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ICES CM 2008/D:02 (Not to be cited without prior reference to the author.) Antibody Against Infectious Salmon Anemia Virus Among Feral Atlantic Salmon (Salmo salar) Rocco C. Cipriano USGS/National Fish Health Research Laboratory, 11700 Leetown Road Kearneysville, WV 25430, P: 304/724-4432; F: (304) 724-4435 E: [email protected] Key Words: Indirect ELISA, Atlantic salmon, Salmo salar, antibody, feral fish Running Title: ISAV antibody in feral Atlantic salmon Journal: ICES CM 2008/D-02 Abstract Archived sera from Atlantic salmon (Salmo salar) that returned to the Penobscot River (Maine), Merrimack River (Massachusetts), and Connecticut River (in Massachusetts) from 1995 until 2002 were analyzed for antibodies against Infectious Salmon Anemia Virus (ISAV)using an enzyme-linked immunosorbent assay (ELISA). A maximum of 60 samples were archived per river system per year. In any given year, the number of fish sampled by ELISA for ISAV-antibodies in the Penobscot River ranged from 2.9–11.2%, whereas the range of salmon sampled was between 31.3–100% and 20.0–67.5% in the Merrimack River and the Connecticut River, respectively. Archived sera were not available for the 1995 and 2002 year classes from the Connecticut River. A total of 1,141 samples were processed and 14 serum samples tested positive for antibodies to ISAV. In the Penobscot River, serum from one fish tested positive in each of the 1995 and 1999 year class returns and sera from two fish tested positive in the 1998 returns. In the Merrimack River, sera from four fish tested positive in each of the 1996 and 1997 returns and sera from two fish were positive in the 2002 return. None of the archived sera from Atlantic salmon that returned to the Connecticut River tested positive. Introduction Infectious Salmon Anemia Virus (ISAV) is a highly contagious disease (Thorud and Djupvik 1988) that has been associated with Atlantic salmon (Salmo salar) in Scotland (Bricknell et. al. 1998), Canada (Lovely et al. 1999; Jones et al. 1999), the United Kingdom (Rodger et al. 1999), the United States (Bouchard et al. 2001), and the Faroe Islands (Anonymous 2000). The virus has also been isolated from coho salmon (Oncorhynchus kisutch) in Chile (Kibenge et al. 2001). Although ISA epizootics have not been documented among wild salmon, Canadian Department of Fisheries and Oceans biologists have detected the virus in a few fish that had escaped from farms and returned to the Magaguadavic River fish trap in the Bay of Fundy, New Brunswick. In one occasion the virus was also found in a limited population of wild Atlantic salmon kept in a land-based facility that was supplied fed with brackish water. The origin of the infection in the latter population was never confirmed, but the fish could either have been infected in the wild or in the fish trap by cohabitation with infected escapees. (Olivier et al. 2002). The virus spreads from fish to fish by shedding of virons from the blood, gut, urine, and epidermal mucus of infected salmon (Totland et al. 1996) and fish may transmit the disease weeks before they themselves show apparent signs of infection. The disease is pronounced within net-pen culture in the marine environment, where it is most often transmitted by cohabitation with infected live salmon or tainted biological materials (Nylund et al. 1994, Vagsholm et al. 1994, Rolland and Nylund 1998a) and contaminated well boats (Shannon 1998; Murray et al. 2002). In November 2000, the U.S. Department of Commerce National Oceanic and Atmospheric Administration – National Marine Fisheries Service Fisheries and the U.S. Department of Interior United States Fish and Wildlife (USFWS) listed Atlantic salmon in Maine rivers as an endangered species (Federal Register 2000). In support of this listing was the threat posed by certain Atlantic salmon diseases, including ISA. After feral Atlantic salmon return from ocean migrations, these fish are captured in their natal rivers and transported to holding facilities where they are held for up to six months before they can be spawned. Failure to detect individual infected ISAV fish upon capture, however, could result in the general contamination of sea run year classes and their communal holding facilities in which they are maintained. Such an event would minimally necessitate the depopulation of one or more restoration facilities and the consequent loss of single or multiple year classes of salmon associated with a given river system. Because these populations are