Source: Sparber, F. et al., Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction. J. Vis. Exp. (2019)
This video demonstrates Malassezia-induced skin infection in mice. Malassezia oil suspension is applied over the wounded ear, triggering inflammation and swelling of the ear tissue. Later, the ears are dissected, homogenized, and spread on an agar plate for growth. The number of colonies indicates the fungal burden resulting from skin inflammation.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Cultivation of Malassezia under laboratory conditions
NOTE: Store all the reagents and media used for this protocol at room temperature (RT, 20 – 25 °C) unless stated otherwise, as the lower temperature can inhibit fungal growth.
2. Preparation of the inoculum for experimental Malassezia infection of mice
3. Infecting mice with Malassezia
4. Analysis of Malassezia-induced skin inflammation
NOTE: This procedure describes the analysis of Malassezia-induced ear swelling during infection which serves as a parameter of skin inflammation. A prerequisite for analyzing the fungus-induced ear swelling is to measure the baseline ear thickness prior to tape-stripping and/or infection (Step 3.5).
5. Analysis of fungal burden in the infected skin
Figure 1: In vitro cultivation of Malassezia. (A) M. sympodialis strain ATCC 42132 grown for 3 days at 30 °C and 180 rpm in liquid mDixon medium (left) next to a control Erlenmeyer flask containing mDixon medium that was not inoculated (right). (B) Colonies of M. sympodialis strain ATCC 42132 on mDixon agar after 5 days of incubation at 30 °C.
Figure 2: Analysis of Malassezia skin infection on the basis of ear thickness and fungal burden. (A) Histology of ear sections obtained from C57BL/6 mice that were treated with olive oil (vehicle, left) or infected with M. furfur strain JPLK23 for 5 days (middle and right). On the right, the ear skin was tape-stripped prior to infection. Sections were stained with hematoxylin and eosin (H&E). (B) Summary graphs showing the increase in ear thickness over time for C57BL/6 mice that were exposed to Malassezia or left uninfected as controls. The absolute thickness of M. pachydermatis strain ATCC 14522-exposed or vehicle-treated ear skin at each time point is displayed on the left; the increase in ear thickness at the indicated time points relative to the baseline on day 0 is shown on the right. (C) Fungal burden in the skin of C57BL/6 mice that were infected with M. pachydermatis strain ATCC 14522 or treated with olive oil as a control (vehicle). In both cases, the skin was tape-stripped. Each symbol in the summary graphs B and C represents one animal. The statistical significance of the differences between groups was calculated using one-way ANOVA (B) or Student's t-test (C). ***p <0.001, ****p <0.0001, D.L.: Detection Limit
The authors have nothing to disclose.
Agar | Sigma-Aldrich | A1296-1KG | |
Attane Isoflurane | Piramal Healthcare | – | |
Biosaftey cabinet (BSC) Faster Ultra Safe | DASIT GROUP | TEC 5594 | BSL2 certified |
Centrifuge | Eppendorf | 5415D | Compatible with 2ml Eppendorf tubes |
Dessicated Ox-bile | Sigma-Aldrich | 70168-100G | |
Eppendorf Tubes (2 ml) | Eppendorf | 0030 120.094 | |
Glucose | Sigma-Aldrich | 49159-5KG | |
Gylcerol (99 %) | Honeywell | 10314830 | |
Heating pad | Eickenmeyer | 648048 | |
Incubator Hereaus B20 | Heraeus | 412047753 | BSL2 certified |
Ketasol (100 mg) | Graeub AG | 6680416 | |
Magentic heating plate MR Hei-Standard | Heidolph Instruments | 442-1355 | |
Malassezia spp. | ATCC | 14522, 14521, 42132 | |
Malt extract | Sigma-Aldrich | 70167-500G | |
Multiply Biosphere Tubes (200 µl) | Sarstedt AG | 7084211 | Safelock |
Native olive oil | – | – | Commerc. available |
Nonidet P40 | Axon Lab | A1694,0250 | |
Oditest measurment devise | Kroeplin | S0247 | Range 0-5 mm |
Oleic Acid | Sigma-Aldrich | 75090-5ML | |
Peptone | Oxoid | LP0037 | |
Petri dishes | Sarstedt AG | 82.1473 | |
Phosphat buffered salt solution (PBS, 1x) | Amimed/Bioconcept | 3-05F39 | |
Rompun (2 %) | Bayer | KP0BFHR | |
Shaking incubator Infors Minitron | Infors | – | BSL2 certified |
Spectrometer | Jenway | 20308 | Optical density measurement at 600nm |
Spectrometer Cuvettes | Greiner Bio-One | 613101 | |
Stainless Steel balls (5mm) | ABF | KU.5G80 1.3541 | |
Syringes 1 ml Sub-Q | BD Bioscience | 305501 | |
Tissue Lyzer II | Quiagen | 85300 | |
Transpore Hypoallergic Tape | 3M | 1527-1 | |
Tween 40 | Sigma-Aldrich | P1504-100ML | |
Vitamin A Retinoli Palmitas Eye Cream | BAUSCH & LOMB | Commerc. available |