Marc Verlinden

Intestinal mucus protects Campylobacter jejuni in the ceca of colonized broiler chickens against the bactericidal effects of medium-chain fatty acids

Campylobacter jejuni is the most common cause of bacterial-mediated diarrheal disease worldwide. Because poultry and poultry products are a major source of C. jejuni infections in humans, efforts should be taken to develop strategies to decrease Campylobacter shedding during primary production. For this purpose, the efficacy of medium-chain fatty acids (MCFA) as feed additives to control C. jejuni colonization in broiler chickens was analyzed. First, the antimicrobial activity of the MCFA caproic, caprylic, and capric acid on C. jejuni was evaluated in vitro. Minimal inhibitory concentrations were 0.25 mM for caproic and 0.5 mM for caprylic and capric acids at pH 6.0 and 4 mM for all 3 compounds at pH 7.5. Time-kill curves revealed strong bactericidal properties of the tested compounds toward C. jejuni at pH 6.0. Concentrations as low as 4 mM caprylic and capric acids and 16 mM caproic acid killed all bacteria within 24 h. Capric acid had the highest activity, with concentrations of 4 mM killing all bacteria within the hour. Together these data show a profound bactericidal, dose-dependent activity of the tested MCFA toward C. jejuni in vitro. For this reason, the effect of these 3 MCFA on C. jejuni was evaluated in vivo. The addition of any of the acids to the feed, from 3 d before euthanization, was not capable of reducing cecal Campylobacter colonization in 27-d-old broilers experimentally infected with C. jejuni at 15 d of age. Using a cecal loop model, sodium caprate was not able to reduce cecal Campylobacter counts. When time-kill curves were conducted in the presence of chick intestinal mucus, capric acid was less active against C. jejuni. At 4 mM, all bacteria were killed only after 24 h. Thus, despite the marked bactericidal effect of MCFA in vitro, supplementing these acids to the feed does not reduce cecal Campylobacter colonization in broiler chickens under the applied test conditions, probably due to the protective effect of the mucus layer.

The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.

Passive immunization to reduce Campylobacter jejuni colonization and transmission in broiler chickens

Campylobacter jejuni is the most common cause of bacterium-mediated diarrheal disease in humans worldwide. Poultry products are considered the most important source of C. jejuni infections in humans but to date no effective strategy exists to eradicate this zoonotic pathogen from poultry production. Here, the potential use of passive immunization to reduce Campylobacter colonization in broiler chicks was examined. For this purpose, laying hens were immunized with either a whole-cell lysate or the hydrophobic protein fraction of C. jejuni and their eggs were collected. In vitro tests validated the induction of specific ImmunoglobulinY (IgY) against C. jejuni in the immunized hens’ egg yolks, in particular. In seeder experiments, preventive administration of hyperimmune egg yolk significantly (P < 0.01) reduced bacterial counts of seeder animals three days after oral inoculation with approximately 104 cfu C. jejuni, compared with control birds. Moreover, transmission to non-seeder birds was dramatically reduced (hydrophobic protein fraction) or even completely prevented (whole-cell lysate). Purified IgY promoted bacterial binding to chicken intestinal mucus, suggesting enhanced mucosal clearance in vivo. Western blot analysis in combination with mass spectrometry after two-dimensional gel-electrophoresis revealed immunodominant antigens of C. jejuni that are involved in a variety of cell functions, including chemotaxis and adhesion. Some of these (AtpA, EF-Tu, GroEL and CtpA) are highly conserved proteins and could be promising targets for the development of subunit vaccines.

Mycotoxins Deoxynivalenol and Fumonisins Alter the Extrinsic Component of Intestinal Barrier in Broiler Chickens

Deoxynivalenol (DON) and fumonisins (FBs) are secondary metabolites produced by Fusarium fungi that frequently contaminate broiler feed. The aim of this study was to investigate the impact of DON and/or FBs on the intestinal barrier in broiler chickens, more specifically on the mucus layer and antioxidative response to oxidative stress. One-day-old broiler chicks were divided into four groups, each consisting of eight pens of seven birds each, and were fed for 15 days either a control diet, a DON-contaminated diet (4.6 mg DON/kg feed), a FBs-contaminated diet (25.4 mg FB1 + FB2/kg feed), or a DON+FBs-contaminated diet (4.3 mg DON and 22.9 mg FB1 + FB2/kg feed). DON and FBs affected the duodenal mucus layer by suppressing intestinal mucin (MUC) 2 gene expression and altering the mucin monosaccharide composition. Both mycotoxins decreased gene expression of the intestinal zinc transporter (ZnT)-1 and regulated intracellular methionine homeostasis, which are both important for preserving the cells critical antioxidant activity. Feeding a DON- and/or FBs-contaminated diet, at concentrations close to the European Union maximum guidance levels (5 mg DON and 20 mg FB1 + FB2/kg feed) changes the intestinal mucus layer and several intestinal epithelial antioxidative mechanisms.

