Necropsy techniques for fish roy p.e. yanong

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Necropsy techniques for fish roy p.e. yanong

  1. 1. Necropsy Techniques for FishRoy P.E. Yanong, VMDFish medicine has become more mainstream within theveterinary profession over the past two decades,Aquarium hobbyists, public aquariums, and private fa-cilities are relying more and more on veterinary exper-tise for fish health problems. Most problems in fishhealth are related to water quality and other husbandryissues; the necropsy is only one important part of acomplete work up. As with necropsy of exotic terres-trial animals, an understanding of the basic anatomyand physiology of a few common fish species can helpthe practitioner gain confidence when working withmore exotic fish. However, because species differencesdo exist, protocols may require modification. Moribundor fresh dead specimens are best. With fish, perhapsmore than any other class of animals, wet mount biop-sies of external and internal tissues are very informa-tive. Parasites are an important contributor to disease,and most easily identified alive. The delicate structureof the gill and the rapidity with which it can break downalso necessitates careful analysis at the outset. Like-wise, other pathology can be identified quickly usingwet mounts. Microbiological culture of kidney, brain,liver, spleen, and other affected organs, and histopa-thology are also important aspects of the fish necropsy.Cultures should be incubated at temperature rangessimilar to those for the fish. Certain viruses, includingSpring viremia of carp (SVC} and koi herpes virus (KHV-not Herpesvirus cyprini, the causative agent of carppox) are important pathogens that require confirma-tory virus isolation and culture. SVC is a notifiable dis-ease.9 2003 Elsevier Inc. Aft rights reserved.Key words: Aquarium, fish, necropsy, biopsy, anatomy,pathology.[ ~ish health is an important and diverse areaof opportunity for the veterinary profession.Aquarium fish are the number one pets in theU.S. in private water gardens, stocked with koiand goldfish, and maintain popularity. Publicand private aquarium facilities are increasing innumbers. Besides traditional stand-alone publicaquaria, large indoor and outdoor exhibits areroutinely integrated into offices, restaurantchains, outdoor stores, hotels, and other facili-ties. Health of breeding populations of endan-gered species is a concern, and fish are impor-tant research animals. Finally, the health ofnatural fish populations continues to provideinformation as an indicator of environmentalhealth.Importance of History and ManagementThe necropsy can help determine causes ofmorbidity and mortality in fish, However, moreso than in other classes of animals, the impor-tance of history and environmental factors can-not be overlooked,a-~The vast majority of diseaseproblems in fish are linked intimately to waterquality and management issues, factors that donot always translate into directly observable pa-thology. Clinicians expecting to determine causeof disease based solely on biopsies or necropsywill be disappointed, as will their clientele. Com-plete information on the life support systems,water quality, husbandry/general management,the fish, and the problem is critical. The follow-ing is not a complete list, but major issues in-clude:Life Support System (LSS)Includes the physical system set up includingdimensions of the tank(s) or pond (s) ; filtrationcomponents; plants; invertebrates; length oftime established; substrate; pipe/system materi-als; and if an outdoor pond, access to birdsand/or predators.Water QualityIncludes water source (municipal, well, other;water treatment protocols); history and mea-surements of ammonia, nitrite, nitrate in marinesystems, pH, temperature, dissolved oxygen,hardness, alkalinity, salinity and heavy metalsand other toxins.Fromthe TropicalAquaculture Laboratory,DepartmentofFish-criesand Aquatic Sciences, Universityof Florida, Ruskin, FL.Address reprint requeststo: Roy P.E. Yanomg, VMD,AssistantProfessor, TropicalAquaculture Laboratory, Universityof Flqrida,1408 24th St. SE, Ruskin, FL 33570.9 2003 ElsevierInc. All rights reserved.1055-937X/03/1202-0005530. 00/0doi:l O.1053/saep.2003.127885Seminars in Avian and Exotic Pet Medicine, Vol 12, No 2 (April), 2003: pp 89-105 89
  2. 2. 90 YanongGeneral Husbandry/ManagementIncludes maintenance protocols for life sup-port systems (water changes, cleaning, filtrationbackwash, and component checks); sanitation,disinfection, and quarantine protocols; nutri-tion (live foods, prepared diet, and storage pro-tocols); chemical/salt usage; general fish keep-ing/breeding experience of client; and overallexpectations/purpose of the fish.FishIncludes specific biology and requirements;familiarity of client with species; other fish of thesame or different species in community systemsand their compatibility; and recent introduc-tions.ProblemIncludes the chief complaint and clients per-spective; previous problems; major changes inany of the categories listed above; chemothera-peutants used so far; morbidity/mortality timecourse; behavioral changes and visible lesions.Indicationsfor NecropsyIf evaluations of history, life support systems,water quality, and husbandry or managementare not adequate to solve the problem, nonle-thal techniques including biopsy of skin, fins,and gills; examination of fresh fecal samples;and blood culture may be all the additional in-formation required. 4 However, if a client is in-terested in a thorough evaluation of a problem,a necropsy is required. Likewise, if a communityor population of fish is at risk, necropsies shouldbe performed to fine-tune treatment and pre-vention.Importance of Sample SelectionGood sample selection is critical. Moribundfish showing typical disease signs are preferable.If several fish can be killed when evaluating apopulation, fish should be chosen at differentstages, including at least one with early stagedisease. Fish with late stage disease often havesecondary complications that mask initiatingcauses.For large populations of low disease preva-lence with mortality as the only sign, randomsampling may result in the selection of healthyfish for evaluation. If necropsy samples reveal nosignificant findings (and environmental andmanagement factors have been carefully evalu-ated and ruled out), evaluation of fresh deadfish can be helpful. In general, fresh dead fishshould have relatively clear eyes, good colora-tion, red to pink gills, and should not have a badodor. Because of the rapidity with which deadfish are cannibalized, autolyze, and/or take onsecondary organisms (such as fungi, bacteria,and parasites including the ciliates Tetrahymenaor Uronema),tile significance of lesions on deadfish should be regarded cautiously.Although moribund fish are ideal specimensfor post mortem work ups, fresh dead, frozen,and formalin-fixed specimens can provide usefulinformation. 