Parasite Immunology, 2003, 25, 185 –188
The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen
ORIGINAL Immunity in calves ARTICLE infected Blackwell Publishing Ltd. with Babesia bovis
W. L. GOFF1, W. C. JOHNSON1, R. H. HORN2, G. M. BARRINGTON3 & D. P. KNOWLES1 1
Animal Disease Research Unit, USDA-ARS, Pullman, WA 99164-6630, USA and 2Moscow, ID 83844-2201, USA and 3Department of Clinical Medicine and Surgery, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA
SUMMARY
RESEARCH NOTE
The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-γ. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-γ and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.
Innate immunity in naïve animals is important, both to the survival of the host and to the type and magnitude of the acquired immune response. Young calves possess a strong innate immunity to B. bovis that is spleen-dependent and involves the monocyte/macrophage as both regulatory and effector cell (1,2). The activation of innate immunity after intravenous inoculation of B. bovis-infected blood stabilate has been characterized as a type-1 response involving the early induction of IL-12 and IFN-γ (2). NK cells have been identified as a source of IFN-γ early in the response of naïve animals to intracellular pathogens, and are activated by IL-12, IL-15 and IL-18 (3–7). In this report, we describe the innate response in calves to Boophilus microplus tick-transmitted B. bovis, and demonstrate the proliferation of an NK-like CD8+ cell population in the spleen. Three Holstein–Friesian steer calves were obtained at 8 weeks of age and were maintained according to the American Association for Laboratory Animal Care procedures with an acceptable bovine ration and with water and mineral block provided ad libitum. Each animal underwent a surgical procedure at 12 weeks of age to marsupialize the spleen to facilitate aspiration of spleen cells (8). At 16 weeks of age they were placed in stanchions approved for experimental tick infestation, and 1 g (approximately 20 000) of Boophilus microplus larvae from 25 pooled females infected (haemolymph-positive) with the virulent T2-Bo isolate of B. bovis (9) were applied under a feeding patch as previously described (10). The larvae were allowed to attach and feed for 4 days before being removed by spraying with a 0·75% solution of the acaracide, chlorpyrifos (Dursban®, Dow AgroSciences). Blood and spleen aspirates were collected from each calf before and then daily beginning at day 4 and 7 post-tick application (PTA), respectively. Splenic aspirates were collected and processed for cytokine mRNA expression
Keywords Babesia bovis, type-1 immunity, innate immunity, NK cells
Correspondence: W.L. Goff, Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, 3003 Animal Disease Biotechnology Facility, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6630, USA (e-mail:
[email protected]). Received: 11 February 2003 Accepted for publication: 12 June 2003 © 2003 Blackwell Publishing Ltd
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Figure 1 Activation of the innate immune response during an initial tick-transmitted B. bovis infection in young calves. (a) Cytokine mRNA expression from spleen cells collected on day 8 post tick application where the expression levels are indicated as a fold increase over the expression level at day zero. (b) In vitro nitric oxide production from mononuclear phagocytes within the total spleen cell preparation collected before and during infection in response to medium (), purified B. bovis merozoites (), exogenous TNF-α (), and exogenous IFN-γ plus TNF-α (). (c) Plasma IFN-γ levels from day 4 through day 15 post tick application.
