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Frontiers in Aquaculture ·

Volume 5 - 2026 | https://doi.org/10.3389/faquc.2026.1805926

Large-scale molecular serogroup analysis of Flavobacterium psychrophilum isolates recovered from captive, wild, and feral fishes in the USA

  • Sean M. G. Lennox1,2
  • Christopher Knupp1,2
  • Esteban Soto3
  • Douglas R. Call4
  • Pierre Nicolas5
  • Thomas P. Loch1,2,6*corresponding author
  1. 1Aquatic Animal Health Laboratory, Michigan State University, East Lansing, MI, United States
  2. 2Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, United States
  3. 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, Davis, CA, United States
  4. 4Paul G. Allen School for Global Health, Washington State University, Pullman, WA, United States
  5. 5Institut National de Recherche pour L'agriculture, L'alimentation et L'environnement (INRAE), Université Paris-Saclay, Mathématiques et Informatique Appliquées du Génome à l’Environnement, Jouy-en-Josas, France
  6. 6Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States
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Abstract

Flavobacterium psychrophilum, causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), is one of the most problematic bacterial pathogens affecting captive-reared salmonids (Family Salmonidae) worldwide. Considerable efforts have been undertaken to characterize the genetic diversity of F. psychrophilum using multilocus sequence typing (MLST) in the United States and elsewhere. In comparison, few studies have sought to characterize the serovariation of F. psychrophilum in the USA. To this end, 320 F. psychrophilum isolates, originating from 17 US states that were recovered from 12 fish species and 1 hybrid from 1981 to 2021, were analyzed using a multiplex polymerase chain reaction molecular serogrouping scheme. Results revealed substantial F. psychrophilum serovariation, with identification of all currently recognized molecular serogroups (i.e., Serogroups -0, -1, -2, -3, and -4). Significant associations between F. psychrophilum molecular serogroup and host fish species were identified: positive associations included rainbow/steelhead trout (Oncorhynchus mykiss) and Serogroup-2, and coho salmon (O. kisutch) and Serogroup-0; negative associations included rainbow/steelhead trout and Serogroup-0 as well as coho salmon and Serogroups-1 and -2. Some regional variation in the distribution of F. psychrophilum molecular serogroups was also apparent. Pairing MLST and molecular serogroup data uncovered further evidence of F. psychrophilum vertical transmission occurring in hatchery environments. Collectively, these findings provide a foundation for future F. psychrophilum vaccine research and further clarify the epidemiology of this problematic bacterium.

Keywords

1 Introduction

Flavobacterium psychrophilum (Family Flavobacteriaceae; Bernardet et al., 1996), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), is one of the most widespread and difficult to control bacterial pathogens affecting captive-reared salmonids (Family Salmonidae; Starliper, 2011; Loch and Faisal, 2017). Although most salmonid species are considered susceptible, rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), and Atlantic salmon (Salmo salar) are most affected (Holt, 1987; Nematollahi et al., 2003; Barnes and Brown, 2011; Fredriksen et al., 2016; Bruce et al., 2021). Chinook salmon (O. tshawytscha; Van Vliet et al., 2015; Knupp et al., 2019) and brown trout (S. trutta; Lorenzen et al., 1997; Knupp et al., 2019), amongst other salmonids, also experience BCWD outbreaks. In addition, a range of non-salmonid species are affected by F. psychrophilum, most notably ayu (Plecoglossus altivelis; Wakabayashi et al., 1994). Although several antibacterial treatment options exist to control active disease outbreaks (US Food and Drug Administration, 2025), F. psychrophilum continues to cause substantial losses in captive-reared fish.

Persistent disease-associated losses in salmonids has driven efforts to resolve the genetic diversity and population structure of F. psychrophilum. A multilocus sequence typing (MLST) scheme devised by Nicolas et al. (2008) has since been applied to >1550 F. psychrophilum isolates originating from 5 continents (i.e., Asia, Australia, Europe, North America, and South America), revealing >260 sequence types (STs; https://pubmlst.org/fpsychrophilum). Consequently, the observed genetic diversity has provided a framework for better understanding host associations (Soule et al., 2005a; Soule et al., 2005b; Nicolas et al., 2008; Fujiwara-Nagata et al., 2013; Nilsen et al., 2014; Avendaño-Herrera et al., 2014; Van Vliet et al., 2016; Knupp et al., 2021a; Li et al., 2021; Knupp and Loch, 2023), virulence (Nilsen et al., 2014; Van Vliet et al., 2016; Knupp et al., 2021b; Knupp and Loch, 2023; Fujiwara-Nagata et al., 2024), and environmental persistence (Knupp et al., 2024). Likewise, MLST-based population-structure reconstructions show that some STs/clonal complexes (CCs; e.g., CC-ST10, CC-ST124) are distributed across continents (Siekoula-Nguedia et al., 2012; Strepparava et al., 2013; Nilsen et al., 2014; Van Vliet et al., 2016; Knupp et al., 2019; Sebastião et al., 2020; Li et al., 2021), whereas others are geographically restricted to single continents, countries, or states (Nilsen et al., 2014; Van Vliet et al., 2016; Knupp et al., 2019; Sebastião et al., 2020).

In contrast to the better-defined genotypic landscape of F. psychrophilum, the serotypic diversity of F. psychrophilum, particularly in the USA, remains less resolved; among other factors, a lack of standardized methods and serological reagents may have contributed to this incongruence. Pacha (1968) analyzed 10 F. psychrophilum isolates from the states of Washington and Oregon (USA) with slide agglutination using rabbit antisera prepared against 3 previously recovered F. psychrophilum isolates. The author noted agglutination for all 10 isolates with each of the 3 antisera preparations, suggesting serotypic similarity. Holt (1972) later analyzed six F. psychrophilum isolates from the USA (n=5 from Oregon; n=1 from New Hampshire) by comparing agglutination titers following exposure to antisera prepared against each isolate. Some degree of serotypic commonality was observed, though some differences between the Oregon and New Hampshire isolates were noted (Holt, 1972). Holt (1987), using similar methods, analyzed six F. psychrophilum isolates from five US states (i.e., Alaska, New Hampshire, Michigan, Oregon, and Washington) and British Columbia (Canada), and found that the Oregon, Washington, and British Columbia isolates appeared serotypically similar; however, differences were noted between those and the remaining isolates – this provided some of the earliest evidence of F. psychrophilum serotypic heterogeneity.

