Prevalence of Pentatrichomonas hominis infections in six farmed wildlife species in Jilin, China.

Pentatrichomonas hominis is an anaerobic flagellated protozoan that primarily parasitizes the gastrointestinal tract and is a conditional pathogen. It has an extensive host range and is well known as a potential causative agent of zoonotic disease. The objective of this study was to provide the first findings of the prevalence of P. hominis in six farmed wildlife species, sika deer (S.D.), Rex rabbits (R.R.), blue foxes (B.F.), silver foxes (S.F.), raccoon dogs (R.D.) and minks (M.), that are commercially important in Jilin Province, China. In this study, 450 faecal samples were tested for P. hominis infection by culturing and nested PCR assays. The average prevalence of P. hominis infections were as follows: S.D. 20% (26/130), R.R. 16.25% (13/80), B.F. 45% (27/60), S.F. 43.33% (26/60), R.D. 53.33% (32/60) and M. 48.33% (29/60). The prevalence in herbivores (18.57% for S.D. and R.R.) was significantly lower than that in non-herbivores (47.5%). PCR product sequencing indicated that infections were mainly caused by the P. hominis strain Changchun Canine 1, and we found a P. hominis strain with a mutated sequence, Changchun-RR, which had three mutations compared with the referenced homologous P. hominis sequences. Morphological observations of the Changchun-RR strain showed that it was similar to P. hominis. Our study suggests that P. hominis is widespread in six farmed wildlife species in Jilin Province and provides baseline information for the presence of this parasite in these animals.

Morphological, ultrastructural, and molecular characterization of intestinal tetratrichomonads isolated from non-human primates in southeastern Brazil.

Non-human primates are our closest relatives and represent an interesting model for comparative parasitological studies. However, research on this topic particularly in relation to intestinal parasites has been fragmentary and limited mainly to animals held in captivity. Thus, our knowledge of host-parasite relationships in this species-rich group of mammals could be considered rudimentary. The current study combined morphological, ultrastructural, and molecular analyses to characterize isolates of intestinal tetratrichomonads recovered from the feces of three species of South American, non-human primates. Fecal samples were collected from 16 animals, representing 12 distinct species. Parabasalid-like organisms were evident in five samples (31%) of feces: two from Alouatta sara, two from Callithrix penicillata, and one from Sapajus apella. The five samples presented morphologies consistent with the description of Tetratrichomonas sp., with four anterior flagella of unequal length, a well-developed undulating membrane, and a long recurrent flagellum. Sequencing of the ITS1-5.8S rRNA-ITS2 region demonstrated that the isolates from A. sara, and C. penicillata were closely related and highly similar to isolates of Tetratrichomonas brumpti, recovered previously from tortoises (Geochelone sp.). The flagellate recovered from S. apella demonstrated a similar morphology to those of the other isolates, however, sequence analysis showed it to be identical to an isolate of Tetratrichomonas sp. recovered from white-lipped peccaries (Tayassu pecari). The findings of this study extend and enhance our knowledge of parasitism of non-human primates by members of the genus Tetratrichomonas and indicate that the host range of these parasites is broader than previously believed.

Long-term in vitro cultivation of Histomonas meleagridis coincides with the dominance of a very distinct phenotype of the parasite exhibiting increased tenacity and improved cell yields.

The majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

BACKGROUND INFORMATION: The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. RESULTS: Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. CONCLUSIONS: The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. SIGNIFICANCE: Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.

Responding to a Zoonotic Emergency with Multi-omics Research: Pentatrichomonas hominis Hydrogenosomal Protein Characterization with Use of RNA Sequencing and Proteomics.

Pentatrichomonas hominis is an anaerobic flagellated protist that colonizes the large intestine of a number of mammals, including cats, dogs, nonhuman primates, and humans. The wide host range of this organism is alarming and suggests a rising zoonotic emergency. However, knowledge on in-depth biology of this protist is still limited. Similar to the human pathogen, Trichomonas vaginalis, P. hominis possesses hydrogenosomes instead of mitochondria. Studies in T. vaginalis indicated that hydrogenosome is essential for cell survival and associated with numerous pivotal biological functions, including drug resistance. To further decipher the biology of this important organelle, we undertook proteomic research in P. hominis hydrogenosomes. Lacking a decoded P. hominis genome, we utilized an RNA sequencing (RNA-seq) data set generated from P. hominis axenic culture as the reference for proteome analysis. Using this in-house reference data set and mass spectrometry (MS), we identified 442 putative hydrogenosomal proteins. Interestingly, the composition of the P. hominis hydrogenosomal proteins is very similar to that of T. vaginalis, but proteins such as Hmp36, Pam16, Pam18, and Isd11 are absent based on both MS and the RNA-seq. Our data underscore that P. hominis expresses different homologs of multiple gene families from T. vaginalis. To the best of our knowledge, we present here the first hydrogenosome proteome in a protist other than T. vaginalis that offers crucial new scholarship for global health, therapeutics, diagnostics, and veterinary medicine research. In addition, the research strategy used here using RNA sequencing and proteomics might inform future multi-omics research in other understudied organisms without decoded genomes.

