Diseased Turbot Research Articles

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1β, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

Rapid pathogen discovery in diseased turbot (Scophthalmus maximus) using 16S rRNA high throughput sequencing

Pathogenic bacterium discovery using 16S rRNA high throughput sequencing has become more commonplace. It offers advantages of being high-throughput screening, cost-effective (less than $15 per sample), and rapid (less than one week). Fish pathogens causing aquatic bacterial diseases outbreak represent a major threat to aquaculture industry. A haemorrhagic disease in turbot (Scophthalmus maximus) outbreaks with unknown etiology in Haiyang, China. Initial analyses suggested it was a bacterial disease based on gross pathology of diseased fish containing ascitic fluid from the distended abdomen, a swollen intestine, an inflamed and red mouth. To fully understand the accurate etiology, distribution of pathogenic bacterium and comprehensive information on the diseased turbots is required. Tested tissues (ascites, heart, eye, mouth, intestine, spleen, and liver) with typical symptoms of diseased turbots were collected and 16 s rRNA sequencing was performed using the Illumina Mi-Seq platform. Sequencing results revealed generally low microbial diversity (< 50 %) in different infected tissues, but the microbiota did exhibit a little higher diversity in the intestine than in the other tissues. A key finding was that Edwardsiella was the most abundant genus detected in all the fish tissues. It was indicated that Edwardsiella spp. was the main causative pathogen in the diseased turbot. The evidence also suggested that Edwardsiella spp. as a severely pathogenic bacteria may cause disruption of the healthy balance in the host. Our study was a new attempt to investigate unknown pathogens in clinical marine fish specimens through the high-throughput next-generation sequencing technology.

Isolation, identification and vaccine development of serotype III Streptococcus parauberis in turbot (Scophthalmus maximus) in China

Streptococcus parauberis is an important disease in farming fish that could infect a series of fish causing streptococcosis, which leads to significant economic losses. In this research, we first characterized the streptococcosis caused by S. parauberis in farming fish in China. In 2019, bacteria were isolated from diseased turbot in 3 farms located in Penglai, Haiyang, and Laizhou city, Shandong Province, and identified as S. parauberis by 16S rRNA sequencing and Biolog GenIII. Capsule polysaccharide (cps) genes of isolates were obtained by whole genome sequencing and aligned with the cps cluster in GenBank. By cps genes analysis, the isolates were all classified as serotype III, which were different from the isolates that infect flounder (Paralichthys olivaceus) in Japan and South Korea, but the same with the isolates in Spain and the USA reported recently. Then the serotype III S. parauberis vaccines were developed for turbot. The specific antibody could be detected in the vaccinated fish, and the specific antibody from oil emulsified vaccine group fish was significantly higher than that of the water-based vaccine group from 14 days post vaccination. The relative percent of survival were 78.00% and 100% for water-based vaccine and Marcol 52 emulsified vaccine, respectively. To our knowledge, this is the first report of S. parauberis infection in farming fish in China, and would provide important information for disease control and prevention for streptococcosis in aquaculture.

Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China.

Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A.salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A.salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A.salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.

Mitochondrial genome sequencing of notorious scuticociliates (Pseudocohnilembus persalinus) isolated from Turbot (Scophthalmus maximus L.)

Previously, a pathogenic ciliate was isolated from the surface ulcer of a diseased Turbot (Scophthalmus maximus L.) at an aquaculture farm in North China. After morphological and molecular biological identification based on 18rRNA, the ciliated was identified as the notorious scuticociliates (Pseudocohnilembus persalinus). In this study, the whole sequence of the mitochondrial genomic gene of P. persalinus was carried out. The sequencing results showed that the complete sequence of P. persalinus mitogenome was 38,375 bp. There were 2 rRNAs, 4 tRNAs, and 34 protein-coding genes (PCGs), respectively, located on both the heavy strand and the light stand. 15 PCGs were on the heavy strand, and 19 PCGs on the light strand. Besides, phylogenetic trees among 11 ciliates were constructed based on the sequences of 17 PCGs located in mitogenome using BI methods. The results of clustering showed that P. persalinus and Uronema marinum was the first cluster belonging to the order Scuticociliatida. Our research results will further provide primary data for evolution and classified study of scuticociliates.

Draft Genome Sequence of Edwardsiellapiscicida Strain ACC35.1 Isolated from Diseased Turbot (Scophthalmus maximus) in Europe.

ABSTRACT Edwardsiella piscicida is a bacterial fish pathogen with a high degree of virulence. The strain ACC35.1 was isolated from diseased turbot in Europe. The draft genome sequence comprises 3.84 Mb with a G+C content of 59.8% and >3,450 protein-coding genes.

