Filtrates of grown in rabbit serum broth supplemented with sodium ribonucleate (Na-RNA) 1.0% contained a haemolysin for sheep, rabbit, and pig erythrocytes. Haemolysin was not detected in the absence of Na-RNA. Haemolytic titres were highest when viable counts were maximal at the end of the logarithmic growth phase. Haemolysin could not be demonstrated in the spirochaete-free supernates of either centrifuged washings from blood-agar cultures, or centrifuged frozen-and-thawed cultures in semi-solid blood agar. In contrast, washed spirochaetes lysed sheep erythrocytes suspended in phosphate-buffered saline (PBS) or PBS-cysteine, provided that bovine serum albumin (BSA) fraction V was present. Haemolysin was produced aerobically as well as anaerobically. Ultrasonically disrupted spirochaetes did not produce haemolysin.

After incubation for 30 min at 37C, haemolysin was present in supernate or filtrate from centrifuged suspensions of resting cells of in PBS containing glucose or maltose, cysteine hydrochloride, Mg, and Na-RNA or ribonucleic acid (RNA)-core. By analogy with its function as a non-specific carrier molecule for the oxygen-stable streptococcal haemolysin streptolysin S, ribonucleic acid can be regarded as a carrier substance for the treponemal haemolysin. RNA-core was a more efficient carrier than Na-RNA; BSA fraction V and Tween 80 were less efficient than Na-RNA. Haemolysin was not produced in the absence of a carrier. Haemolysin production occurred at temperatures up to 42C, but was reduced at 18C and below. The haemolysin was inactivated slowly at 37C and 42C, and more rapidly at 60C. Titres also decreased slightly after 18 h at 4C.

Haemolysin was produced in only small amounts by resting cells of a weakly haemolytic porcine-intestinal spirochaete, and only during a restricted period in the logarithmic phase. A prozone was observed when this haemolysin was titrated.

The demonstration that the treponemal haemolysin can be produced from resting cells will facilitate its purification and thus expedite studies on its interaction with other types of mammalian cell.


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