格氏乳球菌分离株的血清同源性及血清学检验

以分离于病(死)牙鲆(Paralichthys olivaceus L.)的格氏乳球菌(Lactococcus garvieae)的代表菌株(T030817-1株),分别制备全菌(OK)及热处理(121℃作用1h)菌体(O)免疫原,强化免疫接种家兔制备相应抗血清,对10株格氏乳球菌分离株进行了血清同源性检验。结果表明,供试10株菌均存在同种的表面K抗原和同种的菌体O抗原(血清同源),其中O抗原具有强免疫原性、K抗原免疫原性及O凝集抑制阻断作用弱;以相应OK抗血清为第1抗体、标准的羊抗兔IgG荧光抗体为第2抗体,对10株格氏乳球菌的纯培养物及用代表菌株(T030817-1)人工感染病死牙鲆的肝组织进行了间接荧光抗体染色,结果显示了特异的荧光反应;同时,对从人工感染死亡牙鲆分离的6株纯培养菌以凝集试验进行了检验,亦显示特异凝集反应。     

牙鲆迟钝爱德华氏菌病及其病原特征

爱德华氏菌属(Edwardsiella)的迟钝爱德华氏菌(E.tarda),是多种淡、海水养殖鱼类的一种重要病原菌,在世界不少国家和地区均有由该菌引起的、不同程     

牙鲆病原迟钝爱德华氏菌的药物敏感性测定与分析

对分离并经鉴定的牙鲆病原迟钝爱德华氏菌进行了常用抗菌类药物的敏感性测定,结果表明供试的35株迟钝爱德华氏菌,均对所测定的37种抗菌类药物中的头孢唑啉等20种药物高度敏感、对青霉素G等5种药物耐药、对氨苄青霉素等12种药物表现在株间存在差异。     

牙鲆腹水病病原研究

描述了牙鲆腹水病的症状,经对细菌的分离与鉴定表明所检病例均为迟钝爱德华氏菌的单独感染。用分离菌做对健康牙鲆的人工感染试验,表明了相应的原发病原学意义及较强的致病作用。药敏试验表明,对供试17种抗生素中的氨曲南等15种药物敏感,对四环素耐药,对磺胺甲恶唑/甲氧苄啶(复方新诺明)的耐药表现了株间差异。     

牙鲆腹水病病原及免疫学防治效果初步研究

从患有腹水病的牙鲆体内和体表分离纯化菌株,经人工感染实验,证实分离的病原菌为腹水病的致病菌,通过形态学和理化特性研究,确定该病原菌为迟钝爱德华氏菌.用较强毒力的菌株制作福尔马林灭活疫苗,对牙鲆进行3种途径免疫接种,测定血清凝集抗体效价和免疫保护力,3种免疫途径中以腹腔注射效果最佳,免疫保护力达88%.     

牙鲆迟钝爱德华氏菌血清型及荧光抗体检验

以迟钝爱德华氏菌的代表菌株(HC010907-1)为免疫原,制备免疫血清,对分离于牙鲆鱼的130株迟钝爱德华氏菌进行了血清型检定,结果表明:供试的130株迟钝爱德华氏菌均为同种血清型;同时以此免疫血清为第一抗体,以标准羊抗兔IgG荧光抗体为第二抗体,进行了荧光抗体技术检验迟钝爱德华氏菌的可行性试验,表明亦具有较强的特异性。     

抗迟缓爱德华氏菌病牙鲆家系的筛选与分析

牙鲆(Paralichthys olivaceus)养殖病害日益严重,迟缓爱德华氏菌(Edwardsiella tarda)是牙鲆养殖中的主要致病菌,带来了极大的经济损失,为了培育出对该病抵抗能力强的牙鲆新品种,2014年4–6月间,利用我们长期以来建立和筛选出来的牙鲆抗病群体和家系的亲鱼,通过杂交,自交,雌核发育建立47个家系,9–10月间,对其中39个家系进行感染实验,先用少量鱼进行预实验摸索出半致死浓度,然后再进行正式感染实验,感染数量为每个家系80尾。各家系的存活率范围为1.19%~51.19%,最终平均存活率为20.29%,认为存活率高于30%的7个家系为抗病力强家系,存活率在20.29%~30%的9个家系为抗病力一般家系,平均存活率以下的23个家系为抗病力弱家系。1406#家系抗病力最强,它为1005#家系的自交后代,而其他抗病力强家系也大多是1005#、09104#、0915#的后代。而09104#为0768#的后代,并且0768#抗鳗弧菌病能力强。1005#为韩国群体自交后代,0915#为韩国群体和日本群体杂交后代。这些抗病力强的牙鲆可以作为新品系进行推广养殖,可望减少牙鲆腹水病发生。     

