RESUMO
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freund's incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.
Assuntos
Antígenos de Bactérias/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis , Doenças das Cabras/prevenção & controle , Linfadenite/veterinária , Animais , Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/sangue , Infecções por Corynebacterium/imunologia , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Cabras , Immunoblotting , Imunoglobulina G/sangue , Interferon gama/sangue , Linfadenite/prevenção & controleRESUMO
Corynebacterium pseudotuberculosis is the causal agent of caseous lymphadenitis, a chronic illness that attacks goats and sheep characterized by pyogranulomas formation in lymph nodes and organs. Regarding the current knowledge of the pathogenesis of the caseous lymphadenitis, there is evidence that besides the humoral response the induction of a durable cellular response is fundamental for its control. In this sense, research on antigens of C. pseudotuberculosis that are capable to inducing cellular immunity is an important step for the development of diagnosis tests and more efficient vaccines. In the present study, the interferon-gamma production in cultures of whole blood from infected goats stimulated with secreted bacterial antigen or somatic antigen were used to evaluate the cellular response. The results demonstrated a significant difference in the ability of the two antigens to induce a cellular response. That is, IFN-gamma production was high with cells from infected animals in response to the secreted antigen while IFN-gamma production was low when somatic antigen was used. The concomitant use of these antigens with PWM also showed differences. That is, the secreted antigen increased the IFN-gamma production induced by PWM, while the somatic antigen seems not to have altered the response to PWM.
Assuntos
Antígenos de Bactérias/administração & dosagem , Corynebacterium pseudotuberculosis/imunologia , Cabras/imunologia , Interferon gama/biossíntese , Animais , Células Sanguíneas/imunologia , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/patogenicidade , Feminino , Doenças das Cabras/imunologia , Cabras/microbiologia , Técnicas In Vitro , Linfadenite/imunologia , Linfadenite/veterinária , Masculino , Mitógenos de Phytolacca americana/administração & dosagemRESUMO
The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.
Assuntos
Antígenos de Bactérias/imunologia , Corynebacterium pseudotuberculosis/imunologia , Cabras/imunologia , Leucócitos Mononucleares/imunologia , Sulfato de Amônio/química , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Butanóis/química , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Leucócitos Mononucleares/citologiaRESUMO
Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.
Assuntos
Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/imunologia , Doenças das Cabras/imunologia , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Linfadenite/veterinária , Animais , Anticorpos Antibacterianos/sangue , Afinidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Western Blotting/veterinária , Brasil , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/patologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Doenças das Cabras/microbiologia , Cabras , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/sangue , Cinética , Linfadenite/imunologia , Linfadenite/patologia , MasculinoRESUMO
RESUMO: Um meio quimicamente definido para Corynebacterium pseudotuberculosis é descrito. Foram testadas, simultaneamente, a influência de concentrações crescentes de misturas de aminoácidos essenciais, não essenciais e vitaminas, assim como a proporção de sais mono e dibásicos do tampão fosfato, sobre o crescimento de três cepas. Foi observado que o aumento da concentração de vitaminas levou a um maior crescimento, enquanto que o aumento da concentração de aminoácidos não se traduziu em aumento da população bacteriana. Quanto aos tampões utilizados, foi demonstrado que naqueles onde havia maior concentração de sais dibásicos ocorreu um maior desenvolvimento das culturas. O meio de cultura descrito permitiu o crescimento da bactéria e a secreção de proteÃnas livres de macromoléculas heterólogas, abrindo novas perspectivas para o estudo imunológico de C. pseudotuberculosis. PALAVRAS CHAVE :Corynebacterium pseudotuberculosis, meio sintético, proteÃnas. SUMMARY: A chemically defined medium for Corynebacterium pseudotuberculosis is described. We have tested simultaneously the influence of increasing concentrations of mixtures of essential, not essential amino acids and vitamins, as well as the ratio of phosphate buffer mono and dibasics salts, on the growth of three strains. It was observed that the increase of the vitamins concentration led to a
RESUMO
RESUMO: Um meio quimicamente definido para Corynebacterium pseudotuberculosis é descrito. Foram testadas, simultaneamente, a influência de concentrações crescentes de misturas de aminoácidos essenciais, não essenciais e vitaminas, assim como a proporção de sais mono e dibásicos do tampão fosfato, sobre o crescimento de três cepas. Foi observado que o aumento da concentração de vitaminas levou a um maior crescimento, enquanto que o aumento da concentração de aminoácidos não se traduziu em aumento da população bacteriana. Quanto aos tampões utilizados, foi demonstrado que naqueles onde havia maior concentração de sais dibásicos ocorreu um maior desenvolvimento das culturas. O meio de cultura descrito permitiu o crescimento da bactéria e a secreção de proteÃnas livres de macromoléculas heterólogas, abrindo novas perspectivas para o estudo imunológico de C. pseudotuberculosis. PALAVRAS CHAVE :Corynebacterium pseudotuberculosis, meio sintético, proteÃnas. SUMMARY: A chemically defined medium for Corynebacterium pseudotuberculosis is described. We have tested simultaneously the influence of increasing concentrations of mixtures of essential, not essential amino acids and vitamins, as well as the ratio of phosphate buffer mono and dibasics salts, on the growth of three strains. It was observed that the increase of the vitamins concentration led to a