Range Expansion and Increasing Borrelia burgdorferi Infection of the Tick Ixodes scapularis (Acari: Ixodidae) in Iowa,

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1 Vector-Borne Diseases, Surveillance, Prevention Journal of Medical Entomology, 54(6), 2017, doi: /jme/tjx121 Advance Access Publication Date: 17 June 2017 Research article Range Expansion and Increasing Borrelia burgdorferi Infection of the Tick Ixodes scapularis (Acari: Ixodidae) in Iowa, Jonathan D. Oliver, 1,2 Steve W. Bennett, 3 Lorenza Beati, 4 and Lyric C. Bartholomay 5 1 School of Public Health Division of Environmental Health Sciences, University of Minnesota, 420 Delaware St. S.E., Minneapolis, MN (joliver@umn.edu), 2 Corresponding author, joliver@umn.edu, 3 University of St. Thomas, 2115 Summit Ave., Saint Paul, MN (steve.bennett@stthomas.edu), 4 U.S. National Tick Collection, Georgia Southern University, 75 Georgia Ave., Bldg. 204, Statesboro, GA (lorenzabeati@georgiasouthern.edu), and 5 Department of Pathobiological Sciences, University of Wisconsin, 1656 Linden Dr., Madison, WI (lyric.bartholomay@wisc.edu) Subject Editor: Maria Diuk-Wasser Received 3 October 2016; Editorial decision 20 May 2016 Abstract A passive surveillance program monitored ticks submitted by the public in Iowa from Submitted ticks were identified to species and life stage, and Ixodes scapularis Say nymphs and adults were tested for the presence of Borrelia burgdorferi. An average of 2.6 of Iowa s 99 counties submitted first reports of I. scapularis per year over the surveillance period, indicating expansion of this tick species across the state. The proportion of vector ticks infected by B. burgdorferi increased over time between 1998 and In 2013, 23.5% of nymphal and adult I. scapularis were infected with B. burgdorferi, the highest proportion of any year. Active surveillance was performed at selected sites from Ixodes scapularis nymphs collected at these sites were tested for the presence of B. burgdorferi, Anaplasma phagocytophilum, and spotted fever group Rickettsia spp. (likely representing Rickettsia buchneri). Nymphs tested were 17.3% positive for B. burgdorferi, 28.9% for A. phagocytophilum, and 67.3% for Rickettsia spp. The results of these surveillance programs indicate an increasing risk of disease transmission by I. scapularis in Iowa. Key words: tick, Rickettsia buchneri, Anaplasma phagocytophilum, Lyme disease The blacklegged tick, Ixodes scapularis Say, is currently the most important vector of disease to humans in the continental United States. It transmits a variety of disease-causing organisms, including those responsible for Lyme disease, human granulocytic anaplasmosis, babesiosis, and Powassan virus. Of these, Lyme disease is by far the most significant, with about 30,000 cases reported to the CDC per year ( and an estimated 10 times as many cases diagnosed (Hinckley et al. 2014, Nelson et al. 2015). Although very few fatalities result directly from Lyme disease infection, the number of people infected and morbidity inflicted underscores the public health importance of this illness. Furthermore, the expanding distribution of I. scapularis in the midwestern and northeastern United States, and the concomitant expansion of Lyme disease, emphasize the increasing importance of this disease to public health (Lingren et al. 2005, Hanincova et al. 2006, Eisen et al. 2016). Active surveillance programs emphasizing systematic collection and pathogen testing of vector ticks provide great precision in evaluating the spatial and temporal distribution of ticks and patterns of pathogen distribution. These programs, however, are expensive in resources and manpower, which typically results in surveillance efforts that are quite limited in scope and duration. Passive surveillance programs relying on self-reporting by tick-bite victims or their physicians are comparatively easily and cheaply maintained, producing datasets of far greater scope but substantially less precision than active surveillance programs. The result is that passive surveillance programs produce reliable data about whether vector tick species are present in an area, the proportion of ticks infected with a tickborne pathogen (limited in geographic granularity in regions from which few ticks are submitted), and the progress of establishment as a nonendemic tick species spreads into a territory not colonized previously. Negative tradeoffs of passive surveillance programs include a positive correlation of tick submissions with population density, the potential for false negatives in areas with few people to submit samples, uncertainty about acquisition location and timing of some specimens, and the inability to predict the prevalence of human cases in advance (Johnson et al. 2004). Despite these drawbacks, passive surveillance programs remain an important and costeffective tool in vector-borne disease epidemiology. Between 1990 and 2013, a statewide tick-borne disease passive surveillance program was maintained at Iowa State University, with The Authors Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com 1727

