Assessment of the Impact of Humic Acids on Intestinal Microbiota, Gut Integrity, Ileum Morphometry, and Cellular Immunity of Turkey Poults Fed an Aflatoxin B1-Contaminated Diet.

A recent study published data on the growth performance, relative weights of the organs of the gastrointestinal tract, liver histology, serum biochemistry, and hematological parameters for turkey poults fed an experimental diet contaminated with aflatoxin B1 (AFB1) and humic acids (HA) extracted from vermicompost. The negative effects of AFB1 (250 ng AFB1/g of feed) were significantly reduced by HA supplementation (0.25% w/w), suggesting that HA might be utilized to ameliorate the negative impact of AFB1 from contaminated diets. The present study shows the results of the remaining variables, as an extension of a previously published work which aimed to evaluate the impact of HA on the intestinal microbiota, gut integrity, ileum morphometry, and cellular immunity of turkey poults fed an AFB1-contaminated diet. For this objective, five equal groups of 1-day-old female Nicholas-700 turkey poults were randomly assigned to the following treatments: negative control (basal diet), positive control (basal diet + 250 ng AFB1/g), HA (basal diet + 0.25% HA), HA + AFB1 (basal diet + 0.25% HA + 250 ng AFB1/g), and Zeolite (basal diet + 0.25% zeolite + 250 ng AFB1/g). In the experiment, seven replicates of ten poults each were used per treatment (n = 70). In general, HA supplementation with or without the presence of AFB1 showed a significant increase (p < 0.05) in the number of beneficial butyric acid producers, ileum villi height, and ileum total area, and a significant reduction in serum levels of fluorescein isothiocyanate-dextran (FITC-d), a marker of intestinal integrity. In contrast, poults fed with AFB1 showed a significant increase in Proteobacteria and lower numbers of beneficial bacteria, clearly suggesting gut dysbacteriosis. Moreover, poults supplemented with AFB1 displayed the lowest morphometric parameters and the highest intestinal permeability. Furthermore, poults in the negative and positive control treatments had the lowest cutaneous basophil hypersensitivity response. These findings suggest that HA supplementation enhanced intestinal integrity (shape and permeability), cellular immune response, and healthier gut microbiota composition, even in the presence of dietary exposure to AFB1. These results complement those of the previously published study, suggesting that HA may be a viable dietary intervention to improve gut health and immunity in turkey poults during aflatoxicosis.

Evaluation of the Antimicrobial Activity of a Formulation Containing Ascorbic Acid and Eudragit FS 30D Microparticles for the Controlled Release of a Curcumin-Boric Acid Solid Dispersion in Turkey Poults Infected with Salmonella enteritidis: A Therapeutic Model.

The selection of components within a formulation or for treatment must stop being arbitrary and must be focused on scientific evidence that supports the inclusion of each one. Therefore, the objective of the present study was to obtain a formulation based on ascorbic acid (AA) and Eudragit FS 30D microparticles containing curcumin-boric acid (CUR-BA) considering interaction studies between the active components carried out via Fourier transform infrared spectrometry (FTIR) and differential scanning calorimetry (DSC) to minimize antagonistic effects, and comprehensively and effectively treat turkey poults infected with Salmonella enteritidis (S. enteritidis). The DSC and FTIR studies clearly demonstrated the interactions between AA, BA, and CUR. Consequently, the combination of AA with CUR and/or BA should be avoided, but not CUR and BA. Furthermore, the Eudragit FS 30D microparticles containing CUR-BA (SD CUR-BA MP) showed a limited release of CUR-BA in an acidic medium, but they were released at a pH 6.8-7.0, which reduced the interactions between CUR-BA and AA. Finally, in the S. enteritidis infection model, turkey poults treated with the combination of AA and SD CUR-BA MP presented lower counts of S. enteritidis in cecal tonsils after 10 days of treatment. These results pointed out that the use of an adequate combination of AA and CUR-BA as an integral treatment of S. enteritidis infections could be a viable option to replace the indiscriminate use of antibiotics.

The effects of essential oil from Lippia origanoides and herbal betaine on performance, intestinal integrity, bone mineralization and meat quality in broiler chickens subjected to cyclic heat stress.

