Monitoring of Campylobacter jejuni in a chicken infection model by measuring specific volatile organic compounds and by qPCR.

Campylobacter is one of the leading bacterial foodborne pathogens worldwide. Poultry is the host species with this pathogen with the highest clinical impact. Flocks become colonised with Campylobacter, which leads to contamination of product entering the food-chain. Rapid and reliable Campylobacter detection methods could support controls to minimize the risks of contamination within the food-chain, which would easier enable the implementation of a logistical slaughter schedule or other control options. The present study evaluates current and emerging C. jejuni detection technologies on air samples in a unique study set-up of pre-defined C. jejuni prevalences. Both non-invasive detection technologies on air samples by subsequent measuring of volatile organic compounds (VOCs) or by qPCR detected the C. jejuni presence and could additionally distinguish between the number of present C. jejuni-positive birds in the study set-up. Nevertheless, electrostatic air samplers diagnosed fewer birds as C. jejuni-positive compared to the cultivation-based method. By measuring the VOCs, it was possible to detect the presence of two positive birds in the room. This apparent high sensitivity still needs to be verified in field studies. Techniques, such as these promising methods, that can facilitate C. jejuni surveillance in poultry flocks are desirable to reduce the risk of infection for humans.

NanI sialidase contributes to toxin expression and host cell binding of Clostridium perfringens type G strain CP56 in vitro.

Necrotic enteritis, caused by NetB producing Clostridium perfringens type G strains, is a globally important poultry disease. An initial step in the pathogenesis of necrotic enteritis is the colonization and degradation of the intestinal mucus layer, a process in which C. perfringens sialidases - such as NanI sialidase - may play an important role. Sialidases cleave terminal sialic acid from complex carbohydrates on glycoconjugates, such as mucins. This study shows that NE-associated C. perfringens strain CP56 is able to use sialic acid (Neu5Ac) as a carbon source for bacterial growth. It is shown that supplementation of Neu5Ac in the growth medium does not only induce the production of extracellular sialidases of strain CP56, but also increases the production of both alpha toxin and NetB toxin. Moreover, it was found that pre-treating avian hepatocellular carcinoma cells (LMH cells) with the recombinant NanI sialidase increases the adherence of C. perfringens type G strain CP56 to these cells. As such, the data suggest an important role for sialidases in the pathogenesis of the disease.

Protein Truncating Variants of colA in Clostridium perfringens Type G Strains.

Extracellular matrix (ECM) degrading enzymes produced by Clostridium perfringens may play an important role during the initial phases of avian necrotic enteritis by facilitating toxin entry in the intestinal mucosa and destruction of the tissue. C. perfringens is known to produce several ECM-degrading proteases, such as kappa toxin, an extracellular collagenase that is encoded by the colA gene. In this study, the colA gene sequence of a collection of 48 C. perfringens strains, including pathogenic (i.e. toxinotype G) and commensal (i.e. toxinotype A) chicken derived strains and strains originating from other host species, was analyzed. Although the colA gene showed a high level of conservation (>96% nucleotide sequence identity), several gene variants carrying different nonsense mutations in the colA gene were identified, leading to the definition of four truncated collagenase variant types (I-IV). Collagenase variant types I, III and IV have a (nearly) complete collagenase unit but lack parts of the C-terminal recruitment domains, whereas collagenase variant types II misses the N-terminal part of collagenase unit. Gene fragments encoding a truncated collagenase were mainly linked with necrotic enteritis associated C. perfringens type G strains with collagenase variant types I and II being the most prevalent types. Gelatin zymography revealed that both recombinant full-length and variant type I collagenase have active auto-cleavage products. Moreover, both recombinant fragments were capable of degrading type I as well as type IV collagen, although variant type I collagenase showed a higher relative activity against collagen type IV as compared to full-length collagenase. Consequently, these smaller truncated collagenases might be able to break down collagen type IV in the epithelial basement membrane of the intestinal villi and so contribute to the initiation of the pathological process leading to necrotic enteritis.

Elevated faecal ovotransferrin concentrations are indicative for intestinal barrier failure in broiler chickens.

Intestinal health is critically important for the welfare and performance of poultry. Enteric diseases that cause gut barrier failure result in high economic losses. Up till now there is no reliable faecal marker to measure gut barrier failure under field conditions. Therefore, the aim of the present study was to identify a faecal protein marker for diminished intestinal barrier function due to enteric diseases in broilers. To assess this, experimental necrotic enteritis and coccidiosis in broilers were used as models for gut barrier failure. Ovotransferrin was identified as a marker for gut barrier failure using a proteomics approach on samples from chickens with necrotic enteritis. These results were confirmed via ELISA on samples derived from both necrotic enteritis and coccidiosis trials, where faecal ovotransferrin levels were significantly correlated with the severity of gut barrier failure caused by either coccidiosis or necrotic enteritis. This indicates that faecal ovotransferrin quantification may represent a valuable tool to measure gut barrier failure caused by enteric pathogens.

Evaluation of a new enzyme-linked immunosorbent assay for the determination of neopterin.

BACKGROUND: Determination of neopterin especially evaluated for use on the Behring ELISA Processor BEP III highly suited for the demands of blood donation screening in blood banks. METHODS: A new commercially available enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neopterin, a low-molecular-mass pteridine. Neopterin is produced by interferon-activated macrophages or monocytes during the activation of the cellular immune system in various diseases. In Austria testing of neopterin to detect cellular immune activation is mandatory since 1995. The former assay version has been used for the measurement of neopterin at the Medical University Graz. As a result of the cooperation with the blood bank in Graz and the Dade Behring company we have developed a new ELISA kit based on a special coating procedure. For comparison we performed measurements with the current IBL Neopterin ELISA version, the HPLC method and with the ELItest Neopterin ELISA (BRAHMS). The new assay is based on a simple assay procedure with two incubations of 1 h and of 30 minutes. RESULTS: Linear regression analysis showed a significant correlation to the HPLC method. The assay is accurate and precise. CONCLUSIONS: The above mentioned neopterin assay as an alternative method to other ELISA kits and the HPLC is highly suited for automation and was especially evaluated as a simple, rapid and reproducible version for the Behring ELISA Processor BEP III during this study.