The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.     

Evaluation of the contamination degree of roe-deer with cadmium and polychlorinated biphenyls in Warmia and Mazury district

The results of investigations concerning degree the contamination degree with cadmium and polychlorinated biphenyls (PCB's) of roe-deer organs hunt in seven selected areas of Warmia and Mazury voivodship are presented. The highest mean cadmium content in the organs investigated was found in the samples from G?ebock area (liver - 0.0576 mg/kg, kidney - 3.351 mg/kg) and the lowest one was observed in the samples from Pisz area (liver - 0.181 mg/kg, kidneys - 1.653 mg/kg). Mean level of polychlorinated biphenyls residues in the roe-deer fat from Warmia and Mazury district was 0.004 mg/kg. The highest PCB's mean concentration was found in the roe-deer fat samples from Milomlyn area (0.008 mg/kg), and the lowest one in those from G?ebock area (0.002 mg/kg). Presented levels of investigated xenobiotics do not make at present (except cadmium in kidney) any toxicological threats for venison consumers in Warmia and Mazury district.     

家蚕微孢子虫(Nosema bombycis)侵染草地贪夜蛾卵巢细胞(Sf21)体系的建立

家蚕微孢子虫(Nosemabombycis,简称N.b)经KOH处理后,接种于草地贪夜蛾卵巢细胞(Sf21),建立家蚕微孢子感染增殖体系,同时,对孢子与宿主细胞在感染增殖过程中的形态变化进行了跟踪观察。KOH处理的孢子发芽率约为67%,Sf21细胞的初期感染率约为4.3%,接种24h内,细胞感染率变化不明显,但随后逐渐增加,到接种后的第7天达76.9%。接种后第12天起,细胞内充满不同发育阶段的孢子,并可见大量的胞外游动孢子。随后,大量细胞破裂,孢子逸出后,破裂的细胞内出现大量囊泡,且有合胞体(巨型多核融合细胞)出现。另外,将感染孢子的家蚕中肠组织和丝腺组织用胰蛋白酶处理后直接接种Sf21细胞,不经发芽处理,细胞也能感染。     

Evaluation of nested polymerase chain reaction, pepsin/trypsin digest, and histology for the detection of Myxobolus cerebralis in salmonid fishes of Indiana and Michigan.

The objectives of this study were to survey fish from state hatcheries in Indiana and Michigan and to compare the nested polymerase chain reaction (PCR) test with pepsin/trypsin digest (PTD) and histopathology for the diagnosis of whirling disease (WD). One group of 40 and 9 groups of 60 fish heads, for a total of 580 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, PTD, and PCR tests. The heads were hemisectioned, and one half was fixed in 10% neutral-buffered formalin for histopathologic examination. The other half was processed for PTD. Some of the sediment was examined for the presence of myxozoan spores, and the rest was prepared for the nested PCR. Histologic examinations did not reveal Myxobolus cerebralis in any of the 580 samples. One hundred serial step sections, taken at 5-microm intervals, were evaluated for samples with positive spore identification by PTD. Histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 16.9% (98/580) of samples in sediment after PTD. Spores morphologically similar to those of M. cerebralis were observed in 1.0% of PTD samples (n = 6). The nested PCR indicated that M. cerebralis spores were present in 0.5% of samples (n = 3). All 3 nested PCR-positive samples came from the same hatchery, however, spores of M. cerebralis were seen in 1 sample, spores of other myxozoan species were seen in the second sample, and spores were not seen in the third sample. When comparing the PTD to the nested PCR test, the PTD diagnosed 1 true positive, 5 false positives, 2 false negatives, and 572 true negatives, for a sensitivity of 33% and a specificity of 99.1%. Screening for M. cerebrallis infection in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR test was highly specific and was used to differentiate spores of M. cerebralis from similar spores of other species.     

大蜜蜂和东方蜜蜂防御行为的比较研究

在云南西双版纳基诺乡的橡胶林和云南农大东方蜜蜂研究所试验蜂场里,分别选择大蜜蜂(Apisdorsata)和东方蜜蜂(Apiscerana)各1群,观察、记录蜂群在受惊扰后蜜蜂的攻击行为。东方蜜蜂在受惊扰后,每次参加攻击的工蜂平均个体数为38只,攻击时间为1.37min,追逐的距离为22m;而大蜜蜂在受惊扰后,每次参加攻击的工蜂平均个体数为510只,攻击时间为131min,追逐的距离为342m。结果表明:与在洞穴、蜂箱内营巢的东方蜜蜂相比,露天营巢的大蜜蜂具有很强的防御和攻击能力。     

