Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review

After substantial investments in research, the Atlantic halibut Hippoglossus hippoglossus is now being cultivated commercially in Norway, Iceland, Scotland and Canada. As with other domesticated species, disease problems have been experienced. This review summarizes the current state of knowledge of diseases of the Atlantic halibut, and their diagnosis, prophylaxis and treatment. In economic terms, the most important losses have been suffered at the larval and juvenile stages. The most important infections are caused by nodaviruses, causative agents of Viral Encephalopathy and Retinopathy (VER), which are the major reason why Norway's production of halibut fry has been level since 1995. An aquatic birnavirus, Infectious Pancreatic Necrosis Virus, is also an important agent of mortality. Vibrio anguillarum, Flexibacter ovolyticus and atypical Aeromonas salmonicida are the major bacterial pathogens. The protozoan parasites recorded include Ichthyobodo sp., the microsporidium Enterocytozoon sp., Trichodina hippoglossi, and the metazoan pathogens include myxozoans, helminths, Entobdella hippoglossi, Lepeophtheirus hippoglossi and other parasitic copepods. Experimental vaccines have been tested against V anguillarum and atypical A. salmonicida, with good results. A recombinant vaccine against nodaviruses is under development. A few trials have been carried out on non-specific immunostimulants, but no such treatment is currently available. A number of efficacy and pharmacokinetic trials with various antibacterial agents have also been published.     

Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and efficacy of protection induced by vaccination

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings. Serum agglutinating activity demonstrated a rise following vaccination, the mean log2 titre rising from 3.8 to 8.4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture

Aims: Subcellullar localizations and cross‐immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. Methods and Results: Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross‐protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%. Conclusions: Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross‐immunity against different pathogens. Significance and Impact of the Study: Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Screening and characterisation of potentially pathogenic bacteria associated with Atlantic cod Gadus morhua larvae: bath challenge trials using a multidish system

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.     

Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development

Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.     

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.     

Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.     

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.     

Ontogenetic development and composition of the mucous cells and the occurrence of saccular cells in the epidermis of Atlantic halibut

An increase in the number of mucous cells of the epidermis, as well as qualitative changes of the mucus composition from predominantly neutral to a mixture of neutral and sulphated glycoproteins occurred during the development from a pelagic larva to a bottom-dwelling flatfish. Numerous saccular cells were observed in the epidermis of the yolk-sac larvae that disappeared simultaneously as the mucous cells increased in number in the epidermis of the metamorphosed halibut. These findings may help to understand the protective role of the mucus layer of Atlantic halibut during development as compared to other fish species in aquaculture.     

Use of PCR-RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry

Small subunit ribosomal genes were explored using PCR-RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus). Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals. In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp. were subjected to a whole cell PCR protocol amplifying approximately 1500 bp of 16S rDNA. Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles. Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP. Efficient detection of polymorphism and the resolution of species and subspecies were achieved. Using the RFLP on 103 isolates generated as many as 22 genotypes. Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed. Partial sequencing found this tree to be dominated by gamma-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species. Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea. These species have not previously been reported as halibut flora. The most frequently isolated genotype resembled Vibrio salmonicida.     

Study on the immune enhancement of different immunoadjuvants used in the pentavalent vaccine for turbots

In this study, we investigated the immune enhancing effects of different adjuvants used in a pentavalent vaccine for turbots. The pentavalent vaccine consisted of inactive bacterial cells from five common pathogenic strains (Vibrio anguillarum, Vibrio scophtalmi, Edwardsiella tarda, Vibrio harveyi and Vibrio alginolyticus) and the adjuvants were astragalus polysaccharides (APS), propolis, and the Freund’s complete adjuvant (FCA). Turbots were immunized with the pentavalent vaccine alone or with one of the adjuvants, and the immune efficiency was evaluated by measuring the activities of lysozyme (LSZ) and superoxide dismutase (SOD), and serum antibody titers. Fish were also challenged with the pathogens after immunization and the relative percent survival (RPS) was assessed. Our results showed that APS, propolis, and FCA had significant immune-enhancing effects on turbots as shown by the higher titers of antibodies against the pathogens, increased LSZ and SOD activities, and enhanced RPS after challenge with pathogens. Among the three adjuvants, FCA had the most significant immune synergistic effects with the vaccine, and APS and propolis had lower and similar immune synergies.     

水产动物副溶血性弧菌的拮抗菌研究进展

细菌性疾病的爆发常造成水产养殖业的巨大经济损失,其中副溶血性弧菌(Vibrio parahaemolyticus)引起的细菌性疾病更是引起了人们的关注。拮抗菌在代谢活动中通过分泌抗菌物质直接对病原菌产生抑制或竞争作用来抑制或杀死病原菌,在病害防治中发挥着重大作用。对副溶血性弧菌拮抗菌的种类、产生拮抗物质的种类、筛选拮抗菌常用的方法以及拮抗细菌的抑菌机制进行阐述,以期为副溶血性弧菌的生物防治提供参考。