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Veterinary Parasitology Nov 2007Virgin heifers (44) were intravaginally inoculated at estrus with low (10(6)) or high (10(8)) doses of live Tetratrichomonas sp., Pentatrichomonas hominis (P. hominis),...
Virgin heifers (44) were intravaginally inoculated at estrus with low (10(6)) or high (10(8)) doses of live Tetratrichomonas sp., Pentatrichomonas hominis (P. hominis), or Tritrichomonas foetus (T. foetus). Controls were inoculated with Diamond's trypticase yeast extract maltose media. Genital infection was determined by culture of cervico-vaginal mucus (CVM) in Schneider's media and InPouch TF as well as by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). The presence of trichomonads in fecal samples was determined by culture in Schneider's medium and PCR/RFLP. In CVM samples, tetratrichomonads were found by PCR/RFLP and Schneider's culture only sporadically at intermittent weeks. The presence of tetratrichomonads was not associated with the dose in the experimental vaginal inoculation since Tetratrichomonas sp. appeared more frequently in heifers inoculated with a low dose of tetratrichomonads than in heifers inoculated with a high dose of tetratrichomonads. Moreover, Tetratrichomonas spp. were isolated not only in heifers inoculated with tetratrichomonads but also in control heifers and in heifers inoculated with P. hominis. In feces, Tetratrichomonas spp. were frequently identified by culture in Schneider's and by PCR/RFLP in heifers of all groups. P. hominis was never found in CVM or feces by any method. Based on the common appearance of tetratrichomonads in feces and vaginal secretions, it appears that tetratrichomonads were detected periodically in the vagina of heifers as a consequence of repeated contamination from feces and not as a result of experimental infection. In summary, in this study, the strains of Tetratrichomonas sp. and P. hominis did not establish persistent infection in heifers.
Topics: Animals; Cattle; Cattle Diseases; Estrus; Female; Genital Diseases, Female; Protozoan Infections, Animal; Trichomonadida; Vagina
PubMed: 17950533
DOI: 10.1016/j.vetpar.2007.09.007 -
American Journal of Veterinary Research Jul 2007To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces.
OBJECTIVE
To determine the optimum reaction conditions and detection limits of PCR assay for identification of Pentatrichomonas hominis in DNA extracted from canine feces.
SAMPLE POPULATION
DNA extracted from feces of 4 dogs with diarrhea from which trichomonads were observed, 81 dogs that had feces submitted to a diagnostic laboratory, and 19 dogs residing in a laboratory animal facility.
PROCEDURES
Optimum reaction conditions and absolute and practical detection limits of 2 P hominis 18S species-specific primer pairs were determined by use of an in vitro cultivated canine isolate of P hominis in the presence and absence of canine feces. The optimized PCR assay was applied to amplification of P hominis 18S rRNA genes from DNA extracted from the feces of dogs.
RESULTS
Under optimized conditions, a primer pair was identified as able to detect as few as 1 P hominis organism/180-mg fecal sample. The PCR assay identified P hominis in diarrheic feces of 4 dogs in which trichomonads were seen by light microscopy. The P hominis genes were not amplified from other fecal samples examined.
CONCLUSIONS AND CLINICAL RELEVANCE
Molecular identification of P hominis in feces of 4 dogs with trichomonosis and diarrhea reported here validates the identity of this species in such infections. Sensitive and specific PCR amplification of P hominis 18S rRNA genes from DNA extracted from feces will directly facilitate studies examining pathogenicity of this trichomonad and enable differentiation of P hominis from other known or novel species of trichomonads that may infect the gastrointestinal tract of dogs.
Topics: Animals; DNA, Protozoan; Diarrhea; Dog Diseases; Dogs; Feces; Female; Male; Polymerase Chain Reaction; Protozoan Infections, Animal; Species Specificity; Trichomonadida
PubMed: 17605615
DOI: 10.2460/ajvr.68.7.783 -
The Journal of Eukaryotic Microbiology 2007Tritrichomonas 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....
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.
Topics: Animals; Cattle; Cattle Diseases; DNA, Protozoan; Male; Microscopy, Electron, Scanning; Molecular Sequence Data; Phylogeny; RNA, Ribosomal; Sequence Analysis, DNA; Trichomonas; Tritrichomonas foetus
PubMed: 17403157
DOI: 10.1111/j.1550-7408.2007.00247.x -
Veterinary Parasitology Apr 2007Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some...
Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some investigators presumptively identified P. hominis as a causative agent of feline diarrhea. However, molecular identification of P. hominis infection in the cat has not been reported. Another trichomonad, Tritrichomonas foetus, is recognized as an intestinal pathogen in cats and often presumptively diagnosed on the basis of the presence of trichomonads in diarrheic feces. It is of importance to determine if cats are natural hosts for P. hominis, as the presence of this organism could result in inaccurate assumption of T. foetus infection. In this study, we used a species-specific PCR assay to identify P. hominis 18S rRNA genes in fecal samples collected from a convenience population of cats in which a high prevalence of T. foetus infection had been previously identified (cat show) or suspected (submitted for T. foetus diagnostic testing). The prevalence of T. foetus infection in these samples was 31% and 28.6%, respectively. P. hominis infection was identified by PCR of DNA extracted from feces of five cats (1.9% and 2.1% of fecal samples, respectively). All cats in which P. hominis was identified were also infected with T. foetus. PCR identification of P. hominis infection in the cat should facilitate future studies to determine the pathogenicity of this species and enable differentiation of P. hominis from other known or as-yet unidentified species of trichomonads that may infect cats.
Topics: Animals; Cat Diseases; Cats; Feces; Polymerase Chain Reaction; RNA, Protozoan; RNA, Ribosomal, 16S; RNA, Ribosomal, 18S; Trichomonadida; Trichomonas Infections
PubMed: 17127004
DOI: 10.1016/j.vetpar.2006.10.020 -
The Journal of Parasitology Aug 2005Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to...
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.
Topics: Animals; Base Sequence; DNA, Protozoan; Diarrhea; Dog Diseases; Dogs; Electrophoresis, Agar Gel; Feces; Female; Molecular Sequence Data; Polymerase Chain Reaction; Protozoan Infections; Protozoan Infections, Animal; RNA, Ribosomal; Sequence Alignment; Trichomonadida; Tritrichomonas foetus
PubMed: 17089769
DOI: 10.1645/GE-474R.1 -
Sexually Transmitted Infections Apr 2007To investigate the possible involvement of human trichomonads (Pentatrichomonas hominis and Trichomonas tenax) other than Trichomonas vaginalis in the aetiology of...
OBJECTIVE
To investigate the possible involvement of human trichomonads (Pentatrichomonas hominis and Trichomonas tenax) other than Trichomonas vaginalis in the aetiology of vaginal trichomoniasis.
METHODS
Vaginal swabs taken from women attending antenatal clinics were tested for Trichomonas vaginalis by traditional assays (wet-mount microscopy and InPouch culture) and nucleic acid amplification (polymerase chain reaction). These swabs were also tested for the presence of P hominis and T tenax by nucleic acid amplification. Oral and rectal swabs from these women were tested for T tenax and P hominis respectively. Data on sociodemographic characteristics, sexual and anogenital hygiene practices likely to seed P hominis and T tenax into the vagina were collected by a questionnaire.
RESULTS
93% (161) of the 173 samples in which T vaginalis was detected by wet preparation or culture was evaluable by PCR. Of this, T vaginalis was detected in 94% (152) by T vaginalis-specific PCR. Neither P hominis nor T tenax was detected in any of the vaginal swab samples. These included nine samples for which T vaginalis had been detected by wet preparation or culture, but were negative by T vaginalis nucleic acid amplification. P hominis and T tenax were not detected in any of the rectal and oral swabs, respectively.
CONCLUSION
In this group of women, there was no evidence for the involvement of trichomonads other than T vaginalis in the aetiology of vaginal trichomoniasis.
Topics: Adolescent; Adult; Animals; Female; Ghana; Humans; Mouth Diseases; Polymerase Chain Reaction; Rectal Diseases; Trichomonas; Trichomonas Vaginitis; Vaginal Smears
PubMed: 16790560
DOI: 10.1136/sti.2006.020941 -
Cellular Microbiology Jun 2005Trichomonas vaginalis, an ancient protist, colonizes the vaginal mucosa causing trichomonosis, a vaginitis that sometimes leads to severe health complications....
