Free Essay

Plasmid Profile Analysis of Portuguese Borrelia Lusitaniae Strains

In: Science

Submitted By lrvitotino
Words 4638
Pages 19
This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit:

Author's personal copy
Ticks and Tick-borne Diseases 1 (2010) 125–128

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases journal homepage:

Original article

Plasmid profile analysis of Portuguese Borrelia lusitaniae strains
Liliana Vitorino a , Gabriele Margos b , Líbia Zé-Zé c , Klaus Kurtenbach b,1 , Margarida Collares-Pereira d,∗
Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Lisboa, Portugal Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom c Centro de Estudos de Vectores e Doencas Infecciosas Dr. Franscisco Cambournac, Instituto Nacional de Saúde Dr. Ricardo Jorge, Águas de Moura, Portugal ¸ d Unidade de Leptospirose e Borreliose de Lyme, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, and Centro de Recursos Microbiológicos, FCT/UNL, Rua da Junqueira 96, 1349-008 Lisboa, Portugal b a

a r t i c l e

i n f o

a b s t r a c t
Plasmid profiles of 2 Portuguese Borrelia lusitaniae strains, one isolated from a human patient and the other one from an Ixodes ricinus tick, were obtained by pulsed-field gel electrophoresis to evaluate the plasmid diversity in each strain. Overall, a maximum of 6 plasmids were detected that ranged from 19 kb to 76 kb, revealing completely different plasmid profiles from those previously described for other genospecies of B. burgdorferi sensu lato, the causative agents of Lyme borreliosis. The plasmid location of the ospA gene was investigated by hybridization, allowing its allocation to the plasmid of 70 kb instead of the 54 kb linear plasmid described for B. burgdorferi sensu stricto strains. © 2010 Elsevier GmbH. All rights reserved.

Article history: Received 30 March 2010 Received in revised form 6 July 2010 Accepted 14 July 2010 Keywords: Lyme borreliosis Borrelia lusitaniae Genome organization ospA Ixodes ricinus

Introduction Borrelia burgdorferi sensu lato (s.l.) are the causative agents of Lyme borreliosis (LB) which is the most commonly reported arthropod-borne zoonosis in North America, Europe, and the temperate zone of Asia (Steere, 2001; Masuzawa, 2004). These spirochaetes are maintained in nature in enzootic cycles involving ticks of the Ixodidae family (Kurtenbach et al., 2006; Lindgren and Jaenson, 2006). The B. burgdorferi s.l. species complex comprises 17 species (Kurtenbach et al., 2010), of which B. burgdorferi sensu stricto (s.s.), B. garinii, and B. afzelii are recognised as human pathogens (Stanek and Strle, 2009). Over the last few years, however, other Borrelia species have also been associated with human disease, e.g. B. lusitaniae. This species was isolated from 2 Portuguese patients presenting with chronic skin lesions (Collares-Pereira et al., 2004) and a vaculitis syndrome (Carvalho et al., 2008a), respectively, and its pathogenic potential was successfully established in a mouse model (Zeidner et al., 2001). B. lusitaniae is the sole species found in southern Portugal and North Africa (De Michelis et al., 2000; Younsi et al., 2005), and it has been described as being highly diverse based on phylogenies of the 5S–23S intergenic spacer region (IGS) (De Michelis et al., 2000)

∗ Corresponding author. Tel.: +351 213652600; fax: +351 213632105. E-mail addresses:, (M. Collares-Pereira). 1 Deceased in March 2009. 1877-959X/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2010.07.001

and in multilocus sequence typing (MLST) analysis (Vitorino et al., 2008). The most remarkable feature of the Borrelia genome is that it consists of a linear chromosome and several circular and linear plasmids, which may constitute up to 40% of its genomic DNA (Fraser et al., 1997; Glöckner et al., 2004). Borrelia plasmids have been designated according to their shape and size: The plasmid cp32, for instance, is circular and has a molecular weight of 32 kb, whereas lp25 is a linear plasmid of 25 kb. The plasmid repertoire of different B. burgdorferi s.s. isolates can vary considerably, but some extrachromosomal elements appear to be essential, namely the circular plasmid cp26 and the linear plasmid lp54, which are present in most isolates (Casjens, 2000; Glöckner et al., 2004). The reason for this may be that they encode molecules that are critical for Borrelia survival in vitro (Byram et al., 2004; Chaconas, 2005; Stewart et al., 2005). Nevertheless, there is considerable plasmid variation in number and size among Borrelia species and strains (Xu and Johnson, 1995). This and the fact that plasmids can be easily lost during in vitro cultivation (Norris et al., 1995) potentially constitute a drawback for using plasmid fingerprinting for species characterization in Borrelia. Furthermore, the presence of multiple plasmids of similar sizes (e.g. lp28 and cp32) may complicate the accurate determination of the plasmid content (Stevenson et al., 2000). In addition, only linear plasmids are well separated by pulsed-field gel electrophoresis (PFGE) (Xu and Johnson, 1995), and their size and low-copy number can be below PFGE sensitivity, hence the number of plasmids detected by this technique is usually an underestimation. In spite

Author's personal copy
126 L. Vitorino et al. / Ticks and Tick-borne Diseases 1 (2010) 125–128

