Adapted tissue assay for the assessment of ileal granulocyte degranulation following in ovo inoculation with select bacteria or coccidial challenge in chickens.

A previously described heterophil degranulation assay was adapted for use with ileal mucosal tissue via quantification of ß-D-glucuronidase and assay end product 4-methylumbelliferone (4-MU). Three initial experiments evaluated the effect of in ovo inoculations of Citrobacter freundii (CF) or mixed lactic acid bacteria (LAB) on ileal granulocyte degranulation. Inoculations were administered on embryonic d18, body weights (BW) were recorded on day of hatch (DOH) and d10 to calculate body weight gain (BWG), and ileal mucosal scrapings were collected on DOH or d10 for the 4-MU assay. In all experiments, treatments were statistically analyzed relative to control groups. Treatments minimally affected BWG in all in ovo experiments (p > 0.05) relative to respective control groups. Similarly, ileal degranulation in in ovo treatments did not statistically differ (p > 0.05). Based on BWG, in ovo treatments may have induced low-level inflammation unable to elicit detectable changes via the 4-MU assay. Four subsequent experiments were conducted to evaluate effects of Eimeria maxima (EM) on ileal degranulation. Treatments included non-inoculated controls and low, medium, or high EM infection. Across all four experiments, final BW or BWG over the inoculation period were suppressed (p < 0.05) in EM groups relative to respective controls with the exception of EM-low (p = 0.094) and EM-medium (p = 0.096) in one trial. Ileal mucosal scrapings for the 4-MU assay were collected on day of peak lesions. Resulting values were reduced (p < 0.05) for EM treated birds in three experiments with the exception of EM-medium (p = 0.247). No differences were observed in one experiment (p = 0.351), which may have been attributed to a variation in strain of infecting Eimeria. Although refinement for low level inflammation is warranted, results indicate successful adaptation of the 4-MU assay for use with intestinal tissue during significant gastrointestinal inflammation.

An overview of the use of bacteriophages in the poultry industry: Successes, challenges, and possibilities for overcoming breakdowns.

The primary contaminants in poultry are Salmonella enterica, Campylobacter jejuni, Escherichia coli, and Staphylococcus aureus. Their pathogenicity together with the widespread of these bacteria, contributes to many economic losses and poses a threat to public health. With the increasing prevalence of bacterial pathogens being resistant to most conventional antibiotics, scientists have rekindled interest in using bacteriophages as antimicrobial agents. Bacteriophage treatments have also been investigated as an alternative to antibiotics in the poultry industry. Bacteriophages' high specificity may allow them only to target a specific bacterial pathogen in the infected animal. However, a tailor-made sophisticated cocktail of different bacteriophages could broaden their antibacterial activity in typical situations with multiple clinical strains infections. Bacteriophages may not only be used in terms of reducing bacterial contamination in animals but also, under industrial conditions, they can be used as safe disinfectants to reduce contamination on food-contact surfaces or poultry carcasses. Nevertheless, bacteriophage therapies have not been developed sufficiently for widespread use. Problems with resistance, safety, specificity, and long-term stability must be addressed in particular. This review highlights the benefits, challenges, and current limitations of bacteriophage applications in the poultry industry.

Proper Immune Response Depends on Early Exposure to Gut Microbiota in Broiler Chicks.

The successional changes in the early intestinal microbiota occur concomitantly with the development, expansion, and education of the mucosal immune system. Although great attention of researchers has been focused on understanding the linkage between microbiota and immune functions, many essential details of the symbiotic relationship between the intestinal pioneer microbiota and the avian immune system remain to be discovered. This study was conducted to understand the impact of different early life intestinal colonizers on innate and adaptive immune processes in chicks and further identify immune-associated proteins expressed in the intestinal tissue. To accomplish it, we performed an in ovo application of two apathogenic Enterobacteriaceae isolates and lactic acid bacteria (L) to determine their influences on the intestinal proteome profile of broilers at the day of hatch (DOH) and at 10 days old. The results indicated that there were predicted biological functions of L-treated chicks associated with the activation and balanced function of the innate and adaptive immune systems. At the same time, the Enterobacteriaceae-exposed birds presented dysregulated immunological mechanisms or downregulated processes related to immune development. Those findings suggested that a proper immune function was dependent on specific gut microbiota exposure, in which the prenatal probiotic application may have favored the fitting programming of immune functions in chicks.

Technical note: fluorescein as an indicator of enteric mucosal barrier function in preruminant lambs.