Although some antigenic characterization has since been undertaken on F. psychrophilum originating from the USA (Crump et al., 2001; MacLean et al., 2001; LaFrentz et al., 2004, LaFrentz et al., 2007), these studies were mostly in the context of taxonomic differentiation or vaccine development. Dedicated studies on F. psychrophilum serotypic characterization since the work of Holt (1987) have primarily focused on isolates recovered from other regions in the world. Indeed, in-depth serotypic studies have been conducted for F. psychrophilum recovered from Asia, Europe, and South America via the microtiter method, slide agglutination, and enzyme-linked immunosorbent assay (ELISA), describing 3 – 16 F. psychrophilum serotypes (Wakabayashi et al., 1994; Lorenzen and Olesen, 1997; Izumi and Wakabayashi, 1999; Madetoja et al., 2001; Mata et al., 2002; Izumi et al., 2003; Valdebenito and Avendaño‐Herrera, 2009; Avendaño‐Herrera et al., 2020; Ilardi et al., 2023). Lorenzen and Olesen (1997) identified three main F. psychrophilum serotypes (i.e., Th, Fd, and FpT), and two subtypes within Th (i.e., Th-1 and Th-2), though cross-reactivity was observed for some isolates and others were unable to be characterized with this scheme. The serotyping approach of Wakabayashi et al. (1994), after improvements made by Izumi and Wakabayashi (1999); Izumi et al. (2003), identifies four main F. psychrophilum serotypes (i.e., O-1, O-2, O-3, and O-4), with some isolates displaying cross-reactivity. To some extent, these serotyping schemes correlate with one another, for example, O-1 correlates to FpT, O-3 correlates to Th (Th-1), and O-4 correlates to Fd (Izumi et al., 2003).

Flavobacterium psychrophilum is clearly serotypically diverse, but the disparate methods of serocharacterization has made some comparisons across laboratories and regions of the world less certain. To this end, Rochat et al. (2017) performed a comparative genomic analysis of 34 F. psychrophilum isolates and identified 4 main groups based on the differing arrangements of genes within the predicted O-polysaccharide biosynthesis loci (i.e., an important antigenic component of the cell surface lipopolysaccharide of F. psychrophilum; MacLean et al., 2001). The authors subsequently developed a multiplex polymerase chain reaction (mPCR) assay capable of identifying these “molecular serogroups,” including Type-0, Type-1, Type-2, and Type-3, with Type-0 encompassing isolates that did not belong to any of the other Types. Generally, the newly identified molecular serogroups aligned with classically identified serotypes (e.g., Type-0, O-1, and FpT; Type-1, O-4, FpT, and Fd; Type-2, O-3, and Th; Type-3, O-2, and FpT; Rochat et al., 2017). In 2020, Avendaño‐Herrera et al. (2020) described molecular serogroup Type-4. To date, this molecular serogrouping assay has been applied to at least 669 isolates from 22 countries (Rochat et al., 2017; Avendaño‐Herrera et al., 2020; Ilardi et al., 2023; Vaibarova et al., 2024; Calvez et al., 2021; Saticioglu et al., 2018; Li et al., 2021; Sundell et al., 2019; Park et al., 2023; Fujiwara-Nagata et al., 2024), resulting in the discovery of several apparent host associations – specifically, Type-0 with coho salmon, Type-1 and Type-2 with rainbow trout, and Type-3 with ayu (Rochat et al., 2017). However, only 23 of these isolates originated in the USA (Rochat et al., 2017; Saticioglu et al., 2018; Knupp et al., 2021a; Knupp and Loch, 2023), leaving a significant knowledge gap with direct implications for epidemiological surveillance, vaccine development, and cross-protection studies.

Herein and to characterize the serovariation among F. psychrophilum isolates recovered from the USA and inform development of effective BCWD/RTFS prevention measures, the mPCR serogrouping scheme of Rochat et al. (2017) and Avendaño‐Herrera et al. (2020) was used to characterize 320 F. psychrophilum isolates recovered from 12 fish species and 1 hybrid across 17 US states (1981 - 2021). Associations between molecular serogroup, host species, geographic origin, and MLST genotype were then assessed.

2 Materials and methods

2.1 Bacterial isolates

Flavobacterium psychrophilum isolates that were previously typed via MLST (n=46, Van Vliet et al., 2016; n=124, Knupp et al., 2019; n=43, Sebastião et al., 2020; n=36, Li et al., 2021; n=3, Harrison et al., 2022) were analyzed in this study, as well as 68 previously unpublished isolates (Supplementary Table 1). Collectively, the 320 isolates originated from 17 US states and were recovered from fish within 5 genera (i.e., Oncorhynchus, Salmo, Salvelinus, Coregonus, and Acipenser) encompassing 12 fish species and 1 hybrid, including Apache trout (O. apache; n=2), Chinook salmon (n=42), coho salmon (n=49), cutthroat trout (O. clarkii; n=1), steelhead (anadromous O. mykiss) and rainbow trout (n=170), sockeye salmon (O. nerka; n=1), Atlantic salmon (n=21), brown trout (n=23), brook trout (Salvelinus fontinalis; n=2), lake trout (Salvelinus namaycush; n=3), splake (Salvelinus namaycush x Salvelinus fontinalis; n=1), lake whitefish (Coregonus clupeaformis; n=3), and white sturgeon (Acipenser transmontanus; n=1), or were of unknown origin (n=1). These isolates were recovered during active disease outbreaks or routine surveillance between the years of 1981 and 2021. All isolates were confirmed as F. psychrophilum via the endpoint PCR assay of Toyama et al. (1994), following the methods in Van Vliet et al. (2015), and maintained as cryo-stocks at -80°C in tryptone yeast extract salts (TYES; Holt, 1987) broth supplemented with 20% glycerol. Isolates were revived onto TYES agar, incubated for up to 120 hours at 15°C, checked intermittently for growth, verified visually for purity, and then utilized for nucleic acid extraction.

2.2 Nucleic acid extraction

Genomic DNA was extracted from the 320 isolates using a DNeasy® Blood and Tissue Kit (QIAGEN LLC, Germantown, MD) following the manufacturer’s protocols for Gram-negative bacteria, except that bacteria were inoculated directly into the Buffer ATL + Proteinase K solution and centrifuged at 17,200 × g after addition of Buffer AW2. In the final elution step, 50 µL of Buffer AE was passed through the DNeasy membrane twice. Following extraction, DNA was quantified using an Invitrogen™ Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA) on an Invitrogen™ Qubit™ Fluorometer and then diluted to a standard concentration of 20 ng/μL using nuclease-free H2O and stored at -20°C until further use.

2.3 Multilocus sequence typing

For MLST analyses, partial sequences of seven F. psychrophilum housekeeping genes (trpB, gyrB, dnaK, fumC, murG, tuf, and atpA; Nicolas et al., 2008) were amplified via endpoint PCR (Van Vliet et al., 2016) and purified using ExoSAP-IT (Applied Biosystems; Knupp et al., 2019). Amplicons were then bidirectionally sequenced at the Michigan State University - Research Technology Support Facility using a sequencing primer pair described by Fujiwara-Nagata et al. (2013). The quality of resulting chromatograms was verified using an in-house script as described in Van Vliet et al. (2016) before allele and ST assignment. The software goeBURST (www.phyloviz.net/goeburst/; Francisco et al., 2009) was used to infer phylogenetic relationships of F. psychrophilum isolates as they compared to isolates within the pubMLST database and to group isolates into CCs or singletons based on variations within their allelic profiles (Feil et al., 2004).

