Pooled Polymerase Chain Reaction to Detect Tritrichomonas Foetus in Beef Bulls

Abstract

Abstract. Preputial scraping samples from 305 mixed breed beef bulls were examined for the detection of Tritrichomonas foetus infection. All samples were collected by veterinarians and transported in commercial media to an accredited lab. Upon arrival samples underwent microscopic examination for the presence of Tritrichomonas foetus and were then incubated until 5 days postcollection before final microscopic examination. Culture detected 14 samples with Trichomonad spp.; all were confirmed to be Tritrichomonas foetus by polymerase chain reaction (PCR). After final examination samples were randomly placed in groups of 5 samples; technicians were blinded as to culture results of the individual samples constituting each pool. From each sample within a group, a portion of the fluid sediment was removed and pooled with the other samples of the group to form 61 pools. From each of the formed pools an aliquot was removed for PCR. PCR detected 16 positive pools; an additional 2 positive samples were then identified on individual PCR on samples previously diagnosed as culture negative. Relative to culture, the 95% confidence intervals for sensitivity and specificity of PCR pools to detect Tritrichomonas foetus were 76.8% to 100% (mean value: 100%) and 85.5 to 99.5% (mean value: 93.4%), respectively.

Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection ofTritrichomonas foetus–colonized bulls

“…However, a protocol using pooled cultured smegma samples utilizing endpoint PCR has been previously reported. 12 Using the pooling methodology outlined in the present study, a slight difference in sensitivity was observed between the 1/5 and 1/3 sample pools when compared to testing the samples individually. Utilizing the study laboratory's protocol, pooling at a 1/5 dilution produced better sensitivity than the average reported sensitivity (approximately 90%) for a single cultured smegma sample tested by microscopy 5,8,15 and was equivalent to the stated sensitivity of 95% or higher, 21 obtained by collecting and culturing 3 separate smegma samples with 7 days of sexual rest between collections.…”

Tritrichomonas foetus in Beef Bulls

“…A method to lower the cost of using PCR is to pool samples. A published study using a pooled PCR strategy with culture as the criteria showed an increase of diagnostic sensitivity to detect the organism over culture existed, and that the increased diagnostic sensitivity could be accomplished without a significant increase in the price of diagnostic testing [7,[15][16][17]. However further study is required before any conclusion can be drawn.…”

“…In order to confirm a herd is infected with T. foetus it is necessary to demonstrate the organism or it's DNA. This can be done by direct microscopic examination, culture followed by microscopic examination and/or PCR of Preputial Scrapings (PPS) or cervical mucus [3][4][5][6][7].…”

Effects of bacterial contamination of media on the diagnosis of Tritrichomonas foetus by culture and real-time PCR

“…Infected cows often mount an immune response that is effective in clearing T. foetus from reproductive tissues (Cobo et al, 2002;Ondrak, 2010); however, they can act as a source of infection for unaffected bulls in the herd if re-bred before the organisms are eliminated (Kennedy et al, 2008). There are no approved treatments that will clear the infection once it is established; consequently, control involves the recognition and rapid removal of infected animals from the herd, and is the best way to minimize losses due to T. foetus (Cobo et al, 2002;Ondrak, 2010;Perez et al, 2006;Rodning et al, 2008).…”