Alterations in the Profile of Blood Cell Types during Malaria in

1940 • JID 2005:191 (1 June) • Davison et al.

M A J O R A R T I C L E

Alterations in the Profile of Blood Cell Typesduring Malaria in Previously UnexposedPrimigravid Monkeys

Billie B. Davison,1 M. Bernice Kaack,1 Linda B. Rogers,1 Kelsi K. Rasmussen,1 Terri Rasmussen,1

Elizabeth W. Henson,1 Sonia Montenegro,2 Michael C. Henson,3 Fawaz Mzwaek,4 and Donald J. Krogstad4

1Division of Comparative Pathology, Tulane National Primate Research Center, Covington, and 2Alton Ochsner Medical Foundation, 3TulaneUniversity Health Sciences Center, and 4Tulane School of Public Health and Tropical Medicine, New Orleans, Louisiana

Malaria in nonimmune, primigravid women threatens both mother and fetus. We used the Plasmodiumcoatneyi/rhesus monkey model to examine factors associated with this. Clinical and immunologic responsesduring the blood stage of chronic malaria (4 months) were evaluated in 8 malaria-naive primigravid (PMI)and 8 age-matched nulligravid (NMI) infected monkeys, compared with those in 8 primigravid, noninfectedcontrol monkeys. Although parasitemia levels were similar, recrudescence was more frequent and prolonged,and anemia was more severe in PMI than in NMI monkeys. During infection, CD2+, CD4+, and CD8+ lym-phocyte levels were higher in NMI than in PMI monkeys. Monocyte and neutrophil levels were lower in PMIthan in NMI monkeys. During chronic, untreated malaria, NMI monkeys had a B lymphocyte count 23 timesgreater than that of PMI monkeys. Pregnancy-induced immunomodulation, defined as a lack of appropriatecellular responses to malaria, was indiscernible until the immune system was challenged by a pathogen.

Malaria is an overwhelming public health problem—

300–500 million cases and 2–3 million deaths occur

annually [1]. For pregnant women and their fetuses,

malaria presents a particularly severe challenge [2]. The

dynamics of malaria during pregnancy vary with en-

demicity [3, 4]. In highly endemic areas where women

have acquired immunity to malaria, primigravid and

secundigravid women are more susceptible to malaria-

induced pregnancy complications than are multigravid

women [5–8]. In areas of low endemicity, women often

lack sufficient immunity to control severe clinical malar-

Received 13 September 2004; accepted 4 January 2005; electronically published28 April 2005.

Presented in part: 50th Annual Meeting of the American Society of TropicalMedicine and Hygiene, Atlanta, 11–15 November 2001 (abstract 722).

Financial support: National Institutes of Health (NIH; Public Health Service grantP51RR00164 to the Tulane National Primate Research Center); National Instituteof Allergy and Infectious Disease, NIH (grants 5RO1AI42400 and UR 3/CCU 418652-03, University of Mississippi Subcontract Project C).

Reprints or correspondence: Dr. Billie B. Davison, Tulane National Primate Re-search Center, Div. of Comparative Pathology, 18703 Three Rivers Rd., Covington,LA 70433 ([email protected]).

The Journal of Infectious Diseases 2005; 191:1940–52� 2005 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2005/19111-0022$15.00

ia, and all children of women at all gravidities are sus-

ceptible to complications, including intrauterine growth

retardation (IUGR), prematurity, low birth weight (LBW),

failure to thrive, neonatal death, and congenital malaria

[2, 3, 9–12]. Spontaneous abortion during the first tri-

mester [10, 13–15] has been demonstrated in monkeys

with malaria infection [16]. The Plasmodium coatneyi–

infected monkeys that we report represent women in

areas of low transmission who lack malaria immunity

[17, 18]. However, unlike chronically infected, un-

treated monkeys, pregnant women generally receive an-

timalarial treatment, which limits the duration and se-

verity of their illness. Despite treatment, many of these

women’s pregnancies have serious malaria-associated

complications [18].

