Detection of Vibrio Anguillarum Antigen by the Dot Blot Assay

Cipriano, Rocco C.; Pyle, Josephine; Starliper, Clifford E.; Pyle, Stephen W.

doi:10.7589/0090-3558-21.3.211

Cited by 39 publications

(29 citation statements)

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“…The dot-blot analysis was performed as described by Cipriano et al (1985). Only a reaction similar to that exhibited by the homologous strain was scored as positive.…”

Section: Methodsmentioning

confidence: 99%

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Silva-Rubio¹,

Acevedo²,

Magariños

et al. 2008

Dis. Aquat. Org.

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Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The characteristics of isolates were compared with the type strain V. ordalii ATCC 33509 T . The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their antigenic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS electrophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demonstrated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area. KEY WORDS: Vibrio ordalii · Fish vibriosis · Serotyping · Genotyping · Atlantic salmon Resale or republication not permitted without written consent of the publisherDis Aquat Org 79: [27][28][29][30][31][32][33][34][35] 2008 (Colquhoun et al. 2004) using conventional methods and miniaturized systems have demonstrated that Chilean V. ordalii isolates share the same biochemical properties present in the previous description of the species (Schiewe et al. 1981), with the exception of production of acid from trehalose and lack of acid production from mannitol. In addition, these Chilean strains could not be serotyped due to auto-agglutination of the isolates (Colquhoun et al. 2004).However, until now, no discriminative methods based on genetic techniques have been applied to this bacterium. Pulsed-field gel electrophoresis (PFGE) (Skov et al. 1995), randomly amplified polymorphic DNA (RAPD)-PCR, enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR and repetitive extragenic palindromic (REP)-PCR have become accessible and sensitive methods for the intraspecific genetic variability in many pathogens (Williams et al. 1990, Versalovic et al. 1991, Bachellier et al. 1999. Therefore, the objective of this study was to describe in depth the biochemical and antigenic characteristics of the Vibrio ordalii strains isolated from disease outbreaks in Salmo salar in Chile, for future development and/or improvement of the vaccination program against vibriosis. In addition, PFGE and 3 PCR-based methods were employed to analyse the geneti...

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“…The dot-blot analysis was performed as described by Cipriano et al (1985). Only a reaction similar to that exhibited by the homologous strain was scored as positive.…”

Section: Methodsmentioning

confidence: 99%

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Silva-Rubio¹,

Acevedo²,

Magariños

et al. 2008

Dis. Aquat. Org.

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“…Thermostable 'O' antigens from strain CECT 4606 of Vibrio vulnificus biotype 1 were also obtained. Indirect dot blot assays were performed by the method described by Burreson & Frizzell (1986) and Cipriano et al (1985) with some modifications (Amaro et al 1997 were fixed on nitrocellulose papers. Membranes were incubated with eel serum, and immunological reactions were revealed as previously described (Amaro et al 1997).…”

Section: Methodsmentioning

confidence: 99%

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Collado¹,

Fouz²,

Sanjuán³

et al. 2000

Dis. Aquat. Org.

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Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids -inactivated extracellular products -from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum. To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen. The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p. injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies. KEY WORDS: Vibrio vulnificus serovar E · Vibrio vulnificus biotype 2 · Eel vaccines · Vibrio vaccines · Vaccination by injection · Vaccination by prolonged immersion Resale or republication not permitted without written consent of the publisherDis Aquat Org 43: [91][92][93][94][95][96][97][98][99][100][101] 2000 Vibrio vulnificus serovar E is an opportunistic human pathogen (Amaro & Biosca 1996) which is able to survive and spread infection through water (Amaro et al. 1995).To make eel culture profitable, in both brackish and fresh water, alternative strategies are needed to solve the problem, and should focus mainly on preventative measures. The use of vaccines in aquaculture has been shown to successfully protect fish against bacterial diseases, such as vibriosis (caused by Listonella anguillarum [formerly Vibrio anguillarum] and V. ordalii ), edwardsiellosis, furunculosis, streptococcosis and pasteurellosis (Song et al. 1982, Austin 1983, Larsen 1988, Smith 1988, Dec et al. 1990, Roogers & Xu 1992, Magariños et al. 1994, Quentel & Ogier de Baulny 1995, Toranzo et al. 1995, Romalde et al. 1996, Gravningen et al. 1998. In particular, the success achieved in immunization against other vibrioses (Smith 1988) suggests that the best solution would be an effective vaccine against V. vulnificus. Although numerous vaccines against other vibrioses have been developed and licensed to date, no vaccine against V. vulnificus serovar E is available at present. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulatio...

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“…Dot-blot assay. Dot-blot analysis was performed using thermolabile and thermostable antigens as previously described (Cipriano et al, 1985). Briefly, 1 ml volumes of antigens were dotted onto cellulose membranes (0.5 mm; Bio-Rad) and blocked for 1 h with 3 % (w/v) skimmed milk in Tris-buffered saline (TBS) (50 mM Tris/HCl, pH 7.4).…”

Section: Serological Testingmentioning

confidence: 99%

Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality

et al. 2007

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Vibrio vulnificus biotype 3 has been implicated as the causative pathogen of an ongoing disease outbreak that erupted in Israel in 1996. Recent work based on multi-locus sequence typing (MLST) showed that V. vulnificus biotype 3 is genetically homogeneous. The aim of this study was to investigate the existence of subpopulations within this homogeneous biotype by characterizing the surface antigens and analysing the sequence diversity of selected outer-membrane protein (OMP)-encoding genes. Rabbit antisera were prepared against biotype 1, 2 and 3 strains. The results of the slide-agglutination test, dot-blot assay (using fresh and boiled cells), and immunoblotting of lipopolysaccharides (LPS) and OMPs were evaluated. By slide-agglutination and dot-blot assays all biotype 3 strains agglutinated with the selected biotype 3 strain. This homogeneity was supported by immunoblot analysis of the LPS. Analysis of OMP patterns revealed that all three biotypes share a considerable number of common bands that are antigenically related. Cluster analysis of DNA sequence data from selected OMP-encoding genes showed that biotype 3 strains form a genetically distinct and homogeneous clone. The homogeneity of surface antigens and the lack of any sequence diversity among both housekeeping and OMP-encoding genes reaffirms the highly clonal nature of biotype 3 and suggests that it has only recently descended from the parent population of V. vulnificus.

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Detection of Vibrio Anguillarum Antigen by the Dot Blot Assay

Abstract: ABSTRACT:The

Cited by 39 publications

References 0 publications

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality

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