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.     

Detection of Leishmania (L.) chagasi in canine skin

Tritrichomonas foetus is a serious veterinary pathogen that causes bovine trichomonosis, a sexually transmitted disease that eventually leads to abortion and infertility. T. foetus has a simple life cycle that consists of only a trophozoitic form. During unfavorable environmental conditions, the trophozoites, which are polar and flagellated, can adopt a spherical shape and internalize their flagella. These rounded organisms are known as pseudocysts. Although it is currently assumed that T. foetus pseudocyst formation is reversible and that it represents a response to stressful conditions, there are no reports showing the presence of this form in vivo. For this reason, the aim of this study was to verify whether T. foetus pseudocysts are encountered in naturally infected bulls. Towards this goal, fresh preputial samples obtained from seven mature bulls that were naturally infected with T. foetus were analyzed using complementary techniques, such as video microscopy, fluorescence microscopy, scanning and transmission electron microscopy. The analyses revealed that approximately 55% of the parasites were in pseudocyst form in each preputial sample, whereas approximately 25% of T. foetus displayed pear-shaped bodies. Previous research demonstrated that in vitro T. foetus pseudocysts are able to divide by a budding process. Here, this division mode was observed in approximately 20% of fresh T. foetus obtained from preputial bovine samples. Thus, this study shows that in infected bulls, pseudocysts are present and occur more frequently than the pear-shaped parasites.     

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.     

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.     

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.     

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.     

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.     

An improved molecular assay for Tritrichomonas foetus

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.     

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.     

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.     

Use of DNA amplification for the rapid identification of Mycobacterium bovis

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.     

PCR detection of Tritrichomonas foetus in preputial bull fluid without prior DNA isolation

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.     

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.     

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.     

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.     

Detection of Tritrichomonas foetus by polymerase chain reaction in cultured isolates, cervicovaginal mucus, and formalin-fixed tissues from infected heifers and fetuses.

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.     

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.     

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.     

应用多重PCR同时检测牛、羊源性成分

我们选择了3对引物A,B,C.A可扩增大多数脊椎动物(哺乳类、鸟类、爬行类、两栖类和鱼类)细胞色素b基因上一个371 bp的区段,B可扩增位于牛mtDNAD-loop区段内的一个274 bp的区段,C可扩增羊mtDNA基因上一个199bp的区段.由于3对引物扩增的产物分别相差约100 bp左右,可通过琼脂糖凝胶电泳分离来并观察结果,建立了基于内对照的同时检测牛、羊源性成分的三重PCR方法.     

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.