Tritrichomonas foetus: preparation of monoclonal antibodies with effector function

Monoclonal antibodies were prepared against Tritrichomonas foetus and characterized with regard to binding and immune effector activities. Nine of 27 monoclonal antibodies which reacted with T. foetus appeared to bind to the surface of live parasites. Immunoelectron microscopy confirmed the surface binding of two of these. At least six of these surface reactive monoclonal antibodies facilitated complement mediated lysis of T. foetus and one acted as an opsonin for phagocytosis by peripheral blood bovine monocytes. Five surface reactive monoclonal antibodies identified a molecule of approximately 150,000 relative molecular weight on Western blots of whole parasite preparations. These results collectively suggest the 150,000 relative molecular weight molecule may be an important target antigen for the immune response to T. foetus.     

Host-parasite interaction in bovine infection with Tritrichomonas foetus

Tritrichomonas foetus is a parasite of particular veterinary importance causing bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. The present review summarizes the current knowledge on potential mechanisms of pathogenicity of T. foetus, the immunology of host-parasite interaction in bovine tritrichomonosis, and the experimental model systems of this parasitic disease.     

Characterization of antigenic proteins from Tritrichomonas foetus recognized by antibodies in rabbit serum, bovine serum and bovine cervicovaginal mucus

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract and is recognized as 1 of the more common infectious agents causing decreased reproductive efficiency in beef cattle. Infections result in reproductive failure and produce considerable economic loss. Vaccination of heifers with vaccines containing T. foetus induces elevated serological responses to many T. foetus antigens, decreases the rate and/or length of infection with T. foetus, and decreases fetal loss caused by infection. Because T. foetus infections are usually limited to lumen and mucosal surfaces of the reproductive tract, it has been assumed that protection from infection and abortion is partially mediated by immunoglobulins in the uterus and vagina. The objective of this study was to identify and characterize specific antigens of T. foetus that show promise for use in a recombinant vaccine that will generate a protective mucosal immune response in cattle. Surface proteins were identified by using polyclonal rabbit anti-trichomonal sera eluted from paraformaldehyde-fixed cells. Analyses of these proteins, utilizing mucosal antibodies from vaccinated and convalescent cows, have identified proteins involved in generating a local immune response. Western immunoblot analysis indicates that these proteins are well conserved and are excellent candidates for incorporation into a recombinant vaccine.     

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.     

Pyrimidine metabolism in Tritrichomonas foetus

The pyrimidine metabolism of Tritrichomonas foetus (KV 1) was studied using whole cells and cell homogenates. Pyrimidines and pyrimidine nucleosides were readily incorporated into nucleic acids. Orotate and aspartate were not incorporated into pyrimidine bases. Enzymes of the pyrimidine salvage pathway (i.e., thymidine and uridine phosphorylases and uridine kinase) were detected in trophozoite homogenates, but the activities of de novo pyrimidine synthesis enzymes (i.e., carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase) were below the level of detection in these same homogenates. The evidence presented supports the proposal that T. foetus is incapable of synthesizing pyrimidines de novo but is capable of salvaging preformed pyrimidines and pyrimidine nucleosides from the growth medium and that enzymes of this parasite's pyrimidine salvage pathway are not organelle-associated.     

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.     

Lactoferrin-binding proteins of Tritrichomonas foetus.

Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle. It has an essential requirement for iron, which it obtains from host lactoferrin. However, specific lactoferrin-binding protein receptors have not yet been identified in T. foetus. To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T. foetus lactoferrin-binding proteins. Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin. This is relevant because T. foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin. Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T. foetus may also be involved. In the presence of bovine cervical mucus, binding of lactoferrin to T. foetus was diminished, suggesting that mucus has an effect on lactoferrin binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa. Because lactoferrin-binding proteins may be susceptible to digestion by T. foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible. It is possible that there may be multiple proteins or multimers of the same protein. In summary, the data showed that binding of lactoferrin to T. foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.     

The Surface Charge of Tritrichomonas foetus

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of ?1.03 μmμs^?1μV^?1μcm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (> 9.0), there is an increase in the surface charge reaching an EPM of ?2.5 μmμs^?1μV^?1μcm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.     

Osmoregulation in the parasitic protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.     

Osmoregulation in the Parasitic Protozoan Tritrichomonas foetus

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K⁺ concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K⁺ concentrations and was inhibited when K⁺ and/or Cl⁻ was absent from the medium. The results suggested that the well-documented Na⁺-K⁺-2Cl⁻ cotransport system was responsible for the K⁺ influx activated during the RVI. However, inhibitors of Na⁺-K⁺-2Cl⁻ cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K⁺ influx in T. foetus.     

Polysaccharide and glycolipid composition in Tritrichomonas foetus

1. The polysaccharide and glycolipid composition in Tritrichomonas foetus was studied by paper, thin-layer and gas-liquid chromatographic analysis. 2. The carbohydrate components of the polysaccharide were glucose (47%), galactose (34%) and mannose (19%). N-acetylneuraminic acid was the sialic acid derivative characterized in the flagellate whole cells. 3. The sialic acid density was estimated as 2.7 x 10(7) residues/cell. 4. The long-chain base dihydrosphingosine, the carbohydrates galactose (67%), glucose (21%) and mannose (12%) as well as the fatty acids myristic (48%) and palmitic (52%) acids were characterized as components of the total glycolipids of T. foetus. 5. Total glycolipids were fractionated: a galactocerebroside and a ganglioside were identified.     

