88
S.L. Chang
strains of Naegleria exhibited marked CPE on PMK cells while 3 wild strains (3 swimming
pools and 1 grown in PMK cell cultures for quite sometime) exerted no harmful effect.
8. Piaquing in Bottled Cell Cultures
Because of the CPE pathogenic strains of Naegleria can be isolated from nonpathogenic
strains by piaquing in PMK cell sheets. The piaquing technique has been described in another
early report (Berg et αί, 1961), for quantifying virus concentrations. Briefly, it consists of
(a) preparing cell sheets in 4-oz. prescription bottles, (b) placing
l
A ml amoebic suspension
over each cell sheet and allowing a standing period of Vi-l hour, and (c) overlaying the sheet
with melted agar (45°C). The bottles thus prepared are incubated at 35°-37°C and examined
after 34 days of incubation for 7-10 days for appearance of plaques.
9. AnimalPathogenicity Test
This was done by either intranasal or intracerebral inoculation or both into mice of an
amoebic suspension and observing thereafter the development of symptoms and signs of
meningoencephalitis. Sick animals were autopsied for pathogenical examination of the
brain.
PRESENCE OF NAEGLERIA AMOEBAE IN WATER AND
SEWAGE EFFLUENT
With the methods aforedescribed a large number of samples collected from effluents of
aerobic sewage treatment plants, from lakes and rivers, and from outdoor swimming pools
have been processed and examined for types and numbers of amoebae. The results obtained
from these examinations are summarized in Table 1, together with some reported elsewhere
(Chang, 1971).
Table 1 shows that sewage effluent from aerobic treatment processes carry far more
free-living amoebae than large rivers that are used as receiving waters. These rivers, in turn,
carry significantly more amoebae than the lakes that are free of sewage pollution.
Interestingly, even the outdoor swimming pools carry small numbers of free-living amoebae,
probably introduced into the pool by bathers who have walked on the nearby ground.
Another interesting point is the predominance of A canthamoeba over Naegleria amoebae
in sewage effluents and polluted river water and the reversal of the occurrence in lake waters.
Of particular interest is the isolation oiN. gruberizs the only species in 3 out of the 6 lakes in
Florida. However, the Australian cases of amoebic meningoencephalitis were associated
with swimming in a sewage-polluted estuary (Carter, 1968). Since the Acanthamoeba is
better adapted to adverse conditions than the Naegleria amoebae (Chang, 1971), it is
understandable that the former is present in larger numbers than the latter in sewage effluent
and polluted waters.
CYTOPATHIC EFFECT AND PATHOGENICITY OF WILD AND PATHOGENIC
STRAINS OF NAEGLERIA
The CPE on mamalian cells was ascertained by growing both wild and pathogenic strains
of Naegleria amoebae in the respective cell cultures; the pathogenicity of these strains were
ascertained by intranasal and intracerebral inoculations into mice. The results obtained
from these experiments are summarized in Table 2.
S.L. Chang
strains of Naegleria exhibited marked CPE on PMK cells while 3 wild strains (3 swimming
pools and 1 grown in PMK cell cultures for quite sometime) exerted no harmful effect.
8. Piaquing in Bottled Cell Cultures
Because of the CPE pathogenic strains of Naegleria can be isolated from nonpathogenic
strains by piaquing in PMK cell sheets. The piaquing technique has been described in another
early report (Berg et αί, 1961), for quantifying virus concentrations. Briefly, it consists of
(a) preparing cell sheets in 4-oz. prescription bottles, (b) placing
l
A ml amoebic suspension
over each cell sheet and allowing a standing period of Vi-l hour, and (c) overlaying the sheet
with melted agar (45°C). The bottles thus prepared are incubated at 35°-37°C and examined
after 34 days of incubation for 7-10 days for appearance of plaques.
9. AnimalPathogenicity Test
This was done by either intranasal or intracerebral inoculation or both into mice of an
amoebic suspension and observing thereafter the development of symptoms and signs of
meningoencephalitis. Sick animals were autopsied for pathogenical examination of the
brain.
PRESENCE OF NAEGLERIA AMOEBAE IN WATER AND
SEWAGE EFFLUENT
With the methods aforedescribed a large number of samples collected from effluents of
aerobic sewage treatment plants, from lakes and rivers, and from outdoor swimming pools
have been processed and examined for types and numbers of amoebae. The results obtained
from these examinations are summarized in Table 1, together with some reported elsewhere
(Chang, 1971).
Table 1 shows that sewage effluent from aerobic treatment processes carry far more
free-living amoebae than large rivers that are used as receiving waters. These rivers, in turn,
carry significantly more amoebae than the lakes that are free of sewage pollution.
Interestingly, even the outdoor swimming pools carry small numbers of free-living amoebae,
probably introduced into the pool by bathers who have walked on the nearby ground.
Another interesting point is the predominance of A canthamoeba over Naegleria amoebae
in sewage effluents and polluted river water and the reversal of the occurrence in lake waters.
Of particular interest is the isolation oiN. gruberizs the only species in 3 out of the 6 lakes in
Florida. However, the Australian cases of amoebic meningoencephalitis were associated
with swimming in a sewage-polluted estuary (Carter, 1968). Since the Acanthamoeba is
better adapted to adverse conditions than the Naegleria amoebae (Chang, 1971), it is
understandable that the former is present in larger numbers than the latter in sewage effluent
and polluted waters.
CYTOPATHIC EFFECT AND PATHOGENICITY OF WILD AND PATHOGENIC
STRAINS OF NAEGLERIA
The CPE on mamalian cells was ascertained by growing both wild and pathogenic strains
of Naegleria amoebae in the respective cell cultures; the pathogenicity of these strains were
ascertained by intranasal and intracerebral inoculations into mice. The results obtained
from these experiments are summarized in Table 2.