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A 14-month-old intact, female Abyssinian cat was presented for chronic intermittent diarrhea and bilateral enlargement of the mammary glands. Gastrointestinal coccidiosis was diagnosed; therapy with sulfadi-methoxine was unsuccessful in the elimination of Isospora felis and clinical signs. Infection with Tritrichomonas foetus was diagnosed by fecal polymerase chain reaction (PCR) and successfully treated with ronidazole and dietary modification.

Tritrichomonas foetus was found in the uterus of a cat with pyometra and in the faeces of three other cats in the same household, one of which had chronic diarrhoea. This is the first report of a feline uterine infection with T. foetus and also the first time T. foetus has ever been diagnosed in animals in Norway. The diagnosis was made by microscopic examination and sequencing studies.

The sensitivity (Se) and specificity (Sp) of different testing schemes were estimated for detecting Tritrichomonas foetus (T. foetus) in smegma samples from experimentally infected bulls. Culture and polymerase chain reaction (PCR) on smegma samples were evaluated alone and in parallel testing. Mature dairy bulls (n=79) were intrapreputially inoculated with T. foetus (n=19); Campylobacter (C.) fetus venerealis (n=13); both T. foetus and C. fetus venerealis (n=11); Tetratrichomonas spp. (n=9); C. fetus fetus (n=8); or were not inoculated (n=19). For each bull, smegma samples were collected for 6 week post-inoculation and tested for T. foetus by In Pouch TF culture and PCR. Most T. foetus-inoculated bulls became infected, according to culture (86.7%), PCR (90.0%), and both tests together (93.3%). In T. foetus-inoculated bulls, both tests combined in parallel on a single sample had a Se (78.3%) and Sp (98.5%) similar to two cultures (Se 76.0%, Sp 98.5%) or two PCR (Se 78.0%, Sp 96.7%) sampled on consecutive weeks. The PCR on three consecutive weekly samples (Se 85.0%, Sp 95.4%) and both tests applied in parallel on three consecutive weekly samples (Se 87.5%, Sp 95.6%) were similar to the current gold-standard of six weekly cultures (Se 86.7% and Sp 97.5%). Both tests used in parallel six times had the highest Se (93.3%), with similar Sp (92.5%). Tetratrichomonas spp. were only sporadically detected by culture or PCR. In conclusion, we have proposed alternative strategies for T. foetus diagnostics (for the AI industry), including a combination of tests and repeat testing strategies that may reduce time and cost for bull surveillance.

Accuracy of culture for diagnosis of Tritrichomonas foetus was investigated in 2832 naturally exposed range beef bulls from 124 herds. Preputial fluid samples were inoculated into the culture medium, incubated at 37 degrees C, and daily examined. Diagnostic test was evaluated using Bayesian techniques to estimate sensitivity and specificity without a gold standard. Median posterior test sensitivity was 72.04% (95% probability interval: 58.07-86.38%) and specificity was 95.37% (95% probability interval: 94.07-96.65%). Low diagnostic test accuracy may have resulted from host and/or diagnostic test procedure related factors. Under natural range conditions, more accurate methods for T. foetus diagnostic and repeated preputial samplings of bulls may be necessary on trichomonosis control programs.

A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log(10) dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n=159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P<0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples.

Tritrichomonas foetus is a venereal disease of cattle that has a clear pattern of adverse reproductive sequela in the affected female, with a carrier state in the bull in which he shows no outward signs. Given sexual rest and symptomatic treatment, the female returns to reproductive viability. In contrast, the male remains infective and is a risk to other female contacts. Prevention of the disease relies on excluding infected males and females from the population of susceptible cattle. Control of this disease requires a plan to identify, isolate, and treat infected females, and eliminate or cull infected males. Improvements in culture technique and the application of newer methods, such as polymerase chain reaction, enhance the likelihood of detecting infected animals.

