蜜蜂球囊菌的分离鉴定及体外抑菌试验

蜜蜂白垩病在世界各地呈现稳定高发态势,为了找到防治该病的有效方法,对疑似白垩病虫体进行真菌分离鉴定及体外抑菌试验。用点种法对虫体进行真菌分离培养;用显微观察法对分离菌进行形态学鉴定;用PCR扩增法,对分离菌菌丝进行分子生物学鉴定;用PDA液体试管培养法,做枯草芽孢杆菌抑制分离菌菌丝试验;用PDA平板培养法,做枯草芽孢杆菌抑制分离菌菌斑生长及孢子萌发试验。结果得到4株分离株;分离菌菌丝直径、孢子球直径、孢子长宽比均与蜜蜂球囊菌的大小一致;分离菌约580bp处出现目的条带,16S r DNA基因序列同源性与蜜蜂球囊菌最高;在PDA液体试管中,分离菌菌丝停止生长;在PDA平板上,分离菌菌斑逐渐萎缩消失;在PDA平板上,分离菌孢子不萌发。结论分离菌为蜜蜂球囊菌;枯草芽胞杆菌可抑制蜜蜂球囊菌生长,可用于蜜蜂白垩病的防治。     

蜜蜂白垩病病原的分离与鉴定

为了解伊犁河谷蜜蜂白垩病的流行情况及研究快速诊断方法,本试验开展了该病的流行病学调查,并将50份疑似样品处理后采用分离培养法、镜检法及PCR法对病原菌进行了分离与鉴定。结果5份为阳性,阳性率10%,培养基菌落颜色呈白色,较致密,中央凸起,老化菌落颜色呈浅棕色至棕色;镜检可见树枝状分枝的有隔菌丝,二叉分枝间距较大,菌丝体呈疏松的蛛网状,用乳酸棉监染色液染色后菌丝为蓝色;PCR反应结果为阳性,阳性结果测序后所得序列用Blast软件比对,与球囊菌属的蜜蜂球囊菌基因序列相似性最高,达到99%。上述结果表明,从伊犁河谷病蜂中分离的病原菌为蜜蜂白垩病病原。     

蜜蜂白垩病PCR快速检测方法的建立及初步应用

根据Genebank网站公布的蜜蜂球囊菌ITSl.5.8s,ITS2 rDNA保守序列(U68313)设计出一对特异性引物ASCU和ASCD,可特异性的扩增蜜蜂球囊菌ITSI,5.8srRNA,ITS2序列上大小为471 bp的片段.优化了PCR最佳反应条件和体系;特异性试验结果表明与蜜蜂幼虫芽孢杆菌、蜂房蜜蜂球菌、蜜蜂败血杆菌无交叉反应,具有良好的特异性.敏感性试验结果表明敏感性可达7CFU/mL的蜜蜂球囊菌.通过将定量的蜜蜂球囊菌添加到蜂蜜、花粉、蜂胶和蜂王浆等蜂产品中进行模拟检疫,结果显示所建立的PCR检测方法适用于上述蜂产品中蜜蜂球囊菌的检疫.该方法与病原菌分离培养等传统检疫方法相比较,具有快速、灵敏、特异等优点,可用于蜜蜂及蜂产品中白垩病的进出口检疫及快速诊断.     

蜜蜂球囊菌检测分子标记的灵敏性测定

蜜蜂球囊菌(Ascosphaera apis)是蜜蜂白垩病的重要病原物,传统的鉴定方法是以形态学、生物学、培养条件等方面的特征为基础,这些特征差异并不明显,给以形态学特征来鉴定Ascosphaera属下各种的工作带来不可逾越的困难。A.apis检测分子标记可以用于准确检测该病原物。本研究利用该检测分子标记,结合PCR技术,分别检测不同浓度的A.apisDNA溶液,以测定该检测分子标记的灵敏性。试验结果表明,A.apis检测分子标记的最低检测DNA模板浓度为1.74×10-7ng/μl,即在25μl的PCR反应体系中加入4.34×10-6ng的A.apis的DNA样品,检测结果即可呈阳性,初步测定该检测分子标记具有较高的灵敏性。本研究为下一步利用该分子标记构建一套以PCR为基础的快速、可靠的检测方法以及应用于蜜蜂白垩病的快速诊断奠定基础。     

蜜蜂白垩病发病因素的分析与研究

蜜蜂白垩病是西方蜜蜂的恶性传染病。该病的病原为蜂球囊菌（Ａｓｃｏｓｐｈａｅｒａａｐｉｓ）,主要以该菌的孢子形式传播,这种孢子的抗逆性强。通过调查与对比试验观察,总结并研究出蜂王、环境因素、饲养管理措施、雄蜂等为蜜蜂白垩病的发病因素,为饲养管理与药物防治措施的制订提供了科学依据。     

