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The cannulated efferent lymph node in sheep was used to examine the effect of different adjuvants on the antibody and cytokine responses following sub-cutaneous vaccination with a recombinant Taenia ovis antigen (45 W). Vaccination with Quil A elicited relatively higher levels of IgM than did IFA or Al(OH)3. In general, 45 W specific IgG1 and IgG2 titres were higher and maintained for longer periods of time in lymph from sheep vaccinated with IFA and lower and shorter lived in animals which received the Al(OH)3 based vaccine. Interferon-gamma was present within one day in efferent lymph from all sheep which received the Quil A formulation and in only one of the three sheep that received the IFA formulation. GM-CSF was only detected in lymph from sheep vaccinated with the IFA formulation. IL-8 was present in lymph prior to vaccination and only animals which received the Quil A formulation had increased levels of IL-8 after vaccination. Neither of the inflammatory cytokines IL-1 beta and TNF alpha were detected in efferent lymph from any animals in this study. This paper highlights the potential of the lymphatic cannulation model for investigations of the in vivo action of adjuvants.

This review summarises some of the immune evasion tactics adopted by pathogens. They include the antagonism of immune function through the use of homologues of cytokine receptors, expression of viral proteins which interact with cytokine signal transduction and expression of cytokine mimics and host proteins that influence the Type I or II cytokine responses. Some of the viral defense molecules that interfere with the functions of cytokines include the EBV protein BCRF1 (viral IL-10) which blocks synthesis of cytokines such as IFN-gamma, viral IL-17 and IL-8 receptor encoded by the herpesvirus saimiri genome and chemokine receptor homologues of Epstein-Barr virus, herpesvirus saimiri and cytomegalovirus. These immunomodulatory tactics function to protect the host from the lethal inflammatory effects as well as inhibit the local inflammatory response elicited to kill the foreign pathogen. Other strategies include the alterations in cytokine expression such as demonstrated with the hepatitis B virus (HBV) core protein and terminal protein which can inhibit interferon-beta gene expression, the interactions of the hepatitis C virus core protein to lymphotoxin-beta receptor and the effects of the interferon signal transduction pathway by adenovirus EIA oncogene and HBV by reducing levels or activity of the cytosolic latent transcriptional factors (STATS). Immune evasive strategies of helminth parasites related to cytokine activities will also be briefly discussed.

A monoclonal antibody (mAb) specific for ovine IL-1 beta was produced and, in conjunction with a polyclonal rabbit antiserum, used to develop a sensitive enzyme immunoassay (EIA) for ovine interleukin 1 beta (IL-1 beta). The mAb neutralised the activity of recombinant ovine IL-1 beta (rOvIL-1 beta) and native OvIL-1 in an ovine thymocyte proliferation assay. However, it did not neutralise the biological activity of rOvIL-1 beta in the murine NOB1/CTLL assay. The mAb did not react with rOvIL-1 alpha, IL-2, IL-4, IL-8, tumor necrosis factor-alpha, gamma-interferon or recombinant human IL-1 beta in indirect EIA. Immunohistological staining of activated alveolar macrophages and frozen lymph node sections sections demonstrated that the mAb detected IL-1 beta secreted by ovine macrophages (CD11c-positive). The EIA was highly sensitive, detecting less than 50 pg ml-1 of rOvIL-1 beta and low levels of native IL-1 beta in supernatants from lipopolysaccharide-stimulated macrophages. The EIA did not detect heat-inactivated IL-1 beta.

The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction. The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently. Thus, only low yields of biologically active rOvIL-2 were obtained. The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl. An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin. A large proportion of the rOvIL-2 was partitioned to the insoluble fraction. This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained. The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity. Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts. Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.