small and genetically constitute distinct population segments (King et al. 2001; Spidle et al. 2001), individuals cannot be sacrificed to determine the status of ISAV infection. The principal element to manage and control this pathogen within the New England restoration program, therefore, is to ensure that ISAV-positive fish do not enter the prespawning holding facilities. Development of a reliable non-lethal assay that detects ISAV infected fish is critical to the achievement of this end. The virus is cultured in Atlantic salmon head kidney (SHK-1) cells (Dannevig et al. 1995a,b) and on the Chinook salmon embryo (CHSE-214) cell line (Bouchard et. al. 1999). Focal cytopathic effects (CPE) associated with ISAV replication in CHSE-214 cells enables the use of this cell line for diagnostic purposes. Unfortunately, not all lineages of CHSE-214 cells support replication of all ISAV isolates (Kibenge et. al. 2000), and the absence of CPE in SHK-1 cells limits the use of this cell line for diagnostic purposes. Use of both cell lines, therefore, is more sensitive than the use of either cell line alone (Opitz et al. 2000). The recently developed TO (Wergeland and Jakobsen 2001, Grant and Smail 2003) and ASK (Rolland et al. 2002) cell lines may further enhance cell culture-based diagnosis of ISAV because both of these cell lines support viral replication and show defined CPE. Nonculture based diagnostics to detect ISAV include an indirect fluorescent antibody test (Falk and Dannevig 1995a) and a reverse transcriptase-poylmerase chain reaction (RTPCR) procedure (Mjaaland et. al. 1997). Devold et al. (2000) found RT-PCR to be more sensitive for detection of ISAV among carrier sea trout, than either culture or injection of suspect blood samples into naïve fish. However, most existing diagnostics require the sacrifice of the host, which would be unacceptable with the endangered populations of Atlantic salmon listed in the rivers of coastal Maine. Consequently, it is imperative that fish health monitoring on such endangered populations of fish must be accomplished by non-lethal assay. Within such populations, variations of the PCR procedure described by Mjaaland et al. (1997) and the cell culture methods of Falk and Dannevig (1995a) are currently used to diagnose ISAV (Bouchard et. al. 1999, Jones et. al. 1999, Lovely et. al. 1999) from blood samples that do not harm the fish. Atlantic salmon that survive infection with ISAV develop a specific antibody response against the pathogen (Falk and Dannevig 1995b). Researchers and diagnosticians at the Atlantic Veterinary College University of Prince Edward Island and Aquagestion - Fundacion Chile have used this basic immunological principal to develop an indirect enzyme-linked immunosorbent assay (ELISA) that detects antibodies to ISAV in Atlantic salmon sera (Kibenge et al. 2002). Because serum samples can be easily acquired by drawing a small volume of blood from the hemal arch of fish, ELISA the assay is nonlethal and does not adversely affect fish health. This study was conducted to determine if the ISAV-antibody ELISA could detect seroconversion in individually archived samples of sea run Atlantic salmon from New England rivers that predated the first reported occurrences of this disease in North America. Materials And Methods At the end of each annual spawning season, as many as 60 Atlantic salmon from each of the Penobscot, Merrimack, and Connecticut Rivers are sampled for fish health analysis. In addition to other tests, approximately 3.0 mL of blood is drawn from the hemal arch of individual salmon. Blood is allowed to clot and the clot is separated from the walls of a glass test tube using a wooden applicator. The tube is placed at 4oC overnight and the blood is allowed to separate. Serum is removed from each sample with a Pasteur pipette, placed in a cryovial tube, and stored at –70oC. In order to determine whether or not storage at –70oC affected the stability of Atlantic salmon antibodies, microtiter agglutination assays against Aeromonas salmonicida formalin-killed whole cell antigens were conducted on the sera that were obtained from the 1996 year class of salmon, which had returned to the Merrimack River. Formalin- killed antigens were prepared as described by Anderson and Dixon (1989) using an isolate of A. salmonicida that was previously obtained from a population of Atlantic salmon from the Richard Cronin National Salmon Station (Sunderland, MA) in 1990. Microagglutination assays were conducted as described by Rose and Bigazzi (1980). These