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Applied and Environmental Microbiology, December 2003, p. 7435-7446, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7435-7446.2003

Characterization of Vibrio fluvialis-Like Strains Implicated in Limp Lobster Disease

B. D. Tall,^1* S. Fall,¹ M. R. Pereira,¹ M. Ramos-Valle,¹ S. K. Curtis,¹ M. H. Kothary,¹ D. M. T. Chu,¹ S. R. Monday,¹ L. Kornegay,¹ T. Donkar,¹ D. Prince,² R. L. Thunberg,¹ K. A. Shangraw,¹ D. E. Hanes,¹ F. M. Khambaty,¹ K. A. Lampel,¹ J. W. Bier,¹ and R. C. Bayer²

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740,¹ The Lobster Institute, University of Maine, Orono, Maine 04469²

Received 27 March 2003/ Accepted 15 September 2003

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20°C; none could grow at temperatures above 23°C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37°C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37°C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

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* Corresponding author. Mailing address: Microbial Methods Application Branch, HFS 517, Room 3E016, Wiley Building, Division of Microbiological Studies, Office of Plant and Dairy Foods and Beverages, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Pkwy., College Park, MD 20740-3835. Phone: (301) 436-1645, ext. 2553. Fax: (301) 436-2644. E-mail: btall{at}cfsan.fda.gov.

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Applied and Environmental Microbiology, December 2003, p. 7435-7446, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7435-7446.2003

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