Abstract
BackgroundNecrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of C. perfringens produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE.MethodsWe have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-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, purified by metal affinity chromatography, and used to immunize broiler birds.ResultsImmunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. 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 toxoid is a promising vaccine candidate for controlling NE in poultry.
Highlights
Clostridium perfringens, a Gram-positive anaerobic spore-forming bacterium, is the causative agent of necrotic enteritis (NE) in poultry and has become an economicallyHow to cite this article Hunter JGL, Wilde S, Tafoya AM, Horsman J, Yousif M, Diamos AG, Roland KL, Mason HS. 2019
We found that birds immunized against both toxins developed fewer severe lesions after C. perfringens challenge than birds immunized with either antigen alone (Jiang et al, 2015)
The NetB sequence contains a mutation W262A in the rim domain (Savva et al, 2013), which substantially attentuates the toxicity of the protein in cell culture assays
Summary
Clostridium perfringens, a Gram-positive anaerobic spore-forming bacterium, is the causative agent of necrotic enteritis (NE) in poultry and has become an economicallyHow to cite this article Hunter JGL, Wilde S, Tafoya AM, Horsman J, Yousif M, Diamos AG, Roland KL, Mason HS. 2019. Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis. Methods: We have developed a fusion protein combining a non-toxic carboxylterminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. 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 toxoid is a promising vaccine candidate for controlling NE in poultry
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