EPA-600/1-79-018
May 1979
PATHOGENIC NAEGLERIA: DISTRIBUTION IN NATURE
by
Flora Mae Wellings
Philip T. Amuso
Arthur L. Lewis
Mary Jane Farmelo
Dewey J. Moody
Caroline L. Osikowicz
Epidemiology Research Center
Tampa, Florida 33614
Grant Nos. R-803511 and R-804375
Project Officer
Walter Jakubowski
Field Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
This study was conducted
in cooperation with
State of Florida
Central Operations Services
Department of Health and Rehabilitative Services
Tallahassee, Florida 32301
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
11
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FOREWORD
The U.S. Environmental Protection Agency was created because of increas-
ing public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled land
are tragic testimony to the deterioration of our national environment. The
complexity of that environment and the interplay between its components re-
quire a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching for
solutions. The primary mission of the Health Effects Research Laboratory in
Cincinnati (HERL) is to provide a sound health effects data base in support of
the regulatory activities of the EPA. To this end, HERL conducts a research
program to identify, characterize, and quantitate harmful effects of pollu-
tants that may result from exposure to chemical, physical, or biological
agents found in the environment. In addition to the valuable health informa-
tion generated by these activities, new research techniques and methods are
being developed that contribute to a better understanding of human biochemical
and physiological functions, and how these functions are altered by low-level
insults.
This report describes the results of a survey for pathogenic Naegleria
amoebae in freshwater lakes, studies on the source of the amoebae, and
attempts to determine environmental or ecological factors relevant to the
occurrence of the organism in freshwater lakes. A better understanding of
these factors may allow development of measures to reduce exposure.
iii
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ABSTRACT
Human infection with pathogenic Naegleria, a free-living soil amoeba,
results in a usually fatal disease known as primary amoebic meningoencepha-
litis (PAM). Epidemiological data usually included exposure to freshwater
lakes or streams within the week prior to onset. However, no confirmed iso-
lations had been made from the suspected exposure sites. The major objective
of this study was to determine the presence or absence of pathogenic Naegleria
in Florida's freshwater lakes which had been associated with fatal PAM in-
fections. Secondary objectives were to elucidate the source of these amoebae,
i.e., soil, avian, and/or mammals and to determine the environmental and/or
ecological factors related to the presence of pathogenic Naegleria in lake
waters.
Before field studies could be initiated candidate techniques for concen-
trating amoeba from water and sediments were modified or devised. Isolation
and identification techniques were also evaluated and modified.
Results showed conclusively that pathogenic Naegleria are widely dis-
tributed in Florida's freshwater lakes. Examination of samples obtained from
freshwater lakes in Georgia also revealed that the distribution of these
amoebae was extensive. Samples submitted from a lake in South Carolina, which
was associated with a PAM case, also yielded pathogenic Naegleria, proving
that these organisms are not unique to Florida's subtropical climate. These
studies indicate that pathogenic Naegleria are ubiquitous and overwinter in
lake bottom sediments or at the sediment/lake water interface. No significant
differences have been established among lakes supporting large populations of
pathogenic Naegleria and those supporting very limited or undetectable popula-
tions. However, there does appear to be a trend toward increased numbers of
pathogens in lakes with increased levels of gram negative bacteria on which
pathogenic Naegleria feed.
Laboratory manipulation of the organism has led to a better understand-
ing of the alterations in pathogenicity occasioned by passage of these
amoebae on different types of media.
This report was submitted in fulfillment of Grants No. R-803511 and No.
R-804375 from the U.S. Environmental Protection Agency and was funded in part
by the Department of Health and Rehabilitative Services, State of Florida.
This report covers the period 1-1-75 to 12-31-78.
IV
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CONTENTS
Foreward ............................... iii
Abstract ............................... iv
Figures ................................ vii
Tables ................................ viii
Acknowledgments ............................ xi
1. Introduction .......................... 1
Overall objective of the study
Background data
Study rationale and approach
2. Conclusions ........................... 4
3. Recommendations ......................... 6
4. Materials and Methods ...................... 8
Sampling sites ....................... 8
Stock cultures ....................... 8
Cultivation techniques ................... 8
Incubation temperature ................. 9
Antimycotic agent ................... 9
Antibiotic concentration in CSYECM ........... 9
Growth media ...................... 10
Concentration techniques .................. 10
Throat and rectal swabs or feces ............ 10
Lake waters ...................... 11
Small water samples .................. 12
Sediment samples .................... 12
Isolations ......................... 12
Identification ....................... 13
Mouse inoculation ..................... 13
Indirect fluorescent antibody test ............. 13
5. Results and Discussion ..................... 14
Infection experiments ................... 14
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CONTENTS (continued)
Results and Discussion (continued)
Page
Animal isolation attempts 14
Isolation from natural and thermally enriched lakes ... 15
Characterization of seropositive nonpathogenic and
pathogenic Naegleria 22
Anaerobic studies ... 26
Most probable number (MPN) test 27
Standard method development 29
Minimal lethal dose experiments 33
Sodium chloride tolerance 41
Intra-auricular inoculation 42
Axenic culture 43
Electron microscopy 45
Supportive activites 45
References 56
Appendices
A. Tables 58
B. Chang's Medium 79
vi
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FIGURES
Page
1 Percent of positive water and sediment samples as related
to the average water temperature by month 18
2 Percent positive of sediment samples from all natural
lakes compared with water depths 19
3 Number of sediment samples tested and percent positive
from Lake Conway 20
4 ELow sheet and time required for isolation and identification
of pathogenic Naegleria from field specimens 32
5 Percentage of mouse deaths on individual days post inoculation
and after challenge with 5,000 trophozoites instilled
intranasally 35
6 Seropositive nonpathogenic Naegleria cyst cross section.
Print magnification 10,770 46
vii
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TABLES
Page
1 Antibiotic Effect on Growth of Pathogenic Naegleria 10
2 Number and Percent of Lakes Yielding Pathogenic
Naegleria in the Florida Survey 15
3 Relationship of Water Temperature and Sample Size to Positivity
Shown in the Lake Survey 16
4 Relationship of Sample Size and Water Temperature to Positivity
of Sediment Samples in Lakes Surveyed 17
5 Temperature Effect on Pathogenic Naegleria Cyst Survival .... 21
6 Percent of Water Samples Yielding Pathogenic Naegleria Isolates
from a Thermally Enriched and Three Natural Lakes as a
Function of Temperature 22
7 Generation Time Study of Seropositive Nonpathogenic and
Pathogenic Naegleria at Two Temperatures 23
8 Indirect Fluorescent Antibody Test Results Using Various
Antigens and Antisera, September 23, 1976 24
9 Comparison of Shearing Forces Required to Rupture Cysts of the
Seropositive Nonpathogenic and the Pathogenic Naegleria. ... 25
10 Most Probable Number Test Results 27
11 Most Probable Number Studies 28
12 Most Probable Number Test Results Before and After the
Addition of a Bacterial Suspension to Membranes 29
13 Isolation Results from Six Incubation Treatments of Sediment
Samples 31
14 Data on Mouse Minimal Lethal Dose and Challenge Experiment ... 34
15 Percentage of Deaths on Individual Days Post Inoculation:
Initially and After Challenge 33
viii
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TABLES (continued)
Page
16 Results of Mouse Intranasal Inoculation of a Mixed Culture of
Pathogenic and Seropositive Nonpathogenic Naegleria 37
17 Results of Minimal Lethal Dose and Challenge Experiment in Mice. . 39
18 Decrease in Virulence Following Rapid Passage on Inorganic Agar. . 40
19 Plaque Size of Seropositive Nonpathogenic and Pathogenic
Naegleria as a Function of Sodium Chloride Concentration .... 42
20 Results of Pathogenic Naegleria Inoculation of Guinea Pigs by
Various Routes 44
21 Incubation Histories of Samples from Selected Georgia Lakes
for Pathogenic Naegleria Isolation 51
22 Location of Lakes, Water Temperature, and Results of Pathogenic
Naegleria Isolation Attempts 52
23 Bacteriological Data and the Presence or Absence of Pathogenic
Naegleria in Georgia Lakes Sampled 53
24 Bacterial Density Ranges as Related to the Geographical Location
of the Lake with Respect to Atlanta and the Presence or
Absence of Pathogenic Naegleria 54
A-l Sample Data and Results of Tests on Thermally Enriched Lakes . . 58
/
A-2 Sample Data and Results of Tests on Lake Conway 60
A-3 Sample Data and Results of Tests on Lake Hourglass 63
A-4 Sample Data and Results of Tests on Lake Baldwin ,65
A-5 Sample Data and Results of Tests on Lake Spier 67
A-6 Sample Data and Results from Orange County Lakes Sampled
Infrequently 68
A-7 Results of Survey for Pathogenic Naegleria in Lake Water and
Sediment Samples from Lakes Outside of Orange County 69
A-8 Results of Isolation Attempts for Pathogenic Naegleria from
200 ml Sediment Samples from Potential Control Lakes 72
A-9 Sediment Specimen Positivity Based on Water Depth at
Sampling Site 75
A-10 Chemical Data - Positive Lakes in Georgia 76
ix
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TABLES (continued)
Page
A-ll Chemical Data - Negative Lakes in Georgia 77
A-12 Characteristics of Seropositive Nonpathogenic and Pathogenic
Naegleria 78
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ACKNOWLEDGMENTS
The authors acknowledge the invaluable contributions made to this study
by S. L. Chang, M.D., formerly with the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, Cincinnati, Ohio, now retired. His
expertise based on years of experience in the field of protozoology was of
inestimable value during the initial planning and start-up of this project
when staff was being trained and techniques were being developed.
The authors also acknowledge the valuable contributions of Walter
Jakubowski, the Project Officer who provided the opportunity to broaden the
study to include testing field samples from outside of Florida. His actual
participation in the field investigation of the PAM case and his facilitating
the lake survey in Georgia are sincerely appreciated. In addition, the over-
all project has benefited from his critical review of reports and his helpful
suggestions.
The studies conducted in Georgia could not have been accomplished with-
out the able assistance of Dr. John E. McCroan, State Epidemiologist and Dr.
Maurice Patton, Health Officer, and staff members of the Laboratory and
Sanitary Engineering Section of the Georgia Department of Human Resources.
Special recognition must be extended to Mr. W. J. Carroll, owner of the
recreational site where our first investigation took place, who voluntarily
and graciously encouraged the investigation.
In house, Dr. Lillian Stark is recognized for the statistical analyses
of data.
xi
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SECTION 1
INTRODUCTION
OVERALL OBJECTIVES
Infection in man with pathogenic Naegleria, a free living soil amoeba,
results in a disease known as primary amoebic meningoencephalitis (PAM). All
but one of the 34 recognized cases which have occurred in the United States
have been fatal within six days from the onset of symptoms. Sudden death is
always tragic but in the case of PAM, it is compounded by the fact that 32 of
the 33 fatalities have involved young, healthy and active individuals between
the ages of 18 months and 25 years. One 40-year-old male accounts for the
thirty-third fatality.
In the majority of cases, epidemiological evidence indicated that expo-
sure to freshwater lakes or streams had occurred during the week prior to
death. Thus, it was assumed that pathogenic Naegleria had been present in
the waters to which these individuals were exposed. Irrefutable scientific
data to support this hypothesis, i.e., isolation of the organism from expo-
sure sites, had not been published. Therefore, this study was initiated
principally to establish a monitoring program to determine whether or not
pathogenic Naegleria are present in Florida's lakes. Secondary objectives
were to elucidate the source(s) of the amoebae, i.e., soil, avian, or
mammals and to determine the environmental or ecological factors related to
the presence of pathogenic Naegleria in lake waters.
BACKGROUND DATA
The existence of free living soil amoebae has been recognized for years
but their role as a human pathogen was not known until 1965. In 1958, Dr.
C. G. Culbertson identified Acanthamoeba in necrotic brain lesions in monkeys
and mice.(1) Animals were sacrificed four days after inoculation with
amoebae isolated from cell cultures which originally were thought to have
contained a virus. In a 1961 publication, Culbertson prophetically suggested
that these free living amoebae might cause similar lesions in man.(2)
The first clinical cases of PAM were reported from Australia in 1965.(3)
In 1966, Dr. C. G. Butt reported three fatal cases in Florida.(4) Since the
causative agent was not isolated from any of these cases, identification was
based on stained preparations of brain tissue. The amoebae were identified
as Hartmanella strains. In 1968, Butt reported the isolation of amoebae
from the brain and spinal fluid of a 16-year-old male who had died in
Orlando, Florida.(5) At autopsy, several small fragments of brain tissue and
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spinal fluid were inoculated onto agar plates covered with live bacteria.
The plates were incubated at room temperature and at 37°C. At the latter
incubation temperature, the amoebae grew continuously, whereas at room tem-
perature, only an initial growth phase occurred. Culture methods used for
isolating Hartmanella strains, i.e., addition of neomycin or other antibi-
otics, were unsuccessful, which ruled out the possibility that the amoebae
were Hartmanella strains. In addition, the trophozoites could be induced to
form flagellates which is a characteristic of the genus Naegleria but not
Hartmanella. Because of the foregoing and the mitotic characteristics, the
isolated amoebae were considered to be similar to, or possibly identical with
Naegleria gruberi, demonstrating for the first time that free living Nae-
gleria were involved in PAM. It has since been ascertained that the Nae-
gleria species was not gruberi but a previously unrecognized species which
has been referred to in the literature as Naegleria fowleri,(6) Naegleria
aerobia,(7) and Naegleria invadens.(8) Because of the lack of nomenclature
consistency, pathogenic Naegleria will be used in this report.
Since that time, many cases have been reported worldwide. In the United
States, 34 cases have been recognized. In 1977, three were reported, one
each from Georgia, Texas and South Carolina. The Texas and South Carolina
cases were the first reported from those states. In 1978, five cases were
reported, one in California, two in Florida, one in South Carolina, and one
in Pennsylvania. The Pennsylvania case involved a 40-year-old male who had
been swimming in Florida and South Carolina lakes before onset of his fatal.
illness. Only the case reported from California survived following early and
extensive treatment. The increased number of reported cases in 1977 and 1978
point to the possibility that because of the national interest in this dis-
ease and the improved technologies developed through Federal funding, the
disease is more readily recognized and diagnosed.
Based on autopsy and animal data, amoebae enter the brain following in-
tranasal infection. Studies by Chang indicate that pathogenic Naegleria
excrete a cytolytic substance which lyses cells, thus providing nutrients for
the amoebae.(9) Once infection, i.e., lysis of olfactory mucosal epithelial
cells, is initiated, the amoebae pass directly along the olfactory nerve
plexus to the brain.
Before this study began, no data were available on the distribution of
pathogenic Naegleria in the environment. In 1975, de Jonckheere, et al.,
reported that a single pathogenic Naegleria was isolated from a thermally
polluted canal in Belgium(lO) which was considered to have been the exposure
site ror a boy who died of PAM in 1973.(11) During their studies, another
Naegleria species was isolated which was shown by the indirect fluorescent
antibody (IFA) technique(12) to be antigenically closely related to the
pathogenic Naegleria. However, this new isolate did not produce fatal
infections in mice when instilled intranasally.
STUDY RATIONALE AND APPROACH
Florida with its semitropical climate and innumerable freshwater lakes,
both natural and man made, offered a year round study site for this project.
The State of Virginia had reported more cases of PAM(13) than had Florida.
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However, most of the cases in Virginia appeared in epidemic form as opposed
to the endemicity of Florida's cases. Thus it appeared that an ecological
study in Florida should offer a relatively good possibility of success.
Initially, techniques had to be modified or developed for concentrating
amoebae from large volumes (189 to 278 liters) of water and from lake bottom
sediment samples. Isolation media and incubation temperatures were tested
to determine optimal conditions and immune sera prepared for identification
procedures including IFA.
Once reliable techniques were developed for isolating amoebae from
throat and stool swabs, early studies were directed towards elucidating the
role of avians and mammals in the dissemination of the organism. Extensive
sampling was undertaken to determine the distribution of pathogenic Naegleria
in Florida's lakes. Various chemical and physical parameters were investi-
gated in an attempt to characterize those lakes yielding pathogenic Naegleria,
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SECTION 2
CONCLUSIONS
These studies have shown conclusively that pathogenic Naegleria amoebae
are widely distributed in freshwater lakes. Over 50% of the water tested
from lakes in which the temperature was 30°C or greater, yielded the amoeba
from one or more water or bottom sediment samples. This amoeba species has
the propensity to proliferate in warm waters. Therefore, thermal enrichment,
i.e., increasing natural water temperatures by the addition of cooling waters,
has been implicated in a fatal case of PAM in Belgium.(10) The role of ther-
mal enrichment in the proliferation and maintenance of pathogenic Naegleria
in Florida's lakes is unimportant because natural lake water temperatures have
at times surpassed those recorded in the thermally enriched lakes studied.
The number of isolates obtained from measured quantities of water from ther-
mally enriched and natural waters showed larger populations in the latter.
Laboratory studies demonstrated that variations in temperature were more
deleterious to the cysts than were either constant high or low temper-
atures. Under the latter condition, encystation occurred, thus, insuring
survival over time. Since the temperature of the thermally enriched waters
in this study tended to vacillate extensively, this may explain the lower
pathogenic Naegleria population levels demonstrated.
No evidence could be found for implicating avians or mammals in the dis-
semination of this organism in nature. No amoebae could be recovered from
stools of various avians and mammals inoculated with large numbers of patho-
genic Naegleria. This does not rule out the possibility that these and other
avian or mammalian species not tested may play a role in dissemination, per-
haps mechanical if not biological. However, the wide distribution of the
organism as seen in the number of lakes yielding the amoebae in a single
sampling would appear to indicate that these pathogenic amoebae are ubiqui-
tous in Florida. Isolates obtained from lake bottom samples during winter
months demonstrated the mechanism for over-wintering, i.e., encystment and
residence at the lake bottom/water interface or in the upper few centimeters
of the lake bottom sediments.
Two distinct antigenically related Naegleria species have been confirmed.
IFA testing showed extensive antigen sharing but specific antigens were demon-
strable using heterologous absorbed serum. Growth studies showed distinct
differences in motility, plaque size, and colony populations. Sonic disrup-
tion of cysts of the nonpathogens required approximately 50% greater shearing
forces than did cysts from the pathogenic Naegleria strains. Differentiation
of the seropositive nonpathogenic and pathogenic strains is readily accom-
plished. The latter produce luxuriant growth in Chang's calf serum-yeast-
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extract-casein medium (CSYECM)(8) in 24 to 48 hours (see Appendix B), where-
as minimal to no growth results with the former. In addition, intranasal
instillation of the seropositive nonpathogenic Naegleria does not produce a
lethal infection in mice.
Chemical analyses of various lake waters have failed to elicit signifi-
cant differences among lakes supporting as opposed to those not supporting
pathogenic Naegleria growth. There are preliminary indications that the con-
centration of gram negative organisms may be related to population levels of
pathogenic Naegleria.
