Two-step (culture and PCR) diagnostic approach for differentiation of non-T. foetus trichomonads from genitalia of virgin beef bulls in Argentina

Preputial fluids from 567 virgin Angus and Hereford bulls, 1-2 years old, were inoculated into Sutherland medium, and approximately 8.4% produced cultures with a protozoan suggestive of Tritrichomonas foetus. Under brightfield microscopy, large numbers of single-celled motile organisms with multiple anterior flagellae, a posterior flagellum, axostyle, and a visible undulating membrane were detectable. Motility was jerky and rolling, as described for T. foetus. Air-dried smears of cultures stained with Giemsa or Diff-Quick + iodine revealed an organism similar to T. foetus, although somewhat more rounded. Several organisms appeared to have four anterior flagellae. Scanning electron microscopy (5000x) of representative samples revealed four anterior flagellae on most organisms, and an axostyle that was consistently longer than that seen in T. foetus. Using pan-trichomonal primers and T. foetus-specific primers in a polymerase chain reaction (PCR) assay, amplification products of 372bp were detected in all virgin bull isolates, but only with the pan-trichomonal primers. Positive control isolates of T. foetus yielded amplification products of the expected size (372 and 347bp) with the two sets of primers, respectively. We conclude that these protozoa are not T. foetus, and note the similarity of these findings with those reported earlier in North American beef cattle. Because in several countries there is no legal treatment for bovine trichomonosis, veterinarians recommend slaughter of bulls with positive preputial cultures. The existence of easily mis-identified non-T. foetus trichomonads in the bovine prepuce suggests that the current "gold standard" diagnostic test (culture of preputial scrapings or washings) should be augmented with a more specific confirming test, such as the PCR employed in this study.     

Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.

Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity.     

Comparison of two sampling tools for diagnosis of Tritrichomonas foetus in bulls and clinical interpretation of culture results.

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.     

Diagnosis of trichomoniasis in 'virgin' bulls by culture and polymerase chain reaction

The diagnostic test for Tritrichomonas foetus in bulls is microscopic examination of cultured preputial samples. Trichomonads other than T. foetus can be present in a preputial sample. Both a staining technique and a polymerase chain reaction assay were useful in differentiating between T. foetus and another trichomonad observed in samples from virgin bulls.     

Collection of preputial material by scraping and aspiration for the diagnosis of Tritrichomonas foetus in bulls

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.     

The immune response in cattle infected with Tritrichomonas foetus

Holando-Argentina calves (males and females) were experimentally infected with Tritrichomonas foetus var. Belfast (T. foetus) by introducing 10(7) protozoa into the preputial and vaginal cavities, in order to analyse the course of the immune response to infection. Samples of serum, vaginal mucus and preputial secretion were taken periodically and assayed by means of microagglutination of living protozoa. The serum antibody titre, which averaged 32 before infection and was equivalent to titres in a non-infected group, increased to 512 in the heifers 11 weeks later and to 128 in the bulls 4 months post-infection. Agglutinating antibodies were not detected in the preputial cavity, but heifers showed antibodies in the vaginal mucus and became trichomoniasis free after 4 months. Conversely, genital secretions from the bulls gave rise to positive cultures during the whole period of experimentation. The intradermal sensitivity was checked using a soluble antigen from T. foetus. The diameter of the papula increased up to three times in heifers, while in bulls the results were no different than those from the non-infected group. Serum antibodies were of the IgG2 subclass, while those isolated from vaginal mucus were characterized as IgG1, an opsonizing antibody. Heifers were refractory to challenge infection after 1 year. The poor immune response in bulls is consistent with their role as carriers of T. foetus.     

Application of a PCR assay to enhance the detection and identification of Tritrichomonas foetus in cultured preputial samples.

The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination. Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay. The objective of this study was to evaluate a previously described PCR assay for detecting T. foetus in cultured preputial material. The detection limits of the assay for T. foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined. Preputial samples were collected from 13 bulls uninfected with T. foetus. The PCR assay was able to detect 5 T. foetus organisms in the growth medium and the cultured preputial material. Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms. The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination. This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test. In addition, the assay could be used to confirm the identification of T. foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.     

