Detection and Identification of a Strain of Renibacterium salmoninarum Sourced from Cultured Chinook Salmon (Oncorhynchus tshawytscha)

A chronic and infective disease occurred on the farmed Chinook salmon (Oncorhynchus tshaivytscha) in a farm in Huairou district of Beijing and it caused high accumulative mortality.The typical clinical symptoms indicated that it might be bacterial kidney disease (BKD).The purpose of this study was to confirm its pathogen.The typical clinical symptoms of sick fish,morphology observation of bacteria in kidney tissue smears and sequence analysis of bacteria DNA were conducted.A number of gram positive brevi bacteria were observed in kidney tissue print.16S rDNA sequence of K1 strain was more than 99% homology with that of Renibacterium salmoninarum and they were in the same cluster.383 and 320 bp fragment were amplified by PCR and nested PCR.The fragments were sequenced and analyzed.The results showed the fragment was shared 100% nucleotide identity with P57 surface protein gene of Renibacterium salmoninarum. It was indicated that these diseased fish were suffering from BKD caused by Renibacterium salmoninarum.This was the first report about Renibacterium salmoninarum detected in farmed fish in China.     

细鳞鲑疖疮病的病原鉴定及防治药剂筛选

自然发病的细鳞鲑(Brachymystax Lenok)出现鳃盖边缘、鳍条基部充血,鳍条溃烂,肛门红肿等症状。从病灶处分离得到的优势菌株,定名为BJSY-1、BJSY-2、BJSY-3。通过回归感染试验证明BJSY-1为致病菌,其LD_(50)为6.97×10~5CFU/mL。经生理生化鉴定和16S rDNA序列分析最终确定BJSY-1菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.Salmonicida)。该菌对四环素类、喹诺酮类等药物高度敏感。选择氟苯尼考作为治疗药物,按20 mg/kg·bw的用药量拌饵投喂,每天投喂1次,连续投喂有较好的治疗效果,为杀鲑气单胞菌的治疗和防控提供了参考。     

秦岭细鳞鲑消化系统的形态学观察

运用大体解剖和常规组织学方法对秦岭细鳞鲑消化系统的组织形态进行了系统观察。结果表明,秦岭细鳞鲑的消化道由口咽腔、食道、胃和肠等4部分组成,除口咽腔外,管壁由内向外分为黏膜、黏膜下层、肌层和浆膜。口咽腔宽大,上颌、犁骨及腭骨等处布有细齿,黏膜衬以复层扁平上皮,间有较多的黏液细胞和少量的杯状细胞及味蕾;食管粗而短,黏膜向管腔突出形成多个纵行皱襞,上皮由复层扁平上皮逐渐向单层柱状过渡,上皮细胞间可见到数量较多的粘液细胞和杯状细胞,食管腺不发达;胃体积较大,呈“U”形,包括贲门部、胃体和幽门部,胃体部小凹及胃底腺结构明显,肌层发达;胃与肠相接处有63-65个幽门盲囊,肠道粗短而略微盘曲,黏膜上皮为单层柱状,未见明显的肠腺。以上结果显示,秦岭细鳞鲑消化道组织结构与其肉食性功能密切相关。肝小叶界限不清;双列肝细胞围绕中央静脉呈放射状走行,肝细胞个体大、呈多边形,核呈圆形或卵圆形,1-2个。胰脏大部分环绕前肠边缘、呈细长条索状,另可见分散于幽门盲囊及胃周围的弥散部分;其外分泌部为管泡状腺,腺细胞呈锥体形,胰岛结构明显。     

