Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-07-10T13:03:11Z No. of bitstreams: 1 2010 - Daniel da Silva Guedes Junior .pdf: 3397672 bytes, checksum: fea6a394bbb430b666ddc9c054a24c27 (MD5) / Made available in DSpace on 2017-07-10T13:03:11Z (GMT). No. of bitstreams: 1 2010 - Daniel da Silva Guedes Junior .pdf: 3397672 bytes, checksum: fea6a394bbb430b666ddc9c054a24c27 (MD5) Previous issue date: 2010-02-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq, Brasil. / The geographic distribution of bovine borreliosis is determined by the dispersion of its vector. Borrelia theileri is the predominant species in cattle, and B. coriaceae and B. burgdorferi also been reported causing clinical disease. B. theileri cause mild disease in cattle, and still is important for its potential to be confused with the spirochete of Lyme disease, B. burgdorferi, and agents of epizootic bovine abortion, B. coriaceae. In Brazil, as well as in other South American countries, the agent of this disease has not been isolated further confusing the diagnosis. The objective of this study was to identify genotypically Borrelia sp that affects cattle in Brazil. DNA extraction, was performed from blood and ticks of cattle with positive serology by indirect ELISA with crude antigen of Borrelia burgdorferi. Primers were designed for genes of Borrelia burgdorferi and B. theileri groups: 16S, flaA, flaB, GroEL, hbb, recA, 5s-23s, p66, rrs, rpoB and glpq. After the PCR reaction, only the primers amplified rrs and rpoB sequences. The predictive amino acid sequence of RRS3 revealed 99% homology with B. hermsii and B. duttonii and predictive amino acid sequence of RPOB showed 67% homology with B. duttonii and B. recurrentis. This suggests that the species of Borrelia sp present in Brazil is not owned by group B. burgdorferi. Little is known regarding the genetic variability of genes that encode membrane proteins of Brazilian isolates of A. marginale. The products of these genes constitute an important tool, as there may be significant antigen polymorphism, which may damage cross-protection between isolates and the chances of identifying candidate immunogens. The aim of the present study was to determine the degree of conservation of sequences of these genes in a Brazilian isolate of A. marginale comparing with Saint Maries and Florida isolates. For this, primers were designed to amplify the genes omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, am097 (VirB9-1), am956 (PepA), am254 (ef-tu), am854 by PCR. The genes were then sequenced by Sanger method and the predicted amino acid sequences aligned and homology analyzed by the program CLUSTAL W. With the exception of OMP 7 all proteins (OMP1, OMP4, OMP5, OMP8, OMP10, OMP14, OMP15, SODB, OPAG1, OPAG3, VIRB3, VIRB9-1, PepA, EF-Tu, AM854) exhibited homology greater than 92% with other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODB, VIRB9-1 e AM854 showed homology greater than 72% regarding to A. marginale centrale which confers cross-protection against A. marginale. / A distribui??o geogr?fica da borreliose bovina ? determinada pela dispers?o do seu vetor. Borrelia theileri ? a esp?cie predominante em bovinos, sendo que B. burgdorferi e B. coriaceae tamb?m foram relatadas causando doen?a cl?nica. Portanto, B. theileri causa doen?a leve em bovinos, e ainda ? importante pelo seu potencial em ser confundido com a espiroqueta da Doen?a de Lyme, B. burgdorferi, e com agentes do Aborto Epizo?tico bovino, B. coriaceae. No Brasil, assim como em outros pa?ses Sul americanos, o agente desta enfermidade ainda n?o foi isolado prejudicando ainda mais o diagn?stico. O objetivo deste trabalho foi ? identifica??o genot?pica da esp?cie de Borrelia sp que acomete bovinos no Brasil. Foram utilizados para extra??o de DNA, o sangue e carrapatos de bovinos com sorologia positiva ao ELISA indireto com ant?geno bruto para Borrelia burgdorferi. Foram desenhados oligonucleot?deos iniciadores para genes dos grupos Borrelia burgdorferi e B. theileri: 16S, flaA, flaB, groel, hbb, recA, 5s-23s, p66, rrs, rpob e glpq. Ap?s a rea??o de PCR, somente os oligonucleot?deos iniciadores rrs e rpob amplificaram seq??ncias. A seq??ncia preditiva de amino?cidos de RRS3 revelou homologia de 99% com B. hermsii e B. duttonii e a seq??ncia preditiva de amino?cidos de RPOB demonstrou 67% de homologia com B. duttonii e B. recurrentis. Isto sugere que a esp?cie de Borrelia presente no Brasil n?o seja pertencente ao grupo de B. burgdorferi. Pouco se sabe sobre a variabilidade gen?tica dos genes que codificam prote?nas de membrana de isolados brasileiros de A. marginale. O produto destes genes constitui uma ferramenta importante, pois pode haver polimorfismo antig?nico, que pode prejudicar a prote??o cruzada entre os isolados e as chances de identifica??o de candidatos a imun?genos. O objetivo do presente estudo foi determinar o grau de conserva??o das seq??ncias destes genes em um isolado brasileiro de A. marginale frente aos isolados Saint Maries, Florida e A. marginale centrale. Para tanto, oligonucleot?deos foram desenhados para amplificar os genes omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, am097 (VirB9- 1), am956 (PepA), am254 (ef-tu), am854 por PCR. Os genes foram ent?o seq?enciados pelo m?todo de Sanger e as seq??ncias preditas de amino?cidos alinhadas e a homologia analisada atrav?s do programa CLUSTAL W. Com exce??o de OMP 7 todas as demais (OMP1, OMP4, OMP5, OMP8, OMP10, OMP14, OMP15, SODB, OPAG1, OPAG3, VIRB3, VIRB9-1, PepA, EF-Tu, AM854) apresentaram n?veis de homologia de 92 a 100% entre os isolados de A. marginale. Destas, apenas OMP1, OMP5, EF-Tu, VirB3, SODB, VIRB9-1 e AM854 apresentaram homologia superior a 72% em rela??o a A. marginale centrale, o qual confere prote??o cruzada contra A. marginale.

