Understanding the early pathogenesis of necrotic enteritis in chickens

了解产气荚膜梭菌的早期发病机制和毒力日益增加的消费者压力正迫使畜牧业开发替代品，或至少大幅减少抗生素的使用量。我们必须找到有效的工具来预防坏死性肠炎，这是一种影响无抗生素鸡的致命疾病。这种疾病是由产气荚膜梭菌（Clostridium perfringens, Cp）的致病菌株引起的，但需要诱发因素来创造有利于产气荚膜梭菌（C. perfringens, Cp）丰度增加的肠道环境。疾病过程的另一部分被认为依赖于引起ne的菌株取代鸡肠道中非致病性共生分离株的能力。许多革兰氏阳性细菌已被证明能产生各种称为细菌素的蛋白质毒性化合物，并抑制密切相关菌株的生长。Perfrin已在致病性netB阳性ne引起菌株中被发现，并被认为起重要作用。在琼脂斑点试验中，我们观察到并非所有netB阳性的Cp菌株都携带perfrin基因，并且产生了perfrin以外的细菌素，并且一些共生的Cp菌株可以抑制致病性Cp菌株的生长。我们在鸡体内建立了一个模拟坏死性肠炎的结扎肠环模型，并注意到病变严重程度与肠绒毛内Cp棒的数量相关（Parent等，2016）。因此，Cp附着在肠细胞上可能在NE的发病机制中起作用，因此我们对IV型pilins感兴趣，IV型pilins已知在粘附细胞表面起作用。我们最近的研究表明，当注射到鸟类体内时，这些药片也具有免疫原性。所有这些最近的发现，导致了这个建议的研究计划的发展。这项工作是一项创新，利用独特的Cp菌株库和新的动物模型（结扎肠环模型）来培养快速发展的基因组学、转录组学和生物信息学领域的高素质人才。它将为使用抗生素养鸡这一伦理问题提供独特的答案和新的解决方案。这个提议代表了我在过去几年里一直在进行的进一步了解坏死性肠炎的研究计划的逻辑连续性。我的研究目标是更好地了解NE早期感染步骤的致病机制，更准确地了解1)鉴定和表征两种新的Cp细菌素，2)验证三种IV型匹林在坏死性肠炎模型中的保护作用，3)利用我们的结肠炎模型识别参与早期发病机制的基因及其对宿主的影响，利用免疫组织化学，生物标志物和细菌转录组学。

Understanding early pathogenesis and virulence of Clostridium perfringens An increasing consumer pressure is forcing animal agriculture to develop alternatives, or at least to substantially reduce the amount of antibiotics used. We must find effective tools to prevent necrotic enteritis, a deadly disease from affecting antibiotic-free chickens. This disease is caused by pathogenic strains of Clostridium perfringens, but predisposing factors are necessary to create an intestinal environment conducive to an increase in the abundance of C. perfringens (Cp). Another part of the disease process is thought to rely on the ability of NE-causing strains to displace non-pathogenic commensal isolates from the gut of chickens. Numerous Gram positive bacteria have been shown to produce various proteinaceious toxic compounds called bacteriocins, and inhibiting the growth of closely related strains. Perfrin has been identified in pathogenic netB positive NE-causing strains and thought to play an important role. When testing various Cp strains on agar spot test, we observed that not all netB positive Cp strains carry the perfrin gene, that bacteriocins other than perfrin were produced, and that some commensal Cp strains can inhibit the growth of pathogenic Cp strains. We have developed an in vivo ligated intestinal loop model reproducing necrotic enteritis in chickens and noted that lesion severity was correlated with numbers of Cp rods lining the intestinal villi (Parent et al, 2016). Thus, Cp attachment to the enterocyte might play a role in the pathogenesis of NE hence our interest in type IV pilins which are known to play a role in adherence to cell surface. We have recently showed that these pilins are also immunogenic when injected in birds. All of these recent findings, have led to the development of this suggested research program. The proposed work is innovative, using a unique Cp strains bank with access to a new animal model (ligated intestinal loop model) to train highly qualified personal in the rapidly developing field of genomics, transcriptomics and bioinformatics. It will provide unique answers and bring new solutions to an ethical problem, which is the use of antibiotics to grow chickens. This proposal represents a logical continuity of the research program on further understanding necrotic enteritis I have been conducting in the past years. The objectives of my research program are to better understand the pathogenic mechanisms underlying the early infection steps of NE, more precisely 1) identify and characterize two new Cp bacteriocins, 2) verify the protective efficacy of three type IV pilins in a necrotic enteritis model, 3) use our ligated intestinal loop model to identify genes involved in early pathogenesis and their effects on the host, using immunohistochemistry, biological markers and bacterial transcriptomics.