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Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods
International Journal of Food Microbiology 31 (1996) 317-323
Short communication
Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods I. Karunasagaf’,*,
G. Sugumar”, Indrani Karunasagar”, P.J.A. Reilly”,b
“Department of Fishery Microbiology, College of Fisheries, Mangalore - 575002, India bNatural Resources Institute, Chatham Maritime, Kent ME4 4TB, UK
Received 19 April 1995; revised 3 October 1995; accepted 27 December 1995
Abstract Detection of Kanagawa positive strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR) was tested. Primer pairs for specific amplification of tdh gene fragment is described. The assay could detect contamination of seafood homogenate when PCR was performed using lysate prepared directly from fish homogenates. The sensitivity of the assay could be improved to detect less than 10 cells of v. parahaemolyticus by performing PCR after 8 h enrichment in alkaline peptone water. Keywords: Polymerase chain mostable direct hemolysin
* Corresponding
reaction;
Vibrio parahaemolyticus;
Seafoods;
author, Tel: + 91 824 439256; fax: -t 91 824 438366.
016%1605/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PIZ SO168-1605(96)00974-O
tdh Gene; Ther-
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
318
1. Introduction
The majority of Vibrio parahaemolyticus strains isolated from cases of gastroenteritis produce a /?-hemolysin on a special blood agar medium, Wagatsuma agar (Wagatsuma, 1968). The ability to cause hemolysis on this agar has been termed the Kanagawa phenomenon (Joseph et al., 1982). This phenomenon is due to a thermostable hemolysin encoded by the tdh gene which has been cloned and sequenced (Kaper et al., 1984; Nishibuchi and Kaper, 1985). Recently Lee and Pan (1993) described PCR based detection of V. parahaemolyticus in clinical samples. Since rapid techniques are necessary for quality control purposes in the fish processing industry, the possibility of using polymerase chain reaction (PCR) based tests for detection of Kanagawa positive V. parahaemolyticus in seafoods was studied.
2. Materials and methods 2.1. Bacterial strains V. parahaemolyticus ATCC 17802, TY 49, VP-l, and VP-S were Kanagawa positive strains, the latter three isolated from clinical cases at the National Institute of Cholera and Enteric Diseases (NICED) Calcutta, India. Kanagawa negative strains of V. paruhaemolyticus (20 strains) comprised fish and environmental isolates (Karunasagar et al., 1990). For V. vulnzjicus ATCC 27562 and three environmental isolates were included (Karunasagar et al., 1987). Clinical isolates of Non 01 V. cholerae strains (14 strains) were obtained from Christian Medical College, Vellore. Environmental isolates (6 strains) of Non 01 V. cholerae were from this laboratory (Karunasagar et al., 1992). V. cholerae Ogawa was a clinical isolate from this laboratory and V. cholerae 0 139 strains (two strains) were obtained from NICED Calcutta. V. hollisae and V. damsela cultures (four strains each) were isolated from seafood in this laboratory. V. mimicus strains VM 4197, VM 4053 and VM 4203 were obtained from NICED, Calcutta.
Table 1 Nucleotide Primer M 454 M 456 BG 24 M 441
No.
sequence
of primers
used and their binding
sites
Sequence
Position
S-CGTTGATTATTCTTTTACGC-3’ 5’-TTTCATGATTATTCAGTTT-3’ 5’-CTGTCCCTTTTCCTGCCCCC-3’ 5’-TTTGTTGGATATACACAT-3’
