McGowan Janine, De la Mora Alvaro, Goodwin Paul H, Habash Marc, Hamiduzzaman Mollah Md, Kelly Paul G, Guzman-Novoa Ernesto
School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G2W1, Canada.
Departamento de Producción Animal: Abejas, FMVZ, UNAM, Cd. Univ., Mexico DF 04510, Mexico.
J Microbiol Methods. 2016 Dec;131:16-22. doi: 10.1016/j.mimet.2016.09.021. Epub 2016 Sep 29.
The microsporidium fungus Nosema ceranae is an intracellular parasite that infects the midgut of the honey bee, Apis mellifera. A major limitation of research on N. ceranae is that the fungus is non-culturable and thus studying it depends on the seasonal availability of Nosema spores. Also, spore viability and infectivity can vary considerably, and thus there is a need for reliable methods for determining those traits. This study examined different conditions for N. ceranae spore cryopreservation at -70°C, assessing spore viability and infectivity. Viability was determined by a staining procedure counting total spores numbers with bright field microscopy and un-viable spore numbers with the fluorescent dye, propidium iodide. Spore infectivity was determined with a dilution inoculation assay. Infectivity was dependent on the inoculum dose for the proportion of bees with detectable Nosema infections based on the number of spores per bee at 18days after inoculation; 4000 spores per bee or higher were needed to get approx. 100% of the inoculated bees infected. The median infective dose (ID) was 149 spores per bee, and the minimum dose capable of causing a detectable infection was 1.28 spores. The proportion of N. ceranae infected bees correlated significantly with the number of spores per bee (r=0.98, P<0.0001). N. ceranae spores cryopreserved in water or 10% glycerol did not differ in viability compared to fresh spores, but lost infectivity when inoculated into bees. This study shows that while cryopreservation of N. ceranae spores can preserve viability, the spores can have reduced infectivity.
微孢子虫真菌——蜜蜂微孢子虫（Nosema ceranae）是一种细胞内寄生虫，可感染西方蜜蜂（Apis mellifera）的中肠。对蜜蜂微孢子虫研究的一个主要限制是该真菌无法培养，因此对其研究依赖于微孢子虫孢子的季节性可得性。此外，孢子的活力和感染力可能有很大差异，因此需要可靠的方法来确定这些特性。本研究检测了在-70°C下蜜蜂微孢子虫孢子的不同冷冻保存条件，评估孢子的活力和感染力。活力通过染色程序来确定，用明场显微镜计数总孢子数，并用荧光染料碘化丙啶计数无活力的孢子数。孢子感染力通过稀释接种试验来确定。基于接种后18天每只蜜蜂的孢子数，对于有可检测到的蜜蜂微孢子虫感染的蜜蜂比例而言，感染力取决于接种剂量；每只蜜蜂需要4000个或更多的孢子才能使约100%的接种蜜蜂被感染。半数感染剂量（ID）为每只蜜蜂149个孢子，能够引起可检测到的感染的最小剂量为1.28个孢子。感染蜜蜂微孢子虫的蜜蜂比例与每只蜜蜂的孢子数显著相关（r = 0.98，P < 0.0001）。保存在水或10%甘油中的蜜蜂微孢子虫孢子与新鲜孢子相比，活力没有差异，但接种到蜜蜂体内时会丧失感染力。这项研究表明，虽然蜜蜂微孢子虫孢子的冷冻保存可以保持活力，但孢子的感染力可能会降低。
