Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.     

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.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.     

Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.     

Efficacy of different administration routes for vaccination against Vibrio anguillarum in Atlantic halibut (Hippoglossus hippoglossus L.)

Atlantic halibut (Hippoglossus hippoglossus L.) is a potentially important new species to cold-water aquaculture. Development of a viable industrial farming technique has been hampered by continued pathogen problems within the rearing cycle and there are several reports that indicated how susceptible juvenile halibut are to bacterial and viral diseases. Interest has been expressed, within the industry, over the possibility of vaccinating suitably sized animals to protect against the more common aquaculture pathogens. Vibrio spp. are of particular concern due to their ubiquitous nature and the relatively frequent occurrence of these pathogens within marine aquaculture. We have previously investigated the susceptibility of Atlantic halibut to infection by Vibrio anguillarum and the efficacy of intraperitoneal injected delivery of a commercial vaccine in protecting against the disease. Given the very high rate of protection offered by immunisation we wanted to investigate the effect of alternate routes of administration on the efficacy of the vaccine.     

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.     

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.)

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.     

抗鳗弧菌独特型单克隆抗体dsFv的构建及其噬菌体表面呈现

抗鳗弧菌独特型单克隆抗体 1E10是能够模拟鳗弧菌的保护性表位 ,可以作为疫苗使用的一种单克隆抗体 .利用基因工程抗体技术从抗鳗弧菌独特型单克隆抗体杂交瘤细胞株 1E10中克隆出抗体的重链及轻链可变区基因 (VH 和VL) .通过定点突变技术将VH4 4和VL10 5突变为半胱氨酸并且连接到噬菌体表达载体pCANTAB5E中 ,突变后的VH 和VL 基因位于cpⅢ先导序列和cpⅢ基因之间 ,在LacZ启动子调控之下 ,以融合蛋白的形式被导入细胞间隙 ,依靠链间二硫键组装成二硫键稳定型Fv抗体 (dsFv) .加入辅助噬菌体M13K0 7后 ,dsFv以融合蛋白的形式表达在噬菌体表面 .ELISA测定显示 :dsFv噬菌体能够与抗原结合并且这种结合呈噬菌体浓度依赖 .结果表明 :成功构建出了抗鳗弧菌独特型单克隆抗体dsFv基因并使其在噬菌体表面获得了正确呈现 .该dsFv噬菌体有望成为新一代基因工程疫苗用于预防鱼类鳗弧菌感染     

Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides

The objective of this study was to determine the effect of two levels of inclusion of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on growth, feed utilization, immune status and disease resistance of European sea bass (Dicentrarchus labrax). Specimens of 35 g at initial density of 3 kg/m3 were fed during 67 days at 0 per thousand, 2 per thousand and 4 per thousand dietary MOS level of inclusion in a commercial sea bass diet. Food conversion rate, specific growth rate, whole body biochemical composition, phagocyctic index of head kidney macrophages, NBT index, lysozyme and alternative complement pathway (ACP) activities as well as gut and liver histological structure were evaluated. Growth significantly increased at both MOS dietary inclusion levels. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients. No differences were found on gut histological evaluation. Statistical differences (P<0.05) on the phagocytic index were denoted with the inclusion of 4 per thousand Bio-Mos group. A positive correlation was found between the levels of lysozyme and alternative complement pathway activities in blood and the level of inclusion of MOS in diets. After the feeding trial, a cohabitation challenge test and direct gut inoculation were also performed with the pathogen Vibrio alginolyticus in a ratio 3:1. Twenty-one days post-challenge the number of cohabitant fish infected in the control group reached 33% comparing with none on the 0.4 per thousand MOS group. Finally, new fish were infected with V. alginolyticus by gut canalisation. After 24h post-infection no significant difference was denoted between groups and 48 h post-infection total infected fish in the control group was twice that of the 2 per thousand and 4 per thousand MOS groups.     

Glyceraldehyde-3-phosphate dehydrogenase of Edwardsiella tarda has protective antigenicity against Vibrio anguillarum in Japanese flounder

Edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be an effective vaccine candidate against infection by E. tarda in Japanese flounder Paralichthys olivaceus. The GAPDH of E. tarda is highly homologous to that of Vibrio cholerae (91%), and therefore E. tarda GAPDH may have protective antigenicity against Vibrio species. In this study, we immunized Japanese flounder with GAPDH of E. tarda and infected the fish with V anguillarum. The result showed that GAPDH prepared from E. tarda protected Japanese flounder effectively in a challenge of V anguillarum. Therefore, E. tarda GAPDH should be considered as a multi-purpose vaccine candidate against several kinds of pathogenic bacteria.     

Side effects in sea bass (Dicentrarchus labrax L.) due to intraperitoneal vaccination against vibriosis and pasteurellosis

Sea bass (Dicentrarchus labrax L.) were injected intraperitoneally with monovalent (Photobacterium damselae subsp. piscicida or Vibrio anguillarum) and divalent (Ph. damselae subsp. piscicida and V. anguillarum) vaccine formulations, with or without adjuvants (mineral oil, liposome or alginate), to evaluate the short and long-term pathological effects. Eight animals from each group were sampled one, two, four and 11 months after intraperitoneal injection. The acute peritoneal response and the progression to a chronic status were evaluated by analysing peritoneal leucocytes collected during the first days post-injection. To evaluate the chronic response, the late peritoneal leucocyte response was analysed and the peritoneal cavity was examined and the intra-abdominal lesion level scored based on a pre-defined scale. Correlation between leucocyte exudative response, tissue inflammatory response and the development of granuloma were sought. The acute leucocyte response was characterized by an early (24-48 h) mobilization of neutrophils and macrophages, with phagocyte numbers dependent on the formulation, but no significant variations were observed in lymphocytes/small cells and EGCs. Later on, a steady increase occurred in lymphocytes/small cells and EGCs and a high concentration in neutrophils and macrophages was maintained up to 30-60 days in groups i.p. injected with oil adjuvanted formulations with antigen. All the lesions observed were moderate, indicating that in sea bass, the pathological effects due to intraperitoneally injected vaccines are less severe than in other fish species. The divalent oil adjuvanted vaccine induced the most severe side effects, with macroscopic granulomas consistently present up to 11 months.