桑树根际硅酸盐细菌的分离鉴定及解钾能力测定

利用选择性培养基,从桑树根际土样中分离获得29个具有解钾能力的细菌分离株,经rep-PCR DNA指纹分析得到24株硅酸盐细菌。通过解钾能力测定,筛选出FK2、FK3、FK8、FK11、FK4′、FK23和PK187个具有较强解钾能力的菌株,其中FK2菌株的解钾能力最强,有效态钾增长41.79%。对这7株具有较强解钾能力的细菌进行菌落形态特征观察及16S rDNA序列测定和同源性分析:FK2菌株为假单胞菌属(Pseudomonas sp.),FK3和FK23菌株为中华根瘤菌属(Sinorhizobium sp.),FK11和FK8菌株为根瘤菌属(Rhizobiumsp.),FK4′菌株为中慢生根瘤菌属(Mesorhizobium sp.),PK18菌株为屈挠杆菌属(Flexibacter sp.)。     

纤维素降解细菌的筛选、生物学特性及降解效果

为了从东祁连山高寒草甸土壤中分离筛选纤维素分解细菌,本研究根据在羧甲基纤维素钠培养基和滤纸平板培养基上的生长情况,初步筛选出3株具有较强纤维素分解能力的细菌,并对其生长条件进行了初步研究,结果表明,3株菌的最适生长温度范围为25~30℃;最适生长pH因菌种不同位于5~8之间;最适生长盐浓度位于4%~5%。菌株X1-2具有较好降解特性,根据形态观察、革兰氏染色及16SrRNA系统发育比较,鉴定该菌为芽孢杆菌(Bacillus sp.),是一株十分具有开发生产纤维素酶能力的菌株。     

一株纤维素降解菌的筛选、鉴定及对饲料粗纤维降解效果的研究

通过筛选高活性纤维素酶产生菌,为微生物纤维素酶制剂的开发提供理论依据。采用刚果红染色法从绵羊粪便中分离筛选纤维素降解菌,用DNS法测定酶活力,根据酶活力复筛后,分离得到一株能够产生高活力纤维素酶和β-葡萄糖苷酶的目的菌B5。通过形态学观察和16S rDNA序列测定,对B5菌株进行种属鉴定;应用B5菌株对四种纤维性饲料进行消化降解实验,探究菌株B5降解饲料粗纤维的特性。形态学观察确定B5菌落符合放线菌的特征;分子学鉴定其16S rDNA序列全长1 585 bp,在进化关系上与链霉菌属(Streptomyces sp.)聚成一簇,同源性高达99%。鉴定B5菌株属于链霉菌属成员。B5菌株对饲料粗纤维降解效率与酶活力显著相关,对饲料粗纤维降解效率与饲料中纤维素含量相关性不明显。     

4株家蚕丝胶蛋白降解细菌的分类鉴定与培养温度研究

从缫丝厂废水池分离获得4株对家蚕丝胶蛋白具有较强降解活性的细菌。通过菌落的形态特征和生理生化特征分析及16S rDNA序列测定与同源性分析,对4株细菌进行分类鉴定的结果是:S45、Y3菌株为蜡状芽孢杆菌(Bacillus cereus),S68、X10菌株为短小芽孢杆菌(Bacillus pumilus)。温度对丝胶蛋白降解细菌的降解活性有影响,在培养温度为30℃时,S45、Y3、S68、X10菌株对家蚕丝胶蛋白的降解活性最高,其降解率分别为26.52%、20.27%、21.55%和28.86%。由于芽孢杆菌具有很强的耐热等抗逆能力,所以4株丝胶蛋白降解细菌有望应用于缫丝厂排放废水的处理。     

桑树根际固氮细菌的分离鉴定及固氮酶活力测定

利用固氮细菌可降低桑园化肥使用量和提高桑叶产量与品质。采用选择性培养基,从桑树根际分离获得24个具有固氮能力的细菌分离株,以rep-PCR基因指纹分析聚类为18个聚类群。经固氮酶活性测定,PA19、PA2和PK1菌株具有较强的固氮酶活性。利用菌落形态特征观察及16S rDNA碱基序列测定和同源性分析,对3株细菌进行鉴定的结果是:PA19菌株为中慢生根瘤菌属(Mesorhizobium sp.),PA2菌株为假单胞菌属(Pseudomonas sp.),PK1菌株为土壤杆菌属(Agrobacterium sp.)。     