Malaria is not unique in adversely affecting preg-

nancy. Pregnant women have increased susceptibility

to many intracellular pathogens [19, 20]. Pregnancy-

associated immunomodulation, including the down-

regulation of T lymphocyte responses to protect the

fetus from rejection, has been hypothesized as one ex-

planation [19, 20]. Pregnancy-associated immunomod-

ulation has been demonstrated in rodents [21–23] but

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Malaria in Naive Primigravid Rhesus • JID 2005:191 (1 June) • 1941

Figure 1. Daily mean parasitemia in primigravid (PMI) and age-matched nulligravid (NMI) monkeys with malaria. Parasitemia was quantified dailyin Giemsa-stained thick and thin blood smears (collected by ear stick from individual monkeys) by counting the parasitized infected red blood cells(PRBCs) in 10 fields of 200 cells to determine the percentage of infected cells. The daily absolute no. of PRBCs was calculated by multiplying thedaily RBC count (determined by extrapolation of the weekly RBC count determined by a complete blood cell count) by the percentage. Each pointrepresents the daily mean of the total PRBCs occurring in each group of 8 monkeys. PMI monkeys had higher peak parasitemia and more-chronicrecrudescence of parasitemia throughout the first 9 weeks of infection (gestational week [GW] 6–15) than did age-matched NMI monkeys. The secondperiod of recrudescence (GW 12–14) was significantly different in the 2 groups (group, ; time, ; group � time, [2-wayP p .04 P p .0001 P p .02analysis of variance for all]). The difference in the degree of severity (more-chronic recrudescence patterns) of parasitemia reflects the more-severeclinical malaria observed in the primigravid monkeys. All 10 figures follow 2 corresponding timelines on the X-axis, although, for the sake of clarity,only the gestational timeline is represented in weeks. The timeline that is not shown follows weeks after inoculation. GW 6, 8, 10, 12, 14, 16, 18,20, and 22 (shown) are equivalent to weeks postinfection (WPI) 0, 2, 4, 6, 8, 10, 12, 14, and 16 (not shown). The first trimester ends on gestationalday (GD) 55, during GW 8, WPI 2; the second trimester ends on GD 110, during GW16, WPI 10; and the third trimester ends with cesarean-sectiondelivery on GD 155, during GW 22, WPI 16. The parasitemia graph represents daily parasite values but is also displayed along the weekly gestationaltimeline (X-axis). T-1, T-2, and T-3, separated by lightly dotted lines, denote trimesters of rhesus monkey gestation as they would equate to humangestation in terms of fetal development and organogenesis.

remains controversial in humans [24–28]. Aggressive Th1 re-

sponses to malaria during pregnancy may cause fetal death,

IUGR, or neonatal death [29–33].

Although B lymphocytes produce antimalarial antibody [34–

36], there are no reports of altered numbers of B cells in malaria

during pregnancy. Suppression of B lymphopoiesis during nor-

mal pregnancy [37] and selective deletion of B lymphocytes

specific for paternal histocompatibility antigens have been re-

ported [38]. B lymphocyte levels decline in pregnant women

infected with cytomegalovirus, rubella, and toxoplasmosis [39].

It is impossible to perform rigorous (controlled) studies in

malaria-infected pregnant women, because they must be treat-

ed, and confounding variables—such as poor socioeconomic

conditions, malnutrition, and exposure to other pathogens—

are inherent to human studies. To address this problem, we

used the P. coatneyi/rhesus monkey model of malaria during

pregnancy [16, 40, 41]. This model produces the same clinical

consequences (severe maternal anemia, early abortion, IUGR,

prematurity, LBW, high neonatal mortality, and congenital in-

fection) [16] and placental pathology as those observed in hu-

mans [41]. The monkey model provides an opportunity to

monitor malaria-infected pregnant females prospectively with-

out confounding variables and to thus define the effects unique

to malaria.

The present study was based on timed matings with 16 pre-

viously nonpregnant (nulligravid) female rhesus monkeys. Eight

primigravid females were infected with P. coatneyi on gesta-

tional day 42, during gestational week (GW) 6 (the pregnant

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Figure 2. A, Red blood cell (RBC) counts in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) and nulligravid(NMI) monkeys. RBC counts were monitored weekly throughout infection and gestation (automated counts; Coulter T 540 or Bayer AVIDA 120). Eachpoint represents the mean of the total weekly RBC count occurring in each group of 8 monkeys. Counts in PMI monkeys dropped to their nadir atgestational week (GW) ∼12 (weeks postinfection [WPI] 6). Chronic parasite recrudescence during the entire second trimester in PMI monkeys (figure1) resulted in the lower RBC count vs. that in NMI monkeys and failure of RBC counts to recover during the third trimester, similar to RBC countsof NMI monkeys. The gradual decline of RBCs in PC monkeys ( RBCs/mL) occurred as a result of normal pregnancy and has been65.32–4.8 � 10observed in other studies at Tulane National Primate Research Center (unpublished data). B, Hemoglobin levels in PC, PMI, and NMI monkeys. TheRBC count nadir was associated with an average hemoglobin level of 6.75 gm/dL in PMI monkeys vs. 7.87 gm/dL in NMI monkeys. Hemoglobin levelswere lower in PMI than in NMI monkeys from GW 9 to 14 (WPI 3–8), which corresponded with the 2 periods of parasite recrudescence shown infigure 1. See figure 1 for description of the timeline used.