Increased intestinal permeability can be observed during the physiologic stress response and has been linked to suppression of animal health and performance. Previously published data have shown the efficacy of fluorescein isothiocyanate dextran (FITC-d; 4.17 mg/kg) as a marker of enteric inflammation and mucosal barrier function in multiple species. Fluorescein is a smaller, less expensive alternative molecule possessing similar properties. The following two experiments compared FITC-d and fluorescein as potential indicators of intestinal permeability in pre- and postweaned lambs administered daily intramuscular injections of dexamethasone (Dex; 0.1 mg/kg) for 1 wk. Experiment 1 consisted of five preweaned lambs that were placed in one of two treatment groups: fluorescein with Dex (F+Dex) or fluorescein only (F). On day 7, blood was collected before and 1 h after oral administration of fluorescein (50 mg/kg). Experiment 2 included 12 weaned lambs and four treatment groups: F+Dex, F, FITC-d with Dex (Fd+Dex), and FITC-d only (Fd). On day 7, blood was collected before and 2 h after oral administration of FITC-d (4.17 mg/kg) or fluorescein (50 mg/kg). Plasma fluorescence was reported as the ratio between T1h/T0 or T2h/T0 for experiment 1 or 2, respectively. Experiment 1 showed a significant increase in T1h/T0 ratio of F+Dex relative to F lambs (P = 0.05) indicative of increased leaky gut; however, no differences (P = 0.22) were obtained in experiment 2. Results of these experiments suggest fluorescein may serve as a suitable marker of enteric permeability in preruminant lambs, but not in those with functional rumens.

Comparative effectiveness of probiotic-based formulations on cecal microbiota modulation in broilers.

The potential of probiotics to manipulate the intestinal microbial ecosystem toward commensal bacteria growth offers great opportunity for enhancing health and performance in poultry. This study aimed to evaluate the efficacy of five probiotic-based formulations in modulating cecal microbiota in broilers at 21 and 42 days of age. Probiotics investigated included a synbiotic (SYNBIO), a yeast (YEAST), and three single-strain formulations of Bacillus amyloliquefaciens (SINGLE1), B. subtilis (SINGLE2) and B. licheniformis (SINGLE3). Alpha-diversity analyses showed that cecal microbiota of SINGLE1, SINGLE2, and YEAST had low diversity compared to the control diet with no feed additive (CON) at 21d. At the same age, weighted Unifrac distance measure showed significant differences between samples from SYNBIO and CON (P = 0.02). However, by analyzing principal coordinates analysis (PCoA) with unweighted Unifrac, there was no evidence of clustering between CON and probiotic treatments. By 42d, there were no differences in alpha or beta-diversity in the microbiota of probiotic treatments compared to CON. Similarly, taxonomic microbial profiling did not show major changes in cecal microbial taxa. In conclusion, not all probiotic-based formulations tested had a core benefit on the modulation of microbiota. However, based on the quantitative beta diversity results, SYNBIO greatly influenced the cecal microbial community structure attributable to transient variations in relative taxon abundance.

Mode of Action of Dietary Dexamethasone May Not Be Dependent Upon Microbial Mechanisms in Broilers.

Dexamethasone (Dex), a synthetic glucocorticoid (GC), in feed has been shown to increase gut permeability via stress-mediated mechanisms, but the exact mode of action on gut barrier function is not fully understood. Stress has been reported to alter the profile and virulence of intestinal flora predisposing for opportunistic disease. This study aimed to evaluate the relationship between dietary Dex and recoverable intestinal microbial profile in broilers to better understand mode of action and refine future uses of the model. Three experiments were conducted that administered Dex-treated feed for one week in conjunction with the antibiotics BMD (bacitracin methylene disalicylate) or Baytril® (enrofloxacin) to evaluate if enteric microbial mechanisms were important in Dex-induced permeability. Serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT) have been reported to increase after Dex treatment and were used to assess gut epithelial leakage. Shifts in bacterial profiles were also measured on selective agar. Combining Dex with BMD or Baytril resulted in increased (P < 0.05) serum FITC-d versus Dex-only. Additionally, Baytril did not reduce aerobic BT and bacterial profiles remained similar after Dex. These results suggest a minimal role of intestinal microbes in Dex-induced changes to intestinal barrier function.

Intestinal Pioneer Colonizers as Drivers of Ileal Microbial Composition and Diversity of Broiler Chickens.

