Download Age-Associated Decline in Resistance to Babesia microti Is

MAJOR ARTICLE
Age-Associated Decline in Resistance to Babesia
microti Is Genetically Determined
Edouard Vannier,1 Ingo Borggraefe,1,a Samuel R. Telford, III,2,a Sanjay Menon,1 Timothy Brauns,3 Andrew Spielman,2
Jeffrey A. Gelfand,3 and Henry H. Wortis3
1
Division of Infectious Diseases, Tufts–New England Medical Center, 2Department of Immunology and Infectious Diseases, Harvard School
of Public Health, and 3Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts
Background. Although infection by the protozoan Babesia microti is rarely symptomatic in immunocompetent
young people, healthy individuals aged 150 years may experience life-threatening disease. To determine the basis
for this age relationship, we developed a mouse model of babesiosis using a novel clinical isolate of B. microti.
Methods. Mice were infected at 2, 6, 12, or 18 months. Parasitemia was monitored on Giemsa-stained blood
smears or by flow cytometry.
Results. In DBA/2 mice, early and persistent parasitemias increased with age at infection. BALB/c and C57BL/
6 mice were resistant, regardless of age, which indicates that allelic variation determines resistance to B. microti. Unlike
immunocompetent mice, SCID mice, which retain an innate immune system but lack the lymphocytes needed for
adaptive immunity, developed high and persistent levels of parasitemia that were markedly reduced by transfer of
naive BALB/c or DBA/2 splenocytes. BALB/c cells reduced the persistent parasitemia to a greater extent than did
age-matched DBA/2 cells. Of importance, there was an age-associated loss of protection by cells of both strains.
Conclusion. The resistance to B. microti infection conferred by the adaptive immune system is genetically
determined and associated with age. We postulate that there are age-related differences in the expression of alleles
critical for adaptive immunity to B. microti.
Babesiosis is a malaria-like parasitic disease that was
traditionally regarded as an infection of wild and domestic animals. Only in the last 45 years have Babesia
species been acknowledged to be pathogenic in humans
[1]. Babesiosis is now recognized as an emerging infectious disease in New England. Cases of Babesia microti infection have been reported from Nantucket, eastern Connecticut, Block Island [2], Long Island, and
south to New Jersey [3]. As with Lyme disease, babesiosis has been reported in the upper Midwest (Wis-
Received 15 September 2003; accepted 30 October 2003; electronically published
19 April 2004.
Financial support: Earl P. Charlton Research Fund (to E.V.); Tufts–New England
Medical Center Research Fund; Alfond Family Research Fund; Edmund C. Lynch Jr.
Research Fund (to J.A.G.); National Institutes of Health (NIH; grants RO3 AG18076
to J.A.G. and RO1 AG19781 to H.H.W.); Eshe Family Fund (to H.H.W.); Deutscher
Akademischer Austauschdienst (to S.M.).
a
Present affiliations: Center for Developmental Neurology, Children’s Hospital,
University of Munich, Munich, Germany (I.B.); Division of Infectious Diseases,
Department of Biomedical Sciences, Tufts University School of Veterinary Medicine,
North Grafton, Massachusetts (S.R.T.).
Reprints or correspondence: Dr. Henry Wortis, Dept. of Pathology, Tufts University
School of Medicine, 150 Harrison Ave., Boston, MA 02111 ([email protected]).
The Journal of Infectious Diseases 2004; 189:1721–8
2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/18909-0023$15.00
consin, Minnesota, and Missouri) and the coastal
regions of northern California and Washington [1]. The
risk of babesiosis is worldwide and growing [4, 5].
Severe babesiosis is usually encountered in young
immunocompromised patients (who have HIV or who
are receiving immunosuppressive or cancer chemotherapy) [6] or subjects who have undergone splenectomy [7]. However, these cases are rare in the United
States, and severe clinical disease is most often seen in
healthy individuals aged ⭓50 years [2, 8]. This ageassociated increase in morbidity is not caused by an
increased risk of acquiring infection [9]. The severity
of symptoms and the rate of hospitalization are higher
in older individuals [10]. In one study of hospitalized
patients with babesiosis, 25% were admitted to the intensive care unit, 25% required hospitalization for 114
days, and 6.5% died [8]. The mean age upon admission
was 62 years. In a more recent study, 21% of the hospitalized patients developed acute respiratory failure,
18% had disseminated intravascular coagulation, 12%
underwent congestive heart failure, and 9% died [11].
