RESUMO
Nano-emulsions are promising carriers for antigen delivery. Here, we evaluated the efficacy of a water-oil nano-emulsion containing concentrated, inactivated Clostridium novyi (C. novyi) type B supernatant culture (nano-iCnB) in protecting Swiss mice against a lethal dose of alpha toxin concentrated extract. Proteins were confirmed in the nano-iCnB and their stabilities were determined according physical parameters such as Zeta Potential (ZP). Biochemical, hematological parameters and morphological appearance of liver, spleen and thigh muscle alterations were examined to determine the safety of the compound. Partial protection against lethal doses was achieved in immunized mice despite low IgG titers. These data suggest that our nano-emulsion is a simple and efficient method of promoting antigen delivery for toxin-related diseases.
Assuntos
Vacinas Bacterianas/administração & dosagem , Toxinas Botulínicas Tipo A/toxicidade , Clostridium , Animais , Vacinas Bacterianas/imunologia , Clostridium/imunologia , Feminino , Fígado/patologia , Camundongos , Nanopartículas , Baço/patologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologiaRESUMO
Objetivo: establecer las condiciones de producción a nivel de laboratorio de la alfa toxina de Clostridium septicum IRP15 para la formulación de una vacuna veterinaria y la optimización del proceso de producción. Métodos: se caracterizó y estandarizó la edad apropiada del inóculo para los cultivos en un fermentador New Brunswick Scientific 7 L. Las condiciones de cultivo fueron: cepa C. septicum IRP15, medio de cultivo VBH, 5 L/vaso de 7 L, inóculo de 250 mL (5 por ciento), 37 ºC, 24 h, bajo agitaciones prueba de 0, 25 y 50 r.p.m. Se estableció el perfil cinético morfológico, de biomasa, consumo de sustrato y producción de toxina. Resultados: para las fermentaciones de 0 y 25 r.p.m. no se presentó fase de adaptación; el microorganismo creció de manera exponencial hasta las 4 y 6 h de fermentación, consumiendo simultáneamente la mayor cantidad de glucosa presente en el medio. A partir de estas horas y hasta las 24, se realizó la prueba de DL50 en ratones y se destaca que a 25 r.p.m. se obtuvo el mayor título de toxina (1/23). En las fermentaciones a 50 r.p.m. se observó que el microorganismo experimenta una fase de adaptación de 4 h aproximadamente; con un retardo en producción de biomasa, consumo de glucosa y producción de toxina, condición que no resulta óptima para la producción del antígeno. Conclusiones: la producción de toxina se presenta en la fase logarítmica y durante la fase estacionaria, asociándose así al crecimiento y al fenómeno de esporulación(AU)
Objective: to set the laboratory production conditions of Clostridium septicum IRP15 alpha toxin for the formulation of a veterinary vaccine and the optimization of the productive process. Methods: the appropriate inoculum age for the cultures was characterized and standardized in a 7L New Brunswick Scientific biorreactor. The conditions of culturing were C. septicum IRP15 strain, VBH medium at 5 L/7 L glass, 250 mL (5 percent) inoculum, 37 ºC, and 24 h under shaking conditions of 0, 25 y 50 r.p.m. The following kinetic parameters were monitored: morphological changes, biomass production, glucose consumption and toxin production. Results: for the shaking conditions at 0 and 25 r.p.m., C. septicum did not show an adaptation phase growth. The bacteria kept growing at the log phase up to 4-6 hours of fermentation respectively, thus consuming the highest amount of glucose from the medium. As from the growth phase hours till the 24 h of cultivation, the 50 percent lethal dose (LD50) in mice assay was conducted and at 25 r.p.m. condition, the best titre of toxin was reached (1/23). The cultures at 50 r.p.m. condition showed that the bacteria experienced adaptation phase for almost four hours, resulting in delayed biomass production, glucose consumption and toxin production. These results suggested that 50 r.p.m. is not useful for the antigen production. Conclusions : the toxin production occurred at the log phase and during the stationary phase, thus it is associated to growth and to sporulation(AU)
Assuntos
Animais , Vacinas/uso terapêutico , Clostridium/imunologia , Infecções por Clostridium/veterinária , Reagentes de Laboratório/uso terapêutico , ColômbiaRESUMO
Malignant oedema is a fatal disease of several animal species, produced by one or more members of the Clostridium genus. We report here a case of malignant oedema in a 1-year-old Friesian sheep after a blood sample was collected from the jugular vein. Clostridium septicum and Clostridium sordellii were isolated from the lesions and also demonstrated by a fluorescent antibody test. This report stresses the need for maintaining a clean environment for animals and for strict hygienic measures during procedures that generate wounds, together with immunity acquired by proper vaccination, for prevention of malignant oedema.