17-36 85-103 269-288 702-719
1. Karunasagar et al. /ht.
J. Food Microbiology 31 (1996) 317-323
319
2.2. PCR reuction
PCR primers used in this study and their binding sites are indicated in Table 1. These were designed, based on the sequence of tdh2 gene published by Yamasaki et al. (1991). Primers M 454 and M 456 would bind within the non-coding region flanking tdh2 gene while BG 24 and M 441 would bind within the coding region. Primer pairs tested were M 454 and M 441, M 456 and M 441 and BG 24 and M 441. To prepare bacterial cell lysates, 37°C overnight tryptic soy broth (TSB) cultures were centrifuged at 8000 x g for 15 min and the cell pellet resuspended in 100 ~1 distilled water and heated at 110°C for 10 min. PCR reactions were performed in 100 ~1 volume taking 1 ~1 cell lysate, 200 PM dNTPs, 0.5 PM primers and 2.5 U Taq DNA polymerase (Bangalore Genie, India). Thirty cycles of amplification were performed using a Biomed 60-2 programmable Thermocycler with 1 min denaturation, annealing and extension steps at 94, 55 and 72°C respectively. PCR reaction products were resolved on a 1% agarose gel containing 0.5 pg ethidium bromide per ml and visualised by UV induced fluorescence. 2.3. Detection of contamination in fish directly by PCR Samples of fishes such as mackerel (Rastrelliger kanagurta), lactarius (Lactarius luctarius), pink perch (Nemipterus sp.), white sardine (Esculosa thoracata), sciaenid (Johnius sp.) were obtained from Mangalore fish market. Fish samples, 50 g, fish samples were homogenised with 450 ml alkaline peptone water (APW) for 2 min. One millilitre of homogenate was centrifuged at 100 x g to sediment fish particles and the supernatant was again centrifuged at 8000 x g for 10 min and the deposit re-suspended in 20 ~1 distilled water and lysed by heating at 110°C for 10 min. Two microlitres of lysate was used in the PCR reaction. As a positive control, one sample of fish homogenate was contaminated with the TY 49 strain of I’. parahaemolyticus at a level of 1.8 x 10’ cells/ml and lysates prepared as described above. 2.4. Stundard Detection method
Homogenates were enriched in APW for 18 h at 37°C and subcultured into TCBS agar (Hi Media Laboratories, Bombay, India). Confirmation of typical colonies was performed according to Twedt and Stelma (1984). 2.5. Minimum cell number required to give PCR signals To determine the sensitivity of the PCR assay, lysates prepared from fish homogenates contaminated with V. parahaemolyticus TY 49 strain at levels ranging from 106-- < lo/ml were used. To test whether an enrichment step would increase the sensitivity of the PCR reaction, 1 ml fish homogenates containing lo’-10’ I’. parahaemolyticus cells were inoculated into 10 ml alkaline peptone water (APW). After 6, 8 and 10 h, 1 ml
320
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
Fig. 1. PCR amplification of tdh gene fragment in Kanagawa positive V. parahaemolyticus strains. Lane A-D: Kanagawa negative strains of V. parahaemolyticus; lane E: V. parahaemolyticus ATCC 17802; lane F: TY 49. lane G: VPl; lane H: VPS; lane I: molecular weight marker.
aliquots of APW enrichment were centrifuged at 8000 x g for 5 min, the cell deposit lysed and PCR reaction performed using the lysate as described above.
3. Results and discussion Among the various primer pairs tested in the PCR reaction, M 454 and 441 did not result in any amplification signal. The primer pair 456 and 441 yielded a 623 bp amplification signal only in four Kanagawa positive strains of V. parahaemolyticus (Fig. 1). All Kanagawa negative environmental isolates, clinical and environmental isolates of non 01 V. cholerae, V. cholerae 01 Ogawa, V. cholerae 0 139, V. mimicus, V. hollisae and V. damsela strains tested were negative in the assay showing thereby that this primer pair specifically amplified the tdh gene of V. parahaemolyticus. This primer pair bound to sites different from that used by Lee and Pan (1993). The primer pair BG 24 and 441 also produced an amplification signal in Kanagawa positive strains of V. parahaemolyticus but produced non-specific bands in one culture and in a V. hollisae culture. Therefore, this primer pair was not used for further studies. To study the possibility of using the tdh PCR assay for detection of Kanagawa positive V. parahaemolyticus in seafoods, PCR was done directly on lysates of homogenates from 20 fish samples. Simultaneously homogenates were tested for the presence of V. parahaemolyticus by the culture method. None of the 20 fish samples tested harboured Kanagawa positive V. parahaemolyticus as indicated by the standard enrichment method though 12 of them contained Kanagawa negative V. parahaemolyticus. None of the fish homogenates, including the latter, tested positive in tdh PCR assay whereas a fish homogenate spiked with Kanagawa positive V. parahaemolyticus gave an amplification signal in the PCR assay (Fig. 2). When the primer pairs selected were used for detection of V. parahaemolyticus directly from fish homogenate, an amplification signal was obtained with the
I. Karunasagar et al. 1 Int. J. Food Microbiology 31 (1996) 317-323
321
Fig. 2. Detection of Kanagawa positive V. parahaemolyticus in fish homogenate by PCR. Lane A: molecular weight marker; lane B-H: seafood homogenates containing Kanagawa negative V. parahaemolyticus; lane I: seafood homogenate contaminated with TY 49; lane J: broth grown TY 49 (positive control).
sample containing lo4 or more V. parahaemolyticus cfu/ml (Fig. 3). To increase the sensitivity of the reaction, homogenates containing loo-lo5 I/. parahaemolyticus were inoculated to APW (at 1:10) and after 8 h enrichment, PCR was performed with cell lysates. PCR signals could be obtained in APW inoculated with fish homogenate containing less than 10 cells of V. parahaemolyticus (Fig. 4). These results indicate that combination of APW enrichment and PCR can detect less than 10 V. parahaemolyticus cells present in fish samples.
