Activity of disulfiram (bis(diethylthiocarbamoyl)disulphide) and ditiocarb (diethyldithiocarbamate) against metronidazole-sensitive and -resistant Trichomonas vaginalis and Tritrichomonas foetus.
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Clinical resistance of Trichomonas vaginalis to metronidazole is best correlated with MIC values measured under aerobic conditions. Under these conditions both disulfiram (bis(diethylthiocarbamoyl)disulphide), and its first mammalian metabolite, ditiocarb (diethyldithiocarbamate), showed high levels of activity against metronidazole-sensitive (disulfiram MIC, 0.1-0.7 microM; ditiocarb MIC, 0.3-9 microM) and -resistant (MICs 0.2-1.3 microM and 1.2-9 microM respectively) isolates. Tritrichomonas foetus was also sensitive-the MICs for seven metronidazole-sensitive isolates were 0.1-1.0 microM for disulfiram and 1.0-6.9 microM for ditiocarb; those for two highly metronidazole-resistant strains were 0.3-1.3 microM and 0.6-6 microM respectively. Under anerobic conditions most strains became highly resistant to both compounds. Surprisingly, disulfiram was consistently more active than ditiocarb. (+info)
Sialic acid-specific lectin from Tritrichomonas foetus.
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A novel sialic acid-specific lectin (TFL) was isolated from Tritrichomonas foetus culture supernatant and purified by erythrocyte adsorption followed by fetuin-agarose affinity chromatography. According to gel filtration TFL is a protein of 728 kDa, different from the two sialidases of 853 and 254 kDa, secreted by T. foetus into the medium. The lectin is formed by multimeric complexes of 66 kDa subunit according to SDS-PAGE under reducing conditions. TFL is glycosylated with 4.2% of carbohydrates, half of which is represented by glucose. The lectin reacts equally with N-acetyl and N-glycolyl neuraminic acid, free, in alpha2,3- or alpha2,6-linkage. TFL has 7-fold weaker affinity to alpha2,8-linked N-acetylneuraminic acid (Neu5Ac) in colominic acid. Horse erythrocytes containing 4-O-acetyl Neu5Ac are agglutinated equally as compared to the human cells. TFL affinity to 9-O-acetyl Neu5Ac is 4-fold weaker as documented by hemagglutination inhibition with de-O-acetylated bovine submaxillary mucin, and ovine submaxillary mucin. A panel of mono- and oligosaccharides other than Neu5Ac do not inhibit TFL activity at 200 mM. The lectin does not require bivalent cations for activity, shows optimal reactivity at neutral pH and is stable at 4 degrees C. Anti-TFL antibodies identify membrane positivity on T. foetus, suggesting that the lectin functions in adhesion of the parasites. These findings, together with good stability and immunogenicity, make TFL a prospective candidate for further studies, especially in searching for efficient diagnostics and prevention of bovine trichomoniasis. (+info)
Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes.
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Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed. (+info)
Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.
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Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity. (+info)
Iron-induced changes in pyruvate metabolism of Tritrichomonas foetus and involvement of iron in expression of hydrogenosomal proteins.
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The main function of the hydrogenosome, a typical organelle of trichomonads, is to convert malate or pyruvate to H(2), CO(2) and acetate by a pathway associated with ATP synthesis. This pathway relies on activity of iron-sulfur proteins such as pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase and ferredoxin. To examine the effect of iron availability on proper hydrogenosomal function, the metabolic activity of the hydrogenosome and expression of hydrogenosomal enzymes were compared in Tritrichomonas foetus maintained under iron-rich (150 microM iron nitrilotriacetate) or iron-restricted (180 microM 2,2-dipyridyl) conditions in vitro. The activities of PFOR and hydrogenase, and also production of acetate and H(2), were markedly decreased or absent in iron-restricted trichomonads. Moreover, a decrease in activity of the hydrogenosomal malic enzyme, which is a non-Fe-S protein, was also observed. Impaired function of hydrogenosomes under iron-restricted conditions was compensated for by activation of the cytosolic pathway, mediating conversion of pyruvate to ethanol via acetaldehyde. This metabolic switch was fully reversible. Production of hydrogen by iron-restricted trichomonads was restored to the level of organisms grown under iron-rich conditions within 3 h after addition of 150 microM iron nitrilotriacetate. Protein analysis of purified hydrogenosomes from iron-restricted cells showed decreased levels of proteins corresponding to PFOR, malic enzyme and ferredoxin. Accordingly, these cells displayed decreased steady-state level and synthesis of mRNAs encoding PFOR and hydrogenosomal malic enzyme. These data demonstrate that iron is essential for function of the hydrogenosome, show its involvement in the expression of hydrogenosomal proteins and indicate the presence of iron-dependent control of gene transcription in Tt. foetus. (+info)
Antibody responses of cattle immunized with the Tf190 adhesin of Tritrichomonas foetus.
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The antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 of Tritrichomonas foetus were investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding to T. foetus antigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P < 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P < 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P < 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge with T. foetus than controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals. (+info)
An improved polymerase chain reaction assay for the detection of Tritrichomonas foetus in cattle.
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An improved polymerase chain reaction test has been developed to detect Tritrichomonas foetus, the causative agent of trichomoniasis in cattle. The test amplifies a region of the 5.8S ribosomal RNA gene of T. foetus, and it is simple, sensitive, and specific when compared with traditional methods to examine field samples. (+info)
Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase.
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Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket. (+info)