Natural killer cells act as early responders in an experimental infection with Neospora caninum in...

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International Journal for Parasitology 37 (2007) 329–339

Natural killer cells act as early responders in an experimentalinfection with Neospora caninum in calves

Siv Klevar a, Siri Kulberg a, Preben Boysen b, Anne K. Storset b, Torfinn Moldal a,Camilla Bjorkman c, Ingrid Olsen a,*

a Department of Animal Health, National Veterinary Institute, P.O. Box, 8156 Dep., N-0033 Oslo, Norwayb Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box, 8146 Dep., N-0033 Oslo, Norway

c Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-750 07 Uppsala, Sweden

Received 25 August 2006; received in revised form 1 November 2006; accepted 6 November 2006

Abstract

The intracellular protozoan parasite Neospora caninum is a cause of abortion and congenital disease in cattle worldwide. We havepreviously shown that natural killer (NK) cells produce IFN-c in response to N. caninum tachyzoites in vitro. This study aimed to inves-tigate the role of NK cells and other cellular immune responses in an experimental N. caninum infection model in calves. Phenoyping ofperipheral blood lymphocytes showed a drop in the percentage of NK cells at days 4–6 after i.v. inoculation, followed by an increase inthe percentage of both NK cells and CD8+ T cells which peaked at days 11–15. A whole blood flow cytometric assay showed that CD4+T cells were the major IFN-c producing cells, but in the early stages of the infection both NK cells and CD8+ T cells contributed to IFN-c production. We also compared the ability of two different N. caninum antigen preparations – sonicated soluble antigens and intact heat-inactivated parasites – to induce proliferation and IFN-c production in various cell types. Heat-inactivated tachyzoites induced a 3.7times greater increase in the number of IFN-c producing NK cells compared with sonicated soluble antigens. This indicated the presenceof some NK cell-stimulating antigens in the intact tachyzoite that were absent from the sonicated soluble antigens. The heat-inactivatedwhole tachyzoites also inhibited cd T cell proliferation while the soluble antigens from N. caninum did not. We believe this is the first timeNK cells have been demonstrated to be early responders in N. caninum infection in calves.� 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Neospora caninum; Natural killer cells; Immune response; Bovine

1. Introduction

In 1984 Bjerkas et al. (Bjerkas et al., 1984) described anintracellular apicomplexan protozoan parasite closelyrelated to Toxoplasma gondii in a dog brain, and the para-site was classified as a species, Neospora caninum, in 1988(Dubey et al., 1988). Since then, neosporosis has emergedas a serious disease leading to abortion and congenitalinfection in cattle and neuromuscular disorders in dogsworldwide. However, infections with this parasite arenormally benign in healthy non-pregnant cattle. Parasite

0020-7519/$30.00 � 2006 Australian Society for Parasitology Inc. Published b

doi:10.1016/j.ijpara.2006.11.002

* Corresponding author. Tel.: +47 23 21 60 00; fax: +47 23 21 60 01.E-mail address: [email protected] (I. Olsen).

identification and antibody response have been describedin a range of species, often without disease (Dubey, 2003).

Cattle can acquire N. caninum infection either by inges-tion of oocysts that are shed in the faeces of acutely infect-ed dogs or through vertical transmission (McAllister et al.,1998; De Marez et al., 1999). The ingested oocysts developinto rapidly multiplying tachyzoites which enter the bloodwhere they preferentially inhabit cells of the mononuclearphagocytic system and eventually infect different tissues.It is assumed that the host immune response triggers thetachyzoite stage of the parasite to differentiate into brad-yzoites and a persistent tissue cyst infection is established(Buxton et al., 2002; Williams and Trees, 2006). Unlikeovine toxoplasmosis where abortions mainly occur afterprimary infections, these cysts may be reactivated during

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330 S. Klevar et al. / International Journal for Parasitology 37 (2007) 329–339

pregnancies and infect the foetus. Transplacental parasitetransmission is very efficient in infected cattle and willoccur during consecutive pregnancies (Davison et al.,1999; Wouda et al., 1998). However, some degree of pro-tective immunity to transplacental transmission of N. cani-

num develops in both experimentally and naturally infectedcattle (Innes et al., 2002).

As an intracellular parasite, N. caninum incites both aspecific antibody response and a typical cell-mediatedimmunity, inducing production of cytokines such as IFN-c (Lunden et al., 1998; Andrianarivo et al., 2001). This cel-lular immune response can be measured in vitro, usingN. caninum antigens that can induce lymphocyte proliferationand IFN-c production (Innes et al., 2002). IFN-c is impor-tant for host defence and is known to limit N. caninum

multiplication (Innes et al., 1995; Baszler et al., 1999). Thispro-inflammatory cytokine is produced by CD4+ T cellsduring bovine neosporosis (Marks et al., 1998; Tuo et al.,2005). However, infections with other protozoan agentsin cattle have shown that CD8+ T cells also contributeto IFN-c production (Glass, 2001; Voyich et al., 2001).Furthermore, studies from in vivo protozoal infections inhumans (Korbel et al., 2004) demonstrated that naturalkiller (NK) cells are among the first cells to produceIFN-c. Even though knowledge of acquired immunityagainst N. caninum has been obtained from several studies,little information about the innate immune mechanisms inthe early phases of neosporosis has been provided.

Generally, NK cells are important in early innateimmune responses (Carayannopoulos and Yokoyama,2004). They have two major effector functions: the produc-tion of cytokines, and lysis of target cells (Korbel et al.,2004). Several studies have shown that NK cells play akey role as cytokine producers in host resistance againstprotozoan infections, particularly before the onset of Tand B cell-mediated immunity (Scharton-Kersten and Sher,1997). Early in the course of an infection, invading patho-gens are captured by professional antigen-presenting cellswhich release NK cell-activating cytokines, such as IL-12.IL-12 is one of the most potent inducers of NK cell cyto-toxicity, and has been regarded as necessary for IFN-c pro-duction by NK cells during infection with intracellularpathogens (Boehm et al., 1997; Sher et al., 2003; Korbelet al., 2004).

Recently, NK cells in cattle have been characterised(Storset et al., 2004). We have previously shown thatN. caninum triggers bovine NK cells to produce IFN-cin vitro, even in the absence of IL-12, and that IFN-c pro-duction was dependent on an intact parasite (Boysen et al.,2006). However, the role of NK cells and the effect ofdifferent N. caninum antigen preparations on the IFN-cproduction in N. caninum infection in vivo have not beeninvestigated. The purpose of the present study was to inves-tigate cellular immune responses in N. caninum-infectedcalves, in particular the role of bovine NK cells as an earlyresponder in the course of the infection. Furthermore,experiments were performed to examine if different subsets

of lymhocytes from N. caninum-infected calves varied inIFN-c response and proliferation to different preparationsof antigens from N. caninum.