2.4 Assignment of F. psychrophilum molecular serogroup via mPCR

Rochat et al. (2017) originally referred to the serovariation distinguished by their mPCR scheme as “mPCR serotypes,” or simply “Types.” These Types capture some of the major known serovariation that occurs within F. psychrophilum based on the varying genomic organization of polysaccharide encoding loci, but they do not capture some additional variation that is indicative of the at least 20 known O-polysaccharide genetic types or “O-serotypes” noted by Cisar et al. (2019), Cisar et al. (2024) through comparison of 379 F. psychrophilum genomes available in GenBank. Rochat et al. (2017) acknowledged this additional serovariation and originally identified 15 of these O-serotypes, which are presented as they relate to their overarching Types therein. For these reasons and herein, mPCR serotypes or Types are referred to as “molecular serogroups” (i.e., Type-0 as molecular Serogroup-0, Type-1 as molecular Serogroup-1, Type-2 as molecular Serogroup-2, Type-3 as molecular Serogroup-3, and Type-4 as molecular Serogroup-4).

Flavobacterium psychrophilum isolates were assigned to a molecular serogroup using the mPCR scheme of Rochat et al. (2017) and Avendaño‐Herrera et al. (2020). Each mPCR assay was performed in a 25 μL reaction volume consisting of 1X GoTaq™ green master mix (Promega, Madison, WI), 0.1 μM of each control primer, 0.5 μM each of the Type-1, Type-2, Type-3, and Type-4 primers, and 2 μL of template DNA (Rochat et al., 2017; Avendaño‐Herrera et al., 2020; Supplementary Table 2). Reaction mixtures were then subjected to the following cycling parameters in an Eppendorf Mastercycler® pro thermal cycler (Eppendorf, Framingham, MA): initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute, then a final extension step at 72°C for 10 minutes (Rochat et al., 2017). Control DNA representing each molecular serogroup was included in every run: ATCC 49418T (Serogroup-0), FP900406 (Serogroup-1), CSF-259-93 (Serogroup-2), KU060626-4 (Serogroup-3), and RAH-H (Serogroup-4). A no-template negative control (nuclease free H2O) was also included. Resulting PCR amplicons were electrophoresed alongside an Invitrogen™ 1Kb Plus DNA Ladder (Thermo Fisher Scientific, Waltham, MA) on a 1.5% agarose gel in 0.5X TAE buffer (Millipore Sigma, Burlington, MA) and then viewed under ultraviolet (UV) transillumination. A resulting amplicon size of 141 base pairs (bp) was indicative of Serogroup-0, whereas the presence of the 141 bp amplicon in addition to an amplicon of 549 bp, 841 bp, 361 bp, or 992 bp corresponded to Serogroup-1, Serogroup-2, Serogroup-3, and Serogroup-4, respectively (Rochat et al., 2017; Avendaño‐Herrera et al., 2020).

2.5 Statistical association of molecular serogroup and host species

Associations between molecular serogroup and host species for rainbow/steelhead trout, coho salmon, Chinook salmon, brown trout, and Atlantic salmon were analyzed via Fisher’s Exact Test with Monte Carlo simulation using R version 4.5.1. However, molecular Serogroup-3 was excluded from the analysis due to low sample size. Standardized Pearson residuals were calculated post-hoc and the Holm-Bonferroni method was applied to determine which species-serogroup cells were significant (α = 0.05).

3 Results

Using mPCR, the 320 US originating F. psychrophilum isolates analyzed in this study were assigned to molecular Serogroup-0 (29.4%; n=94), Serogroup-1 (26.6%; n=85), Serogroup-2 (23.1%; n=74), Serogroup-3 (1.3%; n=4), and Serogroup-4 (19.7%; n=63; Supplementary Table 1).

3.1 Flavobacterium psychrophilum molecular serogroup and host fish species

Most of the characterized F. psychrophilum isolates (95.3%, n=305) were recovered from five salmonid species: rainbow/steelhead trout (n=170), coho salmon (n=49), Chinook salmon (n=42), brown trout (n=23), and Atlantic salmon (n=21; Figure 1). Rainbow/steelhead trout was the only species from which all five molecular serogroups were recovered, with most belonging to Serogroup-2 (35.9%, n=61) or Serogroup-1 (31.2%, n=53), followed by Serogroup-4 (19.4%, n=33), Serogroup-0 (11.2%, n=19), and Serogroup-3 (2.4%, n=4; Figure 1). Isolates recovered from coho salmon primarily belonged to molecular Serogroup-0 (89.8%, n=44), with a few isolates belonging to Serogroup-4 (8.2%, n=4) and Serogroup-2 (2.0%, n=1; Figure 1). Flavobacterium psychrophilum isolates recovered from Chinook salmon were most frequently molecular Serogroup-1 (50.0%, n=21), followed by Serogroup-0 (33.3%, n=14), Serogroup-4 (11.9%, n=5), and Serogroup-2 (4.8%, n=2; Figure 1). Brown trout isolates included molecular Serogroup-4 (47.8%, n=11), Serogroup-0 (21.7%, n=5), Serogroup-2 (17.4%, n=4), and Serogroup-1 (13.0%, n=3; Figure 1). Lastly, F. psychrophilum isolates recovered from Atlantic salmon were most commonly molecular Serogroup-4 (42.9%, n=9), followed by Serogroup-0 (23.8%, n=5), Serogroup-1 (19.0%, n=4), and Serogroup-2 (14.3%, n=3; Figure 1). The remaining 15 isolates originated from 8 other fish species (n=14) or were of unknown origin (n=1) and belonged to molecular Serogroup-0 (46.7%, n=7), Serogroup-1 (26.7%, n=4), Serogroup-2 (20.0%, n=3), and Serogroup-4 (6.7%, n=1; Table 1; Figure 1).

Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by host species (inner ring) for US-originating *Flavobacterium psychrophilum* isolates. Species with <5 isolates are listed as “other.”.
Figure 1. Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by host species (inner ring) for US-originating Flavobacterium psychrophilum isolates. Species with <5 isolates are listed as “other.”.

Table 1. Flavobacterium psychrophilum multiplex polymerase chain reaction molecular serogroup distribution by host species.

Host speciesSerogroup-0Serogroup-1Serogroup-2Serogroup-3Serogroup-4Total
Salv. namaycush2–1––3
C. clupeaformis–21––3
O. apache2––––2
Salv. fontinalis2––––2
O. clarkii1––––1
O. nerka–1–––1
Salv. namaycush x Salv. fontinalis––––11
A. transmontanus–1–––1
Unknown––1––1
Total743-115

Only species with ≤ 5 isolates are shown. Species with > 5 isolates are presented in Figure 1.

