Clinical isolates of Tritrichomonas foetus in bulls in Wyoming, South Dakota and Montana, USA.

BACKGROUND: Several Tritrichomonas species have been found in mammalian hosts. Among these trichomonads T. foetus is often found in the urogenital tract of cattle and the gastrointestinal tract of the domestic cat, resulting in sexually transmitted bovine trichomonosis and fecal-orally transmitted feline trichomonosis, respectively. The aims of the current study were to molecularly characterize clinical isolates of T. foetus in cattle populations in Wyoming, South Dakota, and Montana of the United States of America and to phylogenetically analyze Tritrichomonas species of mammalian hosts. RESULTS: DNA sequencing of rRNA genes showed over 99% identity of the newly described isolates to other bovine isolates. Further, T. foetus isolates of various mammalian hosts originated in different geographic regions worldwide were clustered into two well-defined clades by phylogenetic analysis of rRNA and cysteine protease 2 genes. Clade I consisted of isolates originated from cattle, pig, and human whereas clade II contained isolates of cat and dog. CONCLUSION: It is concluded that all mammalian Tritrichomonas spp. apparently belong to T. foetus. Analysis of more sequences is warranted to support this conclusion.

Tritrichomonas foetus: characterisation of ecto-phosphatase activities in the endoflagelar form and their possible participation on the parasite's transformation and cytotoxicity.

The protist parasite Tritrichomonas foetus displays a pear-shaped (PS) and a pseudocystic or endoflagellar form (EFF). Here, we characterised the ecto-phosphatase activity on the surface of EFF and compare its biochemical properties to that of the PS regarding rate of substrate hydrolysis, pH activation profile and sensitivity to well-known phosphatases inhibitors. Two strains exhibiting low- and high-cytotoxicity were used. The enzyme activities of PS and EFF exhibited similar characteristics of protein tyrosine phosphatases (PTP). However, the ecto-phosphatase activities for both forms presented distinct kinetic parameters and different inhibition patterns by PTP inhibitors, suggesting the presence of distinct ecto-enzyme activities between PS and EFF, as well, between both strains. Ultrastructural cytochemistry confirmed the differential distribution of the ecto-phosphatase activity during the EFF transformation. An increase in the percentage of the EFF resulted in a proportional increase in the ecto-phosphatase activity. During EFF reversion, ecto-phosphatase activity decreased and was restored to the level found in the parasites before EFF induction. PS and EFF from the high-cytotoxic strain exhibited higher ecto-phosphatase activities than PS and EFF from the low-cytotoxic strain, respectively. In both strains, the EFF was more cytotoxic and exhibited higher ecto-phosphatase activity when compared to the PS. A large part of the ecto-phosphatase activities of EFF from both strains and PS from the high-cytotoxic strain was irreversibly inhibited when the parasites were pre-treated with a specific antibody against amoebic PTP (anti-EhPRL). Immunoreaction assays revealed that the anti-EhPRL antibody cross-reacted with a 24-kDa protein differentially expressed on the cell surface of PS and EFF T. foetus. A positive correlation was observed between the surface expression of 24-kDa protein and ecto-phosphatase activity. Irreversible inhibition of a part of the ecto-phosphatase activities partially blocked the EFF induction and the cytotoxic effects exerted by both forms. These results suggest that the ecto-phosphatase activities could play a role on the EFF transformation and cytotoxicity of T. foetus.

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.

Tritrichomonas foetus from domestic cats and cattle are genetically distinct.

The genetic relationship amongst Tritrichomonas foetus isolated from domestic cats and cattle was investigated by DNA sequencing of the internal transcribed region of the ribosomal DNA unit and the TR7/TR8 variable-length repeat. The results reject the hypothesis that T. foetus from domestic cats is genetically identical to T. foetus in cattle. We suggest recognition of a 'cat genotype' and a 'cattle genotype' of T. foetus. Review of public nucleotide repositories revealed that the 'cat genotype' has not been isolated from cattle nor the 'cattle genotype' recovered from cats. However, at least one cat isolate has been shown to induce disease in experimentally infected cattle. We conclude that these genotypes fall within the single species T. foetus.

Whole genome sequencing of a feline strain of Tritrichomonas foetus reveals massive genetic differences to bovine and porcine isolates.

