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Title: Importance of infectious pancreatic necrosis virus in striped bass, Morone saxatilis /
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Title: Importance of infectious pancreatic necrosis virus in striped bass, Morone saxatilis /
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Full Text















IMPORTANCE OF INFECTIOUS PANCREATIC NECROSIS VIRUS
IN STRIPED BASS, Morone saxatilis






By


SALLY JANET WECHSLER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1986

































Copyright 1986

by

Sally Janet Wechsler















ACKNOWLEDGMENTS


I want to thank Dr. G. L. "Pete" Bullock who made my

research project possible, for which I will be eternally

grateful. I am also grateful to Dr. C. P. Goodyear and

the U. S. Fish and Wildlife Emergency Striped Bass

Committee for providing the funding for the investigation.

Dr. R. Gregory deserves recognition as a gracious liaison

person. My appreciation also goes to Dr. F. M. Hetrick

whose laboratory performed the original viral isolation

and who has provided me with extremely helpful comments and

suggestions. My sincere thanks go to Dr. P. E. McAllister

in whose laboratory I worked, and whose endless patience,

goodwill, and scientific insights helped make it all come

together. I also want to acknowledge Dr. C. L. Schultz who

was very helpful in my orientation at Leetown (WV).

I sincerely acknowledge Dr. J. N. Kraeuter, Dr. L. C.

Woods and other Baltimore Gas and Electric Company

personnel who provided access to their facilities,

expertise, and waterfront living accommodations. My

appreciation also extends to R. Lucakovic, J. G. Boone, J.

H. Uphoff, D. Costen, and other personnel of the Maryland

Department of Natural Resources who provided me with

striped bass from the Chesapeake Bay. I also acknowledge

the generous assistance I received from all the people at










the U. S. F. W. S. National Fish Health Research

Laboratory, Kearneysville, WV. Special mention goes to W.

Owens, R. Owens, G. "Sonny" Wilson, B. Knott, W. B. Shile,

S. R. Phelps, and Drs. D. P. Anderson, K. Wolf, B. C.

Lidgerding, and R. C. Simon. Thanks also go to Dr. R. L.

Herman for providing training in fish histopathology, D.

Bowling for preparing the slides, and to Dr. E. B. May

of the University of Maryland School of Medicine-

Baltimore for assistance with the tissue processing and

histological examination. I want to thank Dr. S. W.

Pyle for help with gel electrophoresis. My thanks go to

Dr. G. R. Gilbert, who kindly agreed to be my major

professor, and also to the other committee members Drs.

L. M. Hutt-Fletcher, J. V. Shireman, and J. M. Gaskin.















TABLE OF CONTENTS


ACKNOWLEDGMENTS .. iii

LIST OF TABLES .viii

LIST OF FIGURES ix

ABSTRACT x

CHAPTERS

ONE INTRODUCTION 1

Background .. 1
Objectives .. 10

TWO MATERIALS AND METHODS 12

Cell Cultures and Virus Isolates 12
Cell Cultures .. 12
Isolates of IPNV. 12

Cultivation and Assays of IPNV 13
Preparation of Virus Stocks 13
Virus Infectivity Plaque Assay 13
Virus Infectivity Assay 14

Characterization of IPNV-Sb 14
Purification of IPNV Isolates 14
Determination of Protein Concentration 16
Electrophoresis of Viral Polypeptides 17
Production of Antiserum to IPNV-Sb 19
Neutralization Kinetics 20

Sample Processing for IPNV Assays 21
Processing of Fish Tissues for IPNV assay 21
Preparation of Striped Bass Blood for
IPNV assay .. 22

Detection of Virus-Neutralizing Antibody .23
Fish Blood Preparation for Neutralization
Assay .. 23
Virus-Neutralizing Antibody Assay 23

Fish .. .. 24










Virus Infection Studies .. 25
Waterborne IPNV Challenge of Striped
Bass Fry 25
Waterborne IPNV Challenge of Striped
Bass Fingerlings .. 27
Virus Inoculation of Striped Bass
Fingerlings .. 28
Histological Examination 29

Virus Transmission Studies .. 29
Oral Transmission of IPNV to Striped Bass 29
Vertical IPNV Transmission in Striped Bass 30
Transmission of IPNV from Striped Bass to
Brook Trout .. 31

Humoral Response of Striped Bass to IPNV .32
Early IPNV Titers and Neutralizing
Antibody 32
Exogenous Steroids and Levels of
Neutralizing Antibody in Striped Bass
Challenged with IPNV 32
Antibody Response of Striped Bass to
Second IPNV Challenge 33

Survey of Chesapeake Bay Striped Bass for
IPNV and Virus-Neutralizing Antibody 34
Sampling Young-of-Year Striped Bass 34
Sampling Yearling Striped Bass 35
Sampling of Adult Striped Bass 35

Procedures That Affect IPNV Recovery 35
Tissue Site of IPNV in Striped Bass 35
Storage Conditions of IPNV-infected
Homogenates 36
Storage Temperature of Whole IPNV-infected
Striped Bass 36
Detection of IPNV-carriers after
Steroid Injection 37

THREE RESULTS .38

Virus Infection Studies of Striped Bass .38

Transmission Studies of IPNV in Striped Bass 50
Oral Transmission of IPNV to Striped Bass 50
Vertical IPNV Transmission in Striped Bass 50
Transmission of IPNV from Striped Bass
to Brook Trout 53

Humoral Response of Striped Bass to IPNV 53
Early Humoral Response to IPNV Challenge. 53
Effect of Steroids on Titers of Circulating
IPNV and Virus-Neutralizing Antibody 55











Antibody Response to a Second IPNV
Challenge 59

Survey of Chesapeake Bay Striped Bass .61

Procedures that Affect IPNV Recovery from
Striped Bass .. 61
Tissue Site of IPNV in Striped Bass .61
Virus Recovery from Steroid Injected
Chronic Carriers 61
Virus Recovery from Stored IPNV-carrier
Tissue Homogenates .. 65
Recovery of IPNV from Stored Whole Fish 65

Comparison of IPNV Isolates 70
Protein Electrophoretic Patterns 70
Neutralization Kinetics 72

FOUR DISCUSSION 76


APPENDIX SOURCES OF SUPPLIES AND EQUIPMENT 89

REFERENCES ... 92

BIOGRAPHICAL SKETCH 104


vii














LIST OF TABLES


1 Percent cumulative mortality in striped bass
fingerlings 46

2 Range in viral titers in striped bass
fingerlings that died .. 47

3 Range in viral titers in surviving fingerlings 48

4 Virus titers in fingerlings subjected to a
change in temperature .. 49

5 Range in virus titers in striped bass following
consumption of IPNV-infected brook trout 51

6 Recovery of IPNV during vertical transmission
studies 52

7 Detection of virus-neutralizing antibodies in
fingerlings .. 54

8 Recovery of IPNV from plasma and buffy coat 56

9 Attempts to isolate IPNV from Chesapeake Bay
striped bass .. 62

10 Detection of virus-neutralizing antibody in
striped bass from the Chesapeake Bay 63

11 Striped bass tissues from which IPNV was isolated 64

12 Recovery of IPNV from steroid injected carriers 66

13 Virus titers in homogenates stored at different
temperatures .. 67

14 Recovery of IPNV from homogenates stored in
different types of containers 68

15 Recovery of IPNV from striped bass stored whole 69

16 Neutralization rates for three IPNV isolates 75


viii













LIST OF FIGURES



1 Percent daily mortality in striped bass fry 40

2 Percent daily mortality in striped bass
fingerlings ... 44

3 Mean virus-neutralizing antibody titers 58

4 Virus-neutralizing antibody titers in striped
bass given a second IPNV challenge 60

5 Electrophoretic profile of IPNV polypeptides 71

6 Neutralization kinetics of three IPNV isolates 73











Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of
the Requirements for the Degree of Doctor of Philosophy



IMPORTANCE OF INFECTIOUS PANCREATIC NECROSIS VIRUS
IN STRIPED BASS, MORONE SAXATILIS


by

Sally Janet Wechsler

December 1986


Chairman: Carter R. Gilbert
Major Department: Forest Resources and Conservation


Infectious pancreatic necrosis virus (IPNV), a pathogen

for Atlantic menhaden (Brevoortia tyrannus), was isolated

recently from striped bass fry (Morone saxatilis) in a

hatchery on the Chesapeake Bay (MD). The major goal of

this study was to investigate the effects of IPNV infection

in striped bass.

No clinical or histopathological signs of disease were

observed in striped bass exposed to IPNV by immersion or

intraperitineal injection. This was true even in IPNV-

exposed striped bass that were subjected to an abrupt drop

of pH or a temperature change. Chronic IPNV infection was

not detected in striped bass challenged with waterborne

virus; however, striped bass that consumed or were

inoculated with IPNV contained infectious virus for at

least eight months, despite the presence of circulating

virus-neutralizing antibody.










Striped bass develop virus-neutralizing antibody by

seven days after IPNV inoculation. This humoral response

could be depressed by exogenous corticosteroids. Striped

bass did not exhibit an anamnestic response, but did have

increased antibody titers after a second intraperitoneal

injection with IPNV.

A few striped bass caught in the Chesapeake Bay had

IPNV-neutralizing antibody, although no IPNV was isolated

from these fish. The source of exposure for the striped

bass is not known. Neutralization kinetics and patterns of

viral polypeptides in SDS-polyacrylamide gel electro-

phoresis demonstrated that the IPNV isolates from striped

bass and menhaden are closely related to each other and to

the salmonid isolate VR-299.

Virus-infected striped bass transmitted IPNV to brook

trout; therefore, striped bass should be assayed for IPNV

prior to their introduction into IPNV-free areas.

Detection of IPNV-carriers was improved if striped bass

received steroids prior to assay. A population of striped

bass from which IPNV has been isolated need not be

destroyed since striped bass appear to be resistent to

IPNV-induced disease.















CHAPTER ONE
INTRODUCTION


Infectious pancreatic necrosis virus (IPNV), a

significant pathogen for salmonids (Wolf et al., 1960), has

been recovered from many fish species (Hill, 1982; Ahne,

1985). Recently IPNV was isolated from striped bass

(Morone saxatilis) fry in a hatchery on the Chesapeake Bay

(Schutz et al., 1984). Efforts to raise striped bass in

hatcheries have increased (Schutz et al., 1984), partly in

response to declining populations of striped bass on the

east coast of the United States (Goodyear et al., 1985).

Because IPNV can devastate hatchery populations of trout

(Wolf et al., 1960), this investigation was initiated to

study the impact of IPNV infection on striped bass.


Background


Early in this century, many North American trout

hatcheries experienced annual epizootics that resulted in

massive losses of young fry, affecting the fastest growing

individuals first (M'Gonigle, 1941). In 1955, Wood et al.

described microscopic lesions of pancreatic necrosis in

affected trout fry and also demonstrated that the

condition could be transmitted to fish located downstream

from affected fish. Wood et al. (1955) named the disease










infectious pancreatic necrosis (IPN). Although Wood et al.

(1955) speculated that the pathogenic agent was a virus,

the viral nature was not demonstrated until 1960 by Wolf et

al. Wolf and coworkers (1960) used filtered homogenates of

clinically affected trout to challenge fish and cell

cultures. Significant numbers of exposed fish died and

cytopathic effects (CPE) were apparent in inoculated fish

cell cultures. Electron microscopy revealed that IPNV is a

naked, icosahedral virus, between 55 75 um in diameter

(Moss & Gravell, 1969; Cohen & Scherrer, 1972; Kelly & Loh,

1972). The virus may exist also as tubular particles (Moss

& Gravell, 1969; Ozel & Gelderblom, 1985). The genome of

IPNV consists of two segments of double-stranded

ribonucleic acid (RNA); the molecular weight of one segment

is 2.5 x 106 and the other is 2.3 x 106 (Dobos, 1976;

Macdonald & Yamamoto, 1977). The latter segment encodes

the largest viral associated polypeptide, and the former

encodes the other proteins (Macdonald & Dobos, 1981;

Mertens & Dobos, 1982).

The viral polypetides of IPNV fall into three general

molecular weight classes--low, medium, and high (Cohen et

al., 1973; Dobos & Rowe, 1977; Chang et al., 1978). The

high molecular weight (90 105 x 103) polypeptide

corresponds to the polymerase, the enzyme that catalyzes

the synthesis of messenger RNA (Macdonald & Dobos, 1981;

Stephens & Hetrick, 1983). The capsid protein, the major

component, is of medium weight (50 57 x 103), and the












internal proteins are of low molecular weight (27 31 x

103) (Macdonald & Dobos, 1981).

Because the viral genome consists of two segments of

double stranded RNA, Dobos et al. (1979) proposed that IPNV

be classified a birnavirus. Included in this proposed

group are infectious bursal disease virus (IBDV), found in

young chickens (Nick et al., 1976); Drosophila X virus,

isolated from fruit flies (Teninges et al., 1979); and

Tellina virus and oyster virus, isolated from bivalve

molluscs (Hill, 1976; Underwood et al., 1977). Although

these viruses are similar morphologically and

biochemically, they can be distinguished serologically and

by comparison of the virion-associated proteins (Dobos et

al., 1979). None of the birnaviruses, except IPNV, have

been demonstrated to be pathogenic for fish.

In young trout IPNV infection may be manifested either

as acute death or by fish that exhibit brief episodes of

violent spinning after which the fish sink to the bottom of

the tank (Wolf, 1981). Death usually occurs within 1 2

days after onset of clinical signs. Upon necropsy, dead or

moribund fish may have multiple petechial hemorrhages on

the internal organs. Wolf (1981) considers the finding of

a clear to cloudy gelatinous material in the stomach and

anterior intestine to be pathognomonic for IPNV in young

trout. Histological lesions include necrosis of pancreatic

acini (Lightner & Post, 1969; Swanson et al., 1982) and










frequently, acute catarrhal enteritis (McKnight & Roberts,

1976). Considerable portions of the pancreas become

fibrotic in trout that survive IPNV infection (McKnight &

Roberts, 1976; Swanson et al., 1982).

