AN ABSTRACT OF THE THESIS OF
SIDNEY JOSEPH HAYES III
M. 5.
(Degree)
for the
(Name)
Date thesis is presented
Title
in MICROBIOLOGY
(Major)
May 12, 1966
OCCURRENCE OF CLOSTRIDILTM BOTULINUM TYPE E IN SHELLFISH, LAKE
FISH AND AQUATIC SEDIMENTS INEORTHNFST
Abstract approv
7
Redacted for Privacy
jor Professor
Comparatively little work has been done to determine the ecolor
of Clostridium botulinum type E since its initial isolation in the
nineteen-thirties.
This spore forming, anaerobic microorganism is
relatively heat labile and has been missed in ecological surveys in
which heat was used to selectively screen for spore formers.
Use of
gentler methods has, however, facilitated its demonstration in marine
sediments throughout the Northern Hemisphere.
The type E organism elaborates a highly potent neurotoxin and
has been isolated as the causative agent in recent fatalities involving the consumption of fish products.
Until recently the organ-
ism was not believed to be present in the United States south of the
Canadian border.
The purpose of this investigation was to determine if the organism could be demonstrated in shellfish, inland lake fish, and sediment samples throughout the Northwest.
Samples of coastal shellfish--
including various species of clams, crabs and oysters, varieties of
smoked fish products, species of inland lake fish and inland lake,
river and coastal sediment samples were examined for the presence of
the type E organism.
The organism was found, to some extent, in almost every type of
sample
tested.
Type E toxin was demonstrated in incubated samples
of shellfish and smoked fish products collected from eleven sites
along the Oregon and Washington coast.
The organism was found in
shore sediments from the tidewater and freshwater areas of the Columbia, Alsea, and Umpqua rivers but could not be demonstrated in sedi-
ments taken from saltwater beaches at the mouths of these rivers.
The type E organism was also demonstrated in fish from inland lakes
in the Oregon Cascade Mountains, in sediments from the shores of these
lakes, and along the shores of a river and three reservoirs in this
area.
These samples were collected between 95 and 120 linear miles
from the coast.
The isolation of the type E botulinum organism in fish and shellfish products demonstrates that the organism does present a potential
hazard which should be recognized by Northwest processors and distributors of these products.
High concentrations of the spores of this
organism, such as those found in bottom sediments of some of the inland lakes and reservoirs may serve to contaminate fish and other
wildlife.
Much of the data collected supports a terrestial distribu-
tion of the spores.
OCCURRENCE OF CLOSTRIDIU14 BOTULINUN TYPE E
IN SHELLFISH, LAKE FISH AND AQUATIC SEDIMENTS
IN THE NORTHWEST
SIDNEY JOSEPH HAYES III
A THESIS
Submitted to
OREGON STATE UNIVERSITY
in partial fulfillment of
the requirements for the
degree of
MASTER OF SCIENCE
June 1966
APPROVED:
Redacted for Privacy
Professor of Microbiology
In Charge of Major
Redacted for Privacy
Chairman of Department of Microbiology
Redacted for Privacy
Dean'of Graduate School
Date thesis is presented
Typed by Connie Hayes
May 12, 1966
ACKNOWLEDGMENT
Surveys of most any type are by nature time consuming and
generally cannot be fully accomplished through the efforts of any
one person.
I am greatful to individuals who helped me and tolerated
the hazard of continuous association with toxic or infectious cul-.
tures and the smells of incubating samples which, at times, freshened
our laboratory.
I am especially greatful to Miss Janet Dougherty
(who was very dependable) and, to Miss Roxie Rhodes who ran the
chloride and moisture tests on the smoked fish.
This present work was an extention of an initial ecological survey, here in the Northwest, begun and conducted by Professor James M.
Craig.
Jim offered valuable advice, assistance and friendship to me;
and they were much appreciated.
I wish to thank members of the Oregon State Game Coimnission, es-
pecially Mr. Leonard Mateison, Director of the Central Area of the
state, and the Oregon State Police for assistance in several phases
of collection of the lake fish and some of the clam samples.
My Trojan wife who typed this thesis may, in the long run, receive ample compensation but, her effort was no less appreciated.
Sometimes a person gets to admire another individual so much they
are afraid they will tell them.
cellent major professor.
I am happy to have had such an ex-
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERTURE REVIEW
3
Reviews on Clostridjun Bo-tulinum and Botdism
World distribution of all types of Clostridiurn
botulinurn
Foods implicated in hutnan hchlism
Outbreaks of type Ebctulis
Ecology of Clostrtdium botulinun type E
Factors conerning the origin of Clost,rjdium
hotulinum type E
Health Hazard of Closifidjum Botuljnurri Type H
Food protection and the prevention of botulirum intoxication
Effect of Physical and Chemical Factors in. Smoked
Fish Products on Growth and Toxin Production by Cl
Botulinum Type E
Characteristics of ClostnidiLm Botulinum Type E
Pharmacological action of hctulinurn toxin
Physical and chemical characteristics
EXPERIMENTAL PROCEDURE AND MATERIALS
Culture Media
TPG medium
BHI medium
Liver.veal-egg agar
Blood agar
Antitoxins
Toxin Diluert
Chemtcals
Collection of Sarrples
Shellfish
Flsti
Smoked fish
Aquatic sediments
Incubation Procedures
Incubation techriqoes
Clams and oysters
(a0)
Crabs
(B.)
Fish
(c.)
Smoked fish
(d.)
Sediment samples
(e0)
3
3
24
24
5
5
8
8
8
11
11
U
13
13
13
13
121
15
15
15
16
18
18
19
19
20
21
23
23
23
224
224
25
Page
Screenin.g Specimens for Toxicity
Tissue samples
Sediment samples
Typing of Toxic Specimens with Antitoxin: Demcn
stration of Botulinun Toxin
Isolation of Clostridium Bat: linum Culture from
Toxic Specimens
Determination of the Sodium Choride Co'tent in
Smoked Fish
Determination of the Moisture Conteet of Smoked
Fish
RESULTS AND DISCUSSION
Introduction: Physical Aspects in Demonstrating
Cl. Botulinum
An Analysis and Approach: Consideration of the
Ecology of Cl. Botulinum Type E in the Northwest
Development of Survey Techniques and Results
Development of the technique for testing fish
Result.s of the lake fish survey
Procedure and results for shellfish
Comparison of methods for detecting Cl. botulinum in mud specimens
Comparison cf incubation temperatures of 20°C
and 28°C for various periods in TPG medium
Comparison of TPG medium with beef heart infu
sion plus neat
Results of survey of inland lake, reservoir,
river, and marine sediments
Comparison of the saltwater, tidewater, and
freshwater areas of three Oregon rivers
toxicity, chloride and
Smoked fish produ:ts:
moisture results
Isolation of Cl. Bo±:iinum Cultures from Toxic
Specimens
Cultural examination of type E isolates
SUI"ll"IARY AND CONCLUSIONS
BIBLIOGRAPETY
NOTE:
26
26
27
28
32
33
35
36
36
3?
141
141
L.5
L8
50
Si
53
55
58
60
6i
66
101
The generic and species name of the microorganism Clostridium
tulinum has not been Latir.ized, i.e., underlined or italicized,
in this section. or in headings throughout the text. This is in
agreement with Section 1, Rule 5 of the International Code of
Nomenclature of Bacteria and Viruses,
LIST OF FIGURES
Page
1.
2.
3.
Li.
5.
Plastic bag incubation technique used with clams,
oysters, lake fish, and crab viscera,
96
Isolated colonies of Clostridjum botulinum type E
grori on blood agar from a cockle clam sample.
97
Isolated colonies of Clostridium botulinum type E
grom on LVE from a cockle clam sample.
98
Isolated colonies of Clostridium botulinum type E
gro
on LVE from a kippered sturgeon sample.
99
Distribution of Clostridium botulinum type E in
the Northwest.
100
LIST OF TABLES
Page
1.
2.
3.
Lake fish samples collected, screened for
toxicity and typed for botulinum toxin
70
Crabs, oysters, and shrimp collected, screened
for toxicity and typed for botulinuin toxin
71
Clams collected, screened for toxicity and
typed for botulinum toxin
72
L Inland lake sediments collected, screened
5.
6.
7.
8.
9.
10.
11.
12.
13.
lL.
for toxicity and typed f or botulinum toxin
73
Inland river and reservoir sediments
collected, screened for toxicity and typed
for botulinum toxin
7L
A comparison of 20°C and 28°C incubation
temperatures for periods of 5, 7, and 1Li days
in TPG medium with and without trypsinization
for detection of toxic mud specimens
75
A comparison of two culture media and incuba-.
tion procedures with and without trypsinization
for detection of toxic mud specimens
76
Marine sediments collected, screened for toxicity
and typed for botulinum toxin
77
Alsea river sediments collected, screened for
toxicity and typed for botulinum toxin
78
Columbia river sediments collected, screened
for toxicity and typed for botulinum toxin
79
Umpqua river sediments collected, screened for
toxicity and typed for botulinum toxin
80
Toxicity tests on cultures derived from isolated
colonies
81
Smoked or kippered fish collected, screened for
toxicity and typed for botulinum toxin
83
Incidence of Clostridium botulinum type E in
smoked fish products
8Li
Page
15.
16.
17.
Chloride and moisture content of smoked fish
samples
85
Results of successful typing of toxic speci-.
mens with antitoxin
86
Results of successful typing of toxic specimens with antitoxin
91-
OCCURRENCE OF CLOSTRIDIUN BOTULINUN TYPE B IN SHELLFISH,
LAKE FISH AND AQUATIC SEDINTS IN THE NORTHt1EST
INTRODUCTION
This investigation represents the result of an ecological survey
to obtain more adequate data on the cocurronce and distribution of
Clostridium botulinum type F in fish and their environments in the
Northwest.
This spore forming anaerobic microorganism produces a
potent exotoxin which is quite lethal when ingested b:, man.
Recent well publicized diagnoses of type S botulinal ieitoxica-
tion, resulting from the consumption of fish products, aroused inIn
terest in the distribution of the organism in the environment0
considering the large and growing fish processIng industry in the
Northwest a survey of the distribution of the microorgansm in fish,
fish products, sediment samples and water was initiated under a con
tract with the U.S. Food and Dnug Administration at Oregon State
in the early part of 196k.
initial work primarily involved
attempts to isolate the microorganism from the gills and viscera of
river and marine fish.
A subsequent attempt to complete the
oologi-
cal picture, concerning the distribution of the microorganims in four
separate areas in Oregon, is presented in this thesis.
Isolation of
the organism has been attempted from some of the samples.
A survey for the presence of the microorganims has been made in
shellfish, including Oregon and Nashington crabs and oysters and,
various types of Oregon clams.
2
Specimens of marine and river sediments have been taken from the
banks of the Columbia, Alsea, and Umpqua rivers.
Examination was
made at three locations along the course of these rivers in order to
shed some light on the possible source of these microorganisms.
lected sites were:
Se-
(a) the saltwater beach adjacent to the river's
mouth, (b) a saltwater or tidewater area of the river, (c) a freshwater zone of the river above tidewater.
Lake fish and environmental sediments from Oregon and Northern
California lakes and man-made reservoirs, have been examined to determine the distribution of the microorganism.
Such results may be of
some direct practical importance in considering that lake fisherman
sometimes preserve these fish by home smoking methods.
Examination of smoked fish products, primarily from producers
along the Oregon and Washington coast has been carried out with regard to the distribution of the microorganism and the chloride and
moisture content of the product.
LITERATURE REVIEW
Reviews on Clostridjum Botulinum and Botulism
Dolman (21), has given an excellent review of the historical
highlights concerning the isolation and immunological characteriza-
tion of Clostridium botulinum types A (21), B (21), C
(), C,B (L9),
D (21), E (L-2), and of the characteristics of a type F (21) strain.
Meyer (26), has reviewed the international literature, the state of
public health knowledge, and statistical data on botulism up to 1956.
World distribution of all types of Clostridium botulinum:
The
highest geographic incidence of type A has been found in the Western
United States and the Soviet Ukraine (21).
Type B is found primarily
in France, Norway, and the Eastern United States (12).
Type Chas
been identified in the Western United States and Canada, South
America, South Africa, and Australia; and type C,
South Africa, Europe and North America (21).
in Austrailia,
Type U appears to be
prevalent in South Africa and Australia (12).
Type F has been iso-
lated from outbreaks in Northern Japan, British Columbia, Labrador,
Alaska, Sweden, Denmark, USSR and recently from the Northwestern
U.S.A. (12,8 ) and the Great Lakes region (12).
Type F has been iso-
lated from an outbreak in Denmark (12), from marine sediments taken
at depths of 725 and 900 fathoms off the Oregon and California coast
(23) and from the viscera of a fish taken in the Columbia River (12,
9,23).
L.
Foods implicated in human botulism:
in Europe, for the most
part, outbreaks have been associated primarily with processed meats-especially ham, sausages, and other pork products, whereas most out-
breaks in the U.S.A. are caused by home preserved vegetables and
fruit; those in the USSR and Japan are caused by fish, and those in
Alaska and Western Canada by fish and marine mammals, such as the
seal, whale and walrus (26,16,to).
Meyer states that the types of
Cl. botulinum and the nature of the foodstuffs implicated greatly influence the local case-fatality rate.
In addition, the amount of
toxin ingested is a critical factor, and this is in turn affected by
the physical and chemical characteristics of the food and the method
of preserving it (26).
Outbreaks of type E botulism:
Cl. botulinum type E, unlike
types A to U, is not known to have been isolated in the southern
hemisphere
i6,L1 ).
Type E spores were similarly thought to occur
rarely in the U.S.A. (33) (excluding Alaska); and only local outbreaks of type E intoxication were reported between 1950 and 1963 involving:
white whale meat (1956), salmon-egg cheese (1959), fermented
seal meat (1960), commercially smoked fish (ciscoes) in a vacuumpacked bag (1960), and salmon eggs (1961).
two cases of type E botulism were reported.
and two deaths were due to canned tuna fish.
However in 1963 twentyIn Detroit three cases
Two fatal cases occurred
in Kalamazoo, Michigan through the consumption of smoked white fish.
Seventeen cases and five deaths occurred in Tennessee, Alabama, and
Kentucky from vacuumpacked smoked white-fish chubs.
In the former
and latter cases the U.S. Food and Drug Admthistration was able to
remove the product from the market and prevent an inorease in the
number of fatalities (ii).
Eco10
of Clost.ridium botulinum trpe E:
The isolation of a
type E strain from soil was first reported by Dolmar. and Kerr in
l9Ll7.
A one to two gram sample was taken from a garden in Naiairno, B.C.
Since then the organism has been. found widely dispersed in marine
sediments and on the shores in certain parts of the world, in particular Japan, Germany, France, Denmark, Greenland and Sweden (21,16,
33,l,14.o).
Recently type E has been shown to occur in aquatic sediments at
three locations in Oregon (a spot along the Alsea River, the Siuslaw
River at Florence, and in Yaquina Bay at Newport) (8); and it has
been demonstrated in marine muds along the west coast of the United
States down to 36° North Latitude, as far as 80 miles off shore and,
at depths of 1070 fathoms.
in addition, the latter survey revealed
that the incidence of type E demonstrated for crabs and fish from
two particular environments paralleled the incidence in sediment
samples from the related environment (23).
Factors concerning the crjgjn of Clostridium botulinum type E:
As soon as it became apparent that Cl. botulinum type E had a predilection for fish the question arose as to whether spores of this
type were primarily telluric or marine in origin (18).
Various work
supports or attests to a terrestrial predisposition of the spores
(2o,12,l6,33,L,l8,16,l,Lio).
Since l9L.? Dolman has stressed a
He
hypothesis which provides for a terrestrial origin of the spore.
has postulated a mechanism whereby type E spores, perhaps widely, but
thinly, scattered over land masses in the northern hemisphere, might
be brought to coastal regions as the result of land erosion, and
through the intercourse of streams, rivers, and ocean currents where
they would tend to he distributed nidally in terraqueous deposits of
the littoral. (16,19).
Germination and multiplication in the marine
and littoral foci where micro-environmental conditions are propitious
could then account for the findings (l6,33,23,L0) that the concentration of type E in lagoon bottom sediment, coastal sand and adjacent
sea sediments may be higher than in neighboring soil.
Physically Clostridium botulinum type E is predisposed to a
marine environment.
Its spores show no significant difference to
those of types A and B with respect to their ability to remain viable
(no decline in number of spores) after eighty days incubation at 3°
to
O
in sterile sea water and in growth (vegetation and multiplica-
tion) in media containing up to four to six percent NAC1 (16).
How-
ever, type E cultures grow and produce toxin over a temperature range
50
to 100 lower than type A or B strains (Lvi).
3.3° and 37.5°C (Ll,L?,18).
Growth occurs between
Maximum growth occurs at 35°C.
toxin production is best at 25°C.
However
Evidence indicates that cultures
groiii at 37°C are frequently nontoxic (L1,l6,L2).
Dolman states (12) that Johannsen's demonstration of type E in
potato peels (33) might reveal a mechanism whereby type E and
possibly other types of Cl. botulinum could be intermittently
replenished in cultivated soil.
It is postulated that such a mecha-
nism would be similar to that shown (13L) for type C organisms which
are able to multiply and produce toxin in decomposing marsh vegetation rich in organic matter.
