Broad diversity of host responses of the white-footed mouse Peromyscus leucopus to Borrelia infection and antigens

Abstract

Peromyscus leucopus, the white-footed mouse, is one of the more abundant mammals of North America and is a major reservoir host for at least five tickborne diseases of humans, including Lyme disease and a newly-recognized form of relapsing fever. In comparison to Mus musculus, which is not a natural reservoir for any of these infections, there has been little research on experimental infections in P. leucopus. With the aim of further characterizing the diversity of phenotypes of host responses, we studied a selection of quantitative traits in colony-bred and -reared outbred P. leucopus adults that were uninfected, infected with the relapsing fever agent Borrelia hermsii alone, or infected after immunization with Lyme disease vaccine antigen OspA and keyhole limpet hemocyanin (KLH). The methods included measurements of organ weights, hematocrits, and bleeding times, quantitative PCR for bacterial burdens, and enzyme immunoassays for serum antibodies against both the immunization proteins and cellular antigens of the infecting organism. The results included the following: (i) uninfected animals displayed wide variation in relative sizes of their spleens and in their bleeding times. (ii) In an experiment with matched littermates, no differences were observed between females and males at 7 days of infection in bacterial burdens in blood and spleen, relative spleen size, or antibody responses to the B. hermsii specific-antigen, FbpC. (iii) In studies of larger groups of males or females, the wide variations between bacterial burdens and in relative spleen sizes between individuals was confirmed. (iv) In these separate groups of males and females, all animals showed moderate-to-high levels of antibodies to KLH but wide variation in antibody levels to OspA and to FbpC. The study demonstrated the diversity of host responses to infection and immunization in this species and identified quantitative traits that may be suitable for forward genetics approaches to reservoir-pathogen interactions.

Keywords: Lyme disease; Relapsing fever; Reservoir; Tickborne; Vaccine; Wildlife.

Figure 1

Four scatter plots of the relationships between body mass and either body length (panel A), liver mass (B), heart mass (C), or spleen mass (D) of 28 uninfected male P. leucopus (experiment 1). The least-squares linear regression line, the number (n) of animals in the dataset, the coefficient of determination (R²), and the power equation for the trend line (where x is the x-axis value and y is the y-axis value) are shown.

Figure 2

Four scatter plots of the relationships between final (day 7) body mass and either initial (day 0) body mass (panel A), liver mass (B), heart mass (C), or spleen mass (D) of 7 female and 7 littermate-matched P. leucopus infected with B. hermsii on day 0 (experiment 2). The least-squares linear regression line, the number (n) of animals in the dataset, coefficient of determination (R²), and the power equation for the trend line (where x is the x-axis value and y is the y-axis value) are shown. In panel A the female and male animals are distinguished by symbols and colors, as shown, and by separate regression lines. In the other panels females and males are not distinguished.

Figure 3

Scatter plot of log-transformed normalized B. hermsii genome copies in spleen on whole blood copies in 30 male P. leucopus at day 7 of infection (experiment 3). Spirochete burdens were estimated by species-specific quantitative PCR (qPCR). The least-squares linear regression with 95% confidence limits and coefficient of determination (R²) are shown.

Figure 4

Combined frequency histograms and scatter plots of the binding of serum antibodies of 30 male P. leucopus (experiment 3) to OspA, FbpC, GlpQ, and keyhole limpet hemocyanin (KLH) proteins. The animals were immunized with OspA and KLH on day 0, infected with B. hermsii on day 21, and euthanized for blood and tissue collection on day 28. Antibody concentrations of the different specificities were determined by enzyme immunoassay (EIA) as described in text. In the histograms the x axes indicate relative EIA values of optical density at 450 nm and the y axes indicate relative counts. In the scatter plots both the x and y axes indicate the relative EIA values in a pairwise fashion. For x axes values rise from left to right, and for y axes values rise from bottom to top of the graphs.

Figure 5

Selective characteristics of 30 female (panel A; experiment 4) and 30 male (panel B; experiment 3) P. leucopus immunized with OspA and keyhole limpet hemocyanin (KLH) and then infected with B. hermsii. The box-whisker plots are of the relative spleen size as percentage (%) of total body mass, the burden of spirochetes in the spleen and blood as determined by qPCR of genome copies of B. hermsii, and antibody levels to KLH and OspA as determined by enzyme immunoassay with readings in optical density at 450 nm. The coefficient of variation (CV) is shown for each determination. The CV for genome copies was calculated from the original and not the log-transformed values. Each horizontal box indicates the first and third quartiles, and the indentation inside the box is the median. The 1.5× interquartile range is indicated by the horizontal line (whiskers) bisecting the box, and values outside this range are indicated by asterisks and ovals.

Figure 6

Paired bleeding times of 30 uninfected male P. leucopus (experiment 3) taken 4 days apart. The least-squares linear regression and coefficient of determination (R²) are shown.