Fig. 3. Detection limit of V. parahaemolyticus by PCR directly on fish homogenates. Lane A-F: homogenates containing varying cell numbers of V. parahaemolyticus; lane A: 1.8 x IO’; lane B: 1.8 x 102; lane C: 1.8 x 10s; lane D: 1.8 x 104; E: 1.8 x 10’; F: 1.8 x 106; lane G: broth grown culture of V. paruhaemolyticus; lane H: DNA molecular weight marker.
322
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
Fig. 4. Detection of V. parahaemolyticus in fish homogenates after 8- 14 h enrichment. Lane A-G: APW inoculated with fish homogenates containing varying cell numbers of V. parahaemolyticus. Lane A-C: 4 x loo cells after 14, 12 and 10 h enrichment; lane D: 4 x IO’ cells after 10 h enrichment; lane E: 4 x 10” cells after 8 h enrichment; lane F: 4 x 10’ cells after 8 h enrichment; lane G: 4 x lo* cells after 8 h enrichment; lane H: fish homogenate without V. parahaemolyticus; Lane I: broth grown V. parahaemolyticus; lane J: DNA molecular weight marker.
Acknowledgements Thanks are due to Prof. Dr. W. Goebel, University of Wiirzburg, Germany for kindly synthesising the primers used in the study. We thank Dr. G.B. Nair, NICED, Calcutta and Dr. M. Jesudasan, Christian Medical College, Vellore for providing some of the clinical isolates. This work was supported by grants from Post Harvest Fisheries Research Programme of the United Kingdom Overseas Development Administration (ODA) project No. R5793 and equipment grant from Alexander Von Humboldt Foundation, Germany.
References Joseph, SW., Colwell, R.R. and Kaper, J.B. (1982) Vibrio parahaemolyticus and related halophilic vibrios. CRC Critical Reviews in Microbiology 10, 77-124. Kaper, J.B., Campen, K.R., Seidler, R.J., Baldini, M.H. and Falkow, S. (1984) Cloning of the thermostable direct or Kanagawa phenomenon associated hemolysin of Vibrio parahaemolyticus. Infect. Immun. 45, 290-292. Karunasagar, I., Mathew, S. and Karunasagar, I. (1987) Vibrio oulniJicus in fish and clams in Mangalore waters, West coast of India. Indian J. Mar. Sci. 16, 136- 137. Karunasagar, I., Suseela, M., Malathi, G.R. and Karunasagar, I. (1990) Incidence of human pathogenic vibrios in seafoods harvested along the coast of Karnataka. FAO Fisheries Rep. 401 (Suppl), 53-56. Karunasagar, I., Rosalind, G., Malathi, G.R. and Karunasagar, 1. (1992) Virulence of seafood associated strains of Non 01 Vibrio cholerae. In: Huss, H.H., Jakobsen, M. and Liston, J. eds. Quality Assurance in The Fish Industry. Elsevier Sci. Publ. Amsterdam. pp. 21 l-216. Lee, C. and Pan, S. (1993) Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction. J. Gen. Microbial. 139, 3225-3231. Nishibuchi, M. and Kaper, J.B. (1985) Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus J. Bacterial. 162, 558-564.
I. Karunasagar
rt al. 1 Int. J. Food Microbiology
31 (1996) 317-323
323
Twedt, R.M. and Stelma, G.N. Jr. (1984) Recovery of Vibrio paraharmolyticus and related halophilic vibrios. In: Bacteriological Analytical Manual P 12.01-12.10, 6th Ed. Washington, DC. Food and Drug Administration. Wagatsuma, S. (1968) A medium for the test of the hemolytic activity of Vibrio parahaemolyticus. Media Circle. 13, 1599161. Yamasaki, S., Shirai, H., Takeda, Y. and Nishibuchi, M. (1991) Analysis of the gene of Vibrio hoNisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio paraharmolykus. FEMS Microbial. Lett. 80, 2599264.
