A. Menconi

Poultry enteric inflammation model with dextran sodium sulfate mediated chemical induction and feed restriction in broilers

Gut inflammation is a cardinal event occurring in various gastrointestinal diseases regardless of etiology. A potential mechanism of action for antibiotic growth promoters and probiotics is alleviation or attenuation of such inflammation. In vivo inflammation models and markers to quantify changes in inflammation, such as paracellular leakage and tight junction function, are necessary tools in the search for methods to reduce enteric inflammation. Dextran sodium sulfate (DSS) and feed restriction (FRS), and fluorescein isothiocyanate dextran (FITC-d; 3 to 5 kDa) marker were evaluated for induction and assessment of enteric inflammation in broilers. Three independent experiments were conducted where birds received an inflammation inducer treatment and an oral gavage of FITC-d (2.2 mg/bird) 2.5 h before killing on d 4, followed by measurement of serum FITC-d levels and release of FITC-d from different regions of gastrointestinal tract (GIT) to evaluate tight junction function. Experiment 1 tested control (CON) and DSS; Experiments 2 and 3 evaluated CON, DSS, and FRS. In all experiments DSS, as well as FRS in Experiments 2 and 3, showed higher (P<0.05) leakage of FITC-d into serum than CON, but FRS was not different from DSS. The amount of FITC-d retained in duodenal and cecal tissue was affected (P<0.05) by FRS in Experiments 2 and 3, and DSS affected FITC-d retention in duodenum only, suggesting differences in gut passage or absorption/adsorption. In conclusion, DSS oral gavage and FRS could induce leaky gut, with changes in serum FITC-d and migration of FITC-d from GIT.

Identification and Characterization of Lactic Acid Bacteria in a Commercial Probiotic Culture

The aim of the present study was to describe the identification and characterization (physiological properties) of two strains of lactic acid bacteria (LAB 18 and 48) present in a commercial probiotic culture, FloraMax®-B11. Isolates were characterized morphologically, and identified biochemically. In addition, the MIDI System ID, the Biolog ID System, and 16S rRNA sequence analyses for identification of LAB 18 and LAB 48 strains were used to compare the identification results. Tolerance and resistance to acidic pH, high osmotic concentration of NaCl, and bile salts were tested in broth medium. In vitro assessment of antimicrobial activity against enteropathogenic bacteria and susceptibility to antibiotics were also tested. The results obtained in this study showed tolerance of LAB 18 and LAB 48 to pH 3.0, 6.5% NaCl and a high bile salt concentration (0.6%). Both strains evaluated showed in vitro antibacterial activity against Salmonella enterica serovar Enteritidis, Escherichia coli (O157:H7), and Campylobacter jejuni. These are important characteristics of lactic acid bacteria that should be evaluated when selecting strains to be used as probiotics. Antimicrobial activity of these effective isolates may contribute to efficacy, possibly by direct antimicrobial activity in vivo.

Evaluation of the respiratory route as a viable portal of entry for Salmonella in poultry via intratracheal challenge of Salmonella Enteritidis and Salmonella Typhimurium

Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (106 cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.

Evaluation of a Bacillus direct-fed microbial candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation in broiler chickens fed on a rye-based diet

Abstract 1. The effects of the dietary inclusion of a Bacillus-based direct-fed microbial (DFM) candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation were evaluated in broilers consuming rye-based diets. 2. In the present study, control mash rye-based diets (CON) or Bacillus-DFM supplemented diets (TRT) were administered ad libitum to male broilers in three independent experiments. 3. In Experiments 1 and 2 (n = 25/group), liver samples were taken to evaluate bacterial translocation, digesta samples were used for viscosity measurements and the intestinal microbial flora was evaluated from different intestinal sections to enumerate total recovered gram-negative bacteria (TGB), lactic acid bacteria (LAB) and anaerobic bacteria (TAB). Additionally, both tibias were removed for assessment of bone quality. 4. In Experiment 3, each experimental group had 8 replicates of 20 chickens (n = 160/group). Weekly, body weight (BW), feed intake (FI) and feed conversion ratio (FCR) were evaluated. At d 28-of-age, samples were taken to determine bacterial translocation, digesta viscosity and bone quality characteristics. 5. In all experiments, consumption of Bacillus-DFM reduced bacterial translocation to the liver and digesta viscosity. Additionally, DFM supplementation improved BW, bone quality measurements and FCR. Moreover, chickens fed on the Bacillus-DFM diet in Experiments 1 and 2 showed a significant reduction in the number of gram-negative and anaerobic bacteria in the duodenal content compared to control. 6. In summary, chickens fed on a rye-based diet without DFM inclusion showed an increase in bacterial translocation and digesta viscosity, accompanied by reduced performance and bone quality variables relative to the Bacillus-DFM candidate group. Hence, incorporation into the feed of a selected DFM ameliorated the adverse anti-nutritional effects related to utilisation of rye-based diets in broilers chickens.

