Antigenic Analysis By Immunofluorescence of In Vitro-Produced Metacyclics of Trypanosoma Brucei and Their Infections In Mice*

SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

Infectivity of Trypanosoma rhodesiense Cultivated at 28°C with Various Tsetse Fly Tissues1

ABSTRACT. Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28°C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7–16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUM 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 545, and TRUM 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes ceased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium “conditioned” by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

Infectivity of Trypanosoma rhodesiense cultivated at 28 degrees C with various tsetse fly tissues

Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28 degrees C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7-16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUm 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 454, and TRUm 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes eased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium "conditioned" by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

Antigenic analysis by agglutination of Trypanosoma brucei brucei parasitemias initiated in mice with in vitro-produced metacyclics.

Trypanosomes from 14 first-peak parasitemias initiated in mice by injection of in vitro-produced metacyclics were stabilated. Strains derived from these stabilates were analyzed for their antigenic composition by cross-agglutination with immune sera produced in rabbits against 12 of the stabilates. The antigenic composition of the 14 stabilates was compared also with two first-peak parasitemias from mice inoculated with fly-derived metacyclics, the variant-specific antigen of the strain used to initiate the cultures that ultimately became infective, and the antigenic variant that was used to infect the flies. One variant-specific, presumably basic, antigen was found, either as the predominant (nine parasitemias) or as a minor (seven parasitemias) antigen, in all first peak-parasitemia strain initiated with culture- or fly-derived metacyclics; it was absent, however, from the strains (not first-peak parasitemias) used to start the cultures or to infect the flies. Only one of the first-peak parasitemias appeared to have the basic antigen alone. The remaining parasitemia populations seemed to have from about two to six antigens, some of which were common to culture- and fly-derived infections. There was very little, if any, antigenic relationship between the foregoing populations and the strains employed for initiation of cultures or for infection of flies. It is evident from the results that much antigenic similarity exists between the culture- and tsetse fly-derived first-peak parasitemias.     

Studies on the development of metacyclic Trypanosoma brucei sspp. cultivated at 27 degrees C with insect cell lines

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T.b. rhodesiense were grown at 27 degrees C in 25-cm2 flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 X 10(5) metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics

Okanla E. O., Stumpf J. L. &; Dusanic D. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics. International Journal for Parasitology12: 251–256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosoma cruzi in three weekly injections of 1 × 10⁸ trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund's complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestans. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestans included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and blood-stream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infestans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector.     

The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.     

Trypanocidal drugs and the role of kidney forms of Trypanosoma (Herpetosoma) musculi in immunity of mice against reinfection

Adrenals, hearts, kidneys, livers, lungs, and spleens were removed from C3H/Anf mice which had been inoculated with Trypanosoma (Herpetosoma) musculi and no longer exhibited parasitemias. Imprints of each organ were examined microscopically, and each was homogenized and injected into recipient mice. It was confirmed that trypanosomes could be detected only in the donor kidneys. Lampit or Ethidium treatment eliminated bloodstream and kidney forms when administration was initiated after the development of patent parasitemias. However, mice treated with Lampit on the same day they were inoculated with T. musculi developed parasitemias later than animals injected with drug after parasites had appeared in their blood. Both Lampit and Ethidium depressed antibody production as detected in enzyme-linked immunosorbent assays of antisera from animals having parasitemias at the time of treatment. The elimination of kidney forms by Lampit or Ethidium treatment did not reduce the resistance of mice to reinfection by T. musculi 12 weeks or 15 and 22 weeks, respectively, after the initial inoculation of these animals with the parasites. Kidney forms were not required for the sustained protective immunity of the mice against reinfection during the intervals of these experiments.     

Studies on the Development of Metacyclic Trypanosoma brucei sspp. Cultivated at 27°C with Insect Cell Lines1

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T. b. rhodesiense were grown at 27°C in 25-cm² flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 × 10⁵ metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei

Clones 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 10(2)--10(3)/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 x 15(5) 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.     

