Real-time PCR for simultaneous detection and quantification of Borrelia burgdorferi in field-collected Ixodes scapularis ticks from the Northeastern United States.
ABSTRACT The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
Article: Short report: density of Lyme disease spirochetes within deer ticks collected from zoonotic sites.[show abstract] [hide abstract]
ABSTRACT: We determined whether the density of Lyme disease spirochetes varied between individual host-seeking deer ticks. Guts were dissected from 30 adult Ixodes dammini collected from three intensely zoonotic coastal Massachusetts sites, and the number of Borrelia burgdorferi present was estimated by a modified counting technique using indirect immunofluorescence. A median of 1,925 spirochetes was observed; ticks from the three sites contained similar numbers of spirochetes. No tick contained more than 4,500 spirochetes. Initial experimental reports establishing the efficiency of spirochetal transmission may have been based on ticks with a uniform spirochetal density, and extrapolations from these studies may thus overestimate the infectivity of host-seeking ticks in nature.The American journal of tropical medicine and hygiene 10/1995; 53(3):300-2. · 2.59 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: At the IV International Conference on Lyme Borreliosis, a workshop was held to identify the unique development of the Lyme disease spirochete, Borrelia burgdorferi, in its established and suspected arthropod vectors. The following is a summary of the panel's discussions of research aspects concerning relationship(s) of this borrelia to its vectors, and the mode(s) of its transmission to animal hosts.Scandinavian journal of infectious diseases. Supplementum 02/1991; 77:35-40.
Article: Field trial of an outer surface protein A (OspA) antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect Borrelia burgdorferi in Ixodes scapularis.[show abstract] [hide abstract]
ABSTRACT: Field-collected adult male Ixodes scapularis from Westchester County, New York were bisected and Borrelia burgdorferi infection rates were ascertained by both a direct fluorescent antibody test and an outer surface protein A (OspA) antigen-capture enzyme-linked immunosorbent assay (ELISA). Both assays gave identical antigen positivity rates with 89% concordance between the two assays. Storing dried ticks before ELISA analysis had no significant effect on the ability of the ELISA to determine the presence of OspA compared with assaying live ticks. The OspA antigen positivity rate for dried ticks was 49% compared with 53% for live ticks, with mean OspA antigen spirochete equivalents of 3,388 and 2,823 for dried and live ticks, respectively.The American journal of tropical medicine and hygiene 04/1994; 50(3):354-8. · 2.59 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2003, p. 4561–4565
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 8
Real-Time PCR for Simultaneous Detection and Quantification of
Borrelia burgdorferi in Field-Collected Ixodes scapularis
Ticks from the Northeastern United States
Guiqing Wang,1Dionysios Liveris,1Brandon Brei,2† Hongyan Wu,1Richard C. Falco,3
Durland Fish,2and Ira Schwartz1,4*
Department of Microbiology and Immunology1and Department of Medicine,4New York Medical College, Valhalla,
New York 10595; Department of Epidemiology and Public Health, Yale School of Medicine, New Haven,
Connecticut 065202; and Vector Ecology Laboratory, Calder Ecology Center,
Fordham University, Armonk, New York 105043
Received 22 January 2003/Accepted 29 May 2003
The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays
based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia
burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of
the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for
detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR
targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states
(Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%)
adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to
197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No
significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected
at different locations in these four states (P ? 0.23 by one-way analysis of variance test) or in ticks infected with
the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P ? 0.39).
A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment
estimate of k ? 0.279) was observed. From the frequency distribution data and previously published trans-
mission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal
tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is
a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected
ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
Hard ticks (Ixodidae) in the Ixodes persulcatus complex, such
as Ixodes scapularis and Ixodes pacificus in North America,
Ixodes ricinus in Europe, and Ixodes persulcatus in far eastern
Russia and Asia, are the principal vectors of the Lyme disease
spirochete, Borrelia burgdorferi, and several other tick-borne
pathogens (2, 25). The incidence of human Lyme disease in areas
where it is endemic is correlated with the abundance and preva-
lence of Ixodes ticks infected with B. burgdorferi (34). Moreover,
studies of laboratory animals have suggested that the efficiency
of host infection is determined in part by the number of spi-
rochetes inoculated using a needle or deposited by the tick at
the time of feeding (21, 27). Thus, monitoring B. burgdorferi
density in host-seeking ticks will provide more accurate data
for assessment of population risk of Lyme disease and the
potential variability of disease manifestations after tick bites.
Currently, the number of spirochetes in field-collected or
experimentally infected Ixodes ticks is estimated mainly by
microscopy-based assays in which the spirochetes are counted
directly on tick midgut smears stained with fluorescently la-
beled specific antibodies (8, 9, 27) or on silver-stained histo-
logical sections of ticks (11, 14). For specific detection of B.
burgdorferi in field-collected adult I. scapularis ticks and mon-
itoring of the spirochete kinetics during the tick life cycle, an
OspA antigen-capture enzyme-linked immunosorbent assay
(ELISA) was also developed (3–5). These methods are labor-
intensive, which limits the number of ticks that can be ana-
lyzed. Moreover, the sensitivities of these methods are rela-
tively low, e.g., a lower detection limit of approximately 150
spirochetes was reported for the antigen-capture ELISA (5).
Real-time PCR has features that allow for rapid detection of
infectious pathogens in environmental or experimentally in-
fected animal tissues and clinical specimens (38). The tech-
nique has recently been employed to detect the presence of
B. burgdorferi DNA in I. ricinus ticks from Switzerland (15), to
quantify the spirochete loads in experimentally infected animal
tissues (23, 24, 35), and to differentiate the three B. burgdorferi
sensu lato species that are pathogenic to humans in Europe
(22, 28, 30). By targeting the B. burgdorferi-specific recA gene,
we have successfully developed a real-time PCR protocol to
quantify the spirochetes in infected animal tissues and in skin
biopsy specimens of patients with erythema migrans (19, 36,
37). The present study evaluates a modified real-time, quanti-
tative PCR (qPCR) protocol for simultaneous detection and
quantification of B. burgdorferi DNA in field-collected I. scapu-
laris ticks from the northeastern United States. The genotypic
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, New York Medical College, Valhalla, NY
10595. Phone: (914) 594-4658. Fax: (914) 594-4176. E-mail: schwartz
distribution of B. burgdorferi in host-seeking ticks and the
spirochete loads of ticks infected with distinct genotypes of
B. burgdorferi are also analyzed.
MATERIALS AND METHODS
Tick collection. I. scapularis ticks were collected from four northeastern states
(New York, Connecticut, Rhode Island, and New Jersey) of the United States by
drag sampling as previously described (6, 32). A total of 292 I. scapularis ticks
(232 nymphs and 60 adults) were collected from five different locations in the
lower Hudson valley of New York during 1998 and 2000. An additional 206
nymphal I. scapularis ticks collected from southeastern Rhode Island (n ? 61),
southern Connecticut (n ? 81), and eastern New Jersey (n ? 64) for a separate
project in 1999 were also included in this study.
Preparation of DNA from ticks. All ticks were preserved in vials containing
70% ethanol until DNA extraction. DNA was prepared from ticks by using a
commercial DNA extraction kit (Isoquick; Orca Research, Bothell, Wash.) as
previously described (31). This method effectively removes potential PCR inhib-
itors from blood-fed ticks (31). Extracted DNA was resuspended in 50 ?l of
sterile water. Five and two microliters of such DNA were used for nested PCR
and real-time PCR, respectively.
Real-time qPCR. Simultaneous detection and quantification of B. burgdor-
feri DNA in ticks were performed with the Lightcycler PCR instrument
(Roche Diagnostics, Mannheim, Germany), for which the B. burgdorferi-
specific chromosome-encoded recA gene was chosen as the target. It is as-
sumed that only a single copy of recA is present per spirochete based on the
genome sequence of B. burgdorferi type strain B31 (10) and a previous
experimental report (23). qPCR was performed in 10-?l reaction mixtures
containing 1? FastStart Taq DNA polymerase mixture (Roche), 4 mM
MgCl2, 1 ?M concentrations of primers nTM17.F (5?-GTG GAT CTA TTG
TAT TAG ATG AGG CTC TCG-3?) and nTM17.R (5?-GCC AAA GTT
CTG CAA CAT TAA CAC CTA AAG-3?) (23), and 2 ?l of tick DNA extract.