链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌多重PCR检测方法的建立及应用

根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。     

家蚕微孢子虫蛋白水解酶的活性检测及质谱鉴定和序列分析

微孢子虫的蛋白水解酶类不仅参与代谢活动,而且可能作为一种潜在的侵染宿主的毒力因子。为了研究家蚕微孢子虫蛋白水解酶的种类及蛋白质结构特点,将家蚕微孢子虫总蛋白经非变性凝胶电泳后与底物酪蛋白孵育,检测到可水解酪蛋白的蛋白酶类的活性。采用质谱法分析该类蛋白酶主要是锌离子依赖型的细胞质型亮氨酰氨肽酶和丝氨酸蛋白酶。细胞质型亮氨酰氨肽酶序列是由1 515个碱基编码的505个氨基酸组成,无信号肽,有2个潜在的锌离子结合部位,属于金属蛋白酶M17家族;丝氨酸蛋白酶具有肽酶S8结构域和跨膜域。通过序列比对发现在兔脑炎微孢子虫、蝗虫微孢子虫、蜜蜂微孢子虫、肠道微孢子虫和比氏肠道微孢子虫中均有这2种酶,且相似度较高,其进化较为保守。     

家蚕微孢子虫原位杂交诊断技术研究

根据家蚕微孢子虫rRNA基因高度保守的特点,设计合成了1对PCR引物,从家蚕微孢子虫基因组DNA上扩增出1个12kb片段,用同位素标记后作为检测家蚕微孢子虫的特异性探针。采用原位杂交的方法,对家蚕卵及感染家蚕微孢子虫4、6、10d的幼虫进行原位杂交检测。实验结果表明,该方法可以从家蚕单粒卵内检测到296粒孢子,对感染家蚕微孢子虫4、6、10d的幼虫均有阳性杂交信号出现。     

家蚕微孢子虫PCR检测的研究

根据家蚕微孢子虫（Nosemebombycis,N．b）孢子的DNA序列，设计合成一对引物，对家蚕微孢子虫孢子及其近缘种孢子的DNA进行PCR检测。结果表明只有家蚕微孢子虫孢子DNA获得特异性扩增区带，大小为317bp，可以区别于Nosemasp.MG1和MG2、柞蚕微孢子虫、Vairimorphanecatrix及Pleistophoraanguillarum等。对N.b孢子DNA检测灵敏度达1ng水平。     

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).     

The effects of hive types (shield and sword) on wintering ability,survival rates and strength of honeybee colonies (<Emphasis Type="Italic">A. mellifera</Emphasis> L.) in spring season

This study was carried out to determine the effects of shield and sword comb orientation hive types on wintering ability, survival rates (in winter) and population growth of honeybee colonies (A. mellifera anatoliaca) in spring season. In ancient Anatolia beekeeping; honeybee colonies were identified sword and shield (the colonies which build up the combs vertical and horizontal according to positions of the hive entrance) before the uses of top-opened hive with movable frames. Total twenty honeybee colonies, which have similar condition according to queen age, genotype, number of frames covered with adult worker bees, brood areas and food stocks, were used in this study. Average wintering ability of colonies in the shield and sword groups were found to be 98.57% and 69.76%; average survival rates were found to be 100% and 100% in shield and sword group colonies respectively. The average number of frames covered with adult worker bees at mid June in shield and sword group colonies were found to be 15.6 ± 1.58, 12.00 ± 1.25 number/colony and the average brood areas were found as 7863.5 ± 402.9, 5997.0 ± 373.3 cm²/colony respectively. Differences between the group means on wintering ability, sealed brood areas and colony strength were found significant (P < 0.01), but differences on survival rates were not found significant (P > 0.05). The colonies living in shield (horizontal) hives have showed better wintering ability and more colony population than colonies living in sword (vertical) hives.     

The prevalence of green muscle disease in turkeys from the Warmia and Mazury province of Poland

The green muscle disease (GMD), described for the first time in 1968, is a cause of significant losses in the production of turkey meat. Pathological changes of different intensity are found in smaller pectoral muscles on one or both sides of the sternal crest. GMD has been more frequently observed in Poland since 1991 in breeding turkeys of heavy and medium-heavy types assigned to slaughter after completing the reproductive period. The present study deals with the occurrence of this disease in the Warmia and Mazury District of Poland. Characteristic green foci located in small pectoral muscles were found in 4090 out of 26,169 slaughtered turkeys, i.e. in 15.63% of the carcasses investigated. It was established that 2430.5 kg of meat were confiscated what gave 0.59 kg of confiscated meat per head on the average.     