Trichomonas vaginalis, an ancient protist, colonizes the vaginal mucosa causing trichomonosis, a vaginitis that sometimes leads to severe health complications. Preparatory to colonization of the vagina is the adhesion to vaginal epithelial cells (VECs) by trichomonads. We hypothesized that VECs alter the gene expression to form a complex signalling cascade in response to trichomonal adherence. In order to identify the genes that are upregulated, we constructed a subtraction cDNA library after contact with parasites that is enriched for differentially expressed genes from the immortalized MS-74 VECs. Sixty cDNA clones were sequenced and to our knowledge for the first time, differentially regulated genes were identified in response to early trichomonal infection. The identified genes were found to encode functional proteins with specific functions associated with cell structure maintenance and extracellular matrix components, proinflammatory molecules and apoptosis. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed expression of selected genes. Further, cyclooxygenase 2 (COX-2) protein expression was analysed using Western blot and immunofluorescence assays. Data suggest that p38 mitogen-activated protein (MAP) kinase and tyrosine kinases play a role in COX-2 induction. Finally, T. vaginalis and Tritrichomonas foetus but not Pentatrichomonas hominis induce expression of COX-2. This is a first attempt at elucidating the basis of interaction of trichomonads with host cells and the corresponding host responses triggered by the parasites.
Topics: Animals; Cell Adhesion; Cells, Cultured; Cyclooxygenase 2; Epithelial Cells; Female; Gene Expression Regulation; Humans; Membrane Proteins; Phosphotransferases; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; Trichomonas vaginalis; Tritrichomonas foetus; Vagina
PubMed: 15888089
DOI: 10.1111/j.1462-5822.2005.00522.x -
Letters in Applied Microbiology 2004To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens.
AIM
To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens.
METHODS
Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs.
CONCLUSIONS
A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well.
SIGNIFICANCE AND IMPACT OF THE STUDY
The presence of P. hominis in vaginal specimens has not been reported before.
Topics: Animals; Bacteria; DNA, Protozoan; DNA, Ribosomal; Eukaryota; Feces; Female; Genes, rRNA; Humans; Leukocytes; Polymerase Chain Reaction; Sensitivity and Specificity; Trichomonadida; Vagina; Yeasts
PubMed: 15130148
DOI: 10.1111/j.1472-765X.2004.01528.x -
Cryobiology Feb 2004Conventional methods for the propagation and preservation of parasites in vivo or in vitro have some limitations, including the need for labor, initial isolation and...
Conventional methods for the propagation and preservation of parasites in vivo or in vitro have some limitations, including the need for labor, initial isolation and loss of strains, bacterial, and fungal contamination, and changes in the original biological and metabolic characteristics. All these disadvantages are considerably reduced by cryopreservation. In this study, we examined the effects of various freezing conditions on the survival of several protozoan parasites after cryopreservation. The viability of Entamoeba histolytica was improved by seeding (p < 0.05, chi2 test), while this was not so effective for Trichomonas vaginalis. Of six cryoprotectants examined, dimethyl sulfoxide (Me(2)SO), and glycerol showed the strongest cryoprotective effects. The optimum conditions for using Me(2)SO were a concentration of 10% with no equilibration, and those for glycerol were a concentration of 15% with equilibration for 2h. The optimum cooling rate depended on the parasite species. Trypanosoma brucei gambiense and Leishmania amazonensis were successfully cryopreserved over a wide range of cooling rates, whereas the survival rates of E. histolytica, T. vaginalis, Pentatrichomonas hominis, and Blastocystis hominis were remarkably decreased when frozen at improper rates. Unlike the cooling rate, exposure of the protozoans to a rapid thawing method produced better motility for all parasites.
Topics: Animals; Blastocystis hominis; Cryopreservation; Cryoprotective Agents; Entamoeba histolytica; Eukaryota; Leishmania mexicana; Movement; Parasites; Trichomonadida; Trichomonas vaginalis; Trypanosoma brucei gambiense
PubMed: 14969677
DOI: 10.1016/j.cryobiol.2003.10.004 -
Journal of Veterinary Diagnostic... Jul 2003Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A...
Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.85 rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.85 rRNA gene and ITSR sequence resultsand PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.
Topics: Animals; Base Sequence; Cattle; Cattle Diseases; DNA, Protozoan; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping; Sequence Analysis, DNA; Trichomonas; Trichomonas Infections
PubMed: 12918825
DOI: 10.1177/104063870301500417