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Journal o f Food Protection, Vol. 77, No. 10, 2014, Pages 1784-1786 doi: 10.4315/0362-028X.JFP-14-175 Copyright © , International Association for Food Protection
Research Note
Occurrence of Vibrio vulnificus and Toxigenic Vibrio parahaemolyticus on Sea Catfishes from Galveston Bay, Texas LESLIE BAUMEISTER,1 MONA E. HOCHMAN,2 JO H N R. SCHWARZ,2 and ROBIN BRINKM EYER1* 'Department o f Marine Sciences and 2Department o f Marine Biology, Texas A&M University, Galveston Campus, 200 Seawolf Parkway, Galveston, Texas 77553, USA MS 14-175: Received 15 April 2014/Accepted 6 June 2014
A BSTRACT Dorsal and pectoral fin spines from two species of sea catfishes (Bagre marinus and Ariopsis felis) landed at 54 sites in Galveston Bay, Texas, and its subbays from June to October 2005 were screened with traditional cultivation-based assays and quantitative PCR assays for Vibrio vulnificus and Vibrio parahaemolyticus. V. vulnificus was present on 51.2% offish (n = 247), with an average of 403 + 337 SD cells g '. V. parahaemolyticus was present on 94.2% (n = 247); 12.8% tested positive for the virulence-conferring tdh gene, having an average 2,039 ± 2,171 SD cells g 1. The increasing trend in seafood consumption of “ trash fishes” from lower trophic levels, such as sea catfishes, warrants evaluation of their life histories for association with pathogens of concern for human consumption.
O nce spum ed as nuisance fish in com m ercial and recreational by-catch (15), the sea catfishes gafftopsail B agre m arinus and hardhead A riopsis fe lis are rapidly gam ing in popularity as seafood, especially in the G ulf of M exico and the southern A tlantic states o f the U nited States. Both species are covered w ith excessive m ucus and have venom ous spines in the dorsal and pectoral fins that can inflict deep tissue w ounds (3-5). A m ong coastal G ulf o f M exico fishing com m unities there are num erous anecdotal accounts describing painful upper and low er extrem ity infections caused by catfish spine punctures. Both Vibrio vulnificus and Vibrio parahaem olyticus have been isolated from soft tissue w ounds inflicted by fish fins (4, 9, 19), including sea catfish (3, 14). W hereas V. parahaem olyticus gastrointestinal or w ound infections are rarely fatal (13), V. vulnificus infections typically have fatality rates o f 30 to 50% (12). The trend tow ard increased com m ercial and recreational landings o f sea catfishes could result in greater incidence o f vibrioses in seafood handlers and, potentially, in consum ers w ho purchase w hole fish at local seafood markets. O ur aim was to determ ine the frequency of occurrence and the concentration o f V. vulnificus and V. parahaem olyticus on sea catfishes to evaluate the likelihood o f infection. M A T E R IA L S A N D M E T H O D S Between June and October 2006, 247 gafftopsail and hardhead catfish landed with nets at 54 sites throughout Galveston Bay, Texas, and its subbays (Upper Galveston Bay, Trinity Bay, West Bay, and East Bay; Fig. 1) were screened for the presence or absence of V. * Author for correspondence. Tel: 409-741-7178; Fax: 409-740-4868; E-mail: [email protected].
