Production and purification of Clostridium perfringens type C beta-toxin and IgG and IgY antitoxins.

OBJECTIVES: This study aimed to produce and purify Clostridium perfringens type C beta-toxin, sheep anti-beta toxin immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY). METHODS: Two methods were used for beta-toxin purification: single-step metal affinity chromatography (MAC) using zinc as a chelator and ion exchange chromatography (IEX). The purified and inactivated beta-toxoids were then administered to sheep and chickens in order to produce IgG and IgY. RESULTS: All assays using the IEX failed. In contrast, MAC purified more than 21 mg of toxin per run in a single-step protocol. The purified and inactivated beta-toxoids were then administered to sheep and chickens, and IgG and IgY were purified with a high yield, medium antibody titer of 50 IU/mL, and high avidity (73.2 %). CONCLUSIONS: C. perfringens type C beta-toxin and sheep or chicken anti-beta toxin IgG and IgY antibodies were successfully produced and purified using a simple protocol. This protocol can be used for the production of components used in the diagnosis and research of necrotic enteritis caused by C. perfringens type C, as well as for the evaluation of existing vaccines and the development of new preventive methods against this disease.

The risk of multiple sclerosis on the Orkney islands. A review of the search for distinctively Orcadian risks, with a hypothesis for further investigations.

The most extensive and meticulous epidemiological study yet to be published on the frequency of multiple sclerosis (MS) across the regions of Scotland has confirmed that the high incidence of MS on the Orcadian islands is unique and is most probably the highest in the world. Environmental and genetic studies of Orcadian MS have been carried out over many years but the results have been discouragingly inconclusive; no convincing explanation of the distinctively high Orcadian MS risks has come to light. However, studies of both prevalence and incidence of MS over a time line of approximately five decades, show that Orcadian MS has steadily increased to significantly exceed the neighbouring genetically related populations including North Eastern Scotland and the Shetland islands. Over this period the islands have progressively expanded occupations related to agriculture and have simultaneously acquired the highest concentration of cattle in Europe. Coinciding high and increasing Orcadian MS risk with increasing agricultural activities including bovine density and dairying, points towards a potential but unexpected causal risk. Raised incidence of MS with farming and in particular with dairy farming have been documented in Australia, Denmark, and more recently in Norway, further pointing to a possible MS risk associated with agricultural activities. A clue to the cause of this curious association has unexpectedly emerged from laboratory studies. Using very rarely available tissues from patients coming to autopsy during an MS attack, a toxin known as beta-haemolysin (sphingomyelinase), which is produced by the bacterium Staphylococcus aureus, has been identified in the affected tissues. Staph aureus is a common inhabitant of the mucosal linings of the human nasal sinuses and sinus mucosal inflammations have been shown to be closely associated with attacks of MS and optic neuritis. Irrespective of origin, human or animal, all strains of Staph aureus carry the beta haemolysin gene. However, the toxin is only sporadically expressed by the strains most commonly isolated from human carriers. Strains carried by bovines nearly always express toxin. Has the increasing high risk of MS in Orcadians been promoted by the nasal transmission and subsequent establishment of the high secreting bovine genotypes of Staph aureus in the Orcadian population? To demonstrate that bovine associated strains of Staph aureus are carried more frequently in the Orcadian population (or even specifically in Orcadian MS cases), would not of itself necessarily explain the high prevalence of Orcadian MS. It would however clearly justify an in-depth exploration of the nasal bacterial microbiome of MS cases. This should include the incidence of beta-toxin secreting Staph aureus genotypes. If MS cases are shown to have a distinctive nasal bacterial microbiome, including beta-toxin secretors, this finding would open up an almost entirely new range of investigations and approaches to the understanding of the pathogenesis of MS.

Ts17, a Tityus serrulatus ß-toxin structurally related to α-scorpion toxins.

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Nav1.1-Nav1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na+-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Nav1.2 and Nav1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Nav1.5. Circular dichroism spectra showed that Ts17 presents mainly ß-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug. Key contribution Purification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, ß-toxin classification.

