Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

Fatal encephalitozoonosis in two koalas

Two young koalas from a fauna park, recently out of the pouch and approximately 6 months old, were found dead with no previous clinical signs or gross lesions. On histopathological examination, large numbers of spores consistent with a microsporidian organism were present intracellularly within the small intestinal mucosa. Electron microscopy and polymerase chain reaction studies (sequencing the 5' end of the SSU RNA gene) identified the organism as Encephalitozoon intestinalis with 100% homology with those of previously reported human isolates. This is believed to be the first report of this organism in a marsupial.     

草鱼三元病原菌融合子的构建及生物学特性

采用细胞融合技术首次将肠型点状产气单胞菌菌株和鱼害粘球菌菌株融合，构建二元融合菌株AM-1，再将AM-1与荧光假单胞菌菌株融合，构建三元融合菌株AM-2。对AM-1和AM-2菌株的形态结构、生理生化特性、致病性、抗原性、抗体效价进行分析。结果表明，AM-1活菌椭圆形，无鞭毛，死菌短杆状，AM-2菌株短杆状，有鞭毛，它们的生理生化性状介于3原始亲本菌株之间；用AM-2菌株对当年健康草鱼种进行人工感染攻毒，被攻毒草鱼种具有烂鳃病、赤皮病及肠炎病的部分症状；AM-2菌株抗原的受免草鱼种对荧光假单胞菌、鱼害粘球菌和肠型点状产气单胞菌菌株的免疫保护率分别达64.90％、68.00％、67.00%，抗体效价最高达1∶256、1∶256、1∶512。测定了AM-1和AM-2菌株及亲本DNA含量并进行了琼脂糖凝胶电泳分析，AM-1和AM-2菌株DNA含量明显高于任一原始亲本。图4表6参23     

6株肠艾美耳球虫繁殖力的比较研究

[目的]为肠艾美耳球虫的早熟选育及兔球虫疫苗的研制提供理论依据。[方法]采用6个不同地区肠艾美耳球虫的孢子化卵囊经口接种45日龄无球虫兔,接种剂量为1×104个卵囊/只,并对6株肠艾美耳球虫的繁殖力进行比较。[结果]衡水株排卵量最多,张家口株次之,胶南株排卵量最少。睢宁株的排卵量接近胶南株,如皋株、通江株相近,在感染后第8.5～9天均有排卵,各虫株的排卵高峰期相似,第13天排卵量达到峰值,第21天接近0。[结论]不同地理株肠艾美耳球虫的排卵量不同,但排卵高峰相一致。     

3种兔球虫18S rDNA部分序列测定与系统发育分析

采用单卵囊分离法从河北某兔场分离大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫,接种无球虫兔后获得大量纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18S rDNA保守引物,PCR扩增3种兔球虫18S rDNA片段,产物纯化后测序。将3种球虫18S rDNA测序结果与GenBank中发布的兔球虫18S rDNA序列用DNAStar软件进行比对。使用MEGA4.0软件对兔球虫18S rDNA进行同源性比较,并绘制遗传进化树。结果表明,大型艾美耳球虫扩增出大小为1 521bp的18S rDNA片段;黄艾美耳球虫及肠艾美耳球虫均扩增出大小为1 520bp的18S rDNA片段。序列比对结果显示,3种河北株兔球虫与GenBank中相应的3种兔球虫18S rD-NA(EF694016、EF694011、EF694012)相似性分别为99.6%、99.6%和100%。3种河北株兔球虫序列和GenBank中兔球虫18S rDNA序列(EF694007-EF694017)位于一个单系集群。     