vulnificus and toxigenic V. parahaemolyticus. Water temperatures and salinities ranged from 24.8 to 31.7°C and 3.0 to 27.5 ppt in Upper Galveston Bay, 25.1 to 32.7°C and 6.0 to 19.7 ppt in Trinity Bay, 25.9 to 27.6°C and 11.8 to 29.8 ppt in West Bay, and 23.1 to 31.0°C and 8.1 to 22.6 ppt in East Bay. Fish (caught and killed in < 4 h) were provided by the Texas Parks and Wildlife Department Marine Lab (Dickinson) and commercial shrimpers. Average fish total length was 22.6 cm, and no external infections were observed. The dorsal and pectoral fin spines were removed with sterilized bone cutters. One pectoral fin spine from each fish was transferred to an autoclaved 2-ml centrifuge tube and was stored at —80°C for later DNA extraction. To screen for presence of V. vulnificus, the other pectoral and the dorsal spines were incubated separately in 10 ml of alkaline peptone water broth for 24 h at 37°C. Broth was streaked onto modified cellobiose-polymyxin B-colistin agar and was incubated for another 16 h at 40°C; finally, two phenotypically indicative colonies from each of these agar plates were screened for presence of the V. vulnificus-specific cytolysin gene, vvhA, by positive hybridization with an alkaline phosphatase-labeled DNA probe (21). A similar protocol was used to screen for presence of V. parahaemolyticus using thiosulfate-citrate-bile salts-sucrose agar and DNA probes targeting the species-indicative thermolabile hemolysin gene till and the pathogenicity-conferring thermostable direct hemolysin gene tdh (6). Probes were purchased from DNA Technology A/S (Risskov, Denmark). For quantitative PCR (QPCR) enumeration of vibrios, DNA from the frozen pectoral fin spines was extracted with 3% cetyl trimethylammonium bromide and 0.25 mg/ml proteinase K at 37°C for 30 m, followed by purification with 25:24:1 phenol-chloro form-isoamyl alcohol. A random subset of spines was examined with 4,6-diamidino-2-phenylindole staining and epifluorescence microscopy to estimate a 99% efficiency of bacterial cell lysis with this DNA extraction method. QPCR assays were performed on a SmartCycler (Cepheid, Sunnyvale, CA) for V. vulnificus (18) and V. parahaemolyticus (16), with primer sets and probes targeting the pathogenicity-conferring genes whA and tdh, respectively. The
J. Food Prot., Vol. 77, No. 10
reaction mixtures (25 pi) for all QPCR assays contained 1 x PCR buffer, 5 mM MgCl2, 200 pM each deoxynucleoside triphosphate, 1.5 U Platinum Taq DNA polymerase (Promega, Madison, WI), 2 pi of template DNA, 2 pi of DNA internal amplification control, and 150 nM each internal amplification control primers (forward and reverse). The vvhA assay contained 200 nM forward and reverse primers and 240 nM DNA probe. Thermocycling steps were 94°C for 120 s, followed by 40 cycles of 94°C for 15 s, 58°C for 15 s, and 72°C for 20 s. The tlh assay contained 150 nM each primer and 150 nM DNA probe. The tdh assay contained 200 nM each primer and 75 nM DNA probe. For both tlh and tdh assays, thermocycling steps were 95°C for 60 s followed by 45 cycles of 95°C for 5 s and 59°C for 45 s. Internal amplification control DNA was provided by the U.S. Food and Drug Administration Gulf Coast Seafood Laboratory (Dauphin Island, AL). Standard curves to calculate cells per gram in QPCR assays were based upon duplicate DNA extracts from 10-fold serial dilutions of positive control strains. Duplicate cell concentrations of control cultures were determined by epifluorescence microscope (Axioskop, Zeiss, Germany) direct count method (10), using 4,6-diamidino-2-phenylindole staining. Statistical analyses were conducted with STATA (StataCorp, College Station, TX).
