[Trypanosomatidae of public health importance occurring in wild and synanthropic animals of rural Venezuela]. / Trypanosomatidae de importancia en salud pública en animales silvestres y sinantrópicos en un area rural del municipio Tovar del estado Mérida, Venezuela.

INTRODUCTION: Chagas disease and leishmaniasis are important public health problems due to their high frequency and broad distribution in Latin America. Understanding of the roles of reservoir animals is crucial for a global assessment of the epidemiology of these diseases. OBJECTIVE: To identify parasites classed as Trypanosomatidae as they occurred in sylvatic animals, and to establish rates of coinfection. MATERIALS AND METHODS: Sylvatic animals were systematically captured in the rural area of El Carrizal, Merida State, Venezuela, betweenJuly, 1998 and February, 2000. The captures were made in Tomahawk type homemade traps, placed 15 nights per month throughout the study period. Blood was extracted from each captured and anesthetized animal by means of cardiac puncture. The search for trypanosomatids was undertaken by fresh blood examination, Giemsa stained blood smears and by means of blood-agar culture. Occasional xenodiagnoses were made to check diagnostic accuracy. The isolates obtained in culture media were identified by restriction fragment analysis and hybridization with specific probes. RESULTS: Three species of sylvatic animals (n = 215) were captured: Rattus spp. (135), Sigmodon hispidus (73) and Didelphis marsupialis (7). From them, three species of Trypanosomatidae were identified: Leishmania (Viannia) guyanensis, Trypanosoma cruzi and Trypanosoma lewisi. Trypanosoma. cruzi was identified in D. marsupialis (4/7), S. hispidus (1/73) and Rattus spp. (1/ 135), whereas L. (V.) guyanensis and T. lewisi were identified only in Rattus spp., 1/135 and 12/ 135, respectively. CONCLUSIONS: The coexistence of these genetically related hemoflagellates in sylvatic hosts was important for understanding the immunological interactions that may be established in reservoir animals, and the possible implications that this may have for the susceptible host. Finally, the identification of L. (V.) guyanensis in Rattus spp and T. cruzi in S. hispidus constituted the first reports of this relationship in Venezuela.

A comparative study of D-lactate, L-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense.

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced D-lactate, but not L-lactate, whereas T. b. gambiense procyclic forms produced L-lactate, but not D-lactate, and T. lewisi produced both isomers.

Metabolic activity of Trypanosoma lewisi cultured in vitro in the presence of normal or ablastinic rat serum.

Trypanosoma lewisi, in cultures supplemented with normal rat serum, synthesized the majority of new RNA during the first 13 hr of the culture, whereas DNA synthesis occurred from the 8th hr onwards and amino acids were incorporated into macromolecules uniformly throughout the 24 hr culture period. Thymidine was taken up by the parasite only between the 7th and 14th hr of culture, unlike uracil and amino acids which were taken up as required. Ablastin, a trypanostatic factor in the serum of infected rats, maximally inhibited DNA synthesis if it was added to the cultures before the 5th hr, partially inhibited synthesis if added between the 5th and 11th hr, but if added after this had no inhibitory effect. Ablastin only partially inhibited RNA synthesis and was without effect on amino acid utilisation.

Trypanosoma lewisi: in vitro oxygen uptake by parasites grown in albino and black rats.

Oxygen consumption of Trypanosoma lewisi grown in albino and black rats was measured by the direct method of Warburg. Respiration rates in buffered Ringer's solution, with and without glucose and in normal serum from the respective hosts, were determined for T lewisi grown in albino and black rats. Significantly higher respiratory rates were obtained for trypanosomes grown in albino hosts: 31 percent higher in buffered Ringer's solution with glucose and 26 percent higher in homologous serum.In this study, in which the development of the parasitemia was observed for 27 days, T lewisi populations were higher in albino rats than in black rats.

[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.

The phospholipases of pathogenic and non-pathogenic Trypanosoma species.