Edwardsiellosis in farmed turbot, Scophthalmus maximus (L.), associated with an unusual variant of Edwardsiella tarda: a clinical, aetiological and histopathological study

AbstractDuring 2005 and 2010, a survey of edwardsiellosis on eight turbot, Scophthalmus maximus (L.), farms was conducted in China. This report presents the detailed results of the study on this disease. Diseased turbot displayed two distinct types of gross signs: black discoloration of the dorsal skin on the posterior portion of the body; and red cutaneous foci on the ventral side. Internally, the most pronounced clinical signs in all fish examined were enlarged kidneys. The causal agent of the disease was finally proved to be one species of bacterium that was identified as Edwardsiella tarda by physiological and biochemical tests, API 32E and 16S ribosomal RNA sequence analysis. It is noteworthy that unlike the commonly described E. tarda strains, the isolates in this study were non‐motile strains without flagella. A histopathological study revealed that E. tarda infection was systemic in turbot and that kidney showed the most significant pathological changes, including acute focal necrosis, an influx of macrophages and formation of granuloma. The most common histopathological characteristics of this disease are the proliferation of macrophage in various organs and formation of granuloma. In addition, this article also gave background information on the disease and presented the results of virulence tests with the E. tarda strain identified in this study.

Turbot reovirus (SMReV) genome encoding a FAST protein with a non-AUG start site

BackgroundA virus was isolated from diseased turbot Scophthalmus maximus in China. Biophysical and biochemical assays, electron microscopy, and genome electrophoresis revealed that the virus belonged to the genus Aquareovirus, and was named Scophthalmus maximus reovirus (SMReV). To the best of our knowledge, no complete sequence of an aquareovirus from marine fish has been determined. Therefore, the complete characterization and analysis of the genome of this novel aquareovirus will facilitate further understanding of the taxonomic distribution of aquareovirus species and the molecular mechanism of its pathogenesis.ResultsThe full-length genome sequences of SMReV were determined. It comprises eleven dsRNA segments covering 24,042 base pairs and has the largest S4 genome segment in the sequenced aquareoviruses. Sequence analysis showed that all of the segments contained six conserved nucleotides at the 5' end and five conserved nucleotides at the 3' end (5'-GUUUUA ---- UCAUC-3'). The encoded amino acid sequences share the highest sequence identities with the respective proteins of aquareoviruses in species group Aquareovirus A. Phylogenetic analysis based on the major outer capsid protein VP7 and RNA-dependent RNA polymerase were performed. Members in Aquareovirus were clustered in two groups, one from fresh water fish and the other from marine fish. Furthermore, a fusion associated small transmembrane (FAST) protein NS22, which is translated from a non-AUG start site, was identified in the S7 segment.ConclusionsThis study has provided the complete genome sequence of a novel isolated aquareovirus from marine fish. Amino acids comparison and phylogenetic analysis suggested that SMReV was a new aquareovirus in the species group Aquareovirus A. Phylogenetic analysis among aquareoviruses revealed that VP7 could be used as a reference to divide the aquareovirus from hosts in fresh water or marine. In addition, a FAST protein with a non-AUG start site was identified, which partially contributed to the cytopathic effect caused by the virus infection. These results provide new insights into the virus-host and virus-environment interactions.

Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA 7) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size.

Genetic characterization of pAsa6, a new plasmid from Aeromonas salmonicida subsp. salmonicida that encodes a type III effector protein AopH homolog

A new plasmid designated pAsa6 from an Aeromonas salmonicida subsp. salmonicida strain isolated from diseased turbot has been characterized. pAsa6 consists of 18536 bp, has a G+C content of 53.8% and encodes 20 predicted open-reading frames (ORFs). Eight ORFs showed homology to transposases, of which six are complete and two are partial IS sequences. Two ORFs showed homology to replication proteins, and six ORFs showed homology to hypothetical proteins. Two ORFs are truncated homologs of putative A. salmonicida sulfatases. Two genes, aopH and sycH encode homologs of an effector protein for which a role in fish colonization by A. salmonicida has been previously reported, and its chaperone, respectively. The results of filter conjugation experiments suggested that pAsa6 is not mobilizable, as it failed to be conjugally-transferred to several species of marine bacteria tested. All the ORFs of pAsa6 with the exception of four copies of a IS 1 transposase gene, have a counterpart in the recently sequenced 155-kb A. salmonicida plasmid pAsa5, suggesting either that pAsa6 is a derivative of pAsa5, or that pAsa5 is the result of the fusion of a pAsa6-like plasmid and a larger plasmid of ca. 135-kb. The pAsa6-encoded repA and aopH genes could be PCR-amplified from strains lacking pAsa6, suggesting presence of a large, possibly pAsa5-like plasmid that was not detected on agarose gels, or the existence of chromosome-integrated plasmid sequences. This study demonstrates that genomic locations for the aopH gene different to pAsa5 or pAsa5-like plasmids exist in A. salmonicida.

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.

