表观健康猪群携带猪链球菌2型毒力因子的分布特征

为了研究钦州市猪链球菌2型(SS2)的毒力因子分布特征,采用多重PCR方法对从我市健康猪扁桃体分离的11株SS2相关毒力基因——荚膜多糖(Cps2J),溶菌酶释放蛋白(Mrp),胞外蛋白因子(EF),溶血素(Sly),进行毒力基因检测。检测结果显示:毒力基因型为Mrp+epf+Sly+共有8株,毒力基因型为Mrp+epf+Sly-共有1株,这两种毒力基因型可归为强毒力基因型,占此次健康猪源毒株的81.8%;另外Mrp-epf-Sly-共有2株,属于弱毒和无毒基因型,占健康猪源毒株的18.2%。通过结果分析,预示我市目前SS2的主要流行菌株是同时具有4种毒力因子的高致病性菌株。     

Smad泛素化调节因子2siRNA的筛选及转染原代肾小管上皮细胞条件的优化简

试验旨在优化Smad泛素化调节因子2（Smurf2） siRNA最佳转染条件,筛选最佳siRNA干扰片段,进而实现对Smurf2基因的瞬时沉默,为研究Smurf2基因在马兜铃酸肾病（aristolochic acid nephrohathy,AAN）中的作用奠定基础。本研究通过培养小鼠原代肾小管上皮细胞（renal tubular epithelial cells,RTECs）,并以脂质体Lipofectamine^TM 2000为转染介质,将Smurf2 siRNA转染入RTECs;通过观察绿色荧光的表达量及Real-time PCR反应优化转染条件,转染后Real-time PCR检测mRNA表达抑制率。CCK-8法检测Smurf2 siRNA复合物对RTECs活性的影响;同时,用Real-time PCR和Western blotting检测不同位点Smurf2 siRNA对Smurf2 表达的影响。结果显示,当Lipofectamine ^TM 2000与siRNA比例为1.5 μL∶30 pmol时,转染效率最高,为70%～80%;Smurf2-619 siRNA干扰效果最明显;与正常组相比,转染siRNA组的Smurf2蛋白表达水平显著下降（P<0.05）。最终确定了Smurf2 siRNA最佳转染条件,其中Smurf2-619 siRNA对Smurf2 表达的抑制率最高。     

猪圆环病毒2型核酸疫苗的小鼠免疫保护试验

为了研究猪圆环病毒2型(PCV2)核酸疫苗在小鼠攻毒试验中的免疫保护效果,以PCV-2 GXWZ-1株为模板,扩增出ORF2基因及其截短基因8个片段(A(ORF2)、B(51-100aa)、C(101-150aa)、D(181-235aa)、E(151-200aa)、F(51-150aa)、G(101-235aa)、H(51-235aa)),将其插入到pcDNA3.0载体中,构建出真核表达质粒,并将其转染至PK-15细胞,用间接免疫荧光试验检测其瞬时表达情况。将试验小鼠随机分成9组,其中免疫组7组,阴阳性对照各1组,将纯化的真核表达质粒对小鼠进行组合免疫;二免后,用经处理过的PCV-2 GXWZ-2株阳性病料悬液腹腔注射免疫组和非免疫对照组小鼠,阴性对照组用生理盐水腹腔注射;其后进行体重记录、病理切片制作及PCR检测。结果表明:共有6个真核表达质粒成功在PK-15细胞中表达。在攻毒后的3周内,阳性对照组小鼠PCR诊断均为阳性;免疫组中,部分组小鼠在攻毒后第1周或在第2周为阳性,到第3周时各免疫组小鼠全部为阴性;阴性对照组始终为阴性。免疫组在病理保护学方面明显优于非免疫对照组,非免疫对照组的体重增长速率略低于免疫组和阴性对照组。由此可见猪圆环病毒2型核酸疫苗在小鼠攻毒试验中有明显的保护作用。     

猪传染性胸膜肺炎放线杆菌和猪圆环病毒2型二重实时荧光定量PCR检测方法的建立

以胸膜肺炎放线杆菌(App)apx ⅣA基因和猪圆环病毒2型(PCV-2)ORF2基因为靶基因,分别设计特异性引物和探针,建立App和PCV-2的二重荧光PCR方法。App引物扩增的目的片段大小为132bp,PCV-2为169bp。建立的方法可在同一反应体系中同时对App和PCV-2进行定量检测鉴别,其敏感性分别为1×101拷贝和1×100拷贝,对其他猪病病原核酸的检测为阴性。利用该方法对10份人工感染临床样品进行检测试验,其结果符合率达100%,说明该方法可用于对临床样品中App和PCV-2的检测。     