of these limitations, plasmid fingerprinting is a useful method for a first approach to compare plasmid profiles between and within Borrelia species. Extensive plasmid content analyses have been reported for several North American B. burgdorferi s.s. strains (Casjens et al., 2000; Palmer et al., 2000) and some European B. garinii and B. afzelii isolates (Glöckner et al., 2004, 2006). The present study was undertaken to elucidate the plasmid profile for 2 Portuguese B. lusitaniae strains, one isolated from a human patient and the other one from a questing tick. Materials and methods Strain cultivation B. lusitaniae strains PoTiBL37 and PoHL1 were grown in liquid Barbour–Stoenner–Kelly-H (BSK-H) medium (Sigma® ) supplemented with gelatin pH 7.6, at 32 ◦ C. Cultures were examined weekly for motile spirochaetes by dark-field microscopy, until the density reached about 1 × 106 bacteria per ml (2–3 weeks). Low passages (≤20) have been used for both strains. For comparative purposes, the Portuguese B. garinii strains (PoTiBGr4, PoTiBGr6, and PoTiBGr20) were also used in this study. DNA extraction DNA from some passages of B. lusitaniae and B. garinii strains was firstly extracted in agarose plots according to Chu et al. (1986). Since this approach displayed low sensitivity to detect Borrelia plasmids, the following methodology was used. Around 100 ml of high density culture was spun down at 8000 rpm for 20 min, resuspended in TES (50 mM Tris–HCl, pH 8.0; 50 mM EDTA, pH 8.0; 15% sucrose) with 200 l of lysozyme (50 mg per ml), and incubated 15 min on ice. 5% sodium deoxycholate and 70 l of DEPC (diethyl pyrocarbonate) were added, and the mixture was left at room temperature for 10 min. After the addition of ammonium acetate (7.5%), the cells were incubated on ice for 15 min and centrifuged for 10 min at 10,000 rpm. The supernatant was recovered, and the nucleic acids were precipitated by adding 0.6 volumes of isopropanol and centrifugation for 6 min at the same speed. The pellet was resuspended in TE buffer and treated with RNAse (10 mg per ml) for 1 h at 37 ◦ C. To remove RNAse, extraction with chloroform was performed twice. The DNA was precipitated by adding 1/10 volume of 2.5 M NaCl and 2.5 volumes of absolute ethanol, left at 20 ◦ C for 1 h, and afterwards centrifuged at 10,000 rpm for 6 min. Finally, DNA was washed in 70% ethanol, dried at 65 ◦ C for 10 min, and dissolved in 150 l of TE buffer. PFGE Counter-clamped homogeneous electric field electrophoresis (CHEF-PFGE) was performed using the Gene Navigator System (Pharmacia). Agarose gels (1%) (Seakem GTG, FMC) in 0.5× TBE Low EDTA (45 mM Tris, 45 mM Boric Acid, 1 mM EDTA, pH 8.0) were used and 10 l of DNA was applied to each well. The electrophoresis was performed at a constant voltage (220 V) at 8 ◦ C using the following parameters: 0.3 s, 1 h; 0.5 s, 1 h; 0.7 s, 1 h; 2.0 s, 5 h; 4.0 s, 5 h. /HindIII ladder marker (125–23,130 bp; New England Biolabs) and Low Range PFG marker (2.03–194 kb; New England Biolabs) were used as molecular size markers (Zé-Zé et al., 1998). Different pulse-field parameters (0.5 s, 4 h; 1.0 s, 4 h; 2.0 s, 4 h; 4.0 s, 4 h; 8.0 s, 5 h; 10.0 s, 3 h) were also used to confirm the obtained plasmid profiles. The gels were stained with ethidium bromide and photographed under UV light (KODAK DC 290). The relative sizes of the identified plasmid fragments were

estimated using the KODAK 1D 2.0 (Kodak) software (Zé-Zé et al., 2008). Plasmid-specific PCR PCR primers to amplify the vls locus (Zhang et al., 1997) were used on B. lusitaniae strains. Two microliters ( l) of boiled culture were used as DNA template in a 50- l PCR mixture containing 1 pmol of each primer, 5 mM (each) dATP, dGTP, dCTP, and dTTP (Invitrogen), 1.75 U of Taq polymerase (Invitrogen), 3 mM MgCl2 , 0.5× BSA, and 1× Taq buffer. The PCR parameters used were as follows: an initial denaturation step at 94 ◦ C for 5 min, 35 cycles at 94 ◦ C for 1 min, 60 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by a final extension step at 72 ◦ C for 5 min. Southern hybridization To detect the linear plasmid that contains the gene encoding the OspA in B. lusitaniae PoTiBL37 and PoHuL1 strains, specific primers, 5 -GAC ACT GCC TCT GGT GAT AGC-3 (forward), 5 -CTT TCC CTT TTC CTT CTT TTG-3 (reverse), were designed against conserved regions of ospA from B. lusitaniae sequences available from GenBank. The amplification was carried out using Digoxigenin (DIG)-high prime DNA labelling kit (Roche) to probe the PCR product. The membrane was prehybridized in hybridization solution [5× standard sodium citrate (SSC), 0.1% sarcosil, 0.02% sodium dodecyl sulphate (SDS), 1× blocking solution, 50% formamide] for 1 h at 37 ◦ C. The denatured DIG-labelled DNA probe (10 min at 100 ◦ C) was added to the hybridization solution, and the membrane was incubated overnight at the same temperature. The membrane was washed twice in 2× SSC, 0.1% SDS at room temperature, twice in 0.5× SSC, 0.1% SDS at 65 ◦ C, rinsed in washing buffer (0.1 M maleic acid; 0.15 M NaCl, pH 7.5; 0.3% Tween 20), incubated for 30 min in blocking solution (provided by the manufacturer), and then in antibody solution (Anti-Digoxigenin-AP 1:10,000 in blocking solution) for 30 min. The membrane was washed twice in washing solution and equilibrated in detection buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5). The membrane was covered with chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD), incubated for 5 min at room temperature and then at 37 ◦ C for 10 min to enhance the luminescent reaction. Exposure to X-ray film lasted no longer that 45 min. Results The plasmid profiles of 2 B. lusitaniae strains, PoTiBL37 and PoHL1, isolated from a vector tick collected in Portugal and from a skin biopsy of a Portuguese patient, respectively, were determined by PFGE (Fig. 1). Several low passages of both strains were used to detect potential plasmid loss due to subsequent subculturing. Few plasmids were detected compared to the number of plasmids known for the B31 strain (12 linear and 9 circular plasmids) as follows: only 5 plasmids in PoTiBL37 (19 kb, 26 kb, 64 kb, 71 kb, and a higher one above the resolution limit of the ladder used) and 6 in PoHL1 (19 kb, 25 kb, 64 kb, 70 kb, 76 kb, and a higher one above the resolution limit of the ladder used). Plasmid profiles in PoTiBL37 and PoHL1 were almost identical, except for the largest plasmids that differ in number and size. The mean size of each fragment/plasmid was estimated from several gels by linear interpolation with 2 flanking size standards (Heath et al., 1992) using Kodak 1D 2.0 software. Profiles obtained with different running parameters displayed the same plasmid number and sizes which indicated that we detected linear plasmids (Beverly, 1988; Smith and Condemine, 1990) and that the additional plasmid found in PoHL1 was not an artifact (data not shown).

