ABSTRACT
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)
Subject(s)
Animals , Toxoids , Enterocolitis, Pseudomembranous/veterinary , Vaccines, Synthetic/therapeutic use , Clostridium perfringens/immunology , Gas Gangrene/veterinary , Horses , Immunization/veterinaryABSTRACT
This study described an outbreak of necrohemorrhagic enteritis in a beef cattle feedlot in Nova Crixás, State of Goiás, Brazil, with emphasis on epidemiological, lesional, and laboratory aspects. Visits to the property were carried out and a necroscopic examination was performed on the bovine cadavers (N=57), which presented similar macroscopic alterations. Epidemiological data were collected, mainly referring to the feeding management of animals, and tissue samples were submitted to histopathological examination. Samples of feces and intestinal contents were also collected for bacterial isolation and PCR genotyping to detect the etiological agent, being confirmed Clostridium perfringens type A strains in 100% of the samples. Furthermore, 33.3% of strains isolated from intestinal contents and 40% of those isolated from feces were positive for beta-2 encoding gene. Considering the history, macroscopic and microscopic findings, as well as bacterial isolation and PCR, the diagnosis of bovine necrohemorrhagic enteritis was determined.(AU)
Descreve-se um surto de enterite necro-hemorrágica em um confinamento de bovinos de corte no município de Nova Crixás, Estado de Goiás, Brasil, com ênfase nos aspectos epidemiológicos, lesionais e laboratoriais. Foram realizadas visitas à propriedade e todos os cadáveres bovinos (N=57) foram submetidos ao exame necroscópico, os quais apresentaram alterações macroscópicas semelhantes. Foram compilados dados epidemiológicos, sobretudo referentes ao manejo alimentar dos animais e amostras de tecido foram submetidas a exame histopatológico. Foram colhidas, também, amostras de fezes e conteúdo intestinal para isolamento bacteriano e genotipagem por PCR para detecção do agente etiológico, sendo confirmadas estirpes de Clostridium perfringens tipo A em 100% das amostras. Ainda, 33,3% das cepas de Clostridium perfringens isoladas no conteúdo intestinal e 40% daquelas isoladas nas fezes foram positivas para o gene codificador da toxina beta-2. Considerando o histórico, os achados macroscópicos e microscópicos, o isolamento bacteriano e o PCR, foi estabelecido o diagnóstico de enterite necro-hemorrágica por C. perfringens tipo A.(AU)
Subject(s)
Animals , Cattle , Clostridium perfringens/isolation & purification , Enteritis/pathology , Enteritis/veterinary , BrazilABSTRACT
Objective@#To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy.@*Methods@#Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal.@*Results@#The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence.@*Conclusions@#The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.
ABSTRACT
Objective To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy. Methods Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal. Results The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence. Conclusions The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.
ABSTRACT
Alfa toxina, una proteína formadora de poros con actividad citotóxica, es uno de los principales factores de virulencia secretados por la mayoría de las cepas de Staphylococcus aureus. Se ha establecido la relevancia de esta proteína en la patogenia de la neumonía asociada a infecciones por S. aureus. Por lo tanto, la inhibición de la secreción de alfa toxina puede ser una alternativa en el control de las infecciones causadas por este microorganismo. En este trabajo mostramos que quercetina, un flavonoide de origen natural, inhibe de manera dosis dependiente la actividad hemolítica y disminuye la secreción de alfa toxina en sobrenadantes de cultivos de S. aureus sensible y resistente a meticilina. Además, quercetina previene de manera significativa el daño de células alveolares humanas cuando se co-cultivan con S. aureus. Nuestros datos sugieren que quercetina puede disminuir la virulencia de S. aureus al afectar la secreción de alfa toxina.
Alpha toxin, a pore-forming protein with cytotoxic activity, is one of the major virulence factors secreted by most strains of Staphylococcus aureus. The relevance of this protein in the pathogenesis of pneumonia associated with S. aureus infections has already been esta blished. Therefore, inhibiting alpha toxin secretion can be an alternative for controlling these infections. This study shows that quercetin, a naturally occurring flavonoid, inhibits hemolytic activity in a dose-dependent manner and reduces alpha toxin secretion in culture supernatants of methicillin-sensitive and methicillin-resistant S. aureus. Furthermore, quercetin significantly prevents damage to human alveolar cells when co-cultured with S. aureus. Our results suggest that quercetin can reduce S. aureus virulence by affecting alpha-toxin secretion.
Subject(s)
Humans , Quercetin , Staphylococcus aureus , Antioxidants , Quercetin/pharmacology , Staphylococcal Infections , Staphylococcus aureus/pathogenicity , Bacterial Toxins/metabolism , Virulence , Virulence Factors , Hemolysin Proteins , Antioxidants/pharmacologyABSTRACT
BACKGROUND AND OBJECTIVES: Staphylococcus Aureus (S. Aureus) is one of the most common and predominant form of bacteria in the nasal airway that roduces toxin. Alpha toxin from S. Aureus, also known as alpha-hemolysin, causes damage to the membrane in many types of cells. The purpose of this study is to develop a rat model of rhinosinusitis induced by the intra-nasally applied alpha-toxin of S. Aureus. MATERIAL AND METHODS: Forty micro-liters of 100 microgram/ml of alpha-toxin was applied intra-nasally to 4-6 weeks-old Sprague-Dawley rats and the same amount of vehicle was applied to control rats. At days 1, 5 and 14 the rats were sacrificed and their nasal cavity prepared for histological investigation. RESULTS: Inflammatory cell clusters were observed in the alpha-toxin applied rats. The number of sinus air spaces occupied by inflammatory cell clusters increased significantly at days 1 and 5 compared with the control rats. Comparisons across the time interval demonstrated statistically significant changes, showing a peak at day 1 among alpha-toxin applied rats. CONCLUSION: Intra-nasally applied alpha-toxin induces acute rhinosinusitis in the rats. The histological evidence of rhinosinusitis revealed the appearance of inflammatory cell clusters in the sinonasal air spaces. These findings indicate that this rat model of alpha-toxin induced rhinosinusitis may be applied for better understanding of the role of bacterial toxin in the pathogenesis of rhinosinusitis.
Subject(s)
Animals , Rats , Bacteria , Membranes , Models, Animal , Nasal Cavity , Rats, Sprague-Dawley , Staphylococcus aureus , StaphylococcusABSTRACT
The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.
ABSTRACT
Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type C by polymerase chain reaction(PCR). PCR product was inserted into vector pGEM-T directively. The cloned recombinant plasmid pXCPA1 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases NcoI/EcoRI from plasmid pXCPA1, and then inserted into an expression vector pET-28c which cleaved with NcoI/EcoRI by blunt-end ligation. The recombinant expression plasmid pETXA1 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pETXA1 possessed a positive alpha-toxin gene sequence and reading frame. BL21(DE3) (pETXA1) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies, and the expression level of the alpha-toxin proteins were about 16.28% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.