Paul R. Wood

In vitro immunodiagnostic assays for bovine tuberculosis

The immune response to mycobacterial infections in cattle is predominantly cellular in nature and current diagnostic tests for M. bovis are based on the measurement of T cell responses. The low sensitivity of serological assays for tuberculosis is therefore not surprising and serological tests will at best be used to complement rather than replace cellular assays. The recently developed bovine interferon gamma (IFN-gamma) assay is a rapid (24 hour) and simple whole blood in vitro assay, which in Australian field trials was found to be significantly more sensitive than the intradermal tuberculin test for the diagnosis of bovine tuberculosis. The problem of false-positive reactions, due to the cross-reactive nature of the antigen preparations used, can largely be overcome by using a comparative assay in which an animals IFN-gamma response to bovine PPD and avian PPD are compared. Although reasonably M. bovis specific proteins have been identified and characterised, their use in either serological or cellular diagnostic assays is likely to be restricted due to the genetic diversity of the bovine immune response to M. bovis infection.

Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep.

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, respectively. Sheep received two vaccinations with various combinations of these two delivery systems and/or purified recombinant 45W protein in a conventional vaccine formulation containing Quil A as adjuvant (protein/Quil A vaccine). Sheep receiving two inoculations of either the nucleic acid or the recombinant adenovirus alone, demonstrated only low levels of 45W‐specific antibody. However, immunization with either nucleic acid or recombinant adenovirus primed animals to mount an enhanced immune response after a subsequent vaccination with the protein/Quil A vaccine. The most striking result was that sheep initially immunized with the nucleic acid vaccine and boosted with the recombinant adenovirus, mounted IgG1 responses >65 fold higher than those of sheep receiving either vaccine alone. The level of antibody in these sheep was commensurate with that observed in animals vaccinated twice with the protein/Quil A adjuvanted vaccine. In both cases, host‐protection from experimental challenge infection with T. ovis was obtained.

Defence against the immune barrage: helminth survival strategies.

Parasites have generated a range of countermeasures against the host immune system which allows their survival long enough for reproduction to occur. Parasite subsistence is enhanced by evasion of the immune response utilizing mechanisms such as antigenic variation of exposed immunogenic proteins, shedding of surface proteins which are the target of an immune response, and protease production to neutralise specific anti‐parasite immune components. Recent advances in the fields of immunology and parasitology have highlighted a range of mechanisms by which the parasite actively modulates the immune response to allow survival. Parasite factors can directly suppress the function of certain subsets of immune cells as well as stimulating other cell populations which have suppressive activity. Strategies such as the skewing of the type 1‐type 2 cytokine profile to that of a less appropriate response, and the mimicry of host immune regulatory proteins are becoming more widely acknowledged as means by which helminths enhance their survival. An illustration of the extent by which parasites can exploit host immune components is emphasized by the use of host cytokines as parasite growth factors. This review will examine some of the strategies developed by helminths which enables them not only to survive in the host, but also to prosper.

Antigenic competition in a multivalent foot rot vaccine.

The antigenic competition that occurs when pilus antigens of different serogroups are combined in multivalent vaccines for foot rot has been investigated using recombinant pilus antigens. Our prototype vaccine contains pili from nine serogroups of Dichelobacter nodosus which are expressed in Pseudomonas aeruginosa. Sheep inoculated with this multivalent vaccine were not as well protected against foot rot as those given the monovalent vaccine. Levels of agglutinating and total antibody specific for any particular pili serogroup were found to be significantly reduced in sheep vaccinated with six or more closely related pili. This effect was more pronounced for agglutinating antibody, which is thought to mediate protection, but was also observed with total antibody levels measured by ELISA. The antigenic competition was not associated with the total antigen load as a tenfold higher dose of monovalent pili induced high titres of antibody. Furthermore, distributing the vaccine to four sites, each draining to a different lymph node, failed to overcome the competition. Experiments with mixtures of monospecific sera indicate that the phenomenon is unlikely to be due to blocking of serogroup-specific protective antibodies by an excess of cross-reactive non-protective antibody elicited by heterologous pili.

