Comparative evaluation of bacterial culture and two ELISA techniques for the detection of Renibacterium salmoninarum antigens in salmonid kidney tissues

Jansson, Eva; Hongslo, T.; Höglund, Johan; Ljungberg, O

doi:10.3354/dao027197

Cited by 33 publications

(29 citation statements)

References 23 publications

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Chase¹,

Pascho²

1998

Dis. Aquat. Org.

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Nucleic acid-based assays have shown promise for diagnosing Renibacter~um salmoninarum in tissues and body fluids of salmonids. Development of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. sahoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. sdmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specdlcity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positi.ve reactions in the enzymelinked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarurn. Kidney samples from 74 naturally infected c h o o k salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43 %, respectively.

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Section: Introductionmentioning

confidence: 99%

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

Chase¹,

Pascho²

1998

Dis. Aquat. Org.

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“…For example, localized infections can occur in the head and skin tissues, which are sites not usually assayed in screening programs (29). Several ELISAs have been described utilizing either polyclonal antibodies (21,24,27,30), monoclonal antibodies (33), or a combination of both types of antibodies (23). Assays relying on polyclonal antisera are limited by reagent availability, lot consistency, and cross-reactivity (29).…”

Section: Discussionmentioning

confidence: 99%

“…Assays relying on polyclonal antisera are limited by reagent availability, lot consistency, and cross-reactivity (29). A potential limitation of the monoclonal ELISA is a lower reported sensitivity than that of several polyclonal ELISAs (24,38). The epitope analysis described here has implications for incorporating additional MAbs to increase sensitivity.…”

Section: Discussionmentioning

confidence: 99%

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Wiens

Owen

2005

Appl Environ Microbiol

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Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.

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“…Soluble Rs antigens were detected by the ELISA performed as previously described (Jansson et al 1996). Kidney samples were prepared with 0.01 M phosphatebuffered saline pH 7.4 (PBS) and the solvent HemoDe (Fisher Scientific, USA) in a stomacher and heated to 98 + 2°C in an autoclave.…”

Section: Methodsmentioning

confidence: 99%

“…Immunological methods, such as immunofluorescence techniques, enzyme-linked irnmunosorbent assay (ELISA) and Western blot for detection of Rs antigens in internal organs, have been used as diagnostic tools (Bullock & Stuckey 1975, Pascho & Mulcahy 1987, Griffiths et al 1991, Hsu et al 1991, Gudmundsdottir et al 1993, Olea et al 1993, Jansson et al 1996. Methods based on nucleic acid techniques have been developed for the detection of Rs in kidneys (Mattsson et al 1993, Leon et al 1994, in the ovarian fluid (Magnusson et al 1994), and in salmonid eggs (Brown et al 1994).…”

Section: Introductionmentioning

confidence: 99%

Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations

Jansson¹,

Ljungberg²

1998

Dis. Aquat. Org.

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Humoral antibodies to heat-stable antigens of Renibacterium salmoninar~~m (Rs) were detected by enzyme-linked immunosorbent assay (ELIS,I) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion A slow antibody response was found: 3%(1/30) was positive 4 wk after immersion and 7 2 % (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to b e infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At G, 8 and 12 wk after immersion the proportion of positives indicated by ELlSA was 58Ytp. The Rs infection was detected by cultivation in 3 6 % of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salinon (59% In 1 farm) and from rainbow trout (20%) in 1 of 5 sampled farms) in naturally exposed populations all of which classified posltive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled expel-iments.

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Comparative evaluation of bacterial culture and two ELISA techniques for the detection of Renibacterium salmoninarum antigens in salmonid kidney tissues

Cited by 33 publications

References 23 publications

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

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

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations

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