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This report describes the vector competence of 3 ixodid tick species, Ixodes scapularis (Say), I. spinipalpis (Nuttall), and Dermacentor andersoni (Stiles), for Borrelia burgdorferi in Colorado. The study was based on preliminary field work performed in 6 Colorado counties, where rodents and ticks were collected and assayed for the presence of B. burgdorferi. Four of the 6 counties produced 52 rodent and 39 I. spinipalpis isolates of B. burgdorferi. Two B. burgdorferi isolates were tested under laboratory conditions and found to be infective to Imperial Cancer Research Fund (ICRF) outbred mice. The 1st, a low-passage strain originating from New York (B-31, passage 6) was used as a control, and the 2nd was isolated from ear tissue of a Neotoma mexicana (Baird) (Mexican wood rat) that was trapped in Colorado. Larvae of I. scapularis, I. spinipalpis. and D. andersoni were fed on infected mice and cultured in Barbour-Stoner-Kelly media to assay for infection at 1, 2, 3, and 4 wk after repletion. The infection rates in replete larvae. were 75, 69, and 8.5%, respectively, whereas transstadial nymphal infection rates were 80, 75, and 0%, respectively. Both I. scapularis and I. spinipalpis were shown to be competent vectors that acquired the infection from the host reservoir mice and subsequently transmitted it to naive mice. Given that I. scapularis are not found in Colorado, I. spinipalpis are restricted to the nests and burrows of rodents, and because of the semiarid environment in Colorado, the risk of human contact with B. burgdorferi appears to be low.