PINKERTON'S stains and with a modified. GIMENEZ stain. It did not react with a monoclonal antibody against the group-specific. LPS chlamydial antigen. On the.
魚 病 研 究Fish
Pathology,25(2),107-114,1990.6
Isolation
of a Rickettsiales-Like
Organism
from
Diseased
Coho Salmon (Oncorhynchus kisutch)in Chile*1 J. L. FRYER*2 C. N.LANNAN*2 and
L. H.GARCTS*3
J. J.LARENAS*3
P. A. SMITH*3
*2Department of Microbiology , Oregon State University Corvallis, Oregon 97331-3804, USA *3Veterinary Sciences Faculty University of Chile Santiago, Chile (Received April 13, 1990)
A
rickettsiales-like
kisutch)
reared
epizootic was
with
net
accompanying
but
ature
for
failed
GIMENEZ
to
stain.
grow
chlamydial
was
did On
Rickettsiales
in
vicinity
cell
organism,
any
of
effect
eleven
for
on
the
stained react
with
the
basis
and
represents
of
but and
monoclonal
available the
in
shape,
in
cell
it
isolation
salmon S. A.
and and
lines
was
cell
The
optimal
Streptomycin, sensitive
to
stains
and
against suggest
the this
of
this
an culture, 1ƒÊm
species
not
of a member
in
four
PINKERTON'S
we
experiencing
approximately
from
CPE.
(Oncorhynchus
and
isolated
media. of
antibody
information, first
fish
bacteriological
GIEMSA a
in
production
organism,
with
not
the
coho Chile
observed
(CPE)
standard
based
from
Montt,
coccoid
cytopathic
15-18•Ž
culture
of Puerto
predominantly
inhibitory
antigen. order
in
organism It
the
The
producing
were
isolated
isolated
in
pleomorphic,
replication
tetracycline The
was pens
mortality.
It replicated,
onids,
of the
organism seawater
GRAM-negative,
diameter.
and
in
of
in
salm
temper gentamicin,
penicillin. with
a
modified
group-specific
organism group
LPS
is a member from
an
aquatic
poikilotherm.
Rickettsiales-like
and
chlamydiales-like
or
ganisms have been reported to infect many species of aquatic animals (HOFFMAN et al., 1969; HARSH BARGER et al.,1977;JOHNSON,1984). These reports have been largely descriptive in nature with detailed accounts of the ultrastructure of the agents and the pathology produced in their aquatic hosts. Until now, however, attempts at in vitro culture of these intracellular microorganisms, observed in fish and shellfish, have been un successful (WOLF,1981;BRADLEY et al.,1988). During 1989,coho salmon (Oncorhynchus kisutch) cultured in seawater net pens in Chile ex perienced an epizootic of unknown etiology. The disease was first observed at the Huito Channel, which is in the vicinity of Puerto Montt, and the site of a large number of salmon farms. The peak mortality occurred in May and was severe, with losses of up to 90% reported at certain *1 This was an invited lecture
at the
paper
annual
Society of Fish Pathology,
, presented as a special meeting of the Japanese Tokyo,March,1990.
locations (BRAVO and CAMPOS,1989). The dis ease was only observed in coho salmon and not in chinook (Oncorhynchus tshawytscha) and At lantic salmon (Salmo salar) or in rainbow trout (Oncorhynchus mykiss), although these species are also reared in the affected area. Disease signs in moribund fish were not diag nostic. They included : lowered hematocrit, swol len kidney, enlarged spleen, and, occasionally, mottled liver. No infectious agents were isolated by BRAVOand CAMPOS(1989) who first described the disease, but they did observe, by both light and electron microscopy, an unidentified parasite in the blood and internal organs of infected fish. As part of an effort to determine the cause of this continuing epizootic, kidney tissue from diseased fish was inoculated onto an established chinook salmon cell line, CHSE-2l4 (ATCC CRL 1681, LANNANet al.,1984),which is routinely used in the diagnosis of salmonid viral diseases. A rickettsiales-like agent was isolated in these cell cultures.
