DOI: 10.5061/dryad.6wwpzgn1r
title: Borrelia burgdorferi strain and host sex influence pathogen prevalence and abundance in the tissues of a laboratory rodent host

description: Experimental infections with different pathogen strains give insight into
 pathogen life history traits. The purpose of our study was to compare
 variation in tissue infection prevalence and spirochete abundance among
 strains of B. burgdorferi in a rodent host (Mus musculus, C3H/HeJ). Male
 and female mice were experimentally infected via tick bite with one of 12
 strains. Ear tissue biopsies were taken at days 29, 59, and 89
 post-infection (PI), and 7 tissues were collected at necropsy. The
 presence and abundance of spirochetes in the mouse tissues were measured
 by qPCR. To determine the frequencies of our strains in nature, their
 MLSTs were matched to published datasets. For the infected mice, 56.6% of
 the tissues were infected with B. burgdorferi. The mean spirochete load in
 the mouse necropsy tissues varied 4.8-fold between the strains with the
 lowest and highest values. The mean spirochete load in the ear tissue
 biopsies decreased rapidly over time for some strains. The percentage of
 infected tissues in male mice (65.4%) was significantly higher compared to
 female mice (50.5%). The mean spirochete load in the 7 tissues was 1.5x
 higher in male mice compared to female mice; this male bias was 15.3x
 higher in the ventral skin. Across the 11 strains, the mean spirochete
 loads in the infected mouse tissues were positively correlated with the
 strain-specific frequencies in their tick vector populations. Our study
 suggests that laboratory-based estimates of pathogen abundance in host
 tissues can predict the strain composition of this important tick-borne
 pathogen in nature.
description: Experimental infection of mice with
 strains of <em>B. burgdorferi</em> via tick bite
 A total of 120
 specific-pathogen-free C3H/HeJ mice (60 male, 60 female) aged 6-8 weeks
 were used in this study. For all 12 strains, 8 mice (4 male, 4 female)
 were used for a total of 96 mice in the infected group, with an additional
 24 (12 male, 12 female) uninfected control mice. To manage the workload,
 the experiment was run in two orthogonal temporal blocks (A and B;
 separated by ~6 months). At 6-8 weeks, experimental mice were infested
 with 3 <em>I. scapularis</em> nymphs putatively infected with
 the strain of interest (See ESM Section 1), whereas control mice were
 infested with 3 uninfected nymphs. Engorged nymphs were recovered and
 tested for <em>B. burgdorferi</em> using qPCR to confirm the
 infectious challenge for each mouse. Another
 objective of the same study was to compare transmission of the 12
 <em>B. burgdorferi</em> strains from infected mice to
 <em>I. scapularis</em> ticks over the first 90 days of the
 infection. For this reason, mice were infested with 50-100 naïve
 <em>I. scapularis</em> larval ticks at days 30, 60, and 90
 post-infection (PI)<em>.</em> Ear tissue
 biopsies were taken from each mouse prior to their experimental infection
 (-2 to -1 weeks PI), and before each larval infestation (days 29, 59, and
 89 PI). Mice were anaesthetized by isoflurane prior to collection of a 2
 mm ear tissue biopsy by punch. The pre-infection tissue biopsy was taken
 from the left ear, and the 3 post-infection tissue biopsies were taken
 from the right ear. Blood samples were taken from each mouse at
 pre-infection (-2 to -1 weeks PI), day 28 PI, and at euthanasia (day 97
 PI). Pre-euthanasia blood samples were taken by submandibular bleeding
 with Goldenrod lancets (Golde et al., 2005) and 10-50 µL of blood were
 collected. At day 97 PI, all mice were euthanized by isoflurane overdose
 followed by cervical dislocation, cardiac puncture, and
 exsanguination.  Necropsy and tissue sample
 collection Mice were
 necropsied immediately following euthanasia, and seven organs/tissues were
 collected including the kidney, left ear, right ear, ventral skin (from
 belly of mouse), right rear tibiotarsal joint, heart, and bladder. Each
 mouse was processed with sterile equipment disinfected with Virkon between
 uses. Tissues were kept at 4 ºC before being trimmed and weighed to the
 nearest 0.1 mg within 24 hours of their collection. Final samples were
 held at -80 ºC prior to DNA extraction. Homogenization and DNA extraction
 of tissue samples Necropsy and ear
 biopsy samples were homogenized by micropestle or by 3.6 mm stainless
 steel beads with the Qiagen TissueLyser II. For all organs, extractions
 were done on partial samples, hereafter also referred to as tissue. DNA
 was extracted from homogenized tissue samples using the Qiagen DNEasy
 Blood and Tissue kit individual spin columns following the manufacturer’s
 instructions (See ESM Section 1). qPCR to
 measure the abundance of <em>B. burgdorferi</em> in mouse
 organs To test for the
 presence and quantity of <em>B. burgdorferi</em>, a probe and
 primer assay targeting the <em>23S rRNA</em> intergenic spacer
 gene was used as described previously (Courtney et al., 2004; see ESM
 Section 1). Quantification cycle (Cq) values were transformed using a
 synthetic gene standard (IDTDNA, gBlock; see ESM Section 2). The
 repeatability of the sample Cq for the <em>23S rRNA</em>
 intergenic spacer gene was 85.3% and was based on 70 necropsy
 samples. A second qPCR
 was performed on each sample to quantify the <em>M.
 musculus</em> housekeeping gene <em>Beta-actin</em>
 using a previously described protocol (Dai et al., 2009; see ESM Section
 1). Quantification cycle (Cq) values were transformed using a synthetic
 gene standard (IDTDNA, gBlock; see ESM Section 2). The repeatability of
 the sample (Cq) for the mouse <em>Beta-actin</em> gene was
 85.8% and was based on 70 necropsy samples.
description: IMPORTANT! In the dataset: Ear punch qPCR testing master file,
 punch 4 is the same as R_ear in the necropsy tissue dataset. If you are
 going to combine the datasets, remove punch 4, or R_Ear.