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.     

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.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.     

The application of a transport and enrichment medium to the diagnosis of Campylobacter fetus infections in bulls

The use of a transport and enrichment medium (TEM) in the diagnosis of Campylobacter fetus infections in bulls is described. The medium significantly improved the diagnosis rate in samples which, because of the length of time between collection and receipt at the laboratory, were unsuitable for processing by direct culture. The TEM was able to support the viability, and subsequent multiplication, of C. fetus in some samples for up to 7 days before the TEM was incubated. The submission of paired samples of TEM, one containing unfiltered preputial washing (PW) and the other containing PW filtered through a 0.60 micron cellulose acetate filter, significantly increased the accuracy of diagnosis.     

The barren camel with endometritis--isolation of Trichomonas fetus and different bacteria.

This study constitutes the first reported isolation of Trichomonas fetus from the uterus of breeding camels. Twenty-four of 68 camels, which were barren for 1-4 years were found to be positive for T. fetus. One preputial washing of a camel bull which had served the herd was found to be positive for T. fetus. The uterine flora of all camels were examined. Ten different bacterial and 2 fungal species were isolated. All samples were negative for Campylobacter sp., Taylorella equigenitalis and Streptococcus zooepidemicus.     

Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium.

One hundred and five bulls from an artificial insemination unit were tested for the presence of Campylobacter fetus subspecies venerealis. The method involved the inoculation of preputial samples into a new transport enrichment medium prior to culture and immunofluorescence tests. Seventeen bulls (16%) were found to be either positive or suspected carriers of C. fetus at one or more sampling times. The average age of these 17 bulls was about two years greater than the average age of all the bulls in the unit. A combined treatment of vaccination and dihydrostreptomycin sulfate injection suppressed or eliminated the organism from carrier bulls. The use of transport enrichment medium has increased our capability and effectiveness to monitor the presence of C. fetus in artificial insemination bulls.     

Malassezia species isolated from the intermammary and preputial fossa areas of horses

BACKGROUND: Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. HYPOTHESIS: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. ANIMALS: Eleven healthy horses (5 mares and 6 geldings). METHODS: Samples of surface material were taken digitally from the intermammary area of 5 mares and the preputial fossa region of 6 geldings. The samples were examined cytologically and were cultured on modified Sabouraud's dextrose agar. The DNA from yeast colonies grown on the agar was extracted, and samples were assayed using fungal generic polymerase chain reaction (PCR) analysis. Amplicons with positive PCR results were sequenced and compared with sequences in the BLAST database search program. RESULTS: Of 44 attempts at culture, 5 yielded a species identified as Malassezia equi, and 2 yielded M slooffiae. In contrast, of 44 cytologic examinations, yeasts with the morphology of Malassezia spp. were seen in 40 samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Due to its presence in healthy horses, finding of Malassezia-type yeast on cytologic examination may not incriminate it as a pathogen. Despite difficulty in culturing, cytologic examination was an effective tool to rapidly demonstrate the organism.     

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.     

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.     

Bovine genital campylobacteriosis (vibriosis): vaccination of experimentally infected bulls

The therapeutic efficacy of a Campylobacter fetus subsp venerealis bacterin was determined in experimentally infected bulls. Ten of twelve 5-year-old Angus bulls became infected after being infused intrapreputially with C fetus subsp venerealis. Of the 10 bulls, 6 were vaccinated with 5 ml of C fetus subsp venerealis vaccine on 2 occasions 4 weeks apart. Preputial washings of the vaccinated bulls were culturally negative by the 8th week after primary vaccination. None of the 18 heifers exposed to the vaccinated bulls became infected. The 4 infected, nonvaccinated bulls remained culturally positive to C fetus (P less than 0.002), and each bull infected at least 1 heifer (P less than 0.001). Two noninfected, nonvaccinated bulls remained culturally negative and did not infect any heifer. The 4 infected, nonvaccinated bulls were then vaccinated. Two bulls remained infected 9 weeks after primary vaccination, as determined by the virgin heifer test and cultural examination of preputial washings. Serologic data from 7 sampling periods were different (P less than 0.001) for vaccinated vs nonvaccinated bulls at 4 (against K antigen) or 6 (against O antigen) weeks after primary vaccination. Vaccination was effective in eliminating the infection in most of the infected bulls, but cannot be recommended as the sole measure of control in infected herds.     

