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Applied and Environmental Microbiology, September 2000, p. 3924-3930, Vol. 66, No. 9
Department of Biochemistry, Microbiology, and
Molecular Biology1 and School of Marine
Sciences,2 University of Maine, Orono, Maine
04469
Received 2 May 2000/Accepted 6 July 2000
Juvenile oyster disease (JOD) causes significant annual mortalities
of hatchery-produced Eastern oysters, Crassostrea
virginica, cultured in the Northeast. We have reported that a
novel species of the Juvenile oyster disease (JOD) refers
to a syndrome of unknown origin that results in seasonal mortalities of
hatchery-produced juvenile Crassostrea virginica raised in
the northeastern United States (9, 11, 17). While the
severity of the annual epizootics has been variable since they first
appeared in the late 1980s, mortalities in some years have exceeded
90% of total production at JOD enzootic sites in Maine, Massachusetts,
and New York (9, 11, 39, 40). Typical external signs of JOD
include a reduction in growth rate, the development of fragile and
uneven shell margins, and cupping of the left valve. Internally, signs
of JOD usually include mantle retraction and lesions and proteinaceous
deposits (conchiolin) on the inner shell surfaces (9, 11,
17). Such signs usually appear within 4 to 6 weeks after
deployment of seed at enzootic sites, and they immediately precede
mortality events during which losses may exceed 50% of total
production in a single week (3, 7).
Several hypotheses concerning the etiology of JOD have been explored,
and evidence indicates that the disease is infectious rather than due
to nutritional and/or abiotic factors (9). Although no
obvious agent has been identified in histological samples (9, 15,
39, 40), the pathology and correlating environmental factors
(e.g., warm temperatures and moderate salinity) have led to
investigations of a possible bacterial (9, 18, 31) or
protistan etiology (14, 33, 44). Vibrio spp., in particular, have been investigated (18, 31) because of the similarities between the signs of JOD and those of brown ring disease
of manila clams, caused by Vibrio tapetis (8, 16, 36,
37). JOD has been reproduced in animals both by injection with
homogenates from affected animals (38) and by proximity to
JOD-affected animals in experimental aquaria (34). These experiments further support the involvement of an infectious agent such
as a bacterium or protozoan.
In 1997, for the purpose of further elucidating the etiology of JOD, we
tested the effect of two antibacterial antibiotics (norfloxacin and
sulfadimethoxine-ormetoprim) on JOD mortalities of cultured juvenile
C. virginica (7). Repeated immersion in either
antibacterial solution resulted in a delay in the onset of JOD
mortalities in treated animals, reduced weekly mortality rates, and a
statistically significant reduction in cumulative mortalities compared
to that of controls. Bacteriological analyses revealed that healthy
oysters generally harbored low numbers of phenotypically diverse
bacteria. In contrast, JOD-affected animals were found to be
extensively colonized by a previously undescribed species of the marine
To date, the CVSP Bacterial cultures.
Bacteria were isolated and maintained on
a seawater-based complex medium (SWT) (6). CVSP strain
CV919-312 was isolated from a JOD-affected animal during the 1997 epizootic (7). All other bacterial isolates originated from
this study.
Oyster deployments.
Approximately 6,000 juvenile C. virginica (2.5-mm shell height) were obtained from a local
hatchery on 26 June 1998. Ten groups of 500 animals each were placed
into mesh bags constructed of fiberglass window screen at a density of
5,000 m Sampling protocols.
Animals were examined weekly for JOD
signs and mortality, and sampling for bacteriological analyses was
conducted at least biweekly. A minimum of three animals were removed
from each tray, sealed in waterproof baggies, and packed in ice.
Animals from any given tray were processed together, and unless
otherwise noted, the entire meats were sampled. After being rinsed with
filter-sterilized seawater, the hinge ligament and adductor muscle of
each oyster were severed using a flame-sterilized scalpel and forceps.
