Control of Disease in Aquaculture

150 Pages·2008·9.28 MB·English

NOAA Technical Report NMFS III
October 1992
Control of Disease in Aquaculture
Proceedings ofthe Nineteenth
U.S.-Japan Meeting on Aquaculture
Ise, Mie Prefecture, Japan
29-30 October 1990
Ralph S. SVIjcek (editor)
u.s.
DepartmentofCommerce
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NOAA Technical Report NMFS III
Control of Disease in Aquaculture
Proceedings ofthe Nineteenth
u.s.-Japan Meeting on Aquaculture
!se, Mie Prefecture,Japan
29-30 October 1990
Satellite Symposium: 2 November
Ralph S. Svrjcek
PublicationsUnit
NorthwestandAlaskaFisheriesScience Centers
PanelChairmen:
Conrad Mahnken, United States
SeijiSakaguchi,Japan
Underthe U.S.-Japan CooperativeProgram
inNaturalResources (U)NR)
October 1992
u.s.
DEPARTMENTOFCOMMERCE
BarbaraHackmanFranklin,Secretary
NationalOceanic andAtmosphericAdministration
JohnA. Knauss,UnderSecretaryforOceansandAtmosphere
National MarineFisheries Service
WilliamW. FoxJr.,AssistantAdministratorforFisheries
PREFACE
The UnitedStatesandJapanesecounterpartpanelson aquaculturewereformed in 1969under the
United States-Japan Cooperative Program in Natural Resources (UJNR). The panels currently in
clude specialists drawn from the federal departments most concerned with aquaculture. Charged
with exploring and developing bilateral cooperation, the panels have focused their efforts on ex
changinginformationrelatedtoaquaculturewhichcouldbeofbenefittobothcountries,
The UJNRwasbegun during theThird Cabinet-LevelMeetingoftheJointUnitedStates-Japan
Committee on Trade and Economic Affairs inJanuary 1964, In addition to aquaculture, current
subjectsin theprogramincludedesalinationofseawater,toxicmicroorganisms,airpollution,energy,
foragecrops,nationalparkmanagement,mycoplasmosis,windandseismiceffects,proteinresources,
forestry, and severaljoint panels and committees in marine resources research, development, and
utilization.
Accomplishmentsinclude increasedcommunication and cooperation among technical special
ists; exchangesofinformation,data, and research findings; annual meetingsofthe panels, a policy
coordinative body; administrative staff meetings; exchanges ofequipment, materials, and samples;
severalmajortechnicalconferences;andbeneficialeffectsoninternationalrelations,
ConradMahnken-UnitedStates
SeijiSakaguchi-Japan
TheNationalMarineFisheriesService (NMFS) doesnotapprove, rec
ommend orendorseany proprietary productor proprietary material
mentioned in thispublication. No reference shall be made to NMFS,
or to this publication furnished by NMFS, in anyadvertisingorsales
promotionwhichwouldindicateorimplythatNMFSapproves,recom
mends or endorses any proprietary product or proprietary material
mentioned herein, or which has as its purpose an intent to cause
directlyorindirectly the advertised product to be used or purchased
becauseofthisNMFSpublication.TheU.S.-JapansubseriesofNOAA
TechnicalReportsonaquacultureisusedtocommunicatepreliminary
results,interimreports,andsimilartimelyinformation.Itisnotsubject
toformalpeerreview.
