A comparison of the effectiveness of the microscopic method and the multiplex PCR method in identifying and discriminating the species of Nosema spp. spores in worker bees (Apis mellifera) from winter hive debris

The objective of this study was to compare the effectiveness of the multiplex PCR method and traditional light microscopy in identifying and discriminating the species of Nosema spp. spores in worker bees from winter hive debris in the Province of Warmia and Mazury (NE Poland). A total of 1000 beesdead after from the bottom of the hive from bee colonies were analyzed. Spores were identified with the use of a light microscope (400-600x magnification). Spores were assigned to species by the multiplex PCR method. The microscopic evaluation revealed the presence of Nosema spp. spores in 803 samples (80.3%). Nosema ceranae spores were observed in 353 positive samples (43.96%), Nosema apis spores were found in 300 samples (37.35%), while 150 samples (19.67%) showed signs of a mixed infection. A multiplex PCR analysis revealed that 806 samples were infested with Nosema spp., of which 206 were affected only by Nosema ceranae, 600 showed signs of mixed invasion, while no samples were infected solely by Nosema apis parasites. In two cases, the presence of spores detected under a light microscope was not confirmed by the PCR analysis. The results of the study indicate that Nosema ceranae is the predominant parasitic species found in post-winter worker bees from the bottom of the hive in the region of Warmia and Mazury.     

Nosema spp. infection and its negative effects on honey bees (Apis mellifera iberiensis) at the colony level

Nosemosis caused by the microsporidia Nosema apis and Nosema ceranae are among the most common pathologies affecting adult honey bees. N. apis infection has been associated with a reduced lifespan of infected bees and increased winter mortality, and its negative impact on colony strength and productivity has been described in several studies. By contrast, when the effects of nosemosis type C, caused by N. ceranae infection, have been analysed at the colony level, these studies have largely focused on collapse as a response to infection without addressing the potential sub-clinical effects on colony strength and productivity. Given the spread and prevalence of N. ceranae worldwide, we set out here to characterize the sub-clinical and clinical signs of N. ceranae infection on colony strength and productivity. We evaluated the evolution of 50 honey bee colonies naturally infected by Nosema (mainly N. ceranae) over a one year period. Under our experimental conditions, N. ceranae infection was highly pathogenic for honey bee colonies, producing significant reductions in colony size, brood rearing and honey production. These deleterious effects at the colony level may affect beekeeping profitability and have serious consequences on pollination. Further research is necessary to identify possible treatments or beekeeping techniques that will limit the rapid spread of this dangerous emerging disease.     

Occurrence of acute paralysis virus of the honey bee (Apis mellifera) in a Hungarian apiary infested with the parasitic mite Varroa jacobsoni.

Viruses of the honey bee have been known for a long time; however, recently the attention of scientists and apiculturalists has turned towards the relationship between these viruses and the parasitic mite Varroa jacobsoni. Although clinical symptoms indicated the presence of some of the viruses of bees in Hungary, none have previously been isolated or identified. During July unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested with Varroa jacobsoni. Large amounts of acute paralysis virus (APV) were detected serologically in healthy honey bee pupae killed by the injection of a bacteria-free extract of diseased adult bees. Crystalline arrays of 30 nm particles were seen in ultrathin sections of the tissues of injected pupae and naturally infected adult bees. In spite of the application of acaricide treatments the bee population in several colonies had collapsed by the end of summer and the apiary suffered severe wintering losses.     

东方蜜蜂微孢子虫对蜜蜂健康的影响

自1996年首次报道以来,东方蜜蜂微孢子虫(Nosema ceranae)已被证实可以影响蜜蜂个体的行为、定向、飞行和寿命等,使其成为蜜蜂健康与保护领域的研究热点.本文整理了近10年N.ceranae的研究情况,从N.ceranae的传播途径、对蜜蜂健康的影响、与其他病原的相互作用进行综述,提出目前N.ceranae研...     

在工蜂产卵超过23天的中蜂群中介绍蜂王

在中蜂的饲养管理中,蜂群失王且工蜂产卵后,一般处理方法都是并入它群;实验是在不得已的情况下,将3群失王且工蜂产卵超过23 d的中蜂群合并的同时,介绍蜂王获得成功.     