Assuntos
Coleta de Amostras Sanguíneas/veterinária , Infecções por Clostridium/veterinária , Edema/veterinária , Doenças dos Ovinos/diagnóstico , Criação de Animais Domésticos , Animais , Coleta de Amostras Sanguíneas/efeitos adversos , Clostridium/imunologia , Clostridium/isolamento & purificação , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/etiologia , Morte Súbita/veterinária , Diagnóstico Diferencial , Edema/etiologia , Feminino , Imunofluorescência/veterinária , Ovinos , Doenças dos Ovinos/etiologiaRESUMO
The immunoprotective capacity of four Clostridium chauvoei strains at different growth stages is reported. In all the strains tested, the cells coming from the stationary phase were those with the highest immunoprotective capacity and, depending on the strain, this protective capacity diminished or even disappeared in other phases. Protein profiles were similar in all the strains and few proteins were differentially expressed during growth as shown by SDS-PAGE. For strain 17, a local strain, a clear relationship was observed between the diminution of immunogenicity and the total loss of protective capacity of sonicated cells at late stationary phase.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Clostridium/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Variação Antigênica , Vacinas Bacterianas/imunologia , Técnicas Bacteriológicas , Western Blotting , Clostridium/crescimento & desenvolvimento , Infecções por Clostridium/imunologia , Infecções por Clostridium/prevenção & controle , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Camundongos , SonicaçãoRESUMO
For more than a century, bacteria and bacterial products have been used for the treatment of cancer. Starting from the practical observation of tumor regression in individuals with concomitant bacterial infection, the field has evolved into some standard clinical practices, such as the use of BCG for the treatment of superficial bladder cancer. However, in the last few years, new applications have started to emerge that may profoundly change the perspective of the field. BCG can be engineered to express cytokines to improve its efficacy. Bacteria such as Salmonella and Listeria can be attenuated by genetically-defined mutations and provide effective vehicles for DNA vaccines encoding tumor-associated antigens. Salmonella and nonpathogenic strains of Clostridium can selectively accumulate in tumors in vivo, providing attractive delivery systems to target immunomodulatory molecules and therapeutic agents to the tumor site. Many of these new developments have been attempted for prophylactic or therapeutic vaccination in several different experimental models of cancer and in many cases, results from clinical trials are now emerging. There is still some way to go before achieving products that could be in routine use, but the field has great promise for the development of more effective immunotherapies for several different cancers. In this paper, we will review the current state of such applications and highlight some of the directions that the field may take.
Assuntos
Bactérias/imunologia , Imunoterapia , Neoplasias/terapia , Adjuvantes Imunológicos , Vacina BCG/imunologia , Vacina BCG/uso terapêutico , Clostridium/imunologia , Engenharia Genética , Humanos , Listeria/imunologia , Salmonella/imunologia , Vacinas Atenuadas/imunologiaRESUMO
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.