In every instance, we verified that animals included in the
bacteriological analyses were alive. This was done by observing the
heartbeat under the dissecting microscope and testing for contraction
of the tissue and mantle in response to pressure. The tissues were then
combined in a preweighed microcentrifuge tube containing 0.35 ml of
sterile 70% seawater (natural seawater diluted to 70% in double-
distilled water [ddH2O]) and were homogenized using a
sterile pellet pestle. After being reweighed, the homogenates were
serially diluted in sterile 70% seawater, and 20 µl of each dilution
was spread onto SWT agar.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Additional Evidence that Juvenile Oyster Disease Is
Caused by a Member of the Roseobacter Group and Colonization
of Nonaffected Animals by Stappia stellulata-Like
Strains
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-proteobacteria Roseobacter group
(designated CVSP) was numerically dominant in JOD-affected animals
sampled during the 1997 epizootic on the Damariscotta River, Maine. In
this study we report the isolation of CVSP bacteria from JOD-affected
oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in
animals raised at a JOD-free site. Animals raised at the JOD enzootic
site that were unaffected by JOD were stably and persistently colonized
by Stappia stellulata-like strains. These isolates
(designated M1) inhibited the growth of CVSP bacteria in a
disk-diffusion assay and thus may have prevented colonization of these
animals by CVSP bacteria in situ. Laboratory-maintained C. virginica injected with CVSP bacteria experienced statistically
significant elevated mortalities compared to controls, and CVSP
bacteria were recovered from these animals during the mortality events.
Together, these results provide additional evidence that CVSP bacteria
are the etiological agent of JOD. Further, there are no other
descriptions of specific marine -proteobacteria that have been
successfully cultivated from a defined animal host. Thus, this system
presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the
invertebrate host in the ecology of these marine -proteobacteria.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-proteobacteria, Roseobacter group. A role for this
bacterium (designated CVSP) as either the primary etiological agent or
an efficient colonizer of JOD-affected animals was further supported by
the fact that they either were not recovered from or were present at
very low levels (<1% of total CFU) in animals that survived the JOD epizootic.
-proteobacterium represents the only organism that
has been definitively correlated with any JOD epizootic. The objectives
of this study were to determine (i) if the presence of CVSP bacteria
was a consistent feature of subsequent epizootics, (ii) if CVSP
bacteria were present in oysters raised at a site where JOD is not
enzootic, and (iii) if JOD could be reproduced under laboratory
conditions by exposure of juvenile C. virginica to a CVSP isolate.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2. Each bag was then attached to a rectangular
frame constructed of 0.5-in. polyvinyl chloride pipe and was placed
into buoyant polyethylene trays. Three trays were deployed at a
commercial shellfish nursery on the Damariscotta River, Maine
(44°1'N, 69°32'W), and the remaining seven trays were deployed at a
commercial nursery on Maquoit Bay, Maine (43°50'N, 70°03'W).
Animals in four of the trays at Maquoit Bay were removed after 1 month
and used for bacterial challenge experiments (see below). A deployment
of juvenile oysters from another local hatchery was performed on 12 August 1998. A total of 2,000 animals were distributed into each of
four bags as described above. Three of these were deployed in trays at
the Damariscotta River site and were monitored and sampled as described below. The fourth bag was deployed at Maquoit Bay for the purpose of
comparing the bacteriology of these animals with those raised on the Damariscotta.
Exposure of juvenile oysters to CVSP bacteria by immersion. Four trays of animals (approximately 2,000 animals total) were removed from Maquoit Bay on 23 July 1998. The animals (mean shell height, 8.4 mm) were divided into 9 groups of 200 animals each and placed into each of 9 randomly assigned 5.5-gal aquaria containing aerated, UV-sterilized artificial seawater (Crystal Sea Marinemix; Marine Enterprises Intl., Baltimore, Md.) at a salinity of 31 ppt. Oysters were maintained on algal spat formula (Innovative Aquaculture Ltd., New Brunswick, Canada), and standard aquarium heaters were used to maintain water temperatures between 19 and 21°C. After a 1-week acclimation period, a culture of CVSP strain CV919-312 (grown to an optical density at 600 nm [OD600] of 1.0) was added to three aquaria at a final concentration of 106 CFU/ml. Another three aquaria each received a diluted inoculum of CV919-312 (103 CFU/ml final concentration), and the remaining three aquaria each received an equivalent volume (40 ml) of SWT medium only. Thereafter, CVSP bacteria (at either 103 or 106 CFU/ml, final concentration) or medium alone was added to the assigned aquarium three times weekly for 5 months. Animals were fed daily and monitored weekly for signs of JOD. One month after the start of the experiment, two animals from a tank receiving the 106 CFU/ml dose of CV919-312 and two animals from a control tank were removed for bacteriological analysis. Each animal was processed individually by removing the tissues, homogenizing, diluting in 70% filter-sterilized seawater, and plating onto agar media as described above.
