Introduction Trichomonads were first reported as a cause of bovine infer- tility in France in 1888.1 In the US, it was first noted in 1932 in Pennsylvania dairy cows2 and in 1958 in western US beef herds.3 Currently, T. foetus has been practically eliminated from intensively managed cattle populations around the world where the management includes limited commingling of cattle and artificial insemination is commonly practiced. However, it remains endemic in some herds in the western US managed under range conditions with natural service.1 Trichomoniasis exerts its most prominent impacts in a cattle operation through its negative effects on herd reproductive performance, resulting in fewer pregnant cows and consequently fewer calves for sale. Additional factors related to trichomoniasis that exert a negative influence on the profitability of a beef cattle operation include feed and other maintenance costs for nonproductive cows, replacement costs of T. foetus-infected bulls and nonproductive females, testing costs to control T. foetus, and reduced weaning weights due to late-born calves.4,5 It is difficult to estimate the prevalence of bovine trichomo- niasis with any degree of confidence. Estimates in California6 and Florida7 had a herd-level prevalence of 15.8 and 28.8%, respectively, whereas bull-level prevalence was 4.1 and 6.0%, respectively. In Colorado and Nebraska abattoirs, only 0.172% of bulls’ samples were culture positive for T. foetus.8 Recent preva- lence estimates of infected bulls in Alabama9 and Tennessee10 were 0.27 and 0.18%, respectively, with a prevalence estimate of 2.17% for Wyoming herds.11 Although the prevalence of bovine trichomoniasis is typically Review of bovine trichomoniasis Jeff Ondrak 6U Ranch, Steele City, NE Abstract Bovine trichomoniasis, caused by the protozoa Tritrichomonas foetus, results in reproductive failure and substantial financial losses. It was first reported in the US in 1932 and in the western beef herds in the US in 1958. Western herds, along with herds in certain areas of Florida, are considered the traditional range for this disease in the US. Since characteristics of the causative organism are well known, its control and even eradication are possible. Background information of T. foetus as it relates to etiology, pathogenesis, diagnosis, and control is provided. Keywords: Tritrichomonas foetus, cattle, trichomoniasis lower in nonendemic areas, its potential for substantial economic loss from this disease makes it a concern for the cattle industry. The following narrative summarizes the basics in bovine trichomoniasis regarding its etiology, pathogenesis, diagnosis, and control. Etiology Tritrichomonas foetus is a spindle- to pear-shaped single-cell protozoa with 3 anterior flagella. It has an undulating membrane along the length of its body containing an accessory filament at its margin and a single posterior flagellum. The organism ranges from 9 - 25 μm in length and 3 -15 μm in width, and replicates through asexual longitudinal binary fission.12 Although the life cycle of this protozoa does not include a cyst form, it can develop into a pseudocyst.13 The organism has an affinity for the bovine reproductive tract due to its limited oxygen concen- trations, ample food supply in host’s red and white blood cells, and bacteria. Hence it is considered an obligate parasite of the bovine reproductive tract.12 In addition, Tritrichomonas foetus is capable of surviving temperatures used to store frozen semen for artificial insemination.14 Pathogenesis Transmission Natural transmission of T. foetus is considered strictly venereal and occurs during coitus.15 The rate of natural transmission is high, with many naïve susceptible females becoming infected after a single exposure through coitus to an infected bull with Clinical Theriogenology 2021; 13: 226 as few as 200 T. foetus organisms.16 Bulls became chronically infected after 104 T. foetus organisms were experimentally inoculated into prepuce and some bulls became infected from inoculates containing as few as 102 organisms.17 Impact in the female Tritrichomonas foetus can be isolated from the female bovine reproductive tract as early as 4 days after introduction.18 However, it does not interfere with conception or maternal recognition of pregnancy19 nor induce any macro- or microscopic lesions in the reproductive tract until 50 days of pregnancy, which are characterized by mild inflammatory changes and eventual fetal loss in a majority of the infected females up to 95 days after exposure.20 Early fetal death and abortion are followed by early return to estrus in females, the most common clinical sign. Fetal loss is followed by 2 - 6 months of infertility, as the immune system cleared the parasite from the reproductive tract.19 This immune response led to a limited amnestic response to repeated T. foetus infection