Enzyme-linked Immunosorbent Assay for a Soluble Antigen of Renibacterium salmoninarum, the Causative Agent of Salmonid Bacterial Kidney Disease

1987 ◽  
Vol 44 (1) ◽  
pp. 183-191 ◽  
Author(s):  
R. J. Pascho ◽  
D. Mulcahy
Keyword(s):  
Immunosorbent Assay ◽  
Incubation Temperature ◽  
Tween 80 ◽  
Broth Culture ◽  
Tween 20 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Soluble Antigen ◽  
Renibacterium Salmoninarum ◽  
Bacterial Kidney Disease ◽  
Wash Buffer

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

scholarly journals Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

2006 ◽  
Vol 72 (4) ◽  
pp. 2672-2678 ◽  
Author(s):  
Alison M. Coady ◽  
Anthony L. Murray ◽  
Diane G. Elliott ◽  
Linda D. Rhodes
Keyword(s):  
Kidney Disease ◽  
Chinook Salmon ◽  
Oncorhynchus Tshawytscha ◽  
Soluble Antigen ◽  
Renibacterium Salmoninarum ◽  
Juvenile Chinook Salmon ◽  
Bacterial Kidney Disease ◽  
Important Virulence Factor ◽  
Chromosomal Loci

ABSTRACT Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.

Genetic effects of ELISA-based segregation for control of bacterial kidney disease in Chinook salmon (Oncorhynchus tshawytscha)

2006 ◽  
Vol 63 (12) ◽  
pp. 2793-2808 ◽  
Author(s):  
Jeffrey J Hard ◽  
Diane G Elliott ◽  
Ronald J Pascho ◽  
Dorothy M Chase ◽  
Linda K Park ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Chinook Salmon ◽  
Oncorhynchus Tshawytscha ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigen Level ◽  
Renibacterium Salmoninarum ◽  
Listonella Anguillarum ◽  
Bacterial Kidney Disease ◽  
Genetic And Phenotypic Correlations ◽  
High Heritability

We evaluated genetic variation in ability of Chinook salmon (Oncorhynchus tshawytscha) to resist two bacterial pathogens: Renibacterium salmoninarum, the agent of bacterial kidney disease (BKD), and Listonella anguillarum, an agent of vibriosis. After measuring R. salmoninarum antigen in 499 adults by enzyme-linked immunosorbent assay (ELISA), we mated each of 12 males with high or low antigen levels to two females with low to moderate levels and exposed subsets of their progeny to each pathogen separately. We found no correlation between R. salmoninarum antigen level in parents and survival of their progeny following pathogen exposure. We estimated high heritability for resistance to R. salmoninarum (survival h2 = 0.890 ± 0.256 (mean ± standard error)) independent of parental antigen level, but low heritability for resistance to L. anguillarum (h2 = 0.128 ± 0.078). The genetic correlation between these survivals (rA = –0.204 ± 0.309) was near zero. The genetic and phenotypic correlations between survival and antigen levels among surviving progeny exposed to R. salmoninarum were both negative (rA = –0.716 ± 0.140; rP = –0.378 ± 0.041), indicating that variation in antigen level is linked to survival. These results suggest that selective culling of female broodstock with high antigen titers, which is effective in controlling BKD in salmon hatcheries, will not affect resistance of their progeny.

scholarly journals 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

1998 ◽  
Vol 10 (1) ◽  
pp. 60-66 ◽  
Author(s):  
Ronald J. Pascho ◽  
Dorothy Chase ◽  
Connie L. McKibben
Keyword(s):  
Polymerase Chain Reaction ◽  
Immunosorbent Assay ◽  
Membrane Filtration ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ovarian Fluid ◽  
Chain Reaction ◽  
Renibacterium Salmoninarum ◽  
Polymerase Chain

Ovarian fluid samples from naturally infected chinook salmon ( Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

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