Characterization and grouping of Trypanosoma cruzi stocks by DNA base-specific fluorochromes and discriminant analysis.

Fluorochromes with G-C and A-T specificity were used for a single-cell DNA analysis of the blood-stream forms of 14 Trypanosoma cruzi stocks in a cytofluorometric assay. The kinetoplast contained 22.3%-37.9% of the total DNA G-C base content and 42.7%-63.5% of the total DNA A-T base content. In spite of these differences, the mean base A-T/G-C ratio of the total DNA was 1.11 and was nearly constant in all stocks. The G-C base ratio of kinetoplast/nucleus resulted in a grouping corresponding with the peanut agglutinin (PNA)- and wheat germ agglutinin (WGA)-type characteristics of the T. cruzi stocks. The discriminant analysis revealed relationships, in that each stock contained some trypanosomes with DNA fluorescence characteristics of common to at least one other stock. After chromomycin A3 staining, the mean hit rates for the classification into group 1 PNA and the WGA group were 99% and 96%, respectively, and the respective rates obtained after DAPI application were 84% and 94%.

Characterization and grouping of Trypanosoma brucei brucei, T.b. gambiense and T.b. rhodesiense by quantitative DNA-cytofluorometry and discriminant analysis.

For characterization and differentiation 13 stocks of Trypanosoma brucei spp. were used for a quantitative cytofluorometric determination of their DNA fluorescence intensities by two base-pair-specific fluorochromes. T. b. gambiense could be distinguished from T. b. brucei by an about 20% smaller G-C, and A-T content in the nuclear DNA and a 4% greater G-C and 15% greater A-T content of the kinetoplast DNA. T. b. gambiense could be differentiated from T. b. rhodesiense by a 20% smaller G-C and a 3.8% smaller A-T nuclear DNA content and a 2% greater G-C and a 18% greater A-T DNA content in the kinetoplast. After chromomycin (G-C specific) staining the DNA ratio kinetoplast/nucleus of T. b. gambiense was 6.8, of T. b. brucei 5.2 and of T. b. rhodesiense 5.4. The corresponding values after DAPI (A-T specific) application were 20.8 for T. b. gambiense, 14.4 for T. b. brucei and 16.6 for T. b. rhodesiense. With the fluorescence intensities a discriminant analysis has been computed. After chromomycin application the gambiense stocks could be separated from T. b. brucei and T. b. rhodesiense by this method with a hit rate of 100%. Such perfect separation could not be observed between T. b. brucei and T. b. rhodesiense. Even if most of them were classified into the corresponding subgroups some trypanosomes would nevertheless pass over into the brucei or rhodesiense subgroup and vice versa.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytofluorometry as a method for the differentiation of trypanosomes.

The DNA binding guanine specific antibiotic, chromomycin A3, has been evaluated for fluorescence intensity measurements of T. cruzi, T. brucei brucei and T. musculi. Optimal fixation and staining conditions have been determined. The fluorometry was performed with a microscope photometer equipped with electronic systems for short time excitation of 7 milliseconds and operation control. The trypomastigote bloodstream forms of these species have a different chromomycin specific DNA content. The total DNA content of T. cruzi was 2.1-fold higher than for T.b. brucei and 2.3-fold higher than for T. musculi. The nuclear DNA content also was higher in T. cruzi. The nuclear DNA values were recorded to be 1.6-fold greater than in T.b. brucei and 2.0-fold greater than in T. musculi. The amount of the kinetoplast DNA of T. cruzi was shown to be 3.2-fold higher than in T. musculi and 11.7-fold higher than in T.b. brucei. The higher total DNA of T.b. brucei in relation to T. musculi was based on the nuclear values because the content of the kinetoplast DNA of T.b. brucei was 3.7-fold smaller than of T. musculi. The kDNA comprised 25% in T. cruzi, 18% in T. musculi and only 4% in T.b. brucei of the total amount of the chromomycin specific DNA. The chromomycin fluorescence intensities of the DNA of trypanosomes were subjected to a statistical model of discriminant analysis. It was possible to get perfect separation of the three trypanosome species. The hit rate was 100%.

Comparative characterization of Venezuelan Trypanosoma cruzi stocks by electron microscopy, isoelectrofocusing and lectin typing.

