Evaluating the stability of Tritrichomonas foetus in various media under simulated environmental conditions of shipment for RT-qPCR assays

Abstract

Tritrichomonas foetus is a notable reproductive pathogen in cattle, and the processes of sample collection, handling, transportation, and testing present considerable challenges for surveillance initiatives. Advancements include the development of a direct detection method for T. foetus using reverse-transcription quantitative real-time PCR. We compared the stability of samples in 3 different media: PBS, sterile saline, and lactated Ringer solution over periods of 24–120 h, with storage temperatures of 0°C, 20°C, 37°C, and 54.5°C to mimic shipping of samples in various environmental temperatures.

Tritrichomonas foetus is an obligate protozoan parasite of the bovine reproductive tract, meaning it has no free-living stage and cannot complete its lifecycle without a host. The parasite is found on the penis and in the prepuce and distal urethra of infected bulls, and from the cranial vagina to the oviduct of infected cows. ¹⁷ It is the causative agent of bovine trichomonosis, 1 of 2 true venereal diseases in cattle, with transmission of the agent only occurring during coitus. ¹³

Bulls have no clinical signs associated with a T. foetus infection and do not mount a sufficient immune response to clear the infection; therefore, an infected bull is considered chronically infected.^2,3,14 T. foetus infection in heifers and cows, on the other hand, can result in endometriosis, cervicitis, and vaginitis; these infections frequently lead to embryonic death, abortion, pyometra, and fetal maceration.^7,15 In a cow–calf operation, such productivity losses can be highly detrimental. Moreover, the absence of clinical signs in males underscores the importance of accurate testing for the maintenance of herd health and biosecurity.

In 2023, cattle generated $101 billion in cash receipts in the United States, making up 40.5% of the animal and animal product revenue (https://www.ers.usda.gov/data-products/chart-gallery/gallery/chart-detail/?chartId=76949). Cattle production in the United States is a big business that relies on successful calf production. The cattle industry, and the entire beef supply chain, are negatively impacted by a reduction in the number of calves produced. Loss of embryos or fetuses increases the cost of production for cow–calf operators, and subsequent increased costs are spread through the industry as fewer animals enter the supply chain. The success of breeding can be severely impeded by the complications associated with trichomonosis. A survey conducted in New Mexico determined that the economic impact on the livestock enterprise of T. foetus infections is >$400 per cow. ¹⁶ Furthermore, because bulls show no clinical signs and are chronically infected by T. foetus, infected bulls must be culled from the herd as there is no legal means of treatment in North America. The strategy of testing and culling positive bulls has demonstrated some success in managing disease transmission in individual state programs, as shown in Wyoming. ¹⁷ To reduce the frequency of replacing breeder bulls—a costly proposition for an operation—biosecurity measures, including the screening of herd sires, are recommended for early detection of T. foetus. ¹⁰

A preputial sample is collected by using a sterile pipette to scrape the preputial surface while aspirating and is submitted for T. foetus testing. This assay typically involves using commercial kits to ensure accurate detection. In addition to the traditional InPouch medium (Biomed), modified Diamond medium (Remel) and physiologic saline have been used for T. foetus testing.^3,12 However, after recognizing the potential for overgrowth of bacteria resulting in false negatives, and the development of reverse-transcription quantitative real-time PCR (RT-qPCR) testing, PBS has been shown to be a more reliable alternative for detecting T. foetus, especially when bacterial contamination is a concern. ⁶ PBS was the original medium tested and validated with RT-qPCR for T. foetus. Sterile saline (SS) has also been validated in some laboratories. ¹¹ In Texas, the Texas Veterinary Medical Diagnostic Laboratory (TVMDL; College Station, TX, USA) mandates that T. foetus samples be submitted for state-regulated testing in either PBS or SS (https://tvmdl.tamu.edu/tests/tritrichomonas-foetus-bovine-rtpcr/). Other states have similar guidelines. PBS and SS are crucial for maintaining the stability of the parasite during transport to the laboratory. We explored the possibility of lactated Ringer solution (LRS) as a potential holding medium for T. foetus samples. LRS is a common intravenous fluid used in both large and small animals and would offer practitioners another suitable medium if PBS or SS were not available at the time of sampling.

Given the vast geographic expanse of the U.S. cattle industry, and subsequently the geographic expanse of T. foetus, there are environmental challenges to consider when submitting samples for testing. T. foetus, a delicate organism with no free-living stage, is highly susceptible to environmental stressors such as temperature extremes. In the Texas Panhandle, where we conducted our study, temperatures can fluctuate dramatically—from as low as −21°C to as high as 20°C within a single day. ⁹ Improper handling or exposure to adverse conditions during transit could compromise the integrity of the sample, leading to inaccurate test results, as demonstrated by a previous study. ⁶ Specifically, degradation of the RNA in samples increases the likelihood of false negatives in qPCR testing. A false negative represents a large threat to a cow–calf operator because the infection can persist in bulls and additional animals in the herd could become infected. Ensuring that samples are adequately protected during shipping and choosing the appropriate shipping timeline can help prevent costly testing errors and complications for cattle producers, ultimately supporting the health and productivity of the cattle industry.

We compared the stability of T. foetus samples maintained in 3 media (PBS, SS, or LRS) over times of 24–120 h, under storage temperatures of 0°C, 20°C, 37°C, or 54.5°C. These conditions were selected to mimic those that samples shipped in Texas are likely to experience. We hypothesized that LRS would perform similarly to SS in preserving T. foetus sample viability. Additionally, we expected that temperatures <20°C and >37°C would affect the samples negatively, leading to false negatives or higher Cq values compared to samples from the same individual shipped at more optimal temperatures.

Materials and methods

Bulls and husbandry

Our study was conducted under a Texas Tech IACUC-approved protocol (2022-1177). Eleven sexually mature bulls previously determined to be naturally infected with T. foetus through preputial samples tested by RT-qPCR at a state diagnostic laboratory were purchased for our study. These bulls were 2–6-y-old, and all were of English or Continental breeding (Angus, Charolais, Hereford). Upon arrival at the research facility, all bulls were reconfirmed positive for T. foetus via RT-qPCR testing. The animals were housed as a herd in a dirt paddock and fed a total mixed ration at the Bushland Research Facility (Bushland, TX, USA). All animals remained infected throughout the study.

Sampling methodology

Preputial samples were collected using the Pizzle Stick Trich testing device (Lane Manufacturing) attached to a sterile 20-mL syringe. The device was inserted into the sheath and guided caudally to just before the preputial fornix. Negative pressure was maintained on the syringe as 20 back-and-forth strokes were performed, focusing on the midshaft and caudal portion of the free penis, where the highest concentration of T. foetus organisms is typically found. ¹³

Once obtained, samples were transferred to a sterile cryovial containing 1.5 mL of LRS, SS, or PBS. For each bull, one sample was collected in each medium, for a total of 33 samples per sampling event. The procedure was repeated every 7 d to allow for the 4 temperature trials. Immediately post-collection, the samples were placed in a Styrofoam cooler to be protected from the sun and other elements.

To simulate the effects of time and temperature during delayed shipping and suboptimal shipping conditions, the samples were packaged according to diagnostic laboratory protocols and exposed to 4 experimental temperatures: 0°C, 20°C, 37°C, or 54.5°C. All shipping boxes were packed at room temperature. The samples held at 0°C were placed in a cardboard sample-collecting box, sealed with tape, and placed within a Styrofoam shipping box. Packing paper was added to minimize dead space and provide insulation to protect from freezing temperatures. The Styrofoam shipping box was placed in its cardboard shipping box, closed, and placed in a frost-free freezer set at 0°C. For the higher temperature trials (20°C, 37°C, or 54.5°C), the protocol was similar, but included ice packs (1 ice pack for 20°C, 2 ice packs for 37°C, 3 ice packs for 54.5°C) to help regulate temperature, then placed in an incubator (PHCbi; PHCNA) set at the designated temperature. A reusable temperature and humidity data logger (RC-5+; Elitech Technology) was placed in each box and set to collect data every 2 min.

All samples were tested by our RT-qPCR assay in-house at our Infectious Disease Diagnostic Laboratory (Texas Tech, Amarillo, TX, USA). Our testing included automated nucleic acid extraction and purification and RT-qPCR following all procedures and controls developed and validated in a previous T. foetus testing study, which is employed as the preferred detection method at the TVMDL. ⁸ We used the MagMax 96 Viral RNA isolation kit (Life Technologies) and an automated magnetic particle processor (KingFisher-96; ThermoFisher). An aliquot (50 μL) of each sample was pipetted into a well in a 96-well deep-well plate with 130 μL of lysis binding solution and 20 μL of bead mix.

The qPCR reaction mix consisted of 5 μL of extracted RNA, 6.25 μL of 4× master mix, 1.25 μL of 20× primer−probe mix, and 12.5 μL of nuclease-free water. The 20× primer−probe mix contained T. foetus primers and probe oligonucleotides. ⁸ Thermocycler conditions were 25°C for 2 min (single cycle), 50°C for 15 min and 95°C for 2 min (single cycles), and 95°C and 15 s and 55°C for 45 s (40 cycles). Samples with a Cq ≤ 35 were considered positive based on published standards. ⁸

Statistical analysis

RT-qPCR results were uploaded into R statistical software (v.4.0.3, https://www.r-project.org/). Model selection was made using adjusted R² and likelihood ratio tests. Two-tailed t-tests were conducted as part of the regression analysis with a significance level set at α = 0.05. We identified individual bull, time, temperature, and medium type as our variables of interest. To explore the relationships among these predictor variables and the response variable (Cq), and control for unobserved differences, we constructed several models. For these models, we used 20°C and PBS as the reference values, as 20°C is a relatively neutral environmental temperature in which samples do not experience extreme heat or cold during shipment, and PBS is the medium recommended by TVMDL for T. foetus storage. The initial model accounted for a nonlinear relationship between time and Cq to account for our hypothesis of increasing RNA degradation over time. Informed by results from the first model, the second model also includes interactions between time and temperature, and time and media, to test our hypotheses that temperature and medium influence the relationship between time and Cq.

$$\begin{array}{c}\mathrm{M}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{l}1:\mathrm{C}\mathrm{q}={\mathrm{\beta }}_0+{\mathrm{\beta }}_1\mathrm{b}\mathrm{u}\mathrm{l}{\mathrm{l}}_i+{\mathrm{\beta }}_2\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\\ +{\mathrm{\beta }}_3\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{s}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{d}+{\mathrm{\beta }}_4\mathrm{t}\mathrm{e}\mathrm{m}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\\ +{\mathrm{\beta }}_5\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\end{array}$$

$$\begin{array}{c}\mathrm{M}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{l}2:\mathrm{C}\mathrm{q}={\mathrm{\beta }}_0+{\mathrm{\beta }}_1\mathrm{b}\mathrm{u}\mathrm{l}{\mathrm{l}}_i+{\mathrm{\beta }}_2\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\\ +{\mathrm{\beta }}_3\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{s}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{d}+{\mathrm{\beta }}_4\mathrm{t}\mathrm{e}\mathrm{m}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\\ +{\mathrm{\beta }}_5\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}+{\mathrm{\beta }}_6\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}*\mathrm{t}\mathrm{e}\mathrm{m}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}\\ +{\mathrm{\beta }}_7\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}*\mathrm{m}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\end{array}$$

Results

Model 1 yielded statistically significant results for all included predictor variables: individual bull, time, time squared, temperature, and media (Suppl. Table 1). Although each variable significantly impacted the Cq value, not all were biologically relevant. For example, although individual bull variation was statistically significant, this variation is expected and does not hold biological relevance for the model.

Surface plots of average (across animal samples) deviations in Cq values were made relative to a baseline value obtained at 0°C and 24 h storage, as time and temperature for sample holding varied (Fig. 1). A statistically significant (p ≤ 0.05) positive correlation between time and Cq values was observed, with a parameter estimate of 0.08 (Suppl. Table 1). This indicates that Cq values generally increased over time (Fig. 1). Lower temperatures (0°C, 20°C) were significantly correlated with lower Cq values (p < 0.05). Positive PCR results occurred earlier in samples stored at these temperatures (Fig. 1). The estimated coefficients in the regression model (parameter estimates) for 0°C and 20°C were −1.23 and −2.16, respectively (Suppl. Table 1).

Figure 1.

Average deviations of Cq values for Tritrichomonas foetus samples from baseline shipping condition (0°C, 24 h) by time and temperature for different media (turquoise 0–1 = deviations from baseline; gray = 1–2 Cq deviations from baseline red = 2–3 Cq deviations from baseline; blue = 3–4 Cq deviations from baseline; brown = 4–5 Cq deviations from baseline; green = 5–6 Cq deviations from baseline; purple = 6–7 Cq deviations from baseline). A. Lactated Ringer solution. B. PBS. C. Sterile saline.

Analysis of media types revealed statistically significant (p < 0.05) positive associations between LRS and SS and increasing Cq values (parameter estimates: 0.62 and 0.48, respectively; Suppl. Table 1). However, no significant differences were observed among PBS, SS, and LRS in their ability to support detection of T. foetus.

In model 2, we explored interactions between time and colder temperatures (0°C, 20°C). These interactions were found to be statistically significant (Suppl. Table 2). The shipping box placed in the 0°C freezer reached 0°C by 21 h and maintained that temperature for the duration of the project. The shipping box that was exposed to 20°C incubation temperatures cooled rapidly to 4°C by 2 h post incubation and maintained that temperature for ~12 h before gradually rising to the incubated temperature of 20°C by 48 h of incubation. The shipping box that was exposed to 37°C was cooled to its lowest temperature of 10°C at 3 h post start of incubation, followed by a gradual rise to 37°C by 24 h of incubation. The shipping box that was exposed to 54.5°C cooled to its lowest temperature of 8°C by 5 h after the start of incubation and reached 54.5°C by 36 h of incubation.

Discussion

We demonstrated that time is a significant factor affecting the viability of T. foetus samples. As storage time increases, the qPCR takes longer to fluoresce a positive Cq value. This finding supports prior research, which emphasized that shipping time is critical for maintaining the stability and integrity of T. foetus samples sent for laboratory testing.^5,11 Our results reinforce the recommendation that T. foetus samples should be submitted for testing as quickly as possible after collection.

We also found that external shipping temperature has the potential to impact T. foetus qPCR results. When evaluating the average Cq values of each medium, we saw the most fluctuation when observing the media performance at 0°C. Samples were stored at 0°C, 20°C, 37°C, or 54.5°C, and all remained viable for up to 120 h. Samples from a subset of bulls had transient “peaks” in Cq values at 72 and 96 h under 0°C conditions, disqualifying them from T. foetus–positive status. However, these bulls later had positive Cq values at 120 h. Although we cannot rule out the possibility of laboratory errors, other factors, such as biological variability and individual differences among the bulls, might also have contributed to these results. The T. foetus infections were of natural source in our cases. The concentration of protozoal organisms per bull was unknown at the start of the study and remained unknown for the duration of the study. Due to infections being natural and not inoculated or of equivalent concentrations, we cannot guarantee an equal number of organisms per bull or per subsequent sample, which may account for some of the variability in our results. We advise exercising caution when drawing conclusions about shipping at 0°C due to the limited number of data points. At the other end of the temperature spectrum, we found that samples stored at 54.5°C produced positive qPCR results, consistent with the results reported in a previous study. ⁶ A different study reported mostly negative qPCR results for samples stored at 42°C; however, that study did not include the higher temperature of 54.5°C. ⁴ We hypothesize that the fluctuation in positive Cq values from the various temperature trials may be due to a nucleic acid degradation effect caused by the freezing and thawing occurring during the simulated shipping.

The parameter estimates for the 3 media tested (PBS, SS, LRS) were statistically significantly different than zero in explaining differences in Cq values; however, there was no meaningful difference in their performance for maintaining T. foetus sample stability. All 3 media, while producing different results, performed equally well in maintaining T. foetus samples from a practical perspective. Diagnostic laboratories could consider accepting a broader range of media for sample submission, thereby increasing testing for T. foetus. In addition to their performance in maintaining sample stability, all 3 media are clear and can be easily assessed for sample suitability and contamination by feces or blood. ¹¹

Based on our results, we believe that PBS, SS, and LRS are all suitable media for the short-term storage and shipping of T. foetus samples for qPCR analysis. Ideally, samples collected for the detection of T. foetus should be delivered to the laboratory as soon as possible.^1,4,6,8 If immediate delivery is not a viable option, samples should be stored at 0–20°C and rush-shipped to account for potential delays and environmental stressors.

Supplemental Material

sj-pdf-1-vdi-10.1177_10406387251330297 – Supplemental material for Evaluating the stability of Tritrichomonas foetus in various media under simulated environmental conditions of shipment for RT-qPCR assays

Supplemental material, sj-pdf-1-vdi-10.1177_10406387251330297 for Evaluating the stability of Tritrichomonas foetus in various media under simulated environmental conditions of shipment for RT-qPCR assays by SaraBeth Boggan, Ryan B. Williams and Jennifer H. Koziol in Journal of Veterinary Diagnostic Investigation