Bayesian estimation of Tritrichomonas foetus diagnostic test sensitivity and specificity in range beef bulls

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.     

Comparison of the 5.8S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity.

Sequence analysis of the 5.8S rRNA gene and the internal transcribed spacer regions (ITSRs) was used to compare trichomonadid protozoa (n = 39) of varying morphologies isolated from the bovine preputial cavity. A multiple sequence alignment was performed with bovine isolate sequences and other trichomonadid protozoa sequences available in GenBank. As a group, Tritrichomonasfoetus isolates (n = 7) had nearly complete homology. A similarity matrix showed low homology between the T. foetus isolates and other trichomonads recovered from cattle (<70%). Two clusters of trichomonads other than T. foetus were identified. Eighteen isolates comprised 1 group. These isolates shared >99% homology among themselves and with Pentatrichomonas hominis. The other non-T. foetus cluster (n = 14) did not exhibit a high degree of homology (<87%) with other bovine isolates or any of the trichomonad sequences available in GenBank. The sequence homology among isolates in that cluster was >99%, except for 1 isolate that varied from the others in both ITSRs (approximately 2% dissimilarity). Sequence analysis of the 5.8S rRNA gene and ITSRs was useful for comparing trichomonadid protozoa isolated from the bovine preputial cavity and demonstrated that 2 distinct types of trichomonads constituted the non-T. foetus isolates recovered from the bovine preputial cavity.     

Identification of trichomonadid protozoa from the bovine preputial cavity by polymerase chain reaction and restriction fragment length polymorphism typing.

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.     

Detection of trichomonad species in the reproductive tracts of breeding and virgin bulls

Trichomoniasis is a sexually transmitted disease of cattle and a large bowel diarrheal disease of cats caused by Tritrichomonas foetus. Recently, other species of trichomonads have been identified from the prepuce of virgin bulls. It is not clear whether these non-T. foetus isolates are common (nor) or is it clear whether they are also present on the prepuce of breeding bulls. To answer these questions, we first developed an immunofluorescent assay (IFA) with T. foetus-specific monoclonal antibodies for comparison with a T. foetus-specific PCR assay. Results showed that all PCR positive isolates were also IFA positive, whether the isolates were from cats or cattle and PCR negative isolates were IFA negative. Bovine non-T. foetus (non-Tf) trichomonad isolates were detected by both assays in 14 virgin bulls, 10 breeding bulls, 21 bulls of undetermined breeding status (presumably breeding bulls) and 2 cows. These isolates from virgin bulls were mostly Tetratrichomonas spp. whereas the non-Tf isolates from most breeding bulls and the two cows were Pentatrichomonas hominis. All T. foetus isolates were from breeding bulls or bulls of undetermined breeding status. This IFA test which discriminates between T. foetus and non-Tf may be useful as a diagnostic assay, since no effective legal treatment is available, bulls positive for T. foetus are culled. With increasing reports of T. foetus large bowel infection in cats, these monoclonal antibodies may also be useful for diagnosis of feline infection. Since two isolates of non-Tf trichomonads were obtained vaginas of breeding cows, it may be that these parasites are sexually transmitted like pathogenic T. foetus.     

Ultrastructural study of a tetratrichomonad species isolated from prepucial smegma of virgin bulls

We present observations on an unusual tetratrichomonad species isolated from preputial smegma of virgin bulls. Ultrastructural studies were performed using scanning and electron microscopy techniques. This protozoan presents four anterior flagella of unequal length and a recurrent one forming the undulating membrane. It shows one anterior nucleus, a Golgi complex, an axostyle, and a costa. The hydrogenosomes are rather elongated, seen in groups, and presenting different electron densities. Vacuoles of different sizes containing bacteria and material in process of digestion were frequently found. PCR was also used in order to compare the species herein described with other trichomonad species. The amplification products were seen only with primers TFR1 and TFR2 (specific to trichomonads), but not with TFR3 and TFR4 (specific to Tritrichomonas foetus), suggesting that although collected from the genital tract of the bull, this protist was not T. foetus. We propose that the appearance of these tetratrichomonads were probably due to the sodomy practiced among bulls. Concomitant contamination of preputial cavity with feces could explain the presence of the opportunistic organism. The observations presented here show the importance of the correct diagnostic when investigating samples obtained from the urogenital tract of cattle. We also suggest that this flagellate belongs to the species Tetratrichomonas buttreyi.     

