NOAA Technical Report NMFS III

October 1992

Control of Disease in Aquaculture
Proceedings of the Nineteenth
U.S.-Japan Meeting on Aquaculture
Ise, Mie Prefecture, Japan
29-30 October 1990
Ralph S. SVIjcek (editor)

u.s. Department of CommerceNOAA Technical Reports NMFS
The m~or responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution of fishery resources, tounderstand and predict fluctuations in the quantity anddlstribution of these resources, and to establish levels fortheir optimum use. NMFS is also charged with the development and implementation of policies for managing national fishing grounds, with the development andenforcement of domestic fisheries regulations, with the surveillance of foreign fishing off U.S. coastal waters, andwith the development and enforcement of international
fishery agreements and policies. NMfS also assists thefishing industry through marketing services and economic
analysis programs and through mortgage insurance andvessel construction subsidies. It collects, analyzes, andpublishes statistics on various phases of the industry.
The NOAA Technical Report NMFS series was established in 1983 to replace two subcategories of the Technical Report series: "Special Scientific Report -Fisheries"and "Circular." The series contains the following types ofreports: scientific investigations that document long-term

continuing programs of NMFS; intensive scientific reports
on studies of restricted scope; papers on applied fisheryproblems; technical reports of general interest intended toaid conservation and management; reports that review, in
considerable detail and at a high technical level, certain
broad areas of research; and technical papers originatingin economics studies and in management investigations.
Since this is a formal series, all submitted papers, except
those of the U.S-:Japan series on aquaculture, receive peer
review and all papers, once accepted, receive professional
editing before publication.
Copies of NOAA Technical Reports NMFS are available free in limited numbers to government agencies, bothfederal and state. They are also available in exchange forother scientific and technical publications in the marine
sciences. Individual copies may be obtained from the
U.S. Department of Commerce, National Technical Information Service, 5285 Port Royal Road, Springfield, VA
22161. Although the contents of these reports have notbeen copyrighted and may be reprinted entirely, reference
to source is appreciated.

Recently Published NOAA Technical Reports NMFS
98.

99.

Marine DlIUlllll&1 straodings in the United States:
proceedings oC the second Dlarine lnaDllnal
stranding workshop; Mi&Dli, Dorida, 3-5 DeceDlber, 1987, edited by John E. Reynolds III and Daniel
K. Odell. January 1991, 157 p.
Marine Bora and Cauna oC the northeastern UnitedStates: erect Bryozoa, by John S. Ryland and Peter
J. Hayward. Fl!bruary 1991, 48 p.

100. Marine Bora aDd fauna oCthe eastern United States:
DicyeDlida, by Robert B. Short. February 1991, 16 p.
101. Larvae oC nearshore fishes in oceanic waters near
Oahu, Hawaii, by Thomas A. Clarke. March 1991,
19 p.
102. Marine ranching: proceedings oC the seventeenth
U.S.-japan Dleeting on aquaculture; Ise, Mie
PreCecture, japan, 16-18 October 1988, edited by
Ralph S. SVJjcek. May 1991, 180 p.
103. Benthic DlacroCauna oC the New York Bight,
1979-89, by Robert N. Reid, David J. Radosh, Ann B.
Frame, and Steven A. Fromm. December 1991,50 p.
104. Incidental catch oC Dlarine DllUDlllals by Coreign
and joint venture trawl vessels in the U.S. EEZ oC

the North Pacific, 1973-88, by Michael A. Perez andThomas R. Loughlin. December 1991, 57 p.
105. Biology, oceanography, and fisheries oC the North
Pacific transition zone and subarctic frontal zone,edited by Jerry A. Wetherall. December 1991,92 p.
106. Marine ranching: proceedings oC the eighteenth
U.S.-japan Dleetiog on aquaculture; Port Ludlow,
Washington, 18-19 SepteDlber 1989, edited by Ralph
S. SVJjcek. February 1992, 136 p.
107. Field guide to the searobins (PriOftOtNs and Bellator) in the western North Atlantic, by Mike Russell,Mark Grace, and Elmer J. Gutherz. March 1992, 26 p.
108. Marine debris survey DlaDual, by Christine A. Ribie,
Trevor R. Dixon, and Ivan Vining. April 1992,92 p.
109. Seasonal cliDlatologies and variability oC eastern
tropical Pacific surface waters, by Paul C. Fiedler.
April 1992, 65 p.
110. The distribution oC KeDlp'S ridley sea turtles(Lepidoclaelys kenap.) along the Tellas coast: anadas, by Sharon A. Manzella and Jo A. Williams. May
1992,52 p.

NOAA Technical Report NMFS III

Control of Disease in Aquaculture
Proceedings of the Nineteenthu.s.-Japan Meeting on Aquaculture!se, Mie Prefecture, Japan
29-30 October 1990
Satellite Symposium: 2 November
Ralph S. Svrjcek
Publications Unit
Northwest and Alaska Fisheries Science Centers

Panel Chairmen:

Conrad Mahnken, United States
Seiji Sakaguchi, Japan

Under the U.S.-Japan Cooperative Programin Natural Resources (U)NR)

October 1992

u.s. DEPARTMENT OF COMMERCE
Barbara Hackman Franklin, Secretary

National Oceanic and Atmospheric Administration
John A. Knauss, Under Secretary for Oceans and Atmosphere

National Marine Fisheries Service
William W. FoxJr., Assistant Administrator for Fisheries

PREFACE

The United States and Japanese counterpart panels on aquaculture were formed in 1969 under theUnited States-Japan Cooperative Program in Natural Resources (UJNR). The panels currently include specialists drawn from the federal departments most concerned with aquaculture. Charged
with exploring and developing bilateral cooperation, the panels have focused their efforts on exchanging information related to aquaculture which could be of benefit to both countries,
The UJNR was begun during the Third Cabinet-Level Meeting of the Joint United States-Japan
Committee on Trade and Economic Affairs in January 1964, In addition to aquaculture, current
subjects in the program include desalination of seawater, toxic microorganisms, air pollution, energy,
forage crops, national park management, mycoplasmosis, wind and seismic effects, protein resources,
forestry, and several joint panels and committees in marine resources research, development, andutilization.
Accomplishments include increased communication and cooperation among technical specialists; exchanges of information, data, and research findings; annual meetings of the panels, a policycoordinative body; administrative staff meetings; exchanges of equipment, materials, and samples;several major technical conferences; and beneficial effects on international relations,
Conrad Mahnken-United States
Seiji Sakaguchi-JapanThe National Marine Fisheries Service (NMFS) does not approve, recommend or endorse any proprietary product or proprietary material
mentioned in this publication. No reference shall be made to NMFS,
or to this publication furnished by NMFS, in any advertising or sales
promotion which would indicate or imply that NMFS approves, recommends or endorses any proprietary product or proprietary material
mentioned herein, or which has as its purpose an intent to cause
directly or indirectly the advertised product to be used or purchased
because of this NMFS publication. The U.S.-Japan subseries of NOAA
Technical Reports on aquaculture is used to communicate preliminary
results, interim reports, and similar timely information. It is not subject
to formal peer review.

ii

CONTENTS_

Studies on diseases of cultured Atlantic halibut

1

T.AKIYAMA

Scoliosis of fishes caused by tryptophan deficiency

7

T. MEYERS

Control of IHN virus in sockeye salmon culture

13

Identification of a conserved antigenic domain in the major
capsid protein of infectious pancreatic necrosis virus

15

T.AOKI
I. HIRONO

Cloning of hemolysin genes of aeromonads

21

T. HONJO

Harmful red tides of Heterosigma akashiwo

27

Impact of the myxosporean parasite Ceratomyxa shasta on survivalof migrating Columeia River Basin salmonids

33

M. YOSHIMIZU
T.KIMURA

Viral infections of cultured fish in Japan

43

K.MOMOYAMA

Some important infectious diseases of kuruma shrimp,
Penaeus japonicus, in Japan49

J. R. WINTON

The application of molecular biology to the detection of
infectious hematopoietic necrosis virus

53

Bacterial and viral diseases of marine fish during seed production

57

An ecological study of the parasitic nematode Hysterothylacium haze in the Japanese63

0. BERGH
G.H.HANSEN
I. HUSE
A.JELMERT

R.J. BARRIE
C. L. MASON
J. C. LEONG

J. L. BARTHOLOMEW
J. L. FRYER
J. S. ROHOVEC

K. MUROGA
T. YOSHINAGA

common goby Acanthogobius flavimanus, in a brackish inlet
Epidemiology of marine fish diseases in the warm waters along the Kuroshio Current

69

Characterization of hematic neoplasia in the softshell clam Mya arenaria

85

M.OTOTAKE
T. NAKANISHI

Kinetics of bovine serum albumin administered by the immersion method
in fishes acclimatized to seawater and to fresh water

95

T. NOMURA
M. YOSHIMIZU
T.KIMURA

The epidemiological study of furunculosis in salmon propagation

101

H.ISHIOKA

P. W. RENO
A. ILLINGWORTH
M.DORITYiii

Functions of hemocytes during the wound healing process in the pearl oyster

109

Skeletal abnormalities of fishes caused by parasitism of Myxosporea

113

Presence of oncogenes in fish tissues and in fish cell lines

119

Streptococcal infection in cultured yellowtail

125

G.MOBERG

Stress induced pathologies in fish: the cost of stress

131

A. MURATA

Control offish disease inJapan

135

T. SUZUKI
YMAENO
M. SORlMACHI
E. READ-CONNOLE
C.A. SMITH
F. M. HETRICK
H.SAKOStudies on Diseases of Cultured Juvenile Atlantic Halibut

0IVIND BERGH', GEIR H0VIK HANSEN", INGVAR HUSE' and ANDERSJELMERT'* Institute of Marine Research
Austevoll Aquaculture Research Station
N-5392 Storeb¢, Norway** Department of Microbiology and Plant Physiology
University of Bergen, Jahnebakkm 5
N-5007 Bergen, Norway

ABSTRACT
Bacterial infections by Flexibacter and Vibrio species are major causes of mortalities inAtlantic halibut (Hippoglossus hippoglossus L.) larviculture. Egg surface disinfection is apossible prophylactic treatment. This article summarizes and reviews several experimel1ls
concerning causes of mortality of Atlantic halibut eggs and larvae.

Introduction_

Cultivation of Atlantic halibut (Hippoglossus
hippoglossus L.) is presently at the verge of a commercial breakthrough in Norway. However, as is the casewith all cultivated species, there are problems emerging concerning diseases related to opportunistic
microorganisms (Bergh et al. 1992; Bergh andJelmert 1990; Opstad and Bergh 1990; Pittman et al.
1990). The purpose of this work is to summarize several experiments by studying the effects of microorganisms
on mortalities of halibut eggs and yolk sac larvae,
possible prophylactic treatment procedures, and effects of some physical stressors.

Methods and Materials_

Eggs from one female were artificially stripped andfertilized with sperm from two males and reared in
250-L upstream incubators between 6 and 7° C until
further processing.
The eggs were transferred to polystyrene multiwelldishes (NUNC, Roskilde, Denmark) for the disinfection trials and the infection experiment. Each wellcontained one egg and II mL of sterilized seawater.
The dishes were incubated in darkness between 5
and 6° C for the duration of the experiment.
For the infection experiment, eggs were divided
into 6 groups, each containing 60 eggs. Four days
before hatching, each well of each of the 5 treatmentgroups was infected with 200 !-LL of a suspension ofan accenic culture of one of the following bacteria:
Flexibacter sp. strains NCIMB 13128 and NCIMB
13127 T, (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) which wereisolated from two different groups of halibut eggs
and otherwise seemingly identical (Hansen andBergh et al. 1992), Vibrio strain HI-I0448 (Institute ofMarine Research, Bergen, Norway) and Vibrio
anguillarum NCMB 6 (National Collection of Marine
Bacteria, Aberdeen, Scotland); and Vibrio fischeri
strain ATCC 7744 (American Type Culture Collection, Rockville, MD). Final total counts of bacteria in
the wells were measured by staining with DAPI (Porter and Feig 1980) and counting in a Nikon
epiflourescence microscope at 600X to be in the order of 2-3 X 106 bacteria x mL-l. One group of 60
eggs was not infected, serving as the control. Within
24 hours after hatching, visible remnants of the eggshell were removed along with 10 mL of the water,and 10 mL of sterile seawater were immediately
added.. Mortality was recorded until Day 37 after
hatching. For a further description of the infection
experiment, see Bergh et al. (1992).
For the disinfection experiment another egg group
was disinfected one day before hatching. The following procedure was followed. Eggs were divided into
four groups, which were exposed to different concentrations of the iodophor disinfectan t Buffodine(Evans Vanodine, Preston, England): 0.5, 0.05, and
0.005%, plus one untreated control group. Applica-

2

NOAA Technical Report NMFS III

Figure 1

Halibut egg showing
large surface wounds.
This appearance istypical of eggs infected with Flexibacter
sp. Egg diameter isapproximately 3 mm.
The photograph wastaken from a Wildzoom binocular microscope operated in
the dark field mode.
Photo by Guri Grung
and Vibeke Valkner.
tion time was 10 minutes. Immediately following thedisinfection, the solution was carefully pipetted off
the eggs, and more sterile seawater was added. T1.isprocedure was repeated three times. The control
group was washed the same way as the other groups.
Thereafter, 60 randomly chosen eggs from eachgroup were incubated in polystyrene multiwell dishes
as previously described. Within 24 hours after hatching, visible remnants of the eggshell were removed
along with 10mL of water, and 10 mL of sterile seawater was added. Mortality was recorded until Day 37
after hatching. The remaining living larvae were examined microscopically under a dissection
microscope for developmental disorders. A further
description of this experiment is given by Bergh andJelmert (1990).

Results_

Figure 1 shows a halibut egg with severe surface damage, an appearance typical for eggs infected with
Flexibacter sp. Figure 2 demonstrates a normal egg,
without visible damage.
Scanning electron microscopy of infected eggs revealed the chorion to be completely dissolved overlarge areas (up to 206 f-Lm in diameter), whereas thezona radiata was severely damaged. Isolation of
epibiotic bacteria from this egg group revealed an
epiflora totally dominated (99% of colony-forming
units) by Flexibacter sp. (Bergh et al. 1992)

The infection experiment revealed three different
types of mortality patterns:
1

The uninfected control group showed very low
mortality throughout the experiment, as only 5
out of 60 larvae died.

2

The two groups infected with Flexibacter sp.
showed high mortalities at hatching; out of 60 larvae per group, 40 and 49 had died in the NCIMB
13127 T and NCIMB 13128 groups, respectively. At
Day 18, these groups were terminated in order togain material for re-isolation of bacteria. Eighty
and 93% of the larvae were dead in Groups
NCIMB 13127 T and NCIMB 13128, respectively.

3

The groups infected with Vanguillarum strains orwith V. jischeri showed an intermediate mortality
pattern. Only 1-4 larvae died per group at hatching, but high mortalities occurred throughout therest of the experiment. When the experiment was
terminated at Day 37, mortalities had risen to
95% in the V. anguillarum NCMB 6 group, 78% in
the V jischeri ATCC 7744 group, and 67% in thegroup infected with V anguillarum 651.

In the disinfection experiment, 7 out of 56 remaining larvae in the group treated with 0.5% Buffodine
were dead when the experiment was terminated, 9·
out of 59 were dead in the 0.05% Buffodine group,
24 out of 59 were dead in the 0.005% Buffodine
group, and 19 out of 58 in the untreated group.

Bergh et al.: Studies on Diseases of Cultured Atlantic Halibut

3

Figure 2

A normal halibut
egg. Diameter is approximately 3 mm.
The photograph istaken from a Wildzoom binocular microscope operated in
the dark field mode.
Photo
by
Guri
Grung and Vibeke
Valkner.
The groups that were disinfected with 0.5% and0.05% Buffodine could not be significantly distinguished for cumulative mortality at the end of theexperiment (P>0.05, t-test with arcus sinus transformation of proportions). The two remaining groups
were not statistically separable, but they both had significantly higher mortalities than the two groups thatwere treated with the highest Buffodine concentrations (P<0.05%, t-test with arcus sinus transformationof proportions).
With respect to developmental disorders, 5 out of
49 living larvae treated with 0.5% Buffodine were
found to posess at least one kind of disorder whenthe experiment was terminated in the group. In theother groups, scores were 16 out of 50 (0.05%
Buffodine), 22 out of 36 (0.005% Buffodine) and 23
out of 39 (control). The most common disorder was
the presence of black, probably necrotic, tissue in thegill, heart, or frontal yolk-sac region. Work is inprogress to characterize the ultrastructural changes
associated with these kinds of disorders.

Discussion_

We have presented initial results showing that bacterial infections may be closely involved in themortality of cultured halibut eggs and yolk-sac larvae.
The infection experiment proves that Flexibacter sp. isa causative agent of mortality at the egg stage, at
hatching, and at the early yolk-sac stage. It has earlier

been shown that bacteria are able to cause at least
minor destruction to the chorion (Hansen andOlafsen 1989), but we are not aware of other reports
showing that bacteria are able to dissolve the chorion
completely. It could not be deduced from the scanning electron micrographs that the zona radiata wascompletely destroyed; however, evidence of severe destruction was clear. This biotype is commonly isolated
from Atlantic halibut eggs and could be considered amajor problem in halibut larviculture.
With the two Vibrio species that were used in theinfection experiment, the situation is different.
These bacteria seemed to be harmless to the egg
stage, causing no significant mortality. However, theprofound mortality of the infected groups throughout the yolk-sac stage indicates that Vibrio-infections
during the yolk-sac stage may help to explain thehigh mortality rates experienced so far.
Recent observations indicate that infectious pancreatic necrosis virus (IPNV), serotype Nl, isprevalent in juvenile stages of the Atlantic halibut(Mortensen et al. 1990). The IPNV is associated withhigh mortalities, but it is not verified whether thevirus is the principal lethal agent. The time of infection has so far not been shown. Also, nematodes
could be present in relatively large amounts (G.
Bristow, Zoological Laboratory, University of Bergen,pers. commun. 1991); these are probably introducedby feeding the larvae with collected zooplankton.
The disinfection experiment demonstrated thatthe bacterial epiflora on halibut eggs is at least partly

4

NOAA Technical Report NMFS III

responsible for many of the developmental disorders
commonly occuring in halibut yolk-sac larvae. Surface disinfection of eggs with iodophors should be anadequate prophylactic treatment, as they have good
pathogen/host differential of toxicity (Amend andPietsch 1972; Ross and Smith 1972; Amend 1974).
However, more work is needed to establish reliable
disinfection procedures.
Mortality rates could be augmented by several sublethal factors (Rosenthal and Alderdice 1976).
Sublethal physical stressors might increase egg andlarvae sensitivity to infectious microorganisms, rather
than per se be the causative agent of death. The effectsof sublethal stressors to early life stages of halibut
have been investigated in several studies.
Jelmert and Naas (1990) reported that lowered 02
concentrations, exposure to H 2S and exposure tohigh light levels led to a higher prevalence of deformed yolk-sac larvae.
Sensitivity of halibut eggs to physical shocks wasinvestigated by Holmefjord and Bolla (1988), whofound that eggs were most sensitive before the closure of the blastopore. In a more extensive study,
eggs of halibut were compared with several other marine fishes, Opstad (I. Opstad, Austevoll Aquaculture
Research Station, pers. commun. 1991) found similar
results, with the addition that eggs during the hatching period were highly sensitive to physical stress.
Effects of water flow on yolk-sac larvae were studied
by Opstad and Bergh (1990), who concluded thathigh rates of water exchange in upstream incubators
significantly increased mortality. Yolk-sac utilization
was inversely related to rate of flow.
Absence of flow, however, caused rapid increase in
the amount of bacteria in the incubators (Opstad
and Bergh 1990; Skiftesvik et al. 1990), and subsequent larval mortality. Thus, these two effects must
be carefully weighed against each other. Althoughnormally not regarded as a sublethal stressor or causat.ive agent of diseases, extreme light regimes havebeen shown to induce reduced yolk-sac utilizationand increased mortality of halibut yolk-sac larvae(Skiftesvik et al. 1990).
Studying development and mortality of Atlantic
halibut eggs and larvae at different temperatures.
Pittman et al. (1990) concluded that 3° C is near the
lower limit for development of halibut eggs and larvae. At this temperature, the larvae often showed
incomplete caudal development and suffered higher
mortality than those reared at 6° C. The groups
reared at 9° C had high egg mortality and quickly
developed abnormalities, such as small hearts andlivers, and large peritoneal and pericardial spaces,indicating that this temperature was sublethal. No
primary cause of larval deat.h could be identified,although four critical periods of high mortality wereidentified for aquaculture systems at temperatures
between 3 and 9° C: hatching, 10-14, 25-35, and45-60 days after hatching. The latter was probably
due to starvation (I. Opstad and A.B. Skiftesvik,
Austevoll Aquaculture Research Station, pers.
commun. 1991). For the three other critical periodsof the yolk-sac stage, evidence presented here indicates that effects of bacteria could not be ruled out.
There are not yet any data available ranking thequantitative importance of the different causes ofdeath of Atlantic halibut eggs and larvae, although
early-life stage mortalities are still a major factor limiting the commercial success of halibut aquaculture.
The data presented here, however, give evidence that
pathogenic or opportunistic microorganisms areclosely involved in some typical mortality and developmental disorder patterns of eggs and yolk-sac
larvae.

Acknowledgments_

This work has been supported by the Royal orwegian Council for Scientific and Industrial Research( TNF) , by the Norwegian Council for Fisheries Research (NFFR), and by Mowi a/so We are grateful to
Guri Grung and Vibeke Valkner who kindly let us use
their photographs.

Citations_

Amend. D. F.
J 974. Comparative toxicity of two iodophors to rainbow
trout eggs. Trans Am. Fish. Soc. 103:73-78.
Amend, D. F., and]. P. Pietsch.
1972. Virucidal activity of two iodophors to salmonid
viruses. J. Fish. Res. Board Can. 29:6165.
Bergh, 0., and A.Jelmert.
1990. Antibacterial treatment prQcedures of eggs of halibut(Hippoglossus hippoglossus L.). Presented at council meeting,International Council for the Exploration of the Sea. ICESCM-1990/F:39, 6 p.
Bergh. 0 .. G. H. Hansen, and R. E. Taxt.
1992. Experimental infection of eggs and yolk sac larvae of
halibut, Hippoglossus hippoglossus L. J. Fish Dis. 15. (Inpress.)
Hansen, G. H., andJ. Olafsen.
1989. Bacterial colonization of cod (Gadus morhua L.) andhalibut (Hippoglossus hippoglossus) eggs in marine
aquaculture. Appl. Environ. Microbiol. 55(6):1435-1446.
Hansen, G. H., 0 Bergh,]. Michaelsen, and D. Knappskog.
1992. Flexibactl'T ovulyticus sp. nov., a pathogen of eggs andlarvae of Atlantic halibut. Hippoglossus hippoglossus L. Int. J.
System. Bacteriol. 42(3). (In press.)
Holmefjord, I., and S. Bolla.
1988. Effects of mechanical stress on Atlantic halibut eggs at
different times after fertilization. Aquacultl:lfe 68:369-371.

Bergh et at.: Studies on Diseases of Cultured Atlantic Halibut
Jelmert, A., and K. E. Naas.
1990. Induced deformities of the Atlantic halibut(Hippoglossus hippoglossus L.) yolk sac larvae. A new experimental approach. Presented at council meeting, International Council for the Exploration of the Sea. ICE5-CMI990/F:45, 8 p.
Mortensen, S. H., B. Hjeltnes, O. R0dseth, j. Krogsrud, and K. E.
Christie.
1990. Infectious pancreatic necrosis virus, serotype N1, isolated from Norwegian halibut (Hippoglossus hippoglossus) ,turbot (Scophthalmus maximus) , and scallops (Pecten
maximus). Bull. Eur. Assoc. Fish. Pathol. 10(2):42.
Opstad, 1., and 0. Bergh.
1990. Effects of continuous flow rate on development andmortality of halibut yolk sac larvae. Presented at council
meeting, International Council for the Exploration of the
Sea. ICE5-CM-1990/F:41, 11 p.
Pittman, K. 0. Bergh, 1. Opstad, A. B. Skiftesvik, L. Skjolddal, and
H. Strand.
1990. Development of eggs and yolk sac larvae of halibut(Hippoglossus hippoglossus L.). j. Appl. Ichthyol. 6:142-160.

5

Porter, K. G., and Y. S. Feig.
1980. The use of DAPI for identifying and counting aquatic
microflora. Limnol. Oceanogr. 25:943-948.
Rosenthal, H., and D. F. Alderdice.
1976. Sublethal effects of environmental stressors, naturaland pollutional on marine fish eggs and larvae. j. Fish Res.
Board Can. 33:2047-2065.
Ross, A.j., and C. A. Smith.
1972. Effect of two iodophors on bacterial and fungal fish
pathogens. j. Fish Res. Board Can. 29:1359-1361.
Skiftesvik, A. B., 1. Opstad, 0. Bergh, K. Pittman, and L. Skjolddal.
1990. Effects of light on the development, activity and mortality of halibut (Hippoglossus hippoglossus L.) yolk saclarvae. Presented at council meeting, International Council for the Exploration of the Sea. ICE5-CM-1990/F:43, 16 p.

Scoliosis of Fishes Caused by Tryptophan Deficiency
TOSHIO AKIYAMANational Research Institute ofAquaculture
Fisheries Agency
Tamaki, Mie 519-04, Japan

ABSTRACT
Scoliosis, caused by dietary tryptophan (Trp) deficiency, has been reported mainly insalmonids. Neither abnormality in the vertebra per se nor microscopically visible damage
in the surrounding tissues was detected in the scoliotic fish, most of which returned tonormal shape within a short period of time after restoration of Trp to the diet. Therefore, serotonin (5-HT), which is one of the Trp metabolites and a known
neurotransmitter, was suspected as a key substance responsible for the symptom. This
paper reviews several feeding studies where purified diets containing various combinations of L-Trp, 5-hydroxy-L- tryptophan (5-HTP, direct precursor of 5-HT) , MK486 (local
inhibitor of 5-HT synthesis only in periphery) and DL-p-chlorophenylalanine (PCPA,
general inhibitor of 5-HT synthesis) were fed to chum salmon fry (Oncorhynchus keta).
The findings indicate that occurrence of the spinal deformity is related to depletion of 5HT in the central nervous system. In addition, the relationship between watertemperature during rearing period and incidence of the scoliosis is also discussed.

Introduction_

Although it is known that 10 amino acids are essential for normal fish growth, all of the quantitativerequirements for essential amino acids have been
determined only for chinook salmon, Oncorhynchus
tschawytscha, coho salmon, O. kisutsch, carp,
Cyprinus carpio, channel catfish, Ictalurus punctatus,
Japanese eel, Anguilla japonica, and Nile tilapia,
Oreochromis niloticus. The author conducted a seriesof dietary studies to determine the amino acid requirements for the fry of chum salmon, O. keta,
which is one of the most important species in thesalmon enhancement project in Japan. In these experiments, spinal deformity was observed in manyof the fish fed a tryptophan(Trp)-deficient diet(Fig. 1; Akiyama et al. 1985). Since the abnormality
was first attributed to Trp deficiency in sockeye
salmon by Halver and Shanks (1960), the same deficiency symptom has been reported in rainbow
trout (Shanks et al. 1962; Kitamura 1969; Kloppel
and Post 1975; Poston and Rumsey 1983; Walton etal. 1984) and coho salmon (Ogata and Arai 1981).
So far, the biochemical pathway resulting in theoccurrence of spinal deformity due to Trp deficiency has not been elucidated.

Characteristics of Spinal Deformity_

The spinal deformity caused by Trp deficiency ismainly scoliotic or slightly lordoscoliotic, and neither
lordosis or kyphosis has been noted. Scoliosis occurs
after 1-2 weeks of feeding a Trp-deficient diet inrainbow trout (Kitamura 1969; Kloppel and Post
1975) and chum salmon (Akiyama et al. 1986a). Most
scoliotic fish return to normal shape after restorationof Trp to their diet; therefore, this symptom is reversible. However, most spinal deformities caused bynutrient deficiency are not reversed even by restoring
nutrients to their optimum level in the diet. For example, ascorbic acid-deficient fish form thermallylabile underhydroxylated collagen which is denatured and digested at higher temperatures; thisresults in connective tissues with a low collagen content and in the development of a fragile bone
structure, which finally results in irreversible symptoms of scurvy, such as lordosis and scoliosis (Sato etal. 1983; Ikeda et al. 1983). Although Kloppel andPost (1975) observed some minor abnormalities suchas protrusions of the fibrous matrix sheath investing
the notochord of scoliotic rainbow trout that werecaused by Trp deficiency, serious lesions of the vertebrae and microscopically visible damage in the
7

8

NOAA Technical Report NMFS 111(%)

100 ..

~

tI.l

a
:3
a(.)
tI.l
~

a

50

..

~

(.)

z

~

0

u
::s

I

-

_

0
(%)

/~

150
Z

:;:
c.:l
f-<

100

:t:

c.:l

1,;1

0:
50

0

/

I

•

•

•

I

•

Requirement
0.29%

Figure 1

I

!
0.2

I

.,

0.4

0.6

TRYPTOPHAN % IN DIET

surrounding tissues have not been recognized inrainbow trout (Kitamura 1969) and chum salmon(Akiyama et al. 1986b).

Construction of Hypothesis_

Trptophan is not only an essential structural elementof protein but also the precursor of nicotinamide adenine dinucleotide (NAD) and niacin in higher
vertebrates (Fig.2). Therefore, attention was focusedon the role of dietary niacin in the early studies of
Trp metabolism. Poston and Combs (1980), however,reported that dietary Trp is not an efficient precursorof niacin in salmonids. Moreover, Poston and Rumsey(1983) showed that the deletion of dietary niacin did
not significantly increase the incidence of scoliosis inrainbow trout fed a diet containing a low level of Trp.
It is possible that the symptom of scoliosis may be
induced by an abnormal and involuntary contraction
or relaxation of muscle due to a defect in the ner-

Relationships between tryptophan levelin diet and weight gain or incidence ofscoliosis; average value of duplicate tanksof 35 fish, each group fed for 4 weeks at
16.0° C (Akiyama et al. 1985).

vous system. Many reports on spinal deformity caused
by a metabolic disfunction or lesion in the nervous
system are available. In mammals, scoliosis develops
in bipedal rats with brain-stem lesions (Tamura 1974)
and in rabbits whose dorsal root in the spinal cord
was removed (MacEwen 1973). In fish, yellowtail
parasitized by cysts of Myxobolus in the 4th ventricleof the brain (Egusa 1985; Sakaguchi et al. 1987)
showed scoliosis. Vertebral. deformity was reported tooccur in yellowtail whose brain was infected by betahemolytic streptococcal bacterium (Shiomitsu 1982;
Kaige et al. 1984). Spinal deformities caused by anabnormality in the peripheral nervous system arewel1 known in fishes exposed to pesticides such asdiazinon, which develop severe spinal curvature together with fracturing (Hirose and Kitsukawa 1976;
Hirose et al. 1979). It is speculated that these symp. toms are induced by excess accumulation ofacetylcholine in the neuromuscular junction. Thus,
abnormality in the nervous system is one of the mostimportant factors for occurrence of spinal deformity.

Akiyama: Scoliosis of Fishes Caused by n-yptophan Deficiency

Protein -Tryptophan -1

95-HydroIy-L-tryptophan_ Serotonin(S-HT)(5-HTP)

Iynurenine

Hel!tonin~

Xanthurenic a c i r ~3-HydroIyanthranic acidl-----·~

Glutaric acid1

Guinolinic acid1

CO2, H20

Figure 2
Map of tryptophan metabolism.

NAD

Among the various Trp metabolites, serotonin (5HT) is known to function as a brain neurotransmitteror modulator and is involved in the regulation of
sleep, body heat, sexual behavior, appetite, pain recognition, secretion of growth hormone andprolactin, besides classical functions such as the contraction of smooth muscle of blood vessels, theuterus, and the digestive tract. In addition, it is
known that torticollis and abnormal posture can be
induced by the destruction of rat midbrain in which
both serotoninergic and dopaminergic neurons arelocated (Tanaka and Kimura 1984), and that 5-HT
modulates the central pattern generator for locomotion in the spinal cords of the lampreys Ichthyomyzon
unicuspis and Petromyzon marinus (Harris-Warrich etal. 1985). These reports suggest that muscular function can be partly controlled by a serotoninergic

(%)
60

_.-

~.

CI.l
H
CI.l

5-HT Involvement in the Occurrenceof Scoliosis _ _._In the first experiment which tested the involvementof 5-HT in the occurrence of scoliosis an oral administration of 5-HT to Trp-deficient chum salmon fry
decreased the incidence of scoliosis, but did not completely inhibit its occurrence (Akiyama et al. 1986a).
Therefore, we fed fry a Trp-deficient diet supplemented with 5-hydroxy-tryptophan (5-HTP, 100-130
mg/l00 g diet), which is a direct precursor of 5-HT

Trp 0.05%
Trp 0.05% + J:ynurenine

0--0 Trp 0.05%@--@

neuron. From these facts, I hypothesized thatscoliosis caused by Trp deficiency would be induced
by an imbalance of muscular tension due to a decreased 5-HT level in the nervous system.

+ 5-HTP

Trp 0.29%...--. Trp 0.29% + PCP!

0

H

~

0
c..J

40

CI.l

Iloo
0

1>:1
c..J
lIO;

1>:1
J:l
H

20

c..J

lIO;

H

O ....= = = = = = = = = d i : = = =.......=-====2

FEEDINGPERIOD.til...._
4(WEEI)

Figure 3
Effects of oral administration of tryptophan (Trp)metabolites to Trp-deficient or sufficient chumsalmon fry. Administration of 5-hydroxy-Ltryptophan(5-HfP) to Trpdeficient fish completely
prevented scoliosis, whereas kynurenine failed to inhibit the occurrence.
DL-p-Chlorophenylalanine (PCPA) developed
scoliosis even in the fishfed Trp-sufficient diet
(Akiyama et aI. 1986b).

i0

NOAA Technical Report NMFS III

Tryptophan hydroxylaae

PCPA

Aroaatic L-aaino aciddecarboxylase..... 1<...~---- MI486~ . I COOH
OH~CO()HOH~~ )J NH2 - - - - . . ~NjJ NH2 - - - - - . . ~)J NH2~

H

Tryptophan

S-Hydroxy-L-tryptophon

Serotonin(Trp)(S-HTP)(S-HT)and can easily pass through a blood-brain barrier incontrast to 5-HT (Akiyama et a!. 1986b, 1989). The
treatment completely prevented the occurrence ofscoliosis and increased the brain 5-HT level in Trpdeficient fish (Fig.3). Kynurenine, a precursor of
niacin, NAD, and xanthurenic acid was fed at 120 mg(as L-kynurenine)/100 gdiet to Trp-deficient fish,
but failed to prevent scoliosis. Moreover, both
scoliosis and decreased brain 5-HT levels were observed in fish fed a Trp-sufficient diet supplemented
with DL-p-chlorophenylalanine (PCPA) (Akiyama et
a!. 1986a; 1986b), which is an inhibitor of tryptophan-hydroxylase (TrpOHase) and a potent depletorof both brain and peripheral stores of 5-HT (Fig. 4).
TrpOHase is a rate-limiting enzyme on the 5-HT
pathway. These findings indicated a relationship between the occurrence of scoliosis and 5-HT levels.
The reduced ability of orally administrated 5-HT to
inhibit the development of scoliosis compared withcomplete prevention with the use of 5-HTP suggested
involvement of the serotoninergic neuron in the central nervous system. Therefore, the author preparedTrp-deficient diets (0.05%) containing various combinations of 5-HTP (10 or 50 mg/l00 g diet) with or
wi thout
L-2-hydrazino-al pha-methyl-beta- (3,4dihydroxyphenylpropionic acid) (MK486, 1 or 5 mg/
100 g diet). MK486 functions ~s an inhibitor of aromatic L-amino acid decarboxylase only in peripheryand inhibits 5-HT biosynthesis from 5-HTP. Thus. it
consequently functions to promote the conversion ofexogenous 5-HTP to 5-HT in the central nervous system. Feeding a Trp-deficient diet supplemented withboth 5-HTP and MK486 resulted in a significantly
lower incidence of scoliosis and higher levels of brain

H

Figure 4
Functions of administrated drugs on serotonin(5-HT) pathway. DL-pCh lorophenylalan ine(PCPA) is an inhibitor oftryptophan-hydroxylase(TrpOHase) and inhibits
a biosynthesis of 5-hyd ro x y-L-tr yp to p h an
(5-HTP) from Trp. L-2h yd razi n o-al fa-m e thy 1be ta- (3, 4-di h ydroxyphenyl propionic acid);(MK486) is an inhibitorof L-amino acid decarboxylase only in peripheryand consequently induces
a conversion of exogenous
5-HTP to 5-HT in central
nervous system.

5-HT and 5-hydroxyindoleacetic acid than did adding
5-HTP alone (Akiyama and Murai, unpub!. data).
The experiment suggests that the deficiency of 5-HTin the central nervous system was related to the occurrence of scoliosis.

Effect of Temperature on the Occurrenceof Scoliosis_
Scoliosis caused by Trp deficiency has been reported
only in salmonids such as sockeye salmon, rainbow
trout, coho salmon and chum salmon, although it
has also been studied in chinook salmon (Halver et
a!. 1957), channel catfish (Dupree and Halver 1970),
eel (Arai et a!. 1972), carp (Nose et a!. 1974; Nose
1979), red sea bream, Pagrus major (Yone 1976) andtilapia, Tilapia zillii (Mazid et a!. 1978). At first I considered scoliosis to be a characteristic Trp deficiency
symptom of all salmonids except chinook salmon.
Arai et a!. (1986), however, reported development ofscoliosis in Trp-deficient Ayu fish (Plecoglossidae)
reared at 16° C, although the incidence was low. I
also found one scoliotic fish when a Trp-deficient
diet was fed to yellowtail at 20° C (Akiyama, un pub!.
data), even though the brain was not infected by
Streptococcus and not parasitized by cysts of Myxobolus.
These facts suggest that scoliosis due to Trp deficiency is unlikely to be peculiar to salmonids.
Because salmonids are coldwater fish, and because
most of the fishes in which scoliosis was not observed
as a symptom of Trp deficiency are warmwater fish(except chinook salmon), I focused my attention on
the influence of environmental temperature. It isAkiyama: Scoliosis of Fishes Caused by Tryptophan Deficiency

likely that among the fishes developing scoliosis, incidences of scoliosis decrease as the optimum
temperature for each species rises. Moreover, spinal
deformity has not been observed to be an external
symptom of dietary Trp deficiency in mammals andbirds, which are warm-blooded animals. In fact, theauthor presumed that the occurrence and incidenceof scoliosis might be influenced by rearing temperatures, and therefore fed the up-deficien t diet tochum salmon fry at three different temperatures: 10,
16, and 20° C. The experiment showed that as therearing temperature was lowered, the incidence ofscoliotic fish increased and brain 5-HT levels decreased (Akiyama and Murai, unpubl. data). It is still
unknown why the brain 5-HT level in Trp-deficient
fish varied under different temperature conditions.

Conclusion
All of these findings described above indicate thatscoliosis caused by Trp deficiency is related to thelevel of 5-HT in the central nervous system. In fishes,
hereafter, the central nervous system, especially the
5-HT neuron, should be considered as one of theimportant factors in an occurrence of idiopathic spinal deformity.

Citations_

Akiyama, T, S. Arai, T Murai, and T Nose.
1985. Tryptophan requirement of chum salmon fry. Bull.
Jpn. Soc. Sci. Fish. 51(6):1005-1008.
Akiyama, T., T Murai, and T Nose.
1986a. Oral administration of serotonin against spinal deformity of chum salmon fry induced by tryptophan
deficiency. Bull. Jpn. Soc. Sci. Fish. 52(7):1249-1254.
Akiyama, T, T Murai, K Mori.
1986b. Role of tryptophan metabolites in inhibition of spinal deformity of chum salmon fry caused by Tryptophan
deficiency. Bull.Jpn. Soc. Sci. Fish. 52(7):1255-1259.
Akiyama, T, H. Kabuto, M. Hiramatsu, T Murai, and K Mori.
1989. Effect of dietary 5-hydroxy-L-tryptophan for prevention of scoliosis in tryptophan-deficient chum salmon
fry. Nippon Suisan Gakkaishi 55(1):99-104.
Arai, S., T. Nose, and Y Hashimoto.
1972. Amino acids essential for the growth of eels, Anguillaanguilla and A. japonica. Bull. Jpn. Soc. Sci. Fish. 38(7):753759.
Arai, S., A. Nakazawa, and Y Deguchi.
1986. Effects of each essential and non-essential amino acids
on free amino acids in whole body of ayu fish. Abstr. of theAutumn Meeting ofJpn. Soc. Sci. Fish., p.147. (lnJapanese.)
Dupree, H.K, andj.E. Halver.
1970. Amino acids essential for the growth of channel catfish, fetalurus punctatus. Trans. Am. Fish. Soc. 99(1):90-92.
Egusa, S.
1985. Myxobolus buri sp. n. (Myxosporea: Bivalvulida) parasitic
in the brain of Seriola quinqueradiata TEMMINCK et
SCHLEGEL. Fish Pathol. 19(4):239-244. (In Japanese; English abstr.)

11

Halver,j.E., and W.E. Shanks.
1960. Nutrition of salmonoid fishes. VIIl. Indispensableamino acids for sockeye salmon. j. Nutr. 72:340-346.
Halver,j.E., D.C. Delong, and E.T Mertz.
1957. Nutrition of salmonoid fishes. V. Classification of essential amino acids for chinook salmon. j. Nutr. 63:95105.
Harris-Warrick, R.M.,j.C. McPhee, andj.A. Filler.
1985. Distribution of serotonergic neurons and processes in
the lamprey spinal cord. Neuroscience 14(4):1127-1140.
Hirose, K, and M. Kitsukawa.
1976. Acute toxicity of agricultural chemicals to seawater teleosts, with special respect to TLM and the vertebral
abnormality. Bull. Tokai Reg. Fish. Res. Lab. 84:11-20. (InJapanese; English abstr.)
Hirose, K, M. Kitsukawa, and A. Ishikawa.
1979. Effects of water temperature on median lethal concentrations (LC50) of a few pesticides to seawater teleosts. Bull.
Tokai Reg. Fish. Res. Lab. 98:45-53. (In Japanese; English
abstr.)
Ikeda, S., M. Sato, and R. Yoshinaka.
1983. Role of vitamin C in collagen formation of fish. Vitamins Oapan) 57(8):433-449. (In Japanese, English abstr.)
Kaige, N., T Miyazaki., and S. Kubota.
1984. The pathogen and the histopathology of vertebral deformity in cultured yellowtail. Fish. Pathol. 19(3):173-179(In Japanese, English abstr.)
Kitamura, S.
1969. Summary on the hypovitaminosis C of rainbow trout,
Salmogairdneri. Fish Pathol. 3:73-85 (InJapanese.)
Kloppel, TM., and G. Post.
1975. Histological alterations in tryptophan-deficient rainbow trout. j. Nutr. 105:861-866.
MacEwen, G.D.
1973. Experimental scoliosis. C1in. Orthop. 93:69-74.
Mazid, M.A., Y Tanaka, T. Katayama, KL. Simpson, and C.O.
Chichester.
1978. Metabolism of amino acids in aquatic animals-Ill Indispensable amino acids for Tilapia zillii. Bull. Jpn. Soc. Sci.
Fish. 44(7) :739-742.
Nose, T
1979. Summary report on the requirements of essential
amino acids for carp. In Finfish nutrition and fishfeed
technology, Vol. I O. E. Halver and K Tiews, eds.), p. 146156. Heenemann Verlagsgesellschaft GmbH, Berlin.
Nose, T, S. Arai, D. Lee, and Y Hashimoto.
1974. A note on amino acids essential for growth of young
carp. Bull. Jpn. Soc. Sci. Fish. 40(9):903-908.
Ogata, H., and S. Arai.
1981. Essential amino acid requirements for coho salmon Il .
Leucine,isoleucine,
tryptophan
andhistidinerequirements. Abstract of the Spring Meeting of Jpn. Soc.
Sci. Fish., p.44. (In Japanese).
Poston, H.A., and G.F. Combs,Jr.
1980. Nutritional implications of tryptophan catabolizing enzymes in several species of trout and salmon. Proc. Soc.
Exp. BioI. Med. 163:452-454.
Poston, H.A., and G.L. Rumsey.
1983. Factors affecting dietary requirement and deficiency
signs of L-tryptophan in rainbow trout. j. Nutr. 113:25682577.
Sakaguchi, S., T Hara, T Matsusato, T Shibahara, Y. Yamagata, H.
Kawai, and Y Maeno.
1987. Scoliosis of cultured yellowtail caused by parasitic
Myxobolus buri. Bull. Natl. Res. Inst. Aquaculture 12:79-86.
(lnJapanese; English abstr.)

12

NOAA Technical Report NMFS 111

Sato, M., T. Kondo, R. Yoshinaka, and S. Ikeda.
1983. Effect of water temperature on the skeletal deformity
in ascorbic acid-deficient rainbow trout. Bull. Jpn. Soc.
Sci. Fish. 49(3) :443-446.
Shanks, W.E., G.D. Gahimer, andj.E. Halver.
1962. The indispensable amino acids for rainbow trout. Prog.
Fish-Cult. 24:68-73.
Shiomitsu, K.
1982. Isolation of Streptococcus sp. from the brain of cultured
yellowtail. Fish Pathol. 17(1):27-31. (In Japanese; English
abstr.)
Tamura, T.
1974. An experimental study on scoliosis in Lipedal rats withbrainstem lesion. j. Jpn. Orthop. Assoc. 48(3):137-158.
(In Japanese. English abstr.)

Tanaka, C., and M. Kimura.
1984. Serotonin (5-hydroxytryptamin). In Neurotransmitters(G. Takagaki and T. Nagatsu, eds.), p.156-191. Kodansha,
Tokyo. (In Japanese.)
Walton, M.J., R.M. Coloso, C.B. Cowey,j.w. Adron, and D. Knox.
1984. The effects of dietary tryptophan levels on growth andmetabolism of rainbow trout (Salmo gairdneri). Br. j. Nutr.
51:279-287.
Yone, Y
1976. Nutritional studies of red sea bream. Rep. Fish. Res.
Lab. Kyushu llniv., 3:87-101.

Control of IHN Virus in Alaskan Sockeye Salmon Culture
THEODORE MEYERS
Alaska Department ofFish and Game
F.R.E.D. Division
P.O. Box 25526
Juneau, AK 99802-5526

A recent review of trends in the prevalence and risk management of Infectious Hematopoietic Necrosis Virus (IHNV) in Alaskan sockeye salmon Oncorhynchus nerka culture has
been reported by Meyers et al. (l 990). The reader is referred to this report for further
details, discussion, and references.
Prior to 19S0, IHNV prevented successful culture of sockeye salmon in Alaska. This led
the Fisheries Rehabilitation, Enhancement, and Development Division (FRED) of the
Alaska Department of Fish and Game to develop a policy to control the negative effectsof the virus in sockeye salmon culture. This policy included procedures for the collection
and incubation of eggs and for the rearing of fry that were based upon the known andsuspected biological characteristics of the virus-host relationship. Many of these criteria
are common sense approaches such as: use of a virus-free water supply; disinfection ofutensils, facilities, and external surfaces of broodfish; separate fertilization of eggs fromeach female using 1 or 2 males; separate water hardening of each family of eggs in a 100
ppm iodine disinfectant for 1 hour; compartmentalization of families into Kitoi Box
incubators or into stacks of Nopad trays at egg densities of 250,000-300,000 (SO-100
females), or into modified Bams Boxes used at one facility that are each loaded with
500,000 eggs; physical isolation of each sockeye stock and isolation of all sockeye stocks
from any nonsockeye species; and release of fry unfed or after short-term rearing (4-6weeks) with pooling of fry in raceways or start tanks according to the date of eggtake.
These criteria nearly eliminate opportunities for horizontal virus transmission from theparents to offspring or from the water supply. They also further reduce the rare occurrence of vertical transmission of the virus within the egg and allow forcompartmentalization of eggs and fry so that the occasional incubators or raceways offish developing IHN can be destroyed and the virus contained to protect the remaining
fish inventory. This "sockeye culture policy" has allowed Alaskan hatcheries a great measure of success in controlling IHNV at several different facilities around the state fornearly 10 years. Based on these guidelines, an average of 2-3 million sockeye salmon eggs
can be spawned in a day and totals of 20-36 miliion eggs may be taken at certain
facilities. Although vertical transmission of the virus generally occurs in fry almost every
year at certain facilities, total losses are minimized to between 1 and 3% of the statewide
fry production. In 1990 only 1% of the sockeye fry were destroyed owing to IHN of 6S
million healthy fish that were released. Production data from various Alaskan sockeye
salmon hatcheries suggest that vertical transmission of IHNV is more dependent upon
the proportion of high virus-titered female fish rather than total virus prevalence. Also,
as one would expect, the risk of vertical virus transmission increases with increasing
numbers of eggs taken from females of a high-titered stock. Hence, IHN outbreaks are
more common at the larger eggtake facilities that have greater prevalences of hightitered broodfish.
During the past 14 years, yearly monitoring of sockeye salmon stocks by ,FRED has
resulted in a data base summarizing IHNV occurren<:e in over 96 wild and hatcherysockeye salmon stocks in Alaska. Yearly prevalence of IHNV ranges fromto 100% in
both ripe and postspawned females with as many as 92% within a stock having high titers(~1 0 4 ). Repeated yearly sampling has shown that all anadromous sockeye salmon tested
in Alaska are positive for IHNV. The data base has been useful for examining generaltrends and has shown some differences from previously reported IHNV-sockeye salmon
relationships. For example, no significant differences in mean IHNV prevalence were

°

13

14

NOAA Technical Report NMFS III
found in ovarian fluids from postspawned female sockeye salmon vs. those from ripe
females. Furthermore there were no significant differences between geometric mean
virus titers of postspawned vs. ripe female fish, but postspawners did have a significantly
greater mean proportion of high-titered fish. The log value of 10' was selected as thebreakpoint for high virus titers owing to the tendency for bimodality of IHNV titers tooccur at this level in most stocks of Alaskan sockeye salmon. The significance of thisphenomenon needs further investigation. As found by other investigators, the mean virus
prevalence in male fish from 27 stocks of sockeye salmon was significantly less (9%) thanin female fish (40.1 %).
This data base is a useful tool for examining general trends for IHNV within a geographic area or statewide. However, these trends may not always be true for certain
individual sockeye salmon stocks that may be unique due to genetic reasons, the strainof the indigenous virus, or environmental factors affecting natural virus exposure andtransmission.

Citation
Meyers, T.R.,J.B. Thomas,J.E. Follett, and R.R. Saft.
1990. Infectious hematopoietic necrosis virus: trends inprevalence and the risk management approach in Alaskan
sockeye salmon culture. J. Aquat. Anim. Health 2:85-9&.

Identification of a Conserved Antigenic Domain in the
Major Capsid Protein of Infectious Pancreatic Necrosis Virus
RJ. BARRIE, C.L. MASON, andJ.C. LEONG*
Department of Microbiology
Oregon State University
Corvallis, Oregon 97331-3804

ABSTRACTThe gene for the major capsid protein, VP2, of the Sp serotype of infectious pancreatic
necrosis virus (lPNV) was cloned and expressed in Escherichia coli. Nonoverlapping fragments of the VP2 gene were recloned in trpE fusion vectors of the pATH series and theexpressed fusion proteins were characterized for reactivity with antisera to three different
serotypes of IPNV. One clone, pBIO, which contained an insert encoding amino acids 99
to 206 of the VP2 protein, produced a fusion protein recognized by antisera for all threeserotypes. In contrast, the pA43 clone, which contained an adjacent region on the VP2gene encoding for amino acids 207 to 315, produced a fusion protein that was only
recognized by homologous antisera in Western immunoblots. A comparison of the derived amino acid sequence for each clone with that reported for two other IPNV clones
indicates that the pBlO region is conserved and the pA43 region is very heterogeneous.

Introduction_

Infectious pancreatic necrosis virus (IPNV) is abirnavirus that causes one of the most serious diseases in trout and salmon farms in North America,
Europe, and Asia. It can also kill a number of
nonsalmonid fish species including striped bass
(Morone saxatilis) , turbot, menhaden, and eels (Wolf
1988), and it has been isolated from a variety of marine fish and molluscs. The ubiquitous nature of thisbirnavirus and its ability to infect such a wide varietyof hosts make this virus important for scientific study.
Most IPNV isolates are closely related antigenicallyand yet, they exhibit marked differences in hostrange in vivo and in vitro, pathogenicity, andtemperature of replication.
The most extensive study of the antigenic relationships of the aquatic birnaviruses compared 175 virus
isolates from 44 fish and shellfish species from 11
countries by reciprocal plaque reduction tests using
polyclonal sera (Hill and Way 1983). From these results, it was proposed that there are 2 major
serogroups: 1 containing 9 serotypes which includes
171 isolates from fish, and the other containing 1
serotype which includes those viruses isolated frommolluscs. These virus isolates also contain common
immunoreactive determinant(s). Other studies haverecognized only three major serotypes characterized
by the following virus isolates: VR299, a North American strain; Sp, a European strain which is pathogenic
for trout; and AB, a European strain which is
nonpathogenic for trout (Wolf 1988). In our paper
we review recent efforts to unravel the mechanismsthat biologically distinguish the different IPNV isolates by characterizing the immunoreactive regions of
the major capsid protein of the Sp serotype of IPNV.

Methods_

The viral genome is composed of two doublestranded RNA segments, A and B. The B segment
encodes the viral RNA polymerase, VPl. The A segment encodes the virion proteins, VP2 and VP3. The
major capsid protein, VP2, is responsible for the induction of neutralizing antibodies (Lipipun et al.
1989 ). In addition, there is a nonstructural protein,
NS, which is an autocatalytic protease responsible forcleavage of the polyprotein, VP2-NS-VP3, encoded by
the viral genome A segment (Duncan et al. 1987;
Manning et al. 1990).

*Send correspondence to this author
15

16

NOAA Technical Report NMFS III

Sma!
BamHl
Xbal
Sail

Pall
HlndlllPal~BamHVClP

ATGolpal~._.Nd.!a.1 _ _.R~I
US Kb

I I

I

II

II II

Pat

Kplll

. . ._ _ , _ , _ ,S8u3A IrBgment8'

225~p3537
GEL ISOLAllON OF ---~----J

EACH FRAGMENT
T4 DNA Ugase

COlONYIMMUNOBlOT

Since the entire VP2 gene has been expressed in
Escherichia coli as part of a trpE fusion protein (Manning and Leong 1990), it was possible to examine
different regions of the VP2 gene for immunoreactivity with a panel of rabbit antisera and monoclonal
antibodies to different serotypes of IPNV. The VP2gene was excised from the plasmid pUCI9/A+SAK
and cut with the restriction enzyme Sau3Al which
generated seven DNA fragments (Fig. 1). These fragments were inserted in-frame with the trpE gene in
one of the three pATH vectors, pATH 1, pATH2, or
pATH3, which put the resulting trpFrVP2 fusion gene
under the control of the tryptophan operator andpromoter (Dieckmann and Tzagaloff 1985). Recombinants expressing a portion of the VP2 gene weredetected by direct colony immunoblot with antiIPNV-Sp sera (Gilmore et al. 1988).

Figure 1

Construction of trpFrVP2 gene fusions.
The 1.5 Kb cloned insert containing
the IPNV-Sp VP2 gene was restrictedwith Sau3Al; the fragments were purified and subcloned into the trpE
fusion expression vectors, pATH 1, 2,
or 3. Recombinants containing fragments in the correct orientation andin the appropriate reading frame wereselected by colony immunoblot as previously described (Gilmore et al. 1988).