results were compared to the data that had been previously obtained in a similar manner from these very same salmon in 1996, when the sera were originally obtained. Means of the antibody titers derived from these fish in 1996 and again in 2003 after they had been archived at –70oC for eight years were calculated and compared by a paired ttest (P=0.05) using SygmaStat software (Jandel Scientific Software, Inc.; San Rafael, CA). The indirect sandwich ELISA that detects ISAV antibodies was conducted as described by Kibenge et al. (2002). Briefly, 96-well ELISA plates are coated with either 100 µl of purified ISAV (positive) antigen (Atlantic Veterinary College., University of Prince Edward Island) or SHK-1 cell (negative) antigen (Atlantic Veterinary College., University of Prince Edward Island) in 0.2M NaHCO3 (Fisher Scientific, Fair Lawn, NJ) per well. Plates were held overnight at 4oC. Wells were then washed three times (10 minutes per wash) using Dulbecco’s phosphate-buffered saline without calcium or magnesium chloride (Gibco, Invitrogen Corp., Carlsbad, CA) containing 0.05% Tween20 (Bio-Rad Laboratories, Hercules, CA; TPBS) and drained by inversion. Wells were then blocked for one hour at room temperature by addition of 100 µl of 3% goat serum in TPBS per well. Dilutions of test sera, positive control serum, and negative control serum were then prepared in TPBS containing 1% goat serum (antibody buffer) and 100 µl of each sample was added in triplicate wells containing either the ISAV antigen or the SHK1 antigen. Plates were then incubated for 90 minutes at 16oC, washed three times in TPBS as described previously, and 100 µl of a 1:500 dilution of monoclonal antiserum to Atlantic salmon immunoglobulin (1PA3D1; Atlantic Veterinary College., University of Prince Edward Island) prepared in antibody buffer was added to each well for 60 minutes at 37oC. Wells were then washed three times in TPBS, drained, and 100 µl of a 1:1500 dilution of goat anti-mouse IgG-1 (gamma) alkaline phosphatase conjugate (Caltag Laboratories, Burlingame, CA) was added to each well for 60 minutes at 37oC. Plates were washed three times in TPBS and 100 µl of alkaline phosphate substrate solution (Bio-Rad Laboratories, Hercules, CA) was added to each well. Plates were allowed to incubate overnight in the dark at 18oC. Color development was arrested by the addition of 100 µl of 0.4N NaOH (Fisher Scientific, Fair Lawn, NJ) and optical density was read at 405 nm in an automatic microtiter ELISA reader equipped with Soft Max Pro software (Molecular Devices, Sunnyvale, CA). This ELISA assay for detection of ISAV antibodies in Atlantic salmon sera is somewhat complex and plates serum samples against two antigens: ISAV (control positive) and SHK-1 (control negative) antigens. If the readings obtained from triplicate samples of the same serum incubated against the ISAV antigen is statistically greater (P<0.05) than the same sample incubated against the SHK-1 antigen and the difference between the means exceeds an optical density of 0.24; then results indicate that the fish possess antibodies against ISAV. were performed using Systat 10 software (SPSS, Inc., Chicago, IL.). Statistical analyses Results and Discussion Although most antibodies are stable for years when stored at –20oC (Harlow and Lane 1988), one could question whether or not antibody stability was affected in the archived sera, some of which dated to 1995 salmon returns. Even the best of assays would be rendered ineffective, if the target of the assay was degraded by the method of sample storage. To address this point, it was fortunate that the salmon that return to the Connecticut and Merrimack Rivers were vaccinated against A. salmonicida when they were moved to captive broodstock facilities where they are held from four to six months until the fish were spawned. A data set (n=60) of titers for antibodies against A. salmonicida was available for the class of Atlantic salmon that had returned to the Merrimack River in 1996. These titers had been determined approximately six months after the fish had been taken from the river and vaccinated against furunculosis. Comparison of these titers with those produced from the same samples, which had been stored at –70oC for eight years, were not statistically different. When the 60 serum samples derived from the Atlantic salmon that had returned to the Merrimack River in 1996 were initially processed, the mean response was 6.433 (std. dev. 3.186, std. error of the mean 0.411) against formalin-killed whole cell