and malaria-infected [PMI] group); the other 8 monkeys re-

ceived intravenous saline—the primigravid, uninfected control

(PC) group. Outcomes observed in the 2 primigravid groups

were compared with those in a third group of age-matched,

malaria-infected nonpregnant females (the nulligravid, malaria-

infected [NMI] group) throughout a timed course of chronic

malaria (16 weeks), to separate the effects of P. coatneyi infec-

tion (malaria) from those of pregnancy (the primigravid state).

We show that, in the context of nonimmune, primigravid

pregnancy challenged by malaria, decreased levels of mono-

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Figure 3. Monocyte responses in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) and nulligravid (NMI)monkeys. Monocyte responses to malaria were significantly lower in PMI than in NMI monkeys. PMI monkeys maintained approximately the samemonocyte levels as did PC monkeys. Each point represents the mean of the total weekly monocyte counts occurring in each group of 8 monkeys(group, ; time, ; group � time, [2-way analysis of variance for all]). During a late parasite recrudescence (gestationalP ! .0001 P ! .0001 P ! .0001week [GW] 16; week postinfection 10), monocytes peaked to their highest levels in NMI but not in PMI monkeys, although the parasitemia in PMImonkeys was higher (65,223 vs. 32,472 PRBC/mL, respectively) (figure 1). See figure 1 for description of the timeline used.

cytes, CD4+ and CD8+ lymphocytes, and, surprisingly, B lym-

phocytes are associated with more-severe systemic parasitemia

and clinical malaria in the mother. Whether this failure leads

to aberrant increases in Th1 immunity at the level of the pla-

centa is currently under investigation by our laboratory and by

others [30, 32, 41, 42].

MATERIALS AND METHODS

Experimental design. The 3 groups of 8 spleen-intact, age-

matched female rhesus monkeys (Macaca mulatta) had no pre-

vious exposure to malaria. They were housed individually under

the same conditions, in accordance with the Guide for the Care

and Use of Laboratory Animals (US Department of Health and

Human Services, National Institutes of Health).

Before each weekly examination, monkeys were anesthetized

with 10 mg/kg of ketamine hydrochloride. Eight time-bred pri-

migravid monkeys were inoculated intravenously with 106 P. coat-

neyi parasites at GW 6 (late first trimester) (PMI group), 8 non-

pregnant monkeys were inoculated with the same inoculum

(NMI group), and 8 primigravid monkeys were inoculated with

saline for use as controls (PC group). Antimalarial treatments

were withheld until 1 week after delivery. The timeline follows

the gestational age of the infant in weeks (GW 6–22) in the

pregnant monkeys, and infection in NMI monkeys was moni-

tored along the same timeline as weeks postinfection (WPI; 0–

16). GW 6 corresponds to infection time 0 on all graphs pre-

sented. Physical examinations were performed weekly, and blood

samples were obtained for serum collection, complete blood cell

(CBC) counts, and monthly flow-cytometric (FACS) analysis.

Daily ear sticks were performed for thick and thin blood smears

for the determination of parasite counts.

Malaria parasite inoculum. The inoculum, obtained from

a simian retrovirus–free monkey infected with P. coatneyi, was

stored in liquid nitrogen. It contained ∼106 parasites/mL and

was thawed in water at 37�C before administration into the

saphenous vein. P. coatneyi is a falciparum-like parasite that

produces serious clinical malaria in rhesus monkeys and has

recrudescence patterns similar to those of Plasmodium falcip-

arum malaria [43]. Because P. coatneyi sequesters in the blood

vascular system, P. coatneyi–infected rhesus monkeys have been

used elsewhere as models for cerebral malaria [44] and malaria

during pregnancy [16].

Parasite counts. Parasitemia levels were estimated by ex-

amination of Giemsa-stained blood smears prepared from daily

ear sticks from the time of inoculation (GW 6) until 1 week

after delivery. Parasitized red blood cells (PRBCs) and unpar-

asitized RBCs were counted in 10 microscopic fields (magni-

fication, �1000) and recorded as the percentage of PRBCs.

Weekly RBC counts from the CBC count were multiplied by

the percentage of PRBCs, to estimate the number of PRBCs

per cubic millimeter of blood.

