淋巴囊肿病毒结构蛋白及其抗原性分析

病鱼为威海水产养殖场感染淋巴囊肿病的牙鲆（Paralichthys olivaceus）,收集病鱼的囊肿组织,匀浆破碎,采用差速离心和蔗糖密度梯度离心方法,分离纯化淋巴囊肿病毒粒子.负染后,电镜观察证实获得的病毒纯度高,杂质极少,病毒粒子呈近似于圆形的多角形,结构完整.纯化的淋巴囊肿病毒粒子经SDS-PAGE,硝酸银染色后,电泳图谱清晰显示病毒结构蛋白带共有22条,且分子量主要集中在123～26 kD.应用Western blotting法分析病毒结构蛋白的抗原性,结果显示,分子量分别为123.55 kD、65.292 kD和54.438 kD的3条蛋白带发生了免疫反应,其中分子量为65.292 kD的蛋白带反应强度明显高于其他2条蛋白带.本研究旨为确定淋巴囊肿病毒主要衣壳蛋白提供基础依据.[中国水产科学,2006,13（3）：415-420]     

Immune responses and gut morphology of Senegalese sole (Solea senegalensis,Kaup 1858) fed monospecies and multispecies probiotics

The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non‐specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1‐month trial. Fish were fed for 1‐month two diets with 1.0 or 4.6 × 10⁶ CFU kg^?1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 10⁵ CFU kg^?1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A₁ the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A₂ versus B₂). The current study indicate that the use of the multispecies probiotic at 1.0 × 10⁶ CFU kg^?1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.     

Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.     

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis,Kaup) at early stages of development

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. ‘Minor’ anomalies were predominant, and some of the detected disorders might be a result of non‐/low‐pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.     

Phylogenetic analysis and molecular methods for the detection of lymphocystis disease virus from yellow perch, Perca flavescens (Mitchell)

Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch.     

Dietary protein, growth, nutrient utilization and body composition of juvenile blackspot seabream, Pagellus bogaraveo (Brunnich)

When considering new marine species for Mediterranean aquaculture, blackspot seabream emerges as a potential candidate. However, there are scarce data on the nutritional requirements and optimal growth conditions of this species. A 12‐week feeding trial was conducted to evaluate the effects of dietary protein on growth, nutrient utilization and body composition of blackspot seabream (23 g). Five isolipidic diets (12%) with graded levels of protein (20–60%) were distributed, twice a day, to duplicate groups of fish, until satiation. Growth increased significantly with increasing dietary protein up to 40%, but higher protein levels induced a similar daily growth index (1.4). Feed conversion ratio (FCR) decreased with increasing levels of dietary protein (4.2–1.6). No significant differences were detected in protein of whole body blackspot seabream among treatments, but fat percentage decreased with increasing dietary protein. Dry matter and energy digestibility showed a concomitant increase with the reduction in dietary wheat meal, attaining maximal values with high protein diets. These results suggested that the most favourable values for growth and FCR are obtained with diets containing 40% protein. However, the excessive lipid deposition reveals that more nutritional studies are necessary before the species can be established in aquaculture.     

Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): evaluation in larval and postlarval stages

The Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.     

Effect of dietary carbohydrate-to-lipid ratios on growth, lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis, Kaup)

A study was undertaken to determine the effect of various dietary carbohydrate‐to‐lipid ratios on growth performance, whole‐body composition and tissue lipid content in Senegalese sole (Solea senegalensis) juveniles. Data on the dietary regulation of key hepatic enzymes of the lipogenic and glycolytic pathways (glucose‐6‐phosphate dehydrogenase, G6PD; malic enzyme, ME; fatty acid synthetase, FAS; pyruvate kinase, PK and glucokinase, GK) were also generated. Four isonitrogenous (crude protein: 52% dry matter (DM)) diets were formulated to contain one of two lipid levels (11% and 21% DM). Within each dietary lipid level, the nature of the carbohydrate fraction (raw or extruded peas) was varied. Triplicate groups of 54 sole (initial body weight: 23.6±1.2 g) were grown in recirculated seawater over 67 days. Fish were fed using automated feeders. At the end of the study, whole‐body, liver, viscera and muscle samples were withdrawn for analyses. During the experimental period, the mean fish weight about doubled in all treatments. No significant differences were found in growth performance (ranging from 1.1% to 1.4% body weight day^?1) among dietary treatments. High‐fat diets increased whole‐body fat content. Similarly, daily fat gain ranged from 0.54 to 0.78 g kg^?1 day^?1 and highest values were found in fish fed high‐lipid diets. Dietary treatments also affected tissue lipid content (liver, viscera and muscle), with highest values in fish fed high‐fat diets. The nature of dietary carbohydrates had little influence on performance criteria, but affected tissue lipid deposition. The activities of G6PD, ME and FAS were depressed by elevated levels of dietary lipid, confirming the inhibitory effect of dietary fats on lipid biosynthesis. At both dietary lipid levels, ME and FAS activities were little affected by dietary carbohydrate. Activities of PK and GK were not affected by the starch level of the diets. In Senegalese sole juveniles, the lipogenic pathway is more susceptible to modulation by dietary means (particularly through lipid intake) than the glycolytic pathway.     