Closed system anaesthesia in dogs using liquid sevoflurane injection; evaluation of the square-root-of-time model and the influence of CO2 absorbent

Objective To determine whether predictable alveolar concentrations of sevoflurane are reliably produced in dogs when liquid sevoflurane is injected into closed circuit breathing systems, as calculated by Lowe's square‐root‐of‐time anaesthetic uptake model, and to confirm the validity of the model using soda lime and calcium hydroxide lime. Study design Prospective clinical study. Animals Eleven healthy dogs with a mean body mass of 34 ± 9 kg scheduled for pelvic limb orthopaedic surgery. Materials and methods Following pre‐anaesthetic medication, anaesthesia was induced with propofol and maintained with sevoflurane in a closed circle system. Epidural anaesthesia was performed with morphine and bupivacaine. Liquid sevoflurane was injected into the circuit by syringe, using dosages and time intervals derived from Lowe's square‐root‐of‐time anaesthetic uptake model. The target alveolar concentration chosen was 1.1 × MAC (2.6% end‐tidal sevoflurane). Either soda lime (group S; n = 6) or calcium hydroxide lime (Amsorb; group A; n = 5) were used for CO₂ absorption. Sevoflurane concentration and the respiratory gas composition were measured with an infrared gas analyser. Results End‐tidal sevoflurane concentrations were close to the predicted value of 2.6% at 9 minutes (2.53 ± 0.1% group S; 2.60 ± 0.26% group A) and 16 minutes (2.55 ± 0.30 group S; 2.52 ± 0.28% group A) but declined thereafter to reach 50% (group S) and 64% (group A) of the predicted value at 121 minutes. There was a constant trend towards higher end‐tidal sevoflurane concentrations in group A but the difference was not statistically significant. Conclusions The square‐root‐of‐time model leads to significantly lower alveolar concentrations than expected, suggesting that the rate of sevoflurane uptake in dogs declines less rapidly than predicted. The use of Amsorb tends to reduce the deviation from predicted concentrations. Clinical relevance The model used in this study provided only an approximate guide to the volume of liquid sevoflurane required. Consequently, the definitive dose schedule must be based on measured anaesthetic concentrations and clinical monitoring.     

褪黑素和光照对鹌鹑白细胞介素-2的影响

采用褪黑素(MLT)完全抗原主动免疫鹌鹑，在不同光照条件下饲养，研究了MLT参与神经-内分泌-免疫网络调控时对IL-2水平的影响。结果表明：主动免疫后，MLT和IL-2水平明显下降；随着光照时间的延长，IL-2水平也显著下降。一定剂量的MLT可促进机体免疫因子IL-2的分泌。     

猪繁殖与呼吸综合征病毒河北地方株ORF6基因的克隆及原核表达

从河北沧州分离到一株疑似猪繁殖与呼吸综合征病毒,接种Marc-145细胞,经4代盲传,出现细胞病变,经鉴定为PRRSV,命名为HB-3(cz)株。根据GenBank公布的PRRSV JXA1株ORF6基因的核苷酸序列,设计并合成一对特异性引物(P1/P2),用RT-PCR方法扩增完整ORF6基因,将扩增产物连接到pGM-T载体并转化克隆菌,阳性重组质粒PGM-M进行序列测定与分析。后将克隆质粒PGM-M双酶切后连接原核表达载体pET-32a(+),在Rosseta-DE3中成功获得表达,经Western-blotting分析表明,表达蛋白与阳性血清发生特异性反应。     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

南阳牛IGF2基因第2外显子多态性及其与生长发育相关性研究

以126头纯种南阳黄牛为研究材料，利用PCR-RFLPs技术对胰岛素样生长因子2（IGF2）第2外显子的多态性及其与生长发育的相关性进行了分析。结果表明：南阳牛在该位点的遗传多态性丰富。检测到2种等位基因A和B，频率分别为0．6865和0．3135，A等位基因在群体中是优势等位基因；BB型个体的6、12、18、24月龄体斜长和胸围，12、18和24月龄的坐骨端宽，初生、6月龄和24月龄体重等体征性状显著地优于AA型；6月龄体斜长、12月龄的胸围、18月龄和24月龄坐骨端宽也显著优于杂合型。初步推断JGF2基因是非常有价值的辅助选择标记，可为南阳牛肉用型方向选择提供理论依据。