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Trichomonas gallinae is a widespread protozoan parasite that primarily affects birds, causing a disease known as avian trichomonosis. The present study aimed to investigate the prevalence and genetic diversity of T. gallinae, a parasite causing avian trichomoniasis in feral pigeons, budgerigars, and finches in Tehran, Iran. The 5.8S ribosomal RNA locus, along with the internal transcribed spacer 2 (ITS2) region, has been extensively utilized for genotype identification and for determining inter- and intra-specific diversity. More recently, the Fe-hydrogenase (Fe-Hyd) gene has been suggested as an additional genetic marker to enhance the accuracy of strain subtyping discrimination. In the present study, a total of 12% (12/100) birds examined were infected with T. gallinae using microscopy and PCR methods. Infection was found in seven of 30 (23.3%) feral pigeons, three of 40 (7.5%) budgerigars, and two of 30 (6.66%) finches. Analysis of the ITS2 region of T. gallinae isolates revealed two highly similar sequences. The first sequence (GenBank: OQ689964-OQ689970) was found in five feral pigeons and two budgerigars, whereas the second sequence (GenBank: OQ689971-OQ689975) was identified in two feral pigeons, one budgerigar, and two finches. Phylogenetic analysis confirmed the presence of two distinct clusters (cluster I and cluster II) within the trichomonads based on the ITS2 region. However, further analysis using Fe-Hyd revealed greater diversity, with three subtypes identified (A1, A2, and C1). One isolate identified in the present study (GenBank accession number: OQ694508.1) belonged to subtype A1. Combining ITS2 and Fe-Hyd markers holds promise for a more comprehensive understanding of the population structure of T. gallinae and the potential role of ITS2 in host adaptation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Trichomonas is a significant protist genus, and includes T. vaginalis, the most prevalent sexually transmitted non-viral infection of humans, and T. gallinae of rock doves (Columba livia), one of the earliest known avian pathogens. New Trichomonas genotypes, including T. vaginalis-like isolates, have been discovered in American columbid hosts, suggesting geographically widespread cryptic diversity of Trichomonas in pigeons and doves. We sampled 319 birds from 22 columbid species in Australia, Papua New Guinea, New Zealand and southern Africa and uncovered 15 novel lineages of Trichomonas, more than doubling the known diversity of this parasite genus and providing evidence for frequent host-switching that eventually gave rise to T. vaginalis in humans. We show that Trichomonas has a columbid origin and likely underwent Miocene expansion out of Australasia. Our chronological topology for Trichomonas is calibrated on the evolution of a host phenotypic trait associated with ecological entrapment of the most basal extant lineage of Trichomonas in Ptilinopus fruit-doves., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates., (Copyright © 2019. Published by Elsevier B.V.)

Background: The methods routinely used to detect trichomonads in the lungs are not sensitive enough, and an effective method is urgently needed., Method: Primers were first designed to match the conserved area of the 18S rRNA gene of trichomonads. Then, nested PCR was carried out to detect trichomonads in bronchoalveolar lavage fluid (BALF). Finally, all positive specimens were subjected to DNA sequencing and phylogenetic analysis., Results: Among 115 bronchoalveolar lavage fluid samples, ten samples tested positive in nested PCR (10/115), while no samples were positive in wet mount microscopy (0/115) (P < 0.01). Among the ten positive specimens, two were identified as Tetratrichomonas spp. and the other eight as Trichomonas tenax in phylogenetic analysis., Conclusions: Nested PCR is an effective way to detect trichomonads in bronchoalveolar lavage fluid.

Trichomonosis is an important cause of mortality in multiple avian species; however, there have been relatively few reports of this disease in owls. Two barn owls (Tyto alba) and four barred owls (Strix varia) submitted for diagnostic examination had lesions consistent with trichomonosis including caseous necrosis and inflammation in the oropharynx. Microscopically, these lesions were often associated with trichomonads and molecular testing, if obtainable, confirmed the presence of Trichomonas gallinae, the species most commonly associated with trichomonosis in birds. The T. gallinae genotype in one barn owl and two barred owls was identified as ITS-OBT-Tg-1 by sequence analysis. Columbids are the primary hosts for T. gallinae, and columbid remains found within the nest box of the barn owls were the likely source of infection. This study is the first to formally describe the strains and genetic variation of T. gallinae samples from clinical cases of trichomonosis in barn and barred owls in the eastern USA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. Characterized by chronic inflammation of the colon and repeated bouts of diarrhea, ICD is largely unresponsive to medical interventions, including corticosteroid, antiparasitic, and antibiotic treatments. Although ICD is accompanied by large disruptions in the composition of the commensal gut microbiome, no single pathogen has been concretely identified as responsible for the onset and continuation of the disease., Results: Fecal samples were collected from 12 ICD-diagnosed macaques and 12 age- and sex-matched controls. RNA was extracted for metatranscriptomic analysis of organisms and functional annotations associated with the gut microbiome. Bacterial, fungal, archaeal, protozoan, and macaque (host) transcripts were simultaneously assessed. ICD-afflicted animals were characterized by increased expression of host-derived genes involved in inflammation and increased transcripts from bacterial pathogens such as Campylobacter and Helicobacter and the protozoan Trichomonas. Transcripts associated with known mucin-degrading organisms and mucin-degrading enzymes were elevated in the fecal microbiomes of ICD-afflicted animals. Assessment of colon sections using immunohistochemistry and of the host transcriptome suggests differential fucosylation of mucins between control and ICD-afflicted animals. Interrogation of the metatranscriptome for fucose utilization genes reveals possible mechanisms by which opportunists persist in ICD. Bacteroides sp. potentially cross-fed fucose to Haemophilus whereas Campylobacter expressed a mucosa-associated transcriptome with increased expression of adherence genes., Conclusions: The simultaneous profiling of bacterial, fungal, archaeal, protozoan, and macaque transcripts from stool samples reveals that ICD of rhesus macaques is associated with increased gene expression by pathogens, increased mucin degradation, and altered fucose utilization. The data suggest that the ICD-afflicted host produces fucosylated mucins that are leveraged by potentially pathogenic microbes as a carbon source or as adhesion sites.

