Abstract
Objective—To determine the sensitivity of a real-time PCR assay for the detection of Tritrichomonas foetus in protozoal cultures of preputial scraping samples pooled from up to 25 bulls and to determine the specificity of that assay for detection of T foetus in cultures for individual animals.
Design—Cross-sectional study.
Animals—188 bulls and 150 steers.
Procedures—Preputial scraping samples were collected, placed in a culture kit, and incubated at 37°C for 7 days. Cultures for individual animals were tested for T foetus by means of a real-time PCR assay. Pools of protozoal cultures were made by including fixed aliquots of samples with known positive and negative results in ratios of 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25. Specificities of the real-time PCR assay and culture for detection of T foetus in samples obtained from individual animals and sensitivity of real-time PCR assay for each evaluated pool ratio were determined.
Results—Specificity estimates for culture and the real-time PCR assay for detection of T foetus in preputial scraping samples for individual animals were not significantly different (98.8% and 100%, respectively). Sensitivities of the real-time PCR assay for the various pooled samples with known positive and negative T foetus results were not significantly different; overall sensitivity of the assay was 94%.
Conclusions and Clinical Relevance—Results indicated the evaluated real-time PCR assay had high specificity and good sensitivity for the detection of T foetus in pooled protozoal cultures of preputial scraping samples obtained from up to 25 animals.
Reproductive performance of animals is one of the most important factors affecting the economic success of cow-calf operations.1–3 Early pregnancy loss and failure to conceive are the primary causes of poor reproductive performance of cows.1 Trichomoniasis is a venereal disease of cattle caused by Tritrichomonas foetus; this disease is commonly identified in herds with prolonged calving seasons and low pregnancy4,5 and calving6 rates. Trichomoniasis has a substantial effect on the profitability of cow-calf operations, with an estimated loss of 5% to 35% return/cow that undergoes breeding.7
Methods for the control of trichomoniasis typically include identification and subsequent elimination of infected carrier bulls from a herd.8 Identification of carrier bulls is commonly accomplished by means of collection of preputial scraping samples, protozoal culture, and microscopic identification. Although protozoal culture is the gold-standard method, the sensitivity of that method for identification of T foetus is variable (range, 67.8% to 98.9%),8–13 depending on factors such as sample collection variables, transport conditions, and culture method. Polymerase chain reaction assays have been developed to replace14 or complement15 culture-based methods for detection of T foetus.
Polymerase chain reaction assays (both conventional and real-time methods) have a lower detection limit than culture methods for T foetus.14 This has led to the use of testing strategies such as the analysis of pooled preputial scraping samples. Results of other studies16,a indicate that the sensitivities of conventional and real-time PCR assays of pooled preputial scraping samples obtained from groups of 5 bulls (1 sample with positive results included in each pool) for detection of T foetus are 100%. However, there is a need for additional information regarding the analysis of pooled samples from a larger number of animals to optimize this testing method. Given the economic consequences of culling a healthy bull from a herd, further studies regarding the potential for false-positive real-time PCR assay results are warranted. The objectives of the study reported here were to determine the specificity of a real-time PCR assay for the detection of T foetus in preputial scraping samples collected by use of a commercially available culture kit and the sensitivity of a real-time PCR assay for detection of that organism after preputial scraping sample collection, protozoal culture, and pooling of culture media for groups of samples obtained from up to 25 bulls.
Materials and Methods
Animals—Preputial scraping samples were collected during June 2010 from 150 steers housed at the University of Saskatchewan Beef Research and Teaching Unit and during March and April 2011 from 187 virgin bulls housed at bull stations (Maple Creek and Spring Creek) of the Agri-Environment Services Branch of Agriculture and Agri-Food Canada. One bull with known positive results for T foetus was also included in the study; this bull was housed at the Western College of Veterinary Medicine Animal Care Unit. The breed and approximate age of each animal were recorded. Animal procedures were performed in accordance with recommendations of the Canadian Council on Animal Care17 and approved by the University of Saskatchewan Protocol Review Committee.
Specificity of a real-time PCR assay for detection of T foetus in protozoal cultures of preputial scraping samples of individual animals—Preputial scraping samples were collected from animals by means of the aspiration method.9,18 Two preputial scraping samples were collected from each virgin bull; 1 sample collected from each of those animals was used for the purposes of the present study. Collection and use of the samples obtained first or second for this study was alternated for each bull (determined by the order in which animals entered the chute) and recorded. One preputial scraping sample was collected from each steer.
