Live attenuated vaccine-based control of necrotic enteritis of broiler chickens

https://doi.org/10.1016/j.vetmic.2005.10.015Get rights and content

Abstract

A vaccine for necrotic enteritis (NE) of chickens would reduce the current need to prevent or treat the disease in broiler chickens with antimicrobial drugs. The objective of this study was to understand aspects of immunity to the disease. The first experiment examined the virulence of six strains of Clostridium perfringens isolated from cases of NE in broiler chickens. Using a 5-day experimental oral infection of 2-week-old broiler chickens, four of the six strains were found to be virulent. Pulsed-field gel electrophoresis and PCR showed that virulence was not associated with a plasmid encoding the beta2 toxin gene, cpb2, since this was present in virulent and one of the two avirulent strains.

In the second experiment, two virulent and one avirulent strains were tested for their ability to immunize (“infection-immunization”) chickens through the oral route. The procedure used experimental infection for 5 days followed by bacitracin treatment for 9 days, and then re-challenge 2 days later with a virulent strain, CP4. Infection-immunization with the virulent isolates protected chickens from subsequent virulent challenge, whereas the infection-immunization with the avirulent isolate did not. In a third experiment, two of four alpha-toxin-negative mutants of CP4 protected birds from experimental NE after oral immunization. These two mutants were also attenuated for virulence. We conclude that it is possible to immunize chickens successfully against NE and that immunogen(s) other than alpha-toxin are important in protective immunity against oral infection.

Introduction

Clostridium perfringens is a Gram-positive, spore-forming anaerobe that is found in the soil and in the intestine of humans and animals. C. perfringens synthesizes and secretes up to 17 different extracellular toxins, 4 of which (alpha, beta, epsilon and iota) are major mouse-lethal toxins used to distinguish five toxin types: A, B, C, D or E (Songer, 1996). Necrotic enteritis (NE) is an enteric disease of birds that is caused predominantly by C. perfringens type A and to a lesser extent type C strains (Songer, 1996), and is an economically significant disease in broiler chickens. The disease is thought to occur when C. perfringens, which is part of the normal intestinal flora, overgrows in the small intestine and produces extracellular, particularly alpha, toxins that damage the intestine. Predisposing factors include infection with coccidia, as well as a high protein, high fibre and/or a wheat-based diet (Truscott and Al-Sheikhly, 1977, Al-Sheikhly and Al-Saieg, 1980, Riddell and Kong, 1992). Necrotic enteritis is commonly controlled in a preventative manner by incorporation of antimicrobial drugs in the feed or water, but this practice is increasingly criticised or has been banned in some jurisdictions. Since NE is such a common disease of broiler chickens, there is a need to investigate alternative approaches to its effective control. Despite the importance of NE, there is very little known about the basis of immunity to this infection, although immunization is an obvious approach to control.

The purposes of this study were therefore to examine aspects of immunity to NE in chickens, specifically: firstly, whether it is possible to immunize broiler chickens against NE; secondly, whether both virulent and avirulent strains of C. perfringens are immunogenic; and thirdly, whether immunity might depend on antigens other than the alpha-toxin.

Section snippets

Bacterial strains and plasmids

Six strains of C. perfringens (CP1–CP6) were obtained from D.A. Barnum, Department of Pathobiology, University of Guelph. These strains were isolated from different field cases of necrotic enteritis in Ontario and were stored frozen at −70 °C in cooked meat medium (CMM) (Difco, Detroit, MI, USA) within one to two passages in vitro following isolation. Strains Cpa 1, Cpa 2, Cpa 3, Cpa 4 are alpha-toxin negative derivatives of CP4 identified by the absence of a zone of turbidity surrounding

PCR toxin typing and pulsed-field gel electrophoresis

All six C. perfringens strains were found by multiplex PCR to be type A and all except CP5 were positive for the cpb2 gene (data not shown). PFGE and Southern blot were performed using the restriction enzyme I-CeuI, which cuts only chromosomal rRNA genes and therefore does not cut plasmid DNA. All strains except CP5 had the cpb2 gene localized to a plasmid of approximately 54 kb (for CP5 no cpb2 hybridization occurred) (data not shown). PCR of the cpb2 gene in CP5 confirmed the Southern blot

Discussion

This study of immunity to necrotic enteritis in chickens has shown, for the first time, that it is possible to immunize broiler chickens successfully against this disease by infection with virulent strains and subsequent treatment, that immunizing ability is associated with virulent and not avirulent strains, and that oral immunization with live alpha-toxin deficient isolates of C. perfringens protects against infection. There therefore appear to be factor(s) other than alpha-toxin that are

Acknowledgements

This work was supported by the Ontario Ministry of Agriculture and Food, the Poultry Industry Council, and the Saskatchewan Chicken Industry Development Fund. We thank J.G. Songer for providing reference strains and D.A. Barnum for providing C. perfringens field isolates.

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    But strains with a spontaneously mutated alpha-toxin did not lose their ability to cause NE (Cooper and Songer, 2009). In addition, Thompson et al. (2006) do not find a clear and particular communication between this toxin and NE, while they mentioned that alpha-toxin is an essential virulence factor (Thompson et al., 2006). Further studies indeed concluded the domination of the cpa+ netB+ genotype (type G) in chickens and NetB (a pore-forming toxin) had a significant role in NE pathogenesis (Cooper et al., 2013; Ezatkhah et al., 2016b; Keyburn et al., 2008, 2010).

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