Kirby C. Stafford

Effect of Tick Removal on Transmission of Borrelia burgdorferi and Ehrlichia phagocytophila by Ixodes scapularis Nymphs

The effect of feeding duration on pathogen transmission was studied for individual ticks infected with either laboratory or field strains of the Lyme disease spirochete Borrelia burgdorferi and field strains of Ehrlichia phagocytophila, an agent of human granulocytic ehrlichiosis. Infected nymphal Ixodes scapularis were allowed to feed individually on mice, and equal numbers were removed at 24-h intervals for < or =96 h. Mice were assayed for infection by culture, serologic testing, and polymerase chain reaction (PCR) analysis. Fed ticks were assayed by culture or PCR analysis. Transmission of B. burgdorferi did not occur during the first 24 h among 66 attempts, with maximum transmission occurring between 48 and 72 h. A model estimating the probability of infection from individual ticks removed by patients in a Lyme disease-endemic area yielded an overall probability of 4.6%. Infected I. scapularis nymphs transmitted E. phagocytophila within 24 h in 2 of 3 attempts, which indicates that daily tick removal may not be adequate to prevent human infection with this agent.

Genetic variants of Ehrlichia phagocytophila, Rhode Island and Connecticut.

Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease.

Reduced abundance of Ixodes scapularis (Acari: Ixodidae) and the tick parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) with reduction of white-tailed deer.

Abstract The principal vector for the pathogens of Lyme disease, human granulocytic ehrlichiosis, and human babesiosis is the tick Ixodes scapularis Say. A chalcid wasp, Ixodiphagus hookeri, in the family Encyrtidae parasitizes populations of the tick on several islands or other geographically isolated sites in New England with high densities of these ticks and white-tailed deer (Odocoileus virginianus), the principal host for adult I. scapularis. Deer densities were reduced at a forested tract in Bridgeport and the Bluff Point Coastal Reserve in Groton, Connecticut, from levels exceeding 90 animals per km2 in 1992 (Bridgeport) and 1994 (Bluff Point) to 17 and 10 animals per km2, respectively, by fall 2001. Tick densities declined with sustained reductions in the population of white-tailed deer. Similarly, prevalence of tick parasitism by Ixodes hookeri declined at both sites from 30 to 25% to <1.0% and was significantly correlated with previous year’s deer density at both sites (rs = 0.933 and rs = 0.867, P ≤ 0.0001) and with nymphal tick densities at Bridgeport (rs = 0.867, P ≤ 0.0001), but was not as well correlated with tick densities in Groton. The virtual disappearance of I. hookeri in this study corresponds with a lack of I. hookeri in mainland I. scapularis at comparable deer and tick densities, suggesting that there is a threshold deer density of ≈10–20/km2, with corresponding tick densities necessary for I. hookeri to successfully parasitize I. scapularis.

Control of immature Ixodes scapularis (Acari: Ixodidae) on rodent reservoirs of Borrelia burgdorferi in a residential community of southeastern Connecticut

Abstract A 3-yr community-based study was conducted on residential properties on Mason’s Island, Mystic, CT, to determine the efficacy of a rodent-targeted acaricide (fipronil) to control immature Ixodes scapularis (Say) on Peromyscus leucopus. Results indicated that modified commercial bait boxes were effective as an acaricide delivery method for reducing nymphal and larval tick infestations on white-footed mice by 68 and 84%, respectively. Passive application of fipronil significantly reduced the infection rate of Borrelia burgdorferi among white-footed mice by 53%. Moreover, the abundance of questing I. scapularis adults on treated properties was reduced by 77% and fewer were infected with spirochetes (31%) compared with untreated sites (47%) after 3 yr of treatment. Likewise, the abundance of host-seeking nymphs was significantly reduced on treated properties by >50%. Finally, infection rates in flagged nymphal ticks for both B. burgdorferi and Anaplasma phagocytophilum were reduced by 67 and 64%, respectively, after only 2 yr of treatment. Results from this 3-yr trial indicate that the use of fipronil passively applied to reservoir animals by bait boxes is an environmentally acceptable means to control ticks, interrupt the natural disease transmission cycle, and reduce the risk of Lyme disease for residents of treated properties.

