【作者】 李霞；

【导师】 于汉寿；

【作者基本信息】 南京农业大学 ， 微生物学， 2012， 硕士

【摘要】 蜜蜂螺原体是一种螺旋状、能运动、无细胞壁的原核生物,主要寄生在青壮年蜂的体内,是引起我国蜜蜂“爬蜂病”的主要病原之一。20世纪80年代以来,蜜蜂螺原体病在我国各地养蜂区普遍蔓延,且常与孢子虫病、麻痹病等混合发生,给养蜂业造成了严重的经济损失。据报道,在蜜蜂螺原体病发病时期,蜜蜂、植物花及其它一些膜翅目、双翅目、鳞翅目昆虫内均能检测到螺原体,在养蜂地区的非发病时期,尤其是冬季和炎热的夏季却均未能检测到。根据研究结果,学者们推测出蜜蜂螺原体在自然界的一些可能的侵染循环途径,但一直缺乏系统的研究和直接的证据。本研究针对引起我国蜜蜂“爬蜂病”的螺原体Spiroplasma melliferum,建立了一种分子生物学快速检测方法,系统地对其宿主范围、可能的传播途径进行了研究,并对分离自同一时期不同宿主中螺原体的基本生物学特性及致病性进行了比较。初步研究了S.melliferum在蜜蜂中肠和胸肌内的定殖与侵染特征。针对引起我国蜜蜂螺原体病的病原菌S. melliferum,通过对Chelex-100DNA提取技术的改进,建立了一种分子生物学快速检测方法。该方法可以在2-4小时内检测出植物花、蜜蜂及其生活环境中是否含有螺原体,最低检测浓度为5～6个/mL。与常规分离培养法比较,该方法具有灵敏、快速、高效等优点,这为蜜蜂螺原体病的快速诊断及螺原体资源调查奠定了基础。为了探索蜜蜂螺原体在自然界中可能的传播途径及其侵染循环,本研究采用分子生物学快速检测方法和分离培养技术定期对蜜蜂及自然界中蜜蜂经常活动的场所中的材料、植物花及其它一些相关昆虫进行检测。从2010年3月到2012年1月,共采集了1339只意蜂(Apis mellifera)、131种昆虫、51种植物花和77个从蜂箱内采集的样本,并在其中92个样本中检测到螺原体,分离获得54株螺原体菌株。在一年四季(包括蜜蜂螺原体病发病时期和非发病时期)采集的蜜蜂样本中均检测到螺原体,以在春季、发病时期病蜂体内检测到螺原体的几率最高。此外,在发病时期采集的蜜蜂幼虫、蛹、植物花、其它昆虫、蜂房、巢门、巢脾等样本中,均检测到螺原体,在非发病时期,则仅在少数蜜蜂体内发现。表明蜜蜂螺原体长期存在于蜜蜂体内,可通过水平传播方式进行传播。为进一步证实蜜蜂螺原体可以通过水平传播方式在蜜蜂和植物花间进行传播,本研究采用人工接种法分别接种螺原体到蜜蜂和植物花上,对其传播途径进行了探索。结果显示：体内含菌的蜜蜂可以通过采食花蜜的方式将螺原体传播到植物花表面,而花表的螺原体又可以通过蜜蜂的采食传播到蜜蜂体内以及其它植物花的表面,传播到蜜蜂体内的致病性菌株可以引起蜜蜂“爬蜂病”的症状,而植物无任何病状。该结果为研究蜜蜂螺原体在自然界的传播途径提供了直接的证据。通过对分离自同一时期相同地点不同宿主(患病蜜蜂、健康蜜蜂、死蜂、打碗花、楝树花、一年蓬、蓼科植物花)中的7株分离菌株的形态学、运动性、基本生物学特性、血清学及分子生物学特性的研究及比较,发现7株菌的特性均符合Spiroplasma属的描述。其中,分离菌株MF1006和LK1001的生长速度最快,倍增时间分别为1.8h和2.4h；MF1008生长最慢,倍增时间为7.8h。菌株MF1006、YNP1001和LK1001在37℃均不能生长,最适温度比其余4株菌略低。代谢抑制试验、菌体变形试验和ELISA试验的结果均一致表明：螺原体S. melliferum CH-1的抗血清对菌株MF1006、YNP1001、LK1001几乎没有抑制作用,对其它4株菌的生长抑制作用却较强；ZHUF0901和MF0905的抗血清则分别与MF1006、YNP1001和LK1001发生较强的反应。根据16SrDNA和ITS序列构建的系统发育树显示：MF1006、YNP1001与Spiroplasma apis聚为一类；LK1001与Spiroplasma clarkii聚为一类；其它4株菌则都与S. melliferum聚类。以上结果表明同一时期同一地区的蜜蜂和植物花样本中所含的螺原体均不止一种；不同宿主中的螺原体均分别与S. apis和S. melliferum聚类,进一步证实螺原体在自然界中可通过水平传播方式进行传播。综上,提出了蜜蜂螺原体在自然界中的传播途径：(1)在蜜蜂螺原体病暴发时期(一般为春季4、5月),蜜蜂螺原体由含菌蜜蜂体内传播到蜂箱内蜜蜂经常活动的场所或蜂箱外界的植物花表面,健康蜜蜂或其它昆虫在含菌的处所和植物花表面活动后可将其携带或感染的螺原体传播到蜂箱内别处或其它的植物花表面,为其它蜜蜂和昆虫的再次感染提供侵染原；(2)在非发病时期,蜂场几乎没有爬蜂,蜜蜂螺原体检测到的几率也极低,推测此时螺原体不易穿过蜜蜂的中肠屏障到达淋巴组织大量繁殖而引起蜜蜂死亡,少量的螺原体在蜜蜂体内可能会长期生存或经消化道排泄在蜂箱内或周围后感染其它健康蜜蜂,当天气变化或其它因素致使蜜蜂抵抗力下降时,螺原体便可穿过蜜蜂中肠屏障导致蜜蜂的死亡,这可能也是在非发病季节蜂场也偶尔出现少量爬蜂的缘故。此外,通过饲喂新鲜菌液的方法对两株与引起蜜蜂“五月病”的S. apis聚类的螺原体MF1006、YNP1001以及一株与引起蜜蜂“螺原体死亡病”的S. melliferum聚类的螺原体MF1008的致病性进行研究,发现感染供试螺原体菌株的意蜂(Apis mellifera)在第5d时均开始出现“爬蜂”症状,但在相同的实验条件下,感染MF1006及YNP1001菌液的蜜蜂的发病速度较快。到第9d时,感染供试螺原体菌液的实验组意蜂的死亡率均明显高于对照组的意蜂(饲喂新鲜培养基),与对照组意蜂相比差异均极显著(1%水平)。从实验组及阳性对照组死亡的蜜蜂中均能分离到螺原体,在培养基对照组的死蜂内则未能分出。通过对死蜂内再分离物及饲喂的螺原体菌株的16S rDNA序列作比对,发现再分离菌株均与原菌株同源性最高,说明螺原体MF1006、MF1008. YNP1001确实是意蜂死亡的病因。菌株MF1006和YNP1001的发现丰富了对我国蜜蜂“爬蜂病”病原的认识,这是在我国蜜蜂和植物花上首次发现的除S. melliferum以外的另一种对蜜蜂致病的螺原体。最后,利用分子生物学检测方法在患病蜜蜂的中肠、淋巴液和胸部肌肉中均检测到螺原体,健康蜜蜂内则没有。应用透射电子显微镜对S. melliferum CH-1在意蜂的中肠和胸部肌肉中的分布、侵染机制及由其引起的宿主细胞的病理变化进行研究,发现螺原体通常大量地聚集在膜包裹的细胞质囊泡内,分布于中肠上皮细胞顶端和内部的核附近、基底面的质膜与基板之间、基板内部及胸部肌肉细胞内。相对于健康蜜蜂肌肉细胞内整齐排列的纤维束,被螺原体侵染的纤维束表现出明显的断裂、松散排列。这些症状都可能是导致“爬蜂”及蜜蜂死亡的原因。更多还原

