灭活菌苗免疫的中华倒刺鲃外周血免疫指标的变化

以温和气单胞菌灭活菌苗为免疫原,平均体重(100±25)g的健康中华倒刺鲃为实验对象,免疫组腹腔注射0.2mL浓度为1.0×108CFU/mL的免疫原,对照组注射等量灭菌生理盐水,分别在单次注射0、1、2、4、7、14、21、28、35d后随机从两组各取6尾实验鱼,尾静脉采血,测定外周血的血细胞数量、白细胞分类计数、吞噬活性、抗体效价和蛋白质含量等免疫指标的变化,第35天活菌攻毒。结果表明:温和气单胞菌灭活菌苗(F-AS)可诱导中华倒刺鲃红细胞和白细胞数量增加,并引起各种白细胞分类百分比变化,提高吞噬活性和抗体效价,血清中总蛋白及球蛋白含量增加,红细胞也具有免疫功能,灭活茵苗的相对免疫保护力达65.21%。免疫早期(第1周)主要是红细胞、单核细胞和中性粒细胞数量明显增加,吞噬细胞的吞噬活性迅速提高,吞噬百分比和吞噬指数第4天达峰值;随后则是淋巴细胞大量增殖,第21天淋巴细胞、抗体效价及球蛋白达峰值。可见灭活菌苗通过促进中华倒刺鲃血细胞增殖、提高吞噬细胞的吞噬活性、产生特异性抗体等方式提高免疫保护力;免疫早期非特异性细胞免疫起重要作用,之后特异性免疫起主要作用。     

嗜水气单胞菌灭活疫苗免疫施氏鲟后的免疫应答反应与保护效果

为了研究嗜水气单胞菌灭活疫苗免疫施氏鲟(Acipenser schrenckii Brandt,1869)后的免疫应答反应与保护效果,本研究采用福尔马林灭活法制备嗜水气单胞菌灭活疫苗。通过腹腔注射途径免疫健康施氏鲟,对施氏鲟外周血的红细胞和白细胞数量、白细胞组成、吞噬活性、吞噬指数、血清酸性磷酸酶活性、血清溶菌酶活性、血清中和抗体效价等免疫指标及疫苗保护效果进行测定。结果显示,免疫组红、白细胞数量迅速上升,于免疫后第4天达峰值,分别为(8.50±0.17)×10~8个/mL和(8.96±0.44)×10~6个/mL;单核细胞和嗜中性粒细胞分类百分比于第7天达峰值,分别为10.50%和15.53%,极显著高于对照组(P0.01),淋巴细胞分类百分比在第21天才达峰值73.51%;吞噬指数和吞噬百分比均于第4天达到最高值,分别为4.53和30%;血清中溶菌酶活性和酸性磷酸酶活性分别在免疫后第7天和第14天达到峰值,极显著高于对照组(P0.01);血清抗体效价于免疫后第21天达到峰值1:203,极显著高于对照组(P0.01)。攻毒感染实验结果表明,免疫组的相对免疫保护率为76.84%。由此可见,嗜水气单胞菌灭活疫苗免疫施氏鲟后,能够显著诱导施氏鲟体内的免疫应答反应,增强鱼体的免疫保护能力。本研究为养殖鲟因嗜水气单胞菌感染引起疾病的免疫预防技术研究奠定了前期基础。     

A3α肽聚糖增强灭活嗜水气单胞菌疫苗免疫效果的研究

为了探讨 A_(3α)肽聚糖对灭活嗜水气单胞菌疫苗免疫效果的影响,用添加肽聚糖的饲料投喂注射接 种经甲醛灭活的嗜水气单胞菌菌苗的彭泽鲫,测定鲫鱼白细胞吞噬活性、血清和体表粘液溶菌酶活性、抗体 效价以及活菌攻毒后的免疫保护率。结果表明,肽聚糖与灭活菌苗联合使用,鱼体表粘液和血清溶菌酶活性、 白细胞吞噬活性、抗体效价以及活菌攻毒后的免疫保护率显著提高,表明 A_(3α)肽聚糖可增强灭活菌苗的免疫效 果。     

