Anatomy of Fish

48

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Basic Anatomy of a Typical Bony Fish

49

SECTION 2 - FINFISH DISEASES

Basic Anatomy of a Typical Bony Fish

SECTION 2 - FINFISH DISEASESF.1 GENERAL TECHNIQUESF.1.1 Gross ObservationsF.1.1.1 BehaviourF.1.1.2 Surface ObservationsF.1.1.2.1 Skin and FinsF.1.1.2.2 GillsF.1.1.2.3 BodyF.1.1.3 Internal ObservationsF.1.1.3.1 Body Cavity and MuscleF.1.1.3.2 OrgansF.1.2 Environmental ParametersF.1.3 General ProceduresF.1.3.1 Pre-Collection PreparationF.1.3.2 Background InformationF.1.3.3 Sample Collection for Health SurveillanceF.1.3.4 Sample Collection for Disease DiagnosisF.1.3.5 Live Specimen Collection for ShippingF.1.3.6 Dead or Tissue Specimen Collection for ShippingF.1.3.7 Preservation of Tissue SamplesF.1.3.8 Shipping Preserved SamplesF.1.4 Record-KeepingF.1.4.1 Gross ObservationsF.1.4.2 Environmental ObservationsF.1.4.3 Stocking RecordsF.1.5 References

VIRAL DISEASES OF FINFISHF.2 Epizootic Haematopoietic Necrosis (EHN)F.3 Infectious Haematopoietic Necrosis (IHN)F.4 Oncorhynchus masou Virus (OMV)F.5 Infectious Pancreatic Necrosis (IPN)F.6 Viral Encephalopathy and Retinopathy (VER)F.7 Spring Viraemia of Carp (SVC)F.8 Viral Haemorrhagic Septicaemia (VHS)F.9 Lymphocystis

BACTERIAL DISEASE OF FINFISHF.10 Bacterial Kidney Disease (BKD)

FUNGUS ASSOCIATED DISEASE FINFISHF.11 Epizootic Ulcerative Syndrome (EUS)

ANNEXESF.AI OIE Reference Laboratories for Finfish DiseasesF.AII List of Regional Resource Experts for Finfish

Diseases in Asia-PacificF.AIII List of Useful Diagnostic Manuals/Guides to

Finfish Diseases in Asia-Pacific

50505050505152525252535353545454555556565757575757

48

5962656872767982

86

90

9598

105

50

infectious disease agent and should besampled immediately. Affected animals shouldbe kept (isolated) as far away as possible fromunaffected animals until the cause of the mor-talities can be established.

F.1.1.2 Surface Observations (Level I)

Generally speaking, no surface observations canbe linked to a single disease problem, however,quick detection of any of the following clinicalsigns, plus follow-up action (e.g., removal or iso-lation from healthy fish, submission of samplesfor laboratory examination), can significantly re-duce potential losses.

F.1.1.2.1 Skin and Fins (Level I)

Damage to the skin and fins can be the conse-quence of an infectious disease (e.g., carperythrodermatitis). However, pre-existing lesionsdue to mechanical damage from contact withrough surfaces, such as concrete raceways, orpredator attack (e.g., birds, seals, etc., or chemi-cal trauma) can also provide an opportunity forprimary pathogens or secondary pathogens (e.g.,motile aeromonads) to invade and establish. Thisfurther compromises the health of the fish.

Common skin changes associated with dis-ease, which should encourage further actioninclude red spots (Fig. F.1.1.2.1a), which maybe pin-point size (petechiae) or larger patches.These tend to occur around the fins, opercu-lum, vent and caudal area of the tail, but maysometimes be distributed over the entire sur-face. Indications of deeper haemorrhaging orosmotic imbalance problem saredarkenedcolouration. Haemorrhagic lesions may precedeskin erosion, which seriously affect osmoregu-lation and defense against secondary infec-tions. Erosion is commonly found on the dorsalsurfaces (head and back) and may be causedby disease, sunburn or mechanical damage. Insome species, surface irritation may be indi-cated by a build up of mucous or scale loss.

Surface parasites, such as copepods, ciliates orflatworms, should also be noted. As with the gills,these may not be a problem under most circum-stances, however, if they proliferate to noticeablyhigher than normal numbers (Fig. F.1.1.2.1b),this may lead to secondary infections or indi-cate an underlying disease (or other stress)problem. The parasites may be attached su-perficially or be larval stages encysted in thefins, or skin. Such encysted larvae (e.g., flat-worm digenean metacercariae) may be de-tected as white or black spots (Fig. F.1.1.2.1c)

F.1 GENERAL TECHNIQUES

General fish health advice and other valuableinformation are available from the OIE Refer-ence Laboratories, Regional Resource Ex-perts in the Asia-Pacific, FAO and NACA. Alist is provided in Annexes F.AI and AII, andup-to-date contact information may be ob-tained from the NACA Secretariat in Bangkok(e-mail: [email protected]). Other useful guidesto diagnostic procedures which provide valu-able references for regional parasites, pestsand diseases are listed in Annex F.AIII.

F.1.1 Gross Observations

F.1.1.1 Behaviour (Level I)

At a time when there are no problems on the farm,normal behaviour of the animals should be ob-served to establish and describe the normalsituation. Any change from normal behaviourshould be a cause for concern and warrants in-vestigation. Prior to the clinical expression of dis-ease signs, individual finfish may exhibit in-creased feed consumption followed by cessa-tion of feeding, or the fish may simply go off feedalone. Taking note of normal feed conversionratios, length/weight ratios or other body-shapesigns described below, is essential in order todetect impending disease.

Abnormal behaviour includes fish swimming nearthe surface, sinking to the bottom, loss of bal-ance, flashing, cork-screwing or air gulping (nonair-breathers) or any sign which deviates fromnormal behaviour. Bursts of abnormal activity areoften associated with a generalised lethargy.Behavioural changes often occur when a fish isunder stress. Oxygen deprivation leads to gulp-ing, listlessness, belly-up or rolling motion. Thiscan be due to blood or gill impairment. Flashingcan indicate surface irritation, e.g., superficialsecondary infections of surface lesions. Cork-screw and other bizarre behaviour may also in-dicate neurological problems that may be diseaserelated (see F.6 - Viral Encephalopathy and Re-tinopathy).

Patterns of mortalities should be closely moni-tored, as well as levels of mortality. If losses per-sist or increase, samples should be sent for labo-ratory analysis (Level II and/or III). Mortalities thatseem to have a uniform or random distributionshould be examined immediately and environ-mental factors during, pre- and post-mortalityrecorded. Mortalities that spread from one areato another may suggest the presence of an

51

F.1 General Techniques

in the skin (or deeper muscle tissue).

Abnormal growths are associated withtumourous diseases, which can be caused bydisease, such as Oncorhynchus masou virus(see F.4 - Oncorhynchus masou Virus Disease)and Lymphocystis (see F.9 - Lymphocystis), orother environmental problems.

The eyes should also be observed closely fordisease indications. Shape, colour, cloudiness,gas bubbles and small haemorrhagic lesions (redspots) can all indicate emerging or actual dis-ease problems. For example, eye enlargementand distension, known as Popeye, is associ-ated with several diseases (Fig. F.1.1.2.1d).

F.1.1.2.2 Gills (Level I)

The most readily observable change to soft tis-sues is paleness and erosion of the gills(Fig.F.1.1.2.2a). This is often associated withdisease and should be of major concern. Redspots may also be indicative of haemorrhagicproblems, which reduce the critical functioningability of the gills. Fouling, mucous build-up orparasites (ciliate protistans, monogeneans,copepods, fungi, etc.) may also reduce func-tional surface area and may be indicative ofother health problems (Fig.F.1.1.2.2b).Thesemay affect the fish directly or render it moresusceptible to secondary infections.

(MG Bondad-Reantaso)

Fig.F.1.1.2.1a. Red spot disease of grass carp.

(JR Arthur)

(K Ogawa)

Fig.F.1.1.2.1c. Ayu, Plecoglossus altivelis, in-fected with Posthodiplostomum cuticola (?)metacercariae appearing as black spots onskin.

(R Chong)

Fig. F.1.1.2.1d. Typical ulcerative, popeye, finand tail rot caused by Vibrio spp.

(SE McGladdery)


Fig.F.1.1.2.2b. Fish gills infected with mono-genean parasites.

Fig.F.1.1.2.2a.Example of gillerosion on Atlan-tic salmon, Salmosalar, due to in-tense infestationby the copepodparasite Salmin -cola salmoneus.

Fig.F.1.1.2.1b. Surface parasites, Lerneaecyprinacea infection of giant gouramy.

>

52

F.1.1.2.3 Body (Level I)

Any deviation from normal body shape in a fishis a sign of a health problem. Common changesinclude pinhead which usually affects young fishindicating developmental problems; lateral ordorso-vental bends in the spine (i.e., lordosis andscoliosis) can reveal nutritional or environmen-tal water quality problems. Another common, andeasily detected, change in body shape isdropsy. Dropsy is a distention of the abdomen,giving the fish a pot belly appearance. This is astrong indicator of disease problems which mayinclude swelling of internal organs (liver, spleenor kidney), build up of body fluids (clear =oedema; bloody fluids = ascites), parasite prob-lems, or other unknown cause. Dropsy is a com-mon element in many of the serious diseaseslisted in the Asia Diagnostic Guide since it is com-monly associated with systemic disruption of os-moregulation due to blood-cell or kidney dam-age.

F.1.1.3 Internal Observations (Level I)

As a follow up to behavioural changes, samplesof sick fish should be examined and cut openalong the ventral surface (throat to anus). Thiswill allow gross observation of the internal organsand body cavity. A healthy-appearing fish shouldalso be opened up the same way, if the personhas little experience with the normal internal work-ings of the fish they are examining. Organ ar-rangement and appearance can vary betweenspecies.

Normal tissues should have no evidence of freefluid in the body cavity, firm musculature, cream-white fat deposits (where present) around thepyloric caecae, intestine and stomach, a deepred kidney lying flat along the top of the bodycavity (between the spinal cord and swim-blad-der), a red liver, a deep red spleen and pancreas.The stomach and intestine may contain food.Gonadal development will vary depending onseason. The heart (behind the gill chamber andwalled off from the body cavity) and associatedbulbous arteriosus should be distinct and shiny.