3种兔球虫ITS-1序列测定与系统发育分析

从河北省兔场分别单卵囊分离孢子化大型艾美耳球虫卵囊、黄艾美耳球虫卵囊及肠艾美耳球虫卵囊,接种无球虫兔后获得纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18SrDNA和5．8SrDNA保守引物,PCR扩增3种兔球虫ITS-1片段,产物纯化后测序。将3种球虫ITS-1测序结果与GenBank发布的兔球虫ITS-1序列进行比对和遗传距离比较,绘制系统发育树。结果表明,大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫河北株分别扩增出424、455、434bp的ITS-1片段。大型艾美耳球虫、黄艾美耳球虫及肠艾关耳球虫河北株与GenBank中发布的同种兔球虫ITS-1序列相似性分别为97．4％、97．9％和96．9％。系统发育树显示兔球虫ITS-1序列形成1个单系群,该单系群根据寄生部位分为2个姊妹群。     

刺参培育池中玻璃海鞘生殖腺组织学的初步研究

2004年2—3月对刺参培育池的玻璃海鞘生殖腺进行组织学观察,结果表明,玻璃海鞘雌雄同体,精巢和卵巢相间排列,两侧生殖管数量不同,右侧4~7条,左侧3条。精巢为分枝状,分散包围在卵巢周围。生殖输送管沿着直肠与肛门相连接,末端开口在围鳃腔中。卵母细胞外有被囊细胞,生殖腺发育可分为4个时期。     

4种海鞘排泄的初步研究

在实验室内对柄海鞘、玻璃海鞘、中国瘤海鞘和史氏菊海鞘的排氨率进行了研究,同时探讨了温度和体重与排氨率的关系。结果表明,温度和体重对海鞘的排氨率有显著的影响。酚海鞘和玻璃海鞘的排氨率与温度呈倒钟形曲线变化趋势,排氨率的最高峰分别出现在２３℃和１８℃。柄海鞘和中国瘤海鞘的单位个体排氨率与体重的关系呈幂函数变化趋势,２３℃时的回归方程分别为：ＱＮ＝５２．６７Ｗ＾０．６６７（Ｒ＾２＝０９８０７）;ＱＮ＝１     

海鞘(Ciona intestinalis)新microRNA基因的识别及其靶标预测

microRNAs(miRNAs)是一类长度约22 nt的非编码小RNA,通过碱基互补配对的方式调控靶基因的表达.本文采用同源搜索的方法,以线虫、果蝇、文昌鱼和人类的miRNA为探针,与海鞘的基因组序列进行比对,同时结合miRNA二级结构特征及SVM假阳性分析共发现10个新的miRNA基因.通过靶基因预测,共找出225个潜在靶基因,这些靶基因主要参与细胞代谢、转录调节、结合活性等功能.新miRNA基因的识别为海鞘miRNA功能研究奠定了基础,并为更进一步揭示脊椎动物miRNA的起源与进化提供了理论依据.     

Biodeposit production and benthic loading by farmed mussels and associated tunicate epifauna in Prince Edward Island

An experimental study was done to evaluate the biodeposition dynamics associated with mussels and two fouling tunicates, Ciona intestinalis and Styela clava, in mussel aquaculture in Prince Edward Island (PEI), eastern Canada. The presence of C. intestinalis on small constructed mussel socks increased biodeposition by a factor of about 2 relative to mussel socks without tunicates. S. clava were small and had a negligible effect on total biodeposition from mussel socks although they increased sedimentation rates relative to that of abiotic control socks. Sinking rates of faecal pellets from large C. intestinalis varied between 1.39 and 6.54 cm s^− 1 (LSMean = 2.35 cm s^− 1). Using biodeposit production and sinking rates and hydrological data obtained in the present study, footprints of benthic loading due to mussel and tunicate biodeposition for a typical mussel farm in PEI were modelled using Shellfish-DEPOMOD. The results show benthic loading below longlines with C. intestinalis to be ca. 2 times greater than those from lines with only mussels with rates of up to 15.2 g m^− 2 d^− 1. However, given the greater settling rate of C. intestinalis biodeposits relative to mussel biodeposits, the extent of the footprint (≥ 1 g m^− 2 d^− 1) is similar or even more restrained.