南宁市商业肉鸭鸭疫里默氏杆菌病的调查研究

2004年12月~2005年12月间,对南宁市及其周边地区26个商业肉鸭群鸭疫里默氏杆菌(R iem erella ana tip estif er)病的发生情况进行了调查。从167羽具有浆膜炎病变的病死鸭中,应用巧克力培养基的分离培养,以及对分离株进行的生化试验,细菌外膜蛋白基因的聚合酶链式反应(PCR)扩增的鉴定,结果分离到74株鸭疫里默氏杆菌;通过玻片凝集试验鉴定,所有分离株均属于血清2型;在鸭疫里默氏杆菌阳性病料中同时分离到2 3株大肠杆菌,混合感染率高达3 1%;脑组织的分离率明显高于肝脏组织的;在2~6周龄的发病时间段里,肉鸭对鸭疫里默氏杆菌的感染表现出明显的年龄抵抗力;常用药物的敏感性试验表明大多数分离株对头孢唑啉、头孢啦啶、头孢哌酮表现高度敏感。     

抗马铃薯晚疫病菌的粘细菌菌株X6-Ⅱ-1的分离鉴定、拮抗活性及发酵条件的优化

马铃薯晚疫病(potato late bright)是马铃薯(Solanum tuberosum)最为严重的病害,其病原菌为致病疫霉(Phytophthora infestans)。为了从土壤样品中筛选出对马铃薯晚疫病具有拮抗作用的粘细菌,本实验利用大肠杆菌(Escherichia coli)划线诱导法从土样中分离菌株,通过平板对峙法筛选具有抗马铃薯晚疫病菌活性的粘细菌,结合形态观察、生理生化特征和16S r RNA基因序列分析确定菌株的分类地位。采用称重法测定菌株生长曲线,滤纸片法确定活性物质的分布,并通过单因素分析与正交优化相结合的方法对菌株的发酵参数进行了研究。共从土壤样品中共分离得到5株粘细菌,其中有3株对致病疫霉表现出拮抗活性,菌株X6-Ⅱ-1的拮抗活性最强,致病疫霉菌丝生长边缘与菌株X6-Ⅱ-1菌饼最短距离可达到5 mm,经鉴定为叶柄粘球菌(Myxococcus stipitatus)。该菌株可以杀死并溶解大肠杆菌,且可以抑制枯草芽胞杆菌(Bacillus subtilis)的生长。拮抗致病疫霉的活性物质主要存在于细胞外,产活性物质的最适发酵条件为:接种量10%,摇床转速180 r/min,培养温度为30℃,发酵时间为7 d。粘细菌菌株X6-Ⅱ-1能够产生抗马铃薯晚疫病菌的活性物质,具有开发成抗马铃薯晚疫病生物农药的潜在价值。本研究为活性物质的分离鉴定以及抗马铃薯晚疫病农药的研发提供基础数据。     

芒果叶提取物体外抑菌及抑制鸡新城疫病毒增殖试验研究

进行了芒果叶水提物对部分畜禽常见致病菌的体外抑制试验以及对10日龄鸡胚人工感染鸡新城疫病毒(NDV)的抑制试验。抑菌试验结果表明,芒果叶水提物对大肠杆菌、多杀性巴氏杆菌、鸡白痢沙门氏菌、猪丹毒杆菌43-6混43-8、金色葡萄球菌86003、鸭疫里默氏杆菌、链球菌等致病菌有良好的抑菌效果。抑制NDV增殖试验结果表明,NDV强毒组在48h时的鸡胚保护率为10.00%,2.500g/mL芒果叶生药+NDV组的鸡胚保护率为63.64%,两者差异显著(P<0.05);1.250g/mL+NDV组、0.625g/mL+NDV组、0.313g/mL+NDV组的鸡胚保护率分别为72.72%、70.00%、69.23%,与强毒组相比,差异极显著(P<0.01),可见芒果叶水提物对NDV增殖具有抑制作用。     

