2株猪流行性腹泻病毒的分离鉴定及ORF3基因的克隆与序列分析

为研究、防控广东地区猪流行性腹泻,首先通过RT-PCR法确定某猪场粪便样品中存在猪流行性腹泻病毒,随后利用Vero细胞进行盲传,最后经核酸类型鉴定、RT-PCR、病毒TCID50的测定、目的基因测序和序列分析等方法,确认分离到2株猪流行性腹泻病毒毒株,命名为PEDV GDKP-2013和PEDV GDKP2-2013,通过ORF3全基因核苷酸序列分析可知,GDKP-2013株与GDKP2-2013株与经典参考毒株的氨基酸序列一致性为 JX261936-CHGD-01,98.2%,98%;JQ039903-CH-GD-2011,98.6%,98.4%;AF353511-CV777,95.4%,95.1%;KC344845-STPED0810,95.1%,94.8%;EU054929-DR13,95.5%,95.2%;KF272920-USA-Colorado-2013,95.1%,94.8%,表明此2毒株与国内分离株的氨基酸同源性高,与韩国、泰国和美国分离株的氨基酸同源性要低一些,同时,与疫苗参考株的氨基酸同源性也相对较低;基因系统进化分析可知,GDKP-2013株与GDKP2-2013株主要处在Ⅰa分支,均与弱毒疫苗株（Ⅰb分支）相互之间遗传进化关系较远。     

一株A型副鸡嗜血杆菌的分离鉴定

从山东省某蛋鸡场发病鸡群中分离到一株细菌,经培养特性观察、生化试验、V因子生长试验、致病性试验和PCR试验,证实分离菌为一株致病性副鸡嗜血杆菌,血清型鉴定该菌为A型。药物敏感性试验表明分离菌对头孢噻肟、头孢曲松、丁胺卡那霉素高度敏感,对硫酸新霉素、强力霉素中度敏感,对磺胺间甲氧嘧啶钠、环丙沙星、庆大霉素耐药。     

大杯伞柄多糖与菌丝体粗多糖的免疫识别方法初探

采用热水(80℃)提取和75%乙醇沉淀、Sepharose CL-4B凝胶过滤及DEAE Sepharose CL-4B阴离子交换等方法分离纯化大杯伞(Clitocybe maxima)柄粗多糖,获得伞柄多糖纯品,制备多糖抗体;采用热水法提取大杯伞菌丝体粗多糖。用碳二亚胺法将纯化伞柄多糖与牛血清白蛋白进行共价化学偶联,并将偶联产物(拟糖蛋白)免疫兔子以制备抗血清,以酶联免疫法检测抗体滴度。依据该抗体与来自相同菌株的菌丝体粗多糖免疫反应差异对这两种多糖进行免疫识别,结果表明:经过亲和层析纯化后,第六次免疫抗血清对伞柄多糖的抗体滴度可达64K,而且对菌丝体粗多糖反应呈阴性,显示该抗体可识别这两种多糖之间的结构差异。     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

2株猪繁殖与呼吸综合征病毒的分离、鉴定及遗传进化分析

Two strains of porcine reproductive and respiratory syndrome viruses (PRRSV) were isolated from serum of some pig farms in Guangdong province and showed PRRSV positive in RT-PCR testing. The two viruses could passage stably and cause typical cenotaphic effect, they were named as LZ-GD and LB-GD. The analysis of variable region sequences of ORF5 and Nsp2 of the two viruses showed that LZ-GD and LB-GD strains were far to Europe strain Lelystad, the homology of nuclear nucleotide sequence were 63.5% and 63.8%, respectively, with classic American strain VR-2332 were 88.7% and 89.1%, respectively, and that with highly pathogenic JXA-1 strain were 99.2% and 99.3%, respectively. There were 30 amino acids deletion in Nsp2. It shared the deletion with JXA-1, HUN4 and other pathogenic variant. Thus, the two strains of PRRSV belonged to highly pathogenic American type.     

