应用抑制性差减杂交筛选鸭疫里默氏杆菌强弱菌株基因组的差异片段

为了分析鸭疫里默氏杆菌(Riemerella anatipestifer,RA)强毒菌株HXb2和弱毒菌株NJ-1基因组之间的差异基因,以HXb2株基因组为待测菌株,以NJ-1株基因组为驱动菌株,构建了两株细菌基因组间的抑制性差减文库。经过dot-blot杂交和PCR验证,共鉴定到34个强弱毒株基因组差异片段。测定的序列经BLAST、DNAStar和PSORTb分析,结果表明有2个片段所在基因编码的蛋白为外膜蛋白,有2个片段所在基因编码的蛋白为胞质膜蛋白如磷酸盐转运蛋白,11个片段所在基因编码的蛋白为胞内蛋白如丙氨酸脱氢酶、β-内酰胺酶及一些假定蛋白等,有19个片段所在基因编码的蛋白(大部分为假定蛋白)定位未知。这些差异基因编码的蛋白可能与鸭疫里默氏杆菌的毒力及HXb2株特异蛋白等有关。本研究为下一步鉴定新的毒力基因及其他特异蛋白的功能奠定了基础。     

猪链球菌2型新毒力相关基因lin缺失菌株的构建

利用温度敏感型穿梭自杀质粒pSET4s,定点敲除猪链球菌2型野生型强毒株ZY458的lin基因,构建基因缺失突变菌株458△lin,利用家兔感染模型对ZY458及其突变菌株的生物学特性进行比较研究.家兔感染试验表明,接种ZY458菌株的家兔体质量下降,出现明显的临床症状,5只家兔4 d内全部死亡;而458△lin感染组家兔生长正常,未出现任何明显临床症状.结果表明,lin基因是猪链球菌2型新发现的一种毒力相关基因,在猪链球菌2型的致病过程中具有重要作用.     

猪链球菌2型分选酶srtC5基因敲除突变菌株的构建及其特性分析

构建猪链球菌2型（Streptococcus suis serotypc 2）强毒株ZY458分选酶srtC5基因敲除突变体并分析其特性。首先通过重叠延伸PCR技术扩增AsrtC5片段,该片段携带srtC5编码基因及其上下游部分序列,但是中间缺失212bp序列。随后构建重组自杀载体pSET4s：：△srtC5,并将其电转化导入ZY458菌株,通过同源重组获得△srtC5突变体。通过体外的细胞黏附试验和体内的家兔攻毒试验证明△srtC5突变体比较野毒株,其细胞黏附能力和对家兔的致病性都显著降低。     

猪链球菌2型对家兔的试验感染及其病理学观察

利用猪链球菌2型四川分离强毒菌株458#,通过腹腔注射接种日本大耳白家兔,观察其临床症状和病理学变化。结果表明,猪链球菌2型强毒株458#可引起家兔发病或死亡,并能从发病家兔的组织样品中分离到所接种毒株。感染家兔的特征性组织病理学变化表现为典型的弥漫性血管内凝血和微血栓形成。猪链球菌2型感染家兔动物模型的建立,对于评价猪链球菌2型的毒力,探索其发病机制具有重要的意义。     

猪链球菌2型前噬菌体基因的检测及其与毒力基因分布的关系

为了探究猪链球菌(Streptococcus suis,SS)2型菌株中前噬菌体基因与毒力因子分布的关系,利用生物信息学方法预测了19株已全基因测序的SS2中的前噬菌体基因分布,并设计前噬菌体保守基因引物2对,通过PCR检测前噬菌体在101株SS2菌株中的分布,并与毒力因子分布进行比较。结果表明,已全基因测序的猪链球菌2型菌株中,弱毒株含有比强毒株更多的前噬菌体片段。PCR检测显示,14株4种代表性毒力因子(sbp2',mrp,ef,sly)为阴性的菌株中均含有前噬菌体解旋酶基因和末端酶基因,而86株不完全含有这4种代表性毒力因子的菌株中不含有上述前噬菌体基因,说明猪链球菌2型菌株中毒力因子分布与前噬菌体基因分布无明显相关性。     

猪链球菌核糖体基因分型研究

从青岛农业大学动物科技学院实验室分离培养的15株猪链球菌中提取基因组DNA,利用PstⅠ消化并进行电泳分析,将电泳分离的DNA片段转移至尼龙膜上并固定;以大肠杆菌C83907 16srDNA两侧保守区域设计引物,利用PCR扩增出16srRNA基因片段,用地高辛标记制成探针;该探针与基因组DNA酶切片段进行杂交,通过对杂交带型分析,进而对猪链球菌进行核糖体基因分型。     

