Microb Eeol (1983) 9:65-75
IYIlCROBIL CCOLOGV 9 1983 Springer-Verlag
Distribution of Vibrio cholerae in Two Florida Estuaries Mary A. H o o d , *z G. E. Ness, 1 G. E. Rodrick, 2 and N. J. Blake 3 ~Department of Biology, University of West Florida, Pensacola, Florida 32504, USA; 2Department of Comprehensive Medicine, University of South Florida, Tampa, Florida 33612, USA; and 3Department of Marine Science, University of South Florida, St. Petersburg, Florida 33701, USA
Abstract. T h e distribution o f Vibrio cholerae was examined in 2 Florida estuaries, Apalachicola and T a m p a Bay. Vibrio cholerae serotype non-01 was the most a b u n d a n t serotype, being isolated from 45% o f the oyster samples, 30% o f the sediments, 50% o f the waters, and 75% o f the blue crabs. Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations between V. cholerae and temperature, salinity, or the other physical/chemical parameters measured, Escherichia coli, or fecal coliforms were not observed, but a range o f temperatures and salinities appeared relevant to the distribution o f the organism. The organism was present in the highest concentrations when salinities were 10%o--25%0 and temperatures were 20~176 In vitro growth curves o f 95 V. cholerae e n v i r o n m e n t a l isolates further supported that 10~176 was an ideal salinity range for the organisms. T h e results suggest that V. cholerae is a widely distributed organism in the nutrient-rich warm waters o f the G u l f Coast estuaries.
Introduction F r o m 1973 to the present, outbreaks o f cholera and cholera-like gastroenteritis have occurred in the G u l f Coast states o f Texas [9, 10, 19], Louisiana [4-6], and Florida [7, 8], and the evidence to date suggests that the most likely vector o f the organism, Vibrio cholerae, is shellfish. In Florida, the outbreaks have been traced to the c o n s u m p t i o n o f raw oysters, with both V. cholerae serotypes 01 and non-01 being implicated as the causative organism [4, 7, 8]. T h e ecology o f V. cholerae in U n i t e d States estuaries, first investigated in Chesapeake Bay by K a p e r et al. [I 5], showed that the organism was present in water, sediment, and shellfish, and that its presence did not correlate to fecal pollution indicators, that is, fecal coliforms. On the basis o f this information, it was concluded that the organism was an a u t o c h t h o n o u s species in the estuary. O t h e r studies, as reviewed by Colwell et al. [11 ], which were conducted in Louisiana and Mary-
* Present address: Bunting Institute, Radcliffe/Harvard, 10 Garden St., Cambridge, MA 02138. 0095-3628/83/0009-0065502.20
66
M.A. Hood et al.
l a n d as well as s t u d i e s c o n d u c t e d i n E n g l a n d [16, 20], h a v e f u r t h e r s u p p o r t e d this o b s e r v a t i o n . It was the p u r p o s e o f this i n v e s t i g a t i o n to e x a m i n e the ecology o f V. cholerae i n 2 F l o r i d a estuaries. T h e s e e s t u a r i e s were selected b e c a u s e t h e y r e p r e s e n t t h e m o s t e c o n o m i c a l l y i m p o r t a n t s o u r c e s o f shellfish i n the state. A p a l a c h i c o l a Bay is the p r i m a r y l o c a t i o n o f the s t a t e ' s o y s t e r i n d u s t r y w i t h a p p r o x i m a t e l y 6 m i l l i o n p o u n d s o f oysters h a r v e s t e d f r o m this b a y i n 1980. T a m p a Bay, i n a d d i t i o n to oysters, s u p p o r t s a n a c t i v e c l a m i n d u s t r y , a n d a p p r o x i m a t e l y 120 t h o u s a n d p o u n d s o f c l a m s were h a r v e s t e d f r o m this b a y i n 1980. T a m p a B a y is also u n i q u e as it is o n e o f the m o s t s o u t h e r l y l o c a t e d e s t u a r i e s i n t h e U n i t e d States a n d has a r e l a t i v e l y high t e m p e r a t u r e r e g i m e . S i n c e oysters h a v e p l a y e d a s i g n i f i c a n t role i n the t r a n s m i s s i o n o f V i b r i o - r e l a t e d g a s t r o e n t e r i t i s b e c a u s e t h e y are e a t e n raw, e m p h a s i s was p l a c e d o n the d i s t r i b u t i o n o f V. cholerae i n t h e oyster, Crassostrea virginica.
Materials and M e t h o d s
Sample Site and Collection The locations of sample sites are given in Fig. 1. Sites were selected representing a range of water qualities. Sites 36 and 3 were approved shellfish harvesting waters, site 19 was a conditionally approved area, and sites 29, 1, and 2 were prohibited shellfish harvesting waters. Oysters, Crassostrea virginica, and clams, Mercenaria campechiensis, were collected with tongs, placed in a Koolatron (Koolatron Industries, Ontario, Canada) with ice packs (Blue Ice), and shipped to the laboratory for analysis within 6 to 8 hours. Temperatures were maintained below 12~ Additional oyster samples, water, sediment, plankton, blue crabs, stone crabs, fiddler crabs, mussels, and algae were collected. All samples were placed in sterile containers, stored at 4~ and returned to the laboratory for analysis within 4 hours. Water samples were collected in sterile glass containers at the surface of the water, approximately 1/2 meter below the surface, and near the bottom of the water column. Sediments were collected using a ponar grab (Wildco, Saginaw, MI). Plankton samples were taken using a .01 mm mesh net for 10 min at approximately 4 knots. A total of 40 oyster samples were examined on a seasonal basis from Apalaehicola and Tampa Bay from 5 sites (19, 29, 36, 1, and 3) from April 1980 through August 1981. Eight clam samples were collected from Tampa Bay (sites 2 and 3); 6 water, 10 sediment, 4 plankton, 3 algal, 4 mussel samples, 6 blue crab samples, and 12 other crab (stone, fiddler, hermit) samples were collected from Apalachicola Bay during June, August, December, and March. Blue crabs were available only in June and August.
Physical and Chemical Parameters Temperature and salinity (Beckham RS5-3 thermister-salinometer), dissolved oxygen [18], and total suspended and volatile suspended matter [2] were determined.
Bacterial Analysis Oysters were washed, scrubbed free of dirt, and shucked with a sterile knife. Approximately 100 g oyster tissue (from 7-10 animals) and shell liquor were weighed, and an equal volume of phosphate buffer saline was added. The mixture was homogenized for 90 sec in a Waring blender and
V. cholerae in Florida Estuaries
67
Fig. 1. Location of collection sites in Apalachicola and Tampa Bay. Classification of the waters for shellfish harvesting is noted as approved, conditionally approved, prohibited, or unclassified.
