B. M. Honigberg

Analysis of surface saccharides inTrichomonas vaginalis strains with various pathogenicity levels by fluorescein-conjugated plant lectins

Certain surface saccharides of organisms from clone-derived cultures of fiveTrichomonas vaginalis strains, JH30A-cl. 1, JH31A-cl. 1, JH32A-cl. 1, JH34A-cl. 1, JH162A-cl 1, and JH384A-cl. 2, which differed in their pathogenicity for women and experimental hosts, were compared with the aid of fluorescein-conjugated plant lectins using a quantitative fluorescence method. The lectins used were: concanavalin A (Con-A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and garden pea agglutinin (GPA). On the basis of experimental results and control experiments, the latter involving incorporation of specific inhibitory sugars in the reaction mixtures, it was concluded that: (1) All five strains had large numbers of Con-A- and WGA-binding saccharide residues. (2) Some also had smaller numbers of SBA- and CBA-binding sites. (3) No strain bound significant amounts of GPA. The differences in CBA binding were not related to pathogenicity of the parasites; however, those in SBA binding could be correlated with the pathogenicity levels of the five strains. The results obtained with SBA in the presence ofN-acetyl-d-galactosamine andd-lactose and those recorded for GPA suggested that the differences between the pathogenic and mildT. vaginalis strains reflected the levels ofd-lactosyl residues on the cell surfaces—these residues were more abundant on strains having higher pathogenicity levels. Possible explanation of the apparent relationships between the presence of the specific sugar residues and pathogenicity are suggested directly or by analogy with other pyranosyls (galactosyls).

Pathogenicity of Trichomonas vaginalis. a clinical and biologic study.

Abstract Pathogenicity for female patients (medical record), pathogenicity for mice (subcutaneous assay), and generation times in vitro of 9 freshly isolated Trichomonas vaginalis strains were compared. High correlation was found between the generation time and pathogenicity levels of each strain for the patients and for mice. There appears to be a close parallelism between the inherent pathogenicity levels of the parasites for the experimental and natural hosts. The results suggest that the high positive correlation between pathogenicity of the trichomonad strains for mice and for patients reflects a causal relationship.

Further studies on the surface saccharides in Trichomonas vaginalis strains by fluorescein-conjugated lectins

Fluorescence emitted by individual cells of severalTrichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in “qualitative” titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41° C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid ofd-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to bed-lactose residues. Similar differences were observed withRicinus communis agglutinin Type I (RCA I), for whichd-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed — more of the lectin bound to a mild than to a virulent strain. The results obtained withRicinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition withN-acetyl-d-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins fromDolichus biflorus andHelix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of allT. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent)T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residuesd-mannose andd-lactose. To render the lectin assay applicable to routine virulence evaluation ofT. vaginalis strains, fluorescence of virulent and avirulent strains was examined visually after incubation of these strains with SBA at concentrations ranging from 50 μg to 1 μg. Differences in virulence were reflected in the SBA titers characteristics of the several strains tested.

Antigenic analysis of Trichomonas vaginalis strains by quantitative fluorescent antibody methods.

Clones of 5Trichomonas vaginalis strains, JH30A, JH31A, JH34A, JH162A, and JH384A, kept in liquid nitrogen (with DMSO serving as the cryoprotectant) after a few weeks of cultivation in axenic cultures, were analyzed for their antigenic properties by quantitative direct fluorescent antibody methods. Measurements of fluorescence of individual cells obtained with the aid of an ultramicrofluorometer indicated that each strain possessed unique antigens as well as antigens it shared with the other strains studied. The relative amounts of common antigens present in each strain were estimated primarily by calculating the percent of fluorescence reduction recorded for organisms stained with their homologous conjugates, nonadsorbed and adsorbed with the homologous and heterologous antigens (strains). Antigenic relationships among the strains were estimated on the basis of statistical analyses of the data obtained in direct, one-step inhibition, and adsorption staining.