沛县鸭传染性浆膜炎的病原分离鉴定及药敏试验

收集沛县具有典型鸭传染性浆膜炎症状的34份病死鸭进行细菌的分离培养。总共得到18株疑似鸭疫里默氏杆菌（RA）,从中随机选取10株RA进行生化鉴定及动物回归试验,经确认后使用本地区常见的抗菌药进行了纸片法药敏试验,结果发现RA分离菌株对头孢噻肟、氧氟沙星、阿莫西林＋棒酸等药物敏感度较好,对氟苯尼考、丁胺卡那、阿奇霉素等药物敏感度很低。研究结果为该地区鸭传染性浆膜炎的药物防治提供了很好的理论依据。     

草鱼肠道中香港海鸥型菌的选择性分离与鉴定

从广东省某市池塘采集商品规格(体重2．0～2．5k)的草鱼(Ctenoharyngodon idellus)，利用加有头孢哌酮的麦康凯琼脂培养基，对其肠道中细菌进行选择性分离与鉴定。结果显示，12尾草鱼的肠道中，5尾可分离到具有以下特征的细菌：两端生鞭毛，菌体弯曲似海鸥状，革兰氏染色阴性，过氧化氢酶、氧化酶、尿酶和精氨酸双水解酶阳性，还原硝酸盐。不发酵、氧化或同化葡萄糖、甘露醇等15种常见的糖醇类，细菌形态、生化特性与报道的香港海鸥型菌相似，阳性率为41．7％。但在肌肉中未分离到此菌。进行分离细菌的16S rRNA基因序列分析，与GenBank中登录的4株香港海鸥型菌序列的同源性为99．7％～99．9％。由菌的培养特征、形态特点、理化特性、16S rRNA基因序列分析等多元鉴定的结果表明，从草鱼肠道中选择分离的细菌为香港海鸥型菌。药敏实验结果表明，所分离菌株对奥格门丁和亚胺培南的敏感性与报道的香港海鸥型菌有一定区别。     

Tanjungides A and B: New Antitumoral Bromoindole Derived Compounds from Diazona cf formosa. Isolation and Total Synthesis

Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey’s analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines.     

Coupled magnetic nanoparticle-mediated isolation and single-cell image recognition to detect Bacillus' cell size in soil

Microbial morphology fundamentally constrains how species interact with their environment, and hence ultimately affects their niche. However, the methodology of functional microbes in the soil ecosystem is still poorly studied since it is difficult to capture and identify the active monospecific community from the complicated environment and enormous number of microbial species in soils. To comprehensively reveal the morphology of active microbes in soil ecosystem, magnetic nanoparticle-mediated isolation (MMI) and single-cell image recognition (SCIR) were employed to study soil active Bacillus community, which functionally boosted the soil fertility in organic fertilisation compared to mineral fertilisation and unfertilised control treatments in our previous study. The results showed that MMI and SCIR can efficiently isolate active Bacillus from soil particles and other microorganisms. High throughput sequencing showed that the captured Bacillus showed similar community structure in different long-term fertilisation soils, while SCIR revealed that the active Bacillus was greater in number and larger in size in organic fertilisation treatment compared to mineral fertilisation and unfertilised control treatments. Our study demonstrates that the combination of MMI and SCIR is a potentially powerful tool to capture and identify the morphology of active and functional microbes in the soil ecosystem.     

一株鸭瘟病毒的分离与鉴定

从湖北省武汉市某种鸭场送检的死亡鸭肝脏中分离到1株病毒,结合流行病学调查,剖检病理变化特征初步诊断为鸭瘟病毒感染。根据Gen Bank上公布的鸭瘟病毒UL6基因序列设计一对引物,提取病毒核酸DNA进行PCR扩增,得到大小约为416 bp的目的片段。测序结果表明与Gen Bank上公布的鸭瘟病毒UL6基因的序列相似性达到99%。鸭胚感染试验表明,感染的鸭胚在第四天开始死亡,并出现典型的鸭瘟病毒感染症状。上述结果表明,该种鸭场种鸭死亡是由鸭瘟病毒所引起的。