1 Fresh dead fish examined imme-diately or kept in a plastic bag in the refrigeratoror on ice for 6 to 12 hours can provide almost asmuch information as moribund fish) Frozenand formalin-fixed fish are much less informa-tive) Common protozoal parasites and monoge-neans often die or leave a dead host. Culturescollected from fish that have been dead in waterfor as little as thirty minutes to sixty minutes maybe contaminated due to decomposition and au-tolysis.Basic Fish Anatomy and PhysiologyFish species vary widely in their anatomy andphysiology. Several popular fish species from twoimportant families, the Cyprinidae (koi-Cyprinuscarpio and goldfish-Carassius auratus) and theCichlidae (discus-Symphysodo,~sp. and Oscar-Astronotus ocellatus) highlight some of the simi-larities and differences in gross anatomy. Severalgood references provide additional species in-formationY,~-9ExternalStructures~OrgansSkinThe skin consists of a mucus layer, epidermis,and dermis. Epidermal layers contain numeroustypes of cells including taste cells, alarm cells,pigment cells, and mucus-producing cells. Inaddition to other functions, mucus acts as a
  3. 3. NecropsyTechniquesfor Fish 91Figure 1. Primary gill lamella (P) are supported bygill cartilage (G); secondary lamella (S) are visible asplate-like structures extending from the primary la-mella Bar - 250 /*m H & E stain.lubricant to decrease friction in swimming,helps provide a barrier against and prevents theattachment of pathogens, and contains biomol-ecules important for immune defense. The fishexterior generally contains either 1) scales (orig-inating from the dermal layer and comprised ofa type of bone) that are covered by the epider-mis (eg, koi, goldfish, cichlids, tetras, and loa-ches-that have very fine scales) ; 2) no scales (eg,channel catfish) ; or 3) bony plates (eg, Corydorassp. catfish, Plecostomus catfish [Hypostomusandrelated genera], seahorses [Hippocampusspp.]).The lateral line system, comprised of a series ofvery small pits, is known as the organ of distanttouch. Among other functions, it facilitatesschooling behavior. Injury or lesions to the skincan result in significant osmoregulatory compro-mise and increased chance of infections.GillThe primary respiratory apparatus in fish, thegills evolved to maximize extraction of oxygenfrom water. The gills are also the sites for carbondioxide and ammonia removal from the body.In most teleosts (bony fishes), there are four gillson each side. Fine structure is comprised ofprimary and secondary lamellae. (Fig 1) Primarylamellae contain a specific type of cartilage (gillcartilage) for structural support while secondarylamellae branch off the primary lamellae. Agrouping of primary and secondary lamella (gillfilaments) can be thought of as having a "Christ-mas tree" (three-dimensional) structure. Be-cause the epithelium on secondary lamellae isone cell thick and the surface area of the gills islarge, gill compromise is a constant threat.PseudobranchThe pseudobranch is anatomically related to,and resembles the gills, but is not involved withrespiration. The pseudobranch is located on thedorsal section of the inner operculum, and mayor may not be superficial enough to be seengrossly. In cyprinids, it is deeply embedded. Thepseudobranch is a hemibranch, only containingfilaments on one side. Proposed functions in-elude provision of gases/oxygen to the eye, os-moregulation, or sensory capability, but theseare still debated. Some clinicians believe patho-physiology of the pseudobranch may contributeto idiopathic exophthalmia in some species.OperculumThe operculum is the gill covering or gill flapwhich is important for respiration. Oxygenatedwater passes through the mouth, enters the oralcavity, passes through the pharynx, enters theopercular cavity (where gills are located), passesover the gills, and then out the opercular valve(gill slit). This direction of water movement overthe gills is important for gas exchange. Theoperculum and gill slit are highly modified insome species (eg, seahorses), making ante-mor-tern biopsies of gills difficult. Rate of opercularmovement is used to monitor respiration. Themales of some fish (eg, Cyprinids, includinggoldfish) develop small white dots/bumpsknown as nuptial tubercles during breeding sea-son on their opercula and anterior pectoralfins. IoFinnageThe types of finnage present depend on spe-cies, and sexual dimorphism exists in many spe-cies (Fig 2). The caudal and dorsal fins areprimarily locomotory. The dorsal fin acts as astabilizer, and in some species, may have venom-ous spines. The pelvic fins are also stabilizersand, in some fish, such as angelfish and goura-mis, are sensory. The adipose fin, or "fatty" fin,present in some fish groups, including most tet-ras, is located behind the dorsal fin. The anal fin
  4. 4. 92 YanongFigure 2. Fin types in an African cichlid. A: anal; C:caudal; D: dorsal; P: pectoral; V: pelvic.is a stabilizer but has become a modified malereproductive intromittent organ in the Poecili-idae family (swordtails, guppies, mollies, platys)and some other groups. Pectoral fins of somemature males may have nuptial tuberclesd ~MouthThe location of the mouth on the head (ie,more ventral, terminal, or dorsal); presence ofjaw teeth and/or pharyngeal teeth (teeth lo-cated in throat region of many types offish) ; andoverall structure of the oral cavity can differsignificantly, depending on type of food/preyeaten. Some species, including many Africancichlids, arowana (Osteoglossum and Scleropagesspp.), and jawfish (Opistognathusspp.) use theirmouth to brood developing eggs or fry. Themouth may also be used for nest building andfor territorial or breeding displays or fighting.Vent/anus/genital poreIf the intestinal tract, urinary tract, and go-nadal ducts empty into a common chamber (clo-aca), then the common opening to the outside isknown as the vent. If the intestine/digestive sys-tem opens out separately, this opening is knownas the anus, and the reproductive opening isknown as the genital pore. Some fish species canbe sexed based on location and form of thesestructures.EyesBecause fish are aquatic, they lack tear glandsand most lack movable eyelids. Muscles attachedto the spherical lens move it toward or away fromthe retina for accommodation. The lens refractslight by means of successive differentially denselayers. Fish eyes are particularly prone to exter-nal injury and physiopathological or infectiousprocesses leading to exophthalmia or buphthal-mia.NaresThe nares are the "nostrils" of fish and areolfactory in function.Internal Structures~OrgansBrainThe central nervous system. Squash preps ofthe brain are not typically informative but areimportant for culture and histopathology.MuscleMuscle of fish is similar to that of other ver-tebrates. There are three major types: skeletal("filet" muscles), smooth (eg, gastrointestinaltract musculature), and cardiac (heart).Swim bladderAlso known as the gas or air bladder, it isimportant for maintaining neutral buoyancy inwater and, thus, conserving energy. In some spe-cies, it may also be used for sound production orrespiration. Swim bladders are either physosto-mus (connection to the esophagus is patent, andair moves in through this opening) or physoclis-tous (there is no connection, and air enters via avascular rete, or "gas gland"). In Cyprinids suchas koi and goldfish, it is bi-lobed (anterior andposterior chamber, separated by a sphincter)and easily removed still inflated from the body.The caudal chamber connects to the esopha-gus. 2,5 In other species, such as discus, an inti-mate association with the lateral body wall,makes it difficult to dissect out without punctur-ing it. Depending on species, it may be moretranslucent or somewhat opaque, thick or thin.It is usually located beneath the kidney.KidneyUsually red/brown in color, kidney functionsinclude hematopoiesis, excretion, and immu-nity. Melanomacrophage centers (MMCs), blackor brown clusters of cells, are commonly seen in