2. Materials and methods

2.1. Animals and study design

Nine clinically healthy calves of the Norwegian Redcattle breed, 3 months of age, were selected for the exper-iment. All animals came from a herd certified free ofbovine virus diarrhoea virus and bovine leukaemia virusand were tested seronegative for antibodies to N. caninum

by iscom ELISA (Bjorkman et al., 1997). They were keptin vermin- and bird-proof pens with deep litter beddingand fed standard hay and commercial cattle concentrate.Calves inoculated with N. caninum and uninfected con-trols were kept at different time-points and in separatepens isolated from other livestock. The calves wereobserved clinically and rectal temperatures were recordeddaily from the day before inoculation and until 10 daysafter inoculation.

Five of the calves were inoculated i.v. with 107 N. cani-

num tachyzoites in 5 ml of 0.9% NaCl. One of the infectedcalves was euthanised 11 days p.i. for the purpose of fur-ther research. Four control calves were inoculated withuninfected Vero cells purified likewise and similarly admin-istrated. Blood was sampled from all calves 1–3 days beforeinoculation and then three times per week for 2 weeks,thereafter twice a week for the rest of the experiment.The project was approved by the Norwegian AnimalResearch Authority.

2.2. Serology

Antibodies to N. caninum were analysed by iscomELISA as previously described (Bjorkman et al., 1997) pri-or to challenge and weekly thereafter for the duration ofthe experiment. An O.D. value of P0.2 was consideredpositive.

2.3. Culture of N. caninum and preparation of tachyzoites for

inoculation

Neospora caninum tachyzoites of the Nc-Liverpool iso-late were propagated in Vero cell monolayers, in RPMI1640 medium supplemented with 10% FCS, 2 mM L-gluta-mine, 100 U penicillin, 100 lg of streptomycin and 0.25 lgof fungizone/ml (all Invitrogen, Carlsbad, California).Tachyzoite-infected cells were harvested after 3 or 4 daysin culture using a rubber policeman and counted in a haem-ocytometer using trypan blue exclusion; passage numberused in this experiment was 9.

Purification of tachyzoites was performed as previouslydescribed (Bjorkman et al., 1994). The purified tachyzoiteswere washed twice and resuspended in PBS at 107 tachyzo-ites/ml and used immediately.

S. Klevar et al. / International Journal for Parasitology 37 (2007) 329–339 331

2.4. Antigen preparations

Purified tachyzoites were heat-inactivated by incubationat 56 �C for 50 min and the efficiency of inactivation wasverified by the inability to re-infect Vero cell cultures. Sim-ilar amounts of cultured Vero cells were purified and heat-inactivated as described for tachyzoites and used as con-trols. Soluble antigens were obtained by sonicating purifiedtachyzoites (approximately 108) in 2.5 ml PBS on ice atmaximum amplitude for 10 · 10 s using a Vibra-cellVCX400 with a tapered microtip (Sonics and materials,Newton, CT, USA). Debris was spun down at 13,000g

for 15 min, and the supernatant was filtered through a0.22 lm filter to remove residual particulate material. Theprotein concentration was determined by measuring theOD at 280 nm. A sonicated lysate of uninfected Vero cellswas prepared in a similar manner. Both N. caninum soni-cated antigens and heat-inactivated tachyzoites were pre-pared from the same batch. All antigen preparations werestored at 4 �C.

2.5. Phenotyping of lymphocyte subpopulations

Phenotyping of lymphocytes was done as previouslydescribed with minor modifications (Kampen et al.,2006). mAbs against various bovine leukocyte surface mol-ecules and the concentration used are shown in Table 1.Secondary anti-mouse isotype-specific antibodies labelledwith phycoerythrin (PE), fluorescein isothiocyanate (FITC)(both from SouthernBiotech, Birmingham, AL), or allo-phycocyanin (APC, Pharmingen, San Diego, California)were used at a final concentrations of 5 lg/ml or 2.5 lg/ml and 1.25 lg/ml, respectively. The samples were analysedusing a FACSCalibur flow cytometer (Becton DickinsonBiosciences). Data were collected from 10,000 cells persample in the gate set for lymphocytes and the analyseswere done using CellQuest Pro� software (Becton Dickin-son Biosciences). The results are reported as the percentageof gated cells positive for each cell surface marker. To esti-mate the total number of the various cell types, total whitecell counts were measured using a Z1 Coulter ParticleCounter (Beckman Coulter, Fullerton, California), fol-lowed by estimation of total numbers of mononuclear cellsusing flow cytometry and gating according to size andgranularity in a forward and side scatter plot.

Table 1The specificity and isotype of the primary antibodies used forflow-cytometric assays

Specificity Clone Isotype Concentration (lg/ml)

CD4 IL-A11 IgG2a 8CD8 7C2B IgG2a 5cd TcR GB21A IgG2b 5NKp46 AKS-1a IgG1 1

All antibodies except AKS-1 were purchased from VMRD, Pullman, WA.a AKS-1 is now available from Serotex, Oxford, UK.

2.6. NK cell cytotoxicity assay

The cytotoxic capacity of bovine NK cells was tested ina 4 h 51Cr redirected lysis cytotoxicity assay as previouslydescribed (Storset et al., 2004) with the following modifica-tions: peripheral blood mononuclear cells (PBMCs) wereseparated by density gradient centrifugation (1150g,20 min) on Lymphoprep (Nycomed Pharma), and the cellswere washed twice in HBSS (GibcoBRL, Paisley, UK).Then PBMCs were cultured with the murine FccR+ targetline P815 in the presence of 1 lg/ml anti-NKp46 mAb(AKS-1), without any addition of cytokines. Specific 51Crrelease was calculated on the basis of the ratio [(samplerelease � spontaneous release)/(total release � spontane-ous release)]. Lytic units (LU), defined as the capacity tokill 20% of targets contained within 107 effectors, was cal-culated as described by Bryant and colleagues (Bryantet al., 1992), using values from the sloping part of the cyto-toxicity curve. Where applicable, NK cells-per-target val-ues were calculated using data from phenotypic assays onthe day of sampling.

