DNA viruses associated with diseases of marine and ...

HELGOL,~NDER MEERESUNTERSUCHUNGEN Helgol~nder Meeresunters . 37, 289-307 (1984)

D N A v iruses a s s o c i a t e d w i th d i s e a s e s of m a r i n e and

a n a d r o m o u s f ish

F. M. Hetr ick

Department of Microbiology, University of Maryland; College Park, AID 20742, USA

ABSTRACT: The associat ion of DNA-conta in ing viruses with diseases of mar ine and anadromous fish is reviewed. One section of the review descr ibes those diseases with a proven viral etiology. Available information on the physical, chemical , and biological propert ies of the viruses is included. Another section deals with those diseases where a viral etiology is suspected but not established. The primary evidence associat ing viruses with many of these diseases is the observation of virus particles in electron micrographs of thin sections of tissue samples from diseased fish. Finally, the possible role of pollutants, and other stress factors, in predisposing fish to viral infection is discussed as are the problems associated with s tudying diseases of wild fish populations.

I N T R O D U C T I O N

T h e s y s t e m a t i c s t u d y of v i r a l d i s e a s e s of f i sh b e g a n w i t h t h e d e v e l o p m e n t of t h e f i rs t

f i sh ce l l l i n e b y Wol f i n 1960. T o d a y t h e r e a r e m o r e t h a n 25 c o n f i r m e d or s u s p e c t e d v i r a l

d i s e a s e s of f i sh [ see r e v i e w s b y M c A l l i s t e r (1979); P i l c h e r & F r y e r (1980); W o l f (1982)]. As

in o t h e r t y p e s of i n t e n s i v e a n i m a l h u s b a n d r y , v i r u s e s c a n c a u s e e x p l o s i v e d i s e a s e

o u t b r e a k s in f i sh f a r m i n g o p e r a t i o n s . W h e n t h e s e e p i z o o t i c s occur , m a n y t i s s u e s a m p l e s

a re a v a i l a b l e for l a b o r a t o r y w o r k u p . A l so f ish, g e n e r a l l y h o m o g e n o u s w i t h r e s p e c t to

s ize a n d age , a re a v a i l a b l e as h o s t s for e x p e r i m e n t a l t r a n s m i s s i o n s t u d i e s to t e s t t h e

p a t h o g e n i c i t y of a n y v i r a l i so la t e s . C o n s e q u e n t l y , m o s t of t h e b e t t e r c h a r a c t e r i z e d v i r u s e s k n o w n t o d a y a r e p a t h o g e n s for f r e s h w a t e r a n d a n a d r o m o u s f i sh s p e c i e s t h a t a r e

c u l t u r e d o n a l a r g e s c a l e a n d h e n c e e c o n o m i c a l l y i m p o r t a n t .

V i r a l d i s e a s e s of m a r i n e f i sh a r e m o r e d i f f i cu l t to s t u d y s i n c e t h e y i n v o l v e w i l d

r a t h e r t h a n c a p t i v e p o p u l a t i o n s . C u r r e n t m e t h o d o l o g y f a v o r s t h e d e t e c t i o n of t h o s e

a g e n t s a s s o c i a t e d w i t h l a r g e f i sh k i l l s , s u c h as t h e a n n u a l s p r i n g e p i z o o t i c s i n t h e

A t l a n t i c m e n h a d e n ( S t e p h e n s e t al., 1980), or w i t h c h r o n i c d i s e a s e s l i k e l y m p h o c y s t i s . It

i s c e r t a i n t h a t m o r e d i s e a s e s w i t h a v i r a l e t i o l o g y o c c u r i n m a r i n e f i sh t h a n a r e c u r r e n t l y

k n o w n . If t h e d i s e a s e s a re e n z o o t i c , r a t h e r t h a n ep i zoo t i c , t h e y w o u l d p r o b a b l y go

u n n o t i c e d . If t h e y o c c u r i n c o m m e r c i a l l y u n i m p o r t a n t " t r a s h " f ish, t h e y w o u l d n o t l i k e l y

b e i n v e s t i g a t e d .

N o n e t h e l e s s , t h e l is t of d i s e a s e s a s s o c i a t e d w i t h m a r i n e f i sh is g r o w i n g ( S i n d e r -

m a n n , 1970). Hil l , i n t h i s s y m p o s i u m , w i l l r e v i e w t h o s e v i r u s e s w i t h a n R N A g e n o m e t h a t

a r e of i m p o r t a n c e in t h e m a r i n e e n v i r o n m e n t . T h i s r e p o r t d e s c r i b e s t h e D N A - c o n t a i n i n g

v i r u s e s t h a t h a v e b e e n a s s o c i a t e d w i t h d i s e a s e s of m a r i n e a n d a n a d r o m o u s f i sh

( T a b l e 1). T h e f i rs t s e c t i o n d e s c r i b e s t h o s e d i s e a s e s w i t h a k n o w n v i r a l e t i o l o g y w h i l e t h e

© Biologische Anstalt Helgoland, Hamburg

290 F . M . Hetr ick

Table 1. Diseases of marine and anadromous fish for which DNA viruses have a proven or suspected etiology

Disease Virus group Species affected

V i r a l e t i o l o g y c o n f i r m e d

Lymphocystis

Oncorhynchus masou virus (OMV)

Salmonid Herpesvirus disease

Erythrocytic necrosis

Iridovirus

Herpesvirus

Herpesvirus

Iridovirus

Wide range of fresh and saltwater fish (> 80 species)

Rainbow trout; masu, chum, coho, and kokanee salmon

Rainbow trout, kokanee or sockeye salmon

Variety of marine and anadromous fish (> 20 species)

V i r a l e t i o l o g y s u s p e c t e d

Carp gill necrosis Iridovirus Carp

Epidermal hyperplasia Adenovirus Cod

Epithelioma papillosum Herpesvirus Carp, chub, roach (carp pox)

Herpesvirus scopthalmi Herpesvirus Turbot (disease of turbot)

Pacific cod Herpesvirus Herpesvirus Pacific cod

Ulcus syndrome Iridovirus, Rhabdovirus Atlantic cod

Papillomatosis (125-150 nm particle)? Atlantic salmon

Eel stomatopapilloma (52-56 nm particle)?, European eels (cauliflower disease) Orthomyxovirus

second sect ion is devo ted to a discussion of those d iseases for which a viral e t io logy is

suspec ted but not proven.

DISEASES WITH A K N O W N VIRAL ETIOLOGY

L y m p h o c y s t i s

Lymphocyst is is a chronic viral d isease of marine, estuarine, and freshwater fish species which has a wor ldwide distribution. The disease was first repor ted in European

f lounder (Pleuronecte$ [lesus); the first ex tens ive study of lymphocyst is was ini t ia ted by

We i s senbe rg (1914) and cont inued for near ly 50 years (Weissenberg, 1965). The characterist ic feature of the d isease is the d e v e l o p m e n t of macroscopica l ly vis ible whit ish

nodules , or groups of nodules , r e s emb l ing warts on the skin and fins of affected fish. A l though the d isease is rarely fatal, fish wi th the d isease are uns ight ly and, if caught, are

d iscarded for aes thet ic reasons. At least 83 species of fish from 33 famil ies in 7 orders are suscept ib le to the disease

(McCosker et al., 1976; Lawler et al., 1977). Some of the species more recent ly shown to

DNA viruses associated with fish diseases 291

be infected include: win te r f lounder (Pseudopleuronectes americanus) reported by Murchelano & Bridges (1976); Baltic f lounder (Platichthys flesus) reported by Vit insh & Baranova (1976) and Russell (1974); Baltic her r ing (Clupea harengus var membras) reported by Aneer & Ljungberg (1976); plaice (Pleuronectes platessa) reported by Russell (1974); At lant ic croaker (Micropogon undula tus) reported by Howse & Chris tmas (1971); yel lowfin sole (Limanda aspers)reported by Alpers et al. (1977); and striped bass (Morone saxatilis) by Krantz (1970).

Outbreaks of lymphocystis also occur in cul tured fish. St ickney & White (1974) reported infection of t ank-cu l tu red southern f lounder (Paralichthys lethostigma and P. dentatus) and Paperna et al. (1982) descr ibed an ou tbreak in Sparus aurata cul tured in the Gulf of Aqaba (Red Sea). Despite the broad host r ange of lymphocyst is virus, the infection has never been reported in sa lmonid fish. Lymphocystis is a par t icular p roblem to aquarists as in t roduct ion of a s ingle infected fish can result in all fish becoming infected because of the close conf inement and the broad host range of the virus. Since the disease is slow in developing, the p rob lem may not be ev ident soon enough to cull out the infected fish.

There appears to be some host in f luence on the virus. The disease can usual ly be t ransmit ted among members of the same genus with ease, however, t ransmiss ion be tween fish from different famil ies is difficult and somet imes impossible. Variat ions in the seasonal p reva lence of the disease have also b e e n reported for different host species. Weissenberg (1945) reported peak occurrences among centrarchids in the win te r whi le Nigrell i (1954) found the disease to be more common in freshwater species dur ing the summer months, vir tual ly d i sappear ing in fall and winter.

The virus is in t roduced into the water via epi thel ia l cells shed from infected fish or directly from dis in tegra t ing cells. The portal of entry for the virus into ne w hosts is probably through abras ions or in jur ies to the skin or fins since the warty growths usual ly appear on the areas most vu lne rab le to injury. The oral route of t ransmiss ion may also occur if the host feeds on infected fish. In these cases, the lymphocyst is cells occur in the gut, heart, and other in terna l organs. Addit ional ly , the large lymphocyst is cells may fall to the bottom where they may be inges ted by bottom feeding fish. There is no ev idence that the virus is t ransmit ted by fish eggs, however, t ransportat ion of fish with the agen t is a means of spreading the disease from one area to another.

There is no known therapy for lymphocystis. Control measures involve restriction of movement of infected fish, removal of dead fish from ponds or aquaria , and good sani tat ion practices in fish cul ture facilities. The disease can be d iagnosed solely on the observation of the characteristic cell hyper t rophy (Weissenberg, 1965). Lymphocystis- infected cells usual ly range from 100-250 ~tM in d iamete r and may at ta in sizes up to 2 mm in flounder. The cells have a thick (8-10 ~tM) hya l ine membrane , large nucle i and nucleoli, and large basophi l ic inclus ions at the per iphery (Wolf, 1968).