【Abstract】 Spiroplasmas isolated from honeybees are characterized by helical, motile and lack of cell wall. They usually parasitize in the young bees and are one of the main pathogens which caused "crawling bee disease" in China. Since the1980s, spiroplasmosis has spread widely all over the country, and often occurs mixed with sporidiosis, palsy disease and so on. It has caused severe economic losses to the beekeeping. It is reported that spiroplasma could be detected in honeybees, plant flowers and other insects such as Hymenoptera, Diptera, Lepidoptera in the onset of spiroplasmosis, but fail to be detected in the non epidemic period, especially in winter and hot summer. Based on the findings, researchers have speculated several infection cycle ways of honeybee spiroplasmas in nature, but still lack of systematic research and direct evidence. In this research, a rapid molecular detecting method was developed and used for the detection of Spiroplasma melliferum which caused "crawling bee disease" in our country. Its host range, possible routes of transmission was systematicly studied. Basic biological characteristics and pathogenicity of spiroplasmas isolated from different hosts during the same period were compared. In addition, we preliminarily studied the location and infection characteristics of S. melliferum in midgut and chest muscles of the honeybee Apis mellifera.A molecular detecting approach, based on the Chelex-100chelating resin DNA extraction method was developed and used for the detection of spiroplasma in plant flowers, honrybees and their living environment. The detection was carried out in2-4hours and its minimum detectable concentration was5-6spiroplasmas per mL. Compared with conventional isolation culture, this method was more sensitive, rapid and efficient. This laid the foundation for the rapid diagnosis of spiroplasmosis and the resource survey of spiroplasmas.In order to explore the possible routes of transmission of honeybee spiroplasmas in nature, we detected honeybees, flowers, other insects and the living environment of honeybees regularly by using molecular detection and isolation culture methods. A total of1339honeybees,131kinds of insects,51kinds of plant flowers and77samples from beehives were collected from March2010to January2012, from which92samples were detected to contain spiroplasmas, and54spiroplasma isolates were obtained totally. Spiroplasmas can be detected in honeybees throughout the year (including the epidemic period and non epidemic period). The detection rate of diseased honeybees collected in spring and the epidemic period was highest. In addition, spiroplasmas were detected in honeybee larvae, pupae, plant flowers, other insects, hive, nest door and honeycomb which were collected in the epidemic period while spiroplasmas only found in few honeybees in the non epidemic period. It was thought that spiroplasmas persisted in