Four species of trypanosome were examined for phospholipase activities using 1-[3H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine and 1-acyl-2[14C]linoleoyl-sn-glycero-3-phosphocholine as substrates. The major activity in each species is a phospholipase A1 (EC 3.1.1.32) which does not require calcium. The most effective of the detergents tested for activation of the enzyme from each species, and the Ph optima, are as follows: Trypanosoma brucei, 0.125% Triton X-100 at pH 6.0-8.5; T. congolense, 0.5 mM linoleate at pH 6.0; T. theileri, 0.1% Triton X-100 at pH 6.75; T. lewisi, 0.2 mM sodium dodecyl sulfate at pH 5.2. The specific activity of the enzyme from a pathogenic species, T. brucei, is very high (145 nmol/min/mg/protein) and could contribute to the tissue damage characteristically caused by this parasite. The level in T. lewisi, a non-pathogenic species, is relatively low (1 nmol/min/mg). The levels in T. theileri (31 nmol/min/mg) and T. congolense (10 nmol/min/mg are intermediate. These results are compatible with the hypothesis that phospholipases contribute to the pathogenicity of trypanosomes.

[Histones from Trypanosoma lewisi nuclei]. / Gistony iader zhgutikovogo prosteishego Trypanosoma Lewisi.

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.

A method for the assay of ablastin in the serum of rats infected with Trypanosoma lewisi.

This paper describes a simple quantitative assay for albastin, a factor present in the serum from rats infected with Trypanosoma lewisi, which prevents the division of the parasite. The assay measures in vitro the inhibition of the incorporation of 3H-TdR into the DNA of T. lewisi in the presence of serum from infected animals. Utilising this method, one can measure the titre of albastin in a particular serum sample and the time and duration of its appearance in the circulation of infected rats.

The generation of phospholipase A and hemolytic fatty acids by autolysing suspensions of Trypanosoma congolense.

When T. congolense undergoes autolysis there is a concomitant appearance of phospholipase A activity and hemolytic fatty acids. The generation of enzyme activity is exponential, and the appearance of hemolytic activity corresponds to a free fatty acid concentration of 0.02 to 0.03 mg. per ml. The concentration of the trypanosome suspension markedly affected the kinetics of the generation process. In contrast, the autolysis of T. lewisi did not generate hemolytic activity unless exogenous phospholipase A was added to the organisms.

Immunologic and fine structure evidence of avidly bound host serum proteins in the surface coat of a bloodstream trypanosome.

Intact, washed Trypanosoma lewisi bloodstream forms, isolated from rats, were agglutinated specifically by antisera against rat whole serum, albumin, alpha2-macroglobulin, and IgG. However, trypsinized bloodstream and intact culture forms lacking surface coat were not agglutinated by these antisera. Trypsinized bloodstream forms, incubated in dilute rat or heterologous host serum proteins, were agglutinated with specific antisera. The characteristic surface coat of intact bloodstream forms was absent from trypsinized cells; however, trypsinized and serum-incubated bloodstream forms reacquired a surface coat similar to that of intact cells. Gel-diffusion and immunoelectrophoretic results showed that rat albumin, alpha2-macroglobulin, and IgG were present in the surface coat of bloodstream forms. Results of quantitative rocket immunoelectrophoresis demonstrated that the adsorbed rat serum proteins constituted by weight about 5% of the trypanosome total surface coat protein.

Adenosine 3',5'-monophosphate in reproducing and differentiated trypanosomes.

Trypanosoma lewisi, a blood protozoan of rats, undergoes differentiation from a rapidly reproducing form to a nonreproducing form in response to the host antibody ablastin. Intracellular concentrations of adenosine 3',5'-monophosphate (cyclic AMP), which has been implicated in controlling reproduction in cultured mammalian cells, was measured in the two developmental forms of T. lewisi. The concentrations were significantly different, and the results support a hypothesis under which ablastin stimulates an increase in intracellular cyclic AMP.