The relatively high numbers of pathogens present in several of the lakes
tested, i.e., one pathogenic Naegleria per 25 ml of water tested and 6 per
25 ml in one of the lakes, would appear to pose a real threat to individuals
swimming in such lakes. However, no cases of PAM have been associated with
these lakes since this study was initiated in spite of the fact that many
individuals were observed swimming in these lakes at the time samples were
obtained.
Samples of water or bottom sediments from lakes associated with fatal
cases reported from several states since July, 1977, were processed for
amoeba isolation. Pathogenic Naegleria were recovered from all exposure
sites with the exception of those in Texas and California. In the latter
instances, samples were in transit for longer periods which may have
encouraged overgrowth of other amoebic and/or bacterial species.
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SECTION 3
RECOMMENDATIONS
There is now ample evidence that pathogenic Naegleria are widely dis-
tributed in lakes in Florida and Georgia and probably throughout the southern
United States. Increased ambient temperatures resulting in increased lake
water temperatures enhance the proliferation of pathogenic Naegleria. Why
this is true in some lakes and not in others should be ascertained. A
thorough evaluation should be made of lakes in which pathogenic Naegleria
proliferate and those in which few, if any, are found. This should include
limnological studies as well as enumerations of gram negative organisms on
which pathogenic Naegleria feed. A determination of all amoeba species and
ciliates present in the study lakes should be made in an attempt to identify
predators.
Lakes should also be carefully evaluated for pollution sources both
direct and indirect. Preliminary evidence that increased populations of gram
negative organisms enhance pathogenic Naegleria proliferation appears to in-
dicate that pollution plays a role in population densities. If this can be
shown conclusively, control measures could then be devised and instituted.
Relatively little data are available on the seropositive nonpathogenic
and pathogenic Naegleria amoebae per se. Nutritional and enzymatic
studies of both species should be undertaken to identify the biochemical
differences between these organisms. The possibility of an endosymbiont in
the pathogenic Naegleria which permits bypassing the growth factor(s) re-
quired by the seropositive nonpathogen should be investigated. More refined
studies of cyst survival under anaerobic conditions should be done to con-
clusively determine whether cysts are facultative or just tolerant under an-
aerobic conditions.
Modes of dissemination of pathogenic Naegleria in nature should be
studied further. Various species of mammals and avians not yet tested and
perhaps, reptiles may play a role in introducing either by mechanical or
biological means, pathogenic Naegleria into lake waters.
Differences in pathogenicity of isolates from surface waters versus
water nearer the bottom should be studied, particularly in lakes where in-
fections had occurred. Quantitation of organisms at the two levels should
also be determined. Epidemiological evidence continues to accrue indicating
that infection occurs in those people who swim near the bottom of lakes.
Whether this is related to the number of organisms to which the swimmer is
exposed or to increased pathogenicity of the organism is not known. Only
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when sufficient data are available to permit a thorough understanding of the
organism per se and the chain of events leading to infection in man can con-
trol measures be instituted.
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SECTION 4
MATERIALS AND METHODS
At the initiation of this study, limited data were available on field
techniques applicable to the concentration and isolation of pathogenic
Naegleria from field specimens, particularly from lake bottom sediments and
large volume water samples. Thus, much time and effort were devoted to de-
vising or adapting and testing techniques before the field work was started.
Data presented in this section include the various approaches used in
the development of techniques and the rationale for them. Studies were not
necessarily done in the order of presentation but rather are grouped on the
basis of relatedness.
SAMPLING SITES
Sampling sites were selected initially on two bases (1) thermal enrich-
ment, and (2) suspected exposure sites for fatal cases of PAM. One thermally
enriched site which received electric plant cooling waters and two natural
lakes which had been considered to be the exposure sites for three fatal
cases of PAM were sampled initially. As the study progressed a larger number
of lakes were tested. Samples of lake water and bottom sediments were ob-
tained from both shallow and deep water areas. Depending on the extent of
sampling at each lake and the geographical distances between sites, one or
several lakes were sampled on a single day. Quantitative data were based on
multiple, measured samples and on the most probable number technique which
was adapted to this study.
STOCK CULTURES
Stock cultures of McM, MO (Australian isolates),HB, RL and GJ (Florida
isolates) were received from S. L. Chang, M.D., Environmental Protection
Agency, Cincinnati, Ohio. All strains had originally been isolated from
fatal cases of PAM. Cultures were received and maintained on inorganic agar
seeded with Enterobacter aerogenes. Routine passages were made as necessary
for maintaining the strains. Strain 43-9 used in these studies is a sero-
positive nonpathogenic Naegleria strain isolated in February, 1976, from a
100 ml sediment sample obtained from a thermally enriched lake.
CULTIVATION TECHNIQUES
To determine optimal conditions for isolation of pathogenic Naegleria
from field specimens, various cultivation parameters were investigated as
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described below.
Incubation Temperature
Temperatures from 35°C to 46°C were chosen as possible incubation temper-
atures. Inoculation of four stock cultures containing both trophozoites and
cysts into CSYECM for axenic cultivation of pathogenic Naegleria resulted in
little or no propagation of the organism at 46°C. However, satisfactory
growth occurred at 35°C. Cysts of four stock cultures induced by holding
trophozoite cultures at 4°C for five days were inoculated into CSYECM and
incubated at 35°C, 42°C, and 44°C. Excystation and trophozoite replication
occurred at all temperatures, but the amount of growth was inversely propor-
tional to the temperature of incubation, few trophozoites being noted at 44°C
and most prolific growth occurring at 35°C.
Two final temperature-growth experiments utilizing five stock cultures
of trophozoites and cysts and four stock cultures of cysts only, showed
excellent growth at 35°C, good growth at 43 to 44°C, and poor growth at 46°C.
From these studies it was concluded that incubation of field samples should
be carried out at 43 to 44°C to favor propagation of Naegleria and to dis-
courage growth of undesirable saprophytes.
Antimycotic Agents
Large numbers of saprophytes and other undesirable microflora that
naturally contaminated field specimens overgrew pathogenic Naegleria at 37°C.
Various antimycotic agents were tested to determine their inhibitory effect
on these interfering species.
Various dilutions of Nystatin, Actidione and 5-Fluorocytosine were in-
corporated into CSYECM. Both Nystatin and Actidione inhibited pathogenic
Naegleria growth at extremely low concentrations and were therefore elimi-
nated. However, concentrations of 5-Fluorocytosine as high as 1,400 yg per
ml permitted good growth of Naegleria. When a saprophytic fungus isolated
from a throat swab taken from a bird was introduced into CSYECM containing
1,400 Hg of 5-Fluorocytosine and incubated at 37°C, saprophytic growth was
not inhibited. Because laboratory strains of Naegleria showed good growth at
43 to 44°C and the growth of temperature sensitive saprophytic microflora was
somewhat inhibited, antimycotic agents were not incorporated into the medium.
Antibiotic Concentration in CSYECM
The effect of various levels of antibiotics on amoebic growth was
determined in anticipation of the use of CSYECM for isolating amoebae from
nasal exudates of swimmers exposed to pathogenic Naegleria contaminated
water. Table 1 shows results of growth studies in which varying doses of
penicillin and streptomycin were added to CSYECM. Even with the highest
levels of antibiotics used, no toxic effects were noted. Therefore, with
exceedingly contaminated specimens, large doses of antibiotics were used
with impunity.
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TABLE 1. ANTIBIOTIC EFFECT ON GROWTH OF PATHOGENIC NAEGLERIA
Pathogenic Total
strain inoculum
Amoeba concentration at 48 hours in media containing
the following U/ml of penicillin and yg/ml of
streptomycin
100
300
400
500
GJ
83-16*
2.9 X 10
5.8 X 10
4.6 X 10"
1.7 X 10
5.1 X 10"
1.8 X 10
5.1 X 10"
1.6 X 10
3.7 X 10'
1.7 X 10
77N-475** 4.4 X 10
8.5 X 10-
4.9 X 10'
6.6 X 10'
6.2 X 10"
* Pathogenic Naegleria isolated from field specimen February 24, 1976.
** Pathogenic Naegleria isolated from field specimen June 28, 1977.
Growth Media
Sensitivities of CSYECM and inorganic agar plates spread with a lawn of
living E_. aerogenes were investigated. Varying two-fold dilutions of an
HB, trophozoite suspension were titrated on both media. Only one tube con-
taining CSYECM and one inorganic agar plate were used for dilutions 10"-'-
through 10~3.6 but four replicate tubes and plates were used for dilutions
3 ft £. £.
j.^ " through 10""'". The 50% end points for the two media were comparable,
10-5.5/0.2 ml for CSYECM and 10-5.4/Q.2 ml for the inorganic agar. Since
sensitivities were relatively equal, inorganic agar with I£. aerogenes was
selected for use with field specimens.
CONCENTRATION TECHNIQUES
Throat and Rectal Swabs or Feces
Before the role of avians or mammals in pathogenic Naegleria amplifica-
tion or dissemination in nature could be evaluated, techniques for concen-
trating the organism from throat and rectal swabs or feces had to be
developed.
Normal duck feces were inoculated with 10 Naegleria. Three simulated
rectal swabs were prepared from the inoculated feces and processed individu-
ally as follows. A swab was placed in 10 ml of sterile antibiotic water
(SAW) containing 200 U penicillin, 200 yg streptomycin, and 50 yg aureomycin
ml. After vigorous agitation the suspension was serially filtered through
45 ym, 10 ym, and 8 ym porosity membranes at a centrifugal force of 1,200 X g.
The latter two membranes were washed by passing 10 ml of SAW through the
membrane in situ using the above relative centrifugal force.
Subsequently, individual membranes were placed in 5 ml aliquots of SAW
and shaken to suspend any membrane-attached organisms. Both the suspending
SAW and membrane (8 ym and 10 ym) were inoculated separately into CSYECM.
Organisms were recovered from both membranes and the suspending medium,
10
-------
demonstrating incomplete elution from the membrane. An enterococci appeared
on day two post-inoculation and subsequently interfered with replication of
the recovered Naegleria.
Similar experiments using centrifugal force filters were conducted; in
these, the organisms were recovered from the suspending SAW in pure culture
but membranes yielded enterococcus contaminated Naegleria cultures. This
contamination was overcome by increasing ten-fold the concentration of peni-
cillin and streptomycin in the SAW, by increasing the time of membrane
agitation on a rotary shaker to one hour at room temperature, and by in-
cubating this suspension for an hour at 37°C prior to inoculation into CSYECM.
To determine the sensitivity of this isolation technique, two serial
ten-fold dilutions of trophozoites were made in distilled water and concen-
trated. Back titration of the original dilutions showed 1(P and 10^
organisms per ml. After centrifugal concentration on 8 ym porosity membranes,
the yield was 10^ and <1CH, respectively, from the two inocula. The question
of trophozoite loss into the filtrate due to G forces was addressed in subse-
quent experiments by examination of the filtrate. No organisms were detected,
and results of these latter experiments also showed a 10% recovery rate.
Although the reduced recovery yield has not been explained, it may be due to
several factors, i.e., trophozoite death due to manipulation, adherence to
glassware, etc.
Lake Waters^
Initially, 3.78 liters (one gallon) of lake water were passed through a
series of nylon mesh cloth screens having pore sizes of 45 ym and 10 ym.
After each filtration, the screens were flushed with sterile distilled water
and the filtrate homogenized in a Waring blender. Organisms present in the
final filtrate were concentrated by centrifugation. This technique (Dr. S. L.
Chang, personal communication) proved to be time consuming and severely
limited the sample size. To facilitate large volume sampling, a sand column
technique was developed.
A column 40 cm in length and 5 cm in diameter containing 600 ml of
Ottawa silica sand was prepared. Cysts and trophozoites of the McM strain of
pathogenic Naegleria were inoculated into a gallon of distilled water and
passed through the column at a pressure of 5 psi. Sand from the column was
removed and deposited in a beaker into which 700 ml of 1% beef extract was
added. After the mixture was stirred vigorously for ca 15 minutes, the sand
was permitted to settle, and the supernate was decanted into centrifuge
bottles. After a series of centrifugations of the supernate, the final
pellet was resuspended in 1 ml of distilled water. Back titration of the
inoculum in CSYECM indicated that 10^3 viable organisms had been present in
the original inoculum, but that only 10% of these (10^-3 amoebae) were re-
covered from the sand column.
To determine distribution within the column, a gallon of Lake Conway
water inoculated with 10?-3 trophozoites was forced through a sand column
which was encased in tygon tubing (15.9 mm internal diameter by 598 mm).
The tubing was sliced at one-inch intervals and the sand contained in each
11
-------
section assayed separately. Organisms were detected in all samples, but per-
cent of recovery was not established.
Because of the extensive distribution of organisms throughout the column,
amoeba losses would be expected during filtration. After it was passed
through the column, the first liter of water from a lake known to contain
pathogenic Naegleria was collected. A second liter was obtained after 378.5
liters had been filtered, and a third, after 1,135.6 liters. Only the last
liter collected yielded pathogenic Naegleria. Based on these data, field
samples were limited to a maximum of 189.2 liters to minimize loss of amoebae
from the column.
The refined technique used during the field studies has been described
in detail elsewhere.(14) A polyvinylchloride (PVC) pipe 38.1 mm in diameter
and 60 cm long was filled with approximately 600 ml of Ottawa silica sand to
within 10 cm of the top. With a small, high volume pump (Myers Mod. #M7
powered by a 3 H.P. gasoline engine, Briggs and Stratton Mod. #80200) on site,
189.2 liters of lake water were forced through the sand column at approxi-
mately 3.78 liters/min. A 45 pirn mesh cloth placed over the bottom of the
column and secured with a hose clamp prevented loss of sand through the
bottom.
Small Water Samples
One to two liter water samples were obtained in sterile glass jars and
returned to the laboratory for processing within four hours. These were
filtered through individual 5 ym porosity membrane filters (Millipore Corp.,
Bedford, Mass.). The filter was inverted onto an inorganic agar plate (IA)
seeded with J£. aerogenes.
Sediment Samples
Lake bottom sediments were obtained in one of three ways(14) depending
on the depth of the water. In shallow areas, a small can was used to scoop
up the desired sample. In depths from 1.2 to 2.4 m, cores were obtained with
a PVC pipe. In deep water, drag samples were obtained using a weighted can.
Sand from the columns and sediment samples were placed in individual
resealable plastic bags for transport to the laboratory. Initially, samples
were processed immediately. As the study progressed, samples were held at
43°C from 18 to 96 hours before processing.
ISOLATIONS
These techniques have been reported in detail elsewhere(14) but briefly,
each of the mud and sand column specimens were vigorously stirred in 1% beef
extract. Following settling of the sand and sediments, the supernates were
decanted into centrifuge tubes and centrifuged at 600 x g for 20 minutes.
The supernate was then discarded and the sediments inoculated onto sufficient
IA plates covered with live E^. aerogenes to permit inoculation of all sedi-
ments, approximately 20 plates per sample. These were incubated in closed
petri dish cans at 43 to 44°C.
12
-------
IDENTIFICATION
These procedures have been described in detail elsewhere.(14) Plaques
were examined for morphology, and those showing a sharp, even border were
tentatively considered to be pathogenic Naegleria if the trophozoites showed
typical limax form and eruptive-like pseudopods. Trophozoites were tested
for flagellate transformation. Cysts were carefully examined to determine
the presence of pores. Final identification was based on the trophozoite
growth pattern in CSYECM, on their antigens as shown in the indirect immuno-
fluorescent antibody (IFA) test, and on their pathogenicity for mice follow-
ing intranasal instillation.
MOUSE INOCULATION
All isolates identified as pathogenic Naegleria based on IFA and growth
in CSYECM were not tested for pathogenicity in mice because of the large
number of isolates obtained. Usually, only one isolate from each positive
field sample was confirmed by mouse inoculation. Details of the actual
procedure have been described elsewhere.(14) In all instances, when intra-
nasal instillation of trophozoites resulted in mouse fatality, brains were
harvested and the cause of death confirmed by microscopic visualization of
the amoebae or reisolation on IA.
INDIRECT FLUORESCENT ANTIBODY TEST
The technique used was a modification of that described by Van Dijck,
et al.(12) Serial dilutions of hyperimmune rabbit antiserum prepared
against the GJ strain were reacted with acetone fixed trophozoites on
a slide. Fluorescein-labeled anti-rabbit globulin was then added. Following
the prescribed reaction period, slides were washed, air-dried, and coverslips
were mounted. Reactions were read through a microscope equipped with an
HBO-200 light source, a dark field condenser and filters (BC-38, OG1 and a
Wrattin 2B eyepiece filter). Normal rabbit serum was used as a control.
The GJ antiserum absorbed with 43-9 trophozoites was included in certain of
the tests. (14)
13
-------
SECTION 5
RESULTS AND DISCUSSION
This study represented the first large scale field investigation ini-
tiated in the United States to determine the natural distribution of patho-
genic Naegleria. Because no background data were available, techniques were
developed or adapted and evaluated. Once techniques were established, in-
fection experiments were carried out and rectal and throat swabs obtained
from wild-caught mammals were examined. This was followed by a full-scale
study of thermally enriched lakes and four natural lakes which had been
considered as exposure sites for PAM cases. Such lakes are referred to as
suspect lakes in this report. When it was recognized that pathogenic
Naegleria might be more widespread than originally believed, a survey of
freshwater lakes in Florida was undertaken.
INFECTION EXPERIMENTS
An Indian Runner duck, Muscovy duck, a raccoon, and an opossum were in-
oculated with pathogenic Naegleria, HB^ strain. The inoculum was deposited
into the crops of the ducks, but was instilled intranasally in the mammals.
All daily rectal and nasopharyngeal swabs were negative for the agent. The
animals' drinking water was also tested and only one specimen, obtained from
the raccoon's cage two days after inoculation, was positive for pathogenic
Naegleria. No immune response was evidenced in the microscopic agglutination
test(l2) of sera from any of the inoculated subjects and no isolations of
pathogenic Naegleria were made from necropsy tissues tested.
ANIMAL ISOLATION ATTEMPTS
Rectal and throat swabs from wild-caught raccoons and opossums chosen
because of their propensity for visiting freshwater shores and shallows were
tested for pathogenic Naegleria. In all, 137 throat and rectal swabs were
collected and processed. Ten yielded amoebae when inoculated onto IA plates.
However, only one plaque obtained from the culture of a pharangeal swab taken
from a raccoon gave rise to amoebae which could be induced into flagellate
transformation. These amoebae were identified as Naegleria gruberi. None of
the isolates could be propagated in CSYECM and none resulted in fatality
following mouse intranasal instillation. A large amoeba (probably Acanth-
anoeba) from each of two raccoons (rectal swabs) was pathogenic for 3 to 4 week
old white Swiss mice by the intracerebral route. Although the concentration
technique was only 10% efficient, the total absence of pathogenic Naegleria
in the 137 specimens tested would appear to indicate that the species tested
do not play a major role in amplification of pathogenic Naegleria in nature.
14
-------
However, these species could possibly play a role in dissemination of the
amoebae by mechanical transfer from lake to lake.
