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Prevalence and risk factors for Trichomonas foetus infection in cattle in northeastern Costa Rica
Preventive Veterinary Medicine, 14 ( 1992 ) 155-165
155
Elsevier Science Publishers B.V., Amsterdam
Prevalence and risk factors for Trichomonas foetus infection in cattle in northeastern Costa Rica Enrique Perez a, Patricia A. Conrad b, David Hird c, Aria Ortuno d, Jorge Chacon d, Robert BonDurant e and Jos Noordhuizen f aHerd Health Section, Dutch lnteruniversity Cooperation Program, School of Veterinary Medicine, Universidad Nacional, P.O.Box 86-3000, Heredia, Costa Rica bDepartment of Veterinary Microbiology and Immunology, University of California, Davis, CA 95616, USA CDepartment of Epidemiology and Preventive Medicine, University of California, Davis, CA 95616, USA dDepartment of Reproduction, School of Veterinary Medicine, UniversidadNacional, P.O.Box 86-3000, Heredia, Costa Rica CDepartment of Veterinary Reproduction, University of California, Davis, CA 95616, USA fDepartment of Animal Husbandry, Epidemiology Division, Agricultural University, P.O.Box 338, 6700 AH Wageningen, Netherlands (Accepted 27 May 1992)
ABSTRACT Perez, E., Conrad, P.A., Hird, D., Ortuno, A, Chacon, J., BonDurant R. and Noordhuizen, J., 1992. Prevalence and risk factors for Trichomonasfoetus infection in cattle in northeastern Costa Rica. Prey. Vet. Med., 14: 155-165. Two studies (one pilot and one cross-sectional) were conducted in the two main livestock-rearing areas of Costa Rica using an improved method (In-Pouch TF) for the detection of Trichomonas foetus infection in bulls. Herd prevalence rates of 6.7% (6/89) and 15.9% (10/63 ), and bull prevalence rates of 3.9% (6/153 ) and 6.2% ( 14/225 ) were found in the pilot and cross-sectional studies, respectively. Adjusted prevalences in the cross-sectional study were 18.4% and 7.2% for farms and bulls, respectively. Breed (Bos taurus) and age (over 4 years) were the main risk factors identified using random-effects logistic regression. The nature of the disease and the high prevalence indicated that trichomoniasis could have a serious economic impact on cattle production in Costa Rica.
INTRODUCTION
The e c o n o m y o f Costa Rica is based primarily on agriculture. With a national cattle population o f approximately 2 million animals, the livestock secCorrespondence to."E. Perez, Herd Health Section, Dutch Interuniversity Cooperation Program, School of Veterinary Medicine, Universidad Nacional, P.O. Box 86-3000, Heredia, Costa Rica.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-5877/92/$05.00
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tor is important for domestic food supplies and for foreign exchange earnings from meat and milk exports. The average annual calving rate in Costa Rica is approximately 50%, with mean calving intervals of 18-22 months (National Census of Costa Rica, 1984). Little is known about the prevalence of reproductive diseases such as trichomoniasis in Costa Rica. However, because natural service is the standard practice in beef herds, infertility due to Trichomonasfoetus infections could be contributing to the low calving rate. Trichomoniasis has a worldwide distribution and is a major cause of infertility in naturally bred cattle in many countries (Johnson, 1964; Clark et al., 1974; Wilson et al., 1979; BonDurant, 1985 ). In published surveys, the percentages of infected bulls varied from 7 to 15.8% (Skirrow and BonDurant, 1988; BonDurant et al., 1990). Little quantitative information is available concerning the risk factors associated with trichomoniasis. Diagnostic difficulties such as the need for a transport medium and maintaining appropriate flesh media, may have contributed to this lack of information. Currently, diagnosis is based on microscopic observation of T. foetus from vaginal secretions or smegma samples, usually after in vitro cultivation of the samples for 12-72 h. Diagnosis by this method, using bull smegma samples, had a sensitivity of 81.6% and a specificity of 100% (Skirrow et al., 1985). However, the requirement for special culture media preparation and storage has restricted the use of this method for diagnosis of trichomoniasis in Costa Rica and many other developing countries. Factors of possible importance (especially in dual purpose and extensive beef herds) for the transmission of T. foetus are present in Costa Rica. These factors include: ( 1 ) the use of natural breeding; (2) bulls over 4 years of age; ( 3 ) communal bulls; (4) year-round breeding; ( 5 ) lack of accurate diagnostic procedures (Skirrow and BonDurant, 1988 ). Although T. foetus has been diagnosed as a putative cause of infertility in some herds in Costa Rica (Amelingmeier, 1981; Zamora, 1982), surveys to determine the prevalence of trichomoniasis or risk factors associated with the disease have not been reported. The objectives of this study were: ( 1 ) to assess the practicability of a new, simple method for the detection of T. foetus (InPouch TF R, BioMed Diagnostics, San Jos6, CA) (Thomas et al., 1990) under tropical conditions; (2) to estimate the prevalence of trichomoniasis in two major cattle-producing regions in Costa Rica; ( 3 ) to evaluate (by a cross-sectional study ) risk factors associated with infection in the northeast region of the country (humid tropical lowlands). MATERIALSAND METHODS
Study population and sampling methodology The 51 260 km z area of Costa Rica is divided politically into seven provinces, each subdivided into cantons and districts. For socio-economic plan-
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ning, the country is divided into five regions. Two of these regions were selected for this study: the Dry Pacific Region (dry tropical forest) and the Northern Region (wet tropical forest).
Tilaran pilot study The Dry Pacific region was chosen for testing of the sampling methodology and for conducting a pilot study. Within the region, the canton of Tilaran was selected for this study because the main income of this region is from livestock production and a Livestock Information Network pilot project had been established there by the Herd Health Section of the School of Veterinary Medicine, National University. Tilaran has mainly cow-calf and dual purpose, medium-sized farms of less than 200 ha each. It was calculated that the sample size required was 145 bulls, based on an estimated 895 breeding bulls in Tilaran (National Census of Costa Rica, 1984), an expected prevalence of 7% for bull trichomoniasis (BonDurant et al., 1990), a tolerable error of 5% and a desired confidence level of 99%. Using a farm list created by the Livestock Information System pilot project in Tilaran, a proportional random sampling within the seven districts was made and 89 farms were selected for sampling during August-September of 1990. Our records indicated that more than 145 bulls should be present on these 89 farms. A smegma sample was collected from each bull with an infusion pipette and a 6 cm 3 syringe. The pipette was inserted into the prepuce and scraped over the epithelium of the fornix and glans penis while applying negative pressure with the syringe. The entire sample was transferred immediately after collection to the InPouch T. foetus test pouch (Thomas et al., 1990). The pouches were kept at ambient temperature until they were transferred, within 6 h of sampling, to an incubator where they were stored in an upright position at 37 °C for 4 days. Each pouch was examined daily for 4 days using a phase-contrast microscope. Positive T. foetus samples were identified based on the presence of flagellated parasites with characteristic morphology and motility (Levine, 1973; Honigberg, 1978 ).