~,LLY APPROVED l
ED (UNAPPROVED)
immediately (within less than 1 minute) added to an alkaline peptone water (APW) enrichment containing peptone (10 g/l) and NaC1 (10 g/l) in a 3-tube most probable number (MPN) series, as described by Hood et al. [12] for the detection and enumeration of V. cholerae, and to lauryl tryptose broth in a 3-tube MPN for the enumeration o f total and fecal coliforms. The procedure for total and fecal coliforms was carried out to the completed test according to standard procedures [1, 2], with 1 modification. Escherichia colt and coliform colonies from eosin methylene blue agar were confirmed by the API 20E Systems (Analytab Products, Inc., Plainview, NY). All media and reagents were obtained from Difco Laboratories, Inc. (Detroit, MI) unless otherwise specified. Oysters were dissected aseptically. Each tissue type (approximately 50 g) was placed into an equal volume of phosphate buffer saline, homogenized, and added to the APW enrichment in a 3-tube MPN series. Tissues examined included the gills, labial palps, mantle, and digestive tract (stomach and hind gut). Shell liquor was also examined. Using a sterile syringe, the gastric contents were withdrawn from the stomach and hind gut. Water samples were filtered through a sterile 0.45 ~ membrane filter (Millipore Corp., Bedford, MA) in aliquots o f one 1 liter, three 100 ml, three 10 ml, and three I ml. Celite (approximately 0.5 g/l) (J. T. Baker Chemical Co., Phillipsburg, NJ) was added to facilitate filtration. Each filter was then added to the APW enrichments. Sediment samples were added directly to the APW enrichments in a 3-tube MPN series in concentrations o f 10 g, 1 g, and 0.1 g. Hemolymph (1 ml aliquots) o f blue crabs (Callinectes sapidus) was collected with a sterile syringe and added to APW enrichments. Surface swabs were made over the entire animal and the individual
68
M . A . Hood et al.
swabs were placed into APW. The animal was then dissected and tissue parts were placed into APW. The tissues examined included the gills, the digestive tract, other visceral tissue and particles of the internal chitin. Whole stone crabs, fiddler crabs, hermit crabs (approximately 50 g), and the macrofilamentous algae (Ulva lactuca, approximately 20 g) were placed into an equal volume of phosphate buffer saline and ground in a homogenizer (Virtis Co., Gardiner, NY) for 1 minute at a moderate speed (40 unit reading). The homogenate was added to the APW enrichment in a 3-tube MPN series. Mussels (Brachiodonta sp.) were opened with a sterile knife, and 30 g of tissue was added to 100 ml of phosphate buffer saline. The tissue was homogenized and added to APW, as in the oyster preparation. After 6 hours of incubation at 35~ 1 loopful of the homogenate was streaked onto thiosulfatc citrate bile salts agar (TCBS) and incubated for 20 hours at 35~ Green and yellow colonies were selected and streaked onto trypticase (Baltimore Biological Laboratories, Cockeysville, MD) (10 g/l), sodium chloride (10 g/l), agar, or trypticase soy agar (TSA) (BBL) plus NaC1. All isolates were screened for cytochrome oxidase using the disk method, 0/129 (Sigma, St. Louis, MO) sensitivity, and growth on and utilization of gelatin with 0% NaCI. All oxidase positive and 0/129 sensitive colonies that grew on and utilized gelatin were placed onto the API 20E System. The only isolates considered to be K choleraewere those that gave typical reactions on API 20E. Serotyping was performed using polyvalent and monovalent V. cholerae antiserum. Isolates were also serotyped by Dr. R. Seibeling, Louisiana State University, Baton Rouge, Louisiana.
Salinity Studies All 1I. choleraeisolates (95 non-01 environmental strains, 2 01 environmental strains, and 1 01 clinical strain obtained from a local outbreak of cholera [8]) were grown in TSB 1% NaC1 at 35"C for 18 hours. Aliquots of 0.1 ml were inoculated into tubes of peptone broth pH 7.6 containing 1% peptone and sea salts (Instant Ocean, Aquarium Systems, Inc., Eastland, OII) at concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 70, and 90~ After 24 hours at 35~ growth was measured turbidimetrically using a Klett-Summersson spectrophotometer. Composite graphs were made by determining the percent of maximum growth at each salinity for each isolate and calculating the mean for all the isolates.
Statistical Analysis Using standard statistical programs (SAS), analysis of variance and regression analysis were performed. Multiple linear correlation coefficients were calculated and tested for significance at the 95% and 99% confidence level by comparing them with tabulated r values [16]. F values were calculated and compared with tabulated values [ 16] to determine if there were significant differences at the 95% confidence level.
Results
Vibrio cholerae s e r o t y p e n o n - 0 1 s t r a i n s w e r e i s o l a t e d f r o m 4 5 % o f all o y s t e r samples. Although fewer samples were taken of sediments, water, and blue c r a b s , 3 0 % o f t h e s e d i m e n t s , 5 0 % o f t h e w a t e r s , a n d 7 5 % o f all b l u e c r a b s c o n t a i n e d V. cholerae n o n - 0 1 ( T a b l e 1). O f 4 7 n o n - 0 1 i s o l a t e s t h a t w e r e ser o t y p e d , 16 d i f f e r e n t s e r o v a r s a n d s e v e r a l s t r a i n s w i t h m u l t i p l e a n t i g e n s w e r e n o t e d ( T a b l e 2). I n c o n t r a s t t o V. cholerae n o n - 0 1 , s e r o t y p e 01 w a s far less a b u n d a n t . O n l y 2 s t r a i n s o f V. cholerae s e r o t y p e 01 w e r e i s o l a t e d f r o m 1 o y s t e r
V. cholerae in Florida Estuaries
69
Table 1. Frequency of Vibrio choleraeisolation in Florida estuaries
Samples Oysters (site) 19 29 36 1 3
No. samples examined
No. samples containing V. cholerae non-01
No. samples containing V. cholerae 01
6 8 16 5 5
5 (83%) 4 (50%) 5 (31%) 3 (60~ 1 (20%)
0 0 1 (6%) 0 0
Clams 2 3
4 4
0 0
0 0
Blue crabs 36
6
4 (67%)
0
Other crabs 29/36
12
0
0
Water 29 36
2 4
1 (50%) 2 (50%)
0 0
Sediment 19 29 36
2 3 5
2 (100%) 0 1 (20%)
0 0 0
Mussels 29/36
4
0
0
Plankton 29/36
4
0
0
Algae 29/36
3
0
0
s a m p l e . Vibrio cholerae w a s n o t i s o l a t e d f r o m o t h e r c r a b s , m u s s e l s , p l a n k t o n , or the filamentous algae commonly found growing attached to oyster beds. S t r o n g l i n e a r c o r r e l a t i o n s b e t w e e n I1. cholerae a n d t h e p h y s i c a l / c h e m i c a l parameters examined were not evident but a range of temperatures and salini t i e s a p p e a r e d r e l e v a n t t o t h e d i s t r i b u t i o n o f t h e o r g a n i s m . S t r a i n s o f V. cholerae serotypes non-01 were isolated from oysters, waters, sediments, and blue crab s a m p l e s c o l l e c t e d f r o m w a t e r s w h o s e s a l i n i t i e s r a n g e d f r o m 6%o-30.6%o a n d whose temperatures ranged from 9.6~176 Vibrio cholerae 01 w a s i s o l a t e d f r o m o y s t e r s c o l l e c t e d f r o m a n a r e a w h o s e s a l i n i t y w a s 12%0 a n d w h o s e t e m p e r a t u r e w a s 29~ F i g u r e 2 p r e s e n t s t h e c o n c e n t r a t i o n o f K cholerae in o y s t e r s c o l l e c t e d a t d i f f e r e n t s a l i n i t i e s . T h e m o s t a b u n d a n t l e v e l s o f V. cholerae w e r e
