Download Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV

Journal of Aquatic Animal Health 16:152–160, 2004
q Copyright by the American Fisheries Society 2004
Early Kinetics of Infectious Hematopoietic Necrosis Virus
(IHNV) Infection in Rainbow Trout
HAMDI OGUT*
Karadeniz Technical University, Faculty of Sürmene Marine Sciences,
Sürmene, Trabzon, Turkey 61530
P. W. RENO
Coastal Oregon Marine Experiment Station, Hatfield Marine Science Center,
Oregon State University, Newport, Oregon 97365, USA
Abstract.—A series of experiments was carried out with infectious hematopoietic necrosis virus
(IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ;1.2 g) to determine the
duration of the patent period and the timing of onset of the infectious periods. We first attempted
to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed.
No infection transfer occurred despite high titers (104.79 to 104.91 plaque-forming units 5–8 d
postexposure (dpe). To determine the number of secondary cases produced by one infectious
individual, we exposed approximately 50 rainbow trout (weight, ;1.5 g) in each of seven replicate
tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence
of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22
cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish
during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4
dpe. There was a positive correlation between the level of infection among donors and prevalence
values among recipient fish (r2 5 0.60). The level of challenge by one infectious fish under the
conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but
was not enough for initiation of a full-scale epizootic among recipients.
Infectious hematopoietic necrosis virus (IHNV),
a rhabdovirus, causes a disease (IHN) characterized by extensive necrosis of hematopoietic tissues
in early life stages of economically important salmonids. Enzootic in the Northwest Pacific of North
America (Parisot et al. 1965; Amend 1975), it has
also been detected in other parts of the world, including Taiwan (Chen et al. 1985), Belgium (Hill
1992), Italy (Bovo et al. 1987), France (Laurencin
1987), and Japan (Sano et al. 1977).
Early phases of an IHNV epizootic have not
been evaluated. Understanding disease initiation
and factors that could affect spread of virus in a
susceptible population would be an essential tool
in preventing and controlling IHN disease. In particular, using cohabitation method and only a single infectious individual would imitate conditions
in nature where serious IHN epizootics occur in
wild salmonids. For example, in a natural epizootic
of IHNV, Fraser River System, British Columbia,
Canada, an estimated 8.3 million of 16.8 million
sockeye fry died of IHN disease within days of
leaving the spawning channel (Traxler and Rankin
1989). Various management strategies, such as decreasing density or increasing flow rate, could significantly decrease infection spread; sometimes,
perhaps, practices such as holding fish to determine the level of prevalence in closed enclosures
could unintentionally exacerbate spread of infection and consequent mortality.
In this study, we examined the early stages of
IHNV infection in rainbow trout Oncorhynchus
mykiss. Our goal was to use the cohabitation method to examine the initiation of infection in a susceptible population of rainbow trout by a single
infectious fish. The results obtained have implications in understanding early infection spread in
populations and in developing control strategies
for wild and cultured fishes.
Methods
* Corresponding author: [email protected]
1 Present address: Karadeniz Technical University,
Sürmene Deniz Bilimleri Fakültesi, 61530 Camburnu,
Trabzon, Turkey.
Received August 26, 2003; accepted June 18, 2004
Fish.—Rainbow trout(size ;1.2 g), kindly provided by the Oregon Department of Fish and Wildlife (ODFW), Oak Springs Fish Hatchery, Oregon,
were used in the experiments described below. No
recorded IHNV isolation has occurred in this
152
EARLY KINETICS OF IHNV SPREAD
hatchery. Stock fish were held in 1-m circular
tanks in a volume of 366 L, aerated with spargers,
and held at a temperature of 16–178C in dechlorinated city water supplied at a flow rate 3.5–4.1
L/min. Fish were fed once a day at a ratio of 1%
body weight with biodiet (Bioproducts, Inc., Astoria, Oregon) pelletized feed. Fish were acclimated for about 1 month before being used in the
experiments.
