一种新的蜜蜂细菌性幼虫病病原的分离鉴定

2005年早春,在浙江部分地区出现了一种严重的蜜蜂细菌性幼虫病,该病导致蜜蜂幼虫颜色发黄,失去光泽;严重时幼虫死亡腐烂。从10批发病死亡幼虫样品中,分离得到并保存了5类纯培养物。通过蜂群接种试验和实验室人工培养的幼虫接种,确定L2菌株能引起与自然发病相似的症状,且能从接种发病的幼虫上再次分离到相同菌株,证明L2菌株是该蜜蜂细菌性幼虫病的致病菌。进一步对分离到的该致病菌从发病特征、病原形态学、生理生化特性、16SrRNA序列等方面进行了分析鉴定,结果显示:该菌株属于肠球菌属的屎肠球菌(Enterococcusfaecium),不是目前报道的任何一种已知蜜蜂细菌性幼虫病的病原。     

中华蜜蜂的欧洲幼虫腐臭病病原研究

采用中华蜜蜂Apis cerana cerana F.临床自然发病的欧洲幼虫腐臭病(European Foulbrood, EFB)幼虫,对其病原体进行了分离鉴定。结果表明：中华蜜蜂EFB的病原系蜂房蜜蜂球菌Melissococcus pluton。该菌为革兰氏阳性的兼性厌氧菌,形态学及染色特性、致病性试验、血清学试验和细菌DNA G+C mol%试验均证明中华蜜蜂和西方蜜蜂的EFB病原属于同属的蜂房蜜蜂球菌。生理生化特性结果与国外的早期研究结果相近似。该试验为不同地理位置、不同种寄主间蜂房蜜蜂球菌的遗传差异的研究,以及中华蜜蜂EFB病的防治学研究打下基础。     

我国蜜蜂主要病原检测技术

蜜蜂是重要的经济昆虫。蜜蜂病害威胁蜜蜂产业的发展。蜜蜂病害检测及病原鉴定是病害防治的基础。文章详细介绍我国蜜蜂常见6种主要病害(美洲幼虫腐臭病,欧洲幼虫腐臭病,囊状幼虫病,慢性麻痹病,微孢子虫病,白垩病)的病原快速准确的检测方法。     

牦牛肠道粪肠球菌的分离和鉴定

试验为获得1株与牦牛肠道共生的粪肠球菌,通过对健康牦牛粪样中的肠球菌进行分离培养、革兰染色、生化试验、16S rRNA基因测序比对等生物学鉴定,初步证实该分离菌株为粪肠球菌。此试验为进一步分析牦牛肠道菌群结构,探索牦牛耐饥、耐渴、耐粗饲、对高山草原极强适应性与其肠道菌群的关系,进一步研究与牦牛共生的粪肠球菌的生物学特性、生理特性、对宿主危害性等提供一些实验依据。     

雏鸡源致病性粪肠球菌的分离鉴定及其耐药性分析

【背景】粪肠球菌(Enterococcus faecalis)是人和动物肠道正常菌群之一,也是一种条件性致病菌。近年来,粪肠球菌引起人和动物感染的报道越来越多。【目的】探明引起某养鸡场雏鸡发病死亡的病原及其致病性和有效治疗药物。【方法】结合临床症状和病理剖检,开展病原菌分离、生理生化特性检测和16S rRNA基因序列分析、致病性试验、耐药分析和对患病鸡群的药物治疗。【结果】患病鸡有昏睡、瘫痪或共济失调等临床症状;肝、脾肿大,肝脏发黄、少量出血点、质脆易碎,肠道粘膜增厚、出血,脑轻微水肿;从肝脏组织分离得到一株革兰氏阳性球菌,经纯化培养后命名为CJ517;依据该菌株形态特征、生理生化特性和16S rRNA基因序列分析鉴定为粪肠球菌;致病性试验显示CJ517菌株能致死小鼠,致死率为66.67%;该菌对头孢噻肟、磷霉素、丁胺卡那等药物敏感,对多西环素、卡那霉素、新霉素、氟苯尼考等药物耐药;经用敏感药物和提高免疫力结合治疗后,鸡群病情得到控制。【结论】研究结果可为临床诊断和治疗动物粪肠球菌感染提供参考。     

神农架川金丝猴源肠道粪肠球菌的分离与鉴定

通过培养特征、形态观察、抑菌性实验,从神农架健康野生金丝猴肠道分离到8株抑菌效果明显的菌株;生理生化鉴定和16S rRNA基因序列同源性分析,该8株菌为粪肠球菌。毒力因子检测和动物急性毒性实验筛选出2株安全性良好的菌株,研究两菌株对胃肠道环境（低pH值、高胆盐）的耐受特性和生长情况。结果表明,粪肠球菌dlt7a和dlt7b株繁殖能力很强,无迟缓期,具有较强的耐受胃酸及肠道高胆盐环境的能力,且安全性好,具有较好的益生特性,可作为金丝猴微生态制剂的候选菌株。     