Tritrichomonas foetus damages bovine oocytes in vitro

Tritrichomonas foetus is an extracellular parasite of the urogenital tract in cattle. It causes infertility and abortion, but there is no documented information on the susceptibility of bovine oocytes to the parasite, except by one article that claimed no effects of T. foetus on oocytes or embryos. The aim of the present study was to study the effects provoked by T. foetus when in interaction with bovine oocytes. Oocytes were obtained from cow ovaries and divided into two groups: (1) one group contained cumulus cells, whereas (2) a second group was denuded from these cells. Light microscopy, video microscopy and scanning electron microscopy (SEM) revealed that exposure of oocytes to T. foetus caused rapid adhesion of the trichomonads to cumulus cells and to the zona pellucida (ZP). Motile parasites were observed for 12 h. The ZP was completely damaged, and the parasites were able to infiltrate beneath the ZP and reached the oocytes directly when the oocytes were denuded of the cumulus cells. Both the oocytes and the cumulus cells exhibited morphological characteristics compatible with apoptosis after interaction with T. foetus, such as chromatin condensation, the presence of several cytoplasmic vacuoles, with intact cellular membranes and organelles. The results from this study demonstrate that when a large number of T. foetus interacts with oocytes in vitro damage and apoptosis are provoked in the cow's reproductive cells. The behavior of this parasite as one of the causes of cattle infertility is discussed.     

Efficacy of ipronidazole against trichomoniasis in beef bulls

Preputial smegma samples from 195 beef bulls were collected repeatedly and cultured for Tritrichomonas foetus. Seventy-five (38.5%) of these bulls were positive for trichomonads on at least 1 culture. Sensitivity of the culture procedure (number of positive cultures/number of total cultures from known-positive bulls) was 81.6%. Storage of preputial smegma in lactated Ringer's solution at 5 C for 24 hours resulted in a 14% loss of sensitivity. Seventy-three of the 75 infected bulls were available for treatment and were alloted randomly to 2 groups. Bulls in both groups were treated with procaine penicillin (7,000 IU/kg, IM) for 2 days before ipronidazole treatment. Thirty grams of ipronidazole powder was dissolved in 60 ml of sterile water, and was given IM to group 1 bulls. Group 2 bulls were given a similar 30-g ipronidazole solution IM on day 1, and were given 15 g of ipronidazole dissolved in 30 ml of sterile water on days 2 and 3. Efficacy of treatment (ie, negative cultures of preputial smegma for trichomonads for 6 consecutive weeks after treatment) was 92.8% for the 42 bulls treated once and 100% for the 31 bulls treated 3 times.     

Measurement of antibody in serum and genital fluids of bulls by ELISA after vaccination and challenge with Tritrichomonas foetus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to Tritrichomonas foetus using both whole cell antigen (WCA) and membrane protein antigen (MPA). The test was used to detect specific antibody in serum, preputial washings and seminal plasma samples from 7 adult bulls which were vaccinated subcutaneously on 3 occasions with a membrane protein vaccine against T. foetus var brisbane in an oil adjuvant, and from 4 unvaccinated control animals. One month after administration of the third dose of vaccine, vaccinated and control bulls were repeatedly challenged with the live vaccine strain of the T. foetus. A steady increase in serum antibody titre was detected after each inoculation of vaccine when both antigens were used in the ELISA. However, MPA was more sensitive. After challenge, vaccinated bulls developed an increased titre. No specific antibody was detected in control bulls, except in one bull after challenge in which seroconversion was detected. The serum antibody titres of both groups of animals were also measured with the microagglutination test which proved less sensitive than the ELISA. Antibody titres to both antigens, although lower than in serum, were detected in the seminal plasma of vaccinated animals. The control bulls remained non-responsive. No antibody was detected by ELISA in preputial washings from either control or vaccinated bulls prior to challenge. Post-challenge, some of the vaccinated bulls were responsive with both antigens whereas the control bulls remained negative.     

