PATHOGENIC FREE-LIVING AMOEBAE AND
RECREATIONAL WATERS
by
Shih L. Chang
Water Hygiene Division
Office of Water Programs
ENVIRONMENTAL PROTECTION AGENCY
Cincinnati, Ohio 1*5268, U.S.A.
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Amoebic MeningoencephalItis and Recreational Waters
Shih L. Chang
The swimming-associated amoebic meningoencephalitis is a relatively
new and uncommon disease, but has attracted increasing attention of
public health workers because of its extremely high fatality and its
occurrence in widely scattered resort areas. In a very recent report
Chang (1971) gathered from the literature and personal communications
a total of ^5 cases - 22 in the United States, 17 in Czechoslavokia,
and 6 in Australia. In addition, five suspected cases were reported in
Britain (Symmers, 1969; Appley et al., 1970; Warhurst et al., 1970).
Two of them were revealed in a retrospective examination of old patho-
logical specimens and the other three occurred in August 19&9 without
a history of swimming.
In April 1971 (Hecht et al., 1970, another case of swimming-
associated anoebic meningoencephalitis occurred near San Bernardino,
California, in a different setting. A girl in a group of about 20
young people was afflicted with the disease after a few days of swimming
In a small, shoulder-deep pool fed by water from a hot spring. The
temperature of the pool water is normally 27.7~39.3°C.
The causative amoebae of this disease is morphologically
indistinguishable from the free-living forms known as Naegleria gruberi.
which are small (15-18u in diameter), actively motile, and capable of
s
trancient flagellate transformation, and are commonly found in sewage
effluents from cerobic treatment processes, in fresh surface waters,
and in soil (Singh, 1952; Chang, 1958, I960, 1971). Cysts of JL. .gruberi
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2
have also been found in municipal water supplies (Chang et al., I960)
and fn outdoor swimming pools (Chang, 1971).
The generally accepted belief in the mode of infection of amoebic
•
menlngoencephalitis is that the amoebae gain their entrance into the
upper part of the nasal cavity through diving or swimming activities.
From there they work their way through the nasal mucosa and the
cribiform plate into the cranial cavity. They first attack the meninges;
expansion of the lesion into the adjacent part of the brain results in
meningoencephalitis.
To understand the epidemiology of this disease a number of questions
must be answered: 1. Are pathogenic strains of Naegleria fundamentally
different from the wild strains of N. orubcri in pathogenic?ty? 2. Can
wild strains of N. gruberi become pathogenic under certain conditions?
3. Can pathogenic strains of Naeoleria establish growth in the midst of
wild N. grubcr? in a natural aquatic environment? 4. Can pathogenic
Naegleria produce asymptomatic infections in man by parasitizing the
nasopharyngeal region and cause the disease only when the amoebae are
washed into the upper nasal cavity? 5. Can wild strains of JL. gruberi.
parasitize human nasopharyngeal region and gain pathogenic!ty through
such a parasitism? 6. Do aquatic or terrestrial animals or birds serve
as carriers of the pathogenic Naegleria? 7. Why should an indoor
swimming pool be involved in the occurrence of the disease?
It is intended in this report to present data that hove been
obtained both in the laboratory and the field to answer some of these
questions and to discuss the direction of future research in order that
preventive measures Caii Le forrnuiatCu.
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3
Materials and Methods
1. Naeqlcria Amoebae
Nine pathogenic strains of Naegleria, all isolated from autopsy or
cerebrospinal fluid, were carried in PMK (primary monkey kidney) cell
cultures. Four of these strains were also-carried on the BST (buffered
sucrose tryptose) agar plates in association with a strain of Bordetella
bronchoseptica. Ten wild strains of N. gruberi, isolated from swimming
pools, river or lake waters, and sewage effluents, were carried on the
BST agar plates in association with a strain of Aerobector aeroqenes.
All cell cultures were kept at 35°C and plate cultures at room
temperature (25°-27°C).