秦岭细鳞鲑物种有效性的探讨

采用PCR技术获得细鳞鲑秦岭亚种25尾个体的线粒体DNA控制区598bp的序列,从Gene Bank下载黑龙江流域的尖吻细鳞鲑与钝吻细鳞鲑的同源序列,以近缘物种哲罗鲑为外类群,构建了中国细鳞鲑属的分子系统发育树,并结合形态特征讨论了细鳞鲑秦岭亚种的有效性。遗传结构分析显示,秦岭亚种与黑龙江流域两种细鳞鲑之间以及后两种细鳞鲑之间的遗传距离分别为:0.0202,0.0207,0.0199,明显大于细鳞鲑属种内遗传距离:0.0023,0.0033,0.0091。以邻接法(NJ)和最大简约法(MP)构建的系统发育树表明,秦岭地区的细鳞鲑单独构成一个单系群,而黑龙江流域的两种细鳞鲑构成一个大的支系。以上结论证明了秦岭细鳞鲑已经达到了亚种水平的分化,该研究的结果支持形态分类上秦岭亚种的分类地位。     

一株山羊源杀鲑气单胞菌的分离、鉴定及药物敏感性检测

对患呼吸系统疾病的病羊进行病原的分离,并对分离菌进行形态观察、致病性试验、16Sr DNA基因序列的测定及药敏试验。结果显示,分离菌对健康Balb/c小鼠具有较强的致病性,其引发小鼠的病理变化与临床剖检患病山羊的病理变化相同,均为急性间质性肺炎。16Sr DNA基因序列测定结果表明其与杀鲑气单胞菌的16Sr DNA基因序列一致性为98%,该菌株仅对头孢噻肟、强力霉素等药物敏感,对氧氟沙星、氟苯尼考、庆大霉素、环丙沙星、恩诺沙星、丁胺卡那霉素、大观霉素、诺氟沙星、克林霉素、氨苄西林、阿莫西林等临床常用药物均呈不同程度的耐药性。     

兰州鲇与秦岭细鳞鲑血清生化指标的比较研究

为了解兰州鲇(Silurus lanzhouensis)与秦岭细鳞鲑(Brachymystax lenok tsinlingensis)在血清生化指标等方面的异同,对兰州鲇和秦岭细鳞鲑血清主要生化指标进行了比较分析。结果表明,兰州鲇总蛋白(TP)、白蛋白(ALB)、白球比(A/G)、尿素(BUN)、葡萄糖(GLU)、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-c)、钙(Ca2)和磷(P)含量显著低于秦岭细鳞鲑(P0.05),血清谷丙转氨酶(ALT)、谷草转氨酶(AST)和低密度脂蛋白胆固醇(LDL-c)含量显著高于秦岭细鳞鲑(P0.05),两种鱼类的球蛋白(GLO)含量无显著差异(P0.05)。研究结果显示,兰州鲇与秦岭细鳞鲑在血清学上反映其在营养代谢上有较大差异,兰州鲇的蛋白质代谢强度相对较大,而秦岭细鳞鲑的糖脂代谢较为旺盛。     

一株猪源乳杆菌的分离鉴定

从猪小肠中分离得到一株乳杆菌,对其进行生理生化鉴定、16S r DNA基因测序和同源性分析。分析结果显示,该菌株的菌落有溶钙圈,白色圆形,边缘光滑,无鞭毛,无荚膜,无芽孢的革兰氏阳性杆菌,16SrDNA序列与NCBI公布的约氏乳杆菌Lactobacillus gasseri的同源性为98.7%,将该菌株鉴定为格氏乳杆菌。     

