AN ABSTRACT OF THE THESIS OF
Patrick John O'Leary for the degree of
in
Microbiology
Title:
Master of Science
February 3, 1977
presented on
ENTERIC REDMOUTH BACTERIUM OF SALMONIDS:
A
BIOCHEMICAL AND SEROLOGICAL COMPARISON OF
SELECTED ISOLATES
Redacted for Privacy
Abstract approved:
.
a_I.
A comparison of the agglutinating and precipatiating
antigens of Enteric Redrnouth Bacterium (ERMB) was made.
There are two major and one minor agglutinating antigens
which describe two serotypes (I and II).
metabolizes sorbitol.
Only serotype I
A bacterin from serotype I cross
protected against a challenge of bacteria of serotype II
in rainbow trout (Salmo gairdneri), but serotype II could
not cross protect from a challenge of serotype I.
Two
patterns of positive agglutination were observed in a
slide agglutination test depending upon the antigenic
composition of the bacterium and agglutinating antisera.
Incubation temperature altered the motility of ERMB.
At 9 C nonfunctional peritrichous flagella were produced.
At 18 and 27 C the bacterium was motile.
No motility was
observed at 37 C due to a complete loss of flagella.
The percent quanine plus cytosine for ERMB was found
to be 47.95 ± 0.45 (95 percent confidence interval).
This
work supports the proposal of Yersinia ruckeri as the genus
and species designation for ERMB.
Enteric Redmouth Bacterium of Salmonids: A Biochemical
and Serological Comparison of Selected Isolates
by
Patrick John O'Leary
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
June 1977
APPROVED:
Redacted for Privacy
oSsor
or I1crob1oiogy
in charge of major
Redacted for Privacy
Department of Micr
Redacted for Privacy
Dean of Gradua
Date thesis is presented
February 3, 1977
Typed by Deanna L. Cramer for Patrick John O'Leary
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to those who
contributed directly or indirectly in this work.
To Dr. J. L. Fryer for his support, patience and
assistance, but most of all for giving me a chance.
To Dr. JoAnn C. Leong for her continued help and
encouragement.
To Dr. R. Seidler, for his excellent technical
assistance.
To Drs. W. H. Ewing and D. Brenner for their counsel
and helpful suggestions.
To the staff of the Oregon Department of Fish and
Wildlife, in particular James Sanders, Rich Holt, and
John Conrad.
To the people who are or were of this lab, especially
Rowan Gould, Dan Mulcahy, Jerry Zinn, and John Rohovec.
To A. Soeldner and J. Knopper of the Oregon State
University Department of Botany, Electron Microscope
Laboratory.
To Mrs. M. Shadow and Ms. S. Suki, for their ever
present trust and affection.
To my parents for their support and understanding.
Above all, to my wife Chris, for enduring.
Funds for these experiments were provided by the Oregon
Department of Fish and Wild1fe and financed in part by
Anadramous Fish Act Fund (PL-89--304).
TABLE OF CONTENTS
Page
INTRODUCTION. ....................
1
LITERATUREREVIEW ............ . ......
3
Enteric Redmouth Bacterium ............
Literature Review of the Genus Yersinia
.
.
.
.
3
13
MATERIALS AND METHODS ................. 20
Solutions and Media Commonly Employed ......
Isolate Location and Selection ..........
Determination of Viable Count versus Optical
Density of an Enteric Redmouth Bacterium.
Experimental Animals ...............
Serological Methods ...............
Bacteria Antisera ..............
Slide Agglutination Tests ..........
20
20
23
23
24
24
25
Adsorption of Rabbit Anti-Enteric
Redmouth Bacterial Antisera ........
25
Determination of Agglutinating
Antibody Titers ..............
Ouchterlony Double Diffusion Plates .....
Biochemical Tests ................
Electron Micrographs ...............
Isolation of Deoxyribonucleic Acid ........
Percent Guanine Plus Cytosine Determination
LD50 Determination of Selected Isotopes of
Enteric Redmouth Bacterium in Rainbow
Trout and Mice .................
26
27
27
29
30
31
33
Determination of Enteric Redmouth Bacterial
Serotype Cross-Protection ...........
34
RESULTS ........................
36
Optical Density versus Viable Count of
Isolate HI-70 ................. 36
Selection of Enteric Redmouth Bacterium
Isolates for Serological Comparison ...... 36
Determination of Serotype Scheme by Cross-
Adsorption ................... 39
Biochemical Tests ................ 47
Slide Agglutination Tests of Six Enteric
Redmouth Bacterium Isolates .......... 53
Ouchterlony Double Diffusion Plates of
Enteric Redmouth Bacterium ........... 58
Table of Contents (continued)
Page
Estimated Lethal Dose of Fifty Percent
(LD50) for Rainbow Trout ............ 61
ERMB LD50 Determination for Mice ......... 62
Determination of Cross-Protection between
Serotypes I and II ............... 63
Effects of Temperature on Flagellation and
Motility of Enteric Redmouth Bacterium as
Ascertained by Electron Microscopy ....... 65
Determination of Percent Guanine plus
Cytosine .................... 68
Comparison of Enteric Redmouth Bacterium to
the Genus Yersinia ............... 71
DISCUSSION ....................... 81
SUMMARY AND CONCLUSION ................. 86
BIBLIOGRAPHY...................... 88
LIST OF TABLES
Page
Table
1
Distinguishing characteristics of the
species within the genus Yersinia ....... 16
2
3
4
5
6
7
Isolation data on enteric redmouth bac-
teria used in these studies .......... 22
Titers of a known enteric redmouth bacterium antisera against selected enteric
redmouth bacteria isolates
...........
38
Serological comparison of enteric redmouth
bacterial isolates BC-74 and HI-70 .......
41
Serological comparison of enteric redmouth
bacterial isolates BC-74, HI-70 and SC-72
.
43
..................
44
.
.
Serological comparison of enteric redmouth
bacterial isolates BC-74, HI-70, SC-72,
and SP-70
Serological comparison of enteric redmouth
bacterial isolates BC-74, HI-70, SC-72,
SP-7OandTH--75 ................
8
Serological comparison of enteric redmouth
bacterial isolates BC-74, HI-70, SC-72,
SP-70, TH-75 and OS-76 .............
9
12
13
50
Result of slide agglutination tests
between six isolates of enteric redmouth
bacterium ...................
11
48
Biochemical comparison of 17 enteric
redmouth bacterial isolates at four dif-
ferent temperatures ..............
10
46
54
Comparison of protection of rainbow trout
to enteric redmouth bacterium by injection
of monovalent and bivalent bacterins ......
64
Individual and average guanine plus cytosine values for enteric redmouth bacterium
as determined by thermal melting ........
68
Table of thermal melting points for each
run of eriteric redinouth bacterium using
E. coli WP2 as standard
...........
71
List of Tables (continued)
Page
Table
14
15
Comparison of enteric redmouth bacterium
to the five tribes of Enterobacteriaceae.
.
.
.
77
Definitive characteristics of the genus
Yersinia as they relate to enteric red-
mouth bacterium ................ 78
16
Distinguishing characteristics between the
species of Yersinia and enteric redmouth
bacterium ................... 8 0
LIST OF FIGURES
Page
Figure
1
Growth curve of isolate HI-70 in BHI
broth at 18 C plotting viable count
versus optical density at 525 nm ....... 37
2
3
Slide agglutination tests of enteric
redmouthbacterium .............. 55
Ouchterlony double diffusion plates of
selected isolates of enteric redmouth
bac te r i unt ................... 5 9
4
Electron micrographs of single cells of
enteric redmouth bacterium incubated at
selected temperatures ............ 66
5
Electron micrographs of enteric redmouth
bacterium cell groups incubated at
selected temperatures ............. 69
6
Deoxyribonucleic acid thermal denaturation
curves for isolates E. coli WP2, HI-70,
and BC-74 ................... 72
7
Deoxyribonucleic acid thermal denaturation
curves for isolates E. coli WP2, BC-74 and
SC-72 ..................... 73
8
Deoxyribonucleic acid thermal denaturation
curves for isolates E. coli WP2, HI-70,
and SC - 7 2 .
.
................
7 4
ENTERIC REDMOUTH BACTERIUM OF SALMONIDS: A BIOCHEMICAL
AND SEROLOGICAL COMPARISON OF SELECTED ISOLATES
INTRODUCTION
Enteric Redmouth Disease (ERMD) is of vital importance to salmonid aquaculture.
It can be easily confused
with diseases caused by Gram negative, oxidase positive
bacteria.
The Enteric Redmouth bacterium (ERMB) responds
poorly to the antibiotic treatments used for most other
diseases, thus necessitating a prompt and accurate
diagnosis.
As with all enteric bacteria, resistant forms occur
that are insensitive to various antibiotics.
To circum-
vent this problem, vaccination as a prophylactic measure
against specific etiological agents has shown great promise.
Recently companies have been formed to research and
produce vaccines and bacterins for salmonids.
The ERNB
has been proposed by various commercial enterprises for
bacterin production and has just recently been licensed by
one of these companies for use in the United States.
Therefore, it has become imperative to obtain knowledge
concerning the serology and especially the protective
antigens of ERNB so that the most efficacious bacterin can
be produced.
This study investigates the antigenic composition of
six ERMB isolates by agglutinating and precipitating
reactions.
These antigens are correlated to protection in
viva utilizing rainbow trout (Salmo gairdneri) and an
antigenic scheme is proposed.
to classify ER
Finally, an attempt is made
taxonomically by various biochemical
reactions and bacterial deoxyribonucleic acid molar percent
guanine plus cytosine.
3
LITERATURE REVIEW
Enteric Redmouth Bacterium
A disease syndrome termed Itredmoutht was first ob-
served in California among salmonids in 1948 (wagner and
Perkins, 1952) and three years later in Colorado.
The
isolated organism was a gram negative, single polarly
flagellated, oxidase positive bacillus, with a typical
convex, entire, smooth, milky-white colony morphology on
Brain Heart Infusion agar.
The bacterium was classified
at the time as Pseudomonas hydrophila and has since been
reclassified as Aeromonas hydrophila (Buchanan and Gibbons,
1974).
Another bacterium presently classified as Pseudo-
monas flourescens has also been shown to be the etiological agent of a very similar malady (Bullock and McLaughlin,
1970).
The pathological symptoms are identical; therefore,
the only means to differentiate between these two bacteria
is by isolation and identification of the causative
organism.
Both A. hydrophila and P. flourescens have been
reviewed extensively (Bullock and McLaughlin, 1970;
Snieszko and Bullock, 1968; and Eddy, 1962).
In the early 1950ts Rucker isolated still another
bacterium from rainbow trout (Salmo gairdneri) at the
Hagerman National Fish Hatchery in Idaho with similarly
accompanying pathological symptoms.
This bacterium was
4
also a gram negative, motile rod, but was oxidase negative.
This represented the first oxidase negative fish pathogen
ever to be isolated.
The first comprehensive paper on the
bacterium (Ross, Rucker and Ewing) did not appear until
1966.
This oxidase negative bacterium will be the subject
of this thesis.
Confusion occurs in the literature due to the various
names given the diseases associated with these bacteria.
They have been referred to as redmouth disease, red vent
disease, bacterial hemorrhagic septicemia, red throat and
pink throat disease.
In an attempt to clarify some of the
nomenclature, McDaniel (1971) proposed this disease be referred to as Hagerman redmouth disease (HRM) after the
locality in which it was isolated and originally endemic.
The disease associated with A. hydrophila or P. flourescens
would thenceforth be called bacterial hemorrhagic septicemia (BHS).
Due to the continued spreading in geographi-
cal distribution and the stigma associated with the Idaho
trout farmers, the Technical Procedures Committee of the
Fish Health Section of the American Fisheries Society
(Denver Workshop, 1974) proposed Enteric Redmouth Disease
(ERMD) as the descriptive title of the disease (Dulin et
al., 1976).
This is the only "redmouth" disease bacterium
belonging to the family Enterobacteriaceae (Ross et al.,
1966) and still has not been given a genus and species
classification.
The only significant taxonomical work done
5
after the original publication with the bacterium was
comparing the deoxyribonucleic acid (DNA) homology relatedness of ERMB to different genera of bacteria (Steigerwalt
et al., 1975).
They found that ERMB was not closely re-
lated to the genera Serratia or Klebsiella.
In the 1950's the geographical distribution of ERMD
was limited to an endogenous focus of infection in the
Hagerman Valley, Idaho.
By the mid 1960's it had spread to
Colorado, Nevada, California and Arizona (Ross et al.).
McDaniel reported in 1971 that the disease had spread to
Alaska, Wyoming, Washington, Oregon and Utah. and Wobeser
(1973) extended the observations to Sasckatchewan, Canada.
To date the disease has been documented in Nebraska
(McElwain, 1975), Missouri (Camenish, 1975), Ohio (Bullock
and Snieszko, 1975) and Tennessee (McCraren and Warren,
1973).
This spread of endemic range for the disease has
been attributed to the transport of live undetected asymptomatic fish and possibly infected eggs (Rucker, 1966;
Wobeser, 1973; Bullock and Snieszko, 1975; Busch and Lingg,
1975)
Busch and Lingg (1975) have documented the carrier
state for ER
in yearling rainbow trout (S. gairdneri) and
have data to support the hypothesis of a recurring cyclic
nature of infection.
They challenged one group of fish
with an intraperitoneal injection (IP) of approximately 18
cells and another group of heat stressed fish with a water
borne challenge of 2.75 x 106 cells/ml.
Mortality started
at 5 and 7 days,respectively,and subsided temporarily on
day 15 postinfection.
Recurring mortalities were then ob-
served between days 59 and 75 postinfection.
The gross
pathology was also recorded for the duration of the experiment which was terminated on day 102.
Attempts were made
to recover the bacterium from various tissues during the
course of the experiment.
It was readily isolated from
all indicator tissues sampled from symptomatic fish.
In
asymptomatic carrier fish, if the bacteria could be cul-
tured, it could only be cultured from the lower intestine
and was also found to occur in a cyclic nature.
Approxi-
mately 50-75 percent of the surviving fish were positive
from the intestine but still negative upon culturing kidney
material which is normally taken in a routine diagnostic
procedure.
On the basis of mortality rates, gross patholo-
gical changes and recovery of the organism, a regular 36-40
day cycle of intestinal shedding of the ERMB has
been hypothesized which preceeds the reappearance of gross
pathological changes and mortality by 3-5 days.
The actual
cycle length could vary depending upon the natural resistance, immunity and stresses imposed upon the fish.
This
hypothesis is substantiated by McDaniel (1971) who documented a similar mortality cycle in yearling rainbow trout
chronically infected with ERMB.
The fact that the bacterium
could only be isolated in the lower intestine of
7
asymptomatic carrier fish and not normally sampled tissue
could explain why the geographical boundaries of the disease progressed as they did.
Enteric redmouth bacterium
could never be detected by the normal sampling procedures
used to certify fish before transport.
Busch (1973) has
proposed the passive hemagglutination test to be standard
procedure for the detection of fish exposed to ERNB because
of it's greater sensitivity.
Water has been shown to be an effective route by which
ERMD can be transmitted in the laboratory (Busch and Lingg,
1974; Ross et al., 1966; Rucker, 1966).
In the natural
environment the disease may be transmitted by the regular
shedding of the bacteria by symptomatic or asymptomatically
infected fish or by fish to fish contact (Busch and Lingg,
1975; Bullock and Snieszko, 1975).
Since the spread of the
disease in nature has been associated with the transport of
infected fish, the bacterium is not suspected of being
ubiquitous.
It has been isolated from rainbow trout
and steelhead trout (S. gairdneri), cutthroat trout (S.
clarki), sockeye salmon (Onchorhynchus tshawytscha), coho
salmon (Oncorhynchus kisutch) and chinook salmon
(Oncorhynchus tshwytscha).
It has never been detected
from other aquatic animals in the headwaters which supply
hatcheries.
This observation is made because no epizootic
or mortalities in the headwaters has occured where ERNB
has been isolated.
A serious effort to detect a natural
reservoir of infection for ERNB has never been effected,
therefore this possibility still exists.
In the original paper Ross et al.
(1966) described
ERNB as a gram negative, peritrichously flagellated,
fermentative bacillus approximately 2-3 pm in length by
1 pm in width.
The cells normally appear singularly but
tend to become filamentous as the culture ages.
The cob-
flies on nutrient agar are cream colored, smooth, circular
and slightly raised with entire edges.
No pigmentation
has been observed and the colonies do not fluoresce under
ultraviolet light but may appear slightly irridescent when
examined by reflected light.
The biochemical characteristics of ERMB have been
documented by various sources (Ross et al., 1966; Busch,
1973; Wobeser, 1973) and reviewed by Dulin (1976).
The
distinguishing biochemical reactions at 25 C, described in
the original paper to separate ERMB from other species of
bacteria, are: oxidase negative, indole negative, methyl red
positive, Voges-Proskauer negative, Simmon's citrate positive, and fermentation of maltose and mannitol but neither
lactose or sucrose.
Ross et al. (1966) also found some of
the bacteria to be motile at 22 C but not at 37 C.
Homo-
geneity among the 14 isolates examined by Ross et al.
(1966)
and the 44 isolates studied by Busch (1973) was observed
in most of the biochemical tests conducted, although minor
discrepancies did occur.
The variation of certain test
results with incubation temperature or test method
under-
lies the difficulties in comparing results of different
authors.
Ross et al.
(1966) incubated his cultures at 37
and 22 C, while Busch incubated his at 25 C and Wobeser at
23 C.
The variability occurred in the hydrogen sulfide,
lysine decarboxylase, and arginine dihydroloase reactions.
Ross and Wobeser never observed hydrogen sulfide production, while Busch detected it in 100 percent of his isolates using lead acetate medium.
Busch also obtained 3
out of 44 negative methyl red tests.
Variability in both
the lysine decarboxylase and arginine dihydrolase was seen
by Busch but not by Ross at semi-comparable temperatures.
Enteric redmouth bacterium is very homogeneous and antigenically stable with only one serotype recognized (Ross et
al., 1966; Bullock and Snieszko, 1975; Busch, 1975).
et al.
Ross
(1966) noted that ERMB cells agglutinated in the pre-
sence of antisera against Arizona somatic antigen groups of
26 and 29.
The pathology and histopathology of ERMD have been
described many times in the literature (Ross et al., 1966;
Rucker, 1966; Busch, 1973; Wobeser, 1973; Dulin, 1976)
The disease is characterized by inflammation and petechial
hemorrhages about the jaws, palate, operculum, head, vent,
isthmus and base of rayed fin regions.
Exopthalmia common-
ly occurs which generally progresses from the unilateral to
a bilateral state with occasional rupturing of the eye.
The fish typically become sluggish and darken in color.
10
Splenomegaly with petechial hemorrhaging in the body fat
and distal portion of the intestine has been noted.
The
stomach may be filled with a watery colorless liquid and
the intestine with a yellowish fluid.
The histopathology of the disease is typified by an
acute septicemia with bacteria and the accompanying inflammatory response of a gram negative bacteria in virtually all tissues.
most detailed report.
Wobeser has the best described and
He stated in his 1973 paper:
The histopathological lesion observed
in fish from which the (ERN bacterium) was
isolated involved primarily the hematopoietic and reticuloendothelial (RE) systems.
In both the anterior and posterior kidney
there was a severe loss of hemapoietic
tissue, due to necrosis, together with
dilation of sinusoids, decrease in size
and numbers of pigment cells and swelling
and vacuolation of RE cells. Nephrons in
the posterior kidney did not appear to be
In the spleen there was a total
altered.
loss of the normal lymphoid follicular
structure, congestion of the sinusoids
with erythrocytes, and RE cell hypertrophy.
In the livers of the apparently healthy
fish, vacuoles were present in the hepatocytes of the diseased fish. The RE
cells lining the hepatic sinusoids of
diseased fish were swollen and prominent,
with vesicular nuclei. Very marked
accumulations of mononuclear cells were
present in the periportal area of livers
of diseased fish....Foci of necrosis, in
association with pooling of erythrocytes
in dilated sinusoids, produced the hemorrhages grossly visible in the livers of
some fish.
Periocular lesions were common, with
retrobulbar edema being present in all of
the fish from which the eyes were examined,
and panopthalmitis, characterized by
II
intraocular hemorrhage and inflammatory
cell infiltration, was present in 4 of
8 fish examined. The inflammatory cells
present within the eye were a mixture of
polymorphonuclear leukocytes and
macrophage-like cells.
The prescribed chemotherapeutic treatment for ERND
has varied, depending upon drug availability and the
author.
Rucker (1966) suggested a combination of sulf a-
merazine at 20 g/lOO kg of fish per day, followed by three
days of chioramphenicoloroxytetracycline at 5.5 g per 100
kg fish per day.
McDaniel (1971) described a treatment
schedule that Vern Bressler had developed consisting of
6.6 g sulfamerizine plus 4.4 g furazolidone per 100 kg
fish per day for five days.
After four days the mortality
declined and the treatment gave excellent results.
Snieszko (1975) reported that sulfamerazine, oxytetracycline and chioramphenicol are the most frequently used
drugs for this disease.
It should be noted,however, that
none of these drugs have been approved for use by the Food
and Drug Administration.
Various investigators have probed the problem of
immunizing fish against ERMD (Anderson and Ross, 1972;
Ross and Klontz, 1965; Anderson and Nelson, 1974).
Ross
and Klontz (1965) showed the first promising signs of
immunizing fish against ERMD in a small laboratory experiment.
A bacterin of 15 ml, wet, packed, phenol-killed
ERNB cells was incorporated and pelleted into 4.54 kg (10
12
ib) Commercial Trout Feed Neal.
Rainbow trout received the
bacterin five times a week for two weeks then weekly for
eight weeks.
The orally immunized fish showed a 90 percent
survival when challenged by intraperitoneal injection of
1-40 LD50ts compared to the controls which had only a 20
percent survival.
Anderson and Ross (1972) compared the
effectiveness of four different oral ERMD bacterins.
The
bacterins were incorporated into Complete Test Diet and it
was calculated that each fish received 1,500 mg of bacterin
during the course of the experiment.
analyzed on the basis of an LD50.
The bacterins were
The experimental groups
are listed in descending order of protection:
3 percent
chloroform killed cells > 3 percent phenol killed,then
dialysed cells > 0.5 percent phenol killed,then washed
cells > sonicated plus 1 percent formalin cells.
In 1974
Anderson and Nelson compared inoculated to orally fed ERMD
bacterins.
The bacterins were prepared by killing ERMB
cells with three percent formalin for four days.
Approxi-
mately 1 mg of lyophilyzed bacterin was injected into
fingerling rainbow trout and the same amount was incorporated into Complete Test Diet and fed over a two week
period.
The bacterins were compared again on the basis of
LD501s.
It was demonstrated that both bacterins provided
protection, but the inoculated fish had a higher level and
duration of protection than did the orally fed fish.
Anderson et al. (1974) found humoral agglutinating
13
antibodies in the injected group of fish,but neither
Anderson et al.
(1974) or Anderson et al. (1972) found
humoral agglutinating antibodies in the orally fed groups
of fish.
Wildlife Vaccines Inc. of Denver, Colorado pre-
sently have the only commercially licensed ERMD bacterin.
They have vaccinated fish by the immersion method and have
shown promising results (personal communication, Dr. John
Rohovec, June, 1976, Wildlife Vaccines Inc., Denver,
Colorado).
Literature Review of the Genus Yersinia
The genus Yersinia is presently comprised of three
species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica.
Due to the previously disputed taxonomical loca-
tion of these bacteria, the species have been referred to
by various synonyms.
Yersinia pestis and Y. pseudotubercu-
losis were originally named Bacterium pestis and Bacillus
pseudotuberculosis,respectively.
By the sixth edition of
Bergeyts Manual of Determinative Bacteriology they had both
been included in the genus Pasteurella (Breed et al., 1948).
Yersinia enterocolitica was first isolated in 1949 by
Hässig et al.
It has been referred to as Bacterium entero-
colitica, Pasteurella X, Germe X, and Pasteurella pseudotuberculosis type b (Nelehn, 1969).
In 1944 van Loghem
proposed the oxidase variable genus Pasteurella be divided.
The oxidase negative bacteria becoming the new genus
14
Yersinia after the French bacteriologist A. J. Yersin, who
first isolated the etiological agent of plague in 1884.
Thai proposed in 1954 that Yersinia be included in the
family Enterobacteriaciae.
A numerical method analysis
supported the emergence cf the new genus Yersinia
(Smith
and Thai, 1965) and the relationship of it to the Entprobacteriaceae (Taibot and Sneath, 1960).
Therefore, a new
tribe Yersinieae has been accepted containing only the
genus Yersinia.
A new bacterium has been isolated from
the muskrat (Ondatra zibthica) and has been tentatively
named Y. philomirgia (Jensen et al., 1969).
The eighth
edition of Bergey's Manual of Determinative Bacteriology
recognizes only three species:
Y. pestis, Y. pseudotuber-
culosis, and Y. enterocolitica (Buchanan and Gibbons,
1974)
The genus Yersinia is described by Mollaret and Thai
in the eighth edition of Bergey's Manual of Determinative
Bacteriology as being gram and oxidase negative.