  5. 5. Necropsy Techniquesfor Fish 93the kidney, spleen, and/or liver, depending onspecies. MMCs are believed to play a role inimmune function and degradation of senescentcells. The kidney is an important part of thereticuloendothelial system (RES), common toall vertebrates. The RES functions to removeparticulate matter, senescent cells, and infec-tious agents from the body. The kidney lies ven-tral to the spinal cord and dorsal to the swimbladder in most species. The kidney can be di-vided into two anatomic and physiologic sectionsin most fish. The anterior is primarily hemato-poietic and the posterior is primarily excretory.LiverThe liver functions are similar to those inother vertebrates. In most teleost species, thisorgan is more properly called the hepatopan-creas (contains exocrine pancreatic tissue whichcan also be found in other areas in many fish).Coloration can vary from pale tan to brown tored, depending on species, diet, nutritional sta-tus, and reproductive status. It is often one of thelargest/most visible organs in the anterior por-tion of the coelomic cavity. In some species suchas koi and goldfish, the liver is intimately inter-twined with the intestinal tract.SpleenThe spleen is important for immune function(MMCs, RES) and hematopoiesis. It is thesmaller, dark red organ frequently located closeto the stomach and may be triangular, oval/rounded, or more elongate in shape.HeartLocated in the pericardial cavity (separatefrom the coelomic cavity), in the throat region,caudal and ventral to the gills. The heart istwo-chambered (atrium, ventricle), but containsa thin walled sinus venosus leading into theatrium, and a thick, elastic bulbus arteriosus,leading out of the ventricle. It is typically notvery infbrmative on squash preps but importantfor histopathology. The heart is also part of theRES.Gonad (ovary and/or testes)Sexual reproduction in fish is diverse andmany species exhibit sexual dimorphism. Sizeand structure of the gonad and developmentalstage of oocytes can vary greatly depending onspecies, age, reproductive status, and season.During breeding periods, size of the gonad, in-creases greatly and often fills the coelomic cav-ity, displacing other organs. Most commonfreshwater aquarium species, and many marinespecies, are dioecious (separate sexes). Manycommon marine fish species are protandrous(male first, then female) or protogynous fie-male first, then male) hermaphrodites. The fe-males of some species, such as the commonlive-bearing Poeciliids (swordtails, guppies, andrelated groups), hold sperm in their ovaries forvarious lengths of time. One common error ismistaking sperm for flagellates, especially sincethe testes are closely apposed to the intestine atthe vent/anus. In general, sperm are muchshorter lived than flagellates on a slide, and willusually die within a minute or two.StomachThe stomach is typically thicker walled thanthe intestine, and gastric glands/mucosa are dis-tinct compared with intestinal tract lining on wetmounts and histology. A true/distinct acid stom-ach is not present in Cyprinid species such asgoldfish and koi. It is very important to examinethe stomach in cichlids for the presence of par-asites and/or granulomas.Intestinal tractUsually thinner walled than the stomach, it istypically shorter in carnivores (eg, Oscars) andlonger in omnivores (koi, goldfish) and herbi-vores (eg, plecostomus).Clinical ObservationsClinical observations of live fish before nec-ropsy can help identify clinically diseased ani-mals and pinpoint location of lesions. Sick fishtypically isolate themselves and are darker orlighter than their conspecific tank mates. Otheraberrant behaviors, such as piping at the surface,may indicate gill pathology; flashing (scratch-ing) may indicate skin irritation and/or parasit-ism; aberrant positioning may indicate swimbladder disease or gastrointestinal disease; andspinning or aberrant swimming may indicateneurologic disease. 8,n
  6. 6. 94 YanongNecropsy Equipment and SuppliesGeneralSuppliesA compound microscope, a dissecting micro-scope or magnifying lamp (helpful for smallspecies) a pathology report form, latex gloves,scissors (small (ophthahnic), medium, and/orlarge, with blunt tips and with pointed tips),scalpel, rat tooth forceps, microdressing forceps,necropsy tray/table (depending on size 9f fish),slides, cover slips, microscope, paper towels, nee-dies/syringes (for blood sampling), dechlori-hated freshwater (or salt water) for wet mounts,tricaine methanesulfonate (TMS or MS-222)(Finquel| Argent, Redmond, WA; or Tricain-S|Western Chemical, Ferndale, WA, USA), andsodium bicarbonate (baking soda) are generalsupplies for performing necropsy. More specificequipment may be required for larger fish orcertain species (for example, larger sturgeonmay require a drill, dremel tool, or saw to cutthrough the neurocranium to culture the brain).MicrobiologicalsuppliesCulture swabs (for submission to a microbiol-ogy laboratory), or microbiological media (tryp-tic soy agar (TSA) with 5% sheeps blood plates;Sabourauds dextrose agar or other media asindicated), sterile loops, sterile gauze, alcoholburner, glass jar/container for instruments, al-cohol pads, matches or barbeque grill lighter arethe basic microbiological supplies.HistopathologyTen percent neutral buffered formalin (forlight histopathology); Trumps solution (forelectron microscopy); specimen jars are the ba-sic histopathology supplies.Wet Mount EvaluationUse of MS-222 before biopsy of external struc-tures has been shown to result in significant lossof some parasites from the body? 2 Before eutha-nasia by lethal dosing with MS-222, biopsies ofthe skin, gills, and fin should be evaluated. Withexperience, the clinician can do this quickly andwith minimal distress to the animal by holdingthe fish in the folds of a net and exposing eachFigure 3. Gill, skin, and fin clips on a slide. Smallsamples are more easily examined.area to be sampled one at a time. Latex glovesshould be worn to avoid additional damage tothe skin and to avoid accidental removal of ex-ternal parasites