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Caracteriza??o genot?pica de Borrelia sp e de genes de Anaplasma marginale que codificam prote?nas de membrana com potencial imunog?nico. / Genetic caracterization of Borrelia sp and membrane protein genes of Anaplasma marginale with imunogenic potential.

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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.

Identiferoai:union.ndltd.org:IBICT/oai:localhost:jspui/1860
Date10 February 2010
CreatorsDaniel da Silva, Guedes Junior
ContributorsFonseca, Adivaldo Henrique da, Ara?jo, Fl?bio Ribeiro de, Fragoso, Stenio Perdig?o, Massard, Carlos Luiz, Botteon, Paulo de Tarso Landgarff, Barreiras, Jairo Dias
PublisherUniversidade Federal Rural do Rio de Janeiro, Programa de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Instituto de Veterin?ria
Source SetsIBICT Brazilian ETDs
LanguagePortuguese
Detected LanguageEnglish
Typeinfo:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis
Formatapplication/pdf
Sourcereponame:Biblioteca Digital de Teses e DissertaƧƵes da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ
Rightsinfo:eu-repo/semantics/openAccess
RelationAra?jo FR, Costa CM, Ramos CA, Farias TA, Souza II, Melo ES, Elisei C, Rosinha GM, Soares CO, Fragoso SP, Fonseca AH 2008. IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu. Mem Inst Oswaldo Cruz 103: 186-190. Barbet AF, Blentlinger R, Yi J, Lundgren AM, Blouin EF, Kocan KM 1999. Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands, and cattle. Infect Immun 67: 102-107. 82 Barbet AF, Lundgren A, Jooyoung yi, Rurangirwa FR, Palmer GH 2000. Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics. Infect Immun 68: 6133?6138. Brayton KA, Knowles DP, Mcguire TC, Palmer GH 2001. Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens. Proc Natl Acad Sci USA 98: 4130-4135. Brayton KA, Palmer GH, Lundgren A, Yi J, Barbet AF 2002. Antigenic variation of Anaplasma marginale MSP2 occurs by combinatorial gene conversion. Mol Microbiol 43: 1151-1159. Brayton KA, Meeus PF, Barbet AF, Palmer GH 2003. Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during Anaplasma marginale persistence in vivo. Infect Immun 71:6627-6632. Brayton KA, Kappmeyer LS, Herndon DR, Dark MJ, Tibbals DL, Palmer GH, McGuire TC, Knowles Jr DP 2005. Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. Proc Natl Acad Sci USA 102: 844-849. Brayton KA, PalmerGH, Brown WC 2006. Genomic and proteomic approaches to vaccine candidate identification for Anaplasma marginale. Expert Rev Vaccines 5: 95?101. Brown WC, Shkap V, Zhu D, Mcguire TC, Tuo W, McElwain TF, Palmer GH 1998. CD41 T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge. Infect Immun 66: 5406-5413. Collins NE, Liebenberg J, Villiers EP, Brayton KA, Louw E, Pretorius A, Faber FE, Heerden HV, Josemans A, Kleef MV, Steyn HC, Strijp MFV, Zweygarth E, Jongejan F, Maillard JC, Berthier D, Botha D, Joubert F, Corton CH, Thomson NR, Allsopp MT, Allsopp BA 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. Proc Natl Acad Sci USA 102: 838-843. Herndon DR, Palmer GH, Shkap V, Knowles Jr DP, Brayton KA 2010. Complete Genome Sequence of Anaplasma marginale ss. Centrale. J Bacteriol 192: 379-80. Hotopp JCD, Lin M, Madupu R, Crabtree J, Angiuoli SV, Eisen J, Seshadri R, Ren Q, Wu M, Utterback TR, Smith S, Lewis M, Khouri H, Zhang C, Niu H, Lin Q, Ohashi N, Zhi N, Nelson W, Brinkac LM, Dodson RJ, Rosovitz MJ, Sundaram J, Daugherty SC, Davidsen T, Durkin AS, Gwinn M, Haft DH, Selengut JD, Sullivan SA, Zafar N, Zhou L, Benahmed F, Forberger H, Halpin R, Mulligan S, Robinson J, White O, Rikihisa Y, Tettelin H 2006. Comparative Genomics of Emerging Human Ehrlichiosis Agents. PLoS Genet 2: 208-223. Lewin, B 2000. The operon. In B. Lewin (ed.), Genes VII. Oxford University Press, Oxford, United Kingdom. p. 275?277. 