Study On Epidemiological Investigation And RDNA-ITS Sequence Analysis Of Parabronema Skrjabini Of Camels

Parabronema skrjabini was a major parasite in the camels abomasum and the diease caused by it was one of the important parasitosisis which brought great damages to camels breeding. In order to know the vectors of Parabronema skrjabini, the epidemiological investigation, amplification of the internal transcribed spacers gene and screening the vectors were done based on the ITS1 and ITS2 sequences, the results were as follows:80 Bactrianus camelus of different ages and species were dissected from the main camel-breeding areas in Inner Mongolia of China, then the infection rate and the infecion density were calculated. They showed that 58 camelus were infected and the infection rate was 72.5%, and the highest infecion density was 1315.The internal transcribed spacer (ITS, ITS1 and ITS2)of ribosomal DNA nucleotide fragments of Parabronema skrjabini from Inner Mongolia region were amplified by PCR using three pairs of conserved primers(NC5-NC13,NC5-NC2,NC13r- NC2), then they were purified and cloned to pGM-T vectors. The inserts were successfully sequence and it showed that ITS, ITS1 and ITS2 were 837bp, 372bp and 484bp respectively. Comparsion with sequences of ITS1, 5.8S and ITS2 from the families in Spirurata reported in Genbank, the homology of the ITS1 was between 48.8% and 61.7%, while they were 65.6%～100% in 5.8S and 6.8%～74.2% in ITS2. Phylogenetic tree based on the 5.8S sequences showed that Parabronema skrjabini was consanguinity with Okapinema japonica, then followed by the three species of Family Habronema.PSp1 and PSp2 primers based on specific region of ITS sequence were designed, and a 600bp fragment was amplified from the genetic DNA of eggs or larvae of Parabronema skrjabini. Sensitivity tests showed that specific fragments could be amplified from DNA template of five eggs, and the specific tests showed that the primers had a good sepcificity to amplify genetic DNA of Parabronema skrjabini from the suspected vectors in vitro.It was the first time that the complete sequence of ITS and 5.8S rDNA of Parabronema skrjabini were reported. The result of the present studies gave a new method to the vectors screening of nematodes and have laid a foundation for further molecular biology study of Parabronema skrjabini.