The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China

An iridovirus-like agent associated with the ‘reddish body syndrome’ (RBS) of farmed turbot Scophthalmus maximus was found in China. By light microscope, many enlarged cells were observed in the spleen and kidney of diseased turbot. Iridovirus-like particles were detected by transmission electron microscopic (TEM) examination in various organs of diseased turbot. The virion had an envelope and viral particles were only present in the cytoplasm of infected cells. TEM images of virions showed them to be icosahedral in symmetry, measuring 120–130 nm from vertex to vertex and 110–116 nm from face to face. Complete virions consisted of three layers: the outer layer was an icosahedral capsid 10–14 nm in thickness, the intermediate layer was an 11–15 nm thick translucent space and the inner spheroid core was a homogeneously electron-dense nucleoid measuring 64–70 nm in diameter. The virus particle acquired an envelope through budding from the cytoplasm into a cytoplasmic vesicle. Infected cells became hypertrophic and the cytoplasm was homogeneous. According to the TEM examination, the virions were mostly in cells of gill, intestinal submucosa, spleen and basement membranes of capillaries in glomeruli of the diseased fish. The spleen was the major target organ for the virus. Further analysis using PCR suggested that the molecular characterization of the virus was different from that of red sea bream iridovirus (RSIV) in Japan, Korea, Taiwan and infectious spleen and kidney necrosis virus (ISKNV) in China. This is the first report of iridovirus infection in turbot in China.

Induction of apoptosis in a carp leucocyte cell line infected with turbot ( Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot ( Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells.

Control of Vibrio alginolyticus in Artemia culture by treatment with bacterial probiotics

In order to determine the influence of six lactic acid bacterial (LAB) strains, lactic acid and major extracellular products (ECPs) of LAB on the bacterial load of Artemia culture, two experiments were carried out. In both experiments, the dominant bacterial species were identified as Vibrio alginolyticus. The treatment with Lactobacillus brevis (1×10 8 bacteria/ml) and lactic acid (0.1 g/l) reduced the load of this bacterium in the Artemia culture water. Biochemical and morphological characteristics of the V. alginolyticus strains isolated from Artemia culture were determined. For comparative purposes, another V. alginolyticus strain isolated from diseased turbot larvae was also included. The in vitro antibacterial activity of extracellular products (ECPs) from LAB, as well as lactic acid (0.1 and 0.05 g/l), was assessed against both V. alginolyticus strains. ECPs from LAB were able to inhibit the growth of V. alginolyticus, especially those from L. brevis. In the case of lactic acid treatments, no significant inhibition of V. alginolyticus growth was observed.

Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment

The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.

Genetic analysis of turbot pathogenic Streptococcus parauberis strains by ribotyping and random amplified polymorphic DNA

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.

Genetic analysis of turbot pathogenicStreptococcus parauberisstrains by ribotyping and random amplified polymorphic DNA

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures.

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.

Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.).

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of viral haemorrhagic septicaemia virus (VHSV), the rhabdovirus which causes viral haemorrhagic septicaemia (VHS) in farmed salmonid fish. Phylogenetic analysis of a 360 nt region of the glycoprotein gene from a range of marine and fresh water VHSV isolates identified three genogroups, I-III. Significantly, two virus isolates recovered from ulcerated North Sea cod caught off the Shetland Islands, and an isolate recovered from diseased turbot farmed on the island of Gigha, Scotland were assigned to the same genogroup. Moreover, a virus isolated from diseased turbot farmed on the Baltic Sea coast shared 99.4% nucleotide sequence similarity with a virus associated with a VHS outbreak in rainbow trout. This is the first time that a genetic link between a VHS outbreak and natural VHSV infections of marine fish species has been demonstrated.

Antigenic characterization of Vibrio anguillarum-related organisms isolated from turbot and cod

This work reports on the antigenic characterization and virulence for rainbow trout Oncorhynchus mykiss of Vibrio anguillarum-related (VAR) strains isolated from diseased turbot Scophthalmus maximus and cod Gadus morhua in Spain and Denmark. These vibrio strains belong to Vibrio splendidus biovar I (serogroups B, C, D, F and G) and V. pelagius biovar I (serogroup A). The pathogenicity assays demonstrated that 10 of 15 (67%) VAR strains tested were virulent for rainbow trout, showing an LD50 (50% lethal dose) ranging tram 8.4 x 103 to 7 x 105 CFU (colony forming units) per fish. Moreover, all the V. splendidus biovar I strains of serogroup F, which is the predominant type among strains isolated from cod, were virulent for rainbow trout, while V. splendidus biovar I strains isolated from turbot were avirulent. Analysis of lipopolysaccharide (LPS) and membrane proteins showed that VAR strains belonging to different serogroups possessed distinct electrophoretic banding patterns. The immunoblot analysis demonstrated a high immunorelation among VAR strains of the same serogroups, while strains from different serogroups were not immunologically related. Immunoblot assays also confirmed the dissimilarities in LPS structure observed among the distinct subgroups of serogroup F. These findings suggest that present vibriosis vaccines for cod could be improved if V. splendidus biovar I representative of serogroup F (subgroups α and β) were tested in trial vaccines.