Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using human and animal trypanocidal drugs.

Beside typical human trypanosomes responsible of sleeping sickness in Africa and Chagas disease in Latin America, there is a growing number of reported atypical human infections due to Trypanosoma evansi, a livestock parasite, or Trypanosoma lewisi, a rat parasite, especially in Asia. Drugs available for the treatment of T. brucei ssp. in humans are obviously of choice for the control of T. evansi because it is derived from T. brucei. However, concerning T. lewisi, there is an urgent need to determine the efficacy of trypanocidal drugs for the treatment in humans. In a recent study, pentamidine and fexinidazole were shown to have the best efficacy against one stock of T. lewisi in rats. In the present study suramin, pentamidine, eflornitine, nifurtimox, benznidazole and fexinidazole, were evaluated at low and high doses, in single day administration to normal rats experimentally infected with a stock of T. lewisi recently isolated in Thailand. Because none of these treatments was efficient, a trial was made with the most promising trypanocide identified in a previous study, fexinidazole 100mg/kg, in 5 daily administrations. Results observed were unclear. To confirm the efficacy of fexinidazole, a mixed infection protocol was set up in cyclophosphamide immunosuppressed rats. Animals were infected successively by T. lewisi and T. evansi, and received 10 daily PO administrations of 200mg/kg fexinidazole. Drastic effects were observed against T. evansi which was cleared from the rat's blood within 24 to 48h; however, the treatment did not affect T. lewisi which remained in high number in the blood until the end of the experiment. This mixed infection/treatment protocol clearly demonstrated the efficacy of fexinidazole against T. evansi and its inefficacy against T. lewisi. Since animal trypanocides were also recently shown to be inefficient, other protocols as well as other T. lewisi stocks should be investigated in further studies.

Zoonotic trypanosomes in South East Asia: Attempts to control Trypanosoma lewisi using veterinary drugs.

A growing number of atypical human infections due to the livestock parasite Trypanosoma evansi, or to the rat parasite Trypanosoma lewisi, are reported in humans in Asia. In some cases, clinical evolutions request treatments, however, so far, there were very few attempts to control T. lewisi using trypanocidal drugs. In a study published elsewhere, the efficacy of human trypanocides is evaluated in laboratory rats, and it concludes that none of them is able to cure rats experimentally infected with T. lewisi. Control of T. lewisi in rat would be a step for identification of drugs against this parasite. In the present study, 4 veterinary drugs: diminazene aceturate, isometamidium chloride, melarsomine hydrochloride and quinapyramine sulfate and chloride, were evaluated at low and high doses, in intra-muscular injections to normal rats experimentally infected with a stock of T. lewisi from Thailand. None of these treatments being efficient, a trial was also made using melarsomine hydrochloride in T. evansi infected rats and in mixed T. lewisi and T. evansi infected rats, in order to demonstrate the efficacy of the drugs under the present protocol. T. evansi was cleared from the rat's blood the day after the treatment, while, T. lewisi remained unaffected until the end of the experiment. These observations clearly demonstrated the efficacy of melarsomine hydrochloride against T. evansi and its inefficacy against T. lewisi. In conclusion none of the veterinary drugs was efficient against this stock of T. lewisi. Other protocols using higher doses or other drugs and T. lewisi stocks should be investigated in further studies. The control of T. lewisi infection in Wistar rats, using veterinary trypanocidal drugs, remains so far unsuccessful.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen.

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Evaluation of trypanocidal drugs used for human African trypanosomosis against Trypanosoma lewisi.

Trypanosomes from animals are potential pathogens for humans. Several human cases infected by Trypanosoma lewisi, a parasite of rats, have been reported. The number of these infections is possibly underestimated. Some infections were self-cured, others required treatment with drugs used in human African trypanosomosis. An in vitro evaluation of these drugs and fexinidazole, a new oral drug candidate, has been performed against T. lewisi in comparison with T. brucei gambiense. All have comparable activities against the two parasites. Suramin was not effective. In vivo, drugs were tested in rats immunosuppressed by cyclophosphamide. The best efficacy was obtained for fexinidazole, and pentamidine (15 mg/kg): rats were cured in 7 and 10 days respectively. Rats receiving nifurtimox-eflornithine combination therapy (NECT) or pentamidine (4 mg/kg) were cured after 28 days, while melarsoprol was weakly active. The identification of efficient drugs with reduced toxicity will help in the management of new cases of atypical trypanosomosis.

