IMPORTANCE OF INFECTIOUS PANCREATIC NECROSIS VIRUS
IN STRIPED BASS, Morone saxatilis
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
Sally Janet Wechsler
I want to thank Dr. G. L. "Pete" Bullock who made my
research project possible, for which I will be eternally
grateful. I am also grateful to Dr. C. P. Goodyear and
the U. S. Fish and Wildlife Emergency Striped Bass
Committee for providing the funding for the investigation.
Dr. R. Gregory deserves recognition as a gracious liaison
person. My appreciation also goes to Dr. F. M. Hetrick
whose laboratory performed the original viral isolation
and who has provided me with extremely helpful comments and
suggestions. My sincere thanks go to Dr. P. E. McAllister
in whose laboratory I worked, and whose endless patience,
goodwill, and scientific insights helped make it all come
together. I also want to acknowledge Dr. C. L. Schultz who
was very helpful in my orientation at Leetown (WV).
I sincerely acknowledge Dr. J. N. Kraeuter, Dr. L. C.
Woods and other Baltimore Gas and Electric Company
personnel who provided access to their facilities,
expertise, and waterfront living accommodations. My
appreciation also extends to R. Lucakovic, J. G. Boone, J.
H. Uphoff, D. Costen, and other personnel of the Maryland
Department of Natural Resources who provided me with
striped bass from the Chesapeake Bay. I also acknowledge
the generous assistance I received from all the people at
the U. S. F. W. S. National Fish Health Research
Laboratory, Kearneysville, WV. Special mention goes to W.
Owens, R. Owens, G. "Sonny" Wilson, B. Knott, W. B. Shile,
S. R. Phelps, and Drs. D. P. Anderson, K. Wolf, B. C.
Lidgerding, and R. C. Simon. Thanks also go to Dr. R. L.
Herman for providing training in fish histopathology, D.
Bowling for preparing the slides, and to Dr. E. B. May
of the University of Maryland School of Medicine-
Baltimore for assistance with the tissue processing and
histological examination. I want to thank Dr. S. W.
Pyle for help with gel electrophoresis. My thanks go to
Dr. G. R. Gilbert, who kindly agreed to be my major
professor, and also to the other committee members Drs.
L. M. Hutt-Fletcher, J. V. Shireman, and J. M. Gaskin.
TABLE OF CONTENTS
ACKNOWLEDGMENTS .. iii
LIST OF TABLES .viii
LIST OF FIGURES ix
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
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
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
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
LIST OF TABLES
1 Percent cumulative mortality in striped bass
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
7 Detection of virus-neutralizing antibodies in
fingerlings .. 54
8 Recovery of IPNV from plasma and buffy coat 56
9 Attempts to isolate IPNV from Chesapeake Bay
striped bass .. 62
10 Detection of virus-neutralizing antibody in
striped bass from the Chesapeake Bay 63
11 Striped bass tissues from which IPNV was isolated 64
12 Recovery of IPNV from steroid injected carriers 66
13 Virus titers in homogenates stored at different
temperatures .. 67
14 Recovery of IPNV from homogenates stored in
different types of containers 68
15 Recovery of IPNV from striped bass stored whole 69
16 Neutralization rates for three IPNV isolates 75
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
Sally Janet Wechsler
Chairman: Carter R. Gilbert
Major Department: Forest Resources and Conservation
Infectious pancreatic necrosis virus (IPNV), a pathogen
for Atlantic menhaden (Brevoortia tyrannus), was isolated
recently from striped bass fry (Morone saxatilis) in a
hatchery on the Chesapeake Bay (MD). The major goal of
this study was to investigate the effects of IPNV infection
in striped bass.
No clinical or histopathological signs of disease were
observed in striped bass exposed to IPNV by immersion or
intraperitineal injection. This was true even in IPNV-
exposed striped bass that were subjected to an abrupt drop
of pH or a temperature change. Chronic IPNV infection was
not detected in striped bass challenged with waterborne
virus; however, striped bass that consumed or were
inoculated with IPNV contained infectious virus for at
least eight months, despite the presence of circulating
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
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.
Early in this century, many North American trout
hatcheries experienced annual epizootics that resulted in
massive losses of young fry, affecting the fastest growing
individuals first (M'Gonigle, 1941). In 1955, Wood et al.
described microscopic lesions of pancreatic necrosis in
affected trout fry and also demonstrated that the
condition could be transmitted to fish located downstream
from affected fish. Wood et al. (1955) named the disease
infectious pancreatic necrosis (IPN). Although Wood et al.
(1955) speculated that the pathogenic agent was a virus,
the viral nature was not demonstrated until 1960 by Wolf et
al. Wolf and coworkers (1960) used filtered homogenates of
clinically affected trout to challenge fish and cell
cultures. Significant numbers of exposed fish died and
cytopathic effects (CPE) were apparent in inoculated fish
cell cultures. Electron microscopy revealed that IPNV is a
naked, icosahedral virus, between 55 75 um in diameter
(Moss & Gravell, 1969; Cohen & Scherrer, 1972; Kelly & Loh,
1972). The virus may exist also as tubular particles (Moss
& Gravell, 1969; Ozel & Gelderblom, 1985). The genome of
IPNV consists of two segments of double-stranded
ribonucleic acid (RNA); the molecular weight of one segment
is 2.5 x 106 and the other is 2.3 x 106 (Dobos, 1976;
Macdonald & Yamamoto, 1977). The latter segment encodes
the largest viral associated polypeptide, and the former
encodes the other proteins (Macdonald & Dobos, 1981;
Mertens & Dobos, 1982).
The viral polypetides of IPNV fall into three general
molecular weight classes--low, medium, and high (Cohen et
al., 1973; Dobos & Rowe, 1977; Chang et al., 1978). The
high molecular weight (90 105 x 103) polypeptide
corresponds to the polymerase, the enzyme that catalyzes
the synthesis of messenger RNA (Macdonald & Dobos, 1981;
Stephens & Hetrick, 1983). The capsid protein, the major
component, is of medium weight (50 57 x 103), and the
internal proteins are of low molecular weight (27 31 x
103) (Macdonald & Dobos, 1981).
Because the viral genome consists of two segments of
double stranded RNA, Dobos et al. (1979) proposed that IPNV
be classified a birnavirus. Included in this proposed
group are infectious bursal disease virus (IBDV), found in
young chickens (Nick et al., 1976); Drosophila X virus,
isolated from fruit flies (Teninges et al., 1979); and
Tellina virus and oyster virus, isolated from bivalve
molluscs (Hill, 1976; Underwood et al., 1977). Although
these viruses are similar morphologically and
biochemically, they can be distinguished serologically and
by comparison of the virion-associated proteins (Dobos et
al., 1979). None of the birnaviruses, except IPNV, have
been demonstrated to be pathogenic for fish.
In young trout IPNV infection may be manifested either
as acute death or by fish that exhibit brief episodes of
violent spinning after which the fish sink to the bottom of
the tank (Wolf, 1981). Death usually occurs within 1 2
days after onset of clinical signs. Upon necropsy, dead or
moribund fish may have multiple petechial hemorrhages on
the internal organs. Wolf (1981) considers the finding of
a clear to cloudy gelatinous material in the stomach and
anterior intestine to be pathognomonic for IPNV in young
trout. Histological lesions include necrosis of pancreatic
acini (Lightner & Post, 1969; Swanson et al., 1982) and
frequently, acute catarrhal enteritis (McKnight & Roberts,
1976). Considerable portions of the pancreas become
fibrotic in trout that survive IPNV infection (McKnight &
Roberts, 1976; Swanson et al., 1982).
The exact mechanisms by which IPNV causes death in
infected fish are not known (Hill, 1982). Correlation
between virus titers and severity of disease has been
reported (Okamoto et al., 1984). Virus titers
progressively rise in trout fry following challenge with
IPNV and the highest titers of virus are recovered from
fish that have died (Okamoto et al., 1984). Swanson and
Gillespie (1982) speculated that key events occurred within
the first few days following viral challenge. Using
experimentally infected Atlantic salmon (Salmo salar),
Swanson and Gillespie (1982) noted that peak viremia
occurred at day two. Swanson and Gillespie (1982) stated
that the Atlantic salmon, unlike trout, are successful, by
some unexplained mechanism, in preventing further increases
in viral titers, thus preventing IPNV-induced mortality.
For reasons yet to be determined, by six months of age
trout lose their susceptibility to IPNV-induced mortality
(Frantsi & Savan, 1971; Wolf, 1972). In addition, trout
species differ in their susceptibility to IPNV-induced
mortality (Hill, 1982; Silim et al., 1982). The resistance
may be mediated genetically. Wolf (1976) reported the
development of IPNV-resistant trout strains, using
Many different factors have been described that affect
the outcome of IPNV infection on trout. Frantsi and Savan
(1971) demonstrated that water temperature affects the
number of deaths associated with IPNV. The authors found
fewest deaths in viral-exposed trout fry kept at 4.50C,
most at 100C, and an intermediate number of deaths in fry
held at 150C. Also, as mentioned earlier, the age at which
fish are exposed to IPNV affects IPNV-induced disease
(Dorson & Torchy, 1981). Young fish (less than six months)
are more susceptible to IPNV-induced mortality, but older
trout do became subclinically infected with IPNV (Frantsi &
Stress was also found to influence IPNV infection,
especially in trout that survived early exposure but
continue to be infected. Frantsi and Savan (1971) found an
increase in IPNV isolation from trout survivors after an
episode of mild stress induced by low oxygen. McKnight and
Roberts (1976) reported 10 20% mortality in IPNV-carrier
rainbow trout (ages 6 to 11 months) at 72 hours following a
stressful event such as handling, transport, overcrowding,
or low oxygen. Higher IPNV titers were obtained from
stressed fish compared to titers from non-stressed fish
(McKnight & Roberts, 1976).
It is not known how IPNV persists in infected trout.
Normally IPNV multiplies intracytoplasmically and is
released by viral-induced cytolysis (Malsberger & Cerini,
1963; Argot & Malsberger, 1972). Defective interfering
particles are produced within cells, but do not cause cell
rupture (Nicholson & Dunn, 1974; Macdonald, 1978).