ISOLATION FROM NATURAL AND THERMALLY ENRICHED LAKES
The results for all Florida lake samples processed are shown in Tables
A-l through A-8 (Appendix A). These data show conclusively that pathogenic
Naegleria are widespread throughout Florida. Each of the four lakes sus-
pected of having been the exposure site for PAM cases supported large popu-
lations of pathogenic Naegleria. One of the two thermally enriched lakes
which was sampled frequently yielded the agent but the other, which was
sampled only once, was negative. Of the ten lakes which were sampled in an
attempt to locate a negative control lake, only Lakes Corner, Jessup and
East Tohopekaliga did not yield pathogenic Naegleria. Of these three only
Lake Corner was sampled frequently and at depths which should have been
positive had the lake been heavily populated. Even so, no samples were
obtained during the summer months which would be necessary before the lake
could be considered as negative. Lake Beauclaire, one of the seven positive
lakes in this group, yielded a single isolate from a sediment sample taken
at a water depth of ca 1.2 m. Summer sampling of this lake would also be
required if this lake were to be considered as a control lake having a low
population of pathogenic Naegleria.
Lakes that were surveyed, i.e., sampled only once or twice to determine
the geographical distribution of the agent, were categorized based on their
location in or outside of Orange County. These lakes were all sampled at
least once between June and October. Lakes in northern Florida were sampled
in June and August because of the lower ambient temperatures that occur in
September and October in that section. Even under these conditions, only
40.4% (21/52) positivity was shown in lakes outside of Orange County, where-
as, 81.2% (13/16) of lakes in Orange County yielded pathogenic Naegleria
(see Table 2).
TABLE 2. NUMBER AND PERCENT OF LAKES YIELDING PATHOGENIC NAEGLERIA
IN THE FLORIDA SURVEY
Number of lakes positive
Number Water Sediment Water and
Surveyed lakes tested only only Sediment Total Percent
Outside Orange County 52 10 5 6 21 40.4
In Orange County 16 8 0 5 13 81.2
Sample size and water temperature may have influenced these results. Because
all sediment samples in this survey were from shallow water sites, the
variability due to depth was held constant.
15
-------
The relationship of water temperature and sample size to positivity in
surveyed lakes is shown in Tables 3 and 4. Four 0.25 ml water samples from
Orange County lakes and one 100 ml sediment sample from a lake outside of
Orange County all obtained in February were excluded from these tables.
Large water samples (189.2 L) from the lakes surveyed outside of Orange
County (see Table 3) yielded positive results in 46.1% (6/13) of the tests,
whereas one and two liter samples were positive in 12.5% (4/32) and 15.4%
(4/26) of the tests respectively. Average and median water temperatures were
lower in lakes from which large samples were obtained, yet a higher percent-
age of positivity was shown. Therefore, a larger number of lakes might have
been positive had large samples been obtained from lakes surveyed outside of
Orange County. The fact that data from Orange County lakes surveyed did not
show this difference would appear to indicate that populations of pathogenic
Naegleria were greater in Orange County than in other Florida lakes. In
addition, results obtained from two liter samples also appear to indicate the
presence of larger populations of this amoeba in Orange County lakes. In
spite of the fact that the water temperatures averaged over 30°C in lakes
outside of Orange County only 4 of 26 two liter samples were positive.
Whereas, in Orange County lakes where the water temperature averaged only
26.7°C, both two liter samples were positive. If these indications of
increased population levels were to be confirmed, it could help to explain
why five of the nine PAM cases occurred in Orange County.
TABLE 3. RELATIONSHIP OF WATER TEMPERATURE AND SAMPLE SIZE
TO POSITIVITY SHOWN IN THE LAKE SURVEY
Sample size
(liters)
Average
temperature
Median
temperature
# Positive/ Percent Positive Negative Positive Negative
// tested positive samples samples samples samples
Lakes outside
of Orlando
1
2
189.2
Orlando lakes
4/32
4/26
6/13
12.5
15.4
46.1
31.9
30.1
29.0
32.0
30.7
29.4
32.0
30.7
28.5
32.2
31.0
29.0
2.0
94.6
189.2
2/2
4/4
8/14
100
100
57.1
26.7
31.1
29.8
31.0
27.8 29.5
29.0
The major variables affecting the sediment sample results were size and
water temperature because, as previously stated, all samples were obtained
from comparable, shallow water sites. In contrast to the large water samples
which appeared to offer a better opportunity for isolating pathogenic
16
-------
Naegleria, large sediment samples appeared to offer the least possibility.
None of ten 600 ml samples obtained in June were positive, whereas 10% of
the 300 ml samples obtained during the same time period yielded pathogenic
Naegleria. In August, 33.3% of the 100 ml samples obtained from lakes out-
side and 75% of those from lakes in Orange County were positive, whereas
none of ten 400 ml samples tested during August yielded the agent. These
findings suggest that elution of the amoeba from large samples may be less
efficient than from the small ones or, amoebae which had been eluted may be
trapped as the larger quantities of sand settle out. The validity of this
observation requires more refined experimentation.
TABLE 4. RELATIONSHIP OF SAMPLE SIZE AND WATER TEMPERATURE TO POSITIVITY
OF SEDIMENT SAMPLES IN LAKES SURVEYED
Month
Average
water
temperature
(°C)
Sample size (ml)
100
Percent
positive
200
Percent
positive
300
Percent
positive
400
Percent
positive
600
Percent
positive
Lakes outside of Orlando
June
July
Aug.
Sept.
Oct.
31.4
N.R.t
30.9
29.6
26.8
A
33.3
75.0
0.0
10.0 (2/20)**
0.0 (1/1)tt
0.0 (0/10)
Lakes in Orlando
Aug.
Sept.
Oct.
31.1
29.9
26.6
(3/9) 7.1 (1/14)
4) 14.3 (1/7) --
(0/4) 25.0 (1/4)
75.0 (3/4)
27.3 (3/11)
0.0 (0/2)
0.0 (0/10)
* No samples.
** Number positive over number tested.
t Not recorded
tt Companion 3.2 liter water sample was positive.
In examining the percent of positive water and sediment samples as
related to temperature from all natural lakes (see Figure 1), there is ample
evidence of a direct correlation between positive water samples and elevated
water temperatures. Positive sediment samples showed less correlation with
temperature, having a slightly higher positivity rate in February and March
when the average water temperatures were 15.3°C and 21°C respectively, than
in September when the average water temperature was 30.5°C.
Positive sediment samples showed a relatively good correlation with
depth. Figure 2 shows this relationship for sediment samples from all lakes
tested excluding the two thermally enriched lakes. It should be noted that
in depths of 6.1 m to 9 m, over 50% of all samples taken yielded pathogenic
17
-------
70
60
20
10
0 x x x ~x?
Jan. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec.
Figure 1. Percent of positive water and sediment samples as related to the
average water temperature by month. () average water tempera-
ture; (xx) percent of water samples positive; (oo) percent of
sediment samples positive.
Naegleria. All positive samples from the 0 m to 1 m depth were obtained from
June through October (see Table A-9, Appendix A). Conversely, positive sam-
ples from all other depths were obtained from October through June except for
the 13 samples obtained from the 2.1 m to 3 m depths in July, all of which
were positive. This would appear to indicate that had more sediment samples
been taken during the warmer months, the overall percentage of positivity
would have increased, particularly for the shallow areas (0 m to 3m). These
findings accentuate the importance of testing sediment samples from deep
18
-------
60
50
40
3 30
en
o
a)
u
S 20
CM
10
0
0.1 1.1-2 2.1-3 3.1-4 4.1-5 5.1-6 6.1-7 7.1-8 8.1-9 9.1-10
Depth (meters)
Figure 2. Percent positive of sediment samples from all natural
lakes compared with water depths.
water sites during the cooler months.
In Lake Conway, over 40% of all sediment samples obtained from water
depths of 4 m to 10 m were positive (Figure 3). A comparison of the dotted
line representing the actual number of samples tested with the solid line
representing the percent of samples which were positive rules out bias in
the sampling procedures; that is, of 96 samples obtained from water depths
of 6.1 m to 8 m, 81.2% (78/96) were positive, whereas of 127 samples obtained
from water depths of 0 m to 4 m, only 6.3% (8/127) were positive. These data
from a single lake confirm the hypothesis that overwintering of the patho-
genic Naegleria does occur in deep water sediments or at the sediment/water
interface. The inability to demonstrate pathogenic Naegleria in the water
column or in sediments obtained from shallow water sites during cool weather
19
-------
100
co
QJ
90
80
70
60
« 50
M
O
01
> 30
4-1
rl
CO
,2 20
g 10
M
cu
cu
0-2
2.1-4 4.1-6
Depth in Meters
6.1-8
8.1-10
Figure 3. Number of sediment samples tested and percent positive from Lake
Conway - January, 1976 - March, 1978. ( ) number of samples
tested; ( ) percent positive.
is no indication that the amoebae have disappeared from the lake.
Some investigators have incriminated thermal enrichment in the mainten-
ance and proliferation of pathogenic Naegleria in nature. However, our data
do not support this hypothesis in Florida. Laboratory studies have shown
that fluctuating temperatures are detrimental to pathogenic Naegleria cysts.
Four comparably heavy plate cultures of the pathogenic GJ strain were pre-
pared and held at 35°C. This resulted in encystment of the trophozoites by
day four. Cysts in a circumscribed, marked area on each plate were counted
and then each plate was subsequently exposed to a different temperature, i.e.,
one plate was held at a constant 35°C, one at 22°C, and another at 4°C. The
fourth plate was subjected to alternating temperatures of 22°C for 8 hours,
then 4°C for 16 hours. Cysts within the marked areas were observed daily
and viable cysts counted. Retractile cysts were considered viable. This
20
-------
criterion was verified by random testing of refractile versus non-refractile
cysts through inoculation onto IA plates seeded with IS. aerogenes. As shown
in Table 5, only the 35°C temperature was more detrimental to the cysts than
the alternating temperatures, 7.7% and 16.7% survival respectively. At 22°'C,
51.2% of the cysts remained viable over the 35 day period while at 4°C, 81.3%
survived. These experiments should be repeated using other strains, partic-
ularly recently isolated ones. Strain differences may alter the data but
because of the gross differences noted, it is doubtful that the deleterious
effect of temperature fluctuation would be negated by strain differences.
These data as they stand, suggest that pathogenic Naegleria population levels
would be lower in thermally enriched lakes than in natural lakes because of
temperature fluctuations in the former. Previously published data showed
that the thermally enriched lake water temperatures fluctuated rapidly and
extensively, depending on the frequency of thermal water discharge. In
natural lakes, a more uniform water temperature curve rather closely re-
flected ambient temperatures.(14)
TABLE 5. TEMPERATURE EFFECT ON PATHOGENIC NAEGLERIA CYST SURVIVAL
No. of days
exposure
7
14
28
35
35°C
64.2
27.9
10.0
7.7
Percent of viable cysts after
22°C for 8 hrs.
4°C for 16 hrs.
54.9
49.2
20.1
16.7
daily exposure
22°C
86.4
83.7
53.8
51.2
at
4°C
90.5
89.0
84.0
81.3
Table 6 shows the number and percent of water samples which yielded
pathogenic Naegleria as a function of temperature in the thermally enriched
and three suspect lakes. The seven positive samples from thermally enriched
Lake Monroe were obtained in April, March, October, November and December,
whereas positive samples from the natural lakes were obtained from June
through mid-October with the majority in July and August, 24 and 33 positive
samples respectively. (Tables A-l through A-4, Appendix A). In May when the
water temperature in Lake Monroe was 30°C, none of ten <2 liter samples were
positive. Conversely, when a comparable water temperature was recorded in
Lake Hourglass, four of five 0.1 liter samples yielded pathogenic Naegleria
isolates. As shown on Table 6, when water temperatures in the natural lakes
were between 25°C and 34°C the number of positive specimens equaled or far
exceeded those of the thermally enriched lakes, thus indicating lower popu-
lation levels in the latter. Supportive evidence for this is discussed below.
Because the purpose of this study was to determine the distribution of patho-
genic Naegleria in Florida's lakes, extensive evaluation of the thermally en-
riched lake was not undertaken. However, it would be of interest to quanti-
tatively follow pathogenic Naegleria population fluctuations in thermally
enriched lakes to determine the real effect of temperature changes due to
21
-------
thermal cooling water discharges under natural conditions.
TABLE 6. PERCENT OF WATER SAMPLES YIELDING PATHOGENIC NAEGLERIA
ISOLATES FROM A THERMALLY ENRICHED AND THREE NATURAL
LAKES AS A FUNCTION OF TEMPERATURE
-__Lakes
Temperature°C
20-24
25-29
30-34
35-39
Percent of water samples positive
Monroe
(thermally enriched) Conway
33.3 (2/6)*
8.3 (2/24)
14.3 (2/14)
20.0 (1/5)
0.0 (0/0)
7.1 (1/14)
20.0 (6/32)
0.0 (0/0)
Hourglass
0.0
15.4
68.3
0.0
(0/0)
(2/13)
(28/41)
(0/0)
Baldwin
100.0
0.0
85.3
0.0
(1/1)
(0/18)
(29/34)
(0/0)
* Number of samples positive/number tested.
As previously discussed, Corner Lake, which is comparable in size to
Lake Spier, has yielded no Naegleria isolates. Although the lake has been
rather extensively studied between October and May (140 samples), findings
during hot summer months should be more conclusive. Lake Beauclaire has
yielded only a single isolate from a depth of 4 m. Lake Jessup and
Tohopekalig have been sampled less extensively but no pathogenic Naegleria
have been isolated. These findings may be misleading since even frequent
grab samples may result in negative findings if concentration of the agent is
at a very low level. As can be noted on Table A-5 (Appendix A), no pathogens
were isolated from Lake Spier during 1976, even though sampling took place
during the summer months. In May, 1977, an isolate was obtained from the water
column when the water temperature was 26°C. Only one plate of 20 inoculated
was positive indicating a very low population level. Severe draught condi-
tions in the spring of 1977, caused a drop of two to three feet in lake water
levels. This resulted in a natural concentration of lake waters and probably
facilitated the isolation of pathogenic Naegleria from the heretofore
"negative" Lake Spier. At no time have the numbers of isolates per sample
approached those found in samples from Lakes Conway, Hourglass, or Baldwin,
indicating the persistence of a relatively low pathogenic Naegleria popula-
tion level in Lake Spier.
CHARACTERIZATION OF SEROPOSITIVE NONPATHOGENIC AND PATHOGENIC NAEGLERIA
An important aspect of this present study has been the elucidation of
the physiologic and antigenic differences between the nonpathogenic and
pathogenic Naegleria trophozoites and structural differences between their
cyst forms. Two of the early, definitive characteristics of pathogenic
Naegleria outgrowth from a field specimen, were the plaque size and the
leading edge. Their plaques were smaller than those shown by seropositive
nonpathogenic Naegleria. The advancing plaque line was remarkably clean and
22
-------
appeared to the naked eye as a line drawn on glass by a diamond marking pen.
When observed under the microscope, all amoebae appeared to be advancing en
masse. Conversely, the seropositive nonpathogenic Naegleria plaque was
larger and the advancing plaque line fuzzy to the naked eye. When viewed
under the microscope the fuzzy line was seen to be due to individual amoebae
advancing before the main plaque line.
The difference in plaque size would lead one to think that the genera-
tion time for seropositive nonpathogenic Naegleria would be less than that
for pathogens. To determine the validity of this assumption a generation
time study was conducted. Eight plaque count plates for each of four strains
were seeded with not more than 20 amoebae. Microscopic examination of each
plate permitted the selection of an isolated amoeba which was designated as
the test organism. The amoeba site was marked on the underside of the plate
with a wax crayon. Four of each species of plaque count plates were incu-
bated at 37°C and the remainder at 43°C. Counts were made every two hours
through the tenth hour. Results are shown in Table 7.
The difference in generation time is more pronounced at the 43°C temper-
ature than at 37°C. This is in keeping with the higher incubation tempera-
ture favored by the pathogenic strain. However, there was an almost two-fold
difference in the population levels at 43°C and 37°C, even for the nonpatho-
gens. As had been noted with field isolates, plaques formed by the nonpatho-
gens were larger, i.e., trophozoites dispersed over a larger area by the
sixth hour, whereas the pathogenic Naegleria trophozites remained aggregated
through the tenth hour. Both reproduction and movement are energy demanding.
Why the seropositive nonpathogenic Naegleria utilizes much of its energy in
motility as opposed to reproduction and the pathogenic Naegleria does the
converse is unclear. However, these data do indicate a distinct physiological
difference between the two species.
TABLE 7. GENERATION TIME STUDY OF SEROPOSITIVE NONPATHOGENIC
AND PATHOGENIC NAEGLERIA AT TWO TEMPERATURES
Tempera- Average plaque counts at time (hours)
Strain ture (°C) 0 2 4 6 8 10 Plaque morphology
43-9*
83-16**
43-9
83-16
37
37
43
43
1
1
1
1
1.75
2.25
2.00
2.25
4
6
6
8
.0
.65
.50
.00
7.
13.
17.
24.
75
00
50
50
17.25
26,50
30.50
54.50
39
63
56
127
.50
.25
.50
.00
Widely dispersed
by t = 8 h
Aggregated
through t = 10 h
Widely dispersed
by t ~ 6 h
Aggregated
through t = 10 h
* Seropositive nonpathogenic Naegleria isolated from lake bottom sediments
on 2-3-76.
* Pathogenic Naegleria isolated from lake bottom sediments on 2-24-76.
23
-------
Antigenic differences between the seropositive nonpathogenic and patho-
genic Naegleria also have been documented, as well as their antigenic dissim-
ilarity with N_. gruberi. Rabbit antisera prepared against the GJ strain were
successfully absorbed with the 43-9 strain, the nonpathogenic seropositive
lake isolate. Table 8 shows the results of the initial test with this anti-
serum. With unabsorbed antiserum, _N. gruberi (381-2) is readily distin-
guished from both the pathogenic (GJ and 375-17) and nonpathogenic (43-9)
strains since it fluoresced poorly and reached a titer of only 1:40. However,
the pathogenic and nonpathogenic strains were positive to within one dilution
of each other, 1:1280 and 1:640, respectively. In the presence of rabbit
anti-GJ serum absorbed with 43-9 amoebae, the titer against GJ amoebae re-
mained unchanged and against 43-9 amoebae only dropped to 1:40, indicating
incomplete absorption. In later tests using completely absorbed 43-9 and GJ
rabbit antisera, no heterologous reactions occurred. The homologous titers
with these antisera before and after absorption dropped from 1:320 to 1:40
for 43-9 and from 1:640 to 1:160 for GJ.(14) Based on the cross absorption
studies, it appears that the specific antigens are minor in the 43-9 strain
and major in the GJ, possibly reflecting the complex enzymatic system
required for cytolysis which permits tissue destruction by the pathogen.