Briefly, an individually wrapped 25-inch plastic pipetteb attached to a 20-mL syringe was inserted into the prepuce and the plastic sheath was pulled back. The scraping was performed by moving the pipette back and forth 10 times during application of suction (syringe volume, 15 mL). Then, the pipette was withdrawn and rinsed into the upper chamber of a self-contained T foetus culture pouch.c Pouches were closed in accordance with manufacturer instructions and transported to the laboratory in a warm (approx 25°C) cooler within 4 to 7 hours after collection.
Preputial scraping samples were tested by means of 2 methods: real-time PCR assay after enrichment in culture and culture with microscopic examination. Culture pouches were placed upright in an aerobic incubator at 37°C (day 0) and incubated for 7 days. All pouches were examined by means of bright-field microscopy (100×) by scanning along the seam of the pouch starting 1 cm from the bottom on one side, continuing to and along the bottom, and up the other side to approximately 1 cm from the bottom of the pouch.5 Microscopic examination of pouches was performed on days 1, 3, 5, and 7 of incubation and the results recorded.
On day 3, a 500-μL aliquot was collected from each culture pouch and submitted for performance of real-time PCR assay at a commercial diagnostic laboratory.d The DNA extraction was performed for a 200-μL aliquot by use of a commercial DNA extraction kite and an automated extraction system.f The assay used was a 5′ Taq nuclease real-time PCR assay with minor groove binder-DNA probes and was performed as previously described.14 Results were considered positive for samples with a Ct value < 40. Further identification of organisms in samples with visible T foetus–like organisms but with negative real-time PCR assay results was performed by means of a conventional PCR assay as previously described,19 with minor modifications. Briefly, reactions were performed in a volume of 50 μL at 94°C for 3 minutes followed by 40 cycles of 94°C for 30 seconds, annealing at 58°C for 20 seconds, and extension at 72°C for 30 seconds. A final extension step of 10 minutes at 72°C was used. The PCR assay products were separated and identified by means of electrophoresis in a 2.5% agarose gel.
Sensitivity of a real-time PCR assay for detection of T foetus in pooled culture medium for preputial scraping samples of multiple animals—Preputial scraping samples collected from steers were used as samples with known negative results for T foetus. After protozoal culture of those samples for 7 days, the contents of each pouch were transferred to 4-mL cryovials and stored at −80°C. Preputial scraping samples were collected from a bull that was naturally infected with T foetus (confirmed on the basis of multiple positive culture and PCR assay results); these were used as samples with known positive results. Preputial samples were collected from that bull once a week for 12 weeks; protozoal culture and microscopic examination were performed for those 2 samples by use of the same methods that were used for samples obtained from other animals. Cultured samples collected from the infected bull were withdrawn from incubation at different times (days 0 to 5) and subsequently tested by real-time PCR assay.
Pools of protozoal culture media were made by mixing fixed aliquots of individual protozoal cultures of preputial scraping samples that had known positive and negative results (known results determined by means of culture and real-time PCR assay). The evaluated pool ratios indicate the number of protozoal cultures with positive results for T foetus to the total number of protozoal cultures included in the pool and were 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25 (total volume of each mixture, 500 μL). The concentrations of organisms in culture pouches of preputial scraping samples collected from the bull with known positive results were determined with real-time PCR assays and distributed to ensure that all ratios contained equal numbers of pools with positive samples ranging from high (107 organisms/mL) to low concentration (103 organisms/mL) samples. Thirty-one pooled samples were prepared for each pool ratio and tested for detection of T foetus by means of real-time PCR assay.
