Molecular-phylogenetic investigation of trichomonads in dogs and cats reveals a novel Tritrichomonas species.

BACKGROUND: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. METHODS: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. RESULTS: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. CONCLUSIONS: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator-prey link in the evolution of this trichomonad.

A case report of pulmonary tritrichomonosis in a pig.

BACKGROUND: Tritrichomonads like porcine Tritrichomonas foetus (previously named Tritrichomonas suis), can commensally live in nasal cavity of pigs, but it is rare to cause pulmonary tritrichomonosis. CASE PRESENTATION: A 40-day-old piglet was presented for persistent labor breathing and diagnosed with parasite infections in the lung by analysis of bronchoalveolar lavage (BAL) under microscope. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infection. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. FINDINGS: Here, we report a case of pulmonary tritrichomonosis in a pig. By taking advantage of next-generation sequencing approach, we found 9611 homologous tags belonging to 50 annotated genes of tritrichomonads by analysis of mRNA of the bronchoalveolar lavage with the parasite infections. Furthermore, RT-PCR and DNA sequencing analysis confirmed the presence of the tritrichomonad. CONCLUSION: Our results demonstrate that tritrichomonads like porcine Tritrichomonas foetus can cause lung infections of pigs and reveal that next-generation sequencing is potential to identify rare diseases like pulmonary tritrichomonosis in clinical.

Multiyear Survey of Coccidia, Cryptosporidia, Microsporidia, Histomona, and Hematozoa in Wild Quail in the Rolling Plains Ecoregion of Texas and Oklahoma, USA.

We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

Tritrichomonas--systematics of an enigmatic genus.

Tritrichomonas spp. are parasitic protozoans that proliferate on mucus membranes of the urogenital, gastro-intestinal or nasal tract. For instance, Tritrichomonas foetus is an important cause of reproductive failure in cattle. Some years ago, T. foetus was also identified as a causative agent of diarrhoea in cats. Previous studies on the morphological, physiological and molecular levels have raised doubts as to the phylogenetic relationship among some Tritrichomonas species, particularly in relation to T. foetus, Tritrichomonas suis, and Tritrichomonas mobilensis. With the advent of molecular genetic tools, it has become clear that these three tritrichomonad species are closely related or may even represent the same species. Indeed, since recently, T. suis and T. foetus are generally considered as one species, with T. mobilensis being a closely related sister taxon. To date, molecular studies have not yet been able to resolve the taxonomic (specific) status of T. foetus from cattle and cats. In the future, novel genomic approaches, particularly those involving next generation sequencing are poised to resolve the taxonomy of Tritrichomonas spp. Here, we review the literature on the current state of knowledge of the taxonomy of T. foetus, T. suis, and T. mobilensis with special reference to the relationship between T. foetus from cattle and cats.

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

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

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

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

A novel EF-hand calcium-binding protein in the flagellum of the protozoan Tritrichomonas suis.

The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41 kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

Conservation of calnexin in the early branching protozoan Tritrichomonas suis.

The cloning and characterization of Ts-p66, a calcium-binding protein representing calnexin of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA expression library was screened with monospecific antibodies affinity-purified on an immuno-reactive 66 kDa antigen in a Triton X-114 membrane-protein fraction. The deduced amino acid sequence of the resulting cDNA clones revealed that Ts-p66 belongs to the calreticulin protein family and represents calnexin of T. suis. The key structural features and sequence motifs of the calnexins were all conserved. By lectin-blotting we demonstrated that the native protein is glycosylated. Northern and Southern hybridizations showed that T. suis calnexin was highly expressed and encoded by a single or low copy number gene. A cDNA encoding Ts-p66 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant proteins bind Ca(2+). Using immunofluorescence with affinity-purified antibodies, a staining pattern was observed which points towards a putative localization of Ts-p66 in the nuclear membrane and endoplasmic reticulum. Demonstration of a structurally conserved calnexin in the amitochondriate protist T. suis indicates the very early evolutionary origin of the machinery for quality control of protein folding in the endoplasmic reticulum and the molecules involved hereby.

Alpha-tubulins of Tritrichomonas mobilensis are encoded by multiple genes and are not posttranslationally tyrosinated.

Reverse-transcriptase polymerase chain reaction cloning of the 3'-ends of the alpha-tubulin cDNAs of Tritrichomonas mobilensis in combination with Southern-blot analysis identified seven to eight distinct alpha-tubulin genes. All seven lack a carboxy-terminal tyrosine and the corresponding sequence compatible with posttranslational tyrosination. This indicates that whereas tyrosination of alpha-tubulin has been found in most species, including humans and trypanosomes, it is absent in trichomonads.

Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis.

Tritrichomonas foetus and Tritrichomonas suis isolates were cultivated axenically in Diamond's medium. Studies on the chromosome numbers of these two species with a light microscope were done by adding different concentrations of colchicine into the medium, incubating at 37 degrees C for 6-8 h and using a hypotonic swelling technique. The diploid chromosome numbers of both T. foetus and T. suis were 2n=10.

Comparative genetic analysis of tritrichomonadid protozoa by the random amplified polymorphic DNA technique.

The taxonomic classification within the genus Tritrichomonas is a subject of controversy, and, potentially, separation of the tritrichomonads from cattle and swine on the species level is not valid. To tackle this hypothesis we comparatively assessed several isolates of protozoan parasites from the three Tritrichomonas species T. foetus, T. suis, and T. mobilensis by the RAPD (random amplified polymorphic DNA) technique. In this method with 20 different primers, all T. foetus and T. suis isolates resulted in identical genomic fingerprints, thus yielding additional evidence for the genetic identity of T. foetus and T. suis. In contrast, it turned out that the species T. mobilensis isolated from the squirrel monkey is genetically distinct and can clearly be discriminated from the other tritrichomonads. Consequently, the results obtained in this study support a possible future revision of the taxonomic classification of the genus Tritrichomonas.

Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples.

Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa.

The taxonomic situation in the genus Tritrichomonas is the subject of controversial discussion: potentially T. foetus and T. suis, the tritrichomonads from cattle and swine, respectively, could belong to the same species. In order to shed some light on this question, a molecular biological analysis was performed. The 5.8S rRNA gene and the flanking internal transcribed spacer regions (ITS1 and ITS2) of 12 different isolates of 3 Tritrichomonas species T. foetus, T. suis and T. mobilensis were enzymatically amplified by PCR and subcloned. Also, the corresponding regions of the trichomonads Trichomonas vaginalis, T. tenax, T. gallinae and Pentatrichomonas hominis were included in this study. Sequence analysis of cloned fragments was used to compare the parasite isolates. The genus Tritrichomonas exhibited an extremely high degree of homogeneity. All T. foetus and T. suis isolates had identical sequences, and only 1 substitution was found in the ITS2 region of T. mobilensis. In contrast, the genus Trichomonas shared more diversity. The results obtained in this study support a possible future revision of the taxonomic classification of tritrichomonads.

A novel mechanism of mycophenolic acid resistance in the protozoan parasite Tritrichomonas foetus.

Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5'-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypoxanthine. We have cloned a T. foetus strain (mpar) that was 50-fold more resistant to mycophenolic acid than wild type (IC50 = 1 mM for mpar vs 20 microM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpar was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpar strains. Mycophenolic acid (100 microM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpar, although no inhibition of T. foetus mpar growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpar so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpar incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpar hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpar: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpar. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpar. The mpar strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.