2. Bacteri a-Associates
The B. bronchoseptica. Gram~negative and rod-shaped, were isolated
from a culture on inorganic agar of one of the pathogenic strains of
Naeqleri'a. It was apparently, a contaminant but found to support the
growth of both wild and pathogenic strains of Naegleria. Stock cultures
were carried on heart infusion agar slants. The A. aeroqenes was
isolated from a se\vage effluent. It supported the growth of wild strains
of Haeqlcria on the BST agar plates and that of the pathogenic strains
on Inorganic agar. Stock cultures were carried on nutrient agar slants.
3. HaT.-i?l ion Cel 1 Cul tures
Primary MK cells were purchased from a commercial source. PrepaVation
of tube and bottle cultures has been described elsewhere (Chang et a).,
1958; Berg et a)., 19&3). Hep-2 cells (oesophageal cancer cell line) was
obtained from Or.' Robert Sullivan of the Milk Sanitation Research Section,
FDA, Public health Service, and Vera cells (African green monkey kidney
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cell line) from Dr. Gerald Berg of the Advanced Waste Treatment Research,
Cincinnati Water Research Laboratory. 'Both cell lines were originally
purchased from the-American Type Culture Collections. Tube and bottle
cultures of each cell line were prepared in the same manner as the PMK
cells except that the EME (medium with Earl's base) was used for growth
and maintenance of the Hep-2 cells.
*K BST Aaar
The BST agar was developed primarily for cultivation of free-living
amoebae in laboratory (Chang, 1958). It is a phosphate-buffered (PH J.k-
7.5) agar containing 1% (w/v) sucrose and 0.2% tryptose. The low concen-
tration of tryptose is necessary because of its inhibitive effect on
amoebae at concentrations commonly employed in bacteriological media.
Agar plates were poured with 25 ml of the medium per petri dish.
5 Concentration of Amebae from Water or Wastewater
The method for concentrating amoebae in water has been previously
described (Chang 1971). In brief, gallon-size samples are allowed to
pass through 25u microstrainers before they are filtered through lOu
membranes. Each membrane is placed on the wall of a sterile beaker and
flushed repeatedly with a small amount of dilution water with the aid
of a capillary pipette. The washings are surveyed for types and concen-
tration of amoebae by examining under low power one or more drops placed
on a microslide. The number of amoebae gal'1 is obtained by multiplying
the average number of amoebae drop'1 by the number of drop in ml and
number of ml in a gallon.
Fiberglass prefiltcrs can be used to replace the lOu membranes. If
. .. , ».._.. M^ .•<- —x* —-^.* .p».«. Q§" * •fiF>r"i"lt«T>S
LUC ^fcTciiii^*' itCfcwi i. fc^j *•- - » « - -
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5
filters may be used for each sample! The washings are pooled before they
are examined for amoebae. If the volume of the pooled washings is too
large, it can be reduced by centrifuging and resuspending the sediment
In a desirec amount of dilution water.
6. Examination of Concentrated Samples for Amoebae bv Plaguing on Plates
This technique is quite similar to the plaque counting of bacteriophage
and has been described elsewhere (Chang,1971). In essence, a concentrated
sample is diluted with a suspension of B. bronchoseptica such that each
drop from a capillary pipette contains not more than 3 amoebae. The
entire sample is plaqued by placing 10 drops on each poured BST agar
plate and smearing the mixed suspension evenly over the whole plate with
a sterile golf club-shaped glass rod. After the liquid is absorbed, the
plates are turned over and incubated at 25°-28°C. Examination of plates
for plaques starts on the 3rd day and covers a period of 2 weeks.
•
Wild strains of N. gruberi and other schizopyrenid amoebae grow
fast on bacteria and generally show plaques in 3 to k days of incubation;
HartTnnel la and Ac?nth:-o.:ba spp. grow slower and take 7-10 days to show
plaques.. The pathogenic strains of Naegleria grow so poorly in the
presence of bacteria growth that their plaques are not visible until
after 12-15 days. These differences have already been observed with
several strains of wild and pathogenic Naegleria and other species of
free-living amoebae (Chang, 1971)•
Microscopic examination of the plaques for trophozoites and cysts
enables one to place the amoebae in general groups mentioned above. To
establish cultures of single strains of amoebae the center of a plaque
if fntir^ftft l.ii th an • «i^/-n I pi- i nn nnorllo f\f I O("»0 pnrf rn<» pmn»na»» SO
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6
up are placed in the center of a BST agar plate preseeded with B^.
bronchoscotica or on an inorganic agar plate preseeded with A^. aerogenes.
