| Goose coccidia are intracellular parasites of the intestinal tract or kidney of domestic goslings and Anseriformes birds, and belong to Apicocomplexa, Sporozoa, Eumeriina and Eimeriidae. There are sixteen species of coccidia reported from domestic goslings and Anseriformes birds and mixed infection with coccidia are the rule. So exact identification of coccidian species by the traditional methods are very difficulty. Here using the isolation techniques of a single oocyst, we isolated and identified four species of goose coccidia that is Eimeria anseris, E. hermani, E. stigmosa and E. fulva. Then using the genus-specific primers, the 18S rDNA and ITS rDNA fragments of these four goose coccidia were amplified, sequenced and analysed. The results from the research layed the foundation for classification of goose coccidia species by molecular method.1. The epidemiological survey of goose coccidia in YangzhouBased on the morphology of the oocysts, the coccidial species were investigated and identified from fecal samples collected from 63 domestic gooseries in Gaoyou, Jiangdu, Hanjiang, Yizheng and Baoying of Yangzhou. The results showed that 57 gooseries were infected with goose coccidia, and the positive rate was 90.47%. Of which, the lowest positive rate was 66.66% in under three-week-old gooseries,100% in 3-9-week-old gooseries was the higest. All of the gooseries (100.00%) in Hanjiang, Yizheng and Baoying were infected with coccidia, the other two area also with a high positive rate. The mixed infection of goose coccidia was commonly, the rate was 85.96%. The six coccidial species were identified, namely Eimeria anseris, E. hermani, E. fulva, E. nocens, E. stigmosa and Tyzzeria parvula. T.parvula and E. hermani were dominant species, with a high incidence 74.60% and 65.08% respectively.2. Isolation and Identification of four species of goose coccidiaUsing techniques of single oocyst, four pure strains were isolated and found to be E. anseris, E. hermani, E. stigmosa and E. fulva. E. anseris:The incubation period was 6 d, young geese with obvious clinical symptoms and usually died. The oocysts were pyriform, the front-end abnormal narrow. The oocyst wall were single layer structure, colourless, smooth, porose, noa-operculate.The sporocysts were ovoid, with residuum. The oocysts size were (18.5～22.7) μm × (14.1～ 19.3) μm. E. hermani:The incubation period was 4 d, young geese without obvious clinical symptoms and no died. The oocysts were ovoid. The oocyst wall were bilayer structure, colourless, smooth, porose, noa-operculate. The sporocysts were ovoid, stieda body not obvious. The oocysts size were (24.0～27.6) μm × (17.5～19.5) μm. E. stigmosa:The incubation period was 4.5 d, young geese without obvious clinical symptoms and no died. The oocysts were wide ovoid. The oocyst wall were thick, yellowish-brown, rough, porose, noa-operculate. The sporocysts were ovoid, with residuum. The oocysts size were (19.7～23.3) μm × (14.1～19.3) μm. E. fulva:The incubation period was 7 d, young geese with obvious clinical symptoms but no died. The oocysts were wide ovoid. The oocyst wall were bilayer structure, yellowish-brown, rough, porose, noa-operculate. The sporocysts were ovoid, with stieda body and residuum. The oocysts size were (25.6～32.4) μm x (20.2～25.2) μm.3. Sequence and Phylogenetic analysis of 18S rDNA from four species of goose coccidiaA pair of Eimeria genus-specific primer was designed based on the analysis of the 18S rDNA genes of different Eimeria sp. Using the primers, the 18S rDNA fragments of E. anseris, E. hermani, E. stigmosa and E. fulva were amplified and sequenced, then they were analysed by DNAstar and aligned with corresponding sequence of the fifteen species of Eimeria sp. from other animals in the GenBank. Among the four goose coccidia and twenty-nine other species of parasitic protozoa that belong to Eimeriina, the phylogenetic tree was obtained using neighbor-joining method. The result showed that the gene fragments of E. anseris, E. hermani, E. stigmosa and E. fulva were respectively amplified with 1695bp,1696 bp,1703bp and 1694bp. The similarities was highest between four goslings coccidia, followed by that between goslings coccidia and crane coccidia. Eimeria sp. and encysted coccidia were respectively located in a monophyletic. Eimeria sp. from fish and other animals was divided into two sister lineages. Eimeria sp. from wetland birds and Eimeria sp. from Galliformes birds and mammals were respectively located in a monophyletic. These results suggest that the 18S rDNA gene could be used to identify goslings coccidia.4. Sequence and Phylogenetic analysis of ITS rDNA from four species of goose coccidiaUsing a pair of Eimeria genus-specific primer of the ITS rDNA genes, the ITS rDNA fragments of E. anseris, E. hermani, E. stigmosa and E. fulva were amplified and sequenced. Then these ITS rDNA genes were analysed by DNAstar and aligned with each other. Among the four goose coccidia and many other species of parasitic protozoa that belong to Eimeriina, four phylogenetic trees were obtained using MP method based on ITS,ITS1 and ITS2. The result showed that the gene fragments of E. anseris, E. hermani, E. stigmosa and E. fulva were respectively amplified with 1051bp, 1000bp,926bp and 1073bp. The similarities of 5.8S rDNA between four goslings coccidia was the highest, followed was ITS and ITS2, ITS1 was the lowest. The phylogenetic tree of ITS rDNA among the four goose coccidia showed that the different goose coccidia with the morphology similarities were closer in the tree. The other three phylogenetic trees could not explain the evolutionary relationships of species from different animals. These results suggest that the ITS rDNA gene could be used to identify goslings coccidian and show the evolutionary relationship of different goslings coccidia. |
PDF Full Text Request