Efficacy of a polyvalent immersion vaccine against Flavobacterium psychrophilum and evaluation of immune response to vaccination in rainbow trout fry (Onchorynchus mykiss L.)

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. © 2017 The Author(s)

Construction of a Vibrio alginolyticus hopPmaJ (hop) mutant and evaluation of its potential as a live attenuated vaccine in orange-spotted grouper (Epinephelus coioides)

Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. However, little is known about the associated effectors (T3SEs) and their physiological role. In this study, the T3SE gene hopPmaJ (hop) was cloned from V. alginolyticus wild-type strain HY9901 and the mutant strain HY9901Δhop was constructed by the in-frame deletion method. The results showed that the deduced amino acid sequence of V. alginolyticus HopPmaJ shared 78–98% homology with other Vibrio spp. In addition, the HY9901Δhop mutant showed an attenuated swarming phenotype and a 2600-fold decrease in the virulence to grouper. However, the HY9901Δhop mutant showed no difference in morphology, growth, biofilm formation and ECPase activity. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901Δhop induced a high antibody titer with a relative percent survival (RPS) value of 84% after challenging with the wild-type HY9901. Real-time PCR assays showed that vaccination with HY9901Δhop enhanced the expression of immune-related genes, including MHC-Iα, MHC-IIα, IgM, and IL-1β after vaccination, indicating that it is able to induce humoral and cell-mediated immune response in grouper. These results demonstrate that the HY9901Δhop mutant could be used as an effective live vaccine to combat V. alginolyticus in grouper

Genetic and serological diversity of Flavobacterium psychrophilum isolates from salmonids in United Kingdom

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)5-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 profile observed within the F. psychrophilum isolates examined was PFGE cluster II − (GTG)5-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

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish

Efficacy and safety of a non-mineral oil adjuvanted injectable vaccine for the protection of Atlantic salmon (Salmo salar L.) against Flavobacterium psychrophilum

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

Skin mucus proteins of rainbow trout (Oncorhynchus mykiss) in response to mucosal vaccination and challenge with Flavobacterium psychrophilum

First paragraph: Flavobacterium psychrophilum is one of the most important pathogens affecting rainbow trout (Oncorhynchus mykiss) worldwide at the fry stage of their life cycle. Studies have shown disruption of the fish's skin mucus to be a critical element in the establishment of F. psychrophilum infection in rainbow trout (Henriksen et al., 2013; Madetoja et al., 2000; Martínez et al., 2004). Mucus is the first barrier (biochemical and physical) of the fish, playing a vital role in the protection against pathogens, and contains various antibacterial and inflammatory factors such as lysozyme, immunoglobulin, complement, lectins, acute-phase proteins and proteases (Shunsuke, 2016; Subramanian et al., 2007). Furthermore, the study of external fish mucus provides non-lethal alternatives for the early detection of infections

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04-A2 and VET03-A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorised as wild type (fully susceptible, WT) or non-wild type (NWT) using normalised resistance interpretation (NRI) determined cut-off values (COWT). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC COWT ≤0.25 mg L-1) and 42% were WT for oxytetracycline (MIC COWT ≤0.25 mg L-1). In contrast, all the isolates tested were WT (MIC COWT ≤2 mg L-1) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion-based COWT values were ≥51 mm for 10 µg amoxicillin, ≥44 mm for 30 µg florfenicol, ≥30 mm for 2 µg oxolinic acid and ≥51 mm for 30 µg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%), and oxolinic acid (99%)

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 β, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia

A T3SS Regulator Mutant of Vibrio alginolyticus Affects Antibiotic Susceptibilities and Provides Significant Protection to Danio rerio as a Live Attenuated Vaccine

Vibrio alginolyticus is a major cause of Vibriosis in farmed marine aquatic animals and has caused large economic losses to the Asian aquaculture industry in recent years. Therefore, it is necessary to control V. alginolyticus effectively. The virulence mechanism of V. alginolyticus, the Type III secretion system (T3SS), is closely related to its pathogenicity. In this study, the T3SS gene tyeA was cloned from V. alginolyticus wild-type strain HY9901 and the results showed that the deduced amino acid sequence of V. alginolyticus tyeA shared 75–83% homology with other Vibrio spp. The mutant strain HY9901ΔtyeA was constructed by Overlap-PCR and homologous recombination techniques. The HY9901ΔtyeA mutant exhibited an attenuated swarming phenotype and an ~40-fold reduction in virulence to zebrafish. However, the HY9901ΔtyeA mutant showed no difference in growth, biofilm formation and ECPase activity. Antibiotic susceptibility test was observed that wild and mutant strains were extremely susceptible to Amikacin, Minocycline, Gentamicin, Cefperazone; and resistant to oxacillin, clindamycin, ceftazidime. In contrast wild strains are sensitive to tetracycline, chloramphenicol, kanamycin, doxycycline, while mutant strains are resistant to them. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes, the results showed that compared with HY9901 wild type, ΔtyeA had increased expression of vscL, vscK, vscO, vopS, vopN, vscN, and hop. Following vaccination with the mutant strain, zebrafish had significantly higher survival than controls following infection with the wild-type HY9901 (71.2% relative percent survival; RPS). Analysis of immune gene expression by qPCR showed that vaccination with HY9901ΔtyeA increased the expression of IgM, IL-1β, IL-6, and TNF-α in zebrafish. This study provides evidence of protective efficacy of a live attenuated vaccine targeting the T3SS of V. alginolyticus which may be facilitated by up-regulated pro-inflammatory and immunoglobulin-related genes