The amoebae will move away from the site of inoculation, and transfer
of the migrated amoebae with the bacteria-associate onto a new plate
results in the formation of a pure amoeba culture with a known
bacter i a-as soc i ate.
7. Preliminary Test for Pathooenicity
This is done by demonstrating the CPE (cytopathic effect) of the
amoebae on PMK cells. The amoebae of an established plate culture were
suspended in sterile dilution or distilled water, and were repeatedly,
centrifuged and washed to remove the associated bacteria. The sediment
was finally suspended in water containing 200 ugm each of tetracycline
and streptomycin. After 2-k hours of storage in a refrigerator the
suspension was centrifuged again and the supernate discarded. The
sediment was transferred to a tube of PMK cell cultures. Generally,
one trial was enough to establish a positive culture. Occasionally,
I or 2'trials were needed before a positive culture was prepared.
Similar procedure was used for preparing amoebic cultures in Hep-2 or
Vera eel 1 tubes.
As soon as the cultures were established, they were observed for the
CPE on the mammalian cells. In the preceding report (Chang, 1971) it
has been shown that k pathogenic strains of Naeglcria exhibited marked
CPE on PMK cells while 3 v/ild strains (3 swimming pools and I grown in
PMK cell cultures for quite sometime) exerted no harmful effect.
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7
8. Plaguing in P~"^ CP!! Cultures"
Because of the CPE pathogenic strains of Naeqleria can be isolated
fr
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8
amoebae than the lakes that are free-of sewage pollution. Interestingly,
even the outdoor swimming pools carry small numbers of free-living
amoebae, p'robably introduced into the pool by bathers who have walked
on the nearby ground.
Another interesting point is the predominance of Acanthe^oeba over
Naegleria enoebae in sewage effluents and polluted river water and the
reversal of the occurrence in Jake waters. Of particular interest is
the isolation of N. gruberi as 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, 19&8).
Since the Acanthamoeba is better adapted to adverse conditions than the
Naeqleria amoebae (Chang, 1971), it is understanable that the former is
present in larger numbers than the latter in sewage effluent and polluted
waters.
Cvtooathic Effect and Pathocenicitv of Wild and
Pathogenic Strains of Macgleria
The CPE on mammalian cells was ascertained by growing both wild
and pathogenic strains of Naeqleria amoebae in the respective cell
cultures; the pathogenicity of these strains were ascertained by intra-
nasal and intracerebral inoculations into mice. The results obtained
from these experiments are summarized in Table 2.
Table 2 shows that 8 strains of the pathogenic Naeqleria exhibited
marked CPE on PMK cells but none grew in either the Vera or Hep-2 cell
cultures. They all produced a fatal meningoencephal!tis in mice by
either route of inoculation. On the other hand, none of the wild strain
of N^ qruhcri showed CPC on Pf'.K or Vera cells but a mild CPE on Hep-2
cells. Ti'icy produi.Ku no liisedSc in mice even by • im inlrcicerebrai
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9
Inoculation, and of the inoculated mice were sacrificed and no
inflammation of, or presence of amoebae, in the brain at the site of
Inoculation.
One of the 9' pathogenic strains of Naeoleria (strain WM from
Richmond, Virginia) was reported to be highly pathogenic to mice
(Duma et al.f 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, Lj and L2> were received
from Dr. David Warhurst of the Liverpool School of Tropical Medicine.
Strain L. was isolated from the ccrebrospinal fluid of the 3 recent
cases in England and L. from the brain at autopsy. The former was i-
dentified'as Naegleria and l_2 as Schizoovrenus. 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 of N. aruberi
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 thesev
cells during the period of cultivation in our laboratory and their lack
of pathogenicity in mice strongly indicate that the cytopathic property
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10
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. rhvsodes that were isolated
from human throats (Wang & Feldman, 19&7) 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 Naealeria in Water in
Coexi s ten ce w i t h W i 1 d S t r a i n s
Taking advantage of the fact that wild strains of N. qruberi 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 Maegleria (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
hoi-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. rcrocrnrs and R. bronchscpt ica
suspensions were added to the water to give each a calculated density
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II
of 5,000 ml"'. The bacteria-laden water was dispensed in J*0-m1 amounts
in 3 petri dishes, and each dish received enough suspensions of both
strains of Nj°gleria to give a calculated amoeba density of 50 ml of
each strain.