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.     

Direct preputial hernia associated with a ventral abdominal wall defect in a two‐year‐old gelding

The case of a 2‐year‐old gelding with acute onset of preputial swelling and prolapse is presented. After initiating conservative management using a penile repulsion device, the horse repeatedly displayed signs of mild abdominal discomfort with sudden deterioration to an episode of violent colic after 5 days of hospitalisation. Ultrasonographic examination of the preputial swelling at that time demonstrated the presence of small intestine between the internal and external laminae of the prepuce and led to the diagnosis of a direct preputial hernia. The contents of the hernia were readily reduced through a defect in the ventral abdominal wall after the anaesthetised horse was placed in dorsal recumbency. The historical information, clinical progression and surgical findings were supportive of an acquired ventral abdominal wall defect. To the authors' knowledge, this is the first reported case of a direct preputial hernia associated with an acquired ventral abdominal wall defect.     

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.     

What is your diagnosis? Preputial mass in a ferret

Abstract: A 7‐year‐old neutered male polecat‐type ferret (Mustela putorius furo) was presented for evaluation of a cutaneous mass close to the preputial orifice. Cytologic examination of a fine‐needle aspirate revealed numerous large clumps of amorphous pink mucinous material and numerous large clumps of slightly pleomorphic epithelial cells. The cells were arranged in papillary structures, palisades, and loosely cohesive sheets with a vaguely honeycomb appearance. Occasional acinar formations were also seen. The cells had moderate to large amounts of finely granular gray to gray–blue cytoplasm. The cells were round to wispy and elongated, with indistinct borders. Often, anuclear cytoplasmic clumps were seen free in the background or adjacent to intact cells. Nuclei were round to oval and usually off‐center. Chromatin was finely stippled and contained 1–3 indistinct nucleoli. Anisokaryosis and anisocytosis were moderate. Binucleated cells were noted occasionally. The cytologic features were consistent with a carcinoma of probable apocrine origin. Histopathologic examination supported a diagnosis of secretory apocrine adenocarcinoma of the preputial skin. Secretory apocrine adenocarcinomas of the prepuce are seen relatively frequently in ferrets, although their cytologic appearance has not been described widely. These neoplasms carry a poor prognosis although prompt surgical removal with wide and deep surgical margins and adjunctive radiotherapy may improve survival.     

Measurement of immunoglobulins in reproductive tract fluids of bulls

Seminal, seminal vesicular, urethral and preputial fluids from bulls of two different age groups were assessed for quantitative differences in immunoglobulins. Selected markers were measured in individual samples to differentiate locally derived immunoglobulins from those present as a result of trauma or secretions from other accessory glands.Immunoglobulin levels in reproductive tract fluids from older bulls (5–6 years) were higher than those of younger bulls (3–4 years) and preputial fluids contained the highest concentration of immunoglobulins of all fluids examined. Similarities existed, however, among all fluids in the relative concentrations of immunoglobulins. IgG was generally in highest concentration, though the predominant subclass varied. A marked predominance of IgG₂ over IgG₁ occurred in preputial fluid samples of older bulls. IgA was in second highest concentration, and levels were often equal to or greater than those in serum. IgM was in low concentration and occasionally undetectable. IgG/IgA ratios did not exceed 5 in most of the reproductive fluids, whereas serum ratios were usually over 100. Proportional contents of albumin and immunoglobulin in reproductive tract fluids by comparison with those in serum indicated that substantial quantities of IgG as well as IgA were synthesized locally or derived by selective transport. Increased numbers of plasma cells in the lamina propria of the preputial and penile mucosa of older bulls were correlated with higher immunoglobulin concentrations in preputial fluid from older bulls, suggesting that differences in local synthesis were responsible.     