牛源A型多杀性巴氏杆菌强弱毒株差异基因的SSH法筛选

近年来由于牛的频繁调运,由牛源A型多杀性巴氏杆菌引起的牛肺炎型疾病呈现出流行趋势。为探讨牛源A型多杀性巴氏杆菌强弱毒株间的基因差异,本研究利用抑制消减杂交技术(SSH)对牛源A型多杀性巴氏杆菌强毒株Pm CQ2和弱毒株Pm CQ6进行试验分析,共得到28个差异基因片段,测序比较发现其中27个与毒力、代谢、转录及DNA合成相关的基因片段,另1个基因片段所编码的为假定蛋白。该结果为牛源A型多杀性巴氏杆菌的后续致病机制研究提供了参考。     

鸡毒霉形体HS株pMGA多基因族的研究

鸡毒霉形体（Mycoplasma gallisekpticum,GM)基因组中存在着编码细胞表面血凝粘附素蛋白（protein of Mycoplasma gallisepticum adhesin,pMGA)相关基因组的多基因族。为筛选出含有完整的pMGA基因片段，并估算出鸡毒霉形体HS株基因组中pMGA多基因族的基因数，本试验以pUC18为载体，用EcoR I限制性内切酶构建了鸡毒霉形体HS株的基因组文库。根据pMGA基因引导序列的保守区和pMGA基因间隔区的序列，人工合成了2个寡核苷酸探针（探针I、探针Ⅱ），经标记后，双筛选所建的基因文库，从600个转化子中共得到的38个含有pMGA基因的阳性克隆子，选其中1个7．5kb的片段（17号阳性克隆子，W17），用4种限制性内切酶（EcoR I,Hind Ⅲ，Pst I,Bgl Ⅱ）酶切消化，绘制了该片段的物理图谱。该片段酶切产物经电泳、转膜后与探针I和探针Ⅱ杂交，发现2个探针杂交图谱基本相同，根据杂交带及放射信号的强度值，估测出该片段含有4个pMGA基因，以这个片段所含的pMGA基因数为标准，估算出鸡毒霉形体HS株含有39个pMGA基因。     

血清Ⅱ型鸭疫里默氏杆菌基因组文库的构建

为研究血清Ⅱ型鸭疫里默氏杆菌的抗原基因,筛选毒力较强的血清Ⅱ型鸭疫里默氏杆菌菌株,提取其基因组,分别用Sau3A I酶切法和shotgun法对基因组进行处理后,选取1.5～3 kb的DNA片段,与PUC118载体连接后电转化JM109大肠杆菌感受态细胞,构建血清Ⅱ型鸭疫里默氏杆菌基因组文库。两种方法得到的库容量分别为40 000和50 000左右,随机挑选阳性克隆鉴定表明有外源DNA片段的插入,说明建库成功。该文库的成功构建为Ⅱ型鸭疫里默氏杆菌新的抗原基因的筛选奠定了基础。     

猪链球菌血清型9株新的毒力相关基因STP有助于降低小鼠死亡率

猪链球菌是一个重要的猪病病原，可造成一系列疾病。猪链球菌血清2型（SS2）和链球菌血清9型（SS9）在中国广泛流行。SS9的毒力凶子还不太清楚，分别从病猪和健康猪身上分离两个菌株GZ0565和SH040917。抑制性消减杂交技术（SSH）用来识别与SS9病原相关的毒力基因。     

犬2型腺病毒SY株的致弱驯化

作者对分离和系统鉴定的一株犬传染性喉气管炎病毒（犬2型腺病毒）SY（沈阳）株，用犬肾传代细胞MDCK进行了致弱驯化，每驯化15代做一次初步的犬的安全性试验。用大剂量不同代次病毒对易感犬进行人工感染试验，每天观察犬的临床症状及白细胞总数，并于接毒后的第5d安乐死，作病理解剖学和病理组织学检查，易感犬的感染试验结果表明，SY株在犬贤细胞上传15代时对犬的致病力已降低，30代时对犬已不引起发烧，精神沉郁和厌食等症状，仅引起轻度的鼻炎，中度的咽炎，剖检肺表现正常，仅见支气管淋巴结肿大，45代时无临床症状，剖检除扁桃体肿大外没有见到其它症状，60代毒已完全失去致病力，未见任何临床症状及病理变化。     

柔嫩艾美耳球虫山西株早熟系的免疫特性

对鸡柔嫩艾美耳球虫山西株早熟系的免疫特性进行了研究。结果表明：该株的早熟系与亲本株具有相同的免疫原性,但致病性大大降低;其最佳免疫剂量和次数：首次免疫为1500个/只,14 d后以2000个/只二免,二免后免疫保护期可达90 d以上,免疫后鸡群的抗球虫能力与血清中特异性IgG的变化规律一致;提示该早熟系具备早熟疫苗株的免疫特性。     

Competition of a sporidesmin-producing Pithomyces strain with a non-toxigenic Pithomyces strain

Sporidesmin, a mycotoxin produced by some strains of Pithomyces chartarum, is responsible for the hepatogenous photosensitisation disease facial eczema, which causes severe losses in agricultural revenue in New Zealand. A sporidesmin-producing strain of P. chartarum, isolated in New Zealand, was grown in co-culture with a South African strain that does not produce the mycotoxin. Competition occurred between the two strains when grown both on agar plates and on dried ryegrass, with a significant decrease in the total amount of sporidesmin produced. Biological control of toxkenic P. chartarum can thus occur under laboratory conditions, raising the possibility of similar control in the field situation.     

