Large-Scale Analysis of Flavobacterium psychrophilum Multilocus Sequence Typing Genotypes Recovered from North American Salmonids Indicates that both Newly Identified and Recurrent Clonal Complexes Are Associated with Disease

Abstract

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

Keywords: Flavobacteriaceae; Flavobacterium psychrophilum; MLST; bacterial coldwater disease; fish disease; genetic diversity; rainbow trout fry syndrome; recombination.

FIG 1

eBURST diagram of the 314 F. psychrophilum isolates genotyped in this study combined with all previously typed North American isolates (n = 107) in the F. psychrophilum MLST database. †, a sequence type (ST) that contains isolates typed in this study; ‡, an ST containing previously genotyped isolates. Italicized numbers accompanying STs indicate the number of states that ST was found in (if no number, ST was recovered from one state only). Clonal complexes (CC) are encircled and the founding ST for a CC is depicted as a number within a rectangle. Founding STs labeled with a star (★) indicate a newly described CC as a result of this study. The founding ST, or founder, is the ST with the highest number of single-locus variants (SLVs). In instances where multiple STs have the same numbers of SLVs, the founder is named after the ST with the most isolates. If both STs have the same numbers of isolates, the CC is named for the earliest found ST.

FIG 2

eBURST diagram of all 1,411 F. psychrophilum isolates worldwide. This includes the 1,097 isolates from the F. psychrophilum MLST database and the 314 newly typed isolates from this study. †, sequence type (ST) is present in North America; ★, ST was found in North America and abroad. The founding ST, or founder, is the ST with the highest number of single-locus variants (SLVs; depicted as gray circles). In instances where multiple STs have the same numbers of SLVs, the founder is named after the ST with the most isolates. If both STs have the same numbers of isolates, the clonal complex (CC) is named for the earliest found ST. The founding ST of a CC is enclosed within a box. The size of each circle is proportional to how many isolates belong to that ST.

FIG 3

Frequency and host of origin among 10 North American F. psychrophilum sequence types (STs). STs are displayed along the x axis with four clonal complexes (CCs) and two singleton STs shown. All 421 currently typed North American F. psychrophilum isolates are presented here.

FIG 4

Map of the United States and Canada. Distribution of 314 F. psychrophilum isolates recovered from the current North American study. These isolates were recovered from 20 U.S. states and 1 province of Canada. AK, Alaska; BC, British Columbia; CA, California; CO, Colorado; ID, Idaho; IN, Indiana; MD, Maryland; MI, Michigan; MN, Minnesota; MT, Montana; NC, North Carolina; NM, New Mexico; OR, Oregon, PA, Pennsylvania; SD, South Dakota; UT, Utah; VA, Virginia; VT, Vermont; WA, Washington; WI, Wisconsin; WV, West Virginia.