The
cells are ovoid or rods, 0.5-1.0 by 1-2 pin and not encapsulated.
Yersiniapestis is always non-motile.
Yersinia
pseudotuberculosis and Y; entercclitica are motile at
temperatures below 30 C.
Biochemically the genus is nega-
tive for lysine decarboxylase, malonate utilization, tetrathinate reduction, and citrate as a sole carbon source.
They test methyl red positive.
Voges-Proskauer is nega-
tive for all species at 37 C but usually positive for Y.
15
enterocolitica at 22 C.
Nitrate is usually reduced to
nitrite and they are catalase positive.
Indole is nega-
tive for Y. pestis and Y. pseudotuberculosis but variable
The genus will attack carbohydrates
Y. enterocolitica.
fermentatively.
Glucose, maltose, mannitol, trehalose,
glycerol, xylose, and fructose are fermented with acid but
no gas.
No fermentation of dulcitol, erythritol, fucose,
inositol, glycogen, raffinose, or melezitose can be detected.
Lactose is usually not fermented, but
sidase is produced.
from -2 to 45 C.
-galacto-
The temperature range is said to be
The colonies are non-pigmented.
The
guanine plus cytosine content of the deoxyribonucleic acid
(DNA) for the genus ranges from 45.8-46.8 moles percent as
ascertained by thermal melting (Mollaret and Thal, 1974).
Distinguishing biochemical characteristics between
species of Yersinia are summarized (Table 1).
The reac-
tions have been compiled from Mollaret and Thai (1974),
Sonnenwirth (1974), and Nilehn (1969).
The differentiation
of the species presently is based on motility, production
of urease, ornithine decarboxylase, indole and H2S; dtartrate utilization; and the fermentation of esculin,
sucrose, cellobiose, melibiose, and adonitol.
The species
can be definitively ascertained by susceptibility to known
bacteriophage, agglutionation with specific antisera,
flourescent antibody staining, and susceptibility of experimentally infected animal hosts.
The results utilizing
Table 1.
Distinguishing characteristics of the species
within the genus Yersinia.a
pestis
Y. pseudotuberculosis
Y.
enterocoliticu
Motility
37°C
+
+
22°C
-
-
Urease
-
+
+
Ornithine
decarboxylase
-
-
+
Esculin
+
+
-
Sucrose
-
Cellobiose
-
-
Melibiose
-
+
Adonital
-
+
d-tartrate
+
-
-
H2S production
+
-
-
+
+
aRevised from Nilehn, 1969; Moliaret and Thai, 1974; and
Sonnerwirth, 1974.
specific antibodies must be carefully interpreted due to
cross-reactions which will be discussed later.
Yersinia enterocolitica has been subdivided further
into five biotypes (Nilehn, 1969).
The biotypes are cate-
gorized according to their indole, nitrate reduction,
ornithine decarboxylase, Voges-Proskauer, B-galactosidase
reactions, and the fermentation of salicin, esculin, lac-
tose, xylose, trehalose, sorbitol, sucrose, and sorbose.
The biotypes tend to group correlating to the host from
which the isolate was taken.
In the original paper (Nilehn,
1969), strains of biotype five were isolated from hares
17
with one from a rabbit.
Ninety percent of all strains iso-
lated from man, the majority of strains isolated from pigs,
one dog strain, and two strains each from a monkey and
guinea-pig were all found to be biotype four.
Biotype
three consisted of most chinchilla strains and 14 out of
218 isolates from man.
Biotypes one and two comprised
strains isolated from various sources; man, chinchilla,
cow, horse, sheep, and guinea-pig.
Yersinia pestis has been shown to possess at least
ten different antigenic components as evaluated by
Ouchterlony gel diffusion plates (Schwartz, 1973).
Schütze
(1932) was the first to show separate envelope and somatic
antigens.
The "VW" antigens and "fraction 1"
the major virulence factors for Y. pestis.
(Fl) comprise
They are both
produced during growth at 37 C but not at 28 C or below
(Schwartz, 1973).
The Fl antigen is a heat-labile protein
corresponding to the capsule of the fully virulent bacteria.
The VW antigen system is comprised of a protein V
fraction (MW 90,000) and a lipoprotein W fraction (MW
90,000) whose coordinate action is antiphagocytic as is
Fl.
The V antigen appears to be cell-bound.while the W
antigen is excreted into the media.
A diagnostic antigenic scheme of 10 serotypes for Y.
pseudotuberculosis based on 15 somatic and 5 flagellar
antigens has been produced (Thai and Knapp, 1971).
Serolo-
gical cross-reactions occur frequently with other organisms.
This may be exemplified by serofactor 1.
This is an R-
somatic antigen which is common to all serotypes of Y.
pseudotuberculosis, Y. pestis, Shigella flexneri, Shigella
sonnei, and Escherichia coil (Wetzler, 1970).
It has also
been shown to be a minor component of Y. entercolitica.
The ability of Y. pseudotuberculosis to immunize against
Y. pestis is due to the fact that serofactor 1 is a protec-
tive antigen for both species (Thai, 1955).
Three different antigenic schemes have been proposed
for Y. enterocolitica.
Wauter (1970) has proposed 17 sero-
types based on the thermostabie somatic antigens (Sonnenwirth, 1974).
A simplified version of this scheme based on
both the somatic antigen and biochemical characteristics
has been proposed by Knapp and Thai (1973) where only six
serotypes are recognized.
Winblad (1968) has also proposed
an antigenic scheme where eight different somatic antigens
were found and incorporated into nine serotypes.
Serotype
nine is comprised of the strains of Y. enterocolitica that
have been untypeable in other schemes.
This serotype has
also been shown to cross-react with Brucella abortus
(Ahvonen et al., 1969).
colitica
The various biotypes of Y. entero-
described previously tend to overlap when com-
pared to the Winblad somatic-antigen system (Nilehn, 1969).
Strains of biotype four appear homogeneous in regard to
their somatic antigen and belong to serotype three.
Bio-
types 1, 2, and 3 are antigenically heterogeneous; strains
19
of biotype 1 belong to serotype 5, 6, 7, or 9; strains of
biotype 2 belong to serotype 5, 8, or 9.
consists of serotypes 1, 2, 4, 5, or 9.
Biotype three
All of the human
isolates from biotype 5 belong to serotype 9, and most of
the chinchilla isolates corresponded to serotype 1.
The reservoirs for each of the three species are pri-
manly found in animals.
Yersinia pestis utilizes rodent
and insect vectors (Schwartz, 1973). Yersinia pseudotuberculosis and Y. enterocolitica have been found in a
wide variety of mammals and birds (Sonnenwirth, 1974;
Wetzler, 1970).
The latter two are thought to be trans-
mitted by fecally contaminated forage or foodstuffs
(Nilehn, 1969).
Experimentally, Y. pseudotuberculosis has
been found to be pathogenic for mice, guinea-pigs, gerbils,
and white rabbits (Sonnenwirth, 1974).
Yersinia enteroco-
litica has not been found to be pathogenic by intraperitoneal, intravenous, or subcutaneous injection of the
above mentioned laboratory animals.
An atypical strain
was isolated in 1973 from man that was shown to be pathogenic in mice by intravenous and oral routes (Carter et al.,
1973).
Chinchillas have recently been shown to be sus-
ceptible to Y. enterocolitica and are presently being considered as a model host (Wetzler, 1970).
20
MATERIALS AND METHODS
Solutions and Media ... oflnonly Ernplyed
Phosphate buffered saline (PBS) was made as described
by Williams and Chase (1968) to pH 7.2 with the following
constituents:
NaC1
KH2
PO1
Na2HPO7H2O
g/lOO ml
Mo 1 an ty
4.38
0.075
2.45
0.018
15.28
0.057
to volume
H2O
The buffer was autoclaved and stored until used in 200 ml
clear glass prescription bottles.
Brain Heart Infusion (BHI) agar and broth were made
with and according to the directions of Difco (Detroit,
Michigan).
The BHI broth was prepared by adding 37 g BHI
broth to one liter of distilled water.
If BHI agar was
desired, then agar was added at a rate of 1.5 percent to
the BHI broth.
The medium was sterilized and dispensed
according to standard aseptic laboratory procedures.
Isolate Location and Selection
The ERNE strains used in this study were furnished by
R. A. Holt and J. E. Sanders of the Oregon Department of
21
Fish and Wildlife and Dr. D. F. Amend of Tavolek Laboratories Incorporated, Seattle, Washington.
Each isolate was cultured on BHI agar and examined
macroscopically for purity, colony morphology and pigmentation.
stain.
Cells were microscopically observed by Gram
Morphology and motility were examined by means of
phase contrast microscopy.
All cultures were subjected to
the eight biochemical tests as recommended by Ross, Rucker,
and Ewing (1966).
Each isolate showed characteristic
INViC reaction, -+-+, fermentation of maltose and mannose
but not lactose or sucrose at 22 C.
The cultures obtained
from diseased fish that conformed to these tests were considered true ERNB isolates.
Cultures of each isolate were
preserved on BHI slants under mineral oil and stored at
room temperature.
The acquisition data for each isolate
has been summarized (Table 2).
The isolates for serological comparison were chosen
on the basis of a difference in titer to a known EPMB antiserum and diversity of geographical location.
Normally a
constant known antigen is maintained and unknown antibody
is then titered against it.
In this experiment the
variability in serology of the bacteria was of concern and
not the antibody.
Therefore, known anti-ERMB antisera was
obtained from J. E. Sanders to be used as a constant antibody source, while different bacterial isolates serve as
the antigen.
Any gross difference in titer could be taken
Table 2.
Isolate
Code
Isolation data onentericredmouth bacteria used in these studies.
Origin
Date
Isolated
Host
Isolated
by
Culture
Obtained from
BC-74
Big Creek Hatchery, Oregon
1974
Fall Chinook
R.
HI-70
Hagerinan, Idaho
1970
Rainbow Trout
G. Post
SC-72
Saskatachwan Potholes, Canada
1972
Rainbow Trout
G.
Wobeser
D.
SP-70
Salem Pond, Oregon
1970
Chinook Salmon
R.
Holt
R.
Holt
TH-75
Trask Hatchery, Oregon
1975
Coho Salmon
R.
Holt
R.
Holt
OS-76
Oak Springs Hatchery, Oregon
1976
Rainbow Trout
J.
Sanders
J. Sanders
RR-70
Roaring River Hatchery, Oregon
1970
Rainbow Trout
R. Holt
R.
Holt
SS-75
South Santiam Hatchery, Oregon
1975
Summer Steelhead
R. Holt
R.
Holt
SH-75
South Santiam Hatchery, Oregon
1975
Summer Steelhead
R.
Holt
R.
Holt
ER-75
Elk River Hatchery, Oregon
1975
Fall Chinook
R.
Holt
R.
Holt
EL-75
Elk River Hatchery, Oregon
1975
Fall Chinook
R.
Holt
R.
Holt
BC-75
Big Creek Hatchery, Oregon
1975
Fall Chinook
R.
Holt
R.
Holt
BH-75
Big Creek Hatchery, Oregon
1975
Fall Chinook
R.
Holt
R.
Holt
WS-75
Willamette Salmon Hatchery, Oregon
1975
Winter Steelhead
R.
Holt
R.
Holt
WS-74
Willamette Salmon Hatchery, Oregon
1974
Spring Chinook
R.
Holt
R.
Holt
WT-75
Willamette Trout Hatchery, Oregon
1975
Rainbow Trout
R.
Holt
R
Holt
GC-70
Gnat Creek Hatchery, Oregon
1970
Steelhead
R0
Holt
R.
Holt
Holt
R.
Holt
D
Pmend
mend
L.J
23
as a difference in agglutinating serological composition of
the bacteria.
The techniques by which the titers were
determined are explained in a later section.
Determination of Viab ....... ount v'ersus Optical Density of an
Enteric Redmouth Bacterium
Side arm flasks with 75 ml of BHI broth were inoculated with 0.2 ml of an overnight culture of isolate HI-70.
Flasks were incubated at 18 C.
At periodic intervals plate
counts were performed in triplicate and the optical density
at 525 nm was measured in a Spectronic 20 using BHI broth
as a reference.
Experimental Animals
Adult New Zealand white rabbits (4 kg/rabbit) and
White Swiss Webster mice (18 g/mouse) were obtained from
the Oregon State University Laboratory Animal Resource
Center.
The rabbits were kept in separate cages while the
mice were maintained at three per cage.
Rainbow trout (4.5 g/fish) were obtained from the
Oregon Department of Fish and Wildlife, Wizard Falls
Hatchery.
The fish were held in fiberglass 69 liter,
rectangular, self-cleaning aquaria equipped with fiberglass
lids containing glass portals to maintain the normal photoperiod.
