A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease.

[Toxins of Clostridium perfringens as a natural and bioterroristic threats]. / Toksyny Clostridium perfringens jako zagrozenie naturalne i bioterrorystyczne.

Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens.

Lipoproteins from Clostridium perfringens and their protective efficacy in mouse model.

Clostridium perfringens is an obligately anaerobic rod-shaped bacterium and etiological agent for several diseases in humans and animals. The pathogen has been listed as Validated Biological Agent and warrants development of medical countermeasures. The homologs of some of the lipoproteins identified from various fractions of C. perfringens in our previous studies were observed to be virulence determinants in other pathogenic bacteria. Three putative virulence associated lipoproteins; polysaccharide deacetylase family protein, probable ion-uptake ABC transporter, and a putative lipoprotein of no known function are reported here with respect to their immuno-protective potentials. The three proteins were over expressed and purified to near homogeneity. The lipoproteins were shown to be exposed on the C. perfringens surface and, hence, accessible to antibodies and potentially visible to the host immune system. Immunization of mice with purified recombinant proteins elicited protective immunity against challenge with C. perfringens in mouse gas gangrene model. Distribution and relationship of orthologous proteins across other bacterial select agents especially among the members of Firmicutes, was carried out to look for conserved antigenic determinants.

Vaccines against Clostridium perfringens alpha-toxin.

Clostridium perfringens alpha-toxin is thought to be an important agent in gas gangrene, which is a lifethreatening infection with fever, pain, edema, myonecrosis, and gas production. The toxin (370 residues) is composed of an N-terminal domain (1-250 residues, N-domain) in which the catalytic site is found and a C-terminal domain (251-370 residues, C-domain) responsible for binding to membranes. During the past decade, recombinant DNA technology has been employed to develop second-generation vaccines, including site-directed mutants and the C-domain of the toxin, to prevent gas gangrene. These immunities have led to protection against the lethal effects of wild-type C. perfringens in mice. C-domain vaccines are capable of protecting against heterologous clostridia causing clostridial myonecrosis. This article summarizes the current knowledge on vaccines against alpha-toxin.

Clostridium perfringens vaccines.

Both Clostridium perfringens spores and toxins have reportedly been considered as a biological warfare agents. The spores may be incorporated into weapons which cause traumatic injury, and the resulting delivery of spores deep into tissues would result in the development of gas gangrene. Of the C. perfringens toxins, the epsilon-toxin is of particular concern and now appears on the list of CDC select agents. Currently there are no licensed vaccines suitable for use in humans which protect against either gas gangrene or epsilon-toxin. However, vaccines being developed for use in animals have the potential to be developed for use in humans.

Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis.

Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa(247-370) polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa(247-370) on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa(247-370)-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.

The management of ballistic trauma: an infection control perspective.

This article discusses the relationship between ballistic trauma, clostridial contamination and potential wound infection and considers the implications for care by nurses and infection control teams. An overview of ballistics is presented followed by an examination of the pathophysiology of wounding and its effects. The philosophy of war surgery is balanced against civilian concepts and the differing management strategies that prevail. It explores the causes of, and relationship between, wound contamination and the seriousness of wound infection. Nurses, inexperienced in dealing and caring for these types of patients, should be aware and understand the beliefs, arguments and controversies that surround ballistic trauma management. By so doing, nurses and infection control teams will be able to provide an enhanced level of holistic nursing care.

Immunization with the C-Domain of alpha -Toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens.

Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene.

[Detection of exotoxins of the pathogens of anaerobic gas infection using enzyme immunoassay]. / Vyiavlenie ékzotoksinov osnovnykh vozbuditelei anaérobnoi gazovoi infektsii s pomoshch'iu immunofermentnogo analiza.

To detect the exotoxins of the causative agents of the main anaerobic gas infection (Clostridium perfringens, C.oedematiens, C.histolyticum, C.septicum) a rapid and easily reproducible variant of the enzyme-linked immunosorbent assay (ELISA), based on the use of the sandwich modification activated due to avidin-biotin interaction, was proposed. The possibility of using the avidin-biotin variant of ELISA for the detection of C.perfringens, C.oedematiens, C.histolyticum and C.septicum toxins in experimental gas gangrene in guinea pigs was shown. The method made it possible to reproducibly detect 0.02-0.2 ng of antigens (gangrene toxins-toxoids) with the immunoassay being highly specific.

Environment, incidence, aetiology, epizootiology and immunoprophylaxis of soil-borne diseases in north-east Mexico.

Within the framework of an extensive research programme, the socio-economic and environmental conditions which influence the emergence of soil-borne diseases in north-eastern Mexico were analysed. Furthermore, specimens collected from carcasses in the field were bacteriologically examined and the causal organisms of soil-borne diseases differentiated by means of gas chromatographic analysis of their metabolic products and the long-chained fatty acids contained in the cell. With experimental clostridial vaccines prepared with the Goettingen Bioreactor Technique, trials to protect cattle and guinea-pigs against gas gangrene were carried out. It was found that the farm structure and the dry climate as well as the specific soil conditions and plant cover favour the emergence of soil-borne diseases. Causal organisms B. anthracis, C. perfringens, C. sordellii, C. haemolyticum, C. chauvoei/septicum, C. novyi A, C. botulinum and site-specific field strains of clostridia were detected. Experimental site-specific vaccines proved to be highly efficient in protecting cattle and guinea pigs.

