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.

1. Vaginal inoculation with Candida albicans
<ol>
	<li>Three days prior to inoculation, while restraining the animal to expose the abdomen, inject 100 &mu;l of sesame oil containing 0.1-0.5 mg of &beta;-estradiol subcutaneously in the lower abdomen. Advance the needle about 5 to 10 mm lateral to the skin to minimize leakage from the injection site. 
	The subcutaneous administration of estrogen in the lower abdomen is optimal in this model due to the close proximity to the genital tract. Effective doses may vary by mouse strains, ages or estrogen derivatives. In previous studies using CBA-J (H-2&kappa;), C3H/HeN (H-2&kappa;), C57BL/6 (H-2b), Balb/c (H-2d), DBA/2 (H-2d), SJL (H-2s) mice at 6-8 weeks of age, 0.1 mg/100 &mu;l was found effective evidenced by thickening of the vaginal wall, reduced vaginal mucus and increased epithelial cell sloughing. Mice above treated with estrogen at this concentration exhibit consistent vaginal colonization with Candida. For inoculation in mice of other strains and ages, a pilot study is recommended to ensure the effectiveness of estrogen under the modified conditions and increase the estrogen dose if necessary. 
	The estrogen solution should be prepared fresh each time on the day of injection. To ensure complete solubility of estrogen in sesame oil, thoroughly mix the solution using a vortex mixer and heat intermittently at 37&deg;C. Repeat the injection weekly throughout the study period.</li>
	<li>To prepare the inoculum, add a loopful of C. albicans blastoconidia from a recent subculture preparation on Sabouraud-dextrose agar (SDA) into 10 ml of Phytone-peptone broth supplemented with 0.1% glucose. Incubate the broth culture to stationary phase for 18 h at 25&deg;C in a shaking water bath.</li>
	<li>Following incubation, collect the broth culture into a 15 ml conical tube and centrifuge at 800 x g for 5 min. Wash the pellet twice with sterile PBS.</li>
	<li>Enumerate viable blastoconidia on a hemocytometer by trypan blue dye exclusion. Adjust the cell concentration to 2.5 x 106/ml (or to a desired inoculum concentration) in sterile PBS.</li>
	<li>To stabilize the mouse, hold the base of the tail with two fingers and lift the hip upward so that the vaginal opening faces toward you (Figure 1A). It is ideal if the mouse is placed on a flat grated surface (e.g. cage top) so that the mouse can provide resistance against the tail restraint.</li>
	<li>Pipette 20 &mu;l (or a desired volume not to exceed 20 &mu;l) of the inoculum suspension by inserting the pipette tip about 5 mm deep into the vaginal lumen (Figure 1B). Complete this step as quickly and gently as possible to minimize distress in the mouse.</li>
</ol>
2. Vaginal lavages
<ol>
	<li>Following euthanasia (or anesthesia), hold the mouse downward by the base of the tail with two fingers so that the vaginal opening becomes exposed.</li>
	<li>Lavage the vaginal lumen by introducing 100 &mu;l of sterile PBS with repeated aspiration and agitation with a pipette tip. The pipette tip may become clogged with cells. If this occurs, dispense the obstructing cells and continue lavaging with remaining PBS in the vagina. Collect the lavage fluid into a microcentrifuge tube.</li>
	<li>Alternatively, vaginal lavages can be performed on anesthetized mice with isoflurane inhalant anesthesia. For this, expose the mice to vaporized isoflurane until they are fully sedated (~30 sec.). Hold the mice downward by the base of the tail and gently lavage the vaginal lumen using 50 &mu;l of sterile PBS. Make sure to avoid harsh agitation with a pipette tip to minimize trauma to the vagina during this procedure. Sedated mice should recover from anesthesia within 30 sec of exposure to ambient air.
	 Isoflurane can be vaporized using an isoflurane vaporizer and O2 (preferred) or a standard drop system closed anesthetic chamber without the vaporizer system (requires close monitoring of the animal while sedated to avoid respiratory distress).
	 Vaginal lavages on anesthetized mice should be the method of choice for longitudinal lavage samples on the same mice. Consecutive lavaging does not influence assessment of fungal burden over time41.</li>
	<li>For wet-mount preparations, transfer 10 &mu;l of the lavage fluid onto a glass slide and observe at 400-1000x magnification by light microscopy. Additionally, cellular fractions of the lavage fluid can be stained to examine cell and nuclear morphologies. For smear preparations, transfer 10 &mu;l of the lavage fluid onto a glass slide and gently spread using the outer wall of a pipette tip. Preserve the smear samples with CytoPrep fixative and stain by the standard Papanicolaou technique (Pap smear). Observe at 400 &times; magnification by light microscopy.</li>
</ol>
3. Quantification of vaginal fungal burden
<ol>
	<li>In a 96 well round-bottom plate, transfer the lavage fluid into one well of the top row and 180 &mu;l of sterile PBS into the following 5 wells of that column (down the plate).</li>