2.7. IFN-c production in whole blood

Heparinised whole blood was dispensed into 24-well tis-sue culture trays (1 ml/well) and stimulated with sonicatedN. caninum (5 lg/ml) and sonicated lysate of Vero cells(0.7 lg/ml) prepared as described above, and incubatedat 37 �C in 5% CO2 in air for 24 h. One well served as anunstimulated control. The plasma was removed andassayed for IFN-c using a bovine IFN-c ELISA-kit(Bovine IFN-c EASIA, Biosource, Nivelles, Belgium)according to the manufacturer’s instructions. Results werecalculated as corrected OD: [(mean antigen stimulatedwells � mean control wells)/(test positive control � testnegative control)]. A regression line was calculated andthe quantity of IFN-c present in each of the test sampleswas determined from the standard curve. The assay wasperformed before challenge, then 4 days after inoculation,and thereafter every week throughout the experiment. Toexclude a possible response to Vero cells in the infectedand control groups, IFN-c production were measured afterstimulation with sonicated Vero cells at the end of thestudy.

2.8. Intracellular staining for IFN-c

Staining for intracellular IFN-c was performed as previ-ously described (Olsen and Storset, 2001) with minor mod-ifications. Briefly, 3 ml of heparinised whole blood wasincubated with soluble N. caninum antigens (5 lg/ml) atdays 11, 18 and 25 p.i. and with heat-inactivated N. cani-

num (10,000 tachyzoites/ml) at days 18 and 25 p.i. in 6-welltissue culture trays at 37 �C in 5% CO2. Positive controlcultures stimulated with 50 lg/ml phorbol 12-myristate13-acetate (PMA, Sigma Chemical Co., St. Louis, MO)and 1 lg/ml Calcium Ionophore (Sigma) were included.


After 9 h incubation, 10 lg/ml Brefeldin A (Sigma) wasadded and samples were incubated for another 12 h.PBMCs were separated as described above. Cells werewashed twice and labelled with mAbs directed againstNKp46, CD4, CD8 and cd T cell receptor (Table 1) fol-lowed by incubation with subtype-specific secondary anti-bodies as described for phenotyping of lymphocytes. Thecells were then fixed and permeabilised and incubated witha mAb (clone 6.19, subclass IgG2a or clone 6.6, subclassIgG2b) raised against affinity purified bovine IFN-c (kind-ly provided by Dr. Gregers Jungersen, Danish Institute forFood and Veterinary Research, Copenhagen, Denmark).After incubation with fluorescence-labelled secondary anti-bodies, the samples were analysed by flow cytometry asdescribed above. One hundred thousand lymphocytes wereread using a forward and side scatter gating. The results aregiven as means ± SEM.

2.9. Lymphocyte proliferation assay

The carboxyfluorescein diacetate succinimidyl ester(CFSE) proliferation assay was performed before theexperimental infection and at 1, 2, 4 and 5 weeks p.i.CFSE-labelled cells give fluorescence in the FL1 channeland the proliferation of cells leads to a twofold reductionin intensity for each cell division. For cell proliferationstudies, PBMC were incubated with 5 lM CFSE in PBSwith 0.2% BSA for 10 min at 37 �C, followed by additionof 5 ml cold RPMI and incubation on ice for 5 min. Thecells were washed three times in RPMI and dissolved inRPMI with 10% FCS (106 cells/ml). The cells were stimu-lated in triplicate with (i) sonicated N. caninum (5 lg/ml),(ii) heat-inactivated N. caninum (10,000 tachyzoites/ml) or(iii) non-stimulated control, and incubated at 37 �C in 5%CO2 in air for 5 or 6 days. In addition Vero cell controls,prepared as described above, were included at one time-point. Following incubation, the cells were stained withmAbs against surface markers (Table 1) and PE or APCconjugated secondary antibodies were added as describedfor phenotyping of lymphocytes, and analysed by flowcytometry. The results are given as means ± SEM.

2.10. Detection of N. caninum DNA in tissues and

histopathology

Tissue samples were collected aseptically for detection ofN. caninum DNA by PCR. From one calf euthanised 11days p.i., samples included the forebrain, midbrain, hindbrain, spleen and lung tissue. From the four infected calveseuthanised on day 32 p.i., samples included the forebrain,midbrain and hind brain. To optimise parasite DNA detec-tion from the brain, samples of 1 cm3 from each part of thebrain were homogenised and DNA was isolated from 50 to100 ll homogenate using the DNeasy� kit (Qiagen, Basel,Switzerland) according to the manufacturer’s recommen-dations or by a DNA extraction machine (Easymag, Bio-meriux). In addition, eight samples of 25 mg were

collected from the spleen and the lungs from the calfeuthanised on day 11 after inoculation and treated asdescribed above. A N. caninum-specific PCR was per-formed as described by Liddell et al. (1999). DNA extract-ed from purified N. caninum tachyzoites from infected Verocells was used as a positive control.

Tissue from the heart, lung, liver, kidney, spleen, jejunaland ileal Peyer’s patches, jejunal lymph node, musculusmasseter, diaphragm, musculus psoas, musculus quadri-ceps femoris and half the brain were sampled for histolog-ical examination, from all the inoculated calves. Inaddition, samples from the bronchial, mediastinal, hepaticand subiliac lymph nodes were taken from the calf that waseuthanaised on day 11 after inoculation. The samples werefixed in 10% neutral buffered formalin, routinely processedand embedded in paraffin wax. Slides were stained withH&E.

2.11. Statistical analysis

Statistical analysis of differences in percentages of lym-phocytes in blood, and of differential LU values from cyto-toxicity assays, were carried out with Wilcoxon matchedpairs signed rank sum test using JMP 5.1 statisticalsoftware (SAS Institute Inc., Cary, NC). P < 0.05 wasconsidered statistically significant.

3. Results

3.1. Clinical symptoms and seroconversion

The inoculated calves showed virtually no clinicalsymptoms, only a slight decrease in food uptake on days6–8 post inoculation. Before inoculation, all calves wereseronegative to N. caninum, (OD < 0.2). Three of theinoculated calves had seroconverted 11 days post inocula-tion, and by day 18 p.i. all of the infected calves had sero-converted. There was no fluctuation in antibody levelswithin the infected group (Fig. 1). The OD values inserum from uninfected controls remained below 0.2(range from 0.05 to 0.06) throughout the study (datanot shown).

3.2. Changes in lymphocyte subpopulations in peripheral

blood after infection

The proportions of the lymphocyte sub-populations inperipheral blood were measured using mAbs against sur-face markers and reported as the percentage of gated cellspositive for each cell surface marker in a flow-cytometricassay.