The mean age was 53 years. Even with adequate treatment, low-grade infection may persist for 12 years and
Aging and B. microti Infection • JID 2004:189 (1 May) • 1721
flare during cancer chemotherapy [2]. Whether aged individuals relapse remains to be studied.
There has been a single, unsuccessful attempt to show agerelated differences in susceptibility to a laboratory mouseadapted strain of B. microti [12]. In the present study, we report
the use of a novel B. microti clinical isolate that reliably infects
several inbred mouse strains. We determined whether mice of
different ages and strains differ in their ability to control infection. In addition, we determined whether age-related differences in the adaptive immune system contribute to the observed differences in age-related susceptibility to babesiosis.
MATERIALS AND METHODS
Mice. DBA/2J and BALB/cBy mice aged 2, 6, 12, and 18
months were purchased from the National Institute on Aging.
C57BL/6 mice at these ages were provided by Dr. Symin Meydani (Jean Mayer USDA Human Nutrition Research Center on
Aging at Tufts University, Boston). For each set of experimental
infections, mice of the 3 inbred lines were obtained at different
ages and infected simultaneously. The experiment was repeated
once or twice, to ensure the reproducibility of our infection
protocol. Two-month-old C.B.17-SCID mice were purchased
from Taconic. All mice were maintained under specific pathogen–free conditions in clean, well-tended quarters at the Division of Laboratory Animal Medicine (Tufts University). All
mice were provided with water and chow ad libitum. For preparation of parasite-infected red blood cells (pRBCs), (C57BL/
6⫻129S6) F1 mice (Taconic) were maintained at the Animal
Facilities of the Harvard School of Public Health (Boston). The
Division of Laboratory Animal Medicine at Tufts University
approved all experimental designs.
Preparation of pRBCs. (C57BL/6⫻129S6) F1 mice were
exposed to 5–10 Ixodes scapularis (also known as “I. dammini”)
nymphs infected with the RM/NS strain of B. microti. This
strain was isolated in 1997 from a Nantucket Island resident
and was directly infectious to laboratory mice (Mus musculus
domesticus); the strain is maintained by alternating blood transfer and tick transmission. Partial sequence analysis of the 18S
and b-tubulin genes indicated that RM/NS belongs to the clade
of B. microti (S. R. Telford, III, in press). Parasitemia was monitored by analysis of Giemsa-stained blood smears beginning
at 7 days after the ticks had detached from their hosts. When
parasitemia reached 1% (sigmoid growth), blood samples were
harvested into Alsever’s solution. Absolute parasitemia was calculated, and blood was diluted in PBS until the desired number
of pRBCs was contained within 0.2 mL. pRBCs were delivered
by intraperitoneal injection.
Assessment of parasitemia. In all experiments but 2, the
degree of infection was assessed by analysis of the Giemsastained blood smears at 3–4-day intervals. We determined the
1722 • JID 2004:189 (1 May) • Vannier et al.
percentage of pRBCs (parasitemia expressed in percentages) by
counting the number of RBCs containing 1, 2, or 4 merozoites/
100 RBCs. A trained clinical laboratory technician, unaware of
the source of each sample, conducted the counting.
