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PubMed1993

[A rapid staining method for the study of the flagellar and round forms of Trichomonas vaginalis from culture].

This method has been developed for the routine observation of the different forms of Trichomonas vaginalis in axenic culture with a low rate of cell loss. This rapid dyeing process is especially suitable for the examination of the ratio between the flagellated and round forms of the parasite. As a result of this staining one can not see fine cell details but can firmly distinguish between flagellated forms and multi-nucleated, multi-flagellated forms (i.e. dividing cells forms).

Animals↗
PubMed1995

[In vitro effect of metronidazole ester on Trichomonas vaginalis and Entamoeba histolytica].

Metronidazole ester, a new nitroimidazole drug, was synthesized by the Department of Physical Chemistry, School of Pharmacy, Shanghai Medical University. The trichomonadicidal and amoebicidal activity of metronidazole ester was evaluated in vitro by the cultivation of Trichomonas vaginalis and Entamoeba histolytica at different drug concentrations. The results indicated that the 100% lethal concentration for T. vaginalis and E. histolytica was 2.6 micrograms/ml and 40 micrograms/ml, respectively after incubation with metronidazole ester for 48 h. In comparison with metronidazole, the effect of metronidazole ester for both protozoa was similar.

Animals↗
PubMed1993

A Trichomonas vaginalis cDNA with partial protein sequence homology with a Plasmodium falciparum excreted protein ABRA.

We have previously isolated two extracellular cysteine proteases (60 kDa, 30 kDa) from the cell filtrate of an isolate of Trichomonas vaginalis. All isolates tested produced a 60 kDa protease as demonstrated by immunoblot with cross-reacting rabbit serum. A T. vaginalis cDNA library was constructed using lambda gt11. A 760 bp T. vaginalis specific cDNA was sequenced and demonstrates partial protein sequence homology with an extracellular cysteine protease of Plasmodium falciparum. These results are consistent with the hypothesis that this protease may be important in the pathogenesis of T. vaginalis infection.

Amino Acid Sequence↗
PubMed1994

The interaction of Lactobacillus acidophilus and Trichomonas vaginalis in vitro.

Recent work with a mouse model of Trichomonas vaginalis infection indicated that estrogenized BALB/c mice that were preinfected with Lactobacillus acidophilus showed a greater duration of T. vaginalis infection as compared to a control group of mice that were not treated with L. acidophilus. To examine the interaction between T. vaginalis and L. acidophilus further we performed in vitro competitive growth assays between the 2 species. Although the addition of L. acidophilus to the T. vaginalis cultures slowed the growth of the protozoa, the added bacteria did not increase trichomonad death. However, T. vaginalis had a deleterious effect on L. acidophilus growth in combined cultures when compared to matched controls. Using an initial inoculum of 10(5)/ml, at 40 hr the control L. acidophilus concentrations had grown to 1.3 x 10(7)/ml. However, in combined culture with T. vaginalis, L. acidophilus concentrations at 40 hr had fallen to 7.8 x 10(5)/ml and 6.1 x 10(4)/ml for the 10:1 (T. vaginalis at 10(4)) and 1:1 (T. vaginalis at 10(5) test ratios, respectively (P < 0.01). This demonstrates that T. vaginalis can cause the concentration of L. acidophilus to fall in vitro and may explain why the concentration of L. acidophilus in the vagina falls in trichomoniasis.

Animals↗
PubMed1993

Biogenesis of the hydrogenosome in the anaerobic protist Trichomonas vaginalis.

Trichomonas vaginalis is a primitive protist that lacks mitochondria and peroxisomes. This aerotolerant anaerobe does, however, contain a specialized organelle involved in carbohydrate metabolism called the hydrogenosome. Hydrogenosomes are the site of fermentative oxidation of pyruvate, coupled to ATP production via substrate level phosphorylation. The organelle is surrounded by 2 membranes and appears to contain no genetic material. Hydrogenosomes are proposed to have arisen either through the conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. Our studies show that hydrogenosomal proteins are synthesized on free polyribosomes and are, thus, posttranslationally translocated into the organelle. The 2 hydrogenosomal proteins we have examined in detail, ferredoxin and the beta subunit of succinyl CoA synthetase, appear to be synthesized as larger precursors that contain an 8- or 9-amino acid amino-terminal extension, respectively. These apparent leader sequences are absent from the mature proteins found in hydrogenosomes. The biochemical properties of the amino-terminal extensions are similar to one another and to mitochondrial presequences. These observations are consistent with the hypotheses that the specificity of protein translocation into hydrogenosomes is conferred by a short amino-terminal signal sequence and that the mechanisms underlying translocation are similar to that of mitochondria. Using in vitro import assays, we have shown that this short leader sequence is absolutely required for association of hydrogenosomal proteins with the organelle and that this association is dependent on ATP, temperature, and the presence of intact organelles.