The exact mechanisms by which IPNV causes death in

infected fish are not known (Hill, 1982). Correlation

between virus titers and severity of disease has been

reported (Okamoto et al., 1984). Virus titers

progressively rise in trout fry following challenge with

IPNV and the highest titers of virus are recovered from

fish that have died (Okamoto et al., 1984). Swanson and

Gillespie (1982) speculated that key events occurred within

the first few days following viral challenge. Using

experimentally infected Atlantic salmon (Salmo salar),

Swanson and Gillespie (1982) noted that peak viremia

occurred at day two. Swanson and Gillespie (1982) stated

that the Atlantic salmon, unlike trout, are successful, by

some unexplained mechanism, in preventing further increases

in viral titers, thus preventing IPNV-induced mortality.

For reasons yet to be determined, by six months of age

trout lose their susceptibility to IPNV-induced mortality

(Frantsi & Savan, 1971; Wolf, 1972). In addition, trout

species differ in their susceptibility to IPNV-induced

mortality (Hill, 1982; Silim et al., 1982). The resistance

may be mediated genetically. Wolf (1976) reported the

development of IPNV-resistant trout strains, using

selective breeding.










Many different factors have been described that affect

the outcome of IPNV infection on trout. Frantsi and Savan

(1971) demonstrated that water temperature affects the

number of deaths associated with IPNV. The authors found

fewest deaths in viral-exposed trout fry kept at 4.50C,

most at 100C, and an intermediate number of deaths in fry

held at 150C. Also, as mentioned earlier, the age at which

fish are exposed to IPNV affects IPNV-induced disease

(Dorson & Torchy, 1981). Young fish (less than six months)

are more susceptible to IPNV-induced mortality, but older

trout do became subclinically infected with IPNV (Frantsi &

Savan, 1971).

Stress was also found to influence IPNV infection,

especially in trout that survived early exposure but

continue to be infected. Frantsi and Savan (1971) found an

increase in IPNV isolation from trout survivors after an

episode of mild stress induced by low oxygen. McKnight and

Roberts (1976) reported 10 20% mortality in IPNV-carrier

rainbow trout (ages 6 to 11 months) at 72 hours following a

stressful event such as handling, transport, overcrowding,

or low oxygen. Higher IPNV titers were obtained from

stressed fish compared to titers from non-stressed fish

(McKnight & Roberts, 1976).

It is not known how IPNV persists in infected trout.

Normally IPNV multiplies intracytoplasmically and is

released by viral-induced cytolysis (Malsberger & Cerini,










1963; Argot & Malsberger, 1972). Defective interfering

particles are produced within cells, but do not cause cell

rupture (Nicholson & Dunn, 1974; Macdonald, 1978).

Therefore, this may be a mechanism by which IPNV persists

in carriers (Nicholson & Dexter, 1975; Hedrick et al.,

1978; Macdonald & Kennedy, 1979). Other researchers have

proposed a relationship between levels of virus-

neutralizing antibodies and titers of IPNV; i. e. fish with

high levels of IPNV-neutralizing antibody would have lower

titers of IPNV (Yamamoto 1975a, 1975b). However, there is

no correlation between the tissue levels of virus and

antibody titers in IPNV-carrier trout (Reno, 1976; Reno et

al., 1978). Another mechanism by which IPNV persists may

be due to an IPNV-induced decrease in the mitogenic

responsiveness of lymphocytes and macrophages (Knott &

Munro, 1986).

Trout survivors present after an episode of IPNV disease

continue to contain, and periodically shed, IPNV (Wolf et

al., 1968; Billi & Wolf, 1969; Yamamoto & Kilistoff, 1979).

Fish located downstream from the effluent of an IPNV-infected

hatchery can become infected with IPNV (Sonstegard et al.,

1972). The virus can be spread by other animal vectors.

Gulls, chickens, and mink, after being fed IPNV-infected

fish, transiently shed virus in their feces (Eskildsen &

Jorgensen, 1973; Sonstegard & McDermott, 1972). Once shed,

IPNV can survive for weeks in dried areas (Wolf, 1966; Ahne,

1982), or for months in aqueous environments (Desautels &










MacKelvie, 1975; Baudouy & Castric, 1977; Wedemeyer et al.,

1978).

Another means by which IPNV may be spread is by the

transport of eggs taken from IPNV-infected stocks (Hill,

1982). Although egg-associated transmission of IPNV was

suggested as early as 1959 (Snieszko et al.), and was

documented in 1963 (Wolf et al.), transport of eggs from

IPNV-infected stocks continued, perhaps resulting in the

international spread of the virus (Sano, 1971).

Disease outbreaks associated with IPNV have been

reported around the world. Although the virus always is

morphologically similar, IPNV has several serotypes (Wolf &

Quimby, 1971; McMichael et al., 1975). Different serotypes

signify that an antibody generated against IPNV isolated

from one disease outbreak may, or may not, react with IPNV

recovered from a different location or disease episode.

The virus has three major serotype groups: (1) most North

American IPNV isolates (Buhl, Reno, Powder Mill, West

Buxton, Cascade Locks, VR-299); (2) isolates from Denmark

and France (d'Honnincthun, Bonnamy, Sp); and (3) IPNV from

Denmark and Japan (Ab, EEV) (Okamoto et al., 1983). The

exact placement of IPNV isolates varies somewhat between

authors (Macdonald & Gower, 1981; Ishiguro et al., 1984).

The differences probably are related to the variation of

methods and antisera used to determine serotypes (Nicholson

& Pochebit, 1981), and to variations in sensitivity of the

isolates to neutralization (Macdonald & Gower, 1981).











Isolates of IPNV differ somewhat in their stability

during storage and freeze-thaw cycles (Wolf & Quimby,

1971; Lientz & Springer, 1973; McMichael et al., 1975).

However, despite the differences in serotype and variation

in storage stability, IPNV isolates induce similar clinical

signs in challenged trout (Wolf & Quimby, 1971; Silim et

al., 1982). The virus has been isolated from many

clinically normal non-salmonid fishes including white

sucker, Catostomus commersoni (Sonstegard et al., 1972);

perch, Perca fluviatilis (Munro et al., 1976); European

eel, Anguilla anguilla (Castric & Chastel, 1980); bream,

Abramis brama (Adair & Ferguson, 1981); Atlantic

silverside, Menidia menidia (McAllister et al., 1984);

tilapia, Tilapia mossambica (Chen et al., 1985); and

goldfish, Carassius auratus (Hedrick et al., 1985). In

addition, IPNV has been recovered from moribund

nonsalmonids, including northern pike (Esox lucius) (Ahne,

1978), sea bass (Dicentrarchus labrax) (Bonami et al.,

1983), and southern flounder (Paralichthys lethostigma)

(McAllister et al., 1983). However, the pathogenicity of

IPNV has not been demonstrated for these species.

Experimental transmission studies using the pike isolate

did not induce viral disease in either pike or rainbow

trout (Ahne, 1978), and similar avirulence was observed for

the flounder isolate in both flounder and brook trout

(McAllister et al., 1983).










The lack of demonstrable pathogenicity of IPNV has

also been reported for other nonsalmonids. Experimental

IPNV infection of various marine species did not cause

clinical disease, although virus multiplication probably

occurred in the french grunt, Haemulon flavolineatum

(Moewus-Kobb, 1965). Vertical transmission of IPNV was

demonstrated in experimentally inoculated zebra fish,

Brachydanio rerio (Seeley et al., 1977), although no

disease was detected in the offspring.

In contrast, IPNV has been shown to be pathogenic for

three nonsalmonid species. An IPNV isolate has been

demonstrated to induce high mortality and brachionephritis

in Japanese eels, Anguilla japonica (Sano et al., 1981).

In yellowtail, Seriola quinqueradiata, experimental

inoculation with IPNV resulted in high mortality in

fingerlings that developed ascites and hepatic hemorrhage

(Sorimachi & Hara, 1985). Altantic menhaden, Brevoortia

tyrannus, injected with IPNV developed dark coloration and

hemorrhage at fin bases, and began swimming in circles

prior to death 3 5 days post inoculation (Sterhens et

al., 1980). Virus was reisolated from the brain, kidney,

spleen, liver, blood and gonadal tissue from menhaden that

died.

In 1984, Schutz et al. reported the isolation of IPNV

from striped bass fry in a hatchery operated by the

Baltimore Gas and Electric, Co. Virus was recovered from

fry exhibiting erratic swimming behavior and high











mortality. Histological examination of moribund fry

revealed areas of necrosis in the epidermis. The virus was

isolated from kidneys taken from surviving striped bass at

three and six months following the original IPNV isolation.

Inflammation around pancreatic acini was observed in

histological sections taken from the survivors at three

months. This constellation of findings resembles that

found in salmonids in which IPNV causes death in young fry

and histopathological lesions in pancreatic acini of

infected fish. Thus, it was hypothesized that IPNV may

cause mortality in striped bass fry (Schutz et al., 1984).

Striped bass traditionally have been important both

as commercial and recreational fish (Morgan & Rasin, 1981);

however, the Chesapeake Bay stocks of striped bass have

been declining (Goodyear et al., 1985). The reasons for

this decline are not known, although many possibilities

have been suggested. These include loss of appropriate

habitat (Kerhehan et al., 1981), overfishing (Coutant,

1985), starvation of fry (Eldridge et al., 1981), pollution

(Hall et al., 1984), and temperature and oxygen levels

(Coutant, 1985). In addition, disease might be

contributing to the decline. "Spinning disease" can be

induced by IPNV in Atlantic menhaden (Stephens et al.,

1980) and a disease episode was occurring in menhaden in

the Chesapeake Bay at the time that IPNV was isolated from

the moribund striped bass fry. Records kept by the










Maryland Department of Natural Resources indicated a

correlation between large outbreaks of "spinning disease"

in menhaden and poor year classes of striped bass in the

Chesapeake Bay (Schutz et al., 1984).


Objectives


Research was initiated to investigate the importance

of IPNV infection in striped bass. The points to be

specifically addressed were (1) whether IPNV induced

mortality in striped bass; (2) what histological lesions

developed in striped bass exposed to IPNV; (3) the

influence of age and strain of striped bass on IPNV

virulence; (4) the effect of water temperature on IPNV-

induced disease in striped bass: (5) the routes (both

vertical and horizontal) by which IPNV is transmitted in

striped bass; (6) the influence of stress, including abrupt

environmental changes and exogenous corticosteroids, on

IPNV infection in striped bass; (7) the humoral response of

striped bass to IPNV; (8) comparison of the striped

bass isolate of IPNV with a menhaden IPNV isolate and the

standard North American salmonid isolate (VR-299); and

(9) the effects of sample handling procedures on viral

infectivity.














CHAPTER TWO
MATERIALS AND METHODS

Cell Cultures and Virus Isolates

Cell Cultures

Chinook salmon embryo (CHSE-214) cells were grown at

180C in Eagle's minimal essential medium (EMEM) containing

10% fetal bovine serum (EMEM-10). For cell transfers,

confluent monolayers were dispersed with 0.25% trypsin.

For virus assays, cells were suspended in EMEM-10

containing antibiotics: 200 IU/ml penicillin and 200 ug/ml

streptomycin (PS). Cells were seeded into eight-well

culture plates and incubated at 180C in ambient air plus 2%

carbon dioxide (C02) until monolayers were confluent.


Isolates of IPNV

The striped bass isolate of IPNV (IPNV-Sb) that was

originally isolated from moribund striped bass fry (Schutz

et al., 1984), was used for all experiments, except where

noted. The virus was passage twice in CHSE-214 cells and

aliquots were stored at -700C.

Three other isolates of IPNV, the standard North

American isolate (VR-299), an isolate from Atlantic

menhaden (IPNV-M), and a European isolate (IPNV-Ab), were

handled as described for IPNV-Sb. Before use, aliquots of

virus were thawed and diluted in phosphate buffered saline

(pH 7.2; PBS).










Cultivation and Assays of IPNV

Preparation of Virus Stocks

Confluent monolayers of CHSE-214 cells grown in 75 cm2

flasks, were drained of medium and inoculated with IPNV

( < 0.01 plaque forming units [pfu] of IPNV per cell).

Following an one hour adsorption period at 150C (with

gentle agitation every 15 minutes), EMEM-10 was added to

the IPNV-inoculated cells. The virus-exposed cells were

incubated at 150C until the monolayers showed extensive

cytopathic effects (CPE) (usually 2 3 days). Cells and

culture fluid were harvested and centrifuged at 1500 x I

for 15 minutes at 40C. The supernatant liquid was stored

in 1 ml aliquots at -700C. The infectivity of stock virus

was determined by plaque assay as described below.


Virus Infectivity Plaque Assay

A modification of a virus infectivity assay (Moss &

Gravell, 1969) was used to determine virus titers.

Aliquots (0.1 ml) of each sample dilution were inoculated

onto duplicate wells of drained CHSE-214 monolayers. The

inoculated monolayers were incubated for 1 hour at 190C to

allow virus adsorption and then were overlayed with EMEM

containing 2% normal calf serum, 0.16 M Tris buffer and PS

(EMEM-2), plus 1% agarose. A second overlay, 2 ml of EMEM-

2 (without agarose), was added. Plates were incubated at

180C in ambient air plus 2% CO2 until cytopathic effects

(CPE) were noted. Cell sheets were fixed with 30% formalin,










and stained with 1% crystal violet in ethanol. Plaques

were counted and infectivity titer was calculated as pfu

per ml or pfu per g of tissue.