Data and statistical analysis—Crude specificity estimates for real-time PCR assay and culture were determined by calculating the proportion of nonbreeding animals (virgin bulls and steers) that had negative results for each of those testing methods. The 95% CIs were calculated with the Wilson score method.20 Those animals had never been exposed to breeding and were assumed to have true-negative results for T foetus infection. Exact logistic regressiong was used to evaluate potential differences in the specificity of the 2 tests. The animal category (steer or bull) was added as a covariate variable in the model. First-order interactions with test type were evaluated and retained in the model if results were significant (P < 0.05). The order of preputial scraping sample collection (first or second scraping) was added to the model and retained if results were significant (P < 0.05). The difference in prevalence of non–T foetus trichomonads between bulls and steers was evaluated with a Fisher exact test.h
Crude sensitivity estimates of real-time PCR assays for each pool ratio were determined by calculating the proportion of the total number of replicates for each pool ratio for which results were positive for T foetus. The 95% CIs were calculated with the Wilson score method.20 Generalized estimating equationsi were used to compare the sensitivity estimates among pool ratios (after accounting for repeated use of the same sample in multiple pools) and to determine whether sensitivity varied by replicate, order of preputial sample collection, number of days a sample with positive results was incubated, and concentration of organisms in a sample with positive results. Variables other than pool ratio were retained in the model if results were significant (P < 0.05) or if they were confounding variables. Factors were considered to be confounding variables if removing or adding the factor from the model changed the regression coefficient for another risk factor of interest by > 10%.21 Analysis of differences in Ct values among pool ratios was performed with a linear mixed model,j after accounting for repeated use of the same sample in multiple pools. Plots of standardized residuals versus predicted values were used to identify extreme outlier values. A commercially available statistical software package was used for all analyses.k Values of P < 0.05 were considered significant.
Results
Specificity of the real-time PCR assay for detection of T foetus in protozoal cultures of preputial scraping samples of individual animals—Of the 187 virgin bulls included in the study, 108 (58%) were 1 year old and 44 (24%) were 2 years old; the age for 35 (19%) bulls was unknown (these bulls were ≤ 2 years old). Breeds included Angus (n = 129 [69%]), Charolais (54 [29%]), Limousin (2 [1%]), and Simmental (2 [1%]). The tested preputial scraping samples were obtained during the first scraping for 92 (49%) bulls and during the second scraping for 95 (51%) bulls. Steers were crossbred animals (age, 14 to 15 months); all tested preputial samples for steers were obtained during the first scraping.
Contamination, determined by observation of distension of culture pouches with gas, was detected for 48 of 337 (14%) protozoal cultures of preputial scraping samples. All preputial scraping samples had negative real-time PCR assay results for T foetus. Four of 337 (1.2%) preputial scraping samples had positive results for non–T foetus trichomonads on the basis of microscopic observation of culture pouches; these were considered false-positive results for T foetus. Such false-positive results were determined on day 5 for 3 steers and day 7 for 1 bull. These organisms had a round shape, an undulating membrane, and ≥ 4 anterior flagella (observed by means of microscopic examination [400×] with phase contrast). Non–T foetus trichomonad organisms were successfully identified by means of conventional PCR assay for only 2 of the 4 preputial scraping samples with false-positive results. Results for one of those protozoal cultures indicated a 142–base pair product consistent with Pentatrichomonas hominis. Results for another culture indicated a product of 200 to 300 base pairs that had not been previously reported (data not shown). The prevalence of non–T foetus trichomonads was not significantly (P = 0.22) different between bulls (1/187 [0.5%]) and steers (3/150 [2.0%]).
No significant (P = 0.94) difference was detected between the specificity of culture (98.8%; 95% CI, 97.0% to 99.5%) and that of real-time PCR assay (100%; 95% CI, 98.9% to 100%) for detection of T foetus. No significant (P = 0.25) difference was detected regarding the specificity of those tests between steers and bulls.
Sensitivity of the real-time PCR assay for detection of T foetus in pooled culture media for preputial scraping samples of multiple animals—No significant (P = 0.70) differences were detected among real-time PCR assay sensitivity estimates for pool ratios of protozoal cultures with known positive and negative results in this study. These included pool ratios 1:10 (sensitivity, 90.3%; 95% CI, 75.1% to 96.6%); 1:2, 1:15, 1:20, and 1:25 (sensitivity, 93.5%; 95% CI, 79.3% to 98.2%); and 1:3 and 1:5 (sensitivity, 96.8%; 95% CI, 83.8% to 99.4%).
