猪生物发酵床保育期垫料中效应细菌的筛选及鉴定

本试验对猪生物发酵床保育期垫料样品中效应细菌的组成和作用进行了研究,共获得了6株效应细菌;经纯培养后观察其个体与群体形态,并进行生化特征鉴定以及16S rRNA基因分析.结果表明,所得的6株效应细菌中,芽孢杆菌属(Bacillus sp.)细菌有4株,分别为枯草芽孢杆菌(Bacillus subtilis)、嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)、蜡样芽孢杆菌(Bacillus cereus)、地衣芽孢杆菌(Bacillus licheniformis);沙雷氏菌属(Serratia sp.)有1株,为粘质沙雷氏菌(Serratia marcescens);假单胞菌属(Pseudomonas sp.)有1株,为假单胞杆菌(Pseudomonas).     

高原早熟禾根际促生菌分离筛选及特性研究

为获得高原早熟禾（Poa alpigena L.）根际优良促生菌资源,从甘南草原采集高原早熟禾样品,从其根际分离筛选具有溶磷、固氮能力的促生菌株,研究其促生特性,并验证其对高原早熟禾种子萌发及幼苗生长的影响,在此基础上,运用16S rRNA基因分析法确定其分类地位。结果发现：共分离出32株具有促生作用的菌株,筛选出的5株优良菌株中溶解无机磷菌株有4株,溶有机磷菌株3株,固氮菌4株;5株菌株均具有分泌吲哚-3-乙酸（Indole-3-acetic acid,IAA）的能力,分泌量在9.62~76.16 μg·mL^-1之间;待测菌均能提高高原早熟禾幼苗的发芽率、发芽指数、幼苗活力指数,并能促进高原早熟禾幼苗的生长;分类地位鉴定结果表明5株菌中有2株枯草芽孢杆菌,1株蕈状芽孢杆菌,1株不动杆菌以及1株荧光假单胞菌。本研究为进一步的促生菌机理研究和功能微生物资源开发奠定了基础。     

青贮玉米促生菌的初步研究

从西宁奶牛场青贮玉米根系分离出促生菌,依据形态、培养性状、生理生化特征,鉴定为蜡状芽胞杆菌(Bacillus cereus),该菌株能明显促进青贮玉米幼苗生长。     

促生菌对Cd胁迫下蒌蒿光合特性的影响

为揭示促生菌对Cd胁迫下蒌蒿光合特性的作用机理,利用具有促生功能的芽孢杆菌(Bacillus sp.,T3),成团泛菌(Pantoea agglomerans,J2),荧光假单胞菌(Pseudomonas fluorescens,Y5)的混合菌剂进行盆栽接种试验,研究促生菌对Cd胁迫下蒌蒿光合气体交换、光合色素、光合矿...     

一种复合芽孢菌制剂对富营养化污水的净化作用研究

以高效去除COD为主要指标,从长沙市郊区某富营养化的养殖小区鱼塘淤泥和污水液中分离筛选出4株目的菌株,发现其中以L菌株活性最好,经初步鉴定为侧孢杆菌(Bacillus laterosporus Laubach).同时,采用地衣芽胞杆菌(Bacillus Subtilis)、短小芽孢杆菌(Bacillus pumilus Meyer and Gottheil)、纳豆芽孢杆菌(Bacillus natto)与菌株L分别培养后,按比例复合制剂,进行污水净化试验.结果表明,与单菌相比,复合菌综合了单菌的优良祛污活性,在对合成废水的处理中,72h后废水中NH4+-N、NO3--N和COD的去除率分别达84.4％、86.91％和81.36％.     

高山芽胞杆菌B901分离鉴定及安全性研究

在细菌分离鉴定的研究过程中,发现了 1株具有抑菌活性的芽胞杆菌,命名为B901.经16SrRNA测序以及同源性比较,鉴定其为高山芽胞杆菌(Bacillus altitudinis).对该菌进行生物学特性分析、抑菌试验和安全性试验.结果表明:B901菌株对金黄色葡萄球菌和大肠杆菌具有抑菌活性,B901抑菌产物产生较快,培...     

