Published Ahead of Print 7 November 2011.
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
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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-
?uence 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 signi?cantly 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--de?cientmice with chronic B. microti infections demonstrated protective responses comparable to those
of immunocompetentmice. Likewise, in vivo NK cell depletion did not signi?cantly impair the protective responses. Conversely,
macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and
cytokines pro?les, 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 ef?cacious
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-af?nity
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 in?amma-
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-speci?c 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 modi?cation 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--de?cient （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 ?xed with glutaraldehyde, were
prepared as previously described （7）. Brie?y, 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 ?xedwith 0.25%glutaraldehyde for 15min at roomtemperature and
then washed three times with sterile PBS. The ?xed 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-?xed B.
microti pRBCs in 0.5 ml of PBS （the test group） or an equivalent amount
of glutaraldehyde-?xed 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 speci?c antibody response to B. microti antigens was determined by
indirect immuno?uorescence 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 ?xed 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 speci?c 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 puri?ed by glutathione-Sepharose 4B columns （Amersham Biosci-
ences）, and the resulting antigen was used in an enzyme-linked immu-
nosorbent assay （ELISA） to detect speci?c antibodies （50）. Brie?y, 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 sacri?ced at days 0, 2, 4, or 6 postchallenge infection with B. rodhiani
（?ve 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
?uorescein 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 ?ow cytometer （Beckman
Coulter）.
IFAT. To differentiate the two parasitic infections, standard IFAT was
performed with speci?c antibodies. Brie?y, IFAT slides were coated with
pRBCs collected from mice at day 6 postchallenge infection with B. rod-
haini （50）. The slides were dried and ?xed in absolute acetone for 10min.
The ?xed 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 ?uorescence 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 ?ow 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 ?ow cytometry by staining
cells derived fromperitoneal ?uid and splenocytes with CD11b and F4/80
MAbs, respectively.
Statistical analysis. Statistical analysis of any signi?cant 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
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ing any statistically signi?cant differences. All results were considered sta-
tistically signi?cant 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 ?rst 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 ?rst 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 signi?cantly 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 signi?cant 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 signi?cant difference com-
pared to those detected before infection with B. microti （data not
shown）. Immuno?uorescence assays for pRBCs collected from
mice 6 days after the challenge infection were performed using
speci?c 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 speci?c 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 ?ve 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. Speci?c antibody to B. rodhaini （rBrP26） was detected at
day 6, whereas the IgG1 and IgG2 levels were signi?cantly 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 signi?cantly 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 signi?cant 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 ?ve 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 signi?cantly different between test
and control mice （Fig. 2I）. In addition, the detectable nitric oxide
（NO） was signi?cantly 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-
ni?cantly different fromthose of immunosuf?cient 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 ef?-
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 signi?cant difference
compared tomice that received control antibody （P0.05）.Con-
sistently, there was no signi?cant difference in mortalities and
survivals between intact and NK cell-depleted mice （Fig. 4A and
B）. In sharp contrast, clodronate liposome-treatedmice hadmore
signi?cant 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 pro?les 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 ?ve 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 signi?cant differ-
ence in the antibody response between clodronate liposome-
treated mice and PBS-liposome-treated mice （Fig. ）, although
the parasitemia pro?le after B. rodhaini challenge infection was
different. On the other hand, cytokine production, including the
production of IL-12 and IL-2, was signi?cantly 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 in?ammatory 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 immunosuf?cientmice 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 in?uenced 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 ?ndings 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 ?ve 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
speci?c 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 speci?c 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 signi?cantly 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 proin?ammatory 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-in?ammatory 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
?nding 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 signi?cant differences （, P0.05; , P0.005; , P0.0001 [compared to the control]）. The results are expressed asmean valuesthe SD of
?ve 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 ?ndings indicated that
the mechanism of resistance to B. rodhaini infection is IFN-,T
cell, and antibody independent. Our ?ndings suggest that both
IFN- and antibody seem to have speci?c 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 speci?c 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 in?amma-
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 ?rst 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-
Del?n 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 bene?cial
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-speci?c 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-speci?cmemory 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 suf?cient 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-speci?c 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 Scienti?cResearch 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-Del?n I, Homer MJ, Wettstein PJ, Persing DH. 2001. Innate
resistance to Babesia infection is in?uenced by genetic background and
gender. Infect. Immun. 69:7955–7958.
2. Aguilar-Del?n 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 con?rmed autochthonous case of human
Babesia microti infection in Europe. Eur. J. Clin. Microbiol. Infect. Dis.
26:595–601.
27. Homer MJ, Aguilar-Del?n 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-
de?cient 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 proin?ammatory 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 ?ve 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 proin?ammatory cytokine with im-
munoregulatory functions that bridge innate resistance and antigen-
speci?c 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
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