RESULTS AND DISCUSSION Screening for presence or absence w ith alkaline p hosphatase-labeled D N A probes detected V. vulnificus (vvhA + ) on 39.0 to 63.2% o f fish in the four subbays (Table 1). V. parahaemolyticus was detected on 84.2 to 95.8% o f fish (tlh +)\ how ever, only 7.5 to 24.8% w ere
PATHOGENIC VIBRIOS ON SEA CATFISHES
1785
toxigenic ( tdh+). T he Q PC R assays were m ore sensitive, detecting both V. vulnificus (i.e., vvhA\ 100 + 350 SD to 850 + 1,490 SD cells g - 1 ) and toxigenic V. parahaemolyticus (i.e., tdh +\ 262 ± 389 SD to 4,182 ± 9,046 SD cells g - 1 ) on all fish specim ens from all landing sites. A one-w ay analysis o f variance determ ined that there were no significant differences betw een V. vulnificus (P = 0.3475) and toxigenic V. parahaemolyticus (P = 0.5474) cells per gram counts on fish am ong subbays. A lso, no significant differences were observed for A .felis versus B. marinus (vvhA+ P = 0.573; tdh+ P = 0.0682). There was a w eak positive correlation betw een fish total length and V. parahaemolyticus cells per gram counts (R 2 = 0.25, P = 0. 05); for V. vulnificus, the relationship was moderate (R 2 = 0.72, P — 0.005). O ur results confirm that both V. vulnificus and V. parahaemolyticus are frequently associated w ith the sea catfishes B. marinus and A. felis. Both pathogens were detected throughout G alveston Bay, using two different m ethods. N ote that, unlike other studies o f shellfish or crustaceans, no prior em ichm ent step o f the sam ples was required for detection w ith Q PCR; this indicates higher initial cell densities, w hich are w ell w ithin the ranges o f infective doses. F or both Vibrio species, the infective dose for gastroenteritis is ~ 1 0 6 CFU for healthy people and —102 CFU for those w hose health is com prom ised (17). There are currently no data regarding the infective doses for w ound exposure in hum ans, but for m ice the levels are < 1 0 3 CFU (7). Recently, G ivens et al. (8), using
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TABLE 1. Average percent occurrence of the toxin-conferring genes vvhA and tdh for V. vulnificus and V. parahaemolyticus, respectively, on sea catfish spinesa Vibrio vulnificus
Subbay
No. of sites
No. of fish
Cultivation whA (%)
Galveston Bay Trinity Bay West Bay East Bay
23 9 10 12
68 16 27 136
39.0 63.2 54.8 47.9
Vibrio parahaemolyticus
QPCR vvhA (cells g~ ')
190 850 100 470
+ ± + ±
370 1,490 350 1,320
Cultivation tlh (%)
Cultivation tdh (%)
93.3 84.2 95.8 94.8
7.5 9.5 9.5 24.8
QPCR tdh (cells g -1)
365 4,182 262 2,071
+ + + +
1,182 9,046 389 6,654
“ Values determined with traditional cultivation-based assays and QPCR cells per gram counts. cultivation-based and QPCR methods of detection, found concentrations of V. vulnificus and V. parahaemolyticus that were several log greater in fish intestines versus oyster, sediment, or water samples, indicating that fish are a significant source of these pathogens. Similar to our results, V. parahaemolyticus was detected less frequently in fish than V. vulnificus, and QPCR was superior to cultivationdependent direct plating and colony hybridization for detection of either pathogen. As fisheries landings of species from higher trophic levels continue to decline, the trend to “ fish down the food web” to lower trophic levels for smaller or low-value “ trash fishes,” such as sea catfish, will necessitate reevaluations of the life histories and seafood safety of these new target species. Sea catfishes are bottom dwellers living in close contact with sediments that harbor higher levels of vibrios than the overlying water column (2, 11, 20). Optimal temperatures for both catfish species are 25°C and higher; however, hardheads tend to avoid temperatures exceeding 37°C (15). Adult hardhead catfish can be found in salinities ranging from 0 to 40 ppt (15). Gafftopsail catfish have been found in freshwater, but they tend to prefer salinities of 5 to 30 ppt (15). These temperature and salinity ranges match well with those required for growth and survival of V. parahaemolyticus and V. vulnificus (2), and the exterior mucus of the catfishes provides a plentiful source of carbon and protection (1). ACKNOWLEDGMENT
parahaemolyticus in Alabama oysters. Appl. Environ. Microbiol. 69: 1521-1526. 7.
8.
9. 10.
11.
12.
13.
14. 15.
16.
We extend a special thank you to T. Hans for assistance with QPCR assays.
