The dynamics of Borrelia burgdorferi infections within its natural hosts are poorly understood. We necropsied four wild-caught western gray squirrels (Sciurus griseus) that were acquired during a previous study that evaluated the reservoir competence of this rodent for the Lyme disease spirochete. One animal was infected experimentally, whereas the others were infected in the wild before capture. To investigate dissemination of B. burgdorferi and concurrent histopathologic lesions in different tissues, blood specimens, synovial and cerebrospinal fluid, ear-punch biopsies, and diverse tissue samples from skin and various organs were taken and examined by culture, polymerase chain reaction, and histology. Borrelia-positive cultures were obtained from three of the squirrels, that is, from skin biopsies (7 of 20 samples), ear-punch biopsies (2 of 8), and one (1 of 5) lymph node. Sequencing of amplicons confirmed B. burgdorferi sensu stricto (s.s.) infection in 9 of 10 culture-positive samples and in DNA extracted from all 10 positive cultures. The experimentally infected squirrel yielded most of the positive samples. In contrast, bodily fluids, all other organ specimens from these animals, and all samples from one naturally infected squirrel were negative for Borrelia for both assays. None of the necropsied squirrels exhibited specific clinical signs associated with B. burgdorferi. Similarly, necropsy and histological examination of tissues indicated the presence of underlying infectious processes, none of which could be ascribed conclusively to B. burgdorferi infection. Based on these results, obtained from a small number of animals investigated at a single time point, we suggest that B. burgdorferi s.s. infection in S. griseus may result in rather localized dissemination of spirochetes, and that mild or nonclinical disease might be more common after several months of infection duration. Since spirochetes could be detected in squirrels 7–21 months postinfection, we conclude that S. griseus can infect Ixodes pacificus ticks with B. burgdorferi s.s. trans-seasonally
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