| Chicken coccidiosis is a protozoan disease caused by apicomplexan Eimeria spp.,which lead to slow growth of chickens and restrict the development of global poultry industry.Control of chicken coccidiosis depends primarily on prophylactic chemotherapy with anticoccidial drugs even though their long-term use has led to development of resistant organisms.In order to control the chicken coccidiosis,it is imperative to study the resistance mechanism of coccidiosis and explore the target of drug action.Ethanamizuril（EZL）was independent developed at the Shanghai Veterinary Research Institute,Chinese Academy of Agriculture Sciences.Previous studies have shown that EZL possesses high anticoccidial activity and has good prospect of application.In order to study the the resistance mechanism of Eimeria tenella aganist ethanamizuril,we explored the relationship between administration dosage and the EZL concentration in cecum,induced resistance strains of EZL,the proteomics and transcriptomics analysis of senstive strain and resistance strains and joint analysis of two groups.In addition,studying the pharmacokinetics of ethanamizuril solution in chickens provides guidance and reference for its rational use in clinic.The results are as follows:1.A ultra high performance liquid chromatography（UPLC）method was used to detect a new anticoccidial drug ethanamizuril in the caecum of chicken,which was to study the relationship between dosage and drug content in the caecum of chicken.The caecum were collected after administration the ethanamizuril mixed in feedstuff,which were homogenated,ethyl acetate extracted and applied the UPLC-UV to determine.Chromatographic separation was performed on ACQUITY UPLC BEH C18column（1.7μm,2.1×100 mm）with gradient elution at 251 nm and the mobile phase consisting of acetonitrile and water at the flow rate of 0.2 mL/min.The column temperature was 30 oC.Linear calibration curves were obtained over concentration range of 0.025-0.5μg/g and 0.5-16.5μg/g,the regression equation were Y=6.2156 X-0.0222（R2=0.9994）and Y=1.3445 X-0.1544（R2=0.9999）respectively.The average recovery from the spiked samples ranged from 85%to 106%with RSD of within groups and between groups were less than 10%.This method was applied to detect the content of ethanamizuril in the caecum of chicken.At the 8th day of dosing,the rate of concentration increasing of ethanamizuril decreased in the caecum,which provide reference for further study the Eimeria tenella resistance mechanism to ethanamizuril.We studied the pharmacokinetics of oral ethanamizuril solution in chickens and developed a method for its detection in plasma.Ethanamizuril was administered as single oral doses at low（0.67mg/kg）,medium（1.33 mg/kg）and high（6.67 mg/kg）levels.The plasma concentration time curve data were analyzed using the pharmacokinetic software DAS 3.0（WSO2,Mountain View,CA）with a non-compartmental model to obtain pharmacokinetic parameters.Peak plasma concentrations of ethanamizuril were 2.16±0.57,3.91±0.71,23.71±5.02 mg/L at 5.17±1.80,4.6±2.12,4.6±2.12 h,respectively.The terminal elimination half-lives(t1/2λz)for ethanamizuril were 10.8,10.7 and 13.3 h after oral administration at low,medium and high doses,respectively.The results suggested that ethanamizuril was absorbed rapidly and eliminated slowly.A comparison across the dose range indicated that Tmax values were similar while Cmax and AUC0-t were positively correlated with increasing dosages.2.Induced the resistance strain of Eimeria tenella to EZL by fixed dosage and gradually increasing dosage methods.After 26 passages,we got two resistance strains,which were completely resistant to 10mg/kg EZL and 200 mg/kg EZL,respectively.The single oocyst was separated and indentified in the fourth generation.The oocyst used to passage were all Eimeria tenella.The resistance reversal strains were induced without add drug in feed to two resistance strains.Determinated the efficacy trial at 10passages.The reversal strain of fixed dose group was resistant to 10 mg/kg EZL.The reversal strain of gradually increasing group was sensitive to 10 mg/kg EZL.It is indicated that the drug resistance acquired by the progressive increasing dose group is more likely to be recovered than that of the fixed dose group in the case of long-term non-drug use.3.We used label-free quantitative proteomics to compare sensitive and resistant strains of Eimeria tenella to EZL,screening the different proteins and analysing the function of different proteins.We identified 1384 proteins and 7618 peptides and found 222,573 and 620 differently expressed proteins in the groups R10 vs C,R200 vs C and R200 vs R10,respectively.There were 96 unique proteins that were found in both resistant strains when compared with the control strain.These were primarily involved in replication,invasion,membrane components and transport as well as glucose metabolism.Our results indicated that ethanamizuril resistance is complex and a single differentially expressed protein responsible for resistance could not be identified.This research provide new ideas for further study the resistance mechanism of coccidiosis.4.We used RNA-Seq technology to study the transcriptome of sensitive Eimeria tenella strain and two resistant strains of Eimeria tenella to EZL,screening the different genes and analysing the function of different genes.The compare of R10 vs C had 118 different genes,42 genes were upregulated and 76genes were downregulated.The compare of R200 vs C had 1,821 significant difference genes,690genes were upregulated and 1131 genes were downregulated.R200 vs R10 had 1,383 significantly different genes,525 genes were upregulated and 858 genes were downregulated.In R10 vs C and R200vs C,there were 48 significant differences which have consistent trend,11 genes were upregulated and37 genes were downregulated.In addition,1359 genes have consistent trends in R10 vs C and R200 vs C,but its difference was not significant in R10 vs C and significant differences in R200 vs C,480 genes were upregulated and 879 genes were downregulated.The number of downregulated differentially expressed genes was significantly higher than that of the upregulated differential gene.The differential genes are mainly involved in cell process,metabolic process,which is the main component of organelles and membranes,which mainly plays the role of binding and catalysis,which lays a foundation for further research on drug resistance mechanism.5.Analysing the proteome and transcriptome results together,the correlation of the two omics was bad.The factors that affect the transcription and protein group correlation may be associated with the formation of drug resistance,such as processing after the transcription and translation after modification and so on.Most of the differentially expressed genes/proteins are related to metabolic processes,cell processes,binding,catalytic activity,the components of organelles and membrane.The differently expressed genes mainly involved in metabolic pathways,biosynthesis of antibiotics,biosynthesis of secondary metabolites,carbon metabolism,microbial metabolism in diverse environments.It is of great significance to provide reference and guidance to explore the mechanism of drug resistance of coccidios.In conclusion,these study have given us a preliminary understanding of drug resistance mechanism of E.tenella and the pharmacokinetic charateritics of oral ethanamizuril solution in chickens.These study also established a foundation for further exploring the drug resistance mechanism and the target of drug action. |