antigens of A. salmonicida. After the same samples had been stored for eight years at –70oC, the mean response was 5.917 (std. dev. 2.204, std. error of the mean 0.285). Statistical analysis indicated that although the mean titer was a little lower after storage, the difference was not statistically significant (paired t-test(59 degrees of freedom) = 0.311). This provided direct evidence that the titers remained stable in the sera samples that were archived in the manner explained in this study. Thus, it was reasoned that antibodies for ISAV would show similar stability and could be detected by the ELISA test as described by Kibenge et al. (2002). Four of these 1996 Merrimack River fish tested positive for ISAV antibodies (Table3). The log(2) microagglutination titers against A. salmonicida formalin-killed antigens in these four fish were 6, 4, 10 and 6. Another 26 sera from this year class produced log(2) agglutinin titers against A. salmonicida equal to or greater than 7, but all of these samples tested negative by ELISA for ISAV-antibodies. The negative results produced with the ELISA test for ISAV-specific antibody on these latter 26 samples, indicated that the monoclonal antibodies and reagents produced for the ISAV–antibody ELISA did not cross-react with trout immunoglobulin developed against another organism (e.g. – A. salmonicida). This point would be further substantiated by the data obtained from other Connecticut River fish. Although those fish were not titrated for A. salmonicida-specific antibodies, all of them had been vaccinated against furunculosis and one could readily infer that injection vaccination stimulated antibody formation, as it did in the 1996 Merrimack River fish. However, none of the Connecticut River salmon yielded a positive ISAV-antibody result. Table 1 indicates the total number of Atlantic salmon that returned to specific river systems during the time that archived samples were processed. Also presented are the numbers of samples that were archived and tested for the presence of ISAV antibodies for respective year and river classes of Atlantic salmon. A maximum of 60 samples were archived per river system per year. Because of the sheer numbers of returns, a much smaller percentage of Atlantic salmon were sampled for ISAV from the Penobscott River than those stocks from the Merrimack and Connecticut Rivers. In any given year, the number of fish sampled by ELISA for ISAV antibodies in the Penobscot River ranged from 2.9–11.2%, whereas the range of salmon sampled was between 31.3– 100% and 20.0–67.5% in the Merrimack River and the Connecticut River, respectively. Archived sera were not available for the 1995 and 2002 year classes from the Connecticut River. A cumulative total of 1,141 samples were processed and 14 samples (about 1.2%) tested positive for antibodies to ISAV. In the Penobscot River, one fish tested positive in each of the 1995 and 1999-year class returns and two fish were positive in the 1998 returns (Table 2). In the Merrimack River, four fish tested positive in each of the 1996 and 1997 returns and two fish were positive in the 2002 return (Table 3). None of the fish that returned to the Connecticut River tested positive. These results, especially those derived from as early as the 1995 salmon returns, suggest indirect evidence of viral exposure which possibly pre-dates the original detection of ISAV in Atlantic salmon from the United States (Maine, U.S.A; Bouchard et al. 2001). Several investigators have now described phylogenetic differences between North American and European isolates of ISAV (Blake et al. 1999, Cunningham and Snow 2000, Kibenge et al 2000, Krossøy et al. 2001). From calculations of mutation rates for the ISAV polymerase gene, Krossøy et al. (2001) have estimated that Norwegian strains of ISAV diverged from North American isolates around 1900; - a period of time that also coincided with transport of certain salmonid species between the continents. Although these authors caution that mutation rates do not perfectly correlate with time, their findings suggested that isolates of ISAV were present in North America long before the first reported outbreaks of disease (Lovely et al. 1999; Jones et al. 1999, Bouchard et al. 2001). Such a suggestion is consistent with the finding, in this study, that ISAV antibodies were detected in three year class returns of feral Atlantic salmon (1995, 1996, and 1997), before the first reports of ISAV disease in the United States. Furthermore, the current study was unable to correlate an