Tritrichomonosis is a widespread, economically important venereal disease caused by Tritrichomonas foetus. The traditional diagnosis of this disease, which causes infertility and abortion in cattle, is based on the culture of the parasite. This process is time consuming, has low sensitivity, and is prone to contamination with intestinal or coprophilic trichomonadid protozoa, resulting in false positive diagnostics of T. foetus. In order to avoid the shortcomings of the traditional method, we developed a simple PCR assay based on TFR3 and TFR4 primers, which does not require parasite culturing. The sensitivity of the PCR assay resulted comparable to that of the classical method, being able to detect as few as five T. foetus parasites. In addition the method is highly specific. The analysis of preputial fluid washing samples showed that 58 out of 203 samples were positive by both, the PCR and the culture method (+/+), 9 samples were positive by PCR and negative by the traditional method (+/-) and only one sample resulted negative by PCR and negative by culture (-/+). The samples for the PCR assay can be stored for a week at 4 degrees C or 72h at room temperature. In summary, our study demonstrated that the PCR assay is an effective method for the diagnosis of T. foetus from preputial samples, and that it compares advantageously to the classical method.

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.

A rapid, reliable polymerase chain reaction (PCR) assay, originally developed for definitive laboratory identification of the bovine venereal pathogen Tritrichomonas foetus from cultures of male reproductive tract fluids, was used for testing the following: 1) cultured, geographically disparate trichomonad isolates, 2) formalin-fixed tissues from infected heifers and naturally infected fetuses, and 3) cervicovaginal mucus (CVM) from experimentally infected females. In 12 of 12 Western Hemisphere isolates of pathogenic T. foetus (isolated from outbreaks of clinical trichomoniasis or from screening surveys) and in 1 of 1 American Type Culture Collection strain of Tritrichomonas suis, PCR yielded a positive result, i.e., a 347-base pair amplicon in the 5.8S ribosomal RNA and internal transcribed spacer (5.8S-ITS) region of the genome, whereas cultures of Trichomonas vaginalis and Trichomonas gallinae did not produce a PCR product. The PCR assay was also positive in formalin-fixed, paraffin-embedded endometrial samples from 4 of 4 experimentally infected heifers, as well as in archived tissues from 2 of 2 T. foetus-infected aborted bovine fetuses that were submitted to the diagnostic laboratory from a natural outbreak. It was negative in fixed, embedded uterine tissues of 2 of 2 uninfected virgin heifers used as negative controls and in archived fixed gut tissue of a T. gallinae-infected pigeon. In another experiment, CVM aspirated from 4 of 4 experimentally infected heifers in the fifth or sixth postinfection week yielded a positive PCR product of the expected size, whereas CVM from 2 of 2 controls were PCR negative. Pending validation in larger clinical studies, the PCR assay for the 5.8S-ITS coding region of the T. foetus genome offers the prospect of definitive identification of this agent directly from CVM or from formalin-fixed tissues or when false-positive culture results are suspected.

The objectives of this study were to determine the detection limit of a PCR test for Tritrichomonas foetus, to investigate the effect of sampling method, guanidinium thiocyanate (GuSCN), and sample storage, and to confirm the accuracy of the test on field samples. Serial 10-fold dilutions of culture material were used to determine the detection limit. For the sample handling trial, five positive bulls were sampled by sheath washing and scraping on six occasions over a period of 18 days (n=29 samples) and eight control bulls were sampled three, four or six times (n=28 samples). Samples were cultured, while portions with and without GuSCN were subjected to DNA extraction within 6h, after 30 h and after 5 days at 4 degrees C. PCR and agarose gel electrophoresis was performed. A two-tailed chi-square test was used to test for differences between treatments. The PCR assay showed a specificity of 98%. Its sensitivity declined with storage time, from 90% at 6h to 31% at 5 days. Sampling method and GuSCN had no effect on test sensitivity. The detection limit of the assay was 100 organisms. Parallel testing of 193 field samples gave complete agreement between culture and PCR results.

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.

