Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus.

RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.

Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes.

To determine whether systemic immunization with plasmid DNA and virus vector against visna/maedi virus (VMV) would induce protective immune responses, sheep were immunized with VMV gag and/or env sequences using particle-mediated epidermal bombardment and injection of recombinant modified vaccinia Ankara. The results showed that immunization induced both humoral and cell-mediated responses prior to and after virus challenge. The vaccination protocol did not prevent infection, but immunization with the gag gene or a combination of gag and env genes resulted in significantly reduced provirus loads in blood and mediastinal lymph node, respectively. Provirus loads in lung and draining lymph node were unaffected, but p25 expression was undetectable in lungs of animals immunized with a combination of gag and env genes. Analysis of target tissues for lesions at post-mortem showed that immunization with the env gene caused a significant increase in lesion score, while the gag gene or a combination of gag and env genes had no effect. Inclusion of the ovine interferon-gamma gene in the initial priming mixture had minimal effect on immune responses, provirus load, or lesion development, although it resulted in a decreased p25 expression in the lung. The results thus show that systemic immunization with gag or a combination of gag and env genes reduces provirus load in blood and lymphoid tissue, respectively whereas env immunization has no effect on provirus load but increased lesion development.

Mucosal immunization against ovine lentivirus using PEI-DNA complexes and modified vaccinia Ankara encoding the gag and/or env genes.

Sheep were immunized against Visna/Maedi virus (VMV) gag and/or env genes via the nasopharynx-associated lymphoid tissue (NALT) and lung using polyethylenimine (PEI)-DNA complexes and modified vaccinia Ankara, and challenged with live virus via the lung. env immunization enhanced humoral responses prior to but not after VMV challenge. Systemic T cell proliferative and cytotoxic responses were generally low, with the responses following single gag gene immunization being significantly depressed after challenge. A transient reduction in provirus load in the blood early after challenge was observed following env immunization, whilst the gag gene either alone or in combination with env resulted in significantly elevated provirus loads in lung. However, despite this, a significant reduction in lesion score was observed in animals immunized with the single gag gene at post-mortem. Inclusion of IFN-gamma in the immunization mixture in general had no significant effects. The results thus showed that protective effects against VMV-induced lesions can be induced following respiratory immunization with the single gag gene, though this was accompanied by an increased pulmonary provirus load.

Diagnostic tests for small ruminant lentiviruses.

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.

Transmission of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLV) are classical slow retroviruses causing chronic inflammatory disease in a variety of target organs. The routes of transmission have been investigated and a large body of evidence has accumulated over many years. The main routes are through ingestion of infected colostrum and/or milk, or through inhalation of respiratory secretions. However, many studies also provide evidence that intrauterine infection may occur, though the extent and significance of this route is controversial. Embryos treated to IETS standards appear to pose very little risk of infection. SRLV have been detected in semen suggesting a potential source of transmission. However, such transmission has not been demonstrated to date. The application of control measures based on this information allows more efficient strategies to be developed which will reduce the rate of transmission.

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

Phenotypic characterisation and infection of ovine microglial cells with Maedi-Visna virus.

Maedi-Visna Virus (MVV) infection of the central nervous system (CNS) results in pathological changes, the mechanisms of which are poorly understood. MVV preferentially infects cell of the monocyte/macrophage lineage in vivo. The neuroparenchymal microglial cells are the resident tissue macrophages in the CNS and therefore likely targets for MVV infection. However, no information is currently available on the susceptibility of these cells to MVV infection or their contribution to neuropathological changes as a result of MVV infection. Highly enriched primary ovine microglial cell cultures were set up from brain tissues of lambs. These cells were amoeboid or bipolar with spikes, a morphology consistent with microglial cells of other species, and stained positive for CD1, CD11a, CD11c, CD14, MHC-class I, MHC-class II, and beta-N-acetyl galactose, but not with markers of astrocytes or oligodendrocytes. These sheep microglial cells were permissive for MVV infection. Productive MVV infection resulted in selective transcriptional up-regulation of the pro-inflammatory cytokines TNFalpha and IL-6. In contrast, there was no change in levels of transcripts for TGFbeta1, IL-1beta, GM-CSF, IL-10, or IL-12. These data provide the first evidence that ovine microglial cells can support productive infection with MVV, and that this leads to a selective up-regulation of proinflammatory cytokines. These may contribute to visna neuropathology.

Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo.

Plasmid DNA administration has revolutionised approaches to vaccination, and many studies have demonstrated the generation of both humoral and cytotoxic T cell responses which confer protection against live pathogen challenge. However, the mechanisms underlying DNA vaccination are poorly understood. Several studies have suggested the involvement of professional antigen presenting cells such as dendritic cells (DC), but direct evidence for this is lacking. We have used the pseudoafferent lymphatic cannulation model in sheep to study the expression of a plasmid encoding enhanced green fluorescent protein (EGFP) by afferent lymph DC following administration to skin. The cells were analysed by flow cytometry. Preliminary studies were carried out to determine if the pEGFP would function in sheep cells in vitro. The results showed that electroporation of sheep skin fibroblasts, primary macrophages, and afferent lymph DC with 30 microg pEGFP resulted in varying degrees of fluorescence in these cells e.g. 35% of skin cells examined at 48 h, and 7% of afferent lymph DC examined after 4 h. Following intradermal injection of 120 microg of pEGFP, small numbers of fluorescent DC (1-5%) were evident by flow cytometry after 1-4 h. The fluorescent DC continued to drain into the lymphatics over a period of 24 h. Analysis by PCR showed that free pEGFP appeared in the afferent lymph plasma within 1 h of injection, peaking at 2 h and becoming undetectable after 6 h. The results suggest that primary immune responses may be initiated by uptake of soluble protein antigen by afferent lymph DC and by free plasmid rapidly draining to the lymphatics where it may be taken up by DC in the lymph plasma and the local lymph node.

Transforming growth factor-beta 1 (TGF-beta1) expression in ovine lentivirus-induced lymphoid interstitial pneumonia.

The aim of this study was to detect the localization of TGF-beta1 protein expression in normal sheep lungs and lungs with interstitial pneumonia associated with infection with maedi-visna virus (MVV). Immunohistochemical localization of TGF-beta1 was determined in 24 lungs of adult sheep naturally infected with MVV and six control lungs of seronegative sheep. The lungs of infected animals showed different lesional degrees: grade 0, no lesions; grade I, mild; grade II, moderate; grade III, severe. In normal lungs, TGF-beta1 was primarily expressed in airway epithelium, bronchial cartilage and glands, endothelial cells and smooth muscle of blood vessels, alveolar macrophages and type II pneumocytes. No staining was observed in alveolar interstitium. In MVV-infected sheep an increased number of positive alveolar and interstitial macrophages and staining of alveolar interstitium was observed in grade I, grade II and some grade III lesions. In grade III lesions an inverse relationship was found between TGF-beta1 staining and smooth muscle hyperplasia. Small lymphoid aggregates, in general, showed strong reactivity, whereas larger ones showed weak reactivity, mainly associated with follicular areas. No significant differences in the staining intensity of airways and blood vessels were observed between control and MVV lungs. The increased expression of TGF-beta1 in early maedi lesions and its down-regulation in more advanced disease suggest the operation of a temporal regulatory mechanism whereby early expression may lead to the smooth muscle hyperplasia which develops during the disease. The striking inverse relationship between TGF-beta1 expression and follicle organization is intriguing and warrants further investigation.

Regulation of the long terminal repeat in visna virus by a transcription factor related to the AML/PEBP2/CBF superfamily.

The long terminal repeats of maedi visna virus strain 1514 contain a consensus AP-1 binding site which has been shown to be important in controlling virus transcription. However, this consensus site is absent in strain EV-1. Here, we have compared the ability of oligonucleotides corresponding to LTR sequences from EV-1 with those from 1514 to bind transcription factors in competitive gel retardation assays and activate reporter gene expression. The experiments demonstrated no observable binding of AP-1 to the EV-1-derived sequences and significant differences in the abilities of the 1514 and EV-1 sequences to activate transcription. However, both viral sequences interacted with a second, previously undetected, transcription factor. This factor gave specific gel shifts which were competed by an oligonucleotide containing the consensus sequence for the AML/PEBP2/CBF family of transcriptional factors, but not by control AP-1 or OCT-1 oligonucleotides. The factor was therefore denoted AML (vis). A second AML (vis) site, noted upstream of the TATA box proximal AP-1 site, gave single shifts which were competed by the downstream AML (vis) oligonucleotide. Both sites were functional in transfection assays. In gel shift retardation assays, polyclonal antisera directed against known runt domain proteins were able to supershift part of the AML (vis) binding activity in nuclear extracts from physiologically relevant cell types. The results thus suggest that the AML (vis) binding factor belongs to the AML/PEBP2/CBF family of transcription factors and may be important in controlling virus replication in these and other strains of ruminant lentiviruses.