TABLE 8. INDIRECT FLUORESCENT ANTIBODY TEST RESULTS USING VARIOUS
ANTIGENS AND ANTISERA - SEPTEMBER 23, 1976
Titers*
Antigen Antiserum 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560
GJ**
375-17tt
381-2§
GJ
43-9 +
GJ
GJ
GJ
GJ
GJ
GJ
GJ
#3t
#3
#3
#1
#7
#7
&
&
&
8
8
8
t-
43-9
Controls
GJ
375-17
38L-2
(absorbed)TT
GJ #7 & 8
(absorbed
None
None
None
4
4+
2
4
3
4
4
4
1
3
4
3
3+
4
3
3
2+
3
3
2+
2
2
2
2
2
1
2
1
1+
1
1
1
1
1
1
1
* Titers scored on a scale of 1 to 4 based on intensity and number of
fluorescent positive sites per slide.
** Pathogenic Naegleria isolated from fatal human case in July, 1973.
t Rabbit anti-GJ serum ampouled on 12-12-75.
tt Pathogenic Naegleria isolated from lake water on 9-9-76.
§ K[. gruberi isolated from lake sediments on 9-13-76.
§§ Rabbit anti-GJ serum ampouled on 5-27-76.
t Seropositive nonpathogenic Naegleria isolated from lake sediments on
tl 2-24-76.
++ Absorbed with an equal volume of 43-9 amoeba.
24
-------
Early observations of cysts of the seropositive nonpathogenic and the
pathogenic Naegleria indicated that the latter had few if any pores and
appeared to have a thinner cyst wall. During sonication of a trophozoite
suspension, it was noted that the few cysts present in the 43-9 (seropositive
nonpathogen) preparation required a longer exposure time to the shearing
forces in order to rupture than did cysts in the GJ (pathogenic Naegleria)
preparation. Thus, an experiment was performed to determine the validity of
this observation and the magnitude of the difference.
Amoebae from each strain were grown in an inorganic solution containing
2.5% bacterial homogenate of E_. aerogenes. After seven days at 43°C, cysts
were harvested by centrifugation at 1,000 X g and washed twice with PBS.
Final cyst concentrations were adjusted to within 10% of each other (1.42 X
106/ml for strain 43-9 and 1.32 X 106/ml for strain GJ) as determined by
triplicate hemocytometer counts.
Aliquots (15 ml) of each suspension were subjected to sonication in a
Branson Sonifier Cell Disruptor, Mdl. No. W-350, at 100 watts using the
No. 419 microtip. After 45 seconds an aliquot was removed and the cysts
counted. Additional aliquots were removed and counted at 60, 80, 100, and
120 seconds. The following day duplicate aliquots (15 ml) were treated and
sampled at various times. Results are shown in Table 9. The sonication time
required to disrupt 50% (DT5Q) of the pathogenic GJ strain cysts was approxi-
mately 65 seconds, whereas the DT^Q for the 43-9 cysts was approximately 100
seconds. Therefore, our data appear to indicate that the seropositive non-
pathogenic Naegleria cysts are more resistant to sonication than the patho-
genic cysts. Because of the difficulty in distinguishing cyst stages from
late pre-cyst stages by low power microscopic examination, there may have
been a small percentage of the latter included in the cyst preparations
tested. However, this would have been true for both populations because
the same individual counted all the cysts. Therefore, it was assumed that
such a variable would be held constant. To determine the validity of that
assumption, cyst cultures will be subjected to sonic disruption on days 6, 9,
12 and 15. If the results are comparable to those of the initial study, the
assumption will be validated.
TABLE 9. COMPARISON OF SHEARING FORCES REQUIRED TO RUPTURE CYSTS OF THE
SEROPOSITIVE NONPATHOGENIC AND THE PATHOGENIC NAEGLERIA
Intact cysts remaining after sonication
(seconds)
Strain
43-9
Number
Percent:
0
142
100
45
102.5
72.2
55
.._
60 65
98.5
69.4
70
94.0
66.2
75
90.0
63.4
80
89.0
62.7
90
77.0
54.2
100
72.0
50.7
105
68.0
47.9
120
66.0
46.5
GJ
Number
Percent
132
100
78
59.1
72
54.5
69
52
. 5
.6
63.0
47.7 --
57
43
.0
.2
45
34
.0
.1
36.0
27.3
25
-------
The foregoing data which are summarized in Table A-12 (Appendix A),
appear to support the contention that the seropositive nonpathogenic Naegleria
is grossly different from the pathogenic strain. When these differences are
added to the inability of the nonpathogenic strain to produce fatalities in
mice following intranasal instillation of large numbers of trophozoites, it
would appear that the differences are sufficiently great to warrant separate
species classification for these two agents. As has been suggested previ-
ously, the seropositive nonpathogen might well be referred to as Naegleria
fowleri and the pathogen as Naegleria invadens.(14) Thus, the species desig-
nations would clearly differentiate the pathogen from the nonpathogen.
ANAEROBIC STUDIES
Sixteen IA plates were inoculated with an agar plug from either an
actively growing or encysted culture of pathogenic strains GJ and 437-2
(field isolate), IJ. gruberi 371-2 and the seropositive nonpathogenic 43-9.
A cyst and a trophozoite culture plate of each species were placed in an
anaerobic jar (Brewer) containing a gas pack (Gas Pak Disposable Hydrogen +
C02 Generator Envelope, BBL #70304) and methylene blue indicator strips (Gas
Pak Disposable Anaerobic Indicators, BBL, Cockeysville, Md.) and incubated
at 35°C. Companion plates of each species were incubated aerobically in the
same incubator. After 48 hours, the anaerobic jar and the plates were main-
tained at room temperature for three weeks. The anaerobic jar was then
opened and all 16 plates placed in the35°C incubator and held for three days,
at which time cultures were examined for evidence of growth.
The four trophozoite cultures held aerobically all supported trophozoite
growth throughout the experimental period. Three of the aerobic cyst cul-
tures showed excystation and heavy trophozoite growth. However, 437-2 cysts
failed to excyst and no trophozoite growth resulted.
Under anaerobic conditions, several of the 371-2 trophozoites encysted.
When they were removed from the jar and incubated at 35°C, excystation and
growth ensued. A few trophozoites of strain 437-2 also encysted but failed
to excyst when placed under aerobic conditions at 35°C. Neither 43-9 nor
GJ showed any encystment under anaerobic conditions.
Three companion cyst culture strains held anaerobically survived. They
excysted and reproduced under aerobic conditions at 35°C. The one exception
was strain 437-2. Actually no cysts were visible after the three week
anaerobic incubation period. The fact that even under aerobic conditions
cysts failed to excyst would appear to indicate an abnormality or nonviability
in the cysts of strain 437-2,
These data appear to indicate that seropositive nonpathogenic and patho-
genic Naegleria cysts can survive under anaerobic conditions. Therefore, if
cysts were to become embedded in anaerobic lake bottom sediments during
winter months, they should survive. However, this would preclude their being
demonstrated in the water column and perhaps in sediment samples as well,
unless sufficient sediment samples were tested. Such negative findings would
not indicate the disappearance of pathogenic Naegleria from the lake, but
26
-------
simply reflect inadequate sampling. As the temperature of the waters in-
creases and the activity in the lake results in agitation of the sediments,
the embedded cysts could be freed to initiate the cycle over another summer.
MOST PROBABLE NUMBER (MPN) TEST
Quantitation of pathogenic Naegleria in the water column was initially
based on small measured samples; 25, 50, 100, and 200 ml samples were
filtered through a cellulose nitrate membrane with an average pore size of
5 ym. Membranes were inverted onto IA plates seeded with E_. aerogenes.
Because plaques coalesced, it was impossible to obtain accurate counts.
Therefore, quantitative results were expressed as one or more pathogenic
Naegleria per unit volume.
In an attempt to refine the data, the MPN methodology routinely used in
bacteriology was applied to the enumeration of pathogenic Naegleria. A sus-
pension of trophozoites was prepared by washing a plate culture of the GJ
strain with PBS (0.4 percent NaCl). The trophozoite concentration was
determined by triplicate hemocytometer counts. Appropriate dilutions were
made to achieve suspensions of approximately 100 per ml and one per ml. The
former was titrated on IA with I£. aerogenes using the MPN methodology at 10^,
10"1, 10~2 and 10~3 dilutions with five plates per dilution. From the sus-
pension containing one trophozoite per ml, five 100 ml, five 10 ml, five 1 ml,
and five 0.1 ml aliquots were filtered through 5 ym porosity cellulose nitrate
membranes at approximately 5 psi. Each membrane was inverted onto IA plates
seeded with .E. aerogenes (Table 10). The results were sufficiently encour-
aging to warrant field trials.
TABLE 10. MOST PROBABLE NUMBER TEST RESULTS
Test
method
Titrated
Filtered
MPN
code
5,5,3,2
5,5,3,2
Index
per ml
140
1.40
Expected
per ml
100
1.0
Three of the suspect lakes were included in the field trial. Water
samples were collected in silicone coated, one liter screw cap bottles at
depths of 0.3 m, 0.6 m, and 2.4 m from each of the three lakes. Samples were
transported to the laboratory and processed within six hours. From each
liter sample, five each of 1 ml, 10 ml, and 100 ml subsamples were filtered
through 5 pm porosity membranes, the filter removed and inverted onto a
seeded IA plate and incubated at 43°C. Amoebae from plaques produced were
tested for flagellation and if positive, transferred to CSYECM. Luxuriant
growth in this medium was considered a confirmation of pathogenic Naegleria.
Values shown on Table 11 were derived from the standard MPN tables. The
relatively high number of positive specimens for Lake Baldwin shown by this
27
-------
method is comparable to that obtained in the summer of 1976 when at least one
pathogenic Naegleria was present in each 25 ml of lake water tested. The
findings at Lake Conway were much lower in 1977 using the MPN test than the
year before when the small measured samples were used. However, in 1976 the
water temperature was 33°C and in 1977, it was 29°C, which may help to account
for the lower population. These studies indicated that the MPN methodology
could be a useful tool for determining pathogenic Naegleria concentration in
lake waters.
TABLE 11. MOST PROBABLE NUMBER STUDIES
Lake
Conway
Baldwin
Spier
Water Sample
temperature depth
(°C) (meters)
30.0 <0.3
0.6
0.6
2.1
29.0 <0.3
<0.3
0.6
0.6
2.4
30.5 <0.3
0.6
2.4
30.0
31.0 <0.3
0.6
Date
07-22-77
07-22-77
07-22-77
07-22-77
08-29-77
08-29-77
08-29-77
08-29-77
08-29-77
08-01-77
08-01-77
08-01-77
08-01-77
08-26-77
08-26-77
08-26-77
08-26-77
08-26-77
08-26-77
08-01-77
08-01-77
Number
with
100
0
0
0
0
2
0
0
0
1
5
5
0
5
5
5
5
5
5
5
2
0
of positive plates
inoculum (ml)
10 1
0
0
0
0
2
0
0
0
0
3
2
0
5
4
3
5
4
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
MPN
index
liter
<2.0
<2.0
<2.0
<2.0
9.0
<2.0
<2.0
<2.0
2.0
79.0
49.0
<2.0
240.0
130.0
79.0
240.0
170.0
49.0
49.0
7.0
<2.0
In a controlled experiment designed to assess whether a direct plaque
count could be used instead of the MPN test to determine population sizes,
it was noted that an amoebic suspension filtered through a 5 vim porosity
cellulose nitrate membrane failed to yield plaques when the membrane was
inverted onto an IA plate seeded with E,. aerogenes. Thus, the following
experiment was carried out to recheck the MPN technique.
A 48-hour culture of a pathogenic field strain was washed from an IA
plate and the concentration established by duplicate hemocytometer counts.
The concentrate was diluted to approximately 1,000 cells per ml and two sets
2-8
-------
0 -1 -2 -3
of 10 , 10 ,10 , and 10 dilutions were made. From Set No. 1, five 1 ml
aliquots of each dilution were inoculated directly onto individual IA plates
seeded with I£. aerogenes. From Set No. 2, five 1 ml aliquots of each dilu-
tion were filtered through individual 5 ym porosity membranes which were
rinsed with distilled water and inverted onto seeded IA plates. From the
suspension containing 10 amoebae/ml, five 100 ml, five 10 ml, five 1 ml, and
five 0.1 ml aliquots were filtered and plated as described above
On day 14, only Set No. 1 showed any correlation with the initial inocu-
lum. Due to the low counts obtained on the filtered samples, it was hypothe-
sized that trophozoites had been trapped within the membranes and the IA
plates were too dry to permit capillary attraction to draw bacteria up into
these areas, therefore the trophozoites had encysted due to lack of nourish-
ment. To test this, 0.2 ml of the bacterial suspension used to seed the IA
plates was placed on each membrane and the plates reincubated at 43°C for an
additional week. Results shown in the last column on Table 12 confirmed the
above hypothesis.
Conditions of the initial test, in which good correlation was noted,
were revised and it was found that the plates had been freshly prepared and
thus contained sufficient moisture to have permitted growth within the inter-
stices. Thus, 0.2 ml of the bacterial suspension is now routinely placed on
each membrane when plated.
TABLE 12. MOST PROBABLE NUMBER TEST RESULTS BEFORE AND AFTER*
THE ADDITION OF A BACTERIAL SUSPENSION TO MEMBRANES
Set No
1
2
3
Direct
cell
count
. (ml)
1,000
1,000
10
Dilutions used
10°
10°
io2
io-1 io-2 io-3
io-1 io-2 io-3
io1 10° io-1
Day
Number of
positive
plates
5,5,5,4
5,2,1,0
5,4,1,0
14
MPN
ml
1,600
7.0
0.17
Day 21
Number of
positive MPN
plates ml
5,5,5,5 >2,400
5,5,5,4 16.0
^Bacterial suspension added at day 14 and read seven days later.
STANDARD METHOD DEVELOPMENT
The development of a standard method for isolating amoeba from specimens
mailed to a laboratory was deemed necessary so that results would be compar-
able. Such a method must designate the type(s) of specimen(s) to be obtained
and how they are to be handled before and during transport to a laboratory.
Because pathogenic Naegleria were isolated from sediment samples and not
water samples shipped to ERG, studies were conducted to determine what if any
procedures could enhance the isolation rate.
29
-------
A large sample of bottom sediment was obtained from Lake Conway on March
20, 1978. This was homogenized and eighteen 200 ml subsamples were taken
from the large sample and assigned to six groups of three samples. Each
group was incubated at room temperature (26°C) or at 43°C for 18 or 96 hours
and then either processed immediately or held at room temperature for varying
time periods before processing. Each sample was processed individually and
plated onto 10 IA plates seeded with E_. aerogenes. Thus, 30 plates were
examined for each treatment regime. Trophozoites from each plaque were tested
for flagellation. Those undergoing flagellate transformation were inoculated
into CSYECM. Non-flagellating amoebae were identified by examination of both
trophozoites and cysts.
Results are shown on Table 13. The most striking finding was the pure
culture of pathogenic Naegleria demonstrated in the samples which had been
incubated for 96 hours and then processed immediately. When comparable
samples incubated for 96 hours at 43°C were permitted to remain at room temp-
erature for 96 hours before processing, N_. gruberi and Hartmanella species
were demonstrated. Even so, only about 30 percent of the pathogenic
Naegleria were lost over the four-day period, which might be similar to the
time required for shipping samples from distant sites to the laboratory.
There was little difference between samples held for 18 hours either at room
temperature or at 43°C before processing. However, when samples were incu-
bated for 18 hours at 43°C and then held at room temperature for 72 or 168
hours, _N. gruberi became the predominant isolate, i.e., 83.3% and 78.6% at
each of the respective temperatures. These data demonstrate that prolifera-
tion of 1J. gruberi is favored when the sediments are held at room temperature.
Based on these data, all sediment samples are held at 43°C for 96 hours
before processing. No quantitative data can be derived from these enriched
specimens, but the probability of isolating pathogenic Naegleria is defi-
nitely enhanced. This is extremely important when specimens from suspect
lakes are to be examined in a laboratory located hundreds of miles away from
the collecting site. This procedure also extends the time required to con-
firm the presence of pathogenic Naegleria in a suspect exposure site. The
enhanced probability of making an isolate using this procedure far outweighs
the disadvantage of delay. However, if a rapid answer were desired, addi-
tional sediment samples could be examined without preincubation but negative
findings would need to be confirmed by processing the companion samples which
had been incubated.
Figure 4 depicts the laboratory tests employed and the time usually
required for processing specimens. Once specimens are processed and incu-
bated, a presumptive positive identification can be made in three to four
days based on the appearance of the plaque line and luxuriant growth in
CSYECM. Confirmation in mice requires at least four additional days.
Amoebae from spinal fluids, in case of human disease, could be identi-
fied by IFA studies within a few hours if sufficient trophozoites were
present. Cerebral spinal fluids obtained aseptically could be inoculated
directly into CSYECM for shipment to a laboratory. Incubation of the sus-
pension at 37°C for at least 18 hours would enhance the probability of
survival during shipment.
30
-------
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Inorganic Agar Plates
Seeded with E. aerogenes
2-3 days
at 43°C
Flagellation Test
4-
+
Non Naegleria
Chang's Calf Serum-Yeast Extract
Caseine Medium
24-48 hours
+ (luxuriant growth)
4-
Mice
3-10 days
Pathogenic
Naegleria
(limited growth)
4-
Cysts
Phase Contrast Microscopy
3 hours
F.A.
Single Walled
with Few or No Pores
Double Walled
Many Pores
Indirect Fluorescent
Antibody (trophozoites)
N. gruberi
Nonpathogenic
Seropositive
Naegleria
N. gruberi
Figure 4. Flow sheet and time required for isolation and identification of
pathogenic Naegleria from field specimens.
32
-------
MINIMAL LETHAL DOSE EXPERIMENTS
From an epidemiological standpoint, knowing the minimal mouse lethal
dose (MLD) would be most important. In a preliminary experiment, a single
pathogenic Naegleria trophozoite inoculated into four tubes of CSYECM pro-
duced an amoeba population in three of the cultures. Therefore, under ideal
circumstances a single trophozoite would be necessary to initiate an infec-
tion in vivo. In view of the numerous variables involved in intranasal (IN)
infections, one would not anticipate the same dose response. Therefore, an
attempt was made to determine the MLD in mice following IN instillation of
the organism.
A suspension containing 100,000 trophozoites per ml was prepared based
on triplicate hemocytometer counts using pathogenic strain 76N-437 which had
been isolated from Lake Bell on October 11, 1976. Its passage history was:
initial isolation on IA, one passage in mouse brain, 18 in CSYECM, two on IA,
one in mouse brain, and five on IA. Thus, in roughly 20 weeks this isolate
had been passaged 28 times before being used in the MLD experiment. Two-fold
dilutions of the suspension were made and carried well beyond the endpoint.
When the initial suspension was back titrated, the original count was found
to be valid. Table 14 (see p. 34) details the initial inoculation and sub-
sequent challenge of survivors. No deaths occurred with less than 78 tropho-
zoites, and only two of five mice succumbed as a result of this dose.