中蜂生产野桂花蜂蜜优质高产技术研究

野桂花是一种特色蜜源,其蜂蜜色、味俱佳,被称为"蜜中之王"。但采野桂花蜂蜜的蜂群产量不高、质量不稳定。根据中蜂生物学特性,改良设计了一种中蜂蜂箱,同时开展蜂群采野桂花蜂蜜优质高产饲养技术的研究工作。结果表明:实验组蜂群繁殖速度、野桂花蜂蜜产量都显著高于对照组,但2组生产的野桂花蜂蜜浓度差异不显著。     

First detection of Kashmir bee virus in Hesse, Germany

We gathered dead bees of 56 Hessian bee colonies following a sudden collapse during winter 2002/03. Viral RNA was purified from ten dead bees per sample. Kashmir bee virus (KBV) was detected by use of a RT-PCR protocol. 13 samples were positive for KBV. The PCR amplicon was sequenced. A BLAST GenBank search clearly identified the Hessian amplicon as a KBV fragment. Similarities of more than 85% were found. Phylogenetic analysis revealed a close genetic relationship of the Hessian isolate to an isolate from New Zealand. The Northamerican, the Russian and Australian notations listed in GenBank did not cluster round with the Hessian isolate. This is the first documented detection of KBV in Middle Europe.     

Vertical transmission of American foulbrood (Paenibacillus larvae) in honey bees (Apis mellifera)

The mode of transmission between hosts (horizontal versus vertical) of disease agents is important for determination of the evolution of virulence in pathogens. For disease management, it is imperative that the epidemiology of the disease is understood and pathogen transmission rates between hosts is a key factor for this understanding. Surprisingly little is known about transmission rates in honey bee pathology. We have studied the rate of vertical transmission of Paenibacillus larvae, the causative agent of American foulbrood (AFB) in honey bee colonies, as colonies reproduce by colony fission (swarming), by culturing for the spores from repetitive samples of adult bees. The results demonstrate vertical pathogen transmission to daughter swarms. The spore density declines over time in both mother colonies and daughter swarms if mother colonies do not exhibit clinical disease symptoms. Occasional positive samples more than a year post swarming, also in daughter swarms, indicate production of infectious spores from diseased larvae, without clinical disease observable by beekeepers, and/or maintenance of infective spores in the hive environment, allowing both horizontal and vertical transmission to be maintained. The results suggest that the virulence of AFB, being lethal at colony level in contrast to other bee diseases shaped by evolution, could be dependent on apicultural practices and that the pathogen probably would be maintained without causing frequent colony mortality in a natural system.     

不同分蜂方法的繁蜂效果研究

为了弄清西方蜜蜂在不同方法下进行分蜂后的繁蜂效果和生产性能,对2011年石榴花期前1个月左右已达11脾的蜂群,采取异地和本地分蜂两种方法进行人工分蜂,并对两种分蜂方法的蜂群增殖情况和蜂蜜产量进行了分析。结果表明,第1种分蜂方法优于第2种分蜂方法。第1种分蜂方法分出的蜂繁殖快、进蜜好,是一种理想的分蜂方法。     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

Development of infestation with Varroa jacobsoni O. in bee colonies in Tunisia

The mite Varroa jacobsoni, an ectoparasite of the honey bee, was imported to Tunisia probably in 1976. Afterwards, this parasitosis caused severe losses of colonies for several years. The continued examination of the level of infestation in colonies of a "GTZ" project stated a steady number of mites since 1980. Only in a few colonies, the infestation was above the limit of damage. Though the colonies in North West Tunisia did not receive any treatment since 1986 there was no increase of infestation. In order to investigate the reason for this the mites' ability of reproduction was examined during two following years. The portion of infertile female mites in the worker brood in most of the colonies was with 50% considerably higher than in Europe. In Brazil, the adaptation between host and mite produced similar low reproduction rates. As, however, in Tunisia the portion of infertile females in the drone brood of the individual colonies corresponded to the one in the worker brood climatic conditions are supposed to be responsible.     

优良浆意蜂DNA分子特异标记(W316bp)的鉴定研究

第一部分分离提取三品系意蜂 (浙农大、萧山、平湖 )的DNA ,使用紫外分光光度计 ,波长 2 6 0nm鉴定DNA纯度 ;第二部分以W (5′ -CGGCCCCGGT - 3′)为随机引物进行PCR ,扩增 ,电泳 ,获得DNA多态性图谱 ;第三部分比较三品系意蜂工蜂与雄蜂的DNA多态性图谱。结果凡是三品系意蜂的工蜂均出现一高产浆特异标记W316pb条带 ,但三品系意蜂的雄蜂均无W316bp条带。则进一步鉴定了W316bp是高产浆意工蜂DNA分子的特异标记     