Author's personal copy
L. Vitorino et al. / Ticks and Tick-borne Diseases 1 (2010) 125–128 127

Fig. 1. Plasmid profiles of B. burgdorferi sensu lato determined by PFGE. 1, 13: /HindIII ladder; 2, 12: low range ladder; 3–5: B. lusitaniae PoHL1, passages P6, P20, and P9, respectively; 6–8: B. lusitaniae PoTiBL37, passages P8, P16, and P17, respectively; 9: B. garinii PoTiBGr20; 10: B. garinii PoTiBGr4; 11: B. garinii PoTiBGr6. B. garinii isolates were used for comparison.

Although a plasmid of 28 kb was not detected in the plasmid profiles, primers designed to amplify the vls locus (encoded on lp28-1 and associated with infectivity of B. burgdorferi s.s.) produced a weak PCR product in PoHL1 (data not shown). In addition, we investigated the location of the ospA gene by southern hybridization. The size of the plasmid that hybridized with the ospA probe was 70 kb (Fig. 2). Discussion The number of plasmids reported here for the 2 Portuguese B. lusitaniae strains is probably lower than the total number of plasmids, mainly because (i) some of them must be in such low-copy number that they could not be detected by PFGE, (ii) the circular plasmids are difficult to detect by this approach, and (iii) we cannot rule out that some plasmids could have been lost during

Fig. 2. OspA hybridization within plasmid profiles. 1, 13: /HindIII ladder; 2, 12: low range ladder; 3–5: B. lusitaniae PoHL1, passages P6, P20, and P9, respectively; 6–8: B. lusitaniae PoTiBL37, passages P8, P16, and P17, respectively; 9: B. garinii PoTiBGr20; 10: B. garinii PoTiBGr4; 11: B. garinii PoTiBGr6. OspA probe is specific for B. lusitaniae, thus no signal was detected in B. garinii isolates.

the DNA extraction, despite using a method that enhances plasmid DNA isolation. In other Borrelia species, very small plasmids in the size range below 10 kb have been described. But even when using another DNA extraction technique (Chu et al., 1986) and completely different PFGE runs including very short pulse times to detect smaller molecules (Smith and Cantor, 1987), the number and size of the detected plasmids remained the same. The plasmid profiles of B. lusitaniae strains ranged from 19 kb to 76 kb, with a higher one above the resolution limit of the ladder used. Thus, it is not possible to accurately estimate its size (Fig. 1). The size range of the plasmids detected in B. lusitaniae greatly differed from the ones detected by PGFE in B. burgdorferi s.s., B. afzelii, and B. garinii, where the largest plasmid was 57.7 kb (Xu and Johnson, 1995). This result is in agreement to both the largest plasmid size of lp60 (GenBank accession no. NC 008277) reported in B. afzelii and other plasmids between 20 kb and 40 kb also present in the genome. In the case of B. lusitaniae, not only the number of plasmids is quite low, but it seems that this species lacks all the small plasmids described so far for the other Borrelia with medical importance in LB. In view of the fact that most of the Borrelia virulence genes are located on plasmids (e.g., genes that encode for OspC, Erps, and CRASP proteins), the low number of these genetic elements in B. lusitaniae strains could be associated with the lower infectivity reported for this species, since only 2 human isolates have been obtained so far (Collares-Pereira et al., 2004; Carvalho et al., 2008a). Furthermore, the number (0.04/100,000 inhabitants) of reported cases in Portugal (Carvalho et al., 2008b) is not as high as in other European countries (Stanek and Strle, 2009) despite a high reported infection prevalence of B. lusitaniae in ticks (De Michelis et al., 2000). Another approach to study the plasmid content is to use plasmid-specific primers and to perform PCR (Purser and Norris, 2000; Iyer et al., 2003). The lp28-1-like plasmid was not detected in both isolates. The vlsE locus and the contiguous array of vls silent cassettes are located adjacent to one another in the linear plasmid lp28-1 (Zhang et al., 1997). Given that the lack of this plasmid has been correlated with the low or intermediate infectivity of B. burgdorferi s.s. clones (Purser and Norris, 2000) and the ability to colonize tissues other than the joints (Labandeira-Rey and Skare, 2001), B. lusitaniae Portuguese isolates could have a low or an intermediate infectivity phenotype due to the absence of the vls locus. However, primers designed to amplify this gene, based on B. burgdorferi s.s. genome sequencing data, yielded a weak PCR amplicon in the PoHL1 strain, indicating that the vls locus is present in that isolate, but being located in a different genomic region than the lp28-1. There are also other plasmids that have been described as essential for Borrelia survival, such as the lp54 and the cp26, encoding the OspA and OspC proteins, respectively (Barbour, 1988; Marconi et al., 1993; Sˇ dziene et al., 1993; Stewart et al., 2005). Both genes a were detected by PCR in B. lusitaniae strains, using primers designed specifically for this genospecies. The absence of a plasmid in the size range of lp54 prompted us to investigate which plasmid would encode ospA. Surprisingly, the probe hybridized with a plasmid of 70 kb which differs considerably from the plasmid known to encode this gene in other Borrelia species (lp54). Taken together, these results underpin that B. lusitaniae is different from other Borrelia species that have been associated with pathogenicity in plasmid number and content (Palmer et al., 2000). As already reported by some authors (De Michelis et al., 2000; Younsi et al., 2005; Vitorino et al., 2008), B. lusitaniae strains display a high intraspecific genetic diversity. Hence, it will be of great interest to evaluate the plasmid profile of other Portuguese B. lusitaniae strains. This species is phylogenetically very distant from

