ABSTRACT This goal of this research was to study, by Spoligotyping, 43 Mycobacterium bovis isolates, from cattle slaughtered in the State of São Paulo, typed in 12 different spoligotypes. Among these isolates 35 were classified in 6 spoligotypes known already, and 8 isolates in 6 new spoligotypes. Within those, 29 isolates corresponded to the SB0295, SB0121 and SB0140 spoligotypes. The SB0140 spoligotype is the major type found in Argentina and is also present in Paraguay and Uruguay. The SB0120 spoligotype is the most frequently observed type in France (26%) and has also benn in Belgium, China, Denmark, Iran, Japan, Portugal, Russia, South Africa, Sri Lanka and the Netherlands. The AR28 spoligotype has also benn found in Argentina and Brazil. SB0122 was only cited in the Netherlands. It is important to emphasize that among the new spoligotypes found in the present study, at the moment, there are 6 unique spoligotypes, 2 of them isolated as N7 spoligotype and 2 isolated as the N8 spoligotype. The results show that there are in Brazil spoligotypes that, up to this moment, seem to be shared between Brazil, Argentina and Europe, and therefore the molecular discrimination of the M. bovis isolates by the Spoligotyping constitutes a tool for surveillance systems in detecting focuses of bovine tuberculosis. study 4 Paulo 1 3 already those SB0295 SB SB012 SB014 Uruguay 26% 26 (26% Belgium China Denmark Iran Japan Portugal Russia Africa Netherlands AR AR2 moment N Europe M tuberculosis SB029 SB01 (26 SB02 SB0 (2 (

RESUMO O objetivo do presente trabalho foi estudar, pela técnica de Spoligotyping, 43 isolados de Mycobacterium bovis, provenientes de bovinos abatidos no Estado de São Paulo, em 12 diferentes espoligotipos. Destes isolados 35 foram classificados em 6 espoligotipos já conhecidos (SB0295, SB0121, SB0140, SB0120, AR28 e SB0122) e os outros 8 em 6 novos espoligotipos. Vinte e nove amostras foram classificados nos espoligotipos SB0295, SB0121 e SB0140, sendo o espoligotipo SB0140 majoritário na Argentina e presente no Paraguai e Uruguai. O espoligotipo SB0120 é o mais freqüente na França (26%), tendo sido encontrado na Bélgica, China, Dinamarca, Iran, Japão, Portugal, Rússia, África do Sul, Espanha, Sri Lanka e Holanda. O espoligotipo AR28, já foi encontrado na Argentina e no Brasil. O espoligotipo SB0122 foi referido apenas na Holanda. Dos novos espoligotipos encontrados no presente estudo, é importante ressaltar, que até o momento 6 deles são espoligotipos únicos, dois isolados como espoligotipo N7 e dois isolados como espoligotipo N8. Estes resultados mostram que existem no Brasil espoligotipos que parecem, até o momento, ser compartilhados entre Brasil, Argentina e Europa. Sendo assim a discriminação molecular de isolados de M. bovis através do Spoligotyping constitui-se numa ferramenta para sistemas de vigilância na detecção de focos de tuberculose bovina. estudar 4 Paulo 1 3 SB0295 SB (SB0295 AR AR2 SB012 SB014 Uruguai 26%, 26 26% , (26%) Bélgica China Dinamarca Iran Japão Portugal Rússia Sul Espanha Holanda estudo ressaltar únicos N N8 parecem Europa M constituise constitui se bovina SB029 (SB029 SB01 2 (26% SB02 (SB02 SB0 (26 (SB0 (2 (SB (

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.