Each aquarium was supplied with aerated, 12 C fish
pathogen free well water.
24
Serological Methods
Preparation of Rabbit Anti-Enteric Redmouth Bacteria
Antisera
Isolates BC-74, SC-72, SP-70, HI-70, TH-75, and OS-76
were taken from stock cultures, inoculated into BHI broth
and incubated for 24 hr at room temperature.
To insure
culture purity, a Gram stain was examined under light
microscopy and the cells were checked for motility by means
of phase contrast microscopy.
The culture was plated out
onto BHI agar to observe colony morphology and homogeneity.
The cells in broth culture were then killed with formalin
(0.3 percent v/v).
After overnight incubation at room
temperature, one ml of the culture was placed into Thioglycolate broth to examine for sterility.
The killed cells were washed three times with PBS and
incorporated with an equal volume of Freund's complete adjuvant.
Approximately 1.5 ml of this preparation was in-
jected into each hind flank of the rabbit.
After three weeks blood was obtained from the marginal
ear vein of each rabbit.
The clot was allowed to retract
for an hour at room temperature and then overnight at 4 C.
The serum was harvested, centrifuged at 5000 x g, filter
sterilized with a 0.45 jim filter and stored at 4 C.
The
rabbits were bled twice more over the next three weeks.
After the final bleeding and processing of the blood, each
appropriate antisera from the three collections were
pooled and filter sterilized through a 0.22 pm filter.
The
25
serum was then distributed into two ml vials and frozen at
-60 C until used.
Slide Agglutination Tests
One loopful of bacteria grown on BHI agar was added to
0.5 ml PBS and stirred on a vortex mixer to achieve a uniform suspension of cells.
To one well of a concavity slide,
one drop of PBS, bacterial cell suspension, and antisera
each were added.
To another well two drops of PBS and one
drop of bacterial cell suspension were added to serve as a
control for evaluation of autoagglutination.
The slide was
then gently rotated for two minutes to mix the solutions.
Agglutination of the cells was determined both macroscopically and microscopically with the aid of a dissecting
microscope.
The agglutination was recorded as either nega-
tive when no discernable agglutination occurred or positive
when agglutination of the cell suspension was observed.
Adsorption of Rabbit Anti-Enteric Redmouth Bacterial
Antis era
Isolates BC-74, HI-70, SC-72, SP-70, TH-75 and OS-76
were cultured on BHI agar in 32 oz prescription bottles at
room temperature.
The cells were harvested and washed
three times in PBS.
Each centrifugation in the cross ad-
sorption employed a relative centrifugal force of 7200 x g
to insure that a minimal amount of diluting would occur
during the procedure.
26
Each isolate was adsorbed against its homologous and
every heterologous antiserum by the following procedure:
a
volume of antiserum was added to an equal volume of packed,
washed cells; the cells were resuspended in the antiserum
and allowed to incubate for two hours at 30 C with periodic
mixing; the cells were centrifuged and the remaining antiserum was adsorbed two additional times in the same fashion.
After the final adsorption each antiserum was titered
against the cells which it was adsorbed against to insure
that all conunon antibodies were adsorbed out.
Determination of Agglutinating Antibody Titers
The bacterial cell antigens were prepared by growing
the appropriate ERMB isolate in BHI broth overnight at
room temperature.
The cells were washed three times in
PBS and resuspended in PBS to an optical density of 0.85
at 525 mu.
The microtiter system (Cooke Engineering,
Alexandria, Virginia) was employed using 0.025 ml dilutors
and pipets.
Two fold dilutions of the samples were made
in disposable clear plastic plates with "U" shaped wells.
Controls with no antisera were run in each plate to detect
any autoagglutination.
After the dilutions were made and
the bacterial cell antigens were added, the plates were
gently mixed and covered to prevent evaporation.
They were
incubated for one hour at room temperature and then over-
27
night at 4 C.
The dilution of the last well to show agglu-
tination was taken as the titer for that sample.
Ouchterlony Double Diffusion Plates
Ouchterlony double diffusion plates employed sterile
0.8 percent agarose in 0.05 M Tris buffer (Sigma).
The
plates were poured to a depth of approximately 5 mm and
allowed to harden.
The center antiserum well was cut with
a number three cork borer and the surrounding antigen wells
with a number two cork borer.
The antigens were prepared
by inoculating each isolate on BHI agar in 32 oz prescripThe cells were harvested and washed
tion bottles at 18 C.
three times in PBS.
They were then resuspended in a mini-
mal volume of PBS and sonicated until the cells were disThe antisera and
rupted as determined by phase microscopy.
antigens were then placed in the appropriate wells and the
plates were allowed to incubate in a moisture chamber.
After the precipitin bands had developed, photographs were
taken using Kodak high contrast copy film.
Biochemical Tests
In general, the biochemical tests used were those recommended by Edwards and Ewing (1972) or Lennette, Spaulding and Truant (1974).
However, some of the tests used
were not mentioned in these references.
The sugars were
incorporated into Purple broth base (Difco) at a
28
concentration of 0.5 percent (w/v) .
The NaC1 concentra-
tion for the salt tolerance test was measured as percent
salt (w/v) added to BHI broth.
All biochemical tests were
incubated at 9, 18, 27, or 37 C.
The assay used for the detection of deoxyribonuclease
(DNase) and ribonuclease (RNase) was determined by the
method of Jefferies et al. (1957).
DNA (Sigma; Type II,
sodium salt, highly polymerized, from calf thymus) and RNA
(Sigma; Type 11-S1 sodium salt from Torula Yeast) were in-
corporated into BHI broth with 1.5 percent Noble agar
(Difco) at a final concentration of 2 mg/ml.
Zones of
clearing after flooding the plate with lN HC1, indicated
hydrolysis of the substrate.
Lipase was assayed by the method of Holding and Collee
(1971).
Tween 20, 40, 60 or 80 (Sigma) were added to give
a final concentration of 1 percent (v/v) with 1.5 percent
(w/v) Noble agar.
Lipolytic activity was indicated by the
presence of a precipitate in the agar around the colony.
Elastase and Fibrinolyase were determined by the
method of Sbarra et al.
(1963).
Each substrate was incor-
porated into a minimal salts preparation and served as the
sole carbon source.
Zones of clearing around the colony
evidenced the enzymatic activity.
Electron Micrographs
Isolate HI-70 was selected for all electron microscope (EM) studies.
The cells were grown in BHI broth at
9, 22, and 37 C for three passages each to acclimate the
bacteria to the respective temperature.
then inoculated into 15 ml
The cells were
BHI broth and grown at the
respective temperature to middle log phase.
Glutaralde-
hyde was added to a final concentration of 1.5 percent
(v/v) and the culture was allowed to incubate for another
two hours.
The cultures from each of the three tempera-
tures were then acclimated to 22 C and dialyzed overnight
against distilled water to remove excess salt.
were centrifuged at 1085 x g.
The cells
The supernatant was dis-
carded and the pellet was resuspended in 2-4 drops of distilled water.
One drop of the cell suspension was placed
on a 300 mesh Formvar coated copper screen.
The excess
liquid was blotted off with filter paper and the screen
was allowed to air dry.
Varian Model yE-b
The sample was shadow cast in a
vacuum evaporator at 1 x l0
Torr.
Platinum-palladium (80:20) was used to shadow cast at
approximately 35° angle with a specimen to source distance
of 6 cm.
The shadow casting and EN operations were per-
formed by A. H. Soeldner and J. A. Knopper of the Oregon
State University Botany Electron Microscope Laboratory.
Images were observed on a Philips EM-300 transmission
30
electron microscope and recorded on Kodak electron image
plates.
The photographs in this thesis were then produced
from the plates by L. Nelson of Scott Photo, Corvallis,
Oregon.
Isolation of Deoxyribonucleic Acid
The deoxyribonucleic acid (DNA) was harvested basically by the method of Marmur (1961) as modified by Seidler
(personal communication).
Two to three grams wet weight of
cells were grown on BHI agar, washed once in saline-EDTA
(0.15 M NaC1 plus 0.1 M ethylenediaminetetra acetate, pH 8)
and then resuspended in 50 mls of the same.
Lysis of the
cells was effected by agitating the cells in the presence
of two percent (w/v) sodium dodecyl sulfate.
Upon complete
lysis of the cells, deproteinization was accomplished by
the addition of an equal volume of saline-EDTA equilibrated
phenol (pH 7.5).
The contents were shaken on a reciprocal
shaker and after 30 minutes, centrifuged at 12,100 x g to
separate the phases.
The upper aqueous phase was collected
and another volume of saline-EDTA was added to the phenol
phase.
The contents were shaken for 30 minutes and centri-
fuged as above.
The upper aqueous phase was collected and
pooled with the first.
Two volumes of 95 percent (v/v)
ethanol were layered on top of the pooled aqueous phases
to precipitate the nucleic acids.
The strands were spooled
around a glass rod and rinsed in 70 percent (v/v) ethanol.
31
The stranded nucleic acids were then dissolved in dilute
saline-citrate (0.1X SSC; 0.015 M NaC1 plus 0.0015 M tnsodium citrate) and adjusted to lx ssc with lOX SSC.
The
next deproteinization was carried out with an equal volume
of chioroforin-isoamyl alcohol (24:1, v/v).
The solution
was shaken for 15 minutes, centrifuged, and precipitated
as above.
The nucleic acids were dissolved in 0.1X SSC
and brought up to lx SSC with lOX SSC.
Heat treated
nibonuclease (Sigma) was added to a final concentration of
100 ig/ml and incubated at 37 C for three hours to destroy
the contaminating nibonucleic acid.
The solution was de-
proteinized as above with saline-EDTA equilibrated phenol,
redissolved, and then precipitated three more times1 as
previously mentioned,with ethanol.
After the last pre-
cipitation, the DNA was dissolved and left in 0.].X SSC.
One ml of acetate-EDTA (3.0 M sodium acetate plus 0.001 M
ethylenediaminetetra acetate, pH 7.0) was slowly added for
every nine mls of 0.lX SSC.
The DNA was precipitated by
the dropwise addition of 0.6 volumes isopropyl alcohol.
The purified DNA was rinsed in 70 percent ethanol, dissolved in 4.5 ml 0.lX SSC and stored for future use in a
stoppered vial at 4 C.
Percent Guanine Plus Cytosifle Determination
The deoxyribonucleic acid (DNA) for the ERMB was
isolated by the previously described procedure.
Eschenicia
32
coli WP2 was used as the reference DNA.
The E. coli WP2
DNA and the actual thermal melt was provided by Dr. Ramond
Seidler of the Oregon State University Department of Microbiology.
Each thermal melt run contained a reference dis-
tilled water cell, the E. coli WP2 DNA cell, and two ERMB
experimental DNA cells.
The thermal melts were performed
in a Gilford Nodel 2000 Recording Spectrophotometer and
each thermal melt was done in duplicate.
The DNA was added to the cuvettes at a final concentration of 20-40 iig/ml.
The temperature of the chamber was
raised quickly to 7-10 C below the expected thermal melting
point (Tm).
The cuvettes were taken out and any condensa-
tion was wiped off.
equilibrate.
They were then replaced and allowed to
The temperature was raised in 0.5-0.7 C
increments with a 5 minute equilibration period.
At the
end of each of these periods the optical density at 260 nm
(0D260) was measured and plotted electronically.
This pro-
cess was halted when no further increase in 0D260 could be
measured after three consecutive rises in temperature.
This point was taken as the maximum increase in 0D260.
The percent guanine plus cytosine (percent GC) was
calculated by the method of Mandel et al.
(1970).
The
0D260 at each temperature was multiplied by the thermal
expansion coefficient of that temperature.
This product
was then divided by the 0D260 of the sample at 25 C to
33
give the relative optical density and plotted against the
respective temperature.
The Tm is defined as the temperature corresponding to
half the increase in the relative adsorbance of the thermal
denaturation curve.
This was obtained by first calculating
the Tm of the sample by graphing.
Four points on both
sides of the graphed Tm were taken and linear regressed by
the method of least squares (Hewlitt-Packard - 65 calculator:
Statistics Package).
the 0D260
The Tm was obtained by entering
corresponding to half the increase in relative
adsorbance into the linear regressed line.
After the Tm'S
of a run were calculated, the %GC could be obtained by the
following formula:
%GCEB = %GCE
coli WP2 + l.99(TmE
TmE
coli WP2
where the %GC for E. coli WP2 is taken to be exactly 51.
Once the %GC for all runs were calculated, a One-Way
Analysis of Variance between the isolates was calculated to
determine any significant difference at the 95 percent confidence intervals (Snedicor and Cochran, 1973).
LD50 Determination of Selected Isolates of Enteric
Redmouth Bacterium i Rainbow Trout and Mice
The LD50ts for isolates BC-74, HI-70 and SC-72 were
determined for rainbow trout and white Swiss Webster mice.