Fc gamma receptor expression on sheep afferent lymph dendritic cells and rapid modulation of cell surface phenotype following Fc gamma receptor engagement in vitro and in vivo.

Afferent lymph dendritic cells were analysed for the presence of Fc gamma receptors by Western blotting and for modulation of surface markers following Fc gamma receptor engagement in vitro and in vivo. The results showed that unstimulated dendritic cells expressed Fc gamma RII constitutively. When dendritic cells were incubated in vitro with antigen/antibody complexes in antibody excess, a marked reduction in surface staining was observed for MHC class II, CD1, CD44, and VLA-4 after 8 h in culture. These changes did not occur with antigen or antibody alone. DC expression of LFA-1 and LFA-3 were slightly reduced after 8 h in culture with Ova alone, but this was enhanced slightly when the cells were cultured with immune complexes. Even more marked reductions in surface staining for MHC class II, CD1, CD44 and VLA-4 were observed on dendritic cells 4-8 h following secondary antigen challenge in vivo. LFA-1 and LFA-3 expression was reduced only slightly. The level of expression of MHC class II, CD1, LFA-1 and LFA-3 was substantially increased over resting values 24 h after Fc gamma R occupancy. The intensity of staining at this time was also significantly elevated for CD44, LFA-1, LFA-3 and VLA-4. These results show that engagement of Fc gamma receptors cause a substantial modulation of the dendritic cell surface phenotype after immune complex uptake. The phenomenon may function to maximize subsequent presentation of the challenge antigen to T cells.

Enhanced proliferation of CD4+ T cells induced by dendritic cells following antigen uptake in the presence of specific antibody.

Afferent lymph dendritic cells bear an Fc gamma receptor which binds antigen/antibody complexes thereby enhancing uptake of antigen. In this report, we have addressed the question of whether the enhanced uptake of antigen results in augmented antigen presentation and T cell proliferation in in vitro secondary responses in sheep. Inclusion of affinity-purified IgG anti-ovalbumin antibody in cultures of afferent lymph dendritic cells, purified CD4+ T cells, and substimulating amounts of ovalbumin resulted in a five- to 169-fold enhancement of T cell proliferation. This effect was antigen-specific as replacement of the anti-ovalbumin antibody with an IgG anti-human serum albumin specific antibody did not cause enhanced T cell responses. The antigen-specific augmentation required intact antibody Fc portions as F(ab')2 fragments of the anti-ovalbumin antibodies were ineffective. The enhanced antigen presentation was found to be maximal with immune complexes in moderate antibody excess (three- to 30-fold), but still occurred at antibody/antigen ratios of 300. The augmented responses were inhibitable with anti-MHC Class II specific antibodies, indicating that at least some of the antigen taken in via Fc gamma receptors entered a Class II processing pathway. The results thus show that antigen uptake via Fc gamma receptors on dendritic cells results in functional augmentation of antigen presentation and T cell proliferation.

Sequence and repeat structure variants in the long terminal repeat of maedi-visna virus EV1.

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i.) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.

T and B cell responses to mycobacterial 65-kDa heat-shock protein in sheep infected with maedi visna virus.

Sheep infected with maedi visna virus were tested for immune reactivity to recombinant HSP65 and tuberculin PPD from mycobacteria. The results showed that both naturally and experimentally infected animals had elevated IgM but not IgG or IgA antibodies to HSP65 from Mycobacterium leprae or M. bovis. In experimentally infected animals, the elevated IgM antibodies appeared in blood from about 3 to 4 weeks postinfection. Increased T cell proliferative responses to HSP65 and PPD were also found in both naturally and experimentally infected sheep. The T cell responses to HSP65 were substantially inhibited by antibodies to ovine major histocompatibility complex class II molecules, indicating that the responses were class II restricted. Increased expression of a putative HSP65 molecule was observed in synovial membranes from sheep infected with maedi visna virus and goats infected with the related, caprine arthritis encephalitis virus. The results thus show that lentivirus infection induces T and B cell anti-HSP65 immune responses and suggest that synovial inflammation may be due, at least in part, to T and B cell recognition of HSP65-like molecules expressed in joints.