In the remaining 2 experiments, pRBCs were detected by use
of a flow cytometry–based assay. This assay, which makes use
of the nucleic-acid staining dye YOYO-1 (Molecular Probes),
is highly reproducible and correlates very well with counts of
B. gibsoni–infected RBCs obtained by microscopy [13]. We have
made some minor modifications in this technique. In brief, 1
drop of blood was collected in 300 mL of PBS containing 16
IU/mL of heparin. Cells were fixed in glutaraldehyde (0.025%)
for 30 min at room temperature, permeabilized in the presence
of Triton X-100 (0.25%) for 5 min at room temperature, and
treated with heat-inactivated pancreatic RNAse (100 mg/mL;
Roche Molecular Biochemicals) for 15 min at 37C. The latter
step minimizes false-positive results that are caused by the presence of RNA in reticulocytes (and other nucleated cells). Then,
cells were incubated for 10 min at room temperature with the
fluorescent nucleic-acid staining dye YOYO-1 (20 nmol). Samples were analyzed in a FACSCalibur flow cytometer (Becton
Dickinson) by use of an Argon-Ion laser for excitation (488
nm). To validate our nucleic acid–based detection method, parasitemia was assessed in 108 blood samples by use of light
microscopy of Giemsa-stained blood smears and by use of flow
cytometry. The coefficient of correlation was 0.96 (99% confidence limits, 0.935–0.976) with a slope of 0.6, which indicates
that the values obtained by microscopy are 40% lower than
those obtained by flow cytometry (data not shown).
Purification and transfer of spleen cells. Spleens were harvested postmortem in a sterile fashion, and spleen cells were
isolated, as described elsewhere [14]. Thirty to 60 min before
spleen cell transfer, mice received an intraperitoneal injection
of heparin (10 IU), to prevent emboli. Mice were gently heated
for 2 min, and the cell suspension (20 million cells in 0.4 mL)
was slowly delivered into a tail vein.
Statistical analysis. In each age group, levels of parasitemia were expressed as mean SEM and were reported as a
function of time (figure 1). Because the levels of parasitemia
in a given group of mice were not always synchronous, we
assessed the parasite burden in each mouse by measuring the
area under the curve or integral. Then, we calculated the mean
daily levels of parasitemia by dividing the integral of parasitemia
by days in the period examined. In experiments comparing the
3 inbred strains of mice (figure 1), we arbitrarily designated
days 7–35 as the “early” and days 35–98 as the “late” phases
of infection. In the transfer experiments, the length of the early
and late phases of infection increased as the parasite load at
time of infection decreased, that is, the early phase ended on
day 35 when mice were infected with 100,000 or 10,000 pRBCs,
on day 42 when infected with 1000 pRBCs, and on day 56
Figure 1. With increasing age, DBA/2 mice lose their ability to control and resolve Babesia microti infection. DBA/2 mice (A–D) were compared with
C57BL/6 (E–H) and BALB/c mice (I–L) for their resistance to B. microti infection. Mice were infected intraperitoneally with 105 parasite-infected red blood
cells (pRBCs) at 2 months (A, n p 6; E, n p 5; and I, n p 6), 6 months (B, n p 5; F, n p 5; and J, n p 6), 12 months (C, n p 8; G, n p 7; and K,
n p 4), or 18 months (D, n p 6; H, n p 6; and L, n p 6) of age. Parasitemia is the percentage of pRBCs, as determined by morphological analysis of
Giemsa-stained blood smears. Parasitemia is plotted against time after infection. Data are mean SEM.
when infected with 100 pRBCs. The late phase ended on day
98 in all experiments, with the exception of the infection with
100,000 pRBCs, which was terminated on day 73. Differences
in mean daily levels of parasitemia among the groups were
analyzed for statistical significance by use of one-way analysis
of variance using Fisher’s least significant difference test for
multiple post hoc comparisons (StatView; Abacus Concepts).
P ! .05 was considered to be statistically significant.
RESULTS
Decreased ability of DBA/2 mice to control and resolve B.
microti infection. Mice were infected with 105 pRBCs at 2,
6, 12, or 18 months of age. Parasitemia was monitored over a
98-day period (figure 1). None of these mice died during the
course of infection. We excluded 24-month-old mice from our
study, because DBA/2 mice are prone to lymphoproliferative
disorders as they age beyond 21 months.