TRYPANOSOMA (NANNOMONAS) CONGOLENSE: ANALYSIS BY FLUORESCEIN-CONJUGATED PLANT LECTINS OF SURFACE SACCHARIDES OF CLONED VARIANT ANTIGEN TYPES DIFFERING IN INFECTIVITY FOR MICE

Surface saccharides of 4 cloned VATs (variant antigen types) of Trypanosoma (Nannomonas) congolense, AmNats (Amherst Nannomonas antigen types) 1.1, 1.2, 2.1, and 3.1, derived from 3 different stocks, were compared by fluorescein-conjugated, plant lectins using a quantitative fluorescence method. It was ascertained by the ID63 assay that the 4 AmNats differed in their infectivity for mice. The lectins employed for AmNats 1.1, 2.1, and 3.1 were concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), garden pea agglutinin (GPA), and gorse seed (Ulex europaeus) agglutinin (UEA). In view of the results obtained with these 3 AmNats, only Con A, WGA, and GPA were used with AmNat 1.2, which was isolated after the lectin analyses of the other cloned VATs were completed. On the basis of experimental results, we concluded that the amounts of saccharide residues binding the several lectins differed among the 4 AmNats. In each instance, the reaction specificity was controlled by inclusion of an appropriate sugar in the incubation mixture. Although the actual numbers of various specific lectin-binding sites differed among the AmNats 1.1, 2.1, and 3.1, all of them were found to have the following sugars on their surfaces: alpha-D-mannose, N-acetyl-D-glucosamine, D-galactose, alpha-D-glucose, and alpha-L-fucose. AmNat 1.2 treated with Con A, WGA, and GPA only had the first 2 sugars named above and alpha-D-glucose residues. The results of the ID63 assay indicated AmNats 1.1 and 2.1 to be significantly more infective for mice than AmNats 1.2 and 3.1. The lectin analysis revealed that the 2, more infective, cloned VATs incubated with Con A or WGA emitted significantly (approximately 39% to approximately 62%) more fluorescence than the less infective ones. Thus there were significantly more numerous Con A and WGA binding sites on the more infective AmNats. The situation was reversed with regard to GPA. Upon treatment with this lectin, fluorescence emitted by AmNats 1.1 and 2.1 was significantly (approximately 56% to approximately 81%) lower than that recorded for the less infective AmNats 1.2 and 3.1. In light of our results, infectivity of T. congolense cloned VATs was correlated with the presence of higher numbers of alpha-D-mannose and N-acetyl-D-glucosamine residues and of lower numbers of alpha-D-glucose residues on the surface of the bloodstream trypanosomes. There appeared to be no correlation between infectivity and the numbers of D-galactose and alpha-L-fucose residues present on these parasites.

INFECTIVITY OF MONOMORPHIC AND PLEOMORPHIC TRYPANOSOMA BRUCEI STOCKS CULTIVATED AT 28 C WITH VARIOUS TSETSE FLY TISSUES

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunninghams liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.

Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages ofLeishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54 cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1∶3, 1∶5, and 1∶10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasites/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1–5 parasites. After 48- and 72-h exposures, the macrophages harbored 6–11 and 11–20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with the amastigote forms, after inoculations of the macrophage cultures with promastigotes at the dilutions previously used for amastigotes, only 48–78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5×106 promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much less virulent for these experimental hosts.

Comparison of virulence of clones of twoTrichomonas vaginalis strains by the subcutaneous mouse assay

No statistical differences in virulence were found among five clones isolated from each of twoTrichomonas vaginalis strains JH31A and Balt 42. The former strain, isolated from a patient showing no cervical epithelial abnormalities, caused relatively small subcutaneous lesions in mice [mean volume for the noncloned strain, 75.45±4.43 mm3 (n=70); mean of means for cloned populations, 77.28±3.14 mm3 (n=230)]. The latter, Balt 42, isolated from a woman with an in situ carcinoma of the cervix uteri, produced large subcutaneous abscesses in mice [mean volume for the noncloned strain, 202.28±12.53 mm3 (n=55); mean of means for cloned populations, 200.48±13.72 mm3 (n=264)]. The apparent homogeneity ofT. vaginalis strains with regard to virulence reinforces the dependability of the subcutaneous mouse assay.