or lesions. Also, be gentle whenusing a net to avoid accidental removal of exter-nal parasites from the body. Covering the eyes ofthe fish can help decrease stimulation and re-duce movement. Any abnormalities includingareas with discoloration, increased mucus, hem-orrhages, erosions, ulceration, masses, or para-sites should be sampled as described in the fol-lowing section.Thorough evaluation of wet mounts is a man-datory part of a good necropsy. Many importantfindings, including external and internal para-sites, are best observed and quantified in thismanner.Slides are prepped to receive biopsy samplesby placing three small drops of water equidistanton a slide. This will allow for three differentbiopsies to be evaluated per slide. Once taken,each biopsy should be placed on one of thesedrops of water with a cover slip. (Fig 3) Forexternal (skin, gill, and fin) samples, it is impor-tant to use dechlorinated freshwater (do not usedistilled water) for freshwater fish. Use salt waterfor saltwater fish. For internal samples, freshwa-ter should suffice for both freshwater and ma-rine fish species. If tile clinician is not yet com-fortable identifying organs on wet mount, slidesshould be labeled with a marker according tothe organ to reduce confusion. In most in-stances, only a small section (2 to 3 mm 3) oforgan is necessary. Larger sections of fins can beexamined. Excess tissue leads to thick preps that
  7. 7. Necropsy Techniquesfor Fish 95are dark and difficult to examine. Larger fishoften have more fibrous tissue in their internalorgans making squash preparations a bit moredifficult. 1During microscopic examination of squashpreps/wet mounts, the clinician should use amethodical search pattern. A quick scan at 40X(low power) will help with orientation and pre-liminary identification of areas of interest. Ma-nipulation of the condenser to decrease the ap-erture will help increase contrast and simplifyobservation of smaller structures/parasites, aswill the use of phase contrast. Most parasites andlesions of interest should be visible at 40x-100xpower. However, at 100x, only the movement ofsome smaller parasites (such as the flagellatesSpironucleus and Ichthyobodo) may be discerned,with more structures visible at 400x. Microspo-ridian parasites are also very small (approximaterange, 1 to 5 • 2 to 8/xm), j3 and although theircysts (sporoblastic vesicles) or xenomas are visi-ble at higher powers, individual spores, whichare nonmotile, are more clearly seen at 200 to400x. Flavobacterium columnare, the cause of co-lumnaris disease, is a long, rod-shaped Gram-negative bacteria that can often be found flexingin haystack formations./, 2 These are most com-monly appreciated at 200 to 400x.Skin, Gill, and Fin BiopsySkinUsing a cover slip or the back of a scalpelblade, gently scrape any areas that appear abnor-mal (increased mucus, erosions, masses) (Figs 4Figure 5. Normal scales and mucus fi-omskin scrape.100x. Note growth rings on ctenoid type scales("teeth" or comb located at bottom margins of thescale; most visible near bottom of figure), and pig-ment ceils.and 5). In addition, scrape areas that are eithersofter, have fewer scales, and/or experience re-duced water flow (such as areas on the head,ventrum, and behind the pectoral fins). Theseareas have a greater chance of harboring para-sites. Scrapes made near the vent/anus may re-sult in expression of sperm in mature males, andshould not be confused with flagellates.GillsLift the operculum (the scissor blades can beused as a lever to do this or forceps can be used)and cut a small section of the gill tips (Fig 6). Ifthere are any specific areas that appear abnor-mal, target these areas. After the fish has beeneuthanized, a section of the gill arch (base of gillwhere filaments connect) should be examinedby wet mount and saved in fixative, especially ifareas of the gill distal to this section appear paleor necrotic. At least one fungus, Branchiomyces,may be found more commonly at this location ifpresent/4 Because of the gills delicate structureand location, gill pathology is often the first signof poor water quality, bacterial disease, or para-sitism.Figtwe 4. Skin scrape using cover slip.FinsCut a small section from the dorsal fin, caudalfin, pectoral fin, and anal fin, and examine each.(Fig 7) For better evaluation of dark, pigmented
  8. 8. 96 YanongFigure 6. A) Gill biopsy. Scissors are used to open theoperculum and cut the tips of the primary lamella; donot cut the gill arch at this step. B) Gill wet mount:Primary lamella are supported by gill cartilage (G).Secondary lamella (S) are shown. Bar - 250/xm.skin or fins, the clinician should flatten the sam-ple completely, increase microscope lighting(but keeping the condenser stopped down forcontrast), and focus up and down through sev-eral different planes to look for parasite move-ment. The novice may mistake pigmented cellsfor pathology. Check along the edges of darkfins, skin, or scales, and in the water immediatelysurrounding these areas.Parasites in FishUnderstanding fish parasites can appear to bea daunting task. External parasitism is a com-mon cause of "flashing" (fish rubbing againstsomething in their environment) and parasitesare frequently encountered during necropsy.Many important groups are uncommon in otherclasses of animals. The following is a very briefdiscussion of a few important groups, andshould only be considered a starting point. Forcomprehensive coverage including pictorials, re-fer to several excellent texts. 1,2,8,13ASA6 It be-hooves the fish clinician to understand the sig-nificance and life cycle of common parasites.The most common group, the ciliated proto-zoans, includes Ichthyophthirius multifiliis (whitespot, Ich) (Fig 8) in freshwater fish and Crypto-cauon irritans in marine fish (marine Ich). Bothspecies have a fairly broad host range, can mul-tiply rapidly, from one to several hundred in onegeneration, and require multiple treatments.Consequently, observation of just a few of theseon a fish is cause for concern. Other ciliates,such as Trichodina and Epistylis (and otherstalked ciliates), can be used as an indication ofwater quality conditions, since both are morecommon in highly organic waters. 1,2,16The flagellated protozoans (including Ichthyo-bodo necator, found externally, and Spironucleusvortens and Cryptobia iubilans (Fig 9), both foundin the gastrointestinal tracts of cichlids, areamong the smallest of the parasites of fish. Forexample, Ichthyobodo necator is approximately10 X 5 /xm and consequently, is much moreeasily identified initially by movement. M1 cancause significant damage to their hosts, althoughgranuloma formation is associated specificallywith Cryptobia iubilans. 17,18 The dinoflagellatesAmyloodinium and Piscinoodinium (Fig 10) arenonmotile, oval to rounded, granular, brown/yellow/or green, and can also be a cause foralarm. Their life cycles are similar to those of theFigure 7. Fin biopsy. Only a small section is requiredfor examination (target lesions).
