A field study on correlations between macroscopic gut health scoring, histological measurements and performance parameters in broilers.

Optimal intestinal health is a prerequisite for sustainable poultry production. Intestinal health can be evaluated by scoring macroscopic intestinal abnormalities and by histological measurements. The aim of this study was to evaluate correlations between macroscopic gut appearance scoring (GAS), coccidiosis lesion scoring, histological measurements and performance parameters in broilers under field conditions. Therefore, a cross-sectional study was performed on 50 broiler farms where birds were sampled at 28 days of age. The GAS was determined by scoring the absence or presence of 10 macroscopically visible parameters of the gastrointestinal tract, yielding a GAS between 0 and 10, with 0 meaning no gut abnormalities. On individual bird level, when birds had a GAS score of 6 or higher, significantly shorter villi were found in the duodenum. Also, CD3+ T-lymphocyte area percentage in the duodenal mucosa was significantly negatively correlated to villus length. On farm level, the average feed conversion ratio (FCR) was 1.59 ± 0.04 [1.52-1.73]. However, no correlations were found between the GAS at the age of 28 days and the prevalence of coccidiosis, nor did coccidiosis lesion scoring correlate with the FCR. Also, a higher GAS could not be associated with a higher FCR. In conclusion, on all farms a certain degree of macroscopic visible gut and coccidiosis lesions were present in birds of 28 days of age, while this did not correlate with the FCR. This suggests that broilers are able to cope with a certain level of gut damage before it influences the overall performance.

The Salmonella Enteritidis TolC outer membrane channel is essential for egg white survival.

Salmonella Enteritidis has developed the potential to contaminate eggs by surviving in the antimicrobial environment of the hen's egg white. This has led to a worldwide pandemic of foodborne salmonellosis infections in humans due to the consumption of contaminated eggs and egg-derived products. The molecular mechanisms of Salmonella Enteritidis egg white survival are not fully clear. Using in vivo expression technology and promoter-reporter fusions we showed that the promoter of the tolC gene, encoding the TolC outer membrane channel that is used by multidrug efflux pumps to export harmful molecules and to secrete bacterial products, is activated by egg white at the chicken body temperature. Using a Salmonella Enteritidis tolC deletion mutant we showed that TolC has an important role in egg white survival. Chromatographic separation techniques and subsequent testing of antimicrobial activities of separated egg white fractions led to the identification of ovotransferrin as the egg white antimicrobial factor which is capable of inhibiting growth of a tolC deletion strain but not the wild type strain. We provide evidence that TolC protects Salmonella Enteritidis against ovotransferrin-mediated growth inhibition in egg white.

Short-chain arabinoxylans prepared from enzymatically treated wheat grain exert prebiotic effects during the broiler starter period.

Carbohydrate-degrading multi-enzyme preparations (MEP) are used to improve broiler performances. Their mode of action is complex and not fully understood. In this study, we compared the effect of water-soluble fractions isolated at the pilot scale from wheat grain incubated with (WE) and without (WC) MEP. The fractions were incorporated in a wheat-based diet (0.1% w/w) to feed Ross PM3 broilers and compared with a non-supplemented control group (NC). The body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) until d 14 were determined. At d 14, ileal and cecal contents and tissue samples were collected from euthanized animals. The intestinal contents were used to measure the short-chain fatty acids (SCFA) concentration using gas chromatography and to determine the abundance and composition of microbiota using 16S sequencing. Villi length of ileal samples was measured, while L-cell and T-cell densities were determined using immuno-histochemistry. The MEP treatment increased the amount of water-soluble arabinoxylans (AX) and reduced their molecular weight while retaining their polymer behavior. The WE fraction significantly (P < 0.05) increased FI by 13.8% and BWG by 14.7% during the first wk post hatch when compared to NC. No significant effect on FCR was recorded during the trial. The WE increased the abundance of Enterococcus durans and Candidatus arthromitus in the ileum and of bacteria within the Lachnospiraceae and Ruminococcaceae families, containing abundant butyrate-producing bacteria, in the ceca. It also increased the concentration of SCFA in the ceca, decreased the T-lymphocyte infiltration in the intestinal mucosa, and increased the glucagon-like-peptide-2 (GLP-2)-producing L-cell density in the ileal epithelium compared with WC and NC. No significant effects were observed on villi length. These results showed that AX present in the WE fraction altered the microbiota composition towards butyrate producers in the ceca. Butyrate may be responsible for the reduction of inflammation, as suggested by the decrease in T-lymphocyte infiltration, which may explain the higher feed intake leading to improved animal growth.

