Identification And Analysis Of Taenia From 6 Regions Of Yunnan Province By Morphological And Molecular Biological Methods

Objective:To identify Taenia cestodes from Nujiang, Diqing, Dali, Lingcang, Chuxiong and Xianggelila of Yunnan province by morphological and molecular biological methods, The genomic distance and phylogenetic tree were constructed;To understand the distribution of three kinds of Taenia in Yunnan Province;To provide a reference orientation for specific categories of Taenia; To provide a scientific basis for prophylaxis and treatment of taeniasis and cysticercosis.Methods:(1) morphological analysis:Scolex and mature proglottid of 14 Taenia cestodes from 6 regions of Yunnan province were dyed by Hydrochloric Acide Staining Red Card.while gravid proglottid were dyed by Malachite Green Staining. To identify Taenia cestodes by morphology identificationand comparing with the Taenia solium and Taenia saginata first time; and combined with the Electron microscopy Scolex picture of Taenia asiatica (Dali strain), to discuss the form of Taenia asiatica scolex;(2) PCR-RAPD analysis of Taenia genomic DNA:After Morphological identification, genomic DNA was extracted from the segments of mature proglottid or gravid proglottid of 14 Taenia cestodes,2 standard strains of Taenia asiatica and 1 standard strains of Taenia saginata, PCR-RAPD were carried out with 12 random primers. Different geography system growth tree was constructed by means of SPSS software;(3) Analysis of Taenia mtDNA COXI gene sequence:the genomic DNA of Taenia cestodes was extracted from proglottid collected in 6 region of Yunnan province, and mtDNA COXIgene fragments were amplified by PCR, and then sequenced. The genomic distance and phylogenetic tree were constructed in comparison with other known mtDNA COXIgene sequences of Taenia.solium Taenia.saginata and Taenia asiatica GenBank using DNAMAN softwareResults:(1) morphological analysis showed that:DL1 have spherical scolex, a top protrusion and two small circle hook, branch section of mature proglottid was Irregular the mature of gravid proglottid were divided into 3 leaves, branches irregular pregnancy section, each side branch is about 7 to 13, more of that were fork on the base,it is Taenia solium; NJ1～NJ4 and DQ1-DQ3 have square scolex, there are small protrusions, no small hook, branch section of mature proglottid was neat,the mature of gravid proglottid were divided into 2 leaves, each side of the branch about 15-25,they are the T.asiaticain; DL2～DL4, LC1, CX1 and BN1 first section was square, havenot the top protrusion and small hook, the mature of gravid proglottid were divided into 2 leaves too, branch section of mature proglottid was Irregular, each side ot the branch about 15～28, according to that,we can not conclude it was Taenia.saginata or Taenia asiatica. (2) 134 DNA fragments were amplified by 12 primers. The number of DNA fragments amplified by single primer was between 3 and 22, and the average was 11.17. Electrophoresis of P1～6, P9 and P11 have specific DNA fragments. The rescaled distance cluster combination demonstrated that NJ1-4, DQ1-3 and DL2-3 occupied one branch with BZ1 and BZ3, which belongs to Taenia asiatica. LC1, BN1 and CX1 occupied one branch with BZ2,which belongs to Taenia.saginata.Two branchs occupied with DL1 (3) Taenia mtDNA COXI gene sequence analysis showed that:the homologie of NJ4, NJ1 and DQ2 was 0.002, DL4 and NJ3 homologie was0.005, NJ2 and DQ3 homologie was0.012; the homologie of DL3 and GB3 was 0.017, while the homologie was 0.037 with GB2; The phylogenetic tree demonstrated that 10 Taenia cestodes including NJ1-4, DL2-3 and DQ1-3 occupied one brance with GB3. BN1, CX1, LC1 and GB2 occupied one brance, then two brance occupied and occupied with other one which was occupied by DL1 and GB1. Taenia cestodes from Nujiang and Diqing were Taenia.asiatica. Taenia cestodes from Xishangbanna, Lincang and Chuxiong were Taenia.saginata.Taenia cestodes from Dali were Taenia. solium or Taenia. asiatica.Conclusions:(1) There are three kinds of Taenia in the area of Yunnan province, Taenia cestodes from Nujiang and Diqing were Taenia.asiatica. Taenia cestodes from XiShangbanna, Lincang and Chuxiong were Taenia.saginata. Dali was repeat popular area of three kinds of Taenia; (2) There are two forms Taenia.asiatica in Yunnan province, the Scolex top of which have processes or not, the traditional morphological method is difficult to identify Taenia. saginata and Taenia. asiatica that havenot processes on the top of Scolex;(3) PCR-RAPD technology and mtCOXI sequencing analysis of all fragments can make up for lack of morphological identification, and provide the molecular basis for identification of three kinds of Taenia; (4) PCR-RAPD technology and mtDNA COXI sequencing analysis of fragments can be used for Genetic polymorphism of the Taenia, and provide a reference orientation for specific categories. (5) PCR-RAPD technology can be used to select the specific gene segments for the classification and identification of Taenia...