エンテロトキシンの研究

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There have hitherto been reports made by many investigators on the enterotoxin poisoning-poisoning elicited by a group of toxins produced in the course of the growth of Micrococcus pyogenes var. aureus-which are claimed to have the highest frequency rate of all the cases of bacterial food poisoning. However, the nature of enterotoxin has not been determined yet because of the fact that animal experiments as well as the preparation of the extract of toxin cannot be easily done,, and the enterotoxin is thermostable, acid fast and alkali fast. With a view to contributing to the fundamental study of enterotoxin poisoning from the standpoint of public health, the author undertook a thorough examination of the toxicological, biochemical and immunological properties of the extract of enterotoxin. Results of the experiments are summarized as follows: 1) In the present experiments were used four strains of Bergey's (1948) socalled M. pyogenes var, aureus (Staphylococcus aureus), of which two were isolated from the food which had caused enterotoxin poisoning and two from a case of cow's mastitis. 2) All four strains ferment glucose, lactose, marmite and glycerin but not starch and inulin. They do not produce indole. They produce hydrosulphide and reduce nitrate ; ammonium phosphate is not utilized as nitrogen source. Milk coagulation and hemolysis are evidencedmarkedly. in the light of these observations, no differences in biological characters have been recognized between enterotoxin producing strains and nonproducing ones. 3) For the culture of enterotoxin production, Dolman's so-called "semisolid" medium, composed of peptone water and nonorganic salts solution, was used. Incubation of the cultures in the air containing 20 % carbon dioxide has been favorable for the production of enterotoxin. 4) The toxicity of enterotoxin can be determined by kitten or puppy test by intravenous or intraperitoneal injections. 5) The extract of enterotoxin in grayish white powder form has been prepared by the following procedure : the cultures were salted out with 60 % ammonium sulphate, dialized against running water for 24 hours, precipitated by adding acetone in 4 time amount, and then treated by pure acetone and ether. 6) The enterotoxin fraction of high toxicity has been isolated in white powder form from the cultures by adsorbing and eliminating at pH 5.0 such impurities as amines, organic basic substances and other positive ions with the aid of ion exchange resin amberlite IRC 50, and then treating the filtrate with alcohol. No action of enterotoxin was evident in the amine fraction. 7) It has been revealed on animal experiments that, when the extract prepared by means of the above two methods was divided into protein and nonprotein fractions by Sevag's method, each fraction almost ceased to be toxic singly, but when recombined they were again capable of eliciting poisoning as severe as regular enterotoxin poisoning. The author, therefore, is of the opinion that as against Hammon and others who have reported that enterotoxin originated solely in nonprotein polysaccharide fraction, formation of toxin, when considered as an agent producing illness characteristic of enterotoxin poisoning clinically, would depend not only on polysaccharide fraction but also on the author's so-called protein fraction, although it is observed that the former demonstrates singly a slight action of arousing vomiting and diarrhea. 8) The extract does not exhibit hemolytic and necrotic activities. 9) The extract was found to be positive for polysaccharide and protein on qualitative examination of various reactions, such as JFehling's, Biuret's, Xanthoprotein's, Nylander's and Ninhydrin's. 10) The presence of glycine, alanine, asparagin, tryptophan, glutamic acid, Ieucine, glucose, galactose and arabinose was definitely evidenced by the following test : the extract was divided into protein and nonprotein fractions, these fractions were hydrolyzed with metallic acid, and their hydrolyzate was examined by means of paper chromatography. It cannot he denied, however, that some of these substances enumerated herein are attributable to culture medium. 11) The flocculation test using the extract of enterotoxin as antigen and the rabbit serum immunized with enterotoxin as antibody, demonstrated specific reaction. This antigenicity was observed in conformity with the extracts of enterotoxin prepared from four strains used in the present experiments, but no antigenic relations were recognized between them and those of cultures of M. pyogenes var, aureus strain 209P and strain E. Coli B which do not produce enterotoxin.
千葉大学の論文
1954-05-28

エンテロトキシンの研究

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