The major changes were seen in the NK cells (defined bythe surface expression of NKp46) and CD8+ T cells(defined as CD8+ NKp46- cells). The NK cells showed amarked decrease 4–6 days post inoculation and thereafterreached a peak on days 11–15 (Fig. 2A). This was seen inall the infected calves while the control group remained

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Fig. 1. Antibody responses to Neospora caninum in infected calves. The figure shows the variation in OD in serum from calves inoculated with 107

tachyzoites. The Y-axis indicates the OD values measured by N. caninum iscom ELISA, the X-axis shows the number of days after inoculation with N.

caninum tachyzoites (infected calves). Day-2 indicates values obtained before inoculation. The calves were inoculated at day 0. OD P 0.20 (dotted line)was considered a positive result.

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Fig. 2. Changes in lymphocyte subpopulations in peripheral blood of Neospora caninum-infected calves. The figure shows the variation of (A) NKp46+cells, (B) CD8+ T cells and (C) CD4+ T cells in infected calves (left) and controls (right). The Y-axis indicates the percentage of gated cells positive foreach cell surface marker, the X-axis shows the number of days after inoculation with N. caninum tachyzoites (infected calves) or control Vero cells (controlcalves). Day 0 (arrow) is the day of inoculation.


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Fig. 3. Natural killer cell-specific cytotoxicity detected within freshlydrawn peripheral blood mononuclear cells (PBMCs) from infectedanimals. Target cells were murine Fc-receptor+ P815 cells, and anti-NKp46 mAb (AKS1) was added to obtain redirected lysis. No cytokineswere added. (A) Pre-infection; (B) 11 days p.i. Results show means oftriplicate samples of all five animals within the infected group.


at a relatively stable level. Both the decrease from day 0 today 6 and the increase from day 6 to day 15 of NK cells inthe infected group were significant (P < 0.031). A relativelystable proportion of the NKp46+ cells expressed CD8(mean = 8.7%). The level of CD8+ T cells started toincrease at day 8 after inoculation and reached a maximumat day 11 in all infected animals (P < 0.031), but with sub-stantial variation between individuals (Fig. 2B). There wasno increase in CD8+ T cells in the control group, and theyremained at a stable level throughout the study. The calcu-lation of absolute numbers of NKp46+ cells and CD8+ Tcells gave similar results as the use of percentage of lym-phocyte subpopulation, except for a more pronouncedincrease of NKp46+ cells in calves 4725 and 4727 at days11–13 (data not shown).

The proportion of CD4+ T cells in each individualremained largely constant, both for the infected groupand for the control group throughout the experiment(Fig. 2C). However, the absolute numbers for CD4+ Tcells revealed a rise in two of the animals at day 11 p.i.(data not shown). The level of cd TCR+ cells ranged from10% to 23% in both groups. There was somewhat morevariation in the percentage of gated cd TCR+ cells withinthe infected group compared with the control group, butno consistent trends were observed.

Within the first 10 days p.i. the white blood cell count(WBC) in infected calves decreased (P < 0.031), thenincreased and finally levelled out for the rest of the exper-iments while the variation in WBC in the control animalswas negligible (data not shown). There were no major dif-ferences in granulocyte recruitment between the infectedanimals and the control animals. Hence, it looks like thevariation in total WBC in the infected animals was primar-ily due to variation in the mononuclear cell population.The variation in WBC was, however, within the referencerange for calves of this age.

3.3. NK cell-specific cytotoxicity detected in PBMC ofinfected calves

To investigate if the NK cells were any different in func-tion before and after N. caninum infection, NK cell cyto-toxicity was assessed at two time points. PBMCs werecultured for 4 h with 51Cr-loaded P815 targets in the pres-ence of anti-NKp46 mAb to obtain a redirected lysis effect.Prior to infection, NK cytotoxicity in PBMCs was weak,but detectable (Fig. 3A), and 11 days p.i., NK cytotoxicitywas more clearly detectable (Fig. 3B). Converted to LUs,the cytotoxicity rose from 2.6 [0.7–5.4] LU pre-infectionto 5.5 [2.3–10.1] LU p.i. (median [range]). More compara-ble data were obtained by correcting the number of effectorcells to NKp46+ cells as analysed in flow cytometry, result-ing in cytotoxicity values pre- (41 [20–103] LU) and post-(99 [31–121] LU) infection. However the rise in cytotoxicitywas not significant. No cytotoxicity was detected in theabsence of mAbs, indicating that the killing was NKp46-specific.

3.4. IFN-c production in blood after in vitro stimulation

The four infected calves that were kept throughoutthe experiment, were tested for IFN-c production.Whole blood was stimulated with sonicated N. caninum

tachyzoites or sonicated Vero cells and IFN-c produc-tion was measured. Within the first 11 days p.i., highlevels of IFN-c were detected in three of the infectedanimals, while the fourth calf had relatively low levelsof IFN-c at 18 and 25 days p.i. (Fig. 4). There wereno detectable IFN-c responses to sonicated N. caninum

tachyzoites in the control group and none of the groupsshowed any IFN-c response to sonicated Vero cells (datanot shown).

3.5. The effect of different antigen preparations on IFN-cproduction from various subsets of lymphocytes

Three infected calves with the highest IFN-c response inblood were examined to identify which subsets of lympho-cytes that were responsible for IFN-c production afterstimulation with sonicated and heat-inactivated N. caninum

tachyzoites. The intracellular staining assay was not per-formed in control calves since no IFN-c was detected inplasma after stimulation with antigens (see above). After1.5 weeks, intracellular IFN-c production in response tosonicated, soluble N. caninum antigens, was seen inNKp46+, CD4+ and CD8+ cells, while CD4+ cells werethe predominant IFN-c producers after 3.5 weeks(Fig. 5). No IFN-c production was seen in cd T cells. At18 and 25 days p.i. the response to heat-inactivated N. cani-num tachyzoites was compared with the response to solubleN. caninum antigens to see whether the antigen prepara-tions had different effects on various subsets of lympho-cytes. An average increase of 3.7 (±1.4) in the number ofIFN-c producing NK cells was seen in response to theheat-inactivated preparation compared with the solubleantigens (Fig. 6). In comparison, the number of CD4+ cellsproducing IFN-c only increased by an average of 1.4(±0.3).

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Fig. 4. IFN-c production detected in whole blood from Neospora caninum-infected calves after in vitro stimulation with sonicated N. caninum tachyzoites.The figure shows the variation of IFN-c production between the four infected calves that were kept throughout the experiment. The Y-axis indicates thequantity of IFN-c (ng/ml) present in each of the test samples, the X-axis shows the number of days after inoculation with N. caninum tachyzoites. Day -2indicates values obtained before inoculation. The calves were inoculated at day 0.