Does release of encapsulated nutrients have an important role in the efficacy of xylanase in broilers?

The non-starch polysaccharides (NSPs) in cell walls can act as a barrier for digestion of intracellular nutrients. This effect is called "cage effect." Part of the success of fibrolytic enzymes in broiler feed is assumed to be attributed to cage effect reduction. Further, changes in viscosity and potential prebiotic action should also be considered. The aim of this study was to gain insight into the relative importance of the cage effect in xylanase efficacy in broilers. Using a 2×2 factorial design, 24 pens with 30 Ross 308 male chicks were fed corn-soy based diets consisting of normal and freeze-thawed (5 d at -18°C) corn, both with and without xylanase. The freeze-thaw method was used to eliminate the cage effect, whereas a corn-based diet was used to exclude viscosity effects. Body weights (BW), feed intake (FI), and feed conversion ratio (FCR) were determined at d 13, 26, and 39. A balance study was executed at the end of the growing phase. These birds were euthanized at d 34 (non-fasted) to determine the viscosity of digesta, blood metabolites, intestinal morphology, and microbiota composition. During the finisher period, there was a significant interaction between enzyme supplementation and freeze-thawing for FCR, in which FCR was improved by freeze-thawed corn and tended to be improved by normal corn+enzyme compared with the control group. The improvement in performance (finisher period) of freeze-thawed corn and xylanase coincided with increased gut absorption of glucose (based on postprandial plasma concentrations) and increased number of Clostridiumcluster IV in the caecum, and agreed with the higher gut villus height. In addition, xylanase inclusion significantly increased the postprandial plasma glycine and triglycerides concentration, and led to elevated bacterial gene copies of butyryl CoA:acetate CoA-transferase, suggesting a prebiotic effect of xylanase addition through more than just the cage effect reduction. The applied model managed to rule out viscosity by using corn, and it was possible to isolate the cage effect by freeze-thawing the dietary corn.

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota.

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.

A review on prebiotics and probiotics for the control of dysbiosis: present status and future perspectives.

Dysbiosis or dysbacteriosis is defined as a shift in the intestinal microbiota composition resulting in an imbalance between beneficial and harmful bacteria. Since the ban on the use of growth-promoting antibiotics in animal feed in the EU, dysbiosis has emerged as a major problem in intensive animal production. Prebiotics and probiotics are currently under investigation as possible alternatives to growth-promoting antibiotics, as their mode of action is thought to be based largely on a modulation of the composition and function of the intestinal microbiota. In this review, we analyse the currently available data from both animal and human nutrition that document the potential and limitations of prebiotics and probiotics for the control of dysbiosis. An impressive number of empirical feeding trials have been carried out in healthy animals, yielding sometimes contradictory results. More in-depth studies have revealed the complexity of the interactions taking place in the lower intestinal tract, thus illustrating that pre- and probiotics cannot be a simple replacement for growth-promoting antibiotics. Although there are indications that the strategic use of pre- and probiotics can provide major benefits, there is still a lack of basic knowledge on the delicate interactions between the microbiota, the host and the feed components, which hampers the widespread use of these valuable feed additives.

Salmonella Gallinarum field isolates from laying hens are related to the vaccine strain SG9R.

Salmonella enterica subspecies enterica serotype Gallinarum can cause severe systemic disease in chickens and a live Salmonella Gallinarum 9R vaccine (SG9R) has been used widely to control disease. Using whole-genome sequencing we found point mutations in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes that likely explain the attenuation of the SG9R vaccine strain. Molecular typing using Pulsed Field Gel Electrophoresis and Multiple-Locus Variable number of tandem repeat Analysis showed that strains isolated from different layer flocks in multiple countries and the SG9R vaccine strain were similar. The genome of one Salmonella Gallinarum field strain, isolated from a flock with a mortality peak and selected on the basis of identical PFGE and MLVA patterns with SG9R, was sequenced. We found 9 non-silent single-nucleotide differences distinguishing the field strain from the SG9R vaccine strain. Our data show that a Salmonella Gallinarum field strain isolated from laying hens is almost identical to the SG9R vaccine. Mutations in the aceE and rfaJ genes could explain the reversion to a more virulent phenotype. Our results highlight the importance of using well defined gene deletion mutants as vaccines.

Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation.

A rapid and sensitive HPLC-UV method for the quantitative determination of four short-chain fatty acids (SCFAs) and lactic acid (LA) produced during in vitro fermentation is presented. Extraction of SCFAs from supernatants of bacterial cultures is aggravated due to their polarity and volatility. Detection can only be performed at a short, non-selective UV wavelength (210nm), due to the lack of any significant chromophore. Therefore special attention was paid to the optimization of the sample preparation procedure and the HPLC-UV conditions. The final extraction procedure consisted of a liquid-liquid back extraction using diethylether. Prior to HPLC-UV analysis the samples were acidified (pH<2) in order to improve retention of the SCFA's and LA on the Hypersil Gold aQ column. Matrix-matched calibration graphs were prepared for all analytes of interest (range 0.5-50mM) and correlation and goodness-of-fit coefficients were between 0.9951-0.9993 and 3.88-8.27%, respectively. Limits of detection and quantification ranged from 0.13 to 0.33mM and 0.5 to 1.0mM, respectively. The results for the within-day and between-day precision and accuracy fell within the ranges specified. The reported validated method has been successfully used for the in vitro screening of supernatants of bacterial cultures for the presence of butyric acid, aiming to select for butyric acid-producing bacteria. In addition, the method has been used to determine the production pattern of selected fatty acids by bacterial species isolated from human feces and chicken caeca.

Hepatic and intestinal CYP3A expression and activity in broilers.

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT-PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra-acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.

Clostridium novyi type B as a causative agent of bovine meat spoilage.

A series of bovine meat spoilage cases in which meat from clinically healthy Belgian Blue cattle showed green discoloration are described. Histology of skeletal muscle revealed numerous spore-forming rods in the discolored areas of the meat. These organisms stained positively for Clostridium novyi by immunohistochemistry. A combination of 16S rDNA and fliC gene sequencing of bacterial DNA, isolated from the spoiled meat samples, revealed the unique presence of C. novyi type B. Although this bacterium has been implicated in clinical necrotic hepatitis in cattle, the cases described here are the first implicating C. novyi type B as a cause of bovine meat spoilage.

From the gut to the peripheral tissues: the multiple effects of butyrate.

Butyrate is a natural substance present in biological liquids and tissues. The present paper aims to give an update on the biological role of butyrate in mammals, when it is naturally produced by the gastrointestinal microbiota or orally ingested as a feed additive. Recent data concerning butyrate production delivery as well as absorption by the colonocytes are reported. Butyrate cannot be detected in the peripheral blood, which indicates fast metabolism in the gut wall and/or in the liver. In physiological conditions, the increase in performance in animals could be explained by the increased nutrient digestibility, the stimulation of the digestive enzyme secretions, a modification of intestinal luminal microbiota and an improvement of the epithelial integrity and defence systems. In the digestive tract, butyrate can act directly (upper gastrointestinal tract or hindgut) or indirectly (small intestine) on tissue development and repair. Direct trophic effects have been demonstrated mainly by cell proliferation studies, indicating a faster renewal of necrotic areas. Indirect actions of butyrate are believed to involve the hormono-neuro-immuno system. Butyrate has also been implicated in down-regulation of bacteria virulence, both by direct effects on virulence gene expression and by acting on cell proliferation of the host cells. In animal production, butyrate is a helpful feed additive, especially when ingested soon after birth, as it enhances performance and controls gut health disorders caused by bacterial pathogens. Such effects could be considered for new applications in human nutrition.

Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains.

Necrotic enteritis is an economically important disease of chickens caused by Clostridium perfringens. Immunity to necrotic enteritis is not fully characterized yet, but previous reports indicate that immunoprotective potential is present in the secreted component of C. perfringens. This study aimed to compare the vaccine potential of the supernatants of eight chicken strains of C. perfringens differing in origin, level of alpha toxin production and presence of netB gene. The supernatant of only one strain provided full protection, while one other strain provided partial protection against a severe infection challenge. Our results indicate that the protective characteristics of the supernatants are not solely based on the presence of NetB or alpha toxin.