ii
CONTENTS _
0.BERGH StudiesondiseasesofculturedAtlantic halibut 1
G.H.HANSEN
I.HUSE
A.JELMERT
T.AKIYAMA Scoliosisoffishes causedbytryptophan deficiency 7
T. MEYERS ControlofIHNvirusinsockeyesalmonculture 13
R.J. BARRIE Identificationofaconservedantigenicdomain in themajor 15
C.L. MASON capsidproteinofinfectiouspancreaticnecrosisvirus
J.C.LEONG
T.AOKI Cloningofhemolysingenesofaeromonads 21
I.HIRONO
T. HONJO Harmfulred tidesofHeterosigmaakashiwo 27
J.L.BARTHOLOMEW Impactofthe myxosporeanparasite Ceratomyxashastaonsurvival 33
J.L.FRYER ofmigratingColumeiaRiverBasinsalmonids
J.S.ROHOVEC
M.YOSHIMIZU Viralinfectionsofculturedfish inJapan 43
T.KIMURA
K.MOMOYAMA Someimportantinfectiousdiseasesofkurumashrimp, 49
Penaeusjaponicus, inJapan
J.R.WINTON Theapplication ofmolecularbiologyto the detectionof 53
infectioushematopoietic necrosisvirus
K. MUROGA Bacterialandviraldiseasesofmarinefish duringseed production 57
T.YOSHINAGA An ecologicalstudyoftheparasiticnematodeHysterothylaciumhazein theJapanese 63
commongobyAcanthogobiusflavimanus, in abrackishinlet
H.ISHIOKA Epidemiologyofmarinefish diseasesin thewarmwatersalongtheKuroshioCurrent 69
P.W. RENO Characterizationofhematic neoplasiain thesoftshellclam Mya arenaria 85
A. ILLINGWORTH
M.DORITY
M.OTOTAKE Kineticsofbovineserumalbuminadministered bytheimmersionmethod 95
T.NAKANISHI infishes acclimatized toseawaterand tofreshwater
T. NOMURA Theepidemiologicalstudyoffurunculosis insalmonpropagation 101
M.YOSHIMIZU
T.KIMURA
iii
T.SUZUKI Functionsofhemocytesduringthewound healingprocessin the pearloyster 109
YMAENO Skeletalabnormalitiesoffishes caused byparasitismofMyxosporea 113
M.SORlMACHI
E. READ-CONNOLE Presenceofoncogenesinfish tissuesandinfish celllines 119
C.A.SMITH
F.M. HETRICK
H.SAKO Streptococcalinfectionin culturedyellowtail 125
G.MOBERG Stressinduced pathologiesinfish: the costofstress 131
A. MURATA ControloffishdiseaseinJapan 135
Studies on Diseases of CulturedJuvenile Atlantic Halibut
0IVIND BERGH', GEIR H0VIKHANSEN", INGVAR HUSE' and ANDERSJELMERT'
*InstituteofMarineResearch
AustevollAquacultureResearchStation
N-5392Storeb¢, Norway
**DepartmentofMicrobiology andPlantPhysiology
University ofBergen,Jahnebakkm 5
N-5007Bergen, Norway
ABSTRACT
Bacterial infections by Flexibacterand Vibrio species are major causes of mortalities in
Atlantic halibut (Hippoglossus hippoglossus L.) larviculture. Egg surface disinfection is a
possible prophylactic treatment. Thisarticle summarizesand reviewsseveral experimel1ls
concerningcausesofmortalityofAtlantic halibuteggsand larvae.
Introduction _
groups was infected with 200 !-LL of a suspension of
an accenic culture of one of the following bacteria:
Cultivation of Atlantic halibut (Hippoglossus Flexibacter sp. strains NCIMB 13128 and NCIMB
hippoglossus L.) is presently at the verge ofa commer 13127T, (National Collection of Industrial and Ma
cial breakthrough in Norway. However, as is the case rine Bacteria, Aberdeen, Scotland) which were
with all cultivated species, there are problems emerg isolated from two different groups of halibut eggs
ing concerning diseases related to opportunistic and otherwise seemingly identical (Hansen and
microorganisms (Bergh et al. 1992; Bergh and Bergh et al. 1992), Vibrio strain HI-I0448 (Institute of
Jelmert 1990; Opstad and Bergh 1990; Pittman et al. Marine Research, Bergen, Norway) and Vibrio
1990). The purpose ofthis work is to summarize sev anguillarum NCMB 6 (National Collection of Marine
eralexperimentsbystudyingthe effectsofmicroorganisms Bacteria, Aberdeen, Scotland); and Vibrio fischeri
on mortalities of halibut eggs and yolk sac larvae, strain ATCC 7744 (American Type Culture Collec
possible prophylactic treatment procedures, and ef tion, Rockville, MD). Final total counts ofbacteria in
fects ofsome physical stressors. the wells were measured by staining with DAPI (Por
ter and Feig 1980) and counting in a Nikon
epiflourescence microscope at 600X to be in the or
Methods and Materials _
der of 2-3 X 106 bacteria x mL-l. One group of 60
eggs was not infected, serving as the control. Within
Eggs from one female were artificially stripped and 24 hours after hatching, visible remnants of the egg
fertilized with sperm from two males and reared in shell were removed along with 10 mL of the water,
250-L upstream incubators between 6 and 7° C until and 10 mL of sterile seawater were immediately
further processing. added..Mortality was recorded until Day 37 after
The eggs were transferred to polystyrene multiwell hatching. For a further description of the infection
dishes (NUNC, Roskilde, Denmark) for the disinfec experiment, see Bergh etal. (1992).
tion trials and the infection experiment. Each well For the disinfection experiment another egg group
contained one egg and II mL of sterilized seawater. was disinfected one day before hatching. The follow
The dishes were incubated in darkness between 5 ing procedure was followed. Eggs were divided into
and 6° Cfor the duration ofthe experiment. four groups, which were exposed to differentconcen
For the infection experiment, eggs were divided trations of the iodophor disinfectant Buffodine
into 6 groups, each containing 60 eggs. Four days (Evans Vanodine, Preston, England): 0.5, 0.05, and
before hatching, each well ofeach ofthe 5 treatment 0.005%, plus one untreated control group. Applica-
2 NOAATechnicalReportNMFS III
Figure 1
Halibut egg showing
large surface wounds.
This appearance is
typical of eggs in
fected with Flexibacter
sp. Egg diameter is
approximately 3 mm.
The photograph was
taken from a Wild
zoom binocular mi
croscope operated in
the dark field mode.
Photo by Guri Grung
andVibekeValkner.
tion time was 10 minutes. Immediately following the The infection experiment revealed three different
disinfection, the solution was carefully pipetted off types ofmortality patterns:
the eggs, and more sterile seawater was added. T1.is
procedure was repeated three times. The control 1 The uninfected control group showed very low
mortality throughout the experiment, as only 5
group was washed the same way as the other groups.
outof60 larvae died.
Thereafter, 60 randomly chosen eggs from each
group were incubated in polystyrene multiwell dishes
2 The two groups infected with Flexibacter sp.
as previously described. Within 24 hours after hatch
showed high mortalities at hatching; out of60 lar
ing, visible remnants of the eggshell were removed
vae per group, 40 and 49 had died in the NCIMB
along with 10mL ofwater, and 10 mL ofsterile sea
13127Tand NCIMB 13128 groups, respectively. At
water was added. Mortalitywas recorded until Day 37
Day 18, these groups were terminated in order to
after hatching. The remaining living larvae were ex
gain material for re-isolation of bacteria. Eighty
amined microscopically under a dissection
and 93% of the larvae were dead in Groups
microscope for developmental disorders. A further
NCIMB 13127T and NCIMB 13128, respectively.
description of this experiment is given by Bergh and
Jelmert (1990). 3 The groups infected with Vanguillarum strains or
with V. jischeri showed an intermediate mortality
pattern. Only 1-4 larvae died per group at hatch
Results _
ing, but high mortalities occurred throughout the
rest ofthe experiment. When the experiment was
Figure 1 shows a halibut egg with severe surface dam terminated at Day 37, mortalities had risen to
age, an appearance typical for eggs infected with 95% in the V. anguillarum NCMB 6 group, 78% in
Flexibacter sp. Figure 2 demonstrates a normal egg, the V jischeri ATCC 7744 group, and 67% in the
withoutvisible damage. group infectedwith V anguillarum651.
Scanning electron microscopy of infected eggs re
vealed the chorion to be completely dissolved over In the disinfection experiment, 7 out of56 remain
large areas (up to 206 f-Lm in diameter), whereas the ing larvae in the group treated with 0.5% Buffodine
zona radiata was severely damaged. Isolation of were dead when the experiment was terminated, 9·
epibiotic bacteria from this egg group revealed an out of 59 were dead in the 0.05% Buffodine group,
epiflora totally dominated (99% of colony-forming 24 out of 59 were dead in the 0.005% Buffodine
units) byFlexibactersp. (Bergh etal. 1992) group, and 19 outof58 in the untreated group.
Berghetal.:StudiesonDiseasesofCulturedAtlanticHalibut 3
Figure 2
A normal halibut
egg. Diameter is ap
proximately 3 mm.
The photograph is
taken from a Wild
zoom binocular mi
croscope operated in
the dark field mode.
Photo by Guri
Grung and Vibeke
Valkner.
The groups that were disinfected with 0.5% and been shown that bacteria are able to cause at least
0.05% Buffodine could not be significantly distin minor destruction to the chorion (Hansen and
guished for cumulative mortality at the end of the Olafsen 1989), but we are not aware ofother reports
experiment (P>0.05, t-test with arcus sinus transfor showing that bacteria are able to dissolve the chorion
mation of proportions). The two remaining groups completely. It could not be deduced from the scan
were not statistically separable, but they both had sig ning electron micrographs that the zona radiata was
nificantly higher mortalities than the two groups that completelydestroyed; however, evidence ofsevere de
were treated with the highest Buffodine concentra struction was clear. This biotype is commonly isolated
tions (P<0.05%, t-test with arcus sinus transformation from Atlantic halibut eggs and could be considered a
ofproportions). major problem in halibutlarviculture.
With respect to developmental disorders, 5 out of With the two Vibrio species that were used in the
49 living larvae treated with 0.5% Buffodine were infection experiment, the situation is different.
found to posess at least one kind of disorder when These bacteria seemed to be harmless to the egg
the experiment was terminated in the group. In the stage, causing no significant mortality. However, the
other groups, scores were 16 out of 50 (0.05% profound mortality of the infected groups through
Buffodine), 22 out of36 (0.005% Buffodine) and 23 out the yolk-sac stage indicates that Vibrio-infections
out of 39 (control). The most common disorder was during the yolk-sac stage may help to explain the
the presence ofblack, probably necrotic, tissue in the high mortality rates experienced so far.
gill, heart, or frontal yolk-sac region. Work is in Recent observations indicate that infectious pan
progress to characterize the ultrastructural changes creatic necrosis virus (IPNV), serotype Nl, is
associated with these kinds ofdisorders. prevalent in juvenile stages of the Atlantic halibut
(Mortensen et al. 1990). The IPNV is associated with
high mortalities, but it is not verified whether the
Discussion _
virus is the principal lethal agent. The time ofinfec
tion has so far not been shown. Also, nematodes
We have presented initial results showing that bacte could be present in relatively large amounts (G.
rial infections may be closely involved in the Bristow, Zoological Laboratory, University ofBergen,
mortality ofcultured halibut eggs and yolk-sac larvae. pers. commun. 1991); these are probably introduced
The infection experiment proves thatFlexibactersp. is byfeeding the larvae with collected zooplankton.
a causative agent of mortality at the egg stage, at The disinfection experiment demonstrated that
hatching, and at the earlyyolk-sac stage. Ithas earlier the bacterial epiflora on halibut eggs is at least partly
4 NOAATechnicalReportNMFS III
responsible for many ofthe developmental disorders although four critical periods ofhigh mortality were
commonly occuring in halibut yolk-sac larvae. Sur identified for aquaculture systems at temperatures
face disinfection ofeggs with iodophors should be an between 3 and 9° C: hatching, 10-14, 25-35, and
adequate prophylactic treatment, as they have good 45-60 days after hatching. The latter was probably
pathogen/host differential of toxicity (Amend and due to starvation (I. Opstad and A.B. Skiftesvik,
Pietsch 1972; Ross and Smith 1972; Amend 1974). Austevoll Aquaculture Research Station, pers.
