DAO 115:139-146 (2015)  -  DOI: https://doi.org/10.3354/dao02881

Comparison of quantitative PCR and ELISA for detection and quantification of Flavobacterium psychrophilum in salmonid broodstock

Amy Long1,3, Douglas R. Call2, Kenneth D. Cain1,*

1Department of Fish & Wildlife Sciences and the Aquaculture Research Institute, University of Idaho, PO Box 441136, Moscow, ID 83844-1136, USA
2Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164-7090, USA
3Present address: Oregon Department of Fish & Wildlife, Department of Microbiology, Nash Hall, Room 220, Oregon State University, Corvallis, OR 97331-3804, USA
*Corresponding author:

ABSTRACT: A quantitative PCR (qPCR) assay was developed for Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease. The assay was targeted to fp1493 as it encodes a putative outer membrane protein (FP1493) that is reactive to the monoclonal antibody (MAb FL43) used in a standardized F. psychrophilum capture enzyme-linked immunosorbent assay (ELISA). The qPCR was specific to F. psychrophilum and was able to detect between 8 and 809000 copies of fp1493. To determine if antigen level in the tissue was indicative of bacterial concentration, kidney samples from 108 steelhead Oncorhynchus mykiss and coho salmon O. kisutch female broodstock were screened by ELISA and qPCR. There was no correlation between ELISA optical density (OD) values and the number of F. psychrophilum cells g-1 of kidney tissue as estimated by qPCR (rS = 0.42; p > 0.05). The median number of F. psychrophilum cells in steelhead samples was 6.11 × 103 cells g-1 of tissue. For coho salmon samples, the median number of cells was 3.95 × 103 cells g-1 of tissue. Agreement between the 2 assays was less than 50%. As fp1493 is a single-copy gene and differential expression of FP1493 has been reported, we hypothesize that the discrepancy between the 2 assays is due to increased expression of FP1493 in the in vivo environment. Therefore, ELISA OD values most likely provide an indication of differential protein expression, while the qPCR assay estimates bacterial load in tissue.


KEY WORDS: Flavobacterium psychrophilum · Quantitative PCR · Kidney tissue · ELISA · FP1493


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Cite this article as: Long A, Call DR, Cain KD (2015) Comparison of quantitative PCR and ELISA for detection and quantification of Flavobacterium psychrophilum in salmonid broodstock. Dis Aquat Org 115:139-146. https://doi.org/10.3354/dao02881

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