70
M. A. Hood et al.
Table 2. Frequency of V. cholerae serotypes non-01 L.S.U. serovar" Unknown DD BB CC Q Id JJ OO N E M U X T LL K Z Multiple antigens Id/W/V/J/P Q/Y Y/NN Id/CC
No. isolated 15 9 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Source oyster, water, blue crab oyster, water oyster oyster oyster oyster water oyster oyster water oyster oyster oyster oyster oyster oyster oyster oyster oyster oyster blue crab
"Serotyping was done by Dr. Ron J. Seibeling, Louisiana State University, Baton Rouge, Louisiana Mean levels of Vibrio cholerae in oysters collected from Apalachicola and Tampa Bay Table 3.
Sites
K cholerae
1 29 19 36 3
424 23 21 10 2
MPN levels/lO0 g oyster tissue
f o u n d at s a l i n i t i e s o f 12%o--25~. In vitro g r o w t h c u r v e s f o r t h e V. cholerae environmental isolates revealed a similar pattern. The composite growth curves for t h e n o n - 0 1 s t r a i n s a n d t h e 01 s t r a i n s ( p l u s t h e s a l i n i t y o p t i m u m f o r t h e c l i n i c a l s t r a i n ) a r e p r e s e n t e d in Fig. 3. A l t h o u g h all i s o l a t e s g r e w a t 0~ V. cholerae s e r o t y p e n o n - 0 1 i s o l a t e s g r e w b e s t a t s a l i n i t i e s o f 1 5 ~ The 2 e n v i r o n m e n t a l 01 i s o l a t e s g r e w o p t i m a l l y a t 5~176 w h e r e a s t h e c l i n i c a l 01 s t r a i n e x h i b i t e d o p t i m u m g r o w t h a t 15~--35~ The results of these studies suggest t h a t V. cholerae g r o w s b e s t a t s a l i n i t i e s t y p i c a l o f e s t u a r i n e w a t e r s . Vibrio cholerae n o n - 0 1 s h o w e d a s e a s o n a l d i s t r i b u t i o n i n o y s t e r s w i t h h i g h e s t concentration occurring during the months of August, September, October, and
V. cholerae in Florida Estuaries
71
20
o
E
.E
2 4
6
8 ].0 12 -14 16--18--20 22 24 26 28-30 T32-34 3~6 38
Salinity (%0) Fig. 2. Bay.
The effect o f salinity on the distribution of Vibrio cholerae in Apalachicola and Tampa
November (Fig. 4). However, serotype 01 strains were recovered only in April 1980 from Apalachicola Bay. Vibrio cholerae non-01 also appeared to follow a similar distribution pattern in sediment, water, and blue crabs. Statistical analysis of grouped distribution data revealed that V. cholerae was significantly higher when environmental water temperatures were above 20~ Concentrations of V. choleare showed no correlation with total presumptive coliforms, total confirmed coliforms, fecal coliforms, or E. coli. However, levels of K cholerae in oysters were significantly highest in prohibited shellfish harvesting waters, intermediate in conditionally approved waters, and lowest in approved waters (Table 3). Shellfish harvesting waters are classified on the basis of historical, bacteriological, and hydrological data collected over many years by the Florida State Department of Natural Resources (DNR). Since the bacteriological data used by DNR to classify waters include fecal coliform levels, the results seem somewhat contradictory. However, nutrient levels are probably higher in the prohibited shellfish harvesting areas, which may account for these distribution patterns.
72
M.A. Hood et al.
100
g
90
80
.=
g
E E
70
o
6o
;
i
tjI' I
l t
Z 1
g
5o
t
40
o
\
3oli 20
10
0| 0
I 10
I 20
I 30
I 40
| 50
I 60
I 70
I 80
I 90
Salinity 1%0)
Fig. 3. The effect of salinity on the growth of Vibrio cholerae. The isolates included 95 strains of V. cholerae non-01 (A), 1 strain of a clinical V. cholerae 01 (B), and 2 strains of environmental V. cholerae 01 (C). Dissections o f the oyster revealed that V. cholerae was present only in the digestive tract. T h e o r g a n i s m could not be r e c o v e r e d f r o m the gills, the labial pulps, the m a n t l e tissue, or the shell liquor. Microscopic e x a m i n a t i o n o f the s t o m a c h contents o f the oysters containing V. cholerae revealed a p r e d o m i n a n c e o f diatoms. Dinoflagellates, algae, a n d small unidentifiable detrital particles were also present. P r e l i m i n a r y u p t a k e studies o f V. cholerae f r o m the oyster sample in o u r l a b o r a t o r y also s u p p o r t e d the finding that the b a c t e r i u m is concentrated in the digestive tract. In the blue crab, the o r g a n i s m was f o u n d on the surface o f the exoskeleton, on the gills, in the h e m o l y m p h , in the digestive tract, and on internal chitin structures.
Discussion In the 2 Florida estuaries examined, V. cholerae non-01 was widely distributed and could be found in oysters, blue crabs, water, a n d sediment. H o w e v e r , K
K cholerae in Florida Estuaries
73
20
~ 2.0
1.0 o
.8
E z
,4
.2
10
o
):
9
-
"J'~z
.
