The limitation of growth of Trypanosoma musculi in vitro

Trypanosoma musculi grow readily in vitro provided their growth is supported by mammalian cells. In the presence of murine spleen cells, or spleen cell-conditioned medium, the parasites increase by 100-fold, or more, in a period of 5–6 days. Growth ceases abruptly and death of the parasites soon follows. The reason for the termination of growth has been obscure and is the subject of this report. Termination of growth is not due to an immunological process; not even of ablastin affecting epimastigote reproduction. Instead it appears that other growth inhibitory substances are responsible. Culture medium, collected from spent cultures on day 8 after initiation, inhibits T. musculi growth in fresh medium in dose-dependent fashion. No inhibitory substances were present in medium collected earlier, during the phase of rapid parasite growth. These inhibitory substances appeared to be derived from the parasites rather than the cocultivated spleen cells.     

The interaction of Trypanosoma congolense and Haemonchus contortus in Djallonké sheep

The interaction between Trypanosoma congolense and Haemonchus contortus was studied in 5 groups of 8 Djallonké sheep. Two groups received a single infection with either H. contortus or T. congolense, and 2 groups were infected with T. congolense followed by H. contortus (TH) or vice versa (HT). One group was kept as uninfected controls. Mortality due to infection was observed only in the dual infection groups. In the TH group, the effects were more acute whereas in the HT group they were more chronic. No significant differences in weight gain could be demonstrated between infected and control groups. Djallonké sheep are able to withstand a single infection with either T. congolense or H. contortus, which confirms their trypanotolerant nature and provides preliminary indication of resistance against helminth infections. However, when exposed to successive infections with both parasites, some of the animals lose this tolerance.     

In vitro antiprotozoal activity of the lipophilic extracts of different parts of Turkish Pistacia vera L.

Thirteen lipophilic extracts prepared with n-hexane from various parts of Pistacia vera L. tree (Anacardiaceae) growing in Turkey were screened for their in vitro activity against four parasitic protozoa, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum. Melarsoprol, benznidazole, miltefosine, artemisinin and chloroquine were used as reference drugs. The cytotoxic potentials of the extracts on rat skeletal myoblast (L6) cells were also assessed and compared to that of podophyllotoxin. The screening method employed was medium-throughput, where the extracts were tested at two concentrations, at 0.8 and 4.8 μg/ml (T. brucei rhodesiense, L. donovani and Plasmodium falciparum), or at 1.6 and 9.7 μg/ml (T. cruzi and L6 cells). At 4.8 μg/ml concentration, the branch extract of Pistacia vera (PV-BR) significantly inhibited (77.3%) the growth of L. donovani, whereas the dry leaf extract (PV-DL) was active against Plasmodium falciparum (60.6% inhibition). The IC₅₀ values of these extracts were determined as 2.3 μg/ml (PV-BR, L. donovani) and 3.65 μg/ml (PV-DL, Plasmodium falciparum). None of the extracts possessed cytotoxicity on mammalian cells.     

Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Platelet activity in immune lysis of Trypanosoma musculi

It was observed that, when blood from CBA/J mice recovering from T. musculi infection was added to parasites in vitro, platelets adhered to the trypanosomes which subsequently died. This platelet adherence trypanosomal lysis (PATL) activity of the blood appeared late in infection and persisted long after parasites had been eliminated from the blood, although showing a cyclical pattern of activity. Trypanosomal lysis in vitro by fresh (non-heat-inactivated) immune plasma free of platelets could also be demonstrated.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

Studies on Trypanosoma vivax: The infectivity of cyclically and mechanically transmitted ruminant infections for mice and rats

The infection of laboratory rodents by cyclically established Trypanosoma vivax infections in ruminants was only successful for a period up to 4 weeks after the first fly feed on the ruminants; rats and mice were equally susceptible. Similar results were obtained when ruminants were inoculated by syringe with infected blood from the early stage of a cyclically transmitted T. vivax infection. The development of antibodies, as measured by fluorescence, in the ruminants had no influence on the outcome of the rodent-inoculations.

When blood was inoculated into susceptible oxen from the later chronic stage of a cyclically and from a mechanically established bovine infection, subsequent rodent infections were achieved only occasionally; parasitaemia in the rodents was low and the patent period short. It was concluded that the infectivity of T. vivax for rodents was predominantly a characteristic of the isolate and not of the donor or recipient mammalian species. The value of blood inoculation of rodents in epidemiological studies for detecting early infections of T. vivax is emphasized.     

