The immune response of cattle to nonliving vaccines derived from Brucella abortims rough strain 45/20 was studied. Vaccines contained trehalose dimycolate and a derivative of muramyl dipeptide, N-acetylmuramyl-L-cx-aminobutyryl-Disoglutamine. A factorial experiment was designed to test the effects of type of antigen, quantity of antigen, and quantity of mineral oil on the immune response to porin. Muramyl dipeptide was kept constant at 5 mg per dose, and 1 part of trehalose dimycolate was incorporated for two parts of dry matter. Over a 10-week period, blastogenesis responses to porin were largest in cattle immunized with outer membranes; the highest antibody titers to the porin-lipopolysaccharide complex were achieved by immunization with detergent-extracted outer membrane proteins. There was no advantage in the use of 25, rather than 5, mg of any of the antigens, but antibody responses were improved by increasing the quantity of oil from 0.6 to 1.8 ml per dose. In other animals, blastogenesis and antibody responses were sustained at high levels longer than 3 months after two vaccinations with outer membrane proteins. Intradermal injection of porin evoked inflammatory reactions histologically consistent with delayed-type hypersensitivity. Cross-reactions in cases of delayed-type hypersensitivity occurred with porin derived from a smooth strain of B. abortils but were less extensive than in the blastogenesis test. The magnitude of the delayed-type hypersensitivity and blastogenesis responses induced by vaccination exceeded those observed after natural or experimental infections. No ill effects were observed after vaccination. These findings provide a basis for the use of trehalose dimycolate and muramyl dipeptide adjuvants in evaluating nonviable vaccines for bovine brucellosis.Development of an effective nonviable vaccine for Brlcella abortus would constitute a major advance toward the goal of eradicating bovine brucellosis from the United States. It is well established that B. abortus is a facultative intracellular parasite for which a cell-mediated immune response associated with delayed-type hypersensitivity (DTH) is crucial for protective immunity (8,14). Such a response can be induced effectively by nonviable vaccines in which trehalose dimycolate (TDM) and muramyl dipeptide (MDP) have been incorporated as adjuvants (for reviews, see references 1 and 7). Woodard et al. (25) have demonstrated that guinea pigs immunized with killed cells of B. t Present address: Syncor International Corp., Berkeley. CA 94710.abortus rough strain 45/20 combined with both TDM and MDP adjuvants developed a relatively high level of resistance to infection with virulent B. abortuls 2308. The use of these adjuvants would be feasible in a commercial vaccine for cattle because the absence of mycobacteria and the low content of mineral oil minimize local reactions and prevent development of tuberculin hypersensitivity.We have isolated and characterized the major outer membrane proteins (OMP) of B. abortlls (21). One of these (group 2)...
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