After a prolonged period of maintenance in Nelson's axenic medium, the HB-1 and C-66 strains of pathogenic Naegleria fowleri were observed to have lost their original virulence for mice. Cloned low virulence substrains were established and compared to cloned high virulence substrains that were maintained on monkey kidney cell (MKC) cultures. Some differences in physiology between the low virulence and high virulence substrains were shown by their growth in Nelson's medium and MKC cultures, cytopathic effects, catalase production, as well as in pathogenicity for mice inoculated intranasally. The importance of the identification of the factors which may contribute to the loss or acquisition of virulence of these free-living amebae is stressed. In the decade since primary amebic meningoencephalitis (PAM) caused by Naegleria spp. was first recognized, over 60 human cases have been reported and over 20 isolations of amebae have been made (Chang, 1974). A salient feature of these isolates is the almost uniform lack of reports of loss of virulence in culture. However, Chang (1971) reported the loss of cytopathic effects (CPE) caused by the WM (Virginia, USA) isolate of N. fowleri in tissue culture. More recently, Visvesvara and Callaway (1974) observed a decrease in the virulence for mice of the HB-1 (Florida, USA) and C-66 (or Carter 66, Australia) strains after intranasal inoculation. In a series of experiments in our own laboratories on the susceptibility of nonhuman primates to N. fowleri, variation in the virulence of axenically maintained HB-1 and C-66 strains was noted (Wong et al., 1975). In order to study some of the factors which might affect the virulence of these two strains, amebae were grown in axenic medium or tissue culture over a period of time and tested for animal virulence. This report summarizes the result of these experiments. MATERIALS AND METHODS The isolation and cultural history of the HB-1 and C-66 strains of N. fowleri used in this study have been reviewed elsewhere (Wong et al., 1975). Received for publication 18 February 1977. * Supported by Research Grant RR00169 from the NIH. t California Primate Research Center, University of California, Davis, California 95616. S Present address: Department of Nutrition and Food Science, University of Kentucky, Lexington, Kentucky 40506. Maintenance of cultures Cultures of the 2 strains of ameba were either m intained by continued passages in Nelson's axenic medium (NM) or in monkey kidney cell (MKC) monolayer cultures. The MKC were maintained in Leibowitz's L-15 medium with 2% v/v of fetal calf serum added. The L-15 was changed every 3 to 4 days. Subcultures in NM were carried out once a week and those in MKC every third day. At the beginning of this experiment, NM subcultures had been passaged about 50 times and those in MKC had been transferred over 170 times. Enhancement of strain virulence Two methods were employed to enhance virulence of uncloned populations of the HB-1 or C-66 amebae which had been maintained in NM: (1) by repeated transfers in MKC which selected out the high virulence population; and (2) by passages through mouse tissues via intranasal or intracerebral inoculation. E tablishment of clones Clones of amebae of HB-1 and C-66 strains were established by spreading dilute suspensions of trophozoites onto nonnutrient agar plates and picking the individual amebae from the surface with a pasteur pipette and the aid of a stereomicroscope. Each of the 10 amebae thus obtained was grown either in NM or MKC cultures and tested for CPE capability in MKC and pathogenicity in mice. Selected clones showing definite high or low virulence were designated as high virulence (HV) or low virulence (LV) substrains of the two strains of amebae.
Read full abstract