Analysis of the miRNA Expression Profiles in the Primary Neurons of Mice Infected with Japanese Encephalitis Virus

The interaction between JEV and the host at the miRNAs level was preliminarily explored by studying the miRNAs expression profiles of primary neurons in mice infected with JEV. Total RNA of JEV-infected and uninfected primary neurons of the suckling mice was extracted individually by Trizol and then analyzed miRNA expression profiles by high-throughput sequencing analysis. Significantly differentially expressed miRNAs were selected for verification by real-time quantitative PCR. Through bioinformatics analysis, 26 miRNAs with significant expression differences were screened out, among which 18 miRNAs were up-regulated and 8 miRNAs were down-regulated. The results of quantitative real-time PCR of the JEV-E gene indicated that the expressions of mir-21a-3p mir-223-5p mir-147-3p mir-155-5p and mir-146a-5p could promote the expression of JEV-E gene in neurons, while the expression of mir-301a was just on the contrast. The expression of miRNAs in primary neurons could affect the replication of JEV. This study provided the theoretical basis and direction for further studies on the regulatory function of miRNAs in the mechanism of neural dysfunction induced by JEV.     

Isolation and Identification of Group I Type 4 Avian Adenovirus and Analysis in Immune Effect of Fiber-2 Protein

The aim of this study was to identify pathogens, which caused pericardial effusion, hepatomegaly and bleeding at a chicken farm in Henan province, and furthermore to analyze effectiveness of immunogenic proteins of the pathogen. This study employed virus isolation, serological assays, PCR and sequencing analysis, animal experimentation, E. coli expression and protein purification, immunogenicity and challenge test and other methods. The results showed that virus was isolated in 7-day-old SPF chicken embryonated eggs, inoculated via the yolk sac route by two blind passages. Viral confirmation was carried out using PCR techniques, and showed a 900-bp-long fragment which shared a 100% homology with the Hexon gene of the serotype C4 strain. Serum neutralization results indicated that this isolate avian adenovirus was the group I type 4 avian adenovirus, named HN-ZK strain. The virus could induce CPE on primary hepatocytes of chicken embryo, and its titer was 10^7.5 TCID₅₀·0.1 mL^-1. Animal experimentation illustrated that the isolated virus caused 100% (10/10) typical symptoms and pathogenicity in 35-day-old SPF chickens. Furthermore, the DNA of the isolate virus was used as a template to express Fiber-2 fragment, with a molecular weight of 33 kDa by using the E. coli expression system, and the protein was concentrated and purified to 300 mg·mL^-1 after centrifugation and purification. SPF chickens were immunized with different doses of the purified Fiber-2 protein, and showed that a dose of 20 μg per chick was completely resistant to challenge of the virulent virus, suggesting the purified Fiber-2 protein has better immunogenicity. This study can provide data for diagnosing the hydropericardium hepatitis syndrome, and developing a genetic engineering subunit vaccine.     

The Effect of Caffeic Acid on Zearalenone-induced Ovarian Granulosa Cell Apoptosis in Mice

Ovarian granulosa cells provide a special microenvironment for follicle formation and maturation through interaction with oocytes and their own secretion. A variety of harmful stimuli can cause granulosa cell apoptosis and metabolic disorders, reduce the quality of oocytes and have a negative impact on embryo formation. Zearalenone (ZEA) is a common cause of ovarian granulosa cells injury in the livestock industry, which is produced by mycotoxins, and lack of effective treatment drug. Therefore, in the current study zearalenone was used to induce ovarian granulosa cell injury and to explore the protective effect of caffeic acid on zearalenone-induced ovarian granulosa cell apoptosis in mice. Mouse ovarian granulosa cells were isolated by mechanical method, and indirect immunofluorescence was used to identify the isolated cells. MTT assay was used to determine the effect of caffeic acid on the activity of normal mouse ovarian granulosa cells.After granulosa cells were co-treated with caffeic acid (200, 100 and 50 μg·mL^-1) and ZEA for 24 hours, and control and ZEA group were set up at the same time, cell morphology and adherence were observed under a microscope. MTT was also used to detect cell viability. Caspase-3 mRNA expression level was detected by qRT-PCR. Cleaved-caspase-3 and cleaved-PARP protein expre-ssion levels were determined by Western blot. The results showed that positive FSHR staining appeared in cell cytoplasm of the test group, which confirmed that the isolated cells were mouse ovarian granulosa cells. The cell viability was above 90% which showed that caffeic acid had no toxic effect on granulosa cells. Compared with control group, ZEA group had smaller cell size, poor adherence, increased cell gap, and significant reduction in cell viability (P<0.001). Furthermore, the relative expression of caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein level were significantly increased (P<0.001) compared with the control group. After caffeic acid treatment, cell gap was reduced, adherence was tight, cell viability was significantly increased (P<0.001). Caffeic acid significantly reduced zearalenone-induced increase in caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein expression level (P<0.001). This study indicated that caffeic acid can restore granulosa cell viability by inhibiting ZEA-induced apoptosis.     