Essential oils (EO) affect performance, intestinal integrity, bone mineralization, and meat quality in broiler chickens subjected to cyclic heat stress (HS). Day-of-hatch Cobb 500 male broiler chicks (n = 475) were randomly divided into four groups. Group 1: No heat stress (Thermoneutral) + control diets with no antibiotics; Group 2: heat stress control + control diets; Group 3: heat stress + control diets supplemented with thymol chemotype (45 ppm) and herbal betaine (150 ppm) formulation EO1; Group 4: heat stress + control diets supplemented with phellandrene (45 ppm) and herbal betaine (150 ppm) formulation EO2. From day 10-42, the heat stress groups were exposed to cyclic HS at 35°C for 12 h (8:00-20:00). BW, BWG, FI, and FCRc were measured at d 0, 10, 28, and 42. Chickens were orally gavaged with FITC-d on days 10 (before heat stress) and 42. Morphometric analysis of duodenum and ileum samples and bone mineralization of tibias were done. Meat quality was assessed on day 43 with ten chickens per pen per treatment. Heat stress reduced BW by day 28 (p < 0.05) compared to thermoneutral chickens. At the end of the trial, chickens that received both formulations of EO1 and EO2 had significantly higher BW than HS control chickens. A similar trend was observed for BWG. FCRc was impaired by EO2 supplementation. There was a significant increase in total mortality in EO2 compared with EO1 EO1 chickens had lower FITC-d concentrations at day 42 than the HS control. In addition, EO1 treatment is not statistically different if compared to EO2 and thermoneutral. Control HS broilers had significantly lower tibia breaking strength and total ash at day 42 than heat-stressed chickens supplemented with EO1 and EO2. Heat stress affected intestinal morphology more than thermoneutral chickens. EO1 and EO2 improved intestinal morphology in heat-stressed chickens. Woody breast and white striping were more common in thermoneutral chickens than heat stress chickens. In conclusion, the EO-containing diet could improve broiler chicken growth during cyclic heat stress, becoming increasingly relevant in antibiotic-free production in harsh climates.

Research Note: Modified serum fluorescein isothiocyanate dextran (FITC-d) assay procedure to determine intestinal permeability in poultry fed diets high in natural or synthetic pigments.

Oral administration of fluorescein isothiocyanate dextran (FITC-d) has been used as an indicator for intestinal permeability in poultry research for several years. Under healthy conditions, tight junctions in the intestinal wall will not allow the 4-6kDa FITC-d to enter the bloodstream. Detection of FITC-d in serum (1-hour post-oral administration of FITC-d) has proven to be a reliable indicator of leaky gut syndrome (increased intestinal inflammation and disruption of tight junctions). Administration of supplementary phytobiotics in feed, particularly products with high beta-carotene levels or other pigments, has resulted in strong serum background fluorescence, which can render this assay unreliable. To account for this increase in background autofluorescence, the FITC-d assay procedure has been modified to accommodate these particular serum samples by including pre-administration serum collection from each treatment group to remove background fluorescence. The modified FITC-d procedure detailed will allow for analysis of intestinal permeability in pigmented serum.

Evaluation of Three Formulations of Essential Oils in Broiler Chickens under Cyclic Heat Stress.

The objective of the present research was to assess the dietary supplementation of three formulations of essential oils (EO) in chickens under heat stress (HS). Day-of-hatch Cobb 500 chicks (n = 500) were randomly distributed into four groups: 1. HS control + control diets; 2. HS + control diets supplemented with 37 ppm EO of Lippia origanoides (LO); 3. HS + control diets supplemented with 45 ppm LO + 45 ppm EO of Rosmarinus officinalis (RO) + 300 ppm red beetroot; 4. HS + 45 ppm LO + 45 ppm RO + 300 ppm natural betaine. Chickens that received the EO showed significant (p < 0.05) improvement on BW, BWG, FI, and FCR compared to control HS chickens. Average body core temperature in group 3 and group 4 was significantly (p < 0.05) reduced compared with the HS control group and group 2. Experimental groups showed a significant reduction in FITC-d at 42 days, a significant increase in SOD at both days but a significant reduction of IFN-γ and IgA compared with HS control (p < 0.05). Bone mineralization was significantly improved by EO treatments (p < 0.05). Together these data suggest that supplemental dietary EO may reduce the harmful effects of HS.

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

BACKGROUND: Interest 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. RESULTS: The 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. CONCLUSIONS: CR 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.

Evaluation of Ascorbic Acid or Curcumin Formulated in a Solid Dispersion on Salmonella Enteritidis Infection and Intestinal Integrity in Broiler Chickens.