分子技术在蜜蜂白垩病病原鉴定和诊断上的应用

蜜蜂白垩病是一种由球囊菌属（Ascosphaera）的真菌寄生而引起蜜蜂幼虫死亡的传染性疾病，对蜂群的群势发展影响极大，近几年又呈现出大面积爆发的趋势。但目前对于该病的诊断和病原鉴定多依赖于典型症状，方法极不可靠。本文介绍了可用于分子鉴定和诊断的的研究基础，探讨利用球囊菌属的各病原rDNA的ITS1—5．8S-ITS2序列资料，分别设计出高特异性引物，用PCR技术扩增出可用于鉴定病原物种的分子标记，从而构建一套快速、准确诊断该病的分子技术。     

用改造式蜂箱饲养中蜂的研究报告

通过通过箱体、实行中蜂贮蜜与繁殖分区管理，解决流蜜期繁殖与贮蜜的矛盾，发挥蜂群繁殖与采集的积极怀，增加蜂蜜的产量，获得纯正的成熟蜂蜜。三个阶段的试验结果表明：中蜂改造式蜂箱与中蜂标准箱、即氏蜂箱相比每群平均多增蜂１．０框，多造牌０．３和，越冬蜂损失少０．２框，提高蜂蜜产量３０％以上，增加浓度１－２Ｂｅ，中囊病病情指数低１３．２％。     

人工饲粮对幼年工蜂生长发育及生理代谢的影响

本试验利用液相色谱-质谱联用技术(LC-MS)对幼年工蜂进行代谢组学分析,旨在探究不同人工饲粮对幼年工蜂生长发育及生理代谢的影响,为蜂王邮寄的人工饲粮配制提供一定理论依据。试验选取1日龄的意大利蜜蜂(Apis mellifera ligustica)300只,分为5组,分别饲喂5种人工饲粮,每组30只工蜂。对照组饲粮:蔗糖粉∶蜂蜜=3∶1;试验组A饲粮:蔗糖粉∶蜂粮∶蜂蜜=6∶5∶1;试验组B饲粮:蔗糖粉∶蜂粮∶蜂蜜=6∶3∶1;试验组C饲粮:蔗糖粉∶花粉∶蜂蜜=6∶5∶1;试验组D饲粮:蔗糖粉∶花粉∶蜂蜜=6∶3∶1。用5种人工饲粮进行室内喂养,记录蜜蜂7 d内的死亡数量;解剖试验组A和对照组第1、3、5、7天的工蜂咽下腺,测定其平均重量、颜色和饱满度;采用LC-MS方法检测试验组A和对照组蜜蜂饲喂7 d后的代谢物差异,对检测数据进行模式识别分析,筛选并鉴别差异代谢物。结果表明:1)对照组和试验组A蜜蜂第1~5天时均没有出现死亡,在第6和7天时试验组A蜜蜂死亡数量均显著低于对照组(P<0.05)。试验组B、C、D的蜜蜂在6 d内全部死亡。2)试验组A第7天时的蜜蜂咽下腺平均重量显著高于对照组(P<0.05),咽下腺颜色为全部乳白色,且咽下腺小体饱满。3)采用主成分分析(PCA)、偏最小二乘法判别方法(PLS-DA)和正交偏最小二乘方-判别分析(OPLS-DA)分析代谢组数据,结果显示试验组A和对照组明显分离,共鉴定出23个差异代谢物,包括氨基酸、脂类、糖类等,其中有10种代谢物上调,13种代谢物下调。差异代谢物通路分析发现,差异极显著的通路有牛磺酸和亚牛磺酸生物合成通路、赖氨酸降解通路、戊糖磷酸盐代谢通路、戊糖和葡萄糖醛酸相互转化通路(P<0.01),差异显著的通路有氨基酸的生物合成通路,色氨酸代谢通路,碳代谢通路,丁酸代谢通路,丙氨酸、天冬氨酸和谷氨酸代谢通路,丙酮酸代谢通路(P<0.05)。由此可见,试验组A的饲粮更适合蜜蜂工蜂的生长发育和生存,在邮寄环境中可代替常用的炼糖饲粮。     

