MAJOR ARTICLE
Inability of a Variant Strain of Anaplasma
phagocytophilum to Infect Mice
Robert F. Massung,1 Rachael A. Priestley,1 Nathan J. Miller,2 Thomas N. Mather,2 and Michael L. Levin1
1
Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; 2Center for Vector-Borne Disease,
University of Rhode Island, Kingston
Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. Polymerase chain reaction
and DNA sequencing were used to determine the presence and prevalence of Anaplasma phagocytophilum
human agent (AP-ha) and a genetic variant not associated with human disease (AP-variant 1). The remaining
ticks from each cohort were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or
DBA/2 (Mus musculus) mice. The engorged ticks and murine blood samples were evaluated for the presence
of AP-ha and AP-variant 1. Although a high percentage of the infecting ticks harbored AP-variant 1, only APha was amplified from the murine blood samples. Additional ticks were fed on immunocompromised SCID
mice, and, again, only AP-ha was capable of establishing an infection, and only AP-ha could be detected by
xenodiagnosis. These data suggest that AP-variant 1 cannot establish an infection in mice, and we propose
that AP-variant 1 has an alternative natural reservoir, possibly white-tailed deer.
Anaplasma phagocytophilum is an obligate intracellular
bacterium that exhibits a tropism for granulocytic leukocytes. Human infections have been referred to as
human granulocytic ehrlichiosis because of the previous
classification of the agent in the genus Ehrlichia [1].
Human ehrlichiosis presents as an acute febrile illness
and can be difficult to diagnose because of the lack of
specificity of the clinical signs and symptoms. In the
United States, the large majority of infections have occurred in 2 regions, the Upper Midwest and the Northeast, where the agent is transmitted to humans by the
tick vector Ixodes scapularis [2–4].
Previous studies have shown that the white-footed
mouse (Peromyscus leucopus) is a competent reservoir
for A. phagocytophilum [5–7]. However, on the basis of
serological assays or the molecular detection of A. phagocytophilum DNA in blood or tissue samples, other
small mammals (e.g., squirrels, voles, rats, and rac-
Received 13 February 2003; accepted 30 May 2003; electronically published 21
November 2003.
Presented in part: International Conference on Rickettsiae and Rickettsial
Diseases, Ljubljana, Slovenia, 4–7 September 2002 (abstract O-11).
Financial support: National Institutes of Health (grant AI-30733).
Reprints or correspondence: Dr. Robert F. Massung, Centers for Disease Control
and Prevention, 1600 Clifton Rd., MS G-13, Atlanta, GA 30333 ([email protected]).
The Journal of Infectious Diseases 2003; 188:1757–63
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18811-0019$15.00
coons) and large mammals (e.g., white-tailed deer and
horses) have also been suggested to be potential reservoirs [7–11]. Most of these molecular-based studies
have been based on polymerase chain reaction (PCR)
amplification of the 16S rRNA gene of the agent. DNA
sequencing of the A. phagocytophilum 16S rRNA gene
amplified from ticks in Connecticut and Rhode Island
and from white-tailed deer (Odocoileus virginianus) in
Maryland and Wisconsin detected a novel genetic variant of A. phagocytophilum, AP-variant 1 [8, 12, 13].
The 16S rRNA sequence of AP-variant 1 differs from
that originally described by Chen et al. [14], by 2 bp,
and the AP-variant 1 sequence has never been amplified
from a confirmed human infection. This suggests that
AP-variant 1 does not cause human infections and that
this strain may show additional biological or genetic
properties distinct from those of the human disease–
causing agent. AP-variant 1 was found to be common
in ticks in Rhode Island, where few cases of human
granulocytic ehrlichiosis have been reported, but rare
in Connecticut, where more cases have been reported
than in any other state [12]. The prevalence of APvariant 1 in Rhode Island may be due to a selective
advantage over the human agent (AP-ha), in either vector or reservoir populations. Despite the analysis of a
large number of blood samples from white-footed mice
from both Rhode Island and Connecticut, AP-variant
A. phagocytophilum Variant in Mice • JID 2003:188 (1 December) • 1757
1 has not been found in white-footed mice [12, 15]. The present
study was designed to test the infectivity of AP-variant 1 for
white-footed mice and for other commonly used strains of
laboratory mice. If infections can be established, these mice
would then provide blood and tissue samples for subsequent
attempts to obtain an isolate of the variant in tissue culture.