The Cytoskeleton of Parabasalian Parasites Comprises Proteins that Share Properties Common to Intermediate Filament Proteins.

Certain protist lineages bear cytoskeletal structures that are germane to them and define their individual group. Trichomonadida are excavate parasites united by a unique cytoskeletal framework, which includes tubulin-based structures such as the pelta and axostyle, but also other filaments such as the striated costa whose protein composition remains unknown. We determined the proteome of the detergent-resistant cytoskeleton of Tetratrichomonas gallinarum. 203 proteins with homology to Trichomonas vaginalis were identified, which contain significantly more long coiled-coil regions than control protein sets. Five candidates were shown to associate with previously described cytoskeletal structures including the costa and the expression of a single T. vaginalis protein in T. gallinarum induced the formation of accumulated, striated filaments. Our data suggests that filament-forming proteins of protists other than actin and tubulin share common structural properties with metazoan intermediate filament proteins, while not being homologous. These filament-forming proteins might have evolved many times independently in eukaryotes, or simultaneously in a common ancestor but with different evolutionary trajectories downstream in different phyla. The broad variety of filament-forming proteins uncovered, and with no homologs outside of the Trichomonadida, once more highlights the diverse nature of eukaryotic proteins with the ability to form unique cytoskeletal filaments.

Pentatrichomonas hominis: first isolation from the feces of a dog with diarrhea in China.

A trichomonad-like parasite isolated from canine fecal samples in Changchun, China was successfully cultivated in vitro using RPMI1640 medium supplemented with 10% heat-inactivated calf serum and antibiotics. These were then subjected to scanning and transmission electron microscopy for ultrastructural study. This parasite has four anterior flagella of unequal length, one independent flagellum, and one recurrent flagellum. It exhibits an anterior nucleus, a Golgi complex, an axostyle, food vacuoles, and hydrogenosomes. These features are consistent with the ultrastructural characteristics of previously described Pentatrichomonas hominis. Polymerase chain reaction and sequence analysis of three genetic loci, including ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α, were also used to compare these samples with other trichomonad species. Molecular identification was also consistent with P. hominis. This is the first time that isolation of P. hominis has been isolated from dog in China, although several other strains of P. hominis have been isolated from human samples.

Molecular and morphologic identification of Pentatrichomonas hominis in swine.

A marasmic pig with watery diarrhea was identified to harbor the human-pathogenic protist Pentatrichomonas hominis by PCR and sequence analysis of three genetic loci (ITS1-5.8S rRNA-ITS2, 18S rRNA, and EF-1α), electron microscopy, and infection experiments. DNA sequencing and phylogenetic analysis indicated the organism isolated in this study was most closely related to P. hominis. SEM and TEM observation of the ultrastructure demonstrated that it had a morphology identical to P. hominis. The result of experimental infections with P. hominis exhibited that the cells had the ability to propagate in the cecum of piglets and fecal-oral route might be the major way in which pigs became infected. The present study confirmed that swine could be a host for P. hominis and might serve as a reservoir for human trichomoniasis.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

Hepatitis and splenitis due to systemic tetratrichomoniasis in an American white pelican (Pelecanus erythrorhynchos).

A free-ranging, young adult, female American white pelican (Pelecanus erythrorhynchos), found dead on the grounds of the San Diego Zoo Safari Park in Southern California, had severe multifocal to coalescing necrotizing hepatitis and splenitis on postmortem examination. Histologically, within the large areas of necrosis were myriad pleomorphic, 5-20 µm in diameter, protozoal organisms with 1 to multiple nuclei. Ultrastructurally, the organisms were consistent with a trichomonad flagellate. Polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene identified nucleotide sequences with 99% identity to Tetratrichomonas gallinarum, which is a common inhabitant of the intestinal tract of galliform and anseriform birds that has occasionally been associated with disease, including typhlitis and hepatitis. Damage to the cecal mucosa in the pelican from trematodes and secondary bacterial infection could have allowed invasion and systemic dissemination of the organism. Exposure of the pelican to a variety of native and exotic anseriform and galliform birds at the zoological institution could have led to cross-species infection and severe manifestation of disease in a novel host.