A juvenile Cinereous Vulture ( Aegypius monachus) fledgling was found disorientated on the roof of a building in Madrid City, Spain, in October 2016. A veterinary examination revealed multiple plaques distributed throughout the oropharyngeal cavity. Lesions were located under the tongue and at the choanal slit, hard palate, and esophagus opening and ranged from 2 to 7 mm, coalescing in areas up to 2 cm, with a yellowish color of the surface. Motile trichomonad trophozoites were detected in fresh wet mount smears from the lesions. Sequence analysis of the internal transcribed spacer (ITS)1/5.8S/ITS2 and small subunit ribosomal RNA confirmed that Trichomonas gypaetinii was the etiologic agent. Microbiologic cultures did not reveal any pathogenic bacteria or fungi. The animal recovered successfully after treatment with metronidazole and trimethoprim-sulfamethoxazole and was later released in a suitable habitat. Avian trichomonosis lesions caused by T. gypaetinii have not been reported.

Introduction: The protozoan Trichomonas tenax is considered to be a human specific flagellate of the oral cavity, found in humans with poor oral hygiene and advanced periodontal disease. Morphological variability and great similarity between species occurring in humans and animals, complicate the specific identification of trichomonads, using microscopic examination and other standard parasitological techniques., Objective: The aim of the study was to search for and identify T. tenax in domesticated animals using molecular methods. The obtained data were assessed in terms of potential effects of a spread of the species deriving from the animals in the human environment., Material and Methods: 301 animals: 142 dogs, 57 cats and 102 horses, were examined in terms of their mouth status and occurrence of trichomonads. ITS1-5.8S rRNA-ITS2 region was amplified and sequenced., Results: Finally, 7 dogs, 3 cats and 1 horse were diagnosed positive for T. tenax by PCR. In the oral cavity of 9 /11 animals, gingivitis and dental plaque accumulation were diagnosed. 9 /11 sequences of trichomonad isolates showed 100% identity with T. tenax sequence derived from the GenBank. The sequences of 2 isolates differed by substitutions., Conclusions: It was proved that T. tenax , considered so far as a human specific parasite, can also inhabit the oral cavity of dog, cat and horse. To summarize, T. tenax was detected in the mouths of different domesticated animals, indicating that in Poland it can colonize a wider range of hosts than previously known. The owners of 3 dogs showed oral tissue inflammation of different intensity and were also positive for T. tenax ; therefore, oral trichomonosis spread from humans to domestic animals and conversely should be taken into consideration.

Avian trichomonosis is a widespread disease in columbids and other birds, caused by ingestion of the unicellular flagellate Trichomonas gallinae which proliferate primarily in the upper respiratory tracts. Studies using genetic analyses have determined some highly pathogenic lineages in birds, but the prevalence and distribution of potentially pathogenic and non-pathogenic T. gallinae lineages in wild birds is still not well known. We examined 440 oral swab samples of 35 bird species collected between 2015 and 2017 in Hesse, central Germany, for Trichomonas spp. infection and for determining the genetic lineages. Of these birds, 152 individuals were caught in the wild and 288 individuals were admitted from the wild to a veterinary clinic. The overall Trichomonas spp. prevalence was 35.6%. We observed significant differences between bird orders, with the highest prevalence in owls (58%) and columbids (50%), while other orders had slightly lower prevalences, with 36% in Accipitriformes, 28% in Falconiformes and 28% in Passeriformes. Among 71 successfully sequenced samples, we found 13 different haplotypes, including two previously described common lineages A/B (20 samples) and C/V/N (36 samples). The lineage A/B has been described as pathogenic, causing lesions and mortality in columbids, raptors and finches. This lineage was found in 11 of the 35 species, including columbids (feral pigeon, woodpigeon, stock dove), passerines (greenfinch, chaffinch, blackbird) and raptors (common kestrel, sparrowhawk, red kite, peregrine falcon and common buzzard). One new lineage (R) was found in a sample of a chaffinch. In conclusion, we found that the prevalence of Trichomonas spp. infection in wild birds was high overall, and the potentially pathogenic lineage A/B was widespread. Our findings are worrying, as epidemic outbreaks of trichomonosis have already been observed in Germany in several years and can have severe negative effects on bird populations. This disease may add to the multiple pressures that birds face in areas under high land-use intensity., Competing Interests: The authors have declared that no competing interests exist.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present paper, an outbreak of trichomonosis in a flock of 15 breeding pairs of canaries is described. Trichomonosis was diagnosed on characteristic clinical signs, microscopic examination of crop/esophageal swabs, gross pathology and histopathology. Trichomonads were successfully grown in culture media and were characterized by multi-locus sequence typing. The three genomic loci ITS1-5.8S-ITS2, 18S rRNA and Fe-hydrogenase were analyzed. Molecular characterization confirmed the finch trichomonosis strain, identical to the strain that caused emerging disease in free-living passerine birds in Europe. Flock treatment with metronidazole (200mg/L) in drinking water for 5days was partially effective. After individual treatment with oral application of metronidazole (20mg/kg SID) for 5days no further clinical signs were observed in the flock over next 30 months., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Avian trichomonosis is known as a widespread disease in columbids and passerines, and recent findings have highlighted the pathogenic character of some lineages found in wild birds. Trichomonosis can affect wild bird populations including endangered species, as has been shown for Mauritian pink pigeons Nesoenas mayeri in Mauritius and suggested for European turtle doves Streptopelia turtur in the UK. However, the disease trichomonosis is caused only by pathogenic lineages of the parasite Trichomonas gallinae. Therefore, understanding the prevalence and distribution of both potentially pathogenic and non-pathogenic T. gallinae lineages in turtle doves and other columbids across Europe is relevant to estimate the potential impact of the disease on a continental scale., Results: We examined 281 samples from four wild columbid species for Trichomonas infection and determined the genetic lineages. The overall prevalence was 74%. There were significant differences between the species (P = 0.007). The highest prevalence was found in stock doves Columba oenas (86%, n = 79) followed by wood pigeons Columba palumbus (70%, n = 61) and turtle doves (67%, n = 65), while three of five collared doves Streptopelia decaocto (60%) were infected. We found seven different lineages, including four lineages present in columbids in the UK, one lineage already described from Spain and three new lineages, one of those found in a single turtle dove migrating through Italy and another one found in a breeding stock dove. Stock doves from Germany and collared doves from Malta were infected with a potentially pathogenic lineage (lineage A/B), which is known to cause lesions and mortality in columbids, raptors and finches., Conclusions: Generally, turtle doves showed high prevalence of Trichomonas infection. Furthermore, the potentially pathogenic lineage A/B (or genotype B according to previous literature) was found in a recovering stock dove population. Both findings are worrying for these columbid species due to the occasional epidemic character of trichomonosis, which can have severe negative effects on populations.