3株分离自高寒草甸根际芽孢杆菌的分子鉴定及其生物活性

为青海省退化草地植被恢复、提高地上生物量筛选优良的生防芽孢杆菌菌源,采用16SrDNA及gyrB基因序列分析法,鉴定分离自青海省黄南州高寒草甸高山嵩草(Kobresia pygmaea)根围的3株芽孢杆菌菌株;平板对峙试验检测菌株拮抗病原真菌活性;菌悬液(细胞浓度为106 cfu·mL-1)浸种以检测菌株对植物催芽及促生效果;CMC、Gram染色法及DNS法检测菌株降解纤维素活性,并扩增菌株降解纤维素酶编码基因。结果表明,菌株HNC3被鉴定为短小芽孢杆菌(Bacillus pumilus),菌株HNC8、HNC11被鉴定为苏云金芽孢杆菌(Bacillus thuringiensis);3株菌株对小麦赤霉病菌(Fusarium graminearum)、瓜类枯萎病菌(Fusarium oxysporum)具有明显的拮抗活性(抑菌圈平均直径均≥11mm);3株菌株对小麦(Triticum aestivum)种子具有明显的催芽及促生效果;小麦种子经菌株HNC3菌悬液处理后,芽长、根长增加率分别达55%、80%;菌株HNC3降解纤维素酶活高达367.51U·mL-1,并在该菌株中扩增到地衣聚糖酶(Lichenase,blg1S)、甘露糖苷酶(mannosidase,ManSig)、木聚糖酶(xylanase,xynD、xynB)4个纤维素酶编码基因。分离自青藏高原高寒草甸根围的3株芽孢杆菌兼具拮抗、催芽、促生及降解纤维素活性,在高原草地植被恢复、生态农牧业中具有一定的研究及应用潜力。     

红三叶根际溶磷菌的筛选与培养基优化

为从岷山红三叶根际土壤中筛选出大量高效溶磷菌株,本研究采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基进行溶磷菌株的初步筛选和优良溶磷菌株16SrRNA基因序列鉴定,并对分离筛选效果较好的PKOC2培养基进行响应面优化。结果表明:采用Pikovaskaia’s、PKOC1和PKOC2三种溶磷培养基,从红三叶根际分离出26株溶磷菌株;经16SrRNA基因序列对7株优良溶磷菌株进行初步比对鉴定,初步鉴定结果为:菌株MHS4为蜡样芽胞杆菌(Bacillus cereus);菌株MHS7和MHS19为枯草芽胞杆菌(Bacillus subtilis);菌株MHS27为苏云金杆菌(Bacillus thuringiensis);菌株MHS30为荧光假单胞菌(Pseudomonas fluorescens);菌株MHS31为盖氏假单胞菌(Pseudomonas gessardii);菌株MHS49为产酸克雷伯菌(Klebsiella oxytoca)。PKOC2溶磷培养基优化配方为:葡萄糖18.19g·L~(-1),Ca_3(PO_4)25g·L~(-1),MgCl2·6H_2O 3.21g·L~(-1),MgSO_4·7H_2O 0.25g·L~(-1),KCl 0.2g·L~(-1),(NH_4)_2SO_40.08g·L~(-1)。红三叶根际溶磷菌资源丰富,优化后的溶磷培养基为高效溶磷菌资源筛选提供资源支持。     

动物粪便中纤维素分解菌的分离和鉴定

纤维素分解菌可用于动物微生态添加剂和除臭剂。此试验采用羧甲基纤维素平板法及刚果红染色法,从鹅的粪便中初步分离筛选出7株能够降解纤维素的细菌,结合摇瓶发酵法进行复筛,最终选出3株羧甲基纤维素酶活性高的菌株,并对其进行形态学和16SrRNA鉴定。结果表明:3株纤维素分解菌的菌落形态不一,但其表面均微粗糙,革兰氏染色均呈阳性,短杆状或细杆状,有芽孢;结合16SrRNA鉴定结果,最终将分离到3个菌株鉴定为芽孢杆菌属的地衣芽孢杆菌、蜡样芽孢杆菌和沙福芽孢杆菌,分别命名为Bacillus licheniformis G-1、Bacillus cereus G-4和Bacillus safensis G-10。     

