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Appl Environ Microbiol, April 1998, p. 1459-1465, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Influence of Water Temperature and Salinity on
Vibrio vulnificus in Northern Gulf and Atlantic Coast
Oysters (Crassostrea virginica)
M. L.
Motes,1
A.
DePaola,1
D. W.
Cook,1,*
J. E.
Veazey,2
J. C.
Hunsucker,3
W. E.
Garthright,4
R. J.
Blodgett,4 and
S.
J.
Chirtel4
Gulf Coast Seafood Laboratory, U.S. Food and
Drug Administration, Dauphin Island, Alabama
36528-01581;
State Cooperative
Programs, Southeast Region, U.S. Food and Drug Administration, Baton
Rouge, Louisiana 708092;
Southeast
Regional Laboratory, U.S. Food and Drug Administration, Atlanta,
Georgia 303093; and
Division of
Mathematics, U.S. Food and Drug Administration, Washington, D.C.
202044
Received 14 November 1997/Accepted 30 January 1998
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ABSTRACT |
This study investigated the temperature and salinity parameters
associated with waters and oysters linked to food-borne Vibrio vulnificus infections. V. vulnificus was enumerated
in oysters collected at three northern Gulf Coast sites and two
Atlantic Coast sites from July 1994 through September 1995. Two of
these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in V. vulnificus
cases. Oysters in all Gulf Coast sites exhibited a similar seasonal
distribution of V. vulnificus: a consistently large number
(median concentration, 2,300 organisms [most probable number] per g
of oyster meat) from May through October followed by a gradual
reduction during November and December to 
10 per g, where it remained
from January through mid-March, and a sharp increase in late March and
April to summer levels. V. vulnificus was undetectable (<3
per g) in oysters from the North and South Carolina sites for most of
the year. An exception occurred when a late-summer flood caused a drop
in salinity in the North Carolina estuary, apparently causing V. vulnificus numbers to increase briefly to Gulf Coast levels. At
Gulf Coast sites, V. vulnificus numbers increased with
water temperatures up to 26°C and were constant at higher
temperatures. High V. vulnificus levels (>103
per g) were typically found in oysters from intermediate salinities (5 to 25 ppt). Smaller V. vulnificus numbers
(<102 per g) were found at salinities above 28 ppt,
typical of Atlantic Coast sites. On 11 occasions oysters were sampled
at times and locations near the source of oysters implicated in 13 V. vulnificus cases; the V. vulnificus
levels and environmental parameters associated with these samples were
consistent with those of other study samples collected from the Gulf
Coast from April through November. These findings suggest that the
hazard of V. vulnificus infection is not limited to brief
periods of unusual abundance of V. vulnificus in Gulf Coast
oysters or to environmental conditions that are unusual to Gulf Coast
estuaries.
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INTRODUCTION |
Between 1989 and 1994, cases of
primary septicemia resulting from shellfish-borne Vibrio
vulnificus averaged 15 per year, with fatality rates averaging
45% (4). Most cases (85%) occurred between May and October
(13, 14, 21), and only oysters harvested from Gulf Coast
states have been implicated. Frequently, implicated oysters are traced
to one of a small number of harvest locations (4). These
facts suggest that oysters harvested from specific areas or harvested
from areas with certain environmental conditions (temperature and
salinity) may have unusually large numbers of V. vulnificus
organisms, thereby increasing the risk of infection for individuals.
The persons at greatest risk for infection are those with liver disease
(16, 23); however, fewer than 1 in 104 persons
from this high-risk group become ill after consuming raw oysters
(8).
The highest concentrations of V. vulnificus in Gulf Coast
oysters have been reported during the warm months (5, 22). In the Chesapeake Bay, V. vulnificus was recovered from
oysters at levels of 103 to 104/g during summer
months but was not recovered during winter months (30).
