randomly selected from 2 rooms of CC (n = 25) and CF (n = 25) at a commercial facility. Birds were given fluorescein isothiocyanate dextran (FITC-D) by oral gavage to measure intestinal permeability. Jejunal and ileal samples were collected to evaluate villus height, crypt depth, and their ratio. Ileal contents were collected for bacterial DNA isolation and 16S rRNA gene sequencing. Serum FITC-D was similar between housing type (P = 0.709). Hens housed in the CF had increased jejunal villus height and crypt depth compared with hens from the CC (P < 0.002). Hens from the CC tended to have a greater villus height to crypt depth ratio in both the jejunum and ileum compared with the CF (P = 0.064; P = 0.091, respectively). Microbial community diversity measurements favored hens housed in the CC as ileal contents tended to have increased species richness (P = 0.059), had greater alpha diversity (P = 0.044), and had an increased number of over represented operational taxonomic units (46/64), including Romboutsia sp. (30.80%), Lactobacillus kitasatonis (17.16%), and Lactobacillus aviarius (11.15%). Correlations between microbial communities with intestinal traits identified significant association with the greatest number of correlations with FITC-D and ileal morphology. Many of these correlations identified microbial communities associated with expected traits; thus, providing limited functional data to microbial communities with limited information. The greater number of correlations of ileal morphology with ileal microbial communities suggesting local microbial communities contribute to the intestinal environment distant. In this limited study, several parameters favored hens from CC suggesting an advantage of this system for intestinal health. However, the lower intestinal health parameters observed in CF were not at levels to indicate detrimental effects.

Assessment of Fermented Soybean Meal on Salmonella typhimurium Infection in Neonatal Turkey Poults.

Simple SummaryFermented soybean meal (FSBM) improved the performance of neonatal turkey poults, and these changes were associated with morphometric changes in the intestinal mucosa, as well as reduction of leaky gut, within turkeys challenged with Salmonella typhimurium (ST) in two experimental infective models. Although the two strains used for the fermentation process showed in vitro activity against ST, no significant effect was observed in vivo. Nevertheless, FSBM reduced the severity of the ST infection in the therapeutic model. The fermentation with different beneficial bacteria and different inclusion rates of FSBM requires further investigation.This study’s objective was to evaluate the effect of the fermented soybean meal (FSBM) on Salmonella typhimurium (ST) to turkey poults using two models of infection. In the prophylactic model, one-day-old turkeys were randomly allocated to one of four different groups (n = 30 turkeys/group): (1) Control group, (2) FSBM group, (3) Control group challenged with ST (Control + ST), and (4) FSBM group challenged with ST (FSBM + ST). On day 9 of age, all poults were orally challenged with 106 colony forming units (CFU) ST and 24 h post-inoculation, intestinal samples were collected to determine ST recovery and morphometric analysis. Blood samples were collected to evaluate serum fluorescein isothiocyanate-dextran (FITC-d). In the therapeutic model, a similar experimental design was used, but turkeys were orally gavaged 104 CFU ST on day 1, and samples were collected at day 7. FSBM improved performance and reduced leaky gut in both experimental infective models. In the prophylactic model, FSBB induced morphology changes in the mucosa. Although the strains (Lactobacillus salivarius and Bacillus licheniformis) used for the fermentation process showed in vitro activity against ST, no significant effect was observed in vivo. The fermentation with different beneficial bacteria and different inclusion rates of FSBM requires further investigation.

Effect of Bacillus–direct-fed microbial on leaky gut, serum peptide YY concentration, bone mineralization, and ammonia excretion in neonatal female turkey poults fed with a rye-based diet

Rye is high in nonstarch polysaccharides (NSP), a complex carbohydrate which cannot be digested by poultry as they lack the endogenous enzymes to do so. Exogenous carbohydrases must therefore be supplemented to avoid the antinutritional effects associated with a high NSP diet. The objectives of the present study were to evaluate the effects of a rye-based diet with and without supplementation of a Bacillus direct-fed microbial (DFM) on body weight, bone mineralization, and leaky gut, as well as its role on influencing serum concentrations of peptide YY (PPY) and the ammonia concentration in turkey manure. Two independent trials were conducted. In each experiment, day-of-hatch female turkey poults were neck tagged and randomly assigned to either a control rye-based diet or a rye-based diet supplemented with the DFM (n = 25 birds/group). At 10 days-of-age, poults in both groups were administered with an appropriate dose of fluorescein isothiocyanate-dextran (FITC-d) by oral gavage. One hour later, all poults were euthanized. Blood was collected to evaluate serum FITC-d and PPY concentrations. Furthermore, in Trial 2 only, both tibias were removed for assessment of bone parameters, and turkey manure was collected to evaluate physicochemical analysis. In both trials, poults treated with the DFM showed a significant increase (P < 0.05) in body weight and body weight gain as compared with control nontreated poults. Poults that received the DFM also had a significant reduction in serum levels of PPY and FITC-d when compared with control nontreated poults. In Trial 2, turkeys treated with the DFM had a substantial increase in tibia strength, tibia diameter, total ash, calcium, and phosphorus when compared with control nontreated turkeys. Their manure was also shown to have a significant reduction in the concentration of ammonia. This is the first report of a commercial DFM reducing the concentration of this compound in turkey manure. In summary, the results of the present study confirm that turkeys fed with a rye-based diet have a significant increase in gut permeability, a reduced body weight, and decreased bone mineralization when compared with turkeys fed with the DFM. Turkeys that received the rye-based diet supplemented with the Bacillus-DFM also had a significant reduction in the serum concentration of PPY when compared with control turkeys. This finding suggests a possible prebiotic effect of rye, warranting future studies to test this effect. Further studies to evaluate the microbiota diversity, as well as the concentration of ceca short-chain fatty acids, are also necessary to confirm the reliability of PPY as a potential metabolomic biomarker in poultry.