The Preliminary Mechanism Study Of Ethanamizuril Against E.tenella

Avian coccidiosis is a protozoan disease caused by Eimeria spp. Among them, E.tenella is one of the most pathogenic Eimeria species and usually leads to the high morbidity and mortality, which brings the huge economic burden to the poultry industry annually. Nowadays the control of coccidiosis is mainly depends on the anticoccidials, but it has become increasingly difficult owe to the emergence of drug-resistance. Therefore, there is a pressing need to search for novel anticoccidials and elucidate the mechanism of action of anticoccidials.Ethanamizuril is a novel triazine anti-coccidiosis compound synthesized by our department and previous studies showed it has a broad development prospect. To understand the preliminary mechanism of action of ethanamizuril, we explored the stage and peak period of action of ethanamizuril against E.tenella, ultrastructural effects of ethanamizuril on E.tenella and the primary site of action of ethanamizuril, and effects of ethanamizuril on two important metabolic enzymes associate with the function of the action site. The results are as followings:1. In the study of the action stage and peak period of ethanamizuril against E.tenella, ethanamizuril was medicated respectively at two days(group1) and one day(group2) before the infection, the day for infection(group3), one day(group4), two days(group5), three days(group6) and four days(group7) after the infection for three days. Meanwhile, the diclazuril control group, infection control group and blank control group were set up. The result showed the anticoccidial indexes of group1, group2 and group3 were 104±11、92±11 and 125±16, respectively, showing almost no anticcidial activity. The anticoccidial indexes of group4, group5, group6 and group7 were 170±8、191±5、171±4 and 161±5, respectively, showing the medium and high anticcidial activity. The anticoccidial indexes of diclazuril control group, infection control group and blank control group were 181±14、73±31 and 200±2, respectively. The above data indicated ethanamizuril has the highest anticoccidial activity when it is medicated at 2th d post the infection for three days, and the corresponding peak period of action of ethanamizuril is the schizogony and gametogony of E.tenella.2. In the study of ultrastructural effects of ethanamizuril on E.tenella and the primary site of action of ethanamizuril, chicken infected by E.tenella were given a single oral dose of 15mg/kg ethanamizuril at different time points and killed at 16 h after medication. The caecum tissues were collected and the ultrathin sections were obtained. Observed with transmission electron microscope, we found that ethanamizuril affected both the schizogony and gametogony of E.tenella: the differentiations of second-generation schizonts and microgamonts were inhibited. Obvious outer membrane blistering and perinuclear space enlargement were occurred in merozoites and microgamonts. Besides, merozoites became irregular in shape with the ethanamizuril treatment. The abnormal evagination of microgametes finally resulted in the degeneration of microgamonts. The micromorphological alteration of WFB2 hindered the formation of oocyst and resulted in the proceeding degeneration of zygotes. In addition, mitochondria, endoplasmic reticulum and Golgi body swelling showed the abnormity in different extents.3. Enolase is a multifunctional protein, which also plays an important role in other biological processes in addition to the glycolysis. To learn about the role of enolase in E.tenella and the effect of ethanamizuril on enolase in second-generation merozoites of E.tenella, Real-time PCR, Western blotting and immunofluorescence were carried out. The results showed that ethanamizuril down-regulated the enolase expressions at the transcriptional and translational levels. The subcellular localization of enolase indicated that it was primarily distributed at the apical end of the second-generation merozoites of E.tenella. Compared with the control group, the fluorescence intensity of enolase was obviously reduced after treated with ethanamizuril.4. LDH is an important proteases in glycolysis and has the great significance in various parasite studies. The present experiment applied Real-time PCR, Western blotting, immunofluorescence, LDH activity assay and caspase3 activity detection to analyze the effects of ethanamizuril on LDH in second-generation merozoites of E.tenella. The results demonstrated LDH was mainly distributed in the cytoplasm of second-generation merozoites. With the ethanamizuril treatment, LDH expressions were down-regulated at the transcriptional and translational levels, LDH activity was decreased in the cytoplasm and nucleus of merozoites, but the caspase3 activity in merozoites was significantly enhanced.In summary, we have a preliminary understanding about the mechanism of action of ethanamizuril with above findings. Meanwhile, these findings established the foundation for further exploring the mechanism of action of ethanamizuril.