J.S. Rothel

A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1%. The most favourable results were obtained with the interferon gamma (IFN-gamma) assay which had a sensitivity of 81.8% and a specificity of 99.1%. Respective figures for the single intradermal tuberculin test were 68.1% and 96.7%. The use of MPB-70 as the antigen in the IFN-gamma assay reduced the sensitivity of this assay, without producing any useful increase in specificity. The IFN-gamma assay was also demonstrated to be a practical diagnostic test for use with large groups of cattle.

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.

Production and characterization of monoclonal antibodies specific for bovine gamma-interferon

Nine stable hybridoma cell lines were established which secreted specific monoclonal antibodies (MAbs) to bovine gamma-interferon (BoIFN-gamma). Specific binding of each of the MAbs to recombinant BoIFN-gamma (rBoIFN-gamma) was demonstrated in an indirect ELISA, whilst none of the MAbs bound to rBoIFN-alpha or rBoIFN-beta. In a Western blot the MAbs reacted with the 16 kDa and 32 kDa polypeptides present in rBoIFN-gamma preparations. Competitive ELISAs showed that four MAbs bound to one epitope on rBoIFN-gamma, and the other five MAbs bound to a separate epitope. Two MAbs, each recognising different epitopes, were shown to neutralise the anti-viral activity of natural BoIFN-gamma.

Nucleic acid vaccination of sheep: Use in combination with a conventional adjuvanted vaccine against Taenia ovis

This report describes the use of a nucleic acid vaccine in a large outbred animal species both alone and in combination with a conventionally adjuvanted vaccine. The gene encoding a host‐protective antigen (45W) from the sheep parasite Taenia ovis was cloned into the expression vector pcDNA3 and the resultant plasmid termed pcDNA3‐45W. Eleven of 15 sheep injected either intramuscularly or intradermally with pcDNA3‐45W mounted a serum antibody response to 45W which for both routes of injection was predominantly IgG1. However, the level of antibody elicited by the nucleic acid vaccine was low and repeated vaccinations did not boost the response. Injection of pcDNA3‐45W into animals in which an immune response had previously been generated by vaccination with recombinant 45W using Quil A as adjuvant (rec45W vaccine), did not result in enhanced antibody levels. Initial vaccination with pcDNA3‐45W and subsequently with the rec45W vaccine resulted in antibody levels significantly higher (P < 0.05) than those obtained in sheep which had only received the rec45W vaccine. This enhanced antibody response was predominantly of the IgG1, subclass (IgG1: IgG2 5:1) in animals injected with the nucleic acid vaccine by the i.m. route. Surprisingly, a second rec45W vaccination of these animals led to little or no increase in IgG1 levels and a 10‐fold increase in IgG2 resulting in a predominance of 45W‐specific IgG2. (IgG1:IgG2, 0.25:1). These studies revealed that nucleic acid vaccination has efficacy, albeit limited, in the sheep and supports previous investigations which showed that antibody responses elicited by immunization are determined by both the route and mode of antigen delivery.

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.

Characterization of monoclonal antibodies to ovine tumor necrosis factor-α and development of a sensitive immunoassay

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.

The use of recombinant ovine IL-1β and TNF-α as natural adjuvants and their physiological effects in vivo

In the present study we have investigated the use of recombinant ovine IL‐1β and TNF‐α both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogenic effects. Even at the maximum dose tested (100 μg) the only significant physiological effect was a transient increase in body temperature of approximately 2°C in sheep injected with TNF‐α. Administration of either cytokine had profound effects on the levels of circulating leucocytes for up to 5 days postinjection. The incorporation of either IL‐1β or TNF‐α in aqueous or Al(OH)3 vaccine formulations enhanced antibody responses to a recombinant antigen from the cestode parasite Taenia ovis. The addition of IL‐1β to aqueous vaccine formulations increased antibody responses 15–20‐fold and in Al(OH)3 formulations by three to six fold. TNF‐α stimulated 1.5 to six‐fold and 2.5 to seven‐fold increases in antibody levels in aqueous and Al(OH)3‐based formulations, respectively, in a dose‐dependent manner. The addition of either cytokine to Quil A or IFA vaccines did not enhance the antibody levels elicited. When 10 μg of both IL‐1β and TNF‐α were incorporated in the aqueous or Al(OH)3 vaccine formulations, increases of 21‐fold and 25‐fold, respectively, were observed in antibody levels. The adjuvant activity of IL‐1β and TNF‐α in combination in the Al(OH)3‐based vaccine resulted in antibody levels commensurate with those obtained using Quil A or IFA.

Soluble Mycobacterium bovis protein antigens: Studies on their purification and immunological evaluation

The eradication of bovine tuberculosis is an ultimate aim of the beef industry and the development of accurate diagnostic tests will expedite eradication. Characterization of Mycobacterium bovis antigens, and a detailed understanding of their immune reactivity will aid in the development of more specific and sensitive diagnostic tests. With this aim, studies were conducted which have resulted in the purification and immunological characterization of the major soluble M. bovis antigens. The purified antigens were found to contain cross-reactive epitopes and immunological responses to these proteins varied among individual animals. Thus if more specific diagnostic tests are to be formulated, they will have to be at the epitope level, using only species-specific epitopes and not whole proteins. Due to the genetic diversity of the response of infected cattle to individual epitopes, a large cocktail of such epitopes will be necessary for the development of a sensitive test.

Mapping of the T and B cell epitopes of the Mycobacterium bovis protein, MPB70.

A clone coding for the entire gene for the Mycobacterium bovis protein antigen MPB70 was used to produce a series of overlapping subclones by making a series of deletions from the 3′ end of the gene. The subclones expressed incomplete MPB70 proteins as fusions with glutathione‐S‐transferase. The insert DNA was sequenced to determine the extent of the deletion and the proteins expressed by the clones were examined for the presence of T cell and B cell epitopes. T cell epitopes were mapped by measuring the ability of recombinant antigens to stimulate gamma interferon (γ‐IFN) production in a whole blood culture system. γ‐IFN production was measured using a sandwich enzyme immunoasssay specific for bovine γ‐IFN. B cell epitopes were mapped with a series of anti‐MPB70 monoclonal antibodies using an indirect enzyme immunoassay.

Antibody and cytokine responses in efferent lymph following vaccination with different adjuvants

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.