Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.
Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.
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DOI：
10.1016/j.parint.2015.06.010
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发表时间：
2015-10
影响因子：
1.9
通讯作者：
Blake DP
中科院分区：
文献类型：
--
作者：
Nolan MJ;Tomley FM;Kaiser P;Blake DP
艾美耳球虫是鸡的高致病性寄生虫。由于缺乏衡量寄生虫在宿主体内复制的非主观定量工具，旨在减少其影响的研究受到阻碍。现有方法耗时且通常对时间敏感，因此无法在大规模遗传、流行病学和进化分析中使用。我们使用实时定量 PCR (qPCR) 来准确定量鸡组织中的柔嫩艾美耳球虫，并表明该方法比传统方法更有效、更灵敏。我们测试了四种鸡特异性参考 qPCR 检测，发现 β-肌动蛋白 (actb) 最适合样本标准化。在实验环境中，鸡接种了 500、1500 或 4500 个柔嫩球虫卵囊，寄生虫复制和感染的影响通过 i) 对感染后 5 天和 8 天 (dpi) 收集的盲肠组织中提取的 DNA 进行 qPCR 分析，ii) 对 6 至 8 dpi 采集的样本进行粪便卵囊计数 (FOC)，以及 iii) 病灶评分caeca 在 5 点和 8 点进行尸检。定量实时 PCR 测试结果表明，对于 5 dpi（即配子发育前）收集的样本，研究组中的寄生虫数量出现显着的剂量依赖性增加（R2 = 0.994）（p < 0.002），但在第八天（大多数卵囊脱落后）收集的样本中寄生虫数量没有显着增加（R2 = 0.006）（p > 0.379）。 FOC (R2 = 0.997) 和病变评分也揭示了寄生虫复制和感染严重程度的强烈剂量依赖性增加。重要的是，qPCR 通过提高统计能力和减少实验研究中的群体规模，显着改善动物福利。所描述的 qPCR 方法克服了粪镜定量的主观局限性，允许对组织、粪便和卵囊进行可重复的中高通量检查，并且是确定艾美耳球虫感染在实验和现场环境中影响的宝贵工具。定量 PCR 表明寄生虫的剂量依赖性显着增加。比粪便卵囊计数/病变评分更灵敏的寄生虫复制测量 对动物福利和实验改进 (3R) 具有重大影响 支持关注抗性/易感性遗传基础的研究 对艾美耳球虫生物学和控制关键方面的研究具有重大影响
The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings. Quantitative PCR indicates significant dose-dependent increases in parasites. More sensitive measure of parasite replication than faecal oocyst count/lesion score Significant implications for animal welfare and experiment refinement (3R's) Support studies focusing on the genetic basis of resistance/susceptibility Major implications for investigations of key aspects of Eimeria biology and control