Exposure of juvenile oysters to CVSP bacteria by injection. Juvenile oysters (C. virginica) (mean shell height, 36 mm) were obtained from Middle Peninsula Aquaculture (North, Va.) in late September 1999. They were acclimated for 2 weeks in laboratory aquaria containing aerated artificial seawater at ambient room temperature (20 to 22°C), and were fed a maintenance diet of algal paste. An inoculum of CVSP strain CV919-312 was prepared by diluting an overnight broth culture 1:100 in fresh SWT broth and incubating with shaking (250 rpm) at 25°C until the OD600 was 1.5 (approximately 7 h). The animals were then randomly assigned to either the treatment or control group and were notched on the right valve margin to facilitate delivery of the inoculum. One hundred microliters of the CVSP culture (approximately 3.3 × 107 cells) was injected through a 23-gauge needle on a repeating pippetor into the mantle cavity of each animal in the treatment group (n = 121). Each animal in the control group (n = 122) was similarly injected through the notch on the valve margin with 100 µl of sterile SWT medium. In all cases, the needle was positioned so that the inoculum was delivered directly into the mantle cavity and not into the soft tissues of the animals. After injection, oysters were kept out of the water for 4 h and then were distributed into six (randomly assigned) aquaria; three held treatment animals and three contained the control animals (n = 44 ± 1 per aquarium). The animals were fed every other day and monitored weekly for JOD signs and deaths. During mortality events in treatment aquaria, two animals (one each from the first and the last treatment aquarium to show mortalities) were removed for bacteriological analyses.
Statistical analysis.
Percent cumulative mortality was
calculated as previously described (7). The data were
subjected to the arcsine transformation (49) prior to a
standard one-way analysis of variance and Dunnet's post-hoc test
( = 0.05) using the Statmost 3.5 statistical package (Dataxiom
Software, Inc., Los Angeles, Calif.).
Phenotypic analyses of bacteria.
Determinations of Gram
reaction, motility (in a wet mount), catalase activity, oxidase
activity, nitrate reductase, and reaction in oxidative-fermentative
media were performed according to standard methods (45). The
capacity for anaerobic growth was tested using an anaerobic-bag system
(Marion Scientific, Kansas City, Mo.). The API 20 NE system
(bioMérieux Vitek, Inc., Hazelwood, Mont.) was used to test
for arginine dihydrolase, urease, esculin hydrolysis, gelatinase,
-galactosidase, and assimilation of 12 different substrates. The
test strips were prepared according to the instructions of the
manufacturer and were incubated at 23°C for 5 days before interpretation. Biolog GN microplates (Biolog Inc., Haywood, Calif.) were also used to test for the ability of strains to utilize 95 different carbon sources. The 96-well plates were inoculated with cells
suspended in sterile 1.5% saline at an OD600 of 0.2. After incubation for 48 h at 23°C, the plates were read with the
Biolog microplate reader.
Molecular characterization of isolates.
Genomic DNA was
prepared from bacterial cultures using the Qiagen tissue kit (Qiagen
Inc., Valencia, Calif.) and was diluted to 10 µg ml1 in
10 mM Tris-HCl-1 mM EDTA (pH 8.0). Primers used for amplification of
16S rDNA were previously described (30) and correspond to Escherichia coli positions 8 to 27 (primer 27F) and 1492 to
1510 (primer 1492R). Each 50-µl PCR mixture contained 50 ng of
genomic DNA, 2 mM MgCl2, amplification primers (100 nM
each), and 200 µM each deoxynucleoside triphosphate in 1×
thermophilic DNA polymerase buffer. After an initial 4-min denaturing
step (94°C), 1.25 U of Taq polymerase (Life Technologies,
Inc., Rockville, Md.) was added to each reaction tube, and the tubes
were held at 94°C for an additional 2 min. The thermocycler was then
set to run 35 cycles under the following conditions: 40 s
denaturing (94°C), 30 s annealing (62°C), and 60 s
elongation (72°C), followed by an additional 7-min elongation period
at the end of the program. PCR products were purified using the
QIAquick gel extraction kit (Qiagen), and the purity and concentration
of DNA were analyzed by electrophoresis on a 1% agarose gel. Purified
products were sequenced at the University of Maine's DNA sequencing
facility and were analyzed using Sequence Navigator software (Applied
Biosystems, Inc., Foster City, Calif.) and BLAST analysis
(1).
