Tritrichomonas Augusta Research Articles

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was >40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.

Design and validation of an oligonucleotide probe for the detection of protozoa from the order Trichomonadida using chromogenic in situ hybridization

Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite–lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads ( Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.

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.

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.

Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads

The flagellate form of Trichomonas vaginalis ( T ν) transforms to amoeboid cells upon adherence to coverslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T ν F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T ν adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum, Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a ‘trichomonas branch’ and a ‘tritrichomonas branch’ is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

Morphogenesis of the hydrogenosome: An ultrastructural study

The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.

Morphogenesis of the hydrogenosome: An ultrastructural study

The morphogenesis of hydrogenosomes in several trichomonad species ( Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.

Cytoskeleton in trichomonads: II. Immunological and biochemical characterization of the preaxostylar fibres and undulating membrane in the genus Tritrichomonas

The production of 3 monoclonal antibodies (MAbs) and the use of immunocytochemical techniques such as immunofluorescence (IF), immunoblotting (IB) and immunogold staining (IGS) reveal that the preaxostylar fibres of Tritrichomonas foetus are composed of at least 3 polypeptides of 55, 53 and 46 kDa. Two of these MAbs cross-react with Tritrichomonas mobilensis and one with Tritrichomonas augusta and Tritrichomonas muris on polypeptides with very similar molecular weights (M.W.) However, no cross-reactivity is seen with the preaxostylar fibres of other trichomonad species tested. These cross-reactivities restricted to the Tritrichomonas genus are similar to those observed with the costa and several explanations are proposed. Similarly, 5 MAbs obtained against Tritrichomonas foetus cytoskeleton label the undulating membrane (UM) by IF and IGS. IB identifies 5 polypeptides of very different M.W. (148, 72, 39, 33 and 23 kDa) in Tritrichomonas foetus. Among them, 2 cross-react by IF and IB in Tritrichomonas mobilensis, one in Tritrichomonas augusta and none in Tritrichomonas muris. These results are in agreement with the electron microscopy studies which have shown that the UM ultrastructure of Tritrichomonas foetus, Tritrichomonas mobilensis and Tritrichomonas augusta are very similar and different from that of Tritrichomonas muris. The lack of cross-reactivity with the lamellar type UM of the Trichomonadinae genera which is very different from the Tritrichomonadinae UM is also demonstrated.

Tritrichomonas mobilensis n. sp. (Zoomastigophorea: Trichomonadida) from the Bolivian squirrel monkey Saimiri boliviensis boliviensis.

A trichomonad flagellate, Tritrichomonas mobilensis n. sp., is described from the large intestine of the squirrel monkey, Saimiri boliviensis boliviensis. The organism has a lanceolate body 7-10.5 micrometers in length; a well developed undulating membrane; a stout, tubular axostyle with periaxostylar rings that terminate in a cone-shaped segment projecting from the posterior end of the cell; and a moderately wide costa. The anterior flagella are about as long as the body, and the recurrent flagellum is of the acroneme type. All its characteristics suggest that the new species belongs in the Tritrichomonas augusta type of the subfamily Tritrichomonadinae.

Electrophoretic analysis of soluble proteins and esterase, superoxide dismutase and acid phosphatase isoenzymes of members of the protozoan family trichomonadidae

1. 1. Polyacrylamide gel electrophoresis was used to compare the proteins and isoenzymes of esterase, superoxide dismutase, and acid phosphatase in soluble, whole-cell extracts of four strains of Trichomonas vaginalis, two strains of Trichomonas gallinae, and one strain each of Tritrichomonas foetus, Tritrichomonas augusta, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. 2. 2. Intraspecific, interspecific, and intergeneric differences were found in protein and isoenzyme profiles. 3. 3. At least four to seven isoenzymes were detected among the ten strains for each of the three enzymes studied. Each strain usually contained one or two isoenzymes of both esterase and acid phosphatase, and two or three isoenzymes of superoxide dismutase.

SEROLOGICAL INVESTIGATIONS OF TRICHOMONADS. I. COMPARISONS OF "NATURAL" AND IMMUNE ANTIBODIES.