Dietary protein/lipid level and protein source effects on growth,tissue composition and lipid metabolism of blackspot seabream (Pagellus bogaraveo)

A study was carried out to determine the effects of fish meal (FM) replacement by plant protein (PP) on growth, body composition and lipid metabolism of blackspot seabream fed different protein/lipid levels. Four experimental diets were formulated to contain two protein (P) and lipid (L) levels (60P/6L or 50P/10L), varying in their protein source (100% FM or 50% FM: 50% PP). Dietary inclusion of PP did not affect growth of fish fed 60P/6L, although fish fed 50P/10L exhibited lower final body weight and daily growth index. Fish fed 60P/6L presented the highest protein and the lowest lipid content. FM replacement by PP has decreased muscle n‐3 whereas the n‐6 fatty acids increased. Glucose‐6‐phosphate dehydrogenase and fatty acid synthetase (FAS) were depressed in fish fed 50P/10L. FAS was significantly increased with 60P/6L PP which was positively correlated with lipid retention data. Those results suggest the conversion of other nutrient than lipid (protein and/or carbohydrates) into corporal fat. Hepatic lipoprotein lipase activity was lowest in fish fed PP diets. Plasma glucose peaked 1–2 h postfeeding, in all groups and was generally higher with 60P/6L FM. This work shown that besides dietary P/L level, protein source has a strong effect on species lipogenesis and lipid retention. Hence, the 50P/10L FM diet was the most cost‐effective for blackspot seabream juveniles.     

Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup)

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.     

Stocking density affects Senegalese sole (Solea senegalensis,Kaup) growth independently of size dispersion,evaluated using an individual photo‐identification technique

To assess how initial size dispersion affects Senegalese sole (Solea senegalensis, Kaup) growth, 128 fish were stocked either under homogeneous (HOM) or heterogeneous (HET) initial size dispersion (standard length, CV 11.8% and 29.7% respectively), and also under low (LD) and high (HD) stocking density (60% and 180% of bottom occupation). After 105 days, growth parameters, evaluated at an individual level using linear mixed‐effects models, were not significantly affected by initial size dispersion, LD fish growing significantly faster than HD fish. Variance of individual growth rate distributions of weight and standard length were lowest for LD‐HOM fish, indicating the most regular growth, and highest for LD‐HET. The diminution of the coefficient of variation along time in HET groups (between 13% and 15.4% for weight, and between 4.3% and 5.8% for standard length), and its slight decrease in the HOM groups (between 1% and 3% for both parameters), is consistent with the absence of strong competition between individuals.     

Effectiveness of a divalent vaccine for sole, Solea senegalensis (Kaup), against Vibrio harveyi and Photobacterium damselae subsp. piscicida

The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.     

淋巴囊肿病毒27.8 ku受体蛋白在牙鲆组织中的分布

应用抗牙鲆淋巴囊肿病毒(lymphocystis disease virus,LCDV)受体蛋白(27.8 ku)的单克隆抗体(2G11和3D9)定位LCDV受体蛋白在牙鲆组织中的分布。通过对牙鲆外周血、白细胞、鳃、胃、肠、表皮、肝脏、头肾、体肾、脾、性腺、脑、心脏等进行LCDV受体蛋白的间接免疫荧光与免疫组织化学定位观察,发现在牙鲆外周血白细胞的细胞膜、鳃上皮细胞、表皮、胃黏膜上皮细胞顶端、肠上皮细胞、肝细胞、脾表层结缔组织细胞及头肾后端的肾小管上皮细胞内均有较强的阳性信号,表明这些部位分布有LCDV的27.8 ku受体蛋白,但在体肾、性腺、脑、心脏及外周血红细胞中未观察到阳性信号。推测LCDV通过与鳃、表皮及消化道上皮的受体结合进入牙鲆体内,通过与外周血白细胞上的受体结合侵染白细胞而进入血液循环,进而感染肝脏、脾脏、头肾等器官。     

牙鲆淋巴囊肿病毒一抗原蛋白的确认及定位

以牙鲆淋巴囊肿病毒（LCDV）为抗原免疫Balb/c小鼠,而后将小鼠脾细胞与P3U1骨髓瘤细胞融合,以囊肿组织冰冻切片的免疫荧光染色筛选杂交瘤细胞,阳性结果显示特异性块状荧光信号集中在囊肿细胞的细胞质边缘部分,且多个荧光信号相连呈现链圈状,有限稀释 法克隆阳性杂交瘤细胞,三次克隆后获得4株稳定产生抗LCDV抗体的单克隆杂交瘤细胞株（1A8、1D7、2B6、2D11）。应用Western-blotting法分析单抗识别蛋白的分子量,结果显示,单抗1D7 和2B6均能特异性结合一条分子量116 kD病毒多肽;应用免疫电镜技术定位单抗识别的抗原决定簇,结果发现胶体金颗粒集中吸附在病毒粒子衣壳周围,且背景清洁,无散在的金颗粒或其他污染物。实验结果说明分子量约为116 kD的蛋白多肽为LCDV病毒衣壳蛋白,且具有线性抗原决定簇。     

Cellular and humoral immune responses of Senegalese sole,Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate‐buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL^?1) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.