Flavobacterium psychrophilum is one of the most important bacterial pathogens affecting cultured rainbow trout (Oncorhynchus mykiss) and is increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries. Little is known about the heterogeneity of F. psychrophilum isolates on UK salmonid farms. A total of 315 F. psychrophilum isolates, 293 of which were collected from 27 sites within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. Seven PFGE groups and 27 singletons were formed at a band similarity of 80%. PFGE group P (n=75) was found to be numerically predominant in eight sites within the UK. Two major PFGE clusters and 13 outliers were found at the band similarity of 40%. The predominant profileobserved within the F. psychrophilum isolates examined was PFGE cluster II - (GTG)-PCR type r1-16S rRNA lineage II - serotype Th (70/156 isolates examined, 45%). Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, confounding the ability to control RTFS outbreaks. The occurrence over time (up to 11 years) of F. psychrophilum pulsotypes in three representative sites (Scot I, Scot III and Scot V) within Scotland was examined, potentially providing important epidemiological data for farm management and the development of site-specific vaccines.

Rainbow trout fry syndrome (RTFS) is a disease caused by the Gram-negative bacterium Flavobacterium psychrophilum, responsible for significant economic losses in salmonid aquaculture worldwide. The diversity of F. psychrophilum isolates and the inherent difficulties in vaccinating juvenile fish has hampered the development of a vaccine for RTFS. Disease episodes tend to occur between 10–14 °C with necrotic lesions often seen on the skin surrounding the dorsal fin and tail. At present no commercial vaccines are available for RTFS in the UK, leaving antibiotics as the only course of action to control disease outbreaks. The current work was performed as a pilot study to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum, to induce protective immunity in rainbow trout fry. Duplicate groups of 30 trout (5 g) were immersed in 1 L of the vaccine for 30 s. Samples were taken 4 h, day 2 and 7 post-vaccination (pv) of skin mucus, tissues for histology and gene expression analysis; serum and histology samples were taken 6 weeks pv. A booster vaccination was given at 315 degree days (dd) also by immersion. Challenge was by immersion with a heterologous isolate of F. psychrophilum 630 dd post primary vaccination. The vaccine provided significant protection to the trout fry with a RPS of 84% (p < 0.0001). Detection of increased numbers of IgT positive cells in systemic organs, up-regulation of IgT expression in hind-gut and an increase in total IgT in serum was observed in vaccinated fish; however a functional role of IgT in the observed protection remains to be demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-017-0448-z) contains supplementary material, which is available to authorized users.

IntroductionAeromonads are ubiquitous in aquatic environments and several species are opportunistic pathogens of fish. Disease losses caused by motile Aeromonas species, particularly Aeromonas hydrophila, can be challenging in intensive aquaculture, such as at striped catfish (Pangasianodon hypophthalmus) farms in Vietnam. Outbreaks require antibiotic treatments, but their application is undesirable due to risks posed by resistance. Vaccines are an attractive prophylactic and they must protect against the prevalent strains responsible for ongoing outbreaks.MethodsThis present study aimed to characterize A. hydrophila strains associated with mortalities in striped catfish culture in the Mekong Delta by a polyphasic genotyping approach, with a view to developing more effective vaccines.ResultsDuring 2013–2019, 345 presumptive Aeromonas spp. isolates were collected at farms in eight provinces. Repetitive element sequence-based PCR, multi-locus sequence typing and whole-genome sequencing revealed most of the suspected 202 A. hydrophila isolates to belong to ST656 (n = 151), which corresponds to the closely-related species Aeromonas dhakensis, with a lesser proportion belonging to ST251 (n = 51), a hypervirulent lineage (vAh) of A. hydrophila already causing concern in global aquaculture. The A. dhakensis ST656 and vAh ST251 isolates from outbreaks possessed unique gene sets compared to published A. dhakensis and vAh ST251 genomes, including antibiotic-resistance genes. The sharing of resistance determinants to sulphonamides (sul1) and trimethoprim (dfrA1) suggests similar selection pressures acting on A. dhakensis ST656 and vAh ST251 lineages. The earliest isolate (a vAh ST251 from 2013) lacked most resistance genes, suggesting relatively recent acquisition and selection, and this underscores the need to reduce antibiotics use where possible to prolong their effectiveness. A novel PCR assay was designed and validated to distinguish A. dhakensis and vAh ST251 strains.DiscussionThis present study highlights for the first time A. dhakensis, a zoonotic species that can cause fatal human infection, to be an emerging pathogen in aquaculture in Vietnam, with widespread distribution in recent outbreaks of motile Aeromonas septicaemia in striped catfish. It also confirms vAh ST251 to have been present in the Mekong Delta since at least 2013. Appropriate isolates of A. dhakensis and vAh should be included in vaccines to prevent outbreaks and reduce the threat posed by antibiotic resistance.

Flavobacterium psychrophilum is the causative agent of Rainbow Trout Fry Syndrome which has had a major impact on global salmonid aquaculture. Recent outbreaks in Atlantic salmon in Scotland and Chile have added to the need for a vaccine to protect both salmon and trout. At present no licensed vaccines are available in Europe, leaving antibiotics as the only course of action to contain disease outbreaks. Outbreaks generally occur in fry at temperatures between 10 and 15 °C. Recently outbreaks in larger fish have given added impetus to the development of a vaccine which can provide long term protection from this highly heterogeneous pathogen. Most fish injectable vaccines are formulated with oil emulsion adjuvants to induce strong and long lasting immunity, but which are known to cause side effects. Alternative adjuvants are currently sought to minimise these adverse effects. The current study was performed to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum to induce protective immunity in Atlantic salmon. The vaccine was formulated with an adjuvant containing squalene and aluminium hydroxide, and was compared to a vaccine formulated with a traditional oil adjuvant, Montanide ISA 760VG, and a non-adjuvanted vaccine. Duplicate groups of salmon (23.5 ± 6.8 g) were vaccinated with each of the vaccine formulations or phosphate buffered saline by intraperitoneal injection. Fish were challenged by intramuscular injection with F. psychrophilum six weeks post-vaccination to test the efficacy of the vaccines. Cumulative mortality reached 70% in the control salmon, while the groups of salmon that received vaccine had significantly lower mortality than the controls (p = 0.0001), with no significant difference in survival between vaccinated groups. The squalene/alum adjuvant was safe, more readily metabolised by the fish and induced less histopathological changes than the traditional oil adjuvant.

22Bacteria from the genus Flavobacteriaceae often show low susceptibility to antibiotics. With the 23 exception of two Chryseobacterium spp. isolates that were positive for the florfenicol resistance 24 gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates 25 from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the 26 floR positive isolates revealed the presence of a region that contained the antimicrobial resistance 27 (AMR) genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a 28 chloramphenicol acetyltransferase gene. In silico analysis of 377 published genomes for 29Flavobacteriaceae isolates from a range of sources, confirmed that well-characterized resistance 30 gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated 31 decreased susceptibility to a range of antimicrobials was confirmed in both floR positive isolates 32 using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floR isolates 33 possessed putative virulence factors, including production of siderophores and, haemolysins, and 34 were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection 35 of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic 36 mechanisms rather than the horizontally acquired resistance genes more normally associated with 37Gram-negative bacterial pathogens such as Enterobacteriaceae. 38 39