Results and Discussion_

Two recombinant plasmids were identified from theclones derived from the pATHI vector/insert ligation
mixture, and the trpFrVP2 fusion proteins expressed bythese plasmids were characterized by Western
immunoblot analysis. The antisera used to detect the
VP2-specific protein in the bacteria had been prepared against purified virions of IPNV-Sp. The
recombinant plasmid, pBI0, was found to produce a
47 kDa fusion protein and the recombinant plasmid,
pA43, was found to produce a 52 kDa fusion protein(Fig. 2). The VP2 derivation of the IPNV sequence inthe fusion proteins was verified by Western
immunoblot using anti-VP2-specific antisera preparedagainst purified VP2 virion protein from the IPNV-Sp
strain (Huang et al. 1986). The cell lysate preparedBarrie et al.: Antigenic Domain in Protein of Infectious Pancreatic Necrosis Virusfrom induced cells contammg pBI0 or pA43 wasfound to contain protein bands that were strongly reactive with the anti-IPNV-Sp/VP2 sera (Fig. 3).
The DNA sequence of the viral insert in pBI0 andin pA43 was determined after subcloning of the insert into the M13 sequencing vectors mp18 andmpl9. Sequence analysis was performed by the modified Sanger dideoxy chain termination method(Davis et a!. 1986). The pBI0 insert comprised 323
nucleotides encoding 108 amino acids and mapped
to amino acid number 99 to 206 of the VP2 proteinof IPNV-Sp (Mason and Leong, unpub!. data). The
pA43 insert comprised two neighboring Sau3A fragments of 297 and 27 nucleotides. This insert
probably originated from a partial cleavage product(Fig. 1) and it was mapped to the adjacent region of
the VP2 protein at amino acid number 207 to 315 of
the VP2 protein. Although both pBI0 and pA43 contained inserts encoding 108 amino acids, striking
differences in the observed migration of the fusion

3

2

4

5

6

.......--50

u

>
Z

Q.

o
,...
ttl
C.

u

Figure 2
Immunoblot showing reactivity of trpEVP2 fusion proteins with anti-IPNV-Sp
sera. E. coli cells containing the recombinant plasmids (pBI0 or pA43) weregrown to mid-log phase before induction
with 15 Jlg/ml indoleacrylic acid. The
cultures were then grown to stationary
phase before the cells were harvested bycentrifugation for protein analysis. The
cells were lysed and the proteins separated on an SDS-polyacrylamide gel as
previously described (Gilmore et al.
1988). The proteins were transferred tonitrocellulose and then exposed to antiIPNV-Sp antisera. Lane 1 contains
IPNV-Buhl infected fish tissue culture celllysate; lane 2, prestained low molecularweight markers from BioRad at 75 kDa,
50 kDa, 39 kDa, 27 kDa, and 17 kDa;
lanes 3 and 4, pBI0 induced bacterial celllysate; lane 5, pA43 induced bacterial celllysate; lane 6, bacterial cells containing
the pATH vector with no insert; lane 7,
bacterial cells without a plasmid; lane 8,
low molecular weight markers; and lane
9, purified IPNV-Buhl. The arrow in lane
5 indicates the position of the trpE-VP2
fusion protein encoded by the recombinant plasmid, pA43. The computed
molecular weight of the trpE-VP2 fusion
protein was 56.5 kDa.

proteins produced by each plasmid were noted. Careful analysis of the 3' terminal sequence of both
plasmids by direct sequence analysis from the recombinant pATH fusion plasmids themselves (Wang et a!.
1988) indicated that the translational termination
codon, TAG, was present immediately after the endof both VP2 cDNA inserts. The calculated isoelectric
points for both fusion proteins was 6.6, and there was7.......--75kDau

17

Figure 3
Immunoblot of trpE-VP2 fusion proteins with antisera
to the VP2 protein of IPNV-Sp. E. coli cells containing
the recombinant plasmid, pBID or pA43, were grown
and prepared as described in Figure 2. Lane I contains purified IPNV-Sp; lanes 2 and 3, lysates fromuninduced and induced cells containing pBIO; lane
4, lysate from uninduced cells containing the pATHI
vector without any VP2 insert; lane 5, prestained low
molecular weight markers from BioRad at 75 kDa, 50
kDa, and 39 kDa; lan.es 6 and 7, lysates fromuninduced and induced cells containing pA43. The
photograph taken for lanes 6 and 7 was a lighter exposure of the immunoblot and these lanes contained
five times as much bacterial lysate as that used inlanes 2, 3, and 4.

18

NOAA Technical Report NMFS III

insert

pB10

•

SP

ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYNSLMSLTT~PQDKA

JA

ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYNSLMSLTT~PQDKVNl

ISRKYDIQSSTLPAGLYALNGTLNAATFEGSLSEVESLTYNSLMSLTTNPQDKV

SP
JA
N1

NNQLVTKGVTVLNLPTGFDKPYVRLEDETPQGLQSMNGARMRCTAAIAPRRYEINNQLVTKGITVLNLPTGFDKPYVRLEDETPQGPQSMNGARMRCTAAIAPRRYEINNQLVTKGVTVLNLPTGFDKPYVRLEDETPQGLQSMNGAKMRCTAAIAPRRYEI

•

pA43

..
. ...

insert.. ..

•. . .... . .

•

SP
JA
N1

DLPSQSLPPVPATGTLTTLYEGNADIVNSTTVTGDINFSLAEQPANETRFDFQLDLPSERLPTVAATGTPTTIYEGNADIVNSTAVTGDITFQLEAEPVNETRFDFILDLPSQRLPPVPATGTLTTLYEGNADIVNSTTVTGDINFSLAEQPANETKFDFQLSPDLMGLDNDVPVVTVVSSVLATNDNYRGVSAKMTQSIPTENITKPITRVKLSYKI
QFLGLDNDVPVVTVTSSTLVTADNYRGASAKFTQS IPTEMITKP ITRVKLAYQLDFMGLDLDVPVVTVVSSVLATNDNYRGASAKMTQSIPTENITKPITRVKLSYKI

JA

Nl

...

• •

• ••

Figure 4
A comparison of the derived amino acid sequence of IPNV-Sp, IPNV- Jasper, and IPNV-Nl
cDNA inserts present in pBI0 and in pA43. The
asterisks indicate differences in amino acids.
The IPNV:Jasper (Ja) sequence was taken from
Duncan and Dobos (1986). The IPNV-Nl sequence was taken from Havarstein et al. (1990).

A

B
2

3 4

5

6

7

8 9

10

2

3

4

130kDa _'-SOkDa

75 - .

'-56.5kDa
50 - .

'-39

Figure 5
Immunoblots of trpFrVP2 fusion proteins with antisera to the heterologous IPNV strains, IPNV-Buhl and IPNV-EVE. E. coli
cells containing trpFrVP2 fusion proteins encoded by the recombinant plasmids, pBlO or pA43, were analyzed for reactivity with antisera prepared to purified virus of the two heterologous IPNV strains. (A) Reactivity with antisera to IPNV-Buhl.
Lanes 1 and 2 contain lysates from uninduced and induced cells containing the pATHl expression vector with no viral
insert; lanes 3 and 4, lysates from uninduced and induced cells containing pA43; lanes 5 and 6, lysates from uninduced
and induced cells containing pBI0; lane 7, prestained low molecular weight markers from BioRad; lanes 8 and 9, lysatesfrom uninduced and induced cells containing pBlO; lane 10, purified IPNV. (B) Reactivity with antisera to IPNV-EVE.
Lanes 1 and 2 contain lysates from uninduced and induced cells containing pBlO; lanes 3 and 4, lysates from uninduced
and induced cells containing pA43.

no dramatic difference in the amino acid composition. Thus far, the only possible explanation for theslower migration of the pA43 fusion protein might be
the series of four prolines found towards the amino
terminus of this insert (Fig. 4).
Polycional rabbit antisera prepared to the different
serotypes of IPNV will crossreact with the heterolo-gous VP2 proteins 10 Western immunoblots (R.
Barrie and J. Leong, unpub!. data). Thus, there areconserved linear epitopes among the IPNV strains.
When the expressed trpE-VP2 fusion proteins were
examined for reactivity with polycional anti-IPNV
sera prepared to three different IPNV serotypes, only
pBIO reacted with the heterologous antisera in West-Barrie et al.: Antigenic Domain in Protein of Infectious Pancreatic Necrosis Virus

19

Acknowledgments_

ern immunoblots (Fig. 5, A and B). The anti-IPNVBuhl sera was prepared against purified virions of
the Buhl virus isolate which had been previously
characterized as a member of the IPNV-VR299 serotype found in rainbow trout (Oncorhynchus mykiss)in North America (Hill and Way 1983). The IPNVEVE isolate was obtained from Japanese eels
(Anguilla japonica) suffering from branchionephritis in Japan (Sano et al. 1981); it has been
antigenically grouped with the AB serotype of
IPNV, which is nonpathogenic for rainbow trout.
The fusion protein encoded by pA43 was completely nonreactive with the heterologous antisera.
Thus, it appears that the VP2 gene region encoded
by pBI0 contains an antigenic determinant(s)which is conserved among the IPNV strains examined and the insert in pA43 encodes a region
which is highly variable.
A comparison of the nucleotide sequence and its
derived amino acid sequence of each insert withthat of other published sequences of the VP2 gene
showed that the pBI0 region was highly conservedat the nucleotide and amino acid level (Fig. 4;
Christie et al. 1988; Havarstein et al. 1990). There
were three amino acid differences between the Sp
and the Jasper isolate (a member of the VR299 serotype), and there were only two amino aciddifferences between Sp and Nl isolates, the latter ofwhich is another IPNV isolate from the Sp serotype(Havarstein et al. 1990). In contrast, 27 amino acidchanges between the Sp and Jasper isolates were observed for the pA43 gene fragment. Only five aminoacid changes were found between isolates Sp and
Nl. The similarity between the Sp and Nl genomes
indicates that these two isolates are highly related, afinding that is consistent with the findings ofChristie et al. (1988).

Conclusion_

In summary, two immunoreactive regions of the viral
major capsid protein, VP2, have been identified. One
region from amino acids 99 to 206 contains a very
conserved epitope(s) which was recognized by neutralizing antisera to three different IPNV serotypes.
Another region from amino acids 207 to 315 contains
a highly divergent epitope(s) that may encode theserotype-specific epitope(s) of an IPNV strain. A suggestion that the amino acid region from 206 to 350
encoded the serotype-specific epitope(s) of IPNV wasmade by Havarstein et al. (1990) when a comparisonof the deduced amino acid sequence of IPNV-Nl and
IPNV- Jasper capsid proteins revealed that this region
was very heterogeneous.

This publication is the result of research sponsoredby the Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration,
Office of Sea Grant, Department of Commerce, under grant no. NA85AA-D-SG095 (project no. R/
FSD-ll) and the United States Department of Agriculture to the Western Regional Aquaculture
Consortium under grant nos. 87-CRSR-2-2319 and 8838500-4027. Oregon Agricultural Experiment Station
Technical Paper No. 9327. We thank L. Bootland forreviewing the manuscript.

Citations_

Christie, KE., L.S. Havarstein, H.O. Djupvik, S. Ness, and C.
Endresen.
1988. Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch.
Virol. 103:167-177.
Davis, L.C., M.D. Dibner, andJ.F. Batty (eds.).
1986. Basic methods in molecular biology. Elsevier SciencePub. Co., NY, 377 p.
Dieckmann, C.L., and A. Tzagaloff.
1985. Assembly of the mitochondrial membrane system. J.
BioI. Chern. 260:1513-1520.
Duncan, R., and P. Dobos.
1986. The nucleotide sequence of infectious pancreatic necrosis virus (lPNV) dsRNA segment A reveals one large ORF
encoding a precursor protein. Nucl. Acids Res. 14:59345935.
Duncan, R., E. Nagy, P.]. Krell, and P. Dobos.
1987. Synthesis of the infectious pancreatic necrosis virus
polyprotein, detection of virus-encoded protease, and finestructure mapping of genome segment A coding regions.
J. Virol. 61:3655-3644.
Gilmore, R.D.Jr, H.M. Engelking, D.S. Manning, andJ.C. Leong.
1988. Expression in Escherichia coli of an epitope of theglycoprotein of infectious hematopoietic necrosis virus
protects against viral challenge. Bio/Technology 6:295300.
Havarstein, L.S., KH. Kalland, K.E. Christie, and C. Endresen.
1990. Sequence of the large double-stranded RNA segmentof the N1 strain of infectious pancreatic necrosis virus: Acomparison with other Birnaviridae. J. Gen. Virol. 71 :299308.
Hill, B.]., and K Way.
1983. Serological classification of fish and shellfish
birnaviruses. Abstract, First international conference ofeuropean association of fish pathology, Plymouth, England,
October, 1983.
Huang, M.T.F., D.S. Manning, M. Warner, E.B. Stephens, and J.C.
Leong.
1986. A physical map of the viral genome for infectious pancreatic necrosis virus Sp: Analysis of cell-free translation
products derived from viral cDNA clones. J. Virol.
60(3):1002-1011.
Lipipun, v., P. Caswell-Reno, Y.-L. Hsu, J.L. Wu, M.-C. Tung, P.w.
Reno, W. Wattanarijarn, and B.L. Nicholson.
1989. Antigenic analysis of Asian aquatic birnavirus isolates using monoclonal antibodies. Fish Pathology 24(3):155-160.

20

NOAA Technical Report NMFS III

Manning, D.S., andJ.C. Leong.
1990. Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus. J. Viro!.
179:1&-25.
Manning, D.S., C.L. Mason, andJ.C. Leong.
1990. Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein. J. Virol.
179:9-15.
Sano, T., N. Okamoto, and T. Nishimura.
1981. A new viral epizootic of Anguilla japonica. J. Fish. Dis.
4:127-139.

Wang, L.M., D.K. Weber, T.Johnson, and A.V. Sakaguchi.
1988. Supercoil sequencing using unpurified templates produced by rapid boiling. Biotechniques 6:839-843.
Wolf, K.
1988. Infectious pancreatic necrosis virus. In Fish viruses
and fish viral diseases (K. Wolf, ed), p. 115-157. Cornell
Univ. Press, NY.

Cloning of Hemolysin Genes of Aeromonads

TAKASHI AOKI and IKUO HIRONO
Department ofBiological Resources
Faculty ofAgriculture
Miyazaki University
Mryazaki889-21,Japan

ABSTRACTThe role of extracellular products is critical in the pathogenic mechanisms of bacterial
infections. In Aeromonas spp., hemolysins may be the most important of these products inestablishing and maintaining infections. This report reviews our knowledge of the structure and expression of hemolysin genes in Aeromonas and discusses preliminary results on
gene homology and ancestry among various Aeromonas spp.

The related species Aeromonas hydrophila (Ljungh
and Wadstrom 1982) and A. salmonicida (Titball andMunn 1985a) produce several extracellular proteinsthat are virulence factors. In the study of pathogenic
mechanisms of these bacteria, there has been interestin the role of these extracellular substances as toxins.
A. hydrophila produces a variety of extracellular products, including a protease, glycerophospholipid
cholesterol acyltransferase (GCAT), cytotoxin, an enterotoxin, acetylcholinesterase (Nieto et al. 1991),
and hemolysins (Ljungh and Wadstrom 1982). Extracellular products of A. salmonicida include hemolytic,
leukocytolytic, proteolytic, and GCAT activities (Elliset al. 1981, 1988). The virulence of A. hydrophila andA. salmonicida is significantly enhanced by their ability to secrete hemolysin. Hemolysin may be the mostimportant of these products in causing tissue damage
and in establishing and maintaining infections with
Aeromonas.
There are many reports describing the number
and nature of hemolysins found in A. hydrophila(Ljungh et al. 1981; Thune and Johnson 1986; Asao
et al. 1986) and A. salmonicida (Titball and Munn
1985a; Rockey et al. 1988). Asao et al. (1986) purified
and characterized two hemolysins from A. hydrophila
which were biologically similar but immunologicallydistinct. Both hemolysins caused fluid accumulation
in infant mouse intestines and rabbit ileal loops andelicited a cytotoxic effect on Vero cells. Two distinct
hemolysins have also been found in A. salmonicida.
One is a broad-spectrum hemolysin with maximum
activity against horse erythrocytes (H-lysin) (Titballet al. 1985b), and the other is active against trout

erythrocytes (T-lysin) (Rockey et al. 1988). H-Iysin
contains GCAT. Nomura et al. (1988) purified
salmolysin, an extracellular hemo lytic toxin from A.
salmonicida. Salmolysin was lethal to rainbow trout
Oncorhynchus mykiss when it was injected intramuscularly.
Almost all isolates of A. hydrophila and A.
salmonicida produce aerolysin, a substance with hemolytic activity. The level of aerolysin production is
known to vary under different growth conditions,and individual isolates can alternate between highand low level phases of production. When this hemolysin gene is cloned into E. coli, the gene's
characteristics can be more easily studied.
For this reason, we cloned the two hemolysin
genes from A. hydrophila and the one hemolysin
gene from A. salmonicida to study their structureand expression (Aoki and Hirono 1991; Hirono andAoki 1992, a and b).
We have previously reported the cloning of two hemolysin genes (for aerolysins AHH-l and AHH-2)
from A. hydrophila ATCC7966 into a plasmid vector in
E. coli K-12 (Aoki and Hirono 1991; Hirono and Aoki
1991, Table 1). Open reading frames (ORF) of theAHH-l and AHH-2 genes were 1,734 and 981 basepairs (bp), respectively. The sequences included the-10 region and the -35 region of a promoter and aribosome binding site (Shine-Dalgarno sequences)
upstream from the ORF. Two palindromic sequenceswere found immediately following the terminationsite. Analysis of the deduced amino acid sequencesindicated a highly hydrophobic N-terminal region in
the AHH-l gene with the characteristics of a leader
21

22

NOAA Technical Report NMFS 111

"

ro

~

~

W

~

ro

~

~

~

OCATOCCOAATCATCCACCTTAOATCOTATCOCOATCCTTGTTCTGTCAACAATAAATOCATTCOCCOCCACACTGTTAATTCACGGCOAOAATAATGAATGTCA
110
120
130
UO
150
160
170
180
190
200
210
TOACAGGCAAGCAOAATAAcoccccaAAATATAACCGTGAATAATATGGTTCTGCCGTATTCOTTCATTGmAAATAGCTTGGCOTOATTCOACAAGGAGATAA
,
~
220
230
240
250
260
270
280
290
300
310
lOMlGAAAAACAAAAAACCACOCAAATTCATCACOCAAGCCCCCACTCTCAGTCTcaroCOCTGGCCTTGTTGGCAGGCAGCGTGCAAGGCOAAGATATTGGCOA
-10
320
330
340
350
260
370
380
390
400
410
420

-

ACGTACCGA~CACCOCCATOCTGGCCAGCCTGCAATCCGAACAGGGTCTGAmACCTCAATOCCGACGTCTGGCTGAAGGGGCAGGGGGCGACOCCOCTSD

IIeILeuAlaSerLeuGInSerGl uOI nG lyLeu IleTyrLeuAsnA IaAspYal TrpLeuLysG lyOInG lyA laThrProLe

ill
ill
~
~
m
ill
~
~
m
~
CATOACCCGGGATCAOCTGCOCOAGCGGGTOCTGGCACGGGGTGAGCGTCTGTTCATCOAmCAGCOCCGTCACCGICCAGATCOAGCOACAACAGGCCAGAAA
ulIelThrArs.\spGInLeuArgGIuArgYslLeuAlaArgG IyO1uArg!.euPhe IleAspPhoSerAIaYa1ThrAspG In II eG IuArgG InG InA laArg!.y

530
5~
550
560
570
580
590
600
610
620
630
GGCCATGGAGCAGCTGGCCGGGATCTCTmGATOCOGACTGGOTOCTGGTGTCCGGCTACAAGGGGGAGCTGCTGTTCACCCCOCTGOOAGGCOTCOATOACCC
sAl allelO 1uOlnLeuA 1aO lyll eSerPheAspAl aAspTrpYalLeuYal SerG lyTyrLysOlyOl uLeuLeupt.eThrProLeuOlyOIyValAspAspPr
640
650
660
670
680
690
700
710
720
730
GGCCTTCTATCAOCTGATOOAOCGGGTCOAOAOCCTGGAAOGGCACCCCAACCCCCACAAGCOCTCOCTGACCCAOCCGCCTGCCGCCOAGGCCOOTCTGCCCCA
cAl aPheTyrO InLeulletGl uArgVa101 uSerLeuGl eGlyO1nOlyAsnG ly81 sLysArgSerLouThrG1nProPrcA1aA IsO1uA laG 1yLouPro/l1
740
750
760
770
780
790
800
810
820
830
840
TOTGGCCTmACCTCAACOTCAACCGCAAOATCAOCOATOCCGAOTGCACCTTcccaCOCTCOCOTACCTGOAOCCGOGGCOACCGOCTGTTCTGCOATTCOCC
sValAlaPheTyrLeuAsnValAsnArgLyslloSerAspAlaOluCysThrPhePrcArgSerArg!hrTrpSerArgGlyAspArgLeuPheCysAspSerPr

rn

m

m

m

~

m

m

m

~

m

GAACATCTCOCTGGTCTACCGAOTCAACCTGGAGCOCTCCCTGCAATTTGGCAACACGGCOTCCGCCACOCCGGATOCCAAGATAGTGCOGATCTCGCTGGACGA
cAsn II oSerLeuValTyrArgValAsnLeuGIuArgSerLeuGInPbeGlyAanThrAlaSerAIaThrPrcAspA. aLys II eValArg II eSerLeuAspG I
950
960
170
980
990
1000
1010
1020
1030
1040
1050
AGAGTCCCCCGGroCCGGTATCCAOCTCAACOAGGATCTGAGCTGGAGCGAAAACATCOCGGACTATCTGCTGCTGOACCCCTGGOcccaCOACTATGCCACCOA
uGluSerAIaGlyAIaGlyileGinLeuAsnGluAspLeuSerTrpSerG1uAanileAlaAspTyrLeuLeuLeuA.pGlyTrpA1aArs.\spTyrAlaThrAs
1060
1070
1080
1090
1100
1110
1120
lISO
IUD
1150
TGCCATCOCCCAGGATTATCGCTTCACOACTGAGGCGTCCAACACCAAGGCGGCGGTOCTCAAGAGCCTGCCGACCAACCTCAACAGCAAGTACOAGCATCGCOA
pAlal1eA1aGInAspTyrArgPheThrThrGIuAlaSerAsnThrLysA 1aAlaYalLeuLysSerLeuProThrA.nLeuAsnSerLyaTyrG1uBI sArgG I
1160
1170
1160
1190
1200
1210
1220
1230
1240
1250
1260
OATCTCOoamCOAGGTGGGGGTCACCGGCCCGGTCGAGGroAACAAGGATGGCCCAAGGCCAAGCTGGAGGCGTCOGCCAAGTTCAGCCAGCAGCOCCAGCTCuII oSerGlyPheG1uValGlyYa1ThrG1y01yYaiGIuYaIAsnLysAspG1yPrcArgProSerTrpArs.\rs.\rgProSerSerAI aSerSerAI aSerSo
1270
1280
1290
1300
1Sl0
1320
mo
1340
1350
1360
GCCTACAACACCCAGGATTACCOGGTTGAACGCTCCGGCCGAGCGCCCAGAAGGTOAGmCAGCTGGGTGCOCOATCAATACOCOACCOCAGAGTCCCTGCTCT
rProThrThrPrcArglleThrG lyLeuAsnAlaPrcA1aG IuArgProG1uG lyGI uPheG InLeuG1yAIaArgSer II eArgAspArs.\rgVaIPrcA 1aLe
1370
1380
1390
UOO
1410
U20
1430
WO
1450
U60
1470
CCTCCAAOACGGCCACOTCTGGOCATGGCTACOACOTGOATCACAACCOCATCCAOCCGCTCAOCTACAAGGoamGroCCGAATCTGATGTCATCTACAAGOC
uLeuOlnAspGlyHlsValTrpAlaTrpLeuArgArgGlySerGlnProHlsPrcAlaAlaG1nLeuOInOlyVaiCysAiaGIuSorAspVailieTyrLysAi
1480
1490
1500
1510
1520
1530
1540
1550
1560
1570
OOcaccoaACOAGACGGOCAOCACCGAOTTCAAOATCOACT=TCAACATCCGCCCCATCTACACCGGCATCTACAAOCACTACTACOTGGTGOOOGCCCA
aAlaPtoAspGluThrGlySerThrGluPheLyslleAspSerSerValAsnlleArgProlleTyrTbrGlylleTyrLysHI sTyrTyrVal ValGlyAlali1
1580
1590
1600
1610
1620
1630
1640
1650
1660
1670
1680
TGTCTCCTTCCACCCCTTCOAAOATACCGACAAGCOCAGACGGGTGGCOOCGTCCACCAGCTTCAAGGTGGACTOOAACCACCCGGTGTTTACCGGCOGTCOCCC
sVal SerPbeGlnG IYPheGIuAspThrAspLysAraArs.\rgVaIA1aAi aSerThrSerPheLysValAspTrpAsnHI sProVaIPheTbrG I10 lyArgPr
1690
1700
1710
1720
1730
1740
1750
1760
1770
1780
OGTCAA.CCTGCAOCTGGGGGGCTTTGACAACCCTTGCCTOAGCOCOOATGCCAACCATOOTCTGAOcacaGroACCTTTGACOAOACCTCOGCCGCCCAOTCCTC
oValAsnLeuG1nLeeG110IYPheAspAsnArgCYsLeuSerAI aAspA IaAsnH IsO 1yLeuSerAlaVa1ThrPheAspGIuThrSerA1aA laO 1nSerSe
1790
1800
1810
1820
1830
IUD
1850
1860
1870
1880
1890
CATCTATOACGAOTACGGCCGCTATOTCAGCOCOCAGOATACCCTCCGCTGCCTGGATOGCAACAACCTTGGCCAOCTGCAOAGCTGCAGCCTGAOCCTGGOTCAr 11eTyrAspG1uTyrG lyArg!yrYa1SerAl aOI nAspThrLeuArgCysLeuAspGI yAsnAsnLeuGlyO InLeuGInSerCysSerLeuSorLeuG lyO 1
1900
1910
1920
1930
1940
1950
1960
1970
1980
1990
OCOCTGGOAGTGGAAAGCGOACAOCOATGCOCTCAGCAACCTGAGTGCCCACCAOCTGCTGGTGCATOACAAOCAOAOCOCCCCOCTCCCOCTCTACOACOAOAA
nArgTrpGIuTrpLysA IaAspSerAspA IaLeuSerAsnLeuSorA1aH IsG InLe.uLeuVa IHlsAspLysOInSerG IyA IaLeuOlyLeuTyrAspG IuAs
2000
2010
2020
2030
2040
2050
2060
2070
2080
2090
2100
COOCAATCCGCAOAATGTGAOCOTACOOACCCTGACCTCCTACACCCGCATCTTCOGGCCACCTGCCAOTCACTOACAOAGCACCCCCTTGCOCTCAAOACAACC
nOlyAsnProGlnAsnValSorValArgThrLeuThrSerTyrTbrArgllePheGlYProPrcAlaSerHls
2110
2120
2130
2UO
2150
2160
2170
21~
2190
2200
ACOACOCCAAGroCCGGCOmmATGAOCCCCOOATCAGGCCTTOTTTTCTATGAGCGGGGCOAGCACOTCCOCCAGCTTGTAGAAGGTCTGCAcccaGGTOG
paiIldr.....lrucb...
2210
2220
2230
2240
2250
2260
2270
2280
2290
2300
2310
TGCTGGGCOCCACACCAGCCCTATCTCOCOGTAGroATGTTCACCGGCAGCGGGATGGCCACCAGATCCOTGTTGTCOAGGATGCCGGCATCOATGGCCATCTOC
2320
2330
2340
2350
2360
2370
2380
2390
2400
2410
GGCAGGAAGGTGGCTGAOCCCOGCOCCGACCATCTGCACCAGGGTGTGCAGGCTGGTGGCGGCAAACGGGTTGATCTTGCTCTTGTCACCCGCAGCCOGCAGGGC
2420
mo
2440
2450
2460
2470
2480
2490
2500
2510
2520
TCACCGCOTGTTCGGTGAGACAGTCOTcccaCTCCAOCAGGAAGATACTCTGOTTGGGCAGCTCCTTGTAGTCOAACGGGGTGTGCAGGCTGGr.r.ATGCTGGr.r.r.

Figure 1
Nucleotide sequence and deduced
amino acid sequence of hemolysin
gene AHH-l from Aeromonas hydrophila
ATCC7966. The deduced amino acidsequence is given under the nucleotide sequence. A putative promoter isindicated by the areas marked -35 and-10, and ribosome binding site is indicated by SD. The palindromic structure
sequence of a terminator-like region is
also indicated downstream from theAHH-l coding region.

Aoki and Hirono: Cloning of Hemolysin Genes of Aeromonads

Table I
Maximum matching comparison of amino acid sequences of cloned hemolysin genes of Aeromimashydrophila and A. salmonicida.

AHH-I
AHH-2
ASH-I
aer*

AHH-I

AHH-2

ASH-I

17.4%
17.0
17.1

17.0
16.9

18.5

aer

* Howard and Buckley 1986.

peptide (Figure 1). However, the N-terminal regionof the AHH-2 gene was not hydrophobic.
Two aerolysin genes were also cloned from A.
hydrophila, Ah65 (Howard and Buckley 1986; Howard
et al. 1987) and AH2 (Chakraborty et al. 1986). The
nucleotide sequence of the Ah65 aerolysin gene
was 1,458 bp. There was very low homology between
the aerolysin gene from Ah65 and each of the
ATCC7966 genes, and there were no indications ofsimilarity in the predicted amino acid sequences(Table 1).
We also cloned one hemolysin gene (ASH-I) from
A. salmonicida ATCC14174 (Hirono and Aoki 1992,
a and b) (Table 2) which had an ORF of 1,716 bp. It
had the -10 region and the -35 region of a putative
promoter and a ribosome-binding site up stream
from the ORF, and the termination codon andpalindromic sequences downstream from the ORF.
The N-terminal region of the ASH-l gene was highlyhydrophobic. Comparative analysis of the fundamental molecular structures of our cloned AHH-l,
AHH-2, and ASH-1 genes, and the previously reported aerolysin gene from Ah65 (Howard et al.
1987) suggests that they have not descended from acommon ancestor.
The recombinant plasmids pAHH-l, pAHH-2, andpASH-l were introduced into a maxicell strain CSR603

of E. coli and the plasmid-encoded proteins examinedby polyacrylamide gel electrophoresis (Sancar et al.
1979). Strains containing pAHH-l and pASH-l produced proteins of 62 kDa and 56 kDa, respectively(Hirono and Aoki 1991, 1992a). These results are inagreement with the size of the proteins predicted by
the DNA-sequenced ORF. However, the molecular sizeof novel protein synthesized by cells harboring thepAHH-2 plasmid was 26 kDa. The molecular size ofthe protein was clearly different from the size predicted by the hemolysin AHH-2 gene sequence which
inferred a protein with a molecular weight of 37.7
kDa. The products of transcription or translation in
AHH-2 may be either greatly truncated or reduced inquantity. As mentioned above, analysis of the deduced
amino acid sequences indicated that the N-terminal
region of the AHH-2 gene was not hydrophobic. As
yet, it is difficult to explain the difference in the molecular size of the final protein.
Hemolysin was released by the E. coli cells containing the AHH-l gene and those containing the ASH-lgene. The hemolytic activity in a supernatant was inactivated by heating to 70° C for 10 minutes.
However, the hemolysin was expressed but was notsecreted from E. coli cells carrying the recombinant
plasmid containing the AHH-2 gene. The aerolysin
cloned by Howard and Buckley (1986) similarly was
not released from the E. coli cells in which they were
cultured. The E. coli hemolysin requires four cistrons
encoded with the hemolysin structured protein for
full expression of active protein to be achieved(Felmlee et al. 1985). These proteins are associatedwith the cell envelope and are involved in transporting the hemolysins out of the cells. This complex
system is unlike the one producing the extracellular
hemolysins in A. hydrophila and A. salmonicida. It isvery interesting that the hemolysin release mechanism produced by E. coli cells containing the hemolysin gene is different from Aeromonas spp.
Using colony hybridization analysis, the cloned hemolysin genes were tested for presence ofhomologous regions in isolates A. hydrophila and A.
salmonicida from humans and fish in Japan (Hirono

Table 2
Cloned hemolysin genes from Aeromonas hydrophila and A. salmonicida.
Hemolysin
genes
AHH-I
AHH-2
ASH-I

23

Sources

Open reading
frame (bp)Predicted molecular size
from 0 A sequences (Oa)

Molecular size
from Maxicell (kOa)

A. hydrophila
A. hydrophila
A. salmonicida

1,734
981
1,716

63,65837,79764,78062 (Hirono and A.oki 1991)
26 (Aoki and Hirono 1991)
56 (Hirono and Aoki 1992a)

24

NOAA Technical Report NMFS III

and Aoki 1992, a and b). Ten of 15 strains of A.
hydrophila isolated from humans and 14 of 33 strainsfrom fish possessed sequences homologous to the hemolysin gene of AHH-1 (Table 3). The AHH-1 gene
was detected in all 38 strains of A. salmonicida. The
AHH-2 gene was detected only in the original strain
ATCC7966 and not in the other strains of A.
hydrophila and A. salmonicida tested. The ASH-1 gene
was detected in two strains of A. hydrophila from fish
and the original ATCC14174 strain. We used strainsof A. hydrophila and A. salmonicida isolated in the
Linited States for cloning the hemolysin gene andtested for homology with strains isolated in Japan.
Homology of the tested strains with the cloned hemolysin was low. We shall attempt to survey the
distribution of our cloned hemolysin genes in moreisolates of aeromonads. The presented data, however,indicate that the hemolysin genes having different
structures occur in A. hydrophila and A. salmonicida
isolates from different geographical locations.

Table 3Detection of DNA sequences homologous to the hemolysin genes from Aeromonas hydrophila and A. salmonicida bycolony hybridization. (Hirano and Aoki 1991, 1992b.;
Strains

Sources

AHH-I

AHH-2

ASH-I

A. hydrophila

Human
Fish
ATCC7966
Fish
ATCCI4174

10/15*
14/33
1/1
38/38
1/1

0/4
0/5
1/1
0/19
0/1

0/15
2/33
0/1
0/38
1/1

A. salmonicida* Number of strains containing hemolysin genes/Number of tested strains.
Hemolysin genes of the AHH-l and AHH-2 of A. hydrophila ATCC7966 and the ASH-I gene of A. salmonicida ATCCI4174 werecloned into plasmid vectors in E. coli K-12.

Citations_Aoki, T, and 1. Hirono.
1991. Cloning and characterization of the hemolysin de':erminants from Aeromonas hydrophila. j. Fish Dis. 14:303-312.
Asao, T, S. Kozaki. K. Kato, Y. Kinoshita, K. Otsu, T Umemllra,
and G. Sakaguchi.
1986. Purification and characterization of an Aeromo''lashydrophila hemolysin. j. Clin. Microbiol. 24:228-232.
Chakraborty, T, B. Huhle, H. Bergbauer. and W. Goebel.
1986. Cloning, expression. and mapping of the Aeromonas
hydrophila aerolysin gene determinan t in Escherichia coli K12. J. Bacteriol. 167:368-374.
Ellis, A.E., TS. Hastings, and A.L.S. Munro.
1981. The role of Aeramonas salmonicida extracellular products in the pathology of furunculosis. J. Fish Dis. 4:41-51.
Ellis, A.E., A.S. Burrows, and KJ. Stapleton.
1988. Lack of relationship between virulence of Aermo"lassalmonicida and the putative virulence factors: A-layer, extracellular proteases and extracellular haemolysins. J. Fish
Dis. 11:309-323.
Felmlee, T, S. Pellett, and R.A. Welch.
1985.
ucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol. 163:94-105.
Hirono, 1., and T Aoki.
1991. Nucleotide sequence and expression of a hemolysin genefrom Aeromonas hydrophila. Microb. Pathog. 11:189-197.
Hirono, 1., and T Aoki.
1992a. Molecular cloning, expression, and nucleotide sequenceof a hemolysin gene from Aeromonas salrrwnicida. The Proceed-

ings of the Symposium on Diseases in Asian Aquaculture.
Asian Fisheries Society. (In press.)
1992b.
ucleotide sequence, expression, and characterization of a hemolysin gene from Aeromonas salmonicida.
Submitted to Infection Immun.
Howard, S.P., andJ.T. Buckley.
1986. Molecular cloning and expression in Escherichia coli of
the structural gene for the hemolytic toxin aerolysin from
Aeromonas hydrophila. Mol. & Gen. Genet. 204:289-295.
Howard, S.P., W.J. Garland, M.J. Green, andJ.T Buckley.
1987. Nucleotide sequence of the gene for the hole-forming
toxin aerolysin of Aeromonas hydrophila. J. Bacteriol.
169:2869-2871.
Ljungh A., and T Wadstrom.
1982. Aeromonas toxins. Pharmacol. & Ther. 15:339-354.
Ljungh. A., B. Wretlind, and R. M611by.
1981. Separation and characterization of enterotoxin andtwo haemolysins from Aeromonas hydrophila. Acta Pathol.
Microbiol. Scand. Sect. B. Microbiol. Immunol. 89:387-397.
l'o;ieto, TP., Y. Santos, L.A. Rodriguez, and A.E. Ellis.
19Y1. An extracellular acetylcholinesterase produced byAcromonas hydmphila is a major lethal toxin for fish. Microb.
Pathog. 11:101-110.
Nomura. S., Fujino, M., Yamakawa, M., and E. Kawahara.
1988. Purification and characterization of salmolysin, andextracellular
hemolysin
toxin
from
Aeromonas
salmoni,ida. J. Bacteriol. 170:3694-3702.
Rockey, D.D.,J.L. Fryer, andJ.S. Rohovec.
1988. Separation and in vivo analysis of two extracelluar
proteases and the T-hemolysin from Aeromonas
salmonicida. Dis. Aquat. Org. 5:197-204.

Aoki and Hirono: Cloning of Hemolysin Genes of Aeromonads
Sancar, A., A.M. Hack, and W.O. Rupp.
1979. Simple method for identification of plasmid-coded
proteins. J. Bacteriol. 137:692-693.
Thune, R.L., and M.e.Johnson.
1986. Aeromonas hydrophila B-haemolysin: purification and examination of its role in virulence in O-group channel catfish, Ictaluruspunctatus (Rafinesque). J. Fish Dis. 9:55-61.

25

Titball, R.W., and e.B. Munn.
1985a. Interrelationships of extracellular products from
Aeromonas salmonicida. In Fish and shellfish pathology (A.E.
Ellis, ed.), p. 61-68. Acad. Press, I\ry'
Titball, R.W., and e.B. Munn.
1985b. The purification and some properties of H-lysin from
Aeromonas salmonicida. J. Gen. Microbiol. 131:1603-1609.

Harmful Red Tides of Heterosigma akashiwo
TSUNEO HONJO
Nansei National Fisheries Research InstituteFisheries Agency
Ohno, Saeki, Hiroshima 739-04,Japan

ABSTRACTThe raphidophyte Heterosigma akashiwo is one of several species of flagellates thatcause harmful red tides. This paper reviews the distribution of H. akashiwo, the relationship between eutrophication and the occurrence of red tides in the Seto Inland Sea, andenvironmental and biological features of red tide development (life cycle, growth dynamics, and allelopathy).

Introduction_

The raphidophyte Heterosigma akashiwo is one of several species of flagellates causing prodigious red
tides. The total damage by H. akashiwo red tides has
amounted to about 2 billion yen over a perioq of 16
years (1972-87) in the Seto Inland Sea of Japan.
Most of this damage affected fish culture operations.
This paper seeks to advance research on red tides byreviewing the known ecological features of Heterosigma red tides.

Morphology_

The cells of Heterosigma akashiwo (HADA) Hara et
Chihara are yellow-brown and ovoid, and slightly
compressed dorso-ventrally. The cell size is 8-25 X
6-15 flom. This organism is thought to lack a cellwall and be limited only by a single membrane. The
delicate structure of the cell has hindered the studyof its surface morphology, although the existence ofan external structure, the glycocalyx, has been demonstrated (Yokote et al. 1985). In Japan, H. akashiwo
has been referred to as Entomosigma sp. and H.
inlandica; in many other countries it has been confused with the chrysophyte Olisthodiscus luteus
CARTER. Recently, Hara and Chihara (1987) reported that Entomosigma sp. and H. inlandica aresynonymous with H. akashiwo, and that most red
tides ascribed to O. luteus actually involve H.
akashiwo.

Distribution_

Heterosigma akashiwo occurs in the temperate and subtropical embayments in Japan (Hara and Chihara
1987), Singapore (Taylor 1990), New Zealand (Taylor
1990), England (Lackey and Lackey 1963), and Belgium (Conrad and Kufferath 1954), and in theeastern (Tomas 1982) and western areas (Lackey andClendenning 1965; Taylor 1990) of North America,
Bermuda (Tomas 1982), and Chile (Taylor 1990)(Fig. 1). Damages to fish have been recorded for the
Seto Inland Sea (yellowtail, Seriola quinqueradiata andred sea bream, Pagrus major), New Zealand (salmon),British Columbia (salmon), and Chile (salmon) (Taylor 1990).

Eutrophication and Red Tides_

In the Seto Inland Sea, nitrate, inorganic nitrogen(ammonia + nitrite + nitrate), and phosphorous concentrations have increased rapidly since themid-1960s (Fig. 2). Nutrient concentrations reacheda maximum in the mid-1970s then gradually decreased thereafter. The total number of red tideoccurrences in the Seto Inland Sea before 1965 wasless than 50 cases per year. Beginning in 1968, thenumber increased year by year, reaching a peak of
299 cases in 1976. After 1976, the number decreased
to 160-170 cases per year. The increase in the 1970s
coincided with a rapid development in the Japanese
economy, with a time lag of 2-3 years. The decreasein frequency after the first oil shock (1973-1974) cor27

28

NOAA Technical ReportNMFS III

Figure 1
Distribution of Heterosigma akashiwo red tide (dotted areas). Fish indicate places where cultured fish have sustained damage.

300

.31

200

1.5 0.6-..

to 0.4E.5 02...III

o

1:
::l

Z

100

o

0

0-0------0

o 0

Owr---........L.I.--_ _.L,L-_ _....u....--L.l...LLJ~......................................L.&-..,.....................................,
1960
1910
1!110
1910
1950

Figure 2
Total number of
red tide cases (openbars) and of Heterosigma akashiwo redtides (black bars)
and concentrationsof nitrogen (opencircles and solidlines) and phosphorous (black circles
and dashed lines)
in the Seto Inland
Sea (1950-89).

Honjo: Red Tides of Heterosigma akashiwo-----Winter - - - - - - l.... Spring - - - - - - - - - - Red Tide ---Summer -~""Autumn,.
diatoms ,

"OJ
o

j

o

-

..0::

~
c

/

/

germination
15°C

rest i ng spores---I".

~

0...:..=..:..:.\

2-5div/day(rapid)oHet~~o_s~tRJ.a_ ----~ti Iization of nutrients

7m

29

lysis
- - - - - ••- - - - - - - - - - - - - - - - -- - Pycnocl i ne

I ------.,\.L

Release from bottom sediments

!t

o

o
o

o
o

and growth-pro mot ing
Water mass with
substances (Fe and org ni cs, • low oxygen content\

J'-----t15m--..z....:'"--""-----~'-------'----.........L----........;:·;..;·~·=-------..J....._

resting spores

BottomFigure 3
Schematic diagram of ecological features during the period of red tide development.

responds with the switchover of the Japanese economy
to a lower rate of development. The patterns ofchange in the total number of tides and the number
of Heterosigma red tides were similar to that of nutrient
concentrations. Thus, the frequency of red tide occurrences is closely related to eutrophication.

Environmental Features During the Period
of Red Tide Development_
Temperatures suitable for the growth of H. akashiwo
have been reported from culture experiments to bein the range of 15-30° C (Tomas 1978a; Mori et al.
1982; Yamochi 1989). This helps to explain why
Heterosigma red tides tend to occur in coastal watersfrom May through late June. However, the range ofsuitable salinity differs among culture strains: 30%0
for a Fukuyama strain (Iwasaki et al. 1968); 10%0 for
a Gokasho strain (Iwasaki and Sasada 1969); 27-28%0
for a Hakata strain (Honjo and Hanaoka 1973); 1240%0 for a Narragansett strain (Tomas 1978a); and12-28%0 for a Tanigawa strain (Mori et al. 1982).
This suggests that these strains have developed aphysiological acclimation to the range of salinity ineach habitat.
Growth of H. akashiwo is usually initiated in earlysummer when a pycnocline is formed in the

middle layer (Fig. 3). Oxygen concentrations inbottom water decrease rapidly after the formationof the pycnocline. The pH of the anoxic bottom
water decreases to about 7. As a result, nutritive
substances (inorganic nutrients, metals, and organic constituents including growth-promoting
substances) are released from the bottom sediments and these substances accumulate to highlevels in anoxic bottom waters (Honjo 1974). The
Heterosigma population has access to these substances in the bottom water through diurnalvertical migration at night.

The Development of Heterosigma
Red Tides_

Growth originates from cell stocks that overwinteredas motile forms (Yamochi 1989) and/or that germinated from resting cells (Imai et al. Nansei National
Fisheries Research Institute pers. commun. 15 October, 1990). Tomas (1978b) and Yamochi (1989)
found that motile cells aggregate and change tononmotile cells. Population growth can be divided
into two phases: rapid and exponential (Fig. 3; Honjo
and Tabata 1985). The rapid phase is defined as theperiod when cells increase more than four-fold each

30

NOAA Technical Report NMFS III

day and the exponential phase as that period when
the correlation coefficient between log cell number
and time is greater than or equal to 0.95 and there
is an obvious increase in the number of cells formore than 4 days. The duration of Heterosigma redtides is shorter than for other flagellates. The
abrupt disappearance of Heterosigma cells requires
further study.

Growth Rate and Division Periodicity_

Blooms of this organism develop so rapidly and dramatically that many workers have studied the growthof H. akashiwo in vitro and in situ as an approach tounderstanding the enigma of red tide formation.
Their results, however, have conflicted (Honjo andHanaoka 1973; Tomas 1978a).
Honjo and Tabata (1985) studied Heterosigma
growth dynamics and division periodicity in outdoor
tanks. They found that H. akashiwo has a potential forhigh growth rates (2-5 divisions per day). There wasa strong tendency for large cells to dominate at the
beginning of this fast growth phase and to be replaced by small cells toward the end of the phase.
During culture experiments, a portion of a H.
akashiwo patch in a tank was cultured in a I-liter volumetric flask hung in the tank. The growth rate in thisexperiment was 2.3 divisions per day. Other H.
akashiwo cells were collected from the tank and individually cultured in small tissue chambelis. In thesechambers the highest growth rate was 3.3 divisions
per day; the growth rates of large cells inoculated
into these chambers were much higher than those ofsmall cells.
In the tank environment, H. akashiwo prefers darkassociated cell division. Cell division first occurred alittle before sunset and continued through the night
until the next morning. Similarly, in the small chambers cell division began just before the onset of thedark period and continued for 4-5 hours into thenext light period. Seven of 24 H. akashiwo cells divided three times during the night in the smallchambers, with a cell division interval of about 6hours. Puiseux-Dao (1981) comprehensively reviewed
the events of the cell cycle and Chisholm (1981) described the chronobiology of cell division inunicellular algae. In their reviews, the cell division
cycle was discussed in terms of the length of the circadian period (1 division per day), but noconsideration was given to the rapid division cycles of
H. akashiwo.

Allelopathy_

The study of allelopathy is important for elucidating
the mechanisms of temporal succession of phytoplankton and of monospecific bloom events.
Allelopathic interactions in which organic metabolites of one plant or microorganism suppress orenhance the growth of other plants or microorganisms are different from competitive interactions
which involve the removal or reduction of certain factors such as water, minerals, foods, and light. Theinteractions among marine and freshwater phytoplankton have been well studied and have beenreviewed by Smayda (1980), Maestrini and Bonin(1981), and Rice (1984). Some of the most dramaticchanges of species composition in marine phytoplankton have been observed during Heterosigma redtide blooms, and allelopathic interactions have been
observed between H. akashiwo and the centric diatom
Skeletonema costatum during in vitro experiments(Pratt 1966; Honjo et al. 1978). Their studies imply
that an alle10pathic relationship between these organisms occurs during high cell densities of H.
akashiwo. Stuart (1972) and Sakshaug (1977) examined physical features of the allelopathic substances
but experienced difficulty in determining their molecular weight.
Recently Honjo et al. (unpubl. data) investigatedthe biological and chemical features of allelopathic
substances from H. akashiwo (Fig. 4). During a bloomof H. akashiwo in an outdoor tank, an abrupt decrease in cell numbers of centric diatoms (dominant
species, S. costatum) and an increase in the dinophyte
Prorocentrum triestinum occurred when high concentrations of dissolved carbohydrates were detected in
the water. Crude polysaccharide extracts from
Heterosigma cells and from filtrates of the bloom seawater were both separated into two main fractions bygel chromatography. In bioassay experiments, a macromolecular fraction from Heterosigma cells greatly
suppressed the growth of S. costatum at concentrations above 1.0 f,Lg glucose per mL, whereas this same
fraction enhanced the growth of P. triestinum andH. akashiwo and had no effect on the growth of
Phaeodactylum sp. The other fraction caused moderate suppression of S. costatum growth. Results ofbioassays using S. costatum and the two fractions from
H. akashiwo bloom water were similar to those using
the fractions from Heterosigma cells. Histochemical
analysis of the crude polysaccharide extracts from
Heterosigma cells indicated a polysaccharide-protein
complex with features analogous to the glycocalyx on
the cell surface of the organism. Results suggest thata polysaccharide-protein complex exfoliated from

Honjo: Red Tides of Heterosigma akashiwo

31

growthsuppression,"",,'----. ... ,/glycocalyx,'
1\(seawate r):'

growthen hancement

excretionpolysaccha ride - _-+--__
exfol iat ion protei n comp lex-+.-..::t....----I~

HeterosigmB
red tid e~

Prorocentrum~

growthen hlnce ment

Figure 4
General scheme of allelopathic relationships between Heterosigma akashiwo and other phytoplankton.

and/or excreted by H. akashiwo is a species-specificallelopathic substance and plays an important role incausing dramatic changes in cell numbers of otherphytoplankton during Heterosigma blooms.

Citations_

Chisholm, S.w.
1981. Temporal patterns of cell division in unicellularalgae. In Physiological bases of phytoplankton ecology (T.
Platt, ed.) p.150-181. Can. Bull. Fish. Aquat. Sci. 210.
Conrad, W., and H. Kufferath.
1954. Recherches sur les eau saumatres des environs de
Lilloo. Perti II. Descrictive. Inst. R. Sci. Nat. Belg. Mem.
127:1-346.
Hara, Y, and M. Chihara.
1987. Morphology, ultrastructure and taxonomy of theraphidophycean alga Heterosigma akashiwo. Bot. Mag. Tokyo 100:151-163.
Honjo, T.
1974. Studies on the mechanisms of red tide occurrence in
Hakata Bay. IV. Environmental conditions during theblooming season and essen tial factors of red tideoccurrence. Bull. Tokai Reg. Fish. Res. Lab. 79: 77-121.
(lnJapanese.)

Honjo, T., and T. Hanaoka
1973. Studies on the mechanisms of red tide occurrence in
Hakata Bay. II. General features of the red tide flagellate,
Heterosigma sp. Bull. Plankton Soc. Jpn. 19:75-81. (InJapanese.)
Honjo, T., and K Tabata.
1985. Growth dynamics of Olisthodiscus luteus in outdoor
tanks with flowing coastal water and in small vessels.
Limnol. Oceanogr. 30:653-664.
Honjo, T., T. Shimouse, N. Ueda, and T. Hanaoka.
1978. Changes of phytoplankton composition and its characteristics during red tide season. Bull. Plankton Soc. Jpn.
25:13-19. (In Japanese.)
Iwasaki, H. and K Sasada.
1969. Studies on the red tide dinoflagellates II. On Heterosigma inlandica appeared in Gokasho Bay, Shima Peninsula. Bull.Jpn. Soc. Sci. Fish. 35:943--947. (In Japanese.)
Iwasaki, H., T. Fujiyama, and E. Yamashita.
1968. Studies on the red tide dinoflagellates 1. On
Entomosigma sp. appeared in coastal area of Fukuyama. ].
Fac. Fish. Anim. Husb. Hiroshima Univ. 7:259-267. (InJapanese.)
Lackey,].B., and E. Lackey.
1963. Microscopic algae and protozoa in the waters near Plymouth in August 1962. ]. Mar. BioI. Assoc. UK 43:797-805.
Lackey,].B. and KA. Clendenning.
1965. Ecology of the microbiota of San Diego Bay,
California. Trans. San Diego Soc. at. Hist. 14:9-40.

32

NOAA Technical Report NMFS 111

Maestrini, S.Y., and D.J. Bonin.
1981. Allelopathic relationships between phytoplankton species. In Physiological bases of phytoplankton ecology (T.
Platt, ed.), p.323-338. Can. Bull. Fish. Aquat. Sci. 210.
Mori, S., Y. Nakamura, M. Watanabe, S. Yamochi, and M.
Watanabe.
1982. The effect of various environmental factors on thegrowth yield of red tide algae. 11. Olisthodiscus luteus. Res.
Rep. Nat!. Inst. Environ. Stud. 30:71-86. (In Japanese.}
Pratt, D.M.
1966. Competition between Skeletonema costatum andOlisthodiscus luteus in Narragansett Bay and in culture. Limnol. Oceanogr. 11:447-455.
Puiseux-Dao, S.
1981. Cell-cycle events in unicellular algae. In Physiologicalbases of phytoplankton ecology (Platt, T. ed.), p.130149. Can. Bull. Fish. Aquat. Sci. 210.
Rice, E. L.
1984. Allelopathy. Acad. Press. London, 422p.
Sakshaug, E.
1977. Limiting nutrients and maximum growth rates for diatoms in Narragansett Bay. J. Exp. Mar. BioI. Ecol. 28:109-123.
Smayda, T. J.
1980. Phytoplankton species succession. In The physiological ecology of phytoplankton (I. Morris, ed.), p.4S3570. Blackwell, London.

Stuart, M.
1972. The effect of Olisthodiscus luteus Carter upon thegrowth of Skeletonema costatum (Grev.) Cleve. M.S. thesis,
Univ. Rhode Island, Kingston, RI, 82p.
Taylor, FJ.R.
1990. Red tides, brown tides and other harmful algal
blooms: the view into the 1990s. In Toxic marine phytoplankton (E. Graneli, B. Sundstrom, L. Edler, and D.M.
Anderson, eds.), p.527-533. Elsevier, NY.
Tomas, C.R.
1978a. Olisthodiscus luteus (Chrysophyceae) I. Effects of salinity and temperature on growth, motility and survival. J.
Phycol. 14:309-313.
1978b. Olisthodiscus luteus (Chrysophyceae) II. Formationand survival ofa benthic stage. J. Phycol. 14:314-319.
1982. Olisthodiscus luteus (Chrysophyceae) V. Its occurrence,
abundance and dynamics in Narragansett Bay, Rhode
Island. J. Phycol. 16:157-166.
Yamochi, S.
1989. Mechanisms for outbreak of Heterosigma akashiwo redtide in Osaka Bay, Japan. Bull. Osaka Pref. Fish. Exp. Stat.
8: 1-11 O. (In Japanese.)
Yokote, M., T. Honjo, and M. Asakawa.
1985. Histochemical demonstration of a glycocalyx on thecell surface of Heterosigma akashiwo. Mar. BioI. 88:295-299.

Impact of the Myxosporean Parasite Ceratomyxa shasta on Survivalof Migrating Columbia River Basin Salmonids
j.L. BARTHOLOMEW,j.L. FRYER, andj.S. ROHOVEC*
Department of Microbiology
Oregon State University
Corvallis, Oregon 97331-3804

ABSTRACT
Columbia River Basin salmon ids are exposed to the parasite Ceratomyxa shasta during
both their seaward and return migrations. The impact of ceratomyxosis on the survival of
migrating fish is difficult to assess because there are few data on causes of fish mortality
once they are released from the hatchery. This study examines the impact of this diseaseon juvenile salmonids by 1) sampling the downstream migrants to determine what percent of fish leaving the basin are infected with the parasite and by 2) determining theeffects of entering salt water on the progress of the infection. Results of comparisons ofmethods for diagnosing a C. shasta infection indicate that serological techniques using
monoclonal antibodies are more sensitive than techniques for examination of spores.

Introduction_

The Columbia River Basin has long supported an important Pacific salmon fishery for Oregon,
Washington, and Idaho. However, the resource has
steadily declined even with supplementation of fishfrom hatcheries. As research efforts are directed toward determining the causes behind the depletion of
Columbia River Basin salmonid stocks, the impact of
diseases must be considered. One disease that is enzootic to the basin is caused by the myxosporean parasite
Ceratomyxa shasta. Although this parasite has devastated certain hatchery productions, it is not normally adisease of hatchery fish; therefore, its impact on survival of fish in the wild is difficult to evaluate. The
parasite first infects and multiplies in the intestinal
tract of the fish and from that site spreads to othertissues. The infection results in tissue necrosis accompanied by a severe host inflammatory reaction. Signs
of infection may include lethargy, darkening of the
body surface, abdominal distension, and hemorrhaging in the area of the vent (Bartholomew et al. I989a).
In addition, the disease has a prolonged incubation
period and current diagnostic methods (Amos 1985)
detect the parasite only in heavily infected fish.
Assessing the impact of ceratomyxosis requires examining a variety of factors. Studies of geographic* Send correspondence to this author

distribution Qohnson et al. 1979; Hoffmaster et al.
1988) indicate that the parasite has spread within the
Columbia River Basin, but the extent and cause of
expansion is not yet known. The degree of resistanceto infection among salmonid fish within the basin has
also been examined and compared with resistanceamong fish populations from watersheds where theparasite is not found (Zinn et al. 1977; Buchanan etal. 1983). All groups offish tested from the ColumbiaRiver Basin were relatively resistant. However, theability of many wild and upriver stocks to resist infection has not been examined.
Further investigations of the impact of
ceratomyxosis on migrating Columbia River salmonids were designed to answer 1) how many ColumbiaRiver salmonids become infected during their downstream migration, 2) how the disease progresses after
the fish enter salt water, and 3) how effective are themethods currently used to diagnose ceratomyxosis.

Materials and Methods_

Examination of Downstream Migrants for
Infection
To estimate how many salmonids become infectedduring their downstream migration, outmigrants
33

34

NOAA Technical Report NMFS 111

Cross section
Rock

cliff

Sand

Puget

Ship

~"',"'
A

beach

channel~~:::::: 9~
I~.

.1

1

950 :00 m I

WASHINGTON

Puget Island...

•,,:._.; .: l!·.~;.:.'i.::'.: :.~. ,0,,:', :;':~'~:'~":~ • •........

Columbia-.. - _.......£':-- --------------nlIJCh.