A non-toxic recombinant protein rSUMO-CPBm4 as a potential vaccine candidate against Clostridium perfringens type C beta enterotoxemia

@#Beta toxin (CPB) is a lethal toxin and plays a key role in enterotoxemia of ruminants caused by Clostridium perfringens type C strain. The existing vaccines based on crude CPB need time-consuming detoxification and difficult quality control steps. In this study, we synthesized the rCPBm4 of C. perfringens type C strain and small ubiquitin-like modifier (SUMO)-tag CPBm4 (rSUMO-CPBm4) by introducing four amino acid substitutions: R212E, Y266A, L268G, and W275A. Compared with rCPBm4, rSUMO-CPBm4 was expressed with higher solubility in Escherichia coli BL21 (DE3). Neither rCPBm4 nor rSUMO-CPBm4 was lethal to mice. Although rCPBm4 and rSUMO-CPBm4 were reactogenic with polyclonal antibodies against crude CPB, rabbits vaccinated with rSUMO-CPBm4 developed significant levels of toxin-neutralizing antibody (TNA) titers that conferred protection against crude toxin challenge. These data suggest that genetically detoxified rSUMO-CPBm4 is a promising subunit vaccine candidate for C. perfringens type C beta enterotoxemia.

Cryo-EM structure of the octameric pore of Clostridium perfringens ß-toxin.

Clostridium perfringens is one of the most widely distributed and successful pathogens producing an impressive arsenal of toxins. One of the most potent toxins produced is the C. perfringens ß-toxin (CPB). This toxin is the main virulence factor of type C strains. We describe the cryo-electron microscopy (EM) structure of CPB oligomer. We show that CPB forms homo-octameric pores like the hetero-oligomeric pores of the bi-component leukocidins, with important differences in the receptor binding region and the N-terminal latch domain. Intriguingly, the octameric CPB pore complex contains a second 16-stranded ß-barrel protrusion atop of the cap domain that is formed by the N-termini of the eight protomers. We propose that CPB, together with the newly identified Epx toxins, is a member a new subclass of the hemolysin-like family. In addition, we show that the ß-barrel protrusion domain can be modified without affecting the pore-forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

Design and Development of a New Method for the Production of Nanotoxoids from Clostridium Perfringens Beta Toxin.

In recent years, a nanoparticle-based strategy has shown that non-denatured protein toxins can be used to enhance the appropriate immune response. Once the toxin reacts between the nanoparticles and the protein (toxin), it loses its toxicity because it does not attach to its ligand at the cell surface. The results of the nanoparticle and toxin complex show that the nanoparticles facilitate the internal release of the toxin. Clostridium perfringens beta toxin is produced by Clostridium perfringens type B and C, and diarrhea is the most important disease caused in newborn lambs. When beta toxin forms a complex with nanoparticles, the reaction between the toxin and the nanoparticle leads to the formation of a new form of nanoparticle in which the toxin loses its lethality due to its involvement; therefore, it becomes a toxoid. The nanoparticles used in this research are of poly lactic-co-glycolic acid (PLGA) type, one of the most developed biodegradable polymers. This study aimed to isolate and purify Clostridium perfringens beta toxin and produce its complex with PLGA nanoparticles to form a non-toxic structure. In this study, Clostridium perfringens beta toxin type B was isolated using ammonium sulfate precipitation and gel filtration chromatography. Toxin assay was performed in vivo (lethal dose [LD50]) and in vitro by sodium dodecyl sulphate-polyacrylamide gel electrophoresis at each stage, and the quantity of purified toxin was calculated to be 10 mg/ml. Afterward, the beta toxin antigen was used as the basis for the preparation of nanotoxoid candidates with nanoparticle formulation. Moreover, the PLGA polymer and water-oil-water methods were used to fabricate nanoparticles. Under optimal conditions, nanoparticles without antigen with an average size of 100 nm and zeta potential of -23.28 mV, as well as nanoparticles containing antigen with an average size of 120 nm and zeta potential of -18.2 mV, were prepared. When nanoparticles are injected into mice with the beta toxin, the toxin becomes a toxoid with no toxicity effects, and it cannot bind to its receptors and reveal its effects. In this study, the mice showed mild symptoms in one case, and none of them died. The beta and PLGA toxin model could also be applied as a candidate to study the release and immunization of the target animal. In order to achieve antigen regulation using natural polymers, it is recommended to conduct a comparative study between nanoparticles based on natural polymers.

