On opportunist infections by Trypanosoma lewisi in humans and its differential diagnosis from T. cruzi and T. rangeli.

Trypanosoma lewisi is a cosmopolitan species originally found in Rattus spp., being nonpathogenic, host-restricted, and transmitted by rat fleas. This species has been recorded as an opportunist blood parasite of human beings mainly in Asia, with a case in Africa. In Brazil, this species was recently recorded in captive monkeys. As T. lewisi can share vertebrate hosts both with Trypanosoma rangeli and Trypanosoma cruzi, some markers for the differential diagnosis of these species were examined and discussed herein. The identification of T. lewisi was based on morphological features of bloodstream stages at the initial phase of infection in mammals, isoenzyme electrophoresis at the MDH locus, and PCR products of kinetoplast DNA (kDNA) minicircles using the primers TC121/TC122.

The occurrence and ultrastructure of Trypanosoma (Herpetosoma) lewisi (Kent, 1880) Laveran and Mesnil, 1901, the parasite of rats (Rattus norvegicus) in Poland.

This study reports the light and electron microscopic examination of Trypanosoma (Herpetosoma) lewisi (Kent, 1880) Laveran and Mesnil, 1901, isolated from rats (Rattus norvegicus) from Poland. Bloodstream trypomastigotes were identified morphometrically from 100 specimens collected from three naturally infected rats Rattus norvegicus. Body length ranged from 15.45-23.64 microm and width from 1.3-2.32 microm while the free flagellum was 8.1 microm long. Electron microscopic study of bloodstream trypomastigotes exhibited typical ultrastructural features similar to those of other stercorarian trypanosomes. The presently determined morphological data have been compared with those provided by other authors.

Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro.

The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T. musculi, the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T. lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T. musculi and T. lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C.

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.

The glycoproteins of the complex surface coat of Trypanosoma lewisi.

Blood stream forms of Trypanosoma lewisi from rats previously infected were labelled in vitro by galactose-oxidase oxidation followed by NaB3H4 reduction and subjected to 7.5% SDS-polyacrylamide gel analysis, by this procedure 8 bands were detected on SDS polyacrylamide gel electrophoresis. We conclude that surface composition of Trypanosoma lewisi resulted more complex than that of Trypanosoma so far studied, suggesting a possible relation with induced immunity and reduced ability of the parasite to survive in its host.

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi.

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.

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.

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.

[Comparative morphological studies on the kinetoplast of various species of trypanosomes, with special reference to trypanosoma cruzi (author's transl)]. / Vergleichende Kinetoplastmorphologie verschiedener Trypanosomenarten unter besonderer Berücksichtigung von Trypanosoma cruzi

Comparative electron microscope studies on the morphology of the kinetoplast DNA (K-DNA) of the epimastigotes in many trypanosome species were carried out under standardized conditions. The K-DNA shows a morphological variation during the cell cycle of culture forms of the trypanosome species under study. In longitudinal sections of the kinetoplast, the K-DNA of T. cruzi appears as a compact trabecular structure; as a relatively disaggregated unit, with a central band or in a transitional stage between these forms. The conspicuous central band of the K-DNA which occurs at the beginning of cell division when the basal body is duplicated, could be demonstrated in all the 8 T. cruzi isolates studied. This has been found in the human-pathogen strains as well as in the T. cruzi-like trypanosomes of wild animals. In contrast, in comparable developmental stages of T. conorhini, T. rangeli and two strains of T. lewisi, this structural configuration of the K-DNA could not be observed. Based on these results, the extent to which the central band of K-DNA may be used in differentiating between trypanosomes is discussed. These findings may also reflect the present state of knowledge, as based on the study of 12 trypanosome isolates, so that corrections or additions may be lateron possible.

Ultracytochemistry of the surface coat/pellicle complex in Trypanosoma brucei.

Ultracytochemistry of polysaccharides and specific sugar residues reveals differences in the surface staining pattern between developmental forms of Trypanosoma brucei. The techniques used were the PA (periodic acid)-TCH (thiocarbohydrazide)-silver albumose reaction for the polysaccharides, and the Concanavalin A (Con A)-perioxdase-DAB coupling method for specific sugar residues. Blood and metacyclic forms, both possessing a surface coat, stain distinctly for carbohydrates at the level of the pellicular membrane. The external portion of the bloodform coat lacks any positive staining. Pellicles of non-coated culture and vector forms react only faintly for polysaccharides, whereas heavy staining of oxidized peroxidase/DAB reaction product, indicative of sugar bound Con A, occurs. It is suggested that the sugar moieties of the coat glycoproteins are located close to the membrane-coat junction.