投喂阿维拉霉素对鲤免疫应答的影响

将阿维拉霉素按照一定的浓度添加到饲料中,投喂经注射接种福尔马林灭活的嗜水气单胞菌(Aerom onashydrophila)菌苗的鲤,于免疫后的第7 d、14 d、21 d、28 d和35 d检测供试鲤血液中的白细胞吞噬活性、血清中溶菌酶活性、抗体效价、补体C3含量及免疫保护率(RPS),探讨了阿维拉霉素对受免鲤免疫应答的影响。结果表明,饲料中添加阿维拉霉素(4 mg/kg和8 mg/kg),投喂较长时间(≥28 d)会降低受免鲤的血液白细胞吞噬活性、血清中的溶菌酶活性和抗体效价,减少受免鲤血清中补体C3的含量,减弱鲤抵抗嗜水气单胞菌人工感染的能力,每公斤饲料中添加8 mg的阿维拉霉素其免疫抑制作用更强。     

嗜水气单胞菌灭活疫苗对虹鳟免疫力和抗病力的影响

为研究嗜水气单胞菌(Aeromonas hydrophila)灭活疫苗对虹鳟(Oncorhynchus mykiss)(250 g±12 g)免疫力和抗病力的影响,给试验鱼腹腔注射0.2 mL的灭活嗜水气单胞菌悬液(1.0×108cfu/mL)。分别在注射前(0 d)和注射后7、14、21、28、35、42 d取样,测定血液白细胞吞噬活性、血清溶菌酶(LZM)活力、补体C3含量以及头肾和脾脏中IgM、IgT基因表达情况。结果表明:注射嗜水气单胞菌疫苗能显著提高白细胞吞噬率(PP)和吞噬指数(PI)、LZM活力和补体C3含量,注射后21 d达峰值,PI、LZM和补体C3在42 d仍显著高于对照组。免疫后21 d头肾和脾脏中IgM基因上调表达,35 d达峰值(分别上升24倍和26倍),42 d下降但仍极显著高于对照组。头肾和脾脏中IgT的上调表达比IgM弱得多,头肾35 d达峰值(上升6.2倍),脾脏28 d达峰值(上升6.1倍)。免疫42 d后嗜水气单胞菌攻毒结果表明,嗜水气单胞菌灭活疫苗对虹鳟有较高的免疫保护作用,免疫保护率达到71.43%。     

嗜水气单胞菌3种疫苗对斑点叉尾免疫原性比较研究

将经福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)、菌体脂多糖(LPS)和菌体外膜蛋白(OMP)作为免疫原,分别接种斑点叉尾(Ictalurus punctatus Rafinsque)后,通过测定受免鱼的凝集抗体效价,头肾和血液中吞噬细胞的吞噬活性和用A.hydrophila活菌攻毒的方法,探讨了斑点叉尾对A.hydrophila的3种疫苗的免疫应答状况和对活菌攻毒的免疫保护效果。试验结果显示:对斑点叉尾经腹腔注射接种3种疫苗均能刺激受免鱼产生较强的免疫应答,3种疫苗的受免鱼均产生了特异性凝集抗体,接种F-Ah灭活菌苗的试验鱼最高,接种OMP疫苗的试验鱼其次,而接种LPS疫苗的试验鱼血清中凝集抗体效价最低;与未接种疫苗的对照鱼相比,受免鱼头肾和血液中吞噬细胞的吞噬活性明显上升,头肾和血液中吞噬细胞活性由高到低依次是LPS、OMP和F-Ah免疫接种的斑点叉尾;活菌攻毒的结果证明接种3种疫苗的受免斑点叉尾A.hydrophila的感染产生了不同程度的相对免疫保护力(RPS),以OMP免疫接种后的斑点叉尾RPS最高,达到72.5%,接种LPS的斑点叉尾稍差,RPS为62.5%,而RPS最低的是接种F-Ah的免疫组,RPS只有24.2%。     