Deaths were erratic and even 2,500 trophozoites failed to produce death in
all mice inoculated. When survivors were challenged with twice the maximal
initial dose, the same irregular death pattern occurred. The challenge sus-
pension was prepared from the 76N-437 culture which had undergone two addi-
tional passages on IA. As shown in Table 15 and Figure 5, incubation periods
were extended beyond day 12 in 12.2% of the mice and flaccid paralysis of the
posterior limbs developed on day 26 in one animal. The latter was sacrificed
and the brain tissue inoculated into CSYECM and onto IA plates seeded with
_E. aerogenes.
TABLE 15. PERCENTAGE OF DEATHS ON INDIVIDUAL DAYS POST
INOCULATION: INITIALLY AND AFTER CHALLENGE
Inoculation
Initial
Challenge
Percentage of deaths on post inoculation days
5 6 7 8 9 10 12 14 16 19 26
35.7 14.3 21.4 7.2 21.4
2.0 38.8 18.4 8.2 8.2 12.2 4.1 2.0 4.1 2.0
Few amoebae were noted after two days in CSYECM. Usually, brain tissue
derived from mice that succumbed to pathogenic Naegleria infections showed
extensive amoebic growth within twenty-four hours after inoculation into
CSYECM. Only three plaques developed on the IA plates; one with a clear,
sharp plaque line typical of pathogenic Naegleria, one with a "fuzzy" plaque
33
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line, and one with a more diffuse line. After progeny from each plaque were
passed onto fresh IA plates, they were incubated at 37°C, and each retained
its characteristic plaque line.
Amoebae tha t formed sharp plaque lines were transferred in three days to
a fresh IA plate. After three days incubation at 37°C, a suspension contain-
ing 4.1 X 105 amoebae per ml was prepared in PBS (0.4% NaCl). Each of six,
three to four week old white Swiss weanling mice received 0.025 ml of the
suspension by IN instillation. Mice were observed over a 14 day period. One
mouse was missing from the cage on day six and may have been eaten by its
litter mates. On day seven, one developed flaccid paralysis of its hind
quarters. On day 10 two mice died, only one of which displayed a comparable
paralysis. On day 12 the fifth mouse died following paralysis of the hind
quarters, and day 14 the last mouse died without paralysis. In none of these
animals was the expected cerebral edema noted. All brains were harvested and
yielded amoebae when inoculated onto IA plates and in CSYECM. All plaques
produced on IA plates were characteristic of pathogenic Naegleria, i.e., had
sharp plaque lines. Progeny from the amoebae isolated on IA and in CSYECM
from the first brain harvested on day seven were instilled IN into weanling
mice. Four of four mice receiving IA grown amoebae died on day seven, and
amoebae were recovered from the brains. Five of five mice which received
progeny from the CSYECM cultures died but the incubation periods were ex-
tended. One died on day seven, three on day ten and one on day 20. Amoebae
were recovered from all brains in both CSYECM and on IA.
IA cultures having a "fuzzy" plaque line were passed on IA weekly for
eight weeks. From that passage an amoebic suspension of 2.7 X 10^ per ml was
prepared in PBS (0.4% NaCl). Each of five, white Swiss weanling mice re-
ceived 0.025 ml of the suspension instilled IN. Two mice died on day 15, one
on day 16, and one was sacrificed on day 21 when posterior limb paralysis was
noted. One mouse survived. Amoebae were isolated from each of the harvested
brains. Isolates grew well in CSYECM and showed a 1:640 IFA titer but plaque
lines were sharp as opposed to the original "fuzzy" plaque line.
After ten IA transfers, progeny from the amoebae showing a diffuse
plaque line on initial isolation were instilled in 0.025 ml amounts at a
concentration of 2.9 X 105 per ml IN into each of five white Swiss weanling
mice. No deaths occurred. These amoebae had failed to grow in CSYECM but had
shown the same 1:640 IFA titer achieved by the other two isolates which proved
to be pathogenic for mice and grew well in CSYECM.
In an effort to explain the isolation from a single infected mouse brain
of a nonpathogen and two other isolates differing in virulence expression,
the following hypotheses were considered:
1) a mixed population, i.e., seropositive nonpathogenic and pathogenic
Naegleria in the culture
2) transfer factor and/or a virus
3) strain selection due to culture techniques
36
-------
4) immunological status of mice
The following studies were conducted to elucidate these possibilities.
Although a mixed culture of seropositive nonpathogens and pathogenic
Naegleria was unlikely because of the differences in their plaque character-
istics, the possibility was investigated. A suspension containing both was
prepared and instilled IN into mice.
A pathogen isolated from lake water, 77N-795 and a nonpathogen, 43-9,
were washed with PBS (0.4 NaCl) and three suspensions prepared as follows.
The number of pathogens was held constant at 1C)6 per ml in all instances.
Nonpathogens were at levels of 2.5 X 105, 1.25 X 105 and 6.25 X 10^ per ml
in suspensions 1, 2, and 3, respectively (see Table 16). Groups of ten,
three-to four-week-old white Swiss weanling mice received 0.005 ml IN from
one of the three suspensions. Mice were observed daily for morbidity. All
suspensions produced death in 70% to 90% of the mice in five to six days,
which is typical of the pathogenic Naegleria. Ill mice were sacrificed and
harvested, brain tissues placed on IA cultures at both 37°C and 25°C and
plates observed daily for plaques. No isolates of 43-9 (seropositive non-
pathogen) were recovered.
Control plates inoculated with the three test suspensions were held at
37°C and room temperature. At both temperatures, the nonpathogens grew out
and masked the presence of the pathogens. When plaques were permitted to
grow out to the edge of the plate, washings contained only the 43-9 strain
based on their failure to multiply after being inoculated into CSYECM. If a
mixed culture had been present in the harvested brain tissues, the nonpatho-
gen, 43-9, should have been isolated under the test conditions, i.e., should
have overgrown the pathogens. These findings did not support the hypothesis
that seropositive, nonpathogenic amoebae isolated from the paralyzed mouse
were in the initial inoculum.
TABLE 16. RESULTS OF MOUSE INTRANASAL INOCULATION OF A MIXED CULTURE
OF PATHOGENIC AND SEROPOSITIVE NONPATHOGENIC NAEGLERIA
Number of
No. mice dead/ Percent trophozoites Percent Day of
Suspension inoculated of deaths inoculated nonpathogenic death
#1
#2
#3
7/10
9/10
8/10
70
90
80
6,250
5,625
5,313
25.0
12.5
6.25
5-6
5-6
5-6
To investigate the possibility of a transfer factor or a virus, a
culture medium exchange experiment was conducted using pathogenic strain
76N-437 and the seropositive nonpathogenic strain 43-9. Thirty ml of CSYECM
were placed in each of two 75 cnr cell culture flasks. Both were inoculated
37
-------
with strain 76N-437 and incubated at 37°C for three days at which time ap-
proximately 106 amoebae per ml were present in each flask based on duplicate
hemocytometer counts. The amoeba suspension was removed from one of the
flasks, sonicated at 100 watts X 15 minutes in a rosette cooling cell, and
filtered through a 0.45 jam porosity cellulose nitrate membrane. The sus-
pension from the second flask was filtered only. Into one of two 75 cm2
flasks was placed 12 ml of the sonicated filtrate, and into the second flask,
12 ml of the nonsonicated filtrate. Twelve ml of CSYECM and 0.5 ml of a
bacterial homogenate were added to each flask to facilitate axenic culture of
the nonpathogenic seropositive 43-9 strain which was inoculated into each
flask. After five days of incubation at 37°C, aliquots were removed, placed
on IA plates, and incubated for subsequent mouse inoculation and IFA study.
None of the progeny produced fatalities in mice following IN instilla-
tion nor did IFA titers differ from the control. These data failed to con-
firm the presence of a transfer factor under the conditions of this experi-
ment. Unfortunately, only a small portion of the amoebic population was
tested. Therefore, if only a small portion of the population were affected,
which would be expected if either a transfer factor or a virus were involved,
the probability of their being detected in the small volume tested is minis-
cule. The experiment will be repeated and the total population will be
inoculated into CSYECM so that even a low-level transfer-positive or virus
infected population could be demonstrated. Even so, the negative data
accrued does not support the initial hypothesis.
In considering the strain selection theory the MLD experiment was
repeated using a field isolate which had been passaged even more often than
had the strain used in the first MLD experiment. Strain 77N-475 had been
passed twice on IA, twice in CSYECM, once in mice and 13 times on IA. It
had been isolated from lake bottom sediments on June 28, 1977, and was used
in the MLD experiment on October 3, 1977. Based on 18 passages over a 14
week period, this strain had been passaged an average of every five days.
A suspension of trophozoites at 350,000 per ml was prepared from an IA
plate culture and dilutions made as shown on Table 17. Each dilution in
quantities of 0.025 ml was instilled IN into each of five, three- to four-
week-old white Swiss mice which were observed over a 21 day period. A direct
dose response relationship was shown. The MLD of 70 amoebae was comparable
to the 78 MLD shown on the first experiment. However, 33 of the 45 mice
survived the initial inoculation and were challenged by IN instillation of
10,000 trophozoites of the same strain (77N-475) which had had four additional
IA passages in the three week interval. Only 9.1% (4/33) of the mice suc-
cumbed to the infection. Because of the low fatality rate produced both
initially (26.7%) and following challenge (9.1%), plus the fact that the
tjrpically sharp plaque line of earlier passages on IA had been replaced by
a more diffuse plaque line, 25 mice were inoculated IN with 0.025 ml of a
suspension of trophozoites (strain 77N-475 which had undergone seven
additional passages on IA over a four-week period) at a concentration of
4.5 X 10^ per ml. Not one fatality occurred among the 25 mice inoculated.
38
-------
TABLE 17. RESULTS OF MINIMAL LETHAL DOSE AND CHALLENGE EXPERIMENT IN MICE
Initial inoculation
(10-3-77)
Challenge with 10,000 amoeba
(10-27-77)
No. of
amoebae No. deaths/ Percent No. deaths/
Dilution instilled No. inoculated survivors No. inoculated Survivors
10°
io~°'7
lo'1'4
lo-2'1
io~2'8
1Q-3.5
io~3'8
io~4-1
lo-4'4
8,750
1,750
350
70
14
2.8
1.4
0.7
0.35
5/5
4/5
2/5
1/5
0/5
0/5
0/5
0/5
0/5
0
20
40
80
100
100
100
100
100
0
0/1
0/3
1/4
1/5
1/5
1/5
0/5
0/5
0
100
100
75
80
80
80
100
100
When 77N-475 was isolated initially, it was a typical pathogenic
Naegleria which killed 80 to 90% of the mice in five to six days. Therefore,
an earlier passage from stock (passage 9) was inoculated IN into five mice
and the high passage 77N-475 which had undergone seven additional passages on
IA over a four-week period was inoculated into five additional mice. Each
was instilled IN with 0.025 ml of a suspension containing 6.5 X 1CP low
passaged trophozoites and 8.5 X 10-5 high passaged trophozoites. Four of five
mice receiving the former died within 11 days, none of five died after re-
ceiving the latter.
It was noted early in the project that repeated transfers of pathogenic
Naegleria in CSYECM resulted in an attenuated strain incapable of killing
mice following IN instillation. However, when these attenuated strains were
administered 1C, five of five mice died within six days and the recovered
brain passaged strains once again were capable of producing fatal infections
by IN instillation. It was also noted that pathogens maintained on IA for
long periods of time with passage every 3 to 5 months did not show any
decrease in pathogenicity. However, both strains used in the MLD experiments
had undergone numerous rapid passages on IA and did show a decrease in mouse
pathogenicity comparable to that seen following CSYECM long-term culture.
Both strains, 76N-437 and 77N-475, had been used frequently as positive
controls for IFA. When first isolated, they were typical pathogens in that
they underwent flagellate transformation, produced sharp plaque lines, grew
well in CSYECM, showed a 1:640 IFA titer, and produced death in weanling mice
within a five-to seven-day period following IN instillation. Because they
39
-------
were used as controls, they had been passaged at least weekly and sometimes
twice weekly. Since an earlier passage of strain 77N-475, when instilled
IN into mice, killed them all and the high passaged strain killed none, it
would appear that rapid passage on IA may select for the less virulent
strains.
To confirm this, strain 78N-470, a field isolate obtained on June 6,
1978, was passaged approximately every five days on IA, and every third
passage, an additional IA plate was inoculated. When grown out, trophozoites
were inoculated into mice IN. Table 18 shows the results.
The decrease in virulence as expressed by an increased incubation period
is not evidenced until passage 15, when four of five mice died on day seven
and one died on day 11. This is much more pronounced by passage 18, when
only two of five mice inoculated died, both on day 12. By passage 22, one
death occurred on day 18. At the same time, the plaque lines began to be
less sharp. Even so, the culture could be maintained in CSYECM, and IFA
titers remained constant. These data confirm the selection process resulting
from rapid passage on IA.
TABLE 18. DECREASE IN VIRULENCE FOLLOWING RAPID PASSAGE ON INORGANIC AGAR
Date
6-16-78
6-30-78
7-14-78
7-28-78
8-10-78
8-24-78
9-08-78
Passage
level
(IA)
3
6
9
12
15
18
22
Trophozoite/
inoculum
(0.025 ml)
14,500
13,750
14,250
14,750
10,750
11,000
13,750
No. deaths/
No. inoculated
5/5
5/5
5/5
5/5
5/5
2/5
1/5
Day of
4,4,4,
4,4,5,
5,5,5,
5,5,6,
7,7,7,
12,12
18
death
4,4
5,5
6,6
6,6
7,11
Trophozoites do not encyst in CSYECM nor did they encyst during rapid
passage on IA. In both cases pathogenicity for mice following Lntranasal
instillation was repressed. Pathogenicity is restored in CSYECM passaged
amoebae following either 1C inoculation in mice or slow passage on IA which
permits encystment. Therefore, it is suggested that pathogenicity for man
may reside only in those trophozoites which have undergone recent excystation,
which most likely would occur on or near the lake bottom. If this is true,
it would support epidemiological data which indicate that infections in man
occur most frequently in those individuals who swim underwater, near the
lake bottom. Perhaps the mechanism (enzyme?) required for excystation is the
same as that required for invasion, i.e., lysis of cell walls. During axenic
culture and rapid passage on IA, such an enzyme would not be required;
40
-------
therefore, it may be repressed. If encystation is permitted the need to ex-
cyst when ideal conditions are present may lead to derepression. Cultures
that have undergone rapid IA passage will be permitted to encyst in an effort
to elucidate this.
It must be emphasized that the seropositive nonpathogenic Naegleria
isolated in nature is different from nonpathogenic strains produced by the
pathogen. First, the IFA titers of the latter are always the same as the
pathogenic parent. The seropositive nonpathogens never achieve a comparable
titer. Secondly, and possibly more important, regardless of the inoculation
route, IN or 1C, the seropositive nonpathogen has never been capable of pro-
ducing fatalities in mice. Conversely, the avirulent amoebae produced by the
pathogenic Naegleria strain quickly revert to virulence following 1C inocu-
lation. Lastly, the seropositive nonpathogeus cannot: be maintained in CSYECM,
whereas the avirulent strains derived from axenically cultured or rapidly
passaged pathogenic Naegleria can be, although growth of the latter is light
compared to that of the parent strain. These data clearly indicate that the
two strains are grossly different (see Table A--12, Appendix A).
There is little doubt that variation in susceptibility among mice would
play a role in the outcome of an infection w.ith these amoebae. Age, sex, and
genetic strain are among the variables. Hag^etty, et a_l,,(15) have elucidated
the relative importance of the latter two variables. Mouse experiments con-
ducted during this study did not address these issues. However, since all
experiments were conducted in three- to four -week-old white Swiss weanling
mice, these two variables were constant. Tin.' oalv exception was the age of
mice at challenge. Test groups included bot'i ruaJes and females with the
overall majority being males. This too was thr same in all experiments so
that results obtained would appear to be related more to the amoeba per se_
than to the susceptibility of the mice.
SODIUM CHLORIDF TOLERANCE
Four seropositive nonpathogenic and Ion, pathogenic Nae_gl_eria were grown
in duplicate cultures on bacteria seeded TA plates containing increasing in-
crements of NaCl from 0.5 to 1%. Concentrations were standardized by counts
and 0.025 ml applied to che center of the pJare. These were incubated at
35°C for 44 hours when plaque sizes were measured on the duplicate plates and
averaged. Table 19 shows the results.
Two of the seropositive nonpathogens, one isolated in Florida and one in
Belgium, grew moderately well in the presence of saline at concentrations of
0,5 to 0.9% with their maximal growth at 0.6% saline. A second nonpathogenic
Belgian isolate showed little growth at or above 0,85% saline, whereas one
local isolate showed very limited growth in the presence of even 0.5% saline
and none at higher concentrations.
Three of the pathogenic Naegle_ria strains grew in the presence of physio-
logical (0.85%) saline but one did not. This was surprising, in that the
latter strain, GJ, had been isolated from a fatal case of PAM. Either this
strain had been altered during passage or higher saline concentrations can be
tolerated in vivo than in vitjrq. Only one of the pathogens, that isolated
41
-------
from Lake Okeechobee, produced growth in the presence of 0,9 to 1.0% saline.
Unfortunately, the saline content of the lake waters was not ascertained when
specimens were obtained. These data would appear to indicate that pathogenic
Naegleria would not be found in a salt water milieu. This is in keeping with
the epidemiological data indicating exposure to freshwater lakes as a link in
the infection chain for PAM.
TABLE 19. PLAQUE SIZE OF SEROPOSITIVE NONPATHOGENIC AND PATHOGENIC
NAEGLERIA AS A FUNCTION OF SODIUM CHLORIDE CONCENTRATION
Average plaque
radius
Nonpathogenic strains
%NaCl 43-9*
0.50 17
0.55 25
0.60 25
0.65 21
0.70 16
0.75 16
0.80 12
0.85 12
0.90 11
0.95 3
1.000 4
76-36-
250**
12
12
15
14
13
12
11
8
10
6
4
LVH**
7
6
6
7
5
4
4
2
1
NG
NG
356*
<3_
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
at 44 hrs. (mm)
Pathogenic
375*
14
14
12
12
8
7
6
5
4
3
-------
hours prior to being separated into four groups of four animals each. Group
1 was inoculated intracardially with 0.025 ml of a suspension containing 1.0
X 10" pathogenic Naegleria per ml (field isolate 78N-198, passage 7). In
Group 2, a 20 guage needle attached to the inoculating syringe was pierced
through the tympanic membrane and the inoculum deposited in the middle ear.
