| Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial poultry production worldwide. At present, drug resistance is the most serious problem in prevention and treatment of coccidiosis, its mechanism is unknown. Knowledge of the gene(s) controlling resistance to a specific antimalarial drug, enables the identifying of resistance to a specific drug, the monitoring of the spread of resistance, as well as potentially increasing the effectiveness of decisions concerning drug policy. Knowing which genes are involved in resistance to a drug can also help us to understand the molecular basis of drug resistance and aid in the design of new versions of drugs that are unaffected by the mutations causing parasite drug resistance.Two drug-resistance strains of E. tenella to diclazuril and maduramycin were studied by genetic recombination to obtain recombinant lines with resistance to both diclazuril and maduramycin, to research the drug-resistant strains among the different isozymes, the drug resistance genes were cloned and analyzed.1. Development by genetic recombination of a line of Eimeria tenella resistance to diclazuril and maduramycinTo develop a line of Eimeria tenella resistance to two drugs by genetic recombination, a mixture of sporulated oocysts from two drug-resistance strains of E. tenella to diclazuril and maduramycin were administered to a group of untreated chicks. The resultant oocysts were given to chicks medicated with diclazuril and maduramycin to select parasites exhibiting the recombinant phenotypes. The biology characteristics of the recombinant line were studied in comparison with the parent strains. The results showed that the proportion of parasites with resistance to both diclazuril and maduramycin is only 0.4%. The recombinant line is resistant to both diclazuril and maduramycin, and sensitive to robenidine and nicarbazin. The reproductive potential of the recombinant line is much higher than that of maduramycin-resistant strain, but less than that of the diclazuril-resistant strain. The pathogenicity of the recombinant line is similar to both of parent strains. This study demonstrated that a line of E. tenella resistance to diclazuril and maduramycin was successfully developed by genetic recombination.2. Study on isozyme of different drug resistance strainsPolyacrylamide gel electrophoresis was used to analyse E. tenella, drug resistance strains and recombinant's lactic dehydrogenase (LDH), glucose phosphoric acid isomerase (GPI)and glucose-6-phosphoric acid dehydrogenase (G6PD) isozyme spectrum. The result indicated that 1 band of LDH in sensitive strain. while bands were not present in 3 resistance strains. The sensitive strain was characterized by 4 GPI isoenzyme band, GPI-2 and GPI-4 in diclazuril-resistant strain, GPI-2, GPI-3 and GPI-4 in maduramycin-resistant strain and recombinant strain.4 G6PD isoenzyme bands of sensitive strain were observed, and G6PD-3 and G6PD-4 were appeared in 3 drug resistance strains. The analysis of isozymes showed that enzyme variation existed between sensitive strain and drug resistance strains, and differences in GPI isozymes occurred among drug resistance strains, while both of LDH and G6PD were not different.3. Cloning and bioinformatics analysis resistance gene of E. tenellaBased on ESTs of differentially expressed genes of sporulated oocysts received by SSH and cDNA microarray, the complete sequences of three novel genes including a complete open reading frame were obtained with RACE technique. Among these novel genes, two new genes were up-regulated in resistance strains of E. tenella and one was down-regulated. Bioinformatics analysis showed M20 protein had 19 kDa molecular weight, no signal peptide or trans-membrane structures. Blast searches show that this protein was homologous with Toxoplasma gondii GT1 putative insulysin. These results showed that this gene failed drugs by interfered cation regulation trans-membrane. E2 protein had 32 kDa molecular weight, one hydrophobic region, one trans-membrane structures and a DUF477 superfamily conserved domain. It had 36% identity with Domain of unknown function DUF477 of Toxoplasma gondii. This gene was down-regulated, its function need further development. H3 protein had 36 kDa molecular weight, no trans-membrane structures and one signal peptide, and a Laminin G domain conserved domain. It had 65% identity with Toxoplasma gondii conserved hypothetical protein. This gene was up-regulated. The result supposed that it promoted parasites into the host cell, reduced parasites damage by high concentrations drug.
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