However, more work is needed to establish reliable commun. 1991). For the three other critical periods
disinfection procedures. of the yolk-sac stage, evidence presented here indi
Mortality rates could be augmented by several sub cates thateffects ofbacteria could not be ruled out.
lethal factors (Rosenthal and Alderdice 1976). There are not yet any data available ranking the
Sublethal physical stressors might increase egg and quantitative importance of the different causes of
larvae sensitivity to infectious microorganisms, rather death of Atlantic halibut eggs and larvae, although
than persebe the causative agent ofdeath. The effects early-life stage mortalities are still a major factor lim
of sublethal stressors to early life stages of halibut iting the commercial success of halibut aquaculture.
have been investigated in several studies. The data presented here, however, give evidence that
Jelmert and Naas (1990) reported that lowered 02 pathogenic or opportunistic microorganisms are
concentrations, exposure to H S and exposure to closely involved in some typical mortality and devel
2
high light levels led to a higher prevalence of de opmental disorder patterns of eggs and yolk-sac
formed yolk-sac larvae. larvae.
Sensitivity of halibut eggs to physical shocks was
investigated by Holmefjord and Bolla (1988), who Acknowledgments _
found that eggs were most sensitive before the clo
sure of the blastopore. In a more extensive study,
eggs ofhalibut were compared with several other ma This work has been supported by the Royal orwe
rine fishes, Opstad (I. Opstad, Austevoll Aquaculture gian Council for Scientific and Industrial Research
Research Station, pers. commun. 1991) found similar ( TNF), by the Norwegian Council for Fisheries Re
results, with the addition that eggs during the hatch search (NFFR), and by Mowi a/so We are grateful to
ing period were highly sensitive to physical stress. Guri Grung and Vibeke Valkner who kindly let us use
Effects of water flow on yolk-sac larvae were studied their photographs.
by Opstad and Bergh (1990), who concluded that
high rates ofwater exchange in upstream incubators Citations _
significantly increased mortality. Yolk-sac utilization
was inversely related to rate offlow.
Amend.D.F.
Absence of flow, however, caused rapid increase in
J974. Comparative toxicity of two iodophors to rainbow
the amount of bacteria in the incubators (Opstad
trouteggs. TransAm. Fish.Soc. 103:73-78.
and Bergh 1990; Skiftesvik et al. 1990), and subse Amend, D. F.,and]. P. Pietsch.
quent larval mortality. Thus, these two effects must 1972. Virucidal activity of two iodophors to salmonid
be carefully weighed against each other. Although viruses. J. Fish.Res. BoardCan. 29:6165.
normally not regarded as a sublethal stressor or caus Bergh,0.,andA.Jelmert.
1990. Antibacterial treatment prQcedures ofeggs of halibut
at.ive agent of diseases, extreme light regimes have
(Hippoglossus hippoglossusL.). Presented atcouncil meeting,
been shown to induce reduced yolk-sac utilization
International Council for the ExplorationoftheSea. ICES
and increased mortality of halibut yolk-sac larvae CM-1990/F:39,6p.
(Skiftesviket al. 1990). Bergh.0..G. H. Hansen,and R. E.Taxt.
Studying development and mortality of Atlantic 1992. Experimental infection ofeggs and yolk sac larvae of
halibut, Hippoglossus hippoglossus L. J. Fish Dis. 15. (In
halibut eggs and larvae at different temperatures.
press.)
Pittman et al. (1990) concluded that 3° C is near the
Hansen,G. H.,andJ. Olafsen.
lower limit for development of halibut eggs and lar 1989. Bacterial colonization of cod (Gadus morhua L.) and
vae. At this temperature, the larvae often showed halibut (Hippoglossus hippoglossus) eggs in marine
incomplete caudal development and suffered higher aquaculture. Appl. Environ. Microbiol.55(6):1435-1446.
Hansen,G. H.,0 Bergh,]. Michaelsen,andD.Knappskog.
mortality than those reared at 6° C. The groups
1992. Flexibactl'T ovulyticus sp. nov., a pathogen of eggs and
reared at 9° C had high egg mortality and quickly
larvaeofAtlantic halibut. HippoglossushippoglossusL. Int.J.
developed abnormalities, such as small hearts and
System. Bacteriol. 42(3). (In press.)
livers, and large peritoneal and pericardial spaces, Holmefjord,I.,andS.Bolla.
indicating that this temperature was sublethal. No 1988. Effectsofmechanical stresson Atlantic halibuteggsat
primary cause of larval deat.h could be identified, differenttimesafterfertilization. Aquacultl:lfe68:369-371.