Blood counts. CBC counts were obtained weekly by use of

either the Coulter T 540 (before September 2001) or the Bayer

AVIDA 120 (after September 2001) devices. Whole-blood sam-

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Figure 4. Nos. of polymorphonuclear neutrophils (PMNs) in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI)and nulligravid (NMI) monkeys. Nos. of PMNs were significantly decreased in both malaria-infected groups through gestational week (GW) 19, withPC monkeys having ∼1.75 times the nos. of PMNs as PMI or NMI monkeys. Each point represents the mean of the total weekly PMN counts occurringin each group of 8 monkeys (group, ; time, ; group � time, [2-way analysis of variance for all]). See figure 1 for descriptionP ! .05 P ! .002 P p NSof the timeline used.

ples in K2 EDTA (Vacutainer; Becton Dickinson) were evaluated

for RBC count, hemoglobin (Hb) concentration, hematocrit

level, leukocyte count, and platelet count. Lymphocyte, mono-

cyte, and neutrophil counts were obtained from these CBC

counts. The Coulter T 540 was calibrated by use of normal and

abnormal controls and fresh whole blood; the Bayer AVIDA

120 was normalized by use of the Coulter T 540.

Evaluations of lymphocyte subsets. Whole-blood samples

in EDTA were evaluated for CD2+, CD20+, CD4+, and CD8+

lymphocytes by staining with fluorochrome-conjugated mono-

clonal antibodies, followed by flow cytometry [45]. The per-

centages of CD4+ lymphocytes were determined by staining

with CD4 fluorescein isothiocyanate (BD Clone M-T477; Phar-

mingen) mouse IgG2a k. The percentages of CD2+ T lympho-

cytes and CD20+ B lymphocytes were determined by use of

double staining with RD1-T11 and FI-B1 (Coulter). The per-

centage of CD8+ lymphocytes was determined by use of FI-Leu

2a (Becton Dickinson). Erythrocytes were lysed and leukocytes

fixed by use of the TQ-Prep machine and ImmunoPrep reagents

(Coulter). Data acquisition and analysis for flow cytometry

were performed on the FACSCalibur instrument by use of

CellQuest software (Becton Dickinson). Samples from the 3

different groups (PMI, NMI, and PC) were evaluated at the

same time points, beginning at GW 6 (day 0 of infection or

of saline administration). Absolute counts for individual lym-

phocyte subsets were estimated by multiplying the total (CBC

count) lymphocyte count by the percentages determined by

FACS analysis.

Ultrasound examinations. All fetuses were monitored

weekly by ultrasound and were delivered by cesarean section

at GW 22, before labor and delivery. The examinations were

performed by use of a digital real-time ultrasound system (To-

shiba PowerVision; Toshiba) with Toshiba microtransducers

(PVK-738F/738H/745V) linked to a database and software pro-

gram (Sonultra Ultra32-OB; Sonultra). This software is based

on established normal values for intrauterine fetal growth in

rhesus monkeys [46, 47]. Gestational age and expected deliv-

ery dates were based on timed breeding dates and ultrasound

measurements.

Cesarean sections. Because the gestation of rhesus monkey

averages 23.5 weeks, elective cesarean sections were performed

at GW 22, to ensure the collection of placental and cord-blood

samples and to avoid the confounding effects of labor and

delivery. Immediately before surgery, blood samples were ob-

tained for CBC count, FACS analysis, standard chemistry pan-

els, and blood smears. Preanesthesia consisted of glycopyrrolate

followed by ketamine hydrochloride; this was maintained by

use of isofluorane. The fetus and placenta were removed after

exteriorizing the uterus through a vertical midline incision. A

sample of amniotic fluid was obtained with a needle and syringe

before the removal of the infant, and a cord-blood sample was

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Figure 5. Total blood platelet counts in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) and nulligravid (NMI)monkeys. Total nos. of blood platelets were significantly lower in PMI than in NMI monkeys and in both groups than in PC monkeys. Each pointrepresents the mean of the total weekly platelet counts occurring in each group of 8 monkeys (group, ; time ; group � time,P ! .005 P ! .0001 P !

[2-way analysis of variance for all]). PC monkeys consistently had platelet counts 1500,000 cells/mL after gestational week (GW) 10 vs. PMI.0001monkeys, which had levels of 300,000–350,000 cells/mL. The lower levels in PMI monkeys lasted throughout the entire final 2 trimesters of pregnancy.The platelet counts in PC monkeys steadily increased during the last 4 weeks of gestation, to reach their highest level on the day of delivery. Seefigure 1 for description of the timeline used.

obtained before extraction of the placenta. Postsurgical recovery

of the dams was uneventful.