as demonstrated in multiple studies, with the longest estimated duration of partially protective immunity being < 15 months.21 With some notable exceptions (namely pyometra and chronic infection) complete clearance of T. foetus from the female reproductive tract typically occurs in 5 - 20 weeks.18 Pyometra may be an earliest clinical sign of T. foetus infection in a cow herd;22 in affected cows, purulent debris in the uterine lumen frequently had large numbers of T. foetus.2 Cows were infective for as long as 300 days23 or 22 months postbreeding.24 Carrier cows had infection through normal pregnancy with T. foetus isolated up to 9 weeks25 or 63 - 97 days26 after delivery of an apparently normal calf. Impact in the male Tritrichomonas foetus is primarily an inhabitant of the superficial layers of the penile and preputial epithelium, but not routinely present in other locations of the male reproductive tract.27,28 The absence of tissue invasion may be the reason for a limited immune response in T. foetus-infected bulls.29 Lack of pathological changes and failure of the immune response to eliminate T. foetus from the preputial cavity leads to chronic infection in older bulls. This relationship between age and T. foetus carrier bull status may be due to development of crypts in penile and preputial epithelium.12 However, a more recent study disputes this longstanding dogma.30 Other unknown individual specific factors may have a role in development of a T. foetus infection and carrier bull status. In summary, the absence of macro and microscopic patho- logical changes and a limited immunological response to T. foetus infection in bulls result in a lack of clinical signs in infected bulls and development of inapparent, chronically infected bulls. Herd-level impact Clinical signs in a herd are the culmination of individual responses. Consequently, there are increases in: number of nonpregnant cows, incidence of pyometra and abortion, and delayed expected calving dates. Percent nonpregnant cows increased substantially (57%31 and 45.3%32) with subsequent decreases in annual calf crop. A computer model predicted a 20 - 40% prevalence of T. foetus infection in bulls would lead to a 14 - 50% reduction in annual calf crop.4 Additionally, a second common herd-based clinical sign is a prolonged calving interval. Cows exposed to T. foetus infected bulls had calving intervals of 96.5 and 98.9 days longer than nonexposed cows during the first and second years of herd infection, respectively.21 Herd records would specifically identify this herd-based clinical sign, or it may be recognized due to an increased number of cows calving late in the calving season or lower weaning weights due to younger calves at weaning. Diagnosis One of the main aspects of controlling bovine trichomoniasis is identifying infected bulls. The following discussion on the diagnosis of bovine trichomoniasis will focus exclusively on the diagnostic process in a bull. Sample collection A modified version of a glass vaginal pipette and rubber bulb technique used in earlier studies to collect samples from female cattle was equally effective for aspirating samples from bull preputial cavities.33 This device has been adapted to utilize a plastic infusion pipette, typically 45 - 53 cm in length and 0.497 - 0.571 cm in diameter, with a 12 or 20 ml disposable syringe. General collection technique includes the following: long hairs at the preputial orifice are clipped short to reduce contamination of the sampling device. A syringe is attached to an infusion pipette and the free end of the pipette is passed into the prepuce. When the free end of the pipette reaches the fornix of the prepuce, suction is applied with the syringe. While maintaining suction with the syringe, the pipette is moved forward and backward over the surface of the penis and prepuce several times to scrape smegma into the pipette. Prior to removing the pipette from prepuce, the negative pressure in the syringe is gently released to avoid pulling sample into the syringe. After this is accomplished, the pipette is removed, and sample examined. A faint pink color in the sample is acceptable as the mucous membrane of the prepuce is mildly abraded during the collection process. However, overly aggressive scraping can lead to bleeding which may reduce the quality of the sample. If the sample is of adequate quality and volume, it is transferred to the appropriate Clinical Theriogenology 2021; 13: 227 transport container. If a larger sample is needed, the pipette is reintroduced into the prepuce following the guidelines listed in the previous steps to collect additional smegma. After an adequate sample is recovered, it should be immediately placed in an appropriate medium for transport and testing. Maintenance of the sample Maintenance of T. foetus preputial specimens has been an impor- tant preanalytical