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Borrelia burgdorferi Not Detected in Widespread Ixodes scapularis
(Acari: Ixodidae) Collected from White-Tailed Deer in Tennessee
Author(s): M. E. Rosen, S. A. Hamer, R. R. Gerhardt, C. J. Jones, L. I. Muller, M. C.
Scott, and G. J. Hickling
Source: Journal of Medical Entomology, 49(6):1473-1480. 2012.
Published By: Entomological Society of America
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VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION
Borrelia burgdorferi Not Detected in Widespread Ixodes scapularis
(Acari: Ixodidae) Collected From White-Tailed Deer in Tennessee
M. E. ROSEN,1,2S. A. HAMER,3,4R. R. GERHARDT,5C. J. JONES,5,6L. I. MULLER,1M. C. SCOTT,1
AND G. J. HICKLING1
J. Med. Entomol. 49(6): 1473Ð1480 (2012); DOI: http://dx.doi.org/10.1603/ME11255
Lyme disease (LD), caused by the bacterium Borrelia burgdorferi and transmitted in
the eastern United States by blacklegged ticks, Ixodes scapularis Say, is classiÞed as nonendemic in
Tennessee and surrounding states in the Southeast. Low incidence of LD in these states has been
attributed, in part, to vector ticks being scarce or absent; however, tick survey data for many counties
spp. prevalence of I. scapularis, we collected ticks from 1,018 hunter-harvested white-tailed deer
160 deer (15.7%) from 35 counties were infested with adult I. scapularis; 30 of these counties were
new distributional records for this tick. The mean number of I. scapularis collected per infested deer
was 5.4 ? 0.6 SE. Of the 883 I. scapularis we removed from deer, none were positive for B. burgdorferi
and one tested positive for B. miyamotoi. Deer are not reservoir hosts for B. burgdorferi; nevertheless,
past surveys in northern LD-endemic states have readily detected B. burgdoreferi in ticks collected
from deer. We conclude that I. scapularis is far more widespread in Tennessee than previously
reported. The absence of detectable B. burgdorferi infection among these ticks suggests that the LD
of the Northeast and upper Midwest.
Lyme disease, Ixodes scapularis, Borrelia burgdorferi, Odocoileus virginianus, Ten-
of humans in the United States, with over 20,000 new
as nonendemic in the Southeast (Centers for Disease
Control and Prevention [CDC] 2008). For the period
2000Ð2009, Tennessee (population 5.6 million; 2000
Census) reported an average of only 19.8 LD cases
annually to the CDC (CDC 2012), with most of these
reports recorded by the Tennessee Department of
Health as ÔprobableÕ rather than ÔconÞrmedÕ (A. Mon-
cayo, personal communication).
The blacklegged tick (Ixodes scapularis Say) is the
vector of B. burgdorferi to humans in the eastern
United States (Piesman 2002). Before this study, I.
scapularis had been reported from ten Tennessee
counties based on opportunistic tick collections (Fig.
1; Dennis et al. 1998, Durden and Kollars 1992). More
recent efforts to map the eastern United States distri-
bution of I. scapularis (DiukÐWasser et al. 2010) did
not survey sites in Tennessee, so the distribution of I.
scapularis throughout the state has been uncertain.
tailed deer (Wilson et al. 1990). Systematic collection
I. scapularis removed from deer also can be tested for
underestimate the prevalence seen in questing ticks
because complement-mediated immune responses of
feeding ticks (Kurtenbach et al. 1998). Nevertheless,
this approach has proven useful for determining the
geographic extent of Borrelia spp. in I. scapularis pop-
ulations (e.g., Magnarelli et al. 1986, 1995; Gill et al.
1993; Keefe et al. 2009).
Reported cases of several tick-borne diseases (in-
cluding spotted fever group rickettsioses and human
ehrlichioses infection) are on the rise in Tennessee
1Center for Wildlife Health, University of Tennessee Institute of
Agriculture, Knoxville, TN 37996.
2Corresponding author: Wildlife Disease Laboratory, Michigan
48910 (e-mail: email@example.com).