In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei,T. rhodesiense,and T. gambiense1

ABSTRACT. A series of new in vitro systems for the cultivation of bloodstream forms of Trypanosoma (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense, and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast-like cells derived from embryos of New Zealand White rabbits (REF) or a mountain vole, Microtus montanus (MEF), with HEPES-buffered Minimum Essential Medium (MEM), with Earle's salts, supplemented with 15% inactivated rabbit serum. These two and other feeder layers were cross-checked with different sera to test for growth support of bloodstream forms of the three trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be used as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some of the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by electron microscopy. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieved with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.     

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei

Variant antigenic types (VATs) represented in a total of 47 first relapse populations of 6 clones of Trypanosoma brucei LUMP 227 were identified by immunofluorescent staining of living trypanosomes, using antiserum raised against purified surface antigens. The relative growth rates of these 6 clones were measured both individually and when grown together in a mixed population, and were found to be different under these two sets of conditions. A pattern emerged in the VATs represented in relapses of each clone, with some types being expressed more frequently than others and certain VATs being only very rarely expressed. It is suggested that new VATs are expressed according to a statistically definable order of priority which is different for each parent VAT, and that some VATs may be able to change to certain others only after passing through an intermediate VAT. The order of priority of appearance of VATs does not appear to correlate with growth rate measured either in individual clones or when clones are grown in a mixed population.     

The activity of nifluridide on reduviids and rodent and human trypanosomes

The ability of nifluridide to kill reduviids was assayed in mice fed 7 ppm in diet and on cattle injected subcutaneously at 5 mg/kg body weight. Nifluridide was systemically active against Triatoma infestans on mice and Rhodnius prolixus on cattle. No effects on Trypanosoma (Schizotrypanum) cruzi could be detected in the intestinal contents of Triatoma infestans killed by the compound. In vitro and in vivo studies were conducted to determine the effects of nifluridide on trypanosomes growing in medium and in experimentally infected mice. Culture forms of Trypanosoma cruzi grown at 27 degrees C that are morphologically similar to epimastigotes found in infected bugs were affected by 2.5 to 10 ppm in the medium. Mice fed nifluridide in the diet simultaneous with infection of Trypanosoma cruzi or Trypanosoma (Herpetosoma) musculi exhibited parasitemias and tissue infections similar to nontreated infected mice. At the concentration tested, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma musculi were unaffected by nifluridide. Only the culture epimastigotes of Trypanosoma cruzi were affected by the drug but not the bloodstream and tissue forms.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Nonvariant Antigens Limited to Bloodstream Forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense1

ABSTRACT. The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and Immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled ～3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Antigenic variation during the developmental cycle of Trypanosoma brucei

During the complex life cycle of Trypanosoma brucei, changes in the exposed surface antigens occur in both the mammalian host and the insect vector (Glossina spp.). These antigenic changes are associated with alterations of the variant surface glycoprotein (VSG) composition or with the loss of the VSG. In the bloodstream of the mammalian host, trypanosomes successfully evade destruction by the host's immune response by continuously expressing alternative VSGs, at low frequency, which are not destroyed by host antibodies. When ingested by the tsetse fly, the bloodstream trypanosomes rapidly lose their surface coat and surface membrane antigens are exposed which are normally covered in the bloodstream. In the salivary glands of the tsetse fly, the trypanosomes differentiate to the metacyclic stage, which reacquires a surface coat. The antigenic composition of the metacyclics is heterogeneous. The same metacyclic types are expressed regardless of the bloodstream antigenic type ingested by the tsetse fly. In the mammal the metacyclics differentiate to long-slender bloodstream forms but continue to express the metacyclic VSG for at least three days. The next VSGs expressed in the mammalian host appear to be influenced by the antigenic type ingested by the tsetse. The ingested antigenic type is often expressed in the first parasitemia following expression of the metacyclic antigenic types.