Therefore, this may be a mechanism by which IPNV persists
in carriers (Nicholson & Dexter, 1975; Hedrick et al.,
1978; Macdonald & Kennedy, 1979). Other researchers have
proposed a relationship between levels of virus-
neutralizing antibodies and titers of IPNV; i. e. fish with
high levels of IPNV-neutralizing antibody would have lower
titers of IPNV (Yamamoto 1975a, 1975b). However, there is
no correlation between the tissue levels of virus and
antibody titers in IPNV-carrier trout (Reno, 1976; Reno et
al., 1978). Another mechanism by which IPNV persists may
be due to an IPNV-induced decrease in the mitogenic
responsiveness of lymphocytes and macrophages (Knott &
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.,
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
In 1984, Schutz et al. reported the isolation of IPNV
from striped bass fry in a hatchery operated by the
Baltimore Gas and Electric, Co. Virus was recovered from
fry exhibiting erratic swimming behavior and high
mortality. Histological examination of moribund fry
revealed areas of necrosis in the epidermis. The virus was
isolated from kidneys taken from surviving striped bass at
three and six months following the original IPNV isolation.
Inflammation around pancreatic acini was observed in
histological sections taken from the survivors at three
months. This constellation of findings resembles that
found in salmonids in which IPNV causes death in young fry
and histopathological lesions in pancreatic acini of
infected fish. Thus, it was hypothesized that IPNV may
cause mortality in striped bass fry (Schutz et al., 1984).
Striped bass traditionally have been important both
as commercial and recreational fish (Morgan & Rasin, 1981);
however, the Chesapeake Bay stocks of striped bass have
been declining (Goodyear et al., 1985). The reasons for
this decline are not known, although many possibilities
have been suggested. These include loss of appropriate
habitat (Kerhehan et al., 1981), overfishing (Coutant,
1985), starvation of fry (Eldridge et al., 1981), pollution
(Hall et al., 1984), and temperature and oxygen levels
(Coutant, 1985). In addition, disease might be
contributing to the decline. "Spinning disease" can be
induced by IPNV in Atlantic menhaden (Stephens et al.,
1980) and a disease episode was occurring in menhaden in
the Chesapeake Bay at the time that IPNV was isolated from
the moribund striped bass fry. Records kept by the
Maryland Department of Natural Resources indicated a
correlation between large outbreaks of "spinning disease"
in menhaden and poor year classes of striped bass in the
Chesapeake Bay (Schutz et al., 1984).
Research was initiated to investigate the importance
of IPNV infection in striped bass. The points to be
specifically addressed were (1) whether IPNV induced
mortality in striped bass; (2) what histological lesions
developed in striped bass exposed to IPNV; (3) the
influence of age and strain of striped bass on IPNV
virulence; (4) the effect of water temperature on IPNV-
induced disease in striped bass: (5) the routes (both
vertical and horizontal) by which IPNV is transmitted in
striped bass; (6) the influence of stress, including abrupt
environmental changes and exogenous corticosteroids, on
IPNV infection in striped bass; (7) the humoral response of
striped bass to IPNV; (8) comparison of the striped
bass isolate of IPNV with a menhaden IPNV isolate and the
standard North American salmonid isolate (VR-299); and
(9) the effects of sample handling procedures on viral
MATERIALS AND METHODS
Cell Cultures and Virus Isolates
Chinook salmon embryo (CHSE-214) cells were grown at
180C in Eagle's minimal essential medium (EMEM) containing
10% fetal bovine serum (EMEM-10). For cell transfers,
confluent monolayers were dispersed with 0.25% trypsin.
For virus assays, cells were suspended in EMEM-10
containing antibiotics: 200 IU/ml penicillin and 200 ug/ml
streptomycin (PS). Cells were seeded into eight-well
culture plates and incubated at 180C in ambient air plus 2%
carbon dioxide (C02) until monolayers were confluent.
Isolates of IPNV
The striped bass isolate of IPNV (IPNV-Sb) that was
originally isolated from moribund striped bass fry (Schutz
et al., 1984), was used for all experiments, except where
noted. The virus was passage twice in CHSE-214 cells and
aliquots were stored at -700C.
Three other isolates of IPNV, the standard North
American isolate (VR-299), an isolate from Atlantic
menhaden (IPNV-M), and a European isolate (IPNV-Ab), were
handled as described for IPNV-Sb. Before use, aliquots of
virus were thawed and diluted in phosphate buffered saline
(pH 7.2; PBS).
Cultivation and Assays of IPNV
Preparation of Virus Stocks
Confluent monolayers of CHSE-214 cells grown in 75 cm2
flasks, were drained of medium and inoculated with IPNV
( < 0.01 plaque forming units [pfu] of IPNV per cell).
Following an one hour adsorption period at 150C (with
gentle agitation every 15 minutes), EMEM-10 was added to
the IPNV-inoculated cells. The virus-exposed cells were
incubated at 150C until the monolayers showed extensive
cytopathic effects (CPE) (usually 2 3 days). Cells and
culture fluid were harvested and centrifuged at 1500 x I
for 15 minutes at 40C. The supernatant liquid was stored
in 1 ml aliquots at -700C. The infectivity of stock virus
was determined by plaque assay as described below.
Virus Infectivity Plaque Assay
A modification of a virus infectivity assay (Moss &
Gravell, 1969) was used to determine virus titers.
Aliquots (0.1 ml) of each sample dilution were inoculated
onto duplicate wells of drained CHSE-214 monolayers. The
inoculated monolayers were incubated for 1 hour at 190C to
allow virus adsorption and then were overlayed with EMEM
containing 2% normal calf serum, 0.16 M Tris buffer and PS
(EMEM-2), plus 1% agarose. A second overlay, 2 ml of EMEM-
2 (without agarose), was added. Plates were incubated at
180C in ambient air plus 2% CO2 until cytopathic effects
(CPE) were noted. Cell sheets were fixed with 30% formalin,
and stained with 1% crystal violet in ethanol. Plaques
were counted and infectivity titer was calculated as pfu
per ml or pfu per g of tissue.
Virus Infectivity Assay
The simultaneous seeding method (McDaniel, 1979) was
used to detect IPNV in striped bass fry and striped bass
sex products. An aliquot (0.05 ml) of each sample dilution
was added to each of four wells of a 96-well tissue culture
plate and then 0.1 ml of CHSE-214 cells (about 2 x 105
cells/ml) was added to each well. Plates were incubated in
ambient air at 180C. If no CPE was observed by 5 days, the
sample was harvested from the wells and inoculated with
fresh cells (blind-passaged). If no CPE was noted after 5
additional days, the sample was considered to be negative
Characterization of IPNV-Sb
Purification of IPNV Isolates
Three isolates of IPNV, the striped bass isolate
(IPNV-Sb), the menhaden isolate (IPNV-M), and the North
American isolate (VR-299), were each purified using a
modification of a procedure previously described by Chang
et al. (1978). Confluent monolayers of CHSE-214 cells were
inoculated with virus ( < 0.01 pfu/cell). The virus was
allowed to adsorb for 1 hour at 150C, then EMEM-2 was
added. After 48 hours incubation at 150C, the cell sheets
showed extensive CPE. The cells and culture fluids were
centrifuged at 7000 x 2 for 20 minutes at 4C. The cell
pellet was resuspended in 5 ml of buffer made up of 0.01 M
Tris, 0.01 M sodium chloride, and 0.001 M disodium
ethylenediamine tetraacetate (TNE; pH 7.5). A equal volume
(5 ml) of trichlorotrifluoroethane (Freon) was added and
the solution was homogenized for two minutes at high speed.
The homogenate was centrifuged at 4500 x 2 for 15 minutes
at 40C. The top layer of TNE was removed and stored at
40C. An additional 5 ml of TNE were added to the Freon-
cell mixture. This solution was homogenized and
centrifuged as described above. The TNE layer was combined
with the first freon-extract. The original cell
supernatant fluid was adjusted to contain 6% (wt/v)
polyethylene glycol (M.W. 20,000), and 2.2% (wt/v) sodium
chloride. This mixture was stirred for 3 hours at 40C.
The solution was centrifuged at 9000 x q for 1 hour at 40C.
The supernatant liquid was discarded and the pellet was
resuspended in the TNE layer (5 8 ml) from the freon
extraction. This suspension was gently layered over a
sucrose or cesium chloride (CsC1) gradient.
For IPNV samples that were used to inoculate rabbits,
the crude virus preparation was purified on a linear
sucrose (10 50%) density gradient in Ultra-clear
centrifuge tubes (25 x 76 mm) that were centrifuged at
97,000 x q for 45 minutes at 40C. The virus band was
withdrawn by side tube puncture using a 20 gauge needle and
5 ml syringe. The virus band was diluted in TNE and
centrifuged at 83,000 x j for 40 minutes at 40C. The viral
pellet was resuspended in 1 ml TNE and stored at -700C.
Protein content was measured by the method described below.
For IPNV samples that were analyzed for virus specific
proteins, the crude virus preparation was purified on a
linear CsCl (20 to 40%) gradient in cellulose nitrate
centrifuge tubes (5/8" x 4") that were centrifuged at
115,000 x q for 16 hours at 40C. The virus band was
removed by side tube puncture with a 22 gauge needle and 5
ml syringe, diluted in TNE, and layered over a second CsCl
gradient. The band containing pure virus was removed,
dialyzed against TNE, and concentrated to 1 ml using
membrane microconcentrators. Protein concentration was
determined as described below and infectivity was
quantified by the plaque assay.
Determination of Protein Concentration
The Lowry method (Lowry et al., 1951), as modified by
Garvey et al. (1977), was used to determine the protein
concentration of the purified IPNV isolates (IPNV-Sb, IPNV-
M and VR-299). Bovine albumin was diluted (1 to 0.01
mg/ml) in phosphate buffered saline for protein standards.
One milliliter of a solution containing 2% sodium
carbonate, 0.02% cupric sulfate in 0.1 N sodium hydroxide
was added to 0.2 ml of each unknown and standard sample.