Each isolate was inoculated into approximately 10 ml of BHI
34
broth and incubated at 18 C overnight.
The culture was
grown to an optical density of 0.7 to 1.0 at 525 rim.
Ten
fold serial dilutions of the culture were made using PBS as
the diluent and plate counts were made to determine the
viable counts.
Ten fish per dilution were injected intra-
peritoneally (IP) with 0.1 ml of the appropriate dilution.
Mortalities were taken once a day and necropsied to ascertain cause of death.
The mice were injected IP with 0.1 ml of the cultures
indicated.
Mortalities were taken twice a day and the ex-
periment was terminated after two weeks.
All LD50ts were
calculated by the method of moving averages (Meynell and
Meynell, 1965)
Determination of Enteric Redflouth Bacteria Serotvte
Cross-Protection
Isolates BC-74, HI-70 and SC-72 were inoculated with
one ml of an overnight culture into 300 ml of BHI broth and
incubated at 18 C.
A fourth flask was inoculated with 0.5
ml each of the same BC-74 and HI-70 cultures and treated in
the same manner.
After 24 hr the cultures were killed by
the addition of 0.3 percent formalin and allowed to set
with periodic mixing overnight.
The culture was examined
for sterility by inoculating one ml into thioglycolate
broth.
The cells were washed three times in PBS and resus-
pended to an optical density of 1.5 at 525 nm.
Rainbow
trout were injected with 0.1 ml of the cell suspension.
35
Each group, consisting of 220 fish,were marked by the
fluorescent pigment technique (Pribble, 1976) with a different color (Scientific Marking Material, Seattle, WashingThe fish were held six weeks before challenging to
ton)
allow time for any stimulation of the immune response that
might occur (Patterson, 1972).
During this time the LD501s
to be used for challenge purposes was determined.
Approximately 25 fish from each vaccinated group and
25 control fish were pooled into three previously described
rectangular tanks.
The appropriate concentration of cells
was estimated by comparing the optical density at 525 rim
of the cells growing at 18 C in BHI to the previously constructed graph plotting optical density at 525 nm versus the
viable cell count of isolate HI-70 cells grown in the same
manner.
Spread plate counts were also done to confirm the
actual number of viable cells injected into each fish.
Mortalities were collected once a day, necropsied, and cultured for the presence of ERMB by streaking kidney tissue
on BHI agar.
RESULTS
Optical Density versus Viable Count of Isolate HI-70
A growth curve for isolate HI-70 was determined in BHI
broth at 18 C.
A graph was constructed plotting optical
density at 525 nm versus viable count of the bacterial culture (Fig. 1).
With the aid of this graph, viable cell
count could easily be estimated by means of spectroscopy.
Selection of Enteric Redmouth Bacterium Isolates for
Serological Comparison
From the titers obtained it can be readily observed
that four isolates had a considerably reduced titer (Table
3).
Three of these isolates BC-74, SP-70 and TH-75 were
chosen for serological comparison.
The fourth isolate,
BC-75, was not chosen because it was obtained from the same
hatchery as isolate BC-74.
Three other isolates, HI-70,
SC-72, and OS-76, were also chosen for their diversity in
geographical location.
Isolate HI-70 was obtained from
Hagerman, Idaho, the site where ERMD was originally endemic.
Isolate SC-72 was isolated by Wobeser (1973) in Canada and
OS-76 represented the most recent outbreak of ERMD in
Oregon.
37
E
U)
w
C-)
I-
z
0
0.
I.
I.
>
0.2
0.4
0.6
0.8
1.0
OPTICAL DENSITY (525 nm)
Figure 1.
Growth curve of isolate HI-70 i: BHI broth at
18 C plotting viable count versus optical
ensity at 525 nm.
38
Table 3.
Titers of a known Enteric redrnouth bacterium
antiseraa against selected enteric redmouth
bacteria isolates.
Isolate
Titer
BC-74
0
HI-70
322
SC-72
256
SP-70
0
TH-75
0
OS-76
645
RR-70
512
SS-75
512
SH-75
1024
ER-75
512
EL-75
645
BC-75
0
BH-75
645
WS-75
512
WS-74
812
WT-75
512
GC-70
645
aThe antiserum was furnished by J. E. Sanders of the Oregon
Department of Fish and Wildlife. The antiserum was prepared in rabbits to an enteric redmouth bacterium isolated
from steelhead trout (Salmo gairdneri) at Gnat Creek
Hatchery, Oregon.
39
Determination of Serotype Scheme by Cross-Adsorption
This experiment was conducted to ascertain 1) the number of agglutinating serotypes among the available isolates;
2) the amount of cross-reactivity between serotypes; and
3) propose an antigenic scheme whereby various isolates may
be typed.
The experiment was performed by taking antisera
against isolates BC-74, HI-70, SP-70, TH-75, SC-72, and
05-76, and adsorbing each exhaustively against the homologous and heterologous cells.
The adsorbed and unadsorbed
antisera from each isolate were then titered against the
cells from each of the six isolates.
and compared in Tables 4 through 8.
The titers are listed
Antigens between two
isolates are compared by cross-adsorbing the antisera from
each isolate against it's homologous and heterologous cells.
If the two isolates do not have any antigens in common, then
no titer should be observed when the unadsorbed antisera are
titered against the heterologous cells.
If one isolate has
all the antigens of the second, then no titer should be obtained if the antisera was adsorbed by cells of the first
isolate.
This is due to the total adsorption and removal
of the specific antibody.
The corollary experiment would
have to be performed to determine if the second isolate
possessed additional antigens.
When the cross-adsorbed
antisera is iitered against homologous or heterologous
cells and a reduction in titer is produced in comparison
40
to the unadsorbed antisera, then at least one, but not all,
corrnon antigens are indicated.
Care must be employed in
interpreting the results because 1) the accuracy of the
test, and 2) dilution of the antisera occurs upon adsorption, therefore an antigenic difference is not always indicated when the titers of the adsorbed antisera are lower
than the unadsorbed antisera.
In order to facilitate explanation of the serotyping
rational, a table of only isolates BC-74 and HI-70 is given
first (Table 4) .
Isolates SC-72, SP-70, TH-75, and OS-76
are then added sequentially (Tables 5 through 8).
should be noted that:
It
1) antisera adsorbed with the homolo-
gous cells did not produce a titer against it's homologous
cells, indicating complete adsorption of the specific
agglutinating antibodies; and 2)
antisera
in no case was an adsorbed
titer greater than the respective unadsorbed
titer.
Unadsorbed antisera BC-74 and HI-70 shows a titer of
1024 and 256 respectively,when titered against their homologous cells, but show no titer against the heterologous cell
types (Table 4).
When each antisera is adsorbed by the
heterologous cells, the titer remains essentially the same
or within the limits of the test.
This indicates that the
two isolates do not have any agglutinating antigens in common.
Therefore,isolate BC-74 has been given antigen 1 and
antigen 2 is designated to HI-70.
41
Table 4.
Serological comparison of enteric redmouth bacterial isolates BC-74 and HI-70.
Titer when Tested
with Organism
Antisera
BC-74
HI-70
Adsorbed with
Organism
Unadsorbed
BC-74
HI-70
tJnadsorbed
BC-74
HI-70
BC-74
1024
0
512
HI-70
0
0
0
0
256
256
0
0
0
Antibodies in
Antisera (and
Antigens in
Corresponding
Organisms)
2
42
Enteric redmouth bacteria isolate SC-72 was considered
next (Table 5).
The SC-72 agglutinating antibody titers
were similar to HI-70.
It produced a titier to unadsorbed
antiserum against HI-70 and itself but only a very low titer
to BC-74.
When any of the three antisera were adsorbed by
HI-70 or SC-72,no titer was produced except when BC-74 was
adsorbed and titered against homologous cells. This suggests
that isolate SC-72 also has antigen 2.
A titer of 4 was
produced when unadsorbed BC-74 was titered against SC-72 and
when unadsorbed SC-72 was titered against BC-74. From these
results a minor antigen was assigned these two isolates.
Isolate SP-70 was added to the discussion next (Table
6).
BC-74 and SP-70 appear to have major antigen 1 in com-
mon because when either isolate was titered against it's
homologous or heterologous cells a titer is produced, but
not against HI-70 and only a very low titer with SC-72.
Conversely, when antisera against BC-74 or SP-70 was adsorbed by either of the two isolates, no titers were ob-
tained supporting the idea that the two isolates are identical.
When antisera against HI-70 or SC-72 were adsorbed
with BC-74 or SP-70, titers were obtained and only slightly
reduced from the unadsorbed antisera when titered against
themselves.
This further indicates that no major antigens
are in common between the two isolates.
A titer of 4 and 8,
respectively,was obtained when unadsorbed SC-72 antisera
was adsorbed against SP-70 and unadsorbed SP-70 was adsorbed
43
Table
5.
serological comparison of enteric redmouth bacterial isolates BC-74, HI-70 and SC-72.
Titer when Tested
with Organism
Antisera
BC-74
Adsorbed with
Organism
SC-72
1024
0
0
HI-70
512
512
0
0
0
4
0
0
0
0
0
0
0
256
256
512
256
0
0
0
4
0
128
64
2048
128
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SC-72
HI-70
BC-74
tjnadsorbed
SC-72
HI-70
BC-74
Unadsorbed
BC-74
HI-70
SC-72
Antibodies in
Antisera (and
Antigens in
Corresponding
Organisms)
1,3
2
0
2,3
44
Table 6
Serological comparison of enteric redmouth bacterial isolates
BC-74, HI-70, SC-72, and SP-70.
SP-70
Antibodies in
Antisera (and
Antigens in
Corresponding
Organisms)
1,3
Titer when Tested
with Organism
Antisera
BC-74
HI-70
SC-72
SP-70
Adsorbed with
Organism
BC-74
HI-70
SC-72
0
0
0
0
0
4
0
0
0
0
512
0
0
0
0
0
256
256
512
256
2
0
0
0
0
128
128
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
4
0
0
0
0
128
64
2048
128
2,3
0
0
0
0
64
64
4
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
2048
0
0
0
0
0
8
256
1,3
0
0
0
0
0
256
128
Unadsorbed
BC-74
HI-70
SC-72
SP-70
1024
Unadsorbed
BC-74
HI-70
SC-72
SP-70
0
512
512
0
0
1024
1024
0
0
512
256
0
0
45
with SC-72, suggesting a minor antigen in common between
SC-72 and SP-70.
At this time there is no evidence to
indicate that the minor antigens of SP-70, BC-74, and SC72 are identical.
The definitive agglutinating titers (un-
adsorbed BC-74 titered against SP-70 and unadsorbed SP-70
titered against BC-74) are masked by the major antigen 1.
For the sake of simplicity, SP-70 will also be given the
minor antigen 3 until it can be shown that this antigen is
different from that of isolate BC-74.
Isolate TH-75 follows the same patterns set by BC-74
and SP-70 (Table 7).
When the antisera against any of the
three isolates were adsorbed out by the cells of the other
three isolates, no titer was achieved regardless of the
cell type that it was titered against.
This suggests that
TH-75 does not have additional antigens exceeding either
BC-74 or SP-70.
When SC-72 or SP-70 antiserum was adsorbed
by BC-74, SP-70 or TH-75 and titered against any of the
three types, a titer of zero was observed.
This indicates
no additional antigens exceeding HI-70 or SC-72.
When the
same adsorbed antiserum was titered against HI-70, or SC72, only a slight reduction in titer was observed, further
supporting the hypothesis that no major antigens are in
common.
A titer of 4 and 8,respectively,was recorded when
unadsorbed TH-75 antisera was titered with SC-72 cells and
unadsorbed SC-72 antisera was titered with TH-75 cells.
There is no verification that this minor antigen is antigen
46
Table 7.
Serological comparison of enteric redmouth bacterial isolates BC-74,
HI-70, SC-72, SP-70, and TH-75.
Titer when Tested
with Organism
Antisera
BC-74
HI-70
SC-72
SP-70
TH-75
Adsorbed with
Organism
BC-74
HI-70
SC-72
SP-70
TH-75
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
1024
0
4
512
1024
0
0
0
0
0
512
512
0
0
0
512
256
256
256
0
0
0
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
0
0
256
256
512
256
0
0
0
0
0
0
0
0
0
0
0
0
128
256
128
64
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
4
128
8
64
2048
128
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
2048
0
0
0
8
256
512
0
0
0
0
0
0
256
128
256
256
0
0
0
0
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
2048
0
0
4
0
1024
512
0
0
512
512
0
0
0
0
256
128
256
512
0
0
0
0
0
0
0
0
0
0
0
0
0
1024
1024
0
0
0
0
64
0
0
64
192
Antibodies in
Antisera (and
Antigens in
Corresponding
Organisms)
1,3
0
2
0
0
0
0
2,3
0
1,3
1,3
47
3 because the definitive titers are again masked by the
major antigens.
