Clostridium Perfringens Alpha-toxin Research Articles

Preparation and Application of Clostridium perfringens Alpha Toxin Nanobodies.

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.

Clostridial Myonecrosis: A Comprehensive Review of Toxin Pathophysiology and Management Strategies.

Clostridial myonecrosis, commonly known as gas gangrene (GG), is a rapidly progressing and potentially fatal bacterial infection that primarily affects muscle and soft tissue. In the United States, the incidence of GG is roughly 1000 cases per year, while, in developing countries, the incidence is higher. This condition is most often caused by Clostridium perfringens, a Gram-positive, spore-forming anaerobic bacterium widely distributed in the environment, although other Clostridium species have also been reported to cause GG. The CP genome contains over 200 transport-related genes, including ABC transporters, which facilitate the uptake of sugars, amino acids, nucleotides, and ions from the host environment. There are two main subtypes of GG: traumatic GG, resulting from injuries that introduce Clostridium spores into deep tissue, where anaerobic conditions allow for bacterial growth and toxin production, and spontaneous GG, which is rarer and often occurs in immunocompromised patients. Clostridium species produce various toxins (e.g., alpha, theta, beta) that induce specific downstream signaling changes in cellular pathways, causing apoptosis or severe, fatal immunological conditions. For example, the Clostridium perfringens alpha toxin (CPA) targets the host cell's plasma membrane, hydrolyzing sphingomyelin and phosphatidylcholine, which triggers necrosis and apoptosis. The clinical manifestations of clostridial myonecrosis vary. Some patients experience the sudden onset of severe pain, swelling, and muscle tenderness, with the infection progressing rapidly to widespread tissue necrosis, systemic toxicity, and, if untreated, death. Other patients present with discharge, pain, and features of cellulitis. The diagnosis of GG primarily involves clinical evaluation, imaging studies such as X-rays, computer tomography (CT) scans, and culture. The treatment of GG involves surgical exploration, broad-spectrum antibiotics, antitoxin, and hyperbaric oxygen therapy, which is considered an adjunctive treatment to inhibit anaerobic bacterial growth and enhance the antibiotic efficacy. Early recognition and prompt, comprehensive treatment are critical to improving the outcomes for patients affected by this severe and life-threatening condition.

Purinergic Receptor Antagonists Inhibit Hemolysis Induced by Clostridium perfringens Alpha Toxin.

Clostridium perfringens alpha toxin (CPA), which causes yellow lamb disease in sheep and gas gangrene and food poisoning in humans, is produced by all types of C. perfringens and is the major virulence determinant of C. perfringens type A. CPA induces hemolysis in many species, including humans, murines, sheep and rabbits, through its enzymatic activity, which dissolves the cell membrane. Recent studies have shown that some pore-forming toxins cause hemolysis, which is achieved by the activation of purinergic receptors (P2). However, the relationship between P2 receptors and non-pore-forming toxin hemolysis has not been investigated. In the present study, we examined the function of P2 receptors in CPA toxin hemolysis and found that CPA-induced hemolysis was dependent on P2 receptor activation, and this was also true for Staphylococcus aureus β-Hemolysin, another non-pore-forming toxin. Furthermore, we use selective P2 receptor antagonists to demonstrate that P2X1 and P2X7 play important roles in the hemolysis of human and murine erythrocytes. In addition, we found that redox metabolism was mainly involved in CPA-induced hemolysis using metabolomic analysis. We further demonstrate that CPA activates P2 receptors and then activates NADPH oxidase through the PI3K/Akt and MEK1/ERK1 pathways, followed by the production of active oxygen to induce hemolysis. These findings contribute to our understanding of the pathological effects of CPA, clarify the relationship between P2 activation and non-pore-forming toxin-induced hemolysis, and provide new insights into CPA-induced hemolysis.