increased spur in either the frequency or distribution of ISAV-specific antibodies among Atlantic salmon returns that post-dated the outbreaks in the Bay of Fundy, Cobscook and Passamaquoddy Bays. Although blood and mucus of infected fish is highly contagious (Rolland and Nylund 1998b), it does not appear that these initial epizootics served as significant focal reservoirs of infection that stimulated antibody production among transient, feral salmon. Possibilities exist for other routes of infection among feral Atlantic salmon returning to New England Rivers that do not necessarily include epizootics at net-pen aquaculture sites. It has been established that the virus may be harbored without apparent disease in other feral anadromous salmonids, which are themselves capable of transmitting the agent to susceptible Atlantic salmon (Nylund and Jakobsen 1995, Nylund et al. 1995, Rolland and Nylund 1998a), and that sea lice (Nylund et al. 1994) may serve as vectors for infection. The degree to which such transmission, effected in the natural environment, evokes the development of disease or production of humoral antibody is not yet understood. It is important to keep in mind some criticisms about these findings. Kibenge et al. (2002) have shown that their antibody ELISA test has correlated well with results from PCR and viral neutralization assays, but this assay has not been certified by any scientific authority for use as a validated diagnostic nor was it incorporated into this study to sanction its use in fish health certification programs. It was believed, however, that this assay was the most practical research tool available that could provide indirect evidence of ISAV exposure using the archived sera of Atlantic salmon that were still available long after the carcasses or other tissues of these fish had been destroyed. At face value, these results suggested that a small number (about 1.2%) of these returning salmon had, over the course of time, developed antibodies. Because only 1.2% of the samples tested were positive and the viral status of the host animals was unknown, however, it is indeed valid to question if whether some or even all of the 14 positive samples were actually false positives. It is impossible for this author to definitively refute such criticism and, therefore, caution is advised when reviewing the degree of sero-conversion that was measured. However, as a federal entity involved in the recovery of Atlantic salmon runs in the northeastern United States and in light of full disclosure about the nature of these returning salmon, the data are presented as positive test results as described by Kibenge et al. (2002). Furthermore, the presence of antibody itself does not necessarily correlate with the presence of active infection and, thus far, no overt disease condition or clinical signs of infection have been noted among feral Atlantic salmon that have returned to New England rivers. In 2001, however, personnel from the U.S. Fish and Wildlife Service (USFWS) Fish Health Unit (Lamar, PA) began to annually sample the blood of Atlantic salmon returning to the Penobscot River for the presence of ISAV using tissue culture and RT-PCR. During their 2001 screen, one Atlantic salmon produced a weak PCR positive-result upon its immediate capture from the Penobscot River (Barbash 2003). This was sequenced and found to be 97% homologous to the Scottish/Norwegian strain of ISAV (Barbash 2003; Blake et al. 1999). Tissue culture assays run from blood samples of this fish were negative. The serum from this particular fish was among the 2001 archived samples that were processed for ISAV antibodies. Unfortunately, there was no way of denoting which of the 60 samples from these returns belonged to the specific PCR positive salmon. Suffice it to say that all of the individuals in this population tested negative for ISAV-antibodies. The USFWS tissue culture and RT-PCR screens for subsequent years have thus far been negative. The collective USFWS findings, however, do suggest that there is a low prevalence of the virus in feral salmon returning to the Penobscot River, which is a finding that is consistent with the low prevalence of ISAV antibodies produced by the ELISA test among the archived sera. In light of this, it would have been more disconcerting if the ELISA test had detected greater prevalences of antibody than those which were actually reported in this study. On the other hand, I did not have sera archived for all of the fish that returned to the specific rivers. It is possible