大庆地区猪场中除臭菌的分离筛选及初步鉴定

为了获得高效除臭菌株,试验在大庆地区不同猪场采集猪粪、土壤和淤泥样本,通过感官初筛试验及定性复筛试验筛选除臭菌株。结果表明:共筛选出2株除臭菌株,X-3菌株的氨气与硫化氢去除率为46.6%、6.8%;X-5菌株的氨气与硫化氢去除率为5.6%、54.5%;经染色镜检与生化鉴定,X-3菌株为短状杆菌属(Brachybacterium sp.),X-5菌株为副球菌属(Paracoccus sp.)。     

桑树内生细菌的分离及生防益菌的筛选

对不同品种健康桑树植株组织中的内生细菌进行分离,共得到229个菌株。探讨了较合理的内生细菌分离纯化方法,对内生细菌数量进行了测定。结果表明,桑树根、茎、叶柄、叶片、花蕾等组织内均存在大量的内生细菌,且不同品种及组织中内生细菌的数量均存在差异。离体抑菌作用测定表明,229株桑树内生细菌中,有42株(18.3%)菌株对桑树炭疽病菌有拮抗作用,有25株(10.9%)菌株对桑粘格孢菌有拮抗作用;以上67株菌种又有8株菌株对多种病原真菌都有抑菌作用,表现出较强抑菌活性,具有作为生防菌的应用潜能。对8株内生拮抗菌株进行了细菌学鉴定,结果表明,菌株G21、G49、Y12和J26归属于芽孢杆菌属(Bacillussp.),G82和J50归属于假单孢菌属(Pseudom onas sp.),Y33归属于欧文氏菌属(Erwinia sp.),B19归属于短小杆菌属(Curtobacterium sp.)。     

玉米秸秆降解真菌的筛选及鉴定

为了筛选到高效玉米秸秆降解真菌。实验以树林、稻田、玉米田等地土层下5 cm取得的腐殖质土壤为样品进行富集培养,通过刚果红染色法进行初步筛选,滤纸崩解法复筛,分别以羧甲基纤维素钠和玉米秸秆粉为唯一碳源,经30℃、120 r/min摇床培养72 h测定纤维素酶活性,液态发酵方法考察实际降解能力,通过分子学方法鉴定菌株。获得2株具有较高纤维素酶活和玉米秸秆降解能力的真菌菌株Z_(2-6)和Z_(3-5),液体发酵15 d后,秸秆降解率分别是59.43%和59.13%,通过分子学鉴定都为尖孢镰刀菌。真菌菌株Z_(2-6)和Z_(3-5)是具有较好研究价值的玉米秸秆纤维素降解真菌。     

单宁降解菌的筛选及降解研究

从自然界中分离、筛选降解单宁的微生物,并研究对单宁降解的影响。在含有梯度浓度单宁的无糖察氏培养基上划线分离,分离出的菌株在250 ml锥形瓶中液体发酵,测定单宁的降解率。对分离出的3株菌表型观察,发现有2株青色霉菌,命名为SL-2和SL-4,1株黑色曲霉菌,命名为SL-5,分别接种含单宁0.5 g/l的无糖察氏培养基,28℃条件下振荡培养72 h,降解率分别达到58.6%、63.9%和49.4%。因此可知,我们成功分离出了3株具有单宁降解能力的微生物菌株。     

一株茶园草甘膦降解菌的筛选与鉴定

采用生物富集、农药驯化等方式从茶园草甘膦污染土壤中筛选出一株潜在降解菌株CGL-1。该菌株经过形态学观察以及构建系统进化树等方法,初步鉴定为微球菌属Micrococcus sp.。菌株CGL-1能在以草甘膦为唯一碳源的无机盐培养基中生长良好,对草甘膦的最高耐受浓度为1200 mg/L,显示对草甘膦具有较好的降解效能。草甘膦降解菌株的筛选有望为草甘膦的生物降解提供新的微生物资源。     