Adenosine Triphosphate↗
PubMed1995

Examination of nonflagellate and flagellate round forms of Trichomonas vaginalis by transmission electron microscopy.

Plasmodium-like round cells without sections of flagella, round flagellar cells with dividing nucleus and binuclear flagellate round forms were represented from the log-phase of Trichomonas vaginalis cultures. The fine-structural characteristics of the round cells support the opinion that the cells without flagella as well as the round multinuclear flagellate cells represent developmental stages preceding the appearance of mononuclear flagellate cells. In the round cells, amitotic division was observed instead of the well-known cryptopleuromitosis, without formation of metaphasic chromosomes and desmosomes. Our investigations onto the fine structure of the parasite support the assumption that the non-flagellate forms are viable cells of the parasite and point to still unknown phases of its biology.

Animals↗
PubMed1995

Characterization of cDNAs encoding adhesin proteins involved in trichomonas vaginalis cytoadherence.

Trichomonas vaginalis cytoadherence is mediated by four adhesins (AP65, AP51, AP33 and AP23). Adhesin gene expression was previously shown to be up-regulated by iron. Therefore, a cDNA library was constructed from mRNA of T. vaginalis grown in a high-iron medium. Ten cDNA clones (three for AP65, one for AP51, and six for AP33) were recognized with polyclonal antiserum or monoclonal antibody raised against these adhesins. No cross-hybridization among cDNAs of the three adhesins was observed in Southern analysis, confirming restriction mapping analysis of representative cDNAs. Southern analysis probed with representative cDNAs of the adhesins indicated multiple copies for each of the three adhesin genes. Northern analysis showed transcripts of 1.8 kilobases (kb), 1.4 kb, and 0.9 kb for AP65, AP51 and AP33, respectively. Consistent with adhesin expression, mRNAs for these adhesins were detected only when parasites were grown in high-iron conditions. Specific antibodies eluted from E. coli expressing recombinant proteins reacted only with the respective parasite adhesins. Recombinant proteins bind to fixed HeLa cells and competed with radiolabeled trichomonad adhesins for binding. Although proteinase activity is required for cytoadherence, recombinant proteins show no detectable proteinase activity. These data show that recombinant proteins from these clones exhibited characteristics of the trichomonad adhesins.

Animals↗
PubMed1995

Two Trichomonas vaginalis surface proteinases bind to host epithelial cells and are related to levels of cytoadherence and cytotoxicity.

Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis require cysteine proteinase activity. Because of the large number of cysteine proteinases synthesized by T. vaginalis, a ligand assay was used to identify specific proteinases which may selectively target host cells. Two cysteine proteinases from trichomonal extracts with relative molecular masses (Mr) of 65,000 daltons (65-kDa) and 30-kDa were found to avidly bind to HeLa cell and vaginal epithelial cell surfaces. The two proteinases were distinguished by differential inhibition with leupeptin and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). Leupeptin pretreatment of live organisms inhibited the 30-kDa proteinase, which concomitantly reduced or eliminated cytoadherence. The T. vaginalis isolates with low levels of cytoadherence also had diminished or no detectable 30-kDa proteinase activity. On the other hand, TLCK pretreatment inhibited both the 30-kDa and 65-kDa proteinases, which resulted in decreased levels of cytoadherence and totally abolished contact-dependent cytotoxicity. Furthermore, isolates capable of attachment but with little or no cytotoxicity toward HeLa cells had no detectable host cell-bound 65-kDa proteinase. Finally, antiserum generated to each proteinase reacted by indirect immunofluorescence with live organisms, suggesting a surface location for both proteinases. This strategy and use of the ligand assay may permit for the deliniation of the role of two specific T. vaginalis surface proteinases in the properties of cytoadherence and cytotoxicity.

Animals↗
PubMedDate not supplied

Lysis of erythrocytes by Trichomonas gallinae.

The hemolytic activity of five live isolates of Trichomonas gallinae was investigated. The isolates were subsequently tested against the erythrocytes of seven adult animal species. Each of the five isolates tested lysed all human blood groups, as well as rabbit, rat, chicken, horse, bovine, and sheep erythrocytes. No hemolysin released by the parasite could be detected. Our preliminary results suggest that the hemolytic activity is not due to the hemolysin release by T. gallinae or to a product of its metabolism. Pretreatment of live trichomonads with concanavalin A reduced levels of hemolysis by 40%.

Adult↗
PubMed1996

Comparable growth of a Trichomonas vaginalis strain in PEHPS and TYI-S-33 media.