MATERIALS AND METHODS
Mouse strains. Animals used for experimental procedures
included the white-footed mouse and 2 in-bred strains of laboratory mice: immunocompetent DBA/2 and immunocompromised CB-17/SCID.
Tick collection, prevalence testing, and mouse infestation.
Questing nymphal black-legged ticks (I. scapularis) were collected
from 4 sites (Corrano, Tower, Trustom, and Webster) near South
Kingston, Rhode Island, in June 2001 by following standardized
sampling procedures [16]. To determine the prevalence of APha and AP-variant 1, ∼30 ticks from each site were immediately
used for DNA extraction, PCR testing, and DNA sequencing.
Additional ticks that were collected from each site were maintained in a humidified chamber. The prevalence of AP-ha and
AP-variant 1 in the flat ticks allowed us to calculate the number
of ticks necessary to ensure ⭐1 infected tick/mouse. This number
of ticks (6–8 Trustom ticks and 10–12 Corrano ticks) were placed
on each mouse and were allowed to feed to repletion. The engorged nymphs were frozen immediately and were stored at
⫺20C until used for DNA extraction.
Xenodiagnosis and collection of murine blood samples.
Uninfected, laboratory-reared I. scapularis larvae were placed
on mice on days 10 and 17 after mice had been subjected to
infestation with field-collected ticks; ticks were allowed to feed
to repletion. Engorged larvae were collected and were stored
in a humidified chamber until they molted to the nymphal
stage. DNA was extracted from these newly molted nymphs
and was used for PCR testing, and the amplicons from positive
samples were used for DNA sequencing. Blood samples were
collected from mice by retroorbital bleeding. Blood samples
were either used for DNA extractions immediately or stored at
⫺20C until DNA was extracted.
DNA extractions. DNA was extracted directly from blood
samples by use of a QIAamp DNA Blood Mini Kit (Qiagen);
the protocol followed was suggested by the manufacturer. In
brief, detergent lysis was performed in the presence of proteinase K, for 10 min at 70C. The lysed material was applied
to a spin column containing a silica gel–based membrane and
was washed twice. Purified DNA was eluted from the columns
in sterile distilled H2O and was stored at 4C until used as a
template for PCR amplification. DNA was extracted from I.
scapularis ticks by a modification of the manufacturer’s protocol
for the DNeasy Tissue Kit (Qiagen), as described elsewhere [13].
1758 • JID 2003:188 (1 December) • Massung et al.
PCR analysis. A nested PCR assay that amplified a 546bp portion of the 5 region of the 16S rRNA gene was used to
identify A. phagocytophilum in tick and murine blood samples
[13]. In brief, primary amplifications consisted of 40 cycles in
an ABI GeneAmp PCR System 9700 thermal cycler (Applied
Biosystems), with each cycle consisting of denaturation for 30
s at 94C, annealing for 30 s at 55C, and extension for 1 min
at 72C. The 40 cycles were preceded by denaturation for 2
min at 95C and were followed by extension for 5 min at 72C.
Primary amplifications used primers ge3a and ge10r and reagents from the Qiagen Taq PCR Master Mix Kit (Qiagen).
Each reaction contained 2.5 mL of purified DNA as template
in a total volume of 25 mL and 200 mmol/L each dNTP (dATP,
dCTP, dGTP, and dTTP), 1.25 U of Taq polymerase, and 0.5
mmol/L each primer. Reaction products were subsequently
maintained at 4C until used as a template for nested reactions.
Nested amplifications used primers ge9f and ge2 and 1 mL
of the primary PCR product as a template, in a total volume
of 50 mL. Each nested amplification contained 200 mmol/L each
dNTP (dATP, dCTP, dGTP, and dTTP), 1.25 U of Taq polymerase, and 0.2 mmol/L each primer. Nested cycling conditions
were as described for the primary amplification, except 30 cycles
were used. Reactions were subsequently maintained at 4C until
analyzed by agarose-gel electrophoresis or purified for DNA
sequencing.