Trichomonads are known to inhabit the oral cavities of various mammals, including dogs, cats and horses. However, little attention has been paid to species identification, prevalence and zoonotic potential of these parasites, although their hosts live in close proximity with humans. According to the original description, oral trichomonads in dogs and cats belong to the genus Tetratrichomonas. Interestingly, later investigations suggested that the oral cavities of dogs and cats could be infected with different species of the genus Trichomonas, including the human oral cavity parasite Trichomonas tenax. Thus, in this study we investigated the occurrence of oral trichomonads in 111 domestic dogs and 122 cats using cell cultivation methods, nested PCR analyses, and the sequencing of ITS1-5.8rRNA-ITS2 regions. We found that both dogs and cats harbour T. tenax, with prevalences of 8.1% and 4.1%, respectively. Considerably more dogs were infected with different species of the genus Trichomonas (30.6%), which we also identified in cats (6.6%). An analysis of the potential risk factors suggested that dogs of more than 3years old or with dental disease signs are more frequently infected with Trichomonas sp. than younger dogs or dogs without the disease signs, and that crossbreed dogs revealed increased rates of infection in comparison with purebred dogs. An analysis of the cat population suggested that Trichomonas sp. infection is lower in younger and crossbreed cats. Although the morphology of Trichomonas sp. is very similar to that of T. tenax, based on a phylogenetic analysis of ITS1-5.8rRNA-ITS2 regions and the ssrRNA genes, we consider Trichomonas sp. to represent a new trichomonad species, for which we propose the name Trichomonas brixi., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Trichomonas gallinae infects the upper digestive tract of pigeons. It is transmitted from mother to young squabs by feeding crop milk. Generally, infection resulted in severe mortalities in young birds. In this study, we examined 3315 pigeons of different ages from the Minoufiya governorate for the clinical infection by T. gallinae. The infection was confirmed in infected birds by microscopical examination of oral swabs, histopathological examination, and PCR of the ITS1/5.8S/ITS2 gene. The prevalence was 63 (1.9%). The parasite was found in 35 (2.04%) from Ashmoun, 15 (1.66%) from Minoof, 8 (1.6%) from Quesna, and 5 (2.5%) from El-Shohada birds. The infection was mainly detected in squabs 60 (1.8%). The sequence of T. gallinae ITS1/5.8S/ITS2 gene from Egypt has high nucleotide sequence identity (up to100%) to T. gallinae from pigeon of USA, Austria, Canada, and Spain. The sequence belongs to genotype B of T. gallinae. Histopathological examination presented the parasites in crop, liver, larynx, and trachea as poorly eosinophilic bodies with severe inflammatory cell infiltration. This is the first study to present the prevalence and genotype of T. gallinae from Minoufiya governorate, Egypt., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oropharyngeal swabs (n = 609) were collected randomly from 80,000 domestic pigeons (Columba livia domestica) on five pigeon farms and at one pigeon slaughterhouse in Shandong Province, China, from September 2012 to July 2013. Trichomonas spp. were detected in 206/609 (33.8%) samples. The prevalence was 14.9-31.1%, depending on different levels of sanitation and management, and was 4.8% in nestling pigeons, 13.6% in breeding pigeons and 35.2% in adolescent pigeons. Trichomonas gallinae genotypes A and B, and Trichomonas tenax-like isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the 5.8S rDNA-internal transcribed spacer (ITS) regions. RFLP analysis with the restriction enzyme BsiEI generated different RFLP band patterns between T. gallinae and T.tenax-like isolates. When BsiEI RFLP analysis was combined with HaeIII RFLP analysis, all infection types of T. gallinae and T.tenax-like isolates could be identified., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the context of an epidemiological study carried out by several wildlife recovery centers in Spain, trichomonads resembling Trichomonas gallinae were found in the oropharyngeal cavity of 2 Egyptian vultures (Neophron percnopterus) and 14 cinereous vultures (Aegypius monachus) which did not show any symptoms of trichomonosis. In order to characterize them, these isolates along with seven other T. gallinae isolates obtained from different hosts and from different geographical origin were analyzed. Genetic analyses were performed by sequencing the small subunit ribosomal RNA (SSU-rRNA) and the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA regions. The morphological study of the isolates in both light and scanning electron microscopy was also performed. The sequences obtained in the genetic analysis coincide with previously published sequences of an isolate named as Trichomonas sp., obtained from a bearded vulture (Gypaetus barbatus), and showed clear differences to the T. gallinae sequences (97 and 90-91% homology, respectively, for SSU-rRNA and ITS regions) and display higher similarity with Trichomonas vaginalis and Trichomonas stableri than with T. gallinae. Multivariate statistical analysis of the morphometric study also reveals significant differences between the trichomonads of vultures and the isolates of T. gallinae. The isolates from vultures presented smaller values for each variable except for the length of axostyle projection, which was higher. These results together with the different nature of their hosts suggest the possibility of a new species of trichomonad which we hereby name Trichomonas gypaetinii, whose main host are birds of the subfamily Gypaetinae.

