Trypanosoma rangeli--in vitro metacyclogenesis and fate of metacyclic trypomastigotes after infection to mice and fibroblast cultures

High metacyclogenesis was induced when freshly-isolated Trypanosoma rangeli from humans were grown in a modified liver-infusion-tryptose medium and transferred into the medium overlaid on mouse fibroblasts at 27 degrees C in a 5% CO2 atmosphere. Such in vitro-generated metacyclic trypomastigotes could induce a significantly high and constant parasitemia in both ICR and SCID mice for a period of about a week but thereafter the parasitemia gradually decreased. Histological examination could not detect any tissue-forms of T. rangeli in various organs of SCID mice. On the other hand, two long-maintained stocks of T. rangeli produced lower metacyclogenesis and only latent parasitemia in both strains of mice. When these populations were incubated in fibroblast cultures at 37 degrees C in a 5% CO2 atmosphere, only trypomastigotes survived for two to three weeks without proliferation, while other forms, mainly epimastigotes, soon began to swell and degenerate. Electron microscopy showed that most surviving trypomastigotes had the basket-like conformation of the kinetoplasts. This is characteristic of the non-dividing trypomastigote stage of T. cruzi, and suggests that T. rangeli trypomastigotes may survive long periods in the blood without proliferation.     

Biological aspects of the Dm 28c clone of Trypanosoma cruzi after metacyclogenesis in chemically defined media

The biological characterization of the Trypanosoma cruzi clone Dm 28c in terms of its growth in LIT medium, cell-cycle, infectivity to mice and interaction with professional and non-professional phagocytic cells shows that it behaves as a bona fide T. cruzi representant. The biological properties of this myotropic clone do not change according to the origin of the trypomastigote forms (i. e., from triatomines, infected mice, cell-culture or from the chemically defined TAUP and TAU3AAG media). In addition Dm 28c metacyclic trypomastigotes from TAU3AAG medium display a high infectivity level to fibroblasts and muscle cells. Experiments on binding of cationized ferritin to trypomastigotes surface show the existence of cap-like structures of ferritin in regions near the kinetoplast, however the nature and role of these anionic sites remain to be determined. The results indicate that metacyclic trypomastigotes from the Dm 28c clone obtained under chemically defined conditions reproduce the biological behaviour of T. cruzi, rendering this system very suitable for the study of cell-parasite interactions and for the isolation of trypanosome relevant macromolecules.     

Developmental stages of Trypanosoma cruzi-like flagellates in Cavernicola pilosa

The developmental stages of Trypanosoma cruzi ssp., found in the intestinal tract of Cavernicola pilosa, are described and measurements given for nine life stages. The frequencies of the various stages in foregut, midgut and hindgut of the triatomines are provided; parasites were rare in the foregut and metatrypomastigotes were seen only in the mid- and hindguts. All adult bugs examined harboured intestinal infections of T. cruzi-like flagellates, large clumps of amastigotes were frequently observed in the midgut. The faeces of C. pilosa, containing metacyclic trypomastigotes, did not produce patent parasitaemia when inoculated into mice. Inoculated mice were not protected against subsequent challenge infections with the highly virulent Tulahuen stock of T. c. cruzi. The blood of bats also failed to produce parasitaemia when inoculated into mice, nor were the mice protected against subsequent challenges with T. c. cruzi. Although the developmental stages described were very similar to those of T. c. cruzi it is presumed that they were stages of T. c. marinkellei because of their failure to infect mice and Rhodnius prolixus, and their failure to protect inoculated mice against challenge with T. c. cruzi.     

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choach -2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.     

Rhodnius prolixus infected with Trypanosoma rangeli: In vivo and in vitro experiments

Studies were carried out on the activation of the prophenoloxidase (proPO) in adults of Rhodnius prolixus infected by short and long epimastigote forms of Trypanosoma rangeli. The in vitro activation of the proPO cascade using l-DOPA as substrate was very low in the absence of fat body extract, hemolymph, and parasites. On the other hand, a higher PO activity was observed when short, but not long, epimastigotes of T. rangeli were incubated with fresh hemolymph, fat body extract, and l-DOPA. Supernatant from lysed long epimastigotes increased the PO activity at levels identical to those observed with supernatants from lysed short epimastigotes. Similarly, the PO activity of hemolymph obtained from inoculated insects with long epimastigotes of T. rangeli showed a very low activity when incubated with l-DOPA compared to the PO activity of hemolymph taken from insects inoculated with short epimastigotes of T. rangeli. Control insects inoculated with sterile PBS showed no PO activity. These data indicate the presence of (a) factor(s) in the hemolymph as well as in the fat body extract that may be released (or induced) by the presence of short epimastigotes of T. rangeli and which results in the activation of the R. prolixus proPO system. The implications of these findings are discussed in relation to the development of T. rangeli and its ability to overcome the proPO system, survive, and successfully colonize the hemolymph of R. prolixus.     