In Group 3, the inoculum was placed directly into the oral cavity and readily
swallowed. The inoculum was deposited on the eyes in Group 4. Table 20
shows the results. No infections followed oral or optic inoculation. Deaths
following intracardial inoculation were not surprising, since the blood
stream permitted widespread dissemination of the organism. Because the
deaths due to middle ear inoculation were unexpected, amoebae dissemination
via the blood stream due to rupture of the tympanic membrane at the time of
inoculation was considered. Therefore, the tympanic membrane was removed
from a guinea pig and allowed to heal. A week later, an inoculum containing
ca 27,000 trophozoites was deposited in the middle ear. The animal was held
on its side to preclude loss of the inoculum. After ca five minutes soft wax
was placed in the ear canal and a bandage applied to hold the inoculum in
place. No temperature elevation or illness was noted over a 27 day period,
therefore it was concluded that deaths in guinea pigs following inoculation
through the intact tympanic membrane resulted from hematological dissemina-
tion of the pathogenic Naegleria. This rules out the ear as the portal of
entry in the Georgia case in which there was evidence of an old tympanic
membrane rupture. However, if the tympanic membrane were ruptured while an
individual were swimming in pathogenic Naegleria contaminated waters, infec-
tion via the circulatory system could conceivably occur.
AXENIC CULTURE
Axenic cultures of both the seropositive nonpathogenic and pathogenic
Naegleria were desireable for absorption of antisera and electronmicroscopic
comparison studies of the two strains. CSYECM provided prime cultures of the
pathogenic strain but an axenic culture medium had to be developed for the
seropositive nonpathogens. These data have been published in detail else-
where (14) but briefly, one liter cultures of E. aerogenes were prepared and
incubated at 37°C for four days, after which the bacteria were removed by
centrifugation. Cells were resuspended, washed three times and disrupted by
sonication in distilled water. One half of the suspension was heat treated
to kill any surviving bacteria and the remainder was recentrifuged and fil-
tered through a 0.45 pm porosity cellulose nitrate membrane and designated as
cell free lysate (CFL). The heat-treated unfiltered portion was designated
bacterial homogenate (BH). These were added in 0.0125 ml increments, 0 to
0.05 ml/ml of CSYECM, Page's malt-yeast extract-amoeba saline medium(16) or
inorganic solution. Regardless of the medium used or the quantity of BH or
CFL, the seropositive nonpathogenic Naegleria grew fairly well. Maximal
growth occurred in CSYECM with 0.05 ml of either BH or CFL per ml of medium.
Good growth was noted in the other media at the same supplement concentration.
The axenic culture requirements of the two strains accentuates the difference
between the nutritional needs of the pathogen and those of the seropositive
nonpathogenic Naegleria.
43
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ELECTRON MICROSCOPY
Cysts of the pathogenic Naegleria were disrupted by sonic forces much
more rapidly than were cysts of the seropositive nonpathogen. This finding
suggested that structural differences in the cyst walls may account for the
difference. Therefore, electronmicroscopic (EM) studies were initiated.
Both the seropositive nonpathogenic and the pathogenic Naegleria were
grown in CSYECM. Bacterial homogenate was added as a nutritional supplement
for the nonpathogens. After two weeks, cysts were harvested and fixed for
one hour at 25°C in a mixture of equal volumes of six percent glutaraldehyde
and 0.12M sodium cacodylate (NaCac) buffer (pH 6.8). They were rinsed with
0.06M NaCac at 4°C, then centrifuged. This was repeated four times. Cysts
were then postfixed for one hour at 4°C in a mixture of osmium tetroxide
and NaCac at final concentration of 2% and 0.06M respectively. The rinsing
procedure was repeated as before. Following the last rinse, the pellet was
embedded in 2% Dif co Noble agar.(17) The tissue was minced into small pieces
and stained at 4°C with 0.25% uranyl acetate solution (aqueous). A slow-
schedule dehydration of the stained pieces was performed using ethyl alcohol
followed by several changes of anhydrous acetone before the pieces were
embedded in Spurr's(lS) low viscosity embedding medium. Thin sections were
cut with glass knives on a Sorval Porter-Blum ultramicrotome. The sections
were mounted on naked 300 mesh copper grids and stained with uranyl acetate
and lead citrate. Electron micrographs were obtained on a Phillips EM200
using Kodak Electron Microscope film.
Unfortunately, none of the prints made of the pathogenic Naegleria cysts
showed a complete cross section of a cyst like that obtained for the sero-
positive nonpathogenic Naegleria (see Figure 6). Partial sections of the
pathogenic Naegleria cyst walls did appear to be much less dense than those
of the nonpathogens but due to the lack of a good print, such a difference
may be an artifact.
Additional studies which actually compare cyst wall thickness should be
done. Verification of gross structural differences would increase the list
of differences between these two amoeba species.
SUPPORTIVE ACTIVITIES
Suspect Site Investigations
As an adjunct to the surveillance studies conducted the Tampa Epidem-
iology Research Center (ERG) has served in a supportive role when cases of
PAM were diagnosed in humans. Studies done and data accrued are shown below
according to the state in which the case was diagnosed.
Georgia
In August, 1977, ERC staff members went to Hephzibah, Georgia, to par-
ticipate in the investigation of a PAM case involving a 14-year-old black
female. She had been swimming at a privately-owned recreational park before
the onset of illness. Since the epidemiological evidence pointed to the
45
-------
Figure 6. Seropositive nonpathogenic i?aegleria cysts cross section. Print
magnification 10,770.
46
-------
probability that this pool was the site of exposure, it was the focal point
of the investigation.
The swimming pool was constructed with concrete sides and a sandy bottom
underlined with clay. Next to the pool was an eight acre lake elevated ca 15
feet above the pool and separated from it by a cement walkway, a terraced
area, and a narrow dirt road. A pipe connected the lake and pool which per-
mitted gravity feed from the lake into the pool during high water. A second
two acre lake was connected to the eight acre lake in much the same fashion
as described above. This produced a "lock" effect with the two acre lake at
the highest elevation and the pool at the lowest. Both lakes had natural
sand-clay bottoms and sides and were surrounded by sparsely populated,
heavily wooded areas. According to local reports, very little swimming was
ever done in either lake.
In all, 62 samples were collected. The samples included small and large
volumes of water, 100 ml or 200 ml of sediments, and approximately 500 ml of
water for the MPN test. The MPN sample in 1 ml to 100 ml aliquots, was
filtered on-site using Swinney filter units equipped with 5.0 ym porosity
cellulose nitrate membranes (Millipore Corp., Bedford, Mass..) . After filtra-
tion, membranes were inverted onto IA plates covered with IJ. aerogenes and
allowed to incubate at 43°C for 14 days. Identification of the isolates was
based on plaque morphology, flagellate transformation, IFA tests, and growth
in CSYECM and was confirmed by mouse intranasal instillation.
Additionally, chemical analysis of the pool water was performed using
an engineer's field kit (Mod. DR-EL/2, Hach Chemical Co., Loveland, Co.).
Bacteriological analysis (total and fecal coliforms) was performed on the
pool waters by the U.S. Army Health and Environmental Activity, Dwight D.
Eisenhower Army Medical Center, Ft. Gordon, Ga., using the membrane filter
technique (Standard Methods, 14th Ed., 1975).
Pathogenic Naegleria amoebae were isolated only from samples obtained
from the swimming pool. Both 50 gallon water samples were positive, as were
three of the four sediment samples and one of the five 20 ml water samples.
The chemical and bacteriological findings were within acceptable ranges.
The apparent absence of pathogenic Naegleria in the two feeder ponds may
simply reflect the limited number of samples obtained,
Texas
In August, 1977, about two weeks after the Georgia case, PAM was reported
in a teenage female who had been swimming in a polluted Texas lake immediately
prior to onset of illness. The Texas State Department of Health submitted
samples from the suspected exposure site to ERC for isolation attempts. The
specimens included five 200 ml water samples and 1 liter of sediment. Two
plate cultures of amoebae isolated by the Texas State Department of Health
were submitted at the same time.
The water specimens were processed by the membrane filter method and the
sediment processed as described earlier. All were negative for pathogenic
Naegleria. The two plate cultures submitted proved to be Naegleria gruberi,
47
-------
a nonpathogen. Specimens were in transit at ambient temperatures for 72
hours, which may account for the negative findings.
South Carolina
In September, 1977, a case of PAM was reported from Charleston, S.C.
The patient was a young boy who had a recent history of swimming in a large,
man-made lake in the Charleston area. The South Carolina Department of
Health submitted two gallons of water and five 200 ml sediment samples that
were collected from the suspect lake.
Only one of the sediment samples yielded pathogenic Naegleria. No
isolates were obtained from the water. This reinforces the axiom that
negative results have little validity. If the sediment samples had not been
submitted the only evidence incriminating the suspect exposure site would
have been circumstantial.
Florida
On July 5, 1978, a 14-year-old boy with a history of swimming was tenta-
tively diagnosed as having PAM based on the observation of amoebae in the
cerebral spinal fluid (CSF). A portion of this CSF was delivered to ERG at
10:00 A.M. Aliquots were inoculated onto an IA plate with E^. aerogenes and
into CSYECM and incubated at 37°C. At 2:00 P.M. amoebae from the CSYECM
culture were examined for flagellate transformation and by the IFA test.
Both were positive for pathogenic Naegleria. Thus, the diagnosis was con-
firmed within four hours of receipt of the specimen. A field team went to
the lake suspected of being the exposure site. Ten 200 ml sediment samples
were collected from various sites on the lake. Two liters of water were ob-
tained for MPN determinations of Naegleria. One liter was taken where the
boy had been seen swimming and the other across the lake at a beach where
most of the swimming activity usually occurs.
Even though the MPN index was <2 pathogenic Naegleria per liter at the
site where the boy had been swimming, three of the four sediment samples from
the same site were positive. Conversely, the MPN index for the beach across
the lake was two pathogenic Naegleria per liter but both sediment samples
were negative. Representative organisms from each positive sample and from
the CSF were confirmed by mouse intranasal instillation.
California
In May, 1978, a case of PAM was diagnosed in a nine-year-old California
girl. One week before onset she had bathed in a hot springs near San
Bernandino. This same hot springs had been implicated in a 1971 case of PAM.
On July 12, 1978, and August 22, 1979, specimens were received from the
California Department of Health for isolation of pathogenic Naegleria. A
subculture of the amoebae reportedly isolated from her initial CSF specimen
was requested but has not been received.
From the first set of specimens obtained from the hot springs, all four
48
-------
sediment samples were negative for pathogenic Naegleria. From the second
set obtained from a stream near the springs, no pathogens were isolated but
seropositive nonpathogenic Naegleria were isolated from two of the six sedi-
ment samples. Once again, these negative samples do not preclude the possi-
bility that pathogens are present in the springs or adjoining streams.
Additional testing should be done.
South Carolina
In August, 1978, a case of PAM was reported from Charleston, S.C., for
the second straight year. The possible exposure sites consisted of a drain-
age ditch and the same man-made lake that had been implicated the previous
year.
Both water and sediment samples submitted from the ditch failed to yield
pathogenic Naegleria. However, seven of the 12 sediment samples representing
all sites sampled from the man-made lake were positive for pathogenic
Naegleria. These data reinforced the validity of the isolate obtained from
this same lake the year before.
Florida
In late August, 1978, a nine-year-old white male from Bakers County,
Florida, succumbed to a fatal PAM infection. A week after his death, ERG
staff members went to Baker County and obtained water and sediment samples
from the four suspect swimming sites. Samples obtained from each site in-
cluded a 50 gallon water sample and four 100 ml sediment samples obtained
from different areas. A MPN test was done on one site at each swimming area.
None of the 50 gallon water samples yielded pathogenic Naegleria, which was
surprising in view of the fact that two of the four sites showed two amoebae
per liter. Only three of the 16 sediment samples yielded pathogenic Naegleria
These were all from Clay Pond which had shown two pathogens per liter in the
MPN test and which was the swimming site nearest to the infected boy's home.
Water temperature in both of the negative sites was 26°C, whereas water temp-
eratures in the two positive sites were 31°C and 33°C.
Additional Supportive Activities
Comparable surveillance studies for pathogenic Naegleria had been ini-
tiated in Virginia under the direction of Richard Duma, M.D. He and members
of his staff therefore spent several days at ERC observing techniques. As a
control for both laboratories specimens were shared.
In May, 1978, environmental specimens were received from Dr. Duma. At
that time, pathogenic Naegleria had not been isolated from any of their
environmental samples. Samples processed at ERC yielded one strain of sero-
positive nonpathogenic Naegleria but no pathogenic Naegleria were obtained.
These data would appear to support their negative findings.
On July 8, four 200 ml sediment samples were collected from different
sites at a known positive lake in Florida. Each sample was well mixed,
divided into two equal samples and placed in resealable plastic bags. These
49
-------
were incubated at 43°C. On the afternoon of July 10, 1978, one of each set
was packed and shipped via United Parcel Service to Dr. Duma's laboratory and
the companion set placed at room temperature. Upon notification that the
samples had arrived and processing was about to be initiated, the four com-
panion samples held at room temperature were processed. Results were identi-
cal in both laboratories; one sample was negative and three were positive.
These data confirm the reliability of techniques employed.
When ERG personnel assisted in the epidemiological investigation of the
fatal PAM case which occurred in Hephzibah, Georgia in July, 1977, Georgia
State officials requested that a survey be taken for pathogenic. Naegleria in
Georgia lakes. With EPA approval and additional funding, two ERG staff
members, utilizing ERC's Mobile Laboratory, surveyed lakes in Georgia from 6
to 18 August, 1978.
Specimens were collected from 20 lakes representing 16 counties. Two
sites were sampled on all but one lake. Specimens obtained from each lake
included two 50 gallon water samples, six or eight 100 ml sediment samples,
and two water samples for the on-site MPN pathogenic Naegleria determination.
Comparable water samples were collected for chemical and bacteriological
studies. The former were done using a Hach DR-EL/2 kit (Hach Chemical Co.,
Loveland, Colorado). The latter analyses were performed by the Chatham
County Department of Health, Chatham County, Georgia, using the multiple tube
method to enumerate total and fecal coliforms and fecal streptococci. Water
samples were delivered to the laboratory within six hours. Results are in-
cluded in this report.
Samples for pathogenic Naegleria isolations were incubated at 43°C until
shipped to the base laboratory, where they were again incubated at 43°C until
processed. These incubation histories are shown on Table 21. Samples were
processed as previously described and amoebae from at least one plaque from
each positive sample were tested for mouse pathogenicity by intranasal instil-
lation. These were all confirmed.
Seven of the 20 lakes (35%) tested yielded pathogenic Naegleria from at
least one of the samples tested. All seven positive lakes were located south
of Atlanta, or below the so-called Fall Line. Thirteen of the 20 lakes
tested were south of Atlanta giving a 53.8% (7/13) positivity for lakes in
that geographic area (see Table 22).
Chemistry data varied little from lake to lake and were within the range
seen in Florida's freshwater lakes. No real differences could be seen be-
tween negative and positive lakes (see Tables A-10 and A-ll, Appendix A).
Bacteriological findings are shown in Table 23 according to lake and
site sampled. Bacterial densities varied extremely aiaong the lakes and be-
tween sites on the same lakes. These data are summarized on Table 24
according to the geographic location of the lake in relation to Atlanta and
the presence or absence of pathogenic Naegleria. In lakes located south of
Atlanta, there does appear to be a trend toward higher bacterial counts in
the positive lakes based on the median. Using the F and t tests designed for
data showing extensive variations, the differences were not significant at
50
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TABLE 21. INCUBATION HISTORIES OF SAMPLES FROM SELECTED GEORGIA LAKES FOR
PATHOGENIC NAEGLERIA ISOLATION
Lake
Griffin
Cypress
Carrol
Olms te ad
Lester
Deltina
Fort Yargo
Yonah
Sky
Dockery
Blackburn
Indian
Spivey
Ransby
Robin
Concharty
Tobesofkee
Houston
Blackshear
Long
Number
43°C in
mobile laboratory
72
72
48
48
72
72
48
48
24
24
120
120
96
96
72
72
48
48
24
24
of hours held at
Various
temperatures
in transit*
30
30
30
30
30
30
30
30
30
30
o**
0
0
0
0
0
0
0
0
0
43°C in
base laboratory
48
48
48
48
12
12
12
12
12
12
96
96
96
96
96
96
120
120
120
120
* Temperature not known but undoubtedly wide variations.
** Returned to base laboratory in mobile laboratory.
51
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TABLE 22. LOCATION OF LAKES, WATER TEMPERATURE, AND RESULTS OF PATHOGENIC
NAEGLERIA ISOLATION ATTEMPTS
Lake
Griffin
Cypress
Carrol
Olms tead
Lester
Deltina
Fort Yargo
Yonah
Sky
Dockery
Blackburn
Indian
Spivey
Ransby
Robin
Concharty
Tobesofkee
Houston
Blackshear
Long
Geographic
location with
respect to Lake water
Atlanta temperature
North South °C
X 29.0
X 29.5
X 30.0
X 32.0
X 28.5
X 31.0
X 27.0
X 25.0
X 24.0
X 21.0
X 24.5
X 30.5
X 27.5
X 33.0
X 28.5
X 29.0
X 29.0
X 33.5
X 31.0
X 34.0
Naegleria Number positive/
MPN* index number tested
per liter Water
of water samples
5, 5
5,<2
5,<2
<2,<2
<2,<2
<2,<2
<2,<2
<2,<2
<2,<2
<2, ND
<2,<2
<2,<2
<2, 2
<2,<2
<2,<2
<2,<2
<2,<2
<2,<2
<2,<2
5,<2
* Two sites tested except when indicated ND (not
** Seropositive nonpathogenic Naegleria isolated.
1/4
0/4
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/1
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2**
0/2
done) .
Sediment
samples
0/6
0/6
3/8
1/8
0/8
0/8
0/8
0/8
0/8
0/4
0/8
0/8
1/8
0/8
1/8
0/8
0/8
0/8
0/8
0/8
52
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TABLE 23. BACTERIOLOGICAL DATA AND THE PRESENCE OR ABSENCE OF PATHOGENIC
NAEGLERIA IN GEORGIA LAKES SAMPLED
Pathogenic Naegleria Bacterial results
Lake
Griffin
Cypress
Carrol
Olmstead
Lester
Deltina
Fort Yargo
Yonah
Sky
Dockery
Blackburn
Indian
Spivey
Ransby
Robin
Concharty
Tobesofkee
Houston
Blackshear
Long
Site
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
Water Sediment Coliform
neg. pos. neg. pos. MPN
+ - 9,200
16,000
141
>24,000
+ 11
+ 23
+ 3,480
3,480
542
211
5,400
172
3,480
2,400
1,600
2,400
BIT*
2,400
2,400
ND** ND 3,480
1,720
9,200
3,480
348
- 33
+ BIT
920
- 79
348
+ 109
- - 70
46
221
221
460
790
- - 22
-t - 23
1,410
+ - BIT
Fecal
coliform
79
1,720
17
9,200
2
2
920
460
<2
14
920
2
45
49
172
109
BIT
109
543
120
130
79
17
348
<2
BIT
8
13
49
5
8
7
49
33
130
23
5
7
46
BIT
Fecal
strep.
260
>24,000
5,420
542
221
33
79
221
33
46
240
175
49
1,600
109
49
BIT
700
16,000
>24,000
49
70
33
17
<2
BIT
348
22
240
17
<2
<2
17
49
7
33
49
5
221
BIT
* BIT - Broken in transit.
** ND - Not done.
t Seropositive nonpathogenic Naegleria isolated.