The Pacific Coast band-tailed pigeon (Patagioenas fasciata monilis) is a migratory game bird of North America that is at risk for population decline. Epidemics of avian trichomonosis caused by upper digestive tract infection with Trichomonas spp. protozoa in these and other doves and pigeons of the United States are sporadic, but can involve tens of thousands of birds in a single event. Herein, we analyze the role of trichomonosis in band-tailed pigeon mortality and relate spatial, temporal and demographic patterns of parasite transmission to the genetic background of the infecting organism. Infections were most common in adult birds and prevalence was high in band-tailed pigeons sampled at mortality events (96%) and rehabilitation centers (36%) compared to those that were hunter-killed (11%) or live-caught (4%). During non-epidemic periods, animals were primarily infected with T. gallinae Fe-hydrogenase subtype A2, and were less often infected with either T. gallinae subtype A1 (the British finch epidemic strain), T. stableri n. sp. (a T. vaginalis-like species), or Tritrichomonas blagburni n. sp.-like organisms. Birds sampled during multiple epidemics in California were only infected with T. gallinae subtype A2 and T. stableri. The non-clonal etiology of avian trichomonosis outbreaks in band-tailed pigeons and the risk of spill-over to raptor and passerine species highlights the need for additional studies that clarify the host range and evolutionary relationships between strains of Trichomonas spp. in regions of trichomonosis endemicity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trichomonas gallinae , the cause of avian trichomonosis, is most commonly found in the order Columbiformes. Racing pigeons are often treated preventively with nitro-imidazoles, which could result in the emergence of resistant isolates, and these isolates can be a threat to wildlife when exchanges occur. The sequence type of 16 T. gallinae isolates obtained from racing pigeons and 15 isolates from wild pigeons was determined based on the ITS1/5.8S rRNA/ITS2 region sequence. In addition, the resistance profiles of these isolates against 5 different nitro-imidazoles (metronidazole, dimetridazole, ronidazole, tinidazole, and carnidazole) were determined. Two different Trichomonas sequence types were isolated. Sequence type A isolates were recovered from racing and wild pigeons, in contrast to sequence type B, which was only isolated from wild pigeons. Isolates with sequence type B were all susceptible to the tested nitro-imidazoles, except for tinidazole resistance in 3 isolates. Resistance to the nitro-imidazoles was observed more frequently in isolates obtained from racing pigeons than from wild pigeons, with most isolates belonging to sequence type A. A higher percentage of the sequence type A isolated from racing pigeons, in comparison with those isolated from the wild pigeons, were resistant to the nitro-imidazoles and displayed higher mean lethal concentration (MLC) values. Two isolates belonging to sequence type A, 1 recovered from a racing pigeon and 1 from a wild pigeon, displayed a similar resistance pattern, suggesting a potential exchange of resistant isolates between racing pigeons and wild pigeons.

In recent years, Trichomonas gallinae emerged as the causative agent of an infectious disease of passerine birds in Europe leading to epidemic mortality of especially greenfinches Chloris chloris and chaffinches Fringilla coelebs. After the appearance of finch trichomonosis in the UK and Fennoscandia, the disease spread to Central Europe. Finch trichomonosis first reached Austria and Slovenia in 2012. In the present study the genetic heterogeneity of T. gallinae isolates from incidents in Austria and Slovenia were investigated and compared with British isolates. For this purpose comparative sequence analyses of the four genomic loci ITS1-5.8S-ITS2, 18S rRNA, rpb1 and Fe-hydrogenase were performed. The results corroborate that one clonal T. gallinae strain caused the emerging infectious disease within passerine birds and that the disease is continuing to spread in Europe. The same clonal strain was also found in a columbid bird from Austria. Additionally, the present study demonstrates clearly the importance of multi-locus sequence typing for discrimination of circulating T. gallinae strains.

