Presentation by scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon.

This paper presents, for the first time, documentation by detailed scanning electron microscopy of the life cycle of microsporidia of the genus Encephalitozoon. Phase 1 is represented by the extracellular phase with mature spores liberated by the rupture of host cells. To infect new cells the spores have to discharge their polar filament. Spores with everted tubes show that these are helically coiled. When the polar tubules have started to penetrate into a host cell they are incomplete in length. The infection of a host cell can also be initiated by a phagocytic process of the extruded polar filament into an invagination channel of the host cell membrane. After the penetration process, the tube length is completed by polar tube protein which passes through the tube in the shape of swellings. A completely discharged polar tube with its tip is also shown. The end of a polar tube is normally hidden in the cytoplasm of the host cell. After completion of the tube length the transfer of the sporoplasm occurs and phase 2 starts. Phase 2 is the proliferative phase, or merogony, with the intracellular development of the parasite that cannot be documented by scanning electron microscopy. The subsequent intracellular phase 3, or sporogony, starts when the meronts transform into sporonts, documented as chain-like structures which subdivide into sporoblasts. The sporoblasts finally transform directly into spores which can be seen in their host cell, forming bubble-like swellings in the cell surface.

Reverse lectin histochemistry: design and application of glycoligands for detection of cell and tissue lectins.

Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The well-documented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (lectin)-carbohydrate recognition in situ. Carrier-immobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous high-affinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namely lectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.

Interspecific differentiation of Trypanosoma cruzi, Trypanosoma conorhini and Trypanosoma rangeli by lectins in combination with complement lysis.

Four-day-old epimastigote culture forms of Trypanosoma cruzi, Trypanosoma rangeli and Trypanosoma conorhini were tested with 21 lectins. Furthermore T. conorhini was incubated with the following sera: rat, Wistar HAN, germ free; normal fresh hen, rat and human serum. T. rangeli was agglutinated only by the D-mannose specific lectins from Canavalia ensiformis and Pisum sativum. T. cruzi and T. conorhini could be distinguished by the lectin from Tridacna crocea. The epimastigote culture forms of T. conorhini were not lysed by normal fresh rat, hen and human sera. Therefore, T. cruzi, T. conorhini and T. rangeli can be distinguished interspecifically by lectins and by the different lytic effect of rat, hen and human sera. It is possible to separate each of the species by complement lysis. The lysis-resistant species can be cultivated for further examinations.

Differentiation of Trypanosoma cruzi, T. cruzi marinkellei, T. dionisii and T. vespertilionis by monoclonal antibodies.

Anti-T. dionisii and anti-T. vespertilionis monoclonal antibodies secreted by 17 hybridoma clones were tested against various strains of T. dionisii, T. vespertilionis, T. cruzi and T. cruzi marinkellei. Strain and species specific antigens were detected for the homologous immunizing strains. The common antigenic determinants of the tested trypanosome species include a component of the flagellum and different cell structures. Seventeen T. cruzi strains could be classified into two groups when tested with anti-T. dionisii monoclonal antibodies. The cross reactions between T. dionisii and T. cruzi demonstrate a strong correlation between T. dionisii and T. cruzi group 2. On the other hand T. cruzi group 1 and T. cruzi marinkellei show very similar antigenic character.

The affinity of the lectins Ricinus communis and Glycine maxima to carbohydrates on the cell surface of various forms of Trypanosoma cruzi and Trypanosoma rangeli, and the application of these lectins for the identification of T. cruzi in the feces of Rhodnius prolixus.

