Use of 8-azaguanine to differentiate leptospires isolated from Iowa surface waters.

Definitive classification of leptospires according to serotype rests with use of the microscopic agglutination and agglutinin-absorption tests. R. C. Johnson and P. Rogers (J. Bacteriol. 88:1618, 1964) observed inhibition of pathogenic serotypes by the purine analogue 8-azaguanine, whereas saprophytic serotypes were able to grow. This report is concerned with the effect of 8-azaguanine on growth of leptospires isolated from Iowa surface waters. A preliminary study of the applicability of 8-azaguanine to a field situation was undertaken because the complex and time-consuming serotype definition of saprophytes unnecessarily complicates epidemiological studies of human cases with a potential water exposure. Leptospires for testing were isolated from water obtained at 12 sampling points on five Iowa streams during August to December 1965. A pathogen control, Leptospira icterohaemorrhagiae, was obtained from Mildred M. Galton, Communicable Disease Center, Atlanta, Ga., and a saprophyte control, L. patoc I, was obtained from the National Animal Disease Laboratory, Ames, Iowa. Stuart's liquid medium without phenol red (Difco) with 10% rabbit serum (Pel-Freeze Biologicals, Rogers, Ark.) was used as the growth medium. This medium was sterilized by Seitz filtration and dispensed in 9-ml amounts in 20 x 125 mm screw-cap tubes. The 8-azaguanine (2amino-6-oxy-8-aza-purine; Calbiochem, Los Angeles, Calif.) was used in a final concentration of 100 jig/ml of medium. All batches of media were used within 3 weeks of preparation. All cultures were incubated at 30 C, with maintenance transfers at 4-day intervals. A 1% inoculum of 4-day-old culture was used to initiate growth for test sets. Each set consisted of duplicate tubes of plain medium and medium containing 8-azaguanine; three trials were conducted for each organism. Growth was measured by use of a Coleman no. 9 Nepho-colorimeter as a nephelometer. H. C. ' Present address: Wisconsin State University, Whitewater, Wis. 5319. Ellinghausen, Jr. (Am. J. Vet. Res. 20:1072, 1959) suggested that growth, as mirrored by relative leptospiral cell turbidities, could be adequately determined by nephelometry. Nephelos readings were made for all cultures at the end of 2 and 4 days of incubation; the cultures were examined microscopically on inoculation and at the end of the 4-day incubation period. Nephelos values which were obtained are summarized in Table 1. Both types of media gave base-line nephelos values of three immediately after the addition of the 1% inoculum. The pathogen control grew noticeably slower than the saprophyte control. The saprophyte control was growing well at the end of 2 days in the 8-azaguanine medium, whereas the pathogen control was almost completely inhibited; the differences in growth in the 8-azaguanine medium are more marked at the end of 4 days of incubation. Although the saprophyte control grew well, a