F.1.1.3.1 Body Cavity and Muscle(Level I/II)

Clues to disease in a body cavity most commonlyconsist of haemorrhaging and a build up of bloodyfluids. Blood spots in the muscle of the body cav-ity wall, may also be present. Body cavity wallswhich disintegrate during dissection may indicatea fish that has been dead for a while and whichis, therefore, of little use for accurate diagnosis,

due to rapid invasion of secondary saprobionts(i.e., microbes that live on dead and decayingtissues).

Necrotic musculature may also indicate amuscle infection, e.g., by myxosporean para-sites. This can be rapidly investigated bysquashing a piece of the affected muscle be-tween two glass slides or between a Petri dishlid and base, and examining it under a com-pound or dissection microscope. If spore-likeinclusions are present, a parasite problem canbe reasonably suspected. Some microsporidianand myxosporean parasites can form cysts inthe muscle (Fig.F.1.1.3.1a), peritoneal tissues(the membranous network which hold the or-gans in place in the body cavity), and organsthat easily visible to the naked eye as clumpsor masses of white spheres. These too, requireparasitology identification. Worms may also bepresent, coiled up in and around the organs andperitoneal tissues. None of these parasites(though unsightly) are usually a disease-prob-lem, except where present in massive numberswhich compress or displace the organs(Fig.F.1.1.3.1b).

F.1.1.3.2 Organs (Levels I-III)

Any white-grey patches present in the liver, kid-ney, spleen or pancreas, suggest a disease prob-lem, since these normally represent patches ofnecrosis or other tissue damage. In organs suchas kidney and spleen, this can indicate disrup-tion of blood cell production. Kidney lesions canalso directly affect osmoregulation and liver le-sions can affect toxin and microbial defensemechanisms. Swelling of any of these organs toabove normal size is equally indicative of a dis-ease problem which should be identified, as soonas possible.

Swollen intestines (Fig.F.1.1.3.2a andFig.F.1.1.3.2b) should be checked to see if thisis due to food or a build up of mucous. The latteris indicative of feed and waste disposal disrup-tion, as well as intestinal irritation, and is com-monly found in association with several seriousdiseases. This may also occur due to opportu-nistic invasion of bowels that have been irritatedby rapid changes in feed, e.g., by the flagellateprotistan Hexamita salmonis. Mucous filled intes-tines can be spotted externally via the presenceof trailing, flocculent or mucous faeces (casts).


53

(H Yokoyama)

(K Ogawa)

Fig.F.1.1.3.1a. Myxobolus artus infection in theskeletal muscle of 0+ carp.

(K Ogawa)

Fig.F.1.3.1b. Ligula sp. (Cestoda) larvae infec-tion in the body cavity of Japanese yellow goby,Acanthogobius flavimanus.

(H Yokoyama)

Fig.F.1.3.2a. Distended abdomen of goldfish.



Fig.F.1.3.2b. Japanese Yamame salmon(Onchorynchus masou) fingerlings showingswollen belly due to yeast infection.

F.1.2 Environmental Parameters(Level I)

Water quality and fluctuating environmentalconditions, although not of contagious concern,can have a significant effect on finfish health,both directly (within the ranges of physiologi-cal tolerances) and indirectly (enhancing sus-ceptibility to infections). This is especiallyimportant for species grown in conditions thatbear little resemblance to the wild situation. Wa-ter temperature, salinity, turbidity, fouling and

plankton blooms are all important factors. Highstocking rates, common in intensive aquacul-ture, predispose individuals to stress as well asminor changes in environmental conditions thatcan precipitate disease. Accumulation of wastefeed indicates either overfeeding or a decreasein feeding activity. In either situation, the break-down products can have a direct toxic effector act as a medium for microbial proliferationand secondary infections. Likewise, other pol-lutants can also have a significant effect on fishhealth.

F.1.3 General Procedures

F.1.3.1 Pre-Collection Preparation(Level I)

Wherever possible, the number of specimensrequired for laboratory examination should beconfirmed before the samples are collected.Larger numbers are generally required forscreening purposes than for diagnosis of mor-talities, or other abnormalities. The diagnosticlaboratory which will be receiving the sampleshould also be consulted to ascertain the bestmethod of transportation (e.g., on ice, preservedin fixative, whole or tissue samples). The labo-ratory will also indicate if both clinically affected,as well as apparently healthy individuals, arerequired for comparative purposes.

Inform the laboratory of exactly what is goingto be sent (i.e., numbers, size-classes or tis-sues and intended date of collection and deliv-ery) so the laboratory can be prepared prior tosample arrival. Such preparation can speed upprocessing of a sample (fixative preparation,labeling of slides, jars, cassettes, test-tubes,Petri-plates, data-sheets, etc.) by as much asa day.

54

Table F.1.3.31 . Sample sizes needed to detect at least one infected host in a population of a givensize, at a given prevalence of infection. Assumptions of 2% and 5% prevalences are most com-monly used for surveillance of presumed exotic pathogens, with a 95% confidence limit.

F.1.3.4 Sample Collection for Disease Diagnosis (Level I)

All samples submitted for disease diagnosis should include as much supporting information aspossible including: reason(s) for submitting the sample (mortalities, abnormal growth, etc.) handling activities (net/cage de-fouling, size sorting/grading, site changes, predators, new spe-

cies/stock introduction, etc.)

1 Ossiander, F.J. and G. Wedermeyer. 1973. Journal Fisheries Research Board of Canada 30:1383-1384.

F.1.3.2 Background Information (Level I)

All samples submitted for diagnosis should include as much supporting information as possibleincluding: reason(s) for submitting the sample (i.e. health screening, certification) gross observations,feed records,and envronmental parameters history and origin of the fish population date of transfer and source location(s) if the stock does

not originate from on-site.

These information will help clarify whether handling stress, change of environment or infectiousagents are causes for concern. It will also help speed up diagnosis, risk assessment, and hus-bandry management and treatment recommendations.

F.1.3.3 Sample Collection for Health Surveillance

The most important factors associated with collection of specimens for surveillance are: sample numbers that are high enough (see Table F.1.3.3 below) susceptible species are sampled sampling includes age-groups and seasons that are most likely to manifest detectable infec-

tions.Such information is given under the specific disease sections.


Prevalence (%)

Population Size 0.5 1.0 2.0 3.0 4.0 5.0 10.0

50 46 46 46 37 37 29 20

100 93 93 76 61 50 43 23

250 192 156 110 75 62 49 25

500 314 223 127 88 67 54 26

1000 448 256 136 92 69 55 27

2500 512 279 142 95 71 56 27

5000 562 288 145 96 71 57 27

10000 579 292 146 96 72 29 27

100000 594 296 147 97 72 57 27

1000000 596 297 147 97 72 57 27

>1000000 600 300 150 100 75 60 30

55

(Clearly indicate the name and telephone num-ber of the person responsible for picking up thepackage, or receiving it at the laboratory).

Where possible, ship early in the week to avoiddelivery during the weekend which may lead toimproper storage and loss of samples.

Inform the contact person(s) as soon as the ship-ment has been sent and provide the name of thecarrier, flight number, waybill number and esti-mated time of arrival, as appropriate.

F.1.3.6 Dead or Tissue Specimen Collec-tion for Shipping (Level I)

In some cases, samples may be unable to bedelivered live to a diagnostic laboratory due todistance or slow transport connections. In suchcases, diagnostic requirements should be dis-cussed with laboratory personnel prior to samplecollection. Shipping of non-preserved tissuesor dead specimens may require precautions toprevent contamination or decay. In addition,precautions should be taken to protect ecto-parasites, if these are of probable significance.

For bacteriology, mycology or virology:

Small fish may be bagged, sealed and trans-ported whole on ice/frozen gel-packs.

For larger fish, the viscera can be asepticallyremoved, placed in sterile containers andshipped on ice/frozen gel-packs.

For bacteriology or mycology examinations ship fish individually bagged and sealed,on ice/frozen gel-packs.

For virology examination - bag fish with fivevolumes of Hanks basal salt solution contain-ing either gentamycin (1,000 mg/ml) or peni-cillin (800 IU/ml) + dihydrostreptamycin (800mg/ml). Anti-fungal agents such as Mycostatinor Fungizone may also be incorporated at alevel of 400 IU/ml.

Note: Intact or live specimens are ideally bestsince dissected tissues rapidly start autolysiseven under ice, making them useless for steriletechnique and bacteriology, particularly for tropi-cal climates. Fish destined for bacteriologicalexamination can be kept on ice for a limited pe-riod. The icing should be done to ensure that theorgans/tissues destined for examination usingsterile technique are kept at temperatures belowambient water (down to 4C is a standard low)but not freezing. Individual bagging is also rec-ommended in order to prevent contamination by


2 Further details are available in Recommendations for euthanasia of experimental animals Laboratory Animals 31:1-32 (1997).

environmental changes (rapid water qualitychanges, such as turbidity fluxes, saltwaterincursion into freshwater ponds, unusualweather events, etc.).

These information will help clarify whether han-dling stress, change of environment or infec-tious agents may be a factor in the observedabnormalities/mortalities. Such information isnecessary for both rapid and accurate diagno-sis, since it helps focus the investigative pro-cedures required.

F.1.3.5 Live Specimen Collection for Ship-ping (Level I)

Collection should take place as close to ship-ping time as possible, to reduce mortalitiesduring transportation. This is especially impor-tant for moribund or diseased fish.

The laboratory should be informed of the esti-mated time of arrival of the sample, in order toensure that the laboratory has the materials re-quired for processing prepared before the fisharrive. This shortens the time between removalof the fish from water and preparation of thespecimens for examination (see F.1.3.1).

The fish should be packed in double plasticbags, filled with water to one third of theircapacity with the remaining 2/3 volume inflatedwith air/oxygen. The bags should be tightlysealed (rubber bands or tape) and packed in-side a styrofoam box or cardboard box linedwith styrofoam. A plastic bag measuring 60 x180 cm is suitable for a maximum of four 200-300 g fish. The volume of water to fish volume/biomass is particularly important for live fishbeing shipped for ectoparasite examination, soadvance checking with the diagnostic labora-tory is recommended. The box should be sealedsecurely to prevent spillage and may be doublepacked inside a cardboard carton. The labora-tory should be consulted about the packagingrequired.

