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 ma...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+neutropenia+decreases+inflammation+associated+with+murine+vaginal+candidiasis+but+has+no+effect+on+the+course+of+infection.">Acute neutropenia decreases inflammation associated with murine vaginal candidiasis but has no effect on the course of infection.</a> Inf. Immun. 66, 1273-1273 (1998).</li><li>Black, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+severity+of+Candida+vaginitis+in+BALB/c+nu/nu+mice+versus+the+parent+strain+is+not+abrogated+by+adoptive+transfer+of+T+cell+enriched+lymphocytes.">Increased severity of Candida vaginitis in BALB/c nu/nu mice versus the parent strain is not abrogated by adoptive transfer of T cell enriched lymphocytes.</a> J. Reprod. Immunol. 45, 1-1 (1999).</li><li>Buchannan, D. 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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 collec...

<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 ...

protocols-for-vaginal-inoculation-sample-collection-experimental

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Immunology and Infection

32.6K vistas.  Louisiana State University Health Sciences Center. Las principales técnicas que se utilizarán en la evaluación de Candida en un modelo animal experimental se describen. Los métodos permitirá una rápida recolección de muestras vaginales y los linfocitos de los ganglios de drenaje linfático lumbar. Estas técnicas pueden dar lugar a modelos de ratón de otras enfermedades en el tracto genital inferior femenino.