分离自蜜蜂(Apis mellifera)的三株螺原体的基本特性

【目的】调查我国蜜蜂螺原体的种类,研究它们的基本生物学特性,初步确定其分类地位,为研究螺原体在自然界中的传播途径提供依据。【方法】螺原体的分离、培养方法,应用暗视野显微镜和透射电子显微镜观察螺原体形态,运用分子生物学方法(选16S rDNA、ITS、rpoB基因进行系统发育分析)和血清学方法(生长抑制试验、代谢抑制试验、菌体变形试验)研究螺原体分离菌株可能的分类地位。【结果】从健康的意蜂(Apis mellifera)体内分离到3株螺原体MF0903、MF0904、MF0905。3株螺原体都呈典型的螺旋状,但菌株MF0905的菌体短小,螺旋数较少;MF0903和MF0904菌落呈规则的圆形,MF0905菌落近圆形、较大;它们都能利用葡萄糖、D-果糖作为碳源,不能利用尿素;菌株MF0903、MF0904能强烈代谢精氨酸、不能利用蔗糖作为碳源,而MF0905不能代谢精氨酸、能利用蔗糖作为碳源;根据16S rDNA、ITS、rpoB基因序列构建系统发育树显示,分离菌株MF0903、MF0904与Spiroplasma melliferum聚类较近,而MF0905与Spiroplasma clarkii聚类较近。生长抑制试验、代谢抑制试验、菌体变形试验结果均表明标准菌株Spiroplasma melliferum CH-1的抗血清对菌株MF0905没有抑制作用,而能抑制菌株MF0903和MF0904生长。【结论】分离菌株MF0903、MF0904属于Spiroplasma melliferum,而MF0905可能是Spiroplasma clarkii,这表明我国蜜蜂中存在的螺原体不仅仅是Spiroplasma melliferum。

Characteristics of three spiroplasma isolates from honeybee (Apis mellifera)

Abstract: [Objective ] To investigate the kinds and characteristics of spiroplasma in honeybees, as well as to study the taxonomy and transmission of honeybee spiroplasma under natural conditions. [Methods] We examined the morphology of spiroplasma isolates by dark field and transmission electron microscopy and studied the biological characteristics by using conventional culture-dependent methods and molecular biology and serological methods. [Results] Three spiroplasma isolates were obtained from healthy Apis mellifera. All isolates exhibited helicity during their growth phase, with one isolate (MF0905) being shorter and having less helicity. This isolate also differed from the other two (MF0903 and MF0904) in having larger colonies with an irregular margin instead of being round. In addition, isolate MF0905 could not hydrolyze arginine whereas MF0903 and MF0904 could. All three isolates could use glucose and D-fructose as a carbon source but did not hydrolyse urea. Phylogenetic analysis based on the 16S rDNA, ITS and the rpoB gene showed that MF0903 and MF0904 had a close relationship with Spiroplasma melliferum, and MF0905 was close to S. clarkii. Serological studies including the growth inhibition test, metabolic inhibition test and deformation test gave the same result as the phylogenetic analysis. [Conclusion] The spiroplasma isolate MF0905 might be S. clarkii and other two isolates were S. melliferum. This result indicated that Spiroplasma melliferum is not the only spiroplasma species in honeybees in China.