Identification and Pathogenicity Analysis of Aeromonas caviae M12 from Fox

We tried to identify the bacteria and explore the mechanism of the bacteria's pathogenicity via housekeeping gene gyrB and in vitro organ culture (IVOC) of ileum and intramuscular injection. Microscope, electron microscope and scanning electron microscope were also used to observe and the structure, pathogen of M12 and changes of infected tissues. The results showed that M12 was short gram-negative bacteria, and both ends of it were obtuse and the size without flagellum was (0.6 to 1.6) μm×(0.6 to 0.7)μm while length of flagellum was about 2 to 3 times of the length of bacteria.It had 100.0% similarity with Aeromonas caviae from GenBank. IVOC test observed M12' adhesion in intestinal epithelial cell, resulting in formulation of biofilm structure and damage in intestinal epithelial cell. Histopathologic examination showed that M12 could cause damage in intestine, liver, lung, kidney, muscle tissue of rabbit.The assay would offer references for researching the pathogenic mechanism of Aeromonas caviae.     

Establishment and Application of Loop-mediated Isothermal Amplification for Rapid Detection of Porcine Epidemic Diarrhea Virus

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of porcine epidemic diarrhea virus (PEDV), and provide a simple, sensitive, accurate and reliable tool for diagnosis of PEDV.The conservative PEDV N gene (GenBank accession number: KT799997) of PEDV was selected as a target to design six specific primers.The reaction system and temperature of LAMP were optimized, and the LAMP method for specific amplification of PEDV was established. Results showed that the PEDV LAMP detection method was established successfully, and it could detect PEDV specifically at 60℃ for 60 min,and the detection limit was 91 copies/μL, which was one hundred-fold higher than conventional RT-PCR method.75 clinical samples were detected by LAMP and PCR, respectively, the coincidence of LAMP and PCR was about 97.3%. All the data suggested that the LAMP assay had strong specificity, high sensitivity, simple operation, low equipment requirement, and was suitable for rapid detection of PEDV clinical samples.     

Effects of UCHL1 Inhibition on Porcine Oocyte Matuation in vitro,Zona Pellucida Ubiquitination and Polyspermy

This study was aimed to examine the effects of UCHL1 inhibition on porcine oocyte maturation in vitro, zona pellucida (ZP) ubiquitination and polyspermy. DAPI staining, Hoechst staining and SDS-PAGE methods were used to detect the matuation rate of porcine oocytes in vitro, the level of ubiquitination of zona pellucida (ZP) and polyspermy. The results showed that after different concentrations UCHL1 inhibitor (10, 20, 25 and 30 μmol/L, DMSO and control group) were added into maturation medium for culturing 46 h in vitro, the mature rate of control group was 86.22%, while the maturation rate of 30 μmol/L group was 15.30%, and the maturation rate of every treatment group had significant difference (P<0.05). Western blotting result showed that every group generated ubiquitin markers of ZP were about 61, 80 and 106 ku in different degree. According to the analysis of gray value, the result had significant difference (P<0.05). Conducting fertilization in vitro, the number of sperm adhered on oocyte ZP in control group was the most, the number of sperm running into oocyte was fewer, the number of sperm adhered on oocyte ZP with 30 μmol/L UCHL1 inhibitor was the fewest, and there was almost no sperm running into the oocyte. The results showed that UCHL1 inhibitor had an impact on maturation of porcine oocytes in vitro. With the higher concentration of UCHL1, the lower degree of ZP protein ubiquitinated, UCHL1 could regulate sperm attachment and polyspermy.     

安淮山羊骨骼肌卫星细胞的分离培养与鉴定

为了分离得到安淮山羊骨骼肌卫星细胞,成功构建山羊骨骼肌卫星细胞系,为相关研究提供实验材料。以安淮山羊背最长肌肌肉组织为材料,采取胶原酶和胰蛋白酶结合的二步消化法,结合差速贴壁法纯化原代骨骼肌卫星细胞。对骨骼肌卫星细胞中特异性基因Pax7用免疫荧光染色法及PCR扩增加以鉴定。通过分离、纯化,得到纯度较高的山羊骨骼肌卫星细胞,其形态为圆形,折光性强。培养一段时间后密度增大,细胞呈梭型。所得骨骼肌卫星细胞生长状态良好,具有较为一致的生长方式和形态特点。经免疫荧光染色法以及PCR鉴定发现细胞系中有Pax7基因的表达。通过上述材料和方法,可获得较纯的山羊骨骼肌卫星细胞,并稳定传代。     