Statistical analysis. RBC, platelet, lymphocyte, monocyte,

and neutrophil counts were analyzed use of the STATISTICA

program for Macintosh computers (StatSoft). Normally distrib-

uted continuous data were compared between groups by use of

a 2-way analysis of variance (ANOVA) with repeated measures.

Post hoc analyses were performed by use of by the Newman-

Keuls test. Analyses of data at individual time points (between

or among groups) were performed by use of 1-way ANOVA.

Parasitemia and FACS data were analyzed with general linear

model repeated-measures procedures by use of the statistical

program SPSS for Windows (SPSS). The daily mean parasitemia

levels of PMI and NMI were compared. There were 6 consec-

utive measurements of each lymphocyte subset for each mon-

key during the period beginning 2 weeks before inoculation

and continuing until the end of pregnancy (GW 22) and/or

infection (WPI 16). The main effects of time and grouping

(PMI, PC, and NMI) were tested, as was the interaction between

the 2 factors.

RESULTS

Parasitemia levels, RBC counts, and Hb levels. There were

no significant differences in mean peak parasitemia levels be-

tween PMI and NMI monkeys (figure 1). The duration of each

recrudescence was longer, with higher parasite density, in PMI

monkeys; this, however, resulted in more-chronic parasitemia

that led to more-severe anemia. In humans, the prevalence and

density of P. falciparum are highest during the first half of

pregnancy, and levels decrease progressively until delivery [17,

48]. This pattern has also been observed in monkeys.

Although a similar decline in RBC counts occurred in PMI

and NMI monkeys (figure 2A), the chronic parasitemia in PMI

monkeys resulted in more-severe and -persistent anemia and

lower Hb levels (figure 2B) in this group, compared with those

in NMI monkeys (group, ; time, ; group �P ! .0001 P ! .0001

time, [2-way ANOVA for all]). The nadir at GW 14P ! .0001

in PMI monkeys was ∼1 million RBCs less than those of NMI

monkeys, and Hb levels were 6.75 and 7.87 g/dL, respectively.

Although Hb levels were similar during the recovery period,

NMI monkeys had more-rapid and -complete recoveries of

their RBC counts than did PMI monkeys at GW 18–22. The

gradual decline of RBCs in PC monkeys (from 5.32 to 4.8 �

106) (figure 2A) occurred as a result of normal pregnancy; this

has been observed in other studies at Tulane National Primate

Research Center (unpublished data).

Monocytes and polymorphonuclear neutrophils. PMI mon-

keys had significantly fewer circulating monocytes than did

NMI monkeys, despite chronic parasitemia in PMI monkeys

throughout the second trimester (group, ; time,P ! .0001 P !

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Figure 6. Absolute no. of total lymphocytes in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) and nulligravid(NMI) monkeys. The absolute no. of lymphocytes was significantly lower in PMI than in NMI monkeys, starting at gestational week (GW) 14 (weekspostinfection [WPI] 8). Post hoc Newman-Keuls evaluation for each time point tested at GW 14 was , that for GW 18 (WPI 12) wasP p .002 P p

, and that for GW 22 (WPI 16) was . Differences in counts in PMI vs. PC monkeys were not significantly different ( ) for any.0002 P p .0005 P p NStime point tested. Although PMI monkeys had severe clinical malaria, their lymphocyte counts were similar to those of PC monkeys. Each point representsthe mean of the monthly lymphocyte counts occurring in each group of 8 monkeys. See figure 1 for description of the timeline used.

; group � time, [2-way ANOVA for all]) (figure.0001 P ! .0001

3). During parasite recrudescence at GW 16, monocytes peaked

in NMI but not PMI monkeys, although PMI monkeys had

65,223 PRBCs/mL, compared with 32,472 PRBCs/mL in NMI

monkeys (figure 1). P. coatneyi infection was also associated

with decreased levels of neutrophils (group, ; time, P!P ! .05

.002 [2-way ANOVA for both]) (figure 4) and platelets (group,

; time, ; group � time, [2-way ANOVAP ! .0007 P ! .0001 P ! .001

for all]) (figure 5) in both PMI and NMI monkeys, compared

with PC monkeys.

Total lymphocyte counts. Primigravid females inoculated

with P. coatneyi had strikingly different lymphocyte patterns

than did age-matched NMI monkeys. The most significant dif-

ferences began late in trimester 2 (GW 14) ( ) (figureP p .002

6), concurrently with the RBC nadir (figure 2A). Moreover, by

GW 18 and at delivery (GW 22), total lymphocyte counts were

lower in PMI than in NMI monkeys ( andP p .0002 P p

, respectively). Interestingly, despite the severe clinical ma-.0005

laria observed in PMI monkeys, their lymphocyte counts did

not differ from those of PC monkeys.

These differences were even more apparent when lymphocyte

subsets were examined separately. Unfortunately, because of the

small group size with individual and daily variation, PMI mon-

keys started with lower numbers of T and CD4+ lymphocytes

than did the PC monkeys at inoculation. However, 2 weeks

before this sample was obtained, another baseline value showed

the 3 groups to be more comparable. Regardless, numbers of

T lymphocytes decreased significantly in PMI monkeys during

the second trimester (by GW 14) and remained significantly

lower, whereas levels increased in NMI monkeys (group � time,

) (figure 7). By repeated-measures analysis, levels ofP p .004

CD4+ lymphocytes were significantly lower throughout the

course of infection in PMI monkeys than in NMI monkeys

(group � time, ) (figure 8). Moreover, there was aP p .003

negative trend in absolute numbers of CD4+ lymphocytes in

PMI monkeys, compared with that in PC monkeys; levels of

CD4+ lymphocytes were significantly lower in PC monkeys at

GW 14 ( ) and GW 22 (delivery) ( ) (figureP p .003 P p .001

8). In contrast to the increase in numbers of CD8+ lymphocytes

in NMI monkeys, PMI monkeys had almost no change (group

� time, ), and their CD8+ lymphocyte profiles wereP p .006

similar to those of PC monkeys (figure 9). Levels of CD8+

lymphocytes were significantly lower in PMI monkeys than in

NMI monkeys at GW 14 ( ) and GW 22 (delivery)P p .007

( ). Pregnancy itself, represented by PC monkeys, wasP p .05

associated with little change in levels of CD4+ and CD8+ lym-

phocytes from baseline until delivery. Despite malaria, the cel-

lular profiles of PMI monkeys were more similar to those of

PC monkeys than to those of NMI monkeys.

Our most striking observation was the failure of B lympho-

cyte (CD20+) counts to increase in PMI monkeys to levels

observed in NMI monkeys, despite chronic malaria infection

of similar duration (figure 10). Even given our small groups,

the difference between PMI and NMI monkeys was significant

throughout gestation and malaria infection. The average CD20+

lymphocyte count in NMI monkeys was ∼23 times that of PMI

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Figure 7. Absolute no. of T lymphocytes (CD2+) in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) and nulligravid(NMI) monkeys, determined by flow-cytometric analysis. The absolute no. of CD2+ lymphocytes is displayed, because they changed over time in the 3monkey groups at 6 consecutive time points during gestation/infection. In repeated-measures analysis, the postinoculation average CD2+ lymphocyte countsin PMI monkeys did not change over time ( , for within-subject factor time). This is similar to the profile observed in PC monkeys ( , forP p .29 P p .148between-subjects factor groups). In contrast to the 2 pregnant groups, however, NMI monkeys showed a positive response in CD2+ lymphocyte counts(the interaction term time � group is significant [ ]). See figure 1 for description of the timeline used. GW, gestational week.P p .004

monkeys (time, group, and time � group, ). AlthoughP ! .001

the absolute number of circulating B lymphocytes in PMI mon-

keys was 5-fold greater than that in PC monkeys, the difference

was not significant.

DISCUSSION

Because we examined leukocyte populations in long-term (16

week) asexual or blood-stage malaria infection in monkeys with

intact spleens and without antimalarial treatment, we obtained

a complete leukocyte profile uncompromised by confounding

variables. Thus, the effects could be identified of primigravid

pregnancy and chronic malaria, the 2 defining variables in the

experiment, on peripheral blood leukocyte levels. Even in severe

malaria, the absolute numbers of peripheral blood monocytes,

neutrophils, and lymphocytes in PMI monkeys resembled those

of PC monkeys more than those of NMI monkeys.

Unlike NMI monkeys, T lymphocyte levels did not increase

in pregnant monkeys in response to malaria. Malaria infection

during pregnancy was associated with lower CD4+ and CD8+

lymphocyte counts, similar to PC monkeys. The higher group

mean CD2+ and CD4+ lymphocyte counts in PC monkeys than

in PMI and NMI monkeys on the day of inoculation was likely

due to individual variation resulting from early unidentified

pregnancy-associated events in specific individuals. However,

levels were similar for all cell types in the 3 groups before

inoculation, at GW 4. Studies have suggested that the num-

bers and types of circulating lymphocytes in healthy pregnant

women are similar to those in nonpregnant women [25, 49],

although some studies have shown changes in various cell types

[24–26, 37, 49–54]. Overall, our findings are consistent with

healthy pregnant women and nonpregnant women having sim-

ilar cellular profiles. Immunomodulation—a decline in CD4+

and CD8+ and B lymphocyte counts during pregnancy—in

pregnant monkeys was apparent only when they were chal-

lenged with Plasmodium infection.

