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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
Creator:
Wechsler, Sally Janet
Copyright Date:
1985
Language:
English

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Blood ( jstor )
Fingerlings ( jstor )
Fish ( jstor )
Freshwater bass ( jstor )
Infectious pancreatic necrosis virus ( jstor )
Mortality ( jstor )
Necrosis ( jstor )
Steroids ( jstor )
Trout ( jstor )

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University of Florida
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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 MedicineBaltimore 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 . . 13.3

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 o67

14 Recovery of IPNV from homogenates stored in
different types of containers . o 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 IPNVexposed 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 electrophoresis 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 EPNV 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 EPNV-induced mortality.

For reasons yet to be determined, by six months of age trout lose their susceptibililty 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 4.5�C, 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 virusneutralizing 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). En 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 IPNVinduced 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 passaged 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 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 EMEM2 (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 40C. 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 Freoncell 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 (CsCl) 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, IPNVM 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 destained 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 IPNVSb 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 2 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 1500j

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 IPNVneutralizing 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/mnin 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


I










(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 posthatch. Georgia fry were exposed to EPNV 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 CC & 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 EPNV 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 IPN.V 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, 10 , 10 , 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 IPNVcontaining 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 virusneutralizing 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

1PNV 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 virusneutralizing 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 IPNVinoculated 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 IPNVneutralizing 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 10 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 10 6 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 virusneutralizing 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 IPNVAb.


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 virusneutralizing 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 lability 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 virusexposed 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 ( 0 ) 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.










7 0 -r . . . . .


60


50


40-


30 -1


20 "" 102


0 4 a 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 a 12 16
DAYS POST CHALLENGE

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

70


60


50
V

40

0
2 30Q 20


10


0 a
0 4 8 12 16
DAYS POST CHALLENGE Figure 1 D. Georgia striped bass fry were exposed to IPNV at ten days post-hatch.































100.


Figure exposed


70 60


50


40

0
2 30


Cl 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 4 812 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 ( I ! ) 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 a 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










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 postinoculation (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.


I












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 10 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.

percentage 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 102d 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


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 meth d 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.


I











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 i04c


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 l04 1.9 x l04 2.8 x 104 4.8 x 103 2.0 x 105

3.6


1.2 X 105 2.6 x l04 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 22�C. 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 1101


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 virusneutralizing 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 IPNVinjected 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


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


500

200-700

500

750-1000


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. fNot 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 IPNVinoculated 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 (leukocytes) 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 IPNVinoculated 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 therefter (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



Controlse 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. injecLion 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 intraperitoneal inoculation with 10 plaque forming units (pfu) of infectious pancreatic necrosis virus (IPNV). Fingerlings were injected with phosphate buffered saline (PBS) ( 0 )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 POSr CHALLUGE


Figure 3 A. beginning 50


30



20 " 10-


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


U


0




o 0
0
0 0
o
0 0
00 00
o 0
00


-- a -


WEEKS POST CHALLEGE


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


at weekly










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, IPNVinjected 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

300- .

(/ 250- , 0 0 0

-200
o
150 -0 10
150 - 1" 00 0
,, - ,, ~ ~.' ,,
100- , .0 , 4




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 ( 0 ). Control fish received an
i.p. injection with phosphate buffered saline ( M ) on day 0. A second IPNV challenge ( L ) was given at 11 weeks after the first injection. Control striped bass were injected i.p. with IPNV at week 11. Virus- 2 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 IPNVinfected striped bass were stored under different conditions 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 container (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 NVd


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.


I











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 x102 b5 x103 3 x104 2x 104 1 x104

2 days

40C NVc 1 x103 6 x103 4x 103 4 x103

-200C NV 7 x103 3 x104 5x 104 7 x104

-700C NV 8 x103 5 x104 5 x104 3 x104

2 weeks

40C NV NV 2 x104 NV 1 x104

-200C 1 x 102 5 x103 6x 104 6 x104 3 x104

-700C NV NV 5 x104 2 x104 lxl103


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


NDf


4 x 102


5 NV


6 8 x i01 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 l03


3 x 102


1 x 102 5 x 101


2 x 102


2 x 103 4 x l02


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 l03


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.

eplaque forming units of IPNV per ml of homogenate. fNot 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



























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 (20,10y). 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 V0 Vt, where D = reciprocal of the dilution of antibody, t 0.25 minutes, V0 = 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> 600 50
40
o. 302010


30



0 2 4
-nME (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

9080

n 7060

D 50- 40A

1. 3020

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


5 e55
IPNV-M 3 x 105 7 x 105 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, V0 = total number if 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 w ith the br ine shr imp naupl ii f ed to the f ish 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.

StriF.jd 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 posthatch, 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 IPNVexposed 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 IPNVisolates 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 IPNVinfected 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 IPNVinfected 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 (-200 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 lability 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 IPNVinjected 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 IPNVinoculated 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 mg/kg 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 IPNVcarriers. 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 (ultrapure 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)




Full Text

PAGE 1

IMPORTANCE OF INFECTIOUS PANCREATIC NECROSIS VIRUS IN STRIPED BASS, Morone saxatilis By SALLY JANET WECHSLER A DISSER TAT ION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIV E RSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1986

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Copyright 1986 by Sally Janet Wechsler

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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 acknowlege 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 othe~ 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 iii

PAGE 4

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. iv

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ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTERS ONE INTRODUCTION Background Objectives TABLE OF CONTE N TS TWO MATERIALS AND METHODS . iii .viii ix X 1 l 10 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 V

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Virus Infection Studies Waterborne IPNV Challenge of Striped Bass Fry Waterborne IPNV Challenge of Striped Bass Fingerlings Virus Inoculation of Striped Bass Finger lings Histological Examination 25 25 27 28 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 Humeral 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 SO Vertical IPNV Transmission in Striped Bass 50 Transmission of IPNV from Striped Bass to Brook Trout 53 Humeral Response of Striped Bass to IPNV 53 Early Humeral Response to IPNV Challenge. 53 Effect of Steroids on Titers of Circulating IPNV and Virus-Neutralizing Antibody. 55 vi

PAGE 7

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 BIOGRAPHICAL SKETCH. vii 92 104

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LIST OF TABLES 1 Percent cumulative mortality in striped bass finger lings 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 finger lings 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 14 Recovery of IPNV from homogenates stored in 67 different types of containers 68 15 Recovery of IPNV from striped bass stored whole 69 16 Neutralization rates for three IPNV isolates 75 viii

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1 2 3 4 5 6 LIST OF FIGURES Percent daily mortality in striped bass fry. Percent daily mortality in striped bass finger lings Mean virus-neutralizing antibody titers Virus-neutralizing antibody titers in striped bass given a second IPNV challenge Electrophoretic profile of IPNV polypeptides Neutralization kinetics of three IPNV isolates ix 40 44 58 60 71 73

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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 (Marone 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 intraperit o neal 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. X

PAGE 11

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. xi

PAGE 12

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 (Merone 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

PAGE 13

2 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 10 6 and the other is 2.3 x 10 6 (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 10 3 ) 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 10 3 ), and the

PAGE 14

3 internal proteins are of low molecular weight (27 31 x 10 3 ) (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 bursa! 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

PAGE 15

4 frequently, acute catarrhal enteritis ( M cKnight & 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, b y six months of age trout lose their susceptibililty 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.

PAGE 16

5 M any 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 4.5c, most at 10c, and an intermediate number of deaths in fry held at 1s 0 c. 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,

PAGE 17

6 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 &

PAGE 18

7 M acKelvie, 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) l PNV 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 ( M acdonald & Gower, 1981).