鮰爱德华氏菌灭活疫苗免疫斑点叉尾鮰后外周血免疫指标的变化

在水温(25±1)℃下,给平均体质量(80±5)g的斑点叉尾鮰Ictalurus punctatus腹腔注射福尔马林灭活的鮰爱德华氏菌(Formalin-killed Edwardsiella ictaluri,FKE)后,第2、4、6、8、14、21,和28d测定外周血液免疫指标,并在免疫后第28天进行攻毒试验。结果显示:注射鮰爱德华氏菌灭活菌苗后,斑点叉尾鮰外周血红细胞和白细胞数量显著升高,白细胞分类组成变化显著,吞噬细胞的吞噬活性与凝集抗体效价显著上升。第4d免疫组红细胞和白细胞达峰值2.52×106/μL和4.67×105/μL,极显著高于对照组(P<0.01);单核细胞、中性粒细胞、吞噬细胞分类百分比和吞噬指数均在第4d达到峰值,分别为22.3%、5.67%、39.3%和4.33,极显著高于对照组(P<0.01);淋巴细胞分类百分比、凝集抗体效价在第21d达到峰值,分别为54.33%和1:341.33,极显著高于对照组(P<0.01)。攻毒试验结果表明:免疫组相对免疫保护率为64.3%。福尔马林灭活的鮰爱德华氏菌免疫后,斑点叉尾鮰获得较强的抗鮰爱德华氏菌感染保护能力,为进一步研究斑点叉尾鮰肠道败血症的免疫奠定基础。     

嗜水气单胞菌疫苗免疫效果研究

选取对鲤鱼毒力强的嗜水气单胞菌菌株制成灭活疫苗,研究疫苗的免疫保护效应.采用甲醛灭活制备疫苗,分别对鲤鱼进行注射和浸泡试验,测定白细胞吞噬活性、淋巴细胞转化能力、血清、皮肤黏液的抗体效价及活菌攻毒后的免疫保护率.结果表明,受免鱼各项指标均明显高于对照组,注射组免疫保护率达90%,其中浸泡组各项略低于注射组.嗜水气单胞菌灭活疫苗对鲤鱼有显著免疫保护效应,可作为预防细菌性败血症感染的疫苗.     

酵母免疫多糖对受免斑点叉尾(鮰)免疫应答的增强作用

在饲料中添加定量的酵母免疫多糖,投喂注射接种了福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)菌苗的斑点叉尾(鮰)(Ictalurus punctatus),28 d后通过测定供试鱼的增重量、血清中凝集抗体效价、溶菌酶活性、谷丙转氨酶活性(SGPT)、血清总蛋白含量、白细胞吞噬活性以及活菌攻毒后的免疫保护率(RPS),探讨了酵母免疫多糖对受免斑点叉尾(鮰) 免疫应答的增强作用.结果表明,用添加200.0 mg/(kg·d)酵母免疫多糖的饲料投喂受免斑点叉尾(鮰),不仅可以使受免斑点叉尾(鮰) 对灭活嗜水气单胞菌的免疫应答水平提高,增强抵抗嗜水气单胞菌人工感染的能力,而且还具有一定的促进生长和改善肝功能的作用.     