母猪卧向行为学的观察

本文通过2个试验对母猪在分娩圈和分娩栏中的卧向行为进行了观察，试验一中，选择10头大白（Yorkshire）经产母猪作为观察对象，试验二中分别选择10头大白（Yorkshire）经产母猪和10头长白（Landrace）经产母猪作为观察对象。观察采用瞬时记录方法，每周观察三次，隔日观察，每天上午、下午各观察一次，每次3h，每次观察间隔5min。观察中发现，母猪卧向以向外为主。分娩栏（四周是栏杆）中的母猪以选择卧向外（向北）为主，其次是卧向内（向南），卧向左和卧向右最少且差异不显著，上栏前和下栏后差异不显著。分娩圈（四周是墙壁）中母猪以选择卧向外为主，卧向内最少，卧向左和卧向右差异不显著。长白母猪比大白母猪选择卧向外的多，妊娠阶段比哺乳阶段选择卧向外的多。     

武冈铜鹅与其他3种鹅肉品质特性的比较

本试验旨在测定武冈铜鹅、四川白鹅、武川鹅以及天府肉鹅的常规肉品质和矿物质元素、氨基酸、风味脂肪酸、肌苷酸含量及肌纤维直径与密度,并进行比较。选取1日龄体格健壮的武冈铜鹅、四川白鹅、武川鹅和天府肉鹅各30只,分为4组,每组5个重复,每个重复6只鹅,公母各占1/2,饲喂相同饲粮。试验期为70 d。结果表明:1)武冈铜鹅胸肌粗蛋白质含量显著高于其他3种鹅(P<0.05),胸肌粗脂肪含量显著高于四川白鹅(P<0.05);而腿肌粗脂肪含量与其他3种鹅差异不显著(P>0.05),腿肌贮存损失率显著低于其他3种鹅(P<0.05)。2)武冈铜鹅肌肉铁、镁、钾含量均显著高于四川白鹅和天府肉鹅(P<0.05)。3)武冈铜鹅肌肉中天门冬氨酸、谷氨酸、丙氨酸、缬氨酸、赖氨酸的含量极显著高于武川鹅和四川白鹅(P<0.01),甘氨酸和脯氨酸含量极显著高于其他3种鹅(P<0.01),而胱氨酸和苯丙氨酸与其他3种鹅差异不显著(P>0.05)。4)武冈铜鹅肌肉中肉豆蔻酸含量极显著高于其他3种鹅(P<0.01);十六烯酸含量极显著高于武川鹅(P<0.01);亚麻酸含量显著高于武川鹅(P<0.05);二十烯酸含量极显著高于天府肉鹅(P<0.01)。5)武冈铜鹅胸肌肌纤维密度显著高于四川白鹅和武川鹅(P<0.05),而各组肌纤维直径没有显著差异(P>0.05)。由此可见,武冈铜鹅肌肉品质以及风味优于武川鹅、四川白鹅以及天府肉鹅。     

实时光电微生物检测技术在乳品质控中的应用

对酸奶中的霉菌酵母菌同时采用实时光电微生物检测系统与国标法进行检测,分析比较两种检测方法所得的检测数据.本实验利用实时光电微生物检测系统的霉菌酵母检测试剂瓶对市售酸奶及阳性添加样品采用半定量的检测方法进行快速检测,能快速地检测到目标菌存在,继而给系统提前预警.结果表明,当霉菌酵母菌含量在10～3.5×105CFU/mL时,实时光电微生物检测方法在1.8～33h内预警.当平板计数法的检测结果小于10CFU/mL时,实时光电微生物检测方法的检测时间均大于33h,在仪器设置运行48h内未出现预警现象.实时光电微生物检测技术与国标平板法两种检测方法的检测结果相当吻合.应用实时光电微生物检测技术能快速便捷,对酸奶产品中霉菌酵母菌进行监测,其检测速度和灵敏度均能满足工厂实验室的快速筛选检测,还利于产品的关键控制点的监测.     

鹿流行性出血病毒分子生物学研究进展

鹿流行性出血病毒是一种在全世界野生及驯养的有蹄动物中广泛存在的重要病原体.该病毒属呼肠病毒科环状病毒属,有12个血清型,是一种有10个节段(L1-L3、M4-M6、S7-S10)的双链RNA病毒,编码10个蛋白(VP1-7和NS1、NS2、NS3/NS3A).VP7蛋白具有很强的抗原性且保守性最高.VP2与病毒型特异性有关,可诱导产生中和抗体.VP3为群特异性抗原,高度保守,具有亲水性保守区域.非结构蛋白NS1、NS2和NS3/NS3A及其编码序列均相当保守.该病毒的分子生物学诊断技术主要有PCR和核酸探针杂交技术.     

