No abstract

A panel of 28 monoclonal antibodies against Piscirickettsia salmonis was produced using a puri-®ed fraction of the bacterium. To determine their speci®city to the pathogen, the antibodies were assayed by ELISA and indirect immuno¯uorescence microscopy. Six monoclonal antibodies were selected based on their strong reaction against P. salmonis and absence of cross-reactivity with other common ®sh pathogens. Western blot analysis showed that the antibodies reacted to several antigens of P. salmonis. Immuno¯uorescence assays revealed that these antibodies reacted with the same speci®city to different isolates of P. salmonis obtained from the south of Chile. This panel of monoclonal antibodies represents an important tool to develop simple, rapid, sensitive and highly speci®c methods for the detection of the pathogen and diagnosis of the disease.

The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Piscirickettsia salmonis in fish tissue samples was developed. The test uses a combination of different monoclonal antibodies specific against P. salmonis in the capture step of the assay. The antibodies 7G4, 6E2 and 2C1 chosen for the capture step are bound to the solid support with an adhesive protein purified from a bivalve mollusc, resulting in a high yield of adsorption and binding stability. The monoclonal antibody 7G4, used as a second antibody, is conjugated to horseradish peroxidase. The resulting ELISA test detected 7 different isolates of P. salmonis and does not cross-react with several other fish pathogens, revealing a high specificity and sensitivity. The test also detects P. salmonis in kidney tissue of infected coho salmon with 98% correlation with the immunofluorescence assay. KEY WORDS: ELISA · Salmon diseases · Piscirickettsia salmonis · Piscirickettsiosis · Monoclonal antibodies Resale or republication not permitted without written consent of the publisherDis Aquat Org 49: [33][34][35][36][37][38] 2002 reports describing detection of P. salmonis with immunofluorescence assays using polyclonal antibodies conjugated with fluorescein isothiocyanate , Barnes et al. 1998. Recently, we have developed a panel of monoclonal antibodies with high sensitivity and specificity like those tested in an immunofluorescence assay (Jamett et al. 2001). These monoclonal antibodies react with several isolates of P. salmonis, and the coccoid immunofluorescent shapes suggest that the antibodies recognize membraneassociated antigens (Jamett et al. 2001). This report describes the development of a highly specific sandwich enzyme-linked immunosorbent assay (ELISA), based on the use of monoclonal antibodies in the capture step of the assay and as a secondary antibody conjugated with horseradish peroxidase. MATERIALS AND METHODSIn addition to Piscirickettsia salmonis LF-89 (ATCC VR 1361), several other isolates of P. salmonis obtained from different places in the south of Chile were used. The names of these isolates and their origins are as follows: (1) an isolate from the kidney of steelhead trout from Calbuco, Puerto Montt, obtained by Fundación Chile and named P. salmonis-Fundación Chile; and (2) isolates from the kidney of coho salmon from Pargua and Ralún, Puerto Montt, obtained by Aquatic Health Laboratories and referred to as P. salmonis-Aquatic Health, Ralún 14 and Ralún 17, 052 and L047. A bank of support inocula was prepared from the P. salmonis isolates by resuspending infected CHSE-214 lysates in a mixture of 10% dimethylsulfoxide in fetal calf serum and storing them in liquid nitrogen.To determine the specificity of the ELISA assay, several bacteria maintained and grown as described previously (Jamett et al. 2001) were used as antigens (see Table 1).Growth and purification of Piscirickettsia salmonis. P. salmonis LF-89 and other isolates listed above were continuously propagated in the chinook salmon embryo cell line CHSE-214 (CR...

Piscirickettsia salmonis is the etiologic agent of Salmonid Rickettsial Septicemia (SRS) a disease responsible for extensive mortalities in the Chilean salmon industry. This agent was identified for the first time in 1990 by Fryer (Fryer et al., 1990). The disease occurs mainly in coho salmon (Oncorhynchus kisutch). It was found later that this patogen affects other farmed salmon species (Gaggero, et al., 1995) including salmonids from other countries besides Chile (Brocklebank et al., 1993; Rodger and Drinan, 1993; Olsen et al., 1997). The pathogen is a Gram-negative obligate i n t r a c e l l u l a r b a c t e r i u m f o u n d i n cytoplasmic vacuoles of infected cells. The organism is pleomorphic, predominantly