A
I~
J
A
J
S
o
9
0
N
~
D
J
~
: l
M
A
M
J
J
A
~.o
I~
.9
"~
.7
"6
.5
~
.4
z~
.3
.8
E
sediment 9 water 9
.2 i
A
M
J
J
A
S
0
N
D'l'~ J
F
M
A
M
I
I
J
Fig. 4. The seasonal distribution of I,'. cholerae in Apalachicola and Tampa Bay. Top figure presents V. cholerae in oysters. Bottom figure presents blue crabs, sediment, and water.
cholerae 01 was far less frequent, being isolated from only 1 oyster sample collected in Apalachicola Bay. In oysters, the organism was recovered only from the digestive tract, whereas in crabs, it could be found throughout the body. Although statistical analysis revealed no strong linear correlations between the distribution of the organism and the parameters, salinity and temperature, the organism was present in the highest concentrations when salinities were 10~176 and temperatures, 20~176 The abundance of the organism in the estuary and the lack of correlation between fecal coliforms and V. cholerae levels support the findings of Kaper et al. [15] that V. cholerae may be autochthonous to United States estuaries. The presence of the organism in oysters when there is an abundance of diatoms, dinoflagellates, and detrital particles in the digestive tract of the animal, coupled with the knowledge that the bacterium readily attaches to chitin [3], suggests that V. cholerae may be concentrated as a result of the oyster feeding on certain plankton to which the bacterium is attached. Although V. cholerae was not
74
M.A. Hood et al.
i s o l a t e d f r o m p l a n k t o n c o l l e c t e d f r o m A p a l a c h i c o l a Bay, o t h e r i n v e s t i g a t o r s h a v e i s o l a t e d F. cholerae f r o m C h e s a p e a k e Bay p l a n k t o n [11]. It is u n c l e a r w h y we h a v e b e e n u n a b l e to i s o l a t e V. cholerae f r o m p l a n k t o n , b u t t h e r e is s t r o n g e v i d e n c e suggesting t h a t F. cholerae specifically a d h e r e s to c o p e p o d s [ 14] a n d t h a t the o r g a n i s m exists i n t h e e n v i r o n m e n t i n a n e p i b i o t i c state [13]. Acknowledgments. We thank the following investigators for helpful discussions: Dr. R. R. ColwelI, Dr. R. J. Siebeling, Dr. R. J. Seidler, and Nell Roberts. Special thanks are extended to Dr. R. J. Siebeling for providing serotype information. We also gratefully acknowledge John Cheney and Kathy Williams for their excellent technical support, Florida State Department of Natural Resources for their logistical support, and Dr. Mike Bundrick and the University of West Florida Institute for Statistical and Mathematical Modeling for statistical assistance. The work is a result of research sponsored by Florida Sea Grant, NOAA, Office of Sea Grant, Department of Commerce, under Grant No. SG #NA 80AA-D-00038. The U.S. Government is authorized to produce and distribute any copyright portion that may appear herein. Acknowledgmentis also given to the BuntingInstitute for support in the preparation of the manuscript.
References 1. Association of Analytical Chemists (1975) Official methods of analysis of the association of analytical chemists, 12th ed. Washington, DC 2. American Public Health Association (1975) Standard methods for the examination of water and wastewater, 14th ed. American Public Health Association, Inc, Washington, DC 3. Belas, MR, ColweU RR: (1982) Adsorption kinetics of laterally and polarly flagellated Vibrio. J Bacteriol 151:1568-1580 4. Blake PA, AUegra DT, Snyder JD, Barrett TJ, McFarland L, Caraway CT, Felley JC, Craig JP, Lee JV, Puhr ND, Feldman RA: (1980) Cholera--a possible endemic focus in the United States. N Engl J Med 302:305-309 5. Center for Disease Control (1978) Vibrio cholerae--Louisiana. Morb Mort Weekly Rpts 27:637. 6. Center for Disease Control (1978) Follow up on Vibrio cholerae serotype Inaba infection-Louisiana. Morb Mort Weekly Rpts 27:388 7. Center for Disease Control (1979) Non-01 Vibrio cholerae infections--Florida. Morb Mort Weekly Rots 28:571-572 8. Center for Disease Control (1980) Cholera--Florida. Morb Mort Weekly Rpts 29:601 9. Center for Disease Control (1981) Cholera--Texas. Morb Mort Weekly Rpts 30:389-390 10. Center for Disease Control (1981) Cholera on a Gulf Coast oil rig--Texas. Morb Mort Weekly Rpts 30:589-590 11. Colwell RR, Seidler RJ, Kaper J, Joseph SW, Garges S, Lockman H, Maneval D, Bradford H, Roberts N, Remmers E, Huq I, and Huq A (1981) Occurrence of l/ibrio cho[erae serotype 01 in Maryland and Louisiana estuaries. Appl Environ Microbiol 41:555-558 12. Hood MA, Ness GE, Rodrick GE (1981) Isolation of Vibrio cholerae serotype 01 from the eastern oyster, Crassostrea virginica. Appl Environ Microbiol 41:559-560 13. Hood MA, Ness GE, Rodrick GE, Blake NJ (1982) The ecology of Vibrio cholerae in two Florida estuaries. In: Colwell RR (ed) Vibrios in the environment, John Wiley and Sons, New York, in press 14. Huq A, Small EB, West PA, Huq MI, Rahman R, Colwell RR (1983) Ecological relationships between Vibrio cholerae and plantonic crustacean copepods. Appl Environ Microbiol 45: 275-283 15. Kaper J, Lockman H, Colweli RR, Joseph SW (1979) Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay. Appl Environ Microbiol 37:91-103
II. cholerae in Florida Estuaries
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16. Lee JV, Bashford DJ, Donovan TJ, Furniso AL, West PA (1982) The incidence of Vibrio cholerae in water, animals and birds in Kent, England. J Appl Bacteriol 52:281-291 17. Snedecor GW, Cochran WG (1967) Statistical methods, 6th ed. Iowa State University Press, Ames 18. Strickland JDH, Parsons TR (1972) A practical handbook for seawater analysis, 2nd ed. Fish Res Bd of Can Bull 19. Weissman JB, DeWitt WE, Thompson J, Muchnick CN, Portnoy BL, Feeley JC, Gangarosa EJ (1974) A case of cholera in Texas, 1973. Am J Epidemiol 100:487-498 20. West PA, Lee JV (1982) Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England. J Appl Bacteriol 52:435--448
IYIlCROBIL CCOLOGV 9 1983 Springer-Verlag
Distribution of Vibrio cholerae in Two Florida Estuaries Mary A. H o o d , *z G. E. Ness, 1 G. E. Rodrick, 2 and N. J. Blake 3 ~Department of Biology, University of West Florida, Pensacola, Florida 32504, USA; 2Department of Comprehensive Medicine, University of South Florida, Tampa, Florida 33612, USA; and 3Department of Marine Science, University of South Florida, St. Petersburg, Florida 33701, USA
Abstract. T h e distribution o f Vibrio cholerae was examined in 2 Florida estuaries, Apalachicola and T a m p a Bay. Vibrio cholerae serotype non-01 was the most a b u n d a n t serotype, being isolated from 45% o f the oyster samples, 30% o f the sediments, 50% o f the waters, and 75% o f the blue crabs. Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations between V. cholerae and temperature, salinity, or the other physical/chemical parameters measured, Escherichia coli, or fecal coliforms were not observed, but a range o f temperatures and salinities appeared relevant to the distribution o f the organism. The organism was present in the highest concentrations when salinities were 10%o--25%0 and temperatures were 20~176 In vitro growth curves o f 95 V. cholerae e n v i r o n m e n t a l isolates further supported that 10~176 was an ideal salinity range for the organisms. T h e results suggest that V. cholerae is a widely distributed organism in the nutrient-rich warm waters o f the G u l f Coast estuaries.