添加缬氨酸和异亮氨酸对哺乳母猪及其仔猪生产性能的影响

本试验旨在探讨在哺乳母猪日粮中添加缬氨酸和异亮氨酸对母猪及其仔猪生产性能、母猪采食量和乳成分的影响.试验选用健康、体况、预产期相近的第2胎法系JALAX母猪144头,分成3个处理组,即对照组、试验A组和试验B组,每个处理48头母猪.对照组饲喂基础日粮,试验A组饲喂添加低水平支链氨基酸日粮(基础日粮 缬氨酸0.26% 异亮氨酸0.06%),试验B组饲喂添加高水平支链氨基酸日粮(基础日粮 缬氨酸0.36% 异亮氨酸0.14%).试验从母猪妊娠108 d进产房开始,到仔猪21日龄断奶时结束.试验结果表明:(1)添加缬氨酸和异亮氨酸提高了仔猪的断奶窝重、断奶窝增重、平均日增重和21日龄仔猪成活率,但以试验A组的效果较好,和对照组比,上述指标分别提高了23.10%(P<0.01)、30.45%(P<0.01)、19.26%(P<0.05)和7.16%(P<0.05);(2)添加缬氨酸和异亮氨酸提高了哺乳母猪哺乳11～15 d,16～21 d以及哺乳全期的采食量,试验A组比对照组分别提高了35.61%(P<0.01)、27.72%(P<0.01)和24.21%(P<0.01),试验B组虽然与对照组之间差异不显著(P>0.05),但有提高的趋势;(3)添加缬氨酸和异亮氨酸极显著提高了泌乳第1天、第7天和第15天乳中乳脂、乳蛋白、总固形物、非乳脂固体的含量(P<0.01),极显著降低了乳糖的含量(P<0.01),但以试验A组的效果最好;(4)添加缬氨酸和异亮氨酸极显著提高了初乳(泌乳第1天)的总必需氨基酸、总非必需氨基酸以及总氨基酸的含量(P<0.01),但对第7天和第15天乳中氨基酸含量影响不显著.因此,日粮中添加缬氨酸和异亮氨酸可以提高哺乳母猪的日采食量和改善母猪乳品质,从而提高哺乳仔猪的生长性能.     

犬腺病毒纤突蛋白基因功能区的比较分析

根据GenBank中已发表的CAV纤突基因序列，设计合成4对8条引物，用已建立的PCR方法，对犬2型腺病毒沈阳分离株第5代强毒经蚀斑克隆驯化的第60代毒弱毒（SY-V60）和犬1型腺病毒长春犬株（CCC-V6）的纤突基因进行了扩增，PCR产物经纯化后进行基因序列测定，测定结果经拼接后得到一个由1632和1629个核苷酸组成的纤突蛋白全基因序列，分别编码543和542个氨基酸。犬2型腺病毒（SY-V60）与犬1型腺病毒（CCC-V6）的纤突基因的同源性达到80.48%。犬2型腺病毒（SY-V60）与犬1型腺病毒（CCC-V6）纤突基因的同源性达到80.48%；根据犬的腺病毒与人2型腺病毒纤突蛋白的序列同源性比较结果，推测了犬腺病毒纤突蛋白的尾（tail)、轴（shaft)，结（knob)三个功能区的氨基酸序列，2个型之间的尾区同源性为76.9%；轴区同源性为78.59%；其结区同源性为83.24%，而同型毒株之间结和尾区同源性较高，轴区同源性较低。     

航天诱变紫花苜蓿第一代植株(SP1)表型变异及基因多态性分析

采用正交设计优化紫花苜蓿(Medicagosativa L.)SSR-PCR反应体系,从17对SSR引物中筛选出6对扩增产物具有稳定多态性的引物,检测第1代植株的基因多态性。结果表明:4个紫花苜蓿品种德福(Defi)、德宝(Derby)、阿尔刚金(Algonguin)、三得利(Sanditi)共检测到25个等位基因,每对引物检测出2～8个等位基因,平均为4.17个。结合植株较高、叶色较深、叶片较大、多叶4个表型突变指标,检测经过表型变异筛选的植株等位基因频率及每个位点的多态性信息量(PIC),PIC在0.2216～0.8328之间变化,平均为0.6366。分析多态基因植株与表型变异的相关性,根据检测结果初步确定了13株突变植株,为后继世代遗传变异的多代跟踪及选育奠定基础。