Virus.—The IHNV isolate (193-110) was originally isolated from rainbow trout in the Hagerman
Valley of Idaho (Roberti 1987). The stock of IHNV
for bath exposure to prepare an infectious donor
fish was prepared by passing the virus once in 1–
2-g rainbow trout by bath exposure. The virus was
then passed once more in vitro on the epithelioma
papillosum cyprini (EPC) cell line, titered, and
held in liquid nitrogen until use.
The presence of IHNV was determined by
plaque assay on EPC cells as described by Burke
and Mulcahy (1980), modified by treatment with
polyethylene glycol (PEG) (Drolet et al. 1995).
Whole-fish homogenates were weighed, diluted 1:
5 (weight : volume) in Eagle’s Minimum Essential
Medium with Earl’s salts (MEM; Sigma Chemical
Co., St. Louis, Missouri) but without serum and
containing pen-strep (20 units of penicillin and
0.02 mg of streptomycin per milliliter; Sigma
Chemical Co.), and centrifuged to remove debris.
Two different dilutions (1022 and 1023) of the
whole-fish homogenates were placed into replicate
wells of 24-well plates containing EPC cells. Virus
homogenates were absorbed to the EPC cells by
gentle rocking for an hour at room temperature.
Exposed cells on 24-well plates were then overlaid
with 0.75% methylcellulose in complete MEM and
incubated at 188C. The wells were examined for
plaque formation at 3 and 7 d postexposure (dpe)
by staining the wells with crystal violet in formalin
(25% formalin, 10% ethanol, 5% acetic acid, and
1% [weight: volume] crystal violet) and counting
the number of plaques present.
Lethal dose experiment.—For determination of
50% infectious dose to be used for subsequent experiments, stockfish (1.52 g) were transferred to
aerated 7.5-L experimental tanks. Groups of 50
fish were exposed to five concentrations of virus
(101, 102, 103, 104, and 105 virus/mL) prepared in
MEM. Three replicates and one control tank were
used per concentration tested. During the 6-h exposure, static city water (;168C) was aerated continuously. After the exposure period, we turned on
flowing water at a rate about 0.25 L/min and after
6 h resumed feeding the fish. Fish were fed once
153
daily at the rate of 1% body weight. For the duration of the experiment (14 d), mortalities and
moribund fish were noted daily, examined for disease signs and tested for virus on plaque assay. At
the end of the experiment, all survivors were examined for disease signs, killed with an overdose
of tricaine methanesulfonate (MS-222; Sigma
Chemical Co.), and tested for the presence of the
viral agent in five fish pools from each tank.
Infection of donor fish for all experiments.—Donor fish, IHNV-infected cohabitants, were obtained
by bath exposure of rainbow trout to 105 virus/mL
in MEM for 6 h at 168C with aeration. Three tanks
(7.5 L), two for exposure to IHNV, the other for
control (exposed to MEM), each containing 100
fish were prepared for production of ‘‘donor fish’’
(diseased fish).
The patent and infectious periods.—The experiment described below was conducted in order to
determine the patent period, the period between
initial exposure to virus and the time when the
exposed donor fish became infectious, and the infectious period, during which pathogen discharge
occurs. The experiment scheme, shown in Figure
1a, was carried out in 7.5-L randomly assigned,
aerated tanks, each containing 50 fish (mean
weight 5 1.5 g). Three replicates for each treatment were used, plus one control (exposed to
MEM only). Considering the completion time of
the bath exposure as day 0, five donor fish were
randomly selected and put into each of three replicate cohabitation cages (15 3 15 3 9 cm) covered
with screening to prevent direct contact between
donor and recipient fish. After immersion in a
clean tank at a high water flow rate for 1 h to rinse
any remnants of agent off the cage and fish, each
cage and donor fish inside were placed into each
of the three replicate tanks. At the end of a 24-h
cohabitation period, donor fish and control fish in
the cages were killed with an overdose of MS-222
and assayed for the presence of IHNV in cell culture as a single pool (five fish per day). The same
procedure was repeated daily with donor fish for
8 d, that is, at 1, 2, 3, 4, 5, 6, 7, and 8 dpe. Recipient
fish from each day were monitored for 17 dpe.