Nucleotide sequence accession numbers. The nucleotide sequences of the strains CV812-530 and CV902-700 rDNAs have been submitted to GenBank and assigned accession no. AF246614 and AF246615, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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JOD-associated mortalities at field deployment sites. No animals from either group developed signs of JOD when maintained at the Maquoit Bay site, nor did any animals from the first group develop signs of JOD at the Damariscotta River site. However, JOD signs and mortalities were observed in animals from the second group deployed on the Damariscotta River beginning on 23 September in one of the trays (no. 3). Mortalities were not observed in the other two trays until over a month later. The last sampling date was 29 October, at which time cumulative mortalities were 0.7, 0.1, and 7.1% for trays no. 1, 2, and 3, respectively.
Bacteriological analysis of oyster groups.
Between
103 and 104 CFU per oyster were recovered from
the meats of animals originating from the first group (Fig.
1A). We have noticed that CVSP bacteria
are more efficiently recovered from swab samples of animal surfaces,
but this is an impractical method for sampling animals with a shell
height of less than 15 mm. After 12 August, the animals were
sufficiently large (15 to 20 mm) that swab sampling replaced the
procedure of sampling of entire meats. The total CFU recovered from
these swab suspensions typically ranged from 103 to
104 CFU per ml (Fig. 1A). Regardless of the sampling
protocol, no CVSP-like bacteria were isolated at any time from animals
in this group. Instead, another specific type of bacterium was
consistently observed to be present at levels of between 10 and 50% of
total CFU (Fig. 1B). These colonies (designated M1) had a very mucoid consistency and a distinctive light-brown pigmentation after 3 days of
incubation on SWT agar. Seventeen M1 isolates recovered from this first
group were stored for subsequent phenotypic characterization. Two of
these M1 strains were also subjected to 16S rDNA analysis. These were
strain CV729-100, which was isolated on 29 July from an animal held at
Maquoit Bay, and strain CV812-530, which was isolated on 12 August from
an animal deployed on the Damariscotta River. No other specific types
of colonies besides the M1 type were consistently isolated from these
animals over the course of the study.
|
,
Damariscotta River samples; 
, Maquoit Bay samples.
1 (Fig.
2A). However, total CFU recovered from
animals removed from tray no. 3 at the Damariscotta River site
increased to approximately 104 CFU mg1 during
the weeks in which JOD mortalities were observed. Similarly, total CFU
in animals from the remaining two trays were elevated on the date when
mortalities in those animals were observed. Bacterial colonies
morphologically identical to those of CVSP bacteria were recovered from
animals exhibiting signs of JOD and coincided with the timing of the
mortalities (Fig. 2B). Additionally, when present, the CVSP-type colony
was abundant. Typically, CVSP-like CFU comprised at least 25% of total
CFU recovered and reached a maximum of 99% of total CFU on one
occasion (tray no. 3, on 14 October). Two isolates from JOD-affected
animals, CV923-115, isolated on 23 September from an animal in tray no.
3, and CV1028-008, isolated on 28 October from an animal in tray no. 1, were subjected to molecular analysis.
|
), Tray 2 (
), Bag 3 (Challenge experiments.
When animals in laboratory aquaria were
exposed to CVSP bacteria via the addition of a culture of CVSP strain
CV919-312 to aquaria water, there was no difference (P 
0.05) in the mortality rate of exposed oysters compared to that of
controls. After 5 months, cumulative mortalities did not exceed 0.5%
in any of the treatment or control tanks, nor were any CFU resembling
CVSP bacteria recovered from animals sampled 1 month after repeated
exposure to the strain. In contrast, all four animals sampled (two each from a treatment and a control aquarium) were extensively colonized by
M1 bacteria. The M1 colonies comprised between 10 and 30% of the total
CFU recovered from each of these animals. One M1 isolate (CV910-004)
was stored and subjected to further phenotypic and molecular characterization.