Morphological and molecular identification of Tetratrichomonas flagellates from the giant anteater (Myrmecophaga tridactyla).

A tetratrichomonad flagellate found in the diarrhoeic faeces of a 5 years-old male giant anteater (Myrmecophaga tridactyla) was characterised by morphological and genetic analysis. This protozoan presents four anterior flagella of unequal length and a recurrent flagellum attached to the undulating membrane without a free end portion, and a broad axostyle projection. Numerous vacuoles of different sizes containing bacteria and digestion products were found. The complete sequence of the DNA coding for the 16S rRNA-ITS1-5.8S rRNA-ITS2 region was also obtained in order to compare this isolate with other tetratrichomonad species. The sequence obtained was identical to others previously obtained by other researchers from bovines and turtles (Geochelone sp.). It is not easily explainable how the same organism could be found in such different hosts and locations; however these results indicate that some tetratrichomonad species could have a wide host range and could survive in a wide range of environmental conditions.

The true diversity of devescovinid flagellates in the termite Incisitermes marginipennis.

More than 40 years ago, ten species of devescovinid flagellates were described to occur in the gut content of the termite Incisitermes marginipennis. Based on light microscopic examinations, the flagellates were then classified into the two genera Devescovina and Metadevescovina. Here, we combined molecular phylogenetic analysis of the small subunit rRNA genes of the gut flagellates with the first ultrastructural investigation of the genus Metadevescovina. Our results suggest that I. marginipennis contains only one species of devescovinid flagellates, Metadevescovina modica, which comprises three variants of the same phylotype (> or =99.5% sequence similarity). Monophyly of all Metadevescovina sequences obtained from Pterotermes and Incisitermes species in this and previous studies and the absence of filamentous bacterial epibionts typical of Devescovina species (M. modica is densely colonized with spirochetes) corroborate the validity of the genus Metadevescovina and allow its differentiation from other genera of devescovinid flagellates.

Fine structure of the bird parasites Trichomonas gallinae and Tetratrichomonas gallinarum from cultures.

The trophozoites of Trichomonas gallinae and Tetratrichomonas gallinarum were studied by means of light and electron microscopy after cloning and cultivating them axenically. T. gallinae trophozoites varied in shape reaching from ovoidal to pyriform and had a size of about 7-11 microm. They were provided with four free flagella and a fifth recurrent one, which did not become free at the posterior pole. The nucleus was ovoid, had a size of about 2.5-3 microm, and was situated closely below the basal bodies of the flagella. The axostyle consisted of a row of microtubules running from the region of the apical basal bodies to the posterior end of the cell. In addition to flagellated stages, which contained food vacuoles, hydrogenosomes, a costa-like structure, and glycogen granules besides lacunes of endoplasmic reticulum, spherical, nonflagellated, and cyst-like stages occurred. The trophozoites of T. gallinarum appeared mostly pear-shaped and ranged in size from 6 to 15 microm. They had also four free anterior flagella and a fifth recurrent one, which became free at the posterior pole in contrast to that of T. gallinae. Another clearly visible difference to T. gallinae was the occurrence of a sphere of lacunes of the endoplasmic reticulum surrounding in a regular distance the nucleus with its typical perinuclear membranes. Furthermore, the food vacuoles appeared very large. However, both species clearly differed from the trophozoites of Histomonas meleagridis.

Light and transmission electron microscopic studies on trophozoites and cyst-like stages of Histomonas meleagridis from cultures.

The present study deals with Berlin strains of Histomonas meleagridis, the specimens of which were cultivated in Dwyer's medium. The light and electron microscopic examination revealed that the cultivated trophozoite stages (reaching about 10 mum in size) appeared more or less spherical, although their surface (covered by a single membrane) showed amoeba-like waves. All stages were uni-nucleated and reproduced by binary fission with an extranuclear spindle apparatus. Some trophozoites appeared ovoid and possessed a single flagellum with a typical microtubular 9 x 2 + 2 arrangement. Furthermore, the latter were characterized by an inner row of typical microtubules (remnant of an axostyle) and a Golgi apparatus (both adjacent to the nucleus), multivesicular structures, hydrogenosomes, and many food vacuoles containing either starch grains or bacteria. Their cytoplasm was densely filled with glycogen granules and ribosomes. Similar stages were also documented in the caeca and cloaca of chicken when being inoculated (via cloaca) with such culture stages. In addition to these typical trophozoites, the cultures contained a low number of 10-mum-sized spherical cyst-like stages with a surrounding amorphous layer. The cytoplasm of some of these cyst-like stages-when studied by electron microscopy-appeared with two membranes or had formed an amorphic, cyst-wall-like layer at their surface, apparently corresponding to their light microscopical appearance. Such stages might be involved in transmission from one host to another and probably have been missed before in microscopical examinations of infected poultry.