An external standard template containing 10 to 105copies of the B. burgdor-
feri recA gene was included in each run to generate a standard curve. The
amplification program was performed as follows: (i) heating at 95°C for 10
min to activate the FastStart Taq polymerase; (ii) 45 cycles, with 1 cycle
consisting of increasing the temperature 20°C/s to 95°C and holding the
temperature at 95°C for 10 s, decreasing the temperature 20°C/s to 60°C and
holding the temperature at 60°C for 5 s, and increasing the temperature
20°C/s to 72°C and holding the temperature at 72°C for 10 s. The fluorescent
product was collected at 82°C at the last step of each cycle to minimize signal
from nonspecific products. A melting curve was acquired by heating the
product at 20°C/s to 95°C, cooling it at 20°C/s to 60°C, and slowly heating it
at 0.2°C/s to 95°C with fluorescence collection at 0.2°C intervals. The number of
spirochetes in each PCR mixture was calculated by comparing the crossing points
of the samples with those of the standards with the Lightcycler software. The
spirochete load in each tick was estimated by multiplying the number of spiro-
chetes determined in each PCR mixture by a factor of 25 (since only 2 ?l of a
50-?l DNA extract was used per reaction mixture). The specificity of qPCR
products was confirmed by melting curve analysis and/or gel-based post-PCR
analysis. A sample was considered positive only if it exhibited a log-linear (ex-
ponential) phase of amplification in the fluorescence curve and either it showed a
specific peak with the melting temperature at 85°C in the melting curve analysis
or, if post-PCR analysis was performed, a specific band with the expected size
was visible on a 1.5% agarose gel.
PCR amplification and RFLP analysis. A nested PCR which amplifies a
portion of the 16S-23S ribosomal DNA spacer of B. burgdorferi was performed as
previously described (18). The primers for the first round of PCR were PA
(5?-GGTAT GTTTA GTGAG GG-3?) and P95 (5?-GGTTA GAGCG CAGGT
CTG-3?) and would yield a 1,014-bp product on the basis of the sequence of
isolate B31 (10). Two microliters of a 1/100 dilution of the first-round PCR prod-
uct was employed as the template in the second round of PCR with primers PB
(5?-CGTAC TGGAA AGTGC GGCTG-3?) and P97 (5?-GATGT TCAAC TC
ATC CTGGT CCC-3?). Positive samples yielded a DNA fragment with a size of
approximately 940 bp that was subjected to restriction fragment length polymor-
phism (RFLP) analysis by digestion with MseI as described elsewhere (17, 18).
Statistical analysis. Quantitative data obtained by real-time qPCR were ana-
lyzed using Minitab (release 12; South College, Pa.) and Excel97 (Microsoft Corp.,
Redmond, Wash.) software. To examine the distribution of B. burgdorferi in infected
ticks, a histogram of spirochete counts from the PCR-positive nymphs (n ? 91) was
generated (Fig. 1A). As the distribution is skewed to the left and overdispersed,
the data were transformed to normally distributed data with a ln(x ? 10) trans-
formation (Fig. 1B) for parametric statistical analysis. A Kolmogorov-Smirnov
test was applied to verify that the transformed data satisfied the assumption of
normal distribution. The degree of spirochete aggregation among both infected
host-seeking ticks and all host-seeking ticks was quantified by calculating the
variance-to-mean ratios (s2/m) and the corrected moment estimates of k [k ?
(m2? s2/n)/(s2? m)] (12).
To determine whether the prevalence of B. burgdorferi infection differed in
nymphs in the geographic locations sampled, the percentage of ticks infected
from each of the eight collection locations was computed, a one-way analysis of
variance (ANOVA) was then performed by Tukey’s method to detect significant
differences in the prevalence of tick infection in different locations. ANOVA
using transformed data was also employed to determine whether the spirochete
load in infected ticks is correlated with geographic location or ribosomal spacer
restriction fragment length polymorphism type (RST) genotype. For all ANOVA
comparisons, the normality of the data (counts) was evaluated with a Kolmog-
orov-Smirnov test or binomial data were examined for central limit theorem
violations. We also evaluated variance equalities with Bartlett’s test. For report-
ing data, the mean number of spirochete loads and 95% confidence intervals
were back transformed. The spirochete numbers present in the 18.4% of infected
ticks with the lowest numbers of spirochetes, which corresponds to the reported
proportion unable to transmit in a previously published laboratory study (16),
was used to estimate a threshold number of spirochetes needed to transmit
infection from infected tick to reservoir host.
FIG. 1. Distribution of spirochete burdens in 91 B. burgdorferi-positive I. scapularis nymphs from the northeastern United States. The spirochete
burdens were determined by a real-time qPCR. (A) Before data transformation, the distribution is skewed to the left and overdispersed. (B) After
data transformation, the distribution is normal. The dark gray bars indicate the 18.4% of ticks with the fewest (?300) spirochetes.
4562WANG ET AL.APPL. ENVIRON. MICROBIOL.
Detection and quantification of B. burgdorferi in field-col-
lected ticks. A total of 498 field-collected I. scapularis ticks
from four northeastern states were tested by qPCR for the
presence of B. burgdorferi-specific DNA. Ninety-one of 438
(20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were
positive (P ? 0.05). The number of spirochetes in individual
ticks varied from 25 to 86,750 per infected nymph and from 350
to 197,200 per infected adult tick. The mean numbers of spi-
rochetes were 1,964 per infected nymphal tick and 5,351 per
infected adult tick (P ? 0.05). No significant differences in the
prevalence of B. burgdorferi infection in host-seeking I. scapu-
laris nymphs on the basis of collection location were detected
(P ? 0.23; df ? 4; n ? 345) (Table 1). Ticks from three sites in
New York (New Rochelle, Rye, and Yonkers) were excluded
from this analysis because these samples did not satisfy central
limit theorem assumptions. The differences in the spirochete
loads of infected nymphal ticks from different locations were
not significant (P ? 0.50; df ? 6; n ? 91).
Spirochete aggregation and threshold to transmit infection.
Of the 438 nymphal I. scapularis ticks analyzed in this study,
the untransformed arithmetic mean B. burgdorferi counts (used
to quantify aggregation) were 7,216 spirochetes in infected
ticks (n ? 91) and 1,499 spirochetes in all ticks (n ? 438).
There was a high degree of spirochete aggregation among
infected ticks (s2/m ? 24,877; k ? 0.279) and extreme spiro-
chete aggregation among all ticks (s2/m ? 30,390; k ? 0.047).
A previous experiment showed that only 81.6% (31 of 38) of
nymphal ticks infected with B. burgdorferi transmitted infection
to laboratory mice (16). The spirochete numbers present in the
18.4% of infected ticks with the lowest number of spirochetes,
which corresponds to the proportion unable to transmit infec-
tion was used to estimate a threshold number of spirochetes
needed to transmit infection from tick to reservoir host. As
shown in Fig. 1B, the 18.4% of infected ticks with the lowest
spirochete burdens harbored ?300 spirochetes each.
Spirochete loads in ticks infected with distinct genotypes of
B. burgdorferi. We have previously reported the typing of
B. burgdorferi based on PCR-RFLP of the 16S-23S ribosomal
DNA intergenic spacer (13). This approach was employed to
assess the genetic diversity among B. burgdorferi sensu stricto
isolates from patients with early Lyme disease; all clinical iso-
lates were classified into three genetically distinct groups, des-
ignated RST1, RST2, and RST3 (13, 17, 18). In this study,
nested PCR amplification and subsequent RFLP analysis of
the 16S-23S rRNA spacer amplicon was performed on 348 of
498 (69.9%) I. scapularis ticks. The distribution of B. burgdor-
feri genotypes and the spirochete load in 73 infected ticks for
which both genotyping and quantitative data were available are
summarized in Table 2. Twenty-six percent (19 of 73) of the
ticks were infected with the RST1 genotype of B. burgdorferi,
20.6% (15 of 73) were infected with RST2, and 39.7% (29 of
73) were infected with RST3; 13.7% of positive ticks had mixed
infection of two or more genotypes. The percentage of ticks
infected with the RST3 genotype was higher than those in-
fected with RST1 (P ? 0.07) and RST2 (P ? 0.01) genotypes.
However, no significant difference was found in the mean num-
ber of spirochetes in ticks infected with distinct genotypes of
B. burgdorferi (P ? 0.39; df ? 2; n ? 63).
Comparison of nested and real-time qPCR. First, both
nested PCR and qPCR were performed on DNA isolated from
348 ticks to determine the relative sensitivities of the methods.
