The Effect of Cultivation and Freezing on the Virulence of Trichomonas vaginalis for Mice

Abstract

The effect of cultivation on the virulence of several T. vaginalis strains for mice injected intraperitoneally was studied. A comparison of 12 strains of T. vaginalis injected intraperitoneally into mice revealed loss of virulence in the majority of strains after 2 to 3 months cultivation that did not appear to be due to random variation. The sex of the mouse host was not a factor in virulence assay via the intraperitoneal route. Preliminary studies indicated that the virulence of a strain via the intraperitoneal route can be preserved by freezing the organism. The pathogenicity of Trichomonas vaginalis for laboratory animals has been investigated recently by Honigberg and Braunthal (1957), Iwai (1957), Reardon and Jacobs (1958), Bogovsky and Teras (1958), Newton et al. (1960), Reardon et al. (1961), Honigberg (1961), and Frost and Honigberg (1962). These workers have observed considerable differences in virulence of various T. vaginalis strains for laboratory animals. Previous studies have shown that many species of pathogenic organisms lose virulence upon cultivation. Whether this occurs with T. vaginalis has not been clearly established. Honigberg (1961) reported no loss of virulence by a T. vaginalis strain after 5 months of cultivation when injected subcutaneously into mice. The present study was undertaken to determine whether cultivation affected the virulence of T. vaginalis for mice injected intraperitoneally. Since these studies revealed that loss of virulence did occur, preliminary experiments were made on the effect of preservation by freezing on the virulence of T. vaginalis. MATERIALS AND METHODS Strains of T. vaginalis were isolated from vaginal smears placed in Diamond's culture medium (1957) containing 200 units of penicillin and 200 ,Ag of streptomycin per ml and were maintained at 35 C. Transfers to fresh medium were made every 48 hr. After six transfers the strains were inoculated into Received for publication 17 May 1963. * This investigation supported in part by U. S. Public Health Service Grant E-3593. 226 medium lacking antibiotics. After two subcultures in medium lacking antibiotics, the cultures were tested for bacterial contamination by inoculating tubes of brain-heart infusion and thioglycollate broth and observing for at least 1 week for the presence of bacteria. Only strains showing no evidence of bacteria contamination were used in these studies. All strains were tested initially for virulence between the 3rd and 4th week after isolation and again 2 to 3 months later. Twenty-fourto 30-hr-old cultures were used for all animal inoculations. The organisms were centrifuged at 375 g for 6 min and washed once in 5 ml of sterile 0.85% saline. After centrifuging again and removing the saline, the organisms were suspended in Diamond's medium minus serum. The suspension was then standardized by hemocytometer counts to 2 X 106 organisms per ml. White mice, 2 to 3 months old (National Lab, St. Louis, Mo.), were injected intraperitoneally with 106 organisms. The mice were observed for 2 weeks, deaths and gross pathological appearance of the dead being recorded during this interval. Checks for possible bacterial contamination of cultures used for inoculation were made using brain-heart infusion broth and thioglycollate broth. Also, the lesions of randomly selected experimental animals were checked for possible bacterial contamination. Strains of T. vaginalis that were to be frozen were inoculated into bottles containing 150 ml of Diamond's medium minus agar. After 48 to 72 hr incubation at 37 C in a nitrogen atmosphere the cultures were centrifuged at 375 g for 6 min and the supernatant fluid discarded. The organisms were suspended in fresh medium containing 10% glycerol to give a final concentration of 200 X 106 organisms per ml. The suspension was dispensed into small ampules in 0.1 to 0.2 ml amounts and the ampules sealed with a gas torch. The ampules were placed in a cotton-lined metal container and stored in a -43 C deepfreeze. The following day the ampules were transferred to precooled vacuum jars. After 8 weeks the frozen strains were thawed This content downloaded from 207.46.13.128 on Tue, 06 Sep 2016 04:36:42 UTC All use subject to http://about.jstor.org/terms LINDGREN AND IVEY-CULTURE AND VIRULENCE IN TRICHOMONAS 227 TABLE I. Effect of prolonged cultivation on the virulence of strains of Trichomonas vaginalis for mice injected intraperitoneally. Strains Weeks in Number of Average number days culture deaths for death to occur 1 3 0/10 1 12 0/10 2 3 6/10 4.7 2 12 3/10 8.3 3 3 7/10 8.6 3 15 1/10 13.0 4 3 3/10 8.7 4 15 6/10 10.0 7 3 5/10 9.6 7 15 5/10 9.4 8 3 9/10 10.3 8 15 1/10 13.0 9 3 8/10 8.5 9 15 1/10 13.0 11 3 0/10 11 15 2/10 12.0 and inoculated into fresh medium. Virulence assays were performed 1 week later.