At weekly intervals of incubation at room temperature 3-ml samples
were taken and processed for plate plaquing for enumerating the wild
Strain of N. grubori and another set of 3-ml samples for cell sheet
plaquing to enumerate the pathogenic Naegleria. Decimal dilutions were
prepared from the samples and used in both plaquing when plaque counts
were expected to be high. The duplicate plaqued plates were incubated
at room temperature and the plaqued cell sheets in bottles were incubated
at 35°C and were examined for appearance of plaques daily for 2 weeks.
To free the samples of bacteria for plaquing on cell sheets, each sample
was washed and centrifuged *4 times with dilution water containing
50 ug/ml' each of tetracylene and streptomycin.
The averages of the duplicate plaque counts during a 6-week study
are plotted graphically in Figure 1. The growth curves of the strains
of Nseglcria in the figure show clearly that while the wild strain was
growing in a manner similar to that ascertained in plate cultures
(Chang, 1958), the pathogenic strain showed a steady decrease in popu-
lation and reached almost its extinction in a 6-week period.
To illustrate the appearance of plaques formed by the pathogenife
strain cell culture bottles, a photograph of a plaque bottle prepared
with a *»th week sample is shown in Plate I (A). A photograph of 3 '
plcqucd plates prepared with a 100-fold dilution of a *Uh week sample is
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12
also shown in the same plate to show the appearance of plaques formed by
the wild strains of N. gruberi (B). Worth mentioning is that the
plaques formed on plate cultures in bacteria-association can be identified
with some degree of certainty by examining the plaques under 10X
magnification. As shown in Plate I the wild N. gruberi plague was
packed with well-defined, pearl-like cysts (C), while that formed by the
pathogenic strain showed the majority of the cysts of "ghose cell"-like
appearance (D).
These findings indicate that under natural aquatic conditions with
bacteria as the chief, if not sole, supply of food, it would be very
difficult for pathogenic strains of Haegleria to maintain colonies in
the midst of overwhelmingly larger numbers of wild strains. This does
not rule out the possibility that under certain conditions, which
favors the survival of the pathogenic, Naegleria may persist in an
aquatic environment over a long period.
Probability of Existence of Hu-nan Carriers and/or
Animal Hosts of Pathogenic Naeglcria.
i
Very recently we received 12 cultures of free-living amoebae from
Or. George R. Healy of the Center for Disease Control, Public Health
Service. These cultures were isolated on inorganic agar with A. aerooenes
by nasal swabs from children before and after swimming in lakes. Of
significance is that eight of these isolates made after swimming and,2
made before swimming have been identified as Naegleria. Attempts will
be made to establish them in cell cultures and test them for pathogenic!ty
in mice. It would be most interesting if any of these strains of
should U-rn out to be patnorenic.
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13
These findings were in contrast to those reported by Wand and
Feldman (196?) who encountered no Naeoleria amoebae in their cell
cultures prepared with throat swabs. This difference could be due to
either preference of Naeoleria amoebae to parasitize the nose to the
throat or a short duration of parasitism by these amoebae.
Comments and Conclusions
The findings from this study provide only a few missing pieces of
swimming-associated meningoencephalitis puzzle. They do indicate that
this disease entity is caused by a species of Naegleria which is
physiologically and pathologically different from the species Jk .qruberi,
commonly found in natural waters and soil. It is logical to hypothesize
that the cytopathic effect of the pathogenic Naegler.ia is essential to
its ability to penetrate the mucosa in the upper nasal cavity and the
cribriform plate to facilitate the cranial invasion.