3Department of Fisheries and Wildlife, Michigan State University,
East Lansing, MI 48824.
4Current AfÞliation: Veterinary Integrative Bioscience Depart-
ment, Texas A&M University, College Station, TX 77802.
5Department of Entomology and Plant Pathology, University of
Tennessee Institute of Agriculture, Knoxville, TN 37996.
0022-2585/12/1473Ð1480$04.00/0 ? 2012 Entomological Society of America
(Tennessee Department of Health 2009). This pat-
tern, evident throughout the Southeast, has been
linked to increasing abundance and geographic range
of several tick species and their hosts (particularly
deer; Paddock and Yabsley 2007). Whether LD is
similarly on the rise in the Southeast remains conten-
tious. To date, there have been no published reports
of B. burgdorferi being detected in I. scapularis in
Tennessee. Our goal in this study was to examine
blacklegged ticks collected from hunter-harvested
relative abundance, and LD-pathogen status.
Materials and Methods
er-harvested deer at 47 Tennessee Wildlife Resource
Agency (TWRA) check stations in November of 2007
and 2008 to quantify the presence of I. scapularis in
the counties from which the deer were harvested.
because tick distributions have previously been re-
ported and mapped by county (e.g., Dennis et al.
1998), and because tick presence in some other states
is known to correlate with LD incidence reported at
the county level (e.g., Kitron and Kazmierczak 1997).
across the state. Before searching for ticks, volunteers
and TWRA employees watched a training video ex-
plaining the purpose of the research and the sampling
protocol. They were instructed to search for ticks on
each deerÕs head and neck to just below the scapula,
on both sides of the animal. All ticks from one deer
were placed in a vial with 70% ethanol. Data on coun-
ty-of-harvest, sex, and age (determined by TWRA
biologists) were recorded for each deer.
All ticks were brought to the University of Tennes-
seeÕs Center for Wildlife Health (CWH) laboratory
and identiÞed to species using dichotomous keys
(Sonenshine 1979). Voucher specimens were submit-
ted to the U.S. National Tick Collection in Statesboro,
of the abdomen) and scutum width (at the widest
point) of intact ticks were measured (nearest 0.01
mm), and an engorgement index (EI) was calculated
as the ratio of body length to scutum width (after
Falco et al. 1996).
Borrelia spp. Assay. Total DNA was extracted from
each tick using a DNeasy Blood and Tissue Kit (Qia-
gen Inc., Valencia, CA). A polymerase chain reaction
(PCR) amplifying the mitochondrial 16S rRNA gene
(Black and Piesman 1994) was run on all samples to
verify DNA extraction. If this ampliÞcation was not
successful the original extraction product was diluted
1:10 and rerun for both Borrelia spp. and mitochon-
drial DNA. This dilution step was included because
blood from the tickÕs meal can inhibit PCR (Schwartz
et al. 1997). All samples were then tested at the CWH
laboratory using a Borrelia genus-speciÞc nested PCR
targeting the 16S-23S rRNA intergenic spacer (IGS)
locus (Bunikis et al. 2004a). These IGS primers will
detect both B. burdorferi senso stricto and B. burdor-
feri senso lato (Bunikis et al. 2004a). Water served as
the negative control and DNA from I. scapularis ticks
infected with strain B31 B. burgdorferi (Centers for
Disease Control and Prevention, Fort Collins, CO)
served as the positive control. PCR amplicons were
visualized by gel electrophoresis. Positive samples
Recovery Kit (Zymoclean, Orange, CA) according to
manufacturerÕs instructions, and submitted for se-
quencing at the University of TennesseeÕs Core Se-
quencing Facility. Sequences were compared with
those published in GenBank (http://blast.ncbi.nlm.
For independent veriÞcation of sample-infection
(Tsao et al. 2004) was run at Michigan State Univer-
sityÕs Insect Microbiology Laboratory on the DNA
from all I. scapularis collected in 2007.
were pooled for analysis and no attempt was made to
percent of deer infested with I. scapularis was visual-
ized on a Tennessee county map (ESRI, Redlands,
CA). Differences in the proportion of deer infested
with I. scapularis among TWRA management regions
were assessed using a Chi-square test of association.