肠球菌E4及其抑菌产物特性的研究

目的获得抑制微生物生长的菌株。方法根据形态学和生理生化学特性进行菌种鉴定;采用牛津杯法测定抑菌谱和抑菌物质的理化特性。结果排除了过氧化氢和有机酸的作用,该菌发酵上清液对苏云金杆菌、枯草杆菌、大肠埃希菌、鸡白痢沙门菌等有抑制作用。根据菌株的生理生化特征,该菌株初步定为肠球菌属,定名为E4（Enterococcus sp．）。所产抑菌物质具有较好的热稳定性,在酸性条件下稳定且活性高。结论分离筛选了1株可产抑菌物质的肠球菌,其产生的抑菌物质具有良好的生物化学特性和广谱的抑菌能力。     

藏灵菇中Enterococcus faecalis RQ15的分离及鉴定

以民间家庭制作酸奶用藏灵菇为试材,通过有氧与厌氧相结合的分离方式,从中分离到1株乳酸球菌。经形态学、生理生化特性研究及分子生物学鉴定,该菌株为粪肠球菌,编号Enterococcus faecalis RQ15。在12%复原脱脂乳中延时培养及冷藏保存该菌株时,产生胨化现象。后续的研究表明Enterococcus faecalis RQ15产生的蛋白酶具有低温蛋白酶的特征。     

泌尿外科住院患者泌尿系感染病原菌分布及其耐药性分析

了解泌尿外科住院患者泌尿系感染病原菌的分布及其对常用抗菌药物的耐药情况。对泌尿外科泌尿系感染住院患者的消毒中段尿培养结果进行回顾性分析,尿培养菌株的鉴定、药敏分析和统计分析采用VITEK2全自动微生物仪。3 a中泌尿外科泌尿系感染住院患者共分离到细菌1 233株,其中革兰阴性杆菌772株,占62.61%,革兰阳性球菌353株,占28.63%,真菌82株,占6.65%。菌株数居前5位的细菌依次为:大肠埃希菌、肠球菌属、洋葱伯克霍尔德菌、假丝酵母菌属和变形杆菌。分离大肠埃希菌产ESBLs率为66.18%,粪肠球菌中未发现VRE菌株,屎肠球菌VRE为0.8%。未发现对美洛培南耐药的大肠埃希菌,对肠埃希菌耐药率在10%以下的有亚胺培南、阿米卡星、哌拉西林/他唑巴坦和呋喃妥因。洋葱伯克霍尔德菌和铜绿假单胞菌对所监测的抗菌药物均有不同程度的耐药。未发现对万古霉素、替考拉宁及氨苄西林耐药的粪肠球菌。泌尿系感染的病原菌以大肠埃希菌和肠球菌为主,非发酵革兰阴性杆菌及假丝酵母菌所占比例超过20%,不容忽视,病原菌的耐药率较高,临床医生应根据尿培养和药敏试验结果合理使用抗菌药物。     

1株虎源致病性肠球菌的分离鉴定及序列分析

从病死老虎肺脏中分离到1株肠球菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。本菌对多种抗生素高度耐药,对小白鼠有强致病性,其LD50为2.7×109.2cfu。并用PCR方法扩增分离菌株16S rDNA基因,获得1 415 bp片段,该片段核苷酸序列提交GenBank,登陆号为HM346186,将分离株的16S rDNA核苷酸序列与GenBank上其他肠球菌进行同源性分析。结果表明,分离株的16S rDNA核苷酸序列与肠球菌(EU285587)的同源性为100%,因此该分离菌株被鉴定为致病性肠球菌,命名为YN-1株(云南-1株)。     

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.     

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera)

American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death.     

Complete genome sequence of Melissococcus plutonius ATCC 35311

We report the first completely annotated genome sequence of Melissococcus plutonius ATCC 35311. M. plutonius is a one-genus, one-species bacterium and the etiological agent of European foulbrood of the honeybee. The genome sequence will provide new insights into the molecular mechanisms underlying its pathogenicity.     

Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey

American foulbrood is a severe bacterial disease affecting larvae of the honeybee Apis mellifera and it is caused by Paenibacillus larvae larvae. The disease is present worldwide and cases have been reported in almost all the beekeeping regions of the five continents. During 2001 and 2002 we carried out a nationwide study to assess the presence and amount of P. l. larvae spores in honey samples from Uruguay, combining classic bacteriological, and molecular approaches. The distribution of P. l. larvae spores in honey of the whole country showed a clear pattern and may provide useful data for a control and prevention strategy of American foulbrood.     

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.     

Adult honeybee's resistance against Paenibacillus larvae larvae, the causative agent of the American foulbrood.

