The Axenic Cultivation of Insect Forms of Trypanosoma (Duttonella) vivax and Development to the Infective Metacyclic Stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27°C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine, hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9–12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.     

Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by 'in vitro' culture

Abstract By culturing Trypanosoma cruzi epimastigotes in modified Grace's medium with 10% foetal bovine serum, a significant quantity of metacyclic forms could be obtained. Transformation was observed after 8 days of culture, with metacyclic forms reaching 75%. Cultured Vero cells were infected with metacyclic forms and maintained until free-amastigote forms were obtained. Additionally, amastigote-like forms could be obtained by subjecting metacyclic cultures to heat shock. Parasites were grown with glucose as the major carbon source. The metabolites produced and excreted during culture were identified by difference proton nuclear magnetic resonance spectroscopy and quantified by enzymatic methods. The final products of glucose catabolism differed not only quantitatively but also qualitatively for the three major life-cycle stages of T. cruzi . The end products of metabolism produced by epimastigote forms were mainly acetate and pyruvate and, to a lesser extend, l-alanine and ethanol. Differences between epimastigotes and metacyclic forms were only quantitative. However, free amastigotes as well as amastigote-like forms, excreted acetate, glycerol, and pyruvate and to a lesser extent succinate, but no l-alanine or ethanol.     

Genetic engineering of Trypanosoma (Dutonella) vivax and in vitro differentiation under axenic conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT 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° 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° 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.     

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.     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.     

Flagellar elongation induced by glucose limitation is preadaptive for Trypanosoma cruzi differentiation

Trypanosomes must sense and respond to environmental change in order to progress through their life cycles. The American trypanosome, Trypanosoma cruzi, differentiates from the noninfective epimastigote form to the infective metacyclic form spontaneously in axenic culture. Here, we investigate the initial stimulus for that change and demonstrate that T. cruzi epimastigotes sense limitation of glucose in the medium and respond by undergoing significant morphological and biochemical change. As part of this change, the mean flagellar length of the population triples, which is correlated with an increased ability to maintain interactions with hydrophobic substrates, a requirement for differentiation to the next life cycle stage.     

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H₂O₂. However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H₂O₂. Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas’ disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H₂O₂ concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H₂O₂ exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.     

Differential behaviour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi

1. Glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) of two morphological forms of Trypanosoma cruzi, epimastigotes and metacyclics, are reported. 2. The kinetic behaviour and some of the kinetic parameters of the enzyme in both forms were studied. The enzymes showed a simple Michaelis-Menten kinetic. 3. The activity in epimastigote forms was alway higher than the metacyclic ones. At subsaturating concentrations of substrate was almost 10-fold higher, whereas at saturating concentrations was about 2-fold higher. 4. In epimastigote forms the specific activity and Km values, at pH 7.5 and 37 degrees C, was found to be 142 mUnits x mg-1 of protein and 0.23 mM, respectively. 5. In the same conditions, the specific activity and Km values in metacyclic forms was 75 mUnits x mg-1 of protein and 1.06 mM, respectively. 6. A possible role in the carbohydrate metabolism of glucose 6-phosphate dehydrogenase in both forms of Trypanosoma cruzi is discussed.     

Crossed-immunoelectrophoretic analyses of Trypanosoma cruzi epimastigote, metacyclic, and bloodstream forms

Sonicated suspensions of epimastigote, metacyclic, or bloodstream forms of Trypanosoma cruzi were emulsified in Freund's complete adjuvant. Rabbits immunized with epimastigotes or metacyclics received five intramuscular (i.m.) injections of 1 x 10(9) sonicated trypanosomes at weekly intervals. Immunization with bloodstream forms included three i.m. injections of 5 x 10(7) and six injections of 2 x 10(8) sonicated trypanosomes. Selected antisera from these rabbits were employed in crossed immunoelectrophoretic studies against the homologous or heterologous extracts of sonicated trypanosomes. Extracts of epimastigote, metacyclic, and trypomastigotes produced 31, 29, and 11 precipitin peaks respectively against the homologous rabbit antisera. Tandem, crossed-immunoelectrophoresis of these extracts against antiepimastigote or antimetacyclic sera revealed that epimastigotes or metacyclics may each have at least four antigens that did not appear to be shared by the other, whereas each of these forms may have at least eight or nine antigens that were not detected with extracts from trypomastigotes. Cross-absorptions of antiepimastigote or antimetacyclic sera with live trypanosomes caused marked reductions in the numbers of precipitin peaks formed against the homologous extracts, but cross-absorptions with sonicated suspensions of epimastigotes or metacyclics showed that epimastigotes or metacyclics each have at least two antigens that were not detected in extracts of the other. Differentiation appeared to be accompanied by antigenic change. More antigens appear to be shared by epimastigotes and metacyclic forms than by trypomastigotes and epimastigotes or metacyclics.     