Recent studies have reported Trichomonas tenax as a cause of pleuropulmonary infections in humans. In this study, sputum and vaginal swab samples were collected from patients suffering from respiratory ailments in Rodriguez, Rizal and sex workers attending the social hygiene clinics in Angeles City in Pampanga, Mandaluyong City and Pasay City in Metro Manila, Philippines, respectively. DNA was extracted from samples and the 18S rRNA gene was amplified and sequenced. Phylogenetic trees were constructed using neighbor-joining, maximum-likelihood, maximum parsimony, and Bayesian inference analyses. Results showed that the new primer sets successfully amplified T. tenax from 14 sputum samples and Trichomonas vaginalis from 19 vaginal swab samples. Consequently, all isolates clustered with high bootstrap support and posterior probability values to their respective reference strains in the phylogenetic tree. Thus, the genus Trichomonas formed a highly supported clade with T. vaginalis in the first clade and T. tenax in the second clade. These findings conclude that T. vaginalis is solely present in the genito-urinary tract of females and that T. tenax can be found in the respiratory tract of humans. To our knowledge, this is the first report of detection and identification of T. tenax from sputum samples in the Philippines. However, further studies are still needed to determine the pathogenicity of this organism in humans., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Because of the common pathology, protein expression and drug resistance mechanisms-though in different hosts- of the luminal trichomonads Trichomonas vaginalis, T. gallinae and T. muris, doubts were raised concerning their phylogenetic relationship. The random amplified polymorohic (RAPD) technique for genetic DNA polymorphism studying was used to genetically characterize and assess their interspecies polymorphism. Results showed a high statistical support for the close relationship between the tested parasites and proved a tight association between Tritrichomonas muris and T. gallinae as well as a high genetic similarity between T. muris and T. vaginalis.

Trichomonas gallinae is an emerging pathogen in wild birds, linked to recent declines in finch (Fringillidae) populations across Europe. Globally, the main hosts for this parasite are species of Columbidae (doves and pigeons); here we carry out the first investigation into the presence and incidence of Trichomonas in four species of Columbidae in the UK, through live sampling of wild-caught birds and subsequent PCR. We report the first known UK cases of Trichomonas infection in 86% of European Turtle Doves Streptopelia turtur sampled, along with 86% of Eurasian Collared Doves Streptopelia decaocto, 47% of Woodpigeons Columba palumbus and 40% of Stock Doves Columba oenas. Birds were more likely to be infected if the farm provided supplementary food for gamebirds. We found three strains of T. gallinae and one strain clustering within the Trichomonas tenax clade, not previously associated with avian hosts in the UK. One T. gallinae strain was identical at the ITS/5.8S/ITS2 ribosomal region to that responsible for the finch trichomonosis epizootic. We highlight the importance of increasing our knowledge of the diversity and ecological implications of Trichomonas parasites in order further to understand the sub-clinical impacts of parasite infection.

Avian trichomonosis, caused by the flagellated protozoan Trichomonas gallinae, is a recently emerged infectious disease of British passerines. The aetiological agent, a clonal epidemic strain of the parasite, has caused unprecedented finch mortality and population-level declines in Britain and has since spread to continental Europe. To better understand the potential origin of this epidemic and to further investigate its host range, T. gallinae DNA extracts were collected from parasite culture and tissue samples from a range of avian species in Britain. Sequence typing at the ITS1/5.8S rRNA/ITS2 region resolved three distinct ITS region types circulating in free-ranging British birds. Subtyping by sequence analyses at the Fe-hydrogenase gene demonstrated further strain variation within these ITS region types. The UK finch epidemic strain was preponderant amongst columbids sampled, however, wide strain diversity was encountered in isolates from a relatively small number of pigeons, suggesting further strains present in columbid populations across the UK are yet to be identified. Fe-hydrogenase gene sequence data in isolates from birds of prey with disease were predominantly identical to the UK finch epidemic strain, demonstrating its presence as a virulent strain in UK birds of prey since at least 2009.

We report the first documented occurrence of an outbreak of trichomonosis in a free-ranging small flock of Eurasian collared doves (Streptopelia decaocto) and African collared dove hybrids (Streptopelia risoria) in the Caribbean. In total, 18 birds were examined, including six African collared dove x Eurasian collared dove hybrids and 12 Eurasian collared doves. The affected age class consisted of adults. Sex distribution was equal. With a flock population size of 200 birds, mortality rate for the outbreak was estimated at 15-20%. Living birds were weak, showing evidence of mucus-stained beaks and open-mouth breathing. Caseous ulcerative yellow lesions were restricted to the upper gastrointestinal tract, with the exception of one bird, which had lesions in the upper gastrointestinal tract and in the liver. Ninety-four percent (17/18) of the affected birds had multiple extensive lesions. Lesions located on the roof of the oral cavity extended in 33% (6/18) into the orbit and in 11% (2/18) into the braincase. Using wet-mount microscopy, we were able to confirm Trichomonas gallinae in 22% (4/18) of the sampled animals. Fifteen samples submitted for PCR analysis tested positive. Sequence analysis of the internal transcribed spacer 1 (ITS-1) region of the ribosomal RNA (rRNA) revealed two distinct genotypes of Trichomonas. One sequence had 100% identity to the prototype T. gallinae isolate, whereas the other sequences had 98-100% identity to recently described Trichomonas-like parabasalid. On the basis of gross and histologic findings, along with the sequence results from the columbids in this report, it is likely that this Trichomonas-like parabasalid is pathogenic.

