Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids.

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.

Comparative determination of paralytic shellfish toxins (PSTs) using five different toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska.

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.

Prevalence and analysis of Renibacterium salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha in North Puget Sound.

Renibacterium salmoninarum causes bacterial kidney disease (BKD), a chronic and sometimes fatal disease of salmon and trout that could lower fitness in populations with high prevalences of infection. Prevalence of R. salmoninarum infection among juvenile Chinook salmon Oncorhynchus tshawytscha inhabiting neritic marine habitats in North Puget Sound, Washington, USA, was assessed in 2002 and 2003. Fish were collected by monthly surface trawl at 32 sites within 4 bays, and kidney infections were detected by a quantitative fluorescent antibody technique (qFAT). The sensitivity of the qFAT was within an order of magnitude of the quantitative real-time PCR (qPCR) sensitivity. Prevalence of infection was classified by fish origin (marked/hatchery vs. unmarked/likely natural spawn), month of capture, capture location and stock origin. The highest percentages of infected fish (63.5 to 63.8%) and the greatest infection severity were observed for fish collected in Bellingham Bay. The lowest percentages were found in Skagit Bay (11.4 to 13.5%); however, there was no difference in prevalence between marked and unmarked fish among the capture locations. The optimal logistic regression model of infection probabilities identified the capture location of Bellingham Bay as the strongest effect, and analysis of coded wire tagged (CWT) fish revealed that prevalence of infection was associated with the capture location and not with the originating stock. These results suggest that infections can occur during the early marine life stages of Chinook salmon that may be due to common reservoirs of infection or horizontal transmission among fish stocks.