Renato Carminati

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.

Serological survey for bovine herpesvirus 1 in cattle from different regions in the state of Bahia, Brazil

A presenca de anticorpos contra Herpesvirus Bovino tipo 1 (HBV-1) foi avaliada em bovinos jovens provenientes de quinze municipios diferentes, distribuidos nas regioes Norte, Sul e Central do estado da Bahia. Foram coletadas amostras de sangue de bovinos de 1 a 4 anos de idade, nao vacinados contra HBV-1 e sem previo diagnostico de infeccao pelo virus HBV-1. O total de amostras correspondeu a 558 soros, equivalente a 10% da populacao bovina de cada fazenda. Os 558 soros foram submetidos a tecnica de Microssoroneutralizacao (MSN) em microplacas, utilizando-se monocamadas de celulas MDBK e a cepa de HBV-1 Los Angeles, e ao teste de ELISA comercialmente adquirido: HerdCheck-IDDEX. Os resultados da amostragem feito pelo teste de ELISA mostraram que 56% (314/558) dos soros foram positivos e 44% (244/558), negativos. A tecnica de MSN mostrou que 48% (269/558) dos soros foram negativos e 52% (289/558) foram positivos. Destes soros positivos, 54% tiveram titulos soroneutralizantes entre 4-16, 35% entre 32-64, e 11% ³ 64. Os resultados sugerem que o HBV-1 esteja circulando ativamente na populacao bovina jovem. Por outra parte, a alta difusao do virus em animais jovens representaria uma fonte continua de infeccao viral para o rebanho.

DETECÇÃO DE ANTICORPOS CONTRA LEPTOSPIROSE EM SORO DE CAPRINOS PELO TESTE DE SOROAGLUTINAÇÃO MICROSCÓPICA (SAM) EM CONTRASTE DE FASE

A leptospirose e uma zoonose de distribuicao cosmopolita de grande importância zoonotica, que acomete animais de todas as especies, ocasionada pelo contato direto ou indireto com urina de animais infectados. O diagnostico baseia-se na pesquisa de aglutininas anti-leptospira em soro de animais suspeitos, utilizando microscopia em contraste de fase ou campo escuro. O presente trabalho objetivou identificar sorogrupos de leptospiras predominantes em caprinos pelo metodo de soroaglutinacao por meio da microscopia de contraste de fase. Foram analisados 38 soros de caprinos com e sem sintomas, pela Soroaglutinacao Microscopica (SAM), submetidos a uma bateria com 23 sorovares de Leptospira. Obteve-se um resultado na triagem na qual todas as 38 amostras foram reagentes para os 23 sorogrupos. Na titulacao verificou-se que os sorogrupos predominantes foram hardjobovis, copenhagen, butembo, bratislava e tarassovi, confirmando dados descritos na literatura que ratificam a presenca desses sorogrupos na especie caprina.

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.