2 1728 Journal of Medical Entomology, 2017, Vol. 54, No. 6 Fig. 1. Ixodes scapularis submissions from Specimens were geographically associated to their submitter s home town address, not corrected by where submitters believed they had acquired the tick. Circle size is graduated to indicate the number of specimens submitted per town, with the smallest circles on the map each representing a single submission, and the largest circle representing 255 submissions. Circle color indicates the proportion of submitted nymphs and adults that tested positive for B. burgdorferi. Asterisks in the labeled counties indicate the active surveillance sites listed in Table 1. Dark gray indicates the extent of towns or cities that submitted ticks to the program. the primary goals of monitoring tick distribution and the infection rate of susceptible ticks with Borrelia burgdorferi. Over this time, >11,000 ticks were submitted from within Iowa, identified, and catalogued, and hundreds of nymphal and adult I. scapularis were tested for the presence of B. burgdorferi. This manuscript provides a summary of these 24 yr, with an emphasis on the changing proportions of ticks submitted to the program, the continuing expansion of I. scapularis across Iowa, and the increasing proportion of ticks infected with B. burgdorferi. Over this time period, I. scapularis was also expanding its range in the states that border Iowa to the north and east. In Minnesota and Wisconsin, states in which I. scapularis has long been endemic, the range of these ticks expanded, covering most of Minnesota and nearly all of Wisconsin (Eisen et al. 2016). In Illinois, a few I. scapularis had been found in the northernmost regions of the state in the early 1990s. By 2005, presence of the ticks was reported in Chicago (Jobe et al. 2006), and by 2015, populations were established in counties throughout the state (Eisen et al. 2016). Active surveillance was also performed in Iowa from 2007 to 2009 to evaluate the accuracy of B. burgdorferi infection rates of ticks acquired through passive surveillance during the same time period. To this end, ticks acquired by active surveillance were tested for the presence of both B. burgdorferi, Anaplasma phagocytophilum, and spotted fever group (SFG) Rickettsia spp. This surveillance effort has provided additional insight into the presence of tick-borne pathogens and symbiotic bacteria in Iowa. Materials and Methods Passive Surveillance Ticks were submitted by mail to the passive surveillance program by medical and veterinary workers and members of the public. Submissions were solicited primarily by fliers posted at state parks and online on a departmental website. In its 24 yr of operation, >11,000 specimens were processed. Ticks were identified to species (Clifford et al. 1961, Keirans and Clifford 1978, Yunker et al. 1986, Keirans and Litwak 1989, Durden and Keirans 1996), life stage, and engorgement status, and all available provenance information was recorded. Submitters were encouraged to send fresh ticks to enhance the ease of identification and pathogen detection. The specimens themselves were retained in 70% ethanol prior to processing. Submitters were asked to include information about where and when they had acquired the tick. If this information was not provided, the submitter s county of residence was listed as the site of acquisition. Active Surveillance Active surveillance for I. scapularis was performed at eight sites between 2007 and 2009 (Fig. 1). Sites consisted mostly of state parks selected for potential tick habitat (Diuk-Wasser et al. 2006) and a high degree of human usage. Some sites were sampled for 3 yr, others for a subset of years. Active surveillance methods were adapted from previously described dragging methodologies