INFECTIONS OF GRANULOCYTIC EHRLICHIAE AND BORRELIA BURGDORFERI IN WHITE-TAILED DEER IN CONNECTICUT

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immuno-sorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1: 5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11(18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.

The United States Department of Agriculture's Northeast Area-Wide Tick Control Project: Summary and Conclusions

From 1997 to 2002, the U.S. Department of Agricultures Northeast Area-wide Tick Control Project used acaricide-treated 4-Poster Deer Treatment Bait Stations in five eastern states to control ticks feeding on white-tailed deer. The objectives of this host-targeted technology were to reduce free-living blacklegged (Ixodes scapularis Say) and lone star (Amblyomma americanum [L.]) tick populations and thereby to reduce the risk of tick-borne disease. During 2002 to 2004, treatments were suspended, and tick population recovery rates were assayed. Subsequently, the major factors that influenced variations in efficacy were extrapolated to better understand and improve this technology. Treatments resulted in significant reductions in free-living populations of nymphal blacklegged ticks at six of the seven sites, and lone star ticks were significantly reduced at all three sites where they were present. During the study, maximal significant (p < or = 0.05) efficacies against nymphal blacklegged and lone star ticks at individual sites ranged from 60.0 to 81.7 and 90.9 to 99.5%, respectively. The major environmental factor that reduced efficacy was the occurrence of heavy acorn masts, which provided an alternative food resource for deer. Although the 4-Poster technology requires 1 or more years to show efficacy, this host-targeted intervention was demonstrated to be an efficacious, economical, safe, and environment-friendly alternative to area-wide spraying of acaricide to control free-living populations of these tick species.

Managing Japanese Barberry (Ranunculales: Berberidaceae) Infestations Reduces Blacklegged Tick (Acari: Ixodidae) Abundance and Infection Prevalence with Borrelia burgdorferi (Spirochaetales: Spirochaetaceae)

ABSTRACT In many Connecticut forests with an overabundance of white-tailed deer (Odocoileus virginianus Zimmermann), Japanese barberry (Berberis thunbergii DC) has become the dominant understory shrub, which may provide a habitat favorable to blacklegged tick (Ixodes scapularis Say) and white-footed mouse (Peromyscus leucopus Rafinesque) survival. To determine mouse and larval tick abundances at three replicate sites over 2 yr, mice were trapped in unmanipulated dense barberry infestations, areas where barberry was controlled, and areas where barberry was absent. The number of feeding larval ticks/mouse was recorded. Adult and nymphal ticks were sampled along 200-m draglines in each treatment, retained, and were tested for Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner) presence. Total first-captured mouse counts did not differ between treatments. Mean number of feeding larval ticks per mouse was highest on mice captured in dense barberry. Adult tick densities in dense barberry were higher than in both controlled barberry and no barberry areas. Ticks sampled from full barberry infestations and controlled barberry areas had similar infection prevalence with B. burgdorferi the first year. In areas where barberry was controlled, infection prevalence was reduced to equal that of no barberry areas the second year of the study. Results indicate that managing Japanese barberry will have a positive effect on public health by reducing the number of B. burgdorferi-infected blacklegged ticks that can develop into motile life stages that commonly feed on humans.

The Relationship Between Deer Density, Tick Abundance, and Human Cases of Lyme Disease in a Residential Community

ABSTRACT White-tailed deer (Odocoileus virginianus Zimmerman), serve as the primary host for the adult blacklegged tick (Ixodes scapularis Say), the vector for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Our objective was to evaluate the degree of association between deer density, tick abundance, and human cases of Lyme disease in one Connecticut community over a 13-yr period. We surveyed 90–98% of all permanent residents in the community six times from 1995 to 2008 to document residents exposure to tick-related disease and frequency and abundance of deer observations. After hunts were initiated, number and frequency of deer observations in the community were greatly reduced as were resident-reported cases of Lyme disease. Number of resident-reported cases of Lyme disease per 100 households was strongly correlated to deer density in the community. Reducing deer density to 5.1 deer per square kilometer resulted in a 76% reduction in tick abundance, 70% reduction in the entomological risk index, and 80% reduction in resident-reported cases of Lyme disease in the community from before to after a hunt was initiated.