The immunomodulatory properties of bovine milk and whey have long been documented. The recent advance of whey protein fractionation technology has now allowed us to study the immunobiological properties of some highly purified components of whey, with a view to exploiting their possible industrial and biomedical applications. The effects of fractionated bovine whey proteins on cellular immune responses were therefore examined using a panel of in vitro assays. Both lactoferrin (LF) and lactoperoxidase (LP) were found to inhibit proliferation and interferon-gamma (IFN-gamma) production of ovine blood lymphocytes in response to mitogenic stimulation. However, their effects in a combined fraction or in whey protein concentrate (WPC) were either diminished or eliminated. LF and LP had no effect on lipopolysaccharide (LPS)-induced ovine blood lymphocyte proliferation, production of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF alpha) by ovine bronchoalveolar lavage (BAL) macrophages, major histocompatibility complex (MHC) Class II antigen expression by ovine BAL macrophages and bovine natural killer (NK) cell activity. However, alpha-lactalbumin (alpha LA) exhibited an enhancing effect on IL-1 beta production. It is noteworthy that as bovine whey fractions become progressively more purified, their modulatory effects on the immune response also become more clear-cut. The effects of LF, LP and alpha LA may be eliminated by their combination in whey or by other minor components of whey. Further investigation of industrial applications for whey proteins of high purity is warranted.

The epitheliotropic parapoxvirus, orf virus, can repeatedly infect sheep skin. A specific immune response is generated as reinfections induce smaller lesions with quicker resolution times than primary lesions. Cyclosporin-A treatment abrogates this partial immunity. Cytokine mRNAs detected in lesion biopsies include the transcripts for IL-1 beta, IL-3 GM-CSF, TNF-alpha and, less reproducibly, IFN-gamma. CD4+ T-cells predominate in afferent lymph draining the site of infection, and are the major source of GM-CSF and IFN-gamma. IL-1 beta and IL-8 are also detected. The orf virus genome contains a homologue of mammalian vascular endothelial growth factor that may enhance virulence and a vaccinia virus E3L-like gene which may inhibit the anti-viral effect of the interferons. A GM-CSF inhibitory activity has also been discovered and has been 'chased' into a 10 kb DNA segment of the orf virus genome. These studies indicate that orf virus may temporarily avoid host immunity by a combination of acute, rapid infection and replication in the epidermis and by producing virulence factors that inhibit protective proteins of the host immune and inflammatory response.

Goats infected with caprine arthritis encephalitis virus (CAEV) develop chronic arthritis sharing many features with human rheumatoid arthritis (RA). TNF is thought to be a key mediator contributing to the formation of the arthritic lesion in RA. We studied this cytokine in goats suffering from chronic arthritis. TNF alpha expressing cells were detected by in situ hybridization in synovial membranes of arthritic carpal joints. Expression of TNF alpha did not correlate with the degree of viral replication as assessed by in situ hybridization for viral RNA. In line with the lack of correlation between the degree of viral replication and TNF expression in vivo, we failed to detect increased cytokine RNA in goat macrophages infected with CAE virus in vitro and no TNF protein was found in culture supernatants. In addition, virus infection failed to prime goat macrophages for enhanced TNF expression in response to lipopolysaccharide. Taken together, these findings argue against a direct role of CAE virus in increasing the expression of TNF alpha in caprine arthritis.

Until recently, work on cytokines has been dominated by the use of murine or human molecules. In the last 5 years we have seen a rapid expansion in the production of bovine, ovine and porcine cytokine reagents. cDNA clones, recombinant proteins and monoclonal antibody probes are not available for a wide variety of cytokines from veterinary species. One of the most interesting recent proposals in immunology has been the division of T helper cells into two classes. Th1 cells have been characterised by the production of gamma-interferon, interleukin (IL)-2, tumour necrosis factor-beta (lymphotoxin-alpha) and the ability to mediate delayed-type hypersensitivity responses, and Th2 cells by their production of IL-4, IL-5, IL-6 and IL-10 and the ability to stimulate production of mast cells, eosinophils and IgE. An important issue for us is to determine whether polarisation of T helper cells to Th1 or Th2 occurs in veterinary species. This paper will attempt to review the status of the Th1 and Th2 debate for sheep, cattle and pigs. It will also discuss the potential for the use of cytokines in modulating the type of immune response following vaccination. By incorporation of particular cytokines into vaccine formulations or the inhibition of production of specific cytokines it may be possible to redirect the nature of the immune response to a particular antigen.

