ABSTRACTThe rodentPeromyscus leucopusis a major natural reservoir for the Lyme disease agentBorrelia burgdorferiand a host for its vectorIxodes scapularis. At various locations in northeastern United States 10 to 15 B. burgdorferistrains coexist at different prevalences in tick populations. We asked whether representative strains of high or low prevalence differed in their infections ofP. leucopus. After 5 weeks of experimental infection of groups with each of 6 isolates, distributions and burdens of bacteria in tissues were measured by quantitative PCR, and antibodies toB. burgdorferiwere evaluated by immunoblotting and protein microarray. All groups of animals were infected in their joints, ears, tails, and hearts, but overall spirochete burdens were lower in animals infected with low-prevalence strains. Animals were similar regardless of the infecting isolate in their levels of antibodies to whole cells, FlaB, BmpA, and DbpB proteins, and the conserved N-terminal region of the serotype-defining OspC proteins. But there were strain-specific antibody responses to full-length OspC and to plasmid-encoded VlsE, BBK07, and BBK12 proteins. Sequencing of additional VlsE genes revealed substantial diversity within some pairs of strains but near-identical sequences within other pairs, which otherwise differed in theirospCalleles. The presence or absence of full-length bbk07 and bbk12 genes accounted for the differences in antibody responses. We propose that forB. burgdorferi, there is selection in reservoir species for (i) sequence diversity, as for OspC and VlsE, and (ii) the presence or absence of polymorphisms, as for BBK07 and BBK12.IMPORTANCEHumans are dead-end hosts forBorreliaagents of Lyme disease (LD), and, thus, irrelevant for the pathogens’ maintenance. Many reports of human cases and laboratory mouse infections exist, but less is known about infection and immunity in natural reservoirs, such as the rodentPeromyscus leucopus. We observed that high- and low-prevalence strains ofBorrelia burgdorferiwere capable of infectingP. leucopusbut elicited different patterns of antibody responses. Antibody reactivities to the VlsE protein were as type-specific as previously characterized reactivities to serotype-defining OspC proteins. In addition, the low-prevalence strains lacked full-length genes for two proteins that (i) are encoded by a virulence-associated plasmid in some high-prevalence strains and (ii) LD patients and field-captured rodents commonly have antibodies to. Immune selection against these genes may have led to null phenotype lineages that can infect otherwise immune hosts but at the cost of reduced fitness and lower prevalence.