酵母免疫多糖对受免斑点叉尾免疫应答的增强作用

在饲料中添加定量的酵母免疫多糖,投喂注射接种了福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)菌苗的斑点叉尾(Ictalurus punctatus),28 d后通过测定供试鱼的增重量、血清中凝集抗体效价、溶菌酶活性、谷丙转氨酶活性(SGPT)、血清总蛋白含量、白细胞吞噬活性以及活菌攻毒后的免疫保护率(RPS),探讨了酵母免疫多糖对受免斑点叉尾免疫应答的增强作用。结果表明,用添加200.0 mg/(kg.d)酵母免疫多糖的饲料投喂受免斑点叉尾,不仅可以使受免斑点叉尾对灭活嗜水气单胞菌的免疫应答水平提高,增强抵抗嗜水气单胞菌人工感染的能力,而且还具有一定的促进生长和改善肝功能的作用。     

盐度驯化下史氏鲟的血液学参数

测定了5个不同盐度梯度(0、10、20、25、28)下分别饲养14d和21d的史氏鲟血液生理学参数。结果表明:在不同的饲养时间下,红细胞总数(RBC)、红细胞压积(HCT)、血栓细胞总数(PLT)、血红蛋白浓度(HGB)均随盐度的升高逐渐减少,红细胞平均体积(MCV)、平均血红蛋白含量(MCH)随盐度的升高逐渐增加,平均血红蛋白浓度(MCHC)无显著变化,红细胞体积分布宽度(RDW)、白细胞总数(WBC)、淋巴细胞数(LYM)、单核细胞数(MON)、嗜酸性粒细胞数(EOS)、中性粒细胞含量(NEUT)、嗜碱性粒细胞含量(BASO)随盐度的增加先升高后下降。在相同的盐度条件下,红细胞总数(RBC)、血栓细胞总数(PLT)、平均血红蛋白含量(MCH)、平均血红蛋白浓度(MCHC)、嗜碱性粒细胞含量(BASO)随养殖天数的增加逐渐下降,其它血液指标随饲养时间的延长逐渐升高。白细胞分类计数(DLC)值显示,在不同的盐度条件下,白细胞中各类细胞比例依次为淋巴细胞(Lym)>单核细胞(Mon)>嗜中性粒细胞(Neut)>嗜碱性粒细胞(Baso)>嗜酸性粒细胞(Eos)。通过盐度驯化后血液生理学分析,史氏鲟具有较强的渗透调节能力,经过驯化可以在28的盐水中生存。     

Differentiation and development of Leydig cell, and induction of spermatogenesis during testicular differentiation in the Japanese eel, Anguilla japonica

The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight^-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.     

Functional partition of cells in the gill epithelium of the Japanese eel, Anguilla japonica, and the role of hormones

Viable chloride and pavement cells were isolated from the gill epithelium of Japanese eel, Anguilla japonica, by a 3-step percoll gradient centrifugation at low speed. Viability of the isolated cells were tested by the trypan blue exclusion test, rhodamine-123, or pre-labelling the cells with fluo-3 Ca²⁺ dye and examined by laser confocal microscopy. Isolated chloride cells responded to ionomycin with a rapid increase in Ca²⁺ fluorescence, which was abolished by chelating external Ca²⁺ with EGTA. Peptide hormones, including arginine vasotocin, isotocin, insulin-like growth factor I and II, and urotensin I increased Ca²⁺ entry, urotensin II had no effect, and eel corpuscles of Stannius extract reduced residual Ca²⁺ fluorescence. Isolated chloride cells and pavement cells from eels were analyzed for their enzymatic activities involved in intermediary and nitrogen metabolism. Chloride cells had high levels of glutaminase I, glutamate dehydrogenase, glutamate oxalacetate transaminase, HCO₃-ATPase and carbamoyl phosphate synthetase II. Pavement cells had highly active glucose-6-phosphate dehydrogenase and AMP deaminase. Both had high levels of lactate dehydrogenase compared with other tissues.     

Establishment and characterization of a liver cell line,ALL, derived from yellowfin sea bream,Acanthopagrus latus,and its application to fish virology

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%–20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus–yellowfin sea bream interaction.     