  9. 9. Necropsy Techniques for Fish 97Figure 8. Ichthyophthirius multifiliis. 100x. Ciliated pro-tozoan, causative agent of freshwater "Ich" or white spot.Ichthyophthi~Jus multifiliis is one of the largest proto-zoan parasites of fish. Note U-shaped macronucleus."Ichs" and one parasite can become hundreds inone generation.The monogenes (Fig 11) are flatworms with adirect life cycle that can cause significant dam-Figure 10. Piscinoodinium on a fin clip. 100x. Notegranular appearance, and oval/tear drop shape. Par-asite is nonmotile.age to skin, fins, and gills. Although many re-quire a microscope for visualization, some arelarge enough to see unaided. Egg-laying speciesare more problematic because eggs are resistantto treatment.The microsporidians and myxozoans are twononmotile, spore-forming groups that are typi-cally found within granular-appearing cysts.These can be found anywhere in the body. Anycysts that appear to have granular structure (anddo not contain a worm) should be squashed torule out one of these two groups. Individualmicrosporidians are very small (requiring highpower magnification) and have a "woodenshoe/clog" appearance (with one structure, theposterior vacuole, resembling the opening toFigure 9. Stomach squash/wet mount from an Afri-can cichlid. 40x. Note granulomas (G) caused by C~yp-tobia iubilans.Figure 11. Monogene (Gyrodactylus sp.). Gyrodactylusis live bearing, as evidenced by the hooks located onthe embryo (E). The opisthaptor (O) contains nu-merous hooks and anchors. Bar -- 100 /xm
  10. 10. 98 YanongFigure 12. Digenean trematodes: encysted metacer-carial stages, coelomic cavity. 100x. Fish was clinicallynormal. Digenes in the gills can be more problematic,if numerous.the shoe). Myxozoans are larger (6 to 65 /xm),and more varied in shape. Different species con-tain 2 to 6 polar capsules. Because microsporid-ians, in general, have a direct life cycle, and mostmyxozoans studied have an indirect life cycle, itis critical to distinguish between the two.Digenean trematodes are flatworms with anindirect life cycle that, in freshwater fish, typi-cally involves a bird and a snail as intermediatehosts (although more complicated life cycles ex-ist). The life stage most commonly found in fishis the encysted metacercaria (Fig 12). Cysts aretypically oval to round, and differ somewhat inappearance from one species to another. Thesecan be found in almost any organ, and becomea problem if they interfere with organ function(such as overabundance in gill tissue). Somewild specimens can have tens or hundreds ofdigenes in their coelomic cavity, with no appar-ent ill effects. Adult digenes are less common,but may be found in fish. One distinguishingfeature of digenes is the presence of both an oral(or anterior) and ventral sucker on their body.Nematodes can be found in the gastrointesti-nal tract or other organs as adults or encystedjuveniles. Cestodes are less commonly found.Lernaea (anchoIworm- resembles one or sev-eral small, long filaments hanging from the fish)and Argulus (fish louse) are two common crus-tacean parasites, and can cause problems inmany species including goldfish and koi, butboth are easily identified and visible grossly.EuthanasiaAfter skin, gill, and fin biopsies have beentaken, provide a lethal dose of MS-222 to thefish. One recommendation 19 is for a dosage rateof 50 to 250 rag/L, buffered with sodium bicar-bonate (baking soda) at a two-part sodium bicar-bonate to one part MS-222 ratio. Fish should bekept at the dosage rate that results in anesthesiawithin 60 seconds 1 and should be kept there forapproximately 10 minutes after breathing (ces-sation of opercular movement) has occurred.However, in practice, some species may requirequite a bit more than 250 mg/L. As much as 600to 800 mg/L or more may be required to pro-duce a surgical plane of anesthesia within 60seconds (eg, for labyrinth fishes such as goura-mis). At these much higher doses, the fish areusually dead within several minutes.The lethal dose can be administered as a bathor administered via squeeze bottle to one set ofgills (left or right), avoiding dripping into theother side (in case additional sampling or his-topathological sections are required). The fishshould be still, no longer responsive to stimuli,and have lost the ocular righting reflex (eyes arein normal position, parallel to body, regardlessof position of the body--the clinician may haveto gently press the eye parallel to check).External ExaminationAfter the fish has been euthanized, a quick,but complete external gross examination shouldbe performed. Record a description of lesionsincluding size, location, and number. Deformi-ties should also be noted. Additional lesionsshould be sampled and wet mounted and exam-ined microscopically after microbiological sam-ples have been taken. Lateral, dorsal, and ven-tral view line drawings of a fish on the pathologyreport can be helpful for reference of lesions.Skin/Fins/Gills/OperculumExamine these structures for erosions, hem-orrhages, parasites, flmgi, and microscopicallyfor microbubbles (indication of supersatura-tion), telangectasia (ballooning of capillaries inthe gills--evidence of high ammonia or toxins),and other masses. Sampling method may also
  11. 11. Necropsy Techniquesfor Fish 99artificially induce telangectasia. Brownish ordark brown gills may indicate nitrite toxicitywhile pale white or pink gills indicate severeanemia in live fish. Be cautious, however, whenevaluating fish found dead. Gills degrade andturn white rapidly. Be sure to examine the innersurfaces of the opercula.MouthCheck external and internal (oral cavity,pharynx) regions for lesions/parasites.Vent~Anus~Genital PoreCheck each (if applicable, depending on thespecies) for swelling, redness, and protrusions(eg, nematodes).EyesExamine for cloudiness, hemorrhages, in-creased mucus, exophthalmia, endophthalmia,and buphthalmia. If indicated, scrape for exter-nal parasites.MuscleAreas that appear discolored, hemorrhaged,have masses, or are othm~ise abnormal shouldbe thinly filleted and wet mounted for examina-tion, following microbiological sampling.Microbiological SamplingAfter the external examination, the brain,kidney, liver (and spleen, if possible) should becultured for bacteria. Other internal organs, thecoelomic cavity, or coelomic fluids may also war-rant culture. Culture of ulcers may be useful andhelpful, but requires a great deal of skill andluck to isolate and differentiate primary initiat-ing bacteria from secondary invaders or water-borne contaminants. 1 Some disease-initiatingbacteria grow much more slowly than secondar-ies or contaminants, and may quickly over runthe plate. Most common bacterial pathogens offish will grow on TSA with 5% sheeps blood.There are some exceptions, including Flavobac-terium columnare, which requires Ordals media.Fungal cultures are useful for positive identifica-tion of external or internal fungal infections,although typical water mold infections can betentatively diagnosed based on microscopic eval-uation and clinical appearance. Corn-meal agarfor Oomycetes ("water mold" fungi) and Sab-ouraud dextrose agar for non-Oomycetes is rec-ommended. For specific procedures, consult afungal or general microbiology laboratory, orrefer to appropriate fish health texts. 