产芽孢木质素降解菌株N13的分离与鉴定

研究旨在从牛粪中筛选出1株对木质素有降解能力的细菌,并对其鉴定到种。对其形态和生理生化特征进行研究,发现其特征与解淀粉芽孢杆菌（Bacillus amyloliquefaciens）很相近。将所测得的16S rDNA序列用BLAST软件在GenBank数据库中进行比对,选取序列相似性高的菌株,用ClustalX（1.8）软件进行16S rDNA相似性分析,Neighbor-Joining法构建系统发育树。N13菌株与Bacillus amyloliquefaciens的相似性达到99.85%,鉴定该菌为解淀粉芽孢杆菌。     

Toxinogenic Bacillus pumilus and Bacillus licheniformis from mastitic milk

To elucidate the occurrence of heat-stable toxin-producing strains among mastitic Bacillus isolates, 100 milk samples of mastitic cows from different parts of Finland were screened. Bacillus was identified as the major organism in 23 samples. Toxinogenic Bacillus isolates identified by sperm cell motility inhibition assay were isolated from six samples. Four isolates belonged to the species Bacillus pumilus and two to Bacillus licheniformis. The toxic substances were heat-stable and soluble to methanol thus being of non-protein nature. The methanol extracted substances disrupted the sperm cell plasma membrane permeability barrier at exposure concentrations of 1-15 microg ml(-1) (B. pumilus) or 20-30 microg ml(-1) (B. licheniformis). The toxic properties of the two mastitic B. licheniformis strains were similar to those of B. licheniformis strains known to produce the lipopeptide lichenysin A and the synthetase genes lchAA, lchAB and lchAC for lichenysin were found in the mastitic strains by PCR. Toxin synthetase genes for the syntheses of lichenysin or surfactin were searched but not found in the toxic B. pumilus strains. The ribopatterns of the mastitic B. pumilus and B. licheniformis isolates were similar to those of the toxinogenic strains described earlier from food poisoning incidents and contaminated indoor air. B. licheniformis and B. pumilus survive pasteurization and other heat treatments as spores. Toxin-producing strains of these species in the dairy production chain may thus be of food safety concern.     

生牛乳中芽孢杆菌的分离鉴定及其耐热性

生牛乳杀菌后残留少数芽孢具有极强耐受性,在一定条件下芽孢大量繁殖,使牛乳发生变质,严重影响产品品质和消费者健康。采集并选择含有芽孢杆菌的生牛乳样品,进行需氧芽孢杆菌和嗜热需氧芽孢杆菌的分离,通过形态学、生理生化和分子生物学方法对分离菌株进行鉴定,同时对分离到的嗜热需氧芽孢杆菌进行耐热性研究。结果表明：共分离出10?株芽孢杆菌,其中3?株为嗜热需氧芽孢杆菌,7?株为需氧芽孢杆菌,其中能耐受115?℃/15?min热处理的有凝结芽孢杆菌和克劳氏芽孢杆菌,121?℃/20?min热处理可以将生牛乳中芽孢杆菌全部杀灭,致死率达100%,芽孢数下降4?个数量级;生牛乳中芽孢杆菌的优势菌有枯草芽孢杆菌、蜡样芽孢杆菌、短小芽孢杆菌、地衣芽孢杆菌、环状芽孢杆菌、明胶芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌和克劳氏芽孢杆菌;耐热性较强的有凝结芽孢杆菌和克劳氏芽孢杆菌,最高能耐受115?℃/15?min热处理。     

几株高产蛋白酶菌株的分离鉴定及初步研究

从采集的245份海洋生物及环境样本中筛选高产蛋白酶菌株,进行形态学和分子生物学鉴定,并对其生物学特性和菌株间的配伍效果进行研究。结果显示,所分离的菌株中Pe、Pc为枯草芽孢杆菌,Ps为地衣芽孢杆菌。培养16 h后进入生长平台期,Pc和Ps菌株生长的最适pH均为7.0,最适生长温度均为35℃;菌株Pe的最适pH为6.5,最适生长温度是30℃。菌株Pe与Ps 1:1配伍后产酶效果最好。     

