Characteristics Of A Precocious Line Of Eimeria Maxima And The Associated Genes

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial poultry production worldwide. Courrent control of the Eimeria species is based primarily on the use of medication. But the extensive use of anticoccidial drugs over the years has led to the emergence drug resistance in the field, and there are concerns about drug residues in poultry products, and there are strong desires of consumers to ban drugs from animal feeds. There is, therefore, a pressing need to move away from chemotherapeutic control of coccidiosis towards vaccination. Immunization with attenuated vaccines is increasingly playing an important role in the control of coccidiosis in the poultry industry.The precocious line of Eimeria was obtained by repeated passages of oocysts first collected from feces of previously infected chickens. Compared with the parent strain, the precocious line had a reduced prepatent period, markedly less pathogenicity, and much lower reproductive potential, but retained immunogenicity. Despite this variety of changes, few clearly discernible differences between the DNA of the parent strain and the precocious line have been identified. Indeed, the limited genetic variability might correspond to significant changes in the genome that affect the biological features of the parasite.Our research focused on analysis gene expressed changes with biochemical and morphological alterations in sporulated oocysts between the precocious line E. maxima and its parent strain using high throughput and high sensitivity approaches. It will help to understand the genetic basis of precocious phenotype in Eimeria and contribute to investigate the mechanism that regulate the life cycles of apicomplexan parasites.1 Selection and Characteristics of a Precocious Line of E. maximaA precocious line of E. maxima was selected by repeated passages of oocysts first collected from feces of previously infected chickens. The prepatent period of parasite was reduced from 142 h to 107 h. The size of sporulated oocysts of precocious strain was remarkably smaller than that of the parent strain. The peak of oocyst production was advanced by 1 day in comparison with the parent strain. Compared with the parent strain, the precocious line had a markedly less pathogenicity, and much lower reproductive potential, but retained immunogenicity. The precocious line of E. maxima was passaged in chickens with 5 generations without selection pressure. Its pathogenicity was significant less than the parent strain, showing the characteristics of precocious line were genetically stable.2 Construction of sporulated oocysts Subtractive cDNA Libraries of the precocious line E. maxima and its parent strain.A forward subtractive cDNA library was constructed by Suppression Subtractive Hybridization, using the sporulated oocysts cDNA of the precocious line as tester and the cDNA of parent strain as driver. A reverse subtractive cDNA library was constructed, using parent strain as tester and precocious line as driver. PCR amplification revealed that the two subtractive cDNA libraries contained approximated 98% and 97% recombinant clones. The size of most insert cDNA was about 500bp. These results indicated the subtractive cDNA libraries could be used to screen differentially expressed genes.3 Identification and analysis the differentially expressed genes of sporulated oocysts between the precocious line E. maxima and its parent strain by cDNA microarray techniqueIn order to screen differentially expressed genes from 2 subtractive cDNA libraries in large scale, 3164 clones were selected to fabricate cDNA microarrays. Each clone repeated 3 times. Microarray hybridization results suggested that 426 clones were identified from 2 subtractive cDNA libraries. Of which, 314 clones down regulated, 112 up regulated. A total of 426 clones were selected and sequenced. 360 valid ESTs were obtained. The results of sequence analysis indicated that these clones represented 54 clusters. Blast searches showed that some proteins encoded by 16 differentially expressed genes of sporulated oocysts shared significant identity with previously described proteins, including Eimeria acervulina serpin and cation-transporting ATPase, Eimeria tenella rhomboid-like protein and transhydrogenase, precore/core protein, tegument protein, retrotransposon protein, zyxin protein, alpha-crystallin-related protein, hemagglutinin esterase, etc. These results suggested that these differentially expressed genes would be related to host cell attachment, invasion and pathogenicity. The others 38 clusters had no homologues to the reported proteins in GenBank, which may be novel genes in Eimeria.4 Cloning and bioinformatics analysis differentially expressed novel genes of the sporoluated oocysts of the precocious line of E maxima and its parent srtainBased on ESTs of differentially expressed genes of sporulated oocysts received by SSH and cDNA microarray, the complete sequences of five novel genes including a complete open reading frame were obtained with RACE technique. Among these novel genes, one new gene was up-regulated in precocious line of E. maxima and four were down-regulated. Bioinformatics analysis showed A29 protein had 18 kDa molecular weight,a signal peptide, two hydrophobic regions and two trans-membrane structures. Blast searches show that this protein was homologous with E. acervulina cation-transporting ATPase. These results showed that this gene was related to cation regulation and drug-resistance. A43 protein had 28 kDa molecular weight, a signal peptide, six hydrophobic regions, six trans-membrane structures and a rhomboid superfamily conserved domain. It had 91% identity with E. tenella rhomboid-like protein. This gene was related to invasion or pathogenicity. A74 protein had 22 kDa molecular weight, one hydrophobic region and a SERPIN superfamily conserved domain. It had 91% identity with E. acervulina serpin. This gene was related to invasion.A78 protein had a signal peptide and two hydrophobic region. B71 protein had a signal peptide. These two proteins had no homolog to the reported proteins in GenBank, which suggested they would be novel genes.5 Expression of serpin genes of E. maxima in E.coliOne differentially expressed novel gene was ligated to prokaryotic expression vector pGEX-4T-2 and constructed a recombinant plasmid 4T-A74. This recombinant plasmid was transformed into BL21for expression. After induction by IPTG, The fusion protein was expressed in the form of inclusion bodies. The fusion protein was purified successfully with GST resin. Western-blot revealed that this protein was a native antigen.