Mortalities were removed daily and examined for
disease signs and virus during this period. At the
end of the experiment, all surviving fish in the
tanks were killed and examined for the presence
of IHNV per 10 fish per pool.
Spread of infection from a single donor fish.—
To determine the daily progress of infection in a
susceptible recipient fish population initiated from
a single infectious individual, we randomly as-
154
OGUT AND RENO
FIGURE 1.—Schematic representation of experimental designs to (a) determine the timing of the onset of the
infectious period in donor fish with infectious hematopoietic necrosis virus (IHNV) and (b) characterize the spread
of infection in recipient fish exposed to a single donor fish.
signed 7.5-L aerated tanks (seven replicates/treatment and one control/treatment) to the various
treatments (Figure 1b). Each replicate contained
approximately 50 fish (mean weight, 1.5 g). At the
end of 3 dpe, one donor fish marked by fin clip
was released into each of the 21 treatment tanks;
to each control tank was added one donor fish,
which had been exposed to MEM only. Every other
day (2, 4, and 6 dpe), seven tanks were randomly
selected and all of the fish were killed with an
overdose of TMS and examined individually for
disease signs and presence of IHNV; one of the
control tanks was also selected randomly and all
fish were killed and tested for the presence of
IHNV as 10 fish per pool. The number of mortalities occurring in all tanks was recorded, and the
fish that died during (i.e., before the end of) the
experiment were examined for disease signs and
presence of virus. Flow rate of the water (16–17 8C)
was adjusted to 0.25 L/min (turnover rate 5 1.68
times/h).
Results
Patent and Infectious Periods
Donor fish at 0 dpe, when cohabited with recipient fish, were able to transmit IHNV to recipient fish in one pool, even though virus was undetected in the donor fish (Figure 2). No transmission was found in 11 other pools (approximately 10 fish per pool) tested on day 1.
None of the fish that died among control or recipient fish during the experiment were positive
for virus. The prevalence of infection from each
treatment was unexpectedly low. At 2, 3, and 4
dpe, the proportion of surviving recipient fish that
were found to be infected ranged from 6.6% to
20% (Figure 2). However, from 5 to 8 dpe, no virus
EARLY KINETICS OF IHNV SPREAD
155
FIGURE 2.—Daily change of titers in recipient and donor fish in the patent and infectious period experiment. A
group of donor fish was exposed to IHNV by bath for 6 h. Immediately after completion of the challenge period,
5 of them were randomly selected and held in cages with approximately 50 recipient fish for 24 h. The following
day, the cages were removed and the donors killed and assessed for IHNV titer; recipient fish were observed for
17 d. Gray bars represent the total number of 10 recipient fish pools from 3 replicate tanks in each treatment day;
black bars represent the number of positive pools; and circles represent the mean (6SE) level of virus (plaqueforming units [pfu]/g) in 5 donor fish.
was detected among recipient fish, whether they
had died or had survived the experiment.
Spread of Infection from a Single Donor Fish
To determine the number of secondary cases
produced by one infectious individual, we exposed
approximately 50 rainbow trout (weight, ;1.5 g)
in replicate tanks to a donor fish that had been
infected with IHNV by bath exposure 3 d earlier.
Typical disease signs among donor fish were noted
by 3 dpe, including darkening, exophthalmia, and
fecal casts. Fish in seven randomly selected replicate tanks were harvested at days 2, 4, or 6 after
initiation of the experiment. As shown in Table 1
and Figure 3, on day 2, only three of seven donor
fish were found to be infected with virus. Virus
titer (plaque-forming units; pfu) ranged from 10 3.3
and 104.1 pfu/g of tissue. Of the three tanks of
recipient fish housed with infected donors, two
were found to have infected recipient fish, although the prevalence in each tank was low (2%
and 4%). By 4 dpe, all seven donor fish were infected with titers ranging between 102.7 and 105.3
pfu/g of tissue. Infected recipient fish were detected in five of seven tanks, at a prevalence ranging between 2% and 16%. Similarly, at 6 dpe, all
seven donor fish were infected with IHNV at titers
approximately the same as at 4 dpe. Again, five
of the seven tanks contained IHNV-infected fish,
but the number of infected recipient fish was higher at 6 dpe than at 4 dpe.