The effects of Tritrichomonas foetus and nutritional status on the fertility of cows on a community pasture in Saskatchewan.

A prospective observational study of a breeding season in a Saskatchewan community pasture was carried out to determine the cause or causes of a chronic infertility problem. There were 774 cows, from 27 herds, divided into 4 breeding groups (A,B,C,D) on the pasture. Cows entering the pasture in May were weighed, had their body condition scored and height measured. All bulls received breeding soundness examinations and a preputial wash, which was cultured for Tritrichomonas foetus and Campylobacter foetus subsp. venerealis. In July, cows were also weighed and had their body condition scored and again when they left the pasture. In addition, cows were pregnancy checked when they left the pasture. Bulls were tested again for Tritrichomonas foetus at the end of the grazing season. Two breeding groups had T. foetus-positive bulls and an average pregnancy of 84%, which was significantly lower than that of the two T. foetus negative groups (93.5%) (P = 0.0001). A cow was 2.97 times less likely to be pregnant if she had been exposed to T. foetus-positive bulls. Cows with average daily gains above the mean for the pasture were 2.12 times more likely to be pregnant. Body condition score upon entering and leaving the pasture, height, age, and breeding group were significant predictors of average daily gain.     

Ultrastructural features of Tritrichomonas mobilensis and comparison with Tritrichomonas foetus

Tritrichomonas mobilensis is an intestinal parasite of squirrel monkeys. There are few reports concerning the morphological aspects of this parasite. In addition, the taxonomic relationship between T. mobilensis and Tritrichomonas foetus, a serious pathogen that causes bovine and feline trichomonosis, has been questioned. For this reason, in the present study, we examined and compared both tritrichomonads with regard to their morphology, ultrastructure, endocytic activity and cytotoxicity when in the presence of host cells. Electron microscopy demonstrated consistent morphological differences between the hydrogenosomes of both parasites. Moreover, T. mobilensis and T. foetus had striking differences in their endocytic behavior. Thus, this work provides additional data that support the hypothesis that T. mobilensis is a distinct species from T. foetus.     

Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity

The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.     

A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls

Seven batches of 25% water-phase, oil-in-water vaccine were prepared from whole cultures of Tritrichomonas foetus. Two inoculations were given, spaced 6 weeks apart, to virgin heifers and infected bulls. A significant reduction (P less than 0.01) in the duration of infection in vaccinated heifers was seen when they were challenged by being bred to a bull infected with the same isolate as that contained in the vaccine. Only 1/12 vaccinated heifers were pregnant 4.5 months after the end of the breeding season compared to 2/12 in the control group. The vaccine, therefore, has no practical advantage. Vaccine was supplied to 2,724 bulls on properties where the infection was present. From these bulls, 110 reliable results were obtained, where bulls had been infected, been inoculated and tested 1 month later. No curative effect was demonstrable with 69/110 (62.7%) bulls, remaining infected after the course of inoculations. There was also no difference between vaccine batches or between bulls of different ages. Further work on improving the vaccine is indicated. Three media suitable for the culture of T. foetus are described in detail.     

Detection of BHV-1 in a Naturally Infected Bovine Fetus by a Nested PCR assay

Bovine herpesvirus 1 (BHV-1) is frequently associated with abortion in naturally and experimentally infected cattle. Most of the virus isolation and immunofluorescent antibody protocols described in the literature for detecting BHV-1 in bovine foetuses are rather laborious, costly and time-consuming. The detection is described of BHV-1 in the tissues of a naturally aborted bovine foetus by a nested PCR assay with no further hybridization procedures.Optimal results were achieved by filtering the foetal tissues on a chromatography column before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels.This nested PCR was faster and easier to perform than the virus isolation test. To our knowledge, this is the first time that BHV-1 has been detected in the tissues of a naturally infected bovine foetus by means of a nested PCR. The test seems to be a practical alternative for rapid detection of BHV-1 in bovine foetus.