Activity of D-carnitine and its derivatives on Trypanosoma infections in rats and mice.

Little progress has been made in the treatment of African trypanosomiasis over the past decades. L-carnitine has a major role in glycolysis-based energy supply of blood trypanosomes for it stimulates constant ATP production. To investigate whether administration of the isomer D-carnitine could exert a competitive inhibition on the metabolic pathway of the L-form, possibly resulting in parasite replication inhibition, several formulations of this compound were tested on Trypanosoma lewisi and T. brucei rhodesiense in rodent models. High oral dosages of D-cornitine inner salt and proprionyl-D-carnitine were not toxic to animals and induced about 50% parasite growth inhibition in reversible, i.e. competitive, fashion. A putative mechanism could be an interference in pyruvate kinase activity and hence ATP production. Considering both, lack of toxicity and inhibitory activity, D-carnitine may have a role in the treatment of African trypanosomiasis, in association with available trypanocidal drugs.

In vitro study of anticancer acridines as potential antitrypanosomal and antimalarial agents.

The requirement for rational drug design in the search for new agents that are active against parasitic protozoa prompted our in vitro studies with a group of 9-anilinoacridines. In vitro growth assays with Trypanosoma lewisi identified a series of C-1' alkylaminoacridines which possess previously unreported potent growth-inhibitory activities against T. lewisi at a concentration range of 0.1 to 1 microM. In contrast, several 9-anilinoacridines that possess acridine ring NH2 substituents at C-3 and C-6 were inactive against T. lewisi, but they possessed strong activity against Plasmodium falciparum at a concentration range of 0.1 to 2.8 microM. In mammalian cells, amsacrine [4'-(9-acridinylamino)methanesulfon-m-anisidide] inhibits DNA topoisomerase II; however, amsacrine was only weakly active against T. lewisi. Such differences in the patterns of susceptibility of mammalian cells, T. lewisi, and P. falciparum to these 9-anilinoacridines may reflect enzyme differences between different parasites and mammalian cells that can be exploited by further improvements in drug design.

Trypanocidal drugs and their effect on parasitaemia, specific IgG production and protective immunity in rats infected with Trypanosoma lewisi.

In rats infected with Trypanosoma lewisi, parasitaemia normally resolves by day 32; thereafter, the rodents become solidly immune to re-infection. Rats treated on day 5 of infection with a single i.m. dose of 35 mg/kg of the trypanocidal drug ethidium bromide (EB) had recovered from parasitaemia by day 12, whereas berenil (BE) given at 100 mg/kg, more than twice the recommended dose, had no effect on parasitaemia. However, rats treated for 4 consecutive days beginning on day 5 of infection with Lampit (LA) and Radanil (RA) at 350 mg/kg showed no parasitaemia on days 16 and 20, respectively. EB was the most effective drug in lowering the total IgG antibody as compared with the control animals, whose specific IgG titres remained elevated for over 200 days after the parasitaemia had been cleared. LA also significantly reduced the antibody levels through day 240, whereas RA only transitorily depressed the antibody levels on days 20 and 30. BE, which had no effect on parasitaemia, correspondingly failed to depress the total IgG levels. Re-challenge infection of the drug-treated, recovered animals on day 240 (208 days after the normal resolution of the infection) revealed that except for the EB group, which displayed transitory parasitaemia for 4 days, other treated and control rats completely resisted the challenge; pre-challenge antibody titres were lower than 1:160 in EB-treated animals in contrast to the levels of 1:320 or higher measured in the other drug-treated and control animals, which resisted the infection.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Interaction of trace metal contaminants on hematological responses: influence of infection with Trypanosoma lewisi.

The influence of trace metal contaminants (lead, cadmium, and mercury) on Sprague-Dawley rats was investigated to determine whether subclinical levels of these metals might have adverse effects on the host's ability to respond to an infective agent.In the present study, a trace metal contaminant-trypanosome system was employed to observe the hematological changes in rats infected with Trypanosoma lewisi. The animal groups were: (1) lead (Pb), (2) cadmium (Cd), (3) mercury (Hg), and (4) controls (animals not exposed to trace metals).Regardless of the trace metals, infected animals showed decreases in total erythrocyte counts and lower hematocrit values. Generally, infected animals also showed increases in total leucocyte counts, decreases in the percentages of lymphocytes and monocytes, increases in percentage of neutrophils, and no changes in percentages of eosinophils and basophils.

Polyamine oxidase-mediated trypanosome killing: the role of hydrogen peroxide and aldehydes.