Flagellates of Trypanosoma cruzi (stock Molino 1), obtained from the intestine of experimentally infected Rhodnius prolixus, grown in cellular or acellular culture, as well as from the blood of infected mice, were examined by a direct fluorescence test using the lectins RCA (Ricinus communis-120) and SBA (soy bean agglutinin; Glycine maxima), conjugated with fluorescein isothiocyanate, for the detection of beta-D-galactose and alpha,beta-N-acetyl-D-galactosamine on the membranes of the flagellates. The same reactions were carried out using Trypanosoma rangeli (stock San Agustin), obtained from the intestine, hemo-lymph or salivary glands of experimentally infected R. prolixus, as well as from cultures and from the blood of experimentally infected CFW mice. The results indicate that the membrane of T. rangeli in the salivary glands of the vector contains beta-D-galactose, but that this sugar is absent from all other developmental stages of this trypanosome. All stages of intestinal and cultured. T. cruzi presented positive reactions with RCA-FITC and SBA-FITC. The high specificity of this technique makes it useful for the examination of R. prolixus, previously used in xenodiagnosis of Chagas' disease and for the examination of intradomiciliary or sylvatic vectors in epidemiological surveys in areas where T. cruzi and T. rangeli coexist. Formaldehyde fixed samples can be examined months later and false reports due to T. rangeli can be avoided.

An enzyme-linked lectin assay for the study of lectin receptors of Leishmania.

An enzyme-linked lectin assay (ELLA) based on the ELISA assay, using intact formalin-fixed promastigotes to coat poly-L-lysine-treated microtiter plates is described. The assay was used to study the lectin receptors of Leishmania donovani chagasi, L. donovani donovani and L. mexicana amazonensis. ConA, RCA, WGA, and PNA receptors were found in the three parasites. SBA receptors were found to be as frequent as the other receptors in L. donovani chagasi but not in the other two parasites which showed little SBA binding. Trypsin treatment of the two L. donovani subspecies did not remove any of the lectin receptors studied.

Septata intestinalis and Encephalitozoon cuniculi: cross-reactivity between two microsporidian species.

An infection with Septata intestinalis was diagnosed in a 35-year-old AIDS patient without diarrhoea. The diagnosis was based on morphological examinations of a duodenal biopsy specimen. Serum antibodies were detected reacting with spores of Encephalitozoon cuniculi. Spores of S. intestinalis and E. cuniculi stained with Brown Hopps Gram stain showed a red colour (Gram negative) and not a blue/black colour which was described for microsporidian spores in tissue.

Interspecies transmission of Enterozytozoon bieneusi supported by observations in laboratory animals and phylogeny.

Enterocytozoon bieneusi is emerging as an important cause of chronic diarrhoea in AIDS patients. Its reservoirs and transmission patterns are unknown. In this study, we have examined E. bieneusi sequences from four Rhesus macaques of different origin, which were kept at one animal facility. The sequences were identical in all animals, which suggested that infection had occurred within the facility. Full sequence agreement of E. bieneusi from macaques was found with an E. bieneusi genotype that occurs frequently in humans. To clarify, the relevance of possible inter-species transmission from man to macaque, a phylogenetic analysis was conducted including all sequences of E. bieneusi deposited in GenBank. The hitherto used system of diverse nomenclatures could be reduced to an outlier group and three main lineages, one of which could be further sub-divided into five subgroups. Based in this phylogeny, an association of parasites and host species could be observed for main lineages 2 and 3, as well as for most of the subgroups of main lineage 1. For confirmation, the phylogeny of main lineage 1 was reconstructed with an alternative method of distance estimation, yielding essentially the same parasite-host associations. Zoonotic potential of E. bieneusi is thus supported on a phylogenetic basis.

Selective lectin reactions of two stocks of Leishmania enriettii with differing pathogenicity.

