A method for the in vitro study of intracellular brucella has been described. Exudative leukocytes containing intracellular brucella have been maintained in vitro in a synthetic tissue culture medium or in human or animal serum. Intracellular brucella are protected in vitro against the lethal action of therapeutic agents or the bactericidal action of serum. This protection of intracellular brucella is dependent upon the presence of an intact, viable host cell. None of the currently available therapeutic agents, whether used alone or in combinations, were capable of killing all intracellular brucella in vitro in 24 hours. A remarkable protection of intracellular brucella against streptomycin has been demonstrated. The most effective reduction in the number of viable intracellular brucella was accomplished by exposure of the host cells to streptomycin plus aureomycin, terramycin, or chloramphenicol. The available evidence suggests that the ability of brucella to localize and remain viable within the cells of an infected host is an important biologic factor in establishing and perpetuating brucella infections, despite therapeutic measures or the operation of the host's humoral defense mechanisms. Reduction of neotetrazolium by leukocytes and brucella in vitro provides a method for assessing the metabolic status of the host cell, but does not discriminate with any degree of certainty a viable from a non-viable intracellular organism.

Summary and Conclusions A chronic, sublethal Brucella melitensis infection was established in mice. Therapy with aureomycin, terramycin, sulfadiazine, or streptomycin, alone and in combinations, was started 4 weeks after establishment of the infection, and continued for 3 or 6 weeks. The results of therapy were evaluated by spleen cultures for brucella at the completion of treatment and 4 weeks after stopping therapy. The experimental results showed that any of the combined types of therapy with streptomycin plus aureomycin, terramycin, or sulfadiazine were definitely superior to any one of the drugs when used alone. Such combined therapy completely eradicated brucella from the spleens of all but 1 of 100 mice treated with any of these combinations. Therapy with aureomycin or terramycin alone in doses of 60 mg per kg per day for 3 weeks resulted in the eradication of brucella from the spleens in only 20 to 30% of the animals, as determined by spleen cultures at the completion of treatment. However, the final result of this therapy, determined by spleen cultures 4 weeks after stopping treatment, was eradication of the organisms in 70 to 80% of the animals. This observation emphasized the significant role of the host's immune mechanisms in the final eradication of brucella infections. Therapy with aureomycin alone in doses of 30 to 60 mg per kg per day for 6 weeks produced, as an immediate result, eradication of brucella from the spleens in 75 to 100% of the animals. However, the final results of this prolonged therapy, as determined 4 weeks after stopping treatment, were no better than the final results with only 3 weeks of treatment.