Large-scale analysis of Flavobacterium psychrophilum MLST genotypes recovered from North American salmonids indicates both newly identified and recurrent clonal complexes are associated with disease

Authors: KNUPP, CHRISTOPHER - Michigan State University; Wiens, Gregory - Greg; FAISAL, MOHAMED - Michigan State University; CALL, DOUGLAS - Washington State University; CAIN, KENNETH - University Of Idaho; NICOLAS, PIERRE - French National Institute For Agricultural Research; VAN VLIET, DANIELLE - Michigan State University; YAMASHITA, COJA - Michigan State University; FERGUSON, JAYDE - Michigan State University; MEUNINCK, DAVE - Michigan State University; HSU, HUI-MIN - Michigan State University; BAKER, BRIDGET - Michigan State University; SHEN, LING - Michigan State University; LOCH, THOMAS - Michigan State University

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2019
Publication Date: 3/6/2019
Citation: Knupp, C., Wiens, G.D., Faisal, M., Call, D.R., Cain, K.D., Nicolas, P., Van Vliet, D., Yamashita, C., Ferguson, J.A., Meuninck, D., Hsu, H., Baker, B.B., Shen, L., Loch, T.P. 2019. Large-scale analysis of Flavobacterium psychrophilum MLST genotypes recovered from North American salmonids indicates both newly identified and recurrent clonal complexes are associated with disease. Applied and Environmental Microbiology. Mar 6;85(6)e02305-18.
DOI: https://doi.org/10.1128/AEM.02305-18

Interpretive Summary: A frequent cause of fish loss at salmonid aquaculture facilities is bacterial cold water disease (BCWD). This disease is caused by the microorganism Flavobacterium psychrophium. This study describes the analysis of 314 Flavobacterium psychrophium isolates obtained from ten fish species originating from twenty US states and one Canadian province. These isolates were collected over a nearly four decade period. To determine the relatedness of the isolates, we utilized multilocus sequence typing (MLST). MLST compares the DNA sequences of internal fragments of seven housekeeping genes amplified and sequenced from each isolate. Different sequences are assigned an allele number and the combination of alleles from an isolate allow assignment of a numerical sequence type (ST). In our study, we identified 66 different ST of which 47 are new. The most common type associated with disease was ST10, and combined with related variants, this strain type had the widest geographical distribution and infected the most different types of fish. We also found the same sequence type in both female broodstock and progeny suggesting that egg disinfection practices are not fully effective. This information will be used to more appropriately develop control measures to monitor and reduce the severity of BCWD.

Technical Abstract: Flavobacterium psychrophilum is the etiological agent of Bacterial Coldwater Disease (BCWD) and Rainbow Trout Fry Syndrome (RTFS), which cause significant economic losses in salmonid aquaculture, particularly to farm-raised rainbow trout (Oncorhynchus mykiss). The genetic heterogeneity of this fish pathogen has recently been explored by numerous investigators, whereby isolates from Europe, Asia, South America, Oceania, and most recently, North America, have been genotyped using multilocus sequencing typing (MLST). These analyses revealed North American F. psychrophilum isolates to be quite genetically heterogeneous; however, much remains to be elucidated in regards to the population structure of this bacterium in North America. In this study, MLST was used to genotype 314 North American F. psychrophilum isolates that were recovered from 10 fish host species from 20 US states and 1 Canadian province and were collected over nearly four decades. Using the phylogenetic program, eBURST, these isolates were placed into 66 sequence types (STs), 47 of which were novel. Furthermore, 7 novel North American CCs were discovered, which brings the total number of North American CCs to 12. Many of the identified CCs have only been detected in North America, whereas others were found in North America and abroad. These CCs were diverse and varied in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified in this study was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. ST275 of CC-ST10 was recovered from wild/feral adult steelhead and the recovery of CC-ST10 from feral/wild fish in North America has not been reported previously. Furthermore, the progeny of infected broodstock were found to harbor the same ST, suggesting some STs are capable of circumventing current egg disinfection practices. The diversification of F. psychrophilum in North America was found to be driven 4 times more frequently by recombination and may be an indication of how new phenotypes emerge within this pathogen.