Tritrichomonas foetus is a protozoan parasite that colonizes the reproductive tract of cattle as well as the gastrointestinal tract of cats. Bovine tritrichomonosis is a sexually transmitted disease whereas feline tritrichomonosis is thought to be transmitted by the fecal-oral route. Furthermore, T. foetus is known as an essentially apathogenic commensal located in the nasal cavity of pigs. Transmission of T. foetus between the different hosts has to be considered a realistic scenario that may have important implications for the epidemiology of infections and disease. In our study, we generated whole genome sequencing (WGS) data from bovine, feline and porcine T. foetus strains to investigate the genetic (dis)similarities among these diverse strains. As a reference, we used a previously released draft assembly from a bovine T. foetus strain K isolated from an infected bull in Brazil. In particular, we identified single nucleotide polymorphisms (SNPs) and the insertion-deletion (indel) variations within the genomes of the different strains. Interestingly, only a low degree of polymorphism (68 SNPs and indels) was found between the bovine and the porcine strains in terms of variants with a predicted impact of moderate or high and where one species is homozygous for one allele and the other homozygous for the other allele. Conversely, however, a 964 times higher number of such differences was detected by comparing the feline with either the bovine (65,569) or the porcine (65,615) strain. These data clearly indicated a close phylogenetic relationship between bovine and porcine T. foetus but a remarkable genetic distinctness of these two strains from the feline strain. The latter observation was confirmed by PCR-based sequencing of 20 in silico-selected indel markers and five in silico-selected SNP markers that uniformly demonstrated a relatively distant phylogenetic relationship of three independent feline T. foetus isolates in comparison to the bovine and porcine strains investigated. In summary, our comparative genome sequencing approach provided further insights into the genetic diversity of T. foetus in relation to the different host origins of the parasite. Furthermore, our study identified a large number of SNP- and indel-containing sequences that may be useful molecular markers for future epidemiological studies aimed at the elucidation of the transmission patterns of T. foetus within different host species.

Multilocus analysis reveals further genetic differences between Tritrichomonas foetus from cats and cattle.

Tritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster.

Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye.

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus.

Characterization of a human isolate of Tritrichomonas foetus (cattle/swine genotype) infected by a zoonotic opportunistic infection.

Tritrichomonas species flagellates (IMC strain) were isolated from the biliary tract of an individual who had developed cholecystitis as a complication of acquired agammaglobulinemia. Sequence analysis of Tritrichomonas sp. (IMC clone 2 (cl2)) was performed for several genetic regions including the ITS1-5.8S rDNA-ITS2 region, the cysteine protease (CP)-1, CP-2 and CP-4 to CP-9 genes, and the cytosolic malate dehydrogenase 1 gene. In addition to comparison of the variable-length DNA repeats in the isolate clone with those in T. foetus (Inui cl2) and the T. mobilensis (U.S.A.: M776 cl2) reference strains, this analysis showed that the Tritrichomonas sp. (IMC cl2) was T. foetus (cattle/swine genotype). Injection of T. foetus (IMC cl2) directly into the livers of CBA mice resulted in liver abscess formation on Day 7. Moreover, inoculation via orogastric intubation caused infection in the cecum on Day 5 in CBA mice co-infected with Entamoeba histolytica (HM-1: IMSS cl6). T. foetus (IMC cl2) was able to grow in YI-S medium for over 20 days, even at 5°C. These results indicate that the T. foetus isolate is able to survive in the feces and edible organ meat of the definitive host for a prolonged period of time, and it is possible that the parasite could infect humans.

Are Tritrichomonas foetus and Tritrichomonas suis synonyms?

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

Molecular characterization of trichomonads from feces of dogs with diarrhea.

Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to opportunistic overgrowth of the commensal, Pentatrichomonas hominis. However, molecular characterization of canine trichomonads has never been reported. This study was performed to determine, by means of rRNA gene sequence analysis, the identity of trichomonads observed in feces from dogs with diarrhea. Total DNA was isolated from fecal samples obtained from a 3-mo-old mixed breed dog and litter of German Shepherd puppies having profuse liquid diarrhea containing numerous trichomonads. Total DNA was subject to PCR amplification of partial 18S rRNA gene or 5.8S, ITS1, ITS2, and partial 18S and 28S rRNA genes using species-specific and universal primers, respectively. Products of 642 and 1864 base-pair length were amplified and cloned. On the basis of rRNA gene sequence, the trichomonads observed in the single dog and the litter of puppies shared 100% identity with Tritrichomonas foetus and P. hominis, respectively. The present study is the first to establish the molecular identity of trichomonads infecting dogs with diarrhea. These studies validate the longstanding assumption that canine trichomoniasis may be attributed to P. hominis. Importantly, these studies additionally recognize that canine trichomoniasis may also be caused by infection with T. foetus.