The samples were mixed, incubated for 10 minutes at 250C,
and then 0.1 ml of 1 N phenol reagent was added. After 1
hour incubation at 250C, the optical density of each sample
at 660 nm was determined by spectrophotometry. All samples
were assayed in duplicate. The mean optical density of
each standard was plotted against the protein
concentration. The protein concentration of the unknown
samples were calculated by interpolation from the standard
Electrophoresis of Viral Polypeptides
Comparison of the structural proteins of three IPNV
isolates (IPNV-Sb, IPNV-M, and VR-299) was performed by
examination of the banding pattern of the viral proteins in
discontinuous sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The Tris-glycine buffer method
described by Laemmli (1971) was used. A 10% resolving gel
was prepared by combining 13.3 ml of 30% acrylamide, 0.4 ml
of 10% sodium dodecyl sulfate, 10.0 ml of 18.5% tris-HCl
(pH 8.8), and 16.2 ml of distilled water. The solution was
degassed under vacuum for 15 to 30 minutes and 0.1 ml of
10% ammonium persulfate and 0.02 ml of N,N,N',N'
tetramethyl ethylenediamine (TEMED) were added. The
mixture was gently swirled and poured into the gel mold.
After polymerization (20 to 30 minutes), the gel was rinsed
with distilled water. The gel was overlayed with a buffer
containing 2.5 ml of 18.5% Tris-HCl (pH 8.8), 0.1 ml 10%
SDS and 7.4 ml distilled water, and allowed to stand
overnight. The 4% acrylamide stacking gel was made by
combining 1.3 ml of 30% acrylamide, 2.5 ml of 6% Tris-HC1
(pH 6.8), 0.1 ml of 10% SDS and 6.1 ml of distilled water.
This mixture was degassed as above; then 0.05 ml of 10%
ammonium persulfate and 0.005 ml of TEMED were added. The
solution was gently swirled and poured on top of the
resolving gel. After 20 minutes, the gel was rinsed with
distilled water. Total gel size was 1.5 mm x 14 x 16 cm.
Ten ug of each IPNV isolate were mixed with a solution that
contained 4% SDS, 20% glycerol, 10% 2-mercaptoethanol,
0.01% bromphenol blue, and 1.5% Tris-HCl. The sample
solution was heated to 1000C for three minutes, cooled to
40C, and loaded onto the gel. Running buffer (3.0 g Tris,
14.4 g aminoacetic glycine and 1 g SDS in one liter of
distilled water) was placed in the upper and lower
chambers. A direct current of fifteen mAmps was applied
until the bromphenol blue dye line passed through the
stacking gel. The current was then raised to 25 mAmps and
held constant until the dye line was 1 cm from the bottom
of the resolving gel. The gel was removed, put into a
solution of 50% methanol and 10% acetic acid for one hour,
and stained overnight in a solution of 0.01% coomassie
blue, 25% methanol and 10% acetic acid. The gel was
destined using a solution of 25% methanol and 10% acetic
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
Production of Antiserum to IPNV-Sb
Antibody to the striped bass isolate of IPNV (IPNV-Sb)
was prepared in New Zealand White rabbits. Purified IPNV-
Sb was diluted to give 1 mg/ml in PBS. Rabbits were
injected intravenously with 0.3 ml of this preparation.
The remaining 0.7 ml was mixed with an equal volume of
Freunds' incomplete adjuvant. Half of this mixture (0.7
ml) was injected intramuscularly; the remaining 0.7 ml was
injected subcutaneously into two different sites. The same
procedure was repeated at two week intervals, for one
month. Two weeks after the third boost, the rabbit was
exsanguinated. The blood was held overnight at 40C and
centrifuged at 1500 x g at 40C for 20 minutes. Serum was
collected, heated at 560C for 30 minutes to inactivate
complement, filtered using a membrane filter (0.45 micron
pore size), and stored in 1 ml aliquots at -700C.
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
Fingerlings weighing less than 5 grams were processed
as whole fish. Larger fish were dissected using sterilized
instruments and the internal organs, blood and feces were
assayed for virus. Samples were weighed and dissociated
using a sterile pestle and alundum. The resultant paste
was suspended 1:10 (wt/v) in PBS and centrifuged at 1500 j
for 30 minutes at 40C to sediment debris. Four serial
dilutions of supernatant fluid was assayed for infectious
virus using the plaque method.
Preparation of Striped Bass Blood for IPNV Assay
Striped bass blood samples were obtained from the
caudal vein, by venipuncture using a 20 to 22 gauge needle
or by severing the caudal peduncle. Blood was collected in
heparinized microhematocrit capillary tubes. Within 2
hours of collection, blood samples were centrifuged and
processed for virus assay as follows. The buffy coat
(about 1 ul) was cut from the microhematocrit tube at the
interface of the packed cells and plasma, placed into 0.5
ml sterile distilled water with PS, vigorously mixed, and
incubated at 190C for 1 hour. An additional 0.5 ml of PBS
was then added to give about a 1:1000 (v/v) dilution of the
buffy coat. An additional dilution (1:10) was made in PBS.
The plasma was expelled into 1.0 ml PBS and a second
dilution (1:10) made. These samples were immediately
assayed for IPNV using the plaque assay.
Detection of Virus-Neutralizing Antibody
Fish Blood Preparation for Neutralization Assay
Fish blood samples were obtained from the caudal vein
as described above and collected in heparinized
microhematocrit capillary tubes or in plain tubes. Tubes
were centrifuged and the supernatant fluid removed.
Because preliminary assays indicated that normal striped
bass blood neutralized IPNV at serum or plasma dilutions
less than 1:100, all striped bass blood samples were
assayed for virus-neutralizing antibodies at 1:100 or
greater dilution. After centrifugation of the blood
samples, 10 ul of fish plasma/serum were added to 1.0 ml
of PBS, heated at 450C for 30 minutes to inactivate
complement (Sakai, 1981), and stored at 4 or -200C.
Virus-Neutralizing Antibody Assay
To detect circulating virus-neutralizing antibodies,
serum or plasma samples are incubated with a known amount
of virus, and then the residual infectivity determined.
The following protocol was used for detection of IPNV-
neutralizing antibody in striped bass. Equal volumes of
fish serum or plasma sample and IPNV (1.6 x 103 pfu/ml)
were mixed, and, with periodic gentle agitation, incubated
at 190C for one hour. Residual infectivity was determined
by plaque assay. Total virus was determined by combining
equal volumes of test virus and PBS and measuring virus
infectivity. Blood samples were tested at the 1:100
dilution and were recorded as being positive for
neutralizing activity if the sample neutralized 50% or more
of total virus. Antibody titer was determined by testing
serial dilutions of plasma against a constant number of
virus and calculating the serum dilution that neutralized
50% of total virus (Reed & Muench, 1938). Unless otherwise
indicated, fish samples were tested only against the
striped bass isolate of IPNV (IPNV-Sb).
Striped bass fry from Maryland (Delmarva Ecological
Laboratories, Elkton, MD) were held at the Baltimore Gas
and Electric Company (MD) striped bass hatchery located on
the Chesapeake Bay (MD). All other experimental fish were
maintained at the U.S. Fish and Wildlife Service, National
Fish Health Research Laboratory (Kearneysville, WV).
Striped bass fry were obtained from Richmond Hill State
Fish Hatchery (GA) and Richloam Fish Hatchery (FL).
Striped bass fry were provided with brine shrimp (Artemia
salina) nauplii as live food.
Striped bass fingerlings obtained from Harrison Lake
National Fish Hatchery (VA) were maintained in 15 L tanks
that received 4 I/min of 220C spring water unless
otherwise noted. Striped bass fingerlings were fed
commercial salmon or trout food. Five-year-old striped
bass obtained from Edenton National Fish Hatchery (NC) were
kept in spring and reservoir water (12 250C). Rainbow
(Salmo gairdneri), brook (Salvelinus fontinalis) and brown
(Salmo trutta) trout were provided as forage.
Brook trout fingerlings were obtained from White
Sulfur Springs National Fish Hatchery (WV) and were held in
120C spring water and fed trout ration.
Virus Infection Studies
Waterborne IPNV Challenge of Striped Bass Fry
This series of virus challenge trials was conducted to
determine if IPNV would induce significant mortality in
striped bass. Several factors, including age at exposure,
strain of striped bass, water temperature, and
environmental stress, were investigated for their effect on
striped bass exposed to a static IPNV-bath.
Striped bass fry from Florida and Georgia were divided
into groups of 60 and held in 500 ml tissue culture
bottles containing spring water (190C). Florida fry were
challenged with IPNV at 1, 3, 5, 7, 10 and 15 days post-
hatch. Georgia fry were exposed to IPNV at 5, 7 and 10
days post-hatch. For each strain and age group, IPNV was
added to three bottles to give 106 pfu/ml of water. A
similar volume of PBS was added to three control bottles.
After 6 hours, and daily thereafter for three weeks, half
of the water in each bottle was replaced, debris was
removed, and newly hatched brine shrimp were provided as
forage for the striped bass fry. All dead fish were
removed, stored at 4 or -200C, and assayed for IPNV using
the simultaneous seeding procedure. The length of storage
varied from 1 90 days.
Maryland strains of striped bass fry (Chesapeake and
Delaware Canal [C & D] and Nanticoke [NAN] River) were
divided into groups of 30 fry that were placed in 200 ml
culture bottles containing Chesapeake Bay estuarine water
(18 230C). The C & D canal striped bass fry were
challenged with IPNV at 1, 5, 15 and 20 days post-hatch.
The NAN striped bass fry were exposed to IPNV at 10, 15 and
20 days post-hatch. The challenge protocol and daily care
were as described above, with the exception that daily water
changes replaced 75% (instead of 50%) of the water. Dead
striped bass were stored for 0 3 days at 40C prior to
being assayed using the simultaneous seeding method. At
the end of three weeks, 98% of the surviving striped bass
were assayed for virus. Remaining survivors were assayed
for virus and virus-neutralizing antibody six months after
To test the effects of an abrupt shift in pH on
mortality in IPNV-exposed striped bass fry, five-day-old
striped bass fry (C & D strain) were challenged with IPNV
and maintained as described above. The only difference
occurred on day five after viral exposure when 50% of the
water (pH 7.1) was replaced with water to which sulfuric
acid had been added to bring the pH of the water to pH 6.3.