Development of Rapid Detection Kit for Necrotic Enteritis Disease in Poultry using Protein A Agglutination

Necrotic enteritis causes significant losses in the global poultry industry, necessitating accurate diagnosis for effective intervention. This study aimed to develop a diagnostic tool for detecting necrotic enteritis in poultry based on the presence of Clostridium perfringens (C. perfringens) Alpha-toxin in poultry feces. The reagent of the detection kit was developed by conjugation of IgG against C. perfringens toxin and Staphylococcus cells containing protein A. The IgG antibody was derived from an 8-month-old thin-tailed male sheep immunized with purified 2 ml of C. perfringens Alpha-toxin. Sensitivity assays were carried out to determine the detection limit, while Escherichia coli (E. coli) and Salmonella enteritidis (S. enteritidis) were used to identify specificity. A purified Alpha-toxin with a protein concentration of 2.8 mg/ml and a specific molecular weight of 43 kDa was successfully obtained. A strong reaction of the hyperimmune antibody (IgG) was also detected in the thin-tailed male sheep serum. The developed rapid detection kit in this study indicated C. perfringens Alpha-toxin with a lower concentration (12 ng/ml). Agglutination reactions could differentiate positive control from negative without significant cross-reactivity towards other bacteria (S. enteritidis and E. coli). Keywords: Agglutination, Clostridium perfringens, Detection, Necrotic Enteritis, Toxin

Co-administration of the prebiotic 1-kestose and the paraprobiotic Lactiplantibacillus plantarum FM8 in magellanic penguins promotes the activity of intestinal Lactobacillaceae and reduces the plc gene levels encoding Clostridium perfringens toxin.

Despite the well-known potential health benefits of prebiotics and non-viable probiotics (paraprobiotics) in various animal species, research regarding their use in penguins is scarce. Our study aimed to investigate the impact of a combined administration of prebiotics and paraprobiotics (referred to here as "parasynbiotics") on the gut microbiome and overall health of Magellanic penguins (Spheniscus magellanicus). The parasynbiotics consisted of 1-kestose, which is a fructooligosaccharide comprising sucrose and fructose, and heat-killed Lactiplantibacillus plantarum FM8, isolated from pickled vegetables. It was administered to eight penguins aged <3 years (Young-group) and nine penguins aged >17 years (Adult-group) for 8 weeks. Results from 16S rRNA sequencing revealed that compared to baseline, parasynbiotic administration significantly decreased the relative abundance of intestinal Clostridiaceae_222000 in both groups and significantly increased that of Lactobacillaceae in the Young-group. Quantitative real-time polymerase chain reaction revealed a significant decrease in the plc gene levels encoding alpha-toxin of Clostridium perfringens in the Young-group after parasynbiotic administration (P=0.0078). In the Young-group, parasynbiotic administration significantly increased the plasma levels of total alpha-globulin (P=0.0234), which is associated with inflammatory responses. Furthermore, exposure of dendritic cells to heat-killed L. plantarum FM8 promoted the secretion of interleukin 10, a major anti-inflammatory cytokine. Overall, parasynbiotic administration enhanced the activity of gut Lactobacillaceae, decreased the levels of C. perfringens and its toxin encoding plc gene, and reduced inflammatory response in penguins. These results provide novel insights into the potential benefits of parasynbiotics for improving penguin health.

Evaluation of Three Vaccination Schemes Against Clostridium perfringens Alpha Toxin and Their Effects on the Performance, Level of Intestinal Lesions, and Serum Antibody Titers in Broilers.

