Necrotic enteritis, disease induction, predisposing factors and novel biochemical markers in broilers chickens

Necrotic enteritis (NE) is an important enteric disease in poultry production that has re-emerged as a major problem following an EU wide ban on the use of in-feed antimicrobials. Although the primary aetiological agent of disease is Clostridium perfringens type A, a commensal in the gastrointestinal tract (GIT) of chickens, numerous additional influential factors have been reported that can predispose chickens to NE. These precipitating factors mainly include diet, co-infection with other pathogens particularly coccidia, as well as environmental and management factors. Despite being first described almost more than 50 years ago, a reliable, consistently reproducible experimental model for NE induction is still lacking. Here, a series of experiments were conducted to investigate the importance of the various potential predisposing factors, in isolation and in combination, that are believed to play a role in sub-clinical NE development: feed withdrawal, dietary protein sources, co- infection with coccidia, C. perfringens dose and contact with reused litter. In addition, chicken breed sensitivity to NE was assessed, and last, but not least, blood and gut tissue samples were used to identify novel biochemical markers for sub-clinical NE. Chapter 3 reports an experiment showing that feed withdrawal up to 24 hrs in experimentally challenged birds did not result in NE specific lesions. Chapter 4 shows that replacing dietary soyabean meal with potato protein concentrate or canola meal or adding synthetic trypsin inhibitor to the soyabean meal control diet did not induce sub-clinical NE in birds housed on reused litter, a natural source of C. perfringens challenge. Chapter 5 describes that in vitro growth of C. perfringens on in vitro digested grower diets was prolonged following the addition of fishmeal, suggesting that the role of fish meal as a predisposing factor for in vivo sub-clinical NE cannot be excluded. All subsequent diets therefore contained high levels of fish meal. When this was used in combination with high dose of coccidial vaccine, a repeated in-feed challenge for three days at 102 colony forming units (cfu) C. perfringens per g feed did not result in sub-clinical NE, though at 109 cfu/g resulted in 10% of challenged birds (3 out of 30) showing NE-specific lesions (Chapter 6). Further study is needed to determine if the two Ross birds with gross NE lesions compared to the one Hubbard bird (out of 15 birds each) was due to a lower level of NE resistance. 3 The failure to significantly induce sub-clinical NE in the previous experiments suggests that challenging the birds with C. perfringens in the isolated presence of suspected predisposing factors may not provide a suitable experimental model. Indeed, when birds were dosed twice daily with 108 cfu C. perfringens for three days in the presence of high levels of fishmeal, canola meal as main protein source, coccidial and IBD vaccinations, and feed withdrawal prior to challenge, 40.6% of the challenged birds developed lesions of sub-clinical NE without inducing mortality (Chapter 7). This concurred with reduced growth performance relative to the sham-infected control birds, and thus is a successful model for induction of sub-clinical NE. Finally this work has, for the first time provided novel information on potential biomarkers (Chapter 8). Whilst challenge did not impact on the expression of genes previously shown to be differentially expressed upon C. perfringens toxin exposure, the serum ceruloplasmin concentration increased, suggesting that monitoring this acute phase protein may indicate the presence of C. perfringens infection in poultry. However, as such markers generally lack specificity, further research confirming its role in response to sub-clinical NE is needed to provide a fully effective diagnostic and prognostic marker for flock health and welfare, as well as ultimately helping to gain better understanding of the pathophysiology of sub-clinical NE. Improved knowledge of the effect of different dietary components on the growth of C. perfringens may help in the formulation of broiler diets to assist in further reducing the incidence of NE particularly in the absence of antimicrobial growth promoters. It is hoped that host responses in terms of acute phase proteins, and possibley gene expression, will also provide greater insight into the pathogenesis of NE. Provided that the developed experimental sub-clinical NE model is reproducible, this will benefit the understanding of this billion dollar disease and enable further investigation of various chemical and non-chemical interventions to reduce its severity and impact on poultry production.