After 24 hours, the acidified water (now pH 6.5) was
replaced with ambient water. Fish that died were collected
daily for three weeks and immediately assayed for virus
using the simultaneous seeding method.
Waterborne IPNV Challenge of Striped Bass Fingerlings
Twenty-six-day-old striped bass were divided into 12
groups of 50 fish each. Six groups were kept at 120C, and
six were kept at 220C. Water flow to all tanks was stopped
for six hours, and the water aerated. At each temperature,
two tanks were seeded with 104 pfu of IPNV per ml water,
two tanks received phosphate buffered saline (sham
controls), and two tanks of striped bass served as
treatment controls. Dead fish were collected twice daily
for three weeks and stored at -200C until assayed for virus
using the plaque assay. Samples were stored for 7 240
Six-month-old striped bass fingerlings were assigned
to three groups of 12 striped bass. One group of striped
bass was exposed to a 6-hour static immersion in 106 pfu of
IPNV per milliliter of water. Phosphate buffered saline
was added to the 6-hour static bath of the sham control
group. The treatment control group of striped bass
underwent six hours of static bath. Tanks were checked
daily for mortality. At four weeks post exposure, striped
bass were assayed for IPNV and for virus-neutralizing
Virus Inoculation of Striped Bass Fingerlings
An alternative method of IPNV exposure was used for
striped bass fingerlings two months and older. Rather than
being immersed in IPNV, each fish received an
intraperitoneal (i.p.) injection with IPNV. For all
injection and sampling procedures, striped bass were
anesthetized with tricaine methanesulfonate (MS-222).
Striped bass, 60, 90, 120, 150 and 180 days old, were
placed into groups of 50 fish. At each age, one group of
striped bass received an injection of 0.05 ml of PBS
containing 0, 103, 105, or 106 pfu of IPNV. Treatment
controls were anesthetized and returned to the tank. Dead
fish were collected daily for four weeks, and stored at
-200C for 1 7 days until they were assayed for infectious
virus. At monthly intervals, survivors were bled for
virus-neutralizing antibody, and tissues were assayed for
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.
The histology of IPNV-infected striped bass was
examined. Striped bass fingerlings were selected at
monthly intervals after IPNV injection, incised along the
ventral abdomen, and immersed in Bouin's fluid fixative
(Luna, 1968). Fingerling tissues were embedded in
paraffin. Striped bass fry (3 6 per day) were fixed in a
solution of formalin and glutaraldehyde (4:1) and embedded
in hydroxyethyl methacrylate. Blocks were sectioned at 4 -
6 microns, stained with hematoxylin, eosin and phloxine
(Thompson, 1966) and examined by light microscopy.
Virus Transmission Studies
Oral Transmission of IPNV to Striped Bass
This study was conducted to ascertain if striped bass
could become infected with IPNV by consuming IPNV-
containing food. Six-month-old striped bass were tagged
and placed into four tanks. Each tank contained six
striped bass. Three-month-old brook trout, each harboring
102 104 pfu of IPNV, were added to the tanks. Each
striped bass was observed to consume one or two trout.
Striped bass that did not eat brook trout were removed from
the experiment. For six months, the striped bass were
assayed periodically for the presence of IPNV and virus-
Vertical IPNV Transmission in Striped Bass
A series of experiments was conducted to investigate
if vertical transmission of IPNV occurred in striped bass.
To determine if striped bass that survived a natural IPNV
episode actually shed IPNV in sex products, the following
study was conducted. A population of two-year-old striped
bass from which IPNV was originally isolated (Schutz et
al., 1984) was sampled. Milt was manually expressed from
males; however, since striped bass females mature at the age
of three plus years (Setzler et al., 1980), eggs were not
available. Because preliminary results showed that IPNV can
be recovered from striped bass kidney and, therefore, might
be shed in the urine; urine was manually expressed and
collected from sexually immature striped bass. Fourteen
urine and 20 milt samples were tested for the presence of
IPNV using the simultaneous seeding assay. Samples were
processed within two hours of collection.
To determine if IPNV-infected striped bass transmit
IPNV in their sex products, five-year-old striped bass were
injected i.p. with 106 pfu of IPNV in December 1984, and
spawned in April and May 1985. Samples of sex products,
fertilized eggs and offspring were assayed for IPNV using
the plaque method.
To investigate whether IPNV-infected striped bass sex
products would result in IPNV-infection of the offspring,
sex products were collected from spawning striped bass
adults caught in the Nanticoke River (MD). Subsamples of
the eggs and milt were tested for the presence of IPNV.
The remaining portions of eggs and milt were used to
produce fertilized eggs. Eggs were dipped once in clean
water and mixed with milt for fertilization. Additional
water was added to the fertilized eggs and the mixture was
placed in buckets and aerated. Striped bass fry hatched 2
days later. Treatment groups included (1) virus-exposed
eggs plus virus-free milt--eggs were briefly mixed with
virus (106 pfu/ml final IPNV concentration) before being
dipped in water and then fertilized; (2) virus-free eggs
plus virus-exposed milt--sperm was mixed with IPNV (106
pfu/ml final concentration of IPNV) and added to the eggs;
and (3) treatment controls--virus-free eggs were mixed
with virus-free milt. Periodically, fertilized eggs and
fry were tested for the presence of IPNV using the
simultaneous seeding assay.
Transmission of IPNV from Striped Bass to Brook Trout
This experiment was performed to ascertain whether
IPNV could be transmitted from IPNV-infected striped bass
to brook trout located downstream from the striped bass.
Brook trout were utilized in this study because they are
extremely susceptible to IPNV infection (Silim et al,
1982). Four-month-old striped bass were inoculated with
106 pfu of virus. At two months post inoculation, the
internal organs from three IPNV-infected striped bass, and
fecal samples from five fish were assayed for IPNV.
Fifteen IPNV-infected striped bass were placed in a tank.
Water (120C) from the tank containing striped bass flowed
into a tank that contained 20 seven-month-old brook trout.
Every two weeks, 3 4 trout were assayed for IPNV using
the plaque assay.
Humoral Response of Striped Bass to IPNV
Early IPNV Titers and Neutralizing Antibody
The tissue levels of IPNV and circulating virus-
neutralizing antibodies during the first ten days of IPNV
infection were monitored in four-month-old striped bass
fingerlings inoculated i.p. with 106 pfu of IPNV. For ten
days, 3 4 fish daily were exsanguinated from the severed
caudal peduncle and dissected. The kidneys, spleen,
intestines, feces, and buffy coat were assayed for IPNV.
Titers of virus-neutralizing antibody were measured in the
blood samples from individual or pools of two fish.
Exogenous Steroids and Levels of Neutralizing Antibody in
Striped Bass Challenged with IPNV
One investigation was conducted to determine the
effect of exogenous corticosteroids on the development of
viremia and virus-neutralizing antibodies in IPNV-
inoculated striped bass. Striped bass yearlings were
weighed, had a blood sample taken, and were divided into
four groups of six fish each. The treatment groups were
(1) sham control--fish were given two i.p. injections of
PBS 24 hours apart; (2) steroid control--fish were
injected i.p. with the corticosteroid triamcinolone
acetomide (100 mg/kg) followed 24 hours later with an i.p.
injection of PBS; (3) virus control--fish were given a
single i.p. injection with 107 pfu of IPNV; and (4) steroid
+ virus--fish were injected i.p. with steroid (100 mg/kg)
24 hours before i.p. inoculation with 107 pfu of IPNV.
Half of the fish in each group were bled at 3 days post
inoculation (dpi) and weekly thereafter for five weeks.
Fish in the other half of each group were bled at 7 dpi and
weekly thereafter, for five weeks. Blood plasma and
leukocytes were assayed for IPNV. Levels of circulating of
virus-neutralizing antibody were also measured.
Another study was conducted to determine if exogenous
steroids affected levels of virus-neutralizing antibodies
in IPNV-carrier striped bass. Striped bass fingerlings
were inoculated i.p. with 105 pfu of IPNV. Eleven months
later, the fish were weighed, bled, and injected i.p. with
triamcinolone acetomide (100 mg/kg). Striped bass were
bled twice weekly for three weeks. Titers of IPNV-
neutralizing antibody were determined.
Antibody Response of Striped Bass to Second IPNV Challenge
The purpose of this study was to investigate the
humoral response of IPNV-inoculated striped bass that were
given a second exposure to IPNV, either by injection or by
immersion challenge. For one part of this experiment,
yearling striped bass were given an i.p. inoculation
containing 107 pfu of IPNV. Blood samples were taken twice
weekly for five and one half weeks. The fish were allowed
to rest for five weeks prior to the second i.p. injection
with IPNV. Three months after the first virus injection,
the striped bass received an i.p. inoculation of 106 pfu of
IPNV. Blood samples were taken twice weekly for three
weeks, and periodically for nine additional weeks. Levels
of virus-neutralizing antibody were measured.
In a second part of the experiment, IPNV-inoculated
striped bass were given a second IPNV challenge by the
waterborne route. Five-month striped bass fingerlings
were given i.p. inoculation with 105 pfu of IPNV. Fourteen
months later, a blood sample was taken from these fish.
The fish were immersed for 5 minutes in water containing
105 pfu of IPNV per ml. Blood samples were taken twice
weekly for three weeks and assayed for levels of virus-
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
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-
Sampling Adult Striped Bass
Adult striped bass were caught in gill nets located in
the Chesapeake Bay. Kidneys, spleen, gonads, and
intestines were excised, placed in sterile plastic bags,
and stored at 40C for 1 3 days prior to assay for IPNV.
Blood samples were collected from the caudal vein in
sterile glass tubes and the serum tested for virus-
neutralizing activity against both IPNV-Sb and IPNV-Ab.
Procedures that Affect IPNV Recovery
Tissue Site of IPNV in Striped Bass
When monitoring fish populations for IPNV, tissue
samples should be taken from which virus can be recovered
most frequently. An investigation was conducted to
ascertain which striped bass tissues harbor IPNV.