Introduction F r o m 1973 to the present, outbreaks o f cholera and cholera-like gastroenteritis have occurred in the G u l f Coast states o f Texas [9, 10, 19], Louisiana [4-6], and Florida [7, 8], and the evidence to date suggests that the most likely vector o f the organism, Vibrio cholerae, is shellfish. In Florida, the outbreaks have been traced to the c o n s u m p t i o n o f raw oysters, with both V. cholerae serotypes 01 and non-01 being implicated as the causative organism [4, 7, 8]. T h e ecology o f V. cholerae in U n i t e d States estuaries, first investigated in Chesapeake Bay by K a p e r et al. [I 5], showed that the organism was present in water, sediment, and shellfish, and that its presence did not correlate to fecal pollution indicators, that is, fecal coliforms. On the basis o f this information, it was concluded that the organism was an a u t o c h t h o n o u s species in the estuary. O t h e r studies, as reviewed by Colwell et al. [11 ], which were conducted in Louisiana and Mary-
* Present address: Bunting Institute, Radcliffe/Harvard, 10 Garden St., Cambridge, MA 02138. 0095-3628/83/0009-0065502.20
66
M.A. Hood et al.
l a n d as well as s t u d i e s c o n d u c t e d i n E n g l a n d [16, 20], h a v e f u r t h e r s u p p o r t e d this o b s e r v a t i o n . It was the p u r p o s e o f this i n v e s t i g a t i o n to e x a m i n e the ecology o f V. cholerae i n 2 F l o r i d a estuaries. T h e s e e s t u a r i e s were selected b e c a u s e t h e y r e p r e s e n t t h e m o s t e c o n o m i c a l l y i m p o r t a n t s o u r c e s o f shellfish i n the state. A p a l a c h i c o l a Bay is the p r i m a r y l o c a t i o n o f the s t a t e ' s o y s t e r i n d u s t r y w i t h a p p r o x i m a t e l y 6 m i l l i o n p o u n d s o f oysters h a r v e s t e d f r o m this b a y i n 1980. T a m p a Bay, i n a d d i t i o n to oysters, s u p p o r t s a n a c t i v e c l a m i n d u s t r y , a n d a p p r o x i m a t e l y 120 t h o u s a n d p o u n d s o f c l a m s were h a r v e s t e d f r o m this b a y i n 1980. T a m p a B a y is also u n i q u e as it is o n e o f the m o s t s o u t h e r l y l o c a t e d e s t u a r i e s i n t h e U n i t e d States a n d has a r e l a t i v e l y high t e m p e r a t u r e r e g i m e . S i n c e oysters h a v e p l a y e d a s i g n i f i c a n t role i n the t r a n s m i s s i o n o f V i b r i o - r e l a t e d g a s t r o e n t e r i t i s b e c a u s e t h e y are e a t e n raw, e m p h a s i s was p l a c e d o n the d i s t r i b u t i o n o f V. cholerae i n t h e oyster, Crassostrea virginica.
Materials and M e t h o d s
Sample Site and Collection The locations of sample sites are given in Fig. 1. Sites were selected representing a range of water qualities. Sites 36 and 3 were approved shellfish harvesting waters, site 19 was a conditionally approved area, and sites 29, 1, and 2 were prohibited shellfish harvesting waters. Oysters, Crassostrea virginica, and clams, Mercenaria campechiensis, were collected with tongs, placed in a Koolatron (Koolatron Industries, Ontario, Canada) with ice packs (Blue Ice), and shipped to the laboratory for analysis within 6 to 8 hours. Temperatures were maintained below 12~ Additional oyster samples, water, sediment, plankton, blue crabs, stone crabs, fiddler crabs, mussels, and algae were collected. All samples were placed in sterile containers, stored at 4~ and returned to the laboratory for analysis within 4 hours. Water samples were collected in sterile glass containers at the surface of the water, approximately 1/2 meter below the surface, and near the bottom of the water column. Sediments were collected using a ponar grab (Wildco, Saginaw, MI). Plankton samples were taken using a .01 mm mesh net for 10 min at approximately 4 knots. A total of 40 oyster samples were examined on a seasonal basis from Apalaehicola and Tampa Bay from 5 sites (19, 29, 36, 1, and 3) from April 1980 through August 1981. Eight clam samples were collected from Tampa Bay (sites 2 and 3); 6 water, 10 sediment, 4 plankton, 3 algal, 4 mussel samples, 6 blue crab samples, and 12 other crab (stone, fiddler, hermit) samples were collected from Apalachicola Bay during June, August, December, and March. Blue crabs were available only in June and August.
Physical and Chemical Parameters Temperature and salinity (Beckham RS5-3 thermister-salinometer), dissolved oxygen [18], and total suspended and volatile suspended matter [2] were determined.
Bacterial Analysis Oysters were washed, scrubbed free of dirt, and shucked with a sterile knife. Approximately 100 g oyster tissue (from 7-10 animals) and shell liquor were weighed, and an equal volume of phosphate buffer saline was added. The mixture was homogenized for 90 sec in a Waring blender and
V. cholerae in Florida Estuaries
67
Fig. 1. Location of collection sites in Apalachicola and Tampa Bay. Classification of the waters for shellfish harvesting is noted as approved, conditionally approved, prohibited, or unclassified.