V. vulnificus has been recovered, although sporadically and
usually in small numbers, from oysters harvested from cooler environments such as the New England Coast (20) and the
Pacific Coast (11). These observations point to temperature
playing a significant role in controlling the numbers of this organism in oysters. The role of water salinity in the abundance of V. vulnificus is less clear (25, 29).
Understanding the relationship between V. vulnificus and
temperature and salinity may help predict the concentrations of
V. vulnificus in shellfish. Many of the early studies that
gathered this type of information were limited by the narrow
geographical scope of sampling (12, 19, 25) or by sporadic
or seasonal sampling schedules. Before 1988, the enumeration
methodology for V. vulnificus was not standardized. Recent
improvements have made enumeration less time-consuming and permit the
examination of larger numbers of test samples (18, 27).
Tamplin (24, 26) investigated the relationship of
environmental factors and V. vulnificus densities in oysters
collected monthly in 14 states. Levels in oysters ranged from none
detected (<0.3 per g) to 1,100,000 per g. This information was used to derive a linear regression model based on water temperature and salinity to predict V. vulnificus levels in oysters
(28).
This study expands on Tamplin's research (26) and reports
on the seasonal distribution and abundance of V. vulnificus
in Northern Gulf and Atlantic Coast oysters in an attempt to further define the relationship between the density of this organism and the
temperature and salinity of the water. The selected sampling sites were
considered representative of the Gulf Coast shellfish-growing areas
with regard to productivity, and they represent more than 90% of the
total harvest for Alabama and Florida and ca. 25% for Louisiana. Sites
in Louisiana and Florida were also chosen based on their history of
shellfish-associated V. vulnificus cases; sites in Alabama
and on the Atlantic Coast were picked based in part on their lack of
association with cases (4). The intensive and methodical
sampling of areas frequently associated with harvest of implicated
oysters was expected to provide information on concentrations of
V. vulnificus in illness-associated oysters at the time they were harvested. This information may be used to determine the extent to
which environmental factors influence the concentration of the pathogen
and incidence of the disease.
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MATERIALS AND METHODS |
Oyster harvesting and handling.
Oysters (Crassostrea
virginica) were harvested weekly (25 July 1994 through 25 September 1995) from three Northern Gulf Coast (Apalachicola Bay, Fla.,
29°44'25"N, 84°53'10"W; Cedar Point, Ala., 30°18'30"N,
88°07'45"W; and Black Bay, La., 29°36'40"N, 89°34'00"W) and two
Atlantic Coast (Folly River, S.C., 32°40'30"N, 79°56'04"W and
Newport River, N.C., 34°45'30"N, 76°45'00"W) shellfish-growing areas. Harvesting was reduced to a monthly schedule beginning in
November on the Atlantic Coast and in January on the Gulf Coast. Weekly
harvesting was resumed in March on both coasts. Collections (lots)
consisted of 25 legal-size culled oysters in the shell harvested with
either oyster tongs or a dredge. A 50-ml volume of surface water was
collected at the harvest site in a screw-cap plastic tube and kept with
the oysters for verification of the salinity in the laboratory.
Temperature and salinity were measured in the upper 0.5 m of the
surface water with a salinometer (Beckman Instruments, Inc., Cedar
Grove, N.J.) or with a calibrated bimetallic dial thermometer and a
refractometer (Cambridge Instruments, Inc., Buffalo, N.Y.). Immediately
after being harvested, the oysters were chilled by contact with bagged
ice in a 48-qt ice chest for approximately 2 h. The chilled
oysters and the tube of water were shipped in insulated shipping
containers (no. 132; FDC Packaging, Inc., Medfield, Mass.) with three
frozen cool packs (no. 405; FDC Packaging, Inc.). Bubble wrap was laid
between the oysters and the ice packs to prevent direct contact. The
oysters were shipped overnight to a laboratory for analysis; the
Atlantic Coast oysters were shipped to the Food and Drug Administration
(FDA) Southeast Regional Laboratory, Atlanta, Ga., and the Gulf Coast oysters were shipped to the FDA Gulf Coast Seafood Laboratory, Dauphin
Island, Ala. Upon receipt, the temperature of the water sample was
recorded to ensure that the temperature in the shipping container had
remained low enough to prevent the growth of V. vulnificus.