In DBA/2 mice infected at 2 months of age, levels of parasitemia peaked on day 17 and returned to levels undetectable
on day 35 (figure 1A). Thereafter, levels of parasitemia remained
low (1%–3%). Parasitemia in DBA/2 mice infected at 6 months
of age followed a similar pattern (figure 1B). A different pattern
emerged in the 2 older groups of mice. In mice infected at 12
months of age (figure 1C), levels of parasitemia peaked on day
17 (21%) and returned to near basal levels by day 24. After
this initial recovery, a protracted parasitemia (4%–11%) was
observed. In mice infected at 18 months (figure 1D), levels of
parasitemia peaked on day 24 (27%) and reached a nadir (6%)
on day 35. Thereafter, parasitemia oscillated between 6% and
14%. Thus, the degree of the initial, but reversible, parasitemia
increases with age at infection. Most dramatically, in older DBA/
Aging and B. microti Infection • JID 2004:189 (1 May) • 1723
Figure 2. Strain-specificity of the age-related loss of resistance to Babesia microti. DBA/2, C57BL/6, and BALB/c mice were infected, as indicated
in figure 1. For each mouse, the mean daily parasitemia in the early phase
of infection (A) was calculated as the integral of parasitemia from days 7
to 35, divided by 28, whereas mean daily parasitemia in the late phase (
B) was calculated as the integral of parasitemia from days 35 to 98, divided
by 63. Mean daily levels of parasitemia are plotted against age at time of
infection. Data are mean SEM. For each strain, the effect of age on
early (A) and late parasitemia (B) was tested by use of a 1-way analysis
of variance. The mean of the 2-month-old mice compared for statistical
significance (Fisher’s least significant difference test) with those of the other
age groups. *P ! .05; **P ! .01; and ***P ⭐ .001.
2 mice, there was a subsequent protracted parasitemia, the
extent of which correlates with age.
Development of persistent parasitemia in old DBA/2 mice,
but not in old BALB/c or C57BL/6 mice. C57BL/6 mice infected at 2 or 6 months of age displayed a marginal parasitemia
that resolved by day 17 (figure 1E and 1F). Aging did not render
C57BL/6 mice highly susceptible to RM/NS. Early parasitemia
peaked at 7% and 5% in 12- and 18-month-old mice, respectively
(figure 1G and 1H). No persistent parasitemia was observed in
C57BL/6 mice, regardless of age (figure 1E–1H).
Similarly, BALB/c mice infected at 2 and 6 months of age
presented with marginal parasitemia (3%–4%), which became
undetectable by day 17 (figure 1I and 1J). Early parasitemia in
BALB/c mice infected at 12 months of age was no higher than
that seen in mice infected at 2 months of age but was prolonged
until day 28 (figure 1K). Early parasitemia in 18-month-old mice
1724 • JID 2004:189 (1 May) • Vannier et al.
peaked at higher levels (9%) and returned to basal levels by day
28 (figure 1L). Neither older BALB/c nor older C57BL/6 mice
exhibited the protracted late parasitemia seen in older DBA/2
mice.
To quantify these differences, we calculated the mean daily
parasitemia (integral of percentage of parasitemia over time,
or area under the curve, divided by days in the period examined) in the early (days 7–35; figure 2A) and late (days 35–98;
figure 2B) phases of infection. In DBA/2 mice, the early parasitemia in the 2-month-old mice did not differ significantly
from that observed in the 6-month-old mice. There was a
marked increase in parasitemia in mice infected at 12 (P p
.001) or 18 (P ! .001; figure 2A) months of age. The effect of
age on the subsequent protracted (or late) parasitemia was
evaluated between days 35 and 98 (figure 2B). In the late parasitemia in 6-month-old mice was 2 times higher than that
recorded in 2-month-old mice, 13 times higher in 12-monthold mice (P p .03), and ∼7 times higher in 18-month-old mice
(P ! .001). In BALB/c mice infected at 18 months of age, early
parasitemia was 3 times higher (P ! .001; figure 2A) than in
mice infected at 2 months of age, whereas the degree of protracted parasitemia remained marginal (figure 2B). In C57BL/
6 mice, there was no significant age-related difference in late
parasitemia (figure 2B), whereas an increase in early parasitemia
was only seen at 12 months of age (P ! .05; figure 2A).
Age-based ability of spleen cells to control and resolve parasitemia.
Spleen cells (2 ⫻ 107 ) obtained from young (2
months) or old (18 months) DBA/2 or BALB/c mice were
transferred into 2-month-old C.B.17-SCID recipient mice.
