2012, 80（1）:311. DOI: 10.1128/IAI.05900-11.  Infect. Immun. 

Igarashi and Xuenan Xuan

Yamagishi, Tatsunori Masatani, Naoaki Yokoyama, Ikuo 

Goo, Longzheng Yu, Shinuo Cao, Yongfeng Sun, Junya

Gabriel Oluga Aboge, Yuzi Luo, Hideo Ooka, Youn-Kyoung 

Yan Li, Mohamad Alaa Terkawi, Yoshifumi Nishikawa,

 

Infection in Mice

Babesia microti against Babesia rodhaini

Cross-Protective Immunity Conferred by 

Macrophages Are Critical for

http://iai.asm.org/content/80/1/311

Updated information and services can be found at: 

These include:

REFERENCES

http://iai.asm.org/content/80/1/311#ref-list-1 at: 

This article cites 57 articles, 23 of which can be accessed free

CONTENT ALERTS

 more» articles cite this article）, 

Receive: RSS Feeds, eTOCs, free alerts （when new

http://iai.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: 

http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: 

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from Macrophages Are Critical for Cross-Protective Immunity Conferred by

Babesia microti against Babesia rodhaini Infection in Mice

Yan Li, Mohamad Alaa Terkawi, Yoshifumi Nishikawa, Gabriel Oluga Aboge, Yuzi Luo, Hideo Ooka, Youn-Kyoung Goo, Longzheng Yu,

Shinuo Cao, Yongfeng Sun, Junya Yamagishi, Tatsunori Masatani, Naoaki Yokoyama, Ikuo Igarashi, and Xuenan Xuan

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan

Although primary infection ofmice with Babesia microti has been shown to protectmice against subsequent lethal infection by

Babesia rodhaini, themechanismbehind the cross-protection is unknown. To unravel thismechanism, we investigated the in-

fluence of primary infection ofmice with nonlethal B. microti using different time courses on the outcome of subsequent lethal

B. rodhaini infection. Simultaneous infections ofmice with these parasites resulted in rapid increases in parasitemia, with 100%

mortality in BALB/cmice, as observed with controlmice infected with B. rodhaini alone. In contrast,mice with acute, resolving,

and chronic-phase B. microti infections were compley protected against B. rodhaini, resulting in low parasitemia and no mor-

talities.Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses

were induced. Interestingly, the protectedmice had significantly decreased levels of antibody response, cytokines （including

gamma interferon [IFN-], interleukin-2 [IL-2], IL-8, IL-10, and IL-12）, and nitric oxide levels after infection with B. rodhaini.

SCIDmice and IFN--deficientmice with chronic B. microti infections demonstrated protective responses comparable to those

of immunocompetentmice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely,

macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and

cytokines profiles, indicating thatmacrophages contribute to the protection against this challenge infection.We conclude that

future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of

macrophages.

Babesiosis is caused by intraerythrocytic parasites of the genus

Babesia. The infection is one of themost important tick-borne

diseases parasitizing a wide range of mammalian hosts, including

humans, worldwide. Babesiosis causes huge economic losses in

the livestock industry. Recently, the disease has become an impor-

tant emerging zoonosis, with Babesia microti being the most im-

portant cause of human babesiosis in America, Europe, and Asia

（23, 26, 27, 35, 55）. Most of the human cases are either from

tainted blood transfusions or from bites of infected Ixodes scapu-

laris nymphs, which inject sporozoites into the bloodstreamof the

host during their feeding. The infection is often asymptomatic in

healthy humans but can occasionally be fatal in immunocompro-

mised individuals （27, 36, 37）.

Abetter understanding of the immune response to infection by

Babesia parasites is important for designing a safe and efficacious

vaccine （9, 28）. Over the past decade, several studies have demon-

strated the importance of T helper （Th） cells in regulating the

immune response to Babesia infection （2, 12, 28）. These cells pro-

duce the cytokines needed for the bothmaturation of high-affinity

immunoglobulin isotype production and the activation ofmacro-

phages for phagocytosis and parasiticidal activity （10, 11）. How-

ever, the timing and magnitude of these cytokines can determine

the outcome of the infection. The early response of the inflamma-

tory cytokines gamma interferon （IFN-） and interleukin-12 （IL-

12） is critical for controlling the initial burst of intraerythrocytic

parasite multiplication. Moreover, the failure to maintain Th1-

predominant response during the acute stage is correlated with a

rapid increase in parasite load. On the other hand, the switch to

the predominance of the Th2 response （IL-4 and IL-10） at the

resolution stage accompanied by elevated antigen-specific immu-

noglobulinG（IgG） appears to be crucial for the control of parasite

replication （2, 12）. Phagocytosis of parasitized erythrocytes by ac-

tivated macrophages occurs in the spleen and is believed to be

essential for the removal of the parasites （48）.

The use of mice, rather than large mammals, infected with

rodent Babesia provides an economic model for investigating the

host immune response to babesiosis （28, 50）. Mice infected with

B. microti reveal transient high parasitemia, followed by complete

recovery from the acute infection, and the cured mice are usually

resistant to the reinfection by the same parasite. This protection is

mainly due to T-cell-mediated immunity in the spleen （28, 44）. In

contrast, B. rodhaini causes a more severe disease, resulting in

100%mortality （34）. Interestingly,mice that had a prior infection

with B. microti are known to be protected against challenge infec-

tion by B. rodhaini Antwerp strain, with a survival rate of up to

83% （58）. However, the mechanism behind this cross-protection

is unknown. We believe that understanding the mechanism be-

hind this protection could provide important clues for the future

design of vaccines against babesiosis.

In the present study, the cross-protection conferred by primary

infection of mice with B. microti against challenge with lethal B.

rodhaini was examined either in the absence or presence of im-

mune effector cells. We show that innate immunity based on the

Received 9 September 2011 Returned for modification 7 October 2011

Accepted 24 October 2011

Published ahead of print 7 November 2011

Editor: J. H. Adams

Address correspondence to Xuenan Xuan, gen@obihiro.ac.jp.

Y. Li and M. A. Terkawi contributed equally to this study.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.05900-11

0019-9567/12/$12.00 Infection and Immunity p. 311–320 iai.asm.org 311

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from macrophage response but not adaptive immunity is crucial to the

cross-protection offered by B. microti against lethal B. rodhaini

infection. However, antibody, B and T lymphocytes, IFN-, and

NK cells did not play a major role in this cross-protection.