In addition he (17) has renewed inter-
est in the idea that fish (and shellfish), birds and animals' may act
as reservoirs and healthy carriers of type E organisms.
Bengston's
demonstration that the Lucilia larvae is a concentrator and reservoir
of type C organisms (3), Johannsen's isolation of types B and C from
the livers of lemmings in Northern Sweden,2 Eklund's isolation of
type E from crabs(23) and repeated demonstrations of the organisms in
fish and marine mammals (16) tend to support this hypothesis.
Thus, type E spores carried by fish or animal reservoirs, land
erosion, or ocean currents might be found, at extensive distances
from their telluric starting point, perhaps accumulating along parts
of the shore and bottom of rivers and lakes, along the coast, or in
sea sediments.
As a result of these accumulations, other marine or
freshwater fish and animals may ingest the spores, or become con-
taminated through skin apertures or wounds, and thus might continuously or transiently harbor the organism.
Upon the death of the host
(presumably brought about through hunters, fishermen or natural
causes), the spores, under favorable conditions of temperature and
1
Burova, A.E. et al.
The role of animals in the distribution of
botulism;
sited in (12).
2
Personal communication in 1963 between Johannsen and Dolman;
sited in (12).
anaerobiosis, may vegetate and migrate from the gut or other integuments into the muscles (12,16,19).
Conditions conducive to the proli-
feration of the microorganism would result in the formation of toxin.
Health Hazard of Clostridium Botulinum Type F
Food protection and the prevention of botulinum intoxications:
For years (24) the canning industry has used as an index of safety
a processing time and temperature which would destroy 60 billion of
the most heat resistant type A and B botulinum spores present in the
part of the can having the greatest temperature lag (43).
It has
been recognized that cultural findings on plant products reflect the
flora of the soil and may indicate the potential botulinal hazard in
a community (39).
It is also a possibility that Clostridium botu-.
linum or its spores can gain entrance to the tissues and organs of
an animal during life and proliferate and produce toxin after
slaughter and processing.
In this regard, better quality raw materi-
als may be necessary for safe food preservation by new methods than
were required for foods commerically sterilized by heat (10).
Effect of Physical and Chemical Factors in Smoked Fish Products on
Growth and Toxin Production by Cl. Botulinum Type E
As a result of recent outbreaks of type E botulinum from smoked
white fish and vacuum-packed smoked chubs (1963) considerable atten-
tion has been given to food packaging methods in addition to prophylactic considerations of the ecology, germination, and developmental
(outgrowth) aspects of the organism.
With regard to the questionable procedure of vacuum packaging
smoked fish, experiments have shown that this seems to be the only
procedure that definitely disturbs the microbial equilibrium in a
mixed microbial population, i.e. as determined by a comparison of
fish exposed to oxygen versus vacuum-packed fish.
Vacuum-packed
fish have a prolonged shelf life which increases the botulinal risk
in the product if spores are present.
The nonproteolytic Cl. botu-
linum type E organism can develop toxin in this environment without
the common organoleptic spoilage indication which is a major safety
factor.
Toxin containing fish in an air atmosphere spoil to a
point where an ordinary consumer (excluding Eskimos) would probably
reject it; however there would be no undesirable organoleptic properties to cause rejection of a toxic vacuum-packed fish (52).
Dolman, however, considers the difference is slightly more subtle.
He states that:
"If during some process between the catching of the fish
and its insertion into the package spores are introduced
into or onto the fish, there appears to be a danger of
the development of toxin. If that contamination is surface contamination, the danger would be enhanced, in my
view, by its enclosure in a vacuum. If, however, the
spores were introduced by pollution of muscle adjacent to
the intestine at the time of gutting (which is a pretty
crude procedure under any circumstances) or if it were
introduced from immersion of the fish in brine or pickling solution so that spores therein could penetrate to
a depth of, I should think, not more than perhaps a few
millimeters and certainly not more than half an inch, one
would have toxin production whether the vacuum pack was
there or not." (13).
10
With respect to outgrowth, important influencing factors are
temperature, salt or sugar concentrations, (or perhaps water
activity), pH, and substrate (L6).
ture has been considered.)
(The minimum outgrowth tempera-
Dolman has stated (12) that too little
is known about the minimum amounts of water in foods that are corn-
patible with toxigenesis.
In addition there appears to be no data
available in the literature on the sugar concentration required to
inhibit outgrowth of type E spores (246).
The literature of pH in-
hibition varies with growth reported down to pH
60°c.
A pH of
at 23°C or at
has been considered a satisfactory level for
public health safety, with respect to type A and B spores of Cl.
botulinurn (Li6).
0
0
Findings that at L6 F and 50 F growth of type E
0
0
occurs at L.O but not at L.5 percent salt and that at 60 F, 70 F and
85°F growth occurs at a salt concentration of LI.5 but not at 5 percent have indicated that type E spores are considerably less salt
tolerant than types A and B spores at optimal incubation temperature
(L6).
Dr. Slocum of the U.S. Food and Drug Administration in con-
sidering this aspect has stated (so) that the composition of fish
products range between some which are not vastly different from the
raw materials in salt and moisture content to others which are very
dry and high in salt.
The latter group he believes represent no
substantial hazard but between these two groups are all gradations.
11
Characteristics of Clostridjum Botulinum Type E Toxin
Pharmacolojcal action of botulinurn toxin:
acts in a well-defined manner:
Botulinum toxin
it paralyzes cholinergic synapses
(i.e., the acetyichoLine release mechanism at the nerve terminals).
After an initial latency of about 20 minutes, transmission across
individual synapses becomes impaired.
Once junctional block becomes
complete, it is irreversible by any knom procedure (5,6).
differences of various types of toxin (A
-
Antigenic
E) are reflected only in
susceptibilities of animal species and in dose-response relationships
(6,36).
The toxins are distributed in the body through the blood
stream, and death usually results from respiratory paralysis (6).
These types are quite toxic when administered via oral, respiratory
or intraperitoneal routes; Unactivated (see below) cultures containing type E toxin are most toxic when administered via oral and
respiratory pathways (7).
Physical and chemical characteristics:
Type E toxin is a simple
protein which possesses hemagglutinating properties and is considered
highly purified when it acts like a single substance in the toxin antitoxin reaction, in the ultracentrifuge, and in electrophoresis.
It has a strong absorption at 278 millimicrons and contains no nucleic
acids.
The ratio of absorbance at 260 millimicrons to that at 278
millimicrons has been used as an index of purity (L45).
weight of the protein has been found to be 18,6000.
activation the toxicity was 7.5 x 10
The molecular
After trypsin
mouse minimum lethal dose
MLD)
12
per milligram of nitrogen (29).
From this activated protein a pep-
tide has been isolated with a maximum absorbance at 280 millimicrons
and a toxicity of 2.8 x 10
mouse MLD per milligram of nitrogen (30).
(It should be noted that the toxicity of type E toxin is increased
50 to 100 fold by controlled treatment with trypsin at pH 6.2 (53);
however, uncontrolled trypsin digestion destroys (activated) type E
toxin (32).
The activation procedure allows attainment of culture
toxicities of the same order of magnitude as those obtained with
types A, B, C, and D; and provides an explanation for the lower toxicity of type E cultures .-- an observation that had been difficult to
understand in view of the high mortality associated with outbreaks
of type.E botulism (7).The toxicity of any botulinum toxin is destroyed if heated for 10 minutes in a boiling-water bath (32).
13
EXPERIMENTAL PROCEDURE AND MATERIALS
Culture Media
TPG Medium:
1.
Trypticase -- Baltimore Biological Laboratory
2.
BactoPeptone -- 'Difco'
3.
Bacto-Dextrose -- 'Djfco'
4.
Sodium thioglycoilate -- 'Difco'
The above reagents were used in the preparation of trypticase
-
proteose - glucose medium (TPG medium) which had the following composition:
50 gram (gm.) trypticase; .5 gm. peptone; 4 gm. glucose;
2 gm. sodium thioglycollate; made up to 1000 milliliter (ml.) using
distilled water.
The pH of this solution was adjusted to 7.0 before
sterilization in an autoclave for 15 minutes (at 15 pounds pressure
and l2l0C).3
BEl Meat Medium:
3
5.
Bacto - Beef heart for infusion -_ 'DiSco'
6.
Ground round steak
7.
Sodium chloride -- "Baker Analyzed" reagent grade
The method is a modification of Schmidt et al. procedure (48) in
which sterile concentrated solutions of sodium thioglycollate and
glucose are added to a trypticase (5%) - peptone (o.%) medium
just prior to inoculation.
The trypticase - peptone medium which
has been used for the preparation of spore suspensions of the proteolytic types A and B Cl. botulinum yields only slight growth
and poor sporulation with the predominantly saccharolytic type E
strains. Addition of 0.4 percent glucose to the trypticase - peptone medium provides a satisfactory growth and sporulation medium
for type E strains.
lL
The above reagents were used in the preparation of a beef heart
infusion with minced meat medium (BHI meat medium) (25).
50 gm. of
Bac-bo-Beef heart for infusion was made up to 1000 ml. with H20 and
allowed to infuse for one hour, heated to 80°C to coagulate some of
the protein, and clarified by several filtrations through layers of
gauze and cotton.
To the filtrate 1.5 percent Bacto - peptone and
0.5 percent NaCl were added.
To 5 gm. of minced meat in a
ounce
(oz.) screw-capped bottle 30 ml. of the above solution was added.
The meat suspension in each bottle was adjusted to approximately pH
6.9 - 7.0 by addition of N/i NaOH using pH paper.
The bottles of
medium were autoclaved 15 minutes and the caps screwed tightly shut
while they were still hot.
Liver Veal Eg Agar:
8.
Bacto - Liver - Veal agar base -- 'Difco'
9.
Fresh eggs
The liver-veal agar base was used in preparing liver-veal-egg
agar plates.
The base was rehydrated by suspending 97 gm. in 1000 ml.
cold distilled water.
The suspension was heated to boiling to dis-
solve the medium completely and autoclaved for 15 minutes.
The eggs
were scrubbed and disinfected by allowing them to set for an hour in
solutions of 1:10,000 mercuric chloride and 70 percent ethanol respectively.
The egg-yolks were aseptically separated from the white
by the usual culinary technique, dropped into a sterile beaker, and
mixed with an equal volume of sterile 0.85 percent saline.
The
15
autoclaved liver-veal-egg agar base was cooled to L5°_5O°C and the eggyolk suspension was added to a final concentration of 8-10 percent.
Plates were poured immediately and kept at Li°C where they remained us-
able for several months (53).
Blood Aar:
10.
Bacto - Blood - agar base -- 'Difco'
11.
Outdated defibrinated human blood
12.
Defibrinated sheep blood
The blood-agar base was used in preparing blood-agar-plates.
The
base was rehydrated by suspending LIO gm. in 1000 ml. cold distilled
water.
The suspension was heated to boiling to dissolve the medium
completely and, autoclaved for 15 minutes.
The sterilized medium was
allowed to cool to Lj.5°C and 5 percent sterile dêfibrinated blood was
added.
Plates were poured immediately and kept at Li,°C.
Antitoxins:
13.
Type A botulinum antitoxin, equine serum, 10 units per ml.
lLi.
Type B botulinum antitoxin, rabbit serum, 28 units per ml.
15.
Type C botulinum antitoxin, equine serum, 10 units per ml.
16.
Type E botulinum antitoxin, equine serum, 10 units per ml.
17.
Glycerol -- 'Baker Analyzed' reagent grade
Vaccine vials containing 120 units of lyophilized antitoxin (sent
to us by U.S. Food and Drug Admin.) were reconstituted with so% (v/v)
sterile glycerol-water (pH 7.0).
Toxin Diluent:
18.
Bacto - Gelatin -- 'Difco'
19.
Sodium phosphate, dibasic -- 'Baker Analyzed' reagent grade
16
The above reagents have been used at concentrations of 0.2 per.-
cent gelatin and 0.L percent phosphate
as a toxin diluent and animal
injection menstrum which was suggested by Duff et al. (22).
The pH
of the diluent was adjusted to 6.2 with N/lU hydrochloric acid.
20.
Trypsin (1:250) -- 'Djfco'
A concentrated trypsin solution, which it was hoped would approximate 10 percent, was made up by suspending 10 gm. of trypsin
(1:250) in enough distilled water to obtain 100 ml. of suspension.
This was covered and allowed to sit at room temperature overnight to
effect a better solution of the trypsin.
The solution was filtered
under negative pressure through a Seitz EK filter pad and distributed
in 30 ml. quantities into vaccine vials.
0
These were stored at -12 C.
Chemicals:
21.
Potassium chromate -- 'Baker Analyzed' reagent grade
A 5 percent solution of K2Cr0
in deionized water was used as an
indicator in the standardization of silver nitrate against an NaCl
solution containing a known number of equivalents.
22.
Ferric ammonium sulfate -- 'Baker Analyzed' reagent grade
An indicator was made up by preparing a saturated solution of
FeNH(SO1)2.l2H2Q in deionized water.
The saturated solution was
The toxicity of various botulinal toxin preparations in saline
which is the classic diluent for the extraction and dilution of
toxin from suspected specimens and cultures (10) or in simple
buffers, has been found to be lower than in buffered systems containing gelatin (37).
17
prepared by 'dissolving' 80 gm. of the reagent in enough water to
make 1000 ml.
Silver nitrate -- 'Baker Analyzed' reagent grade
23.
A N/b
solution of silver nitrate was prepared by dissolving
16.989 gin. AgNO3 in deionized water and diluting to 1000 ml.
solution was standardized against a N/b
This
NaCl solution containing
5.8Li5 gm. of pure dry NaC1 per 1000 ml.
Ammonium thiocyanate -- 'Baker Analyzed' reagent grade
21L.
A N/b
solution was prepared from a reagent that contained only
a trace of Cl by dissolving 7.613 gin. NHSCN in enough deionized
water to make 1000 ml.
This solution was standardized against a
standard N/mo AgNO3 solution.
25.
Ethyl alcohol (95 percent CH3CH2OH)
26.
Nitric acid (69.0 - 71.0 percent HNO3) -- Baker and Adamson,
reagent grade
27.
Acetone -- United States Pharrnacopeia grade
Acetone was used for cleaning needles, syringes and Milbipore
'Swinney' syringe type filters.
28.
Saline solution (0.85 percent NaC1 in distilled water)
29.
Plastic poultry bags 9 x 3 x 18 inches
These bags were used for collecting and incubating fish and
shellfish samples.
18
Collection of Samples
Independently, specimens of shellfish, lake fish, marine and.
river sediments, and inland lake and reservoir sediments have been
collected.
The methods, which for some cases differ only slightly,
are given below.
Shellfish:
Six different species of clams are included in the
collection as indicated. in Table 3.
Specimens of razor clams, in
the main, were collected by the Oregon State Police (from violators
of undersize and overlimit regulations) along the beach at Seaside,
Oregon.
The remaining clam specimens were dug along the Oregon
coast; the sites from which they were taken are also indicated in
Table 3.
Most of the Washington or littleneck clams were obtained
from fish markets along the coast.
Five to ten clams of the same
species were placed in a clean plastic bag after most of the adherent
sand had been rinsed away in seawater.
The plastic bags were sealed
with rubber bands and kept on ice until returned to the laboratory,
where they were refrigerated at 7°C until they could be tested.
Some
of the last samples to be incubated were held at this temperature for
18 days.
The oysters, which had been washed free of mud were picked from
oyster buckets at canneries in the towns indicated in Table 2.
Four
individual oysters were included per plastic bag and were held. under
the same conditions described for the clams.
A few crabs were caught by hand; however, the majority of samples
19
were purchased live from the crab canneries indicated in Table 2.
These were held in their paper containers at 5°C for not more than
three days before they were incubated.
Fish:
The specimens of trout, kokanee (land-locked salmon),
perch, chubs and catfish were taken by means of gill nets from the
inland and mountain lakes:
to a depth of six feet in Kiamath lake,
thirty-six feet in Elk Lake and one-hundred feet in Odell Lake.
The
fish taken by hand from the gill nets were transferred to plastic bags,
one to three fish per bag depending upon their size.
Surface slime
was rinsed from the collectors hands between each group of two or
three fish in an attempt to minimize cross contamination.
The fish
from South Twin Lake were picked from the water by hand or with a net
and rinsed in the water adjacent to the boat before they were inserted
into individual plastic bags.
Rotenone had been added to the lake;
as a result of the poison the fish lost their equilibrium and came to
the surface.
All lake trout and kokanee specimens were held (up to
a week) on ice until incubated; most of the chubs and perch were
frozen at -12°C until they could be incubated and tested.
These
samples and the lakes from which they were taken are indicated in
Table 1.
Smoked fish:
Samples of smoked and kippered fish products were
purchased from various processors along the coast of Oregon and
Southern Washington.
kets in Corvallis.
A few samples were collected from retail marIndividually wrapped and labeled items were
placed in plastic bags and held at 7°C until they could be tested.
20
The identity of these specimens and their sources are shown in Tables
l3 and lL1, respectively.
Aquatic sediments:
Three rivers draining the Oregon coastal
watershed were selected as sources of sediment specimens to be examined for the presence of Cl. botulinum type E.
These are the
Columbia on the north, the Alsea about midway between the northern
and southern borders, and the Umpqua, about 100 miles further south.
Each of the rivers flows from east to west and empties directly into
the Pacific Ocean.