PEHPS medium, developed for axenic cultivation of Entamoeba histolytica and E. invadens, was also capable of supporting the growth of a Trichomonas vaginalis strain, with an inoculum of 1 to 100 trichomonads/ml. The logarithmic growth phase in PEHPS or in TYI-S-33 medium lasted 72 h; yield (3.33 +/- 0.56 x 10(6) trichomonads/ml), duplication time (4.27 h), number of duplications (16.85), or increase ratio (33,328) in PEHPS medium showed no significant differences with those obtained in TYI-S-33 under similar culture conditions. Accordingly, PEHPS medium might be used for the axenic cultivation of T. vaginalis.

Adult↗
PubMed1997

Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis.

Heat shock protein (Hsp) homologs of Trichomonas vaginalis belonging to Hsp70 and Hsp60 families with Mr 72 and 58 kDa, respectively, were detected by cross-reacting polyclonal antibody to DnaK of Escherichia coli. polyclonal antibody to Hsp60 of Heliothis virescens, and polyclonal antibody to GroEL/Hsp60 of cyanobacteria. A diffuse and finely granular intracellular staining of T. vaginalis was obtained with anti-DnaK on frozen 4-micron sections of the parasite as revealed by light microscopy. This antibody also recognized T. vaginalis antigen(s) present mainly in the Golgi apparatus, endoplasmic reticulum, and hydrogenosomes, as demonstrated by immunoelectron microscopy. Both antibodies against Hsp60 variants localized a homolog antigen in light microscopic granules, corresponding to hydrogenosomes of T. vaginalis.

Animals↗
PubMed1997

Chromosome number of Trichomonas vaginalis.

Chromosome number of an axenically grown Trichomonas vaginalis isolate was studied using a 1 mM solution of colchicine and a hypotonic swelling technique. The diploid chromosome number was 2n = 6 [corrected]. Each pair of chromosomes can be identified by its morphology and size. This observation could be important with respect to gene mapping and molecular cloning for genes of T. vaginalis.

Animals↗
PubMed1997

Conservation of mitochondrial targeting sequence function in mitochondrial and hydrogenosomal proteins from the early-branching eukaryotes Crithidia, Trypanosoma and Trichomonas.

Kinetoplastid protozoa are the earliest-branching eukaryotes to possess a true mitochondrion. This organelle is host to a variety of intriguing and unique features, including RNA editing. We examined the characteristics of protein import into mitochondria of Trypanosoma brucei. Dihydrofolate reductase (DHFR) carrying a yeast mitochondrial targeting signal was correctly translocated into trypanosome mitochondria in vivo, as were DHFR fusion proteins bearing two unusually short (7-9 amino acids) presequences from trypanosomatids. The short trypanosomal targeting signals were functional in Saccharomyces cerevisiae as well, but their targeting efficiency was lower and processing was absent. Trichomonads branched even earlier than kinetoplastids in eukaryotic evolution and contain energy-generating organelles called hydrogenosomes. The origin of hydrogenosomes has been controversial, but most evidence suggests that they are related to mitochondria. Putative hydrogenosomal targeting signals from Trichomonas vaginalis are short (5-12 amino acids). Three such sequences were capable of targeting a passenger protein to mitochondria both in yeast and in trypanosomes, and one of the hydrogenosomal presequences was efficiently processed in both organisms. These findings suggest a resemblance between the import machineries of mitochondria and hydrogenosomes.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of cytosolic malate dehydrogenase from Trichomonas vaginalis.

Malate dehydrogenase (EC 1.1.1.37.) (MDH) was purified to apparent homogeneity from the cytosolic fraction of the protozoan Trichomonas vaginalis Donné. The four step purification included ion-exchange chromatography (DEAE-Sephacel and Q-Sepharose, elution with NaCl) and affinity chromatography (Reactive Red Agarose, elution with NADH and NaCl). The enzyme was purified about 132-fold (30.6% yield) to a specific activity of 352 units mg-1. The Km values determined at pH 7.8 (pH optimum from 7.5 to 8.3) for oxaloacetate and NADH were 16.2 microM and 10.6 microM, respectively. The MDH activity was inhibited by the substrate, decreasing to 50% at about 1 microM concentration of oxaloacetate. The reverse reaction from malate to oxaloacetate showed a pH optimum around pH 9.5. The Km for malate and NAD+ (determined at pH 7.8) were 1220 microM and 69.9 microM, respectively. SDS-PAGE analysis of the purified MDH revealed a single band with an apparent size of 34.5 kDa. The native molecular weight was estimated by HPLC gel filtration to be 60 kDa, which indicates that the T. vaginalis MDH exists as a dimer.