that there may (or may not) have been even more fish that seroconverted to ISAV among those returns for which there were no archived samples. This is further compounded by the fact that due to the larger Atlantic salmon runs in the Penobscot River, only 2.9 – 11.2% of the salmon from any given return were sampled and these would have been the closest feral fish to net-pen cultures of Atlantic salmon that sustained frank disease. Because none of the Atlantic salmon within the United States restoration program are vaccinated against ISAV, it was presumed that the antibodies detected by the ELISA test resulted from some form of natural exposure to the virus. Occasionally, small numbers of salmon that escape aquaculture pen sites are found in Maine rivers and aquaculture salmon are vaccinated against ISA. However, a commercial ISA vaccine was not licensed in Canada or the United States until 1999 (Kibenge et al. 2003) and, therefore, one could assume that antibodies detected before 1999 (Penobscot River returns in 1998) did not result from vaccination. Through combinations of visual inspections at the site of capture, tagging and marking, and age determinations by scale analysis, all efforts are expended by biologists within the Penobscot River program to ensure that any possible aquaculture escapees are excluded from the captive population of sea-run fish. Consequently, it is highly improbable that any of the current results were impacted by aquaculture escapees. Furthermore, there are no commercial aquaculture operations that impact feral returns as far south as either the Merrimack or Connecticut Rivers. These data highlight the need to further investigate correlations between diagnostic assays that detect infection (e.g. - PCR or tissue culture) versus those which provide indirect evidence of exposure based on production of specific antibodies. As developers of the ISAV-antibody ELISA (Kibenge et al. 2002) work toward its validation, studies should be initiated to assess how results from the ISAV-antibody ELISA correlate with results from tissue culture and PCR analysis among populations of salmon that have been infected over a course sufficient to induce antibody development. Until such time as this assay undergoes an impartial validation, the results of the current study should be interpreted to provide presumptive but not definitive evidence of exposure. 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Dis Aquat Org 44:183-190. Table 1: Total and archived sera samples for Atlantic salmon returning to New England Rivers. Percent of return screened for ISAV antibodies River Year of return Total number of returns Number of sera archived Penobscott 2002 2001 2000 1999 1998 1997 1996 1995 779 785 535 958 1,210 1,355 2,045 1,342 60 60 60 60 60 60 60 60 Merrimack 2002 2001 2000 1999 1998 1997 1996 1995 57 84 85 192 123 71 76 34 57 60 60 60 60 60 60 30 100.0 71.4 70.5 31.3 48.8 84.5 78.9 88.2 Connecticut 2002 2001 2000 1999 1998 1997 1996 1995 44 40 77 154 300 199 260 188 Not available 27 37 60 60 60 60 Not available --67.5 48.1 39.0 20.0 30.2 23.0 --- 7.7 7.6 11.2 6.3 5.0 4.4 2.9 4.5 Table 2: Summary of positive ELISA results for detection of Infectious Salmon Anemia Virus (ISAV) antibodies in feral Atlantic salmon returning to the Penobscot River Year of salmon return Number of salmon Number of salmon positive Mean ISAV o.d. of positive samples Mean SHK-1 o.d. of positive samples Difference P Value of the of positive means for samples positive samples 2002 60 0 --- --- --- --- 2001 60 0 --- --- --- --- 2000 60 0 --- --- --- --- 1999 60 1 0.359 0.044 0.315 0.033 1998 60 1 2 0.594 0.222 0.288 -0.016 0.306 0.240 0.012 0.023 1997 60 0 --- --- --- --- 1996 60 0 --- --- --- --- 1995 60 1 0.142 -0.182 0.324 0.05 Table 3: Summary of positive ELISA results for detection of Infectious Salmon Anemia Virus (ISAV) antibodies in feral Atlantic salmon returning to the Merrimack River Year of salmon return Number of salmon Number of salmon positive Mean ISAV o.d. of positive samples Mean SHK-1 o.d. of positive samples Difference P Value of the of positive means for samples positive samples 2002 60 1 2 1.308 0.568 0.695 0.316 0.613 0.252 0.028 0.002 2001 60 0 --- --- --- --- 2000 60 0 --- --- --- --- 1999 60 0 --- --- --- --- 1998 60 0 --- --- --- --- 1997 60 1 2 3 4 1.773 1.149 0.604 0.515 0.108 0.296 0.252 0.170 1.665 0.853 0.352 0.345 0.033 0.041 0.005 0.041 1996 60 1 2 3 4 0.316 0.596 0.319 0.318 0.068 0.223 0.056 -0.061 0.248 0.373 0.263 0.378 0.012 0.029 0.041 0.016 1995 30 0 --- --- --- ---