0.05).
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Molecular characterization of isolates.
A total of six CVSP
strains isolated from JOD-affected animals were analyzed. Two of these
were from animals in the second group (CV923-115 and CV1028-008), two
strains were isolated from JOD-affected oysters given to us by
commercial growers (CV910-103 and CV1123-045), and two were isolated
from laboratory-maintained animals that had been exposed to CV919-312
by injection (CV102-1001 and CV211-3001). The 16S rRNA genes of each
isolate were amplified and sequenced, and all sequences were shown to
be identical across the resulting 1,390-bp region. BLAST analysis
revealed that these sequences were also 100% identical to the CVSP
bacteria (CV919-312 and CV1010-352) isolated from the 1997 JOD episode
(7). This sequence (GenBank accession no. AF114484 and
AF114485) also shares extensive similarity with Roseobacter
sp. strain ISM, marine -proteobacterium MBIC3951, and
Roseovarius tolerans (accession no. AF098485, AB018689, and
Y11551, respectively). The percent sequence identities (excluding gaps)
of the CVSP sequence with these strains are 96.1, 95.5, and 94.6%, respectively.
-proteobacteria MBIC1535 and MBIC3993. The sequence was
also identical (excluding gaps) to that of Agrobacterium stellulatum (the new designation for this species is Stappia
stellulata) (25, 47). The 16S rRNA sequences from the
two M2 isolates were identical to each other and were almost identical
to the M1 strain, with the exception of an unambiguous difference
between the M1 and M2 sequence at nucleotide position 1043. The 16S
rDNA nucleotide sequences of M1 isolate CV812-530 and M2 isolate
CV902-700, have been submitted to GenBank (see "Nucleotide sequence
accession numbers" above).
Phenotypic characterization of M1 and M2 isolates.
Eighteen M1
isolates (CV729-100, CV812-530, CV910-004, and 15 others) and two M2
isolates (CV902-700 and CV923-700) were phenotypically characterized.
Like S. stellulata, all were gram-negative motile rods, were
oxidase positive, grew (albeit poorly) under anaerobic conditions, and
formed star-shaped-like aggregates when grown in liquid media. Further,
all isolates tested produced urease and -galactosidase, reduced
nitrate to gas, and hydrolyzed esculin. No strains were positive for
arginine dihydrolase or gelatinase. Interestingly, all M1 isolates
exhibited a very strong catalase activity, while this activity was
barely detectable in the M2 isolates. Other differences were observed
between the M1 and M2 isolates with respect to utilization of some
carbon sources, and only M1 strains inhibited the growth of CVSP strain
CV919-352 in a disk-diffusion assay (Table
1).