MATERIALS AND METHODS

Mice. Six-week-old female BALB/c and C.B-17/Icr-scid/scid （SCID）mice

were purchased from CLEA Japan. IFN--deficient （IFN-/） mice de-

rived from BALB/c background were bred and maintained as previously

described （28, 49）. All animal experiments were conducted in accordance

with the Standards Relating to the Care andManagement of Experimental

Animals promulgated by the Obihiro University of Agriculture and Vet-

erinaryMedicine of Japan. All experiments were repeated at least twice to

obtain reproducible data.

Maintenance of the parasites and mouse infections. The B. microti

Munich strain and B. rodhaini Australian strain were maintained in mice

by intraperitoneal （i.p.） passage as previously described （28, 50）. Initial

infection ofmicewith B.microtiwas done by i.p. inoculations ofmicewith

107 of parasitized erythrocytes （pRBCs）. Test mice were infected with B.

microti prior to challenge infection with B. rodhaini, while control mice

were either inoculated with nonparasitized murine erythrocytes

（npRBCs） or not inoculated （mock mice） before the challenge infection.

Preparation of dead B. microti for mice immunization. The inocula

of B. microti pRBCs and npRBCs, both fixed with glutaraldehyde, were

prepared as previously described （7）. Briefly, B. microti-infected murine

blood was harvested when the parasitemias reached 50%, treated with

Histopaque-1077 （Sigma）, and then washed with sterile phosphate-

buffered saline （PBS; pH 7.2） three times. After the last wash, the RBCs

were fixedwith 0.25%glutaraldehyde for 15min at roomtemperature and

then washed three times with sterile PBS. The fixed RBCs were stored at

4°C in sterile PBS supplemented with penicillin and streptomycin. Before

inoculation, the cells were washed twice with sterile PBS, counted, and

reconstituted at the desired concentrations. Mice were immunized i.p.

three times at 2-week intervals with either 2108 glutaraldehyde-fixed B.

microti pRBCs in 0.5 ml of PBS （the test group） or an equivalent amount

of glutaraldehyde-fixed npRBCs in 0.5 ml of PBS （the control group）.

Blood samples were collected from the tail vein 2 weeks after the last

inoculation and before the challenge with B. rodhaini pRBCs. Thereafter,

the specific antibody response to B. microti antigens was determined by

indirect immunofluorescence test （IFAT） （50）.

Determination of parasitemia and survival rates. Thin blood smears

were made by using blood obtained from tail veins of mice. The smears

were fixed inmethanol and stained for 45minwith 10%Giemsa diluted in

Sörensen buffer （pH 6.8）. Thereafter, parasitemia was determined by ex-

amining 103 erythrocytes, under an oil immersion microscope, for the

presence of intraerythrocytic Babesia. The examination was performed at

2-day intervals after the initial infection. In addition, the infected mice

were observed daily for anymortality until the experimentwas terminated

at day 20 postchallenge infection. For hematological evaluation, 10 lof

blood collected from each mouse at 2-day intervals was transferred into

plastic tubes containing 2ml of premixed solution. A full blood cell count

was made using an automatic cell counter （Nihon Kohden, Japan）.

Detection of specific antibody to B. rodhaini P26. Recombinant B.

rodhaini P26 （rBrP26） was expressed as a glutathione S-transferase fusion

protein with a molecular mass of 57.7 kDa. The expressed fusion protein

was purified by glutathione-Sepharose 4B columns （Amersham Biosci-

ences）, and the resulting antigen was used in an enzyme-linked immu-

nosorbent assay （ELISA） to detect specific antibodies （50）. Briefly, 96-well

microtiter plates （Nunc, Roskilde, Denmark） were coated overnight at

4°C with 50 l of rBrP26 at a concentration of 0.2 g/per well in a coating

buffer （50 mM carbonate-bicarbonate buffer [pH 9.6]）. The plates were

washed once with 0.05% Tween 20-PBS （PBST） and then incubated with

100 l of a blocking solution （3%skimmilk in PBS） for1hat 37°C. After

one wash with PBST, the antigen-coated wells were incubated with 50 l

of mice sera diluted 1:100 with the blocking solution for1hat 37°C. The

plates were washed six times with PBST and then incubated with 50 l

of the horseradish peroxidase-conjugated goat anti-mouse immuno-

globulins IgG1 and IgG2 （Bethyl Laboratories） diluted to 1:4,000 with

the blocking solution for1hat 37°C as a secondary antibody. The

plates were washed six times as described above, and then 100 lofa

substrate solution {0.1 M citric acid, 0.2 M sodium phosphate, 0.3 mg

of ABTS [2,2=azinobis（3-ethylbenzthiazolinesulfonic acid）]/ml

[Sigma, St. Louis,MO], 0.01%of 30%H2O2} per well was added. After

incubation for1hat room temperature, the optical density was mea-

sured by an MTP-500 microplate reader （Corona Electric, Tokyo, Ja-

pan） at a wavelength of 415 nm.

Detection of serum cytokines. To obtain serum for cytokine detec-

tion, blood was collected from each mouse by cardiac puncture and then

processed to get serum. Test and control mice （20 mice for each group）

were sacrificed at days 0, 2, 4, or 6 postchallenge infection with B. rodhiani

（five mice for each day）. The concentrations of the individual sample

cytokines were determined by ELISA kits using standard curves prepared

with known concentrations ofmouse recombinant IFN-, tumor necrosis

factor alpha （TNF-）, IL-2, IL-4, IL-10, and IL-12 （Endogen） and IL-8

（Cusabio Biotech Co., Germany） according to the manufacturer’s in-

structions.

Measurement of nitric oxide （NO）. The levels of nitrate and nitrite

production in themice serumweremeasured using a nitrate/nitrite assay

kit （Cayman Chemical Co.） according to themanufacturer’s instructions.

The NO levels were calculated with a standard absorbance curve derived

from tests run on the same plate.

Flow cytometry assays. Splenocytes and peritoneal cells from three

mice were obtained and resuspended in cold PBS containing 0.5%bovine

serum albumin. Thereafter, the cells were incubated with the respective

fluorescein isothiocyanate-labeled anti-mouse monoclonal antibodies

（MAbs）CD49b/Pan-NKcell （DX5）,CD11b, or F4/80 （BDBiosciences, La

Jolla, CA） at 4°C for 30 min （41）. After three washes with cold PBS, the

cells were analyzed using an EPICS XL flow cytometer （Beckman

Coulter）.

IFAT. To differentiate the two parasitic infections, standard IFAT was

performed with specific antibodies. Briefly, IFAT slides were coated with

pRBCs collected from mice at day 6 postchallenge infection with B. rod-

haini （50）. The slides were dried and fixed in absolute acetone for 10min.

The fixed slides were incubated with either anti-rBmP94/CT or anti-

rBrP26 rabbit antibody （29, 43） diluted at 1:100 in PBS in amoist chamber

at 37°C for 1 h. After the slides were washed four times with PBS contain-

ing 0.05%Tween 20 （PBST）, Alexa Fluor 488-conjugated goat anti-rabbit

IgG （Molecular Probes） was applied as a secondary antibody （1:250）, and

then the slides were incubated at 37°C for 1 h. The slides were washed four

times with PBST and examined using a fluorescence microscope （E400

Eclipse; Nikon, Japan）.

In vivo depletion ofNKcells andmacrophages. To examine the effect

of NK cell depletion on the outcome of the Babesia infections, 50 lof

anti-asialo-GM1 antibody diluted in 200 l of PBS （Wako, Japan） was

administered tomice i.p. on days2,3, and6 relative to the infection

with B. rodhaini （20）. This protocol resulted in effective depletion of NK

cells in the spleen when examined by flow cytometry using anti-mouse

DX5 （BD Biosciences, La Jolla, CA）. In separate experiments, macro-

phages were depleted by i.p. administration of 300 l of clodronate lipo-

somes 2 days before and 3 days after challenge infection with B. rodhaini

（20）. Clodronate encapsulated in liposomes （54） was a gift from Roche

Diagnostics,GmbH,Netherlands. Seven days after the last injection,mac-

rophage depletion was determined by using flow cytometry by staining

cells derived fromperitoneal fluid and splenocytes with CD11b and F4/80

MAbs, respectively.

Statistical analysis. Statistical analysis of any significant differences

between the means of all variables was done by one-way analysis of vari-

ance （GraphPad Prism 5; GraphPad Software, Inc.）. Tukey’s multiple-

comparison test was used for pairwise comparison of data from the mul-

tiple groups. Survival analysis was done by using log-rank andWilcoxon

Li et al.

312 iai.asm.org Infection and Immunity

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from tests incorporating the Kaplan-Meier nonparametricmodel for establish-

ing any statistically significant differences. All results were considered sta-

tistically significant when P was 0.05.

RESULTS

Primary infection with B. microti offers complete protection

against lethal B. rodhaini challenge infection. Infection with B.

rodhaini caused severe parasitemia resulting in mortalities of all

mice within the first week of infection. In contrast, infection with

B. microti caused transient parasitemia for about 3 weeks and,

thereafter, the infection became persistent in mice with low level

of parasitemia （data not shown）. To determine whether primary

infection of mice with B. microti at the acute, resolving, and

chronic stages could provide protection to lethal B. rodhaini in-

fection, BALB/cmice were initially inoculated with B. microti and

then challenged with B. rodhaini at days 0, 7, 14, 28, and 56 after

primary infection. Strikingly,mice thatwere simultaneously coin-

fected with B. rodhaini exhibited rapid increase in the B. rodhaini

parasitemia with a peak of more than 80% （Fig. 1A）. These mice

eventually succumbed to the infection within the first week, as did

controlmice infected with B. rodhaini alone （Fig. 1B）. In contrast,

mice at acute （day 7）, resolving （day 14）, and chronic （days 28 and

56） stages of B. microti infection showed complete protection,

resulting in 100%survival against lethal infection with B. rodhaini

（Fig. 1D, F, H, and J）. These mice developed significantly lower

levels of parasitemia, which resolved within the third week of in-

fection （Fig. 1C, E, G, and I）. Hematological kinetics were moni-

tored during the infection course of B.microti and after B. rodhaini

challenge infection. Notably, a significant reduction in the total

number of RBCs and hematocrit coincided with the parasitemia

increase. Mice that recovered from B. microti infection showed

normal hematological values with no significant difference com-

pared to those detected before infection with B. microti （data not

shown）. Immunofluorescence assays for pRBCs collected from

mice 6 days after the challenge infection were performed using

specific antibodies to differentiate the two infections from each

other. The results revealed that the majority of pRBCs were in-

fected by B. rodhaini （data not shown）. These results indicated

that primary infection of mice with B. microti offered complete

cross-protection against lethal infection of B. rodhaini.

Immunization with dead B. microti fails to protect the mice

from lethal B. rodhaini challenge infection. To examine

whether the dead parasites could offer protective immunity

against B. rodhaini infection, B. microti-infected RBCs were

administered i.p. into BALB/c mice, followed by two consecu-

tive boosters at 14-day intervals. The immunized mice devel-

oped high titers of specific antibody against B. microti （1:3,200

to 1:6,400）. Control mice, which were inoculated with murine

noninfected RBCs did not show antibody response to B.microti

（data not shown）. Strikingly, after challenge infection with B.

rodhaini, all of the mice showed rapid increases in parasitemia

and consequently succumbed to the infection within a week

（Fig. 1K and L）. These results indicated that immunization of

FIG 1 Parasitemia and survival rates after B. microti inoculation and B. rodhaini challenge infection of BALB/c mice. Parasitemia course （A, C, E, G, and I） and

survival rates （B, D, F, H, and J） of mock and test mice are presented. Test mice were initially infected with B. microti then challenged with B. rodhaini on days

0, 7, 14, 28 and 56 after primary infection.Mockmice received B. rodhaini alone. Arrows indicate the time of challenge infection with B. rodhaini （A, C, E, G, and

I）. The parasitemia course （K） and survival rate （L） ofmice immunized with either dead B.microti （pRBCs） or nonparasitizedmurine RBCs （npRBCs） and then

challenged with B. rodhaini. The results are expressed as a mean percent parasitemias the standard deviations （SD） of five mice.

Macrophages Mediate Protection against Babesia in Mice

January 2012 Volume 80 Number 1 iai.asm.org 313

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from mice with dead B. microti did not confer protective immunity

against challenge infection with B. rodhaini.

Protected mice induce low levels of antibody and cytokine

production. Serum antibody and cytokines were measured in

mice acuy （7 days） and chronically （28 days） infected with B.

microti at days 2, 4, and 6 after challenge infections with B. rod-

haini. Specific antibody to B. rodhaini （rBrP26） was detected at

day 6, whereas the IgG1 and IgG2 levels were significantly lower in

mice initially infected with B. microti and then challenged with B.

rodhaini than those detected in either mock or control mice （Fig.