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8 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, Perea fluviatilis (Munro et al., 1976); European eel, Anguilla anguilla (Castric & Chaste!, 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).

PAGE 20

9 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 (Ster.hens 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

PAGE 21

10 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

PAGE 22

11 M aryland 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 humeral 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.

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CHAPTER TWO MATERIALS AND METHODS Cell Cultures and Virus Isolates Cell Cultures Chinook salmon embryo (CHSE-214) cells were grown at 1a 0 c 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 E M E M -10 containing antibiotics: 200 IU/ml penicillin and 200 ug/ml streptomycin (PS). Cells were seeded into eight-well culture plates and incubated at 1a 0 c in ambient air plus 2% carbon dioxide (CO 2 ) 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 p a ssaged twice in CHSE-214 cells and aliquots were stored at -7o 0 c. 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). 12

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Cultivation and Assays of IPNV Preparation of Virus Stocks 13 Confluent monolayers of CHSE-214 cells grown in 75 cm 2 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 15c (with gentle agitation every 15 minutes), EMEM-10 was added to the IPNV-inoculated cells. The virus-exposed cells were incubated at 15c until the monolayers showed extensive cytopathic effects (CPE) (usually 2 3 days). Cells and culture fluid were harvested and centrifuged at 1500 x for 15 minutes at 4c. The supernatant liquid was stored in 1 ml aliquots at -7o 0 c. The infectivity of stock virus was determined by plaque assay as described below. Virus Infectivity Plaque Assay A modification of a virus infectivity assay ( M oss & 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 19c 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 EMEM2 (without agarose), was added. Plates were incubated at 1a 0 c in ambient air plus 2% co 2 until cytopathic effects (CPE) were noted. Cell sheets were fixed with 30% formalin,

PAGE 25

14 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 10 5 cells/ml) was added to each well. Plates were incubated in ambient air at 1a 0 c. 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 1s 0 c, then EMEM-2 was added. After 48 hours incubation at 1s 0 c, the cell sheets

PAGE 26

15 showed extensive CPE. The cells and culture fluids were centrifuged at 7000 x 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 for 15 minutes at 4c. The top layer of TNE was removed and stored at 4c. 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 4c. The solution was centrifuged at 9000 x for l hour at 4c. 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 (CsCl) 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 for 45 minutes at 4c. The virus band was withdrawn by side tube puncture using a 20 gauge needle and

PAGE 27

--------------------------16 5 ml syringe. The virus band was diluted in TNE and centrifuged at 83,000 x for 40 minutes at 4c. The viral pellet was resuspended in 1 ml TNE and stored at -7o 0 c. 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 for 16 hours at 4c. 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 2s 0 c,

PAGE 28

17 and then 0.1 ml of l N phenol reagent was added. After l hour incubation at 25c, 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-polyacrylarnide 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

PAGE 29

18 overnight. The 4% acrylamide stacking gel was made by combining 1.3 ml of 30% acrylamide, 2.5 ml of 6% Tris-HCl (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 100c for three minutes, cooled to 4c, 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 l cm from the bottom of the cesolving 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 destained 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

PAGE 30

19 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 logarithm 10 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 4c and centrifuged at 1500 x 9. at 4c for 20 minutes. Serum was

PAGE 31

20 collected, heated at 56c for 30 minutes to inactivate complement, filtered using a membrane filter (0.45 micron pore size), and stored in 1 ml aliquots at -70c. 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 4c. 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

PAGE 32

21 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 V 0 / Vt, where D = the reciprocal of the dilution of antibody, t = 0.25 minute, V 0 = 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.

PAGE 33

22 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 for 30 minutes at 4c 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 l 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 19c for l 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.

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23 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 45c for 30 minutes to inactivate complement (Sakai, 1981), and stored at 4 or -20c. 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 10 3 pfu/ml) were mixed, and, with periodic gentle agitation, incubated at 19c 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

PAGE 35

24 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 22c 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 2s 0 c). Rainbow

PAGE 36

25 (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 12c 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 (19c). 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 10 6 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 -20c, and assayed for IPNV using

PAGE 37

26 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 23c). 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 str iped bass were stored for O 3 days at 4c 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

PAGE 38

27 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 12c, and six were kept at 22c. Water flow to all tanks was stopped for six hours, and the water aerated. At each temperature, two tanks were seeded with 10 4 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 -20c 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 10 6 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.

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28 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, 10 3 10 5 or 10 6 pfu of IPNV. Treatment controls were anesthetized and returned to the tank. Dead fish were collected daily for four weeks, and stored at -20c for l 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 12c and an additional 24 fish were maintained at 22c. All the fish were anesthetized and inoculated i.p. with 10 6 pfu of virus. Two weeks later, half of the fish held at half of the fish held at 12c were transferred to 22c, and half of the fish held at 22c were transferred to 12c. Fish were observed daily for mortality. After one month, survivors from each group were bled and assayed for virus.

PAGE 40

29 Histological Examination T~e 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 10 2 10 4 pfu of IPNV, were added to the tanks. Each striped bass ~as 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.

PAGE 41

30 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. processed within two hours of collection. Samples were To determine if IPNV-infected striped bass transmit IPNV in their sex products, five-year-old striped bass were injected i.p. with 10 6 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

PAGE 42

31 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 (10 6 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 (10 6 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 :PNV 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 10 6 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.

PAGE 43

32 Fifteen IPNV-infected striped bass were placed in a tank. Water (12c) 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. Humeral 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 10 6 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

PAGE 44

33 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 10 7 pfu of IPNV; and (4) steroid + virus--fish were injected i.p. with steroid (100 mg/kg) 24 hours before i.p. inoculation with 10 7 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 10 5 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 humeral 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

PAGE 45

34 containing 10 7 pfu of IPNV. 3lood 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 10 6 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 10 5 pfu of IPNV. Fourteen months later, a blood sample was taken from these fish. The fish were immersed for 5 minutes in water containing 10 5 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

PAGE 46

35 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 4c 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

PAGE 47

36 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 lability 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 -7o 0 c. 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 -7o 0 c) 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

PAGE 48

37 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 -7o 0 c for two to fourteen days. After storage, frozen fish were allowed to soften at 4c, 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 d u ys following steroid injection.

PAGE 49

CHAPTER T HREE 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 1to 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. M ortality 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 22c exhibited no difference in mortality compared to control groups (p < 0.01) (Figure 2). 38

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-----------------Figure 1: Percent daily mortality of strip 5 d bass fry during the 21 days following exposure to 10 plaque forming units of infectious pancreatic necrosis virus (IPNV) per milliliter of water ( 0-0) 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.

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40 70 -, ... ... . . --.. -1 f t i\ o i I\ 50 \ j I 40 / \ I i i l I I \ i, I I I. I I I 30 ""1 I \ i I I I I \ I i \\ 20 1 ,! \ \ ', f \ i 10 1 1 \ ~ \ .. 11: --a _ ./ ib \ .;' \'. 0 +~ ;> ~~ ~~-; ~-0 4 a 12 16 20 DAYS P0S7 CHALLENGE Figure 1 A. Chesapeake and Delaware Canal (MD) striped were exposed to IPNV at one day post-hatch. bass fry 70 60 50 40 J 0:: 0 30 0 20 10 0 0 Figure 1 fry were 4 8 12 16 20 DAYS POST CHALLENGE B. Chesapeake and Delaware Canal striped bass exposed to IPNV at five days post-hatch.

