Equivalent Mortality in Normal and Athymic Mice Infected with Naegleria fowleri

Abstract

Naegleriafowleri, a normally free-living amoeba, is the causative agent of primary amoebic meningoencephalitis (PAM), and since its first description in 1965 over 100 cases have been reported worldwide (John, 1982, Annual Review of Microbiology 36: 101-123). A perplexing aspect of PAM is that pathogenic N. fowleri are often isolated from recreational waters, yet exposed individuals only rarely develop PAM and it occurs in apparently healthy humans. Although affected individuals might have some underlying immune dysfunction it is most likely PAM is the result of environmental or genetic factors that serve to enhance virulence of amoebae. Studies have been directed at elucidating the role of the immune system in resistance to PAM. In laboratory animals, immunization by a variety of routes with Naegleria antigen can heighten resistance to a subsequent lethal infection (John et al., 1977, Infection and Immunity 16: 817820; Thong et al., 1978, The American Journal of Tropical Medicine and Hygiene 27: 238-240). Investigations of cellular immune components showed that Naegleria antigen could elicit a delayed hypersensitivity reaction in guinea pigs (Diffley et al., 1976, Zeitschrift fur Parasitenkunde 49: 133-137) and macrophage inhibition by lymphokine macrophage inhibition factor (Cursons et al., 1980, Infection and Immunity 29: 408-410). Although Naegleria antigen can evoke an immunological response which may lessen susceptibility to a lethal infection, little is known about the role of the humoral and cellular immune systems in innate resistance to PAM. Congenitally athymic (nude [nu/nu]) mice are deficient in T-cell-dependent cell-mediated immunity and in the humoral response to Tcell-dependent antigens (Wortis, 1974, Contemporary Topics in Immunobiology 3: 243-263). Investigations on the pathogenicity of N. fowleri in nude mice would serve as an useful aid for delineating the role of T-cell-dependent activities in the innate resistance against development of PAM. Providing that the T-cell-dependent arm of the immune system is a significant factor in innate resistance, it would be expected nude mice would show increased susceptibility and the course of PAM would be more rapid compared to normal mice. The purpose of this study was to determine if the development of PAM and the course of the disease was more rapid in congenitally athymic mice than in phenotypically normal mice. Mice were also infected with trophozoites of Naegleria lovaniensis, an amoeba physiologically and morphologically similar to N. fowleri, but nonpathogenic in immunocompetent mice (Stevens et al., 1980, International Journal for Parasitology 10: 51-64). The Lee strain of N. fowleri and N. lovaniensis were cultivated in tubes containing Chang's medium (De Jockheere, 1977, Applied and Environmental Microbiology 33: 751-757). Amoebae in late exponential growth were enumerated by hemacytometer counts, centrifuged (1,000 g for 15 min), and adjusted to the desired density in Chang's medium so that 25 1l delivered the appropriate number of amoebae (Fig. 1). Mice were of Swiss Webster background and congenitally athymic mice were obtained by mating homozygous (nu/nu) males with heterozygous (+/nu) females. Mice were maintained under specific pathogen free conditions in an area separate from other laboratory animals. For infection, mice of either sex and approximately 12 wk old were anesthetized with ether and 25 Al of an amoebae suspension was delivered into the nares with a micropipet. Development of PAM was evident by typical symptoms of anorexia, excitability, and ruffling of neck hair, which were followed by convulsions and death. PAM was