Until further proof, this minor antigen
will also be considered as antigen 3.
The last serotyping isolate, OS-76,was added so
all six isolates may be compared at once.
Isolate OS-76
compares strongly with HI-70 and SC-72, and the arguments
put forth for their antigenic analysis will also hold for
OS-76.
No titers were observed when unadsorbed antisera
having antigens 1 and 3 were titered against OS-76 and when
unadsorbed OS-76 antisera was titered against isolates with
antigens 1 and 3.
Therefore, it can be concluded that OS-
76 does not have antigen 3.
In summary, isolates BC-74, SP-70, and TH-75 have
antigens 1 and 3; isolates HI-70 and OS-76 have only antigen 2; and isolate SC-72 has both antigens 2 and 3.
From
this data it can be concluded that ERNB has two serotypes,
I and II, with some cross reactivity which has been attributed to antigen 3.
One should bear in mind that this
technique assays only for agglutinating antibodies and does
not preclude common antigens among ERMB isolates of a nonagglutinating nature.
Biochemical Tests
Seventeen isolates oE ERMB were tested by various biochemical tests at 9, 18, 27, and 37 C.
Homogeneity among
the isolates was demonstrated with regard to carbohydrate
48
Table 8.
Serological comparison of enteric redmouth bacterial isolates BC-74, HI-70,
SC-72, SP-70, TH-75, and OS-76.
Titer when Tested
with Organism
Antisera
BC-74
81-70
SC-72
SP-70
TH-75
OS-76
Adsorbed with
Organism
BC-74
HI-70
SC-72
SP-70
TH-75
OS-76
Unadsorbed
BC-74
81-70
Sc-72
SP-70
TH-75
OS-76
1024
0
4
512
1024
0
0
0
0
0
0
0
512
512
0
256
256
0
0
0
0
512
256
0
0
0
0
0
0
0
0
0
0
0
0
0
512
0
0
512
512
0
Unadsorbed
BC-74
HI-70
sC-72
SP-70
TH-75
OS-76
0
256
256
512
256
0
0
0
0
0
512
256
0
0
0
0
0
0
0
0
0
0
128
256
128
64
0
0
0
0
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
OS-76
4
0
128
64
2048
128
4
0
8
0
0
0
192
128
0
0
0
0
0
0
0
0
0
0
0
64
64
64
192
128
192
0
0
0
0
0
0
0
0
Unadsorbed
BC-74
HI-70
SC-72
SP-70
TH-75
OS-76
2048
0
8
256
512
0
0
0
0
0
0
0
1024
1024
0
0
0
256
128
256
256
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
256
384
Unadsorbed
BC-74
81-70
SC-72
SP-70
TH-75
OS-76
2048
0
0
4
0
1024
512
0
0
512
512
0
0
0
256
128
256
512
0
0
0
0
0
0
0
0
0
0
0
0
512
0
0
256
512
0
Unadsorbed
BC-74
81-70
SC-72
SP-70
TH-75
OS-76
0
0
1024
512
2048
1024
0
0
0
0
2048
1024
0
0
0
0
0
0
0
0
0
0
0
0
0
512
1024
0
0
0
512
512
0
0
1024
768
0
0
0
0
0
0
0
2
256
384
0
2048
1,3
0
0
0
0
0
Antibodies in
Antisera (and
Antigens in
Corresponding
Organisms)
2,3
0
1,3
0
0
0
0
0
1,3
0
0
2
49
fermentation (Table 9).
A plus sign in the table denotes
100 percent of the isolates gave a positive reaction and a
minus sign denotes 100 percent of the isolates with a negative reaction.
Parenthesis around a plus sign indicates
that the reaction was positive but delayed with respect to
changes of other biochemical reactions at said temperature.
A time period for a biochemical reaction was not enforced
among the different temperatures.
This was due to the
fact that a reaction at 37 C will normally proceed at a
faster rate than the same reaction at a lower temperature.
If a time period based at 37 C was used, then most of the
reactions at 9 C would be delayed.
Therefore, each reac-
tion is considered delayed only with respect to other biochemical reactions at the same temperature and are not
compared to reactions at other temperatures.
The carbohydrate metabolism of ERNB showed no variation at the different temperatures tested.
Glucose, lac-
tose, xylose, arabinose, cellubiose, dulcitol, salicin,
inositol, raffinose, rhamnose, melibiose, and esculin
could not be degraded.
Sorbitol gave a mixed reaction
which corresponded to the serotype of the isolate.
All
isolates that were serotype I could utilize sorbitol and
all isolates that were serotype II could not.
Only four
of the isolates were positive out of 17 for sorbitol.
Three of these isolates were included in the serotyping
experiment.
To test the above statement, the fourth
50
Table 9.
Biochemical comparison of 17 enteric redmouth
bacterial isolates at four different temperatures.
9°C
18°C
27°C
37°C
Carbohydrate Metabolism
glucose
mannitol
maltose
mannose
glycerol
trehalose
galactose
sorbitol
sucrose
lactose
xylose
arabinose
cellubiose
dulcitol
salicin
inositol
raffinose
rhamnose
melibiose
esculin
indole
methyl red
Voges Proskauer
citrate (Sirnmon's)
tartrate (Jordan's)
malonate
urease (Christensen)
lysine decarboxylase
ornithine decarboxylase
arginine dihydrolase
phenylalanine deaminase
nitrate reduction
nitrite reduction
GI motility
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(+)
(+)
(+)
(+)
d
d
d
d
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
Tolerance to NaCl
0%
0.5%
1%
3%
7%
10%
51
Table 9 (continued)
9°C
18°C
27°C
37°C
Extracellular enzymes
DNase
RNase
hyaluronidase
chrondroitin sulfatase
lipase tweens 20
40
60
80
gelatinase
fibrinolyase
elastase
hemolysis
pigment
oxidase
catalase
H2S production
TS1
-
+
+
d
-
-
+
-
d
+
+
+
d
+
+
+
+
-
+
-
y
-
+
+
I
-
-
-
-
+
+
+
+
-
-
-
-
K/A
K/A
K/A
K/A
+ = 100 percent positive reaction
- = 100 percent positive reaction
(+)= delayed reaction in comparison to the rest of the tests
at temperature indicated.
d = variable reaction
K = alkaline
A = acid
52
isolate, BC-75, was tested against antisera representing
the serotypes I and II. It was found,as expected, that it
reacted only with serotype I.
The IMViC reactions (indole, methyl-red, VogesProskauer, and citrate) for ERNB were -+-+ at all four
temperatures.
Tartrate could also be utilized as a sole
carbon source, but malonate could not.
Urease was not
produced and nitrite was not reduced but nitrate was.
The first influence of temperature on reactions of
the bacterium was exhibited in the decarboxylation of
amino acids.
Lysine and argine could be decarboxylated
only at 18 C while temperature showed no effect on
ornithine decarboxylase (positive) and phenyalamine deaminase (negative).
The bacteria were nonmotile at 9 and
37 C, but were motile at 18 and 27 C exhibiting luxurious
growth throughout the culture media.
The Triple Sugar
Iron agar slants (TSI) were alkaline over acid with no gas
or hydrogen sulfide production as would be expected from
the glucose, sucrose and lactose reactions given above.
The tolerance of ERMB to various concentrations of
NaCl did not vary with temperature.
The bacteria could
tolerate concentrations of three percent or less but not
concentrations of seven percent or greater.
The assays for various extracellular enzymes also
displayed a marked temperature dependency.
DNase was
produced only at 37 C and RNase was not produced at any
53
Fibrinolyase and elastase were not
of the temperatures.
produced by any of the isolates at the temperatures tested.
Hyaluronidase and chondroitin sulfatase could also be produced at 18 and 37 C,but hyaluronidase was not produced at
either temperature.
The lipases as assayed by Tween 20,
All
40, 60 and 80 also showed some temperature dependency.
of the isolates could utilize Tweens 20 and none of the
isolates could utilize Tweens 80.
Tweens 40 and 60 could
be degraded at 9, 18 and 27 C but not at 37 C.
Gamma hemoGela-
lysis was observed on five percent sheep blood agar.
tin was not liquefied at 9, 18, or 37 C.
At 27 C only the
top one third of the tube was liquefied.
The tube was al-
lowed to incubate another three weeks but no further
liquefication was observed.
Slide Agglutination Tests of Six Enteric Redmouth
Bacterium Isolates
The six isolates, BC-74, HI-70, SC-72, SP-70, TH-75
and OS-76 chosen for the cross adsorption experiment were
also compared by slide agglutination (Table 10).
The
antiserum to each isolate was tested against the homologous
and heterologous cell types.
Two kinds of positive agglu-
tination reactions occurred (Figures 2-c and 2-d).
The
type of agglutination seen in Figure 2-d has been designated
in Table 10.
The cells were agglutinated giving
a fine granular appearance.
Figure 2-c depicts the
Table 10.
Results of slide agglutination tests between six isolates of enteric
redmouth bacterium..
Cells
agglutinated
against
Antigens on cells
(and antibodies
in corresponding
Antisera
BC-74
HI-70
SC-72
SP-70
TH-75
OS-76
antisera)
BC-74
+
-
±
+
+
-
1,3
HI-70
-
+
+
-
-
+
2
SC-72
±
+
+
±
±
+
2,3
SP-70
+
-
±
+
+
-
1,3
TH-75
+
-
±
+
+
-
1,3
OS-76
-
+
+
-
-
+
2
+ = the agglutination reaction seen in Figure 2c.
± = the agglutination reaction seen in Figure 2d.
- = the agglutination reaction seen in Figure 2a or Figure 2b.
U,
55
Figure 2.
Slide agglutination tests of enteric redmouth
bacterium.
a.
Control without serum.
b.
Control with serum.
c.
Positive agglutination of major antigens.
d.
Positive agglutination of minor antigens.
1]
rDj
'4
4
I
57
agglutination of a "+" reaction.
This produced a more com-
plete agglutination where all cells were clumped together.
The antigens of the various isolates have been added to
Table 10 to facilitate comparison of agglutination to presence or absence of the various antigens.
It can be seen
that when antibody containing a major antigen is agglutinated with cells of the same major antigen, the complete
or "+" type of agglutination occurs.
When antibody and
cells with only the minor antigen 3 in common were agglutinated, the lesser or "+" type of agglutination was observed.
When the antibody and cells have no antigens in
common, then no agglutination occurs (Figure 2b).
The
cells do not agglutinate when non-specific rabbit serum
is added (Figure 2b).
Supportive evidence was obtained that indicate that
minor antigen 3 in isolates BC-74, SC-72, SP-70, and TH75 is identical in all cases.
Isolates BC-74, SP-70 and
TH-75 have antigens 1 and 3, while SC-72 has antigens
2 and 3.
When any of the three former isolates are reacted
with SC-72, the "±" type of reaction occurs.
Either all
four isolates have the common and identical antigen 3,or
isolate SC-72 has to have three different antigens to
react separately with each isolate, or a combination of
the two. In my opinion, the first is most probable.
Ouchterlony Double Diffusion Plates
Redmouth Bacterium
f Enteric
Ouchterlony double diffusion plates of the six ERMB
isolates to which antisera were prepared are diffused
against sonicated cells from each of the six isolates
(Figure 3a-3f).
The center well contained the antisera,
which was different and appropriately labeled in each of
the figures.
The antisera from the isolates of serotype I
are in the left hand column, while the antisera of the serotype II isolates are in the right hand column.
The six
surrounding wells contain the sonicated cellular antigens
and are identical throughout.
The pattern is as follows:
top well is BC-74, upper left well is HI-70, lower left
well is SP-70, bottom well is SC-72, lower right well is
TH-75, and the upper right well is OS-76.
The most obvious conclusion to be drawn from the
figures is the diversity of antigens among the isolates.
The precipitin lines do not form a readily discernible
pattern.
The only band possessed by all of the isolates
is the innermost precipitin line, which is assumed to be
the endotoxin.
Antisera against isolates BC-74 and TH-75
(Figure 3a and 3e) show a precipitin band that corresponds
to antigen 1 as indicated by the arrows.
Antisera against
HI-70 and OS-76 (Figure 3b and 3f) also show distinct precipitin bands with the isolates containing antigen 2
(arrows).
In other Ouchterlony plates the bands
59
Figure 3.
Ouchterlony double diffusion plates of selected
isolates of enteric redmouth bacterium.
a.
Isolate BC-74
b.
Isolate HI-70
c.
Isolate SP-70
d.
Isolate SC-72
e.
Isolate TH-75
f.
