DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 63: 33–41, 2005 Published January 25
INTRODUCTION
Herpes-like viral infections were first reported in the
eastern oyster Crassostrea virginica Gmelin in 1972
from the east coast of the USA (Farley et al. 1972). In
1991 in New Zealand and in 1992 in France, larval
Pacific oysters C. gigas Thunberg were reported with
herpes-like viral infections (Hine et al. 1992, Nicolas et
al. 1992) that caused significant mortality. More limited
evidence suggests that herpes-like viruses may infect
and kill juvenile oysters, but the pathological effects of
the virus on juveniles are not well documented. Since
that time, larval Pacific oyster losses have recurred
annually in France and adjacent countries during the
summer months (Renault et al. 1994a,b, 2000b). The
same or similar herpes-like virus(es) has been reported
in other oyster species including flat oysters Ostrea
angasi and Ostrea chilensis in Australia and New
Zealand (Hine & Thorne 1997, Hine et al. 1998) and
Ostrea edulis in France (Renault et al. 2000b). Other
© Inter-Research 2005 · www.int-res.com*Email: carolynf@u.washington.edu
Herpes virus in juvenile Pacific oysters Crassostrea
gigas from Tomales Bay, California, coincides with
summer mortality episodes
Carolyn S. Friedman1,*, Robyn M. Estes1, Nancy A. Stokes2, Colleen A. Burge1,
John S. Hargove1, Bruce J. Barber3, 5, Ralph A. Elston4, Eugene M. Burreson2,
Kimberly S. Reece2
1School of Aquatic and Fishery Sciences, University of Washington, PO Box 355020, Seattle, Washington 98195, USA
2Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA
3340 Hitchner Hall, University of Maine, Orono, Maine 04469, USA
4Pacific Shellfish Institute, PO Box 687, Carlsborg, Washington 98324, USA
5Present address : Department of Marine Science, Eckerd College, 4200 54th Ave S, St. Petersburg, Florida 33711, USA
ABSTRACT: Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June
2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus
(OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific
oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have
occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination
using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis
indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a
single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French
OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay,
suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A
viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-
infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear
pycnosis and fragmentation were observed in infected oysters, which is consistent with previous
observations of OsHV infections in France. Together these data suggest that OsHV may be associated
with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of
a causal relationship warrants further investigation.
KEY WORDS: Pacific oyster · Herpes virus · Mortality · Tomales Bay · California
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Dis Aquat Org 63: 33–41, 2005
bivalve species including manila clams Venerupis
(=Ruditapes) philippinarum and Suminoe oysters
Crassostrea rivularis (=C. ariakensis) are also suscepti-
ble to herpes-like viral infection (Arzul et al. 2001,
Renault et al. 2001). Both intra- and inter-specific
transmission of the virus have been demonstrated (Le
Deuff et al. 1994, Arzul et al. 2001). Davison (2002)
demonstrated that the French OsHV is a member of
the family Herpesviridae and suggested that the
apparent lack of host specificity of OsHV is unique
among members of this family.
Seasonal and severe losses of juvenile Pacific oysters
have occurred annually in Tomales Bay, California,
USA, between mid-April and mid-October beginning
in 1993 and appear to be associated with elevated
spring and summer water temperatures (Cherr &
Friedman 1998, Righetti 2002). Multiple environmental
stressors have been investigated as possible causes of
these losses but no definitive explanation has yet been
found. In the process of conducting a national survey
for the presence of oyster herpes-like virus (OsHV),
and cognizant of the association of OsHV productive
infections with temperatures of 25 to 26°C (Le Deuff et
al. 1994, 1996), we included a preliminary investiga-
tion for the presence of OsHV in Tomales Bay, Califor-
nia. We examined the distribution of this virus in the
USA using the PCR primers developed by Renault et
al. (2000a) and Renault & Arzul (2001).
