Production and utilization of interleukin-15 in malignant catarrhal fever.

Malignant catarrhal fever (MCF) is an often fatal lymphoproliferative disease of ungulates caused by either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). The pathogenesis of MCF is poorly understood, but appears to involve an auto-destructive pathology whereby cytotoxic lymphocytes destroy areas of a variety of tissues. The cytokine interleukin-15 (IL-15) is involved in the development and maintenance of cytotoxic lymphocytes and may therefore have a role in the pathogenesis of MCF. Virus-infected large granular lymphocytes (LGLs) were obtained from the tissues of rabbits infected with AlHV-1 or OvHV-2. These cells exhibited a similar proliferative response to IL-15 and to IL-2 in culture, but their content of the activated cytotoxic enzyme (BLT-esterase) was maintained at higher levels in the presence of IL-15 compared with IL-2. The LGLs did not express IL-15 mRNA or produce IL-15 protein. By contrast, there was abundant expression of IL-15 mRNA and protein in affected tissues. IL-15 production was associated with necrotic lesions of the mesenteric lymph node and appendix of OvHV-2-infected rabbits, but was not found in the same tissues of rabbits infected with AlHV-1 in which there were no necrotic lesions. The cellular source of the IL-15 was predominantly lymphoid cells that did not express B cell or monocyte-macrophage markers. Only a few IL-15+ cells (<10%) co-localized with pan-T cells or CD8+ T cells. The abundance of IL-15 in tissue with lesions of MCF suggests that this cytokine may have a role in the pathogenesis of MCF.

Immunohistochemical study of experimental malignant catarrhal fever in rabbits.

Malignant catarrhal fever (MCF) is an often-fatal lymphoproliferative disease of a variety of ungulates that occurs worldwide. It is caused by either of the highly related but distinct gammaherpesviruses alcelaphine herpesvirus-1 (AlHV-1, wildebeest reservoir) or ovine herpesvirus-2 (OvHV-2, sheep reservoir). MCF in rabbits is an excellent model as it closely resembles the disease in susceptible ungulates that include cattle, deer and bison. In this study, newly available and previously characterized monoclonal antibodies specific for rabbit leucocyte differentiation molecules were used to perform a detailed immunohistochemical examination of both AlHV-1 MCF and OvHV-2 MCF in rabbits. Differences in the MCF caused by the two viruses included: less tissue necrosis and more lymphoid cell accumulations in AlHV-1 MCF compared with OvHV-2 MCF, and in particular marked tissue necrosis in the mesenteric lymph node, appendix and liver of OvHV-2-infected animals when compared with either other tissues in OvHV-2 MCF or AlHV-1 MCF lesions in any tissue. In both AlHV-1 MCF and OvHV-2 MCF, lymphoid cell accumulations in lymphoid and non-lymphoid tissues consisted mainly of T-cells with a corresponding absence of B-cells. CD8(+) T-cells accounted for a proportion of these in the non-lymphoid tissues, but there was evidence for the accumulation of an unidentified T-cell subset/subsets as well. This study extends our understanding of the mechanisms of immuno-pathogenesis of MCF.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

A field vaccine trial in Tanzania demonstrates partial protection against malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle that, in East Africa, results from transmission of the causative virus, alcelaphine herpesvirus 1 (AlHV-1), from wildebeest. A vaccine field trial involving an attenuated AlHV-1 virus vaccine was performed over two wildebeest calving seasons on the Simanjiro Plain of northern Tanzania. Each of the two phases of the field trial consisted of groups of 50 vaccinated and unvaccinated cattle, which were subsequently exposed to AlHV-1 challenge by herding toward wildebeest. Vaccination resulted in the induction of virus-specific and virus-neutralizing antibodies. Some cattle in the unvaccinated groups also developed virus-specific antibody responses but only after the start of the challenge phase of the trial. PCR of DNA from blood samples detected AlHV-1 infection in both groups of cattle but the frequency of infection was significantly lower in the vaccinated groups. Some infected animals showed clinical signs suggestive of MCF but few animals went on to develop fatal MCF, with similar numbers in vaccinated and unvaccinated groups. This study demonstrated a baseline level of MCF-seropositivity among cattle in northern Tanzania of 1% and showed that AlHV-1 virus-neutralizing antibodies could be induced in Tanzanian zebu shorthorn cross cattle by our attenuated vaccine, a correlate of protection in previous experimental trials. The vaccine reduced infection rates by 56% in cattle exposed to wildebeest but protection from fatal MCF could not be determined due to the low number of fatal cases.