Influence of antigens and adjuvants on the production of gamma-interferon and antibody by ovine lymphocytes

The production of gamma‐interferon (γ‐IFN) and antigen‐specific immunoglobulin isotypes was monitored in sheep given primary and secondary inocula of protein and polysaccharide antigens with or without adjuvants. In efferent lymph from prefemoral nodes draining the site of inoculation, γ‐IFN levels increased within 24 h after injection of adjuvants Quill A, dextran sulfate (DXS) and 6 days after oil adjuvants in saline. The increased synthesis of γ‐IFN was prolonged by 1–2 days by the presence of adjuvanted antigen, but the major stimulus to γ‐IFN production was provided by the adjuvant. Alhydrogel (AH) did not initiate γ‐IFN synthesis. Following a second inocula of antigen in saline, increased levels of γ‐IFN were detected in efferent lymph after 3–4 h, and persisted for at least 2 days. Following cultivation of leucocytes from immunized sheep with specific antigen in vitro, strong positive correlations between levels of interleukin (IL) IL‐2, ‐IFN, proliferative responses and antibody titres were observed. However, after analysis of the antigen‐specific isotypes of immunoglobulin (Ig) there was no correlation between the production of γ‐IFN and particular Ig isotypes. Together with findings that AH did not preferentially induce IgG1 or IgG2, these results suggest that the specific subpopulations of helper T lymphocytes which regulate the production of antibody isotypes by differential secretion of γ‐IFN or IL‐4, are not as clearly defined in sheep as in mice.

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA.

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.

Immunological parameters associated with antigenic competition in a multivalent footrot vaccine.

A murine model for antigenic competition with multivalent D. nodosus pili vaccine has been established that parallels the phenomenon observed in sheep where levels of antibody, specific for any particular serogroup of pili, are significantly lower following vaccination in the presence of multiple serogroups of pili than with that serogroup alone. This competition was observed in both high and low responder strains of mice and was not dependent on the multiplicity of the antigens in the multivalent vaccine but could be observed with a large excess of a single heterologous serogroup. Competition was manifest by a reduction in the number of serogroup-specific antibody secreting cells elicited in response to vaccination. The antibody response to a single serogroup of pili reached a plateau at high doses and it was at these doses that antigenic competition was most pronounced, under conditions where both B- and T-cell responses were limiting. The limit in T-cell responsiveness was not imposed at the level of presentation of antigen. Pili-specific T cells were largely cross-reactive for different serogroups, and under conditions of limiting T-cell stimulation within a lymph node the available T cells would have to be shared between B cells specific for each serogroup of pili, which may in turn result in the decrease of serogroup-specific antibody induced following inoculation with the multivalent vaccine.

An analysis of cellular proliferation, and synthesis of lymphokines and specific antibody in vitro by leucocytes from immunized cattle.

The relationships between the production of lymphokines, cellular proliferation and antibody synthesis by immune bovine PBL in vitro was examined to identify the cellular reactions responsible for differences in the titres of serum antibodies in calves from selected sire lines and MHC Class I phenotypes. Leucocytes from 22 calves immunized with ovalbumin and DNP-BSA proliferated specifically in vitro in the presence of 1-10 micrograms/ml ovalbumin 7-28 days after the second vaccination. Significant correlations between the production of IL-2, IFN-gamma and maximum proliferation were observed for the total group. The quantity of specific antibody produced when PBL were incubated alone or with 10(-1)-10(-2) micrograms/ml ovalbumin was also correlated significantly with the maximum proliferation and the serum antibody titre between 7 and 14 days. Anti-ovalbumin IgG was also synthesized in MLRs where the quantity of antibody was significantly correlated with the magnitude of proliferation. The responses in vitro to DNP-BSA were too low to provide meaningful comparisons. The results indicate that at intervals during the period of increasing serum titres, events in the bovine antibody response in vivo can be replicated in vitro. In addition, assays for proliferation, IL-2 or gamma-IFN, or specific antibody can be used to assess the magnitude of the immune response in vivo in experimental groups of cattle. Significant sire line differences in the serological responses to ovalbumin were observed but DNP-BSA was a poorer antigen and differences in the responses to this antigen were not significant. However, the sire line differences in vivo were not reflected in vitro in proliferative and lymphokine assays; only the production of antibody in vitro was significantly correlated with the in vivo serum titre.

Identification of Mycobacterium bovis isolates using a monoclonal antibody

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.

Urea/DTT solubilization of a recombinant Taenia ovis antigen, 45W, expressed as a GST fusion protein results in enhanced protective immune response to the 45W moiety

Vaccination and challenge infection experiments were conducted in sheep using different forms of a recombinant protein (45W) from the cestode parasite Taenia ovis. 45W was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (45W-GST) and was produced as both soluble protein and insoluble inclusion bodies. Vaccination of animals with either the soluble or inclusion body derived protein resulted in the immune response being predominantly directed to the GST moiety of 45W-GST. Conversely, vaccination with 45W-GST which had been solubilized/treated with urea and dithiothreitol (DTT), elicited enhanced responses to the 45W moiety and significantly reduced responses to GST. Vaccination with all forms of 45W-GST protected sheep against experimental T. ovis infection. However, protection was highly correlated with anti-45W antibody levels and these were significantly higher in animals vaccinated with the urea/DTT treated form of 45W-GST. It is suggested that recombinant proteins expressed either with or without fusion partners may stimulate enhanced immune responses when incorporated in vaccine formulations in a denatured/reduced state.