Application of medium-chain fatty acids in drinking water increases Campylobacter jejuni colonization threshold in broiler chicks

Campylobacteriosis is the most reported bacterial-mediated gastroenteritic disease in many developed countries. Broiler chickens are a natural host for Campylobacter spp., and contaminated poultry meat products are a major source for transmitting pathogenic Campylobacter strains to humans. Currently, no intervention measure efficiently and effectively controls this pathogen in poultry flocks. Medium-chain fatty acids (caproic, caprylic, capric, and lauric acids) show a marked anti-Campylobacter activity in vitro. However, in recent trials using our in vivo models, administering these acids to the feed of broiler chicks neither prevented nor reduced cecal C. jejuni colonization in broilers. In the present study, we examined whether a drinking water application of medium-chain fatty acids might be more effective in combating Campylobacter colonization in poultry. Although Campylobacter colonization and transmission was not reduced, we demonstrate that adding an emulsion of a mixture of caproic, caprylic, capric, and lauric acids to the drinking water of broiler chicks reduces their colonization susceptibility and prevents C. jejuni survival in drinking water. Thus, the merit of water applications of medium-chain fatty acids is the reduction of the probability of Campylobacter entry into and transmission throughout a flock.

Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs

Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities.

Antimicrobial Susceptibility Pattern of Brachyspira intermedia Isolates from European Layers

A broth microdilution method was used to determine the antimicrobial susceptibility of 20 Brachyspira intermedia isolates obtained from different layer flocks in Belgium and The Netherlands between 2008 and 2010. The antimicrobial agents used were tylosin, tilmicosin, tiamulin, valnemulin, doxycycline, and lincomycin. The minimal inhibitory concentration (MIC) distribution patterns of tylosin, tilmicosin, lincomycin, and doxycycline were bimodal, demonstrating acquired resistance against doxycycline in three strains, against the macrolides in two strains, and against lincomycin in one strain. The MICs of tiamulin and valnemulin showed a monomodal distribution, but with tailing toward the higher MIC values, possibly suggesting low-level acquired resistance in six isolates. Sequencing revealed a G1058C mutation in the 16S rRNA gene in all doxycycline-resistant strains. The strain resistant to tylosin, tilmicosin, and lincomycin had an A2058T mutation in the 23S rRNA gene.

Biocheck.UGent: A quantitative tool to measure biosecurity at broiler farms and the relationship with technical performances and antimicrobial use

The Biocheck.UGent scoring system has been developed to measure and quantify the level of biosecurity on broiler farms. This tool is composed of all relevant components of biosecurity on broiler farms and is subdivided into external (purchase of 1-d-old chicks, off-farm movements of live animals, feed and water supply, removal of manure and dead birds, entrance of visitors and personnel, supply of materials, infrastructure and biological vectors, location of the farm) and internal (disease management, cleaning and disinfection, materials, and measures between compartments) biosecurity. The unique feature of this scoring system is that it takes the relative importance of the different biosecurity aspects into account, resulting in a risk-based weighted score. The Biocheck.UGent scoring system and accompanying questionnaire can be filled in for free at www.Biocheck.UGent.be. The obtained biosecurity scores are provided immediately after completion of the questionnaire, and the scores for each subcategory can be compared with national averages to allow the farmer to benchmark the obtained results to his colleagues. Preliminary results (n = 15) show a huge range in the biosecurity level on broilers farms in Belgium, with internal biosecurity scores ranging from 54/100 to 87/100 and external biosecurity scores ranging from 55/100 to 72/100. These first results show that despite the well-known importance of biosecurity, theres a lack of implementation of many biosecurity measures and room for improvement.

Occurrence of viable Brachyspira spp. on carcasses of spent laying hens from supermarkets

Brachyspira spp. are frequent inhabitants of the chickens intestine and some have been associated with enteric disease in humans. We studied contamination with Brachyspira spp. of carcasses of spent laying hens as a possible source of infections for humans and animals that may eat this meat. Eleven batches of hen carcasses, for a total of 110 carcasses, were bought in Belgian supermarkets during 2009-2010. Carcass rinse samples were examined for the presence of Brachyspira. Brachyspira spp. were cultured from some carcass in all batches. Besides presumably non-pathogenic species such as Brachyspira murdochii and Brachyspira innocens, the poultry pathogen Brachyspira intermedia and the poultry and suspected human pathogen Brachyspira pilosicoli were identified in 7/11 and 1/11 carcass batches, respectively, at high numbers, as shown using quantitative polymerase chain reactions. Multilocus sequence typing (MLST) demonstrated the presence of 2 and 13 MLST types of B. pilosicoli and B. intermedia, respectively, with all strains belonging to novel MLST types. The findings show that carcasses of spent laying hens are commonly contaminated with high numbers of Brachyspira spp., including the suspected zoonotic agent B. pilosicoli.