San Carlos study A second study was carried out from January-August of 1991 to determine the prevalence and risk factors associated with trichomoniasis in the Northern Region (San Carlos), 78 km north of San Jose. This region produces more than 60% of the daily milk production of the country ( 1.2 million kg day-1 ) and is also important in beef production. There are two main types of farms: small farms with approximately 20-60 cows each, which are used mainly for dairy production, and medium-sized farms with 100-200 animals, principally used for beef production. In addition, there are some larger beef farms. The population of reproductive bulls in the Northern Region was approximately 5124 (National Census of Costa Rica, 1984). The sample size was
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TABLE 1 Descriptive statistics and crude odds ratios (OR) of possible farm-level risk factors for bovine trichomoniasis in Costa Rica, 1990-1991 Variable
Farming Experience (years)
San Carlos
Tilaran
Description No. 1 Pos. 2 Rate (%) OR
Description No. ~ P o s ] Rate (%) OR
1-10 49 11-19 60 20-24 46 >25 70 Full-time Yes 194 Farmers No 31 Stocking rate 0.43-0.84 56 (head ha -1 ) 0.85-1.26 56 1.27-1.74 50 > 1.75 63 Feed Yes 204 supplement No 21 Breeding Continuous 155 Seasonal 70 Production Beef 102 system Dual 111 Milk 12 Communal Yes 57 bull No 168 Veterinary Yes 117 services No 108
1 4 5 4 11 3 4 5 2 3 13 1 11 3 3 9 2 2 12 8 6
2 7 11 6 6 10 7 9 4 5 6 5 7 4 3 8 17 3 7 7 6
0.26 1.10 2.30 0.88 1 1.78 1.22 1.75 0.56 0.68 1 0.73 1 0.58 0.30 1.92 3.34 1 2.11 1 0.80
1-9 10-19 20-29 >30 Yes No 0.15-0.70 0.71-0.95 0.96-1.24 >1.25 Yes No Continuous Seasonal
23 44 21 65 130 23 37 39 36 41 63 90 137 16
1 1 1 3 4 2 1 2 1 2 3 3 5 1
4 2 5 5 3 9 3 5 3 5 5 3 4 6
1.13 0.48 1.27 1.37 1 3.03 0.61 1.48 0.64 1.38 1 0.85 1 1.72
~Number of bulls sampled 2Number of bulls positive to T. foetus. For variables with more than two levels, the reference level for each variable was all other levels combined.
calculated based on an expected prevalence of 4% (prevalence found in the pilot study), with a tolerable error of 5% and a desired confidence of 99%. The computerized database for herds in the National Brucellosis Control Program developed by the Ministry of Agriculture and Livestock was used to select the farms for sampling. The database contained information on farm location, herd size, and number of animals by age group, sex and type of management. A proportional random sampling within 16 districts was made for the required sample of 102 bulls. The same sampling methodology described in the first study was employed, by trained veterinary officers of the Ministry of Agriculture and Livestock (Northern Region Branch). After sample inoculation, the pouches were sent to the Reproduction Laboratory of the School of Veterinary Medicine, Heredia, arriving 24-36 h after sampling. Microscopic examination was performed upon arrival and daily for 4 days. The potential risk factors evaluated for T. foetus infection rates in the San
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Carlos study are described in Table 1. In the table, veterinary services means reproductive herd health, mating system means one bull with a group of cows or multiple bulls with the cows and supplemental feeding means molasses or minerals versus nothing. The risk factor data were collected through a questionnaire administered by the investigators to the owners/operators. These data were entered into R-BASE for DOS (Microrim, 1987 ) together with the laboratory results, and converted to ASCII files for statistical analysis.
Statistical analysis The units of analysis were either the individual bull or the individual farm. Continuous variables were categorized using quartiles. Crude odds ratios were calculated for risk factors using PROC FREQ (Statistical Analysis Systems Institute, 1989 ), using the first listed category of each dichotomous variable as the reference level. For variables with three or more levels, each level of the variable was compared with all other levels combined as a reference level. Multivariate analysis was performed in the San Carlos study, using EGRET (Statistics and Epidemiology Research Corporation, 1990 ). In the multivariate analysis, the reference level for the variables with three or more classes was chosen using the pattern (protection or risk) of the crude odds ratio. To control for possible herd effects, a logistic-binomial regression model for distinguishable (non-interchangeable) data was used (Mauritsen, 1984). This model was selected because the data had variables measured both at herd level (herd effects) and bull level (individual effects) associated with the individual responses rather than just with the herds. RESULTS AND DISCUSSION
Pouch collection technique In both studies the most positive results were obtained within the first 48 h of culturing. Similarly, the incubation time required for the detection of T. foetus ( 1- 10 trichomonads m l - 1), in laboratory conditions after a single inoculum was 20 h (Borchardt et al., 1992). Only seven samples in the San Carlos study had excessive bacterial and fungal contamination. Positive samples (four) arriving late at the laboratory (36 h) after sampling were still viable. Thomas et al. (1990) described the successful cultivation of parasites in pouches that were sent by mail and not received until 5 days after collection. The InPouch test was convenient both for sampling and diagnosis with the minimal laboratory facilities available in Costa Rica. The pouches could be carried easily to the field in a vehicle without refrigeration, and subsequently be examined directly under the microscope for parasites. This re-
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duced the cost of preparing and maintaining fresh media, as well as the cost of microscope slides and pipettes. Sensitivity for the detection of T. foetus using Diamond's medium (Diamond, 1957) is reportedly 81.6% (Skirrow et al., 1985), with an assumed specificity of 100% (BonDurant et al., 1990). There is only one report of sensitivity for the InPouch TF R system (Thomas et al., 1990). Statistical analysis of these authors' results showed a x statistic (Martin et al., 1987) of 0.77, indicating a close agreement between Diamond's media and the InPouch system. Therefore, the same sensitivity and specificity (Skirrow et al., 1985 ) was utilized in this study to determine the adjusted prevalence rates.
