Borrelia burgdorferi strain and host sex influence pathogen prevalence and abundance in the tissues of a laboratory rodent host

Abstract

Experimental infections with different pathogen strains give insight into pathogen life history traits. The purpose of the present study was to compare variation in tissue infection prevalence and spirochete abundance among strains of Borrelia 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 postinfection, and seven tissues were collected at necropsy. The presence and abundance of spirochetes in the mouse tissues were measured by quantitative polymerase chain reaction. To determine the frequencies of our strains in nature, their multilocus sequence types were matched to published data sets. 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. 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 seven tissues was 1.5× higher in male mice compared to female mice; this male bias was 15.3× 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. The 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.

Keywords: Borrelia burgdorferi; Lyme borreliosis; pathogen abundance; pathogen life history; tick-borne disease.

Figure 1.

The tissue spirochete load in the right ear tissue samples over the course of the infection separated by strain. The spirochete loads were originally calculated as the log10-transformed ratio of the number of B. burgdorferi 23S genes/10⁶ copies of the mouse Beta-actin gene. As B. burgdorferi and mice both have 2 copies of their respective genes, their ratio is equivalent to the number of spirochetes/diploid mouse cell. The right ear was sampled on days 29, 59, 89, and 97 post-infection (PI). The spirochete loads are EMMs based on a total of 256 infected ear samples from the 84 infected mice. Only 6 of 11 strains had positive ear samples at day 97 PI (euthanasia). The spirochete loads in the right ear tissue biopsies (days 29, 59, and 89 PI) were generally consistent for most strains except for strains 66, 57, and 54, which showed significant decreases.

Figure 2.

The prevalence (A) and abundance (B) of B. burgdorferi in the mouse necropsy tissues on day 97 PI is shown for the 7 necropsy tissues and the two sexes. The 7 mouse necropsy tissues include the kidney, left ear, right ear, ventral skin, tibiotarsal joint, heart, and bladder. (A) Infection prevalence is the proportion of necropsy tissues infected with B. burgdorferi. The infection prevalence was higher in the internal tissues (bladder, heart, tibiotarsal joints) compared to external tissues (ventral skin, left ear, right ear). The infection prevalence of the tibiotarsal joint, ventral skin, and right ear was significantly higher in males compared to females. (B) Abundance is the log10 transformed ratio of the number of spirochetes / 10⁶ diploid mouse cells in the necropsy tissues. The abundance of B. burgdorferi was highest in the bladder and heart and lowest in the kidney and ears. Males had significantly more (1.45x) spirochetes in their tissues compared to females. Each bar is based on 43 male and 41 female mice, respectively. The estimated marginal means were generated from the final models from which non-significant interactions had been removed. Error bars represent the 95% confidence intervals.

Figure 3.

The prevalence (A) and abundance (B) of B. burgdorferi in the mouse necropsy tissues on day 97 PI is shown for 11 B. burgdorferi strains and the two sexes. The 7 mouse necropsy tissues include the kidney, left ear, right ear, ventral skin, tibiotarsal joint, heart, and bladder. (A) Infection prevalence is the proportion of necropsy tissues infected with B. burgdorferi. The tissue infection prevalence was significantly higher in male mice compared to female mice for all strains except strain 126. (B) Abundance is the log10-transformed ratio of the number of spirochetes / 10⁶ diploid mouse cells in the necropsy tissues. Strain 66 had the lowest tissue abundance, and strain 174 had the highest tissue abundance. Males had consistently higher tissue abundance than females for all 11 strains. The estimated marginal means were generated from the final models from which non-significant interactions had been removed. Error bars represent the 95% confidence intervals.

Figure 4.

Correlation between the spirochete load in the necropsy tissues versus the spirochete load in the third right ear biopsy across the 11 strains of B. burgdorferi. The necropsy tissue spirochete load is based on the 7 mouse tissues tested at necropsy (day 97 PI) using qPCR. The ear biopsy spirochete load is based on the third right ear biopsy (day 89 PI) using qPCR. Both values are expressed as the log10-transformed (number of spirochetes per 10⁶ diploid mouse cells). The correlation between the two variables is positive and highly significant (r = 0.923, df = 20, t = 10.73, p = 9.551*10⁻¹⁰).