Compared with PC monkeys, pregnant monkeys with ma-

laria also showed a significant decrease in neutrophil levels

starting at GW 9. Throughout normal human pregnancy, there

is a consistent increase in neutrophil levels [55]. Declining neu-

trophil levels might interfere with a malaria-infected pregnant

woman’s ability to control bacterial infections associated with

chorioamnionitis (CA). CA is the most common cause of pre-

term delivery [56], is often associated with malaria [57], and,

like concomitant HIV and malaria [58, 59], has been linked to

increased vertical transmission of HIV [60].

Thrombocytopenia was noted in both malaria-infected

groups. PMI monkeys had one-half the platelet count of PC

monkeys. Low platelet levels have been associated with severe

preeclampsia and other pregnancy complications that cause

premature delivery and LBW [61, 62], and they may contribute

to malaria-associated pregnancy complications.

Levels of monocytes, which are essential to innate immunity

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Figure 8. Absolute no. of helper T lymphocytes (CD4+) in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI)and nulligravid (NMI) monkeys, determined by flow-cytometric analysis. In pregnant monkeys, the average no. of CD4+ lymphocytes actually showeda negative trend after gestational week (GW) 10 (week postinfection 4), in contrast to the positive response observed in NMI monkeys. Moreover,the average no. of CD4+ lymphocytes in PMI monkeys was considerably less than that in PC monkeys for the whole period after inoculation (time,

; group, ; time � group, ). Because of the small group size with individual and day-to-day variation, PMI monkeys startedP p .65 P p .077 P p .003with lower nos. of T lymphocytes and CD4+ lymphocytes than did PC monkeys at the time of inoculation. However, 2 weeks before this sample wasobtained, another baseline value showed the 3 groups to be more comparable. See figure 1 for description of the timeline used.

and effective immune responses to malaria, were lower in PMI

than in NMI monkeys. Monocytes and macrophages are key

cells in controlling malaria through the phagocytosis of PRBCs,

resulting in parasite killing and antigen presentation to CD4+

lymphocytes [63, 64]. Peripheral blood monocyte responses

have not been reported in prospective studies of pregnant

women with malaria. The decrease that we observed may con-

tribute to the increased susceptibility of primigravid women to

severe clinical malaria. Increased macrophage levels within the

placenta have been associated with poor fetal outcome [65, 66],

but it remains to be shown whether decreasing monocyte levels

in the peripheral blood of the monkeys is associated with a

shifting of these cells into the uterine or placental compartment.

Importantly, our studies show a profound affect of pregnancy

on the host’s ability to increase numbers of B lymphocytes, as

does the nongravid host who is chronically infected with ma-

laria. Antimalarial antibody–producing B cells are essential to

clear blood-stage parasites [67]. Antibody studies under way

in monkeys should determine whether B cells effectively pro-

duce antimalarial antibodies. We suspect, as has been reported

for humans [68], that this is a malaria-induced polyclonal ac-

tivation of B lymphocytes; perhaps, if it is not suppressed during

pregnancy, it could lead to increased maternal hyperreactivity

to fetal antigens. Alterations in the reactivity of the maternal

immune system toward paternal alloantigens or foreign anti-

gens might contribute to the reduction in the intrauterine de-

velopment of the placenta and fetus [69]. Conceivably, even

the 5-fold increase in B lymphocyte levels observed in PMI

versus PC monkeys may cause malaria-associated pregnancy

complications. Studies of human and rodent pregnancy have

correlated increased maternal B lymphocyte counts with del-

eterious effects on the fetus, such as IUGR, preterm delivery,

and LBW [26, 27, 37, 38, 69–71]. Individual differences in the

reactivity of the maternal immune system to malaria antigens

may account for variability in fetal outcome, with poor out-

comes correlating with high B cell production. Importantly, we

hypothesize that the individual reactivity of the maternal im-

mune system to malaria antigens may change in successive

pregnancies. Thus, our findings with respect to B lymphocytes

may bear directly on the poor fetal outcome of a woman’s first

pregnancy, compared with those of later pregnancies. Exami-

nation of these same monkeys, reinfected with malaria during

subsequent pregnancies, will be the subject of future reports to

verify or repudiate this possibility.

