P.R. Wood

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.

Serological reactivity to Mycobacterium bovis protein antigens in cattle

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass. The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.

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.

T cell cytokines and disease prevention

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.

Purification of a Major Mycobacterium bovis Antigen for the Diagnosis of Bovine Tuberculosis

A Mycobacterium bovis antigen has been purified from culture filtrate by chromatofocusing. This antigen is a major component of culture filtrate and cell extracts and shows a considerable degree of micro‐heterogeneity in electric charge and molecular weight. Studies with monoclonal and polyclonal antibodies raised against the purified antigen show that some of its antigenic determinants also occur in higher molecular weight species in culture filtrate and particularly in whole cell preparations. Immunoblotting and ELISA studies, using sera from M. bovis‐infected animals, showed that this antigen is one of the most immunoreactive components of M. bovis, recognized by the majority of animals with detectable antibody response to M. bovis. The specificity of the purified antigen is far superior to that of the crude culture filtrate, with very few false positive results. The purified antigen also elicits strong in vivo and in vitro cell‐mediated responses The amino acid compositions of two variants of this antigen have been determined and found to be similar to that of MPB‐70.

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.

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.