细鳞鲑幼鱼对饲料中维生素E的需求量

本试验旨在研究饲料中不同水平的维生素E对细鳞鲑幼鱼生长性能、血清生化指标和机体维生素E积累量的影响,并确定细鳞鲑幼鱼对饲料中维生素E的需求量。选取初均质量为(40.2±3.6)g的细鳞鲑幼鱼270尾,随机分成6个组,每组3个重复,每个重复15尾。各组分别投喂不同维生素E水平(实测值分别为16.6、64.9、165.5、316.2、615.5和1 214.7 mg/kg)的6种等氮等能的试验饲料。试验期120 d。结果表明:1)细鳞鲑的末均质量(FW)、增重率(WGR)和特定生长率(SGR)均随着饲料维生素E水平的升高呈先升高后降低的趋势,165.5 mg/kg组细鳞鲑的FW显著高于其他各组(P0.05),165.5 mg/kg组细鳞鲑的WGR和SGR显著高于16.6和1 214.7 mg/kg组(P0.05)。各组间细鳞鲑的摄食率、饲料系数和蛋白质效率没有显著差异(P0.05)。2)随着饲料维生素E水平的升高,血清甘油三酯(TG)和总胆固醇(TC)含量均呈逐渐降低的趋势,且615.5和1 214.5 mg/kg组显著低于其他各组(P0.05)。随着饲料维生素E水平的升高,血清高密度脂蛋白胆固醇(HDL-C)含量呈先升高后降低的趋势,且165.5 mg/kg组显著高于其他各组(P0.05),64.9和316.2 mg/kg组显著高于16.6、615.5和1 214.5 mg/kg组(P0.05);而血清低密度脂蛋白胆固醇(LDL-C)含量与HDL-C含量呈相反的趋势,且165.5和316.2 mg/kg组显著低于其他各组(P0.05)。各组间血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性没有显著差异(P0.05)。3)随着饲料维生素E水平的升高,细鳞鲑肝脏维生素E积累量除615.5与1 214.5 mg/kg组间无显著差异(P0.05)外,呈显著升高的趋势(P0.05);肌肉维生素E积累量也呈升高的趋势,在饲料维生素E水平大于165.5 mg/kg时恒定在一个水平,且165.5、316.2、615.5和1 214.5 mg/kg组肌肉维生素E沉积量显著高于16.6和64.9 mg/kg组(P0.05)。由此可见,饲料中添加适宜水平的维生素E可以改善细鳞鲑的生长性能和血清生化指标,增加肝脏和肌肉维生素E积累量。以增重率和肌肉维生素E积累量为评价指标,根据折线模型得出,细鳞鲑对饲料中维生素E的需求量分别为145.87和180.98 mg/kg。     

一株棕熊源鲍曼不动杆菌的分离鉴定

1头10月龄圈养雌性棕熊,在无任何征兆情况下突然死亡,为查找死因,进行动物尸体剖检、细菌培养、细菌形态学观察、16SrDNA分析、药敏试验等.结果显示:肝、脾、肾病料组织接种于哥伦比亚血琼脂平板37℃培养24 h后,血平板上均见灰白色、黏稠、圆形、表面光滑的菌落,分离株革兰染色阴性,呈两端钝圆的短杆菌,无芽孢;分离株1...     

一株猪源化脓隐秘杆菌的分离与鉴定

四川内江某猪场发生疾病,病猪的肝、肺出现化脓性结节,脾出血性肿大,无菌采集病猪的病变组织进行细菌分离,得到1株革兰氏阳性菌,形态多样,呈球状、杆状或棒状,单在或成丛排列,该菌在TSA平板上生长缓慢。经细菌分离培养、16S rDNA鉴定、动物致病性试验等,结果显示该株分离菌为致病性化脓隐秘杆菌。药敏试验显示,该分离菌对头孢呋肟、氨苄西林、卡那霉素、四环素、氟苯尼考等药物有不同程度的敏感性,对于指导临床用药具有积极的意义。     

Effect of dietary α‐tocopherol + ascorbic acid,selenium, and iron on oxidative stress in sub‐yearling Chinook salmon (Oncorhynchus tshawytscha Walbaum)

A three‐variable central composite design coupled with surface‐response analysis was used to examine the effects of dietary α‐tocopherol + ascorbic acid (TOCAA), selenium (Se), and iron (Fe) on indices of oxidative stress in juvenile spring Chinook salmon. Each dietary factor was tested at five levels for a total of fifteen dietary combinations (diets). Oxidative damage in liver and kidney (lipid peroxidation, protein carbonyls) and erythrocytes (erythrocyte resistance to peroxidative lysis, ERPL) was determined after feeding experimental diets for 16 (early December) and 28 (early March) weeks. Only TOCAA influenced oxidative stress in this study, with most measures of oxidative damage decreasing (liver lipid peroxidation in December and March; ERPL in December; liver protein carbonyl in March) with increasing levels of TOCAA. We also observed a TOCAA‐stimulated increase in susceptibility of erythrocytes to peroxidative lysis in March at the highest levels of TOCAA. The data suggest that under most circumstances a progressive decrease in oxidative stress occurs as dietary TOCAA increases, but higher TOCAA concentrations can stimulate oxidative damage in some situations. Higher levels of TOCAA in the diet were required in March than in December to achieve comparable levels of protection against oxidative damage, which may have been due to physiological changes associated with the parr‐smolt transformation. Erythrocytes appeared to be more sensitive to variation in dietary levels of TOCAA than liver and kidney tissues. Using the March ERPL assay results as a baseline, a TOCAA level of approximately 350–600 mg/kg diet would provide adequate protection against lipid peroxidation under most circumstances in juvenile Chinook salmon.     

