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Major finfish diseases in Asia and Major finfish diseases in Asia and practical measures adopted in practical measures adopted in

controlling diseasescontrolling diseases

S Chi b tS Chi b tSupranee ChinabutSupranee ChinabutDepartment of Fisheries, Department of Fisheries,

THAILANDTHAILAND

What is disease?What is disease?

““Any abnormality of structure or functionAny abnormality of structure or function””The inability to perform physiological The inability to perform physiological function at normal levels, nutritional and function at normal levels, nutritional and environmental factors are provided at an environmental factors are provided at an adequate leveladequate levelDiseased animals are not just sick or dyingDiseased animals are not just sick or dyingDiseased animals are not just sick or dyingDiseased animals are not just sick or dyingDiseased animals are those performing Diseased animals are those performing below expectationsbelow expectations

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How disease occurred in fish?How disease occurred in fish?

Fish are poikilothermal animal which mean Fish are poikilothermal animal which mean th i b d t t h dith i b d t t h ditheir body temperature change according their body temperature change according to the water temperatureto the water temperatureTheir biological systems are tremendously Their biological systems are tremendously altered by water temperature and other altered by water temperature and other environmental factors; pH, DO etc.environmental factors; pH, DO etc.; p ,; p ,

Environmental EffectsEnvironmental EffectsTemperatureTemperatureOxygenOxygenOxygenOxygenAmmoniaAmmoniaNitrite and nitrateNitrite and nitrateCarbon dioxideCarbon dioxideChlorineChlorineS t t dS t t dSupersaturated gasSupersaturated gasSuspension solidsSuspension solidsToxic organic compoundsToxic organic compounds

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Fish Disease DiagnosisFish Disease Diagnosis

Get information from fish farmersGet information from fish farmersGet necessary water quality parameters Get necessary water quality parameters Pathogen examinationPathogen examination

ParasitesParasitesBacteriaBacteria HistopathologyHistopathologyF ngiF ngiFungiFungiVirusesViruses

Rickettsia

Pathogenic DiseasesPathogenic Diseases

Infectious disease is a disease cause by Infectious disease is a disease cause by th i i i l di itth i i i l di itpathogenic organisms including: parasites, pathogenic organisms including: parasites,

bacteria, fungus and virusbacteria, fungus and virus

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Parasitic DiseasesParasitic Diseases

A great number and diversity of animal A great number and diversity of animal i bl f iti i fi hi bl f iti i fi hspecies are capable of parasitizing fish, species are capable of parasitizing fish,

ranging from microscopic protozoan to ranging from microscopic protozoan to grossly visible crustaceans and annelidsgrossly visible crustaceans and annelidsIn the wild, there is a big range of In the wild, there is a big range of parasites but small in numbersparasites but small in numbersparasites but small in numbersparasites but small in numbersIn cultured, there is limited range of In cultured, there is limited range of parasites but larger numbersparasites but larger numbers

Oodiniasis (Velvet/Rust/GoldOodiniasis (Velvet/Rust/Gold--dust dust Disease)Disease)

The disease is cause by The disease is cause by OodiniumOodinium sp sp ((Pi i di iPi i di i )) A l di iA l di i((Piscinoodinium spPiscinoodinium sp) or ) or AmyloodiniumAmyloodinium spspDinoflagellate, spherical and pyriform Dinoflagellate, spherical and pyriform shape, 30 X 100 micronshape, 30 X 100 micronTremendous numbers of vacuoles in Tremendous numbers of vacuoles in cytoplasmcytoplasmcytoplasmcytoplasmOne nucleiOne nucleiFound on skin and gillsFound on skin and gills

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Ichthyobodiasis (CostiasisIchthyobodiasis (Costiasis))

Disease of freshwater and marine fishDisease of freshwater and marine fishCause by Cause by IchthyobodoIchthyobodo sp or sp or CostiaCostia spspTwo species found in SEA:Two species found in SEA:

I. necatrixI. necatrixI. pyriformisI. pyriformis

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Oval or kidney shape Oval or kidney shape 10 10 X X 20 20 micron with micron with i t d d di t d d d 22 fl llfl lla pointed end and possesses a pointed end and possesses 2 2 flagellaflagella

More problem for fry and fingerling and More problem for fry and fingerling and weaken the adultweaken the adult

Clinical signsClinical signs

Excess mucus production that forms a Excess mucus production that forms a blbl fil th b d f dfil th b d f dblueblue--gray film over the body surface and gray film over the body surface and gillsgillsFish do not feed in case of heavy infectionFish do not feed in case of heavy infectionMay swim in rapid, erratic movements or May swim in rapid, erratic movements or flashingflashingflashingflashingFresh skin scrapping and gill squashes Fresh skin scrapping and gill squashes can demonstrate parasites under can demonstrate parasites under microscopemicroscope

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Ichthyophthiriosis/Ich/WhitespotIchthyophthiriosis/Ich/Whitespot

Ichthyophthyrias multifiliisIchthyophthyrias multifiliis is the causative agentis the causative agentL ili t tL ili t t 11Large ciliate protozoa Large ciliate protozoa 11mmmmFeed and grow under epidermis of skin and gillsFeed and grow under epidermis of skin and gillsMature trophont leave fish and transforms into Mature trophont leave fish and transforms into reproductive cyst; tomontreproductive cyst; tomontDivisions within tomontDivisions within tomontTheronts released and infect fishTheronts released and infect fishTheronts cannot survive long away from hostTheronts cannot survive long away from host

Clinical signsClinical signs

Infected fish showed white spot on bodyInfected fish showed white spot on bodyHaemorrhagic lesionsHaemorrhagic lesions

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PreventionPrevention

Leave the pond or tank empty for a day or Leave the pond or tank empty for a day or moremorePrevent wild fish or amphibian come into Prevent wild fish or amphibian come into the pond/ quarantine newly arrive fish the pond/ quarantine newly arrive fish before stockbefore stockRaise water temperatureRaise water temperature 11--22ooC may killC may killRaise water temperature Raise water temperature 11--22 C may kill C may kill trophozoitestrophozoites

TricodinosisTricodinosis

Causative agents are Tricodinids Causative agents are Tricodinids S b ll h ith ki di k fS b ll h ith ki di k fSaucer or bell shape with sucking disk from Saucer or bell shape with sucking disk from lateral viewlateral viewDorsal view shows round shapeDorsal view shows round shapeMultiply by binary fissionMultiply by binary fissionFeed on bacteriaFeed on bacteriaInfected fish shows abnormal coloration, Infected fish shows abnormal coloration, sluggish and lost weightsluggish and lost weightFound on skin and gillsFound on skin and gills

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TetrahymeniasisTetrahymeniasis

The causative agent is The causative agent is TetrahymenaTetrahymena spspDisease of freshwater fish, guppyDisease of freshwater fish, guppyOval shape Oval shape 3030--50 50 micron with micron with 3030--40 40 rows rows of cilia through the whole cellof cilia through the whole cellCytostome at anterior part with cilia Cytostome at anterior part with cilia around itaround itaround itaround itIt has both macro and micro nucleusIt has both macro and micro nucleusReproduce by binary fissionReproduce by binary fission

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Infected fish show white patch on the bodyInfected fish show white patch on the bodyFocal necrosis and haemorrhage at Focal necrosis and haemorrhage at infected areasinfected areasScales protrudedScales protruded

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Bacterial DiseasesBacterial Diseases

Motile Aeromonad SepticaemiaMotile Aeromonad SepticaemiaStreptococosisStreptococosisVibriosisVibriosis

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Motile Aeromonad SepticaemiaMotile Aeromonad Septicaemia

Aeromonas hydrophilaAeromonas hydrophila, , A. sobriaA. sobria and and A. A. caviaecaviaecaviaecaviaeThese bacteria had been found in healthy These bacteria had been found in healthy / diseased fish and aquatic environment/ diseased fish and aquatic environmentmight serve as a water pollution indicatormight serve as a water pollution indicator

Clinical signsClinical signs

petechiae haemorrhagepetechiae haemorrhagehaemorrhagehaemorrhagehaemorrhagehaemorrhagecongestioncongestionulcerationulcerationascitic fluid in clinical dropsyascitic fluid in clinical dropsyabdominal swellingabdominal swellingggtissue necrosistissue necrosisfin / tail rotfin / tail rot

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Predisposing causesPredisposing causes

Under stressful conditionsUnder stressful conditions-- crowdingcrowding-- low water qualitylow water quality

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IsolationIsolation

grow on most general purpose mediumgrow on most general purpose mediumR.S. medium is a selective mediaR.S. medium is a selective media

Motile aeromonadsMotile aeromonads

Active motility with a single polar flagellumActive motility with a single polar flagellumproduce gas and acid from carbohydratesproduce gas and acid from carbohydratesproduce gas and acid from carbohydratesproduce gas and acid from carbohydratesgram negative, short rod gram negative, short rod 00..5 5 um x um x 11..0 0 -- 11..5 5 umumaerobic & facultative anaerobicaerobic & facultative anaerobicoxidase positiveoxidase positiveresistance to vibriostat resistance to vibriostat 00//129129

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Differentiation between the motile Differentiation between the motile AeromonasAeromonas species by biochemical species by biochemical

properties (Adapted from Popoff properties (Adapted from Popoff 19841984))Characteristics Characteristics A. hydrophila A. caviae A. sobriaA. hydrophila A. caviae A. sobria

Motility + + +Motility + + +Motility Motility Aesculin hydrolysis Aesculin hydrolysis + + + + --Growth in KCN broth + + Growth in KCN broth + + --LL--Histidine & LHistidine & L--arginine arginine + + + + --utilizationutilizationLL--Arabinose utilization Arabinose utilization + + + + --Fermentation of salicin + + Fermentation of salicin + + --Acetoin production (VP test) + Acetoin production (VP test) + -- + + Gas from glucose Gas from glucose + + -- ++HH22S from cysteine + S from cysteine + -- ++

ControlControl

Health managementHealth management

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TreatmentTreatment

varietvarietiesies of antibioticof antibioticss

StreptococcosisStreptococcosis

Gram positiveGram positiveGram positive Gram positive bacteriabacteriaCoccusCoccusNonmotileNonmotile

Streptococcus spp.

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Colony morphology of Colony morphology of StreptococcusStreptococcus

StreptococcosisStreptococcosisTThere are several other closely related here are several other closely related

f b t i th t i ilf b t i th t i ilgroups of bacteria that can cause similar groups of bacteria that can cause similar disease, including disease, including LactococcusLactococcus, , EnterococcusEnterococcus, and , and VagococcusVagococcus..Many Many species of fish have been found species of fish have been found susceptible to infection, including salmon,susceptible to infection, including salmon,susceptible to infection, including salmon, susceptible to infection, including salmon, mullet, golden shiner, pinfish, eel, trout, mullet, golden shiner, pinfish, eel, trout, tilapia, sturgeon, striped bass tilapia, sturgeon, striped bass and and variety variety of ornamental fishof ornamental fish..

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Some stressors that have been Some stressors that have been associated with Strepassociated with Streptococcosis tococcosis

outbreaksoutbreaks

high water temperatures high water temperatures high stocking densitieshigh stocking densitiesharvesting or handlingharvesting or handlingpoor water qualitypoor water quality

high ammonia or nitrite concentrations.high ammonia or nitrite concentrations.

Clinical signsClinical signsabnormal swimming behavior abnormal swimming behavior ((spinningspinning))loss of buoyancy controlloss of buoyancy controlloss of buoyancy controlloss of buoyancy controllethargylethargydarkeningdarkeninguniuni-- or bilateral exophthalmia or bilateral exophthalmia corneal opacity (whitish eyes)corneal opacity (whitish eyes)hemorrhages in or around eye, gill plate, base of hemorrhages in or around eye, gill plate, base of fi t/ l h th b dfi t/ l h th b dfins, vent/anus, or elsewhere on the bodyfins, vent/anus, or elsewhere on the bodyascites(i.e., distended abdomen/dropsy/bloating)ascites(i.e., distended abdomen/dropsy/bloating)ulcerationsulcerationsIn some cases, the fish may show no obvious In some cases, the fish may show no obvious signs before death.signs before death.

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DiagnosisDiagnosis

Bacteria isolation from kidney and brain Bacteria isolation from kidney and brain follow by identificationfollow by identificationfollow by identificationfollow by identificationHistologyHistologyClinical signsClinical signs

CultivationCultivation

TSA supplemented with TSA supplemented with 00..5 5 % glucose % glucose BHIABHIATodd Hewit broth agarTodd Hewit broth agarBlood agarBlood agarsmall colonies (small colonies (00..5 5 -- 11..0 0 mm)mm)

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TreatmentTreatment

Erythomycin is common use in the fieldErythomycin is common use in the field

VaccineVaccine

Formalin killed Formalin killed S. iniaeS. iniae cell preparation by cell preparation by immersionimmersionimmersionimmersionChallenged intraperitoneal with live Challenged intraperitoneal with live bacteriabacteria

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A

VibriosisVibriosis

The most common bacterial disease in The most common bacterial disease in marine fishesmarine fishesmarine fishesmarine fishesfrequently act as opportunistic pathogens frequently act as opportunistic pathogens causing disease in fish which are under causing disease in fish which are under stressstressproblem in seabass and grouperproblem in seabass and grouperproblem in seabass and grouperproblem in seabass and grouper

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Clinical signsClinical signs

acute without clinical signacute without clinical signdarkening of body colorationdarkening of body colorationloss of appetiteloss of appetitehaemorrhagic septicaemiahaemorrhagic septicaemiaulcerative lesion on the skinulcerative lesion on the skinexopthalmiaexopthalmia

Causative agentsCausative agents

ViVibbriorio spp.spp.V. parahaemolyticusV. parahaemolyticusV. alginolyticusV. alginolyticusV. choleraeV. cholerae

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VibrioVibrio sppspp..

TCBS is selective mediaTCBS is selective mediagrow on general purpose media withgrow on general purpose media with % NaCl% NaClgrow on general purpose media with grow on general purpose media with 2 2 % NaCl% NaClGram negativeGram negativestraight or slightly curved rodsstraight or slightly curved rodsmotile, facultative anaerobicmotile, facultative anaerobicoxidase and catalase positiveoxidase and catalase positiveppsensitive to vibriostat sensitive to vibriostat 00//129129

ControlControl

maintenance of high water qualitymaintenance of high water qualitygood husbandrygood husbandry

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Viral DiseasesViral Diseases

11. . Viral nervous necrosisViral nervous necrosis22 Koi herpesvirusKoi herpesvirus22. Koi herpesvirus. Koi herpesvirus

Viral Nervous Viral Nervous NecrosisNecrosis (VNN)(VNN)VViral encephalopathy and retinopathy (VER)iral encephalopathy and retinopathy (VER)AA di ff i l d j il fdi ff i l d j il fAAn disease affecting larvae and juveniles of many n disease affecting larvae and juveniles of many farmed marine fish in Asia farmed marine fish in Asia and and AustraliaAustraliaRReported in Australia, eported in Australia, China, Japan, China, Japan, IndonesiaIndonesia, , Philippines,Philippines, SingaporeSingapore, Taiwan and Thailand, Taiwan and ThailandIInfect more than nfect more than 30 30 marine fish species, especially at marine fish species, especially at h l l d j il d h i f ih l l d j il d h i f ithe larval and juvenile stages, and the infection the larval and juvenile stages, and the infection

usually results in high mortalityusually results in high mortality Mass mortality Mass mortality were reportedwere reported in larval sea bass in larval sea bass Lates Lates calcarifercalcarifer in a in a hatchery associated with clinical signs hatchery associated with clinical signs such as such as

b l i i b h ib l i i b h i

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Viral Nervous Viral Nervous NecrosisNecrosis

Histological investigations in moribund fish Histological investigations in moribund fish l d k d l ti i th ti dl d k d l ti i th ti drevealed marked vacuolation in the retina and revealed marked vacuolation in the retina and

brainbrain

Cytopathic effects (CPE) were observed in Cytopathic effects (CPE) were observed in SSNSSN--1 1 cells inoculated with the tissue filtrate of cells inoculated with the tissue filtrate of ff t d bff t d baffected sea bass.affected sea bass.

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ControlControl

Disinfection of fertilized eggs by ozonized Disinfection of fertilized eggs by ozonized ttwaterwater

Disinfection of water, feeds, ponds, facilities Disinfection of water, feeds, ponds, facilities and toolsand toolsFrequent taking off feces and dead fishFrequent taking off feces and dead fish

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Koi herpesvirus disease (KHVD)Koi herpesvirus disease (KHVD)

Causative agentCausative agentPathogen type: Pathogen type: DNA herpesDNA herpes--like like virusvirus

Pathogen common name: Pathogen common name: Koi Koi herpesvirusherpesvirus (KHV) ((KHV) (170 170 –– 230 230 nm) nm)

Family Family HerpesviridaeHerpesviridae

Host range: Host range: Common carp, Common carp, Koi carpKoi carp ((Cyprinus carpioCyprinus carpio))

Virions KHV as present in infected KF-1 cells.

List of countries and dates of first reports of KHV List of countries and dates of first reports of KHV (Crane (Crane et alet al., ., 20042004))

EuropeEurope AmericaAmerica AsiaAsia AfricaAfricaUK (1998)UK (1998) USAUSA Israel (1998)Israel (1998) S AfricaS AfricaUK (1998)UK (1998) USA USA

(1998)(1998)Israel (1998)Israel (1998) S. Africa S. Africa

(2003)(2003)Belgium (1999)Belgium (1999) Indonesia (Indonesia (20022002))Denmark (Denmark (20022002)) China (China (20022002))Germany (Germany (20022002)) Taipei China Taipei China

((20022002))Netherlands Netherlands ((20022002) )

Japan (Japan (20032003))

Switzerland Switzerland ((20032003))

Thailand (Thailand (20052005))

Luxemburg Luxemburg ((20032003))

SingaporeSingapore ((20062006))

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Outbreak in Japan

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JapanKasumigaura lake,

Ibaraki Prefecture

over ½ of the total carp production in Japan1,200 MT of carp loss in November 2003 (¼ of the total carp production in the lake)

DiagnosisDiagnosisVVirus isolation and confirmed by PCR of irus isolation and confirmed by PCR of the viral isolates. Even without isolation the viral isolates. Even without isolation of the virus PCR can be used to detect the of the virus PCR can be used to detect the DNA of virus in fresh, infected tissuesDNA of virus in fresh, infected tissues

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Koi fin (KFKoi fin (KF--1) cells 7 days following infection with 1) cells 7 days following infection with KHV at 20KHV at 20ooC. Large and frequent cytoplasmic C. Large and frequent cytoplasmic vacuolation is present in several cells most of which vacuolation is present in several cells most of which are involved in syncytium formation. Bar = 100 µmare involved in syncytium formation. Bar = 100 µm

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M

KHV Sensitivity TestNested PCRNested PCR

Increasing Increasing specificity : specific specificity : specific

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M

p y pp y pbinding of 2binding of 2ndnd primer primer pairpairIncreasing Increasing sensitivity: 2sensitivity: 2ndnd round round of PCR amplificationof PCR amplification

1st Step PCR 484 bp

M = 100 bp ladderNo. 1-14 = series dilution 10-1 – 10-14

(un-diluted stock 6,000 ug/ml) No. 15 = - veNo. 16 = + ve

Nested PCR 292 bpUsed to detect Used to detect organism present in organism present in a very low copy a very low copy numbernumber

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Hepatocytes with signet-ring of the nucleus.