108
J. L. FRYER, C. N.LANNAN,
Materials
L. H.GARCES,J.
J.LARENAS, and P. A.SMITH
were
and Methods
scraped
1000•~g with
Isolation Two
year
length
old
and
on
October
the
Eicosal
Puerto
900
to
17,
Montt,
1200
sanitized
with taken
cells
(Automod, MO),
Sigma
(FBS)
cultures
Oregon
were
Disease
Upon 15•Ž.
the
these
and
serum
transport
cultures
to
were effect
incubated
4,
10,
vitro
15 mm
bottom
tissue
plates
At
removed (BSS). and
culture
and
routine
Chemical
The
(both
CCL
55)
prepared slides
(Permount, for
New
Co.,
coverslips
were
with
observation
microscopy flasks, 2.5 %
at
were and
the
glutaraldehyde
cell
sheet
methanol (Allied GIEMSA NJ). on
Fair
under
for grown
solution
mounting
100•~
prepared
oil
was
(Sigma)
micro medium
Lawn,
electron
25cm2 tissue fixed in
NJ)
immersion.
transmission in
at
Phillipsburg,
Co.,
in
were
and
mounted
Chemical
The
salt
NY)
histological
Fisher
Cultures
York,
et
flat
incubated
in absolute GREENWALD
Chemical
Glass,
in
BSS.
culture
removed
After
fixation
were
60/40
critical
wt•“ SEM
Au/Pd, operated
al.,
LANNAN
1983);
and
CCL
from
59)
nebulosus)
(WOLF
(Lepomis
macrochirus) soy
salmon
91)
with
1 % dextrose
MI)
MEM-10,
made
agar up
broth
with
Biologics,
cells
with
spent
originating promelas)
S.
medium
et al.,
Manual,
1984), of
malachite
and
from
medium Detroit, Petragnani HERROLD'S
Agriculture,
1974)
green,
serum
CA) cell
KDM-2
(EVELYN,
STEVENSON, (Myoctrim-TC,
Alameda,
5 % coho
OF
medium,
Dept.
BF-2 bluegill
1966). with
Laboratories,
egg
bovine
and the
agar
(ATCC (Ictalurus
1969);
soy
Difco
medium Inc.,
BB
thioglycolate,
Difco (U.
(DALY
mycoplasma
The
(all
%
agar
place
fluid
without
with 10
charcoal
42),
from
tryptic
EPC (FIJAN
bullhead
QUIMBY,
Dorset
(both
yolk
(Oncor
1962);
carpio),
CCL
(WOLF
agar,
blood,
medium
(ATCC
trout
(Pimephales
derived
for were:
(ATCC
QUIMBY,
brown
and
CCL
lines
RTG-2
(Cyprinus
the
also
(Oncorhynchus
MALSBERGER,1965);
(ATTC
Tryptic
and
(ATCC
lines
were
CHH-l
rainbow
minnow
cell
observed cell
et al.,1984);
FHM
fathead
(GRAVELL
and
salmon
carp
Three
species
salmon;
(WOLF
isolation
four
These
from
common
the
and fish
chum
for
organism.
lines
CPE.
from
mykiss)
used
the
organism
derived
the
was of
cell
of
1680)
from
egg
same
sensitivity
line
coho
keta)
coverslips
balanced
cells were fixed with MAY
Baker
scope
in
antibiotic cell
CSE-ll9,from
18
cells,
and
intervals,
rinsed
Corp.,
T.
CHSE-2l4
and
development
CRL
24-well
were
cells
nm
micro the
microscopy.
BSS.
Amray 1000A
propagation
CHSE-2l4
(Corning).
organism,
hour
and The stained
with
the
24
in
an
the
(Bellco
placed plates
seeded
with
15•Ž.
coverslips were
culture
were
oculated
(J.
round NJ)
with
CHSE-2l4
from
Vineland,
in
200
with
Microscopy Sterile
with
inoculated
and
in
kV.
hynchus
Inc.,
coated
warmwater
15,
60 kV
in light
the
from
21•Ž.
in and
electron
coverslips
rinsed
originating
spent
at
for
the
and
Oregon
of
1963),
prepared
salmonid
fresh
stained
glutaraldehyde,
The
the
sectioned,
coverslips
additional
was
in
(REYNOLDS,
scanning
Science
(CPE)
to at
the
incubated
aliquots
transferred
In
by on
intervals,
dried,
20
UT).
Newport,
cultures,
were
Louis,
viewed
plates
viewed
at
fixed
1969).The
CMl2/STEM
grown those
2.5%
and
St.