1,2,16Before culturing the brain, use an alcoholpad to disinfect the dorsal part of the head,midline, but slightly caudal to the eyes. The sitecan be wiped dry with a sterile gauze pad, orallowed to evaporate. After the alcohol has evap-orated, a small, sharp, sterile scalpel blade (ethylalcohol dipped, flamed, and cooled) is used tocut through the top of the head into the braincase. Frequently a small amount of cerebrospi-hal fluid will leak out. The cut area can then besampled with a fine, sterile inoculating needle orswab and streaked onto a TSA with 5% sheepsblood plate or sent to a laboratory. Certain bac-teria, such as Streptococcus sp., can be readilyisolated from brain tissue.The kidney can be approached dorsally, ven-trally, or laterally. The ventral approach is pre-ferred if liver and spleen will also be sampled.For the dorsal approach the dorsal fin shouldfirst be cut off with scissors, and the dorsal areaof the fish wiped with isopropyl alcohol, espe-cially around the area to be cut. Flaming thewiped area with a barbeque lighter or allowing itto evaporate will help prevent excess alcoholfrom entering the cut section. The mid-region ofthe insertion of the dorsal fin is a good landmarkfor the cut in most fish, although some species,including swordtails, Corydorascatfish, and ple-costomus catfish, have the majority of their kid-ney located anteriorly (slightly caudal and dorsalto the opercular cavity). A sterilized pair of scis-sors (ethyl alcohol dipped, flamed, and cooled)is used to cut through skin and muscle to thekidney (Figs 13 and 14).The spinal column should be severed duringthe dorsal approach, since the kidney is directlybeneath the spine. Blood will ooze out of thearea, an indication that the kidney and near byvessels have been cut. A sterile loop should thenbe inserted into this area and streaked onto aTSA with 5% sheeps blood plate, or a cultureswab should be used to remove a sample. Care
  12. 12. 1O0 YanongFigure 13. Kidney culture, dorsal approach. The cutshould be around the level of the lateral line, only tothe area immediately ventral to the spine.should be taken to avoid cutting too deeply andinto other organs, especially the stomachand/or intestine, as contamination will result.The cut should be made at the level of the spine(the lateral line can be a good approximatelandmark).In some species, visualization of an intactswim bladder is a good indication that the gas-trointestinal tract has not been breached. Inother specms, cutting the swim bladder usingthis approach is unavoidable, but not a problemif the cut remains dorsal to the other organs.For the ventral approach, the fish should beplaced in right lateral recumbency, and the lat-eral body should be wiped down with alcohol. AFigure 14. Kidney culture dorsal approach. The cutis directly below the spine, without cutting into theswim bladder. The posterior kidney and spine areexposed.precise, shallow incision with a sterile scalpelblade should be made caudal to the operculum,to serve as an opening for blunt dissection scis-sors to enter and cut out an area of the bodywall. This step will expose most of the coelomiccavity, swinl bladder, and kidney. Cut from theinsertion point dorsally and caudally, and thenfrom the same point, toward the ventrum andcaudally. Care should be taken to avoid cuttinginto any organs, especially the intestines/analarea, to prevent contamination. The swim blad-der (or the air space of the swim bladder forthose species with delicate bladders) is moved tothe side (or deflated) and the kidney (visible asa red or dark red area immediately below thespine) is punctured with a sterile scalpel. A ster-ile culture swab or inoculating loop is then in-serted to take the sample. (Fig 15) After allcultures have been taken, this window should beextended cranially, including cuts through (orremoval of) the cleithrum (crescent-shaped, flatbone caudal to the operculum, that supports theshoulder girdle) to expose the anterior sectionof the coelomic cavity, as well as the pericardialcavity and heart.The lateral approach is similar to the ventralapproach except that instead of completelyopening up a window into the body cavity, ascalpel is used to cut a small slit directly into theswim bladder and kidney, just big enough forinsertion of a sterile swab or loop. This approachSFigure 15. Ventral approach, female koi. This koi isfull of eggs. Care must be taken to avoid contamina-tion by contact with the gastrointestinal tract. Notethe kois saddle shaped area of posterior kidney, lo-cated at the junction of the anterior and posteriorchambers of the swim bladder.
  13. 13. Necropsy Techniquesfor Fish 1O1iiiiiiiiiiiiiiiiiiiiiii!{i!!ii!ii!ii!iiiiii{ii!iii!!ii!i!!iii!i~i}ii!i~!!!i!iiiiiiiii!ili!!i!!iiiii!ii!!iili!i!i!i!ilili~ili!i:!il~ii5: i !il84 Z! 15 ;& i i!ii! i;!!ii!ii!ii!!iliiiiii!iii!iiiiiii!iiiilii~iiiiiiiiiii!~i!~ii~!i~i~i~!~i!i~!!~ii~ii~!i~ii~i!~i~i~i!!~ii~ii~ii~!~!i~!~iiii!~i!~i!~ii~!i~i~!~i~!!~ii~i!i%~i!iii~i!i~i!ii~!~i~i~~7 ~i~!~!~?i~ i!!~!~!ii!i!i!i~i~i!!ilili!iii!i!!iiiiiiiiiiiiiiii~ii~i~i!i~i}i!i~i}!i~!~i!i~!i~i!~!~i~i!!~ii~!i~!!~i!~}!!~!~i~i;i~!~!!~i!~ii!~i~!!i~i~!~!!!i!~!!~i~i~!~!~i~!~i~i~i~!~!ii~i!~i!~i!~i~!:~!~ii!iiii~ii!iiii!i?!!iiii!!iiiii!i!iiiiiiiiiii!iiiiiiiiiiiiiiiiiiiiiiiFigure 16. Koi, internal organs. Egg mass has beenremoved. Note intact, bi-lobed swim bladder, and sad-dle shaped section of posterior kidney. Liver is inti-Inately intertwined with the intestine, there is nostomach, and the spleen is a dark red elongate organ.assumes a good understanding of anatomy ofthe species examined.After the kidney of the fish has been asepti-cally sampled, the liver should be stabbed with asterile scalpel and the cut section sampled. Ifcontamination of the outer portion of the liver issuspected, the outer surface can be seared withthe fiat end of a heated scalpel blade beforestabbing. Splenic samples can be more challeng-ing. For larger specimens, the sample can betaken as per the liver, with careful deflection ofoverlying viscera. For smaller spleens, a verysmall piece can be crushed and pulled out witha sterile pair of forceps and this sample thenstreaked onto a plate or placed on a swab.Culture of ulcers can be complicated. Onegood recommendation is to culture early lesionsby placing a 1 /xL sterile loop into the leadingedge of the lesion (aseptically removing scales atthe leading edge to increase likelihood of cul-turing initiating pathogens) and place on to asmall 4 mm 2 area of a culture plate. Next, use asterile Mini-Tip Culturette (Becton-Dickinson,Sparks, MD, USA), to swab the inoculated areaonto half the plate. Finally, pull the streak acrossone quarter of the plate, and then across the lastquarter. This should help isolate individual col-onies, a Externally found saltwater bacterial spe-cies may require marine agar, or blood agar withadditional salt, for growth.Cultures should be incubated according tonormal temperature ranges for the species ex-amined (25 to 30~ for most warm water spe-cies). If mycobacterial infections are suspected,an appropriate mycobacterial media such as Lo-wenstein -Jensen media or Dorsets egg agarshould be used (in addition to TSA with 5%sheeps blood agar for other bacteria). Somerapid, less fastidious mycobacterial strains willgrow on blood agar in 4 to 7 days.Internal ExaminationAfter cultures have been taken internal or-gans should be examined grossly and microscop-ically by means of wet mount preparations. Asdescribed previously, differences among species,and even among varieties can be striking, so theclinician should be familiar with normal anat-only of some common species as a starting point.Koi (Figs 15 and 16) and fancy goldfish (Fig 17)are very popular water garden species and canbe quite expensive. Oscar (figs 18, 19, 20) anddiscus (Fig 21) are two very popular cichlid spe-cies that often foster strong owner-pet bonds.Small sections (approximately 1 cm-~) of eachorgan described in the "Basic Fish Anatomy andPhysiology" section, as well as specific lesions,should be fixed in 10% neutral buffered forma-lin for histological preparation and examina-tion. (The heart, brain, skin, muscle, eye, oper-cula and pseudobranch are often forgotten, sobe sure to include samples of each of these.)Samples for electron microscopy (EM) shouldFigure 17. Oranda (fancy goldfish), internal organs.Note intimate attachment of liver with intestine. Msonote small posterior chamber of the swim bladderrelative to the anterior chamber. This is genetic in thisvariety, and, along with the body shape, makes the fishprone to buoyancy control problems. Goldfish alsolack true stomachs.