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Fig. 5. Intracellular IFN-c production from different subsets of lympho-cytes. IFN-c production from NKp46+, CD8+ and CD4+ cells; (A) 1.5weeks and (B) 3.5 weeks after experimental infection with Neospora

caninum. Whole blood was stimulated with sonicated soluble N. caninum

antigens overnight with subsequent staining for surface markers andintracellular IFN-c and analysed by flow-cytometry. The figures show thepercentages of total lymphocytes of the different subsets producing IFN-cin three different animals.

Fig. 6. Increased number of IFN-c-producing natural killer (NK) cells inresponse to heat-inactivated tachyzoites. IFN-c production from (A)NKp46+ cells and (B) CD4+ cells in response to Neospora caninum

antigens. On average there was a 3.7 (±1.4)-fold increase in IFN-cproduction from NK cells when stimulated with heat-inactivated wholetachyzoites compared with sonicated, soluble antigens, while an averageincrease of 1.4 (±0.3) was seen in the CD4+ cells. The assay wasperformed twice in three infected animals and the figure shows arepresentative animal. The plots are gated on approximately 5000NKp46+ or CD4+ cells, and the number in the upper right quadrantsshow the percentages of NKp46+ cells and CD4+ cells producing IFN-cin one animal.


3.6. Heat-inactivated tachyzoites inhibited proliferation of cdT-cells

Proliferation of various lymphocyte subsets after stimu-lation with N. caninum antigens was measured by flowcytometry after labelling the cells with CFSE. The cells

were incubated with heat-inactivated or soluble antigensfor 5–6 days, followed by staining for surface markers toidentify the proliferating subsets. Proliferating CD4+ cellswere seen in three of the infected animals after 2 weeks,while all the infected animals had proliferating CD4+ cellsafter 4 weeks. Two of the infected animals also had prolif-erating CD8+ cells at 2 weeks and three had proliferatingCD8+ cells at 4 weeks p.i. The response was similar to boththe soluble and heat-inactivated N. caninum antigens,although somewhat stronger in response to the heat-inacti-

Table 2Proliferation of CD4+ and cd T cells in response to Neospora caninum antigens 4 weeks p.i.

Infected calves Controls calves

CD4+ cells cd T-cells CD4+ cells cd T-cells

Medium control 8% (±3) 35% (±4) 3% (±0.4) 17% (±1.3)Soluble sonicated antigens 32% (±8) 33% (±5) 3% (±0.3) 11% (±1.3)Heat-inactivated tachyzoites 74% (±8) 15% (±3) 3% (±0.5) 9% (±1.0)

Peripheral blood mononuclear cells were labelled with carboxyfluorescein diacetate succinimidyl ester and proliferation was measured as a reduction influorescence intensity after 6 days of incubation. The numbers show the mean percentages (±SEM) of each subpopulation that proliferated in response totwo different antigen preparations.


vated fraction. No response was seen in response to soni-cated or heat-inactivated Vero cells and no response wasseen in the control animals.

The cd T cells proliferated in non-stimulated control cul-tures from the infected animals at 2 and 4 weeks p.i. and toa lesser extent in control calves (Table 2). The addition ofsoluble N. caninum antigens did not affect this response.However, surprisingly, addition of heat-inactivated tach-yzoites inhibited the proliferation of cd T cells, especiallyin the infected calves (Table 2). There was a clear differencebetween the effect of soluble antigens and heat-inactivatedtachyzoites on CD4+ cells and cd T cells in infected ani-mals. The CD4+ cells were stimulated by both antigenpreparations while the heat-inactivated tachyzoites clearlyinhibited the proliferation of cd T cells.

3.7. PCR and histopathology

Neospora caninum DNA was not detected in any of thesampled tissues from the experimentally infected calves.

Histological examination of the musculus masseter ofthe calf that was euthanised 11 days post inoculationrevealed a small inflammatory focus with predominatelymacrophages and lymphocytes and some polymorphonu-clear leukocytes. In some muscle fibres, hyaline degenera-tion was found, and there were structures suspected to bedegenerated protozoans in the lesion. In the liver therewere multifocal necroses with infiltration by macrophagesand lymphocytes. There was mild and multifocal inflam-mation characterised by necrotising vasculitis and perivas-cular infiltration of macrophages and lymphocytesthroughout the brain parenchyma of the calves that wereeuthanasied 32 days after inoculation. Protozoans werenot detected in the lesions.

4. Discussion

This study aimed to investigate cellular immuneresponses in an experimental N. caninum infection incalves, in particular, the role of NK cells in early innateimmune responses and the effect of different N. caninum

antigen preparations on the IFN-c production of differentlymphocytes.

Interestingly, the percentage of NK cells of total lym-phocytes in blood decreased at days 4-6 after i.v. inocula-tion of calves, followed by an increase in the percentage

of both NK cells and CD8+ T cells, which both peakedat days 11-15. Little is known about the mechanisms regu-lating NK cells mobilization during infections (Wald et al.,2006). One possible explanation for the decrease in NKcells in blood after 4-6 days may be that antigen-presentingcells in N. caninum-infected tissues produced NK cell-acti-vating cytokines and chemokines, causing NK cells tomigrate into affected tissues (Amichay et al., 1996; Bironet al., 1999; Loetscher et al., 1996). An example of para-site-induced NK cell migration is the migration of NK cellsinto the skin of mice after s.c. infection with Leishmania

major promastigotes, due to up-regulating of the NK cellactivating chemokine CXCL10 (Muller et al., 2001). Otherstudies have demonstrated that stimulated NK cells wererecruited from the peripheral blood to the sites of viralinfection (Salazar-Mather et al., 2002). The migration ofNK cells and release of chemokines, e.g., CXCL10, in lungtissue, has been observed in Mycobacterium tuberculosis

infection (Sadek et al., 1998; Lande et al., 2003). Further-more, protozoan pathogens can release factors chemotaticto leukocytes as observed in in vitro studies of T. gondii

(Nakao and Konishi, 1991). Hence, location of parasitesand activated antigen presenting cells may be importantfor NK-cell migration.