Of the host species examined, there were statistically significant positive associations for rainbow/steelhead trout and molecular Serogroup-2, as well as coho salmon and Serogroup-0 (P < 0.05). Conversely, significant negative associations included rainbow/steelhead trout and molecular Serogroup-0, as well as coho salmon, Serogroup-1, and Serogroup-2 (P < 0.05; Table 2).

Table 2. Standardized Pearson residuals and Holm-Bonferroni adjusted P-values from post-hoc pairwise analysis of F. psychrophilum molecular serogroup distribution across the five main fish species examined in the present study (i.e., Oncorhynchus mykiss, Salmo salar, O. kisutch, S. trutta, and O. tshawytscha; α = 0.05).

Host speciesSerogroup-0Serogroup-1Serogroup-2Serogroup-4
O. mykiss-4.18(0.0006)1.25(1.0000)3.49(0.0082)-0.20(1.0000)
O. kisutch7.93(0.0000)-3.63(0.0051)-3.11(0.0299)-1.92(0.6034)
O. tshawytscha0.53(1.0000)2.88(0.0597)-2.51(0.1570)-1.24(1.0000)
S. trutta-0.64(1.0000)-1.28(1.0000)-0.61(1.0000)2.88(0.0597)
S. salar-0.43(1.0000)-0.69(1.0000)-0.88(1.0000)2.25(0.2934)

Molecular Serogroup-3 was omitted from the analyses due to low sample size. Positive residual values indicate positive associations, whereas negative residual values indicate negative associations. Significant associations are bolded.

3.2 Flavobacterium psychrophilum molecular serogroup and geographic distribution

The F. psychrophilum isolates analyzed in this study originated from 17 US states, with the majority being recovered from fish in Michigan (n=168), California (n=45), Pennsylvania (n=26), Utah (n=21), Oregon (n=18), and Washington (n=11; Table 3). Molecular Serogroup-2 was the most widespread (10/17 states), followed by Serogroup-0 and Serogroup-1 (9/17 states each), Serogroup-4 (5/17 states), and lastly Serogroup-3 (2/17 states; Table 3). Michigan was the only state from which at least 1 F. psychrophilum isolate from each of the 5 molecular serogroups was found, whereas 2–3 molecular serogroups were found from 8/17 states and the remaining 8 states only had isolates in the present dataset that belonged to 1 molecular serogroup (Table 3).

Table 3. Flavobacterium psychrophilum multiplex polymerase chain reaction molecular serogroup distribution by US state of origin.

OriginSerogroup-0Serogroup-1Serogroup-2Serogroup-3Serogroup-4Total
Michigan603434139168
California12717––45
Pennsylvania–36–1726
Utah4107––21
Oregon142––218
Washington632––11
Wisconsin52––18
Missouri–22––4
New Jersey––––44
New Mexico22–––4
Minnesota–––3–3
Colorado––2––2
New York––2––2
Alaska1––––1
Idaho––1––1
Indiana1––––1
Wyoming––1––1
Total948574463320

3.2.1 Michigan

Within the present dataset, Michigan accounted for the largest number of isolates (52.5%, n=168), which were collected over >75 distinct sampling events and from 12 locations. Because of this more extensive dataset, available metadata was used to explore potential links between F. psychrophilum molecular serogroup and fish gamete sources, rearing facilities, life stage, and wild/feral vs. captive (i.e., farm or hatchery-reared) status. Most Michigan isolates were recovered from fish being reared in six state fish hatcheries (n=75) or from wild/feral fish (n=89) returning to spawn at four weirs (i.e., gamete collection stations), all being operated by the Michigan Department of Natural Resources (MDNR; Figure 2). These isolates were recovered from seven fish species, including rainbow/steelhead trout (n=56), Chinook salmon (n=38), coho salmon (n=26), brown trout (n=21), Atlantic salmon (n=19), lake trout (n=3), and brook trout (n=1). In addition, two isolates were recovered from rainbow trout being reared in a hatchery not operated by MDNR and two were recovered from wild lake whitefish that were collected from Whitefish Bay, Lake Superior (Supplementary Table 1; Table 4).

Hatchery and weir locations within Michigan from where *Flavobacterium psychrophilum* isolates within this study were recovered. MI-SFH1, Marquette County; MI-SFH2, Emmet County; MI-SFH3, Benzie County; MI-SFH4, Van Buren County; MI-SFH5, Wexford County; MI-SFH6, Schoolcraft County; MI-WE1, Manistee County; MI-WE2, Chippewa County; MI-WE3, Presque Isle County; MI-WE4, Benzie County. MI-SFH3 and MI-WE4 occupy the same site. Surface water includes diverted spring water that is open to the environment before entering fish rearing units.
Figure 2. Hatchery and weir locations within Michigan from where Flavobacterium psychrophilum isolates within this study were recovered. MI-SFH1, Marquette County; MI-SFH2, Emmet County; MI-SFH3, Benzie County; MI-SFH4, Van Buren County; MI-SFH5, Wexford County; MI-SFH6, Schoolcraft County; MI-WE1, Manistee County; MI-WE2, Chippewa County; MI-WE3, Presque Isle County; MI-WE4, Benzie County. MI-SFH3 and MI-WE4 occupy the same site. Surface water includes diverted spring water that is open to the environment before entering fish rearing units.

Table 4. Flavobacterium psychrophilum multiplex polymerase chain reaction molecular serogroup distribution within Michigan by species.

HostSerogroup-0Serogroup-1Serogroup-2Serogroup-3Serogroup-4Total
O. mykiss1472511158
O. tshawytscha11202–538
O. kisutch22–––426
S. trutta523–1121
S. salar542–819
Salv. namaycush2–1––3
C. clupeaformis–11––2
Salv. fontinalis1––––1
Total603434139168

When examining F. psychrophilum molecular serogroup as it relates to Michigan state fish hatchery (MI-SFH) and weir (MI-WE), at least two serogroups were recovered from each (Figure 3): two serogroups were identified at MI-SFH1, MI-SFH3, MI-SFH5, and MI-SFH6, whereas four serogroups were present at MI-SFH2 and MI-SFH4 (Figure 3). In contrast, molecular serogroup diversity was greater at most Michigan weirs, with all five serogroups identified at MI-WE1, four serogroups (i.e., Serogroup-0, -1, -2, and -4) identified at MI-WE2 and MI-WE3, and two serogroups (Serogroup-0 and -4) present at MI-WE4 (Figure 3). Of note, MI-WE1 is the only weir where gametes of two species are collected and at different points during the year (i.e., steelhead trout in spring, Chinook salmon in fall). Of the identified molecular serogroups, Serogroup-0 and Serogroup-4 were most widespread, with each observed at five of the six state fish hatcheries and all four weirs at varying frequencies (Figure 3). Similarly, molecular Serogroup-2 was found at four state fish hatcheries and three weirs, whereas Serogroup-1 was observed at two state fish hatcheries and three weirs (Figure 3). Molecular Serogroup-3 was least prevalent, with detection at only one weir (Figure 3).

