Fine structure and molecular characterization of two new parabasalid species that naturally colonize laboratory mice, Tritrichomonas musculus and Tritrichomonas casperi.

Tritrichomonas muris is a common flagellated protist isolated from the cecum of wild rodents. This commensal protist has been shown previously to alter immune phenotypes in laboratory mice. Other trichomonads, referred to as Tritrichomonas musculis and Tritrichomonas rainier, also naturally colonize laboratory mice and cause immune alterations. This report formally describes two new trichomonads, Tritrichomonas musculus n. sp., and Tritrichomonas casperi n. sp., at the ultrastructural and molecular level. These two protists were isolated from laboratory mice and were differentiated by their size and the structure of their undulating membrane and posterior flagellum. Analysis at the 18S rRNA and trans-ITS genetic loci supported their designation as distinct species, related to T. muris. To assess the true extent of parabasalid diversity infecting laboratory mice, 135 mice bred at the National Institutes of Health (NIH) were screened using pan-parabasalid primers that amplify the trans-ITS region. Forty-four percent of mice were positive for parabasalids, encompassing a total of eight distinct sequence types. Tritrichomonas casperi and Trichomitus-like protists were dominant. T. musculus and T. rainier were also detected, but T. muris was not. Our work establishes a previously underappreciated diversity of commensal trichomonad flagellates that naturally colonize the enteric cavity of laboratory mice.

Ultrastructural features of Tritrichomonas mobilensis and comparison with Tritrichomonas foetus.

Tritrichomonas mobilensis is an intestinal parasite of squirrel monkeys. There are few reports concerning the morphological aspects of this parasite. In addition, the taxonomic relationship between T. mobilensis and Tritrichomonas foetus, a serious pathogen that causes bovine and feline trichomonosis, has been questioned. For this reason, in the present study, we examined and compared both tritrichomonads with regard to their morphology, ultrastructure, endocytic activity and cytotoxicity when in the presence of host cells. Electron microscopy demonstrated consistent morphological differences between the hydrogenosomes of both parasites. Moreover, T. mobilensis and T. foetus had striking differences in their endocytic behavior. Thus, this work provides additional data that support the hypothesis that T. mobilensis is a distinct species from T. foetus.

Are Tritrichomonas foetus and Tritrichomonas suis synonyms?

Tritrichomonas suis, a tritrichomonad of pigs, and the related species Tritrichomonas foetus, a tritrichomonad of cattle, are morphologically identical. The taxonomic relationship between these two tritrichomonads has been questioned ever since they were established as distinct species in 1843 and 1928, respectively. Here, we compare the similarities of morphology, ultrastructure, distribution, host specificity, characteristics of in vitro cultivation, immunology, biochemistry and analysis of molecular data from published sources between these two species. All data indicate that these two tritrichomonad species are identical. Thus, we propose that T. foetus and T. suis are synonyms.

Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus.

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 microm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.

Scanning electron microscopy study of Tritrichomonas augusta.

Tritrichomonas augusta is a flagellated protozoan that parasitizes amphibians and reptiles. According to scanning electron microscopy (SEM), the cell shape of T. augusta varies from slender pyriform to ovoidal. Our data show the morphological features of the trophozoites: the emergence of the anterior flagella, the structure of the undulating membrane and the position and shape of the pelta, axostyle and posterior flagellum. In addition, herein we describe spherical forms which are probably pseudocysts. The description of the external structure of T. augusta, as demonstrated by SEM, contributes to the understanding of the biology of this parasite.

Pseudocysts in trichomonads--new insights.

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with antitubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.

Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

Glutamylated and tyrosinated tubulin were localized in Giardia intestinalis and selected trichomonads of the Tritrichomonadinae subfamily, using specific monoclonal antibodies directed at each of the post-translational modifications. Analysis was carried out using indirect immunofluorescence microscopy. Although trichomonad tubulins remained unlabeled by anti-tyrosine tubulin (TUB-1A2), the presence of the glutamylation motif (GT 335) was confirmed and found to differ in distribution among tritrichomonads. Tritrichomonas muris was most heavily labeled with GT 335, while T. foetus was the least so. Like trichomonads, Giardia was unreactive to anti-tyrosine tubulin; however, the GT 335 antibody produced marked fluorescence in Giardia trophozoites. This study is the first to report immunofluorescent localization of tubulin glutamylation in Giardia and confirms previously reported mass spectrometry data.

Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis.

In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.

Fine structure and isozymic characterization of trichomonadid protozoa.

Tritrichomonas suis and T. foetus are characterized herein at the ultrastructural and biochemical levels. Microcinematography and measurements, scanning and transmission electron microscopy, cytochemistry for carbohydrate detection (Thiéry technique), and isozyme electrophoresis analysis were performed. In all, 11 different strains from 5 species of parasites were studied (T. foetus, T. suis, Trichomonas gallinae, T. vaginalis, and Monocercomonas sp.). A total of 11 enzymes were scored. Fine-structure study using scanning and transmission electron microscopy demonstrated that T. suis and T. foetus are identical morphologically. The high degree of isozymatic similarity noted between T. suis and T. foetus is consistent with the hypothesis that they may be different strains of the same species.

