Detecting Flavobacterial Fish Pathogens in the Environment via High-Throughput Community Analysis

Abstract

Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult-to-control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In this study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum over 5 orders of magnitude, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout (Oncorhynchus mykiss) aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence. IMPORTANCE Early detection of a deadly disease outbreak in a population can be the difference between mass mortality or mitigated effects. In the present study, we evaluated and compared two molecular techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two freshwater fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while the droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.

Keywords: 16S rRNA; Flavobacterium; aquaculture; detection; droplet digital PCR; metagenomics; pathogen.

FIG 1

16S rRNA gene cladogram for the genus Flavobacterium. Sequences of the 253-bp V4 region of the 16S rRNA gene from Flavobacterium type strains were aligned, and a phylogenetic tree was constructed using Fasttree. F. columnare (red) is 4 bp away from F. verecundum, and F. psychrophilum (blue) is 6 bp away from F. limicola.

FIG 2

Comparison of MiSeq and droplet digital PCR (ddPCR) assay results. Estimated cell counts of F. columnare (red) and F. psychrophilum (blue) are shown for each of the five 10-fold dilutions assayed. Counts are shown on the y axis as log values, while dilutions are shown on the x axis and increase from the lowest concentration sample (5) to the highest concentration sample (1). (A to C) Samples inoculated with F. columnare alone (A), F. psychrophilum alone (B), or F. columnare and F. psychrophilum combined (C). The coinoculated samples in panel C show F. columnare (Fc) counts with circles and F. psychrophilum (Fp) counts with triangles. Black circles indicate the total estimated bacteria within the sample calculated using quantitative PCR.

FIG 3

Class and genus level taxonomic barplots of water samples with spiked flavobacteria removed. (A) Class level taxonomic breakdown for each sample excluding reads from F. columnare and F. psychrophilum. (B) Genus level taxonomic breakdown for each sample excluding reads from F. columnare and F. psychrophilum. Labels on the y axis correspond to the dilution series. Barplots are presented as relative abundances. Fc, Fp, and FcFp indicate the addition of F. columnare alone, F. psychrophilum alone, or their combination, respectively. NA indicates that no assignment could be made at this taxonomic level. Uninoculated 1 and 2 are samples without inoculum added.