Studies on Trypanosoma rangeli Tejera, 1920. VI. Developmental pattern in the haemolymph of Rhodnius prolixus

The morphological sequence of Trypanosoma rangeli development in the body cavity of Rhodnius prolixus is described. The metacyclic trypanosome is the product of successive division and transformation during the intra and extracellular development in the haemocoele. The significance of the early invasion of T. rangeli into the haemolymph is discussed. The epidemiological importance of the developmental pattern of T. rangeli in the vectors haemolymph and the host-response to the parasite are considered.     

Effects of eicosanoid biosynthesis inhibitors on the prophenoloxidase-activating system and microaggregation reactions in the hemolymph of Rhodnius prolixus infected with Trypanosoma rangeli

Investigations on the effects of eicosanoid biosynthesis inhibitors on the hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, parasitemia and mortality of Rhodnius prolixus infected with Trypanosoma rangeli were performed. Hemocoelic injection of live T. rangeli epimastigotes into fifth-instar larvae of R. prolixus that previously fed on blood containing an inhibitor of phospholipase A(2) (dexamethasone), a specific inhibitor of the cyclooxygenase pathway (indomethacin), and a non-selective lipoxygenase inhibitor (NDGA) (i) reduced the hemocyte microaggregation, (ii) attenuated the proPO system in the hemolymph and (iii) enhanced parasitemia and mortality induced by the parasite challenge in these insects. The effects obtained by dexamethasone administered orally were counteracted by inoculation of the insects with arachidonic acid. We suggest that the infectivity of T. rangeli can be increased by interference with the R. prolixus immune system. This is the first demonstration that the triatomine's immune responses to a parasite infection are modulated by a physiological system that includes eicosanoid biosynthesis.     

Trypanosoma (Herpetosoma) rangeli Tejera, 1920: mouse model for high, sustained parasitemia

The Neotropical mammalian parasite Trypanosoma (Herpetosoma) rangeli Tejera, 1920 is difficult to study due to the scarcity of blood forms in the vertebrate host. High and persistent parasitemias (up to 7 times the original inoculum at the peak, and persisting for up to 2 wk) were obtained by i.p. inoculation of infant (6.0 g) male white mice (NMRI strain) with 15 X 10(3) trypomastigotes/g body weight from 12-day-old cultures of the "Dog-82" strain of T. rangeli. This strain was cultured 15 mo at ambient temperature in LIT medium, modified by substituting defibrinated adult rabbit blood for fetal calf serum. These results underlined the importance of host age and time of culture of the parasite as factors influencing levels of parasitemia. The abrupt decline in the parasitemias may be due to an early development of a strong immunological response. Negative xenodiagnoses with Rhodnius prolixus may be due either to sterile immunity in the host mice, or to the low susceptibility of the strain of Rhodnius used. Concurrent experiments established that the T. rangeli strain was not naturally contaminated with T. cruzi.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Evolution of infection in mice inoculated by the oral route with different developmental forms of Trypanosoma cruzi I and II

Oral infection has become the most important transmission mechanism of Chagas disease in Brazil. For this study, the development of Trypanosoma cruzi infection in mice, induced by the oral and intraperitoneal (IP) routes, was compared. Four groups of Swiss mice were used to evaluate the influence of parasite genetics, number of parasites, inoculation volume and developmental stages on the development of the orally induced infection: 1 – blood trypomastigotes (BT) via oral; 2 – BT via IP; 3 – culture metacyclic trypomastigotes (MT) via oral; and 4 – culture MT via IP. Animals inoculated orally showed levels of parasitemia, as well as infectivity and mortality rates, lower than animals inoculated via IP, regardless of DTU (discrete typing unit) and inoculum. Animals infected with TcII showed higher levels of these parameters than did animals infected with TcI. The larger volume of inoculum showed a greater capacity to cause an infection when administered via the oral route. BT infection was more virulent than culture MT infection for both routes (oral and IP). However, mice inoculated orally with BT showed lower levels than via IP, while mice inoculated orally with culture MT showed similar levels of infection to those inoculated via IP. Mice inoculated with culture MT showed more histopathological changes than those inoculated with BT, regardless of the inoculation route. These results indicate that this alternative experimental model is useful for evaluating infection by T. cruzi isolates with subpatent parasitemia and low virulence, such as those belonging to the TcI and TcIV DTUs, which are prevalent in outbreaks of orally transmitted Chagas disease.     