SYNOPSIS. Active, normal sera or other body fluids from many vertebrate species were found to cytolyze Tritrichomonas augusta; heat inactivated sera agglutinated the organisms. Techniques were devised for quantitative determination of “natural” agglutinating and lytic activity against a number of trichomonads. Amphibian, avian, mammalian, and reptilian sera all possessed activity, as did chicken egg yolk, or yolk sac contents, human ascitic fluid, and cow's milk. For individuals of a given species, levels of “natural” agglutinin did not vary significantly. Among donor species, there were wide and consistent differences. The serum of any one individual, tested against 6 strains of T. augusta, showed constant activity. Agglutinating activity was relatively unaffected by changes of physical factors in the reaction milieu. Lytic activity was restored to heat‐inactivated serum by addition of guinea pig serum as a source of complement. When serum was fractionated, agglutinating activity was found primarily in the γ globulins and on into the β. On the basis of these reactions and the activity of specific serum fractions, an antibody mechanism has been postulated.By means of reciprocal agglutination tests with absorbed sera, both “natural” and immune agglutinins have been shown to be specific for Tritrichomonas augusta, T. foetus, and T. suis (?), as well as for Trickomonas gallinarum and Trichomonas vaginalis. Absorption with one species does not remove activity against others. Among strains of T. augusta, absorption with any one removes some antibody against others, but not necessarily all. On the basis of reciprocal absorption and test of 6 strains, 3 antigenic groups have been established: 2 groups are well‐separated; the 3rd intermediate between the others and cross‐reacting with both.Lack of agglutinin absorption by 2 common enterobacteria, a yeast, and Forssman antigen (sheep erythrocytes) showed that these, at least, were not involved as sources of heterophil stimulation for “natural” antibody. Whether “natural” antibody results from heterophil stimulation, however, or is genetically determined, has not been answered. Direct stimulation of “natural” antibody production by occult infection with trichomonads could not have occurred in the case of trichomonad‐free, laboratory reared chicks, or mammalian sera active against T. augusta.

Trichomonads from poikilotherms: nutritional and physiological notes.

SYNOPSIS. Several trichomonads from poikilotherms (Hypotrichomonas acosta, Monocercomonas colubrorum and M. sp., Trichomonas batrachorum, and Tritrichomonas augusta) grew in a nearly‐defined medium to a level of 10–40% that permitted with media containing casein digests; T. augusta (4 strains) grew best. The defined medium contained TEM‐4T (a semisynthetic fat based on beef‐tallow fatty acids) and ascorbyl “palmitate” as sources of fatty acids. Besides ascorbyl palmitate, thiomalic and ascorbic acids, and tocopherol served as reducers and antiöxidants. High concentrations of nucleotides (adenylic, cytidylic, guanylic, and uridylic acids) seemed necessary. Indigo carmine was a useful redox indicator; the flagellates never grew in the oxidized zone. Extra trace elements and amino acids or rich natural media permitted better growth at about 34.5°C—the upper temperature limit for these trichomonads, raising the issue of a biochemical basis of a thermal barrier to the invasion of warmblooded hosts. Except for the limited carbohydrate in the nucleotides, carbohydrate could be omitted, the main substrates being a combination of glycerol, acetate, and gylcero‐phosphate, posing the question of the identity of the anaerobic, presumably non‐glycolytic, energy‐yielding pathway.

Agar Techniques for Colonizing and Cloning Trichomonads

SYNOPSIS. Agar media have been used in plates, Burri tubes, and sealed slide preparations to grow trichomonads as clonal colonies. Periodic observation of slide‐culture colonies confirmed their origin from single organisms as clones. Axenic cultures of Trichomonas vaginalis (3 strains), T. gallinae (2 strains), T. gallinarum (1 strain), Tritrichomonas augusta (5 strains), Trit. foetus (4 strains), Trit. suis (?) (6 strains), Pentatrichomonas hominis (1 strain), and an undescribed porcine form were used successfully. Urinary and vaginal specimens with T. vaginalis were also plated directly from patients. In all trials, organisms were recovered from colonies for broth culture and re‐plating.Colonies of the two strains of T. gallinae showed constant differences in their growth under the same conditions. Plated T. vaginalis have shown zones of growth inhibition in tests against drug‐impregnated discs.

Morphological variations in tritrichomonas augusta (alexeieff) from amphibia

Journal of MorphologyVolume 94, Issue 1 p. 125-163 Article Morphological variations in tritrichomonas augusta (alexeieff) from amphibia† Benton W. Buttrey, Benton W. Buttrey Department of Zoology, University of Pennsylvania, PhiladelphiaSearch for more papers by this author Benton W. Buttrey, Benton W. Buttrey Department of Zoology, University of Pennsylvania, PhiladelphiaSearch for more papers by this author First published: January 1954 https://doi.org/10.1002/jmor.1050940105Citations: 8 † A portion of a dissertation in zoology presented to the faculty of the Graduate School of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume94, Issue1January 1954Pages 125-163 RelatedInformation