Hemolytic Activity of Leptospira interrogans Serovar canicola Cultured in Protein‐Free Medium

Abstract

Hemolytic activity of the culture supernatant of Leptospira interrogans serovar canicola strain Moulton grown in protein-free medium was demonstrated. The activity began to appear in the late logarithmic phase of growth of the organism and reached a plateau after 2 weeks of cultivation. It was inactivated by the addition of dipotassium ethylenediaminetetraacetate, but was effectively restored by Mg . Hemolysis by the culture supernatant was stimulated by "hot-cold" incubation. Sheep erythrocytes treated with the culture supernatant of the organism were transformed into spherocytes, in which invagination was observed. A hemolysin inhibitor in rabbit serum was found to be in the chloroform-methanol soluble fraction of the serum. The hemolysin of Leptospira may be phospholipase C.

Characterization of Inhibitor to Leptospiral Hemolysin Present in Bovine Serum

“…Stalheim (11) reported the presence of PLase C produced by Leptospira inter rogansserovar pomona and noted that PdC was more susceptible than Spm as a sub strate.In this experiment, PLase C activity against Spin was detected in the culture supernatant of Leptospira interrogans serovar canicola and lower activity to PdC was also observed with TLC. As reported previously (15), leptospiral hemolysin was of the hot-cold type of hemolysin and required Mg2+, but not Ca2+, for mani festationof hemolytic activity. SRBCs treated with leptospiral culture superna tantwere transformed into spherocytes and invagination was observed in the sphero cytes.Moreover, hemolysis by leptospiral hemolysin was inhibited by PLs and hemolytic sensitivity of mammalian erythrocytes depended on Spm content in erythrocyte membrane.…”

“…Intrinsic features of leptospiral hemolysin and the inhibitor in rabbit serum remain to be elucidated. We (15) already revealed that the inhibition was not caused by the immune reaction of the sera. In this study, it was proved that the hemolysis was inhibited by PLs in animal sera.…”

“…From the above results, inhibitors of leptospiral hemolysin were identified as PLs, and the hemolysin produced by Leptospira was presumed to be PLase C, especially sphingomyelinase (15).…”

Leptospirosis

“…Plasma has been shown to prevent hemolysis (576). Phospholipase C activity has been reported in serovar canicola (655). A hemolysin from serovar lai is not associated with sphingomyelinase or phospholipase activity and is thought to be a poreforming protein (343).…”

Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia

“…Thiol-activated toxins are produced by four genera of Gram-positive bacteria [8,12,19^22]. Hoshi et al [13] reported that the mode of action of the hemolysin from P. gingivalis was 'hotcold' lysis, similar to hemolysin from Leptospira canicola [11], Staphylococcus aureus [23] or Clostridium welchii [12]. However, hemolytic activity by prevolysin O did not increase with chilled treatment after incubation at 37 ‡C for 1 h. Some hemolytic substances from Gram-negative bacteria are activated by divalent cations [9].…”