Serotypes and anti-microbial susceptibility of Plesiomonas shigelloides isolates from humans, animals and aquatic environments in different countries

A total of 73 strains of Plesiomonas shigelloides isolated from humans (24 strains) animals (21 strains) and aquatic environment (28 strains) were determined for their O:H serotype and susceptibility to 18 anti-microbial substances and to the vibriostatic agent O/129. Of all strains, 86.3% were typeable by the O and 94.5% by the H anti-sera used. The serotype distribution was heterogeneous within a country and between the countries. Of the 57 different serotypes identified, O11:H2 (2 strains), O22:H3 (4 strains), O35:HH11 (2 strains), O52:H3 (2 strains) and O90:H6 (2 strains) were found among isolates from humans and animals (mainly in cats) in Finland and Cuba, and O23:H1a1b (3 strains) among isolates from environmental sources in Slovak Republic and Italy. Most (93-100%) of all strains were susceptible to all anti-microbials tested but resistant (92-96%) to the broad-spectrum penicillins (ampicillin, mezlocillin). No correlation between anti-microbial resistance patterns and serotypes was found.     

The pathogenicity and cultural characteristics of virulent, intermediate and benign strains of Bacteroides nodosus causing ovine foot-rot

The relationship between the cultural and biochemical characteristics of 22 strains of Bacteroides nodosus and their virulence for sheep was examined. Virulent, intermediate and benign strains were recognised. Although there was some relationship between virulence and colony morphology on hoof medium with 4% agar, colonies of one virulent and 4 intermediate strains resembled those of benign strains. However, on hoof medium with 2% agar and on blood Euonagar, colonies of this virulent and one intermediate strain differed from each other and the other 3 intermediate strains, which in turn differed from the benign. The degree of piliation, as assessed by electron microscopy, was not a reliable indicator of virulence in strains not possessing a beaded colony type. Together, the results of colony morphology and proteolytic tests such as zymogram, degrading proteinase and elastin-agar tests allowed better discrimination of virulent and benign strains. Intermediate strains generally possessed virulent protease activity. In strains with benign zymogram patterns, activity bands 2 and 3 were more labile than in strains with virulent patterns. The addition of CaCl2 to the culture medium resulted in greater stability of proteolytic activity, particularly with benign strains, and prevented the disappearance of protease activity in the band 5 position in virulent, intermediate and benign strains during prolonged incubation. There were slight differences in the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns of outer membrane proteins of some benign strains but those of intermediate category resembled virulent strains. There was some relationship between the apparent Mr of the pilin monomer on SDS-PAGE gels and serogroup specificity.     

A method to differentiate between virulent and benign isolated of Bacteroides nodosus based on the thermal stability of their extracellular proteinases

A rapid modification of the hide powder azure proteinase degrading test was devised to distinguish between benign and virulent strains of Bucteroides nodosus grown as cultures on either liquid or solid media. The assay was based on the difference in stability, at 60 degrees C, of the enzymes released in the growth medium. The assay can be completed in four hours. Results for ovine virulent strains and their culture variants were similar and were clearly distinguishable from those of the ovine benign, bovine, deer and goat isolates which were similar to each other. The proteinase isozymes from cultures of the deer and goat isolates were separated by polyacrylamide gel electrophoresis and their molecular weights determined.     

水貂出血性肺炎大肠杆菌毒力基因测定和H基因分型

为了分离鉴定引起水貂出血性肺炎的大肠杆菌,并对其致病性、血清型和毒力基因进行鉴定.本研究主要通过细菌分离鉴定试验对具有典型肺炎症状的死亡水貂的病原体进行分离,利用16S rRNA对进行细菌鉴定,并通过PCR方法对分离细菌的血清型和毒力基因进行检测,分析分离菌对动物的致病性.结果显示:28例水貂肺炎病例中分离出8株大肠杆...     