Molecular serogroup distribution of *Flavobacterium psychrophilum* in Michigan by source location and host species. Number of isolates is shown within each bar for each serogroup. Findings shown for Michigan state fish hatcheries and weirs only. MI-SFH, state fish hatchery; MI-WE, weir.
Figure 3. Molecular serogroup distribution of Flavobacterium psychrophilum in Michigan by source location and host species. Number of isolates is shown within each bar for each serogroup. Findings shown for Michigan state fish hatcheries and weirs only. MI-SFH, state fish hatchery; MI-WE, weir.

3.3 Multilocus sequence typing

The F. psychrophilum isolates analyzed by MLST in this study (n=62) belonged to CC-ST10 (n=17), CC-ST375 (n=10), CC-ST256 (n=6), CC-ST9 (n=3), and CC-ST191 (n=1), or were singletons (n=25; Supplementary Table 1; Figure 4). Ten novel genotypes (i.e., STs) were identified: ST369, ST370, ST371, ST372, ST373, ST375, ST376, ST379, ST380, and ST381. Of these, ST380 and ST381 belonged to CC-ST10, and ST371 and ST375 formed a new CC (i.e., CC-ST375) with ST286 and ST349. The remaining six novel genotypes were singletons (Supplementary Table 1; Figure 4).

eBURST diagram depicting inferred genetic relatedness (as determined by multilocus sequence typing; MLST) of *Flavobacterium psychrophilum* isolates typed in the present study (n=62) compared to previously typed isolates in the pubMLST database (n=1556). Clonal complexes (CC) are defined as groups of sequence types (ST) connected by single locus variant (SLV) links and are encircled. Each CC is named after the most likely founding ST (i.e., ST with the greatest number of SLVs). If multiple STs have the same number of SLVs within a CC, the CC is named after the ST with the largest number of isolates. If both STs have the same number of isolates, the CC is named after the earliest identified ST. The founding ST of each CC appears within a box. Previously identified STs that do not contain isolates included in the present study are shown in black. Newly reported STs are shown in red. Previously reported STs containing newly typed isolates are shown in blue. Previously reported STs containing previously typed isolates included in the present study are shown in purple.
Figure 4. eBURST diagram depicting inferred genetic relatedness (as determined by multilocus sequence typing; MLST) of Flavobacterium psychrophilum isolates typed in the present study (n=62) compared to previously typed isolates in the pubMLST database (n=1556). Clonal complexes (CC) are defined as groups of sequence types (ST) connected by single locus variant (SLV) links and are encircled. Each CC is named after the most likely founding ST (i.e., ST with the greatest number of SLVs). If multiple STs have the same number of SLVs within a CC, the CC is named after the ST with the largest number of isolates. If both STs have the same number of isolates, the CC is named after the earliest identified ST. The founding ST of each CC appears within a box. Previously identified STs that do not contain isolates included in the present study are shown in black. Newly reported STs are shown in red. Previously reported STs containing newly typed isolates are shown in blue. Previously reported STs containing previously typed isolates included in the present study are shown in purple.

Most isolates (n=111) included in this study belonged to CC-ST10 and were recovered from rainbow/steelhead trout (n=104), coho salmon (n=2), Chinook salmon (n=1), Apache trout (n=2), and white sturgeon (n=1), or were unknown in origin (n=1; Supplementary Table 1). Within CC-ST10, isolates were assigned to molecular Serogroup-2 (48.6%, n=54), Serogroup-1 (38.7%, n=43), Serogroup-0 (11.7%, n=13), and Serogroup-3 (0.9%, n=1); no isolates within CC-ST10 belonged to molecular Serogroup-4 (Figure 5; Supplementary Table 1). The CC containing the second most isolates herein (CC-ST256, n=18 total) were assigned to molecular Serogroup-0 (94.4%, n=17) and Serogroup-1 (5.6%, n=1; Figure 5; Supplementary Table 1). Similarly, CC-ST9 (n=16 total) was predominately molecular Serogroup-0 (93.8%, n=15), with only one Serogroup-2 isolate (6.3%; Figure 5; Supplementary Table 1). Clonal complexes CC-ST375 (n=14 total), CC-ST281 (n=11 total), and CC-ST11 (n=4 total) were exclusively molecular Serogroup-4 (Figure 5; Supplementary Table 1).

Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by multilocus sequence typing clonal complex (inner ring) for 320 US-originating *Flavobacterium psychrophilum* isolates. Clonal complexes with <5 isolates are listed as “other.” CC, clonal complex; ST, sequence type.
Figure 5. Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by multilocus sequence typing clonal complex (inner ring) for 320 US-originating Flavobacterium psychrophilum isolates. Clonal complexes with <5 isolates are listed as “other.” CC, clonal complex; ST, sequence type.

4 Discussion

Several studies have characterized the serodiversity of F. psychrophilum in Asia, Europe, and South America (Wakabayashi et al., 1994; Lorenzen and Olesen, 1997; Izumi and Wakabayashi, 1999; Mata et al., 2002; Izumi et al., 2003; Valdebenito and Avendaño‐Herrera, 2009; Rochat et al., 2017; Ngo et al., 2017; Saticioglu et al., 2018; Avendaño‐Herrera et al., 2020; Ilardi et al., 2023; Vaibarova et al., 2024). Some of the resultant data can and has been utilized to guide the development of vaccine preparations against predominating F. psychrophilum variants in a particular region, with promising results (Hoare et al., 2017a; Hoare et al., 2017b; Hoare et al., 2019). However, a large-scale analysis of F. psychrophilum serodiversity in the USA had yet to be conducted. Thus, the present study was undertaken to address this knowledge gap, and to investigate the potential implications of the newly derived F. psychrophilum molecular serogroup information in the context of host associations, sero-/genovariation, and geographic distribution, particularly as it pertains to future vaccine research.