Marine
cytopathic
medium cultures
for
point
bovine
be
as
the
CHSE-2l4
Logan,
Hatfield
arrival,
in
culture
4•Ž
Laboratory,
When
observed
Co.,
to were
selected
in
Minimum
Inc.,
at
from
(MEM-10)
10 % fetal
University
Fish
USA.
kept
At
Works,
of
salts
Laboratories,
State
Center
at
with
(Hyclone
The
Earle's
Chemical
supplemented
Glass
Eagle's
with
was
at
post
embedded
(SPURR,
then
Philips
was
and
citrate
a
centrifuged
mode.
manner
into
tissue directly
were
with
Cells
pellet
medium
lead
transmission
scopy
samples
a monolayer
antibiotic-free
Medium
in
and
cell
dehydrated,
cells
viewed
flask
The
embedding
REYNOLD'S
were
virological
kidney
(Corning
containing
in
Essential
and
inoculated
flasks
fish
and
time,
and
near was
the
iodophor,
same
Guar
at
the
5 min.
osmium,
SPURR'S fixed
pen
epizootic of
in
collected net
Isla
bacteriological
25 cm2 tissue culture NY)
an
surfaces
50 ppm
the
41 cm
were
on
where
removed
Corning,
weight,
farm
outer
At
to
a saltwater
salmon
for
aseptically
g in from
Chile
The
analysis.
salmon,37
1989
S. A.
progress.
were
coho
from
for
were cultures
1977),
1985);
and Hana
inoculated showing
Rickettsiales-like
extensive
CPE.
incubated the
at
The
15•Ž
appearance
at
added
seeded
to
in
incubated
for
the
was in
an
flasks from
of
at
additional
14 days,
CPE.
complete
cell
cultures
These
observed
for
fixed
manner
for
each
for
demonstration
the :
a
organism. cell
culture
incubated viewed
were
and
with with
of
heat-fixed
stained
the
a
the
fluo-
stains
PINKERTON'S
developed and
chlam
(MACCHIAVELLO, or
for
a
of
Rickettsia 1981).
were
formalin stain
1937),
modification
staining
coverslips
buffered
GRAM
appropriate
rickettsiae
(BARTHOLOMEW, on
the
following of
stain
grown
and
in
(GIMENEZ,1964),
neutral
days.
were
MACCHIAVELLO
GIMENEZ
cells
CPE
the
from
and
smears
or
tsugamushi
flasks
CPE
stained
the
medium
antibody
GIMENEZ
each
to characterize
of
microscope.
ydiae
14
from
fresh
and
and
After
medium.
15•Ž
14
for
medium
onto
antibiotic-free
incubated
for
The medium
15•Ž
showing
rescence
or
109
a reagent
smears
Additional
Sigma).
of
0.5ml
inoculated
prepared
cells
of spent at
as
monoclonal
50ƒÊg/ml;
appearance
incubation,
were
(all 0.1ml
used
Salmon
Airdried
strep
gentamicin,
with
cuture,
flask
for
CHSE-214
penicillin,100IU/ml;
15ƒÊg/ml
observed
days
from Coho
was
were
30
indicated
25 cm2 flasks of
inoculated
a lysed
for
concentrations
100ƒÊg/ml;
tetracycline,
days
the
MEM-10:
tomycin,
were
media
observed
of growth.
Antibiotics were
inoculated
and
Organism
CHSE-214 fixed
and
(SIMMONS
tsu
in
10%
stained
and
with
GENTZKOW,
1944). Titration The
infectivity titer
dilution
assay
wells
per
plates
dilution
with
were
MUENCH For
wells. for
cells
were
FBS
and
cells
were
fixed
with
crystal
endpoint
in
the
six
24-well
dilution.
by
Results
with
assay
per
Isolation No
Dilution
method
of
REED
and
medium
a
0.1
organism or
overlayed
24
for
to
was
14
formalin
the
from
infected
cells,
CPE
5%
stained
5-6
1%
and
Freeze-thaw
was
medium
complete
was
assay.
Aliquots
frozen
at -70•Ž
with
the
20•Ž
titered
of
frozen water
bath
culture by
the
in
no
either
were
and
the titer
CPE
dilution
medium
It
10% (both
or
Sigma).
rapidly again
10%
A
and
(genus-specific) Integrated
prepared
in
determined.
monoclonal
chlamydial Diagnostics,
the LPS Inc.,
17
days
and
it
in
entire
a delay
in
the
30
to
days
the
At
of
of in
before
CPE at
60-day
apparent
culture
was
appear
incubated
during
4•Ž
observa
temperature the
All
were
sheet days.
appearance
CPE
15-18•Ž.
cultures
cell
15-18•Ž,i.e.10
for was
fresh within
14
below
the
vitro
was
perimental
the
to
monolayer
Cultures CPE
Thus, for
organism
a
coho
salmon
subsequent
incubated
ex
at
15•Ž.