REFERENCES Benhamed, S., F. A. Guardiola, M. Mars, and M. A. Esteban. 2014. Pathogen bacteria adhesion to skin mucus of fishes. Vet. Microbiol. 171:1-12. 2. Blackwell, K. D„ and J. D. Oliver. 2008. The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina estuaries. J. Microbiol. 46:146-153. 3. Bonner, J. R„ A. S. Coker, C. R. Berryman, and H. M. Pollock. 1983. Spectrum of Vibrio infections in a Gulf Coast community. Ann. Intern. Med. 99:464-469. 4. Calif, E., B. Kaufman, and S. Stahl. 2003. Vibrio vulnificus infection of the lower limb after fish spine injuries. Clin. Orthop. Relat. Res. 411:274-279. 5. Carry, M. J., R. H. Kutz, R. L. Finley, Jr., J. Upston, and G. F. Rogers. 2010. Digital catfish envenomation mimicking necrotizing fasciitis. Plast. Reconstr. Surg. 126:226e-230e. 6. DePaola, A., J. L. Nordstrom, J. C. Bowers, G. Wells, and D. W. Cook. 2003. Seasonal abundance of total and pathogenic Vibrio
17.
1.
18.
19.
20.
21.
DePaola, A., J. L. Nordstrom, A. Dalsgaard, A. Forslund, J. Oliver, T. Bates, K. L. Bourdage, and P. A. Gulig. 2003. Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers. Appl. Environ. Microbiol. 69:4006-4011. Givens, C. E., J. C. Bowers, A. DePaola, J. T. Hollibaugh, and J. L. Jones. 2014. Occurrence and distribution of Vibrio vulnificus and Vibrio parahaemolyticus—potential roles for fish, oyster, sediment and water. Lett. Appl. Microbiol. 58:503-510. Hlady, W. G., and K. C. Klontz. 1996. The epidemiology of Vibrio infections in Florida, 1981-1993. J. Infect. Dis. 173:1176-1183. Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:1225-1228. Johnson, C. N., A. R. Flowers, N. F. Noriea in, A. M. Zimmerman, J. C. Bowers, A. DePaola, and D. J. Grimes. 2010. Relationships between environmental factors and pathogenic vibrios in the Northern Gulf of Mexico. Appl. Environ. Microbiol. 76:7076-7084. Mahmoud, B. S. M. 2009. Reduction of Vibrio vulnificus in pure culture, half shell, and whole shell oysters (Crassostrea virginica) by X-ray. Int. J. Food Microbiol. 130:136-139. Mahmoud, B. S. M„ and D. D. Burrage. 2009. Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray. Lett. Appl. Microbiol. 48:572-578. Midani, S., and M. H. Rathmore. 1994. Vibrio species infection of a catfish spine puncture wound. Pediatr. Infect. Dis. J. 13:333-334. Muncy, R. J., and W. M. Wingo. 1983. Species profiles: life histories and environmental requirements of coastal invertebrates (Gulf of Mexico)—sea catfish and gafftopsail. U.S. Fish and Wildlife Service, Division of Biological Services, FWS/OBS-82/11.5. U.S. Army Corps of Engineers, TR EL-82-4. Nordstrom, J. L., M. C. L. Vickery, G. M. Blackstone, S. L. Murray, and A. DePaola. 2007. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl. Environ. Microbiol. 73:5840-5847. Ohio Department of Health. 2014. Vibriosis: non-cholera Vibrio spp. In Ohio Department of Health infectious disease control manual. Available at: http://www.odh.ohio.gov/pdf/idcm/vibrio.pdf. Accessed 30 March 2014. Paniker, G., and A. K. Beij. 2004. Real time PCR detection of Vibrio vulnificus in oysters: a comparison of oligonucleotide primers and probes targeting vvhA. Appl. Environ. Microbiol. 71:5702-5709. Ralph, A., and B. J. Currie. 2007. Vibrio vulnificus and V. parahaemolyticus necrotising fasciitis in fishermen visiting an estuarine tropical northern Australian location. J. Infect. 54:111-114. Vezzulli, L., E. Pezzati, M. Moreno, M. Fabiano, L. Pane, and C. Pruzzo. 2009. Benthic ecology of Vibrio spp. and pathogenic Vibrio species in a coastal Mediterranean environment (La Spezia Gulf, Italy). Microb. Ecol. 58:808-818. Wright, A. C„ G. A. Miceli, W. L. Landry, J. B. Christy, W. D. Watkins, and J. G. Morris, Jr. 1993. Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe. Appl. Environ. Microbiol. 59:541-546.
