Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis
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SUBMITTER: Loutet S
PROVIDER: S-EPMC10909890 | biostudies-literature | 2024 Feb
REPOSITORIES: biostudies-literature
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Project description:Necrotic enteritis (NE), caused by Clostridium perfringens, is an intestinal disease with devastating economic losses to the poultry industry. NE is a complex disease and predisposing factors that compromise gut integrity are required to facilitate C. perfringens proliferation and toxin production. NE is also characterized by drastic shifts in gut microbiota; C. perfringens is negatively correlated with Lactobacilli. Vaccines are only partially effective against NE and antibiotics suffer from the concern of resistance development. These strategies address only some aspects of NE pathogenesis. Thus, there is an urgent need for alternative strategies that address multiple aspects of NE biology. Here, we developed Limosilactobacillus (Lactobacillus) reuteri vectors for in situ delivery of nanobodies against NetB and α toxin, two key toxins associated with NE pathophysiology. We generated nanobodies and showed that these nanobodies neutralize NetB and α toxin. We selected L. reuteri vector strains with intrinsic benefits and demonstrated that these strains inhibit C. perfringens and secrete over 130 metabolites, some of which play a key role in maintaining gut health. Recombinant L. reuteri strains efficiently secreted nanobodies and these nanobodies neutralized NetB. The recombinant strains were genetically and phenotypically stable over 480 generations and showed persistent colonization in chickens. A two-dose in ovo and drinking water administration of recombinant L. reuteri strains protected chickens from NE-associated mortality. These results provide proof-of-concept data for using L. reuteri as a live vector for delivery of nanobodies with broad applicability to other targets and highlight the potential synergistic effects of vector strains and nanobodies for addressing complex diseases such as NE.
| S-EPMC8924699 | biostudies-literature
Project description:A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.
| S-EPMC2797654 | biostudies-literature
Project description: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.
| S-EPMC2233674 | biostudies-literature
Project description: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.
| S-EPMC3992141 | biostudies-literature
Project description:Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.
| S-EPMC6372158 | biostudies-literature
Project description:Clostridium perfringens type G strains cause necrotic enteritis (NE) in poultry, an economically important disease that is a major target of in-feed antibiotics. NE is a multifactorial disease, involving not only the critically important NetB toxin but also additional virulence and virulence-associated factors. We previously identified a C. perfringens chromosomal locus (VR-10B) associated with disease-causing strains that is predicted to encode a sortase-dependent pilus. In the current study, we sought to provide direct evidence for the production of a pilus by C. perfringens and establish its role in NE pathogenesis. Pilus structures in virulent C. perfringens strain CP1 were visualized by transmission electron microscopy (TEM) of immunogold-labeled cells. Filamentous structures were observed extending from the cell surface in wild-type CP1 but not from isogenic pilin-null mutant strains. In addition, immunoblotting of cell surface proteins demonstrated that CP1, but not the null mutant strains, produced a high molecular weight ladder-like pattern characteristic of a pilus polymer. Binding to collagen types I, II, and IV was significantly reduced (Tukey's test, P < 0.01) in all three pilin mutants compared to CP1 and could be specifically blocked by CnaA and FimA antisera, indicating that these pilins participate in adherence. Furthermore, fimA and fimB null mutants were both severely attenuated in their ability to cause disease in an in vivo chicken NE challenge model. Together, these results provide the first direct evidence for the production of a sortase-dependent pilus by C. perfringens and confirm its critical role in NE pathogenesis and collagen binding.IMPORTANCE In necrotic enteritis (NE), an intestinal disease of chickens, Clostridium perfringens cells adhere tightly to damaged intestinal tissue, but the factors involved are not known. We previously discovered a cluster of C. perfringens genes predicted to encode a pilus, a hair-like bacterial surface structure commonly involved in adherence. In the current study, we have directly imaged this pilus using transmission electron microscopy (TEM). We also show that inactivation of the pilus genes stops pilus production, significantly reducing the bacterium's ability to bind collagen and cause disease. Importantly, this is the first direct evidence for the production of a sortase-dependent pilus by C. perfringens, revealing a promising new target for developing therapeutics to combat this economically important disease.
