Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing

Abstract

The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n ‫؍‬ 63) or were singletons (n ‫؍‬ 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.T he causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS), Flavobacterium psychrophilum, is an important bacterial pathogen of wild and farmed salmonids worldwide (1). In addition to horizontal transmission, F. psychrophilum is suspected of being vertically transmitted (2-5) and appears to resist standard povidone-iodine treatment during egg disinfection (4, 6, 7), which make efforts to control this bacterium particularly problematic. Since the initial isolation of F. psychrophilum in North America (8), F. psychrophilum infections have been reported in Europe, South America, Asia, and Australia (9-11) and from all of...

Multiplex PCR for genotypingFlavobacterium columnare

“…LaFrentz et al () redesigned the primers developed by Ashrafi et al () to be able to amplify the six housekeeping genes of F. columnare isolates from all four genetic groups, which will facilitate such research. Similar research has been conducted with F. psychrophilum which has increased knowledge of molecular diversity, bacterial population structure, epidemiology and host associations (Avendano‐Herrera et al, ; Knupp et al, ; Nicolas et al, ; Siekoula‐Nguedia, Blanc, Duchaud, & Calvez, ; Strepparava, Nicolas, Wahli, Segner, & Petrini, ; Van Vliet, Wiens, Loch, Nicolas, & Faisal, ).…”

First isolation of Flavobacterium psychrophilum associated with reports of moribund wild European eel (Anguilla anguilla) in Scotland

“…Nicolas et al (2008) performed a multilocus sequence analysis and showed the existence of 33 distinct sequence types, with the bacterium isolates from eels grouped in ST15. Individual sequence types within the species F. psychrophilum have been observed as both host specialists and host generalists (Avendaño-Herrera et al, 2014;Nilsen et al, 2014;Van Vliet, Wiens, Loch, Nicolas, & Faisal, 2016). According to Nicolas et al (2008), this sequence type seems to be distinct from those associated with salmonid aquaculture.…”

Assessment of cross‐protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live‐attenuated coldwater disease immersion vaccine

“…For effective development and commercialization of a vaccine to BCWD/RTFS it must be demonstrated that conferred protection is to a variety of bacterial strains. Although F. psychrophilum strain diversity continues to be characterized, it is known that at least seven F. psychrophilum serotypes exist (Dalsgaard & Madsen, ; Mata, Skarmeta, & Santos, ), and intraspecific genetic heterogeneity has been described using phage typing (Castillo, Espejo, & Middelboe, ), random amplified polymorphic DNA (RAPD) analysis (Chakroun, Grimont, Urdaci, & Bernardet, ), pulsed field gel electrophoresis (Arai, Morita, Izumi, Katagiri, & Kimura, ), restriction digestion/sequence analysis of ribosomal and housekeeping genes (Dalsgaard & Madsen, ), and MLST (Ngo et al., ; Nicolas et al., ; Van Vliet et al., ).…”

“…Bacterial strains used in this study included nine virulent strains of F. psychrophilum and were compared to a standard virulent strain, CSF-259-93 as a positive control (Table 1). All 10 F. psychrophilum strains were genotyped via multilocus sequence typing (MLST; Nicolas et al, 2008) in previous (Duchaud et al, 2018;Van Vliet et al, 2016) or the current/ongoing studies (Table 1) using previously described methods (Van Vliet et al, 2016) and represent seven different sequence types belonging to three clonal complexes. For virulence challenges, bacteria were cultured in tryptone yeast extract salts broth (TYES, 0.4% tryptone, 0.04% yeast extract, 0.05% calcium chloride, 0.05% magnesium sulphate, pH 7.2); following previously published protocols (LaFrentz et al, 2008;Long et al, 2013;Sudheesh & Cain, 2016).…”

“…BCWD outbreaks may drastically decrease production in rainbow trout rearing operations, a cultured species of importance that provides more than 800,000 tons of annual, global production (FAO, ). Since the initial isolation of F. psychrophilum in North America (Borg, ), F. psychrophilum infections have been reported in Europe, South America, Asia, and Australia and from all of the major areas of intensive salmonid aquaculture that have been studied (Nematollahi, Decostere, Pasmans, & Hasesbrouck, ; Ngo et al., ; Van Vliet, Wiens, Loch, Nicolas, & Faisal, ). Currently, there is not a commercial BCWD vaccine that is available for use in the USA, leaving treatment with antibiotics as a primary means of controlling mortality due to this disease.…”