Genetic caracterization of Borrelia sp and membrane protein genes of Anaplasma marginale with imunogenic potential

Autor: Guedes Junior, Daniel da Silva
Přispěvatelé: Fonseca, Adivaldo Henrique da, Ara?jo, Fl?bio Ribeiro de, Fragoso, Stenio Perdig?o, Massard, Carlos Luiz, Botteon, Paulo de Tarso Landgarff, Barreiras, Jairo Dias
Jazyk: portugalština
Rok vydání: 2010
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFRRJ
Universidade Federal Rural do Rio de Janeiro (UFRRJ)
instacron:UFRRJ
Popis: 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 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.
Databáze: OpenAIRE
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