Platelet Endothelial Cell Adhesion Molecule 1 (CD31) Is Essential for Clostridium perfringens Beta-Toxin Mediated Cytotoxicity in Human Endothelial and Monocytic Cells.

Beta toxin (CPB) is a small hemolysin beta pore-forming toxin (ß-PFT) produced by Clostridium perfringens type C. It plays a central role in the pathogenesis of necro-hemorrhagic enteritis in young animals and humans via targeting intestinal endothelial cells. We recently identified the membrane protein CD31 (PECAM-1) as the receptor for CPB on mouse endothelial cells. We now assess the role of CD31 in CPB cytotoxicity against human endothelial and monocytic cells using a CRISPR/Cas9 gene knockout and an antibody blocking approach. CD31 knockout human endothelial and monocytic cells were resistant to CPB and CPB oligomers only formed in CD31-expressing cells. CD31 knockout endothelial and monocytic cells could be selectively enriched out of a polyclonal cell population by exposing them to CPB. Moreover, antibody mediated blocking of the extracellular Ig6 domain of CD31 abolished CPB cytotoxicity and oligomer formation in endothelial and monocytic cells. In conclusion, this study confirms the role of CD31 as a receptor of CPB on human endothelial and monocytic cells. Specific interaction with the CD31 molecule can thus explain the cell type specificity of CPB observed in vitro and corresponds to in vivo observations in naturally diseased animals.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry's production of vaccines against the action of C. perfringens in equine species.

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production.

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

Clostridium perfringens-Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus).

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets (P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

Acute hemorrhagic necrotizing enteritis: a case report and review of the literature.

Acute hemorrhagic necrotizing enteritis (AHNE) is a potentially fatal infection, triggered by beta toxin produced by Clostridium perfringens type C and characterized by extensive hemorrhagic, inflammatory, or ischemic necrosis that mainly affects the small bowel, clinically presenting as diarrhea, hematochezia, abdominal pain and hypotensive shock. AHNE is rarely reported in humans nowadays, we present a case of AHNE in a 51-year-old man presenting as watery diarrhea, hematochezia and abdominal pain along with shortness of breath who unfortunately died of the disease despite active medical treatment and multiple surgical interventions. We aim to improve awareness of clinicians on this fulminant disease, associated with high mortality rates. This is the first case report that attempts to summarize the pathogenesis, clinical characteristics, diagnostic methods, treatment and prognosis of AHNE based on the current English literature. AHNE, which is exceedingly rare in clinical practice, has been associated with poorly specific clinical manifestations, high rates of misdiagnosis in its early stages and mortality rates in severe cases. In patients with a history of ingesting contaminated food and presenting with sudden progressively worsening abdominal pain, diarrhea, hematochezia, accompanied by hypotensive shock or ileus, AHNE should be highly suspected. In order to reduce the mortality of this disease, emphasis should be laid on early recognition and timely surgical intervention in AHNE. In severe cases, death cannot be avoided despite adopting active supportive treatment and timely surgical intervention.

Molecular docking analysis of Clostridium perfringens beta toxin model with potential inhibitors from the ZINC database.

Beta toxin from Clostridium perfringens after being secreted in gut is capable of causing necrotic enteritis in humans and several other animal species and does not respond to routinely used antibiotics. Therefore, there is a need to design an effective inhibitor for the Clostridium perfringens beta toxin (CPB) using cutting edge drug discovery technologies. Hence, potential CPB inhibitors were identified using computer aided screening of compounds from the ZINC database. Further, we document the molecular docking analysis of Clostridium perfringens beta toxin model (that revealed 4 binding pockets, A-D) with the identified potential inhibitors. We show that ZINC291192 [N-[(1-methylindol-3-yl) methyl eneamino]-7,10-dioxabicyclo[4.4.0]deca-2,4,11-triene-8- carboxamide] has optimal binding features with calculated binding energy of -10.38 kcal/mol and inhibition constant of 24.76 nM for further consideration.

