Immersion challenge model for Flavobacterium psychrophilum infection of Atlantic salmon (Salmo salar L.) fry.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWBD) and rainbow trout fry syndrome (RTFS), which affect salmonids. To better understand this pathogen and its interaction with the host during infection, including to support the development of resistant breeds and new vaccines and treatments, there is a pressing need for reliable and reproducible immersion challenge models that more closely mimic natural routes of infection. The aim of this present study was to evaluate a challenge model developed previously for rainbow trout for use in Atlantic salmon. First, preliminary challenges were conducted in Atlantic salmon (n = 120) and rainbow trout (n = 80) fry using two F. psychrophilum isolates collected from each fish species, respectively; fish had been pretreated with 200 mg/L hydrogen peroxide for 1 h. Thereafter, the main challenge was performed for just one F. psychrophilum isolate for each species (at 2 × 107 CFU/mL) but using larger cohorts (Atlantic salmon: n = 1187; rainbow trout: n = 2701). Survival in the main challenge was 81.2% in Atlantic salmon (21 days post-challenge) and 45.3% in rainbow trout (31 days post-challenge). Mortalities progressed similarly during the preliminary and main challenges for both species, demonstrating the reproducibility of this model. This is the first immersion challenge model of F. psychrophilum to be developed successfully for Atlantic salmon.

A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus.

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus.

Aeromonas hydrophila ST251 and Aeromonas dhakensis are major emerging pathogens of striped catfish in Vietnam.

Introduction: Aeromonads 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. Methods: This 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. Results: During 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. Discussion: This 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.

In silico analysis of AhyI protein and AI-1 inhibition using N-cis-octadec-9z-enoyl-l-homoserine lactone inhibitor in Aeromonas hydrophila.

AhyI is homologous to the protein LuxI and is conserved throughout bacterial species including Aeromonas hydrophila. A. hydrophila causes opportunistic infections in fish and other aquatic organisms. Furthermore, this pathogennot only poses a great risk for the aquaculture industry, but also for human public health. AhyI (expressing acylhomoserine lactone) is responsible for the biosynthesis of autoinducer-1 (AI-1), commonly referred to as a quorum sensing (QS) signaling molecule, which plays an essential role in bacterial communication. Studying protein structure is essential for understanding molecular mechanisms of pathogenicity in microbes. Here, we have deduced a predicted structure of AhyI protein and characterized its function using in silico methods to aid the development of new treatments for controlling A.hydrophila infections. In addition to modeling AhyI, an appropriate inhibitor molecule was identified via high throughput virtual screening (HTVS) using mcule drug-like databases.The AhyI-inhibitor N-cis-octadec-9Z-enoyl-l-Homoserine lactone was selected withthe best drug score. In order to understand the pocket sites (ligand binding sites) and their interaction with the selected inhibitor, docking (predicted protein binding complex) servers were used and the selected ligand was docked with the predicted AhyI protein model. Remarkably, N-cis-octadec-9Z-enoyl-l-Homoserine lactone established interfaces with the protein via16 residues (V24, R27, F28, R31, W34, V36, D45, M77, F82, T101, R102, L103, 104, V143, S145, and V168), which are involved with regulating mechanisms of inhibition. These proposed predictions suggest that this inhibitor molecule may be used as a novel drug candidate for the inhibition of auto-inducer-1 (AI-1) activity.The N-cis-octadec-9Z-enoyl-l-Homoserine lactone inhibitor molecule was studied on cultured bacteria to validate its potency against AI-1 production. At a concentration of 40 µM, optimal inhibition efficiency of AI-1 was observedin bacterial culture media.These results suggest that the inhibitor molecule N-cis-octadec-9Z-enoyl-l-Homoserine lactone is a competitive inhibitor of AI-1 biosynthesis.

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-1ß, 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.

First Succinylome Profiling of Vibrio alginolyticus Reveals Key Role of Lysine Succinylation in Cellular Metabolism and Virulence.