河南省猪等孢球虫的分子变异规律研究

[目的]掌握河南省猪等孢球虫的分子变异规律。[方法]从河南省豫东、豫南、豫西、豫北及豫中地区的部分规模化猪场采集粪便样品,进行猪球虫卵囊的分离与纯化;从分离到的8株猪球虫分离株中提取核酸并进行测序,并与猪等孢球虫扩增株基因序列及其他相关原虫基因序列进行比对。[结果]河南省不同地区的8个猪球虫株均为猪等孢球虫,并且没有明显遗传差异。I.suis与其他7种原虫差异较大,不属于同一个生物型,同一种动物所感染的不同种类原虫之间的同源性较低。[结论]该研究结果可为有效预防和控制仔猪球虫病提供科学依据。     

橡子壳色素抗氧化活性研究

本研究以常见的抗氧化剂Vc为对照,对橡子壳色素主要成分和体内外抗氧化活性进行了研究分析。结果表明,橡子壳色素中含丰富的多酚类和黄酮类物质,其中总多酚含量达到27.8%,总黄酮含量8.6%。体外抗氧化实验结果表明,橡子壳色素对DPPH·的清除能力和对Fe3+的还原能力均与其质量浓度呈正相关,其中对DPPH·清除率的半数有效剂量(IC₅₀=10 μg/mL)大于Vc组(IC₅₀=5 μg/mL),当橡子壳色素质量浓度在40 μg/mL时达到了Vc的清除能力;当橡子壳色素质量浓度为1 mg/mL时,橡子壳色素还原能力(A_{710 nm}为0.98)接近Vc的还原能力(A_{710 nm}为1.17)。体内抗氧化实验采用对除空白组外的各组实施60Coγ照射进行氧化损伤实验,分析各组的抗氧化能力。结果表明,模型组小鼠体质量和各器官质量与空白组相比显著下降(P＜0.05),并且辐照的各组小鼠都出现了食欲不振、精神萎靡等不良现象,表明氧化应激模型建立成功;橡子壳色素组的小鼠器官质量显著高于模型组(P＜0.05),低于空白组但无显著差异(P＞0.05),该结果表明橡子壳色素对经60Coγ射线照射的小鼠器官有一定的修复作用。实验小鼠血清中的抗氧化性相关酶以及氧化产物的测定结果进一步表明,橡子壳色素低、中、高剂量组均能显著提高小鼠血清中SOD和CAT的活性,并且显著降低MAD含量(P＜0.05),其中100 mg/(kg·d)剂量组橡子壳色素的作用效果最好,与同质量浓度Vc作用效果相当。橡子壳色素具有较好的体内和体外抗氧化活性,并且体内100 mg/(kg·d)剂量的橡子壳色素与同剂量下Vc的抗氧化效果相当,作为天然抗氧剂具有较好的应用前景。      

益生菌安全性和有效性评价方法的研究进展

益生菌是指一类活的、摄入足够量就能够对人体产生有益作用的微生物,目前广泛应用于食品和饲料工业领域。随着市场上商品化益生菌的不断出现,如何评价一个菌株的安全性及有效性成为人们关注的问题。目前,益生菌的评价主要从安全性和有效性入手,安全性和有效性评价又包括体内和体外的评价。很多企业在开发益生菌产品时很少对菌株做出全面的安全性评价,而且各国研究者采用的评价指标和评价方法也不尽相同。对于有效性评价,同样也没有统一的评价标准。针对目前益生菌评价存在的问题,亟需建立一套完善的评价方法,对益生菌的安全性和有效性进行系统、科学地评价。作者从益生菌可能产生的潜在危害及功效方面出发,着重论述了益生菌的溶血性、毒力因子、抗生素抗性和产有毒代谢产物的体外安全性评价方法,耐酸、耐胆盐、耐人工胃肠液、胃肠黏膜黏附性和抑菌活性的体外有效性评价方法,及体内的安全性和有效性评价方法的研究进展,期望为建立完善的益生菌评价技术体系和标准提供参考。     