Comparative single‐dose pharmacokinetics of orally administered florfenicol in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792) at health and experimental infection with Streptococcus iniae or Lactococcus garvieae

This study evaluates changes in the pharmacokinetic behavior of a single oral dose of florfenicol in rainbow trouts experimentally infected with Lactococcus garvieae or Streptococcus iniae. One hundred and fifty fish were randomly divided into three equal groups: 1—healthy fish, 2—fish inoculated with S. iniae (2.87 × 10⁷ CFU/ml, i.p.), and 3—fish inoculated with L. garvieae (6.8 × 10⁵ CFU/ml, i.p.). Florfenicol was administered to all groups at 15 mg/kg by oral gavage. Blood sampling was performed at 0, 2, 3, 6, 8, 12, 24, 48, 72, and 120 hr after drug administration to each group, and plasma concentration of florfenicol was assayed by HPLC method. The MICs of florfenicol were 1.2 μg/ml and 5 μg/ml against L. garviae and S. iniae, respectively. Healthy fish showed higher values for most of the PK/PD parameters as compared to fish infected with L. garvieae which was reversed in fish infected with S. iniae. Fish infected with L. garvieae showed decreased relative bioavailability accompanied by increased volume of distribution at steady‐state (Vd_ss) and total body clearance (Cl_B)_. Infection with S. iniae increased the peak concentration of drug after administration (Cmax) and decreased elimination half‐life (T_{1/2 β})_, central compartment volume (V_c), and Vd_ss. In conclusion, infection with these bacteria can affect the pharmacokinetic behavior of florfenicol in rainbow trouts as shown by decreased bioavailability and increased total body clearance and volume of distribution in L. garvieae infection and decreased volume of distribution accompanied by increased Cmax in S. iniae‐infected fish.     

Preparation and Identification of Monoclonal Antibody against Min-A Strain of Pseudorabies Virus (PRV)

With purified pseudorabies virus (PRV) to immune BALB/c mice, spleen cells from imunized mice were fused with SP2/0 myeloma cells by application of lymphocyte hybridoma technique, followed by double antibody sandwich ELISA screening, four times cloning by limiting dilution method to obtain two stable secreting anti-PRV monoclonal antibody hybridoma cell lines, 2C6E6 and 3D5F1.After identification, these two monoclonal antibodies belonged to IgG1 and IgG2a subtypes, respectively, the average number of chromosomes of hybridoma cells was 84, ELISA titers of these two hybridoma cell culture supernatants and mouse ascites monoclonal antibodies were 1:512 and 1:1 638 400, 1:1 024 and 1:819 200.These two monoclonal antibodies showed no cross-reaction with classical swine fever virus, porcine reproductive and respiratory syhdrome virus and porcine parvovirus, which indicated that they had high specificity.The hydridoma cells could passage for 30 generations which could still secrete monoclonal antibody against PRV, which indicated that these two monoclonal antibodies had good stability.The results in the assay laid the foundation for establishing rapid diagnostic methods of PRV.     

