A tissue culture assay system to detect cytotoxin was developed, and the effect of culture conditions on cytotoxin production was examined. Broth-culture filtrates from clinical isolates were used. The expression of cytotoxicity was dependent on the presence of serum in the culture medium. Although the titre of cytotoxin was low, the greatest cytotoxicity was revealed when newborn calf (NCS), adult bovine or goat serum was added to minimum essential medium. Moderate or weak cytotoxicity was observed when fetal calf (FCS), swine or human serum was added, and no cytotoxicity was seen with the addition of horse, rabbit, cat or chicken serum. Furthermore, the cytotoxic titre in serum-free culture (SFC) for Chinese hamster ovary (CHO) cells was higher than that with serum supplementation. CHO cells showed the highest sensitivity in FCS, NCS and SFC assay systems, whereas HeLa, Vero, HEp-2 and Intestine 407 cells were sensitive only in the NCS assay. Brucella broth was excellent for cytotoxin production, but iron supplementation had no effect. Cytotoxic expression in the three assay systems differed between stationary and stirred cultures. Our results on cytotoxin production by reference strains of did not agree with the original reports. These findings suggest that several different cytotoxins may be produced by , and that the assay system may be important for the expression of cytotoxic activity.


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