Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study

Authors


  • Disclosures: None.

David A. Schneider, Animal Disease Research Unit, USDA-ARS, 3003 ADBF, Washington State University, Pullman, Washington 99164-6630 USA (e-mail: das@vetmed.wsu.edu).

Summary

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cell-rich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

Introduction

Babesiosis is a tick-borne disease affecting cattle in much of the world, with Babesia divergens, B. bigemina and B. bovis the economically important species. Babesia bovis is the most virulent, often causing death in susceptible animals because of the development of anaemia, cerebral vascular congestion and pulmonary and renal failure (1). The virulent nature of the disease is attributed in part to the sequestration of parasitized erythrocytes to capillary endothelium, but overproduction of inflammatory cytokines has also been suggested (2–4). Young calves are relatively resistant to clinical infection (5) and demonstrate a strong innate immunity composed of a type-1 inflammatory response (6). In comparison with adult cattle, we have previously demonstrated that the immune response of calves involves early IL-12 expression with consequent IFN-γ production, a nitric oxide burst and modulation by IL-10 (6–9). This age-related immunity is dependent upon cellular events within the spleen as splenectomy of calves renders them equally susceptible (5,10).

Our studies have utilized a technique to marsupialize the spleen of calves (11) so that cells could be acquired for ex vivo analysis (microplate assays and flow cytometry) (12–16). Such analyses have proven valuable in determining the function of various splenic cell phenotypes but lack the ability to place these cell populations within their anatomical context which include the marginal zone, red and white pulp (17). Amongst many factors that comprise an effective immune response to haemoparasitic infection, trafficking and interaction of cells within such domains are central (18).

Intravital imaging techniques have been used to dynamically study such factors within superficial lymphoid organs (19,20) and, to a limited extent, also within deeper structures including the spleen of mice (21). But current techniques are not well suited to study the spleen of large mammals because of the limits on depth resolution (22). An approach readily applied to the spleen of large mammals is the serial analysis of the distribution of phenotyped cells in tissue sections. Similar to a recent study on the acute immune response of naïve mice to haemoparasitic infection (23), we have applied this technique to the spleen of naïve calves infected with Babesia bovis. The results document acute change in the distribution of several cells thought to be important to the spleen-dependent response of naïve calves to B. bovis and serve to underscore common themes in the acute response to haemoparasitic infections. In addition, this is the first documented use of magnetic resonance imagery to measure spleen volume in calves.

Materials and methods

Animals and experimental B. bovis infection

Twelve Holstein–Friesian steer calves were obtained at 8 weeks of age, vaccinated against pathogenic Clostridium species, castrated and dehorned. All animals were cELISA seronegative for Anaplasma marginale (VMRD, Pullman WA, USA) and B. bovis and B. bigemina (24–26). The care and use of these calves were approved by the Institutional Animal Care and Use Committee at Washington State University (Pullman, WA, USA). At 12 weeks of age, all calves underwent a surgical procedure to marsupialize the spleen (11). When necessary, spleen cell aspirates were obtained under local lidocaine anaesthesia into 60cc syringes containing ACD and prepared for in vitro studies as previously described (14,27).

Ten of the twelve calves were inoculated intravenously with 1 × 105 erythrocytes infected with the T2Bo virulent isolate of B. bovis (7). The acute response to infection was studied 7–14 days postinfection (dpi). As described below, repeated measures of spleen volume and cell content were made in four inoculated calves whereas change in regional distribution of phenotyped cells was determined by sequential euthanasia of six inoculated calves in comparison with two un-inoculated calves.

Determination of spleen volumes and differential cell counts

Magnetic resonance imagery was performed with a 1·0 Tesla machine (Philips Intera, Andover, MA, USA). Sequences were acquired in a dorsal plane. The area imaged was from the spine to the ventral abdominal wall. A 40 cm field-of-view ensured that the entire spleen could be visualized. One-centimetre-thick slices with a 2 mm gap were acquired using a short tau inversion recovery (STIR) sequence. This sequence resulted in a hyperintense spleen on a low intense background. The volume was calculated by tracing the outline of the spleen for the area on each slice and multiplying by the number of slices plus gap thickness (3D-DOCTOR; Able Software Corporation, Lexington, MA, USA). Each calf’s spleen volume was calculated on the day prior to infection and then at 11 or 12 dpi, 2 calves each.

