Bacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR)

Abstract

NOTEBacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR) ABSTRACT: Genomc DNA was extracted from 4 strains of Carnobacterium p~scicola and 2 strains of Corynebacterium aquaticum that had previously been reported to produce a 57 kDa protein that reacted with polyclonal antiserum against Renibacterium salmoninarum. Genomic DNA was also extracted from a Gram-negative bacterium isolated from the ludney tissue of a mature female coho salmon Oncorhynchus kisutch. The bacterium, tentatively identified as Pseudomonas maltophila, cross-reacts with 2 polyclonal antisera, one of which is used in an enzyme-linked immunosorbent assay and the other in a fluorescent antibody test to identlfy R. sal~noninarum The Isolate of P. maltophila, and the Carnobacterium piscicola and Corynebacterium aquahcum strains, were negative by a polymerase chain reaction (PCR) that was designed to amplify a segment of the gene encoding p57, a major protein of R. salmon~narum. These results suggest that although antibodies directed against R. salmoninarum crossreact with antigens of bacterial species other than R. salmoninarum, the cross-reacting antigen(s) is clearly not the same protein, as the non-R. salmoninarum bacteria lacked the gene encoding p57. These findings highlight some of the shortcomings of immunodiagnostic tests for detecting R. salmoninarum and indicate the high degree of specificity associated with a PCR-based diagnostic technique.

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 criteria by which we selected bacteria to confirm the specificity of the primers used in the nested PCR were that a given bacterium must either be a common bacterial pathogen of salmonids, or a species reported previously to produce false-positive reactions in the ELISA. Our results support those of others (Mattsson et al 1993, Brown et al 1995, Hariharan et al 1995) that nucleic acid-based assays may be used to accurately diagnose R. salmoninarum if one selects primers that amplify a DNA sequence that is unique to the kidney disease bacterium. Primer specificity, however, is only one factor in avoiding false-positive reactions in a nested PCR.…”

“…The accuracies of serologically based diagnostic methods for R. salmoninai-um, such as the ELISA and the FAT, have been under question because of reports of cross-reacting bacteria (Austin et al 1985, Dixon 1985, Bandin et al 1993, Brown et al 1995, Wood et al 1995. The criteria by which we selected bacteria to confirm the specificity of the primers used in the nested PCR were that a given bacterium must either be a common bacterial pathogen of salmonids, or a species reported previously to produce false-positive reactions in the ELISA.…”

A Single Ala139-to-Glu Substitution in theRenibacterium salmoninarumVirulence-Associated Protein p57 Results in Antigenic Variation and Is Associated with Enhanced p57 Binding to Chinook Salmon Leukocytes

“…Although the role of p57 in pathogenesis is unclear, this protein is a well-accepted marker of infection by R. salmoninarum (6,11,13,34,37,41). The correlation of cell-associated p57 with isolate virulence, the high levels of synthesis by R. salmoninarum, the in vitro binding activity with fish leukocytes, and the maintenance of duplicated msa genes support the hypothesis that p57 plays an important role in the pathogenesis of bacterial kidney disease.…”

“…Diagnosis of R. salmoninarum infection in salmon is often based on detection of the 57-kDa (p57) protein, mRNA, or DNA (6,11,13,34,37,41). p57 is a good marker of active infection as this protein is the predominant cell surface and secreted protein produced by R. salmoninarum (23,26,51).…”

Comparison of the Membrane-Filtration Fluorescent Antibody Test, the Enzyme-Linked Immunosorbent Assay, and the Polymerase Chain Reaction to Detect Renibacterium Salmoninarum in Salmonid Ovarian Fluid

“…Antisera against R. salmoninarum can cross-react with other bacterial species, resulting in false-positive reactions. 1,5,43 The same polyclonal antibody preparation was used in the MF-FAT and the ELISA, although the tests differ because the MF-FAT detects antigens of R. salmoninarum in association with the bacterial cell, whereas the ELISA detects an extracellular antigenic fraction. The sensitivity of the MF-FAT, about 25 bacteria/ml or the observation of only 1 bacterium in 150 microscope fields, means that careful attention is necessary to avoid cross-contamination of the ovarian fluid samples.…”