Competition of a sporidesmin-producing Pithomyces strain with a non-toxigenic Pithomyces strain

Sporidesmin, a mycotoxin produced by some strains of Pithomyces chartarum, is responsible for the hepatogenous photosensitisation disease facial eczema, which causes severe losses in agricultural revenue in New Zealand. A sporidesmin-producing strain of P. chartarum, isolated in New Zealand, was grown in co-culture with a South African strain that does not produce the mycotoxin. Competition occurred between the two strains when grown both on agar plates and on dried ryegrass, with a significant decrease in the total amount of sporidesmin produced. Biological control of toxigenic P. chartarum can thus occur under laboratory conditions, raising the possibility of similar control in the field situation.     

野生亚洲象解没食子酸链球菌巴氏亚种和解没食子酸链球菌的分离

解没食子酸链球菌巴氏亚种和解没食子酸链球菌是一种人兽共患的细菌传染性疫病。已报道的感染动物有鸽子、火鸡、鹅、鸭,尚未见野生亚洲象感染的报道。2014年11月17日,本研究从患有严重胸膜炎、腹膜炎的野生亚洲象中采集病料,分离到解没食子酸链球菌巴氏亚种和解没食子酸链球菌,特此报道。     

Bone strain in the equine tibia: an in vivo strain gauge analysis

Rosette strain gauges were bonded to the cranial, caudal, and medial surfaces of the tibia in the middiaphyseal region of 6 adult ponies. While the ponies were walking, the cranial side was mainly subjected to tension, and the caudal side, to compression. The compression strain on the caudal side was 1.5 times greater than the tension strain on the cranial side. None of these principal strains was aligned along the long axis of the bone; both deviated laterally from the long axis. On the medial surface, the principal strain deviated caudally about 40 degrees from the long axis. From analysis of the strain patterns on the 3 sides of the bone, it could be concluded that during loading of the tibia, torsion was superimposed on craniocaudal bending. The strain pattern was not affected after transection of the cranial tibial muscle, as determined by measuring with the same gauges before and after surgical interference. The contribution of the cranial tibial muscle in reducing the strain in the tibial cortex was therefore very small.     

Genomic comparison of foot-and-mouth disease virus R strain and its chick-passaged attenuated strain

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus that is the causative agent of porcine circovirus associated disease (PCVAD), a disease complex affecting swine around the world. Although this virus is believed to negatively affect the host's immune system, the mechanism by which PCV2 induces disease is not completely understood. This report describes a series of PCV2 experiments using the gnotobiotic pig model in which a relationship was demonstrated between abnormal leukograms and development of clinical disease in PCV2-infected pigs. When compared to control pigs the leukogram was characterized by a decrease in lymphocytes within 14 days post inoculation (dpi) followed by an increase in neutrophils 7-14 days later. No significant changes in the circulating monocyte, basophil, and eosinophil cell populations were detected. The combination of an absolute neutrophilia and lymphopenia produced a neutrophil/lymphocyte ratio that was predictive of clinical disease and was inversely correlated with the presence of neutralizing antibodies. Based on previous reports, the lymphopenia may be attributed to a direct cytolytic effect of the virus and could negatively affect the pig's immune response. The role of the neutrophilia in the pathogenesis of PCVAD in gnotobiotic pigs is unknown.     

山羊痘病毒疫苗株和野毒株ORF121基因比较分析

山羊痘(goat pox,GP)是由山羊痘病毒(goat poxvirus,GPV)感染引起山羊的一种烈性传染病,弱毒疫苗接种是预防和控制GP的重要手段之一。ORF121是GPV编码和GPV转录、复制及细胞内包装相关的基因之一,在GPV突破宿主细胞免疫系统过程中扮演重要角色。为比较分析GPV疫苗弱毒株和野毒株编码的ORF121基因特征,本研究扩增并测定了GPV疫苗株和野毒株的ORF121基因序列,比较分析了二者核酸水平和氨基酸水平的变异及其蛋白质二、三级结构差异。结果表明,GPV疫苗弱毒株和野毒株编码的ORF121基因核苷酸序列相似性为97.9%,氨基酸序列相似性为98.2%。本研究结果为揭示GPV异原宿主致弱的机制积累了资料。