Detection of Tritrichomonas foetus in the Bull

Abstract

Bovine trichomoniasis is a venereal disease in cattle that causes severe reproductive loss and can have devastating economic impact on the cattle industry. Bovine trichomoniasis is caused by a protozoal organism that inhabits the reproductive tract of both female and male cattle. Infected females suffer clinically with inflammatory gynecologic disease and resultant reproductive losses. Bulls are considered clinically asymptomatic carriers. Infected bulls can spread the disease readily throughout cattle herds. Thus, it is important to ensure bulls are tested and determined to be negative for Tritrichomonas foetus (T. foetus) prior to being utilized for breeding purposes. The importance of being able to consistently identify T. foetus in breeding bulls is crucial for control and eradication of T. foetus from cattle herds. The focus of this study was to determine if there are additional reservoir sites of T. foetus in portions of the reproductive tract, specifically the urethra and the secondary sex glands in bulls chronically infected with T. foetus. The identification of reservoir sites for the organism in the proximal reproductive tract of the bull has the potential to have detrimental effects on the possibility of a topical curative treatment in bulls. Eight naturally infected T. foetus positive adult Bos taurus bulls ranging in age from four to 14 years of age were donated to Auburn University College of Veterinary Medicine (AUCVM) for research purposes. Five of the bulls were Angus and/or Angus cross bulls and three were Charolais. Upon admission to AUCVM, all bulls were tested for T. foetus. A preputial scraping using a Pizzle Stick (Lane Manufacturing, Denver, CO) was performed to obtain a reliable smegma sample for T. foetus testing. All smegma samples were placed in individual labeled vials containing Modified Diamond’s Media (MDM) (Thermo Fisher Scientific; Waltham, MA) and submitted to the Thompson-Bishop-Sparks-Alabama State Diagnostic Laboratory (TBSASDL) for testing for live organisms via culture, and testing for T. foetus DNA via Real Time Polymerase Chain Reaction (RTPCR) (VetMAX™-Gold Trich Detection kit, Thermo Fisher Scientific; Waltham, MA). All bulls were tested at least five times over the course of five months via culture and RTPCR to determine the chronicity of the T. foetus infection. Following testing, the bulls were sedated, euthanized, and submitted to the TBSASDL for necropsy. The reproductive tracts in their entirety were removed and placed on clean tables for sectioning. The tracts were dissected from the area of least potential contamination to the area of the most potential contamination. Sterile technique was used during dissection and sampling of the urogenital tracts. Three bulls had 1.0 cm x 1.0 cm sections cut from the right and left ampullae, right and left vesicular glands, the prostate, and sections of urethra, penis, and prepuce. The sections of the urethra included 5 cm proximal from the distal end of the urethra, 23 cm proximal from the distal end of the urethra, 5 cm distal to the last bend of the sigmoid, 13 cm proximal to the sigmoid at the level of the trigone of the bladder. The five other bulls had tissue collected via scrapings of the inside surface of the glands, urethra at the specified locations as described above, and the penis and the prepuce. Following the collection of the tissues, all samples were placed in individual vials of Modified Diamonds media (MDM) to remove the smegma and any tissue collected by the scraping. All samples were submitted to the TBSASDL for testing for T. foetus via culture for live organisms and RTPCR for T. foetus DNA. The prostate, vesicular glands, ampullae, bladder, and the urethra proximal to the sigmoid flexure were all found to be negative on culture for live organisms and for T. foetus DNA. All bulls were determined to be chronically infected with T. foetus. All smegma samples from bulls were found to be positive for T. foetus on culture. All smegma from the preputial scraping, penis, and the urethral sections 5 cm proximal from the distal end of the urethra, and 23 cm proximal from the distal end of the urethra were all found to be positive for T. foetus DNA via RTPCR with a cycle threshold (CT) of 35.0, 33.5, 34.3, and 36.0, respectively for Bull 1 and 35.6, 30.6, 32.2 and 32.0 respectively for Bull 2 and 30.1, 30.6, 36.4, and 35.5, respectively for Bull 3. Bull 2 and Bull 3 were positive 5 cm distal to the last bend of the sigmoid with CT values of 37.4 and 35.0, respectively. Additionally, Bulls 6, 7, and 8 were determined to be positive for T. foetus DNA at the level of the distal urethra 5 cm from the distal end of the penis with CT values of 38.0, 33.8, and 35.0 with preputial scrapings having CT values of 29.6, 34.5 and 31.2, respectively. However, Bulls 4 and 5 were only found to be positive for T. foetus DNA only on preputial scrapings with CT values of 30.0 and 33.5, respectively with all other areas of the reproductive tract sampled negative for T. foetus DNA via RTPCR. A total of six of the eight (75%) bulls sampled in this study were found to be positive for T. foetus DNA in the distal urethra. Three of the eight (37.5%) bulls had T. foetus DNA detected in the lower half of the urethra. Two of the eight (25%) bulls were positive for T. foetus DNA near the distal bend of the sigmoid flexure. Eleven of 24 tests for T. foetus DNA in the urethra of naturally infected T. foetus bulls were found to be positive. The significance of this finding was evaluated utilizing a one tailed, one sample proportion z test with an α = 0.05, utilizing Excel 2016 (Microsoft Co; Redmond, WA). It was determined that the proportion of urethral samples positive for T foetus DNA was a significant finding (p <0.05, z =2.441). The study herein built upon the findings of Rush et al., and further documents T. foetus DNA in a more proximal location in the urethra in naturally infected Bos taurus bulls (Rush et al., 2020). There are few reports stating that T. foetus can be found in the distal urethral orifice of some bulls (Michi, 2016). These studies were performed prior to the development and wide use of RTPCRs so it is possible that false negatives and false positives may have occurred during testing procedures. This study provides important information for researchers looking into the clearance of T. foetus from chronically infected bulls.