柔嫩艾美耳球虫莫能菌素耐药株筛选及其与敏感株基因组SNP密度分布差异分析

doi:10.16431/j.cnki.1671-7236.2024.11.045

摘要/Abstract

摘要： 【目的】明确莫能菌素耐药虫株与敏感虫株在基因水平的差异，进而解析莫能菌素耐药性产生的分子机制。【方法】通过模拟田间感染的方式诱导柔嫩艾美耳球虫对莫能菌素的耐药性。第1次诱导试验，将 400 只 14 日龄无球虫鸡平均分为2组，分别为Ⅰ组(接种不加药组)和Ⅱ组(莫能菌素推荐剂量组),按照100 mg/kg 在基础饲粮中添加莫能菌素。从Ⅰ组中随机选择 10 只鸡，接种对莫能菌素敏感的柔嫩艾美耳球虫豪顿株，剂量为5 000卵囊/鸡。自接种后 8 d开始，将Ⅰ组新鲜粪便抛洒到Ⅱ组垫料上模拟球虫田间感染，连续抛洒17 d。接种后33 d，随机剖检Ⅱ组10只鸡，收集盲肠内卵囊。第2次诱导试验，将100只14日龄无球虫鸡作为Ⅲ组(莫能菌素高剂量组)，按照133 mg/kg在饲料中添加莫能菌素，随机选择10只鸡接种收集的卵囊，剂量为 5 000 卵囊/鸡。接种后17 d，莫能菌素添加量提高至400 mg/kg。接种后27 d，收集盲肠卵囊。以抗球虫指数(ACI)、病变记分减少率(RLS)、相对卵囊产量(ROP)和最适抗球虫活性百分率(POAA)四项指标综合判定诱导株的耐药性。提取耐药株或敏感株孢子化卵囊基因组并进行基因组重测序，通过单核苷酸多态性(SNP)密度分布解析耐药株与敏感株之间的差异。【结果】第1次诱导试验，接种后33 d获得耐100 mg/kg莫能菌素的卵囊(1×莫能菌素诱导株)。第2次诱导试验,接种后27 d获得耐400 mg/kg莫能菌素的卵囊(4×莫能菌素诱导株)。4×莫能菌素诱导株的ACI为 121，POAA为 25.4%，RLS为33%，POP为 64%。与参考基因组相比，敏感株基因组SNP共12 191个，耐药株基因组SNP共74 931个，耐药株在第11号染色体3.3～4.2 Mb 区间内SNP富集分布，该区间内3个基因ETH2_1113700、ETH2_1113500和ETH2_1113600的SNP位于编码区。【结论】本研究获得了对莫能菌素完全耐药的柔嫩艾美耳球虫耐药株。在第11号染色体，耐药株与敏感株的SNP分布具有显著差异，筛选到 3个耐药候选基因。

关键词: 球虫病; 柔嫩艾美耳球虫; 莫能菌素; 耐药性; SNP密度

Abstract: 【Objective】 The purpose of study was to determine the genetic differences between Eimeria tenella monensin-resistant and monensin-sensitive strains,and elucidate the molecular mechanism of monensin resistance. 【Method】 Resistance to monensin was induced in Eimeria tenella by simulating field infection.400 broilers were equally divided into two groups,group Ⅰ (inoculated and untreated group) and group Ⅱ (monensin recommended dose group). Monensin was added to the basal diet at 100 mg/kg.10 birds in group Ⅰ were inoculated with 5 000 oocysts on the first day.Litter containing feces with shed oocysts was randomly sampled from group Ⅰ and dispersed to the litter of group Ⅱ from the 8th day to the 25th day.33 days post inoculation,the caeca of 10 chickens in group Ⅱ were removed to collect the resistant oocysts.In the second experiment,100 broilers formulated the group Ⅲ (monensin higher dose group).10 chickens of group Ⅲ were inoculated with 5 000 resistant oocysts.The chickens of group Ⅲ were fed with gradient monensin (from 133 to 400 mg/kg).27 days post inoculation,the caeca of 10 chickens were removed to collect the resistant oocysts.The resistance was evaluated by ACI,POAA,RLS and POP.The genomes of resistant and sensitive strains were sequenced,then single nucleotide polymorphism (SNP) density was compared with the reference genome. 【Result】 In the first experiment,the resistant oocysts against 100 mg/kg monensin (1×monensin induced strain) were generated 33 days post inoculation.In the second experiment,the resistant oocysts against 400 mg/kg monensin 4×monensin induced strain were harvested 27 days post inoculation.Evaluation result of resistance showed ACI,POAA and ROP of 4×monensin induced strain were 121,25.4%,33% and 64%,respectively.Compared with the reference genome,there were 12 191 SNPs in the genome of the sensitive strain and 74 931 SNPs in the genome of the drug-resistant strain.The SNPs of the drug-resistant strain were enriched and distributed in the region of 3.3-4.2 Mb on chromosome 11,and the SNPs of three genes ETH2_1113700,ETH2_1113500 and ETH2_1113600 in the region were located in the coding region. 【Conclusion】 This study obtained a fully resistant strain of Eimeria tenella to monensin.On chromosome 11,there were significant differences in SNP distribution between resistant and sensitive strains,and three candidate genes for drug resistance were identified.

Key words: coccidiosis; Eimeria tenella; monensin; resistance; SNP density

中图分类号:

S852.72

顾小龙, 薛毅, 方素芳, 班成怿, 施宇博, 杨舰航, 杜芳辰, 齐珍珍, 崔平. 柔嫩艾美耳球虫莫能菌素耐药株筛选及其与敏感株基因组SNP密度分布差异分析[J]. 中国畜牧兽医, 2024, 51(11): 5108-5116.

GU Xiaolong, XUE Yi, FANG Sufang, BAN Chengyi, SHI Yubo, YANG Jianhang, DU Fangchen, QI Zhenzhen, CUI Ping. Selection of a Monensin-resistant Strain of Eimeria tenella and Differences Analysis Between Resistant and Sensitive Strains by SNP Density Distribution[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(11): 5108-5116.

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