| Protozoan parasites of the genus Eimeria which infect chickens develop in the intestine cause great economic losses in the poultry industry. At present, coccidiosis is still mainly controlled by the use of chemotherapeutic agents; thus, new drugs are urgently needed due to the rising problem of drug-resistant strains of Eimeria. In this study, to evaluated the potential mechanism of diclazuril action on the second-generation merozoites of E. tenella, two treatment conditions were employed: (1) chickens were challenged with E. tenella oocysts and received no diclazuril treatment, as an Infected group or (2) chickens were challenged with E. tenella oocysts and administered a regular diet containing 1 mg/kg diclazuril continuously for 24 h, from the 96th h to 120th h after inoculation, as a Diclazuril group. Chickens were inoculated by oral gavage with an 8×104 oocysts per chicken suspended in 1ml of distilled water. Ultrastructural changes were monitored by transmission electron microscopy (TEM). Apoptosis and mitochondrial transmembrane potential were determined by flow cytometry (FCM). Differential expressed genes in second-generation merozoites induced by diclazuril were isolated and analyzed using suppression subtractive hybridization and microarray technologies. The findings in the present study help to elucidate the complex mechanism underlying diclazuril effects on coccidiosis at the molecular level. The merozoite organelles and its genome would appear to represent valuable targets for development of new therapeutic drugs. The results are as the followings:1 Establishment of animal moded infected by E. tenella and preparation of second-generation merozoitesFine Chinese Yellow Broiler male chickens were inoculated by oral gavage with E. tenella sporulated oocysts. By this way, the coccidiosis animal model had been reproduced successfully. Purified second-generation merozoites were obtained from infected chicken caecal tissue at the 120th h after inoculation by a combination of enzymatic digestion, centrifugation, erythrocytes disruption and percoll density gradient centrifugation. Scanning electron microscopy (SEM) examination showed a high degree of purity of the merozoites preparation.2 Observation of ultrastructural changes in second-generation merozoites of E. tenella induced by TEMIn the Infected group, considerable numbers of viable merozoites showed normal features under TEM. However, in the Diclazuril group, merozoites that were characterised by obvious apoptotic features were observed. Apoptotic merozoites had morphological changes including reduced amounts of chromatin, which was condensed and compressed against the nuclear envelope and aggregated into large dark, compact masses and cytoplasm association with abundant vacuolisation. These findings which have not been reported previously lead to new drug targets for chemotherapeutic intervention in this parasite. 3 Determination of apoptosis and mitochondrial transmembrane potential in second-generation merozoites of E. tenella by FCMApoptosis and mitochondrial transmembrane potential in second-generation merozoites of E. tenella were determined using double staining method by FCM. The results showed that Rh123+PI- in the Diclazuril group was decreased by 97.58% (P < 0.01), compared with the Infected group. Rh123-PI- and Rh123-PI+ in the Diclazuril group were increased by 45.04 % (P < 0.01) and 266.67% (P < 0.01), respectively, in comparison with the Infected group. With the changes of mitochondrial membrane potential, diclazuril treatment also significantly increased early apoptosis by 180.75% (P < 0.01) and late apoptosis by 86.82% (P < 0.05). A positive correlation was suggested between the collapse of mitochondrial transmembrane potential and apoptosis. The mitochondrial dysfunction induced by diclazuril in E. tenella was most likely associated with the mitochondrial dependent apoptosis pathway.4 Isolation and analysis of differentially expressed genes in second-generation merozoites of E. tenella using SSH and cDNA microarrayIn the forward-subtracted library, the cDNA from the Diclazuril group was used as the"tester group"; and the cDNA from the Infected group was used as the"tester group"in the reverse-subtracted one. PCR amplification showed that the recombinant clones were 98% both in the two subtractive library, and the inserts were about 500bp. Rapid and high throughput screening of differentially genes in SSH subtracted libraries were used by cDNA microarray. The microarray hybridization results showed 881 differentially expression genes were obtained in all and 532 clones from the T1-H cDNA library and 268 from the T1-H cDNA library. Further QRT-PCR test on the chip hybridization results showed that clone differences is the same degree of similarity. A total of 229 ESTs from 251 differentially clones sequence were group into 55 contigs that contain 23 Contigs and 32 Singletons. All contigs then were submitted to Blast P for homologous searching with the nucleotide database in GeneBank. The homologous proteins including E. tenella microneme proteins, E. tenella surface antigens, E. tenella actin-depolymerizing factor (ADF), E. acervulina heat shock protein 90, T. gondii receptor for activated C kinase, T. annulata glyceraldehyde-3-phosphate dehydrogenase, etc.. These coding proteins identified in this study are related closely to the parasite discern, attach, motility, host-cell invasion, metabolism, signal transmission and so on. The results showed that diclazuril may be involved in the regulation of key molecules essential to parasite invasion and development in the clinical action of anticoccidiosis. These differential genes identified in this study can be acted as attractive target for control coccidiosis, remains to be investigated.5 Analysis of differential expression genes by QRT-PCRQRT-PCR analysis showed that diclazruil treatment resulted in downregulation of EtMICs and ADF genes of second-generation merozoites in E. tenella: EtMIC1 by 65.63% (P < 0.01), EtMIC2 by 64.12% (P < 0.01), EtMIC3 by 56.82% (P < 0.05), EtMIC4 by 73.48% (P < 0.01), EtMIC5 by 78.17% (P < 0.05) and ADF by 63.86% (P < 0.01). This indicated that downregulation of invasion-related EtMICs and ADF genes by diclazuril had interfered with the formation of biological complexes acting as important role during the course of invasion. As the same, the attenuation of damage to caecal mucosa induced by merozoite infection under SEM and the 65.13% (P < 0.01) decrease in merozoite number in the Diclazuril group also support our speculation.6 Cloning, expression and analysis of differential genesBased on the ESTs of differentially expressed genes, E. tenella receptor for activated C kinase (EtRACK) full-length cDNA was amplified using the RACE technology. G3PDH sequence was obtains by the electronic clone method. Recombinant expression plasmids pET-28a-mz-ADF and pET-28a-EtRACK were constructed successfully, and expressed in E. coli BL21(DE3) cell with the IPTG induction, respectively. The fusion protein purified using nickel-ion metal chelating column have been finished preparing the antibody and this pave a road to the study of protein expression in developmental stage, location of protein expression, protein-protein interaction and protein biological function, etc..In conclusion, the effects of diclazuril treatment on the normal development of second-generation merozoites in E. tenella are as the followings: First, decreased the mitochondrial transmembrane potential and induced apoptosis. Second, affect the skeleton-related protein and lost the living capacity. Third, interfere with the normal reorganization between parasite and hose cell. Forth, inhibit the gliding motor complex overhauling and formation. Fifth, shutoff the signal transduction of the parasite. However, the molecular mechanism of diclazuril action on the anticocidiosis will need further investigation and verification. |
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