Inhibition of hemocyte microaggregation reactions in Rhodnius prolixus larvae orally infected with Trypanosoma rangeli

Hemocoelic inoculation of epimastigotes of Trypanosoma rangeli strain H14 into 5th-instar larvae of Rhodnius prolixus previously fed on blood containing the same parasites, showed reduced number of hemocyte microaggregates in the hemolymph, enhanced number of flagellates in the hemolymph as well as increased mortality of these insects. All these effects were counteracted by combined inoculation of R. prolixus with T. rangeli and arachidonic acid. In vitro assays using hemolymph taken from insects previously fed on blood containing parasites showed that hemocyte microaggregation reactions were also attenuated when T. rangeli is used as inducer of the reaction, and that simultaneous applying T. rangeli with arachidonic counteracted the hemocyte microaggregation inhibition. We suggest that arachidonic acid pathway can be a mediator of hemocyte microaggregation reactions in the hemolymph of insects inoculated with T. rangeli, and that oral infection with this protozoan inhibits the release of arachidonic acid.     

Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus

Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus.     

Studies on Trypanosoma rangeli Tejera, 1920. VII--Its effect on the survival of infected triatomine bugs

The pathological effects of Trypanosoma rangeli on Rhodnius prolixus and R. robustus, and the relation of mortality to infection, were studied under laboratory conditions. Frequent observations revealed that when the first instar nymphs of R. prolixus and R. robustus were infected with T. rangeli, survival of the bugs during the stages of development to the adult stage decreased. This decrease was statistically significant when compared with uninfected control-bugs, indicating that T. rangeli is pathogenic for both species of triatomine. In R. prolixus the most affected nymphal stages were the first, second and fifth instars, where a higher mortality was also observed. In R. robustus a progressive increase of the mortality from the first to fifth instars, was observed. The pathogenicity of T. rangeli as measured by overall mortality was the same in R. prolixus and R. robustus. The possible pathogenic mechanism of T. rangeli in triatomine-bugs and its epidemiological implications, are discussed.     

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.     

Infectivity and route of penetration in rats after oral and intraperitoneal inoculations of bloodstream and in vitro-cultured metacyclic forms of Trypanosoma lewisi

Morphological changes of Trypanosoma lewisi blood trypomastigotes cultured in Schneider's Drosophila medium (SDM), supplemented or not with uric acid (SDM + UA), were compared to those that occurred in a control medium (M-199). No difference in trypanosome morphology and numbers was observed between SDM + UA and SDM cultures; there was little transformation into metacyclic stages in M-199. No difference was observed between the capacity of SDM- or SDM + UA-cultured metacyclic stages to infect rats. The infectivity of bloodstream forms was always higher than that of the SDM- or SDM + UA-cultured forms, whether inoculated orally or intraperitoneally. The oral inoculation of rats with tritium-labeled culture and bloodstream forms showed that the metatrypanosomes from the cultures remained longer in the salivary glands and tongue of the animal than the blood trypanosomes.     

Trypanosoma (Herpetosoma) leeuwenhoeki in Choloepus hoffmanni and Didelphis marsupialis of the Pacific Coast of Colombia

Trypanosoma (Herpetosoma) leeuwenhoeki, originally described in Panamanian sloths, was isolated from Didelphis marsupialis (Marsupialia) and Choloepus hoffmanni (Edentata) inhabiting the Pacific coast of Colombia. Trypanosomes were characterized by their large blood forms (total length 51-53 microns), poor infectivity for mice, and lack of development in Rhodnius prolixus. Isoenzyme studies, with either strains or clones, revealed homogeneous profiles clearly distinct from Trypanosoma cruzi and Trypanosoma rangeli reference strains. The present report extends the geographical distribution of T. leeuwenhoeki to South America and broadens its known host range to another order of mammals.     

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.