Microscopy GIEMSA-stained
staining
against
was
period.
and
ten
medium
After
thawed
reactions
fluorochrome-labeled
commercially
fornia
staining
there
completion.
the
and
after
When
approximately
took
develop
cultures Serological
by
did
optimum
or
glycerol
18•Ž,
CPE
transferred
in
cell
appeared
cultures
(Fig.1). was
lysed
reached not
cells
cultures
cultures,
tion
were
cryopreservative
(DMSO) cultures
which
endpoint
remaining
with
sulfoxide
days,
a cell
of
addition
dimethyl 6
from
However,
rounded
the
in
of
15•Ž
15 or
from
produced
CHSE-214
above
21•Ž,
CPE. these
Spent
at
isolated
was
antibiotics.
clusters
at
temperatures
violet.
CPE
appeared
completely
then
with
days
were
no
antibiotic-free
to
The
15•Ž,
of
incubation
(Sigma). at
form
days
infected
and
containing
the
in
pathogens
fish,
of
containing
days
and
the
each
allowed
then
MEM
in
from
added
methylcellulose
incubated
removed
aliquot
hours;
with
0.75%
was
was
hour
10%
ml
dilution
The one
bacterial
diseased cultures
assay,
sample
adsorb
plaque
wells
plate
appropriate three
by
calculated
plaque
the
by plates
(1938).
the
from
determined in 96-well
or
three
endpoints and
was
(TCID50)
antibody group-specfic antigen Benicia,
(Cali CA)
were
of
large
microorganisms. pleomorphic,
forms,
and
varied
in
1.5ƒÊm.
smears
contained
frequently diameter
Microscopic
fluid
These occurring
as
appeared from
from
numbers
infected
of
darkly
microorganisms coccoid in
or
pairs.
approximately
examination
ring They
0.5 of
fixed
to and
JLFRYER,CNLANNAN,LHGARCES,JJLARENAS,and
110
PASMITH
2
1 Figs.
1
and
2.
1,
214
Figs.
Cytopathic
cells
effect
Phase
cytoplasmic
inclusion
organisms
MAY
3-6.
3, Ultrathin within
within
free form
of
in
micrograph
being (arrow)
of
6,
released and
CHSE-214
cell
three
an
variation
CHSE
post
within
inoculation
Note
a
paired
Bar=10 ƒÊm
6 CHSE-214 the
cell
rippled
24
sizes
CHSE-214
wall
CHSE-214 hours
are
after
attached
eight
among
undergoing
binary
with
of
rickettsiales-hke
forms.
fission
5, the
exterior
inoculation
he
spherical
Bar=1 ƒÊm
the
after
coccoid
organisms
electron-lucent
inoculation to
days the
and
cell
micrograph
cells size
Rickettsiales-like
cell organism
infected
election
in
cultured
5
cells varied
in
organisms
4
of an
Scanning from
the
days
rickettsiales-hke
CHSE-214
organism
Rickettsiales-like
3
Note A
of
stain
infected
cytoplasm
Organisms
rickettsiales-like 2,
4,
the
Bar=1 ƒÊm or
the itm
vacuoles
Bar=1 ƒÊm
oigamsm
cells
a
by
Bar=100
GREENWALD-GIEMSA
section
a vacuole
electron like
in
membrane-bound
structures
produced
contrast
Scanning
rickettsiales
surfaces
of
the
host
organisms Note
Bar=10 ƒÊm
the
ring
Rickettsiales-like
stained to
cell
be
cultures
showed
replicating
infected
within
cells
inclusions
microscopy
revealed
paired
organisms
enclosed
bound
vacuoles
(Fig.3).