The current diagnostic test for Tritrichomonas foetus involves the culture of collected preputial or vaginal samples. In an earlier study, which evaluated sampling tools for use with bulls, it was observed that the sensitivity of the diagnostic test was higher for 2nd samples collected from the right side of the prepuce than it was for samples collected 1st from the left side. The study described in this paper was conducted to evaluate which of these factors was responsible for the effect on diagnostic sensitivity. Twenty-nine bulls infected with T. foetus were repeatedly sampled in a 2-factor cross-over design. Samples taken from the right side of the prepuce were 4 times as likely to be positive as samples taken from the left side (P = 0.03). Other factors did not have a significant effect on the outcome of the diagnostic test. Unexpected factors may affect the sensitivity of the diagnostic test for T. foetus.

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).

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.

Two trials were carried out to assess the diagnostic sensitivity and practicability of preputial scraping as a method of collecting preputial material from bulls infected with Tritrichomonas foetus. In the 1st trial, preputial material was collected by simultaneous scraping and aspiration from 3 infected and 1 uninfected bull 10 times over a 5-week period. In the 2nd trial, samples from 5 infected bulls were collected by both sheath washing and scraping on 6 occasions, while 8 uninfected animals were sampled 3 times. Samples were cultured using a modified Trichomonas culture medium (Oxoid). In the first trial, 29 of 30 samples from infected bulls were found to be positive. In the second trial, 83 % of samples collected by both methods tested positive. In neither trial were any samples from the control bulls found to be positive. Scraping was found to be quick and safe, and offered advantages over preputial washing in that urine contamination was easily avoided, samples were smaller and more concentrated and contamination was reduced. It may, however, be subject to greater operator variability than sheath washing. It is concluded that preputial scraping is as effective as washing and represents a suitable alternative for the collection of material for direct examination and culture of Tritrichomonas foetus.

An improved polymerase chain reaction test has been developed to detect Tritrichomonas foetus, the causative agent of trichomoniasis in cattle. The test amplifies a region of the 5.8S ribosomal RNA gene of T. foetus, and it is simple, sensitive, and specific when compared with traditional methods to examine field samples.

The growth rates of 16 isolates of Tritrichomonas foetus from three distinct geographic regions were investigated in modified Diamond's medium, liver infusion broth medium and a commercially available culture kit. While some differences in growth characteristics were detected for different isolates and in the three different media, all isolates grew. Trichomonads reached peak concentrations from an initial concentration of 10(4) trichomonads/ml on Days 2, 3 and 4 in modified Diamond's medium, on Days 2-6 (excluding CAPTF102) in the commercial culture kit and on Days 2-7 in liver infusion broth medium. Viable parasites were detectable for longer periods in liver infusion broth medium and the commercial culture kit than in Diamond's medium. Peak concentrations for isolates tended to be higher in modified Diamond's medium than in liver infusion broth medium or the commercial culture kit. Results show that these three media are suitable for the growth of all 16 T. foetus isolates from three continents and suggest that these media could be used effectively throughout the world.

Objective: To compare sensitivity for diagnosing Tritrichomonas foetus infection in bulls using 2 sampling tools and to calculate negative predictive values for infection., Design: Randomized clinical trial., Animals: 30 Bos taurus bulls naturally or experimentally infected with T foetus., Procedure: Preputial scrapings were obtained once/wk for 6 weeks using an artificial insemination pipette and a metal brush; which tool was used first for each bull was randomly determined. Samples were collected first from the left side of the prepuce and then from the right side and placed in commercially available transport media chi 2 Values and confidence limits were adjusted for effect of clustering of results by bull., Results: Significant differences in sensitivity of results were not found between samples collected using the brush or pipette. Using the pipette, sensitivity was estimated to be 91.6% (95% confidence interval, 84.3 to 95.7%); negative predictive values ranged from 41 to 99% for prevalence of infection of 90 to 5%, respectively. Sensitivity was 88.8% for first sample obtained and 96.1% for second sample obtained., Conclusions and Clinical Relevance: Collection of preputial scrapings with an artificial insemination pipette or a metal brush and use of a commercially available culture system can provide a sensitive diagnostic test for T foetus infection in bulls. Calculated negative predictive values indicated that 1 or 2 tests would suffice in most clinical situations. For bulls from herds in which T foetus is endemic, 2 to 4 tests/bull may be required to ensure that each bull is not infected.