Immediately following each MRI procedure, a 1 cm3 biopsy of marsupialized spleen was removed under local lidocaine anaesthesia for determining differential cell counts. Each biopsy was immediately processed into a single cell suspension using a tissue grinder (Tenbroek; Bellco Glass, Inc., NJ, USA), suspended in 50 mL of PBS and enumerated for differential cell counts by standard methods used for whole blood (28).

Immunohistochemistry (IHC)

Six inoculated calves were euthanized by captive bolt and jugular exsanguination for collection of spleen tissue: one calf each on dpi 7, 8, 9 (fever day 1) and 14 (fever day 5), and two calves at 13 dpi (fever days 4 and 5). In this way, the spleens from three calves each were examined from two periods: a period just prior to, or including, the initiation of fever (7, 8 and 9 dpi) and a period several days after fever initiation (13 and 14 dpi). Spleen tissue from two uninfected calves was similarly collected. Multiple 15 × 15 × 5 mm sections of spleen were collected from each calf immediately posteuthanasia. Each section was placed into a cryostat mould containing Tissue-Tek® O.C.T.™ Compound (Sakura Fineteck USA, Inc., Torrance, CA, USA), snap frozen by floating on liquid nitrogen, and stored at −80°C. Cryostat sections (15 μm) were mounted on standard SuperFrost™ Plus slides (Electron Microscopy Services, Hatfield, PA, USA), fixed in 95% EtOH for 10 min and allowed to air dry overnight at room temperature. Formalin-fixed, paraffin-embedded samples of spleen were also collected from each calf and routinely stained in haematoxylin and eosin (H&E).

Immunolabelling was carried out at room temperature in a humidified chamber. A Super PAP Pen HT™ (Research Products International Corp., Mt. Prospect, IL, USA) was used to create a hydrophobic margin to retain fluid reagents on slides. Thin sections on slides were preblocked for 1 h using 10% goat serum/0·1% Triton-X 100/PBS. After three 5-min washes in PBS, thin sections were exposed (2–4 h) to primary antibody (Table 1) diluted in 10% goat serum/PBS. Unbound primary antibody was removed with three 5-min washes in PBS and then exposed (2 h) to fluorophore-conjugated secondary antibody, all diluted 1 : 200 in 10% goat serum/0·1% Triton-X 100/PBS. After three 5-min washes in PBS, the slides were coverslipped using ProLong® Gold antifade mounting media with DAPI (Molecular Probes, Inc., Eugene, OR, USA). DAPI staining aided in follicle localization, especially in the presence of a greatly expanded red pup postinfection.

Table 1.   Primary antibodies
TargetNameSpecies isotypeWorking concentration (μg/mL)Source
CD3MM1aMouse IgG12·5VMRD, Inc., Pullman, WA, USA
CD4IL-A11Mouse IgG2a2·5VMRD, Inc., Pullman, WA, USA
CD335 (NKp46)MCA2365 (clone AKS1)Mouse IgG15AbD Serotec, Oxford, UK
CD13 (aminopeptidase N)MCA2338 (clone CC81)Mouse IgG12·5AbD Serotec, Oxford, UK
CD172a (SIRPα)DH59bMouse IgG15VMRD, Inc., Pullman, WA, USA
MCA2041G (clone CC149)Mouse IgG2b2·5AbD Serotec, Oxford, UK
TcR1-N24 (δ chain)GB21aMouse IgG2b5VMRD, Inc., Pullman, WA, USA
WC1IL-A29Mouse IgG12·5VMRD, Inc., Pullman, WA, USA
MCA838G (clone CC15)Mouse IgG2a5AbD Serotec, Oxford, UK
Babesia bovis MSA-1BABB35Mouse IgG2a5Goff WL, et al. (29)

Immunohistochemistry (IHC) controls for these experiments included substitution of primary or secondary antibodies with antibody diluent, and substitution of primary antibodies with isotype-matched irrelevant antibodies. Dual-labelling experiments were performed by co-incubation of primary antibodies followed by co-incubation of selective secondary antibodies. Nonspecific staining and cross-reactions between secondary antibodies or between a primary antibody and nonrelevant secondary antibody were not observed. Note: Attempts were made to localize CD8+ cells by IHC (primary antibody = BAQ111a, isotype = IgM; VMRD, Inc., Pullman, WA, USA). CD8 localization was precluded, however, by significant background mediated by anti-IgM secondary antibody.

