Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA)

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune y-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clin~call diseased fish within 30 minutes of preparing a kidney sam le, and thus because of its ease of use, ti; ELISA could be employed as a rapid field test for bacterial Ldney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish. Zusammenfassung Identifizierung von Renibacterium salmoninarum mit Hive des ELISA-TestsZur Identifizierung des Renibacterium salmoninarum, dem Erreger der bakteriellen Nierenkrankheit (BKD), wurde ein ELISA-Test entwickelt, der sich als Schnelltest unter Feldbedingungen bewahrt hat. Das in diesem Test verwendete Immun y-globulin ist an kreuzreagierenden Bakterien adsorbiert und mit alkalischer Phosphatase konjugiert worden. Mit diesem Reagenz gelang es bei klinisch an der BKD erkrankten Fischen Renibacterium salmoninarum in der Niere innerhalb von 30 Minuten nachzuweisen. Bei Carriern erwies sich dagegen die Erregeranzucht als aussagekraftiger. Resume Detection de Renibacterium salmoninarum i l'aide du test ELISAUn test (ELISA) a it6 develo p i our dktecter et identifier le Renibacterium salmoninarum. L'immunoglobuline y utilisie Bans test a CtC absorbee sur deux espkces de bactiries contreriagissantes dans le but de creer un systkme de test s Ccifique. Renibacterium salmoninarum a pu ktre dkcouvert en I'espace de 30 minutes dans un khantieon pris sur le rein chez des poissons portant des sym tomes cliniques et, en raison de sa facilitk d'emploi, le test ELISA a pu ktre employe comme test rapiie pour la maladie bacterielle du rein (BKD), bien que I'isolation de R. salmoninarum ait 6t6 plus sensible que le test ELISA pour la detection du poisson porteur.

Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

“…The FAT relies on observation of R. salmoninarum stained by fluorescein-labelled specific immunoglobulin, whereas the ELISA uses irnmunoglobulin to quantify the levels of the p57 protein (Rockey et al 1991) or components of the ECP fraction , Meyers et al 1993). The ELISA is considered more sensitive than bacteriological culture or the FAT for detecting R. salmoninarum (Dixon 1987, Gudmundsd6ttir et al 1993, Jansson et al 1996 and is more likely to identify fish with asymptomatic infections than the FAT (Meyers et al 1993).…”

Comparison of serological techniques for the identification of Renibacterium salmoninarum

“…132 Antisera The y-globulin fraction of an antiserum against R. salmoninarum, absorbed with Rothia dentocariosa and Bacillus sphae~icus as previously described (DIXON 1987), was used in all serological tests. An antiserum prepared against Aeromonas salmonicida for use as a negative control in agglutination tests was provided by C. Rodgers of this laboratory.…”

“…To circumvent the need to culture R. salmoninarum, serological techniques such as immunodiffusion (ID) (CHEN et al 1974;KIMURA et al 1978), the fluorescent antibody test (FAT) (BULLOCK and STUCKEY 1975;BULLOCK et a1.1980), staphylococcal co-agglutination (CoA) , counterimmunoelectrophoresis (CIE) (CIPRIANO et al 1985) and more recently the enzyme-linked immunosorbent assay (ELISA) (DIXON 1987) have been used to detect the organism directly in fish samples. However, culture of the bacterium, especially for the detection of the very low levels of bacteria present in some sub-clinical carrier fish, may be more sensitive than serological methods, and it has been recommended that culturing R .…”

“…However, culture of the bacterium, especially for the detection of the very low levels of bacteria present in some sub-clinical carrier fish, may be more sensitive than serological methods, and it has been recommended that culturing R . salmoninarum should be used in conjunction with another confirmatory test to provide a definitive diagnosis of BKD (FRYER and SANDERS 1981;AUSTIN and RAY-MENT 1985;CIPRIANO et al 1985;DIXON 1987). Thus, there are still circumstances where the protracted diagnostic method of culture of the bacterium and its subsequent identification is advisable.…”

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1988 Final Report.

“…The bacterium is dependent on cysteine for its growth which can survive and multiply intracellularly within phagocytic cells (Young and Chapman, 1979;. A number of methods have been developed to detect the organism in diseased fish, they include: the use of selective medium for solation , fluorescent antibody techniques (Bullock and Stuckey, 1975;Laidler, 1980;Elliott and Barila, 1987), counter immunoelectrophoresis (Groman, 1975), co-agglutination (Kimura and Yoshimizu, 1981), enzyme linked immunosorbent assays (ELISAs) Dixon, 1987; and Western blot analysis .…”