Veterinary and medical laboratories engaged in the cultural diagnosis of bovine or human tuberculosis were requested to supply samples of the media that they routinely use for the primary isolation of M. bovis. Fourteen laboratories supplied 7 basic media types; these were Lowenstein-Jensen, Stonebrink's, modified Middlebrook 7H11 agar, tuberculosis bovine blood agar, egg yolk agar, Gerloff's egg and Herrold's egg yolk. Two strains of M. bovis were used to test the media, strain AN5, a glycerol-tolerant laboratory strain and M86/90 a glycerol-sensitive wildtype strain. AN5 grew well on all media with the exception of Herrold's and strain M86/90 did not grow on media containing glycerol and grew poorly on Herrold's medium. It is recommended that Lowenstein-Jensen with pyruvate (but without glycerol), Stonebrink's, modified Middlebrook 7H11 and tuberculosis bovine blood agar should be considered the media of choice for the primary isolation of M. bovis. Egg yolk agar also proved adequate for this purpose in the trial. This medium may be suitable for routine use but to date experience with its use is limited.

In the present study we have investigated the use of recombinant ovine IL-1beta and TNF-alpha 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 microg) the only significant physiological effect was a transient increase in body temperature of approximately 2 degrees C in sheep injected with TNF-alpha. Administration of either cytokine had profound effects on the levels of circulating leucocytes for up to 5 days postinjection. The incorporation of either IL-1beta or TNF-alpha 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-1beta to aqueous vaccine formulations increased antibody responses 15-20-fold and in Al(OH)3 formulations by three to six fold. TNF-alpha 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 microg of both IL-1beta and TNF-alpha 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-1beta and TNF-alpha in combination in the Al(OH)3-based vaccine resulted in antibody levels commensurate with those obtained using Quil A or IFA.

Complete desensitization to tuberculin skin-testing (anergy) was produced in cattle by repeated intravenous injections of living BCG organisms into animals sensitized by a prior subcutaneous dose of BCG. Two levels of desensitization were produced; complete desensitization following 10 i. v. doses and partial desensitization following 5 i. v. doses of 100 mg BCG. Intradermal tuberculin testing at the end of the experiment stimulated the appearance of reactive blood lymphocytes in sensitized cattle as measured by in vitro ³H-thymidine uptake. The cattle which were partially desensitized showed this response but the completely desensitized cattle did not. Reactivity of blood lymphocytes in vitro to PHA and Brucella abortus antigen was not depressed in the anergic cattle. Serum antibody titres to BCG polysaccharide, PPD, or whole BCG organisms showed remarkably little change during the i. v. desensitizing injections of BCG. Differential blood leucocyte counts also remained within normal limits during this period. The production of MIF by blood lymphocytes from the anergic cattle appeared to be unimpaired. Using ³H-thymidine uptake by lymphocytes from sensitized cattle, it was found that serum from desensitized cattle did not inhibit lymphocyte stimulation with tuberculin. It was concluded that the lack of tuberculin-sensitized lymphocytes in the blood of anergic cattle may have been due to their removal from the recirculating pool and their continued suppression in lymphoid tissue.