Image and data analysis

Immunohistochemistry (IHC) slides were viewed and photographed using an Axio Imager M1 microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) equipped with an LED illuminator for bright field microscopy and an X-Cite 120 Fl Illuminating system (EXFO Photonic Solutions, Mississauga, ON, Canada) for epi-fluorescence microscopy. Digital images were captured using an AxioCam MRc5 digital camera connected to a desktop computer running AxioVision (version 4.7.1.0) and prepared for presentation using Photoshop Elements (version 4.0; Adobe Systems Inc., San Jose, CA, USA). Figure images are representative, and variation within or between time points (dpi) is noted in the Results section. In particular, the term ‘progressive’ is used to indicate appreciation of an ordered change over time. Measurements of the splenic marginal zone included the region extending from its follicle junction (indicated in figures by a dashed curved line) to a width of ∼100 μm, and measurements of the red pulp included regions furthest away from neighbouring white pulp. IHC measurements must be considered approximate as uncontrolled changes in tissue dimensions are expected to have occurred during euthanasia and preparation of thin frozen sections. All data were tabulated in Microsoft Office Excel 2003 and are reported as mean ± standard error. Splenic volume (MRI) and differential cell count data were analysed for significant (< 0·05) postinfection increases by paired T-test (SAS® for Windows 9.2; SAS Institute Inc., Cary, NC, USA).

Results

As previously described (7), intravenous inoculation of 105 T2Bo isolate of B. bovis into 6 month-old naïve Holstein calves consistently induced fever (>39·5°C) between 8 and 10 dpi. The rare presence of B. bovis-infected erythrocytes was noted in each animal by examination of Giemsa stained blood films just prior to euthanasia. Although calves were necropsied at different intervals, each was experiencing a decrease in haematocrit from their normal pre-infection levels. At 7 dpi the haematocrit was decreased 19% and by 13–14 dpi had decreased 45 ± 6·7% (n = 3).

The spleen of naïve calves doubled in volume by 11–12 dpi and was associated with significant increases in the total splenic content of small leucocytes (approximately twofold), large leucocytes (approximately eightfold) and total leucocytes (approximately twofold) (Table 2). As determined by FACS analysis (data not shown), the large leucocyte population included monocytes, macrophages, dendritic cells (DCs) (12) and large granular natural killer (NK) cells (15). As viewed in H&E sections, splenomegaly 7–14 dpi was associated with a progressive basophilic hyperplasia within the red pulp and histological reduction in the white pulp (w) and trabeculae (t) elements (Figure 1, 1·25×), and also a loss in zonal distinction between marginal zone and red pulp (Figure 1, 10×). The regional distributions of phenotyped cells were further investigated by IHC.

Table 2.   Effects of acute Babesia bovis infection on naïve calf spleen volume and leucocyte contenta
SpleenPre-infectionPostinfectionDifference (post–pre)t-value (paired, upper-tail, df = 3)P-value
  1. aValues reported are mean ± standard error; * indicates ‘difference’ significantly >0.

  2. bDetermined by MRI.

  3. cDetermined by differential cell counts on dissociated splenic biopsies.

  4. dCalculated as the paired product of spleen volume and differential cell count.

Volumeb (cm3)506 ± 49996 ± 91491 ± 49* 9·970·0011
Leucocyte concentrationc (×103 cells/cm3)
 Small57·5 ± 6·753·3 ± 6·1−4·3 ± 5·9−0·720·7385
 Large1·4 ± 0·45·5 ± 1·34·1 ± 1·5* 2·710·0365
 Total58·9 ± 7·058·8 ± 6·0−0·1 ± 6·0−0·020·5078
Leucocyte contentd (×106 cells/spleen)
 Small29·3 ± 4·852·8 ± 7·523·5 ± 6·1* 3·840·0156
 Large0·7 ± 0·25·8 ± 1·75·2 ± 1·8* 2·840·0327
 Total30·0 ± 4·758·8 ± 8·628·8 ± 7·0* 4·100·0131
Figure 1.