by reverse transcription polymerase chain reaction (RTPCR) and stimulating spleen cell cultures for nitric oxide production (2), and flow cytometry (11). Serum antibody was detected using immunofluorescence and a competitive ELISA (12,13), and plasma IFN-γ levels were determined by ELISA (14). Calves at this young age possess a strong innate immunity and as expected, the clinical disease was mild in each animal. The onset of fever was between 7 and 10 days, and duration between 4 and 8 days. The individual percentage decrease in
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haematocrit from pre-exposure levels was 46, 54, and 62, but was of short duration. All animals survived the acute infection without drug intervention and all demonstrated crisis forms of B. bovis in stained peripheral blood films (9). The response of each calf included an increase in proinflammatory cytokine mRNA expression in the spleen. On day 8 PTA, IL-12, IL-15, and IFN-γ message was from three to six times the basal expression levels of day 0 (Figure 1a). IL-10 mRNA expression on day 8 PTA was also nearly twice the level as day 0, and increased even more over the next few days (data not shown). IL-18 and TNF-α message appeared to be constitutively expressed with a slight increase in IL-18 expression on day 8 PTA and a slight decrease in TNF-α expression from days 8–12 PTA (data not shown). Each cytokine message returned to pre-infection levels at about 14 days PTA. There was no evidence of IL-4 expression in the spleen at any time. The importance of IFN-γ in the type-1 response to babesial infection is becoming increasingly clear. Nitric oxide has been shown to be babesiacidal (15), and is produced by bovine monocytes exposed to B. bovis merozoites in the presence of IFN-γ and TNF-α (16,17). IFN-γ and TNF-α act as co-stimulatory signals for inducible nitric oxide synthase in bovine monocytes and therefore, nitric oxide produced in response to merozoites alone is not only a measure of cellular activation but also an indicator of the presence of endogenous IFN-γ and TNF-α (17). In addition, nitric oxide induction in response to in vitro stimulation with TNF-α alone is evidence for the endogenous presence of IFN-γ and vice versa (17). Figure 1(b,c) provides evidence that splenic monocytes/macrophages from each calf were activated in response to infection, that IFN-γ and TNF-α were influential in the spleen, and that a significant level of IFN-γ was produced systemically. CD8+ lymphocytes have a role in type-1 innate immunity in bovines, and NK cells appear to be important. NK cells that are defined as CD8+, CD3–, and CD2+ have been identified in young calves (18). Both NK cells and a CD8+, WC1– γδT-cell subpopulation that is prominent in the bovine spleen (11,19) have been documented as a source of IFN-γ (18,20). However, Figure 2 shows that NK-like cells rather than CD8+ γδT-cells were the predominant phenotype in the proliferating population, increasing from about 15% of blasted cells before infection to about 55% at day 10 PTA. The CD8 molecule can exist as either a αβ heterodimer or a αα homodimer (21). Since the MoAb used in our study binds the α chain, it was not possible to determine the exact CD8 phenotype of these NK-like cells. Both immunohistology and flow cytometry will be used to characterize further these NK-like cells and determine whether they are an important source of IFN-γ during the response to B. bovis infection. © 2003 Blackwell Publishing Ltd, Parasite Immunology, 25, 185–188
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Immunity in calves infected with Babesia bovis
expression pattern in the spleen of our calves supports a similar situation in response to tick-transmitted B. bovis infection. Interestingly, IL-10 does not appear capable of inhibiting IFN-γ production from human NK cells when co-stimulated with IL-12 and IL-15 (25). Although we have demonstrated the ability of IL-10 to down-regulate bovine IL-12 and IFN-γ mRNA expression and cytokine production (26), we have yet to investigate the role of IL-10 with bovine NK-like cells. Our efforts are now focused on further characterization of splenic NK-like cells and the role of IL-10 in modulating both cytokine production and effector cell receptor expression on monocyte/macrophages and NK cells. This work is published in memoriam to David Horn, a special person who provided technical support during this study. We also thank Pete Steiner, Duane Chandler and Robert Finch for excellent animal care, Paul Lacy for technical assistance and Shirley Sandoval for assistance in surgery. This study was supported by USDA-ARS-ADRU-CRIS no. 5348-32000-010.
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Figure 2 Phenotype of CD8+ spleen cells in a representative calf before (a) and at day 10 (b) post tick application; 5 × 105 spleen cells were stained with monoclonal antibodies BAQ-111 A for CD8+ (27), GB21A for TcR1+ (28), BAQ95A for CD2+ (29) and MM1A for CD3+ (30). Cells were gated for size using forward and side scattering properties before examining the phenotypes of proliferating blast cells (R2).
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