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assay performed by G.A.E. Gall, Department of Animal Science, University of
California at Davis. All experiments involving live fish were conducted at 12°C
in pathogen-free well water.

Exposure of Fish to Virus

Dilutions of virus were prepared in MEM-5 to achieve concentrations of
10 ^-10^ TCID50 ml when virus was added to containers with 750 ml aerated
water. Each container held 20 juvenile chinook salmon or 30 of the smaller
steelhead trout or sockeye salmon. In each experiment, control fish were
exposed to MEM-5 without virus. After 12-h exposure, the contents of each jar
were placed into a separate 68-1 aquarium receiving single-pass water flowing at
a rate of 1 l/min. Fish were fed twice daily and observed for signs of IHNV
infection for 14 d. Dead or moribund fish were removed daily. The 50% lethal
concentration (LC 50) was calculated from the accumulated mortality according
to the method of Reed and Muench (1938); however, in cases where mortality
was insufficient to determine the LC50, an LC25was similarly calculated. At the
conclusion of each experiment at least 10 control fish were divided into pools
of five fish each and examined for virus as previously described ( Hetrick, Fryer,


and Knittel 1979) to ensure that the fish were originally free of virus. Five dead
fish were pooled from each dilution series and examined for virus to verify that
virus was present in dead fish exposed to each isolate.

Virulence Testing

Juvenile chinook and sockeye salmon and steelhead trout were exposed to
second-passage TR, ER, RB and MP isolates to determine virulence of the
isolates in each species (Table 1 ). In all three chinook salmon stocks tested, the
TR and ER isolates were more virulent (lower LC,^) than the RB and MP
isolates, while the TR, RB and MP isolates were more virulent in the sockeye
salmon and steelhead trout groups. For the latter two species, the LCso's of TR,
RB, and MP isolates were similar, while the ER isolate was less virulent.
Although RB and MP isolates were virulent for sockeye salmon and steelhead
trout, they were comparatively avirulent for all three chinook salmon stocks.
Virus was recovered from dead or dying fish in each combination of virus
isolate and fish stock, except in the case of Elk River chinook salmon. No Elk
River chinook salmon died after exposure to RB or MP. Virus was not found in
control fish and mortality did not exceed 5% in any control fish group.

TABLE 1. Virulence of Four Isolates of Infectious Hematopoietic Necrosis Virus in Five Oregon Salmonid
stocks. Virulence is Measured by the Logm Virus Concentration (LC) Required to Produce 25%
or 50% Mortality Following a 12-h Exposure.

Virus Source
Fish tested Trinity Elk Round Mendenhall

(size)* River River Butte Ponds

(TR) (ER) (RB) (MP)

Log LC,,, in TCID,o/ml

Round Butte chinook (0.42) 3.3 4.0 5.0 4.8

Trask River chinook (0.54) 2.3 3.2 4.8 4.3

Elk River chinook (0.57) 2.1 4.2 > 5.0 > 5.0

Log LC.,„ in TCID rjm\

Round Butte steelhead (0.23) 2.8 3.5 2.7 2.7

Wizard Falls sockeye (0.18) , 2.4 2.9 2.2 2.0

• Average weight of fish In grams at time of exposure. Variation in weight stemmed from average size differences
at hatching.

Resistance of Coho Salmon-Rainbow Trout Hybrids

Groups of 10 each of coho salmon, rainbow trout, and coho salmon rainbow
trout hybrids were exposed to the TR isolate at concentrations of 10-10^
TCID^o/ml. The small number of fish used in this experiment was due to the
high prehatching and posthatching mortality (98%) of diploid coho salmon
rainbow trout hybrids. Coho salmon and coho salmon rainbow trout hybrids
were more resistant than rainbow trout (Table 2). Virus was recovered from
coho salmon, coho salmon X rainbow trout hybrids, and rainbow trout which
died during the 14-d exposure, and also from surviving coho and hybrid salmon.