- --:""!!!'-'

14 m depth 200 m width __

I

I

I

Kilometersr

N
I

Figure 1
Jones Beach sampling site. The beach and purse seining areas are indicated qy the two asterisks on the main map. From
Dawley et al. (1984a).

were collected just prior to entering the estuary andwere maintained in fresh water to monitor diseasedevelopment. Outmigrating juvenile salmonids of different species and year classes were obtained bybeach and purse seine from a collection facility operated by the National Marine Fisheries Service at
Jones Beach (75 river km, measured upstream fromthe mouth of the Columbia River) on the Oregonside of the Columbia River (Fig. 1). Seining procedures were those described by Dawley et al. (1984a).
A purse seine 206 m long and 11 m deep with a meshof 1-2 em was used in water about 9 m deep. The
seine was set drifting with the current, then towed
upstream for 5 minutes before closing and pursing.
Beach seining with a net 95 m long, 5 m deep, with amesh of 1-2 em, was done in water about 6 m deep at
the outer end of the net sweep. Fish were collected
on 12 occasions between 20 May and 8 September in1983 and on six occasions between 5 July and 20 Sep-

tember in 1984. In 1984, fish were collected by beachseine only. All fish were transported to the Round
Butte Hatchery Isolation Facility on the Deschutes
River, a facility operated by the Oregon Departmentof Fish and Wildlife. Holding tanks were I-m
circulars with a 375-L capacity. The water supply was
free of pathogens and the water temperature was
10° C. All groups of fish were fed an Oregon Moist
Pellet diet containing 3% terramycin in the form of
TM so (Pfizer) as a prophylactic measure against bacterial fish pathogens (Udey et al. 1975). Groups were
held for at least 150 days and observed for development of ceratomyxosis. Fish deaths occurring within
10 days after transportation were attributed to handling mortality and were not included in the results.
In all experiments, dead fish were collected daily,
and either immediately necropsied or frozen for later
examination. All fish held in tanks that remained at
the end of the observation period were killed andBartholomew et a1: Impact of Ceratomyxa shasta on SaImonid Survival

supplied with 12° C pathogen-free water until theyreached smolt stage. Fish were exposed to the infectious stage of C. shasta in the Willamette River at
Corvallis, Oregon, on three occasions. On the firstoccasion, two groups of 50 Alsea steelhead trout
were exposed: one group for 3 days and the otherfor 5 days. On the second occasion, groups of 50
Alsea steelhead trout and 50 Big Creek coho salmon
were held for 5 days. On the third occasion, 100
Alsea steelhead trout and 100 Round Butte chinook
salmon were exposed for 3 days. Control groups ofan equal number of each species were not exposedin the river. Mter exposure, the groups were divided
and half of the fish were transported to freshwater
holding facilities at the OSU-FDL and the other half
to ultraviolet-treated saltwater facilities at the MarkO. Hatfield Marine Science Center in Newport, Oregon. The fish were fed Oregon Moist Pellets
containing 3% terramycin in the form of TM so(Pfizer). All groups were held for at least 100 days.

examined for C. shasta. Wet mounts of intestinal tract
scrapings were examined microscopically (as detailed
later) for up to 5 minutes, and samples containing
spores of C. shasta were considered infected.

Determining Saltwater Effects
Three strains of salmonid smolts were used to determine the effects of salt water on the progress ofceratomyxosis.
Big
Creek
coho
salmon(Oncorhynchus kisutch) were from Big Creek Hatchery located on the lower Columbia River and Round
Butte chinook salmon (0. tshawytscha) were fromthe Round Butte Hatchery on the Deschutes River, aColumbia River tributary. Both strains migrate
through waters enzootic for C. shasta and are relatively resistant to the parasite (Zinn et al. 1977;
Johnson et al. 1979). Alsea steelhead trout (0.
mykiss) were taken from the Alsea Hatchery. The
Alsea River empties directly into the Pacific Ocean
and does not harbor the infectious stage of C.
shasta. Salmonids from this system have not developed resistance. All fish were held at the Oregon
State University-Fish Disease Laboratory (OSU-FDL)
in I-m diameter tanks of 375 L capacity and wereComparison of Detection Methods
The sensitivity of the standard detection method of
examining wet mounts of material from the lower

Table I
Prevalence of Ceratomyxa shasta in chinook salmon smolts beach seined from the Columbia River at Jones Beach,
Oregon.

Mortalities
infected with
C. shasta

Percent offish collected
infected with
C. shasta

3
6

3

3

I

I

8

2

I

I

36
21

3
3

3
2
5
2

113
141
68

24
46
33

17
17
2

15
12
3

Aug. 12
26

109
34

36
20

13
8

12
24

Sept.

112

39

25

22

1072

273

95

9

Date
collected(1983)

Numbercollected"

Holding
mortalities

May 20
27

91
81

3
10
17
24

75
53
65
130

1
15
29

June

July

Total

8

35

"Number collected minus holding mortality during the first 10 days.

36

NOAA Technical Report NMFS III

Table 2
Prevalence of Ceratomyxa shasta in chinook salmon smolts purse seined from the Columbia River at Jones Beach,
Oregon.

Holding
mortalities

Mortalities
infected with
C. shasta

Percent offish collected
infected with
C. shasta

9
37

5
6

3
2

33
5

June 10
17
24

58
47
98

17
18
25

0
4
8

0
9
8

I

15
29

128
43
47

46
38
33

21
5

16
12
23

Aug. 12
26

47
21

45
18

4
I

9
5

Sept.

14

12

2

14

549

263

61

11

Date
collected(1983)

Numbercollected"

May 20
27

July

8

Total

II

"Number collected minus holding mortality during the first 10 days.

Table 3
Prevalence of Ceratomyxa shasta in coho salmon smolts purse seined from the Columbia River atJones Beach, Oregon.

Holding
mortalities

Mortalities
infected with
C. shasta

Percent offish collected
infected with
C. shasta

13
4

4
3

5
8

I

4
2
2

0
1
0
1

0
20
0
8

0
0

0
0

0
0

2

0

0

0

179

26

9

5

Date
collected(1983)

Numbercollected"

May 20
27

82
38

3
10
17
24

23
5

June

July

1
15

Aug. 26
Total

I

12

"Number collected minus holding mortality during the first 10 days.

intestinal wall (Amos 1985) was compared with detection of C. shasta by serological methods (Bartholomew et al. 1989b). The lower portion of the intestinewas excised from 121 adult salmon that had diedprior to spawning. Two smears were made from eachfish. The first smear was examined for spores as awet mount at 400X magnification for a maximum of
5 minutes. The second smear was' air-dried, fixed in
I: 1 acetone:xylene solution, and incubated for 15
minutes with a monoclonal antibody specific forBartholomew et a1: Impact of Ceratomyxa shasta on Salmonid Survival

37

Table 4
Prevalence of Ceratomyxa shasta in steelhead trout smolts purse seined from the Columbia River at Jones Beach,
Oregon.

Holding
mortalities

Mortalities
infected with
C. shasta

Percent offish collected
infected with
C. shasta

11
13

5
4

2
0

18
0

45
4
1

13
2
1

6
0
1

13
0
100

0

0

9

12

Date
collected(1983)

Numbercollected"

May 20
27
3
10
24

June

July
26

75

Total

"Number collected minus holding mortality during the first 10 days.

Table 5
Prevalence of Ceratomyxa shasta in chinook salmon smolts beach seined from the Columbia River at Jones Beach,
Oregon.
Percent offish collected
infected with
C. shasta

Numbercollected"

Holding
mortalities

Mortalities
infected with
C. shasta

5
26
31

88
75
84

76
22
31

6
12
14

7
16
17

Aug. 16
23

82
87

34
74

11
13

13
15

Sept. 20

47

29

9

19

Total

463

266

65

14

Date
collected(1984)
July"Number collected minus holding mortality during the first 10 days.

prespore stages of C. shasta. Specific antibodies weredetected using biotinylated horse anti-mouse IgG
and fluorescein-conjugated avidin D (Vector Laboratories, Burlingame, CA). Methyl green dye (1 % indistilled water) was used as a counterstain. Smears
were examined with a Zeiss standard microscopecontaining an IV epifluorescence condensor at
250X magnification until prespore or spore stagesof C. shasta were detected or for three minutes. All
microscopic examinations were made by the same
individual.

Results_

Infections in Downstream Migrants
Chinook salmon were the largest group collected in
1983; 1072 subyearling smolts were captured inbeach seines and 549 yearling chinook salmon weretaken in purse seines. Smaller numbers of coho
salmon (179) and steelhead trout (75) were also captured in purse seines. Ceratomyxa shasta was present in

38

NOAA Technical Report NMFS 111

Table 6

Effects of salt water on steelhead trout and coho and chinook salmon exposed to the infectious stage of Ceratomyxa
shasta.
Fresh water

Exposure

Salmonid
Alsea steelhead trout

length(days)

No. offish
recovered'No. offish
infected

Percent
infected

No. of fishrecovered'No. offish
infected

Percent
infected

3'
3'

21
24
25
23
18
25

21
24
0
23
18
0

100
100
0
100
100
0

6
37
13
9
16

3
37
0
7
8
0

50
100
0
54
89
0

25
25

I

0

4
0

25
25

0
0

0
0

27
25

0
0

0
0

30
27

0
0

0
0

control d5'5'control d
Big Creek coho salmon

5'
control dRound Butte
chinook salmon

Salt water3'
control d

11

a Number of fish exposed minus number of fish that died before spores were detected.
, Fifty fish exposed; 25 were transferred to fresh water and 25 to salt water.
'One hundred fish exposed; 50 were transferred to fresh water and 50 to salt water.
d Con trol fish were not exposed to the infectious stage of C. shasta.

1-24% of the individuals of the chinook salmon
groups caught by beach seine (Table 1). The prevalence of infection was between 1-3% in groups
collected from May· through June but increased to
12-24% from July to the final sample period in September. The prevalence of infection among yearling,
purse-seined chinook salmon also showed a tendency
to increase during the later collection periods (Table
2). Among the yearling chinook salmon, the prevalence of infection in groups collected from May
through June was generally less than 10%, except fora 20 May collection where three of nine fish developed ceratomyxosis. Infection incidence after July
averaged 15%. The total incidence of C. shasta infection among chinook salmon was 9% for beach-seinedgroups and 11 % for purse-seined groups. Smaller
numbers of coho salmon and steelhead trout werecollected in May and early June, during the peak of
their migration. The prevalence of infection for these
species was 5 and 12%, respectively (Tables 3 and 4).
In 1984, 463 chinook salmon smolts were caught inbeach seines between 5 July and 20 September. Infection incidence among these subyearling salmon
averaged 14% (Table 5).

Effects of Salt Water
All Alsea steel head trout held in fresh water after exposure to C. shasta died from ceratomyxosis (Table6). In groups of Alsea trout transferred to salt water,
between 26 and 76% of the fish died prior to development of the disease; losses of 36 to 56% of
unexposed, control fish held in salt water indicatedthat these deaths were caused by inability of the fish
to adjust to saltwater conditions. The prevalence of
infection among fish surviving the prepatent losses insalt water was lower in two of the four groups thanamong fish transferred to fresh water. Big Creek
coho and Round Butte chinook salmon were resistant to infection when held in either fresh or salt
water after exposure to C. shasta.

Sensitivity of Detection Methods
Detection of C. shasta infections by standard wet
mount examination of intestinal tract scrapings wasless sensitive than detection by indirect fluorescentantibody techniques (IFATs) in which a monoclonal

Bartholomew et al: Impact of Ceratomyxa shasta on Salmonid Survival

39

Table 7Comparison of detection sensitivity between wet mount examination for spores and indirect fluorescent antibody
techniques (IFAT) using monoclonal antibodies against Ceratomyxa shasta prespore stages.
IFAT

Wet mount
No. samples

No. positive% positive

No. positive% positive

Spring chinook salmon
Willamette Hatchery
Clackamas Hatchery

20
27

8
16

40
59

19
25

95
93

Fall chinook salmon
Bonneville Hatchery

40

7

17

14

35

Coho salmon
Big Creek HatcheryCascade Hatchery

14
20

14
17

100
85

14
20

100
100

Total

121

62

51

92

76

Sample origin"" Adult prespawning mortalities.

antibody was used. Of 121 intestinal samples examined where both methods were used, 51 % of thesamples were diagnosed as positive for the presenceof spores by the wet mount technique and 76% werediagnosed positive for the presence of prespore andspore stages using IFATs (Table 7).

Discussion_

Evaluating the impact of ceratomyxosis on salmonid
populations requires consideration of a variety of factors. Two of these, geographic distribution of theparasite and resistance to infection of resident salmonids, have been examined in previous studies (Zinn
et al. 1977; Johnson et al. 1979; Buchanan et al. 1983;
Hoffmaster et al. 1988). Past studies have demonstrated the presence of the infectious stage of C.
shasta in the mainstem of the Columbia and Snake
Rivers and have shown that parasite presence andconcentration vary between locations. There is also
evidence that its distribution has increased within thebasin. Because all salmonids migrating in the Columbia River Basin are exposed to C. shasta, native strains
have developed some resistance to infection. However, resistance is not complete in the fish
populations studied and little is known about the susceptibility of feral and upper Columbia and Snake
River salmonids to infection by C. shasta.
The prevalence of infection among outmigratingfish was higher than the incidence predicted fromsusceptibility studies. In studies by Zinn et al. (1977)
and Buchanan et al. (1983), the infection incidence

among Columbia River strains of chinook salmon
and steelhead trout was generally less than 5%, evenwhen fish were exposed to the infectious stage for120 days. However, of 2084 chinook salmon smolts
collected at Jones Beach during 1983-84, 221 fish or
10.6% died from ceratomyxosis. The number of fish
infected did not vary significantly between year
classes, as the prevalence in yearling purse-seinedchinook salmon paralleled that of the subyearlingchinook salmon collected in beach seines. The prevalence also did not vary significantly between the 2years of this study, although the number and type ofsamples collected were limited during 1984, butthere was a trend toward increasing numbers of infected fish from the later collection dates.
The dates of peak migration at Jones Beach were
determined by Dawley et al. (1984b) for each species.
Coho salmon and yearling chinook salmon and steelhead trout migrated through the lower ColumbiaRiver during May and early June. Migration ofsubyearling chinook salmon past Jones Beach began
to increase during May but reached its peak fromearly June to midjuly. This means that the majorityof subyearling chinook salmon were migrating during periods of increasing water temperature when
the impact from C. shasta is greater (Udey et al.
1975). Dawley et al. (1984b) also determined that
migration rates were 22, 18, 17, and 35 km/day forsubyearling chinook, yearling chinook and coho
salmon, and steelhead trout, respectively. Therefore,
the duration of exposure to C. shasta for subyearlingand yearling salmon and steelhead trout migratingfrom Oxbow Dam on the Snake River, a distance of

40

NOAA Technical Report NMFS III

over 1000 river miles from the Columbia Estuary,
could be as long as 43, 53, and 27 days, respectively.
Coho salmon originate from the lower ColumbiaRiver and therefore have a shorter time of exposure
to the parasite. Long periods of exposure to C. shasta
combined with the stress of migration andsmoltification may explain why the numbers ofchinook salmon and steelhead trout infected in ourstudy are higher than numbers predicted from susceptibility studies. Also, it was not possible in thisstudy to determine the strain origin of the infectedfish so their resistance status cannot be assumed.
Studies by Ratliff (1981) and Ching and Munday(1984) demonstrate that fish originating from rivers
enzootic for the parasite can still be highly susceptible to infection. Ching and Munday exposedchinook salmon representing six stocks from theFraser River, British Columbia, to the infectious sta.ge
of C. shasta in the lower Fraser River. Between 87 and100% of the fish in each group died fromceratomyxosis. Ratliff (1981) further demonstrated
that prevalence of infection may increase with increasing length of exposure to C. shasta and hecalculated that 50-70% of all chinook salmon released into the Deschutes River may become
infected. Prior to changes in the Columbia River Basin that resulted from the building of dams, upriversalmonids may also have avoided ceratomyxosis by
migrating through infectious areas before parasite
concentrations were high. However, the presence ofdams and reservoirs has impeded outmigration,raised water temperatures (Raymond 1979) and may
have created conditions favorable for proliferation of
C. shasta (Ratliff 1981). These changes may havecaused the range and numbers of the parasite to increase more rapidly in the upper portions of thebasin than fish could adapt by developing resistanceor avoidance strategies. Therefore, strains of salmonids from the upper Columbia and Snake Rivers may
have lower or more variable levels of resistance than
the strains that have been examined.
Because ceratomyxosis has a long incubation period, most salmonids that become infected during
their downstream migration enter the ocean before
the disease results in death. Acute ceratomyxosis has
been reported in juvenile chum salmon (0. keta) captured off the coast of British Columbia (Margolis andEvelyn 1975). This finding indicates that the disease
is not attenuated when fish enter salt water. To demonstrate this under laboratory conditions, Ching andMunday (1984) exposed chinook salmon to the infectious stage of C. shasta for 10 days, then held the fishin either fresh or salt water. They found that mortal-

ity was 100% in both groups. Similar experiments
were conducted in this study using shorter exposure
lengths and both susceptible and resistant strains ofsalmonids. Although survival was poor, in C. shastasusceptible steelhead trout transferred to salt water
after exposure, two of four groups had a lower incidence of infection than groups transferred to fresh
water. It appears that migration to salt water may reduce the progress of ceratomyxosis if the fish are notoverwhelmed by a large infectious dose. Entry into
salt water did not impair the ability of resistant fish toblock the infection.
While all of the studies described here show the
impact of ceratomyxosis on captured fish held underlaboratory conditions, many field studies rely on sampling fish prior to overt signs of ceratomyxosis.
Reports by Yasutake et al. (1986) and Bartholomew etal. (1989a) indicate that spores do not form until latein the infection and that, in cases of acuteceratomyxosis, fish may die before spore development. Therefore, diagnosis made on the basis ofidentifying spores is likely to underestimate theprevalence of the parasite. To demonstrate this, an
IFAT utilizing monoclonal antibodies specific forprespore stages of C. shasta was compared with examination by standard wet mount procedures. Whenboth methods were used to examine samples collected from prespawning adult salmon mortalities,
there was a 25% increase in the number of infections
detected by using the serological method. An evengreater increase in sensitivity could be expected
when techniques are developed which can detect theearlier stages of C. shasta infection. Monoclonal antibodies and DNA probes that specifically recognize alllife stages of the parasite are necessary for an accurate evaluation of the numbers of fish infected with
C. shasta.

Acknowledgmen~__

The authors acknowledge the technical help of J.E.
Sanders, C.A. Arakawa, W. Moynihan, D.E. Ratliff,and E.M. Dawley. This publication is the result of research sponsored by the Bonneville Power
Administration under contract No. DE-AI79-83 BP
11987; G.R. Bouck, Contracting Officer's TechnicalRepresentative; and, in part, by Oregon Sea Grantwith funds from the National Oceanic and Atmospheric Administration, Office of Sea Grant,
Department of Commerce under grant No. NA89AAD-SGI08. This is Oregon Agricultural Experiment
Station Technical Paper No. 9491.

Bartholomew et al: Impact of Ceratmnyxa shasta on Salmonid Survival

Citations_

Amos, K.H., (ed.).
1985. Procedures for the detection and identification of certain fish pathogens, 3rd ed. Am. Fish. Soc. Fish Health
Section, 114 poo
Bartholomew,J.L., C.E. Smith,J.S. Rohovec, andJ.L. Fryer.
1989a. Characterization of a host response to the
myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy and immunological
techniques. J. Fish Dis. 12:509-522.
Bartholomew,J.L.,J.S. Rohovec, andJ.L. Fryer.
1989b. Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean,
Ceratomyxa shasta. J. Protozoo!. 36:397-401.
Buchanan, D.V.,J.E. Sanders,J.L. Zinn, andJ.L. Fryer.
1983. Relative susceptibility of four strains of summer steelhead to infection by Ceratomyxa shasta. Trans. Am. Fish.
Soc. 112:541-543.
Ching, H.L., and D.R. Munday.
1984. Susceptibility of six Fraser chinook salmon stocks to
Ceratomyxa shasta and the effects of salinity on
ceratomyxosis. Can.J. Zoo!' 62:1081-1083.
Dawley, E.M., R.D. Ledgerwood, TH. Blahm, R.A. Kim, A.E.
Rankis, and FJ. Ossiander.
1984a. Migrational characteristics and survival of juvenile
salmonids entering the Columbia River estuary during
1982. Annual report of research by the Coastal Zone andEstuarine Studies Division, NMFS,January 1984, 49 p.
Dawley, E.M., R.D. Ledgerwood, TH. Blahm, R.A. Kim, and A.E.
Rankis.
1984b. Migrational characteristics and survival of juvenile
salmonids entering the Columbia River estuary during
1983. Annual report of research by the Coastal Zone andEstuarine Studies Division, NMFS,July 1984,88 p.

41

Hoffmaster, J.L., J.E. Sanders, J.S. Rohovec, J.L. Fryer, and D.G.
Stevens.
1988. Geographic distribution of the myxosporean parasite,
Ceratomyxa shasta Noble, 1950, in the Columbia River basin,
U.S.A. J. Fish Dis. 11:97-100.
Johnson, K.A.,J.E. Sanders, andJ.L. Fryer.
1979. Ceratomyxa shasta in salmonids. U.S. Fish Wildl. Serv.,
Fish Dis. Leafl. 58, 11 p.
Margolis, L., and TP.T. Evelyn.
1975. Ceratomyxa shasta (Myxosporida) disease in chumsalmon (Oncorhynchus ketal in British Columbia. J. Fish.
Res. Board Can. 32:1640-1643.
Ratliff, D.E.
1981. Ceratomyxa shasta: epizootiology in chinook salmon ofcentral Oregon. Trans. Am. Fish. Soc. 110:507-513.
Raymond, H.L.
1979. Effects of dams and impoundments on migrations ofjuvenile chinook salmon and steelhead from the Snake
River, 1966 to 1975. Trans. Am. Fish. Soc. 108:505-529.
Udey, L.R.,J.L. Fryer, and K.S. Pilcher.
1975. Relation of water temperature to ceratomyxosis inrainbow trout (Salmo gairdneri) and coho salmon(Oncorhynchus kisutch). J. Fish. Res. Board Can. 32: 15451551.
Yasutake, W.T,J.D. Mclntyre, and A.R. Hemmingsen.
1986. Parasite burdens in experimental families of coho
salmon. Trans. Am. Fish. Soc. 115:636-640.
Zinn,J.L., K.A.Johnson,J.E. Sanders, andJ.L. Fryer.
'
1977. Susceptibilty of salmonid species and hatchery strainsof chinook salmon (Oncorhynchus tshawytscha) to infections
by Ceratomyxa shasta. J. Fish. Res. Board Can. 34:933-936.

Viral Infections of Cultured Fish in Japan
MAMORU YOSHIMIZU and TAKAHISA KIMURA
Laboratory of Microbiology
Faculty ofFisheries
Hokkaido University, Minato 3-1-1
Hakodate, Hokkaido 041,Japan

ABSTRACT
Since infectious pancreatic necrosis virus and infectious hematopoietic necrosis virus
were first isolated in the 1970s, more than 20 fish viruses have been isolated and at least 5
viruses have been observed by electron microscopy. Viral diseases are major problems
and cause economic losses among cultured fishes in Japan and other countries. This
paper reports our current understanding of the extent of viral infection in the cultured
fishes ofJapan.

Introduction
A virological study of cultured fishes in Japan was
initiated when an unknown disease occurred among
rainbow trout Oncorhynchus mykiss in the 1960s. The
causative agent was identified as infectious pancreatic
necrosis virus (IPNV) (Sano 1971). Subsequently, infectious hematopoietic necrosis virus (IHNV) was
isolated from kokanee salmon O. nerka (Kimura andAwakura 1977). Since then, various viral infectionsof fish have been reported. At present, more than 20
fish viruses have been isolated and at least 5 viruseshave been observed by electron microscopy studies(Table 1).

Viral Diseases of Salmonid Fishes_

Infectious Pancreatic Necrosis
Infectious pancreatic necrosis is an acute systemic
disease affecting the fry and fingerlings of rainbow
trout. Its occurrence is widespread in Japan. Susceptibility of fish to IPNV depends on body weight;
smaller fry are more susceptible. The signs of thisdisease are darkening of body coloring, moderate
exophthalmia, and abdominal distention. Internally,
the spleen, heart, liver, and kidneys are pale and thedigestive tract is almost always devoid of food. Rainbow trout is the fish most affected by IPNV, but thevirus has also been isolated from amago (0. rhodurus)

and masu salmon (0. masou). Recently, the fry of
rainbow trout were observed to be less susceptible to
IPNV and, consequently, damage attributed to IPN
has decreased (Okamoto et al. 1987).

Infectious Hematopoietic Necrosis
Infectious hematopoietic necrosis is an acute systemic disease which mainly affects the fry of rainbow
trout and masu and kokanee salmon but which has
also been isolated from moribund ayu Plecoglossus
altivelis (Yoshimizu et al. 1987c). The characteristic
sign of IHNV infection is V-shaped hemorrhages
located in muscle tissue. Recently, a large rainbow
trout, with a body weight of 50-80 g was found to be
infected with IHNV and subsequently died (Mori etal. 1987). In this case, petechiae were observed in thefatty tissues and on the wall of the body cavity. Thisvirus is widespread and especially prevalent in thecentral part of Honshu, the Japanese mainland (Sanoet al. 1977). In several districts, river waters havebeen contaminated with IHNV and are now unsuitable for rainbow trout culture. Although the verticaltransmission of IHNV is doubtful (Yoshimizu et al.
1988, a and b), it can be controlled by disinfecting
eggs with iodine during the early eyed stage. Fish at
the fry stage are very. susceptible to IHNV. They
should be reared in either well water or ultra-violet
irradiated river water. When fish are past this sensitive stage, they can be transferred to the usual
rearing ponds.
43

44

NOAA Technical Report NMFS III

Table I

Viral infection in cultured fishes in Japan.
Isolated virus
DNA virus
Nerkavirus in Towada Lake,
Aomori and Akita
Prefecture (NeVTA)
Oncorhynchus masvu virus (OMY)
Yamame tumor virus (YfV)
Icosahedral cytoplasmic
deoxyribovirus (ICDV)Herpesvirus cyprini
Herpesvirus
Unidentified small virus
RNA virus
Infectious pancreatic necrosis
virus (IPNV)Infectious hematopoietic
necrosis virus (IHNV)Chum salmon virus (CSV)
Yellowtail ascitic virus (YAV)
Rhabdovirus olivaceus (HRV)
Eel virus from European eel (EVE)
Eel virus of America (EVA)
Eel virus of Europe X (EVEX)
Papovavirus
Birnavirus
Birnavirus
Birnavirus
Coronavirus cypnni virus (CACV)
Picornavirus
Reovirus
Reovirus
Observed by electron microscopy
Viral erythrocytic necrosis virus
Lymphocystis virus
Paramyxovirus
Herpesvirus
Picornavirus

Host

Kokanee salmon
Masu salmon
Yamame (masu salmon)
Japanese eel
Fancy carp
Japanese eel
Tiger puffer

Salmonid fish
Salmonid fish
Masu salmon
Yellowtail
Various marine fish
European eel
American eel
European eel
Japanese eel
Yellowtail
Japanese flounderRed sea breamCommun carp
Japanese eel
Common carp
Japanese eel
Various marine fish
Various marine fish
Black rockfish
Japanese flounder
Ishidai

Herpesvirus Infection
A herpesvirus, nerkavirus in Towada Lake Akita and
Aomori Prefecture (NeVfA), was first isolated from
diseased kokanee salmon in Towada Lake (Sano1976). In 1978, another herpesvirus was isolated
from the ovarian fluid of apparently normal maturemasu salmon (Kimura et al. 1980). This virus wasnamed Oncorhynchus masou virus (OMY) from the scientific name of the host fish. Oncorhynchus masou
virus was found to be pathogenic and significantly
more oncogenic in young masu salmon and several
other salmonid fish (Kimura et al. 1981, a and b;
Yoshimizu et al. 1987a). In 1983, a similar herpesvirus were isolated from tumor tissue. of yamame(landlocked O. masou) and was named yamame tumor virus (Sano et al. 1983). Subsequent study

showed that OMV is enzootic in the northern parcof
Japan (Yoshimizu et al. 1988b) and that the characteristics of these three herpesviruses are similar
except that NeVfA lacks oncogenicity (Hedrick et al.
1987; Sano et al. 1988). In 1983, we recommended
the disinfection of fish eggs with iodine at the early
eyed stage in Hokkaido. Now OMY cannot be detected in most of the hatcheries in this area
(Yoshimizu et al. 1988b). Although the host speciesof this virus is primarily masu salmon, OMY has also
been isolated from the tumor tissues of pen-cultured
coho salmon, O. kisutch.

Chum Salmon Virus Infection
In 1978, a reovirus was isolated from an apparently
normal adult chum salmon, O. keta returning to its
hatchery in Hokkaido (Winton et al. 1981). After initial isolation and characterization, it was named
chum salmon virus (CSV). This virus was not observed again until 1986, during an episode of massmortalities of masu salmon fry for which it was responsible. Since then, the virus has been detected instocks of adult masu salmon at new locations in
Hokkaido (Yoshimizu 1988). Artificial infection studies of this virus showed no significant mortality inseveral species of salmonid fishes (Winton et al.
1989) .

Viral Erythrocytic Necrosis
Inclusion bodies stained with Giemsa were observedin the erythrocytes of chum and pink salmon, O.
gorbusrha, collected in Okhotsuku and along thenorth Pacific coast of Hokkaido. The causative agentof viral erythrocytic necrosis (VEN), an iridovirus,
was subsequently observed by electron microscopy(\oshimizu et al. 1988b).

Viral Infections of Eels_

Many viruses have been isolated from cultured eels
(Anguilla anguilla, A. japonicus, and A. rostrata) by
Sano (1976) and Sano and Fukuda (1987). They include a birnavirus, eel virus from the European eel(EVE); the rhabdoviruses, eel virus of America (EVA)
and eel virus of Europe X (EVEX); papovavirus; herpesvirus; picornavirus; and a reovirus. These viruses
are not recognized as pathogenic against eel except
for EVE (Nishimura et al. 1981). Sorimachi (1982,
1984) reported having isolated icosahedral cytoplasmic deoxyribovirus (ICDV) from a diseased eel. This

Muroga and Yoshimizu: Viral Infections of Cultured Fish inJapan

45

virus was shown to be pathogenic against Japaneseeels following artificial infection. Mortality was 4075% at water temperatures of 14.5-18.5° C, 15% at
22.8° C, and 0% at 24.1 ° C. Infected fish showedsigns of decoloration; congestion of the anal, pectoral, and dorsal fins; and an increase of mucus on the
body surface.

Sebastes inermis, and also for salmonid species, especially rainbow trout and masu salmon (Yoshimizu etal. J 987b). Signs of HRV infection are gonadal congestion, focal hemorrhage of skeletal muscle andfins, and accumulation of ascitic fluid. Hirame rhahdovirus is distributed widely from Hokkaido to
Honshu in Japan.

Viral Infections of Carp

Kuchishiroshou of Tiger Puffer_

Herpesvirus cyprini was isolated from papilloma tissueof cultured fancy carp (Cyprinus carpio, also called
common or asagi carp) and confirmed as the agentof infection by induction of epithelial tumors by artificial infection (Sano et al. 1985). Coronavirus cyprini,
carp coronavirus (CACV), was isolated from diseased
common carp raised in the laboratory. Fish infectedwith CACV showed acute mortality showing no external signs except erythematous skin on the abdomen.
Experimentally, CACV was virulent for carp fry at
20° C. Cumulative mortality for 3-week-old fry was
72.5%. The affected fish manifested swollen andhemorrhagic abdomens filled with ascites and eventually died. Reovirus was also isolated from common
carp (Sano and Fukuda 1987).

Viral Infection of Other Marine Fishes_

Viral Pancreatic-Hepatic Necrosis of Yellowtail
A yellowtail ascites virus (a birnavirus) was isolated
from the fry of yellowtail Seriola quinqueradiata(Sorimachi and Hara 1985). This epizootic is anacute viral infection of both naturally grown andhatchery-raised fry. The epizootic period occurs from
May to June at water temperatures of 18 to 22° C.
The moribund fry typically show anemic gills, hemorrhaging in the liver, and ascites and petechiae in thepyloric caea. The disease name, viral pancreatic-hepatic necrosis, was proposed by Egusa and Sorimachi(1986).

Rhabdovirus Infection of Japanese FlounderThe rhabdovirus, Rhabdovirus olivaceus-also referred
to as hirame rhabdovirus (HRV)-was isolated from
diseased hirame Uapanese flounder), Paralichthys
olivaceus, and black sea bream, Milio macrocephalus(Gorie et al. 1985; Kimura et al. 1986). This virus ispathogenic for marine fish such as hirame, black sea
bream, red sea bream Pagrus major and black rockfish

From cultured tiger puffer, FUfJ'h rubripes, an unidentified, small virus was isolated (Inoue et al. 1986).
The epizootic period is from May to June during water temperatures of 18-22° C. Moribund fish had
necrosis around the mouth and had been observed
to be fighting with each other. From the signs of thisinfection, the disease was named "Kuchishiroshou",from the Japanese words "kuchi", meaning mouth,"shiro", meaning white, and "shou", meaning disease.
Viral particles were observed in the brain by electron
microscopy. Kuchichiroshou occurs in southwest Japan where tiger puffer are cultured.

Epidermal Hyperplasia of Japanese Flounder.
Outbreaks of a disease resulting in mass mortalitiesof larval and juvenile Japanese flounder was reported
by Iida et al. (1989). Once the disease occurs in apond, the resident fish populations usually become
extinct within one month. Mfected fish are characterized by opaque fins. Histopathologically, hyperplasia
is observed in the epidermal layer of the fins andskin. In the epidermal tissues of infected fish, hexagonal virus particles were observed by electron
microscopy. Japanese flounder larvae experimentally
exposed to the filtrate of infected tissue homogenate
suffered 18-50% mortalities with 93-100% of the survivors exhibiting epidermal hyperplasia. This virus
has not been isolated in any of the 33 fish cell lines inwhich culture has been attempted, including that of
the host species.
Birnaviruses were also isolated from Japaneseflounder and red sea bream (Yoshimizu and Kimura,
unpub!. data). These viruses were neutralized with
antibody against IPNV; the pathogenicities of thesebirnaviruses have not been clarified.

Lymphocystis Disease
In several species of marine fishes, suzuki, Lateolabrax
japonicus, yellowtail, red sea bream, Japaneseflounder, and others, lymphocystis disease was re-

46

NOAA Technical Report NMFS III

ported and iridovirus was observed by electron microscopy (Matsusato 1975; Miyazaki and Egusa 1972;
Tanaka et al. 1984). Seasonal variation in the prevalence of lymphocystis was noted with increased
prevalence in summer. Lymphocystis cells were observed mainly on the fins or body SUf::iCc. The virus
particles were polyhedral, presenting hexagonal or
pentagonal profiles in tissue sections. They may beseen in a crystalline array and are always located in
the cytoplasm.

Other Diseases
Yoshikoshi and Inoue (1988) reported picornavirus
in moribund fry of ishidai, Oplegnathus fasciatus, andMiyazaki et al. (1989) reported herpesvirus in theepidermal necrosis of Japanese flounder andparamyxovirus in the epithelial necrosis of black sea
bream. To date, these viruses have not been isolated.

Citations_

Egusa, S., and M. Sorimachi.
1986. A histopathological study of yellowtail ascites virus(YAV) infection of fingerling yellowtail, Seriola
quinqueradiata. Fish Patho!' 21:113-122.
Corie, S., K Nakamoto, K Katashima.
1985. Disease of cultured hirame Oapanese flounder,
Paralichthys olivaceus)-1. Preliminary report on a disease ofmarine pen cultured hirame may be caused by viral
infection. Bul!. Hyogo Pref. Fish. Exp. Stn. 23:66-68. (InJapanese.)
Hedrick, R.P., T McDowell, W.D. Eaton, T Kimura, and T Sano.
1987. Serological relationships of five herpesviruses isolated
from salmonid fishes. ]. Appl. lchthyo!. 3:87-92.
Iida, Y., K Masumura, T Nakai, M. Sorimachi, and H. Matsuda.
1989. A viral disease occurred in larvae and juveniles ofJapanese flounder, Paralichthys olivaceus. Aquatic Animal
Health 1: 7-12.
Inoue, K, S. Yasumoto, N. Yasunaga, and I. Takami.
1986. Isolation of a virus from cui tured tiger puffer, Tah~(ugurubripes, infected with "Kuchishiro-sho" and it's pathogenicity. Fish Patho!' 21:129-130.
Kimura, T, and T Awakura.
1977. Current status of disease of cultured salmonids in
Hokkaido, Japan. In Proceedings from the international
symposium on disease of cultured salmonids, sponsored by
Tavolek Inc., Seattle, WA, p. 124-160.
Kimura, T, M. Yoshimizu, and M. Tanaka.
1980. Salmonid viruses: a syncytium-forming herpesvirus
from landlocked Oncorhynchus masou. Fish Health News
9:iii.
1981a. Studies on a new virus (OMV) from Oncorhynchus
masou-1. Characteristics and pathogenicity. Fish Patho!.
15:143-147.
1981b. Studies on a new virus (OMV) from Oncorhynchus
masou-ll. Oncogenic nature. Fish Pathol. 15:149-153.

Kimura, T, M. Yoshimizu, and S. Corie.
1986. A new rhabdovirus isolate in Japan from cultured
hirame Oapanese flounder) Paralichthys olivaceus and ayu
Plecoglossus altivelis. Dis. Aquat. Org. 1:209-217.
Matsusato, T
1975. On the lymphocystis disease in cultured yellowtail. Fish Patho!' 10:90-93. (In Japanese.)
Miyazaki, T., and S. Egusa.
1972. A histopathological observation on lymphocystis disease in sea bass Lateolabrax japonicus (Cuvier andValenciennes). Fish Patho!' 6:83-87. (In Japanese.)
Miyazaki, T, K Fujiwara,]. Kobara, and N. Matumoto.
1989. Histopathology associated with two viral diseases of larval and juvenile fishes: epidermal necrosis of Japaneseflounder Paralichthys olivaceus epithelial necrosis of black sea
bream Acanthopagrus schlegeli. ]. Aquatic Animal Health
1:85-93.
Mori, S., F. Iketani, T Komatsu, and T Nishimura.
1987. IHN of large size of rainbow trout. Proceedings of
the Annual Meeting of Japanese Society of Fish Pathology,
p.9. (In Japanese.)
Nishimura, T, M. Toba, F. Ban, N. Okamoto, and T Sano.
1981. Eel rhabdovirus, EVA, EVEX and their infectivity tofishes. Fish Patho!. 15:173-183. (In Japanese.)
Okamoto, N., T Matsumoto, N. Kato, S. Tazaki, M. Tanaka, N. Ai,
H. Hanada, Y. Suzuki, C. Takamatsu, T Tayama, and T. Sano.
1987. Difference in susceptibility to IPN virus among rainbow trout populations from three hatcheries inJapan. Nippon Suisan Cakkaishi 58:1121-1124. (InJapanese.)
Sano, T
1971. Studies on viral diseases ofJapanese fishes-I. Infectious
pancreatic necrosis of rainbow trout; first isolation from
epizootics in Japan. Bull. Jpn. Soc. Sci. Fish. 37:405-408.
(In Japanese.)
1976. Viral diseases of cultured fishes in Japan. Fish Patho!.
10:221-226.
Sano, T, and H. Fukuda.
1987. Principal microbial diseases of mariculture in
Japan. Aquaculture 67, Vo!. 1:59-69.
Sano, T, H. Fukuda, and M. Furukawa.
1985. Herpesvirus cyprini: Biological and oncogenic
properties. Fish Patho!. 20:381-388.
Sano, T, N. Fukuda, N. Okamoto, and F. Kaneko.
1983. Yamame tumor virus: lethality and oncogenicity. Bull.
Jpn. Soc. Sci. Fish. 49:1159-1163.
Sano, T., H. Fukuda, T Inokari, F. Tichiya, and H. Hosoya.
1988. Comparison of three representative strains of salmonid herpesvirus. Proceedings of the Annual Meeting ofJapanese Society of Fish Pathol, p.21.
Sano, T, T. Nishimura, N. Okamoto, T Yamazaki, H. Hanada, andY. Watanabe.
1977. Studies on viral disease of Japanese fish. IV. Infectious
hematopoietic necrosis (IHN) of salmonids in the mainlandofJapan. ]. Tokyo Univ. Fish. 63:81-85.
Sorimachi, M.
1982. Characteristics and distribution of viruses isolated
from pond-cultured eels. Bul!. Nat!. Inst. Aquaculture
3:97-105. (In Japanese.)
1984. Pathogenicity of ICD virus isolated from Japanese
ee!. Bull. Nat!. Inst. Aquaculture 6:71-75. (In Japanese.)
Sorimachi, M., and T Hara.
1985. Characteristics and pathogenicity of a virus isolated
from yellowtail fingerling showing ascites. Fish Patho\.
19:231-238.

Muroga and Yoshimizu: Viral Infections of Cultured Fish in Japan
Tanaka, M., M. Yoshimizu, M. Kusakari, and T. Kimura.
1984. Lymphocystis disease in kurosoi Sebastes schlegeli andhirame Paralichthys olivaceus in Hokkaido, Japan. Bull. Jpn.
Soc. Sci. Fish. 50:37-42. (In Japanese.)
Winton,]. R., C. N. Lannan,]. L. Fryer, and T. Kimura.
1981. Isolation of a new reovirus from chum salmon in
Japan. Fish Pathol. 15:155-162.
Winton,]. R., C. N. Lannan, M. Yoshimizu, and T. Kimura.
1989. Response o£ salmonid fish to artificial infection withchum salmon virus. In Viruses of lower vertebrates (W.
Ahne and E. Kurstak, eds.), p. 270-278. Springer-Verlag,
Berlin.
Yoshikoshi, K., and K. Inoue.
1988. Viral neuron necrosis of ishidai. Proceedings of theAnnual Meeting of Nippon Suisan Gakkai, p.121.
Yoshimizu, M.
1988. Chum salmon virus isolated from masu salmon,
Oncorhynchus masou. Fish and Eggs, Techn. Rep. Hokkaido
Salmon Hatchery 157:36-38.

47

Yoshimizu, M., M. Tanaka, and T. Kimura.
1987a. Oncorhynchus masou virus (OMV): incidence of tumor
development among experimentally infected representative
salmonid species. Fish Pathol. 22:7-10.
Yoshimizu, M., N. Oseko, T. Nishizawa, and T. Kimura.
1987b. Rhabdovirus disease of Japanese flounder. Fish
Pathol. 22:54-55.
Yoshimizu, M., M. Sami, T. Kimura, and M. Ugazhin.
1987c. Characteristics of the virus isolated from cultured ayu(Plecoglossus altivelis). Proceedings of the Annual MeetingofJapanese Society of Fish Pathology, p. 12.
Yoshimizu, M., M. Sami, and T. Kimura.
1988a. Survival and inactivation of infectious hematopoietic
necrosis virus (IHNV) in fertilized eggs of masu salmon
Oncorhynchus masou and chum salmon O. heta. Nippon
Suisan Gakkaishi 54:2089-2097.
Yoshimizu, M., T. Nomura, T. Awakura, and T. Kimura.
1988b. Incidence of fish pathogenic viruses among anadromous salmonid in northern part of Japan. Sci. Rep.
Hokkaido Salmon Hatchery 42:1-20.

Some Important Infectious Diseases of Kuruma Shrimp,
Penaeus japonicus, in JapanKAZUO MOMOYAMA
Yamaguchi Prefectural Naikai Fisheries Experimental Station
Yamaguchi 754, Japan

ABSTRACTThe kuruma shrimp, Penaeus japonicus, is the most widely cultured crustacean in Japan.
Shrimp farming operations for this species have been greatly affected by several infectious diseases including baculoviral midgut gland necrosis (BMN), vibriosis, and blackgill disease. This paper discusses our knowledge of the BMN virus in detail, and brieflly
reviews the latter two diseases with respect to the culture of crustaceans.

Introduction

---=-

_

It may safely be said that kuruma shrimp, Penaeus
japonicus, is the only crustacean species cultured on anindustrial scale in Japan. About six to seven hundred
million post-larval kuruma shrimp are produced at
public and private hatcheries every year for stocking
and farming (Japan Sea Farming Assoc. 1990). Annual
production of cultured kuruma shrimp reached 3,020
metric tons in 1988 (DSI 1990).
The infectious diseases responsible for high mortalities to cultured kuruma shrimp in Japan are baculoviral
midgut gland necrosis (BMN), vibriosis, and black gilldisease. Baculoviral midgut gland necrosis is a very severe viral disease that occurs during the seedproduction process which usually causes 90% or higher
mortalities in the population of the rearing tank. Recently, the frequency of BMN outbreaks has decreased
greatly, probably because of the preventive measures executed at hatcheries. Vibriosis is a bacterial diseaseaffecting the lymphoid organ that has been responsible
for a great deal of damage to the shrimp farming industry over the past several years. Black gill disease iscaused by Fusarium solani, a ubiquitous pathogen of
shrimp and lobster, invading the gills, appendages, andvarious parts of the exoskeleton.
In the present paper BMN is discussed in detail, andvibriosis and black gill disease are touched briefly.

Baculoviral Midgut Gland Necrosis_

To date, four baculoviruses have been recorded In
penaeid shrimp worldwide (Lightner 1985; Lester etal. 1987; Johnson and Lightner 1988). They exhibit
higher pathogenicity to larval or postlarval shrimp
than to adults. Baculoviral midgut gland necrosis was
first noticed in 1971, and since then it has oftencaused mortalities of over 90% during the mass production of kuruma shrimp larvae in Japan(Momoyama 1981; Sano et al. 1981). Heavy losses
due to baculovirus infection during the larval production of P aztecus (Couch 1978) and P monodon(Ligh tner et al. 1983) have also been reported inother countries.
The results of our histological and epizootiologicalstudies on BMN in kuruma shrimp are summarized
as follows.

Histopathology
Midgut glands of diseased shrimp become soft anddevelop a white turbid appearance at the advanced
stage of infection, whereas those of healthy
postlarvae appear brown or colorless during the early
developmental stages (Momoyama 1981). From histological examinations, it was confirmed that only the
midgut gland and the intestine are affected by thedisease. Disarrangement and exfoliation of epithelial
cells are remarkable in the midgut gland of the diseased shrimp. Nuclear hypertrophy and chromatolysis of infected epithelial cells are the mostcharacteristic cytopathological changes in BMN(Momoyama 1981). In contrast with other penaeid
shrimp baculovirus infections, no inclusion bodies
are present in the hypertrophied nuclei (Lightner
1985; Lester et al. 1987;Johnson and Lightner 1988).
49

50

NOAA Technical Report NMFS 111

Electron microscopy observations of the hypertrophied nuclei and the midgut gland lumen haverevealed many rod-shaped particles having outer andinner envelopes, which represent virions of thebaculovirus group. The average length and diameterof the virions are 310 nm and 72 nm, respectively(SanoetaI.1981).
Baculoviral midgut gland necrosis can be diagnosed by detecting the hypertrophied nuclei ofaffected midgut gland epithelial cells in both freshand stained squash preparations. In stained squas:lpreparations homogeneous hypertrophied nuclei
about 20 to 30 /-Lm in diameter appear among thesmaller normal ones which are about 10 /-Lm in diameter. The Feulgen reaction makes the difference
clearer between hypertrophied and normal nuclei.
The diagnostic technique of using a dark field microscope with a wet-type condenser has the advantagesof precision and rapidity. Hypertrophied nuclei areclearly seen as white bodies of 10 to 30 /-Lm diameterin fresh squash preparations from diseased samples
under the dark field microscope. The reason why theinfected nuclei appear white is thought to result froman increased number of reflected or diffracted rays
due to the numerous virus particles in the nucleus(Momoyama 1983).

Source of Infection
Epizootiological investigations indicate that mature
female kuruma shrimp spawners with latent BMN-virus infections and cultured young kuruma shrimpthat have recovered from infection with the virus are
the main source of infection in hatchery epizootics(Momoyama 1988). Histological examinations reveal
nuclear hypertrophy of the midgut gland epithelial
cells in both spawners and young cultured survivorsof the disease. Fluorescent antibody techniques havebeen used to reveal the presence of BMN-specific virus antigen in the hypertrophied nuclei of spawners(Momoyama 1988).

The Effect of Developmental Stage of the Hoston the Susceptibility to BMN Virus
The susceptibility of kuruma shrimp to BMN virus bywaterborne infection (Momoyama and Sano 1988)
was determined for fertilized eggs, nauplii, zoeae,
mysis larvae, and postlarvae (2, 4, 6, 8, and 10 dayold) (Momoyama and Sano 1989). When the fertilized eggs and nauplii were exposed to the virus, they
showed no evidence of infection. But when thezoeae, mysis larvae, and postlarvae were exposed,some or all of the shrimp were diagnosed to be infected on the final day of the test period (6 to 16days). Susceptibility to infection tended to decrease
with the advance in developmental stages from zoea
to P-10 (lO-day-old postlarva). Cumulative mortalities
decreased from 100% in zoeal stage larvae to 0% in
P-6 larvae. The growth rates of test shrimp inoculated
at stages P-2 through P-6 were lower than those ofcontrols, but there were no differences in mortality
or growth rates between test shrimp and controls inoculated at stages P-8 and P-10. These results indicatethat kuruma shrimp from the zoeal to the P-6 stage
are highly susceptible to BMN virus, but stage P-8 orolder postlarvae become refractory to this disease.
The route of baculovirus infection in shrimp has
generally been considered to be by oral ingestion ofvirus contaminated sediments or by cannibalism of
diseased shrimp (Lightner et al. 1983; Couch 1974).
In the previously mentioned study on waterborne susceptibility, kuruma shrimp were not administered any
food during the inoculation period. However, peristaltic movements were frequently observed in theoesophageal part of the shrimp, suggesting that test
shrimp ingest the seawater containing the virus particles through the mouth. This hypothesis was
supported by an observation that azocarmine G accumulated in the stomach and midgut gland lumen of
shrimp when they were dipped in seawater containing this dye (Momoyama and Sano 1989).

Inactivation and Survival of BMN Virus
The effects of disinfectants, heating, and ultraviolet
irradiation on BMN virus, and the survival time ofthe virus in seawater at different temperatures were
examined by waterborne infectivity experiments using larval and postlarval kuruma shrimp. The virus
was inactivated by 10-minute exposure at 25° C to anyof the following chemicals: 5 ppm chlorine, 25 ppm
iodine, 100 ppm benzalkonium chloride andbenzethonium chloride, 30% ethyl alcohol, and 0.5%
formalin (Momoyama 1989a). The virus was still active after 2 hours of heat treatment at 35 and 40° C,
but was inactivated within 2 hours at 45° C, 30 minutes at 50 and 55° C, and 5 minutes at 60° C(Momoyama 1989b). The virus was also inactivatedwithin 20 minutes by ultraviolet irradiation with a 15watt ultraviolet lamp at a distance of 30 cm(Momoyama 1989b). In seawater, the virus could notsurvive longer than 4 days at 30° C, 7 days at 25° C,
12 days at 20° C, and 20 days at 15° C (Momoyama
1989c).

Momoyama: Infectious Diseases of Penaeusjapunicus

Prevention
The following two preventive measures are now usedagainst this epizootic in some hatcheries. One prevents vertical infection from spawners by rinsing thefertilized eggs with virus-free seawater then transferring them to a disinfected rearing tank. The otherprevents horizontal infection by adding chlorine in
the rearing tank to kill infected populations. Since
1985, the measure to prevent vertical infection has
been carried out on an industrial scale, and BMN has
never occurred in the hatcheries where this treatment has been practiced.

Vibriosis_

Vibriosis has caused a great deal of damage to theshrimp farming industry over the last several years.
Although some dead shrimp infected with vibriosis
develop white turbid muscle at the 6th abdo.minal
segment, shrimp suffering from this disease do not
usually show any specific external clinical signs.
The lymphoid organ (Oka 1969) is intensively invaded by the causative bacterium resulting in
extensive necrosis and nodule formation (Egusa etal. 1988). The nodules are seen by the unassisted eye
as very small black spots and are composed of a bacterial colony in the center, a melanized zone aroundthe bacterial colony, and multiple layers of hemocytes
encapsulating the melanized zone. Small nodules arealso frequently observed in other organs such as thegills, heart, midgut gland, and abdominal musculature, but extensive necrotic lesions can not be foundin these organs.
The Vibrio sp. isolated from kuruma shrimp has
been identified as a new species (Takahashi et al.
1985a), and was tentatively named Vibrio sp. PJ (PJ is
the abbreviation of the scientific name of kuruma
shrimp Penaeus japonicus). Vibrio sp. PJ has very highpathogenicity to kuruma shrimp. LD so values obtained by intramascular injection were about 20 to
100 cells/g body weight of shrimp.
Now, two antibiotics, oxytetracycline (Takahashi etal. 1985b) and oxolinic acid, are on the market withthe government's approval. Although mortalities are
decreased significantly by administering these antibiotics, disease often returns shortly after treatment.

Black Gill Disease_

Black gill disease often occurs in the intensive culture
systems in the Okinawa and Kagoshima districts, butrarely in those of the Chugoku district.

51

Black gill disease is caused by Fusarium solani,
which is a member of the imperfect fungi. This fungus is a ubiquitous pathogen and infects penaeid
shrimp (Cook 1971; Lightner 1975) as well as lobster(Lightner and Fontaine 1975; Alderman 1981) andfreshwater shrimp (Burns et al. 1979). The fungus
usually invades the gills, appendages, and various
parts of the exoskeleton.
In kuruma shrimp, gills are most susceptible to thefungus and infected gills always become black(Ishikawa 1968; Bian and Egusa 1981). In the degenerating gill filaments, hemal channels are foundextremely congested with hemocytes, encapsulated
hyphae, and tissue debris. The fungus also often penetrates into the thoracic central nerve and sometimes
into the ventral thoracic artery (Momoyama 1987).
Tissue destruction, cellular inflammation, andhyphae encapsulated by multiple layers of hemocytes
are always observed in the lesion. Failure to exchange gas in the gills and damage to the central
nerve and ventral thoracic artery are thought to be
responsible for death.
As blackening of the gills in shrimp is often induced by various causes, detection of macro- andmicro-conidiospores is necessary for diagnosis of thisdisease (Egusa and Veda 1972).

Citations
Alderman, OJ.
1981. Fusarium solani causing an exoskeletal pathology incultured lobsters, Homarus vulgaris. Trans. Brit. Mycol.
Soc. 76:25-27.
Bian, B.Z., and S. Egusa.
1981. Histopathology of black gill disease caused by Fusarium
solani (Martius) infection in the kuruma prawn, Penaeus
japonicus Bate. j. Fish. Dis. 4: 195-201.
Burns, C.D., M.E. Berrigan, and G.E. Henderson.
1979. Fusarium sp. infections in the freshwater prawn Macrom-achium rosenbergii (De Mann). Aquaculture 16: 193-198.
Cook, H.L.
1971. Fungi parasitic on shrimp. FAO Aquaculture Bull.
3:13.
Couch,j.A.
1974. An enzootic nuclear polyhedrosis virus of pink shrimp:
ultrastructure, prevalence and enhancement. j. Invertebr.
Pathol. 24:311-331.
1978. Diseases, parasites and toxic responses of commercial
penaeid shrimps of the Gulf of Mexico and South Atlantic
coasts of North America. Fishery Bull, U.S. 76:1-44.
DSI (Department of Statistics Information Division).
1990. Annual statistics on the fishery and culture production
1988. DSI, Ministry of Agriculture, Forestry, and Fisheries,
Tokyo, 296 p. (In Japanese.)
Egusa, S., and T. Ueda.
1972. A Fusarium sp. associated with black gill disease of the
kuruma prawn, Penaeus japonicus Bate. Bull. Jpn. Soc. Sci.
Fish. 38:1253-1260.

52

NOAA Technical Report NMFS III

Egusa, S., Y. Takahashi, T Itami, and K. Momoyama.
1988. Histopathology of vibriosis in the kuruma prawn,
Penaeus japonicus Bate. Fish Pathol. 23:59-65. (In Japanese; English abstr.)
Ishikawa, Y.
1968. Preliminary report on black gill disease of the kuruma
prawn, Penaeus japonicus Bate. Fish Pathol. 3:34-38. (InJapanese.)Japan Sea Farming Association.
1990. Annual seed production and stocking record for s~a
farming 1988, p421. (In Japanese.)
Johnson, P.T, and D.V. Lightner.
1988. Rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Org. 5:123-141.
Lester, R.G., A. Doubrovsky, J.L. Paynter, S.K. Sambhi, andJ.G. Atherton.
1987. Light and electron microscope evidence of baculovirus
infection in the prawn Penaeus plebejus. Dis. Aquat.
Org.3:217-2] 9.
Lightner. D.V.
1975. Some potentially serious disease problems in the culture of penaeid shrimp in North America. In Proceedingsof the third U.S.japan meeting on aquaculture; 15-16 Oct.
1974, Tokyo, Japan, p. 75-97. Special Publ. Fish. Agency
Japanese Government and Japan Sea Reg. Fish. Res. Lab.,
Niigata.
1985. A review of the diseases of cultured penaeid shrimps
and prawns with emphasis on recent discoveries and developments. In Proceedings of the first international conference on the culture of penaeid prawns/shrimps; 4-7 December 1984, lloilo City, Philippines (Y. Taki, J.H.
Primavera, J.A. Llobera, eds.), p. 79-103.
Lightner, D.V., and C.T. Fontaine.
1975. A mycosis of the American lobster, Homarus
americanus, caused by Fusarium sp. J. Invertebr. Pathol.
25:239-245.
Lightner, DV, RJv1. Redman, and TA. Bell.
1983. Observations on the geographic distribution, pathogenesis and morphology of the baculovirus from Penaeus
monodon Fabricius. Aquaculture 32:209-233.
Momoyama, K.
1981. Studies on infectious mid-gut gland necrosis of
kuruma shrimp (Penaeus japonicus)-I. Occurrences andsymptoms. Bull. Yamaguchi Pref. Naikai Fish. Exper. St.
8:1-11. (In Japanese.)