American foulbrood is a widespread disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae subsp. larvae. Spores represent the infectious stage; when ingested by a larva they germinate in the midgut. The rod-shaped vegetative forms penetrate the larva's intestinal tissue and start multiplying rapidly, which finally kills the larva. Spores fed to adult honeybees, however, do not harm the bees. We investigated this phenomenon. Specifically, we studied the influence of the adult honeybee midgut on the vegetative growth and on the germination of spores of P. larvae larvae. We focused on two groups of adult workers that are likely to have large numbers of spores in their gastrointestinal tracts in infected colonies: middle-aged bees, which are known to remove or cannibalize dead larvae and clean brood cells, and winterbees, which do not have frequent chances to defecate. We found that midgut extract from winterbees and worker-aged bees of different colonies almost completely inhibited the growth of the vegetative stage of P. larvae larvae and suppressed the germination of spores. The inhibiting substance or substances from the adult midgut are very temperature stable: they still show about 60% of their growth-inhibiting capacity against this bacterium after 15 min at 125 degrees C. We established a method to test growth-inhibiting factors against P. larvae larvae in vitro.     

Antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases and finding out new strain of Serratia marcescens via DGGE-PCR,as a secondary infection on honeybee larvae

American foulbrood (AFB) and European foulbrood (EFB) are the two major bacterial diseases affecting honeybees, leading to a decrease in viability of the hive, decreasing honey production, and resulting in significant economic losses to beekeepers. Due to the inefficiency and/or low efficacy of some antibiotics, researches with nanotechnology represent, possibly, new therapeutic strategies. Nanostructure drugs have presented some advantagesover the conventional medicines, such as slow, gradual and controlled release, increased bioavailability, and reduced side-effects. In this study, different infected larvae were collected from two apiaries; the combs that had symptoms of American and European foulbrood were isolated. In vitro antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases were characterized using UV–Vis spectrophotometry and scanning electron microscope (SEM) that proves the formation of silver nanoparticles with size range 160–660 nm. The antimicrobial activity of the silver nanoparticles was tested using agar diffusion assay and proved their ability to effectively cease the pathogenic bacterial growth in both AFB and EFB. DGGE-PCR technique has been applied for the identification of un-common bacterial infections honeybees depending on 16S rRNA amplification from their total extracted DNA and has been identified as Serratia marcescens (TES), deposited in GenBank with a new accession number (MT240613). The results were confirmed strain has been detected by DGGE-PCR analysis causing uniquely infected brood that was attacked by the American Foulbrood It could be concluded that greenly synthesized silver nanoparticles is projected to be used as effective treatment for honeybee bacterial diseases. These material need more investigations under field conditions and study the possibility of its residues in honeybee products such as honey, and beeswax.     

Preliminary investigations into possible resistance to oxytetracycline in Melissococcus plutonius,a pathogen of honeybee larvae

AIMS: To investigate the occurrence of oxytetracycline (OTC) resistance in Melissococcus plutonius, which causes European foulbrood in honeybee colonies. METHODS AND RESULTS: Strains of M. plutonius were isolated from diseased colonies in England and Wales and tested for resistance to OTC. The minimum inhibitory concentration (MIC) of OTC was also determined for selected isolates. No resistance to the antibiotic was found in any isolate and the average MIC was found to be 3.9 microg ml-1. Melissococcus plutonius was found to be susceptible to both chlortetracycline and tetracycline. CONCLUSIONS: No resistance to OTC was found in M. plutonius. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that OTC can continue to be used to treat European foulbrood and that resistance may not explain why some treatments fail.     

Low-Molecular-Weight Metabolites Secreted by Paenibacillus larvae as Potential Virulence Factors of American Foulbrood

The spore-forming bacterium Paenibacillus larvae causes a severe and highly infective bee disease, American foulbrood (AFB). Despite the large economic losses induced by AFB, the virulence factors produced by P. larvae are as yet unknown. To identify such virulence factors, we experimentally infected young, susceptible larvae of the honeybee, Apis mellifera carnica, with different P. larvae isolates. Honeybee larvae were reared in vitro in 24-well plates in the laboratory after isolation from the brood comb. We identified genotype-specific differences in the etiopathology of AFB between the tested isolates of P. larvae, which were revealed by differences in the median lethal times. Furthermore, we confirmed that extracts of P. larvae cultures contain low-molecular-weight compounds, which are toxic to honeybee larvae. Our data indicate that P. larvae secretes metabolites into the medium with a potent honeybee toxic activity pointing to a novel pathogenic factor(s) of P. larvae. Genome mining of P. larvae subsp. larvae BRL-230010 led to the identification of several biosynthesis gene clusters putatively involved in natural product biosynthesis, highlighting the potential of P. larvae to produce such compounds.