The overall sensitivity of the real-time PCR assay for detection of T foetus was 94% (95% CI, 90.0% to 96.5%). However, the sensitivity was significantly (P < 0.01) associated with the concentration of T foetus in protozoal cultures of preputial scraping samples with known positive results included in each pool. For each log increase in the concentration of organisms, the odds of a positive test result increased 3.7 times (95% CI, 2.3 to 6.1 times). The number of days a protozoal culture of a preputial scraping sample with known positive results had been incubated was also significantly (P < 0.01) associated with the real-time PCR assay sensitivity. For each additional day a sample was incubated, the odds of a negative result increased by 9.8 times (95% CI, 3.9 to 24.7 times). The mean real-time PCR assay Ct values were not significantly (P = 0.052) different among pool ratios (Table 1).
Means and 95% CIs of real-time PCR assay Ct value estimates for various ratios of pooled protozoal cultures of preputial scraping samples collected from a bull with known positive results for Tritrichomonas foetus and steers with known negative results for that organism.
| Pool ratio | Mean Ct | 95% CI |
|---|---|---|
| 1:2 (n = 29) | 30.8 | 29.3–32.3 |
| 1:3 (n = 30) | 30.1 | 28.7–31.6 |
| 1:5 (n = 30) | 30.8 | 29.3–32.3 |
| 1:10 (n = 28) | 31.3 | 29.8–32.9 |
| 1:15 (n = 29) | 32.4 | 30.9–33.9 |
| 1:20 (n = 29) | 32.6 | 31.1–34.1 |
| 1:25 (n = 29) | 33.1 | 31.6–34.6 |
The number of replicates for each pool are indicated.
Pooled samples for 13 animals had negative T foetus real-time PCR assay results for all evaluated pool ratios; 9 of these samples contained the same sample with known positive results. Visual assessment of the culture pouch of that sample with known positive results revealed evidence of contamination, including change in color of the media to green and accumulation of a large amount of gas. In addition, pools that included that sample with known positive results had significantly (P < 0.01) higher Ct values (39.0; 95% CI, 35.7 to 42.3) than pools that included another sample with known positive results (31.4; 95% CI, 30.8 to 31.9).
Discussion
The results of the present study indicated that a real-time PCR assay for detection of T foetus in preputial scraping samples was highly specific (100%), and results were comparable to those obtained by means of microscopic examination of protozoal culture pouches (specificity, 98.8%). In addition, the use of a real-time PCR assay for testing of pooled T foetus cultures of preputial scraping samples obtained from up to 25 bulls seemed to have high sensitivity.
Culture methods used for the detection of T foetus carrier bulls have typically been assumed to have 100% specificity.22 However, results of other studies15,23 indicate trichomonads that are morphologically similar to T foetus may be detected in preputial scraping samples. The origin of these nonpathogenic organisms is believed to be feces, and the presence of such organisms in preputial scraping samples is likely the result of sodomy and riding activity of bulls, especially for young animals.24,25 Results of another study15 indicate the prevalence of non–T foetus trichomonads in preputial scraping samples obtained from virgin bulls is 8.4%. That prevalence is higher than the prevalence of such organisms determined in the present study. This difference in results could have been attributable to differences in the type of culture media used in each study, use of individually wrapped pipettes in the present study (which may have decreased the chance of fecal contamination), or geographic variability in prevalence of such organisms in animals.
To the author's knowledge, the present study is the first in which non–T foetus trichomonads were identified in preputial scraping samples obtained from steers. No significant difference was detected in the prevalence of false-positive culture results between steers and virgin bulls in this study. The use of steers may be useful for studies of trichomoniasis, in particular for determination of test specificity. However, further research is warranted to validate such methods by means of anatomic and histologic examination of the prepuce and penis of bulls and steers. Future studies are warranted to determine whether steers can acquire T foetus infection and become carriers of that organism.
