Effects of Biological Materials and Collection Media on PCR Detection of Tritrichomonas foetus

Tritrichomonas foetus is an important pathogen of the bovine reproductive tract causing early embryonic death and abortion in cows and persistent, asymptomatic infection in bulls. PCR detection methods have greatly enhanced diagnostic accuracy over culture; however, pre-analytical sample handling is just as critical as technical performance in detecting this pathogen and is not well studied. The purpose of this study was to investigate the effects of biological materials present in the prepuce on PCR detection of T. foetus in a variety of collection media. Simulated preputial samples were created using InPouch (IP) media, lactated ringers solution (LRS), or sterile saline (SAL); inoculated with low numbers of one of three T. foetus strains; and spiked with either blood, semen, urine, or sham treatment. Samples were transported to the lab, placed in growth media (LRS and SAL samples), incubated, and tested for T. foetus by PCR. Samples containing urine had statistically significantly greater mean Ct values (P = 0.008) than samples containing other materials, seen most dramatically in IP (P < 0.0001.) Urine contamination resulted in significantly (P = 0.037) fewer samples being identified as “positive” for T. foetus. Overall, SAL collections also had significantly higher mean Ct than IP or LRS (P < 0.001), and were less likely (P = 0.018) to results in classification as a “positive” sample. Results of this study indicate that collection media and biological materials can affect T. foetus PCR detection. The presence of urine in preputial samples can result in false negative results, while blood had no detrimental effects.


Introduction
Tritrichomonas foetus is an important pathogen of the bovine reproductive tract causing early embryonic death and abortion [1] [2]. This protozoal organism colonizes the epithelium of the penis and prepuce in the bull and has been identified in the distal urethra; however, not in the penile or prostatic urethra, accessory sex glands, nor epididymis [1] [3]. Attachment to urogenital epithelium is thought to occur via glycoprotein adhesins which results in minimal immune recognition or site-specific inflammation; consequently, clearance of the organism is rare, particularly in older bulls [1] [3] [4] [5].
Transmission of T. foetus to cows primarily occurs during breeding and is highly efficient, with up to 95% of females becoming infected after a single mating [6] [7] [8]. Economic impacts of T. foetus on a herd include loss of income from fewer and lighter-weight calves to market at weaning, loss of genetic potential from culled animals, costs of replacing infected bulls, laboratory fees for diagnostic testing, and feed costs for non-productive animals [1] [7] [9]. No approved treatments exist to clear the infection; consequently, identification and removal of infected bulls is the best way to minimize T. foetus impacts [8] [9].
Molecular diagnostic methods with high sensitivity and specificity have greatly improved the analytical detection of T. foetus [10] [11]. Pre-analytical sample handling, however, can account for up to 62% of diagnostic errors and it is critical to address sample collection and transport when evaluating diagnostic accuracy [9] [12]. The purpose of the present study was to evaluate the presence of biological materials that are commonly present in bull preputial samples on the PCR detection accuracy of T. foetus utilizing three different collection media.

Sample Preparation
The collection media used in this study included InPouch TM (IP) media (individual pouch), 1.5 mL of LRS, or 1.5 mL sterile saline (SAL); the three T. foetus strains used were the laboratory quality control strain, a clinical isolate from 1986 and confirmed by phenotypic and genotypic methods [

Real-Time PCR Assay
Testing on IP was conducted as previously described [11]. Briefly, a 1 mL volume of media was centrifuged at 3500 rpm at room temperature for 3 min, decanted, and re-suspended in 300 µL of 1 × PBS. Due to the presence of agar in DPM that can interfere with PCR reactions [14], testing performance in this medium was optimized using centrifugation of the 1mL aliquot at 3500 rpm at room temperature for 3 min followed by decanting of supernatant, and re-suspension of the pellet in 200 µL of 1 × PBS. DNA extraction was performed using MagMAX 96 Viral RNA Isolation Kit for total nucleic acid from low-cellularity biological samples (Life Technologies, Grand Island, NY) modified for T. foetus extraction with a final volume of 85 µL. Real time PCR was performed in a 33 µL reaction mixture containing 8 µL of template DNA; 1 µL of VetMAX T. foetus primer-probe reagent (Life Technologies, Grand Island, NY); 12.5 µL of VetMAX Plus qPCR Master Mix, (Life Technologies, Grand Island, NY); 3.5 µL of sterile water, and 8 µL of internal amplification control (IAC) DNA (Life Technologies, Grand Island, NY). PCR was conducted on the ABI 7500 Fast (Sunnyvale, CA); reactions included initial polymerase activation at 95˚C for 10 min, followed by 40 cycles of DNA dissociation at 97˚C for 5 s and annealing/extension at 55˚C for 40 s. Results were expressed as cycles to threshold (Ct) values, and simulated samples were PCR-tested in triplicate to obtain mean Ct values for each. Non-T. foetus, internal amplification control (IAC) DNA was included with all reactions to ensure that the PCR reactions performed as expected and no PCR inhibition was present in tested samples. Positive and negative extraction and amplification controls were tested with each run to ensure consistent performance of the assay.

Evaluation of Biological Materials and Collection Media on T. foetus Detection by PCR
The effects of the biological material on T. foetus PCR detection are shown in Figure 1.

Assessment of Biological Material and Collection Media on T. foetus Classification
Percentage of positive samples detected using the laboratory cut-off is shown in

Discussion
Identification of bulls, which are colonized with T. foetus, continues to be a costly and frustrating problem to the cattle industry [1] [3]. As a fastidious organism which is susceptible to DNA degradation in the face of imperfect environmental conditions, T. foetus detection is critical to any control program [15] [16]. The implementation of diagnostic methods with high sensitivity and specificity such as real-time PCR can dramatically improve identification of infected bulls, particularly those that are colonized with low numbers of organisms expected during natural infection [15] [17]. In the present study, samples were created with a low number of T. foetus (50 -60 organisms/ml in IP system) to assess how sample conditions affect DNA detection in these challenging samples.
This study demonstrates the importance of mimicking true sample conditions, including the use of field isolates, when evaluating diagnostic procedures for accuracy. The presence of urine interfered with DNA detection, resulting in higher mean Ct values for all three T. foetus strains evaluated in this study; however, the effects were seen most dramatically in field strains, with one-third (FS1) to one-half (FS2) of samples producing false negative results. T. foetus has been shown to be susceptible to extremes in pH, with optimal survival and DNA detection at 6.8 -7.0, and can tolerate ranges of 6.0 -8.5 [16] [18]. The urinalysis on this bull showed his urine pH to be 9.0, which may account for the greater Ct values and lower detection in SAL samples compared to IP and LRS which are designed to maintain a neutral pH. Additionally, our laboratory classifies bulls with high Ct values (37 -39) as "inconclusive" since these values are near the limit of detection for the assay. PCR testing in this study identified two samples each spiked with blood or semen but seven samples spiked with urine fell into in this category.
Overall, samples created in SAL had significantly greater mean Ct values for T. foetus, even though samples were inoculated into DPM culture media within four hours of collection. Isotonic saline appears to do a poor job of maintaining conditions that preserve T. foetus for detection. LRS provides superior buffering capacities under physiologic conditions and was shown to maintain pH significantly better than isotonic saline even within the first 2 hours of administration [19].
There were several limitations to the present study. The T. foetus spiking concentration was selected to mimic bulls colonized with low numbers of organ- The present study demonstrates that to maximize T. foetus detection, particularly from bulls harboring low numbers of organisms, collection procedures should include use of a media which provides an environment simulating physiologic conditions and avoiding urine collection at the time of preputial sampling. Even though T. foetus strains collected into SAL had only four hours of contact before DPM inoculation, DNA detection was significantly impaired over IP and LRS collections, which may indicate that a more complex and balanced support media is needed to support T. foetus prior to arrival at a diagnostic facility.