The inflammatory response, as measured by the accumulation of leukocytes and ovine serum albumin, induced by interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), interleukin-beta (IL-8), and granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in lactating ovine udders, and in test cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns. In the lactating udders, IL-1 beta and TNF-alpha, but not IL-8 and GM-CSF, induced significant accumulation of cells. In the teat cisterns, all four cytokines IL-1 beta, TNF-alpha, IL-8, and GM-CSF induced significant cell accumulation. IL-1 beta was the most potent cytokine. A slight increase in serum albumin, paralleling the changes in leukocyte numbers, was observed after infusion of IL-1 beta and, to some extent, TNF-alpha. The cell accumulation induced by IL-1 beta or TNF-alpha was dose and time dependent in lactating udders, and time-dependent in teat cisterns. The cell numbers were considerably higher in lactating udders than in teat cisterns after infusion with IL-1 beta. The first influx of cells was observed earlier, and the cell numbers peaked earlier in the teat cisterns than in the lactating udders. IL-8 and GM-CSF induced dose and time dependent cell accumulation in teat cisterns only. The differences between lactating udders and teat cisterns may be attributable to the differences in tissue area involved and the number of receptors available, or to dilution of cytokines in milk, or to presence of inhibitory factors. Differences between cytokines in their inflammatory effects may be explained by their modes of action. IL-1 beta and TNF-alpha have a wide range of cellular functions enabling them to induce a more prominent response than IL-8 and GM-CSF. Furthermore, receptors for IL-1 beta and TNF-alpha are present on a larger number of cell types. Finally, the results indicated that the teat cisterns, being the port of entry for udder infections, as well as the lactating udders are capable of a diversified inflammatory response which is important in the defence against udder infections.

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) was isolated and two forms of recombinant ovine GM-CSF were produced. A glycosylated form was produced in mammalian cells infected with a recombinant vaccinia virus encoding ovine GM-CSF. Recombinant ovine GM-CSF was also produced in Escherichia coli and purified by affinity chromatography. Both forms of the protein were detected by ovine GM-CSF-specific monoclonal antibodies, and exhibited activity on ovine bone marrow haemopoetic progenitor cells.

Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.

An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed. This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T. The level of expression of fusion protein in the soluble fraction was approximately 20% of the total accumulated proteins. Affinity purification by glutathione-Sepharose yielded a fusion protein and subsequent thrombin cleavage of this material yielded rOvIL-1 beta. The specific activity of the purified recombinant protein was 10(3)-10(4) times higher than the fusion protein. The rOvIL-1 beta was 10-100 times more potent than human interleukin-1 beta (HuIL-1 beta) in an ovine thymocyte proliferation assay, although they were of equal potency in the NOB-1/CTLL assay. This simple purification method, which produces purified rOvIL-1 beta with a high specific activity (approximately 10(8) U mg-1), will now make it possible to evaluate the in vivo effects of IL-1 beta in sheep.

Monoclonal antibodies (mAbs) and a polyclonal rabbit antiserum were raised against recombinant ovine tumor necrosis factor-alpha (rovTNF alpha). Ten mAbs specific for rovTNF alpha were isolated and designated TNF1-10. All mAbs were of the IgG1 isotype and reacted with rovTNF alpha in Western blot analysis. Eight of the ten mAbs, TNF1, TNF3-7 and TNF9 and 10, completely blocked the activity of rovTNF alpha and macrophage derived native ovTNF alpha, as measured by their ability to inhibit TNF alpha-mediated lysis of WEHI-164 or L929 cells. In addition, TNF3, -7, -9 and -10 blocked the cytolytic activity of recombinant human TNF alpha (rhuTNF alpha). However, when tested for the ability to inhibit TNF alpha induced thymocyte proliferation, only mAbs TNF1, -3, -5, -7, -9 and -10 could completely block activity. Competitive binding analysis using unlabelled and horseradish peroxidase (HRPO) labelled mAbs indicated that the mAbs could be divided into five groups based on their reactivity with rovTNF alpha. The mAbs were used to develop a sensitive sandwich immunoassay for the detection of ovTNF alpha. All combinations of mAbs and the polyclonal antiserum were tested to determine which pair of antibodies gave the most sensitive assay. The combination of TNF5 as the capture antibody and the polyclonal antiserum gave the most sensitive result, detecting less than 0.24 ng rovTNF alpha ml-1. A similar sensitivity was obtained when TNF4 was used as the capture antibody and TNF10 HRPO labelled mAb as the second antibody. The immunoassay was more sensitive than the WEHI-164 bioassay which had a detection limit of 1 ng ml-1 for rovTNF alpha. This immunoassay also detected glycosylated ovTNF alpha in the supernatant of COS-7 cells which had been transfected with an ovTNF alpha cDNA.