Herein, molecular serogrouping analyses for 320 F. psychrophilum isolates revealed significant positive associations between Serogroup-2 and rainbow/steelhead trout, and Serogroup-0 and coho salmon; additionally, and although not statistically significant, Serogroup-1 was commonly associated with rainbow/steelhead trout as well as Chinook salmon. Likewise, molecular Serogroup-4 isolates were commonly associated with brown trout and Atlantic salmon (Figure 1). These apparent molecular serogroup-host associations are not without precedent, as previous studies have noted associations between F. psychrophilum serovariation and host species (Wakabayashi et al., 1994; Izumi and Wakabayashi, 1999; Izumi et al., 2003; Lorenzen and Olesen, 1997; Mata et al., 2002; Rochat et al., 2017). By examining the present findings amongst previously published and collated molecular serogrouping results (i.e., F. psychrophilum isolates recovered from 22 countries on 5 continents) multiple broadly consistent host associations become evident (Table 5; Figure 6). For example, molecular Serogroups-1 and -2 comprise the vast majority (n=467, 78.1%) of F. psychrophilum isolates recovered from rainbow/steelhead trout across five continents, although all five molecular serogroups have been recovered from this species (Table 5, Figure 6); Rochat et al. (2017) originally identified significant positive associations between rainbow/steelhead trout and molecular Serogroups -1 and -2, which clearly continues to be well supported. Similarly, F. psychrophilum isolates recovered from Atlantic salmon inhabiting four continents frequently belong to molecular Serogroup-4 (n=42, 42%; Figure 6). Strikingly, >90% of F. psychrophilum isolates (n=70; Figure 6) recovered from coho salmon across three continents in this and previous studies belong to molecular Serogroup-0, though a subset of previously typed Serogroup-0 isolates (n=23) are undifferentiated Serogroup-0/4. Several significant negative associations were also identified in the present study and include rainbow/steelhead trout and molecular Serogroup-0, in addition to coho salmon, Serogroup-1, and Serogroup-2. These are likewise well supported by the aggregate molecular serogrouping findings to date in which 10% of rainbow/steelhead trout F. psychrophilum isolates (n=60) belonged to Serogroup-0, and no coho salmon isolates were Serogroup-1, while just 3.9% (n=3) were Serogroup-2 (Figure 6).

Table 5. Flavobacterium psychrophilum multiplex polymerase chain reaction molecular serogroup distribution by species from previous studies.

HostSerogroup-0Serogroup-1Serogroup-2Serogroup-3Serogroup-4TotalReference
O. mykiss2359609N/A151Rochat et al., 2017
O. kisutch22–1–N/A23
O. tshawytscha1–––N/A1
S. salar1223N/A8
S. trutta221–N/A5
O. mykiss41450–1785Avendaño‐Herrera et al., 2020
S. salar2114–1532
O. kisutch––1––1
O. mykiss–227––29Ilardi et al., 2023
S. salar16–5–1839
O. mykiss–43191366Vaibarova et al., 2024
S. trutta––4––4
O. mykiss9814–N/A31Calvez et al., 2021
O. mykiss19113N/A24Saticioglu et al., 2018
O. kisutch1–––N/A1
O. mykiss21111–N/A24Sundell et al., 2019
O. mykiss2131–7Fujiwara-Nagata et al., 2024
O. kisutch3––––3
O. mykiss–10––N/A10Li et al., 2021
O. mykiss––1–N/A1Park et al., 2023

Only the five main species examined in the present study (i.e., O. mykiss, S. salar, O. kisutch, S. trutta, and O. tshawytscha) are presented.

Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by species (inner ring) for 850 *Flavobacterium psychrophilum* isolates characterized by Rochat et al. (2017); Avendaño‐Herrera et al. (2020); Ilardi et al. (2023); Vaibarova et al. (2024); Calvez et al. (2021); Saticioglu et al. (2018); Sundell et al. (2019); Fujiwara-Nagata et al. (2024); Li et al. (2021); Park et al. (2023), and the present findings. Note that only Avendaño‐Herrera et al. (2020); Ilardi et al. (2023); Fujiwara-Nagata et al. (2024); Vaibarova et al. (2024), and the present study used the molecular Serogroup-4 primers described by Avendaño‐Herrera et al. (2020); as such, only n=25 *O. mykiss*, n=23 *S. salar*, n=47 *O. kisutch*, n=14 *O. tsawytscha*, and n=5 *S. trutta* isolates are true molecular Serogroup-0, all other isolates within Serogroup-0 for all species displayed here are actually undifferentiated Serogroup-0/4. Only the five main species examined in this study (i.e., *O. mykiss, S. salar, O. kisutch, O. tsawytscha*, and *S. trutta*) are presented.
Figure 6. Sunburst figure depicting multiplex polymerase chain reaction molecular serogroup distribution (outer ring) by species (inner ring) for 850 Flavobacterium psychrophilum isolates characterized by Rochat et al. (2017); Avendaño‐Herrera et al. (2020); Ilardi et al. (2023); Vaibarova et al. (2024); Calvez et al. (2021); Saticioglu et al. (2018); Sundell et al. (2019); Fujiwara-Nagata et al. (2024); Li et al. (2021); Park et al. (2023), and the present findings. Note that only Avendaño‐Herrera et al. (2020); Ilardi et al. (2023); Fujiwara-Nagata et al. (2024); Vaibarova et al. (2024), and the present study used the molecular Serogroup-4 primers described by Avendaño‐Herrera et al. (2020); as such, only n=25 O. mykiss, n=23 S. salar, n=47 O. kisutch, n=14 O. tsawytscha, and n=5 S. trutta isolates are true molecular Serogroup-0, all other isolates within Serogroup-0 for all species displayed here are actually undifferentiated Serogroup-0/4. Only the five main species examined in this study (i.e., O. mykiss, S. salar, O. kisutch, O. tsawytscha, and S. trutta) are presented.

Among these molecular serogroup-host associations, Serogroup-0 and coho salmon warrants specific consideration – within the mPCR scheme, Serogroup-0 isolates do not possess any of the identified key molecular determinants of the other serogroups and have markedly more diverse arrangement of their polysaccharide encoding loci (Rochat et al., 2017). This highlights a need for further elucidation of the serovariation within molecular Serogroup-0, particularly given the importance of farmed coho salmon in other regions of the world, such as Chile (FAO, 2026). Most immediately, prioritizing whole genome sequencing to identify the at least 20 purported F. psychrophilum O-serotypes (Cisar et al., 2019, Cisar et al., 2024) may aid selection of candidate isolates for vaccine development, and inform rapid diagnostic tools to guide targeted vaccine deployment, particularly for species predominantly or regularly affected by molecular Serogroup-0 variants (e.g., coho salmon, Atlantic salmon, Chinook salmon). Also noteworthy is the rare occurrence of molecular Serogroup-3 isolates from salmonids in the USA and globally, whereby none have been reported from coho salmon, Chinook salmon, or brown trout, and only 3% each of rainbow trout and Atlantic salmon isolates published thus far belong to Serogroup-3 (Figure 6). Recently, Fujiwara-Nagata et al. (2024) experimentally investigated the apparent predilection of molecular Serogroup-3 for ayu, revealing that although only Serogroup-3 isolates were virulent to this species, varying degrees of virulence were observed amongst heterologous and homologous Serogroup-3 MLST genetic variants. Although identifying the mechanisms behind the apparent host associations of F. psychrophilum molecular serogroups was not a goal of this study, others have suggested that expression of proteases, including collagenase (Nakayama et al., 2016) and elastase (Rochat et al., 2019), in addition to differences in O-polysaccharide structure (Rochat et al., 2017; Fujiwara-Nagata et al., 2024) and complement resistance (Fujiwara-Nagata et al., 2023) may be key factors. Collectively, this and previous studies further illustrate that additional factors beyond what is captured by current MLST genotyping and mPCR serogrouping schemes influence host-F. psychrophilum interactions.