Evaluation of a commercially available organic acid product on body weight loss, carcass yield, and meat quality during preslaughter feed withdrawal in broiler chickens: A poultry welfare and economic perspective

The effect of a commercial organic acid (OA) product on BW loss (BWL) during feed withdrawal and transportation, carcass yield, and meat quality was evaluated in broiler chickens. Two experiments were conducted in Brazil. Commercial houses were paired as control groups receiving regular water and treated groups receiving OA in the water. Treated birds had a reduction in BWL of 37 g in experiment 1 and 32.2 g in experiment 2. In experiment 2, no differences were observed in carcass yield between groups. Estimation of the cost benefit suggested a 1:16 ratio by using the OA. In experiment 3, conducted in Mexico, significant differences on water consumption, BWL, and meat quality characteristics were observed in chickens that were treated with the OA (P < 0.05). These data suggest this OA product may improve animal welfare and economic concerns in the poultry industry by reducing BWL and improving meat quality attributes.

Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens

Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 10(6) spores/g of feed, whereas a second group was gavaged with a single dose of 10(6) spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75°C for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 10(5) spores per gram of digesta, which is about a 1-log10 reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10(2) spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable.

Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments

Experimental and epidemiological evidence has indicated the respiratory route to be a potential portal of entry for salmonellas in poultry. The purpose of this study was to evaluate and compare the infectivity of Salmonella enterica serovar Senftenberg following oral gavage, intratracheal or intravenous challenge in chickens. Seven-day-old chicks were challenged with either 104 or 106 colony-forming units of S. Senftenberg per chick by oral gavage, intratracheal or intravenous challenge, respectively, in two independent trials. Chickens were humanely killed 24 h post challenge and S. Senftenberg was cultured and enumerated from caecal contents, caecal tissue–caecal tonsils and liver and spleen. In both trials, intratracheal delivery of S. Senftenberg was the only route that allowed colonization of the caeca of chickens when compared with oral gavage or intravenous challenge in a dose response fashion (P < 0.05). Liver and spleen samples yielded no S. Seftenberg after the lower dose challenge by the oral or intratracheal route and only low levels following the high-dose administration by these routes, whereas intravenous challenge resulted in recovery of the organisms after both doses. The results of the present study suggest that S. Senftenberg entering the blood is likely to be cleared and will not be able to colonize caeca to the same extent as compared with intratracheal challenge. Clarification of the potential importance of the respiratory tract for transmission of salmonellas under field conditions may be of critical importance to develop intervention strategies to reduce the transmission in poultry.

Histopathological and morphometric changes induced by a dextran sodium sulfate (DSS) model in broilers

Oral administration of dextran sodium sulfate (DSS) is commonly used as an inducer of enteric inflammation in rodents. However, there is a dearth of knowledge regarding appropriate dosage, timing, or ageresponses in broilers for this potential inducer of inflammation without necrosis. Two experiments were conducted in day-of-hatch chicks to analyze clinical parameters and enteric histological changes induced by DSS when administered via drinking water ( DW: ). In both experiments, birds were distributed into nontreated control or varying concentrations of DSS in DW. For both experiments, only 0.75% DSS in DW was histologically evaluated. In Experiment 1, chicks received DSS from day 3 to 11, and at 3, 6, and 8 d of treatment, chicks were weighed, and sections of the duodenum, ileum, and ceca were formalin fixed. The addition of 0.75% DSS caused depression, anemia, and watery bloody diarrhea, plus significantly (P < 0.05) decreased BW gain at all times. Shortened ileal villi at 6 d and duodenal villi at 8 d of treatment, reduced duodenal and ileal epithelial cell height at 3, 6, and 8 d, and increased duodenal goblet cell density at 6 and 8 d were observed in response to DSS administration (P < 0.05). In Experiment 2, birds received DSS from days 10 to 16 and were sampled at 3 and 6 d of treatment. Similar changes were found in ceca of treated birds. There was no significant change in the duodenal villus height and goblet cell density by 6 d of treatment, suggesting that 6 d of 0.75% DSS in DW was not sufficient for the reproduction of duodenal symptoms in these older birds. However, there was a significant decrease in ilealvillus height and decreased ileal epithelial cell height at 3 and 6 d of treatment, as well as a significant decrease in BW compared to the control group. These findings indicate that DW administration of 0.75% DSS caused generalized mild and non-necrotic enteritis in broilers and that this compound may be useful for enteric inflammation modeling in poultry.

Physiological Properties and Salmonella Growth Inhibition of Probiotic Bacillus Strains Isolated from Environmental and Poultry Sources.

The objective of the present study was to describe the physiological properties of seven potential probiotic strains of Bacillus spp. Isolates were characterized morphologically, biochemically, and by 16S rRNA sequence analyses for identification. Tolerance to acidic pH, high osmotic concentrations of NaCl, and bile salts were tested. Isolates were also evaluated for their ability to metabolize different carbohydrates sources. The antimicrobial sensitivity profiles were determined. Inhibition of gastrointestinal Salmonella colonization in an avian model was also evaluated. Five strains of Bacillus were tolerant to acidic conditions (pH 2.0) and all strains were tolerant to a high osmotic pressure (NaCl at 6.5%). Moreover, all strains were able to tolerate concentration of 0.037% bile salts after 24 h of incubation. Three strains were able to significantly reduce Salmonella Typhimurium levels in the crop and in the ceca of broiler-type chickens. Among the 12 antibiotics tested for antibiotic resistance, all strains were resistant to bacitracin and susceptible to gentamycin, neomycin, ormethoprim, triple sulfa, and spectinomycin. Bacterial spore formers have been shown to prevent gastrointestinal diseases in animals and humans. The results obtained in this study show important characteristics to be evaluated when selecting Bacillus spp. candidates to be used as probiotics.

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.