应用Dot-ELISA快速检测牙鲆腹水病病原的研究

选用硝酸纤维素膜作固相载体,建立检测迟钝爱德华氏菌的斑点酶联免疫吸附试验诊断方法。根据棋盘试验,确定迟钝爱德华氏菌免疫血清最佳工作浓度为1∶800,酶标抗体最佳工作浓度为1∶200,以出现明显清晰斑点者判定为阳性。用该方法检测时,迟钝爱德华氏菌呈阳性,温和气单胞菌、嗜水气单胞菌、河流弧菌、溶藻弧菌、鳗弧菌、腐败希瓦氏菌、产碱普罗威斯登菌、阪崎肠杆菌和大肠杆菌均呈阴性。试验结果表明,Dot-ELISA方法简便、特异、快速、结果直观,便于在基层推广使用。     

牙鲆腹水病病原免疫血清的制备和纯化试验研究

用迟钝爱德华氏菌免疫家兔,制备出高效价迟钝爱德华氏菌免疫血清.先制备迟钝爱德华氏菌抗原, 耳缘静脉注射免疫家兔.经微量反应板法检测血清效价, 效价达到1 ∶ 2560;用Millipore Montage抗体纯化试剂纯化抗体,纯化的抗体经SDS-PAGE电泳,杂带较少,条带主要集中于50 kD处;用斑点酶联免疫吸附试验检测时, 迟钝爱德华氏菌呈阳性, 温和气单胞菌、嗜水气单胞菌、河流弧菌、溶藻弧菌、鳗弧菌、腐败希瓦氏菌、产碱普罗威斯登菌、阪崎肠杆菌和大肠杆菌均呈阴性.试验结果表明,特异、高效价的迟钝爱德华氏菌免疫血清,为快速检测牙鲆腹水病病原奠定了基础.     

牙鲆NOD2基因的表达分析及在抗迟缓爱德华氏菌感染过程中的功能

本研究利用实验室已建牙鲆(Paralichthys olivaceus)转录组数据库预测得到牙鲆NOD2基因(PoNOD2),并利用PCR技术进行序列验证。同时,设计迟缓爱德华氏菌注射感染牙鲆成鱼和体外免疫刺激牙鲆鳃细胞系实验,探究PoNOD2基因在抗菌免疫反应中的作用。PoNOD2基因的开放阅读框长度为2964 bp,编码988个氨基酸。PoNOD2蛋白有3种保守结构域,包括C-末端LRR,中心NACHT和N-末端CARD结构域。实时荧光定量PCR结果显示,在所检测牙鲆组织中,迟缓爱德华氏菌的侵染能显著上调PoNOD2的表达。体外免疫刺激牙鲆鳃细胞系实验显示,在PGN、PolyⅠ:C和迟缓爱德华氏菌刺激下,PoNOD2表达上调。亚细胞定位显示,PoNOD2蛋白定位于牙鲆鳃细胞的细胞质中。在迟缓爱德华氏菌侵染牙鲆鳃细胞过程中,PoNOD2基因的过表达能够抑制细菌生长,并引起IL-1β、IL-6和IL-8等炎性细胞因子的表达上调。结果表明,PoNOD2在抑制迟缓爱德华氏菌生长以及调节牙鲆对病原菌的免疫应答中发挥重要作用。     

Cytological studies on induced meiogynogenesis in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel)

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major . After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed.     

Optimum dietary protein level for maximum growth of juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to determine the optimum dietary protein level for juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel) fed a white fish meal and casein‐based diets for 8 weeks. Olive flounder with an initial body weight of 4.1 ± 0.02 g (mean ± SD) were fed one of the six isocaloric diets containing 35%, 45%, 50%, 55% and 65% crude protein (CP) at a feeding rate of 4–5% of wet body weight on a dry‐matter basis to triplicate groups of 20 fish per aquarium. After 8 weeks of feeding, per cent weight gain (WG) and feed efficiency ratios of fish fed the 55% CP diet were not significantly higher than those from fish fed the 50% and 65% CP diets, but significantly higher than those from fish fed the 35% and 45% CP diets. Fish fed the 50%, 55% and 65% CP diets had significant higher specific growth rates than did fish fed the 35% and 45% CP diets; however, there was no significant difference among fish fed the 50%, 55% and 65% CP diets. The protein efficiency ratio was inversely related to the dietary protein level; that is, maximum efficiency occurred at the lowest dietary protein level. Broken‐line model analysis indicated that the optimum dietary protein level was 51.2 ± 1.8% for maximum weight gain in juvenile olive flounder. The second‐order polynomial regression analysis showed that the maximum WG occurred at 57.7% and it revealed that the minimum range of protein requirement was between 44.2% and 46.4%. These findings suggest that the optimum dietary protein level for maximum growth could be greater than 46.4%, but less than 51.2% CP in fish meal and casein‐based diets containing 17.0 kJ g^?1 energy for juvenile olive flounder.     