Serotypes and intimin types of intestinal and faecal strains of eae+ Escherichia coli from weaned pigs

Enteropathogenic Escherichia coli (EPEC) that are known to cause severe diarrhoea in children and young rabbits are well characterized, but there are few reports on the serotypes and intimin (eae) types of EPEC in weaned pigs. Based on detection of the eae gene by PCR and by DNA-hybridisation with LEE specific gene probes, 20 intestinal and 17 faecal eae(+) strains from diarrhoeal (164) and non-diarrhoeal (57) weaned pigs from 13 Hungarian farms, representing 12.8% of diarrhoeal and 14.0% of non-diarrhoeal pigs, were identified. The dominant serotype was O123:H11 (40%) among intestinal, and O108:H9 (23%) among faecal strains. The majority (85%) of the intestinal strains possessed eae-beta and 10% carried eae-gamma gene. In contrast, significantly (p<0.025) fewer faecal strains (53%) harboured the eae-beta gene, and 23% were eae-gamma positive. In vitro adhesion tests of intestinal and faecal eae(+) strains indicated adhesion of 20/37 of the strains to PK15 (porcine kidney) cells while only 3/37 strains adhered to HeLa cells. The ultrastructure of intimate bacterial attachment of representative porcine eae(+) strains to PK15 cells showed no pedestal formation, in contrast to the human EPEC (O127:H5, eae-alpha) strain. In conclusion, the data do not demonstrate a significant role for the eae(+)E. coli in porcine post-weaning diarrhoea, but provide new information on a dominant porcine serotype (O123:H11, eae-beta), and on differences of serotypes and intimin types of porcine eae(+) strains according to their site of isolation. Furthermore there was an indication that the PK15 cell line could be used as a model to study in vitro adherence of eae(+)E. coli of some human and porcine origin.     

3株H9亚型禽流感病毒的分离与鉴定

为了获得H9亚型禽流感病毒(AIV)流行毒株并掌握流行毒株的分子特征和致病性,采用病毒分离、血凝性试验、鸡胚半数感染量(EID₅₀)测定、HA基因序列分析、致病性试验、交叉保护性试验等对3份临床疑似H9亚型AIV感染病料进行了研究。结果:3份临床病料样品可引起10日龄SPF鸡胚规律性死亡,3株分离毒株对1%鸡红细胞的凝集效价分别10log₂、11log₂和10log₂;对SPF鸡胚的EID₅₀分别为10^-8.83/mL、10^-9.50/mL和10^-9.0/mL;与2018年上海分离毒株亲缘关系较近,进化树处于同一分支;对SPF雏鸡的发病率分别为80%、100%和90%;均未引起SPF雏鸡死亡;彼此之间具有100%的交叉保护率,商品化禽流感(H9亚型)灭活疫苗对3株分离毒株的保护率分别为100%、90%和100%。本试验成功分离鉴定到了3株低致病性H9亚型AIV流行毒株,并证实当前商品化疫苗对H9亚型AIV流行毒株仍具有较好的保护效果。     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.     

牛源大肠杆菌O157：H7的分离及毒力基因鉴定

从2个牛场采集新鲜粪便,增菌后,免疫磁珠富集,涂布筛选性培养基,挑取可疑菌落用rfbE/fliC二重PCR和血清学方法鉴定。设计毒力基因stx1、stx2、eae、hlyA和tccp相应引物,针对O157：H7对分离株进行PCR鉴定。口服攻毒链霉素处理的BALB/c小鼠明确分离株致病性。结果显示,成功分离到7株出血性大肠杆菌O157：H7,并且有1株迟缓性发酵山梨醇麦康凯培养基。毒力基因检测显示,其中6株毒力因子表型为stx1-stx2＋eae＋hlyA＋tccp＋,另有1株表现型为stx1＋stx2＋eae＋hlyA＋tccp＋,各分离株tccp基因均为阳性,但携带的重复片段数量有差异。所采集样品中肠出血性大肠杆菌O157：H7的检出率高达12%。1×1010 CFU同剂量口服接种经PBS洗涤的5株O157：H7分离株全菌,小鼠存活率有差异分别为40%,50%,60%,20%,50%,各分离株在小鼠体内排菌时间也有差异分别为攻毒后7,9,13,13,15d。     

Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.