Analysis.
Each lot of oysters was divided into two
subsamples of 12 oysters each, and the subsamples were analyzed
separately. These subsamples were considered to be replicate oyster
samples from the same growing area, harvested at the same time. The
oysters were washed, shucked, diluted 1:1 in phosphate-buffered saline (PBS), and homogenized for 90 s in a blender at 14,000 rpm. To prepare the first dilution, 20 g of the homogenate was weighed into a sterile jar and diluted to 100 g with PBS. Subsequent
dilutions in PBS were made on a volume basis. V. vulnificus
was enumerated by procedures described in the Bacteriological
Analytical Manual (7). A three-tube
most-probable-number (MPN) series was used for enumeration. Isolates
were confirmed by enzyme immunoassay with a monoclonal antibody
specific to V. vulnificus (27). Visual readings
of the enzyme immunoassay plates were confirmed with a microplate
reader (Bio-Tek Instruments, Winooski, Vt.). Normal inoculation sizes
for the MPN determinations were 10
1 through
106 g, with the exception of the winter months, when the
analysis included 1.0-g amounts (Gulf Coast oysters only). An
additional 25 g was inoculated into 2,475 ml of alkaline peptone
water for a presence/absence test.
Statistical analyses.
For statistical analysis, MPN values
that were indeterminate (<0.3 or <3.0) as a result of no positive
tubes in any of the series were assigned a value halfway between the
maximum value and zero (i.e., 0.15 or 1.5, respectively). MPN counts
were converted to base 10 logarithms before being subjected to
analysis. Variance due to the three-tube MPN method was estimated by
five sets of eightfold replicate measurements; each eightfold set was
performed on the same dozen-oyster homogenate (6).
Regression analysis and analysis of variance were performed with
Statistical Analysis Systems from SAS Institute, Cary, N.C.
Plotting of the V. vulnificus counts against temperature
employed a smoothing technique (moving average), which was necessary because in some instances there were few measurements at a particular temperature. This technique involved the use of 3°C increments, derived a geometric mean of the V. vulnificus count for all
values corresponding to the three temperatures (e.g., 19, 20, and
21°C), and plotted this against the mean of the temperatures (i.e.,
20°C for the above temperatures). A similar procedure was applied
with salinity in which 3-ppt salinity increments were used.
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RESULTS |
Oysters analyzed.
A total of 226 lots of oysters (52 from
Alabama, 50 from Louisiana, 47 from Florida, 38 from South Carolina,
and 39 from North Carolina) were received and analyzed in duplicate for
V. vulnificus within 28 h of harvest. Of these, only 13 lots were received with temperatures above 13°C and none had V. vulnificus counts higher than might be expected based on
temperature and salinity at the harvest sites. No oysters were received
at temperatures above 19°C.
Oysters from Gulf Coast sites.
Only 16.9% of the paired
oyster subsamples from a lot produced identical MPN counts, but in
83.1% of the cases, the MPN counts of the second subsample fell within
the 95% confidence limits of the MPN counts of the first. The average
variance between Gulf Coast pairs was 0.170. Although the sets of
eightfold replicate measurements showed that the MPN method contributes
0.12 to the log variance, the variance due to the difference from one
dozen to the next dozen dredged at the same place and time is 0.05 log unit.
Geometric means were calculated from the paired
V. vulnificus values for each lot of oysters and are presented
by site in
Fig.
1. MPN counts at
all three sites appear to be similar and
seasonally influenced, ranging
from 10
3 to 10
4 organisms per g from May to
October and falling to <10 per g
from late December through mid-March.
