草鱼铜绿假单胞菌的鉴定及药物敏感性分析

从患病草鱼C tenopharyngodon idella体表病灶上分离到一株优势菌,经分离纯化培养制成细菌悬液,分别以腹腔注射和浸泡两种方式感染草鱼。结果表明：该菌株能感染草鱼,且具较强毒力,腹腔注射0.2mL该菌液（3×109cfu/mL）,可导致受试草鱼100%死亡;创伤浸泡感染（3×108cfu/mL）,死亡率也达100%,受感染鱼出现与自然感染相似的类似赤皮病症状。对该病菌进行形态特征观察和主要理化特性分析,初步鉴定为铜绿假单胞菌Pseudom onas aeruginosa。进一步对该菌进行分子鉴定,共选用16S rRNA、RNA聚合酶β亚单位（rpoB）和促旋酶亚单位蛋白（gyrB）3个基因进行鉴定分析。序列克隆与BLAST分析结果显示,这3个基因均与GenBank上登录的铜绿假单胞菌的相应序列具有很高的同源性,16S rRNA、rpoB和gyrB基因与已知铜绿假单胞菌相应序列的同源性分别在99%、98%和93%以上。综合生理生化与分子鉴定结果,可判定所分离的菌株为铜绿假单胞菌。药敏试验结果显示：该菌对阿米卡星、菌必治、氟哌酸和妥布霉素等11种药物高度敏感,对链霉素和庆大霉素中度敏感,对罗红霉素、红霉素、四环素、苯唑青霉素、新霉素和头孢氨苄等16种药物具有耐药性。     

双重PCR快速鉴别无乳链球菌和海豚链球菌

根据无乳链球菌的cfb基因和海豚链球菌16SrRNA基因序列,设计、合成2对引物,优化扩增条件,建立了快速鉴别无乳链球菌和海豚链球菌的双重PCR方法.用该方法扩增无乳链球菌和海豚链球菌,可分别获得474、296bp的特异性片段,扩增嗜水气单胞菌、铜绿假单胞菌等其他常见鱼病原菌无特异性片段.该方法可实现对无乳链球菌和海豚链球菌的快速鉴别,具较高的灵敏度,可检测到基因组含量分别为3.2×10-3ng/μL的无乳链球菌和3.0×10-2ng/μL的海豚链球菌.对采集自广东与海南两省养殖罗非鱼病鱼的11份病原样品进行检测,均可获得474bp片段,测序与BLAST分析结果表明,扩增到的序列均为无乳链球菌cfb基因序列,可从分子水平上确定这些样品为无乳链球菌,与生化鉴定结果一致.     

转mapk双链RNA干扰表达载体黄瓜对根际土壤细菌多样性的影响

随着RNA干扰技术的发展,通过植物表达病原物特异的dsRNA来防治植物病害的转基因作物越来越多.转根结线虫mapk双链RNA表达载体的黄瓜能够通过RNA干扰作用沉默线虫的mapk基因,对根结线虫具有良好的防治效果.为了评价该转基因黄瓜的安全性,明确mapk双链RNA干扰表达载体转基因黄瓜植株对根际土壤细菌多样性的影响,采用16S rRNA基因克隆文库方法对非转基因黄瓜和转基因黄瓜土壤细菌群落多样性进行分析.结果表明,非转基因黄瓜土壤细菌文库包含124个OTU(可操作分类单元),转基因黄瓜土壤细菌文库包含122个OTU.2个文库共同拥有的OTU为115个.2个文库都包含1 3个类群细菌,Acidobacteria、Actinobacteria、Armatimonadetes、Bacteroidetes、candidate division BRC1、Chloroflexi、Firmicutes、Gemmatimonadetes、Nitrospira、Planctomycetes、Proteobacteria、Verrucomicrobia、unclassified_Bacteria. 其中 Proteobacteria、Bacteroidetes 、Chloroflexi和Acidobacteria是优势菌群.其他细菌类群数量相对较少.在纲分类水平上,两个文库包含的细菌类群一致,且各类群细菌比例差异不大.在Acidobacteria门中,Acidobacteria_Gp6为优势菌群.在Bacteroidetes门中,Sphingobacteria纲细菌数量最多.在Chloroflexi门中,unclassified Chloroflexi细菌最多.在Proteobacteria门中,其中Betaproteobacteria纲的细菌数量最多.从多样性指数角度分析,两种土壤细菌群落的Shannon、Simpson和Chao值差异不大.总体来看,两种土壤细菌类群差异不显著,转基因黄瓜未对根际土壤细菌群落产生明显影响.     

乌江网箱养殖花鲈体表细菌分离鉴定与感染草鱼试验

利用微生物学常用的细菌分离纯化方法,从患病花鲈体表分离出了5株菌株,分别编号为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ。进行生化试验和细菌16S rRNA 基因序列测定,并将测序结果输入到NCBI（http://www.ncbi.nlm.nih.gov/）中,利用BLAST 工具对序列进行同源性对比分析。结果发现,5株细菌中,初步鉴定Ⅰ为野菊微小杆菌,Ⅱ为乙酰微小杆菌,Ⅲ~Ⅳ为巨型球菌,Ⅴ为细菌MM5。用5 株细菌分别感染健康草鱼,发现5株细菌均具有致病性,其中巨型球菌对草鱼的危害较大。     