These SCID mice (BALB/c genetic background) retain an intact
innate immune system but lack T and B cells; that is, the cellular
components of adaptive immunity [see 15]. Our experiments
took advantage of the fact that DBA/2 and BALB/c mice share
the same major histocompatibility complex (MHC) haplotype,
H-2d. Unlike BALB/c, DBA/2 carry Mtv-7, the provirus coding
for the strong superantigen Mls-1a [16]. In the absence of host
T and B cells in SCID mice, this presents no difficulties.
Seven days after adoptive cell transfer, recipient mice were
infected with 105 pRBCs (figures 3A and 3E). Parasitemia in SCID
mice that received vehicle only (culture medium) became detectable on day 21 and rapidly plateaued at high levels (35%–
50%), at and beyond day 32. Mean daily levels of parasitemia
(see above) were calculated for the early (days 7–35) and late
(days 35–73) phases of infection. Early parasitemia was reduced
by the transfer of spleen cells from young BALB/c mice
(7.1% 0.9% in SCID plus BALB/c cells vs. 16.9% 3.0% in
SCID plus medium; P ! .01), but not from young DBA/2 mice
(12.2% 1.8%). Late parasitemia was reduced by transfer of
either young BALB/c cells (2.7% 0.9% in SCID plus BALB/c
cells vs. 53.1% 0.8% in SCID plus medium; P ! .001) or young
DBA/2 cells (12.9 0.7%; P ! .001, vs. SCID plus medium).
Figure 3. Age decreases the ability of transferred DBA/2 and BALB/c spleen cells to control persistent parasitemia in SCID mice: importance of the
parasite load at infection. SCID mice (aged 2 months) were injected intravenously with 2 ⫻ 107 spleen cells obtained from “young” (aged 2 months; )
or “old” (aged 18 months; ) DBA/2 mice (A–D). A similar transfer was performed with spleen cells from young or old BALB/c mice (E–H). SCID mice
in the control group (n p 3–4; ) received cell-free vehicle. After 7 days, recipient mice were infected intraperitoneally with 105 parasite-infected red
blood cells (pRBCs) (A, 3 recipients of young cells and 4 recipients of old cells; E, 5 young and 4 old), 104 pRBCs (B, 6 young and 4 old; and F, 4 young
and 4 old), 103 pRBCs (C, 4 young and 4 old; and G, 4 young and 4 old), or 102 pRBCs (D, 4 young and 3 old; H, 2 young and 3 old). Parasitemia is the
percentage of pRBCs, as determined by morphological analysis of Giemsa-stained blood smears (A plus B and E plus F) or by flow cytometry of wholeblood cells stained with the fluorescent nucleic-acid staining dye YOYO-1 (see Materials and Methods; C plus D and G plus H). Levels of parasitemia are
plotted against time after infection. Data are mean, and SEM was omitted for clarity.
When SCID mice were given old BALB/c spleen cells (figure 3E),
mean daily levels of parasitemia in the early and late phases of
infection did not differ from those seen in mice receiving young
BALB/c spleen cells (both P 1 .05). Similarly, transfer of old DBA/
2 cells conferred the same degree of protection as young DBA/
2 cells (figure 3A), whether from early or late parasitemia (both
P 1 .05). Under these experimental conditions, the number of
spleen cells was limited (2 ⫻ 107 cells transferred, rather than the
typical spleen content of 10 ⫻ 107 cells). These results suggested
that, if we used fewer parasites to infect SCID mice or transferred
more spleen cells, the magnitude of sustained parasitemia might
be comparable with that seen in wild-type (wt) mice.
