Vibrio vulnificus has been shown to be the causative agent of two types of human illnesses with distinguishing routes of infections and manifestations (5). The first entails dermal infection via open wound, which can become necrotic in some cases. The second and more severe type results in disease and often death of immunocompromised individuals following the ingestion of raw shellfish. The victim most often has a preexisting condition that results in high levels of serum iron. These high iron levels have been shown to enhance the pathogenicity of V. vulnificus. The iron mouse model was implemented to mimic the septicemic state of a patient with high serum iron levels, demonstrating the increase of virulence potential in the presence of excess iron (36).While the extent of infection in both disease states is reliant upon the expression of numerous virulence factors, the negatively charged polysaccharide capsule has been shown to protect this organism from the immune system in animal models (31). As a result, the expression of capsular polysaccharide (CPS) is believed to be a primary virulence factor of V. vulnificus that is essential for pathogenicity. Although clinical strains of V. vulnificus are encapsulated, the ability to express CPS is often lost, as witnessed in the laboratory, resulting in a nonencapsulated spontaneous mutant that is no longer virulent and appears translucent. Nomenclature commonly used in the laboratory and herein to describe colony morphology is that of opaque (O) for an encapsulated strain and translucent (T) for a nonencapsulated strain. The event leading to or resulting in the loss of capsule expression is not understood; it is seemingly a spontaneous, random happening that can be visualized on the surface of an agar plate.The capsule of V. vulnificus has been partially described, both phenotypically and genetically. Multiple capsule types exist, with the carbohydrate composition characteristically acidic, containing many amino sugars, including galactosamine, fucosamine, glucosamine, and quinovosamine (7). The repeating unit is believed to consist of four sugars, with common residues shared among encapsulated strains expressing different capsule types (24). Recently, Wright et al. (37) identified a genetic locus that encodes the transport of a group 1-like CPS in an encapsulated strain of V. vulnificus. Because of these previous studies, it is anticipated that the capsule gene complex of V. vulnificus is comprised of genes that correspond to previously defined characteristics of the CPS, such as carbohydrate composition and grouping.We proposed to locate the capsule gene complex by using transposon mutagenesis. Selection and genetic analysis of four nonencapsulated transposon mutants has led to the identification of two types of loci. Numerous genes involved in capsular synthesis and expression have been identified which do correspond to possible capsule typing and phenotypic characteristics. In addition, an integron-like region has been located, similar to that of the super integr...

V~b n o vuln~ficus was isolated in 1996 f~o m 2 d~s e a s e outbreaks on a Danish eel f a~m lvhich used brackish water A charactenstic clinical sign was extensive deep muscle necrosis in the head region V vulnlficus was isolated from k~d n e y mucus spleen gill and intestlne of diseased eels Th~rty-two isolates were examined phenotypically and serologically for pathogenic~ty to eels and for correlat~on to nbotype and plasmid profile B~ochemically, the ~solates showed propert~es slm~lar to those described previously for eel-pathogenic strains of V i/ulnlficus w~t h the exception of lndole prod u c t~o n Virulence was evaluated by LDSo (the 50 '0 lethal dose) which ranged from < 9 4 X 103 to 2 3 X 105 CFU (colony-formng units) per f~s h The isolates which were lethal for eels showed identical nbotypes and serotypes A relat~onship between certain plasm~ds and virulence \4as not found A serotyping system based on l~popolysacchande (LPS)-associated O antigen type and on carbohydrate capsule antigens showed that the eel-virulent ~solates shared a common LPS-based homogeneous O serogroup and a capsule antigen V rulnlficus serovar 0 4 and capsule type 9 was identical serologically to the Japanese isolate ATCC 33149 and was the agent responsible for the disease outbreaks that occurred on the Danish eel farm Despite absence of ant~biotic resistance treatment had llttle effect and disease reoccurred

We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae 01 in clinical specimens. Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V. cholerae 01 in less than 15 min, without cumbersome laboratory equipment. A total of 120 clinical and environmental bacterial strains, induding both 01 and non-Ol serotypes of V. cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V. cholerae 01. Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100%o specificity and 96% sensitivity compared with conventional culture methods. Another field trial, in Mexico, showed that Cholera SMART was 100%o in agreement with a recently described coagglutination test when 108 stool specimens were tested.

A collection of 143 Vibrio cholerae non-O1 strains isolated from shrimp farms in Thailand were characterized and grouped by ribotyping. Sixty-four ribotypes were distinguished following digestion of chromosomal DNA with the restriction enzyme BglI, and the reproducibility of the method was 100%. There was no correlation between specific ribotype distributions and the locations of the shrimp farms. Ribotype similarity was examined by cluster analysis, and two main groups with 10 and 54 ribotypes, respectively, were found. Correlation between ribotype and O-antigen expression was shown to exist among those isolates tested. Ribotyping appears to be a suitable method for differentiating environmental V. cholerae non-O1 strains, and comparison of ribotype patterns showed a high degree of genetic divergence within V. cholerae non-O1.

A serotyping scheme for Vibrio vulnificus predicated on the detection of lipopolysaccharide (LPS) antigens is proposed. The serovar 0 typing scheme used to type V. vulnificus employs polyclonal antisera raised in rabbits immunized with heat-killed whole-cell vaccines. Polyclonal typing sera produced in this manner cross-react with heterologous strains. Affinity purification of polyclonal antisera with LPS affinity columns resolved some of these cross-reactions; however, affinity-purified polyclonal antisera still showed cross-reactions that were nonreciprocal. On the basis of the serological patterns that were obtained with affinity-purified polyclonal antisera, V. vulnificus strains were selected as vaccine strains for production of monoclonal antibody. Spleen cells harvested from BALB/c mice immunized with formalin-killed V. vulnificus cells were fused with SP2/O-Ag 14 myeloma cells. Hybridomas were screened by using LPS and whole-cell enzyme-linked immunosorbent assay to identify clones secreting LPS-specific antibodies. Monoclonal antibodies identified five LPS serological varieties of V. vulnificus and a single serovar each for Vibrio damsela and Vibrio hollisae. No cross-reactions between V. vulnificus and V. hollisae or V. damsela were observed.