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INTRODUCTION: Producing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. OBJECTIVE: Considering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. METHODS: Subcutaneously, 18 Texel sheep received two doses (200 µg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). RESULTS: Both formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. CONCLUSION: While adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.
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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)
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Animales , Toxoides , Enterocolitis Seudomembranosa/veterinaria , Vacunas Sintéticas/uso terapéutico , Clostridium perfringens/inmunología , Gangrena Gaseosa/veterinaria , Caballos , Inmunización/veterinariaRESUMEN
OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.
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Escherichia coli/genética , Formaldehído/farmacología , Resistencia a la Kanamicina/efectos de los fármacos , Plásmidos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Vacunas contra Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/genética , Transferencia de Gen Horizontal/efectos de los fármacos , Plásmidos/genética , Vacunas de Productos Inactivados , Vacunas SintéticasRESUMEN
Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) comprises some opportunistic pathogens associated with infectious outbreaks in hospital settings. A. baumannii is the most relevant species owing to its capacity to develop resistance to the different classes of antimicrobials. The aim of this study was to identify the species, establish the genetic patterns, resistance and biofilm profiles in ACB isolates associated with nosocomial infection in a hospital of Pelotas, Rio Grande do Sul, Brazil. Twenty-two clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA51-like genes, and the genetic relationship was determined through pulsed-field gel electrophoresis (PFGE). Their antibiotic resistance profiles and carbapenemases synthesis were evaluated following CLSI guidelines. PCR was carried out to evaluate the presence of carbapenemases genes and the isolates were classified for their biofilm-forming ability. All isolates obtained in the study were identified as A. baumannii and 72.7% of the isolates were classified as strong biofilm formers. In the class carbapenems, 95.4% and 77.3% of the isolates were resistant to meropenem and imipenem, respectively. The blaVIM gene was identified in 90.9% of isolates and carbapenemases synthesis were confirmed in 95.4% of the isolates. Fourteen genetic patterns were confirmed through PFGE analyses. The isolates collected within a time gap of 2 years demonstrated a genetic relationship, and the same clone was identified in different departments in the hospital. To the best of our knowledge, this is the first report of identification and characterization of A. baumannii nosocomial isolates in Pelotas, RS, Brazil.
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Infecciones por Acinetobacter , Acinetobacter baumannii , Infección Hospitalaria , Infecciones por Acinetobacter/epidemiología , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Brasil , Carbapenémicos/farmacología , Infección Hospitalaria/epidemiología , Electroforesis en Gel de Campo Pulsado , Humanos , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
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⯵g 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.
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Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Proteínas de Unión al Calcio/inmunología , Infecciones por Clostridium/veterinaria , Portadores de Fármacos , Escherichia coli/genética , Fosfolipasas de Tipo C/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antitoxinas/sangre , Toxinas Bacterianas/genética , Vacunas Bacterianas/administración & dosificación , Proteínas de Unión al Calcio/genética , Infecciones por Clostridium/prevención & control , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ovinos , Fosfolipasas de Tipo C/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaAsunto(s)
Antitoxinas/sangre , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/prevención & control , Infecciones por Clostridium/veterinaria , Enfermedades de las Ovejas/prevención & control , Toxemia/veterinaria , Animales , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Bovinos , Infecciones por Clostridium/prevención & control , Femenino , Conejos , Ovinos , Toxemia/prevención & control , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A-E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals.