Virulence assay of Coccidioides immitis in embryonated eggs.

During work on Coccidioides immitis the need arose for a relatively rapid method for the determination of the virulence of a number of strains of this microorganism. A satisfactory method has been described by Friedman, Smith, and Gordon (1955) in which a standard dose, usually 100 arthrospores, is inoculated intraperitoneally into mice. The virulence was determined from (i) the percentage of animals dead after a fixed interval of time or (ii) the number of days after inoculation when a specified percentage of the animals died. Karrer (1953) suggested that intracerebral injections in mice could be used for virulence assays. This method may be too sensitive, however, because the LD5o for the two strains tested was less than 10 arthrospores. Characteristic of such virulence assays, considerable time and numbers of animals are required. Several workers have shown that C. immitis could be subcultured in the developing chick egg. Moore (1941) used the chorioallantoic membrane for cultivation and histopathological study of pathogenic fungi. Newcomer, Wright, and Tamblyn (1952) studied the growth and development of the spherule phase following injections into the allantoic cavity or the yolk sac. Brueck and Buddingh (1951) used the yolk sac of embryonated eggs for the isolation and growth of pathogenic fungi and Burke, Salvin, and Gerloff (1952) and Vogel and Conant (1952) studied the growth and morphology of C. immitis in embryonated eggs using various sites of inoculation. Goodman, Fountaine, and Vincent (1953) reported that LDW% values for chick embryos were not obtained even with injections of 106 organisms unless the eggs were cooled immediately prior to and following injection. The site of inoculation, however, was not specified in Goodman's paper. Vogel, Peace, and Koger (1957) studied the histopathological reactions of the yolk sac tissue to C. immitis. On the basis of these reports, a