Glycosylation of IgG during potentially arthritogenic lentiviral infections.

Agalactosyl IgG [Gal(0)] was first discovered in patients with rheumatoid arthritis (RA). However, the proportion of this glycoform is also raised in tuberculosis and leprosy. This has helped reinforce the suggestion that RA may be triggered by a mycobacterium-like slow bacterial infection. On the other hand, arthritis can occur in mycobacterial diseases, so raised Gal(0) could be associated with a tendency to arthritis, rather than with a particular type of infection. Therefore, we wished to find out whether the percentage of Gal(0) [%Gal(0)] is increased in sheep and goats following infection with maedi visna virus or caprine arthritis encephalitis virus (CAEV), both of which can lead to inflammatory synovitis. We found that the normal level of Gal(0) in these species is much lower than in humans. Goats infected with CAEV or Mycobacterium paratuberculosis (used as a control mycobacterial infection) had a significant increase in %Gal(0), though it was still below the level seen in normal humans. Studies by Western blot confirmed the presence of terminal N-acetylglucosamine on heavy chains, and percentages of Gal(0) comparable to those seen in human RA could be generated by exposing goat IgG to streptococcal beta-galactosidase. The rise in %Gal(0) was greatest in members of infected herds that were just starting to manifest arthritis, and tended to be lower in those in which severe carpitis had developed at the time of bleeding, implying the possibility that raise %Gal(0) may be an early or predisposing event for the development of arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoglobulin deposits in synovial membrane and cartilage and phenotype analysis of chondrocyte antigens in sheep infected with the visna retrovirus.

Synovial membranes and cartilage slices from sheep infected with the maedi-visna retrovirus were examined for immunoglobulin deposits by immunohistology. Granular deposits of IgM and IgG were observed in the synovial membranes and upper layers of cartilage from about 40% of virus-infected sheep. These deposits were present in animals with subclinical joint disease, as well as those affected clinically. No significant deposits were found in the synovial membrane or cartilage from normal sheep. Infected animals tended to have reduced cartilage proteoglycan staining. Altered expression of MHC class II, CD1 and adhesion molecules by chondrocytes in cartilage from infected sheep with clinical or subclinical synovitis was observed suggesting that in vivo cell activation is an early event in cartilage degradation in these infections. Exogenously derived antiviral antibodies exhibited molecular mimicry towards chondrocyte antigens, but no in vivo evidence for cross-reactivity was observed. The results showed that IgM and IgG deposits, putatively containing either virus/antivirus immune complexes or autoantibodies were formed in the joints of sheep with clinical or subclinical synovitis. These immune deposits may initiate and perpetuate chronic inflammation with concomitant activation of chondrocytes leading to pannus formation and cartilage destruction.

Quantitative analysis of immunohistological changes in the synovial membrane of sheep infected with Maedi-Visna virus.

We have carried out a quantitative immunohistological analysis of synovial membrane from the joints of clinically arthritic sheep naturally infected with Maedi-Visna virus (MVV) and compared the results to subclinically affected joints (carpal and tarsal) from infected sheep and to joints from a control population. Significantly elevated numbers of all three T lymphocyte subsets (CD4+, CD8+ and gamma delta) were found in the synovia from clinically arthritic sheep compared to controls. There was also a significant increase in the number of CD8+ T lymphocytes in the carpal synovium of subclinically arthritic animals. In both clinically arthritic and subclinical disease states CD8+ T cells predominated over CD4+ T cells and T cells bearing the gamma delta T cell receptor. Significant increases were also observed in the numbers of cells staining for MHC class II antigens in the synovial lining cell layer and subintimal cell populations of synovia from clinically arthritic sheep. These increases were apparent in the subintimal cell population at the subclinical stage of disease. Macrophage-like cells staining for the viral core protein p15 were observed in some of the most inflamed samples. The data are thus consistent with a disease process driven by chronic viral antigen presentation to infiltrating T cells, and could serve as a model for elucidating the mechanisms underlying some types of inflammatory joint disease in man.

Autoimmune reactivity in sheep induced by the visna retrovirus.

Sheep, either naturally or experimentally infected with visna virus, were examined for the production of autoantibodies. Elevated titres of IgM and IgG antiglobulins and rheumatoid factors as well as antibodies to ssDNA, cardiolipin or histones were found in serum of both groups of animals using ELISA techniques. IgA autoantibodies were usually absent or present in low titres. The specificity of the autoantibody reactivities was confirmed by inhibition assays, affinity chromatography, Western blotting and agglutination methods. Antiglobulin titres were slightly elevated in synovial fluids, although the levels of the other autoantibodies were not raised. No significant correlations were observed between autoantibody and immunoglobulin concentrations. In a separate group of sheep infected experimentally, small but significant rises in the levels of all IgM autoantibodies, except anti-histone antibodies, were observed during and subsequent to the primary immune response to the virus. The autoantibody profiles tended to parallel each other, suggesting a common stimulus for their induction and/or a high degree of polyreactivity. The results thus show that the visna retrovirus induces, either directly or indirectly, autoimmune reactivities that are commonly observed in human autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosis.

Modulation of T-cell reactivity to synthetic peptide analogues of foot-and-mouth disease virus in sheep by amino acid substitutions.

The ability of synthetic peptide analogues of foot-and-mouth disease virus VP1 capsid protein to induce T-cell proliferation in vitro following immunization of sheep with the uncoupled peptides was assessed. Elevated T-cell responses were obtained to a 21-residue peptide containing VP1 residues 141-158, and a 40-residue peptide containing residues 200-213 and 141-158 linked via a diproline-serine spacer. In contrast, no significant T-cell response was obtained with a 19-residue peptide containing residues 200-213 alone. In an attempt to engineer T-cell reactivity to this peptide, a sequence motif found in many peptides recognized by human or mouse T-cells was introduced by amino acid substitution. Substitution of a glycine or an aspartic acid for an alanine at position 207 in the 19-residue peptide resulted in the introduction of two such motifs running consecutively. Immunization of sheep with these peptides resulted in significant T-cell proliferative responses and elevated antibody responses. Analysis of further sequence variants showed that T-cell responsiveness was maintained with peptides containing single amino acid changes within these motifs, provided position 207 was glycine. The results thus suggest that increased T-cell reactivity, might be engineered via sequence manipulation of the 200-213 component of the 40-residue synthetic peptide. Such an additional T-cell epitope in the 40-residue peptide could potentially result in superior neutralizing antibody responses directed against the major epitope in residues 141-160 of VP1.

Retroviral arthritis: phenotypic analysis of cells in the synovial fluid of sheep with inflammatory synovitis associated with visna virus infection.

A phenotypic analysis on synovial fluid cells from the carpal and tarsal joints of sheep with visna virus-induced inflammatory synovitis was performed. The results showed increased representation of cells bearing lymphocyte, macrophage, and dendritic cell markers compared to equivalent synovial fluid cells from normal uninfected age-matched controls. In infected sheep, CD8+ T cells tended to predominate over CD4+ cells, while the numbers of gamma delta T cells varied from being absent in some samples to constituting the major T cell subset in others. B cells were found in relatively smaller numbers. Analysis of the large mononuclear cells showed that they stained with monoclonal antibodies that recognize macrophages and afferent lymph dendritic cells. Major histocompatibility complex (MHC) class II+ macrophage/dendritic cells were found in normal joints, but significantly elevated proportions of such cells were present in the carpal joints of infected sheep. The intensity of MHC class II staining was also significantly elevated in infected animals compared to control animals. A high proportion of these cells also stained for CD1 in both normal and infected animals, but were significantly elevated in number in the carpal joints of infected sheep. The elevated proportion of cells expressing molecules associated with accessary cell function and the increase in the numbers of accessory molecules per cell suggests an enhanced capacity for presenting antigen to a variety of T cell subsets within the joints of infected sheep, which could initiate or perpetuate potentially damaging local synovial inflammatory responses.