The seasonal trend is emphasized
when the data from all three sites are
combined (Fig.
2). Data
from oysters
collected between days 1 and 15 of each month were
combined, as were
data from day 16 to the end of each month. The
data fell into four
clearly defined periods, a cold-weather period
(January, February, and
early March), a warm-weather period (May
through October), and two
transitional periods, in the spring
(late March and April) and fall
(November and December), when
counts increased or decreased with time.
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FIG. 1.
Weekly densities of V. vulnificus in oysters
from Gulf Coast sites in Alabama (+), Florida ( ), and Louisiana
( ) during a 14-month period. Each point represents the geometric
mean (n = 2) of the MPN of bacteria per gram of oyster
meat.
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FIG. 2.
Seasonal distribution of V. vulnificus in
Gulf Coast oysters. Each point represents the geometric mean of the MPN
of V. vulnificus organisms per g of oyster meat for all
observations recorded within a half-month period. Transitional periods
represent periods of change.
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Table
1 presents the summary statistics
of
V. vulnificus counts in oysters and their associated
environmental parameters
by period for each site and all Gulf Coast
sites.
V. vulnificus was recovered from all of the oysters
sampled during the warm-weather
period, with 70% of the samples
exceeding 10
3 organisms per g. The median value for each of
the three sites
during the warm-weather period was 2.3 × 10
3 per g. Only 3.7% of the samples had MPN counts
exceeding 10
4 per g, and none had MPN counts exceeding
4.3 × 10
4 per g.
The transitional periods were characterized by water temperatures
averaging
20°C and
V. vulnificus MPN counts averaging
10
2 per g. The counts increased during the spring and
decreased during
the fall.
In the cold-weather period, the water temperatures averaged <15°C.
V. vulnificus MPN counts in all Gulf Coast oyster samples
examined during this period were <10 per g, with a median of <1
per
g. Counts of <0.3 per g (i.e., no isolates from tubes inoculated
with
1-g portions) were observed in 38% of the cold-weather period
samples,
but only 2 of 24 were negative for
V. vulnificus when
25-g
portions were examined. The lowest water temperature at which
V. vulnificus was recovered, 10.8°C, was the lowest observed during
the study.
Differences among the three Gulf sites with respect to temperature,
salinity, and log MPN data were analyzed by a two-way
analysis of
variance, using the sample week as a blocking factor.
There was
evidence that the mean log MPN values were not the same
at the three
sites (
P = 0.0003). After all three pairwise
comparisons
were performed and the Bonferroni adjustment were made, the
Louisiana
site had a higher mean log MPN value than did the Florida and
Alabama sites at the
P < 0.05 level. No differences
were observed
between the Florida and Alabama sites (
P < 0.05). A two-way analysis
of variance of temperature with the week
as a blocking factor
did not demonstrate any overall difference among
the sites (
P = 0.33). The two-way analysis of salinity
demonstrated clear evidence
of a site effect (
P = 0.0001). All three pairwise comparisons
had
P values well
below the Bonferroni critical value of 0.0167.
The Florida site had the
highest average salinity, and the Louisiana
site had the lowest.
The Gulf log MPN data were regressed on temperature and salinity data
to determine whether these were good predictors of
V. vulnificus concentrations. The linear-regression model which best
fits the data is: log MPN =
9.6823 + (0.5855 × temperature)
(0.0092 × temperature
2) + (0.6804 × salinity)
(0.0355 × salinity
2) + (0.00054 × salinity
3). The
r2
for the model is 0.705 (
r = 0.84). When the site factor
is added
to this model, the
P value indicates that site is
not significant
(
P = 0.43). Thus, after adjustment for
temperature and salinity,
there is no evidence of any independent
effect of the sampling
site.