Given that recent advances in metagenomics have highlighted the importance of intestinal microbes for poultry health, there has been a corresponding search for early manipulation strategies of intestinal microbiota in order to advance immune system development and optimize functional properties of growth. In this study, we used the in ovo technique as an experimental model to address how early bacterial intestinal colonization could affect the development and establishment of the mature ileal microbiota. Inoculations containing one of the following: 0.2 mL of 0.9% sterile saline (S), approximately 102 cells of Citrobacter freundii (CF), Citrobacter species (C2) or lactic acid bacteria mixture (L) were administered via in ovo into the amnion. Results showed that Enterobacteriaceae abundance was negatively correlated with aging, although its high population at day of hatch affected the microbiota composition, delaying mature microbiota establishment. L treatment increased colonization of butyrate-producing bacteria by 3 and 10 days, and segmented filamentous bacteria in the lower ileum by 10 days. On the other hand, L-probiotic decreased the population of Enterococcaceae. In addition, L and C2 microbial communities were less diverse at 10 than 3 days of age in the upper ileum. Importantly, these findings provide a valuable resource for a potential study model for interactions between microbial colonization and associated immune responses. In conclusion, our analysis demonstrates that intestinal pioneer colonizers play a critical role in driving the course of microbial community composition and diversity over time, in which early life exposure to L-based probiotic supported selection alongside greater colonization of symbiotic populations in the ileum of young broilers.

Crude Inactivated Influenza A Virus Adjuvated with a Bispecific Antibody Complex Targeting Chicken CD40 and AIV M2e Confers Protection Against Lethal HPAI Challenge in Chickens.

In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

Evaluation of the Epithelial Barrier Function and Ileal Microbiome in an Established Necrotic Enteritis Challenge Model in Broiler Chickens.

Necrotic enteritis (NE) is a recognized multifactorial disease that cost annually to the poultry industry around $2 billion. However, diverse aspects related to its presentation are not completely understood, requiring further studies using known induction experimental models. Therefore, the purpose of this study was to measure the changes occurring in performance, intestinal integrity and ileal microbiome using a previously established NE-challenge model. Chickens were assigned to a negative control group (NC) or a positive control group (PC). In the PC, broilers were orally gavaged with Salmonella Typhimurium (ST) (1 × 107 cfu/chick) at day 1, Eimeria maxima (EM) (2.5 × 104 oocyst/chick) at day 18 and Clostridium perfringens (CP) (1 × 108 cfu/chick/day) at 23-24 days of age. Weekly, body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were evaluated. Morbidity and mortality were determined throughout the study, and NE lesion scores were recorded at day 25. Additionally, blood and liver samples were collected to measure gut permeability as determined by levels of serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT). Ileal contents were processed for 16S rRNA gene-based microbiome analysis. Performance parameters and intestinal permeability measurements were negatively impacted in the PC resulting in elevated serum FITC-d and BT with a -6.4% difference in BWG. The NE lesion score in PC (1.97 vs. 0.00) was significantly higher in comparison to NC, although there was no difference in mortality. The microbiome analysis showed a dramatic shift of ileal microbiomes in PC groups as compared to NC (ANOSIM: R = 0.76, P = 0.001). The shift was characterized by reduced abundance of the phylum Actinobacteria (P < 0.01), and increased abundance of the genera Butyrivibrio, Lactobacillus, Prevotella and Ruminococcus in PC compared to NC (P < 0.05). Expectedly, Clostridium was found higher in PC (2.98 ± 0.71%) as compared to NC (1.84 ± 0.36%), yet the difference was not significant. In conclusion, results of the present study showed the different intestinal epithelial and microbiological alterations occurring in an established NE-challenge model that considers paratyphoid Salmonella infections in young chicks as an important predisposing factor for presentation of NE.

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.

A Fast and Inexpensive Protocol for Empirical Verification of Neutralizing Epitopes in Microbial Toxins and Enzymes.

In vivo targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, Clostridium perfringens alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC) enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected in silico using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for in vitro toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin's hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and de facto identification of neutralizing epitopes in microbial toxins and enzymes within 2 weeks from in silico analysis of a putative target sequence.

Optimizing Fluorescein Isothiocyanate Dextran Measurement As a Biomarker in a 24-h Feed Restriction Model to Induce Gut Permeability in Broiler Chickens.