honeybees and were transmitted by horizontal transmission in nature.To further confirm spiroplasmas could spread through horizontal transmission between honeybees and plant flowers, we explored its routes of transmission though artificial inoculation of spiroplasmas to honeybees and plant flowers, respectively. The results showed that spiroplasmas in honeybees could spread from honeybees to flower surface, and the spiroplasmas in flower surface aslo could spread to the honeybees and other plant flowers though feeding of honeybees. The honeybee infected by spiroplasma showed symptoms of "crawling bee disease", and plant flowers did not show any symptoms. This result provided direct evidence to study the route of transmission of spiroplasmas in nature.Morphology, motility, basic biological characteristics, serological and molecular biological characteristics of seven spiroplasma isolates isolated from different hosts (diseased Apis mellifera, healthy A. mellifera, dead A. mellifera, Calystegia hederacea, Melia azedarach, Erigeron annuus, Polygonaceae) for the same period and the same location were studied and compared. The result showed that all traits above of7spiroplasma isolates accorded completely with the descriptions of genus Spiroplasma. The growth velocity of isolates MF1006and LK1001was the fastest, their doubling time were1.8h and2.4h, respectively. MF1008was the slowest, its doubling time was7.8h. Isolates MF1006, YNP1001and LK1001could not grow at37℃, the optimum temperature was slightly lower than the remaining four strains. The results of metabolic inhibition test, deformation test and ELISA were consistent. S. melliferum CH-1antiserum could not inhibit MF1006, YNP1001, LK1001. But it showed much stronger reaction with the other four strains and could inhibit the growth of the strains. ZHUF0901antiserum and MF0905antiserum showed strong reaction with isolates MF1006, YNP1001and LK1001, respectively. Phylogenetic analysis based on the16S rDNA and ITS revealed that MF1006and YNP1001had a close relationship with Spiroplasma apis, and LK1001was close to Spiroplasma clarkii. Other four strains were all close to S. melliferum. These results suggest that the honeybees and flowers of the same area during the same period contained more than one kind of spiroplasma. The relationship of spiroplasmas isolated from different hosts was close, which further confirmed that spiroplasmas spread through horizontal transmission in nature.In summary, we proposed the route of transmission of honeybee spiroplasmas in nature. First, in the epidemic period (usually in April and May for the spring), spiroplasmas were spread from honeybees to the living eviroment or plant flower surface outside the beehives. Healthy honeybee or other insects were infected by spiroplasmas during feeding in the spiroplasma-infected places or flowers, and then transported spiroplasmas to other