DNA sequencing and data analysis. All samples producing positive PCR products were subjected to DNA sequencing
reactions by use of fluorescent-labeled dideoxynucleotide technology (BigDye Terminator Cycle Sequencing Ready Reaction
Kit; Applied Biosystems). Sequencing reaction products were
separated, and data were collected by use of an ABI 3100 Genetic Analyzer automated DNA sequencer (Applied Biosystems). To ensure maximum accuracy of the data, the full sequence was determined for both strands of each DNA template.
Sequences were edited and assembled by the Staden software
programs [17] and were analyzed by the Wisconsin Sequence
Analysis Package (Genetics Computer Group) [18]. The partial
16S rRNA sequence for AP-variant 1 has been deposited in
GenBank (accession no. AY193887).
RESULTS
Determination of prevalence of AP-ha and AP-variant 1 in
ticks. PCR and DNA sequencing of the 16S rRNA gene was
used to determine the prevalence of AP-ha and AP-variant 1
in a subset of cohorts of questing nymphal I. scapularis ticks
(n p 116) that had been collected from 4 sites in Rhode Island.
Each site tested had previously been shown to have high tick
and reservoir densities. The results of this preliminary testing
are shown in table 1. The Trustom site had both the highest
total prevalence of infected ticks (25.8%) and the highest prev-
alence of AP-variant 1 (22.6%). No variants of A. phagocytophilum other than AP-variant 1 were found in any of the ticks.
Because AP-ha was not detected at either the Tower or Webster
sites, ticks from these 2 sites were not used for subsequent
feedings on mice.
Tick infestations of mice. Nymphal ticks collected from the
Corrano (10–12 ticks/mouse) and Trustom (6–8 ticks/mouse)
sites were placed on A. phagocytophilum–naive laboratory-bred
mice (white footed [n p 32] or DBA/2 [n p 30]), were allowed
to feed to repletion, and were collected in water pans. A total of
337 engorged ticks were collected and tested by PCR and DNA
sequencing for the presence of AP-ha and AP-variant 1 (tables
2–5). The number of ticks collected from individual mice ranged
from 1 to 10. The DNA sequencing results from the engorged
ticks identified which agents were presented to each mouse. A
total of 32 of the engorged nymphs were positive for AP-variant
1, and 15 were positive for AP-ha. A total of 3 of the AP-ha–
infected mice (DBA-7, DBA-8, and DBA-24) had ticks without
molecular evidence of AP-ha infection (tables 3 and 4).
Detection of A. phagocytophilum in murine blood samples. EDTA-anticoagulated whole blood samples were collected from each mouse on days 10 and 17 after tick attachment
and were tested for AP-ha and AP-variant 1. The results of this
testing are shown in tables 2 (Trustom ticks fed on white-footed
mice), 3 (Trustom ticks fed on DBA/2 mice), and 4 (Corrano
ticks fed on either white-footed or DBA/2 mice). Although 12
white-footed mice were fed upon by PCR-positive Trustom
ticks (table 2), only 1 of these ticks was positive for AP-ha, and
none of the mice had positive results for either agent, by PCR
testing of blood samples. Five DBA/2 mice were positive by
PCR after having been fed upon by Trustom ticks (table 3),
and the blood samples from each of these mice contained only
AP-ha. Four white-footed and 3 DBA/2 mice were positive by
PCR after having been upon by Corran ticks (table 4), and the
blood samples from each of these mice contained only AP-ha.
In total, 12 mice (4 white footed and 8 DBA/2) became infected,
and each of these infections was with AP-ha. Of the 12 mice
exposed to AP-ha, on the basis of the engorged-tick results, 9
became infected with AP-ha. In contrast, of the 22 mice exposed
to AP-variant 1, none became infected.
Infections in SCID mice. Considering that the mouse immune response may be responsible for our inability to detect
an infection by AP-variant 1 in immunocompetent whitefooted and DBA/2 mice, additional field-collected ticks were
placed on 5 SCID mice (8 Trustom ticks/mouse, 3 mice; and
13 Corrano ticks/mouse, 2 mice). As described above, ticks and
blood samples from mice were tested for AP-ha and AP-variant
1, by PCR and DNA sequencing (table 5). Although each of
the 5 mice was fed upon by at least 1 infected tick, 2 mice were
fed upon by ticks that were positive for both AP-ha and APvariant 1 (SCID-3 and SCID-4), and both of these mice became
positive for AP-ha only. These 2 mice had positive results for
AP-ha in both blood samples collected (day 5, both mice; day
12, SCID-3; day 14, SCID-4). Although each of the 5 SCID
mice was fed upon by a tick positive for AP-variant 1, only 1
mouse (SCID-1) became positive for AP-variant 1, and this
positive result by PCR was of the blood sample drawn at the
early time point (day 5) only. The sample drawn on day 14
yielded negative results.