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Journal o f Food Protection, Vol. 77, No. 10, 2014, Pages 1784-1786 doi: 10.4315/0362-028X.JFP-14-175 Copyright © , International Association for Food Protection
Research Note
Occurrence of Vibrio vulnificus and Toxigenic Vibrio parahaemolyticus on Sea Catfishes from Galveston Bay, Texas LESLIE BAUMEISTER,1 MONA E. HOCHMAN,2 JO H N R. SCHWARZ,2 and ROBIN BRINKM EYER1* 'Department o f Marine Sciences and 2Department o f Marine Biology, Texas A&M University, Galveston Campus, 200 Seawolf Parkway, Galveston, Texas 77553, USA MS 14-175: Received 15 April 2014/Accepted 6 June 2014
A BSTRACT Dorsal and pectoral fin spines from two species of sea catfishes (Bagre marinus and Ariopsis felis) landed at 54 sites in Galveston Bay, Texas, and its subbays from June to October 2005 were screened with traditional cultivation-based assays and quantitative PCR assays for Vibrio vulnificus and Vibrio parahaemolyticus. V. vulnificus was present on 51.2% offish (n = 247), with an average of 403 + 337 SD cells g '. V. parahaemolyticus was present on 94.2% (n = 247); 12.8% tested positive for the virulence-conferring tdh gene, having an average 2,039 ± 2,171 SD cells g 1. The increasing trend in seafood consumption of “ trash fishes” from lower trophic levels, such as sea catfishes, warrants evaluation of their life histories for association with pathogens of concern for human consumption.
O nce spum ed as nuisance fish in com m ercial and recreational by-catch (15), the sea catfishes gafftopsail B agre m arinus and hardhead A riopsis fe lis are rapidly gam ing in popularity as seafood, especially in the G ulf of M exico and the southern A tlantic states o f the U nited States. Both species are covered w ith excessive m ucus and have venom ous spines in the dorsal and pectoral fins that can inflict deep tissue w ounds (3-5). A m ong coastal G ulf o f M exico fishing com m unities there are num erous anecdotal accounts describing painful upper and low er extrem ity infections caused by catfish spine punctures. Both Vibrio vulnificus and Vibrio parahaem olyticus have been isolated from soft tissue w ounds inflicted by fish fins (4, 9, 19), including sea catfish (3, 14). W hereas V. parahaem olyticus gastrointestinal or w ound infections are rarely fatal (13), V. vulnificus infections typically have fatality rates o f 30 to 50% (12). The trend tow ard increased com m ercial and recreational landings o f sea catfishes could result in greater incidence o f vibrioses in seafood handlers and, potentially, in consum ers w ho purchase w hole fish at local seafood markets. O ur aim was to determ ine the frequency of occurrence and the concentration o f V. vulnificus and V. parahaem olyticus on sea catfishes to evaluate the likelihood o f infection. M A T E R IA L S A N D M E T H O D S Between June and October 2006, 247 gafftopsail and hardhead catfish landed with nets at 54 sites throughout Galveston Bay, Texas, and its subbays (Upper Galveston Bay, Trinity Bay, West Bay, and East Bay; Fig. 1) were screened for the presence or absence of V. * Author for correspondence. Tel: 409-741-7178; Fax: 409-740-4868; E-mail: [email protected].