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.

Characterization of antigenic proteins from Tritrichomonas foetus recognized by antibodies in rabbit serum, bovine serum and bovine cervicovaginal mucus.

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract and is recognized as 1 of the more common infectious agents causing decreased reproductive efficiency in beef cattle. Infections result in reproductive failure and produce considerable economic loss. Vaccination of heifers with vaccines containing T. foetus induces elevated serological responses to many T. foetus antigens, decreases the rate and/or length of infection with T. foetus, and decreases fetal loss caused by infection. Because T. foetus infections are usually limited to lumen and mucosal surfaces of the reproductive tract, it has been assumed that protection from infection and abortion is partially mediated by immunoglobulins in the uterus and vagina. The objective of this study was to identify and characterize specific antigens of T. foetus that show promise for use in a recombinant vaccine that will generate a protective mucosal immune response in cattle. Surface proteins were identified by using polyclonal rabbit anti-trichomonal sera eluted from paraformaldehyde-fixed cells. Analyses of these proteins, utilizing mucosal antibodies from vaccinated and convalescent cows, have identified proteins involved in generating a local immune response. Western immunoblot analysis indicates that these proteins are well conserved and are excellent candidates for incorporation into a recombinant vaccine.

Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline trichomonal diarrhea.

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, a n organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Survival of a feline isolate of Tritrichomonas foetus in water, cat urine, cat food and cat litter.

Feline intestinal trichomoniasis caused by Tritrichomonas foetus is associated with large bowel diarrhea in cats from many parts of the world. It has long been recognized as an economically important sexually transmitted disease that causes early abortion in cattle. Isolates of T. foetus from cattle are infectious for the large intestine of cats and isolates of T. foetus from cats are infectious for the reproductive system of cattle. The parasite is maintained by fecal-oral transmission in cats. The present study was conducted to examine the survival of a feline isolate of T. foetus, AUTf-12, under various conditions that are relevant to fecal-oral transmission in cats. Trophozoites were grown in TYM medium and then exposed to water, cat urine, dry cat food, canned cat food, clumping cat litter, or filter paper for various lengths of time and then re-cultured in TYM medium. Trophozoites survived exposure to distilled or tap water for 30 but not 60 min, while they survived for at least 180 min in urine. Trophozoites survived for 30 min on dry cat food but survived for 120-180 min in canned cat food. No survival of trophozoites was observed on cat litter but trophozoites survived for 15 min when placed on filter paper. Our results indicate that T. foetus can survive and be potentially infectious in water, urine, dry cat food and canned cat food.

Intestinal Tritrichomonas foetus infection in cats in Switzerland detected by in vitro cultivation and PCR.

Tritrichomonas foetus, a parasite well known for its significance as venereally transmitted pathogen in cattle, has recently been identified as a cause of chronic large-bowel diarrhea in domestic cats in the US, UK, and, more recently, also in Norway. In a period of 3 months (October to December 2007), 45 cats of Switzerland suffering from chronic diarrhea were investigated for intestinal infections, including a search for trichomonads. A commercially available in vitro culture system was used to screen for infection, complemented with a PCR and subsequent amplicon sequencing to support speciation. The PCR is based upon amplification of a sequence derived from the internal transcribed spacer region 1 (ITS1) on the ribosomal RNA gene (rRNA) using primers designed to detect a broad range of genera and species belonging to the family of Trichomonadidae. The method was furthermore adapted to the uracil DNA glycosylase (UDG) system in order to prevent carry-over contamination and it included a recombinant internal control to track for inhibitory reactions. Eleven out of the 45 cats were culture-positive, as revealed by microscopic identification of trichomonadid organisms. One of the isolates was subjected to scanning electron microscopy and findings revealed the presence of three flagella, thus placing the isolate into the gender Tritrichomonas sp. PCR and subsequent amplicon sequencing were carried out with ten of the 11 isolates. A total homology with published T. foetus sequences was confirmed in all of the cases. T. foetus therefore appears to range among those organisms that can cause chronic diarrhea in cats in Switzerland.

