一类新兽药沙咪珠利对柔嫩爱美尔球虫诱导耐受及耐受逆转机制研究

Eimeria tenella is an obligate in tracellular apicomplexan(coccidian) parasite that infects chickens and causes a severe form of coccidiosis. E. tenella parasitizes in the caeca of chicken during the endogenous developmental stage, which leads to intestinal lesions, nutrient malabsorption, hemorrhage, and even death in severe cases. Coccidiosis is one of the most detrimental and lethal disease and is a major welfare and economic problem in the poultry industry. However, certain Eimeria strains, the causative agent of coccidiosis, exhibit or develop resistance to most drugs, thereby rendering the anticoccidials inefficient and leading to clinical failure.Ethanamizuril(EZL) is a novel anticoccidial triazine compound developed using systematic structure-activity relationship studies of these compounds in our laboratories. EZL has an excellent efficacy in preventing coccidiosis in broiler chickens and no cross-resistance was observed between DZL and EZL or TZL.In this study, EZL resistance will be induced in Eimeria tenella by exposing it to a series of EZL concentrations. The EZL-resistant E. tenella strains were obtained. We investigated the proteome of second-generation merozoites of EZL-resistant E. tenella using two-dimensional gel electrophoresis (2-DE), and investigated the RNA of second-generation merozoites of EZL-resistant E. tenella using high throughput sequencing.Using Western Blot and real-time PCR to identify different expressed protein or DNA.Find the difference between the control and EZL-resistant strains and reversion resistant strains. Taken together, our data provide insights into the molecular mechanisms of E. tenella resistance to EZL.This study may provide useful guidance for the safe and rational use of EZL.

每年因鸡球虫病造成的直接和间接经济损失惨重，球虫已对现有的抗球虫药物均产生耐药性！对课题组自主研发的新抗球虫药-Ethanamizuril，在发现其具有良好的抗球虫活性和无交叉耐药性的同时，为了延缓球虫对沙咪珠利产生耐药性，本项目通过选择Ethanamizuril一系列的固定有效剂量，人工体内多代稳定诱导鸡柔嫩爱美尔球虫，得到诱导耐受株，考察耐受株是否存在交叉耐药性。将耐受株在无药物压力下继续传代，获得耐受逆转株。研究沙咪珠利给药剂量-时间-耐药性的内在关系。运用双向电泳技术和高通量测序研究诱导耐受前后、耐受逆转后的蛋白水平和RNA水平的变化，通过Western Blot和RT-PCR验证差异因子，再运用免疫共沉淀和酵母双杂交系统筛选差异因子互作蛋白。初步探索球虫对沙咪珠利耐受及耐受逆转的分子机制。该项目为该药的临床安全合理使用和阐明球虫对抗球虫药物耐药及耐药逆转机制提供理论参考.

鸡球虫病是由艾美尔属球虫引起的一种原虫病，能够导致鸡的生长发育缓慢并制约养禽业的发展。随着药物的广泛使用，鸡球虫几乎对所有的抗球虫药物都产生了耐药性。为了更好的控制鸡球虫病，研究鸡球虫对抗球虫药物的耐药机制和探索药物作用靶点势在必行。本项目主要通过固定剂量和逐渐递增剂量诱导并筛选了对沙咪珠利耐药的柔嫩艾美尔球虫虫株，并对耐药虫株逆转诱导；建立了鸡盲肠组织中沙咪珠利的含量测定方法，确定了随着给药剂量的增加，给药剂量-药效-时间-耐药性内在的定性定量关系；通过蛋白质组学和转录组学对诱导耐受前/后的柔嫩艾美尔球虫孢子化卵囊进行研究，筛选了差异基因和差异蛋白，并用RT-PCR对其进行验证，对其中的差异蛋白进行了多抗的制备，初步分析了可能参与耐药的蛋白和基因。重要结果：经过26次传代分别获得了对10 mg/kg 沙咪珠利和对200 mg/kg沙咪珠利完全耐药的虫株。将耐药虫株在饲料中不添加任何药物的情况下传代，诱导获得耐药逆转株。不同给药剂量下盲肠组织中沙咪珠利在给药后第8 d，随着给药剂量的增加，盲肠组织中药物浓度增加幅度减小，为进一步研究球虫对沙咪珠利产生的耐药性机制提供了理论参考。对敏感株和耐药株进行蛋白组学分析，总共鉴定了1384个蛋白和7618条肽段；固定剂量耐药株（R10）和敏感株（C），逐渐递增剂量耐药株（R200）与敏感株，逐渐递增剂量耐药株和固定剂量耐药株分别有222，573，620个差异蛋白。主要与复制、入侵、膜组分、运输和糖代谢等相关。对敏感株和耐药株进行转录组分析，R10 vs C有差异基因118个，上调42个，下调76个；R200 vs C有1821个显著差异基因，上调690个，下调1131个。差异基因主要参与细胞过程，代谢过程，是细胞器和膜的主要组成部分，主要发挥结合和催化功能等，为进一步研究球虫的耐药机制奠定基础。将蛋白组和转录组结果进行整合分析，两组学的相关性差，说明转录后加工，翻译后修饰等影响转录组和蛋白组相关性的因素可能与耐药性的形成有关。