*
The isolation of Naeqleria amoebae only in some of the Florida
lakes is an interesting fact, but its significance cannot be ascertained
without a further study of the presence of pathogenic strains among
these amoebae. Such a study can be made with the methods described in
this report and will be carried out in the near future. It is hoped
that similar studies will be made in other involved areas. In this
^S
connection it is worth noting that Dr. Lubor Cerva has already
v
initiated such a study at the indoor swimming pool in Czechoslavokia
(Cerva, 1971). He reported the presence of Haenleria amoebae in the
water and in all areas of the swimming pool in contact with the water and
failed to isolate £ny pctl-.o^nic strains of Mncglcria in any part of
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14
the pool. Since the method used by terva could allow the wild strains
of Ncsglcrin to overgrow the pathogenic individuals which, if present,
would be so few in numbers, a different approach may be necessary to
give the latter a chance to be isolated.
The possible presence of pathogenic Naegleria in human carriers or
animal reservoirs should be investigated. The method employed should
allow the pathogenic individuals to be detected. A fluorescent antibody
Straining method, if developed, would be ideal for such a survey.
Summary
1. The swimming-associated cases of amoebic meningoencephalitis were
caused by a species of Haegleria which is distinct from the wild strains
of N. gruberi. This is based on the facts that the pathogenic strains
exhibited profound CPE on PMK cells and produced a fatal meningoencephalitis
in mice by intranasal and intracerebral inoculations, while the wild
•
strains of N. oruberi exerted no CPE on PMK cells and produced no disease
In mice by both inoculations.
2. Both Acanthe.fTioeba and Noegleria were frequently found in sewage
effluents and fresh surface waters, and in small numbers in outdoor
..swimming pools. The Acantha.r.oeba was predominant in sewage effluents and
polluted waters, while the Nacglcria was predominant in small warm lakes.
3. There was no evidence that wild strains of N. qruberi could acquire CPE
or bccc-'o pathogenic after a long period of cultivation in cell
cultures.
I*. Under simulated natural aquatic environment the pathogenic Naegleria,
in coexistence with a wild strain of H. grubori reduced its population
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Table I
Small Free-Living Amoebae in Aerobic Sewage Effluents,
Fresh Surface Waters, and Outdoor Swimming pools
Samples examined
No. of Amoebae
Sal'1
Amoebae isolated in cultures
and % in total amoebae
Waste effluents
Cincinnati trickling
filters
50,000 * 6,000
Lebanon stabilization
ponds . 35,000 * it,000
A., rhvsodes (65%)
Hi. qruberi ( 18%)
Others (17%)
A., rhvsodes (60%)
Ik qruberi (20%)
Others (20%)
Surface waters
Kentucky lake 1
lake 2
Florida lake 1*
. lake 2
lake 3
lake **
lake 5
lake 6
Ohio River near
Cincinnati
Missouri River near
-- Kansas «»».->-
Swinminq pools
Cincinnati pool 1
pool 2
pool 3
150 -
350 65
130
190
185
200
110
140
320 i 60
18 - 7
21 1 8
11 +5
N. qruberi (**2%)
A., rhvsodes (30%)
Others (28%)
A. rhvsodes (^5%)
Hi qruhcri (U0%)
Others (15%)
ils. qruberi
Hs. qruberi
Nj qruberi
Hi. qrur-er.i. A_._ radios a
Hi. qruberi , FL_ aoricola
Schizopyrenus russel1i
800 i 167 A_.. rhvsodes (80%)
N. qruberi (18%)
Others ( 2%)
Aj. rhvsodes (60%)
N. qrubcri (15%)
Others (25%)
A., rhvsodes (83%)
fL.5L*i^£l (17%)
/L. rhvsodes (86%)
N. Oj.-v.Serl (10%)"
Hi mr icnla (
A_j. rhysoHos. (90%)
N. grii^ri (10%)
« Ihcsc results x.'2r.i oDl.Tir.ca rro-n a coopcrotivc s iuay wi in LIIC uivibiun 01
Heal III, Florida State Department of Health and Rehabilitation Service.
More samples will be examined andxresults will be reported in detail
when the study is completed.