Morphological and molecular identification of non-Tritrichomonas foetus trichomonad protozoa from the bovine preputial cavity.

Tritrichomonas foetus is the causative agent of bovine trichomonosis. This protozoan is found in the preputial cavity of bulls and is transmitted to cows during coitus. Currently, the diagnosis of this parasite is based on microscopic examination of preputial washings or scrapings, but it was recently recognized that other trichomonads similar in size, shape, and motility to T. foetus can be present in preputial samples. Despite the serious consequences of an incorrect diagnosis for bovine trichomonosis, the precise speciation of these other trichomonads has remained uncertain. Here, a total of 12 non-T. foetus isolates were microscopically examined. On the basis of morphological criteria, seven of these isolates were identified as Tetratrichomonas sp., whereas four other isolates coincided with the description of Pentatrichomonas hominis. In the last isolate, a third non-T. foetus species was identified as belonging to the genera Pseudotrichomonas or Monocercomonas: the first time that species of either of these genera have been reported in preputial samples. To confirm these data, small subunit rRNA gene sequences were obtained by PCR from the 12 trichomonad isolates. These new sequences were analysed in a broad phylogeny including 72 other parabasalid sequences. From our phylogenetic trees, we confirmed the taxonomic status of non-T. foetus organisms isolated from preputial samples (Tetratrichomonas, Pentatrichomonas, and Pseudotrichomonas) and suggested the existence of two Tetratrichomonas species, despite their morphological similarity. The route of transmission of the non-T. foetus organisms identified in the bovine preputial cavity is discussed and we confirm that the PCR assay using the previously described T. foetus-specific primers TFR3 and TFR4 could be a useful alternative method for the diagnosis of bovine trichomonosis.

Intra-strain clonal phenotypic variation of Tritrichomonas foetus is related to the cytotoxicity exerted by the parasite to cultured cells.

As observed in most of the investigated trichomonads, a strain of Tritrichomonas foetus includes different parasite subpopulations. Such population diversity might account for important properties such as the ability of the parasite to destroy host cells. The aim of this study was to characterize the cytotoxicity exerted by subpopulations (named as K1, K2, K3, K4 and K5) of an isolate of T. foetus on epithelial cultured cells. The five populations studied here destroyed epithelial monolayers at different rates (from 25% to 55%), even though the cytoadhesion level and whole-cell protease activity were closely related among them. We were also able to detect differences in contact-dependent and contact-independent cytotoxicity mechanisms among the five populations. An extracellular parasite protease had varying activity among the parasite populations. The intensity of contact-independent cytotoxicity was strictly related to the degree of enzyme activation, suggesting that such a protease might be involved in the cytotoxicity mediated by T. foetus.

Fine structure and isozymic characterization of trichomonadid protozoa.

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

Cattle pathogen tritrichomonas foetus (Riedmüller, 1928) and pig commensal Tritrichomonas suis (Gruby & Delafond, 1843) belong to the same species.

A number of reports suggest that the sexually transmitted pathogen of cattle, Tritrichomonasfoetus, and a gastrointestinal commensal of pigs, Tritrichomonas suis, are very similar and may be co-specific. A conclusive review of the taxonomic and nomenclatural status of these species has not been presented so far. Toward this end, we reexamined and compared porcine and bovine trichomonads with regard to their morphology, pathogenic potential, and DNA polymorphism. Using light and electron microscopy, no distinguishing features between T. foetus and T. suis strains were found in size, general morphology, and karyomastigont structure. Both bovine and porcine trichomonads showed pathogenic potential in the subcutaneous mouse assays and did not separate into distinct groups according to strain virulence. Three DNA fingerprinting methods (i.e. RFLP, RAPD, and PCR-based analysis of variable-length DNA repeats) that produce species-specific DNA fragment patterns did not distinguish between the bovine and porcine strains. Sequencing of a variable 502-bp DNA fragment as well as comparison of 16S rRNA gene sequences did not reveal species-specific differences between the cattle and porcine strains. Therefore, we conclude that T. foetus and T. suis belong to the same species. To prevent confusion that may arise from T. foetus-T. suis synonymy, we propose to suppress the older name suis and maintain its accustomed junior synonym foetus as a nomen protectum for both cattle and porcine trichomonads. The case has been submitted to the International Commission on Zoological Nomenclature for ruling under its plenary power.