Author's personal copy
128 L. Vitorino et al. / Ticks and Tick-borne Diseases 1 (2010) 125–128 Heath, J.D., Perkins, J.D., Sharma, B., Weinstock, G.M., 1992. NotI cleavage map of Escherichia coli K12 strain MG1655. J. Bacteriol. 174, 558–567. Iyer, R., Kalu, O., Purser, J., Norris, S., Stevenson, B., Schwartz, I., 2003. Linear and circular plasmid content in Borrelia burgdorferi clinical isolates. Infect. Immun. 71, 3699–3706. Kurtenbach, K., Hoen, A.G., Bent, S.J., Vollmer, S.A., Ogden, N.H., Margos, G., 2010. Population biology of Lyme borreliosis spirochetes. In: Robinson, D.A., Falush, D., Feil, E.J. (Eds.), Bacterial Population Genetics in Infectious Disease. John Wiley & Sons, Inc.. Kurtenbach, K., Hanincová, K., Tsao, J.I., Margos, G., Fish, D., Ogden, N.H., 2006. Fundamental processes in the evolutionary ecology of Lyme borreliosis. Nat. Rev. Microbiol. 4, 660–669. Labandeira-Rey, R., Skare, J.T., 2001. Decreased infectivity in Borrelia burgdorferi strain B31 is associated with loss in linear plasmid 25 or 28-1. Infect. Immun. 69, 446–455. Lindgren, E., Jaenson, T.G.T., 2006. Lyme borreliosis in Europe: Influences of Climate and Climate Change, Epidemiology, Ecology and Adaptation Measures. World Health Organization, Copenhagen, Denmark, 34 pp. Marconi, R.T., Samuels, D.S., Garon, C.F., 1993. Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes. J. Bacteriol. 175, 926– 932. Masuzawa, T., 2004. Terrestrial distribution of the Lyme borreliosis agent Borrelia burgdorferi sensu lato in East Asia. Jpn. J. Infect. Dis. 57, 229–235. Norris, S.J., Howell, J.K., Garza, S.A., Ferdows, M.S., Barbour, A.G., 1995. High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Infect. Immun. 63, 2206–2212. Palmer, N., Fraser, C., Casjans, S., 2000. Distribution of twelve linear extrachromosomal DNAs in natural isolates of the Lyme disease spirochetes. J. Bacteriol. 182, 2481–2491. Purser, J.E., Norris, S.J., 2000. Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proc. Natl. Acad. Sci. U.S.A. 97, 13865–13870. Richter, D., Matuschka, F.R., 2006. Perpetuation of the Lyme disease spirochete Borrelia lusitaniae by lizards. Appl. Environ. Microbiol. 72, 4627–4632. Sˇ dziene, A., Wilske, B., Ferdows, M.S., Barbour, A.G., 1993. The cryptic ospC gene a of Borrelia burgdorferi B31 is located on a circular plasmid. Infect. Immun. 61, 2192–2195. Smith, C.L., Cantor, C.R., 1987. Purification, specific fragmentation, and separation of large DNA molecules. Methods Enzymol. 155, 449–467. Smith, C.L., Condemine, G., 1990. New approaches for physical mapping of small genomes. J. Bacteriol. 172, 1167–1172. Stanek, G., Strle, F., 2009. Lyme borreliosis: a European perspective on diagnosis and clinical management. Curr. Opin. Infect. Dis. 22, 450–454. Steere, A.C., 2001. Lyme disease. N. Engl. J. Med. 345, 115–125. Stevenson, B., Zückert, W.R., Akins, D.R., 2000. Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species. J. Mol. Microbiol. Biotechnol. 2, 411–422. Stewart, P.E., Byram, R., Grimm, D., Tilly, K., Rosa, P.A., 2005. The plasmids of Borrelia burgdorferi: essential genetic elements of a pathogen. Plasmid 53, 1–13. Vitorino, L., Margos, G., Feil, E.J., Collares-Pereira, M., Zé-Zé, L., Kurtenbach, K., 2008. Fine-scale phylogeographic structure of Borrelia lusitaniae revealed by multilocus sequence typing. PLoS ONE 3 (12), e4002, doi:10.1371/journal.pone.0004002. Xu, Y., Johnson, R.C., 1995. Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains. J. Clin. Microbiol. 33, 2679–2685. Younsi, H., Sarih, M.H., Jouda, F., Godfroid, E., Gern, L., Bouattour, A., Baranton, G., Postic, D., 2005. Characterization of Borrelia lusitaniae isolates collected in Tunisia and Morocco. J. Clin. Microbiol. 43, 1587–1593. Zeidner, N.S., Núncio, M.S., Schneider, B.S., Gern, L., Piesman, J., Brandão, O., Filipe, A.R., 2001. A Portuguese isolate of Borrelia lusitaniae induces disease in C3H/HeN mice. J. Med. Microbiol. 50, 1055–1060. Zé-Zé, L., Tenreiro, R., Brito, L., Sanfos, M.A., Paveia, H., 1998. Physical map of the genome of Oenococcus oeni PSU4 and localization of genetic markers. Microbiology 144, 1145–1156. Zé-Zé, L., Chelo, I.M., Tenreiro, R., 2008. Genome organization in Oenococcus oeni strains studied by comparison of physical and genetic maps. Int. Microbiol. 11, 237–244. Zhang, J.R., Hardham, J.M., Barbour, A.G., Norris, S.J., 1997. Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89, 275–285.