Sediment samples were initially collected along the shore at the
waters edge and sometimes up to a depth of one foot at three different
locations along each watercourse:
to the rivers
(a) the salt water beach adjacent
mouth, (b) the tidewater zone of the river, and (c) a
freshwater zone well upstream from the tidewater.
Samples of 100 to
150 grams were taken by pushing a sterile open wide mouth, screw cap
bottle into the bottom mud or sand.
The bottles were capped and
stored at 5°C until the sediments could be incubated and tested.
Samples from areas of the beach and adjacent land were taken from some
of the areas in a similar manner.
Sediment samples were also obtained from eight lakes in the
Cascade mountain region of Central Oregon (several of which provided
the fish specimens) and from Tule Lake in Northern California.
These
were collected by the method just given for obtaining river samples.
A number of specimens were also taken with an Ekman dredge from depths
of 35 and 12 feet respectively in Elk and Kiamath lakes.
The contents
21
of the dredge were emptied into plastic bags or into a well rinsed
bucket from which a sterile screw capped bottle was filled.
specimens were held at
All
°C until they could be tested.
In addition sediment samples taken at the waters edge from three
man made reservoirs in Central Oregon were obtained.
These are the
Haystack reservoir, the Round Butte Dam reservoir, and the Pelton
Dam reservoir.
All three of them are of recent origin, having been
constructed within the past five years; they thus represent a some-
what different environmental situation from that of the eight natural
lakes.
The specimens were collected in sterile wide mouth screw
0
capped bottles and held at 5 C until tested.
Incubation Procedures
Heat is the ecological selective agent that has been most frequently employed in the isolation of Clostridium botulinum from
habitats that usually contain a preponderance of concomitant microorganisms.
Heat treatments of soil-enrichment cultures for 1 to 2
hours at 70°C and for 30 minutes to 1 hour at 8000 have in the past
been routine general methods; however, recent data indicate that many
botulinum strains, in particular type E, cannot survive such drastic
treatment.
Pedersen (L2) found that exposure of soil and mud speci-
mens to a temperature of 80°C for 5 minutes prevented isolation of
many type E strains.
Dolman (T1J4) has successfully isolated type E
organisms by heating food or soil enrichments to 60°C for 15 minutes.
Foster (26) found that heating his samples to 60°C for 30 minutes
22
reduced the number of his cultures with type E toxin.
He also found
that drying the samples before testing gave no benefit -- but he
found that incubating the enrichment cultures at 20°C for lL days
gave a slightly higher percentage of cultures with type E toxin than
incubation at 2°C (for 8 days), which had previously been considered
the optimum temperature for the elaboration of toxin by the organism.
In this investigation we have not used a heat activation technique in
any of our incubation procedures.
Crisley (10) has reviewed the enrichment media which have been
employed for cultivating the various types of Clostridium botulinum.
The most common enrichment media are those into which cooked meat is
incorporated.
Many employ 0.1 or 0.2 percent starch which has been
shown (5L) to adsorb unsaturated fatty acids in the recovery medium,
which may delay germination of the spores (28).
Others contain thio-.
glycollate and agar which help prevent the diffusion of oxygen into
germinating spore cultures which may be especially oxygen-sensitive.
In this investigation we have used beef heart infusion medium
with minced meat (BHI meat medium) to recover Clostridium botulinum
type E from most of our sediment samples.
Foster (27) has found the
BHI meat medium superior to six commonly used enrichments for spore
forming anaerobes.
In addition we have used TPG medium for incu-
bating fish, shellfish, and smoked fish products.
The U.S. Food and
Drug Administration in work with several (type E) laboratory strains
has found more complete and better sporulation in this medium than in
cooked-liver or cooked-meat medium (51).
23
Incubation techniques:
(a)
Clams and oysters:
testing in the following way:
Clams and oysters were prepared for
From three to ten individuals of a
single species were taken from the refrigerated plastic bag and the
shells removed with a single sterile knife or pair of scissors.
The
tissues of each individual in the group of shellfish were transferred
to a new clean plastic bag, and sufficient sterile TPG medium added
to cover the tissue.
Most of the air was squeezed out of the bag,
and the open end twisted and closed with a rubber band.
Bags con-
taining the prepared specimens were then placed in metal pans and
incubated for five days at 27°C - 28°C.
The plastic bag incubation
is illustrated in Figure 1.
(b)
Crabs:
For the crab specimens, the top shell was cracked
and the intestines and gills were transferred to a plastic bag and
incubated in TPG medium in the same manner.
Muscle tissue from each
crab, amounting to three grams, was picked with sterile forceps from
the joints between the legs and body and placed in a four ounce screw
capped bottle containing 50 ml. TPCT.
A similar sample to which three
grams of mud containing type E organisms was added was used as a control.
The caps of such bottles were placed on loosely and the bottles
were placed in Case-Anaero jars and evacuated to 27 inches of mercury
and flushed with an atmosphere of nitrogen containing L percent CO2.
The jars were evacuated and flushed at least three times.
method has been used by several investigators (51,1).
A similar
2L4
(c)
Fish:
In the case of the trout and other lake fish, the
plastic bag containing one or more fish was cut open and used to hold
the fish while the peritoneal cavity and gill region were opened and
the viscera and gills cut into fragments with a sterile pair of
scissors.
The fish (one large fish or up to three small fish) were
then transferred to a clean plastic bag and sufficient sterile TPG
The rest of the procedure was the same as
added to cover the tissue.
that described for the shellfish.
For the handling of both fish and shellfish specimens sterile
rubber gloves were worn.
After the preparation of each specimen, the
gloves were dipped in a 1.25 percent hypochlorite solution, dried
with paper towels, and the excess chlorine rinsed off in a tap water
bath.
They were then dried again with paper towels before handling
the next specimen.
(d)
Smoked fish:
The smoked (or kippered5) fish samples (which
were held on ice in plastic bags or refrigerated in toto in grocery
bags) were removed aseptically from their paper wrappers and placed
on a piece of sterile paper.
A large section of skin and some tissue
representing in all 5 - 10 grams was placed in each of two four ounce
bottles containing 50 ml. TPG.
The remaining skin and tissue was
placed in an empty, sterile, L ounce bottle, tightly capped, ref rig-
erated, and held for moisture and chloride determinations.
Kippering implies a semi-cooked product (120°F or higher for up
to two hours). Plain smoked fish (1cc smoked) do not reach this
temperature.
25
A control was used to determine whether micro-flora on the
smoked product would competitively outgrow, repress or elaborate enzymes which could destroy or in some way inactivate the type E toxin.
This was prepared by the addition of 3 grams of mud known to contain
Clostridium botuilnum type E to one of the two bottles containing the
fish specimen in TPG.
Use of a fish control is an extention of a
sediment control procedure described by Foster (26).
The organism is
considered absent from the specimen only if the control culture contains type E toxin and the specimen culture does not.
The loosely
capped sample and control bottles were evacuated in a Case jar and
incubated for five days at 28°C.
The five day incubation temperature
was adopted for TPG medium after previous experiments with pure and
mixed cultures showed a significant toxicity developed at 28°C during
this period.
A few of the earlier samples were incubated at this temperature
and duration after placing a 20 to
O gram sample in 150 ml. of TPG
in an 8 ounce screw cap bottle or plastic bag.
Some of these cultures
were incubated without special anaerobic precautions while others
were held in Case anaerobic jars.
(e)
Sediment samples:
Sediment samples were not heat shocked
and were incubated using a modified version of a method suggested by
Foster (26).
Instead of inoculating three, 10 ml. cultures of the
BUT meat medium with one gram samples of a soil sediment, a single
screw cap bottle containing 30 ml. of the medium was inoculated with
a three gram sample of the sediment.
A second control bottle of the
26
medium received three grams of the sample being tested plus three
grams of control mud known to contain the type E organism.
These
bottles were incubated anaerobically in Case jars for fourteen days
at 20°C, as recommended by Dr. Foster.
Where a comparison of sediments was made between the 881 meat
medium and TPG or where the TPG medium was used independently to
cultivate sediment samples, the procedure was the same except that
the mud was added to 50 ml, TPG and these bottles were incubated anaerobically for five days at 28°C.
Screening Specimens for Toxicity
Tissue Samples:
Fish, clams, oysters, crabs, and smoked fish
products were tested or screened, i.e., incubated suspensions were
tested for toxicity, in the following manner.
Eight ml. of the incu-
bated culture was transferred to a centrifuge tube and then centrifuged between 2500 and
20 minutes.
000 revolutions per minute (rpm) for 15 to
In some cases in which the suspension was quite clear
centrifugation was omitted.
An additional LQ ml. of the suspension
which had been incubated in plastic bags was removed to sterile
bottles.
All bottles containing incubated suspensions were frozen
at -12°C.
(Additional aliquots of the incubated suspensions for
transfers to TPG (at a level of 10 percent), rescreening, or antitoxin procedures, were withdrawn, after thawing, from the frozen suspension bottles.)
A portion of the supernatant fluid was removed to
a sterile vaccine vial and the pH adjusted to 6.2 with N/l or N/b
HC1
27
(depending on the sample).
The pH was measured with pH paper.
NaOH was used when the pH was overshot.
N/i
One-tenth ml. of a 10 per-
cent sterile trypsin solution was added per ml. of culture supernatant
to give a solution containing approximately 1 percent trypsin.
The
specimen was mixed and held at 37°C for L5 minutes. Cultures of fish,
clams, oysters, and the first several crab specimens were diluted 1:5
with gel- phosphate buffer at p1-i 6.2, and 0.5 ml. was injected intra-
peritoneally in two Swiss-Webster strain white mice, which were then
observed at intervals over a four day period.
The later clams and
all the smoked fish, for which moisture and chloride tests were run,
were treated in the same manner except that the trypsinized. specimens
were diluted 1:1 with gel- phosphate.
Consideration of the dilution
in regard to non-botulinal deaths is treated in the results and discussion section.
Sediment Samples:
The sediment samples run in all comparison
tests were tested for toxicity by centrifuging, diluting 2 ml. of the
supernatant with 2 ml. of gel- phosphate at pH 6.2 and injecting 0.5
ml. into each of two mice.
Where trypsinization was carried out, the
supernatarit fluid was adjusted to pH 6.2 with N/l HC1, sterile tryp-
sin added to a concentration of 1 percent, and the mixture held at
37°C for 45 minutes.
It was then diluted with gel- phosphate at
pH 6.2 and 0.5 ml. injected intraperitoneally in two mice.
A modi-
fication was used on all sediment specimens for which controls were
run.
Two ml. of the surface fluid from the sample or control was
withdrawn from the incubation bottle, placed in a sterile injection
28
bottle and diluted with 2 ml. of gel-phosphate at pH 6.2.
zation was not employed.
Trypsini-
Two mice were each injected intraperito-
neally with 0.5 ml. of the diluted specimen and one mouse was simi
larly injected with the diluted control suspension.
observed over a four day period.
The mice were
The centrifugation was eliminated
due to the increased time required for screening the controls in addi-.
tion to the sediment samples.
The effect of this omission is con-
sidered in the results and discussion section.
In all procedures the undilu Led and diluted suspensions in the
injection bottles were kept refrigerated at 5°C when not in use.
dilutions were made with 5°C buffer.
The injection bottles were
capped with rubber hoods after dilution.
out aseptically.
trays,
All
All procedures were carried
The prepared injection bottles, syringes, pij
. were autoclaved at the end of the run.
Unused incubated
suspensions were distributed in gallon paint cans and autoclaved along
with cultures which were being discarded.
of Toxic Specimens with Antitoxin:
Typjn
Demonstration of Botulinum
Toxin
Antitoxin is a serum antibody produced by immunization of animals
with small amounts of a toxin or a detoxified preparation of the
toxin called a toxoid.
A specific antitoxin can combine
vivo or
vitro with its homologous toxin and in so doing neutralize the
toxic character of the toxin.
Specific antitoxins can be prepared
against the toxins produced by Clostridiuxn botulinum types A, B,
29
0, E, and F.
Each type of antitoxin reacts specifically with its
homologous toxin.
Type Cproduces a toxin whose antitoxin neutra-
lizes the toxin produced by type C,B as well as its homolog.
Specific
neutralization may be demonstrated by in vitro mixing of toxin and
antitoxin preparations and injection of the suspension into a mouse
(or other test animal); or by injection of the antitoxin preparation
into the mouse, followed after a period of time, by injection of the
homologous toxin into the same mouse.
In both cases, if there is
enough antibody present to neutralize the toxin the mice will remain
alive.
The latter method is preferred because it requires less anti-
An International Unit for
toxin and permits dilutions of the toxin.
relating the strength of the botulism antitoxin to the strength of
In terms of MLD's per unit
its homologous toxin has been established.
or anti - MLD's the unit for types A, B, C, and 0 antitoxin is con-
sidered to be the smallest quantity of antitoxin which will protect
a mouse given 10,000 MLD of toxin and, for type E antitoxin the
smallest quantity that will protect against 1,000 NLD of toxin (32).
In this investigation an aseptic technique was used to withdraw
an aliquot from the stock antitoxin preparation, which was diluted
to one unit per ml. with gel- phosphate at pH 6.2 and, 0.5 ml. (or
0.5 unit) was injected intraperitoneally into each of four mice for
each toxic specimen tested.
A minimum of 20 to 30 minutes was allowed
to pass before the mice were challenged.
As a standard routine only
type E protected mice and unprotected mice were originally challenged.
One-half ml. of 1:5 and 1:50 dilutions of the toxic specimen was
30
injected intraperitoneally into two protected and two unprotected
mice for each dilution.
If both protected and unprotected mice died
the antitoxin test was rerun using type A and B antitoxin protected
mice and additional unprotected mice.
In the original screening injected mice which died in more than
1 hour but less than 2L and showed progressive evidence of respira-.
tory paralysis before death were presumptively considered to have
died from botulism.
These specimens were tested for the presence of
botulinum toxin by performing antitoxin tests as quickly as possible
(several days) on the incubated suspensions which had been held at
0
.
-12 C.
The incubated suspension was thawed, an aliquot was centri.-
fuged, 2 ml. of the supernatant was transferred to a sterile dilution
bottle and the pH was adjusted to 6.2 with N/i Rd. After adding
sufficient sterile trypsin to give a 1 percent solution, the specimen
was mixed and held at 37°C for L5 minutes.
It was then diluted 1:5
with gel- phosphate; this was mixed and 2 ml. was transferred to a
second bottle which was diluted up to 1:50 using the gel- phosphate
menstrum.
The diluted suspensions were refrigerated until needed and
injected as indicated.
Before dilution or trypsinization, the supernatants (of a centrifuged aliquot) of toxic clam or lakefish cultures were filtered.
This was done by using either a millipore 'Swinney' syringe filter
with a 0.22 micron cellulose acetate pad, or by using a 2 ml. Boerner
centrifuge filter with a Seitz asbestos 'germicidal' filter pad.
Filtration of the supernatant was not employed in the case of smoked
31
fish or for sediment samples.
For samples in which the screening test was toxic (positive) but
for which toxicity could not be demonstrated (loss of toxicity) in
the antitoxin test, an aliquot of the incubated suspension was transferred to 20 ml. TPG in a screw capped tube and incubated anaero-
bically for five days at 28°C.
A similar trpe of transfer was made
for those samples in which the injected mice died after 24 hours in
the initial screening test.
All transfer cultures were rescreened
for toxicity before antitoxin procedures were employed by centrifugin
preparing a 1:1 gel- phosphate dilution of the supernatant, and injecting 0.5 ml. intraperitoneally in two mice.
Nontoxic (negative)
or questionably toxic transfers were not considered further.
The designation of a sample as questionably toxic is based on
the time of death and symptoms of the injected mice.
Mice usually
die within 20 minutes after the intraperitoneal injection of a suspension containing a high concentration of a proteiriaceous material.
Such deaths were considered nonspecific in that mice injected with
the most concentrated botulinum toxin rarely die before 45 minutes.
In addition, samples were considered questionable when one of the
two injected mice died, or when both mice died late in the observation period (between 40 and 96 hours).
An unsterile suspension in-
jected into the peritoneum of a mouse may initiate a bacterial infec-
tion (peritonitis); the mouse may become inactive and will generally
die between 40 and 96 hours.
32
Isolation of Clostridium Botulinum Cultures from Toxic Specimens
Meyer (:38) has pointed out that the percentage of toxic cultures
in soil surveys may be as high as L0 to 50 percent, and typing or isolation may be achieved in only 5 percent.
Ultimate isolation of
Clostridium botulinum has been described as "an art in which the judicious use of enrichment cultures, and plating from toxic cultures
are carried out, frequently
without assurance that success in any of
the steps will subsequently be rewarded" (10).
Crisley (10) has reviewed a number of the plating methods which
have been used successfully for the isolation of Clostridium botulinum
in pure culture.
In this investigation we have used a slightly modi-
fied procedure which was developed by the U.S. Food and Drug Administration (51).
To a 2 ml. aliquot from the toxic culture (transferred
to a sterile tube) 2.2 ml. of 95 percent ethyl alcohol was added and
the suspension allowed to stand at room temperature for a minimum of
1 hour with frequent mixing.
Serial 1:10 dilutions were made (from
an initial 1:2 to at least 1:2000) using 0.85 percent sterile saline.
A loop (approximately 0.01 ml.) of each dilution mixture was streaked
over liver-veal-egg agar plates and a loop of the 1:20 or 1:200 dilution streaked over a blood agar plate.