3 Journal of Medical Entomology, 2017, Vol. 54, No Fig. 2. Range expansion of I. scapularis across Iowa (A) Map of Iowa showing counties with reported (blue) and established (red) I. scapularis populations based on previously accepted definitions (Dennis et al. 1998, Eisen et al. 2016). Counties with established populations had either six or more ticks or two of the three motile life stages submitted within a calendar year. Counties with fewer ticks submitted within a year were deemed reported only. The year listed in the red counties indicates the year of establishment. The white number associated with each reported county denotes the number of years in which I. scapularis were submitted. (B) Chart showing the cumulative number of counties that had submitted an I. scapularis specimen. The 1986 data point is based on presence of ticks described in the literature (Installation Pest Management Consultation 1988), with a dashed line extrapolating through the intervening years. (Gatewood et al. 2009). Each site encompassed ten 100-m transects arranged in five groups of parallel lines. Each of these five double transects was positioned within a suitable habitat at each site. Transects were sampled by dragging a 1-m 2 white drag cloth, sampling an area of 1,000 m 2 at each site per visit. Drag clothes were checked for the presence of ticks at 20-m intervals. Sampling sites were visited at intervals over the summer season, from three to six times per year from mid-may through mid-august limited by flooding and wet weather. The number and location of surveyed sites varied between years. In 2007, seven sites were surveyed (Clayton Co., Delaware Co., Dubuque Co., Johnson Co., Linn Co., Muscatine Co., and Winneshiek Co.). In 2008, six sites were surveyed (Allamakee Co., Clayton Co., Johnson Co., Linn Co., Muscatine Co., and Winneshiek Co.). Exceptional amounts of rain and flooding in 2009 limited surveillance substantially, and only five sites were surveyed three times each (Allamakee Co., Clayton Co., Johnson Co., and Muscatine Co.). Pathogen Testing Over the course of the passive surveillance program, several protocols were used to identify infection of adult and nymphal I. scapularis with B. burgdorferi, changing as more efficient or accurate techniques for detection became available. From (Lingren et al. 2005), ticks were dissected and examined by indirect fluorescence assay for the presence of spirochetes, as previously described (Telford and Spielman 1989, Sharon et al. 1992). From , the State Hygienic Laboratory at The University of Iowa tested ticks by nested PCR (Lebech et al. 1995; F1 F3, F6 F8) or later by reverse transcriptase qpcr using a proprietary primer set. For these assays, tick samples were ground using a bead beater, and DNA was extracted using a Qiamp viral RNA kit (Qiagen Inc., Valencia, CA). Samples originating from were dissected in lysis buffer prior to DNA extraction (Beati et al. 2012) using a DNeasy blood and tissue kit (Qiagen) and evaluated for B. burgdorferi infection, again using Lebech s nested PCR primers (Lebech et al. 1995). All PCR assays included positive and negative control samples for each set tested. Ticks acquired through active surveillance were evaluated for infection with B. burgdorferi, A. phagocytophilum, and SFG Rickettsia spp. by PCR. The rrfa rrlb intergenic spacer gene was used to detect B. burgdorferi (Derdakova et al. 2003), A. phagocytophilum was detected by nested PCR as described (Massung et al. 1998), and Rickettsia was detected by amplifying a portion of the ompa gene (Fournier et al. 1998). Statistical Analysis Where noted in the text, Student s t-tests were performed. All t-tests were two-tailed and assumed unequal variance. When used on proportional data, values were normalized by arcsine square-root transformation. Proportions of 0 were excluded from the comparison. Two-tailed t-tests or one-way ANOVA tests were used to test for statistical significance comparing active and passive surveillance I. scapularis infection rates with B. burgdorferi. Analyses were performed using the data analysis package for Microsoft Excel. Results Passive Surveillance At the initiation of the passive surveillance program in 1990, I. scapularis had been reported in nine of Iowa s 99 counties (Fig. 2). Few I. scapularis were submitted in any given year in the 1990s, but the number of counties reporting their first example of this species remained fairly constant (Fig. 2). Beginning in 2000, a higher proportion of I. scapularis relative to the total number of submitted ticks was reported, and a greater number of specimens were submitted to the passive surveillance program for identification, overall (Fig. 3). Range expansion continued at a fairly steady rate through the mid- 2000s when it began to level off (Fig. 2). By the closure of the passive surveillance program in 2013, 72 of Iowa s 99 counties had submitted at least one I. scapularis specimen, an average of 2.7 new counties per year submitting first reports over the 23 yr of operation since Adult I. scapularis demonstrated two separate periods of activity, the first peaking in May and the second peaking in late October