In SCID mice infected with 104 pRBCs, the transfer of young
BALB/c spleen cells achieved a near complete eradication of
pRBCs (figure 3F). However, SCID mice receiving old BALB/
c spleen cells displayed a recrudescent parasitemia. Furthermore, the sustained parasitemia in SCID mice receiving young
DBA/2 cells (figure 3B) remained higher than in young wt DBA/
2 mice (compare with figure 1A). When SCID mice were infected with 103 pRBCs, young BALB/c cells conferred a greater
degree of protection from late parasitemia than did old BALB/
cells (figure 3G). However, young DBA/2 mice remained more
effective in resolving parasitemia (figure 1A) than SCID mice
receiving young DBA/2 cells (figure 3C). In SCID mice infected
Aging and B. microti Infection • JID 2004:189 (1 May) • 1725
Figure 4. Age and genetic make-up of transferred spleen cells affect control of persistent parasitemia in young SCID recipient mice. Data shown in figure
3 were divided into 2 time zones, and mean daily levels of parasitemia were derived (see Materials and Methods). The early phase ended on day 35 in mice
infected with 104 parasite-infected red blood cells (pRBCs; figure 3B and 3F), on day 42 in mice infected with 103 pRBCs (figure 3C and 3G), or on day 56 in
mice infected with 102 pRBCs (figure 3D and 3H). Late phases all ended on day 98. In SCID mice infected with 103 or 102 pRBCs, mean daily levels of
parasitemia were multiplied by 0.6, to correct for the difference in absolute parasitemia detected by microscopy on Giemsa-stained blood smears and by flow
cytometry using the nucleic-acid staining dye YOYO-1 (see Materials and Methods). Data are mean SEM. *P ! .05; and **P p .01.
with 102 pRBCs (figures 3D and 3H), transfer of young DBA/
2 cells reduced the persistent parasitemia to nearly undetectable
levels (figure 3D), whereas a persistent parasitemia was observed at and beyond 8 weeks in SCID mice injected with old
DBA/2 spleen cells (figure 3D).
We could best see the overall pattern of parasitemia in SCID
mice by assessing mean daily levels of parasitemia in the early
and late periods (figure 4). The level of parasitemia in the late
period was related to age and number of parasites used for
infection (figure 4, right panels). When recipient SCID mice
were infected with 104 pRBCs, old BALB/c splenocytes were
less protective than young BALB/c cells (P ! .05). Old BALB/
c cells also conferred less protection to SCID mice infected with
103 pRBCs (P ! .05). This age-associated difference in protection was lost when the infectious load was further decreased
to 102 pRBCs. At this lower infectious load, young DBA/2 splenocytes were excellent at reducing late parasitemia, compared
1726 • JID 2004:189 (1 May) • Vannier et al.
with old DBA/2 cells (P ! .01). In SCID mice infected with 103
pRBCs, young DBA/2 cells remained more protective than old
DBA/2 cells (P ! .05), but the deleterious effect of age was
reduced (figure 4). When the early phase of infection was considered (figure 4, left panels), age had no effect on the ability
of DBA/2 splenocytes to reduce parasitemia, regardless of the
infectious load. On the other hand, young BALB/c cells were
not consistently better than old BALB/c cells at reducing early
parasitemia. Young BALB/c cells were better when SCID mice
were infected with 103 pRBCs (P ! .05). However, this ageassociated difference was not seen when SCID mice were infected with 104 or 102 pRBCs (both P 1 .05).
DISCUSSION
Age is clearly a risk factor for severe B. microti infection in
humans [17]. There are only a handful of studies, most of them
in cattle [18, 19], that have begun to characterize the effect of
age on the resistance to Babesia species. These studies may have
limited value for understanding human disease, because B. bovis
is not pathogenic in people. There has been only 1 study [12]
assessing the effect of age on the resistance of laboratory mice
to the human pathogen B. microti. Although infection tended
to resolve faster in younger BALB/c mice, peak levels of parasitemia were higher in older BALB/c mice. The goal of the
present study was to develop a mouse model of B. microti
infection that displays the age-related loss of resistance seen in
humans. Using this model, our aim was to determine whether
the age-related loss of resistance was genetically determined.
Inbred mice were infected with a novel B. microti clinical
isolate, RM/NS. Young mice of all 3 strains (DBA/2, BALB/c,
and C57BL/6) cleared infection. We have recently observed that
(DBA/2 ⫻ BALB/c) F1 mice are resistant and that the frequency
of susceptibility in F2 mice suggests that several loci contribute
to resistance (authors’ unpublished data). In DBA/2 mice, there
was a gradual loss in control and clearance of parasites with
increasing age, as reflected by the gradual increase in early and
late parasitemia. In contrast, old BALB/c and C57BL/6 mice
cleared parasites at the end of the early phase and did not suffer
from recrudescent parasitemia. Although adaptation was required to increase the virulence of the Peabody strain used by
Habicht et al. [12], infection with our clinical isolate RM/NS
resulted in high levels of parasitemia, without the need for
adaptation. When infected with a number of organisms similar
to the inoculum delivered by naturally infected ticks, perhaps
on the order of ⭓25,000 sporozoites [20], BALB/c mice were
as resistant as C57BL/6 mice to RM/NS. As reported by Habicht
et al. [12], a 500–1000 fold increase in the infectious load clearly
overcomes the intrinsic resistance of BALB/c mice but also may
overcome age-related differences in resistance.