 Splenomegaly in calves following acute infection with Babesia bovis is associated with expansion and increased cellularity of the red pulp and loss of zonal definition around white pulp as shown in representative macroscopic (1·25× objective) and microscopic (10× objective) images. Haematoxylin and eosin; r, red pulp; w, white pulp; cyan [, periarteriolar lymphatic sheath; yellow outline, central arteriole; f, follicle; mz, marginal zone; dashed curve, follicle–marginal zone junction; {, band of cells within marginal zone; t, trabeculae; bars = 1 mm (1·25×), 200 μm (10×); dpi, days postinfection.

Small leucocyte populations of the spleen: CD3+, CD4+ cells and γδ T cells

Examples of the splenic cellular immunoreactivity to monoclonal antibodies specific for CD3 and CD4 are shown in Figure 2a–f. Two cell populations were clearly evident in this dual-labelling experiment: CD3+/CD4+ and CD3+/CD4 cells. In the uninfected calf, CD3+/CD4+ cells were always most dense within the periarteriolar lymphatic sheath (PALS; see ‘[’ in Figure 2a,d). A band of CD3+/CD4+ and CD3+/CD4 cells was consistently present within the marginal zones of uninfected spleens, extending 185 ± 29 μm away from the follicle [see ‘{’ in Figure 2a,d]. Both populations were relatively scarce within the red pulp. During the acute response to infection, the distinctive presence of this marginal zone band was obscured by a progressive red pulp increase in CD3+/CD4 cells and a more modest increase in CD3+/CD4+ cells (Figure 2b,c,e,f).

Figure 2.

 Changes in the parafollicular distribution of phenotyped small leucocyte populations in the spleen of calves during acute infection with Babesia bovis. Shown are spleen sections dually immunolabelled for the T-cell markers CD3 (a–c) and CD4 (d–f), or for the γδ T-cell markers TcR1-N24 (δ chain) [TcR1; (g–i)] and WC1 (j–l). In particular, note the presence of bands (demarked by ‘{’) of CD3+/CD4+ cells, CD3+/CD4 cells, and TcR1+/WC1+ cells within the marginal zone of the uninfected spleen (a/d, g/j). Arrows (g/j) indicate the red pulp location of TcR1+/WC1 cells in uninfected spleen. Postinfection, note the progressive hyperplasia of CD3+/CD4 and CD3+/CD4+ cells (b/e and c/f) and TcR1+/WC1 cells (h/k and i/l) which obscures these zonal distinctions. Also note the relative loss of TcR1+/WC1+ cells from the marginal zone. Cyan [, margins of periarterial lymphatic sheath; yellow outline, central arterioles; dashed curve, follicle–marginal zone junction; {, band of cells within marginal zone; bars = 200 μm; dpi, days postinfection.

The localization of γδ T cells in the spleen is shown in Figure 2g–l. Two major γδ T-cell phenotypes were observed in this dual-labelling experiment: TcR1+ cells that were either WC1+ or WC1. WC1+ cells were generally small and round in appearance whereas WC1 cells were larger angular cells. In the uninfected calf, WC1+ cells densely populated the marginal zone (900–2500 cells/mm2, see ‘{’ in Figure 2j) but were relatively scarce in the red pulp (100–150 cells/mm2) whereas brightly fluorescent TcR1+/WC1 cells were predominately observed within the red pulp, often appearing clustered (see arrow, Figure 2g). During acute infection, the density of TcR1+/WC1 cells progressively increased in the red pulp and marginal zone (Figure 2g–i) whereas the density of marginal zone WC1+ cells progressively dropped to a similar density as observed in the red pulp (Figure 2j–l).