The role of salinity in determining
V. vulnificus levels
becomes clear when the full model (described above) is compared with
one lacking the salinity terms. The model log MPN =
5.5031 +
(0.5391 × temperature)
(0.0081 × temperature
2) has an
r2 of 0.60. (The nature of this association with temperature can
be seen in Fig.
3; the numbers of
V. vulnificus organisms increased
slowly over the range from 10 to
18°C, increased more rapidly
from 18 to 26°C, and then stopped
increasing above 26°C.)
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FIG. 3.
Influence of water temperature on the concentration of
V. vulnificus in Gulf Coast oyster meats. Each point
represents the geometric mean of all observations recorded within a
3°C temperature range. See Materials and Methods for an explanation
of the smoothing technique.
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The addition of the salinity terms explains about 10% more of the
total variability in the log MPN data and also explains
the difference
between the sites. Figure
4 shows the
residuals
[(actual values)
(model predicted values)] from the
model lacking
salinity terms plotted against salinity. This graph
demonstrated
that the residuals (from the temperature effects model)
are low
at both the high and low extremes of salinity and that the
three
states seem to fall on the same curve. Figure
4 also shows the
residuals from the model that includes both temperature and salinity.
The evenness and linearity of the remaining residuals are evident
from
the graph. The model is not intended to represent other estuaries,
and
it should not be extrapolated beyond the range of temperatures
and
salinities shown in the data from the three Gulf estuaries.
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FIG. 4.
Residuals (measured log V. vulnificus count
minus model predictions) plotted against salinity for three Gulf
estuaries.
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If one makes an arbitrary division of the residuals into two groups at
salinities of
15 and >15 ppt, there is a positive
correlation
(
r = 0.43,
P = 0.0001,
n = 74) between salinity and
the residuals in the
low-salinity group and a negative correlation
(
r =
0.44,
P = 0.0001,
n = 71) in the
high-salinity group.
Oysters from Atlantic Coast sites.
Weekly geometric means of
V. vulnificus counts in oysters from the Atlantic Coast
sites during the 14-month study period are presented in Fig.
5. MPN counts were below the lower limit
of sensitivity for the method (3 per g) in 87% of the South Carolina oysters and 42% of the North Carolina oysters. MPN counts of <10 per
g occurred in 86% of the combined set of samples from the two Atlantic
Coast sites. Oysters from the North Carolina site collected in July and
August had MPN counts exceeding 102 per g on three
occasions and exceeding 103 per g once. There was a
significant difference (P < 0.05) in the geometric
means of the counts in oysters between the North and South Carolina
sites and between the Atlantic and Gulf Coast sites.
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FIG. 5.
Weekly densities of V. vulnificus in oysters
from Atlantic Coast sites in North Carolina () and South Carolina
() during a 14-month period. Each point represents the geometric
mean (n = 2) of the MPN of bacteria per gram of oyster
meat.
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Seawater temperatures at the Atlantic Coast sites (Table
1) were
similar to those recorded at the Gulf Coast sites and, during
the warm
period, were within the range associated with the recovery
of
V. vulnificus from shellfish. The water temperatures exceeded
20°C
from May through October, and temperatures above 30°C were
common
during late July and in August.
Water salinities at the Atlantic Coast sites were significantly higher
(
P < 0.01) than at the Gulf Coast sites and normally
ranged from 22 to 36 ppt; 73.4% of samples exceeded a salinity
of 26 ppt. The only occasion when
V. vulnificus was detected in
large numbers occurred during the summer months when water salinities
were below normal.
V. vulnificus illnesses.
There were 13 oyster-associated V. vulnificus illnesses during the period
of this study in which the most probable harvest site was identified as
one of our sampling sites. None of the illnesses were linked to the
Atlantic Coast sites. Table 2 presents environmental and bacteriological data for the closest sampling date to
the harvest date. Of the 11 salinities associated with the cases, 7 were within 1 standard deviation of the mean for the warm period, 3 were above, and 1 was below. Of the 11 temperatures associated with
cases, 9 were within 1 standard deviation of the warm period mean and
the other 2 were below. Therefore, the environmental conditions at
times implicated by cases were no more conducive to V. vulnificus abundance than were conditions at other times in the
warm period.