Disposition of mirosamicin in honeybee hives

Disposition of mirosamicin, a macrolide antibiotic, to honeybee adults, larvae, honey and royal jelly in the beehive after in-feed administration to adult bees was studied. Treatment was initiated at the end of July when the availability of natural pollen and nectar was poor. The drug was mixed with pollen-substitute paste and administered to honeybee colonies continuously for a week at a dossage of 200 mg/hive/week. High distributions in adult bees, jelly, larvae and a relatively low distribution in honey, of mirosamicin were observed. One day dosing of microsamin in sucrose syrup, a nectar substitute, resulted in a very high and long lasting residue in honey. Both royal and worker jelly, secreted from the jelly glands of adult bees, are acidic, so that a high distribution of a basic drug, such as mirosamicin, in jelly can be expected. This mechanism was considered to be responsible for a high concentration of mirosamicin in honeybee larvae, the host of Paenibacillus-larvae infection (American foulbrood), as primary larval food is jelly.     

Antimicrobials in beekeeping

The bee diseases American and European foulbrood and nosemosis can be treated with anti-infectious agents. However, in the EU and the USA the use of these agents in beekeeping is strictly regulated due to the lack of tolerance (e.g. Maximum Residue Limit) for residues of antibiotics and chemotherapeutics in honey. This article reviews the literature dealing with antimicrobials of interest in apiculture, stability of these antimicrobials in honey, and disposition of the antimicrobials in honeybee hives.     

氟氯苯氰菊酯条蜂场休药期规定的验证研究

将治螨药物氟氯苯氰菊酯条应用于蜂场,6周后抽取蜂蜜样品检测其残留状况。结果所有样品均未发现残留超标情况,验证了2005版《兽药使用指南》中关于氟氯苯氰菊酯条的休药期规定。     

Emerging and re-emerging viruses of the honey bee (Apis mellifera L.)

Until the late 1980s, specific viral infections of the honey bee were generally considered harmless in all countries. Then, with the worldwide introduction of the ectoparasite mite Varroa destructor, beekeepers encountered increasing difficulties in maintaining their colonies. Epidemiological surveys and laboratory experiments have demonstrated that the newly acquired virulence of several viruses belonging to the family Dicistroviridae (acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus) in Europe and the USA had been observed in relation with V. destructor acting as a disseminator of these viruses between and within bee colonies and as an activator of virus multiplication in the infected individuals: bee larvae and adults. Equal emphasis is given to deformed wing virus (DWV) belonging to the Iflaviridae. Overt outbreaks of DWV infections have been shown to be linked to the ability of V. destructor to act not only as a mechanical vector of DWV but also as a biological vector. Its replication in mites prior to its vectoring into pupae seemed to be necessary and sufficient for the induction of a overt infection in pupae developing in non-viable bees with deformed wings. DWV in V. destructor infested colonies is now considered as one of the key players of the final collapse. Various approaches for combating bee viral diseases are described: they include selection of tolerant bees, RNA interference and prevention of new pathogen introduction. None of these approaches are expected to lead to enhanced bee-health in the short term.     

Levels of mercury in samples of bees and honey from areas with and without industrial contamination]

Increasing numbers of specialists have been concerned with the problem of friendly environment in relation to man as well as to farm and wild animals. Greater interest in the biological monitoring of environment and ecosystem contamination can be observed. Determination of residues of organic and inorganic substances in bees (Apis mellifera) and in their products is one of effective possibilities of environmental pollution monitoring. Our work was aimed at the study of mercury levels in bees and their products. Mercury levels were determined in the head, abdomen and thorax of bees (Apis mellifera) from 20 bee populations coming from industrially contaminated areas with a dominant load of mercury (10 populations) and from uncontaminated areas. Mercury levels were determined simultaneously in honey coming from both contaminated and uncontaminated areas. The following mercury levels were found in bees from the contaminated area: heads 0.029-0.385 mg/kg, thorax 0.028-0.595 mg/kg and abdomen 0.083-2.255 mg/kg. Mercury levels in samples from uncontaminated areas ranged from 0.004 to 0.024 mg/kg in the heads, from 0.004 to 0.008 mg/kg in the thorax and from 0.008 to 0.020 mg/kg in the abdomen. In honey samples from the contaminated and uncontaminated areas mercury levels ranged from 0.050 to 0.212 mg/kg and from 0.001 to 0.003 mg/kg, respectively. The results of sample analyses for mercury loads in bees and honey from both contaminated and uncontaminated areas are given in Tab. I. Mean mercury levels in the single parts of the body in Apis mellifera and in honey from contaminated and uncontaminated areas are given in Figs. 1, 2, 3.(ABSTRACT TRUNCATED AT 250 WORDS)