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Lyme disease (Borrelia burgdorferi) and human granulocytic ehrlichiosis (Ehrlichia phagocytophila) are perpetuated in a natural cycle involving the black-legged tick (Ixodes scapularis) and its vertebrate hosts. Using I. scapularis nymphs as the mode of infectious challenge, we studied how infection with one pathogen in white-footed mice (Peromyscus leucopus) affects their ability to acquire the other agent and subsequently to infect larvae, which these agents would do in nature. Two groups of mice were infected with either B. burgdorferi or E. phagocytophila. One week later, B. burgdorferi-infected mice were challenged with E. phagocytophila, and E. phagocytophila-infected mice were challenged with B. burgdorferi. Simultaneously, two control groups of uninfected mice were infected with each agent from the same tick cohorts used on the first groups of mice. Uninfected I. scapularis larvae were fed on all mice for xenodiagnosis at weekly intervals lasting 2 months. For the B. burgdorferi challenge, all control and E. phagocytophila-infected mice acquired B. burgdorferi. However, fewer xenodiagnostic larvae acquired B. burgdorferi from mice with mixed infections compared with mice infected with B. burgdorferi only. For the E. phagocytophila challenge, all five control mice acquired E. phagocytophila, but only two of five mice infected with B. burgdorferi subsequently acquired E. phagocytophila. Consequently, mice with both infections produced fewer xenodiagnostic ticks infected with E. phagocytophila than mice infected with E. phagocytophila only. Thus, a primary infection with either B. burgdorferi or E. phagocytophila in mice inhibited transmission of a second agent, suggesting interference between these two agents.