The culture fluids of nine nontoxigenic strains of Clostridium botulinum types C and D were treated with trypsin and tested for toxigenicity. Toxigenicity was demonstrated in six strains only after trypsin treatment. In these strains 20 to 935 LD50/ml of toxin was produced in egg-meat, Segner's fortified egg-meat (SFEM), cooked-meat and TYG media, but not in LYG medium. Activation of toxicity was not demonstrated in the toxigenic strains, C-Stockholm and D-1873. The toxin titer was elevated five to tenfold by trypsinization in the D-South African sfrain. The trypsin-activated toxin produced by the nontoxigenic strains seemed to be different from that produced by the D-South African strain. The effects of carbon sources and nitrogen sources were studied in TYG medium with strain D-139, one of the strains producing trypsin-activated toxin. The most concentrated toxin was produced when the glucose concentration was 0.2%. The concentration of toxin decreased with an increase in glucose concentration. The same tendency was observed when glycerol was used as a carbon source, in place of glucose. Addition of 0.5% (NH4)2SO4 induced a marked increase in the production of trypsin-activated toxin in glycerol medium, but did not clearly in glucose medium. The titer of toxin was elevated about fifteenfold by addition of 0.5% NH4Cl to glycerol medium. The production of toxin, however, was inhibited by addition of 0.5% NaNO3. The productivity of trypsin-activated toxin was influenced by carbon sources and nitrogen sources in media.
The present study was performed to elucidate the anti-tumor mechanism of Corynebacterium anaerobium (CA) against Ehrlich ascites tumor by using athymic nude mice (nu/nu) and hetero (nu/+) mice of the BALB/c strain. 1) The anti-tumor effect of CA on both nu/nu and nu/+ groups injected intraperitoneally with a mixture of tumor cells and CA was dose dependent. The most effective dose was 250μg per mouse. The overdose administration was much less effective or ineffective. 2) Treatment with carrageenan, which preferentially suppresses the macrophage function, decreased the anti-tumor effect of CA on both groups. 3) A remarkable enhancement was demonstrated in the anti-tumor effect of CA on the nu/nu group to which had been transferred the thymic cells of the nu/+ group. 4) No significant enhancement was shown in the anti-tumor effect of CA on the nu/nu group with CA-sensitized splenic cells transferred from the nu/+ group exhibiting delayedtype hypersensitivity to CA. 5) The nu/+ group, as well as the nu/nu group, to which had been transferred thymic cells, the tumor of which had been completely suppressed by CA, was highly resistant to rechallenge with the tumor cells, presenting a complete rejection. 6) Non-adherent cells derived from the spleen and lymph node of the nu/+ group being resistant to rechallenge with the tumor cells, as well as peritoneal macrophages derived from the CA-treated nu/nu group, exerted a strong cytotoxicity on the tumor cells in vitro. From the findings mentioned above, it was concluded that the anti-tumor activity of CA against Ehrlich ascites tumor was effectuated by CA-activated macrophages and cell-mediated immunity to tumor induced by administration with CA and the tumor cells. Another possible anti-tumor mechanism due to the bystander effect of delayed-type hypersensitivity to CA could not be ascertained.
Investigation was made on the biological properties of purified Asp-hemolysin isolated from the mycelium and culture filtrate of Aspergillus fumigatus. Some differences were found in sensitivity of erythrocytes from various animal species to the hemolysin. Chicken erythrocytes were the most sensitive (MHD, 3μg), whereas frog erythrocytes were slightly hemolyzed (MHD, 2.5mg). The minimum lethal dose of the toxin by the intravenous route was shown to be 750 and 250μg/kg of body weight for mice and chickens, respectively. Even a large dose (1g/kg) of the toxin had no lethal effect on mice when administered orally. The purified hemolysin was free from phospholipase C activity, as well as from dermonecrotic and hemorrhagin-like activities, which were found in crude extracts. The hemolytic activity of Asp-hemolysin was activated a little by Zn2+, cholesterol and G-strophanthin. It was inhibited slightly by lecithin and Cu-chlorophyll, and remarkably by Hg2+, iodine and anti-Asp-hemolysin sera. The hemolytic activity inhibited specifically by Hg2+ could be restored after addition of reducing agents, such as β-mercaptoethanol, cysteine and hydrosulfite. The hemolytic activity of this toxin was prevented distinctly when antitoxic serum was mixed with the toxin prior to the addition of an erythrocyte suspension, but indistinctly when antitoxic serum was added to a mixture of the toxin and this suspension. These biological activities of Asp-hemolysin were not lost by treatment with any of such proteolytic enzymes as trypsin, pepsin, papain and subtilin.
Staphylococcal food poisoning is caused by ingestion of enterotoxin preformed in foodstuffs. Diagnosis of staphylococcal food poisoning may, therefore, depend upon the detection of enterotoxin in the incriminated foodstuff or in cultures of staphylococci isolated from it or both. For a highly sensitive immunological detection of enterotoxin, specific anti-enterotoxin sera are necessary. Staphylococcal enterotoxin has been classified into five types, A through E, on the basis of antigenicity. Purification of enterotoxin E was first reported by Bergdoll et al. (1971), who stated that enterotoxin E was neutralized with anti-enterotoxin A serum and formed a precipitin line against this serum. Terayama et al. (1974) also purified enterotoxin E and reported that enterotoxin E did not react to anti-enterotoxin A serum. The authors also succeeded in purifying enterotoxin E by essentially the same method as those utilized to purify enterotoxin A, B, and C. Essentially pure enterotoxin E was obtained by three consecutive steps, chromatography on CM-1 and DEAE-Sephadex and twice-repeated gel filtration on Sephadex G-100. Enterotoxin E at a concentration of 120μg/ml formed a distinct precipitin line against anti-enterotoxin A serum. This precipitin line fused with that of enterotoxin E appearing against anti-enterotoxin E serum. At a low concentration of 20μg/ml of the toxin, the precipitin line against anti-enterotoxin A serum and that against anti-enterotoxin E serum coalesced with each other. In contrast, enterotoxin A at 120μg/ml reacted to anti-enterotoxin E serum, against which a precipitin line was formed and fused with that against anti-enterotoxin A serum. When 20μg/ml of this toxin was used, no precipitin line was formed against anti-enterotoxin E serum.
To explore the toxicity of Bacillus cereus, the fate of spores injected into the mouse peritoneal cavity was compared between this organism and Bacillus subtilis. One hour after injection of about 1.8×109 spores of B. cereus, most of the spores were found to have been phagocytized by phagocytic cells and a few as germinated ones in the peritoneal cavity. Two hours after injection, the germinated spores began to outgrow. Outgrown vegetative cells started to proliferate. Three hours after injection, they showed a marked proliferation, while some of them and some germinated spores were engulfed by phagocytic cells. By that time the peritoneal cavity was filled with proliferating vegetative cells. All the mice died during a period from 4 to 6 hours after injection. The MLD of B. cereus spores for mice by the intraperitoneal route was calculated as 4.35×108. In contrast, when about 1.6×109 spores of B. subtilis were injected intraperitoneally to mice, some of them germinated in the peritoneal cavity, and only a few of them were able to outgrow. Outgrown vegetative cells hardly proliferated. Thus no death occurred to the mice injected with B. subtilis spores.