Cell Surface Carbohydrates in Tritrichomonas foetus1

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid-thiosemicarbazide-silver proteinate, concanavalin A-horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.     

Methodological approaches to immunohistochemical demonstration of beta-hexosaminidase in human placental and renal tissue with monoclonal antibodies

The lysosomal enzyme, beta-hexosaminidase (Hex), was studied in full-term human placentas and in renal tissue using monoclonal antibodies raised against Hex purified from human placentas. The immunohistochemical reaction for Hex was pronounced in trophoblastic cells and macrophages of the basal plate and the smooth chorion, but was faint or negative in the amnion as well as in the syncytiotrophoblast and Hofbauer cells of the chorionic villi. The maternal decidual cells of the basal plate were negative. Biochemical enzyme analysis showed the highest activity in basal plate cells (containing trophoblast, decidual cells, macrophages and neutrophils) and a low activity in the chorionic villi. Placental tissue was less positive with monoclonal antibodies specific for Hex A, compared with antibodies reacting with both Hex A and Hex B. Epithelial cells of the renal proximal tubules were positive to the same degree with antibodies recognizing both Hex A and Hex B as well as those recognizing only Hex A.     

Adenosine triphosphatase activity of Tritrichomonas foetus.

Homogenates of Tritrichomonas foetus exhibited a Mg2+-dependent adenosine triphosphatase (ATPase) activity, with a pH optimum in Tris buffers of 8.2 to 8.3. The activity was not sensitive to oxygen. At high concentrations, quercetin and 4-chloro-7-nitrobenzofurazan inhibited ATPase activity in the cytoplasmic extract by 20 and 70%, respectively, whereas oligomycin, venturicidin, triethyltin, leucinostatin, dibutylchloromethyltin chloride, spegazzinine, efrapeptin, citreoviridin and sodium azide had no effect and N,N'-dicyclohexylcarbodi-imide stimulated the activity somewhat. The activity was localized in a population of small cytoplasmic particles which also contained an acid phosphatase. There was no indication of an association of ATPase with hydrogenosomes. The ATPase activity (or activities) in this aerotolerant anaerobe is different from the ATPases characteristic of mitochondria or of anaerobic bacteria.     

Surface Domains in the Pathogenic Protozoan Tritrichomonas foetus

ABSTRACT The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus . The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.     

Identification of tissue histiocytes on paraffin sections by a new monoclonal antibody

A mouse monoclonal antibody (MAC 387) with specificity for monocytes and tissue histiocytes was produced by immunization of a BALB/c mouse with peripheral blood monocyte components derived by affinity chromatography of detergent-solubilized monocyte material on Sepharose 4B coupled to rabbit anti-monocyte antibodies. MAC 387 strongly stained the cytoplasm of cells of the monocyte/macrophage series on paraffin sections after controlled trypsinization of sections. The antibody showed broad reactivity for a variety of tissue histiocytes, including infiltrating and reactive histiocytes, alveolar macrophages, Kupffer cells, follicle-center macrophages, splenic red pulp macrophages, tumor-infiltrating macrophages, sinus histiocytes, epithelioid giant cells (variably), and cases of histiocytosis X and dermatopathic lymphadenopathy. Molecular weight data obtained by Western blotting, immunoprecipitation, and immunoaffinity-purification revealed that the antigen was present in different forms in the monocyte and granulocyte. In the granulocyte, free alpha (Mr 12 KD) and beta (Mr 14 KD) chains expressing the MAC 387 epitope were found together with associations of one alpha and one beta chain linked by disulfide bonds to yield a heterodimer of Mr 26 KD. In the monocyte, free alpha and beta chains are not found, but instead the heterodimer and associations of two (Mr 56 KD) and four (Mr 112 KD) heterodimers are disulfide-linked together. This new monoclonal reagent should have particular value for identification of tissue histiocytes in routine paraffin sections and particularly for demonstration of histiocytes in malignant lymphomas.     

Ultrastructural characterization of the isolated hydrogenosome in Tritrichomonas foetus

In the present study we show new aspects of the hydrogenosome ultrastructure as well alterations induced by the fractionation technique. The morphology of freshly isolated hydrogenosomes as well those found in whole cells of Tritrichomonas foetus were examined in thin-sections, in replicas of fast-freezing, and conventional freeze-fracture, freeze-etching, and by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). The true surface as well the concave and convex fracture faces of the inner and outer membranes are shown. We showed that after fractionation procedures the hydrogenosome ultrastructure can be changed, since isolated hydrogenosomes present patchwork-like structures, rosettes and the inner hydrogenosomal membrane is displaced. The peripheral vesicle is seen as a distinct compartment, since its content and morphological appearance is quite different from the rest of the organelle. The peripheral vesicle shows a smooth surface but presenting pores with 20 nm in diameter with a density of 7/micron 2 when observed after freeze-etching. We report the existence of characteristic intramembrane particles distribution and density on hydrogenosome membranes of isolated and whole T. foetus, suggesting that this organelle can have its morphology changed as consequence of technical modifications or as expression of its metabolic state.     

Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.     

Surface domains in the pathogenic protozoan Tritrichomonas foetus.

The quick-freezing and freeze-etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.