Prevalence In the Tilaran pilot study, 153 bulls were found on the 89 farms, and all were tested. The prevalences of T. foetus infection were 6.7% ( 6/89 ) by farm and 3.9% (6/153) by bull. The herd prevalence rate was lower than those reported in other studies, i.e. 26% in several western states in the USA (Johnson, 1964), 40% in the Northern Territory, Australia (Dennett et al., 1974) and 15.78% in California (BonDurant et al., 1990). The bull prevalence in Tilaran (3.9%) was similar to that (4.1%) found in California (BonDurant et al., 1990). In the present study, within-herd prevalence ranged from 50 to 100% of bulls (mean, 83.3%; median, 75%). For infected herds, the median herd size was one bull (range 1-2 ) and for uninfected herds it was three bulls (range 1-14). The California survey described a within-herd prevalence that ranged from 4 to 38.5% (median, 12.5%; mean, 15.4%), with a median herd size of 14 bulls (range 6-114) and seven (range 1-210) in infected and uninfected herds, respectively. Studies from tropical locations reported withinherd prevalence rates that ranged from 30.6% in Australia (Clark et al., 1974) to 100% in Nigeria (Akinboade, 1980). Perhaps the differences between our results and those of other authors could be attributed to differences in herd size, age when bulls begin sexual activity, population dynamics, and/or the sampling procedure. To our knowledge, the most recent studies are based on convenience samples, with the exception of the studies in California (BonDurant et al., 1990) and San Carlos, which are based on random sampling (making the two latter studies comparable). In the San Carlos study, the overall prevalences were 6.22% ( 14/225 ) for bulls and 15.87% (10/63) for farms. The farm prevalence in this study was equal to that in the California survey (BonDurant et al., 1990); however, within-herd prevalence was higher ( 12.5-100%; mean, 39%; median, 29%). A median herd size was four bulls (range 1-13) in infected and uninfected herds. The difference between the present study and the Californian study may be due to intrinsic differences in the production systems. For example, almost all bulls in San Carlos were over 3 years old (206/225) with all cases
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TABLE2 Descriptive statistics and crude odds ratios (OR) of possible individual-bull risk factors for bovine trichomoniasis in Costa Rica, 1990-1991 Variable
San Carlos
Tilaran
Description No. l Pos. 2 Rate (%) OR
Description No. t
Pos. 2 Rate (%) OR
B. indicus B. taurus
108 45
1 5
1 11
Single Multiple 5-19 20-24 25-34 >35 1-2.9 3-3.9 4-4.9 >5 313-469 470-596 597-697 >698
101 52 29 27 57 31 34 37 29 53 37 38 37 38
5 2 3 4 5 3 0 3 3 7 0 3 5 8
1 0.37 0.78 0.93 1.72 0.78 NC 0.61 0.85 4.00 NC 0.59 1.60 3.19
Breed
B. indicus B. taurus Cross Mating Single Multiple Cows per bull 1-19 20-29 30-39 >40 Bullage 1-2.9 (years) 3-3.9 4-4.9 >5 Bullweight 270-499 (kg) 500-599 600-699 >700 Bull in Yes service No
137 72 16 96 129 34 60 74 57 19 61 56 89 43 44 76 62 190 35
3 10 1 4 10 0 7 5 2 0 1 2 11 2 4 5 3 9 5
2 14 6 4 8 0 12 7 4 0 2 4 13 5 9 7 5 5 14
O. 15 6.02 1.01 1 1.93 NC 3
2.98 1.14 0.47 NC 0.19 0.48 6.25 0.69 1.70 1.09 0.70 1 3.35
5 1 1 1 3 1 0 1 1 4 0 1 2 3
1 13
tNumber of bulls sampled. ZNumber of bulls positive to T. foetus. 3Not calculated. For variables with more than two levels, the reference level for each variable was all other levels combined.
found in these bulls: in California, 46% of the bulls were older than 3 years (328/724) with an infection rate in the older bulls of 6.7%. San Carlos herds were also smaller than Californian herds (Table 2 ). The prevalences reported here are only estimates of the true prevalence for these regions. The true prevalence (TP) can be found by correcting the observed San Carlos prevalences by the method described by Marchevsky (1974)
Tp_Ap+ Sp- 1 Sp+ Se-1 where apparent prevalence (Ap) is taken from the results of culture, sensitivity (Se) is taken to be 81.6% (Skirrow et al., 1985 ) and specificity (Sp) 100% (BonDurant et al., 1990). Using this formula, the true prevalences for San Carlos were calculated as 18.43% and 7.22% for farms and bulls, respectively. Factors that potentially favor transmission of T. foetus were present in San Carlos with 66% of the farms having multiple bulls with their cows, 25.39%
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of the farms using communal bulls and 65% of the bulls in use being older than 4 years. Assuming that after 1 year of exposure to one carrier bull, 42.5% of cows in the herd may become infected (Clark et at., 1983 ), T. foetus infection could make an important contribution to the reduced or low fertility rate in this region.
Risk factors associated with infection Age The infection rate of trichomoniasis usually increases with age (Clark et al., 1974; Skirrow et al., 1985), as was shown in our study (Table 3). This association with age is thought to be due to the development of deeper epithelial crypts in the prepuce of older bulls (Clark, 1971 ). (Although bulls less than 4 years of age are less often carriers of T.foetus (BonDurant, 1985 ), they can be infected (Kimsey et al., 1980, Skirrow et at., 1985; BonDurant et al., 1990).)
Breed A strong association was found (Table 3) between the Bos taurus breed types and risk of T. foetus infection, as was also the case in the California TABLE 3 Logistic-binomial regression model for possible risk factors of bovine trichomoniasis in San Carlos, 1991 Term
fl
SE (fl)
P-value
OR
95% CI (OR)
Intercept Stocking rate ( < 1.26 vs. > 1.27) ~ Production system (beef vs. other) Communal bull (No vs. Yes) Veterinary services (No vs. Yes) Cows per bull (1-9, >40 vs. other) Breeding policy (Cont. vs. Seas. ) Mating policy (single vs. multiple) Bull in service (Yes vs. No) Breed (B. indicusvs. B. taurus) Bull age ( < 3 yearsvs. > 3 years) Bull weight (270-499 kg, > 700 kg vs. other) Farming experience ( 1-10 years, > 25 years vs. other) Farming only (No vs. Yes) Feed supplement (No vs. Yes) Intercept of random effects
- 11.71 -0.99 1.61 1.71 - 1.43 1.93 1.38 -0.75 2.48 1.94 2.84 0.67
2.91 0.85 1.68 1.14 0.97 1.11 1.11 1.67 1.06 0.79 1.25 0.75
< 0.001 0.24 0.34 0.14 0.14 0.08 0.21 0.65 0.02 0.02 0.02 0.37
0.37 4.99 5.51 0.24 6.92 3.96 0.47 11.88 6.94 17.15 1.96
0-1.9 0.1-134.9 0.5-51.4 0-1.6 0.7-60.4 0.4-34.7 0-12.3 1.4-94.2 1.4-32.8 1.4-199.8 0.4-8.6
1.27
0.95
0.19
3.55
0.5-23.2
1.26 0.11 0
1.23 1.88 0.60
0.31 0.95 -
3.54 1.12 -
0.3-39.6 0-44.23 -
IFirst level considered as reference level.