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41 70 60 50 i: 40 :J 0:: 0 30 20 10 0 0 4 8 12 1 6 DAYS POST CHALLENGE Figure 1 C Florida striped bass fry were exposed to IPNV at seven days post-hatch. 70 60 50 .._,, i: 40 0:: 0 30 a 20 10 0 0 4 a 12 16 DAYS POST CHALLE N GE Figure l D. Georgia str i ped bass fry were exposed to IPNV at ten days post-hatch. 20 20

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42 70 60 50 40 '"j 0:: 0 JO <{ 0 20 10 0 0 4 8 12 16 20 DAYS POST CHALLENGE Figure 1 E. Nanticoke River (MD) striped bass fry were exposed to IPNV at 15 days post-hatch. 70 60 50 40 :::i 0:: 0 :::::E JO < a 20 10 0 0 4 8 12 16 DAYS POST CHALLENGE Figure 1 F. Nanticoke River striped bass fry were exposed to IPNV at 20 days post-hatch. 20

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Figure 2: Percent daily mortality of striped ba!s 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 < 0.0lr tested by analysis of variance) at either temperature.

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44 13 12 1 1 10 9 8 7 a:: 0 6 :::! 5 Q 4 3 2 0 2 4 6 8 10 1 2 14 16 18 DAYS POST EXPOSURE Figure 2 A. Striped bass fingerlings were held at 12c. 1J 12 1 10 9 8 7 0 6 :::e 5 < a 4 3 2 0 2 4 6 8 10 12 DAYS POST CHALLENGE Figure 2 B~ Fingerlings were held at 22c~ 14 16 18

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45 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 10c 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.

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46 Table 1: Percent cumulative mortality in striped bass fingerlings following intraperitoneal injection of infectious pancreatic necrosis virus (IPNV). 60 90 120 150 180 CONTROL TREAT MENT 28 20 0 8 SHAM 26 18 0 0 VIRUS INOCULUM (pfu) 38 40 ND 24 26 14 2 16 14 0 2 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 22c. bNot done. cPercentage of striped bass that died in the 28 days following inoculation.

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47 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) TREATSHAM MENT 10 3 10 5 10 6 60 d 10 2 10 3 -10 5 ND 90 NV NV NV ~-v 10 2 -10 4 120 NV NV NV 10 3 -10 6 10 5 -10 7 150 e 10 5 180 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 22c. Dead fish were assayed for virus using the plaque assay me 1 hod that detected titers greater than or equal to 5 x 10 pfu/g. ~ot 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.

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48 Table 3: Range of virus titers in striped bass fingerlings surviving intraperitoneal injection of infectious pancreatic necrosis virus. VIRUS INOCULUM (PFU)a MONTHSb 1 2 3 4 NV 6 12 14 ND astriped bass fingerlings received an intraperitoneal inoculation with the indicated plaque forming units (pfu) of infectious pancreatic necrosis virus (IPNV). Fish were maintained at 22c. bMonths following intraperitoneal injection that surviving striped bass were assayed for IPNV using the plaque methd 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.

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49 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. TEMPERATURE a 22 12 22 --> 12 12 --> 22 2.7 X 10 4 9.7 X 10 4 2.3 X 10 5 1.2 X 10 5 2.9 X 10 4 2.9 X 10 4 4.9 X 10 3 2.6 X 10 4 3.9 X 10 4 4.5 X 10 4 1.1 X 10 6 3.3 X 10 4 3.0 X 10 4 1.4 X 10 4 2.2 X 10 5 NVb NV 3.2 X 10 4 1.9 X 10 4 8.2 X 10 3 7.2 X 10 4 2.0 X 10 6 l.9x 10 4 9.2 X 10 4 NV 5.0 X 10 6 2.8 X 10 4 8.0 X 10 3 1.2 X 10 5 6.4 X 10 5 4.8 X 10 3 2.1 X 10 4 ------------------------------------4.0 X 10 4 C 9.8 X 10 5 2.0 X 10 5 3.9 X 10 4 4.od l. 7 3.6 4.4 asix-month-old gtriped bass were given an intraperitoneal injection of 10 plaque forming units (pfu) of infectious pancreatic necrosis virus (IPNV). Fish were held at ei ~ ner 12 or 22c. 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 methpd that detected titers equal to or greater than 5 x 10 pfu/g. Virus titers were expressed as pfu per gram of tissue. ~o 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.

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50 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 10 2 10 4 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 (10 1 -10 3 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

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51 Table 5: Range in virus titer in striped bass following ingestion of brook trout that contained infectious pancreatic necrosis virus. WEEKS POSTa EXPOSURE 1 2 3 4 12 33 VIRUS TITERb 10 1 10 1 -10 3 10 1 -10 3 10 1 -10 3 10 1 -10 2 10 1 NUMBER TESTED 1 3 2 2 2 1 asix-month-old striped bass were fed brook trout, each of which contained 10 2 10 4 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.

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52 Table 6: Recovery of infectious pancreatic necrosis virus (IPNV) from samples taken during investigations of vertical transmission of IPNV in striped bass. SAMPLES Adults inoculated with IPNVa Internal Organs Sex Products (Eggs and Milt) Fertilized Eggs Fry Noninoculated Adultsb Sex Products IPNV added to Eggsc Fertilized Eggs Fry IPNV added to Miltd Fertilized Eggs Fry VIRUS RECOVERED Yes No No No No Yes No No No aFive-year-old, hatchery-reared stri~ed 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 10 5 pfu of IPNV/ml immediately prior to mixing with virus-free milt. dMilt was exposed to 10 5 pfu of IPNV/ml immediately prior to mixing with virus-free eggs.

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53 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 10 4 10 5 pfu/g. One of four trout tested after six weeks had 10 2 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 10 6 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 magni 4:. ude 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).

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54 Table 7: Detection of infectious pancreatic necrosis virus (IPNV) and virus-neutralizing antibody in IPNV injected striped bass fingerlings. ANTIBODyd 1 3 I 3 2 3 I 3 o I 3 3 4 I 4 o I 2 4 4 / 4 o I 3 5 4 / 4 o I 2 6 3 I 3 o I 3 7 4 / 4 1 / 3 500 8 4 / 4 2 I 3 200-700 9 4 / 4 2 I 3 500 10 4 / 4 2 / 4 750-1000 aDays post injection (intraperitoneal) of 10 6 plaque forming units (pfu) of IPNV. 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. fNot tested.

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55 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 bas~ that did not receive stero i d. Virus was recovered more frequently from the buffy coat (leukocytes) 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 obser v ed between the two groups IPNV-injected striped bass that did not receive steroid. Striped bass that were bled at three dpi and weekly therefter (Figure 3a) had somewhat higher

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56 Table 8: Recovery of infectious pancreatic necrosis virus (IPNV) from the plasma and buffy coat of virus inoculated striped bass fingerlings. TREAT MENT DAYS POST INJECTIONa Steroid+ Controlse SAMPLE buffy coat plasma buffy coat plasma buffy coat plasma 3 7 3/3c 3/3 1/3 0/3 1/3 3/3 0/3 0/3 0/3 0/3 0/3 0/3 10 0/3 0/3 0/3 0/3 0/3 0/3 14 1/3 0/3 0/3 0/3 0/3 0/3 17 0/3 0/3 0/3 0/3 0/3 0/3 astriped bass fingerlings received 10 7 plaque forming units (pfu) of IPNV or phosphate buffered saline by in tr aper i toneal ( 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. injec t ion 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. 21 0/3 0/3 0/3 0/3 0/3 0/3

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Figure 3: Titers of virus-neutralizing antibody in striped bass fingerlings that 5eceived an intra peritoneal inoculation with 10 plaque forming units (pfu) of infectious pancreatic necrosis virus (IPNV). Fingerlings were injected with phosphate buffered saline (PBS) ( 0 ) or with triamcinolone acetomide (100 mg/kg) ( O ) 24 hours prior tf viral inoculation. Antibody titers are expressed as 10the 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.