Objective: To compare sensitivity of a generic trypticase-yeast extract-maltose (TYM) medium versus a commercial nutrient medium in the diagnosis of Tritrichomonas foetus infection in heifers and to assess sensitivity when incubation of samples inoculated into commercial medium pouches is delayed overnight., Design: Prospective study., Animals: 30 virgin beef heifers., Procedures: 20 heifers vaccinated with a trichomonad antigen and 10 unvaccinated control heifers were exposed at synchronized estrus by intravaginal instillation of 10(6) T foetus organisms. Cervicovaginal mucus samples were collected every other week for 10 weeks from controls and once (10 weeks after exposure) from vaccinated heifers. Samples were inoculated into both media and immediately incubated at 37 C (98.6 F). A duplicate inoculation from controls was made into commercial medium, and the pouch was shipped overnight to a diagnostic laboratory without prior incubation., Results: For 40 of 50 samples from control heifers, there was agreement on diagnoses between media. There was agreement on a positive diagnosis for 3 of 20 samples from vaccinated heifers and on a negative diagnosis for 15 of these 20 samples. For samples shipped overnight before incubation, there were 10% fewer positive diagnoses, compared with samples incubated immediately in commercial medium and 10% more positive diagnoses, compared with samples immediately incubated in TYM., Clinical Implications: Use of the commercial medium is a more sensitive indicator of current infection in heifers than use of generic TYM medium. In herds where infection prevalence is high, this method is likely to identify more infected females, an important consideration when control programs include culling of infected cows.

The purpose of this study was to investigate the prevalence of Tritrichomonas foetus infection in beef bulls in north-western Spain. The study area comprised of 350 herds (5909 cows and 102 bulls) managed under extensive or semi-extensive systems where natural mating alone or alternated with artificial insemination are used. The targets of this survey were bulls of 1 year of age or older. Preputial smegma samples were taken from 70 bulls (68.6% of bull population) servicing a total of 184 herds (52.6%) and 4136 cows (69.9%). Data collected at sampling included farm location, herd size, age, breed, private or communal bull and previous infertility problems in the herd. The percentage of infected bulls was 2.9% (2 of 70). Age of infected bulls was 5 and 7 years and both were of the local breed, Asturiana de los Valles. These results confirm the presence of T. foetus infection in Spain and the necessity to include this disease in the differential diagnosis of reproductive failure in rangeland grazing cattle.

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.

We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds.

Bovine trichomoniasis is a prevalent sexually transmitted disease of cattle caused by the protozoan Tritrichomonas foetus. Currently, diagnosis is most often made by culture. In order to provide a faster immunodiagnostic approach, a specific enzyme-linked immunosorbent assay (ELISA) was investigated. A protective surface antigen (TF1.17 antigen) of T. foetus was immunoaffinity purified and used in an ELISA to detect antibodies in vaginal mucus from heifers inoculated with T. foetus. In preliminary studies, antibodies of the immunoglobulin A (IgA) isotype were detected in mucus from all experimentally infected heifers which were tested at 6 weeks postinoculation, whereas IgG1 and IgG2 were not. In addition, IgA responses detected in postinoculation samples were all greater than those detected in preinoculation samples, unlike those detected by a whole-cell antigen ELISA. For these two reasons, IgA antibodies appeared to be useful diagnostically. Further investigation of IgA antibodies used vaginal mucus collected weekly from heifers inoculated intravaginally with 10(2), 10(4), or 10(6) T. foetus organisms. Heifers with positive cultures for T. foetus had similar IgA responses to TF1.17 antigen over the 10 weeks of infection regardless of the initial inoculum dose. This indicates that if the dose is sufficient to establish infection, the magnitude and duration of the immune response are no longer dependent on dose. All of the infected animals receiving all dosages responded with high absorbance values in the IgA anti-TF1.17 antigen ELISA by 6 weeks postinoculation, and all absorbance values remained high at 10 weeks. To determine the duration of the IgA response, four other heifers inoculated with 7 x 10(6) T. foetus organisms were studied through 24 weeks postinoculation. IgA antibody responses to TF1.17 antigen were still high at 24 weeks, even though some heifers cleared the infection several weeks earlier. These results indicate that experimentally infected heifers produced detectable long-lasting IgA responses to TF1.17 antigen in vaginal mucus, which may be useful in a herd test for the diagnosis of bovine trichomoniasis.