蜜蜂白垩病在宁波地区发生情况的初步调查及其防治新技术

蜜蜂白垩病(chalk-brood),又名石灰质病,是蜜蜂幼虫的一种顽固性传染病。最早于1955年在美国鉴定出它的致病微生物是蜂球囊菌(Ascosphaera apis)。以后,该病传播到欧洲、苏联、新西兰等国。1979年开始在北美州蔓延为害,到了80年代,白     

不同辐照剂量对蜂球囊菌杀灭效果的影响

本试验以蜂球囊菌(Ascosphaera apis)为研究对象,探究不同辐照剂量对蜂球囊菌的杀灭效果,以期明确有效杀灭蜂球囊菌的最低辐照剂量,为Co60γ射线辐照蜜蜂饲料(特别是蜂花粉)时的辐照剂量选择提供依据。利用细菌纯化技术从患白垩病意蜂幼虫体内分离纯化得到蜂球囊菌,并结合形态学、乳酸酚棉兰染色及5.8SrDNA序列分析技术进行鉴定;同时制备不同梯度浓度的蜂球囊菌孢子悬液加入到蜜蜂幼虫饲料中,以饲喂方式侵染3日龄意蜂幼虫,确定半数致死浓度(LC50);蜜蜂幼虫饲料中添加上述确定LC50的孢子,以不同辐照剂量处理,并通过侵染幼虫试验,确定有效杀灭蜜蜂饲料中孢子的最低辐照剂量。结果表明,通过形态学和分子生物学鉴定,从患白垩病的意蜂幼虫体内分离纯化得到真菌即为蜜蜂白垩病的病原——蜂球囊菌;蜂球囊菌对人工饲养条件下的意蜂幼虫的LC50为9.5×104个/mL;当Co60γ射线辐照剂量为7.0kGy时,幼虫患病率与未辐照组差异显著(P0.05),由此确定对添加半数致死浓度的蜂球囊菌孢子饲料的最低有效辐照剂量为7.0kGy。     

蜜蜂白垩病综合防制

正1病原导致蜜蜂白垩病的蜜蜂球囊菌属真菌界较低等的原始菌种,蜜蜂球囊菌菌丝的生长和子囊孢子的形成与其他低等菌类无异,其成熟的子囊孢子囊在条件适合的环境下破壁长出菌丝体,菌丝体的营养生长期由有丝分裂来完成。蜜蜂幼虫体表的充分暴露和大量进食使它     

蜜蜂白垩病综合防治初探

蜜蜂白垩病多年来对蜂群的危害相当严重 ,威胁着养蜂业的发展 ,因此 ,对该病的防治非常重要。1　白垩病的病原蜜蜂白垩病原主要是蜂球囊菌 (Ａｓ ｃｏｓｐｈａｅｒａａｐｉｓ)所引起。蜂球囊菌的实体里面有许多孢囊 ,孢囊内充满大小的孢囊孢子 ,孢子的抗逆性很强 ,在适宜条件下孢囊萌发雌、雄菌丝。雌菌丝形成藏卵器并与雄菌丝生长形成藏精器结合 ,再形成膨大球形的子囊 ,子囊具有很强的生命力 ,在干燥状态下 ,存活时间很长。蜜蜂白垩病主要是通过孢囊孢子和子囊孢子传播。患病幼虫、病死幼虫的尸体 ,以及被污染的饲料 ,蜂具等都是该病的传…     

蜜蜂白垩病发病特点及其防治

蜜蜂白垩病是真菌蜜蜂球囊菌(Ascophaera apis)引起的,容易感染西方蜜蜂的幼虫,成年蜂虽不易感染,但是病原的携带者和传播者。1991年我     

患白垩病蜂群的温湿度研究

以蜜蜂白垩病致病菌蜜蜂球囊菌(Ascosphaera apis)感染西方蜜蜂(Apis mellifera).通过记录染病蜂群的温湿度和发病幼虫数,研究了养蜂生产中,温湿度变化对感染该病的西方蜜蜂蜂群(中等群势)的影响.结果显示:当蜂群内的温度降低到某个阈值范围时,不论是试验组还是对照组都会出现不同程度地增加发病幼虫数.     

