Additional files supporting conclusions in manuscript on development of an experimental necrotic enteritis model in turkeys

Manuscript title: Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions. Authors: Simon P. Hardy1+ ([email protected]), Sylvie L. Benestad2+ ([email protected]), Inger Sofie Hamnes2 ([email protected]), Torfinn Moldal2 ([email protected]), Bruce David3 ([email protected]), John R. Barta4 ([email protected]), Jean-Michel Reperant5 ([email protected]), Magne Kaldhusdal2 * ([email protected]) Conclusions: This study represents a first and major step forward in the development of in vivo experimental models of necrotic enteritis in turkeys

Pharmacokinetics and clinical assessment of amoxicillin for the control of necrotic enteritis in broiler-breeders under field conditions

Necrotic enteritis (NE) is an acute clostridium infection characterised by sudden onset, high mortality and severe necrosis of intestinal mucosa. The present study was conducted to clinically assess the efficacy of amoxicillin to control naturally occurring NE caused by Clostridium perfringens type A in broiler-breeders and to further support the clinical outcome of drug administration with evidence of efficacy based on the pharmacodynamic/pharmacokinetic correlation. A flock of 34,000 broiler-breeders exhibited an abrupt increase in mortality and a mild decrease in egg production, together with a reduction in body weight (BW). No signs of gross lesions were apparent, except a necrotic pseudomembrane covering the intestinal mucosa. Microbiological cultures from the intestine and PCR revealed the presence of Clostridium perfringens type A. After the administration of an amoxicillin compound (Animox (R) 500 water soluble powder50%, PHARMAQUA) at the dose of 20 mg kg(-1) BW diluted in the drinking water for five consecutive days, the flock recovered normally, the production data reached levels prior to induction of the disease and the intestine tissue was rapidly improved. The minimal inhibitory concentrations (MIC) of the isolated pathogen was determined and a pharmacokinetic study was performed under field conditions. The pharmacodynamic/pharmacokinetic correlation revealed a C-max/MIC ratio of 167.9, well above the value of 10-12 for a bactericidal and eliminatory effect to be expected. Furthermore, the AUC/MIC ratio was 1383.2, supporting the efficacy of the drug even if used in immunocompromised patients. Based on both clinical and pharmacological evidence amoxicillin can be used for the control of NE during an outbreak

Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens

Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin

Entero-hepatic pathobiology: histopathology and semi-quantitative bacteriology of the duodenum

Duodenum swabs were harvested from 80 broilers in 15 flocks of 8 broiler farms and were submitted to bacteriological evaluation. Five broiler breeders were sampled to obtain comparative data. Macro and microscopic lesions in the gut and the liver were evaluated in all birds submitted to swab harvesting and also broilers of two slaughter plants. Semiquantitative bacterial examination in duodenum swabs demonstrated higher microorganism growth in blood agar than in MacConkey medium. Staphylococcus sp, Enterococcus sp and Escherichia coli were found in some broiler flocks. An increased cellularity of the muscular mucosae of duodenum villi and/or the presence of some gametocytes or schizonts of coccidia were found in the lesioned gut in addition to high bacterial growth in blood agar. In some flocks, enlarged yellowish livers with vacuolated hepatocytes and/or increased reticuloendothelial response were seen in some birds. Liver and gut samples were collected in two slaughter plants in order to establish a pattern of response in broilers after withdrawal of growth promoters. Livers with macroscopic lesions presented microscopic lesions in the parenchyma such as increased leukocyte response in the sinusoidal space, vacuolar degeneration of hepatocytes or focal necrosis in the perilobular region, increased size or number of lymphoid nests in the perilobular region, the perivascular space and surrounding the biliary duct and increased extramedular granulocytopoiesis. Duodenum with hyperemic patches showed increased cellular response in the muscular mucosa of villi. These observations support the concept that semi-quantitative bacteriology and liver lesion evaluation could be used as a model to study gut bacterial imbalance in chickens