Species differences in the adrenergic responses of fish red cells: studies on whitefish,pikeperch, trout and carp

The occurrence and pH dependence (pHe 7–8) of the adrenergic red cell responses of two salmonids, trout and whitefish, and a percinid, pikeperch were studied. These are all species that live in well-oxygenated waters. The responses were compared to those of carp, which tolerates oxygen-deficient waters.The adrenergic responses of trout and whitefish red cells were pronounced. In these species red cell swelling, the accumulation of sodium and chloride in the cell, and the increase in red cell oxygen content at atmospheric oxygen tension were maximal at pH 7.3. In contrast, pikeperch red cells responded to -adrenergic stimulation only at extracellular pH 7.1. In carp, the adrenergic response, occurring below extracellular pH 7.5, was small as compared to the two salmonids. In each case the onset of the adrenergic response coincided with the onset of the Root effect.The differences in the adrenergic responses between the two salmonids and pikeperch suggest that the occurrence of the adrenergic response is not directly related to the environmental oxygen requirements of the species, but may be linked to the activity pattern.     

Establishment,characterization, virus susceptibility and transfection of cell lines from cobia,Rachycentron canadum (L.), brain and fin

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.     

Identification of a heparin-binding,mesoderm-inducing peptide in the swim-bladder of the red seabream,Pagrus major: a probable fish fibroblast growth factor

The brain, pituitary, liver, ovary and swim-bladder of the red seabream (Pagrus major) were screened for the presence of a growth factor capable of stimulating the proliferation of BHK-21 cells, and the swimbladder extract was found to exert the most powerful activity. Swim-bladder derived growth factor (SBDGF) with heparin-binding ability was isolated by heparin-affinity and size-exclusion chromatography using heparin-Sepharose CL-6B and Superose 12, respectively. The relative molecular mass of SBDGF was 22,500 and the isoelectric point was 9.4. The growth of mammalian fibroblasts (BALB/3c 3T3, BHK-21) and endothelial cells (CPAE) was markedly stimulated by SBDGF. Its growth-promoting activity was also effective in fish fibroblasts (BF-2 and RTG-2), and was heat- and acid-labile. In the mesoderm-induction assay, explants of presumptive ectoderm dissected from Xenopus laevis blastulae (stage 9) were incubated with SBDGF. SBDGF induced ventral mesoderm in the explants, including hemocyte-like cells, mesenchyme and coelomic epithelium. These properties suggest that SBDGF is a fish fibroblast growth factor and that the swim-bladder is the source.     

不同摄食状态下南方鲇幼鱼肠道黏液细胞的量化分析

量化分析了南方鲇(Silurus meridionalis)幼鱼在正常摄食节律下摄食前(S0d)、摄食后64 h(S0d-64h)、饥饿16d(S16d)、饥饿32 d(S32d)以及饥饿后首次恢复摄食64 h(S16d-64h,S32d-64h)肠道各类型黏液细胞的反应特征。结果发现,摄食后肠道各部位黏液细胞总数均有一定程度减少,其中前肠减少最为显著(P<0.05);中肠和后肠II型黏液细胞数量显著减少(P<0.05),中肠的III型黏液细胞数量显著增多(P<0.05);肠腔中黏液增多,黏液细胞呈空泡结构。S16d组和S16d-64h组前肠和中肠的I型细胞数量显著减少(P<0.05),III型细胞数量显著增多(P<0.05)。S32d组和S32d-64h组前肠各类型细胞数量以及黏液细胞总数变化不明显(P>0.05);中肠的II型细胞数量显著增多(P<0.05),IV型细胞数量显著减少(P<0.05);饥饿和恢复摄食后肠各类型黏液细胞数目变化不明显(P>0.05)。研究表明,南方鲇幼鱼肠道黏液细胞的摄食反应明显;饥饿胁迫以及恢复摄食后肠道黏液细胞的反应特征与肠道各段的生理功能相适应;面对营养胁迫时,肠道前段能迅速调节黏液细胞的数目,而后肠基本不变,可能是该鱼应对营养缺乏的保护性适应机制。     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.