猪繁殖与呼吸综合征病毒RT-LAMP快速检测方法的建立

为建立一种快速、灵敏、简便的猪繁殖与呼吸综合征病毒（PRRSV）早期检测方法,本研究利用逆转录环介导恒温扩增技术（RT-LAMP）,针对PRRSV的ORF5基因片段设计了4条引物,利用Bst DNA聚合酶在65 ℃恒温条件下进行逆转录扩增,通过1.0%琼脂糖凝胶电泳和加入SYBR Green Ⅰ染料肉眼判断结果,建立了PRRSV RT-LAMP检测方法。结果表明,该检测方法具有良好的特异性,与其他常见病毒如猪瘟病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒等无交叉反应,较普通RT-PCR灵敏性高100倍。采用RT-LAMP和RT-PCR分别对10份临床样本同时进行检测,符合率为100%。因此,本研究建立的RT-LAMP是一种可适用于临床PRRSV检测的快速、简单、灵敏、特异的检测方法。     

应用CNCPS方法和体外产气法研究豆腐渣饲料的营养价值

为揭示豆腐渣对反刍动物的饲用价值,本试验采用康奈尔净碳水化合物-蛋白质体系（CNCPS）和体外产气法对豆腐渣营养价值进行评定。测定豆腐渣的CNCPS营养成分与体外发酵参数,应用CNCPS公式计算碳水化合物（CHO）和粗蛋白质（CP）中各组分含量。结果显示,豆腐渣的CP含量为14%~25%,粗脂肪（EE）含量为1.9%~2.9%,代谢能（ME）为10~12 MJ/kg DM;易在瘤胃中降解的蛋白质（PA＋PB₁）和在瘤胃中缓慢降解的粗蛋白质（PB₂＋PB₃）的含量相当,不可降解真蛋白质（PC）含量较少;潜在可利用碳水化合物（CA＋CB₁＋CB₂）较高;体外发酵产气量均达到70 mL,氨态氮（NH₃-N）浓度在8~23 mg/dL,总挥发性脂肪酸（TVFA）在46~70 mmol/L。而不同来源的豆腐渣营养成分和发酵情况存在显著差异,且CNCPS碳水化合物、蛋白质组分的优劣不同。凌源豆腐渣的最大产气量（B）最高,极显著高于其他3种苹果渣（P＜0.01）,而各豆腐渣的产气速度（C）无显著差异（P＞0.05）。辽源豆腐渣氨态氮（NH₃-N）含量最高,极显著高于其他饲料（P＜0.01）。不同地方豆腐渣瘤胃液挥发性脂肪酸比例差异极显著（P＜0.01）。不同来源的豆腐渣的总可消化养分（TDN）及其他能值差异较大,以新民豆腐渣的能值最低,极显著低于其他苹果渣（P＜0.01）。综上可知,作为反刍动物非粮饲料,豆腐渣为反刍动物提供的可利用能值高,利于瘤胃微生物蛋白质合成。     

黄顶菊种子萌发过程中DNA甲基化的MSAP分析

为明确入侵植物黄顶菊Flaveria bidentis生长发育与DNA甲基化之间的相互关系,采用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP)方法研究了黄顶菊种子萌发过程中DNA甲基化的动态变化。结果表明,DNA甲基化与去甲基化两者共同调控黄顶菊生命初期的生长发育,且去甲基化的变化在种子萌发过程中占主导。筛选出的12对引物共扩增出998条MSAP条带,其中多态性条带为951条,多态性百分比为95.37%。黄顶菊种子萌发过程中胞嘧啶发生甲基化主要以双链甲基化形式为主,位点数为94个,而单链甲基化位点数仅为50个;多态性位点数占总位点数比率为48.95%,表明有近一半的位点发生了DNA甲基化和去甲基化的变化;发生去甲基化变化的多态性片段有73个,而发生甲基化变化的有21个,说明黄顶菊种子萌发阶段DNA甲基化的变化主要以去甲基化形式为主,且在萌发第4天后去甲基化数目持续快速上升。