Bruno Jean Adrien Paule

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-γ production, IgG avidity and antigen recognition by Western blotting

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.

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Caninde´ goats and their potential protection against caseous lymphadenitis

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 Freunds 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.

Investigação de áreas de risco como metodologia complementar ao controle da leishmaniose visceral canina

Risk areas of canine visceral leishmaniasis in the city of Camacari, Bahia, Brazil, were investigated. A total of 278 dogs from 141 homes pertaining to 20 investigated risk areas was serologically screened (ELISA). The general seroprevalence was 21.7% (56/258) after exclusion of 20 dogs used at the beginning of the survey to limit the study area. The respective results of the univariated and multivariated analysis of factors related to infection of dogs by Leishmania chagasi, to vector distribu-tion pattern in the area and to the methodology used to localize the canine focuses are discussed.

Análise por SDS-PAGE e Western blot de antígenos somáticos e extracelulares de Corynebacterium pseudotuberculosis

Different techniques to isolate secreted, cell-surface located and somatic fractions of Corynebacterium pseudotuberculosis have been studied by SDS-PAGE and immunoblotting, by using a pool of sera from naturally infected goats. The secreted antigens were obtained from culture supernatant of the bacteria grown in a chemically defined medium or in Brain Heart Infusion medium. The surface fraction was obtained by NaCl 1 M treatment and the somatic ones were obtained by using distinct procedures (detergents and ultrasounds). By Coomassie blue staining, we observed 20 bands in the secreted fraction, 35 bands in the surface extract and 40 to 50 bands in the somatic fraction, depending on the extraction procedure. Sixteen immunoreactive proteins were detected among the studied fractions. Bands with molecular weights of 125, 108, 75, 68, 41, 40, 31 and 24 kDa were recognized with higher intensity and all of them were found in the secreted fraction. The use of the chemically defined medium allowed us to evidence the presence of proteins with high molecular masses in the secreted fraction of C. pseudotuberculosis, which had not been previously described.