B. burgdorferi s.s., and its distinct plasmid content is consistent with this notion. The divergent plasmid content of B. lusitaniae may reflect its adaptation to particular hosts, such as lizards, which are now believed to be important reservoir hosts of B. lusitaniae (Dsouli et al., 2006; Richter and Matuschka, 2006). Acknowledgments The ICAT/FCUL research team involved in this work strongly acknowledges its scientific leader, Prof. Rogério Tenreiro, for the general scientific supervision and engagement in providing the needed resources and facilities. We are also very grateful to Dr. Susana Baptista for providing the tick isolate (PoTiBL37) used in this study. L. Vitorino is the recipient of FCT research grants SFRH/BD/10676/2002. References
Barbour, A.G., 1988. Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. J. Clin. Microbiol. 26, 475–478. Beverly, S.M., 1988. Characterization of ‘unsusual’ mobility of large circular DNAs in pulsed-field gradient electrophoresis. Nucleic Acids Res. 16, 925–939. Byram, R., Stewart, P.E., Rosa, P., 2004. The essential nature of the ubiquitous 26kilobase circular replicon of Borrelia burgdorferi. J. Bacteriol. 186, 3561–3569. Carvalho, I.L., Fonseca, J.E., Marques, J.G., Ullmann, A., Hojgaard, A., Zeidner, N., Núncio, M.S., 2008a. Vasculitis-like syndrome associated with Borrelia lusitaniae infection (case report). Clin. Rheumatol. 27, 1587–1591. Carvalho, I.L., Milhano, N., Santos, A.S., Almeida, V., Barros, S.C., De Sousa, R., Núncio, M.S., 2008b. Detection of Borrelia lusitaniae, Rickettsia sp. IRS3, Rickettsia monacensis, and Anaplasma phagocytophilum in Ixodes ricinus collected in Madeira Island, Portugal. Vector Borne Zoonotic Dis. 8, 575–579. Casjens, S., 2000. Borrelia genomes in the year 2000. J. Mol. Microbiol. Biotechnol. 2, 401–410. Casjens, S., Palmer, N., van Vugt, R., Huang, W.M., Stevenson, B., Rosa, P., Lathigra, R., Sutton, G., Peterson, J., Dodson, R.J., Haft, D., Hickey, E., Gwinn, M., White, O., Fraser, C.M., 2000. A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 35, 490–516. Chaconas, G., 2005. Hairpin telomeres and genome plasticity in Borrelia: all mixed up in the end. Mol. Microbiol. 58, 625–635. Chu, G., Vollrath, D., Davsi, R.W., 1986. Separation of large DNA molecules by counter-clamped homogeneous electric fields. Science 234, 1582–1585. Collares-Pereira, M., Couceiro, S., Franca, I., Kurtenbach, K., Vitorino, L., Goncalves, L., Baptista, S., Vieira, M.L., Cunha, C., 2004. First isolation of Borrelia lusitaniae from a human patient. J. Clin. Microbiol. 42, 1316–1318. De Michelis, S., Sewell, H.-S., Collares-Pereira, M., Santos-Reis, M., Schouls, L.M., Benes, V., Holmes, E.C., Kurtenbach, K., 2000. Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal. J. Clin. Microbiol. 38, 2128–2133. Dsouli, N., Younsi-Kabachii, H., Postic, D., Nouira, S., Gern, L., Bouattour, A., 2006. Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi sensu lato (Spirochaetaceae) in Tunisia. J. Med. Entomol. 43, 737–742. Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G., Clayton, R., Lathigra, R., White, O., Ketchum, K.A., Dodson, R., Hickey, E.K., Gwinn, M., Dougherty, B., Tomb, J.F., Fleischmann, R.D., Richardson, D., Peterson, J., Kerlavage, A.R., Quackenbush, J., Salzberg, S., Hanson, M., van Vugt, R., Palmer, N., Adams, M.D., Gocayne, J., Weidman, J., Utterback, T., Watthey, L., McDonald, L., Artiach, P., Bowman, C., Garland, S., Fuji, C., Cotton, M.D., Horst, K., Roberts, K., Hatch, B., Smith, H.O., Venter, J.C., 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580–586. Glöckner, G., Lehmann, R., Romualdi, A., Pradella, S., Schulte-Spechtel, U., Schilhabel, M., Wilske, B., Suhnel, J., Platzer, M., 2004. Comparative analysis of the Borrelia garinii genome. Nucleic Acids Res. 32, 6038–6046. Glöckner, G., Schulte-Spechtel, U., Schilhabel, M., Felder, M., Sühnel, J., Wilske, B., Platzer, M., 2006. Comparative genome analysis: selection pressure on the Borrelia vls cassettes is essential for infectivity. BMC Genomics 7, 211.…...

Similar Documents

Free Essay

Theory of Strain

...Theory of Strain The strain theory explains delinquency as being caused by the strain or frustration of not having an equal opportunity or means to achieve commonly shared goals such as economic or social success. Persons with little formal education and few economic resources are denied the ability to acquire the goals of American society, thus producing a sense of alienation, hopelessness and frustration. Because opportunities for success are more open for the middle – and upper classes, strain is experienced most by those in the lower socioeconomic class, where quality education and employment opportunities are more limited. Strain is more common among lower-class persons, who live in inner-city urban areas that are characterized more by social problems and crime. As a way to enhance educational and employment opportunities and reduce delinquency, the government funded policies such as “Head Start” and job programs for the lower-class youths. The Head Start program is a federal program for preschool children three to five years of age in low-income families. This program promotes school readiness by enhancing the social and cognitive development of children through the provision of educational, health, nutritional, social and other services to enrolled children and families. They engage parents in their children’s learning and help them in making progress toward their educational, literacy and employment goals. Significant emphasis is placed on the involvement......

Words: 754 - Pages: 4

Premium Essay

Borrelia Burgdorferi

...All known organisms need iron to survive. All except one - Borrelia burgdorferi, the bacteria that causes Lyme disease, has evolved to use manganese instead. For almost all organisms, iron is essential in processes like making new enzymes and other proteins. Perhaps the most obvious example is its role in haemoglobin, the oxygen-transporting protein found in the red blood cells of almost all vertebrates. Researchers knew that the bacteria B. burgdorferi did not need iron, but have now discovered that it requires high levels of manganese instead. The bacteria's use of manganese rather than iron helps explain how it can evade the immune system. One of the immune system's responses against invaders is to lower the amount of iron in the blood. This starves the pathogen of the iron it needs (as well as making us feel terrible) and works against almost every pathogen. But if the invader doesn't need iron, like B. burgdorferi, this attack is quite ineffective. Antibiotics are currently the only treatment for Lyme disease. Penicillin is generally effective if Lyme disease is caught early, but it works by attacking a bacterium's cell walls - something certain forms of Borrelia don't have. The team behind the study hope knowledge of manganese's role will aid the manufacture of new treatments. "We'd like to find targets inside pathogenic cell that could thwart their growth," Valerie Culotta (John Hopkins University Bloomberg School of Public Health and involved in the......