A common quail (Coturnix coturnix) from a private keeping died unexpectedly and showed a moderate lymphocytic infiltration of the colonic mucosa associated with numerous protozoa-like objects at the pathological examination. These organisms were further identified using chromogenic in situ hybridization (ISH) and gene sequencing. ISH was performed on paraffin embedded tissue sections and produced a positive signal using a probe specific for the 18S ribosomal RNA (rRNA) gene of the order Trichomonadida, but remained negative with probes specific for the 18S rRNA gene of the common bird parasites Histomonas meleagridis, Tetratrichomonas gallinarum or Trichomonas gallinae. The trichomonads were found on the mucosal surface, inside the crypts and also immigrating into the lamina propria mucosae. DNA was extracted from the paraffin embedded tissue and the entire 18S rRNA gene, ITS-1 region, 5.8S rRNA gene, ITS-2 region and a part of the 28S rRNA gene were sequenced using primer walking. The acquired sequence showed 95% homology with Tritrichomonas foetus, a trichomonad never described in birds. A phylogenetic analysis of a part of the 18S rRNA gene or of the ITS-1, 5.8S and ITS-2 region clearly placed this nucleotide sequence within the family of Tritrichomonadidae. Therefore, the authors propose the detection of a putative new Tritrichomonas sp. in the intestine of a common quail., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Trichomonas gallinae is a protozoan parasite that is well characterised as a cause of trichomonosis in columbid and raptor species world-wide. The parasite emerged as a novel infection of British passerines in 2005, leading to epidemic mortality associated with significant declines of breeding populations of greenfinches (Carduelis chloris) and chaffinches (Fringilla coelebs). We characterised the extent of T. gallinae genotypic heterogeneity within the affected wild British avifauna by analysing individual isolates from 17 of the species affected. To do so, we employed improved platform-based multilocus typing tools as well as the hydrogenosomal Fe-hydrogenase gene as a single marker locus for fine-typing. We found no evidence of heterogeneity amongst the parasites infecting British passerines, indicating that a clonal strain of T. gallinae is the causative agent of this emerging infectious disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Trichomonas gallinae is a flagellated protozoon which parasitizes in the upper digestive tract of different birds, especially columbiformes (doves and pigeons) and falconiformes. The parasite is also a common inhabitant of the crop of psittacine birds and is frequently detected in budgerigars. The lesions associated with T. gallinae infection of the upper digestive tract range from mild inflammation of the mucosa to large caseous lesions that block the lumen of the oesophagus. Nitroimidazoles are considered to be the drugs of choice for the treatment of trichomonosis. However, only a few studies report the existence of resistant strains of T. gallinae to these drugs. Thus, in the present investigation cloned cultures of T. gallinae obtained from budgerigars and pigeons were analysed for the first time for their in vitro susceptibilities against four 5´-nitroimidazole derivates, including metronidazole, dimetridazole, ronidazole and ornidazole. Significantly different minimal lethal concentrations (MLCs) were observed for them against all four drugs. The lowest MLCs revealed the Trichomonas isolates obtained from two budgerigars, ranging from 2.0 ± 0.3 to 3.0 ± 0.7 μg/ml for metronidazole and dimetridazole, and from 2.0 ± 0.6 to 6.7 ± 1.7 μg/ml for ornidazole and ronidazole. Contrary to this, the highest MLCs were recorded for one Trichomonas isolate obtained from a pigeon, ranging from 83.3 ± 6.7 (for dimetridazole and ronidazole) to 103.3 ± 3.3 μg/ml (for metronidazole and ornidazole). The data obtained for the resistance testing were further compared with already available genetic data of the small subunit rRNA gene sequences and ITS-1, 5.8S rRNA and ITS-2 sequences, indicating a certain correlation between in vitro results and strain relationships., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The oral cavity is suitable for invasion of many microorganisms. Entamoeba gingivalis (E.gingivalis) and Trichomonas tenax (T.tenax) settle in the oral cavity of patients with poor oral hygiene and gingival disease. In the present study, two slide specimens were prepared from the cole region of the teeth of 46 persons for investigation of the parasites. One of the slide specimens was dried in the air while the other one put into fixative and they were stained with trichrome and Giemsa stains. The two staining methods were used for 36 samples and only Giemsa, for 10 samples. E. gingivalis was positive in 7 (19.44%) out of 36 samples stained by the trichrome stain while T. tenax was positive in one (2.17%) out of 46 samples stained by Giemsa stain. Parasitic infections were found to be positive in seven (21.2%) specimen from 33 patients with periodontal disease and in one (7.69%) specimen from 13 healthy controls. Dental policlinics are generally far from parasitology laboratories and microscopical wet mount examination can not be performed. Therefore dentists can send the specimens and have the parasites diagnosed with Giemsa and trichrome staining methods as an alternative to wet mount examination.

Background: Trichomonas vaginalis is a human urogenital pathogen responsible for trichomonosis, the number-one, non-viral sexually transmitted disease (STD) worldwide, while T. tenax is a commensal of the human oral cavity, found particularly in patients with poor oral hygiene and advanced periodontal disease. The extent of genetic identity between T. vaginalis and its oral commensal counterpart is unknown., Results: Genes that were differentially expressed in T. vaginalis were identified by screening three independent subtraction cDNA libraries enriched for T. vaginalis genes. The same thirty randomly selected cDNA clones encoding for proteins with specific functions associated with colonization were identified from each of the subtraction cDNA libraries. In addition, a T. vaginalis cDNA expression library was screened with patient sera that was first pre-adsorbed with an extract of T. tenax antigens, and seven specific cDNA clones were identified from this cDNA library. Interestingly, some of the clones identified by the subtraction cDNA screening were also obtained from the cDNA expression library with the pre-adsorbed sera. Moreover and noteworthy, clones identified by both the procedures were found to be up-regulated in expression in T. vaginalis upon contact with vaginal epithelial cells, suggesting a role for these gene products in host colonization. Semi-quantitative RT-PCR analysis of select clones showed that the genes were not unique to T. vaginalis and that these genes were also present in T. tenax, albeit at very low levels of expression., Conclusion: These results suggest that T. vaginalis and T. tenax have remarkable genetic identity and that T. vaginalis has higher levels of gene expression when compared to that of T. tenax. The data may suggest that T. tenax could be a variant of T. vaginalis.