Later, another 150 ticks were added to increase the sample size
from underrepresented locations for qPCR comparisons
among geographic locations. Of the 348 I. scapularis (288
nymphal and 60 adult) ticks subjected to both nested PCR of
the 16S-23S rRNA gene spacer and qPCR analysis, 78 (22.4%)
ticks were positive and 239 (68.7%) ticks were negative by both
methods, resulting in a concordance of 91.1% between nested
and real-time qPCR. Of the remaining 31 ticks, 13 ticks were
positive by nested PCR and 18 ticks were positive by qPCR.
The overall positivity rates of ticks were 26.1% (91 of 348) by
nested PCR and 27.6% (96 of 348) by qPCR (P ? 0.05),
indicating that the sensitivities of the two approaches were
similar for detection of B. burgdorferi DNA in infected ticks.
TABLE 1. Prevalence of B. burgdorferi infection and spirochete
loads in field-collected I. scapularis ticks from
northeastern United States
Tick stage and
Spirochete load in
New York sites
New Jersey6411 (17)1,675949 340–2,621
Total43891 (21) 2,925 1,9641,329–2,900
New York60 15 (25) 5,7505,351 2,470–11,592
aThe median numbers of spirochetes in infected ticks are based on original
data from real-time qPCR. Means and 95% confidence intervals (95% CI) were
TABLE 2. Genotype and spirochete loads in I. scapularis
nymphs infected with B. burgdorferia
No. of ticks
Spirochete load in infected ticksb
Median Mean95% CI
aSeventy-three (94%) of 78 PCR-positive ticks with RFLP data available were
bThe median numbers of spirochetes in infected ticks are based on original
data from real-time qPCR. Means and 95% confidence intervals (95% CI) were
VOL. 69, 2003QUANTITY OF B. BURGDORFERI IN TICKS4563
In this study, the prevalence of infection and quantity of
B. burgdorferi in 438 I. scapularis nymphal ticks and 60 adult
ticks collected from various locations in the northeastern
United States where Lyme disease is endemic were assessed
using a real-time qPCR targeting the B. burgdorferi-specific
recA gene. The sensitivity, specificity, and reproducibility of the
qPCR targeting the recA gene have been assessed and reported
previously (36). It is estimated that approximately 2,000 and
5,300 spirochetes were harbored in infected nymphal and adult
I. scapularis ticks, respectively. Assuming that the efficiency of
DNA extraction is not 100%, the actual number of spirochetes
in infected ticks may be somewhat higher than those calculated
in this study after measurement of B. burgdorferi-specific DNA
by qPCR. Despite this, the spirochete density detected by
qPCR in these field-collected I. scapularis ticks was compara-
ble to those previously determined by microscopy-based assay
and OspA antigen-capture ELISA (1, 3). In two early studies,
approximately 2,000 spirochetes were reported to be harbored
per nymphal tick collected from three sites in Massachusetts
where Lyme disease is endemic (1), and 4,000 spirochetes were
detected in adult ticks collected from Westchester County,
New York (3). More recently, Rauter et al. reported that there
were approximately 4,000 spirochetes per nymphal I. ricinus
tick from southern Germany as determined by qPCR (30).
Taken together, the data suggest that 2,000 to 4,000 spiro-
chetes on average are present in the midguts of host-seeking
nymphal ticks from a variety of areas where Lyme borreliosis is
In the present study, the sensitivity of the real-time qPCR
protocol was comparable to that of a well-established nested
PCR. However, the qPCR approach has certain advantages.
First, the specificity of the qPCR product can be verified simply
by analyzing the melting temperature after the PCR run. Ad-
ditionally, real-time PCR can be performed in less than 1 h in
a single capillary tube without opening the reaction tube,
thereby significantly reducing the PCR running time and prob-
ability of cross-contamination. Thus, real-time qPCR analysis
provides a rapid and reliable tool for the qualitative and quan-
titative analysis of B. burgdorferi sensu lato, and potentially
other vector-borne pathogens, in field-collected ticks and
should be useful for ecological and epidemiological surveil-
lance of tick-borne diseases.