Effect of temperature and salinity on virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Experiments were designed to determine the effects of temperature and salinity on the virulence of Edwardsiella tarda to Japanese flounder, Paralichthys olivaceus. In the temperature experiment, a two‐factor design was conducted to evaluate the effects of both pathogen incubation temperature and fish cultivation temperature on pathogen virulence. E. tarda was incubated at 15, 20, 25 and 30±1°C, and the fish (mean weight: 10 g) were reared at 15, 20 and 25±1°C respectively. The fish reared at different temperatures were infected with the E. tarda incubated at different temperatures. The results of a 4‐day LD₅₀ test showed that temperature significantly affected the virulence of E. tarda (P<0.01) and the interaction between the two factors was also significant (P<0.01). For fish reared at 15°C the virulence of E. tarda was the highest at 25°C of pathogen incubation, followed by 20, 15 and 30°C. When the fish rearing temperature was raised to 20 and 25°C, the virulence of E. tarda incubated at all temperatures increased. Isolation testing demonstrated results similar to those of LD₅₀. The higher rearing temperature increased the proliferation rate of the pathogen in fish. In the salinity experiment, the incubation salinity of E. tarda was at 0, 10, 20 and 30 g L^?1, respectively, and the fish with mean weight of 50 g were cultured in natural seawater of 30 g L^?1. The results of one‐way anova in 4‐day LD₅₀ test showed that incubation salinity significantly affected virulence. Virulence was lower when the salinity of the incubation medium was at 0 and 30 g L^?1, higher at 10 and 20 g L^?1. The results of isolation test were in accordance with those of LD₅₀. At 20 g L^?1E. tarda had a faster proliferation rate than that at 10 g L^?1.     

复合诱食剂对牙鲆摄食生长的影响

以初始体质量为(3.01±0.04)g的牙鲆(Paralichthys olivaceus)为实验对象,进行为期70 d的摄食生长实验。共配制5种等氮等能饲料,其中以鱼粉和豆粕为蛋白源(豆粕蛋白替代45%的鱼粉蛋白)配制出基础饲料,分别在基础饲料中添加0.0%、0.5%、1.0%和1.5%的复合诱食剂配制成4种豆粕取代饲料,同时以全鱼粉饲料为对照饲料,研究复合诱食剂对牙鲆摄食生长的影响。结果表明,当豆粕取代饲料中诱食剂添加量为1.0%时,牙鲆的摄食率(P<0.05)、饲料效率(P<0.05)、鱼体脂肪含量(P<0.01)以及肝脏、肠胰蛋白酶活力和肠氨肽酶活力(P<0.01)显著高于不添加诱食剂组;当豆粕取代饲料中诱食剂的添加量为0.0%和0.5%时,牙鲆的特定生长率显著低于全鱼粉组(P<0.05);而当添加量为1.0%和1.5%时,牙鲆的特定生长率与全鱼粉组比较没有出现显著差异(P>0.05)。结论认为,当以豆粕蛋白替代45%的鱼粉蛋白时,添加1.0%的复合诱食剂能够显著提高牙鲆幼鱼对饲料的摄食率和生长率。     

Infection dynamics of Neoheterobothrium hirame (Monogenea) on juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel), in coastal waters of Japan

A recent epidemic of the invasive monogenean Neoheterobothrium hirame is suspected to have caused the significant decline of the commercial catch of olive flounder, Paralichthys olivaceus, in Japan. To clarify the causal link between parasite invasion and host depletion, we monitored the infections of N. hirame on juvenile flounder for 2 years at two widely separate bays, Obama Bay, experiencing a devastating reduction in flounder catches, and Miyako Bay with a stable catch. Prevalence, mean intensity and abundance of developmental stages of N. hirame were compared between the bays and between fish year classes. Fishes captured at Obama Bay harboured three times more worms than those at Miyako Bay and had a significantly higher overall prevalence of infection. In addition, there was a negative correlation between parasite intensity and host condition factor at Obama Bay, suggesting a causal link between the N. hirame epidemic and the reduction of local flounder populations.     

Effect of phenanthrene on haematological parameters in olive flounder, Paralichthys olivaceus (Temminch et Schlegel)

Effects of phenanthrene on haematological parameters in olive flounder, Paralichthys olivaceus, were studied by exposing fish to water‐borne phenanthrene (0.5, 1 and 2 μM) for 4 weeks. Red blood cell count, haemoglobin concentration, haematocrit levels decreased with increasing exposure time of phenanthrene to the fish. Plasma potassium level increased significantly in the experimental group but the levels of sodium and chloride were significantly decreased compared with control. There was no significant change in plasma magnesium, calcium and phosphate in fish of all treated groups compared with the control. The most profound physiological disturbance in phenanthrene‐exposed fish was a decrease in blood osmolality. Malondialdehyde level in the plasma in the phenanthrene group (2 μM) was 1.4‐fold higher following 4‐week exposure compared with the control group. Acetylcholinesterase activity decreased as the concentration of phenanthrene increased, reaching 65.9% of the control value at the highest dose at the 4th week. A key finding from this study is that olive flounder exposed to phenanthrene at concentrations more than 1.0 μM are likely to experience negative impacts on haematological parameters.     