Differentiation of Trypanosoma cruzi epimastigotes: metacyclogenesis and adhesion to substrate are triggered by nutritional stress

Differentiation of Trypanosoma cruzi epimastigotes to metacyclic trypomastigotes occurs in the insect rectum, after adhesion of the epimastigotes to the intestinal wall. We investigated the effect of the nutritional stress on the metacyclogenesis process in vitro by incubating epimastigotes in the chemically defined TAU3AAG medium supplemented with different nutrients. Addition of fetal bovine serum induced epimastigote growth but inhibited metacyclogenesis. In this medium, few parasites attached to the substrate. Ultrastructural analysis demonstrated reservosomes at the posterior end of the epimastigotes. Incubation of the cells in TAU3AAG medium containing gold-labeled transferrin resulted in high endocytosis of the marker by both adhered and free-swimming epimastigotes. No intracellular gold particles could be detected in trypomastigotes. Addition of transferrin gold complexes to adhered epimastigotes cultivated for 4 days in TAU3AAG medium resulted in decrease of both metacyclogenesis and adhesion to the substrate, as compared with parasites maintained in transferrin-free medium. Adhesion to the substrate is triggered by nutritional stress, and proteins accumulated in reservosomes are used as energy source during the differentiation. A close relationship exists among nutritional stress, endocytosis of nutrients, adhesion to the substrate, and cell differentiation in T. cruzi epimastigotes.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

Trypanosoma cruzi: characterization of an intracellular epimastigote-like form.

Almeida-de-Faria, M., Freymüller, E., Colli, W., and Alves, M. J. M. 1999. Trypanosoma cruzi: Characterization of an intracellular epimastigote-like form. Experimental Parasitology 92, 263-274. A detailed study of transient epimastigote-like forms as intermediates in the differentiation of Trypanosoma cruzi amastigotes to trypomastigotes inside the host cell cytoplasm was undertaken using the CL-14 clone grown in cells maintained at 33 degrees C. Several parameters related to these forms have been compared with epimastigotes and other stages of the parasite. Consequently, the designation of intracellular epimastigotes is proposed for these forms. Despite being five times shorter (5.4 +/- 0.7 micrometer) than the extracellular epimastigote (25.2 +/- 2.1 micrometer), the overall morphology of the intracellular epimastigote is very similar to a bona fide epimastigote, when cell shape, position, and general aspect of organelles are compared by transmission electron microscopy. Epimastigotes from both sources are lysed by human complement and bind to DEAE-cellulose, in contrast to amastigotes and trypomastigote forms. A monoclonal antibody (3C5) reacts with both epimastigotes either isolated from axenic media or intracellular and very faintly with amastigotes, but not with trypomastigotes. Some differences of a quantitative nature are apparent between the two epimastigote forms when reactivities with lectins or stage-specific antibodies are compared, revealing the transient nature of the intracellular epimastigote. The epitope recognized by 3C5 monoclonal antibody reacts slightly more intensely with extracellular than with intracellular epimastigotes, as detected by immunoelectron microscopy. Also a very faint reaction of the intracellular epimastigotes was observed with monoclonal antibody 2C2, an antibody which recognizes a glycoprotein specific for the amastigote stage. Biological parameters as growth curves in axenic media and inhability to invade nonphagocytic tissue-cultured cells are similar in the epimastigotes from both origins. It is proposed that the epimastigote-like forms are an obligatory transitional stage in the transformation of amastigotes to trypomastigotes with a variable time of permanency in the host cell cytoplasm depending on environmental conditions.     

In vitro effects of citral on trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.     

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.     

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

, , and 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology 16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH₄Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms.

The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.     

Inhibition of mammalian host cell infection by insect-derived, metacyclic forms of Trypanosoma cruzi in the presence of human or rabbit anti-T. cruzi antibodies

The presence of serum from chronic chagasic patients or rabbits immunized with killed epimastigote forms of Trypanosoma cruzi inhibited infection of rat heart myoblasts by insect-vector (Triatoma infestans)-derived, metacyclic forms of Trypanosoma cruzi. The effect was produced even after diluting the chagasic serum to non-agglutinating levels and was evidenced by marked reductions in both the percentage of infected myoblasts and the number of parasites per 100 cells. Human IgG or IgM purified from chronic chagasic serum and serum from rabbits immunized with killed T. cruzi epimastigotes also reduced both parameters. While previous work has shown that immunological destruction of invasive forms of T. cruzi may underlie the protective effects of the humoral immune response against this parasite, the present in vitro results suggest that specific anti- T. cruzi antibodies could also contribute to protection via inhibition of host cell infection by the vectortransmissible form of the parasite.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Trypanosoma brucei: in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans

1 Metacyclic forms of Trypanosoma brucei obtained from the salivary glands of the tsetse fly, Glossina morsitans have been cultured for the first time in their infective forms for more than 200 days in continuous culture. The parasites were grown at 25 C and 30 C on a bovine embryonic spleen (BESP) feeder layer in buffered RPMI 1640 medium supplemented with 20% heat-inactivated bovine fetal serum (BFS) and 5% lactalbumin hydrolysate. Initial growth rate was enhanced when normal, noninfected, salivary glands were added to the cultures. The parasites thus cultured appeared like slender or intermediate blood stream forms which were infective to rats and mice. Addition of rat anti-T. brucei specific antiserum to the cultures caused agglutination of the parasites and rendered them noninfective. This study opens up new areas of investigating sleeping sickness. The cultured metacyclic parasites have the potential of being applied as antigens for controlling African trypanosomiasis.     

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.