Proposed methods for decreasing the risk of false-positive results (determined by means of culture) include staining of culture samples26 and PCR assay.15,27 Although real-time PCR assays have high sensitivities, such assays may yield a higher number of false-positive results than do conventional PCR assays; nonviable organisms or extremely low numbers of viable organisms may be detected by use of PCR assays with high analytic sensitivity.28 However, 5′ Taq nuclease real-time PCR assays with minor groove binder-DNA probes have higher specificity than conventional PCR assays for detection of T foetus in bovine diagnostic samples.14
In another study,5 the use of protozoal culture, conventional PCR assay, and real-time PCR assay were compared for detection of T foetus in preputial scraping samples obtained from bulls in a herd in Nebraska. The authors of that study suggested that false-positive results were more common with real-time PCR assay versus other methods; they considered conventional PCR assay and culture to be the best methods for detection of T foetus. Those findings were not supported by results of the present study. The bulls used in that other study5 originated from a herd that included animals infected with T foetus; therefore, results may not have been falsely positive. In the present study, the animals used in experiments for determination of assay specificity had no previous exposure to breeding; we assumed that such animals did not have T foetus. Results of another studyl also indicate that real-time PCR assay is specific for detection of that organism; diagnostic samples with high real-time PCR assay Ct values have true-positive results for T foetus as determined by means of product sequencing.l
The real-time PCR assay results of the present study regarding pooled culture media for preputial scraping samples obtained from up to 25 animals were similar to results for pooled preputial samples obtained from groups of 5 animals in other studies.16,a In addition, results of the present study indicated no significant differences in sensitivity among various pool ratios for groups of samples obtained from up to 25 animals. In this study, samples with known positive results that had various concentrations of T foetus were evaluated; this method was used to determine the potential effects of bulls with various severities of infection on the testing of pooled culture media for preputial scraping samples. A long sample incubation time (up to 5 days) resulted in a decreased chance of identification of a preputial scraping sample with T foetus in a pool. However, this finding should be interpreted cautiously because the source of preputial scraping samples with known positive results for T foetus was a single bull, and differences in results for various strains of organisms were not evaluated.
Most of the pools of culture media for preputial scraping samples with negative T foetus real-time PCR assay results contained the same sample with known positive results, regardless of the pool ratio. The media in the culture pouch of this sample turned green, the pouch contained a large amount of gas, and the culture had the lowest concentration of organisms (5.5 × 103 organisms/mL) of any protozoal culture of preputial scraping samples in the study. The finding that this sample was contaminated suggested that PCR inhibitors might have affected the performance of the PCR assay. Several substances commonly found in preputial fluid are potential PCR inhibitors, including hemoglobin in blood, urea in urine, and various substances in feces. Although the mechanism of such inhibition is poorly understood, such substances typically decrease the analytic sensitivities of PCR assays.29 Considering that characteristics of each sample included in a pool could alter the result of a PCR assay (depending on the concentration of PCR inhibitors), further research is warranted regarding testing of pooled preputial scraping samples by means of PCR assay without prior enrichment of organisms in culture. We advise that T foetus cultures of preputial scraping samples with evidence of contamination should not be included in pools for testing; such samples should be individually tested.
Results of the present study suggested that real-time PCR assays may be used to detect T foetus in pooled protozoal culture media for a large number of preputial scraping samples in a short time with low cost. Such methods may have lower possibility of artifactual results versus testing methods that require a greater amount of sample manipulation. Results indicated a real-time PCR assay was highly specific for detection of T foetus in protozoal cultures of preputial scraping samples obtained from bulls, and testing of pooled samples obtained from up to 25 bulls seemed to be a sensitive method, provided samples were collected and cultures were individually incubated before pooling.
ABBREVIATIONS
| CI | Confidence interval |
| Ct | Cycle threshold |
Naikare HK, Hampton LD, Reinisch AJ, et al. Evaluation of a real-time PCR assay of pooled specimens for the detection of Tritrichomonas foetus infection (abstr), in Proceedings. 51st Annu Meet Am Assoc Vet Lab Diagn 2008;62.
Continental Plastics, Cambridge, ON, Canada.
InPouch TF, Biomed Diagnostics, San José, Calif.
Prairie Diagnostic Services, Saskatoon, SK, Canada.
Biosprint 96 One-for-all Vet kit, Qiagen, Mississauga, ON, Canada.
MagMAX Express-96 Standard Magnetic Particle Processor, Applied Biosystems, Life Technologies Inc, Burlington, ON, Canada.
PROC LOGISTIC, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC FREQ, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC GENMOD, SAS, version 9.2, SAS Institute Inc, Cary, NC.
PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
Leyva Baca I, Simunich M, Peddireddi L, et al. Sequence confirmation of Tritrichomonas foetus samples previously tested with MagMAX sample preparation system and VetMAX T. foetus reagents (abstr), in Proceedings. 55th Annu Meet Am Assoc Vet Lab Diagn 2012;105.
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