Molecular docking analysis of P2X7 receptor with the beta toxin from Clostridium perfringens.

Clostridium perfringens beta-toxin (CPB) is linked to necrotic enteritis (over proliferation of bacteria) in several species showing cytotoxic effect on primary porcine endothelial and human precursor immune cells. P2X7 receptor on THP-1 cells is known to bind CPB. This is critical to understand the mechanism of pore formation for effective drug design. The structure of CPB and P2X7 receptor proteins were modeled using standard molecular modeling procedures (I-TASSER and Robetta server). This is followed by protein-protein docking (HADDOCK server) to study their molecular interaction. Interacting residues (19 residues from CPB and 21 residues from P2X7) were identified using the PISA server. Thus, we document the molecular docking analysis of P2X7 receptor with the beta toxin from Clostridium perfringens towards drug design and development of drugs to control necrotic enteritis.

Effectiveness of Chitosan Nanoparticles in Development of Pentavalent Clostridial Toxoid Vaccine in Terms of Clinical Pathology Elements and Immunological Responses.

Toxoid vaccines can provide protective immunity against clostridial diseases. Since the duration of the toxoid vaccine immunogenicity is short, these vaccines need to contain an adjuvant. The nanoparticles of chitosan can stimulate humoral and cell-mediated immune responses. In the present study, the effect of chitosan nanoparticles was investigated on the immunogenicity of the pentavalent clostridial toxoid vaccine containing Clostridium perfringens types D, C, and B, Clostridium septicum, as well as Clostridium novyi. Rabbits were immunized by two injections with 3-week intervals and checked clinically and through autopsy 2 weeks after the last injection. Hematological changes were investigated during immunization, including the changes of white and red blood cell counts, hemoglobin, packed cell volume, platelet, neutrophil, lymphocyte, eosinophil, basophile, monocyte, and Neut/Lymph. Biochemical factors, namely creatinine, blood urea nitrogen, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, and albumin, were also studied. The changes in immune responses during the immunization period were investigated through indirect enzyme-linked immunosorbent assay (ELISA). The results of ELISA showed that chitosan significantly enhanced immunogenicity when accompanied with in the pentavalent clostridial toxoid vaccine. During the immunogenicity period and following that, no changes were observed in clinical behavior and internal organs after autopsy. The hematological and biochemical factors were reported with no significant pathologic changes during immunization in the control and vaccinated groups (p &lt;0.05). The obtained findings revealed that the toxoid vaccines could not induce significant physiological changes in the body. The vaccine containing chitosan could stimulate humoral immunity 2-3 times higher than the nonchitosan vaccine. The humoral immune response was significantly duplicated due to the chitosan effect. Chitosan not only had no local or general side effects but also could be a good help with the enhancement of the immune system; therefore, it can be recommended as an appropriate safe adjuvant in the development of toxoid vaccines.

Production, immunogenicity, stability, and safety of a vaccine against Clostridium perfringens beta toxins.