None of the mortalities that occurred at 2 or 4
dpe was positive for virus, whereas two fish that
died 6 dpe were positive for IHNV. All of the fish
that died in the three control tanks during the experiment were free of IHNV.
The amount of virus in both recipient and donor
fish increased in the period from 2 to 4 dpe (Figure
3). The increase from 4 to 6 dpe was slight. The
pattern of mean titer increase among donor fish
was reflected in the mean titers obtained from recipient fish. The titer of virus from donors increased 12.3 times from 2 to 4 dpe, whereas prevalence in the recipient fish increased 7.6 times in
the same period. Between 2 and 6 dpe, the level
of infection in donors increased 34.3 times, whereas IHNV prevalence in recipient fish increased 9.5
times. The mean titer was significantly higher in
cohabitant donor fish than in the recipient fish
(two-tailed t-test, P 5 0.0059). There was a significant relationship (nonlinear regression analysis, P 5 0.0006) between the level of infection
among the donors and the prevalence of infection
in the recipient fish with which they cohabitated
(Figure 4). As intensity of infection among donors
increased, prevalence among recipient fish also in-
156
OGUT AND RENO
TABLE 1.—Titers in donors and infection and mortality in recipient fish in an experiment to study initiation of infection
with infectious hematopoietic necrosis virus (IHNV). Twenty-one tanks (50 rainbow trout each) were challenged with
a single IHNV-infected fish by cohabitation. At 2, 4, and 6 d postexposure, fish from seven randomly selected tanks
were sampled along with a tank of control fish. All cohabited fish were individually tested for the presence of virus;
controls were tested as pools of 10 fish.
Days
Replicate
n
Donor titer a
2
1
2
3
4
5
6
7
Control
Average
1
2
3
4
5
6
7
Control
Average
1
2
3
4
5
6
7
Control
Average
50
53
47
53
53
50
51
52
51.13
50
44
50
50
51
50
50
50
49.38
49
51
54
49
45
46
49
51
49.25
10,500
0
0
3,500
2,000
0
0
0
2,290
6,500
37,500
44,500
500
142,000
1,000
200,000
0
61,710
100,000
88,000
200,000
94,000
12,500
9,500
500
0
72,070
4
6
a
Cumulative
mortality (%)
Number
infected
Infected
mortality (%)
0
0
1
2
0
0
0
1
1
0
0
0
2
0
0
0
0.375
1
5
3
0
5
0
8
0
2.75
8
4
10
3
0
2
0
0
3.375
0
0
2
2
1
1
1
2
0
1
3
4
0
2
2
1
2
0
0
0
0
0
0
0
0
0
0
2
0
0
0
% Infected
2
0
0
0
4
0
0
0
0.73
2
11
6
0
10
0
16
0
5.6
16
8
19
6
0
4
0
0
6.9
Plaque-forming units per gram of fish weight.
creased. Even though mean titers among recipient
fish was low, the majority of incidences, that is,
the number of new cases (22), occurred between
2 and 4 dpe (Figure 3).
Discussion
These laboratory experiments, designed to emulate the consequences of the introduction of an
IHNV-infected fish into a naive population of fingerling trout, are the first study of the initiation of
the infectious process by IHNV. The dosage we
used (105.7pfu/mL) was greater than amounts recorded in earlier field studies (7 pfu/mL) in river
water (Batts and Winton 1989); in addition, to assure high infection rates, we also halted the water
flow for the 6-h exposure period. The focus of the
experiments was the potential initiation of infection by a single fish as the infectious unit. As expected, the bath exposure method for donor production was efficient in producing infected fish
(.95% infected), accompanied by high mortality
(75%). We placed single bath-infected fingerlings
in tanks with unexposed fish and monitored the
infectious process in the previously unexposed
fish. When infectious trout were cohabited with
susceptible recipient fish, the level of virus released was sufficient to initiate a primary infection
but not frank disease in cohabitants. Virus titers
in bath exposed donor fish were generally between
104 and 105 pfu/g. Thus, under the conditions of
these experiments, a single infected trout introduced into a susceptible population did spread infection, and subsequent secondary and tertiary infections could lead to epizootics of IHNV. We were
particularly interested in the first 6 d of the process; after the primary infection took place, the
outbreak of secondary and tertiary infections
would have made it extremely difficult to assess
which infected fish derived from which ‘‘pulse’’
of infection (primary, secondary). Further experiments should be undertaken to expand these studies.