Quorum sensing in veterinary pathogens: mechanisms, clinical importance and future perspectives.

Under certain circumstances the individuals of a bacterial population may find advantages in acting together and making "collective decisions". This phenomenon is better known as quorum sensing. When the concentration of signal molecules produced by the surrounding bacteria exceeds a certain threshold, the bacterial population acts as a single organism, collectively expressing virulence genes, biofilm forming genes, etc. Several mechanisms of quorum sensing are discussed, each with its distinct signal molecules and respective receptors. Some of these mechanisms are restricted to sensing intraspecies signalling, but interspecies and even interkingdom signalling have also been described. Several veterinary pathogens such as Staphylococcus aureus, Staphylococcus pseudintermedius, Pseudomonas aeruginosa and Salmonella Typhimurium use quorum sensing as a means to optimize virulence gene expression and host colonization. Therefore, targeting of the QS mechanisms may provide a novel strategy for combating bacterial infections, also in veterinary medicine.

Arabinoxylooligosaccharides from wheat bran inhibit Salmonella colonization in broiler chickens.

The objective of this in vivo experiment was to evaluate the influence of arabinoxylooligosaccharides (AXOS) on shedding and colonization of Salmonella Enteritidis in broilers. Arabinoxylooligosaccharides, which are oligosaccharides derived from arabinoxylans by partial hydrolysis, have a beneficial effect on feed conversion ratios when added to broiler diets. Additionally, AXOS have been shown to promote the growth of bifidobacteria in the cecocolonic compartment of the gastrointestinal tract. To investigate the impact of AXOS on colonization of broilers with Salmonella, 224 one-day-old chicks were divided into 4 groups and given either unsupplemented feed or feed supplemented with 0.2% AXOS-3-0.25, 0.2% AXOS-9-0.25, or 0.4% AXOS-9-0.25 throughout the experiment. The AXOS-3-0.25 and AXOS-9-0.25 both have an ara-binose-to-xylose ratio of 0.25 and have an average degree of polymerization of 3 and 9, respectively. At 14 d posthatch, each animal was orally inoculated with 2.5 x 10(9) cfu of Salmonella Enteritidis. Cloacal swabs, taken at regular times, showed a significant reduction of Salmonella presence in the group given 0.4% AXOS-9-0.25 compared with the control group. This reduction was observed in the 1- to 11-d postinfection period. Colonization of the ceca as well as the translocation of Salmonella to the spleen was significantly reduced at 3 and 7 d postinfection in the 0.4% AXOS-9-0.25 group. A similar, although more moderate, decrease in colonization was observed in the group given 0.2% AXOS-9-0.25. It was concluded that dietary addition of AXOS provides dose-dependent protection against oral infections with Salmonella Enteritidis in poultry.

A comparative study on the pathogenesis of egg contamination by different serotypes of Salmonella.

Salmonella enterica serotype Enteritidis is the predominant serotype associated with egg-borne salmonellosis in humans. Apparently this serotype possesses particular characteristics that increase its chance to contaminate eggs. To identify these characteristics, two Salmonella serotype Enteritidis strains as well as one strain of each of the serotypes Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Virchow and Salmonella Hadar strain were used to examine different aspects related to egg contamination. After an intravenous infection of laying hens, it was observed that the ability of the serotype Enteritidis strains to colonize the reproductive organs was significantly higher compared with the Salmonella Heidelberg, Salmonella Virchow and Salmonella Hadar strains but not with the Salmonella Typhimurium strain. Inoculating low numbers of the different Salmonella serotypes in egg albumen at 42 degrees C demonstrated that the growth of the strains belonging to the Salmonella serotypes Virchow and Hadar was seriously repressed. The other serotypes, however, survived in albumen for 24 h. Furthermore, using two different specifically designed egg infection models, it was shown that all strains used in this study were able to penetrate into and multiply inside the yolk at 25 degrees C. These findings indicate that the ability to grow in eggs post lay is not specific for the serotype Enteritidis. In conclusion, comparing strains belonging to different Salmonella serotypes has revealed that most probably a preferential colonization of the reproductive organs and an enhanced survival at 42 degrees C allows the serotype Enteritidis to contaminate eggs.