Isolate OS-76
BC-74
OS-76
HI-70
Antiserum
TH-75
SP-70
SC-72
--
ci
k.
Q
S P -70
AntiH 1-70
c/o
fl'
61
corresponding to antigens 2 or 3 were shown to be continuous with no spurs.
This demonstrates that BC-74, SP-
70 and TH-75 contain an identical antigen not possessed by
the other three isolates.
This is thought to be antigen 1.
Isolates HI-70, SC-72 and OS-76 contain another separate
but identical antigen not possessed by the original three
isolates which may be antigen 2.
No explanation can be
put forth to explain the absence of these bands when anti-
sera from SP-70 or SC-72 were employed.
Estimated
Rainbow Trout
Fifty Percent (LD5o): for
The LD50 was estimated for purposes of challenging
fish to ascertain levels of relative resistance.
Isolates
BC-74, HI-70 and SC-72 were injected IP with 0.1 ml of
dilutions from l0
to lO
the appropriate bacterium.
of a log phase broth culture of
Mortality occurred only in the
SC-72 injected fish with no mortalities in the controls.
From the plate count, the o iginal culture was estimated
to be 9.37 x 108 cells/ml and the LD50 for this isolate was
calculated to be 9.37 x iO3 cells/fish.
Since no mortality
occurred in the BC-74 and HI-70 injected fish, the experi-
ment was repeated using dilutions of 10_i to 10.
The
plate counts of the undiluted cultures were 9.93 x 108 and
8.00 x 108 cells/ml,respective1y.
From the mortalities in-
curred, the LD50ts for isolates BC-74 and HI-70 are calculated at 5.95 x 106 and 4.00 x 106 cells/f ish,respectively.
Busch and Lingg (1975) obtained an LD5O value of 30
cells/fish and Anderson and Ross (1972) obtained values of
The isolates used
8.5 x i05 cells/fish upon IP injection.
in this study were not previously passed through the exThis may account
perimental hosts to increase virulence.
for the discrepancies between the LD50 values of the above
two papers and these results.
ERME LD50 Determination for Mice
This experiment was conducted to evaluate the pathogenicity of ERMB in a host whose body temperature range is
similar to man.
Five white Swiss Webster mice per group
were injected with isolates BC-74, HI-70, or SC-72.
Each
injection group was injected with a serial dilution of an
overnight ERMB culture ranging from l02 to 10_6.
talities in any group occurred.
No mor-
An attempt was then made
to increase the virulence of the bacterium by injecting
five mice with 0.3 mls IP of an undiluted log phase broth
culture.
After five days one mouse died.
The bacterium
was reisolated upon necropsy from a peritoneal washing and
injected directly into five more mice.
The peritoneal
washing was also cultured on a BHI agar plate and then
reinjected into a group of five mice with an estimated dose
in excess of l0
twice more.
cells/mi.
This procedure was carried out
No reproduceable mortality in mice could be
63
obtained by this route of injection.
It was concluded that
ERMB is not pathogenic for White Swiss Webster mice by IP
injection.
Determination of Cross-Protection between
Serotypes I and II
The purpose of this experiment was twofold:
1) to de-
termine if the major serotypes of ERMB are cross-protective
and 2) if they are not cross-protective, to determine if a
bivalent bacteria consisting of the two major serotypes
would provide cross-protection.
As with all experiments,
isolates BC-74 and HI-70 were chosen to represent the
major serotypes I and II; isolates SC-72 was also added to
determine the significance of antigen 3.
The fish were
challenged in duplicate with the three isolates selected
(Table 11), however4solate SC-72 failed to produce an
adequate level of challenge.
The data indicates that the major agglutinating serotype I (isolate BC-74) will cross-protect against major
serotype II (isolate HI-70), but serotype II cannot crossprotect against serotype I.
Isolate SC-72 has previously
been assigned antigens 2 and 3.
Antigens 1 and 3 have
been designated to isolate BC-74,while isolate HI-70 has
only antigen 2.
The only antigen in common between BC-74
and SC-72 is antigen 3.
When both are challenged by BC-74,
they react similarly having mortalities of 18 percent.
This would indicate that antigen 3 alone is a protective
64
Table 11.
Vaccinated
Groups
Comparison of protection of rainbow trout to
enteric redmouth bacterium by injection of
monovalent and bivalent bacterins.
Mortality Fraction
due to Challenge
in Duplicatea
HI-70
BC-74
7/25b
2/25
Combined
% Mortality
HI-70
BC-74
7/25
0/25
18
14
20/25
18/25
3/25
0/25
76
6
sp_72c
7/25
2/25
2/25
1/25
18
6
BC-74
plus
HI-70
6/25
1/25
2/25
1/25
14
6
Control
21/25
17/25
22/25
21/25
76
86
BC-74
HI-70
aEJB was isolated from all mortalities.
bNthers indicate fraction of challenge group that died.
Clsolate SP-72 failed to produce an adequate challenge.
antigen assuming the protective antigen is agglutinating in
nature. Isolates HI-70 and BC-74 do not have any antigens
in common.
When challenged with BC-74, there is no protec-
tion in the HI-70 vaccinated group, but protection is
achieved with the other groups.
Conversely, even though
no antigens are in common between BC-74 and HI-70, both are
protected when challenged by HI-70.
This would indicate
that either the antigenic analysis is correct and/or there
is a nonagglutinating type of antigen which is protective.
The fish were protected against challenge by both
65
representative serotypes when the two were incorporated
into a bivalent bacterin.
Statistically, the HI-70 vac-
cinated groups do not show significant protection when
challenged by BC-74 (95 percent confidence interval;
Snedecor and Cochran, 1973).
In contrast, monovalent bac-
ten, BC-74 and SP-72 and the bivalent bacterin do show
significant protection although no significant difference
between groups can be detected.
When challenged by iso-
late HI-70 each vaccinated group demonstrated significant
protection, but there is no significant difference between
these groups.
Effects of Temperature on Flagellation and Motility of
Enteric Redmouth Bacterium as Ascertained by Electron
Microscopy
It has already been determined that ERMB changes motility when exposed to different environmental temperatures
(Ross et al., 1966).
It has been demonstrated in this work
that at 37 and 9 C the bacteria are nonmotile, while at 18
and 27 C they are motile.
The question now arises, are the
bacteria nonmotile due to a lack of flagella production or
are the flagella produced but nonfunctional?
This study
was instituted to answer this question and reiterate the
fact that ERMB is peritrichously flagellated.
The bacteria
were grown at 9 and 37 C where ERMB is not motile (Figure
4a and 4d).
An incubation temperature of 22 C,where ERMB
is motile,has also been included (Figure 4b and 4c).
It
Figure 4.
Electron micrographs of single cells from cultures of enteric redmouth bacterium incubated at
selected temperatures (Bar = 1 pm).
9 C.
a.
Incubation temperature,
b.
Incubation temperature, 22 C.
c.
Incubation temperature, 22 C.
d.
Incubation temperature, 37 C.
I
-
/
L.'
I JI.,'
/
A.
-
e- -.
).
..;-.
f
11 .a.PEr *.
.-:-'
pu
r:1
,-
-.
-'.
-
?:
..
1
:i
can be seen that both 9 and 22 C cells do have flagella and
are peritrichously flagellated, while at 37 C cells do not
have flagella.
An attempt was made to show that Figures
4a and 4d are not isolated cells but are representative
cells of the bacteria at these growth temperatures (Figure
5a-d).
Figures 5a and 5b are clumps of bacteria at 9 and
22 C respectively and are inundated with flagella.
Figure
5c is a chain of bacteria cells and 5d is a pellet of bac-
teria both grown at 37 C.
It should be noted that in the
latter two figures no intact oz remnants of flagella can
be seen.
Even though no quantitative data can be put
forth, empirically the number of flagella attached to the
9 C cells appeared to be less than the 22 C cells.
GC% Determination
The percent guanine plus cytosine (GC%) for isolates
BC-74, HI-70 and SC-72 were found to be 48.45, 47.77, and
47.64,respectively (Table 12).
Table 12.
Average
The thermal melting point
Individual and average guanine plus cytosine
values for enteric redmouth bacterium as determined by thermal melting (Tm),
BC-74
HI-70
SC-72
48.20
48.70
47.89
47.65
47.74
47.54
48.45
47.77
47.64
Figure 5.
Electron micrographs of enteric redmouth bacterium cell groups incubated at selected
temperatures (Bar = 1 pin).
a.
Incubation temperature,
b.
Incubation temperature, 22 C
c.
Incubation temperature, 37 C.
d.
Incubation temperature, 37 C.
9 C.
71
(Tm) from which the GC% were calculated have been listed
(Table 13).
The denaturation curves of Runs 1, 2, and 3,
respectively, used to estimate the Tm's for the three ERMB
Table 13.
Table of thermal melting points (Tm) for each
run of enteric redmouth bacterium using E. coli
WP2 as standard.
Runi
Run2
Run3
E. coli WP2
73.94
73.52
73.54
BC-74
72.76
72.16
HI-70
72.36
---
72.86
71.89
71.81
SC-72
isolates and Eschericia coli WP2 are plotted (Figures 6
through 8).
A One-Way ANOVA table was constructed
(Snedecor and Cochran, 1973) and no significant dif-
ferences between the GC% of the isolates could be predicted at the 95 percent confidence level.
The average
GC% for ERMB from all six determinations is 47.95 ± 0.45
(95 percent confidence interval).
Comparison of Enteric Redmouth Bacterium to the Genus
Yersinla
Upon analysis it was found that ER!4B compares closely
to the newly formed genus Yersinia.
This section will re-
late ERMB to Yersinia in an attempt to designate the previously unassigned ERNB a genus and species.
IA
>-
I.3
Cl)
z
w
a
-J
I
a0
I.2
ILl
>
I
IJ
-J
w
67
69
71
73
75
77
TEMPERATURE (°C)
Figure 6.
Deoxyribonucleic acid thermal denaturation curves for isolates
E. coli WP2, HI-70, and BC-74.
79
E
C
0
c'J
>(I)
2
0
Iii
-J
4
o 1.2
Fa-
0
w
>
I-
<ii
_J
Ui
a::
Of
O
(I
(3
(D
(1
TEMPERATURE (°C)
Figure 7.
Deoxyribonucleic acid thermal denaturation curves for isolates E. coli WP2, BC-74, and SC-72.
-.4
(.)
E
C
0
(0
c'J
>I-
1.3
U)
z
a
LU
-J
.
0
H
a0
1.2
Iii
>
H
4
-J
LU
(I
Tb
TEMPERATURE (°C)
Figure 8.
Deoxyribonucleic acid thermal denaturation curves for isolates
E. coli WP2, HI-70, and SC-72.
75
The eighth edition of Bergey's Manual of Determinative
Bacteriology (Buchanan and Gibbons, 1974) is the accepted
definitive source for classifying bacteria.
The advances
in systematic microbiology have exceeded the ability of
The Manual to keep current, especially in relation to the
taxonomy of the Enterobacteriaceae.
For this reason even
though The Manual was published in 1974, the section dealing
with Enterobacteriaceae was written before 1970.
Therefore,
sources other than The Manual may be used in an attempt to
obtain the most current classification for ERMB.
The family Enterobacteriaceae is defined by The Manual
as:
Small Gram-negative rods; motile by
peritrichate flagella or non-motile.
Capsulated or non-capsulated. Not
spore-forming; not acid-fast. Aerobic
and facultatively anaerobic. Grows
readily on meat extract media but some
members have special growth requirements.
Chemoorganotrophic; metabolism respiratory and fermentative. Acid is produced from the fermentation of glucose,
other carbohydrates and alcohols; usually aerogenic but anaerogenic groups and
Catalase positive with
mutants occur.
the exception of one serotype of Shigella;
oxidase negative. Nitrates are reduced
to nitrites except by some strains of
39-59
Erwinia. G+C content of DNA:
moles%.
No discrepancies are found between the definition of an
Enterobacteriaceae and ERMB.
The family Enterobacteriaceae is further subdivided
into five tribes.
When ERMB is compared to the distinguish-
ing characteristics of these tribes, it can be seen that
ERMB corresponds only to the tribe Yersinieae (Table 14).
This holds true especially when compared by the most stable
The average GC% of ERNB over six runs
characteristic, GC%.
is 47.95, which is less than one unit from the accepted
range of 45-47 GC%.
The fermentation pattern is assumed
to be mixed acid, but analysis of the acids produced is required.
The tribe Yersinieae is composed of only one genus,
Yersinia.
This in turn is presently comprised of three
species; Y. pestis, Y. pseudotuberculosis and Y. enterocolitica.
It should be noted that agreement occurs when
ERME is compared to the definitive tests for the species
Yersinia.
