Growth Requirements of Pathogenic Leptospira

Abstract

Nutritional requirements for growth at :30 C of' Leptospira pomona and L. canicola have been determined. Both pathogenic serotypes initially required bovine serum albumin (BSA) for growth in a medium (SM-4) which permitted growth of the water isolate B-16. Requirement for BSA was eliminated by (i) removing much of' the apparent toxicity of free fatty acids in Tween 80 on an anion exchange column, (ii) decreasing extended lag periods observed from small inocula by incorporation of' pyruvate into the medium, (iii) the addition of acetate to permit full utilization of substrate fatty acids in Tween 80, and (iv) the addition of' glycerol to decrease generation times. Physiologic signiticance of' these findings is discussed, and the possibility is suggested that apparent toxicity of fatty acids for leptospires may result from their auto-oxidation products. The resulting protein-free medium (SM-5) permitted the growth of pathogens at :30 C to high cell yields in low inocula. Highly virulent and avirulent strains from the same clone of L. canicola Moulton were used to determine additional growth requirements associated with virulence. As incubation temperatures were increased from 30 C to those of mammalian hosts, virulent cells required biotin at 35 C and higher levels of K+ and Mg2+ at 37 C. Additional Fe2+ eliminated the

Protein-free and low-protein media for the cultivation of Leptospira

“…An albumin or serum medium containing long-chain fatty acids (11,26), vitamins BI (21), and B12 (1), and inorganic salts (8,10,12) will support good growth of these spirochetes (7,13). Pyruvate, although not an essential nutrient, augments the initiation of growth of parasitic leptospires (16,24). Our results confirm these observations.…”

“…Unsaturated fatty acids are more toxic to the leptospires than are the saturated acids in protein-free media (22,24). However, Tween 80 (major fatty acid, oleic acid) is superior to Tweens 60 and 40 (major fatty acids, stearic and palmitic, respectively) in the 1% BSA medium.…”

“…Staneck et al (24) reported that albumin was not necessary for cultivation of canicola and pomona if the Tween 80 was passed through an anion-exchange column to reduce its free-fatty acid content. They also reported that treatment of the Tween with the anion-exchange resin was not essential if an adequate concentration of iron was present in the medium.…”

“…A number of chemically defined media for the cultivation of leptospires have been described previously (2,9,19,(22)(23)(24)27). However, a protein-free (PF) medium that will grow a large variety of pathogenic serovars to high cell densities is not presently available.…”

“…to remove any remaining charcoal from the Tween. The Tween concentrations of the CT Tweens were determined (24), and the stock solutions of the CT Tweens were stored at -20°C.…”