Thoroughly dry plates were
employed, as recommended by Dolman (lLJ), to limit growth-spreading
and film formation which is aggravated by condensation in the an-
aerobic jcs.
he plates were incubated anaerobically in the Case
jars by evacuating and flushing the jars at least three times with
:3:3
nitrogen gas containing a
4
percent CO2, and holding them for 48
0
hours at 28 C.
Following incubation the plates were removed from the jars and
immediately examined under a 3tereo-microscope.
Colonies were Gram
stained and those composed of gram-positive or gram-variable spore
forming rods were cut out and transferred to screw capped tubes con-
taming 20 ml. of TPG medium.
With their caps loose, the tubes were
incubated anaerobically in Case jars for four or five days at 28°C.
The tubes were screened for toxicity using a method similar to that
employed for screening sediment samples (cultures were not trypsinized).
The toxic cultures were then typed with antitoxin using the
procedure which has been outlined.
Positive identification of a particular type of botulinal toxin
in the antitoxin test confirmed the presence of that botulinal type
organism in the original sample.
Additional plating, isolation,
screening, and antitoxin procedures were necessary in order to guarantee the isolation of a bacteriologically pure culture.
or alcohol fermentation tests were run.
No sugar
Consideration of the pro-
blems involved in picking 'typical' type E colonies is treated in
the results and discussion section.
Determination of the Sodium Chloride Content in Smoked Fish
The procedure was taken from section 18.008 of Methods of
Analysis A.0.A.C. (2) and is the official method.
From the smoked fish specimens refrigerated in sterile 4 ounce
34
screw capped bottles a sample between 2.5 to 13.0 grams was taken and
weighed (accurate to four decimal places) into a 250 ml. Erlenmeyer
flask.
Exactly 30 ml. of standard N/b
silver nitrate (AgNO3) was
added and the fish was broken up with a glass rod, which was with.-
drawn carefully from the beaker so that no fish particles were withdrawn.
The rod was rinsed with the subsequent addition of 20 ml. of
69 to 71 percent nitric acid.
The mixture was swirled several times
and the flask placed on a stove burner; the solution was allowed to
boll gently for 15 minutes until all the solids except the grey silver
chloride precipitate were dissolved.
The solution was allowed to
cool and 50 ml. of deionized water and 5 ml. of the ferric ammonium
sulfate indicator were added.
dard N/b
The suspension was titrated with stan-
ammonium thiocyanate (NH4SCN) solution until a light brown-
orange color was obtained.
A measure of the variation in replicate
analyses run on different portions of the same specimen is given in
the results and discussion section.
The f-b?. wing formula was used to calculate the percent of NaCl
in the sample:
millieguivalents of NaCl in sample X 58.45 gram
Sample Weight X 103
(boo) =
equals percent NaC1 in sample
The number of milliequivalents (meq.) of NaC1 in the sample equals
the meq. of AgNO3 which reacted with the NaCl.
This is determined
by subtracting the meq. of NH4SCN used in the titration from the
meq. of AgNO3 added originally to the fish suspension.
35
Determination of the Moisture Content of Smoked Fish
The procedure was taken from section 23.003 of Methods of
Analysis A.O.A.C. (2).
From the smoked fish specimens refrigerated in sterile Li
ounce
screw capped bottles a sample between 3.0 to 5.0 grams was taken
and weighed (accurate to four decimal places) into an aluminum dish
50 millimeters in diameter and 20 millimeters deep.
A tight fitting
aluminum lid was fitted over the dish and the sample was placed in
an oven (no mechanical convection) for 16 to 18 hours at 1000 to
102°C.
The dish was cooled in a desicator and weighed.
The weight of the aluminum dish plus the fish sample minus the
weight of the aluminum dish plus the dried fish sample equals the
weight of the moisture in the fish sample.
The weight of the mois-
ture in the sample divided by the weight of the moist fish sample
times 100 equals the percent moisture in the sample.
36
RESULTS AND DISCUSSION
Introduction:
Physical Aspects in Demonstrating Clostridium
Botulinum
There is no known quick simple procedure for demonstrating the
presence of Clostridium botulinum in a specimen.
Procedures that
have been tried or are in the process of being evaluated include
hemagglutination, the opsoriic index, the hypersensitive animal, selec-
tive media, and fluorescent antibody techniques; however, no simple
Workers are tied to a time
succssful procedure has been achieved.
honored, but slower than time, mouse bioassay procedure (L4).
In order to demonstrate the presence of any botulinal type organi.sm in a specimen it is first necessary to stimulate the growth
of the organism and consequent production of toxin by incubation of
the suspected specimen in an enrichment medium.
The success of the
enrichment medium depends on its ability to activate, or break the
dormancy of the botulinal spores, which may contaminate the specimen,
and stimulate germination of the active spores as well as outgrowth
and subsequent vegetative multiplication.
The concept of 'dormancy', as applied to the bacterial endospore
implies the apparent metabolic inactivity characteristic of the spore
stage, as compared to the vegetative cell.
The concept of 'breaking
the dormancy' by 'activating' the spore describes the fact that spore
suspensions respond poorly or not at all to germination agents under
conditions that permit rapid germination of aged or heat-activated
3?
spores.
Activation does not occur in all spores, and the need for
this process depends on the spore itself, its history, and on the
circumstances inducing its transformation into a vegetative cell.
Subsequent irreversible germination of the activated spore results
in a process of degradation of spore macromolecules, resulting in
the excretion of many typical spore substances into the medium.
The
germinated spore then undergoes outgrowth, which is a process of syn-
thesis of new macromolecules, new classes of proteins, and new struc-
tures which result in the emergence of a new vegetative cell (3).
An Analysis and Approach: Consideration of the Ecologr of Clostridium
Botulinum Type E in the Northwest
Since Dolman's isolation of Clostridium botulinum type E from
the soil in l9L9 numerous procedures have been used to cultivate and
isolate the organism; however, no comparative evaluation of methods
has been reported.
Investigators have tended to use media and condi-
tions which they have found to work without suggesting that they are
the best for all circumstances or even for the purpose for which they
have chiefly used them.
Considering this situation, in this investi-
gation, procedures were employed which, it was hoped, would incorpor-
ate some of the best techniques for demonstrating the presence of
botulinum in a specimen.
Even the methods which were adopted, how
ever, were not considered inflexible, and, where it was felt that a
procedure could be improved or simplified, such improvements were
tested.
In previous work done on this contract major emphasis was placed
on investigating possible association between the type E botulinum
The gills and viscera
organism and Northwest river and ocean fish.
were aseptically removed from fresh fish, as they were received at
canning and processing points throughout the Northwest, and placed
in plastic bags.
frozen.
The samples were held on ice until they could be
It was found that the incidence of type E botulinum in these
viscera samples increased as the source of the fish approached the
Of
mouth of the Columbia river from either the north or the south.
the samples collected, most were from salmon which migrate from
freshwater streams into the ocean and then return to spawn.
Since
the incidence was higher in fish taken from the Columbia river as
compared to fish taken from adjacent ocean areas it became of interest
to determine if the river fish were being contaminated from the
bottom sediment.
As means were not available for testing the pre-
valence of the organism in river bottom sediment, indirect methods
were employed for testing areas which might suggest the organism was
in the river sediment and support hypotheses as to how it gets there.
A definitive ecology has not been determined for Clostridium
botulinum type E.
Although the organism has been found widely dis-
persed in marine sediments on the shores in certain parts of the
world, aside from Dolman and Johannsen's isolation of the organism
from garden soil and potato peelings respectively, there is little
or no evidence for a terrestial distribution of the organism or to
indicate that it is a common soil contaminant.
The occurrence of
39
the organism was virtually unknown in the United States until several
years ago when cases of type E botulinum poisoning began occurring
from Great Lakes fish.
This indicated that the organism might be of
fresh water as well as of marine origin.
The supposition that the type E organism could be of telluric
origin instituted a survey of lakefish and of the popular inland
fishing lakes, reservoirs and river sediments in Central Oregon.
These sites, as well as Kiamath Lake in Southern Oregon and Tule Lake
in Northern California are in the migratory bird flyway and as such
might provide points for disseminating or acquiring the organism.
Clostridium botulinum type C is known to occur in sediments of some
waterfowl refuges and at times causes serious outbreaks of limberneck
in ducks.
The possibility of the presence of other botulinal types,
in particular type E, was considered of theoretical interest.
In
addition to the general ecological information a survey of these sedi-
ments would provide, it appeared to have some practical importance in
that lake trout fishermen sometimes preserve these fish by home
smoking methods.
It was also hoped that a search for the organism along the shoreline of the saltwater beach near the mouth, tidewater, and freshwater
areas of ocean flowing rivers might provide direct evidence for the
possible source of these organisms.
The demonstration of the organism
in soil adjacent to the shore of the rivers, but not in direct or
intermittent contact with any body of water, would lend support to
the hypothesis that the spores are not primarily aquatic in origin
LO
but that they are carried to the bottom and shore of rivers and into
coastal areas through the erosion and water run off of land masses.
In view of the fact that no known survey of shellfish for
botulinum, other than crabs, has been reported in the literature,
and in view of the large number and variety of crabs, clams and oys-
ters that are distributed from the Oregon and Washington coast to
many points within the United States, it was considered important to
determine the incidence of the organism in these shellfish, even
though no known botulinal outbreaks have been attributed to them.
Ultimately and perhaps of major interest the incidence of the
organism in smoked fish products from Oregon and Washington processors was assayed.
Samples which were sufficiently contaminated
to produce toxic specimens on assay might he assumed to reflect con-
tamination present in the fish at collection or that introduced from
curring brines or in handling during processing and distributing
processes.
Moisture and chloride determinations on some of these
products were run to determine the range of these values in the market products.
The moisture and chloride content of the fish reflect
the conditions under which they have been processed and probably their
ability to resist the growth of type E organisms.
However, the con-
centrations of each which are incompatible with the growth of
Clostridium botuilnum type E are not clearly understood.
The results of this survey in terms of the identity and source
of the toxigenic samples which have been successfully typed with
botulinum antitoxins are presented in Tables 16 and 17.
Figure 5
gives the approximate geographical distribution of these samples.
Development of Survey Techniques and Results
Development of the technique for testing fish:
When the collec-
tion of lake fish was first contemplated in mid- July of 1965 no
clear idea had been formulated as to whether the whole fish or merely
viscera would be collected.
Collection of viscera necessitates the
use of an aseptic technique requiring sterile knives, sterile paper,
and sterile gloves.
When collection can be made at canneries during
the time the fish are brought in by the fishing trawlers the operation is not too difficult and provisions for resterilizing knives and
gloves can be made.
in a boat.
The procedure is not easily carried out, however,
Although we were primarily interested in the flora of the
gills and gut of the fish, because of the field difficulties, the
whole fish was collected.
The Oregon State Game Commission kindly permitted us to accompany
them on several of their surveys of the lake fish in the Cascade mountairis-.
They would set up gill nets in the lakes which generally
netted more fish than they required.
The excess were removed from
the nets by hand over the side of the boat and placed into individual
plastic bags.
In this manner brook and rainbow trout and kokanee
salmon were collected from the Deschutes National Forest lakes in midJuly and in mid- October and blue and tui chubs and perch were taken
from Klamath lake in the early part of August and October.
The col-
lection of less than fifty fish per trip facilitated their quick
14'2
testing.
Freezing and lengthy storage of a sample introduces an unknown
factor and may result in the destruction of some of the microorganism
or a tendency for one of the species present to dominate the popula-
This factor was eliminated except for a few of the Klamath lake
tion.
chubs.
Freezing and storage problems are almost unavoidable when
large numbers of viscera samples are collected.
The sampling of raw fish has several inherent limitations.
The
surface and viscera of all the fish tested were observed to contain
highly proteolytic organisms.
shrimp samples.
The same results were observed for
Using the plastic bag proäedure for incubating
samples, which is only a semi-anaerobic procedure, viscera samples
were extensively digested and shrimp were completely so and appeared
as a flocculent sediment.
However when the samples were incubated
under complete anaerobiosis under a nitrogen atmosphere and in Case
jars, extensive proteolysis was nearly eliminated.
Shrimp, for ex-
ample, remained firm and retained their characteristic appearance.
in general, previous incubation procedure for viscera samples
consisted of adding a volume of T
weight of the sample.
equivalent to about l/LI the
After incubation, the highly viscous organic
samples, removed from the plastic bags, required centrifugation at
3000 rpm for at least 25 minutes.
In the most favorable circumstances
the result was a centrifuge tube containing three fractions:
a
compact organic lower layer, a fairly clear to extremely turbid middle
layer, and a 1/2 to 3/LI- inch surface layer of fatty products and
L3
light apparently proteinatious material.
From such a tube it was
seldom that more than a millilitr of the central layer could be removed.
Filtration was difficult and time consuming and was accom-
plished with Millipore 'Swinriey' syringe filters or by transferring
the supernatant to the 2 ml. cup of a Boerner asbestos disc centrifuge
filter.
After this step many times less than 1/2 ml. of filtrate was ob-
tamed which was not always sterile.
Filtration and dilution of the
sample at no less than 1:5 was considered necessary because of the
possible presence in some unsterile filtrates of a variety of organisms able to produce peritonitis and to nonspecific toxic materials
in fish specimens.
in addition, two mice were injected at each of
two serial 1:10 dilutions due to uncertainties concerning the signi-
ficance of mortality occurring beyond 2L hours, especially when only
one di1uton was used.
It was observed in early experiments that it takes longer f or a
mouse to die which has been injected with a highly diluted filtrate
from a pure type E culture than for an animal receiving a more concentrated one and that though the symptoms of mice dying from type E
toxin all seem to be consistent, those dying from more dilute solutions have less pronounced symptoms which closely resemble deaths
produced by peritonitis.
Because the level of the toxin present in
incubated suspensions may be quite low, recovery is best obtained using
the lowest dilution which may be employed without producing death
from causes other than botulism.
Although type E spores may be present in a sample, toxin may not
be evident subsequent to incubation.
Foster (27), in reviewing
Johannsen's work (in Swedish), wrote that Johannsen suggested that
competing organisms may influence type E in four ways:
(i) to pre-
vent growth of type E, (2) to prevent toxin formation, (3) to destroy
toxin as it is produced, and (L) to bring about mutation of type E to
a non-toxic form.
Toxicity could not be demonstrated in split 50 gm.
shrimp samples in 150 ml. TPG, seeded with 920 type E spores and in-
cubated in plastic bags and in Case jars for five days at 28°C in our
laboratory.
Attempts to demonstrate toxicity by transferring these
incubated suspensions to fresh TPG followed by reincubation and
screening were not successful.
After three transfers the sample
originally incubated in the Case jar was only questionably toxic and
toxicity was never demonstrated for material which had been originally
incubated in the plastic bags.
Evidence such as this, and results
obtained from similarly treated sediment samples formed an impirical
basis for a view that samples that show high levels of toxicity initially most probably contain an appreciable amount of type E contamination.
In an attempt to simplify the procedure of injecting two dilutions of the incubated suspensions per sample and to eliminate the
sterility test, fish samples were incubated with an excess of TPG
and a comparison was made between 58 fish specimens using a 1:5 dilu-
tion of unfiltered centrifuge supernatants versus injection of dilutions of 1:5 and 1:50 of the filtered supernatent.
The results did not
show sufficient advantage in filtration or injecting two dilutions to
45
justify the extra time and effort involved.
In addition, by using
an excess of TFG the suspensions were little different from bacterial
cultures with heavy growth and lent themselves readily to centrifugation procedures.
Elimination of the filtration step allowed us to
increase the number of samples which could be screened at any one
time from a maximum of 20 to between 30 and 70.
Results of the Lake Fish Survey:
A summary of the screening
results for lake fish using the method previously described is given
The only fish which could be obtained from
for each lake in Table 1.
Odell lake were ten small, four inch kokanee salmon.
The toxicity
results for this lake which these fish represent cannot be considered
to be of much significance.
The samples of small frogs were taken
from the only marsh that any type of samples were collected from, at
the edge of Davis lake in quite warm water.
They were nontoxic and
should represent to some extent the land they were taken from since
sediment and reeds were also scooped up with them and the whole thing
incubated.
The level of type E contamination is quite high in fish
taken from Elk lake and the rotenone poisoned fish from South Twin
lake.
These results indicate that lake fish, as they are pulled
from the lake, may be highly contaminated with type F botulinum organisms and as such represent a special hazard to those sportsmen who
smoke and consume them.
Three types of scrap fish and perch were
collected from Klamath lake.
The lake has an average depth of twelve
feet and is considered to be too warm for trout.
Incubated specimens
of these varieties were not toxic enough to type and the toxicity
L6
could not be increased on subculture.
The clams, small crabs,
Procedure and Results for Shellfish:
and oysters which were collected in the last part of August in 1965
were arbitrarily incubated by the same procedure and with similar
time and temperature as for the lake fish.
The best medium and tern.-
perature for incubation along with the length of incubation which
produces the highest titer toxin is not known0
Luckily, the method
worked and the matter was not pursued further.
Type E botulinum has been found (typed) in oysters from each of
the four collection sites.
The beds from which they were taken lie
adjacent to the canneries from which they were collected.
all situated in the near shore area of bays:
These are
samples from Newport,
Oregon were taken from Yaquina Bay; samples from Tillamook and Bay
City, Oregon were taken from Tillamook Bay; and samples from Nahcotta,
Washington were taken from Willapa Bay.