vulnificus and toxigenic V. parahaemolyticus. Water temperatures and salinities ranged from 24.8 to 31.7°C and 3.0 to 27.5 ppt in Upper Galveston Bay, 25.1 to 32.7°C and 6.0 to 19.7 ppt in Trinity Bay, 25.9 to 27.6°C and 11.8 to 29.8 ppt in West Bay, and 23.1 to 31.0°C and 8.1 to 22.6 ppt in East Bay. Fish (caught and killed in < 4 h) were provided by the Texas Parks and Wildlife Department Marine Lab (Dickinson) and commercial shrimpers. Average fish total length was 22.6 cm, and no external infections were observed. The dorsal and pectoral fin spines were removed with sterilized bone cutters. One pectoral fin spine from each fish was transferred to an autoclaved 2-ml centrifuge tube and was stored at —80°C for later DNA extraction. To screen for presence of V. vulnificus, the other pectoral and the dorsal spines were incubated separately in 10 ml of alkaline peptone water broth for 24 h at 37°C. Broth was streaked onto modified cellobiose-polymyxin B-colistin agar and was incubated for another 16 h at 40°C; finally, two phenotypically indicative colonies from each of these agar plates were screened for presence of the V. vulnificus-specific cytolysin gene, vvhA, by positive hybridization with an alkaline phosphatase-labeled DNA probe (21). A similar protocol was used to screen for presence of V. parahaemolyticus using thiosulfate-citrate-bile salts-sucrose agar and DNA probes targeting the species-indicative thermolabile hemolysin gene till and the pathogenicity-conferring thermostable direct hemolysin gene tdh (6). Probes were purchased from DNA Technology A/S (Risskov, Denmark). For quantitative PCR (QPCR) enumeration of vibrios, DNA from the frozen pectoral fin spines was extracted with 3% cetyl trimethylammonium bromide and 0.25 mg/ml proteinase K at 37°C for 30 m, followed by purification with 25:24:1 phenol-chloro form-isoamyl alcohol. A random subset of spines was examined with 4,6-diamidino-2-phenylindole staining and epifluorescence microscopy to estimate a 99% efficiency of bacterial cell lysis with this DNA extraction method. QPCR assays were performed on a SmartCycler (Cepheid, Sunnyvale, CA) for V. vulnificus (18) and V. parahaemolyticus (16), with primer sets and probes targeting the pathogenicity-conferring genes whA and tdh, respectively. The
J. Food Prot., Vol. 77, No. 10
reaction mixtures (25 pi) for all QPCR assays contained 1 x PCR buffer, 5 mM MgCl2, 200 pM each deoxynucleoside triphosphate, 1.5 U Platinum Taq DNA polymerase (Promega, Madison, WI), 2 pi of template DNA, 2 pi of DNA internal amplification control, and 150 nM each internal amplification control primers (forward and reverse). The vvhA assay contained 200 nM forward and reverse primers and 240 nM DNA probe. Thermocycling steps were 94°C for 120 s, followed by 40 cycles of 94°C for 15 s, 58°C for 15 s, and 72°C for 20 s. The tlh assay contained 150 nM each primer and 150 nM DNA probe. The tdh assay contained 200 nM each primer and 75 nM DNA probe. For both tlh and tdh assays, thermocycling steps were 95°C for 60 s followed by 45 cycles of 95°C for 5 s and 59°C for 45 s. Internal amplification control DNA was provided by the U.S. Food and Drug Administration Gulf Coast Seafood Laboratory (Dauphin Island, AL). Standard curves to calculate cells per gram in QPCR assays were based upon duplicate DNA extracts from 10-fold serial dilutions of positive control strains. Duplicate cell concentrations of control cultures were determined by epifluorescence microscope (Axioskop, Zeiss, Germany) direct count method (10), using 4,6-diamidino-2-phenylindole staining. Statistical analyses were conducted with STATA (StataCorp, College Station, TX).