One point of interest was to determine the duration of the patent period—that is, the time after
exposure during which the pathogen is not transmitted by indirect contact to susceptible trout—
EARLY KINETICS OF IHNV SPREAD
157
FIGURE 3.—Level of IHNV (mean 6 SE pfu/g in replicate experiments) at 2, 4, and 6 d postexposure (dpe).
Each of 21 tanks holding approximately 50 fish was challenged with a single infected rainbow trout that had been
bath-exposed to IHNV. At 2, 4, and 6 dpe, the experiment was terminated in seven randomly selected tanks. Every
fish used in the experiment was tested for IHNV by cell culture; n 5 the total number of recipient fish from seven
replicates, and NI 5 the total number of infected recipient fish.
FIGURE 4.—Relationship between the titers of donor fish and viral prevalence in recipient fish. Fish in each of
21 tanks holding approximately 50 fish were challenged with a single infected rainbow trout that had been bathexposed to IHNV. At 2, 4, and 6 dpe, the fish in seven randomly selected tanks were killed and assessed for
infection.
158
OGUT AND RENO
and the infectious period. The results obtained here
suggest that 2–3 d were necessary for rainbow
trout exposed to IHNV by bath to become infectious and start shedding virus. However, one of
four pools was positive at day 0. Similar results
were also obtained from the same type of experiments with Aeromonas salmonicida, the agent of
furunculosis (Ogut 2001). It is unlikely that donor
fish became infected and that the IHNV replicated
rapidly and began shedding virus 1 d after exposure to IHNV by bath. If that had happened, more
fish would have been infected 48 h after the initiation of cohabitation. In addition, none of four
pools was positive for the virus 1 d after initiation
of cohabitation experiment. Hence, the single positive pool at day 0 may have resulted from the
presence of the virus on the skin, gill, or mucous,
which then sloughed off to infect others. Yamamoto and Clermont (1990) found that virus was
present in gills and intestine of 2-month-old rainbow trout 16 h postexposure. Virus was still present in the gills 24 h postexposure but not in any
other organs. They did not test mucus. Yamamoto
et al. (1990) also detected virus on the body skin
(ventral) of rainbow trout 24 h after exposure. In
the latter two studies the same IHNV isolate (193110) isolate was used. Thus, it is possible to observe virus presence shortly after exposure of the
host. Presence of the virus on the host may indicate
virus replication, as the authors above suggest, or
perhaps remnants of a recent exposure, as our
study suggests.
Unexpectedly, no virus was detected in recipient
fish after 5 dpe, even though the mean level of
infection of donors continued to rise, reaching
104.91 pfu/mL (Figure 3). Earlier studies had indicated that the presence of virus in infected fish
is transient. The virus could be isolated up to 50
d after an epizootic in young fish (Amend 1975;
Bootland et al. [paper given at the International
Symposium on Aquatic Animal Health, 1995];
Drolet et al. 1995). Similarly, Amend (1975) reported that the virus could not be isolated within
3 weeks after an epizootic or over the next 2 years
until sexual maturity, at which time 33% of the
fish were infected with the virus. Note that being
infected does not mean shedding virus. Our experiment differs from the studies mentioned above
in that we used cohabitation challenge rather than
bath or injection. Moreover, we did not observe
IHN disease in recipient fish, an indications that
the contagiousness of this virus is low relative to
other pathogens. For example, in similar experiments with furunculosis, severe epizootic (.75%)
occurred in 6 d when a single infectious fish was
released with susceptible fish (Ogut, unpublished
data). It was demonstrated with IHNV, however,
that an infection was established and still increasing at day 6. Although this could suggest that an
epizootic might have occurred if allowed more
time, we do not believe that is the case. In a following experiment aimed to determine the density
dependence of the spread of IHNV infection (Ogut
and Reno, in press), donor fish were released indefinitely, yet no full-scale epizootic was observed
and infection levels were similar to those observed
in this study. This outcome is an important phenomenon and needs to be further investigated because immersion or injection methods may lead to
wrong conclusions about epizootiological aspects
of IHN.