Virus Infectivity Assay

The simultaneous seeding method (McDaniel, 1979) was

used to detect IPNV in striped bass fry and striped bass

sex products. An aliquot (0.05 ml) of each sample dilution

was added to each of four wells of a 96-well tissue culture

plate and then 0.1 ml of CHSE-214 cells (about 2 x 105

cells/ml) was added to each well. Plates were incubated in

ambient air at 180C. If no CPE was observed by 5 days, the

sample was harvested from the wells and inoculated with

fresh cells (blind-passaged). If no CPE was noted after 5

additional days, the sample was considered to be negative

for IPNV.


Characterization of IPNV-Sb


Purification of IPNV Isolates

Three isolates of IPNV, the striped bass isolate

(IPNV-Sb), the menhaden isolate (IPNV-M), and the North

American isolate (VR-299), were each purified using a

modification of a procedure previously described by Chang

et al. (1978). Confluent monolayers of CHSE-214 cells were

inoculated with virus ( < 0.01 pfu/cell). The virus was

allowed to adsorb for 1 hour at 150C, then EMEM-2 was

added. After 48 hours incubation at 150C, the cell sheets











showed extensive CPE. The cells and culture fluids were

centrifuged at 7000 x 2 for 20 minutes at 4C. The cell

pellet was resuspended in 5 ml of buffer made up of 0.01 M

Tris, 0.01 M sodium chloride, and 0.001 M disodium

ethylenediamine tetraacetate (TNE; pH 7.5). A equal volume

(5 ml) of trichlorotrifluoroethane (Freon) was added and

the solution was homogenized for two minutes at high speed.

The homogenate was centrifuged at 4500 x 2 for 15 minutes

at 40C. The top layer of TNE was removed and stored at

40C. An additional 5 ml of TNE were added to the Freon-

cell mixture. This solution was homogenized and

centrifuged as described above. The TNE layer was combined

with the first freon-extract. The original cell

supernatant fluid was adjusted to contain 6% (wt/v)

polyethylene glycol (M.W. 20,000), and 2.2% (wt/v) sodium

chloride. This mixture was stirred for 3 hours at 40C.

The solution was centrifuged at 9000 x q for 1 hour at 40C.

The supernatant liquid was discarded and the pellet was

resuspended in the TNE layer (5 8 ml) from the freon

extraction. This suspension was gently layered over a

sucrose or cesium chloride (CsC1) gradient.

For IPNV samples that were used to inoculate rabbits,

the crude virus preparation was purified on a linear

sucrose (10 50%) density gradient in Ultra-clear

centrifuge tubes (25 x 76 mm) that were centrifuged at

97,000 x q for 45 minutes at 40C. The virus band was

withdrawn by side tube puncture using a 20 gauge needle and











5 ml syringe. The virus band was diluted in TNE and

centrifuged at 83,000 x j for 40 minutes at 40C. The viral

pellet was resuspended in 1 ml TNE and stored at -700C.

Protein content was measured by the method described below.

For IPNV samples that were analyzed for virus specific

proteins, the crude virus preparation was purified on a

linear CsCl (20 to 40%) gradient in cellulose nitrate

centrifuge tubes (5/8" x 4") that were centrifuged at

115,000 x q for 16 hours at 40C. The virus band was

removed by side tube puncture with a 22 gauge needle and 5

ml syringe, diluted in TNE, and layered over a second CsCl

gradient. The band containing pure virus was removed,

dialyzed against TNE, and concentrated to 1 ml using

membrane microconcentrators. Protein concentration was

determined as described below and infectivity was

quantified by the plaque assay.


Determination of Protein Concentration

The Lowry method (Lowry et al., 1951), as modified by

Garvey et al. (1977), was used to determine the protein

concentration of the purified IPNV isolates (IPNV-Sb, IPNV-

M and VR-299). Bovine albumin was diluted (1 to 0.01

mg/ml) in phosphate buffered saline for protein standards.

One milliliter of a solution containing 2% sodium

carbonate, 0.02% cupric sulfate in 0.1 N sodium hydroxide

was added to 0.2 ml of each unknown and standard sample.

The samples were mixed, incubated for 10 minutes at 250C,










and then 0.1 ml of 1 N phenol reagent was added. After 1

hour incubation at 250C, the optical density of each sample

at 660 nm was determined by spectrophotometry. All samples

were assayed in duplicate. The mean optical density of

each standard was plotted against the protein

concentration. The protein concentration of the unknown

samples were calculated by interpolation from the standard

line.


Electrophoresis of Viral Polypeptides

Comparison of the structural proteins of three IPNV

isolates (IPNV-Sb, IPNV-M, and VR-299) was performed by

examination of the banding pattern of the viral proteins in

discontinuous sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). The Tris-glycine buffer method

described by Laemmli (1971) was used. A 10% resolving gel

was prepared by combining 13.3 ml of 30% acrylamide, 0.4 ml

of 10% sodium dodecyl sulfate, 10.0 ml of 18.5% tris-HCl

(pH 8.8), and 16.2 ml of distilled water. The solution was

degassed under vacuum for 15 to 30 minutes and 0.1 ml of

10% ammonium persulfate and 0.02 ml of N,N,N',N'

tetramethyl ethylenediamine (TEMED) were added. The

mixture was gently swirled and poured into the gel mold.

After polymerization (20 to 30 minutes), the gel was rinsed

with distilled water. The gel was overlayed with a buffer

containing 2.5 ml of 18.5% Tris-HCl (pH 8.8), 0.1 ml 10%

SDS and 7.4 ml distilled water, and allowed to stand










overnight. The 4% acrylamide stacking gel was made by

combining 1.3 ml of 30% acrylamide, 2.5 ml of 6% Tris-HC1

(pH 6.8), 0.1 ml of 10% SDS and 6.1 ml of distilled water.

This mixture was degassed as above; then 0.05 ml of 10%

ammonium persulfate and 0.005 ml of TEMED were added. The

solution was gently swirled and poured on top of the

resolving gel. After 20 minutes, the gel was rinsed with

distilled water. Total gel size was 1.5 mm x 14 x 16 cm.

Ten ug of each IPNV isolate were mixed with a solution that

contained 4% SDS, 20% glycerol, 10% 2-mercaptoethanol,

0.01% bromphenol blue, and 1.5% Tris-HCl. The sample

solution was heated to 1000C for three minutes, cooled to

40C, and loaded onto the gel. Running buffer (3.0 g Tris,

14.4 g aminoacetic glycine and 1 g SDS in one liter of

distilled water) was placed in the upper and lower

chambers. A direct current of fifteen mAmps was applied

until the bromphenol blue dye line passed through the

stacking gel. The current was then raised to 25 mAmps and

held constant until the dye line was 1 cm from the bottom

of the resolving gel. The gel was removed, put into a

solution of 50% methanol and 10% acetic acid for one hour,

and stained overnight in a solution of 0.01% coomassie

blue, 25% methanol and 10% acetic acid. The gel was

destined using a solution of 25% methanol and 10% acetic

acid.

The molecular weights of the IPNV structural proteins

were determined by comparison to the electrophoretic










mobility of proteins of known molecular weight run in the

same gel. The following molecular weight markers were

used: phosphorylase B (97,400), bovine albumin (66,000),

egg albumin (45,000), glyceraldehyde-3-phosphate

dehydrogenase (36,000), carbonic anhydrase (29,000),

trypsinogen (24,000), and tryspin inhibitor (20,100). The

relative mobility (Df) of each polypeptide band was

determined by dividing the distance traveled by each

protein band by the distance traveled by the dye front.

The logarithml0 of the molecular weight of the marker

proteins were plotted against the Df. The molecular weight

of each viral protein was determined from this standard

line.


Production of Antiserum to IPNV-Sb

Antibody to the striped bass isolate of IPNV (IPNV-Sb)

was prepared in New Zealand White rabbits. Purified IPNV-

Sb was diluted to give 1 mg/ml in PBS. Rabbits were

injected intravenously with 0.3 ml of this preparation.

The remaining 0.7 ml was mixed with an equal volume of

Freunds' incomplete adjuvant. Half of this mixture (0.7

ml) was injected intramuscularly; the remaining 0.7 ml was

injected subcutaneously into two different sites. The same

procedure was repeated at two week intervals, for one

month. Two weeks after the third boost, the rabbit was

exsanguinated. The blood was held overnight at 40C and

centrifuged at 1500 x g at 40C for 20 minutes. Serum was










collected, heated at 560C for 30 minutes to inactivate

complement, filtered using a membrane filter (0.45 micron

pore size), and stored in 1 ml aliquots at -700C.


Neutralization Kinetics

Antigenic differences in closely related viral

isolates can be determined from analysis of the patterns

and rates of neutralization (neutralization kinetics) of

virus isolates in reactions with homologous and

heterologous antisera. A modification of the procedure

described by Macdonald and Gower (1981) was used to

determine the antigenic relationships between three IPNV

isolates(IPNV-Sb, IPNV-M, VR-299). Rabbit antisera to

IPNV-M and VR-299 were available at the U. S. Fish and

Wildlife Service, National Fish Health Research Laboratory,

Kearneysville, WV. Antibody to IPNV-Sb was prepared in

rabbits as described above. Each antibody was diluted to a

concentration that neutralized 50% more homologous virus at

5 minutes than at 0.25 minutes after combination of

antibody and virus. Each IPNV isolate (IPNV-Sb, IPNV-M,

VR-299) was diluted in PBS to give a final concentration of

100 200 pfu/well as determined by plaque assay.

For each trial, antibody was assayed with its

homologous and the two heterologous IPNV isolates. Equal

volumes of antibody and IPNV were combined and incubated at

40C. A 25 uL sample was removed at 0.25, 0.5, 1.0, 1.5,

2.0, 3, 4 and 5 minutes. The 25 ul sample was expelled










into 2.5 ml of PBS to stop the reaction and tested for

residual infectivity using the plaque method. The mean

plaque count of four replicate wells was calculated for

each time point. The percent of residual infectivity was

plotted against reaction time for each combination of IPNV

isolate and antiserum. For the purpose of calculation of

the rate of neutralization (K), K was assumed to be linear

for the first 0.25 minute of the reaction and was

determined by the formula K = D/t 2.3 log Vo / Vt, where

D = the reciprocal of the dilution of antibody, t = 0.25

minute, Vo = total virus and Vt= the number of virus

plaques at 0.25 minute (Macdonald & Gower, 1981).


Sample Processing for IPNV Assays


Processing of Fish Tissues for IPNV Assay

Striped bass fry and striped bass sex products were

processed using the following protocol prior to being

assayed for IPNV. Five to 10 fry were washed twice in PBS

and blotted on paper towels to remove excess water.

Striped bass fry, or sex products, were added to 1 ml of

PBS. The mixture was pulled into a 3 or 5 ml syringe

through a 20 or 22 gauge needle, forcibly expelled several

times to disrupt the tissues, and filtered using a membrane

filter (0.45 micron pore size). Four ten-fold dilutions of

each sample were assayed for IPNV using the simultaneous

seeding method.











Fingerlings weighing less than 5 grams were processed

as whole fish. Larger fish were dissected using sterilized

instruments and the internal organs, blood and feces were

assayed for virus. Samples were weighed and dissociated

using a sterile pestle and alundum. The resultant paste

was suspended 1:10 (wt/v) in PBS and centrifuged at 1500 j

for 30 minutes at 40C to sediment debris. Four serial

dilutions of supernatant fluid was assayed for infectious

virus using the plaque method.


Preparation of Striped Bass Blood for IPNV Assay

Striped bass blood samples were obtained from the

caudal vein, by venipuncture using a 20 to 22 gauge needle

or by severing the caudal peduncle. Blood was collected in

heparinized microhematocrit capillary tubes. Within 2

hours of collection, blood samples were centrifuged and

processed for virus assay as follows. The buffy coat

(about 1 ul) was cut from the microhematocrit tube at the

interface of the packed cells and plasma, placed into 0.5

ml sterile distilled water with PS, vigorously mixed, and

incubated at 190C for 1 hour. An additional 0.5 ml of PBS

was then added to give about a 1:1000 (v/v) dilution of the

buffy coat. An additional dilution (1:10) was made in PBS.

The plasma was expelled into 1.0 ml PBS and a second

dilution (1:10) made. These samples were immediately

assayed for IPNV using the plaque assay.










Detection of Virus-Neutralizing Antibody


Fish Blood Preparation for Neutralization Assay

Fish blood samples were obtained from the caudal vein

as described above and collected in heparinized

microhematocrit capillary tubes or in plain tubes. Tubes

were centrifuged and the supernatant fluid removed.

Because preliminary assays indicated that normal striped

bass blood neutralized IPNV at serum or plasma dilutions

less than 1:100, all striped bass blood samples were

assayed for virus-neutralizing antibodies at 1:100 or

greater dilution. After centrifugation of the blood

samples, 10 ul of fish plasma/serum were added to 1.0 ml

of PBS, heated at 450C for 30 minutes to inactivate

complement (Sakai, 1981), and stored at 4 or -200C.


Virus-Neutralizing Antibody Assay

To detect circulating virus-neutralizing antibodies,

serum or plasma samples are incubated with a known amount

of virus, and then the residual infectivity determined.

The following protocol was used for detection of IPNV-

neutralizing antibody in striped bass. Equal volumes of

fish serum or plasma sample and IPNV (1.6 x 103 pfu/ml)

were mixed, and, with periodic gentle agitation, incubated

at 190C for one hour. Residual infectivity was determined

by plaque assay. Total virus was determined by combining

equal volumes of test virus and PBS and measuring virus

infectivity. Blood samples were tested at the 1:100










dilution and were recorded as being positive for

neutralizing activity if the sample neutralized 50% or more

of total virus. Antibody titer was determined by testing

serial dilutions of plasma against a constant number of

virus and calculating the serum dilution that neutralized

50% of total virus (Reed & Muench, 1938). Unless otherwise

indicated, fish samples were tested only against the

striped bass isolate of IPNV (IPNV-Sb).