Because T cells play a central role in the resolution and
clearance of B. microti in young mice [21–23], we hypothesized
that an age-associated decline in adaptive immunity may account for the age-related susceptibility to B. microti. Our experiments took advantage of the observation that BALB/c.scid
mice, unlike C57BL/6.scid mice [24], are unable to control and
clear B. microti infection [22]. Using a transfer protocol, we
established that there is an age-related decline in the ability of
spleen cells to protect from late-phase infection. Specifically,
the effect of age on the protection conferred by transferred
BALB/c spleen cells is best revealed when the initial parasite
load is high. In contrast, the effect of age on the protection
conferred by DBA/2 spleen cells is best seen when mice are
infected with a small number of organisms. Overall, we have
established the experimental conditions of a SCID transfer system that optimally reveals strain differences in the age-associated loss of protection conferred by naive spleen cells against
late-phase B. microti infection.
Spleen cells from neither the young nor the old DBA/2 mice
affected the early parasitemia in SCID mice infected with 105
pRBCs, which is the parasite load used for infection of DBA/
2 mice. This observation may seem at odds with the demonstrated greater susceptibility of old DBA/2 mice. Because the
recipient SCID mice were young and had an intact innate immune system, the age-associated loss of adaptive immunity in
the transferred cells might have been masked. This suggests
that an age-associated decrease in adaptive immunity may be
central to diminished resistance to persistent infection, whereas,
in the early phase of infection, aging of both innate and adaptive
immunity may contribute to decreased resistance. From our
data, we cannot assess the relative contribution of innate and
acquired immunity to early resistance. To test for affects of
aging on the innate response to B. microti, a comparison of
young and aged lymphocyte-deficient mice is needed.
Because T cells, unlike B cells, are required for resolution
and clearance of B. microti infection [21–23], the impairment
of T cell functions may account for most of the age-associated
decline in protection conferred by transferred spleen cells. CD4
T cells, in particular, have been identified as important for
resistance to B. microti [25]. Although numerous studies have
documented a loss of T cell function with age, the present study
firmly establishes that the age-related loss of immune protection
conferred by spleen cells contributes to the loss of resistance
to an infectious pathogen. Because of the clear difference in
susceptibility between ages and strains, we anticipate an approach combining genetics and genomics to unravel the genes
associated with resistance and/or susceptibility to B. microti.
Acknowledgments
We thank Adriana Salazar-Montes, Robert Altman, and Sohela Shah, who contributed to the experimental work, and
Rouette Hunter, who performed many of the parasite counts.
William Dietrich and Dania Richter were valuable discussants
of our experiments.
References
1. Homer MJ, Aguilar-Delfin I, Telford SR 3rd, Krause PJ, Persing DH.
Babesiosis. Clin Microbiol Rev 2000; 13:451–69.
2. Krause PJ, Spielman A, Telford SR 3rd, et al. Persistent parasitemia
after acute babesiosis. N Engl J Med 1998; 339:160–5.
3. Herwaldt BL, McGovern PC, Gerwel MP, Easton RM, MacGregor RR.
Endemic babesiosis in another eastern state: New Jersey. Emerg Infect
Dis 2003; 9:184–8.
4. Shih C-M, Liu L-P, Chung W-C, Ong SJ, Wang C-C. Human babesiosis
in Taiwan: asymptomatic infection with a Babesia microti–like organism
in a Taiwanese woman. J Clin Microbiol 1997; 35:450–4.
5. Hunfeld KP, Lambert A, Kampen H, et al. Seroprevalence of Babesia
infections in humans exposed to ticks in midwestern Germany. J Clin
Microbiol 2002; 40:2431–6.
