A comparative analysis of alternative splicing patterns in Atlantic salmon (Salmo salar) in response to Moritella viscosa and sea lice (Lepeophtheirus salmonis) infection.

Moritella viscosa (M. viscosa) and sea lice (Lepeophtheirus salmonis) are severe pathogens that primarily infect the skin of Atlantic salmon (Salmo salar), which cause significant economic losses in the farming industry. However, the pathogenesis and molecular mechanisms underlying the host's immune defence at the post-transcriptional level remain unclear. Alternative splicing (AS) is an evolutionarily conserved post-transcriptional mechanism that can greatly increase the richness of the transcriptome and proteome. In this study, transcriptomic data derived from skin tissues of Atlantic salmon after M. viscosa and sea lice infections were used to examine the AS profiles and their differential expression patterns. In total, we identified 33,044 AS events (involving 13,718 genes) in the control (CON) group, 35,147 AS events (involving 14,340 genes) in the M. viscosa infection (MV) group, and 30,364 AS events (involving 13,142 genes) in the sea lice infection (LC) group, respectively. Among the five types of AS identified in our study (i.e., SE, A5SS, A3SS, MXE, and RI), SE was the most prevalent type in all three groups (i.e., CON, MV, and LC groups). Decreased percent-spliced-in (PSI) levels were observed in SE events under both MV- and LC-infected conditions, suggesting that MV or LC infection elevated exon-skipping isoforms and promoted the selection of shorter transcripts in numerous DAS genes. In addition, most of the differential AS genes were found to be associated with pathways related to mRNA regulation, epithelial or muscle development, and immune response. These findings provide novel insights into the role of AS in host-pathogen interactions and represent the first comparative analysis of AS in response to bacterial and parasitic infections in fish.

Isolation and characterization of an antigen from the fish pathogen Moritella viscosa.

AIMS: Moritella viscosa is a Gram-negative psychrophilic bacterium that causes winter ulcer disease in farmed fish. The aim of the study was to describe an outer membrane protein of roughly 20 kDa in pathogenic M. viscosa and to compare the coincident protein of strains isolated from different fish species and geographical locations. METHODS AND RESULTS: The protein was isolated from a pathogenic strain of M. viscosa. An oligopeptide sequence obtained with MS/MS analysis showed homology to Escherichia coli OmpA and Neisseria surface protein A. The protein was named Moritella viscosa outer membrane protein 1 (MvOmp1), and sequence analysis confirmed that it is an integral membrane protein consisting of eight antiparallel ß-strands, three short periplasmic turns and four long hydrophilic extracellular loops. The encoding gene, mvomp1, was fully sequenced in nine strains representing different serotypes and phenotypes. The results revealed some differences in the extracellular loops between strains. The mvomp1 gene was cloned and expressed in E. coli, and the recombinant product was recognized by anti-M. viscosa polyclonal antisera. CONCLUSIONS: The results indicate that MvOmp1 is a major protective antigen of M. viscosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The results open up possibilities for use of the protein as a part of a subunit vaccine in the future.

Effects of Moritella viscosa antigens on pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) cell line (SHK-1).

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1 beta and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1 beta expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1 beta and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-beta and IL-8 expression.

Atlantic salmon bath challenged with Moritella viscosa--pathogen invasion and host response.

The Gram-negative bacterium Moritella viscosa is considered to be the main causative agent of winter ulcer, a disease that primarily affects salmonid fish in sea water during cold periods. The disease is initially characterised by localised swelling of the skin followed by development of lesions. To gain more knowledge of the role of M. viscosa in the pathogenesis of winter ulcer, 159 Atlantic salmon (80-110 g) were exposed to a bath challenge dose of 7 x 10(5) cfu ml(-1) for 1 h at 8.9 degrees C. The first mortalities were registered two days post-challenge and the mortality rate increased rapidly. Multi-organ samples were taken throughout the challenge for culture, immunohistochemistry and PCR analysis. Using real-time PCR, M. viscosa DNA was first detected in the gills of all fish examined 2, 6 and 12 h after challenge. From day 2, the bacterium was detected in the muscle/skin, head kidney, spleen and liver. This was in correlation with positive cultured samples and confirmed systemic infection. The early and consistent detection of M. viscosa DNA in gill samples, and less or not in muscle/skin or intestine, could suggest gills as a port of entry for the bacterium. Immunohistochemical analysis using a polyclonal antiserum against M. viscosa demonstrated generalised staining in the lumen of blood vessels and some positive mononuclear cells. The antigens recognised by the antiserum may have originated from extracellular bacterial products and be part of a bacterial invasion strategy. To better understand the immune response in salmon to M. viscosa infection, the expression profiles of the immune genes IL1 beta, C3, ISG15 and CD83 were studied. Increased expression of IL1 beta and C3 was not induced until day 7, which may suggest that M. viscosa might utilize escape mechanisms to evade the host's immune system by suppressing relevant immune responses.

A time-course study of gene expression and antibody repertoire at early time post vaccination of Atlantic salmon.

The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0-35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.

Antigen profiles of the fish pathogen Moritella viscosa and protection in fish.