Five days old promastigote culture forms of two stocks of Leishmania enriettii pathogenic and non-infective for Cavia procellus, were tested with the lectins of Canavalia ensiformis, Ricinus communis-120, Soja hispida (Glycine maxima), Arachis hypogaea, Ulex europaeus, Ulex europaeus I, Ulex europaeus II, Laburnum alpinum, Lotus tetragonolobus, Euonymus europaeus and with a monoclonal antibody against blood group H. The pathogenic stock reacted with the anti-H lectin of Lotus tetragonolobus and the monoclonal anti-H and the monoclonal anti-H but not with anti-H of U. europaeus I and E. europaeus. The non-infective stock reacted with none of these anti-H specific agglutinins. They had strong agglutination reactions to C. ensiformis (500 micrograms/ml, 1,000 micrograms/ml, 10 mg/ml) R. communis-120 (1:10), S. hispida (1,000 micrograms/ml) and A. hypogaea (500 micrograms/ml, 1,000 micrograms/ml, 2,000 micrograms/ml). They showed moderate agglutinations to U. europaeus, L. alpinum and U. europaeus II. The non-infective stock showed only moderate reactions to C. ensiformis (10 mg/ml), R. communis-120 (1:10), A. hypogaea (2,000 micrograms/ml) and S. hispida (1,000 micrograms/ml). Neither N-acetylneuraminic acid nor neuraminidase was detected on the cell surface of both stocks. This result demonstrates clearly that both stocks of L. enriettii differ in their cell surface carbohydrates. The agglutination reactions with lectins of the non-infective stock of L. enriettii are very similar to L.t. major.

Neoglycoproteins as tools for the detection of carbohydrate-specific receptors on the cell surface of Leishmania.

Promastigote and amastigote forms of human pathogenic Leishmania from the Old and New World, including promastigotes of L. enrietti, were tested with neoglycoproteins to ascertain the existence of endogenous lectins. These tools expose the chemically coupled sugar that is attached to the inert carrier as a potential ligand for the binding reaction. Agglutination tests demonstrated that the promastigotes of human Leishmania reacted only with the neoglycoproteins N-acetyl-D-galactosamine-para-aminophenyl-bovine serum albumin (gal-NAc-BSA) and N-acetyl-D-glucosamine-para-amino-phenyl-bovine serum albumin (glcNAc-BSA), whereas the amastigote forms failed to react with the neoglycoproteins. In contrast, the promastigotes of L. enriettii were agglutinated by the neoglycoprotein D-mannose-bovine serum albumin (man-BSA). The agglutination reactions could be inhibited by the homologous sugars N-acetyl-beta-D-glucosamine, N-acetyl-beta-D-galactosamine, and alpha-D-mannose. Fluorescence tests yielded the same results. The incubation of the promastigotes with ethylenedinitrolotetraacetic acid (EDTA) prevented their reaction with the neoglycoproteins, whereas the addition of calcium restored it. This result demonstrates that Leishmania express calcium-dependent lectins that are accessible on their surface.

Neuraminidase fluorescence test for the differentiation of Trypanosoma cruzi and Trypanosoma rangeli.

The cell free supernatants of 13 stocks of Trypanosoma rangeli/T. rangeli-like, 50 stocks of T. cruzi/T. cruzi-like and 3 stocks of T. conorhini grown in culture were examined by using the 2-(4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid) test. It could be demonstrated that only the supernatants of the T. rangeli/T. rangeli-like stocks showed a white-yellow fluorescence under UV light. In contrast, the cell free supernatants of the T. cruzi/T. cruzi-like and T. conorhini stocks showed an ash-grey colour under UV light undistinguishable from that of the control reactions. The 4-MUAc test could be a method of choice for the routine detection of T. rangeli/T. rangeli-like flagellates in culture.

Lectin binding strain-specific carbohydrates on the cell surfaces of Leishmania strains from the Old World.