2A to C）. Likewise, detectable IL-8, IL-12, IL-2, and IFN- levels

were significantly lower in B. microti-infected mice （P 0.05）

FIG 2 Kinetics of serumIgGs and cytokines of protected and susceptiblemice after B. rodhaini challenge infection. The production of IgG （A）, IgG1 （B）, IgG2a

（C）, IL-8 （D）, IL-12 （E）, IL-2 （F）, IFN- （G）, IL-10 （H）, TNF- （I）, andNO（J） inmice after challenge infection with B. rodhaini was determined. Testmice with

acute and chronic B. microti infection, control mice （which received npRBC）, or mock mice （which received no injection） were infected with B. rodhaini.

Detection of IgGs, cytokines, and NO was performed in themice at days 2, 4 and 6 after challenge infection. Asterisks indicate statistically significant differences

（, P 0.05; , P 0.005; , P 0.0001 [compared to mock and control mice]）. The results are expressed as mean values the SD for five mice.

Li et al.

314 iai.asm.org Infection and Immunity

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from than those detected in eithermock or controlmice at days 4 and 6

postchallenge infection （Fig. 2D to G）. IL-10 cytokine was only

detected in the sera of either mock or control mice 6 days after

challenge infection with B. rodhaini. However, IL-10 level was

below the detection limit in B. microti-infected mice （Fig. 2H）.

TNF- production was detected at day 6 after the challenge infec-

tion, and these levels were not significantly different between test

and control mice （Fig. 2I）. In addition, the detectable nitric oxide

（NO） was significantly lower （P 0.05） in B. microti-infected

mice than those detected in either mock or control mice （Fig. 2J）.

Moreover, the levels of IL-4 in all mice were below the detection

limit of the ELISA kit （data not shown）. These results indicated

that B.microti-infectedmice during either the acute or the chronic

stage had diminished their antibody and cytokine production in

response to lethal B. rodhaini infection.

The absence of IFN- and B and T lymphocytes does not im-

pair the complete protection conferred by B. microti infection.

To determine the role of IFN- and B and T lymphocytes in the

protection against lethal infection with B. rodhaini, IFN-/

mice and SCID mice were primarily infected with B. microti, fol-

lowed by challenge infection with B. rodhaini after 28 days. The

kinetics of parasitemia in IFN-/ mice and SCID mice was sig-

nificantly different fromthose of immunosufficient BALB/cmice.

Indeed, IFN-/ and SCID mice demonstrated higher para-

sitemia levels persisting over 28 days after the primary infection

（Fig. 3A and C）. After challenge infection with B. rodhaini, control

IFN-/ and SCID mice developed rapid increases in para-

sitemia approaching80%, and allmice eventually succumbed to

the infection within the second week of challenge infection （Fig.

3B and D）. In contrast, IFN-/ mice and SCID mice with

chronic B.microti infection survived over a period of 3 weeks （Fig.

3B and D）, although they persistently maintained high para-

sitemia ranging between 20 and 30%（Fig. 3A and C）.Notably, the

majority of erythrocyteswere found to be infectedwith B. rodhaini

when the blood smears of protectedmice were examined by IFAT

（data not shown）. These results indicated that the protective status

induced by B. microti infection against the lethality of B. rodhaini

challenge infection was not impaired in the absence of IFN- and

B and T lymphocytes.

The absence of macrophages/monocytes but not natural

killer cells impairs the protection conferred by B.microti infec-

tion in BALB/cmice. To examine the possible contribution ofNK

cells and macrophages/monocytes in protection against lethal in-

fection with B. rodhaini, anti-asialo-GM1 antibody and clodro-

nate liposomewere administered i.p. to BALB/cmicewith chronic

B. microti infections, respectively. The depletion experiment was

performed prior to challenge and optimized for long-lasting effi-

ciency （data not shown）. The parasitemia levels in mice with de-

pleted NK cells were slightly elevated and consequently resolved

within the second week of challenge with no significant difference

compared tomice that received control antibody （P0.05）.Con-

sistently, there was no significant difference in mortalities and

survivals between intact and NK cell-depleted mice （Fig. 4A and

B）. In sharp contrast, clodronate liposome-treatedmice hadmore

significant rapid parasite growth than did PBS-liposome-treated

mice. Consequently, 80% of these mice died within the second

week of challenge infection （Fig. 4C and D）. These results indi-

cated the importance ofmacrophages in offering immune protec-

tion against challenge infection with B. rodhaini. Furthermore, to

examine whether the absence ofmacrophages/monocytes impairs

FIG3 Parasitemia and survival rates of IFN-/ mice and SCIDmice after B. rodhaini challenge infection. Parasitemia profiles and survival rates of chronically

B. microti-infected IFN-/ mice （A and B） and SCID mice （C and D）, respectively, over a period of 20 days after challenge infection are presented. Test mice

were initially infected with B. microti and then challenged with B. rodhaini on day 28 after the primary infection.Mock mice received B. rodhaini alone. Arrows

indicate the time of challenge infection with B. rodhaini. The results are expressed as mean percent parasitemias the SD for five mice.

Macrophages Mediate Protection against Babesia in Mice

January 2012 Volume 80 Number 1 iai.asm.org 315

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from the immune response to the infection, serum IgG antibody and

cytokine productions were measured at day 8 after challenge in-

fection with B. rodhaini. Notably, there was no significant differ-

ence in the antibody response between clodronate liposome-

treated mice and PBS-liposome-treated mice （Fig. ）, although

the parasitemia profile after B. rodhaini challenge infection was

different. On the other hand, cytokine production, including the

production of IL-12 and IL-2, was significantly higher in clodro-

nate liposome-treated mice than in control mice （Fig. 5B and C）.

In sharp contrast, macrophage/monocyte-depleted mice did not

produce TNF- （below the detection limit）, whereas high levels

were detected in PBS-liposome-treated mice （Fig. 5B）. The pro-

duction of IL-8, IL-10, IFN-, and NO was generally higher in

clodronate liposome-treatedmice than in untreatedmice （Fig. 5B

to D）. These results showed that the absence of macrophages/

monocytes not only resulted in higher parasitemia and mortality

but also caused changes in inflammatory cytokine production.