places within the beehives or other flower surface. This aslo provided the pathogens for the re-infection of other honeybees and insects. Second, in the non epidemic period, there was few crawling bees could be seen and the detection rate of spiroplasma was aslo very low. We speculated that it was difficult for spiroplasmas to traverse the midgut barrier to enter the hemolymph, and then multiply in the hemolymph and result in the death of honeybees at this period. A few spiroplasmas may be existed in honeybees for a long time or infect other healthy honeybees though gastrointestinal excretion. With the weather changed or other factors resulted in the honeybee resistance decline, spiroplasmas would pass through the midgut barrier, and then cause the death of honeybees. This may be the possible reason why few crawling bees occasionally appeared in the apiary during the non epidemic period.In addition, the pathogenicity of isolates MF1006, YNP1001, MF1008to Apis mellifera was investigated by feeding spiroplasma cultures. The spiroplasma-infected honeybees showed symptoms of spiroplasmosis around5days after feeding. At the same experimental conditions, the incidence speed of honeybees infected MF1006and YNP1001was faster. After9days, the mortality rates of honeybees fed by spiroplasmas were significantly higher than those fed with fresh medium. Compared with the honeybees in the control group, the honeybees in the experimental group were very significant at1%significance level. Spiroplasmas could be isolated from dead bees in the experimental group and positive control group, but could not be in the control group with fresh medium.16S rDNA sequences comparison showed that the homology of re-isolates and the feeding spiroplasma strains were the highest. It is aslo indicated that isolates MF1006, MF1008, YNP1001were indeed the cause of honeybee death. The discovery of pathogenic strains MF1006and YNP1001enriched the understanding of the pathogen of "crawling bee disease" in China. This was the second pathogenic spiroplasma to honeybees discovered in China. Finally, spiroplasmas were detected in the midgut, lymph and chest muscles of diseased honeybees by using molecular biology methods, but no one was detected in healthy bees. S. melliferum CH-1distribution, infection mechanisms and cytopathological effects in the intestines and chest muscles of Apis mellifera were investigated by transmission electron microscopy. Spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the nuclear at the top and internal of intestinal epithelial cells, between the plasma membrane and basal lamina at the basal part. In addtion, spiroplasmas aslo found in the basal lamina and accumulated at high numbers in chest muscle cells. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. These symptoms were likely to be the reasons leading to the "crawling bee" and the death of honeybees.更多还原