Necrotic enteritis (NE), a devastating enteric disease caused by Clostridium perfringens type A, contributes to the losses of 6 billion dollars worldwide per year and is currently being considered as a major global threat to the poultry industry. In past decades, it has been well-controlled by ineed antimicrobial growth promoters (AGPs). The withdrawal of AGPs due to antibiotic-resistance concerns resulted in a spike in NE incidence and led to the re-emergence of NE in the modern broiler production system. To unveil the association of toxin genes of C. perfringens, particularly for netB, with clinical NE, a self-designed qPCR primer set targeting netB was developed to qualify and quantify netB in NE-producing and non-NE-producing isolates. The netB was demonstrated to exist in the majority of C. perfringens type A isolates. The presence and the amount of netB were not significantly different between two types of isolate, indicating that those indicators are insufficient to predict an association with the pathogenicity of NE. The virulence of netB is suggested to be expressed or triggered under certain conditions, further promoting NE. A side by side trial was implemented with different combinations of netB-positive C. perfringens (CP1) and two predisposing factors to assess their role in NE development. Both CP1 and predisposing factor(s) are required for consistent NE reproduction, and particularly, Eimeria exerts significant effects on NE induction. The use of CP1 without a predisposing factor failed to induce NE. The severity and incidence of NE were positively correlated with the number of predisposing factors given in the NE induction. Analyzing gut microbiota in chickens challenged with CP1 and/or Eimeria by metagenomic sequencing, significant overgrowth of Clostridium sensu stricto 1, the genus contains C. perfringens, was associated with NE. Eimeria infection precedent to CP1 challenge had a synergistic effect on the overrepresentation. In addition to C. perfringens, the other member under Clostridium sensu stricto 1 was found to participate in NE development. Given supplementary dose of 0.4 kg/ton in feed, lauric acid neither exerted the inhibitory effect against proliferation of Clostridium sensu stricto 1 and C. perfringens nor reduced the incidence and severity of NE.

abstract: Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens, leading to an estimated 2 billion dollar global economic loss in the poultry industry annually. Traditionally, NE has been effectively controlled by antibiotics added to the diet of poultry. Concerns about increasing antibiotic resistance of poultry and human based pathogens have led to the consideration of alternative approaches for controlling disease, such as vaccination. NE causing strains of C. perfringens produce two major toxins, α-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. We have developed a fusion protein combining a non-toxic carboxy-terminal domain of the α-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system. The fusion protein was purified by metal affinity chromatography and used to immunize broiler birds. Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. However, the PlcC-NetB fusion had antibody titers four times that of the bacterially produced toxoids alone. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB is a promising vaccine candidate for controlling NE in poultry. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

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