It has been reported that the virulence of axenically cultivated Entamoeba histolytica increases following growth with cholesterol. The purpose of this study was to determine whether cholesterol would enhance the virulence of axenically cultivated Naegleria fowleri. Amoebae were cultivated in axenic medium with (100 pg/ml) or without cholesterol for 6 months and tested in mice for changes in virulence. After 6 months of continuous cultivation, N. fowleri grown with cholesterol was less virulent for mice than the same strain grown without cholesterol.
Several conditions of isolation were evaluated to determine which yielded the greatest number of thermotolerant and pathogenic freeliving amebae. Swab samples, easier to obtain and process, produced more pathogenic amebae than water samples. If water samples are required, 50-ml volumes gave the greatest percentage of pathogenic isolates. An incubating temperature of 42"C yielded the most thermotolerant amebae. A total of 11 pathogenic isolates were obtained from 762 environmental samples and were Acanthamoeba (55 %), Naegleria fowleri Carter, 1970 (27 %), and N. australiensis De Jonckheere, 1981(18%).
Primary amoebic meningoencephalitis (PAM) was induced in mice by intranasal inoculation of Naegleria fowleri (Singh et Das, 1970) to study the role of the blood vessels and lungs in the early and later stages in this disease. Upon culturing blood and lung tissue obtained at 24-, 36-, 48-, 72-, 96-, and 120-hour time periods, it was found that amoebae grew only from blood and lung tissue obtained at the 96 and 120 hour time periods. Paraffin sections of the head revealed small foci of acute inflammation and amoebae within the olfactory bulb of the central nervous system (CNS) at 24 hours. Amoebae were not observed within blood vessels of the CNS until 96 and 120 hours. Also, amoebae were observed within the connective tissue surrounding blood vessels and sutures of the skull, bone marrow, and venous sinusoids between the skull bone tables at 96 and 120 hours. No amoebae or acute inflammatory reactions were observed in the lung sections from any time period and indirect immunofluorescence microscopy was negative for N. fowleri. This study provides evidence that neither blood vessels nor lungs provide routes for N. fowleri to the CNS during the early stages of PAM and that amoebae enter veins of the CNS and bone marrow during later stages of the disease.
This is a followup report on the viability of pathogenic Acanthamoeba castellami, Naegleria australiensis, and N. fowleri during 5 years of cryopreservation and the virulence of N. fowleri during 30 months of cryostorage, all at -70’C. The greatest decrease in viability occurred during the first year of freezing and was 10-fold greater than the average yearly decrease during years 2-5. At 5 years of cryostorage, viability was 33 % for A. castellana, 38 % for N. fowleri and 51% for N. australiensis. Virulence of N. fowleri did not decrease during 30 months of freezing and what appeared to be an increase in virulence during cryopreservation may be the result of reduced viability of the less virulent amebae in a culture.