The analyses conducted herein also revealed several notable associations between F. psychrophilum molecular serogroup and MLST CC and/or ST. For instance, and within the largest and globally distributed CC-ST10 that is strongly associated with rainbow/steelhead trout under natural (Nilsen et al., 2014; Van Vliet et al., 2016; Duchaud et al., 2018; Knupp et al., 2019; Sebastião et al., 2020; Li et al., 2021) and experimental conditions (Knupp et al., 2021a; Knupp and Loch, 2023), US isolates belonging to molecular Serogroup-4 were strikingly absent from this CC (Figure 5) despite nearly 20% of US rainbow/steelhead trout isolates belonging to Serogroup-4 (Figure 1). To our knowledge, molecular Serogroup-4 isolates belonging to CC-ST10 have likewise not been reported elsewhere (Vaibarova et al., 2024; Fujiwara-Nagata et al., 2024). In contrast, all four remaining molecular serogroups (i.e., Serogroup-0, -1, -2, and -3) were identified within CC-ST10, although Serogroup-1 and -2 predominated, in keeping with the strong association of this CC and rainbow/steelhead trout.

In some cases, MLST CCs were predominated by or comprised exclusively of one molecular serogroup (e.g., CC-ST9 and Serogroup-0, CC-ST256 and Serogroup-0, CC-ST281 and Serogroup-4, CC-ST288 and Serogroup-1, CC-ST375 and Serogroup-4; Supplementary Table 1; Figure 5). In contrast, other MLST CCs contained isolates belonging to three or more molecular serogroups (e.g., CC-ST10, CC-ST276). Of particular interest, multiple genotypes within CC-ST10 (i.e., ST10, ST78, ST85, and ST275) encompassed isolates of three different molecular serogroups, revealing substantial intragenotypic serodiversity within CC-ST10 (Supplementary Table 1), which may complicate vaccine development against this predominant F. psychrophilum lineage. Previous studies have also reported the occurrence of multiple molecular serogroups within a single ST (Sundell et al., 2019; Calvez et al., 2021), highlighting the multifactorial nature of the determinants of virulence and host specificity (Sundell et al., 2019; Fujiwara-Nagata et al., 2024). Recent advancements in F. psychrophilum genetic manipulation capabilities (Sloboda et al., 2025) may help unravel the key determinants of virulence, host specificity, and immunogenicity, among numerous other factors of relevance for effective prevention and control of F. psychrophilum associated disease.

The implications for the observed associations between F. psychrophilum molecular serogroup, genotype, and host species are potentially multifold. First and foremost are possible implications for BCWD vaccine development and efficacy testing. Various vaccine formulations against BCWD, including live-attenuated (Álvarez et al., 2008; LaFrentz et al., 2008; Long et al., 2013; Sudheesh et al., 2016; Sudheesh and Cain, 2016; Ma et al., 2018), inactivated whole-cell (Rahman et al., 2000; LaFrentz et al., 2002; Madetoja et al., 2006; Högfors et al., 2008; Fredriksen et al., 2013; Hoare et al., 2017a; Hoare et al., 2017b; Hoare et al., 2019; Marana et al., 2022), and subunit vaccines (Rahman et al., 2002; LaFrentz et al., 2004; Dumetz et al., 2006; Högfors et al., 2008; Plant et al., 2014), as well as passive immunization approaches (LaFrentz et al., 2003), have been developed and tested, with varying degrees of success. Among these, LaFrentz et al. (2008) produced a rifampicin-mediated attenuated strain of F. psychrophilum (CSF-259-93-B.17) that has been well studied as a vaccine candidate. After additional improvements (Long et al., 2013; Sudheesh and Cain, 2016) and administration via immersion, the vaccine conferred significant but variable protection (relative percent survival; RPS 51-72%) against multiple genetically heterologous F. psychrophilum MLST variants (Ma et al., 2018). These authors noted that the specific immune response generated following vaccination and shared antigenicity among the evaluated strains supports the importance of antibodies for sufficient cross protection. Although these results are promising, it is noteworthy that none of the isolates evaluated by Ma et al. (2018) belonged to molecular Serogroup-3 or -4, and only one isolate belonged to the antigenically diverse Serogroup-0.

In the context of further vaccine development and evaluation, Cisar et al. (2019) and Cisar et al. (2024) experimentally demonstrated that differences in the F. psychrophilum wzy gene (i.e., the gene encoding for the polymerase that links trisaccharide repeats of O-polysaccharide and forms the basis of the mPCR typing scheme; Rochat et al., 2017) govern the major genetic determinants of antibody binding specificity for this bacterium. Likewise, concomitant variations in F. psychrophilum R-group genes and the occurrence of a 3.8 kB insertion within the O-polysaccharide loci can also affect O-polysaccharide structure and, in turn, serological reactivity (Cisar et al., 2019, Cisar et al., 2024). This, in conjunction with a high F. psychrophilum recombination rate (Nicolas et al., 2008; Duchaud et al., 2018), particularly amongst US F. psychrophilum (Knupp et al., 2019), suggests that the extensive and expanding intraspecific diversity may, in part, explain the variable efficacy of previous vaccination approaches. In the United Kingdom, Ngo et al. (2017) characterized the sero-/genodiversity of predominating F. psychrophilum variants and their findings were then utilized by Hoare et al. (2017a) to select genetically and serotypically diverse F. psychrophilum variants from rainbow trout and Atlantic salmon for use in a polyvalent, whole-cell killed vaccine preparation. After administration via immersion, the polyvalent vaccine conferred significant protection against a heterologous F. psychrophilum isolate (RPS 84%). In light of those findings, and based on the data generated herein, it may be prudent to emphasize polyvalent preparations containing the molecular serogroups predominating amongst certain host species for which efficacious vaccination protection is being sought (e.g., Serogroup-1 and -2, for rainbow/steelhead trout, Serogroup-2 and -4 for Atlantic salmon and brown trout, Serogroup-1 for Chinook salmon). This approach, paired with utilization of adjuvanted preparations (Hoare et al., 2017a) and inclusion of F. psychrophilum extracellular components (Semple et al., 2020) could prove advantageous for robust immune stimulation and resultant protection.

