Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany.
J Invertebr Pathol. 2013 May;113(1):1-6. doi: 10.1016/j.jip.2013.01.004. Epub 2013 Jan 24.
Nosema apis and Nosema ceranae are two microsporidian pathogens of the European honey bee, Apis mellifera. There is evidence that N. ceranae is more virulent than N. apis subject to environmental factors like climate. This makes N. ceranae one of the suspects in the increasing colony losses recently observed in many regions of the world. Correct differentiation between N. apis and N. ceranae is important and best accomplished by molecular methods. So far only protocols based on species-specific sequence differences in the 16S rRNA gene are available. However, recent studies indicated that these methods may lead to confusing results due to polymorphisms in and recombination between the multi-copy 16S rRNA genes. To solve this problem and to provide a reliable molecular tool for the differentiation between the two bee pathogenic microsporidia we here present and evaluate a duplex-PCR protocol based on species-specific sequence differences in the highly conserved gene coding for the DNA-dependent RNA polymerase II largest subunit. A total of 102 honey bee samples were analyzed by the novel PCR protocol and the results were compared with the results of the originally published PCR-RFLP analysis and two recently published differentiation protocols, based on 16S rRNA sequence differences. Although the novel PCR protocol proved to be as reliable as the 16S rRNA gene based PCR-RFLP it was superior to simple 16S rRNA based PCR protocols which tended to overestimate the rate of N. ceranae infections. Therefore, we propose that species-specific sequence differences of highly conserved protein coding genes should become the preferred molecular tool for differentiation of Nosema spp.
微孢子虫 Nosema apis 和 Nosema ceranae 是欧洲蜜蜂 Apis mellifera 的两种微孢子虫病原体。有证据表明，在气候等环境因素的影响下，N. ceranae 的毒力比 N. apis 更强。这使得 N. ceranae 成为世界许多地区最近观察到的蜂群损失增加的可疑原因之一。正确区分 N. apis 和 N. ceranae 非常重要，最好通过分子方法来完成。到目前为止，仅基于 16S rRNA 基因的种特异性序列差异的方案可用。然而，最近的研究表明，由于多拷贝 16S rRNA 基因之间的多态性和重组，这些方法可能会导致混淆的结果。为了解决这个问题，并为区分这两种蜜蜂致病性微孢子虫提供可靠的分子工具，我们在此提出并评估了一种基于高度保守的 DNA 依赖性 RNA 聚合酶 II 大亚基编码基因的种特异性序列差异的双重 PCR 方案。总共分析了 102 个蜜蜂样本，该新 PCR 方案的结果与最初发表的 PCR-RFLP 分析以及基于 16S rRNA 序列差异的两种最近发表的区分方案的结果进行了比较。虽然新型 PCR 方案被证明与基于 16S rRNA 基因的 PCR-RFLP 一样可靠，但它优于简单的基于 16S rRNA 的 PCR 方案，后者往往高估了 N. ceranae 感染的比率。因此，我们建议高度保守的蛋白质编码基因的种特异性序列差异应成为区分 Nosema spp.的首选分子工具。
