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

Summary

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.

Abstract

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 years^18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies^33,34. 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 Candida^{3,4,11,16,17,19,21,25,37}. This model has also been employed to test potential antifungal therapies in vivo^13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection^6,14,23. 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 properties^3,16,21. 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.

Protocol

1. Vaginal inoculation with Candida albicans

1. Three days prior to inoculation, while restraining the animal to expose the abdomen, inject 100 μl of sesame oil containing 0.1-0.5 mg of β-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^κ), C3H/HeN (H-2^κ

Discussion

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 therapies^{3,4,11,13,16,17,19,21,24,25,37}. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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%
Dispase	Invitrogen	17105-041	1.7 U/ml
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