Since cell-free filtrates of infected t issues produced the disease w h e n inocula ted into fish, a viral etiology for the disease was suspected (Weissenberg, 1951; Wolf, 1962). Electron microscopic ev idence for virus invo lvement was reported by Walker (1962) who observed numerous virions, about 200 nm in diameter , in lymphocyst is tumor cells of perch. Walker & Wolf (1962) found s imilar part icles in lymphocyst is cells of b luegi l l s (Lepomis macrochirus), often in crystal l ine arrays. The virus is most l ikely a m e m b e r of the Iridovirus group.

292 F . M . Hetrick

Isolation of lymphocyst is disease virus (LDV) was first accomplished by Wolf et al. (1966) from infected b luegi l l s in centrarchid fish cell lines. The virus can be serially propagated in b lueg i l l fry (BF-2) cell cul tures in which hypertrophy occurs within 2 to 3 weeks at 25 °C. Virus propagated in cell cul tures produced lymphocystis disease when inocula ted into normal b luegi l l s and virus was subsequen t ly isolated from the lesions (Wolf et al., 1966). The incuba t ion period for lymphocyst is is usual ly several weeks in exper imenta l ly infected bluegi l ls . Progeny virus was first detected at 5 days and increased in titer up to 12 days fol lowing exposure. The deve lopment of lymphocystis lesions para l le led the virus mul t ip l ica t ion and they con t inued to increase in size up to 30 days post- infect ion (Dunbar & Wolf, 1966; Wolf & Carlson, 1965).

Electron microscopy of infected BF-2 cells (Zwil lenberg & Wolf, 1968) showed polyhedra l vir ions about 300 n m in diameter . Mature vir ions do not form in the ma in subs tance of the cytoplasmic inclusions but are located at its surface or scattered elsewhere. The inclus ions are Feu lgen posit ive and show ye l low-green fluorescence after acr id ine orange s ta in ing (Walker, 1965) ind ica t ing the presence of DNA, presum- ably viral in nature.

Size est imates of LDV from different laboratories have var ied from 130-150 nm (Walker & Weissenberg , 1965) to 300 nm (Zwi l lenberg & Wolf, 1968) in diameter d e p e n d i n g on the mater ia ls and host species examined . Robin & Berthiaume (1981) noted two dist inct bands w h e n LDV was purif ied by isopycnic centr i fugat ion in metriza- mide gradients. Examina t ion of the bands by electron microscopy showed two sizes of particles: small, dense particles measur ing 100-150 nm and lymphocystis virions that measured 300-350 nm in diameter . Since all of the infectivity was associated with the b a n d con ta in ing the larger particles, one hypothesis p resen ted was that the smaller part icles were defect ive- interfer ing {DI) particles.

Madely et al. (1978) s tudied the fine structure of LDV from European f lounder and plaice. The virus particles were 175 to 260 nm across and 200-300 nm be tween the vertices. The surface was devoid of clearly ident i f iable subuni ts . With storage at 4 °C, the hexagona l out l ine was lost and knob- l ike subuni ts became visible. They were about 4.5 nm in d iameter and appeared to be connected to the rest of the virion by a narrow stalk. Also seen in the b roken-down virus was a spherical core with a tubular compo- nent , 13 n m in diameter , wi th in it. This tubular mater ia l appeared to have a periodicity sugges t ing a hel ical conformation and may be a nucleocapsid. Analyses of the LDV genome has recent ly b e e n reported (Darai et al., 1983). The genome is a double- stranded, l inear DNA molecule with a molecular weight of 93 ___ 44 × 106. Denaturat ion and r e a n n e a l i n g of the DNA resul ted in the formation of circular molecules sugges t ing that t e rmina l r edundancy is present. Cytosine res idues appear to be methylated. Restric- t ion enzyme pat terns and Southern blot hybr idizat ions separated the isolates into two classes: one found in f lounder and plaice, and the other in lesions of dabs.

Flfigel et al. (1982) character ized the proteins from LDV isolates made from flounder, dab, and plaice. At least 33 structural proteins, with molecular weights r ang ing from 14 to 220 K, were detected by PAGE analysis of pur i f ied virus preparations. Although the protein pat terns were similar for the different isolates, sl ight but distinct differences were also noted.

Walker & Hill (1980) reported a deta i led s tudy on the growth of LDV in a cell l ine (BF-W) der ived from the fins and caudal t issue of b luegi l l sunfish. The virus was

DNA viruses associa ted with fish diseases 293

quant if ied by an en la rged-ce l l enumera t ion assay. Growth s tudies ind ica ted that maxi-

mum virus titers of about 106 ce l l - en la rg ing uni t s /ml were ob ta ined after 14 to 21 days

incubat ion at 25 °C. Virus repl ica t ion also occurred at 20 °C but the f inal titers a t ta ined were approximate ly one log lower. At 15 °C, there was only min ima l viral mul t ip l ica t ion

and the virus not only fa i led to mul t ip ly at 30 °C but s e e m e d to be inac t iva ted at this

temperature . Viral infect ivi ty was not affected by ul t rasonic t reatment , f reez ing and thawing, or

long- term storage in m a i n t e n a n c e m e d i u m at 4 or -- 70 °C. Infect ivi ty was r educed by

t rea tment with ether and viral repl ica t ion was inh ib i ted in the p resence of a 1 mM concentrat ion of the pyr imid ine ana log 5-bromodeoxyur id ine .

Oncorhynchus masou v i r u s

Kimura et al, (1981a) repor ted the isolat ion of a virus in ra inbow trout gonad (RTG-2)

cell cultures from ovar ian fluids of normal adult masu sa lmon (Oncorhynchus masou) during a survey of cul tured sa lmon in Hokkaido, Japan. The agen t was found to have

characterist ics of the Herpesv i rus group and has b e e n provis ional ly n a m e d O. masou virus (OMV). The virus was subsequen t ly shown to be le thal for chum sa lmon (0. keta), coho salmon (0. kisutch), k o k a n e e salmon (O. nerka) and ra inbow trout (Salmo gairdneri). Infected fish were anorexic and exoptha lmia and pe tech ia t ion of the body surface

occurred in some fish. His topa thologica l examina t ion of l iver sections showed mul t ip le necrotic foci and syncyt ium deve lopment . Electron microscopy of infected l iver t issue

showed both empty capsids and nuc leocaps ids in the nucle i of infected cells.

Exper imenta l infect ion of chum salmon showed that fry could be infected by

immers ion for 1 h in 10°C wate r conta in ing 100 TCIDs0/ml of OMV. The age of the fish employed was inf luent ia l in the ou tcome of expe r imen ta l infection. E ighty-day-o ld fry

began to show mortal i t ies at 11 to 12 days pos t -exposure and 60 % of t hem succumbed

within 65 days. OMV was isolated from most mor ibund fish e x a m i n e d with virus titers

be ing approximate ly 106 TCIDs0/g of infected tissue. W h e n 150-day-old fry were used, deaths did not occur until 20 days post- infect ion and 35 % of them died in the ensu ing

120 days. There were no deaths among 240-day-old fish immersed in virus and addit io- nally in jec ted in t raper i toneal ly with 200 TCID50 of virus (Kimura et al., 1981a). In a

subsequent study, Kimura et al. (1983) eva lua ted the suscept ibi l i ty of fry of five sa lmonid species (chum salmon, masu salmon, kokanee salmon, coho salmon, and ra inbow trout)

to OMV. Using one-month-o ld fry, the re la t ive sensi t ivi ty in terms of pe rcen t mortal i ty was kokanee sa lmon 100 %, masu sa lmon 87 %, chum sa lmon 83 %, coho sa lmon 39 %,

and ra inbow trout 29 %, respect ively.

The virus repl ica ted in all seven sa lmonid cell l ines in wh ich it was tested (Kimura

et al., 1981c), but it did not g row in four non-sa lmonid cell l ines -b rown bu l lhead (BB), fa thead m i n n o w (FHM), ep i the l ioma of carp (EPC), and sea b ream k idney (SBK). The

virus repl ica ted opt imal ly at 15 °C but not at all at 25 °C or higher . Dist inct ive CPE and

syncytium formation occurred wi th in 5-7 days at 15°C with m a x i m u m titers a t ta ined

be ing approximate ly 106 TCIDs0/ml. Electron microscopy of infected cells r evea led hexagonal capsids in the nuc leus approx imate ly 115 nm in diameter . Enve loped virions,

200-250 nm in diameter , were seen at the cell surface and inside cytoplasmic structures.

The virus was found to be heat (50°C for 30 min) and acid (pH 3) labile. It was

294 F . M . Hetrick

inact iva ted by t rea tment with ether, and was inh ib i t ed by 50 ~tg/ml of the pyr imidine ana logue , 5- iododeoxyuridine. Replicat ion was also inh ib i ted by phosphonoacet ic acid and acycloguanosine , two newly deve loped ant i -herpesvi rus drugs. The virus was found to be serological ly dist inct from Herpesv/rus salmonis. Taken together, the above virus characterist ics clearly indicate that OMV is a n e w fish herpesvirus.

In addi t ion to producing acute disease in juven i le salmon, OMV has also been reported to induce epi thel ia l tumors among survivors of the acute infect ion (Kimura et al., 1981b, c). Fifty-two 5-month-old chum sa lmon that survived exper imenta l infection were held for addi t ional observation. After 4 months, neoplasms appeared around the mouth and subsequen t ly on the eyebal l and caudal fin. Tumors were noted in more than 60 % of the fish by 8 months post-infection. Some fish deve loped mul t ip le tumors and one had a tumor in the kidney. Viral part icles were not seen in the tumor cells, however, OMV isolat ions were made from one tumor t issue sample that appeared necrotic on day 275 and from primary cell cultures prepared from the tumor tissue of another fish sampled on day 296 postinfection.

A repeat exper iment in chum sa lmon gave the same genera l results, i.e. tumors deve loped in survivors approximate ly 4 months post-infection. Similar results were ob ta ined in exper iments with coho sa lmon and ra inbow trout. Thus, OMV shows pa thogenic i ty for sa lmonids not on ly in caus ing hepat ic necrosis and death, but also in i n d u c i n g tumors among m a n y survivors of the acute disease. These e legan t studies by Kimura and his coworkers are the first descript ion of a bonaf ide oncogenic virus for fish.