Fluorescein isothiocyanate dextran (FITC-d) is a 3-5 kDa marker used to measure tight junction permeability. We have previously shown that intestinal barrier function can be adversely affected by stress, poorly digested diets, or feed restriction (FR), resulting in increased intestinal inflammation-associated permeability. However, further optimization adjustments of the current FITC-d methodology are possible to enhance precision and efficacy of results in future. The objective of the present study was to optimize our current model to obtain a larger difference between control and treated groups, by optimizing the FITC-d measurement as a biomarker in a 24-h FR model to induce gut permeability in broiler chickens. One in vitro and four in vivo independent experiments were conducted. The results of the present study suggest that by increasing the dose of FITC-d (8.32 versus 4.16 mg/kg); shortening the collection time of blood samples (1 versus 2.5 h); using a pool of non-FITC-d serum as a blank, compared to previously used PBS; adding a standard curve to set a limit of detection and modifying the software's optimal sensitivity value, it was possible to obtain more consistent and reliable results.

Evaluation and Selection of Bacillus Species Based on Enzyme Production, Antimicrobial Activity, and Biofilm Synthesis as Direct-Fed Microbial Candidates for Poultry.

Social concern about misuse of antibiotics as growth promoters (AGP) and generation of multidrug-resistant bacteria have restricted the dietary inclusion of antibiotics in livestock feed in several countries. Direct-fed microbials (DFM) are one of the multiple alternatives commonly evaluated as substitutes of AGP. Sporeformer bacteria from the genus Bacillus have been extensively investigated because of their extraordinary properties to form highly resistant endospores, produce antimicrobial compounds, and synthesize different exogenous enzymes. The purpose of the present study was to evaluate and select Bacillus spp. from environmental and poultry sources as DFM candidates, considering their enzyme production profile, biofilm synthesis capacity, and pathogen-inhibition activity. Thirty-one Bacillus isolates were screened for in vitro relative enzyme activity of amylase, protease, lipase, and phytase using a selective media for each enzyme, with 3/31 strains selected as superior enzyme producers. These three isolates were identified as Bacillus subtilis (1/3), and Bacillus amyloliquefaciens (2/3), based on biochemical tests and 16S rRNA sequence analysis. For evaluation of biofilm synthesis, the generation of an adherent crystal violet-stained ring was determined in polypropylene tubes, resulting in 11/31 strains showing a strong biofilm formation. Moreover, all Bacillus strains were evaluated for growth inhibition activity against Salmonella enterica serovar Enteritidis (26/31), Escherichia coli (28/31), and Clostridioides difficile (29/31). Additionally, in previous in vitro and in vivo studies, these selected Bacillus strains have shown to be resistant to different biochemical conditions of the gastrointestinal tract of poultry. Results of the present study suggest that the selection and consumption of Bacillus-DFM, producing a variable set of enzymes and antimicrobial compounds, may contribute to enhanced performance through improving nutrient digestibility, reducing intestinal viscosity, maintaining a beneficial gut microbiota, and promoting healthy intestinal integrity in poultry.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100µg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4µg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4µg of antigen.

Leaky Gut and Mycotoxins: Aflatoxin B1 Does Not Increase Gut Permeability in Broiler Chickens.

Previous studies conducted in our laboratory have demonstrated that intestinal barrier function can be adversely affected by diet ingredients or feed restriction, resulting in increased intestinal inflammation-associated permeability. Two experiments were conducted in broilers to evaluate the effect of three concentrations of Aflatoxin B1 (AFB1; 2, 1.5, or 1 ppm) on gastrointestinal leakage and liver bacterial translocation (BT). In experiment 1, 240 day-of-hatch male broilers were allocated in two groups, each group had six replicates of 20 chickens (n = 120/group): Control feed or feed + 2 ppm AFB1. In experiment 2, 240 day-of-hatch male broilers were allocated in three groups, each group had five replicates of 16 chickens (n = 80/group): Control feed; feed + 1 ppm AFB1; or feed + 1.5 ppm AFB1. In both experiments, chickens were fed starter (days 1-7) and grower diets (days 8-21) ad libitum and performance parameters were evaluated every week. At day 21, all chicks received an oral gavage dose of FITC-d (4.16 mg/kg) 2.5 h before collecting blood samples to evaluate gastrointestinal leakage of FITC-d. In experiment 2, a hematologic analysis was also performed. Liver sections were aseptically collected and cultured using TSA plates to determine BT. Cecal contents were collected to determine total colony-forming units per gram of Gram-negative bacteria, lactic acid bacteria (LAB), or anaerobes by plating on selective media. In experiment 2, liver, spleen, and bursa of Fabricius were removed to determine organ weight ratio, and also intestinal samples were obtained for morphometric analysis. Performance parameters, organ weight ratio, and morphometric measurements were significantly different between Control and AFB1 groups in both experiments. Gut leakage of FITC-d was not affected by the three concentrations of AFB1 evaluated (P > 0.05). Interestingly, a significant reduction in BT was observed in chickens that received 2 and 1 ppm AFB1. An increase (P < 0.05) in total aerobic bacteria, total Gram negatives, and total LAB were observed in chickens fed with 2 and 1.5 ppm of AFB1 when compared with Control and 1 ppm chickens. The integrity of gut epithelial barrier was not compromised after exposure to the mycotoxin.