53
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the 5% level. With 2.201 being significant for the 5% level, the t value for
total coliforms was 1.91, for fecal coliforms, 1.305 and for fecal strepto-
cocci, 1.28. Although significance cannot be established, there does appear
to be a trend toward higher bacterial densities in the positive lakes as
opposed to the negative ones. If more extensive testing were done on selected
lakes, significance may be demonstrated. If so, perhaps more frequent bac-
terial analyses of public swimming areas during July and August and strict
adherence to swimming restrictions in case of increased bacterial counts,
could represent a first line control against human PAM infections.
TABLE 24. BACTERIAL DENSITY.RANGES AS RELATED TO THE GEOGRAPHICAL LOCATION
OF THE LAKE WITH RESPECT TO ATLANTA AND THE PRESENCE OR ABSENCE
OF PATHOGENIC NAEGLERIA
Lake
location in
relation to
Atlanta
North (7)*
South (7)
South (6)
Pathogenic
Naegleria
Absent
Present
Absent
Total coliforms
Range
172- 9,200
11-24,000
22- 3,480
Median
2,400
531
221
Fecal coliforms
Range Median
<2- 920 109
<2-9,200 47.5
5- 348 15
Fecal streptococci
Range
33->24,000
<2->24,000
<2 348
Median
109
221
19.5
*Number of lakes sampled shown in parentheses.
Ample data show a positive relationship between increased water temper-
atures and the presence of pathogenic Naegleria. This was evidenced also in
this study. The average water temperature of the positive lakes was 30.1°C,
whereas that of the negative lakes was 28.2°C. However, exceptions were
noted. The water temperature in one negative lake was 33.5°C and in one
positive lake, only 27.5°C Other factors, perhaps even sampling sites and
frequency, play a role in the isolation of pathogenic Naegleria amoebae from
any given lake, particularly from those lakes in which the water temperature
is >28°C. All study lakes situated north of Atlanta were negative and had an
average water temperature of 25.9°C. Conversely, 53.8% of the study lakes
located south of Atlanta were positive and the average water temperature was
30.5°C. This would appear to indicate that water temperature played an
important role in the negative status of lakes located north of Atlanta.
The one enigma encountered in this study was the failure to demonstrate
pathogenic Naegleria in large water samples from sites where a positive MPN
test was found. Previous experiences in Florida have resulted in negative
MPN tests and positive large water samples. One possible explanation for
this may be the on-site inoculation of the MPN concentrate, as opposed to the
long-term incubation of the large water sample sand column containing the
concentrates. The latter were incubated under crowded conditions, which
perhaps resulted in temperature fluctuations due to poor circulation. As has
54
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been shown in laboratory situations, vacillating temperatures are much more
deleterious to these organisms than is a constant temperature, even room
temperature. Temperature fluctuations may help to account for the poor yield
from the large water samples.
Laboratory studies had shown that a 96-hour incubation period at 43°C
increased the probability of isolating pathogenic Naegleria from sediment
samples. It was assumed that the same treatment should be beneficial for
large water sand column concentrates, but the procedure had not been field
tested. In view of the poor yield (one positive sample) from large water
samples and the relatively good yield (six positive samples) from the MPN
water samples, one questions whether or not predators or toxic substances
were also concentrated and brought into close proximity with the amoebae.
The long enrichment period could have enhanced predation and/or deleterious
toxic effects due to the concentration or proximity. In the case of sedi-
ments, a more natural state exists and incubation apparently enhances
multiplication of the pathogens.
This survey has confirmed data accrued in Florida, indicating that
pathogenic Naegleria amoebae are widely distributed in freshwater lakes in
which the water temperature is elevated over a period of time. The occur-
rence of cases in Virginia precludes limiting the geographical distribution
to the more southern United States.
55
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REFERENCES
1 Culbertson, C. G., Smith, J. W., and Minner, J. R. Acanthamoeba:
observations on animal pathogenicity. Science, 127:1506, 1958.
2 Culbertson, C. G. Pathogenic Ac an thamoeb a (Hartmanella). Am. J. Clin.
Path., 15:195-202, 1961.
3 Fowler, M., and Carter, R. F. Acute pyogenic meningitis probably due to
Acanthamoeba sp.: A preliminary report. Brit. M. J.., _2:740-742, 1965.
4 Butt, C. G. Primary amebic meningoencephalitis. New Eng. J. Med., 274;
1473-1476, 1966.
5 Butt, C. G., Bardo, C., and Knorr, R. W. Naegleria (sp.) identified in
amebic encephalitis. Am. J. Clin. Path., _50:568-574, 1968.
6 Carter, R. F. Description of a Naegleria isolated from two cases of
primary amoebic meningo-encephalitis, and of the experimental pathologi-
cal changes induced by it. J. Path., 100:217-244, 1970.
7 Singh, B. N., and Das, S. R. Studies on pathogenic and non pathogenic
small free-living amoebae and the bearing of nuclear division on the
classification of the order Amoebida. Phil. Trans. Roy. Soc. London,
^59^:435-476, 1970.
8 Chang, S. L. Small, free-living amebas: cultivation, quantitation,
identification, classification, pathogenesis and resistance. Curr. Top.
Comp. Pat'hobiol., !1:201-254, 1971.
9 Chang, S. Etiological, pathological, epidemiological, and diagnostical
considerations of primary amoebic meningoencephalitis. CRC Grit. Rev.
Microbiol., 3^:135-159, 1974.
10 DeJonckheere, J., VanDijck, P., and van de Voorde, H. The effect of
thermal pollution on the distribution of Naegleria fowleri,. J. Hyg.,
Camb., ^5:7-13, 1975.
11 van den Driessche, E., Vandepitte, J., van Dijck, P. J., de Jonckheere,
J., and van de Voorde, H. Primary amoebic meningoencephalitis after
swimming in stream water. Letter to Editor, Lancet, October 27, 1973,
p. 971.
56
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REFERENCES (continued)
12 Van Dijck, P., de Jonckheere, J., Reybrouck, G., and van de Voorde, H.
Rapid identification of Naegleria species by sero-agglutination and
fluorescent antibodies - new possibilities for the water supply service.
Zentralbl. Bakteriol. Parasitenkd. Infektinoskr. Hyg. Abt. 1 Orig.
Reihe B, 158:541-551, 1974.
13 Willaert, E. Primary amoebic meningo-encephalitis a selected bibliog-
raphy and tabular survey of cases. Am. Soc. beige Med. trop. 54:429-
440, 1974.
14 Wellings, F. M., Amuso, P. T., Chang, S. L., and Lewis, A. L. Isolation
and identification of pathogenic Naegleria from Florida lakes. Appl. &
Envir. Microbiol., jJ4_:661-667, 1977.
15 Haggerty, R. M., and John, D. T. Innate resistance of mice to experi-
mental infection with Naegleria fowleri. Inf. and Immun., 20:73-77,1978.
16 Page, F. C. Taxonomic criteria for Limax amoebae with descriptions of
three new species of Hartmanella and three of Vahlkampfia. J. Protozool,
1.4:499-521, 1967.
17 Gowans, E. J. An improved method of agar pelleted cells for electron
microscopy. Med. Lab. Tech., 30^:113-115, 1973.
18 Spurr, A. R. A low viscosity epoxy resin embedding for electron
microscopy. J. Ultrastructure Research, 26:31-43, 1969.
57
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APPENDIX A
TABLES
TABLE A-l. SAMPLE DATA AND RESULTS OF TESTS ON THERMALLY ENRICHED LAKES
(January, 1976 - March, 1978)
Water sample
Date
01-27-76
02-03-76
02-10-76
02-24-76
03-08-76
03-30-76
05-04-76
05-10-76
06-07-76
09-27-76
10-25-76
11-04-76
11-20-76
12-27-76
01-31-77
03-01-77
03-28-77
04-14-77
05-2.6-77
06-23-77
07-27-77
Water
temp.°C
25.0
23.0
16.0
26.0
32.0
35.0
25.0
30.0
34.0
36.0
26.5
28.0
31.0
23.0
21.5
31.0
26.0
23.0
37.0
38.0
41.0
Volume
(liters)
757.1
378.5
1.89.2
0,125
189.2
0.15
0.10
189.2
0.15
0.10
0.60
378.5
0.1
0.2
0.2
0.16
0.10
2.0
189.2
189 , 2
189.2,
189.2
189.2
189.2
189 . 2
189.2.
189.2
189.2
189.2
Depth
(meters)
Lake
<0.3
<-0.3
<0.3
-------
TABLE A-l (continued)
Water sample Sediment sample
Number Number
positive/ positive/
Water Volume Depth number Volume Depth number
Date temp.°C (liters) (meters) tested (ml) (meters) tested
Lake Sanford
02-20-78 17.0 200 1.5 0/6
17.0 200 2.4 0/6
18.0 200 4.3 0/9
59
-------
TABLE A-2. SAMPLE DATA AND RESULTS OF TESTS ON LAKE CONWAY
(January, 1976 - March, 1978)
Water sample
Pate
02-03-76
03-08-76
03-16-76
03-23-76
04-06-76
04-19-76
05-17-76
06-14-76
06-21-76
07-12-76
07-19-76
07-26-76
08-02-76
08-09-76
09-28-76
10-18-76
10-25-76
11-02-76
11-08-76
11-30-76
12-02-76
12-13-76
01-10-77
01-24-77
02-10-77
03-03-77
Water
temp.°C
19.0
26.0
24.0
22.0
24.0
26.0
31.0
29.0
31.0
30.0
31.0
32.0
33.0
34.0
29.0
26.0
23.5
25.0
20.5
18.0
16.0
15.5
16.0
12.0
14.0
17.5
Volume
(liters)
378.54
0.002
.0015
283.9
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
94.6
0.2
0.1
0.05
0.025
94.6
2.0
2.0
2.0
189.2
189.2
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0,3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/5
1/5
1/5
1/5
1/1
0/1
0/1
0/1
0/1
0/1
Sediment sample
Volume
(ml)
100
100
100
200
300
300
200
100
100
100
100
200
100
100
100
100
100
100
200
200
200
100
100
100
100
100
100
200
200
200
200
200
100
100
100
100
100
100
Depth
(meters)
<0.3
<0.3
<0.2
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
2.4
2.1
6.1
7.6
2.1
2.4
7.6
2.1
2.4
7.3
2.4
5.5
8.2
5.5
2.4
6.7
9.7
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/1
1/1
1/1
0/1
0/1
0/3
0/3
1/10
0/2
1/2
2/2
0/4
1/4
2/2
0/6
0/4
4/4
0/5
5/6
2/3
1/2
0/4
4/4
1/4
(continued)
60
-------
TABLE A-2 (continued
Water sample
Date
03-14-77
03-24-77
03-28-77
03-31-77
04-07-77
04-11-77
04-14-77
04-25-77
05-02-77
05-12-77
05-16-77
05-19-77
05-26-77
06-01-77
06-08-77
06-13-77
06-16-77
06-20-77
06-23-77
07-11-77
07-14-77
07-21-77
07-22-77
07-28-77
08-29-77
12-08-77
Water
temp. °C
22.0
26.0
26.0
23.5
25.0
23.5
24.0
26.0
26.0
25.0
26.0
28.0
30.0
30.5
29.0
30.5
31.0
29.5
31.5
32.5
32.0
32.0
30.0
32.0
29.0
19.0
19.0
19.5
19.5
19.5
Volume
(liters)
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
1.0
1.0
1.0
189.2
6.0
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
0.6
2.1
<0.3
2.4
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
Sediment sample
Volume
(ml)
100
100
100
100
100
100
100
200
100
100
100
100
200
200
200
200
100
100
100
100
200
200
350
300
300
300
150
150
150
150
300
300
300
200
200
200
200
200
200
200
200
Depth
(meters)
2.4
7.3
8.2
8.5
<0.3
<0.3
<0.3
<0.3
2.3
2.4
6.4
7.3
<0.3
<0.3
<0.3
<0.3
2.4
6.4
7.3
8.2
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
2.1
4.9
6.4
8.2
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
2.4
2.1
6.1
9.4
2.4
Number
positive/
number
tested
0/4
4/4
2/3
3/3
0/2
0/1
0/1
0/1
0/3
0/6
4/6
2/3
0/1
0/1
0/1
0/1
0/4
4/4
4/4
3/4
0/1
0/1
0/1
0/1
0/1
0/1
2/4
1/4
5/8
0/4
0/1
0/1
0/1
0/1
0/1
0/1
0/4
0/4
4/4
0/4
0/4
61
(continued)
-------
TABLE A-2 (continued)
Water sample Sediment sample
Date
01-05-78
01-23-78
02-02-78
02-13-78
03-16-78
Water Volume
temp.°C (liters)
15.5
16.0
15.5
16.0
16.0
16.0
15.2
14.5
14.0
14.0
14.0
14.5
14.0
14.0
16.5
15.0
14.5
16.5
15.0
14.5
18.0
Number
positive/
Depth number Volume
(meters) tested (ml)
200
200
200
200
200
200
400
200
200
200
200
200
200
200
200
200
200
200
200
200
200
Depth
(meters)
5.2
6.7
2.3
5.5
8.5
9.4
7.9
2.1
6.1
6.4
7.9
2.1
6.4
7.9
2.3
6.1
8.2
2.3
6.1
8.2
6.7
Number
positive/
number
tested
2/3
0/3
0/3
2/3
0/3
1/3
4/4
0/3
3/3
3/3
4/6
0/3
3/3
1/3
0/3
3/3
1/3
0/3
3/3
3/3
14/18
62
-------
TABLE A-3. SAMPLE DATA AND RESULTS OF TESTS ON LAKE HOURGLASS
(January, 1976 - March, 1978)
Water sample
Date
01-13-76
03-16-76
03-23-76
04-19-76
05-17-76
06-14-76
06-21-76
06-28-76
07-12-76
10-11-76
10-18-76
11-02-76
11-08-76
11-18-76
12-09-76
01-03-77
01-17-77
02-03-77
02-24-77
03-10-77
04-04-77
Water Volume
temp.°C (liters)
17.0
25.0
24.0
26.0
28.5
29.5
30.0
30.0
33.0
26.0
26.0
20.0
19.0
21.0
15.5
16.0
14.5
14.0
18.0
21.0
27.5
1,135.6
0.5
189.2
94.6
94.6
0.1
0.05
94.6
0.2
0.1
0.05
0.025
2.0
2.0
189.2
189.2
Sediment
Number
positive/
Depth number Volume
(meters) tested (ml)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
0/1
0/1
0/1
1/1
1/1
4/5
0/1
1/1
5/5
4/5
5/5
5/5
1/1
0/1
0/1
0/1
(cattail roots)
1
63
200
300
200
100
100
100
100
100
100
200
200
200
200
200
100
100
100
100
150
150
150
150
200
200
200
200
100
100
100
100
100
100
400
,000
sample
Number
positive/
Depth number
(meters) tested
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
4.9
2.4
2.1
2.7
3.0
3.6
3.3
1.2
1.8
2.1
2.4
1.2
3.9
3.6
0/1
0/1
0/1
0/2
1/1
0/1
2/2
0/1
0/1
0/1
0/3
0/8
0/4
0/2
0/2
0/2
0/3
0/5
0/2
0/2
0/4
0/4
0/1
0/2
0/4
Shoreline 0/1
3.0
3.0
2.7
3.0
2.7
2.4
3.3
3.0
0/10
0/12
0/3
0/3
1/3
0/3
0/4
0/9*
(continued)
-------
TABLE A-3 (continued)
Water sample
Date
04-07-77
04-14-77
04-25-77
05-09-77
05-12-77
05-19-77
05-26-77
06-01-77
06-06-77
06-08-77
06-13-77
06-16-77
06-23-77
07-11-77
07-14-77
07-18-77
07-21-77
07-28-77
02-02-78
Water
temp.°C
25.0
24.0
26.0
29.0
26.0
29.0
31.0
31.5
28.5
**
31.0
32.0
31.0
33.0
32.0
30.5
32.0
33.5
15.0
Volume
(liters)
189.2
189.2
5.0
189.2
189.2
189.2
189.2
1.0
1.0
1.0
1.0
189.2
189.2
189.2
189.2
189.2
1.0
1.0
1.0
189.2
189.2
Depth
(meters)
<0.3
<0.3
2.7
<0.3
<0.3
<0.3
<0.3
3.0
3.3
2.7
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
3.0
2.7
<0.3
<0.3
<0.3
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/2
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/2
1/1
0/1
0/1
Sediment sample
Volume
(ml)
400
200
200
1,800
200
200
200
375
400
400
400
300
300
300
300
300
300
400
400
400
200
200
200
200
200
Depth
(meters)
<0.3
<0.3
<0.3
2.7
<0.3
<0.3
<0.3
<0.3
3.0
3.3
2.7
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
3.0
2.7
<0.3
<0.3
<0.3
3.0
3.3
1.8
Number
positive/
number
tested
0/2
0/1
0/1
0/18t
0/1
0/1
0/1
0/1
0/4
0/4
0/8*
0/1
0/1
0/1
0/1
0/1
1/1
4/4
8/8*
0/2
0/1
0/1
0/9
0/3
0/3
* Taken at two different sites.
t Taken at six sites.
