Effect of Certain Laboratory Procedures on Virulence of the Jones' Barn Strain of Trichomonas gallinae for Pigeons

Abstract

Continued in vitro cultivation at 37.5 C of the highly virulent Jones' Barn strain of Trichomonas gallinae was accompanied by gradual loss of pathogenicity for Trichomonas-free, nonimmune domestic pigeons. This virulence loss was about twice as rapid when the organisms were isolated with the use of penicillin and dihydrostreptomycin as when they were isolated without such agents. Pathogenicity was rapidly restored, however, to its original virulence level by serial pigeon-topigeon passage of the trichomonads. Whereas attenuation occurred in organisms maintained at incubator temperatures, virulent trichomonads frozen and maintained at subzero temperatures (-19 C and -72 C) showed no decrease in pathogenicity when thawed and placed in appropriate pigeons following 52 weeks at -72 C. Trichomonads maintained at 37.5 C for 98 weeks became so attenuated that they failed to elicit an immune response in infected birds, a reaction characteristic of normal, avirulent T. gallinae. Both Bos (1934) and Gloor (1943) noted that Trichomonas gallinae lost its virulence for pigeons following long-term in vitro cultivation. Gloor stated that this lost virulence could be partly recovered by the passage of the trichomonads through squabs. Meleney, Frye, and Leathers (1939) felt that long-term cultivation had not been shown to deprive completely any strain of Entamoeba histolytica of its virulence for kittens. The freezing and maintenance of protozoan cultures at subzero temperatures were initiated in an attempt to avoid the chores and consequences of long-term in vitro cultivation. In general, the results have been successful. Manwell (1943) and Wolfson (1945) maintained various species of bird malaria at temperatures as low as -78 C, and for periods up to 1 year. They noted only mild changes in infectivity of the thawed organisms from the controls, and Wolfson reported restored virulence following a few animal passages. Jeffery and Rendtorff (1955), using three malaria species from man, Received for publication 3 September 1963. * This investigation was supported by Research Grant AI-00742 from the National Institute of Allergy and Infectious Diseases, Public Health Service. froze sporozoites and asexual stages to about -70 C and kept them for over a year. The thawed sporozoites showed only slightly longer prepatent periods than those derived from mosquito bites. Weinman and McAllister (1947) maintained a variety of protozoa at -70 C, finding that some of the thawed organisms successfully infected laboratory animals after 20 months freezing. They failed, however, in their experiments with Entamoeba histolytica. On the other hand, Diamond, Meryman, and Kafig (1961, 1963) successfully cultured this ameba (as well as certain trichomonads and trypanosomatids) following its freezing and holding in liquid nitrogen (-196 C) up to 14 months. Much of the work on the freezing and subzero maintenance of trichomonads has been done by McEntegart (1954-Trichomonas vaginalis, T. gallinae, Pentatrichomonas hominis, Tritrichomonas foetus); Levine and Marquardt (1955-T. foetus); Levine and Andersen (1961-T. foetus); Levine, Andersen, Losh, Notzold, and Mehra (1962-T. foetus); Honigberg and King (1962-T. vaginalis, T. gallinae); and Diamond, Meryman, and Kafig (1963-T. vaginalis, T. gallinae, P. hominis, T. foetus). The results indicate that the quick-