Khalifa Refaat M A, El-Hady Hanaa A, Omran Eman K, Ahmed Noha S
Department of Medical Parasitolgy, Faculties of Medicine, Assiut.
J Egypt Soc Parasitol. 2013 Apr;43(1):23-32. doi: 10.12816/0006364.
Identification of liver fluke species cannot be achieved by clinical, pathological, coprological or immunological methods. However, the differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different pathological manifestations. Moreover, in countries where the two species co-exist, morphologically intermediate forms were reported. The present study aimed to identify these forms by the use of molecular characterization of DNA sequence. Based on morphometric criteria, adults of Fasciola hepatica, F. gigantica and intermediate forms were collected from naturally infected sheep and cattle from various regions of Sohag Governorate. A simple and rapid new method (QIAamp DNA Mini Kit) was used to isolate DNA from the worms and their RELP patterns were obtained after digestion of the PCR products with AvalI restriction enzymes. The result of a regular PCR experiment for the amplification of the selected 28S rDNA fragment with the designed primer set yielded identical 618- bp-long PCR products for the three types of Fasciola where the RFLP profile obtained from F. hepatica revealed four fragments of 628, 575, 165 and 95 bp, and F. gigantica generated three fragments corresponding to 628, 358 and 300 bp fragments whereas the intermediate forms revealed four fragments of 628, 541, 358 and 300 bp, which were similar to those ofF. gigantica but with a distinctive fragment of 541. These results confirmed that three species are present in our locality: F. hepatica, F. gigantica and an intermediate form which was named F. hepatogigantica n.sp. on basis of having few morphometric characters from F. hepatica (length and pattern of uterine coils) but genetically they were more related to F. gigantica.
通过临床、病理、粪便学或免疫学方法无法鉴定肝吸虫的种类。然而，由于肝片吸虫和巨片吸虫感染的病理表现不同，两者之间的鉴别诊断非常重要。此外，在这两种吸虫共存的国家，曾报道过形态学上的中间类型。本研究旨在通过DNA序列的分子特征鉴定这些中间类型。根据形态测量标准，从索哈杰省不同地区自然感染的绵羊和牛体内收集肝片吸虫、巨片吸虫的成虫及中间类型。采用一种简单快速的新方法（QIAamp DNA Mini试剂盒）从虫体中分离DNA，并用AvalI限制性内切酶消化PCR产物后获得其RELP图谱。用设计的引物对扩增选定的28S rDNA片段进行常规PCR实验，结果显示三种肝吸虫均产生相同长度为618 bp的PCR产物，其中肝片吸虫的RFLP图谱显示为628、575、165和95 bp的四个片段，巨片吸虫产生对应于628、358和300 bp的三个片段，而中间类型显示为628、541、358和300 bp的四个片段，与巨片吸虫的片段相似，但有一个独特的541 bp片段。这些结果证实我们当地存在三种吸虫：肝片吸虫、巨片吸虫和一种中间类型，基于其具有一些来自肝片吸虫的形态测量特征（子宫盘绕的长度和模式），将其命名为肝巨片吸虫新种，但在基因上它们与巨片吸虫的关系更为密切。