  14. 14. 102 YanongFigure 18. Oscar (cichlid), internal organs, level one.Note large liver.be saved in fixative recommended by the pro-cessing laboratory. Trumps fixative is good formost EM studies.OperculumNote if pseudobranch is grossly visible/super-ficial, and general appearance.Swim BladderExamine for thickening, hemorrhages, necro-sis, fungi (some are pigmented), and parasites.K/dneyAnterior and posterior sections should besquashed separately. The anterior section canlook very much like the spleen on wet mountFigure 20. Oscar (cichlid), internal organs, levelthree, digestive tract exposed, swim bladder deflected.Note relatively short gastrointestinal tract length. Alsonote posterior kidney.examination. Tubules should be seen on poste-rior sections, and both sections often haveMMCs. Check for uniformity, granulomas, andparasites.L/verCheck for color, uniformity, fat content (doesit float in 10% buffered formalin?), MMCs, par-asites, and granulomas.Gall bladderLocated near or within the liver, it containsgreenish to yellowish fluid that is normally clear.Figure 19. Oscar (cichlid), internal organs, level two,u4th liver reflected. Note thick walled swim bladder,spleen. Stomach and intestine are surrounded by fat.Figure 21. Discus (cichlid), internal organs. Notelarge caxdtywhere swim bladder was located, dorsally.Kidney is visible. Also note location of heart andrelatively small area occupied by viscera.
  15. 15. Necropsy Techniquesfor Fish 103Figure 22. Koi, spleen, wet mount. MMC = melano-macrophage centers. 40x. Note absence of clear/on-ion skin appearance to area immediately surroundingdark MMCs (cf. Figure 24)It can be relatively large, especially if fish isanorexic.SpleenCheck for color, size, uniformity, MMCs, andgranulomas.GonadNote, by presence of eggs or testes, whetherthe fish is female or male. Check gonads for size,maturity, granulomas, or hardened structures(in females, degenerating eggs/"egg-binding").Note if sperm are present, active, and motile.Figure 23. Tilapia, cut section. Note grossly visiblegranulomas/nodules in kidney (K) and spleen (S).One of the major rule-outs is mycobacteriosis.or undesirable (pathogen) or foreign body.Granulomas are commonly associated with cer-tain bacterial, fungal, and parasitic infections,although these infections may occur withoutchronic, granulomatous lesions. Bacteria that of-ten cause granuloma formation include Mycobac-terium sp., Nocardia sp., and rickettsia-like organ-isms.Parasites that can cause granuloma formationinclude C~yptobiaiubilans in cichlids, nematodes,and digenean trematodes. Foreign bodies, suchas scales, spines, or plant material can causegranulomas as well. Ovaries that are attemptingto reabsorb over ripe eggs can have a granulo-matous-type reaction.StomachCheck for parasites and granulomas. (Re-member, true stomach not present in koi, gold-fish, or other cyprinids).Intestinal tractCheck for tile presence of food or fluid, hem-orrhages, ulcers, and parasites (especially nem-atodes and flagellates).MMCs (Fig 22) should not be confused withgranulomas (Figs 23 and 24), which are typicallymore "rocky" or sharp edged in appearance andsurrounded by a clear/onion skin area, or withoocytes (Fig 25). Granulomas in fish, as in otheranimals, are an attempt to wall off an irritatingFigure 24. Tilapia, spleen, wet mount, granulomas(G). 40x. Note clear area/halo/onion skin appear-ance around "rock-like," irregularly shaped darkstructures. Granulomas are often brownish in color.