	<li>Make 1:10 serial dilutions of vaginal lavage fluid by transferring the 20 &mu;l of the fluid to the next well in the column. Mix thoroughly by repeated aspiration before each transfer. Serial dilutions of up to 12 lavage samples (one full horizontal row) can be performed simultaneously using a 12-channel pipette.</li>
	<li>Starting with the lowest dilution, transfer 10 &mu;l of the sample onto Sabouraud-dextrose agar (SDA). Plating of up to 36 samples can be performed on 1 plate using SDA prepared in square petri dishes with grid and an adjustable spacing multichannel pipette.</li>
	<li>Enumerate colony forming units (CFUs) after incubation at 34&deg;C for 48 h.</li>
</ol>
4. Vaginal tissue extraction
<ol>
	<li>Following the vaginal lavage procedure, lay the euthanized mouse on its back and saturate the groin area with 70% ethanol. Using a pair of forceps, lift the urinary orifice upward so that the vaginal opening becomes exposed.</li>
	<li>Insert a pair of curved forceps into the vaginal lumen and locate the cervix. While maintaining a firm grip with the forceps, extract the cervix through the vaginal cavity (Figure 2A-C).</li>
	<li>Excise the vagina at the base of the vaginal opening and then remove the cervix from the vagina with surgical scissors. Keep in mind that the vaginal tissue is involuted (inner epithelium-side of the vagina is exposed outward). The tissue can be either laterally inverted to maintain the original orientation of the vagina or opened into a sheet by making a lateral incision.
	 Excised vaginal tissues can be used for 1) lymphocyte extraction following collagenase digestion (~1 &times;104/mouse)40, 2) epithelial cell isolation following dispase digestion (~5 &times;104/mouse)28, 3) frozen or paraffin-embedded preparations for histological analyses25.</li>
</ol>
5. Lumbar lymph node excision
<ol>
	<li>Following the vaginal lavage procedure, lay the euthanized mouse on its back and saturate the abdomen with 70% ethanol. Make a lateral incision starting from the lower abdomen to the chest and expose the internal organs. Using a pair of forceps in both hands, move the intestines upward so that the central blood vessels become visible.</li>
	<li>Locate the inferior vena cava and abdominal aorta. Normally, a pair of lumbar lymph nodes can be identified adjacent to the abdominal aorta, located about halfway between the origin of the renal and common iliac arteries39. These lymph nodes can be visually distinguished from fat tissue by the elastic texture and are lighter and more opaque in color compared to fat tissue (Figure 3). These lymph nodes are noticeably larger in infected animals compared to uninoculated animals.</li>
	<li>Excise the lymph nodes by placing microforceps under the node and then pull up gently to separate from surrounding tissue.</li>
</ol>
6. Isolation of lymphoid cells in single-cell suspensions
<ol>
	<li>Transfer lymph nodes onto a sterile wire mesh screen (approximately 3 x 3 cm2 in size) placed inside a sterile glass petri dish containing &tilde;10 ml of Hanks' balanced salt solution (HBSS) (Figure 4).</li>
	<li>With the petri dish inclined slightly, press the lymph nodes against the screen with a syringe plunger head. Make sure to break all lymph nodes so that the cellular contents of the nodes pass through the screen while the non-cellular components of the nodes (i.e., membranes, stroma, fat) remain on the screen.</li>
	<li>Using the same plunger and syringe, aspirate the HBSS containing cells. Wash the screen with &tilde;5 ml HBSS and collect the remaining fluid into a 15 ml conical tube.</li>
	<li>Centrifuge at 800 x g for 10 min. Aspirate any fatty deposits at the top of the fluid with a pipette prior to discarding the fluid. Wash the cell pellet three times with HBSS. Resuspend the pellet in 1 ml of HBSS and enumerate viable cells by trypan blue dye exclusion.</li>
</ol>
7. Representative Results:
The cellular fractions of vaginal lavage fluid from &gt;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 &times; 105/uninoculated mouse to 5 &times;106/inoculated mouse. In addition to lumbar lymph nodes, inguinal, popliteal and mesenteric lymph nodes can also be used.
<img alt="Figure 1" src="/files/ftp_upload/3382/3382fig1.jpg" /> 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.
<img alt="Figure 2" src="/files/ftp_upload/3382/3382fig2.jpg" /> 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.
<img alt="Figure 3" src="/files/ftp_upload/3382/3382fig3.jpg" /> 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.
<img alt="Figure 4" src="/files/ftp_upload/3382/3382fig4.jpg" /> 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.
<img alt="Figure 5" src="/files/ftp_upload/3382/3382fig5.jpg" /> 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&times; (A) or 400&times; (B, C) magnification. The insert in B shows the nuclear morphology of vaginal neutrophils at 1000&times; . Candida yeast (Y) and hyphae (H), epithelial cells (EC) and neutrohils (N) are indicated.
<img alt="Figure 6" src="/files/ftp_upload/3382/3382fig6.jpg" /> 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 &mu;l of lavage fluid from inoculated mice was assessed on indicated time points.

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Gynecol. 178 (2), 203-203 (1998).</li><li>Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+humoral+immune+responses+against+primary+and+repeat+Neisseria+gonorrhoeae+genital+tract+infections+of+17&#946;-estradiol-treated+mice.">Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17&#946;-estradiol-treated mice.</a> Vaccine. 26, 5741-5741 (2008).</li><li>Taylor, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+virulence+of+Candida+albicans+isolates+causing+mucosal+infections+in+people+infected+with+the+human+immunodeficiency+virus.">In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus.</a> J. Infect. Dis. 182, 955-955 (2000).</li><li>Taylor, B. N., Saavedra, M., Fidel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+Th1/Th2+cytokine+production+during+experimental+vaginal+candidiasis.">Local Th1/Th2 cytokine production during experimental vaginal candidiasis.</a> Med. Mycol. 38, 419-419 (2000).</li><li>Tirabassi, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mucosal+vaccination+approach+for+herpes+simplex+virus+type+2.">A mucosal vaccination approach for herpes simplex virus type 2.</a> Vaccine. 29 (5), 1090-1090 (2011).</li><li>Broeck, W. V. a. n. d. e. n., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+descriptive+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: descriptive and nomenclatory standardization in BALB/cAnNCrl mice.</a> J. Immunol. Methods. 312 (1-2), 12-12 (2006).</li><li>Wormley, F. L., Chaiban, J., Fidel, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+and+lymphocyte+activation+marker+expression+during+experimental+vaginal+candidiasis.">Cell adhesion molecule and lymphocyte activation marker expression during experimental vaginal candidiasis.</a> J. Immunol. Methods. 69, 5072-5072 (2001).</li><li>Yano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell-derived+S100+calcium-binding+proteins+as+key+mediators+in+the+hallmark+acute+neutrophil+response+during+Candida+vaginitis.">Epithelial cell-derived S100 calcium-binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis.</a> Infect. Immun. 78 (12), 5126-5126 (2010).</li></ol>

No conflicts of interest declared.

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 (&gt; 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 &mu;l 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.

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments</td>
</tr>
<tr>
<td>Female CBA/J mice</td>
<td>Charles River Laboratories</td>
<td>01C38</td>
<td>5-6 weeks of age</td>
</tr>
<tr>
<td>Candida albicans (3153A)</td>
<td>National Collection of Pathogenic Fungi, UK</td>
<td>NCPF3153</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sesame oil</td>
<td>Sigma-Aldrich</td>
<td>S3547</td>
<td>Does not need to be pre-sterilized before use</td>
</tr>
<tr>
<td>&Beta;-estradiol 17-valerate</td>
<td>Sigma-Aldrich</td>
<td>E1631</td>
<td>0.1-0.5mg in sesame oil</td>
</tr>
<tr>
<td>Phytone peptone</td>
<td>Becton Dickinson</td>
<td>211906</td>
<td>Supplement with 0.1% glucose</td>
</tr>
<tr>
<td>Trypan blue solution</td>
<td>Sigma-Aldrich</td>
<td>T8154</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Sabouraud dextrose agar</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Collagenase type IV</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>0.25%</td>
</tr>
<tr>
<td>Dispase</td>
<td>Invitrogen</td>
<td>17105-041</td>
<td>1.7 U/ml</td>
</tr>
<tr>
<td>Wire mesh screens</td>
<td>TWP</td>
<td>060X060S0065W36T </td>
<td>No. 60 mesh, stainless</td>
</tr>
<tr>
<td>Hanks&rsquo; balanced salt solution</td>
<td>Invitrogen</td>
<td>24020-117</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>CytoPrep fixative</td>
<td>Fisher Scientific</td>
<td>12-570-10</td>
<td>Preserves smear slides</td>
</tr>
<tr>
<td>Papanicolaou stain EA-65</td>
<td>EMD Chemicals</td>
<td>7054X-85</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Papanicolaou stain OG-6</td>
<td>EMD Chemicals</td>
<td>7052X-85</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Harris&rsquo; Alum hematoxylin</td>
<td>EMD Chemicals</td>
<td>638A-85</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>Baxter Healthcare</td>
<td>NDC 10019-773-60</td>
<td>Used with isoflurane vaporizer or in a drop system closed anesthetic chamber</td>
</tr>
</table>

protocols for vaginal inoculation and sample collection in the experimental mouse model of candida vaginitis

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.

Watch this Scientific Journal Video about Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis at JoVE.com

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

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.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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Immunology and Infection

32.9K Views.  Louisiana State University Health Sciences Center. 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.

Video: Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...