Two experimental models were conducted to evaluate and compare the effect of ascorbic acid (AA) or curcumin formulated in a solid dispersion (SD-CUR) as prophylactic or therapeutic alternatives to prevent or control S. Enteritidis (SE) infection in broiler chickens. In the prophylactic model, dietary administration of AA showed a significant reduction in SE counts in crop compared to the positive control (PC) group (p < 0.05), whereas in cecal tonsils (CT), SD-CUR significantly reduced SE recovery. Superoxide dismutase (SOD) activity was significantly higher in chickens supplemented with AA or SD-CUR, and total intestinal IgA levels were significantly lower in both treatments when compared to the PC group. Serum fluorescein isothiocyanate-dextran (FITC-d) levels were reduced by SD-CUR compared to PC, while AA presented significantly lower total aerobic bacteria. In the therapeutic model, only the dietary administration of AA significantly decreased SE in crop and CT on days 3 and 10 post-challenge. FITC-d levels were significantly lower in both treated groups in comparison to PC, but IgA levels were significantly reduced only by AA. The results suggest that dietary AA and SD-CUR have different modes of action to reduce SE intestinal colonization in two different challenge models in broiler chickens.

Evaluation of the Dietary Supplementation of a Formulation Containing Ascorbic Acid and a Solid Dispersion of Curcumin with Boric Acid against Salmonella Enteritidis and Necrotic Enteritis in Broiler Chickens.

Two experiments were conducted to evaluate the effect of the prophylactic or therapeutic administration of a 0.1% mixture containing ascorbic acid and a solid dispersion of curcumin with polyvinylpyrrolidone and boric acid (AA-CUR/PVP-BA) against Salmonella Enteritidis (S. Enteritidis) in broiler chickens. A third experiment was conducted to evaluate the impact of the dietary administration of 0.1% AA-CUR/PVP-BA in a necrotic enteritis (NE) model in broiler chickens. The prophylactic administration of 0.1% AA-CUR/PVP-BA significantly decreased S. Enteritidis colonization in cecal tonsils (CT) when compared to the positive control group (PC, p < 0.05). The therapeutic administration of 0.1% AA-CUR/PVP-BA significantly reduced the concentration of S. Enteritidis by 2.05 and 2.71 log in crop and CT, respectively, when compared with the PC on day 10 post-S. Enteritidis challenge. Furthermore, the serum FITC-d concentration and total intestinal IgA levels were also significantly lower in chickens that received 0.1% AA-CUR/PVP-BA. Contrary, the PC group showed significantly higher total intestinal IgA levels compared to the negative control or AA-CUR/PVP-BA groups in the NE model. However, 0.1% AA-CUR/PVP-BA showed a better effect in reducing the concentration of S. Enteritidis when compared to the NE model. Further studies with higher concentration of AA-CUR/PVP-BA into the feed to extend these preliminary results are currently being evaluated.

Evaluation of the antimicrobial and intestinal integrity properties of boric acid in broiler chickens infected with Salmonella enteritidis: Proof of concept.

The objectives of the present study were to evaluate the antimicrobial effect of boric acid (BA) on Salmonella enteritidis colonization, intestinal permeability, total intestinal IgA levels, and cecal microbiota composition in broiler chickens. For this purpose, sixty day-old-chicks were randomly allocated to one of two groups: 1) non-treated control group and 2) 0.1% (w/w) BA in feed. All chicks were challenged with 104 cfu of S. Enteritidis per bird at 1-d old. On day 3 and 10 post-challenge, samples of crop and ceca-cecal tonsils (CCT) were aseptically collected to evaluate overall aerobic and lactic acid bacteria, as well as S. Enteritidis recovery. Blood and intestinal samples were also collected to evaluate intestinal permeability and total intestinal IgA production, respectively. On day 10 post-challenge, the concentration of S. Enteritidis in crop and CCT significantly decreased (P < .05) by 2.60 log10 and 5.26 log10, respectively, in the BA group compared to the control group. Furthermore, chickens treated with 0.1% BA had a significant reduction in serum fluorescein isothiocyanate dextran (FITC-d) concentration and lower total intestinal IgA levels when compared to the control group. Interestingly, in the microbiota analysis, only the abundance of Actinobacteria phylum was significantly lower in the BA group in comparison with the control group and significant differences in beta diversity were also observed between comparing groups. The results of the present study suggest that BA could maintain intestinal homeostasis, as well as the balance in the microbiota due to its effectiveness in controlling S. Enteritidis infection.

High Molecular Weight Polymer Promotes Bone Health and Prevents Bone Loss Under Salmonella Challenge in Broiler Chickens.