The objective of the trial was to evaluate three vaccination schemes against Clostridium perfringens (CP) alpha-toxoid through drinking water to determine if they can protect against clinical signs of necrotic enteritis and coccidiosis in broiler chickens. Three hundred 1-day-old Cobb 500 male chicks were used in 4 treatments with 10 repetitions. Each group received 1 of the following treatments over the course of 29 days: T1, no vaccination; T2, vaccination on Day 1; T3, vaccination on Day 7; and T4, vaccination on Days 7 and 17. The birds were vaccinated with inactivated CP toxoid type A, administered via drinking water. During the first 14 days, a high-protein diet (27%) consisting of corn, soy, and fish meal was fed. On Day 14 Eimeria acervulina (EA), Eimeria maxima (EMx), Eimeria tenella (ET), Eimeria necatrix, and Eimeria brunetti were used in a coccidial challenge. The field isolate CP type A was then inoculated on Days 18, 19, and 20. Ten birds were slaughtered by treatment to obtain serology samples for antibody titers and intestine samples for CP and Eimeria lesion score and gut integrity indicators. Productive performance was assessed using complete randomized design and compared statistically using the Tukey test, whereas intestinal integrity variables and antibodies against CP alpha toxin were assessed using a Kruskal-Wallis nonparametric method. The results revealed that the treatments had an effect on productive performance (P < 0.05); T3 had better body weight and weight gain than T1. In terms of lesion score at Day 21, T4 had a lower lesion score by EA, EMx, and ET than T1. Cell desquamation in T2 was lower than in T4, and excess mucus (EM) in T1 was the worst in gut integrity indicators at Day 21. On the other hand, T2 had more EM than T3 and T4 at Day 25. In the measurement of antibodies, no statistical differences (P > 0.05) were found. These findings indicate that vaccination on Day 7 (T3) outperformed double vaccination on Days 7 and 17 (T4) and single on Day 1 (T2), in terms of productive performance, gut integrity, and lesion scores; and on the last day of the experiment T3 had the best performance in immunology response.

The in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.

Immunogenicity of a newly developed vaccine against Clostridium perfringens alpha-toxin in rabbits and cattle.

Background and Aim:Clostridium perfringens type A is an anaerobic bacterium that produces four major toxins (alpha, beta, epsilon, and iota) that cause various diseases. Most of the important C. perfringens-associated diseases of farm animals are caused by alpha-toxin. This study aimed to produce a vaccine against alpha-toxin using C. perfringens type A (ATCC 13124) and investigate its potency, stability, and safety.Materials and Methods:The vaccine was formulated of its constituents for 1 h. Each milliliter of the final vaccine product contained alpha toxoid 15 lecithovitellinase activity (Lv) by adding (0.375 mL containing 40 Lv) and approximately 0.2 mL from 3% concentrated aluminum hydroxide gel, <0.001% W/V thiomersal, <0.05% W/V formaldehyde, and nearly 0.425 mL phosphate-buffered saline (pH 7.2). The vaccine efficacy was evaluated in rabbits and cattle by performing potency, stability, and safety tests.Results:The vaccine produced approximately 8.8 and 4.9 IU/mL neutralizing antibodies in rabbits and cattle, respectively. These concentrations were higher than the lowest concentration recommended by various international protocols and the United States Department of Agriculture by 2.20-fold in rabbits and 1.23-fold in cattle. Interestingly, the formulated vaccine enhanced immune responses by 1.80-fold in rabbits compared with that in cattle; the difference was statistically significant (p < 0.0001). The vaccine was stable for 30 months. In vaccinated rabbits, the body temperature slightly increased temporarily during the first 10 h of vaccination; however, the temperature difference was not statistically significant (p > 0.05).Conclusion:This study describes a manufacturing process to obtain sufficient amounts of a vaccine against C. perfringens alpha-toxin. The formulated vaccine effectively elicited a higher level of neutralizing antibody response than the international standards. Furthermore, the vaccine was found to be stable, safe, and effective in preventing C. perfringens-related diseases in rabbits and cattle. Further studies are necessary to evaluate the efficacy of this vaccine in other farm animals.

Novel Lethal Clostridial Infection in Florida Manatees (Trichechus manatus latirostris): Cause of the 2013 Unusual Mortality Event in the Indian River Lagoon