Cloning and expression of a cDNA encoding ovine granulocyte-macrophage colony-stimulating factor.

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been cloned using the polymerase chain reaction. The nucleotide sequence is approx. 93% identical to the published bovine GM-CSF-encoding sequence, 84% to the human sequence and 73% to the murine sequence. The deduced amino acid sequence of the ovine GM-CSF protein was found to be 80% identical to both the human and bovine proteins and 57% to the murine protein. Transient expression of recombinant ovine GM-CSF in COS-1 cells was obtained and its biological activity investigated in a bone-marrow colony-forming assay. Ovine GM-CSF was found to promote the formation of granulocyte-macrophage colonies as well as eosinophil, neutrophil and monocyte/macrophage colonies, an activity characteristic of GM-CSF in other species. Recombinant human GM-CSF was found to have no proliferative effect on ovine bone-marrow cells.

The immune and inflammatory response to orf virus.

Orf virus is a zoonotic, epitheliotropic DNA parapox virus that principally infects sheep and goats. The fact that the virus can repeatedly reinfect sheep has provoked an interest in the underlying cellular, virological and molecular mechanisms for its apparent escape from the host protective immune response. The local immune and inflammatory response in skin and the cell phenotype and cytokine response in lymph analysed around a single lymph node are characteristic of an anti-viral response. An unusual feature is the dense accumulation of MHC Class II+ dendritic cells in the skin lesion. The function of these cells is not known. Orf virus virulence genes and activities have been identified that may interfere with the development of the host protective immune and inflammatory response.

Poxvirus interference with the host cytokine response.

Poxviruses have evolved successfully to survive and replicate in a variety of species in the presence of an active host immune and inflammatory response. They manage this, at least in part, by the acquisition and modification of host immune and inflammatory response modulating genes. A proportion of these virulence genes encode homologues of host cytokines and cytokine receptors. These include soluble interferon, IL-1 beta, and TNF-alpha receptor homologues, that block the host cytokines. Other virulence gene products interfere with interferon signalling within cells and prevent the cleavage of biologically active IL-1 beta from its precursor protein. The parapoxvirus orf virus encodes a homologue of ovine IL-10 and a novel GM-CSF-binding protein. By studying poxvirus virulence proteins that interfere with host cytokine effector responses, important and novel aspects of the host immune and inflammatory response to infection have been revealed.

Immunomodulation by virulence proteins of the parapoxvirus orf virus.

Three orf virus putative virulence proteins are described that exhibit immunomodulatory functions. The OVIFNR gene at the left terminus of the viral genome encodes an interferon resistance protein with homology to the E3L gene of vaccinia virus. OVIFNR functions by preventing a dsRNA-dependent kinase from inhibiting virus and cell protein synthesis as part of the interferon-induced anti-viral state within infected cells. The orf virus orthologue of the ovine interleukin-10 (vIL-10) gene is located at the right terminus of the viral genome. Both vIL-10 and host (ovine) IL-10 function in vitro as inhibitors of pro-inflammatory cytokine production by keratinocytes and macrophages, and both inhibit IFN-gamma production from activated peripheral blood lymphocytes. Both the orf virus vIL-10 and ovine IL-10 stimulate mast cell and thymocyte proliferation. In this respect the orf virus IL-10 differs from Epstein Barr virus IL-10 which does not exhibit cell proliferative activity. Finally, the orf virus GM-CSF inhibitory factor gene (GIF) at the right terminus of the viral genome encodes an inhibitor of GM-CSF that also binds IL-2. Together, these viral proteins are capable of inhibiting key components of the ovine anti-virus immune and inflammatory response.

Reactivity of the workshop monoclonal antibodies with ovine bone marrow cells and bone marrow-derived monocyte/macrophage and mast cell lines.

The workshop monoclonal antibodies were tested by flow cytometry for reactivity against: (1) ovine bone marrow cells, (2) cultured bone marrow-derived monocyte/macrophage cell lines and (3) cultured bone marrow-derived mast cell lines. Both single and two-colour immunofluorescence tests were performed. The results of these analyses are presented and discussed.

Identification of ovine interferons: differential activities derived from fibroblast and lymphoid cells.

A bioassay has been developed to detect interferon (IFN) activity in supernatant fluids of cultured sheep cells by measuring their ability to inhibit the cytopathic effect of Semliki Forest Virus on an immortalised ovine fibroblastic cell line. IFN produced by spleen or lymph node cells stimulated with mitogen was labile at pH 2 as was a similar activity produced by CD4 lymphocytes stimulated with mitogen or specific antigen. In contrast, the IFN produced by fibroblasts stimulated with synthetic double-stranded RNA was stable at that pH. These results demonstrate the existence of an ovine IFN with properties similar to those described for IFN-gamma in mouse and man.