Words: 303 - Pages: 2

Free Essay

Hamstring Strain

...Hamstring strain Have you ever experience a hamstring strain? Do you know how unsafe it can be? One of the common groups of people to go through hamstring injuries, are athletes who indulge in sports that involve jumping and explosive sprinting. In addition of hamstring injuries, they can be very frustrating to deal and treat with. The hamstrings are composing of tendons that attach three large muscles, the biceps femoris, semitendinosus, and semimembranosus. These three muscles helps one knee to bend and extend to his or her hip; however, when one or more of these muscles gets stretched too far and starts to tear, it may cause plenty of pain due to the pulled hamstring. In the circumstance of having to go through a hamstring strain, his or her may experience many symptoms and signs. For example, if his or her feels a sharp pain and possibly a popping sensation at the back of his or her leg, it is probably a sign of a hamstring strain injury. Some symptoms that one may go through during this incident, is pain in the back of his or her thigh when they flex or extend their leg, tenderness, swelling, and bruising in the affected area, and lastly weakness in his or her leg that lasts for a long time after the injury. In the event of a particularly severe strain or complete tear, the victim injured may feel a gap in the torn muscle, which may cause difficulties to run, jump, stretch and possibly walk. In......

Words: 752 - Pages: 4

Premium Essay

Strain Theory

...The strain theory foundation was laid by a well known sociologist Robert Merton. Merton believed that when groups of people do not have access to particular resources within the community, they are faced with the issues of obtaining those achievements and/or resources. Often times the process by which these achievements and/or resources are obtained will lead to criminal action. This places tremendous pressure on individuals which can be described as strain. Merton also refers to the inability to obtain the achievements and/or resources honestly as blocked opportunity structure (Agnew 2006). Over time many studies regarding strain theory have been conducted which has altered the meaning of strain theory. Merton’s foundation of strain theory was built upon by a modern sociologist named Robert Agnew. Agnew took Merton’s strain theory and changed the theory behind which Merton believed that strain led to criminal and deviant behavior. Agnew’s newly defined definition of strain theory including the inability to obtain the achievements and/or resources need; but the loss of property and negative behavior displayed by others created strains (2006). Agnew also provided a structuralized approach to the strain theory by providing categories which the different behaviors can be classified. Agnew’s classification of the various form of strain theory can be determined by the resources available to an individual, behavior of others, and the experiences (both anticipated and......

Words: 1539 - Pages: 7

Free Essay

Roark's Formulas for Stress and Strain

...Roark’s Formulas for Stress and Strain WARREN C. YOUNG RICHARD G. BUDYNAS Seventh Edition McGraw-Hill New York Chicago San Francisco Lisbon London Madrid Mexico City Milan New Delhi San Juan Seoul Singapore Sydney Toronto Cataloging-in-Publication Data is on file with the Library of Congress. Copyright # 2002, 1989 by the McGraw-Hill Companies, Inc. All rights reserved. Printed in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a data base or retrieval system, without the prior written permission of the publisher. 123456789 DOC=DOC 07654321 ISBN 0-07-072542-X The sponsoring editor for this book was Larry Hager and the production supervisor was Pamela A. Pelton. It was set in Century Schoolbook by Techset Composition Limited. Printed and bound by R. R. Donnelley & Sons Company. McGraw-Hill books are available at special quantity discounts to use as premiums and sales promotions, or for use in corporate training programs. For more information, please write to the Director of Special Sales, Professional Publishing, McGraw-Hill, Two Penn Plaza, New York, NY 10121-2298. Or contact your local bookstore. This book is printed on recycled, acid-free paper containing a minimum of 50% recycled, de-inked fiber. Information contained in this work has been obtained by The McGraw-Hill Companies, Inc.......

Words: 289317 - Pages: 1158

Premium Essay

British and Portuguese The British and Portuguese have had a tremendous impact on many things and the development of Africa. The things the Portuguese brought to Africa, mainly West Africa, consisted of the negative and positive things including culture, religion, cultivation, and slavery. British also had a significant role in many parts of the developing Africa. It seems as if the Portuguese had more of an effect on Africa than Britain did. The reason that the Portuguese had such an impact on Africa in the aspect of culture because of language, instruments, music, and dances. They are the reason why a lot of the African colonies speak Portuguese as their official language. Africans adopted the flute, clarinet, guitar, violin, cello, accordion, tambourine, and piano from the Portuguese. When the Portuguese arrived in Africa, they also brought the tradition of familiar rhythms, including the polka, the waltz, and the march, creating an entirely new kind of music in West Africa (Nosotro 1). I believe the most important tradition passed onto the Africans by the Portuguese was the religion of Christianity. Before the Portuguese most Africans didn’t practice a religion and they were killing each other off by cannibalism, murdering twins, and witchcraft. They started by going to the chiefs of villages and converting them first, hoping that the rest of the tribe would follow. Today there are others that have had an influence on religion in West Africa but it was the Portuguese who did it first......