Assuntos
Doenças dos Bovinos , Infecções por Clostridium/veterinária , Clostridium/isolamento & purificação , Meio Ambiente , Microbiologia do Solo , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/normas , Vacinas Bacterianas/uso terapêutico , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/etiologia , Doenças dos Bovinos/imunologia , Cromatografia Gasosa/veterinária , Clostridium/classificação , Clostridium/imunologia , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/etiologia , Gangrena Gasosa/imunologia , Gangrena Gasosa/prevenção & controle , Gangrena Gasosa/veterinária , Cobaias , Incidência , México/epidemiologia , Pasteurella/imunologia , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/prevenção & controle , Infecções por Pasteurella/veterinária , Fatores SocioeconômicosRESUMO
A peroxidase-antiperoxidase (PAP) technique was used to detect Clostridium chauvoei in tissue sections from sheep inoculated intramuscularly with a pure culture of this microorganism. Samples of various tissues were taken for bacteriology, histopathology and immunohistochemistry. A primary antiserum against C. chauvoei for use in the PAP technique was produced in rabbits. Formalin-fixed, paraffin-embedded sections of muscle samples were positively and specifically stained by the PAP technique. The results were consistent with those obtained by bacteriology, but the PAP test was simpler, quicker and less expensive than the bacteriological procedures. The use of the PAP technique would be appropriate for detecting clostridial infections without the constraints of conventional identification methods, especially where laboratory conditions for anaerobic procedures are not readily available.
Assuntos
Infecções por Clostridium/veterinária , Clostridium/isolamento & purificação , Doenças dos Ovinos/diagnóstico , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/imunologia , Clostridium/imunologia , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Formaldeído , Técnicas Imunoenzimáticas/normas , Técnicas Imunoenzimáticas/veterinária , Imuno-Histoquímica , Injeções Intramusculares/veterinária , Masculino , Músculo Esquelético/microbiologia , Inclusão em Parafina/veterinária , Coelhos , Ovinos , Doenças dos Ovinos/microbiologiaRESUMO
The effect of culture conditions on growth and immunogenicity of Clostridium chauvoei were examined. The pH control and partial feeding of the carbon source at high concentrations were beneficial for growth. The biomass yield was significatively improved, however the butanol concentration reached toxic levels hampering further growth. For each experimental condition the immunogenicity of cells was tested. No differences were found with cells obtained at different temperatures, but it decreased significatively with the partial supply of the carbon source and pH control.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Clostridium/crescimento & desenvolvimento , Clostridium/imunologia , Animais , Proteínas de Bactérias/isolamento & purificação , Vacinas Bacterianas/isolamento & purificação , Técnicas Bacteriológicas , Bovinos , Doenças dos Bovinos/prevenção & controle , Clostridium/metabolismo , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Ovinos , Doenças dos Ovinos/prevenção & controle , TemperaturaRESUMO
On smears of marrow-bone death bovines, presumptively diagnostic of black-leg, we have carried out the direct immunofluorescence test (IF). We used two labelled immunosera with fluorescein isothiocyanate. The immunosera wer prepared with the reference strain 5078 of Clostridium chauvoei as following: a) a cellular extract obtained with veronal buffer 0.045 M pH = 8.6, and b) a flagellar suspension obtained by agitation with glass beads, centrifuged at 3,500 x g, and centrifuged again at 16,000 x g during 20 min at 4 degrees C. Both antigenic preparations were injected into rabbits, five doses (0.2, 0.4, 0.8, 1.6 and 3.2 ml) each by i.v. route. Control positive strain of C. chauvoei were used, and control negative strains of C. septicum and C. perfringens were used too. Of all 56 examined samples, 26 (47.5%) gave positive IF test. These results had a 100% of correlation by culture and biochemical identification.
Assuntos
Exame de Medula Óssea/métodos , Doenças dos Bovinos/diagnóstico , Infecções por Clostridium/veterinária , Imunofluorescência , Animais , Antígenos de Bactérias/isolamento & purificação , Medula Óssea/microbiologia , Bovinos , Doenças dos Bovinos/patologia , Clostridium/classificação , Clostridium/imunologia , Clostridium/isolamento & purificação , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/patologia , Coelhos , Especificidade da EspécieRESUMO
The production of a protein insoluble at low temperature ("cryoprotein"), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptide chain of approximately Mr = 180,000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.