The intraspecific variation among culture forms of 14 Venezuelan Trypanosoma cruzi stocks were examined by kDNA configuration, isoenzymes, and agglutination behaviour of lectins. The results have shown that in all of the stocks the central band of kDNA is present, showing that the stocks are parasites of the subgenus Schizotrypanum. By isoenzymes and lectin typing it has been found that the stocks belong to the isoenzyme group I and the lectin-type PNA which were already described for other Venezuelan stocks. The homogeneous results of intraspecific characterization contrast to those found in other countries in South America south of the Amazon basin and seem to be a further evidence that in countries north of the Amazon basin mainly on T. cruzi-type exists.

The occurrence of N-acetyl- and N-glycoloylneuraminic acid in Trypanosoma cruzi.

Different strain of Trypanosoma cruzi were analysed to have 65--105 micrograms of sialic acids per 10(10) cells. By thin-layer chromatography, and in part by gas liquid chromatography and gas-liquid chromatography-mass spectrometry, all strains were found to contain N-acetyl- and N-glycoloylneuraminic acid in various ratios. After incubation of the parasites with either [3H]acetate or N-acetyl-[3H]mannosamine, no radioactivity was found in the sialic acids, thus leading to the suggestion that the parasites are unable to synthesize sialic acids from their precursors.

[Comparative electron microscope studies on the kinetoplast morphology of bat-trypanosomes and Trypanosoma cruzi (author's transl)]. / Vergleichende elektronenmikroskopische Untersuchung der Kinetoplastmorphologie von Fledermaustrypanosomen und Trypanosoma cruzi.

Comparative studies on the ultrastructural morphology of kinetoplasts of trypanosomes belonging to the subgenus Schizotrypanum have been made. Three isolates of Trypanosoma vespertilionis and two strains of Trypanosoma dionisii derived from European bats were tested. Comparison was made also with two isolates from Brasilian bats characterized as T. cruzi and two strains which were derived from two patients suffering from Chagas' disease. In the epimastigote culture form of T. cruzi a typical configuration of kDNA becomes obvious, appearing as a central band at the beginning of cell division. It was investigated whether this morphological character can be used in differentiating between bat-trypanosomes and T. cruzi and on the species characterization of bat-trypanosomes. The central band of kDNA could be demonstrated in all cases of the trypanosomes examined. The special configuration of kDNA makes it possible to distinguish exactly between trypanosomes of the subgenus Schizotrypanum and other trypanosomes. Nevertheless this pecularity alone is not sufficient for characterizing species of that subgenus.

Leishmania donovani: ultrastructural localization of diaminobenzidine reactivity in the amastigotes.

Intracellular amastigotes of Leishmania donovani obtained from spleens of infected hamsters were studied by means of the diaminobenzidine technique for the presence of cytochromes and the activities of cytochrome oxidase and peroxidase. In the absence of H2O2, the oxidation of DAB, evidenced by electron-dense deposits localized on the cristae, inclusions, and enveloping membranes of the mitochondria and kinetoplast, revealed the activity of the cytochrome oxidase and the presence of the cytochromes. The increased deposition of DAB oxidation especially on the enveloping membranes in the presence of H2O2 suggests the activity of a peroxidase, probably cytochrome c peroxidase.

Protein typing by disc electrophoresis of some species of trypanosomes with special emphasis to Trypanosoma cruzi.

Proteins extracted from culture forms of T. cruzi, T. cruzi-like strains, T. rangeli, T. conhorrini and T. dionisii were separated by disc electrophoresis. The electrophoretic protein patterns of all strains examined were highly reproducible. The results indicate that each of these strains have their own "fingerprints" and the trypanosoma species could be identified by some typical specific protein bands. However, it was not possible to distinguish between the human strains of T. cruzi and the T. cruzi-like strains isolated from other sources.

Ultrastructural localization of diaminobenzidine reactivity in leishmania donovani promastigotes.

The ultrastructural localization of the activities of two enzyme systems in the culture forms of Leishmania donovani was shown by means of the diaminobenzidine techniques. The consistent deposition of electron dense reaction product of DAB oxidation without H2O2 in the kinetoplast and mitochondrial cristae and membranes was taken as evidence of the presence of cytochrome oxidase activity and cytochrome c. In the presence of H2O2, a more intense DAB oxidation was attributed to the activity of a peroxidase, possibly cytochrome c peroxidase. Mitochondrial and kinetoplast reactions to DAB were completely inhibited by KCN, methanol-nitroprusside, and by heating to 50 degrees C for 10 min. On the other hand, no inhibitory effect was observed with 100 mM 3-amino-1,2,4-triazole. Under all conditions of incubation tested, the microbodies were completely unreactive to DAB staining, which was utilized as the basis for their identification. These organelles are rounded, moderately electron-opaque bodies with a finely granular matrix and fine tubules or cores and are limited by a single membrane. Under normal staining method, the microbodies were indistinguishable from the rounded sections of mitochondria.