Evaluation of Gastrointestinal Leakage in Multiple Enteric Inflammation Models in Chickens.

Enteric inflammation models can help researchers' study methods to improve health and performance and evaluate various growth promoters and dietary formulations targeted to improve performance in poultry. Oral administration of fluorescein isothiocyanate-dextran (FITC-d; 3-5 kDa) and its pericellular mucosal epithelial leakage are an established marker to evaluate enteric inflammation in multiple species. The present study evaluated different methods to induce gut inflammation in poultry based on FITC-d leakage. Four independent experiments were completed with different inflammation treatment groups, and serum FITC-d and/or retention of FITC-d in GI tract were determined. In experiment 1 (n = 10 birds/treatment, broilers, processed at 14 days), groups included control (CON), dextran sodium sulfate (DSS; drinking water at 0.75%) and feed restriction (FRS; 24 h before processing). Experiment 2 (n = 14 birds/treatment, leghorns, processed at 7 days) included CON, DSS, FRS, and rye-based diet (RBD). In experiments 3 and 4 (n = 15 birds/treatment, broilers, processed at 7 days), groups were CON, DSS, high fat diet (HFD), FRS, and RBD. In all experiments, FRS and RBD treatments showed significantly higher serum FITC-d levels compared to the respective CON. This indicates that FRS and RBD results in disruption of the intact barrier of the gastrointestinal tract (GIT), resulting in increased gut permeability. DSS and HFD groups showed elevation of serum FITC-d levels although the magnitude of difference from respective CON was inconsistent between experiments. FRS was the only treatment which consistently showed elevated retention of FITC-d in GIT in all experiments. The results from present studies showed that FRS and RBD, based on serum FITC-d levels, can be robust models to induce gut leakage in birds in different age and species/strains.

Selection of Bacillus spp. for Cellulase and Xylanase Production as Direct-Fed Microbials to Reduce Digesta Viscosity and Clostridium perfringens Proliferation Using an in vitro Digestive Model in Different Poultry Diets.

Previously, our laboratory has screened and identified Bacillus spp. isolates as direct-fed microbials (DFM). The purpose of the present study was to evaluate the cellulase and xylanase production of these isolates and select the most appropriate Bacillus spp. candidates for DFM. Furthermore, an in vitro digestive model, simulating different compartments of the gastrointestinal tract, was used to determine the effect of these selected candidates on digesta viscosity and Clostridium perfringens proliferation in different poultry diets. Production of cellulase and xylanase were based on their relative enzyme activity. Analysis of 16S rRNA sequence classified two strains as Bacillus amyloliquefaciens and one of the strains as Bacillus subtilis. The DFM was included at a concentration of 10(8) spores/g of feed in five different sterile soybean-based diets containing corn, wheat, rye, barley, or oat. After digestion time, supernatants from different diets were collected to measure viscosity, and C. perfringens proliferation. Additionally, from each in vitro simulated compartment, samples were taken to enumerate viable Bacillus spores using a plate count method after heat-treatment. Significant (P < 0.05) DFM-associated reductions in supernatant viscosity and C. perfringens proliferation were observed for all non-corn diets. These results suggest that antinutritional factors, such as non-starch polysaccharides from different cereals, can enhance viscosity and C. perfringens growth. Remarkably, dietary inclusion of the DFM that produce cellulase and xylanase reduced both viscosity and C. perfringens proliferation compared with control diets. Regardless of diet composition, 90% of the DFM spores germinated during the first 30 min in the crop compartment of the digestion model, followed by a noteworthy increased in the intestine compartment by ~2log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in progress.

Utilization of rye as energy source affects bacterial translocation, intestinal viscosity, microbiota composition, and bone mineralization in broiler chickens.