4 1730 Journal of Medical Entomology, 2017, Vol. 54, No. 6 Fig. 3. Proportion of tick species submitted by year. Lines illustrate the proportion of submission events for the three most commonly submitted species of ticks. Bars indicate the total number of submission events per year. Multiple ticks of the same species and life stage submitted from the same address on the same date are considered a single submission event. Notable is the overall increase in the proportion of I. scapularis submissions beginning in (Fig. 4). These periods of activity were divided by an interlude of almost 2 mo in which very few adult I. scapularis were observed. Nymphs were submitted over a more limited duration, with activity peaking in June. Most (87.5%) nymphal submissions occurred in May through July. Larval activity was more distributed than nymphal activity, with 97.5% of submissions occurring from May to September and peaking in July. Active Surveillance The number of I. scapularis collected at the active surveillance sites varied substantially between locations despite the sites being situated in closed-canopy deciduous forests and in counties that had previously reported the regular presence of the ticks. The combined results of active surveillance performed between 2007 and 2009 are found in Table 1, including data from sites that produced few nymphs (Delaware Co., four nymphs; Dubuque Co., one nymph). Pathogen Infection of Ticks The statewide infection rate of I. scapularis with B. burgdorferi fluctuated from year to year for both nymphs and adult ticks (Fig. 5). Ticks submitted in 2013, the final year of the surveillance program, had the highest rate of infection (23.5%), owing to the high rate of adult infection that year (30%). As adult ticks had undertaken two feeding events while nymphs had only undertaken one, it was expected that adult ticks would have a higher rate of infection, overall. This was generally the case, though in 2003 and 2007, the proportion of infected nymphs exceeded that of the adult ticks by <2% (2003 adults 12.2%, nymphs 14.5%; 2007 adults 15.7%, nymphs 16.3%), and in 1998, 1999, and 2008 exceeded it by >5% (1998 adults 0%, nymphs 12%; 1999 adults 8.3%, nymphs 14.3%; 2008 adults 8.3%, nymphs 25.3%). Adult I. scapularis demonstrated two distinct periods of activity in Iowa based on submissions to the passive surveillance program. The first period spanned March July, peaking in May, and the second spanned September December, peaking in October (Fig. 4). To determine if tick activity period had an effect on B. burgdorferi infection, the proportion of infected ticks submitted during the first period of activity was compared with the proportion submitted during the second annual period of activity, The results were not significantly different (two-tailed P-value > 0.392; df ¼ 26). The overall rate of nymphal I. scapularis infection with B. burgdorferi for the actively surveyed sites from was 17.3% (Table 1). Passive surveillance of the counties in which the active surveillance sites were located yielded an average infection rate of 23.5% counting only the years when the individual sites were surveyed. Statewide, from 2007 to 2009, 18.8% of nymphs were infected with B. burgdorferi (n ¼ 136). The proportion of infected ticks from each site was compared against the proportion of infected ticks acquired by passive surveillance from the site s host county over the same time period. Variation in infection results was not statistically significant across all counties, probably owing to low sample size. The exception was in Clayton Co., where active surveillance produced 27.8% infected nymphs and passive surveillance found 26% infected nymphs from 2007 to Clayton Co. produced the most nymphs of any site for the active surveillance program (n ¼ 54) and the most nymphs of any county for the passive surveillance program (n ¼ 100) over this time period. No significant differences were found between the infection rates derived from the two surveillance methods. Ixodes scapularis nymphs collected through active surveillance were also tested for infection with A. phagocytophilum and SFG Rickettsia spp. Anaplasma phagocytophilum was detected in 28.9% of ticks tested. SFG Rickettsia, most likely the symbiotic Rickettsia buchneri (Kurtti et al. 2015), were found in 67.3% of nymphs.

