Parasite Immunology, 2011, 33, 34–44 DOI: 10.1111/j.1365-3024.2010.01249.x
Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study
D. A. SCHNEIDER,1 H. YAN,2 R. G. BASTOS,2 W. C. JOHNSON,1 P. R. GAVIN,3 A. J. ALLEN,3 G. M. BARRINGTON,3 L. M. HERRMANN-HOESING,1 D. P. KNOWLES1 & W. L. GOFF1*
1Animal Disease Research Unit, USDA-ARS, Pullman, WA, USA, 2Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA, 3Department of Veterinary Clinical Medicine and Surgery, Washington State University, Pullman, WA, USA
SUMMARY INTRODUCTION The spleen is a critical organ in defence against haemopara- Babesiosis is a tick-borne disease affecting cattle in much sitic diseases like babesiosis. Many in vitro and ex vivo stud- of the world, with Babesia divergens, B. bigemina and ies have identified splenic cells working in concert to activate B. bovis the economically important species. Babesia bovis mechanisms required for successful resolution of infection. is the most virulent, often causing death in susceptible ani- The techniques used in those studies, however, remove cells mals because of the development of anaemia, cerebral vas- from the anatomical context in which cell interaction and cular congestion and pulmonary and renal failure (1). The trafficking take place. In this study, an immunohistological virulent nature of the disease is attributed in part to the approach was used to monitor the splenic distribution of sequestration of parasitized erythrocytes to capillary endo- defined cells during the acute response of na ve calves to thelium, but overproduction of inflammatory cytokines Babesia bovis infection. Splenomegaly was characterized by has also been suggested (2–4). Young calves are relatively disproportionate hyperplasia of large versus small leucocytes resistant to clinical infection (5) and demonstrate a strong and altered distribution of several cell types thought to be innate immunity composed of a type-1 inflammatory important in mounting an effective immune response. In par- response (6). In comparison with adult cattle, we have pre- ticular, the results suggest that the initial crosstalk between viously demonstrated that the immune response of calves NK cells and immature dendritic cells occurs within the mar- involves early IL-12 expression with consequent IFN-c ginal zone and that immature dendritic cells are first redi- production, a nitric oxide burst and modulation by IL-10 rected to encounter pathogens as they enter the spleen and (6–9). This age-related immunity is dependent upon cellu- then mature as they process antigen and migrate to T-cell- lar events within the spleen as splenectomy of calves ren- rich areas. The results of this study are remarkably similar ders them equally susceptible (5,10). to those observed in a mouse model of malarial infection, Our studies have utilized a technique to marsupialize suggesting these dynamic events may be central to the acute the spleen of calves (11) so that cells could be acquired for response of na ve animals to haemoparasitic infection. ex vivo analysis (microplate assays and flow cytometry) (12–16). Such analyses have proven valuable in determin- Keywords Babesia bovis, dendritic cells, immunohistochemis- ing the function of various splenic cell phenotypes but try, lymphocytes, macrophages, NK cells, spleen, cd T cells lack the ability to place these cell populations within their anatomical context which include the marginal zone, red Correspondence: David A. Schneider, Animal Disease Research and white pulp (17). Amongst many factors that comprise Unit, USDA-ARS, 3003 ADBF, Washington State University, an effective immune response to haemoparasitic infection, Pullman, Washington 99164-6630 USA trafficking and interaction of cells within such domains (e-mail: [email protected]). Disclosures: None. are central (18). Received: 19 March 2010 Intravital imaging techniques have been used to dynami- Accepted for publication: 06 July 2010 cally study such factors within superficial lymphoid organs *Retired Research Immunologist, Animal Disease Research Unit (19,20) and, to a limited extent, also within deeper struc- USDA-ARS, 4181 W. Upriver Drive, Coeur d’Alene, ID 83814, tures including the spleen of mice (21). But current tech- USA. niques are not well suited to study the spleen of large
