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NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.

Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, Di Rubbo A, Rood JI, Moore RJ - PLoS Pathog. (2008)

Bottom Line: The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE.This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE.Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

ABSTRACT
For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

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Virulence of C. perfringens Strains in the NE Challenge ModelThe lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. Each group consisted of ten birds. The solid horizontal bars represent the average lesion score in each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported [9]: 0, no gross lesions; 1, thin or friable walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2–3 cm long; 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: EHE-NE18, wild-type; ΔnetB, NE18ΔnetB1; ΔnetB Comp, NE18ΔnetB1(pALK20); ΔnetB1 + pJIR1457, NE18ΔnetB1(pJIR1457). One-tailed, nonparametric t-test analysis of the challenge (EHE-NE18) and complemented mutant derivatives against NE18ΔnetB1 showed a statistical difference (**, p < 0.01 and *, p < 0.05) but no statistical significance was observed between the mutant and the plasmid control.
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ppat-0040026-g008: Virulence of C. perfringens Strains in the NE Challenge ModelThe lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. Each group consisted of ten birds. The solid horizontal bars represent the average lesion score in each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported [9]: 0, no gross lesions; 1, thin or friable walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2–3 cm long; 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: EHE-NE18, wild-type; ΔnetB, NE18ΔnetB1; ΔnetB Comp, NE18ΔnetB1(pALK20); ΔnetB1 + pJIR1457, NE18ΔnetB1(pJIR1457). One-tailed, nonparametric t-test analysis of the challenge (EHE-NE18) and complemented mutant derivatives against NE18ΔnetB1 showed a statistical difference (**, p < 0.01 and *, p < 0.05) but no statistical significance was observed between the mutant and the plasmid control.

Mentions: To determine if the netB mutant could cause NE, groups of 10 Ross 308 broiler chickens were challenged with wild-type strain, the isogenic NE18ΔnetB1 mutant, or the complemented NE18ΔnetB1 strains in a NE disease induction model. No NE lesions were detected in birds infected with the netB mutant or the mutant complemented with the vector plasmid pJIR1457. By contrast, significant levels of disease were detected in birds infected with the wild-type parent strain (p < 0.01) or the strain complemented with the netB+ plasmid pALK20 (p < 0.05) (Figure 8). Similar results were obtained in independent virulence trials. Bacteria were cultured from the intestines of birds from all groups and with one exception only the infecting strain was isolated from the lesions. In one of the trials, two birds in the netB mutant challenge group showed a small number of lesions typical of NE. However, no thiamphenicol resistant C. perfringens were isolated from these birds and it was concluded that these lesions were likely to have been caused by environmental or endemic C. perfringens isolates and therefore are not included in our analysis.


NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.

Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, Di Rubbo A, Rood JI, Moore RJ - PLoS Pathog. (2008)

Virulence of C. perfringens Strains in the NE Challenge ModelThe lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. Each group consisted of ten birds. The solid horizontal bars represent the average lesion score in each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported [9]: 0, no gross lesions; 1, thin or friable walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2–3 cm long; 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: EHE-NE18, wild-type; ΔnetB, NE18ΔnetB1; ΔnetB Comp, NE18ΔnetB1(pALK20); ΔnetB1 + pJIR1457, NE18ΔnetB1(pJIR1457). One-tailed, nonparametric t-test analysis of the challenge (EHE-NE18) and complemented mutant derivatives against NE18ΔnetB1 showed a statistical difference (**, p < 0.01 and *, p < 0.05) but no statistical significance was observed between the mutant and the plasmid control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233674&req=5

ppat-0040026-g008: Virulence of C. perfringens Strains in the NE Challenge ModelThe lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. Each group consisted of ten birds. The solid horizontal bars represent the average lesion score in each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported [9]: 0, no gross lesions; 1, thin or friable walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2–3 cm long; 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: EHE-NE18, wild-type; ΔnetB, NE18ΔnetB1; ΔnetB Comp, NE18ΔnetB1(pALK20); ΔnetB1 + pJIR1457, NE18ΔnetB1(pJIR1457). One-tailed, nonparametric t-test analysis of the challenge (EHE-NE18) and complemented mutant derivatives against NE18ΔnetB1 showed a statistical difference (**, p < 0.01 and *, p < 0.05) but no statistical significance was observed between the mutant and the plasmid control.
Mentions: To determine if the netB mutant could cause NE, groups of 10 Ross 308 broiler chickens were challenged with wild-type strain, the isogenic NE18ΔnetB1 mutant, or the complemented NE18ΔnetB1 strains in a NE disease induction model. No NE lesions were detected in birds infected with the netB mutant or the mutant complemented with the vector plasmid pJIR1457. By contrast, significant levels of disease were detected in birds infected with the wild-type parent strain (p < 0.01) or the strain complemented with the netB+ plasmid pALK20 (p < 0.05) (Figure 8). Similar results were obtained in independent virulence trials. Bacteria were cultured from the intestines of birds from all groups and with one exception only the infecting strain was isolated from the lesions. In one of the trials, two birds in the netB mutant challenge group showed a small number of lesions typical of NE. However, no thiamphenicol resistant C. perfringens were isolated from these birds and it was concluded that these lesions were likely to have been caused by environmental or endemic C. perfringens isolates and therefore are not included in our analysis.

Bottom Line: The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE.This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE.Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia.

ABSTRACT
For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

Show MeSH
Related in: MedlinePlus