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TABLE 2.
V. vulnificus illnesses that occurred during
the study and for which one of the study areas was identified as
the most likely harvest area for the implicated oysters
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DISCUSSION |
Examination of oysters from three geographically distinct
estuaries on the northern Gulf Coast demonstrated a similarity in V. vulnificus counts throughout the year. During
warm-weather months, when >85% of the shellfish-associated V. vulnificus cases occur, MPN counts were usually 103 to
104 per g. During cold-weather months, when infections have
not been reported, MPN counts were <10 per g. Jackson et al.
(9) reported similar V. vulnificus densities in
Apalachicola Bay oysters during the summer of 1991 but found much
higher levels in 1992 and 1993 (>105 per g). These
differences may be due to year-to-year variation; we observed much
lower counts in Apalachicola Bay oysters in August and September 1995 (10 to 100 per g) than in the same months in 1994. The low counts in
1995 coincided with unusually high salinity. In the present study, we
rarely found densities greater than 104 per g. The sample
handling procedures used by Jackson et al. before shipment were not
specified (9). The multiplication of V. vulnificus in summer harvest oyster shellstock held without refrigeration has been shown to be rapid (2). If shellstock are chilled immediately after harvest and stored at temperatures of
13°C, the numbers of V. vulnificus organisms do not
differ significantly from those at the time of harvest through 30 h of storage (1). While prolonged storage of shellstock at 0 to 4°C brings about a significant reduction in numbers of V. vulnificus organisms, storage for up to 48 h at these
temperatures brings about no reduction in numbers (3).
Procedures to handle oysters between harvest and analysis were designed
to minimize changes in V. vulnificus density (1).
These procedures consisted of chilling oysters immediately after
harvest and maintaining them at a low temperature during overnight
transport to the analytical laboratory.
This study demonstrates the ubiquitous occurrence of culturable
V. vulnificus organisms in Gulf Coast oysters throughout the year (>99% detection in 298 samples). Failure to detect V. vulnificus by the MPN procedure in seven of nine samples collected
during the winter was reversed by increasing the sample size eightfold to 25 g. Previous studies reporting a low incidence of V. vulnificus in Gulf Coast oysters during the winter have relied on
MPN procedures similar to those used in the present study (6, 9,
25, 29). Since one oyster contains about 25 g of tissue,
culturable V. vulnificus would probably overwinter along the
Gulf Coast and reseed the estuary when the waters become warmer in the
spring. In the present study, densities up to 9.3 per g were observed during January or February. No primary septicemia cases have been reported in these months (4, 8). Further investigation is needed to determine a safe level of V. vulnificus, because
levels may change between harvest and consumption and virulence may be seasonal.
Although the presence of V. vulnificus in Gulf Coast
estuarine environments is favored by relatively high water temperatures (12, 25), Wright et al. (30) reported no
correlation between water temperature and V. vulnificus
concentration in Chesapeake Bay waters during the warmer months. Our
Gulf Coast data suggests that the number of V. vulnificus
organisms in oysters is strongly correlated with water temperature
until the temperature reaches 26°C, above which there appears to be
no additional increase in the number of bacteria. Investigators
developing models for predicting V. vulnificus levels in
estuarine environments need to be aware of this break at 26°C, as
water temperature normally exceeds 26°C from May through October,
when the majority of cases occur.
