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.

The blacklegged tick, Ixodes scapularis Say, first reported in Indiana in 1987, has now been detected in more than half of Indiana's counties. The first case of human granulocytic ehrlichiosis (human anaplasmosis) in Indiana was reported in 2002. We now report the detection of Anaplasma phagocytophilum and Babesia odocoilei (Emerson and Wright 1968) in I. scapularis ticks collected in northern Indiana. Using polymerase chain reaction analysis, 41 of 193 adult ticks (21.2%) collected from deer were positive for A. phagocytophylum, and 22 (11.4%) were positive for Babesia sp. Restriction fragment analysis of 12, and sequencing of another five of the amplified products identified these parasites as B. odocoilei. Five ticks (2.6%) were coinfected. Eight of 68 questing adult ticks (11.8%) were positive for A. phagocytophilum; seven (10.3%) were positive for Babesia sp. Six of the latter seven positive samples were determined to be B. odocoilei by restriction fragment analysis and sequencing of two samples. None of 39 pools of nymphs was positive for Babesia sp. Three of 15 ticks (20%) collected from a dog were positive for A. phagocytophilum and three ticks (20%) were positive for Babesia sp. One was confirmed as B. odocoilei. One tick was coinfected. This is the first report of the presence of these two agents in ticks in Indiana.

BackgroundMosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence.Methodology/Principal FindingsSix lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3–5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise FST indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively.Conclusions/SignificanceWe demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).

To continue monitoring the prevalence and distribution of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaeceae) in southern Indiana, a total of 498 Amblyomma americanum (L.) ticks (262 adults and 292 nymphs) was collected from five southern Indiana counties during May and June 1998. Ticks were pooled and examined for the presence of E. chaffeensis using nested polymerase chain reaction and primers specific for the 16S rRNA gene of E. chaffeensis. The average minimum infection rate for adult ticks collected in 1998 was 3.8% (ranging from 0 to 7.7% in various counties) as compared with previous average minimum infection rates of 1.6% in 1995 and 4.9% in 1997. None of the pools of A. americanum nymphs tested positive. In addition, blood samples were collected from 325 white-tailed deer taken in Indiana and 327 taken in Ohio in November 1998. Serum samples were tested for the presence of E. chaffeensis-like organisms reactive to antibodies using an indirect immunofluorescence assay (IFA). Antibodies were found in deer from six Indiana counties where infection rates ranged from 42.6 to 66.7% and in four Ohio countries where infection rates ranged from 4.4 to 25%. The results of this study reconfirm that E. chaffeensis is well established in southern Indiana and also provide the first evidence of E. chaffeensis-like organisms infecting white-tailed deer in Ohio, suggesting the need to survey Ohio ticks for the presence of Ehrlichia.