Containers should be clearly labeled as follows:

LIVE SPECIMENS, STORE AT ___ to ___C,DO NOT FREEZE(Insert temperature tolerance range of fish beingshipped)

If being shipped by air also indicate

HOLD AT AIRPORT AND CALL FOR PICK-UP

56

The most suitable fixative for preservation offinfish samples for histopathology is Phos-phate Buffered Formalin.

Phosphate Buffered Formalin

37-40% formaldehyde 100.0 ml Tap water 900.0 ml NaH2PO4.H20 4.0 g Na2HPO4 6.5 g

Note: Formaldehyde is a gas soluble in waterand is supplied in a concentrated form of 40%by weight. In concentrated solution, formalde-hyde often becomes turbid during storage dueto the production of formaldehyde, thus warm-ing the solution or adding a small amount ofNaOH will aid depolymerization of the paraform-aldehyde. Formaldehyde is not suitable for fixa-tion in its concentrated form. All formaldehyderegardless of purity, will be acid when pur-chased (usually within the pH range of 3-5). Careshould be taken to check the final pH of anyformalin-based fixative.

F.1.3.8 Shipping Preserved Samples(Level I)

Samples should be transported in sealed, un-breakable, containers. It is usual to double packsamples (i.e. an unbreakable container within asecond unbreakable or well-padded container).Many postal services and transport companies(especially air couriers) have strict regulationsregarding shipping chemicals, including pre-served samples. If the tissues have been ad-equately fixed (as described in F.1.3.7), mostfixative or storage solution can be drained fromthe sample for shipping purposes. As long assufficient solution is left to keep the tissues fromdrying out, this will minimise the quantity ofchemical solution being shipped. The carriershould be consulted before samples are col-lected to ensure they are processed and packedaccording to shipping rules.

Containers should be clearly labeled with theinformation described for live specimens(F.1.3.5).

The name and telephone number of the per-son responsible for picking up the package,or receiving it at the laboratory, should beclearly indicated.

Where possible, ship early in the week to avoiddelivery at the weekend, which may lead toimproper storage and loss of samples.

Inform the contact person as soon as the ship-ment has been sent and provide the name ofthe carrier, flight number, waybill number and


one individual within a sample.

F.1.3.7 Preservation (Fixation) of TissueSamples (Level I)

Fish should be killed prior to fixation. With smallfish, this can be done by decapitation, how-ever, this causes mechanical damage to the tis-sues and is unsuitable for larger fish. Alterna-tively, euthanasia with an overdose of anaes-thetic is a better (unless examination is for ec-toparasites, which may be lost) option. Ben-zocaine or Etomidate, administered at triple therecommended dose is usually effective foranaesthesizing fish. Injection of anaestheticshould be avoided, wherever possible, due tohandling induced tissue trauma2 . Putting fishin iced water is also recommended prior to kill-ing of fish.

Very small fish, such as fry or alevins, shouldbe immersed directly in a minimum of 10:1(fixative:tissue) volume ratio.

For large fish (>6 cm), the full length of the bodycavity should be slit open (normally along themid-ventral line) and the viscera and swim blad-der gently displaced to permit incision of eachmajor organ, at least once, to allow maximumpenetration of the fixative. Ideally, the organ, orany lesions under investigation, should be re-moved, cut into blocks (

57

salinity turbidity (qualitative evaluation or Secchi disc) algal blooms human activity (handling, neighbouring land

use/water activities) pH

The frequency of these observations will vary withsite and fish species. Where salinity or turbidityrarely vary, records may only be required duringrainy seasons or exceptional weather condi-tions. Temperate climates may require more fre-quent water temperature monitoring than tropicclimates. Human activity(ies) should also be re-corded on an as it happens basis, since theremay be time-lag effects. In all cases, date andtime should be recorded, as parameters suchas temperature and pH can vary markedly dur-ing the day, particularly in open ponds and in-ter-tidal sites.

It may not always be possible to monitor oxygenlevels in the pond. However, the farmer shouldbe aware that in open non-aerated ponds, oxy-gen levels are lowest in the early morning whenplants (including algae) have used oxygen over-night. Photosynthesis and associated oxygenproduction will only commence after sunrise.

F.1.4.3 Stocking Records (Level I)

All movements of fish into and out of a hatch-ery or site should be recorded, including: the source of the broodstock/eggs/larvae/ju-

veniles and their health certification the volume or number of fish condition on arrival date and time of delivery and name of person

responsible for receiving the fish date, time and destination of stock shipped-

out from a hatchery or site.

Such records are also applicable (but less criti-cal) to movements between tanks, ponds, cageswithin a site. Where possible, animals from dif-ferent sources should not be mixed. If mixing isunavoidable, keep strict records of which sourcesare mixed and dates of new introductions intothe holding site or system.

F.1.5 References

Chinabut, S. and R.J. Roberts. 1999. Pathologyand histopathology of epizootic ulcerative syn-drome (EUS). Aquatic Animal Health ResearchInstitute. Department of Fisheries, Royal ThaiGovernment. Bangkok, Thailand. 33p.

estimated time of arrival, as appropriate.

F.1.4 Record-Keeping (Level I)

It is critical to establish, and record, normalbehaviour and appearance to compare with ob-servations made during disease events. Record-keeping is, therefore, an essential component ofeffective disease management. For fish, manyof the factors that should be recorded on a regu-lar basis are outlined in sections F.1.4.1, F.1.4.2and F.1.4.3.

F.1.4.1 Gross Observations (Level I)

These can be included in routine records of fishgrowth that, ideally would be monitored on aregular basis, either by sub-sampling from tanksor ponds, or by estimates made from surfaceobservations.

For hatcheries, critical information that should berecorded include: feeding activity growth mortalities

These observations should be recorded daily, forall stages, including date, time, tank #, broodstock(where there are more than one) and food source.Dates and times of tank and water changes, pipeflushing/back-flushing and/or disinfection,should also be recorded. Ideally, these recordsshould be checked (signed off) regularly by theperson responsible for maintaining the facility.

For pond or net/cage sites, observations whichneed to be recorded include: growth fouling mortalities

These should be recorded with date, site loca-tion and any relevant activities (e.g., sample col-lection for laboratory examination). As elsewhere,these records should be checked regularly bythe person responsible for the facility.

F.1.4.2 Environmental Observations(Level I)

Environmental observations are most applicableto open water, ponds, cage and net culture sys-tems. Information that should be recorded in-clude: weather water temperature oxygen


58

Close, B., K. Banister, V. Baumans, E. Bernoth,N. Bromage, J. Bunyan, W. Erhardt, P.Flecknell, N. Gregory, H. Hackbarth, D.Morton and C. Warwick. 1997. Recommen-dations for euthanasia of experimental ani-mals: Part 2. Lab. Anim.31:1-32.

Ossiander, F.J. and G. Wedermeyer. 1973.Computer program for sample size requiredto determine disease incidence in fishpopulations. J. Fish. Res. Bd. Can. 30: 1383-1384.

Tonguthai, K., S. Chinabut, T. Somsiri, P.Chanratchakool, and S. Kanchanakhan.1999. Diagnostic Procedures for Finfish Dis-eases. Aquatic Animal Health Research In-stitute, Department of Fisheries, Bangkok,Thailand.


59

F.2.1 Background Information

F.2.1.1 Causative Agent

Epizootic Haematopoietic Necrosis (EHN) iscaused by a double-stranded DNA, non-envel-oped Iridovirus known as EpizooticHaematopoeitic Necrosis Virus (EHNV). Thisvirus shares at least one antigen with iridovirusesinfecting sheatfish (Silurus glanis) and the cat-fish (Ictalurus melas) in Europe and with amphib-ian iridoviruses from North America (frog virus3) and Australia (Bohle iridovirus). Recently, theOIE included the two agents, European catfishvirus and European sheatfish virus, as causativeagents of EHN (OIE 2000a; http://www.oie.int).Current classification in the genus Ranavirus isunder review (see http://www.ncbi.nlm.nih.gov/ICTV). More detailed information about the dis-ease can be found in the OIE Diagnostic Manualfor Aquatic Animal Diseases (OIE 2000a).

F.2.1.2 Host Range

EHNV infects redfin perch (Perca fluviatilis) andrainbow trout (Oncorhynchus mykiss). Other fishspecies found to be susceptible to EHNV afterbath exposure are Macquarie perch (Macquariaaustralasica), mosquito fish (Gambussia affinis),silver perch (Bidyanus bidyanus) and mountaingalaxias (Galaxias olidus).

F.2.1.3 Geographic Distribution

Historically, the geographic range of EHNVinfections has been restricted to mainlandAustralia. However, a recent OIE decision toinclude sheatfish and catfish iridoviruses ascauses of EHN, increased the geographicdistribution to include Europe. A related virusrecently isolated from pike-perch in Finland, wasfound to be immunologically cross-reactive butnon-pathogenic to rainbow trout.

F.2.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System(1999-2000)

Australia reported the occurrence of EHN inVictoria (last year 1996), New South Wales (lastyear 1996) and South Australia (1992). It wasalso known to have occurred in New South Walesduring first quarter of 2000, with annualoccurrence in the Australian Capital Territory(without laboratory confirmation) (OIE 1999,2000b).

India reported EHN during last quarter of 1999affecting murrels and catfishes (OIE 1999).

F.2.2 Clinical Aspects

There are no specific clinical signs associatedwith EHN. Mortalities are characterised bynecrosis of liver (with or without white spots),spleen, haematopoietic tissue of the kidney andother tissues. Disruption of blood function leadsto osmotic imbalance, haemorrhagic lesions,build up of body fluids in the body cavity. Thebody cavity fluids (ascites) plus enlarged spleenand kidney may cause abdominal distension(dropsy).

Clinical disease appears to be associated withpoor water quality, as well as water temperature.In rainbow trout, disease occurs at temperaturesfrom 11 to 17C (in nature) and 8 - 21C(experimental conditions). No disease is foundin redfin perch at temperatures below 12Cunder natural conditions. Both juvenile and adultredfin perch can be affected, but juvenilesappear more susceptible (Fig.F.2.2a). EHNV hasbeen detected in rainbow trout ranging from fryto market size, although mortality occurs mostfrequently in 0+ - 125 mm fork-length fish.

F.2.3 Screening Methods

More detailed information on methods forscreening EHN can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000a), at http://www.oie.int, or selectedreferences.

As with other disease agents, screening for thepresence of an infectious agent in a sub-clinicalpopulation requires larger sample numbers thanfor a disease diagnosis. Numbers will varyaccording to the confidence level required (seeF.1.3.3).

F.2.3.1 Presumptive

F.2.3.1.1 Gross Observations (Level 1)and Histopathology (Level II)

It is not possible to detect infections in sub-clinicalfish, using gross observations (Level I) orhistopathology (Level II).

F.2.3.1.2 Virology (Level III)

EHNV can be isolated on Bluegill Fin 2 (BF-2) orFathead Minnow (FHM) cell lines. This requiressurveillance of large numbers (see Table F.1.3.3)

VIRAL DISEASES OF FINFISHESF.2 EPIZOOTIC HAEMATOPOIETIC

NECROSIS (EHN)

60

of sub-clinical fish to detect low percentagecarriers.

F.2.3.2 Confirmatory

F.2.3.2.1 Immunoassays (Level III)

Suspect cytopathic effects (CPE) in BF-2 or FHMcell-lines require confirmation of EHNV as thecause through immunoassay (indirect fluorescentantibody test (IFAT) or enzyme linkedimmunosorbent assay (ELISA) or PolymeraseChain Reaction (PCR) (Level III).

F.2.4 Diagnostic Methods

More detailed information on methods fordiagnosis of EHN can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000a), at http://www.oie.int, or selectedreferences.

EHNV is a highly resistant virus that canwithstand freezing for prolonged periods, thus,fish may be stored and or/transported frozenwithout affecting the diagnosis.

F.2.4.1 Presumptive

F.2.4.1.1 Gross Observation (Level I)

As described under F.2.2, mass mortalities ofsmall redfin perch under cool water conditions(< 11 C), which include cessation of feeding,abdominal distension, focal gill and finhaemorrhage, as well as overall skin darkening,should be considered suspect for EHNV infec-tion. Similar observations in rainbow trout finger-lings (11-17 C) may also be considered sus-pect, but the conditions are not specific to EHNin either host.

Necropsy may reveal liver and spleenenlargement or focal pale spots on the liver, butthese, again, are non-specific.

F.2.4.1.2 Histopathology (Level II)

Histopathology in haematopoietic kidney, liver,spleen and heart tissues are similar in bothinfected redfin perch and rainbow trout, althoughperch livers tend to have larger focal or locallyextensive areas of necrosis. Gills of infectedperch show focal blood clots, haemorrhage andfibrinous exudate. Focal necrosis occurs in thepancreas and intestinal wall. In the former tissuesite necrosis can become extensive.


Whole alevin or juvenile perch (

61

F.2.6 Control Measures

Prevention of movement of infected fishbetween watersheds, and minimising contactbetween trout farms and surrounding perchpopulations is recommended. In addition,reducing bird activity at farm sites may beeffective in reducing the chances of exposureand spread. Precautionary advice andinformation for recreational fishermen usinginfected and uninfected areas may also reduceinadvertent spread of EHN.

F.2.7 Selected References

Gould, A.R., A.D. Hyatt, S.H. Hengstberger, R.J.Whittington, and B.E.H. Couper. 1995. A poly-merase chain reaction (PCR) to detect epi-zootic necrosis virus and Bohle iridovirus. Dis.Aquat. Org. 22: 211-215.

Hyatt, A.D., B.T. Eaton, S. Hengstberger,G.Russel. 1991. Epizootic haematopoieticnecrosis virus: detection by ELISA, immuno-histochemistry and electron microscopy. J.Fish Dis.14: 605-618.

OIE. 1999. Regional Aquatic Animal DiseaseYearbook 1999 (Asian and Pacific Region).OIE Representation for Asia and the Pacific.Tokyo, Japan. 35p.

OIE. 2000a. Diagnostic Manual for Aquatic Ani-mal Diseases, Third Edition, 2000. Office In-ternational des Epizooties, Paris, France.237p.

OIE. 2000b. Regional Aquatic Animal DiseaseYearbook 1999 (Asian and Pacific Region).OIE Representation for Asia and the Pacific.Tokyo, Japan. 40p.

Whittington, R.J. and K.A. Steiner. 1993. Epi-zootic haematopoietic necrosis virus (EHNV):improved ELISA for detection in fish tissuesand cell cultures and an efficient method forrelease of antigen from tissues. J. Vir.Meth.43: 205-220.

Whittington, R.J., L.A. Reddacliff, I. Marsh, C.Kearns, Z. Zupanovic, Z. and R.B.Callinan.1999. Further observations on theepidemiology and spread of epizootichaematopoietic necrosis virus (EHNV) infarmed rainbow trout Oncorhynchus mykiss insoutheastern Australia and a recommendedsampling strategy for surveillance. Dis.Aquat. Org. 35: 125-130.F.3

F.2 Epizootic HaematopoieticNecrosis (EHN)

(AAHL)

Fig.F.2.2a. Mass mortality of single species ofredfin perch. Note the small size of fish affectedand swollen stomach of the individual to thecentre of the photograph. Note thecharacteristic haemorrhagic gills in the fish onthe left in the inset.

(EAFP)

Fig.F.3.2a. IHN infected fry showing yolk sachaemorrhages.

(EAFP)

Fig.F.3.2b. Clinical signs of IHN infected fishinclude darkening of skin, haemorrhages on theabdomen and in the eye around the pupil.

62

F.3 INFECTIOUS HAEMATOPOIETICNECROSIS (IHN)



Infectious Haematopoietic Necrosis (IHN) iscaused by an enveloped single stranded RNA(ssRNA) Rhabdovirus, known as InfectiousHaematopoietic Necrosis Virus (IHNV). It is cur-rently unassigned to genus, but the InternationalCommittee on Taxonomy of Viruses (ICTV) iscurrently reviewing a new genus Novirhabdovi-rus which is proposed to include VHSV andIHNV (see http://www.ncbi.nlm.nih.gov/ICTV).More detailed information about the disease canbe found in the OIE Diagnostic Manual for AquaticAnimal Diseases (OIE 2000a).

F.3.1.2 Host Range

IHNV infects rainbow or steelhead trout(Oncorhynchus mykiss), sockeye salmon(O. nerka), chinook (O. tshawytscha), chum(O.keta), yamame (O. masou), amago(O. rhodurus), coho (O. kisutch), and Atlanticsalmon (Salmo salar). Pike fry (Esox lucius),seabream and turbot can also be infected underexperimental conditions.


Historically, the geographic range of IHN waslimited to the Pacific Rim of North America but,more recently, the disease has spread tocontinental Europe and Asia.

F.3.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System(1999- 2000)

India reported occurrence of IHN during lastquarter of 1999 affecting murrels and catfishes;Korea RO reported IHN among rainbow troutduring 3rd and 4th (September) quarters of 2000while Japan reported occurrence of IHN everymonth during 1999 and 2000 (OIE 1999, 2000b).


Among individuals of each fish species, there isa high degree of variation in susceptibility toIHNV. Yolk-sac fry (Fig.F.3.2a) are particularlysusceptible and can suffer 90-100% mortality. Inrainbow trout, such mortalities are correlated withwater temperatures

63

conditions). The nucleocapsids are coiled andshow cross-banding (4.5 5.0 nm apart) innegative stain and TEM. Viral replication takesplace in the cytoplasm with particle maturationat the cell membrane or the Golgi cisternae.

F.3.5 Modes of Transmission

IHNV is usually spread by survivors of infections,which carry sub-clinical infections. When suchfish mature, they may shed the virus duringspawning. Clinically infected fish can also spreadthe disease by shedding IHNV with faeces, urine,spawning fluids and mucus secretions. Othersources of infection include contaminatedequipment, eggs from infected fish, and bloodsucking parasites (e.g., leeches, Argulus spp.).Fish-eating birds are believed to be anothermechanism of spread from one site to another.

The most prominent environmental factoraffecting IHN is water temperature. Clinicaldisease occurs between 8C and 15C undernatural conditions. Outbreaks rarely occur above15C.


Control methods currently rely on avoidancethrough thorough disinfection of fertilised eggs.Eggs, alevins and fry should be reared on virus-free water supplies in premises completelyseparated from possible IHNV-positive carriers.Broodstock from sources with a history of IHNoutbreaks should also be avoided whereverpossible. At present, vaccination is only at anexperimental stage.

As with viral haemorrhagic septicaemia virus(VHSV, see F.8), good over-all fish healthcondition seems to decrease the susceptibilityto overt IHN, while handling and other types ofstress frequently cause sub-clinical infection tobecome overt.


Enzmann, P.J., D. Fichtner, H. Schuetze, andG. Walliser. 1998. Development of vaccinesagainst VHS and IHN: Oral application,molecular marker and discrimination ofvaccinated fish from infected populations. J.Appl. Ichth.14: 179-183.

Gastric, J., J. Jeffrey. 1991. Experimentallyinduced diseases in marine fish with IHHNVand a rhabdovirus of eel. CNEVA Laboratoirede Pathologie des Animaux Aquatiques B.P.

F.3.4.1 Presumptive

F.3.4.1.1 Gross Observations (Level I)

Behavioural changes are not specific to IHN butmay include lethargy, aggregation in still areasof the pond with periodic bursts of erraticswimming (see F.3.2) and loss of equilibrium.

Changes in appearance include darkdiscolouration of the body (especially the dorsalsurface and tail fin regions), especially in yolk-sac fry stages (90-100% mortality). Theabdomen can be distended due toaccumulation of fluids in the body cavity(dropsy) and haemorrhaging may be visible atthe base of the fins, on the operculum andaround the eyes. The eyes may also show signsof water imbalance in the tissues by bulging(pop-eye). There may be vent protrusion andtrailing white/mucoid casts.


Tissue sections show varying degrees ofnecrosis of the kidney and spleen(haematopoietic) tissues, as well as in the brainand digestive tract.


Whole alevins (body length 4 cm), visceraincluding kidney (fish 4 6 cm in length) or kidney,spleen and brain tissues from larger fish, arerequired for isolating the virus on EPC or BF-2cell lines. Confirmation of IHNV being the causeof any resultant CPE requires immunoassayinvestigation, as described below.


F.3.4.2.1 Immunoassays (IFAT or ELISA)(Level III)

Diagnosis of IHNV is achieved via immunoassayof isolates from cell culture using IFAT or ELISA,or immunological demonstration of IHNV antigenin infected fish tissues.

F.3.4.2.2 Transmission Electron Micros-copy (TEM) (Level III)

TEM of cells infected in cell-culture reveals en-veloped, slightly pleomorphic, bullet shaped viri-ons, 45-100 nm in diameter and 100-430 nm long.Distinct spikes are evenly dispersed over mostof the surface of the envelope (although thesemay be less evident under some cell-culture

F.3 Infectious HaematopoieticNecrosis (IHN)

64

70 29289 Plouzane, France. EAS Spec.Publ. No. 14.

Hattenberger-Baudouy, A.M., M. Dabton, G.Merle, and P. de Kinkelin. 1995. Epidemiologyof infectious haematopoietic necrosis (IHN) ofsalmonid fish in France: Study of the courseof natural infection by combined use of viralexamination and seroneutralisation test anderadication attempts. Vet. Res. 26: 256-275 (inFrench).




Park, M.S., S.G. Sohn, S.D. Lee, S.K. Chun,J.W. Park, J.L Fryer, and Y.C. Hah. 1993.Infectious haematopoietic necrosis virus insalmonids cultured in Korea. J. Fish Dis. 16:471-478.

Schlotfeldt, H.-J. and D.J. Alderman. 1995.What Should I Do? A Practical Guide for theFreshwater Fish Farmer. Suppl. Bull. Eur.Assoc. Fish Pathol. 15(4). 60p.

F.3 Infectious HaematopoieticNecrosis (IHN)

65

Four months after the appearance of clinicalsigns, some surviving fish may developepitheliomas (grossly visible tumours) around themouth (upper and lower jaw) and, to a lesserextent, on the caudal fin operculum and bodysurface. These may persist for up to 1 year. In 1yr-old coho salmon, chronic infections manifestthemselves as skin ulcers, white spots on theliver and papillomas on the mouth and bodysurface. In rainbow trout, however, there are few(if any) external symptoms, but intestinalhaemorrhage and white spots on the liver areobserved.

Survivors of OMVD develop neutralisingantibodies which prevent re-infection, however,they can remain carriers of viable virus.


More detailed information on screening methodsfor OMV can be found in the OIE DiagnosticManual for Aquatic Animal Diseases (OIE 2000a),at http://www.oie.int or selected references.

F.4.3.1 Presumptive


Persistent superficial tumours are rare, butindicative of a potential carrier of viable OMV.Species, such as rainbow trout show no suchlesions. Sub-clinical carriage cannot normally bedetected using histology.


OMV can be isolated from reproductive fluids,kidney, brain and spleen tissue samples onChinook salmon embryo-214 (CHSE-214) orrainbow trout gonad-2 (RTG-2) cell lines. Anyresultant CPE requires further immunological andPCR analyses to confirm the identity of the virusresponsible (see F.4.3.2.1).


F.4.3.2.1 Immunoassays and Nucleic AcidAssays (Level III)

Cytopathic effect (CPE) from cell cultures, as wellas analyses of reproductive fluids, kidney, brainand spleen tissue samples from suspect fish canbe screened using specific neutralisation anti-body tests, indirect immunofluorescent anti-body tests (IFAT) with immunoperoxidase stain-ing, ELISA or Southern Blot DNA probe assays.

F.4 ONCORHYNCHUS MASOUVIRUS (OMV)



Oncorhynchus masou virus disease (OMVD) iscaused by Oncorhynchus masou virus (OMV)is believed to belong to the FamilyHerpesviridae, based on an icosahedraldiameter of 120-200 nm, and enveloped,dsDNA properties. OMV is also known asYamame tumour virus (YTV), Nerka virusTowada Lake, Akita and Amori prefecture(NeVTA), coho salmon tumour virus (CSTV),Oncorhynchus kisutch virus (OKV), coho salmonherpesvirus (CSHV), rainbow trout kidney virus(RKV), or rainbow trout herpesvirus (RHV). OMVdiffers from the herpesvirus of Salmonidae type1, present in the western USA. Currently thissalmonid herpesvirus has not beentaxonomically assigned (see http://www.ncbi.nlm.nih.gov/ICTV). More detailedinformation about the disease can be found inthe OIE Diagnostic Manual for Aquatic AnimalDiseases (OIE 2000a).

F.4.1.2 Host Range

Kokanee (non-anadromous sockeye) salmon(Oncorhynchus nerka) is most susceptible,followed, in decreasing order of susceptibility, bymasou salmon (O. masou), chum salmon(O. keta), coho salmon (O. kisutch) and rainbowtrout (O. mykiss).


OMVD is found in Japan and, probably (as yetundocumented) the coastal rivers of eastern Asiathat harbour Pacific salmon.

F.4.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System (1999-2000)

Japan reported OMVD during all months of 1999and 2000; and suspected by Korea RO for 1999,and during first two quarters of 2000 (OIE 1999,2000b).


OMV infects and multiplies in endothelial cells ofblood capillaries, spleen and liver, causingsystemic oedema and haemorrhaging. One-month-old alevins are the most susceptibledevelopment stage. Kidney, spleen, liver andtumours are the sites where OMV is mostabundant during the course of overt infection.

66

F.4 Oncorhynchus Masou Virus (OMV)

(M Yoshimizu)

Fig.F.4.4.1.1a. OMV-infected chum salmonshowing white spots on the liver.

(M Yoshimizu)

Fig.F.4.4.1.1b. OMV-induced tumourdeveloping around the mouth of chum salmonfingerling.

(M Yoshimizu)

Fig.F.4.4.1.3. OMV particles isolated frommasou salmon, size of nucleocapsid is 100 to110 nm.


More detailed information on diagnosticmethods for OMV can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000b), at http://www.oie.int or selectedreferences.

F.4.4.1 Presumptive


Behavioural changes include lethargy andaggregation around the water inflow by youngsalmonids of susceptible species. Pin-pointhaemorrhaging or ulcers may be visible on theskin, along with darkened colouration. Popeyemay also be present. Internally, white spots maybe present on the liver (Fig.F.4.4.1.1a). Afterapproximately 4 months, surviving fish may showsigns of skin growths around the mouth(Fig.F.4.4.1.1b) or, less commonly, on theoperculum, body surface or caudal fin area.


Tissue sections from suspect fish may showlesions with enlarged nuclei in the epithelialtissues of the jaw, inner operculum and kidney.


Whole alevin (body length 4 cm), viscera in-cluding kidney (4 6 cm length) or, for larger fish,skin ulcerative lesions, neoplastic (tumourous tis-sues), kidney, spleen and brain are required fortissue culture using CHSE-214 or RTG-2 cell-lines. The cause of resultant CPE should be con-firmed as viral using the procedures outlined inF.4.3.2.1.


Detection of virions in the nuclei of affected tis-sues and tumours by TEM. The dsDNA virionsare enveloped and icosahedral, measuring 120-200 nm in diameter (Fig.F.4.4.1.3).



Gross behaviour and clinical signs at the onsetof OMVD are not disease specific. Thus, con-firmatory diagnosis requires additional diagnos-tic examination or occurrence with a docu-

67


Yoshimizu, M., T. Nomura, Y. Ezura, Y. and T.Kimura. 1993. Surveillance and control of in-fectioushaematopoietic necrosis virus (IHNV)and Oncorhynchus masou virus (OMV) of wildsalmonid fish returning to the northern part ofJapan 1976-1991. Fish. Res.17: 163-173.

F.4 Oncorhynchus Masou Virus (OMV)

mented history of OMVD on-site or mortalitiesseveral months preceding the appearance ofepithelial lesions and tumours.


As described for F.4.3.1.2

F.4.4.2.3 Immunoassays and Nucleic AcidAssays (Level III)

As described for F.4.3.2.1.


Virus is shed with faeces, urine, external andinternal tumours, and, possibly, with skinmucus. Reservoirs of OMV are clinically infectedfish as well as wild or cultured sub-clinicalcarriers. Maturation of survivors of early life-history infections may shed virus with theirreproductive fluids (egg associated, ratherthan true vertical transmission). Egg-associatedtransmission, although less frequent than othermechanisms of virus release, is the most likelysource of infection in alevins.


Thorough disinfection of fertilised eggs, in additionto rearing of fry and alevins, in water free ofcontact with contaminated materials or fish, hasproven effective in reducing outbreaks ofOMVD.Water temperatures

68

F.5 INFECTIOUS PANCREATICNECROSIS (IPN)



Infectious pancreatic necrosis (IPN) is causedby a highly contagious virus, Infectious pancreaticnecrosis virus (IPNV) belonging to theBirnaviridae. It is a bi-segmented dsRNA viruswhich occurs primarily in freshwater, but appearsto be saltwater tolerant. More detailed informationabout the disease can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000a).

F.5.1.2 Host Range

IPN most commonly affects rainbow trout(Oncorhynchus mykiss), brook trout (Salvelinusfontinalis), brown trout (Salmo trutta), Atlanticsalmon (Salmo salar) and several Pacific salmonspecies (Oncorhynchus spp.). Serologicallyrelated are reported from Japanese yellowtailflounder (Seriola quinqueradiata), turbot(Scophthalmus maximus), and halibut(Hippoglossus hippoglossus). Sub-clinicalinfections have also been detected in a widerange of estuarine and freshwater fish speciesin the families Anguillidae, Atherinidae, Bothidae,Carangidae, Cotostomidae, Cichlidae, Clupeidae,Cobitidae, Coregonidae, Cyprinidae, Esocidae,Moronidae, Paralichthydae, Percidae, Poecilidae,Sciaenidae, Soleidae and Thymallidae.


The disease has a wide geographical distribu-tion, occurring in most, if not all, major salmonidfarming countries of North and South America,Europe and Asia.

F.5.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System(1999-2000)

IPN was reported by Japan and suspected inKorea RO for 1999; in 2000, reported by Japanfor the whole year except for the month ofFebruary, and by Korea RO in April (OIE 1999,2000b).


The first sign of IPN in salmonid fry is the sud-den onset of mortality. This shows a progressiveincrease in severity, especially following intro-duction of feed to post-yolk-sac fry. IPN also af-fects American salmon smolt shortly after trans-fer to sea-cages. Clinical signs include darken-

ing of the lower third of the body and smallswellings on the head (Fig.F.5.2.a) and a pro-nounced distended abdomen (Fig.F.5.2b andFig.F.5.2c) and a corkscrewing/spiral swimmingmotion. Some fish may also show pop-eye de-formities. Cumulative mortalities may vary fromless than 10% to more than 90% dependingon the combination of several factors such asvirus strain, host and environment. Survivorsof the disease, at early or late juvenile stages,are believed to be carriers of viable IPNV forlife. Mortality is higher when water temperaturesare warm, but there is no distinct seasonalcycle.

The pancreas, oesophagus and stomach be-come ulcerated and haemorrhagic. The intestinesempty or become filled with clear mucous (thismay lead to white fecal casts).


More detailed information on screening methodsfor IPN can be found in the OIE DiagnosticManual for Aquatic Animal Diseases (OIE 2000b),at http://www.oie.int or selected references.

As with other disease agents, screening for thepresence of an infectious agent in a sub-clinicalpopulation requires larger sample numbers thanfor a disease diagnosis. Numbers will varyaccording to the confidence level required (seeF.1.3.3).

F.5.3.1 Presumptive

F.5.3.1.1 Gross Observations (Level I)and Histopathology (Level II)

Carriers of sub-clinical infections show noexternal or internal evidence of infection at thelight microscope level.


Screening procedures use viral isolation onChinook Salmon Embryo (CHSE-214) or BluegillFin (BF-2) cell lines. The cause of any CPE,however, has to be verified using confirmatorytechniques (F.5.3.2.2). Fish material suitable forvirological examination include whole alevin(body length 4 cm), viscera including kidney(fish 4 6 cm in length) or, liver, kidney and spleenfrom larger fish.

69


F.5.3.2.1 Immunoassays and MolecularProbe Assays (Level III)

The viral cause of any CPE on CHSE-214 orBF-2 cell lines has to be confirmed by either animmunoassay (Neutralisation test or ELISA) or

PCR techniques, including reverse-transcriptase PCR (RT-PCR) and in situhybridization (ISH).


More detailed information on diagnosticmethods for IPN can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000b), at http://www.oie.int or selectedreferences.

F.5.4.1 Presumptive


Clinical signs in salmonid fry and parr includelying on the bottom of tanks/ponds, or showingcork-screw swimming behaviour. High mortalitiesmay occur when fry are first fed or in smolt shortlyafter transfer to seawater. Chronic low mortalitiesmay persist at other times. Dark discolouration(especially of the dorsal and tail surfaces) maybe accompanied by swollen abdomens, pop-eyeand/or pale faecal casts.

F.5 Infectious Pancreatic Necrosis(IPN)

(EAFP)

Fig.F.5.2a. IPN infected fish showing darkcolouration of the lower third of the body andsmall swellings on the head.

(J Yulin)

Fig.F.5.2b. Rainbow trout fry showing dis-tended abdomen characteristic of IPN infec-tion. Eyed-eggs of this species were importedfrom Japan into China in 1987.

(EAFP)

Fig.F.5.2c. Top: normal rainbow trout fry, be-low: diseased fry.

(J Yulin)

Fig.F.5.4.1.3. CPE of IHNV.

(J Yulin)

Fig.F.5.4.1.4. IPN Virus isolated from rainbowtrout fimported from Japan in1987. Virusparticles are 55 nm in diameter.

70



Prevention methods include avoidance offertilised eggs from IPNV carrier broodstock anduse of a spring or borehole water supply (freeof potential reservoir fish). Surface disinfectionof eggs has not been entirely effective inpreventing vertical transmission.

Control of losses during outbreaks involves re-ducing stocking densities and dropping watertemperatures (in situations where temperaturecan be controlled).

Vaccines are now available for IPN and theseshould be considered for fish being grown in IPNVendemic areas.


Frost, P. and A. Ness. 1997. Vaccination ofAtlantic salmon with recombinant VP2 ofinfectious pancreatic necrosis virus (IPNV),added to a multivalent vaccine, suppressesviral replication following IPNV challenge. FishShellf. Immunol. 7: 609-619.

Granzow, H., F. Weiland, D. Fichtner, and P.J.Enzmann. 1997. Studies on the ultrastructureand morphogenesis of fish pathogenic virusesgrown in cell culture. J. Fish Dis. 20: 1-10.

Lee, K.K., T.I. Yang, P.C. Liu, J.L. Wu, and Y.L.Hsu. 1999. Dual challenges of infectiouspancreatic necrosis virus and Vibrio carchariaein the grouper Epinephelus sp.. Vir. Res. 63:131-134.




Seo, J-J., G. J. Heo, and C.H. Lee. 1998.Characterisation of aquatic Birnavirusesisolated from Rockfish (Sebastes schlegeli)cultured in Korea. Bull. Eur. Assoc. Fish Pathol.18: 87-92.


Tissue pathology is characterised by necroticlesions and ulcers in the pancreas, oesophagusand stomach. The intestines may be empty orfilled with clear mucus (NB difference fromparasite infection by Hexamita inflata(Hexamitiasis), where there is a yellowish mucusplug).


As described for screening (F.5.3.1.2), fish ma-terial suitable for virological examination includewhole alevin (body length 4 cm), viscera in-cluding kidney (fish 4 6 cm in length) or, liver,kidney and spleen for larger fish. The virus(Fig.F.5.4.1.3) can be isolated on CHSE-214 orBF-2 cell lines, but the cause of resultant CPEhas to be verified using confirmatory techniques(F.5.3.2).


The ultrastructural characteristics of IPNV areshared by most aquatic birnavididae, thus,immunoassay or nucleic acid assays are requiredfor confirmation of identity. Birnaviruses are non-enveloped, icosahedral viruses, measuringapproximately 60 nm in diameter (Fig.F.5.4.1.4).The nucleic acid component is bi-segmented,dsRNA, which can be distinguished usingstandard histochemistry.


F.5.4.2.1 Virology and Immunoassay(Level III)

As described for screening (F.5.3.2.1), the viralcause of any CPE on CHSE-214 or BF-2 celllines has to be confirmed by either animmunoassay (Neutralisation test or ELISA) orPCR techniques, including RT-PCR and ISH.


The disease is transmitted both horizontallythrough the water route and vertically via the egg.Horizontal transmission is achieved by viral up-take across the gills and by ingestion. The virusshows strong survival in open water conditionsand can survive a wide range of environmentalparameters. This, in addition to its lack of hostspecificity, provides gives IPNV the ability to per-sist and spread very easily in the open-waterenvironment.

71


Schlotfeldt, H.-J. and D.J. Alderman. 1995.What Should I Do? A Practical Guide for theFreshwater Fish Farmer. Suppl. Bull. Eur.Assoc. Fish Pathol. 15(4). 60p.

Wang, W.S., Y.L. Wi, and J.S. Lee. 1997. Singletube, non interrupted reverse transcriptasePCR for detection of infectious pancreatic ne-crosis virus. Dis. Aquat. Org. 28: 229-233.

Yoshinaka, T., M. Yoshimizu, and Y. Ezura. 1998.Simultaneous detection of infectioushaematopoietic necrosis virus (IHNV) and in-fectious pancreatic necrosis virus(IPNV) by reverse transcriptase (RT) poly-merase chain reaction (PCR). Fish. Sci. 64:650-651.

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F.6.1.1 Causative Agents

Viral Encephalopathy and Retinopathy (VER) iscaused by icosahedral, non-envelopednodaviruses, 25-30 nm in diameter. Theseagents are also known as Striped Jack NervousNecrosis Virus (SJNNV), Viral Nervous Necrosis(VNN) and Fish Encephalitis Virus (FEV). Allshare serological similarities with the exceptionof those affecting turbot (F.6.1.2). More detailedinformation about the disease can be found inthe OIE Diagnostic Manual for Aquatic AnimalDiseases (OIE 2000a).

F.6.1.2 Host Range

The pathology of VER occurs in larval and,sometimes, juvenile barramundi (sea bass, Latescalcarifer), European sea bass (Dicentrarchuslabrax), turbot (Scophthalmus maximus), halibut(Hippoglossus hippoglossus), Japaneseparrotfish (Oplegnathus fasciatus), red-spottedgrouper (Epinepheles akaara), and striped jack(Pseudocaranx dentex). Disease outbreaks withsimilar/identical clinical signs have been reportedin tiger puffer (Takifugu rubripes), Japaneseflounder (Paralichthys olivaceus), kelp grouper(Epinephelus moara), brown spotted grouper(Epinephelus malabaricus), rock porgy(Oplegnathus punctatus), as well as othercultured marine fish species.


VER occurs in Asia, the Mediterranean and thePacific.

F.6.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System (1999-2000)

Australia reported VER occurrence during 8 of12 months in 1999, and 7 of 12 months in 2000.Japan reported VER in 6 of 12 months in 2000,and 3 of 12 months in 1999. Last major outbreakreported by Singapore was in 1997 and recentlyin April 1999 and November 2000 amongseabass. Korea RO suspected VER occurrencefor whole year of 1999 and half year of 2000(OIE 1999, OIE 2000b).


VER affects the nervous system. All affectedspecies show abnormal swimming behaviour(cork-screwing, whirling, darting and belly-up

motion) accompanied by variable swim bladderhyperinflation, cessation of feeding, changes incolouration, and mortality (Fig.F.6.2). Differencesbetween species are most apparent with relationto age of onset and clinical severity. Earlierclinical onset is associated with greater mortality,thus onset at one day post-hatch in striped jackresults in more severe losses than suffered byturbot, where onset is up to three weeks post-hatch. Mortalities range from 10-100%.

Two forms of VER have been induced withexperimental challenges (Peducasse et al.1999):i) acute induced by intramuscular inoculation,

andii) sub-acute by intraperitoneal inoculation, bath,

cohabitation and oral routes.


More detailed information on screening methodsfor VER can be found in the OIE DiagnosticManual for Aquatic Animal Diseases (OIE 2000b),at http://www.oie.int or selected references.

F.6.3.1 Presumptive

There are no obvious diagnostic lesions that canbe detected in sub-clinical carriers.



The nodavirus from barramundi has beencultured on a striped snakehead (Channastriatus) cell line (SSN-1) (Frerichs et al. 1996).The applicability of this cell line to othernodaviruses in this group is unknown.

F.6.3.2.2 Nucleic Acid Assays (Level III)

A newly developed polymerase chain reaction(PCR) method has shown potential for screeningpotential carrier striped jack and other fishspecies (O. fasciatus, E. akaara, T. rubripes, P.olivaceus, E. moara, O. punctatus and D. labrax).


More detailed information on diagnosticmethods for VER can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000b), at http://www.oie.int or selectedreferences.

F.6 VIRAL ENCEPHALOPATHY ANDRETINOPATHY (VER)

73

F.6 Viral Encephalopathy andRetinopathy (VER)

F.6.4.1 Presumptive

F.6.4.1.1 Gross Observations

Abnormal swimming behaviour and swim-blad-der inflation in post-hatch larvae and juvenilestages of the host. Species described above,along with associated mortalities are indicativeof VER. Different species show different grossclinical signs (Table F.6.4.1.1). Non-feeding,wasting and colour changes in association withbehavioural abnormalities, should also be con-sidered suspect.


Normal histological methods may reveal varyingdegrees of vacuolisation in the brain or retinaltissues (Fig.F.6.4.1.2a and Fig.F.6.4.1.2b). Smalllarvae can be embedded whole in paraffinblocks and serially sectioned to providesections of brain and eyeballs. Larger fish(juvenile) usually require removal and fixationof eyes and brain.

All the diseases described/named under F.6.1.1demonstrate vacuolisation of the brain, althoughsome species (e.g., shi drum, Umbrina cirrosa)may show fewer, obvious, vacuolar lesions. Inaddition, vacuolisation of the nuclear layers ofthe retina may not be present in Japaneseparrotfish or turbot. Intracytoplasmic inclusions( 5 m diameter) have been described insections of European sea bass and Australianbarramundi, Japanese parrotfish and brown-spotted grouper nerve tissue. Neuronal necrosishas been described in most species.Vacuolisation of the gut is not caused by VERnodaviruses, but is typical.



As described under F.6.3.2.1.


Immunohistochemistry protocols for tissue sec-tions fixed in Bouins or 10% buffered formalinand direct fluorescent antibody test (DFAT) tech-niques use antibodies sufficiently broad in speci-ficity to be able to detect at least four other vi-ruses in this group. An ELISA test is only appli-cable to SJNNV from diseased larvae of stripedjack.


Virus particles are found in affected brain andretina by both TEM and negative staining.Positive stain TEM reveals non-enveloped,icosahedral, virus particles associated withvacuolated cells and inclusion bodies. Theparticles vary from 22-25 nm (European seabass) to 34 nm (Japanese parrotfish) and formintracytoplasmic crystalline arrays, aggregatesor single particles (both intra- and extracellular).In negative stain preparations, non-enveloped,

(J Yulin)

Fig.F.6.2. Fish mortalities caused by VER.

(S Chi Chi)

Figs.F.6.4.1.2a, b. Vacuolation in brain (Br) andretina (Re) of GNNV-infected grouper in ChineseTaipei (bar = 100 mm).

74

hatcheries may assist in controlling VNNinfection. Culling of detected carrier broodstockis one control option used for striped bass,however, there is some evidence that reducedhandling at spawning can reduce ovarianinfections and vertical transmission in somecarrier fish. Control of clinical disease in stripedbass using the following techniques has alsoshown some success: no recycling of culture water chemical disinfection of influent water and larval

tanks between batches, and reduction of larval density from 15-30 larvae/

litre to

75


Anderson, I., C. Barlow, S. Fielder, D. Hallam,M. Heasman and M. Rimmer. 1993. Occur-rence of the picorna-like virus infecting barra-mundi. Austasia Aquacult. 7:42-44.

Arimoto, M., J. Sato, K. Maruyama, G. Mimuraand I. Furusawa. 1996. Effect of chemical andphysical treatments on the inactivation ofstriped jack nervous necrosis virus (SJNNV).Aquac. 143:15-22.

Boonyaratpalin, S., K. Supamattaya, J.Kasornchandra, and R.W. Hoffman.1996.Picorna-like virus associated with mortalityand a spongious encephalopathy in grouper,Epinephelus malabaricus. Dis. Aquat. Org. 26:75-80.

Bovo, G., T. Nishizawa, C. Maltese, F.Borghesan, F. Mutinelli, F. Montesi, and S.De Mas. 1999. Viral encephalopathy andretinopathy of farmed marine fish species inItaly. Vir. Res. 63: 143-146.

Chi, S.C., W.W. Hu, and B.L. Lo. 1999.Establishment and characterization of acontinuous cell line (GF-1) derived fromgrouper, Epinephelus coioides (Hamilton): acell line susceptible to grouper nervousnecrosis virus (GNNV). J. Fish Dis. 22: 172-182.

Comps, M., M. Trindade, and C. Delsert. 1996.Investigation of fish encephalitis virus (FEV)expression in marine fishes using DIG-labelledprobes. Aquac. 143:113-121.

Frerichs, G.N., H.D. Rodger, and Z. Peric.1996.Cell culture isolation of piscine neuropathynodavirus from juvenile sea bass,Dicentrarchus labrax. J. Gen. Vir. 77: 2067-2071.

Munday, B.L. and T. Nakai. 1997. Special topicreview: Nodaviruses as pathogens in larvaland juvenile marine finfish. World J. Microbiol.Biotechnol. 13:375-381.

Nguyen, H.D., K. Mushiake, T. Nakai, and K.Muroga. 1997. Tissue distribution of stripedjack nervous necrosis virus (SJNNV) in adultstriped jack. Dis. Aquat. Org. 28: 87-91.

Nishizawa, T., K. Muroga, K. and M.Arimoto.1996. Failure of polymerase chainReaction (PCR) method to detect striped jacknervous necrosis virus (SJNNV) in Striped

jack Pseudocaranx dentex selected asspawners. J. Aquat. Anim. Health 8: 332-334.

OIE. 1997. OIE Diagnostic Manual for AquaticAnimal Diseases. Second Edition.OfficeInternational des Epizooties, Paris, France.252p.




Peducasse, S., J. Castric, R. Thiery, J.Jeffroy,A. Le Ven, and F. Baudin-Laurencin,. 1999.Comparative study of viral encephalopathy andretinopathy in juvenile sea bass Dicentrarchuslabrax infected in different ways. Dis. Aquat.Org. 36: 11-20.

Thiery, R., R.C. Raymond, and J. Castric. 1999.Natural outbreak of viral encephalopathy andretinopathy in juvenile sea bass,Dicentrarchus labrax: study by nested reversetranscriptase-polymerase chainreaction. Vir. Res. 63: 11-17.

F.6 Viral Encephalopathy andRetinopathy (VER)

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F.7.1.1 Causative Agents

Spring viraemia of carp (SVC) is caused byssRNA Vesiculovirus (Rhabdoviridae), known asSpring viraemia of carp Virus (SVCV) or Rhab-dovirus carpio (RVC) (Fijan 1999). More detailedinformation about the disease can be found inthe OIE Diagnostic Manual for Aquatic AnimalDiseases (OIE 2000a).

F.7.1.2 Host Range

SVCV infects several carp and cyprinid species,including common carp (Cyprinus carpio), grasscarp (Ctenopharyngodon idellus), silver carp(Hypophthalmichthys molitrix), bighead carp(Aristichthys nobilis), crucian carp (Carassiuscarassius), goldfish (C. auratus), tench (Tincatinca) and sheatfish (Silurus glanis).


SVC is currently limited to the parts of continentalEurope that experience low water temperaturesover winter.

F.7.1.4 Asia-Pacific Quarterly AquaticAnimal Disease Reporting System(1999 2000)

No reported case in any country during reportingperiod for 1999 and 2000 (OIE 1999, 2000b).


Young carp and other susceptible cyprinids(F.7.1.2), up to 1 year old, are most severelyaffected. Overt infections are manifest in springwhen water temperatures reach 11-17 C. Poorphysical condition of overwintering fish appearsto be a significant contributing factor. Mortalitiesrange from 30-70%.

Viral multiplication in the endothelial cells of bloodcapillaries, haematopoietic tissue and nephroncells, results in oedema and haemorrhage andimpairs tissue osmoregulation. Kidney, spleen,gill and brain are the organs in which SVCV ismost abundant during overt infection. Survivorsdemonstrate a strong protective immunity,associated with circulating antibodies, however,this results in a covert carrier state.


More detailed information on methods for

F.7 SPRING VIRAEMIA OF CARP(SVC)

screening SVC can be found in the OIE Diag-nostic Manual for Aquatic Animal Diseases (OIE2000a), at http://www.oie.int or selected refer-ences.

F.7.3.1 Presumptive

There are no methods for detection of sub-clinicalinfections using gross observations or routinehistology.


Screening for sub-clinical carriers uses tissuehomogenates from the brain of any size fish orthe ovarian fluids from suspect broodstock fish.Cell lines susceptible to SVCV are EPC andFHM. Any resultant CPE requires molecular-based assays as described under F.7.3.2.



CPE products can be checked for SVCV using avirus neutralisation (VN) test, indirect fluorescentantibody tests (IFAT), and ELISA. IFAT can alsobe used on direct tissue preparations.


More detailed information on methods fordiagnosis of SVC can be found in the OIEDiagnostic Manual for Aquatic Animal Diseases(OIE 2000a), at http://www.oie.intor selected references.

F.7.4.1 Presumptive


Sudden mortalities may occur with no otherclinical signs. Behavioural clues are non-specificto SVC and include lethargy, separation from theshoal, gathering at water inlets or the sides ofponds and apparent loss of equilibrium.

External signs of infection are also non-specific,with fish showing varying degrees of abdominaldistension (dropsy), protruding vents and trailingmucoid faecal casts. Haemorrhaging at the basesof the fins and vent, bulging eye(s) (pop-eye orexophthalmia), overall darkening and pale gillsmay also be present (Figs.F.7.4.1.1a, b, c andd).

Internal macroscopic signs of infection includean accumulation of body cavity fluids (ascites)

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which may lead to the dropsy visible asabdominal distension, bloody and mucous-filled intestines, swim-bladder haemorrhageand gill degeneration.


Detection of enveloped, bullet-shaped, viralparticles measuring 90-180 nm in length and witha regular array of spicules on the surface inspleen, kidney and brain tissues, or in isolatesfrom CPE in the cell-lines described underF.7.4.1.3, should be considered indicative of SVCin susceptible carp species showing other clinicalsigns of the disease. Viral replication takes placein the cytoplasm with maturation in associationwith the plasma membrane and Golgi vesicles.


Whole fish (body length 4 cm), or visceraincluding kidney (fish 4 - 6 cm in length) or kidney,spleen and brain of larger fish, can be preparedfor tissue culture using Epithelioma papulosumcyprinae (EPC) or FTM cell lines. Resultant CPEshould be examined using the diagnostictechniques outlined below and under F.7.3.2.1to confirm SVCV as the cause.



As described under F.7.4.1.3, SVCV can beconfirmed in CPE products using a virusneutralisation (VN) test, indirect fluorescentantibody tests (IFAT), and ELISA. IFAT can alsobe used on direct tissue preparations.

F.7.4.2.2 Nucleic Acid Assay (Level III)

RT-PCR techniques are under development.


Horizontal transmission can be direct (contactwith virus shed into the water by faeces, urine,reproductive fluids and, probably, skin mucous)or indirectly via vectors (fish-eating birds, the carplouse Argulus foliaceus or the leech Piscicolageometra). Vertical transmission is also possiblevia SVCV in the ovarian fluids (however, the rar-ity of SVC in fry and fingerling carp indicatesthat this may be a minor transmission pathway).

SVCV is hardy and can retain infectivity afterexposure to mud at 4C for 42 days, stream

water at 10C for 14 days, and after drying at4-21C for 21 days. This means that avenuesfor establishing and maintaining reservoirs ofinfection are relatively unrestricted. This, plusthe broad direct and indirect mechanisms fortransmission, makes this disease highly conta-gious and difficult to control.


No treatments are currently available althoughsome vaccines have been developed. Most effortis applied to optimising the overwinteringcondition of the fish by reducing stocking density,reduced handling and strict maintenance ofhygiene. New stocks are quarantined for at leasttwo weeks before release into ponds for grow-out.

Control of spread means rapid removal anddestruction of infected and contaminated fishimmediately on detection of SVC. Repeatoutbreaks may allow action based onpresumptive diagnosis. First time outbreaksshould undertake complete isolation of affectedfish until SVC can be confirmed.


Dixon, P.F., A.M. Hattenberger-Baudouy, andK. Way. 1994. Detection of carp antibodies tospring viraemia of carp virus by competitiveimmunoassay. Dis. Aquat. Org. 19: 181-186.

Fijan, N. 1999. Spring viraemia of carp and otherviral diseases and agents of warm-water fish,pp 177-244. In: Woo, P.T.K and Bruno, D.W.(eds).Fish Diseases and Disorders. Vol 3.Viral, Bacterial and Fungal Infections. CABIPublishing, Oxon, UK.




Oreshkova, S.F., I.S. Shchelkunov, N.V.Tikunova, T.I. Shchelkunova, A.T. Puzyrev,and A.A. Ilyichev. 1999. Detection of spring

F.7 Spring Viraemia of Carp (SVC)

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viraemia of carp virus isolates byhybridisation with non-radioactive probesand amplification by polymerase chainreaction. Vir. Res. 63: 3-10.

Rodak, L., Z. Pospisil, J. Tomanek, T. Vesley, T.Obr, and L. Valicek. 1993. Enzyme-linkedimmunosorbent assay (ELISA) for thedetection of spring viraemia of carp virus(SVCV) in tissue homogenates of the carpCyprinus carpio L. J. Fish Dis. 16: 101-111.

Schlotfeldt, H.-J. and D.J. Alderman. 1995. WhatShould I Do? A Practical Guide for the Fresh-water Fish Farmer. Suppl.Bull. Eur. Assoc. FishPathol. 15(4). 60p.

(EAFP)

F.7 Spring Viraemia of Carp (SVC)

Figs.F.7.4.1.1a, b, c, d. Non-specific clinical signs of SVC infected fish, which may include swollenabdomen, haemorrhages on the skin, abdominal fat tissue, swim bladder and other.

(EAFP)

Fig.F.8.4.1.1. Non-specific internal sign(petechial haemorrhage on muscle) of VHSinfected fish.

a b

c d

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F.8 VIRAL HAEMORRHAGICSEPTICAEMIA (VHS)



Viral haemorrhagic septicaemia (VHS) is causedby ssRNA enveloped rhabdovirus, known as vi-ral haemorrhagic septicaemia virus (VHSV).VHSV is synonymous with Egtved virus. Al-though previously considered to fall within theLyssavirus genus (Rabies virus), the ICTV haveremoved it to unassigned status, pending evalu-ation of a proposed new genus Novirhabdovirus to include VHSV and IHNV (see http://www.ncbi.nlm.nih.gov/ICTV). Several strains ofVHSV are recognised. More detailed informationabout the disease can be found in the OIE Diag-nostic Manual for Aquatic Animal Diseases (OIE2000a).

F.8.1.2 Host Range

VHS has been reported from rainbow trout(Oncorhynchus mykiss), brown trout (Salmotrutta), grayling (Thymallus thymallus), white fish(Coregonus spp.), pike (Esox lucius) and turbot(Scophthalmus maximus). Genetically distinctstrains of VHSV have also been associated withdisease in Pacific salmon (Oncorhynchus spp.),Pacific cod (Gadus macrocephalus) and Pacificherring (Clupea pallasi). These strains show littlevirulence in rainbow trout challenges (OIE2000a). VHSV has also been isolated from At-lantic cod (Gadus morhua), European sea bass(Dicentrarchus labrax), haddock (Melano-grammus aeglefinus), rockling (Rhinonemuscimbrius), sprat (Sprattus sprattus), herring(Clupea harengus), Norway pout (Trisopterusesmarkii), blue whiting (Micromesistiuspoutassou), whiting (Merlangius merlangius) andlesser argentine (Argentina sphyraena)(Mortensen 1999), as well as turbot(Scophthalmus maximus) (Stone et al. 1997).Among each species, there is a high degree ofvariability in susceptibility with younger fish show-ing more overt pathology.


VHSV is found in continental Europe, the Atlan-tic Ocean and Baltic Sea. Although VHSV-likeinfections are emerging in wild marine fish inNorth America, VHS continues to be consid-ered a European-based disease, until the phy-logenetic identities of the VHSV-like viruseswhich do not cause pathology in rainbow troutcan be clearly established.

F.8.1.4 Asia-Pacific Quarterly Aquatic Animal Disease Reporting System(1999-2000)

Japan reported the disease during second quar-ter of 2000, no other reports from other countries(OIE 1999, 2000b).


The virus infects blood cells (leucocytes), theendothelial cells of the blood capillaries,haematopoietic cells of the spleen, heart, neph-ron cells of the kidney, parenchyma of the brainand the pillar cells of the gills. Spread of the viruscauses haemorrhage and impairment of osmo-regulation. This is particularly severe in juvenilefish, especially during periods when water tem-peratures ranging between 4 14C.


More detailed information on methods for screen-ing VHS can be found in the OIE DiagnosticManual for Aquatic Animal Diseases (OIE 2000a),at http://www.oie.int or selected references.

F.8.3.1 Presumptive

F.8.3.1.1 Gross Observations (Level I)and Histopathology (Level II)

There are no gross visible clues (Level I) or his-topathology clues (Level II) to allow presumptivediagnosis of sub-clinical VHS infections. Sub-clinical carriers should be suspected, however,in populations or stocks which originate fromsurvivors of clinical infections or from confirmedcarrier broodstock.


VHSV can be isolated from sub-clinical fish onBluegill Fry (BF-2), Epithelioma papulosumcyprinae (EPC) or rainbow trout gonad (RTG-2).Any resultant CPE requires further immunoas-say or nucleic acid assay to confirm VHSV asthe cause (F.8.3.2).

F.8.3.1.3 Immunoassay (Level III)

Immunohistochemistry can be used to highlightVHSV in histological tissue samples (which ontheir own cannot be used to screen sub-clinicalinfections). Due to the wide range of hosts andserotypes, however, any cross-reactions needto be confirmed via tissue culture and subsequentviral isolation as described under F.8.3.1.2.

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Identification of VHSV from cell-line culture canbe achieved using a virus neutralisation test,indirect fluorescent antibody test (IFAT) or ELISA.

F.8.3.2.2 Nucleic Acid Assay (Level III)

RT-PCR techniques have been developed.

F.8.4 Diagnostic Procedures

More detailed information on methods for diag-nosis of VHS can be found in the OIE DiagnosticManual for Aquatic Animal Diseases (OIE 2000a),at http://www.oie.int or selected references.

F.8.4.1 Presumptive


There are no VHS-specific gross clinical signs.General signs are shared with bacterialsepticaemias, IHN, osmotic stress, handlingtrauma, etc., and include increased mortality,lethargy, separation from the shoal, gatheringaround the sides of ponds, nets or water inlets.

The skin may become darkened andhaemorrhagic patches may be visible at the caseof the fins, the vent and over the body surface.Gill may also be pale. Internal organ changes mayor may not be present depending on the speedof onset of mortalities (stressed fish die quicker).Where present these include an accumulation ofbloody body cavity fluids (ascites), mucous-filledintestines and pale rectal tissues. Pin-pointhaemorrhages may also be present throughoutthe muscle (Fig.F.8.4.1.1), fat (adipose) tissueand swim-bladder.


VHSV can be isolated from whole alevin (bodylength 4 cm), viscera including kidney (fish 4 6 cm in length) or kidney, spleen and braintissue samples from larger fish, using BF-2, EPCor RTG-2 (as described under F.8.3.1.2). Anyresultant CPE requires further immunoassay ornucleic acid assay to confirm VHSV as the cause(F.8.3.2.1/2).


Immunohistochemistry can be used to highlightVHSV

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