Pathogenic Free-Living A moebae and Recreational Waters
89
Table 2 shows that 8 strains of the pathogenic Naegleria exhibited marked CPE on
PMK cells but none grew in either the Vera or Hep-2 cell cultures. They all produced a
fatal meningoencephalitis in mice by either route of inoculation. On the other hand, none
of the wild strain of N. gruberi showed CPE on PMK or Vera cells but a mild CPE on
Hep-2 cells. They produced no disease in mice even by the intracerebral inoculation,
and no inflammation of, or presence of amoebae, in the brain at the site of
inoculation.
One of the 9 pathogenic strains of Naegleria (strain WM from Richmond, Virginia) was
reported to be highly pathogenic to mice (Duma et al, 1971); it exhibited a questionable
CPE on PMK cell and grew well in both Vera and Hep-2 cells. Since this strain has been
established in cell cultures for only a few months, its behavior in these cell cultures will
be followed and its pathogenicity in mice will be retested.
Two additional strains of patient origin, L^ and L2, were received from Dr. David
Warhurst of the Liverpool School of Tropical Medicine. Strain Lj was isolated from the
cerebrospinal fluid of the 3 recent cases in England and L2 from the brain at autopsy.
The former was identified as Naegleria and L2 as Schizopyrenus. Both grew in all 3 types
of cell cultures but exhibited only a mild CPE on Hep-2 cells. No pathogenicity was
observed of either strain in mice by intranasal or intracerebral inoculation. It appears that
both strains of amoebae could have been contaminants rather than the etiologic agents.
Also of interest is the fact that all the wild strains ofiV. gruberi employed in the study
had been grown in PMK cell cultures for at least 15 months prior to the pathogenicity
tests. Their lack of CPE on these cells during the period of cultivation in our laboratory
and their lack of pathogenicity in mice strongly indicate that the cytopathic property and
pathogenicity of the Naegleria are genetically related and cannot be acquired through
prolonged cultivation in cell cultures.
These results indicate a definite correlation between the CPE on PMK cells and
pathogenicity in mice. In this connection it is of interest to note that several strains of A.
rhysodes that were isolated from human throats (Wang and Feldman, 1967) and carried
in PMK cell cultures exhibited a mild CPE on the cells and produced localized
granulomatous lesions with presence of amoebae in the brains of mice at the site of
inoculation, although the infected mice showed no ill effect from the amoebae (Chang et
al., 197-).
SURVIVAL OF PATHOGENIC NAEGLERIA IN WATER IN
COEXISTENCE WITH WILD STRAINS
Taking advantage of the fact that wild strains of N. gruberi form plaques on plates in
bacteria-association much earlier than the pathogenic strains of Naegleria, and that only
the pathogenic strains of Naegleria form plaques in PMK cell sheets, these plaquing
methods were employed to study the survival of a pathogenic Naegleria (Richmond,
Virginia, strain) in coexistence with a wild strain (a swimming pool strain) in a simulated
natural aquatic environment in the following manner.
A suspending water was prepared by mixing 50 mg "Purina" fishfood powder in
200 ml dilution water. The fishfood was so sterilized in a hot-air oven that the powder
was partially charred. This treatment made the powder quite resistant to bacterial
decomposition thus permitting a limited amount of bacterial growth and maintaining an
aerobic condition throughout the period of study. A. aerogenes and B. bronchoseptica
89
Table 2 shows that 8 strains of the pathogenic Naegleria exhibited marked CPE on
PMK cells but none grew in either the Vera or Hep-2 cell cultures. They all produced a
fatal meningoencephalitis in mice by either route of inoculation. On the other hand, none
of the wild strain of N. gruberi showed CPE on PMK or Vera cells but a mild CPE on
Hep-2 cells. They produced no disease in mice even by the intracerebral inoculation,
and no inflammation of, or presence of amoebae, in the brain at the site of
inoculation.
One of the 9 pathogenic strains of Naegleria (strain WM from Richmond, Virginia) was
reported to be highly pathogenic to mice (Duma et al, 1971); it exhibited a questionable
CPE on PMK cell and grew well in both Vera and Hep-2 cells. Since this strain has been
established in cell cultures for only a few months, its behavior in these cell cultures will
be followed and its pathogenicity in mice will be retested.
Two additional strains of patient origin, L^ and L2, were received from Dr. David
Warhurst of the Liverpool School of Tropical Medicine. Strain Lj was isolated from the
cerebrospinal fluid of the 3 recent cases in England and L2 from the brain at autopsy.
The former was identified as Naegleria and L2 as Schizopyrenus. Both grew in all 3 types
of cell cultures but exhibited only a mild CPE on Hep-2 cells. No pathogenicity was
observed of either strain in mice by intranasal or intracerebral inoculation. It appears that
both strains of amoebae could have been contaminants rather than the etiologic agents.
Also of interest is the fact that all the wild strains ofiV. gruberi employed in the study
had been grown in PMK cell cultures for at least 15 months prior to the pathogenicity
tests. Their lack of CPE on these cells during the period of cultivation in our laboratory
and their lack of pathogenicity in mice strongly indicate that the cytopathic property and
pathogenicity of the Naegleria are genetically related and cannot be acquired through
prolonged cultivation in cell cultures.
These results indicate a definite correlation between the CPE on PMK cells and
pathogenicity in mice. In this connection it is of interest to note that several strains of A.
rhysodes that were isolated from human throats (Wang and Feldman, 1967) and carried
in PMK cell cultures exhibited a mild CPE on the cells and produced localized
granulomatous lesions with presence of amoebae in the brains of mice at the site of
inoculation, although the infected mice showed no ill effect from the amoebae (Chang et
al., 197-).
SURVIVAL OF PATHOGENIC NAEGLERIA IN WATER IN
COEXISTENCE WITH WILD STRAINS
Taking advantage of the fact that wild strains of N. gruberi form plaques on plates in
bacteria-association much earlier than the pathogenic strains of Naegleria, and that only
the pathogenic strains of Naegleria form plaques in PMK cell sheets, these plaquing
methods were employed to study the survival of a pathogenic Naegleria (Richmond,
Virginia, strain) in coexistence with a wild strain (a swimming pool strain) in a simulated
natural aquatic environment in the following manner.
A suspending water was prepared by mixing 50 mg "Purina" fishfood powder in
200 ml dilution water. The fishfood was so sterilized in a hot-air oven that the powder
was partially charred. This treatment made the powder quite resistant to bacterial
decomposition thus permitting a limited amount of bacterial growth and maintaining an
aerobic condition throughout the period of study. A. aerogenes and B. bronchoseptica