微孢子虫O-甘露糖糖基化通路相关酶基因序列的比较分析

糖蛋白在免疫识别、信号转导等生命进程中扮演重要角色。为研究微孢子虫糖蛋白及其糖基化类型,采用比较基因组学方法对微孢子虫O-甘露糖糖基化通路进行分析。真核生物的O-甘露糖糖基化通路中有6种主要酶类,包括己糖激酶、磷酸甘露糖酶、GDP甘露糖磷酸化酶、Dol-P甘露糖合成酶、甘露糖转移酶、Dol-P甘露糖蛋白甘露糖转移酶。通过生物信息学方法分析家蚕微孢子虫(Nosema bombycis)、蜜蜂微孢子虫(Nosema ceranae)、兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠脑炎微孢子虫(Encephalitozoon intestinalis)、比氏肠道微孢子虫(Encephalitozoon bieneusi)和柞蚕微孢子虫(Nosema antheraea)的这6种酶基因编码氨基酸序列特征及结构域类型,发现这些酶的垂直同源基因比较保守,多重序列比对表明在6种微孢子虫之间,6种酶的氨基酸序列相似性比较高,系统进化分析显示同属的微孢子虫多聚为一簇。研究结果表明,N.bombycis、N.ceranae、E.cuniculi、E.intestinalis和N.antheraea 5种微孢子虫的O-甘露糖糖基化的蛋白质修饰通路是完整、保守的,然而在E.bieneusi中,其关键酶Dol-P甘露糖蛋白甘露糖转移酶的氨基酸序列比较短,只有342个氨基酸,缺失了关键结构域PMT,因此可能无法完成O-甘露糖糖基化修饰。以上微孢子虫的O-甘露糖糖基化通路中6种主要酶的序列信息,为后续解析家蚕微孢子虫糖基化通路及对糖蛋白糖基化类型与功能的研究提供了线索。     

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.     

蜂群崩溃失调病(CCD)研究进展

2006年冬到2007年春,蜂群崩溃失调病(colony collapse disorder,简称CCD)在世界范围内流行,已造成巨大损失。CCD是指意蜂蜂群中大量的成年工蜂短时间内突然在巢外失踪,没有发现尸体,只剩下蜂王、卵、一些未成年的工蜂和大量蜜粉残留于巢脾内的症状。到目前为止,经调查发现以色列急性麻痹病毒(Israeliacute paralysis virus,IAPV)是导致CCD的最大嫌疑,但CCD的确切致病机理尚未揭开。本文综述了CCD的主要症状、可能的致病原因等方面的研究进展,提出若干需要高度关注的问题,以及防范CCD的措施。     

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。     

维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响

本试验旨在研究维生素B_2对中华蜜蜂工蜂寿命及学习记忆能力的影响。试验分为工蜂饲养试验和学习记忆试验2部分。工蜂饲养试验:将1日龄中华蜜蜂工蜂分成3组,每组5个重复,每个重复约200只。对照组(Ⅰ组)饲喂1∶1的糖水,2个试验组在1∶1糖水的基础上分别添加100(Ⅱ组)、400 mg/kg(Ⅲ组)的维生素B_2,每天记录各组工蜂的死亡情况,直到全部死亡。工蜂学习记忆试验:分组方法同工蜂饲养试验,并按照工蜂饲养方法对1日龄工蜂喂养7 d后,利用吻伸反应方法测定工蜂短时学习记忆及长时学习记忆能力,并通过荧光定量PCR检测学习成功后工蜂学习记忆相关基因的相对表达量。结果表明:工蜂的平均寿命随维生素B_2添加量的升高而延长,而且试验组(Ⅱ组、Ⅲ组)的平均寿命显著高于对照组(P0.05),但Ⅱ组与Ⅲ组间差异不显著(P0.05)。Ⅲ组工蜂的长时学习记忆及短时学习记忆能力均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的长时学习记忆能力也显著高于对照组(P0.05)。Ⅲ组工蜂的多巴胺受体基因2(Acdop3)、c AMP反应元件结合蛋白(Ac CREB)相对表达量均显著高于对照组和Ⅱ组(P0.05),而且Ⅱ组工蜂的Acdop3相对表达量也显著高于对照组(P0.05)。由此得出,维生素B_2影响中华蜜蜂工蜂的寿命及学习记忆能力,在人工饲喂蜂群时需要提供适量的维生素B_2。     

中国柞蚕微粒子病病原的研究

从辽宁柞蚕分离出的两种微孢子虫,经光镜和电镜形态、寄生习性的研究结果,确定为柞蚕微粒子虫新种和修氏内网虫,在中国柞蚕微粒子病蚕中为首次发现。     

南川金佛山东方蜜蜂的生物学特性研究

为选育金佛山优良中蜂品种提供科学依据,我们把不同地理海拔分布的27群重庆南川金佛山东方蜜蜂集中在同一蜜源点进行饲养,开展金佛山东方蜜蜂主要生物学特性的研究。研究表明：金佛山东方蜜蜂和我国优良蜂种阿坝中蜂比较相似。     

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazi? province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.