AIMS: Moritella viscosa is a gram-negative bacterium that causes winter ulcer disease in salmonid fish cultured in sea water below 8 degrees C. The aim of this study was to study the antigen profiles of these bacteria and to reveal the protection which the antigens induce in fish. METHODS AND RESULTS: Lipooligosaccharides (LOS) and an approximately 17-19 kDa outer membrane antigen were shown to be the major specific antigens of M. viscosa. The size of the wall antigen differed between strain groups and even between strains reacting positively in the same sera. Four different serotypes of M. viscosa were determined by producing polyclonal sera. Western blot analysis revealed that sera from vaccinated fish groups that had good or fair protection reacted against the LOS and the 17/19 kDa antigen, while no antibody response was observed with sera from groups that showed no efficacy. CONCLUSIONS: The study provides evidence that LOS and an approximately 17-19 kDa outer membrane antigen are the major specific protective antigens of M. viscosa, and that the M. viscosa species consists of many different serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are important for the production of vaccines against winter ulcers and should also lead to better methods of verifying the bacteria and monitoring winter ulcers.

A DM9-containing protein from oyster Crassostrea gigas (CgDM9CP-2) serves as a multipotent pattern recognition receptor.

DM9 is a novel protein domain with unknown function originally discovered in Drosophila melanogaster. Recently, a protein harboring DM9 repeats was identified as mannose-specific lectin (CgCGL1, renamed as CgDM9CP-1) from the Pacific oyster Crassostrea gigas. In the present study, another DM9 containing protein was identified from oyster C. gigas (designated as CgDM9CP-2). The open reading frame of CgDM9CP-2 gene was of 432 bp, encoding a polypeptide of 143 amino acids with two tandem DM9 repeats. The deduced amino acid sequence of CgDM9CP-2 shared 60.8% identity with that of CgDM9CP-1. In the unrooted phylogenetic tree, CgDM9CP-2 was closely clustered with CgDM9CP-1, and then assigned into the branch of invertebrate DM9CPs. The mRNA transcripts of CgDM9CP-2 were expressed in all the tested tissues, including mantle, gonad, gills, adductor muscle, hemocytes, and hepatopancreas, with the highest expression level in gills. CgDM9CP-2 protein was mainly distributed on the cytomembrane of oyster hemocytes. After mannose stimulation, the mRNA expression of CgDM9CP-2 in gills was up-regulated to the peak level (5.90-fold of that in SSW group, p < 0.05) at 24 h, and kept at a significantly higher level compared with that in control group at 6-48 h. It significantly increased at 6 h (2.33-fold, p < 0.05), and 12 h (3.08-fold, p < 0.05) post Vibrio splendidus stimulation, and then gradually decreased from 48 to 72 h (p < 0.05) with significant difference comparing with that in control group. The recombinant CgDM9CP-2 protein (rCgDM9CP-2) displayed higher binding affinity to D-(+)-mannose while lower binding affinity to lipopolysaccharide and peptidoglycan. rCgDM9CP-2 also exhibited binding activity towards fungi (Pichia pastoris and Yarrowia lipolytica), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), and gram-negative bacteria (Escherichia coli, Vibrio anguillarum, Aeromonas hydrophila and V. splendidus). It could agglutinate fungi P. pastoris and Y. lipolytica, and inhibit the growth of P. pastoris, S. aureus, V. anguillarum, and V. splendidus. These results collectively indicated that CgDM9CP-2 not only served as a pattern recognition receptor with a broad range of recognition spectrum, but also involved in inhibiting the growth of invading microbe in the innate immune response of oyster, which would provide further evidence for the function of DM9 domain in the innate immune system.

Humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Moritella viscosa.

Immunization of Atlantic salmon, Salmo salar L., with experimental vaccine for protection against M. viscosa was tested. The antigen preparation used for the comparative studies was sonicate formalin killed M. viscose unwashed bacteria. Vaccination was carried out by intraperitoneal injection of bacterin in Biojec mineral oil as an adjuvant. When accumulated mortality in the control group with PBS of fish challenged by i.p. injection of M. viscose reached 100% with a challenge dose of 2.3 x 10(6) CFU, mortality in the experimental group was 71.23%. A positive correlation between the survival rates and corresponding median antibody levels of each group was found at the challenge time.

Vaccination against atypical furunculosis and winter ulcer disease of fish.

Atypical furunculosis is a problem in farming of salmonids and various other fish species caused by a heterogeneous group of atypical Aeromonas salmonicida strains. Winter ulcer is a disease of salmonids and cod caused by Moritella viscosa, but a number of fish species are susceptible to the infection. Vaccines are available against atypical furunculosis of salmonids, but their efficacy is dependent on the characteristics of the infective strain. Vaccines for non-salmonid fish are currently not commercially available. Furunculosis vaccines for salmon can induce cross protection against some atypical A. salmonicida infections and only in some fish species. Polyvalent injection vaccines based on inactivated bacterial cells are available against winter ulcer disease of salmonids. Outbreaks of winter ulcer disease in vaccinated salmon are, however, continuously reported.

Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects.

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.