Patton, Dorothy L. PhD*; Sweeney, Yvonne T. Cosgrove BA*; Agnew, Kathy J. BS*; Balkus, Jennifer E. MPH*; Rabe, Lorna K. BS†; Hillier, Sharon L. PhD†
TRICHOMONAS VAGINALIS CAUSES TRICHOMONIASIS, a common and treatable nonviral sexually transmitted infection (STI). The World Health Organization estimates that over 15 million STIs occur annually in the United States and that an estimated 5 million are caused by T. vaginalis.1 Worldwide, the annual incidence of trichomoniasis is reported to be over 170 million cases.2 Several epidemiologic studies indicate that T. vaginalis infection may predispose individuals to HIV infection.3–5
One of the difficulties encountered in studying the pathogenicity of T. vaginalis is the lack of a suitable animal model that reflects trichomoniasis of the human genital tract. Early animal experiments reported in the 1940s concluded that T. vaginalis isolated from human cases of trichomonas vaginitis did experimentally infect monkey vaginas.6 The number of animals infected was small, 3 of 12. A second study in the 1950s also used Rhesus macaques but failed to report the actual number of monkeys in the experiment.7 The most notable observations were that peak numbers of trichomonads were present at or near menses. Perhaps a better understanding of the menstrual cycle and seasonal cycling of the Rhesus macaque at that time would have promoted further experimentally induced T. vaginalis studies. From these initial studies, other nonhuman primate species were evaluated as possible models for T. vaginalis studies.8 The squirrel monkey was shown to be most susceptible to intravaginal inoculation with T. vaginalis; however, further studies in this species have not been forthcoming.
Since the early 1980s, this laboratory has used the female pigtailed macaque (Macaca nemestrina) to investigate pathogenesis of experimentally induced Chlamydia trachomatis cervicitis and salpingitis/pelvic inflammatory disease.9–13 Topical microbicide development studies began in this model in 1995.14–17 This particular macaque was chosen for these studies because of the remarkable similarities to the human in regard to the menstrual cycle, vaginal microflora, anatomy and physiology of the reproductive tract, and natural susceptibility to the human biovars of C. trachomatis.18,19 These studies led us to investigate whether the pigtailed macaque might be susceptible to infection by T. vaginalis, the cause of another prevalent human STI. Before initiating experiments with T. vaginalis, we established that T. vaginalis was not endogenous in the microflora of the pigtailed macaque. Vaginal swabs were collected from 56 randomly selected Macaca nemestrina and tested for the presence of T. vaginalis by culture. All specimens were negative.
In the current study, we first established that the pigtailed macaque was naturally susceptible to infection by a human strain of T. vaginalis. Second, we characterized the course of T. vaginalis infection using culture, pH, microflora, cytokine response, and colposcopy as study measures. From these results, we propose that the pigtailed macaque provides a reliable animal model to further investigate T. vaginalis infection.
Materials and Methods
In this study, 15 sexually mature female pigtailed macaques (Macaca nemestrina) were used to develop a model for the study of trichomoniasis. All the macaques were housed at the Washington National Primate Research Center. Protocol approval was obtained from the University of Washington’s Animal Care Committee. All experiments were performed within National Institutes of Health Animal Use Guidelines.
Trichomonas vaginalis (ATCC 50148) organisms were removed from −80°C freezer storage and inoculated into an InPouch TV culture system (Biomed Diagnostics, White City, OR).20 The culture was incubated for 48 hours and then passed to a new InPouch bag. This procedure was repeated 2 more times to ensure the Trichomonas organisms were viable and growing well after freezer storage. Approximately 0.5 mL of suspended trichomonads were then subcultured into each of 4 new InPouch culture systems and allowed to incubate 24 hours. The InPouch supernatants were then titered. The inocula used in this study contained 6.6 × 105 to 7.1 × 105 trichomonads per milliliter. Trichomonad titration was conducted each day animals were challenged.
Inoculation and Specimen Collection
Experiment 1: Can the Pigtailed Macaque Be Infected With a Human Strain of Trichomonas vaginalis?
Seven animals were enrolled in a 2-week study to determine whether infection with T. vaginalis could be established. Before sedation of the animals, the trichomonads were placed in a warmed incubator chamber and transported to the Primate Center. Just before inoculation, 2 baseline vaginal swabs were collected from each animal, one for cytokine analysis and the other to document the absence of T. vaginalis. At each inoculation, 1.0 mL of inoculum or growth media alone was dispensed into the vagina with a 1-mL syringe. Four animals received a single intravaginal inoculation of 6.6 × 105 trichomonads per milliliter. Three animals received T. vaginalis growth media alone. After inoculation, vaginal specimens were collected with Dacron-tipped swabs on days 2, 7, 9, and 14 for detection of T. vaginalis by culture in the Seattle laboratory. Wet mount slides were read on days 9 and 14 postinfection. At day 14, a second vaginal swab was collected for cytokine detection. A 3-day treatment of banana-flavored metronidazole (35 mg/kg per day) followed immediately. Ten days posttreatment, vaginal swabs for detection of T. vaginalis (test of cure) were collected.
Experiment 2: Further Characterization of Trichomonas vaginalis Infection.
Eight animals were enrolled in a 2-arm (T. vaginalis vs. sham-inoculated) crossover design study that followed animals for 5 weeks before initiating treatment. At baseline, for each animal, a colposcopic examination of the vaginal and cervical tissues was performed. In addition, vaginal swabs for T. vaginalis culture, microflora, pH, and cytokine detection were obtained. Group 1 (n = 4) received a single intravaginal, 1.0-mL inoculation of 7.1 × 105 trichomonads per milliliter, and group 2 (n = 4) was sham-inoculated with 1.0 mL of T. vaginalis growth media alone. After inoculation, vaginal swabs were collected on days 2, 7, 14, 21, 28, and 35 for detection of T. vaginalis by culture, pH, microflora, and cytokine analysis. Colposcopy was also performed to assess for presence of “strawberry cervix” on this same timeline. After the day 35 sample collections were completed, a 3-day course of metronidazole treatment (35 mg/kg per day) was initiated. Ten days posttreatment, colposcopy was completed and vaginal swabs for T. vaginalis detection (test of cure), pH, microbiology, and cytokine analyses were collected. One month later, the experiment was repeated with opposite enrollment of test and control animals in each arm of the study.
Trichomonas vaginalis Detection
Vaginal swab specimens were immersed into the media of the InPouch TV culture system20 immediately after collection. This system provides T. vaginalis-specific growth media in a closed system that inhibits growth of potential contaminants. Cultures were incubated for 48 hours, and then evaluated microscopically under low power (10×) for the presence of motile trichomonads and confirmed at 40×. A negative test was reported if no trichomonads were seen after 72 hours at 37°C incubation.
The pH measurement was recorded at the time of collection using pH paper graded in 0.5 steps (Baker pHix).
For cytokine analysis, cervical samples were collected with Dacron-tipped swabs. The swabs were placed into aliquoted samples of Dulbecco phosphate-buffered saline, without calcium or magnesium, and stored at −70°C. Samples were batched for overnight delivery to Magee-Womens Research Institute at Pittsburgh, Pennsylvania, for cytokine detection utilizing the Luminex 100 Instrument (Luminex Co., Austin, TX). Concentrations were extracted from an 8-point standard curve using the Luminex 100 IS software.
All samples for microbiologic analysis were immediately placed in Port-a-Cul anaerobic transfer tubes (Becton Dickinson and Co., Sparks, MD) for overnight transport to the research microbiology laboratory at Magee-Womens Research Institute in Pittsburgh, Pennsylvania. This transport device has been shown to preserve the viability of aerobic and anaerobic bacteria in clinical samples21 for up to 24 hours.
Briefly, for quantitative culture, the vaginal swabs were placed into 1.5 mL of prereduced balanced salt solution and vortexed. The resultant 1:10 dilution of vaginal fluid was used to prepare serial 1:10 dilutions from 101 to 107 with sterile prereduced saline. A 100-μL volume of the original suspension and of each dilution was inoculated onto Columbia agar supplemented with 5% sheep blood (PML Microbiologicals, Portland, OR), 2 plates of human blood bilayer Tween agar (HBT; Becton-Dickinson, Cockeysville, MD), one prereduced laked blood Kanamycin agar plate (PML), and one Rogosa agar plate (made in house). The Columbia agar and one set of HBT plates were incubated at 36°C in 5% to 7% CO2 for at least 48 hours; the remainder was incubated within an anaerobic chamber (Bactron IV; Sheldon Manufacturing Inc., Cornelius, OR) at 36°C for a minimum of 5 days. Bacteria were identified by biochemical tests as previously described.22 Lactobacilli were tested for production of H2O2 in a qualitative assay on tetramethylbenzidine agar plates.23 Species belonging to the genera Bacteroides, Porphyromonas, and Prevotella were grouped together as pigmented or nonpigmented anaerobic Gram-negative rods.
The median paired difference test was applied to multiple cytokine measures to compare the median difference in the control group with the infected group. The Wilcoxon test was used to compare the overall microflora present in the T. vaginalis-infected group as compared with the uninfected control group at each point of measurement. Friedman’s test was used to examine shift over time in each microorganism and the microflora values over time within each treatment group.
Experiment 1: Can the Pigtailed Macaque Be Infected With a Human Strain of Trichomonas vaginalis?
Detection of Trichomonas vaginalis.
Trichomonas vaginalis infection developed in all 4 test animals inoculated with 6.6 × 105 trichomonads per milliliter (ATCC 50148) and none of 3 control animals. The InPouch cultures were positive by day 2 postinoculation and persisted through day 14 of the study (Table 1). Wet mount slides were read shortly after specimen collection on days 9 and 14. Two of 4 test animals had positive wet mounts on day 9 and 3 of 4 on day 14, indicating a high-density population of trichomonads present in their vaginal swabs. InPouch cultures from all 4 challenged animals were positive on days 9 and 14.
Cervical swabs were collected at baseline (preinfection) and day 14 from test and control animals for assessment of cytokine profiles. Samples were tested in the Luminex system. In this small study, T. vaginalis-infected animals developed slightly increased levels of interleukin-4, GM-CSF, and MIP-1α compared with control animals, but none of these changes reached statistical significance (data not shown).
Experiment 2: Further Characterization of Trichomonas vaginalis Infection
The detection of T. vaginalis infection from the 8 animals enrolled in the 2-arm crossover study is shown in Table 2. Six of 8 challenged animals consistently tested positive for trichomoniasis after inoculation. One animal tested culture positive on days 2 and 7 but negative thereafter. Only one animal (no. 3) was not infected. After oral treatment with metronidazole (35 mg/kg per day for 3 days), infection was resolved in all animals. As expected, no evidence of trichomoniasis was detected in the sham-inoculated arm.
Vaginal pH varied from 5.5 to 8.0 over the menstrual cycle whether infected or not. The higher values were present at menses.
Strawberry cervix was not documented by colposcopy throughout the 5-week study. However, in 3 of 8 infected animals, tissue irritation (erythema) was noted by colposcopy. Friability of cervical tissue, defined as swab-induced cervical bleeding, was documented in one animal at week 5 of infection.
In 6 persistently infected animals, slightly decreased cytokine levels (interleukin-4, GM-CSF, and MIP-1a) were noted at week 2, but were not statistically different from variations seen in the same animals when enrolled in the control arm (P = 0.89, 0.35, and 0.08, respectively). At week 5, cytokine variations were even less pronounced. As a result of the small sample size and the wide range of values, no significant trends or associations were detected.
Vaginal microflora remained similar in the T. vaginalis-infected group as compared with the control group at each respective time point. No statistically significant differences attributable to T. vaginalis infection were detected (Fig. 1). A single incidence of statistically significant difference was noted when the presence of diphtheroids decreased at week 3 in the T. vaginalis-infected group as compared with the control group. This population rebounded the next week.
Trichomonas vaginalis is the world’s most common cause of nonviral STIs yet little is known about the pathogenesis of this widespread protozoan. A reliable animal model of T. vaginalis infection would enable investigators to characterize trichomoniasis in greater detail than clinical studies allow. The current study was designed to evaluate the usefulness of the pigtailed macaque as such a model.
In this study, 11 of 12 macaques challenged with a human strain of T. vaginalis tested positive for trichomoniasis using the InPouch culture system. One of these animals had transient positive culture results (days 2 and 7 postchallenge), testing negative for infection on weeks 2 through 5. In these studies, we did not collect vaginal smears for leukocyte assessment. It will be interesting to document the polymorphonuclear response in future studies using this model. A single intravaginal inoculation with T. vaginalis resulted in persistent colonization with this pathogen.
Colposcopic observations were done to assess clinical findings of T. vaginalis infection. The classic presentation of strawberry cervix seen in approximately 2% of patients24 was not documented in any animals. Tissue irritation was noted more frequently in T. vaginalis-infected macaques than those sham-inoculated during the 5-week study. The vaginal microflora and pH remained constant after infection with T. vaginalis.
Previous studies have used the pigtailed macaque as an experimental animal model to study the pathogenesis of acute and chronic C. trachomatis cervicitis, salpingitis, and pelvic inflammatory disease. The effects of antimicrobial agents on the course of disease have been investigated as well. These studies support using this same animal as a model for T. vaginalis infection. Potential uses of such a model include trichomoniasis pathogenesis, investigations of host immune response, including mechanisms of enhanced HIV acquisition, as well as treatment and prevention strategies.
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