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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. References Amend, D. F. 1975. Detection and transmission of infectious hematopoietic necrosis virus in rainbow trout. Journal of Wildlife Diseases 11:471–478. 159 Batts, W. N., and J. R. Winton. 1989. Concentration of infectious hematopoietic necrosis virus from water samples by tangential flow filtration and polyethylene glycol precipitation. Canadian Journal of Fisheries and Aquatic Sciences 46:964–968. Bovo, G., G. Giorgetti, P. E. V. Jorgensen, and N. J. Nelson. 1987. Infectious hematopoietic necrosis: first detection in Italy. Bulletin of the European Association of Fish Pathologists 7:124. Burke, J., and D. Mulcahy. 1980. 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