concern for trichomoniasis diagnostic testing since the early days of investigation. Specimens not subjected to direct examination were frequently collected and maintained in a solution whose purpose was to preserve T. foetus viability until they could be inoculated into laboratory media for incubation or placed directly into the laboratory incubator. However, the use of various solutions, broths and media strictly for specimen transport has been eliminated in the US following availability of direct inoculation into a growth medium at the collection location. This commercial transport and enrichment medium has a plastic cultivation envelope containing a proprietary medium (InPouch™TF, Biomed Diagnostics, Inc., White City, OR). Inoculated samples should be protected from extreme temperatures, as cold or heat may influence ability to detect T. foetus.34,35 Culture The InPouch™TF is examined in a wet mount fashion,36 by fixing the lower portion of the pouch in a plastic clip (size of a microscope slide and provided by the manufacturer) and placing the clip on a compound microscope. Systematic scanning of the pouch for motile organisms morphologically consistent with T. foetus should be done daily for several minutes and repeated for 6 consecutive days before declaring the specimen negative.37 Using bright field microscopy (100 - 400 x) to examine samples collected with InPouch™TF from virgin bulls, trichomonads were observed with morphological and motility characteristics consistent with T. foetus. Further testing of the trichomonads through staining, scanning electron microscopy, and PCR revealed that the organisms had 4 anterior flagellae, similar to lower-bowel commensal trichomonads (Tetratrichomonas pavlovi or Tetratrichomonas buttreyi). Perhaps these organisms were transferred to the prepuce in feces during sodomy.38 Therefore, culture specificity is no longer regarded as 100%. Polymerase chain reaction To overcome concerns with T. foetus culture sensitivity and specifi- city, investigators examined the value of PCR as a trichomoniasis diagnostic assay, based on the assumption that amplification of DNA segments specific to T. foetus would reduce or eliminate false positives. Furthermore, testing specificity is increased by reducing false negatives, as the test is not influenced by number of organisms.39 Control The specific steps to a trichomoniasis control program should be guided by the T. foetus infection status of the herd, the local prevalence of trichomoniasis, the herd owner’s aversion to the risk of a disease incursion and balancing the cost and benefits of the various aspects of a trichomoniasis control program. Infected herds Based on the information provided above, recommendations for T. foetus elimination from infected herds include the following. Sample and test all herd bulls 3 times, regardless of the test used, and cull all test positive bulls or all herd bulls in positive herd. This limits the time and money spent on testing while eliminates any risk of misidentifying a bull as T. foetus-negative that would allow the organism to remain in the herd. However, this option carries a financial burden that may be unacceptable to the herd owner. Cull all nonproductive females (i.e. those nonpregnant at the end of the breeding season or those that fail to deliver a live calf prior to the next breeding season) or establish 2 distinct female management groups based on their potential for T. foetus infection, with virgin heifers and cows delivering live calves in 1 group and nonproductive cows in the other. This option involves the risk of maintaining T. foetus in the herd through nonproductive cows and requires fastidious management, including absolute isolation of nonproductive cows from all other cattle and utilization of artificial insemination or bulls exclusive to this group. Consider vaccinating all females with an approved trichomo- niasis vaccine (TrichGuard®, Boehringer Ingelheim Vetmedica, Inc., Ingelheim, Germany). This will not prevent infection; however, it may reduce fetal losses associated with infection and the period of infection.40,41 Finally, implement prevention strategies for high-risk herds (refer below). High-risk uninfected herds Herds are considered high risk for the introduction of T. foetus based on a relatively high local prevalence of the disease and utilization of management practices that increase the risk of introduction. Suggested strategies for prevention of T. foetus in high-risk herds include the following. Plan a pasture utilization program to minimize contact with neighboring cattle. Utilize proper artificial insemination protocols with semen from a reputable source in specific management groups or eliminate the entire herd to greatly reduce the risk of T. foetus transmission. Maintain a young bull battery to reduce the rate of transmission and potential development of chronic carrier bulls. Isolate and/or test cattle if unplanned commingling with Clinical Theriogenology 2021; 13: 228 neighboring herds has occurred. Females should be isolated from the rest of the herd until after the breeding season and their pregnancy status can be confirmed. Bulls should be isolated and tested (3 tests at weekly intervals) to ensure that they are T. foetus negative. Restrict the length of the breeding season to < 120 days to reduce the opportunity for transmission of the disease within the herd and to more easily monitor reproductive performance. Institute a surveillance testing program. Surveillance testing should occur between the end of a breeding season and the beginning of the next and include a single test of all bulls in the herd. The advantage of surveillance testing performed closely after the breeding season is early detection of infection that allows time to develop and implement a complete trichomoniasis control program before the next breeding season. Bulls tested under this program must not be exposed to cows prior to the next breeding season. Alternatively, test all bulls immediately before the breeding season. This the advantage of timing the test to coincide with an annual breeding soundness examina- tion, thereby reducing the number of times bulls are handled. However, this may not allow sufficient time for appropriate management of the disease before the start of the breeding season. Finally, implement prevention strategies for low-risk herds, as described below. Low-risk uninfected herds Herds are considered low risk for the introduction of T. foetus based on a low local prevalence of the disease and the utilization of management practices that reduce the risk of the introduc- tion of trichomoniasis into the herd. Suggested strategies for prevention of T. foetus in low-risk herds include the following. Develop communication networks with neighbors to ensure rapid notification if trichomoniasis is diagnosed in a neighbo- ring herd. Monitor fences and cattle to rapidly identify when unplanned commingling with another herd has occurred so immediate steps can be taken to address the problem and reduce the risk of introducing trichomoniasis. Maintain herd records to monitor herd reproductive performance and identify animals within management groups for early detection of a potential trichomoniasis incursion and efficient management of the outbreak. Observe inter- and intra-state animal health regulations regarding trichomoniasis and other diseases. Regulations to protect the livestock industries of each state should be considered as a barrier to trichomoniasis introduction and not as a complete prevention program at the herd level. Purchase replacement animals, preferably virgin bulls and heifers, from a reputable source. Purchase of nonvirgin bulls and cows, especially, from herds with unknown reproductive performance, increases the risk of trichomoniasis introduction. Accepted risks in purchasing nonvirgin replacements may be greatly reduced by having a source that has excellent herd reproductive performance with bulls tested negative or by purchasing pregnant cows. Conclusion Bovine trichomoniasis has plagued the US cattle population since the 1930’s and continues to inflict reproductive loss and subsequently financial loss in the industry. Understanding the characteristics of this disease associated with its etiology, pathogenesis, diagnosis provides a logical and practical approach to control of the causative organism, Tritrichomonas foetus. Conflict of interest None to report. References 1. Skirrow SZ, BonDurant RH: Bovine trichomoniasis. Vet Bull 1988;58:591-603. 2. Emmerson MA: Trichomoniasis in cattle. J Am Vet Med Assoc 1932;81:636-640. 3. Fitzgerald PR, Johnson AE, Thorne J, et al: Trichomoniasis in range cattle. Vet Med 1958;53:249-252. 4. Rae DO: Impact of trichomoniasis on the cow-calf producer’s profitability. J Am Vet Med Assoc 1989;194:771-775. 5. Villarroel A, Carpenter TE, BonDurant RH: Development of a simulation model to evaluate the effect of vaccination against Tritrichomonas foetus on reproductive efficiency in beef herds. J Am Vet Med Assoc 2004;65:770-775. 6. BonDurant RH, Anderson ML, Blanchard P, et al: Prevalence of trichomoniasis among California beef herds. J Am Vet Med Assoc 1990;196:1590-1593. 7. Rae DO, Crews JE, Greiner EC, et al: Epidemiology of Tritrichomonas foetus in beef bull populations in Florida. Theriogenology 2004;61:605-618. 8. Grotelueschen DM, Cheney J, Hudson DB, et al: Bovine tricho- moniasis: results of a slaughter survey in Colorado and Nebraska. Theriogenology 1994;42:165-171. 9. Rodning SP, Wolfe DF, Carson RL, et al: Prevalence of Tritrichomonas foetus in several subpopulations of Alabama beef bulls. Theriogenology 2008;69:212-217. 10. Jones BM, Whitlock BK, Strickland LG, et al: Prevalence of Tritrichomonas foetus in Tennessee beef bulls. Clinical Theriogenology 2015;7:333. 