It is not known in which organs the parasite locates inthe early stages of experimental infection in calves and noN. caninum DNA was detected in the present study whichcould be attributed to small quantities of parasite DNA(Kritzner et al., 2002). Lymphoid organs such as the spleenand liver are alternative candidates for trapping of bothparasites and NK cells (Sardinha et al., 2006). Anotheralternative is lung tissue since most particles injected i.v.in calves are removed by pulmonary intravascular macro-phages, and N. caninum-infected mice demonstrated detec-tion of parasite DNA in the lungs in the acute phase(Winkler, 1988; Chitko-McKown et al., 1992; Collantes-Fernandez et al., 2006).

The increase in NK cells observed after the decrease inperipheral blood was possibly caused by recruitment ofcells from the bone marrow and by proliferation of NKcells in lymphoid organs, such as the spleen, as observedin other parasite infections (Goff et al., 2003; Abou-Bacaret al., 2004; Antunez and Cardoni, 2004). It was previouslyshown that peak NK cell cytotoxicity and IFN-c produc-tion occurred within the first several hours to days afterprimary infection (Biron et al., 1999). In our study, the


apparently higher NK cell cytotoxicity observed at day 11p.i. was largely explained by the increased number of NKcells in peripheral blood. However, there was also a slight,but not significant, rise in cytotoxicity per NK cell follow-ing infection. These results demonstrated that the NK cellsmobilised to blood following infection were fully functionaland therefore presumably mature.

The increase of CD8+ T cells on day 11 p.i. suggeststhat naıve CD8+ T cells responded to N. caninum tachyzo-ites and developed into activated CD8+ T cells. Further-more, we showed that these cells produced IFN-c in theearly course of the infection. The importance of IFN-c pro-duction in a N. caninum infection has been well document-ed (Innes et al., 2002). We observed that NK cells andCD8+ T cells, together with CD4+ cells, contributed toIFN-c production in the early stages, while the CD4+ Tcells dominated later on. An important role of NK cellsas an early IFN-c producer in vivo have been verified inother protozoan infections such as leishmaniasis (Schar-ton-Kersten and Sher, 1997), and malaria (Artavanis-Tsakonas and Riley, 2002). IFN-c production from in vitrostimulated NK cells was detected in N. caninum-infectedcalves, but not in the control calves, suggesting that infec-tion may lead to activated NK cells in blood that morereadily produces IFN-c. A recent study in T. gondii alsodemonstrated a pivotal role of NK cells in the inductionof CD8+ T-cell immunity in the absence of CD4+ T cells(Combe et al., 2005). They showed that the CD8+ T-cellresponse was dependent on IFN-c production from NKcells. Based on this background, it could be argued thatearly activation of NK cells, as observed in our study, pro-motes a stronger CD8+ T-cell response in N. caninum

infections.The ability of the two antigen preparations to induce

proliferation in various cell types was also tested, andCD4+ and CD8+ T cells proliferated in response to bothantigen preparations. The cd T cells proliferated vigorouslyin non-stimulated control samples from the infected ani-mals at 2–4 weeks p.i., suggesting that these cells are moreactivated in infected calves. We have quite often seen pro-liferation in non-stimulated samples in ruminants whenusing the standard 3H thymidine incorporation assay.Another laboratory has also noted that cd T cells can pro-liferate without stimulation (Waters et al., 2000). Surpris-ingly, we found that the heat-inactivated wholetachyzoites clearly inhibited cd T cell proliferation whilethe soluble antigens from N. caninum had no such effect.A balanced immune response is necessary to avoid exces-sive inflammation and tissue damage. The proliferationand apoptosis of activated T cells are closely linked witheach other and activation-induced apoptosis of lympho-cytes has been seen in other intracellular infections (Man-nering et al., 2002; Das et al., 2004). This provides alikely explanation of the observed inhibiting effect on cdT cells in the present study.

We have previously observed that cultured NK cellsstimulated with heat-inactivated tachyzoites produced

IFN-c, while soluble antigens did not elicit such a response(Boysen et al., 2006). Similar results were detected in thecurrent study where the heat-inactivated N. caninum tach-yzoites induced a greater increase in the number of IFN-c producing NK cells compared with the sonicated, solublefraction. The antigens responsible for the observed effecton NK cells and cd T cells were not identified. Likely can-didates are antigens present in the cell membrane andabsent from the sonicated fraction such as unsoluble lip-id-rich antigens present in the tachyzoite stage of N. cani-

num (Hemphill et al., 1999), but further studies will beundertaken. Activation of NK cells may occur through sev-eral mechanisms, in particular stimulation by cytokines butalso directly by the involvement of Toll-like receptors(TLR) (Kopp and Medzhitov, 2003). Stimulation of TLRby T. gondii in dendritic cells (Yarovinsky et al., 2005),and by glycosyl phosphatidyl inositol-derived lipophophos-glycan from L. major in human NK cells (Becker et al.,2003) has been described. Furthermore, direct activationof bovine NK cells by N. caninum tacyzoites was shownin vitro (Boysen et al., 2006). On this basis, it could behypothesised that the response of bovine NK cells toN. caninum depends on membrane antigens on the tachyzoitesurface and a combination of cytokine-mediated and directactivation of bovine NK cells.

In summary, we have demonstrated for the first time thatN. caninum-infected calves developed both NK cell andCD8+ T cell responses in the early stages of the infection,and that these cell types contributed to IFN-c productionduring this period. Furthermore, we have shown a distinctdifference in the effect of heat-inactivated N. caninum tach-yzoites and soluble antigens on IFN-c production by NKcells, and on proliferation of cd T cells. These results suggestthat intact N. caninum tachyzoites are necessary for an opti-mal NK cell response and that both NK cells and CD8+ Tcells, together with CD4+ T cells, are important IFNc-pro-ducing cells in calves during N. caninum infection.

Acknowledgements

We thank Elisabeth Dahl Nybø for technical assistanceand blood sampling, and Knut Erik Witberg for skilledanimal handling. Furthermore, we wish to thank Hilde Sin-dre and Inger Austerheim Heffernan for their assistance incell culture and cytokine assays. Finally, we thank JorunTharaldsen and Annette H. Kampen for their supportand critical reading of the manuscript. This work was car-ried out under Grant No. 14656603 from the NorwegianResearch Council.

References

Abou-Bacar, A., Pfaff, A.W., Georges, S., Letscher-Bru, V., Filisetti, D.,Villard, O., Antoni, E., Klein, J.P., Candolfi, E., 2004. Role of NKcells and gamma interferon in transplacental passage of Toxoplasma

gondii in a mouse model of primary infection. Infect. Immun. 72, 1397–1401.