Large leucocyte populations of the spleen: CD335+ (NK cells), CD172+ (monocytes/macrophages) and CD13+ (immature DC)

Fluorescent immunoreactivity mediated by a CD335-specific antibody, a specific marker for natural killer (NK) cells, is shown in Figure 3a–c. In the uninfected calf, CD335+ cells were typically present as a dense marginal zone band extending approximately 250 μm from the follicle–marginal zone junction (600–1400 cells/mm2, see ‘{’ in Figure 3a) and were less dense in the red pulp (140–480 cells/mm2). By 7 dpi and continuing through 14 dpi, the density of CD335+ cells within the marginal zone was reduced to approximate that found in the red pulp (Figure 3b,c).

Figure 3.

 Changes in the parafollicular distribution of phenotyped large leucocyte populations in the spleen of calves during acute infection with Babesia bovis. Shown are representative sequential sections immunolabelled by a marker for NK cells (CD335; a–c) and a marker for immature dendritic cells (CD13; d–f). Also shown are sections immunolabelled by a marker of monocyte/macrophage cells (CD172a; g–i). (a–c) Note the postinfection decrease in CD335+ cells present as a band [see ‘{’ in (a)] within the marginal zone. (d–e) Note the postinfection replacement of the vast honeycomb-like network of CD13+ cells by the appearance of a distinct row of CD13+ cells at the marginal zone junctions with the follicle and periarterial lymphatic sheath. (g–i) Finally, note the progressive postinfection increase in red pulp CD172a+ cells with expansion into the marginal zone. Cyan [, margins of periarterial lymphatic sheath; yellow outline, central arterioles; dashed curve, follicle–marginal zone junction; {, band of cells within marginal zone; bars = 200 μm; dpi, days postinfection.

MCA2338 is a monoclonal antibody directed towards CD13, a marker for immature splenic dendritic cells (iDCs) (12). In all calves the vast majority of CD13+ cells were ‘dendritic’ in shape; however, thin parallel CD13+ structures resembling small-vessel walls were occasionally observed but were not further evaluated. In the uninfected calf (Figure 3d), CD13+ cells were mostly organized as a discontinuous honeycomb-like network that spanned the red pulp and marginal zone with little zonal distinction. More sparsely stained CD13+ cells were also located at the follicle–marginal zone junction and occasionally within the PALS. An unambiguous change in the distribution of CD13+ cells was already evident at 7 dpi and persisted to 14 dpi (Figure 3e,f), wherein the majority of CD13+ cells formed a distinct band at the follicle–marginal zone junction. Sparsely stained CD13+ cells were also observed within the PALS and outer margin of follicles between 7 and 14 dpi. Postinfection CD13+ cells surrounding the PALS were more sparsely stained and scattered by 14 dpi.

Immunoreactivity specific for the myeloid marker CD172a (12) is shown in Figure 3g–i. CD172a+ cells were numerous in the red pulp of the uninfected spleen. The apparent density of CD172a+ cells increased from 7 to 14 dpi, and progressively obscured distinction between marginal zone and red pulp.

Immunoreactivity for merozoite surface antigen-1 (MSA-1)

MSA-1 was localized in the spleen of B. bovis-infected calves using monoclonal antibody BABB35 (29,30). Immunoreactivity for BABB35 was not observed in uninfected or 7–9 dpi spleens. At 13 and 14 dpi, BABB35 immunoreactivity was consistently observed within the outer margins of all splenic follicles, being most dense near its junctions with the marginal zone and PALS (Figure 4a). BABB35 immunoreactivity was generally punctate and appeared to be distributed along fine ‘dendritic’ structures (Figure 4b) but never clearly highlighted any round cell bodies. Immunoreactivity for BABB35 was frequently co-distributed with structures having sparse immunoreactivity for CD13. In contrast, well-labelled CD13+ cells at the follicle–marginal zone junction were not immunoreactive for BABB35.

Figure 4.

 Parafollicular distribution of merozoite surface antigen-1 (MSA-1) in the spleen of a calf 14 dpi with Babesia bovis. MSA-1 was localized using monoclonal antibody BABB35. (a) Note the distribution of punctate Babb35 immunoreactivity (red) within the outer margin of the follicle juxtaposed to the marginal zone and periarterial lymphatic sheath. Higher magnification (b) [corresponds to red box in (a)] shows BABB35 immunoreactivity is not associated with brightly fluorescent CD13+ (green) cells located at the follicle–marginal zone junction but instead follows a similar ‘dendritic’ course as the more weakly CD13-stained cell processes. Cyan [, margins of periarterial lymphatic sheath; yellow outline, central arterioles; dashed curve, follicle–marginal zone junction; bars = 100 μm (a), 25 μm (b).

