Abstract
Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.
Full Text
The Full Text of this article is available as a PDF (400.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amaro C., Biosca E. G., Fouz B., Alcaide E., Esteve C. Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels. Appl Environ Microbiol. 1995 Mar;61(3):1133–1137. doi: 10.1128/aem.61.3.1133-1137.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amaro C., Biosca E. G., Fouz B., Garay E. Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting. Curr Microbiol. 1992 Aug;25(2):99–104. doi: 10.1007/BF01570967. [DOI] [PubMed] [Google Scholar]
- Amaro C., Biosca E. G., Fouz B., Toranzo A. E., Garay E. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect Immun. 1994 Feb;62(2):759–763. doi: 10.1128/iai.62.2.759-763.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amaro C., Biosca E. G. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl Environ Microbiol. 1996 Apr;62(4):1454–1457. doi: 10.1128/aem.62.4.1454-1457.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Arias C. R., Garay E., Aznar R. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water. Appl Environ Microbiol. 1995 Sep;61(9):3476–3478. doi: 10.1128/aem.61.9.3476-3478.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Amaro C., Marco-Noales E., Oliver J. D. Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2. Appl Environ Microbiol. 1996 Feb;62(2):450–455. doi: 10.1128/aem.62.2.450-455.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Amaro C. Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity. Appl Environ Microbiol. 1996 Jul;62(7):2331–2337. doi: 10.1128/aem.62.7.2331-2337.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Fouz B., Alcaide E., Amaro C. Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2. Appl Environ Microbiol. 1996 Mar;62(3):928–935. doi: 10.1128/aem.62.3.928-935.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Garay E., Toranzo A. E., Amaro C. Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2. FEMS Microbiol Lett. 1993 Mar 1;107(2-3):217–222. doi: 10.1111/j.1574-6968.1993.tb06033.x. [DOI] [PubMed] [Google Scholar]
- Biosca E. G., Llorens H., Garay E., Amaro C. Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels. Infect Immun. 1993 May;61(5):1611–1618. doi: 10.1128/iai.61.5.1611-1618.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biosca E. G., Oliver J. D., Amaro C. Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus. Appl Environ Microbiol. 1996 Mar;62(3):918–927. doi: 10.1128/aem.62.3.918-927.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brauns L. A., Hudson M. C., Oliver J. D. Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Appl Environ Microbiol. 1991 Sep;57(9):2651–2655. doi: 10.1128/aem.57.9.2651-2655.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Martin S. J., Siebeling R. J. Identification of Vibrio vulnificus O serovars with antilipopolysaccharide monoclonal antibody. J Clin Microbiol. 1991 Aug;29(8):1684–1688. doi: 10.1128/jcm.29.8.1684-1688.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Massad G., Oliver J. D. New selective and differential medium for Vibrio cholerae and Vibrio vulnificus. Appl Environ Microbiol. 1987 Sep;53(9):2262–2264. doi: 10.1128/aem.53.9.2262-2264.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morris J. G., Jr, Wright A. C., Roberts D. M., Wood P. K., Simpson L. M., Oliver J. D. Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene. Appl Environ Microbiol. 1987 Jan;53(1):193–195. doi: 10.1128/aem.53.1.193-195.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishibuchi M., Seidler R. J. Demonstration of a common antigen in sonicated cells for identification of Vibrio vulnificus serotypes. J Clin Microbiol. 1985 Jan;21(1):97–101. doi: 10.1128/jcm.21.1.97-101.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishibuchi M., Seidler R. J. Rapid microimmunodiffusion method with species-specific antiserum raised to purified antigen for identification of Vibrio vulnificus. J Clin Microbiol. 1985 Jan;21(1):102–107. doi: 10.1128/jcm.21.1.102-107.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver J. D., Bockian R. In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl Environ Microbiol. 1995 Jul;61(7):2620–2623. doi: 10.1128/aem.61.7.2620-2623.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver J. D., Guthrie K., Preyer J., Wright A., Simpson L. M., Siebeling R., Morris J. G., Jr Use of colistin-polymyxin B-cellobiose agar for isolation of Vibrio vulnificus from the environment. Appl Environ Microbiol. 1992 Feb;58(2):737–739. doi: 10.1128/aem.58.2.737-739.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oliver J. D., Nilsson L., Kjelleberg S. Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl Environ Microbiol. 1991 Sep;57(9):2640–2644. doi: 10.1128/aem.57.9.2640-2644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parker R. W., Lewis D. H. Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens. Appl Environ Microbiol. 1995 Feb;61(2):476–480. doi: 10.1128/aem.61.2.476-480.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schloter M., Assmus B., Hartmann A. The use of immunological methods to detect and identify bacteria in the environment. Biotechnol Adv. 1995;13(1):75–90. doi: 10.1016/0734-9750(94)00023-6. [DOI] [PubMed] [Google Scholar]
- Shimada T., Sakazaki R. On the serology of Vibrio vulnificus. Jpn J Med Sci Biol. 1984 Oct-Dec;37(5-6):241–246. doi: 10.7883/yoken1952.37.241. [DOI] [PubMed] [Google Scholar]
- Simonson J., Siebeling R. J. Rapid serological identification of Vibrio vulnificus by anti-H coagglutination. Appl Environ Microbiol. 1986 Dec;52(6):1299–1304. doi: 10.1128/aem.52.6.1299-1304.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stelma G. N., Jr, Reyes A. L., Peeler J. T., Johnson C. H., Spaulding P. L. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl Environ Microbiol. 1992 Sep;58(9):2776–2782. doi: 10.1128/aem.58.9.2776-2782.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Swaminathan B., Feng P. Rapid detection of food-borne pathogenic bacteria. Annu Rev Microbiol. 1994;48:401–426. doi: 10.1146/annurev.mi.48.100194.002153. [DOI] [PubMed] [Google Scholar]
- Tamplin M. L., Martin A. L., Ruple A. D., Cook D. W., Kaspar C. W. Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters. Appl Environ Microbiol. 1991 Apr;57(4):1235–1240. doi: 10.1128/aem.57.4.1235-1240.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tison D. L., Kelly M. T. Virulence of Vibrio vulnificus strains from marine environments. Appl Environ Microbiol. 1986 May;51(5):1004–1006. doi: 10.1128/aem.51.5.1004-1006.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tison D. L., Nishibuchi M., Greenwood J. D., Seidler R. J. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol. 1982 Sep;44(3):640–646. doi: 10.1128/aem.44.3.640-646.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Veenstra J., Rietra P. J., Stoutenbeek C. P., Coster J. M., de Gier H. H., Dirks-Go S. Infection by an indole-negative variant of Vibrio vulnificus transmitted by eels. J Infect Dis. 1992 Jul;166(1):209–210. doi: 10.1093/infdis/166.1.209. [DOI] [PubMed] [Google Scholar]
- Wright A. C., Miceli G. A., Landry W. L., Christy J. B., Watkins W. D., Morris J. G., Jr Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe. Appl Environ Microbiol. 1993 Feb;59(2):541–546. doi: 10.1128/aem.59.2.541-546.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]