Fish


Striped bass fry from Maryland (Delmarva Ecological

Laboratories, Elkton, MD) were held at the Baltimore Gas

and Electric Company (MD) striped bass hatchery located on

the Chesapeake Bay (MD). All other experimental fish were

maintained at the U.S. Fish and Wildlife Service, National

Fish Health Research Laboratory (Kearneysville, WV).

Striped bass fry were obtained from Richmond Hill State

Fish Hatchery (GA) and Richloam Fish Hatchery (FL).

Striped bass fry were provided with brine shrimp (Artemia

salina) nauplii as live food.

Striped bass fingerlings obtained from Harrison Lake

National Fish Hatchery (VA) were maintained in 15 L tanks

that received 4 I/min of 220C spring water unless

otherwise noted. Striped bass fingerlings were fed

commercial salmon or trout food. Five-year-old striped

bass obtained from Edenton National Fish Hatchery (NC) were

kept in spring and reservoir water (12 250C). Rainbow










(Salmo gairdneri), brook (Salvelinus fontinalis) and brown

(Salmo trutta) trout were provided as forage.

Brook trout fingerlings were obtained from White

Sulfur Springs National Fish Hatchery (WV) and were held in

120C spring water and fed trout ration.


Virus Infection Studies


Waterborne IPNV Challenge of Striped Bass Fry

This series of virus challenge trials was conducted to

determine if IPNV would induce significant mortality in

striped bass. Several factors, including age at exposure,

strain of striped bass, water temperature, and

environmental stress, were investigated for their effect on

striped bass exposed to a static IPNV-bath.

Striped bass fry from Florida and Georgia were divided

into groups of 60 and held in 500 ml tissue culture

bottles containing spring water (190C). Florida fry were

challenged with IPNV at 1, 3, 5, 7, 10 and 15 days post-

hatch. Georgia fry were exposed to IPNV at 5, 7 and 10

days post-hatch. For each strain and age group, IPNV was

added to three bottles to give 106 pfu/ml of water. A

similar volume of PBS was added to three control bottles.

After 6 hours, and daily thereafter for three weeks, half

of the water in each bottle was replaced, debris was

removed, and newly hatched brine shrimp were provided as

forage for the striped bass fry. All dead fish were

removed, stored at 4 or -200C, and assayed for IPNV using










the simultaneous seeding procedure. The length of storage

varied from 1 90 days.

Maryland strains of striped bass fry (Chesapeake and

Delaware Canal [C & D] and Nanticoke [NAN] River) were

divided into groups of 30 fry that were placed in 200 ml

culture bottles containing Chesapeake Bay estuarine water

(18 230C). The C & D canal striped bass fry were

challenged with IPNV at 1, 5, 15 and 20 days post-hatch.

The NAN striped bass fry were exposed to IPNV at 10, 15 and

20 days post-hatch. The challenge protocol and daily care

were as described above, with the exception that daily water

changes replaced 75% (instead of 50%) of the water. Dead

striped bass were stored for 0 3 days at 40C prior to

being assayed using the simultaneous seeding method. At

the end of three weeks, 98% of the surviving striped bass

were assayed for virus. Remaining survivors were assayed

for virus and virus-neutralizing antibody six months after

IPNV challenge.

To test the effects of an abrupt shift in pH on

mortality in IPNV-exposed striped bass fry, five-day-old

striped bass fry (C & D strain) were challenged with IPNV

and maintained as described above. The only difference

occurred on day five after viral exposure when 50% of the

water (pH 7.1) was replaced with water to which sulfuric

acid had been added to bring the pH of the water to pH 6.3.

After 24 hours, the acidified water (now pH 6.5) was

replaced with ambient water. Fish that died were collected











daily for three weeks and immediately assayed for virus

using the simultaneous seeding method.


Waterborne IPNV Challenge of Striped Bass Fingerlings

Twenty-six-day-old striped bass were divided into 12

groups of 50 fish each. Six groups were kept at 120C, and

six were kept at 220C. Water flow to all tanks was stopped

for six hours, and the water aerated. At each temperature,

two tanks were seeded with 104 pfu of IPNV per ml water,

two tanks received phosphate buffered saline (sham

controls), and two tanks of striped bass served as

treatment controls. Dead fish were collected twice daily

for three weeks and stored at -200C until assayed for virus

using the plaque assay. Samples were stored for 7 240

days.

Six-month-old striped bass fingerlings were assigned

to three groups of 12 striped bass. One group of striped

bass was exposed to a 6-hour static immersion in 106 pfu of

IPNV per milliliter of water. Phosphate buffered saline

was added to the 6-hour static bath of the sham control

group. The treatment control group of striped bass

underwent six hours of static bath. Tanks were checked

daily for mortality. At four weeks post exposure, striped

bass were assayed for IPNV and for virus-neutralizing

antibody.










Virus Inoculation of Striped Bass Fingerlings

An alternative method of IPNV exposure was used for

striped bass fingerlings two months and older. Rather than

being immersed in IPNV, each fish received an

intraperitoneal (i.p.) injection with IPNV. For all

injection and sampling procedures, striped bass were

anesthetized with tricaine methanesulfonate (MS-222).

Striped bass, 60, 90, 120, 150 and 180 days old, were

placed into groups of 50 fish. At each age, one group of

striped bass received an injection of 0.05 ml of PBS

containing 0, 103, 105, or 106 pfu of IPNV. Treatment

controls were anesthetized and returned to the tank. Dead

fish were collected daily for four weeks, and stored at

-200C for 1 7 days until they were assayed for infectious

virus. At monthly intervals, survivors were bled for

virus-neutralizing antibody, and tissues were assayed for

infectious virus.

To determine the effect of an abrupt temperature shift

on mortality in IPNV-infected striped bass, 24 six-month-old

striped bass were acclimated for two weeks to 120C and an

additional 24 fish were maintained at 220C. All the fish

were anesthetized and inoculated i.p. with 106 pfu of virus.

Two weeks later, half of the fish held at half of the fish

held at 120C were transferred to 220C, and half of the fish

held at 220C were transferred to 120C. Fish were observed

daily for mortality. After one month, survivors from each

group were bled and assayed for virus.










Histological Examination

The histology of IPNV-infected striped bass was

examined. Striped bass fingerlings were selected at

monthly intervals after IPNV injection, incised along the

ventral abdomen, and immersed in Bouin's fluid fixative

(Luna, 1968). Fingerling tissues were embedded in

paraffin. Striped bass fry (3 6 per day) were fixed in a

solution of formalin and glutaraldehyde (4:1) and embedded

in hydroxyethyl methacrylate. Blocks were sectioned at 4 -

6 microns, stained with hematoxylin, eosin and phloxine

(Thompson, 1966) and examined by light microscopy.


Virus Transmission Studies


Oral Transmission of IPNV to Striped Bass

This study was conducted to ascertain if striped bass

could become infected with IPNV by consuming IPNV-

containing food. Six-month-old striped bass were tagged

and placed into four tanks. Each tank contained six

striped bass. Three-month-old brook trout, each harboring

102 104 pfu of IPNV, were added to the tanks. Each

striped bass was observed to consume one or two trout.

Striped bass that did not eat brook trout were removed from

the experiment. For six months, the striped bass were

assayed periodically for the presence of IPNV and virus-

neutralizing antibody.











Vertical IPNV Transmission in Striped Bass

A series of experiments was conducted to investigate

if vertical transmission of IPNV occurred in striped bass.

To determine if striped bass that survived a natural IPNV

episode actually shed IPNV in sex products, the following

study was conducted. A population of two-year-old striped

bass from which IPNV was originally isolated (Schutz et

al., 1984) was sampled. Milt was manually expressed from

males; however, since striped bass females mature at the age

of three plus years (Setzler et al., 1980), eggs were not

available. Because preliminary results showed that IPNV can

be recovered from striped bass kidney and, therefore, might

be shed in the urine; urine was manually expressed and

collected from sexually immature striped bass. Fourteen

urine and 20 milt samples were tested for the presence of

IPNV using the simultaneous seeding assay. Samples were

processed within two hours of collection.

To determine if IPNV-infected striped bass transmit

IPNV in their sex products, five-year-old striped bass were

injected i.p. with 106 pfu of IPNV in December 1984, and

spawned in April and May 1985. Samples of sex products,

fertilized eggs and offspring were assayed for IPNV using

the plaque method.

To investigate whether IPNV-infected striped bass sex

products would result in IPNV-infection of the offspring,

sex products were collected from spawning striped bass

adults caught in the Nanticoke River (MD). Subsamples of











the eggs and milt were tested for the presence of IPNV.

The remaining portions of eggs and milt were used to

produce fertilized eggs. Eggs were dipped once in clean

water and mixed with milt for fertilization. Additional

water was added to the fertilized eggs and the mixture was

placed in buckets and aerated. Striped bass fry hatched 2

days later. Treatment groups included (1) virus-exposed

eggs plus virus-free milt--eggs were briefly mixed with

virus (106 pfu/ml final IPNV concentration) before being

dipped in water and then fertilized; (2) virus-free eggs

plus virus-exposed milt--sperm was mixed with IPNV (106

pfu/ml final concentration of IPNV) and added to the eggs;

and (3) treatment controls--virus-free eggs were mixed

with virus-free milt. Periodically, fertilized eggs and

fry were tested for the presence of IPNV using the

simultaneous seeding assay.


Transmission of IPNV from Striped Bass to Brook Trout

This experiment was performed to ascertain whether

IPNV could be transmitted from IPNV-infected striped bass

to brook trout located downstream from the striped bass.

Brook trout were utilized in this study because they are

extremely susceptible to IPNV infection (Silim et al,

1982). Four-month-old striped bass were inoculated with

106 pfu of virus. At two months post inoculation, the

internal organs from three IPNV-infected striped bass, and

fecal samples from five fish were assayed for IPNV.










Fifteen IPNV-infected striped bass were placed in a tank.

Water (120C) from the tank containing striped bass flowed

into a tank that contained 20 seven-month-old brook trout.

Every two weeks, 3 4 trout were assayed for IPNV using

the plaque assay.


Humoral Response of Striped Bass to IPNV


Early IPNV Titers and Neutralizing Antibody

The tissue levels of IPNV and circulating virus-

neutralizing antibodies during the first ten days of IPNV

infection were monitored in four-month-old striped bass

fingerlings inoculated i.p. with 106 pfu of IPNV. For ten

days, 3 4 fish daily were exsanguinated from the severed

caudal peduncle and dissected. The kidneys, spleen,

intestines, feces, and buffy coat were assayed for IPNV.

Titers of virus-neutralizing antibody were measured in the

blood samples from individual or pools of two fish.


Exogenous Steroids and Levels of Neutralizing Antibody in
Striped Bass Challenged with IPNV

One investigation was conducted to determine the

effect of exogenous corticosteroids on the development of

viremia and virus-neutralizing antibodies in IPNV-

inoculated striped bass. Striped bass yearlings were

weighed, had a blood sample taken, and were divided into

four groups of six fish each. The treatment groups were

(1) sham control--fish were given two i.p. injections of

PBS 24 hours apart; (2) steroid control--fish were











injected i.p. with the corticosteroid triamcinolone

acetomide (100 mg/kg) followed 24 hours later with an i.p.

injection of PBS; (3) virus control--fish were given a

single i.p. injection with 107 pfu of IPNV; and (4) steroid

+ virus--fish were injected i.p. with steroid (100 mg/kg)

24 hours before i.p. inoculation with 107 pfu of IPNV.

Half of the fish in each group were bled at 3 days post

inoculation (dpi) and weekly thereafter for five weeks.

Fish in the other half of each group were bled at 7 dpi and

weekly thereafter, for five weeks. Blood plasma and

leukocytes were assayed for IPNV. Levels of circulating of

virus-neutralizing antibody were also measured.

Another study was conducted to determine if exogenous

steroids affected levels of virus-neutralizing antibodies

in IPNV-carrier striped bass. Striped bass fingerlings

were inoculated i.p. with 105 pfu of IPNV. Eleven months

later, the fish were weighed, bled, and injected i.p. with

triamcinolone acetomide (100 mg/kg). Striped bass were

bled twice weekly for three weeks. Titers of IPNV-

neutralizing antibody were determined.


Antibody Response of Striped Bass to Second IPNV Challenge

The purpose of this study was to investigate the

humoral response of IPNV-inoculated striped bass that were

given a second exposure to IPNV, either by injection or by

immersion challenge. For one part of this experiment,

yearling striped bass were given an i.p. inoculation











containing 107 pfu of IPNV. Blood samples were taken twice

weekly for five and one half weeks. The fish were allowed

to rest for five weeks prior to the second i.p. injection

with IPNV. Three months after the first virus injection,

the striped bass received an i.p. inoculation of 106 pfu of

IPNV. Blood samples were taken twice weekly for three

weeks, and periodically for nine additional weeks. Levels

of virus-neutralizing antibody were measured.

In a second part of the experiment, IPNV-inoculated

striped bass were given a second IPNV challenge by the

waterborne route. Five-month striped bass fingerlings

were given i.p. inoculation with 105 pfu of IPNV. Fourteen

months later, a blood sample was taken from these fish.

The fish were immersed for 5 minutes in water containing

105 pfu of IPNV per ml. Blood samples were taken twice

weekly for three weeks and assayed for levels of virus-

neutralizing antibody.


Survey of Chesapeake Bay Striped Bass
for IPNV and Virus-Neutralizing Antibody


Sampling Young-of-Year Striped Bass

Young-of-year striped bass were caught using a 100

foot, 50 mm mesh seine at sites in traditionally important

nursery areas in the Chesapeake Bay (MD). Striped bass

were either placed immediately on ice, or a 0.04 ml blood

sample was collected by venipuncture of the caudal vein.

Fish that were bled were returned to the water. Striped











bass tissues were assayed for IPNV. Plasma samples were

assayed for neutralizing activity against the striped bass

isolate of IPNV (IPNV-Sb) and the european IPNV isolate

(IPNV-Ab).


Sampling Yearling Striped Bass

Yearling striped bass from northern Chesapeake Bay

were caught by hook and line. A 0.04 ml blood sample was

obtained by venipuncture of the caudal vein, and the fish

were returned to the water. Plasma samples were tested for

virus neutralizing activity against both IPNV-Sb and IPNV-

Ab.


Sampling Adult Striped Bass

Adult striped bass were caught in gill nets located in

the Chesapeake Bay. Kidneys, spleen, gonads, and

intestines were excised, placed in sterile plastic bags,

and stored at 40C for 1 3 days prior to assay for IPNV.

Blood samples were collected from the caudal vein in

sterile glass tubes and the serum tested for virus-

neutralizing activity against both IPNV-Sb and IPNV-Ab.


Procedures that Affect IPNV Recovery


Tissue Site of IPNV in Striped Bass

When monitoring fish populations for IPNV, tissue

samples should be taken from which virus can be recovered

most frequently. An investigation was conducted to










ascertain which striped bass tissues harbor IPNV.

Individual organs, fat, feces, and blood, were removed from

IPNV-infected striped bass, and assayed for infectious

virus.


Storage Conditions of IPNV-infected Homogenates

The liability of the striped bass isolate of IPNV

(IPNV-Sb) in homogenates of IPNV-infected striped bass

was studied. Pools of internal organs from IPNV-inoculated

striped bass were homogenized and clarified as previously

described. Aliquots of the supernatant fluid were placed

in sterile glass vials and stored at 4, -20 or -700C.

An experiment was conducted to investigate whether the

type of container in which the homogenate of the internal

organs from IPNV-infected striped bass was stored altered

the amount of IPNV detected. The tissue homogenate from

individual IPNV-carrier striped bass was divided into 7

aliquots. One aliquot was assayed immediately for IPNV

using the plaque method. Three aliquots were stored in

plastic bags and three were stored in glass vials. Two

aliquots (one in glass vial, one in plastic) from each fish

homogenate were stored at each of three temperatures (4,

-20 and -700C) prior to virus assay.


Storage Temperature of Whole IPNV-Infected Striped Bass

Sampling fish for IPNV frequently involves taking

whole fish or tissue samples in the field and storing the

samples until they can be assayed for virus. An











investigation was conducted to determine the effect of

different storage temperatures on recovery of infective

IPNV from virus-infected striped bass. For this

experiment, IPNV-infected striped bass were placed in

individual plastic bags and stored at either 4, -20 or

-700C for two to fourteen days. After storage, frozen fish

were allowed to soften at 40C, and then the internal organs

from all fish were excised, and assayed for virus

infectivity.


Detection of IPNV-Carriers after Steroid Injection

A study was conducted to investigate whether

exogenous corticosteroids would enhance recovery of IPNV

from chronic IPNV-infected striped bass. Fifteen months

after IPNV-inoculation, three striped bass were placed in

each of five tanks. All fish were weighed and injected

i.p. with triamcinolone acetomide (10 mg/kg). One group

was immediately exsanguinated and assayed for IPNV. The

other groups were assayed for IPNV and virus-neutralizing

antibody at 3, 7, 14 and 21 days following steroid

injection.















CHAPTER THREE
RESULTS

Virus Infection Studies of Striped Bass

A series of IPNV challenge trials was conducted to

determine if IPNV induces increased mortality in virus-

exposed striped bass. When 1- to 20-day-old striped bass

from four different strains were immersed in IPNV the

resulting mortality was not significantly different from

that of the unchallenged controls (p < 0.01, analysis of

variance [ANOVA]). Even when five-day-old IPNV-exposed fry

were subjected to an abrupt drop in pH (0.8 units), no

statistical difference was observed in the mortality of

control and IPNV-challenged fry. Mortality in different

trials was unpredictable, but within a trial the pattern of

mortality of virus-challenged and control fish were not

significantly different (Figure 1). Virus was recovered

from virus-exposed fish that died but was never isolated

from control fish. When survivors were assayed for virus

three weeks post-challenge, IPNV was recovered only from

fry that had been challenged at one day post-hatch (data

not shown). Virus was not recovered any of the surviving

fish six months after waterborne challenge.

Twenty-six-day-old striped bass exposed to IPNV by

immersion and held at 12 or 220C exhibited no difference in

mortality compared to control groups (p < 0.01) (Figure 2).
























Figure 1: Percent daily mortality of striped bass fry
during the 21 days following exposure to 10 plaque
forming units of infectious pancreatic necrosis virus
(IPNV) per milliliter of water ( E-I ) or to
phosphate buffered saline ( +--- ). Sixty or 180
striped bass were exposed to virus in each trial. There
was no significant difference between the mortality in
IPNV-exposed and unchallenged striped bass (p < 0.01;
tested by analysis of variance). Results from
representative trials are presented.










70 -r .. .. -. .... .. ... .......-.


60 -j


50 -


40-


30 -


20 -


10-


0 4 8 12 16 20
DAYS POST CHALLENGE
Figure 1 A. Chesapeake and Delaware Canal (MD) striped
bass fry were exposed to IPNV at one day post-hatch.


0 4 8 12 16 20
DAYS POST CHALLENGE

Figure 1 B. Chesapeake and Delaware Canal striped bass
fry were exposed to IPNV at five days post-hatch.



































0 4 8 12 16
DAYS POST CHALLENGE

Figure 1 C. Florida striped bass fry were exposed to
IPNV at seven days post-hatch.

70


60


50-


40

0
2 30


20


10-


0-
0 4 8 12 16
DAYS POST CHALLENGE

Figure 1 D. Georgia striped bass fry were exposed to
IPNV at ten days post-hatch.
































10-


0


Figure
exposed


70


60


50


40

050
2 30
/-J





20


10


0


0 4 8 12 16 20
DAYS POST CHALLENGE
1 E. Nanticoke River (MD) striped bass fry were
to IPNV at 15 days post-hatch.


0 4 8 12 16 20
DAYS POST CHALLENGE
Figure 1 F. Nanticoke River striped bass fry were
exposed to IPNV at 20 days post-hatch.



















Figure 2: Percent daily mortality of striped baas
fingerlings exposed at 26 days post-hatch to 10 plaque
forming units of infectious pancreatic necrosis virus
per milliliter of water ( 0-0 ). Sham controls ( ---+ )
were exposed to virus-free phosphate buffered saline
(PBS). Treatment controls ( x--x ) were held for four
hours in a static, aerated bath without PBS or virus.
Each experimental group contained 100 striped bass.
There was no significant difference between mortality of
virus-exposed and nonexposed controls (p < 0.01; tested
by analysis of variance) at either temperature.





































0 2 4 6 8 10 12 14 16 1
DAYS POST EXPOSURE
Figure 2 A. Striped bass fingerlings were held at 120C.


0 2 4 6 8 10 12
DAYS POST CHALLENGE
Figure 2 B. Fingerlings were held at 220C.


14 16 18


i










In addition, no virus was recovered from any fish that

died. No deaths occurred in six-month-old striped bass

that were challenged with IPNV by immersion and no virus

was recovered from any of these fish.

Mortality did not increase in striped bass fingerlings

that were given IPNV by intraperitoneal (i.p.) inoculation

at either 60, 120, 150 and 180 days post-hatch (Table 1).

Even among IPNV-injected striped bass that underwent an

abrupt 100C change in water temperature, mortality was not

significantly different from that of controls. Virus was

recovered from IPNV-injected fish that died but was not

isolated from any controls (Table 2). At one month after

IPNV injection, virus titers of survivors were similar to

those of IPNV-inoculated striped bass that died during the

first month after injection (Table 3). Even levels of IPNV

in virus-inoculated striped bass subjected to changes in

water temperature were not significantly different

(p < 0.01, ANOVA) (Table 4). Virus was isolated from

surviving IPNV-inoculated striped bass for 14 months post-

inoculation (Table 3). Circulating IPNV-neutralizing

antibody was found in more than 75% of the IPNV-carrier

striped bass tested during the 14 months after IPNV

exposure. No IPNV-induced histological lesions were

observed in any sections examined from IPNV-exposed striped

bass, regardless of age or route of exposure.












Table 1: Percent cumulative mortality in striped bass
fingerlings following intraperitoneal injection of
infectious pancreatic necrosis virus (IPNV).



CONTROL VIRUS INOCULUM (pfu)

TREAT- SHAM
AGEa MENT 103 105 10



60 NDb 48c 38 40 ND


90 28 26 24 26 14


120 20 18 2 16 14


150 0 0 0 2 0


180 8 0 0 0 0




aAt the indicated days post-hatch, groups of 50 striped
bass were anesthetized and given intraperitoneal (i.p.)
injections containing the indicated plaque forming units
(pfu) of IPNV. Treatment controls were anesthetized
only. Sham controls were injected with phosphate
buffered saline (virus diluent). Fish were maintained
at 220C.

bNot done.

cPercentage of striped bass that died in the 28 days
following inoculation.











Table 2: Range of virus titers detected from striped
bass fingerlings that died following intraperitoneal
injection of infectious pancreatic necrosis virus.


CONTROL VIRUS INOCULUM (PFU)
TREAT- SHAM
AGEa MENT 103 105 106



60 NDb NVc 102 103-105 ND


90 NV NV NV NV 102-104


120 NV NV NV 103-106 105-107


150 _e 105


180 NV -




aAt the indicated days post-hatch, striped bass were
given intraperitoneal (i.p.) injections of the indicated
plaque forming units (pfu) of infectious pancreatic
necrosis virus (IPNV). Treatment controls were
anesthetized only. Sham controls were injected i.p.
with phosphate buffered saline (virus diluent). Fish
were maintained at 220C. Dead fish were assayed for
virus using the plaque assay me hod that detected titers
greater than or equal to 5 x 10 pfu/g.

bNot done.

CNo IPNV was recovered from striped bass that died
during the first 28 days following injection.

dMagnitude of IPNV titers (pfu/g of tissue) that were
recovered from striped bass that died during the first
28 days following injection with IPNV.


eNo fish died in this group.







48



Table 3: Range of virus titers in striped bass
fingerlings surviving intraperitoneal injection of
infectious pancreatic necrosis virus.


MONTHSb


VIRUS INOCULUM (PFU)a

103 105 106


NVc


102


103-104


103-106


103-106


103-104


104


101-102


NV-101


NDe


NV-102


NV-102


ND


NV-103


NV-101


NV-102


aStriped bass fingerlings received an intraperitoneal
inoculation with the indicated plaque forming units
(pfu) of infectious pancreatic necrosis virus (IPNV).
Fish were maintained at 220C.

bMonths following intraperitoneal injection that
surviving striped bass were assayed for IPNV using the
plaque method that detected titers equal to or greater
than 5 x 10 pfu/g.

CNo IPNV was recovered from surviving fingerlings.

dRange in IPNV titers (pfu/gram of tissue) in striped
bass fingerling survivors that were assayed for virus.


eNot done.











Table 4: Virus titers of individual striped bass that were
given an intraperitoneal injection of infectious pancreatic
necrosis and subjected to a change in water temperature.


TEMPERATUREa


22 --> 12


12 --> 22


2.7 X 104

2.9 X 104

3.9 x 104

3.0 x 104

NV

7.2 x 104

NV

1.2 x 105


4.0 x 104


9.7 X 104

2.9 X 104

4.5 x 104

1.4 x 104

3.2 x 104

2.0 x 106

5.0 x 106

6.4 x 105


9.8 x 105


1.7


2.3 X 105

4.9 x 103

1.1 x 106

2.2 x 105

1.9 x 104

1.9 x 104

2.8 x 104

4.8 x 103


2.0 x 105

3.6


1.2 X 105

2.6 x 104

3.3 x 104


NVb


8.2 x 103

9.2 x 104

8.0 x 103

2.1 x 104


3.9 x 104

4.4


aSix-month-old striped bass were given an intraperitoneal
injection of 10 plaque forming units (pfu) of
infectious pancreatic necrosis virus (IPNV). Fish were
held at either 120 or 220C. Two weeks after the IPNV
inoculation, some fish were transferred into water of a
higher or lower temperature. Two weeks after the
temperature change, fish were assayed for IPNV using the
plaque method that detected titers equal to or greater
than 5 x 10 pfu/g. Virus titers were expressed as pfu
per gram of tissue.

bNo virus was detected.

cMean IPNV titer for each group. Group means were not
significantly different (p < 0.01) as determined by
analysis of variance.

dStandard deviation of mean IPNV titer.


--










Transmission Studies of IPNV in Striped Bass

Oral Transmission of IPNV to Striped Bass

To demonstrate that IPNV can be transmitted to

striped bass by contaminated forage, six-month-old striped

bass were allowed to consume brook trout carrying between

102 104 pfu of IPNV. Virus was recovered from apparently

healthy striped bass eight months after exposure (Table 5).

Virus-neutralizing antibody was detected in all striped

bass that consumed IPNV-infected brook trout.


Vertical Transmission of IPNV in Striped Bass

To determine if striped bass survivors from natural

IPNV infection shed virus in their urine or milt, samples

were taken from the population of two-year-old striped bass

from which IPNV had originally been isolated (Schutz et

al., 1984). No IPNV was detected in any of the urine and

milt samples.

Experiments were conducted to investigate whether

IPNV-infected striped bass adults would transmit IPNV via

their sex products to their offspring. The eggs, milt,

fertilized eggs and offspring from striped bass adults that

had received i.p. inoculation with IPNV were tested for

virus. Virus (101-103 pfu/gram of tissue) was recovered

from the internal organs of the adults, but no IPNV was

detected in any other samples (Table 6). When IPNV was

added to eggs or milt, virus was not be recovered from the

resultant offspring. Virus was only recovered from











Table 5: Range in virus titer in striped bass following
ingestion of brook trout that contained infectious
pancreatic necrosis virus.


WEEKS POSTa VIRUS TITERb NUMBER
EXPOSURE TESTED



1 101 1


2 101-103 3


3 101-103 2


4 101-103 2


12 101-102 2


33 101 1




aSix-month-old stripeI bass were fed brook trout, each
of which contained 10 104 plaque forming units (pfu)
of infectious pancreatic necrosis virus (IPNV). At the
indicated weeks after virus ingestion, striped bass were
assayed for IPNV by the plaque method.

bMagnitude of titer expressed as pfu of IPNV per gram of
striped bass tissue.










Table 6: Recovery of infectious pancreatic necrosis
virus (IPNV) from samples taken during investigations of
vertical transmission of IPNV in striped bass.


SAMPLES VIRUS RECOVERED


Adults inoculated with IPNVa

Internal Organs Yes

Sex Products (Eggs and Milt) No

Fertilized Eggs No

Fry No

Noninoculated Adultsb

Sex Products No

IPNV added to Eggsc

Fertilized Eggs Yes

Fry No

IPNV added to Miltd

Fertilized Eggs No

Fry No


aFive-year-old, hatchery-reared striped bass were given
an intraperitoneal injection with 10 plaque forming
units (pfu) of IPNV. Fish were spawned six months
later. Samples were assayed for virus using the plaque
method.

bSex products (eggs and milt) were obtained from
spawning striped bass caught in the Chesapeake Bay (MD).
Homogenates of eggs, milt, fertilized eggs, and larvae
were assayed for IPNV using the simultaneous seeding
method. Samples were considered positive for IPNV if
cytopathic effects (CPE) was observed during two blind
passages. If no CPE developed, the sample was recorded
to be negative.

cEggs were exposed to 105 pfu of IPNV/ml immediately
prior to mixing with virus-free milt.

dMilt was exposed to 105 pfu of IPNV/ml immediately
prior to mixing with virus-free eggs.















fertilized eggs when virus-exposed eggs were fertilized

with virus-free milt. None of the offspring started to

feed and all died within three weeks.


Transmission of IPNV from Striped Bass to Brook Trout


To determine whether IPNV-infected striped bass shed

sufficient virus to infect susceptible fish located

downstream, brook trout were placed below IPNV-infected

striped bass whose feces contained 104- 105 pfu/g. One of

four trout tested after six weeks had 102 pfu of IPNV/g of

pooled internal organs. Virus was not recovered from trout

tested at two, four and eight weeks of IPNV-exposure.


Humoral Response of Striped Bass to IPNV


Early Humoral Response to IPNV Challenge

To monitor early levels of IPNV and circulating virus-

neutralizing antibodies in striped bass, four-month-old

striped bass were inoculated i.p. with 106 pfu of IPNV and

3 4 fish were assayed each day for 10 days. Titers of

virus remained relatively constant during the first ten

days (Table 7) and were of the same magnitude as IPNV

titers in IPNV-inoculated striped bass tested two months

after injection (Table 3). Virus-neutralizing antibody was

first detected seven days post inoculation (dpi) (Table 7).










Table 7: Detection of infectious pancreatic necrosis
virus (IPNV) and virus-neutralizing antibody in IPNV-
injected striped bass fingerlings.


DPIa IPNVb TITERc ANTIBODYd TITERe


1 3/3


2 3/3


3 4/4


4 4/4


5 4/4


6 3/3


7 4/4


8 4/4


9 4/4


10 4/4


105-106


103-106


105-106


105-106


NTf


0/3


0/2


0/3


0/2


0/3


1/3


2/3


2/3


2/4


500


200-700


500


750-1000


aDays post injection (intraperitoneal)
forming units (pfu) of IPNV.


of 106 plaque


bNumber of four-month-old striped bass that had IPNV
in their tissues per number of fish assayed for virus
virus using the plaque method.

CRange in IPNV titer (pfu per gram of tissue).

dNumber of blood samples (diluted 1:100) that
neutralized more than 50% of total IPNV plaques per
number of blood samples tested.

eRange in titer of IPNV-neutralizing antibody.

Not tested.


104-105










Effect of Steroids on Titers of Circulating Virus and
IPNV-Neutralizing Antibody

The effect of exogenous steroid on viremia and on the

development of virus-neutralizing antibody was investigated

using yearling striped bass that received an i.p. injection

of steroid 24 hours prior to receiving an i.p. injection

with IPNV. Blood samples were taken weekly from individual

fish. Viremia was detected for two weeks in IPNV-

inoculated striped bass that had received steroid (Table

8), but for only one week in IPNV-inoculated striped bass

that did not receive steroid. Virus was recovered more

frequently from the buffy coat leukocytess) than from the

plasma (Table 8). Circulating IPNV-neutralizing antibody

was first detected at 10 dpi in IPNV-inoculated fish that

received steroid (Figure 3) compared to 7 dpi in IPNV-

inoculated fish not treated with steroid (Figure 3).

Levels of virus-neutralizing antibody in the IPNV-injected

striped bass treated with steroid were generally lower than

those in virus-injected fish that did not receive steroid.

Also, antibody titers peaked later (about 4 weeks post

inoculation) in steroid treated fish compared to a peak at

about 3 weeks in virus-injected striped bass that did not

receive steroids. A noticeable, but not statistically

significant, difference in antibody titers was observed

between the two groups IPNV-injected striped bass that did

not receive steroid. Striped bass that were bled at three

dpi and weekly thereafter (Figure 3a) had somewhat higher











Table 8: Recovery of infectious pancreatic necrosis virus
(IPNV) from the plasma and buffy coat of virus inoculated
striped bass fingerlings.


TREAT- DAYS POST INJECTIONa

MENT SAMPLE 3 7 10 14 17 21



Steroid + buffy coat 3/3c 3/3 0/3 1/3 0/3 0/3

IPNVb plasma 1/3 0/3 0/3 0/3 0/3 0/3



IPNVd buffy coat 1/3 3/3 0/3 0/3 0/3 0/3

plasma 0/3 0/3 0/3 0/3 0/3 0/3



Controls buffy coat 0/3 0/3 0/3 0/3 0/3 0/3

plasma 0/3 0/3 0/3 0/3 0/3 0/3


aStriped bass fingerlings received 107 plaque forming
units (pfu) of IPNV or phosphate buffered saline by
intraperitoneal (i.p.) inoculation. Blood samples,
taken at the indicated days after IPNV injection, were
assayed for IPNV using the plaque assay.
b Striped bass fingerlings that received an i.p.
injection with triamcinolone acetomide (100 mg/kg) 24
hours before i.p. inoculation with IPNV.

cNumber of fish that were positive for IPNV per
number of fish tested using the plaque assay.
dStriped bass fingerlings that received only IPNV
by i.p. injection.
eStriped bass fingerlings that did not receive an
injection of IPNV, but were injected i.p. with either
PBS or steroid and PBS.




















Figure 3: Titers of virus-neutralizing antibody in
striped bass fingerlings that received an intra-
peritoneal inoculation with 10 plaque forming units
(pfu) of infectious pancreatic necrosis virus (IPNV).
Fingerlings were injected with phosphate buffered saline
(PBS) ( O ) or with triamcinolone acetomide
(100 mg/kg) ( 0 ) 24 hours prior tp viral inoculation.
Antibody titers are expressed as 10' the serum dilution
that neutralized 50% of total IPNV (about 80 plaques per
well). The mean antibody titer for striped bass that
received PBS is indicated by + and by for fish that
received steroid.




























0 2 4 6
WEEKS POST CHALLENGE


Figure 3 A.
beginning

S0


40-



30-



20 -



10-


Fish that were sampled at weekly intervals
three days after IPNV inoculation.


WEEKS POST CHALLENGE


Figure 3 B. Striped bass that were sampled
intervals after IPNV injection.


at weekly


0




O O
0o 0
O +
0 0


0
00
oo


0o
S "a


-r *










antibody titers than fish sampled at seven dpi and then

weekly (Figure 3b).

Steroid treatment of chronic IPNV-carrier striped bass

did not cause any change in levels of IPNV-neutralizing

antibodies in these fish. Antibody titers remained between

100 and 500.

Administration of 100 mg/kg of triamcinolone

acetomide resulted in a 96% loss among all steroid-injected

striped bass over a three month period. The spleen and

anterior kidneys of these fish were extremely hypocellular.


Antibody Response of Striped Bass Following a Second
IPNV Challenge

To determine the humoral response of striped bass to a

second IPNV exposure, two sets of experiments were

performed. In one, IPNV-injected striped bass were given a

second viral challenge by immersion. In the other, IPNV-

injected striped bass received a second i.p. injection of

IPNV. When IPNV-inoculated striped bass were given a

waterborne IPNV challenge, antibody levels (100 800)

remained unchanged after the second viral exposure.

In contrast, in IPNV-carrier fish that received a

second injection with IPNV, antibody titers increased

(Figure 4). Antibody levels in striped bass began to rise

at seven dpi after the second IPNV inoculation and were

considerably higher than levels detected after the first

IPNV injection. After the second injection, antibody

titers rose and fell twice over a nine week period.









400 -


350-







?- 200 -
0

| 150-

100

50 -



1 2 3 4 5 6 11 12 13 14 15 16 19 20 21 23
WEEKS


Figure 4: Virus-neutralizing antibody titers in striped
bass fingerlings injected with infectious pa ncreatic
necrosis virus (IPNV). Striped bass received 10 plaque
forming units (pfu) of IPNV by intraperitoneal (i.p.)
inoculation on day 0 ( 3 ). Control fish received an
i.p. injection with phosphate buffered saline ( S3 ) on
day 0. A second IPNV challenge ( J, ) was given at 11
weeks after the first injection. Control striped bass
were injected i.p. with IPNV at week 11. Virus-
neutralizing antibody titers are expressed as 10-2 the
dilution of fish serum that neutralized 50% of the total
viral plaques (about 80 plaques per well). Each bar
represents the mean (n = 1 to 6) virus-neutralizing
antibody titer of fish.










Survey of Chesapeake Bay Striped Bass

To determine whether wild striped bass have been

exposed to IPNV, Chesapeake Bay (MD) striped bass of

various ages were sampled. The tissues from some were

assayed for virus. Blood samples were assayed for the

presence of antibodies that would neutralize the striped

bass isolate of IPNV (IPNV-Sb) but not the European Ab

serotype. Virus was not recovered from any wild striped

bass tested (Table 9). Specific IPNV-Sb neutralizing

antibody was detected in 1- to 3-year-old striped bass

caught during the winter of 1984 and in one young-of-year

striped bass caught in 1985 (Table 10).


Procedures that Affect IPNV Recovery from Striped Bass

Tissue Site of IPNV in Striped Bass

Tissues from IPNV-infected striped bass were assayed

individually to determine those from which IPNV could be

recovered most frequently. Virus was recovered from the

anterior kidneys of all striped bass that were positive for

IPNV but was never isolated from brain (Table 11). Virus

was also recovered from other tissues but none with the

consistency found for the anterior kidney (Table 11).

Tissue virus titers ranged in magnitude from undetectable

(< 5 x 101 pfu/g) to 106 pfu/g.


Virus Recovery from Steroid Injected Chronic Carriers

This study was conducted to determine if exogenous

corticosteroids would increase the percentage of virus











Table 9: Attempts to isolate infectious pancreatic
necrosis virus (IPNV) from striped bass caught in the
Chesapeake Bay (MD).


SURVEY
DATE LOCATION YEAR-CLASS VIRUS/SAMPLESa



Aug. 1984 Upper Bay 1984 0 / 39


Sept. 1984 Upper Bay 1984 0 / 66


Dec. 1984 Choptank River 1982 83 0 / 15


Feb. 1985 Upper Bay 1982 83 0 / 30



aNumber of striped bass positive for IPNV per number of
individual fish tested. Individual whole fish, or
samples of kidney, spleen and feces were assayed for
IPNV by the plaque method.











Table 10: Detection of neutralizing antibody specific
for the striped bass isolate of infectious pancreatic
necrosis virus (IPNV-Sb) in Chesapeake Bay striped bass.


SURVEY DATE LOCATION YEAR-CLASS SAMPLESa



Dec. 1984 Choptank River 1982 83 9 / 49 (18%)


Feb. 1985 Upper Bay 1982 83 6 / 94 ( 6%)


July 1985 Upper Bay 1985 0 / 5


Aug. 1985 Choptank River 1985 1 / 45 ( 2%)


Aug. 1985 Upper Bay 1985 0 / 3


Sept. 1985 Upper Bay 1985 0 / 6


Sept. 1985 Upper Bay 1984 0 / 20



aNumber of blood samples positive for IPNV-Sb
neutralizing antibody per number of samples tested.
Serum samples, diluted 1:100, were tested by the plaque
method for neutralizing activity against IPNV-Sb and
against the European isolate (IPNV-Ab). Samples were
considered to be positive for specific IPNV-Sb
neutralizing antibody if they neutralized more than
50% of IPNV-Sb, but did not neutralize IPNV-Ab. Total
virus contained about 80 plaques per well.









Table 11: Striped bass tissues from which
infectious pancreatic necrosis virus was isolated.

TISSUEa # POSITIVE/ # TESTEDb

Anterior kidney 29 / 29 (100%)

Spleen 20 / 25 ( 80%)

Blood 4 / 8 ( 50%)

Fat 2 / 4 ( 50%)

Liver 9 / 20 (45%)

Intestine 2 / 9 ( 22%)

Posterior kidney 4 / 20 ( 20%)

Heart 2 / 11 ( 8%)

Brain 0 / 13 ( 0%)

aTissues from infectious pancreatic necrosis virus
(IPNV) infected striped bass were assayed
individually for virus by the plaque method.
bNumber of tissues positive for IPNV per number
assayed for IPNV.










isolation from striped bass that had been injected with

IPNV 15 months earlier. Injection with triamcinolone

acetomide (10 mg/kg) into these striped bass increased the

percentage of virus-positive fish detected over time (Table

12); the peak occurred two weeks after steroid

administration.


Virus Recovery from Stored IPNV-carrier Tissue Homogenates

Aliquots of tissue homogenates from individual IPNV-

infected striped bass were stored under different condi-

tions to determine if the stability of IPNV infectivity was

affected. Virus infectivity was reduced in homogenates

stored at 40C (Table 13) but IPNV infectivity was not

significantly different (p < 0.01, ANOVA) in homogenate

samples stored at either -200 or -700C. The type of con-

tainer (glass vial or plastic bag) did not result in a

significant difference in virus infectivity (p < 0.01,

ANOVA) (Table 14), although virus titers from homogenates

stored at 40C were significantly different (p < 0.01,

ANOVA) from titers from homogenates stored at -20 or -700C.


Recovery of IPNV from Stored Whole Striped Bass

In an additional study designed to determine if storage

conditions affect the recovery of infectious IPNV from

virus-infected striped bass, whole fish were stored for 2

to 14 days at 4, -20, and -700C. Table 15 shows the virus

titers of IPNV-infected striped bass fingerlings determined

after storage. Virus titers were best maintained in IPNV-












Table 12: Recovery of infectious pancreatic necrosis
virus (IPNV) from chronic virus-carrier striped bass
following injection with steroid.


WEEKSa # POSITIVE/# TESTEDb TITERc



0 0/4 Vd


0.5 0 / 3 NV


1 1 /4 102


2 3 / 4 102- 103


3 1 /4 102


aNumber of weeks following intraperitoneal (i.p.)
injection of triamcinolone acetomide (10 mg/kg) into
striped bass that been injected i.p. with IPNV 14 15
months previously.
bNumber of striped bass from which IPNV was recovered
per number of striped bass assayed for IPNV by the
plaque method.

CRange in magnitude of virus titers expressed as
plaque forming units of IPNV per gram of tissue
(anterior kidney).
dNo virus detected.











Table 13: Titers of virus of infectious pancreatic
necrosis virus-infected striped bass tissue homogenates
that were stored at different temperatures.


STORAGEa FISH NUMBER
1 2 3 4 5


O days

5 x 102 5 x 103 3 x 104 2 x 104 1 x 104

2 days

40C NVc 1 x 103 6 x 103 4 x 103 4 x 103

-200C NV 7 x 103 3 x 104 5 x 104 7 x 104

-700C NV 8 x 103 5 x 104 5 x 104 3 x 104

2 weeks

40C NV NV 2 x 104 NV 1 x 104

-200C 1 x 102 5 x 103 6 x 104 6 x 104 3 x 104

-700C NV NV 5 x 104 2 x 104 1x 103



aLength of time and temperature at which aliquots of
tissue homogenates from striped bass infected with
infectious pancreatic necrosis virus (IPNV) were stored
prior to assay for virus by the plaque method.

bplaque forming units of IPNV per gram of tissue (pooled
internal organs) recovered from homogenates.


cNo virus was recovered.






68



Table 14: Recovery of infectious pancreatic necrosis
virus (IPNV) from striped bass tissue homogenates stored
for 48 hours in different types of containers.

a
40C -200C -700C


Vb pc V P V P


Sample

1 NVd


2 8 x 101


3 5 x 101


4 3 x 102


e
3 x 101


1 x 102


ND


4 x 102


5 NV


6 8 x 101


7 3 x 101


3 x 102


4 x 10 2


2 x 102


6 x 102 1 x 103 4 x 102


3 x 103 3 x 103


3 x 102


1 x 102


5 x 101


2 x 102


2 x 103


4 x 102


2 x 103 2 x 103 1 x 103


2 x 103


1 x 103


9 x 102


4 x 102


6 x 102


2 x103


2 x 102


2 x 103


1 x 103


aTemperature at which aliquots of homogenates of pooled
internal organs from striped bass were stored.for 48
hours prior to being assayed for virus by the plaque
method.

bAliquots of striped bass tissue homogenates were stored
in sterile glass vials.

CAliquots of tissue homogenates were stored in sterile
plastic bags.

dNo IPNV was detected.

plaque forming units of IPNV per ml of homogenate.

Not done.












Table 15: Recovery of infectious pancreatic necrosis
virus (IPNV) from IPNV-infected striped bass stored
whole.


LENGTH OF STORAGE
STORAGE
TEMP(oC)a 0 DAYS 2 DAYS 14 DAYS


9 / 10b


(102-105)c


4 10 / 11 3 / 3


(102-104) (103-104)


-20 4 / 13 1 / 3


(102-103) (103)


-70 0 / 17 0 / 3

(NVd) (NV)


aTemperature at which IPNV-infected striped bass
fingerlings were stored intact in plastic bags.
bNumber of fish that were positive for IPNV per
number of fish that were assayed for virus by the
plaque method.

cRange of IPNV titer expressed as plaque forming
units per gram of pooled internal organs.
dNo IPNV was recovered.










carrier striped bass that were stored intact in the

refrigerator at 40C. All virus infectivity was lost in

IPNV-carrier striped bass that were stored at -700C (Table

15). The loss was evident after only 48 hours of storage.

In fish that were stored at -200C, loss of infectivity was

intermediate between that observed at 40C and -700C.


Comparison of IPNV Isolates

Protein Electrophoretic Patterns

Three IPNV isolates, one from striped bass (IPNV-Sb),

one from Atlantic menhaden (IPNV-M), and the North American

reference salmonid isolate (VR-299), were purified over

discontinuous CsCl gradients. Viral proteins were analysed

using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Similar protein profiles were demonstrated (Figure 5). The

relative mobility (Df) of each viral polypeptide and

molecular weight standard was determined by dividing the

actual distance traveled by each protein band by the

distance moved by the dye front. A standard curve was

developed by plotting the Df of each molecular weight

standard against the logarithml0 of its molecular weight.

The molecular weight of each viral protein in the

polyacrylamide gel was determined from the standard curve.

For IPNV-Sb and IPNV-M, there were polypeptide bands

corresponding to molecular weights of 95, 53, 51, 31, and

29 K. For VR-299, there were proteins with molecular

weights corresponding to 95, 53, 51, and 29 K.











M S V


97
9 7 ..
















24




20




Figure 5: Electrophoretic profile of polypeptides from
three isolates of infectious pancreatic necrosis virus
(IPNV) fractionated on a discontinuous 10%
polyacrylamide gel. The three IPNV isolates are: striped
bass (S), menhaden (M), and the North American VR-299
(V). The left lane contains the following molecular
weight markers: phosphorylase b (97,400), bovine albumin
(66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate
dehydrogenase (36,000), carbonic anhydrase (29,000),
trypsinogen (24,000), and trypsin inhibitor (20,10g).
Numbers on the gel indicate molecular weights x 10
The lowest band represents the dye front.










Neutralization Kinetics

Neutralization kinetics reveals the pattern and rate

at which virus becomes neutralized in the presence of

excess antibody. Each of three IPNV isolates (IPNV-Sb,

IPNV-M, and VR-299) was reacted with homologous and

heterologous antibody, and the residual infectivity at

several time points was measured. The neutralization

kinetic curves for the three IPNV isolates were similar for

homologous and heterologous antibody reactions (Figure 6).

The rate of neutralization (K) was calculated using the

formula K = D/t 2.3 log Vo / Vt, where D = reciprocal of

the dilution of antibody, t = 0.25 minutes, Vo = total

virus, and Vt = number of virus plaques at 0.25 minutes

(Macdonald & Gower, 1981). The calculated neutralization

rates (K) were of the same magnitude for most combinations

of IPNV isolates and antibodies (Table 16). The only

exception was the increased rate detected for the reaction

of VR-299 with its homologous antibody.










100

90

80

C 70

> 60

O 50-

z
i 40-

a 30

20-

10-
10-
0-- i I I
0 2 4
TIME (minutes)



Figure 6 A. The IPNV isolates were tested with antibody
against the striped bass IPNV isolate.





Figure 6: Comparison of the neutralization kinetics of
three isolates of infectious pancreatic necrosis virus
(IPNV); striped bass (0), menhaden ( + ) and the North
American isolate VR-299 ( A ). Equal volumes of
diluted antibody and virus were mixed, incubated at 40C,
and sampled at the indicated times. Residual
infectivity at each time point was determined by the
plaque method, and expressed as the percentage of total
number of IPNV plaques.










100

90

80-

n 70-

60-

D 50-





0,-
5U 40-

3 30-

20-

10-


0 2 4
TIME (minutes)
Figure 6 B. The IPNV isolates were tested with antibody
against the North American isolate VR-299.


0 2
TIME (minutes)
Figure 6 C. The IPNV isolates were tested with antibody
against the menhaden IPNV isolate.










Table 16: Neutralization rates for three infectious
pancreatic necrosis virus (IPNV) isolates reacted with
homologous and heterologous antisera.


Virusa
ANTISERUM

IPNV-Mb IPNV-Sbc VR-299d


I e
IPNV-M 3 x 105 7 x 10 5 x 105


IPNV-Sb 9 x 105 4 x 105 4 x 105


VR-299 9 x 105 6 x 105 24 x 105



aEach IPNV isolate was reacted individually with rabbit
antiserum against each of the isolates. Total virus and
residual infectivity at 0.25 minutes were determined by
plaque assay.

bThe menhaden isolate of IPNV.

CThe striped bass isolate of IPNV.

dThe standard North American isolate of IPNV.

e The rate of neutralization was assumed to be linear
for the first 0.25 minutes of the reaction between
antibody and virus. The neutralization rate (K) was
calculated for each trial using the formula
K = D/t x 2.3 x log Vo/ Vt, where D = dilution of the
antiserum, t = 0.25 minutes, V = total number of viral
plaques, and Vt = number of viral plaques at 0.25
minutes.















CHAPTER FOUR
DISCUSSION


Infectious pancreatic necrosis virus was isolated from

moribund striped bass fry in a hatchery on the Chesapeake

Bay (MD) (Schutz et al., 1984). Efforts to rear striped

bass in hatcheries have increased recently (Schutz et al.,

1984), partly because numbers of striped bass in the

Chesapeake Bay have been declining (Goodyear et al., 1985).

The reasons for the observed decline are not known. It is

known, however, that IPNV virus causes significant losses

in salmonids raised in hatcheries (Wolf et al., 1960) and

is pathogenic for Atlantic menhaden in Chesapeake Bay

(Stephens et al., 1980). The current study was initiated

to investigate what effects IPNV infection has on striped

bass, how IPNV can be transmitted, and whether the IPNV

recovered from striped bass is related to the IPNV isolate

from menhaden.

In IPNV infection trials using 1- to 20-day striped

bass, mortalities in different strains of striped bass

challenged with water borne IPNV were not higher than in

controls. Efforts were made to duplicate the conditions

that existed when IPNV was originally isolated from striped

bass (Schutz et al., 1984). However, none of the clinical

signs or histopathological lesions described by Schutz et










al. (1984) were observed in the experimental striped bass.

The etiology of the mortalities and histological

abnormalities described by Schutz et al. (1984) is not

known.

Peaks of mortality in IPNV-challenged and control fish

coincided within trials but were not predictable between

trials. The reasons for the deaths are not known.

Possibly contaminants (e.g. bacteria, ammonia) introduced

with the brine shrimp nauplii fed to the fish may have

accounted for the mortality pattern.

It is clear, however, that immersion exposure to virus

did not cause predictable mortality in striped bass, even

in IPNV-challenged fish subjected to an abrupt pH change.

In contrast, young brook trout showed increased mortality

after immersion challenge with the striped bass isolate of

IPNV (P. E. McAllister, National Fish Health Research

Laboratory, Kearneysville, WV; unpublished data). The

reasons for the difference in IPNV pathogenicity in fishes

are not known.

Striped bass demonstrated age-related differences in

susceptibility to IPNV infection after waterborne

challenge. Three weeks after immersion IPNV challenge,

only striped bass that had been exposed at one day post-

hatch, contained virus. No virus was recovered from

striped bass that were exposed at 26 days or older to water

borne IPNV. Explanations for these findings probably

involve the nature of the integument in very young fish,











and the speed with which effective defense mechanisms

develop in these fish. The external integument of newly

hatched fry performs exchange functions that are later

performed by the gills and other organ systems (Johansen,

1982; Roberts et al., 1973). Possibly the immature

integument might provide a site to which exogenous virus

can attach, enter and multiply--a site that later becomes

inaccessible to virus. In addition to physical changes in

the integument, fish may quickly develop other nonspecific

defense mechanisms such as inteferon and cellular defense

systems, that may protect fish from waterborne

microorganisms (de Kinkelin & Dorson, 1973; Manning et al.,

1982; Tatner & Manning, 1985). A specific humoral response

probably is not a major factor in protecting very young fry

(Manning et al., 1982; Manning & Mughal, 1985). None of

the experimental striped bass immersed in IPNV developed

virus-neutralizing antibodies.

In contrast to the lack of infectivity of IPNV in all

but the youngest striped bass exposed to waterborne IPNV,

experimental inoculation of IPNV into striped bass resulted

in asymptomatic carriers that contained infectious virus

for longer than one year. No overt signs of disease, such

as "spinning" or increased mortality, were seen in virus

infected striped bass, even in IPNV-injected striped bass

that were subjected to environmental stress. Similarly, no

histopathology was detected in experimental IPNV-infected










striped bass. Atlantic salmon, Salmo salar, develop

subclinical IPNV infections like striped bass; however,

unlike striped bass, Atlantic salmon do develop

degenerative pancreatic lesions (Swanson & Gillespie,

1979). The significance of the IPNV-induced histological

lesions is not known.

Striped bass did become infected with IPNV after

ingesting IPNV-carrier brook trout. Like brook trout,

menhaden are susceptible to IPNV-induced disease (Stephens

et al., 1980). The virus can be isolated from menhaden

during their annual spring epizootic in the Chesapeake Bay

(Stephens et al., 1980). Possibly striped bass may be

exposed to IPNV by consuming IPNV-infected menhaden. The

source of IPNV infection of menhaden in Chesapeake Bay has

not been reported, but brook trout, as well as other fish,

probably can become infected with IPNV via the sex products

(Wolf & al., 1963; Bullock et al., 1976; Seeley et al.,

1977; Dorson & Torchy, 1985).

Experimental transmission studies did not demonstrate

the spread of IPNV from striped bass sex products to

offspring. Virus was not recovered from the milt or urine

of survivors in the population of striped bass from which

the original IPNV isolate was obtained. In addition, IPNV

was not isolated from the offspring from experimentally

infected striped bass adults or from offspring of IPNV-

exposed sex products. The virus was isolated from striped

bass sperm and larvae collected from the Chesapeake Bay in











1984, but not in 1985 or 1986 (F. M. Hetrick, University of

Maryland, unpublished data). The significance of these

findings is unclear.

Although no virus was recovered from any striped bass

organs sampled from Chesapeake Bay, virus-neutralizing

antibody was found in the older fish caught in the winter

of 1984 and young-of-the-year fish sampled in the summer of

1985. Possibly the older fish were exposed to the virus

during the spring IPNV-epizootics in menhaden. Results

from neutralization kinetics and SDS-PAGE of viral proteins

demonstrate the close relationship between IPNV isolated

from striped bass (IPNV-Sb) and from menhaden (IPNV-M).

Neutralization kinetics are sensitive tests for

comparing antigenic relatedness between viruses (Ashe &

Scherp, 1963) and have been used to categorize IPNV-

isolates into distinct serotypes (Macdonald & Gower,

1981). In the current study, the neutralization curves

and the rates of neutralization of the striped bass

isolate of IPNV (IPNV-Sb) were virtually identical to

those of the menhaden isolate (IPNV-M). Use of the same

analytical technique revealed that both isolates are

closely related to the standard North American isolate

(VR-299). The neutralization rate of the reaction of

the VR-299 isolate with its homologous antibody was one

magnitude higher than rates determined for the other

neutralization reactions. This probably indicates that










the antiserum either recognized or was more avidly bound

by some antigenic determinant on VR-299 that was not

present on the other two isolates tested. However, in

all other neutralization reactions, VR-299 patterns were

like those for IPNV-Sb and IPNV-M, indicating a close

relationship between the three isolates.

In addition, polyacrylamide gel electrophoresis of

the three IPNV isolates demonstrated similar viral

polypeptide bands. The calculated molecular weight of

proteins of IPNB-Sb and IPNV-M were 95000, 53000, 51000,

31000 and 29000. All but one (51000) protein band were

demonstrated for the North American isolate (VR-299).

The molecular weights of the viral proteins of IPNV-M

and VR-299 have been reported to be 86000, 56000, 30000,

and 27000 (Stephens, 1981; Stephens & Hetrick, 1983).

The actual molecular weights attributed to the viral

polypeptides has varied, even within the same laboratory

(Dobos, 1977; Dobos & Rowe, 1977; Dobos et al., 1977).

The variation probably is related to differences in the

experimental protocols used (Dobos & Rowe, 1977). For

purposes of the present study, the important finding is

the demonstrated similarity between the menhaden and the

striped bass isolates of IPNV.

As previously discussed, striped bass did not become

infected with IPNV after waterborne challenge, but became

inapparent IPNV-carriers after inoculation or ingestion of

IPNV. Virus-infected striped bass did shed sufficient IPNV











to infect brook trout that were located in tanks downstream

from the striped bass. Previous reports have implicated

IPNV-infected trout as the source of IPNV infection of

nonsalmonids, such as Catostomus commersoni (Sonstegard et

al., 1972). The present results demonstrate that IPNV can

be transmitted from a nonsalmonid species to trout.

The spread of IPNV from healthy appearing (both

grossly and histologically) striped bass to a susceptible

fish species has practical implications. If IPNV-carrier

striped bass are transported to areas that were previously

IPNV-free, the striped bass pose a potential threat to fish

species in the watershed. The virus is relatively stable

in the environment, remaining infective for months in

aqueous environments (Tu et al., 1975; Toranzo & Hetrick,

1982). Therefore, testing a population of striped bass for

IPNV prior to introduction into IPNV-free areas would seem

advisable. A series of experiments were performed to

determine what samples should be taken and how the samples

should be handled to improve the recovery of IPNV from

virus-infected striped bass.

Virus was reisolated from IPNV-infected striped bass

most often from anterior kidney and from the spleen, but

never from the brain. A similar pattern of IPNV recovery

is found in trout (Wolf & Quimby, 1969; Yamamoto, 1974).

These results differ from those of Dorson (1982) who










contended that the brain of trout IPNV-carriers may be the

only tissue containing IPNV.

When IPNV was detected in blood samples from striped

bass, the virus was found associated with the leucocytes.

Only occasionally was IPNV recovered from plasma. Swanson

and Gillespie (1982) reported similar findings from IPNV-

infected brook and rainbow trout. Swanson and Gillespie

(1982) separated the blood cells over a Ficoll gradient

prior to virus assay. The current study developed a simple

separation procedure consisting of removal of the buffy

coat from centrifuged hematocrit tubes. This procedure

permitted virus assay of smaller blood volumes than have

been reported previously (Swanson & Gillespie, 1982; Yu et

al., 1982). To detect levels of virus lower than those

recovered in this study white blood cells can be cocultured

with virus-susceptible cells (Yu et al, 1982). Another

sensitive assay permits recovery of IPNV from the

supernatant fluid from mitogen stimulated lymphocytes from

IPNV-infected Atlantic salmon (Knott and Munro, 1986).

Whatever procedure is utilized to isolate IPNV from blood

and blood components, the samples should be assayed as

quickly as possible (Swanson & Gillespie, 1982).

Previous investigations on the effects of storage

of samples on IPNV recovery have used virus-containing

culture fluids, or tissue homogenates to which IPNV was

added (Malsberger and Cerini, 1963: Wolf, 1964; Wolf et

al., 1969; McMichael et al., 1975). Use of liquid samples












permits obtaining initial levels of infective virus in the

samples. Data from stored IPNV-infected striped bass

tissue homogenates were similar to those reported by the

other authors. An increased loss of virus titer was

observed in homogenate samples stored at 40C, compared to

values obtained from samples stored at -200 or -700C.

Therefore, for best virus recovery, striped bass tissue

homogenates should be stored frozen (-20 or -700C).

In contrast, data from trials storing whole IPNV-

infected striped bass fingerlings gave different results.

All viral infectivity was lost in fish samples stored at

-700 but was retained at 40C. Retention of virus

infectivity was intermediate in samples stored at -200C.

The retention of viral infectivity in whole striped bass

stored at 40C and loss of infectivity in fish stored at

-700C was somewhat unexpected. The disadvantage of using

whole fish is that an initial virus titer can not be

obtained. However, because there was a high ( > 90%)

incidence of IPNV-carriers in the experimental striped bass

used in this study, the lack of virus infectivity in fish

stored at -700C must be a result of events associated with

storage at the lower temperatures. Perhaps the different

rates at which freezing and thawing occurs in whole fish

compared to aqueous solutions may affect viral infectivity.

The observed differences of IPNV stability in stored

whole fish and in stored homogenates were not due to a










difference in storage containers. When IPNV-infected

tissue homogenates were stored in plastic bags similar to

those in which whole fish were stored, homogenate samples

again lost infective virus at 40C, but IPNV remained

infective when stored frozen (-20 and -700C). The reasons

for the variation in IPNV infectivity from intact and

homogenized fish tissues are not known. However,

demonstration of the variation emphasizes the importance of

experiments that attempt to replicate field conditions.

Previous investigations that utilized IPNV-infected

cell cultures or tissue homogenates may, or may not,

accurately reflect practical field conditions. Different

IPNV isolates vary in stabilities during storage (Dorson et

al., 1978; McMichael et al., 1975; Malsberger & Cerini,

1963). Further studies are needed to determine if the

liability of various IPNV isolates is similar to that

demonstrated for the striped bass isolate of IPNV in stored

striped bass samples. Current data demonstrate that for

detection of IPNV-Sb infectivity in striped bass, samples

should be stored intact at 40C and assayed within two

weeks.

Bullock and Stuckey (1975) reported that steroids

increase the recovery of infectious agents from inapparent

trout carriers. Increased detection of IPNV was observed

in IPNV-inoculated fish that were given an intraperitoneal

injection of steroid fifteen months after IPNV injection.










Apparently the steroids temporarily reversed the observed

decline of virus titers in IPNV-infected striped bass.

Tissue titers in IPNV-inoculated striped bass remained

relatively constant over the first 10 days. In contrast,

IPNV levels peak at three days after IPNV inoculation in

Atlantic salmon (Swanson & Gillespie, 1979), and in rainbow

trout that are susceptible to IPNV-induced mortality, IPNV

titers reach high titers at seven days after exposure

(Okamoto et al., 1984). Although Yamamoto (1975b)

suggested a correlation between virus-neutralizing antibody

and IPNV titers in trout, no such relationship was observed

in striped bass. Antibody was not detected in IPNV-

injected striped bass until seven days post inoculation and

reached peak values at approximately three weeks. During

this same time period, levels of virus recovered from IPNV-

inoculated striped bass remained uniform, apparently

unaffected by the presence of the antibody. In fact, virus

titers remained uniform during the first two months after

IPNV-injection and gradually declined over a 15 month

period.

The virus was frequently recovered from the anterior

kidney, spleen, and leucocytes--tissues that are

immunologically active in fish (Ellis, 1982). The effects

of IPNV on the immune system of fish are just beginning to

be investigated. For instance, Knott and Munro (1986)

reported that lymphocytes from IPNV-infected Atlantic

salmon demonstrate decreased mitogen activity. Work with











another birnavirus, infectious bursal disease virus (IBDV)

in chickens, has shown that IBDV is directly

immunosuppressive (Faragher et al., 1972), and bursal (B)

lymphocytes are the target cells for the virus (Hirai &

Calnek, 1979). Because a bursa-equivalent has not been

identified for fish, demonstration of IPNV-induced

immunomodulation in fish will probably be more difficult to

elucidate.

Depression of the humoral response of striped bass to

IPNV was not observed in the current study. In fact, IPNV

was strongly antigenic to striped bass and induced

significant antibody titers after IPNV-injection. A rise

in antibody levels was detected in IPNV-injected striped

bass at 7 days post injection after both a primary and

secondary inoculation. In a classical anamnestic response,

antibody levels rise faster in the secondary response

(Eisen, 1980). The controversy over whether fish

demonstrate a true anamnestic response has been reviewed

(Dorson, 1984). In the current experiments, striped bass

did not appear to exhibit an anamnestic response but they

did mount a humoral response to IPNV infection.

Immersion in IPNV, however, was not sufficient to

stimulate detectable levels of virus-neutralizing antibody.

Even in virus-carrier striped bass that were given a second

IPNV exposure by immersion, antibody levels did not

increase after the second challenge.










Chronic IPNV-carrier striped bass that received

exogenous steroid did not demonstrate any change in the

titers of virus-neutralizing antibody. Administration of

steroids prior to IPNV injection, did delay and reduce the

humoral response of striped bass. Steroid induced

immunosuppression has been described also in trout

(Anderson et al., 1982). In addition, Anderson et al.

(1982) reported that trout given a steroid dose of 200

mgkg did not appear unhealthy during the 23 days of the

experiment. Steroid, administered at a rate of 100 mg per

kg body weight, was lethal to striped bass observed over a

three month period. However, none of the IPNV-injected

striped bass that were treated with steroids developed any

clinical signs or histological lesions attributable to

IPNV.

In conclusion, then, IPNV is not a major pathogen for

striped bass, but striped bass can be inapparent IPNV-

carriers. Virus-carriers may pose a potential threat to

IPNV-susceptible fish species. Therefore, prior to

transport of striped bass into IPNV-free areas, the striped

bass should be tested for IPNV. However, isolation of IPNV

from a population of striped bass should not be used as a

reason to destroy the fish since striped bass apparently

are resistant to IPNV-induced disease.













APPENDIX
SOURCES OF SUPPLIES AND EQUIPMENT


Aldrich Chemical Corporation, Inc. (Milwaukee, WI)
Glycerol

American Scientific Products (McGraw Park, IL)
Heparinized microhematocrit capillary tubes

Ames Company (Elkhart, IN)
N,N,N',N' tetramethyl ethylenedianine (TEMED)
Coumassie blue

Amicon Corporation (Danvers, MA)
Microconcentrators (CENTRICON)

Armour Pharmaceutical (Tarrytown, NY)
Fetal bovine serum

Beckman Instruments, Incorporated (Palo Alto, CA)
Cellulose nitrate centrifuge tubes (5/8" x 4")
Ultra-clear centrifuge tubes
Ultracentrifuge (Beckman L5-50B Ultracentrifuge)

Becton, Dickinson & Co. (Rutherford, NJ)
Syringes and needles

Bellco Glass Inc. (Vineland, NJ)
Multi-stir

Bethesda Research Laboratory (Gaithersburg, MD)
Sucrose ultrapuree enzyme grade)

Biorad (Richmond, CA)
Bis-acrylamide

CGA Corporation (Chicago, IL)
Precision low temperature incubator

Corning Glass Works (Corning, NY)
Tissue culture flasks and bottles

Commercial Products Corporation (Manitowoc, WI)
Kelvinator Series 500 Freezer

Crescent Research Chemicals (Paradise Valley, AZ)
Tricaine methanesulfonate (MS-222)




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