Changes in growth rate and infectivity ofLeishmania donovani subjected to various laboratory procedures

Two substrains ofLeishmania donovani strain 3S were used in a study of grwoth rates of promastigote and amastigote stages as well as of infectivity of the latter stages in the course of cultivation, animal passages, and heat adaptation. One of these substrains, 3S-25A, was initiated with amastigotes of strain 3S maintained by serial passages in golden hamsters since 1962. The 3S-25A promastigotes were transferred 24 times (about 142 generations), then passaged once in a hamster. The amastigotes derived from this hamster on postinoculation day 30 were employed for initiation of culture promastigotes designated as substrain 3S-25A′. This culture was transferred 20 times (about 141 generations). The third substrain, 3S-37, was isolated in culture in 1974, then adapted to 37° C. All cultures were grown in modified Tobies medium (Tm). Young C57BL/6J mice were employed in the estimation of generation times (G) of amastigote stages and infectivity of promastigotes.Upon isolation in culture, substrain 3S-25A promastigotes grew poorly and inconsistently during the first five transfers (about 20 generations). Between the fifth and 13th transfers (about 20th and 65th generations), the populations increased from 1.9×107 to 1.2×108 organisms/ml. They remained approximately constant until the 24th serial transfer (about 142nd generation) at which time some of the promastigotes were inoculated into hamsters while others were stabilated in liquid nitrogen. The initial increase in the promastigote yields appeared to depend upon decrease inG (from 12 to 7 h). Promastigotes of substrain 3S-25A′ reached the maximum numbers in the primary cultures.High infectivity was characteristic of the early in vitro transfers of substrain 3S-25A′, 25% for the fifth serial passage (about 30 generations), but the infectivity decreased rapidly on cultivation, reaching a 0.5% level by the tenth transfer (about 100 generations). In contrast, relatively low infectivity levels were observed for 3S-25A promastigotes during the first few passages, e.g., 1.2% for those from cultures obtained after five serial transfers. These levels increased up to the 12th transfer, when they reached 8%. Further cultivation, however, was accompanied by infectivity decrease — after 24 passages in Tm, it was estimated at 4%.TheGs estimated from counts of amastigotes in livers of hamsters inoculated with substrains 3S-25A and 3S-25A′ were closely comparable; for infections with tenth-transfer promastigotes of these strains, the times were, respectively, 34 and 36 h. On the other hand, theG for amastigotes in livers of hamsters inoculated with strain 3S-37 promastigotes from the tenth serial in vitro passage was much longer, 53 h.

Exometabolites of Leishmania donovani promastigotes. II. Spontaneous changes of exometabolite after isolation.

The defined medium of Steiger and Steiger conditioned by growth ofLeishmania donovani strain 3S promastigotes served as the primary source of parasite exometabolites. Four fractions of the conditioned medium were recovered by Sephadex G-25 column chromatography. These fractions shared immunologic determinants, but differed in their molecular weights, affinity for Sephadex G-25, and absorption spectra. Degradation and/or aggregation of one of the fractions (Fraction IV) appeared to yield substances found in the remaining three fractions. Furthermore, by appropriate manipulations, Fraction IV could be transformed into substances resembling those described by previous workers, eg., excretory factor (EF) or antigenically active glycoproteins (AAGP). The present findings suggest that the primary exometabolite released byL. donovani is a small glycopeptide-like molecule; however, in media conditioned by growth of promastigotes and during isolation procedures, aggregation and/or degradation processes yield higher molecular weight substances which vary in size and physico-chemical properties.The defined medium of Steiger and Teiger conditioned by growth oif Leishmania domovani strain 3S promastigotes served as the primary source of parasite exometabolites. Four fractions of the conditioned medium were recovered by Sephadex G-25 column chromatography. These fractions shared immunologic determinants, but differed in their molecular weights, affinity of Sephadex G-25, and absorption spectra. Degradation and/or aggregation of one of the fractions (Fraction IV) appeared to yield substances found in the remaining three fractions. Furthermore, by appropriate manipulations, Fraction IV could be transformed into substances resembling those described by previous workers, eg., excretory factor (EF) or antigenically active glycoproteins (AAGP). The present findings suggest that the primary exometabolite released by L. donovani is a samll glycopeptide-like molecule; however, in media conditioned by growth of promastigotes and during isolation procedures, aggregation and/or degradation processes yield higher molecular weight substances which vary in size and physico-chemical properties.