Vibrio fluvialis, An Emerging Pathogen Responsible For Limp Lobster Disease In The American Lobster     Ben D. Tall1, S. Fall1, M. R. Pereira1, S. R. Monday1, M. H. Kothary1, K. A. Lampel1, L. Kornegay1, S. K. Curtis1, F. M. Khambaty1, D. T. Chu1, B. M. McCardell1, D. Prince2, and R. C. Bayer2   1CFSAN, US FDA, Washington, D.C. 20204 and 2University of Maine, Orono, ME 04469.     Evidence is accumulating that Vibrio fluvialis is the etiological agent responsible for Limp Lobster Disease in Homarus americanus (American lobster). Infection leads to a rapid onset of symptoms such as weakness, lethargy, and slow or ineffectual responses to sensory stimuli; death occurs within hours. We studied the pathogenic mechanisms involved in this syndrome using 18 isolates obtained from ill lobsters representing five closely related groups as defined by Pulsed-Field Gel Electrophoresis (PFGE). Based on biochemical analyses, these strains appear to embody a new biotype. Sixteen of the isolates were found to harbor a ~12-kb plasmid. Challenge studies using lobsters in which a plasmid-positive outbreak strain was injected via the tail hemocoel revealed that the LD50 dosage was a thousand-fold lower than that for a plasmid-negative strain. This suggests that the 12-kb plasmid may escalate the pathogenicity of these microorganisms in lobsters. Microorganisms recovered from experimentally infected lobsters exhibited API 20E and PFGE profiles that were indistinguishable from those of the outbreak challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and mid-gut tissues, as well as in the hemolymph. In summary, these data support the hypothesis that V. fluvialis cause Limp Lobster Disease, satisfying Koch's postulates at the organismal and molecular level.  Upon further analyses of these strains in mammalian systems, bacteria-free culture supernatants caused Chinese hamster ovary cells to elongate, suggesting the presence of a hitherto unknown enterotoxin. To determine whether these microorganisms can cause disease in warm-blooded vertebrates, suckling mice were orally challenged with either plasmid-positive or plasmid-negative strains.  Significant accumulation of intestinal fluid was observed in mice receiving a dose of 109 CFU/ml of either plasmid- positive or plasmid-negative strains; however, no microorganisms were recovered from intestinal contents in either case. These data suggest that the mechanism resulting in fluid accumulation observed in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of the 12-kb plasmid. Our findings imply that V. fluvialis is capable of causing diarrhea in mice and hence should be treated as a potential mammalian pathogen.