Individual organs, fat, feces, and blood, were removed from
IPNV-infected striped bass, and assayed for infectious
Storage Conditions of IPNV-infected Homogenates
The liability of the striped bass isolate of IPNV
(IPNV-Sb) in homogenates of IPNV-infected striped bass
was studied. Pools of internal organs from IPNV-inoculated
striped bass were homogenized and clarified as previously
described. Aliquots of the supernatant fluid were placed
in sterile glass vials and stored at 4, -20 or -700C.
An experiment was conducted to investigate whether the
type of container in which the homogenate of the internal
organs from IPNV-infected striped bass was stored altered
the amount of IPNV detected. The tissue homogenate from
individual IPNV-carrier striped bass was divided into 7
aliquots. One aliquot was assayed immediately for IPNV
using the plaque method. Three aliquots were stored in
plastic bags and three were stored in glass vials. Two
aliquots (one in glass vial, one in plastic) from each fish
homogenate were stored at each of three temperatures (4,
-20 and -700C) prior to virus assay.
Storage Temperature of Whole IPNV-Infected Striped Bass
Sampling fish for IPNV frequently involves taking
whole fish or tissue samples in the field and storing the
samples until they can be assayed for virus. An
investigation was conducted to determine the effect of
different storage temperatures on recovery of infective
IPNV from virus-infected striped bass. For this
experiment, IPNV-infected striped bass were placed in
individual plastic bags and stored at either 4, -20 or
-700C for two to fourteen days. After storage, frozen fish
were allowed to soften at 40C, and then the internal organs
from all fish were excised, and assayed for virus
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
Virus Infection Studies of Striped Bass
A series of IPNV challenge trials was conducted to
determine if IPNV induces increased mortality in virus-
exposed striped bass. When 1- to 20-day-old striped bass
from four different strains were immersed in IPNV the
resulting mortality was not significantly different from
that of the unchallenged controls (p < 0.01, analysis of
variance [ANOVA]). Even when five-day-old IPNV-exposed fry
were subjected to an abrupt drop in pH (0.8 units), no
statistical difference was observed in the mortality of
control and IPNV-challenged fry. Mortality in different
trials was unpredictable, but within a trial the pattern of
mortality of virus-challenged and control fish were not
significantly different (Figure 1). Virus was recovered
from virus-exposed fish that died but was never isolated
from control fish. When survivors were assayed for virus
three weeks post-challenge, IPNV was recovered only from
fry that had been challenged at one day post-hatch (data
not shown). Virus was not recovered any of the surviving
fish six months after waterborne challenge.
Twenty-six-day-old striped bass exposed to IPNV by
immersion and held at 12 or 220C exhibited no difference in
mortality compared to control groups (p < 0.01) (Figure 2).
Figure 1: Percent daily mortality of striped bass fry
during the 21 days following exposure to 10 plaque
forming units of infectious pancreatic necrosis virus
(IPNV) per milliliter of water ( E-I ) or to
phosphate buffered saline ( +--- ). Sixty or 180
striped bass were exposed to virus in each trial. There
was no significant difference between the mortality in
IPNV-exposed and unchallenged striped bass (p < 0.01;
tested by analysis of variance). Results from
representative trials are presented.
70 -r .. .. -. .... .. ... .......-.
0 4 8 12 16 20
DAYS POST CHALLENGE
Figure 1 A. Chesapeake and Delaware Canal (MD) striped
bass fry were exposed to IPNV at one day post-hatch.
0 4 8 12 16 20
DAYS POST CHALLENGE
Figure 1 B. Chesapeake and Delaware Canal striped bass
fry were exposed to IPNV at five days post-hatch.
0 4 8 12 16
DAYS POST CHALLENGE
Figure 1 C. Florida striped bass fry were exposed to
IPNV at seven days post-hatch.
0 4 8 12 16
DAYS POST CHALLENGE
Figure 1 D. Georgia striped bass fry were exposed to
IPNV at ten days post-hatch.
0 4 8 12 16 20
DAYS POST CHALLENGE
1 E. Nanticoke River (MD) striped bass fry were
to IPNV at 15 days post-hatch.
0 4 8 12 16 20
DAYS POST CHALLENGE
Figure 1 F. Nanticoke River striped bass fry were
exposed to IPNV at 20 days post-hatch.
Figure 2: Percent daily mortality of striped baas
fingerlings exposed at 26 days post-hatch to 10 plaque
forming units of infectious pancreatic necrosis virus
per milliliter of water ( 0-0 ). Sham controls ( ---+ )
were exposed to virus-free phosphate buffered saline
(PBS). Treatment controls ( x--x ) were held for four
hours in a static, aerated bath without PBS or virus.
Each experimental group contained 100 striped bass.
There was no significant difference between mortality of
virus-exposed and nonexposed controls (p < 0.01; tested
by analysis of variance) at either temperature.
0 2 4 6 8 10 12 14 16 1
DAYS POST EXPOSURE
Figure 2 A. Striped bass fingerlings were held at 120C.
0 2 4 6 8 10 12
DAYS POST CHALLENGE
Figure 2 B. Fingerlings were held at 220C.
14 16 18
In addition, no virus was recovered from any fish that
died. No deaths occurred in six-month-old striped bass
that were challenged with IPNV by immersion and no virus
was recovered from any of these fish.
Mortality did not increase in striped bass fingerlings
that were given IPNV by intraperitoneal (i.p.) inoculation
at either 60, 120, 150 and 180 days post-hatch (Table 1).
Even among IPNV-injected striped bass that underwent an
abrupt 100C change in water temperature, mortality was not
significantly different from that of controls. Virus was
recovered from IPNV-injected fish that died but was not
isolated from any controls (Table 2). At one month after
IPNV injection, virus titers of survivors were similar to
those of IPNV-inoculated striped bass that died during the
first month after injection (Table 3). Even levels of IPNV
in virus-inoculated striped bass subjected to changes in
water temperature were not significantly different
(p < 0.01, ANOVA) (Table 4). Virus was isolated from
surviving IPNV-inoculated striped bass for 14 months post-
inoculation (Table 3). Circulating IPNV-neutralizing
antibody was found in more than 75% of the IPNV-carrier
striped bass tested during the 14 months after IPNV
exposure. No IPNV-induced histological lesions were
observed in any sections examined from IPNV-exposed striped
bass, regardless of age or route of exposure.
Table 1: Percent cumulative mortality in striped bass
fingerlings following intraperitoneal injection of
infectious pancreatic necrosis virus (IPNV).
CONTROL VIRUS INOCULUM (pfu)
AGEa MENT 103 105 10
60 NDb 48c 38 40 ND
90 28 26 24 26 14
120 20 18 2 16 14
150 0 0 0 2 0
180 8 0 0 0 0
aAt the indicated days post-hatch, groups of 50 striped
bass were anesthetized and given intraperitoneal (i.p.)
injections containing the indicated plaque forming units
(pfu) of IPNV. Treatment controls were anesthetized
only. Sham controls were injected with phosphate
buffered saline (virus diluent). Fish were maintained
cPercentage of striped bass that died in the 28 days
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)
AGEa MENT 103 105 106
60 NDb NVc 102 103-105 ND
90 NV NV NV NV 102-104
120 NV NV NV 103-106 105-107
150 _e 105
180 NV -
aAt the indicated days post-hatch, striped bass were
given intraperitoneal (i.p.) injections of the indicated
plaque forming units (pfu) of infectious pancreatic
necrosis virus (IPNV). Treatment controls were
anesthetized only. Sham controls were injected i.p.
with phosphate buffered saline (virus diluent). Fish
were maintained at 220C. Dead fish were assayed for
virus using the plaque assay me hod that detected titers
greater than or equal to 5 x 10 pfu/g.
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.
Table 3: Range of virus titers in striped bass
fingerlings surviving intraperitoneal injection of
infectious pancreatic necrosis virus.
VIRUS INOCULUM (PFU)a
103 105 106
aStriped bass fingerlings received an intraperitoneal
inoculation with the indicated plaque forming units
(pfu) of infectious pancreatic necrosis virus (IPNV).
Fish were maintained at 220C.
bMonths following intraperitoneal injection that
surviving striped bass were assayed for IPNV using the
plaque method that detected titers equal to or greater
than 5 x 10 pfu/g.
CNo IPNV was recovered from surviving fingerlings.
dRange in IPNV titers (pfu/gram of tissue) in striped
bass fingerling survivors that were assayed for virus.
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.
22 --> 12
12 --> 22
2.7 X 104
2.9 X 104
3.9 x 104
3.0 x 104
7.2 x 104
1.2 x 105
4.0 x 104
9.7 X 104
2.9 X 104
4.5 x 104
1.4 x 104
3.2 x 104
2.0 x 106
5.0 x 106
6.4 x 105
9.8 x 105
2.3 X 105
4.9 x 103
1.1 x 106
2.2 x 105
1.9 x 104
1.9 x 104
2.8 x 104
4.8 x 103
2.0 x 105
1.2 X 105
2.6 x 104
3.3 x 104
8.2 x 103
9.2 x 104
8.0 x 103
2.1 x 104
3.9 x 104
aSix-month-old striped bass were given an intraperitoneal
injection of 10 plaque forming units (pfu) of
infectious pancreatic necrosis virus (IPNV). Fish were
held at either 120 or 220C. Two weeks after the IPNV
inoculation, some fish were transferred into water of a
higher or lower temperature. Two weeks after the
temperature change, fish were assayed for IPNV using the
plaque method that detected titers equal to or greater
than 5 x 10 pfu/g. Virus titers were expressed as pfu
per gram of tissue.
bNo virus was detected.
cMean IPNV titer for each group. Group means were not
significantly different (p < 0.01) as determined by
analysis of variance.
dStandard deviation of mean IPNV titer.
Transmission Studies of IPNV in Striped Bass
Oral Transmission of IPNV to Striped Bass
To demonstrate that IPNV can be transmitted to
striped bass by contaminated forage, six-month-old striped
bass were allowed to consume brook trout carrying between
102 104 pfu of IPNV. Virus was recovered from apparently
healthy striped bass eight months after exposure (Table 5).
Virus-neutralizing antibody was detected in all striped
bass that consumed IPNV-infected brook trout.