Spores of Ascosphaera apis contained in wax foundation can infect honeybee brood

Chalkbrood disease in honeybees (Apis mellifera L.) is caused by an infection with Ascosphaera apis. Disease expression requires the consumption of fungal spores and a predisposing condition in the susceptible brood. A. apis spores within sheets of wax foundation could be a source of inoculum leading to chalkbrood, but it is also possible that these spores remain confined in the wax and do not contribute to disease. We have resolved this topic by chilling susceptible brood within wax combs built on contaminated foundation (using treatments of spores from 1 mummy and spores from 10 mummies) versus uncontaminated foundation. We found significantly higher levels of chalkbrood in brood exposed to the higher dosage. Our results demonstrate that foundation wax contaminated with spores of A. apis spores may be a source of chalkbrood in honeybee colonies.     

Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood

Chalkbrood (Ascosphaera apis) and stonebrood (Aspergillus flavus) are well known fungal brood diseases of honeybees (Apis mellifera), but they have hardly been systematically studied because the difficulty of rearing larvae in vitro has precluded controlled experimentation. Chalkbrood is a chronic honeybee-specific disease that can persist in colonies for years, reducing both brood and honey production, whereas stonebrood is a rare facultative pathogen that also affects hosts other than honeybees and can likely survive outside insect hosts. Hive infection trials have indicated that accidental drops in comb temperature increase the prevalence of chalkbrood, but it has remained unclear whether virulence is directly temperature-dependent. We used a newly established in vitro rearing technique for honeybee larvae to test whether there are systematic temperature effects on mortality induced by controlled infections, and whether such effects differed between the two fungal pathogens. We found that increasing spore dosage at infection had a more dramatic effect on mortality from stonebrood compared to chalkbrood. In addition, a 24h cooling period after inoculation increased larval mortality from chalkbrood infection, whereas such a cooling period decreased mortality after stonebrood infection. These results raise interesting questions about honeybee defenses against obligate and facultative pathogens and about the extent to which stress factors in the host (dis)favor pathogens with lesser degrees of specialization.     

意大利蜜蜂不同蜂群抗白垩病能力差异的研究

白垩病是意大利蜜蜂（Apis mellifera ligustica,简称意蜂）的一种常见真菌性疾病,不同蜂群间的抗白垩病能力强弱差异检测是抗病育种的基础.研究通过实验比较分析意蜂不同蜂群抗白垩病能力的差异.采用DPS软件方差分析系统分组实验统计分析和Tukey法多重比较,对4群患病蜂群和4群健康蜂群自然状态和接种蜜蜂球囊菌病原状态下的3次检测结果分别进行统计分析.结果表明：自然情况下不同蜂群间的抗病能力有差异;1号蜂群在接种蜜蜂球囊菌前、后蜂群患病个体数均显著高于其他蜂群（p＜0.05）;用白垩病死尸粉末拌入花粉饲喂的方法可以有效诱发蜂群发生白垩病;实验筛选出抗白垩病能力强、弱的蜂群为抗白垩病相关分子标记提供素材.     

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.     

Coprodiagnosis of Hammondia heydorni in dogs by PCR based amplification of ITS 1 rRNA: differentiation from morphologically indistinguishable oocysts of Neospora caninum

Hammondia heydorni is thought to be a non-pathogenic coccidian parasite of dogs that is closely related to Neospora caninum, an important parasite of cattle and dogs. Oocysts of these two species are morphologically indistinguishable from each other. A population of 2240 dogs in the Czech Republic was screened for the presence of H. heydorni/N. caninum oocysts and five (0.22%), represented by five of 3135 faecal samples (0.16%), were positive. The internal transcribed spacer 1 region of the rRNA gene (ITS1) from two isolates were cloned and the DNA sequences were identical with those of the ITS1 of H. heydorni. Based on the rRNA sequences available for H. heydorni and related coccidia, the primer pair JS4-JS5 was designed to amplify the 3' end of the small subunit (SSU) rRNA gene and ITS1 of H. heydorni. When tested on DNA extracted from a variety of parasites, the primers amplified a specific 267 bp fragment in our isolates only. The presence of DNA equivalent to 10 oocysts was sufficient for the amplification of the ITS1. We present a PCR-based diagnostic method as the only fast and reliable method for the diagnosis of H. heydorni in dogs.     

Characterization of <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions

In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3’end of 18S and at the 5’start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.     

新型分子诊断技术在动物疫病检测中的应用与发展

快速有效的病原体检测对于动物疫病的防控和治疗有着非常重要的作用。近年来基于PCR扩增技术的新型分子诊断技术与传统的分子诊断技术相比,对病原体的检测更加快速、特异、敏感、准确,逐步向着分子检测的自动化、高通量和现地使用方向发展。日渐成熟的新型分子诊断技术对于动物疫病检测与鉴别以及出入境检验检疫等领域有着广阔的应用前景,同时可进行大范围的流行病学调查和分析,为动物疫病的科学防控奠定基础。