34 2010 Blackwell Publishing Ltd Volume 33, Number 1, January 2011 Spleen cell dynamics during babesial infection mammals because of the limits on depth resolution (22). sequence. This sequence resulted in a hyperintense spleen An approach readily applied to the spleen of large mam- on a low intense background. The volume was calculated mals is the serial analysis of the distribution of pheno- by tracing the outline of the spleen for the area on each typed cells in tissue sections. Similar to a recent study on slice and multiplying by the number of slices plus gap the acute immune response of na ve mice to haemoparasit- thickness (3D-DOCTOR; Able Software Corporation, ic infection (23), we have applied this technique to the Lexington, MA, USA). Each calf’s spleen volume was cal- spleen of na ve calves infected with Babesia bovis. The culated on the day prior to infection and then at 11 or results document acute change in the distribution of sev- 12 dpi, 2 calves each. eral cells thought to be important to the spleen-dependent Immediately following each MRI procedure, a 1 cm3 response of na ve calves to B. bovis and serve to under- biopsy of marsupialized spleen was removed under local score common themes in the acute response to haemopar- lidocaine anaesthesia for determining differential cell asitic infections. In addition, this is the first documented counts. Each biopsy was immediately processed into a sin- use of magnetic resonance imagery to measure spleen vol- gle cell suspension using a tissue grinder (Tenbroek; Bellco ume in calves. Glass, Inc., NJ, USA), suspended in 50 mL of PBS and enumerated for differential cell counts by standard meth- ods used for whole blood (28). MATERIALS AND METHODS
Animals and experimental B. bovis infection Immunohistochemistry (IHC) Twelve Holstein–Friesian steer calves were obtained at Six inoculated calves were euthanized by captive bolt and 8 weeks of age, vaccinated against pathogenic Clostridium jugular exsanguination for collection of spleen tissue: one species, castrated and dehorned. All animals were cELISA calf each on dpi 7, 8, 9 (fever day 1) and 14 (fever day 5), seronegative for Anaplasma marginale (VMRD, Pullman and two calves at 13 dpi (fever days 4 and 5). In this way, WA, USA) and B. bovis and B. bigemina (24–26). The care the spleens from three calves each were examined from and use of these calves were approved by the Institutional two periods: a period just prior to, or including, the initia- Animal Care and Use Committee at Washington State tion of fever (7, 8 and 9 dpi) and a period several days University (Pullman, WA, USA). At 12 weeks of age, all after fever initiation (13 and 14 dpi). Spleen tissue from calves underwent a surgical procedure to marsupialize the two uninfected calves was similarly collected. Multiple spleen (11). When necessary, spleen cell aspirates were 15 · 15 · 5 mm sections of spleen were collected from obtained under local lidocaine anaesthesia into 60cc syrin- each calf immediately posteuthanasia. Each section was ges containing ACD and prepared for in vitro studies as placed into a cryostat mould containing Tissue-Tek previously described (14,27). O.C.T. Compound (Sakura Fineteck USA, Inc., Tor- Ten of the twelve calves were inoculated intravenously rance, CA, USA), snap frozen by floating on liquid nitro- 5 with 1 · 10 erythrocytes infected with the T2Bo virulent gen, and stored at )80 C. Cryostat sections (15 lm) were isolate of B. bovis (7). The acute response to infection was mounted on standard SuperFrost Plus slides (Electron studied 7–14 days postinfection (dpi). As described below, Microscopy Services, Hatfield, PA, USA), fixed in 95% repeated measures of spleen volume and cell content were EtOH for 10 min and allowed to air dry overnight at made in four inoculated calves whereas change in regional room temperature. Formalin-fixed, paraffin-embedded distribution of phenotyped cells was determined by samples of spleen were also collected from each calf and sequential euthanasia of six inoculated calves in compari- routinely stained in haematoxylin and eosin (H&E). son with two un-inoculated calves. Immunolabelling was carried out at room temperature in a humidified chamber. A Super PAP Pen HT (Research Products International Corp., Mt. Prospect, IL, Determination of spleen volumes and differential cell USA) was used to create a hydrophobic margin to retain counts fluid reagents on slides. Thin sections on slides were pre- Magnetic resonance imagery was performed with a 1Æ0 blocked for 1 h using 10% goat serum ⁄ 0Æ1% Triton-X Tesla machine (Philips Intera, Andover, MA, USA). 100 ⁄ PBS. After three 5-min washes in PBS, thin sections Sequences were acquired in a dorsal plane. The area were exposed (2–4 h) to primary antibody (Table 1) imaged was from the spine to the ventral abdominal wall. diluted in 10% goat serum ⁄ PBS. Unbound primary anti- A 40 cm field-of-view ensured that the entire spleen could body was removed with three 5-min washes in PBS and be visualized. One-centimetre-thick slices with a 2 mm gap then exposed (2 h) to fluorophore-conjugated secondary were acquired using a short tau inversion recovery (STIR) antibody, all diluted 1 : 200 in 10% goat serum ⁄ 0Æ1%