~,LLY APPROVED l
ED (UNAPPROVED)
immediately (within less than 1 minute) added to an alkaline peptone water (APW) enrichment containing peptone (10 g/l) and NaC1 (10 g/l) in a 3-tube most probable number (MPN) series, as described by Hood et al. [12] for the detection and enumeration of V. cholerae, and to lauryl tryptose broth in a 3-tube MPN for the enumeration o f total and fecal coliforms. The procedure for total and fecal coliforms was carried out to the completed test according to standard procedures [1, 2], with 1 modification. Escherichia colt and coliform colonies from eosin methylene blue agar were confirmed by the API 20E Systems (Analytab Products, Inc., Plainview, NY). All media and reagents were obtained from Difco Laboratories, Inc. (Detroit, MI) unless otherwise specified. Oysters were dissected aseptically. Each tissue type (approximately 50 g) was placed into an equal volume of phosphate buffer saline, homogenized, and added to the APW enrichment in a 3-tube MPN series. Tissues examined included the gills, labial palps, mantle, and digestive tract (stomach and hind gut). Shell liquor was also examined. Using a sterile syringe, the gastric contents were withdrawn from the stomach and hind gut. Water samples were filtered through a sterile 0.45 ~ membrane filter (Millipore Corp., Bedford, MA) in aliquots o f one 1 liter, three 100 ml, three 10 ml, and three I ml. Celite (approximately 0.5 g/l) (J. T. Baker Chemical Co., Phillipsburg, NJ) was added to facilitate filtration. Each filter was then added to the APW enrichments. Sediment samples were added directly to the APW enrichments in a 3-tube MPN series in concentrations o f 10 g, 1 g, and 0.1 g. Hemolymph (1 ml aliquots) o f blue crabs (Callinectes sapidus) was collected with a sterile syringe and added to APW enrichments. Surface swabs were made over the entire animal and the individual
68
M . A . Hood et al.
swabs were placed into APW. The animal was then dissected and tissue parts were placed into APW. The tissues examined included the gills, the digestive tract, other visceral tissue and particles of the internal chitin. Whole stone crabs, fiddler crabs, hermit crabs (approximately 50 g), and the macrofilamentous algae (Ulva lactuca, approximately 20 g) were placed into an equal volume of phosphate buffer saline and ground in a homogenizer (Virtis Co., Gardiner, NY) for 1 minute at a moderate speed (40 unit reading). The homogenate was added to the APW enrichment in a 3-tube MPN series. Mussels (Brachiodonta sp.) were opened with a sterile knife, and 30 g of tissue was added to 100 ml of phosphate buffer saline. The tissue was homogenized and added to APW, as in the oyster preparation. After 6 hours of incubation at 35~ 1 loopful of the homogenate was streaked onto thiosulfatc citrate bile salts agar (TCBS) and incubated for 20 hours at 35~ Green and yellow colonies were selected and streaked onto trypticase (Baltimore Biological Laboratories, Cockeysville, MD) (10 g/l), sodium chloride (10 g/l), agar, or trypticase soy agar (TSA) (BBL) plus NaC1. All isolates were screened for cytochrome oxidase using the disk method, 0/129 (Sigma, St. Louis, MO) sensitivity, and growth on and utilization of gelatin with 0% NaCI. All oxidase positive and 0/129 sensitive colonies that grew on and utilized gelatin were placed onto the API 20E System. The only isolates considered to be K choleraewere those that gave typical reactions on API 20E. Serotyping was performed using polyvalent and monovalent V. cholerae antiserum. Isolates were also serotyped by Dr. R. Seibeling, Louisiana State University, Baton Rouge, Louisiana.
Salinity Studies All 1I. choleraeisolates (95 non-01 environmental strains, 2 01 environmental strains, and 1 01 clinical strain obtained from a local outbreak of cholera [8]) were grown in TSB 1% NaC1 at 35"C for 18 hours. Aliquots of 0.1 ml were inoculated into tubes of peptone broth pH 7.6 containing 1% peptone and sea salts (Instant Ocean, Aquarium Systems, Inc., Eastland, OII) at concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 70, and 90~ After 24 hours at 35~ growth was measured turbidimetrically using a Klett-Summersson spectrophotometer. Composite graphs were made by determining the percent of maximum growth at each salinity for each isolate and calculating the mean for all the isolates.
Statistical Analysis Using standard statistical programs (SAS), analysis of variance and regression analysis were performed. Multiple linear correlation coefficients were calculated and tested for significance at the 95% and 99% confidence level by comparing them with tabulated r values [16]. F values were calculated and compared with tabulated values [ 16] to determine if there were significant differences at the 95% confidence level.
Results
Vibrio cholerae s e r o t y p e n o n - 0 1 s t r a i n s w e r e i s o l a t e d f r o m 4 5 % o f all o y s t e r samples. Although fewer samples were taken of sediments, water, and blue c r a b s , 3 0 % o f t h e s e d i m e n t s , 5 0 % o f t h e w a t e r s , a n d 7 5 % o f all b l u e c r a b s c o n t a i n e d V. cholerae n o n - 0 1 ( T a b l e 1). O f 4 7 n o n - 0 1 i s o l a t e s t h a t w e r e ser o t y p e d , 16 d i f f e r e n t s e r o v a r s a n d s e v e r a l s t r a i n s w i t h m u l t i p l e a n t i g e n s w e r e n o t e d ( T a b l e 2). I n c o n t r a s t t o V. cholerae n o n - 0 1 , s e r o t y p e 01 w a s far less a b u n d a n t . O n l y 2 s t r a i n s o f V. cholerae s e r o t y p e 01 w e r e i s o l a t e d f r o m 1 o y s t e r
V. cholerae in Florida Estuaries
69
Table 1. Frequency of Vibrio choleraeisolation in Florida estuaries
Samples Oysters (site) 19 29 36 1 3
No. samples examined
No. samples containing V. cholerae non-01
No. samples containing V. cholerae 01
6 8 16 5 5
5 (83%) 4 (50%) 5 (31%) 3 (60~ 1 (20%)
0 0 1 (6%) 0 0
Clams 2 3
4 4
0 0
0 0
Blue crabs 36
6
4 (67%)
0
Other crabs 29/36
12
0
0
Water 29 36
2 4
1 (50%) 2 (50%)
0 0
Sediment 19 29 36
2 3 5
2 (100%) 0 1 (20%)
0 0 0
Mussels 29/36
4
0
0
Plankton 29/36
4
0
0
Algae 29/36
3
0
0
s a m p l e . Vibrio cholerae w a s n o t i s o l a t e d f r o m o t h e r c r a b s , m u s s e l s , p l a n k t o n , or the filamentous algae commonly found growing attached to oyster beds. S t r o n g l i n e a r c o r r e l a t i o n s b e t w e e n I1. cholerae a n d t h e p h y s i c a l / c h e m i c a l parameters examined were not evident but a range of temperatures and salini t i e s a p p e a r e d r e l e v a n t t o t h e d i s t r i b u t i o n o f t h e o r g a n i s m . S t r a i n s o f V. cholerae serotypes non-01 were isolated from oysters, waters, sediments, and blue crab s a m p l e s c o l l e c t e d f r o m w a t e r s w h o s e s a l i n i t i e s r a n g e d f r o m 6%o-30.6%o a n d whose temperatures ranged from 9.6~176 Vibrio cholerae 01 w a s i s o l a t e d f r o m o y s t e r s c o l l e c t e d f r o m a n a r e a w h o s e s a l i n i t y w a s 12%0 a n d w h o s e t e m p e r a t u r e w a s 29~ F i g u r e 2 p r e s e n t s t h e c o n c e n t r a t i o n o f K cholerae in o y s t e r s c o l l e c t e d a t d i f f e r e n t s a l i n i t i e s . T h e m o s t a b u n d a n t l e v e l s o f V. cholerae w e r e