| S-EPMC8088525 | biostudies-literature
Project description:Necrotic enteritis (NE) in poultry is an opportunistic infection caused by Clostridium perfringens. Well-known as a multifactorial disease, NE development is under the influence of a wide range of environmental risk factors that promote the proliferation of pathogenic C. perfringens at the expense of nonpathogenic strains. Current in vivo NE challenge models typically incorporate pre-exposure to disease risk factors, in combination with exogenous C. perfringens inoculation. Our goal was to enhance current models using a natural uptake of C. perfringens from the barn environment to produce a subclinical infection. We incorporated access to litter, coccidial exposure (either 10× or 15× of the manufacturer-recommended Coccivac B52 Eimeria vaccine challenge; provided unspecified doses of E. acervulina, E. mivati, E. tenella, and two strains of E. maxima), feed composition, and feed withdrawal stress, and achieved the commonly observed NE infection peak at 3 weeks post-hatch. NE severity was evaluated based on gut lesion pathology, clinical signs, and mortality rate. Under cage-reared conditions, 15× coccidial vaccine-challenged birds showed overall NE lesion prevalence that was 8-fold higher than 10× coccidial vaccine-challenged birds. NE-associated mortality was observed only in a floor-reared flock after a 15× coccidial vaccine challenge.
| S-EPMC9251903 | biostudies-literature
Project description:BackgroundNecrotic enteritis is a devastating economic disease caused by Clostridium perfringens in poultry. NetB toxin from C. perfringens type G is the major responsible cause of necrotic enteritis. After the ban on growth-promoting antibiotics, alternative effective intervention approaches such as the vaccination of birds were considered critical to control necrotic enteritis. To date, no commercial vaccines with proven efficacy have been approved against necrotic enteritis. In this study, we evaluated the efficacy of the oral and parenteral vaccines based on NetB antigen from C. perfringens to choose the best prime-boosting vaccination strategy against necrotic enteritis. The broiler chickens were orally vaccinated with either previously developed recombinant probiotic bacterium, Lactobacillus casei strain expressing NetB toxoid, followed by a parenteral booster by the purified recombinant NetB toxoid (oral/parenteral), or the recombinant NetB toxoid alone (parenteral-only).ResultsImmunizations of birds with these vaccines elicited strong specific anti-NetB antibody responses and provided significant protection against the infectious challenge. Additionally, the vaccinated birds represented significant mean body weight gains compared with birds in control groups during the experiment.ConclusionsThe current study showed that oral and parenteral vaccines using NetB antigen from C. perfringens could provide significant protection against necrotic enteritis in broiler chickens.
| S-EPMC11907976 | biostudies-literature
Project description:BackgroundNecrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. The NetB toxin plays a critical role in the pathogenesis of NE. We previously demonstrated that netB is located within a 42 kb plasmid-encoded pathogenicity locus (NELoc-1), which also encodes 36 additional genes. Although NetB clearly plays a role in pathogenesis, the involvement of the other NELoc-1 genes has not yet been established. The current study was to provide experimental evidence to confirm the involvement of these genes in NE pathogenesis.ResultsThe present study has characterized a virulent C. perfringens strain (CP1) that has spontaneously lost the NELoc-1-encoding plasmid, pCP1netB. When assessed for cytotoxicity on Leghorn Male Hepatoma (LMH) cells, the culture supernatant of the pCP1netB-deficient CP1 variant (CP1ΔpCP1netB) demonstrated significantly reduced cytotoxicity compared to the wild-type. In addition, CP1ΔpCP1netB was unable to cause intestinal lesions in chickens in a NE disease model. When netB alone was introduced into CP1ΔpCP1netB, in vitro cytotoxicity was restored to the wild-type level; however, it did not completely restore virulence when used to challenge broiler chickens [mean lesion score of 0.71 compared to 3.23 in the wild type control group (n = 14)].ConclusionsThe results of this study suggest that other genes present in NELoc-1, in addition to netB, are required for full virulence in the chicken challenge model.
| S-EPMC5251324 | biostudies-literature
Project description:Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples (n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.
| S-EPMC7081503 | biostudies-literature