Isolation of Entomopathogenic Fungi From Soils and Ixodes scapularis (Acari: Ixodidae) Ticks: Prevalence and Methods

ABSTRACT Entomopathogenic fungi are commonly found in forested soils that provide tick habitat, and many species are pathogenic to Ixodes scapularis Say, the blacklegged tick. As a first step to developing effective biocontrol strategies, the objective of this study was to determine the best methods to isolate entomopathogenic fungal species from field-collected samples of soils and ticks from an Eastern deciduous forest where I. scapularis is common. Several methods were assessed: (1) soils, leaf litter, and ticks were plated on two types of media; (2) soils were assayed for entomopathogenic fungi using the Galleria bait method; (3) DNA from internal transcribed spacer (ITS) regions of the nuclear ribosomal repeat was extracted from pure cultures obtained from soils, Galleria, and ticks and was amplified and sequenced; and (4) DNA was extracted directly from ticks, amplified, and sequenced. We conclude that (1) ticks encounter potentially entomopathogenic fungi more often in soil than in leaf litter, (2) many species of potentially entomopathogenic fungi found in the soil can readily be cultured, (3) the Galleria bait method is a sufficiently efficient method for isolation of these fungi from soils, and (4) although DNA extraction from ticks was not possible in this study because of small sample size, DNA extraction from fungi isolated from soils and from ticks was successful and provided clean sequences in 100 and 73% of samples, respectively. A combination of the above methods is clearly necessary for optimal characterization of entomopathogenic fungi associated with ticks in the environment.

Topical Treatment of White-Tailed Deer with an Acaricide for the Control of Ixodes scapularis (Acari: Ixodidae) in a Connecticut Lyme Borreliosis Hyperendemic Community

The 4-Poster device for the topical treatment of white-tailed deer, Odocoileus virginianus (Zimmermann), against ticks using the acaricide amitraz, was evaluated in a Lyme borreliosis endemic community in Connecticut. As part of a 5-year project from 1997 to 2002, 21-24 of the 4-Posters were distributed at residential sites in Old Lyme, CT, in a core treatment area of approximately 5.2 km(2) in fall 1997. The 4-Posters were active October to mid-December and March into May, corresponding to the peak periods of activity for adult Ixodes scapularis in this particular area. Corn consumption ranged from 361 to 4789 kg/month for October and November and 696-3130 kg/month during April. Usage of 4-Posters by deer generally was high (>90%), except during acorn masts in fall 1998 and 2001. Amitraz was applied by rollers at the estimated rate of 1.3 g active ingredient/ha/year. The abundance of host-seeking I. scapularis nymphs declined significantly (p < 0.001) in the core treatment area, as compared to a control community in Old Saybrook, CT, through 2004, over the project period from 1998 to 2003, from 9.3/100m(2) to 0.97/100m(2), rising to 1.90/100m(2) in 2004. From 1999 through 2003, there were 46.1%, 49.6%, 63.4%, 64.6%, and 70.2% reductions, respectively, in the nymphal tick population in comparison with the untreated community and initial tick abundance in 1998. Control of I. scapularis adults declined to only 19.1% in 2004; 2 years after the treatment of deer was discontinued. Differences in nymphal tick abundance between the control and core treatment area were significant in 1999 (p = 0.042) and highly significant in 2001 (p < 0.001) and 2002 (p = 0.002). The passive topical application to deer of the acaricide amitraz resulted in a significant decrease in the population of free-living I. scapularis nymphs in the treated core in Connecticut.