Two independent trials were conducted to evaluate the utilization of rye as energy source on bacterial translocation (BT), intestinal viscosity, gut integrity, gut microbiota composition, and bone mineralization, when compared with a traditional cereal (corn) in broiler chickens. In each experiment, day-of-hatch, broiler chickens were randomly assigned to either a corn or a rye diet (n = 20 chickens/group). At 10 d of age, in both experiments, 12 chickens/group were randomly selected, and given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood samples were collected to determine the passage of FITC-d. The liver was collected from each bird to evaluate BT. Duodenum, ileum, and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with rye showed increased (p < 0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that chickens fed with rye had increased the number of total lactic acid bacteria in all three sections of the gastrointestinal tract evaluated when compared to chickens fed with corn. Chickens fed with rye also had significantly higher coliforms in duodenum and ileum, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in chickens fed with rye when compared with corn fed chickens. In conclusion, rye evoked mucosal damage in chickens that alter the intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition as well as bone mineralization. Studies to evaluate dietary inclusion of selected DFM candidates that produce exogenous enzymes in rye fed chickens are currently being evaluated.

Evaluation of respiratory route as a viable portal of entry for Salmonella in poultry.

With increasing reports of Salmonella infection, we are forced to question whether the fecal-oral route is the major route of infection and consider the possibility that airborne Salmonella infections might have a major unappreciated role. Today's large-scale poultry production, with densely stocked and enclosed production buildings, is often accompanied by very high concentrations of airborne microorganisms. Considering that the upper and lower respiratory lymphoid tissue requires up to 6 weeks to be fully developed, these immune structures seem to have a very minor role in preventing pathogen infection. In addition, the avian respiratory system in commercial poultry has anatomic and physiologic properties that present no challenge to the highly adapted Salmonella. The present review evaluates the hypothesis that transmission by the fecal-respiratory route may theoretically be a viable portal of entry for Salmonella in poultry. First, we update the current knowledge on generation of Salmonella bioaerosols, and the transport and fate of Salmonella at various stages of commercial poultry production. Further, emphasis is placed on survivability of Salmonella in these bioaerosols, as a means to assess the transport and subsequent risk of exposure and infection of poultry. Additionally, the main anatomic structures, physiologic functions, and immunologic defense in the avian respiratory system are discussed to understand the potential entry points inherent in each component that could potentially lead to infection and subsequent systemic infection of poultry by Salmonella. In this context, we also evaluate the role of the mucosal immune system as essentially one large interconnected network that shares information distally, since understanding of this sort of communication between mucosal sites is fundamental to establish the next phase of disease characterization, and perhaps immunization and vaccine development. Further characterization of the respiratory tract with regard to transmission of Salmonella under field conditions may be of critical importance in developing interventional strategies to reduce transmission of this important zoonotic pathogen in poultry.

Effect of chitosan on Salmonella Typhimurium in broiler chickens.

Public concern with the incidence of antibiotic-resistant bacteria, particularly among foodborne pathogens such as Salmonella, has been challenging the poultry industry to find alternative means of control. The purposes of the present study were to evaluate in vitro and in vivo effects of chitosan on Salmonella enterica serovar Typhimurium (ST) infection in broiler chicks. For in vitro crop assay experiments, tubes containing feed, water, and ST were treated with either saline as a control or 0.2% chitosan. The entire assay was repeated in three trials. In two independent in vivo trials, 40 broiler chicks were assigned to an untreated control diet or dietary treatment with 0.2% chitosan for 7 days (20 broiler chicks/treatment). At day 4, chicks were challenged with 2×105 colony-forming units (CFU) ST/bird. In a third in vivo trial, 100 broiler chicks were assigned to untreated control diet or dietary treatment with 0.2% chitosan for 10 days (50 broiler chicks/treatment) to evaluate ST horizontal transmission. At day 3, 10 birds were challenged with 105 CFU ST/bird, and the remaining nonchallenged birds (n=40) were kept in the same floor pen. In all three in vitro trials, 0.2% chitosan significantly reduced total CFU of ST at 0.5 and 6 h postinoculation compared with control (p<0.05). In two in vivo trials, at 7 days, dietary 0.2% chitosan significantly reduced total CFU of recovered ST in the ceca in both experiments. Dietary 0.2% chitosan significantly reduced total ST CFU recovered in the ceca of horizontally challenged birds in the third in vivo trial. Chitosan at 0.2% significantly reduced the CFU of recovered ST in vitro and in vivo, proving to be an alternative tool to reduce crop, ceca, and consequently carcass ST contamination as well as decreasing the amount of ST shed to the environment.