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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. 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