Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria. Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios. In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals. Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates. All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios. Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS. Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.

Vibrio choleraeis the etiological agent of the severe diarrheal disease-cholera and natural inhabitant of estuarine and coastal waters. The proximity of the Florida Indian River Lagoon (IRL) to areas affected by recent cholera outbreaks makes this estuary ideal to investigate the environmental dynamics and their potential role in V. cholerae's pathogen emergence. We identified two locations in the IRL, Feller's House UCF Field Station and Shepard Park, as our collection sites. We collected samples from three different fractions - water, plankton, and sediment - and recorded data for several water parameters such as pH, temperature, and, turbidity. In the laboratory, we enriched samples in alkaline peptone water and isolated V. choleraeusing widely used selective media Thiosulfate-Citrate Bile Salts-sucrose agar (TCBS) and CHROMagar Vibrio. From our study, we isolated 100 potential V. cholerae isolates, which were confirmed using biochemical tests such as oxidase and Kligler's Iron Agar. V. cholerae has allelic variations in the core genes such as ompU, which provide pre-adaptation to virulence. We investigated the allelic variations within ompU to characterize V. cholerae isolates. We elucidated the sequenced allele of ompU and built a neighboring-joining phylogeny tree to view the differentiation among vibrios. Our findings provide insight into the endemic population of V. cholerae in the Eastern Coast of Florida. Further studies include a screen for additional virulence genes and investigate the role of environmental dynamics on the distribution of V. cholerae and emergence as a human pathogen.