The results are indicated in
Table 2.
Since the oysters had been scrupulously washed in the canneries
and were not subject to recontamination on incubation it seems likely
that the type E organisms are present in the gut of the oysters.
Clams have been typed from 7 of 8 beach spots along the Oregon
and Washington coast.
Interestingly, no samples containing type E
were found in any of a large number of clams obtained from beaches at
Seaside and Garibaldi, which are south of but near to the Columbia
river; however, the organism has been found in oysters taken from
beds at Bay City, just four miles south of Garibaldi.
None of the
i47
clams taken from Garibaldi which represent four of the six types
(except razor and softshell) indicated iii Table 3 were toxic.
Of the
71 samples of razor clams collected from Seaside (many of which were
heavily covered with sand) only 12 percent were tox-i.c and, of these
one contained type A toxin, another one typed B.
Further south at
Yaquina Bay at Newport. at the mouth of the Yaquiria river 13 out of 13
incubated samples of cockle, littheneck, and softshell clams contained
type E toxin.
From the beath south of Yachats 3 of 6 samples of
cockle and horseneck clams were type E.
At Florence at the mouth of
the Siuslaw river 3 of 3 toxic samples of softshell and horseneck
clams were typed E.
Two of L
samples of' softshell clams at Reedsport
and 3 of L razor and softshell clams at Winchester Bay gave cultures
containing F toxin.
river.
These sites are both at the mouth of the [Jmpqua
Winchester Bay is the most southern point from which clams
were collected.
Except for the clam samples obtained at Seaside all mud and sand
had been washed free of the shell and exposed areas; many of the
specimens remained alive at least a week after collection arid the
majority were free of cuts or' punctures.
It is felt that in most of
the samples containing type E the organism was present in the gut of
the clam.
Except for several small specimens, the crab samples are seen
to be quite toxic as noted in Table 2.
Ten of 12 crab viscera col-
lected from Chinook, Washington were toxIc and of these 3 were typed
as Cl. botulinum F and 1 as type B.
Four viscera of the 6 crabs
L8
obtained at Winchester Bay were toxic.
One muscle tissue culture was
also toxic which was taken from a crab whose viscera culture was not
toxic.
Several of these toxic viscera samples could not be typed
after storage of the suspensions at minus 12°C for two and one-half
days.
This rapid loss of toxicity might indicate that highly active
competitive organisms were present.
For that one crab, the finding
of a high level of toxicity in the muscle tissue but not in the vis..
cera is hard to understand.
Since the top shell was incubated along
with all the viscera samples it is possible that the type E organisms
were present on the outer shell.
The organisms may or may not be in
the viscera.
Comparison of methods for detecting Cl. botulinum in mud specimens:
Sediment or mud samples in previous work were collected with
a gravity coring device consisting of a foot long bronze pipe with
an inner diameter of two inches and a sharpened end.
A sterile plas-
tic tube was inserted inside which was held in place by a core catcher
with fine leaves of sheet bronze that allowed a sediment sample to be
forced into the plastic liner but held it from coming out.
The sam-
pler, which was designed for collection of underwater sediment, was
too bulky for use in collecting shoreline samples.
The plastic core
liners themselves were used, for a time, by stabbing the end of the
tube into various parts of the shore.
This device had drawbacks in
that there were only enough tubes for twenty core samples and, addi-
tional samples could not be col]ted until these were released; in
addition large samples were taken from small areas and, it was
Li9
impossible to bacteriologically correlate supposedly identical samples
from closely related portions of sand from the core.
This method was
used to collect some of the samples from Odell lake in the Deschutes
National Forest but was quickly abandoned.
It was felt that a more
satisfactory survey of a large area could be made by using small
sterile bottles to collect the sediment.
This procedure, which has
been used throughout the survey, has been described in the section of
this report concerning the methods and materials.
Although the method of collection had been worked into a simple
routine, the sediments from the inland lakes (Table L) and rivers
(Table 5) in the Deschutes National Forest area and those from
Kiamath lake in Oregon and Tule lake, California, which had all been
collected between July and mid- October, sat in the cold room for two
months until a method for incubation and testing could be devised.
Several procedures including the heating of sediment samples or
their treatment with alcohol, incubation temperatures varying from
0
0
12 C to 35 C, trypsinization procedures, and various media, such as
Cardella's medium, Dolman's GPI3I medium, Johannsen's beef heart infu.-
sion medium, Kanzawa's fish medium, Pederson's medium, Reinforced
Clostridium medium, and TPG medium have been proposed for the demonstration of type E botulinum in soil samples.
Initial sediment experiments (method considered above along with
the shrimp experiment) in which natural sediments were seeded with
type E showed that an incubation temperature of 12°C for a seventeen
day period was inferior to incubation temperatures of 20°C and 28°C
50
using TPG medium.
Comparison of incubation temperatures of 20°C and 28°C for
various periods in TPG medium:
A second experiment was initiated to
determine which of the above temperatures gave the highest toxicity
for a given incubation time.
Fourteen samples of mud taken from
three widely separated sites near the shore of Elk lake in the
Deschutes National Forest area, and one sample taken at a depth of
35 feet, were selected for this experiment because preliminary work
had shown Clostridium botulinum type E was present.
After thoroughly
mixing each mud sample (which had been collected in sterile bottles)
two 3 gram portions were weighed out and each portion inoculated into
60 ml. of TPG medium in a
ounce wide mouth screw cap bottle.
One
bottle of each pair was incubated at 20°C in a Case anaerobic jar,
the other in a similar jar at 28°C.
Samples of 5 ml. were withdrawn
aseptically from each culture bottle at the intervals required for
testing, and the bottles returned to the anaerobic jar.
Samples were prepared for toxicity testing by centrifuging at
2500 rpm for twenty minutes.
The supernatant fluid was then removed
and diluted with an equal volume of gel- phosphate at pH 6.2; 0.5 ml.
was injected into each of two mice.
Where trypsinization was carried
out, the supernatant fluid was adjusted to pH 6.2 with N/i HC1,
sterile trypsin added to a concentration of 1 percent, and the mixture held at 37°C for L5 minutes.
It was then diluted and injected
as above.
The results obtained in this experiment are shown in Table 6.
51
Although the number of cultures examined was small and the variation
in toxicities between the two temperatures is not great enough to be
highly significant, the 28°C incubation is seen to be equal or superior to the 20°C incubation in each testing interval.
The figures
suggested that incubation at 28°C for five or seven days and testing
the cultures without trypsinization resulted in the greatest number
of toxic cultures.
Neither a lower temperature, a long incubation
period, trypsinization, or the preparation of subcultures offered any
advantage over the above simple procedure.
Comparison of TPG medium with beef heart infusion plus meat:
Shortly after we had finished the TPG experiment and had begun using
the suggested method to test the inland lake, and river, and several
of the coastal sediments, Dr. E.M. Foster of the University of
Wisconsin kindly made available to us the data assembled by his group
on this problem (27).
In summary, the results indicated that beef
heart infusion medium with minced meat detected a somewhat greater
number of toxic mud specimens than did TPCI or other media that were
included in the comparison.
In view of this information it was de-
cided that the next fifty specimens examined would be tested in both
TPG medium incubated at 28°C for five days and the BHI medium incu-
bated (using a slight modification of Fosters method) at 20°C for
fourteen days and, the proportion of toxic cultures obtained by the
two methods compared.
The procedure used in the comparison and the consideration of
the use of a control has been described in the section on methods and
52
materials.
A summary of the results obtained with the fifty mud
specimens tested by both methods is presented in Table 7.
These
specimens were collected from nine different lakes and rivers in the
Deschutes National Forest in Central Oregon, from four shore sites
and seven dredge sites across the center of Kiamath lake, and from
the perimeter of Tule lake in Northern California.
It is probable
that they show considerable variation in mineral and organic composition and microflora.
Differences in the medium and methods are reflected in the resuits.
Twenty-five of the fifty specimens yielded toxic cultures in
the BHI test, without trypsinization, while the parallel tests with
TPG medium produced nineteen toxic cultures out of fifty.
Six speci-
mens yielded toxic cultures in the beef heart infusion but not in
TPG, while there were no specimens which developed toxicity in TPG
and not in BHI.
The data also indicate that trypsinization of the
BHI cultures reduced the number of toxic specimens detected.
In the
case of the cultures in TPG medium, some specimens were toxic only
after trypsinization but an approximately equal number lost toxicity
after the enzyme treatment.
The reason why the toxin molecule from
sediment cultures is more delicate and is destroyed by trypsinization,
whereas the molecule from fish cultures is generally increased in
toxicity by 100 fold is not readily apparent.
in the fifty tests made by the TPG method there were five in
which the control cultures failed to develop toxicity.
In these
cases the negative or questionable results obtained with the test
53
specimens alone would not be consred to show significant inhibition by competitive organisms in the test mud as all five of these
specimens gave control cultures which were toxic in the BHI method.
Only one control was nontoxic with this method, while the corresponding sediment test on the same specimen in the TPG medium was
toxic.
The results of this comparison of the two methods for detecting
toxicity in incubated mud specimens supported the findings of Foster's
group by further suggesting the superiority of the BRI medium with
minced meat, and incubation at 20°C for fourteen days.
Thus, this
work provided the basis for the testing of sediments from the three
areas of three ocean flowing rivers and the artificial reservoir sediments.
Results of survey of inland
$iments
lakes
reservoir,
jver, and marine
A summary of the results of the toxicity testing of the
inland lake sediments, and the rivers associated with them and the
results for the artificial reservoirs, may be found in Tables
5, respectively.
samples.
aud
No attempt was made to type all of the toxic
Those which have been typed were selected arbitrarily.
The
lakes in the Deschutes National Forest are the same as those from
which trout and kokanee salmon had been collected and are all in the
Cascade Mountains of Oregon which lie between 70 and 120 miles from
the coast (map distance).
Toxic cultures developed from sediment
specimens taken from all of these lakes and in some cases over half
of such cultures were toxic.
Five of five toxic cultures from Elk
5L.
lake contained type E toxin,
This lake lies approximately 95 miles
inland and more than 4,000 feet above sea level.
Although only a
small number of samples were taken from each of five points along
four of the fast flowing streams in this area, samples from three
of the five show toxicities of greater than 30 percent.
The toxin in
one sample, taken not far from the source of a branch of the Deschutes
river was found to be type E.
Several shore samples from Kiamath lake were toxic; however, the
bottom sediments were atoxic.
Although no samples from this lake
were typed, several of the shore samples initially exhibited toxicities and symptoms of death in mice which are typical of botulinum
toxins; however, after a period of holding at minus 12°C subcultures
of these suspensions would not yield toxic cultures.
Interestingly,
duplicate tests run on Tule lake sediments which ranged from quite
moist to very dry yielded only a few questionably toxic cultures.
Subcultures of these were all negative.
The screening results of these lake sediments seem to closely
agree with and support the toxicity results in Table 1 for the fish
from the same lakes.
The sediments in Elk lake found to contain
type E support the results of typing 16 of the 22 fish collected.
in addition a large sediment sample collected from this lake along
the shore just beneath the surface of the water has given 290 toxic
controls out of the last 29
controls run (using BHI meat medium);
this result attests to the abundance of type E organisms which may
occur in some areas of the lake and serve to contaminate fish and
55
other wildlife.
Conversely, the low incidence of toxicity in the
sediments from Kiamath lake agrees with the observation that only 6
of the 88 fish samples tested were toxic.
Incubated sediments from three man made reservoirs located about
65 miles north east of the Elk lake area were found to contain type
E toxin.
Haystack reservoir is fed by underground streams and is used
as a source of irrigation water.
Much of the shoreline consists of
a volcanic type shale and there are few places where it is fine enough
to push any amount of it into the mouths of our collecting bottles.
The samples were collected (in the last part of December, 1965) at a
depth of several inches below the water just past a foot-wide periphery of ice along the waters edge; and 80 percent of these samples
were quite toxic.
The toxicities were not so high for samples taken
along the shore at Round Butte Dam reservoir, which is fed by the
Deschutes and the Crooked rivers or from Pelton Dam reservoir which
is fed by the Metolus in addition to these.
The shores of these
latter reservoirs varied between sand and mud in consistency and
appeared to have much higher organic content than some parts of the
shale type shoreline found at the Haystack reservoir.
A higher con-
tent of organic matter would probably support a greater variety of
microorganisms.
Comparison of the saltwater. tidewater, and freshwater areas of
three Oregon rivers:
Table 8 presents the screening results on
marine sediment samples collected from three sites north of the mouth
of the Columbia and four sites to the south.
Although 2 of the 6
56
samples collected north of the river and 5 of the 19 collected further
south were toxigenic none of the cultures, even on subculture, were
ever toxic enough to be considered worth typing, i.e., produce toxins
which on injection will kill mice in less than 16 hours.
The survey work which has been accomplished along saltwater,
tidewater, and freshwater areas of the Columbia, Alsea, and Umpqua
rivers is presented in Tables 9, 10, and 11, respectively.
The
mouth of the Alsea river is about 100 miles south of the mouth of the
Columbia and, the Umpqua is L0 miles south of the Alsea.
The Alsea
is the shortest and is surrounded in its entire length by coastal
mountains which are part of the Siuslaw National Forest.
The Columbia
and Umpqua are straddled only in spots by mountain ranges.
It may be seen from the tables that beach sediments taken along
the shore at the waters edge at the saltwater mouths of each of these
rivers were not toxic, whereas sediments containing the type E organism were obtained at the waters edge from the beaches in the tide-
water areas, where the water contains only a trace of salinity and,
from freshwater shore areas of each of the rivers.
At least half of
the samples from the tidewater areas of each of the rivers were toxic.
In addition similar high toxicities may be observed for the freshwater
areas of the rivers.
In an attempt to determine if only samples taken along the shoreline at the waters edge were toxic, sediments from areas of the shore
not usually in contact with the water and from land further back
which was above the water level even during floods, were examined.
57
This was done on land adjacent to the previously sampled shoreline
in the freshwater area along the Columbia and the tidewater area of
the Alsea.
The land adjacent to the shore in the freshwater area along the
Columbia was predominantly sand and few of the samples were toxic;
however, a dry sand sample taken three yards up a sandy bank which
was five yards from the waters edge (considered eight yards in all)
No type E sediment cul-
was shown to contain type A botulinum toxin.
tures were found from the land adjacent to the river.
In the Alsea tidewater area the picture is quite different.
Half or more of all the sediment samples taken from the land adjacent
to the shoreline from the waters edge up to forty-five yards back
were toxic.
Sediment samples taken at 3_L yards, 12-15 yards, and
at 35 and L5 yards from the waters edge were typed.
The sampling area
consisted of a strip of flat land about fifty yards in width extending
several hundred yards between the river and State Highway 3L (only an
area about 100 yards in length was sampled), just west and across the
river from a fishing charter service called Taylors Landing and about
five miles east of the town of Waldport, Oregon.
Along this area the
shore is never more than a yard wide and gives way to an almost perpendicular bank between three and four yards high leading up to the
area where many of the samples were taken.
Much of this area was
covered with jagged bushes and grass, and contained a few trees, a
few samll puddles of water, and a dirt pile and was in no way marshy.
The samples were collected (in the winter when the surface was damp
yet the ground was hard) at the distances indicated in Table 9, in
rows parallel with the shoreline.
The land has remained dry during
several periods of very high flooding of the river.
The demonstra-
tion of type E at distances of 35 and 245 yards from the shore in
grassy land strongly supports hypotheses which assume a terrestrial
distribution of the organisms.
A survey of the tidewater and fresh-
water areas of the Umpqua river is currently being undertaken but the
results are not included in this thesis..
Smoked fish products:
toxicity
chloride, and moisture results:
The samples of smoked fish grouped according to type, are listed in
Table 13 along with the results of screening tests for toxicity.
Only 19 of 161 samples screened were toxic.
Antitoxin typing was
attempted on 18 of these; however, only 14 samples could be success-
fully typed and all proved to be type E.
These include:
a highly
contaminated piece of smoked herring salted and smoked in Bay City,
packaged in cellophane and distributed at Newport, Oregon; a kippered
salmon tip processed and distributed at Seaside; a piece of bc smoked
salmon processed and distributed at Astoria, and a piece of sturgeon
kippered by a wholesale processor in Chinook, Washington and distributed by a second retailer in Astoria, Oregon.
Eleven of the 19 collection sites indicated in Table 114 had one
or more toxic samples.
tam
Two of the 24 samples which were found to con-
type E organisms were processed at the site they were sold; the
other 2 were distributed at sites (where they were purchased) other
than where they were processed (one of these processors is not listed
59
in Table iLi).
At least 6 of the 19 distributors leave their products
uncovered and at room temperature throughout the selling day and 5 of
the 19 package some or all of their products in plastic bags, piofllm
containers or cellophane.
A surmnary of the chloride and moisture contents which have been
determined for more than half of the samples screened is presenc1
Table 15.
Grinding or homogenization was not attempted; however, some
randomization occurred in cutting, selecting and preparing the sample
for the test.
Nevertheless, the mean variation of the difference in
duplicate chloride analyses run on 32 fish was only 0.13% which indicates the samples were fairly homogenous with respect to salinity.
The finding that the base salinity of raw salmon may range up to perhaps 0.25% may be used to estimate the level of salt added to the products in processing.
