Live and dead qPCR detection demonstrates that feeding of Nosema ceranae results in infection in the honey bee but not the bumble bee

As the honey bee and bumble bee may suffer from the same or related microbial pathogens, cross contamination from commercially reared Bombus spp. to honey bees and wild bumble bees and vice versa is a major concern. Honey bee-collected pollen to feed commercially reared Bombus spp. is a potential risk. Nosema spp. is a fungal pathogen in bees. In this study, we developed new quantitative detection tools based on the detection of RNA using a TaqMan-based RT-qPCR for Nosema ceranae and Nosema apis, with extraction controls based on the actin gene of honey bees and bumble bees, respectively. These tools were subsequently applied to study the epidemiology of N. ceranae, a main disease in honey bees. We screened gamma radiation and cold treatment sterilisation for their efficacy to kill N. ceranae spores fed in sugar water and in pollen to honey bees and bumble bees, respectively. N. ceranae infection in adult bumble bees was checked. Spores passing the inter-alimentary track were found but no infection was observed. N. ceranae spores were fed to honey bees. Their presence and multiplication were demonstrated, showing the spores were both viable and infectious. Our results indicate that N. ceranae found in honey bees cannot infect commercially reared bumble bees (Bombus terrestris) and, that gamma radiation effectively kills N. ceranae. The highly specific and sensitive molecular assays developed, were exploited to detect N. ceranae in pollen and faeces, which would allow more comprehensive epidemiological studies on this important pathogen.

鉴于西方蜜蜂（honey bee）与熊蜂（bumble bee）可能感染相同或相关的微生物病原，商业繁育的熊蜂属（Bombus spp.）与西方蜜蜂、野生熊蜂之间的交叉污染，以及反之的情况，均为备受关注的重大问题。用于饲喂商业繁育熊蜂属昆虫的西方蜜蜂采集花粉，是一项潜在的病原传播风险源。微孢子虫属（Nosema spp.）是蜜蜂的真菌性病原体。本研究针对东方蜜蜂微孢子虫（Nosema ceranae）与蜜蜂微孢子虫（Nosema apis），开发了基于TaqMan探针的实时荧光定量反转录PCR（TaqMan-based RT-qPCR）技术的RNA定量检测新工具，分别以西方蜜蜂与熊蜂的肌动蛋白基因（actin gene）作为提取对照。随后利用该检测工具开展东方蜜蜂微孢子虫的流行病学研究，该病原是西方蜜蜂的主要致病原之一。我们筛选评估了γ射线辐照（gamma radiation）与低温处理灭菌（cold treatment sterilisation）两种手段的杀灭效力，以分别杀灭添加于糖水基质中用于饲喂西方蜜蜂，以及添加于花粉基质中用于饲喂熊蜂的东方蜜蜂微孢子虫孢子。同时检测了成虫熊蜂的东方蜜蜂微孢子虫感染情况：检出了可通过消化道屏障的孢子，但未观察到感染发生。将东方蜜蜂微孢子虫孢子饲喂给西方蜜蜂后，可检测到孢子的存在与增殖，证明该孢子兼具活性与侵染性。本研究结果表明，西方蜜蜂体内检出的东方蜜蜂微孢子虫，无法侵染商业繁育的地熊蜂（Bombus terrestris），且γ射线辐照可有效杀灭东方蜜蜂微孢子虫。本研究开发的高特异性、高灵敏度分子检测方法，可用于检测花粉与粪便中的东方蜜蜂微孢子虫，从而为该重要病原的更全面流行病学研究提供技术支撑。