Infectivity of Trypanosoma brucei-subgroup Flagellates Maintained in Culture

Abstract

Virulence and infectivity of 4 Trypanosoma brucei-subgroup pleomorphic strains: T. brucei EATRO (E) 795 and 1587, and T. rhodesiense E1895 and Bechuanaland (B) 182A, cultivated in modified Tobie's medium (Tm), were tested in C57BL/6J mice. The 3 freshly isolated EATRO strains could be maintained in Tm by serial transfers, but B182A strain, with a history of , 29 syringe passages in laboratory rodents, was difficult to cultivate. Populations of this latter strain contained many rounded and multinucleate forms both in the bloodstream of mice and in culture. Inocula with identical trypanosome numbers were used in all experiments. Strains E795 and E1895 lost all infectivity for normal mice by the 3rd day of cultivation and strain E1587 remained infective for , 40% of the inoculated animals on the 4th day; even 5-day cultures of strain B182A caused lethal parasitemia in ~ 40% of the infected mice. In all instances, infectivity and virulence of cultured trypanosomes could be correlated with percentages of nontransformed bloodstream forms (LS, ISS, SS) present in the cultures, no infection apparently being caused by cultures with less than 20% of such forms. Judging from the survival lengths of mice, the 3 EATRO strains were about equally virulent, while virulence of T. rhodesiense B182A trypanosomes was significantly higher. Essentially identical results were obtained with strain E1895 after 1, 2, or 3 syringe passages in mice. Among hydrocortisone-treated mice, lethal parasitemias (in , 40% of animals) resulted from inoculations of strains E795 and E1895 maintained in Tm medium for up to 6 days, less than 10% of bloodstream forms present in cultures being sufficient for the development of infection in animals whose immune mechanisms were affected by the hormone. The period for which cultures of strain E1895 remained infective and virulent for normal mice could be extended to 5 days by maintaining the parasites in the presence of L cell cultures. Under these conditions, transformation of bloodstream to culture forms appeared to be delayed. The implications of all the results are discussed in the light of the available morphologic, biochemical, and immunologic data. Trypanosoma brucei-subgroup pleomorphic strains rapidly lose infectivity and virulence for mice in the course of in vitro cultivation (for pertinent references see Honigberg, 1967). According to certain early reports (Behrens, 1914; Novy and MacNeal, 1904) these attributes were not lost completely in trypanosomes grown in NNN-type media. Several more recent workers were able to maintain or restore virulence and infectivity for mice in trypanosomes cultivated in Weinman's (1946) medium, but only after addition of trehalose (Weinman, 1957, 1960), arabinose, or inositol (Geigy and Received for publication 25 May 1972. * This investigation was supported by Research Grants AI00742-16, 17, from NIAID, U. S. Public Health Service. t Dr. Yolanda Mendez was a Research Associate in Zoology at the University of Massachusetts, Amherst, from September 1970 to June 1972. Her present address is P. O. Box 3410, Panama City 4, Panama. + Reprint requests should be sent to Dr. B. M. Honigberg, Department of Zoology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01002. Kauffmann, 1964). Other investigators, however, were unable to verify these results (Lehmann, 1961; Williamson in Geigy and Kauffmann, 1964, among others), and according to Bowman et al. (1960) the culture forms could not utilize trehalose. The source of blood used in the preparation of Weinman's medium and the age of cultures also were found to affect infectivity and virulence of the parasites (Amrein and Hanneman, 1969; Amrein et al., 1965). Until quite recently, it has been assumed that the infectivity and virulence loss among T. brucei-subgroup trypanosomes was primarily a physiologic phenomenon, not necessarily related to structural changes. Indeed, light-microscopic observations indicated that trypomastigotes, virtually indistinguishable in structure from the metacyclic forms occurring in tsetse salivary glands, developed in cultures of Trypanosoma rhodesiense and Trypanosoma gambiense strains maintained in different media (Lehmann, 1961; Weinman, 1953). The question still remains, however, whether trypano-