It is evident from the table that about half of
the fish products obtained had salinities below 2.5% and, two-thirds
have salinities below 3.5%.
bc6
in
Many of the higher salinities were found
smoked salmon products almost three-fourths of which had
salinities above
L'.5%.
All chloride values are in terms of NaC1.
Duplicates were not run for the moisture tests.
Twenty percent
of the samples have moistnre contents comparable to raw fish.
6
More
Loc smoking implies that large salmon fillets are processed in
their entirety rather than cut into small pieces, which is done
in kippering fish. In bc smoking the fish fillets are held in
30% to 90% salt brine from around two days to two or more weeks
(whereas kippered samples are held up to a day); some of the
salt may be leached out, the samples are hung at room temperature
or below (65 F is supposedly considered optimum), and subjected
to dry smoke (no heat) at various intervals between one and three
weeks.
than sixty percent of the samples contain between L5% and 65% water.
The samples with moisture contents between 35% and Li5% appeared
fairly dry and those below 35% were very dry and could not be bent,
or cut with a sharp knife.
Isolation of Cl. Botuljnujyi Cultures From Toxic Specimens
Isolation of Cl. botulinum was attempted from the mixed cultures
of twenty-seven toxic specimens which had been typed.
contained type E toxin, one type A and one type B.
Twenty-five
The specimens were
various species of lake fish, clams, oysters, crabs, kippered and
smoked fish products and river and reservoir sediments.
The method
used has been described in the section on methods and materials.
The
results are presented in Table 12.
In the isolation of cultures from specimens (No. 2 through 296)
almost all type of colonies were picked and subcultured in TFG tubes.
Duplicate isolation was attempted for cultures No. 93 through 296.
An attempt was made to pick colonies which resembled stock type E
colonies for cultures No. 7L6 through 911; and of the twentj'.three-
colonies picked, nine did not grow -- which is perhaps an indication
of the marked oxygen sensitivity of the anaerobic organisms which may
be present.
In all, one hundred and eighty-five colonies were picked
and subcultured, of which forty were toxic; nineteen were from blood
agar plates and twenty-one from LVE plates.
Some of the toxic
colonies from the blood agar were selected because they resembled
stock type E colonies; however, the recovery of the toxic colonies
61
from the LVE plates may be attributed to luck.7
The toxic colonies
from blood represent twelve mixed cultures while those from LVE represent nine.
Isolation was successful for thirteen of the twenty-seven mixed
cultures, or an average of slightly less than
O percent.
of Cl. botulinum were isolated from the following samples:
Cultures
trout from
Elk lake in the Deschutes National Forest area of the Oregon Cascade
Mountains; horseneck and softshell clams from Florence, Oregon; a
cockle clam from Yachats, Oregon; oysters and smoked herring from
Newport, Oregon; kippered sturgeon from Astoria, Oregon; a crab (vis-
cera) purchased from Chinook, Washington; and a sediment sample from
3-4 yards above the waters edge from the tidewater area of the Alsea
river in Oregon.
Cultural examination of type E isolates:
The twelve type E
colony isolates representing specimen numbers 146 through 224 (except
No. 156; the twelve isolates were from seven mixed cultures) in Table
16 were examined for purity and colonial uniformity by streaking on
LVE and blood agar plates.
Seven of the twelve appeared to yield
only a single type of colony when examined microscopically (45x: using
a Bausch andLomb stereo-disecting microscope) by reflected and transmitted light.
Five of these seven isolates giving uniform colonies
(on LVE and blood agar,i.e., ten plates in all) were then selected for
Apparently luck was as effective as selection by appearance
62
further examination.
(Three of these represented isolations from
distinct mixed cultures; two had been isolated from the same mixed
culture, i.e., all told they were from four mixed cultures).
A colony
from the blood agar and LVE plates of each culture was transferred
to a tube of TPG (after the plates had been intermittently removed
from and replaced in their Case jars and reevacuated -- in all they
were exposed to air approximately six hours).
colonies (from each of the above ten plates
None of these ten
one colony was picked
from each of the five blood agar plates and one from each of the five
LVE plates) were heat shocked.
After incubation these cultures were
tested for toxicity and typed.
Four of the five cultures from the
blood agar (which represented each of the four mixed cultures) produced type E toxin,
failed to grow.
Of the five transfers from the LVE agar, four
The one which grew produced no toxin.
Gram stained smears of the colonies on blood agar showed a pre.-
dominance of highly refractile mature spores, while the colonies on
LVE contained mostly pleomorphic gram negative rods containing
emerging spores suggestive of the forespore stage and only a few
typically refractile spores.
Other workers (Dr. J. Hobbs, Professor of Bacteriology, School
of Medicine, Leeds, England .-- personal communication:
Jan. lL, 1965)
have observed the tendency of type E cultures to incompletely sporu..
late on LVE agar.
The inability of the four LTE colonies to grow may
be due to any of a number of single or interrelated factors; however,
one hypothesis is that some aspect of sporulation is repressed and
6
as a result many of the filamentous vegetative cells do not pass
beyond the forespore stage and develop into completely mature highly
refractile spores.
These repressed forms may be more sensitive to
oxygen than mature spores and might be killed on exposure of the
plates to air as they are taken out of the anaerobic incubator.
Re-
pressed colonies might also he 'killed' by their own autolytic enzymes
during the anaerobic incubation due to the lack of a spore capsule.
Possible explanations for the survival and maintenance of toxi-
city of four of the five colonies from blood agar might include autooxidizable substances in this medium, such as hemoglobin,which might
foster a lower oxidation reduction potential in the blood agar than
would be present in the LVE and would enhance the viability of the
anaerobe; or perhaps an amount of iron is required in sporulation
which may be supplied by the blood agar but not by the LVE.
Although these ideas may be of some interest a major stumbling
block for all workers who have tried to identify Clostridium botulinuni type E is exemplified by the two colonies from the blood and
LVE plates which grew but were completely nontoxic.
Strains of or-
ganisms which present a similar morphological cultural and biochemical
picture but differ in one important respect:
ability to produce type
E toxin, have been worked on by various groups without any success.
The nontoxic cultures are merely referred to as 'E-like'.
Dolman has presented detailed pictures and biochemical data for
three mutational types, two of which he considers are nontoxic.
He
distinguishes these as TOX for the toxigenic mutant and TP for the
transparent proteolytic mutant and, he considers these colonies are
mutational derivatives of a primordial organism represented by an
opaque, sporulating (Os) colony.
He states that these "strains ex-
hibit very different mutational tendencies -- some persist in the
'TOX' phase for long periods, whereas some quickly degrade to the '05'
form and do not revert; and still others, though remaining toxigenic,
are found to be heterogeneous in a state of complex mutational equilibrium ... any given mutant, once separated, is of rather unpredictable stability; but in general the 'TP' mutants are very unstable and
tend to degrade into the relatively stable '05' form." (12a).
He recommends that they be distinguished by inspecting the
colonial growth with a hand lens or low-power microscope objective.
Careful analysis of Figure 2 reveals that all the colonies, although
they have slight density differences, appear to be of a similar type.
The colonies are magnified five times and represent one of four of
the five cultures from the blood agar which produced type E toxin.
Figure 3 shows the colonies (X) from the LVE plate of this same culture; these were the only colonies of the five LVE plates which grew
and, as has been mentioned, were not toxic.
These colonies do not
produce a dense opalescence in the egg yolk medium around the peri-
phery of the colony (the lecithovitellin reaction, or Nagler reaction
when human serum is substituted for the egg) which has been thought
to be characteristic of type F botulinum as well as many other
clostridia.
Figure L depicts the colonies (5X) of one of the four (of the
65
five LVE plates) cultures which did not grow when suhcultured.
show a medium lecithovitellin reaction.
showed medium to weak colonial reactions.
These
The other three LVE plates
The difficulty of recog-
nizing Dolman's three mutant types on each of these photographic
plates may be evident,
Perhaps, through the app1icaton of much of the receob successful work with the aerobic sporeformers to the anaerobic sporeformers
more intimate physiological and less qualitative experiments may bring
answers to many of the cultural questions arising around Clostridium
botulinum type E.
66
SUWIARY AND CONCLUSIONS
Studies were conducted to deteciine the occurcecce cf the toxin
producing, anaerobic, spore forming microorganism
num type E in shellfish, fresh water fish, and aquatic sediments from
the state of Oregon and from adjacent areas in Washi --- on and Califcr.
nia.
The samples collected inolude
coastal species of clams9 oysers
and crabs; sediments from coastal beach areas and from locations along
the shore at the mouth and in the tidewater and freshwater areas of
of three ocean-flowing rivers; and, fish and sediments from inland
mountain lakes and reservoirs0
Smoked fish products were also ex
amined with regard to the distribution of the microorganism and the
chloride and moisture content of the products.
The samples were incubated anaerobically, at temperatures9 and
in media designed to promote favorable outgrowth and optimum prodo:
tion of toxin by the type E organisms.
Preparations of these inru-
bated cultures were tested for toxicity by injection into mice0
Specific neutralization of an aliquot of the cultures found to be
toxic was attempted using botulmnuui antitoxin.
A sample9 which on in-
cubation was found to be toxic and in which a hotulinom toxi:: was
demonstrated, was considered to cothain cells of
of that toxin type.
Ii:i
An attempt was made to isolate the organism from
a number of the samples in which the presence of the toxin was demon
strated.
Clostridium botulinum type E was isolated from inland lake trout;
67
from clams collected at Florence, Yachats, and Newport, Oregon; from
oysters and smoked herring from Newport; from kippered sturgeon from
Astoria, Oregon; from crab viscera collected in Chinook, Washington;
and from a sediment sample collected 3..4 yards from the waters edge
(i
a bank) in the tidewater area of the Alsea river in Oregon.
Clam samples from 7 of the 9 sites along the coast from which
they were collected showed botulinum contamination.
Of 116 clam
samples incifoated, 28 were found to contain botulinum toxin (26 con-
tained type E toxin, 1 type A, and 1 type B).
Of the 16 incubated oyster samples from four collection sites
along the coast, 5 of the samples, representing each of the sites,
were shown to contain type E botulinum toxin.
harvested from three bay areas along the coast.
These samples were
Type E botulinum
organisms could not be demonstrated in 52 sediment samples from 8
sites along the coast.
Three of these sites were at the mouths of the
Coluribia, Alsea and flmpqua rivers.
The presence of type E organisms
was, however, demonstrated at sites along the shores in the tidewater
and freshwater areas of each of these rivers.
Four Dungeness crabs collected off the coast were typed for the
presence of B and B hotulinum toxins.
type E.
One was typed B and 3 were
Approximately 80 percent of all incubated viscera samples
from crabs collected at Chinook, Washington and Winchester Bay, Oregon
yielded toxic cultures.
Only one of the muscle tissue samples from
these crabs was toxic however.
The incidence of type E organisms in smoked fish products was
SI.
found to be low.
Of 11 varieties of products collected from 19 dis-
tributors along the coast, arid representing 161 individual samples,
only Li
of the 19 toxic samples were shown to contain type E botulinum
organisms.
Chloride and moisture determinations on 99 of the samples
revealed that two-thirds of these had a total NaCl content below 3.5
percent and about the same number were shown to contain between Lj5 and
65 percent water.
Clostridium hotulinum type E has been demonstrated in trout taken
from two high lakes in the Oregon Cascade Mountain range which lies
approximately 95 miles (map distance) from the coast; in sediments
from one of these lakes and from the bank of a nearby river; and along
the shores of three recently constructed reservoirs located nearly 120
miles inland.
The possibility of the existence of type E organisms in the gut
of clams, crabs and oysters has been discussed.
Experiments to determine the optimum incubation time and temperature for sediment samples in TPG medium, and a comparison between this
medium and beef heart infusion with minced meat medium were presented.
id
Fifty sediment samples from mountain lakes were run in paral-
in the latter comparison and the results indicated that beef heart
infusion medium with minced meat detected a somewhat greater number of
toxic mud specimens than did the optimum incubation procedure found
for the TPG medium.
The data also indicated that a trypsinization
procedure, which has been used to increase the toxicity of type E
toxin from fish cultures, reduced the number of the toxic beef heart
69
infusion cultures and was of questionahie value when used on the TPG
sediment cultures.
The demonstration of the type E organism
inland lake
river9
and reservoir sediments; along the shores in the tidewater and fresh
water areas of three ocean-flowing rivers; and in land up to 45 yards
from the waters edge in the tidewater area along the Alsea river,
supports theories of terrsiriaT origin of the organism.
Findings of the organism in shellfish, smoked fish products and
coastal and inland sediments indicate that this organism does indeed
present a potential hazard for individuals who process ar.d consume
fish arid shellfish products in the Northwest without adequate pre
cautions to prevent growth and toxin formation.
TABLE 1
Lake Fish Samples Collected,
Screened for Toxicity1 and
Typed for Botulinum Toxin
Identity of
Specimen
LAKE FISH
1. Elk Lake
a. Trout, eastern
brook
b. Trout, rainbow
c. Kokanee Salmon
2. Davis Lake
a. Frogs, small
Location
Where
Obtained
Samples2
Screened
Samples
Toxic
% of
Samples
Toxic
2
Questibnably
Toxic Samples
Fraction of
Samples Typed
Found to Contain
Botulinuni Toxin
D.N.F.
10
8
80
0
8E/8
D.N.F.
D.N.F.
1
11
1
8
100
63
0
0
1E/i
7E/7
JJ.M.F.
1
0
0
0
-
D.N.F.
10
0
0
0
--
5
0
0
0
3. OcIell Lake
a. Kokanee Salmon
1. Klamath Lake
a. Catfish
b. Chubs, blue
c. Chubs, tut
d. Perch
5. South Twin Lake
a. Trout, rainbow
,
S.S.L.
S.S.L.
S.S.L.
S.S.L.
D.N.F.
5Li
5
9
2L
Li'
5
5
0
17
10
0
0
30
5
17
3
--
5E/5
All samples incubated in TPG medium for five days at 28°C in Case anaerobic jars
3 fish incubated per sample
Samples not considered definitely toxic: Results considered questionable if only one of two mice
died or if two mice died between LI0 and 96 hours
D.N.F. - Deschutes National Forest in Oregon Cascade Mountain range
S.S.L. - Samples obtained from southern section of Kiamath Lake
0
TABLE 2
Crabs, Oysters, and Shrimp Collected,
Screened for Toxicity' and
Typed for Botulinum Toxin
Identity of
1. Crahs Dungeriess
a. one-half inch
h. 3-5 inches4
c. above 5 inches
I. muscle tissue
2. viscera
Individuol: Collected
Per Number of
Samples
Quesionabiy
Toxi.
Toxic Samples
2
1
50
3/3
2
0
0
18
1
lL
1
2
BotulinrmTccn
0
0
---
78
0
1
0/1
3E91B/6
0
0
0
--
15
6
O
8
0
0
18/18
10/2
3. Oysters, Pacific7
50/13
Shrimp8
% Of
Samples
21-/2
2. Oysters, Eastern6
,
Samples
Toxic
Fraction of
Samples Typed
Found to Contain
1 lb./l
6
5E/5
2
--
All samples incubated in TPG medim for five days at 28°C in Case anaerobic jars
Samples not considered definitely toxic:
Results considered questionable if only one of two nice
died or if two mice died between LQ and 96 hours
Samples collected from beach at Garibaldi, Oregon
Samples collected from beach at Garibaldi and Yaquina Bay, Oregon
Samples collected live from seafood dealers in Chinook, Washington and Winchester Bay, Oregon
Samples collected from cannery at Newport
Samples collected from canneries at Nahootta, Washington and Bay City and Tillamook, Oregon
Uncooked sample obtained from shrimp boat at Charleston, Oregon
H
TABLE 3
Clams Collected, Screened for Toxicity
And Typed for Botulinum Toxin
1
Fraction of
Identity of
Specimen
Individuals Collected
Per Number 'F
Collection Groups
I.
Clams9 razor1
230/29
2.
Clams,
113/10
3. Clams,
cockles
soft shell5
Clams, littleneck6
.
Clems, horseneck7
5.
6. Clams,
1
blue8
Samples
Screened
Samples
Toxic
% of
Samples
Toxic
Questionabiy
Toxic Samples
Samples Taped.
Found to Con':ain
Botulinum Toxin
3E9IAJB/6
12
63
16
11
79
2
lIE/li
12
8
67
2
9E/9
11
3
27
5
3E/3
6/2
3
1
33
1
IE/l
3/1
1
0
0
0
6/9
185/10
75
All samples incubated in FPG medium for five days at 28°C in Case anaerobic jars
Results considered questionacle if only one aS two mice
Samples non considered delinitely noxic
died or if two mice died between L3 and 96 hours
Samples collected from beach at Seaside and Winchester Bay, Oregon
Samples collected from beach at Garibaldi, Yaquina Bay, and Yachats, Oregon
Samples collected from beach at Yaquina Bay, Yachats, Florence, Reedsport, and Winchester Bay9 Ores
Samples collerned from beach at Garibaldi, Yaquina Bay and Florence, Oregon and from seafood
dealers at Nahcotta and Ilwaoo, Washington
Samples collected from beach at Garibaldi, and Florence, Oregon
e
Samples collected from beach at Garibaldi, Oregon
TABLE 4
Inland Lake Sediments Collected,
Screened for Toxicity1 and
Typed for Botulinum Toxin
Identity of
Specimen
Location
here
2
Obtaired
Samples
Screened
Samples
Toxic
% of
Samples
Toxic
Questionably
Toxic Samoles
Fraction of
Samples Typed
Found to Contain
Botulinun Toxin
INLABD LAKES
I.