Vertical Transmission of IPNV in Striped Bass
To determine if striped bass survivors from natural
IPNV infection shed virus in their urine or milt, samples
were taken from the population of two-year-old striped bass
from which IPNV had originally been isolated (Schutz et
al., 1984). No IPNV was detected in any of the urine and
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
1 101 1
2 101-103 3
3 101-103 2
4 101-103 2
12 101-102 2
33 101 1
aSix-month-old stripeI bass were fed brook trout, each
of which contained 10 104 plaque forming units (pfu)
of infectious pancreatic necrosis virus (IPNV). At the
indicated weeks after virus ingestion, striped bass were
assayed for IPNV by the plaque method.
bMagnitude of titer expressed as pfu of IPNV per gram of
striped bass tissue.
Table 6: Recovery of infectious pancreatic necrosis
virus (IPNV) from samples taken during investigations of
vertical transmission of IPNV in striped bass.
SAMPLES VIRUS RECOVERED
Adults inoculated with IPNVa
Internal Organs Yes
Sex Products (Eggs and Milt) No
Fertilized Eggs No
Sex Products No
IPNV added to Eggsc
Fertilized Eggs Yes
IPNV added to Miltd
Fertilized Eggs 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
bSex products (eggs and milt) were obtained from
spawning striped bass caught in the Chesapeake Bay (MD).
Homogenates of eggs, milt, fertilized eggs, and larvae
were assayed for IPNV using the simultaneous seeding
method. Samples were considered positive for IPNV if
cytopathic effects (CPE) was observed during two blind
passages. If no CPE developed, the sample was recorded
to be negative.
cEggs were exposed to 105 pfu of IPNV/ml immediately
prior to mixing with virus-free milt.
dMilt was exposed to 105 pfu of IPNV/ml immediately
prior to mixing with virus-free eggs.
fertilized eggs when virus-exposed eggs were fertilized
with virus-free milt. None of the offspring started to
feed and all died within three weeks.
Transmission of IPNV from Striped Bass to Brook Trout
To determine whether IPNV-infected striped bass shed
sufficient virus to infect susceptible fish located
downstream, brook trout were placed below IPNV-infected
striped bass whose feces contained 104- 105 pfu/g. One of
four trout tested after six weeks had 102 pfu of IPNV/g of
pooled internal organs. Virus was not recovered from trout
tested at two, four and eight weeks of IPNV-exposure.
Humoral Response of Striped Bass to IPNV
Early Humoral Response to IPNV Challenge
To monitor early levels of IPNV and circulating virus-
neutralizing antibodies in striped bass, four-month-old
striped bass were inoculated i.p. with 106 pfu of IPNV and
3 4 fish were assayed each day for 10 days. Titers of
virus remained relatively constant during the first ten
days (Table 7) and were of the same magnitude as IPNV
titers in IPNV-inoculated striped bass tested two months
after injection (Table 3). Virus-neutralizing antibody was
first detected seven days post inoculation (dpi) (Table 7).
Table 7: Detection of infectious pancreatic necrosis
virus (IPNV) and virus-neutralizing antibody in IPNV-
injected striped bass fingerlings.
DPIa IPNVb TITERc ANTIBODYd TITERe
aDays post injection (intraperitoneal)
forming units (pfu) of IPNV.
of 106 plaque
bNumber of four-month-old striped bass that had IPNV
in their tissues per number of fish assayed for virus
virus using the plaque method.
CRange in IPNV titer (pfu per gram of tissue).
dNumber of blood samples (diluted 1:100) that
neutralized more than 50% of total IPNV plaques per
number of blood samples tested.
eRange in titer of IPNV-neutralizing antibody.
Effect of Steroids on Titers of Circulating Virus and
The effect of exogenous steroid on viremia and on the
development of virus-neutralizing antibody was investigated
using yearling striped bass that received an i.p. injection
of steroid 24 hours prior to receiving an i.p. injection
with IPNV. Blood samples were taken weekly from individual
fish. Viremia was detected for two weeks in IPNV-
inoculated striped bass that had received steroid (Table
8), but for only one week in IPNV-inoculated striped bass
that did not receive steroid. Virus was recovered more
frequently from the buffy coat leukocytess) than from the
plasma (Table 8). Circulating IPNV-neutralizing antibody
was first detected at 10 dpi in IPNV-inoculated fish that
received steroid (Figure 3) compared to 7 dpi in IPNV-
inoculated fish not treated with steroid (Figure 3).
Levels of virus-neutralizing antibody in the IPNV-injected
striped bass treated with steroid were generally lower than
those in virus-injected fish that did not receive steroid.
Also, antibody titers peaked later (about 4 weeks post
inoculation) in steroid treated fish compared to a peak at
about 3 weeks in virus-injected striped bass that did not
receive steroids. A noticeable, but not statistically
significant, difference in antibody titers was observed
between the two groups IPNV-injected striped bass that did
not receive steroid. Striped bass that were bled at three
dpi and weekly thereafter (Figure 3a) had somewhat higher
Table 8: Recovery of infectious pancreatic necrosis virus
(IPNV) from the plasma and buffy coat of virus inoculated
striped bass fingerlings.
TREAT- DAYS POST INJECTIONa
MENT SAMPLE 3 7 10 14 17 21
Steroid + buffy coat 3/3c 3/3 0/3 1/3 0/3 0/3
IPNVb plasma 1/3 0/3 0/3 0/3 0/3 0/3
IPNVd buffy coat 1/3 3/3 0/3 0/3 0/3 0/3
plasma 0/3 0/3 0/3 0/3 0/3 0/3
Controls buffy coat 0/3 0/3 0/3 0/3 0/3 0/3
plasma 0/3 0/3 0/3 0/3 0/3 0/3
aStriped bass fingerlings received 107 plaque forming
units (pfu) of IPNV or phosphate buffered saline by
intraperitoneal (i.p.) inoculation. Blood samples,
taken at the indicated days after IPNV injection, were
assayed for IPNV using the plaque assay.
b Striped bass fingerlings that received an i.p.
injection with triamcinolone acetomide (100 mg/kg) 24
hours before i.p. inoculation with IPNV.
cNumber of fish that were positive for IPNV per
number of fish tested using the plaque assay.
dStriped bass fingerlings that received only IPNV
by i.p. injection.
eStriped bass fingerlings that did not receive an
injection of IPNV, but were injected i.p. with either
PBS or steroid and PBS.
Figure 3: Titers of virus-neutralizing antibody in
striped bass fingerlings that received an intra-
peritoneal inoculation with 10 plaque forming units
(pfu) of infectious pancreatic necrosis virus (IPNV).
Fingerlings were injected with phosphate buffered saline
(PBS) ( O ) or with triamcinolone acetomide
(100 mg/kg) ( 0 ) 24 hours prior tp viral inoculation.
Antibody titers are expressed as 10' the serum dilution
that neutralized 50% of total IPNV (about 80 plaques per
well). The mean antibody titer for striped bass that
received PBS is indicated by + and by for fish that
0 2 4 6
WEEKS POST CHALLENGE
Figure 3 A.
Fish that were sampled at weekly intervals
three days after IPNV inoculation.
WEEKS POST CHALLENGE
Figure 3 B. Striped bass that were sampled
intervals after IPNV injection.
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
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.
?- 200 -
1 2 3 4 5 6 11 12 13 14 15 16 19 20 21 23
Figure 4: Virus-neutralizing antibody titers in striped
bass fingerlings injected with infectious pa ncreatic
necrosis virus (IPNV). Striped bass received 10 plaque
forming units (pfu) of IPNV by intraperitoneal (i.p.)
inoculation on day 0 ( 3 ). Control fish received an
i.p. injection with phosphate buffered saline ( S3 ) on
day 0. A second IPNV challenge ( J, ) was given at 11
weeks after the first injection. Control striped bass
were injected i.p. with IPNV at week 11. Virus-
neutralizing antibody titers are expressed as 10-2 the
dilution of fish serum that neutralized 50% of the total
viral plaques (about 80 plaques per well). Each bar
represents the mean (n = 1 to 6) virus-neutralizing
antibody titer of fish.
Survey of Chesapeake Bay Striped Bass
To determine whether wild striped bass have been
exposed to IPNV, Chesapeake Bay (MD) striped bass of
various ages were sampled. The tissues from some were
assayed for virus. Blood samples were assayed for the
presence of antibodies that would neutralize the striped
bass isolate of IPNV (IPNV-Sb) but not the European Ab
serotype. Virus was not recovered from any wild striped
bass tested (Table 9). Specific IPNV-Sb neutralizing
antibody was detected in 1- to 3-year-old striped bass
caught during the winter of 1984 and in one young-of-year
striped bass caught in 1985 (Table 10).
Procedures that Affect IPNV Recovery from Striped Bass
Tissue Site of IPNV in Striped Bass
Tissues from IPNV-infected striped bass were assayed
individually to determine those from which IPNV could be
recovered most frequently. Virus was recovered from the
anterior kidneys of all striped bass that were positive for
IPNV but was never isolated from brain (Table 11). Virus
was also recovered from other tissues but none with the
consistency found for the anterior kidney (Table 11).
Tissue virus titers ranged in magnitude from undetectable
(< 5 x 101 pfu/g) to 106 pfu/g.
Virus Recovery from Steroid Injected Chronic Carriers
This study was conducted to determine if exogenous
corticosteroids would increase the percentage of virus
Table 9: Attempts to isolate infectious pancreatic
necrosis virus (IPNV) from striped bass caught in the
Chesapeake Bay (MD).
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
Virus Recovery from Stored IPNV-carrier Tissue Homogenates
Aliquots of tissue homogenates from individual IPNV-
infected striped bass were stored under different condi-
tions to determine if the stability of IPNV infectivity was
affected. Virus infectivity was reduced in homogenates
stored at 40C (Table 13) but IPNV infectivity was not
significantly different (p < 0.01, ANOVA) in homogenate
samples stored at either -200 or -700C. The type of con-
tainer (glass vial or plastic bag) did not result in a
significant difference in virus infectivity (p < 0.01,
ANOVA) (Table 14), although virus titers from homogenates
stored at 40C were significantly different (p < 0.01,
ANOVA) from titers from homogenates stored at -20 or -700C.