83 L?hr CV, Brayton KA, Shkap V, Molad T, Barbet AF, Brown WC, Palmer GH 2002. Expression of Anaplasma marginale major surface protein 2 operon-associated proteins during mammalian and arthropod infection. Infect Immun 70: 6005-6012. Lopez JE, Siems WF, Palmer GH, Brayton KA, Mcguire TC, Norimine J, Brown WC 2005. Identification of Novel Antigenic Proteins in a Complex Anaplasma marginale Outer Membrane Immunogen by Mass Spectrometry and Genomic Mapping. Infect Immun 73: 8109?8118. Lopez JE, Palmer GH, Brayton KA, Dark MJ, Leach SE, Brown WC 2007. Immunogenicity of Anaplasma marginale type IV secretion system proteins in a protective outer membrane vaccine. Infect Immun 75: 2333-2342. Lopez JE, Beare PA, Heinzen RA, Norimine J, Lahmers KK, Palmer GH, Brown WC 2008. High-throughput identification of T-lymphocyte antigens from Anaplasma marginale expressed using in vitro transcription and translation. J Immunol Methods 332: 129-141. Mavromatis K, Doyle CK, Lykidis A, Ivanova N, Francino MP, Chain P, Shin M, Malfatti S, Larimer F, Copeland A, Detter JC, Land M, Richardson PM, Yu XJ, Walker DH, McBride JW, Kyrpides NC 2006. The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies. J Bacteriol 188: 4015-4023. Niu H, Rikihisa Y, Yamaguchi M, Ohashi N 2006. Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway. Cell Microbiol 8: 523-534. Noh SM, Brayton KA, Knowles DP, Agnes JT, Dark MJ, Brown WC, Baszler TV, Palmer GH 2006. Differential Expression and Sequence Conservation of the Anaplasma marginale MSP2 gene superfamily outer membrane proteins. Infect Immun 74: 3471?3479. Noh SM, Brayton KA, Brown WC, Norimine J, Munske GR, Davitt CM, Palmer GH 2008. Composition of the Surface Proteome of Anaplasma marginale and Its Role in Protective Immunity Induced by Outer Membrane Immunization. Infect Immun 76: 2219-2226. Ohashi N, Zhi N, Lin Q, Rikihisa Y 2002. Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agents. Infect Immun 70: 2128-2138. Palmer GH, Brown WC, Rurangirwa FR 2000. Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale. Microbes Infect 2: 167-176. Palmer GH, Bankhead T, Lukehart SA 2009. Nothing is permanent but change - antigenic variation in persistent bacterial pathogens. Cell Microbiol 11: 1697-1705. Pipano E 1995. Live vaccines against hemoparasitic diseases in livestock. Vet Parasitol 57: 213-231. 84 Ramos CA, Ara?jo FR, Os?rio AL, Madruga CR, Rosinha GM, Soares CO, Elisei C 2007. Transcription of genes of membrane proteins of Brazilians isolates of Anaplasma marginale. Rev Bras Parasitol Vet 16:152-155. Sutten EL, Norimine J, Beare PA, Heinzen RA, Lopez JE, Morse K, Brayton KA, Gillespie JJ, Brown WC 2010. Anaplasma marginale Type IV Secretion System proteins VirB2, VirB7, VirB11, and VirD4 are immunogenic components of a protective bacterial membrane vaccine. Infect Immun 78: 1314 ? 1325. Tamura K, Dudley J, Nei M, Kumar S 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599. Vidotto MC, Venancio EJ, Vidotto O 2008. Cloning, sequencing and antigenic caracterization of rVirB9 of Anaplasma marginale isolated from Paran? state, Brazil. Genet Mol Res 7:460- 466.

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