70
M. A. Hood et al.
Table 2. Frequency of V. cholerae serotypes non-01 L.S.U. serovar" Unknown DD BB CC Q Id JJ OO N E M U X T LL K Z Multiple antigens Id/W/V/J/P Q/Y Y/NN Id/CC
No. isolated 15 9 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Source oyster, water, blue crab oyster, water oyster oyster oyster oyster water oyster oyster water oyster oyster oyster oyster oyster oyster oyster oyster oyster oyster blue crab
"Serotyping was done by Dr. Ron J. Seibeling, Louisiana State University, Baton Rouge, Louisiana Mean levels of Vibrio cholerae in oysters collected from Apalachicola and Tampa Bay Table 3.
Sites
K cholerae
1 29 19 36 3
424 23 21 10 2
MPN levels/lO0 g oyster tissue
f o u n d at s a l i n i t i e s o f 12%o--25~. In vitro g r o w t h c u r v e s f o r t h e V. cholerae environmental isolates revealed a similar pattern. The composite growth curves for t h e n o n - 0 1 s t r a i n s a n d t h e 01 s t r a i n s ( p l u s t h e s a l i n i t y o p t i m u m f o r t h e c l i n i c a l s t r a i n ) a r e p r e s e n t e d in Fig. 3. A l t h o u g h all i s o l a t e s g r e w a t 0~ V. cholerae s e r o t y p e n o n - 0 1 i s o l a t e s g r e w b e s t a t s a l i n i t i e s o f 1 5 ~ The 2 e n v i r o n m e n t a l 01 i s o l a t e s g r e w o p t i m a l l y a t 5~176 w h e r e a s t h e c l i n i c a l 01 s t r a i n e x h i b i t e d o p t i m u m g r o w t h a t 15~--35~ The results of these studies suggest t h a t V. cholerae g r o w s b e s t a t s a l i n i t i e s t y p i c a l o f e s t u a r i n e w a t e r s . Vibrio cholerae n o n - 0 1 s h o w e d a s e a s o n a l d i s t r i b u t i o n i n o y s t e r s w i t h h i g h e s t concentration occurring during the months of August, September, October, and
V. cholerae in Florida Estuaries
71
20
o
E
.E
2 4
6
8 ].0 12 -14 16--18--20 22 24 26 28-30 T32-34 3~6 38
Salinity (%0) Fig. 2. Bay.
The effect o f salinity on the distribution of Vibrio cholerae in Apalachicola and Tampa
November (Fig. 4). However, serotype 01 strains were recovered only in April 1980 from Apalachicola Bay. Vibrio cholerae non-01 also appeared to follow a similar distribution pattern in sediment, water, and blue crabs. Statistical analysis of grouped distribution data revealed that V. cholerae was significantly higher when environmental water temperatures were above 20~ Concentrations of V. choleare showed no correlation with total presumptive coliforms, total confirmed coliforms, fecal coliforms, or E. coli. However, levels of K cholerae in oysters were significantly highest in prohibited shellfish harvesting waters, intermediate in conditionally approved waters, and lowest in approved waters (Table 3). Shellfish harvesting waters are classified on the basis of historical, bacteriological, and hydrological data collected over many years by the Florida State Department of Natural Resources (DNR). Since the bacteriological data used by DNR to classify waters include fecal coliform levels, the results seem somewhat contradictory. However, nutrient levels are probably higher in the prohibited shellfish harvesting areas, which may account for these distribution patterns.
72
M.A. Hood et al.
100
g
90
80
.=
g
E E
70
o
6o
;
i
tjI' I
l t
Z 1
g
5o
t
40
o
\
3oli 20
10
0| 0
I 10
I 20
I 30
I 40
| 50
I 60
I 70
I 80
I 90
Salinity 1%0)
Fig. 3. The effect of salinity on the growth of Vibrio cholerae. The isolates included 95 strains of V. cholerae non-01 (A), 1 strain of a clinical V. cholerae 01 (B), and 2 strains of environmental V. cholerae 01 (C). Dissections o f the oyster revealed that V. cholerae was present only in the digestive tract. T h e o r g a n i s m could not be r e c o v e r e d f r o m the gills, the labial pulps, the m a n t l e tissue, or the shell liquor. Microscopic e x a m i n a t i o n o f the s t o m a c h contents o f the oysters containing V. cholerae revealed a p r e d o m i n a n c e o f diatoms. Dinoflagellates, algae, a n d small unidentifiable detrital particles were also present. P r e l i m i n a r y u p t a k e studies o f V. cholerae f r o m the oyster sample in o u r l a b o r a t o r y also s u p p o r t e d the finding that the b a c t e r i u m is concentrated in the digestive tract. In the blue crab, the o r g a n i s m was f o u n d on the surface o f the exoskeleton, on the gills, in the h e m o l y m p h , in the digestive tract, and on internal chitin structures.