斜带石斑鱼病原菌(哈维氏弧菌)的分离与鉴定

从患病斜带石斑鱼 (Epinepheluscoioides)肝脏组织分离到EcGY0 2 0 4 0 1菌株 ,经人工感染、回归感染实验证实为致病菌。通过API系统和菌体常规形态特征、培养特性和生理生化反应指标测定以及 16SrRNA测序分析等综合鉴定 ,Ec GY0 2 0 4 0 1菌株为弧菌属哈维氏弧菌 (Vibrioharveyi) ,其半致死剂量LD50 为 2 .7× 10 6CFU/g鱼体重。药敏试验结果表明 ,EcGY0 2 0 4 0 1菌株对利福平、四环素、喹诺酮类及头孢曲松等抗生素较为敏感     

Soybean meal alters autochthonous microbial populations, microvilli morphology and compromises intestinal enterocyte integrity of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout were fed either a diet containing fishmeal (FM) as the crude protein source or a diet containing 50% replacement with soybean meal (SBM) for 16 weeks. An enteritis-like effect was observed in the SBM group; villi, enterocytes and microvilli were noticeably damaged compared with the FM group. The posterior intestine microvilli of SBM-fed fish were significantly shorter and the anterior intestine microvilli significantly less dense than the FM-fed fish. Electron microscopy confirmed the presence of autochthonous bacterial populations associated with microvilli of both fish groups. Reduced density of microvilli consequently led to increased exposure of enterocyte tight junctions, which combined with necrotic enterocytes is likely to diminish the protective barrier of the intestinal epithelium. No significant differences in total viable counts of culturable microbial populations were found between the groups in any of the intestinal regions. A total of 1500 isolates were tentatively placed into groups or genera, according to standard methods. Subsequent partial 16S rRNA sequencing revealed species that have not been identified from the rainbow trout intestine previously. Compared with the FM group levels of Psychrobacter spp. and yeast were considerably higher in the SBM group; a reduction of Aeromonas spp. was also observed.     

养殖刺参溃疡病病原菌RH2的鉴定及其生物学特性分析

从患溃疡病养殖刺参(Apostichopus japonicus)病灶处分离出3株优势菌(RH1、RH2、RH3),以浸浴、体腔注射、体壁肌肉注射等方式进行感染实验,证实菌株RH2为养殖刺参溃疡病的病原菌,其对刺参的LD50(半数致死量)每尾为5.68×106CFU,并证实浸浴、体腔注射的方式无法感染刺参,该菌可能通过体表创伤侵入的方式感染刺参。经形态学观察、生理生化特性分析、16SrRNA和HSP60基因测序确定菌株RH2为溶藻弧菌(Vibrio alginolyticus)。基于16S rRNA基因构建的系统发育树显示,菌株RH2与溶藻弧菌(AF513447)亲缘关系最近,置信度为50%;HSP60基因序列分析表明RH2与溶藻弧菌(AY332570、AF230931)聚为一个分支,且置信度高达99%。菌株RH2的最适生长盐度为25~35,最适生长pH为7.2~8.0,最适生长温度为14~22℃。对18种药物的敏感实验证实菌株RH2对诺氟沙星、复方新诺明、壮观霉素等较为敏感。     

微生物菌群培养组学在动物医学中的应用

实验室条件下可培养的微生物约占自然界中微生物总数的1%，这限制了人们对99%未知微生物的认识和利用，而研究表明，那些“不可培养的微生物”是可以被开发和利用的，未能被纯培养的微生物才是未知微生物的主体。微生物培养组学探索利用多种培养条件和长时间的培养，结合基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF-MS）和16S核糖体RNA（rRNA）测序可以大规模鉴定各种微生物，同时利用全基因组测序和宏基因组测序手段对未知微生物进行深入分析。本文综述了国内外近年来微生物菌群培养组学在反刍动物胃肠道、禽类盲肠及家畜鼻腔微生物菌群研究中的最新进展，探讨将动物体内菌群培养组学方法应用于动物疾病防治领域的可行性。作为一个新兴的研究方法，尽管该培养组学还存在一些不够成熟的方面，但它的发展前景十分广阔，微生物菌群培养组学方法和其他研究方法的互补已经逐渐成为发展兽医微生物学新的突破口。     

Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by geographic features

Here, we examine soil-borne microbial biogeography as a function of the features that define an American Viticultural Area (AVA), a geographically delimited American wine grape-growing region, defined for its distinguishing features of climate, geology, soils, physical features (topography and water), and elevation. In doing so, we lay a foundation upon which to link the terroir of wine back to the soil-borne microbial communities. The objective of this study is to elucidate the hierarchy of drivers of soil bacterial community structure in wine grape vineyards in Napa Valley, California. We measured differences in the soil bacterial and archaeal community composition and diversity by sequencing the fourth variable region of the small subunit ribosomal RNA gene (16S V4 rDNA). Soil bacterial communities were structured with respect to soil properties and AVA, demonstrating the complexity of soil microbial biogeography at the landscape scale and within the single land-use type. Location and edaphic variables that distinguish AVAs were the strongest explanatory factors for soil microbial community structure. Notably, the relationship with TC and TN of the <53 μm and 53–250 μm soil fractions offers support for the role of bacterial community structure rather than individual taxa on fine soil organic matter content. We reason that AVA, climate, and topography each affect soil microbial communities through their suite of impacts on soil properties. The identification of distinctive soil microbial communities associated with a given AVA lends support to the idea that soil microbial communities form a key in linking wine terroir back to the biotic components of the soil environment, suggesting that the relationship between soil microbial communities and wine terroir should be examined further.     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.