[Agglutination reaction of T. cruzi, T. cruzi like Strains, T. rangeli and T. conorhini with Soja hispida lectin and Aaptos papillata protectin (author's transl)]. / Agglutinationsverhalten von T. cruzi-, T. cruzi like Stämmen, T. rangeli und T. conorhini mit dem Lektin von Soja hispida und dem Aaptos papillata-Protektin

Protectin from the sponge Aaptos papillata (Keller) was used in the characterization of five strains of T. cruzi (Venezuela, Guatemala, Y. Brasilien, Peru, Wien) and six T. cruzi like strains (Triatoma, Maryland, ITMAP 943, FH4, FH5, LN). Based upon their membrane receptors, these T. cruzi and T. cruzi like isolates could be differentiated from rangeli (Venezuela Strain) and T. conorhini (Hawai Strain) by agglutination reaction to the proctectin. Furthermore, after pronase treatment T.rangeli could also be distinguished from T. conorhini by agglutination test with A. papillata protectin and also Soja hispida lectin. It is not possible to differentiate the T. cruzi complex with S. hispida lectin, because it did not agglutinate T. cruzi (Vienna Strain) and T. cruzi like (Maryland Strain). However, after treating this human pathogenic strain with pronase the pseudocrypt antigen of the first order is made available to the S. hispida lecting thereby producing agglutination. The T. cruzi like strain however did not agglutinate with this treatment. On the other hand, while T. rangeli did not agglutinate even after pronase treatment, T. conorhini showed the agglutination reaction. This observed reaction is explained by the availability of the pseudocrypt antigens of the first order after pronase treatment.

[Strain related differences in the immunodiagnosis of malaria (author's transl)]. / Stammbedingte Titerdifferenzen in der Immundiagnostik der Malaria

In infections with P. falciparum and P. vivax antibody titers were found to differ in relation to strains. Various strains of both Plasmodium species used as antigens showed differences in their sensitivity in reactions with one patient serum. In P. vivax infections it is supposed that some antibody titer differences are caused by antigen variations.

[Comparative electron microscope studies on the labelling Leishmania donovani, Leishmania tropica and Leishmania braziliensis with ferritin (author's transl)]. / Vergleichende elektronenmikroskopische Untersuchung über die Markierung von Leishmania donovani, Leishmania tropica und Leishmania braziliensis mit Ferritin

The labelling of negative charges on the cell surface of the developing promastigote forms in Leishmania donovani, L. tropica and L. braziliensis was determined using cationized ferritin and electron microscopy. Ferritin deposits were seen exclusively on the pellicle, since they cannot penetrate the cell membrane. The ferritin particles were distributed in regular rows covering the whole cell surface. The mean diameter of the particles was 8.76 nm and at a magnification of 104.000 an average of 9.8 particles was detected on 1 cm cell surface. Only in the membrane of the proximal part of the reservoir labelling was frequently absent. The possible explanations for this observation were discussed. There was no difference in the pattern size of the ferritin particles or in their number per cm cell surface among the various strains of L. donovani on the one hand or between L. donovani, L. tropica and L. braziliensis on the other.

[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.

[An electron microscopic study on a strain of Trypanosoma vivax maintained in cattle (author's transl)]. / Elektronenmikroskopische Untersuchung eines im Rind gehaltenen Trypanosoma vivax-Stammes

The ultrastructure of the blood forms of Trypanosoma vivax from its natural host, cattle, is described. The pellicula, consisting of an unit membrane with a superimposed surface coat, the structure and attachment of the flagellum and the sub-pellicular microtubules showed the usual structural and organizational features. Cell organelles and cytoplasmic inclusions such as cell nucleus, mitochondria, kinetoplast, Golgi-complex, endoplasmic reticulum and membrane-isolated vacuoles, which occur in trypanosomidae, are presented and described. The ultrastructure of the trypomastigote forms of T. vivax has been compared to that of other trypanosome species and similarities and deviations are discussed. The mitochondrion shows a striking difference in dimension and number of microtubules from other trypanosome species. The kinetoplast, the K-DNA containing part of this mitochondrion, differs in its relative position to the basal body and in the mitochondrion from other flagellate species. It is marginally situated and the K-DNA is frequently lying parallel to the basis of the flagellum.