The only deviations are utilization of citrate
as a sole carbon source, and the amino acid reactions of
lysine and arginine.
The above reactions for ERMB,with
the exception of citrate utilization, have been shown to be
temperature dependent.
If the reactions are conducted at
37 C,only the utilization of citrate remains in disagreement.
The distinguishing characteristics between the species
of Yersinia have been comp ared to ERMB and is found to be
most closely related to Y. pseudotuberculosis and Y.
enterocolitica (Table 15).
Yersinia enterocolitica is
highly infectious for man, but white mice have been found
to be refractile (Wetzler, 1970; Carter et al., 1973) .
In
this study white mice were also found to be refractile to
Table 14.
Comparison of enteric redmouth bacterium to the five tribes of Enterobacteriaceae.a
Fermentation Pattern
Escherichieae
Klebsielleae
Mixed acid
2,3-Butanediol
Proteeae
Yersinieae
Erwinieae
ERMB
Mixed acid
Mixed acid
& 2,3butanediol
Mixed
acid
Methylred
+
D
+
+
Voges Proskauer
-
D
D
-
D
-
Phenylalanine
deamination
-
-
+
-
D
-
Nitrate reduction
+
+
+
+
D
+
Urease
-
D
D
D
-
-
KCN, growth in
D
+
+
-
D
-
50-53
52-59
39-42
45-47
50-58
48
GC%
D
+
variable
aRevised from Buchanan and Gibbon, 1974.
-J
Table 15.
Definitive characteristics of the genus Yersinia
as they relate to enteric redmouth bacterium.a
Yersinia
If motile:
22 C
ERMB
+
+
+
+
fructose
+
+
glucose
+
+
glycerol
+
+
maltose
+
+
mannitol
+
+
mannose
+
+
trehalose
+
+
methyl red
Voges-Proskauer
+
+
citrate
malonate
-
+
37C
encapsulated
-galactosidase
Fermentation of:
lactose
dulcital
erythrital
inositol
raffinose
gelatine hydrolysis
nitrate reduced
lysine decarboxylase
phenylalanine deaminase
-
+
+
arginine dihydrolase
aRevised from Buchanon and Gibbon, 1974; and Sonnenwirth,
1974.
79
Brenner et al.
ERNB.
(1976) conducted DNA homology studies
and found that ERNB possessed a relative binding ratio
(RBR) of 29-31 percent when compared with Y. enterocolitica
501-70.
The RBR for Y. pestis was 43 percent.
When tested
against Y. pseudotuberculosis P105 the RBR for various isolates are as follows:
Y. pseudotuberculosis, 81-100 per-
cent; Y. enterocolitica, 42-53 percent; and ERNB, 29-30
percent.
Steigerwalt et al.
(1975) found that his ERNB
isolates had a RBR of 24-28 percent when compared to
Serratia marcescens 868-57; 16.9-17.5 percent when compared to Serratia liquifaciens 446-68, and 25 percent when
compared to Serratiarudideae 937-72.
This would indicate that ERNB is indeed related by DNA
homologies, although not closely, to the existing species
of Yersinia and two of Serratia.
Due to the biochemical
relatedness of ERMB to Yersinia and the fact that it does
not correlate to an existing species either by DNA homologies or biochemical reactions, a new species designation
for ERMB in the genus Yersinia would appear applicable.
Table 16.
Distinguishing characteristics between the species of Yersinia and enteric
redmo,uth bacterium. a
Y. p:e:s:t:i.s,.Y. p:s:e:udo:tub:er:c'ulosis.
Y. enterocolitica
ERMB
Motility 22 C
-
+
+
37C
-
-
-
urease
-
+
+
esculin
+
+
-
-
(d)
+
-
-
salicin
d
+
-
sucrose
-
-
+
-
cellobiose
-
-
+
-
melibiose
-
+
-
-
ornithine
decarboxylase
-
-
+
+
K/A
K/A
A/A
K/A
rhamnose
TSI
gelatin
hydrolysis
-
phenylalanine
deaminase
-
aRevised from Buchanon and Gibbon, 1974; and Sonenwirth, 1974.
-
+
DISCUSSION
The work expressed in this thesis can be divided into
three parts.
The first deals with some of the biochemical
reactions of ERMB which were performed: 1) not to greatly
extend the knowledge of the biochemical pathways of ERMB,
but to confirm that we did indeed have true ERNB isolates
with no contaminants; 2) to look at discrepancies which
have been reported in the literature; and 3) to look for
possible patterns in the biochemical mechanisms of ERMB in
relation to temperature and/or antigenic composition.
The
second topic delved into the partial serological analysis
to determine if ERMB was truely composed of only one serotype as has been reported (Ross et al., 1966; Bullock and
Snieszkc
,
1975; Busch, 1975) .
Since ERMB has eluded
taxonomical classification, the third section discusses
the classification into a genus and species.
The biochemical reactions observed for all isolates
were homogeneous with very few exceptions and corresponded
quite well with the reported literature (Ross et al., 1966;
Busch,. 1973; Wobeser, 1973).
The tests under dispute are
hydrogen sulfide production, lysine decarboxylase, and
arginine dihydrolase.
Incubation temperature has been
offered as a partial explanation for the variability in
results.
Therefore,all biochemical tests were conducted
at 9, 18, 27, and 37 C.
It was found, as one might expect,
82
that most of these reactions have a temperature optimum
whereby the biochemical reactions will or will not proceed.
The dispute with lysine decarboxylase and arginine dihydro-
lase can be resolved on these grounds.
The variability in
the production of hydrogen sulfide relies on the sensitivity of the test.
Busch (1973) obtained a positive reac-
tion in 100 percent of his isolates using lead acetate
media.
Others, including work reported here, relied on the
gross production with the aid of triple sugar iron agar
slants (TS1).
The sensitivity of lead acetate media ex-
ceeds TS1 slants.
Therefore, the media employed could ex-
plain the discrepancies observed.
Only the fermentation of
sorbitol differs from that reported in the literature.
Previously, ERI'1B was thought to be 100 percent negative,
while four out of seventeen isolates were found to be positive in this study.
The significance of this observation
will be made more apparent later.
The reactions of the
extracellular enzymes also showed a dependence on temperature.
Each of these enzymes:
DNase, RNase, hyaluronidase,
chondroitin sulfatase, lipase, gelatinase, fibrinolyase,
and elastase, could be a virulence factor and contribute
to the pathogenisis of the bacterium.
Unfortunately, none
of the isolates were passed through an appropriate host
prior to testing.
Had this been done, some of the nega-
tive reactions might possibly have converted to positive,
especially in the cases of fibrinolyase, elastase, DNase
83
and RNase which were negative in all isolates regardless of
temperature.
The serological analysis provided two distinct major
agglutinating antigens (1 and 2) and one minor crossreacting antigen, 3.
The two major antigens provided the
distinction for the two serotypes, I and II.
have either antigen 1 or 2.
The bacteria
Antigen 3 was always found to
be present with antigen 1 but antigen 2 was found with and
without 3.
Neither 1 or 2 can cross-react, but if the
minor antigen 3 is present on the cells and the corresponding antibody in the antisera, then cross-agglutination can
occur.
The type of agglutination in a slide agglutination
test is readily discernible between an antigen-antibody
reaction involving the major or minor antigen.
Agglutina-
tion involving a major antigen is more complete and macroscopically visible (Figure 2c).
The minor antigen agglu-
tination is definitely a positive reaction, but is less
complete, giving a granular appearance (Figure 2d).
The utilization of sorbitol was found to be a variable
reaction depending upon the isolate.
The reaction was
later correlated to the presence of a major antigen.
It
was found that isolates of serotype I were sorbitol positive, whereas isolates of serotype II were sorbitol negative.
At present this observation may be coincidence due
to the inadequate number of isolates tested.
Only four
isolates out of seventeen were found to be serotype I and
84
sorbitol positive.
The remainder are serotype II and
sorbitol negative. Previous work reported indicate only one
serotype and it is always sorbitol negative (Ross et al.,
The second serotype
1966; Busch, 1973; Bullock, 1975).
was not observed by the earlier workers which explains why
the variability in sorbitol was also never discovered.
The major agglutinating antigens were correlated to
the presence of precipitin bands in Ouchterlony plates,
in which the various isolates were double diffused against
homologous and heterologous antisera.
It is not presently
known if the antigens correspond to somatic, capsular, or
flagellar antigens.
This work is in progress.
Bacteria from the two major serotypes were vaccinated
separately and in combination into coho salmon and challenged by homotypic and heterotypic organisms.
It was
found that isolates of serotype I could cross protect
against the isolates of serotype II.
Isolates from sero-
type II were found to protect only against bacteria from
serotype II but not from serotype I.
The minor antigen 3
was found to be protective when in combination with either
serotype.
No attempt was made to discover a protective
antigen that is non-agglutinating and non-precipitating.
If one were present, then the correlation of serotype to
protection would be totally negated.
The situation was
and could be avoided by incorporating the different antigens into a polyvalent bacterin.
Isolates BC-74 and HI-70
85
were combined to form a bivalent bacterin.
The protection
against challenge by both isolates equaled or exceeded
that of the homologous monovalent bacterin.
The amount of
the different antigens in the bacterin was not quantitatively ascertained.
Enteric redmouth bacterium was found to conform to all
of the definitions of the family Enterobacteriaceae, and
definitive biochemical reactions of the tribe Yersinieae
and genus Yersinia, with the exceptions of the utilization
of citrate as a sole carbon source.
Enteric redmouth bac-
terium was also shown to possess the typical motility pattern of Yersinia, but not the biochemical reactions or DNA
homologies to include it into an existing species of
Yersinia.
Drs. W. H. Ewing, D. Brenner, and A. 3. Ross
(personal communication) performed the biochemical tests,
GC%, and DNA homologies comparing ERME to Yersinia and
other Enterobacteriaceae.
They have come to the conclu-
sion that ERNB is a separate species in the genus Yersinia
and have proposed the name Yersiflia ruckeri after R. R.
Rucker, the microbiologist who first isolated ERMB (in
press).
The work expressed here was done independently of
the above researchers and the same conclusions were reached.
This thesis is therefore offered in support of the new
genus and species designation, Yersinia ruckeri.
SUMMARY AND CONCLUSION
1.
A serotyping scheme is proposed for enteric redmouth
bacterium.
2.
Two major agglutinating antigens (antigens 1 and 2)
and one minor agglutinating antigen (antigen 3) were
determined.
3.
Two serotypes were found (serotype I and II) based on
the possession of antigens 1 or 2.
Antigen 3 could be
present in either serotype.
4.
The utilization of sorbitol was found to correlate
with serotype I.
Serotype II could not metabolize
this sugar.
5.
Two types of positive agglutination patterns were
found in the slide agglutination test of ERMB corresponding to the presence or absence of the major or
minor antigens.
6.
Precipitin bands in Ouchterlony double diffusion
plates could be attributed to antigens 1 and 2.
7.
Enteric redmouth bacteria from serotype I can cross
protect against a challenge from an isolate of serotype II in rainbow trout.
Bacterins of serotype II
could not cross protect against a challenge from bacteria of serotype I.
87
8.
Temperature could effect not only some of the biochemi-
cal tests but the motility of the bacterium.
ERNB possessed nonfunctional flagella.
At 9 C
At tempera-
tures 18 and 27 C the bacterium was motile with pentrichous flagella.
At 37 C the bacterium was not
motile due to the loss of all flagella.
9.
The percent guanine plus cytosine in the DNA for ERMB
is 47.95 ± 0.45 (95 percent confidence interval).
10.
This work supports Yersinia ruckeni as the genus and
species designation for ERMB.
BIBLIOGRAPHY
1975.
American Fisheries Society, Fish Health Section.
Suggested procedures for the detection of certain infectious diseases of fishes. Washington, D.C., U.S.
Fish and Wildlife Service. Various paging.
Anderson, D. and A. J. Ross. 1972. Comparative study of
Prog. Fish
Hagerman redmouth disease and bacterins.
Cult. 34:226-228.
Anderson,
tion
with
Res.
D. and J. Nelson. 1974. Comparison of protecin rainbow trout (Salmo gairdneri) inoculated
and fed Hagerman redmouth bacterins. J. Fish
Bd. Can. 31:214-216.
1948. Bergey's
Breed, R., E. Murray, and A. Hitchens.
Manual of Determinative Bacteriology, sixth edition.
The Williams and Wilkins Co., Baltimore, Maryland,
1529 p.
Bergey's Manual
Breed, R., E. Murray, and N. Smith. 1957.
The
of Determinative Bacteriology, seventh edition.
Williams and Wilkins Co., Baltimore, Maryland, 1094
p.
Brenner, D., A. Steigerwalt, D. Falcao, R. Weaver, and
1976.
Characterization of Yersinia
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