Koi herpesvirus disease (KHVD)Koi herpesvirus disease (KHVD)

There is anecdotal evidence that koi There is anecdotal evidence that koi "heated" to at least "heated" to at least 30 30 ooC (C (86 86 ooF) may F) may be resistant to an outbreak since the virus be resistant to an outbreak since the virus appears restricted to environmental appears restricted to environmental temperatures of temperatures of 1818--26 26 ooC (C (6464..44--86 86 ooF). F). Whil thi h t "th " thWhil thi h t "th " thWhile this heat "therapy" may save the While this heat "therapy" may save the fish, it could produce subfish, it could produce sub--clinical carriers clinical carriers of the disease.of the disease.

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Epizootic Ulcerative Syndrome (EUS): Epizootic Ulcerative Syndrome (EUS): Southeast AsiaSoutheast Asia

EUSEUS l t dl t d A hA h (ERA)(ERA)

EUS

EUSEUS--related related AphanomycesAphanomyces (ERA):(ERA):PhilippinesPhilippines

Red Spot Disease (RSD): Red Spot Disease (RSD): AustraliaAustraliaMycotic Granulomatosis (MG): Mycotic Granulomatosis (MG): JapanJapan

Ulcerative Mycosis (UM):Ulcerative Mycosis (UM): East Coast East Coast USAUSAUSAUSA

Introduction• Epizootic ulcerative syndrome = EUS (FAO, 1986)

• EUS is “a seasonal epizootic condition of freshwater and estuarine warm water fish of complex infectious etiology characterized by the presence of invasive Aphanomycesinfection and necrotizing ulcerative lesions typicallyinfection and necrotizing ulcerative lesions typically leading to a granulomatous response” (ODA, 1994).

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•Epizootic ulcerative syndromeSingapore 1977*, Malaysia 1979-81, Indonesia 1980, Thailand 1982-83, Laos Cambodia and Myanmar 1984, Vietnam 1984, Philippines 1985-86, Sri Lanka 1987, South China 1987, Bangladesh February 1988, India May 1988, Nepal 1989, Bhutan 1989, Pakistan 1996

EUS distributions

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Mass mortality Mass mortality of wild and of wild and l d fi h il d fi h icultured fish in a cultured fish in a new locationnew location

Small red Small red areas or areas or ulcers on ulcers on the bodythe body

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Deep haemorrhagic ulcers with Deep haemorrhagic ulcers with fungal mycelium on surfacefungal mycelium on surface

Skull erosion, loss of eyes and part of the brain

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Fungus Species Found from EUS Infected FishesFungus Species Found from EUS Infected Fishes

Aphanomyces invadansAphanomyces invadansAphanomyces piscicidaAphanomyces piscicida

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Susceptible fish species

EUSA. invadans Exposure of

dermis

Possible causes includePossible causes include::

abrasionabrasionacidified water acidified water exposureexposureexposureexposureviral viral dermatitisdermatitisbacterial bacterial dermatitis dermatitis

H & E staining

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Glocott staining

UV-Tech staining

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Prevention and controlPrevention and control

Keep eyes on the health of the wild fish during Keep eyes on the health of the wild fish during the cool seasonthe cool seasonthe cool seasonthe cool seasonIf the wild fish show EUS clinical signs, please If the wild fish show EUS clinical signs, please notify Fisheries Officersnotify Fisheries OfficersStop water exchange if the disease occurs in the Stop water exchange if the disease occurs in the natural waternatural waterIf th EUS d t th fi h d t tIf th EUS d t th fi h d t tIf the EUS spread to the fish pond, stop water If the EUS spread to the fish pond, stop water exchange, remove the moribund and death fish.exchange, remove the moribund and death fish.Improve water quality using agriculture lime or Improve water quality using agriculture lime or hydrate limehydrate limeSalt can be applied to minimize the toxic of HSalt can be applied to minimize the toxic of H22SS

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An accurate disease diagnosis is An accurate disease diagnosis is f di i fi fi hf di i fi fi hnecessary for diseases in finfish.necessary for diseases in finfish.

www.oie.intwww.oie.int and and www.enaca.orgwww.enaca.org are are good sources of aquatic animal disease good sources of aquatic animal disease diagnosis techniques. diagnosis techniques.