Production and application of antibodies produced against the flagella and hemolysin of Vibrio parahaemolyticus
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Vibrio parahaemolyticus is a marine bacterium which is the leading cause of bacterial gastroenteritis due to consumption of raw or undercooked bivalve molluscan shellfish in United States and is considered an important seafood-borne pathogen throughout the world. The conventional methods for enumerating this bacteria from seafood is laborious and time consuming, hence the objective of our study was to develop antibody based method which would be sensitive, user-friendly and less time consuming to detect and enumerate Vibrio parahaemolyticus from oysters. In order to condense time and material two rapid and specific antibody based test methods were developed. In the first method, monoclonal antibodies were produced against the polar flagella of Vibrio parahaemolyticus. The monoclonal antibody was employed in Immunomagnetic separation (IMS) protocol and was then coupled with Real-time PCR (q-PCR). IMS coupled to q-PCR was able to detect bacterial numbers in less than 3 hrs with a sensitivity of 1,000 CFU/g of oyster homogenate. In the second method anti-hemolysin serum was produced and employed in Direct colony immunoblot (DCI) protocol to detect pathogenic Vibrio parahaemolyticus. DCI when compared with the currently used conventional method, DNA hybridization (DNAH) showed good correlation and was a faster and simpler method requiring less equipment and stringent laboratory conditions than DNAH. From our results we have found that IMS+q-PCR or DCI could be an useful addition to the methods available for enumerating pathogenic V. parahaemolyticus in seafood.Keywords:
Hemolysin
Immunomagnetic separation
Pathogenic bacteria
Ostreidae
Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct haemolysin (TDH) or TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus.
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V. parahaemolyticus 종류들이 gelatin분해 확인 실험에서 큰 clear zone을 형성했고, V. alginolyticus 분리균주도 V. parahaemolyticus와 비슷한 clear zone을 형성했다. V. fluvialis KCTC 2473은 clear zone을 형성하지 못했다. Hemolytic activity측정 실험에서는 V. parahaemolyticus 분리균주(2)가 가장 활성이 크게 나타났고, V. parahaemolyticus ATCC 17802, V. parahaemolyticus 분리균주(1)과 V. mimicus ATCC 33653도 높은 활성을 나타냈으며, 나머지 다른 비브리오균은 활성이 나타나지 않았다. Azocasein분해 활성 실험에서는 V. parahaemolyticus ATCC 17802, 분리균주(1), 분리균주(2)는 200EU/m1전후로 활성이 높게 나타났고, 나머지는 110EU/ml전후로 활성이 상대적으로 낮게 나타났다. 【Nine strains of pathogenic Vibrio sp. of clinical and environmental origin were examined for the degradation of gelatin, casein and hemolysin which is important to the virulence of this bacterium. Culture filtrates of all nine strains of Vibrio exhibited proteolytic activities. Especially, four strains of V. parahaemolyticus and one V. alginolyticus showed strong gelatin-degrading activity. In terms of hemolytic activity, three V. parahaemolyticus and V. mimicus showed strong $\beta$ -hemolysis whereas those of strains of V. alginolyticus, V. fluvialis, V. vulnificus examined lacked this activity.】
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Vibrio parahaemolyticus is one of the most important enteropathogens in Taiwan, Japan, and other coastal regions. The pathogenesis of V. parahaemolyticus disease is not clearly understood. The expression of some factors by V. parahaemolyticus in iron-rich and iron-limited media was analyzed. In the clinical hemolytic strains, the production of a siderophore, two outer membrane proteins (77 and 80 kDa), and thermostable direct hemolysin was significantly enhanced in iron-limited culture, and hemolytic activities, cell hydrophobicity, HEp-2 cell adherence, and lethality for mice were also enhanced. The environmental nonhemolytic strain CCRC12958 that was cultured in iron-limited medium exhibited lethal activity for mice, and other factors except hemolysis were also enhanced like the responses of clinical strains were. These results suggested that a virulent factor(s) of V. parahaemolyticus may be induced or enhanced under iron-limited conditions. The iron-regulated factors reported in this paper may be important in the pathogenesis of V. parahaemolyticus disease.
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Pathogenesis
Vibrio Infections
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A hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus 06: K46 was purified by 55% ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, Sepharose 4B and Mono Q. The purified hemolysin was physicochemically and immunologically identical with the Vp-TRH (V. parahaemolyticus thermostable direct hemolysin related hemolysin) recently described in V. parahaemolyticus 03: K6 (Honda et al. Infect. Immun. 56: 961–965, 1988). This indicates that V. parahaemolyticus of Kanagawa-negative clinical isolates possessing not only 03: K6 but also different serotypes such as 06: K46 produce Vp-TRH. Production of Vp-TRH by most clinical isolates of Kanagawa-negative V. parahaemolyticus was also demonstrated. These results suggest the importance of Vp-TRH among clinical isolates of Kanagawa-negative V. parahaemolyticus.
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Some phenotypically non-haemolytic strains of V. parahaemolyticus were observed to produce haemolysins after two passages in mices. Depending on the strain, both heat stable and heat labile haemolysins were induced. The heat stable haemolysin so induced, was immunologically different from the thermostable direct haemolysin produced by Kanagawa positive strains of V. parahaemolyticus.
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Thermolabile
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Vibrio parahaemolyticus is known to produce several types of hemolysin, the most document is the TDH and TRH. A new type of hemolysin whose expression is repressed by the presence of phosphate in the medium is reported.
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A simple method involving immunoaffinity column chromatography to purify the thermostable direct hemolysin of Vibrio parahaemolyticus was developed. The thermostable direct hemolysin purified from the culture supernatant of a strain isolated from the first reported case of V. parahaemolyticus infection in China in 1985 was indistinguishable from the hemolysins purified from strains isolated in Japan.
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