Differences among these regions in the mean number
of adult I. scapularis detected on infested deer were
assessed using a KruskalÐWallis nonparametric anal-
ysis of variance (ANOVA). Tick engorgement may
in only Anderson, Bedford, Campbell, Fentress, Davidson, Lake, Marion, Rutherford, Scott, and Shelby Counties (Dennis
et al. 1998, Durden and Kollars 1992).
Current distribution of I. scapularis by Tennessee county. Prior published reports listed I. scapularis as present
1474JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 6
decrease the likelihood of detecting Borrelia spp.
through lysis of spirochetes and inhibition of PCR, so,
DNA extraction/PCR outcome was inßuenced by the
EI of the tick.
Tick Infestation. In total, 1,018 deer from 71 Ten-
nessee counties were inspected; ticks of any species
4,237 ticks of three species were collected from deer,
with the most common being Dermacentor albipictus
(Packard) (n ? 3,296) and least common being Am-
blyomma americanum (L.) (n ? 70; Table 1). The
prevalence of tick infestation in deer was highest in
the Cumberland Plateau TWRA Management region
and lowest in East Tennessee region (Fig. 2, Table 1;
?2? 391.2, 4 df, P ? 0.0001).
Adult I. scapularis (n ? 871) were collected from
15.7% of deer inspected. Deer were most frequently
Table 1); these differences in I. scapularis infestation
among regions were statistically signiÞcant (?2?
147.9; 4 df; P ? 0.0001).
I. scapularis were found on deer harvested from 35
infestation prevalence ranging from 2Ð100% (Fig. 2).
I. scapularis was not detected in some well-sampled
counties (e.g., most counties in East Tennessee), but
was found in some sparsely sampled counties (e.g., in
some Middle Tennessee and Cumberland Plateau
counties; Fig. 2).
The mean number of I. scapularis removed per in-
fested deer ranged from ?2 per deer in East Tennes-
see to 11 in Middle Tennessee (Table 2); this regional
variation in burden was signiÞcant (KruskalÐWallis
nonparametric ANOVA; H ? 42.4; 4 df; P ? 0.0001).
Borrelia spp. Infection. PCR was performed on 883
I. scapularis (502 females, 381 males; this sample com-
prised the 871 I. scapularis listed in Table 2 plus an
was recorded). Of the 833 ticks that were sufÞciently
intact for an engorgement index to be calculated, 40
(4.8%) required 1:10 dilution of the original extract to
produce PCR amplication. There was no indication
that a tickÕs EI (low ? ?2; high ?5 EI) inßuenced
whether this dilution step was required (?2? 0.80; 2
df; P ? 0.37). All males and 124 females (i.e., 58% of
total ticks tested) had an engorgement index of ?3.
(TWRA) region in Nov. 2007 and Nov. 2008 (years pooled)
Numbers and percentages of hunter-harvested deer infested with ticks in each Tennessee Wildlife Management Agency
No. of deer
All tick speciesI. scapularis D. albipictus A. americanum
Oak Ridge WMA
Region boundaries are shown in Figure 2; Oak Ridge Wildlife Management Area (OR WMA) spans two TWRA regions and is reported
Agency regions (thick lines). The numerator in each county is the number of deer found infested with I. scapularis, the
denominator is the number of deer checked. The map is based on 1,018 deer harvested by hunters in November 2007 and
November 2008 (years pooled; median number of deer per checked county ? 8, range 1Ð196). There were nine deer where
Plateau: four deer (one with I. scapularis); East Tennessee: three deer (one with I. scapularis). For the purposes of this map,
Oak Ridge WMA is considered part of Anderson County.