TABLE 2. Mortality of Rainbow Trout, Coho Salmon, and Rainbow Trout x Coho Salmon Hybrids Ex-
posed to the Trinity River Isolate of Hematopoietic Necrosis Virus.

Mortalities/ W fish


Stock (g) 0' /(?"' 10 ■* 10^ LC,„'

Oak Springs rainbow trout 0.21 6 8 10''"

Sandy River coho salmon 0.35 6 10""

Coho X rainbow hybrid 0.19 1 1 3 lO'^"

■' Control fish were exposed to the virus dilutent, MEM-5.

''Concentration of Trinity River IHNV used in 12-h exposure, expressed in TCIDjo/ml.

' Median lethal concentration expressed in TCIDr,(,/ml.

Infective Dose Determination in Cell Lines

Third-passage virus isolates grown in CHSE-214 cells were titered by the
endpoint dilution assay in three salmonid cell lines to determine if the titer of an
individual isolate varied according to the cell line used (Table 3). The titer of
TR and ER isolates in SSE-5 cells was equal to or less than the titer in CHSE-214
cells. In contrast, the RB and MP isolates produced titers three-to twelve-fold
higher in SSE-5 cells than in CHSE-214 cells. Titers of TR and MP isolates were
much lower in CSE-119 cells than in the other cell lines. The virus-induced
cytopathic effect (CPE) in coho salmon cells was similar to that in the other cell
lines, but appeared 2-4 d later and often did not proceed to completion.

TABLE 3. Titer of Four Virus isolates Determined by Endpoint Dilution Assay in Cell lines Derived
from Chinook (CHSE), Sockeye (SSE), and Coho Salmon (CSE) Embryos.

Virus isolates by chinook salmon stock

Trinity I7J( Round Mendenhall

Cell line River (TR) River (ER) Butte (RB) Ponds (MP)

CHSE-214 lO""'" 10"^ 10'"' 10'"'

SSE-5 10"" lO'"'' 10"' 10'"

CSE-119 10'" nd'' nd 10" ■*

■' Virus titer expressed in TCIDr.o units/ml of undiluted stock virus.
'* nd = not done.

Growth Curves

Replication of IHNV in CSE-1 19 cells was indicated by the appearance of CPE
in the TCID50 assays. To confirm replication, the virus titer was determined after
infection of CSE-119 and CHSE-214 cells (Figure 1 ). An increased titer of virus
was detectable 1 d after infection in both flasks of CHSE-214 cells. Release of
virus from infected CSE-119 cells was demonstrated 2-4 d after inoculation of
virus. Virus titers increased to 10'' TCID5o/ml in both CHSE-214 cultures, but did
not reach 10'' TCIDso/ml in CSE-119 cells. The CPE in the CSE-119 cells did not
reach completion 12 d after inoculation of the virus. The TR isolate appeared to
be capable of only limited replication in coho salmon cells.

Efficiency of Plaguing in Cell Lines

The ratio of the titer in CHSE-214 and SSE-5 cells was determined as an index
of the relative plaguing efficiency of each isolate (Table 4). The CHSE-214:SSE-
5 ratio was 1:1.3 and 1:0.96 for TR and ER, respectively. Ratios for RB and MP
isolates were 1:7.4 and 1:2.8, respectively. Thus, the two isolates which were



comparatively avirulent in chinook salmon, RB and MP, plaqued less efficiently
in chinook than in sockeye salmon cells, as compared to TR and ER. No clearly
definable plaques were observed when the four isolates were plaqued in coho
salmon cells (CSE-119).



o ■<













A 6

Time (days)


— r




— r-



Tlmo (days)

FICURE 1. Concentration in Log ,„ of infectious virus detected in the supernatant fluids of
CFHSE-214 and CSE-119 cell lines after infection with Trinity River infectious
hematopoietic necrosis virus. Each curve represents the titers determined in one of a
replicate pair of cell cultures.

TABLE 4. Efficiency of Plaquing of Four Virus Isolates in Cell Lines Derived from Chinook (CHSE)
and Sockeye Salmon (SSE) Embryos.