Amichay, D., Gazzinelli, R.T., Karupiah, G., Moench, T.R., Sher, A.,Farber, J.M., 1996. Genes for chemokines MuMig and Crg-2 areinduced in protozoan and viral infections in response to IFN-gammawith patterns of tissue expression that suggest nonredundant rolesin vivo. J. Immunol. 157, 4511–4520.

Andrianarivo, A.G., Barr, B.C., Anderson, M.L., Rowe, J.D., Packham,A.E., Sverlow, K.W., Conrad, P.A., 2001. Immune responses inpregnant cattle and bovine fetuses following experimental infectionwith Neospora caninum. Parasitol. Res. 87, 817–825.

Antunez, M.I., Cardoni, R.L., 2004. Trypanosoma cruzi: the expansion ofNK, T, and NKT cells in the experimental infection. Exp. Parasitol.106, 85–94.

Artavanis-Tsakonas, K., Riley, E.M., 2002. Innate immune response tomalaria: rapid induction of IFN-gamma from human NK cells by livePlasmodium falciparum-infected erythrocytes. J. Immunol. 169, 2956–2963.

Baszler, T.V., Long, M.T., McElwain, T.F., Mathison, B.A., 1999.Interferon-gamma and interleukin-12 mediate protection to acuteNeospora caninum infection in BALB/c mice. Int. J. Parasitol. 29,1635–1646.

Becker, I., Salaiza, N., Aguirre, M., Delgado, J., Carrillo-Carrasco, N.,Kobeh, L.G., Ruiz, A., Cervantes, R., Torres, A.P., Cabrera, N.,Gonzalez, A., Maldonado, C., Isibasi, A., 2003. Leishmania lipophos-phoglycan (LPG) activates NK cells through toll-like receptor-2. Mol.Biochem. Parasitol. 130, 65–74.

Biron, C.A., Nguyen, K.B., Pien, G.C., Cousens, L.P., Salazar-Mather,T.P., 1999. Natural killer cells in antiviral defense: function andregulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220.

Bjerkas, I., Mohn, S.F., Presthus, J., 1984. Unidentified cyst-formingsporozoon causing encephalomyelitis and myositis in dogs. Z. Para-sitenkd. 70, 271–274.

Bjorkman, C., Lunden, A., Holmdahl, J., Barber, J., Trees, A.J., Uggla,A., 1994. Neospora caninum in dogs: detection of antibodies by ELISAusing an iscom antigen. Parasite Immunol. 16, 643–648.

Bjorkman, C., Holmdahl, O.J., Uggla, A., 1997. An indirect enzyme-linked immunoassay (ELISA) for demonstration of antibodies toNeospora caninum in serum and milk of cattle. Vet. Parasitol. 68, 251–260.

Boehm, U., Klamp, T., Groot, M., Howard, J.C., 1997. Cellular responsesto interferon-gamma. Annu. Rev. Immunol. 15, 749–795.

Boysen, P., Klevar, S., Olsen, I., Storset, A.K., 2006. The protozoanNeospora caninum directly triggers bovine NK cells to produce gammainterferon and to kill infected fibroblasts. Infect. Immun. 74, 953–960.

Bryant, J., Day, R., Whiteside, T.L., Herberman, R.B., 1992. Calculationof lytic units for the expression of cell-mediated cytotoxicity. J.Immunol. Methods 146, 91–103.

Buxton, D., McAllister, M.M., Dubey, J.P., 2002. The comparativepathogenesis of neosporosis. Trends Parasitol. 18, 546–552.

Carayannopoulos, L.N., Yokoyama, W.M., 2004. Recognition of infectedcells by natural killer cells. Curr. Opin. Immunol. 16, 26–33.

Chitko-McKown, C.G., Reddy, D.N., Chapes, S.K., McKown, R.D.,Blecha, F., 1992. Immunological characterization of pulmonaryintravascular macrophages. Reg. Immunol. 4, 236–244.

Collantes-Fernandez, E., Lopez-Perez, I., Alvarez-Garcia, G., Ortega-Mora, L.M., 2006. Temporal distribution and parasite load kinetics inblood and tissues during Neospora caninum infection in mice. Infect.Immun. 74, 2491–2494.

Combe, C.L., Curiel, T.J., Moretto, M.M., Khan, I.A., 2005. NK cellshelp to induce CD8(+)-T-cell immunity against Toxoplasma gondii inthe absence of CD4(+) T cells. Infect. Immun. 73, 4913–4921.

Das, S.D., Subramanian, D., Prabha, C., 2004. Cell proliferation andapoptosis: dual-signal hypothesis tested in tuberculous pleuritis usingmycobacterial antigens. FEMS Immunol. Med. Microbiol. 41, 85–92.

Davison, H.C., Otter, A., Trees, A.J., 1999. Estimation of vertical andhorizontal transmission parameters of Neospora caninum infections indairy cattle. Int. J. Parasitol. 29, 1683–1689.

De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasbarre, L., 1999.Oral infection of calves with Neospora caninum oocysts from dogs:

humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657.

Dubey, J.P., 2003. Review of Neospora caninum and neosporosis inanimals. Korean J. Parasitol. 41, 1–16.

Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988.Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med.Assoc. 192, 1269–1285.

Glass, E.J., 2001. The balance between protective immunity and patho-genesis in tropical theileriosis: what we need to know to design effectivevaccines for the future. Res. Vet. Sci. 70, 71–75.

Goff, W.L., Johnson, W.C., Horn, R.H., Barrington, G.M., Knowles,D.P., 2003. The innate immune response in calves to Boophilus

microplus tick transmitted Babesia bovis involves type-1 cytokineinduction and NK-like cells in the spleen. Parasite Immunol. 25, 185–188.

Hemphill, A., Fuchs, N., Sonda, S., Hehl, A., 1999. The antigeniccomposition of Neospora caninum. Int. J. Parasitol. 29, 1175–1188.

Innes, E.A., Panton, W.R., Marks, J., Trees, A.J., Holmdahl, J., Buxton,D., 1995. Interferon gamma inhibits the intracellular multiplication ofNeospora caninum, as shown by incorporation of 3H uracil. J. Comp.Pathol. 113, 95–100.

Innes, E.A., Andrianarivo, A.G., Bjorkman, C., Williams, D.J., Conrad,P.A., 2002. Immune responses to Neospora caninum and prospects forvaccination. Trends Parasitol. 18, 497–504.

Kampen, A.H., Olsen, I., Tollersrud, T., Storset, A.K., Lund, A., 2006.Lymphocyte subpopulations and neutrophil function in calves duringthe first 6 months of life. Vet. Immunol. Immunopathol. 113, 53–63.