Discussion

The results of this study demonstrate that the spleen of calves doubles in volume and total cell content by 11–12 dpi. While retention of erythrocytes is a functionally important contributor to splenomegaly, here we document that an acute hyperplasia of nucleated cells, which obscures histological appreciation of zonal boundaries, also occurs and is similar to a recent report of the acute response of naïve mice to infection with Plasmodium chabaudi (23). The acute hyperplasia in calves was characterized by an approximate fourfold greater increase in large versus small splenic leucocytes. The variation noted in these leucocyte measurements probably results from sampling a relatively small piece of spleen and the disproportionate increase in red pulp postinfection. Despite this limitation, these results are consistent with a local immune response of naïve animals to acute haemoparasite infection and reflect the central importance of large leucocytes – monocytes/macrophages, DCs and NK cells – to the spleen-dependent immune response of naïve calves to B. bovis infection (31).

In addition to these gross changes in splenic composition, changes were also observed in the distribution of phenotyped cells within and between the domains of the spleen (summarized in Table 3). Regarding the splenic distribution of large leucocyte populations during acute B. bovis infection, observations with functional implications include the following: (i) loss of the relative accumulation of NK cells (CD335+) within the marginal zone, (ii) unambiguous early redistribution of iDCs (CD13+) to the junction of the marginal zone with follicles and PALS, (iii) subsequent juxtaposed appearance of an immunologically important B. bovis surface antigen (MSA-1+), and (iv) restriction of monocyte/macrophage (CD172a+) hyperplasia to within the red pulp.

Table 3.   Changes in the distribution of phenotyped leucocytes within the spleen of naïve calves during acute Babesia bovis infectiona
Cell phenotypesSplenic zonesb7–9 dpic (pre-fever days and first fever day)13–14 dpi (fever days 4 and 5)
  1. aArrows indicate change in phenotyped cellularity relative to the uninfected spleen. Blank cells indicate no appreciable change noted.

  2. bMZ, marginal zone; PALS, periarteriolar sheath; RP, red pulp.

  3. cdpi, days postinfection.

  4. dne, not examined (cellularity too dense for evaluation).

CD3+/CD4+MZ
PALSnedne
RP
CD3+/CD4MZ
PALSnene
RP↑↑
TcR1+/WC1+MZ↓↓
PALS  
RP  
TcR1+/WC1MZ↑↑
PALS  
RP↑↑
CD335+MZ
PALS  
RP  
CD13+MZ↓ overall but with ↑ at junctions with follicle and PALS↓ overall but with ↑ at junctions with follicle and PALS
PALS↑, weakly labelled↑, weakly labelled
RP↓↓
CD172a+MZ 
PALS  
RP↑↑

The marginal zone is a complex environment in which cell trafficking and interaction with blood-borne foreign antigens takes place (32,33). Within the spleen of uninfected calves, iDCs were present as a network-like distribution covering the marginal zone and red pulp whereas NK cells appeared to accumulate only within the marginal zone. Crosstalk between NK cells and DCs is crucial to the innate immune response (34–37), and in mice involves secretion of IL-15 by DCs (38), which primes NK cells to produce IFN-γ, which in turn increases DC activity (39). Similarly, we have previously shown up-regulation of IL-15 mRNA in the spleen of calves early after infection with B. bovis (15) and in vitro crosstalk that requires NK cell-iDC contact (13). Given the initial co-population of the marginal zone with CD335+ and CD13+ cells, it is possible that the first 4–6 days of B. bovis infection in calves involves critical NK/iDC crosstalk and activation. The unambiguous redistribution of iDCs to the junction between marginal zones, follicles and PALS is consistent with the immunological importance placed on NK/DC crosstalk. As such, early activation with narrow redistribution to these junctions by 7 dpi may optimally position iDCs to encounter B. bovis merozoites and infected erythrocytes as they enter the parenchyma of the spleen.