DISCUSSION

The main goal of immunological research on babesiosis has been

the development of a safe and effective vaccine to minimize the

morbidity andmortality of the infected hosts. Better knowledge of

the host immune response to Babesia infection is undoubtedly

needed to achieve this （11, 27）. Rodent babesiosis has been widely

utilized as an experimental model to investigate the host immune

response to Babesia infection （2, 50）. The causative agents of babe-

siosis in rodent are B. microti and B. rodhaini, which cause differ-

ent diseases in mice. B. microti Munich strain initiates a self-

limiting infection in immunosufficientmice that resolves within 3

weeks, and the mice later become resistant to reinfection even

when high challenge dose is given （28）. In contrast, B. rodhaini is

highly virulent and causes lethal infection with 100% mortality

even when one parasite is injected （18）. Moreover, the suscepti-

bility and the outcomes of the infection may be influenced by the

genetic backgrounds of the mice; for instance, the A/J, C3H, and

BALB/cmouse strains are highly susceptible to infection, whereas

C57BL/6mice are resistant to rodent babesiosis. This difference is

most probably dependent on the innate immunity mechanisms

（1）. The global emergence of human babesiosis has spurred an

interest in developing effective strategies for a bettermanagement

and control of the infection. As a step toward better understand-

ing the immunological aspects required for the control of babesi-

osis, we investigated the possible protection conferred by primary

infection of mice with B. microti against lethal infection with B.

rodhaini and the immune cells involved in the protection mecha-

nism.

BALB/c mice with acute, resolving, and chronic stages of B.

microti infection were compley protected against lethal infec-

tion by B. rodhaini. In contrast,mice simultaneously infected with

the two parasites displayed high parasitemia levels and diedwithin

a week. These findings match those of a previous study in which

monkeys chronically infected with B. microti were protected

against Plasmodium cynomolgi infection （53）. In addition, latent

infection with nonlethal murine malaria parasites suppresses

pathogenesis caused by lethal P. berghei NK65 challenge infection

and prolongs the survival of mice （40）. In sharp contrast to our

observations, simultaneous infections by nonlethal malaria have

shown suppressive effects against lethalmalaria infection, and the

resistance developed in this model is impaired in the absence of

IL-10 （40）. The failure in the protection after the simultaneous

FIG 4 Parasitemia and survival rates of NK cell- and macrophage/monocyte-depleted mice after B. rodhaini challenge infection. （A and B） BALB/c mice with

chronic B.microti infection were treated by i.p. injections with either anti-asialo-GM1 antibody to depleteNK cells or control rabbit antibody. （C andD） BALB/c

mice were treated by i.p injections with either clodronate liposome to deplete macrophages or PBS-liposome as a control. The results are expressed as mean

percent parasitemias the SD for five mice monitored over a period of 20 days after challenge infection with B. rodhaini.

Li et al.

316 iai.asm.org Infection and Immunity

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from coinfection of mice with the rodent Babesia might be due to the

initiation of rapid and progressive parasitemia by B. rodhaini in

the bloodstream before B. microti parasites could initiate appro-

priate immune responses that suppress the pathogenesis of the

lethal infection. Moreover, mice immunized with dead B. microti

were not protected against the lethal infection, although highly

specific antibodywas induced. These observations suggest that the

host antibodies elicited after infection with live organisms are

more functional and are probably directed against critical neutral-

ization epitopes exposed during RBC entry. Generally, the impor-

tance of antibody responses in control and protection has been

documented inmany infectious diseases. In Babesia infection, the

possible function of specific antibodies is to neutralize free para-

sites, preventing their entry into the host erythrocytes and result-

ing in the lysis of the parasites by either complement or phagocy-

tosis.On the other hand, the effects of antibodiesmay be restricted

after the parasites enter the erythrocytes, which are devoid of ma-

jor histocompatibility complex molecules. However, certain

merozoite antigens can be expressed on the surfaces of pRBCs,

making them targets for antibody and complements. Thereafter,

the opsonizing antibodies make the pRBCs recognizable and vul-

nerable to phagocytosis （4, 10, 13, 27, 32）.

Controlmice infected with B. rodhaini developed rapid prolif-

eration of parasites associated with increases in the levels of serum

antibody, cytokines, including IFN-, IL-2, IL-8, IL-10, and IL-12,

and NO; these levels were gradually elevated and peaked when the

mortality started. Conversely, immune mice displayed very low

levels of IFN-, 8-fold less than those of the controls, and the IL-10

levels were nearly not detectable after challenge infection with B.

rodhaini. In a related study, the expression of IL-10, IFN-, and

TNF- was significantly elevated in mice infected with lethal

Babesia strain WA1 but not in nonlethal B. microti-infected mice

（24, 25）. These results support the concept that the severe patho-

genicity of babesiosis depends on the timing and magnitude of

particular cytokines （3, 11, 12）. In general, successful resolution of

rodent Babesia is dependent on the ability of mice to mount an

early proinflammatory cytokine response （IL-12 and IFN-） and

the appropriate maintenance of their kinetics during acute stage of

infection, thereby preventing the parasitemia fromescalating to over-

whelming levels. During the resolution stage, the predominance of

these cytokines shifts to the Th2-based cytokines IL-4 and IL-10 ac-

companied by IgG responses （2, 27）. Therefore, the rapid prolifera-

tion of B. rodhaini in mice might be due to the early elevation of an

anti-inflammatory cytokine （IL-10） that inhibits the activity of Th1

cells,NKcells, andmacrophages, thereby preventing the resolution of

the infection （21）. The lethality of rodent malaria parasite is thought

to be due to the overproduction of IL-10 in an early stage of infection

associated with the impairment of parasitemia clearance （38, 42）.

Further study on the differences in cytokine production in mice in-

fected with lethal and nonlethal rodent Babesia is needed to under-

stand the mechanism of host-parasite interactions, which is an im-

portant prerequisite for vaccine design （11）.

The important roles of IFN- and B and T lymphocytes as the

key inducers of the immune effector mechanisms needed for ini-

tial control of the rodent Babesia infection are supported by the

finding that IFN-/ and SCID mice are unable to resolve the

FIG 5 Kinetics of serum IgGs and cytokines of macrophage/monocyte-depleted and control mice after B. rodhaini challenge infection. （A） Production of IgGs.

（B to D） Production of IL-8, IL-12, TNF-, IL-2, IFN-, and IL-10 （B and C） and NO （D） at day 8 postchallenge infection with B. rodhaini. Asterisks indicate

statistically significant differences （, P0.05; , P0.005; , P0.0001 [compared to the control]）. The results are expressed asmean valuesthe SD of

five mice.

