Studies On Construction Of Pure Strains And DNA Barcoding Of Eimeria In Chicken

DNA barcoding is a new method and has become hotspot on biology taxonomy in last several years. As a universal tragt gene for classification and identification, mitochondrial cytochrome c oxidase subunit I (mt CO I ) was chosen. To investigate the feasibility of using CO I gene as the DNA barcoding of Eimeria in chicken, different pure strains of Eimeria were chosen and analyzed sequences of CO I gene. This study was based on construction of pure strains by single-oocyst isolation, biology identification and PCR amplification identification by species-specific primers. 18S rRNA sequences were amplified by PCR at the same time. It is the first time to classify Eimeria by CO I gene in this study and it will provid a new tendency to taxonomy for Eimeria in chicken.To obtain pure strains, 11 strains of 6 Eimeria species were chosen to isolate single-oocyst by small piece of agar-gel and infected a 1～5-days-old chicken, including E. cervulina, E. tenella, E. maxima, E. mivat, E. necatrix and E. brunetti. 179 chickens were infected and 42 of them got isolated-strains successfully. The achievement average rate was 23.46%. Then 11 of these 42 isolated-strains were propagated and identified according to classical indexes such as the prepatent period, location of coccidia in the intestine, the shortest sporulation time, size and shape of oocysts and sporocysts. The rusluts showed that all of them were pure strains, including 1 strain of E. acervulina, 2 strains of E. tenella, 6 strains of E. maxima, 1 strain of E. mivati and 1 stain of E. necatrix. In order to confirm whether the isolations were monospecific, another method was used based on PCR amplification by species-specific primers which were designed according to E. acervulina, E. tenella, E. maxima genome. The results proved that one isolated-strain of E. maxima strain Townsends was contaminated by the E. tenella during the process of propagation.To investigate the feasibility of CO I sequence as the DNA barcoding in chicken, 13 strains of Eimeria were amplified by PCR respectively, including 11 isolated-strains and 2 strains which were preserved in laboratory. The molecular-size of the obtained DNA fragments was 811 bp after PCR amplification. All sequences compared with each other and the results showed that there was some difference among these species but no difference between strains in same species. The similarity between E. tenella and E. necatrix was 98.4%, which was the highest. The similarity between E. maxima and E. acervulina was 86.1%, which was the lowest. The distance between E. maxima and E. necatrix was 0.171, and between E. teneUa and E. necatrix was 0.017. The base composition of A+T was 64.8%, which was higher to G+C contents obviously. The genetic distances among the 5 species revealed that E. tenella was mostly close to E. necatrix, and distant from E. acervulina, E. mivati and E. maxima. Cluster of Eimeria and the other 4 protozoon parasites published in the GenBank could classified obviously.To prove the CO I sequence analysis is accurate in the taxonomy of Eimeria, 18S rRNA sequences of 8 strains in 3 species were amplified by PCR respectively. The molecular-size of the obtained fragments were from 1746 bp to 1756 bp after PCR amplification. Results of aligment showed that there was discrepancy not only among species but also among strains. And the discrepancy between species was greater. Similarity was 98.7%～99.3% in 3 strains of E. maxima and was 99.7%～99.9% in 4 strains of E. tenella. And this number between 3 species is 96.5%～98.1%. The distance between E. maxima and E. tenella was 0.038, and between E. maxima and E. acervulina was 0.021. Phylogenetic tree showed that 3 strains of E. maxima were closed to each other, and so did the 4 strains of E. tenella. E. tenella was close to E. necatrix, and the variability with E. acervulina,E. mivati,E. mitis,E. praecox and E. brunetti was obvious. These results were agreeed with the conclusion of sequence analysis by mt CO I.The results of these studies indicated that the mt CO I sequences which could identify different species of Eimeria in chicken might be better than 18S rRNA. The mt CO I sequences could be an ideal target gene for the DNA barcoding of parasites.