Furthermore, immunoreactivity for merozoite surface antigen-1 (MSA-1) – an antigen important to infectivity (40) – was first observed at 13–14 dpi and only in close proximity to these junctions with the marginal zone. It was noted that the punctate immunostaining for MSA-1 was accompanied by sparse CD13 staining and always in juxtaposition to redistributed iDCs. We have previously shown that maturation of splenic iDC from naïve calves in vitro results in the loss of CD13 expression and gain in capacity to present antigen (12,41). Thus, similar to the P. chabaudi model in mice (23), these results support the hypothesis that iDC mature during processing of the parasite and migrate as antigen-presenting cells to lymphocyte-rich domains.

The spleen-dependent innate response of naïve calves to infection with B. bovis is also characterized by early IL-12 production with subsequent IL-10 modulation (6), the major sources of which in cattle are iDCs and monocytes/macrophages, respectively (8,14,42). We have also shown that monocytes/macrophages of cattle can produce NO with direct babesiacidal activity (14,27,43). It was interesting to note that following haemoparasitic infection, intense acute hyperplasia of monocytes/macrophages is restricted to the red pulp of both mice (23) and calves (present study). Thus, in addition to regulatory function through cytokine production, our collective findings are consistent with monocytes/macrophages acting as effector cells in close juxtaposition with infected erythrocytes as they enter the splenic sinuses.

Regarding the distribution of small leucocytes, dual-labelling experiments demonstrated acute progressive accumulation of numerous CD3+ CD4 cells and TcR1+ WC1 cells within the red pulp. Thus, it is likely that at least a portion of these accumulated lymphocytes were WC1γδ T cells. The role of these cells is still not clear but as bovine WC1γδ T cells express CD2 and CD8, can produce IFN-γ in response to cytokine stimulation, and are found in largest proportion in the spleen and intestine (15,16,44,45), it is intriguing to consider the possibility that cells with this phenotype might be the bovine functional equivalent of NKT cells (46–48). If so, then the observed accumulation of these cells in the red pulp of naïve calves infected with B. bovis is consistent with their expected role in the transition from innate to acquired immunity.

Our results are in agreement with previous reports (49,50) that demonstrate relatively small accumulations of WC1+γδ T cells within the splenic marginal zones of uninfected calves. The splenic decrease in WC1+γδ T cells during the acute response of calves to B. bovis infection may indicate their activation within the marginal zone is followed by redistribution to effector sites outside of the spleen. Indeed, several reports indicate WC1+γδ T cells are most numerous and reactive within the blood of young calves (45,49,51–53).

The observations made in this study were initiated by events occurring within the first few hours and days of inoculation as Babesial antigen entered the spleen via the circulation. The marginal sinus is an important route by which blood-borne particles and nonlymphoid cells first enter the spleen (17). Our observations in naïve calves are consistent with recent intravital imaging studies in rodent models (54–56) which document the early interactions and trafficking of several marginal zone cell types and the importance of these events to the splenic immune responses. Our results, however, do not exclude the potential relevance of initial antigen interaction with other zonal cell populations (e.g., PALS lymphocytes) to the acute response of naïve calves to B. bovis.

In summary, the results of this immunohistological investigation have demonstrated dynamic change in the distribution of several cell types thought to be important to the acute spleen-dependent response of calves to B. bovis infection. In particular, unambiguous redistribution of iDC to regions where parasites first enter the spleen and evidence for further maturation and antigen processing seem noteworthy. The remarkable similarity of these acute splenic responses of calves to B. bovis and those reported in mice responding to P. chabaudi indicates that redistribution of splenic cells is central to the acute immune response of naïve animals to haemoparasite infection.

Acknowledgements

This work was supported by USDA-ARS-CWU-5348-32000-010-00D. The authors especially recognize the expert technical contributions of Sallie Bayly who assisted in the splenic transposition surgeries, Tom Truscott for immunohistochemical advice, and Thomas Wilkinson and Rob Houston for MRI techniques. We thank Duane Chandler and Amy Hetrick for their contributions to the care and use of the animals. The authors thank Dr William C. Davis for his critical review of the manuscript. Mention of trade names or commercial products or enterprises in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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