Xenodiagnosis of SCID mice. Because it appeared that
AP-variant 1 may have transiently infected 1 of the 5 SCID
mice, xenodiagnosis was performed on each of these mice.
Uninfected, laboratory-reared I. scapularis larvae were placed
on mice on day 7 after nymphal attachment and were allowed
to feed to repletion. Engorged larvae were allowed to molt to
nymphs, and DNA was extracted from pools of 5 nymphs and
was tested by PCR and DNA sequencing. All 4 pools of ticks
that had fed on the mouse positive for AP-variant 1, SCID-1,
were negative. In contrast, each of the 8 pools that had fed on
the mice positive for AP-ha, SCID-3 and SCID-4, were positive
for AP-ha. Tick pools that had fed on the PCR-negative mice,
SCID-2 and SCID-5, were negative.
DISCUSSION
Several apparent discrepancies between the results for engorged
ticks and those for murine blood samples are evident in tables
3 and 4. For example, 3 mice (DBA-7, DBA-8, and DBA-24)
became positive for AP-ha, even though each of the engorged
nymphs tested were negative for AP-ha. Although a fixed number of ticks were placed on each mouse, only ticks that fed to
repletion and dropped off unassisted were collected and tested.
Numerous partially fed ticks were not included in the analysis
because the mice removed them while grooming themselves.
Therefore, the infections in these 3 mice were likely to have
been introduced by partially fed ticks that were not collected.
Three additional mice (PL-6, PL-23, and DBA-9) were negative
for AP-ha when tested on both days 7 and 14 but had been
Table 1.
Prevalence of Anaplasma phagocytophilum human
agent (AP-ha) and A. phagocytophilum variant 1 (AP-variant 1),
in nymphal Ixodes scapularis ticks collected from 4 sites in Rhode
Island.
Collection site
Corrano
No. tested
Total positive
by PCR
AP-ha–positive
nymphs
AP-variant
1–positive
nymphs
31
3 (9.7)
2 (6.5)
1 (3.2)
Tower
26
1 (3.8)
0
1 (3.8)
Trustom
31
8 (25.8)
1 (3.2)
7 (22.6)
Webster
28
4 (14.3)
0
4 (14.3)
NOTE.
reaction.
Data are no. (%), unless otherwise noted. PCR, polymerase chain
A. phagocytophilum Variant in Mice • JID 2003:188 (1 December) • 1759
Table 2. Results from ticks collected at the Trustom site that
fed to repletion on naive white-footed mice and from blood samples from white-footed mice.
a
Mouse
Engorged
nymphs
collected
AP-ha–positive
nymphs
AP-variant
1–positive
nymphs
Results of PCR
testing of murine
b
blood samples
AP-ha
AP-variant 1
PL-3
5
0
2
⫺
⫺
PL-4
6
0
1
⫺
⫺
PL-5
7
0
1
⫺
⫺
PL-6
7
1
2
⫺
⫺
⫺
PL-7
8
0
1
⫺
PL⫺8
5
0
1
⫺
⫺
PL-10
7
0
1
⫺
⫺
PL-12
4
0
1
⫺
⫺
PL-13
6
0
2
⫺
⫺
PL-15
4
0
2
⫺
⫺
PL-17
6
0
2
⫺
⫺
PL-21
6
0
1
⫺
⫺
NOTE. ⫺, Negative; AP-ha, Anaplasma phagocytophilum human agent;
AP-variant 1, A. phagocytophilum variant 1; PCR, polymerase chain reaction;
PL, white-footed mice.
a
Only mice fed upon by ticks that were positive or mice that had positive
results by PCR testing of blood samples are included.
b
Blood samples collected on day 10 and day 17 were tested by PCR; a
positive result on either sample was considered to be positive.
fed upon by at least 1 tick that tested positive for AP-ha. These
negative PCR results of murine blood samples may be due to
collecting samples outside the window of infection, inefficient
transmission of the agent from the tick to the rodent during
feeding, or lack of sensitivity of the assay used for analysis.
