| Coccidiosis is an acute epidemic of protozoa caused by one or more kinds of coccidia parasitizing on the host’s intestinal epithelial cells.There are few studies on the coccidia of wild animals in the world.To understand the prevalence of coccidia of wild animals and analyze the molecular characteristic of coccidia from wild animal,we collected feces samples of wild amur hedgehogs,migratory whooper swan and captive blue peafowl,using a combination of morphology and molecular methods to identify coccidia in the feces,and carried out molecular epidemiological investigations,aiming to laid the foundation on molecular differential diagnosis and provide scientific basis for the prevention and treatment of coccidiosis.(One)A total of 24 fresh fecal samples were collected from wild amur hedgehogs in Zhengzhou and Shangqiu,and microscopic examined by washing precipitation method and the sheather’s sugar floatation method.The infection rate of wild amur hedgehogs in some areas of Henan Province was 62.50%(15/24),Cystoisospora rastegaievae and a new species(named C.yuensis n.sp.)were identified.C.rastegaievae and C.yuensis n.sp.infection rates were 50.00%(12/24)and 12.50%(3/24),respectively,and there was no mixed infection.Based on the SSU rRNA,28S rRNA and ITS gene sequence analysis,the sequence homology of two species were 96.50%,98.00%and 99.30%,respectively.The construction of a phylogenetic tree based on the above three gene sites showed that C.yuensis n.sp.and C.rastegaievae were both sat on the Cystoisospora branch and they formed an independent sub-clade.(Two)The migrating whooper swans in the Swan Lake National Urban Wetland Park in Sanmenxia City were sampled 5 times in batches.A total of 858 fresh fecal samples were collected.The results of microscopy showed that the total infection rate of migrating whooper swans in Sanmenxia area of Henan was 39.63%(340/858),a total of 4 species of Eimeria spp.were identified:E.hermani(21.45%,184/858),E.nocens(17.25%,148/858),E.stigmosa(17.13%,147/858)and E.striata(15.38%,132/858).There are 2～4 species of Eimeria mixed infection,with the 2 species of Eimeria mixed infection rate highest.The infection rates of coccidia of migratory whooper swan in the early(October-November),mid-term(December-January)and late(February-March)wintering period were 36.71%(87/237)and 41.85%(231/552)and 31.88%(22/69),the dominant species are E.hermani.Based on the SSU rRNA,28S rRNA and COI gene sequence analysis,the sequence homology among the 4 species of Eimeria was 93.40%～99.00%,95.90%～98.30%and 90.5%～98.80%,respectively.The construction of a phylogenetic tree based on the above three gene sites showed that E.herman,E.nocensi,E.stigmosa and E.striata and Eimeria spp.from domestic geese of Anseriformes gathered in one independent clade.The single oocyst selection technology was used to isolate all coccidia with different morphological features from 45 positive samples of coccidia,a total of 171 samples of single oocyst coccidia were extracted from the whole genomic DNA of the oocyst,and the molecular epidemiology test of the SSU rRNA gene locus was carried out,a total of 4 species of Eimeria were identified,namely E.hermani,E.nocens,E.stigmosa and E.striata,with E.hermani dominant species in every period.Analysis based on the nucleic acid sequence of the SSU rRNA gene locus showed that the systematic relationship analysis of the gene loci showed that E.herman,E.nocensi,E.stigmosa,and E.striata and Eimeria from the domestic geese of Anseriformes gathered in an independent clade.(Three)We randomly collected 255 fresh fecal samples(240 blue peacocks and 15 white peacocks)from peafowls in Zhengzhou,Xinmi,Kaifeng and Qinyang.The results of microscopic examination showed that the total infection rate of coccidia of peafowl was 65.00%(156/240),there are 2～6 coccidia mixed infections,of which the mixed infection rate of the 2 species of Eimeria was the highest.A total of 7 species of Coccidia were identified,namely E.arabic(40.00%,96/240),E.pavonis(51.25%,123/240),E.riyadhae(37.08%,89/240),E.mayurai(14.17%.34/240),E.mandali(22.92%,55/240),I.mayuir(10.83%,26/240)and I.lacazei(8.50%,21/240),with E.pavonis dominant infection species in both peafowls.Analysis of the SSU rRNA,28S rRNA and COI gene sequences of 5 species Eimeria and 2 species of Isospora in peafowl showed that the sequence homology among the 5 species of Eimeria was 96.00%～98.60%,90.70%～98.20%and 85.00%～94.90%,respectively.The sequence homology between the two species Isospora was 98.80%,99.10%and 95.40%,respectively.The construction of a phylogenetic tree based on the above three gene sites showed that all 5 species Eimeria and E.pavonina from peafowl were clustered in a clade,followed by the turkey from Phasianidae,which gathers in a large branch with Galliformes as a whole.The two species of Isospora were clustered with Isospora spp.from Passeriformes.The single oocyst selection technology was used to isolate all coccidia with different morphological features from 43 positive samples of coccidia,a total of 125 samples of single oocyst coccidia were extracted from the whole genomic DNA of the oocyst,and molecular epidemiological detection of SSU rRNA gene locus,the molecular epidemiological results showed totally 7 species coccidia have been identified,namely:E.arabic,E.pavonis,E.riyadhae,E.mayurai,E.mandali,I.mayuir and I.lacazei.The coccidia dominant infection species of blue peafowl and white peafowl was E.pavonis.Based on the SSU rRNA gene locus,the sequence homology analysis between 7 species of peafowl coccidia geographical isolates shows that there were some differences between the geographic strains,but the homology between geographic strains was very high,and the variation between geographic strains was small.This study analyzed the coccidia infection of wild amur hedgehogs,migratory whooper swan and captive blue peafowl in Henan for the first time,and based on the sequence analysis of the SSU rRNA,28S rRNA and ITS regions of the coccidia,it was confirmed that the 2 species of wild Far amur hedgehog form an independent phylogenetic relationship;Based on the sequence analysis of the SSU rRNA,28S rRNA and COI genes of the coccidia,it was confirmed that the 4 species of Eimeria of migratory whooper swan constitute an independent phylogenetic relationship;Based on the sequence analysis of the SSU rRNA,28S rRNA and COI genes of the coccidia,it was confirmed that the 7 coccidia of the captive blue peafowl constitute an independent phylogenetic relationship.This study analyzed the coccidian infection of wild amur hedgehogs,migratory whooper swan and captive blue peafowl in Henan,and based on SSU rRNA,28S rRNA,ITS and COI gene loci to study the coccidia isolated from wild amur hedgehogs,migratory Cygnus cygnus and captive blue peafowl.It not only provides the coccidia infection status and molecular epidemiological data of these animals,but also provides molecular biological identification methods for the research of the wild amur hedgehogs,migratory whooper swan s and captive blue peafowl origin coccidia in various regions of the world,laying a foundation for molecular epidemiological investigations basis. |