Virus isolate by so urce
Cell line









6.2 X lO*'-'

5.3 X 10"

8.0 X 10 '

1.2 X 10'

8.2 X 10"

5.1 X 10"

5.9 X 10"

3.3 X 10'


1 :0.96



CHSE-214 6.2 X 10 "


■' Virus titer in plaque forming units/ml averaged from three replicate flasks.
'' Ratio of titer in CHSE-214 cells:titer in SSE-5 cells.


The IHNV isolates examined in this study could be divided into two types:
those comparatively avirulent in chinook salmon (RB and MP), and those
virulent in chinook salmon, sockeye salmon, and steelhead trout (TR and ER).


This grouping agrees with the natural occurrence of the disease in chinook
salmon at the sites of isolation. Although the Alaskan isolate (MP) was isolated
from chinook salmon and grown in chinook salmon cells, this isolate possessed
far less virulence for chinook salmon than TR or ER. Isolation of IHNV in
Alaskan chinook salmon is comparatively rare and may result from exposure to
heavily infected sockeye or chum salmon, O. keta (Follett, Thomas, and Hauk
1987). Species-specific virulence of IHNV has been previously reported. Virus
associated with naturally occurring sockeye salmon epizootics possessed little
virulence for chinook salmon experimentally exposed (Rucker et al. 1953,
Wingfield et al. 1970). In contrast, IHNV isolated from Sacramento River
chinook salmon (SRCV) was found to be pathogenic in chinook and sockeye
salmon and steelhead trout (Wingfield and Chan 1970). The TR virus may be
similar to SRCV because of its virulence for all species of fish tested. The ER
isolate was less virulent in sockeye salmon and steelhead trout than the TR.
Strains of IHNV with differing host specificities might act as different agents if
present in the same watershed. For this reason, transfer of fish even between
two areas where IHNV is present should be regarded with caution.

Wertheimer and Winton (1982) reported variation in susceptibility of
chinook salmon stocks to IHNV. Although some variation was found among the
three stocks of chinook salmon tested, the results indicated that the lack of
IHNV-caused mortality in chinook salmon reared at Round Butte Hatchery is a
result of host preference of that strain of virus, rather than an exceptional
resistance of Round Butte chinook salmon to IHNV.

Although coho salmon are considered resistant to IHNV, the virus has been
found in asymptomatic adult coho salmon at the Trinity River Hatchery
(LaPatra et al. 1987). Mortality has not been previously observed following
waterborne exposure of juvenile coho salmon to IHNV (Wingfield and Chan
1970, Wingfield et al. 1970). Injection of juvenile coho salmon with virus from
the Trinity River resulted in a 3% mortality in one study (Hedrick et al. 1987),
but no mortality was observed in other injection experiments (Watson,
Guenther, and Rucker 1954; Parisot and Pelnar 1962). The results here may
differ from previous studies because of different experimental conditions.
Susceptibility to IHNV decreases as the fish grow or the temperature rises
(Pilcher and Fryer 1980). We tested coho salmon at a temperature (12°C)
conducive to IHNV pathogenesis (Amend 1970). Although some mortality was
observed when coho salmon were exposed to very high virus concentrations
(10^ TCID5Q/ml), the coho salmon were more resistant than the rainbow trout.
Further studies using larger sample sizes should be done to confirm that IHNV
can cause disease or mortality in young coho salmon. The TR isolate should be
included in such a study since all reports of virus isolation and mortality in coho
salmon have involved virus from the Trinity River system.