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-D-glucose, m-inositol,
-D-lactose, maltose, D-mannitol,
D-mannose, D-melibiose, -methyl
D-glucoside, psicose, L-rhamnose,
D-sorbitol, sucrose, D-trehalose, turanose,
methyl pyruvate, monomethyl succinate, cis-aconitic acid,
citric acid, formic acid, D-gluconic acid, D-glucuronic acid, -hydroxybutyric acid,
-hydroxybutyric acid, p-hydroxyphenylacetic acid,
itaconic acid, -ketobutyric acid, -ketoglutaric acid,
-ketovaleric acid, DL-lactic acid, malonic acid,
propionic acid, D-saccharic acid, succinic acid,
alaninamide, L-alanine, L-alanyl-glycine,
L-asparagine, L-aspartic acid,
L-glutamic acid, glycyl-L-glutamic acid,
L-histidine, hydroxy L-proline, L-leucine, L-ornithine,
L-phenylalanine, L-proline,
L-pyroglutamic acid, L-serine,
L-threonine, DL-carnitine, 
-aminobutyric
acid, urocanic acid, inosine, thymidine, and glycerol. Substrates that were weakly oxidized included Tween 40, -lactose, acetic acid, D-galactonic acid lactone, -hydroxybutyric acid, quinic
acid, succinamic acid, glucuronamide, uridine, phenylethylamine,
and 2-aminoethanol. Variable reactions were observed with bromosuccinic acid and glycyl-L-aspartic acid. The isolates did not
utilize -cyclodextrin, Tween 80, D-raffinose, xylitol,
D-glucosaminic acid, sebacic acid, D-serine,
putrescine, or 2,3-butanediol.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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While the severity of the 1998 annual JOD epizootic was low
compared with those of previous years, it impacted at least three separate culture sites on Maine's Damariscotta River. Bacteriological analysis of JOD-affected animals from each of these epizootics revealed
the presence of CFU morphologically indistinguishable from those of the
CVSP marine -proteobacterium that was found to be associated with
the 1997 epizootic (7). The 16S rRNA genes from isolates in
the present study were amplified, and the sequence was determined to be
identical to that of the previously described CVSP strains. CVSP-type
CFU were not isolated from nonaffected animals raised at the JOD
enzootic site or in cohorts raised at a JOD-free site. Thus, these
results demonstrate that the presence of CVSP bacteria is a reliable
indicator of JOD and constitutes strong correlative evidence for the
role of these marine -proteobacteria in JOD mortality.
An attempt to reproduce JOD signs and mortalities in laboratory-maintained C. virginica via the addition of CVSP bacteria to aquaria water was unsuccessful. However, when cells of CVSP strain CV919-312 were injected directly into the mantle cavities of oysters, the inoculated animals exhibited a reduced capacity to filter the algal food suspension (after 6 weeks) and suffered earlier and significantly elevated mortalities compared to that of controls (Fig. 3). CVSP-type CFU were recovered from animals during these mortality events, and their identity was confirmed by amplification and sequencing of the 16S rDNAs.
While these results support the hypothesis that CVSP bacteria are the etiological agent of JOD, the case would be more convincing if the characteristic conchiolin deposits had also been observed. However, if the oysters are not in good health and actively growing prior to the onset of JOD, mortalities can occur in the absence of this host response (32, 38). Because no net growth was observed during our experiment (data not shown), it is possible that the animals similarly may not have possessed the metabolic resources to produce the characteristic conchiolin deposits. Prolonged lack of a natural diet may also partially account for the unexpected mortalities in the control aquaria after 20 weeks.
In a previous study where JOD signs were reproduced by injection of animals with homogenates from affected animals, JOD signs developed within 1 month and researchers noted a higher prevalence of JOD signs in animals receiving more than one inoculation (38). Thus, the timing and extent of laboratory-induced JOD signs may depend upon the aggressiveness of the exposure regimen, as well as the infectivity of the source material. We suspect that the effect of CVSP bacteria will be observed sooner in animals exposed to homogenates from CVSP-inoculated animals than in those injected with cultured cells. This hypothesis will be addressed in subsequent experiments that will also include additional controls (e.g., heat-killed CVSP bacteria and a known nonpathogenic bacterium) and an increased and/or supplemented algal ration to improve the likelihood of a vigorous metabolic host response.
The apparent avirulence of CVSP when provided to juvenile C. virginica as a suspension in aquaria water should also be
addressed. One possibility is that the cells experienced a rapid loss
of viability upon addition to the artificial seawater. However, in preliminary experiments, we observed that CVSP bacteria survive and
multiply in this water at rates approaching those observed in SWT
medium (data not shown). Another possibility is that the animals did
not concentrate the CVSP bacteria in the course of their normal
filter-feeding activity. Given that oysters are very efficient at
clearing bacterial cells from suspension (26, 35), this is
considered unlikely. An alternative explanation may center on the
production of factors important for bacterial colonization of the
juvenile oysters. Like Sagittula stellata (another marine -proteobacterium) (22), CVSP bacteria produce a polar
holdfast structure in addition to polar flagella (data not shown). The method of exposure (immersion versus injection) may be significant if
these potential colonization factors are not produced or are lost
before the oysters encounter CVSP cells when present in suspension.
