Pure spherules of Coccidioides immitis in continuous culture

Abstract

Investigation of host-parasite relationships involving the parasitic form of Coccidioides immitis has been difficult because, previously, spherules and endospores have not been grown continuously in tissue culture medium without detectable formation of hyphae. Arthroconidia were harvested from mycelial cultures and inoculated into tissue culture flasks which contained RPMI 1640 medium supplemented with 10% calf serum and N-Tamol (Rohm & Haas Co., Philadelphia, Pa.). Flasks were purged with 5% C02, sealed, and placed on a reciprocating shaker at 35°C. Hyphae which arose during incubation were removed by filtration. Arthroconidia readily converted to the spherule-endospore form within 12 days. Six days after complete conversion, spherules and endospores were transferred to RPMI 1640 without N-Tamol. The spheruleendospore cycle was maintained in tissue culture medium for 84 days without the formation of detectable hyphae.

Characterizing in vitro spherule morphogenesis of multiple strains of both species of Coccidioides

“…11,12,14 To date there are few publications detailing the complex methodology for culturing this organism, the most recent published in 1988. 10,[15][16][17] Converse media, a complex chemically defined salt/glucose media, is widely used for growing the parasitic phase of Coccidioides. 10 In the context of the mammalian respiratory system, the composition of Converse media poses two distinct problems for in vitro studies of host-pathogen interactions.…”

“…[18][19][20] A previous study used a supplemented Roswell Park Memorial Institute (RPMI) media (RPMI-sph) to produce continuous cultures of spherules. 15 The components of the supplemented media are similar to commonly used cell culture media, namely, RPMI-1640 and fetal bovine serum (FBS), which would be amenable to the study of host-pathogen interactions. Thus, to advance a media supporting both in vitro spherule production and growth of host immune cells, we compared spherule development in both Coccidioides species grown in supplemented RPMI-sph media and Converse media under different conditions.…”

“…Several obstacles hinder the progress of Coccidioides research, two of which we sought to address. Our specific objectives were to (1) investigate the adequacy of a previously published growth media that simulates growth conditions encountered by fungus in the host environment; 15 (2) identify char-acteristics of an RG2 strain that could be used as an alternative model for risk-group three (RG3) wild-type Coccidioides, which must be worked with in a biosafety level 3 (BSL3) laboratory. 21 To assess the utility of an RG2-strain in modeling fungal growth and development, we chose the cts2/ ard1/ cts3 strain, which was derived from the clinical isolate C735 of C. posadasii, originally created for use as a vaccine candidate.…”

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

“…Morphogenesis is triggered by exposure to high temperatures (>37°C), gas concentrations (5% to 15% CO 2 ), and other factors that have not been precisely defined (Breslau & Kubota, 1964;Klotz, Drutz, Huppert, Sun, & DeMarsh, 1984). In vitro, spherules can be obtained using either chemically defined Converse medium (Converse, 1955) or RPMI 1640 supplemented with 10% fetal bovine serum (FBS; RPMI-sph medium) (Petkus, Baum, Ellis, Stern, & Danley, 1985). Most literature reports the use of Converse medium; therefore, it may be necessary to use this medium to replicate some previously published results.…”

The mysterious desert dwellers:Coccidioides immitis and Coccidioides posadasii, causative fungal agents of coccidioidomycosis

“…Once infection is established, the spherule life stage predominates in the host, with endospores developing internally and the outer cell wall rupturing to release mature endospores. Each endospore can develop into a new spherule, and endospores are likely recognized and engulfed by host immune cells [7][8][9]. Once the spherule matures and enlarges, it cannot be engulfed and can rupture the host cell.…”