2.
3.
4.
Cultus Lake
Davis Lake
Devils Lake
Elk Lake
a. bottom sediment
5. Kiamath Lake
D.N.F,4
4
D.N.F,
6
1
2
---
D.NOF.
2
1
67
50
D.N.F.
D.N.F.
32
1
24
1
100
1
1
1
0
0
near Klamath Falls,
16
4
25
3
14
0
0
1
4
1
25
1
-
13
1
2
0
8
4
--
25
2
-
0
4
--
75
5E/5
---
Ore.
a. bottom sediment
l
"
6, Little Lava Lake
D.N,F.
(source Desehutes R.)
7. Odell Lake
D.N.F.
8. South Twin Lake
D.N.F.
9. Tule Lake, Calif.
Northern California
2
8
16
All samples incubated in TPG medium for five days at 28°C in Case anaerobic jars
Samples collected at the waters edge to one foot below the water surface
Samples not considered definitely toxic:
Results considered questionable if only one of two mice
died or if two mice died between 40 and 96 hours
D,N.F,
Desohutes National Forest in Oregon Cascade Mountain range
TABLE 5
Inland River and Reservoir Sediments Collected,
Screened frr Toxicity1 and
Typed for Botulinum Toxin
Identity
INLAND RIVERS
I. Culbus River
2, Deschutes River
3. Desohutes River
N. Davis Creek
5. Quinn River at
source
Location
Where
2
D.N.F.
Samples
Screened
2
Century Drive
D.N.F.
Cow Camp DNF.
Samples
% of
Samples
Toxic
Questlonab1y
Tcxj.c Samples Bcnclnmx1
0
0
2
50
0
1
2
2
1
0
50
0
Wickinp Res
0
0
D.N,F.
D.N.F.
6
2
53
2
10
8
10
8
80
0
2
25
10
0
0
INLAND RESERVOIRS
6. Haystack Res.
near Madras Ore.
7. Pelton Dam Res.
near Madras, Ore.
3. Round Butte Dam Res. near Madras, Ore.
1
Fraction of
Samples Typed
Found to Contain
-
IE/1
2E/3
IE/l
IE/l
medium for five days at 28°C in Case anaerobic jars
All samples incubated in T
Srnples collected at the waters edge to one foot below the water surface
Samples not considered definitely toxic: Results considered questionable if only one of two mice
died or if two mice died between 14Q and 96 hours
D.N,F. - Deschutes National Forest in Oregon Cascade Mountain range
TABLE 6
0
0
A Comparison of 20
C and 28
C Incubation Temperatures for Periods of 5, 7 and
lL Days in TPG Medium With and Without Trypsinization for Detection of Toxic
Mud Specimens
Incubation
Fraction of Specimens Toxic
2/2 Mice 1/2 Mice
Total
Dead
Dead
20°C
Incubation Time
and Sample Treatment
5 days incubation;
not trypsinized
7 days incubation;
not trypsinized
7 days incubation;
plus trypsinization
Incubation
Fraction of Specimens Toxic
2/2 Mice 1/2 Mice
Dead
Total
Dead
L1/1L
2/1J
6/lLi
l0/lLl
0/lLi
l0/lL4
5/lLi
2/lL
7/lLI
8/l2--
/lL
ll/lLi
--1
7/11
1Li days incubation;
not trypsinized
28°C
6/1L
2/1Ll
8/lL
2/1Ll
1/114
io/i
5/lL
/1Li
8/iLl'
7/1Ll'
8/lLI
11/114-
2/iLl'
1/iLk
10/iLl-
8/iLl
Subcultures made by trans
ferring 5 ml of original
culture of specimen after
5 days incubation at 20°C
or 28 C to 50 ml fresh TPG
and incubating 5 days more
Not trypsinized
Trypsinized
s/i'-i
?/1L4
12/lLl
antitoxin oi the 7th day (using
Several toxic samples (2/2) on the fifth day were typed
a trypsinization procedure) and the results are included as total toxic
-0
TABLE 7
A Comparison of Two Culture Media and Incubation Procedures With and Without
Ti'ypsinization for Detection of Toxic Mud Specimens
Culture Medium
and Incubation
Fraction of
Mud Specimens
Found Toxic
Untrypsinized
3 gram mud specimen
incubated in 50 ml
TPG at 28°C for
5 days in Case
anaerobic jar.
3 gram mud specimen
incubated in 30 ml
of Beef Heart
Infusion plus meat
for lL days at 20 C
in Case anaerobic
Fraction of Mud
Specimens Trypsinized
That Were Toxic Only
After Trypsinizatior
Fraction of Mud
Specimens Losing
Toxicity After
Tryspinization
Total Fraction of
Mud Specimens
Toxic After
Trypsinization
Fraction of
Control
Specimens
Found Toxic
(1)
19/50
8/L0
7/L0
20/L0
L5/50
25/50
1/36
7/36
19/50
L9/50
jar.
(1)
(2)
Nine of the toxic specimens killed 2/2 mice; 10 killed 1/2.
Sixteen of the toxic specimens killed 2/2 mice; 9 killed 1/2.
TABLE 8
Marine Sediments Collected,
Screened for Toxicityl and
Typed for Botulinum Toxin
Identity of
Specimen
Location
Nhere
Obtained2
Samples
Samples
_creened Toxic
% of
Samples
Toxic
Questionably3
Toxic Samples
Fraction of
Samples Typed
Found to Contain
Bctulinum Toxin
LAY SEDIMENTS
1. N. Fork Nehalem
River
NehlaLn, Ore.
3
1
33
1
2. Salmonherry River
Mohler, Ore.
3
0
0
0
I. Sandy beah
Oceanpark, Wash.
3
0
0
0
--
2. Public boat dock
Chinook, Wash.
3
1
33
0
--
3. Shore oyster bed
S. of Chinook, Wash.
3
1
33
0
--
L, Sandy beach
Seaside, Ore.
10
3
30
3
--
3
1
33
1
--
BEACH SEDIMENTS
. Sandy beach
Oceanside
Ore.
All samples incubated in TPG medium for five days at 28°C in Case anaerobic jars
Samples collected at the waters edge to one foot below the water surface
2
So1es not considered definitely toxic: Results considered questionable if only one of two mice
died or if two mice died between LiO and 96 hours
-u
-u
TABLE 9
Alsea River Sediments Collected,
Screened for Toxicity1- and
Typed for Botulinum Toxin
Identity of
Specimen
1. Saltwater beach
near mouth s3 ws
Location
Where
Obtained
Samples
Screened
Samples
Toxic
of
Samples
Toxic
Waldport, Ore.
10
1
10
Taylors Landing
20
10
12
16
8
2
6
8
80
60
75
100
86
80
3
3
2
10
8
2
Questionably
Toxic Samples
Fraction of
Samples Typed
Found to Contain
Botulinum Toxin
2. Tidewater study
site
a. WE
b. 3- yards AWE5
c. 12-15 yards AWE
d. 20 yards AWE
e. 25 yards AWE
f. 30 yards AWE
g. 35 yards AWE
2
7
10
6
h. Li5 yards AWE
3. Freshwater study
site WE
Alsea, Ore.
6
3E / 3
1
1
0
0
1
2E/2
2E/2
50
67
0
IE/l
0
2E / 2
80
1
3E/3
All samples incubated in BHI meat medium for fourteen days at 20°C in Case anaerobic jars
Samples not considered definitely toxic: Results considered questionable if only one of two mice
died or if two mice died between LIO and 96 hours
S - Samples collected from southern shore
WE - Samples collected at the waters edge to one foot below the water surface
AWE - Samples collected above the waters edge at distance indicated
TABLE 10
Columbia River Sediments Collected,
Screened for Toxicity1 and
Typed for Botulinum Toxin
Location
Identity of
Specimen
Jhere
Samples
Screened
Obtained
Samples
Toxic
% of
Samples
Toxic
2
Questionably
Toxic Samples
Fraction of
Samples Typed
Found to Contain
Botulinun Toxin
COLUMBIA RIVER
1. Saltwater beach
near mouth S3 WE2
2. Tidewater study
site WE
3. Freshwater study
site No. 1
a. WE
b. 3.4 yards AWE
c. 8-12 yards AWE
LJ Freshwater study
site No. 2 WE
Fort Stevens, Ore.
10
0
0
1
--
Tongue Point, Ore.
10
5
50
2
1E/3
Rainier, Ore.
20
10
10
5
1
1
2
25
10
10
5
1E/3
1
1
lA/i
100
0
0/1
"
ft
"
Scappoose Bay
(near St. Helens,
Oregon)
2
--
All samples incubated in BHI meat medium for fourteen days at 20°C in Case anaerobic jars
Samples not considered definitely toxic:
Results considered questionable if only one of two mice
2 died or if two mice died between Li,0 and 96 hours
S - Samples collected from southern shore
WE - Samples collected at the waters edge to one foot below the water surface
AWE - Samples collected above the waters edge at distance indicated
TABLE 11
Umpqua River Sediments Collected,
Screened for Toxicity1 and
Typed for Botulinum Toxin
Identity of
Specimen
Location
Where
Obtained
Samples
Screened
Samples
Toxic
% of
Samples
Toxic
,
Questionably
Toxic Samples
Fraction of
Samples Typed
Found to Contain
Botulinum Toxin
UNPQUA RIVER
1. Saltwater beach
near mouth s3 WE4
2. Tidewater study
site AWES
3. Freshwater study
site WE
Winchester Bay
10
0
0
1
--
above Reedsport, Ore.
10
6
60
3
2E/3
Elkton, Ore.
10
5
50
1E/l
All samples incubated in BHI meat medium for fourteen days at 20°C in Case anaerobic jars
Samples not considered definitely toxic: Results considered questionable if only one of two mice
died or if two mice died between L0 and 96 hours
S - Samples collected for southern shore
WE - Samples collected at the waters edge to one foot below the water surface
AWE - Samples collected above the waters edge
TABLE 12
Toxicity Tests on Cultures Derived from Isolated Colonies
T00
Type of Toxin
Demonstrated
iio Mixed
Specimen Cultro
Toxic Cultures
Single Colony
Cultures
Tested
from Blood ag_ar
Toxic Cultures
from LVE1
Type of Toxin
in Culture
from Isolated
Colonies2
Location
Where
Obtairoed
Identity of
Specimen
No
2
E
7
2/0
E
Elk lake, Ore.
3
E
7
2/0
E
Elk lake, Ore.
6
E
9
2/3
E
Elk lake, Ore.
8
E
E
A
B
E
E
E
E
E
E
E
5
0/0
0/0
0/0
0/0
0/3
1/0
2/2
1/1
2/3
--
15
93
110
lL6
152
155
156
159
160
170
5
9
5
10
10
l-
13
12
17
13
3/Lb
i/o
Trout, eastern
brook
Trout, eastern
brook
Trout, eastern
brook
---
E
E
3E
-3E
2E
E
LVE - liver-veal-egg agar
The TPO subcultures from isolated colonies were stored at 5°C
Florence, Ore.
Florence, Ore.
Newport, Ore.
Softshell clam
Horseneck clam
Cockle clam
Newport, Ore.
Newport, Ore.
Yachats, Ore.
Pacific oyster
Pacific oyster
Cockle clam
TABLE 12 (continued)
Toxicity Tests on Cultures Derived from Isolated Colonies
Foxic
Specimen
Type of Toxin
Demonstrated
in Mixed
Culture
Single Colony
Cultures
Tested
Toxic Cultures
from Blood agar
Toxic Cultures
from LVE1
Type of Toxin
in Culture
from Isolated
Colonies2
Location
Where
Obtaired
lEcieritity of
Specimen
No.
224
E
2/49
8/40
E
E
E
F
F
F
F
F
866
895
F
E
900
911
E
E
296
7/46
755
805
82/4
838
11
1/3
E
2
o/o
0/0
o/o
-------F
E
--
13
2
2
2
2
2
2
2
3
/4
2
0,/0
0/0
0/1
0/0
0/1
1/0
o/o
0/0
1/0
E
LVE - liver-veal-egg agar
The TPG subcultures from isolated colonies were stored at 5°C
Astoria, Ore.
Kippered
sturgeon
Chinook, Wash.
Newport, Ore.
Crab viscera
Smoked herring
Tidewater area,
Alsea river,
Oregon
Sediment from
bank 3-/4
yards from
waters edge
TABLE 13
Smoked or Kippered Fish Collected
Screened for Toxicity1
Typed for Botulinum Toxin
Tdentity of
Specimen 2
1. Kippered Black Cod
2. Kippered Halibut
3. Smoked Herring
5.
6.
7.
8.
9.
10.
II.
/4
Kippered Perch
Kippered Red Snapper
Kippered Salmon
Smoked Salmon
Smoked Smelt
Kippered Sturgeon
Smoked Sturgeon
Kippered Tuna
Sauples
Screened
Samples
Toxic
L.
Found to Coo,.ajr.
Botulinum Toxin
0
0
9
1
12
9
LI
0
lE/9
lE/2
1
1
0
1
0
3,.
0
lL
50
3
0
0
201
1
31
0
5
0
8
l3
Questionably3
Toxic Samples
0/2
0/1
1E/1
1
2
1
67
30
Fraction of
Samples Typed
2
1
0
0
0
36
ll;.
6
% of
Samples
Toxic
1
I/2
0/1
All samples incubated in TPG medium for five days at 28°C in Case anaerobic jars
Samples obtained from smoked fish processors indicated in Table lL
Results considered questionable if only one of two
Samples not considered definitely toxic:
mice died or if two mice died between /40 and 96 hours
In some instances more than one sample was taken from a single piece of smoked fish
TABLE 14
Incidence of Clostridium botulinum type E
In Smoked Fish Products
Source
Nahcotta, Wash.
Long Beach, Wash.
liwaco, Wash.
Chinook, Wash.
Joseph., Astoria
Thomp., Astoria
Seaside
Roackaway
Garibaldi
Lincoln City
Lincoln Beach
Newport
Corvallis
Waldport
Yachats
Florence
Winchester Bay
Charleston
Wedderburn
TOTAL
1
No. Samples
Screened 1
Samples
Toxic
11
1
6
-
2
Questionably
Toxic Samples
3
4
4
13
16
1
2
1
---1
31
(
5
1
9
1
2
1
15
4
--
1
9
8
1
4
--
-
3
-1
--
--
1
161
19
-1
1
1
--
2
2
1
9
8
3
3
Siiples Shown
to Contain
Type E Toxin
15
1
--
4
Samples represent the 11 types of smoked, fish products adurubrated
in Tables 13 and 15 which have been collected from the individual
processors at the above spots along the Washington and Oregon
coast.
2
Samples not considered definitely toxic: Results considered questionable if only one of two mice died or if two mice died between
40 and 96 hours.
TABLE 15
Chloride and Moisture Content of Smoked Fish Samples
1.
2.
3.
L
5.
6.
7.
8.
9.
10.
11.
Identity of
anp1es
Sneo-ne'-
estec1
--'
-_2
-'
8
1
6
1
0
0
0
0
3
3
1
1
1
1
0
0
0
0
0
0
0
0
0
0
3
1
1
3
0
1
0
0
1
2
1
1
1
1
1
1
0
lLI
2
0
0
0
1
3
Kippered
Black Cod
Kippered
Halibut
Smoked Herring
Kippered Perch
Kippered
Red Snappei
Kippered Salmon
Smoked Salmon
Smoked Smelt
Kippered Sturgeon
Smoked Sturgeon
Kippered Tuna
TOTAL
1
percent NaC1 Content 2, 3
1
0
3
0
0
1
28
21
13
1
3
2
0
1
0
11
5
5
9
3
0
6
0
0
99
1Li
33
21
3
16
1
0
6
1
i'
_L4
.
.L4
Percent Moisture Content
b & U
& uo
0
1
1
1
0
1
0
0
0
2
0
16
10
5
0
5
2
2
1
6
0
0
0
0
0
1
2
Li-S
20
3
0
1
2
0
0
0
1
1
3
9
22
6
8
20
6
These smoked fish products were collected from processors along the Washington and Oregon coast and,
The points of collection are
do not include all the samples which have been screened for toxicity.
given in Table iLl.
2
,
Samples had chloride contents which ranged between 0.9 and 12.3 percnet. The mean NaCl content from
four determinations on each of two raw salmon was 0.097% and 0.188%.
The mean variation of the differences in duplicate chloride analyses run on 32 fish is 0.133%.
The respective moisture content of
Samples had moisture contents which ranged between 23 and 77%.
the raw salmon in (2) was 73% and 66%.
TABLE 16
Results of Successful Typing of Toxic Specimens with Antitoxin
Type of Antitoxin Used
ype
Dilution of Fiitem1e
Identity cf
Source.
1:50
Individuals No. mice No. mice
dead
in,j ected
1
0/2
0/2
1. Trout, Rainbow
Elk Lake
2. Trout, East Brook
Elk Lake
1
0/2
3.