青海省海晏县线叶嵩草内生细菌的生物功能鉴定及测定

对线叶嵩草（Kobresia capillifolia）内生细菌部分生物功能进行鉴定及测定;采用组织分离法从青藏高原青海省海西线叶嵩草根部分离出10株内生细菌。结果表明,10株内生细菌均能固氮,2G1和2G10能够产IAA;其中2G2、2G3、2G4、2G5、2G6、2G7、2G8和2G9对指示菌均有拮抗作用,对马铃薯炭疽菌Colletotrlchum coccodes的拮抗作用最强,抑菌率达88.58%。通过培养性状和形态特征,结合16S rDNA序列相似性分析,将2G1和2G10分别鉴定为苏云金芽孢杆菌Bacillus thuringiensis和Bacillus simpix,2G2和2G9鉴定为亲甲基醇芽孢杆菌Bacillus methylotrophicus,2G3和2G6鉴定为解淀粉芽孢杆菌Bacillus amyloliquefaciens,2G4和2G5鉴定为云南苏铁芽孢杆菌Bacillus siamensis,2G7和2G8鉴定为枯草芽孢杆菌Bacillus subtilis。该研究结果为青藏高原极端生境牧草根部内生细菌的多样性及功能菌株的开发提供依据。     

Identification of Mycobacterium bovis isolates using a monoclonal antibody

A rapid immunoperoxidase slide assay for the identification of Mycobacterium bovis culture isolates is described. The monoclonal antibody used in this assay is specific for the M. tuberculosis complex of organisms. All M. bovis isolates tested, including 151 separate field isolates of M. bovis were positive as were 11 out of 12 M. tuberculosis strains and 4 out of 6 Bacillus Calmette Guerin (BCG) strains. One strain each of M. africanum and M. microti was negative. This assay provides a considerable improvement in both time and expense over the conventional methods of biochemical typing of M. bovis.     

奶牛子宫内膜炎致病菌的分离与鉴定及耐药性分析

本试验通过对山东省4个大型奶牛场的60头子宫内膜炎患牛进行调查、采样,并用常规方法对细菌进行分离、培养、鉴定。共分离到细菌59株,其中分离率最高的是埃希氏大肠杆菌10株(16.95%)、葡萄球菌属8株(13.6%)、芽孢杆菌属6株(10.2%)、链球菌属5株(8.5%)(其中溶血性链球菌2株)、棒状杆菌属5株(8.5%)、不动杆菌属5株(8.5%)、双球菌5株(8.5%)、肠杆菌属4株(6.8%)、沙门氏菌3株(5.1%)、克雷伯氏杆菌属2株(3.4%)、耶尔森菌属2株(3.4%)、摩拉杆菌属1株(1.7%),而且分离到了梭菌属3株(严格厌氧菌)。药敏试验分析结果显示,大多数致病菌对喹诺酮类药物、阿莫西林、土霉素及氨苄青霉素等耐药率较高,而对头孢菌素类和氨基糖苷类药物耐药率则较低。     

Use of mixed cultures of biocontrol agents to control sheep nematodes

Biological control is a promising non-chemical approach for the control of gastrointestinal nematodes of sheep. Use of combinations of biocontrol agents have been reported to be an effective method to increase the efficacy of biological control effects. In this study, combinations of either two Bacillus thuringiensis (Bt) or Clonostachys rosea (C. rosea) isolates and Bt+C. rosea isolates were evaluated in vitro in microtitre plates for their biocontrol activity on sheep nematodes. The Baermann technique was used to extract the surviving L3 larval stages of intestinal nematodes and counted under a dissecting microscope to determine the larval counts. Results indicate that there was a significant reduction of nematode counts due to combination of biocontrol agents (P<0.001). Combinations of Bt isolates reduced nematodes counts by 72.8%, 64% and 29.8%. The results revealed a control level of 57% when C. rosea isolates P3+P8 were combined. Combination of Bt and C. rosea isolates B10+P8 caused the greatest mortality of 76.7%. Most combinations were antagonistic, with only a few combinations showing an additive effect. None were synergistic. The isolate combinations were more effective than when isolates were used alone.     