Purification and adhesion receptor phenotype of ovine bone marrow-derived haemopoietic colony-forming cells.

Ovine haemopoietic progenitor cells that form colonies (CFC) in soft agar cultures were compared to more mature bone marrow cells for their level of expression of the adhesion receptor molecules ovine (ov) CD44, ov CD11a (LFA-1) and ov CD58 (LFA-3) as well as the 175-antigen using specific monoclonal antibodies. Ov CD44, ov CD11a and ov CD58 were expressed on all CFC of the myeloid (non-erythroid) series, whereas ov CD44 and ov CD11a expression was very low or absent from a small number of blast and erythroid series CFC. Within the mature non-erythroid population of myeloid cells, neutrophils retained a low level of expression of ov CD11a. Most CFC representing all lineages strongly expressed the ov CD44 antigen. In contrast, the majority of CFC lacked the 175-antigen, as did bone marrow lymphocytes, basophils and mast cells. This property of CFC was exploited in a negative selection technique using panning and immunomagnetic beads to select CFC from other bone marrow cells with a 116-125-fold enrichment, 12-14% purity and 29-40% yield. These results demonstrate that ovine CFC express some of the molecules necessary to allow adhesion to haemapoietic stromal cells and vascular endothelium in the tissues. Future studies will concentrate on the function of the adhesion receptor molecules in medullary and extra-medullary haemopoiesis and inflammatory cell development in sheep.

Ovine haemopoiesis: the development of bone marrow-derived colony-forming cells in vitro in the presence of factors derived from lymphoid cells and helper T-cells.

Techniques for the development of ovine bone marrow-derived haemopoietic progenitor cells and in situ identification of colony morphology are described. Both mitogen stimulated lymphoid cells and antigen stimulated helper T-cells generated potent colony-stimulating activity in conditioned medium. Monocyte/macrophage, neutrophil, eosinophil, basophil/mast cell, neutrophil/monocyte and mixed phenotype colonies developed in stimulated bone marrow cultures in a conditioned medium dose-dependent manner. Neutrophil, monocyte/macrophage and eosinophil colonies were detected in greater numbers than the other types, with mixed colonies representing only around 1% of the total. Eosinophil colonies were particularly abundant when compared to published reports of the numbers obtained with similar cultures of 'normal' mouse or human bone marrow cells. This culture technique will allow a detailed analysis of both ovine colony-stimulating factors and of the distribution of haemopoietic progenitor cells in vivo.

Phenotypic analysis of ovine bone marrow cells using monoclonal antibodies and lectins.

Three major subpopulations of ovine bone marrow cells were identified by flow cytometry on the basis of differences in forward (FSC) versus right angle (SSC) light scattering properties and binding of monoclonal antibodies. Region 1 (low FSC, low SSC) contained erythroid series cells and some small lymphocytes. Region 2 (high FSC, low SSC) contained monocytes, myeloid blast cells, medium-large lymphocytes and virtually all of the progenitor/stem cells capable of forming colonies in soft agar cultures. Region 3 (high SSC) contained granulocytes at various stages of development, predominantly (greater than 90%) neutrophils and eosinophils. Using this technique it was possible to identify several cell-surface antigens on bone marrow cells of different lineages using specific monoclonal antibodies and lectins. Amongst the haemopoietic stem/progenitor cell population, immature colony-forming cells were leucocyte common antigen (LCA) negative while more mature colony-forming cells expressed LCA. A proportion of progenitor cells were MHC class I positive. This analysis is an important first step in characterising ovine haemopoietic cells for future studies on: the development of inflammatory cells, the migration of stem/progenitor cells in vivo and the tropism of pathogens.

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma.

An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF-alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.

Kinetics of ovine interferon-gamma production: detection of mRNA and characterisation of biological activity.

The kinetics of interferon-gamma (IFN-gamma) production were studied in sheep mesenteric lymph node (MLN) cells at the molecular level using an ovine IFN-gamma cDNA probe and by bioassay which was verified by blocking antiviral activity with a monoclonal antibody (Mab) against recombinant bovine IFN-gamma IFN-gamma mRNA appeared in MLN cells within 4 h of stimulation with phorbol ester and Concanavalin A and was not detectable by 72 h after stimulation. Biologically active IFN-gamma appeared in the culture supernatants 8 h after stimulation and was still present 96 h later when de novo synthesis had terminated. Acid dialysis and Mab neutralisation demonstrated conclusively that native ovine IFN-gamma is a pH 2 labile cytokine.

