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Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.
Yano, J., Fidel, Jr., P. L. Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis. J. Vis. Exp. (58), e3382, doi:10.3791/3382 (2011).
Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.
1. Vaginal inoculation with Candida albicans
2. Vaginal lavages
3. Quantification of vaginal fungal burden
4. Vaginal tissue extraction
5. Lumbar lymph node excision
6. Isolation of lymphoid cells in single-cell suspensions
7. Representative Results:
The cellular fractions of vaginal lavage fluid from >4-day inoculated mice typically consist of Candida, epithelial cells and cellular infiltrates (Figure 5). By wet-mount microscopy, Candida can be visually identified by the presence of hyphae as well as yeast (Figure 5A). Smear preparations of vaginal lavage fluid can be stained by Papanicolaou technique to examine epithelial cells and infiltrating leukocytes, of which the principal cells are neutrophils identified by the tri-nuclear lobes (Figure 5B). Very few neutrophils, if any, are detected in uninoculated mice (Figure 5C)41.
An example of vaginal fungal burden is shown in Figure 6. Vaginal lavage fluid collected at specific time points are cultured for CFU enumeration (Figure 6A). Vaginal colonization/infection with Candida persists for weeks in estrogen-treated inoculated mice (Figure 6B), while Candida fails to establish vaginal colonization in non-estrogen-treated inoculated mice (Figure 6C). Estrogen-treated uninoculated mice remain negative for Candida throughout the time (data not shown). In addition, vaginal lavages can be performed either one time on separate mice at each time point or longitudinally in the same mice under anesthesia.
The lumbar lymph nodes are the primary draining lymph nodes of the genital tract and the most relevant site to evaluate for systemic immune responses to a vaginal challenge. Note that these lymph nodes may become enlarged in inoculated mice while they normally appear quite small in uninoculated mice. Leukocyte cellular recoveries typically range from 8 × 105/uninoculated mouse to 5 ×106/inoculated mouse. In addition to lumbar lymph nodes, inguinal, popliteal and mesenteric lymph nodes can also be used.
Figure 1. Vaginal inoculation with Candida. A) A mouse restrained for inoculation. The mouse is placed on a wire cage insert and held by the base of the tail, slightly upward to lift the legs and expose the vaginal opening. The hip of the mouse can be stabilized with the same hand as it attempts to resist the tail restraint. B) Introduction of the inoculum into the vaginal lumen. A pipette tip is gently inserted about 5 mm deep into the vaginal lumen. The suspension inoculum is then deposited.
Figure 2. Vaginal tissue extraction. A-B) Extraction of the cervix. The cervix is located with curved forceps and exposed outward through the vaginal cavity. Once out of the vaginal cavity, the cervix is further pulled outward to fully expose the vagina. C) Extraction of the vagina. The vagina is excised from the vulva with scissors. Once detached, remove the cervix from the vagina.
Figure 3. Identification of the lumbar lymph nodes. The location of the lumbar lymph nodes among the surrounding organs/blood vessels in the vicinity of the pelvis is indicated. A, abdominal aorta. B, urinary bladder. C, common iliac artery. I, intestines. L, liver. R, rectum. S, spleen. U, Uteri.
Figure 4. The lumbar lymph nodes placed on a wire mesh screen. The lymph nodes are pooled onto the screen placed in a petri dish with HBSS. The lymph nodes are pressed against the screen with a syringe plunger to obtain lymphoid cells in single-cell suspensions.
Figure 5. Cellular fractions of vaginal lavage fluid from inoculated mice. A) Wet-mount and B) Pap smear preparations of vaginal lavage samples collected 4 days post-inoculation and C) from uninoculated mice. Images are shown at 1000× (A) or 400× (B, C) magnification. The insert in B shows the nuclear morphology of vaginal neutrophils at 1000× . Candida yeast (Y) and hyphae (H), epithelial cells (EC) and neutrohils (N) are indicated.