S a l m o n i d h e r p e s v i r u s d i s e a s e

An agent , with the genera l characteristics of a herpesvirus, was isolated from broodstock ra inbow trout suffering pos t spawning losses at the Nat ional Fish Laboratory in Winthrop, Washington, USA (Wolf, 1976}. Infected ra inbow trout became lethargic before death and exoptha lmia was common. Many fish da rkened in color, showed abdomina l distension, and some had thick fecal pseudocasts. Histological examina t ion showed major pathological changes in the gills, heart, kidneys, and liver. There was widespread fatty infi l t rat ion and edema in the liver. In some cases, Cowdry Type A in t ranuc lea r inclus ions were seen in l iver cells and syncytia were observed in pancreat ic t issue (Wolf, 1979}.

Character iza t ion of the virus, n a m e d Herpesvirus salmonis, was first reported by Wolf et al. {1978}. Infect ion of RTG-2 cells resul ted in the formation of syncytia and Type A in t ranuc lea r inclusions. Studies on the growth kinet ics of the virus revealed an u n u s u a l characteristic of the virus, namely, an opt imal tempera ture for repl icat ion b e t w e e n 5-10 °C. Syncytia formed at 0 °C but the infect ion was otherwise abortive. Viral repl icat ion was depressed at 15 °C and complete ly inh ib i ted at h igher temperatures. The virus was acid, heat, and chloroform labi le bu t s table to freezing and thawing. Viral DNA had a buoyan t dens i ty of 1.709 g/crn 3 and a ~uan ine-cy tos ine value of 50 %. Electron microscopic examina t ion of infected cells showed hexagonal nucleocapsids approximate ly 90 n m in d iameter which were first seen in nuc le i at 36 h. Enveloped vir ions measured 150 n m in d iameter and occurred both cytoplasmical ly and extraceltu- larly. Infectivity studies showed that cel l -associated virus t i tered approximately one log h igher than that ob ta ined with fluids from infected cells.


The disease can be reproduced in ra inbow trout fry and small f inger l ings by inoculat ion of cell culture grown virus (Wolf et al., 1975a, b, 1978). In several trials at temperatures from 6 to 12 °C, 3 to 6 weeks e lapsed before the first dea th wi th addi t iona l mortalities occurring dur ing the next 3 to 4 week period. Virus was readi ly isolated from the tissues of mor ibund fish. As with the na tura l disease, the major pathological changes in exper imenta l ly- infected fish were found in the gills (epithelial edema), heart (edema and necrosis), k idney (edema), and liver (fatty vacuola t ion and edema). The rectal portion of the in tes t ine showed mucosal necrosis and s loughing of cells which contr ibuted to the formation of fecal casts (Wolf, 1979).

In a more recent study, Wolf & Smith (1981) s tudied the pathological changes in parenteral ly- infected ra inbow trout. The virus produced a genera l i zed infect ion wi th in 2 to 3 weeks. Highest virus levels (108 TCIDs0/g of tissue) were found in the k idney with lesser amounts be ing present in the stomach, liver, a nd intest ine. Visceral organs a nd the heart showed major pathological changes, and syncytia in the pancreas were considered to be pathognomonic .

Rainbow trout fry and f inger l ings and kokanee or sockeye sa lmon are the only known susceptible species. Since H. salmonis was or iginal ly isolated from ovar ian fluid samples, vertical t ransmission of the agen t may occur. Fish to fish t ransmiss ion is assumed dur ing epizootics. However, in a control led s tudy (Wolf & Smith, 1981), the disease failed to spread to normal ra inbow trout cohabi ta ted with infected fish for 9 days.

A very similar agent has b e e n isolated in Japan. Beg inn ing in 1970, epizootics among fry of another sa lmonid species (Oncorhynchus merka) were noted a n n u a l l y in Japanese fish farms from June to Sep tember (Sano, 1976). In 1972, a virus was isolated which formed syncytia in RTG-2 cells and which was shown to have typical herpesvirus morphology. The agent was named NeVTA virus for nerka virus in Towanda Lake, Akita and Amori prefectures.

Sano (1976) bel ieves that NeVTA virus is different from H. salmonis even though it has the same low tempera ture r equ i remen t for replication. He reported an 80 % mortality rate among na tura l ly infected O. nerka. In contrast, the North Amer ican isolate ki l led ra inbow trout but no young O. nerka. Studies u t i l iz ing more modern techniques , such as DNA-DNA hybridizat ions and an t igen ic characterizat ions with monoc lona l antibodies, are needed to clarify the re la t ionships be t w e e n H. salmonis and NeVTA and with OMV as well.

V i r a l e r y t h r o c y t i s n e c r o s i s

Infections of erythrocytes character ized by cytoplasmic inc lus ion bodies and nuc lea r al terat ions have been reported in a wide range of poiki lothermic ver tebrates i nc lud ing amphib ians (Bernard et al., 1968) and repti les (Stehbens & Johnston, 1966). However, the disease has been most s tudied in mar ine and anadromous fish and this discussion wil l be confined to those infections.

Piscine erythrocytic necrosis {PEN} was the n a m e given by Laird & Bullock (1969) to describe a pathological condi t ion in the erythrocytes of three fish species, cod CGadus morhua), seasnai l (Liparia attanticus) and scu lp in CMycocephalus scorplus] collected in coastal waters of eastern C a n a d a and the no r theas t em Uni ted States. The infect ion is easily ident if ied by the presence in affected erythrocytes of characteristic acidophilic,

296 F . M . Hetrick

in t racytoplasmic inclus ion bodies (Appy et al., 1976; Walker & Sherburne, 1977). Nuclear degenera t ion is also f requent ly observed, especial ly in Atlantic cod (Walker & Sherburne, 1977). Since there is r e d u n d a n c y in referr ing to PEN in specific fish, the infect ion is now more appropr ia te ly known as VEN, for viral erythrocytic necrosis (Evelyn & Traxler, 1978).

The host range of VEN is broad and inc ludes over 20 species of mar ine and anadromous fish. There is only l imited dis t r ibut ion data on VEN. To date, the major species involved are cod and herr ing off the eastern coast of North America, salmon and herr ing from the Pacific coast of North America, dogfish from the Bay of Naples, and cod and b l e n n y in coastal and offshore waters of the United Kingdom (Small & Egglestone, 1980). The p reva lence of the infect ion ranges b e t w e e n 1 and 90 % d e p e n d i n g on the species and geographic location sampled. The in tens i ty of infect ion in any indiv idual may vary from only a rare inc lus ion body to vi r tual ly 100 % of the erythrocytes examined (Nicholson & Reno, 1981).

In Pacific salmon, the disease has b e e n d iagnosed over a wide range of temperatures (6.5-19°C) but appears to be most severe dur ing the summer (Evelyn & Traxler, 1978). With the except ion of a n e m i a in na tura l ly and exper imenta l ly infected salmonids, no cl inical s igns of disease are apparen t (Evelyn & Traxler, 1978). In this regard, Reno & Nicholson (1980) have demons t ra ted that erythrocytes from affected Atlantic cod were s ignif icant ly more suscept ib le to lysis w h e n m a i n t a i n e d in vitro than were erythrocytes from uninfec ted cod.

As yet, erythrocytic necrosis virus (ENV) has not b e e n associated with massive mortal i t ies of fish. Evelyn & Traxler (1978) reported mortali t ies up to 0.3 % per day in chum sa lmon that were a t t r ibutable to VEN, however, the natura l outbreaks were usua l ly complicated by the presence of vibriosis and bacter ia l k idney disease. Rohovec & A m a n d i (1981) reported cytoplasmic inclusions in the erythrocytes of spawning coho and chinook sa lmon and s tee lhead trout in Oregon hatcheries. They also reported inc lus ions in juven i l e coho sa lmon which were suffering mortalities, the first such observat ion of VEN in fish reared solely in fresh water. Since the deaths could not be a t t r ibuted to other viral or bacter ia l pathogens, toxic agents, or env i ronmenta l factors, this may be the first ins tance reported in which mortali ty was directly a t t r ibutable to VEN. Sherburne (1977) reported a higher inc idence of VEN in p re - spawning than in the pos t - spawning alewife (Alosa pseudoharengus) and MacMil lan & Mulcahy (1979) observed a h igher p reva lence in young Pacific herr ing (Clupea harenguspallasi) than in older fish. Both of these observat ions could be exp la ined by a loss of infected fish from the popula t ion.

Evidence is accumula t ing which indicates that whi le VEN itself may not directly cause large fish losses, it may w e a k e n and predispose infected fish to secondary problems. MacMi l l an et al. (1980) descr ibed some secondary consequences of erythrocytic necrosis virus (ENV) infect ion of chum salmon. Under exper imenta l conditions, they reported that fish infected with VEN averaged 2.6 t imes greater mortali ty than control fish w h e n cha l l enged with Vibrio anguillarum and they showed a shorter t ime-to-death as wel l after bacter ia l exposure. Re-isolation of V. anguillarum was possible in 98 % of the fish with VEN but in only 5 % of the control fish. They also reported that fish with VEN had a s ignif icant ly decreased tolerance to oxygen deplet ion, and a decreased abi l i ty to regula te serum sodium and potass ium in saltwater. That env i ronmenta l stress


can have a signif icant impact on the outcome of hos t -pa thogen confrontat ions has b e e n reviewed by Snieszko (1974) and Wedemeye r (1976). Thus, VEN infect ion of fish may be important in l imi t ing their survival in the wild by render ing them more suscept ib le to mortali t ies from other factors.

The first ev idence of a possible viral et iology for VEN was reported by Walker (1971). Electron microscopic observat ion of erythrocytes from affected cod revea led the presence of large cytoplasmic part icles with a hexagona l profile. The part icles were similar in appearance to virions of the Iridovirus group. Subsequen t electron microscopic studies have shown that, at the ul trastructural level, virions associated with VEN in different species show signif icant differences. Walker & Sherburne (1977) reported that the virus associated with VEN in Atlant ic cod has an average d iameter of 330 nm. The electron dense nucleoid was spheroidal and approximate ly 230 n m in diameter . All the virions were cytoplasmic and ad jacent to the viroplasm as an inclus ion complex. Reno et al. (1978) s tudied the ul trastructure of virus in erythrocytes from VEN-infected Atlant ic herring. The virions were hexagona l or pen tagona l and 145 nm in d iameter w h e n measured edge to edge. Structural detai ls reported were an outer e lectron dense layer 8 nm wide, a less electron dense layer 16 n m wicle and a dense ly s ta in ing core approximately 100 n m in d iameter which conta ined a 40 n m central e lectron t rans lucent area. The ENV of herr ing then more closely resembles that descr ibed in sa lmonids (Evelyn & Traxler, 1978) and is distinct from that in cod or b l e n n y (Reno & Nicholson, 1981).

In summary of the electron microscopic studies, the viruses seen in the cytoplasm of affected cells appear to fall into three size classes and are characterist ic for the host species involved: 140-190 nm in Atlant ic her r ing (Reno et al., 1978) and Oncorhynchus spp. (Evelyn & Traxler, 1978); 200-250 n m in b l e n n y (Johnston & Davies, 1973); and 300-350 nm in Atlant ic cod (Appy et al., 1976; Walker & Sherburne, 1977).

There may be marked al terat ions in the ul t rastructural appearance of erythrocytes from affected fish. In YEN-infected herring, the surface of affected erythrocytes was irregularly lobated and often lost its el l ipt ical shape (Reno et al., 1978). Another morphologic change noted was marg ina t ion of nuc lear chromatin, ev ident as an accumula t ion of e lec t ron-dense mater ia l at the per iphery of the nucleus . Reno et al. (1978) report two types of cytoplasmic inclus ions w h e n affected erythrocytes were rev iewed by electron microscopy. Type 1 were round, granular , e lectron dense, and up to 1.5 ptM in diameter. Virus particles were associated with this type of inc lus ion either at the per iphery or occasionally in the interior. Type II inclus ions were m e m b r a n e - b o u n d structures which had the same appearance as the su r round ing cytoplasm. Virions were not seen in these inclusions.

Evelyn & Traxler (1978) reported successful t ransmiss ion of the disease to chum and pink sa lmon following in t raper i toneal (i. p.) inocula t ion of fil tered extracts p repared from infected chum salmon k idney tissue. In exposed p ink salmon, VEN was ev iden t at 3 weeks post- inoculat ion in 9 of 10 fish exposed to the agent. In chum salmon, the proportion of VEN-infected fish increased from 50 % at 12 days post - inocula t ion to 100 % by Day 48. Seven of the fish escaped bacter ia l infect ion and survived for 7 months at which time four had become VEN nega t ive ind ica t ing that fish may recover from the disease and not be l ife-long carriers. Fish inocu la ted with hea ted (60 °C for 15') extract did not develop the infection ind ica t ing that VEN is relat ively heat labile. Transmiss ion of VEN in adult cod by in t raper i toneal in jec t ion of washed, whole erythrocytes has b e e n

298 F .M. Hetrick

reported by Nicholson & Reno (1981). After 3 to 4 weeks, the characteristic inclusions appeared reaching an in tens i ty of 45-50 % infected erythrocytes after several months and dec l in ing thereafter.

Since all a t tempts to cul ture VEN have thus far b e e n unsuccessful , Reno & Nicholson (1980) deve loped a method for m a i n t a i n i n g erythrocytes in vitro from infected Atlantic cod for use in s tudying VEN infect ion in erythrocytes. The optimal condit ions for m a i n t e n a n c e employed static cul tures at 4 °C in a cell cul ture m e d i u m (MEM) with 10-15 % fetal calf serum. Under these conditions, erythrocytes could be held for at least two weeks with less than 50 % lysis. To study the effect of VEN on macromolecular syntheses, the incorporat ion of rad io- labe l led precursors by erythrocytes from infected and un in fec ted cod was followed. In these exper iments the percent of VEN-infected erythrocytes was in the range of I0 -20 %. W h e n 3H-uridine was employed, there was no difference in up take by the two cell populat ions, however, w h e n trit iated amino acids were used, the level of incorporat ion by infected erythrocytes was double that of normal cells. The most p ronounced effect noted was on DNA synthesis. There was no appreci- able incorporat ion of 3H-thymidine over the course of the exper iment (13 days) by cells from VEN-nega t ive fish. In contrast, there was a con t inu ing uptake of label by infected cells unt i l the sixth day, at which t ime the level of thymid ine incorporat ion was 10 times that seen in un in fec ted cells. This is the first ev idence ind ica t ing that the virus may he repl ica t ing in infected erythrocytes.

Despite repeated at tempts by m a n y investigators, VEN virus has not been success- fully grown in a cell cul ture system. Nevertheless, the f inding of Ir idovirus-l ike particles in m a n y species and the successful t ransmiss ion of the disease makes it qui te l ikely that VEN has a viral etiology. Cer ta in ly the ev idence is as compel l ing as that which l inked viruses to hepat i t is in h u m a n s before laboratory hosts became available.

DISEASES WITH SUSPECTED VIRAL ETIOLOGY

C a r p g i l l n e c r o s i s

Little informat ion is ava i lab le on this disease a nd what is descr ibed here was taken from a review by Wolf (1982). Popkova & Schelkunov (1978) reported the isolation of a large cytoplasmic Ir idovirus-t ike agen t from carp on farms in Russia suffering from gill necrosis. The agen t was isolated in FI-IM cells us ing mater ia ls from the gills and k idney of infected fish as inocula. Cytopathic effects were seen with most of the samples but, except for one isolate, passage of the mater ia ls resul ted in a loss of cytopathology. Electron microscopy showed icosahedral part icles approximately 200 n m in diameter. Exper imenta l t ransmiss ion of the infect ion was apparen t ly successful. No other informa- t ion is avai lable .

E p i d e r m a l h y p e r p l a s i a of cod

Dur ing a s tudy of the u lcus-syndrome in cod (Gadus morhua), Jensen & Larsen (1979) noted a few cases of an u n k n o w n but characterist ic skin disease in cod in Danish waters. The lesions appeared as t ransparen t round spots and they occurred over the ent i re body surface. Histopathological examina t ion showed the epidermis in the lesion


to be about four t imes the normal th ickness and it con ta ined few or no mucosal glands. When the affected tissue was examined by electron microscopy, virus- l ike part icles were seen in the nucle i of some epi thel ia l cells. The part icles showed both hexagona l and pen tagona l outlines, the characteristic shape of icosahedral viruses in thin sections. The particles were approximate ly 77 n m in d iamete r and had 20-25 nm long th in fibers project ing from the corners (Jensen & Bloch, 1980). Taken together these characteristics, i.e. size, morphology, presence of fibers, and nuc lea r location, s trongly indicate that the agent is a m e m b e r of the Adenovi rus group. This is the first report of Adenovi rus- l ike particles in fish. The agent has not b e e n grown in cell cul tures nor has the disease b e e n exper imenta l ly t ransmit ted among fish.

C a r p pox

Carp pox (Epithelioma papillosum) has long b e e n k n o w n as a disease of cul tured carp. Common carp (Cyprinus carpio) and several other cyprinids in Europe, Asia, and the Middle East develop the disease. The ep idermal hyperplas ia is character ized by raised, white verrucose p laques that occur on m a n y parts of the body par t icular ly the gills, skin, eyes, and fins. The tumor is b e n i g n as there is no ev idence of visceral metastasis (Sonstegard & Sonstegard, 1978). Since the disease is innocuous, it has received scant at tention.

A viral etiology is suspected but not proven. Herpesvirus- type part icles have b e e n observed in infected tissues (Schubert, 1966). The vir ions become enve loped as they pass from the nuc leus to the cytoplasm, a genera l feature associated with herpesvi rus replication. Cowdry Type A nuc lea r inc lus ions have b e e n reported but syncytia have not been seen in sections of infected tissue.

The infect ion is readi ly t ransmit ted to normal carp ei ther by r ubb i ng tumor t issue against the abraded ep i the l ium of normal carp or by hold ing normal carp in the same tank as infected fish. Tumors develop in about 60 days at 10 °C. Wi th in the nuc leus of tumor cells, large aggregates of n o n - e n v e l o p e d vir ions are seen (Sonstegard & Sonste- gard, 1978). The virus has not yet b e e n isolated in cell culture.

Herpesvirus scophthalmi i n f e c t i o n of t u r b o t

The first outbreak of this disease was noted in turbot (Scophthalmus maximus) which were moved from a hatchery in Scotland to a warm-wate r ongrowing site which used heated effluent from a power station. Affected fish were le thargic and did not feed. Mortalities in the affected stock reached 30 % a l though a proport ion of these losses were at t r ibutable to other causes (Richards & Buchanan, 1978). Most deaths occurred wi th in 5 days of the or iginal t ransporta t ion and increased dur ing periods of t empera ture change and elevated chlorine levels in the power p lan t effluents.

There were no lesions in any organs except the gills and skin where g ian t cells, some as large as 170 pM in diameter , were dis t r ibuted among ep idermal cells. No bacteria were isolated from in terna l organs but electron microscopy revealed herpesvirus-type part icles wi th in both the nuc leus and cytoplasm of the g ian t cells (Buchanan et al., 1978). The particles in the nuc leus were hexagona l in shape and approximate ly 100 n m in diameter. Those in the cytoplasm had an outer enve lope which was covered

300 F .M. Hetrick

with peplomers approximate ly 18 nm long and measured 200-220 nm in diameter. The nucle i wi th in the syncytia fused to form a s ingle g iant oval nuc leus with a maximum width of 60 ~M and a length of 100 pM (Buchanan & Madely, 1978). Paracrystall ine arrays of the 100 n m particles were seen in these large nuclei . A subuni t structure was clearly vis ible in nega t ive ly - s ta ined preparat ions. The capsomeres appeared "hollow" and were approximate ly 9 nm in diameter . The agen t has yet to be isolated in cell culture.

Release of the virus from infected fish probably occurs dur ing giant cell destruction and the s lough ing of ep idermal cells. Reexamina t ion of his topathological sections from wild turbot caught in previous years showed the presence of similar giant cells sugges t ing that the virus is endemic in wild fish (Buchanan et al., 1978).

H e r p e s v i r u s of Pac i f i c cod

McArn et al. (1978) and McCain et al. (1979) reported the occurrence of two main types of skin lesions on the Pacific cod (Gadus macrocephalus) collected in the Bering Sea. One of the lesions was ulcerative, the other was a raised, r ing-shaped lesion. In the ep idermal areas of the r ing-shaped lesion were large cyst-like bodies, or giant cells, conta ined a basophi l ic center sur rounded by an eosinophi l ic margin. Electron-microscopic examina t ion of the g iant cells showed the presence of herpesvirus- l ike particles. The part icles seen in the nucle i were 80-100 nm in d iameter whereas larger particles, 120-170 n m in diameter, were observed in the cytoplasm (McArn et al., 1978). The agent has not b e e n isolated and its role in the etiology of the disease is unknown.

C o d u l c u s s y n d r o m e

The cod ulcus disease is an u lcera t ing condi t ion in the skin of Atlant ic cod (Gadus morhua) t aken from the coastal waters of Denmark. J ensen & Larsen (1979) described various stages of progression of the disease from e x a m i n i n g lesions on wild fish. The first stage is the appearance of mul t ip le dermal papules, some of which may be hemorrhagic. These may develop into grey to yel low crater-l ike perforations of the skin which in turn may lead to vary ing degrees of u lcerat ion with the lesions be ing 2-8 cm in diameter. The disease can result in s ignif icant mortalities.

Two different viruses have b e e n isolated from affected cod (Jensen et al., 1979). One has a rhabdovirus- l ike morphology be ing a rod-shaped part icle 55 nm wide and 175 nm in length. Blind passage of cod materials in a pike sarcoma l ine at 15 °C resul ted in the isolat ion of the agent . It caused s loughing of the cells in 3-4 days and reached a titer of approximate ly 105 TCIDs0/ml. The other agen t was an icosahedral particle, with an Ir idovirus-l ike morphology. The virus had an overall d iameter of 150 nm and a central nuc leo id about 100 n m in diameter . This virus was isolated by b l ind passage in EPC cells. W h e n adapted, it p roduced cytopathic effects wi th in 20 h post- inoculat ion and a t ta ined titers of 105 TCIDs0/ml.

Some exper imenta l results suggest that the disease is infectious. Transmiss ion was noted w h e n homogena tes of the early papu la r t issues were in jec ted in t raper i toneal ly or appl ied to scarified skin. Also, w h e n heal thy cod were placed in a tank with cod bear ing the early dermal papules , they all deve loped the disease wi th in I1 days. When 36 cod


were inocula ted int racardia l ly with the Iridovirus, 8 d e v e l o p e d the early papu la r s tage of

the disease as did one control fish (Jensen & Larsen, quo ted by Wolf, 1982). D e v e l o p m e n t

of the disease in the control fish i l lustrate the difficulty in t ransmission studies whe re wi ld fish n e e d to be emp loyed since noth ing is known of their i m m u n e status or gene ra l heal th history.

A t l a n t i c s a l m o n p a p i l l o m a t o s i s

Al though recognized for years in wi ld sa lmon (Salmo salar], ep ide rma l hyperp las ia

or papi l lomatosis was first repor ted in ha tchery fish in S w e d e n (Wiren, 1971). More

recently, the d isease was desc r ibed in mar ine cul tured ra inbow trout (Roberts & Bullock, 1979). The growth beg ins as a whitish, raised p l a q u e which g radua l ly increases in size

and deve lops a verrucose surface. Lesions appea r in la te s u m m e r and the inc idence may approach 50 %. Mature lesions may unde rgo necrosis but they are usual ly reso lved by a

graf t - re ject ion- l ike s lough (Carlisle & Roberts, 1977). Morta l i ty is low and w h e n it does

occur it is usual ly associated with secondary mycot ic infections.

Virus- l ike par t ic les are regula r ly seen in infected t issues and eos inophi l ic inc lus ion bodies are common. The virus par t ic les are 125-150 nm in diameter . An outer coat is

separa ted from an e lec t ron-dense nucleoid, 70-95 nm diameter , by an e lec t ron- lucen t

area (Carlisle, 1977). Four passages of pap i l loma mater ia l in Atlant ic sa lmon cell

cultures fai led to yield a cytopathic agent . Similarly, a t tempts to expe r imen ta l ly t ransmit the tumor have been unsuccessful .

Bylund et al. (1980) descr ibed an in te res t ing outbreak in cul tured At lant ic sa lmon in

Finland. The salmon were kept in 22 tanks and at w e e k l y intervals were sub jec ted to prophalyt ic baths of 2-3 % NaC1 or formal in (1-4000) a l ternat ively. Two tanks were exc luded from the prophylaxis for the first severa l weeks . Dur ing the rea r ing season,

papi l lomatosis occurred in only these two tanks wi th 80-90 % of the fish be ing affected.

Since the fish in all the tanks were hand led s imilar ly in all respects, the early prophylac-

tic t reatment seems to have p reven ted the infection. The t endency of the disease to occur in epizoot ics suggests a microbia l et iology.

Whatever the agent, it is w idesp read in nature as the disease has b e e n noted in severa l

countries, in fresh and salt water, and in ha tchery and wi ld fish.

S t o m a t o p a p i l l o m a of e e l s ( c a u l i f l o w e r d i s e a s e )

This d isease of the European eeI (Anguilla anguilla] is charac te r ized by pap i l loma- tous growths which can occur anywhere on the ee l ' s body but most f requent ly form about

the head and mouth. The term caul i f lower d isease is descr ip t ive of the growth which

results from the infold ing of the surface of the papi l loma. The growth can resul t in complete obstruct ion of the mouth which leads to s tarvat ion of the affected eels. The

disease occurs main ly in European coastal waters. The p r e v a l e n c e of the d i sease appears to be increas ing (Deys, 1976) wi th the inc idence approach ing 40 % in some areas.

At least three viruses have b e e n isolated from eels with the disease. A virus wi th

polyhedral symmetry and a d i ame te r of 52-56 nm has b e e n isola ted in RTG-2 and F H M cells from the blood of infected eels. It is b e l i e v e d to rep l ica te in the nuc leus and,

therefore, be a DNA virus (Pfitzner, 1969; Pfitzner & Schubert , 1969; Schmid, 1969;

302 F .M. Hetrick

Koops et al., 1970). However, the cell culture grown virus has not produced the disease w h e n inocula ted into eels.

Wolf & Qu imby (1970) isolated a s imilar virus from tumor homogenates . This virus, te rmed EV-1, p roduced large syncyt ia and pyknot ic loci in RTG-2 cells. The virus has not b e e n further characterized. Nagabayash i & Wolf (1979) reported the isolation of a second virus (EV-2) in FHM cells from eels with s tomatopapil loma. From its biological, physical, and chemical properties, they concluded that it was most probably an orthomyxovirus. W h e n in jec ted into elvers, about half of them died wi th in 2 months but virus could be recovered from only 25 %.

It should also be po in ted out that Peters & Peters (1970) have reported the induct ion of similar proliferations in eels fol lowing their t rea tment with certain chemicals. Whet- her any of the three viruses, ei ther a lone or synergis t ical ly with a chemical agent, are involved in the etiology of this disease remains to be determined.

POLLUTION AND DISEASE

While this topic is covered in depth e l sewhere in this symposium, a few comments are appropria te here. In recent years, chemical pol lutants have been increas ingly impl ica ted in fish disease outbreaks [see reviews by Snieszko (1974) and S inde rmann (1979)]. Much of the ev idence is c i rcumstant ia l such as increased f requencies of diseases in pol lu ted waters or diseases occurr ing in fish be i ng used for toxicity studies. However, in the last few years, control led laboratory studies have demonst ra ted al tered susceptibi- lity to pa thogens or impai red immunolog ica l responses in fish exposed to pollutants, mostly heavy metals.

Zinc, copper, and methy lmercury have b e e n reported to reduce the ant ibody response of two exotic fish species, zebrafish (Sarot & Perlmutter, 1976) and blue gourami (Roales & Perlmutter, 1977) to bacter ial and viral ant igens. Hetrick et al. (1979) found that young ra inbow trout were more suscept ible to infectious hematopoiet ic necrosis virus fol lowing exposure to sub- le tha l copper levels. Increased susceptibi l i t ies of s tee lhead trout to Yersinia ruckeri (Knittel, 1981) and chinook sa lmon and ra inbow trout to Vibrio anffuillarum (Baker et al., 1983) have also b e e n noted fol lowing sub- le thal copper exposure. In coho salmon, Sugat t (1980) reported increased suscept ibi l i ty and reduced agg lu t in in titers to V. anguillarum fol lowing chromium exposure.

To date, controlled studies of pa thogen-po l lu tan t interaction, such as those described above, have not b e e n reported with mar ine fish. Nevertheless, natura l chemical "stresses" are u n d o u b t e d l y be ing imposed on wi ld fish populat ions . These stresses, which can predispose the fish to infect ion with opportunist ic pathogens, will only increase unless "'the solut ion to pol lu t ion is d i lu t ion" practice is a b a n d o n e d by coastal communit ies . One ha l lmark of the Herpesviruses is that they f requent ly establish latent infect ions in their hosts, be ing act ivated only w h e n some stress is appl ied to an individual . Half of the diseases descr ibed in this review have a know n or putative herpesvirus associated with their etiology. This might not be s imply fortuitous but could indicate that stress factors caused a s ignif icant increase in the inc idence of these diseases so that they became apparent .

While m a n y fish kills have b e e n directly t raceable to the introduct ion of toxic pol lutants , the effects of low levels of pol lutants on fish species are probably more


important than the occasional "large spill" since the effects are less l ikely to be obvious and the source more difficult to detect in t ime to save the envi ronment . Documenta t ion of these subtle effects has b e e n difficult to achieve. Attempts to obta in indirect ev idence of stress effects have inc luded measu remen t of specific enzyme activities, steroid levels, and abil i ty to suppress the i m m u n e response.

One aspect of the i m m u n e system that has not b e e n exploi ted in at tempts to find a sui table assay is the phagocyt ic system. For a n u m b e r of reasons, this system seems to be a point of vulnerabi l i ty which warrants eva lua t ion as an indicator of adverse effects of pollutants on fish. Phagocytosis has b e e n clearly es tabl i shed as a major defense mecha- n ism in fish. In addi t ion to the inges t ion and k i l l ing of microorganisms, the phagocyt ic system is also impor tant in the s t imula t ion of ant igen-speci f ic i m m u n e responses (Rijkers, 1982) and it is e n h a n c e d by act ivat ion of the complemen t system. Since several defense mechanisms focus in the phagocyt ic system, it follows that factors which interfere with normal phagocytosis in fish would be accompan ied by adverse effects inc lud ing lowered resis tance to infect ion and increased mortality, par t icular ly from opportunist ic pathogens.

Measurement of phagocytosis has b e e n great ly s implif ied s ince Al len et al. (1972) reported that cells phagocyt iz ing bacteria exhib i ted a chemi luminescence (CL) response due to the genera t ion of oxygen radicals du r ing a respiratory burst. We have deve loped a CL assay for s tudying the effect of various factors and condi t ions on the phagocyt ic activity of cells from the pronephros of str iped bass (Stave et al., 1983) us ing modifica- tions of a method reported by Scott & Klesius (1981). Evidence to date indicates that certain heavy metals and pest icides depress the abi l i ty of str iped bass phagocytes to engulf bacterial pa thogens (Stave et al., unpubl .) . We p lan to expand these studies to other finfish and shellfish species to de te rmine if the CL assay has predict ive va lue in ident i fying those env i ronmen ta l pol lutants that predispose fish to infection. The relat ive simplicity, inexpens ive nature and rapidi ty with which results are ob ta ined are attrac- tive features of the CL assay.

The product ion of farmed fish is increas ing on a global scale in an a t tempt to produce high qual i ty protein for b u r g e o n i n g populat ions, par t icular ly in deve lop ing nations. The Food and Agricul tural Organiza t ion (FAO) predicts that there will be a 5- fold increase over 1975 product ion levels by the end of the century (Hill, 1981). While most of the world 's product ion of farmed fish is current ly reared in earth ponds, recent improvements in hatchery methods, cage construction, knowledge of migratory habits, and demonstra t ion of fiscal v iabi l i ty have promoted interest in " sea - ranch ing" as an al ternative approach.

Intensive mar ine fish farming has b e e n par t icular ly successful with salmon, red seabream, and yellowtail . Mass cul ture of other species will undoub t e d l y be a t tempted in the near future. Control of disease problems wil l be pa ramoun t in de t e r mi n i ng the success of these ventures. In addi t ion to descr ib ing specific et iologic agents for diseases of cul tured mar ine fish, research workers wil l also n e e d to address the equa l ly impor tant problem of e luc ida t ing those env i ronmen ta l factors which predispose fish to infection.

3 0 4 F . M . H e t r i c k

L I T E R A T U R E C I T E D

Al ien , R. C., S t e m h o l m , R. L. & Steele , R. H., 1972. E v i d e n c e for g e n e r a t i o n of a n e lec t ron ic exc i t a t ion s ta te in h u m a n p o l y m o r p h o n u c l e a r l e u k o c y t e s a n d its pa r t i c ipa t ion in bac te r i c ida l act ivi ty . - B i o c h e m . b i o p h y s . Res. C o m m u n . 47, 679--691.

Alpers , C. E,, M c C a i n , B. B., M e y e r s , M. S. & W e l l i ngs , S. B., 1977. L y m p h o c y s t i s d i s e a s e in y e l l o w f i n so le (Limanda aspers) in the Be r ing Sea. - J. Fish. Res. Bd Can . 34, 611-618 .

Anee r , G. & L j u n g b e r g , O., 1976. Lymphocyst . i s d i s e a s e in Balt ic h e r r i n g (Clupea harengus var membras). - J. F i sh Biol. 8, 345-350 .

Appy , R. G., Burr, M. D. B. & Morris , T. J., 1976. Vira l n a t u r e of p i s c i n e e ry th rocy t i c nec ros i s (PEN) in t h e b lood of A t l an t i c cod (Oadus morhua). - J. Fish. Res. Bd Can . 33, 1380-1385.

Baker , R. J., Kni t te l , M. D. & Fryer, J. L., 1983. Suscep t ib i l i t y of c h i n o o k s a l m o n (Oncorhynchus tshawytoscha) a n d r a i n b o w t rout (Salmo gairdneri) to in fec t ion w i th Vibrio anguil larum follow- i n g s u b l e t h a l c o p p e r e x p o s u r e . - J. F ish Dis. 6, 267-276 .

Bernard , G. W., Cooper , E. L. & M a n d e l l , M. L., 1968. L a m e l l a r m e m b r a n e enc i r c l ed v i ruses in the e r y t h r o c y t e s of Rana pipiens. - J. Ul t ras t ruct . Res. 26, 8-16 .

B u c h a n a n , J. S. & M a d e l e y , C. R., 1978. S t u d i e s o n Herpesvirus scophthalmi in fec t ion of turbot Scophthalmus max imus - Ul t ra s t ruc tu ra l obse rva t ions . - J. F i sh Dis. 1, 283-295.

B u c h a n a n , J. C., Richards , R. H., M a d e l e y , C. R. & Somerv i l l e , C. S., 1978. A H e r p e s - t y p e v i rus f rom t h e turbot , Scophthalmus maximus. - Vet. Rec. 102, 527-528 .

By lund , G., V a l t o n e n , E. T. & Niemel / i , E., 1980. O b s e r v a t i o n s on e p i d e r m a l p a p i l l o m a t a in wi ld c u l t u r e d A t l a n t i c s a l m o n (Salmo salar) in F in land . - J. F i sh Dis. 3, 525-528 .

Car l i s le , J. C., 1977. A n e p i d e r m a l p a p i l l o m a of t h e A t l an t i c s a l m o n . II. U l t r a s t ruc tu re a n d et iology. - J. Wildl . Dis. 13, 235-239 .

Car l i s le , J. C. & Roberts , R. J., 1977. A n e p i d e r m a l p a p i l l o m a of t he A t l an t i c s a l m o n {Salmo salar). - I. Epizoot io logy , p a t h o l o g y , a n d i m m u n o l o g y . - J. Wildl . Dis. 13, 230-234 .

Darai , G., A n d e r s , K., Koch, H., Del ius , H., G e l d e r b l o m , H., S a m a l e c o s , C. & Fliigel, R., 1983. A n a l y s i s of the g e n o m e of f ish l y m p h o c y s t i s d i s e a s e v i rus i so la ted d i rec t ly f rom e p i d e r m a l t u m o u r s of P l e u r o n e c t e s . - V i r o l o g y 126, 466-479 .

Deys , B. F., 1976. A t l an t i c ee l s a n d cau l i f lower d i s e a s e ( O r o c u t a n e o u s pap i l loma tos i s ) . - Prog. exp. T u m o r Res. 20, 94-100 .

D u n b a r , C. E. & Wolf, K., 1966. T h e cy to log ica l course of e x p e r i m e n t a l l y m p h o c y s t i s in the b luegi l l . - J. infecL Dis. 116, 466-475 .

Eve lyn , T. P. T. & Traxler , G. S., 1978. Vira l e ry throcy t ic necrosis ' . N a t u r a l o c c u r r e n c e in Pacif ic s a l m o n a n d e x p e r i m e n t a l t r a n s m i s s i o n . - J. Fish. Res. Bd Can . 35, 903-907.

F ldge l , R. M., Darai , G. & G e l d e r b l o m , H., 1982. Vira l p r o t e i n s a n d a d e n o s i n e t r i p h o s p h a t e p h o s p h o h y d r o l a s e ac t iv i ty of f ish l y m p h o c y s t i s d i s e a s e vi rus . - V i ro logy 122, 48-55 .

Het r ick , F. M., Knit te l , M. D. & Fryer, J. L., 1979. I n c r e a s e d s u s c e p t i b i l i t y of r a i n b o w trout to in fec t ious h e m a t o p o i e t i c nec ros i s v i rus af ter e x p o s u r e to copper . - Appl . env i ron . Microbiol . 37, 1 9 8 - 2 0 1 .

Hill, B, J., 1981. V i rus d i s e a s e s of fish, In: Vi rus d i s e a s e s of food a n i m a l s . Ed. by E. P. J. Gibbs . Acad. Press , London , 231-261 .

H o w s e , H. D. & C h r i s t m a s , J. Y., 1971. O b s e r v a t i o n s on t h e u l t r a s t r u c t u r e of l y m p h o c y s t i c v i rus in t h e A t l an t i c c roaker , Micropogon undulatus. - Viro logy 44, 211-214 .

J e n s e n , N. J. & Larsen , J. L., 1979. T h e u l c u s s y n d r o m e in cod {Gadus morhua). I. A pa tho log i ca l a n d h i s t o p a t h o l o g i c a l s tudy . - Nord. V e t M e d . 31, 221-228 .

J e n s e n , N. J. & Bloch, B., 1980. A d e n o v i r u s - l i k e pa r t i c l e s a s s o c i a t e d wi th e p i d e r m a l h y p e r p l a s i a in cod (Gadus morhua}. - Nord. V e t M e d . 32, 173-175.

J e n s e n , N. J., Bloch, B. & Larsen , J. L., 1979. T h e u l c u s s y n d r o m e in cod (Gadus morhua). III. A p r e l i m i n a r y v i ro logica l report . - Nord. V e t M e d . 31, 436--442.

J o h n s t o n , M. R. L. & Davies , A. S., 1973. A Pirhemocyton-l ike pa ra s i t e of the b l e n n y , Blenniusphi l is . (Teleos te i BlennHdae) a n d its r e l a t i o n s h i p to lmmanoplasma N e w m a n , 1909. - I n t . J. Parasitol . 3, 235-241 .

K i m u r a , T., Y o s h i m i z u , M., T a n a k a , M. & S a n n o h e , H., 1981a. S tud i e s on a n e w v i rus (OMV) from Oncorhynchus masou - I. C h a r a c t e r i s t i c s a n d p a t h o g e n i c i t y . - F ish Path. 15, 143-147.

D N A v i r u s e s a s s o c i a t e d w i t h f i s h d i s e a s e s 3 0 5

Kimura , T,, Yosh imizu , M. & T a n a k a , M., 1981b. S t u d i e s on a n e w v i rus (OMV) f rom Oncorhynchus masou - II. O n c o g e n i c na tu re . - F ish Path. 15, 149-153.