Resistance to IHNV was apparently transferred to rainbow trout eggs by coho
salmon sperm. The use of pooled rainbow trout sperm, pooled coho salmon
sperm, and eggs from one rainbow trout female in this study indicates that the
difference in mortality between rainbow trout and coho salmon x rainbow trout
hybrids was not due to between-family variation. The transfer of IHNV
resistance by coho salmon sperm was also demonstrated by Parsons et al.
(1986) using triploid coho salmon x rainbow trout hybrids. Ord et al. (1976)
conferred resistance to viral hemorrhagic septicemia by fertilizing eggs of


susceptible rainbow trout with coho salmon sperm. Similarly, the susceptibility
of sockeye salmon to IHNV was decreased by fertilization with sperm from
more resistant sockeye salmon (Mclntyre and Amend 1978). In contrast, coho
salmon sperm did not confer resistance to injected IHNV in coho salmon x
Chinook salmon hybrids, nor were chinook salmon male x coho salmon female
hybrids resistant to injected IHNV (Hedrick et al. 1987). Further studies are
needed to better define the nature of the resistance factor, and the effects of
waterborne or injected virus exposure on hybrid salmonids. Also, future studies
with hybrids should use multiple family groups (Amend and Nelson 1977).
Another possible problem in our hybrid study was that different sizes of fish
were used in the exposure experiments. The fish hatched at different sizes due
to the smaller size of the rainbow trout eggs. Smaller fish are more susceptible
to IHNV than larger ones, as previously noted. We chose to test all species at
a similar stage of maturity: the time of initial feeding.

Evidence for two virulence groups was also found when the four virus isolates
were used to infect salmonid cell lines. The titer of RB and MP was lower in
chinook than sockeye salmon cells in both plaque and endpoint dilution assays,
but TR and ER showed similar titers in cell lines from the two species. Species
specificity was also reported by Nims, Fryer and Pitcher (1970), who found that
a sockeye salmon cell line produced 10-^ — 10^ times more virus than a chinook
salmon cell line after infection by an IHNV isolate from sockeye salmon. Host
specificity of IHNV should be considered in the selection of cell lines for
diagnostic purposes. In areas where the virus is endemic in sockeye salmon or
steelhead trout, the use of CHSE-214 cells may result in reduced chances of
detection of virus. Still, the SSE-5 cell line was highly susceptible to the four
isolates studied.

Wingfield et al. (1970) found no increase in IHNV titer in CSE-119 cells
exposed to an isolate from sockeye salmon, but we found virus titer increased
after CSE-119 cells were inoculated with TR. Some of this increase may have
been due to absorbed virus releasing from the cell surface, but the appearance
of CPE indicates true replication did occur. Possible reasons for the contrasting
results may be in the use of the virulent TR isolate, or that the age of cell
monolayers and temperature of incubation were lower in the present study. No
IHNV plaques were observed by Phillipon-Fried (1980) in CSE-119 cells. We
obtained similar results when the four IHNV isolates were inoculated in CSE-1 19
cells. The limited replication and incomplete CPE seen in infected coho salmon
cells may explain the low mortality in coho salmon experimentally exposed to
IHNV. The relative resistance of CSE-119 cells suggests that the resistance of
coho salmon is in part determined at the cellular level.


This research was supported by the Oregon Department of Fish and Wildlife
under Public Law 89304, Anadromous Fish Act, and also appears as Oregon
Agriculture Experiment Station Technical Paper No. 7956.


Amend, D. F. 1970. Control of infectious hematopoietic necrosis virus disease by elevating the water temperature.
). Fish. Res. Bd. Canada, 27:265-270.


Amend, D. F., and ). R. Nelson 1977. Infectious hematopoietic necrosis: variation in susceptibility of sockeye
salmon (Oncorhynchus nerka) to disease. ). Fish. Biol., 11 (6):567-573.

Fendrick, |. L., W. |. Groberg, Jr., and |. C. Leong. 1982. Comparative sensitivity of five fish cell lines to wild type
infectious haemotopoietic necrosis virus from two Oregon sources. J. Fish Dis., 5:87-95.

Follett, ). E., ). B. Thomas, and A. K. Hauck. 1987. Infectious haemotopoietic necrosis virus in moribund and dead
juvenile chum Oncorhynchus keta (Walbaum), and chinook, O. tshawytscha (Walbaum), salmon and
spawning adult chum salmon at an Alaskan hatchery, j. Fish Dis., 10:309-313.