However, the latter seems unlikely, because the PCR assay used
for analysis has been shown to detect !2 copies of the 16S
rRNA gene [13].
In the attempts to infect SCID mice, 1 mouse was positive
by PCR for AP-variant 1 on day 5 after nymphal attachment.
This was the only mouse that was positive by PCR for APvariant 1 throughout the course of the present study. However,
this mouse was negative by PCR on day 14, and xenodiagnostic
tick testing also yielded negative results. Therefore, it may be
that the AP-variant 1 DNA that was amplified on day 5 was
derived from the organisms that were injected by a feeding tick
and not cleared, probably because of the compromised immune
system of this mouse rather than replication of the agent in
the mouse. This inference is supported by the negative results
by xenodiagnosis. Alternatively, a situation may have occurred
wherein AP-variant 1 underwent limited replication in this
SCID mouse and the infection was cleared before day 14 and
xenodiagnostic testing.
Our results demonstrating the inability of ticks harboring
AP-variant 1 to transmit the agent to mice can lead to 1 of 2
conclusions: (1) that mice are not susceptible to infection or
1760 • JID 2003:188 (1 December) • Massung et al.
(2) that AP-variant 1 in the ticks was not viable. However,
several factors strongly suggest that the latter was not the case.
Quantitative PCR on the flat nymphal ticks indicated that the
number of copies of AP-variant 1 DNA in the AP-variant 1–
infected ticks was comparable to the amount of AP-ha in APha–infected ticks, 120,000 copies/tick (data not shown). Any
agent detected in a flat nymphal tick could come only from
the previous life stage (larvae). The nymphs in the present study
were collected in early June, which means that they had fed as
larvae in the late summer or early fall of the previous year.
Therefore, the agent that was detected in the flat nymphs was
maintained in these ticks for at least 8 months and through
the molt from larval to nymphal stage. Additionally, quantitative PCR showed significantly higher average amounts (17fold increase) of AP-variant 1 in engorged ticks than in flat
ticks, suggesting replication of the agent induced by tick feeding
(data not shown). On the basis of these factors, we conclude
that the AP-variant 1 that was present in the ticks was viable
and capable of infecting a susceptible host.
Because A. phagocytophilum is not transmitted transovarially
from 1 tick generation to the next, the maintenance of this
species in nature requires competent reservoirs and efficient
vectors. I. scapularis ticks have been shown to be an efficient
vector [2, 4, 6], and various species of large and small mammals
have been examined as potential reservoirs [7–11, 19]. AlTable 3. Results from ticks collected at the Trustom site that
fed to repletion on naive DBA/2 mice and from blood samples
from DBA/2 mice.
a
Mouse
Engorged
nymphs
collected
AP-ha–positive
nymphs
AP-variant
1–positive
nymphs
Results of PCR
testing of murine
b
blood samples
AP-ha
AP-variant 1
DBA-2
4
0
2
⫺
⫺
DBA-3
5
0
3
⫺
⫺
DBA-4
5
0
1
⫺
⫺
DBA-5
5
1
2
+
⫺
DBA-7
1
0
0
+
⫺
DBA-8
3
0
1
+
⫺
DBA-9
4
1
0
⫺
⫺
DBA-11
5
0
1
⫺
⫺
DBA-14
5
1
0
+
⫺
DBA-15
4
0
1
⫺
⫺
DBA-16
6
0
2
⫺
⫺
DBA-17
2
1
0
+
⫺
DBA-18
5
0
1
⫺
⫺
NOTE. +, Positive; ⫺, negative; AP-ha, Anaplasma phagocytophilum human agent; AP-variant 1, A. phagocytophilum variant 1; PCR, polymerase chain
reaction.
a
Only mice fed upon by ticks that were positive or mice that had positive
results by PCR testing of blood samples are included.
b
Blood samples collected on day 10 and day 17 were tested by PCR; a
positive result on either sample was considered to be positive.
Table 4. Results from ticks collected at the Corrano site that
fed to repletion on either naive white-footed or DBA/2 mice and
from blood samples from white-footed or DBA/2 mice.