伪狂犬病病毒闽A株单克隆抗体的制备与鉴定

本试验应用细胞杂交瘤技术,取健康BALB/c小鼠脾细胞与SP2/0 骨髓瘤细胞进行细胞融合,经双抗体夹心ELISA筛选,4次有限稀释法克隆,得到2株能稳定分泌抗伪狂犬病病毒(PRV)的单克隆抗体杂交瘤细胞株:2C6E6、3D5F1。2株杂交瘤细胞培养上清的效价分别为1:512、1:1 024。鉴定结果显示这2株单克隆抗体分别为IgG1亚类和IgG2a亚类,杂交瘤细胞的平均染色体数目为84条。用纯化的PRV免疫经产健康的BALB/c小鼠,采用ELISA方法检测获得的腹水的效价分别为1:1 638 400和1:819 200。经检测这2株单克隆抗体与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪细小病毒均不发生交叉反应,特异性良好。杂交瘤细胞连续培养30代,仍能稳定分泌抗PRV的单克隆抗体,说明这2株单克隆抗体的稳定性良好。本试验研究结果为PRV的快速诊断方法的建立奠定了理论基础。     

犬肾（DK）传代细胞中霉形体的分离与鉴定

取经电镜观察证实有霉形体污染的ＤＫ传代细胞，分别接种于霉形体检验用液体、半流体和固体培养基，证明为霉形体混合感染，即该培养物在液体培养基和半流体培养基上，同时显示出水解精氨酸和发酵葡萄糖２种特性，前者使培养基ｐＨ上升，后者使ｐＨ下降。通过反复挑选单个菌落进行克隆传代和有目的地加入单一种属的霉形体抗血清的方法，将混合的２株霉形体纯化分开。经鉴定，一株为精氨酸霉形体，一株为莱氏无（需）胆甾醇霉形体。     

鸵鸟源新城疫病毒TN株F基因的克隆与序列分析

应用PCR技术对1株鸵鸟源新城疫病毒TN株融合蛋白基因(F基因)进行扩增,将其克隆到pMD18-T载体后进行序列测定,并与国内外NDV毒株对应序列进行比较分析.结果表明,TN株F基因的长度为1 662 bp,可编码553个氨基酸,其裂解位点的氨基酸序列为112G-R-Q-G-R-L117,为1株新城疫弱毒株;与贵州省其他禽源(包括肉鸡、蛋鸡、越南斗鸡、七彩山鸡和鸽子等)毒株间的核苷酸同源性为84.0%～89.6%,氨基酸同源性为87.5%～92.1%;与国内外NDV代表株(Lasota株、B1株、F48E9株、CH2000株和TW2000株)的核苷酸同源性为84.4%～98.6%,氨基酸同源性为88.3%～98.0%.经系统发生树分析,TN株与NDV弱毒株Lasota株和B1株在同一分支上,属于基因II型NDV.     

1株传染性法氏囊病病毒的分离鉴定及VP2基因序列分析

【目的】 研究1株传染性法氏囊病病毒（Infectious bursal disease virus,IBDV）广西分离株的毒力特征及其与VP2基因序列特征的关系,为IBDV的流行病学研究和疫病防控提供参考。【方法】 通过PCR、鸡胚传代培养、DF-1细胞的适应培养及琼脂扩散试验等方法分离鉴定病毒,并扩增其VP2基因,利用Mega 7.0、MegAlign软件将其与其他不同毒力IBDV毒株进行核苷酸及氨基酸序列比对分析,并进行SPF鸡致病性试验,用实时荧光定量PCR方法测定IBDV拷贝数变化情况。【结果】 成功分离到1株IBDV毒株,命名为GX20210126株。该毒株接种SPF鸡胚可导致鸡胚出现出血、矮化和肝脏发黄、出血及针尖状坏死;且GX20210126对DF-1具有良好的细胞适应性,可引起显著细胞病变。琼脂扩散试验显示,该毒株具有良好的抗原性,病毒液能与IBDV阳性血清之间形成白色沉淀线。VP2基因进化分析显示GX20210126株与国际标准超强毒株在同一个大的分支上,氨基酸序列相似性在86.8%~99.6%之间,其中与UK661株相似性最高,为99.6%,仅有2处氨基酸位点发生改变,即D279N和I272T。以EID₅₀为10^-3.8/0.1 mL,0.5 mL/只的剂量点眼、滴鼻接种8日龄SPF鸡,攻毒后鸡出现羽毛蓬松、精神萎靡、拉白绿色水样粪便等临床症状,剖检可见肌肉、肝脏出血,肾脏尿酸盐沉积,法氏囊萎缩,IBDV拷贝数在感染后第5天达到峰值。【结论】 IBDV广西流行株GX20210126具有超强毒株基因序列特征,人工感染鸡群可导致IBDV典型临床症状和病理变化。     