Each
bound
by
outer
two
membrane
membrane These for
wall
and
1965).
a
plasma
structures
localized
in
the
(ANDERSON
contained structures.
near
or
more
Organisms fission
were
tetracycline,
used
electron
microscopy
irregular m
cells
coccoid
in
diameter
surfaces
host
incubation, from
24
the
found
attached
However,
many
observed
host
were
cells
(Fig.6).
or
These
outer
membranes
scanning
observed
free
in
the
ƒÊ
Titers
spilling
culture
before
had
If
and
in
from
lines
highly and
the
or could these
was the
the
from
was
period,
lysis
there
was
a
endpoint when
for
overlay
a
strong material.
by
cells
added.
hour
ten-fold
the
24-hours
was
one
host
the
assay
the
after
the underesti
of
obtained
to
added
of an
cellular
plaque
methylcellulose
overlay
associated
be
the
those by
CPE.
remained
may
adsorbed
CHSE-214
complete
because
to
after
of 106-107TCID50/
for
observed
were
organism
following
organism
were
assay
the
agents
actual titer
comparable
tion
and
tested
adsorp
reduction
in
Tablle
cells, cultures.
1.
Freeze-thaw
(Table
fish,
the
but
none
Cytopathic EPC
BF-2
sensitivity
cytopathic
warmwater
variable.
and
antibiotic was
cell
FHM BB
and
organism
salmonid
was
in
gentamicin,
plaque
of
this titer
the
organism
intercellular
size
the
organisms
medium
debris
the of
dilution
organisms varied
of
developed
containing
undergone
of
cellular
of
affinity
days
in had
Therefore,
1
and
Titers
detected
the
mate
exterior
eight
exception
plaque titer.
vitro The
lines
were
cells.
incubation,
the
flasks
culture.
that
to
infectious
the titer
cell
cultures
morphology.
In
in
ml
After
CPE
dilution
determine
growth
approximately
(Fig.5).
endpoint
apparently
by
no
the
replication
No
from
electron-lucent
hours of
organisms
ruptured
folded
after
cells
the
spaces
examined
organisms were
of
were
and
to
with infected
vitro
streptomycin,
subcultured
Both
was of
frequently
with
in 2).
organism
Titration
(Fig.4). When
inhibited
inocu
the
parasite.
antibiotics,
containing
antibiotics.
that
intracellular
(Table
cell
media
suggested
of
penicillin,
plasma
Many
bacteriological
al.,
material
region.
one
binary
the
obligately
be
et
the
presence
de
ribosome
DNA-like
central
spherical
rippled
of
111
results
organism
inner
a
an
any
cultures
been
containing
concentrated
fibrillar
organisms
undergoing
as
areas
and
was undulate
have
membrane
were
membrane,
These
of
closely-apposed
Rickettsiales
Electron-dense
like
in
lated.
The
membrane
an
Coho Salmon
strated
indi
organism
membranes
other a
in
layers,
and
membrane. scribed
in
from
was
electron
or
microorganisms
cytoplasmic
(Fig.2).
Transmission vidual
the
Organism
all
of
the
1),
but,
in
cell
induction
effect
No
in
was was
growth
Cell lines inoculated pearance
of cytopathic
of
CPE
induced apparent was
with effect
demon
One
cycle
the titer the
of
addition
in
decrease
in
DMSO preservative
the rickettsiales-like
of
freeze-thaw
the
organism
of
10%
in titer. in
at -70•Ž
glycerol
freezing effect
organism
the medium
and
3),
caused
However,
the
decreased
by>99%(Table
increased
to determine
an
even
presence
of
provided
a
survival
growth
by ap
and
further 10% cryo of
the
112
Table
J. L. FRYER, C. N. LANNAN, L. H. GARCES,J. 2.In
vitro antibiotic ettsiales-like
Table
3.Titer after
of
the
six
days
presence
sensitivity
of the
rick
organism
of
rickettsiales-like storage selected
organism
at -70•Ž
in
the
cryopreservatives*1,2
Rickettsiales-like and chlamydiales-like mi croorganisms have been observed in numerous aquatic animals. They have been found world wide in molluscs (BUCHANAN,1978; MORRISON and SHUM,1982;ELSTON and PEACOCK,1984) and have been reported to infect crustaceans (JOHNSON, 1984; SPARKS et al.,1985; BROCK et al.,1986), and amphibians (DESSER and BARTA,1984); but few of these agents have been associated with fishes. A review by WOLF (1981) of the chlamydia and rickettsia of fish indicates that the only example in the literature of rickettsia in fish is an unconfirmed case report from Egypt in 1939 where small coccoid forms, staining pink with GIEMSA, were found within monocytes and in plasma fish.
*1
Starting titer
*2
Frozen
was
107.4
J. LARENAS, and P. A. SMITH
of blood
smears
from
a dead tetradonid
TCID50/ml.