It is also important to consider the source of the animals used in each challenge experiment. Animals exposed to the CVSP bacteria by immersion were members of the first group that was deployed in the 1998 field study. Both groups used that year were progeny of oysters that had been bred for fast growth and had increased resistance to JOD mortalities (4, 12), but only animals in the second group became colonized by CVSP bacteria and experienced any JOD-associated mortalities. In addition, animals from the first group were stably colonized by the M1 genotype of an S. stellulata-like bacterium, while the related M2 genotype was only isolated from the animals in the second group. The ability of the M1 strains (but not the M2 strains) to inhibit the growth of CVSP strain CV919-312 in a disk-diffusion assay raises the possibility that prior colonization of C. virginica by this species may prevent colonization by CVSP bacteria. A probiotic effect of the M1 strains could explain the apparent immunity of the first group to JOD both in the field and when challenged with CVSP bacteria in the immersion experiment. Animals used in the injection experiment had not been bred for JOD resistance, and no M1-like CFU were detected upon bacteriological analyses of the animals.
The body surfaces of oysters, as filter feeders, are in near-continuous
contact with bacteria present in the ambient water. However, bacterial
diseases of postmetamorphic oysters are relatively rare
(13), and when examined by transmission electron microscopy, the external surfaces of the Pacific oyster (Crassostrea
gigas) have been shown to be essentially free of any bacterial
colonization (19). It is also generally accepted that if
oysters do have a "normal microbiota," these organisms are
predominantly associated with the gut and digestive gland (24,
27). In this study, both the CVSP and S. stellulata-type -proteobacteria were consistently isolated from
samples of the external tissue and the inner shell surfaces of C. virginica. These bacteria are not normal microbiota in the sense
that they were not isolated from all animals, yet there is no question
that they can initiate and persist in the association. This raises the
interesting question of how oysters (and perhaps other bivalves)
prevent colonization of tissues by most bacteria, and what factors
allow these -proteobacteria to overcome such defenses.
The marine -proteobacteria are common and abundant in coastal
ecosystems (20). Often referred to as the
Roseobacter group (20, 21), these organisms also
inhabit "extreme" environments such as hypersaline lakes
(28), Antarctic ice (23), and hydrothermal vents
(48). Several species of marine -proteobacteria are known to be associated with a variety of marine plant (2, 29, 43) and animal species (5, 10, 42), although, to our knowledge, none of the animal symbionts have been successfully cultivated and
characterized. S. stellulata (originally described as a
marine Agrobacterium) has been isolated from marine
sediments and seawater (41, 46), but this study provides the
first evidence that this bacterium can also associate with a defined
animal host. Conversely, CVSP bacteria have been isolated only from
JOD-affected C. virginica (7 and this
study), but they too are likely to be a normal member of marine
bacterioplankton and sediment communities.
In sum, our results are not consistent with a multifactorial etiology
for JOD and, instead, support the hypothesis that the CVSP
-proteobacterium is the sole etiological agent of JOD. This system
represents a unique opportunity to study the bacterium-host interactions of both CVSP bacteria and the S. stellulata-like strains with C. virginica. As members
of the marine -proteobacteria, these bacteria are unrelated
taxonomically to other common fish and shellfish pathogens. In
addition, there is little known about the environmental role of this
entire group of bacteria, and this study demonstrates that it will be
important to consider host associations as factors influencing the
abundance and distribution of planktonic marine -proteobacteria. We
are currently developing a PCR-based molecular beacon assay for the
detection of CVSP bacteria in the environment and for use as a
diagnostic tool.
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ACKNOWLEDGMENTS |
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This study was funded by a Maine/New Hampshire Sea Grant development award.
We are grateful to Chance Along Farm, Dodge Cove Marine Farms, Mook Sea Farms, and Pemaquid Oyster Company for their very generous assistance with this study. We also thank P. B. Singer and K. Williams for sequencing services, L. Costello and R. B. Carnegie for assistance in the field, K. Mitchell for help with the challenge experiments, and M. A. Killam for assistance with the phenotypic characterizations.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, ME 04469. Phone: (207) 581-2822. Fax: (207) 581-2801. E-mail: boettche{at}maine.maine.edu.
This is Maine Agriculture and Forestry Experiment Station
Publication number 2434.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 2000, p. 3924-3930, Vol. 66, No. 9
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