Forty-two Trichomonas gallinae isolates were molecularly characterized to determine whether isolates differed in genetic sequence of multiple gene targets depending on host species or geographical location. The 5.8S ribosomal RNA (rRNA) and flanking internal transcribed spacer (ITS) gene regions were amplified by polymerase chain reaction, and the sequences were analyzed phylogenetically. The results of the sequence analysis strongly suggest at least 2 species may exist within the T. gallinae morphologic complex. Based on ITS sequences, one group demonstrated high nucleotide identity to the 3 T. gallinae sequences available in GenBank, whereas the second group was more closely related to T. vaginalis (98%) than to T. gallinae (92%). Two common ground-dove (Columbina passerina) isolates shared a 95% identity with T. vaginalis and a 92% identity with T. gallinae and T. tenax. Sequence analysis of both the 18S rRNA and alpha-tubulin genes from a subset of the isolates supports the 5.8S-ITS sequence results. All of the T. vaginalis-like isolates originated from Arizona, California, or Texas, whereas T. gallinae isolates were found in all sampled states. Both T. vaginalis-like and T. gallinae isolates were involved in trichomoniasis outbreaks in California and Arizona.

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.

Tetratrichomonas gallinarum is a widespread intestinal parasite of galliform and anseriform birds. The pathogenicity of this species is controversial, presenting an unsettled problem as yet. We analysed the polymorphism and genetic relationship among 29 isolates of T. gallinarum obtained from eight bird species and five T. gallinarum-like isolates from the oral cavity and lower respiratory tract of human patients. Two methods were used for the analyses: RAPD and sequencing of 16S rRNA, 5.8S rRNA, ITS1 and ITS2 genes, both producing consistent and well-supported results. The isolates were divided into five groups, A-E, with eleven subgroups. The distance between groups E, D and the cluster A-B-C considerably exceeded usual intraspecific polymorphism seen in trichomonads. Moreover, the largest subgroup, A2 (containing 18 isolates), was divided into three branches according to the host specificity. All isolates from humans were placed into avian subgroups A2 and B2. We conclude that our isolates represent, at least, three morphospecies or rather complexes of several cryptic species. Since certain species of the T. gallinarum complex can differ in their biological characteristics and some of them can infect humans, the problem of T. gallinarum pathogenicity should be re-examined with regard to specific genetic groups and zoonotic potential of some of these lineages should be considered.

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.

The molecular phylogeny of parabasalids has mainly been inferred from small subunit (SSU) rRNA sequences and has conflicted substantially with systematics based on morphological and ultrastructural characters. This raises the important question, how congruent are protein and SSU rRNA trees? New sequences from seven diverse parabasalids (six trichomonads and one hypermastigid) were added to data sets of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, alpha-tubulin and beta-tubulin and used to construct phylogenetic trees. The GAPDH tree was well resolved and identical in topology to the SSU rRNA tree. This both validates the rRNA tree and suggests that GAPDH should be a valuable tool in further phylogenetic studies of parabasalids. In particular, the GAPDH tree confirmed the polyphyly of Monocercomonadidae and Trichomonadidae and the basal position of Trichonympha agilis among parabasalids. Moreover, GAPDH strengthened the hypothesis of secondary loss of cytoskeletal structures in Monocercomonadidae such as Monocercomonas and Hypotrichomonas. In contrast to GAPDH, the enolase and both tubulin trees are poorly resolved and rather uninformative about parabasalian phylogeny, although two of these trees also identify T. agilis as representing the basal-most lineage of parabasalids. Although all four protein genes show multiple gene duplications (for 3-6 of the seven taxa examined), most duplications appear to be relatively recent (i.e., species-specific) and not a problem for phylogeny reconstruction. Only for enolase are there more ancient duplications that may confound phylogenetic interpretation.

The diagnostic test for Tritrichomonas foetus in bulls is microscopic examination of cultured preputial samples. Trichomonads other than T. foetus can be present in a preputial sample. Both a staining technique and a polymerase chain reaction assay were useful in differentiating between T. foetus and another trichomonad observed in samples from virgin bulls.

Preputial fluids from 567 virgin Angus and Hereford bulls, 1-2 years old, were inoculated into Sutherland medium, and approximately 8.4% produced cultures with a protozoan suggestive of Tritrichomonas foetus. Under brightfield microscopy, large numbers of single-celled motile organisms with multiple anterior flagellae, a posterior flagellum, axostyle, and a visible undulating membrane were detectable. Motility was jerky and rolling, as described for T. foetus. Air-dried smears of cultures stained with Giemsa or Diff-Quick + iodine revealed an organism similar to T. foetus, although somewhat more rounded. Several organisms appeared to have four anterior flagellae. Scanning electron microscopy (5000x) of representative samples revealed four anterior flagellae on most organisms, and an axostyle that was consistently longer than that seen in T. foetus. Using pan-trichomonal primers and T. foetus-specific primers in a polymerase chain reaction (PCR) assay, amplification products of 372bp were detected in all virgin bull isolates, but only with the pan-trichomonal primers. Positive control isolates of T. foetus yielded amplification products of the expected size (372 and 347bp) with the two sets of primers, respectively. We conclude that these protozoa are not T. foetus, and note the similarity of these findings with those reported earlier in North American beef cattle. Because in several countries there is no legal treatment for bovine trichomonosis, veterinarians recommend slaughter of bulls with positive preputial cultures. The existence of easily mis-identified non-T. foetus trichomonads in the bovine prepuce suggests that the current "gold standard" diagnostic test (culture of preputial scrapings or washings) should be augmented with a more specific confirming test, such as the PCR employed in this study.