Fryer, J. L., J. S. Rohovec, C. L. Tebbit, J. S. McMichael, and K. S. Pilcher. 1976. Vaccination for control of
infectious diseases in Pacific salmon. Fish Pathol., 10:155-164.

Crischowsky, R. S., and D. F. Amend. 1976. Infectious hematopoietic necrosis virus: prevalence in certain Alaskan

sockeye salmon, Oncorhynchus nerka. ). Fish. Res. Bd. Canada, 33:186-188.
F^edrick, R. P., J. C. Leong, and J. L. Fryer. 1978. Persistent infections in salmonid fish cells with infectious

pancreatic necrosis virus (IPNV). ). Fish Dis., 1:297-308.

Hedrick, R. P., and J. L. Fryer. 1981. Persistent infection of three salmonid cell lines with infectious pancreatic
necrosis virus (IPNV). Fish Pathol., 15:163-172.

Hedrick, R. P., S. E. LaPatra, ). L. Fryer, T. McDowell, and W. H. Wingfield. 1987. Susceptibility of coho
Oncorhynchus kisutch and chinook Oncorhynchus tshawytscha salmon hybrids to experimental infections
with infectious hematopoietic necrosis virus (IHNV). Eur. Ass. Fish Pathol., Bull., 7:97.

FHetrick, F. M., j. L. Fryer, and M. D. Knittel. 1979. Effect of water temperature on the infection of rainbow trout
5<3/mo ^<?//'c//7er/ Richardson with infectious hematopoietic necrosis virus. J. Fish Dis., 2:253-257.

Lannan, C. N., J. R. Winton, and ). L. Fryer. 1984. Fish cell lines: establishment and characterization of nine cell
lines from salmonids. In Vitro., 20:671-676.

LaPatra, S. E., ). L. Fryer, W. Wingfield, and R. P. Hedrick. 1987. Transmission of infectious hematopoietic necrosis
virus (IHNV) between adult species of salmon: management strategies for anadromous broodstock. Fish
Health Section, Am. Fish. Soc. Newsletter, 15(2):7.

Mclntyre, J. D., and D. F. Amend. 1978. Heritability of tolerance for infectious hematopoietic necrosis in sockeye
salmon Oncorhynchus nerka. Am. Fish. Soc, Trans., 107:305-308.

Mulcahy, D. M., G. L. Tebbitt, W. ). Groberg, Jr., ). S. McMichael, |. R. Winton, R. P. Hedrick, M. Philippon-Fried,
K. S. Pilcher, and ). L. Fryer. 1980. The occurrence and distribution of salmonid viruses in Oregon. Oregon
State University Sea Grant College Program, Corvallis, ORESU-T-80-004:1-71.

Nims, L., J. L. Fryer, and K. S. Pilcher. 1970. Studies of replication of four selected viruses in two cell lines from
salmonid fish. Proc. Soc. Exp. Biol. Med., 135:6-12.

Ord, W. M., M. LeBerre, and P. de Kinkelin. 1976. Viral hemorrhagic septicemia: comparative susceptibility of
rainbow trout Salmo gairdneri and hybrids 5. gairdneri x Oncorhynchus kisutch to experimental infection. J.
Fish. Res. Board Canada, 33:1205-1208.

Parsons, J. E., R. A. Busch, G. H. Thorgaard, and P. D. Sheerer. 1986. Increased resistance of triploid rainbow trout
x coho salmon hybrids to infectious hematopoietic necrosis virus. Aquaculture, 57:337-343.

Parisot, T. J., and j. Pelnar. 1962. An interim report on Sacramento River chinook disease: a virus-like disease of
chinook salmon. Prog. Fish Cult., 24:51-55.

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University, Corvallis. 58 p.

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etiology. Crit. Rev. Microbiol., 7:287-364.

Reed, L. J. and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. j. Hygiene,

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Watson, S. W., R. W. Guenther, and R. R. Rucker. 1954. A virus disease of sockeye salmon: interim report. U.S.
Fish Wildl. Serv. Spec. Sci. Rep., 138:1-36.