Recovery of IPNV from Stored Whole Striped Bass
In an additional study designed to determine if storage
conditions affect the recovery of infectious IPNV from
virus-infected striped bass, whole fish were stored for 2
to 14 days at 4, -20, and -700C. Table 15 shows the virus
titers of IPNV-infected striped bass fingerlings determined
after storage. Virus titers were best maintained in IPNV-
Table 12: Recovery of infectious pancreatic necrosis
virus (IPNV) from chronic virus-carrier striped bass
following injection with steroid.
WEEKSa # POSITIVE/# TESTEDb TITERc
0 0/4 Vd
0.5 0 / 3 NV
1 1 /4 102
2 3 / 4 102- 103
3 1 /4 102
aNumber of weeks following intraperitoneal (i.p.)
injection of triamcinolone acetomide (10 mg/kg) into
striped bass that been injected i.p. with IPNV 14 15
bNumber of striped bass from which IPNV was recovered
per number of striped bass assayed for IPNV by the
CRange in magnitude of virus titers expressed as
plaque forming units of IPNV per gram of tissue
dNo virus detected.
Table 13: Titers of virus of infectious pancreatic
necrosis virus-infected striped bass tissue homogenates
that were stored at different temperatures.
STORAGEa FISH NUMBER
1 2 3 4 5
5 x 102 5 x 103 3 x 104 2 x 104 1 x 104
40C NVc 1 x 103 6 x 103 4 x 103 4 x 103
-200C NV 7 x 103 3 x 104 5 x 104 7 x 104
-700C NV 8 x 103 5 x 104 5 x 104 3 x 104
40C NV NV 2 x 104 NV 1 x 104
-200C 1 x 102 5 x 103 6 x 104 6 x 104 3 x 104
-700C NV NV 5 x 104 2 x 104 1x 103
aLength of time and temperature at which aliquots of
tissue homogenates from striped bass infected with
infectious pancreatic necrosis virus (IPNV) were stored
prior to assay for virus by the plaque method.
bplaque forming units of IPNV per gram of tissue (pooled
internal organs) recovered from homogenates.
cNo virus was recovered.
Table 14: Recovery of infectious pancreatic necrosis
virus (IPNV) from striped bass tissue homogenates stored
for 48 hours in different types of containers.
40C -200C -700C
Vb pc V P V P
2 8 x 101
3 5 x 101
4 3 x 102
3 x 101
1 x 102
4 x 102
6 8 x 101
7 3 x 101
3 x 102
4 x 10 2
2 x 102
6 x 102 1 x 103 4 x 102
3 x 103 3 x 103
3 x 102
1 x 102
5 x 101
2 x 102
2 x 103
4 x 102
2 x 103 2 x 103 1 x 103
2 x 103
1 x 103
9 x 102
4 x 102
6 x 102
2 x 102
2 x 103
1 x 103
aTemperature at which aliquots of homogenates of pooled
internal organs from striped bass were stored.for 48
hours prior to being assayed for virus by the plaque
bAliquots of striped bass tissue homogenates were stored
in sterile glass vials.
CAliquots of tissue homogenates were stored in sterile
dNo IPNV was detected.
plaque forming units of IPNV per ml of homogenate.
Table 15: Recovery of infectious pancreatic necrosis
virus (IPNV) from IPNV-infected striped bass stored
LENGTH OF STORAGE
TEMP(oC)a 0 DAYS 2 DAYS 14 DAYS
9 / 10b
4 10 / 11 3 / 3
-20 4 / 13 1 / 3
-70 0 / 17 0 / 3
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
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
9 7 ..
Figure 5: Electrophoretic profile of polypeptides from
three isolates of infectious pancreatic necrosis virus
(IPNV) fractionated on a discontinuous 10%
polyacrylamide gel. The three IPNV isolates are: striped
bass (S), menhaden (M), and the North American VR-299
(V). The left lane contains the following molecular
weight markers: phosphorylase b (97,400), bovine albumin
(66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate
dehydrogenase (36,000), carbonic anhydrase (29,000),
trypsinogen (24,000), and trypsin inhibitor (20,10g).
Numbers on the gel indicate molecular weights x 10
The lowest band represents the dye front.
Neutralization kinetics reveals the pattern and rate
at which virus becomes neutralized in the presence of
excess antibody. Each of three IPNV isolates (IPNV-Sb,
IPNV-M, and VR-299) was reacted with homologous and
heterologous antibody, and the residual infectivity at
several time points was measured. The neutralization
kinetic curves for the three IPNV isolates were similar for
homologous and heterologous antibody reactions (Figure 6).
The rate of neutralization (K) was calculated using the
formula K = D/t 2.3 log Vo / Vt, where D = reciprocal of
the dilution of antibody, t = 0.25 minutes, Vo = total
virus, and Vt = number of virus plaques at 0.25 minutes
(Macdonald & Gower, 1981). The calculated neutralization
rates (K) were of the same magnitude for most combinations
of IPNV isolates and antibodies (Table 16). The only
exception was the increased rate detected for the reaction
of VR-299 with its homologous antibody.
0-- i I I
0 2 4
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.
0 2 4
Figure 6 B. The IPNV isolates were tested with antibody
against the North American isolate VR-299.
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.
IPNV-Mb IPNV-Sbc VR-299d
IPNV-M 3 x 105 7 x 10 5 x 105
IPNV-Sb 9 x 105 4 x 105 4 x 105
VR-299 9 x 105 6 x 105 24 x 105
aEach IPNV isolate was reacted individually with rabbit
antiserum against each of the isolates. Total virus and
residual infectivity at 0.25 minutes were determined by
bThe menhaden isolate of IPNV.
CThe striped bass isolate of IPNV.
dThe standard North American isolate of IPNV.
e The rate of neutralization was assumed to be linear
for the first 0.25 minutes of the reaction between
antibody and virus. The neutralization rate (K) was
calculated for each trial using the formula
K = D/t x 2.3 x log Vo/ Vt, where D = dilution of the
antiserum, t = 0.25 minutes, V = total number of viral
plaques, and Vt = number of viral plaques at 0.25
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
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
Peaks of mortality in IPNV-challenged and control fish
coincided within trials but were not predictable between
trials. The reasons for the deaths are not known.
Possibly contaminants (e.g. bacteria, ammonia) introduced
with the brine shrimp nauplii fed to the fish may have
accounted for the mortality pattern.
It is clear, however, that immersion exposure to virus
did not cause predictable mortality in striped bass, even
in IPNV-challenged fish subjected to an abrupt pH change.
In contrast, young brook trout showed increased mortality
after immersion challenge with the striped bass isolate of
IPNV (P. E. McAllister, National Fish Health Research
Laboratory, Kearneysville, WV; unpublished data). The
reasons for the difference in IPNV pathogenicity in fishes
are not known.
Striped bass demonstrated age-related differences in
susceptibility to IPNV infection after waterborne
challenge. Three weeks after immersion IPNV challenge,
only striped bass that had been exposed at one day post-
hatch, contained virus. No virus was recovered from
striped bass that were exposed at 26 days or older to water
borne IPNV. Explanations for these findings probably
involve the nature of the integument in very young fish,
and the speed with which effective defense mechanisms
develop in these fish. The external integument of newly
hatched fry performs exchange functions that are later
performed by the gills and other organ systems (Johansen,
1982; Roberts et al., 1973). Possibly the immature
integument might provide a site to which exogenous virus
can attach, enter and multiply--a site that later becomes
inaccessible to virus. In addition to physical changes in
the integument, fish may quickly develop other nonspecific
defense mechanisms such as inteferon and cellular defense
systems, that may protect fish from waterborne
microorganisms (de Kinkelin & Dorson, 1973; Manning et al.,
1982; Tatner & Manning, 1985). A specific humoral response
probably is not a major factor in protecting very young fry
(Manning et al., 1982; Manning & Mughal, 1985). None of
the experimental striped bass immersed in IPNV developed
In contrast to the lack of infectivity of IPNV in all
but the youngest striped bass exposed to waterborne IPNV,
experimental inoculation of IPNV into striped bass resulted
in asymptomatic carriers that contained infectious virus
for longer than one year. No overt signs of disease, such
as "spinning" or increased mortality, were seen in virus
infected striped bass, even in IPNV-injected striped bass
that were subjected to environmental stress. Similarly, no
histopathology was detected in experimental IPNV-infected
striped bass. Atlantic salmon, Salmo salar, develop
subclinical IPNV infections like striped bass; however,
unlike striped bass, Atlantic salmon do develop
degenerative pancreatic lesions (Swanson & Gillespie,
1979). The significance of the IPNV-induced histological
lesions is not known.
Striped bass did become infected with IPNV after
ingesting IPNV-carrier brook trout. Like brook trout,
menhaden are susceptible to IPNV-induced disease (Stephens
et al., 1980). The virus can be isolated from menhaden
during their annual spring epizootic in the Chesapeake Bay
(Stephens et al., 1980). Possibly striped bass may be
exposed to IPNV by consuming IPNV-infected menhaden. The
source of IPNV infection of menhaden in Chesapeake Bay has
not been reported, but brook trout, as well as other fish,
probably can become infected with IPNV via the sex products
(Wolf & al., 1963; Bullock et al., 1976; Seeley et al.,
1977; Dorson & Torchy, 1985).
Experimental transmission studies did not demonstrate
the spread of IPNV from striped bass sex products to
offspring. Virus was not recovered from the milt or urine
of survivors in the population of striped bass from which
the original IPNV isolate was obtained. In addition, IPNV
was not isolated from the offspring from experimentally
infected striped bass adults or from offspring of IPNV-
exposed sex products. The virus was isolated from striped
bass sperm and larvae collected from the Chesapeake Bay in
1984, but not in 1985 or 1986 (F. M. Hetrick, University of
Maryland, unpublished data). The significance of these
findings is unclear.
Although no virus was recovered from any striped bass
organs sampled from Chesapeake Bay, virus-neutralizing
antibody was found in the older fish caught in the winter
of 1984 and young-of-the-year fish sampled in the summer of
1985. Possibly the older fish were exposed to the virus
during the spring IPNV-epizootics in menhaden. Results
from neutralization kinetics and SDS-PAGE of viral proteins
demonstrate the close relationship between IPNV isolated
from striped bass (IPNV-Sb) and from menhaden (IPNV-M).