5 Journal of Medical Entomology, 2017, Vol. 54, No Fig. 4. Seasonal pattern of I. scapularis submission as a percentage of total yearly submissions of each life stage based on passive surveillance data collected in The separate peaks in adult I. scapularis activity are comparable with those observed elsewhere in the midwest and eastern United States. Based on n ¼ 1,263 adults, n ¼ 807 nymphs, and n ¼ 157 larvae. Table 1. Ixodes scapularis nymphs acquired through active surveillance were PCR tested for the presence of B. burgdorferi (Bb), A. phagocytophilum (Ap), and Rickettsia spp. (R or R spp.) Site location Total nymphs Nymphs/m 2 Bb Ap R spp. Bb/Ap Bb/R Ap/R Bb/Ap/R %Bb %Ap %R spp. %Bb passive Delaware Co % 75% 75% 14.3% Clayton Co % 25.9% 64.8% 26% Johnson Co % 25% 75% 0% Dubuque Co % 0% 100% 100% Linn Co % 16.7% 58.3% 0% Winneshiek Co % 37.1% 82.9% 25% Muscatine Co % 15.8% 63.2% 20% Allamakee Co % 36.8% 47.4% 33.3% Total % 28.9% 67.3% 23.5% Co-infection % 5.8% 10.9% 18.6% 3.9% Whole numbers indicate the absolute number of ticks that tested positive for a given bacterium or combination of bacteria. Percentages indicate the proportion of the total nymphs per site that tested positive. The proportion of passive surveillance-acquired I. scapularis infected with B. burgdorferi for the host county of each active surveillance site is indicated in the column labeled %Bb passive. Coinfection percentages represent proportions from all active surveillance-acquired nymphs (156), combined. Nymphs per m 2 was calculated combining all visits to the sites. A number of active surveillance-collected I. scapularis nymphs harbored two or three of the assayed bacteria species. Borrelia burgdorferi and A. phagocytophilum coinfected 5.8% of nymphs, B. burgdorferi and SFG Rickettsia 10.9%, A. phagocytophilum and SFG Rickettsia 18.6%, and 3.9% were infected by all three bacteria. Discussion The midwestern vector of B. burgdorferi, I. scapularis, was first reported in the northeastern-most county of Iowa in 1908 (Banks 1908). The tick seems to have been absent from the literature until 1986 when the first case of Lyme disease in the state as well as the presence of the tick were reported in three counties adjacent to the Mississippi River (Installation Pest Management Consultation 1988). Data from the passive surveillance program initiated in 1990 indicate that substantial range expansion of I. scapularis occurred in Iowa over the 24 yr that the program was active. Besides the actual abundance of ticks in the environment, other factors may have affected the submission of tick samples over time. Factors that affected individuals submitting ticks could have changed over the course of the program and may have included awareness of tick-borne disease risks, patterns of outdoor activity, use of insect repellants, recommendations for regularly doing tick checks, availability and

6 1732 Journal of Medical Entomology, 2017, Vol. 54, No. 6 Fig. 5. Proportion of B. burgdorferi-infected I. scapularis for each year from Infection rates are considered for nymphs and adults separately and overall was the first year in which >20 I. scapularis were submitted to the program. Only ticks which were tested and provided definitive positive or negative results are included. The numbers at the top are the total number of ticks tested for the presence of B. burgdorferi per year, A adults and N nymphs. awareness of tick identification services, and a variety of other hard to quantify influences. Despite these complications, passive surveillance does provide a substantial mass of information regarding where and when tick specimens originated, as well as B. burgdorferi infection status and seasonality. The passive surveillance program clearly