We have previously reported on the genotypic distribution
of B. burgdorferi sensu stricto clinical isolates recovered from
skin biopsy specimens of patients with early Lyme disease in
Westchester County, New York, over a 7-year period (18). Of
183 skin isolates, 46 (25.1%) were type 1 (RST1), 70 (38.3%)
were type 2 (RST2), and 55 (30.1%) were type 3 (RST3); the
remaining 6.6% (12 of 183) were mixed cultures composed of
at least two genotypically distinct isolates. RST2 isolates were
cultured from skin biopsy specimens more frequently than
either of the other two genotypes (P ? 0.07 for comparison
between type 1 and type 2; P ? 0.013 for comparison between
type 2 and type 3) (18). In this study, 26% (19 of 73) of the ticks
were infected with RST1, 20.6% (15 of 73) were infected with
RST2, 39.7% (29 of 73) were infected with RST3 and 13.7%
were infected with a mixture of genotypes. The number of ticks
infected by the RST3 genotype was higher than the number of
ticks infected with either the RST1 (P ? 0.07) or RST2 (P ?
0.01) genotype. Although these differences in the distribution
of B. burgdorferi genotypes in isolates from ticks and patients
may simply be due to sampling error, these results may also
reflect differences in transmission of the distinct genotypes
from ticks to humans. An additional explanation for the ob-
served discrepancy may be differences in the pathogenicity of
distinct genotypes of B. burgdorferi. This would be consistent
with differences in pathogenicity observed for RST1 and RST3
isolates in a C3H/HeJ mouse model (37). As shown in this
study, comparable numbers of spirochetes were detected in
ticks infected with RST1 and RST3 genotypes of B. burgdorferi
(with means of 1,980 and 2,212, respectively; P ? 0.05). In
contrast, the spirochete load, which correlates directly with the
severity of disease, was at least twofold higher in the skin
biopsy specimens of patients and in heart and joint tissues of
mice infected with RST1 isolates than in those infected with
RST3 isolates (19, 36, 37).
Although aggregation of macroparasites is well-known from
the literature, microparasite aggregation has not been studied
in great detail (20). Here we report a high degree of B. burg-
dorferi spirochete aggregation in host-seeking I. scapularis
nymphs, with most infected ticks harboring few spirochetes
(?5,000) and a small proportion of ticks with very large spi-
rochete loads (?20,000) (Fig. 1A). Overdispersion is also the
predominant pattern for macroparasites, including ticks, which
are highly aggregated in nature (7, 29, 33). Our findings thus
suggest a pattern of nested aggregation, with B. burgdorferi
spirochetes aggregated in ticks and ticks aggregated in the
Aggregation of spirochetes in infected ticks may have im-
portant epidemiologic consequences. Heavily infected individ-
uals in the tail end of aggregated distributions typically play
a disproportionately large role in parasite transmission and
natural maintenance (39). Similarly, the more heavily infected
nymphs in the right tail of the frequency distribution (Fig. 1A)
may play a more important role in maintaining B. burgdorferi in
nature. Conversely, the nymphs with very few spirochetes may
play little or no role in the natural maintenance of B. burgdor-
feri. Levin and Fish (16) found that 18.4% of B. burgdorferi-
infected nymphs did not transmit spirochetes while feeding on
white-footed mice (Peromyscus leucopus), which are natural
reservoirs of infection. The number of organisms found in the
18% percent of infected ticks with the lowest number of spi-
rochetes is ?300 spirochetes (Fig. 1B), which may represent a
transmission threshold, since ticks infected with few spiro-
chetes cannot transmit infection (9). The mechanisms gener-
ating heterogeneous B. burgdorferi spirochete loads in ticks
remain to be discovered.
We conclude that geographic and RST differences are not
significant determinants of spirochete burden in ticks in north-
eastern United States. Pinchon et al. (26) found high hetero-
geneity in the number of Plasmodium falciparum gametocytes
in the blood meal of mosquitoes feeding on the same host and
an overdispersed distribution of gametocytes ingested by mos-
quitoes. Similarly, feeding behavior and spirochete mainte-
nance may vary in ticks feeding on the same host in a way that
generates an aggregated distribution of spirochete loads. Al-
ternatively, the species and spirochete burden of the vertebrate
host may influence the number of spirochetes acquired and
4564 WANG ET AL.APPL. ENVIRON. MICROBIOL.
maintained by ticks. Further studies are needed to evaluate the
influence of these possible mechanisms.
We thank Terry Schulze, Thomas Mather, and Kirby Stafford for
assistance in collection of ticks from Rhode Island, Connecticut, and
New Jersey and Michele Papero for preparation of DNA from these
This study is supported in part by NIH grant R01AR41511 (to I.S.),
USDA-ARS cooperative agreement 1265-32000-063-02S (to D.F.),
and CDC training fellowship in vector-borne diseases (to B.B.).
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