During a 2-year study, samples of various types of soils were collected from 115 fields that had not previously been tested with Bacillus thuringiensis and which were remote from any large-scale aggregations of lepidopterous insects in rearing or grain-storage areas. An average of about 400 isolates were examined from each soil, and, of 46 373 isolates examined, only 250 (0.5%) were identified as B. thuringiensis. While it was almost impossible to insure that a field had never been treated with B. thuringiensis or that drift from some nearby application had not reached the field, it is noteworthy that of the 250 isolates, 156 (62.4%) were not var. kurstaki, the only variety that has been used commercially in the United States in about 10 years. This is a strong indication that the B. thuringiensis isolates observed were present naturally. To verify the procedures used, samples were taken from two adjacent experimental plots which had been treated about 12 months previously with formulations of var. kurstaki and var. galleriae, respectively. With practically no exception, the variety recovered from each plot was the variety applied, indicating that the varietal status of B. thuringiensis is stable in the soil.
Summary Vibrio parahaemolyticus is ubiquitous in estuarine environments and can be commonly found in seafood products. This bacterial pathogen continues to emerge as an important cause of foodborne illness, and several foodborne disease outbreaks caused by V. parahaemolyticus have been linked to the consumption of contaminated seafood, in particular those consumed raw such as oysters. In response to these outbreaks, especially during the 1990s, several cultural, immunological‐based and molecular detection methods have been developed, which allow for rapid detection and quantification of total and pathogenic V. parahaemolyticus. The development of molecular methodology has allowed for clinical and environmental isolates of V. parahaemolyticus to be subtyped, thus providing the framework for risk‐based strategies aimed at controlling foodborne outbreaks cause by this pathogen. It is important that the detection and typing methods strive to accomplish detection and differentiation of the pathogenic strains from environmental (non‐pathogenic) ones, as well as to detect the presence of the organism and not just the presence of V. parahaemolyticus produced toxins, which can also be produced by closely related species. This review covers the current status of detection and typing methodology for identification and characterisation of V. parahaemolyticus from seafood.
Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0. 1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation. Because of the rapid progression and high mortality rates of Vibrio vulnificuts infections in humans, especially those victims with underlying chronic disease, seawater and seafood are being scrutinized for the presence and level of this organism. The recovery, identification, and enumeration of the Vibrio pathogens in the marine environment and in the shell fish which inhabit it can be overwhelming in logistics, material, and time, while the expeditious and specific identification of V. vulnificuts in the laboratory setting is desirable. One approach to condense the time required presently to confirm the presence of V. vulnificits in the marine environment, seafood, or clinical specimens is through the use of a rapid serological test which detects the speciesspecific flagellar (H) antigens. The premise for serological identification is predicated upon the observation of Gardner and Venkatraman (5), who reported that Vibrio cholerae isolates, both 01 and non-O1 serological varieties, express an H antigen unique to the species. This observation was confirmed independently by Sakazaki et al. (13) and Bhattacharyya and Mukerjee (1, 2); the latter reported that both Vibrio mnetschnikovii and Vibrio anguiillarumin each express species-specific H antigens. As additional species of Vibrio were examined serologically, it was reported that V.
Opaque colony morphology has been correlated to Vibrio vulnificus virulence. However, the number of capsular serotypes expressed by virulent isolates is unknown. In an effort to produce anticapsule sera, capsular polysaccharide (CPS) from three opaque V. vulnificus strains was purified and characterized. Purified CPSs were acidic and contained considerable amounts of hexosamine and trace quantities of protein and nucleic acid. CPS purified from strain C7184 was poorly immunogenic for rabbits and mice, since repeated injection produced little detectable anticapsular antibody. To improve immunogenicity, CPS-protein conjugates were prepared from adipic acid hydrazide derivatives of CPS purified from each strain and carbodiimide as a coupling reagent. The immunogenicity of C7184 CPS was enhanced by conjugation to keyhole limpet hemocyanin, since injection into mice elicited production of anticapsular antibodies, the level of which was dependent on the dose and time since initial immunization. Injection of rabbits with CPS-protein conjugates also produced anticapsular antibodies. Staphylococcus aureus cells armed with each of the three anticapsular antibodies coagglutinated only the homologous opaque strain, indicating the existence of at least three capsular types. Further screening of 32 opaque and translucent V. vulnificus isolates revealed only three cross-reacting strains. These results suggest the presence of numerous V. vulnificus capsular types.
Mice were immunized by injection of Vibrio parahaemolyticus ATCC 17802 polar flagellin in order to produce monoclonal antibodies (mAbs). mAbs were analyzed by anti-H enzyme-linked immunosorbent assay using V. parahaemolyticus polar flagellar cores. The mAb exhibiting the highest anti-H titer was coated onto Cowan I Staphylococcus aureus cells at a concentration of 75 g/ml cell suspension and used for slide coagglutination. Of 41 isolates identified genetically as V. parahaemolyticus, 100% coagglutinated with the anti-H mAb within 30 s, and the mAb did not react with 30 isolates identified as Vibrio vulnificus. A strong coagglutination reaction with V. parahaemolyticus ATCC 17802 was still observed when the S. aureus cells were armed with as little as 15 g of mAb/ml S. aureus cell suspension. At this concentration, the mAb cross-reacted with three other Vibrio species, suggesting that they share an identical H antigen or antigens. The anti-H mAb was then used to optimize an immunomagnetic separation protocol which exhibited from 35% to about 45% binding of 10 2 to 10 3 V. parahaemolyticus cells in phosphate-buffered saline. The mAb would be useful for the rapid and selective isolation, concentration, and detection of V. parahaemolyticus cells from environmental sources.Vibrio parahaemolyticus is a naturally occurring marine bacterium responsible for the majority of seafood-associated human gastroenteritis cases in the United States and is considered an important seafood-borne pathogen throughout the world (14,28,43). Conventional bacteriological methods for the detection and enumeration of V. parahaemolyticus bacteria can be costly in labor, materials, and time (25), while the expeditious identification of V. parahaemolyticus in the laboratory is desirable. Ideally, a method is needed which can easily detect and enumerate V. parahaemolyticus cells by the direct examination of shellfish, seafood, or water and which does not involve lengthy enrichment steps or overnight incubation.After several V. parahaemolyticus outbreaks in the United States (8, 9, 16), the Interstate Shellfish Sanitation Conference (ISSC) implemented a plan for monitoring the levels of V. parahaemolyticus bacteria in freshly harvested oysters. Since the standard most probable number (MPN)/biochemical method for enumerating V. parahaemolyticus bacteria (17) was so labor-intensive and time-consuming, the procedure the ISSC recommended involved plating oyster homogenates directly onto agar plates and, after an overnight incubation, transferring resultant colonies to filters that could be hybridized with DNA probes to detect total (tlh) and pathogenic (tdh) strains of V. parahaemolyticus (12). The probes were successfully used for the direct examination of total and pathogenic V. parahaemolyticus in oysters harvested from Washington, Texas, and New York (16). Gooch et al. (20) compared two direct plating methods to the MPN protocol using probes specific for tlh to confirm V. parahaemolyticus isolates in Alabama oysters. They concluded that both di...
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