-
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 the American Veterinary... May 2003To evaluate the efficacy of and optimize a commercially available culture system for sensitive and specific in-clinic culture of Tritrichomonas foetus from cat feces.
OBJECTIVE
To evaluate the efficacy of and optimize a commercially available culture system for sensitive and specific in-clinic culture of Tritrichomonas foetus from cat feces.
DESIGN
Prospective study.
SAMPLE POPULATION
Samples of freshly voided feces from 117 purebred cats and pure cultures of T. foetus obtained from a cat with chronic diarrhea.
PROCEDURE
Optimal conditions for use of the culture system, such as quantity of fecal inoculum (0.025 to 0.2 g) and cultivation temperature (25 or 37 degrees C [98.6 or 77.0 degrees F]), were determined. Specificity of the system was examined by attempted culture of Giardia lamblia and Pentatrichomonas hominis. Sensitivity of the system to detect T. foetus was determined by inoculation of culture system pouches with serially diluted T. foetus suspensions with and without feces.
RESULTS
Detection limit of the culture system was 1 and 1,000 T. foetus organisms without and with feces from cats, respectively. Optimal fecal inoculum was < 0.1 g of feces. At 37 degrees C, cultures yielded positive results in 24 hours; organisms remained viable for 1 to 6 days, and bacterial overgrowth was common. At 25 degrees C, cultures yielded positive results in 1 to 11 days; organisms were long-lived, and bacterial overgrowth was uncommon. Neither G. lamblia or P. hominis survived in the culture system.
CONCLUSIONS AND CLINICAL RELEVANCE
The culture system was sensitive and specific for culture of T. foetus in feces of cats. Performance was optimal when test kits were inoculated with < or = 0.1 g of freshly voided feces and cultured at 25 degrees C.
Topics: Animals; Cat Diseases; Cats; Feces; Female; Prospective Studies; Protozoan Infections; Protozoan Infections, Animal; Sensitivity and Specificity; Temperature; Time Factors; Tritrichomonas foetus
PubMed: 12762381
DOI: 10.2460/javma.2003.222.1376 -
Journal of Clinical Microbiology Nov 2002Tritrichomonas foetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea....
Tritrichomonas foetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity with T. foetus in cats as well as misdiagnosis of the organisms as Pentatrichomonas hominis or Giardia sp. when visualized by light microscopy. The diagnosis of T. foetus presently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. As T. foetus organisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of feline T. foetus infection. A single-tube nested PCR was designed and optimized for the detection of T. foetus in feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by using P. hominis, Giardia lamblia, and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms.
Topics: Animals; Cat Diseases; Cats; DNA Primers; DNA, Protozoan; DNA, Ribosomal; Feces; Polymerase Chain Reaction; Protozoan Infections; Protozoan Infections, Animal; RNA, Ribosomal, 5.8S; Sensitivity and Specificity; Tritrichomonas foetus
PubMed: 12409385
DOI: 10.1128/JCM.40.11.4126-4130.2002 -
Journal of the American Veterinary... Apr 2000Four purebred domestic cats examined because of diarrhea were found to have Pentatrichomonas hominis, a rarely reported trichomonad parasite, in their feces. Treatment...
Four purebred domestic cats examined because of diarrhea were found to have Pentatrichomonas hominis, a rarely reported trichomonad parasite, in their feces. Treatment with a combination of metronidazole and enrofloxacin tended to improve consistency of the feces, whereas treatment with metronidazole alone reduced the number of P hominis trophozoites in fecal smears but did not necessarily result in an improvement in clinical signs. Two cats were euthanatized. Necropsy revealed lymphoplasmacytic enterocolitis with eosinophils and eosinophilic globular leukocytes, neutrophils in the mucosa of the colon and within intraluminal contents of the cecum, and P hominis trophozoites in intraluminal contents of the colon and cecum.
Topics: Animals; Anti-Infective Agents; Antitrichomonal Agents; Cat Diseases; Cats; Colon; Diarrhea; Enrofloxacin; Feces; Female; Fluoroquinolones; Male; Metronidazole; Protozoan Infections; Protozoan Infections, Animal; Quinolones; Trichomonadida
PubMed: 10767968
DOI: 10.2460/javma.2000.216.1270 -
Journal of Clinical Microbiology Jan 1997A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA...
A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.
Topics: Animals; Female; Humans; Polymerase Chain Reaction; Trichomonas Infections; Trichomonas vaginalis; Vagina; Vaginal Discharge
PubMed: 8968894
DOI: 10.1128/jcm.35.1.132-138.1997 -
Journal of Clinical Microbiology Apr 1991Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic...
Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method.
Topics: Animals; Cloning, Molecular; DNA Probes; DNA, Protozoan; Female; Humans; Trichomonas Vaginitis; Trichomonas vaginalis; Vaginal Smears
PubMed: 1890171
DOI: 10.1128/jcm.29.4.702-706.1991 -
Infection and Immunity Mar 1989Complement pathway activity in the killing of Pentatrichomonas hominis was investigated in this study. At 10(5) organisms per ml, P. hominis was completely killed by the...