Evaluation of the trypanocidal and leishmanicidal in vitro activity of the crude hydroalcoholic extract of Pfaffia glomerata (Amarathanceae) roots

Three different concentrations (1, 10 and 50 μg/ml) of lyophilized hydroalcoholic crude extract of Pfaffia glomerata roots were assayed in vitro against strains of Trypanosoma cruzi (Y) and Leishmania braziliensis. It was observed that P. glomerata hydroalcoholic extract was relatively active within the tested concentrations for L. (V.) braziliensis, but inactive against T. cruzi. Despite the fact that both protozoans belong to the Trypanosomatidae family, we suggest that the difference observed for activity should be related to the biological differences between the two parasite species.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Major acute phase response of haptoglobin and serum amyloid-P following experimental infection of mice with Trypanosoma brucei brucei

Investigation of the pathophysiological role of the systemic cytokines, including interleukin-1, interleukin-6 and tumour necrosis factor , in the host response to infection with African trypanosomes is hampered by the low and transient concentrations of these cytokines in plasma. One of the actions of these cytokines is the stimulation of hepatocyte production of acute phase proteins such as serum amyloid-P and haptoglobin. These acute phase proteins are more stable in the circulation than the cytokines and can be measured as a means of assessing the systemic cytokine response in the trypanosome-infected host. The plasma concentrations of serum amyloid-P and haptoglobin were measured in an experimental mouse model of Trypanosoma brucei brucei infection. Both serum amyloid-P and haptoglobin, increased markedly following infection. Peak concentrations of serum amyloid-P at 125 μg/ml and haptoglobin at 2 g/l were attained 10 to 12 days after infection. Thereafter, serum amyloid-P concentration decreased to approximately 40 μg/ml while the haptoglobin concentration remained elevated at approximately 1.5 g/l. The reactions of the serum amyloid-P and haptoglobin following experimental Trypanosoma brucei brucei infection in mice demonstrate that a major acute phase response has occurred indicating that the systemic cytokine network has been activated. Further studies are required to identify whether the response is stimulated by the parasite or indirectly by tissue damage.     

Synthesis and biological activivity of juvenele hormone analogues (JHA) for Trypanosoma cruzi

Several derivatives of geraniol, geranylacetone, and farnesol bearing carbonate and thiolcarbonate functional groups as well as several derivatives of 4-phenoxyphenol were synthesized and tested for their respective biological activity as growth inhibitors for Trypanosoma cruzi, and for inhibition of tritium-labelled thymidine incorporation in T. cruzi cells. The results indicated that some JHA showed important activity against the development of the cells.     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiophen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reductase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 μM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 °C against trypomastigotes in murine blood as well against the enzyme trypanothione reductase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.     

Serological Comparison of the Three Morphological Phases of Coccidioides immitis by the Agar Gel Diffusion Method

Hyperimmune sera against spherules and against arthrospores of Coccidioides immitis were prepared by inoculation of rabbits. The antibody content of these sera was studied by the agar gel diffusion method. It was observed that antispherule pooled sera formed multiple precipitin bands with extracts of spherules and of arthrospores. The antiarthrospore pooled serum, however, failed to precipitate with the spherule extract, and formed a single band in the presence of an arthrospore solution. When the spherule and the arthrospore extracts were tested with a variety of different antisera, it was observed that the spherule preparation formed bands only in combination with anti-purified spherule pooled serum, whereas the arthrospore extract precipitated with anti-purified spherule, antiarthrospore, and anti-Histoplasma capsulatum pooled sera. It was also observed that a spherule culture supernatant solution formed five precipitin bands in combination with anti-spherule pooled sera, formed one band with pooled antiserum from rabbits with coccidioidomycosis, and did not precipitate in the presence of antiarthrospore pooled serum. Coccidioidin, however, formed two bands in the presence of any of these antisera. It was therefore concluded that extracts from the spherule phase of C. immitis differed from solutions obtained from the arthrospore and mycelial phases.     

Chelating agent inhibition of Trypanosoma cruzi epimastigotes In vitro

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 μg/mL. The most effective compounds included N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included -penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 μg/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interface with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.     

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.     

Accessibility of Trypanosoma brucei procyclic glycosomal enzymes to labeling agents of various sizes and charges

The high efficiency of glycolysis in Trypanosoma brucei has been attributed to impermeability of the glycosomal membrane to most metabolites. However, the strong stimulation of the glycolytic rate by exogenous metabolites and coenzymes in intact glycosomes is only compatible with their accessibility to the internal space. The accessibility of glycosomal enzymes to protein labeling agents of varying charge and size has been investigated. The results show that the glycosomal membrane is permeable to small molecules of the size of metabolites, but impermeable to larger molecules.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Detection of polymorphism in the Trypanosoma cruzi TcP2β gene family by single strand conformational analysis (SSCA)

Single strand conformation analysis (SSCA) is a technique that has been used to detect point mutations. We explored its usefulness in the analysis of four different members of the Trypanosoma cruzi TcP2β gene family and its suitability for detection of polymorphism in different parasite strains. The availability of primers covering a 97-bp sequence at the 5′ end of the genes allowed assessment of the effect of a single base substitution, while the analysis of a 321 bp long sequence permitted the evaluation of sequences differing in several bases. PCR products were analysed under four different electrophoretic conditions: with or without the addition of 10% glycerol in a 6% polyacrylamide gel run at room temperature or at 4°C. Shifts in mobility were radically dependent on the migration condition. Both 97-bp and 321-bp amplicons were best resolved at 4°C, without glycerol. Amplification products derived from total genomic DNA showed a pattern that resembled closely a combination of the products derived from the cloned genes. The results herein demonstrate the usefulness of SSCA to differentiate forms of a complex protozoan gene family, and to scan its polymorphic nature. Furthermore, due to the remarkable sensitivity of the technique it can generate genomic markers, such as Sequence Tagged Sites (STS), of great need in the T. cruzi genome project