Short communication
Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods I. Karunasagaf’,*,
G. Sugumar”, Indrani Karunasagar”, P.J.A. Reilly”,b
“Department of Fishery Microbiology, College of Fisheries, Mangalore - 575002, India bNatural Resources Institute, Chatham Maritime, Kent ME4 4TB, UK
Received 19 April 1995; revised 3 October 1995; accepted 27 December 1995
Abstract Detection of Kanagawa positive strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR) was tested. Primer pairs for specific amplification of tdh gene fragment is described. The assay could detect contamination of seafood homogenate when PCR was performed using lysate prepared directly from fish homogenates. The sensitivity of the assay could be improved to detect less than 10 cells of v. parahaemolyticus by performing PCR after 8 h enrichment in alkaline peptone water. Keywords: Polymerase chain mostable direct hemolysin
* Corresponding
reaction;
Vibrio parahaemolyticus;
Seafoods;
author, Tel: + 91 824 439256; fax: -t 91 824 438366.
016%1605/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PIZ SO168-1605(96)00974-O
tdh Gene; Ther-
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
318
1. Introduction
The majority of Vibrio parahaemolyticus strains isolated from cases of gastroenteritis produce a /?-hemolysin on a special blood agar medium, Wagatsuma agar (Wagatsuma, 1968). The ability to cause hemolysis on this agar has been termed the Kanagawa phenomenon (Joseph et al., 1982). This phenomenon is due to a thermostable hemolysin encoded by the tdh gene which has been cloned and sequenced (Kaper et al., 1984; Nishibuchi and Kaper, 1985). Recently Lee and Pan (1993) described PCR based detection of V. parahaemolyticus in clinical samples. Since rapid techniques are necessary for quality control purposes in the fish processing industry, the possibility of using polymerase chain reaction (PCR) based tests for detection of Kanagawa positive V. parahaemolyticus in seafoods was studied.
2. Materials and methods 2.1. Bacterial strains V. parahaemolyticus ATCC 17802, TY 49, VP-l, and VP-S were Kanagawa positive strains, the latter three isolated from clinical cases at the National Institute of Cholera and Enteric Diseases (NICED) Calcutta, India. Kanagawa negative strains of V. paruhaemolyticus (20 strains) comprised fish and environmental isolates (Karunasagar et al., 1990). For V. vulnzjicus ATCC 27562 and three environmental isolates were included (Karunasagar et al., 1987). Clinical isolates of Non 01 V. cholerae strains (14 strains) were obtained from Christian Medical College, Vellore. Environmental isolates (6 strains) of Non 01 V. cholerae were from this laboratory (Karunasagar et al., 1992). V. cholerae Ogawa was a clinical isolate from this laboratory and V. cholerae 0 139 strains (two strains) were obtained from NICED Calcutta. V. hollisae and V. damsela cultures (four strains each) were isolated from seafood in this laboratory. V. mimicus strains VM 4197, VM 4053 and VM 4203 were obtained from NICED, Calcutta.
Table 1 Nucleotide Primer M 454 M 456 BG 24 M 441
No.
sequence
of primers
used and their binding
sites
Sequence
Position
S-CGTTGATTATTCTTTTACGC-3’ 5’-TTTCATGATTATTCAGTTT-3’ 5’-CTGTCCCTTTTCCTGCCCCC-3’ 5’-TTTGTTGGATATACACAT-3’