1983. Studies on baculoviral mid-gut gland necrosis of
kuruma shrimp (Penaeus japonicus)-lII
Presumptive diagnostic techniques. Fish. Pathol. 17:263-268. (In Japanese;
English abstr.)
1987. Distributions of the hyphae in kuru rna shrimp, Penaeus
japonicus, infected with Fusarium solani. Fish Pathol.
22:]5-23. (In Japanese; English abstr.)
1988. Infection source of baculoviral mid-gut gland necrosis
in mass production of kuruma shrimp larvae, Penaeus
japonicus. Fish Pathol. 23:105-110. (In Japanese; English
abstr.)
1989a. Virucidal effect of some disinfectan ts on baculoviral
mid-gut gland necrosis (BMN) virus. Fish Pathol. 24:4749. (In Japanese; English abstr.)
1989b. Inactivation of baculoviral mid-gut gland necrosis(BM ) virus by ultraviolet irradiation, sunlight exposure,heating and drying. Fish Pathol. 24:115-118. (In Japanese; English abstr.)]989c. Survival of baculoviral mid-gut gland necrosis virus(BMNV) in infected tissues and in seawater. Fish Pathol.
24:] 79-]81. (In Japanese; English abstr.)~omoyama, K., and T Sano.
1988. A method of experimental infection of kuruma shrimp
larvae, Penaeus japonicus Bate, with baculoviral mid-gut
gland necrosis (BMN) virus. J. Fish Dis. 11:105-111.
1989. Developmental stages of kuruma shrimp, Penaeus
japonicus Bate, susceptible to baculoviral mid-gut gland necrosis (BMN) virus. J. Fish Dis. 12:585-589.
Oka. M.

1969. Studies on Penaeus orientalis Kishinoue-VlII. Structureof the newly found lymphoid organ. Bull. Jpn. Soc. Sci.
Fish. 35:245-250.
Sano, T, T Nishimura, K. Oguma, K. Momoyama, and N. Takeno.
1981. Baculovirus infection of kuruma shrimp, Penaeus
japonicus in Japan. Fish Pathol. 15:]85-]91.
Takahashi, Y., Y. Shimoyama, and K. Momoyama.
] 985a. Pathogenicity and characteristics of Vibrio sp, isolated
from cultured kuruma prawn Penaeus japonicus Bate. Bull.
Jpn. Soc. Sci. Fish. 51:721-730, (In Japanese; English abstr,)
Takahashi, Y., T Itami, A. Nakagawa, H. Nishimura, and T Abe.
] 985b. Therapeutic effects of oxytetracycline trial tablets
against vibriosis in cultured kuruma prawns Penaeus
japonicusBate. Bull. Jpn. Soc. Sci. Fish. 51:1639-1643.

The Applica ion of Molecular Biology to the Detection of Infectious
Hematopoietic Necrosis Virus

JAMES R. WINTON

u.s. Fish and Wildlife Service
National Fisheries Research Center
Building 204, Naval Station
Seattle, Washington 98115

ABSTRACT
Traditionally, the detection of most fish viruses, including infectious hematopoietic
necrosis virus (IHNV), has relied upon isolation of the virus in cell culture and identification by serum neutralization or fluorescent antibody assays using polyclonal rabbit
antisera. In recent years, the techniques of molecular biology have provided new strategies for rapid and sensitive detection of antigens, antibodies, and nucleic acids. This
paper reviews the application of these powerful new methods for the detection of IHNV.
Included, are discussions on the creation of monoclonal antibodies specific for viralproteins, development of enzyme-linked immunoassays for detection of viral antigens
and antibodies, electrophoretic identification of radiolabeled viral proteins, a nonradioactive DNA probe specific for the nucleoprotein (N) gene messenger RNA of IHNV, anda polymerase chain reaction (peR) based method for amplification of genomic or messenger RNA of the virus. As these methods become more widely accepted, they will resultin significant improvements in the speed, precision, and sensitivity of IHNV detection.

Introduction_

Infectious hematopoietic necrosis (IHN) is the mostimportant viral disease of trout and salmon in western North America. Depending upon the species,
stock, and size of the fish, strain of the virus, andenvironmental conditions such as temperature, anoutbreak of IHN may result in losses approaching
100% when fish are infected at a small size. Amonggroups of larger fish, mortality is reduced, often becoming chronic, with a typical loss of 25% or less
(Wolf 1988). Presently, no control measure for IHN isavailable other than avoidance of exposure to thecausative rhabdovirus, infectious hematopoietic necrosis virus (IHNV). This has not proven practical atmany large hatcheries with open water supplies or atcommercial trout operations where production offish occurs on a continuous basis. The chemical,
physical, and serological characteristics of IHNV, essential features of the biology of the virus, and a
description of the host and geographic range, havebeen extensively reviewed (McAllister 1979; Pilcher
and Fryer 1980; Nicholson 1982; Wolf 1988).

Virological examinations are regularly conductedon stocks of trout and salmon. These include routine
health checks, diagnostic examinations to identify
causes of mortality, and pathogen-free certification
examinations, which are required prior to moving
fish from one location to another. Traditional methods for isolation and identification of IHNV rely
upon cell cultures for recovering the virus and serum
neutralization or fluorescent antibody assays to identify the agent, a time-consuming and expensive
process (Amos 1985). Recently, new techniques frommolecular biology utilizing monoclonal antibodies,
enzyme-linked immunosorbent assays, nonradioactive
DNA probes, and the polymerase chain reaction,
promise to provide new tools for detection and identification of IHNV that are more rapid, sensitive,
specific, and cost-effective.

Electrophoresis of Structural Proteins_

Leong et al. (1981) described a method for identification of different strains of IHNV using
53

54

NOAA Technical Report NMFS 111

polyacrylamide gel electrophoresis (PAGE) and autoradiography of the virion structural proteins that had
been radioactively labeled with 35S-methionine. The
method required approximately 48 hours and provided a type of fingerprint that would not only
confirm the presence of IHNV, but yielded data
about the strain of the virus, giving researchers additional epizootiological information. Hsu and Leong(1985) compared this electrophoretic method with
two immunoblotting techniques using either 125I_la_beled Protein A or peroxidase to detect the rabbit
anti-IHNV serum bound to structural proteins separated by PAGE and transferred to nitrocellulose
membranes. When used to detect and confirm thepresence of IHNV, the direct electrophoretic method
was the most sensitive and rapid of the three. Although the need to use radioactive materials has
limited the field application of this method, it has
proven useful in analyzing the structural proteins of
IHl\'V (Hsuet al. 1985) and in identifying strains of
the virus (Hsu et al. 1986). This electropherotypic
analysis (based upon variation in the molecularweights of structural proteins) has become widelyused in various epizootiological studies of IHNV.

Monoclonal Antibodies_

Monoclonal antibodies (MAbs) are useful for bothresearch and diagnostic applications as they arehighly reactive, very consistent, and do not cross-react with other antigens to any significant extent(Harlow and Lane 1988). The first monoclonal antibody against IHNV was developed by Schultz et al.
(1985). While the antibody lacked neutralizing activity, it could be biotinylated and used to develop an
immunoblot assay for IHNV (Schultz et al. 1989). For
maximum sensitivity, the assay required initial amplification of the virus in cell culture where as few as
100 plaque forming units of IHNV could provide a
positive diagnosis of IHNV as early as 36 hours after
infection.
A monoclonal antibody was compared withpolyclonal rabbit serum in a rapid fluorescent antibody
test (FAT) for IHNV by LaPatra et al. (1989). The FAT
used fish cell cultures grown on coverslips and infectedwith IHNV for approximately 48 hours at which time
the test approached the plaque assay in sensitivity andoffered a significant improvement in speed. One advantage of the MAb over the polyclonal serum was that it
did not require extensive adsorption before use.
Arnzen et al. (1991) used a monoclonal antibody thatwas conjugated with fluorescein isothiocyanate to produce a direct FAT that detected IHNV antigens in cell
cultures within 6-8 hours after infection.

Another application of monoclonal antibodies for
detection of IHNV includes an immunohistochemical
staining method developed by Yamamoto et al.
(1989). The method proved useful for in situ detection of fish cells initially infected with IHNV
following waterborne exposure to the virus(Yamamoto et al. 1990). Monoclonal antibodies havealso been use to detect neutralization variants among
strains of IHNV (Winton et al. 1988) and in determining important epizootiological information about
the distribution of IHNV strains (Ristow and Arnzen
1989) .

Enzyme-Linked Immunosorbent Assay_

The enzyme-linked immunosorbent assay (ELISA) is
an important tool for the rapid and sensitive detection of antigens. An ELISA was used to detect IHNV
antigens present in infected cell cultures and fish tissues (Dixon and Hill 1984; Way and Dixon 1988).
Because the assays used polyclonal antisera, low levelcross-reactions were observed with other fish rhabdoviruses and with uninfected fish cell cultures orfish tissue extracts. While differences between antisera affected the results, the IHNV ELISA appeared
to be quite specific and could detect infections in cell
culture as early as 48 hours after infection at the tiinethat cytopathic effect was first noted. The assay was
also used to detect IHNV in acutely infected fry.
A modification of the ELISA, the dot blot, where
IHNV antigens were spotted onto nitrocellulose paper and detected by labeled antisera, was reported by
McAllister and Schill (1986). This assay was rapid andrequired no special instrumentation, but thepolyclonal IHNV antiserum required extensive adsorption with both uninfected cells and fetal bovine
serum to remove cross-reacting antibodies before
use. While the assay was not suitable for use with tissues or fluids because the filter matrix became
clogged, it was able to detect IHNV in cell culture
supernatant fluids at concentrations of 105 to 10 6
plaque forming units (PFU) /mL when initial cytopathic effect was observed.
Although fish lack the complex immune system of
the higher vertebrates, it is possible to confirm past
infection with IHNV using methods that detect thepresence of antibodies in serum. Amend and Smith(1974) reported that rainbow trout Oncorhynchus
mykiss developed good titers of neutralizing antibodies that could be detected at least 90 days after
immunization with live virus. Techniques that havebeen used to assay for antibodies to IHNV in fishserum include neutralization, FAT, and ELISA(Amend and Smith 1974; Hattenberger-Baudouy etal. 1989; Jorgensen et al. 1991). Of these, the ELISA

Winton: Molecular Biological Methods for Detection of IHNV

appeared to have the best combination of sensitivity
and ease of processing the numerous samples needed
for serological screening of fish populations.

Nonradioactive DNA Probe_

A biotinylated DNA probe for rapid detection of
IHNV was developed by Deering et al. (1991). Thisprobe was designed to detect the nucleoprotein (N)gene messenger RNA (mRNA) of IHNV because thismolecule is synthesized early and in high abundance
during viral replication. A 30 nucleotide target site
for the probe was chosen by computer search of the
published sequence of the N gene of IHNV (Gilmore
and Leong 1988). A synthetic oligon ucleotide,complementary to this sequence, was made by automated chemical synthesis, coupled with biotin, anddetected with a streptavidin-peroxidase conjugate.
The probe was shown to be specific for IHNV mRNAand detected as little as one picogram of target sequence. It did not react with mRNA of· viralhemorrhagic septicemia virus or hirame rhabdovirus,
but it did recognize strains of IHNV representing
each of the electropherotypes described by Hsu et al.
(1986). The method required initial amplification of
the virus mRNA in fish cell cultures, but at high multiplicities of infection, detectable levels of mRNA
could be extracted from these cells after less than 24hours yielding a positive diagnosis in less than 48
hours.

Polymerase Chain Reaction_

The polymerase chain reaction (PCR) uses two DNA
primers and repeated cycles of DNA synthesis performed by a thermostable polymerase to amplify low
copy numbers of a specific nucleic acid sequence tolevels that can be easily detected (Erlich 1989; Innis
et al. 1990). Arakawa et al. (1990) used PCR to amplify a 252 nucleotide portion of the N gene of IHNV
that included the site for the DNA probe used by
Deering et al. (1991). The primers directed the synthesis of large amounts of DNA from all strains of
IHNV tested. The DNA produced was confirmed to
be of the appropriate sequence by Southern and dot
blot assays. Sufficient messenger RNA for cDNA synthesis using reverse transcriptase and subsequent
PCR amplification could be extracted from cell cultures infected for as little as 24 hours. The PCR wasable to amplify target sequences from IH~'N-infected
rainbow trout to levels that were easily detected witha biotinylated probe. Recent tests using clinical material from IHNV-infected fish revealed that themethod approached cell culture in sensitivity and was55

able to detect IHNV infections in rainbow trout for
up to 32 weeks post-infection.

Discussion_

Control of IHNV continues to rest upon avoidance ofthe virus through use of virus-free fish reared in virus-free water supplies (Wolf 1988). Currently,
assurance of the virus-free status of salmonid fish requires a series of time-consuming, expensive, andlabor-intensive procedures (Amos 1985). Many of themethods reviewed in this paper will be important improvements in the speed, sensitivity, and precision ofthese assays. These novel assays and procedures willfind increasing use in examination of fish and fish
eggs to be moved to new locations and in providing
early diagnosis of IHNV outbreaks allowing prompt
management actions.
One area of concern has been the ability to transfer these new methods to field stations where muchof the fish diagnostic work is being performed. In
this regard, the use of radioactive material, expensive
equipment, or technically demanding procedures
have not proven attractive. While it seems likely that
improvements in speed and sensitivity of diagnostic
methods are still possible, they must be developedwith these limitations in mind.
In the future, additional methods will become
available for control of IHNV. These include the useof antiviral drugs (Hasobe and Saneyoshi 1985;
Kimura et al. 1990), chemicals (Batts et al. 1991), or
modern vaccines based upon recombinant DNA technology (Leong et al. 1988). The combination ofnewer detection methods and improved control strategies promises to provide substantial reduction in thelosses caused by this important virus.

Acknowledgment__

Some of the research appearing in this review was
supported by the Cooperative State Research Service,
U.S. Department of Agriculture to the Western Regional Aquaculture Consortium under Agreements
87-CSRS-2-3219, 88-38500-4027, 89-38500-4287, 9038500-5025, or 91-38500-6078.

Citations_

Amend. D.F., and L.S. Smith.
1974. Pathophysiology of infectious hematopoietic necrosis
virus disease in rainbow trout (Salma gairdneri): earlychanges in blood and aspects of the immune response after
injection of IHN virus. J. Fish. Res. Board Can. 21:13711378.

56

NOAA Technical Report NMFS 111

Amos, KH.
1985. Procedures for the detection and identification of certain fish pathogens, 3rd ed. Fish Health Section, American
Fisheries Society, Corvallis, Oregon, 114 p.
Arakawa, C.K., R.E. Deering, KH. Higman, KH. Oshima, P.J.
O'Hara, andJ.R. Winton.
1990. Polymerase chain reaction (PCR) amplification of a
nucleoprotein gene sequence of infectious hematopoietic
necrosis virus. Dis. Aquat. Org. 8: 165-170.
Arnzen,J.M., S.S. Ristow, C.P. Hesson, andJ. Lientz.
1991. Rapid fluorescent antibody tests for infectious hematopoietic necrosis virus (IHNV) utilizing monoclonal antibodies to the nucleoprotein and glycoprotein. J. Aquat. Anim.
Health 3:109-113.
Batts, WN, M.L. Landolt, andJ.R. Winton.
1991. Inactivation of infectious hematopoietic necrosis vir.rs
by low levels of iodine. Appl. Environ. Microbiol. 57:13791385.
Deering, R.E, C.K Arakawa, KH. Oshima, P.J. O'Hara, M.L.
Landolt, andJ.R. Winton.
1991. Development of a biotinylated DNA probe for dettction and identification of infectious hematopoietic necrosis
virus. Dis. Aquat. Org. 11:57-65.
Dixon, P.F., and B.J. Hill.
1984. Rapid detection of fish rhabdoviruses by the enzymelinked immunosorbent assay (ELISA). Aquaculture. 42:
1-12.
Erlich, H.A.(ed.).
1989. PCR technology. Stockton Press, NY.
Gilmore, RD., andJ.C. Leong.
1988. The nucleocapsid gene of infectious hematopoietic
necrosis virus, a fish rhabdovirus. Virology 167:644-648.
Harlow, E., and D. Lane.
1988. Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Hasobe, M., and M. Saneyoshi.
1985. On the approach to the viral chemotherapy against
infectious hematopoietic necrosis virus (IHNV) in vitro andin vivo on salmonid fishes. Fish Pathol. 20:343-351.
Hattenberger-Baudouy, A.-M., M. Danton, G. Merle, C. Torchy, andP. de Kinkelin.
1989. Serological evidence of infectious hematopoietic necrosis in rainbow trout from a French outbreak of
disease. J. Aquat. Anim. Health 1:126-134.
Hsu, Y.L., andJ.C. Leong.
1985. A comparison of detection methods for infectious I·.ematopoietic necrosis virus. J. Fish Dis. 8:1-12.
Hsu, Y.L., H.M. Engelking, andJ.C. Leong.
1985. Analysis of the quantity and synthesis of the virion proteins of infectious hematopoietic necrosis virus. Fish
Pathol. 20:331-338.
1986. Occurrence of differen t types of infectious hematopoietic necrosis virus in fish. Appl. Environ. Microbio!.
52:1353-1361.
Innis, M.A., D.H. Gelfand,J.J. Sninsky, and T.J. White (eds.).
1990. PCR Protocols. Acad. Press, NY.
Jorgensen, P.E.Y., N.J. Olesen, N. Lorenzen, J.R. Winton, and S.
Ristow.
1991. Infectious hematopoietic necrosis virus: detection ofhumoral antibodies in rainbow trout by plaque neutralization, immunofluorescence, and enzyme-linked immurosorbent assays. J. Aquat. Anim. Health 3: 100-108.
Kimura, T., M. Yoshimizu, Y. Ezura, and Y. Kamei.
1990. An antiviral agent (46NW-04A) produced by Pseudo'ltOllas sp. and its activity against fish viruses. J. Aqu"-l.
Anim. Health 2: 12-20.

LaPatra, S.E., KA. Roberti,J.S. Rohovec, andJ.L. Fryer.
1989. Fluorescent antibody test for the rapid diagnosis ofinfectious hematopoietic necrosis. J. Aquat. Anim. Health
1:29-36.
Leong,J., YL. Hsu, H.M. Engelking, and D. Mulcahy.
1981. Strains of infectious hematopoietic necrosis (lHN)
virus may be identified by structural protein differences. Dev. BioI. Stand. 49:43-55.
Leong,J.C.,J.L. Fryer, andJ.R Winton.
1988. Vaccination against infectious hematopoietic necrosis.
In Fish vaccination (A.E. Ellis, ed.), p. 193-203. Acad.
Press, London.
McAllister, P.E.
1979. Fish viruses and viral infections. In Comprehensive
virology, Vol. 14 (H. Fraenkel-Conrat and R.R Wagner,eds.), p. 401-470. Plenum, New York, NY.
McAllister, P.E., and WB. Schill.
1986. Immunoblot assay: a rapid and sensitive method for
identification of salmonid viruses. J. Wildl. Dis. 22:468474.
Nicholson. B.L.
1982. Infectious hematopoietic necrosis (LH.N.). In Antigens of fish pathogens: Development and production for
vaccines and serodiagnostics (Anderson, D.P., M. Dorson,
and P. Dubourget, eds.), p. 63-69. Merieux, Lyon.
Pilcher, K.S., andJ.L. Fryer.
1980. The viral diseases of fish: a review through 1978. Part
I: Diseases of proven viral etiology. CRC Crit. Rev.
Microbiol. 7:287-364.
Ristow, S.S., andJ.M. Arnzen.
1989. Development of monoclonal antibodies that recognize
a type-2 specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus. J. Aquat.
Anim. Health 1:119-125.
Schultz, C.L., B.C. Lidgerding, P.E. McAllister, and EM. Hetrick.
1985. Production and characterization of monoclonal antibody against infectious hematopoietic necrosis virus. Fish
Pathol. 20:339-341.
Schultz, C.L., P.E. McAllister, WB. Schill, B.C. Lidgerding, andF.M. Hetrick.
1989. Detection of infectious hematopoietic necrosis virus incell culture fluid using immunoblot assay and biotinylated
monoclonal antibody. Dis. Aquat. Org. 7:31-37.
Way, K, and P.F. Dixon.
1988. Rapid detection of VHS and IHN viruses by the enzyme-linked immunosorbent assay (ELISA). J. App!.
Ichthyol. 4:182-189.
Winton,J.R., C.K Arakawa, C.N. Lannan, andJ.L. Fryer.
1988. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic
necrosis virus. Dis. Aquat. Org. 4: 199-204.
Wolf. K
1988. Infectious hematopoietic necrosis. In Fish viruses andfish viral diseases, p. 83-114. Cornell Univ. Press, Ithaca,
NY.
Yamamoto, T., C.K Arakawa, W.N. Batts, andJ.R Winton.
1989. Comparison of infectious hematopoietic necrosis innatural and experimental infections of spawning salmonids
by infectivity and immunohistochemistry. In Viruses oflower vertebrates (W. Ahne and E. Kurstak, eds.), p. 411429. Springer, Berlin.
Yamamoto, T., W.N. Batts, C.K Arakawa, andJ.R. Winton.
1990. Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: Wholebody assay and immunohistochemistry. J. Aquat. Anim.
Health 2:271-280.

Bacterial and Viral Diseases of Marine Fish
During Seed Production
KIYOKUNIMUROGA
Faculty ofApplied Biological Science
Hiroshima University
Higashi-Hiroshima 724, Japan

ABSTRACTIn Japan, seed production techniques have been developed for about 40 species ofmarine fishes; however, mass mortalities due to infectious and noninfectious diseases
have often occurred. Among these problems are the bacterial and viral diseases reviewed
in the present paper. Vibriosis and other bacterial diseases have occurred in various
marine fishes during their juvenile stage. These diseases have essentially Ole same pathology in both juvenile and adult fish in their tendency to terminate in systemic infection,
except for Flexibacter infections. On the other hand, larvae most frequently develop intestinal infections, such as bacterial enteritis with Vibrio sp. INFL in Japanese flounder. It
has been suspected that live diets contaminated with pathogenic bacteria serve as animportant source of these intestinal infections. Recently, some new viral infections, suchas viral epidermal hyperplasia and viral nervous necrosis, have been reported in larval
and juvenile marine fishes. Although isolation of the causative viruses in cultured cells
has not been successful, except for yellowtail ascites virus (YAV) , these diseases wereconfirmed to occur only in the larval, or larval and juvenile, stages by infection experiments and epidemiological surveys.

Introduction_

In Japan, demand for coastal fisheries and aquaculture products has been increasing drastically over the
past few decades, especially since the establishmentof the 200-mile economic zone. Consequently, governmental programs have concentrated on the
improvement and development of coastal fisheries
stocks, including mass release programs for important species. At present, more than 100 million
juveniles of about 40 marine finfish species are produced annually at national, prefectural, and private
hatcheries. Red seabream (Pagrus major), Japaneseflounder (Paralichthys olivaceus), and black seabream(Acanthopagrus schlegeli) are the representative species
produced in southern Japan. Fish production techniques, particularly for the three species mentioned
above, are apparently well established (Fukuhara
1987). However, during fish production, difficulties
are often encountered in controlling diseases ofknown and unknown etiology. The present paper will
review bacterial and viral diseases of marine fishes in
the course of seed production inJapan.

Bacterial Diseases
A wide range of pathogenic bacteria have been isolated from cultured fishes during their juvenile stage(Table 1). Vibrio anguillarum infection has been reported in red seabream, tiger puffer (Takifugu
rubripes), and Japanese flounder (Muroga and Tatani
1982; Muroga et al. 1987b ; and Yamanoi et al. 1988;
respectively) and Vibrio ordalii infection has been observed in juvenile rockfish (Sebastes schlegeli) (Muroga
et al. 1986). Pasteurella piscicida was isolated from juveniles of black seabream and red grouper(Epinephelus akaara) as a disease agent (Muroga et al.
1977; Ueki et al. 1990; respectively). Flexibacter
maritimus infection has been reported in red
seabream, black seabream, and flounder (Wakabayashi et al. 1984, 1986; Baxa et al. 1986). Except
for Flexibacter infections, these bacterial infections terminate in septicemia, or systemic infection injuvenile fishes as they do in adult fishes. On the other
hand, these systemic infections have rarely been reported during the larval stage. Instead, highmortalities during the larval stage are usually asso€i57

58

NOAA Technical Report NMFS III

Table 1Bacterial diseases in larval and juvenile marine fishes.
Stage
Larval stage

Juvenile stagearne of disease

Pathogen

Host fish

Abdominal swelling

Vibrio alginolyticus
Other pathogenic organisms

Red seabreamBlack seabream

Bacterial enteritis

Vibrio sp. I FL

Japanese flounderVibriosis

Vibrio anguillarum

Red seabream
Japanese flounder
Tiger puffer
Rockfish"Vibrio ordalii

Pasteurellosis

Pasteurella piscicida

Black seabream
Red grouper

Flexibacter infection

Flexibacter maritimus

Red seabreamBlack seabream
Japanese flounder"Sebastes schlegeli.

ated with intestinal infections. These intestinal infections occur in the larvae of various marine fishes.
Abdominal swelling ("Fukubu-boman-sho" in Japanese) occurs in red and black seabreams (Iwata et al.
1978; Kusuda et al. 1986; respectively). Vibrio
alginolyticus has been reported to be the causative
agent or to be associated with the disease in both fish
species. In an investigation of this disease in red
seabream reared in extensive nursery ponds, various
species of the genus Vibrio, including V. alginolyticus
and V. vulnificus, were the predominant isolates fromdifferent batches of diseased larvae. The role of these
vibrios was not made clear, however, because significant mortality or abdominal swelling could not be
produced by oral challenge in red seabream larvae
with these isolates (Yasunobu et al. 1988). A marine
turbellarian (Allostoma sp.) was also reported to be
associated with abdominal swelling of black seabream(Yamaguti 1987). At present, abdominal swelling of
seabreams can only be interpreted as being a syndrome caused by ingesting some pathogenic or
toxicogenic organisms.
A disease called "Chokan-hakudaku-sho" in Japanese, which means a disease condition characterizedby opaque intestine, occurs in larval Japaneseflounder. The causative agent is thought to be a Vibrio
species isolated from an affected intestine (Murata
1987). According to Murata (1987), this disease occurs in 14 to 30- or 40-day-old larval flounder;
symptoms include darkening of body color andopaqueness and shrinkage of the intestine. Mortalities sometimes reach 90% or higher, especially when
it occurs in younger fish. The causative bacterium is agram-negative, short rod, which is motile by its single
polar flagellum. It grows well on ZoBell's 2216e agar
and heart infusion or brain heart infusion agar with
3% ~aCl, forming small, circular, gray-colored colonies. Based on biochemical characteristics and GC(guanine and cytosine) value (44.1 mol%) of DNA,the organism was placed in the genus Vibrio. However, the characteristics of this pathogen differ fromthose of previously described fish-pathogenic Vibrio
species. The pathogen was tentatively named Vibrio
sp. INFL after the disease it causes: intestinal necrosisof flounder larvae (Masumura et al. 1989b).
Infection experiments using Vibrio sp. INFL werecarried out by oral administration via rotifers(Brachionus plicatilis) and brine shrimp (Artemia
salina) nauplii, by addition to rearing tanks, and by
intraperitoneal injection (Masumura et al. 1989b).
The disease was reproduced in flounder larvae only
by oral challenge. On the contrary, V. anguillarum,
which was used for comparison, killed flounder juveniles by intraperitoneal injection, but did not killflounder larvae by oral challenge. The pathogenicityof Vibrio sp. INFL therefore seems to be quite different from that of V. anguillarum.
Six different age groups of flounder (11, 16, 17,
27, 41, and 60 day olds) were submitted to an oral
challenge test with Vibrio sp. INFL (Muroga et al.
1990). The bacteria .were incorporated into brine
shrimp nauplii or rotifers and were given for threedays. As a result, mortality of the test groups was significantly higher than that of the respective controls
in the groups of 16, 17, and 27-day-old fish. The characteristic clinical sign of the disease, an opaqueMuroga: Bacterial and Viral Diseases of Marine Fish During Seed Production

intestine, occurred and subsequently Vibrio sp. INFL
was isolated from the intestine in these test groups.
In the older fish groups (41- and 60-day olds), no
apparent changes were observed in either the test
groups or the controls. Based on these results andthe before-mentioned epidemiological data on natural outbreaks of the disease, it was concluded that
this bacterial enteritis is confined to the larval stageof flounder. Histopathological and electron microscopic examinations revealed that pathogen
multiplication and resultant pathological changes occurred only in the intestine (Miyazaki et al. 1990;
Muroga et al. 1990). Although pili-like structures
were not observed on the cells of the pathogen, anadhesive property was demonstrated on a chinook
salmon embryo (CHSE-214) cell line. These findings
led us to the conclusion that the necrotic enteritis
caused by Vibrio sp. INFL in flounder larvae is similar
to enteric colibacillosis of young mammals (Muroga
et al. 1990).
Investigations of the intestinal bacterial flora 9f larval and juvenile marine fishes seem to be essential toelucidate the pathogenesis of the above-mentioned
intestinal infections in larval fishes. A method for isolating and enumerating the aerobic intestinalbacteria of larval and juvenile fish was devised by
Muroga et al. (1987a); the intestinal bacterial flora ofseabreams, flounder, and other fishes was subsequently investigated (Tanasomwang and Muroga
1988, 1989a). The compositions of the intestinal
flora of these marine fishes were characterized by two
predominating groups, Vibrio and Pseudomonas, whichwere thought to be derived from the diets of live rotifers and brine shrimp nauplii. The same genera,
Vibrio and Pseudomonas, were most frequently isolated
from these live diets (Tanasomwang and Muroga
1990). Sodium nifurstyrenate bath proved effective in
reducing bacterial contamination of rotifers (Hayashi
et al. 1976; Yamanoi and Sugiyama 1987; Tanasomwang

and Muroga 1989b), and recently, it was also reported that the bacterial contamination of rotifers
and brine shrimp was significantly reduced by freezing at -15 0 C for 1 month (Yamanoi and Katayama
1989) .

Viral Diseases
Recently, several viral diseases have been reported in
the larvae and juveniles of marine fishes (Table 2).
These include viral ascites of yellowtail (Seriola
quinqueradiata), viral epidermal hyperplasia of Japanese flounder, viral epithelial necrosis of black
seabream, and viral nervous necrosis of Japaneseparrotfish (Oplegnathus fasciatus) , red grouper andstriped jack (Pseudocaranx dentex). Except for the firstvirus mentioned, the causative agents have not been
isolated in cultured cells.
In 1983, an acute disease characterized by ascites
occurred among yellowtail juveniles cultured in a
hatchery, and an IPNV (infectious pancreatic necrosis virus) -like virus, which was named YAY (yellowtail
ascites virus), was isolated in several cell lines including RTG-2 and CHSE-214. It was confirmed by an
immersion challenge that the disease could be reproduced in yellowtail juveniles and that the mortality
was higher in smaller fish (Sorimachi and Hara 1985;
Sorimachi and Egusa 1986). The same disease occurred among wild juveniles of yellowtail which werecaught as seedlings for net cage culture (Isshiki et al.
1989). Histopathological observations of naturally
and experimentally infected fish suggest that acinous
tissues of the pancreas and parenchymal tissue of theliver are the primary tissues involved in YAY infection(Egusa and Sorimachi 1986).
Since 1985, outbreaks ofa disease resulting in massmortalities have occurred in larvae and juveniles of
the Japanese flounder cultured at several hatcheries.

Table 2
Viral diseases in larval and juvenile marine fishes.
Stage
Larval stage

Juvenile stage•Lates catcanJer.

Name of disease59

Pathogen

Host fish

Viral epidermal hyperplasia(Viral epidermal necrosis)
Viral epithelial necrosis
Viral nervous necrosis

Herpesvirus

Japanese flounderParamyxovirus-like virus
Picornavirus-like virus

Viral asci tes

YAV (Yellowtail ascites
virus=IPNV-like)

Black seabream
Japanese parrotfish
Red grouper
Striped jackSeabass·
Yellowtail

60

NOAA Technical Report NMFS III

The disease occurred in 10- to 30-day-old fish rearedat 18-20° C, and mortality usually reached 80-90% ina few weeks. The affected fish had opaque fins and a
hyperplastic epidermis on the fins and skin. Electron
microscopy revealed hexagonal virus particles in the
nucleus and cytoplasm (diameter in enveloped stIte:
200 nm) of the affected epidermal cells. Althoughisolation of the causative agent by the use of several
fish-cell cultures was not successful, the disease wastransmitted to healthy larval flounder by exposing
them to a 0.45 f1m filtrate of diseased fishhomogenate. Morphological and physiological characteristics of the virus indicate that the agent is aherpesvirus (Iida et al. 1989). When immersion challenge tests of flounder larvae were made at threedifferent temperatures (15, 20, and 25° C), the disease progress was apparently delayed at the lowest
temperature (15° C), though the cumulative mortality was the same for all three temperatures. Larvae
younger than 20 days old (smaller than 9.5 mm intotal length) were highly susceptible, but the susceptibility was significantly decreased in fish 23 days old
or older (larger than 11.0 mm). Therefore, this disease proved specific to the larval stage of flounder(Masumura et al. 1989a). A similar disease was reported in Japanese flounder larvae and juveniles
under a name of viral epidermal necrosis (Miyazaki
et al. 1989); however, a precise comparison of theseherpesvirus infections has not been made owing to
the unsuccessful in-vitro culture of the agents. Another epithelial necrosis due to a paramyxovirus-likevirus was reported from larval black seabream(Miyazaki et al. 1989).
Mass mortalities of hatchery-reared Japaneseparrotfish larvae and juveniles have occurred in
Nagasaki Prefecture. the westernmost part of Japan.
Light and electron microscopic examinations revealed an extensive necrosis of the nervous system inthe spinal cord, spinal ganglia, and brain. Numerous
nonenveloped virus particles, icosahedral in morphology and measuring 34 nm in diameter, were
found in the cytoplasm of the affected neurones andglial cells. The disease was named viral nervous necrosis (Yoshikoshi and Inoue 1990). A similar disease
was found in red grouper larvae in 1989 and stripedjack larvae in 1990 (Yoshikoshi 1990). Similar
picorna-like viral particles were observed in degenerative areas of the brain and retina in seabass (Lates
calcarifer) larvae cultured in Australia (Glazebrook et
al. 1990). A comparative study on these new diseaseagents is a matter of great interest because traffic oflive fishes has been increasing among south andsoutheast Asian countries. The frequent occurrencesof these new viral diseases remind us of the necessityof rapid improvement in the current seed production

system for marine fishes from the standpoints ofquarantine and hygiene, both of which have almost
been established for salmon hatcheries.

Citations_Baxa, D.V., K. Kawai, and R. Kusuda.
1986. Characteristics of gliding bacteria isolated from diseased cultured flounder, Paralichthys olivaceus. Fish Pathol.
21:251-25S.
Egusa, S., and M. Sorimachi.
1986. A histopathological study of yellowtail ascites virus(YAV) infection of fingerling yellowtail, Seriola quinqueradiata. Fish Pathol. 21:113-121. (In Japanese; English
abstr.)
Fukuhara, O.
1987. Seed production of red sea bream Pagrus major(Sparidae) in Japan. In Reproduction, maturation, and seedproduction of cultured species: proceedings of the twelfth
U.S.japan meeting on aquaculture (CJ. Sindermann, ed.),p. 13-20. U.S. Dep. Commer., NOAA Tech. Rep. NMFS 47.
Glazebrook,J.s., M.P. Heasman, and S.W. de Beer.
1990. Picorna-like viral particles associated with mass mortalities in larval barramundi, Lates calcarifer Bloch. ]. Fish
Dis. 13:245-249.
Hayashi, K., T. Kimura, and I. Sugahara.
1976. Microbiological studies on the artificial seedling production of ayu fish, Plecoglossus altivelis (Temminck et
Schlegel)-lV. On the elimination of bacteria contaminatedin Brachionus plicatilis and Moina macrocopa. Bull. Fac. Fish.
Mie Univ. 3:87-99. (In Japanese; English abstr.)lida, Y., K. Masumura, T. Nakai, M. Sorimachi, and H. Matsuda.
1989. A viral disease in larvae and juveniles of Japaneseflounder Paralichthys olivaceus. ]. Aquatic Animal Health
1:7-12.
Isshiki, T., K. Kawai, and R. Kusuda.
1989. Incidence of yellowtail ascites virus (YAV) in wild yellowtail fingerling. Nippon Suisan Gakkaishi 55:633-637.
(In Japanese; English abstr.)
Iwata, K., Y. Yanohara, and O. Ishibashi.
1978. Studies on factors related to mortality of young red
seabream (Pagrus major) in the artificial seed production. Fish Pathol. 13:97-102. (In Japanese; English
abstr.)
Kusuda, R.,]. Yokoyama, and K. Kawai.
1986. Bacteriological study on cause of mass mortalities incultured black sea bream fry. Nippon Suisan Gakkaishi
52: 1745-1751. (In Japanese; English abstr.)
Masumura, K., Y. Iida, T. Nakai, and T. Mekuchi.
1989a. The effects of water temperature and fish age on aherpesvirus infection of Japanese flounder larvae,
Paralichthys olivaceus. Fish Pathol. 24:111-114. (In Japanese; English abstr.)
Masumura, K., H. Yasunobu, N. Okada, and K. Muroga.
1989b. Isolation of a Vibrio sp., the causative bacterium ofintestinal necrosis of Japanese flounder larvae. Fish
Pathol. 24:135-H1. (In Japanese; English abstr.)
Miyazaki, T., K. Fujiwara.]. Kobara, N. Matsumoto, M. Abe, andT. Nagano.
1989. Histopathology associated with two viral diseases of larval and juvenile fishes: epidermal necrosis of the Japaneseflounder Paralichthvs olivaceus and epithelial necrosis ofblack sea bream Acanthopagrus schlegeli. ]. Aquatic Animal
Health 1:85-93.

Muroga: Bacterial and Viral Diseases of Marine Fish During Seed Production
Miyazaki, T, N. Kajihara, I<.. Fujiwara, and S. Egusa.
1990. A histopathological study on intestinal necrosis of larval Japanese flounder. Fish Pathol. 25:7-13. (In Japanese;
English abstr.)
Murata, O.
1987. Infectious intestinal necrosis in flounder. Fish
Pathol. 22:59-61. (In Japanese.)
Muroga, 1<.., and M. Tatani.
1982. Isolation of Vibrio anguillarum from juvenile red
seabream (Pagrus major). Fish Patho!. 16:2Il-·214. (InJapanese; English abstr.)
Muroga, 1<.., T Sugiyama, and N. Ueki.
1977. Pasteurellosis in cultured black seabream (Mylio
macrocephalus). J. Fac. Fish. Anim. Husb. Hiroshima Univ.
16:17-21.
Muroga, 1<.., Y Jo, and I<.. Masumura.
1986. Vibrio ordalii isolated from diseased ayu (Plecoglossus
altivelis) and rockfish (Sebastes schlegeli). Fish Patho!.
21:239-243. (In Japanese; English abstr.)
Muroga, 1<.., M. Higashi, and H. Keitoku.
1987a. The isolation of intestinal microflora of farmed red
seabream (Pagrus major) and black seabream (Acanthopagrus
schlegeli) at larval and juvenile stages. Aquaculture, 65:7988.
Muroga, 1<.., V. Tanasomwang, and I<.. Momoyama.
1987b. Vibrio anguillarum infection in tiger puffer (Takifugu
rubripes) fingerlings. Fish Pathol. 22:29-30. (In Japanese.)
Muroga, 1<.., H. Yasunobu, N. Okada, and I<.. Masumura.
1990. A bacterial enteritis of cultured flounder Paralichthys
olivaceus larvae. Dis. Aquat. Org. 9: 121-125.
Sorimachi, M., and T Hara.
1985. Characteristics and pathogenicity of a virus isolated
from yellowtail fingerlings showing ascites. Fish Pathol.
19:231-238. (In Japanese; English abstr.)
Sorimachi, M., and S. Egusa.
1986. Experimental infection of yellowtail ascites virus (YAV)
to yellowtail fingerlings. Fish Pathol. 21:133-134. (InJapanese.)
Tanasomwang, v., and I<.. Muroga.
1988. Intestinal microflora of larval and juvenile stages inJapanese flounder (Paralichthys olivaceus). Fish Pathol.
23:77-83.
I989a. Intestinal microflora of rockfish Sebastes schlegeli, tiger
puffer Takifugu rubripes and red grouper Epinephelus akaara
at their larval and juvenile stages. Nippon Suisan
Gakkaishi 55:1371-1377.

61

1989b. Effects of sodium nifurstyrenate and tetracycline on
the bacterial flora of rotifer (Brachionus plicatilis). Fish
Pathol. 24:29-35.
1990. Intestinal microflora of marine fishes at their larval
and juvenile stages. In Proceedings of the second Asian
fisheries forum (R. Hirano, and 1. Hanyu, eds.), p.647650. Asian Fisheries Soc., Manila, Philippines.
Ueki, N., Y Kayano, and I<.. Muroga.
1990. Pasteurella piscicida infection in juvenile red grouper(Epinephelus akaara). Fish Pathol. 25:43-44. (In Japanese.)
Wakabayashi, H., M. Hikida, and I<.. Masumura.
1984. Flexibacter infection in cui tured marine fish in
Japan. Helgol. Meeresunters. 37:587-593.
1986. Flexibacter maritimus sp. nov., a pathogen of marine
fishes. Int. J. Syst. Bacteriol. 36:396-398.
Yamaguti, M.
1987. Abdominal swelling in black seabream. Fish Pathol.
22:58--59.' (In Japanese.)
Yamanoi, H., and T Sugiyama.
1987. Effects of sodium nifurstyn:nate bath and ultraviolet
irradiation on the elimination of bacteria associated withrotifer. Suisanzoshoku (Aquiculture) 25:191-195. (In Japanese.)
Yamanoi, H., and K. Katayama.
1989. Effects of freezing on bacterial flora of rotifer andbrine shrimp nauplii. Nippon Suisan Gakkaishi 55:2207.
(In Japanese.)
Yamanoi, H., I<.. Momoyama, H. Yasunobu, and I<.. Muroga.
1988. Vibrio anguillarum infection in flounder (Paralichthys
olivaceus) fingerlings. Fish Pathol. 23:69-70. (In Japanese.)
Yasunobu, H., I<.. Muroga, and I<.. Maruyama.
1988. A bacteriological investigation on the mass mortalities
of red seabreamPagrus major larvae with intestinal
swelling. Suisanzoshoku (Aquiculture), 36: Il-20. (In Japanese; English abstr.)
Yoshikoshi, I<..
1990. A virus found in larval marine fishes asssociated withmass mortalities. In Abstracts of workshop of the JapaneseSociety of Fish Pathology, p.5. Japanese Society of Fish Pathology, Tokyo. (In Japanese.)
Yoshikoshi, 1<.., and I<.. Inoue.
1990. Viral nervous necrosis in hatchery-reared larvae andjuveniles of Japanese parrotfish, Oplegnathus fasciatus(Temminck & Schlegel). J. Fish Dis. 13:69-77.

An Ecological Study of the Parasitic Nematode
Hysterothylacium haze in the Japanese Common Goby
Acanthogobius flavimanus, in a Brackish Inlet

TOMOYOSHI YOSHINAGA
National Research Institute ofFisheries Science
Kachidoki, Chou-ku
Tokyo 104, Japan

ABSTRACTThe unique life cycle of the parasitic nematode Hysterothylacium haze in the Japanesecommon goby Acanthogobius flavimanus is presented. It is a direct cycle in which the fish
become infected by ingesting fully developed eggs or hatched larvae of the nematode or
by ingesting invertebrates that can act as transport hosts. Infections by the nematodewere investigated monthly in a wild population of common goby in a brackish inlet overa period of five years. Seasonally, the rate of infection in the body cavity peaked in
November in fish age 0+ and in May in fish age 1+. Annually, the infection rate declined
during the investigation period. The intensity of infection in individual fish tended tofluctuate with the population's overall infection rate for both age 0+ and age 1+ fish. The
influence of the infection on the host population is briefly discussed.

Introduction_

Mass mortalities of the Japanese common goby
Acanthogobius flavimanus (yellowfin goby) occurred in
the inner part of Tokyo Bay every summer from 1973
to 1975. The body cavities of the dead fish wereheavily infected with the eggs, larvae, and adults of anematode; visceral adhesions were often noted(Takahashi et al. 1976; Takahashi et al. 1977).
Takahashi et al. (1977) demonstrated that the heavily
infected fish were intolerant of low dissolved oxygen
concentrations and concluded that the mass mortalities of the goby were caused by the nematodeinfections in combination with the scarcity of dissolved oxygen. The nematode was identified as a new
anisakid, Thynnascans haze, by Machida et al. (1978)and later assigned to the genus Hysterothylacium as H.
haze, a new combination by Deardorff and Overstreet(1981).
This nematode possesses unique features among
the anisakid nematodes. Adult worms and eggs of H.
haze were found in the body cavity of the host fish,
whereas the adults of other anisakid nematodes harbor and deposit their eggs in the digestive tract of
their definitive hosts. It also appears that H. haze hasa unique life cycle and that infection is apparently

harmful to the host goby, heavy infections causing
the deaths of some hosts. There is no other known
case in which an anisakid nematode infection is so
harmful as to kill the host fish. From the extensive
damage observed in heavily infected fish, parasitism
by the nematode may even reduce the host population size.
A previous study of the biology of H. haze has already elucidated its peculiar life cycle (Yoshinaga et
al. 1989). An ecological investigation was made forfive years. In this paper, the seasonal and annualchanges in H. haze infections in goby inhabiting abrackish inlet and the life cycle of this parasite aredescribed. In addition, the influence of its infection
on the population of the host is discussed briefly.

Life Cycle
In general, nematodes molt four times prior to becoming adults. The first-stage larvae are worms
before the first molting. Following the first, second,
and third moltings, the worms are called the second,
third, and fourth-stage larvae, respectively, and after
the fourth molting (the fifth stage), they are adults.
It was reported by Yoshinaga et al. (1988) that all
63

64

NOAA Technical Report NMFS III

~I

..

1- 2

EGG

Ecological Study
2

Invertebrates(transport host)\~

ADULT -•

L4 -L 3+..

L3 -L 2

EGG(Ll -L 2 >.. C2
body cavity

digestive
tract wall

Japanese common goby
(definitive host)Figure 1

Life cycle of Hysterothylacium haze. L1, L2 , L3 , and L4 represent the first-, second-, third-, and fourth-stage larvae,
respectively. The development from L2 to L3 in the body
cavity of the japanese common goby (dotted arrow) is uncertain. (From Yoshinaga et al. 1989.)

stages of H. haze are harbored in the body cavity ofheavily infected fish; this is an exceptional feature
among anisakid nematodes. Detailed observations of
H. haze infections in goby and experiments with infected goby and invertebrates resulted in theelucidation of the H. haze life cycle (Yoshinaga et al.
1989). It is summarized as follows (Fig. 1): when agoby eats fully developed worm eggs, second-stage
larvae in the eggs hatch and penetrate the digestive
tract wall of the fish. The fish may also become infected by eating invertebrate transport hosts
containing second-stage larvae in their body cavities;
various invertebrates (e.g., polychaetes, amphipods,
and mysids) can act as transport hosts. Second-stage
larvae develop to the third stage in the tissue of thegoby's digestive tract wall, then migrate into the body
cavity. In the body cavity, third-stage larvae develop
through the fourth stage to become adults, that mature and deposit eggs in the body cavity of th.e host.
These eggs are released into the water after the deathof heavily infected goby. It should be noted that thedeaths of heavily infected goby are essential for egg
dispersion and subsequent invasion of new hosts. The
eggs hatch in the body cavity of the host. Although it
is uncertain whether the second-stage larvae can develop to third-stage larvae in the body cavity, it seems
that a large nl,lmber of larvae are harmful to the hostand contribute to its death even when the host is
infected with only a small number of adults.

_

An ecological study of H. haze was carried out in
Lake Shonai, a brackish inlet of Lake Hamana, anestuary located on the Pacific coast of central Japan (Fig. 2). The Japanese common goby has a life
span of one year and spawns in the spring(Miyazaki 1940); however, some fish do not become fully mature during the spawning season,probably because of delayed growth. These fish survive to become age 1+ in some areas. Thus, thegoby population in Lake Shonai consists of twoyear classes from late spring to autumn.
Hysterothylacium haze infections in the Japanesecommon goby were investigated monthly from June
1981 to November 1982 and from December 1983 toSeptember 1986. Fish captured commercially by trap
net were sampled. They were examined for H. haze
infection in the body cavity or in the digestive tract
wall, or both, after age determination by scale readings. Infections in the body cavity were examinedA

B

R. Hana·:•. ::1 .... :•. : ..•.

t •.

:: :. The PacHi c'; '.::

R. Shino

2 Iqn

Figure 2

Map of Lake Hamana (A) and Lake Shonai (B).
Lake Shonai is a brackish water inlet of Lake
Hamana, an estuary located on the Pacific Coast in
Shizuoka Prefecture, Honshu.

Yoshinaga: Ecological Study of the parasitic nematode Hysterothylacium haze

100

1983

1980

50

0
d(l

I

I I

100

I

,

I

~
1 b~ 1
'.
,

1981

s:::

I

. :::~·~:k·.

,,

I

I

I!

!

1984

cLl/~
,

50

..-l
tl
H

(

, ,

I

'

CIl
tl

s:::
CIl
'tl

65

0

I I I I I

I I I

100

I I I I

I I I

1985

50

Figure 3
o
I

I

I

I

I'

,

!

,

'"

!

I

I

I

I

I

I

J

,

!

I

I

I

I

I

I

I

I

I

I

1'1

I

I

MonthlyI

1986[mID 2: moderately infected
ID 3: heavily infected

the

level

of

cavities of the Japanese common goby by year
class from 1980 to 1986. Asterisks indicate
months when sample sizes were less than 30.
ID = intensity index determined by macroscopical observation; ID 1 = lightly infectedwith 1-9 worms: ID 2 = moderately infectedwith 10-99 worms; ID 3 = heavily infectedwith more than 100 worms.

o ID 1: lightly infected•inHysterothylacium haze infection in the body

M J J A50ND JFMAM J J A50N 0

Monthchanges~I I I I I I I I I I I I I I I I I I I I
M J J A 5 ON 0 J F M AM J J A 50 NO

Monthwith a stereomicroscope during the entire investigation period. Infections in the digestive tract wall were
examined microscopically by compressing the tract
between glass slides.

iii]

50
fHl

Seasonal Changes
Q)

U

Infections in the body cavItIes occurred in a clear
seasonal pattern, although there were slight differences in the pattern between years (Fig. 3).
Infections in age 0+ fish were first noticed in earlysummer. The incidence of infected fish peaked in
November and subsequently declined to a minimum
level between January and March. A second peak occurred in May in age 1+ fish that had survived after
their spawning season, and then the peak declined.
When the prevalence of the nematode was high,heavily infected fish containing worm eggs in the
body cavity were frequently found.
Although infection of the digestive tract walls wasinvestigated for only two years, it was observed thatthe year when the infection level in the body cavity
was lower and showed a less clear seasonal pattern, aseasonal pattern was still discernible in the incidence
in the two sites of infection (Fig. 4). Characteristically, infection in the digestive tract wall preceded

1984

lOaf

d

0

lOaf

~
-r-!

50

U

0

d

,

0

,

,

1985

]

H

lOO[
50

1986

DBC

~

o ......

EilBC+DTW

.DTW
!

•

!

••

,

!

!

.J

M J JA50NDJFMAMJ J A SO NO

MonthFigure 4

Monthly changes in the incidence of infection with
Hysterothylacium haze in the two infection sites in the
1984-86 year classes of the Japanese common goby.
Blank, semidark, and dark areas represent fish infected only in the body cavity (BC), both in the body
cavity and digestive tract wall (BC+DTW), and onlyin the digestive tract wall (DTW), respectively.

66

NOAA Technical Report NMFS III

that in the body cavity. Infection in the digestive tract
wall began in June or July prior to or in the samemonth as the infection in the body cavity. The incidence of nematode infection in the digestive tract
walls peaked in August and declined to its lowest levels in November and December (1985 year class)when the incidence of infection in the body cavities
began to peak. The incidence of nematodes in thedigestive tract walls continued to rise until April anddeclined in May, when it reached a second peak inthe body cavity.

Age ofthe goby

1+

0+
II_II

Invasion to
tbe digestive
tract "allII

Migration to
the body cavity

age 1+ to age 0+ fish occurred in summer, suggestingthat the survival of part of the goby population after
the spawning season is indispensable to the nematode life cycle.

Annual Changes
During this 5-year investigation, the prevalence andintensities of H. haze infection in goby body cavities
showed sharp annual declines when infection peaks
were observed (Fig. 6). The declines were observed
over the whole life span of the goby (Fig. 3). Also thebeginning of the invasion of the body cavity was delayed to July in 1985 and to August in 1986, whereas
it first occurred in June in the 1982 and 1984 year
classes.

2

El100

100

2

'"'"

1+

ii

---><
Q)

't:l

Deposition of
the eggs

=

'M

Q)

u

=
r-i 50

Infection levelin the body cavity

Q)

50

~Q)

1

I

MJ

I

I

,

,

,

,

,

,

JASONDJF

,

,

,

,

MAMJ

,

,

=
Q)

=

~

'M

=
10

J

Figure 5
A diagram of the seasonal pattern of Hysterothylacium
haze infection in the Japanese common goby in Lake
Shonai.

The seasonal pattern of H. haze infection is summarized from these results as follows (Fig. 5):
second-stage larvae invade the digestive tract wall ofage 0+ goby in summer and develop to the third
stage there. They migrate to the body cavity fromsummer to autumn, develop to the adult stage, mature, and then deposit their eggs there in lateautumn through early winter when the infection levelin the body cavity in the body cavity first peaks. Between December and March, new invasions into thedigestive tract wall occur again and the infectionlevel in the body cavity reaches a second peak in May
in age 1+ fish.
Hysterothylacium haze has never been reported to occur in any fish other than the Japanese common
goby; therefore it appears that the biology of thegoby is closely related to the biology of the nematode. It is also apparent, from the seasonal pattern
above, that the transmission of the nematode from

~

+J

H

,

>.
+J
'M

0'
,
,
,
, 0
'81'82'83'84'85

Year class

O'

,

!

I

•

'81' 82' 83' 84' 85a ~Year class

Figure 6
Annual changes in the level of Hysterothylacium haze in the
body cavities of the Japanese common goby by year classfrom 1981 to 1985. The incidence and mean intensity index (total intensity indexes/number of fish examined) are
shown for the first and second peaks in age 0+ and age 1+
goby, respectively. See Figure 3 caption for definition of
intensity index. Vertical bars are 95% confidence limits.

If H. haze infection had regulated the host population size, some relationship would have been foundbetween the levels of the host population and thenematode population. Figure 7 shows that the goby
catch in Lake Shonai from 1982 to 1986 (data fromthe Shirasu Branch, Hamana Fisheries Cooperation).
Though the total catch of goby showed considerable
annual fluctuation, there was no apparent relationship between the infection levels and catch. It seems
likely that the H. haze infection has little influence
upon the popUlation's size, despite the lethal damage

Yoshinaga: Ecological Study of the parasitic nematode Hysterothylacium haze

Another question arose from the annual decline of
the H. haze infection level. Why did the nematode
population decrease during the investigation? Although there were no data available to interpret it,
two possible reasons may be assumed from the biology of the nematode: one is that the populations of
the invertebrate transport hosts may have decreased,
and another is that the biological conditions of theJapanese common goby in Lake Shonai (e.g., spawning season, survival after the spawning season, andgrowth) may have changed. Nevertheless, some ecological changes should have occurred in Lake
Shonai, based on Price's (1980) contention that parasites generally exist in nonequilibrium states, where
the departure of anyone element in the patch may
render the population inviable.

1982
0- 1983
If)

c::

E
....u
....$.lI)

a

:I:

u

0

2

A

1984

Citations

1

le:(

u
:I:
(J)

LL

0

r
0

67

A

M

J

M

J

98S
A

:t 1986
J

Figure 7
Changes in the catch (metric tons) of the Japanese common
goby in Lake Shonai from 1982 to 1986 (based on data from
Shirasu Branch, Hamana Fisheries Cooperation). Blank anddark areas represent age 0+ and age 1+ fish respectively. In
the semidark area, age was not determined.

to heavily infected hosts. This is probably due to thefact that the Japanese common goby has a life span of
only 1 year and the influence of the infection does
not accumulate year by year, and because the goby
population is regulated more by environmental factors than by the population size of the previous
generation (Tokyo Metropolitan Fisheries Experimen tal Station 1981).