Trypanosoma lewisi and T. musculi were lysed when incubated with bovine serum in the presence of either spermine or spermidine. Similar results were obtained when a fraction from bovine serum containing polyamine oxidase (PAO) activity or a commercially available purified beef plasma PAO were used in lieu of bovine serum. Trypanosomes treated with cytotoxic concentrations of PAO-spermine failed to establish infection in rats. These results are similar to those from our previous studies with African trypanosomes. We now extend the properties of PAO by showing that human retroplacental serum (RPS) containing PAO activity was also capable of mediating trypanosome killing. This is of significance because the macrophage PAO resembles the human RPS PAO. In addition, our preliminary studies, in which an attempt was made to characterize the factors responsible for cytotoxicity, suggested that a number of products of the PAO-polyamine reaction display trypanocidal properties. These included hydrogen peroxide (H2O2), the aldehyde acrolein, and possibly aminoaldehydes. No evidence was obtained that the oxygen intermediates, superoxide and hydroxyl radicals, play a role in the PAO-mediated trypanosome killing. Ammonia, an additional product of PAO-polyamine reaction, was not trypanocidal. Furthermore, the data suggested that less than 30 min exposure to the reaction mixture (and possibly to aminoaldehydes) was adequate to cause irreversible damage to trypanosomes.

Externally disposed membrane polypeptides of intact and protease-treated Trypanosoma lewisi correlated with sensitivity to alternate complement pathway-mediated lysis.

Reproducing forms of Trypanosoma lewisi isolated from X-irradiated rats and adult forms from intact rats were not lysed by fresh mammalian sera. Treating parasites with trypsin or chymotrypsin, but not with neuraminidase, under conditions which did not impair viability rendered the parasites sensitive to lysis by rat, mouse, rabbit, and human sera. Serum from animal strains or humans genetically deficient in complement component C3, C5, or C6 did not lyse protease-treated parasites. The lytic factors in serum displayed the heat sensitivity and the Mg2+ requirement characteristic of the alternate complement pathway. Lysis was resolved into two phases, Mg2+-dependent binding of serum factors to parasites and subsequent C5-dependent, Mg2+-independent lysis. Allowing protease-treated parasites to readsorb host proteins did not block lysis by serum. Protease-treated parasites regenerated components which prevented complement-mediated lysis during 2 h in culture at 37 degrees C. This regeneration was inhibited by cycloheximide but not by tunicamycin. Ten major components were resolved in radioautographs of sodium dodecyl sulfate-polyacrylamide gels of extracts of radioiodinated intact cells. Protease treatment before radioiodination reduced the amount of radioactivity associated with these components disproportionately. Components with apparent molecular weights of 102,000, 88,000, and 47,000 were strongly labeled in intact cells, poorly labeled after enzyme treatment, and again labeled in cells that were cultured at 37 degrees C after enzyme treatment. Cycloheximide blocked the reappearance of these components on cultured cells. The presence of these three components was therefore correlated with resistance to complement-mediated lysis.

[Trypanosome sensitivity to polyene antibiotics]. / Izuchenie chuvstvitel'nosti tripanosomid k polienovym antibiotikam.

Swelling of the plasma membrane is one of the mechanisms of resistance to damages in pathogenic Protozoa. Polyenic antibiotics induce reconstruction of the cytoplasma membrane of unicellular eukaryotic organisms, i. e. fungi and Protozoa by binding the membrane lipids. The effect of 5 heptaene polyenic antibiotics, such as amphotericin B, mycoheptin, levorin, its sodium salt and levoridone on the growth of trypanosomides of Trypanosoma lewisi and Crithidia oncopelti was studied. The MIC and IC50 of these antibiotics were determined. It was found that these antibiotics were inhibitors of the trypanosomide growth and development. Levorin and amphotericin were most active with respect to C. oncopelti and levorin was most active with respect to T. lewisi. Physiological and morphological changes in the trypanosomides induced by the polyenic antibiotics were noted. The effect of levorin and amphotericin B on the content of lipids in the trypanosomide cells was studied. A decrease in the total content of the intracellular lipids due to the effect of the polyenes was shown. Differences in the rate of the inhibitory effect as dependent on the structure of the hydrophile part of the lactone ring of the heptaene polyenic antibiotics were found.

Properties of ablastin--a factor in the serum of rats infected with Trypanosoma lewisi which inhibits the parasites' division.