RESULTS AND DISCUSSION Screening for presence or absence w ith alkaline p hosphatase-labeled D N A probes detected V. vulnificus (vvhA + ) on 39.0 to 63.2% o f fish in the four subbays (Table 1). V. parahaemolyticus was detected on 84.2 to 95.8% o f fish (tlh +)\ how ever, only 7.5 to 24.8% w ere
PATHOGENIC VIBRIOS ON SEA CATFISHES
1785
toxigenic ( tdh+). T he Q PC R assays were m ore sensitive, detecting both V. vulnificus (i.e., vvhA\ 100 + 350 SD to 850 + 1,490 SD cells g - 1 ) and toxigenic V. parahaemolyticus (i.e., tdh +\ 262 ± 389 SD to 4,182 ± 9,046 SD cells g - 1 ) on all fish specim ens from all landing sites. A one-w ay analysis o f variance determ ined that there were no significant differences betw een V. vulnificus (P = 0.3475) and toxigenic V. parahaemolyticus (P = 0.5474) cells per gram counts on fish am ong subbays. A lso, no significant differences were observed for A .felis versus B. marinus (vvhA+ P = 0.573; tdh+ P = 0.0682). There was a w eak positive correlation betw een fish total length and V. parahaemolyticus cells per gram counts (R 2 = 0.25, P = 0. 05); for V. vulnificus, the relationship was moderate (R 2 = 0.72, P — 0.005). O ur results confirm that both V. vulnificus and V. parahaemolyticus are frequently associated w ith the sea catfishes B. marinus and A. felis. Both pathogens were detected throughout G alveston Bay, using two different m ethods. N ote that, unlike other studies o f shellfish or crustaceans, no prior em ichm ent step o f the sam ples was required for detection w ith Q PCR; this indicates higher initial cell densities, w hich are w ell w ithin the ranges o f infective doses. F or both Vibrio species, the infective dose for gastroenteritis is ~ 1 0 6 CFU for healthy people and —102 CFU for those w hose health is com prom ised (17). There are currently no data regarding the infective doses for w ound exposure in hum ans, but for m ice the levels are < 1 0 3 CFU (7). Recently, G ivens et al. (8), using
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TABLE 1. Average percent occurrence of the toxin-conferring genes vvhA and tdh for V. vulnificus and V. parahaemolyticus, respectively, on sea catfish spinesa Vibrio vulnificus
Subbay
No. of sites
No. of fish
Cultivation whA (%)
Galveston Bay Trinity Bay West Bay East Bay
23 9 10 12
68 16 27 136
39.0 63.2 54.8 47.9
Vibrio parahaemolyticus
QPCR vvhA (cells g~ ')
190 850 100 470
+ ± + ±
370 1,490 350 1,320
Cultivation tlh (%)
Cultivation tdh (%)
93.3 84.2 95.8 94.8
7.5 9.5 9.5 24.8
QPCR tdh (cells g -1)
365 4,182 262 2,071
+ + + +
1,182 9,046 389 6,654
“ Values determined with traditional cultivation-based assays and QPCR cells per gram counts. cultivation-based and QPCR methods of detection, found concentrations of V. vulnificus and V. parahaemolyticus that were several log greater in fish intestines versus oyster, sediment, or water samples, indicating that fish are a significant source of these pathogens. Similar to our results, V. parahaemolyticus was detected less frequently in fish than V. vulnificus, and QPCR was superior to cultivationdependent direct plating and colony hybridization for detection of either pathogen. As fisheries landings of species from higher trophic levels continue to decline, the trend to “ fish down the food web” to lower trophic levels for smaller or low-value “ trash fishes,” such as sea catfish, will necessitate reevaluations of the life histories and seafood safety of these new target species. Sea catfishes are bottom dwellers living in close contact with sediments that harbor higher levels of vibrios than the overlying water column (2, 11, 20). Optimal temperatures for both catfish species are 25°C and higher; however, hardheads tend to avoid temperatures exceeding 37°C (15). Adult hardhead catfish can be found in salinities ranging from 0 to 40 ppt (15). Gafftopsail catfish have been found in freshwater, but they tend to prefer salinities of 5 to 30 ppt (15). These temperature and salinity ranges match well with those required for growth and survival of V. parahaemolyticus and V. vulnificus (2), and the exterior mucus of the catfishes provides a plentiful source of carbon and protection (1). ACKNOWLEDGMENT
parahaemolyticus in Alabama oysters. Appl. Environ. Microbiol. 69: 1521-1526. 7.
8.
9. 10.
11.
12.
13.
14. 15.
16.
We extend a special thank you to T. Hans for assistance with QPCR assays.
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