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The minimal growth temperature of the pathogenic leptospires is between 13 and 15 C. The saprophytic leptospires have a minimal growth temperature between 5 and 10 C, or approximately 5 C below that of the pathogens. The capability of the saprophytic leptospires to grow at temperatures below those which allow the growth of the pathogenic leptospires provides a simple method of discrimination. With an inoculum yielding approximately 8 × 107cells per ml in the test medium and an incubation temperature of 13 C, the saprophytic leptospires were easily differentiated from the pathogenic leptospires. All 13 saprophytic leptospires tested grew in the 10% rabbit serum medium at 13 C, whereas none of the 20 pathogens grew during the 30-day incubation period.

A definite relationship exists between the resistance of leptospires to the antibody-complement system and virulence. Leptospires capable of producing either lethal or renal infections in hamsters or guinea pigs were resistant to the leptospiricidal action of antibody and complement. Avirulent leptospires, in contrast to the virulent organisms, were rapidly immobilized and killed by these serum substances. The change of a virulent culture to the avirulent state as a result of growth in culture media was accompanied by the loss of resistance to antibody and complement. Virulent leptospires were phenotypically altered when grown in the presence of the purine analogue, 8-azaguanine. The cells became sensitive to antibody and complement without a corresponding decrease in virulence. The basis for a leptospiral virulence factor, the ability to multiply in vivo, appears to reside in their capacity to resist the leptospiricidal activity of the host antibody-complement system. The immune leptospiricidal assay provides a simple and rapid method of determining the virulence of a culture.