The present study investigated the nature of ablastin, a factor present in the serum of rats infected with Trypanosoma lewisi and which inhibits the parasites' division. Ablastin was unstable to dialysis at pH 1X8, was not adsorbed from serum by trypanosomes and could not be induced in vivo by means other than a natural infection. Attempts to purify ablastin from serum by conventional chromatographic techniques were unsuccessful. Removal of over 90% of the immunoglobulins from ablastinic serum did not reduce the ablastin titre. It is concluded that ablastin is unlikely to be an immunoglobulin as has been previously suggested.

Effects of lysozyme on Trypanosoma lewisi.

Daily lysozyme (hen egg) injections, beginning on day 6 of Trypanosoma lewisi infections in rats, significantly reduced the number of circulating trypanosomes. The effect was dose dependent. Maximum reduction (50%) occurred 24 hours after one treatment of 80 mg was given intraperitoneally (I.P.). The same dose of lysozyme was more effective when divided equally into two injections per day. Controls consisting of appropriate buffers as well as human serum albumin had no effect on trypanosome populations. Animals receiving lysozyme exhibited a weight loss of 5% 24 hours following the first injection, but not other ill effects of the treatment were observed. In vitro experiments indicated that lysozyme did not cause lysis or immobilization alone or in combination with fibrinogen or rat antitrypanosomal serum. These results suggest that the cellular immune response of the host and lysozyme's cationic properties may be important in mediating the anti-trypanosomal response. Lysozyme may thus be an effective trypanocide against trypanosomes whose membranes resemble T. lewisi, such as T. cruzi, or as an adjunct to chemotherapy.

Cell surface saccharides of Trypanosoma lewis i. II. Lectin-mediated agglutination and fine-structure cytochemical detection of lectin-binding sites.

Bloodstream (BSF) and culture forms (CF) of Trypanosoma lewisi were specifically agglutinated with the plant lectins concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and fucose-binding protein (FBP). Lectin-mediated cell agglutination was inhibited, and reversed in the presence of specific lectin-binding saccharides. Cells were agglutinated randomly with all lectins suggesting a uniform distribution in the trypanosome cell surface of the lectin-binding saccharide ligands. The BSF and CF were not agglutinated with phytohaemagglutinin-M, phytohaemagglutinin-P, or influenza virions. Living trypsinized BSF, which lacked a surface coat, gave agglutination results with the lectins identical to those obtained with living intact BSF. Glutaraldehyde- or formalin-fixed intact and trypsinized BSF gave results similar to those obtained with living cells and SBA, WGA, and FBP. However, intact, fixed BSF gave much lower agglutination levels with Con A than trypsinized-fixed, living intact, or living trypsinized BSF cells. Intact and trypsinized living and fixed CF gave identical agglutination results with each of the lectins. Living and fixed cells treated extensively with the glycoside hydrolases alpha-amylase, dextranase, and neuraminidase gave results with the lectins identical to those obtained with untreated cells. Con A bound at the cell surface was visualized with an iron-dextran (Fe-Dex) conjugate. Dense iron marker particles were distributed randomly in the intact BSF surface coat. The Con A-bound Fe-Dex marker was present on the pellicular and flagellar membrane outer lamina of trypsinized BSF and intact CF cells. Horseradish peroxidase (HRPO)-diaminobenzidine (DAB) coupled reactions also were used to visualize surface-bound Con A. Dense Con A-HRPO-DAB deposits were present uniformly in the BSF surface coat, and on the membranes of trypsinized BSF and intact CF trypanosomes. SBA and WGA were conjugated to HRPO and these used in DAB-coupled reactions at the ultrastructure level. Results obtained with the HRPO-conjugated lectins were similar in surface localization and distribution to those obtained with the Con A-HRPO-DAB preparations. Treatment of BSF and CF with the several glycoside hydrolases produced no apparent enhanced or reduced reactivity for the lectins in any of the fine-structure cytochemistry experiments. The cumulative results indicate that ligands similar or identical to alpha-D-mannose, N-acetylgalactosamine, and N-acetylglucosamine, and alpha-L-fucose are constituents in the extracellular surface coat matrix of T. lewisi BSF. Similar conclusions also pertain to the pellicular and flagellar membrane ligands of the BSF and CF cells. Moreover, results obtained with the glycoside hydrolases and influenza virions suggest that the T. lewisi cell surface ligands are not associated directly with repetitively bonded alpha-I,4- and alpha-I,6-D-glucans or sialic acid moieties.

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.