Wertheimer, A. D., and J. R. Winton. 1982. Difference in susceptibility among three stocks of chinook salmon,
Oncorhynchus tshawytscha, to two isolates of infectious hematopoietic necrosis virus. NOAA Tech. Memo.,
Nat. Mar. Fish. Serv., F/NWC-22:1-11.

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in S.F. Snieszko (ed.), A symposium on diseases of fishes and shellfishes. Am. Fish Soc. Spec. Publ.
Washington, D.C., 5:307-318.

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(Oregon strain) and its cytopathic effects in salmonid cell lines, in S.F. Snieszko (ed.), A symposium on
diseases of fishes and shellfishes. Am. Fish. Soc. Spec. Pub. Washington, D.C., 5:319-326.


Calif. Fish and Game 76 ( 3 ): 1 46- 1 60 1 990






Department of Fisheries and Wildlife

Oregon State University, Corvallis, OR 97331



Southwest Fisheries Center Honolulu Laboratory

National Marine Fisheries Service, NOAA

2570 Dole St., Honolulu, HI 96822-23%

and Joint Institute of Marine

and Atmospheric Research,

University of Hawaii, Honolulu, HI 96822

Different ageing techniques affect not only the estimates of length at age but also
estimates of population growth and mortality rates. This study considers these
effects for the splitnose rockfish, Sebastes diploproa, and canary rockfish, S.
pinniger, based on ages determined from the surfaces and sections of otoliths
collected during a trawl survey off the west coast of North America in 1980.
Estimates of growth based on surface rather than section ages were nearly identical
for S. diploproa but were higher for S. pinniger; slightly different whole otolith
ageing techniques are suspected of producing these interspecific differences. For
both species, however, estimates of mortality were reduced by more than half when
section rather than surface ages were used.


Ages of fish are needed to estimate two vital parameters of exploited fish
populations, namely growth and mortality rates. Many fish species typically are
aged by interpreting rings on the otolith, as is the case for rockfishes (Sebastes)
for which the otolith is the preferred ageing structure (Six and Norton 1977,
Chilton and Beamish 1982). Various techniques have been developed to
facilitate the detection and the interpretation of otolith patterns used in age
determination (Chilton and Beamish 1982). Two methods to determine ages
are counting the number of rings or annuli viewed on the exterior of the whole
otolith (surface ages) or on a lateral cross section of the otolith (section ages).
Section ages are often greater than surface ages for older specimens of many
long-lived, slow-growing species (Beamish 1979a, 1979b, Boehlert and Yoklav-
ich 1984), and recent evidence demonstrates that the section ages represent the
true ages of fish (Bennett et al. 1982, Leaman and Nagtegaal 1987, Campana et
al. in press).

Because present evidence supports the validity of section ages for older fish,
important biological and management implications could result if many
demersal fish stocks are underaged due to the more common surface ageing

Accepted for publication April 1990.

Present Address: Southwest Fisheries Center Honolulu Laboratory, National Marine Fisheries Service, NOAA,
Honolulu, HI and Department of Oceanography, Uniyersity of Hawaii, 1000 Pope Road, Honolulu, HI 96822.


technique. Archibald et al. (1981) found that estimates of instantaneous
mortality (Z) were reduced by as much as 50% when the otolith sections were
used to age 10 species of rockfishes. Even for fishes that are not long lived, age
length data without older fish affect the estimation of von Bertalanffy growth
parameters (Hirschhorn 1974). Because population size, growth, and mortality
are the basic data required for most production modeling, systematic bias in age
determination may lead to serious errors in determining stock production
estimates (Le Cren 1974). In the present study, we determine whether
significant differences in the estimates of growth and mortality rates are affected
by the method of otolith age determination for two species of Sebastes — the
splitnose rockfish, 5. diploproa, and canary rockfish, 5. pinniger.


Otoliths from 5. diploproa and 5. pinniger were collected during a 1980 trawl
survey conducted off the west coast of North America (lat 36° 49' to 50° OO'N).

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