Prevalence of coagulase-negative staphylococci in bovine mastitis in Zimbabwe

This study was carried out to determine the prevalence of coagulase-negative staphylococci in clinical and subclinical mastitis in commercial and small-scale farms in Zimbabwe. Thirty five quarter milk samples from clinical mastitis cases and 371 quarter milk samples from cows with subclinical mastitis were cultured for bacterial pathogens. The most frequent pathogens isolated in clinical mastitis were the enteric bacteria (31.4%), followed by coagulase negative staphylococci (22.9%) and then Staphylococcus aureus (17.1%), whereas in subclinical mastitis S. aureus (34.2%) and coagulase-negative staphylococci were (33.2%) the most common. Bacillus species were only isolated in milk samples from subclinical mastitis. Coagulase-negative staphylococci were observed in mixed infections with other bacteria in only 2.2 of the 406 milk samples from clinical and subclinical mastitis where they were isolated together with Bacillus species in 6 of the 9 mixed infection cases. About 95% of the milk samples from which 131 coagulase-negative staphylococci were isolated had correspondingly high somatic cell counts. The coagulase-negative staphylococci isolated most frequently were S. chromogenes (7.9%), S. epidermidis (7.4%) and S. hominis (5.9%). They were all associated with high somatic cell counts. All the coagulase-negative staphylococci isolates were susceptible to cloxacillin and erythromycin, and more than 90% of the isolates were susceptible to neomycin, penicillin and streptomycin. The highest resistance was to tetracycline (17.6%), followed by lincomycin (13.7%). About 8% of the isolates were resistant to both penicillin and streptomycin.     

Ribosomal RNA-based analysis of the bacterial flora from the conjunctivae of cattle with bovine keratoconjunctivitis (BKC)

Bovine keratoconjunctivitis (BKC), colloquially referred to as 'pinkeye', is a disease affecting cattle worldwide; it costs cattle producers millions of dollars in economic loss annually. While Moraxella spp. are the primary etiologic agent of pinkeye, surveys of flora from the conjunctivae of livestock from around the world have indicated that a variety of bacterial commensals occupy this niche. We used molecular biology-based methods to determine the composition of bacterial flora in the conjunctivae of normal dairy and beef cattle from Maryland (n=113), and beef cattle with clinical BKC from Louisiana (n=42). Three regimens were used: 16S rRNA PCR and DGGE analysis of amplicons; 16S rRNA PCR and cloning of amplicons into Escherichia coli followed by screening and sequencing of clones harboring inserts; and culture of bacteria on chromogenic agar followed by 16S rRNA PCR and sequencing. Most taxa were comprised of saprophytes found in the environment, such as Bacillus, Pantoea, E. coli, and Exiguobacterium. Moraxella spp. were infrequently observed. Some species, such as Propionibacterium acnes, represent taxa not previously associated with the conjunctivae. Bacillus pumilus and Bacillus licheniformis isolates from the conjunctivae of Maryland cattle were genetically distinct from isolates previously implicated in septic infections in cattle at the same location. We conclude that employing 16S rRNA-based methods for bacterial identification can be useful in defining the flora present in the conjunctivae of normal cattle, and those with BKC.     

家蚕肠道细菌种群结构分析

用普通培养基和稀释培养基从家蚕4龄幼虫肠道中分离到109株细菌,通过16S rDNA-RFLP分析后,得到14个不同类群。从各个类群中随机选取代表菌株进行16S rDNA序列测定和系统发育分析,结果表明,家蚕肠道细菌主要分布在芽孢杆菌属(Bacillus)、节杆菌属(Arthrobacter)、肠杆菌属(Enterobacter)、微杆菌属(Microbacteri-um)、葡萄球菌属(Staphylococcus)、短杆菌属(Brevibacterium)、纤维微杆菌属(Cellulosimicrobium)、肠球菌属(Entero-coccus)、短状杆菌属(Brachybacterium)、棒状杆菌属(Corynebacterium)10个已知细菌属和另外1个分支中,揭示了家蚕肠道细菌种群的多样性。