Words: 626 - Pages: 3

Free Essay

Consumer Analysis and Profile

...Consumer Analysis and Profile Insert Name Institution Instructor Date Consumer Analysis and Profile Introduction Apparently, marketing is one among the most important aspects of business success in the world today. The needs of the customer ought to be reflected in the product that is being marketed. Worth pointing out also is the actuality that the level of satisfaction that a person obtains from a product depends on the extent to which the product meets their needs. The mobile phone industry is one among the most competitive markets in the world today. The fierce war among the key players is manifested in the competition between Apple Inc and Samsung. This paper is a customer analysis and profile paper for the case of the iPhone. The Primary Target According to the most recent research, the average owner of an iPhone is a heavy browser, who accesses the internet on a daily basis, sending mails and carrying out various financial transactions on the internet. This will be the primary target for quite some time, especially now that the world is fast embracing technology and e-commerce. The primary target constitutes the people mainly aged between ages 20and 30 years (O'Grady, 2009). The most recent research, carried out in china, arguably the biggest iPhone market, indicates that the age bracket that constitutes the biggest portion of iPhone purchases is between 18 and 25 years. This age bracket is closely......

Words: 965 - Pages: 4

Free Essay

Stress and Strain

...MIT - 16.20 Fall, 2002 Unit 2 Loads and Design Considerations Readings: Rivello (Ch. 1) Cutler book (at leisure) G 7.1 Paul A. Lagace, Ph.D. Professor of Aeronautics & Astronautics and Engineering Systems Paul A. Lagace © 2001 MIT - 16.20 Fall, 2002 Sources of Stresses and Strains Depends on type of structure Aircraft Launch Vehicles Space Structures General Other Considerations Paul A. Lagace © 2001 Unit 2 - p. 2 MIT - 16.20 Fall, 2002 Can generally divide these into: • Normal operational effects (regular use) • Environmental effects (internal stresses, material property degradation) • Isolated effects (lightning, impact) In a (large company) • “Design” group does general management • “Loads” group determines operating conditions • This is passed on to “stress” group that analyzes stresses and deformations • “Materials” group provides material ultimates, etc. ⇒ Need to understand each part NOTE: New approach in companies: IPT (Integrated Product Teams) DBT (Design Build Teams) - people from each branch including manufacturing and marketing ⇒ even more important to understand various factors Paul A. Lagace © 2001 Unit 2 - p. 3 MIT - 16.20 Fall, 2002 Factors, Margins, etc. Two important definitions for static considerations Limit Load/Stress/Condition: Maximum load/stress/condition where structure shows no permanent deformation. Ultimate Load/Stress/Condition: Maximum load/stress/condition where...

Words: 756 - Pages: 4

Free Essay

Chinese to Portuguese

...shénme? Olá ! Como te chamas? Tu bom ! Tu chamar o quê? Tiago : 我叫 Tiago Sousa. 你 呢? Wǒ jiào Tiago Sousa. Nǐ ne? Eu chamo-me Tiago Sousa. E tu? Eu chamar Tiago Sousa. Tu (partícula)? Loony : 我叫 Lonilzo,但王老师叫我 Lo. Wǒ jiào Lonilzo, dàn wáng lǎoshī jiào wǒ Lo. Eu chamo-me Lonilzo, mas a professora Wang chama-me Ló. Eu chamar Lonilzo, mas Wang Professora chamar eu Ló. Tiago : 你是葡萄牙人? Nǐ shì pútáoyá rén? Tu és Português? Tu ser Portugal população? Loony : 不,我是几内亚人. 你 呢? Bù, wǒ shì jǐnèiyǎ rén. Nǐ ne? Não, Eu sou Guineense. E tu? Não, Eu ser Guiné população. Tu (partícula)? Tiago : 我是葡萄牙人,你的父母是葡萄牙人? Wǒ shì pútáoyá rén, nǐ de fùmǔ shì pútáoyá rén? Eu sou português, os teus pais são portugueses? Eu ser Portugal população, os teus pais ser Portugal população? Loony : 我的父母是也几内亚人,你有兄弟姐妹吗? Wǒ de fùmǔ shì yě jǐnèiyǎ rén, nǐ yǒu xiōngdì jiěmèi ma? Os meus pais também são guineenses. Tu tens irmãos? Meus pais ser também Guiné população. Tu ter irmãos (abrange os 4 casos) (partícula)? Tiago : 我没有。你有一个姐姐吗? Wǒ méiyǒu. Nǐ yǒu yīgè jiějiě ma? Não. Tens uma irmã mais velha? Eu nãoter. Tu ter uma (partícula) irmã mais velha (partícula)? Loony : 我有一个姐姐。你的父母很忙吗? Wǒ yǒu yīgè jiějiě. Nǐ de fùmǔ hěn máng ma? Tenho. Os teus pais estão muito ocupados? Eu ter uma (partícula) irmã + velha. Teus pais muito ocupado (partícula)? Tiago : 是的,你是很忙吗? Shì de,  nǐ shì hěn máng ma? Sim. Tu és muito ocupado? Sim, tu......

Words: 627 - Pages: 3

Premium Essay


...Strain-Testing to Determine Genera Class Name University/School Name Cytoplasm is generally transparent, making it difficult to study under a microscope. Various methods of staining microbes are used to differentiate them and draw attention to their distinguishing features. In the case of a patient whose symptoms are consistent with an infection of the Bacillus, Escherichia and Mycoplasma genera, a sputum sample can be analyzed with staining procedures to narrow down possibilities to one genera. All tests begin by applying a sample to a slide and allowing it to dry. Most tests then require that the culture be passed over a flame to kill the cells and allow them to accept dyes. Some tests don’t require heat fixing due to the damage it causes to fine structures. The Bacillus, Escherichia and Mycoplasma genera can be distinguished by their distinct features anatomically as well as their reactions to various solutions. The acid-fast test, spore test, flagella test, and the negative strain technique, taken together, will eliminate two of the possibilities and leave one. Mycoplasma can be distinguished from other genera using the acid-fast test. In this process, sputum is applied to a slide and an initial bright red dye is applied to the sample via heat or through a solvent. The dye is then rinsed off with an acid-alcohol solution; Mycoplasma cells retain the red dye after being washed, while Escherichia and Bacillus cells would take on a blue stain. If Mycoplasma is ruled......