Figure 6. Detection of vaginal fungal burden. A) Representative C. albicans colonies grown on a SDA plate. Neat (N) lavage samples from six different inoculated mice (top row) were serially diluted and cultured for CFU enumeration. B) Quantification of vaginal fungal burden in estrogen-treated and C) non-estrogen-treated mice. CFU/100 μl of lavage fluid from inoculated mice was assessed on indicated time points.
An experimental mouse model of Candida vaginitis has been established and historically used for the past few decades to study mucosal host response to Candida as well as for testing antifungal therapies3,4,11,13,16,17,19,21,24,25,37. The protocols presented here incorporate efficient and less labor-intensive methods, and appear to be one of the most optimized model systems of Candida vaginitis described to date. These techniques enable rapid quantification of fungal burden and collection of vaginal specimens. Furthermore, previous studies testing several haplotypic strains of mice (n=13) and various time points post-inoculation showed the similar levels of variability in fungal burden and host responses to vaginal colonization with Candida2,6,41. Hence, this model can be adapted to existing protocols without restriction on mouse strains or the duration of infection. However, one should recognize that the variability in vaginal fungal burden can be high between animals given the same inoculum (Figure 6B). There is evidence supporting that the variability occurs due to differing degrees of early adherence to vaginal epithelium41. Therefore, 7-10 mice/group is suggested for statistical purposes.
Variability in vaginal fungal burden has also been evidenced between different strains of C. albicans, suggesting that not all strains of C. albicans have the capacity to similarly colonize the mouse vagina. For instance, C. albicans 3153A (lab strain) used in this protocol has been reported to show higher vaginal colonization than SC5314 (clinical isolate), where a higher inoculum (> one log) would be required to obtain equivalent levels of vaginal fungal burden seen with 3153A27. In fact, a highly variable vaginal colonization with several clinical isolates has been documented36. Hence, care should be taken when determining an optimal inoculum for each C. albicans strain to ensure consistent colonization in mice. Typically, mean CFUs ranging from 5 x 104 to 5 x 105/100 μl lavage fluid should be detected for consistent colonization. For assessment of vaginal fungal burden, quantification of CFUs by lavages is appropriate for this model as Candida blastoconidia and hyphae normally do not penetrate beyond the superficial layer of vaginal epithelium. Our previous histological evaluation of post-lavage vaginal tissues rarely showed residual Candida25,41. However, we recognize the possibility that hyphae could be miscounted as they grow as a single colony on the agar plate and might not reflect accurate fungal burden. As an alternative method to CFU enumeration, a newly developed in vivo imaging technique has been reported to successfully assess vaginal fungal burden when using genetically engineered luminescent C. albicans9,29. Furthermore, the protocol can be modified to induce C. glabrata colonization in diabetic animals15,20. To date, mouse models for other non-C. albicans-induced vaginitis have not been reported.
Estrogen administration is critical for this model, initiating robust vaginal colonization with Candida6,14,23. In addition to subcutaneous injection of estradiol in sesame oil suspension, injection of water-soluble estradiol in PBS and intradermal implantation of a controlled-release estradiol pellet are alternative methods for estrogen administration and have been employed in other mouse models of female genital tract infections10,35. Requirement of high estrogen in this model may be explained by two physiological factors. One is that elevated estrogen promotes stratification of vaginal epithelium5,8,30. Thickened epithelium from increased epithelial cell proliferation may allow Candida to gain better adherence to the vaginal wall and subsequent colonization. Secondly, vaginal epithelial cells are known to have high glycogen content. An increased tissue estrogen level results in deposition of glycogen into the vaginal walls30. Elevated glycogen in the vaginal microenvironment may in turn allow Candida to flourish by providing additional nutrients. Previous immune analyses showed that exogenous estrogen did not affect cell adhesion molecule expression40. A drawback of treating the animals with exogenous estrogen is that elevated estrogen has also been known to promote overgrowth of vaginal commensal flora30. Since the composition of flora may vary significantly between animals, this may add a variable where commensal organisms could influence Candida growth or modulate host responses. In consideration of this issue, the model requires estrogen-treated control (uninoculated) animals to be included in all experiments and analyzed in parallel.
In this mouse model, the efficacy of anti-fungal agents can be accurately assessed by the protocols described herein, which may provide crucial in vivo testing leading to the development of potential therapies for clinical use. In fact, the robust nature of the model provides good indications of clinical efficacy. In addition to the use of estrogen, neutral vaginal pH in mice promotes growth of hyphae, a pathogenic form of Candida seen in all inoculated mice. Similar to clinical observations, a robust vaginal neutrophil migration occurs in a subset of mice without affecting fungal burden31,41. The presence of neutrophils is prominent in women during symptomatic vaginitis and appears to parallel mice following inoculation12,41. Since other clinical symptoms (i.e., itching, swelling) cannot be precisely measurable in mice, the assessment of vaginal neutrophils serves as a simple yet reliable indication of inflammation (symptoms) in this model.
The microscopy of the vaginal lavage fluid shown in Figure 5 is routinely performed in our laboratory to assess Candida colonization and vaginal inflammation. Moreover, hyphal scoring can also be used as a measure of infection. Thereafter, the cellular and soluble fractions of the lavage fluid can be preserved and cryo-archived for future analyses. Of note, an advantageous feature of the protocol is that vaginal lavages can be performed longitudinally in the same mice under anesthesia. Our previous studies confirmed that longitudinal evaluation does not influence assessment of vaginal fungal burden. This approach is particularly advantageous in cases where longitudinal analyses of infection are desired.
Finally, we have developed a less-invasive excision method for the vagina. This effective and quick technique requires no incision in the abdomen or any internal organs, leaving the rest of the genital tract intact. Another useful feature of this model is the lack of requirement for immunosuppressive agents to initiate Candida colonization. This is particularly important because maintaining natural immune status of the host is a critical aspect of immunological studies and host responses to a microbial challenge. Hence, tissues and cells isolated by these methods could be applied in various in vitro immune assays.
In conclusion, we provide several important features and representative results of the experimental model of vaginal candidiasis. In addition to C. albicans-induced vaginitis, infections by other pathogens of the female lower genital tract, including Neisseria gonorrhoeae, Trichomonas vaginalis and Herpes simplex virus have been studied using mouse models where the organisms are intravaginally introduced in hormone-treated mice1,7,10,22,26,35,38. Therefore, the techniques that are useful for studies involving Candida vaginitis can be applied to and potentially advance methodologies to study pathogenesis and host immune responses for other infectious diseases of the female lower genital tract.
No conflicts of interest declared.
This work was supported by R01 AI32556 (NIAID, National Institute of Health). This work was also supported in part by Louisiana Vaccine Center and South Louisiana Institute for Infectious Disease Research sponsored by the Louisiana Board of Regents.
|Female CBA/J mice||Charles River Laboratories||01C38||5-6 weeks of age|
|Candida albicans (3153A)||National Collection of Pathogenic Fungi, UK||NCPF3153|
|Sesame oil||Sigma-Aldrich||S3547||D–s not need to be pre-sterilized before use|
|Β-estradiol 17-valerate||Sigma-Aldrich||E1631||0.1-0.5mg in sesame oil|
|Phytone peptone||BD Biosciences||211906||Supplement with 0.1% glucose|
|Trypan blue solution||Sigma-Aldrich||T8154|
|Sabouraud dextrose agar||BD Biosciences||211584|
|Collagenase type IV||Sigma-Aldrich||C5138||0.25%|
|Wire mesh screens||TWP||060X060S0065W36T||No. 60 mesh, stainless|
|Hanks’ balanced salt solution||Invitrogen||24020-117|
|CytoPrep fixative||Fisher Scientific||12-570-10||Preserves smear slides|
|Papanicolaou stain EA-65||EMD Millipore||7054X-85|
|Papanicolaou stain OG-6||EMD Millipore||7052X-85|
|Harris’ Alum hematoxylin||EMD Millipore||638A-85|
|Isoflurane||Baxter Internationl Inc.||NDC 10019-773-60||Used with isoflurane vaporizer or in a drop system closed anesthetic chamber|
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