Development of a Quantitative Real-Time PCR (qPCR) Assay for the Beta-Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus namaycush) During an Epitheliocystis-Associated Mortality Event
Epitheliocystis is a multi-etiologic gill condition in which a single cell is expanded by an intracytoplasmic inclusion filled with gram negative bacteria. My research focused on characterizing one purported agent, Blue Jay Creek Burkholderia (BK-BJC), and its relationship to epitheliocystis-associated annual mortality events and histopathologic gill lesions in economically valuable Ontario reared lake trout. Based on the 1503 bp 16S rRNA gene sequence of BK-BJC identified by Contador et al. (2016), two primer sets were developed for detection of this bacterium. From the first primer set (BKBJCV8F/R) with hydrolysis probe, a qPCR assay was developed and used to quantify BK-BJC amounts in the gills of Blue Jay Creek Fish Culture Station lake trout and tank water during an epitheliocystis-associated mortality event in 2013 and a non-outbreak winter in 2015. A second primer set (BKBJCV3) was created to differentiate BK-BJC from ‘Candidatus Branchiomonas cysticola’. In addition, BK-BJC-infected gills were streaked onto a variety of agar plates. Colony growth was assessed by Gram stain, MALDI-TOF mass spectrometry, and qPCR. The BKBJCV8 was found to be non-specific in silico and experimentally, potentially amplifying at least 33 non-target bacteria from GenBank. Conventional PCR with the BKBJCV3 confirmed that all BKBJCV8 qPCR positive samples were positive for BK-BJC. BK-BJC was present in 1 % of the samples collected from fish in 2015. There was a significant direct relationship (p=0.035) between BK-BJC and BK-BJC-like, non-target bacterial loads and mortality rates, and there was an observable direct relationship with interlamellar hyperplasia and single cell necrosis of the gills. No relationship was discovered between epitheliocystis inclusions and bacteria loads in either 2013 or 2015 lake trout. Further, BK-BJC or BK-BJC-like bacteria were not found in the water samples, nor did they grow on any culture medium. In conclusion, we developed two PCR assays and confirmed that BK-BJC was present in all lake trout from an EP outbreak at Blue Jay Creek in 2013. Future research should focus on the development of a more sensitive and specific, qPCR assay for the rapid diagnosis of BK-BJC and further investigations into the ability of BK-BJC to cause EP.