Experimental Infection And Analysis Of Apicoplast Genome Of Sarcocystis Wenzeli In Chickens

Sarcocystis is an apicomplexan parasitic protozoa with public health and veterinary values and a regulatory effect on wildlife population density.Apicoplast is a unique organelle of apicomplexan.The comparative analysis of its genome is of great significance for understanding the evolution of different groups of apicoplast and the evolution process of protists from autotrophy to heterotrophy.Chickens are the intermediate host of S.wenzeli.After infection,chickens will experience symptoms such as muscle weakness,weight loss,necrotizing encephalitis and even death.At present,there is no international data on the apicoplast genome of Sarcocystis.This study used experimental infection,molecular biology and bioinformatics methods to study the life cycle of S.wenzeli,the composition and structure of the apicoplast genome,and the potential antigenicity of the genes encoding apicoplast proteins.The results include:(1)10 cats and 10 dogs fed infected chicken muscle,and the morphological and molecular test results were negative.(2)The apicoplast genome of S.wenzeli was shown to be a circular DNA,with a size of 35028 bp and A+T content of77.74%.Its apicoplast genome contains 4 rRNAs,32 t RNAs and 28 protein-coding genes.4rRNAs are composed of LSU rRNA,SSU rRNA and their inverted repeats LSU rRNA,SSU rRNA;28 protein-coding genes include 1 translation elongation factor(Tuf A),1 Fe S cluster assembly protein(Suf B),1 CLP protease chaperone gene(Clp C),4 DNA-dependent RNA polymerase genes(Rpo B,Rpo C1,Rpo C2.1,Rpo C2.2),16 ribosomal proteins [10 ribosomal small subunit proteins(rps-)and 6 ribosomal large subunit protein(rpl-)] and 5 open reading frames(ORF B-F).The start codons of the protein coding sequence are ATG and ATA,and the stop codons are TAA and TAG.(3)The similarity between the rRNAs(LSU rRNA and SSU rRNA),Rpo B,Rpo C1,and Tuf A in the apicoplast genome of S.wenzeli and the corresponding genes of the other 6 families of apicomplexan preserved in Gen Bank was 57.7 %-93.1%,57.7%-92.5%,21%-74%,24%-76.9% and 53.4%-78.8%.(4)Phylogenetica analysis inferred from the sequences of apicoplast genome and genes showed S.wenzeli formed a individual clade with cyst-forming coccidia within a group containing species of Eimeriidae.(5)Among the 28 apicoplast-encoded proteins,21 proteins had potential antigenic properties,and most of them are ribosomal proteins.Based on the characteristics of antigen index and the presence of signal peptides,ribosomal proteins S3,S5,S17,L11 and open reading frame ORF E were selected for secondary and tertiary structure,B cell epitopes,MHC-I/II epitope sites and post-translational modification analysis,suggesting that they can serve as candidate proteins for serological assays and vaccines.This experiment shows that cats and dogs are not the terminal hosts of S.wenzeli,and the composition and structure of the apicoplast genome of S.wenzeli were analyzed for the first time,phylogenetic analysis inferred from the sequences of apicoplast genome and genes suggested that S.wenzeli had more closely related with species of cyst-forming coccidia and five kinds of apicoplast-encoded proteins with potential application value were found.