Phylogenomic Diversity, Serotype Heterogeneity and In Vivo Virulence of Flavobacterium psychrophilum Isolates From Rainbow Trout Farms in Türkiye

ABSTRACT Flavobacterium psychrophilum , the causative agent of bacterial cold‐water disease (BCWD) and rainbow trout fry syndrome (RTFS), remains one of the most important bacterial pathogens affecting salmonid aquaculture. In this study, 24 F. psychrophilum isolates recovered during a 12‐month surveillance programme from two rainbow trout farms in Türkiye were characterized using phenotypic, serological, antigenic, phylogenomic and experimental infection approaches. Phylogenomic analyses showed that most isolates belonged to CC‐ST10, whereas four isolates were assigned to the CC‐ST90/ST108 lineage, representing the first report of this lineage in Türkiye. Comparative genomic analysis revealed extensive conservation of virulence‐associated loci, with only FP0006 and FP0506 showing variable distribution among isolates. Notably, FP0506 was absent exclusively from ST108/CC‐ST90 isolates. Experimental immersion challenge assays demonstrated that all selected isolates were able to induce mortality in rainbow trout under the conditions tested. However, isolate‐dependent differences in survival were not statistically significant in the standard immersion model and became more evident following H 2 O 2 pre‐treatment, with the FP1 group showing the lowest survival. These findings suggest that in vivo pathogenicity may be influenced by host barrier status and cannot be fully predicted by phylogenomic lineage, serotype or in vitro virulence‐associated phenotypes alone. Phenotypic serotyping identified Th as the predominant serotype, although discrepancies were observed between phenotypic and genome‐based serotype classifications. Whole‐cell protein profiling and Western blot analyses revealed both antigenic heterogeneity and conserved immunoreactive structures across phylogenetically distinct isolates. Antimicrobial susceptibility testing showed a high proportion of non‐wild‐type phenotypes for oxytetracycline, florfenicol and enrofloxacin; however, no acquired antimicrobial resistance genes were detected by genome‐based screening. Collectively, these findings demonstrate that Turkish F. psychrophilum populations are phylogenomically diverse but retain largely conserved virulence‐associated loci and immunoreactive antigenic structures. This study provides the first evidence of the CC‐ST90/ST108 lineage in Türkiye and highlights the value of integrating genomic, serological, phenotypic, antigenic and experimental infection data for understanding the population structure, serotype heterogeneity and pathogenic potential of F. psychrophilum in rainbow trout aquaculture.