As a consequence of rapid growth, broiler chickens are more susceptible to infection as well as bone fractures that result in birds being culled. Intestinal infection/inflammation has been demonstrated to promote bone loss in mice and humans. Given this link, we hypothesize that therapeutics that target the gut can benefit bone health. To test this, we infected broiler chickens (7 days old) with Salmonella and treated the birds with or without MDY, a non-absorbable mucus supplement known to benefit intestinal health, from day 1-21 or from day 14-21. Chicken femoral trabecular and cortical bone parameters were analyzed by microcomputed tomography at 21 days. Birds infected with Salmonella displayed significant trabecular bone loss and bone microarchitecture abnormalities that were specific to the femoral neck region, a common site of fracture in chickens. Histological analyses of the chicken bone indicated an increase in osteoclast surface/bone surface in this area indicating that infection-induced bone resorption likely causes the bone loss. Of great interest, treatment with MDY effectively prevented broiler chicken bone loss and architectural changes when given chronically throughout the experiment or for only a week after infection. The latter suggests that MDY may not only prevent bone loss but reverse bone loss. MDY also increased cortical bone mineral density in Salmonella-treated chickens. Taken together, our studies demonstrate that Salmonella-induced bone loss in broiler chickens is prevented by oral MDY.

Effect of Morinda citrifolia (Noni)-Enriched Diet on Hepatic Heat Shock Protein and Lipid Metabolism-Related Genes in Heat Stressed Broiler Chickens.

Heat stress (HS) has been reported to alter fat deposition in broilers, however the underlying molecular mechanisms are not well-defined. The objectives of the current study were, therefore: (1) to determine the effects of acute (2 h) and chronic (3 weeks) HS on the expression of key molecular signatures involved in hepatic lipogenic and lipolytic programs, and (2) to assess if diet supplementation with dried Noni medicinal plant (0.2% of the diet) modulates these effects. Broilers (480 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to two environmental conditions (heat stress, HS, 35°C vs. thermoneutral condition, TN, 24°C) and fed two diets (control vs. Noni) in a 2 × 2 factorial design. Feed intake and body weights were recorded, and blood and liver samples were collected at 2 h and 3 weeks post-heat exposure. HS depressed feed intake, reduced body weight, and up regulated the hepatic expression of heat shock protein HSP60, HSP70, HSP90 as well as key lipogenic proteins (fatty acid synthase, FASN; acetyl co-A carboxylase alpha, ACCα and ATP citrate lyase, ACLY). HS down regulated the hepatic expression of lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (LIPC), but up-regulated ATGL. Although it did not affect growth performance, Noni supplementation regulated the hepatic expression of lipogenic proteins in a time- and gene-specific manner. Prior to HS, Noni increased ACLY and FASN in the acute and chronic experimental conditions, respectively. During acute HS, Noni increased ACCα, but reduced FASN and ACLY expression. Under chronic HS, Noni up regulated ACCα and FASN but it down regulated ACLY. In vitro studies, using chicken hepatocyte cell lines, showed that HS down-regulated the expression of ACCα, FASN, and ACLY. Treatment with quercetin, one bioactive ingredient in Noni, up-regulated the expression of ACCα, FASN, and ACLY under TN conditions, but it appeared to down-regulate ACCα and increase ACLY levels under HS exposure. In conclusion, our findings indicate that HS induces hepatic lipogenesis in chickens and this effect is probably mediated via HSPs. The modulation of hepatic HSP expression suggest also that Noni might be involved in modulating the stress response in chicken liver.

Effect of a selected Lactobacillus spp.-based probiotic on Salmonella enterica serovar enteritidis-infected broiler chicks.

The effect of a Lactobacillus spp.-based probiotic (FM-B11) on Salmonella enterica serovar Enteritidis (SE) recovery was evaluated in liquid (Expt. 1) and lyophilized (Expt. 2) forms in two separate experiments with two trials each. For each trial, 80 broiler chicks were randomly allocated into two treatments: control and probiotic culture. All chicks were challenged with SE (approximately 10(4) colony-forming units [cfu]) upon arrival at our laboratory. In both experiments, probiotic culture was administered in the drinking water for 3 consecutive days at a final concentration of approximately 10(6) cfu/ml, beginning 1 hr after SE challenge. Cecal tonsils were aseptically removed at 24 and 72 hr postchallenge, followed by enrichment and plating on xylose lactose deoxycholate (XLD) agar for the presence or absence of Salmonella-typical colonies. In Expt. 1, a significant reduction (P < 0.05) in SE-positive samples was observed in both trials at 24 and 72 hr postchallenge. Additionally, in Expt. 2, the lyophilized probiotic decreased (P < 0.05) SE recovery at both 24 and 72 hr postchallenge compared with the control group in trial 1. In trial 2, SE evaluation was performed only at 72 hr after challenge and fewer (P < 0.001) treated samples were positive for SE. Results showed that application of either liquid or lyophilized probiotic culture in the drinking water for 3 consecutive days can help to reduce SE recovery from young birds, although further research is needed to elucidate the mechanism of this response.