Deer sampling effort and Ixodes scapularis infestation by county (thin lines) and Tennessee Wildlife Resource
November 2012ROSEN ET AL.: I. scapularis COLLECTED FROM WHITE-TAILED DEER
B. burgdorferi was not detected in any of the I.
scapularis tested (i.e., 883 at full strength, plus 40
PCR. Furthermore, no B. burgdorferi was detected in
the subset of 431 samples retested using a B. burgdor-
feri-speciÞc quantitative PCR at the independent
one adult female produced a band at ?500 bp (rather
than the ?1,000 bp expected for B. burgdorferi). This
B. miyamotoi with 100% sequence homology (NCBI
I. scapularis is far more widely distributed in Ten-
mapping effort, while based on the greatest geo-
graphic coverage and largest sample sizes to date in
this state, did not sample all counties and so remains
tick. Earlier distribution maps (Dennis et al. 1998,
Durden and Kollars 1992) were derived from sparse,
passively collected, nonstandardized data, so the ex-
panded distribution shown in Fig. 1 reßects, in part,
our improved surveillance effort. Nevertheless, we
consider it likely that I. scapularis distribution has
expanded in recent years in response to changes in
land use, increasing deer numbers, and perhaps cli-
mate change. For example, one of us (R.G.) has sur-
veyed for ticks in Cumberland County since the early
1990s, and has never collected I. scapularis. In the
process of this study (2008), we collected I. scapularis
from deer harvested close to R.G.Õs research area. In
addition, in the spring and fall of 2009, Harmon et al.
(2011) obtained the Þrst collections of questing I.
a study of ticks on white-tailed deer from Steward
We identiÞed signiÞcant variation in abundance of
I. scapularis in different parts of Tennessee. This is a
strong indication that I. scapularis distribution is het-
erogeneous across the state at regional and county
scales. Nationally, it has been shown that there are
signiÞcant associations of I. scapularis with certain
habitat, landform, and climate patterns (e.g., Brown-
stein et al. 2005, DiukÐWasser et al. 2010). One useful
source of information on habitat variation in Tennes-
see is the ecoregion classiÞcation created by the U.S.
Environmental Protection Agency to aid state agen-
cies in management, research, and monitoring of eco-
are based on the abiotic and biotic factors that inßu-
ence ecosystem charactersÑincluding geology, phys-
iography, vegetation, climate soils, land use, wildlife,
and hydrology (GrifÞth et al. 2009). A comparison of
our deer infestation prevalence data with a map of
TennesseeÕs ecoregions indicates that I. scapularis is
Plains ecoregion of Middle Tennessee. The hills and
plains in this ecoregion are composed of a diverse
mixture of sandstone, siltstone, and shale associated
with oak-hickory (Quercus spp. and Carya spp.) for-
ests with some areas of bluestem (Schizachyrium sco-
(GrifÞth et al. 2009). Conversely, I. scapularis were
least abundant in the Blue Ridge Mountains ecore-
gion, and the eastern parts of the Ridge and Valley
ecoregion, suggesting higher-elevations habitats are
unfavorable for this tick (as has been reported else-
where; Jouda et al. 2004). I. scapularis was also largely
ysis of I. scapularisÕ ecological associations in Tennes-
see could help guide studies of B. burgdorferi and
other associated pathogens such as Anaplasma phago-
cytophilum and Babesia spp.
On average, 15.7% of the harvested Tennessee deer
level of infestation relative to most other published
surveys of hunter-harvested deer, in both endemic
and nonendemic areas (e.g., Amerasinghe et al. 1993;
Cortinas and Kitron 2006; Gill et al. 1993; Keefe at al.
2009; Kitron et al. 1992; Loken et al. 1985; Magnarelli
et al. 1986, 1995; Riehle and Paskewitz 1996). In these
cited studies, deer infestation averaged 57% in Þve
LD-endemic states and 35% in three nonendemic
We collected an average of 5.4 I. scapularis per
burdens reported both from endemic states and from
other nonendemic states (these averaged 5.5 and 6.2
ticks per deer, respectively; Amerasinghe et al. 1993;
2007 and Nov. 2008 (years pooled, noninfested deer excluded)
Numbers of I. scapularis (mean ? SE) collected from infested deer brought by hunters to Tennessee check-stations in Nov.
Oak Ridge WMA
13 (1.0 ? 0.2)
211 (5.9 ? 0.9)
217 (3.1 ? 0.4)
18 (1.3 ? 0.3)
33 (1.2 ? 0.3)
492 (3.1 ? 0.3)
15 (1.2 ? 0.4)
176 (4.9 ? 0.8)
154 (2.2 ? 0.4)
8 (0.6 ? 0.2)
26 (1.0 ? 0.2)
379 (2.4 ? 0.3)
28 (2.2 ? 0.5)
387 (10.8 ? 1.6)
371 (5.3 ? 0.8)
26 (1.9 ? 0.4)
59 (2.2 ? 0.4)
871 (5.4 ? 0.6)
Oak Ridge Wildlife Management Area (WMA) spans two TWRA regions and is reported separately. No immature I. scapularis were found
on the sampled deer.
1476JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 6
Cortinas and Kitron 2006; Gill et al. 1993; Keefe et al.
2009; Kitron et al. 1992; Magnarelli et al. 1986, 1995;
Riehle and Paskewitz 1996). Lack of variation in the
intensity of infestation may reßect low search efÞ-
ciency given the time pressure when examining deer
for ticks at busy check stations. It may also be that
low-density tick populations are aggregated, so that
deer that encounter ticks tend to pick up several at a
time, whereas deer in less favorable habitats encoun-
ter no ticks at all.
nessee, and in other Midwestern states (e.g., Cortinas
and Kitron 2006). This is a one-host tick; after the
larvae attach to a host, all subsequent life stages are
completed on that same host. Consequently, it has no
ecological capacity to transmit pathogens to another
host species, including humans. A. americanum is a
very abundant tick in Tennessee, but was not com-
monly found on deer in our fall surveys; this is not
surprising as its life stages are mostly inactive in No-
al. 2000, Goddard 2007). A. americanum is an incom-
petent vector for B. burgdorferi as its saliva lyses the
spirochetes (Piesman and Happ 1997, Ledin et al.
We know of no previous reports of B. burgdorferi
from I. scapularis in Tennessee, and we did not detect
is in marked contrast to similar deer-check surveys
undertaken in LD-endemic states, all of which de-
Gill et al. 1993; Magnarelli et al. 1986, 1995). The
average Borrelia spp. prevalence in these surveys in
other states (14%) was lower than is typically seen in
Qiu et al. 2002, Piesman et al. 1999), which is to be
expected, given complement-mediated lysis of B.
burgdorferi in ticks feeding on deer (Telford et al.
1988). Nevertheless, B. burgdorferi is readily detected
by PCR among male ticks (that do not engorge) and
in female ticks with low engorgement (EI ?3; D.
tested were in one of these two categories.
1986, Kitron et al. 1992, Keefe at al. 2009), except for
among ticks collected from a small number of deer in
Mississippi. To our knowledge, this is the Þrst large
(?500) sample of adult I. scapularis, tested with spe-
cies-speciÞc molecular techniques, to report a zero
prevalence of B. burgdorferi.
B. burgdorferi from 2 of 18 pooled samples of D. al-
bipictus removed from white-tailed deer in Cheatham
County. Similarly, Jordan et al. (2009) reported the
pathogen from 14% of wild turkey and 17% of migra-
tory waterfowl blood samples collected from a site in
middle Tennessee and reported further detections
from these species in ten surrounding counties. A.
americanum was the most common tick collected in
that study; no I. scapularis was observed. However,
a hybridization technique that did not provide se-
quence conÞrmation of B. burgdorferi presence. A
similar survey 1 yr later detected high levels of B.
miyamotoi, but zero prevalence of B. burgdorferi, in
wild turkeys from similar habitats in eastern and cen-
tral Tennessee (Scott et al. 2010).
Sequence-conÞrmed B. burgdorferi has been re-
ported from one red wolf on the Tennessee/North
Carolina border of Great Smoky Mountains National
Park (Penrose et al. 2000). Other wolves in the same
vicinity tested positive for B. burgdorferi antibodies
but were PCR negative for the organism. ELISA-test-
ing of canine serum (using LymeCHEK, San Diego,
in 1996 suggested that 14.5% had been exposed to B.
of the test is uncertain and the vaccination and travel
histories of these dogs were incomplete. More re-
cently, highly speciÞc SNAP 3Dx and 4Dx testing for
the B. burgdorferi C6peptide in 18,891 pet dogs in
Tennessee produced 47 positive samples (a 0.02%
prevalence), with the travel histories of the positive
dogs again unknown. In comparison, SNAP testing of
dogs in known LD-endemic areas typically produces
seroprevalences ?1.0% among dogs (Bowman et al.
2009). SNAP 4Dx tests of blood samples from 20 deer
one deer as positive for B. burgdorferi, and another
three as positive for an unknown Borrelia spp. (Mur-
dock et al. 2009). We conclude that, although B. burg-
wildlife hosts and human-biting ticks appears to be
exceedingly low relative to LD-endemic states.
for B. miyamotoi. Finding a Borrelia spp. other than B.
burgdorferi in our tick sample emphasizes the impor-
tance of using species-speciÞc pathogen tests. B. miy-
amotoi is closely related to the relapsing fever group
of Borrelia, a separate group from the LD Borrelia
(Bunikis et al. 2004b). In 2006, B. miyamotoi was de-
tected in 15 of 36 (42%) adult I. scapularis collected
opportunistically from white-tailed deer at three
TWRA East Tennessee check stations (G. H., unpub-
Tennessee ticks thus appears to be variable in both
time and space, which warrants further study.
Implications for Human-Disease Risk. The re-
ported LD-case rate in Tennessee is very low com-
pared with endemic areas of the northeastern United
States, and it has commonly been argued that this was
because I. scapularis is absent from most of the state.
Our Þndings indicate, however, that I. scapularis is
without detectable infection with the LD pathogen.
This latter Þnding is noteworthy, as there have been
November 2012ROSEN ET AL.: I. scapularis COLLECTED FROM WHITE-TAILED DEER
few published reports of well-established I. scapularis
tick populations without concurrent B. burgdorferi
infection. We know of only one such publication to
date: Maggi et al. (2010) identiÞed a population of I.
adult I. scapularis tested harbored B. burdorferi, al-
with inßuenza-like illness, relapsing fever, and ery-
thema migrans (Platonov et al. 2011). Russian strains
of B. miyamotoi differ slightly from those in North
America, and the eco-epidemiological settings differ
also, so the zoonotic potential of B. miyamotoi in the
southeastern United States remains uncertain.
At a national level, one approach taken to provide
the public with information and advice regarding
maps (CDC 1999) and, more recently, a distribution
map of Borrelia-infected nymphal ticks (that are con-
sidered the key life-stage for transmission to humans;
no survey work was undertaken in Tennessee. There-
on I. scapularis distribution, relative abundance, and
LD pathogen prevalence presently available for Ten-
nessee. We conclude that I. scapularis is far more
widespread in Tennessee than previously reported;
however, the absence of detectable B. burgdorferi
infection among these ticks indicates that the LD risk
they pose in the areas of Tennessee surveyed is pres-
ently far lower than is the case in the LD-endemic
areas of the Northeast and upper Midwest.
We thank the student volunteers who collected ticks: J.
Harmon, E. Baker, R. Dutkowski, H. Edwards, N. Hiles, R.
and the Student Chapters of the Wildlife Society at Univer-
sity of Tennessee Knoxville, University of Tennessee Martin
and Tennessee Technological University. We thank J. Evans
and his many check-station colleagues at TWRA. D. Paulsen
assisted with tick identiÞcations. J. Piesman and G. Dietrich
at the Centers for Disease Control and Prevention provided
Borrelia-positive controls. J. Sidge assisted with the diagnos-
tic assays at Michigan State University. We gratefully thank
was funded by the University of Tennessee Institute for
Agriculture. G.J.H. received support from the NIMBioS, the
National Institute of Mathematical and Biological Synthesis.
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