17-36 85-103 269-288 702-719
1. Karunasagar et al. /ht.
J. Food Microbiology 31 (1996) 317-323
319
2.2. PCR reuction
PCR primers used in this study and their binding sites are indicated in Table 1. These were designed, based on the sequence of tdh2 gene published by Yamasaki et al. (1991). Primers M 454 and M 456 would bind within the non-coding region flanking tdh2 gene while BG 24 and M 441 would bind within the coding region. Primer pairs tested were M 454 and M 441, M 456 and M 441 and BG 24 and M 441. To prepare bacterial cell lysates, 37°C overnight tryptic soy broth (TSB) cultures were centrifuged at 8000 x g for 15 min and the cell pellet resuspended in 100 ~1 distilled water and heated at 110°C for 10 min. PCR reactions were performed in 100 ~1 volume taking 1 ~1 cell lysate, 200 PM dNTPs, 0.5 PM primers and 2.5 U Taq DNA polymerase (Bangalore Genie, India). Thirty cycles of amplification were performed using a Biomed 60-2 programmable Thermocycler with 1 min denaturation, annealing and extension steps at 94, 55 and 72°C respectively. PCR reaction products were resolved on a 1% agarose gel containing 0.5 pg ethidium bromide per ml and visualised by UV induced fluorescence. 2.3. Detection of contamination in fish directly by PCR Samples of fishes such as mackerel (Rastrelliger kanagurta), lactarius (Lactarius luctarius), pink perch (Nemipterus sp.), white sardine (Esculosa thoracata), sciaenid (Johnius sp.) were obtained from Mangalore fish market. Fish samples, 50 g, fish samples were homogenised with 450 ml alkaline peptone water (APW) for 2 min. One millilitre of homogenate was centrifuged at 100 x g to sediment fish particles and the supernatant was again centrifuged at 8000 x g for 10 min and the deposit re-suspended in 20 ~1 distilled water and lysed by heating at 110°C for 10 min. Two microlitres of lysate was used in the PCR reaction. As a positive control, one sample of fish homogenate was contaminated with the TY 49 strain of I’. parahaemolyticus at a level of 1.8 x 10’ cells/ml and lysates prepared as described above. 2.4. Stundard Detection method
Homogenates were enriched in APW for 18 h at 37°C and subcultured into TCBS agar (Hi Media Laboratories, Bombay, India). Confirmation of typical colonies was performed according to Twedt and Stelma (1984). 2.5. Minimum cell number required to give PCR signals To determine the sensitivity of the PCR assay, lysates prepared from fish homogenates contaminated with V. parahaemolyticus TY 49 strain at levels ranging from 106-- < lo/ml were used. To test whether an enrichment step would increase the sensitivity of the PCR reaction, 1 ml fish homogenates containing lo’-10’ I’. parahaemolyticus cells were inoculated into 10 ml alkaline peptone water (APW). After 6, 8 and 10 h, 1 ml
320
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
Fig. 1. PCR amplification of tdh gene fragment in Kanagawa positive V. parahaemolyticus strains. Lane A-D: Kanagawa negative strains of V. parahaemolyticus; lane E: V. parahaemolyticus ATCC 17802; lane F: TY 49. lane G: VPl; lane H: VPS; lane I: molecular weight marker.
aliquots of APW enrichment were centrifuged at 8000 x g for 5 min, the cell deposit lysed and PCR reaction performed using the lysate as described above.
3. Results and discussion Among the various primer pairs tested in the PCR reaction, M 454 and 441 did not result in any amplification signal. The primer pair 456 and 441 yielded a 623 bp amplification signal only in four Kanagawa positive strains of V. parahaemolyticus (Fig. 1). All Kanagawa negative environmental isolates, clinical and environmental isolates of non 01 V. cholerae, V. cholerae 01 Ogawa, V. cholerae 0 139, V. mimicus, V. hollisae and V. damsela strains tested were negative in the assay showing thereby that this primer pair specifically amplified the tdh gene of V. parahaemolyticus. This primer pair bound to sites different from that used by Lee and Pan (1993). The primer pair BG 24 and 441 also produced an amplification signal in Kanagawa positive strains of V. parahaemolyticus but produced non-specific bands in one culture and in a V. hollisae culture. Therefore, this primer pair was not used for further studies. To study the possibility of using the tdh PCR assay for detection of Kanagawa positive V. parahaemolyticus in seafoods, PCR was done directly on lysates of homogenates from 20 fish samples. Simultaneously homogenates were tested for the presence of V. parahaemolyticus by the culture method. None of the 20 fish samples tested harboured Kanagawa positive V. parahaemolyticus as indicated by the standard enrichment method though 12 of them contained Kanagawa negative V. parahaemolyticus. None of the fish homogenates, including the latter, tested positive in tdh PCR assay whereas a fish homogenate spiked with Kanagawa positive V. parahaemolyticus gave an amplification signal in the PCR assay (Fig. 2). When the primer pairs selected were used for detection of V. parahaemolyticus directly from fish homogenate, an amplification signal was obtained with the
I. Karunasagar et al. 1 Int. J. Food Microbiology 31 (1996) 317-323
321
Fig. 2. Detection of Kanagawa positive V. parahaemolyticus in fish homogenate by PCR. Lane A: molecular weight marker; lane B-H: seafood homogenates containing Kanagawa negative V. parahaemolyticus; lane I: seafood homogenate contaminated with TY 49; lane J: broth grown TY 49 (positive control).
sample containing lo4 or more V. parahaemolyticus cfu/ml (Fig. 3). To increase the sensitivity of the reaction, homogenates containing loo-lo5 I/. parahaemolyticus were inoculated to APW (at 1:10) and after 8 h enrichment, PCR was performed with cell lysates. PCR signals could be obtained in APW inoculated with fish homogenate containing less than 10 cells of V. parahaemolyticus (Fig. 4). These results indicate that combination of APW enrichment and PCR can detect less than 10 V. parahaemolyticus cells present in fish samples.