The general pancytopenia we observed, along with throm-

bocytopenia and anemia, suggests generalized bone marrow sup-

pression (BMS). In adult mammals, steady-state production of

B cells occurs within the bone marrow [71], so the decreased B

lymphocyte levels in the pregnant monkeys challenged with ma-

laria also suggest BMS. In mice, B lymphopoiesis in the bone

marrow is markedly down-regulated during pregnancy, which

affects all precursor populations beyond the pro–B cell stage,

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Figure 9. Absolute no. of cytotoxic/suppressor (CD8+) lymphocytes in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid(PMI) and nulligravid (NMI) monkeys, determined by flow-cytometric analysis. In contrast to the increase observed in the CD8+ lymphocytes in NMImonkeys, PMI monkeys showed no change or a slight decrease, and their CD8+ lymphocyte profile was similar to that of PC monkeys (time, P p

; group, ; time � group, ). See figure 1 for description of the timeline used. GW, gestational week..074 P p .211 P p .006

Figure 10. Absolute no. of B lymphocytes (CD20+) in primigravid control (PC) monkeys and age-matched, malaria-infected primigravid (PMI) andnulligravid (NMI) monkeys, determined by flow-cytometric analysis. Nos. of CD20+ B lymphocytes were different throughout gestation/infection in all3 groups. PMI monkeys had significantly fewer B cells than did NMI monkeys throughout gestation, starting at gestational week (GW) 10. Towardthe end of pregnancy, the average no. of CD20+ B lymphocytes in the PMI monkeys was ∼5 times that in PC monkeys. In comparison, the averageCD20+ B lymphocyte count in NMI monkeys was ∼23 times that in PC monkeys (for time, group and time � group, ). See figure 1 for de-P ! .001scription of the timeline used.

[37] whereas most mature B cells are exempt. BMS occurs in

malaria and contributes to anemia, but the mechanisms are un-

clear [72]. Pregnancy, in combination with malaria, may exac-

erbate BMS though undefined processes. Bone-marrow biopsies

in the pregnant monkey model may answer these questions.

Thus, in the face of Plasmodium infection, levels of granu-

locytes, monocytes, and lymphocytes decrease throughout ges-

tation. PMI monkeys apparently tried to maintain a cellular

profile similar to that of PC monkeys, allowing fetal survival.

However, decreased leukocyte levels may contribute to severe

recrudescing parasitemia, thus affecting fetal outcome. Future

reports will present data not only correlating maternal malaria

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and leukocyte populations with fetal/infant/placental growth,

development, and well being but also examining the monkeys’

peripheral and placental cytokine patterns and their association

with differing numbers of pregnancies and levels of immunity.

Given the complex patterns observed, conflicting reports of

increased or decreased levels of lymphocyte subsets during “nor-

mal” human pregnancy are not surprising. We have demon-

strated that it is the system under challenge by a pathogen that

reveals the nature of the modifications induced by pregnancy,

not as a loss of leukocytes through a generalized pregnancy-

induced phenomenon but as a lack of appropriate leukocyte

increases in response to disease. Both successful pregnancy and

survival of the malaria parasite depend on a carefully balanced

immune system. A selective species-survival advantage is likely

when pregnancy dominates, ensuring that immune responses do

not harm fetal growth, development, and survival. Because severe

clinical malaria is manifested by immunopathologic symptoms,

perhaps the inappropriately low leukocyte numbers in response

to malaria are “protective,” allowing fetal survival. The same

protection afforded to the fetus may also be advantageous to

both host and parasite survival, thus making the primigravid

woman a perfect host for Plasmodium species.

The results reported here demonstrate one aspect of the im-

munomodulation occurring in primates challenged with ma-

laria during pregnancy. This was illustrated by the failure of

pregnant monkeys, compared with age-matched nulligravid mon-

keys under the same conditions of severe Plasmodium infection,

to appropriately increase cell numbers in multiple leukocyte

populations. In our study, it was the host immune system under

the influence of pregnancy that was responsible for the in-

creased severity of clinical malaria. These findings could have

important ramifications for the treatment strategies for mater-

nal malaria and other diseases.

Acknowledgments

We thank Calvin Lanclos and Julie Bruhn, Division of Microbiology andImmunology, Tulane National Primate Research Center (TNPRC), for theirexcellent technical support performing the flow-cytometric analysis; GailPlauche and Nancy Hartzog, for their tireless efforts in the clinical pa-thology laboratory, Division of Comparative Pathology, TNPRC; and Dr.Marion Ratterree, the veterinary technicians Frankie Anders and the tech-nicians of H building, and Gloria Jackson and the nursery staff, Divisionof Veterinary Medicine and Research, TNPRC, for outstanding animal careand technical assistance.

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