兔出血症病毒镇江株的分离鉴定及VP60蛋白活性表达

试验旨在分离兔出血症病毒（rabbit hemorrhagic disease virus,RHDV）镇江株,分析其遗传进化变异,并表达具有良好活性的重组VP60蛋白。通过排除细菌感染、血凝（HA）和血凝抑制（HI）检测、动物攻毒试验与病毒传代、LD₅₀测定等方法,自江苏省镇江市某兔场发病死亡动物肝脏组织样品中分离病原并鉴定;RT-PCR方法获得VP60基因,通过分析VP60基因核苷酸及氨基酸序列研究其遗传进化;将VP60基因与pCold-Sumo载体连接,构建低温诱导、融合Sumo标签原核表达载体,15℃、IPTG诱导表达重组VP60蛋白并对表达产物进行反应原性鉴定。结果显示,分离鉴定获得RHDV ZJ2015毒株,该毒株能凝集人"O"型红血球,HA效价为11log2,其血凝性能被RHDV （AV33）抗血清抑制,该毒株的LD₅₀为10^-6.38/mL,具有较强的毒力;RT-PCR扩增得到大小约为1 740 bp的特异性条带,系统进化树分析显示,该毒株属于RHDV1抗原遗传变异株（RHDVa）,与RHDV1和RHDV2 VP60基因核苷酸序列同源性分别为89.4%~97.6%和81.1%~81.5%,氨基酸序列同源性分别为93.8%~98.3%和87.4%~87.6%。构建的低温原核融合表达质粒pCold-VP60在大肠杆菌Rosetta （DE3）中成功表达,通过SDS-PAGE及Western blotting分析,在分子质量74 ku处有特异性表达蛋白条带,且与抗血清发生特异性抗原抗体结合反应,说明重组蛋白具有良好的反应原性。本研究为开展兔病毒性出血症流行病学研究、开发新型重组疫苗与诊断试剂提供参考依据。     

一株猪伪狂犬病病毒变异株的分离鉴定

为了解广东省猪伪狂犬病病毒（pseudorabies virus,PRV）野毒株的基因变异及遗传演化的情况,本试验对广东佛山疑似暴发伪狂犬病的猪场采集的病料（脑、肺脏、扁桃体、肝脏、脾脏）进行了PCR鉴定,初步鉴定为PRV毒株后,将阳性病料接种非洲绿猴肾细胞（Vero）,进行毒株传代培养,对分离毒株进行PCR检测及小鼠感染试验,证实该病毒为PRV,并命名为PRV FS-2015株;并对该毒株进行细胞病变观察、病毒TCID₅₀测定、毒株gC和TK基因扩增及序列分析。结果显示,PRV FS-2015株TCID₅₀为10^-7.5/0.1 mL。PRV FS-2015株的gC和TK基因序列与国内外PRV参考毒株进行同源性比对分析发现,其核苷酸序列同源性分别为95.8%~100.0%和99.4%~100.0%,氨基酸同源性分别为92.3%~100.0%和98.7%~100.0%。遗传进化分析表明,PRV FS-2015株与国内近几年分离的PRV变异株GY、ZJ01、HB1201、HN1201、JS2012、BJ/YT和BP属于同一分支,同源性较高,亲缘关系更近;但与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha、Yangsan、Min-A、SS和SL株的同源性较低,基因变异较大,表明PRV FS-2015毒株属于近几年流行的变异株。本研究结果可为广东省伪狂犬病的防控工作和疫苗株的选择提供科学依据。