Animals↗
PubMed1998

Axenic cultivation of Trichomonas vaginalis in a serum-free medium.

Mammalian serum or bovine serum albumin are essential for Trichomonas vaginalis cultivated under axenic conditions. Unfortunately, these components inhibit several biological properties of these parasites. PACSR is a serum replacement, free of bovine serum albumin. used for axenic cultivation of Entamoeba histolytica. We show that PACSR is also useful for axenic cultivation of T. vaginalis. Tubes containing 5.5 ml PEHP, or TYI basal media, plus 8% PACSR (v/v), were inoculated with 10(3) trichomonads/ml from 3 strains (GT-3, GT-13, and GT-15) and incubated at 36.5 C for 72 hr. Depending on the strain, cultures grown in PEHP plus PACSR reached densities of 50% (GT-13), 63% (GT-3), or 82% (GT-15) as compared to controls grown in PEHPS. On the other hand, yields of GT-3, GT-13, and GT-15 maintained in TYI plus PACSR were, respectively, 53%, 57%, and 67% among those of cultures grown in TYI-S-33. In all experiments, PEHPS and TYI-S-33 contained 8% bovine serum. Yields reached in PEHPS were 2.07+/-0.275 to 4.83+/-4.41 x 10(6) trichomonads/ml, whereas in TYI-S-33, densities were 1.68+/-0.315 to 4.16+/-8.07 x 10(6) trichomonads/ml. In conclusion, PACSR added to PEHP or TYI media is useful for axenic cultivation of T. vaginalis in the absence of serum or bovine serum albumin. PACSR could be useful in performing analyses of biological properties that are inhibited by serum or any of its components.

Animals↗
PubMed1994

A study on the in vitro susceptibility of Trichomonas vaginalis to metronidazole.

The susceptibility of 32 clinical isolates of Trichomonas vaginalis to metronidazole has been studied under aerobic and anaerobic conditions. Of 32 patients with vaginal trichomoniasis, 27 (84%) were cured by a standard metronidazole treatment regimen (200 mg thrice daily for seven days). The geometric means of minimum inhibitory concentration (MIC) under aerobic and anaerobic conditions for these isolates were 2.0 and 0.4 micrograms/ml, respectively; the geometric means of minimum lethal concentration (MLC) under aerobic and anaerobic conditions were 7.4 and 2.0 micrograms/ml, respectively. However, for those five patients with treatment failure, the geometric means of MIC for these isolates under aerobic and anaerobic conditions were 6.8 and 1.1 micrograms/ml, respectively; the geometric means of MLC under aerobic and anaerobic conditions were 18 and 5.5 micrograms/ml, respectively. Trichomonads reisolated from patients after treatment failure had similar susceptibility to metronidazole as before treatment. However, all five women were cured by a second course of metronidazole treatment. Although primary treatment failure was common when isolates of T. vaginalis had aerobic MLC values of > 18 micrograms/ml or anaerobic MLC values > 5.5 micrograms/ml, two cases with isolates having high MLC values (aerobic: 20 micrograms/ml, anaerobic: 5 micrograms/ml) responded well to the standard treatment. It was evident that no metronidazole-resistant trichomonads were found in this study.

Animals↗
PubMed1995

[The action of an aqueous extract of Aloe barbadensis Miller in an in-vitro culture of Trichomonas vaginalis].

The antiparasitic action of an aqueous extract of Aloe barbadensis Miller against and in vitro culture of Trichomonas vaginalis was studied for the first time. Three strains of this parasite were used for the study. Taking an initial concentration of 400 mg/mL of the extract, double serial dilutions were performed, and final concentrations based on the dried weight of the extract were 10.4, 20.8, 41, 83, and 160 mg/mL. Within 24 hours, percentages of inhibition greater than 50% were obtained from concentrations of 20.8 micrograms/mL. Similar results were obtained at 48, and 72 hours, with a lower concentration, the inhibition of growth was greater than 50%.

Aloe↗
PubMed1998

[Morphology and diagnosis of the oral protozoans Trichomonas tenax and Entamoeba gingivalis using the Giemsa-Romanovsky stain].

In the microscopic diagnosis of Trichomonas tenax and Entamoeba gingivalis is the technically and time not demanding native preparation of a culture, in which both protozoans can be detected according to their typical motility, determining. In the permanent preparation of the culture stained according to Giemsa-Romanovsky, which has also documentary character, are all of the characteristic cell organelles stainable, enabling so their detection without their typical motility. Staining according to Giemsa-Romanovsky is technically simple and not time consuming, not very laborious, low cost and the coloration is permanent, that means optimal for the diagnostic of oral protozoans in permanent preparations. (Fig. 5, Ref. 4.)

Adolescent↗