_

Deardorff, T.L., and R.M. Overstreet.
1981. Reviews of Hysterothylacium and Iheringascaris (both previously = Thynnascaris) ( ematoda: Anisakidae) from thenorthern Gulf of Mexico. Proc. BioI. Soc. Wash 93:10351079.
Machida, M., K. Takahashi, and S. Masuuchi.
1978. Thynnascaris haze n. sp. (Nematoda, Anisakidae) fromgoby in the Bay of Tokyo. Bull. Natl. Sci. Mus., Ser. A(Zool.) 4:241-'-244.
Miyazaki, I.
1940. Studies on the Japanese common goby, Acanthogobius
flavimanus (Temminck et Schlegel). Bull. Jpn. Soc. Sci.
Fish. 9:159-180. (In Japanese; English abstr.)
Price, P.W.
1980. Evolutionary biology of parasites. Princeton Univ.
Press, Princeton, NJ.
Takahashi, K., S. Masuuchi, T. Nakamura, and M. Saito.
1976. On a nematode from Acanthogobius flavimanus. In
Annual report of ecological survey of fisheries and shellfishes in the inner area of Tokyo Bay (1974). Publicationof the Tokyo Metropolitan Fisheries Experimental Station
267:42-50. (In Japanese.)
Takahashi, K., S. Masuuchi, T. Nakamura, M. Ogura, and T. Arima.
1977. On a nematode from Acanthogobius flavimanus (2). In
Annual report of ecological survey of fisheries and shellfishes in the inner area of Tokyo Bay (1975). Publicationof the Tokyo Metropolitan Fisheries Experimental Station
276:89-93. (In Japanese.)Tokyo Metropolitan Fisheries Experimental Station.
1981. Annual report of ecological survey of fisheries andshell-fishes in the inner area of Tokyo Bay (19761979). Publication of the Tokyo Metropolitan Fisheries Experimental Station 30, 73 p. (In Japanese.)
Yoshinaga, T., K. Ogawa, and H. Wakabayashi.
1988. Developmental morphology of Hysterothylacium haze
(Nematoda: Anisakidae). Fish Pathol. 23:19-28.
Yoshinaga, T., K. Ogawa, and H. Wakabayashi.
1989. Life cycle of Hysterothylacium haze (Nematoda:
Anisakidae: Raphidascaridinae). J. Parasitol. 75:756-763.

Epidemiology of Marine Fish Diseases
in the Warm Waters Along the Kuroshio Current

HIROKO ISHIOKA
Nansei National Fisheries Research Institute2-17-5 Maruishi, Ohn(rCho, Saeki-Gun
Hiroshima, 739-04,Japan

ABSTRACTIn Japan, epidemiological studies have been carried out on the diseases of marine fishfor many years. From 1981 to 1989, daily clinical record cards were used in every prefecture to obtain information on the outbreak of diseases in cultured marine fish.
Observations were recorded on a standard form, translated into codes, and stored on adata base. During the analysis of this data, fish diseases were classified into six groups
according to the causal agent: bacterial, viral, fungal, parasitic, nutritional, or unidentified disease. The most frequently reported group was bacterial disease, while the least
common one was fungal disease. This paper describes the species-specific features andseasonal variations in outbreaks of these disease groups and discusses the relationshipbetween fish size and diseases. Also, the relationship between locality and diseases areanalyzed for yellowtail, Seriola quinqueradiata. From this epidemiological examination, it isclear that disease outbreaks by bacteria that are normally nonvirulent, such as streptococci and gliding bacteria, suggest that fish cultivation itself disturbs the natural
relationship between fish and the environment. In other words, deteriorated environmental quality and poor fish health led to a beneficial situation for the growth ofpathogens.

Introduction_

Japan is surrounded by two major oceanic currents:
the Kuroshio Uapan) Current and the Oyashio(Okhotsk) Current. The Kuroshio Current is warmer
and its surface temperature varies from about 10° Cin winter to about 30° C in summer (Fig. 1). Most
marine fish cultures in Japan are carried out in the
coastal waters along this current. The marine environment in these coastal waters is suitable for marine
fish cultivation because of its warmer temperature,milder weather, abundant sunlight, and lower chlorinity. Nitrogenous and phosphorous nutrients
supplied by many rivers make this area highly fertile.
These factors support the high production of phytoplankton and zooplankton, which in turn assure foodfor fish and shellfish larvae. Also, the rias coastline iswell suited for the culturing of marine fish because
culture equipment can remain set up throughout theyear and the daily work routine of fish culturing canbe easily performed in the small gulfs and bays.
Fish species chosen for cultivation must be 1)
adaptable to the culture environment, 2) easily cul-

tured, 3) produce readily available seedlings, and 4)
must be commercially valuable as sashimi (raw fish).
Among the 30 species now being cultured in Japan
are Seriola quinqueradiata (yellowtail), Pagrus major(red sea bream), Paralichthys olivaceus 'Uapaneseflounder), and Takifugu rubripes (tiger puffer). Yellowtail is the most popular and familiar species
among these and accounts for about 70% of the total
production of cultured marine fish.
The production of marine fish by artificial cultures
and traditional methods is shown for the period
1976-86 in Figure 2 (SID 1981-90). Recently, the annual catch by marine fisheries (excluding shellfish
and seaweeds) has amounted to about 10 million
metric tons (t) yielding a gross income of about 210
billion yen. The harvest from marine cultivation has
totaled about 0.2 million t valued at 20 billion yen.
Although the net production of fish both by traditional marine fisheries (A) and marine cultures (B)has increased slightly since the early 1980s, the ratio
between them (B/A) has remained fairly constant at
about the 2% level. In contrast, the ratio of grossincome (b/a, Fig. 2) has steadily increased and69

70

NOAA Technical Report NMFS 111

PREFECTURESMIE
2 WAKAYAMA
3. HYOGO
4. YAMAGUCHI
5. KAGAWA
6. TOKUSIMA
7. KOCHI
8. EHIME
9. MIYAZAKI
10. KUMAMOTO
11. KAGOSIMA
12. NAGASAKI
13.01TA
14. SAGA

C: Cold waler

W: Warm waterand are fed nonliving raw food, such as minced sandeels.
In the case of fish species such as red sea breams,
where seedling production is done mainly in landbased ponds, fertilized eggs are collected by net andthen transferred into a rearing tank. After hatching,fish are fed with live feed suitable for their size, suchas oyster eggs, rotifers, brine shrimp, and zooplankton, which are collected from the sea. As they
develop, they are eventually fed nonliving feed suchas minced raw fish, chopped shrimp, or shellfish until
development to the young fish stage is almost completed.
Intensive rearing in the marine net pen is thencarried out for about 1 or 2 years until the fish growto a marketable size. During this rearing period, fish
are fed principally with sliced or cut, raw or frozen
fish meat.
Intensive culture techniques have an adverse effectnot only on fish health, but also on the conditions of
the surrounding environment. Food containing highlevels of protein and fat, such as raw fish meat, is
supplied in large amounts in expectation of more
rapid growth. The leftover food and fish excrement
deposited on the sea bottom cause a deterioration ofwater quality, which is believed to be a cause of red
tides and diseases. In addition, excessive feeding andrestricted activity in the limited confines of the rearing pens also make fish fatty.

Figure 1

Currents around Japan.

Investigating Disease Outbreakamounted to about 10% in 1986. This suggests thatthe market not only demands cultured fish as a protein source but also holds their species-specificquality and freshness in high regard.
Because of the importance of cultured marine
fishes to the Japanese economy, an epidemiologicalstudy of the fish diseases affecting the industry was
undertaken. Data compiled by the various prefectural
fisheries institutes from 1981 to 1989 were examined
to establish trends in disease outbreaks and relationships between fish, their culture environment, anddisease organisms.

Marine Fish Cultivation:
Technical Features_

The techniques used to culture marine fishes in Japan vary little between species and localities. In the
popular case of yellowtail, fry or seed fish migratingwith drifting algae in the surface of the Kuroshio
Current are captured by a small purse seine. The fish
are transferred to floating net pens located in a bay_

At the request of an aquaculturist fish diseases are
diagnosed at the prefectural experimental stations,where daily clinical records are maintained. Diagnostic data are recorded on a standard form (Table 1),translated into codes, and stored in data bases of marine fish diseases at the Nansei National Fisheries
Research Institute. These data can then be enteredinto a personal computer (NEC 9801) and be statistically sorted and calculated using DATA BASE IV
software (Nihon Integrated Software Co., Ltd.). The
program for compiling, sorting, calculating, andprinting disease case data was previously reported(Umezawa and Ishioka 1988). The data base of thepresent study represents diagnoses conducted by 20
prefectures during the period from 1981 to 1989.
The standard form consists of five parts, as shownin Table 1. The first part includes information essential for registering one diagnostic case: fish species
name, region where the disease occurred, name of
the fish farm or fish culturist, diagnosis date, and thedisease name. Diagnosis relies upon physical observations, parasitology, and bacteriology of the diseased

Ishioka: Epidemiology of Marine Fish Diseases

7I

A: GROSS CATCH QUANTITY
OF SEA FISHERIES
, 10
_100

"",,1:)"""0,·,,,,0

z

~

90-

"o

80

0---- 0-0-"-0--0-,•• ,-0,.··,,0------0,·,··,0-

B: PRODUCTION

>70
I--

BY
RATIO

MARINE FISH CULTURE

I-Z

.-

«

3

,.....

...-...-,.....

a/A,.....

:::>

0
I
U
I--

«
u

2
1
0

a . GROSS MARINE FI SHERIES INCOME

25

z
w
>-

N

~~

~

20

w
::>

0

b :MARINE CULTURE INCOME

u

~

RATIO

en

w

-

a:

w

-

b/a

...-

,.....

,.....

10.

8

I

en

6

u.

en
en

2

0

2

a:
Cl

0

'76

'71

'78

'79

'10

'81

'82

'83

'8.5

~

0

YEARS

fish. Because fish are cultured in a group, one diseaseis defined as one diagnostic case of one species in
one fish culture establishment in a region.
The second part of the form contains specific information on the diseased fish; date of observation ofunusual behavior or other abnormal symptoms, suchas body color change or appetite loss; date of appearance of mortality; number of dead fish per pen;whether the progress towards death was sudden or
prolonged; fish size (body length, and weight); andany treatment administrated to the diseased fish.
The third part details cultivation conditions suchas food type and species, region of food production,
size of healthy fish in the pen, the basic informationnecessary for calculating fish density, pen type, andseed information.
The fourth part contains environmental information on the cultivation area, that is, water depth,
weather, degree of rain, temperature conditions,n umber of years the area was used as a fish farm, andFigure 2

Catch quantity and gross income by marine fisheries.

years of fish cultivation experience accumulated by
the aquaculturist.
The fifth part is a memorandum for recording
other information, such as the results of bacterial
checks, symptoms of unidentified diseases, or information on the occurrence of red tide.

Results_

Number of diseases reported
The rate of fish farms requesting disease diagnosis ineach prefecture was calculated by summing the total
number of establishments requesting diagnosis by using
the standard forms for that prefecture and dividing itby the total number of marine fish culture establishments quoted from governmental statistics (SID
1981-90). While both the number of disease cases reported and the diagnostic rate differed from one

72

NOAA Technical Report NMFS III

Table 1
Standard data form used to compile disease diagnosis
information.
Category

Content

Essential
Information

Species nameRegionName of fish farm
Date of investigation
Name of disease
Date abnormality was detected
Date dead fish was observed
Number of dead fish per pen
Was death sudden or prolonged?
Size of dead fish (cm)Weight of dead fish (g)Treatment used
Food type
Region of food production
Age of the fish
Size of healthy fish (cm)Weight of healthy fish (g)Number of the fish per pen
Square measure of the pen (m 2 )Length of the pen (m)Pen type
Days after pen renewing
Place of seed production
Age of seed in troduced
Date of the seed supplied
Water depth (m)Weather
Degree of rain
Temperature conditions
Period the area was applied
for fish cultivation (years)
Length of experience of
aquaculturist
Other information such asbacterial checks, symptoms
of unidentified diseases,
occurrence of red tide, etc ...

Informationon thediseased fish

Information on
cultivation
conditions

Informationon thecultivation
environment

Memorandum

prefecture to another, a considerably high correlation
was calculated between the numbers of disease cases
and numbers of marine fish culture establishments ineach prefecture. Spearman's rank correlation coefficient between number of establishments requesting
diagnoses by their prefectural experimental station andthe total number of establishments in each prefecture
was about 0.9 for yellowtail, and 0.7 for red sea bream
in 1985. These results may suggest that collected data
represent the trend of disease outbreaks in the fish of
the warm current on a smaller scale. Mean diagnostic
rates were about 20% for yellowtail, about 8% for red
sea bream, and about 15% for total marine fishes cultured. Based on these values red sea breams appear tobe less sensitive to disease than yellowtail.

During the 9 years from 1981 to 1989, 56 species
fish were reported to be diseased and over 20,000
disease cases were registered in 20 prefectures. About
three quarters of these cases involved yellowtail. Governmental statistics (SID 1981-90) indicate anincreased number of fish species in marine cultivation. This increase in cultivation may contribute to
the increased number of fish with disease; an especially high incidence of disease originated from therecent increase in the culture ofJapanese flounder.

Disease Diversity
Fish disease was classified into six groups according
to the causal agent: bacteria, parasite, virus, nutriUNIDENTIFIED(6.4%)\

10THERS(0.6%)I

BACTERIAL(76.5% )PARASITIC(6.3 %)

Figure 3
Disease groups observed in marine fish cultures locatedalong the Kuroshio Current.

tion, fungus, and unidentified disease. When the diseases occurred simultaneously, the causal agents wereclassified into both partitions, so the total amount ofrelative frequencies were over 100%. Bacterial diseases were most frequently reported, accounting for
approximately 80% of all disease cases (Fig. 3). Parasitic (8.3%), viral (3.9%), nutritional (6.5%), andunidentified (8.4%) diseases were less frequent. The
partitioning of these values seems to be closely related to the fact that diagnoses were made during theadvanced levels of disease progression. The numberof dead fish recorded in diagnostic cards suggeststhat the diagnoses had been performed after a considerable number of dead fish were noticed.

Species-specificity of diseases
The relative frequencies of the six disease groups var-Ishioka: Epidemiology of Marine Fish Diseases[I]] BACfERIAL~ VIRAL73~ UNIDENTIFIED(I PARAsmc [I NUTRITIONAL ~ OTHER
Paralichthys olivaceus

N=37

Sebastes schleaeli

N=901

Takifugu rubripes

Seriola du.erili

N=428

Seriola au reov i ttata

Seriola quinqueradiata

N = 171

Caranx delicatissiMUs

Trachuru8 japonicus

Pagrus major

N =104

Acanthopagrus schleaeli

Lateolabrax japoniCU8

N=S9Oplegnathus PUCtatU8

Oplegnathus fasciatu8

120110100 90 80 70 60 50 40 30 20 100

Relative Frequency (%)

Figure 4

Composition of disease varieties in each species.

ied with fish species (Fig. 4). Over 90% of the diseased yellowtail experienced bacterial disease. In red
sea bream, a high rate (22.1 %) of parasitic disease
resulted from parasitism by Bivagina tai on the gill, byLongicollum pagrosom in the rectum, andby other parasites. The high rate (33.4%) of nutritional disease involves yellow fat disease and peroxidative lipid intoxication caused by lipid metabolism
disorders.
In tiger puffer, several serious parasitic diseases(Heterobothrium tetradonis, Trichodina sp., andCryptocaryon irritans) break out frequently (39.2%).
The specific viral disease (9.0%), named kuchijiro
sho (white mouth symptom) is often reported; its

pathogen has not yet identified officially. The rate ofunidentified disease was highest among the 13 fish
species listed in Figure 4.
In Japanese flounder, bacterial disease accounted
for about 67% of all the cases registered, while therate of parasitic disease, mainly due to a Trichodina
sp. and Cryptocaryon irritans, was 17%.
The relative frequency of the six disease groups
was not related to any taxonomical order nor to theecological habitat of the cultured fish species.
The relative frequency of diseases caused by different pathogenic bacteria, are shown in Figure 5 for 13
fish species. Yellowtail, purple yellowtail (Seriola
aureovittata) , and gold striped yellowtail (Seriola

74

NOAA Technical Report NMFS 111

ORDER

Tetradontifo.-es

StJalRDER

lpleulonectifor~~

Percoidei
Carall&idae

FAMILY'FT~=~ifl

Oplegrthidae

Opn

SUBFAMILY

hUS

GENUS

120
110
~100

>- 90

~::l 80go... 70

~

60

~
.....
..., 50...al 40
~

IX

30
20
10
0

2l
....l'll
~...
~

::l

....S::l0'.......0
al

...
Jl

....,..,....
...
I
'I:l

...al0.......
Jl

2l
2l
...,
'>
...~::l
al
al

...0
.....

...
~

VJ

til

OIl

G
....

....i

8.

....
...,

rJl
::l

.......

::l

'tl

U

~

l:

al
....,

...

..c:

...
al

""

......

Ol...,::l

i...
.c

l'll

'u

rJl
rJl

~

rJl

B

rJl
::l

.c
...,

~

...!

...al

~

~

U

....al

8

til

rJl

.E...,

...~
.fj

! ...
... ...,~
al

~

8

~

rJl

...::l...

Ol.......~....,0

i

.fj

rJl

C
i::l
....
....

...::lall

~

j
...,

..>C

al

""

s::

.......
~.....c:
~

U
til
rJl

...,~
til

.!

Jl

~
«:

•

Gliding bacterial Disease

dumerili), showed higher rates of pseudotuberculosis
and streptococcosis diagnoses, whereas jack mackerel(Trachurus japonicus) , taxonomically closely related toyellowtail, had high rates of vibriosis andstreptococcosis.
Red sea bream were frequently infected with Vilmo
spp., Edwardsiella tarda, and gliding bacteria (mainly
Flexibacter sp.), but less so with Streptococcus spp. andPasteurella piscicida. No edwardsiellosis was reported
from black sea bream (Acanthopagrus schlegeli) , which
are related to red sea bream.
Japanese flounder were seriously infected with
Edwardsiella tarda and suffered heavy damage; therelative frequency of edwardsiellosis in Japaneseflounder amounted to 41.8% of all bacterial disease
cases. They were affected to a lesser extent by gliding~...."~0til>~.c.......

U

...al

~

o Vibr·i08il!l [) Pseudotubel'cl1losi.s ~ Streptococcosis§I Edoerdsiellosis

tiluFigure 5
Composition of bacterial
diseases in each species.

bacteria and Streptococcus spp.
Tiger puffer suffered mainly from vibriosis andgliding bacteria infections.
With some exceptions, the relative frequency ofbacterial disease seems to be related to the host by
the host's ecological niche and related physiologicalcharacteristics. As shown in Figure 5, pelagic or migratory fish species such as yellowtail were more
susceptible to Pasteurella piscicida, while bottom fishes
like tiger puffer, Japanese flounder, and Japanese
rockfish (Sebastes schlegeli), were more susceptible togliding bacteria. Other species in Figure 5 fell between these two extremes.
A species' ecological habits are closely related to its
anatomical, physiological, and biochemical characteristics. Pelagic and migratory fishes have a typicalIshioka: Epidemiology of Marine Fish Diseases

75

35
30

......./"----x~,d:----5./-._----:.~-......................

o-J....--...----r----.-__~---.~__~__r---r__-r---r__-,..
JAN

FEB

MAR

APR

MAY

JUN

JLY

AUG

SEP

OCT

NOV

MONTH
-YELLOWTAIL---- RED SEA BREAM.... ·JAPANESEFLOUNDER

spindle shape suitable for swimming, developed cerebellum and vascular system, abundant superficial
dark muscle, and an abundance of myoglobin in
their ordinary muscle. On the other hand, the bottom fishes of coastal areas, such as Japanese flounder,
rockfish, puffers, and sea breams have flat orrounded shapes, less-developed cerebellums, andcharacteristics that indicate that these fishes are generally less active. Their white ordinary muscle has lessmyoglobin and hemoglobin, and their superficial
dark muscle is not as developed.
Because Pasteurella piscicida, a causative bacterium forpseudotuberculosis, migrates in the bloodstream of infected fish, active fish, with well-developed vascularsystems, may be more likely to reach an exhausted state.
Infections by gliding bacteria tended to break out
in benthic fish or fish living near the beach. Because
these fish are less active than the pelagic species andmay have a susceptible body surface, gliding bacteria,
which move by means of surface mucus, may have an
advantage in expanding their living area. Also, because these fish are more likely to come into contact
with other individuals via their body surface, new infections and propagation of gliding bacteria areprevalent.
It is believed that the accumulation and integration of information on fish species and pathogens(such as cell or tissue susceptibility to pathogens; anatomical, histological, and physiological aspects offish; environmental conditions; and characteristics of~- TIGERPUFFER

DEC

Figure 6
Seasonal changes in
number of diseases
registered between
1981 and 1989.

the pathogen) are very important to understand thephenomenon or mechanism of bacterial disease outbreak.

Seasonal Variations of Disease Outbreaks
Monthly variation in the relative frequency of disease
cases reported is shown in Figure 6, for four fish species: over 900 cases for each species were reported
during the 9-year period 1981-89.
The highest evidence of disease occurred during
July for yellowtail (30%) and tiger puffer (17%), during August for Japanese flounder (15.5%), andduring September for red sea bream (13%). Reportsof disease for the latter three species peaked in thesummer from June through September when the water temperature is higher than 20° C. These peaks
were not as steep as that of yellowtail, yet the summer
peaks suggest that fish under cultivation are exposed
to physiological and environmental conditions favorable to infection by pathogens, in spite of possible
efforts to promote the defensive activities of fish,
such as immunological resistance.
In yellowtail, viral and bacterial diseases, especiallypseudotuberculosis, show significantly sharp seasonalchanges (Fig. 7). Since the official identification(Sorimachi and Hara 1985) of the viral disease viral
ascites (VA), the rate of this viral disease has increased. VA disease breaks out in late May, reaches a

76

NOAA Technical Report NMFS 111

30

:.

25

-VIRAL

I

I
I
"

I
I

I

"

I
I

o J..;.';:"':':"':;:"~-~":.;'-:;':-~-;';"""F=r=9'-'-'""'t'-..,.........,--,-

<J
Z

w

.,

1.2

5 1.1

-VIRAL

~

1

LI..

0.9

...... PARASITIC

~

0.8

oJ

0.6
0.5

i

3
2
1

...'

\

I

I

I

\

I

. ..

\

//

..

.................

",\k......

~~/:.:::.: ••-::;.....

o-Y~~=;==r=;=:::r-....,.-..."f"""-~:.=,........;r:4JAN

FEB

MAR

APR

MAY

JUN

JLY

AUG

SEP

OCT

NOV

DEC

MONTH
-VIR~L'< 0.7
~

I

5
4

"

\,

"
I

/

6

~~~

>-

I

7

I
I

10

r-------

8

I

15

/

10
9

I

··,···BACTERIAL

r---'

11

I
I

..·········NUTRITIONAL

20

12

/\

0.4

.. ··PARASlT)C- - -NUTRITIONAL -UNIDENTlfIED- .... _- BACTERIAL

Figure 9
Seasonal changes in diseases ofJapanese flounder.

0.3
0.2
0.1

0...Lr-.,........;.-.f.--,..-.,....-,..---:"""';;;;;;;=_.....-,...
JAN FEB MAR

APR MAYJUN

JLY

AUG SEP OCT

NOV

DEC

MONTH

Figure 7
Seasonal changes in yellowtail diseases.

6
\

.' ,

\
\
\
\
\
\
\
\

\

\

•

\

",..I

• I
\
\

• I
,J

•

:..------\~/ .......
0...L,---r--t--r-~--,r----,.---r--::;=---,.---r-----.
APR
MAY
JUN
JLY
AUG
SEP OCTNOV
0 C~
FEB MAR

MONTH
-

VIRAL

••••. PARASITIC - - -NUTRITIONAL-

UNIDENTIFIED •• _. BACTERIAL

Figure 8
Seasonal changes in diseases of red sea bream.

peak in June, and ends in mid:July. The rate of VA
disease diagnoses was very low and account for only
1 % of yellowtail disease reported in June; its occurrence fluctuates yearly. The rate at which the major
bacterial diseases were reported showed similar seasonal variations. Vibriosis accounted for about 10%of all the bacterial diseases reported and the numberof cases peaked in June. Cases of pseudotuberculosis
peaked sharply in July (see Fig. 11), and streptococcosis epidemics seem to occur between July andOctober. Parasitic, nutritional, and unidentified diseases showed gentle seasonal changes in theiroccurrence. Parasitic and unidentified diseases brokeout during summer, and the level of nutritional diseases peaked in autumn.
In red sea bream, viral diseases represented only
3.6% of the total di ease cases reported (Fig. 8), andfor the most part were observed as lymphocystis disease every month of the year. Parasitic diseases werereported least in February and March and most frequently between May and December. Nutritional
disease outbreaks appeared between May and September, indicating that they are chronic in nature.
Half of the bacterial disease cases reported werevibriosis (53.2%), which occurs all year round with apeak at winter (see Fig. 14). Gliding bacterial diseasealso occurred all year round but the higher rates
were found in January and from May through July(see Fig. 15). Edwardsiellosis accounted for about
10% of all disease cases in red sea bream and wasprevalent between July and November (see Fig. 13).
Since the successful development of seedling production methods, production of the Japaneseflounder has gradually increased. The main viral disease observed in cultural flounder was epidermal
necrosis, which occurred in spring during the fry
stage and accounted for only 0.8% of all disease cases(Fig. 9). Japanese flounder suffered from parasitism
by protozoa such as Cryptocaryon spp., Trichodina spp.,
apd other Ciliata. Because there are no effective
treatments for these parasites, infected fish are left
untreated, resulting in a great deal of infection anddeath. A trial is now being conducted to see if conditions leading to infection can be effectively removed

Ishioka: Epidemiology of Marine Fish Diseases

77

by increasing the water supply to rearing areas. It is
unknown why nutritional diseases are rare in Japanese flounder. Bacterial diseases are serious for thisfish accounting for about 67% of all disease cases
reported. The most virulent, edwardsiellosis,
amounts to about 28% of all disease cases.
Edwardsiellosis is most prevalent during the summer,from June through October, with the number of reports peaking in August. Streptococcosis is mostprevalent from July to October and vibriosis and gliding bacterial disease are both most prevalent between
April and August. Unidentified disease accounts for
16% of all disease cases reported.

6

5
JAN FEB MAR

4

APR MAYJUN

JLY AUG SEP OCT NOV DEC

M 0 N TH-'.

...........

oJ..;:::::::;::::::;=::::;:::::::::;:::...,.-.--..,......--.----r-~~
JAN

FEB

MAR

APR

YELLOWTAI L----- RED SEA BREAM

JAPANESEFLOUNDER-

TIGER PUFFER

Figure 11
Seasonal changes in pseudotuberculosis for each species.

MO NTH-VIRAL•.•• PARASITIC- - - NumlTlQNAL-

UNIDENTIFIED ---.BACTERIAL

Figure 10
Seasonal changes in diseases of tiger puffer.

10
~

>u

z

7

0

6

W
::l

In tiger puffer (Fig. 10), kuchijiro sho was registered often throughout the year. Parasitic disease
amounted to about 39% of all disease cases (Fig. 10).
Fatal parasites included a Trichodina spp., a protozoa
which was prevalent between April and October, andHeterobothrium tetrodonis, a monogenea prevalent
mainly from April through August. Nutritional disease was also prevalent from spring through autumn.
Higher levels of bacterial diseases were diagnosed
from June to September: most were cases of vibriosis
and gliding bacterial disease.
Pasteurella piscicida was a virulent pathogen forpseudotuberculosis in yellowtail and to a less extent
in other cultured fish species (Fig. 11). This diseasebroke out seasonally, appearing during warmer
months in all fish species.
Streptococcus sp. was most common in yellowtail andJapanese flounder and occurred less frequently in
red sea bream and tiger puffer, showing similar seasonal variation patterns that peaked in September(Fig. 12). Edwardsiella tarda was virulent for Japaneseflounder and red sea bream and most prevalent from

9

~ 8

w

i1: 5
w

:::I- 4

:sw 3
a:

2

1

a
JAN

FEB

MAR

APR

MAY

JUN

JLY

AUG

SEP

OCT

NOV

DEC

MONTH
-YELLOWTAIL-----RED SEA BREAM .. ···JAPANESE -TIGER PUFFERFLOUNDER

Figure 12
Seasonal changes in streptococco-sis for each species.

June through October; the level of reported cases
peaked during August in Japanese flounder andshowed a plateau from July to November in red sea
bream (Fig. 13). The optimal water temperature
ranges from 15 to 24° C for Japanese flounder and isslightly broader from 13 to 28° C for red sea bream.
Cultured Japanese flounder suffer from exposure to

78

NOAA Technical Report NMFS 111

7...................................

>-

i' 5

ii/i./··

w

:::>

~

4

IE
w 3

>

52
w
a:

JAN

FEB

MAR

APR

MAY

JUN

JLY

AUG

SEP

OCT

NOV

DEC

MONTH
-----RED SEA BREAM·• .. ·JAPANESE FLOUNDER

Figure 13

Seasonal changes in edwardsiello-sis for each species.

5

~ 4
><.J

z

w
::>

3

~

...a:

high temperatures above 25° C in summer and become
more susceptible to bacterial infection. On the other
hand, red sea bream grow well during summer and suffer from nutritional disorders in autumn. Because
Edwardsiella tarda can multiply more actively during thesummer season, Japanese flounder, which are less tolerant of higher temperatures, are more susceptible to thispathogen. Conversely, red sea bream, which are moretolerant of higher temperatures, are less susceptible tothis pathogen than Japanese flounder.
Seasonal variations in the relative frequency ofvibriosis are complicated and differ according to fish
species (Fig. 14). This complexity may result not only
from the difference in susceptibility of each fish species but also from the presence of different varieties ofVibrio. Vibrio anguillarum is known to have varied seratypes. Susceptibility of fish to this pathogen would varywith the serotype and fish species involved.
The term "gliding bacteria" implies several bacterial species. The gliding bacteria resulting in marine
fish diseases are mostly Flexibacter, Cytophaga, andSporocytophaga spp. The unique characteristics of eachgliding bacterium may contribute to the complicatedpatterns of seasonal variations in reported infection
among the different fish species (Fig.15).

2

Fish size and disease
JAN

FEB

MAR

APR

MAy

JUN

JlY

AUG

SEP

OCT

NOV

OEC

MONTH
-YElLOWTAll-----REO SEA BREAM ······JAPANESE · - -..TIGER PUFFERFLOUNDER

Figure 14

Seasonal changes in vibriosis for each species.

6

5

:/\"

/

4

.

/A

3
2

~ ~:.:./.;;/._-----<:~:. ~o--l..f:-r:::.,.;....."T--r--r--r--r--=r=T=:::::;:~'T""
JAN

FEB

MAR

APR

MAY

JUN

JLY

AUG

SEP

OCT

NOV

DEC

MO NTH-YELLOWTAil ----RED SEA BREAM ····JAPANESEFLOUNDER••- TIGER PUFFER

Figure 15

Seasonal changes in gliding bacterial diseases for eachspecies.

The variation In relative frequency of disease cases
reported in yellowtail is illustrated according to fish
size (body weight) in Figure 16. For this analysis, relative frequency was calculated by dividing the total
number of reports for each disease in the definedsize class by the total number of disease cases withbody weight (bw) data for each species. Viral disease
was more frequent in younger fish «50g bw), while
nutritional diseases were more prevalent in older fish(>250g bw) and season also an influencing factor. Occurrence of bacterial disease appears closely related
to body weight. Yellowtail seemed to be successively
attacked by VA virus first, then Vibrio and Pasteurella
piscicida, and finally Streptococcus. This sequence of infection suggests that susceptibility of yellowtail todifferent pathogens varies with body weight or
growth. This may imply not only the development ofdefense mechanisms to the pathogens but may also
represent the changes in the overall physiology of the
fish under cultivation.
In red sea bream, parasitic diseases occurred more
frequently in fish between 50 and 250 g bw while
reports of nutritional disease increased in fish over
250 g bw (Fig. 17). The rate of gliding bacteria diagnoses was highest in young fish below 10 g bw andthat of vibriosis was highest in fish between 50 and
100 g bw. The diagnostic rate of edwardsiellosis wasIshioka: Epidemiology of Marine Fish Diseases

1.4

OJ VIRAL

1.1
1

1.1
1.1

~

PARASITIC•

NUTRITIONAL

~

1.4
\.1
_

1

>'1

- 0.1

~Ol
~0.4

601

°

w
a:

u.

~ 10

lITI VIBRIOSIS!;t 11
.J
W

a:

1&

ESI PSEUDOTUBERCULOSIS

14•

STREPTOCOCCOSIS

11
10

=<10
=<SI

=<111·=<SII=<1111=<IHI=<4110=<1110=<1110=<IS0=<IS0=<ISI1=<3111=<S"O=<1101
BODY WEIGHT

(9)

13
11
11

Figure 16
Relationship between fish size and disease
outbreaks in yellowtail.

VIRAL

10
9

PARASITICI

NUTR IT IONAl

1
>'1

6
S

UNIDENTIFIED5

VIBRIOSIS

>u

z

w
:::>

o

w
a:
u.
w

>

>=
«
.J
~.

PS EUDOTUBERCUlOSIS
STREPTOCOCCOSISEDWARDSIELLOSISBACTERIA

=<10

=<110
=<\1

=<SII
=<ISI

=<1111=<ISI=<1110=<ISIOBODY WEIGHT (9)=<4111=<3100Figure 17
Relationship between fish size and disease
outbreaks in red sea bream.

79

80

NOAA Technical Report NMFS 111

El

VIRAL

ES PARASITIC•m

NUTRITIO~AL

UN IDENTI FlED

lot

>-

()

z

w

::>
0

w
a:

u.

21
VIBRIOSIS

11

w
>

II

«

14

;::
..J

w
a:

\2

[Sl

PSEUDOTUBERCULOSIS•STREPTOCOCCOSISoEDWARDSIELLOSISIImGLIDING BACTERIAL

=<11

=<111

=<HI

;

511

=<111

BODY

WEIGHr(9)=<1111 =<1111=<2111Figure 18
Relations ip between fish size and disease
outbreaks in Japanese flounder.

40

3D

EI VIRAL
~

•

20
lot

~

>-

PARASITIC
NUTRITIONAL
UN IDENTIFIED

~"
w

::>
0

w
a:

u.

w

:::
~

«

..J

w
a:

OJ VIBRIOSIS[S3 PSEUDOTLBERCULOS I S•

STREPTOCOCCOSIS

FE! GLIDING BACTERIA

51 ;<1111 =<1500=<1000=<3000
BODY

WE IGHT(g)

Figure 19
Relationship between fish size and disease
outbreaks i tiger puffer.

Ishioka: Epidemiology of Marine Fish Diseases

higher in larger fish, especially those over 2S0 g bw.
All ages of Japanese flounder experienced outbreaks of parasitic diseases, while viral diseases were
found only in fish below 10 g bw (usually herpes virus) (Fig. 18). Vibriosis and gliding bacterial disease
outbreaks were more common in younger fish«SOg). Although streptococcosis and edwardsiellosis
were both more prevalent in larger fish (>2S0 g),
edwardsiellosis was more common than streptococcosisin the fish of SO-1 00 g bw.
In tiger puffer, a unique feature was found: fish
2S0 to SOO g bw appear to be most susceptible topathogens (Fig. 19). This may be dependent on themethod used to culture this species. Though thisspecies is in great demand by the market, its cultivation from the egg through the spawning stage isvery difficult. The eggs for seedling production are
generally raised by the public sector and are in mostcases supplied from the natural resource byfisheries, which also provides some large sized seedlings. Because the aquaculturist cultures mainly
large-size seedlings, diagnoses may concentrate on
the larger sized fish.
In all species, nutritional disease tended to occur
in larger fish, probably as a result of prolonged nutritional treatment. Outbreaks of edwardsiellosis andstreptococcosis were synchronized with nutritional
disorders.

Locality and Disease
As shown in Figure 20, for yellowtail in 1986, somecharacteristics are common to all the prefectureslisted. Although the relative frequency of bacterial
disease was about 80% in all prefectures, the compositions of different bacterial diseases were to someextent different by locality. Mie Prefecture is themost northern of all the prefectures listed in Figure
20, while Kagoshima Prefecture is located in mostsouthern part of Japan. Interprefectural analysis suggests that the composition of different bacterial
diseases seems not to be related to geographical locality. A possible similarity exists between thecomposition of bacterial infections observed and thetype of cultivation management used in some prefectures. There are two types of management:
cultivation of large seedlings and production of fishof marketable size. Streptococcosis tended to be reported at a higher rate in the prefectures managing
the larger fish (2-year-olds) of marketable size.
No characteristic difference in seasonal outbreak

139

164

45

72

100

~ UNIDENTIF lED~Jm1tJ OTHERS

80

m

~

>-

NUTRiTIONAL~ VIRAL
60~PARASITICU
Z[IIllll]
0 T H ER. . .. BACTERIAL DISEASE

w
::;)

0

w
a:

u.

. . NOCARDIOSIS40wI){{id PSEUDOTUBERCULOSIS

>
«

I-

~ VIBRIOSIS

...J

W

a:

20

DISEASES:=:.::.: COMBINEDWITH STREPTOCOCCOSIS

c=J
w

0

i

0

:E

LU

~

>-

J:

i

u

0

>::

«
~
«
Cl
«
>::

«

0

b

:E

:E

«

en

:E

Cl

::;)

>::

J:

-w

:E

0

«

>::

~

«
«

N

>-

:E

~

«
en
«
Cl
«
z

81

«

I-

0

«
«
en

Cl

«

:E
J:

en

::;)

>::

0

I-

«
:E
«
>«
>::
«
~

J:

u

::;)

Cl

«
«

:E

>-

PREFECTURE

Figure 20
Composition of diseases of yellowtail by prefecture during 1986.

STREPTOCOCCOSIS

82

NOAA Technical Report NMFS 111

50

40

M IE

40

KOCH I3

N= 327

30

2

20

10

N=288

10
Ol.,....................--.-....................................L,-I..L,-I..L,-I~

WAKAYAMA

::!

N=19

o....,---..--.-..............

; ::[ HY::~'9

NAGASAKI

Dou-::

N=139~L1J ~:o [. "-r-Y-A"'~-~"':-~"'C-H"'I-. , . . . ,. . Jn n :~'-r-. . . --r--.-,......_.....-......

L..,J..L,.J"""""",

> 40
~

:} 30
L1J
C(

, ,

, , , , Llol.lo....,
2

KUMAMOTOTOKUSIMA

N=81

10

N= 72

20
10
rI

o

40

30

In 213~01[l"
48 ...-,.......-.-.........-.-.........-.-.&;.;;.1-0~_

KAGAWA
N=100

20

n

10
0
40

30

OITA

N=15'

MIYAZAKI

30,....,

N=49

20

EHIME
N= 78320

00......

In ......

of virulent disease was found according to geographical location. In the example of 1986 shown in Figure
21, pseudotuberculosis broke out earlier in warmer
areas. This disease was reported to occur at a higher
rate in June and July in Kagoshima, Miyazaki, andKochi Prefectures, while the rates were higher in Julyand August in other prefectures. Because the optimal
range (20 to 25° C) for multiplication of Pasteurella
piscicida is strict, the location of outbreaks of this disease could be determined by calendar and locality orlatitude.
As a whole, no characteristic local feature was observed that could explain particular difleaseoutbreaks in the area surrounded by warm oceanic
current, except perhaps slight differences. This sug-

Figure 21
Patterns of the outbreak ofpseudotuberculosis in yellow-tailin each prefecture in 1986.

gests that all fish culture fields along the warm current are in an area favorable to the multiplicationand propagation of pathogens.

Discussions_

From this epidemiological examination, it is clearthat the natural and delicate relationship between
fish and their environment is disturbed on a local
basis by fish cultivation in coastal areas and induces
the propagation of pathogens and the outbreak of
disease in the cultured animals. As shown in Figure
22, the means and methods of fish culture affect not
only fish health but also water quality and pathogen

Ishioka: Epidemiology of Marine Fish Diseases

CULTIVATIONTECHNIQUE

Figure 22
Disease outbreak flowchart for marine fish cultured in the
Kuroshio Current.

distribution; major factors include fish density in apen, location of the pen, pen density, handling, excess food supply, food quality, sanitary treatment ofnets, type of food and other materials, and treatment
of dead fish. Supplying unsuitable or unsanitary foodinduces the degeneration of fat in fish, whereas supplying excessive food makes them generate fat.
Unsuitable handling techniques injure the body surface, especially in younger fish, and allows pathogens
to invade. Fish are also susceptible to pathogens under some nutritional conditions and during certain
developmental stages. Culturing fish under high density adds additional stress.
A high density of pens interrupts the water current
and causes water quality to deteriorate. As excess
food and fish excrement are deposited on the bottom and dissolve into seawater, the quality of water inthe cultivation area degrades. These changes in environmental conditions are beneficial for pathogen
multiplication, which, in turn, lead to infection of
the fish and to outbreak of disease.
As previously described, the seasonal changes of
disease cases are very remarkable, and it is thoughtthat temperature is the most influential environmen-

83

tal factor. Higher temperature is beneficial in activating various physiological functions, including
immunological processes in healthy fish. But this factor can also be a harmful to unhealthy fish underpoor environmental conditions.
The results of this study help us to direct future
study and research in this field. Unidentified diseases
must be clarified as soon as possible, especially in
new fish species introduced for cultivation. In thistype of research, it is most important to have a thorough knowledge of the anatomy, physiology, ecology,
pathology, and immunology of the fish and pathogens. This may lead to the development of improved
cultivation techniques.
Effective methods to diagnose fish health should
be developed, not only to prevent mass mortality by
pathogens that are generally nonvirulent, but also toregulate cultivation techniques. A large amount ofresearch is needed in this area, for example: physiological studies on poikilothermal fish, pathologicalstudies that clarify the mode of action of pathogen
within their hosts during infection, and immunological studies to explain fish defense mechanisms
against pathogens.
Epidemiological research in a small area such as
one bay unit or a fisheries cooperative association
unit should be carried out in detail to clarify the direct relationships among disease outbreaks,
environmental factors, and cultivation techniques.
Such research could provide knowledge for a more
successful cultivation of healthy fish.

Citations_

Sorimachi, M., and T. Hara.
1985. Characteristics and pathogenecity of a virus isolated
from yellowtail fingerlings showing ascites. Fish Pathol.
19:231-238. (In Japanese.)
SID (Statistics and Information Department).
1981-90. Annual statistical report on fisheries and culture
production of Japan. SID, Jpn. Min. Agri. Forest. Fish.,Tokyo. (In Japanese)
Umezawa, S., and H. Ishioka.
1988. Program for compiling disease cases diagnosed in prefectures. In Personal computer programs for fish population dynamics, vol. 1, p. 314-339. Tokai Regional Fisheries
Research Laboratory, Tokyo. (In Japanese.)\Characterization of Hematic Neoplasia in theSoftshell Clam Mya arenaria•
PAUL W. RENO*, ANDREA ILLINGWORTH**, and MICHAEL DORITY****Coastal Oregon Marine Experiment Station/Microbiology
Mark O. Hatfield Marine Science Center
2030 S. Marine Science Dr.
Newport, Oregon 97365-5296**lnstitute Jor Cellular Research
345 State St.
Bangor, ME 04401***Department oj Microbiology
181 Hitchner Hall
University oj Maine
Orono, ME 04469-0131

ABSTRACT
A leukemia-like disease, variously termed hemic, hematopoietic, or hematic neoplasia,(HN) has been detected in bivalves from diverse geographic locales. The wide geographic range of the disease, coupled with mortalities due putatively to HN, makes it
important to determine the nature of the abnormal cells characteristic of the disease.
The studies reported here determined certain biochemical and genomic characteristics
of HN in the softshell clam Mya arenaria. No significant difference in the total amount oflipid of normal and abnormal hemocytes was noted. Fatty acids of the omega 3 andomega 6 families were elevated in clams with the highest HN intensities. Similarly, invitro incorporation of acetate into phospholipids and neutral lipids by normal lymphocytes was significantly different from HN hemocytes but unlike the pattern seen in
mammalian leukemias. Examination of the DNA content of normal and HN cells by flow
cytometry indicated a high degree of aneuploidy in the HN cells. This consisted of clams
with hyperdiploid DNA content of 1.6 to 2.0 times normal, and a hypodiploid population
with DNA 0.85 times normal. Hyperdiploid cells had significantly higher DNA synthesisthan diploid cells. In this respect, HN cells are similar to true leukemias of mammals, but
the combination of hypodiploid and hyperdiploid cells in one individual is uncharacteristic of mammalian neoplasia. In summary, hemocytes of M. arenaria affected with HN
have some features coincident with mammalian leukemias but also several unique characteristics.

Introduction_

The harvest of bivalves from commercial and cultured sources is of considerable economic importance on both worldwide and national scales. Disease affects natural as well as artificially propagatedpopulations. Unfortunately, our knowledge of diseases in bivalves is quite restricted in comparison to
the information base available for finfish. It is important that we establish a more detailed database fordiseases of cultured and feral bivalves in order to predict sustainable harvests of this significant marine
resource accurately.
Among the most widespread diseases of bivalves
are the putative neoplastic diseases which have been
detected in a wide variety of commercially important
species and from a geographically disparate range*Send correspondence to this author.
85

86

NOAA Technical Report NMFS III(Couch 1969; Farley 1969; Brown et al. 1977). The
primary tissue distribution of these diseases appears to be gonadal ("germinomas") (Yevich andBarszcz 1976, 1977) and hematic (variously termed
hematopoietic neoplasms, hematic neoplasms, or
hemic neoplasms). Hematic neoplasia (HN) has
been associated with elevated levels of mortality infield and laboratory studies with the softshell clam
Mya arenaria (Appeldoorn and Oprandy 1980; Cooper et al. 1982) and the edible oyster Ostrea edulis(Alderman et al. 1977). The prevalence rates for
HN in M. arenaria range from 0.02 to 84% , andintensities of HN in individuals span the range
from less than 0.01 % to greater than 99% of hemocytes affected (Couch 1969; Mix 1986; Reno,
unpubl. observations). The pervasive nature of thisdisease and its potential for killing feral and cultured bivalves makes it essential to understand thenature of the disease and its etiology. The results
reported here reflect an initial attempt to define
more clearly the nature of the disease in M.
arenaria.
Hematic neoplasia is characterized by its proliferative nature and the resulting cytological morphological abnormalities assessed by microscopic
examination of either unstained, or fixed andstained cells (Couch 1969; Farley 1969; Cooper etal. 1982). These criteria, however, are not adequate
for accurately defining HN in bivalves as neoplastic
because these invertebrates are evolutionarily distant from the mammals, the phylum on which
virtually all definitive work has been done on thecharacteristics of neoplasia. This lack of definition isnoted in two prominent reviews of presumed neoplasms of bivalves: "Some of the neoplasms may beof questionable validity ... and little is known about
the exact nature of the cells involved but, in general, they are considered neoplastic" (Mix 1986);
and "In spite of intensive study, the true nature ofthese neoplastic cells is not yet quite clear."(Lauckner 1983). There are scores of structural,
biochemical, immunological, and genomic alterations in mammalian cells associated with thetransformed or neoplastic state (reviewed in Wood
1972; Marchesi 1976; Vasiliev and Gelfand 1981;
Heim and Mitelman 1987; Iversen 1988). Amongthe most well characterized biochemical alterations·that occur in transformed cells are those occurringin two fundamental classes of molecules: the nucleic
acids and the lipids. The present work was designed
to monitor potential differences between normal
hemocytes and HN cells from M. arenaria comparedby flow cytometric analysis and in vivo and in vitro
lipid constitution.

Materials and Methods_

Sample Collection, Evaluation of HN, andAnimal Maintenence
Softshelled clams ranging in size from 45 to 120-mm
valve length were collected from Long Cove,
Searsport, Maine, during the summer, placed on ice,and returned to the laboratory for evaluation of HNlevels. When necessary, animals were held in the wetlaboratory at the University of Maine in a recirculating artificial seawater system at 14° C until
needed. Animals were generally evaluated within 1
week of collection. Evaluation of HN was performed
by a modification of the method of Cooper (1982).
Approximately 100 f.LL of hemolymph was removed
from the cardiac or anterior adductor sinus, 50 f.LL
was placed in one well of a 96-well, flat-bottomed
microtitration plate, and hemocytes were allowed toadhere for 20 to 30 minutes. Samples were thenevaluated for the levels of HN by microscopic examination under phase optics at 200X with an inverted
cell culture microscope. The number of nonadherant
or loosely adherant, rounded cells in the hemolymph(HN cells) was compared to the number of fully adherent spreading normal hemocytes by counting 500
to 1000 cells and converting the proportion topercen tages.

Lipid Analysis
The major lipids that are altered in neoplasia are thefatty acids, the neutral lipids, and the phospholipids.
Several methods were used to evaluate lipid changes
in HN cells, including gravimetric total lipid content,
fatty acid methyl ester (FAME) profiles, and in vitro
incorporation of radiolabelled acetate into neutral
lipids and phospholipids.
Gravimetric analysis was carried out on washed hemocytes from normal and abnormal clams using themethod of Sasaki and CapuzlO (1984). This involveda modified Folch procedure employing several extraction cycles with chloroform:methanol. Total lipid
by weight was obtained using an ultramicrobalance(Cahn model 25 automatic electrobalance). Fatty acids were esterified to fatty acid methyl esters (FAMEs)
to stabilize them prior to analysis, which was carriedout on hemocytes from clams with various levels of
HN and clams with no evidence of HN. Fatty acidseparation was carried out using the modified Blighand Dyer (1959) method of Jerkofsky and DeSiervo(1986). Washed, pelleted hemocytes were extractedwith chloroform:methanol followed by a saline phaseReno et al.: Hematic Neoplasia in the SoftsheU Clam Mya arenaria

separation, and final suspension in chloroform. A
known quantity of a 19-carbon fatty acid was added tothe cell pellet prior to extraction to serve as an internal standard for the extraction process. Samples weretransesterified with a methanolic base reagent followed by methylene chloride and final suspension inhexane. Esterified samples were then applied to a gas
chromatograph (Hewlett Packard model HP5890A)
equipped with a 30 m DB carbowax capillary column.
Peak areas were calculated with a disc integrator, andretention times were compared with known standard
FAMEs.
In vitro assimilation of radiolabelled acetate into
neutral and polar lipids was carried out using hemocytes that were removed from the clams within 8hours of capture. To each of four replicate tubes containing the hemocytes, an equal volume of 0.45
fl.m-filtered sterile seawater containing 10 fl.Ci of 14C_
acetate was added. The cell-acetate mixture wasincubated at 15° C for 24 hours and extracted as described for FAMEs, except that, in order to improve
the precipitation of labelled lipid, non-labelled carrier lipid was added. The carrier lipid was preparedfrom two shucked clams by removing the digestive
diverticula and skin of the siphons, blending them ina blender, and extracting as previously described.
The lipid extract was weighed and resuspended inchloroform to a concentration of 300 mg/mL; 30 mg
of carrier lipid was added to each sample. A final
suspension in solvent was divided into 2 aliquots
which were then evaluated for either neutral lipids by
one-dimensional silicic acid paper chromatography(Wuthier 1966) or for polar lipids by two-dimensional silicic acid paper chromatography, followed byautoradiography Uerkofsky and DeSiervo 1986).

Flow Cytometric Analysis
In order to determine the configuration of the DNA
in the genome of HN cells versus normal hemocytes,
flow cytometric analysis of the total DNA content of
the cells as well as the cell cycle status of the cells was
carried out. Preliminary experiments were performed to determine whether live or fixed hemocytes
were optimal for flow cytometric analysis. Fixation of
HN hemocytes in either 2% formalin-seawater or 2%
glutaraldehyde-seawater led to reduced resolution of
DNA peaks compared to unfixed materials suspended in the anticoagulant 0.01 M cysteine inseawater. Consequently, fresh, unfixed, washed hemocytes were used for the flow cytometric analysis in all
further studies. The processing of hemocytes for flow
cytometric analysis of DNA followed standard methods used in human clinical oncology (Raber 1988).

87

Cells were washed in buffered saline with 0.1 % bovine serum albumin, counted, and suspended in asolution of 1 fl.g/mL RNAse and 0.01 % NP-40 detergent to increase membrane permeability. This wasfollowed by the addition of propidium iodide
fluorochrome at the ratio of 10fl.g/l06 cells in a solution of NP-40 and 0.1 % polyethylene glycol 8000 at
pH 5.0. Examination of the cells under a ultraviolet
light microscope confirmed that nuclear morphology
was normal and that no artifacts were present. The
cells were examined in a flow cytometer (BectonDickinson, EPICS model CS) and the relative
fluorescence (DNA content) and cell cycle analysis
was performed using a Modfit analysis program (Verity Software Corp.), which integrated the area underthe various peaks and estimated the percentage of
total area for each peak, as well as their coefficientsof variation.

Results_

Lipid Analysis
Comparison of hemocytes from HN-affected and normal clams by gravimetric analysis indicated that there
was no significant difference between the total extractable lipid in the cells (normal cells = 15.97±8.51
pg lipid/cell; HN cells = 23.2l±11.88 pg lipid/cell,
P = 0.223). In order to more fully define any differences that might exist between the two cell types,
both the basic fatty acid components, as well as thecomplex lipid moieties derived from them were analyzed.
Gas chromatographic analysis of FAMEs from hemocytes taken from HN-affected clams was comparedwith that of normal clams as well as with the patternfound in normal and SV40 virus-transformed WI-38
cells (human diploid fibroblasts) (Table 1). When
compared to normal clams, only clams with rates of
HN in excess of 90% abnormal cells exhibited significant alterations in FAME composition. The saturated
class of FAMEs remained stable in affected clams,with the exception of a decrease in palmitic acid andin stearic acid. Abnormal cells had significantly lower
levels of monounsaturated fatty acids, as well as significantly higher levels of the longer chain fatty acids(20 and 22 carbon polyunsaturated groups). Thechanges found in the FAME profiles of HN cells were
opposite in direction from those found in SV40-transformed human cells.
Evaluation of the in vitro incorporation of lipid
precursors (acetate) into the more complex lipids
also indicated significant changes in hemocytes from88

NOAA Technical Report NMFS III

Table I
A summary comparison of lipid composition of normal and SV40-virus transformed mammalian cells and normaland HN cells from Mya arenaria. Levels of HN ranged from 5 to >95%.

Lipid class
Total lipid/cell (pg)
Neutral lipids (% of total cpm)
Triacylglycerides (% of neutral)
Free fatty acids (% of neutral)
Cholesterol (% of neutral)
Cholesterol esters (% of neutral)
Phospholipids (% of total cpm)
Cardiolipin (% of phospholipids)
Phosphotidylethanolamine(% of phospholipids)
Phosphotidylinositol(% of phospholipids)
Phosphotidylcholine(% of phospholipids)
Fatty acid methyl esters
Palmitic acid (16:0) (% of FAME)
Stearic acid (18:0) (% of FAME)
18:1 Fatty acids (% of FAME)
20:4 Fatty acids (% of FAME)

WI-38"

SV-40
infected
WI-38 aNormalhemocytes

65.0
29.0
27.0
21.0
35.0
5.0
71.0
6.1
12.0

59.0
41.0*
25.0*
26.0*
35.0
4.0
59.0*
2.7*
13.0

16.0
5.0
40.0
36.0
6.2
18.0
95.0
6.0
75.0

23.2 (P=0.223)
8.0 (P<0.05)
62.0 (P<0.005)
22.0 (P<O.OI)
4.2 (P<0.05)
8.0 (P<0.05)
92.0 (P<O.OOI)
2.0 (P<O.OOI)
71.0 (P<O.OOI)

13.0

10.0*

1.0

2.0 (P<O.OOI)

57.0

57.0

2.0

8.0 (P<0.005)

44.0
37.0
63.0
37.0

50.0*
30.0*
78.0*
17.5*

70.0
20.0
25.6
1.2

59.0 (P<O.OI)
25.0 (P<O.05)
22.5 (P<0.05)
4.0 (P<O.OI)

HN
hemocytes

a Data from Howard et al. 1977.
*Significant difference between WI-38 and SV40-transformed WI-38.

diseased clams relative to normal ones. The percentage of labelled acetate incorporated into the total
neutral lipid fraction of the HN cells was significantly
higher, especially the triacylglycerols (P<0.005), theprimary storage form of lipids (Table 1). In fact,
while the level of the other classes of neutral lipids
evaluated, including free fatty acids, cholesterol, andcholesterol esters, decreased significantly, the elevation in triacylglycerides masked the depressed
synthesis of these compounds in the total neutral
lipid fraction. Oil red 0 staining, which preferentially stains triacylglycerols (Luna 1986), specificallystained the vacuoles or "droplets" seen in HN cells byphase contrast microscopy; these droplets are not apparent in normal hemocytes.
As with the neutral lipids, there were also significant changes in the incorporation of acetate into thepolar lipids, which consist almost entirely of themembrane-associated phospholipids. The overalllevel of incorporation into the total phospholipid
class decreased significantly (P<O.OOI). Likewise, decreases occurred in the amount of labelled
phosphatidylglycerol and cardiolipin, which is derived from phosphatidylglycerol (P<O.OOI) (Table 1).
By contrast, there were significant increases in thelevels of incorporation into the phosphotidylinositols

and the phosphotidylcholines(P<O.OOl and P<0.05 respectively). Many of the changes occurring in thelipids of HN hemocytes differ with those documented
in mammalian neoplastic cells (Howard et al. 1973).

Flow Cytometric Analysis
Preliminary experiments indicated that using formalin or glutaraldehyde fixatives for flow cytometricanalysis of hemocytes altered the chromosomal material and .resulted in poor separation of the normal
diploid and aneuploid peaks in HN-positive clams.
Therefore, for all of the experiments reported here,
unfixed hemocytes were rapidly (within 5 minutes ofremoval from the clam) processed for flow
cytometric analysis. This resulted in a clear separation of DNA peaks in the cells.
Flow cytometric analysis indicated that the DNAcontent of the abnormal hemocytes was distinctly
alleupoid. As shown in Figure 1, there was a strong
positive linear correlation (r = 0.95) between the abnormal microscopic appearance of the nonadherent
cells and abnormal DNA content as detected by flow
cytometry. Two forms of aneuploid cells were detected: those with a hypodiploid DNA content andReno et al.: Hematic Neoplasia in the Softshell Clam Mya arenaria

higher (mean = 16.4%, P = 0.0001) proportion ofcells which were either undergoing DNA synthesis, in
preparation for mitosis, or actually in the process ofmitosis (Fig. 3). The proportion of HN cells in somephase of replicative activity ranged from 10 to 25%;
most of this activity was in the hyperdiploid population, but the hypodiploid and even the diploid
populations also were undergoing more extensive
multiplication than norma!.

those with a hyperdiploid DNA content. Examples of
the DNA contents of normal and abnormal hemocytes are shown in Figure 2. The amount of DNA in
diploid clam hemocytes was approximately 3.2 pg,
compared with 6.8 pg in a human lymphocyte standard. In contrast to diploid clam hemocytes, DNA
profiles of cells from abnormal clams invariably had apopulation of cells containing hyperdiploid DNA
comparable in number to the percentage of abnormal cells as assessed by microscopic observation. The
DNA index (DI)-the quantity of aneuploid DNA
relative to the DNA content of the normal diploid
hemocyte-of clams showing hyperdiploidy varied
from 1.6 to 2.0 in 12 HN-positive individuals. In most,but not all clams that were HN-positive, a hypodiploid cell population also occurred which comprised
between 0.5 and 10% of the cells (Fig. 2). The DI of
the hypodiploid cell population, ranging from 0.82
to 0.86, was more constant than the DI of thehyperdiploid population. This hypodiploid peak was
not noted in the hemocytes of normal clams.
One of the hallmarks of neoplastic transformationis the proliferative nature of the cells. Cell cycle
analysis of hemocytes from HN-positive clams indicated that this was indeed the case for this particularmalady. Normal cells showed some indication of proliferative activity; 2 to 5% (mean = 4.4%) were in the
S (DNA synthesis) phase and an approximately equal
percentage were in the G 2 /M (premitotic and mitotic) phases of the cell cycle. Hemocytes from clams
that were aneuploid, however, had a significantly

y = 1.1029 + 0.91207x

89

Discussion_

The present study was designed to help characterize
the abnormal hemocytes in M. arenana with hematic
neoplasia. This disease, and similar diseases in a variety of other bivalves, have attracted much attention
in the scientific community for two reasons. In themarine community, they can cause significant mortalities in the affected animals (Cooper et a!. 1982;
Brousseau and Baglivo 1991; Reno, Leavitt, andCapuzzo, unpub!. observations). In addition, these
diseases have become the focus of epidemiologicalinvestigations assessing the potential causal interaction between anthropogenic pollutants in marine
environments and the generation of neoplasia, andthe potential for using HN disease in bivalves assentinals in appraising the potential carcinogenic activity of these pollutants in humans. While therationale of the first reason still holds' true irrespective of the true nature of the disease, the assumption

RI\2 = 0.907

100
III

80

60

:2
0
ii
::J

CI)

c

40

c(

'#.
20

20

40

60

"10 HN (microscopy)

80

100

Figure 1
Relationship between the percentage of HN
by microscopic evaluation and the percentage of DNA aneuploidy by flow cytometry.

90

NOAA Technical Report NMFS 111

1000

A

a
800

..

HN-

600

C

:::I
0

0

400

200
b

c

0
20

40

60

100

80

120

140

160

180

IRFL (DNA)

B

1000
e

78% HN+

800

..

600

c

:::I
0

0

400

a
200
9

0
20

40

60

80

100

120

IRFL (DNA)on which the second is based is subject to invalidation if the disease is not a true neoplasm, but israther a hyperplastic or anaplastic disease-or evendue to an exogenous source such as a protozooan
parasite.
Information is readily available in the literature
with respect to the characteristics of neoplasia andtransformed cells in mammals and birds. Relatively
little is present that deals stringently with the disease
and nature of the altered cell in poikilothermic vertebrates; virtually none is available with respect to the"diseased cell" in invertebrates. Mix's comprehensive
review (1986) of the nature of the interaction between anthropogenic pollutants and "neoplastic"
diseases in aquatic vertebrates and invertebrates represents the most critical analysis of the extant data.
The author, as well as others (Harshbarger et al.

140

160

180

Figure 2
Examples of DNA distrib ution
and cell-cycle analysis of normal
and abnormal hemocytes. (A)Normal clam; (B) clam with approximately 78% abnormal
cells. IRFL = integrated red
fluorescence (DNA content).
Key to letters on graphs:
a: resting stage of cell cycle (Goand G 1 phases) for diploidcells
b: DNA synthesis phase of cell
cycle (5 phase)c: Premitotic and mitotic phasesof cell cycle (G 2 and M
phases) for diploid cells.
d: Hypodiploid DNA peake: Hyperdiploid DNA peakf: 5 phase for hyperdiploid cells
g: G 2 /M phases for hyperdiploid cells

1979; Lauckner 1983), prudently note that the criteria presently used to de.termine the nature of thedisease are inadequate when compared with the
manifold criteria that are demonstrable in mammals,
and that it may be premature or unwarranted to describe the leukemia-like disease in bivalves as a true
neoplasm without further investigations into the nature of the disease.
Mammalian and avian neoplastic cells derived fromtumor tissue or transformed by biological, physical,
or chemical agents have several dozen characteristics
in common that differ significantly from normal
cells. These characteristics are most readily evaluatedwhen the cells are separated from their normal residence in vivo and are cultured in vitro, if only forshort periods. This process removes the major confounding factor of the large number and wide variety

Reno et al.: Hematic Neoplasia in the SoftsheU Clam Mya arenaria

III % 5.. %G2-M%Go-G1

o

A

100

.!!!

80

u

60

Qi

"C

o·

40

o

20

Q.

O-+'--........- - - ' - _ -.......-

o

62

88

........L...---'........-

92

94

95

94

95

......

% HN

B

o~

o

62

88

92

% HN

c
100

.!!!
Qi
u

"C

80

'0

60

:9"C

..

40

~

20

G)

c.

cf!.

O+----.J'-----'.......-o62.......- - . J . ,.......-..L,L----.I,

88

92

94

95

% HN

Figure 3

Percent of diploid and aneuploid cells in various phases of
the cell cycle. (A) diploid cells; (B) hypodiploid cells;(C) hyperdiploid cells. % S = cells in DNA synthesis phaseof cell cycle; G2 /M = cells in premitotic and mitotic phasesof the cell cycle; GO/G 1 = cells in the resting stages of thecell cycle.

91

of normal cells that are present in tumor tissue invivo. Mfected cells of the circulatory system are most
readily divested of these contaminants by simply
drawing off hematic fluids which contain the abnor-.
mal cells and placing them in culture for further
analysis. This is the approach taken in the current
investigations.
Lipids are the major constituents of cellular membranes, as well as a source of stored energy, and thespecific lipid components that are present have amarked effect on the properties of the membrane(Wood 1972; Shinitzky 1984). Because a wide varietyof the alterations occurring in neoplastically transformed cells are associated with the plasma
membrane surface, it is not surprising that many alterations in the lipid composition of neoplastic cells
have been documented in mammalian systems. Wood
(1972) and others have published a variety of papers
dealing with the alterations in lipid metabolism andbiochemistry in mammalian neoplasia (e.g., Awadand Spector 1976; Yau et al. 1976; Montaudon et al.
1981; Schroeder and Gardiner 1984; Kier et al. 1988;
Calorini et al. 1989). The SV40-induced transformation of human WI-38 lung fibroblast cells (Howard etal. 1973; Perkins and Scott 1978) is an in vitro system
resembling HN of M. arenaria because transformation by viruses produces a homogeneous populationof altered cells, as is found with HN hemocytes, andbecause it has been indicated that HN in M. arenaria
is also viral-induced (Oprandy et al. 1981).
There are significant alterations in more than one
half of the lipid moieties examined in the SV40-WI-38
cells in culture. With the essential building blocks oflipids, the fatty acids, there are increases in the levelsof the saturated fatty acid palmitic acid (16:0), themonounsaturated fatty acid oleic acid (18:1il9), andthe diunsaturated fatty acid linoleic acid (l8:2il6). In
contrast, in M. arenaria HN cells show a decrease inpalmitic acid but a similar increase in the two unsaturated fatty acids. In the virally transformed WI-38
cells, a significant decrease occurs in the levels ofstearic (18:0), palmitoleic (16:1il7), linolenic(l8:3il3), and arachidonic (20:4il6) acids. In M.
arenaria HN, however, there is a marked increase in
the levels of stearic acid in conjunction with decreases in the levels of 16:1il7, 18:3il3, and 20:4il6
fatty acids. The changes seen in mammalian and bivalve cells are at variance in their levels of two major
essential fatty acids derived from de novo sources:
palmitic and stearic acids. These variations may reflect a difference in the process of synthesis of thefatty acids in invertebrates compared with mammals
or may reflect a basic difference in the nature of theabnormal HN cell and the transformed WI-38 cell.
With respect to the more complex lipids, a significant

92

NOAA Technical Report NMFS III

increase is seen in the levels of cholesterol in bothvirally transformed WI-38 and in murine hepatoma
cells (Steele and Jenkin 1977). Cholesterol is one of
the most important lipids in the cellular membrane
and contributes much to the attributes of the cellsurface. In studies with transformed mammalian cells
there is a significant increase in the levels of cholesterol in the cytoplasmic membrane (Howard et al.
1973). In our study, the incorporation of labelled acetate into cholesterol, and its derivatives, thecholesterol esters, of HN cells was significantly lower
than the levels of incorporation seen in normal hemocytes. This is in contrast to uptake observed in
mammalian neoplastic cells. Likewise, the changes inanother class of neutral lipids, the free fatty acids,
remained unchanged while in mammalian cells there
is an increase in free fatty acids. Taken together,
these major differences between the lipid biochemistry of HN cells and that of neoplastically transformed
mammalian cells indicate that it would bepresumptious to declare HN cells neoplastic on thebasis of their lipid biochemistry.
A fundamental change that occurs in neoplasticallytransformed cells is the alteration of the genome of
the cell, which is heritable and leads to the initiationand progression of tumors. These alterations may beconsiderable, such as heteroploidy, which can be easily detected by flow cytometric analysis, or more
subtle, such as chromosomal rearrangements or alterations in specific gene sequences which must be
detected by karyotyping or molecular DNA analysis.
In this study, flow cytometric analysis was used to determine the potential lesions in the DNA content of
HN cells and also to assess the proliferative potentialof the disease. It was found that HN cells were distinctly aneuploid in nature and that the mostconsistent change in the DNA content in HN cells
was to a hyperdiploid state. The level of this
hyperdiploid population correlated well with the proportion of cells which appeared abnormal by
microscopic examination. There was significant variation, however, in the amount of aberrant DNA in thecells, which ranged from 1.6 to 2.0 times normal.
This would be consistent with the clonal developmen t of the transformed cell (Iversen 1988).
However, it was also found that most of the clams thatwere HN positive had a second, markedly stable hypodiploid population of cells, a finding that isinconsistent with the clonal development of transformed cells. This finding is problematic if One ismaking the assumption that HN in M. armaria is atrue neoplastic disease because it is unlikely that astable hypodiploid population would coexist with amore variable hyperdiploid population in the same
animal if this were true.

The available data on the etiology of HN inbivalves is scant. There is evidence that the abnormal
hemocytes of HN clams possess unique surface antigens as detected with both polydonal andmonoclonal antibodies (Reinish et al. 1983; Miosky etal. 1989). In Smolowitz et al. (1989), this same group
has also produced a monoclonal antibody that reacts
both with a subpopulation of cells in the gut of normal clams and with HN cells, indicating an internalsource for HN cells. However, with the well-known
possibility of epitopic crossreactivity, or even with active mimicry of epitopes by parasites, the reaction ofa single monoclonal antibody with a subpopulationof M. arenaria hemocytes is insufficient to confirm
the endogenous source of the HN cells. As mentioned above, several authors have noted that theavailable information on the origin of the HN cell inclams does not preclude the possibility that the cells
are parasitic rather than neoplastic in nature(Lauckner 1983; Mix 1986). Unfortunately, the data
presented here does not alleviate this problem because, for example, the heteroploid cells may beparasitic protozooans or even algae, which would
have a considerably different chromosomal makeup
than clam hemocytes. Further work on genetic analysis of these cells using cytogenetic techniques, as well
as molecular biological techniques, must be carriedout before the resolution of this problem can beachieved.

Citations_

Alderman, D.G., P. Van Banning, and A. Perez-Colomer.
1977. Two European oyster (Os/rea edulis) mortalities associated with abnormal hemocytic condition. Aquaculture.
10:35-340.
Appledoorn, RS., and]]. Oprandy.
1980. Tumors in soft-shell clams and the role played by avirus. Maritimes. August, 1980:4-6.
Awad, A.B., and A.A. Spector.
1976. Modification of the fatty acid composition of Erhlich
ascites tumor cell plasma membranes. Biochim. Biophys.
Acta. 426:723-731.
Bligh, E.G., and WJ. Dyer.
1959. A rapid method of total lipid extraction andpurification. Can.]. Biochem. Physiol. 37:911-917.
Brousseau. D.]., and].A. Baglivo.
1991. Field and laboratory comparisons of mortality in normal and neoplastic Mya arenaria. ]. Invertebra. Pathol.
57:59-65.
BlOwn, RS., RE. Wolke, S.B.N. Saila, and C.w. Brown.
1977. Prevalence of neoplasia in 10 New England populations of the soft shell clam (Mya arenaria). Ann. N.Y. Acad.
Sci. 198:522-532.
Calorini, L., A. Fallani, D.Tombaccini, E. Barletta, G.Magnai, M. Di
Renzo. P. Comoglio, and S. Ruggieri.
1989. Lipid characteristics of RSV-transformed Balb/C 3T3

Reno et al.: Hematic Neoplasia in the SoftsheU Clam Mya arenariacell lines with different spontaneous metastatic
potentials. Lipids. 24:685-690.
Cooper, KR, R.S.Brown, and P.W. Chang.
1982. Accuracy of blood cytological screening techniques forthe diagnosis of a possible hematopoietic neoplasm in thebivalve mollusc, Mya arenaria. J. Invertebra. Pathol.
39:281-289.
Couch,J.A.
1969. Sarcoma-like disease in a single specimen of theAmerican oyster. Comparative Leukemia Research Bibliography: Haematology. No. 36.
Farley, C.A.
1969. Probable neoplastic disease of the hematopoietic system in oysters, Crassostrea virginica and Crassostrea
gigas. Natl. Cancer Inst. Monogr. 31:541-555.
Harshbarger,J.C., S.Y. Otto, and S.C. Chang.
1979. Proliferative disorders in Crassostrea virginica and Mya
arenaria from the Chesapeake bay and intranuclear viruslike inclusions in Mya arenaria with germinomas from a
Maine oil-spill site. Haliotis. 8:243-248.
Heim, S., and F. Mitelman.
1987. Cancer Cytogenetics. Alan R. Liss, Inc., New York,NY.
Howard, B.Y.,J.D. Butler, andJ.M. Bailey.
1973. Lipid metabolism in normal and tumor cells in culture. In Tumor lipids: biochemistry and metabolism (R.
Wood, ed.), p. 200-214. American Oil Chemists' Society
Press,lL.
Iversen, O.H.
1988. Theories of carcinogenesis: facts, fashion or
fiction? Hemisphere Publishing Co., Washington, DC,
327 p.
jerkofsky, M., and AJ. DeSiervo.
1986. Differentiation of strains of varicella-zoster virus bychanges of neutral lipid metabolism of infected cells. J.
Virol. 57:809-815.
Kier, A.B., M.T. Parker, and F. Schroeder.
1988. Local and metastatic tumor growth and membrane
properties of LM fibroblasts in athymic (nude)
mice. Biochim. Biophys. Acta. 938:434-446.
Lauckner, G.
1983. Diseases of Molluska:Bivalvia. In Diseases of marine
animals. (0. Kinne, ed.), p. 477-962. BioI. Anst.
Helgoland, Hamburg, FRG. Vol n.
Luna, L.G.
1968. Manual of histological staining methods of the Armed
Forces Institute of Pathology, 3rd ed. McGraw-Hill Inc.,New York, NY.
Marchesi, Y.T (ed.)
1976. Membranes and neoplasia: new approaches and strategies. Alan R Liss, Inc., New York, NY.
Miosky, D.L., R.M. Smolowitz, and C.L. Reinisch.
1989. Leukemia cell-specific protein of the bivalve mollusc,
Mya arenaria. J. Invertebra. Pathol. 53:32-40.
Mix,M.C.
1986. Cancerous diseases in aquatic animals and their association with environmental pollutants: a critical literature
review. Mar. Environ. Res. 20:1-92.

93

Montaudon, D.,J.C. Louis, andJ. Rober.
1981. Phospholipid acyl group composition in normal andtumoral nerve cells in culture. Lipids. 16:293-297.
Oprandy, jJ., P.W. Chang, A.D. Pronovost, KR. Cooper, R.S.
Brown, and VJ. Yates.
1981. Isolation of a viral agent causing hematopoietic
neoplasia in the soft-shell clam, Mya arenaria. J. Invertebra.
Pathol. 38:45-51.
Perkins, RG., and R.E Scott.
1978. Differences in the phospholipid, cholesterol, and fatty
acyl composition of 3T3 and SV3T3 plasma
membranes. Lipids. 13:653-657.
Raber, M.
1988. Clinical applications of flow cytoinetry. Oncology.
2:35-43.
Reinisch, C.L., A.M. Charles, andJ. Troutner.
1983. Unique antigens on neoplastic cells of the soft shellclam, Mya arenaria. Dev. Compo Immunol. 7:33-39.
Sasaki, G.C., andJ.M. Capuzzo.
1984. Degradation of Artemia lipids under storage. Compo
Biochem. Physiol. B. Compo Biochem. 78:525-531.
Schroeder, F., andJ.M. Gardiner.
1984. Membrane lipids and enzymes of cultured high andlow-metastatic B16 melanoma variants. Cancer Res.
44:3262-3269.
Shinitzky, M.
1984. Membrane fluidity in malignancy, adversative andrecuperative. Biochim. Biophys. Acta. 738:251-261.
Smolowitz, R.M., D. Miosky, and C.L. Reinisch.
1989. Ontogeny of leukemic cells of the soft shell clam. J.
Invertebra. Pathol. 53:41-51.
Steele, w., and H.M.jenkin.
1977. Lipids and lipid metabolism of Novikoff rat hepatoma
cells. In Tumor lipids: biochemistry and metabolism (R.
Wood, ed.), p. 215-225. American Oil Chemists' Society
Press,IL.
Vaseleiv,J.M., and I.M. Gelfand.
1981. Neoplastic and Normal Cells in Culture. Cambridge
Univ. Press, Cambridge, U.K
Wood, R., ed.
1972. Tumor lipids: biochemistry and metabolism. American
Oil Chemists' Society Press, Champaign, IL.
Wuthier, RE..
1966. Two-dimensional chromatography on silica gel-loaded
paper for the microanalysis of polar lipids. J. Lipid Res.
7:544-550.
Yau, TM., T Buckman, A.H. Hale, and M.J. Weber.
1976. Alterations in lipid acyl group composition and membrane structure in cells transformed by Rous sarcoma
virus. Biochemistry. 15:3212-3219.
Yevich, P., and C. Barszcz.
1976. Gonadal and hematopoietic neoplasms in Mya
arenaria. U.S. Natl. Mar. Fish. Servo Mar. Fish. Rev. 38:
42-43.
1977. Neoplasia in soft-shell clams, Mya arenaria, collectedfrom oil-impacted sites. Ann. N.Y. Acad. Sci. 298:409-426.

Kinetics of Bovine Serum Albumin Administered by the Immersion
Method in Fishes Acclimatized to Seawater and to Fresh Water
MITSURU OTOTAKE and TERUYUKI NAKANISHI
Inland Station of National Research Institute ofAquaculture
Tamaki, Mie 519-04, Japan

ABSTRACTTo clarify plasma BSA kinetics in marine fish after hyperosmotic infiltration (HI) treatment, chum salmon (Oncorhynchus keta) and blue tilapia (Tilapia aurea) were acclimatized
to seawater or to fresh water, and were administered bovine serum albumin (BSA) by HI
treatment or intravenous i~ection. First, by using a least squares program, regression
curves were computed from the plasma BSA levels of fish i~ected with BSA to obtain theclearance rate of BSA from plasma. Then, by using a deconvolution method, the BSA
release rate from the organ of BSA entry to plasma after HI treatment was calculated fromplasma BSA levels after HI treatment and the clearance rate. The results showed that theclearance rates of both fishes acclimatized to seawater were higher than those of the fish
acclimatized to fresh water. On the other hand, the release rates of both fishes acclimatized
to seawater were much lower than those of the respective fish acclimatized to fresh water.
Thus, it is quite plausible that the high clearance rates and low release rates of fish inseawater work synergistically, resulting in lower plasma BSA levels of fish in seawater compared with fish in fresh water after HI treatment.

Introduction
The immersion method, first reported by Amend andFender (1976), is a useful method for mass immunization in aquaculture. Many papers have shown theeffectiveness of this method on freshwater fish (Ellis
1988). On the other hand, there is a little knowledge
as to the immersion method's effectiveness on marine fish. In a preliminary study (unpub!. data), theauthors used hyperosmotic infiltration (HI) treatment to administer bovine serum albumin (BSA) toyellowtail Senola quinqueradiata, which is the most important aquaculture species in Japan, and rainbow
trout Oncorhynchus mykiss, and then measured the
BSA levels in their plasma. We found that plasma BSA
levels were much lower in yellowtail than in rainbow
trout after HI treatment. This difference may havebeen caused by two factors: species specificity and thetype of environmental water-fresh water or seawater.
To analyze this difference it is necessary to compare
the kinetics of BSA in the plasma of several species offish that can reside in both seawater and fresh water.
Thus, in the present study, chum salmon (0. keta)
and blue tilapia (Tilapia aurea) were acclimatized toseawater or to fresh water, and then administered
BSA by HI treatment or injection. By using theplasma BSA levels of these animals, the rate of BSA
release into plasma and BSA clearance from theplasma were calculated.

Materials and Methods_

Experimental Animals
Six-month-old chum salmon, 16 ± 1 g (mean ± standard deviation) in body weight, and I-year-old blue
tilapia, 157 ± 20 g in body weight, were used in thisexperiment. Tilapia were fed commercial floating drypellets in a I-ton tank supplied with running fresh
water at 25° C, whereas chum salmon were kept in a
I-ton tank supplied with running fresh water at
15° C, and fed commercial dry pellets for rainbow
trout. These tanks were kept indoors.

Acclimatization to Seawater
Before the administration of BSA, one half of thetilapia and chum salmon were acclimatized to seawater, and the other half were kept in running fresh
95

96

NOAA Technical Report NMFS III

water throughout the experiment. Fish were acclimatized to seawater (salinity: 34 ppt) by increasing the
proportion of seawater day by day to 50% on the firstday, 75% on the second day, 87.5% on the third day,and 100% on the fourth day. Mterwards the fish were
kept in the tank supplied with running seawater for 3
weeks before the experiment. During the acclimatizing period, no fish died or showed abnormal
behavior. Experiments were carried out with fourgroups of fishes: chum salmon in fresh water (freshwater salmon); chum salmon acclimatized to seawater(seawater salmon); tilapia in fresh water (freshwatertilapia); and tilapia acclimatized to seawater (seawater tilapia). Water temperature was maintained at
15° C for freshwater or seawater salmon and at 25° C
for freshwater or seawater tilapia.

Administration of BSA by HI Treatment
Fifty fish of each group were administered BSA (fraction V, Sigma Co.) by the two-step hyperosmotic
infiltration technique according to the method ofAmend and Fender (1976) with slight modification.
Distilled water was used to prepare the two bath solutions; 5.3% NaCI solution and 2% BSA solution. Fishwere immersed in 5.3% NaCI solution for 3 minutes
and then placed directly into 2% BSA solution for 3
minutes. Mter the bath treatment, the treated fish
were placed in tanks supplied with either runningseawater or fresh water for 3 minutes. Mterwards
they were returned to their original tanks. Blood
samples were taken 0.5, 1, 2, 4, and 8 hours after thebath treatment. At each sampling time six fish weresacrificed and the blood was withdrawn from the caudal blood vessels using heparinized syringes.

Administration of BSA by Injection
Fifty fish of each group were administered BSA by
intravenous injection. Bovine serum albumin was dissolyed with sterilized 0.85% NaCI solution to 2 mg/
mL and then injected into fish via the caudal bloodvessels at the dose of 1 mg/kg body weight. Blood
samples were taken 0.5, 1, 2, 4, 8, and 24 hours after
the administration by the same method as mentioned
above.

1978). The gel plate was run at 1 V/mm and 15° C
for 3 hours. The exact amounts of BSA in normal fish
plasma were run in parallel with all tests for standard
reference.

Calculations
Plasma BSA Clearance-By means of an unweighted
least squares program, the plasma BSA levels CpinJ(t)at each time t after injection were fitted to following
exponential equations:

For the index of BSA clearance from plasma, themean residence time (MRT) of each experimentalgroup was given by

The BSA distribution volume Vof each experimentalgroup was calculated bywhere D is the amount of BSA administered by injection (1 mg/kg body weight).
BSA Release into Plasma-The BSA release rate into
the plasma Vet) at each time t was given by

where CpH'(t) is the plasma BSA level after the HI
treatment and T is the time constant. The gross BSA
release into the plasma after the HI treatment wascalculated from Vet) by integration.

Statistics
Data were analyzed using the F test, and then by theStudent's t test to detect significant differences.

Results_

Immunoelectrophoresis
Plasma BSA Levels Mter the HI Treatment
Bovine serum albumin content in the plasma wasquantitatively assayed by rocket immunoelectrophoresis (Laurell 1966; Wallenborg and Andersson

Concentration of plasma BSA in the freshwatersalmon increased until 4 hours after the HI treatment

Ototake and Nakanishi: Kinetics of Bovine Serum in Fish

A

B

120

20

~100

E
'0> 80

fI" "

3-

"

""t----- ---+
"

I

/

,
'

\\~

r----------f

!
!

!Xl

11

J",

J"
I

(}j10

/
/

Figure 1

Changes in plasma concentrations ofbovine serum albumin (BSA) after
hyperosmotic infiltration treatment infreshwater-acclimatized fish (A) andseawater-acclimatized fish (B). Key:(e) = tilapia; (+) = chum salmon. Data
are presented as means ± standard
errors.

I

t

I

5

I
I
I

I

2

4

6

8

HOURS AFTER BATH

2

4

6

8

HOURS AFTER BATH

and reached a maximum of90.6 ± 6.5 f.Lg/mL (mean ±standard error), and then decreased gradually (Fig.
1A). On the other hand, plasma BSA levels of the seawater salmon (Fig. IB) were always significantly lower(P<O.Ol) than those of freshwater salmon. A maximum
plasma BSA level of 18.8 ± 2.6 f.Lg/mL was observed ,inthe seawater salmon 2 hours after the bath; from thenon, plasma BSA stayed at a rather constant level of 1119 f.Lg/mL. The plasma BSA level of freshwater tilapia
increased until 4 hours following the bath when it
reached a maximum of 119.0 ± 8.1 f.Lg/mL and subsequently decreased to 64.1 ± 2.8 f.Lg/mL 8 hours after
the treatment (Fig. 1A). In contrast, plasma BSA levelsof seawater tilapia· (Fig. 1B) were always significantly
lower (P<O.OOl) than those offreshwater tilapia. A lowlevel of BSA (0.09-1.1 f.Lg/mL) was detected in theplasma of the seawater tilapia for the initial 2 hours,
but no BSA was detected in the plasma after 4 hours(minimum detectable level, 0.01 f.Lg/mL).

was adopted for express plasma BSA concentration
for freshwater salmon after injection. On the otherhand,

was adopted as the regression equation for seawatersalmon, freshwater tilapia, and seawater tilapia(Fig. 2). The MRT and the distribution volume of
BSA after the injection are shown in Table 1. Both in

60

A

50

Cp'~=31.o-exp(-1.20·t)+26.9·exp(-O.027-t)

Plasma BSA Levels Mter the Injection
Following BSA injection, the plasma BSA levels werealways lower in seawater salmon than in freshwatersalmon, and the difference was significant (P<O.Ol)
0.5, 1, 2, 8, and 24 hours after the injection (Fig. 2A).
The difference between seawater and freshwater acclimatized fish was more distinct in tilapia than insalmon, and the plasma BSA levels of seawater tilapia
were always significantly lower (P<O.OOl) than those
offreshwater tilapia (Fig. 2B).

Plasma BSA Clearance
The regression equation97

Cp~I=26.9·exp( -O.964·t)

oL..::::::"_ _
o 4 8

B

~_----=:::=:===*=

12 16 20 24
HOURS AFTER INJECTION

Figure 2

Changes in plasma concentrations of bovine serum
albumin (BSA) after injection of BSA into chumsalmon (A) and tilapia (B). Open circles indicate thefish in fresh water and closed circles indicate the fish
acclimatized to seawater. Data are presented asmeans ± standard errors. Regression equations were
calculated with a least squares program.

98

NOAA Technical Report NMFS III

Table 1
Kinetics of plasma BSA administered by hyperosmoticinfiltration treatment.
Maximum BSA
level in plasma(lLg/mL)

Mean residence
time (MRT)(hours)Gross release
into plasma(lLg/mL)

Distribution
volume(mL/100g b.w.)"

Freshwater salmon

84

36
(37)'

Seawater salmon
Freshwater tilapia
Seawater tilapia

19
119

II

I

I

167
(95)'
24
146
14

1.7
(3.7)'
3.4
2.7
3.7

Experimental
group

6

"b.w. = body weight.
'Calculated by the modified regression equation.

chum salmon and in tilapia, the plasma BSA clearance, as represented by the MRT, was more rapid inseawater acclimatized fish than in respective freshwater acclimatized fish. The MRT of seawater salmon
and seawater tilapia were 1/3 and 1/6 of that ofrespective freshwater acclimatized fish. The distribution volume of freshwater salmon was smaller thanthose of other experimental groups.

-;::60~A
6 40.c......

E 20
3a......

Cl

w 100

~

B

c: 80
w

en
0«
w
..J

BSA Release into the PlasmaWc:

The BSA release rate into the plasma, calculated
from the clearance rate and the plasma BSA levels,
is shown in Figure 3. The BSA release rate was always lower in seawater acclimatized fish than in .
freshwater acclimatized fish. In all experimentalgroups, the BSA release rate was high during theearly period, and the peak of the rate was observed
within one hour after the bath treatment. The rate
rapidly decreased after one hour and became low
six hours after the bath. The gross BSA release into
plasma, which is given by the area under the curvein Figure 3, is shown in Table 1. Both in chumsalmon and tilapia, the gross BSA release of freshwater acclimatized fish was larger than that of seawater
acclimatized fish.

Discussion_

In the present study, BSA clearance from the plasma
after the intravenous injection differed greatly within
the same species; in each species, the BSA clearance
was much more rapid in seawater-acclimatized fish
than in freshwater-acclimatized fish. These results
strongly suggest that the environmental water affects
fish physiology and consequently, by some mechanism(s), changes the plasma BSA clearance. Smith

0«

en
co

O~=---~-~-~-::;:::::=oQ

o

2

3

4

5

6

HOURS AFTER BATH

Figure 3
Changes in the BSA release rate into plasma after
hyperosmotic infiltration treatment in chumsalmon (A) and tilapia (B). Open circles indicate
the fish in fresh water and closed circles the fish
acclimatized to seawater. Data are presented asmean ± standard errors.

(1982) reported that plasma BSA administered by theimmersion method was trapped in the spleen and thekidney of rainbow trout. Similarly, the trapping ofplasma BSA by the spleen and the kidney has been
observed in plaice administered BSA by intravenous
injection (Ellis 1980). The kidney is one of the mostimportant organs for osmoregulation in fish, and its
function changes following seawater acclimatization(Hickman and Trump 1969). Thus it is possible thatthe seawater acclimatization mechanism influenced
the BSA trapping in the kidney and affected theplasma BSA clearance. The plasma BSA clearance wasOtotake and Nakanishi: Kinetics of Bovine Serum in Fish

also different between species. Tilapia showed a rapid
BSA clearance, while the chum salmon showed a slow
BSA clearance. This difference may not only be attributed to species specificity, but also to thetemperature of the rearing water.
The estimated gross BSA release into the plasma of
seawater salmon and seawater tilapia was muchsmaller than the amount released into respective
freshwater acclimatized fish. This indicates that thephysiological changes that resulted from seawater
residence reduced the gross BSA release after the HI
treatment. Amend and Fender (1976) suggested that
hyperosmotic pretreatment had a hydrating effect onmembranes of fish tissues, and thus madeBSA infiltrate into the fish more easily. However, fish residing
in seawater are adapted to a hyperosmotic environment, and their membranes are more resistant tohydration than those of fish in fresh water. Therefore, it is reasonable to consider that hyperosmotic
pretreatment stimulates BSA uptake more strongly infish in fresh water than in fish in seawater. In any
case, it is quite plausible that some factors in seawater
influenced the fish physiology and induced the low'
BSA release rate and high BSA clearance rate. These
two rates of seawater-acclimatized fish worked synergistically to make their plasma BSA levels after the HI
treatment much lower than those of freshwateracclimatized fish.
The BSA distribution volume after the intravenous
injection is expected to agree with the whole plasma
volume estimated by tracer-dilution methods using
Evans blue dye, serum albumin, or red blood cells, allof which have been widely used to determine wholeplasma (or blood) volumes. Using these methods,
whole plasma volumes of fish have been determined
to be 2.5-4.7 mL/I00 g body weight in salmonids(Conte et al. 1963; Smith 1966; Huggel et al. 1969,
Nikinmaa et al. 1981; Gingerich et al. 1987) and 3.5
mL/I00 g body weight in yellowtail (Itazawa et al.
1983). The distribution volumes of the present study
for seawater salmon, freshwater tilapia, and seawatertilapia agree well with those reported values. In freshwater salmon, however, the distribution volumes in
the present study were smaller than the whole plasma
volumes reported so far in the literature. Smith(1966) suggested that blood sampling periods of lessthan two hours after tracer injection tended to produce low plasma volume and high clearance rateestimates in salmonids because these periods did notallow for adequate circulation of tracers in the vascular system. Therefore, in the case of freshwatersalmon in the present study, there is a possibility thatthe plasma BSA clearance rate was overestimated. Insuch a case, the gross BSA release is likely to be overestimated. Therefore, the BSA release and clearance

99

rates in freshwater salmon were examined by modifying the regression equation to ignore the first term,
which is the main component for the two hours after
the injection. The distribution volume, the MRT, andthe gross BSA release were recalculated by using thismodified regression equation and the deconvolution
method. Recalculation with the modified regression
equation also showed more rapid BSA clearance andless gross BSA release in seawater salmon than in
freshwater salmon (Table 1).
It has been reported that the plasma kinetics ofseveral ions, such as Na+, Ca 2+, Mg 2+, and Cl-, aredifferent in seawater- and freshwater-acclimatized fish
(Hirano and Uchida 1972; Bentley 1971). There havealso been reports comparing the kinetics of medicines in plasma of seawater- and freshwateracclimatized fish, but the results are contradictory
among the chemicals. Endo and Onozawa (1987)
and Ishida (1990) reported that when oxolinic acidwas administered to ayu, Plecoglossus altivelis, andcoho salmon, O. kisutch, by bath and oral methods
the seawater-acclimatized fish of both species showed
lower oxolinic acid concentration and shorter retention time than freshwater-acclimatized fish. On theother hand, Bergsjo and Bergsjo (1978) reported
that when sulfanilamide or sulfadimidine was administered by bath treatment to rainbow trout, theplasma concentration of seawater-acclimatized fish
was higher than that of freshwater-acclimatized fish.
It still remains unknown why there are such differences between medicines. In any case, from the viewof vaccine application to marine fishes, it is quite important to clarify whether the phenomenon observedin the present study for BSA (seawater-acclimatizedfish have a lower release rate and higher clearancerate) is common to protective antigens such as lipopolysaccharides and proteases.

Acknowledgmen~___

Financial assistance for this study was provided in partby a grant-in-aid (Bio-Media Program) from the Ministry of Agriculture, Forestry, and Fisheries (BMP-91IV-I-9). We would like to thank Yasuo Inui for his critical review of this manuscript. We also acknowledge thestaff of the Hokkaido Salmon Hatchery for supplying uswith the chum salmon eggs used in this study.

Citations_

Amend, D.F., and D.C Fender.
1976. Uptake of bovine serum albumin by rainbow trout
from hyperosmotic infiltration: a model for vaccinatingfish. Science 192:793-794.

100

NOAA Technical Report NMFS 111

Bentley, PJ.
1971. Endocrines and osmoregulation. Springer-Verlag, Berlin/Heidelberg/New York, 300 p.
Bergsjo, T., and T.H Bergsjo.
1978. Absorption from water as an alternative method forthe administration of sulphonamides to rainbow trout,
Salrnogairdneri. Acta. Vet. Scand. 19:102-109.
Conte, F.P., H.H. Wagner, and T.O. Haris.
1963. Measuremen t of blood volume in the fish (Salrno
gairdneri gairdneri). Am. J. Physiol. 205:533-540.
Ellis, A.E.
1980. Antigen trapping in the spleen and kidney of the plaice Pleuronectes platessa L. J. Fish Dis. 3:413-426.
1988. Current aspects of fish vaccination. Dis. Aquat. Org.
4(2):159-164.
Endo, T., and M. Onozawa.
1987. Effects of bath salinity and number of fish on the uptake of oxolinic acid by ayu. Bull. Jap. Soc. Sci. Fish
53(4):557-562. (In Japanese; English abstr.)
Gingerich, W.H., R.A. Pityer, andJJ. Rach.
1987. Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout. Compo Biochem.
Physiol. A Compo Physiol., 87A(2):251-256.
Hickman, C.PJr. and B.F. Trump.
1969. The kidney. In Fish physiology 1 (W.S. Hoar and DJ.
Randall, eds.) p. 91-241. Acad. Press, New York/London,
Hirano, T., and S. Uchida.
1972. Water and ion movements in the intestine and the urinary bladder of the teleosts and their hormonal
control. Medical Science 23(2):56-68. (lnJapanese.)
Huggel, HJ., H.C. Lane, and C.G. Ducrel.
1969. Determination de la courve D'homogeneisation et duvolume sanguin circulant de la truite Salmo gairdneri Rich.

_par la method de la dilution isoto-pique de 1'1311 et du
51Cr. J. Physiol. (Paris), 61:145-154. (In French.)Ishida, N.
1990. Comparison of tissue level of oxolinic acid in freshand seawater fishes after the oral administration. Bull. Jap.
Soc. Sci. Fish. 56(2):281-286. (lnJapanese; English abstr.)
Itazawa, y., T. Takeda, K Yamamoto, and T. Azuma.
1983. Determination of circulating blood volume in threeteleosts, carp, yellowtail and porgy. Jpn. J. Ichthyol.
30(1):94-101. (In Japanese.)
Laurell, C.B.
1966. Quantitative estimation of proteins by electrophoresis
in agarose gel containing antibodies. Anal. Biochem.
15:45-52.
Nikinmaa, M., A. Soivio, and E. Aiolo.
1981. Blood volume of Salrno gairdneri: influence of ambient
temperature. Compo Biochem. Physiol. A Compo Physiol.
69A(4):767-769.
Smi th, L.S.
1966. Blood volumes of three salmonids. J. Fish. Res. Board
Can. 23:1439-1446.
Smith, P.D.
1982. Analysis of the hyperosmotic and bath methods forfish vaccination: Comparison of uptake of particulate andnon particulate antigens. Dev. Compo Immunol., Suppl.
2:81-186.
Wallenborg, B., and U.B. Andersson.
1978. Immunoelectrophoretic techniques with the LKB 21] 7
multiphor. LKB Application Note, 249:1-12. (Pharmacia
LKB Biotechnology Inc., 800 Centennial Ave., Piscataway, NJ
08854.)

The Epidemiological Study of Furunculosis
in Salmon Propagation

TETSUICHI NOMURA*, MAMORU YOSHIMIZU**, and TAKAHISA KIMURA***Hokkaido Salmon Hatchery, Fisheries Agenl)'
Nakanoshima 2-2, Toyohiraku
Sapporo, Japan.

** Faculty ofFisheries, Hokkaido University
Minatomachi 3-1
Hakodate, Japan

ABSTRACTThe authors attempted to determine the distribution and prevalence of Aeromonas
salmonicida in mature chum (Oncorhynchus keta), pink (0. gorbuscha), and masu
salmon (0. masou) in Hokkaido that showed no apparent clinical signs of furunculosis. From September 1979 to November 1989, a total of 12,891 chum, pink, and masu
salmon were collected from 30 rivers. The changing pattern of the annual prevalenceof A. salmonicida in salmon was closely related to changes in fish density in theholding ponds: the prevalence of A. salmonicida increased in proportion to the increase in the number of fish in the ponds. We concluded from the results ofhistological and bacteriological examinations that fish with A. salmonicida in the kidney were not diseased but were carriers of A. salmonicida. The agent could not be
isolated from the immature fish examined. A. salmonicida was also isolated from theovarian fluid of fish showing no apparent clinical sign of furunculosis. Few A.
salmonicida were found on the surface of. the eggs one hour after fertilization. A
survey of agglutination titers against A. salmonicida in sera of chum, pink, and masu
salmon showed great variability within the species. The isolated strains were identified as A. salmonicida subsp. salmonicida and were pathogenic to salmonids. We
concluded that the A. salmonicida carrier state in fish poses a serious problem in theprevention of furunculosis and its reduction plays a key role in salmon propagation.
Both maturation of fish under conditions of low density in ponds, and disinfection of
their eggs, are necessary to prevent fish furunculosis during artificial propagation of
salmon.

Introduction_

Furunculosis of salmonid fishes, caused by Aeromonas
salmonicida, was first reported in 1890s by Emmerichand Weibel (1890, a and b). Since these first reports,
furunculosis has been reported in virtually all partsof the world where wild or cultured salmonids occur(Smith 1960; Herman 1968; Snieszko 1972;· Austin
and Austin 1987).
Furunculosis is not a serious problem in rainbow
trout (Oncorhynchus mykiss) culture in Japan because this species is resistant to the causative agentof the disease. However, serious mortality has been

documented in juvenile amago salmon (0. rhodurus) and masu salmon (0. masou) in accordancewith increased production of these fish. In
Hokkaido, outbreaks of furunculosis have been reported to occur in chum salmon (0. keta) by
Nishino (1967), and in masu and pink salmon (0.
gorbuscha) by Kimura (1970) during the maturationof these species in holding ponds. Nomura andKimura (1981), Nomura (1983), and Nomura et al.
(1983, 1991, a and b) reported isolating A.
salmonicida from the kidneys of mature chum, pink,
and masu salmon that showed no apparent clinicalsigns of furunculosis.

101

102

NOAA Technical Report NMFS 111_

Sea of Okhotsk

Sea of Japan

Nemuro Strait

Figure 1
Rivers where chum, pink, and masu salmon were collected (after Nomura et al. 1991a).

Cape Erimo

Pacific Ocean

Recently, no systematic epidemiological studies
have been done to establish control measures for furunculosis in salmonids from which quarantine anddisease control policies could be based.
In this paper, we report the recent epidemiologicalstudy of A. salmonicida which was carried out for the
purpose of establishing control measures for the disease.

Distribution of A. salmonicida
in Salmonids in Hokkaido_

We attempted to determine the distribution andprevalence of A. salmonicida in mature chum, pink,
and masu salmon populations in Hokkaido thatshowed no apparent clinical signs of furunculosis(Nomura et al. 1991a).
From September 1979 to November 1989, a total of
12,891 chum, pink, and masu salmon were collectedfrom 30 rivers (Fig. 1). At each sampling, a total of 60
fish of each species were randomly selected from therivers' salmonid populations in accordance with Amos
(1985). The fish were separated by species and riverand held in individual ponds at each river for about 1
month until maturity. Mter spawning, they were processed for examination. Kidney materials werestreaked onto nutrient agar plates (Eiken Co., Tokyo,
Japan) and cultured at 20°C for 7 days. No clinicalsigns of furunculosis were observed in the examinedfish. Bacterial colonies that produced a soluble brown
pigment and showed the following characteristics wereclassified as A. salmonicida: Gram-negative staining,lack of motility, failure to grow at 37°C, tested positive
for cytochrome oxidase, and had the ability to fermenton oxidative fermentative basal medium.

I Moheji
5 Shiribetsu
9 Teshio
13 Horonai
17 Abashiri
21 Shibetsu
25 Bettouga
29 Niikappu

2 Hekirichi
61shikari
10 Tonbetsu
14 Shokotsu
18 Shari
22 Tohoro
26 Kushiro
30 Yurappu

3 Shiriuchi
7 Shokanbetsu
II Hrobetsu
15 Yuubetsu
19 Iwaobetsu
23 Nishibetsu
27 Tokachi

4 Shubuta
8 Nabusha
12 Takushibetsu
16 Takoro
20lchani
24 Furen
28 Shizunai

We isolated A. salmonicida from chum salmon in 11 of
the 22 rivers examined; the percent occurrence of thebacterium in this species of fish ranged from 0.6 to49.2%. Populations of pink salmon, from 13 rivers were
tested and A. salmonicida was isolated from 60f theserivers with percent occurrence ranging from 0.2 to
13.3%. In masu salmon the bacterium was isolated from
5 of 10 rivers examined and the percent occurrence
ranged from 1.0 to 5.6%. Hence, A. salmonicida was determined to be distributed widely in the salmonid
populations of Hokkaido, except those of rivers locatedbetween Tsugaru Strait and Cape Erimo (Fig. 2).
In the Ishikari, Shari, Iwaobetsu, Shibetsu, andTokachi rivers, the prevalence of A. salmonicida wasfound to vary yearly. In the Ishikari river, the prevalence of A. salmonicida in chum salmon was high from1979 to 1984 but has gradually been decreasing since1985 (Fig. 3). In the chum salmon of the TokachiRiver and in all three species in the Shibetsu River,the prevalence of the bacterium remained highthroughout the examination period. In the Shari
river from 1979 to 1988, A. salmonicida was not isolated from any of the fish examined; however, it was
isolated from 4 of 60 fish examined in 1989.
From 1979 to 1984, changes in the monthly prevalence of the agent could be observed in fishes in the
Ishikari river. The incidence of A. salmonicida increased until the middle of October and thendecreased thereafter. The pattern of change wasclosely related with changes in fish density in theholding pond; the prevalence of the bacterium appeared to increase proportionately as density of fishin the pond increased (Fig. 4).
The number of A. salmonicida bacteria found in
kidney tissues ranged from 10 1 to 105 colony forming
units per gram (cfu/g) (Nomura et al. 1991a).

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Nomura et al.: Furunculosis in Salmon Propagation

103

Sea of Okhotsk

Sea of Japao

Figure 2

Pacific Oceao

5 Shiribetsu
9 Teshio
13 Horonai
18 Shari
23 Nishibetsu

Cape Erimo
Tsugaro Strait

Year

Year

1979

50

0=240

1984

0=263

0

0

~ I

IOn

I

1980

0=274

0=360

50 I-

0 00
0

q

10

I

I

1981

0=274

0

50

0

-

0

....o

I

0
I
1982

0

1

I

I

~~....L..._""""O

0=300

0=300

so f0
I

001

,...

1983

so -

I

1988

0=469

oI Cbo b -0=541

0

000
106W

Sep. Oct.

I

O~_...........

I

Nov.

Rivers where Aeromonas salmonicida was isolated fromchum, pink, and masu salmon from 1979 to 1989. C:
isolated from chum salmon; P: isolated from pinksalmon; M: isolated from masu salmon (after
Nomura et al. 1991a).

Sep. Oct. Nov.

Month

Figure 3

Changes in the monthly incidence of Aeromonas salmonicida
in chum salmon collected from the Ishikari river, and held
for maturation periods during the period September toNovember, 1979-88 (Modified from Nomura et al. 1991a).

6 Ishikari
10 Tonbetsu
16 Tokora
20lchani
26 Kushira

8 Nobusha
12 Tokushibetsu
17 Abashiri
21 Shibetsu
27 Tokachi

The kidney materials of chum salmon in which A.
salmonicida was isolated were fixed with Bouin's solution for histopathological examination. The kidneyorgans were dehydrated and embedded in Paraplast,and sections of the samples were made and stainedwith HE and Gimsa stain. \Histopathological examination of the infected fish
did not, however, reveal colonies of A. salmonicida
typically observed in fishes with furunculosis. Also,
no outbreaks of furunculosis were recorded in theexamined populations during the research period(Nomura et al. 1991a).
There are few reports examining the prevalenceof A. salmonicida in the organs of apparently normal mature fish. In fact, as far as we know, there is
only one report by Daly and Stevenson (1985) .
They reported that A. salmonicida was detected in
31 of 286 brown trout (Salmo trutta) sampled from
spawning runs in the Ganaraska River, Ontario,
Canada, over a period of two years. Our results
showed that the incidence of this agent in apparently normal chum salmon was higher compared tothat of Daly and Stevenson's (1985) estimated forbrown trout, and that A. salmonicida is distributed
widely in the river populations of salmonids in
Hokkaido.
Morikawa et al. (1981) reported that the numberof A. salmonicida in the kidneys of moribund amago
salmon was 108 to 109 cfu/g. The reason why diseasedfish were not found in the population we examined,even though they had A. salmonicida in their kidneys,
was that the degree of infection was not highenough. From the results of histological and bacteriological examinations, we conclude that fish with A.
salmonicida in the kidney are not diseased fish but
carriers of A. salmonicida.

· 104NOAA Technical Report NMFS III

_

-

---~

01981

'-'

~ 40 .~
J::

Q

~
""
~

~

01980

... 20 I-

0 1983
01979

Q

Q,l

Col

=

01982

-

Q,l

~

'0

0

01986

oS

I

0

1984

1985

d
2

0 1987 01988
I
I

4

Fish stock density (ftSb/m

6
2)

Figure 4
The relationship between average density of the fish inholding ponds and the incidence (% occurrence) of
Aeromonas salmonicida in fish taken from the Ishikari River.
Numbers in the figure indicate the year of examination(unpubl. data).

A. salmonicida in Immature Fish_

We attempted to isolate A. salmonicida from immaturechum and masu salmon (Nomura et al. 1991b). A
total of 680 fish were collected in four coastal set nets
off Hokkaido, and a total of 1,200 juvenile masu
salmon and chum salmon fry were collected from II
hatcheries of the Hokkaido Salmon Hatchery system.
The bacterium was not isolated from any of theexamined fish.

A. salmonicida in Ovarian Fluid_

In 1989 in Hokkaido, we attempted to isolate A.
salmonicida from the ovarian fluids of mature chum,pink, and masu salmon.
Ovarian fluids were collected according to themethod of Yoshimizu et al. (1985). The ovarian fluids
were streaked onto nutrient agar plates (Eiken Co.,Tokyo, Japan) and the plates were cultured at 20°C
for 7 days. Number of viable counts of A. salmonicida
in ovarian fluid and kidney were measured in accordance with the method of Nomura et al. (1991a).
A. salmonicida was isolated from the ovarian fluid offish showing no apparent clinical signs of furunculosis. For example, A. salmonicida was isolated from the·ovarian fluid of 22 of 120 fish examined from theTokachi River. The number of A. salmonicida bacteria

in ovarian fluids ranged from 10 1 to 10 7 cfu/mL in
the populations from the Shibetsu and Teshio rivers.
Ovarian fluid containing A. salmonicida flows out of
the fish at the time eggs are stripped or during theprocess of maturation in the pond. Consequently, infected water and infected ovarian fluid are expelled
into the river because the sewage from the egg stripping areas and the holding ponds is not disinfectedin Hokkaido. We suspect from these results that theagent drained from the fish may be a source of infection for other anadromous salmon that ascend theriver for spawning.
Horne and Maj (1928), McCraw (1952), andHastein and Lindstad (1991) stated that the most important source of A. salmonicida in the spread of
furunculosis is the existence of fish carrying thisagent. Fish carrying the bacterium pose a serious
problem to the prevention of furunculosis, and their
reduction in fish plays a key role in salmon propagation.

A. salmonicida on Egg Surfaces_

The existence of the bacterium in the ovarian fluidsuggests that the surface of eggs taken from the fish
will also be contaminated. Contaminated eggs may
spread the agent to areas where the eggs will betransplanted. We studied the existence of A.
salmonicida on the surface of eggs by artificially contaminating chum salmon eggs with A. salmonicida.
The A. salmonicida 20-1, strain, which was isolated
from the kidneys of chum salmon in the TokachiRiver, was used as innoculum. The strain was cultured
and harvested, then was suspended in phosphatebuffer saline (PBS). The chum salmon eggs werebathed in PBS containing the agent for an hour. The
eggs were incubated in well water at 8° C in the laboratory. At one hour and at 24 hours after fertilization,
we took 20 eggs from the incubator and put theminto sterilized water. The flask was shaken strongly for
5 minutes, we then measured the viable number of A.
salmonicida in the water according to the method of
I'\omura et al. (1991b).
Egg surfaces were initially bathed with 1.1 x 104 to
4.3 X 106 cfu/egg of A. salmonicida. The number of A.
salmonicida present on the egg surfaces decreased
from 68 to 4.6 cfu/egg an hour after fertilization andA. salmonicida could not be isolated from the eggs
cultured on plates 24 hours after the initial bathtreatment.
We also attempted to isolate A. salmonicida fromeggs in the incubation boxes at the Satsunai,
Nakagawa, Nemuro, and Tokachi hatcheries. These
eggs were taken from brood fish in which the preva-

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Nomura et al.: Furunculosis in Salmon Propagation

lence of A. salmonicida was high (Nomura et al.
1991a). Fortunately, A. salmonicida was not isolated
from any of the 15 hatcheries' eggs (Nomura et al.
1991b) .
In Hokkaido, fertilized eggs are transported to ahatchery from the egg collection location 1 hour after fertilization. From the results of our experiment,
it appears that A. salmonicida is able to exist on thesurface of an egg. This makes us concerned that wemay be transporting the bacteria to the hatchery withthe fertilized egg. We believe that it is necessary to
prevent the transfer of A. salmonicida via eggs in order to control furunculosis.

Survival of A. salmonicida in Water_

By definition, A. salmonicida is considered to be an
obligate pathogen (Popoff 1984) and is never foundin surface water. Its ability to survive and remain infectious in the external environment may be a major
determinant in the spread of furunculosis. We studied the viability of A. salmonicida in nonsterile, sterile
filtered, and autoclaved fresh water and in salt water.
A. salmonicida strain 20-1 isolated from chumsalmon in the Tokchi River was used in this experiment. The strain was cultured at 20°C and harvested,
then suspended in fresh water or in sea water. The
suspended cells were inoculated into 200 mL ofnonsterile, sterile filtered, and autoclaved fresh waterand saltwater and were incubated at 10° C.
In sterilized fresh water, A. salmonicida survived for
60 days and in nonsterile water, only 4 days. The survival of A. salmonicida in sterile salt water was 8 days;
this was a shorter survival period than that in sterile
fresh water.

105

It is believed that A. salmonicida is not able to exist
for long time in water without fish, but McCarthy(1980) studied the survival of the agent in water using an antibiotic-resistant strain of A. salmonicida andfound the agent could survive for 8 days in water.
The results of McCarthy (1980) and this study indicate that A. salmonicida survives long enough to infectother fish in the water.

Variation of Agglutination Titer
Against A. salmonicida in the SerumA serological survey of adult salmon was made fromblood samples collected in 1988 in Hokkaido, frommature chum, pink, and masu salmon. Blo<,>d was
aseptically extracted from the donsal artery with 10
mL of Vacteinor (Terumo Co., Tokyo, Japan). The
resulting serum was separated from the blood-cellclot by centrifugation and was stored at -90°C until
assayed. The serum was tested for agglutinating antibody titers individually, by test-tube methods with A.
salmonicida ATCC14174.
Agglutinin titers against A. salmonicida in the serum of mature chum, pink, and masu salmon in
Hokkaido in 1988 are shown in Table 1.
Of a total of 75 serum samples taken from maturechum salmon, 73.3% did not have 'the agglutinin,
and the range of titers was 8 to 32. In pink salmon,
10% of the sample did not have the agglutinin, andthe range was 4 to 32. In masu salmon, 16.6% of theexamined serum did not have the agglutinin, and therange was 4 to 128.
The diversity in the incidence of agglutination titerwithin each of the three species indicates a continuous, widespread interaction between individuals ofTable I

Prevalence and ranges of agglutinin titers against A. salmonicida in the serum of mature chum, pink, and masu salmon
ascending various rivers in Hokkaido.
.
SpeciesRiver_

No. of fish examined

Agglutinin titers
Negative (%)

Modes

Range

Chum salmon

IchaniTokachiShizunai

15
30
30

80.0
76.6
66.6

32
8
16

16- 32
8- 32
8- 32

Pink salmon

Shokotsu
Iwaobetsu
ishibetsu

30
30
30

16.6
6.7
6.7

16
16
16

4- 32
4- 32
4- 32

Masu salmon

Shiribetsu
Shari
Nishibetsu

39
18
27

15.4
0
29.6

8
16
8

4-128
4-128
4- 32

106

NOAA Technical Report NMFS 111_

the host populations and A. salmonicida. The difference in the amount of agglutination titer is
proportionate to the period of A. salmonicida infection.
In general, the percentage of serologically reactive
salmon increased as their length of freshwater residency increased. Weber and Zwicker (1979) reported
that of a total of 43 serum sampled from Atlantic
salmon (Salmo salar) in the Miramichi or Margareen
rivers in Canada, none had A. salmonicida agglutinin, but of 27 Restigouche River salmon, four had atiter of 10, five had a titer of 20, and one had a titerof 640. They confirmed that Atlantic salmon havepreviously contacted A. salmonicida III the
Restigouche River.
In our study, agglutinate titers in the serums were
low. It was suggested that the fish were infected with
A. salmonicida shortly before their eggs were stripped.
Kimura (1970) reported that the immunologicalmethod of preventing furunculosis was useful inadult masu salmon during the holding period because these salmon stay in fresh water for a longenough period to allow them to produce antibodies
after antigen inoculation. In chum salmon, however,the freshwater residency period is short, so thismethod of prevention would not be practical.

Isolation of the Bacteriophageof A. salmonicida from Water_

There is no sensitive medium for selecting A.
salmonicida. This means that when the number of A.
salmonicida in water is low, the isolation of A.
salmonicida from the water will be difficult. This isbecause A. salmonicida cannot grow on the culture
medium under competitive conditions with othernatural bacteria populations. We attempted to isolate
the bacteriophage of A. salmonicida to ascertain theexistence of A. salmonicida in the water.
Water samples, from 11 hatcheries and 4 rivers
were examined. Nutrient agar (Eiken Co., Tokyo, Japan) was employed for the routine culture, dilution,and enumeration of A. salmonicida and its phagestrain. One hundred mL of sample was added to 500
mL of cultured A. salmonicida Ar-32, Ar-43, Ar-71 , andH-70 strains in the logarithmic phase. Detection andenumeration of phage were achieved using the medium and double agar layer technique (Paterson etal. 1969). The results are shown in Table 2.
McCraw (1952) stated that when the bacteriophageof A. salmonicida exists, its presence may indicate theexistence of the bacterium. The bacteriophage wasisolated from two samples of river water and fivesamples from hatchery water. From this result, it wassuspected that A. salmonicida can survive for a longtime in the river water in Hokkaido after leaving thefish and that its existence may be a source of infection to salmonid fish. The results suggest that suchbacteriophage could be very useful for studying theexistence of the agent in water.

Table 2Isolation of Aeromonas salmonicida bacteriophagefrom the samples of river water and hatchery water.
SampleRiver water
Hatchery water
Sewage of hatchery

SamplesNumbers examined

Isolated
containing phage

4
9
13

2

Pathogenicity of the
Isolated A. salmonicida

2
3_

The isolated strains were identified as A. salmonicida,
subspecies salmonicida, by their biological, biochemical, and immunological characteristics. All of theisolated A. salmonicida strains showed auto-agglutination and produced protease in the medium, so we
also expected them to be pathogenic.
In order to examine the pathogenicity of the isolated strain, we injected it into chum and masu
salmon fry and adult chum salmon.
A. salmonicida 20-1 was cultured for 48 hours at
20°C. The cells were washed three time in PBS andwere suspended in PBS. The strain was injected into
chum salmon fry, yearling masu salmon, and chumsalmon brood fish at concentrations of 1.7 X 102 , 1.8
X 10 3 and 6.0 X 105 cfu/fish, respectively.
All of the examined fish showed typical signs offurunculosis 3 to 4 days after injection. The numberof A. salmonicida in the kidneys of moribund fish wasaround 108 cfu/g kidney tissue, the same number reported by Morikawa et al. (1981) in the kidneys ofmoribund amago salmon. On the basis of these results, we suspect the isolate is a pathogenic strain.

Control of A. salmonicida
on the Surface of Egg_

To establish a method of controlling A. salmonicida
on the eggs, the bactericidal effect of popidon-iodine(Isodine), and the toxicity of this agent to the chumsalmon egg were studied.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Nomura et al.: Furunculosis in Salmon Propagation

The bactericidal effects of popidon-iodine to A.
salmonicida were determined in accordance with themethod of Amend and Fryer (1972).
A. salmonicida was completely killed by treatment
with 25 ppm isodine for five minutes and this solution was not toxic to the chum salmon eggs fortreatments lasting up to one hour. Thus, the authors
confirmed that isodine solution has a sanitizing effect on the agent, and that it does not have adverse
effects on chum salmon eggs.

Method for Decreasing the Prevalenceof A. salmonicida in Chum Salmon_

From the results of our epidemiological study, we suspected that the incidence of A. salmonicida wasaffected by the density of fish during their maturation
period in the holding ponds; as the average density ofbrood fish stocked in ponds decreased, the incidenceof A. salmonicida in examined fish also decreased (Fig.
4). Therefore, we examined the relationship between
the stocking density of fish in the pond and the prevalence of A. salmonicida in the fish.

Chum salmon in the Ishikari River were randomly
assigned to experimental holding ponds and held under low (4.9 fish/m 2 ) and high density (14.7 fish/m 2 )conditions until maturation. The kidney tissues of all
the fish used in experiment were cultured on nutrient agar in accordance with the method of Nomura
e t al. (199 I ) .
As we expected, we found that 12.4% of the fish
examined harbored A. salmonicida when they were
stocked at a high density, but no examined fish contained the agent when they were stocked at a low
density (Fig. 5A). The incidence of the bacterium infish that were he1dunder low dissolved oxygen conditions was higher than that of fish held under highdissolved oxygen levels (Fig. 5D). These results
clearly indicate that high stocking densities and low
dissolved oxygen levels in holding ponds have amarked effect on the prevalence of the agent in thefish. We concluded that fish maturation in the pond
under low density conditions and disinfection of theeggs, are necessary to prevent fish furunculosis in theartificial propagation of salmon.

Citation
2

FisMn
15

A

Fish stock density

B'

Dissolved oxygen

10
5

o
ppm

10

8

o

6

5

OL--------Ie Survival rate

100%

50

~

o

~

L

Io_c_id_eo_c_.

tL...D

Lot A

Lot B

Figure 5
The relationship between the densityof fish stock in holding ponds, theconcentration of dissolved oxygen in
the water, survival rate, and the incidence (% occurrence) of Aeromonas
salmonicida (unpub\. data).

107_

Amend. D.F.• andJ.L. Fryer.
1972. Virucidal activity of two iodo-phors to salmonid
virus. J. Fish. Res. Board Can. 29:61-65.
Amos. K.H .. ed.
1985. Fish health blue book-procedures for the detection ofcertain fish pathogens. Fish health section. Am. Fish. Soc.•
114 p.
Austin. B.• and D.A. Austin.
1987. Bacterial fish pathogens: disease in farmed and wild
fish. Ellis Horwood Ltd.• Chichester. West Sussex, England. 195 p.
Daly.J.G.• and R.M.W. Stevenson.
1985. Importance of culturing several organs to detect
Aeromonas salmonicida in salmonid fish. Trans. Am. Fish.
Soc. 114:909-910.
Emmerich. R., and C. Weibel.
1890a. Ube. eine durch Bacterien verursachte Infectionskrankheit del' Forellen. Allg. Fish. Zgt. 15:73-77.
1890b. Uber eine durch Bacterien verursachte Infectionskrankheit del' Forellen. Allg. Fish. Zgt. 15:85-92.
Hastein. T.. and T. Lindstad.
1991. Disease in wild and cultured salmon: possible
interaction. Aquaculture. 98:277-288.
Herman, R.L.
1968. Fish furunculosis 1952-1966. Trans. Am. Fish. Soc.
97:221-230.
Horne.J.H .• and I.M.S. Maj.
1928. Furunculosis in trout and the importance of carriersin the spread of the disease. J. Hyg. 28:67-78.
Kimura, T.
1970. Studies on a bacterial disease occurred in the adult"Sakuramasu" (Oncorhynchus masou) and pink salmon (0.
gorbuscha) rearing for maturity. Sci. Rep. Hokkaido Salmon
Hatchery. 24:9-100. (In Japanese; English abstr.)

108

NOAA Technical Report NMFS III

McCraw, B.M.
1952. Furunculosis of fish. U.S. Fish and Wildl. Servo Spec.
Sci. Rep. 84:87 p.
McCarthy, D.H.
1980. Some ecological aspects of the bacterial fish pathogen-Aeromonas salmonicida. In Aquatic microbiology: symposium of the Society of Applied Bacteriology 6:299-324.
Morikawa, S., S. Miki, and F. Tashiro.
1981. Changes in hemato logical properties and viable cellnumber of bacteria in amago salmon artificially infectedwith Aeromonas salmonicida. Fish Pathol. 16:43-49. (InJapanese; English abstr.)
Nishino, K.
1967. On a bacterial disease of adult "sake" Oncorhynchus
keta, in captivity. Fish Pathol. 2(1):73-74. (In Japanese.)
Nomura, T
1983. Incidence of Aeromonas salmonicida among anadromous salmonids, 1980-1982. Sci. Rep. Hokkai- do Salmon
Hatchery. 37:63-65. (In Japanese; English abstr.)
Nomura, T, T Kimura.
1981. Incidence of Aeromonas salmonicida among anadromous salmonids. Fish Pathol. 16:69-74. (In Japanese; English abstr.)
Nomura, T, T Kimura, I. Shimizu, and K. ara.
1983. Incidence of Aeromo.nas salmonicida among chumsalmon, Oncorhynchus keta, in Chitose River. Sci. Rep.
Hokkaido Salmon Hatchery. 37:53-61. (In Japanese; English abstr.)
Nomura, T., M. Yoshimizu, and T Kimura.
1991a. Prevalence of Aeromonas salmonicida in the chumsalmon (Oncorhynchus keta), pink salmon (0. gorlmscha) andmasu salmon (0. masou) returning to rivers in
Hokkaido. Fish Pathol. 28. 139-147. (In Japanese: English abstr.)..;.....

_

Nomura, T, M. Yoshimizu and T Kimura.
1991 b. Prevalence of Aeromonas salmonicida in the kidney ofchum salmon (Oncorhynchus ketal and masu salmon (0.
masou) at various life stages. Fish Pathol., 26: 149153. (In Japanese; English abstr).
Paterson, W.O., R.J. Douglas, I. Grinyer, and L.A. McDermott.
1969. Isolation and preliminary characterization of some
Aeromonas salmonicida bacteriophages. J. Fish. Res. Board
Can. 26:629-632.
Popoff, M.
1984. Family II. Vibrionaceae. In Bergey's manual of systematic bacteriology, Vol. I (Noel R. Krieg and John G. Holteds.), p. 545-550. Williams and Wilkins, Baltimore.
Smith,I.W.
1960. Furunculosis in salmon kelts. Nature 186:733-734.
Snieszko, S.F.
1972. Panel review on furunculosis of salmonidae. In Reportof the symposium on the major communicabable fish diseases in Europe and their control; 20-22 April 1972,
Amsterdam (William A. Dill, ed.): p 157-163. European Inland Fisheries Advisory Commitee EIFA Technical paper 17(suppl. 2).
Yoshimizu, M., T Kimura, andJ.R. Winton.
1985. An improved technique for collecting reproductive
fluid samples from salmonid fish. Prog. Fish-Cult. 47:199200.
Weber,J.M. and B.M. Zwicker.
1979. Aeromonas salmonicida in Atlantic salmon (Salmosalar). J. Fish. Res. Board Can. 36:1102-1107.

Functions of Hemocytes During the Wound Healing Process
in the Pearl Oyster

TOHRU SUZUKI
National Research Institute ofAquaculture
Nansei, Mie 516-01,Japan

ABSTRACT
Morphological evidence from previous studies suggests that bivalve hemocytes functionin hemostasis and extracellular matrix production during the wound healing process.
The present paper describes the use of an in vitro cell culture system to show that theagranular hemocytes of the pearl oyster Pinetada fueata have the ability to perform these
healing functions. A possible wound healing system is also presented.

Introduction_

Defense reactions against infection and the removal oftissue debris are the responses of the immune systemthat occur after wounding. On the other hand, tissue
regeneration at the wound site is an organogenetic
event in which cell-eell and extracellular matrix (ECM)cell interactions occur. Thus, the wound healing
process can be a useful model to study the functions ofproteins and other cell components in both the immune and organogenesis systems. Because bivalves haveno humoral clotting factors, platelets, or capillary vessels, it is assumed that their wound healing systemdiffers from that of vertebrates and is probably less complex. The wound healing process has been describedfor several bivalves at the morphological level. It is
known that the hemocytes prevent blood loss at thewound site by forming a cellular sheath and producing
an ECM (DesVoigne and Sparks 1968; Pauley andHeaton 1969; Ruddell 1971). The healing process inthe pearl oyster Pinetada fueata and the associated functions of hemocytes have been demonstrated (Suzuki etal. 1991). In this paper, the author describes a study
using cultured hemocytes from the pearl oyster inwhich agranular hemocytes show the ability to form acellular sheath and to produce an ECM. A possible
wound healing system is also presented.

Methods_

muscle with a plastic syringe attached to a 24 gauge
needle (Fig. 1). Approximately 2 mL of blood wascollected from each animal. Blood pooled from fiveanimals was centrifuged at 100 X g for 8 minutes anda pellet of hemocytes was obtained. The pellet waswashed twice with a balanced salt solution for marine
molluscs (MMBSS) prepared according to Machii
and Wada (1989). The pellet was suspended on 2 mLof culture medium PJ35 (Machii and Wada 1989),
developed specifically for pearl oyster tissue. A 0.5mL sample of the suspension was pipetted into aplastic culture flask (50 mL: Nunc). Mter an hour, toallow the hemocytes to adhere to the plastic surface,
2.0 mL of Pf35 was added to the flask. The cells werethen incubated at 25 D C. The culture medium was renewed on the fourth day of culture.

Microscopy
The cultured cells were fixed in Karnovsky's fixative
containing 8% sucrose at 4 D C for 1 hour. Mter washing in 0.1 M phosphate buffer (PB; pH 7.2), they were
post-fixed in 1% osmic acid in PB for 1 hour, washed
twIce in PB, dehydrated through graded alcohols, andembedded in Taab 812 resin. Ultrathin sections were
stained with uranyl acetate and lead acetate. All transmission electron microscopy was performed with a
JEOLJEM- I200EX electron microscope.

Results and Discussion_

Hemocyte Culture

Aggregate Formation

While the valves of P fueata were held slightly openwith a wedge, blood was drawn from the adductor

The hemocytes formed cellular aggregates during
blood collection and rinsing. Three hours after the
109

110

NOAA Technical ReportNMFS 111_

viewed under phase-contrast microscopy (Fig. 5).
The matrix continued to develop until about the seventh day of culture, after which time the number ofliving hemocytes rapidly decreased. Agranular hemocytes of 1-3 layers thickness were attached to thematrix, which was composed of fine fibrils andflocculent substances (Figs. 6 and 7). The fibrils were
20 nm in diameter and showed a faint banding pattern indicating that they are collagenous fibrils. The
flocculent substances are probably proteoglycans. It
has also been ascertained at the biochemical levelthat the matrix includes collagen (Suzuki et al.
1991) .

Blood collection

Suspension

Wound Healing System
Standing for 1h

Culture at 25·C

I

Figure 1

Procedure used for hemocyte culture.

beginning of culture, the aggregates were 80-200 ~min diameter and adhered well to the plastic surface ofthe culture flask (Fig. 2). In bivalves, two categories
of hemocytes occur-agranular and granular hemocytes (Feng et al. 1971). It has been demonstrated
that the former are macrophage-like cells with active
phagocytic capabilities (Reade and Reade 1972;
Moore and Lowe 1977). One hemocyte of the pearl
oyster is able to take in 1-9 vertebrate erythrocytes(Suzuki and Mori 1990). As shown by transmission
electron microscopy, the aggregate is formed only byagranular hemocytes (Fig. 3). They adhere to one
another at an adhesion plaque from which microfilaments can be seen running into the cytoplasm (Fig.
4). Thus, agranular hemocytes can form a cellularaggregate in a relatively short time (perhaps several
minutes if stimulation is provided), although it is notunderstood what factors stimulate the reaction. It is
possible that this aggregate formation is a homologous reaction to the cellular sheath formation at thewound site.

ECM Production
During the first 2-3 days of culture, deposition of an
ECM started in the agranular hemocyte aggregate.
The matrix was observed as a transparent gel whenIn the pearl oyster, four noticeable cellular reactions
occur during the healing p'rocess (Suzuki et al.
1991): 1) removal of tissue debris, 2) cellular sheathformation, 3) ECM deposition, and 4) epithelial regeneration. The first reaction is attributed to thephagocytic ability of agranular hemocytes. In addition, these cells also perform the second and third ofthese healing reactions, cellular sheath formationand ECM deposition, as suggested by the results ofthis paper.
Based on these results, a possible wound healing
system in the pearl oyster is presented in Figure 8.
When the pearl oyster is wounded, agranular hemocytes infiltrate the injured area to remove tissuedebris (and possibly foreign particles) and then forma cellular sheath to prevent blood loss. After sheathformation, they begin to produce an ECM, whichfunctions as a template for the regenerating epithelium. It is possible that the wound site is repaired
through this sequence of processes. Assuming this explanation is accurate, the wound healing system ofthis bivalve is simple compared with that of vertebrates.
It is well known that in vertebrates growth factors
secreted from the platelets, macrophages, andfibroblasts are the humoral factors that control cell
growth and ECM production at the wound site. In
bivalves, however, neither a growth factor nor its
production cell has been identified. The agranularhemocyte is the most likely candidate to be a growth
factor production cell, because, like macrophages andfibroblasts in vertebrates, it is the primary cell typein bivalves that appears at the wound site. In addition, platelets are absent from bivalve hemocytes.
Future studies with in vitro cultures will improve ourunderstanding of both the wound healing systemand organogenesis in bivalves.

Suzuki: Function of Hemocytes During Wound Healing in the Pearl Oyster

III

Figure 2 Hemocyte aggregate after 3 hours of culture. X 135.
Figure 3 Electron microscopy of aggregate after I day of culture. X 2,000.
Figure 4 Adhesion plaque formed at contacting site of agranular hemocytes. X 6,700.
Figure 5 Matrix (arrowhead) formed in a hemocyte aggregate after 7 days of culture. X 135.
Figure 6 Electron microscopy of matrix and agranular hemocyte layer. X 2500.
Figure 7 Fibrils in the matrix at high magnification. X 31,000.

Citation_

DesVoigne, D.M., and A.K. Sparks.
1968. The process of wound healing in the pacific oyster,
Crassostrea gigas. j. Invertebr. Pathol. 12:53-65.
Feng, S.Y.,j.S. Feng., C.N. Burke, and L.H. Khairallah.
1971. Light and electron microscopy of the leucocytes of
Crassostrea virginica (mollusca: pelecypoda). Z. Zellforsch.
120:222-245.
Machii, A., and K.T. Wada.
1989. Some marine invertebrates tissue culture. In Invertebrate cell system application, Vol. 11 (j. Mitsuhashi, ed.) p.
225-223. CRC Press, Boca Raton, Florida.

Moore, M.N., and D.M. Lowe.
1977. The cytology and cytochemistry of the hemocytes of
Mytilus edulis and their responses to experimentally injectedcarbon particles. j. Invertebr. Pathol. 29:18-30.
Pauley, G.B., and L.H. Heaton.
1969. Experimental wound repair in the freshwater mussel
Anodonta oregonensis. j. Invertebr. Pathol. 13:241-249.
Reade, P., and E. Reade.
1972. Phagocytosis in invertebrates 11-the clearance of carbon particles by the clam, Tridacna maxima. J.
Reticuloendothel. Soc. 12:349-360.

112

NOAA Technical Report NMFS III

Infiltration o! agranular hemocytes
Removal of tissue debris by them_

Cellular sheath !ormation
by agranular hemocytes---Regeneration of epithelium
along the new ECM

Secretion of ECM
by agranular hemooytes
Growth !actors?

Ruddell, C.L.
1971. The fine structure of oyster agranular amebocytes
from regenerating mantle wounds in the pacific oyster,
Crassostrea gigas. J. 1nvertebr. Pathol. 18:260-268.
Suzuki, T., and K. Mari.
1990. Hemolymph lectin of the pearl oyster, Pinetada fucata
martensii: a possible non-self recognition system. Dev.
Compo 1mmunol. 14:161-173.

Figure 8
A possible wound healing system in the pearl oyster: ECM=extracellular matrix.

Suzuki, T., R. Yoshinaka, S. Mizuta, S. Funakoshi, and K. Wada.
1991. Extracellular matrix formation by amebocytes during
epithelial regeneration in the pearl oyster Pinctada
fucata. Cell Tissue Res. 266:75-82.

Skeletal Abnormalities of Fishes Caused by
Parasitism of Myxosporea

YUKIO MAENO and MINORU SORIMACHI
National Research Institute of Aquaculture
Fisheries Agency
Nansei-cho, Mie 516-01,Japan

ABSTRACTThe relationship between skeletal abnormalities and parasitic infection was studied for
deformed cultured yellowtail, Seriola quinqueradiata, Japanese bluefish, Scombrops boops,
and mullet, Mugil cephalus. Soft radiographic observations indicated that the deformities
were due to skeletal abnormalities: deformed yellowtail, Japanese bluefish, and mullet
were characterized by scoliosis, lordosis, and lordo-scoliosis, respectively. Myxosporean
cysts were found in various parts of the brain of deformed fish such as the fourth ventricle, the cavity of the optic tectum, the surface of the olfactory lobe and bulb, and theoptic lobe. The cysts were observed in the fourth ventricle in all deformed fish, but not innormal fish. From morphological characteristics, myxosporeans from the deformed yellowtail and Japanese bluefish were identified as Myxobolus bun, and that from deformed
mullet was identified as M. spinacurvatura. These results suggest that Myxobolus species are
responsible for the skeletal abnormalities observed in these deformed fish.

Introduction_

Skeletal abnormalities of fish have been reported tobe caused for various reasons, such as dietary deficiencies (Halver and Shanks 1960), pesticide
exposure (Couch et al. 1977), heavy metal exposure(Holcombe et al. 1976), and bacterial (Kaige et al.
1984) and parasitic infections (Halliday 1976). Many
studies report sporulation of myxosporeans in fish
brains. However, only a few studies have described apossible correlation between skeletal abnormalities
and myxosporean parasitism in the brain. While
studying skeletal abnormalities in three species offish, cultured yellowtail (Seriola quinqueradiata) , Japanese bluefish (Scombrops hoops), and mullet (Mugil
cephalus), we found spores of a myxosporean in thebrains of these fish. The present paper elaborates on
the possible relationship between skeletal abnormalities and parasitism by myxosporeans with specialreference to parasitized regions of the brain.

Materials and Methods_

Seventeen yellowtail (age 1+), including 7 deformed
ones, were collected from a fish farm in Mie Prefec-

ture from 1987 to 1989.Four deformed Japanese
bluefish and four deformed mullet were captured bya set net in Mie Prefecture in 1989.
After external observation, radiography of all fish
was performed with Fuji Lx film at 40 milliamps and
50kV for 20 seconds and subsequently examined forparasitism in viscera by necropsy. From these animals,
three deformed and three normal yellowtail, three
Japanese bluefish, and three mullet were used for
subsequent histological examination.
The brain, liver, kidney, and spleen were excisedfrom each fish and fixed in 10% formalin, embedded
in paraffin, cut serially at 4 j..lm, stained with Giemsa
or hematoxylin and eosin, and then observed for thepresence and location of cysts.
. For the study of spore morphology, some myxosporean cysts were removed from the brain and crushed inphosphate buffer saline to get a suspension of fresh
spores. Light microscopic observations were made bothon fresh spores and those stained with Giemsa.
For the scanning electron microscopy, spore-suspensions were fixed in Karnovsky's fixative for 2
hours at room temperature. They were then dehydrated in a graded ethanol series, treated withisoamyl acetate three times, each for 15 minutes, processed in a critical point dryer, sputter-coated with
113

114

NOAA Technical Report NMFS 111_

1

Figure 1
Scoliosis found in yellowtail
Seriola quinqueradiata (dorsal view).
Figure 2
A cyst of Myxobolus buri foundin the brain of yellowtail(Seriola quinqueradiata) Giemsa stain (X300).

gold, and examined with the JEOL Uapan Electronics Optical Limited) T220A scanning electron
microscope.

Results_

Cultured Yellowtail
The deformities were externally apparent as curvatures from the trunk to caudal region. Radiographyof the deformed specimens revealed extreme skeletal

curvatures consisting of a smooth S-like bend in thelateral plane, scoliosis (Fig. 1).
In the seven deformed yellowtail, myxospore an
cysts were found in one or more of the following
regions: beneath the meninges of the olfactory bulb
and lobe, surface of optic lobe, cerebellum, medulla
oblongata, inferior lobe, the cavity of optic tectum,
and the fourth venticle. In particular, the cysts were
found in the fourth ventricle of the brain in all deformed fish examined (Table 1). In the normal fish,on the other hand, no cysts were found in the fourth
ventricle of three fish, even though seven fish had

Maeno and Sorimachi: Skeletal Abnormalities of Fishes Caused by Myxosporea

115

Table I
Distribution of cysts in the brains of deformed (n=7) and apparently normal (n=1O) yellowtail Seriola quinqueradiata.
(I=olfactory bulb; ll=olfactory lobe; lll=optic lobe; IV=the cavity of optic tectum; V=cerebellum; Vl=fourth ventricle;
VlI=inferior lobe.)

Deformed fish
Normal fish
Infected
Non-infected

II

III

IV

V

VI

VII

5/7

5/7

2/7

3/7

1/7

7/7

2/7

2/5
0/5

3/5
0/5

2/5
0/5

1/5
0/5

0/5
0/5

0/5
0/5

1/5
0/5

cysts in some other region of the brain. No cysts wereobserved in regions other than the brain.
The cysts range from 0.5 to 3 mm in diameter andwere enveloped by a collagenous capsule withfibroblasts (Fig. 2). Most cysts were filled with nearlymature or mature myxosporean spores. Morphological observations showed spores to be broadly
ellipsoidal and symmetrical in shape and to possess aprominent sutural ridge in the front view. In the sideview they were lenticular and symmetrical and consisted of two shell valves. The polar capsules were
pyriform and nearly equal in size, and a smallintercapsular appendix was clearly seen. The length,
width, and thickness of spores were 10.5,9.0, and 6.3/lm, respectively. From these morphological characteristics they were identified as Myxobolus bun Egusa,
1985.

In deformed Japanese bluefish, cysts were always
found in the fourth ventricle and less frequently
found in the cavity of the optic tectum. In some cases
cysts were found attached to the surface of the olfactory bulb and olfactory lobe and in the cavity of the
optic tectum. No cysts were found in tissue otherthan the brain. Both light and scanning electron microscopy revealed that the spores from these fish
were morphologically similar to those obtained fromdeformed yellowtail. The spores were broadly ellipsoidal in frontal view and broadly lenticular in side
view; the shell valve of the spore had a sutural ridgewith fold; each spore had two polar capsules nearly
equal in shape and size; and an intercapsular appendix was presen t (Figs. 4 and 5). The average length of
fresh spores was lOA /lm, width 9.1 /lm, and their
thickness 6.2 /lm.

Japanese Bluefish

MulletRadiographic observations demonstrated that theabnormality in the four Japanese bluefish was
dorso-ventral deformity of the vertebral column
and flexure of the neural spine and hemal spine(Fig. 3).

The four deformed mullet showed spinal curvatureand lordo-scoliosis in radiography (Figs. 6 and 7).
Histologicalexamination
revealedmany
myxosporean cysts on the surface of the gut and invarious visceral organs such as the liver, kidney,

116

NOAA Technical Report NMFS III

4a_

Figure 3
Soft radiograph of lordosis in a Japanese bluefish (Scomm-ops boops) (side view).
Figure 4
Fresh spore of Myxobolus sp. obtained from thebrain of Japanese bluefish. (a) frontal view (arrow indicates intercapsular appendix); (b) sideview (bar=lO 11m). Phase contrast microscopy.
Figure 5
Scanning electron micrographs of spores ofMyxobolus sp.obtained from the brain of Japanese bluefish. (a) frontal view; (b) side view.

spleen, pancreas, and brain of the deformed mullet.
As in yellowtail and Japanese bluefish, cysts were
found in various regions of the brain, such as the
fourth ventricle, the cavity of the optic tectum, thesurface of the olfactory lobe and bulb, and the optic
lobe. Cysts were observed in the fourth ventricle of
all four fish examined (Fig. 8) .The average length of
fresh spores was 11.5 Ilm, their width was 9.8 Ilm, andthickness 6.7 Ilm. From the morphological characteristics of the spores, such as their rounded frontal
view, lenticular side view, two smooth shell valves, two
teardrop-shaped polar capsules situated on the su-

tural plane, and lack of an intercapsular appendix(Fig. 9), these myxosporean spores were considered
to belong to the genus Myxobolus (Lorn and Noble
1984) .

Discussion_

The morphological characteristics of the spores
clearly indicate that the myxosporean found in deformed yellowtail of the present study is M. bun. The
light and scanning electron microscopic examina-

Maeno and Sorimachi: Skeletal Abnormalities of Fishes Caused by Myxosporea

117

Figure 6
Soft radiograph of lordosis in a mullet (Mugil
cephalus) (side view).

8

Figure 7
Soft radiograph of scoliosis in a mullet (Mugil
cephalus) (dorsal view).
Figure 8
Diagrammatic view of the distributions of thecysts in the brain of mullet Mugil cephalus(*=loci of the cyst).

9a

9b

Figure 9
Fresh spore of Myxobolus spinacurvatura obtained from the brain of mullet. (a) frontal
view; (b) side view (bar=lO 11m).

118

NOAA Technical Report NMFS 111

tions also demonstrated a complete similarity in themorphology between the spores from the deformed
Japanese bluefish and those from deformed yellowtail. Thus, the spores parasitic in the brain of thedeformed Japanese bluefish are considered to be
those of M. bun.
To date, more than 400 species of Myxobolus havebeen described. The morphological characteristics ofMyxobolus from the deformed mullet differ fromthose of most species described. Only three Myxobolus
species, M. achmerovi Shulman, 1966, M. bun, and M.
spinacurvatura Maeno et aI., 1990, have morphological similarities to those found in deformed mullet.
However, M. achmerovi, which has been found fromthe gill, fin, and mesentery of common carp
(Cyprinus carpio) and mullet (Mugil cephalus)(Shulman 1966) is different from the present
Myxobolus in that the former spore has an ellipsoidal
frontal view, and a distinct intercapsular appendix.
M. bun also differs from the present Myxobolus inmullet because the spore of M. bun has a broadly
ellipsoidal frontal view, distinct folds around theedge, an intercapsular appendix, and a distinct polar
filament inside the polar capsule. On the other hand,the spores from the deformed mullet were quite similar to those of M. spinacurvatura in bothmorphological characteristics and in spore dimensions. Thus, the Myxobolus found in deformed mullet
in the present study is considered to be M.
spinacurvatura.
In the present study the myxosporean cysts wereinvariably observed in the fourth ventricle of thebrain in all deformed fishes examined. Myxosporeanparasitism in the central nervous system of fish havebeen studied in the bullhead Cottus gobio (Lom et al.
1989), the redfin perch Perea fluviatilis (Langdon
1987), the yellowtail Senola quinqueradiata (Sakaguchi
et al. 1987), the fathead minnow Pimephales promelas(Mitchell et al. 1985), and the Japanese river goby
Acanthogobius flavimanus (Hoshina 1952). However,even in a heavily myxosporean-infected bullhead, motor or sensory disturbances were not observed. Also,in myxosporean-infected fathead minnow and Japanese river goby, no clear external signs of deformity
were found. On the other hand, skeletal abnormalities were observed in the myxosporean-infected
redfin perch and yellowtail. In the redfin perch, cysts
were located in the regions of the mesencephalon,
diencephalon, third ventricle, and medulla oblongata, while in the yellowtail, cysts were found in thecavity of the optic tectum, the surface of the optic
and olfactory lobes, and in the fourth ventricle.

_

These studies, as well as the present study, indicate
some correlation between skeletal abnormalities andmyxosporean parasitism in the brains of fish. In addition, the present study strongly suggests that skeletal
abnormalities occur when cysts infect particular regions of the brain such as the fourth ventricle. Thus,
it is quite possible that myxosporean infection in thisregion of the brain mechanically affects central nervous system function to produce skeletal
abnormalities in fish.

Citations•_

Couch,j.A.,j.T. Winstead, L.R. Goodman.
1977. Kepone-induced scoliosis and its histologicalcosequences in fish. Science 197:585-587.
Halver,j.E., and W.E. Shanks.
1960. Nutrition of salmonid fishes VIII. Indispensableaminoacids for sockeye salmon. j. Nutrition 72:340-346.
Halliday, M.M.
1976. The biology of Myxosoma cerebralis: the causative organism of whirling disease of salmonids. j. Fish BioI. 9:339357.
Holcombe, G.W., D.A. Benoit, E.N. Leonard, andj.M. McKim.
1976. Long-term effects of lead exposure on three generations of brook trout (Salvelinus jontinalis). j. Fish. Res.
Board Can. 33:1731-1741.
Hoshina, T.
1952. Notes on some myxosporidian parasites on fish ofJapan. j. Tokyo Univ. Fish. 39:69-89.
Kaige, N., T. Miyazaki, and S. Kubota.
1984. The pathogen and the histopathology of vertebral deformity in cultured yellowtail. Fish Pathol. 19: 173-179. (InJapanese; English abstr.)
Langdon,j.S.
1987. Spinal curvatures and encephalotropic myxosporean,
Triangula pereae sp. nov. (Myxozoa: Ortholineidae), enzootic
in redfin perch, Perea fluviatilis L., in Australia. j. Fish Dis.
10:425-434.
Lom,j., and E.R. Noble.
1984. Revised classification of the class Myxosporea Butschi,
1881. Folia Parasitologia (Praha). 31:193-205.
Lom,j., S.W. Feist, I. Dykova, and T. Kerp.
1989. Brain myxoboliasis of bullhead, Cottus gomo L., due to
Myxobolus jiroveci sp. nov.: light and electron microscopeobservation. j. Fish Dis. 12:15-27.
Mitchell, L.G., C.L. Seymour, andj.M. Gamble.
1985. Light and electron microscopy of Myxobolus
hendricksoni sp. nov. (Myxozoa: Myxobolidae) infecting thebrain of the fathead minnow, Pimephales promelas
Rafinesque. j. Fish Dis. 8:75-89.
Sakaguchi, S., T. Hara, T. Matsusato, T. Shibahara, Y. Yamagata, H.
Kawai, Y. Maeno.
1987. Scoliosis of cultured yellowtail caused by parasitic
Myxobolus buri. Bull. Nat\. Res. lnst. Aquaculture. 12:79-86.
(In Japanese; English abstr.)
Shulman, S.S.
1966 Myxosporidian fauna of the USSR. Nauka, Moscow and
Leningrad, 251 p.

Presence of Oncogenes in Fish Tissues and in FISh Cell Lines

E. READ-CONNOLE, C. A. SMITH, and F. M. HETRICK*
Department of MierolJiowgy
University ofMarylandCollege Park, MD 20742

ABSTRACTWork during the past decade has shown that avian and mammalian proto-oncogenes
are centrally involved in cell transformation in vitro and in the formation of tumors invivo. The fish systems in which oncogenes have been described are reviewed as are thegeneral methodologies used to detect oncogenes and their gene products. Also discussed
is preliminary work on the development of a test system that measures oncogene activation in fish cell lines in order to evaluate carcinogenic chemicals in the environment.
Sequences related to the rat, raS', v-raj, v-erlrB, c-src, c-myc, c-abl, and c-fos protooncogenes, and to the p53 suppressor gene, were detected by Southern, Northern, andWestern blots. Taken together, the above findings indicate that proto-oncogenes are wellconserved evolutionarily in vertebrates and may be responsible for development of thetransformed phenotype in fish.

Introduction_

Evidence has been accumulating in recent years thatin mammals, particularly humans, tumor development and progression are correlated with changes inthe structure or expression of cellular genes. Yet in a
number of higher vertebrates, oncogenic retroviruseshave long been known to be causally involved in theinitial appearance and subsequent growth of a varietyof naturally arising tumors (Bishop 1985; Klein and
Klein 1985). These two statements are not contradictory. The retroviral oncogenes (v-ones) responsible
for the transformation event are now recognized tohave arisen from the capture and processing of wholeor portions of essential cellular genes termed protooncogenes or c-oncs (Bishop 1983; Stehelin et al.
1976). It is now considered important to understandthe extent of evolutionary conservation of these cellular genes in order to fully characterize their normal
function and delineate their involvement in thetumorigenic conversion of normal cells either directly or through retrovirus-induced transformation.
The family of ras proto-oncogenes, raS' (Harvey),ral (Kirsten), and N-ras are highly conserved frommammals to yeast. The 21 kilo dalton ras protein isexpressed in a wide variety of non transformed cells,suggesting a normal cellular function. Xenopus laevis
oocytes espress the ras protein throughout oogenesis
and embryonic development (Baum and Bebernitz

1990). However, wild type ras protein can be transformed in a variety of carcinogen-induced anirrial
tumor model systems. For example, in rats almost
90% of mammary carcinomas induced by a single
dose of nitrosomethylurea possess a c-H-ras-l
oncogene, which is the activated form of the c-H-ras
gene having a G to A transition at codon 12(Barbacid 1986, 1987). In humans, highly amplified
N-ras DNA sequences were found in small cell lungcancer, and this amplification seems to correlate withtumor progression and prognosis Uohnson et al.
1987). Changes in the structure (mutation) of one ormore oncogenes, their amplification, and the level ofexpression of their mRNA, are all potential indicatorsof tumorigenic initiation and progression.
In view of the widespread occurrence of neoplasia
in freshwater and saltwater fish species, and because
fish are becoming recognized as convenient model
systems for the study of natural and environmentallyinduced diseases (Powers 1989), the search for protooncogene-like sequences in teleosts is warranted.
Experimental evidence indicates that some fish
species, when compared with rodents, are less sensitive to the toxic effects of chemical agents and more
susceptible to their carcinogenic effects. Because of
their aquatic habitat, fish are fully exposed to chemicals in the water at the gill, eye, skin, and gut levels,*Send correspondence to this author.
119

120

NOAA Technical Report NMFS 111_

and therefore may be good indicators of carcinogens
in the environment. Thus, it is worth investigating
oncogenes and their expression in tumorigenesis infish, especially when they are induced by chemicals.
Such studies have now begun. Here we describe thegeneral methodology used in detecting oncogenes
and their expression and review those systems where
oncogenes have been found in fish tissues and incontinuously cultivated fish cell lines.

Methodology_

Oncogene Detection
For the detection of oncogenes at the DNA level, it isimportant to isolate high molecular weight genomic
DNA with a minimal size of 100-200 kb. Standardized
procedures are available for the rapid isolation ofgood quality DNA from either cell cultures or directlyfrom tissues excised from organisms. One procedure(Smith et al. 1988) employs a gentle sodium dodecyl
sulfate (SDS) and proteinase-K lysis step followed byphenol extraction, ethanol precipitation, and spooling of DNA (contaminating RNA can be removed byRNAse treatment). Tissue samples are processed bythis method by including liquid nitrogen freezing
and tissue crushing steps prior to the lysis. Methods
for isolating genomic DNA from fish cell lines aredescribed in Smith et al. (1988) and Read-Connole etal. (1990).
Samples of DNA from the various sources are thendigested to completion with one or more various restriction endonucleases (e.g., EcoRI, Hind III and BamHI), and the resulting DNA fragments are resolved
by size by agarose gel electrophoresis. Mter transferof the DNA fragments to nitrocellulose or nylon
membrane supports, the DNA samples can then be
screened by Southern blotting for the presence ofsequences homologous to nick-translated labelled
DNA probes specific for mammalian and viraloncogenes. In addition, serial dilutions of samples ofrestriction-endonuclease-treated DNA can be directly
spotted onto nitrocellulose or nylon membranes.
These dot blots can then be probed in parallel with
the Southern blots to aid in the quantification of
hybridizable DNA in each sample, and to estimate
any gene amplification that may occur in the tumor
tissues.
Comparisons between normal and tumor tissues
can be made from the banding patterns obtained inSouthern blots probed by the different oncogenes. In
this way one can obtain some idea on the similarity of
the gross structure of the various fish oncogenes thatare detected. Comparisons of the number and size of
oncogene homologous fragments from the Southernblots with the more quantifiable data from the dot
blot experiments should reveal whether any of the
fish genes detected have been rearranged or amplified in the tumor tissues under study.

Detection of Oncogene Expression
For any gene to exert an effect on cells and tissues,mR..'\JA must be transcribed and eventually translated
into protein. One can therefore screen RNA isolated
from the various sources for the presence bf transcription products homologous to those of
mammalian oncogenes. Enhanced expression of various oncogenes following the amplification of DNA
may contribute to malignant progression as is suggested by the human c-N-myc gene involvement in the
progression of human neuroblastomas (Schwab et al.
1984), or it may involve the loss of a gene and its
expression as has been observed in the case of thehuman retinoblastoma susceptibility gene (Friend et
aI.1986;LeeetaI.1987).
Described briefly, cells and tissues being examinedfor oncogene transcription products are first processed by the guanidinium thiocyanate/hot phenol
procedure to extract the total cellular RNA. The total
cellular RNA obtained is subjected to oligo-dT cellulose affinity chromatography to purify or enrich thepoly A+ RNA fraction (putative mRNA). The poly A+
RNA species are then separated by size by denaturing(formaldehyde) agarose gel electrophoresis. Mtertransfer of the RNA to nitrocellulose or nylon membranes, mature mRNA species with homology to
oncogene probes can be detected by northern blotting procedures. In addition, RNA dot blot
experiments can also be performed to quantify thelevels of mature mRNA hybridizing with theoncogene probes (Smith et al. 1987; Louis et al.
1988). The mRNA studies give valuable information
on whether any of the specific oncogenes detected in
the various tumor tissue preparations are of different
sizes (suggesting either truncation of the gene or rearrangements) and whether they are overexpressedor aberrantly expressed when compared with the normal counterparts.

Detection of Oncogene Products
The third approach to monitor oncogenes in cells is
to assay for their gene products (e.g., tyrosine kinase
for c-src expression, epidermal growth factor receptor
for c-erb-B expression). Monoclonal an ti bodies

Read-Connole et al.: Oncogenes in Fish Tissues and Cell lines(Mabs) have been raised against synthetic peptides
spanning the active site regions of the src and ras
gene families and are available commercially.
Nonidet P-40 (non-ionic detergent) lysates of cells
are cleared by high speed centrifugation and the proteins immunoprecitated with Mab mixtures.
Following electrophoresis on 10% SDS-polyacrylamide gels, the bands are transferred to nitrocellulose
membranes, and immunoblotted with an appropriate
antibody. These western hybridizations, in which
Mabs are used against oncogene products, can reveal
the size and level of expression of these proteins.

Oncogenes Detected in Fish Tissues_

A certain population of platyfish (Xiphophorus
maculatus) carry special genes (Tu or tumor genes)
for macromelanophores. When they are interspecifically hybridized with swordtails (X. helleri), theF, offspring carrying these genes develop apreneoplastic state. When the F 1 offspring are backcrossed with swordtails, a certain percentage of the
backcrossed offspring develop a heritable form of
melanoma with a characteristic inheritance pattern(Ozato and Wakamatsu 1983; Anders et al. 1984). Expansion of these classic and elegant genetic studies
involves the use of modern biotechnology techniques
to define the molecular basis of the melanoma formation. For example, elevated levels of cellular src
mRNA and phosphoprotein (pp 60) kinase activity
are detected in melanomas or tumors induced by carcinogens in Xiphophorus (Schartl et al. 1985) andmore recent research indicates that the signal to start
the events leading to melanoma development may
come from the platyfish erbB region (Zechel et al.
1988). They have named this gene Xmrk. An additional copy of the Xmrk gene is found linked to either
sex chromosome of the platyfish when hybridized toa probe specific for this region (Schartl 1990). The
cellular myc (c-myc) gene has been cloned from rainbow trout Oncorhynchus mykiss and sequenced (vanBeneden et al. 1986). The results of this work wereused for evolutionary comparison with other vertebrate c-myc genes. Using the rainbow trout c-myc
clone as a probe, DNA was examined from various
fish tumors, including hepatocellular carcinomas induced in the medaka Oryzias latipes by dimethylnitrosamine. Compared with DNA isolated
from normal tissues, there was no apparent increasein the intensity of hybridizing bands or differences in
restriction patterns noted in Southern blots (vanBeneden et al. 1988). However such experiments
would not detect mutations, which could be detected
only by cloning and sequencing the entire c-myc

121gene. It was also reported (van Beneden et a1.1988)
that the blots, when rehybridized to other viraloncogenes, gave no indication of gross amplification
or rearrangement. Clearly, cloning and sequencingof the entire gene is necessary to show putative differences.
Similarly c-ras-related sequences were cloned fromthe genomic libraries of the goldfish (Cerassius
auratus) by Nemoto et al. (1986). Comparison of thenucleotide sequences of one of these clones withthose in mammalian c-ras genes showed extensive homology to the gene coding for the mammalian p21
protein (96% homology to the c-raS' protein). Subsequent work (Nemoto et al. 1987) involved
sequencing and comparison of the first exon and its
flanking regions in the c-ras-related genes from normal goldfish liver tissues with those from goldfish
erythrophoroma cells cultured in vitro. No differences were apparent in the first 245 nucleotides
which covered the first exonic region and whose
length was identical to the first exonic region in
mammalian c-ras genes. Enhanced expression of c-ras
gene was seen in the erythiophoroma cells.
The high incidence of hepatomas present in winterflounder Pseudopleuronectes americanus in Boston harbor has been associated with the high levels ofpolycyclic aromatic hydrocarbons (PAHs) found in
the harbor's sediment. Genomic DNA from the liver
tumors produced foci in the NIH-3T3 transfection
assay and a c-rask oncogene was identified in atransforman t derived from one of the tumors(McMahon et al. 1988). Sequencing of c-raS' sequences from the tumor and from the transformants
indicated the oncogene to be of flounder origin. Sequencing of the tumor cell DNA amplified bypolymerase chain reaction (McMahon et al. 1987)
showed GC to AT or GC to TA conversions in the
