Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)

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Bloodstream forms of Trypanosoma congolense (2 clones: ILNat3.1 and IL3000, and 4 stocks: IL2079, IL2466, IL3266 and CP-81) were continuously cultivated in vitro at 34-36 degrees C in the absence of feeder cell layers, using HMI-93 medium which was modified from Iscove's modified Dulbecco's MEM (Flow Laboratories, Irvine, Scotland). The modification was done by supplementing the medium with 0.05 mM bathocuproine sulphonate, 1.5 mM L-cysteine, 0.5 mM hypoxanthine, 0.12 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.16 mM thymidine, 20% (v/v) heat-inactivated young goat serum and 5% (v/v) Serum Plus (Hazleton Biologics, Lenaxa, KS, USA). Trypanosomes obtained from two different sources were used to initiate primary cultures: (1) metacyclic forms which were produced in vitro at 27 degrees C, and (2) bloodstream forms obtained from Balb/c mice which had been infected with the bloodstream forms transformed in vitro from the metacyclic forms. Metacyclic forms placed in 25 cm2 T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4-5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 microliters of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 10(6) bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice.

SummaryClones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 102–103/well on days 4–16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2–5 × 105 trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1–2 ml of the trypanosome suspension to a new culture flask at 4–5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1·4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8·7, 14·5 and 15·5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8–32 days from cloning. One cloned population of TC52 consisted of 99·8% VAT 052 and 0·2% VAT 221 at the time when the first VAT test was made on day 18.

STJMMAEYEighteen clones of variable antigen type 052 of Trypanosoma brucei stock S. 427 were derived and maintained as animal-infective bloodstream forms in vitro for up to 60 days of cultivation. The antigenic composition of such clones was monitored weekly by immunofluorescent analysis of viable trypanosomes, using antisera raised to isolated variant-specific surface glycoproteins of both 052 and a variable antigen type (221) which consistently appeared in the first relapse population of type 052 in vitro. The appearance of new variants was detected in 9 of the 18 clones 18-46 days following initiation of the clone and variable antigen type 221 was found in all 9 clones. On one or more occasions in 8 of such clones, viable trypanosomes were found which did not react with either antiserum but were mouse-infective on the 4 occasions tested and probably represent other variable antigen types. The process of antigen, variation in vitro appears to resemble the process in vivo except that new variant types are detected earlier in vivo. This possibly results from different growth rates of the trypanosomes in vivo and in vitro, together with the fact that elimination of the initial variant population by the host's immune response facilitates the detection of newly arising variable antigen types in vivo.