** Not recorded.
64
-------
TABLE A-4. SAMPLE DATA AND RESULTS OF TESTS ON LAKE BALDWIN
(January, 1976 - March, 1978)
Water sample
Date
07-26-76
08-02-76
08-09-76
09-27-76
10-18-76
10-25-76
11-08-76
11-15-76
11-29-76
12-06-76
12-16-76
01-13-77
01-27-77
02-14-77
03-07-77
03-21-77
04-07-77
04-14-77
04-18-77
04-21-77
05-05-77
05-12-77
Water
temp.°C
31.0
32.0
31.0
32.5
24.5
25.0
18.0
21.0
21.0
17.0
19.0
14.5
12.0
16.0
21.5
26.0
26.0
24.0
26.0
26.0
26.0
25.0
Volume
(liters)
94.6
0.2
0.1
0.05
0.025
94.6
2.0
2.0
2.0
189.2
189.2
189.2
1.0
3.0
189.2
189.2
189.2
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
2.3
5.5
<0.3
<0.3
<0.3
Number
positive/
number
tested
1/1
5/5
5/5
5/5
5/5
1/1
1/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
Sediment sample
Volume
(ml)
100
100
100
100
100
200
200
200
200
200
200
200
100
100
100
100
200
200
200
200
200
200
200
100
100
100
100
100
100
100
100
200
200
100
100
200
200
200
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
1.8
1.2
1.8
2.1
<0.3
2.1
4,6
1.2
2.4
2.1
5.5
2.1
1.2
5.5
2.4
2.1
5.2
2.1
2.1
5.5
2.1
5.5
5.8
<0.3
<0.3
<0.3
2.3
5.5
<0.3
<0.3
<0.3
Number
positive/
number
tested
1/1
1/1
0/1
1/1
0/1
0/1
0/3
0/2
0/2
0/2
5/8
0/3
5/6
0/1
0/2
3/10
1/4
3/4
0/2
0/2
2/6
0/2
1/4
2/6
2/6
2/4
4/8
0/4
0/4
2/4
0/4
0/1
0/1
1/3
3/9
0/1
0/1
0/1
65
(continued)
-------
TABLE A-4 (continued)
Water sample
Date
05-26-77
06-01-77
06-08-77
06-13-77
06-15-77
06-23-77
07-11-77
07-14-77
07-21-77
07-28-77
08-01-77
08-26-77
09-26-77
11-14-77
01-16-78
Water
temp.°C
27.0
29.0
27.0
29.0
29.0
29.5
31.0
31.0
30.5
32.5
30.5
30.0
32.0
20.0
19.0
19.0
19.0
19.0
13.2
13.5
15.2
Volume
(liters)
1.0
1.0
1.0
1.0
1.0
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
1.0
1.0
1.0
2.0
2.0
2.0
189.2
Depth
(meters)
3.0
3.0
3.0
3.0
2.1
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
0.6
2.4
<0.3
0.6
2.4
<0.3
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/1
1/1
1/1
1/1
1/1
0/1
Sediment sample
Volume
(ml)
100
100
100
100
100
350
300
300
300
300
300
300
200
200
200
200
200
200
200
200
200
200
200
200
Depth
(meters)
2.4
4.6
5.8
5.5
2.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
2.1
5.8
5.3
6.1
5.2
2.4
5.5
Number
positive/
number
tested
0/4
1/4
2/4
1/4
0/4
0/1
0/1
0/1
0/1
0/1
1/1
1/1
1/1
0/1
1/1
0/2
0/4
0/2
0/4
3/4
2/4
4/6
0/3
2/6
66
-------
TABLE A-5. SAMPLE DATA AND RESULTS OF TESTS ON LAKE SPIER
(January, 1976 - March, 1978)
Water sample
Number
Sediment sample
positive/
Date
01-20-76
07-26-76
08-02-76
08-09-76
08-16-76
09-02-76
09-27-76
10-11-76
10-18-76
10-25-76
04-14-77
04-21-77
04-25-77
05-05-77
05-12-77
05-19-77
05-26-77
06-01-77
06-08-77
06-16-77
06-23-77
07-11-77
07-14-77
07-21-77
07-28-77
08-01-77
Water
temp. °
14.0
32.0
31.5
32.0
34.5
31.0
33.0
26.0
25.0
26.5
24.0
26.0
24.0
26.0
26.0
26.5
29.0
29,0
27.5
29.5
30.0
31.0
31.0
30.0
31.0
31.0
Volume
C (liters)
378.5
94.6
189.2
189.2
189.2
189.2
113.6
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
1.0
1.0
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
0.6
number
tested
0/3
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1*
0/1
0/1
0/1
0/1
0/1
1/1
0/1
1/1
0/1
1/1
0/1
Volume
(ml)
100
100
100
100
200
100
100
100
100
200
200
200
200
200
200
200
200
300
300
300
300
300
200
200
Depth
(meters)
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
Number
positive/
number
tested
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1*
0/1
0/3
l/2t
0/2
1/2
0/1
0/1
* Only one
of 20
plates yielded a pathogenic
Naegleria
plaque.
t Only one of 40 plates were positive with a single plaque.
67
-------
TABLE A-6. SAMPLE DATA AND RESULTS FROM ORANGE
COUNTY LAKES SAMPLED INFREQUENTLY
(January, 1976 - March, 1978)
Lake
Killarney
Virginia
Bell
Osceola
Fairview
Mary Jane
Clear
Holden
Mann
Apopka
Mary Gem
Telfer
Lucern
Turkey
Bear Gully
Irma
Date
sampled
02-10-76
08-16-76
02-10-76
08-23-76
02-10-76
10-11-76
02-10-76
10-07-76
08-23-76
08-23-76
09-02-77
09-02-77
09-02-77
09-22-77
09-29-77
09-22-77
09-22-77
09-26-77
10-17-77
09-26-77
10-06-77
10-10-77
10-10-77
Water
temp.°C
18.0
32.0
20.5
30.5
21.0
25.5
18.0
28.0
30.5
31.5
27.5
28.0
27.5
30.5
30.0
29.0
34.5
30.0
21.0
32.0
30.0
27.0
28.0
Water
Volume
(liters)
.25
94.6
.25
94.6
.25
2.0
.25
2.0
94.6
94.6
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
sample
Number
positive/
number
tested
0/1
1/1
0/1
1/1
0/1
1/1
0/1
1/1
1/1
1/1
1/1
1/1
0/1
1/2
1/1
1/1
1/1
0/1
0/1
1/1
0/1
1/1
0/1
Sediment sample
Volume
(ml)
100
100
100
100
100
100
100
100
100
100
200
200
200
200
200
200
200
200
200
200
200
200
200
Number
positive/
number
tested
0/1
1/1
0/1
0/1
0/1
0/1
0/1
0/1
1/1
1/1
0/1
0/1
0/1
1/2
1/1
0/1
0/1
0/2
0/3
1/1
0/1
0/1
0/1
68
-------
TABLE A-7. RESULTS OF SURVEY FOR PATHOGENIC NAEGLERIA IN LAKE WATER AND
SEDIMENT SAMPLES FROM LAKES OUTSIDE OF ORANGE COUNTY
(January, 1976 - March, 1978)
Samples
Water Sediment
County
Collier
Gadsden
Hernando
Highlands
Hills-
borough
Indian
River
Levy
Marion
Okeechobee
Pasco
Polk
Putnam
Lake
Traf ford
Seminole
Talquin
Large Camp-A-Wyle
Small Camp-A-Wyle
Placid
Thonotosassa
Brotherhood Union
Magdaline
Egypt
Hiawatha
Keystone
Blue Cypress
Stafford
Richard
Rousseau
Half Moon
George
Okeechobee
Bell
Rudy
Buff ram
Parker
Oklawaha
Date
10-03-76
09-13-76
06-29-77
08-30-76
08-30-76
09-09-76
08-30-76
10-13-77
09-20-76
09-20-76
09-20-76
10-13-77
10-07-76
10-07-76
08-30-76
08-30-76
10-03-76
07-09-77
08-30-76
09-30-76
09-30-76
09-09-76
08-30-76
08-30-76
08-30-76
02-10-76
10-11-76
09-30-76
x- s*i
Water
temp.°C
27.5
28.0
30.0
32.0
32.0
32.0
33.0
24.0
30.0
33.0
32.5
26.0
28.0
28.5
29.0
30.0
-
28.0
30.0
26.0
32.0
32.0
31.0
32.0
19.0
24.0
28.0
Size
(L)
189.2
2
2
2
189.2
2
189.2
189.2
189.2
189.2
189.2
189.2
189.2
2
2
0.1
3.2
2
2
2
189.2
2
1
2
2
2
Size
Results (ml) Results
+ 100
200
300
+ 100
100
200
100
200
+ 200
+ 200
200
+ 200
+ 100
100
+ 100
100
100
+ 300
100
+ 100
100
+
+ 100
100
100
100
100
(continued)
_
-
-
+
+
-
-
-
+
-
-
-
-
-
+
-
+
-
-
-
+
69
-------
TABLE A-7 (continued)
Samples
Water Sediment
County
Putnam
Es cambia
Santa
Rosa
Okaloosa
Walton
Holmes
Jackson
Washington
Bay
Gulf
Calhoun
Liberty
Leon
Lake
Crescent
Escambia River
Black Water Bayou
Black Water River
Yellow River
Sykes
Juniper
Cassidy
Harriett
Blue
Sunnyhill
White Western
Merial
Alice
Dead
Dead
Apalachacola River
Bradford
Ella
Date
09-30-76
06-28-77
08-16-77
06-28-77
08-15-77
06-28-77
08-16-77
06-28-77
06-28-77
08-16-77
06-28-77
08-16-77
06-28-77
08-16-77
06-28-77
08-17-77
06-28-77
08-17-77
06-28-77
08-17-77
06-28-77
08-17-77
06-28-77
06-29-77
08-17-77
06-29-77
08-17-77
06-29-77
08-17-77
09-13-76
06-29-77
08-18-77
09-13-76
06-29-77
08-18-77
Water
temp.°C
27.0
29.0
27.0
27.5
30.0
27.5
26.0
32.5
33.0
29.5
33.0
31.0
31.0
30.0
30.0
31.5
31.5
32.0
31.0
32.5
31.0
32.0
32.5
31.0
32.0
31,5
32.0
31.0
29.5
28.5
33.5
30.5
28.0
36.0
32.0
Size
(L)
2
2
2
2
2
1
1
1
2
2
2
2
1
1
1
1
1
1
1
1
1
1
I
1
1
1
1
2
2
189.2
1
1
189.2
1
1
Size
Results (ml) Results
100
600
400
600
400
300
200
300
600
400
600
400
300
200
300
200
300
200
300
200
300
200
+ 300
300
200
300
200
600
400
200
300
200
200
300
200
(continued)
+
-
-
-
+
-
-
_
_
+
-
_
-
-
_
-
-
-
70
-------
TABLE A-7 (continued)
Samples
County
Jefferson
Taylor
Madison
Hamilton
Suwanee
Columbia
Baker
Union
Clay
Alachua
Seminole
Lake
Miccosukee
Andrews
Francis
Suwanee River
Suwanee
Alligator
Ocean Pond
Palestine
Kingsley
Geneva
Lockloosa
Newnans
Brantley
Bear
Date
06-29-77
08-18-77
06-30-77
08-18-77
06-30-77
08-18-77
06-30-77
08-18-77
06-30-77
06-30-77
08-18-77
06-30-77
06-30-77
08-18-77
06-30-77
08-18-77
06-30-77
06-30-77
08-18-77
08-27-77
10-05-77
10-05-77
Water
temp.°C
32.0
31.5
31.0
32.0
32.0
35.0
25.5
27.0
32.0
37.0
36.0
33.0
35.0
28.5
31.0
31.0
30.5
30.5
32.0
29.0
29.5
N.T.*
Water
Size
(L) Results
1
1
2
2
2
2
2
2
1 +
j^ _
1
1
1 +
2
2
1
1
1 +
2
2
1
189.2 +
189.2
Sediment
Size
(ml)
300
200
600
400
600
400
600
400
300
300
300
200
300
600
400
300
200
300
600
400
200
200
200
Results
-
_
-
-
+
-
-
-
_
-
-
-
+
-
Not taken
71
-------
TABLE A-8. RESULTS OF ISOLATION ATTEMPTS FOR PATHOGENIC NAEGLERIA FROM
200 ML SEDIMENT SAMPLES FROM POTENTIAL CONTROL LAKES
(January, 1976 - March, 1978)
Lake
Howell
Little Pickett
Big Pickett
Corner
Date
10-31-77
10-20-77
10-06-77
10-17-77
10-20-77
10-06-77
10-17-77
10-24-77
11-17-77
12-01-77
12-01-77
Water temp.°C
Bottom Surface
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
22.5
28.0
24.0
22.5
22.5
22.5
22.5
22.5
27.0
22.0
22.0
22.0
22.0
22.0
22.0
19.1
19.1
19.1
19.1
19.1
19.1
22.5
22.5
22.5
22.5
22.5
Sediment
Depth No.
(meters) no.
1.9
3.0
4.9
5.2
2.7
6.4
5.2
7.3
2.2
6.1
6.4
5.5
2.7
0.3
0.3
7.0
7.3
4.0
6.1
7.6
0.3
0.3
4.9
4.0
3.3
4.3
5.5
4.9
4.3
4.0
3.3
5.8
4.6
4.9
3.3
4.0
4.3
5.5
sample
positive/
tested
0/3
1/3
0/3
0/3
0/3
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/1
0/3
0/2
0/4
1/4
0/2
0/2
0/1
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/6
0/3
0/3
0/3
(continued)
-------
TABLE A-8 (continued)
Sediment sample
Lake
Corner (continued)
Jessup
Little Lake Mary
Beauclaire
Date
12-14-77
01-16-78
04-06-78
10-05-77
12-05-77
03-30-78
10-05-77
11-07-77
11-28-77
12-12-77
01-09-78
02-06-78
Water
Bottom
14.0
16.2
14.0
17.5
21.0
21.0
21.0
18.5
18.5
22.0
21.0
21.5
15.0
15.0
16.0
16.5
15.8
16.0
12.0
12.0
12.5
16.5
15.8
temp.°C
Surface
19.0
19.0
19.0
19.0
13.1
15.0
14.0
25.5
27.0
20.5
20.5
21.8
22.5
21.8
27.0
23.5
22.5
22.5
18.5
19.0
19.0
19.0
19.0
15.0
15.0
15.0
15.0
15.0
15.5
Depth
(meters)
1.8
2.7
5.2
5.5
4.9
5.2
6.7
4.6
6.4
4.6
6.4
<0.3*
1.8
1.5
1.8
1.8
1.5
<0.3*
2.1
4.0
1.1
3.3
3.3
3.6
3.3
2.4
3.0
3.0
3.6
2.7
1.5
3.0
3.3
3.3
3.4
3.3
2.7
3.0
3.3
3.3
No. positive/
no. tested
0/3
0/6
0/3
0/6
0/3
0/6
0/6
0/3
0/3
0/3
0/3
0/1
0/16
0/4
0/6
0/6
0/6
0/1
1/4
6/12
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/18
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
73
(continued)
-------
TABLE A-8 (continued
Lake
Beauclaire
(continued)
Lawne
East Lake
Tohopekaliga
Harris
Water
Date Bottom
02-06-78 16.0
12.0
12.0
16.0
02-23-78 16.0
16.5
11.8
03-16-78 19.8
12-19-77 19.0
19.0
18.5
20.0
17.0
18.0
03-27-78 18.0
17.0
01-12-78 12.0
13.0
13.8
13.0
13.0
13.8
01-19-78 14.0
temp.°C
Surface
19.0
19.0
19.0
19.0
19.0
19.0
12.0
13.0
13.0
13.0
13.2
13.2
13.0
,SedimenL
Depth No.
(meters) no.
3.4
3.3
2.7
3.3
3.6
4.0
3.0
4.0
3.6
2.1
4.9
2.4
6.1
4.9
4.9
7.3
3.0
3.0
4.3
3.3
3.0
4.0
5.8
sample
positive/
tested
0/3
0/3
0/3
0/3
0/3
1/3
0/3
0/9
0/3
0/3
0/3
0/3
0/3
0/3
1/8
1/4
0/3
0/3
0/3
0/3
0/3
0/3
1/12
* 189.2 liters of water tested on same date were negative.
74
-------
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APPENDIX B
CHANG'S MEDIUM - CALF SERUM-YEAST-EXTRACT-CASEIN MEDIUM (CSYECM) FOR AXENIC
CULTIVATION OF PATHOGENIC NAEGLERIA
#1 Per
500 ml 1000 ml
Isoelectric Casein 5.0 g 10.0 g
Na2HP04-7 H20 1.25 g 2.5 g
(If Na2HP04anhydrous) (0.6625) (1.325)
Dissolve in 400 ml 800 ml
of distilled water using a 2000 ml flask
#2
Dextrose 1.25 g 2.5 g
KH2P04 0.4 g 0.8 g
Dissolve in 40 ml 80 ml
of distilled water using a 250 ml flask
Autoclave #1 and #2 for 15 minutes and allow to cool.
#3
Yeast extract 2.5 g 5.0 g
Dissolve in 10 ml 20 ml
of distilled water
Note: Due to loss in filtration procedure for sterilization, use 10 g yeast
and 40 ml distilled water. Use 20 ml of the filtrate for preparation
of medium.
#4
Fetal Calf serum 50 ml 100 ml
#5
Penicillin (stock) 200,000 U/ml 0.5 ml 1.0 ml
Streptomycin (stock) 200,000 yg/ml 0.5 ml 1.0 ml
Using aseptic technique, pour the yeast extract preparation into the dextrose,
add the antibiotics and swirl. Pour this mixture into the casein and add
fetal calf serum. Mix well. Distribute aseptically, 5 to 10 ml amounts into
16 X 125 mm sterile screw-capped tubes. Incubate for 2 days at 37°C to check
sterility.
79
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-79-018
2.
4. TITLE AND SUBTITLE
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
May 1979 issuing date
6. PERFORMING ORGANIZATION CODE
PATHOGENIC NAEGLERIA: DISTRIBUTION IN NATURE
7. AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.
F.M. WELLINGS, P. T. AMUSO, A.L. LEWIS, M.J. FARMELO,
D.J. MOODY, C.L. OSIKOWICZ
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Epidemiology Research Center
4000 West Buffalo Avenue
Tampa, Florida 33614
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research & Development
U.S. Environmental Protection Agency
Cincinnati. OH 45268
10. PROGRAM ELEMENT NO.
1BA607
11. CONTRACT/GRANT NO.
R-804375
13. TYPE OF REPORT AND PERIOD COVERED
Final; Jan. 1975-Dec. 1978
14. SPONSORING AGENCY CODE
EPA/ 600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Infection in man with pat
a usually fatal disease enti
)logical data usually includ
jeek prior to onset. Howeve
axposure sites. The major o
ibsence of pathogenic Naegle
ed with human cases of PAM.
imoebae, i.e., soil, avian,
ecological factors related t
Results showed conclusive
Ln Florida's freshwater lake
Ln Georgia also showed exten
i lake in South Carolina, wh
Jaegleria, indicating that t
climate. These studies indi
:er in lake bottom sediments
lifferences have been establ
Lc Naegleria and those suppo
17.
a. DESCRIPTORS
hogenic Naegleria, a free-living soil amoeba, results in
ty known as primary amoebic meningo encephalitis. Epidemi-
ed exposure to freshwater lakes or streams within the
r, no confirmed isolations had been made from the suspected
bligation of this study was to determine the presence or
ria in freshwater lakes in Florida which had been associat-
Secondary objectives were to elucidate the source of these
and/or mammals and to determine the environmental and/or
o the presence of pathogenic Naegleria in lake waters.
ly that pathogenic Naegleria amoebae are widely distributed
s. Examination of samples obtained from freshwater lakes
sive distribution of these amoebae. Samples submitted from
ich was associated with a PAM case, also yielded pathogenic
hese organisms are not unique to Florida's subtropical
cate that pathogenic Naegleria are ubiquitous and over win-
or at the sediment/lake water interface. No significant
ished among lakes supporting large populations of pathogen-
rting very limited or undetectable populations.
XEY WORDS AND DOCUMENT ANALYSIS
Amoeba, protozoal diseases, aquatic micro-
biology, surface waters, lakes, swimming
pools
18. DISTRIBUTION STATEMENT
Release to Public
EPA Form 2220-1 (9-73)
b.lDENTIFIERS/OPEN ENDED TERMS
PAM, primary amoebic
meningoencephalitis ,
Naegleria
19. SECURITY CLASS (This Report)
unclassified
20. SECURITY CLASS (This page)
unclassified
c. COSATI Field/Group
06F
48G
57H, J, N, U
68D
21. NO. OF PAGES
92
22. PRICE
80
«. S.GOVEMIMENT PRINTING OFFICE: 1979-657-060/1667 Region No. 5-11
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