Kimura , T., Y o s h i m i z u , M. & T a n a k a , M., 1981c. F i sh v i ruses : T u m o r i n d u c t i o n in Oncorhynchus keta by t h e h e r p e s v i r u s . In: Phy le t i c a p p r o a c h e s to cance r . Ed. by C. J. Dawe l J. C. H a r s h b a r g e r , S. Kondo, T~ S u g i m u r a & S. T a k a y a m a . Jap . Scient . Soc. Press , Tokyo , 59~58.

Kimura , T., Y o s h i m i z u , M. & T a n a k a , 1983. Suscep t ib i l i t y of d i f fe ren t fry s t a g e s of r e p r e s e n t a t i v e s a l m o n i d s p e c i e s to Oncorhynchus masou v i rus (OMV). - F i sh Path . 17, 251-258 .

Knittel , M. D., 1981. Suscep t ib i l i t y of s t e e l h e a d t rout Salmo gairdneri R i c h a r d s o n to r e d m o u t h in fec t ion Yersinia ruckeri fo l l owing e x p o s u r e to copper . - J. F i sh Dis. 4, 33-40 .

Koops, H., M a n n , H., Pfi tzner, I., S c h m i d , O. J. & Schuber t , G., 1970. T h e cau l i f l ower d i s e a s e of eels . In: A s y m p o s i u m on d i s e a s e s of f i shes a n d she l l f i shes . Ed. by S. F. Sn ieszko . W a s h i n g t o n , D.C., 291-295 . (Spec. PubL A m . Fish. Soc. 5.)

Krantz , G. E., 1970. L y m p h o c y s t i s in s t r i ped bass , Roccus saxatilis, in C h e s a p e a k e Bay. - C h e s a - p e a k e Sci. 11, 137-139.

Laird, M. & Bullock, W. L., 1969. M a r i n e f i sh h e m a t o z o a f rom N e w B r u n s w i c k a n d N e w E n g l a n d . - J. Fish. Res. Bd Can . 26, 1075-1102.

Lawler, A. R., Ogle , J. T. & D o n n e s , C., 1977. Dascyllusspp.: N e w hos t s for l y m p h o c y s t i s a n d a l ist of r ecen t hosts . - J. Wildl , Dis. 13, 307-312 .

MacMi l l an , J. R. & M u l c a h y , D., 1979. Art i f ic ia l t r a n s m i s s i o n to a n d s u s c e p t i b i l i t y of P u g e t S o u n d f ish to viral e ry th rocy t i c n e c r o s i s (VEN). - J . Fish. Res. Bd Can . 36, 1097-1101.

M a c M i l l a n , J. R., M u l c a h y , D. & Landol t , M., 1980. Viral e ry th rocy t i c nec ros i s : S o m e p h y s i o l o g i c a l c o n s e q u e n c e s of in fec t ion in c h u m s a l m o n (Oncorhynchus keta). - Can . J. Fish. aqua t . Sci. 37, 799-804.

M a d e l e y , C. R., Smai l , D. A. & E g g l e s t o n e , S. I., 1978. O b s e r v a t i o n s on t he f ine s t ruc tu re of l y m p h o c y s t i s v i rus f rom E u r o p e a n f l o u n d e r s a n d p la ice . - J. gen . Virol. 40, 421-431 ,

McAll is ter , P. E., 1979. F i sh v i r u s e s a n d vi ra l in fec t ions . In: C o m p r e h e n s i v e v i ro logy. Ed. by H. F r a e n k e l - C o n r a t & R. R. W a g n e r . P l e n u m Press , N e w York, 14, 401-470 .

McArn, G. E., M c C a i n , B. & W e l l i n g s , S. R., 1978. S k i n l e s ions a n d a s s o c i a t e d v i rus in Pacif ic cod {Gadus macrocephalus) in t h e Be r ing Sea. - F e d n Proc. F e d n Am. S o t s exp. Biol. 37, 937.

McCa in , B. B., G r o n l u n d , W. D , Myers , M. S. & W e l l i n g s , S. R., 1979. T u m o r s a n d mic rob ia l d i s e a s e s of m a r i n e f i shes in A l a s k a n wate rs . - J. F i sh Dis, 2, 111-130.

McCoske r , J. E., Lagios , M. D. & Tucke r , T., 1976. U l t r a s t ruc tu re of l y m p h o c y s t i s v i rus in q u i l l b a c k rockf ish (Sebastes maliger) w i t h records of in fec t ion in o the r a q u a r i u m f i shes . - Trans . Am. Fish. Soc, 105, 333-344.

M u r c h e l a n o , R. A. & Br idges , D. W., 1976. L y m p h o c y s t i s d i s e a s e in t he w i n t e r f l o u n d e r (Pseudopleu- ronectes americanus). - J. Wildl . Dis. 12, 1 0 t - 1 0 6 .

N a g a b a y a s h i , T. & Wolf, K., 1979. C h a r a c t e r i z a t i o n of EV-2, a v i rus i so la t ed f rom E u r o p e a n ee l s (Anguilla anguitla) wi th s t o m a t o p a p i l l o m a . - J. Virol. 30, 358-364 .

Nicholson , B. L. & Reno, P. W., 1981. Vira l e ry th rocy t i c n e c r o s i s (VEN) in m a r i n e f i shes . - Fish. Path. 15, 129-133.

Nigrel l i , R. F., 1954. T u m o r s a n d o the r a typ ica l cel l g r o w t h s in t e m p e r a t e f r e s h w a t e r f i shes of Nor th Amer ica . - Trans . Am. Fish. Soc. 83, 262-271 .

Paperna , I., Sabna i , I., Colorni , A., 1982. A n o u t b r e a k of l y m p h o c y s t i s in Sparus aurata L. in t he Gul f of A q a b a , Red Sea. - J. F i sh Dis. 5, 433-437 .

Peters, N. & Peters , G., 1970. T u m o r g e n e s e , e i n E n e r g i e p r o b l e m de r Ze l le? U n t e r s u c h u n g e n a n P a p i l l o m e n d e s e u r o p ~ i s c h e n Aals , AnguilIa anguilla. - Arch. F i schWiss . 21, 238-247 .

Pfitzner, I., 1969. Zur A e t i o l o g i e der B l u m e n k o h l k r a n k h e i t de r Aa le . - Arch . F i schWiss . 20, 24-49 . Pfitzner, I. & Schuber t , G., 1969. Ein Vi rus a u s d e m Blut m i t B l u m e n k o h l k r a n k h e i t b e h a f t e t e r Aale .

- Z. Natur forsch . 24b, 70. Pilcher, K. S. & Fryer, J. L., 1980. T h e viral d i s e a s e s of fish: A r e v i e w t h r o u g h 1978. Part. 1 : D i s e a s e s

of p r o v e n viral e t io logy. - C R C crit. Rev. Microbiol . 9, 287-363 , Popkova, T. I. & S c h e l k u n o v , I. S., 1978. I so la t ion of v i rus f rom ca rp a f f l ic ted wi th gi l l necros is .

( V e d e l e n i e v i rusa ot ka rpov , b o l ' n y k h z h a b e r n y m nek rozom) . - VNIIPRKH Ryb. Khoz. 4, 34-38 . Reno, P. W. & Nicho l son , B. L , 1980. Viral e ry th rocy t i c n e c r o s i s (VEN) in A t l an t i c cod (Gadus

morhua, L.): In vi tro s tud ies . - Can . J. Fish. aqua t . ScL 37, 2276-2281 . Reno, P. W. & Nicholson , B, L., 1981. U l t r a s t ruc tu re a n d p r e v a l e n c e of v i ra l e ry th rocy t i c nec ro s i s

306 F . M . H e t r i c k

(VEN) v i rus in Atlantic cod (Gadus morhua L.) from the Nor the rn Atlant ic Ocean. - J. Fish Dis. 4, 361-370.

Reno, P. W., Phi l l ippon-Fried, M., Nicholson, B. L. & S h e r b u m e , S. W., 1978. Ultrastructural s tudies of p isc ine erythrocytic necrosis (PEN) in Atlantic he r r ing (Clupea harengus harengus}. - J. Fish. Res. Bd Can. 35, 148-154.

Richards, R. H. & Buchanan , J. S., 1978. Studies on Herpesvirus scophthalmi infection of turbot Scophthalmus maximus: His topa tho log ica l observat ions . - J. Fish Dis. I, 251-258.

Rijkers, G. T., 1982. N o n - l y m p h o i d defens ive m e c h a n i s m s in fish. - Dev. comp. ImmunoL 6, 1-12. Roales, R. R. & Peflmutter , A., 1977. The effects of sub le tha l doses of me thy lmercu ry and copper,

app l ied s ingly and jointly, on the i m m u n e r e sponse of the b lue gourami (Trichogaster trichopte- rus) to viral and bacter ia l ant igens . - Archs environ. Contam. Toxicol. 5, 325-331.

Roberts, R, J. & Bullock, A. M., 1979. Papi l lomatos is in mar ine cul tured r a inbow trout Salmo gairdneri Richardson. - J. Fish Dis. 2, 75-77.

Robin, J. & Ber th iaume, L., 1981. Purification of lymphocys t i s d isease virus (LDV) g rown in t issue culture. Evidences for the p re sence of two types of viral part icles. - Rev. Can. Biol. 40, 323-329.

Rohovec, J, S. & Amandi , A., 1981. Inc idence of viral erythrocytic necrosis a m o n g ha tchery reared sa lmon ids of Oregon . - J. Fish Path. I5, 135-141.

Russell, P. H., 1974. Lymphocys t i s in wi ld p la ice [Pleuronectesplatessa)and f lounder (Platichthys flesus) in British coastal wa te r s - A his tological and serological study. - J. Fish Biol. 6, 771-778.

Sano, T., 1976. Viral d i seases of cul tured f ishes in Japan , - Fish. Path. 10, 221-226. Sarot, D. A. & Perlmutter , A., 1976, The toxicity of zinc to the i m m u n e re sponse of the zebrafish

(Brachydanio rerio), in jected wi th viral and bacter ia l ant igens . - Trans. Am. Fish. Soc. 105, 456-459.

Schmid, O. J., 1969. Beitrag zur His to logie u n d Aet iologie der B lumenkoh lk rankhe i t der Aale. - Arch. FischWiss. 20, 16-22.

Schuber t , G. H., 1966. The infective agen t of carp pox. - Bull. Off. int. Epizoot. 65, 1011-1022. Scott, A. L & Klesius, P. H., 1981. Chemi luminescence : A novel analys is of phagocy tos i s in fish. -

Devs biol. Stand. 49, 243-256. Sherburne , S. W., 1977. Occur rence of p isc ine erythrocytic necrosis (PEN) in the blood of the

a n a d r o m o u s alewife, Alosa pseudoharenffus, from Maine coastal s t reams. - J. Fish. Res. Bd Can. 34, 281-286.

S inde rmann , C. J., 1970. Principal d i seases of mar ine fish and shellfish. Acad. Press, N e w York, 369 pp.

S inde rmann , C. J., 1979. Pol lu t ion-associa ted d i seases and abnormal i t i es of fish and shel lf ish - Review. - Fish. Bull. U. S. 76, 717-749.

Smail, D. A. & Eggles tone , S. I., 1980. Virus infections of mar ine fish erythrocytes: p reva lence of p isc ine erythrocytic necrosis in cod Gadus morhua L. and b l enny Blennius phol is L. in coastal and offshore wa te r s of the Uni ted Kingdom. - J. Fish Dis. 3, 41-46.

Snieszko, S. F., 1974. The effects of env i ronmen ta l stress on ou tb reaks of infect ious d iseases of fish. - Fish Biol. 6, 197-208.

Sons tegard , R. A. & Sonstegard , K. S., 1978. Herpesv i rus - assoc ia ted ep ide rma l hyperp las ia in fish (carp). - Scient. Publ. int. Ag. Res. Cance r 24, 863-868.

Stave, J., Roberson, B. S. & Hetrick, F. M., 1983. C h e m i l u m i n e s c e n c e of phagocyt ic cells isolated f rom the pronephros of s t r iped bass. - Dev. comp. Immuno t . 7, 269-276.

S tehbens , W. E. & Johns ton , M. R. L., 1966. The viral na tu re of Pirhemocyton tarentolae. - J. Ultrastruct. Res. 15, 543-554.

S tephens , E. B., N e w m a n , M. W., Zachary, A. L. & Hetrick, F. M., 1980. A viral aet iology for the annua l sp r ing epizootics of Atlantic m e n h a d e n in C h e s a p e a k e Bay. - J. Fish Dis. 3, 387-398.

Stickney, R. R. & White, D. B., 1974. Lymphocys t i s in t ank-cu l tu red f lounder. - Aquacul tu re 4, 307-308.

Sugatt , R. H., 1980. Effects of sod ium d ichromate exposu re on the i m m u n e r e sponses of juveni le coho sa lmon, Oncorhynchus kisutch, aga ins t Vibrio anguillarum. - Archs environ. Contain. Toxicol. 9, 207-216.

Vit insh, M. & Baranova, T., 1976. Lymphocys t i s d isease of Baltic f lounder (Platichthys flesus), - C.M./ICES P6.

D N A v i r u s e s a s s o c i a t e d w i t h f i s h d i s e a s e s 3 0 7

Walker , D. P. & Hill, B. J., 1980. S t u d i e s o n t h e cu l ture , a s s a y of infect iv i ty , a n d s o m e in vitro proper t ies of l y m p h o c y s t i s virus. - J. g e n . Virol. 51, 383-395 .

Walker , R., 1962. F ine s t ruc tu re of l y m p h o c y s t i s v i rus of f ish. - V i ro logy 18, 503-505 . Walker , R., 1965. Viral D N A a n d c y t o p l a s m i c R N A in l y m p h o c y s t i s ce l ls of f ish. - Ann . N. Y. Acad .

Sci. 126, 386-395 . Walker , R., 1971. PEN, a v i ra l l e s i o n of f i sh e ry th rocy tes . - Am. Zool. I1, 707. Walker , R. & S h e r b u r n e , S. W., 1977. P i sc ine e ry th rocy t i c n e c r o s i s in A t l an t i c cod, Gadusmorhua,

a n d o ther fish: U l t r a s t ruc tu re a n d d i s t r ibu t ion . - J. Fish. Res. Bd Can . 34, 1188-1195. Walker , R. & W e i s s e n b e r g , R., 1965. C o n f o r m i t y of l igh t a n d e l ec t ron mic ro scop i c s t u d i e s on v i rus

par t ic le d i s t r ibu t ion in l y m p h o c y s t i c t u m o u r cel ls of fish. - Ann . N. Y. Acad . Sci. 126, 375-385 . Walker , R. & Wolf, K., 1962. Vi rus a r r ay in l y m p h o c y s t i s ce l ls of sunf i sh . - Am. Zool. 2, 566. W e d e m e y e r , G, A., Meyer , F. P. & Smith , L., 1976. E n v i r o n m e n t a l s t r e ss a n d f ish d i s ea se s . In:

D i s e a s e s of fish. Ed. b y S. F. S n i e s z k o & H. R. Axel rod . T. F. H. Pub l i ca t ions , H o n g Kong , 5, 73-88.

W e i s s e n b e r g , R., 1945. S t u d i e s o n v i rus d i s e a s e s of fish. IV. L y m p h o c y s t i s d i s e a s e in c e n t r a r c h i d a e . - Zoolog ica N. Y. 30, 169-176.

W e i s s e n b e r g , R., 1951. Pos i t ive r e su l t of a f i l t ra t ion e x p e r i m e n t s u p p o r t i n g t he v i e w tha t t he a g e n t of t h e l y m p h o c y s t i s d i s e a s e of f ish is a t rue vi rus . - A n a t . Rec. I I I , 166-171.

W e i s s e n b e r g , R., 1965. Fifty y e a r s of r e s e a r c h o n t h e l y m p h o c y s t i s v i rus d i s e a s e of f i she s {1914-1964). - Ann . N. Y. Acad. Sci. 126, 362.

Wiren , B., 1971. War t d i s e a s e in A t l a n t i c s a l m o n (Salmo salar) - His to log ica l s t u d i e s of e p i d e r m a l p a p i l l o m a in r e a r ed s a l m o n . - Rep. Swed . S a l m o n Res. Inst., A l v k a r l e b y 7, 1-9.

Wolf, K., 1962. E x p e r i m e n t a l p r o p a g a t i o n of l y m p h o c y s t i s d i s e a s e of f i shes . - V i ro logy 18, 249-256 . Wolf, K., 1966, T h e f ish v i ruses . - Adv. V i rus Res. 12, 35-101 . Wolf, K., 1968. L y m p h o c y s t i s d i s e a s e of fish. F i sh Dis. Leafl. - F i sh Wildl . Serv. U. S. 13, 1-4. Wolf, K., 1976. F ish viral d i s e a s e s in Nor th A m e r i c a , 1971-75, a n d r e c e n t r e s e a r c h of t he Eas t e rn

Fish D i s e a s e Labora to ry USA. - F i sh Path . 10, 135-154. Wolf, K., 1979. C o m p a r a t i v e p a t h o g e n e s i s a n d v i r u l e n c e of p o i k i l o t h e r m v e r t e b r a t e H e r p e s v i r u s e s .

In: M u n i c h S y m p o s i u m o n M i c r o b i o l o g y of v i ra l p a t h o g e n e s i s a n d v i ru l ence . Ed. b y P. A. B a c h m a n n . W H O C o l l a b o r a t i n g C e n t r e for Co l l ec t i on a n d E v a l u a t i o n of Da t a on C o m p a r a t i v e Virology, M u n i c h , 183-200.

Wolf, K., 1982. N e w l y d i s c o v e r e d v i r u s e s a n d viral d i s e a s e s of f i shes , 1977-1981 . In: Mic rob i a l d i s e a s e s of fish. Ed. b y R. J. Rober ts . Acad . Press, London , 59-90 .

Wolf, K. & Car l son , C. P., 1965. M u l t i p l i c a t i o n of l y m p h o c y s t i s v i rus in t he b l u e g i l l (Lepomis macrochirus], - Ann . N. Y. Acad. Sci. 126, 414-419 .

Wolf, K. & Q u i m b y , M. C., 1970. Vi ro logy of ee l s t o m a t o p a p i l l o m a . In: P rog re s s in spor t f i she ry r e s e a r c h 1969. R e s o u r c e Publ . F i sh Wildl . Serv., U. S. 106, 94-95 .

Wolf, K. & Smith , C, E., 1981. Herpesvirus salmonis: p a t h o l o g i c a l c h a n g e s in p a r e n t e r a l l y - i n f e c t e d r a i n b o w trout, Salmo gairdneri Richa rdson , fry. - J. F i sh Dis. 4, 445-457 .

Wolf, K., Gravel l , M. & M a l s b e r g e r , R. G., 1966. L y m p h o c y s t i s v i rus : i so la t ion a n d p r o p a g a t i o n in c e n t r a r c h i d f i sh cell l ines . - Sc ience , N. Y. 151, 1004-1005.

Wolf, K., N a g a b a y a s h i , T. & Q u i m b y , M. C., 1975a. Herpesvirus salmonis: t e s t s of s p e c i e s spec i f i - city, s u g g e s t i o n s for i so la t ion , g e o g r a p h i c d i s t r ibu t ion , a n d cont ro l m e a s u r e s . - F i sh H l th N e w s 4, 6.

Wolf, K., H e r m a n , R. L., Da r l i ng ton , R. W. & Taylor , W. G., 1975b. S a l m o n i d v i rus : e f fec ts of Herpesvirus salmonis in r a i n b o w trout fry. - F i sh Hl th N e w s 4, 8.

Wolf, K., Dar l ing ton , R. W., Taylor , W. G., Q u i m b y , M. C. & N a g a b a y a s h i , T., 1978. Herpesvirus salmonis: c h a r a c t e r i z a t i o n of a n e w p a t h o g e n of r a i n b o w trout. - J. Virol. 27, 659-666 .

Zwi l l enberg , L. O. & Wolf, K., 1968. U l t r a s t ruc tu re of l y m p h o c y s t i s v i rus . - J. Virol. 2, 393-399.

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