The Win95/98/NT program FreeTree for computation of distance matrices and construction of phylogenetic or phenetic trees on the basis of random amplified polymorphic DNA (RAPD), RFLP and allozyme data is presented. In contrast to other similar software, the program FreeTree (available at http://www.natur.cuni.cz/~flegr/programs/freetree or http://ijs.sgmjournals.org/content/vol51/issue3/) can also assess the robustness of the tree topology by bootstrap, jackknife or operational taxonomic unit-jackknife analysis. Moreover, the program can be also used for the analysis of data obtained in several independent experiments performed with non-identical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 42 strains of 10 species of trichomonads. On the phylogenetic tree constructed using FreeTree, the high bootstrap values and short terminal branches for the Tritrichomonas foetus/suis 14-strain branch suggested relatively recent and probably clonal radiation of this species. At the same time, the relatively lower bootstrap values and long terminal branches for the Trichomonas vaginalis 20-strain branch suggested more ancient radiation of this species and the possible existence of genetic recombination (sexual reproduction) in this human pathogen. The low bootstrap values and the star-like topology of the whole Trichomonadidae tree confirm that the RAPD method is not suitable for phylogenetic analysis of protozoa at the level of higher taxa. It is proposed that the repeated bootstrap analysis should be an obligatory part of any RAPD study. It makes it possible to assess the reliability of the tree obtained and to adjust the amount of collected data (the number of random primers) to the amount of phylogenetic signals in the RAPD data of the taxon analysed. The FreeTree program makes such analysis possible.

Tetratrichomonas didelphidis (Hegner & Ratcliffe, 1927) Andersen & Reilly, 1965 is a flagellate protozoan found in the intestine, cecum, and colon of Didelphis marsupialis. The parasitic protozoa used in this study was found and isolated in the intestine of opossums in Pavlova starch-containing medium in Florianópolis, State of Santa Catarina, Brazil, from D. marsupialis and Lutreolina crassicaudata. The strains were cultivated in Diamond medium without maltose and with starch solution, pH 7.5 at 28 degrees C. The specimens were stained by the Giemsa method and Heidenhain's iron hematoxylin. The light microscopy study of the trophozoites revealed the same morphologic characteristics as specimens previously described.

Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).

Protozoa and fungi of the oral cavity, although frequently occuring and connected with considerable clinical adverse effects, are as yet insufficiently known. The aims of the study were to estimate the prevalence of common invasions of Trichomonas tenax and fungi, to associate the symptoms with the diagnosis of trichomonosis complicated by mycosis and to determine trichomonacidal properties of ornidazole, tinidazole and metronidazole. 936 dentist patients with different diagnoses were included into the study. The collected material consisted of rinsings, with simultaneous application of selective media, different for protozoa and for fungi 6ultures. Among the examined patients T. tenax was found in 90 cases including 85 cases where it occurred together with fungi (11 species), on the basis of which the diagnosis of trichomonosomycosis of the oral cavity was established. It was the most often in patients with leukoplakia and Lichen Wilsoni. We received a statistically significant association (0.001

Small subunit (16S-like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to the early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S-like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free-living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free-living ancestral trichomonad.

Trichomonads of the oral cavity were found in 9 of 60 investigated horses. Apart from dental tartar, the oral cavity showed no clinical signs in all positive horses. The clinical investigation of these horses additionally revealed colic in 4 and coughing in 4 horses as well as lumbago in 1 animal. By means of scanning electron microscopy the trichomonads were shown to be round or piriform with an average length of 7.6 microns and greatest width of 6.3 microns. They had 4 anterior flagella with an average length of 8.3 microns, an undulating membrane measuring 8.7 microns with no trailing flagellum as well as an axostyle extending on average 7.8 microns beyond the body, and therefore have to be placed into the genus Trichomonas.

Clones of 32 strains of Trichomonas vaginalis isolated from patients attending a venereal diseases clinic were compared among themselves and with authentic Pentatrichomonas hominis on the basis of their isoenzyme patterns for eight enzymes by thin-layer starch-gel electrophoresis. The enzymes examined were: glucose phosphate isomerase (GPI); phosphoglucomutase (PGM); malic enzyme (NADP+) (ME); hexokinase (HK); malate dehydrogenase (NAD+) (MDH); glucose-6-phosphate dehydrogenase (G6PD); aldolase (ALD); and lactate dehydrogenase (LDH). From the isoenzyme patterns of four enzymes (LDH, MDH, HK, and GPI) the strains of T vaginalis could be divided clearly into five groups. PGM showed differences in only one strain, while two other enzyme patterns (ME and ALD) were the same for all the strains of T vaginalis tested. All isolates were clearly distinguishable from P hominis. Although G6PD patterns were not sharp some differences were evident among T vaginalis strains.

Isoenzyme electrophoresis was used to study levels of genetic differentiation among strains and clones of Trichomonas gallinae, Trichomonas vaginalis, Tritrichomonas foetus, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. Strain variation was found within T. gallinae, T. vaginalis, and T. foetus, however, levels of enzyme polymorphism were greater in T. gallinae than in T. vaginalis or T. foetus. Isoenzyme genotypes were not a stable property of T. gallinae clones cultivated in vitro. Retrospective studies of T. gallinae SG and JB6 clones revealed that mutation occurred during in vitro cultivation. Heterozygotes of hexokinase-1 and phosphoglucomutase displayed 2 allomorphs in equal dosage, indicating that trichomonads are diploid for these protein loci. Phenetic clustering of the biochemical data suggests that levels of genetic divergence among the species studied are extensive.