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prevalence survey. Several researchers have suggested that certain breeds may be more susceptible to trichomoniasis (Abbitt, 1980; Kimsey et al, 1980; Ball et al., 1987), but others have not found any evidence for breed predisposition (Skirrow and BonDurant, 1989). Dennett et al. (1974) did not find differences between Bos indicus and B. taurus breeds in 689 animals studied, but possible confounding factors (such as age) were not taken into account in their study. In our study, B. taurus bulls had at least 6.93 times the risk of infection with T. foetus than B. indicus bulls. While intrinsic differences in susceptibility amongst breeds may exist (Abbitt, 1980; Ball et al., 1987), the prevalence difference could be due to the increased number of matings accomplished in B. taurus bulls compared with B. indicus bulls in the same period of time (Galina and Arthur, 1991 ), thus increasing the risk of exposure to infection. Bull in service In the San Carlos study, this variable was assessed, because few bulls could be sampled during sexual rest, as generally recommended (BonDurant et al., 1990). As expected, bulls in sexual rest (no virgin bulls were sampled) at the moment of sampling had a greater risk of being infected than bulls in service. Clark et al. (1983) suggested that a depletion of the preputial T. foetus population may occur as a result of repeated matings. For this reason, the true prevalence of trichomoniasis in the present survey may have been underestimated. CONCLUSION
The InPouch test proved to be a,practical and convenient method for detecting T. foetus in samples from infected bulls in Costa Rica. The studies assessed risk factors for bovine trichomoniasis under tropical conditions for the first time. Species-specific risk of infection of B. taurus bulls and age were the most meaningful risk factors detected. Trichomonasfoetus infection may be an important contributor to reproductive failure in Costa Rican beef and dual purpose herds, and its economic impact should be assessed. ACKNOWLEDGMENTS
This research was supported by the Dutch Interuniversity Cooperative Program (Perez) and University of California, Davis, Livestock Disease Research Laboratory (Conrad and Hird). The authors thank Jim Self and Dr. Mike Thomas for making available the InPouch tests and the veterinarians of the North Region Branch of the Ministry of Agriculture and Livestock, Costa Rica, San Carlos and Tilaran for their assistance.
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REFERENCES Abbitt, B., 1980. Trichomoniasis in cattle. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. WB Saunders, Philadelphia, PA, pp. 482-488. Akinboade, O.A., 1980. Incidence of bovine trichomoniasis in Nigeria. Rev. Elev. Med. Vet. Pays Trop., 33: 381-384. Amelingmeier, R.C., 1981. Estudio preliminar de diagnosis de Trichomonasfoetus en Costa Rica. Tesis. Mrdico veterinario. Heredia, Costa Rica. Escuela de Medicina Veterinaria, Universidad Nacional, 32 pp. Ball, L., Dargatz, D., Cheney, J.M. and Mortimer, R.G., 1987. Control of venereal disease in infected herds. Vet. Clin. N. Am., Large Anim. Pract., 3: 561-564. BonDurant, R.H., 1985. Diagnosis, treatment and control of bovine trichomoniasis. Comp. Contin. Educ., 7: S 179-$216. BonDurant, R.H., Anderson, M.L., Blanchard, P., Hird, D., Danaye-Elmi, C., Palmer, C., Sischo, W.M., Suther, D., Utterbach, W. and Weighler, W., 1990. Prevalence of trichomoniasis among California beef herds. J. Am. Vet. Med. Assoc., 196:1590-1593. Borchardt, K.A., Norman, B.B., Thomas, M.W. and Harmon, W.M., 1992. An evaluation of the InPouchTFTM culture system for the diagnosis of Trichomonasfoetus infection. Vet. Med., in press. Clark, B.L., 1971. Venereal diseases of cattle. Veterinary Review. No. 1. University of Sidney, pp. 5-26. Clark, B.L., Parsonson, I.M. and Dufty, J.H., 1974. Experimental infection of bulls with Trichomonasfoetus. Aust. Vet. J., 50: 189-191. Clark, B.L., Dufty, J.H. and Parsonson, I.M., 1983. The effect of Trichomonasfoetus infection on calving rates in beef cattle. Aust. Vet. J., 60:71-74. Dennett, D.P., Reece, R.L., Barasa, J.O. and Johnson, R.H., 1974. Observations on incidence and distribution ofserotype of Trichomonasfoetusin beef cattle in North-Eastern Australia. Aust. Vet. J., 50: 427-431. Diamond, L.S., 1957. The establishment of various trichomonads of animals and men in axenic cultures. J. Parasitol., 43: 488-490. Galina, C.S. and Arthur, G.H., 1991. Review of cattle reproduction in the tropics. Part 6: The male. Anita. Breed. Abstr., 59: 403-412. Hird, D.W. and Robinson, R.A., 1982. Dairy farm wells in southeastern Minnesota: The relation of water source to calf mortality rate. Prev. Vet. Med., 1: 53-64. Honigberg, B.M., 1978. Trichomonasfoetus. In: J.P. Kreier (Editor), Parasitic Protozoa. Academic Press, New York, pp. 207-273. Johnson, A.E., 1964. Incidence and diagnosis of trichomoniasis in western beef bulls. J. Am. Vet. Med. Assoc., 145: 1007-1010. Kimsey, P.B., Darien, B.J., Kendrik, J.W. and Franti, C.E., 1980. Bovine trichomoniasis: diagnosis and treatment. J. Am. Vet. Med. Assoc., 177:616-619. Levine, N.D., 1973. The trichomonad. In: N.B. Levine (Editor), Protozoan Parasites of Domestic Animals and of Man. Burgess, Minneapolis, MN, pp. 88-110. Marchevsky, N., 1974. Errors in prevalence estimates in population studies: a practical method for calculating real prevalence. Zoonosis, 6:98-109. Martin, S.W., Meek, A.H. and Willeberg, P., 1987. Veterinary Epidemiology, Principles and Method. Iowa State University Press, Ames, pp. 73-74. Mauritsen, R.H., 1984. Logistic regression with random effects. Ph.D. Thesis, Department of Biostatistics, University of Washington, 84 pp. Microrim Inc, 1987. R: BASE for DOS, User's Guide, version 2.0. National Census of Costa Rica, 1984. Oficina de Estadistica y Censos, Ministerio de Planificacion Nacional, San Josr, Costa Rica.
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Skirrow, S.Z. and BonDurant, R.H., 1988. Bovine trichomoniasis. Vet. Bull. (London), 58: 592-603. Skirrow, S.Z., BonDurant, R.H. and Farley, J., 1985. Efficacy of ipronidazole against trichomoniasis in beef bulls. J. Am. Vet. Med. Assoc., 187: 405-407. Statistical Analysis Systems Institute, 1989. User's Guide: Statistics, Version 6 edn. SAS Institute, Cary, NC. Statistics and Epidemiology Research Corporation, 1990. EGRET Statistical Package Users Manual. SERC, Seattle, WA. Thomas, M.W., Harmon, W.M. and White, B.S., 1990. An improved method for the detection of Trichomonasfoetus infection by culture in bulls. Agri-Practice, 11: 13-17. Wilson, S.K., Kocan, A.A., Gaudy, E.T. and Goodwin, D., 1979. The prevalence of trichomoniasis in Oklahoma beef bulls. Bovine Pract., 14:109-110, Zamora, J.L., 1982. Diagn6stico laboratorial de tricomoniasis en toros de varias zonas de Costa Rica mediante la t6cnica de cultivo. Tesis. M6dico Veterinario. Heredia, Costa Rica, Escuela de Medicina Veterinaria, Universidad Nacional, 44 pp.
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Prevalence and risk factors for Trichomonas foetus infection in cattle in northeastern Costa Rica Enrique Perez a, Patricia A. Conrad b, David Hird c, Aria Ortuno d, Jorge Chacon d, Robert BonDurant e and Jos Noordhuizen f aHerd Health Section, Dutch lnteruniversity Cooperation Program, School of Veterinary Medicine, Universidad Nacional, P.O.Box 86-3000, Heredia, Costa Rica bDepartment of Veterinary Microbiology and Immunology, University of California, Davis, CA 95616, USA CDepartment of Epidemiology and Preventive Medicine, University of California, Davis, CA 95616, USA dDepartment of Reproduction, School of Veterinary Medicine, UniversidadNacional, P.O.Box 86-3000, Heredia, Costa Rica CDepartment of Veterinary Reproduction, University of California, Davis, CA 95616, USA fDepartment of Animal Husbandry, Epidemiology Division, Agricultural University, P.O.Box 338, 6700 AH Wageningen, Netherlands (Accepted 27 May 1992)
ABSTRACT Perez, E., Conrad, P.A., Hird, D., Ortuno, A, Chacon, J., BonDurant R. and Noordhuizen, J., 1992. Prevalence and risk factors for Trichomonasfoetus infection in cattle in northeastern Costa Rica. Prey. Vet. Med., 14: 155-165. Two studies (one pilot and one cross-sectional) were conducted in the two main livestock-rearing areas of Costa Rica using an improved method (In-Pouch TF) for the detection of Trichomonas foetus infection in bulls. Herd prevalence rates of 6.7% (6/89) and 15.9% (10/63 ), and bull prevalence rates of 3.9% (6/153 ) and 6.2% ( 14/225 ) were found in the pilot and cross-sectional studies, respectively. Adjusted prevalences in the cross-sectional study were 18.4% and 7.2% for farms and bulls, respectively. Breed (Bos taurus) and age (over 4 years) were the main risk factors identified using random-effects logistic regression. The nature of the disease and the high prevalence indicated that trichomoniasis could have a serious economic impact on cattle production in Costa Rica.
INTRODUCTION
The e c o n o m y o f Costa Rica is based primarily on agriculture. With a national cattle population o f approximately 2 million animals, the livestock secCorrespondence to."E. Perez, Herd Health Section, Dutch Interuniversity Cooperation Program, School of Veterinary Medicine, Universidad Nacional, P.O. Box 86-3000, Heredia, Costa Rica.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-5877/92/$05.00
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tor is important for domestic food supplies and for foreign exchange earnings from meat and milk exports. The average annual calving rate in Costa Rica is approximately 50%, with mean calving intervals of 18-22 months (National Census of Costa Rica, 1984). Little is known about the prevalence of reproductive diseases such as trichomoniasis in Costa Rica. However, because natural service is the standard practice in beef herds, infertility due to Trichomonasfoetus infections could be contributing to the low calving rate. Trichomoniasis has a worldwide distribution and is a major cause of infertility in naturally bred cattle in many countries (Johnson, 1964; Clark et al., 1974; Wilson et al., 1979; BonDurant, 1985 ). In published surveys, the percentages of infected bulls varied from 7 to 15.8% (Skirrow and BonDurant, 1988; BonDurant et al., 1990). Little quantitative information is available concerning the risk factors associated with trichomoniasis. Diagnostic difficulties such as the need for a transport medium and maintaining appropriate flesh media, may have contributed to this lack of information. Currently, diagnosis is based on microscopic observation of T. foetus from vaginal secretions or smegma samples, usually after in vitro cultivation of the samples for 12-72 h. Diagnosis by this method, using bull smegma samples, had a sensitivity of 81.6% and a specificity of 100% (Skirrow et al., 1985). However, the requirement for special culture media preparation and storage has restricted the use of this method for diagnosis of trichomoniasis in Costa Rica and many other developing countries. Factors of possible importance (especially in dual purpose and extensive beef herds) for the transmission of T. foetus are present in Costa Rica. These factors include: ( 1 ) the use of natural breeding; (2) bulls over 4 years of age; ( 3 ) communal bulls; (4) year-round breeding; ( 5 ) lack of accurate diagnostic procedures (Skirrow and BonDurant, 1988 ). Although T. foetus has been diagnosed as a putative cause of infertility in some herds in Costa Rica (Amelingmeier, 1981; Zamora, 1982), surveys to determine the prevalence of trichomoniasis or risk factors associated with the disease have not been reported. The objectives of this study were: ( 1 ) to assess the practicability of a new, simple method for the detection of T. foetus (InPouch TF R, BioMed Diagnostics, San Jos6, CA) (Thomas et al., 1990) under tropical conditions; (2) to estimate the prevalence of trichomoniasis in two major cattle-producing regions in Costa Rica; ( 3 ) to evaluate (by a cross-sectional study ) risk factors associated with infection in the northeast region of the country (humid tropical lowlands). MATERIALSAND METHODS
Study population and sampling methodology The 51 260 km z area of Costa Rica is divided politically into seven provinces, each subdivided into cantons and districts. For socio-economic plan-
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ning, the country is divided into five regions. Two of these regions were selected for this study: the Dry Pacific Region (dry tropical forest) and the Northern Region (wet tropical forest).
Tilaran pilot study The Dry Pacific region was chosen for testing of the sampling methodology and for conducting a pilot study. Within the region, the canton of Tilaran was selected for this study because the main income of this region is from livestock production and a Livestock Information Network pilot project had been established there by the Herd Health Section of the School of Veterinary Medicine, National University. Tilaran has mainly cow-calf and dual purpose, medium-sized farms of less than 200 ha each. It was calculated that the sample size required was 145 bulls, based on an estimated 895 breeding bulls in Tilaran (National Census of Costa Rica, 1984), an expected prevalence of 7% for bull trichomoniasis (BonDurant et al., 1990), a tolerable error of 5% and a desired confidence level of 99%. Using a farm list created by the Livestock Information System pilot project in Tilaran, a proportional random sampling within the seven districts was made and 89 farms were selected for sampling during August-September of 1990. Our records indicated that more than 145 bulls should be present on these 89 farms. A smegma sample was collected from each bull with an infusion pipette and a 6 cm 3 syringe. The pipette was inserted into the prepuce and scraped over the epithelium of the fornix and glans penis while applying negative pressure with the syringe. The entire sample was transferred immediately after collection to the InPouch T. foetus test pouch (Thomas et al., 1990). The pouches were kept at ambient temperature until they were transferred, within 6 h of sampling, to an incubator where they were stored in an upright position at 37 °C for 4 days. Each pouch was examined daily for 4 days using a phase-contrast microscope. Positive T. foetus samples were identified based on the presence of flagellated parasites with characteristic morphology and motility (Levine, 1973; Honigberg, 1978 ).
San Carlos study A second study was carried out from January-August of 1991 to determine the prevalence and risk factors associated with trichomoniasis in the Northern Region (San Carlos), 78 km north of San Jose. This region produces more than 60% of the daily milk production of the country ( 1.2 million kg day-1 ) and is also important in beef production. There are two main types of farms: small farms with approximately 20-60 cows each, which are used mainly for dairy production, and medium-sized farms with 100-200 animals, principally used for beef production. In addition, there are some larger beef farms. The population of reproductive bulls in the Northern Region was approximately 5124 (National Census of Costa Rica, 1984). The sample size was
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TABLE 1 Descriptive statistics and crude odds ratios (OR) of possible farm-level risk factors for bovine trichomoniasis in Costa Rica, 1990-1991 Variable
Farming Experience (years)
San Carlos
Tilaran
Description No. 1 Pos. 2 Rate (%) OR
Description No. ~ P o s ] Rate (%) OR
1-10 49 11-19 60 20-24 46 >25 70 Full-time Yes 194 Farmers No 31 Stocking rate 0.43-0.84 56 (head ha -1 ) 0.85-1.26 56 1.27-1.74 50 > 1.75 63 Feed Yes 204 supplement No 21 Breeding Continuous 155 Seasonal 70 Production Beef 102 system Dual 111 Milk 12 Communal Yes 57 bull No 168 Veterinary Yes 117 services No 108
1 4 5 4 11 3 4 5 2 3 13 1 11 3 3 9 2 2 12 8 6
2 7 11 6 6 10 7 9 4 5 6 5 7 4 3 8 17 3 7 7 6
0.26 1.10 2.30 0.88 1 1.78 1.22 1.75 0.56 0.68 1 0.73 1 0.58 0.30 1.92 3.34 1 2.11 1 0.80
1-9 10-19 20-29 >30 Yes No 0.15-0.70 0.71-0.95 0.96-1.24 >1.25 Yes No Continuous Seasonal
23 44 21 65 130 23 37 39 36 41 63 90 137 16
1 1 1 3 4 2 1 2 1 2 3 3 5 1
4 2 5 5 3 9 3 5 3 5 5 3 4 6
1.13 0.48 1.27 1.37 1 3.03 0.61 1.48 0.64 1.38 1 0.85 1 1.72
~Number of bulls sampled 2Number of bulls positive to T. foetus. For variables with more than two levels, the reference level for each variable was all other levels combined.
calculated based on an expected prevalence of 4% (prevalence found in the pilot study), with a tolerable error of 5% and a desired confidence of 99%. The computerized database for herds in the National Brucellosis Control Program developed by the Ministry of Agriculture and Livestock was used to select the farms for sampling. The database contained information on farm location, herd size, and number of animals by age group, sex and type of management. A proportional random sampling within 16 districts was made for the required sample of 102 bulls. The same sampling methodology described in the first study was employed, by trained veterinary officers of the Ministry of Agriculture and Livestock (Northern Region Branch). After sample inoculation, the pouches were sent to the Reproduction Laboratory of the School of Veterinary Medicine, Heredia, arriving 24-36 h after sampling. Microscopic examination was performed upon arrival and daily for 4 days. The potential risk factors evaluated for T. foetus infection rates in the San
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Carlos study are described in Table 1. In the table, veterinary services means reproductive herd health, mating system means one bull with a group of cows or multiple bulls with the cows and supplemental feeding means molasses or minerals versus nothing. The risk factor data were collected through a questionnaire administered by the investigators to the owners/operators. These data were entered into R-BASE for DOS (Microrim, 1987 ) together with the laboratory results, and converted to ASCII files for statistical analysis.
Statistical analysis The units of analysis were either the individual bull or the individual farm. Continuous variables were categorized using quartiles. Crude odds ratios were calculated for risk factors using PROC FREQ (Statistical Analysis Systems Institute, 1989 ), using the first listed category of each dichotomous variable as the reference level. For variables with three or more levels, each level of the variable was compared with all other levels combined as a reference level. Multivariate analysis was performed in the San Carlos study, using EGRET (Statistics and Epidemiology Research Corporation, 1990 ). In the multivariate analysis, the reference level for the variables with three or more classes was chosen using the pattern (protection or risk) of the crude odds ratio. To control for possible herd effects, a logistic-binomial regression model for distinguishable (non-interchangeable) data was used (Mauritsen, 1984). This model was selected because the data had variables measured both at herd level (herd effects) and bull level (individual effects) associated with the individual responses rather than just with the herds. RESULTS AND DISCUSSION
Pouch collection technique In both studies the most positive results were obtained within the first 48 h of culturing. Similarly, the incubation time required for the detection of T. foetus ( 1- 10 trichomonads m l - 1), in laboratory conditions after a single inoculum was 20 h (Borchardt et al., 1992). Only seven samples in the San Carlos study had excessive bacterial and fungal contamination. Positive samples (four) arriving late at the laboratory (36 h) after sampling were still viable. Thomas et al. (1990) described the successful cultivation of parasites in pouches that were sent by mail and not received until 5 days after collection. The InPouch test was convenient both for sampling and diagnosis with the minimal laboratory facilities available in Costa Rica. The pouches could be carried easily to the field in a vehicle without refrigeration, and subsequently be examined directly under the microscope for parasites. This re-
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duced the cost of preparing and maintaining fresh media, as well as the cost of microscope slides and pipettes. Sensitivity for the detection of T. foetus using Diamond's medium (Diamond, 1957) is reportedly 81.6% (Skirrow et al., 1985), with an assumed specificity of 100% (BonDurant et al., 1990). There is only one report of sensitivity for the InPouch TF R system (Thomas et al., 1990). Statistical analysis of these authors' results showed a x statistic (Martin et al., 1987) of 0.77, indicating a close agreement between Diamond's media and the InPouch system. Therefore, the same sensitivity and specificity (Skirrow et al., 1985 ) was utilized in this study to determine the adjusted prevalence rates.
Prevalence In the Tilaran pilot study, 153 bulls were found on the 89 farms, and all were tested. The prevalences of T. foetus infection were 6.7% ( 6/89 ) by farm and 3.9% (6/153) by bull. The herd prevalence rate was lower than those reported in other studies, i.e. 26% in several western states in the USA (Johnson, 1964), 40% in the Northern Territory, Australia (Dennett et al., 1974) and 15.78% in California (BonDurant et al., 1990). The bull prevalence in Tilaran (3.9%) was similar to that (4.1%) found in California (BonDurant et al., 1990). In the present study, within-herd prevalence ranged from 50 to 100% of bulls (mean, 83.3%; median, 75%). For infected herds, the median herd size was one bull (range 1-2 ) and for uninfected herds it was three bulls (range 1-14). The California survey described a within-herd prevalence that ranged from 4 to 38.5% (median, 12.5%; mean, 15.4%), with a median herd size of 14 bulls (range 6-114) and seven (range 1-210) in infected and uninfected herds, respectively. Studies from tropical locations reported withinherd prevalence rates that ranged from 30.6% in Australia (Clark et al., 1974) to 100% in Nigeria (Akinboade, 1980). Perhaps the differences between our results and those of other authors could be attributed to differences in herd size, age when bulls begin sexual activity, population dynamics, and/or the sampling procedure. To our knowledge, the most recent studies are based on convenience samples, with the exception of the studies in California (BonDurant et al., 1990) and San Carlos, which are based on random sampling (making the two latter studies comparable). In the San Carlos study, the overall prevalences were 6.22% ( 14/225 ) for bulls and 15.87% (10/63) for farms. The farm prevalence in this study was equal to that in the California survey (BonDurant et al., 1990); however, within-herd prevalence was higher ( 12.5-100%; mean, 39%; median, 29%). A median herd size was four bulls (range 1-13) in infected and uninfected herds. The difference between the present study and the Californian study may be due to intrinsic differences in the production systems. For example, almost all bulls in San Carlos were over 3 years old (206/225) with all cases
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TABLE2 Descriptive statistics and crude odds ratios (OR) of possible individual-bull risk factors for bovine trichomoniasis in Costa Rica, 1990-1991 Variable
San Carlos
Tilaran
Description No. l Pos. 2 Rate (%) OR
Description No. t
Pos. 2 Rate (%) OR
B. indicus B. taurus
108 45
1 5
1 11
Single Multiple 5-19 20-24 25-34 >35 1-2.9 3-3.9 4-4.9 >5 313-469 470-596 597-697 >698
101 52 29 27 57 31 34 37 29 53 37 38 37 38
5 2 3 4 5 3 0 3 3 7 0 3 5 8
1 0.37 0.78 0.93 1.72 0.78 NC 0.61 0.85 4.00 NC 0.59 1.60 3.19
Breed
B. indicus B. taurus Cross Mating Single Multiple Cows per bull 1-19 20-29 30-39 >40 Bullage 1-2.9 (years) 3-3.9 4-4.9 >5 Bullweight 270-499 (kg) 500-599 600-699 >700 Bull in Yes service No
137 72 16 96 129 34 60 74 57 19 61 56 89 43 44 76 62 190 35
3 10 1 4 10 0 7 5 2 0 1 2 11 2 4 5 3 9 5
2 14 6 4 8 0 12 7 4 0 2 4 13 5 9 7 5 5 14
O. 15 6.02 1.01 1 1.93 NC 3
2.98 1.14 0.47 NC 0.19 0.48 6.25 0.69 1.70 1.09 0.70 1 3.35
5 1 1 1 3 1 0 1 1 4 0 1 2 3
1 13
tNumber of bulls sampled. ZNumber of bulls positive to T. foetus. 3Not calculated. For variables with more than two levels, the reference level for each variable was all other levels combined.
found in these bulls: in California, 46% of the bulls were older than 3 years (328/724) with an infection rate in the older bulls of 6.7%. San Carlos herds were also smaller than Californian herds (Table 2 ). The prevalences reported here are only estimates of the true prevalence for these regions. The true prevalence (TP) can be found by correcting the observed San Carlos prevalences by the method described by Marchevsky (1974)
Tp_Ap+ Sp- 1 Sp+ Se-1 where apparent prevalence (Ap) is taken from the results of culture, sensitivity (Se) is taken to be 81.6% (Skirrow et al., 1985 ) and specificity (Sp) 100% (BonDurant et al., 1990). Using this formula, the true prevalences for San Carlos were calculated as 18.43% and 7.22% for farms and bulls, respectively. Factors that potentially favor transmission of T. foetus were present in San Carlos with 66% of the farms having multiple bulls with their cows, 25.39%
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of the farms using communal bulls and 65% of the bulls in use being older than 4 years. Assuming that after 1 year of exposure to one carrier bull, 42.5% of cows in the herd may become infected (Clark et at., 1983 ), T. foetus infection could make an important contribution to the reduced or low fertility rate in this region.
Risk factors associated with infection Age The infection rate of trichomoniasis usually increases with age (Clark et al., 1974; Skirrow et al., 1985), as was shown in our study (Table 3). This association with age is thought to be due to the development of deeper epithelial crypts in the prepuce of older bulls (Clark, 1971 ). (Although bulls less than 4 years of age are less often carriers of T.foetus (BonDurant, 1985 ), they can be infected (Kimsey et al., 1980, Skirrow et at., 1985; BonDurant et al., 1990).)
Breed A strong association was found (Table 3) between the Bos taurus breed types and risk of T. foetus infection, as was also the case in the California TABLE 3 Logistic-binomial regression model for possible risk factors of bovine trichomoniasis in San Carlos, 1991 Term
fl
SE (fl)
P-value
OR
95% CI (OR)
Intercept Stocking rate ( < 1.26 vs. > 1.27) ~ Production system (beef vs. other) Communal bull (No vs. Yes) Veterinary services (No vs. Yes) Cows per bull (1-9, >40 vs. other) Breeding policy (Cont. vs. Seas. ) Mating policy (single vs. multiple) Bull in service (Yes vs. No) Breed (B. indicusvs. B. taurus) Bull age ( < 3 yearsvs. > 3 years) Bull weight (270-499 kg, > 700 kg vs. other) Farming experience ( 1-10 years, > 25 years vs. other) Farming only (No vs. Yes) Feed supplement (No vs. Yes) Intercept of random effects
- 11.71 -0.99 1.61 1.71 - 1.43 1.93 1.38 -0.75 2.48 1.94 2.84 0.67
2.91 0.85 1.68 1.14 0.97 1.11 1.11 1.67 1.06 0.79 1.25 0.75
< 0.001 0.24 0.34 0.14 0.14 0.08 0.21 0.65 0.02 0.02 0.02 0.37
0.37 4.99 5.51 0.24 6.92 3.96 0.47 11.88 6.94 17.15 1.96
0-1.9 0.1-134.9 0.5-51.4 0-1.6 0.7-60.4 0.4-34.7 0-12.3 1.4-94.2 1.4-32.8 1.4-199.8 0.4-8.6
1.27
0.95
0.19
3.55
0.5-23.2
1.26 0.11 0
1.23 1.88 0.60
0.31 0.95 -
3.54 1.12 -
0.3-39.6 0-44.23 -
IFirst level considered as reference level.
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prevalence survey. Several researchers have suggested that certain breeds may be more susceptible to trichomoniasis (Abbitt, 1980; Kimsey et al, 1980; Ball et al., 1987), but others have not found any evidence for breed predisposition (Skirrow and BonDurant, 1989). Dennett et al. (1974) did not find differences between Bos indicus and B. taurus breeds in 689 animals studied, but possible confounding factors (such as age) were not taken into account in their study. In our study, B. taurus bulls had at least 6.93 times the risk of infection with T. foetus than B. indicus bulls. While intrinsic differences in susceptibility amongst breeds may exist (Abbitt, 1980; Ball et al., 1987), the prevalence difference could be due to the increased number of matings accomplished in B. taurus bulls compared with B. indicus bulls in the same period of time (Galina and Arthur, 1991 ), thus increasing the risk of exposure to infection. Bull in service In the San Carlos study, this variable was assessed, because few bulls could be sampled during sexual rest, as generally recommended (BonDurant et al., 1990). As expected, bulls in sexual rest (no virgin bulls were sampled) at the moment of sampling had a greater risk of being infected than bulls in service. Clark et al. (1983) suggested that a depletion of the preputial T. foetus population may occur as a result of repeated matings. For this reason, the true prevalence of trichomoniasis in the present survey may have been underestimated. CONCLUSION
The InPouch test proved to be a,practical and convenient method for detecting T. foetus in samples from infected bulls in Costa Rica. The studies assessed risk factors for bovine trichomoniasis under tropical conditions for the first time. Species-specific risk of infection of B. taurus bulls and age were the most meaningful risk factors detected. Trichomonasfoetus infection may be an important contributor to reproductive failure in Costa Rican beef and dual purpose herds, and its economic impact should be assessed. ACKNOWLEDGMENTS
This research was supported by the Dutch Interuniversity Cooperative Program (Perez) and University of California, Davis, Livestock Disease Research Laboratory (Conrad and Hird). The authors thank Jim Self and Dr. Mike Thomas for making available the InPouch tests and the veterinarians of the North Region Branch of the Ministry of Agriculture and Livestock, Costa Rica, San Carlos and Tilaran for their assistance.
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REFERENCES Abbitt, B., 1980. Trichomoniasis in cattle. In: D.A. Morrow (Editor), Current Therapy in Theriogenology. WB Saunders, Philadelphia, PA, pp. 482-488. Akinboade, O.A., 1980. Incidence of bovine trichomoniasis in Nigeria. Rev. Elev. Med. Vet. Pays Trop., 33: 381-384. Amelingmeier, R.C., 1981. Estudio preliminar de diagnosis de Trichomonasfoetus en Costa Rica. Tesis. Mrdico veterinario. Heredia, Costa Rica. Escuela de Medicina Veterinaria, Universidad Nacional, 32 pp. Ball, L., Dargatz, D., Cheney, J.M. and Mortimer, R.G., 1987. Control of venereal disease in infected herds. Vet. Clin. N. Am., Large Anim. Pract., 3: 561-564. BonDurant, R.H., 1985. Diagnosis, treatment and control of bovine trichomoniasis. Comp. Contin. Educ., 7: S 179-$216. BonDurant, R.H., Anderson, M.L., Blanchard, P., Hird, D., Danaye-Elmi, C., Palmer, C., Sischo, W.M., Suther, D., Utterbach, W. and Weighler, W., 1990. Prevalence of trichomoniasis among California beef herds. J. Am. Vet. Med. Assoc., 196:1590-1593. Borchardt, K.A., Norman, B.B., Thomas, M.W. and Harmon, W.M., 1992. An evaluation of the InPouchTFTM culture system for the diagnosis of Trichomonasfoetus infection. Vet. Med., in press. Clark, B.L., 1971. Venereal diseases of cattle. Veterinary Review. No. 1. University of Sidney, pp. 5-26. Clark, B.L., Parsonson, I.M. and Dufty, J.H., 1974. Experimental infection of bulls with Trichomonasfoetus. Aust. Vet. J., 50: 189-191. Clark, B.L., Dufty, J.H. and Parsonson, I.M., 1983. The effect of Trichomonasfoetus infection on calving rates in beef cattle. Aust. Vet. J., 60:71-74. Dennett, D.P., Reece, R.L., Barasa, J.O. and Johnson, R.H., 1974. Observations on incidence and distribution ofserotype of Trichomonasfoetusin beef cattle in North-Eastern Australia. Aust. Vet. J., 50: 427-431. Diamond, L.S., 1957. The establishment of various trichomonads of animals and men in axenic cultures. J. Parasitol., 43: 488-490. Galina, C.S. and Arthur, G.H., 1991. Review of cattle reproduction in the tropics. Part 6: The male. Anita. Breed. Abstr., 59: 403-412. Hird, D.W. and Robinson, R.A., 1982. Dairy farm wells in southeastern Minnesota: The relation of water source to calf mortality rate. Prev. Vet. Med., 1: 53-64. Honigberg, B.M., 1978. Trichomonasfoetus. In: J.P. Kreier (Editor), Parasitic Protozoa. Academic Press, New York, pp. 207-273. Johnson, A.E., 1964. Incidence and diagnosis of trichomoniasis in western beef bulls. J. Am. Vet. Med. Assoc., 145: 1007-1010. Kimsey, P.B., Darien, B.J., Kendrik, J.W. and Franti, C.E., 1980. Bovine trichomoniasis: diagnosis and treatment. J. Am. Vet. Med. Assoc., 177:616-619. Levine, N.D., 1973. The trichomonad. In: N.B. Levine (Editor), Protozoan Parasites of Domestic Animals and of Man. Burgess, Minneapolis, MN, pp. 88-110. Marchevsky, N., 1974. Errors in prevalence estimates in population studies: a practical method for calculating real prevalence. Zoonosis, 6:98-109. Martin, S.W., Meek, A.H. and Willeberg, P., 1987. Veterinary Epidemiology, Principles and Method. Iowa State University Press, Ames, pp. 73-74. Mauritsen, R.H., 1984. Logistic regression with random effects. Ph.D. Thesis, Department of Biostatistics, University of Washington, 84 pp. Microrim Inc, 1987. R: BASE for DOS, User's Guide, version 2.0. National Census of Costa Rica, 1984. Oficina de Estadistica y Censos, Ministerio de Planificacion Nacional, San Josr, Costa Rica.
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Skirrow, S.Z. and BonDurant, R.H., 1988. Bovine trichomoniasis. Vet. Bull. (London), 58: 592-603. Skirrow, S.Z., BonDurant, R.H. and Farley, J., 1985. Efficacy of ipronidazole against trichomoniasis in beef bulls. J. Am. Vet. Med. Assoc., 187: 405-407. Statistical Analysis Systems Institute, 1989. User's Guide: Statistics, Version 6 edn. SAS Institute, Cary, NC. Statistics and Epidemiology Research Corporation, 1990. EGRET Statistical Package Users Manual. SERC, Seattle, WA. Thomas, M.W., Harmon, W.M. and White, B.S., 1990. An improved method for the detection of Trichomonasfoetus infection by culture in bulls. Agri-Practice, 11: 13-17. Wilson, S.K., Kocan, A.A., Gaudy, E.T. and Goodwin, D., 1979. The prevalence of trichomoniasis in Oklahoma beef bulls. Bovine Pract., 14:109-110, Zamora, J.L., 1982. Diagn6stico laboratorial de tricomoniasis en toros de varias zonas de Costa Rica mediante la t6cnica de cultivo. Tesis. M6dico Veterinario. Heredia, Costa Rica, Escuela de Medicina Veterinaria, Universidad Nacional, 44 pp.