In total, 394 questing adult blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), collected at four sites were analyzed by polymerase chain reaction (PCR) for five microbial species: Anaplasma phagocytophilum, Babesia microti, Babesia odocoilei, Borrelia burgdorferi, and the rickettsial I. scapularis endosymbiont. Identities of genetic variants of A. phagocytophilum were determined by sequencing a portion of the 16S DNA. In 55% of infected ticks (193/351), a single agent was detected. In 45% (158/351), two or more agents were detected; 37% harbored two agents and 8% harbored three agents. One male tick, collected from Ft. McCoy, WI, harbored all four microbial genera The highest rates of co-infection were by the Ixodes endosymbiont and B. burgdorferi (95/351). Two species of Babesia co-occurred within a single tick population in Wells National Estuarine Research Reserve, Wells, ME, whereas only B. odocoilei was found in other tick populations. Only A. phagocytophilum human anaplasmosis variant was detected in questing ticks from Tippecanoe River State Park, IN; from Wells; and Ft. McCoy, whereas a single infected tick from Presque Isle, PA, was infected by AP-Variant 1. Partially engorged ticks from deer in Tippecanoe River State Park were all infected with AP-Variant 1. Frequency of infections with each agent varied among populations. Rates and types of co-infections were not significantly different from random except for the Ixodes endosymbiont and B. burgdorferi in male ticks, which co-occurred less frequently than expected. Thus, I. scapularis hosts an array of pathogenic and symbiotic agents and potential evidence of interactions among microbial species was observed.

In 1994 and 1995, 8 cases of human monocytic ehrlichiosis were confirmed. These cases originated from southern counties where the putative tick vector Ambylomma americanum (L.) is well established. To confirm the presence of Ehrlichia chaffeensis in ticks in southern Indiana and to determine the minimum infection rate, specimens of A. americanum were collected from 5 counties (7 sites). Nucleic acid was isolated from 88 pools of ticks (430 individuals) using an optimized phenol/CTAB (cetyltrimethylammonium bromide) extraction procedure and subjected to polymerase chain reaction analysis using species-specific 16S rRNA gene bacterial primers. Twenty-one of 88 pools (a minimum of 21 of 430 individuals) were positive for the presence of E. Chaffeensis, yielding an average minimum infection rate of 4.9%. Minimum infection rates at individual sites ranged from 0 to 9.4%. These data extend the known distribution of the bacterium to 3 southern counties of Indiana and suggest a higher prevalence of E. chaffeensis than previously reported for Missouri, North Carolina, or Kentucky.

The vector competency of Ixodes cookei Packard and Amblyomma americanum (L.) for Borrelia burgdorferi was studied using Syrian hamsters. Ixodes dammini Spielman, Clifford, Piesman & Corwin were used as controls. Darkfield and immunofluorescent examinations of midgut diverticula revealed B. burgdorferi spirochetes in 32 of 36 (88.9%) I. dammini larvae, 5 of 36 (13.9%) I. cookei larvae, and 7 of 36 (19.4%) A. americanum larvae within 48 h after feeding on infected Syrian hamsters. B. burgdorferi were also observed in the midguts of 94 of 107 (87.8%) I. dammini nymphs that developed from the fed larvae. However, none of 30 I. cookei nymphs was positive for spirochetes and only 1 of 60 (1.7%) A. americanum nymphs was found positive for B. burgdorferi. Nymphs of each tick species, reared from larvae that had fed on infected hamsters, were allowed to feed on uninfected hamsters to determine their ability to transmit B. burgdorferi. Transmission was demonstrated only by I. dammini nymphs.

To monitor the percentage and stability of Ehrlichia chaffeensis-infected ticks in southern Indiana over time, pools of Amblyomma americanum (L.) ticks were screened for infection in southern Indiana for a 2nd time. Nested polymerase chain reaction (with 6% DMSO included only in the 2nd reaction) was performed on 920 ticks in pools of 5 individuals from 9 sites (5 sites previously examined and 4 new ones) in 6 counties. The average minimum infection rate for all sites for 1997 was 1.6%, lower than that of 4.9% previously observed for 1995. However, when only the 5 sites that were positive for infected ticks in 1995 were reexamined, the average minimum infection rate was even more disparate (1.4% in 1997 and 5.1% in 1995). To correlate the presence of infected ticks with the presence of exposed deer, which serve as a reservoir, dried blood samples collected from hunter-killed deer at 2 locations in southern Indiana were tested for E. chaffeensis-reactive antibodies using an indirect immunofluorescent assay. Antibodies were detected in 45 and 47% of 98 samples examined from the 2 stations. These data provide support to our previous report of a population of E. chaffeensis-infected A. americanum in southern Indiana and the high proportion of deer previously exposed to E. chaffeensis suggests a stable maintenance of E. chaffeensis in this tick-vertebrate zoonotic system.

Collection records for the adult black legged tick, Ixodes scapularis Say, in Indiana for the period 1991-1994 are presented and a new, established population of Borrelia burgdorferi-infected I. scapularis is described. The number of I. scapularis adults collected in Indiana increased progressively from 19 in 1991 to > 200 in 1994, and the number of Indiana counties reporting at least 1 adult increased from 13 to 29. Also, during this period, 4 countries in northwestern Indiana yielded > 10 specimens each, and B. burgdorferi-infected ticks were collected in 2 of these counties. An established population of I. scapularis, as evidenced by the presence of questing larvae, nymphs, and adults, was discovered in Jasper County in 1993. Twelve of 39 adults (31%) and 4 of 44 nymphs (9%) collected with cloth drags were infected with B. burgdorferi. Three of 49 (6%) white-footed mice, Peromyscus leucopus, collected from the site were also infected with B. burgdorferi. We believe that this focus was established at least 8 yr ago, and that a tick originating from this focus was responsible for a case of Lyme disease reported from this county in 1985.