Recent studies have shown that a key strategy of many pathogens is to use post-translational modification (PTMs) to modulate host factors critical for infection. Lysine succinylation (Ksuc) is a major PTM widespread in prokaryotic and eukaryotic cells, and is associated with the regulation of numerous important cellular processes. Vibrio alginolyticus is a common pathogen that causes serious disease problems in aquaculture. Here we used the affinity enrichment method with LC-MS/MS to report the first identification of 2082 lysine succinylation sites on 671 proteins in V. alginolyticus, and compared this with the lysine acetylation of V. alginolyticus in our previous work. The Ksuc modification of SodB and PEPCK proteins were further validated by Co-immunoprecipitation combined with Western blotting. Bioinformatics analysis showed that the identified lysine succinylated proteins are involved in various biological processes and central metabolism pathways. Moreover, a total of 1,005 (25.4%) succinyl sites on 502 (37.3%) proteins were also found to be acetylated, which indicated that an extensive crosstalk between acetylation and succinylation in V. alginolyticus occurs, especially in three central metabolic pathways: glycolysis/gluconeogenesis, TCA cycle, and pyruvate metabolism. Furthermore, we found at least 50 (7.45%) succinylated virulence factors, including LuxS, Tdh, SodB, PEPCK, ClpP, and the Sec system to play an important role in bacterial virulence. Taken together, this systematic analysis provides a basis for further study on the pathophysiological role of lysine succinylation in V. alginolyticus and provides targets for the development of attenuated vaccines.

Acetylome profiling of Vibrio alginolyticus reveals its role in bacterial virulence.

It is well known that lysine acetylation (Kace) modification is a common post-translational modification (PTM) that plays an important role in multiple biological and pathological functions in bacteria. However, few studies have focused on lysine acetylation modification in aquatic pathogens to date. In this study, the acetylome profiling of fish pathogen, Vibrio alginolyticus was investigated by combining affinity enrichment with LC MS/MS. A total of 2883 acetylation modification sites on 1178 proteins in this pathogen were identified. The Kace modification of several selected proteins were further validated by Co-immunocoprecipitation combined with Western blotting. Bioinformatics analysis showed that seven conserved motifs can be enriched among Kace peptides, and many of them were significantly enriched in metabolic processes such as biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and biosynthesis of amino acids, which was similar to data previously published for V. parahaemolyticus. Moreover, we found at least 102 acetylation modified proteins predicted as virulence factors, which indicate the important role of PTM on bacterial virulence. In general, our results provide a promising starting point for further investigations of the biological role of lysine acetylation on bacterial virulence in V. alginolyticus. BIOLOGICAL SIGNIFICANCE: Lysine acetylation (Kace) modification, is well known to play important roles on diverse biological functions in prokaryotic cell, whereas few studies focused on aquatic pathogens to date. In this study, the acetylome profiling of fish pathogen, Vibrio alginolyticus was investigated by combining affinity enrichment with LC MS/MS. A total of 2883 acetylation modification sites on 1178 proteins in this pathogen were identified. The further bioinformatics analysis showed that seven conserved motifs can be enriched among Kace peptides, and many of them were significantly enriched in metabolic processes, which was similar to data previously published for V. parahemolyticus. Moreover, we found at least 102 acetylation modified proteins predicted as virulence factors, which indicate the important role of PTM on bacterial virulence. In general, our results provide a promising starting point for further investigations of the biological role of lysine acetylation on bacterial virulence in V. alginolyticus.

Survival and immune response of white shrimp Litopenaeus vannamei following single and concurrent infections with WSSV and Vibrio parahaemolyticus.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV - Vibrio co-infection.

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 ß, 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.

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.

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-1ß 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 profileobserved 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.

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

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

Identification of DLD, by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides.

Vibrio spp. represent a serious threat to the culture of Epinephelus coioides (Orange-spotted Grouper) in Southeast Asia. In this study we used two-dimensional electrophoresis (2-DE) and Western blotting to identify common immunogenic proteins of Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. Membranes were probed with orange-spotted grouper anti-V. alginolyticus sera and accordingly 60, 58 and 48 immunogenic protein spots were detected. By matching analysis for the three Western blotting membranes, 6 cross immunogenic spots for the three Vibrio species were identified. They were Outer membrane protein W (OmpW), dihydrolipoamide dehydrogenase (DLD), succinate dehydrogenase flavoprotein subunit(SDHA), elongation factor Ts(Ts), peptide ABC transporter periplasmic peptide-binding protein and phosphoenolpyruvate carboxykinase(PEPCK). One of the proteins, DLD, was used to evaluate the cross protective function for E. coioides with a bacterial immunization and challenge method. The relative percent survival rate of E. coioides against V. alginolyticus, V. harveyi and V. parahaemolyticus was 90%, 86% and 80%, respectively. This work may provide potential cross protective vaccine candidate antigens for three Vibrio species, and DLD may be considered as an effective cross-protective immunogen against three Vibrio species.