Relapsing fever spp. as well as other genetic loci (spp. were
Relapsing fever spp. as well as other genetic loci (spp. were cultivated in Barbour-Stoenner-Kelly medium (BSKII) (isolates (Ly, La, Lw, Ma, Ku, and Wi) and 18 isolates (A1CA18) were investigated. Cultures were diluted in BSKII medium, and the one with the lowest growth was added to media from which DNA was extracted. Because these spirochetes are fastidious, recovery from a single 315183-21-2 cell cannot be guaranteed. For comparison, additional spp. analyzed included Nearctic relapsing fever strains (HS1), sensu stricto (B31). Lice Lice were collected from your clothing of individuals with louseborne relapsing fever, kept in 70% ethanol, and transferred to the United Kingdom (import license not required). Total DNA was extracted by using a DNeasy Cells kit (Qiagen) from swimming pools of 4 to 6 6 lice. Ticks Ticks were collected from traditional dwellings in 4 villages, Mvumi Makulu (MK), Iringa Mvumi (IM), Ikombolinga (IK), and Mkang’wa (MA), in the Dodoma Rural Area, central Tanzania. Scoops of earth were collected and approved through a sieve, and ticks were collected into containers containing 70% ethanol. Dead ticks were imported under license (AHZ/2074A/2001/13) and heat inactivated (>80C for 30 min) before DNA extraction. After manual homogenization of 1 1 to 6 ticks with a sterile pestle, samples were digested overnight in SNET lysis buffer (20 mmol/L Tris-HCl pH 8.0, 5 mmol/L EDTA, 400 mmol/L NaCl, 1% sodium dodecyl sulfate, 55C), supplemented with proteinase K (400 g/mL final concentration). Debris was pelleted, lysate was transferred to a fresh tube, and total DNA extracted by using standard phenolchloroform extraction or the automated MagnaPure DNA extraction robot with the LC DNA isolation kit II for tissues (Roche, Lewes, UK). DNA extracted from 2 purification rounds was pooled, concentrated, and resuspended in sterile distilled water. PCR A gene and the genes, respectively (5-GTATGTTTAGTGAGGGGGGTG-3 and 5-GGATCATAGCTCAGGTGGTTAG-3 for forward and reverse, respectively, while the inner nested primers were 5-AGGGGGGTGAAGTCGTAACAAG-3 and 5-GTCTGATAAACCTGAGGTCGGA-3, again for forward and reverse). Amplicons were resolved with 1% agarose gels, bands were excised, and DNA was purified by using a Wizard SV gel and PCR clean-up system (Promega, Southhampton, UK). DNA Sequencing Purified DNA was sequenced directly or cloned into pGEMT-easy (Promega) and sequenced. Sequencing reactions were performed according to manufacturer’s recommendations by using BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Warrington, UK) and analyzed on an Applied Biosystems Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Data Analysis Nucleotide sequences were analyzed by 315183-21-2 using Chromas (version 1.45) and DNA Star software (Lasergene 6). Multiple alignments were performed by using ClustalW. Results produced by IGS fragment typing were compared with those obtained using the PLA2B gene with sequences held in GenBank. Phylogenetic Trees The phylogenetic relationships of sequence data were compared by using Mega software (version 3) and neighbor-joining methods for compilation of the tree. A bootstrap value of 250 was used to determine confidence in tree-drawing parameters. Results Clinical Isolates Cultivable isolates of were identical over the 587-bp portion of the IGS sequenced and, consequently, the Ly strain was selected to represent these isolates. This isolate has been used by others as representative for (types were compared with types ICIV, differences appeared negligible (Table 1, Figure 1). The IGS fragment sequences of type II and type I were identical. Table 1 Sequence heterogeneity among the 568- to 587-bp intragenic spacer sequence of the Borrelia duttonii/B. recurrentis group* Figure 1 Neighbor-joining phylogenetic tree (bootstrap value 250) showing clustering of intergenic spacer (IGS) fragment generated within this study and compared with IGS downloaded from GenBank. Accession nos. “type”:”entrez-nucleotide-range”,”attrs”:”text”:”DQ000277-DQ000287″,”start_term”:”DQ000277″,”end_term”:”DQ000287″,”start_term_id”:”66276901″,”end_term_id”:”66276911″ … A comparison of gene sequences confirmed 315183-21-2 the difference between the 2 groups of gene sequences, with the exception of the types, produced single.