We were also interested in determining whether
cohabitation with donor fish would be sufficient
to transfer IHNV to recipient fish. The cohabitation
method for infection transfer is the more natural
way of infection transfer. It emulates arrival of an
infected individual into a naı̈ve population. This
type of infection could be especially important at
the spawning grounds because IHNV is shed in
sexual products at spawning (Burke and Grischkowsky 1984). Spawning fish occupy areas of low
water flow and interactions with other fish in the
same area are often at high density. Added stress
because of physiological changes related to spawning make the fish especially vulnerable to infections. Increased susceptibility is especially important in a river system, where smaller fish in
groups utilize pools to evade predators, resulting
in enhanced virus transfers.
Our results strongly demonstrate that transfer of
virus is possible through cohabitation with even a
single donor fish. Under the conditions of this experiment, infection was transferred but no disease
was observed. It is known from the literature that
IHNV can be transmitted to other fish by cohabitation (Pilcher and Fryer 1980). In a study by
Traxler et al. (1993), sockeye salmon O. nerka and
Atlantic salmon Salmo salar were cohabited in the
same holding area; injecting the sockeye salmon
with IHNV type 3 isolate led to no virus transfer
between fish of the same species but the Atlantic
salmon did become infected. In the same study,
Chinook salmon were injected intraperitoneally
with virus and cohabited with Atlantic salmon and
chinook salmon; no virus was transferred among
cohabited fish. Lastly, Atlantic salmon were infected with the virus and cohabited with naı̈ve Atlantic, Chinook, and sockeye salmon; no virus was
EARLY KINETICS OF IHNV SPREAD
transferred between injected Atlantic salmon and
susceptible Chinook salmon, but the virus was
found in recipient Atlantic salmon and sockeye. In
that experiment, infected fish were fin-clipped and,
as in our experiment, kept in the same tank without
cage or any other separator. The cohabitation
method for exposure in studies testing for risk factors of IHN should especially be encouraged because it involves no artificial stress originating
from exposure to virus by bath or infection method.
In the initiation of infection experiment, one infectious fish (donor) could infect on average of
1.56 fish before it died of IHN. This would indicate
that the IHNV infection would spread among susceptible fish, albeit slowly. In contrast, other studies in our laboratory revealed that one donor infected with Aeromonas salmonicida was capable
of infecting more than 3 recipient fish on average
(Ogut 2001). The amount of virus released from
a single infected individual would probably not be
sufficient to initiate a widespread epizootic, although secondary and tertiary infections might
start an epizootic after longer periods of time. The
low prevalence and lack of frank disease was not
a result of inherent resistance on the part of the
fish used in the experiment; mortality in donor fish
reached 90% after 14 d. Consequently, the absence
of disease and low prevalence in recipient fish
probably reflected the use of a single infectious
fish, which released only small quantities of IHNV
relative to the volume and flow rate. Thus dosage
of pathogen, as anticipated, and duration of exposure may be important factors in initiation of an
IHN epizootic.
To summarize, these experiments indicate that
a single infectious individual housed with susceptible fish is capable of transmitting IHNV, and the
infection spread among recipients is closely related to the level of infection in donor fish. A fullscale epizootic may be observed at greater densities of infectious fish or a slightly more stressful
environment.
Acknowledgments
Funding for this study was provided by the Agricultural Experiment Station of Oregon State University (ORE080) and the Western Regional Aquaculture Center (USDA, Contract #897526). We
also thank two anonymous referees for providing
invaluable comments and advice that helped improve the manuscript.
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