Regional differences in F. psychrophilum serovariation may also warrant consideration during vaccine development. Although systematic examination of geographic associations across F. psychrophilum molecular serogroups was not possible given the opportunistic recovery of the analyzed isolates, some regional variation within the USA was nevertheless apparent. Indeed, nearly all (n=61/63) molecular Serogroup-4 isolates originated from eastern states (i.e., Michigan, n=39; Pennsylvania, n=17; New Jersey, n=4; and Wisconsin, n=1), compared to only two being recovered from a western state (Oregon). This is despite nearly one third (32.5%, n=104) of isolates examined in this study originating from western states. Furthermore, and based upon data generated herein, it appears that some molecular serogroups are widely distributed across US states (e.g., Serogroups-0, -1, and -2), whereas others appear more geographically limited (e.g., Serogroups-3 and -4). Characterization of additional isolates from these and other US states will determine if true regional variation exists or if the differences reported herein are biased by the sampling methods.

The more extensive Michigan dataset was further examined for potential trends that may provide deeper insight into F. psychrophilum population dynamics within hatchery systems and inform potential BCWD prevention strategies. In several instances, identical F. psychrophilum sero-/genovariants were recovered from wild/feral broodstock and the fish hatcheries populated by their gametes, further implicating vertical transmission as a driver of some BCWD outbreaks therein. For example, F. psychrophilum isolates belonging to ST257, ST267, and ST342 and found to be molecular Serogroup-4, -0, and -2, respectively, were recovered from wild/feral steelhead trout broodstock at MI-WE1 and from the hatchery-reared steelhead trout at MI-SFH4, which are derived from the gametes of broodfish at MI-WE1 (Supplementary Table 1; Figure 2). Similarly, isolates belonging to ST13, ST258, and ST350 and belonging to molecular Serogroup-0, -0, and -4, respectively, were each recovered from wild/feral spawning coho salmon broodstock at MI-WE4 and were also isolated from coho salmon being reared at MI-SFH3, which is populated using gametes from MI-WE4 (Supplementary Table 1). Notably, vertical transmission of F. psychrophilum has been implicated as an important source of BCWD outbreaks in the USA (Knupp et al., 2019) and elsewhere (Kumagai and Takahashi, 1997), and is hypothesized to have contributed to the global dissemination of the bacterium from North America via trade of infected eggs (Wakabayashi et al., 1994; Avendaño-Herrera et al., 2014; Duchaud et al., 2018; Li et al., 2021). Although the extent to which vertical transmission contributes to continued BCWD prevention and control challenges within Michigan state fish hatcheries is unclear, this transmission pathway warrants specific management consideration.

In addition to vertical transmission, there are likely myriad factors influencing the dynamics of F. psychrophilum serovariation within Michigan state fish hatcheries. For example, multiple salmonid species are often reared within a single facility, whereby fish are maintained in partial reuse water systems that share water between rearing units that may contain different salmonid species. Nevertheless, and despite such complexity, the molecular serogroup data generated herein could help to inform the development of autogenous bacterins based on the historically recurrent and problematic F. psychrophilum variants affecting species reared at a given facility. For example, at MI-SFH5, molecular Serogroup-4 F. psychrophilum isolates predominate (Figure 3) across all three species reared (i.e., brown trout, Atlantic salmon, and rainbow trout). Amongst these molecular Serogroup-4 isolates, ST375 was the most common MLST genotype and was recurrent in multiple years, potentially illustrating a promising vaccine candidate. Conversely, at MI-SFH4, more variability in molecular serogroup was observed, despite all isolates having originated from a single species (i.e., steelhead trout). Although CC-ST10 was predominant at MI-SFH4, many STs within CC-ST10 were recovered in addition to a variety of other STs, indicating that a polyvalent autogenous bacterin preparation may be most beneficial at this facility.

In conclusion, the findings herein provide the first large-scale analysis of the molecular serogroup distribution of F. psychrophilum isolates originating from the USA. Several notable associations between molecular serogroup and host species were identified and compared with previous findings from several continents, revealing that certain associations (e.g., rainbow trout with Serogroup-1 and Serogroup-2, coho salmon with Serogroup-0) are seemingly consistent regardless of global region. Multiple trends were also identified in which the molecular serogroup composition of some MLST CCs and/or STs appeared largely homogenous, whereas others (e.g., CC-ST10) were markedly more diverse. Collectively, these data can be used to support vaccine development, subsequent efficacy and cross-protection studies, and, more immediately, can be used to guide selection of candidate isolates for development of autogenous bacterins against BCWD. Furthermore, these efforts highlight that the mPCR scheme devised by Rochat et al. (2017) is a straightforward and reproducible method for differentiating F. psychrophilum isolates based on the major molecular determinants of O-polysaccharide structure. Future studies seeking to further elucidate the serotypic diversity of F. psychrophilum in the USA and abroad can build upon these and the recent findings of Cisar et al. (2019) and Cisar et al. (2024) through whole genome sequencing and the development of novel molecular tools to aid identification of the purported F. psychrophilum O-serotypes within each molecular serogroup.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/faquc.2026.1805926/full#supplementary-material

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Footnotes

Edited by: Fabiana Pilarski, São Paulo State University, Brazil

Reviewed by: Shabihul Fatma, Jazan University, Saudi Arabia

Pedro Ilardi, Farmacología en Aquacultura Veterinaria FAV S.A., Chile

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor FP declared a past co-authorship with the author ES.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was funded by the USDA National Institute of Food and Agriculture (grant number: 2020-70007-32410).

Data availability

The data presented in this study are deposited in the PubMLST repository, https://pubmlst.org/fpsychrophilum.

Author contributions

SL: Writing – original draft, Writing – review & editing, Formal analysis, Project administration, Validation, Conceptualization, Investigation, Data curation. CK: Resources, Conceptualization, Investigation, Writing – review & editing, Supervision. ES: Investigation, Resources, Writing – review & editing. DC: Investigation, Writing – review & editing, Resources. PN: Writing – review & editing, Formal analysis, Investigation. TL: Project administration, Validation, Methodology, Data curation, Formal analysis, Conceptualization, Supervision, Writing – review & editing, Funding acquisition, Writing – original draft.

Acknowledgements

We would like to thank all contributors of Flavobacterium psychrophilum isolates, including Dr. Trista Becker, Wade Cavender, Larry Durham, Geofrey Eckerlin, Dr. Jayde Ferguson, Dave Meuninck, Ling Shen, Carl Smith, Dr. Danielle Van Vliet, and Coja Yamashita. We are likewise grateful to Dr. Erina Fujiwara-Nagata and Dr. Ruben Avendaño-Herrera for providing nucleic acid for use as our initial mPCR standards, the Michigan Department of Natural Resources Fisheries Division for their continued support and collaboration, and to past and present members of the Michigan State University - Aquatic Animal Health Laboratory.

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Citation

Lennox, S. M. G., et al. (2026). Large-scale molecular serogroup analysis of Flavobacterium psychrophilum isolates recovered from captive, wild, and feral fishes in the USA. Frontiers in Aquaculture, Volume 5. https://doi.org/
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5 - 2026

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Copyright © 2026 Lennox, Knupp, Soto, Call, Nicolas and Loch.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Thomas P. Loch

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