Macrophages Mediate Protection against Babesia in Mice

January 2012 Volume 80 Number 1 iai.asm.org 317

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from primary infection （2, 19, 28, 57）. Here, IFN-/ and SCID mice

were compley protected against the lethality of B. rodhaini in-

fection, resulting in the survival of allmice. Although the primary

infection was not compley resolved, the findings indicated that

the mechanism of resistance to B. rodhaini infection is IFN-,T

cell, and antibody independent. Our findings suggest that both

IFN- and antibody seem to have specific effects on the control

and resolution of B. microti infection. An appropriate production

of IFN- is needed to initiate an effective killing mechanism, as is

the production of specific IgGs that neutralize the parasites and

opsonize the pRBCs; in this way the parasite burden of B. microti

can be reduced （12）. The lack of anymajor role of lymphocytes or

IFN- in controlling the virulentwave of B. rodhaini raised a ques-

tion as to whether the innate immune responses play any role in

limiting the resistance. Our data indicate that host resistance to B.

rodhaini is impaired after macrophage/monocyte depletion in

vivo, as evidenced by the rapid increase in parasitemia and high

mortality rates in macrophage/monocyte-depleted mice. In con-

trast, after depletion of NK cells, the mice displayed a slight in-

crease in parasitemia, with subsequent resolution of the infection

resulting in a survival rate of 80%. These results provide strong

evidence for the critical role of the innate response, particularly by

macrophages and not NK cells in the protective mechanism con-

ferred by B. microti infection against challenge infection with le-

thal B. rodhaini.

Macrophage/monocyte-depleted mice revealed levels of IL-8,

IL-12, and IL-2 cytokines produced after infection with B. rod-

haini comparable to those of control mice. The IL-8 and IL-12

cytokines detected in macrophage/monocyte-depleted mice may

be derived from other type of cells, including epithelial and den-

dritic cells, respectively （52）. The elevation of the IL-2 level in

macrophage/monocyte-depletedmice ismost probably due to the

increase in B. rodhaini burden and the absence of macrophages

that selectively inhibit IL-2 production, as supported by a previous

study on murine malaria （39）. Moreover, the failure to detect

TNF- in clodronate-treatedmicemight be due to the absence of

macrophages/monocytes, which are known to produce this cyto-

kine in response to IFN- activation （17）. TNF- is an inflamma-

tory cytokine that has been implicated in the regulation of Th1

responses, stimulation of NO production, enhancement of the

production of IL-6 cytokine, and inhibition of erythropoiesis （8）.

Furthermore, in murine malaria models, TNF- has shown dual

roles inmediating the protection and in contributing to highmor-

tality and cerebral malaria （17, 31）. On the other hand, our study

found that macrophage/monocyte-depleted mice had levels of

NO comparable to those of controls, although the parasitemia

reached an overwhelming level. We hypothesize that NO may

have been derived from active granulocytes in a TNF--

independent pathway. Indeed, granulocytes such as neutrophils

are considered to be the first line of defense against infections that

rapidly accumulate at the site of infection to ingest and kill the

invaders, including blood parasites, as a response to several cyto-

kines such as IFN- and TNF-, and lymphotoxin （5）. Collec-

tively, our observations suggest that macrophages are critical for

the suppression of B. rodhaini parasitemia and that this suppres-

sion is independent of the action ofNO.On the contrary, Aguilar-

Delfin et al. （2） have documented that resistance to primary infec-

tion of the rodent Babesia WA1 is correlated with an increase in

NO production. Macrophage-mediated control of blood-stage

malaria infection has been well described in rodents and humans

（20, 47, 56）. The mechanisms by which macrophages kill or crip-

ple malaria blood-stage parasites are proposed to be through re-

active oxygen and nitrogen intermediates. These intermediates are

utilized in the body as oxidative and cytotoxic agents that are

produced by phagocytic cells during the oxidative burst induced

by the infection. Their effects on malaria can be both beneficial

and pathological, depending on the amount and place of produc-

tion （15, 16）. Their importance as babesiacidal agents has been

demonstrated in vitro, in which Babesia replication was inhibited,

inducing degeneration of the parasites that display crisis forms

inside the erythrocytes （15, 23, 33）.

Classical cross-protection occurs when effector lymphocytes

respond to the initial infection and secrete IFN-, thereby activat-

ing bystander macrophages and generating a heightened state of

innate immunity to the secondary infection. In this regard, immu-

nization with either bacillus Calmette-Guerin （BCG） or killed

Propionibacterium acnes protects mice against Babesia and Plas-

modium infections （14, 30）. On the other hand, mice that recov-

ered from infection with Corynebacterium parvum are protected

against lethal P. vinckei （22）. Barton et al. （6） reported that her-

pesvirus latent infection conferred symbiotic protection against

Listeria monocytogenes and Yersinia pestis infections. In a related

study,micewith chronic-phase B.microti infectionswere resistant

to challenge infection with the virulent malaria parasite P. vinckei

（22）. The common mechanism of protection reported in these

studies is most likely not antigen-specific immunity but rather

relies on systemic activation of macrophages and chronic secre-

tion of IFN- （6）. Conversely, in heterologous immunity, cross-

reactive antigenic epitopes between the primary and secondary

pathogens result in antigen-specificmemory lymphocyte that can

be activated after secondary infection based on adaptive immunity

（6, 45）. Macrophages produce cytokines, including IL-12 and

TNF-, that are critical for generating and regulating innate and

acquired immune responses against many pathogens. IL-12 acti-

vates NK cells to produce IFN- and contributes to the develop-

ment of acquired immunity by promoting the differentiation of

Th cells to enhance IFN- production by effector CD4 T cells.

The sufficient production of IFN- and TNF- facilitates the

functions of phagocytosis by macrophages and neutrophils （46,

51）. In our data, therefore, the prolonged activation of macro-

phages caused by the primary infection can not only mediate the

removal of pRBCs but also regulate the consequent response of

effector cells to B. rodhaini challenge.

A greater understanding of the immunological mechanisms

evoked or inhibited during infectionmay provide important clues

regarding the type of response that needs to be induced by vacci-

nation. We clearly showed here that innate immunity based on

macrophages, but not adaptive immunity based on antibody and

B and T lymphocytes, contributes to the resistance induced by B.

microti infection to lethal B. rodhaini infection inmice. The estab-

lishment of strategies for activating macrophage-specific re-

sponses to the parasites may be essential for developing effective

vaccines against Babesia infection.