Discussion In the 2 Florida estuaries examined, V. cholerae non-01 was widely distributed and could be found in oysters, blue crabs, water, a n d sediment. H o w e v e r , K
K cholerae in Florida Estuaries
73
20
~ 2.0
1.0 o
.8
E z
,4
.2
10
o
):
9
-
"J'~z
.
A
I~
J
A
J
S
o
9
0
N
~
D
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~
: l
M
A
M
J
J
A
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I~
.9
"~
.7
"6
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~
.4
z~
.3
.8
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sediment 9 water 9
.2 i
A
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J
J
A
S
0
N
D'l'~ J
F
M
A
M
I
I
J
Fig. 4. The seasonal distribution of I,'. cholerae in Apalachicola and Tampa Bay. Top figure presents V. cholerae in oysters. Bottom figure presents blue crabs, sediment, and water.
cholerae 01 was far less frequent, being isolated from only 1 oyster sample collected in Apalachicola Bay. In oysters, the organism was recovered only from the digestive tract, whereas in crabs, it could be found throughout the body. Although statistical analysis revealed no strong linear correlations between the distribution of the organism and the parameters, salinity and temperature, the organism was present in the highest concentrations when salinities were 10~176 and temperatures, 20~176 The abundance of the organism in the estuary and the lack of correlation between fecal coliforms and V. cholerae levels support the findings of Kaper et al. [15] that V. cholerae may be autochthonous to United States estuaries. The presence of the organism in oysters when there is an abundance of diatoms, dinoflagellates, and detrital particles in the digestive tract of the animal, coupled with the knowledge that the bacterium readily attaches to chitin [3], suggests that V. cholerae may be concentrated as a result of the oyster feeding on certain plankton to which the bacterium is attached. Although V. cholerae was not
74
M.A. Hood et al.
i s o l a t e d f r o m p l a n k t o n c o l l e c t e d f r o m A p a l a c h i c o l a Bay, o t h e r i n v e s t i g a t o r s h a v e i s o l a t e d F. cholerae f r o m C h e s a p e a k e Bay p l a n k t o n [11]. It is u n c l e a r w h y we h a v e b e e n u n a b l e to i s o l a t e V. cholerae f r o m p l a n k t o n , b u t t h e r e is s t r o n g e v i d e n c e suggesting t h a t F. cholerae specifically a d h e r e s to c o p e p o d s [ 14] a n d t h a t the o r g a n i s m exists i n t h e e n v i r o n m e n t i n a n e p i b i o t i c state [13]. Acknowledgments. We thank the following investigators for helpful discussions: Dr. R. R. ColwelI, Dr. R. J. Siebeling, Dr. R. J. Seidler, and Nell Roberts. Special thanks are extended to Dr. R. J. Siebeling for providing serotype information. We also gratefully acknowledge John Cheney and Kathy Williams for their excellent technical support, Florida State Department of Natural Resources for their logistical support, and Dr. Mike Bundrick and the University of West Florida Institute for Statistical and Mathematical Modeling for statistical assistance. The work is a result of research sponsored by Florida Sea Grant, NOAA, Office of Sea Grant, Department of Commerce, under Grant No. SG #NA 80AA-D-00038. The U.S. Government is authorized to produce and distribute any copyright portion that may appear herein. Acknowledgmentis also given to the BuntingInstitute for support in the preparation of the manuscript.
References 1. Association of Analytical Chemists (1975) Official methods of analysis of the association of analytical chemists, 12th ed. Washington, DC 2. American Public Health Association (1975) Standard methods for the examination of water and wastewater, 14th ed. American Public Health Association, Inc, Washington, DC 3. Belas, MR, ColweU RR: (1982) Adsorption kinetics of laterally and polarly flagellated Vibrio. J Bacteriol 151:1568-1580 4. Blake PA, AUegra DT, Snyder JD, Barrett TJ, McFarland L, Caraway CT, Felley JC, Craig JP, Lee JV, Puhr ND, Feldman RA: (1980) Cholera--a possible endemic focus in the United States. N Engl J Med 302:305-309 5. Center for Disease Control (1978) Vibrio cholerae--Louisiana. Morb Mort Weekly Rpts 27:637. 6. Center for Disease Control (1978) Follow up on Vibrio cholerae serotype Inaba infection-Louisiana. Morb Mort Weekly Rpts 27:388 7. Center for Disease Control (1979) Non-01 Vibrio cholerae infections--Florida. Morb Mort Weekly Rots 28:571-572 8. Center for Disease Control (1980) Cholera--Florida. Morb Mort Weekly Rpts 29:601 9. Center for Disease Control (1981) Cholera--Texas. Morb Mort Weekly Rpts 30:389-390 10. Center for Disease Control (1981) Cholera on a Gulf Coast oil rig--Texas. Morb Mort Weekly Rpts 30:589-590 11. Colwell RR, Seidler RJ, Kaper J, Joseph SW, Garges S, Lockman H, Maneval D, Bradford H, Roberts N, Remmers E, Huq I, and Huq A (1981) Occurrence of l/ibrio cho[erae serotype 01 in Maryland and Louisiana estuaries. Appl Environ Microbiol 41:555-558 12. Hood MA, Ness GE, Rodrick GE (1981) Isolation of Vibrio cholerae serotype 01 from the eastern oyster, Crassostrea virginica. Appl Environ Microbiol 41:559-560 13. Hood MA, Ness GE, Rodrick GE, Blake NJ (1982) The ecology of Vibrio cholerae in two Florida estuaries. In: Colwell RR (ed) Vibrios in the environment, John Wiley and Sons, New York, in press 14. Huq A, Small EB, West PA, Huq MI, Rahman R, Colwell RR (1983) Ecological relationships between Vibrio cholerae and plantonic crustacean copepods. Appl Environ Microbiol 45: 275-283 15. Kaper J, Lockman H, Colweli RR, Joseph SW (1979) Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay. Appl Environ Microbiol 37:91-103
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16. Lee JV, Bashford DJ, Donovan TJ, Furniso AL, West PA (1982) The incidence of Vibrio cholerae in water, animals and birds in Kent, England. J Appl Bacteriol 52:281-291 17. Snedecor GW, Cochran WG (1967) Statistical methods, 6th ed. Iowa State University Press, Ames 18. Strickland JDH, Parsons TR (1972) A practical handbook for seawater analysis, 2nd ed. Fish Res Bd of Can Bull 19. Weissman JB, DeWitt WE, Thompson J, Muchnick CN, Portnoy BL, Feeley JC, Gangarosa EJ (1974) A case of cholera in Texas, 1973. Am J Epidemiol 100:487-498 20. West PA, Lee JV (1982) Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England. J Appl Bacteriol 52:435--448
