Preparation of the vascular permeability factor of Vibrio cholerae

In previous papers (J. P. Craig, Nature 207 :614, 1965; J. P. Craig, Proc. Cholera Res. Symp. Public Health Serv. Publ. 1328, 1965), I reported the finding of a heat-labile, nondialyzable vascular permeability factor (PF) in sterile filtrates both of rice-water stools from human cases of Asiatic cholera and of liquid peptone cultures of Vibrio cholerae. Such filtrates, when injected intracutaneously in guinea pigs and rabbits, evoked erythema, localized firm swelling, and a delayed and prolonged increase in permeability of the small blood vessels of the skin to water and plasma protein. The skin response, including the permeability effect, was abolished specifically by convalescent cholera sera. To simplify terminology, I proposed "cholera skin toxin" as a convenient term for this neutralizable vascular permeability factor elaborated by V. cholerae. In accordance with these findings, I postulated (i) that the primary lesion in human cholera might be an increase in permeability of the small blood vessels of the intestinal villi caused by absorption of PF from the gut lumen; (ii) that this increase leads to an accumulation of water, salts, and plasma protein in the subepithelial spaces of the villi; and (iii) that the water and salts pass into the gut lumen whereas protein is retained by the intestinal epithelium. This hypothesis would be consistent with the fact that rice-water stools are essentially free from protein. After these initial findings, I investigated skin toxin production in a number of culture media and under a variety of growth conditions in an attempt to improve toxin yield. The present note describes the method of cultivation which has thus far yielded the highest PF potency with the simplest nutrient and growth requirements. All skin toxin preparations described in this report were produced by V. cholerae Ogawa B1307, which was maintained on stock slants of tryptose-agar under paraffin oil at room temperature. For toxin production, the following modification of a basal medium described by R. A. Finkelstein and C. E. Lankford (Bacteriol. Proc., p. 49, 1955) was used: ammonium chloride, 1.19 g; potassium phosphate, dibasic, anhydrous, 5.0 g; sodium phosphate, dibasic, anhydrous, 5.0 g; sodium sulfate, 0.2 M, 3.13 ml; magnesium chloride, 0.1 M, 2.04 ml; ferric chloride, 0.01 M, 1.78 ml; manganese chloride, 0.01 M, 1.85 ml; saccharose (Difco), 1.0 g; and deionized distilled water to 1,000 ml. This medium was dispensed in 100-ml amounts in Roux flasks with closures allowing maximal gas exchange, and was autoclaved at 15 lb of pressure for 15 min in the vertical position. In the horizontal position, such flasks provided a surface-volume ratio of 2 cm2/ml. The initial pH was 8.25 to 8.30, falling to 7.6 after 24 hr of incubation at 30 C. Such Roux flasks of basal medium (BM) were used both for seed and for toxin production. Organisms were transferred from stock slants to 2% peptone water and, after 6 hr at 37 C, were subcultured to seed flasks of BM and incubated for 18 hr at 30 C. Toxin production flasks were seeded with 0.02 volume of undiluted 18-hr BM culture and incubated for 48 hr at 30 C in the horizontal position. The bulk of the bacteria were removed by centrifugation at 23,000 x g for 30 min at 4 C, and the supernatant fluid was sterilized by passage through a 220-m,u membrane filter (Millipore Filter Corp., Bedford, Mass.) under positive pressure with nitrogen. The PF potency of the sterile crude filtrate remained stable for at least 9 months at 4 C. In preliminary experiments with small batches of media, it was shown that PF yield was greater in 2% peptone than in 5% peptone or basal medium, but also that the yield was inversely proportional to incubation temperature within the range of 30 to 41 C. Because of the desirability of using a totally dialyzable medium for future toxin studies, however, basal medium and incubation at 30 C have been selected as the optimal growth conditions. Pertinent data on several larger lots of cholera filtrate are summarized in Table 1. Data on two batches of BM culture incubated at 30 and 37 C are given in Table 2. Viable counts are expressed in colony-forming units (CFU) per milliliter. PF potency of filtrates is expressed in blueing doses