ACKNOWLEDGMENTS

This study was supported by a grant from the Global COE Program and a

Grant-in-Aid for ScientificResearch fromtheMinistry of Education,Culture,

Sports, Science, and Technology of Japan and by a grant for Research on

Regulatory Science of Pharmaceuticals and Medical Devices of Ministry of

Health, Labor, and Welfare of Japan （H23-iyaku-ippan-003）. M.A.T.,

Li et al.

318 iai.asm.org Infection and Immunity

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from G.O.A., and Y.-K.G. were supported by a research grant fellowship from the

Japanese Society for the Promotion of Science for young scientists.

REFERENCES

1. Aguilar-Delfin I, Homer MJ, Wettstein PJ, Persing DH. 2001. Innate

resistance to Babesia infection is influenced by genetic background and

gender. Infect. Immun. 69:7955–7958.

2. Aguilar-Delfin I, Wettstein PJ, Persing DH. 2003. Resistance to acute

babesiosis is associated with interleukin-12- and gamma interferon-

mediated responses and requires macrophages and natural killer cells.

Infect. Immun. 71:2002–2008.

3. Ahmed JS. 2002. The role of cytokines in immunity and immunopatho-

genesis of pirolasmoses. Parasitol. Res. 88（Suppl）:S48–S50.

4. Allred DR, Cinque RM, Lane TJ, Ahrens KP. 1994. Antigenic variation

of parasite-derived antigens on the surface of Babesia bovis-infected eryth-

rocytes. Infect. Immun. 62:91–98.

5. Appelberg R. 2007. Neutrophils and intracellular pathogens: beyond

phagocytosis and killing. Trends Microbiol. 15:87–92.

6. Barton ES, et al. 2007. Herpesvirus latency confers symbiotic protection

from bacterial infection. Nature 447:326–330.

7. Benach JL, Habicht GS, Holbrook TW, Cook JA. 1982. Glucan as an

adjuvant for amurine Babesiamicroti immunization trial. Infect. Immun.

35:947–951.

8. Beutler B, Grau GE. 1993. Tumor necrosis factor in the pathogenesis of

infectious diseases. Crit. Care Med. 21:S423–435.

9. Brown WC, Palmer GH. 1999. Designing blood-stage vaccines against

Babesia bovis and B. bigemina. Parasitol. Today 15:275–281.

10. Brown WC. 2001. Molecular approaches to elucidating innate and ac-

quired immune responses to Babesia bovis, a protozoan parasite that

causes persistent infection. Vet. Parasitol. 101:233–248.

11. Brown WC, Norimine J, Knowles DP, Goff WL. 2006. Immune control

of Babesia bovis infection. Vet. Parasitol. 138:75–87.

12. Chen D, Copeman DB, Burnell J, Hutchinson GW. 2000. Helper T cell

and antibody responses to infection of CBA mice with Babesia microti.

Parasite Immunol. 22:81–88.

13. Clark IA, Richmond JE, Wills EJ, Allison AC. 1975. Immunity to intra-

erythrocytic protozoa. Lancet ii:1128–1129.

14. Clark IA, Alloson AC, Cox FE. 1976. Protection of mice against Babesia

and Plasmodium with BCG. Nature 259:309–311.

15. Clark IA, Hunt NH. 1983. Evidence for reactive oxygen intermediates

causing hemolysis and parasite death in malaria. Infect. Immun. 39:1–6.

16. Clark IA, Hunt NH, Cowden WB. 1986. Oxygen-derived free radicals in

the pathogenesis of parasitic disease. Adv. Parasitol. 25:1–44.

17. Clark IA, Rockett KA, Cowden WB. 1992. TNF in malaria, p 303–328.

InB. Beutler （ed.）, Tumor necrosis factors: themolecules and their emerg-

ing role in medicine. Raven Press, Ltd, New York, NY.

18. Clark IA. 2001. Heterologous immunity revisited. Parasitology

122（Suppl）:S51–S59.

19. Clawson ML, et al. 2002. Cellular immunity, but not gamma interferon,

is essential for resolution of Babesia microti infection in BALB/c mice.

Infect. Immun. 70:5304–5306.

20. Couper KN, et al. 2007. Macrophage-mediated but gamma interferon-

independent innate immune responses control the primary wave of Plas-

modium yoelii parasitemia. Infect. Immun. 75:5806–5818.

21. Couper KN, Blount DG, Riley EM. 2008. IL-10: the master regulator of

immunity to infection. J. Immunol. 180:5771–5777.

22. Cox FE. 1978. Heterologous immunity between piroplasms and malaria

parasites: the simultaneous elimination of Plasmodium vinckei and Babe-

siamicroti fromthe blood of doubly infectedmice. Parasitology 76:55–60.

23. Gubernot DM, et al. 2009. Transfusion-transmitted babesiosis in the

United States: summary of a workshop. Transfusion 49:2759–2771.

24. Hemmer RM, Ferrick DA, Conrad PA. 2000. Upregulation of tumor

necrosis factor-alpha and interferon-gamma expression in the spleen and

lungs ofmice infectedwith the human Babesia isolateWA1. Parasitol. Res.

86:121–128.

25. Hemmer RM, Ferrick DA, Conrad PA. 2000. Role of T cells and cyto-

kines in fatal and resolving experimental babesiosis: protection in

TNFRp55/ mice infected with the human Babesia WA1 parasite. J.

Parasitol. 86:736–742.

26. Hildebrandt A, et al. 2007. First confirmed autochthonous case of human

Babesia microti infection in Europe. Eur. J. Clin. Microbiol. Infect. Dis.

26:595–601.

27. Homer MJ, Aguilar-Delfin I, ford SR, III, Krause PJ, Persing DH.

2000. Babesiosis. Clin. Microbiol. Rev. 13:451–469.

28. Igarashi I, et al. 1999. Roles of CD4 T cells and gamma interferon in

protective immunity against Babesia microti infection inmice. Infect. Im-

mun. 67:4143–4148.

29. Igarashi I, et al. 2000. Immunization with recombinant surface antigens

p26 with Freund adjuvants against Babesia rodhaini infection. J. Vet.Med.

Sci. 62:717–723.

30. Iseki H, et al. 2008. Babesia: the protective effects of killed Propionibac-

teriumacnes on the infections of two rodent Babesia parasites inmice. Exp.