demonstrated an increase over time in the proportion of I. scapularis infected with B. burgdorferi, a factor associated with increased incidence of Lyme disease in humans. By the closure of the program in 2013, 30% of adult ticks and 23.5% of ticks overall carried the organism, a high proportion of infection indicative of serious human risk. Future public health efforts should include increased surveillance efforts for B. burgdorferi and other tick-associated bacteria. At this time, no formal tick surveillance program is operating in Iowa. The seasonal abundance and activity of I. scapularis acquired through passive surveillance corresponds to the 2-yr life cycle previously described in the Midwestern and eastern United States (Daniels et al. 1989, Platt et al. 1992). Unfed adult ticks that have overwintered seek hosts in the spring and adults unable to feed die by the early summer (Yuval and Spielman 1990). Nymphs that have successfully fed in spring or summer molt into adult ticks whose activity in the fall corresponds to the second yearly peak in adult activity. Long, flexible life cycles and the ability to survive harsh winter conditions at a variety of life stages aid ticks in becoming established in areas with appropriate habitat (Lindsay et al. 1995, Vandyk et al. 1996). Evaluation of active surveillance-collected I. scapularis nymphs for A. phagocytophilum, yielded a high proportion of infected ticks (28.9%). In comparison, a study evaluating infection of I. scapularis with A. phagocytophilum at military bases in Minnesota and Wisconsin indicated that only 11.7% and 3.5% of adults were infected, respectively (Stromdahl et al. 2014). Anaplasma phagocytophilum strains were not specifically identified, but it seems likely that not all detections represent human-infectious strains (Massung et al. 2003). Although human granulocytic anaplasmosis is not a notifiable disease in Iowa, the Iowa Department of Public Health has received fewer than nine reports of infection per year (Sun et al. 2012), supporting the hypothesis that most A. phagocytophilum detected in Iowa ticks is not transmissible to humans. Antibodies against A. phagocytophilum had previously been detected in the sera of white-tailed deer from Iowa (Rainwater et al. 2006), but most deer-infecting strains of A. phagocytophilum likely do not affect humans (Massung et al. 2005, Dugan et al. 2006), although there is some evidence for white-tailed deer susceptibility to a human-derived isolate (Tate et al. 2005). The presence of SFG Rickettsia spp. in the majority of I. scapularis nymphs acquired thorough active surveillance is not surprising, as these likely represent the rickettsial endosymbiont of I. scapularis, Rickettsia buchneri (Kurtti et al. 2015). Previous studies have demonstrated that the proportion of ticks positive for rickettsiae, primarily representing R. buchneri, varies substantially between life stages and geographic locations (Noda et al. 1997, Benson et al. 2004, Swanson and Norris 2007). Other species of Rickettsia have occasionally been identified in I. scapularis, but at much lower rates than R. buchneri (Trout Fryxell et al. 2015). Factors likely to have contributed to the range expansion of I. scapularis in Iowa included progressive changes in land cover and a thriving white-tailed deer population. Insufficient humidity limits the dispersal of I. scapularis ticks by reducing questing height (Vail and Smith 2002), activity (Berger et al. 2014), and survival (Rodgers et al. 2007). Nymphs require at least 85% relative humidity (RH) at 27 C to avoid increased mortality (Stafford 1994). In an environment exhibiting variation in humidity levels, nymphs may withstand 40% RH for 4 h, with a >80% survival rate (Rodgers et al. 2007).

