The Western Progression of Lyme Disease: Infectious and Nonclonal Borrelia burgdorferi Sensu Lato Populations in Grand Forks County, North Dakota

Animal care and use.Infection experiments were performed according to a University of North Dakota Institutional Animal Care and Use Committee (UND IACUC)-approved protocol (1101-2) at the University of North Dakota Center for Biomedical Research. Four- to 6-week-old BALB/c mice (Harlan, Madison, WI) were cared for in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines (Animal Welfare Assurance A3917-01) and the National Research Council of the National Academies Guide for the Care and Use of Laboratory Animals, 8th ed. Wild rodents were collected, euthanized, and necropsied in the field as described in the UND IACUC-approved protocol 1304-3. All efforts were made to minimize animal suffering.

Sample collection and culturing of spirochetes.Live trapping of rodents was conducted during June and July 2012 in the two largest forested areas within an otherwise agricultural landscape in Grand Forks County, i.e., Turtle River State Park (lat 47.94, long −97.50; ca. 254 ha) and Forest River Biological Station and Wildlife Management Area (lat 48.17, long −97.66; ca 349 ha). Sherman live traps (H. B. Sherman Traps, Tallahassee, FL) were baited with peanut butter and oatmeal, supplied with cotton bedding, set in the evening, and recovered in the morning. Captured mammals were identified as M. gapperi and Peromyscus spp. based on morphological characteristics. This method makes it difficult to identify Peromyscus spp. in the field; thus, Peromyscus mice were identified only to the genus level. However, all of the Peromyscus mice in this study were trapped deep in deciduous forests, a preferred habitat for Peromyscus leucopus, while Peromyscus maniculatus prefers more open terrain and/or coniferous forest, strongly suggesting the captured Peromyscus mice were P. leucopus (35; Robert Seabloom, personal communication). Peromyscus spp. and M. gapperi were euthanized with isoflurane and necropsied, and the hearts were immediately inoculated into modified Barbour-Stoenner-Kelly II (BSK-II) medium containing 6% rabbit serum and 50 μg/ml rifampin (36). Surgical tools were sterilized with 95 to 99% ethanol prior to each necropsy to prevent possible cross-contamination between animals. Three days later, uncontaminated cultures were blind passed into modified BSK-II with 6% rabbit serum without rifampin, incubated for three additional days, and then examined for spirochetes via dark-field microscopy.

Amplification and sequencing of ospA, ospC, flaB, the 16S rRNA gene, the 16S rRNA gene-ile tRNA IGS, and p66.ospA, ospC, p66, and the 16S rRNA gene-ile tRNA intergenic spacer region (IGS) were obtained under amplification conditions previously described (37), except that the annealing temperature was adjusted to 48°C (Table 1 lists the primers used). The 16S rRNA gene and flaB were amplified under the following conditions: 1 cycle of 94°C for 5 min; 40 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 1 min; and 1 cycle of 72°C for 5 min. Sequencing was performed at Davis Sequencing (Davis, CA). The chromatograms were visually inspected, and then the forward (coding) and reverse (template) strand sequences were aligned to obtain a double-stranded consensus sequence. 16S rRNA gene sequences were queried using the Ribosomal Database Project's Sequence Match (Seqmatch) program (38) and the Nucleotide Collection (nr/nt) BLAST database (blast.ncbi.nlm.nih.gov) to obtain genus and species identifications. flaB, ospA, ospC, p66, and the IGS nucleotide sequences were queried using BLAST.

NCBI Nuccore and BLAST database searches.Sequences were obtained by searching the NCBI Nuccore database (http://www.ncbi.nlm.nih.gov/nuccore) using the following terms: Borrelia plus p66, Borrelia plus ospC, Borrelia plus “outer surface protein C,” Borrelia plus ospA, Borrelia plus “outer surface protein A,” Borrelia plus “intergenic spacer region,” Borrelia plus IGS, Borrelia plus 16S plus 23S plus IGS, and Borrelia plus 16S plus 23S plus “intergenic spacer region.” A search was performed in the nonredundant protein sequence (nr) BLAST database using the complete B. burgdorferi B31 protein sequences for p66 (chromosome accession no., NC_001318.1), OspC (cp26 accession no., NC_001903.1), and OspA (lp54 accession no., NC_001857.2).