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4x10-
1x10-
1x10
u.
o
Ul
o
IxlO1
1x1 Oc
O .... WiId strain
O .... Pathogenic strain
1x10
-1
I
I
I
TIME (week)
FIGURE I. GROUTH OF WILD AND PATHOGENIC STRAINS OF N, GRUBER1
|M SIMIIIATCn HATIIDAI »mi«Tl»> ptiliinnin
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Table II
Pathogen!city of Wild and Pathogenic Strains of Naeqleria
Strains and Origin
CPE on mammalian cells Pathogenicity on mice*
Mild strains of N. oruberi
2 strains from sewage
effluents
1 strain from Ky lake
and 2 from Ohio and
Missouri Rivers
3 swimming pool strains
No CPE on PMK and vera
cells; some CPE on
Hep-2 cells
No CPE on PMK and Vera
cells; some CPE on
Hep-2 cells
No CPE on PMK and Vera
eel Is; some CPE on
Hep-2 cells
Nonpathogenic to
mice by either intra-
nasal or intracerebral
inoculation
Nonpathogenic to mice
by either fntranasal
or intracerebral
inoculation
Nonpathogenic to mice
by either intranasal
or intracerebral
Inoculation
Pathogenic strains of Neenleria
2 strains from
Florida
Marked CPE on MK cells;
no growth in Vera or
Hep-2 eel 1 cultures
1 strain from
Czcchoslavokia
2 strains from Australia
3 strains from Richmond,
Virginia
ii
ii
ii
ii
Causing fatal meningo-
encephalitis in mice
by either intranasal
or intracerebral
inoculation
n
n n
n n n
n n n
* Most of these results v/crc obtained in cooperative studies with
Or. C. G. Culberison of Lilly Research Laboratories and Or. Richard J.
Dur.;i of Virginia ::^icol Center. These results will be presented in
detail in another report.
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15
steadily and reached almost extinction in 6 weeks of standing at room
temperature, while the wild strains exhibited a growth curve similar
to that observed in plate cultures.
5. Of 12 cultures of amoebae isolated from nasal swabs of children
made before and after swimming in lakes, 2 preswimming 8 postswimming
cultures were identified as Nreoleria. Their growth characteristics
in cell cultures and their pathogenicity in mice remain to be ascertained.
6. Of samples taken from 6 Florida lakes, Naegleria was isolated alone
in three and Naeoleria and Schizopyrenus were isolated in one. The
growth characteristics in cell cultures and pathogenicity of these
Naegleria strains remain to be studied.
From these findings it is concluded that much research is needed
In the laboratory as well as in the field to ascertain the origin of
• the pathogenic Naeoleria in fresh surface waters and in the Czechoslavokian
indoor swimming pool. This requires investigations looking into the
presence of pathogenic Maegleria in waters and pools where cases of
amoebic meningoencephalitis have occurred, into the existence of human
carriers and into the possibility of aquatic and terrestrial animals,
_and birds serving as hosts of pathogenic Nocgleria. With the methods
described in the preceding and this report, it is hoped that such
investigations wi11 be carried out in the "endemic" areas.
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•
Chang.S.L., M-i tchell ,R. B. , and Schneider, N.J. Free-living amoebae in Florida
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"Mori. Wkly. Rep. .20:217.
_Syir.rncrs,W.St.C. .(1S69). Primary amoebic meningorencephal i ti s in Britain.
Brit. Med. J.
Singh, B.N. (1952). Nuclear division in nine species of small freeliving amoebae
and its bearing on the classification of the order Amoebeda. Phil. Trans.
— Roy. Soc. London, Ser. B ^36:^05-
Wann.S.S. «nd Feldr.ian.H. A. (19&7). Isolation of Hartmanncl la species from
hur.ian throats.' New Eng. J. Med., 277:1 17**- 11 79.
rrri-iirosni na I fiuiri. Tran^. kny. -snr. Trop. /-"^". nyy. 6^: i^-2 i .
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"71
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Explanation of Plate 1
A. ..A PM-C cell bottle culture showing plaques formed bv
patnosenic ^eoTeria after an ll^da'y incubl?ion ^
bo,de cdKure was plaqued on the 3rd week of testi
"
^
»- (t"cil? l97') and is shown here for the
or comparison. Notice the "ghost-eel ["-like
h"°9en1e «olerU aar"
the :f8!°9en1e 5«olerU, aparony due
- 1$ not so
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