  16. 16. 104 YanongFigure 25. Koi, oocytes. 40x. Oocytes in various stagesof maturity, from the ovary of an adult female. Notethe relatively rounded shape, and grainy appearancein the large, mid to late stage maturity eggs. Immatureoocytes are much smaller and have clear cytoplasm.VirologyViruses in ornamentals are not well studied.Because of the lack of necessary cell lines forvirus isolation and research, diagnosis of a viraldisease is often based solely on history, clinicalsigns, rule out of other causes, and presence ofclear histopathologic lesions and electron micro-scopic (EM) visualization of viral particles withinthese lesions. Some viral diseases, such as lym-phocystis (caused by an iridovirus) and koi pox(caused by a Herpesviruscyprini), have somewhatpathognomonic lesions, and may only requirehistology or virus isolation for absolute certainty.Certain viruses in fish are considered of concernby the international community. Spring viremiaof carp (SVC), recently reported in the U.S., is anotifiable disease of koi and several other re-lated species and requires isolation and culturefor verification. 2~ There are only three labora-tories in the U.S. that are presently USDA ac-credited to test for SVC (see appendix 1). Clin-ical signs of SVC are nonspecific and includedarkening, external hemorrhages, exophthal-mia, ascites, pale gills, and a protruding vent.Edema and generalized inflammation and pete-chiae may be present in multiple organs inter-nally including the swim bladder. Splenomegalymay be apparent. Concurrent infection withAeromonas sp. or other bacteria may lead to in-accurate diagnoses. 2~ Contact your state veter-inarian and one of these local laboratories forproper sample submission protocols if you sus-pect SVC. Koi herpes virus (KHV--not the samevirus as the etiologic agent of carp pox)is also avery pathogenic virus with high associated mor-talities that should be isolated and cultured forpositive diagnosis. 2a The only consistent sign infish with KHV is pale or irregularly coloredgills.23SummaryThe mastery of necropsy techniques impliesan understanding of pathological findings.There are a number of excellent references thatshould be a part of any fish veterinarians li-brary. 1,z,8,15,16Water quality problems are alwayshigh on the differential list, at the very least as acontributing factor. Dissolved oxygen problems,excess ammonia and nitrite, improper or fluctu-ating pH or temperatures, and supersaturationcan all lead to acute or chronic disease. Some ofthese problems can manifest themselves in gill,skin, or fin pathology. Introduction of toxins,including chlorine and copper, and improperuse of chemotherapeutants can also result invisible pathology.Infection by bacteria and parasites is oftensecondary to husbandry or management prob-lems. External examination, microscopic evalu-ation, and a good history and assessment of hus-bandry practices are often enough to determinecourse of action.Although ornamental fish virology is still in itsinfancy, viruses in some species, such as koi, havegained more research attention. Clinical signs ofdisease and pathology associated with SVC andKHV are not specific and virus isolation is re-quired. SVC is a notifiable disease in the U.S.22Forother viruses, presence of histopathologic lesionsand visualization of viral particles in these lesionsusing transmission electron microscopy is corrob-orating evidence but not definitive.Appendix 1: USDA approveddiagnostic laboratories capable oftesting for spring viremia of carp.1. University of Arkansas-Pine Bluff, Coopera-tive Extension Program, PO Box 4912 or1200 University Drive, Pine Bluff, AR 71611,Phone: (870) 543-8537.2. Pennsylvania Animal Diagnostic Laboratory
  17. 17. Necropsy Techniques for Fish 105System, State Veterinary Laboratory, 2305North Cameron Street, Harrisburg, PA17110, Phone: (717) 787-8808.3. Washington Animal Disease Diagnostic Lab-oratory, College of Veterinary Medicine,Washington State University, PO Box 647034,Pullman, WA 99164-7034, Phone: (509)335-9696.AcknowledgmentsSpecial thanks to Deborah Britt Pouder for her expel--rise and assistance with photography and dissection.References1. Noga EJ: Fish Disease: Diagnosis and Treatment. St.Louis, MO, Mosby, 19962. Stoskopf MK: Fish Medicine. Philadelphia, SaundersCo., 19933. Stoskopf MK: Tropical fish medicine. Vet Clin N Am(Sm Attim Pract) 18 (2):entire edition 19884. Klinger R, Francis-Floyd R, Riggs A: A nonlethal ap-proach to diagnosing bacterial disease, in: 2001 Proceed-ings, Annual Conference, International Association forAquatic Animal Medicine. Tampa, FL, International As-sociation for Aquatic Animal Medicine, 2001, p. 925. Evans HE: Anatomy of tropical fishes, in Gratzek JB,Matthews JR (eds): Aquariology Master Volume: TheScience of Fish Health Management, Morris Plains, NJ,Tetra Press, 1992, pp. 71-936. Moyle PB, CechJrJJ: Fishes: an Introduction to Ichthy-ology (ed 2). Englewood Cliffs, NJ, Prentice-Hall, Inc.19887. Walker WF Jr, Liem KF: Functional Anatomy of theVertebrates, An Evolutionaiy Perspective (ed 2). Or-lando, FL, Saunders College Publishing, Harcourt BraceJovanovich, 19948. Wildgoose WH (ed): BSAVAManual of ornamental fish,(ed 2). Gloucester, England, British Small Animal Vet-erinary Association, 20019. Ferguson H: Systemic Pathology of Fish. Ames, IA, IowaState University, 198910. Pdnzes B, T61g I: Goldfish and Ornamental Carp. Haup-page, NY: Barrons Educational Series, Inc., 198611. Francis-Floyd R: Behavioral diagnosis, in Stoskopf, M(ed): Tropical Fish Medicine Vet Clin N Am (Sm AnimPract) 18(2):303-314, 198812. Callahan HA, Noga EJ: Tricaine dramatically reducesthe ability to diagnose protozoan ectoparasite (Ichthyo-bodonecator)infections. J Fish Dis 25(7):433-438, 200213. Hoffman GL: Parasites of North American FreshwaterFishes (ed 2). Ithaca, NY, Cornell University Press, 199914. Khoo L, Leard AT, Waterstrat PR, Jack SW, Camp KL:Branchiomyces infection in farm-reared channel catfish,Ictaluruspunctatus (Rafinesque). J Fish Dis 21:423-431,199815. Lewbart CA: Self Assessment Color Review of Ornamen-tal Fish. Ames, IA, Iowa State University, 199816. Woo PTK: Fish Diseases and Disorders Vol 1: Protozoanand Metazoan Infections. Cambridge, UK, UniversityPress, CAB International, 199517. Francis-Floyd R, Roese H, Klinger R, Yanong R, Blazer V:Diagnosis and clinical management of gyanulomatousgastritis in African cichlids, in: Proceedings, 29thAnnualConference, International Association for Aquatic Ani-mal Medicine. San Diego, CA, 1998, pp 13%13118. Yanong R, Curtis E, Francis-Floyd R, et al: Granuloma-tous gastritis in juvenile discus (Symphysodon sp.), in:Proceedings, 30th Annual Conference, International As-sociation for Aquatic Animal Medicine. Boston, MA,1999, pp 163-16419. Report of the AVMA Panel on Euthanasia, 2000.JAVMA2i8(5):669-696, 200120. Fijan N: Spring viraemia of carp and other viral diseasesand agents of warm-water fish, in Woo PTK, Bruno DW(eds) : Fish Diseases and Disorders Vol 3: Viral, Bacterial,and Fungal Infections. New York, NY, CABI Publishing,1999, pp 177-24421. Goodwin AE: First report of spring viremia of carp virus(SVCV) in North America. J Aquatic kalim Health 14:161-164, 200222. Hedrick RP, Gilad O, Yun S, et al: A herpesvirus associ-ated with mass mortality of juvenile and adult koi, astrain of common carp. J Aquatic Anim Health 12:44-57,200023. Petty BD, Riggs AC, Klinger RE, Yanong RPE, Francis-Floyd R: Spring viremia of carp. Fact Sheet VM-142.University of Florida Cooperative Extension Service,Gainesville, FL, University of Florida, July 2002 (availableon the web at: http://edis, ifas. ufl. edu/VM106)

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