12th codon of this gene (McMahon et al. 1990). Suchmutations in the 12th codon can activate c-ras protooncogenes to oncogenic forms (Barbacid 1987).
DNA samples from livers taken from fish at a lesspolluted site did not transform NIH-3T3 cells, andonly wild-type sequences (GGT) were seen at the 12codon of c-raS'.
A cellular 53 kilo dalton nuclear phosphoprotein,denoted p53, was discovered more than a decade ago
in SV40 virus transformed mouse cell lines (Chang etal. 1979). Because p53 was found complexed to thetransformation antigen (T antigen) of SV-40 virus,
and because the p53 gene was thought by many investigators to funct}on as a dominantly acting oncogene,it has been intensively investigated during the past
decade. Current work on mutations in the p53 geneindicates that the wild-type gene product actuallyfunctions like a tumor suppressor gene (Finlay et al.

122

NOAA Technical Report NMFS 111_

Table 1
Oncogenes Detected in Fish Tissues.
Transforms
NIH-3T3 Cells

Fish Species

Tissue Tested

Oncogene

Platyfish - swordtail
hybrid (Xiphophorus sp.)

Hereditary melanoma

Xmrk (tu)Schartl et al. 1985
Witthrodt et al. 1989

crb-B

Zechel et al. 1988

Reference

Rainbow Trout(Oncorhyncus mykiss)

Normal liverc myc

van Beneden et al. 1986

Goldfish(Carassius auratus)

Normal liverras

Nemoto et al. 1986

Winter flounder(Pseudopleuronectes
amcricanus)

Liver tumor

ral-

+

McMahon et al. 1988, 1990

Tomcod(Microgadus tomcod)

Liver tumor

ral-

+

Wirgin et al. 1989

Northern pike
(Esox lucius)

External
lymphomas+

van Beneden et al. 1990

1989). Mutations in the pS3 gene are frequently detected in diverse human tumor types (Nigro et al.
1989), and currently it is believed that pS3 gene mutations play an important role in the development ofmany common human cell malignancies. The hypothesis is that the mutated pS3, which develops
during the process of tumorigenesis, binds to thewild-type pS3 gene product, creating an inactive complex. Further loss of growth control can occur when
the wild-type allele is deleted. Thus, pS3, like theretinoblastoma gene, is under intensive study bymany investigators as a potential tumor suppressor
gene (Sager 1989). The tumor suppressor genes arewild-type alleles of genes that play regulatory roles incell proliferation and differentiation, and it is theirloss or inactivation that is oncogenic. The pS3 gene ishighly conserved in vertebrates including fish (Smithet al. 1988).

-Oncogenes D-etected in Fish Cell Lines _ _A number of oncogenes have been described in someof the more commonly used fish cell lines (ReadConnole et al. 1990). These have been detected bySouthern, Northern, and Western blots (Table 2).
The presence of many bands in Southern blots suggests that, as in mammals, some of the fish
proto-oncogenes may exist as families of homologous
sequences that share common functions. Probing

Southern blots with v-erb-b, v-src, and v-raS' revealed astriking similarity in the banding patterns of homologous sequences between the rainbow trout O. my kissand the chinook salmon O. lshawytscha cell line. Thissuggests that, as expected, these genes in the rainbow
trout and chinook salmon are more closely linked inan evolutionary sense than they are to the homologous genes in the other fish species studied. A very
high degree of homology has been shown in the protamine genes of chum salmon (0. keta) and rainbow
trout (Moir and Dixon 1988).
Many of the monoclonal antibodies raised against
mammalian viral or cellular oncogene protein products recognized fish proteins in Western blots.
Immunoprecipitation followed by Western blotting is
considered a highly sensitive and discriminatory procedure, especially when monoclonal antibodies areused that recognize discrete and specific epitopes on
the proteins of interest. The apparent recognition offish proteins by this technique shows that these regions of the oncoproteins may be well conserved
from viruses to mammals and fish.
Both the c-raS' and c-raS' gene products were detected in all the fish cells, showing that these proteinsare conserved in vertebrates and may be essential. It
is possible, however, that the c-raS' and c-raS' antibodies recognized the same fish proteins, but the
monoclonal antibodies used were raised against different peptides representing regions of the two viralproteins and did not cross-react.

Read-Connole et al.: Oncogenes in Fish Tissues and Cell Lines123

Table 2

Oncogenes Detected in Fish Cell Lines.
Detection Method

Oncogene (s) Detected

Southern blots
Using mammalianviral one probes

rat', raj,
erb-B, srep53

Western blots
Using Mabs against
human and viraloncogene productsc-mye, c-abl, c%~~s,v-rat', p53 (murine), v-rat,
v-raj, v-sre

Cellline(s)*BB, RTG-2,
EPC, CHSE-214
EPC
BB, EPC,
CHSE-214High expression in BB and EPC
Moderate expression in RTG
Low in CHSE-214

Northern blots
Using mammalianviral one probes

ablHigh expression in CHSE-214
Moderate in RTG

src, abl, mye, rat

BB, EPC,
RTG-2, CHSE-214

p53

EPC, CHSE-214*BB = Brown bullhead catfish line (Wolf and Quimby 1969); RTG-2 = rainbow trout gonad (Wolf and Quimby 1962); EPC =epithelioma papulosum cyprini (Fijan et al. 1983); and CHSE-214 = chinook salmon embryo (Fryer et al. 1965).

SignificanceThe mechanisms by which normal cells acquire themalignant phenotype form the central focus of cancer research. Transforming genes have been detected
in a variety of tumors, including human tumors, andin cell lines derived from tumors by their ability to
tranform NIH-3T3 mouse cells in culture. Genetic alternations also accompany tumorigenesis in feral
populations of aquatic organisms exposed to environmental carcinogens. Any unambigious signal
regarding oncogene or tumor-repressor-gene expression that correlates with tumor formation could serveas the basis for a test system for evaluating carcinogenic chemicals in the environment. Changes in the
structure (mutation) of one or more oncogenes,
their amplification, or the level of expression of theirmRNA are all potential indicators of tumorigenic initiation and progression.
The number of genes homologous to either theviral oncogenes or the mammalian cell counterparts
found in fish genomes suggests that these genes arehighly conserved evolutionarily, and therefore theirrole in both normal cells and those cells in various
stages of tumorigenic transformation may be common to all vertebrates. The brown bullhead Ictalurusnebulosus is of particular interest to us because of thehigh prevalence of tumors that occur in them during
the late spring and summer in certain areas of the
Chesapeake Bay (unpub!. data). We are now attempt-

ing to establish tissue culture cell lines from thesetumors with the intent of comparing the genomic
DNA of the tumor cells to that of the normal skintissue and the brown bullhead (BB) cell line to determine if any of the potential fish oncogenes arealtered or expressed differently in the tumor cells. If
mutations identified in specific fish oncogenes can beshown to result from chemically-induced DNA damage, then such mutations could serve as indicatorsfor environmental disease.

Citations_

Anders, F., M. Schartl, A. Barkenow, and A. Anders.
1984. Xiphophorus as an in vivo model for studies on normal
and defective control of oncogenes. Adv. Cancer Res. 42:
191-275.
Barbacid, M.
1986. Involvement of ras oncogenes in the initiation of carcinogen-induced tumors. Int. Symp. Princess Takamatsu
Cancer Research Fund. 17:43-53.
1987. Ras genes. Annu. Rev. Biochem. 56:779-827.
Baum, E.Z., and G.A. Berbernitz.
1990. K-ras oncogene expression in Xenopus laevis. Oncogene
5:763-767.
Bishop,J.M.
1983. Cellular oncogenes and retroviruses. Annu. Rev.
Biochem.52:301-354.
1985. Viral oncogenes. Cell 42:23-38.
Chang, C., D.T. Simmons, M.A. Martin, and PT. Mora.
1979. Identification and characterization of new antigens
from SV,o-transformed mouse cells. J. Virol. 31:463-471.

l24

NOAA Technical Report NMFS III

Fijan, N., D. Sulimanovic, M. Bearzotti, D. Muzinic, L. Zwillenberg,
S. Chilmonezyk,j. Vautherot, and P. de Kinkelin.
1983. Some properties of the epithelioma papulosum cyprini(EPC) cell line from carp. Annales Virologie (Institut
Pasteur) 134:207-220.
Finlay, C.A., P.w. Hinds, and AJ. Levine.
1989. The p53 Proto-oncogene can act as a suppressor oftransformation. Cell 57:1083-1090.
Friend, S.H., R. Bernards, S. Rogelj, R.E. Weinberg, j.M. Rapaport,
D.M. Albert, and T.P. Drya.
1986. A human DNA segment with properties of the genethat predisposes to retinoblastoma and osteosarcoma. Nature 323:643-646.
Fryer,j.L., A. Yusha, and KS. Pilcher.
1965. The in vitro cultivation of tissue and cells of Pacific salmon
and steelhead trout. Ann. N.V. Acad. Sci. 126:566-586.
Johnson, B.E., D.C. Ihde, R.W. Makuch, A.F. Gazdar, andj. Minna.
1987. Myc family oncogene amplification in tumor cell lines
established from small cell lung cancer patients and its relationship to clinical status and course. j. C1in. Invest.
79:1629-1634.
Klein, G. and E. Klein.
1985. Evolution of tumors and the impact of molecularbiology. Nature 315:190-195.
Lee, W.H., R. Bookstein, F. Hong, LJ. Young, j.Y. Shew, !.Y.M.P.
Lee.
1987. Human retinoblastoma susceptibility gene: cloning.
identification, and sequence. Science 235:1394-1399.
Louis, MJ., V.W. McFarland, P. May, and PT. Mora.
1988. The phosphoprotein p53 is down-regulated p'lSttranscriptionally during emybrogenesis in vertebrates. Biochimica. Biophysica Acta. 956:395-402.
McMahon, G., E. Davis, and G.N. Wogan.
1987. Characterization of c-Ki-ras oncogene alleles by direct
sequencing of enzymatically amplified DNA from carcinogen-induced tumors. Proc. Nat!. Acad. Sci. (USA),84:4974-4978.
McMahon, G., L.j. Huber,JJ. Stegeman, and G.N. Wogan.
1988. Identification of a c-Ki-ras oncogene in a neoplasm isolated from winter flounder. Marine Environ. Res. 24:345350.
McMahon, G., L.J. Huber, MJ. Moore, J.J. Stegeman, andG.N. Wogan.
1990. Mutations in c-Ki-ras oncogenes in diseased livers of
winter flounder from Boston Harbor. Proc. Nat\. Acad.
Sci. (USA) 87:841-845.
Moir, R.D., and G.H. Dixon.
1988. Characterization of a protamine gene from chumsalmon Oncorhynchus keta. j. Mol. Evo\. 27:8-16.
Nemoto, N., K Kodama, A. Tazawa, Prince Masahito, andT Ishikawa.
1986. Extensive sequence homology of the goldfish ras geneto mammalian genes. Differentiation 32: 17-23.
Nemoto, N., K Kodama, A. Tazawa, J. Matsumoto, Prince
Masahito, and T Ishikawa.
1987. Nucleotide sequence comparison of the predicted firstexonic region of goldfish ras gene between normal and neoplastic tissues. j. Cancer Res. C1in. Oncol. 113:56-60.
Nigro,j.M., S. Baker, A. Preisinger, et al.
1989. Mutations in the p53 gene occur in diverse humantumour types. Nature 342:705-708.
Ozato, K, and Y. Wakamatsu.
1983. Multi-step genetic regulation of oncogene expressionin fish hereditary melanoma. Differentiation 24:181-190.
Powers, D.A.
1989. Fish as model systems. Science 246:352-358.

_
Read-Connole, E., C.D. Smith, and F.M. Hetrick.
1990. Nucleotide sequence homologous to mammalian
proto-oncogenes and their expression in fish cell lines. j.
Aquat. Animal Health 2:77-84.
Sager, R.
1989. Tumor suppressor genes: the puzzle and thepromise. Science 246:1406-1412.
Schartl, M.
1990. Homology of melanoma-inducing loci in XiphophoTUs.
Genetics 126:1083-1091.
Schartl, M., C.R. Schmidt, A. Anders, and A. Barkenow.
1985. Elevated expression of the cellular src gene in tumorsof different etiology in XiphophoTUS. Int. j. Cancer 36:199207.
Schwab, M., j. Ellison, M. Busch, W. Rosenau, H. Varmus, andJ .M. Bishop.
1984. Enchanced expression of the human gene N-myc consequent to amplification of DNA may contribute to malignant progression of neuroblastoma. Proc. Natl. Acad. Sci.
81 :4940-4944.
Schwab, M., R. Oehlmann, S. Bruderlein, and Y. Wakamatsu.
1989. Amplified DNA in cells of genetic melanoma of
XiphophoTUs. Oncogene 4:139-144.
Smith, C.A., M.j. Louis, and F.M. Hetrick.
1988. A sequence homologous to the mammalian p53
oncogene in fish cell lines. J. Fish. Dis. II :525 -530.
Smith, C.A.D., DJ. Winterbourne, V.W. McFarland, and PT. Mora.
1987. Changes in Heparan Sulfate pattern but not in
oncogene expression correlate with tumor growth in spontaneous transformation of cells. Oncogene Research
1:325-341.
Stehelin, D., H.E. Varmus,j.M. Bishop, and P. Vogut.
1976. DNA related to the transforming genes of avian sarcoma virus is present in normal avian DNA. Nature~60:170-173.

van Beneden, RJ., D.R. Watson, TT Chen, j.A. Lautenberger, and
TS. Papas.
1986. Cellular myc (c-myc) in fish (rainbow trout): its relationship to other vertebrate myc genes and to the transforming genes of the MC29 family of viruses. Proc. Natl. Acad.
Sci. (USA) 83:3698-3702.
1988. Teleost oncogenes: evolutionary comparison toother vertebrate oncogenes and possible roles in teleost
neoplasms. Mar. Environ. Res. 24:339-345.
van Beneden, RJ., KW. Henderson, D.G. Blair, TS. Papas, and
H.S. Gardner.
1990. Oncogenes in hematopoietic and hepatic fishneoplasms. Cancer Res. 50:567Is-5674s.
Wirgin, 1., D. Currie, and SJ. Garte.
1989. Activation of the K-ras oncogene in liver tumors of
Hudson River tomcod. Carc;inogenesis 10:2311-2315.
Wittbrodt, j., D. Adam, B. Malitschek, W. Maueler, F. Raulf,
A. Telling, S.M. Robertson, and M. Schartl.
1989. Novel putative receptor tyrosine kinase encoded by themelanoma-inducing Tu locus in Xiphophorus. Nature
341:415-421.
Wolf, K., and M. Quimby.
1962. Established eurythermic line of fish cells in vitro. Science (Washington, D.C.) 135:1065-1066.
1969. Fish cell and tissue culture. In Fish physiology, vol. 3(W.S. Horr and DJ. Randall, eds.), p. 253-305. Acad.
Press, N.Y.
Zechel, C., U. Schleenbecker, A. Anders, and F. Anders.
1988. v-erb-B sequences in Xiphophorus that map to melanoma
determining Mendelian loci and overexpress in a melanoma cell line. Oncogene 3:605-617.

Streptococcal Infection in Cultured Yellowtail

HIROSHI SAKO
Nansei National Fisheries Research InstituteOhno, Saeki
Hiroshima, 739-04,Japan

ABSTRACTStreptococcal infection is widespread in yellowtail (Seriola quinqueradiata) cultured insouthwestern Japan and results in extensive losses. It is urgent that control measures beestablished against this disease. This paper presents the etiology, prophylaxis, and treatment of streptococcal infections in cultured yellowtail.

Introduction_

Yellowtail (Seriola quinqueradiata) culture developed
rapidly in the 1960s, mainly in southwestern Japan.
By 1988, the farming of yellowtail was being practiced
in 27 prefectures (Fig. 1), the total production was
about 166,000 metric tons, or 68% of the total production of marine fish cultured in Japan (Fig. 2).
The incidence of diseases in cultured yellowtail is
gradually increasing as the culture of this fish becomes more popular. About 30 diseases have been
identified so far. Figure 3 shows the loss of cultured
yellowtail due to disease in 1988; the cost to fish
farmers was about 11,300 million yen. Bacterial diseases are responsible for the majority of the losses,
whereas those due to viruses and parasites are minor.
Among the bacterial diseases, streptococcosis was responsible for the highest losses (64%), pasteurellosis
ranking second (21 %). Thus, with yellowtail culture,
it is very important to control the incidence of streptococcal infection.
The author describes here some aspects of streptococcal infection in cultured yellowtail with special
emphasis on the etiology of the causative bacteria, .
prophylaxis, and methods used to treat the disease.

Causative Bacteria

clearly distinguishable by their biochemical andphysiological characteristics (Table 1; Sako, unpubl.
data). The species reported by Kusuda et al. is generally called alpha-hemolytic Streptococcus sp. Almost allof the streptococci isolated from yellowtail were confirmed to be the alpha-hemolytic Streptococcus sp.
(Ki tao 1982). Recen tly, this species was transferred togenus Enterococcus (Riichi Kusuda, Kochi Univ., pers.
commun., April 1990). Another species isolated froma yellowtail juvenile in 1976 by Minami et al. (1979)displayed beta-hemolysis on sheep blood agar, so it
has been called beta-hemolytic Streptococcus sp. This
species is classified with pyogenic streptococci basedon its biochemical characteristics. Its biochemical
characteristics also agreed well with those of S. iniae
ATCC 29178 isolated from the Amazon freshwater
dolphfin, Inia geoffrensis (Pier and Madin 1976). The
last species is a nonhemolytic Streptococcus reported
by !ida et al. (1986), which has biochemical characteristics similar to those of S.agalactiae and has theLancefield grouping antigen B, type lb.
A species that shows the same characteristics as alpha-hemolytic Streptococcus sp. was also isolated from
cultured yellowtail in the Republic of Korea (Park et
al. 1987). The nonhemolytic, group B, type Ib Streptococcus was also isolated from several marine fishes in
the United States (Plumb et al. 1974).

_

Streptococcal infection in cultured yellowtail was first
identified by Kusuda et al. in 1974 (Kusuda et al.
1976). Since then, three species of fish pathogenic
Streptococcus have been reported. These bacteria are

Host Range_

Large numbers of Enterococcus sp. and S. iniae havebeen isolated from wild and cultured marine fishes as
125

126

NOAA Technical Report NMFS III_

their minced meats, are a major feed for yellowtail, it
is possible that they are the origin' of streptococcal
infection in cultured fish.

Total production
166,000 metric tons•••

Pathogenicity

10,000 - 40,000
1,000 - 9,tl9
1tl9
metric tons

Figure 1_

Each of the Streptococcus spp. and their strains exhibit different degrees of virulence. Enterococcus sp.
produces both intra- and extracellular toxins(Kimura and Kusuda 1979). In artificially induced infections, even small amounts of Enterococcus sp. cells
killed yellowtail (Taniguchi 1982a). However, S. iniae
killed yellowtail only occasionally although it frequently caused vertebral deformity (Sako, un pub!.
data) .
Streptococci is an indigenous bacteria in warmblooded animals and grows rapidly at 30-35° C. S.
iniae isolated from yellowtail have been confirmed tobe pathogenic to mice by intraperitoneal injection.
However, Enterococcus sp. and nonhemolytic group B,
type Ib Streptococcus isolated from yellowtail were
nonpathogenic to mice (Sako, unpub!. data).

Yellowtail production in Japan by prefecture, 1988. Source:
Statistics and Information Department, Ministry of Agriculture, Forestry, and Fisheries, Japan.

Diagnosis

well as in small quantities from cultured freshwater
fishes in Japan (Table 2; Kusuda et a!. 1976; Kusuda
et a!. 1978; Minami 1979; Minami et a!. 1979; Kitao et
a!. 1981; Ohnishi and Jo 1981; Kusuda and Kawai
1982; Kusuda et a!. 1982; Ogawa et a!. 1982; Yasunaga
1982; Hatai et al. 1983; Nakatsugawa 1983; Kaige eta!. 1984; Iida et a!. 1986; Sakai et a!. 1986; Atsuta etal. 1990). Because sardines and anchovies, as well as

Major clinical signs caused by Enterococcus sp. are
exophthalmos, petechiae, and ulceration on the inside of the opercula, and congestion of the pectoraland caudal fins. Signs such as congestion andhaemorrhagia are also found in the intestine, liver,spleen, and kidney (Kusuda et al. 1976). Epicarditis isalso seen often. Pathogenic Streptococcus is frequently
isolated from the brains of diseased yellowtail_

JAPANESE FLOUNDER
JAPANESE
iJt,

COHO

YELLOWTAILS

Figure 2

SEA BREAMS

166,000 metric tons

Marine fish culture production for 1988. Source: StatlstIcs and Information
Department, Ministry of Agriculture, Forestry, and Fisheries, Japan.

Sako: Streptococcal Infection in Cultured Yellowtail

127

Nocardiosis

Streptococcosis
Figure 3
Loss of cultured yellowtail todiseases in 1988. *This classification contains cases that thecause of a disease are two or
more: e.g. mixed infection.
Source: Research Division, Fisheries Agency ofJapan.

7,270 million ywI(Shiomitsu 1982; Kaige et a!. 1984; Iida et a!. 1986).
Immunological identification using fluorescent antibody techniques is also useful for the rapid
identification of yellowtail pathogenic streptococci(Kusuda and Kawahara 1987).
A rapid identification system is also commerciallyavailable for the identification of fish pathogenic
streptococci. Three species of streptococci from yellowtail can be easily distinguished using API 20 Strep
System (BIO MERIEUX SA) within 4 hours (Table
3; Sako, unpub!. data).

Prevention
It was reported that fish meats used as feed for yel-lowtail have fish pathogenic Streptococcus (Minami
1979; Yasunaga 1982). The organisms grow quickly inthe minced meat of fish within several hours under
optimal temperature (Fig. 4; Sako, unpubl. data). In
laboratory experiments, it was demonstrated that yellowtail can be infected orally by Streptococcus throughfeed such as minced meat (Taniguchi 1982a). In order to prevent contamination by the pathogen and to

Table 1Biochemical characteristics of n, f3 and non hemolytic Streptococcus sp. isolated from yellowtail.

Characteristics
Hemolysis
Voges-Proskauer reaction
Esculin hydrolysis
Starch hydrolysis
Hippurate hydrolysis
Pyrrolidonylarylamidasel3-galactosidase
Alkaline phosphatase
Sensitivity to bacitracincAMP test
Lancefield group antigen

a-hemolytic
Strt1Jtococcus sp.
(Enterococcus sp.)l3-hemolytic
Strt1Jtococcus sp.
(S. iniae)

nonhemolytic
Strt1Jtococcus sp.
(s. agalactiae)

a,+

+

+

+
+

+

+
+
+
+

+

+
+

+

not A, B, C, D, F, Ga

aNo reaction at least against the antisera of A-G.
'BIb denotes type of group an tigen.

not A, B, C, D, F, Ga

BIb'

128

NOAA Technical Report NMFS 111_

Table 2Fish species in Japan from which a pathogenic streptococci was isolated (Kusuda et al. 1976; Kusuda et al. 1978;
Minami 1979; Minami et al. 1979; Ohnishi and Jo 1981; Kitao et al. 1981; Yasunaga 1982; Kusuda and Kawai 1982;
Kusuda et al. 1982; Ogawa et al. 1982; Nakatsugawa 1983; Hatai et al. 1983; Kaige et al. 1984; !ida et al. 1986;
Sakai et aI. 1986; Atsuta et al. 1990).
Marine fish
Isolates

CulturedFreshwater
cui tured fish

Wild

Enterococcus sp. Yellowtail (Senola quinqueradiata)
Japanese horse mackerel (Trachurus japonicus)
Purplish amber jack (Senola dumerill)
Goldstriped amber jack (Smola lalandl)
Largescale blackfish (Girella punctata)Black scraper (Thamnaconus modestus)

Sardine (Sardinops melanosticta)
Anchovy (Engraulis japonica)
Round herring (Etrumeus microps)
Sand lance (Ammodytes personatus)
Chub mackerel (Scomber japonicus)
Moon dragonets (Repomucenus lunatus)
Black scraper (Thamnaconus modestus)
Multicolorfin (Halichoeres poecilopterus)
Silver jewfish (Pennahia argentata)
Red sea bream (Pagrus major)

Eel (Anguillajaponica)

Streptococcus
iniae

Yellowtail (Smola quinqueradiata)
Japanese horse mackerel (Trachurus japonicus)
Chub mackerel (Scomber japonicus)
Sardine (Sardinops melanosticta)
Red sea bream (Pagrus major)

Ayu (Plecoglossus altivelis)
Rainbow trout

Streptococcus
agalactiae

Yellowtail (Smola quinqueradiata)
Japanese horse mackerel (Trachurusjaponicus)
Japanese flounder (Paralichthys olivaceus)
Knifejaw (oplegnathus fasciatus)
Rabbit fish (Siganus fuscescens)
Largescale blackfish (Girella punctata)
Triggerfish (Aluteres monoceros)
Jacopever (Sebastes schlegell)
Coho salmon (Oncorhynchus kisutch)(Oncorhynchus mykiss)
Amago salmon(Oncorhynchus rhodurus)
Tilapia (Tilapia nilotica)

Yellowtail (Smola quinqueradiata)

slow its growth, the following techniques are recommended: 1) washing feed fish in uninfected water, 2)
feeding fish without defrosting, and 3) feeding processed pellets (Taniguchi 1982b).
Yellowtail can acquire immunity after streptococcal
infection (Kusuda and Takagi 1983). Indeed, many
attempts have been made to prevent streptococcal infection using bacterin. Iida et al. (1982) reported on
the efficacy of vaccination for control of streptococcal infection.

Therapy_

It is not easy to cure yellowtail that are in the advanced stages of streptococcal infection. Minimal
inhibitory concentrations (MIC) of antimicrobial
agents against Streptococcus from yellowtail are shownin Figure 5 (Sako, unpubl. data). Sensitivities to several drugs are different between Enterococcus sp. and
S. iniae, but both species have high sensitivities topenicillins, macrolides, tetracyclines, and lincomycin.

Table 3Differential characteristics among Enterococcus sp., Streptorocrus iniae, and Streptococcus agalactiae isolated fromyellowtail by API 20 Strep System incubated at 35° C for four hours (Sako, unpubl. data). VP = Voges-Proskauer reaction;
HIP = Hippurate hydrolysis; ESC = Esculin hydrolysis; PYRA = Pyrrolidonylaryl amidase; PAL = Alkaline phosphatase.
Test (characteristics)
Species

VP

Enterococcus sp.
Streptococcus iniae
Streptococcus agalactiae

+
+

HIP

+

ESC

PYRA

+
+

+
+

PAL

+
+

Sako: Streptococcal Infection in Cultured Yellowtail

129

Table 4

7

Drugs used to cure yellowtail streptococcal infection
inJapan. Source: Fisheries Agency ofJapan.

30·C

!:as

~

.as
u
I 4

20·c

3

___.------10 ·c

Daily dose (mg/kg body weight)

Drugs

2

3

0

• •

11

50

Alkyl trimethyl ammonium
calcium oxytetracycline
Doxycycline hydrochloride
Spiramycin embonate
Kitasamycin
Erythromycin
Josamycin
Oleandomycin polystyrene
sulfonic acid
Lincomycin hydrochloride
FlorfenicolhrFigure 4
Growth of Streptococcus iniae in minced meat of sand

20-50
25--40
80
25-50
30-50
25
20--4040

lance (Sako, un pub\. data).

Citations

At present, two kinds of tetracyclines, five kinds ofmacrolides, lincomycin, and florfenicol are permitted as the chemotherapy of streptococcal infection inyellowtail in Japan (Table 4). Recently, streptococcithat have aquired resistance to macrolides and tetracyclines were found in many yellowtail farms (M.
Fukudome, Kagoshima Pref. Fish. Exp. Sta., pers.
commun., July 1990). Therefore, chemotherapy
treatments for yellowtail infected with drug-resistant
streptococci are likely to be ineffective.

Dr-~g:sPenicillin G
Amp i c i l l in

Atsuta, S.,]. Yoshimoto, M. Sakai, and M. Kobayashi.
1990. Streptococcosis occurred in the pen-cultured coho
salmon, Oncorhynchus kisutch. Suisanzoshoku 38:215-219.
(In Japanese.)
Hatai, K., S. Yasumoto,]. Tsukahara, E. Hirakawa, N. Yasunaga,
and T. lchiki.
1983. Drug sensitivity of various fish-pathogenic bacteria isolated from cultured fish in Nagasaki Prefecture,
1982. Bull. Nagasaki Pref. Inst. Fish. 9:1~22. (In Japanese; English abstr.)

I

IVI I

10.00625; 0.0125! 0.025 i 0.05!

0.10!

0.20;

C

0.39;

( I-L g/mU
0.78;

•
•

o
o

_

o

Erythromycin

1.56 i 3.1300Chloramphenicol

• •
•
• o

Oxytetracycline

o

•

0

Spiramycin

0

Josamycin

12.5:

25

50

100

;;:200!.o !

Oleandomycin
Kitasamycin

6.25;

Doxycycline•.0 •Streptomycin
Lincomycin.0

Col istin
Sulfadimethoxine
Sodium nifurstyrenate
Trimethoprim
Oxolinic

o

o

acid

Figure 5

Sensitivities of Enterococcus sp. and Streptococcus iniae isolated from yellowtail against various drugs: • Enterococcus sp.; 0Streptococcus iniae (Sako, unpub\.data).

130

NOAA Technical Report NMFS III

Iida, T., H. Wakabayashi, and S. Egusa.
1982. Vaccination for control of streptococcal disease in cultured yellowtail. Fish Pathol. 16:201-206. (In Japanese; English abstr.)
Iida, T., K. Furukawa, M. Sakai, and H. Wakabayashi.
1986. Non-hemolytic Streptococcus isolated from the brain of
the vertebral deformed yellowtail. Fish Pathol. 21:33-38.
(In Japanese; English abstr.)
Kaige, N., M. Miyazaki, and S. Kubota.
1984. The pathogen and the histopathology of vertebral deformity in cultured yellowtail. Fish Pathol. 19:173-179. (InJapanese; English abstr.)
Kimura, H., and R. Kusuda.
1979. Studies on the pathogenesis of streptococcal infection
in cultured yellowtail Smola spp.: effect of the cell-free culture on experimental streptococcal infection. J. Fish Dis.
2:501-510.
Kitao, T.
1982. The methods for detection of Streptococcus sp., causative bacteria of streptococcal disease of cultured yellowtail(Smola quinqueradiata), especially their cultural, biochemical and serological properties. Fish Pathol. 17: 17-26. (InJapanese; English abstr.)
Kitao, T., T. Aoki, and R. Sakoh.
1981. Epizootic caused by l3-haemolytic Streptococcus species
in cultured freshwater fish. Fish Pathol. 15:301-307. (InJapanese; English abstr.)
Kusuda, R., and K. Kawai.
1982. Characteristics of Streptococcus sp. pathogenic toyellowtail. Fish Pathol. 17:11-16. (In Japanese; English
abstr.).
Kusuda, R., and S. Takagi.
1983. Antibody production against Streptococcus sp. in naturally infected yellowtail Smola quinqueradiata. Rep. USA.
Mar. BioI. Inst. Kochi Univ. 5:21-28. (In Japanese; English
abstr.)
Kusuda, R., and E. Kawahara.
1987. Direct and indirect fluorescent antibody identificationof yellowtail pathogens. Bull. Jpn. Soc. Sci. Fish. 53:389394. (In Japanese; English abstr.)
Kusuda, R., K. Kawai, T. Toyoshima, and I. Komatsu.
1976. A new pathogenic bacterium belonging to the genus
Streptococcus, isolated from an epizootic of cultured yellowtail. Bull. Jpn. Soc. Sci. Fish. 42:1345-1352. (In Japanese;
English abstr.)
Kusuda, R., I. Komatsu, and K. Kawai.
1978. Streptococcus sp. isolated from an epizootic of cultned
eels. Bull. Jpn. Soc. Sci. Fish. 44:295.
Kusuda, R., K. Kawai, and T. Shirakawa.
1982. Serological study of Streptococcus sp. pathogenic to cultured yellowtail. Bull. Jpn. Soc. Sci. Fish. 48:1731-1738.
(In Japanese; English abstr.)_
Minami, T.
1979. Streptococcus sp., pathogenic to cultured yellowtail, isolated from fishes for diets. Fish Pathol. 14:15-19. (InJapanese; English abstr.)
Minami, T., M. Nakamura, Y Ikeda, and H. Ozaki.
1979. A beta-hemolytic Streptococcus isolated from cultured
yellowtail. Fish Pathol. 14:33-38. (In Japanese; English
abstr.)
Nakatsugawa, T.
1983. A Streptococcal disease of cultured flounder. Fish
Pathol. 17:281-285. (In Japanese; English abstr.)
Ogawa, S., N. Yasunaga, K. Hatai, and E. Hirakawa.
1982. A beta-haemolytic Streptococcus isolated from epizootics
of cultured seawater fishes. Bull. Nagasaki Pref. Inst. Fish
8:81-90. (In Japanese; English abstr.)
Ohnishi, K., and YJo.
1981. Studies on streptococcal infection in pond-cultured
fishes-I. Characteristics of a beta-hemolytic streptococcus
isolated from cultured ayu and amago in 1977-1978. Fish
Pathol. 16:63-67. (In Japanese; English abstr.)
Park, S.w., D.S. Sim, and YC. Yoo.
1987. Pathogenic Streptococcus sp. isolated from an epizooticof cultured yellowtail. Seriola quinqueradiata. Bull. Nat.
Fish. Res. Dev. Agency 40:75-79. (In Korean; English abstr.)
Pier, G.B., and S.H. Madin.
1976. Streptococcus iniae sp. nov., a beta-hemolytic streptococcus isolated from an Amazon freshwater dolphin, Inia
geoffrensis. In t. J. Syst. Bacteriol. 26:545-553.
Plumb,J.A.,J.H. Schachte, andJ.L. Gaines.
1974. Streptococcus sp. from marine fishes along the Alabama
and Northwest Florida Coast of the Gulf of Mexico. Trans.
Am. Fish. Soc. 103:358-361.
Sakai, M., S. Atsuta, and M. Kobayashi.
1986. A streptococcal disease of cultured jacopever Sebastes
schlegeli. Suisanzoshoku 34:171-177. (In Japanese; English
abstr.)
Shiomitsu, K.
1982. Isolation of Streptococcus sp. from the brain of cultured
yellowtail. Fish Pathol. 17:27-31. (In Japanese; English
abstr.)
Taniguchi, M.
1982a. Experiment on peroral inoculation via food to induce yellowtail streptococcicosis. Bull. Jpn. Soc. Sci. Fish.
48: 1717-1720. (In Japanese; English abstr.)
1982b. Studies on the streptococcicosis of yellowtail and its
preventions in Kochi Prefecture. Fish Pathol. 17:55-59.
(In Japanese; English abstr.)
Yasunaga, N.
1982. Occurrence of Streptococcus sp., a pathogen of cultured
yellowtail, in muscle of sardine for diets. Fish Pathol.
17:195-198. (In Japanese; English abstr.)

Stress Induced Pathologies in Fish: The Cost of StressGARY P. MOBERG
Department ojAnimal ScienceandFisheries Program
University oj California, Davis
Davis, CA 95616

ABSTRACT
Pathology is the consequence of prolonged stress. Maintaining fish under aquaculture
conditions intensifies the problems of stress by adding the impact of stressors that are
unique to culture conditions. Unlike terrestrial domestic animals, fish have notbenefitted from the genetic pressures of generations of domestication that favor thoseindividuals most suitable for culture. The physiological mechanisms by which stress induces the development of pathology remain unknown, although it is recognized that oneof the major factors affecting health during stress is the response of the neuroendocrine
system, a stress responsive system that directly regulated growth, reproduction, and theimmune system. During stress, the neuroendocrine system shifts biological resourcesfrom pre-stress activities to new functions at a biological cost to the fish. As the biological cost of shifting these resources rises during stress, the fish is placed into a
prepathological state, rendering it vulnerable to the development of pathology. It is byfocusing on this biological cost of stress that it is possible to develop strategies to reducestress in aquaculture and to understand the biological basis for the development ofdisease.

Introduction_

Pathology is the consequence of prolonged stress.
While we can not identify the exact mechanisms responsible for the development of pathologies during
stress, there is indisputable evidence that stress canresult in the development of a diverse number of pathologies that include not only disease, but the lossof reproduction, the failure to grow normally, andeven the development of abnormal, deleterious behaviors (Moberg 1985).
Fish in culture are especially at risk to the adverse
effects of stress. Unlike domesticated mammals, fish
have not benefitted from generations of genetic selection for traits that would assist them in adapting to
the restraints of confinement. In essence, fish are stillwild animals, and when culture conditions do not duplicate their natural habitat, they are forced to makebiological adjustments to survive. Exacerbating thisproblem is the failure of aquaculturists to make any
serious effort to systematically define those culture
conditions that the fish would find least stressful.

Practical considerations have dictated most culture
conditions with emphasis on such factors as the easeof cleaning, maximizing the number of fish, or using
any holding facility that is readily available. Little
concern is given to the fish, at least until stress becomes so severe that pathology occurs. Certainly thisis an area of aquaculture that needs to be addressed,
especially as increasingly intensified culture conditions are adopted.

Problem of Defining Stress_

Although there is considerable evidence demonstrating the disruptive effects of stress on the well-beingof fish (Schreck 1982; Pickering and Pottinger 1987;
Maule et al. 1989; Vijayan and Leatherland 1990), it
is surprising that there is not a better understandingof how stress actually leads to the development ofpathologies. One reason for the difficulty in discovering the mechanisms involved is a lack of consistent
responses to stress. Most fish do not develop a pathol131

132

NOAA Technical Report NMFS 111

ogy during stress. In fact, it is not possible to predict
which fish will become vulnerable to a pathology due
to stress. This is because stress, per se, is not necessarily harmful to an individual. Animals have developed
defenses for coping with stress. Stress is a part of life,and for a species to be successful, it must have
evolved biological defenses to stress. Therefore, stressresponses are not bad. In fact, they are desirable andnecessary. Pathology occurs only when the animal isconfronted with stress of such a magnitude that thevery biological responses that evolved for defense result in a biological cost to the individual and renders
it vulnerable to pathology. To understand how thebiological cost of stress leads to the development ofpathology, it is necessary to first examine how fish
respond to stress.

Model of the Stress Response
Numerous studies have identified the various biological responses that fish use in responding to andcoping with stress (for monographs summarizing this

RECOGNITION OF A
THREAT TO HOMEOSTASISSTRESS RESPONSECONSEQUENCES
OF STRESS

Figure 1
Model for the response of animals to a stressful event(from Moberg 1985).

_

work see Pickering 1981 and Adams 1990). Because
these responses are so varied and complex, I will use
a model of animal stress (Fig. 1) to organize current
concepts of stress biology and examine how the biological cost of stress results in the development ofpathology. The development of this model has been
discussed elsewhere (Moberg 1985, 1987), but, in
brief, the model divides the biological response tostress into three major components: recognition of athreat to homeostasis, the stress response, and theconsequences of stress. It is the central nervous system that perceives a threat to homeostasis andorganizes the fish's biological defense. If a threat(also referred to as a stressor) is perceived, three general types of biological responses are available to thefish: behavioral, autonomic nervous system, and neuroendocrine system responses. In the wild, behavior
can be the most biologically efficient way for a fish
to cope by simply allowing the animal to remove
itself from the stressor. However, under the confinement of culture conditions, this option is severely
limited. If the fish is unable to avoid the stressor,
then the roles of the autonomic nervous and neuroendocrine systems become critical. Whileactivation of the autonomic nervous system represents an important way for a fish to avoid or copewith a stressor, its effects are rapid in onset, of shortduration, and relatively specific. For this reason, theimportance of the autonomic nervous system in inducing stress-related pathologies is questionable. In
contrast to the autonomic nervous system, the hypophyseal hormones of the neuroendocrine system
undoubtedly play an important role in stress-induced pathology (Moberg 1985). These hormones
have a widespread action on the animal and longlasting effects on such diverse biological functions
as reproduction, growth, metabolism, resistance todiseases, and behavior. Each of these functions arevital to the fish's well-being.
When the central nervous system organizes the
biological defense to a stressor, it is a combination ofthese three general biological systems that alters
function. In mammals, we have found that a numberof factors influence the pattern of this biological response, and we have found it impossible to predict
how the individual systems will respond (Moberg
1985). While the data for fish is limited, there is evidence indicating that in fish there are also a numberof factors that can alter the stress response. For example, water quality and temperature (Pickering andPottinger 1987, 1989), genetics (Refstie 1982), andsocial interactions (Peters et al. 1988) have all beenfound to influence the stress response. Thus, measurement of the three general biological systems havenot proved to be reliable as an indicator of stress_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Moberg: Stress Induced Pathologies in Fish: The Cost of Stress(Moberg 1987). However, what is important to thefish is not the pattern of the biological response but
the resulting change in biological function-the ultimate consequence of stress.
Regardless of which of the general biological systems the fish chooses in response to the stressor, theresult is a change in biological function. Depending
upon the appropriateness of the response, thischange in biological function may alleviate, or eveneliminate the stressor. But the change in biological
function can also lead to the development of aprepathological state which can eventually result in
the development of a pathology (Fig. 1). For example, if the change in biological function during
stress results in the suppression of the immune system (a prepathological state), then the animal isvulnerable to pathogens, and the opportunity for disease (a pathological state) exists. The longer the fish
is stressed, the longer the immune system is suppressed and the greater the opportunity for disease
to occur. However, disease is only one possible pathological state. Other examples would be the inability
to reproduce, abnormal behavior, or the failure to
grow normally.

Biological Cost of Stress_

The change in biological function that occurs as partof the stress response results in a biological cost to
the fish. The fish's resources are diverted from such
pre-stress activities as growth to new activities. For example, the glucocorticosteroid hormone cortisol
secreted during stress will induce gluconeogenesis,
diverting metabolic resources supporting such functions as growth to the production of glucose. Barton
and Schreck (1987) found in juvenile steelhead(Oncorhynchus mykiss) that acute stress reduced byabout 25% the amount of energy available for otheractivities. Likewise, the biological cost of responding
to a stressor may suppress the ability of the immune
system to respond to pathogens, rendering the fish
vulnerable to disease (Maule et al. 1989). It does notmatter which pattern of responses the animalchooses; a change in biological function occurs that
imposes a cost, whether or not the change in function is effective in helping the animal to cope withthe stressor.
Fortunately, most stressors last for only a brief time
and the biological cost of coping with them is relatively small. However, if a stressor is severe, if it
persists, or if the fish experiences a series of stressors,
the resulting biological cost may be sufficient to induce a prepathological state which in turn can leadto the development of pathology.

133

The concept that there is a biological cost associated with coping with stress also explains how theeffects of subclinical stressors can accumulate, resulting in a significant stress that results in pathology(Moberg 1985, 1992). Separately, none of the
subclinical stressors would result in a significant expenditure of the fish's resources, but combined, thesubclinical stressors could cost the animal sufficient
resources to induce a prepathological state and leadto the development of a pathology. For example,
Jarvi (1989) found a greater mortality rate in Atlantic
salmon smolts (Salmo salar) that were exposed simultaneously to both osmotic stress and the presence of
predators than if the smolts experienced only one of
the stressors. Because of the accumulation of biological costs, a series of apparently innocuous events canlead to disease, loss in reproduction, diminishedgrowth, or mortality.

Conclusion
The biological cost of stress is the key to understanding why fish exposed to stress develop
pathologies. For a fish to cope with a stressor, it
must expend biological resources. The more prolonged the stressor or the greater the effort needed
to cope with a severe stressor, the greater the diversion of biological resources, and thus the greater
the biological cost of the stress. This diversion of
resources occurs at the expense of other biological
functions, leading to a prepathological state where
the fish is at risk to the development of pathology.
For some fish, the biological cost of coping with astressor is greater than for other fish and these are
the first to succumb to the stressor. As we manage
fish in culture, every effort must be made to reducestress and, as a result, lower the biological cost forfish living in culture.

Citations
Adams, S.M.
1990. Biological indicators of stress in fish. Am. Fish. Soc.
Symposium 8, Bethesda, Maryland, 191 p.
Barton, B.A. and C.B. Schreck.
1987. Metabolic cost of acute physical stress in juvenile
steelhead. Trans. Am. Fish. Soc. 116:257-263.
Jarvi, T.
1989. Synergistic effect on mortality in Atlantic salmon,
Salrno salar, smoIt caused by osmotic stress and presence of
predators. Environ. BioI. Fishes 26:149-152.
Maule, A.G., R.A. Tripp, S.L. Kaattari, and C.B. Schreck.
1989. Stress alters immune function and disease resistance inchinook salmon (Oncorhynchus tshawytscha). J. Endocrinol.
120:135-142.

134

NOAA Technical Report NMFS 111

Moberg, G.P.
1985. Biological response to stress: key to assessment of animal well-being? In Animal stress (G.P. Moberg, ed.), p 2429. American Physiological Society, Bethesda, MD.
1987. Problems in defining stress and distress in animals. J.
Am. Vet. Med. Assoc. 191:1207-1211.
1992. Stress: diagnosis, cost and management. In The wellbeing of agricultural animals in biomedical and agriculturalresearch (l.A. Mench, SJ. Mayer and L. Krulisch, eds.), p
58-61. Scientists Center for Animal Welfare, Bethesda,
MD.
Peters, G., M. Faisal, T. Lang, and I. Ahmed.
1988. Stress caused by social interaction and its effect on
susceptibility to Aeromonas hydrophila infection in rainbow
trout Salmo gairdneri. Dis. Aquat. Org. 4:83-89.
Pickering A.D.
1981. Stress and fish. Acad. Press. New York, NY, 354 p.
Pickering, A.D., and T.G. Pottinger.
1987. Poor water quality suppresses the cortisol response of
salmonid fish to handling and confinement. J. Fish BioI.
30:363-374.

_
Pickering, A.D. and T.G. Pottinger.
1989. Stress responses and disease resistance in salmonid
fish: effects of chronic elevation of plasma cortisol. Fish
Physiol. and Biochem. 7:253-258.
Refstie, T.
1982. Preliminary results: differences between rainbow trout
families in resistance against vibriosis and stress. Dev.
Compo Immunol. Supplement 2: 205-209.
Schreck, C.B.
1982. Stress and rearing of salmon ids. Aquaculture 28:241249.
Vijayan, M.M., andJ.F. Leatherland.
1990. High stocking density affects cortisol secretion and tissue distribution in brook charr, Salvelinus Jontinalis. J.
Endocrinol. 124:311-318.

Control of Fish Disease in JapanAKIRA MURATA
Fisheries Agency, Research Division
Kasumigaseki 1-2-1, Tiyoda-ku
Tokyo, Japan

ABSTRACTThe damage to Japanese aquaculture by fish disease in 1988 was estimated at 20,000
metric tons, worth 144 million dollars, and amounted to 6% of the total aquaculture
production. The Fisheries Agency of the Government of Japan financially supports fish
disease control projects carried out by prefectural governments and fisheries cooperative
associations. It also entrusts the Japan Fisheries Resource Conservation Association
UFRCA) with the following tasks; 1) voluntary pathogen inspection of imported larvalfish and fish eggs; 2) training of advisory personnel on fish disease control; 3) improvement of diagnostic methods; and 4) improvement of disease prevention methods and the
publication of their findings. The main body of the Agency is augmented by the
Shimonoseki University of Fisheries and nine national research institutes. One of these is
the National Research Institute of Aquaculture, which has a Fish Pathology Division
totalling 11 researchers. Each of the 47 prefectural govern men ts in Japan has at least one
Fishery Experimental Station. There are a total of 96 stations. Prefectural governments,
with their institutions, undertake disease diagnosis and drug residue testing and provide
guidance on fish disease control and drug use. Fisheries co-operative associations play
important roles in fish disease prevention by giving guidance to fishermen and controlling local fishery practices. There are 17 universities that have a fishery faculty and 16
universities with a veterinary faculty. All these universities have curriculums for fish disease and conduct research on it.

Aquaculture and Fish Disease_

Fishery Products and Aquaculture Production
In 1988, fishery production in Japan totaled
12,785,000 metric tons (t) valued at $19.4 billion (1dollar = 140 yen) (MAFF 1988). Changes in amountand value of aquaculture products over the last 23
years are shown in Figure 1. Aquaculture productionin 1988 reached 1,426,000 t or II % of the total fishery output. These products were valued at $4.9
billion or 25% of the total fishery value.
Aquaculture has been rapidly developing and has
become more important because 1) consumers aredemanding higher quality and more variety in fisheryproducts, 2) aquaculture techniques have improved,and 3) the total amount of aquacultural grounds has
increased owing to the development of new aquacultural facilities, and the expansion of older ones.

There are two types of aquaculture; feeding aquaculture (i.e., fish culture) and aquaculture without
feeding (i.e., shellfish culture). Production by feeding aquaculture has grown markedly in recent years
and has reached 343,000 t or $2.5 billion in 1988about 7 and 19 times the same values for those In
1965, respectively.
The production of marine fish by aquaculture in1988 was valued at $1,811 million and amounted to245,000 t. Yellowtail Seriola quinqueradiata production
was greatest at $949 million (165,900 t), followed by
red sea bream, Pagrus major, and coho salmon,
Oncorhynchus kisutch. (Table 1). Coho salmon, which
is cultured in fresh water until the smolt stage
and then transferred to seawater, is one of thespecies the production of which has increased rapidly
during recent years. Likewise the production ofhorse mackerel, Trachurus japonicus, striped jack,
Caranx delicatissimus, and tiger puffer, Takifugu
rubripes, have increased markedly. Increased variety
135

136

NOAA Technical Report NMFS 111_

Total fishery

~ 10

g
u

-.:::
Gi
E
c:

o

::

i

5

Aquacultureo .b::;::::;:=;;;:;;;;;~;~;;~ Feeding aquacu~ure
1965

1970

1975

1980

1985

Year

20

Total fishery

.
III

.!!

'0
'tJ

c:

~

m 10
Aquacultureo .,I::;:;;;;;~==:;::::~~~-........____,~_,_-_._~F.eeding aquaculture

1965

1970

1975

1980

1985

1990

Year

of cultured species IS a current trend in Japaneseaquaculture.
Production of freshwater fish by aquaculture was$732 million (97,800 t) in 1988. Japanese eel,Anguilla japonica, production was largest at 39,600 t($417 million), followed by Ayu, Plecoglossus altivelis,salmonids, and common carp, Cyprinus carpio. A diversity of species is also seen in freshwateraquaculture. Among these are Japanese native salmonids such as landlocked salmon, O. masou, dwarf rill
trout, O. rhodurus, and mountain trout, Salvelinus
pluvius.

Fish Disease and Its Damage
The rapid development of aquaculture has alsobrought about a problem, namely with the occurrence of diseases. These are caused partly by the

Figure 1
Cultured fish production (feeding aquaculture and aquaculture without feeding) andthe total fishery production since 1965
(MAFF 1988) in metric tons and dollars. 0$=
140 yen.)decline in the health of the cultured fish owing toenvironmental deterioration and overpopulation,and partly by the recent entry of foreign pathogens.
For example, infectious pancreatic necrosis (IPN),
infectious hematopoietic necrosis (IHN), and bacterial kidney disease (BKD) are diseases considered tohave been introduced from abroad. Both IHN and
BKD are thought to have entered through eggs imported for propagation, while IPN probably enteredthrough eggs imported for culture. These diseases
have become prevalent in salmonid farms and are
causing severe damage to salmonid production.
Based on a questionnaire sent to all aquaculture
farmers through the prefectural governments every
year, the Fisheries Agency has estimated the annual
damage by fish diseases to feeding aquaculture. The
results are shown in Figure 2.
The damage to aquaculture by fish disease in 1988
was estimated at 20,000 t or $144 million which is

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Murata: Control of Fish Disease in Japan

137

Table 1
Cultured fish production by species and damage due to disease in 1988 (MAFF 1988).
DamageProduction

Ratio (Damage/Production)

Amount a(1,000 t)

Value b(million dollars)

Amount(1,000 t)

Value(million dollars)

Amount

Value

(%)

(%)

Marine fishyellowtail
red sea breamcoho salmon
flounderhorse mackerel
tiger puffer
kuruma prawn
others

245.0
165.9
45.5
16.5
3.1
6.5
1.2
3.0
3.3

1811
949
431
109
64
37
37
140
44

17.3
14.4
0.5
1.0
0.4
0.4
0.3
0.2
0.1

119
81
5
7
6
2
7
10
1

7
9
1
6
13
6
27
7
3

7
9
6
10
6
19
7
4

Freshwater fisheelayu
salmonids
common carp
others

97.8
39.6
13.6
19.1
18.1
7.4

732
417
120
73
57
65

2.8
1.1
0.3
1.0
0.2
0.2

25
14
3
5
1
2

3
3
2
5
1
3

3
3
3
7
1
4

Total

342.8

2,543

20.1

144

6

6

Fish

I

a in metric tons, t.
b 1$ = 140 yen.

about 6% of the total value of all aquaculture products. In 1979 estimated damage was $137 million andincreased year by year to reach a peak of $190 million
in 1982. It then decreased and has been maintainedat around $140 million since 1985. During the 1980s,
the total value of aquaculture products increased rapidly while the rate of damage by diseases either
decreased or remained nearly constant. It is thoughtthat two factors have worked effectively in reducing
damage by diseases in recent years. One is the practice of control measures, such as disinfection,
disposal of dead fish, and suitable layout of ponds.
The other is the improvement of husbandry techniques, such as improvement of food and the control
of population levels and pen location with regard to
the environmental capacity.
The rate of disease damage (estimated damage
value/total product value) for each species is shownin Table 1. Disease reduced yellowtail production by
9% in 1988. The damage rates of tiger puffer, Japanese flounder (Paralichthys olivaceus) and kuruma
prawn (Penaeus japonicus) were 19, 10, and 7%, respectively, while only 1% of red sea bream
production was affected. As for freshwater finfish,rainbow trout (0. mykiss) showed a high damage rate
of 7%, while that of common carp was only 1%. In
marine aquaculture, species with a long history ofculture generally showed low damage rates, whIle
species with a short history of culture, such as tiger
puffer and japanese flounder, showed high damage
rates. This difference may be caused by the lack ofexperience and less-developed management techniques for culturing new species.
In recent years, fish disease problems have become
more diversified and complicated. In yellowtail culture, streptococcosis and pseudotuberculosis are thetwo major diseases. When an episode of mass mortality occurs, both of these agents are often found in asingle fish. Nocardiosis, viral ascite disease (diseases
for which treatments are not yet established), and adisease causing peduncle curvature have recently become an additional problem.

Fish Disease Control
System-Because of the complexity and variety of diseases occurring in Japanese aquaculture, theFisheries Agency is carrying out systematic fish disease con trol in cooperation with prefectural
governments, fisheries cooperative associations, andsome universities in order both to decrease diseasedamage and to ensure the safety of cultured fish forhuman consumption. It is important for aquaculture

138

NOAA Technical Report NMFS 111.....,...

VALUE OF PRODUCTION
2500

III

~

.!!

'0
'1:l

c 2250

~
i

2000
1979

1961

1963

1965

1967

VALUE OF DAMAGE

III

175

~

.!!

'0
'1:l

tural governments and the fisheries cooperative associations. It also entrusts the Japan Fisheries Resource
Conservation Association (JFRCA) with the following
responsibilities: 1) voluntary pathogen inspection ofimported live fish and eggs; 2) training of advisorypersonnel on fish disease control; 3) improvement of
diagnosis methods; and 4) improvement of disease
prevention methods and the publication of theirfindings (Fig. 4).
The Fisheries Agency oversees nine national research institutes, the Shimonoseki University ofFisheries, and the Hokkaido Salmon Hatchery. In
these facilities either research or education infisheries is carried out, but among these national research facilities, only the National Research Instituteof Aquaculture, which was established in 1979 tocarry out basic and leading research for aquaculture,
has a fish pathology division. This division has 11 researchers and consists of 4 research sections;Pathogens, Pathophysiology, Pharmacology, and Immunology (Fig. 5).

150

c

~

i

_

125

100
1979

1961

1963

1965

1967

DAMAGE RATE
(%)
6

6

4+--....--r--...--,........-----r-~-~-~

1979

1961

1963

1965

1967

YEAR

Figure 2
Changes in cultured fish production (feeding aquaculture)and damage by fish disease. (1$=140 yen.)
farmers to practice proper feeding and fish disease
control. Thus, prefectural governments and fisheries
cooperative associations have been requested to establish a system to assist the aquaculture farmer's
practice of disease control (Fig. 3).
Fisheries Agency-This agency financially supports
fish disease control projects carried out by prefec-

Prefectural Government-Control measures on
the aquaculture grounds are very important for disease prevention. Prefectural governments play important roles in guiding aquaculture farmers either
directly or indirectly through the fisheries cooperative associations.
Prefectural governments and their research institutions undertake disease diagnosis and drug residue
testing, and provide guidance on fish disease control
and drug use (Fig. 6).
There are 96 prefectural fisheries experimental stations in Japan: at least one station in each prefecture.
Investigation and research for aquaculture and Ashdisease is carried out in their propagation and aquaculture divisions. In most of these prefectures, fish
disease control centers (20 in total) have been established, either as a part of the fishery experimental
station or as an independent establishment. Prefectural governments have also established fisheries
extension offices to give technical guidance to aquaculture farmers. These offices and fisheryexperimental stations undertake fish disease prevention measures in a cooperative manner.
The number of staff engaged in disease control
measures in prefectural governments was 470 in
1986. In addition, 30 staff members from various cities, towns, and villages were also engaged in thesemeasures.
Fisheries Cooperative Association-Fisheries cooperative associations playa very important role in fish
disease control by giving guidance to aquaculture
farmers and controlling local fishery grounds. How-

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Murata: Control of Fish Disease in Japan139• FundingI

Fisheries
Agency•• Guidance
Financial support

I

I•• Report

11

Japan Fisheries
Resource
Conservation
Association• Infonnation.rtp o //• Re• Guidance, AssistancePrefectural
government• Infonnation• Guidance• Financial support

I1• Report

Fisheries
Co-operative
Association

Il
I

Farmer• Guidance. Assistancet• Infonnation

Fisheries Agency

Figure 3
Fish disease control system inJapan.

Fisheries Agency

National Research Institute of Aquaculture
Fish Pathology Division

Japan Fisheries Resource
Conservation Association

National Fisheries Research Institute (8)Shimonoseki University of Fisheries• Voluntary pathogen examination of imported live fish and eggs
• Training on fish disease characteristics

Figure 5
Fish disease research system of the Fisheries Agency.

• Improvement of diagnosis and disease prevention methods• Extension (Films. Video tape recordings, Textbooks)

Figure 4
Role of the Japan Fisheries Resource Conservation Association in the control of disease in aquaculture.

Universities-Fish disease education is carried out
by 17 fishery faculties in universities (including the
Shimonoseki University of Fisheries,, Fisheries
Agency) and 16 veterinary faculties. AlI1.hese faculties also have fish disease research facilities.

ever, many of these associations are small in scale,and the level of guidance varies widely. Only a few ofthem carry out drug residue testing, diagnosis of fish
disease, and observation of environmental conditionsin their aquaculture grounds (Fig. 7). The number ofstaff engaging in disease control from those associa:
tions was only 80 in 1986.

Prevention of the Introduction and Outbreak of
Pathogens-In Japan, neither laws nor regulations
have been established concerning fish quarantine.
However, in order to avoid the introduction of fish
pathogens from foreign countries, the Fisheries
Agency requests importers (through prefectural governors) to take the following steps. Importers arerequested 1) to import living eggs or fish in conjunc-

140

NOAA Technical Report NMFS III...,.....

tion with health certificates issued by the government
authority of the exporting country, 2) to receive an
examination for pathogens in the fish or eggs at thetime of import, and 3) to disinfect eggs immediately
after importation.
Examination for pathogens at the time of import iscarried out by the JFRCA as entrusted by the Fisheries
Agency. Examination is done on viral diseases such as
VHS (viral hemorrhagic septicemia), BKD and whirling disease of salmonid fish (Mxyosoma cerebralis), andon viruses, bacteria, and parasites for eel. Examinations are also conducted for other species.
To prevent the spread of fish diseases, prefectural
fish disease control centers and fisheries experimental stations examine live fish and eggs for pathogens
upon the request of the aquaculture farmer. The
Fisheries Agency encourages farmers to transport live
fish and eggs with records of species, place of production, name and address of producer, dates offertilization or hatching, history of fish disease, andmedication history.

Prefectural
Government..

Prefectural Governments (47)

Fisheries Experimental Stations (96)Fish Disease Control Centers (20)Fisheries Extension Offices• Disease diagnosis• Drug residue testing• Providing guidance on fish disease control and drug use• To survey and observe environmental conditions in aquaculture grounds• Research

Figure 6
Role of the prefectural governments in the fish disease control system.

• Guidance. Assistance• Infonnation• Guidance• Financial support

Fisheries
Co-operative
Association

_

I
I
I

Farmer·0"""""."",,_J..
• Infonnation• Drug residue testing• Diagnosis of fish disease• Observation of environmental conditions in aquaculture groundTraining-Fish disease specialists are indispensable
for carrying out adequate control measures such asproviding advice to the aquaculture farmers and diagnosing and disinfecting facilities and equipment.
Since 1973, the Fisheries Agency has been training
the staffs of prefectural governments-in 1984 staffs
of fisheries cooperative associations were included in
the training program. This training was aimed at educating new fish disease specialists and at improving
the knowledge of the current specialists. In recent
years, the Fisheries Agency has entrusted this training to JFRCA.

Figure 7
Role of fisheries cooperative associations in the control of fish disease.

There are various trammg courses offered by the
JFRCA for fish disease technical workers. A basic classconsists of 20 day-lectures and practical exercises
each year for 3 years. In 1989, there were 30 to 40
trainees for each year course; altogether, 100 people
have participated in the training. Other training onfish disease is also carried out. Since 1974, the Japan
Veterinary Medical Association has been providing afish disease course for veterinarians every year as
postgraduate education. In Japan, training for fish
disease technical workers is carried out separately inthe fishery and veterinary fields. On the other hand,

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Murata: Control ofFish Disease in Japanaquacultural guidance and leadership are mainly carried out in the fishery field.
Investigation and Extension-Under the present circumstances where fish disease can cause large-scale
damage and is becoming more complicated to prevent, diagnose, and treat, it is becoming more
important to promote research on fish disease and to
give aquaculture farmers information and techniques
to control this threat.
Studies on fish disease by the Fisheries Agency aremainly conducted at the National Research Instituteof Aquaculture in cooperation with six of the nine
regional national fisheries research institutes and the141

Fisheries Agency
Japan Fisheries Resource
Conservation Association

textbooks
films
video tapes
Prefectural government

guidance
textbooks and pamphlets

FarmerI

Figure 9
Fisheries Agency
National Fisheries Research Institutes (8)National Research Institute of Aquaculture (1)Universities

L

Fishery faculties (17)Veterinary faculties (16)Prefectural government
Fisheries Experimental Stations

I

Figure 8

Major organizations involved in fish disease researchin Japan.

Hokkaido Salmon Hatchery. The Agency entrusts
some universities with fundamental research, such as
exploring the mechanisms of outbreak and infectionof various fish diseases. It also entrusts prefectural
fisheries experimental stations with applied research
for fish disease control techniques, such as disinfection and vaccines (Fig. 8).
Through its extension program, the Agency distributes textbooks about disease diagnosis andtechniques of disease prevention and lends films
and video tape recordings through the JFRCA to theprefectural staffs who are in charge of guiding
aquaculture farmers. Prefectural governments alsooffer guidance courses and distribute textbooks or
pamphlets on fish disease to the aquaculture farmer.
These measures are used to impart knowledge onfish disease characteristics, disease prevention,aquaculture management, and use of drugs, andhow to put all of this knowledge into practice appropriately (Fig. 9).

The Fisheries Agency's extension system forproviding fish disease information in Japan.

Drugs-Appropriate aquaculture management is important to prevent or to reduce damage by fish
disease. Drugs are also important to reduce damage
when disease breaks out.
At present, there are 26 antiba~terial medicines
available for fish in Japan. In addition, there are vaccines for vibriosis, insecticides, anesthetics, andnutritive drugs. In 1988, all the fishery drugs sold inJapan amounted to 1,540 t, which was worth $55 million.
All drugs are strictly evaluated for safety, efficacy, residue accumulation in fish tissues, andother properties by the Central Pharmaceutical Mfairs Council prior to receiving manufacture
approval from the Ministry of Agriculture, Forestry,
and Fisheries, based on the Pharmaceutical Mfairs
Law. Use of antibacterial drugs for fish as well as
livestock is regulated by "The Standard to be Observed by User," which is based on the same law(Table 2).
Staffs of prefectural governments and fisheries cooperative association assist all aquaculture farmers touse drugs properly by visiting, distributing pamphlets, or carrying out guidance courses at regularintervals. The public health divisions of prefectural
governments carry out drug residue testing by sampling cultured fish from shops. The fisheries divisionsof prefectural governments and fisheries cooperative
associations also examine aquaculture products submitted on a voluntarily basis for drug residues before
harvest, in order to ensure that foods are safe forhuman consumption.

142

NOAA Technical Report NMFS III_

Table 2The standard to be observed by user of drug (MAFF 1980).
Drug

Subject animal

Feed additive
containing
alkyltrimethylammoniumcalciumoxytetracycline

Administration and dosage

Withdrawal period·

Yellowtail

Administer orally, mixing not more than
50 g (potency) in 1 t of feed'

20 daysFeed additive
containing
ampicillin

Yellowtail

Administer orally, mixing not more than
20 g (potency) in 1 t of feed

5 daysFeed additive
containing
chlortetracyclinehydrochloride

Yellowtail

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

10 daysEel

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

15 daysFeed additive
containing
erythromycin

Yellowtail

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

30 daysFeed additive
containing
florphenicol

Yellowtail

Administer orally, mixing not more than
109 in 1 t of feed

5 daysFeed additive
containing
kitasamaycin

Yellowtail

Administer orally, mixing not more than
80 g (potency) in 1 t of feed

20 daysFeed additive
containing
oxytetraccyclinehydrochloride

Yellowtail

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

20 days

Red sea bream

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

30 days

Coho salmon

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

30 daysEel

Administer orally, mixing net more than
50 g (potency) in 1 t offeed

30 days

Rainbow trout

Administer orally, mixing not more than
50 g (potency) in 1 t of feed

30 days

Yellowtail

Administer orally, mixing not more than
30 g in 1 t of feed

16 days

Coho salmon

Administer orally, mixing not more than
20 g in 1 t of feed

21 daysEel

Administer orally, mixing not more than
20 g in 1 t of feed

20 days

Rainbow trout

Administer orally, mixing not more than
20 g in 1 t of feed

21 days

Ayu

Administer orally, mixing not more than
20 g in 1 t of feed

14 days

Carp

Administer orally, mixing not more than
109 in 1 t of feed

28 days

Yellowtail

Administer orally, mixing not more than
20 g in 1 t of feed

16 daysFeed additive
containing
oxolinic acid(except liquid)

Feed additive
containing
oxolinic acid(liquid)

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Murata: Control of Fish Disease in Japan

143

Table 2 (Continued)
Administration and dosage

Withdrawal period"

Drug

Subject animal

Bath agent
containing
oxolinic acid

Eel

Give bath, dissolving not more than
5 g in 1 t of water

25 days

Ayu

Give bath, dissolving not more than
10 g in 1 t of water

14 daysFeed additive
containing
spiramaycin
embonate

Yellowtail

Administer orally, mixing not more than
40 g (potency) in 1 t of feed

30 daysFeed additive
containing
sulfadimethoxine
or its sodium salt

Rainbow trout

Administer orally, mixing not more than
100 g in 1 t of feed

30 daysFeed additive
containing
sulfamonomethoxine
or its sodium salt

Yellowtail

Administer orally, mixing not more than
200 g in 1 t of feed

15 daysEel

Administer orally, mixing not more than
200 g in 1 t of feed
Administer orally, mixing not more than
100 g in 1 t of feed
Administer orally, mixing not more than
100 gin 1 t offeed

30 days

Rainbow trout

Administer orally, mixing not more than
150 g in 1 t offeed

30 days

Bath agent
containing
sulfamonomethoxine
or its sodium salt

Rainbow trout

Give bath, dissolving not more than
10 g in 1 liter of salt watet (lessthan 1% concentration)

15 daysFeed additive
containing
sulfamonomethoxine
and ormetoprim(compound drug)

Ayu

Administer orally, mixing not more than
15 g of sulfamonomethoxine and5 g of ormetoprim in 1 t of feed

15 daysFeed additive
containing
thiamphenicol

Yellowtail

Administer orally, mixing not more than
50 g in 1 t of feed

15 days

Coho salmon
Ayu

30 days
15 daysa Withdrawal period defined as the required waiting period following drug administration prior to use offish for human consumption.

b t = metric ton.

Citations_

MAFF (Ministry of Agriculture, Forestry, and Fisheries)
1980. Ministerial ordinance regarding the control and use ofdrugs for animals. MAFF Ordinance No. 42, Sept. 30, 1980,
MAFF, Government ofJapan, Tokyo, Japan. (In English.)1988. Annual statistics of fisheries and aquaculture production, MAFF, Government ofJapan. (In English.)-u.S. GOVERNMENT PRINTING OFFICE 1992-0-790-258/80000