Alignments and phylogeny.Sequences for ospA, ospC, p66, and the IGSs from eastern North Dakota isolates were aligned, along with BLAST and NCBI database sequences, in ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The shaded alignment was generated using BoxShade (ExPASy [http://www.ch.embnet.org/software/BOX_form.html]). Duplicate sequences (identified as the same species and found to be 100% identical) were represented in the analyses by a single sequence. Sequences obtained from BLAST were included in analyses if the query coverage was greater than 90%. The obtained sequences were manually trimmed to conserved regions aligning with the shortest sequence obtained for the eastern North Dakota isolates. Trimmed sequences meeting the above criteria were then used in phylogenetic analyses. For OspA and OspC, a subset of sequences from each clade was chosen to create representative phylogenetic trees. Final phylogenetic analyses for OspA, OspC, and p66 were performed using the PHYLIP programs SeqBoot, Proml, and Consense (version 3.695 [http://evolution.genetics.washington.edu/phylip/]). Briefly, sequence files were put into SeqBoot and analyzed with 1,000 bootstrap replicates. The SeqBoot output file was analyzed in Proml using the Jones-Taylor-Thornton model (48) with multiple data sets, slower analysis, a random number seed of 9, data sets jumbled 5 times, and an outgroup root when appropriate. The resulting file was input into Consense to obtain a single consensus tree using the majority rule (extended) consensus type, and the tree was treated as rooted when appropriate. DNA trees were created using Dnaml in PHYLIP. The trees were visualized using FigTree (version 1.4 [http://tree.bio.ed.ac.uk/software/figtree]) and labeled using Adobe (San Jose, CA) Illustrator CS3.

OspC typing.To sequence ospC from mixed populations, PCR products for ospC from samples M6, M7, and M9 were gel purified and cloned into Escherichia coli using the pCR2.1 TOPO TA cloning kit according to the manufacturer's instructions (Life Technologies, Carlsbad, CA). Plasmids were purified using the Qiagen (Valencia, CA) Plasmid Mini Prep kit and sequenced at Davis Sequencing. OspC amino acid sequences for previously typed isolates (21, 25) were obtained from NCBI. Nucleotide sequences from the eastern North Dakota isolates were translated (ExPASy [http://web.expasy.org/translate/]) and aligned with the previously typed isolates in ClustalW2. A percent identity matrix (PIM) was obtained, and an OspC type group was assigned to each eastern North Dakota isolate if the sequence did not diverge more than 2% from a particular group (25).

MLST.MLST was performed for each eastern North Dakota sample (34). Primer sequences for the eight housekeeping genes used (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) were obtained from the Imperial College London's B. burgdorferi MLST website (http://borrelia.mlst.net). For amplification and sequencing, the outer forward and outer reverse primers for each gene were used. For each gene, a 50-μl reaction was set up using the HotStarTaq Plus master mix (Qiagen) according to the manufacturer's instructions. Primers were added to a final concentration of 1 μM, and 1 μl of purified genomic DNA was added. Previously described amplification conditions were used (34) with the following modifications: (i) the initial denaturing step was decreased to 5 min according to the HotStarTaq Plus master mix instructions, and (ii) the annealing temperature for recG was decreased to 48°C. Sequencing was performed at Eton BioSciences, Inc. (San Diego, CA). Chromatograms were inspected for double peaks, which indicated a mixed population. Chromatograms indicating mixed populations were omitted from further analyses. Single-locus queries were performed for each sequence to obtain an allele number. An allelic-profile query was performed with the available loci for each eastern North Dakota sample using the B. burgdorferi MLST website. When data for eight loci were available, the query type chosen was “exact or nearest match.” When fewer loci were available, the query type chosen was n − 1, where n is the number of available loci.

Infectivity of B. burgdorferi M3.Six female 4- to 6-week-old BALB/c mice were each subcutaneously injected with 10⁶ spirochetes/ml. Two weeks postinjection, infection was preliminarily determined by assaying pre- and postinfection sera by enzyme-linked immunosorbent assay (ELISA). Larval I. scapularis ticks were allowed to feed on infected mice as previously described (39). Briefly, approximately 200 to 300 uninfected larval I. scapularis ticks (Oklahoma State University—Stillwater) were placed on infected BALB/c mice and allowed to attach and feed. Four to 7 days after attachment, engorged larval I. scapularis ticks dropped off and were collected and placed in a humidified chamber until they molted to nymphs. The mice were euthanized 24 h after all I. scapularis ticks detached (i.e., day 8). One tibiotarsal joint and ear pinna and the heart were collected for quantitative PCR (qPCR) analysis. The second tibiotarsal joint and ear pinna were cultured in BSK-II medium with 6% rabbit serum and 50 μg/ml rifampin, blind passed, and examined by dark-field microscopy as described above. After molting, approximately 15 infected nymphal I. scapularis ticks were placed on 6 naive female 4- to 6-week-old BALB/c mice. Engorged I. scapularis ticks, mouse tissues, and cultures were treated as described above.

