Coho Salmon (Oncorhynchus kisutch)in Chile*1

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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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