Results of PCR
testing of murine
b
blood samples
Mouse
Engorged
nymphs
collected
PL-22
10
1
0
+
⫺
PL-23
7
3
0
⫺
⫺
a
AP-ha–positive
nymphs
AP-variant
1–positive
nymphs
necticut and Rhode Island and in white-tailed deer in Maryland
and Wisconsin [8, 12, 13]. AP-variant 1 has not been reported
from studies of blood samples from white-footed mice. However, most of these studies were based on serological detection
methods that would likely not differentiate AP-ha from APvariant 1. Our results clearly demonstrate that AP-variant 1
does not infect either white-footed mice or a commonly used
laboratory strain, DBA/2. These data suggest that white-footed
mice are not a natural reservoir for AP-variant 1 and that other
reservoir species are required for maintenance of this strain in
the wild. The detection of AP-variant 1 DNA in white-tailed
deer suggests that they may serve as a primary reservoir for
this agent. In a previous study of 32 blood samples collected
from white-tailed deer in Maryland, each of the 3 samples with
positive results contained AP-variant 1 [13]. In contrast, the
human agent, AP-ha, has never been detected in a white-tailed
deer [8, 13]. Maintenance of a natural cycle of infection of APvariant 1 with deer as the only reservoir is a distinct possibility,
because white-tailed deer are a host for all active stages of I.
scapularis ticks (larval, nymphal, and adult) [21].
The known cocirculation of the AP-ha and AP-variant 1
strains of A. phagocytophilum in I. scapularis ticks has several
implications. Because AP-variant 1 seems to be the predominant strain in areas where both strains are found, it has been
previously suggested that the variant may have a competitive
advantage in nature, in either the reservoir or vector [12]. Because we have shown that AP-variant 1 will not infect whitefooted mice, the presumed primary reservoir of AP-ha, I. scapularis ticks, remain the only known common factor in the
natural cycles of both strains. Therefore, if a competitive advantage does exist for AP-variant 1, it would likely occur in
the tick. However, testing of this hypothesis in the laboratory
AP-ha
AP-variant 1
PL-27
8
1
0
+
⫺
PL-28
6
1
0
+
⫺
PL-30
8
1
0
+
⫺
PL-31
4
0
1
⫺
⫺
DBA-23
8
1
0
+
⫺
DBA-24
7
0
0
+
⫺
DBA-26
7
2
0
+
⫺
NOTE. +, Positive; -, negative; AP-ha, Anaplasma phagocytophilum human
agent; AP-variant 1, A. phagocytophilum variant 1; PCR, polymerase chain
reaction; PL, white-footed mice.
a
Only mice fed upon by ticks that were positive or mice that had positive
results by PCR testing of blood samples are included.
b
Blood samples collected on day 10 and day 17 were tested by PCR; a
positive result on either sample was considered to be positive.
though these studies have shown that deer, raccoons, squirrels,
chipmunks, dogs, and other mammals can be infected with A.
phagocytophilum in nature, only the white-footed mouse has
been involved in controlled laboratory studies of reservoir
competence, and A. phagocytophilum strains used in these
studies were either isolated from a human patient or later classified as AP-ha by DNA sequencing [6, 20]. More-recent studies
have suggested that a mixed population of A. phagocytophilum
strains, with varying host tropisms, may exist in nature [12].
AP-variant 1 has been described in I. scapularis ticks in Con-
Table 5. Results from field-collected nymphal ticks that fed on SCID mice, from blood samples from
SCID mice, and from xenodiagnostic ticks.
Mouse
Collection site
Results of PCR
testing of murine
a
blood samples
Engorged
nymphs
collected
AP-ha–positive
nymphs
AP-variant
1–positive
nymphs
AP-ha
AP-variant 1
⫺
+
c
Xenopositive
b
tick pools
SCID-1
Trustom
4
0
1
0/4
SCID-2
Trustom
3
0
1
⫺
⫺
0/4
SCID-3
Trustom
6
1
1
+
d
⫺
4/4
SCID-4
Corrano
11
1
1
+d
⫺
4/4
SCID-5
Corrano
9
0
1
⫺
⫺
0/4
NOTE.+, Positive; ⫺, negative; AP-ha, Anaplasma phagocytophilum human agent; AP-variant 1, A. phagocytophilum variant
1; PCR, polymerase chain reaction.
a
Blood samples collected on day 5 and day 14 were tested by PCR; a positive result for either sample was considered to
be positive.
b
Xenodiagnostic larvae were placed on mice on day 7 after nymphal tick infestation; each pool tested contained 5 ticks
that had successfully molted to nymphs; each of the PCR-positive tick pools contained only AP-ha.
c
Positive on day 5 and negative on day 14.
d
Positive on both day 5 and day 14.