[The experimental validation of methods for the emergency immunoprophylaxis of gas gangrene]. / Eksperimental'noe obosnovanie metodov ékstrennoi immunoprofilaktiki gazovoi gangreny.

The effectiveness, both immunological (by an increase in the titers of antitoxins) and protective (by resistance to the inoculation of the absolute lethal dose of infective agents), of the regional (wound) revaccination with tetratoxoid (Clostridium perfringens, C. oedematiens, C. septicum, C. histolyticum) was demonstrated on the experimental model of wound infection (gas gangrene) of guinea pigs. The schedule of rapid immunization with tetratoxoid was developed, which made it possible to create good immunological preparedness (basic immunity) for subsequent revaccination in case of traumas within 6 days. The effectiveness of rapid immunization by the application of tetratoxoid on the wound was shown. This immunization ensured a considered increase in the titers of antitoxins within the first 6 days, which increased the protection of the animals from infection with each of the four causative agents of gas gangrene.

A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens protects mice against experimental gas gangrene.

Fragments of the alpha-toxin of Clostridium perfringens have been produced using genetic manipulation techniques. Antibody which cross-reacted with the alpha-toxin was induced after immunization with fragments representing the N- (Cpa1-249) and C-terminal (Cpa247-370) domains of the toxin. Smaller fragments of the alpha-toxin did not induce cross-reacting antibody. Anti-Cpa1-249 serum neutralized phospholipase C activity but not haemolytic activity of the toxin. Anti-Cpa247-370 serum neutralized both the phospholipase C and haemolytic activities. Only immunization with Cpa247-370 induced protection against the lethal effects of the toxin. Immunization with Cpa247-370 also provided protection in a mouse model against at least 10 LD100 doses of C. perfringens type A. This result confirms the essential role of this toxin in the pathogenesis of gas gangrene.

Recognition, management, and prevention of Clostridium septicum abscess in immunosuppressed patients.

Spontaneous gas gangrene due to Clostridium septicum is a rapidly progressing disease that usually ends in fatal toxemia. We report three cases of asymptomatic C septicum abscesses to document the clinical course of this entity and to establish guidelines for its prevention and treatment. In contrast to previously reported data, C septicum infections can produce abscesses in solid organs, the retroperitoneum, and the extremities. These lesions often occur in patients with cancer, producing liver abscesses without gas formation that may be misinterpreted as metastatic carcinoma. Symptoms may be minimal or nonspecific before fulminant toxemia. Asymptomatic bacteremia should prompt a search for unsuspected cancer and an abscess. Computed tomography is the diagnostic modality of choice. The treatment consists of surgical débridement of necrotic tissue in concert with an appropriate course of antibiotics. We have found recurrences after adequate débridement and short-term antibiotic therapy, suggesting that prolonged and even lifelong prophylactic oral penicillin G potassium may be necessary to prevent further recurrences.

Clostridial myonecrosis.

The best treatment for gas gangrene is prevention by following the principles of thorough debridement, immediate effective parenteral antibiotic therapy, and delayed closure of open fractures.

[Fatal appendectomy caused by postoperative gas gangrene of the abdominal wall. 2 cases]. / Appendicectomie mortelle par gangrène gazeuse pariétale post-opératoire. 2 cas.

The authors report two cases of post-appendicectomy gas gangrene with a fatal outcome. Based on the study of these two cases they analyse the incidence, mechanism and prognosis of this serious post-operative complication. They stress the prophylactic measures necessary for prevention.

[Gas gangrene after aseptic orthopedic surgery]. / Gangrène gazeuse après chirurgie orthopédique aseptique.

Gas gangrene following scheduled orthopedic surgery is not uncommon. In order to assess its frequency and prognosis, identify possible predisposing factors and suggest preventive measures, we reviewed the records of 22 patients (14 males and 8 females, mean age 40 +/- 20 years) admitted between 1969 and 1987 who developed gas gangrene in the wake of orthopedic surgery. In all cases the lower limbs were the site of operation: the knee in 9, the hip in 4, the femur in 4 and the leg in 5 cases. Surgical procedures included on-site foreign material in 19 cases, pneumatic tourniquet in 6 and prolonged vascular stretch in 9. Infection was diagnosed within 1.4 +/- 1.1 days of surgery; local signs, especially crepitants and pain, were prominent for the diagnosis. Pathologic findings consisted of myonecrosis in 18 patients and cellulitis in 4. Local bacteriological studies, carried out in 19 patients, yielded organisms in 14, including 12 with Clostridia perfringens. Four patients (one despite surgical treatment) died within 24 hours of admission. The remainder were treated with a combination of surgery, antimicrobial therapy (18) and hyperbaric oxygen (17). Subsequently, 13 patients had severe functional disability, while 5 recovered without sequelae. In view of the poor prognosis of gas gangrene, several preventive measures are suggested during aseptic surgery of the lower limbs. Careful skin preparation, cleaning of the anal region and short-term prophylactic antibiotic therapy with cefamandole or amoxycillin-clavulanic acid, are among them.