Energy budget of the Japanese flounder Paralichthys olivaceus (Temminck & Schlegel) larvae fed HUFA-enriched and non-enriched Artemia nauplii

The energy budget of the Japanese flounder Paralichthys olivaceus (Temminck & Schlegel) larvae fed enriched (EA) and non‐enriched (NEA) Artemia nauplii was determined by equating energy intake (EI) with the summation of energy channelled to faeces (F), metabolism (M), excretion (U) and growth (G). Larvae (21 days post hatching, 2.2 mg mean wet wt) were reared in six 80‐L circular tanks with three replicates of 160 larvae per tank and fed EA and NEA for 20 days. EI was calculated from the energy content of consumed nauplii, M from the summation of energy for routine, feeding and active metabolisms, U from ammonia excretion and G from energy gained based on weight gain, while F was the difference between EI and the total of other components. The heat increment of larvae was calculated from the difference of O₂ consumption at post‐prandial and routine conditions. Except for G and F, variables were correlated to the dry body weight (W) of larvae in a power function: Y=aW^b. Coefficients a and b were estimated by regression after a logarithmic transformation of the raw data. Overall, growth and survival rates of the larvae fed EA were higher than those fed NEA. For a larval flounder growing from 2 to 20 mg wet wt, the ingested energy was partitioned as follows: 22.8% to faecal loss, 38.3% to metabolism, 1.5% to urinary loss and 37.4% to growth for the EA group, whereas 35.4% to faecal loss, 28.4% to metabolism, 1.3% to urinary loss and 34.9% to growth for the NEA group. Gross conversion and assimilation efficiencies were higher, but the net conversion efficiency was lower in EA‐fed larvae than NEA‐fed larvae. This study suggests that the higher growth and survival rates of the EA‐fed group compared with the NEA‐fed group were attributed to their higher intake of essential fatty acids, higher metabolism and lower energy loss of faeces.     

Effects of dissolved oxygen concentration and stocking density on the growth,energy budget and body composition of juvenile Japanese flounder,Paralichthys olivaceus (Temminck et Schlegel)

An energetic method was used to investigate the effects of stocking density and dissolved oxygen (DO) concentration on the growth of juvenile Paralichthys olivaceus. Fish, with initial weights of 14±2.1 g, were exposed to a normal and a high DO of 5.5±0.5 and 14±2 mg L^?1, as well as four stocking densities per DO concentration (100, 200, 300 and 400 ind m^?2 for the normal DO and 200, 400, 600 and 800 ind m^?2 for the high DO). The feed efficiency (FE_W) decreased significantly with increasing stocking density and increased significantly with increasing DO concentration. The maximum weight was achieved at 400 ind m^?2 under a high DO depending on our rearing conditions. The stocking density and DO concentration change energy ingestion and its allocation for respiration, growth and excretion lost in nitrogen excretion, but do not affect the energy loss through faeces. The results of the body composition of fish indicated that the stocking density and DO concentration had no significant effects on the moisture, lipid content and gross energy, but are affected by the same. Energetic analysis demonstrated that high DO concentrations could alleviate the growth depression caused by high stocking densities, decrease energy loss in respiration and nitrogen excretion and increase the energy proportion allocated to growth.     

牙鲆淋巴囊肿病的病理和病原分离

应用常规显微和亚显微技术观察和分析患淋巴囊肿病养殖牙鲆(Paralichthys olioaceus)的病理学变化，利用差速离心和蔗糖密度梯度离心技术分离其病原——淋巴囊肿病毒，并利用牙鲆细组织细胞系FG—9307为感染基质，观察淋巴囊肿病毒引起的细胞病理变化。结果表明，患病牙鲆的囊肿组织是一些淋巴囊肿细胞的集合体，这些囊肿细胞排列紧密，直径为10—100μm，细胞近圆形，细胞质内散布有大量的嗜碱性包涵体，且多数集中在细胞的边缘部分；囊肿细胞内含有大量病毒粒子，其衣壳外形呈六角或五角形，直径为150—230nm，大多数病毒粒子中央有一致密的核，核外周包围着一双层核衣壳，核衣壳的表面可见一圈把手样亚单位。以患病牙鲆囊肿物制备的上清液接种细胞，7d内未见细胞异常，经盲传2—3代后，细胞出现较明显的细胞病变效应。