Proper Care and Feeding of Coccidioides: A Laboratorian's Guide to Cultivating the Dimorphic Stages of C. immitis and C. posadasii

Abstract

Coccidioidomycosis ("Valley fever") is caused by Coccidioides immitis and C. posadasii. These fungi are thermally dimorphic, cycling between mycelia and arthroconidia in the environment and converting into spherules and endospores within a host. Coccidioides can cause a broad spectrum of disease that can be difficult to treat. There has been a steady increase in disease, with an estimated 350,000 new infections per year in the United States. With the increase in disease and difficulty in treatment, there is an unmet need to increase research in basic biology and identify new treatments, diagnostics, and vaccine candidates. Here, we describe protocols required in any Coccidioides laboratory, such as growing, harvesting, and storing the different stages of this dimorphic fungal pathogen. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and harvest of liquid mycelia cultures for extractions Alternate Protocol 1: Large-volume growth and harvest of liquid mycelia cultures Basic Protocol 2: Mycelial growth on solid medium Alternate Protocol 2: Maintaining mycelial growth on solid medium Basic Protocol 3: Harvesting and quantification of arthroconidia Alternate Protocol 3: Long-term storage of arthroconidia Basic Protocol 4: Parasitic spherule growth and harvest Alternate Protocol 4: Obtaining endospores from spherules Basic Protocol 5: Intranasal infection of murine models.

Keywords: BSL-3; Coccidioides; arthroconidia; dimorphic fungi; endospores; mycelia; spherules.

Figure 1

Graphical protocol overview across the Coccidioides lifecycle. Illustration created with BioRender.

Figure 2

Small mycelia cultures are ideal for nucleic acid extractions. (A) Inoculate 2× GYE in a vented 50-ml conical tube. (B) Incubate at 30°C for 5 to 7 days. (C) Healthy cultures are fluffy, rather than stringy. (D) Pour mycelia through a nested filter. (E) Rinse fungal material with PBS until white.

Figure 3

Maintenance of active culture on solid medium. Fungal growth covers the plate in ~2 weeks (left). A fungal transfer tube can be used to subculture a fresh agar plug onto a new plate (right).

Figure 4

Long-term storage of solid mycelia cultures promotes development of arthroconidia. (A) Stack plates, tape them tightly together, and store them in Unicorn bags for 4 to 6 weeks. (B-C) Vented flasks are an alternative storage option. Mycelia that are fluffy or moist are not ready for harvest (B). Notably, dry cultures demonstrate small aerial structures. This indicates the presence of arthroconidia (C).

Figure 5

Arthroconidia harvest process. (A) Pour PBS onto dry cultures. (B) Scrape the fungal material into solution. (C) Use a serological pipet to transfer the fungal material and PBS solution. (D) Gently release the material into the filter, allowing time for the solution to pass into the conical tube. (E) Arthroconidia in solution are often yellow/brown in color. (F) After the solution passes through the filter, rinse the filter with small volumes of PBS to maximize collection.

Figure 6

Quantification of viable arthroconidia. Plate multiple dilutions to obtain accurate numbers. The 10⁻⁵ plate has too many colonies to count accurately. There are 21 colonies on the 10⁻⁶ plate, 5 colonies on the 10⁻⁷ plate, and 2 colonies on the 10⁻⁸ plate.

Figure 7

Spherule growth. (A) Culture arthroconidia at ~1 × 10⁶ cells/ml in RPMI-sph medium at 39°C, 10% CO₂, for ≥3 days. (B) Pour cultures into a 50-ml conical tube and pellet cells by centrifugation. (C) After resuspension of the pellet, confirm the presence of spherules. Shown is C. posadasii Silveira on day 4, after growth in RPMI-sph medium. (D) Packets of endospores are present after medium change. Shown is C. posadasii Silveira on day 7 after changing the RPMI-sph medium on day 4.

Figure 8

Graphical overview of an intranasal murine Coccidioides infection. Using thumb and index finger, with the nondominant hand, hold the mouse skin behind the neck and cradle the mouse in hand. Using the dominant hand, pipet arthroconidia slowly, alternating between the mouse’s left and right nares. Illustration created with BioRender.

Figure 9

Survival of CD1 mice infected with a range of dosages of the C. posadasii strain Silveira. CD1 mice received an intranasal infection with 100, 1 × 10³, 1 × 10⁴, or 1 × 10⁵ arthroconidia of strain Silveira (n = 5 to 7) or were given control PBS (n = 3). Mice were observed for up to 17 days post-infection for survival analysis.