"
"
1
0/2
ft
ft
U
ft
"
"
ft
U
U
6. "
"
"
0/2
0
E
l:.5
J
None jnprotected
1:5
1:O
Bot.uljnum
No. mLce No. mice
dead
in,j ected
S anple
Type of
Toxin in
hr 2/2;L_12hr
E
0/2
2/2;L12hr 2/2;Ll2hr
E
l/2;L8_
72 hr
2/2;24 hr 2/2;2-L hr
E
0/2
2/2;L_l2hr 0/2
E
2/2;2_24
1
0/2
0/2
2/2;Lk.l2hr 2/2;12-3Ohr
E
1
0/2
0/2
2/2;Ll2hr
2/2;LL_12hr
B
1
0/2
0/2
2/2;2L. hr 2/2;2-LL hr
B
7
"
8.
"
"
"
"
"
1
0/2
0/2
2/2;2_Ll hr 2/2;l2-3Ohr
E
9. "
"
"
"
"
1
0/2
0/2
2/2;2...4 hr 0/2
B
2
0/2
0/2
2/2;2-4 hr 2/2;2_L hr
E
0/2
0/2
2/2;2-4 hr 2/2;2-L hr
E
0/2
0/2
2/2;2-L hr 2/2;Ll-l2hr
E
"
12. Kokanee Salmon
13.
"
15.
"
Elk Lake
"
"
2
TABLE 16 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
No.
Identity of
Dilution
Specimen
27. Kokanee Salmon
of Fi1terele_
Individuals
Source
Elk Lake
"
29
"
32.
"
"
"
139. Clams, Softshell
Newport, Ore.
1143.
Florenne, Ore.
"
Type
Type of Antitoxin. Used
E
NoneUnproteo ted
l:O
Trpe of
Botulinim
ToxIn in
2
No, iiice No. mice
No. mice No. mioe
injecied
dead
injpted
dead
0/2
2/2;2 hr 2/2;2 hr
0/2
3
0/2
0/2
2/2;2L hr 2/2;2_Ll hr
E
2
0/2
0/2
2/2;L4_lhr 2/2;Ll5hr
E
2
0/2
0/2
2/2;LI_l5hr 2/2;L-l5hr
E
0/2
0/2
2/26-l5hr 0/2
E
l/2;23- 0/2
2/2;2_l6hr 2/2;2-l6hr
E
i/i;31- 1/2;3172hr 72hr
2/2;2-l6hr 2/2;2-l6hr
E
per sample
3
31 hr
E
1L4.
"
Newport, Ore.
Li-
lLi6.
"
Florence, Ore.
Li-
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
E
1Li7.
"
Newport, Ore.
Li-
0/2
0/2
2/2;2-8 hr 0/2
E
Li-
0/2
0/2
2/2;2-3lhr 2/2;2-l6hr
E
l/1;LI_15hr
E
0/2
E
11i-8. "
162.
"
i6Li-.
"
165.
"
I'
Winchester Bay
3
0/2
0/2
l/2;15-2Li-
Reedsport, Ore.
3
0/2
0/2
2/2;Ll-6Llhr
3
0/2
0/2
hr
2/2;1536 0/2
TABLE 16 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
N.
Identity of
Specimen
140, Clams9
cockle
141. "
U
Dilution of Filtered Sample
Individuals
Sourc.e
per sample
Newport, Ore,
Type of Antitoxin Used
Type E
None: Unprotected
1:50
1:5
1:50
1: 5
No. mice/No. mice
No. nice No. mice
dead
injected
dead / ipjeoted
4
0/2
0/2
4
0/2
142. "
4
145.
4
151, "
l/2;1524
Type of
Botulinum
Toxin in
SaU.
B
0/2
0/2
hr
7/2;4.24hr 0/2
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
B
0/2
2/2;2.16hr 1/2;2-l6hr
F
4
1/2;216 hr
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
F
154. "
4
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
F
155
"
7
0/2
0/2
2/2;2.-l6hr 2/2;2-l6hr
E
157. "
5
0/2
2/2;L4-15hr 2/2;4-lShr
E
4
0/2
1/2;415 hr
0/2
2/2;2-8 hr 0/2
E
4
1/2;415 hr
1/2;215 hr
0/2
0/2
2/2;4-l5hr 1/2;15-24hr
E
0/2
2/2;2-l6hr 2/2;2-l6hr
E
0/2
2/2;6-lShr l/2;6-lShr
E
0/2
0/2
2/2;2-l5hr 0/2
E
U
Yachats, Ore.
169. "
170. "
153. Clams, littleneck
Florence, Ore.
156.
Newport, Ore.
176.
"
"
Nahcotta, Wash.
8
10
7
E
TABLE 16 (continued)
Results of Successful Typing of Toxic Specimens with Antioxin
Dilution of Filtered
Identity of
Nc. Specimen
152
Clams, I-ioreseneck
Individuals
mle
Source
Floren:e
Type of Antitoxin Used
Type E
None Unprotected
5O
1 5O
1: 5
1 5
No, mice/No. mice
Nc, mice/No, mice
dead
dead
injected
/ injtedj
/
Type of
Bc tulinuin
Toxin in
S amole
Ore.
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
E
171. Clams, razor
Winchester Bay
0/2
0/2
2/2;2-l5hr 2/2;2-cthr
E
JJL. ft
II
5
0/2
0/2
2/2;L_15hr 2/2;L_15hr
E
3
2/2;Ll
2/2;L-
2/2;Lk.l6hr 2/2;L_l6hr
-l.1
Il
Seaside, Ore.
93. "
l6hr
U
ft
ft
3
Type B Antitoxin
1/2;L_
2/2;14--
3
Type A Antitoxin
0/2
0/2
3
2/2;LI._
l6hr
93. "
110.
ft
ft
ft
ft
ft
I,
l6hr
l6hr
0/2
16 hr
110.
I,
if
ft
ft
ft
I
3
Type B Antitoxin
0/2
0/2
Type A Antitoxin
3
2/2;14_
21l hr
0/2
A
TABLE 16 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
of
Identity of
No. Soecimen
on of Filered Sample
Individuals
Source
per sample
159. Oysters, Pacific
Newport, Ore.
160. "
n
189.T2"
Tillamook, Ore.
l93.T1"
Bay City, Ore.
L.
Type E
1:5
1:50
No. mice/No. mice
dead / injected
0/2
Type of
Botulinum
Toxin in
Samicle
2/2;2-6 hr 2/2;26 hr'
F
0/2
0/2
2/2;L.2Lhr 2/2;Lk2Lhr
E
L.
0/2
0/2
2/2;lL hr 2/2;16-32hr
E
Li
1/2;15-.
0/2
2/2;15-96 l/2;15-28hr
hr
E
28 hr
1
itoxin Used
None: Unprotected
1:5
1:50
No. mice No. mice
dead
inected
For both the cotected and unprthe:ted nice, 2, at each of the dilutions, were injected with 0.5 ml.
of the diluted (see methods and materials section of text) centrifuged supernatant of a toxic culture,
These injected mice were held in jars which were layered with wood chips and which contained a source
of food and water; and they were observed over a Li day period. The intervals in tine between which
the mice died are indicated in hours. The antitoxin test on the samples presented ic Table 17 was
La'rLed oo ad
e rec2r were recded in the same nan-er
TABLE 17
Results of Successful Typing of Toxic Specimens with Antitoxin
Typof_Antitoxin_Used
Type E
31
Sa'rJe
Stz of
Sample
o
Identity cf
No, Specimen
Source
1
I
No, rriie/N
None
Q
I
rctected
1
0
Type of
Bc u nUrr
Toxin in
mi:o
I ii1ected
No. mice/No, mice
0/2
l/2;1L
hr
2/2;114 hr 2/2;1-L4 hr
E
dead
deadjjted
Sampe
218. Clams, razor
Winchester Bay
223. Pacific oysters
Nahcotta, Wash, 2 oysters
l/2;14--
0/2
2/2;LI-_l2hr
2/2;Ll2hr
E
Astoria, Ore,
20 gm
12 hr
l/2;L_
16 hr
0/2
2/2;1_Ll hr
1/2;l632hr
E
Astoria, Ore.
20 gm
0/2
0/2
1/2;L_12hr 0/2
E
0/2
0/2
2/2;lLI hr 2/2;lJ-- hr
E
1 "
0/2
0/2
2/2;l-L hr 2/2;1-l5hr
E
1
0/2
0/2
2/2;l-L4 hr
1 "
0/2
0/2
2/2;2-8 hr 2/2;2-8 hr
E
22L. Kippered sturgeon
2LJ9.
Smoked salmon
275. Trout, Rainbow
"
285. "
ft
clams
South Twin Lake 1 fish
278. "
29L.
L
ft
ft
"
2/2;Ll5 hr
E
296. "
U
1 "
0/2
0/2
2/2;1-4 hr 2/2;1-LI hr
E
381. Shore sediment
Elk Lake
3 gm
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
E
3 gm
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
E
387. "
3 gm
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
E
L23. "
3 gm
0/2
0/2
2/2;l-8 hr 2/2;8_LLhr
E
385.
"
H
TABLE 17 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
Identity of
Dilution of Filtered Sa1e
No. Specimen
518. Shore sediment
Size of
2
Columbia
3 1E Alsea
742. FW
Type of
Botulinum
3 gin
2.5 ml
0/2
0/2
2/2;8...l6hr 2/2;8-l6hr
E
Tongue Point
3 gin
0/2
0/2
F
Alsea, Ore.
3 gin
0/2
0/2
l/2;l8_40 0/2
hr
Sample
settled sediment
TW1
1:5
No. mice/No. mice
No. mice No. mice
Toxin in
injcted Sap
dead I in.lected
dead
E
0/2
0/2
2/2;1-8 hr 2/2;1-8 hr
Source
Elk Lake
525. Muddy liquid above Elk Lake
688.
Type of Antitoxin Used
E
None: Unprotected
1:50
1:5
1:50
Type
l/2;l-6 hr
1/2;l8-LlOhr
E
7L6.
"
"
"
3 gin
0/2
0/2
2/2;l-6 hr 2/2;l-6 hr
E
7Li9.
"
"
"
3 gin
0/2
0/2
1/2;TL-6 hr
2/2;l-6 hr
E
1/2;618 hr
2/2;1-6 hr 2/2;1-6 hr
E
0/2
2/2;l-6 hr 1/2;6-l8hr
E
755. TW IE Alsea
Taylors Landing
3 gin
0/2
758
"
"
"
"
"
3 gin
0/2
760.
"
"
"
"
3 gin
0/2
0/2
2/2;1-6 hr 2/2;6-LI0hr
E
3 gin
2/2;28 hr
0/2
2/2;2-8 hr 2/2;2-8 hr
E
788. TW IE Umpqua
above Reedsport
TW - sediment sample from tidewater area of river
WE - sediment sample taken at waters edge
FW - sediment sample from
river
freshwater area of
TABLE 17 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
Dilution of Filtered Sample
Size of
Source
Sappe
Identity of
No.Eecimen
Type of Antitoxin Used
None: Unprotected
Type E
1:50
1:50
l
l_
No, mice/No. mice
No. mice/No. mice
dead / iected
dead
/ injected
Type of
Botulinurri
Toxin in
Sanjle
789. TW1 NE2 Umpqua
above Reedspert
3
0/2
0/2
2/2;28 hr 2/2;28 hr
E
793, FW0 NE Umpqua
Elkton, Ore,
3 gm
0/2
0/2
2/2;2_8 hr 2/2;8-l6hr
E
803, NE Haystack
near Madras
3 gm
0/2
0/2
l/2;6-lBhr 2/2;6..l8hr
E
"
3 gm
0/2
0/2
2/2;1-6 hr 2/2;l-6 hr
E
gm
0/2
0/2
E
1/2;618 hr
2/2;l8-
0/2
2/2;l8-L0 1/2;l8.4Ohr
hr
2/2;l-6 hr 2/2;l-6 hr
reservoir
805. NE "
"
8lL. NE Round Butte Dam
"
"
3
82Ll. WE Pelton reservoir "
"
3 gm
835. Dungeness Crab
Chinook Wash,
viscera
L0hr
835."
835. "
"
"
"
TW - sediment sample from tidewater area of river
WE - sediment sample taken at waters edge
1/2;18L,0hr
Type B Antitoxin
0/2
0/2
E
2/2;6_l8hr 0/2
B
Type A Antitoxin
2/2;60/2
18 hr
FW - sediment sample from freshwater area of
river
TkBLE 17 (continued)
Results of Successful Typing of Toxic Specimens with Antitixin
Identity of
No, Specimen
Dilution of Filtered Sample
Size of
Souroe
Sample
Type of Antitoxin Used
Type E
None: Unprotected
1:50
1:5
1!5
No. mice/No. mice
No. mice/No. mice
dead
dead /
/ in.jected
ected
10
Type of
Botulinum
Todn in
Sample
837, Dungeness Crab
Chinook, Wash.
viscera
0/2
0/2
2/2;2-8 hr 2/2;8-l6hr
F
838. "
U
viscera
0/2
0/2
2/2;2-28hr 0/2
E
viscera
0/2
0/2
2/2;2l8hr 0/2
E
866. Smoked herring
Newport, Ore.
(1.0% NaC1; Li3% moisture)
5-10 gm
0/2
0/2
2/2;2-l6hr 2/2;2-l6hr
F
873. FW1 8-12 yards
AWE2 Columbia
3 gm
2/2;2-
2/2;2-
2/2;28 hr 2/2;8-l6hr
E
8hr
8hr
8L0. "
U
ft
873."
U
Rainier, Ore.
U
Type B Antitoxin
2/2;22/2;8-
II
8hr
l6hr
ype A Antitoxin
873. "
"
"
895. FW WE3 Columbia
"
Rainier, Ore.
3
i
0/2
0/2
0/2
0/2
FW - sediment sample from freshwater area of river
AWE - sediment sample taken above
ters edge,
distance indicated
A
2/2;2-l2hr 0/2
E
WE - sediment sample taken at waters edge
TABLE 17 (continued)
Results of Successful Typing of Toxic Specimens with Antitoxin
Identity of
No. Specimen
900, TW1
Dilution of Filtered Sample
Size of
Source
Sample
l2l5 yards
Type of Antitoxin Used
None: Unprotected
Type E
1:5
1:50
No. mice/No. mice
No. mice/No. mice
dead / -nected
dead
I uYected
j5
Type of
Botulinum
Toxin in
Sainrie
Taylors Landing
3
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
F
"
3 gin
l/2;212 hr
0/2
2/2;2-l2hr 2/2;2-l2hr
E
3 gm
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
E
"
3 gm
0/2
0/2
2/2;2-l2hr 2/2;2-l2hr
F
964. TW 45 yds ANE Aisea "
3 gm
0/2
0/2
2/2;2-8 hr 0/2
E
966. TW 35 yds AWE Alsea "
3 gm
0/2
0/2
2/2;2-8 hr 0/2
E
968. TW 45 yds AWE Alsea "
3 gm
0/2
0/2
2/2;2-8 hr 0/2
E
1063.Kippered salmon
5-10 gm
0/2
0/2
l/2;22-30 0/2
hr
E
AWE' Alsea
911. TW 3_4 yards AWE
Alsea
"
915. TW 12...15 yards AWE
Alsea
923. TW 3_4 yards AWE
Al sea
Seaside, Ore.
TW - sediment sample from tidewater area of river
AWE - sediment sample taken above waters edge, distance indicated
FIGURE 1.
Plastic bag incubation technique used with clams, oysters,
lake fish, and crab viscera. The specimen was placed in
a plastic bag, TPG medium added, and the sample incubated
for 5 days at 28 C.
FIGURE 2.
Isolated colonies of ClostMdiuni botulinum type E grown (in
Case anaerobic .iars for 2 days at 28uC) On blood agar from
a cockle Olam sample. On subculture colonies from this
plate grew and produced type E toxin. (5X.)
a.
FIGURE 3.
Isolated colonies of Clostridium botulinum type E grown (in
Case anaerobic jars for 2 days at 28"C) on LVE (liver-vealegg agar) from a cockle clam sample. A comparison of this
figure with Figure 2 reveals the different colony characteristics exhibited by this organism streaked from the same
source on 2 medium. These colonies do not exhibit a ledthovitellin precipitate. On subculture colonies from this
plate grew but did not produce toxin. (5X.)
99
S.
*
I
a..
FIGURE 4,
Isolated col onies of Clgstridiuin botulinum type E grown
(in Case ans erobic jars for 2 days at 28C) on LVE from a
kippered sti .rgeon sample. Colonies of this sample isolated on blc od agar appeared very similar to those in
1 hese colonies exhibited a weak to medium ledFigure 2.
thovitellin precipitate (around periphery of colonies darlens on setting) not demonstrated by the colonies shown
in Figure 3. On subculture these colonies did not grow. (5X).
100
-- --- -
f11,V (cii4ili
Oss4rb'LiOr
L'A ,-')ibi
,The. NorL.hW
tE
E,Crb.
Beach
L0
0
e
Se
)ASH I4GTO4
See
\ES
:, Sed.
scp.
(
C
tr
/
a
OR
C
L
qt!v
3E,C(.
s
/
I,5ed
Sa4y
a. s.&.
1o.hats
ASea.
7E, KS.
2E,c1.
,1r.
Elk
Lava L..
Ct5pr
ovt0(
(vlt. L'4
eti-
Fort
CfttO.'\\
/
;
eerd
Ci.
Cd,.
Crab
Sonce
045. Oj
2
SnF.
Tr.
Tule L
$orcrvt Corvo
FIGURE 5
5el
s. S,4t
- Trejt.
101
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