The Indian River Lagoon (IRL) on Florida’s east coast is a biologically diverse estuary and an important habitat to the threatened Florida manatee (Trichechus manatus latirostris). An unusual mortality event (UME) was declared by the Working Group on Marine Mammal Unusual Mortality Events in 2013 after a marked increase in manatee deaths in the IRL of an unknown cause. This UME followed a dramatic reduction of seagrass coverage in the IRL due to chronic non-toxic phytoplankton blooms, with a resultant ecosystem shift to mixed macroalgal dominance. At least 199 manatee deaths fitting the UME case definition were documented in and adjacent to the IRL during 2012–2019; mortality was highest in 2013, when 111 of these deaths were documented. The case definition included carcasses in good nutritional condition, with multiorgan congestion or wet lungs consistent with drowning without trauma. The gastrointestinal compartments of manatee carcasses were filled with diverse macroalga species, and the contents were notably more fluid than usual. Gross intestinal findings included blebbing to segmental thickening of the wall. Microscopic lesions were primarily intestinal, including necrosis, edema, hemorrhage, mucosa-associated lymphoid changes, and inflammation, sometimes associated with Gram-positive bacterial rods. A multidisciplinary approach of environmental and carcass sampling found no causative evidence through tests for micro- and macroalgal biotoxins, trace metals, general toxin screening, or vitreum biochemistry. Microbiological, cytological, immunohistochemical, and molecular analyses of Clostridiales from intestinal samples identified Clostridioides difficile toxin A, toxins A/B and toxin A gene; Paeniclostridium sordellii lethal gene (and other potential virulence factors from a sequenced strain); and Clostridium perfringens alpha and epsilon toxin genes. The results from this 8 year-long investigation are indicative that the cause of death in this manatee UME was associated with clostridial infection, initiated by a shift to a predominantly macroalgal diet.

ENGINEERING OF STRAIN PRODUCING C-TERMINAL FRAGMENT OF CLOSTRIDIUM PERFRINGENS ALPHA-TOXIN

An Esсherichia coli 42cpa-c strain synthesizing C-terminal fragment of Clostridium perfringens alpha-toxin was engineered. The strain is distinguished by productive capacity 1.2 mg of target protein per 1 L of cultural liquid. The protein contains octahistidine peptide at C-end of the molecule, enabling to carry out its one-stage purification by metal-affine chromatography on Ni2+-NTA resin. The resulting CPA-C protein preparation is potentially suitable for formulation of the derived vaccine.

Prevalence of Clostridium perfringens alpha toxin and enterotoxin in the faeces of dogs with acute haemorrhagic diarrhoea syndrome.

To investigate the prevalence of Clostridium perfringens alpha toxin encoding gene and C.perfringens enterotoxin encoding gene in dogs with acute haemorrhagic diarrhoea syndrome. Retrospective study looking at the prevalence of C. perfringens alpha toxin and C. perfringens enterotoxin in the faeces of three groups of dogs - those with acute haemorrhagic diarrhoea syndrome (n=16), those with haemorrhagic diarrhoea from another cause (n=17) and those without haemorrhagic diarrhoea (n=10). Correlation between the presence of C. perfringens alpha toxin and/or C. perfringens enterotoxin and Acute Patient Physiological and Laboratory Evaluationfast scores, acute haemorrhagic diarrhoea index scores and length of hospitalisation in dogs with acute haemorrhagic diarrhoea syndrome was assessed. Prevalence of C. perfringens alpha toxin was not higher in dogs with acute haemorrhagic diarrhoea syndrome (43.75%) than dogs with haemorrhagic diarrhoea from another cause (58.82%) (difference in prevalence 15.07%; 95% CI -37% to 32%) or in dogs without haemorrhagic diarrhoea (60%) (difference in prevalence 16.25%; 95% CI -4% to 36%). Dogs with acute haemorrhagic diarrhoea syndrome did not have a significantly higher prevalence of C. perfringens enterotoxin (18.75%) compared to dogs without haemorrhagic diarrhoea (11.76%) (difference in prevalence 6.99%; 95% CI -18% to 32%). Prevalence of C. perfringens enterotoxin was similar in dogs with acute haemorrhagic diarrhoea syndrome and dogs without haemorrhagic diarrhoea (20%) (difference in prevalence 1.25% 95% CI -33% to 30%). The presence of C. perfringens alpha toxin did not correlate with increased Acute Patient Physiological and Laboratory Evaluationfast scores, acute haemorrhagic diarrhoea index scores or length of hospitalisation in dogs with acute haemorrhagic diarrhoea syndrome. This study does not demonstrate increased prevalence of C. perfringens alpha toxin or C. perfringens enterotoxin in dogs with acute haemorrhagic diarrhoea syndrome compared to dogs with haemorrhagic diarrhoea from another cause or dogs without haemorrhagic diarrhoea.