Made available in DSpace on 2016-04-28T20:15:30Z (GMT). No. of bitstreams: 1 2008- Jania de Resende.pdf: 467062 bytes, checksum: cf6acc7c0e32afbfa397b5b1c07d0d87 (MD5) Previous issue date: 2008-02-22 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Embryonic cells of tick in vitro constitute an important one tool for culture and study of the biology of B. burgdorferi. Spirochetes Borrelia burgdorferi is the aetiologic agent of borreliose of Lyme in U.S.A. and Europe, where it is transmitted by tick of the Ixodes genus. The aim of this work was in vitro to cultivate B. burgdorferi (American Cepa G39/40) in primary culture of embryonic cells of Rhipicephalus (B.) microplus and the A. cajennense. From the primary culture of embryonic cells of R. (B.) microplus were performed subculture maintained with medium Leibovitz's (L-15) free of antibiotic, that later it were changed by medium Barbour-Stoener-Kelly (BSK). After the addition of the BSK inoculated B. burgdorferi of and also in Tube of Leighton (TL) with free BSK of cells (control) in a final concentration of 6x106 spirochetes/mL. The embryonic cells of the A. cajennense initially were cultivated in medium L-15 with antibiotic, which was substituted by the BSK. Later, 1,1x107 spirochetes/mL in medium BSK cultivated was inoculated, and also in TL controlled free of cells. All the culture was incubated at 34?C. The development of the culture was observed in microscope of inverted contrast of phase, as well as the countings of B. burgdorferi performed in chamber of neubauer. Cover glass of the TL had been stained with Giemsa. It was evidenced by the observation in microscope of inverted contrast of phase the survival, attach and multiplication of B. burgdorferi, in the embryonic cells of R. (B.) microplus and A. cajennense. It did not have differences in the finale counting of spirochetes cultivated in cells of R. (B.) microplus when compared with the free control of cells, but on with cells of the A. cajennense, the number amount approximately was 1,9x107 spirochetes/mL, and while in the tube it has controlled free of cells was 1x106 spirochetes /mL. The culture of cells of tick R. (B.) microplus and the A. cajennense have potential to be used as substratum for culture of B. burgdorferi, and study of its development. / C?lulas embrion?rias de carrapatos mantidas in vitro constituem uma importante ferramenta para cultivo e estudo da biologia de Borrelia burgdorferi. A espiroquetas B. burgdorferi ? o agente etiol?gico da borreliose de Lyme nos EUA e Europa, onde ? transmitida por carrapatos do g?nero Ixodes. O objetivo deste trabalho foi cultivar in vitro Borrelia burgdorferi (Cepa Americana G39/40) em cultura prim?ria de c?lulas embrion?rias dos carrapatos Rhipicephalus (Boophilus) microplus e Amblyomma cajennense. A partir da cultura prim?ria de c?lulas embrion?rias de R. (Boophilus) microplus foram realizados subcultivos mantidos com meio Leibovitz s L-15 livre de antibi?tico, que posteriomente foi trocado pelo meio Barbour-Stoener-Kelly (BSK). Ap?s a adi??o do BSK na cultura, inoculou-se B. burgdorferi e tamb?m em Tubo de Leighton (TL) com BSK livres de c?lulas (controle), numa concentra??o final de 6x106 espiroquetas/mL. As c?lulas embrion?rias de A. cajennense foram inicialmente cultivadas em meio L-15 com antibi?tico, o qual foi substitu?do pelo BSK. Posteriormente, inoculou-se 1,1x107 espiroquetas/mL cultivadas em meio BSK, e tamb?m em TL controle livre de c?lulas. Todos os cultivos foram incubados em estufa bacteriol?gica a 34?C. O desenvolvimento dos cultivos foram observados em microsc?pio de contraste de fase invertido, assim como as contagens de B. burgdorferi realizados em c?mara de neubauer. As lam?nulas dos TL foram coradas com Giemsa. Foi constatado pela observa??o em microsc?pio de contraste de fase invertido a sobreviv?ncia, ader?ncia e multiplica??o de B. burgdorferi, nas c?lulas embrion?rias de R. (Boophilus) microplus e A. cajennense. N?o houve diferen?as na contagem final de espiroquetas cultivadas em c?lulas de R. (Boophilus) microplus quando comparada ao controle livre de c?lulas, mas sobre c?lulas de A. cajennense o valor total de aproximadamente 1,9x107 espiroquetas/mL, e enquanto no tubo controle livre de c?lulas foi 1x106 espiroquetas/mL. O cultivo de c?lulas do carrapato R. (Boophilus) microplus e A. cajennense t?m potencial para ser utilizado como substrato para cultivo de B. burgdorferi e para estudo de seu desenvolvimento.

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...