Fig. 3. Detection limit of V. parahaemolyticus by PCR directly on fish homogenates. Lane A-F: homogenates containing varying cell numbers of V. parahaemolyticus; lane A: 1.8 x IO’; lane B: 1.8 x 102; lane C: 1.8 x 10s; lane D: 1.8 x 104; E: 1.8 x 10’; F: 1.8 x 106; lane G: broth grown culture of V. paruhaemolyticus; lane H: DNA molecular weight marker.
322
I. Karunasagar et al. / Int. J. Food Microbiology 31 (1996) 317-323
Fig. 4. Detection of V. parahaemolyticus in fish homogenates after 8- 14 h enrichment. Lane A-G: APW inoculated with fish homogenates containing varying cell numbers of V. parahaemolyticus. Lane A-C: 4 x loo cells after 14, 12 and 10 h enrichment; lane D: 4 x IO’ cells after 10 h enrichment; lane E: 4 x 10” cells after 8 h enrichment; lane F: 4 x 10’ cells after 8 h enrichment; lane G: 4 x lo* cells after 8 h enrichment; lane H: fish homogenate without V. parahaemolyticus; Lane I: broth grown V. parahaemolyticus; lane J: DNA molecular weight marker.
Acknowledgements Thanks are due to Prof. Dr. W. Goebel, University of Wiirzburg, Germany for kindly synthesising the primers used in the study. We thank Dr. G.B. Nair, NICED, Calcutta and Dr. M. Jesudasan, Christian Medical College, Vellore for providing some of the clinical isolates. This work was supported by grants from Post Harvest Fisheries Research Programme of the United Kingdom Overseas Development Administration (ODA) project No. R5793 and equipment grant from Alexander Von Humboldt Foundation, Germany.
References Joseph, SW., Colwell, R.R. and Kaper, J.B. (1982) Vibrio parahaemolyticus and related halophilic vibrios. CRC Critical Reviews in Microbiology 10, 77-124. Kaper, J.B., Campen, K.R., Seidler, R.J., Baldini, M.H. and Falkow, S. (1984) Cloning of the thermostable direct or Kanagawa phenomenon associated hemolysin of Vibrio parahaemolyticus. Infect. Immun. 45, 290-292. Karunasagar, I., Mathew, S. and Karunasagar, I. (1987) Vibrio oulniJicus in fish and clams in Mangalore waters, West coast of India. Indian J. Mar. Sci. 16, 136- 137. Karunasagar, I., Suseela, M., Malathi, G.R. and Karunasagar, I. (1990) Incidence of human pathogenic vibrios in seafoods harvested along the coast of Karnataka. FAO Fisheries Rep. 401 (Suppl), 53-56. Karunasagar, I., Rosalind, G., Malathi, G.R. and Karunasagar, 1. (1992) Virulence of seafood associated strains of Non 01 Vibrio cholerae. In: Huss, H.H., Jakobsen, M. and Liston, J. eds. Quality Assurance in The Fish Industry. Elsevier Sci. Publ. Amsterdam. pp. 21 l-216. Lee, C. and Pan, S. (1993) Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction. J. Gen. Microbial. 139, 3225-3231. Nishibuchi, M. and Kaper, J.B. (1985) Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus J. Bacterial. 162, 558-564.
I. Karunasagar
rt al. 1 Int. J. Food Microbiology
31 (1996) 317-323
323
Twedt, R.M. and Stelma, G.N. Jr. (1984) Recovery of Vibrio paraharmolyticus and related halophilic vibrios. In: Bacteriological Analytical Manual P 12.01-12.10, 6th Ed. Washington, DC. Food and Drug Administration. Wagatsuma, S. (1968) A medium for the test of the hemolytic activity of Vibrio parahaemolyticus. Media Circle. 13, 1599161. Yamasaki, S., Shirai, H., Takeda, Y. and Nishibuchi, M. (1991) Analysis of the gene of Vibrio hoNisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio paraharmolykus. FEMS Microbial. Lett. 80, 2599264.