Download Antibody Against Infectious Salmon Anemia Virus Among Feral

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. However, the
evidence that some of these fish presented with ISAV-specific antibodies, even among
those year classes that pre-dated the first known outbreaks of ISAV in the United States,
is consistent with the scientific belief that the virus is prevalent and has evolved with feral
Atlantic salmon for a considerably long period of time (Krossøy et al. 2001, Nylund et al.
2003).
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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
---
---
---
---