Ultrastructural study of a tetratrichomonad isolated from pig fecal samples.

Trichomonads found in pigs include the commensal Tritrichomonas suis (more well known because of its synonymy to Tritrichomonas foetus, a trichomonad parasite of cattle and other animals) and Tetratrichomonas buttreyi, which appear similar to Tritrichomonas suis under the light microscope. A trichomonad isolated from pig fecal samples was subjected to scanning and transmission electron microscopy for ultrastructural study. The organism's ultrastructure revealed features commonly found in trichomonads; however, features such as the number and length of flagella, type of undulating membrane, general body form, and shape and location of organelles such as the nucleus, Golgi complex, and hydrogenosomes indicated that the isolated trichomonad is not Tritrichomonas suis nor Tritrichomonas foetus. Polymerase chain reaction (PCR) corroborated these results. Moreover, the ultrastructure was similar to the ultrastructure of previously described tetratrichomonads. It is especially suggested that the isolate is T. buttreyi. These findings could be of significance in the differentiation among different porcine trichomonads in diagnostic procedures. In addition, this is the first known detailed ultrastructural study of T. buttreyi isolated from pigs; thus, this can serve as an aid for future comparison between porcine and bovine T. buttreyi.

Transmission electron microscopic studies of stages of Histomonas meleagridis from clonal cultures.

Histomonas meleagridis is a 10-20 microm-sized flagellated protozoan, causing histomoniasis in gallinaceous birds. Different strains of H. meleagridis from different origins were used to establish clonal cultures, which can be traced back to a single cell. Cells from these clonal cultures were examined by transmission electron microscopy. Results gave detailed insights in the ultrastructure showing a single flagellum, a band of microtubules remnants of an axostyle or of a costa, respectively pelta, hydrogenosomes, nucleus, spindle apparatus, and other organelles of the trophozoites of H. meleagridis.

Unsolved motility looking for answer.

A rod-like axostyle complex turns the anterior end of a termite flagellate, including the plasma membrane, continually in the same direction relative to the rest of the cell at speeds up to approximately 1 Hz. This motility provides direct visual evidence for the fluid nature of cell membranes. Torque is generated along the length of the axostyle complex by an unknown mechanism. Here I describe findings not published before and promising experiments that may help to solve this remarkable motility.

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation.

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.

Morphological aspects of Monocercomonas sp. and investigation on probable pseudocysts occurrence.

Monocercomonas sp. is a flagellate protozoan found in the large intestine of snakes and in insects. Light microscopy revealed the measurements of morphological features of the trophozoites. Scanning electron microscopy showed in detail the emergence of the three anterior flagella, the recurrent flagellum, the axostyle, and the absence of undulating membrane. In addition, we described spherical forms which are probably pseudocysts. The investigation on the occurrence of this process was carried out through the incubation of Monocercomonas sp. trophozoites in several stressful conditions, such as pH change, nutrient depletion and different temperatures. Results revealed high pseudocyst formation at acidic pH values (4.0, 5.0, and 6.0), in absence of serum and in incubation at 37 degrees C. The occurrence of these pseudocystic forms in trichomonads life cycle is under investigation. This study describes the external structure of Monocercomonas sp., as demonstrated by light and scanning electron microscopy. Moreover, to our knowledge, this is the first time that formation of probable pseudocysts is shown in Monocercomonas sp., contributing to the research field on termite protozoa biology.

Morphological and molecular diversity of the monocercomonadid genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov.

The family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane; both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of the SSU rDNA and the ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: (1) Monocercomonas colubrorum, (2) Monocercomonas ruminantium together with a strain ATCC 50321 designated as Pseudotrichomonas keilini, and (3) Hexamastix. Twenty isolates of Monocercomonas colubrorum split into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of the Monocercomonadidae confirmed that the absence of a costa and an undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters - infrakinetosomal body, comb-like structure, marginal lamella, and the type of axostyle - are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters.