2009: Bovine richomoniasis: a review Bovine richomoniasis: a review 1 L. Strickland 2 Department of Clinical Sciences, College of Veterinary Medicine, Auburn 3 University, Auburn, AL, USA 4 5 Abstract 6 Trichomoniasis is a bovine venereal disease that causes substantial economic 7 losses. Bulls serve as asymptomatic carriers for the protozoan Tritrichomonas foetus, 8 whereas infection in females may result in early embryonic death, abortion, pyometra, 9 fetal maceration, or infertility, all of which negatively influence the profitability of a 10 cattle operation. When allowed adequate recovery time following infection most females 11 mount an immune response and return to normal reproductive status. However, the male 12 can remain infected and remain a risk to a producer’s breeding program. Currently no 13 legal treatment for this disease exists in the United States therefore veterinarians and 14 cattle producers must focus on preventive management and surveillance measures such as 15 testing, identification and removal of positive animals. Understanding the pathogenesis, 16 prevalence, economic impact, and diagnosis of trichomoniasis will assist with 17 implementation of appropriate prevention and control programs. This paper reviews the 18 pathogenesis, prevalence, economic impact, and diagnosis of trichomoniasis in cattle, as 19 well as common guidelines for the prevention and control of trichomoniasis. 20 Keywords: Trichomoniasis, Tritrichomonas foetus, epithelial crypts. 21 22 23 289 Introduction 24 The bovine venereal disease trichomoniasis is caused by the protozoan 25 Tritrichomonas foetus (T. foetus). Bulls serve as asymptomatic carriers when the 26 organism colonizes the epithelium of the penis or prepuce with no clinical signs. 27 Following coitus or artificial insemination with contaminated semen females develop 28 uterine infections that may lead to early embryonic death, abortion, fetal maceration, 29 pyometra or infertility. This manuscript reviews the pathophysiology of bovine 30 trichomoniasis as well as common guidelines for diagnosis 31 Venereal Disease 32 T. foetus is an obligate parasite of the bovine reproductive tract. Similar to most 33 venereal diseases, the male is an asymptomatic carrier while the female suffers 34 identifiable consequences of infection. T. foetus in bulls localizes in the smegma 35 (secretions) of the epithelial lining of the penis, prepuce, and distal urethra.1 The 36 organism does not invade the epithelium, and therefore does not invoke an immune 37 response in the bull.2 T .foetus causes no penile or preputial lesions and does not affect 38 libido.3,4 There are no observable changes in semen quality attributable to the organism’s 39 presence, however in a recent study by Benchimol, et al, exposure to T. foetus resulted in 40 decreased spermatozoal motility, agglutination of sperm cells, and eventual 41 phagocytosis.5-7 The only clinical sign that may be observed in an infected bull is a mild 42 transient preputial discharge during the first two weeks of an infection.4 43 Infection in young bulls (less than 3-4 years of age) is purportedly most often 44 transient, with disease transmission only occurring if sexual contact with a non-infected 45 cow occurs within minutes to days following breeding of an infected cow.8,9 Studies by 46 290 Morgan8 and Clark9 indicate that clearance of the organism in a young bull is possible 47 within 20 minutes following breeding an infected cow. Transmission of T. foetus by a 48 young bull is therefore likely to be a passive, mechanical transmission that differs from 49 transmission associated with a chronically infected older bull. 50 T. foetus infection in the cow occurs during coitus with an infected bull. The 51 organism transverses the cervix and colonizes the entire reproductive tract within 1-2 52 weeks,10 and as the organism multiplies in the uterus it can cause death of the embryo or 53 fetus, most commonly between gestational days 15 to 80.11 Pyometra and abortion are 54 often the first physical signs of trichomoniasis noticed in a herd, but these signs occur in 55 fewer than 5% of infected animals.12 Infertility due to embryonic death is the most 56 economically damaging clinical sign and occurs in a larger percentage of infected cows. 57 An affected cow’s interestrus interval is usually prolonged because the embryonic loss 58 typically occurs after maternal recognition of pregnancy (days 15-17 of gestation).10 59 Unlike the bull, the cow typically mounts an effective immune response to T. foetus,4 but 60 the time it takes to clear T. foetus from the cow’s reproductive tract is quite variable. 61 Primary infections may be cleared from the reproductive tract in as little as 95 days13 or 62 as long as 22 months.8 Subsequent infections are cleared in about 20 days, indicating an 63 anamnestic response.9 Immunity does not persist, however, and the anamnestic response 64 is only significant if re-infection occurs within about 15 months of the primary 65 infection.14,15 A cow in a herd with a long breeding season could therefore become 66 pregnant and infected with T. foetus early in the breeding season, lose that embryo, be 67 infertile for several months, clear the initial T. foetus infection, rebreed, conceive, and 68 carry a calf to term as a result of temporary immunity. The result is that more cows will 69 291 calve later in the calving season than desired, and there is a resultant wide variety in 70 weaning weights rather than just a reduced calving percentage. The later-born calves are 71 then marketed at lighter weights, or the cattle producer will incur increased feeding costs 72 to achieve a desired market weight. In either case the cattle producer will sustain 73 substantial economic losses. 74 Economic aspects 75 Economic losses due to venereal disease result from culling and replacement of 76 infected animals, a decreased percentage of cows calving or calving later than desired 77 with subsequent reduced calf crop and lower weaning weight caused by failure or delay 78 of conception.14 Fitzgerald, et al16 estimated in 1958 that each infected bull in a large 79 herd was responsible for an $800 loss per year. Wilson, et al17 estimated a $2.5 million 80 annual calf loss in 1979 due to trichomoniasis in Oklahoma replacement heifers. In 1986, 81 Fitzgerald estimated that the total economic impact in the USA was $65 million 82 annually.18 In a 1991 study, Speer, et al estimated that annual losses could reach near 83 $650 million.19 Recently the state of Louisiana estimates that current losses exceed $100 84 million for that state alone, so the economic loss is likely considerably greater than earlier 85 studies.20 86 During the 1990’s the Idaho legislature approved statutes that prescribe 87 regulations for identifying and eliminating T. foetus bulls within the state and for 88 importation of bulls into that state.21 Since that time other states have adopted similar 89 legislation and currently the states of Nebraska, North Dakota, South Dakota, Montana, 90 Wyoming, Idaho, Washington, Oregon and Utah require bulls be test negative for 91 trichomoniasis before being transported into the state, sold, or used on public land.22-29 92 292 The Texas Animal Health Commission has recently passed similar requirements30. These 93 regulations reflect the growing concern for control of this bovine venereal disease which 94 is so economically important in the United States. 95 Diagnosis of Bovine Trichomoniasis 96 Diagnosis of T. foetus has traditionally relied upon microscopic identification of 97 key morphological characteristics in preputial smegma or cervicovaginal mucus (CVM) 98 incubated in various culture media. Such characteristics include three anterior flagella, 99 one posterior flagellum, and an undulating membrane resulting in a jerky movement 100 pattern. However, accurate microscopic identification of T. foetus can be complicated by 101 the presence of other trichomonadid protozoa.31-35 Contamination of the preputial orifice, 102 prepuce, or penis with fecal material probably explains the presence of these 103 opportunistic trichomonads. Several non-pathogenic protozoa are normal inhabitants of 104 the bovine gastrointestinal tract,36-38 and therefore proper cleaning of the preputial orifice 105 and proper sampling techniques are critical to avoid fecal contamination of diagnostic 106 samples. None of the contaminating trichomonads, however, results in reproductive 107 pathology in cows or bulls.39 Therefore, research has recently focused on molecular-108 based assays to accurately differentiate T. foetus33,40-42 from other trichomonads. Given 109 the lack of legal therapy for bulls infected with T. foetus in the United States the only 110 reasonable course of action is to slaughter an infected bull. It is therefore imperative to 111 correctly identify T. foetus-infected bulls and not misdiagnose based on the presence of 112 non-pathogenic fecal trichomonads. 113 At present, molecular-based assays are most commonly used as confirmatory tests 114 for bovine trichomoniasis because of the relatively low cost of in vitro cultivation 115 293 compared to molecular-based assays. However, molecular-based assays are currently 116 very effective in diagnosing human trichomoniasis caused by Trichomonas vaginalis, 117 with a sensitivity of 95% and a specificity of 98%.43 It is therefore very likely that in the 118 future the preferred diagnostic test for bovine trichomoniasis will be a molecular-based 119 assay, and some researchers have already advocated their use as an independent 120 diagnostic test for bovine trichomoniasis.44,45 121 Sampling techniques for detection of trichomoniasis in the male 122 Several sampling techniques are utilized for obtaining diagnostic specimens in the 123 bull including: 1) a swab technique;46 2) a dry pipette technique;9,47 3) a wet pipette 124 technique;48 and 4) the douche technique.48 Fitzgerald, et al compared the swab and 125 pipette techniques and reported that the number of parasites recovered via the swab 126 technique is only 20% of the number of parasites recovered via pipette scraping.49 The 127 swab technique is therefore rarely used in the United States. The dry pipette technique is 128 one of the most common sampling methods in the U SA, while the douche method is the 129 preferred technique in Europe.47 Schönmann, et al reported that the two methods are not 130 statistically different.47 131 Regardless of technique used, it is generally recommended that bulls be sexually 132 rested 1-2 weeks before testing for T. foetus; otherwise, false-negative results are more 133 likely because breeding mechanically removes many of the organisms from a bull’s penis 134 and prepuce. Given the sensitivity of T. foetus cultures, false-negative results are also 135 possible even if a bull has been sexually rested. Only with three negative tests at weekly 136 intervals (Figure 1) can a veterinarian or producer be 99% sure that a bull is T. foetus 137 negative.50 138 294 139 Figure 1. Sensitivity (in series) of T. foetus cultures.50 140 Sampling techniques for diagnosis of trichomoniasis in the female 141 Researchers investigating diagnostic sampling methodologies for T. foetus have 142 focused primarily on optimizing sample collection and culture from bulls because of their 143 propensity to develop chronic infections. The technique most commonly used to sample 144 female cattle for T. foetus is a dry pipette technique.48 An infusion pipette is used to 145 aspirate CVM from the vaginal fornix or near the external cervical os. Alternatively, in 146 the case of a post-coital pyometra, an infusion pipette can also be used to aspirate some 147 of the content of the uterus. Either sample is then examined directly or placed into 148 appropriate culture medium. Culturing T. foetus from CVM has a reported sensitivity of 149 58 to 75%.51 Samples can also be evaluated with appropriate molecular-based assays. 150 In vitro culture of Trichomoniasis foetus 151 Direct microscopic examination of specimens for T. foetus may be diagnostic, but 152 a far more sensitive method for the detection of T. foetus is in vitro culture of preputial 153 smegma in a selective nutrient medium for up to a week.51-53 In vitro culture allows the 154 proliferation of T. foetus to more readily detectable levels. All cultures containing 155 Result Sensitivity (in series) First test Negative 80% Second test (one week later) Negative 96% Third test (one week later) Negative 99 295 organisms resembling T. foetus should be confirmed with appropriate molecular-based 156 assays to avoid false-positive results due to fecal trichomonad contamination of culture 157 media.31,32,54 Alternatively, samples may be submitted directly for molecular-based 158 evaluation. If polymerase chain reaction-based evaluations are not available, a current 159 study by Corbeil, et al suggest that immunofluorescent assay may be useful in the 160 diagnosis of T. foetus.55 161 In vitro culture media 162 Various culture and transport media systems have been used including 163 Kupferberg medium and broth, Claussen’s medium, Sutherland medium, trypticase-yeast 164 extract-maltose (TYM) medium, Diamond’s medium, and most recently the InPouch® 165 TF (BioMed Diagnostics, White City, OR, USA) Tritrichomonas foetus culture pouch. In 166 vitro cultivation using either Diamond’s medium or the InPouch® TF is currently the 167 most common method used to diagnose T. foetus in the United States. Both culture 168 systems are fairly equal in sensitivity.47,56-58 However, the InPouch® TF is somewhat 169 more convenient than Diamond’s medium.59 The InPouch® TF has a 12-month shelf-life 170 at room temperature, compared to a much shorter refrigerator-life for Diamond’s 171 medium. Also, the plastic pouch design of the InPouch® TF is less likely to break or leak 172 than tubes containing Diamond’s medium. Unfortunately, the InPouch® TF is more 173 expensive than Diamond’s medium. 174 For many years, cultivation of microorganisms with motility and morphology 175 resembling T. foetus in either the InPouch® TF or Diamond’s medium was considered to 176 be 100% specific. However, accurate microscopic identification of T. foetus has since 177 been shown to be complicated by the presence of other contaminating trichomonadid 178 296 protozoa. All cultures containing organisms resembling T. foetus should therefore be 179 confirmed with appropriate molecular-based assays, or samples should be submitted 180 directly to a laboratory for molecular analysis. Contact the laboratory prior to sample 181 collection to verify the appropriate transport medium. 182 Treatment of cattle infected with Tritrichomoniasis foetus 183 One of the complicating factors associated with bovine trichomoniasis is that 184 there are currently no effective treatments with U.S. Food and Drug Administration 185 approval. Historically, the most successful treatment for bulls with trichomoniasis 186 involved systemic treatment with nitromidazole derivatives.51,60-62 Despite its 187 effectiveness, the use of nitromidazole derivatives is now illegal in food-producing 188 animals in the U.S. because of their mutagenic and carcinogenic properties, and no 189 alternative treatments are available. However, a recent study by Carvalho, et al63 found 190 that T. foetus exposed in vitro to mebendazole resulted in internalization of the flagella, 191 disruption of the nucleus, and cytoplasmic vacuolization. These findings suggest new 192 possibilities in the treatment of trichomonasis. Still, the lack of effective approved 193 therapies for bovine trichomoniasis emphasizes the need for appropriate preventive and 194 control measures. 195 Prevention and Control of Bovine Trichomoniasis 196 Preventing the introduction of T. foetus into a cattle herd and controlling 197 trichomoniasis in an infected herd follow many of the same management strategies and to 198 a large extent focus on herd biosecurity. Ideally, every cattle operation should focus on 199 preventing the introduction of T. foetus. 200 297 Recommended practices to prevent the introduction of T. foetus into a cattle herd 201 include: 202 1) When possible, avoid grazing cattle on public lands where both bulls and cows 203 have a much greater risk of exposure through coitus with other T. foetus-infected 204 animals.64 205 2) Utilize artificial insemination when possible. 206 3) Cull all open cows and heifers. 207 4) Control animal movement into a herd. Maintain good fences to prevent T. foetus-208 infected animals from inadvertently entering a herd, or to prevent uninfected 209 animals from temporarily entering a T. foetus-infected herd and then returning 210 with T. foetus to their uninfected herd of origin. 211 5) Purchase virgin bulls and heifers as replacements. Buying older bulls and cows as 212 replacements greatly increases the chance of purchasing a T. foetus-infected 213 animal. While older bulls are much more likely to become chronically infected 214 with T. foetus than cows, a small percentage of cows will also become chronically 215 infected. 216 6) Test bulls for T. foetus at least once before introducing them into a new herd 217 7) The test should be performed after two weeks of sexual rest. Ideally, a bull should 218 have three negative cultures at weekly intervals. 219 8) Maintain as young a bull battery as possible. Older bulls are considered more 220 likely to develop chronic T. foetus infections. However, any bull exposed to T. 221 foetus in a natural breeding situation is capable of becoming chronically infected, 222 regardless of age. 223 298 9) Breed purchased cows and heifers in a separate herd, and cull all open animals. 224 Ideally, continue to keep the pregnant animals segregated from the rest of the herd 225 through the next breeding season. 226 10) Consider immunization against T. foetus in high-risk herds. 227 Recommendations for control of trichomoniasis in an infected herd includes: 228 1) Test and cull all infected bulls. Infected bulls should be sold for slaughter only. 229 2) Decrease the number of bulls per breeding unit. Single-sire herds offer the lowest 230 exposure potential. However, single-sire units may not always be practical. 231 3) Reduce the average age of the bull herd. Older bulls are considered more likely to 232 develop chronic T. foetus infections. However, any bull exposed to T. foetus in a 233 natural breeding situation is capable of becoming chronically infected, regardless 234 of age. 235 4) Test bulls for T. foetus at least once before introducing them into a new herd. The 236 test should be performed after two weeks of sexual rest. Ideally, a bull should 237 have three negative cultures at weekly intervals. 238 5) Utilize artificial insemination when possible. 239 6) Reduce the breeding season to 60-90 days and cull all open cows and heifers. If 240 there are too many open cows for culling to be economically feasible, then at least 241 these animals should be separated into a high-risk herd. A long breeding season not 242 only allows propagation of T. foetus, but it may also hide production losses due to 243 reduced weaning weights because of delayed conception. 244 7) Culture all cases of pyometra diagnosed in cows or heifers during pregnancy 245 examinations. 246 299 8) Submit all aborted fetuses and placental tissue to a diagnostic laboratory. 247 Immunization against T. foetus is an extremely important management tool for herds 248 infected with T. foetus. Research trials clearly demonstrate the benefit of T. foetus 249 vaccination.64-66 TrichGuard® (Fort Dodge Animal Health, Fort Dodge, IA, USA) and 250 TrichGuard® V5L (Fort Dodge Animal Health) are currently the only T. foetus vaccines 251 available in the United States. The vaccines require an initial subcutaneous dose followed 252 by a booster dose two to four weeks later. The second injection should precede the 253 breeding season by four weeks. Annual revaccination four weeks prior to the breeding 254 season is recommended. 255 References 256 1. Parsonson IM, Clark BL, Dufty JH: The pathogenesis of Tritrichomonas foetus 257 infection in the bull. Aust Vet J 1974;50:421-3. 258 2. Peter D: Bovine venereal diseases. In: Youngquist RS, editor. Current therapy in 259 large animal theriogenology. 1st ed. Philadelphia: W.B. Saunders; 1997. p. 355-63. 260 3. Johnson AE: Incidence and diagnosis of trichomoniasis in western beef bulls. J Am 261 Vet Med Assoc 1964;145:1007-1010. 262 4. Anderson ML, Barr BC, Conrad PA: Protozoal causes of reproductive failure in 263 domestic ruminants. Vet Clin North Am Food Anim Pract 1994;10:439-61. 264 5. Hammond DM, Bartlett DE: The distribution of Trichomonas foetus in the 265 preputial cavity of infected bulls. Am J Vet Res 1943;4:143-149. 266 267 300 6. Roberts SJ: Infectious diseases causing infertility in cows. Veterinary obstetrics and 268 genital diseases. Ann Arbor, MI: Edward Bros; 1986. p. 447. 269 7. Bennchimol M, Rosa IA, Fontes RS, et al: Trichomonas adhere and phagocytose 270 sperm cells: adhesion seems to be a prominent stage during interaction. Parasitol 271 Res 2008;102:597-604. 272 8. Clark BL, Dufty JH, Parsonson RD, et al: Studies on the transmission of 273 Tritrichomonas foetus. Aust Vet J 1977;53:170-2. 274 9. Morgan BB: Studies on the trichomonad carrier-cow problem. J Anim Sci 1944; 275 3:437. 276 10. BonDurant RH: Diagnosis, treatment, and control of bovine trichomoniasis. 277 Compend Contin Educ Pract Vet 1985;7:S179-188. 278 11. Rae DO: Impact of trichomoniasis on the cow-calf producer's profitability. J Am 279 Vet Med Assoc 1989;194:771-775. 280 12. Anderson ML, Barr BC, Conrad PA: Protozoal causes of reproductive failure in 281 domestic ruminants. Vet Clin North Am Food Anim Pract 1994;10:439-461. 282 13. Parsonson IM, Clark BL, Dufty JH: Early pathogenesis and pathology of 283 Tritrichomonas foetus infection in virgin heifers. J Comp Pathol 1976;86:59-66. 284 14. Clark BL, Dufty JH, Parsonson IM: The effect of Tritrichomonas foetus on calving 285 rates in beef cattle. Aust Vet J 1983;60:71-74. 286 301 15. Abbitt B, Meyerholz GW: Trichomonas foetus infection of range bulls in South 287 Florida. Vet Med Sm Anim Clin 1979;74:1339-42. 288 16. Fitzgerald PR, Johnson AE, Thorne JL, et al: Trichomoniasis in range cattle. Vet 289 Med 1958;53:249-52. 290 17. Wilson SK, Kocan AA, Gaudy ET, et al: The prevalence of trichomoniasis in 291 Oklahoma beef bulls. Bov Pract 1979;14:109-10. 292 18. Fitzgerald PR: Bovine trichomoniasis. Vet Clin North Am Food Anim Pract 293 1986;2:277-82. 294 19. Speer CA, White MW: Better diagnostics and control could save beef industry $650 295 million annually. Large Animal Vet 1991:46:18. 296 20. Louisiana Board of Health LAC 7:XXI.339 297 21. Idaho IDAPA 02.04.03 Rules Governing Animal Industry 298 22. Utah Regulations R58-21-3 Trichomoniasis Rules Prevention and Control. 299 23. Nebraska Animal Importation Act and Animal Importation Regulation 54-701. 300 24. North Dakota State Board Animal Health Order #2007-03. 301 25. Montana Department Livestock Sec 32.3.502 302 26. Wyoming Livestock Board W.S. 11.18.103 303 27. Washington Administrative Code WAC 16.154.086 304 302 28. Oregon Department of Agriculture 603-011-0610. 305 29. South Dakota 12:68:27:03 Intrastate Trichomoniasis Testing Requirements. 306 30. Texas Animal Health Commission, Chapter 38 TAC 38.1-38.7 307 31. BonDurant RH, Gajadhar A, Campero CM, et al: Preliminary characterization of a 308 Tritrichomonas foetus-like protozoan isolated from preputial smegma of virgin 309 bulls. Bov Pract 1999;33:124-127. 310 32. Cobo ER, Campero CM, Mariante RM, et al: Ultrastructural study of a 311 tetratrichomonad species isolated from prepucial smegma of virgin bulls. Vet 312 Parasitol 2003;117:195-211. 313 33. Felleisen RSJ, Lambelet N, Bachmann P, et al: Detection of Tritrichomonas foetus 314 by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences. J 315 Clin Microbiol 1998;36:513-519 316 34. Hayes DC, Anderson RR, Walker RL: Identification of trichomonadid protozoa 317 from the bovine preputial cavity by polymerase chain reaction and restriction 318 fragment length polymorphism typing. J Vet Diagn Invest 2003;15:390-394. 319 35. Taylor MA, Marshall RN, Stack M: Morphological differentiation of 320 Tritrichomonas foetus from other protozoa of the bovine reproductive tract. Brit Vet 321 J 1994;150:73-80. 322 303 36. Castella J, Munoz E, Ferrer D, et al: Isolation of the trichomonad Tetratrichomonas 323 buttreyi (Hibler et al., 1960) Honigberg, 1963 in bovine diarrhoeic faeces. Vet 324 Parasitol 1997;70:41-45. 325 37. Hibler CP, Hammond DM, Caskey FH, et al: The morphology and incidence of 326 the trichomonads of swine, Tritrichomonas suis (Gruby & Delafond), 327 Tritrichomonas rotunda n.sp. and Trichomonas buttreyi n.sp. J Protozool 328 1960;7:159-171. 329 38. Levine NP: Veterinary protozoology. Ames: Iowa State University Press; 1985. 330 39. Cobo ER, Canton G, Morrell E, et al: Failure to establish infection with 331 Tetratrichomonas sp. in the reproductive tracts of heifers and bulls. Vet Parasitol 332 2004;120:145-150. 333 40. Riley DE, Wagner B, Polley L, et al: PCR-based study of conserved and variable 334 DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada. J 335 Clin Microbiol 1995;33:1308-1313. 336 41. Felleisen RSJ, Schimid-Lambelet LN, Walubengo J: Comparative evaluation of 337 methods for the diagnosis of bovine Tritrichomonas foetus infection. J Protozool Res 338 1997;7:90-101. 339 42. Ho MSY, Conrad PA, Conrad PJ, et al: Detection of bovine trichomoniasis with a 340 specific DNA probe and PCR amplification system. J Clin Microbiol 1994;32:98-341 104. 342 304 43. Patel SR, Wiese W, Patel SC, et al: Systematic review of diagnostic tests for 343 vaginal trichomoniasis. Infect Dis Obstet Gynecol 2000;8:248-257. 344 44. Nickel DD, Olson ME, Schultz GA: An improved polymerase chain reaction 345 assay f for the detection of Tritrichomonas foetus in cattle. Can Vet J 346 2002;43:213-216. 347 45. Grahn RA, BonDurant RH, Van Hoosear KA, et al: An improved molecular assay 348 for Tritrichomonas foetus. Vet Parasitol 2005;127:33-41. 349 46. Morgan BB: Bovine trichomoniasis. Minneapolis: Burgess Publishing Company; 350 1946. 351 47. Schönmann MJ, BonDurant RH, Gardner IA, et al: Comparison of sampling and 352 culture methods for the diagnosis of Tritrichomonas foetus infection in bulls. Vet 353 Rec 1994;134:620-622. 354 48. Kimsey PB: Bovine trichomoniasis. In: Morrow DA, ed. Current therapy in 355 theriogenology. Philadelphia: W.B. Saunders Company, 1986. p. 275-279. 356 49. Fitzgerald PR, Hammond DM, Miner MC, et al: Relative efficacy of various 357 methods of obtaining preputial samples for diagnosis of trichomoniasis in bulls. 358 Am J Vet Res 1952;13:452-457. 359 50. Food Animal Reproduction and Herd Health Services, University of California at 360 Davis: Bovine trichomonosis: essential facts and testing. 361 www.vetmed.ucdavis.edu/vetext/INF-BE_Bov-trichomonosis.html. 2006. 362 305 51. Skirrow S, BonDurant RH: Bovine trichomoniasis. Vet Bull 1988;58:592-603. 363 52. Johnson AE The diagnosis of trichomoniasis in the bull. Proc of the US Livestock 364 Sanitary Assoc 1965;69:183-189. 365 53. Tedesco LF, Errico F, Baglivi LP: Diagnosis of Tritrichomonas foetus infection 366 in bulls using two sampling methods and a transport medium. Aust Vet J 367 1979;55:322- 324. 368 54. Campero CM, Rodriguez Dubra C, Bolondi A, et al: Two-step (culture and PCR) 369 diagnostic approach for differentiation of non-T. foetus trichomonads from 370 genitalia of virgin beef bulls in Argentina. Vet Parasitol 2003;112:167-175. 371 55. Corbeil LB, Campero CM, Hoosear KV, et al: Detection of trichomonad species 372 in the reproductive tracts of breeding and virgin bulls. Vet Parasitol 373 2008;154:226-232. 374 56. BonDurant RH: Pathogenesis, diagnosis and management of trichomoniasis in 375 cattle. Vet Clin North Am Food Anim Pract 1997;13:345-361. 376 57. Appell LH, Mickelsen WD, Thomas MW, et al: A comparison of techniques used 377 for the diagnosis of Tritrichomonas foetus infections in beef bulls. Agri Pract 378 1993;14:30-34. 379 58. Skirrow SZ, BonDurant RH, Farley J, et al: Efficacy of ipronidazole against 380 trichomoniasis in beef bulls. J Am Vet Med Assoc 1985;187:405-407. 381 306 59. Borchardt KA, Norman BB, Thomas MW, et al: Evaluation of a new culture 382 method for diagnosing Tritrichomonas foetus infection. Vet Med 1992;87:104-383 112. 384 60. Ball L, Dargatz DA, Cheney JM, et al: Control of venereal disease in infected 385 herds. Vet Clin North Am Food Anim Pract 1987;3:561-574. 386 61. Dawson LJ: Diagnosis, prevention, and control of campylobacteriosis and 387 trichomoniasis. Bov Pract 1986;21:180. 388 62. Gasparini G, Vaghi M, Tardani A: Treatment of bovine trichomoniasis with 389 metronidazole (8823 R.P.). Vet Rec 1963;75:940. 390 63. Carvalho KP, Gadelha APR: Effects of three benzimadazoles on growth, general 391 morphology and ultrastructure of Trichomonas foetus. FEMS Microbiolgy Lett 392 2007;275:292-300. 393 64. Gay JM, Ebel ED, Kearley WP: Commingled grazing as a risk factor for 394 trichomonosis in beef herds. J Am Vet Med Assoc 1996;209:643-646. 395 65. Rae DO, Chenoweth PJ, Genho PC, et al: Prevalence of Tritrichomonas fetus in 396 a bull population and effect on production in a large cow-calf enterprise. J Am 397 Vet Med Assoc 1999;214:1051-1055. 398 66. Kvasnicka WG, Hall MR, Hanks DR: Bovine trichomoniasis. In: Howard JL, 399 editor. Current veterinary therapy: food animal practice. 4th ed. Philadelphia: WB 400 Saunders; 1999. p. 420-425. 401 307 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages false /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness false /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages false /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Average /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Average /MonoImageResolution 300 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages false /MonoImageFilter /FlateEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ENU ([Based on 'No Compression'] [Based on 'No Compression wbleeed'] [Based on '[High Quality Print]'] Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames false /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /BleedOffset [ 0 0 0 0 ] /ConvertColors /NoConversion /DestinationProfileName () /DestinationProfileSelector /NA /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure true /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles true /MarksOffset 6 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> << /AllowImageBreaks true /AllowTableBreaks true /ExpandPage false /HonorBaseURL true /HonorRolloverEffect false /IgnoreHTMLPageBreaks false /IncludeHeaderFooter false /MarginOffset [ 0 0 0 0 ] /MetadataAuthor () /MetadataKeywords () /MetadataSubject () /MetadataTitle () /MetricPageSize [ 0 0 ] /MetricUnit /inch /MobileCompatible 0 /Namespace [ (Adobe) (GoLive) (8.0) ] /OpenZoomToHTMLFontSize false /PageOrientation /Portrait /RemoveBackground false /ShrinkContent true /TreatColorsAs /MainMonitorColors /UseEmbeddedProfiles false /UseHTMLTitleAsMetadata true >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice