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Lyme borreliosis is caused by infection with the spirochete Borrelia burgdorferi. Nonhuman primates inoculated with the N40 strain of B. burgdorferi develop infection of multiple tissues, including the central (CNS) and peripheral nervous system. In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins. To further investigate this, we inoculated four immunosuppressed adult Macaca mulatta with 1 million spirochetes of the N40 strain of B. burgdorferi, and compared them with three infected immunocompetent animals and two uninfected controls. The brain, spinal cord, peripheral nerves, skeletal muscle, heart, and bladder were obtained at necropsy 4 months later. The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene. Immunohistochemistry was used to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and to characterize the inflammatory response. Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury. The results showed that the number of spirochetes was significantly higher in immunosuppressed animals. B. burgdorferi in the CNS localized to the leptomeninges, nerve roots, and dorsal root ganglia, but not to the parenchyma. Outside of the CNS, B. burgdorferi localized to endoneurium and to connective tissues of peripheral nerves, skeletal muscle, heart, aorta, and bladder. Although ospA, ospB, ospC, and flagellin were present at the time of inoculation, only flagellin was expressed by spirochetes in tissues 4 months later. Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis. Plasma cells were abundant in inflammatory foci of steroid-treated animals. We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.