Kopp, E., Medzhitov, R., 2003. Recognition of microbial infection byToll-like receptors. Curr. Opin. Immunol. 15, 396–401.

Korbel, D.S., Finney, O.C., Riley, E.M., 2004. Natural killer cells andinnate immunity to protozoan pathogens. Int. J. Parasitol. 34, 1517–1528.

Kritzner, S., Sager, H., Blum, J., Krebber, R., Greif, G., Gottstein, B.,2002. An explorative study to assess the efficacy of toltrazuril-sulfone(ponazuril) in calves experimentally infected with Neospora caninum.Ann. Clin. Microbiol. Antimicrob. 1, 4.

Lande, R., Giacomini, E., Grassi, T., Remoli, M.E., Iona, E., Miettinen,M., Julkunen, I., Coccia, E.M., 2003. IFN-alpha beta released byMycobacterium tuberculosis-infected human dendritic cells induces theexpression of CXCL10: selective recruitment of NK and activated Tcells. J. Immunol. 170, 1174–1182.

Liddell, S., Jenkins, M.C., Dubey, J.P., 1999. A competitive PCR assay forquantitative detection of Neospora caninum. Int. J. Parasitol. 29, 1583–1587.

Loetscher, P., Seitz, M., Clark-Lewis, I., Baggiolini, M., Moser, B., 1996.Activation of NK cells by CC chemokines. Chemotaxis, Ca2+

mobilization, and enzyme release. J. Immunol. 156, 322–327.Lunden, A., Marks, J., Maley, S.W., Innes, E.A., 1998. Cellular immune

responses in cattle experimentally infected with Neospora caninum.Parasite Immunol. 20, 519–526.

Mannering, S.I., Zhong, J., Cheers, C., 2002. T-cell activation, prolifer-ation and apoptosis in primary Listeria monocytogenes infection.Immunology 106, 87–95.

Marks, J., Lunden, A., Harkins, D., Innes, E., 1998. Identification ofNeospora antigens recognized by CD4+ T cells and immune sera fromexperimentally infected cattle. Parasite Immunol. 20, 303–309.

McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A.,McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum.Int. J. Parasitol. 28, 1473–1478.

Muller, K., van Zandbergen, G., Hansen, B., Laufs, H., Jahnke, N.,Solbach, W., Laskay, T., 2001. Chemokines, natural killer cells andgranulocytes in the early course of Leishmania major infection in mice.Med. Microbiol. Immunol. (Berl.) 190, 73–76.

Nakao, M., Konishi, E., 1991. Neutrophil chemotactic factors secretedfrom Toxoplasma gondii. Parasitology 103 Pt 1, 29–34.

Olsen, I., Storset, A.K., 2001. Innate IFN-gamma production in cattle inresponse to MPP14, a secreted protein from Mycobacterium avium

subsp. paratuberculosis. Scand. J. Immunol. 54, 306–313.


Sadek, M.I., Sada, E., Toossi, Z., Schwander, S.K., Rich, E.A., 1998.Chemokines induced by infection of mononuclear phagocytes withmycobacteria and present in lung alveoli during active pulmonarytuberculosis. Am. J. Respir. Cell Mol. Biol. 19, 513–521.

Salazar-Mather, T.P., Lewis, C.A., Biron, C.A., 2002. Type Iinterferons regulate inflammatory cell trafficking and macrophageinflammatory protein 1alpha delivery to the liver. J. Clin. Invest.110, 321–330.

Sardinha, L.R., Elias, R.M., Mosca, T., Bastos, K.R., Marinho, C.R.,D’Imperio Lima, M.R., Alvarez, J.M., 2006. Contribution of NK, NKT, gammadelta T, and alphabeta T cells to the gamma interferonresponse required for liver protection against Trypanosoma cruzi.Infect. Immun. 74, 2031–2042.

Scharton-Kersten, T.M., Sher, A., 1997. Role of natural killer cells ininnate resistance to protozoan infections. Curr. Opin. Immunol. 9, 44–51.

Sher, A., Pearce, E., Kaye, P., 2003. Shaping the immune responseto parasites: role of dendritic cells. Curr. Opin. Immunol. 15,421–429.

Storset, A.K., Kulberg, S., Berg, I., Boysen, P., Hope, J.C., Dissen, E.,2004. NKp46 defines a subset of bovine leukocytes with natural killercell characteristics. Eur. J. Immunol. 34, 669–676.

Tuo, W., Fetterer, R., Jenkins, M., Dubey, J.P., 2005. Identification andcharacterization of Neospora caninum cyclophilin that elicits gammainterferon production. Infect. Immun. 73, 5093–5100.

Voyich, J.M., Palecanda, A., Burgess, D.E., 2001. Antigen-specific T-cellresponses in cattle immunized with antigens of Tritrichomonas foetus.J. Parasitol. 87, 1040–1048.

Wald, O., Weiss, I.D., Wald, H., Shoham, H., Bar-Shavit, Y., Beider, K.,Galun, E., Weiss, L., Flaishon, L., Shachar, I., Nagler, A., Lu, B.,Gerard, C., Gao, J.L., Mishani, E., Farber, J., Peled, A., 2006. IFN-gamma acts on T cells to induce NK cell mobilization and accumu-lation in target organs. J. Immunol. 176, 4716–4729.

Waters, W.R., Palmer, M.V., Pesch, B.A., Olsen, S.C., Wannemuehler,M.J., Whipple, D.L., 2000. Lymphocyte subset proliferative responsesof Mycobacterium bovis-infected cattle to purified protein derivative.Vet. Immunol. Immunopathol. 77, 257–273.

Williams, D.J., Trees, A.J., 2006. Protecting babies: vaccine strategies toprevent foetopathy in Neospora caninum-infected cattle. ParasiteImmunol. 28, 61–67.

Winkler, G.C., 1988. Pulmonary intravascular macrophages in domesticanimal species: review of structural and functional properties. Am. J.Anat. 181, 217–234.

Wouda, W., Moen, A.R., Schukken, Y.H., 1998. Abortion risk in progenyof cows after a Neospora caninum epidemic. Theriogenology 49, 1311–1316.

Yarovinsky, F., Zhang, D., Andersen, J.F., Bannenberg, G.L., Serhan,C.N., Hayden, M.S., Hieny, S., Sutterwala, F.S., Flavell, R.A., Ghosh,S., Sher, A., 2005. TLR11 activation of dendritic cells by a protozoanprofilin-like protein. Science 308, 1626–1629.

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