Complement pathway activity in the killing of Pentatrichomonas hominis was investigated in this study. At 10(5) organisms per ml, P. hominis was completely killed by the presence of 1% normal human serum. In contrast, no killing effect on P. hominis was observed when specific antibodies were absorbed or when the complement was destroyed. Moreover, Mg2+-ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated serum had no killing effect on P. hominis, while serum heated at 50 degrees C or treated with zymosan killed P. hominis as well as did normal human serum. Further study using gel filtration (Sephacryl S-300) and affinity chromatography (protein A) revealed that immunoglobulin M (IgM; 20 micrograms/ml) alone was responsible for the complement activation in the killing of P. hominis, but both IgA (24 micrograms/ml) and IgG (180 micrograms/ml) had no effect on complement-mediated lysis. On the other hand, IgG at 1,260 micrograms/ml completely inhibited complement-mediated killing by IgM, suggesting that a blocking factor is present in IgG. The results of this study indicate that a mechanism of IgM-dependent classical complement pathway activation contributes to the killing effect of normal human serum on P. hominis.
Topics: Animals; Antibodies, Protozoan; Complement Activation; Complement Pathway, Classical; Eukaryota; Humans; Immunoglobulin M; In Vitro Techniques
PubMed: 2917791
DOI: 10.1128/iai.57.3.902-906.1989 -
The Journal of Parasitology Oct 1988Isoenzyme electrophoresis was used to study levels of genetic differentiation among strains and clones of Trichomonas gallinae, Trichomonas vaginalis, Tritrichomonas...
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.
Topics: Animals; Culture Media; Electrophoresis, Starch Gel; Genotype; Isoenzymes; Phenotype; Polymorphism, Genetic; Trichomonas
PubMed: 3418458
DOI: No ID Found -
Journal of Clinical Microbiology Aug 1988Tritrichomonas mobilensis is a recently described enteric protozoon of squirrel monkeys. An earlier report identified one of the metabolic products of this organism as a...
Tritrichomonas mobilensis is a recently described enteric protozoon of squirrel monkeys. An earlier report identified one of the metabolic products of this organism as a lectinlike hemagglutinin. Its further properties were determined in this study. Culture supernatants of T. mobilensis FP4190 were concentrated by ultrafiltration through a membrane with 100,000-molecular-weight cutoff. High titers of agglutinin against human erythrocytes were obtained. Incubation at 70 degrees C for 15 min resulted in complete inactivation. Exposure to 56 degrees C for 30 min was without effect, and only partial loss of activity was obtained during incubation for up to 18 h. Maintenance at pH 4 to 9 for 4 h at room temperature had no deleterious effect. Apparent degradation of the hemagglutinin was achieved by 18 h of contact with proteinase K, but trypsin and collagenase were essentially ineffective. Papain increased the sensitivity of the test. In the presence of this enzyme hemagglutinin was demonstrated also in cultures of Tritrichomonas foetus and Tritrichomonas augusta but not in those of Pentatrichomonas hominis or Trichomonas vaginalis.
Topics: Animals; Hemagglutination Tests; Hemagglutinins; Hot Temperature; Humans; Hydrogen-Ion Concentration; Papain; Species Specificity; Tritrichomonas
PubMed: 3170709
DOI: 10.1128/jcm.26.8.1460-1463.1988 -
Infection and Immunity Sep 1982Human cervicovaginal secretions were obtained from patients at the Gynecology and Obstetrics Clinics at National Taiwan University Hospital and Cathay General Hospital,...
Human cervicovaginal secretions were obtained from patients at the Gynecology and Obstetrics Clinics at National Taiwan University Hospital and Cathay General Hospital, Republic of China. Among the 500 patients examined, 33 (6.6%) were infected with Trichomonas vaginalis as determined by the culture method. Secretions from 24 of the infected patients and 30 noninfected women were assayed for anti-T. vaginalis immunoglobulins by the indirect immunofluorescent antibody technique. A few serum samples from both infected and noninfected persons were also included in this study. Immunoglobulin G (IgG) antibody against T. vaginalis was detected in 17 (70.8%) secretions from the infected women. Among the 17 positive secretions, anti-parasite IgA was found in two specimens, IgE was found in three, and IgM was found in one. Of the 30 secretions, 7 (23.3%) from noninfected women also contained anti-parasite IgG. Low levels of natural anti-trichomonad IgG and IgM were detected in the sera of normal persons. Infection with T. vaginalis caused an increase in the serum IgG antibody titer. Cross-reaction between T. vaginalis and Pentatrichomonas hominis was also observed.
Topics: Animals; Cervix Uteri; Cross Reactions; Female; Humans; Immunoglobulin A; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Trichomonas; Trichomonas Vaginitis; Trichomonas vaginalis; Vagina
PubMed: 6982231
DOI: 10.1128/iai.37.3.852-857.1982