The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus.

Sampling methods for preputial secretions and culture methods for Tritrichomonas foetus were compared in a two-by-two factorial experiment. Fourteen bulls were confirmed as infected by repeated weekly sampling and culture on Diamond's medium over the course of 10 months. In a six-week test of alternative methods of diagnosis, the bulls were randomly assigned to have their preputial contents sampled weekly either by washing or by scraping of the preputial membrane. The samples were cultured on Claussen's medium and in InPouch packets at 37 degrees C in air for seven days. The results were compared by McNemar's chi 2 test for symmetry. Washing yielded 69 positive cultures from 84 samples whereas scraping yielded 65 from 83. These differences were not statistically significant. InPouch culture detected 73 of 83 samples from positive bulls, whereas Claussen's medium detected 61 of 83. The difference in sensitivity between the two culture methods was highly significant (P = 0.0027), with the InPouch method 'missing' two positive samples that were detected by Claussen's medium and Claussen's medium missing 14 positive samples that were detected by InPouch culture.

Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.

Bovine trichomoniasis is a sexually transmitted disease caused by the flagellated protozoan Tritrichomonas foetus. A protective surface antigen was previously identified and immunoaffinity purified from T. foetus isolate D1 with cross-reactive monoclonal antibodies (MAbs) TF1.15 and TF1.17 (BonDurant, R. H., R. R. Corbeil, and L. B. Corbeil, Infect. Immun. 61:1385-1394, 1993). This antigen elicited antibody responses in the serum and cervicovaginal mucus of heifers. Thus, it may be useful as an immunodiagnostic reagent as well as a subunit vaccine. Conservation of the antigen in all strains would be crucial for either application. We investigated the conservation of this antigen among 36 isolates of T. foetus from Argentina, Costa Rica, and the United States using MAbs TF1.15 and TF1.17 in an enzyme-linked immunosorbent assay. MAb TF1.17 reacted with 32 of the 36 isolates, whereas MAb TF1.15 reacted with all of the isolates tested. One of the isolates which did not react with MAb TF1.17 (i.e., D1#3) was investigated further by Western blotting (immunoblotting) to determine the reason for the lack of reactivity with one of the two cross-reactive MAbs. The antigenic band that was reactive with MAb TF1.15 had a molecular mass slightly lower than that of the corresponding band from isolate D1, which reacted with both MAbs TF1.15 and TF1.17. Thus, at least a major portion of the antigen appeared to be conserved. This was confirmed in a study of heifers infected with isolate D1#3. The vaginal immunoglobulin A antibodies of these infected heifers reacted with the antigen of isolate D1 that was immunoaffinity purified with MAb TF1.17. Therefore, even though the epitope recognized by MAb TF1.17 was missing in the challenge isolate (D1#3), the heifers developed an immune response to the rest of the molecule. These results indicate that the major portion of the previously described protective antigen is conserved in different isolates of T. foetus. This portion contains the epitope that reacts with MAb TF1.15. Most isolates express the whole antigen, which possesses both TFl.15 and TF1.17 epitopes, but the few isolates that are missing the portion containing the TF1.17 epitope may still elicit an immune reponse to the conserved portion. Thus, the protective surface antigen is promising for use in immunodiagnosis or vaccination against bovine trichomoniasis.