BACKGROUND AND AIM: The beta toxin is causing the most severe Clostridium perfringens-related diseases. This work was dedicated to developing a vaccine against beta toxin using C. perfringens type C (NCTC 3180). MATERIALS AND METHODS: The crude toxoid harvest contained 710 limits of flocculation (Lf)/mL. The vaccine was formulated. Each 1 mL of the final vaccine product contained at least 50 Lf/mL of beta toxoids, 0.2 mL 3% aluminum hydroxide gel (equivalent to 5.18 mg of aluminum), <0.001% W/V thiomersal, formaldehyde <0.05% W/V, and ~0.7 mL phosphate-buffered saline (pH 7.2). The efficacy of the vaccine was evaluated by potency, stability, and safety tests. RESULTS: The vaccine demonstrated 24.36 IU/mL (standard deviation, ±0.56) and 14.74 IU/mL (±0.36) of neutralizing antibodies in rabbits and cattle, respectively. Indeed, these levels were above the minimum recommended by international protocols since the obtained antibody levels had 2.43- and 1.47-fold increase in both rabbits and cattle, respectively, over the minimum antitoxin level suggested by the United States Department of Agriculture. Interestingly, our formulation was capable of inducing 1.65-fold higher immune responses in rabbits than that stimulated in cattle (65% increase) with a significant difference (p<0.0001). The vaccine was stable up to 30 months. The vaccinated rabbits were suffered from a temporarily slight increase in temperatures in the first 10 h without any significant difference (p>0.05). CONCLUSION: The research showed a procedure for the manufacturing process of the vaccine against C. perfringens beta toxins with a feasible quantity and the vaccine described here showed to be effective in eliciting levels of neutralizing antibodies higher than required by international standards. In addition, The vaccine was stable up to 30 months. Thus, it may represent an effective and safe for preventing C. perfringens-related diseases in rabbits and cattle, although further studies to prove its efficacy in the field on other farm animals are still needed.

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Evidence That VirS Is a Receptor for the Signaling Peptide of the Clostridium perfringens Agr-like Quorum Sensing System.

Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics.IMPORTANCEC. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections.

How a Scorpion Toxin Selectively Captures a Prey Sodium Channel: The Molecular and Evolutionary Basis Uncovered.

The growing resistance of insects to chemical pesticides is reducing the effectiveness of conventional methods for pest control and thus, the development of novel insecticidal agents is imperative. Scorpion toxins specific for insect voltage-gated sodium channels (Navs) have been considered as one of the most promising insecticide alternatives due to their host specificity, rapidly evoked toxicity, biodegradability, and the lack of resistance. However, they have not been developed for uses in agriculture and public health, mainly because of a limited understanding of their molecular and evolutionary basis controlling their phylogenetic selectivity. Here, we show that the traditionally defined insect-selective scorpion toxin LqhIT2 specifically captures a prey Nav through a conserved trapping apparatus comprising a three-residue-formed cavity and a structurally adjacent leucine. The former serves as a detector to recognize and bind a highly exposed channel residue conserved in insects and spiders, two major prey items for scorpions; and the latter subsequently seizes the "moving" voltage sensor via hydrophobic interactions to reduce activation energy for channel opening, demonstrating its action in an enzyme-like manner. Based on the established toxin-channel interaction model in combination with toxicity assay, we enlarged the toxic spectrum of LqhIT2 to spiders and certain other arthropods. Furthermore, we found that genetic background-dependent cavity shapes determine the species selectivity of LqhIT2-related toxins. We expect that the discovery of the trapping apparatus will improve our understanding of the evolution and design principle of Nav-targeted toxins from a diversity of arthropod predators and accelerate their uses in pest control.

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand.

Case history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.Diagnosis: Lamb dysentery due to C. perfringens type B.Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.

The First Identification and Antibiogram of Clostridium perfringens Type C Isolated from Soil and The Feces of Dead Foals in South Korea.

Clostridium (C.) perfringens was isolated from 25 (11.1%) of 225 sampled horses and from 16 (35.56%) of 45 farms. All of the samples were negative for cpe, etx, itx, NetF genes and cpa gene were detected in 100% (25 of 25) of the samples that were positive for C. perfringens. cpb and cpb2 were detected in 40.0% (10 of 25) and 60.0% (15 of 25) of the samples that were positive for C. perfringens, respectively. Of the 25 C. perfringens isolates, 15 (60%) were type A and 10 (40%) were type C. Type C was observed on all the farms where the foals' deaths occurred. None of the isolates were positive for type B, type D, or type E. The MIC Evaluator strips antimicrobial susceptibility test showed meropenem (96%), ampicillin (92%), amoxicillin/clavulanic acid (84%), and tetracycline (8%) sensitivity.