Visual Detection of Clostridium perfringens Alpha Toxin by Combining Nanometer Microspheres with Smart Phones.

Clostridium perfringens α toxin (CPA) is an important virulence factor that causes livestock hemorrhagic enteritis and food poisoning by contaminated meat products. In this study, the nano-silica microspheres combined with smartphone image processing technology was developed to realize real-time CPA detection. First, the N-terminal and C-terminal domain of the CPA toxin (CPAC3 and CPAN) and their anti-sera were prepared. The silica microspheres coupled with the antibody of CPAC3 was prepared to capture the toxin that existed in the detection sample and the fluorescent-labeled antibody of CPAN was incubated. Moreover, the fluorescent pictures of gray value were performed in a cell phone app, corresponding to toxin concentration. The new assay takes 90 min to perform and can detect CPA as little as 32.8 ng/mL. Our results showed a sensitive, stable, and convenient CPA detection system, which provides a novel detection method of native CPA in foods.

Faecal PCR panel results and clinical findings in Western Australian dogs with diarrhoea.

To describe faecal PCR (fPCR) results and clinical findings of dogs seen at a university teaching hospital for diarrhoea. Retrospective case series (April 2015 to July 2018). Data were collected from the hospital electronic medical records. Data extracted included signalment, history, clinical signs, treatment, fPCR panel results, other faecal diagnostic test results and antimicrobial use. One hundred and sixty-eight dogs with diarrhoea had a fPCR panel submitted. Most dogs (115, 68.5%) had diarrhoea of 3 days or less duration. Clostridium perfringens alpha toxin gene was most frequently detected (156, 92.9%) by fPCR, followed by Campylobacter spp. (55, 32.7%), canine parvovirus (CPV) (29, 17.3%), Salmonella spp. (14, 8.3%) and Giardia spp. (9, 5.4%). For the 45 dogs that had a negative point-of-care CPV test, 13 were CPV fPCR positive; some of which were adult dogs with current vaccination status. A total of 94/168 (56%) dogs received antimicrobials at some time during the treatment of diarrhoea. Faecal PCR panels can identify dogs with enteric organisms in their faeces that traditional faecal diagnostics may miss, thus contributing additional information to the diagnostic process. Nonetheless, fPCR results should be interpreted in light of the clinical findings, and particular consideration given to avoiding inappropriate use of antimicrobials. This study highlights that testing for C. perfringens alpha toxin gene is not likely to be diagnostically helpful, and that adult dogs with diarrhoea might be identified as CPV positive with PCR testing, despite a negative point-of-care CPV test result and a current vaccination status.

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Molecular structure and function of the carboxy-terminus of the alpha-toxin from Clostridium perfringens type A.

In order to interpret the molecular structure and biological characteristics of Clostridium perfringens alpha-toxin (CPA), the CPA251-370 gene was cloned and the 120 amino acid carboxy terminal of CPA (CPA251-370) was obtained. The secondary and three-dimensional (3D) structures of CPA251-370 were predicted. The secondary structure of CPA251-370 consisted primarily of 35.48% β-sheets and 44.35% random coils. Compared with the CPA toxin consisting of 10 α-helices and eight β-sheets, the 3D structure of CPA251-370 only contained eight β-sheets. The circular dichroism (CD) spectrum detection showed that the CD spectrum of CPA251-370 changed slightly compared with the CD spectrum of CPA. Biological activity assays showed that CPA251-370 had lost the phospholipase C (PLC) activity and haemolytic activity of CPA. More importantly, the mice immunized with CPA251-370 were protected against a challenge with 1 MLD C.perfringens type A strain C57-1. This study laid a solid foundation for explaining the relationship between molecular structure and biological characteristics of CPA in the future. Our research also provides CPA251-370 as a candidate strains for genetic engineering subunit vaccines of C.perfringens type A.