Parasitol. 118:543–548.

31. Jacobs P, Radzioch D, Stevenson MM. 1996. A Th1-associated increase

in tumor necrosis factor alpha expression in the spleen correlates with

resistance to blood-stage malaria in mice. Infect. Immun. 64:535–541.

32. Jacobson RH, Parrodi F, Wright IG, Fitzgerald CJ, Dobson C. 1993.

Babesia bovis: in vitro phagocytosis promoted by immune serum and by

antibodies produced against protective antigens. Parasitol. Res. 79:

221–226.

33. JohnsonWC, Cluff CW, GoffWL,Wyatt CR. 1996. Reactive oxygen and

nitrogen intermediates and products from polyamine degradation are

babesiacidal in vitro. Ann. N. Y. Acad. Sci. 791:136–147.

34. Kawabuchi T, et al. 2005. Isolation of a human erythrocyte-adapted

substrain of Babesia rodhaini and analysis of themerozoite surface protein

gene sequences. J. Vet. Med. Sci. 67:901–907.

35. Krause PJ, et al. 2003. Increasing health burden of human babesiosis in

endemic sites. Am. J. Trop. Med. Hyg. 68:431–436.

36. Krause PJ, et al. 2008. Persistent and relapsing babesiosis in immuno-

compromised patients. Clin. Infect. Dis. 46:370–376.

37. Leiby DA. 2011. Transfusion-transmitted Babesia spp.: bull’s-eye on

Babesia microti. Clin. Microbiol. Rev. 24:14–28.

38. Li C, Sanni LA, Omer F, Riley E, Langhorne J. 2003. Pathology of

Plasmodiumchabaudi chabaudi infection andmortality in interleukin-10-

deficient mice are ameliorated by anti-tumor necrosis factor alpha and

exacerbated by anti-transforming growth factor beta antibodies. Infect.

Immun. 71:4850–4856.

39. Luyendyk J, Olivas OR, Ginger LA, Avery AC. 2002. Antigen-presenting

cell function during Plasmodium yoelii infection. Infect. Immun. 70:

2941–2949.

40. Niikura M, Kamiya S, Kita K, Kobayashi F. 2008. Coinfection with

nonlethal murine malaria parasites suppresses pathogenesis caused by

Plasmodium berghei NK65. J. Immunol. 180:6877–6884.

41. Nishikawa Y, et al. 2010. Roles of CD122 cells in resistance against

Neospora caninum infection in a murine model. J. Vet. Med. Sci. 72:

1275–1282.

42. Omer FM, de Souza JB, Riley EM. 2003. Differential induction of TGF-

beta regulates proinflammatory cytokine production and determines the

outcome of lethal and nonlethal Plasmodiumyoelii infections. J. Immunol.

171:5430–5436.

43. Ooka H, et al. 2011. Babesia microti: molecular and antigenic character-

izations of a novel 94-kDa protein （BmP94）. Exp. Parasitol. 127:287–293.

44. Ruebush MJ, Hanson WL. 1979. Susceptibility of five strains of mice to

Babesia microti of human origin. J. Parasitol. 65:430–433.

45. Selin LK, et al. 2006. Memory of mice and men: CD8 T-cell cross-

reactivity and heterologous immunity. Immunol. Rev. 211:164–181.

46. Shoda LK, et al. 2000. Babesia bovis-stimulated macrophages express

interleukin-1, interleukin-12, tumor necrosis factor alpha, and nitric

oxide and inhibit parasite replication in vitro. Infect. Immun. 68:

5139–5145.

47. Stevenson MM, Ghadirian E, Phillips NC, Rae D, Podoba JE. 1989. Role

of mononuclear phagocytes in elimination of Plasmodium chabaudi AS

infection. Parasite Immunol. 11:529–544.

48. Stich RW, et al. 1998. Stimulation of nitric oxide production in macro-

phages by Babesia bovis. Infect. Immun. 66:4130–4136.

49. Tagawa Y, Sekikawa K, Iwakura Y. 1997. Suppression of concanavalin

A-induced hepatitis in IFN-/ mice, but not in TNF-/ mice: role

for IFN- in activating apoptosis of hepatocytes. J. Immunol. 59:

1418–1428.

50. TerkawiMA, et al. 2008. C3 contributes to the cross-protective immunity

induced by Babesia gibsoni phosphoriboprotein P0 against a lethal B. rod-

haini infection. Parasite Immunol. 30:365–370.

51. Trinchieri G. 1995. Interleukin 12: a proinflammatory cytokine with im-

munoregulatory functions that bridge innate resistance and antigen-

specific adaptive immunity. Annu. Rev. Immunol. 13:251–276.

Macrophages Mediate Protection against Babesia in Mice

January 2012 Volume 80 Number 1 iai.asm.org 319

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from 52. Trinchieri G. 2003. Interleukin-12 and the regulation of innate resistance

and adaptive immunity. Nat. Rev. Immunol. 3:133–146.

53. Van Duivenvoorde LM, et al. 2010. Suppression of Plasmodium cyno-

molgi in rhesus macaques by coinfection with Babesia microti. Infect. Im-

mun. 78:1032–1039.

54. Van Rooijen N, Sanders A. 1994. Liposome mediated depletion of

macrophages: mechanism of action, preparation of liposomes and appli-

cations. J. Immunol. Methods 174:83–93.

55. Wei Q, et al. 2001. Human babesiosis in Japan: isolation of Babesia microti-like

parasites from an asymptomatic transfusion donor and from a rodent from an

area where babesiosis is endemic. J. Clin.Microbiol. 39:2178–2183.

56. Wood PR, Clark IA. 1984. Macrophages from Babesia and malaria in-

fected mice are primed for monokine release. Parasite Immunol.

6:309–317.

57. Yoneto T, et al. 1999. Gamma interferon production is critical for pro-

tective immunity to infection with blood-stage Plasmodium berghei XAT

but neither NO production nor NK cell activation is critical. Infect. Im-

mun. 67:2349–2356.

58. Zivkovic D, Speksnijder JE, Kuil H, Seinen W. 1984. Immunity to

Babesia in mice. II. Cross-protection between various Babesia and Plas-

modium species and its relevance to the nature of Babesia immunity. Vet.

Immunol. Immunopathol. 5:359–368.

Li et al.

320 iai.asm.org Infection and Immunity

 on April 27, 2012 by SHANGHAI JIAOTONG UNIVERSITY http://iai.asm.org/ Downloaded from