Prevalence and morphology of tritrichomonas suis from the nasal cavity of domestic pigs, sus scrofa, without atrophic rhinitis in the southern Brazil

The Tritrichomonas suis (Gruby and Delafond) that occur in the nasal cavity of domestic pigs. Sus scrofa, was isolated, described and its prevalence studies in healthy swine of different areas of Rio Grande do Sul, Brazil. The parasite protozoa was found in 2 of 208 cultures of nasal washings, representing a prevalence of 0.96 porcent. The morphology study of the live specimens done by phase contrats microscopy, dark field microscopy and by examination of fresh and stained specimens, showed that the T. suis isolated has the same morphological characteristics as the nasal cavity of trichomonads described by othe authors

The heat shock response and major heat shock proteins of Tritrichomonas mobilensis and Tritrichomonas augusta.

The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes.

Intestinal trichomoniasis due to Tritrichomonas mobilensis in tree shrews (Tupaia belangeri).

Intestinal trichomoniasis was observed in 156 of 202 Tupaia belangeri (77.2%). The parasites were located principally in the cecum (75%) and were far less common in the proximal portion of the colon (19%) or terminal portion of the ileum (6%). Advanced trichomoniasis was associated with liquid cecal contents but not diarrhea. The trichomonads had a tendency to penetrate the mucosal epithelial layer, causing desquamation of entire crypts. They never penetrated the epithelial basement membrane and never triggered inflammatory responses. The trichomonads were characterized by three anterior flagella and one trailing flagellum that extended over the entire parasite body, connected to it by an undulating membrane. The capitulum of the straight axostyle formed a small but well defined pelta, and the stout costa had distinct banding. The parasites thereby matched the description of Tritrichomonas mobilensis.

Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.

The interaction of Tritrichomonas foetus and Trichomonas vaginalis with macrophages.

The process of interaction between macrophages and Tritrichomonas foetus and Trichomonas vaginalis was analysed using light microscopy, scanning and transmission electron microscopy. The parasites attach to the macrophage surface and are ingested through a phagocytic process. Parasite-macrophage association index was higher for activated than for resident macrophages. Previous incubation of the parasites in the presence of Concanavalin A rendered their surface less negative and more hydrophobic, as evaluated by measurement of the zeta potential and contact angle, respectively. This treatment significantly increased parasite ingestion by resident, but not activated macrophages.

Fibronectin- and laminin-mediated endocytic activity in the parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus.

The pathogenic protozoan Tritrichomonas foetus is able to ingest polystyrene particles with a diameter up to 1.0 micron. Trichomonas vaginalis, however, ingest particles as large as 4.4 microns in diameter. The particles are found within cytoplasmic membrane-bounded vacuoles. Morphometrical analysis showed that T. vaginalis presents a higher endocytic activity than T. foetus. Coating of the polystyrene particles with cationized ferritin increases their binding to the parasite surface but does not interfere with their ingestion. In contrast, coating with laminin significantly increased the uptake of the particles by both parasites while coating with fibronectin potentiates the ingestion of the particles only by T. foetus. These observations suggest the presence of laminin- and fibronectin-binding sites on the surface of trichomonads, an observation which is in agreement with the recent description of a receptor for laminin on the surface of trichomonads.

Electron spectroscopic imaging of calcium in the hydrogenosomes of Tritrichomonas foetus.

Electron spectroscopic imaging (ESI) of very thin unstained sections of Tritrichomonas foetus fixed in an osmium tetroxide solution containing calcium and potassium ferrocyanide showed the presence of calcium in the form of electron-dense deposits located in the hydrogenosomes.

Tritrichomonas foetus: freeze-fracture cytochemistry using polymyxin B.

The general structure of Tritrichomonas foetus incubated in the presence of the peptide antibiotic polymyxin B, which interacts specifically with anionic phospholipids, was analyzed using transmission electron microscopy of thin sections, and freeze-fracture replicas. Polymyxin B induced morphological changes in the plasma membrane of the parasites with the formation of membrane blebs with a diameter varying from 65 nm to 1.5 micron. Freeze-fracture images of the membrane lining the blebs showed that their inner membrane half is smooth. However, membrane particles, with a density similar to that observed on the E face of the plasma membrane, were seen on the outer half of this membrane.

[The presence of cysts in Tritrichomonas muris]. / Presencia de quistes en Tritrichomonas muris.

Cysts of Tritrichomonas muris are reported. The morphology of this evolution stage is described under light and electron microscopy. The biologic and epidemiologic importance of this finding is discussed.