11. Yao C, Bardsley KD, Litzman EA, et al: Tritrichomonas foetus infection in beef bull populations in Wyoming. J Bacteriol Parasitol 2011;2:117. 12. BonDurant RH, Honiberg BM: Trichomonads of veterinary importance. In: Kreier JP: editor. Parasitic Protozoa. 9th edition, San Diego; Academia Press: 1994. p. 112-188. 13. Granger BL, Warwood SJ, Benchimol M, et al: Transient invagination of flagella by Tritrichomonas foetus. Parasitol Res 2000;86:699-709. 14. Clark BL, White MB, Banfield JC: Diagnosis of Trichomonas foetus infection in bulls. Aust Vet J 1971;47:181-183. 15. Bartlett DE: Trichomonas foetus infection and bovine reproduction. Clinical Theriogenology 2021; 13: 229 J Am Vet Med Assoc 1947;8:343-352. 16. Clark BL, Dufty JH, Parsonson IM. Studies on the transmission of Tritrichomonas foetus. Aust Vet J 1977;53:170-172. 17. Clark BL, Parsonson IM, Dufty JH: Experimental infection of bulls with Tritrichomonas foetus. Aust Vet J 1974;50:189-191. 18. Murname D: Field and laboratory observations on trichomoniasis of dairy cattle in Victoria. Aust Vet J 1959;35:80-83. 19. BonDurant RH: Diagnosis, treatment and control of bovine tricho- moniasis. Compend Contin Educ Pract Vet 1985;7:179-186. 20. Parsonson IM, Clark BL, Dufty JH: Early pathogenesis and patho- logy of Tritrichomonas foetus infection in virgin heifers. J Comp Path 1976;86:59-66. 21. Clark BL, Dufty JH, Parsonson IM: The effect of Tritrichomonas foetus infection on calving rates in beef cattle. Aust Vet J 1983;60:71-74. 22. Rae DO, Crew JE. Tritrichomonas foetus. Vet Clin North Am Food Anim Pract 2006;22:595-611. 23. Mancebo OA, Russo AM, Carabajal LL, et al: Persistence of Tritrichomonas foetus in naturally infected cows and heifers in Argentina. Vet Parasitol 1995;59:7-11. 24. Alexander GI: An outbreak of bovine trichomoniasis in Queensland and its control. Aust Vet J 1953;29:61-66. 25. Skirrow SZ: Identification of trichomonad-carrier cows. J Am Vet Med Assoc 1987;191:553-554. 26. Goodger WJ, Skirrow SZ: Epidemiologic and economic analysis of an unusually long epizootic of trichomoniasis in a large California dairy herd. J Am Vet Med Assoc 1986;189:772-776. 27. Parsonson IM, Clark BL, Dufty J: The pathogenesis of Tritrichomonas foetus infection in the bull. Aust Vet J 1974;50:421-423. 28. Rhyan JC, Wilson KL, Wagner B, et al: Demonstration of Tritrichomonas foetus in the external genitalia and of specific anti- bodies in preputial secretions of naturally infected bulls. Vet Pathol 1999;36:406-411. 29. Soto P, Parma AE: The immune response in cattle infected with Tritrichomonas foetus. Vet Parasitol 1989;33:343-348. 30. Strickland L, Edmondson M, Maxwell H, et al: Surface architectural anatomy of the penile and preputial epithelium of bulls. Clinical Theriogenology 2014;6:445-451. 31. Barling KS, Field RW, Snowden KF, et al: Acute trichomoniasis and sub-optimal fertility in a cow/calf herd: an investigation and case management. Bov Pract 2005;39:1-5. 32. Alstad AD, Krogh D, Fischer K et al: Trichomoniasis in a beef herd. Vet Med 1984;79:708-709. 33. Hammond DM, Bartlett DE: Establishment of infection with Trichomonas foetus in bulls by experimental exposure. Am J Vet Res 1943;4:61-65. 34. Clavijo A, Erol E, Sneed L, et al: The influence of temperature and simulated transport conditions of diagnostic samples on real-time polymerase chain reaction for detection of Tritrichomonas foetus DNA. J Vet Diagn Invest 2011;23:982-985. 35. Bryan LA, Campbell JR, Gajadhar AA: Effects of temperature on the survival of Tritrichomonas foetus in transport, Diamond’s and InPouch TF media. Vet Rec 1999;144:227-232. 36. Cobo ER, Favetto PH, Lane VM, et al: Sensitivity and specificity of culture and PCR of smegma samples of bulls experimentally infect with Tritrichomonas foetus. Theriogenology 2007;68:853-860. 37. Parker S, Campbell J, Gajadhar A: Comparison of the diagnostic sensitivity of a commercially available culture kit and a diagnostic culture test using Diamond’s media for diagnosing Tritrichomonas foetus in bulls. J Vet Diagn Invest 2003;15:460-465. 38. BonDurant RH, Gajadhar A, Campero CM, et al: Preliminary characterization of a Tritrichomonas foetus-like protozoan isolated from preputial smegma of virgin bulls. Bov Pract 1999;33:124-127. 39. Morgan UM, Thompson RCA, Smith HV, et al: Molecular detection of parasitic protozoa. Parasitology 1998;117:73-85. 40. Kvasnicka WG, Taylor REL, Huang JC, et al: Investigations of the incidence of bovine trichomonasis in Nevada and of the efficacy of immunizing cattle with vaccines containing Tritrichomonas foetus. Therio 1989;31:963-971. 41. Hall MR, Kvasnicka WG, Hanks D, et al: Improved control of tricho- moniasis with Trichomonas foetus vaccine. Agri Pract 1993;14:29-34. Clinical Theriogenology 2021; 13: 230 Review of bovine trichomoniasis