Seasonal temperature change explains most of the variability in the
Vibrio levels in the Gulf. Salinity explains an additional 10% of the variability in these levels and also explains the
differences among the three sites. The Louisiana site is in a large
estuarine area, remote from direct river discharge and buffered from
the salt water of the Gulf of Mexico by barrier islands and marshes. The Florida site is heavily influenced by freshwater from the Apalachicola River during periods of high river flow and by salt water
from the wide inlets into the Gulf of Mexico during periods of low
river flow. This area showed the largest range in salinities and had
the highest salinities. The Alabama site is greatly influenced by the
Mobile River; its lowest salinities were also the lowest for the three
estuaries. Figure 4 shows that Louisiana experienced neither the lower
salinities that suppressed Vibrio levels in Alabama nor the
higher salinities that did the same in Florida. These results are
consistent with the experience with the Atlantic estuaries in this
study, because the higher Atlantic salinities are associated with
temperature-driven Vibrio concentrations that are lower than
those in the Gulf.
Water temperatures at the Atlantic and Gulf Coast sites were not
different, but salinities at the Atlantic Coast sites were higher
(P 0.01) and averaged >26 ppt. Work conducted by
Kaspar and Tamplin (10) in seawater microcosms containing
pure cultures of V. vulnificus suggested that survival was
adversely affected by exposure to elevated salinities (>25 ppt). In a
separate study conducted by Motes and DePaola (17), summer
Gulf Coast oysters relayed to high-salinity offshore waters (>32 ppt)
showed a significant reduction in V. vulnificus numbers
after a 2-week period. These results suggest that salinity extremes may
play a pivotal role in the survival and growth of V. vulnificus. High salinities for extended periods were not observed
at the Gulf Coast sites; however, when salinities increased above 25 ppt, V. vulnificus counts were reduced. A rapid increase in
salinity to >25 ppt at the Apalachicola Bay site in mid-August 1994 and again in late August and September 1995 coincided with rapid
declines in V. vulnificus levels within the oysters.
A clear relationship between salinity and V. vulnificus
counts has not been established. We suggest that within the range of
salinities normally encountered in northern Gulf estuaries characterized by high oyster production, salinity plays little role in
controlling V. vulnificus numbers. Results obtained with regression models generated from our salinity and temperature data
support this observation. However, the majority of observations in this
Gulf data were made when the salinity was between 5 and 25 ppt;
observations at both the upper and lower ends of the range were
minimal. Salinities higher than 25 ppt do have a negative effect on
V. vulnificus numbers in oysters. The data from the Atlantic
Coast sites support this, as do observations on a few samples from the
Florida site which had salinities of >25 ppt.
V. vulnificus has been isolated from oysters from numerous
Atlantic Coast sites (19, 20, 30); however, there have not been any documented illnesses associated with Atlantic Coast oysters (4, 21). We observed that V. vulnificus was
isolated less frequently and at lower densities from the Atlantic Coast
oysters than from the Gulf Coast oysters. During warm-weather months, the levels of V. vulnificus in Atlantic Coast oysters were
nearly 2 log units lower than in Gulf Coast oysters. On the other hand, Wright et al. (30) reported that levels of V. vulnificus in Chesapeake Bay oysters were similar to the levels in
Gulf Coast oysters during the warm months. If equal levels occurred, it
may be expected that Chesapeake Bay oysters would be the source of some
cases. It should be noted that the enumeration procedures used in the
Chesapeake Bay study employed a gene probe technique. It is possible
that the enumerating techniques are measuring different portions of the
V. vulnificus population or that the strains of V. vulnificus in the two locations differ in virulence. In
addition, the sharp decline in the Chesapeake harvest in recent years
(15) might have made it hard to observe this rare type of
infection.
In 1994 and 1995, 39 V. vulnificus illnesses were traced to
specific oyster harvest areas (4). Of these, 20 were traced to either Black Bay, La., or Apalachicola Bay, Fla. The contribution of
these areas to the total Gulf harvest during the warm periods of the
year, when most cases occur, is a possible linkage of oyster-associated V. vulnificus cases to these locations. Unfortunately, there
is no statistical data to accurately determine the harvest from
specific areas and the percentage of oysters from each area that are
consumed raw.