MATERIALS AND METHODS
Oysters. Pacific oyster larvae and seed were col-
lected from 4 locations along the US west coast
between June 2002 and April 2003 (Table 1). Up to
5 groups of juvenile (0 year class) Pacific oysters
(4 diploid and 1 triploid group) were examined at these
sites. The seed oysters were produced by the Mollus-
can Broodstock Program of Oregon State University
(n = 2 lines: OR-1 and OR-2) and 2 commercial farms in
Washington State (n = 3 lines: WA-1, WA-2-2N and
WA-3-3N) in the late winter of 2002. Juvenile oysters
(<1 cm in shell height) from each cohort were planted
in 0.64 cm mesh oyster culture bags simultaneously in
California (Tomales Bay) and Washington state (Totten
Inlet) in the spring of 2002. Commercially cultured
eastern oysters from Grand Terre, Louisiana, Fort
Pierce, Florida, Gloucester Point, Virginia, and Mus-
congus Bay, Maine, were also examined (Table 1). Lar-
vae and spat were either preserved in 95% ethanol for
storage until DNA extraction or tissues were excised
for immediate DNA extraction (see below). Larvae
were pooled in aliquots of 1 to 2 ml and spat were
pooled (3 to 10 per group) until evidence of OsHV was
found, after which oysters were sampled individually.
Groups of 30 juvenile oysters from each family were
removed from individual mesh bags and the percent-
age of live versus dead juvenile oysters was enumer-
ated by calculating the number of dead individuals
(gaping oysters or empty shells). All west coast sam-
ples were processed at the University of Washington
(UW) and all east and Gulf Coast samples were pro-
cessed at the Virginia Institute of Marine Science
(VIMS). Any OsHV PCR-positive sample was sent to
the alternate laboratory for confirmation.
Histology. A standard cross section that included
mantle, gills, gonad and digestive tissues was excised
ventral to the labial palps from all oysters at each sam-
pling site, placed in invertebrate Davidson’s solution
(Shaw & Battle 1957) and processed for routine paraf-
fin histology. Deparaffinized 5 µm tissue sections from
a subset of oysters that were PCR positive for the OsHV
were stained with hematoxylin and eosin (Luna 1968)
and viewed by light microscopy to characterize mor-
phological changes within these individuals. We were
particularly interested in determining if herpes-like
intranuclear inclusion bodies or other lesions that
might be related to OsHV infection could be found.
Nucleic acid extraction. Initially, samples were pro-
cessed for nucleic acid extraction according to the
methods of Renault et al. (2000a). Briefly, oyster tissues
were homogenized in a Tenbrook homogenizer in dis-
tilled H2O (0.1 g tissue ml–1 dH2O) and incubated in a
boiling water bath for 10 min. Samples were then
mixed and centrifuged at 9000 × g for 10 min. The
supernatant was collected and stored at –20°C. DNA
quality was poor in these samples as evidenced by gel
electrophoresis, and nucleic acid was extracted from
subsequent samples using the QIAamp DNA Mini Kit
(Qiagen) according to the manufacturer’s protocol.
Polymerase chain reaction. Herpes-like virus: Posi-
tive control OsHV DNA was provided by T. Renault
(IFREMER, La Tremblade, France). We employed the
primers and PCR test conditions of Renault et al.
(2000a) and Renault & Arzul (2001) (primers A3/A4,
A5/A6 and C2/C6). Briefly, each 20 µl reaction con-
tained 10× PCR buffer (10 mM Tris, pH 8.3; 50 mM
KCl), 2.5 mM MgCl2, 8 µg BSA, 0.2 mM dNTP mix,
4 pmol each primer, 1 U Taq polymerase (Promega)
and 0.4 µl of template DNA. Following amplification
with primers A3/A4, 0.4 µl of the reaction mixture was
added to the A5/A6 reaction mixture for the nested
reaction.
Duplicate nucleic acid amplifications were per-
formed with an initial denaturation step at 94°C for
2 min, followed by 35 cycles at 94°C for 1 min, 50°C for
1 min, and 72°C for 1 min, with a final elongation at
72°C for 5 min. Products were separated on 1.0%
agarose gels containing 0.1 µg ml–1 ethidium bromide
and visualized using a UV transilluminator. The nested
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