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

Inactivated recombinant Escherichia coli as a candidate vaccine against Clostridium perfringens alpha toxin in sheep

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric (“yellow lamb disease”) disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 μg of rCPA used for inoculation, induced 13.82 IU.mL−1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL−1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL−1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

Detection of alpha toxin and enterotoxins of Clostridium perfringens isolated from minced meat by real time polymerase chain reaction (PCR)

Clostridium perfringens is one of the most widespread pathogen producing toxins related to variable pathogenic conditions, particularly food poisoning in humans. Thus, this study described the prevalence, enumeration, toxigenic types and antibiotic susceptibility of C. perfringens strains isolated from minced meat in Egypt as well as the validation of a real-time polymerase chain reaction (PCR) test for the identification of C. perfringens toxin genes. A high prevalence of C. perfringens (98/105, 93.3%) was detected in minced meat samples. The total count of viable C. perfringens in 23 samples was 2.0 × 102 to 4.5 × 102 with a mean value 3.7 × 102 ± 1.07 × 102 CFU/g. The toxin typing of C. perfringens based on lecithinase activity and dermonecrotic reactions in albino guinea pig exhibited 33 (33.7%) as toxigenic strains of C. perfringens type A and 65 (66.3%) as non-toxigenic strains. Antibiotic susceptibility testing of isolates against 15 different antimicrobial agents indicated that C. perfringens was extremely sensitive to penicillin, followed by erythromycin, tetracycline, doxycycline and amoxicillin. All the other drugs were relatively less effective against the isolates. The real time PCR (RT-PCR) was performed for screening of alpha (cpa), beta, epsilon, iota toxins and enterotoxin (cpe) genes in toxigenic isolates of C. perfringens type A. All toxigenic strains of C. perfringens type A (33, 33.7%) were positive for alpha toxin (cpa) and enterotoxin (cpe) genes, while none of these isolates carried beta, epsilon and iota toxin genes. To the best of the authors’ knowledge, this is one of the studies that used RT-PCR for the determination of toxigenic strains of C. perfringens in Egypt. It is suggested that RT-PCR could be used instead of the conventional culture procedures for identification of C. perfringens in minced meat in Egypt. Key words: Clostridium perfringens, minced meat, alpha toxin (cpa) gene, enterotoxin (cpe) gene, real time-polymerase chain reaction (RT-PCR).

Expression of the alpha toxin of Clostridium perfringens in Lactobacillus casei genome and evaluation of its immune effects in mice

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine.

Immunization with a nontoxic naturally occurring Clostridium perfringens alpha toxin induces neutralizing antibodies in rabbits

Clostridium perfringens alpha toxin, encoded by plc gene, has been implicated in gas gangrene, a life threatening infection. Vaccination is considered one of the best solutions against Clostridium infections. Although studies have identified many low quality clostridial vaccines, the use of recombinant proteins has been considered a promising alternative. Previously, a naturally occurring alpha toxin isoform (αAV1b) was identified with a mutation at residue 11 (His/Tyr), which can affect its enzymatic activity. The aim of the present study was to evaluate whether the mutation in the αAV1b isoform could result in an inactive toxin and was able to induce protection against the native alpha toxin. We used recombinant protein techniques to determine whether this mutation in αAV1b could result in an inactive toxin compared to the active isoform, αZ23. Rabbits were immunized with the recombinant toxins (αAV1b and αZ23) and with native alpha toxin. αAV1b showed no enzymatic and hemolytic activities. ELISA titration assays showed a high titer of both anti-recombinant toxin (anti-rec-αAV1b and anti-rec-αZ23) antibodies against the native alpha toxin. The alpha antitoxin titer detected in the rabbits' serum pool was 24.0 IU/mL for both recombinant toxins. These results demonstrate that the inactive naturally mutated αAV1b is able to induce an immune response, and suggest it can be considered as a target for the development of a commercial vaccine against C. perfringens alpha toxin.