Epe C, von Samson-Himmelstjerna G, Stoye M, Schnieder T
Institut für Parasitologie, Tierärztliche Hochschule Hannover.
Berl Munch Tierarztl Wochenschr. 1996 Jun-Jul;109(6-7):227-31.
For a comparative characterization of the lungworm species D. viviparus and D. eckerti which is not generally accepted as a separate species, the restriction fragment length polymorphism (RFLP) of the PCR amplified ribosomal second internal transcribed spacer (ITS2) and their sequences of both species have been examined. Ribosomal ITS2 DNA was amplified from genomic DNA of individual worms using primer that correspond to the conserved 3' and 5' ends of the ITS2 flanking 5.8S and 28S regions of Caenorhabditis elegans. PCR products were digested with restriction endonucleases AluI, NspI, SspI, BclI and MseI and separated electrophoretically on a 1% agarose gel. Each restriction enzyme produced a species specific fragment length pattern. PCR products were cloned into pCRII and sequenced. The length of the ITS2 varied between 403 (D. viviparus) and 481 bases (D. eckerti) with a GC content ranged from 25 to 33%. Intraspecific variations were low (0-1.5%). Interspecific differences occur at 112 bases. The sequence homology between D. viviparus and D. eckerti was found with 76.7%. ITS2 sequence differences between D. viviparus and D. eckerti by far exceeded intraspecific variations. Therefore both methods showed distinct differences between the lungworm species examined, thus proving that D. eckerti is correctly described as a separate species. Both species occur in deer and specific primers have been designed for both species that will be used in prevalence studies to investigate the actual role of deer in the transmission of D. viviparus to cattle.
为了对一般不被认为是独立物种的胎生网尾线虫（D. viviparus）和埃氏网尾线虫（D. eckerti）这两种肺线虫进行比较特征分析，已检测了聚合酶链反应（PCR）扩增的核糖体第二内部转录间隔区（ITS2）的限制性片段长度多态性（RFLP）及其序列。使用对应于秀丽隐杆线虫（Caenorhabditis elegans）ITS2侧翼5.8S和28S区域保守3'和5'端的引物，从单个虫体的基因组DNA中扩增核糖体ITS2 DNA。PCR产物用限制性内切酶AluI、NspI、SspI、BclI和MseI消化，并在1%琼脂糖凝胶上进行电泳分离。每种限制性酶产生物种特异性的片段长度模式。PCR产物被克隆到pCRII中并进行测序。ITS2的长度在403（胎生网尾线虫）和481个碱基（埃氏网尾线虫）之间变化，GC含量范围为25%至33%。种内变异较低（0 - 1.5%）。种间差异出现在112个碱基处。胎生网尾线虫和埃氏网尾线虫之间的序列同源性为76.7%。胎生网尾线虫和埃氏网尾线虫之间的ITS2序列差异远远超过种内变异。因此，这两种方法在所检测的肺线虫物种之间显示出明显差异，并证明埃氏网尾线虫被正确描述为一个独立物种。这两种物种都存在于鹿体内，并且已经为这两种物种设计了特异性引物，这些引物将用于流行率研究，以调查鹿在胎生网尾线虫向牛传播中的实际作用。