Four-day-old promastigote culture forms of L. tropica major from USSR (LV-252, LV-253, LRC L-38) and Saudi Arabia (Kö, Ha), L. tropica minor from USSR (LV-239, LRC L-39) Leishmania sp. from Israel (Ro) and Saudi Arabia (Ve-322, Schwe, Ne), L. donovani from Sudan (3S, 1-S, LV-139) and India (LV-125, LRC L-51), L donovani infantum from Israel (LV-140), and L. aethiopica from Ethiopia (LV-1, LV-16, LV-24, LV-26) were tested using the following lectins: C. ensiformis, R. communis-120, A. polypoides, P. vulgaris, E. europaeus, D. biflorus, L. tetragonolobus, U. europaeus, L. alpinum, A. papillata II, A. hypogaea, and S. hispida. All strains reacted with C. ensiformis, R. communis-120, and A. polypoides. No agglutination reactions were observed with P. vulgaris, D. biflorus, E. europaeus, and L. tetragonolobus. Agglutination differences were detected by reactions with A. papillata II, U. europaeus, L. alpinum, A. hypogaea, and S. hispida. L. tropica minor (LRC L-39, LV-249), L. donovani (LV-239, 1-S, 3S, LRC L-51), L. aethiopica (LV-1, LV-15, LV-24, LV-26), and L. tropica major (LB-242, LV-253, LRC LK-38, Kö, Ha) are distinguishable with lectins. From L. tropica major two intraspecific forms can be identified: USSR-type (LV-252, LV-253, LRC L-38) and a Near East-type (Kö, Ha). The Leishmania sp. strains (Ve-322, Ne, Ro, Schwe) belong to the Near East-type. The strains L. donovano LV-140 and L. donovani LV-125 react as L. tropica minor, a fact which cannot be elucidated. The L. donovani strains from Sudan cannot be distinguished from the Indian strain L. donovani LRC L-51.

[The diagnostic value of lectins for the epidemiology of Chagas' disease and the differentiation of Leishmania from the Old and New World]. / Der diagnostische Wert der Lektine für die Epidemiologie der Chagas-Krankheit und die Differenzierung von Leishmanien aus der Alten und Neuen Welt.

An interspecific differentiation between T. cruzi, T. rangeli and T. conorhini can be done with the lectin II from the marine sponge Aaptos papillata. Independently from the origin the T. cruzi strains can intraspecifically subdivided into two strain-groups from WGA-type and PNA-type. The Leishmania strains from the Old World can inter- and intraspecifically distinguished by lectins while the strains from the New World form only two groups. A comparison of the agglutination-behaviour of the Leishmania parasites shows that strains of L. m. mexicana, L. b. braziliensis, L. aethiopica and L. m. amazonensis belong to the same type. The second agglutination-type includes strains of L. t. major (Near East), L. m. amazonensis and L. m. pifanoi.

[Latex-Chagestest-reactions of immunsera against Bartonella bacilliformis, Haemobartonella muris and Eperythrozoon coccoides (author's transl)]. / Reaktionen der Immunseren von Bartonella bacilliformis-, Haemobartonella muris- und Eperythrozoon coccoides-Infektionen im Latex-Chagastest.

Sera from 10 patients positive for Carrion's disease were analysed by means of the latex Chagas test. Sera from 40 SPF-rats experimentally infected with Haemobartonella muris and 5 SPF-mice experimentally infected with Eperythrozoon coccoides were similarly tested. Both the human and mice sera were negative whereas positive reaction was observed with rat sera. The nature of this positive reaction has not been clarified.

Differentiation between Trypanosoma cruzi and Trypanosoma rangeli on the basis of their sialic acid content.

Four day-old culture forms of Trypanosoma rangeli strain San Augustin were tested with the following lectins: Canavalia ensiformis, Pisum sativum, Ricinus communis-120, Sojahispida, Aaptos papillata II, Triticum vulgaris, Limulus polyphemus, lberis amara and Arachis hypogaea. These flagellates were only agglutinated by the lectins from Canavalia ensiformis and Pisum sativum. Furthermore four day-old culture forms of the T. cruzi strains Morcego 1354, P-60, Téhuantépéc and WA 301/130 as well as the T. rangli strains San Augustin, Venezuela and DA-3412 were treated with neuraminidase and the supernatant examined by the Aminoff TBA assay. While the supernatant of the T. cruzi strains showed a red colour complex the supernatant of the T. rangeli strains was weak yellow-green. In contrast to the T. cruzi strains no N-acetylneuraminic acid was detectable on the cell surface of T. rangeli. Therefore the Aminoff test can be used for the differentiation between T. cruzi and T. rangeli.

Differentiation between Trypanosoma cruzi and T. rangeli by their different complement sensitivity.