Words: 581 - Pages: 3

Premium Essay

Andromeda Strain

...ANDROMEDA STRAIN 1. Name and type of microorganism (actual disease mimic) indicate the name and the type of microorganism that caused the pandemic in each movie and what actual disease they mimic Code name: “andromeda”. Andromeda Strain is a deadly extraterrestrial virus. I mutates with each growth cycle, changing its biologic properties. The microbe contains chemical elements required for terrestrial life and appears to have a crystalline structure, but lacks DNA, RNA, proteins, and amino acids, yet it directly transforms matter to energy and vice versa. The scientists learn that Andromeda grows only within a narrow pH range; in a too-acid or too-basic growth medium, it will not multiply—Andromeda's pH range is 7.39–7.43, like that of human blood. Leptospirosis. 2. Origin and how it spread state where the organisms came from and explain how they were spread Project Scoop was one of several attempts to investigate a singularity, or a worm hole, that has mysteriously appeared in the Solar system. Sent specifically collect biological samples, the satellite malfunctioned upon approaching the worm hole and fell back to Earth. It was picked up and released the deadly agent. Piedmont, Utah. Further investigation determines that the bizarre deaths were caused by a crystal-structured, extraterrestrial microbe on a meteor that crashed into the satellite, knocking it from orbit. 3. Cause and......

Words: 592 - Pages: 3

Premium Essay


...comp#1 unit 4, lab#1 Ms. Anderson Profile A loving mother is always a blessing but a loving and understanding mother can be hard to find. In one instant as kid I decided to take her car for a joy ride in the middle of the. On my way home I decided to take the 1996 honda civic off-road. Before I can get ten feet from the concrete the car is stuck teetering. I go home and try to find a way to bring it back before she goes to work. Being a kid I was worried about getting in more trouble then I could hand so I grab some tools and try to pop the steering colum to make it seem like it was stolen. Maybe a year later I decide to take my grandfather’s truck for a ride. I was gone for a half an hour. In the mist of being gone I was distracted will driving and whent up a curb into the trunk of a parked car. After leaving the seen I end up going to make sure no one got hurt and was arrested. Later released to my grandparent’s custody. I end up staying there although I was dreading going home. I could not pop the steering colum so I went home and went to sleep. When morning came my stepdad went in the garage to smoke a cigarette and found the car missing. He woke up my mom and the where frantic looking for the car. After about ten minutes they come in my room asking what did I with the car I told the I didn’t know what happen. So the end up reporting the car stolen after an hour of lies I finally said “I know were the car is because I took it and got it stuck.” Even after all the...

Words: 522 - Pages: 3

Premium Essay

The Strain Theory

...The Strain Theory Professor Jaske CRM 3407 15 February 2016 Thus, the Social Learning Theory appears to be the best theory to explain how people of different cultures and origins are able to co-exist in a ship since the theory gives credibility to the ability of people to live cordially as they learn a particular living environment or system. The Strain Theory does an excellent job of explaining white collar crime, along with the contextual anomie/ strain theory. While examining this theory, it was discovered as the most compelling in our constant battle with white collar and corporate crimes. Ever since the beginning of recorded history man/woman has tried to achieve a better life, but not much has changed in today’s standards, since it can be said it is more now than ever. From television, and radio we see the American dream of home ownership, the ability to fit into the society with the latest gadgets. At the same time trying to further their education has its roots in the American dream of more pay, a better job, and the ability to retire all these things is associated with the American dream of more money. While reading this theory I took a closer look into what fuels the human mind in our society. White collar crime in most cases, according to the Strain Theory and my own beliefs is what fuel the human desire to be successful. Greed has always and always will be a driving force in our society, I see no relief in sight unless we as a society find some way......

Words: 911 - Pages: 4

Free Essay

Field Experience Analysis Profile

...Rosa Teal BSHS/355 July 18, 2016 Field Experience Agency Profile Analysis What is the agency’s name? The agency that I chose was Clayton County Community Services Authority. This agency is located in Forest Park Georgia and they offer services to residences within their county. On what days and at what times is the agency open? The agency operates Monday thru Friday from the hors of 7:00am to 5:00pm. All state and federal holidays are observed in which the agency will be closed as well as following the Clayton County School system in the event of implement weather. What type of assistance does the agency provide? This agency provides sort term financial assistance and along with this assistance there are case management service in order to help their clients to a successful place I their lives. They refer clients that in such crisis basis like eviction, homeless, foreclosures, utility disconnections as well as emergency shelter and food assistance. Though the case management they aid families in financial counseling and how to budget finances. Support services are also referred such as Social Security, Supplemental Security Income, Veterans Benefits and Unemployment Benefits. List the resources this agency has made available for marginalized populations, as: homeless, literacy, tribal issues, teen issues, mental health, veterans issues, runaways, and domestic violence. At this particular agency services that are rendered usually financial assistance but......

Words: 433 - Pages: 2

Free Essay

Study Case Analysis - Pepsico (in Portuguese)

... | | | | |Quaker Foods North America | | | | | Calculated from case Exhibit 7. texto. 7. Based on the preceding analysis, what is your overall evaluation of PepsiCo's business portfolio in 2008? Does the portfolio provide the company's shareholders with an opportunity for above-average market returns? texto. 8. What strategic actions should Indra Nooyi take to sustain the corporation's impressive financial and market performance? Should its free cash flows be used to fund additional share repurchase plans, pay higher dividends, make acquisitions, expand internationally, or for other purposes? texto. Índice Figuras Página Figura 1 – Assessment of Strategic Fit Potentials X Índice Tabelas Página Tabela 1 – Industry Attractiveness Assessment X Tabela 2 – Competitive Position/Business Strength Calculations X Tabela 3 – Estimated Cash Flow (2004-2007) X Tabela 4 – Operating Profit Margins (2004-2007) X Bibliografia 1] SINGH, N; Business Strategy - Analysis on strategies adopted by Pepsi; Rustomjee Business School; 2007. 2] THOMPSON; PETERAF; GAMBLE; STRICKLAND; Crafting & Executing Strategy - Concepts and Readings; 18.ª Edição; McGraw Hill; 2010. 3] 4]......

Words: 1590 - Pages: 7