One rickettsia that is associated with fish but does not replicate in fish tissues is Neorickettsia water bath. helminthoeca, the cause of the "salmon poisoning" disease of canines (NOONAN,1973). This ricket organism by a magnitude of ten over cultures tsia is carried by a digenetic trematode, Nanophye frozen without additives. tus salmonicola, a parasite of salmonids in the Pacific Northwest USA (MILLEMAN and KNAPP, Serological and staining reactions 1970). The isolated organism was GRAM-negative. It One chlamydia-like agent that has been reported did not stain with the MACCHIAVELLO or GIMENEZ to infect fishes is the cause of epitheliocystis in stains for rickettsiae and chlamydiae. It was numerous species (HOFFMAN et al., 1969; MORRI stained positively by PINKERTON'Smethod and SON and SHUM,1983; PAPERNA and ALVES DE with a modification of the GIMENEZstain developed MATOS,1984; ROURKE et al.,1984; ZIMMERet al., to demonstrate R. tsutsugamushi. The organism 1984; BRADLEY et al.,1988). A possible second failed to react with the anti-chlamydial LPS chlamydia-like organism, morphologically similar monoclonal antibody, a reagent used to identify but substantially smaller than that causing ep organisms of the genus Chlamydia. itheliocystis, was observed in lake trout (Salvelinus namaycush) by MCALLISTER and HERMAN (1987). Discussion None of these pathogens of fish or shellfish has been propagated in vitro. Many of the infectious agents observed in In the study reported here, a microorganism cultured salmonids in Chile were introduced with associated with the Chilean coho salmon disease the eggs shipped from salmon producing areas in was isolated, and preliminary characterization of the northern hemisphere. An example of this is the replication of this microorganism in cell Renibacterium salmoninarum, the causative agent culture was made. The microorganism was an of bacterial kidney disease. The disease currently obligately intracellular parasite, and replicated in affecting coho salmon, however, is believed to cultured salmonid fish cells but did not grow in have had its origins in Chile. It has not been any of the standard bacteriological media tested. reported in fish held in fresh water, and the onset Growth of the microorganism was inhibited by of the disease has not been observed until 6-12 streptomycin, gentamicin and tetracycline. Its weeks after the transfer of fish to saltwater rearing apparent penicillin resistance was an unexpected pens. It is possible the source of the microorgan observation as in vitro replication of the Chlam ism responsible for this disease may be one or cultures
were
rapidly
thawed
more native species of aquatic animal(s).
in
a
20•Ž
ydiales (MOULDER, 1984) and most species of the
Rickettsiales-like
Organism
Rickettsiales is inhibited by this antibiotic (WEISS and MOULDER,1984). In vitro sensitivity of the organism to tetracycline suggests this drug may have application in the treatment of the infection. Tetracycline is an accepted therapeutant in fish culture. The fact that no other fish pathogen was isolated from the coho salmon experiencing this epizootic suggests that the rickettsiales-like micro organism, observed in fish and isolated in cell culture, is the causative agent of the coho salmon disease. In vivo studies are planned to test this hypothesis. The potential pathogenicity of the organism, its apparent virulence, and the fact that it is not known to occur outside of Chile dictate that these studies be conducted in the area where the disease is endemic. For these reasons, the agent will be returned to Chile for infection experiments in coho salmon. The exact taxonomic placement of this micro organism is yet to be determined. Although it was an obligate intracellular parasite and repli cated in vitro within cytoplasmic inclusions in host cells, it did not share other characteristics of the Chlamydiales. It did not react with the monoclonal antibody against the group-specific LPS chlamydial antigen, and although polymor phic, did not appear to develop the small in fectious elementary bodies and the large replica ing initial bodies characteristic of these t micro organisms. It did, however, contain the rippled cell wall and electron-lucent spherical structures described for certain rickettsial species (ANDERSON et al.,1965). For these reasons, and until precise placement can be determined, we suggest the organism be associated with the order Ricket tsiales (tribe Ehrlichieae) rather than with the more limited and closelyd efined Chlamydiales .
from
Coho Salmon
113
This manuscript was presented in its entirety by one of the authors (JLF) at the annual meeting of the Japanese Society of Fish Pathology, March 30, 1990, at the Tokyo University of Fisheries, Japan. The work was sponsored by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U. S. Department of Commerce, under Grant NA89AA-D-SG108 , by the Salmon Growers Association of Chile, and by the Veterinary Sciences Faculty of the University of Chile. It is Oregon Agricul tural Experiment Station Technical Paper No . 9170. References
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