Four-day-old culture forms of T. cruzi strains (Col-83, Y, OPS-31, OPS-32, PF, V, P-60), T. cruzi-like strains (WA-301, CL, FL, MR, Castro Alves, Téhuantépéc, P), T. rangeli strain V and T. rangeli-like strain DA 3412 were tested with the following sera: mouse, NMRI-HAN, germ-free; rat, Wistar HAN, germ-free; guinea-pig, PBW:HAN, SPF; normal fresh guinea-pig, hen, rabbit and human sera, heat inactivated guinea-pig serum and normal fresh guinea-pig serum plus EDTA. Incubation of normal fresh guinea-pig, rabbit, rat and human sera lysed the epimastigote forms of T. cruzi and T. cruzi-like strains but not the culture forms of the T. rangeli and T. rangeli-like strains. Mouse serum did not lyse the culture forms of both trypanosome species while the hen serum lysed all culture forms of T. cruzi, T. cruzi-like as well as T. rangeli and T. rangeli-like. Using DEAE 52 cellulose it was possible to separate the metacyclic forms of T. cruzi and T. cruzi-like from the lysed epimastigote stages. A small number of epimastigote forms of T. cruzi and T. cruzi-like are lysis-resistant against normal fresh guinea-pig serum. Fresh normal guinea-pig, rat, rabbit and human sera can be used for the distinction between T. cruzi, T. cruzi-like and T. rangeli, T. rangeli-like.

[The course of trypanosoma cruzi infection in nonsplenectomized SPE rats with and without Haemobartonella muris infection (author's transl)]. / Der Verlauf einer Trypanosoma cruzi-Infektion in nicht entmilzten SPF-Ratten mit und ohne zusätzliche Haemobartonella muris-Infektion.

Rats were infected with H. muris from the first to the second week of their life. Four weeks later 20 of the infected rats were inoculated with 1.5 x 10(5) T. cruzi. Control animals were given the same number of parasites. In both experimental groups no hemoflagellate could be seen in blood smears four weeks after infection. The comparison of the course of infection of T. cruzi in rats with and without H. muris was made using t- and Wilcoxon-Test for Pair Differences. Rats with H. muris infections showed a low development of T. cruzi up to the 16th day of infection with this flagellate (P less than 0.0005). This difference in the development of the trypanosomes is lost from the 17th to the 27th day after infection.

Comparative studies of Trypanosoma cruzi and T. cruzi-like stocks from different South American countries using lectins.

The agglutination behaviour of four-day-old epimastigote culture forms of 34 Trypanosoma cruzi, and T. cruzi-like stocks from Brazil, Chile, Colombia, Ecuador and Peru were tested with 15 carbohydrate-specific lectins. We distinguished intraspecifically two groups of agglutination reactions: Group 1 includes stocks which react with Triticum vulgaris and Aaptos papillata II (wheat germ agglutinin: WGA-type). Group 2 includes stocks agglutinated by Arachis hypogaea and Aaptos papillata II (peanut lectin: PNA-type). The agglutination reactions with lectins from Triticum vulgaris and Aaptos papillata II correlate with the presence of N-acetylneuraminic acid on the cell surface. After treatment with neuraminidase, the WGA-type is agglutinated by PNA but not by lectins from Triticum vulgaris and Aaptos papillata II. Further results demonstrate that a certain zymodeme pattern can be correlated with carbohydrate determinants.

[Differentiation by microimmunofluorescence of T. cruzi and T. cruzi-like strains from T. conorhini and T. rangeli, using protection from the sponge Aaptos papillata (author's transl)]. / Immunfluoreszenzmikroskopische Unterscheidung zwischen T. cruzi-, T. cruzi-like Stämmen, T. conorhini und T. rangeli mit dem Protektin des Schwammes Aaptos papillata.

Carbohydrates containing terminal nonreducting D N-acetylgalactosamin or D N-acetylglucosamin were identified at the cell surface of Trypanosoma cruzi by an indirect immunofluorescence test using the lectin of the sponge Aaptos papillata. With the method described in this study T. cruzi and T. cruzi-like strains are easy to distinguish from T. rangeli, but not from T. conorhini. The immunofluorescence test presented here may be very helpful for certain diagnostic problems.