PAGE 69

58 ea-.---------~.,_ _____________ 70 + 0 0 0 IO 0 0 40 + 0 20 D 10 0 .a C 0~-C!!&-,-Mll .... --,,--~L--......,---------------J 0 2 4 WEEKS POST CWI! e DIGE Figure 3 A. Fish that were sampled at weekly intervals beginning three days after IPNV inoculation. IO ,--------------4!l-~-----------40~2010 0 + 00 0 0 0 + 0 0 0 + + 0 0 0 0 C C D C C D D r""I I I I 2 4 WEEKS POST CH:61 1 DICE I 0 C 0 C Figure 3 a. Striped bass that were sampled at weekly intervals after IPNV injection.

PAGE 70

59 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.

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60 400 350 300 Ul 250 a:: w Ii== >200 0 0 al i== 150 100 50 0 2 J 4 5 6 11 12 13 14 15 16 19 20 21 WEEKS Figure 4: Virus-neutralizing antibody titers in striped bass fingerlings injected with infectious paicreatic necrosis virus (IPNV). Striped bass received 10 plaque for ming units (pfu) of I PNV by intraper i toneal ( i.p.) inoculation on day O ( ea ). Control fish received an i.p. injection with ph:Jsphate buffered saline ( ) on day O. 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 102 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. 23

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61 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 1to 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 10 1 pfu/g) to 10 6 pfu/g. Virus Recovery from Steroid Injected Chronic Carriers This study was conducted to determine if exogenous corticosteroids would increase the percentage of virus

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62 Table 9: Attempts to isolate infectious pancreatic necrosis virus (IPNV) from striped bass caught in the Chesapeake Bay (MD). SURVEY DATE Aug. 1984 Sept. 1984 Dec. 1984 Feb. 1985 LOCATION YEAR-CLASS Upper Bay 1984 Upper Bay 1984 Choptank River 1982 83 Upper Bay 1982 83 VIRUS/SAMPLES a o I 39 o I 66 o I 15 o I 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.

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63 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 I 49 (18%) Feb. 1985 Upper Bay 1982 83 6 I 94 ( 6%) July 1985 Upper Bay 1985 0 I 5 Aug. 1985 Choptank River 1985 l I 45 ( 2%) Aug. 1985 Upper Bay 1985 0 I 3 Sept. 1985 Upper Bay 1985 0 I 6 Sept. 1985 Upper Bay 1984 o I 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.

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64 Table 11: Striped bass tissues from which infectious pancreatic necrosis virus was isolated. TISSUEa # POSITIVE/ # TESTEDb Anterior kidney 29 I 29 (100%) Spleen 20 I 25 80%) Blood 4 I 8 50%) Fat 2 I 4 50%) Liver 9 I 20 45%) Intestine 2 I 9 22%) Posterior kidney 4 I 20 20%) Heart 2 I 11 8%) Brain 0 I 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.

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65 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 4c (Table 13) but IPNV infectivity was not significantly different (p < 0.01, ANOVA) in homogenate samples stored at either -20 or -7o 0 c. 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 4c were significantly different (p < 0.01, ANOVA) from titers from homogenates stored at -20 or -7o 0 c. 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 -7o 0 c. Table 15 shows the virus titers of IPNV-infected striped bass fingerlings determined after storage. Virus titers were best maintained in IPNV

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---------------------------. 66 Table 12: Recovery of infectious pancreatic necrosis virus (IPNV) from chronic virus-carrier striped bass following injection with steroid. # POSITIVE/# TESTEDb 0 0 I 4 NVd 0.5 0 I 3 NV 1 1 / 4 10 2 2 3 I 4 10 2 10 3 3 1 / 4 10 2 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.

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67 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 2 days 4c -20c -7o 0 c 2 weeks 4c -20c l -7o 0 c NVc NV NV NV X 10 2 NV NV NV 6 X 10 4 2 X 10 4 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.

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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 4c -20c -7o 0 c vb pC V p V p Sam,ele 1 NVd 3 X e 10 1 NV NV NV NV 2 8 X 10 1 l X 10 2 3 X 10 2 4 X 10 2 2 X 10 2 4 X 10 2 3 5 X 10 1 NDf 6 X 10 2 1 X 10 3 4 X 10 2 6 X 10 2 4 3 X 10 2 4 X 10 2 3 X 10 3 3 X 10 3 2 X 10 3 2 xl0 3 5 NV NV 3 X 10 2 2 X 10 2 4 X 10 2 2 X 10 2 6 8 X 10 1 l X 10 2 2 X 10 3 2 X 10 3 l X 10 3 2 X 10 3 7 3 X 10 1 5 X 10 1 2 X 10 3 1 X 10 3 9 X 10 2 l x 10 3 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. ePlaque forming units of IPNV per ml of homogenate. fNot done.

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6 9 Table 15: Recovery of infectious pancreatic necrosis virus (IPNV) from IPNV-infected striped bass stored whole. LENGTH OF S'I'ORAGE STORAGE TEMP( 0 c)a 0 DAYS 2 DAYS 14 DAYS 4 10 / 11 3 I 3 (10 2 -10 4 ) (10 3 -10 4 ) -20 4 I 13 1 / 3 (10 2 -10 3 ) (103) -70 o I 17 o I 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.

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70 carrier striped bass that were stored intact in the refrigerator at 4c. All virus infectivity was lost in IPNV-carrier striped bass that were stored at -7o 0 c (Table 15). The loss was evident after only 48 hours of storage. In fish that were stored at -20c, loss of infectivity was intermediate between that observed at 4c and -7o 0 c. 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 logarithm 10 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.

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88 28 24 20 M 71 s V 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), ovalburnin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), and trypsin inhibitor (20,lOg). Numbers on the gel indicate molecular weights x 10. The lowest band represents the dye front.

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72 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 v 0 / Vt, where D = reciprocal of the dilution of antibody, t = 0.25 minutes, V 0 = 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.

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73 100 90 80 (/) 70 ::::, a:: 5 60 J 0 50 IIz 40 w u a: w 30 a. 20 10 0 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 ( D), menhaden ( + ) and the North American isolate VR-299 ( ). Equal volumes of diluted antibody and virus were mixed, incubated at 4c, 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.

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74 100 90 80 (I) 70 :::::, 0:: 5 60 0 50 .... z 40 I.LI u 0:: w 30 a. 20 10 0 0 2 4 Figure TIME (minutes) with antibody 6 B. The IPNV isolates were tested against the North American isolate VR-299. 100 90 80 Ill 70 :::::, 0:: 5 60 50 0 .... .... z 40 uJ (.J a:: uJ 30 ll. 20 10 0 0 2 4 TIME (minutes) Figure 6 c. The IPNV isolates were tested with antibody against the menhaden IPNV isolate.

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75 Table 16: Neutralization rates for three infectious pancreatic necrosis virus (IPNV) isolates reacted with homologous and heterologous antisera. ANTISERUM IPNV-Mb IPNV-Sbc VR-299d e IPNV-M 3 X 10 5 IPNV-Sb 9 X 10 5 VR-299 9 X 10 5 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 V 0 / Vt, where D = dilution of the antiserum, t = 0.25 minutes, V 0 = total number Jf viral plaques, and Vt= number of viral plaques at 0.25 minutes.

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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 Chesapeak e 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 1to 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 76

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77 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. Strip~d 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,

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78 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

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79 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 I PNV 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

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80 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 SOS-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

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81 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

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82 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

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83 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

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84 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 4c, compared to values obtained from samples stored at -20 or -7o 0 c. Therefore, for best virus recovery, striped bass tissue homogenates should be stored frozen (-20 or -7o 0 c). 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 -70 but was retained at 4c. Retention of virus infectivity was intermediate in samples stored at -20c. The retention of viral infectivity in whole striped bass stored at 4c and loss of infectivity in fish stored at -7o 0 c 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 -7o 0 c 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

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85 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 4c, but IPNV remained infective when stored frozen (-20 and -7o 0 c). 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 !ability 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 4c 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.

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86 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

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87 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 humeral 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 humeral 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.

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88 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 humeral 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 mg;kg 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.

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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 LS-SOB Ultracentrifuge) Becton, Dickinson & Co. (Rutherford, NJ) Syringes and needles Bellco Glass Inc. (Vineland, NJ) Multi-stir Bethesda Research Laboratory (Gaithersburg, MD) Sucrose (ultrapure 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) 89

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Difeo Laboratories (Detroit, MI) Freunds' incomplete adjuvant DuPont Company (Wilmington, DE) Trichlorotrifluoroethane (FREON) Fisher Scientific Company (Fairlawn, NJ) Alundum (90-mesh) Bovine albumin Cesium chloride Crystal violet 90 Disodium ethylenediamine tetraacetate (EDTA) Ethanol Formalin Glacial acetic acid Methanol Phenol reagent (Folin Ciocalteau Reagent) Plastic bags (WHIRL-PAC) Sodium bicarbonate Sodium carbonate Sodium hydroxide Sodium phosphate Sodium potassium tartrate Flow Laboratories (McLean, VA) Eagle's minimal essential medium Normal calf serum 96-well tissue culture plates FMC Corporation (Rockville, ME) Agarose Gelman (Ann Arbor, MI) Membrane filters (ACRODISC) GIBCO (Grand Island, NY) Trypsin Harleco (Philadelphia, PA) Bromophenol blue Heath Company (Benton Harbor, MI) Regulated HV Power Supply Hewlett-Packard (Ft. Collins, CO) Series 9800 Desktop computer International Equipment Company (Needham Heights, MS) IEC Centra-7R Centrifuge International micro-capillary centrifuge LKB-Produkter AB (Brooma, Sweden) Hydroxyethyl methacrylate (HISTORESIN)

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-------------------Miles Laboratories (Naperville, IL) Eight-well culture plates 91 Nutrition Biochemical Corporation (Cleveland, OH) Glycine, aminoacetic Sigma Chemical Company (St. Louis, MO) Acrylamide 2-Mercaptoethanol (2-ME) Molecular weight markers Penicillin Polyethylene glycol Sodium chloride Sodium lauryl sulfate (SDS) Streptomycin Trizma base Trizma hydrochloride Sorvall (Ivan) Incorporated (Newtown, CT) Sorval RC2-B Centrifuge Squibb (E. R.) and Sons, Inc. (Princeton, NJ) Triamcinolone acetonide (Kenalog-40) Thomas (Arthur H.) Company (Philadelphia, PA) Dialysis tubing (1/4"; Thomas tubing) Varian Associates Inc. Instrument Group (Palo Alto, CA) Spectrophotometer (Cary 219) Virtis Company (Gardiner, NY) VirTis "23" homogenizer

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REFERENCES Adair, B. M. and Ferguson, H. w. 1981. Isolation of infectious pancreatic necrosis (IPN) virus from non salmonid fish. J. Fish Dis. 4: 69-76. Ahne, w. 1978. Isolation and Characterization of infectious pancreatic necrosis virus from pike (Esox lucius). Arch. Virol. 58: 65-69. Ahne, W. 1982. Comparative studies on the stability of four fish pathogenic viruses. Zbl. Veterinarmed. Reiche B. 29: 457-476. Ahne, w. 1985. Viral infection in fishes: Aetiology, diagnosis and control. Zbl. Vet. Med. 32: 237-264. Anderson, D. P., Roberson, B. s., and Dixon, O. w. 1982. Immunosuppression induced by a corticosteroid or an alkylating agent in rainbow trout (Salmo gairdneri) administered a Yersinia ruckeri bacteria. Dev. Comp. Immunol. Supple. 2: 197-204. Argot, J. and Malsberger, R. G. 1972. Intracellular replication of infectious pancreatic necrosis virus. Can. J. Microbiol. 18: 865-867. Ashe, w. K. and Scherp, H. w. 1963. Antigenic analysis of herpes simplex virus by neutralization kinetics. Immunol. 91: 658-665. Baudouy, A. M. an~ Castric, J. 1977. Persistence du pouvoir pathogene du virus de la necrose pancreatique infectieuse apres un sejour prolonge dans l'eau. Bull. Off. Int. Epiz. 87: 409-413. Billi, J. L., and Wolf, K. 1969. Quantitative comparison of peritoneal washes and feces for detecting infectious pancreatic necrosis virus in carrier brook trout. J. Fish. Res. Bd. Can. 26: 1459-1465. Bonami, J. R., Cousserans, F., Weppe, M., and Hill, B. J. 1983. Mortalities in hatchery-reared sea bass fry associated with a birnavirus. Bull. Euro. Ass. Fish Path. 3: 41. 92

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93 Bullock, G. L. and Stuckey, H. M. 1975. Aeromonas salmonicida: Detection of asymptomatically infected trout. Prog. Fish Cult. 37: 237-239. Bullock, G. L., Rucker, R. R., Amend, D., Wolf, K., and Stuckey, H. M. 1976. Infectious pancreatic necrosis: Transmission with iodine treated and nontreated eggs of brook trout (Salvelinus fontinalis). J. Fish. Res. Bd. Can. 33: 1197-1198. Castric, J. and Chastel, C. 1980. Isolation and characterizaqtion attempts of three viruses from European eel, Anguilla anguilla: preliminary results. Annal. Virol. 131E: 435-448. Cerini, c. P. and Malsberger, R. G. 1965. Morphology of infectious pancreatic necrosis virus. Annal. N. Y. Acad. Sci. 126: 315-319. Chang, N., Macdonald, R. D., and Yamamoto, T. 1978. Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates. Can. J. Microbial. 24: 19-27. Chen, S. N., Kon, G. H., Hedrick, R. P., and Fryer, J. L. 1985. The occurrence of viral infections of fish in Taiwan. In: Ellis, A. E. (ed). Fish and shellfish pathology. Acad. Press. New York. pp. 313-319. Cohen, J., Poinsard, A., and Scherrer, R. 1973. Physico chemical and morphological features of infectious pancreatic necrosis virus. J. Gen. Virol. 21: 485-498. Cohen, J. and Scherrer, R. 1972. Structure de la capside du virus de la necrose pancreatique infectieuse (IPN) de la truite. c. R. Acad. Sci. Paris. 274: 1222-1225. Coutant, C. C. 1985. Striped bass, temperature, and dissolved oxygen: a speculative hypothesis for environmental risk. Trans. Am. Fish. Soc. 114: 31-61. de Kinkelin, P. and Dorson, M. 1973. Interferon production in rainbow trout (Salmo gairdneri) experimentally infected with egtved virus. J. Gen. Virol. 19: 125-127. Desautels, D. and MacKelvie, R. M. 1975. Practical aspects of survival and destruction of infectious pancreatic necrosis virus. J. Fish. Red. Bd. Can. 32: 523-531. Dobos, P. 1976. Size and structure of the genome of infectious pancreatic necrosis virus. Nucl. Acid Res. 3: 1903-1924.

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------------------------94 Dobos, P. 1977. Virus-specific protein synthesis in cells infected with infectious pancreatic necrosis virus. J. Virol. 21: 242-258. Dobos, P., Hallett, R., Kells, D. T. C., Sorenson, 0., and Rowe, D. 1977. Biophysical studies of infectious pancreatic necrosis virus. J. Virol. 22: 150-157. Dobos, P., Hill, B. J., Hallett, R., Kells, D. T. C., Becht, H., and Teninges, D. 1979. Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J. Virol. 32: 593-605. Dobos, P. and Rowe, D. 1977. Peptide map comparison of infectious pancreatic necrosis virus-specific polypeptides. J. Virol. 24: 805-820. Dorson, M. 1982. Infectious pancreatic necrosis in salmonids: overview of current problems. In: Anderson, D. P., Dorson, M. M., and Dubourget, P. (ed). Antigens of fish pathogens. Fond. Marcel Marivex. Lyons, France. pp. 7-32. Dorson, M. 1984. Applied immunology of fish. In: de Kinkelin, P. and Michel, C. (ed). Symposium on fish vaccination. Off. Int. Epizooties. Paris, France. pp. 3974. Dorson, M., Castric, J., and Torchy, C. 1978. Infectious pancreatic necrosis virus of salmonids: biological and antigenic features of a pathogenic strain and of a non pathogenic variant selected in RTG-2 cells. J. Fish Dis. 1: 309-320. Dorson, M. and Torchy, C. 1981. The influence of fish age and water temperature on mortalities of rainbow trout, Salmo gairdneri, caused by a european strain of infectious pancreatic necrosis virud. J. Fish Dis. 4: 213-221. Dorson, M. and Torchy, C. 1985. Experimental transmission of infectious pancreatic necrosis virus via the sexual products. In: Ellis, A. E. (ed). Fish and shellfish pathology Acad. Press. New York. pp. 251-260. Eisen, H. N. 1980. Immunology. In: Davis, B. D., Dullbecco, R., Eisen, H. N., and Ginsberg, H. S. (ed). Microbiology. 3rd edition. Harper and Row Publishers. Hagerstown, MD. pp. 290-547.

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95 Eldridge, M. B., Whipple, J. A., Eng, D., Bowers, M. J., and Jarvis, B. M. 1981. Effects of food and feeding factors on laboratory-reared striped bass. Trans. Am. Fish. Soc. 110: 111-120. Ellis, A. 1982. Differences between the immune mechanisms of fish and higher vertebrates. In: Roberts, R. J. (ed). Microbial diseases of fish. Acad. Press. New York. pp. 1-29. Eskildsen, U. K. and Jorgensen, P. E. V. 1973. On the possible transfer of trout pathogenic viruses by gulls. Riv. Piscic. Ittiop. 8: 104-105. Faragher, J. T., Allan, W. H., and Cullen, G. A. 1972. Immunosupressive effects of the infectious bursal agent in chickens. Nat. 237: 118-119. Frantsi, C. and Savan, M. 1971. Infectious pancreatic necrosis virus: Comparative frequencies of isolation from feces and organs of brook trout (Salvelinus fontinalis). J. Fish. Res. Bd. Can. 28: 1064-1065. Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. 1977. Methods in immunology. 3rd edition. W. A. Benjamin, Inc. Reading, MA. pp. 87-89. Goodyear, C. P., Cohen, J. E., and Christensen, S. W. 1985. Maryland striped bass: recruitment declining below replacement. Transact. Am. Fish. Soc. 114: 146-151. Hall, L. W., Horseman, L. 0., and Zeger, S. 1984. Effects of organic and inorganic chemical contaminants on fertilization, hatching success, and prolarval survival of striped bass. Arch. Environ. Contam. Toxicol. 13: 723729. Hedrick, R. P., Eaton, w. D., Fryer, J. L., Hans, Y. C., Park, J. w., and H-..1ng, S. W. 1985. Biochemical and serological properties of birnaviruses isolated from fish in Korea. Fish. Path. 20: 463-468. Hedrick, R. P., Leong, J.C., and Fryer, J.C. 1978. Persistent infections in salmonid fish cells with infectious pancreatic necrosis virus. J. Fish Dis. 1: 297-308. Hill, B. J. 1976. Properties of a virus isolated from the bi-valve mollusc Tellina tenuis. In: Page, L.A. (ed). Proc. 3rd Internl. Wildlife Dis. Conf. Plenum Press. New York. pp. 445-452.

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96 Hill, B. J. 1982. Infectious pancreatic necrosis virus and its virulence. In: Roberts, R. J. (ed). Microbial diseases of fish. Acad. Press. New York. pp. 91-114. Hirai, K. and Calnek, B. w. 1979. In vitro replication of infectious bursa! disease virus in established lymphoidcell lines and chicken B lymphocytes. Inf. Imm. 25: 964970. Ishiguro, S., Izawa, H., Kodama, M., Onuma, M., and Mikami, T. 1984. Serological relationships among five strains of infectious pancreatic necrosis virus. J. Fish Dis. 7: 127-135. Johansen, K. 1982. Respiratory gass exchange in vertebrate gills. In: Houlihan, D. F., Rankin, J. C. and Shuttleworth, T. J. (ed). Gills. Cambridge Univ. Press. New York. pp. 99-128. Kaufer, I. and Weiss, 1980. Significance of bursa of fabricius as target organ in infectious bursal disease of chickens. Inf. Immun. 27: 364-367. Kelly, R. K. and Loh, P. C. 1972. Electron microscopical and biochemical characterization of infectious pancreatic necrosis virus. J. Virol. 10: 824-834. Kernehan, R. J., Headrick, M. R., and Smith, R. E. 1981. Early life history of striped bass in the chesapeake and delaware canal and vicinity. Trans. Am. Fish. Soc. 110: 137-150. Knott, R. and Munro, A. L.S. 1986. The persistence of IPNV in Atlantic salmon. Vet. Imrnunol. Immunopath. 12: 359-364. Laemmli, U. K. 1971. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nat. 227: 680-685. Lientz, J.C. and Springer, J. I. 1973. Neutralization tests of infectious pancreatic necrosis virus with polyvalent antiserum. J. Wild. Dis. 9: 120-124. Lightner, D. and Post, G. 1969. Morphological characteristics of infectious pancreatic necrosis virus in trout pancreas tissue. J. Fi sh. Res. Bd. Can. 26: 2247-2250. Loh, P. C., Lee, M. H., and Kelly, R. K. 1974. The polypeptides of infectious pancreatic necrosis virus. J. Gen. Virol. 22: 421-423.

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97 Lowry, o. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurements with the folin phenol reagent. J. Biol. Chern. 193: 265-275. Luna, L. G. (ed). 1968. Manual of histological staining methods of the Armed Forces Institute of Pathology. 3rd Edit ion. McGraw-Hill Book Co. New York. 258 p. Macdonald, R. D. 1978. Ringed plaque formation in infectious pancreatic necrosis virus correlates with defective interfering particle production. J. Gen. Virol. 41: 623-628. Macdonald, R. D. and Dobos, P. 1981. Identification of the proteins encoded by each genome segment of infectious pancreatic necrosis virus. Virol. 114: 414-422. Macdonald, R. D. and Gower, D. A. 1981. Genomic and phenotypic divergence among three serotypes of aquatic birnavirus (IPNV). Virol. 114: 187-195. Macdonald, R. D. and Kennedy, J. C. 1979. Infectious pancreatic necrosis virus persistently infects chinook salmon embryo cells independent of interferon. Virol. 95: 260-264. Macdonald, R. D. and Yamamoto, T. 1977. The structure of in fectious pancreatic necrosis virus RNA. J. Gen. Virol. 34: 235-247. Malsberger, R. G. and Cerini, C. P. 1963. Characteristics of infectious pancreatic necrosis virus. J. Bact. 86: 1283-1287. Manning, M. J., Grace, M. F., and Secombes, C. J. 1982. Developmental aspects of immunity and tolerance in fish. In: Roberts, R. J. (ed). Microbial diseases of fish. Academic Press. New York. pp. 31-46. Manning, M. J. and Mughal, M. S. 1985. Factors affecting the immune responses of immature fish. In: Ellis, A. E. (ed). Fish and shellfish pathology. Academic Press. New York pp. 27-40. McAllister, P. E. 1979. Fish viruses and viral infections. In: Fraenkel-Conat, H., Wagner, R. R. (ed). Comprehensive Virology. Plenum Press. New York pp. 401-470. McAllister, P. E., Newman, M. w., Sauber, J. H., and Owens, W. J. 1983. Infectious pancreatic necrosis virus: Isolation from southern flounder, Paralichthys lethostigma, during an epizootic. Bull. Europ. Ass. Fish Pathol. 3: 37-38.

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98 McAllister, P. E., Newman, M. W., Sauber, J. H., and Owens, W. J. 1984. Isolation of infectious pancreatic necrosis virus (serotype Ab) from diverse species of estuarine fish. Helg. Meer. 37: 317-328. McDaniel, D. (ed). 1979. Procedures for the detection and identification of certain fish pathogens. Am. Fish. Soc. Bethesda, MD. 118 pp. McKnight, I. J. and Roberts, R. J. 1976. The pathology of infectious pancreatic necrosis. I. The sequential histopathology of the naturally occurring condition. Br. Vet. J. 132: 76-84. McMichael, J., Fryer, J. L., and Pilcher, K. S. 1975. An antigenic comparison of three strains of infectious pancreatic necrosis virus of salmonid fishes. Aquac. 6: 203-210. Mertens, P. P. c. and Dobos, P. 1982. Messenger RNA of infectious pancreatic necrosis virus is polycistronic. Nat. 297: 243-246. M'Gonigle, R.H. 1941. Acute catarrhal enteritis of salmonid fingerlings. Trans. Am. Fish. Soc. 70: 297-303. Moewus-Kobb, L. 1965. Studies with infectious pancreatic necrosis virus in marine hosts. Annal. N. Y. Acad. Sci. 126: 328-342. Morgan, R. P. and Rasin, V. J. 1981. Temperature and salinity effects on development of striped bass eggs and larvae. Trans. Am. Fish. Soc. 110: 95-99. Moss, L. H. and Gravell, M. 1969. Ultrastructure and sequential development of infectious pancreatic necrosis virus. J. Virol. 3: 52-58. Munro, A. L. S., Liversidge, J., and Elson, K. G. R. 1976. The distribution and prevalence of infectious pancreatic necrosis virus in wild fish in Lock Awe. Proc. Roy. Soc. Edin. 75: 223-232. Nicholson, B. L. and Dexter, R. 1975. Possible interference in the isolation of infectious pancreatic necrosis virus from carrier fish. J. Fish. Res. Bd. Can. 32: 1437-1439. Nicholson, B. L. and Dunn, J. 1974. Homologous viral interference in trout and Atlantic salmon cell culture infected with infectious pancreatic necrosis virus. J. Virol. 14: 180-182.

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--------------------------99 Nicholson, B. L. and Pochebit, S. 1981. Antigenic analysis of infectious pancreatic necrosis virus (IPNV) by neutralization kinetics. Devel. Biol. Stand. 48: 3541. Nick, H., Cursiefen, D., and Becht, H. 1976. Structural and growth characteristics of infectious bursal disease virus. J. Virol. 18: 227-234. Okamoto, N., Sano, T., Hedrick, R. P., and Fryer, J. L. 1983. Antigenic relationships of selected strains of infectious pancreatic necrosis virus and eel virus european. J. Fish Dis. 6: 19-25. Okamoto, N., Taniguchi, N., Seno, Y., and Sano, T. 1984. The relationship between the change in quantities of infection pancreatic necrosis virus in infected rainbow trout fry and the disease process. Fish Path. 19: 1-4. Ozel, M. and Gelderblom, H. 1985. Capsid of symmetry of viruses of the preposed birnavirus group. Arch. Virol. 84: 149-161. Reed, L. J. and Muench, H. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27: 493497. Reno, P. w. 1976. Qualitative and quantitative aspects of the infectious pancreatic necrosis virus carrier state in trout. Ph.D. thesis. University of Guelph, Ontario. 152 p. Reno, P. w., Darley, S., and Savan, M. 1978. Infectious pancreatic necrosis: experimental induction of a carrier state in trout. J. Fish. Res. Bd. Can. 35: 1451-1456. Roberts, R. J., Bell, M., and Young, H. 1973. Studies on the skin of plaice (Pleuronected platessa). II. The development of larval plaice skin. J. Fish Biol. 5: 103108. Sakai, D. K. 1981. Heat inactivation of complements and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, goldfish and tilapia. Bull. Jap. Soc. Sci. Fish. 47: 565-571. Sano, T. 1971. Studies of viral diseases of japanese fishes. II. IPN of rainbow trout. pathogenicity of the isolates. Bull. Jap. Soc. Sci. Fish. 37: 499-503. Sano, T., Okamoto, N., and Nishimura, T. 1981. A new viral epizootic of Anguilla japonica. J. Fish Dis. 4: 127-139.

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100 Schutz, M., May, E. B., Kraeuter, J. N., and Hetrick, F. M. 1984. Isolation of infectious pancreatic necrosis virus from an epizootic occurring in cultured striped bass Merone saxatilis. J. Fish Dis. 7: 505-507. Seeley, R. J., Permutter, A., and Seeley, V. A. 1977. Inheritance and longevity of infectious pancreatic necrosis virus in the zebra fish, Brachydario rerio. Appl. Environ. Micro. 34: 50-55. Setzler, E. M., Boynton, W. R., Wood, K. V., Zion, H. H., Lubbers, L., Mountford, N. K., Frere, P., Tucker, L., and Mihursky, J. A. 1980. Synopsis of biological data on striped bass, Morone saxatilis. FAO Synopsis 121: 12-13. Silim, A., Elazhary, M. A. S. Y., and Lagace, A. 1982. Susceptibility of trouts of different species and orgins to various isolates of infectious pancreatic necrosis virus. Can. J. Fish. Aquat. Sci. 39: 1580-1585. Snieszko, S. F., Wolf, K., Camper, J.E., and Pettijohn, L. L. 1959. Infectious nature of pancreatic necrosis. Trans. Am. Fish. Soc. 88: 289-293. Sons tegard, R. A. and McDermott, L. A. 197 2. Epidemiological model for passive transfer of infectious pancreatic necrosis virus by homeotherms. Nat. 237: 104105. Sons tegard, R. A., Mc Der mot t, L. A., and Sons tegar d, K. S. 1972. Isolation of infectious pancreatic necrosis virus from white suckers, Catastomus commersoni. Nat. 236: 174-175. Sorimachi, M. and Hara, T. 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Path. 19: 231-238. Stephens, E. B. 1981. Isolation and molecular characterization of infectious pancreatic necrosis virus from "spinning" Atlantic menhaden in the Chesapeake bay. Ph.D. dissertation, Univ. of MD, College Park. 132 p. Stephens, E. B. and Hetrick, F. M. 1983. Molecular characterization of infectious pancreatic necrosis virus isolated from a marine fish. In: Crosa, J. E. (ed.), Bacterial and viral disease of fish. Wash. Sea Grant. Seattle. pp. 72-86. Stephens, E. B., Newman, M.W., Zachary, A. L., and Hetrick, F. M. 1980. A viral aetiology for the annual spring epizootics of Atlantic menhaden Brevoortia tyrannus in the Chesapeake Bay. J. Fish Dis. 3: 387-398.

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101 Swanson, R. N., Carlisle, J. C., and Gillespie, J. H. 1982. Pathogenesis of infectious pancreatic necrosis virus infectious virus in brook trout, Salvelinus fontinalis, following intraperitoneal injection. J. Fish Dis. 5: 449-460. Swanson, R. N. and Gillespie, J. H. 1979. Pathogenesis of infectious pancreatic necrosis in Atlantic salmon (Salmo salar). J. Fish. Res. Bd. Can. 36: 587-591. Swanson, R. N. and Gillespie, J. H. 1982. Isolation of infectious pancreatic necrosis virus from the blood and blood components of experimentally infected trout. Can. J. Fish. Aquat. Sci. 39: 225-228. Tatner, M. F. and Manning, J. H. 1985. The ontogenic development of the reticulo-endothelial system in rainbow trout, Salmo gairdneri. J. Fish Dis. 8: 189-195. Teninges, D., Ohanessian, A., Richard-Molard, C., and Contamine, D. 1979. Isolation and biological properties of drosphilia X virus. J. Gen. Virol. 42: 241-254. Thompson, s. w. 1966. Selected histochemical and histological methods. Charles c. Thomas. Springfield, MA. p. 24-160. Toranzo, A. E. and Hetrick, F. M. 1982. Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters. J. Fish Dis. 5: 223231. Tu, K. c., Spendlove, R. s., and Goede, R. w. 1975. Effect of temperature on survival and growth of infectious pancreatic necrosis virus. Inf. Immun. 11: 1409-1412. Underwood, B. o., Smale, C. J., Brown, F., and Hill, B.J. 1977. Relationship of a virus from Tellina tenuis to infectious pancreatic necrosis virus. J. Gen. Virol. 36: 93-109. Wedemeyer, G. A., Nelson, N. C., and Smith, C. A. 1978. Survival of the salmonid virus infectious hemorrhagic necrosis and infectious pancreatic necrosis in ozonated, chlorinated, and untreated water. J. Fish. Res. Bd. Can. 35: 87 5-879. Wolf, K. 1964. Characteristics of viruses found in fishes. Devel. Ind. Microb. 5: 140-148. Wolf, K. 1966. The fish viruses. Ad. Vir. Res. 12: 35101.

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--------------102 Wolf, K. 1972. Advances in fish virology: a review. Symp. Zoo. Soc. Lond. 30: 305-331. Wolf, K. 1976. Fish viral diseases in North American, 1971-75, and recent research of the eastern fish disease laboratory, u. S. A. Fish Path. 10: 135-154. Wolf, K. 1981. Viral diseases of fish and their relation to public health. CRC Handbook Series Zoo. B 2: 403-437. Wolf, K. and Quimby, M. C. 1969. Infectious pancreatic necrosis: clinical and immune response of adult trouts to inoculation with live virus. J. Fish. Res. Bd. Can. 26: 2511-2516. Wolf, K. and Quimby, M. C. 1971. Salmonid viruses: infectious pancreatic necrosis virus. Archiv. Fur. Dis. Ges. Vir. 34: 144-156. Wolf, K., Quimby, M. C., and Bradford, A. D. 1963. Egg associated transmission of infectious pancreatic necrosis virus of trouts. Virol. 21: 317-321. Wolf, K., Quimby, M. C., and Carlson, c. P. 1969. Infectious pancreatic necrosis virus: lyophilization and subsequent stability in storage at 4c. Appl. Micro. 17: 623-624. Wolf, K., Quimby, M. C., Carlson, C. P., and Bullock, G. L. 1968. Infectious pancreatic necrosis: selection of virus-free stock from a population of carrier trout. J. Fish. Res. Bd. Can. 25: 383-391. Wolf, K., Snieszko, S. F., Dunbar, C. E., and Pyle, E. 1960. Virus nature of infectious pancreatic necrosis in trout. Proc. Soc. Exp. Biol. Med. 104: 105-108. Wood, E. M., Snieszko, S. F., and Yasutake, W. T. 1955. Infectious pancreatic necrosis in brook trout. Arch. Path. 60: 26-28. Yamamoto, T. 1974. Infectious pancreatic necrosis virus occurrence at a hatchery in Alberta. J. Fish. Res. Bd. Can. 31: 397. Yamamoto, T. 1975a. Frequency of detection and survival of infectious pancreatic necrosis virus in a carrier population of brook trout (Salvelinus fontinalis) in a lake. J. Fish. Res. Bd. Can. 32: 568-570.

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103 Yamamoto, T. 1975b. Infectious pancreatic necrosis virus carriers and antibody produ c tion in a population of rainbow trout, Salmo gairdneri. Can. J. i 1icro. 21: 13431347. Yamamoto, T. and Kilistoff, J. 1979. Infectious pancreatic necrosis virus: quantification of carriers in lake populations during a 6-year period. J. Fish. Res. Bd. Can. 36: 562-567. Yu, K. K. Y., Macdonald, R. D., and Moore, A. R. 1982. Replication of infectious pancreatic necrosis virus in trout leukocytes and detection of the carrier state. J. Fish Dis. 5: 401-410.

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BIOGRAPHICAL SKETCH Sally J. Wechsler was born and raised in the Washington, DC, metropolitan area. She graduated from Walt Whitman High School (Bethesda, MD) and attended Michigan State University where she obtained a B.S. with high honors and a D.V.M. with honors. She practiced small animal veterinary medicine for five years. She then bought and operated a small animal veterinary clinic in Lakewood, CO, for five years. After selling the clinic, she entered a National Institutes of Health Laboratory Animal Medicine training program at the University of Florida. Concurrently, she started graduate school at the University of Florida where she obtained a M.S. in the School of Medicine, Department of Pathology. During this period she authored several publications concerning health matters in various animal species, including fish. Immediately after receiving her M.S., Sally began her doctoral research. Papers describing the results from the doctoral research have been published. 104

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Carter R. t, Chairman Associate sor of Forest Resources and Conservation I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. rome V. S ireman rofessor of Forest Resources and Conservation I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Linds~ M. Hutt-Fletcher t.. Associate Professor of Pathology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. I' ,.--...n "(_A_ i< ack M. Gas n ssociate Professor of Immunology and Microbiology

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly pres en tat ion and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Frank M. Hetrick Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. illipC McAllister U.S.F.W.S. Research Scientist This dissertation was submitted to the Graduate Faculty of the School of Forest Resources and Conservation in the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Ppilosophy. December 1986 Director, Forest Resources and Conservation Dean, Graduate School

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