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Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

ABSTRACT: Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.

SUBMITTER: Timbermont L

PROVIDER: S-EPMC3992141 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

Timbermont Leen L De Smet Lina L Van Nieuwerburgh Filip F Parreira Valeria R VR Van Driessche Gonzalez G Haesebrouck Freddy F Ducatelle Richard R Prescott John J Deforce Dieter D Devreese Bart B Van Immerseel Filip F

Veterinary research 20140405

Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut les ...[more]

PMID: 24708344

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Project description:High necrotic enteritis (NE) incidence and mortality rates in poultry can be caused by Clostridium perfringens (CP) coinfected with Eimeria spp., a causative agent of coccidiosis. Banning of prophylactic use of antibiotics in feed has been accompanied by increased NE outbreaks, resulting in economically devastating losses to the broiler industry. To determine alternatives for controlling NE, we isolated CP-specific bacteriophages (BP), characterized their properties, evaluated their inhibitory effects on pathogenic CP, selected a highly effective phage (φCJ22), and used φCJ22 as a dietary supplement in experimental NE-afflicted broiler chickens. Male broilers (n = 780) were randomly assigned to 60 pens (n = 13 broilers/pen) and into 5 groups [CP-uninfected negative control (NC), basal diet (BD) without CP and BP; CP-infected positive control (PC), BD + CP; and 3 BP groups receiving low- (LP; BD + CP+105 BP), medium- (MP; BD + CP+106 BP), and high-phage (HP; BD + CP+107 BP plaque-forming units/kg) concentrations]. The results showed that MP and HP groups presented an antimicrobial activity toward clinical CP isolate strains, and the groups decreased NE lesions and mortality rates without changes in chicken performance at the end of the experimental period. After CP-challenge body weight gain and feed efficiency were significantly lower in phage-fed groups than that in the PC group (P < 0.05), and NE-associated mortality was the lowest in the HP group (P < 0.001). Moreover, histopathology revealed lesser gastrointestinal mucosal damage in the NC and BP-treated (LP, MP, and HP) groups than that in the PC group, and MP and HP significantly lowered viable CP number in the cecum content by up to 1.24log10 relative to only CP-infected PC group (P < 0.05). These findings suggest that addition of φCJ22 to chicken feed might effectively ameliorate NE, which is accompanied by reduced CP strains in the gut and compensate the performance of NE-afflicted broilers.

Project description:OBJECTIVE:The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed. METHODS:Eighteen-day-old broiler embryos were injected with 100 ?L of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured. RESULTS:Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-? factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird. CONCLUSION:These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.

Project description:BACKGROUND:Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome. RESULTS:Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis. CONCLUSIONS:The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

Project description:Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic pilR, agrB, and virR null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1pilR mutant, while production in its complement (CP1pilR+) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (P ≤ 0.05), whereas this activity was restored in the complemented strain (P > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (P > 0.05), whereas the complemented strains exhibited significantly reduced binding (P ≤ 0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon. IMPORTANCE Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.

Project description:Necrotic enteritis (NE) is a poultry disease caused by Clostridium perfringens and characterized by severe intestinal necrosis. The incidence of avian NE has been progressively increasing following the removal of antibiotics from poultry feed. We evaluated the effect of diets supplemented with the thermally-processed clays, calcium montmorillonite (CaMM) on clinical signs, immunopathology, and cytokine responses in broiler chickens using an experimental model of NE consisting of co-infection with Eimeria maxima and C. perfringens. In Trial 1, Ross/Ross chickens were fed from hatch with a normal basal diet or a CaMM-supplemented diet with or without a fermentable fiber, an organic acid, and/or a plant extract, and co-infected with E. maxima and C. perfringens under conditions simulating clinical infection in the field. Chickens fed a diet supplemented with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, reduced gut lesions, and increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with chickens fed the basal diet alone. Levels of transcripts for interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase, and tumor necrosis factor-α superfamily-15 were significantly altered in the intestine and spleen of CaMM-supplemented chickens compared with unsupplemented controls (p<0.05). In Trial 2, Cobb/Cobb chickens were fed an unsupplemented diet or a diet supplemented with CaMM or Varium®, each with a fermentable fiber and an organic acid, and co-infected with E. maxima and C. perfringens under subclinical infection conditions. Compared with unsupplemented controls, broilers fed with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, and reduced feed conversion ratio, mortality, and intestinal lesions, compared with chickens fed an unsupplemented diet (p<0.05). Dietary supplementation of broiler chickens with CaMM plus a fermentable fiber and an organic acid might be useful to control avian NE in the field.

Dataset Information

Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

Publications

Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis.

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