ELISA.Anti-B. burgdorferi IgG in sera from inoculated mice was detected as previously described (39). Briefly, 96-well plates were coated with 10 μg/ml B. burgdorferi lysate in a carbonate coating buffer and incubated overnight at 4°C. All washes were performed with phosphate-buffered saline (PBS)-Tween. Serum samples were diluted 1:100 in PBS. Anti-mouse IgG was diluted 1:5,000 in PBS. Each serum sample was analyzed in triplicate.

DNA extraction.DNA from bacterial cultures was extracted with a 25:24:1 phenol-chloroform-isoamyl alcohol extraction. The DNA was further purified with two consecutive ethanol precipitations. Total (mouse and spirochete) DNA for use in qPCR was extracted from tibiotarsal joints using a phenol-chloroform-isoamyl alcohol protocol and further purified with the Qiagen DNeasy blood and tissue kit according to the manufacturer's specifications. Total DNA from hearts and ear pinnae was extracted using a modified DNeasy blood and tissue kit protocol. Briefly, minced tissues were suspended in buffer ATL (Qiagen) with proteinase K and incubated overnight at 56°C. Samples were further purified according to the manufacturer's specifications and as previously described (39).

qPCR.The primers used in qPCR are listed in Table 1. Reactions were performed using Bio-Rad (Hercules, CA) iQ SYBR green Supermix. Mouse DNA was detected using primers for nidogen (nid1) and quantified against 500-, 50-, 5-, 0.5-, 0.05-, and 0.005-ng mouse DNA standards. The amplification conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 49°C for 1 min, 1 cycle of 95°C for 1 min, and 50°C for 1 min; and 1 cycle of 49°C for 1 min and 49 to 95°C at 0.5°C for 10 s for each step. B. burgdorferi DNA was detected using primers for recA and quantified against six B. burgdorferi DNA standards ranging in concentration from 10⁻⁶ to 10⁻¹ copy number. The amplification conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s and 50°C for 1 min; 1 cycle of 95°C for 1 min and 50°C for 1 min; 1 cycle of 50°C for 1 min and 50 to 95°C at 0.5°C for 10 s for each step. Each sample and a no-template control were run in triplicate.

Detection of B. burgdorferi DNA in nymphal I. scapularis.Total DNA was extracted from I. scapularis larvae allowed to molt to nymphs after feeding as larvae using a modified Qiagen DNeasy blood and tissue kit protocol. Ten molted I. scapularis nymphs per mouse were homogenized in buffer ATL (600 μl) with proteinase K (20 μl) and incubated overnight at 56°C. Buffer AL (200 μl; Qiagen) was added, and the tubes were vortexed and incubated at 70°C for 10 min. Wheat germ tRNA, type V (1 μl; 10-mg/ml; R-7876; Sigma-Aldrich, St. Louis, MO) was added, and the tubes were vortexed. Ethanol (230 μl; 95%) was added, and the tubes were vortexed, transferred to DNeasy spin columns, and centrifuged for 1 min at 8,000 rpm. Buffer AW1 (500 μl; Qiagen) was added, and the tubes were centrifuged for 1 min at 8,000 rpm. Buffer AW2 (500 μl; Qiagen) was then added, and the tubes were centrifuged at 14,000 rpm for 3 min. DNA was eluted from the spin column with 100 μl nuclease-free water twice. PCR was performed using primers for the I. scapularis 16S rRNA gene, the B. burgdorferi 16S rRNA gene, and B. burgdorferi flaB (Table 1) under the following amplification conditions: initial denaturation at 94°C for 3 min; 40 cycles of 94°C for 30 s, 50°C for 30 s, and 65°C for 30 s; and final elongation at 65°C for 5 min. The reactions were run on a 2.5% NuSieve gel.

Nucleotide sequence accession numbers.The sequences obtained in this study have been deposited in GenBank with the following accession numbers: KM676013, KM676014, KM676015, KM676016, KM676017, KM676018, KM676019, KM676020, KM676021, KM676022, KM676023, KM676024, KM676025, KM676026, KM676027, KM676028, KM676029, KM676030, KM676031, KM676032, KM676033, KM676034, KM676035, KM676036, KM676037, KM676038, KM676039, KM676040, KM676041, KM676042, KM676043, KM676044, KM676045, KM676046, KM676047, KM676048, KM676049, KM676050, KM676051, KM676052, KM676053, KM676054, KM676055, KM676056, KM676057, KM676058, KM676059, KM676060, KM676061, KM676062, KM676063, KM676064, KM676065, KM676066, KM676067, KM676068, KM676069, and KM676070.