Neutralization kinetics are sensitive tests for
comparing antigenic relatedness between viruses (Ashe &
Scherp, 1963) and have been used to categorize IPNV-
isolates into distinct serotypes (Macdonald & Gower,
1981). In the current study, the neutralization curves
and the rates of neutralization of the striped bass
isolate of IPNV (IPNV-Sb) were virtually identical to
those of the menhaden isolate (IPNV-M). Use of the same
analytical technique revealed that both isolates are
closely related to the standard North American isolate
(VR-299). The neutralization rate of the reaction of
the VR-299 isolate with its homologous antibody was one
magnitude higher than rates determined for the other
neutralization reactions. This probably indicates that
the antiserum either recognized or was more avidly bound
by some antigenic determinant on VR-299 that was not
present on the other two isolates tested. However, in
all other neutralization reactions, VR-299 patterns were
like those for IPNV-Sb and IPNV-M, indicating a close
relationship between the three isolates.
In addition, polyacrylamide gel electrophoresis of
the three IPNV isolates demonstrated similar viral
polypeptide bands. The calculated molecular weight of
proteins of IPNB-Sb and IPNV-M were 95000, 53000, 51000,
31000 and 29000. All but one (51000) protein band were
demonstrated for the North American isolate (VR-299).
The molecular weights of the viral proteins of IPNV-M
and VR-299 have been reported to be 86000, 56000, 30000,
and 27000 (Stephens, 1981; Stephens & Hetrick, 1983).
The actual molecular weights attributed to the viral
polypeptides has varied, even within the same laboratory
(Dobos, 1977; Dobos & Rowe, 1977; Dobos et al., 1977).
The variation probably is related to differences in the
experimental protocols used (Dobos & Rowe, 1977). For
purposes of the present study, the important finding is
the demonstrated similarity between the menhaden and the
striped bass isolates of IPNV.
As previously discussed, striped bass did not become
infected with IPNV after waterborne challenge, but became
inapparent IPNV-carriers after inoculation or ingestion of
IPNV. Virus-infected striped bass did shed sufficient IPNV
to infect brook trout that were located in tanks downstream
from the striped bass. Previous reports have implicated
IPNV-infected trout as the source of IPNV infection of
nonsalmonids, such as Catostomus commersoni (Sonstegard et
al., 1972). The present results demonstrate that IPNV can
be transmitted from a nonsalmonid species to trout.
The spread of IPNV from healthy appearing (both
grossly and histologically) striped bass to a susceptible
fish species has practical implications. If IPNV-carrier
striped bass are transported to areas that were previously
IPNV-free, the striped bass pose a potential threat to fish
species in the watershed. The virus is relatively stable
in the environment, remaining infective for months in
aqueous environments (Tu et al., 1975; Toranzo & Hetrick,
1982). Therefore, testing a population of striped bass for
IPNV prior to introduction into IPNV-free areas would seem
advisable. A series of experiments were performed to
determine what samples should be taken and how the samples
should be handled to improve the recovery of IPNV from
virus-infected striped bass.
Virus was reisolated from IPNV-infected striped bass
most often from anterior kidney and from the spleen, but
never from the brain. A similar pattern of IPNV recovery
is found in trout (Wolf & Quimby, 1969; Yamamoto, 1974).
These results differ from those of Dorson (1982) who
contended that the brain of trout IPNV-carriers may be the
only tissue containing IPNV.
When IPNV was detected in blood samples from striped
bass, the virus was found associated with the leucocytes.
Only occasionally was IPNV recovered from plasma. Swanson
and Gillespie (1982) reported similar findings from IPNV-
infected brook and rainbow trout. Swanson and Gillespie
(1982) separated the blood cells over a Ficoll gradient
prior to virus assay. The current study developed a simple
separation procedure consisting of removal of the buffy
coat from centrifuged hematocrit tubes. This procedure
permitted virus assay of smaller blood volumes than have
been reported previously (Swanson & Gillespie, 1982; Yu et
al., 1982). To detect levels of virus lower than those
recovered in this study white blood cells can be cocultured
with virus-susceptible cells (Yu et al, 1982). Another
sensitive assay permits recovery of IPNV from the
supernatant fluid from mitogen stimulated lymphocytes from
IPNV-infected Atlantic salmon (Knott and Munro, 1986).
Whatever procedure is utilized to isolate IPNV from blood
and blood components, the samples should be assayed as
quickly as possible (Swanson & Gillespie, 1982).
Previous investigations on the effects of storage
of samples on IPNV recovery have used virus-containing
culture fluids, or tissue homogenates to which IPNV was
added (Malsberger and Cerini, 1963: Wolf, 1964; Wolf et
al., 1969; McMichael et al., 1975). Use of liquid samples
permits obtaining initial levels of infective virus in the
samples. Data from stored IPNV-infected striped bass
tissue homogenates were similar to those reported by the
other authors. An increased loss of virus titer was
observed in homogenate samples stored at 40C, compared to
values obtained from samples stored at -200 or -700C.
Therefore, for best virus recovery, striped bass tissue
homogenates should be stored frozen (-20 or -700C).
In contrast, data from trials storing whole IPNV-
infected striped bass fingerlings gave different results.
All viral infectivity was lost in fish samples stored at
-700 but was retained at 40C. Retention of virus
infectivity was intermediate in samples stored at -200C.
The retention of viral infectivity in whole striped bass
stored at 40C and loss of infectivity in fish stored at
-700C was somewhat unexpected. The disadvantage of using
whole fish is that an initial virus titer can not be
obtained. However, because there was a high ( > 90%)
incidence of IPNV-carriers in the experimental striped bass
used in this study, the lack of virus infectivity in fish
stored at -700C must be a result of events associated with
storage at the lower temperatures. Perhaps the different
rates at which freezing and thawing occurs in whole fish
compared to aqueous solutions may affect viral infectivity.
The observed differences of IPNV stability in stored
whole fish and in stored homogenates were not due to a
difference in storage containers. When IPNV-infected
tissue homogenates were stored in plastic bags similar to
those in which whole fish were stored, homogenate samples
again lost infective virus at 40C, but IPNV remained
infective when stored frozen (-20 and -700C). The reasons
for the variation in IPNV infectivity from intact and
homogenized fish tissues are not known. However,
demonstration of the variation emphasizes the importance of
experiments that attempt to replicate field conditions.
Previous investigations that utilized IPNV-infected
cell cultures or tissue homogenates may, or may not,
accurately reflect practical field conditions. Different
IPNV isolates vary in stabilities during storage (Dorson et
al., 1978; McMichael et al., 1975; Malsberger & Cerini,
1963). Further studies are needed to determine if the
liability of various IPNV isolates is similar to that
demonstrated for the striped bass isolate of IPNV in stored
striped bass samples. Current data demonstrate that for
detection of IPNV-Sb infectivity in striped bass, samples
should be stored intact at 40C and assayed within two
Bullock and Stuckey (1975) reported that steroids
increase the recovery of infectious agents from inapparent
trout carriers. Increased detection of IPNV was observed
in IPNV-inoculated fish that were given an intraperitoneal
injection of steroid fifteen months after IPNV injection.
Apparently the steroids temporarily reversed the observed
decline of virus titers in IPNV-infected striped bass.
Tissue titers in IPNV-inoculated striped bass remained
relatively constant over the first 10 days. In contrast,
IPNV levels peak at three days after IPNV inoculation in
Atlantic salmon (Swanson & Gillespie, 1979), and in rainbow
trout that are susceptible to IPNV-induced mortality, IPNV
titers reach high titers at seven days after exposure
(Okamoto et al., 1984). Although Yamamoto (1975b)
suggested a correlation between virus-neutralizing antibody
and IPNV titers in trout, no such relationship was observed
in striped bass. Antibody was not detected in IPNV-
injected striped bass until seven days post inoculation and
reached peak values at approximately three weeks. During
this same time period, levels of virus recovered from IPNV-
inoculated striped bass remained uniform, apparently
unaffected by the presence of the antibody. In fact, virus
titers remained uniform during the first two months after
IPNV-injection and gradually declined over a 15 month
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
Depression of the humoral response of striped bass to
IPNV was not observed in the current study. In fact, IPNV
was strongly antigenic to striped bass and induced
significant antibody titers after IPNV-injection. A rise
in antibody levels was detected in IPNV-injected striped
bass at 7 days post injection after both a primary and
secondary inoculation. In a classical anamnestic response,
antibody levels rise faster in the secondary response
(Eisen, 1980). The controversy over whether fish
demonstrate a true anamnestic response has been reviewed
(Dorson, 1984). In the current experiments, striped bass
did not appear to exhibit an anamnestic response but they
did mount a humoral response to IPNV infection.
Immersion in IPNV, however, was not sufficient to
stimulate detectable levels of virus-neutralizing antibody.
Even in virus-carrier striped bass that were given a second
IPNV exposure by immersion, antibody levels did not
increase after the second challenge.
Chronic IPNV-carrier striped bass that received
exogenous steroid did not demonstrate any change in the
titers of virus-neutralizing antibody. Administration of
steroids prior to IPNV injection, did delay and reduce the
humoral response of striped bass. Steroid induced
immunosuppression has been described also in trout
(Anderson et al., 1982). In addition, Anderson et al.
(1982) reported that trout given a steroid dose of 200
mgkg did not appear unhealthy during the 23 days of the
experiment. Steroid, administered at a rate of 100 mg per
kg body weight, was lethal to striped bass observed over a
three month period. However, none of the IPNV-injected
striped bass that were treated with steroids developed any
clinical signs or histological lesions attributable to
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.
SOURCES OF SUPPLIES AND EQUIPMENT
Aldrich Chemical Corporation, Inc. (Milwaukee, WI)
American Scientific Products (McGraw Park, IL)
Heparinized microhematocrit capillary tubes
Ames Company (Elkhart, IN)
N,N,N',N' tetramethyl ethylenedianine (TEMED)
Amicon Corporation (Danvers, MA)
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)
Bethesda Research Laboratory (Gaithersburg, MD)
Sucrose ultrapuree enzyme grade)
Biorad (Richmond, CA)
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)