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.

Experimental infection of mice with strains of B. burgdorferi 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 I. scapularis 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 B. burgdorferi using qPCR to confirm the infectious challenge for each mouse.

Another objective of the same study was to compare transmission of the 12 B. burgdorferi strains from infected mice to I. scapularis ticks over the first 90 days of the infection. For this reason, mice were infested with 50-100 naïve I. scapularis larval ticks at days 30, 60, and 90 post-infection (PI).

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 B. burgdorferi in mouse organs

To test for the presence and quantity of B. burgdorferi, a probe and primer assay targeting the 23S rRNA 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 23S rRNA intergenic spacer gene was 85.3% and was based on 70 necropsy samples.

A second qPCR was performed on each sample to quantify the M. musculus housekeeping gene Beta-actin 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 Beta-actin gene was 85.8% and was based on 70 necropsy samples.

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.