Characterisation of ovine mast cells derived from in vitro culture of haemopoietic tissue.

Ovine mast cells generated in vitro from bone marrow (BMMC) were compared with mucosal mast cells (MMC) isolated from parasitised abomasum. Ultrastructurally, the granules of BMMC were partially developed and immature. Both cells types contained beta-hexosaminidase, arylsulfatase, histamine, dopamine and sheep mast cell proteinase (SMCP). Greater amounts of beta-hexosaminidase, but less SMCP, histamine and arylsulfatase were present in BMMC. Stimulation with calcium ionophore A23187 caused the secretion of granule constituents and generation of leukotriene C4 by BMMC in a dose-dependent manner. An additional [3H]diisopropylfluorophosphate-binding 31,500 mol. wt. serine esterase, antigenically related to SMCP (27,000 mol. wt.) was present in cultures of BMMC but was not detected in isolated MMC. Both enzymes were detected in BMMC by Day 7 of culture and were secreted concomitantly following stimulation of BMMC with ionophore.

In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep.

In vivo lymphocyte subset depletion offers a unique opportunity to study the roles of different cellular components of the immune system of sheep during infection with orf virus. Lambs were depleted of specific lymphocyte subsets by the intravenous administration of monoclonal antibodies against ovine lymphocyte surface markers and then challenged with orf virus. The skin lesions that developed were scored visually as to their severity. Blood samples were collected to monitor the lymphocyte depletions and to measure orf-virus-specific antibody levels. Skin biopsies were collected from the lesion site and studied to determine the course of the infection and the presence of various cell types and orf virus. All the sheep developed orf virus lesions after infection. All three of the CD4-depleted lambs were unable to clear virus from their skin and did not have an antibody response to the virus. Virus was also detected in the skin of one each of the three CD8-depleted, WC1-depleted and control sheep on the final day of the trial. CD8(+) lymphocytes did not appear to be essential for viral clearance later in the infection. Depletion of the majority of gammadelta(+) T-cells did not affect the outcome of orf virus infection. In sheep with high orf-virus-specific antibody titres at the time of infection, orf lesions healed faster than lesions in sheep with low antibody levels, and this occurred regardless of the lymphocyte depletion status of the animals. This study suggests that the presence of CD4(+) T-cells and orf-virus-specific antibodies are important for the control of viral replication in the skin of infected sheep.

Eosinophil-specific biological activity of recombinant ovine interleukin-5.

Recombinant ovine interleukin-5 (rovIL-5) expressed from Chinese hamster ovary (CHO) cells was tested for cell-specific bioreactivity, in vitro, in a soft agar clonogenic assay and in an enzyme-based microassay for eosinophil potentiating activity (EPA). In soft agar assays, colony and cluster formation from sheep bone marrow cells (SBMC) incubated with rovIL-5 was significantly enhanced compared with SBMC incubated with control supernatants from mock-transfected CHO cells. Colony analysis at 14 days demonstrated that for three separate rovIL-5 preparations 45%, 61% and 66% of colonies were eosinophilic, as were 55%-71% of clusters. In contrast, no eosinophil colonies were detected in parallel control cultures. RovIL-5 was also shown to possess potent and dose-responsive EPA, on the basis of eosinophil peroxidase (EPO) and arylsulphatase (EAS) assay in 7 day SBMC cultures. This activity was inhibited in a dose-responsive manner by TRFK-5, a rat anti-murine IL-5 monoclonal antibody (MAb) previously shown to have cross-reactivity in the ovine EPA assay. The results demonstrate that rovIL-5 exhibited eosinophil-specific properties similar to those of IL-5 derived from other mammalian species.

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

Development of a sandwich ELISA for ovine granulocyte/macrophage colony-stimulating factor.

Recombinant ovine granulocyte/macrophage colony-stimulating factor (rOv GM-CSF) has been expressed in Chinese hamster ovary cells. A stable, cloned line of these cells has been established which secretes high levels (40 mu g ml(-1)) of rOv GM-CSF. Three murine monoclonal antibodies (mAbs) were produced which reacted with rOv GM-CSF on Western blots. These mAbs also neutralised the activity of both recombinant and native Ov GM-CSF in a bone marrow haemopoietic progenitor cell assay. Two of the mAbs, which recognise mutually exclusive epitopes, were selected for the development of a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GM-CSF in biological samples of ovine origin.