7 Journal of Medical Entomology, 2017, Vol. 54, No In Iowa, nymphal I. scapularis activity peaks in June. Typical RH levels during this month vary from 80% RH in the early morning when nymphs are most active to 50% RH in the mid-afternoon. In the afternoon, nymphs tend to be less active and avoid the temporary decrease in RH by moving into moist leaf litter. It appears that a high-humidity leaf-litter retreat from low humidity conditions may be prerequisite for establishment of a viable I. scapularis population in Iowa. A study modelling habitat suitability for I. scapularis in Minnesota, found that land cover was the ecological variable of the highest importance in predicting the distribution of the tick, and cool temperate forest was the land cover formation most predictive to the presence of I. scapularis (Johnson et al. 2016). Of the seven states in the north central region of the United States (IA, KS, MN, MO, ND, NE, and SD), Iowa has the least acreage of forested land cover after Nebraska. However, the area of forested land has expanded from a low of 1.6 million acres in 1974 to nearly 3 million acres in 2013 (USDA Forest Service 2011, Nelson and Brewer 2014). Forest distribution in Iowa continues to be very patchy, with only 27% of forested land located >90 m from the forest edge and 45% of forested land comprising patches of <100 acres (Flickinger 2010). Movement between disjoined islands of habitat presents a challenge to colonization by tick populations. Because they only move a few meters while unattached (Falco and Fish 1991), ticks are entirely reliant on the movement of their hosts to spread between areas of suitable habitat. White-tailed deer, Odocoileus virginianus, with large home ranges and the ability to feed and transport mated female ticks, are likely the most important host for dispersing I. scapularis (Madhav et al. 2004, Werden et al. 2014). The number of white-tailed deer in Iowa has increased rapidly since 1950 when the statewide estimate of deer numbers was just over 10,000 animals. In 1985, around the time that I. scapularis made its reappearance in Iowa, deer populations had expanded to an estimated 90,000 animals, and by 2009, estimates based on a variety of surveillance methods indicated 350, ,000 deer statewide before hunting season (DNR Deer Study Advisory Committee 2009). The high numbers of deer in Iowa provide abundant hosts and vehicles for transporting eggladen female I. scapularis to patchy areas of otherwise inaccessible habitat. Long-range dispersal of immature ticks by birds may also be contributing to the spread of immature I. scapularis into previously uncolonized regions of acceptable habitat (Scott et al. 2012, Schneider et al. 2015). Tick-borne diseases continue to be a public health issue in the United States, particularly in those parts of the country occupied by I. scapularis. There is substantial evidence that I. scapularis has been expanding its range in the eastern and Midwestern United States into areas in which this species was previously absent (Eisen et al. 2016). This, coupled with high rates of B. burgdorferi infection in ticks observed in the course of this study, emphasize that tick-borne disease transmission is a present and growing problem in Iowa. Data Availability Statement Data from this study are available from the Dryad Digital Repository: (Oliver et al. 2017). Acknowledgments Iowa State University Professor Emeritus Wayne Rowley founded the passive surveillance program. Partners in this project included the Iowa Department of Public Health and the State Hygienic Laboratory at The University of Iowa. Heathar Adolfs also contributed to this study by managing aspects of the surveillance program between 2011 and Funding and support for this project were provided by the Iowa Department of Public Health, by the Agriculture and Home Economics Experiment Station (Ames, IA) project 5111, supported by the Hatch Act and State of Iowa funds, and by NSF Grant to L.B. References Cited Banks, N A revision of Ixodoidea, or ticks, of the United States. U.S. Dep. Agric. Bur. Entomol. Tech. Ser. Washington DC. Beati, L., J. Patel, H. Lucas-Williams, H. Adakal, E. G. Kanduma, E. Tembo- Mwase, R. Krecek, J. W. Mertins, J. T. Alfred, S. Kelly, et al Phylogeography and demographic history of Amblyomma variegatum (Fabricius) (Acari: Ixodidae), the tropical bont tick. Vector Borne Zoonotic Dis. 12: Benson, M. J., J. D. Gawronski, D. E. Eveleigh, and D. R. 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