Failure to establish infection with Tetratrichomonas sp. in the reproductive tracts of heifers and bulls

Experimental infection of the reproductive tracts of heifers and bulls with Tetratrichomonas sp. isolated from preputial smegma of virgin bulls was attempted. Nine heifers and four bulls were challenged by inoculation of 7 x 10(6) Tetratrichomonas sp. into the vaginal lumen and preputial cavity, respectively. Vaginal mucus and preputial smegma samples were collected and cultured for Tetratrichomonas sp. Heifers were slaughtered in groups of three at 2, 9 and 21 days after inoculation. Two heifers and two bulls infected with Tritrichomonas foetus and two uninfected heifers were used as controls for the model infection. Tetratrichomonas sp. were only isolated in vaginal mucus of 7/9 inoculated heifers at 6h post-inoculation, and genital secretions taken at slaughter time from vagina, uterus and oviduct were cultural negative. Bulls challenged with Tetratrichomonas sp. remained cultural negative. Since Tetratrichomonas sp. survived only a few hours in the female genitalia and did not survive in the male genitalia after experimental challenge, Tetratrichomonas sp. did not colonize the genital tract. These were likely trichomonads from the digestive tract. Collection of clean samples without fecal contamination from the reproductive tract is proposed as a measure to avoid Tetratrichomonas sp. transitory genital infection.     

Effect of sample pooling and transport conditions on the clinical sensitivity of a real-time polymerase chain reaction assay for Campylobacter fetus subsp. venerealis in preputial samples from bulls

The diagnosis of bovine genital campylobacteriosis (BGC) presents significant challenges, as traditional methods lack sensitivity when prolonged transport of samples is required. Assays of preputial samples by means of real-time polymerase chain reaction (PCR) provide good sensitivity and high throughput capabilities. However, there is limited information on the acceptable duration of transport and temperature during transport of samples. In addition, the use of pooled samples has proven to be a valuable strategy for the diagnosis of other venereal diseases in cattle. The objectives of the present study were to determine the effect of sample pooling and of transport time and temperature on the clinical sensitivity of a real-time quantitative PCR (qPCR) assay for Campylobacter fetus subsp. venerealis in preputial samples from beef bulls. Eight infected bulls and 176 virgin yearling bulls were used as the source of samples. The qPCR sensitivity was comparable for unpooled samples and pools of 5 samples, whereas sensitivity was decreased for pools of 10 samples. Sensitivity for the various pool sizes improved with repeated sampling. For shorter-term transport (2 and 48 h), sensitivity was greatest when the samples were stored at 4°C and 30°C, whereas for longer-term transport (96 h) sensitivity was greatest when the samples were stored at −20°C. The creation of pools of 5 samples is therefore a good option to decrease costs when screening bulls for BGC with the qPCR assay of direct preputial samples. Ideally the samples should be stored at 4°C and arrive at the laboratory within 48 h of collection, but when that is not possible freezing at −20°C could minimize the loss of sensitivity.     

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.     

The origin and species of yeasts in commercial preparations of bovine semen.

Whereas yeasts were not normally isolated from raw semen samples 13% of commercial frozen semen samples and 71% of preputial washings contained yeasts. Nine genera and 25 species of yeasts have been identified from these two sources. Yeasts originating in the preputial cavity were generally saprobic members of the genera Candida, Cryptococcus, Rhodotorula, Saccharomyces, Torulopsis and Trichosporon. Those originating as contaminants during processing were more likely to be opportunistic pathogens of the genus Candida. Conception was not necessarily affected by the presence of large numbers of Candida krusei or C. macedoniensis in the uterus.