6. Benezra D, Brown AE, Polsky B, Gold JW, Armstrong D. Babesiosis
Aging and B. microti Infection • JID 2004:189 (1 May) • 1727
7.
8.
9.
10.
11.
12.
13.
14.
15.
and infection with human immunodeficiency virus (HIV). Ann Intern
Med 1987; 107:944.
Teutsch SM, Etkind P, Burwell EL, et al. Babesiosis in post-splenectomy
hosts. Am J Trop Med Hyg 1980; 29:738–41.
White DJ, Talarico J, Chang HG, Birkhead GS, Heimberger T, Morse
DL. Human babesiosis in New York State: review of 139 hospitalized
cases and analysis of prognostic factors. Arch Intern Med 1998; 158:
2149–54.
Krause PJ, Telford SR 3rd, Pollack RJ, et al. Babesiosis: an underdiagnosed disease of children. Pediatrics 1992; 89:1045–8.
Krause PJ, McKay K, Gadbaw J, et al. Increasing health burden of
human babesiosis in endemic sites. Am J Trop Med Hyg 2003; 68:
431–6.
Hatcher JC, Greenberg PD, Antique J, Jimenez-Lucho VE. Severe babesiosis in Long Island: review of 34 cases and their complications.
Clin Infect Dis 2001; 32:1117–25.
Habicht GS, Benach JL, Leichtling KD, Gocinski BL, Coleman JL. The
effect of age on the infection and immunoresponsiveness of mice to
Babesia microti. Mech Ageing Dev 1983; 23:357–69.
Fukata T, Ohnishi T, Okuda S, Sasai K, Baba E, Arakawa A. Detection
of canine erythrocytes infected with Babesia gibsoni by flow cytometry.
J Parasitol 1996; 82:641–2.
Cong YZ, Rabin E, Wortis HH. Treatment of murine CD5⫺ B cells
with anti-Ig, but not LPS, induces surface CD5: two B-cell activation
pathways. Int Immunol 1991; 3:467–76.
Medzhitov R, Janeway CA Jr. Innate immunity: impact on the adaptive
immune response. Curr Opin Immunol 1997; 9:4–9.
1728 • JID 2004:189 (1 May) • Vannier et al.
16. Festenstein H, Berumen L. BALB.D2-Mlsa: a new congenic mouse
strain. Transplantation 1984; 37:322–4.
17. Krause PJ. Babesiosis. Med Clin North Am 2002; 86:361–73.
18. Levy MG, Clabaugh G, Ristic M. Age resistance in bovine babesiosis:
role of blood factors in resistance to Babesia bovis. Infect Immun
1982; 37:1127–31.
19. Goff WL, Johnson WC, Parish SM, Barrington GM, Tuo W, Valdez
RA. The age-related immunity in cattle to Babesia bovis infection involves the rapid induction of interleukin-12, interferon-gamma and
inducible nitric oxide synthase mRNA expression in the spleen. Parasite
Immunol 2001; 23:463–71.
20. Piesman J, Spielman A. Babesia microti: infectivity of parasites from
ticks for hamsters and white-footed mice. Exp Parasitol 1982; 53:242–8.
21. Ruebush MJ, Hanson WL. Thymus dependence of resistance to infection with Babesia microti of human origin in mice. Am J Trop Med
Hyg 1980; 29:507–15.
22. Matsubara J, Koura M, Kamiyama T. Infection of immunodeficient
mice with a mouse-adapted substrain of the gray strain of Babesia
microti. J Parasitol 1993; 79:783–6.
23. Clawson ML, Paciorkowski N, Rajan TV, et al. Cellular immunity, but
not gamma interferon, is essential for resolution of Babesia microti
infection in BALB/c mice. Infect Immun 2002; 70:5304–6.
24. Aguilar-Delfin I, Homer MJ, Wettstein PJ, Persing DH. Innate resistance to Babesia infection is influenced by genetic background and
gender. Infect Immun 2001; 69:7955–8.
25. Igarashi I, Suzuki R, Waki S, et al. Roles of CD4+ T cells and gamma
interferon in protective immunity against Babesia microti infection in
mice. Infect Immun 1999; 67:4143–8.