Establishment Of Multiplex PCR For Three Coccidian Species Detection And Comparison Of ITS-1 Sequence From Ten Eimeria Maxima Strains

Poultry coccidiasis is caused by protozoan parasites of the genus Eimeria. It caused severe economic losses to the poultry industry. Currently, seven species of Eimeria are confirmed. Tradictional methods to identify species rely on morphological features of sporulated oocysts, sites and disease pathology, and so on, which is time-consuming and unreliable. PCR method may be a viable alternatives. The internal transcribed spaces (ITS) of eukaryote consist of ITS-1 and ITS-2, and its sequences appear species-specific and strain-polymorphic, which can be used to identify different species and study the phylogenetic relationships among strains.E.maxima, E.tenetta and Eacervulina are three common coccidian species. In the present study, a multiplex PCR for detecting the three species coccidia were established and the ITS-1 sequences of 10 strain E.maxima was analysised, which provides a powerful new tool for diagnosis and studying the epidemiology of coccidiosis, and shows the phylogenetic relationships of E.maxima isolated from China.1 Isolation and identification of Eimeria acervulinaUsing single oocyst infectioned technique, one pure species was isolated and found to be Eimeria acervulina. The oocyst is ovoid, with a smooth wall and a polar granule., and without a residuum. The average length and width of 100 oocysts and sporocysts were 18.26 μm× 13.54 μm and 9.48 μm×5.15 μm, respectively. The prepationt period is 97 h. Sporulation time is 17 h.2 Establishment of multiplex PCR to detect E.maxima, E.tenella and E. acervulina.Three sets of spectific primers were designed according to the previously published ITS-1 sequences of E.maxima, E.tenella and E.acervulina in the GenBank. A single PCR and multiplex PCR method were established, and used to detect the mixed samplewhich contained E.maxima, E.tenella and E.acervulina. The results showed that both methods amplified the special fragments with 151 bp, 463 bp and 303 bp from E.maxima, E.tenella and E.acervulina, respectively, and there were no cross rections with Sarcocystis fusiformis, Toxoplasma gondii and E. nocens. It is 0.5ng.that the lowest concentration of DNA could be detected. The results indicated that the multiplex PCR is a rapid, sensitive and special method for detecting Eimeria spp.3 Ribosomal DNA ITS-1 sequence comparisons of Eimeria maxima from different geogriphical regionsA pair spectific primers were designed according to the previously published ITS-1 sequence of E.maxima AF065095 in the GenBank. The ITS-1 sequences of 10 strain E.maxima were amplified by PCR. The products were checked by agrose gel electrophoresis and purified. The purified products were cloned into pGEM-T Easy vector successfully. Then the cloned plasmids were transformed into E.coli DH5 a . PCR and restriction endonuclease were used to identify the recombinant plasmid containing ITS-1 gene. The results indicated that the plasmid were correctly constructed. The fragments inserted of 10 strain E.maxima were sequenced and analysised by DNAStar. The results showed that the .ITS-1 sequences of Yangzhou strain, Longyan strain Fuzhou strain are 157 bp, the others are all 156 bp. The similarities were 65.4%~100%. Eleven strain E.maxima consist of two major groups. Yangzhou strain, Longyan strain and Fuzhou strain clustered together and belong to one clade, and the other eight strains clustered together and belong to another clade. This showed that the derived relationships can't reflect completely the genetic drift in these coccidia.