The 13 illnesses traced to oysters harvested from the study areas
occurred from late April to early November, the times when cases
typically occur. Four of these cases occurred in May 1995; May is the
month with the highest incidence of reported cases from 1992 to 1996 (4). The median V. vulnificus level in oyster samples harvested in May was 2,300 organisms per g, the same as during
other warm months. The V. vulnificus MPN counts (Table 2)
obtained from oysters sampled within 5 days of the date when the
illness-associated oysters were harvested were not unusually high for
the time of year (462 to 9,300 per g) and were consistent with those
reported by Jackson et al. (9). Salinity and temperature data did not indicate that any unusual hydrographic or climatic events
or conditions coincided with the harvest. However, the system for
tracing oysters back to a specific harvest site is not foolproof, and
the harvest areas are large and may contain microenvironments in which
oysters contain different concentrations of V. vulnificus.
Further, the V. vulnificus counts presented here are those
present in the oysters at harvest and probably represent only a minimum
count because of the possible multiplication that occurs after harvest
(1, 2). The total numbers of V. vulnificus may be
less relevant than the presence or proportion of virulent strains
(9).
In conclusion, the concentration of V. vulnificus in oysters
was similar in harvest areas across the northern Gulf Coast. Their
abundance was influenced primarily by the water temperature. The
numbers were at their highest from May through October, when the
majority of shellfish-associated V. vulnificus illnesses
occur. The two Atlantic estuaries sampled showed low levels of V. vulnificus even though the temperatures during the warm months
were similar to Gulf temperatures; this could be due to the much higher
salinity at these sites. The only high levels in the two Atlantic
estuaries occurred in North Carolina, when flooding lowered the
salinity to levels similar to those in the Gulf. Although salinities
below 25 ppt are alike in permitting high V. vulnificus
densities, salinities higher than 25 ppt appear to suppress the
densities. Conversely, variations in surface water temperature above
26°C have little effect on densities, but the densities decline
rapidly as temperatures decline below 26°C. Densities in Gulf Coast
oysters at harvest rarely exceed 104 MPN per g, suggesting
that oysters with counts higher than this level represent oysters that
have been temperature abused. Numbers were <10 MPN per g during
January and February, months when no illnesses have been recorded. This
research establishes a baseline level for V. vulnificus in
northern Gulf Coast oysters at harvest and reinforces findings that
salinities of >25 ppt suppress V. vulnificus levels even in
warm waters.
| |
ACKNOWLEDGMENTS |
We express our appreciation to the following individuals for
assistance in sample collection and shipping: B. Perkins and N. Scarborough (Alabama State Health Department, Seafood Division, Mobile,
Ala.); S. Moore and J. Shields (Florida Department of Environmental
Protection, Bureau of Marine Resources, Regulation and Development,
Apalachicola, Fla.); G. Mercadal and R. Anzalone (Louisiana Department
of Health and Hospitals, Molluscan Shellfish Program, New Orleans,
La.); W. Mobley (North Carolina Department of Environment, Health and
Natural Resources, Shellfish Sanitation Branch, Moorehead City, N.C.);
T. Yarborough (South Carolina Department of Health and Environmental
Control, Shellfish Sanitation, Charleston, S.C.); and T. Previto (FDA,
Office of Seafood, Gulf Coast Seafood Laboratory, Dauphin Island,
Ala.). We are also grateful to R. Creasy, M. Glatzer, and D. Hesselman
(FDA, Office of Regulatory Affairs, State Cooperative Programs,
Southeast Region, Atlanta, Ga.) for technical advice and to R. M. McPhearson (FDA, Office of Seafood, Gulf Coast Seafood Laboratory) for
reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Gulf Coast
Seafood Laboratory, Dauphin Island, AL 36528-0158. Phone: (334)
694-4480. Fax: (334) 694-4477. E-mail:
DWC{at}vm.cfsan.fda.gov.
| |
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