A. phagocytophilum Variant in Mice • JID 2003:188 (1 December) • 1761
will require an isolate of AP-variant 1, which is currently not
available. The tick source for infecting laboratory-reared mice
was host-seeking I. scapularis nymphs that had fed a single time
as larvae, collected from either of 2 sites in Rhode Island. Both
AP-ha and AP-variant 1 were present at each of these sites, as
evidenced by the detection of both strains in flat ticks. However,
no dual infections were detected in any of the flat (n p 116)
or engorged nymphs (n p 337) that were tested. These results
suggest several possibilities, one being that the larvae fed on
reservoirs harboring only 1 strain. In this scenario, ticks that
had fed on either white-footed mice or other reservoirs that
harbored only AP-ha would be positive for AP-ha, and ticks
that had fed on white-tailed deer or other reservoirs that contained only AP-variant 1 would be positive for the AP-variant
1. Alternatively, larvae may have fed on reservoirs that support
both AP-ha and AP-variant 1, but 1 strain precludes growth
of the other in ticks. The latter may result from a competitive
advantage of 1 strain over the other, as mentioned above, or
could be an artifact related to the relative number of organisms
of each strain that were ingested by the tick. In the case of a
mixed infection, the PCR assay used for detection will favor
the predominant agent. For example, when templates are mixed
at a 1:9 ratio and amplified in a single reaction, sequencing of
the amplicon will not detect the lower-concentration template
(data not shown). Because these ticks were field-collected and
likely to have fed as larvae on various hosts, a combination of
these factors may be involved in our inability to detect dual
infections in ticks. However, in a previous study in which 232
adult I. scapularis ticks from the Trustom site in Rhode Island
were tested, no dual infections were detected among the 52
ticks with positive results by PCR that were analyzed by DNA
sequencing [12]. Because these were adult ticks, they had been
fed 2 blood meals and were potentially exposed to infection
twice. This suggests that ticks may be resistant to superinfection
by 11 A. phagocytophilum strain. There is a precedent for resistance to superinfection among Anaplasma species, because
a recent study by de la Fuente et al. [22] has demonstrated that
Anaplasma marginale, the etiologic agent of bovine anaplasmosis, resists superinfection in both cultured tick cells and
bovine erythrocytes. In the case of AP-variant 1, if white-tailed
deer are the primary reservoir and infected ticks are resistant
to superinfection, larvae feeding on deer positive for AP-variant
1 will acquire the agent and be resistant to AP-ha at subsequent
stages. If this is true, an increased prevalence of AP-variant 1
in the deer population, combined with the steadily increasing
deer population in many areas of the United States, could affect
the distribution and prevalence of AP-ha in nature. However,
additional studies are needed to confirm the reservoir competence of white-tailed deer for AP-variant 1, to examine the
reservoir potential of other species of small and large mammals,
and to understand the interaction of AP-ha and AP-variant 1
1762 • JID 2003:188 (1 December) • Massung et al.
in I. scapularis ticks and in both known and potential reservoir
populations.
Last, the 2-bp difference in the 16S rRNA gene sequences,
between AP-ha and AP-variant, 1 serves as a molecular marker
that allows us to differentiate these 2 strains but is unlikely to
result in any biological significance. The altered reservoir competence of these strains more likely results from changes in
genes encoding structural and outer-membrane proteins that
may affect the tissue tropism and infectivity of the agent in a
particular host. An isolate of AP-variant 1 will be instrumental
for facilitating future genetic and biological studies that will
allow us to assess the factors that make AP-variant 1– and APha–unique strains of A. phagocytophilum.
Acknowledgments
We thank Kim Slater, Danielle Ross, and Khadeja Haye, for
excellent technical assistance; Gregory Dasch, for manuscript
review; and the Centers for Disease Control and Prevention
Biotechnology Core Laboratory, for the synthesis of DNA primers used in this study.
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