Comprehensive analysis of Yersinia ruckeri serotype O1 biotype 1 and biotype 2 isolates from rainbow trout in Turkey: Proteomic, serologic, antigenic, genomic, and virulence characteristics

Abstract

Enteric Red Mouth (ERM) disease caused by Yersinia ruckeri first emerged in rainbow trout in the United States in the 1950s. Despite the development of effective vaccines in the 1970s, ERM continues to spread rapidly worldwide (Europe, Asia, Africa, Australia, and New Zealand), causing significant mortality, particularly in salmonid fish. In this study, a total of 69 Y. ruckeri isolates collected from rainbow trout between 2013 and 2023, along with two reference strains (NCTC 12266 and the field strain Biotype 2 [BT2]), were subjected to phenotypic tests such as motility, lipase activity, and sorbitol fermentation to determine biotypes (BT). The isolates were characterized as serotype O1 Biotype 1 (BT1) (n = 46) and BT2 (n = 23) using motility test to detect bacterial motility, SDS-PAGE and Western blot analyses to profile whole-cell proteins and antigenic properties, and slide and microagglutination tests to assess serological relationships. While general similarities were observed in the whole-cell protein profiles, significant differences were found in the antigenic protein patterns. Y. ruckeri strains of serotype O1 BT1 and BT2 showed strong cross-reactions in slide agglutination and quantitative agglutination tests, with titers ranging from 1/160 to 1/1280, except for the Y2022-239 strain. It was determined that the isolates Y2022-266 from O1 BT1 and Y75-2013 from O1 B2 displayed a distinct (1/1280) antibody titer compared to other isolates. Additionally, comprehensive genomic analyses were performed to compare the overall genomic relatedness, virulence genes, and antimicrobial resistance patterns of these biotypes. Genomic analyses revealed that the Turkish Y. ruckeri serotype O1 BT1 and BT2 strains formed a common cluster distinct from isolates of different geographical origins. Furthermore, the Y. ruckeri O1 BT1 (n = 3) and O1 BT2 (n = 5) strains diverged into two subclusters. The AMR genes among these strains were completely similar. The virulence capabilities of pathogenic isolates exhibiting serotype O1 BT1 and BT2 characteristics were also investigated in vivo. The Kaplan-Meier survivorship curves indicate that specific strains within the serotype O1 BT1 and BT2 groups exhibit varying levels of virulence, with BT2 strains showing significantly lower survival rates in rainbow trout than the BT1 strains (p < 0.05). This study is the first to determine the serological, antigenic, and virulence characteristics of Yersinia ruckeri serotype O1 BT1 and BT2 strains isolated in Turkey.