近年来大肠杆菌K88ac菌毛的变异和生物学特性

2000~2005年，从猪大肠杆菌病 (colibacillosis)中分离到一些表达K88菌毛的大肠杆菌(Escherichia coli )，这些分离株只与K88 a因子单抗反应，而不与K88 b、c和d因子单抗反应。分离的菌株 (13/16)以O149为常见血清型，且全部拥有STb毒素基因，通过K88常规血清交叉吸收试验、SDS-PAGE和Western印迹，表明这些菌株不仅与K88ac参考菌株C83907制备的c因子血清反应，而且与以分离株SEC586制备且经K88ab、K88ac和K88ad参考菌株吸收后的血清也反应，表明这些分离株仍为K88ac大肠杆菌。对新近分离的SEC464、SEC525、SEC586、SEC799和EC910株及80年代我国分离的TM128株的K88主要亚单位结构基因faeG进行克隆、测序，发现新近分离株的faeG基因由846对核苷酸组成，编码菌毛主要亚单位的261个氨基酸及21个氨基酸的信号肽，比国内外报道的K88ac FaeG亚单位 (262个氨基酸)少了一个氨基酸，比K88ab、K88ad (264个氨基酸)少了3个氨基酸。TM128株的FaeG氨基酸序列与K88ac(M29375)的同源性为100.0%，SEC464、SEC525、SEC586、SEC799和SEC910株的FaeG亚单位氨基酸序列的同源性为97.7%~99.6%，它们与K88ac的同源性为94.6%~96.6%；与K88ab的同源性为90.0%~91.6%；与K88ad的同源性为87.0%~88.9%。 结果表明新分离的K88ac大肠杆菌黏附素主要亚单位已发生了部分变异。     

我国部分地区奶牛乳房炎源大肠杆菌生物学特性及耐药性分析

调查奶牛乳房炎源大肠杆菌的某些生物学特性及其耐药状况,以提高药物疗效,减少牛乳中药物的残留。本研究从国内7个省、市、自治区部分地区患乳房炎奶牛的乳样中分离纯化与鉴定出95株大肠杆菌(Escherichia coli),并对大肠杆菌分离株进行O血清型鉴定、小鼠(Mus musculus)致病性试验以及抗菌药物敏感性分析。研究结果显示,95株大肠杆菌共鉴定出37种血清型,覆盖了54株分离株,另有2株自凝,39株未鉴定出型,较常见血清型为093和09;大肠杆菌分离株接种小白鼠剖检可见明显病变;95株大肠杆菌对16种抗菌药物中的8种药物耐药率超过50%,青霉素的耐药率甚至达到100%,同一菌株最多耐药14种,最少耐药2种,耐药6种以上菌株占到51.58%。表明,奶牛乳房炎源大肠杆菌分离株血清型比较复杂,且对多种药物产生不同程度的耐药性,存在着严重的多重耐药情况。本研究为奶牛乳房炎疫苗的研制和乳房炎的临床治疗提供理论依据。     

烟草黑胫病拮抗菌的筛选及其生物效应

烟草黑胫病是烟草生产中危害最严重的土传病害之一,生物防治具有抗病和环保的作用,获得高效拮抗菌株是进行生物防治研究的基础。本研究采用平板对峙法,在烟草黑胫病发生严重的田块中选取健康烟株,从其根际土壤中分离筛选到12株对烟草黑胫病病原菌Phytophthora parasitica var.nicotianae具有拮抗效果的菌株,抑菌率59.4%～71.1%。选择抑菌率最高的C-5菌株进行试验。经鉴定C-5菌株为多粘类芽孢杆菌(Paenibacillus polymyxa)。抗菌谱试验结果表明:C-5菌株不仅对黑胫病病原菌有较强的拮抗作用,同时对甜瓜?黄瓜枯萎病病原菌和辣椒疫病病原菌也具有拮抗作用。苗期盆栽试验结果表明:利用C-5菌株发酵制备的微生物有机肥能抑制烟草黑胫病的发生,苗期防治率达80%。本文首次报道了多粘类芽胞杆菌菌株对烟草疫霉有拮抗作用。     

土壤中细菌HC5的分离鉴定及抑菌活性测定

为了筛选出对马铃薯晚疫疫霉菌(Phytophthora infestans)有较强拮抗作用的生防细菌,从马铃薯种植地的土壤中分离细菌,并通过平板对峙法,测定分离细菌对马铃薯晚疫疫霉菌的拮抗作用。经大量拮抗实验,筛选出一株细菌HC5,经形态特征观察、生理生化特性测定和16S rRNA基因序列分析,确定该菌株为假单胞菌(Pseudomonas sp.)。抑菌试验结果表明,HC5菌株对辣椒疫霉菌(Phytophthora capsici)、黄瓜尖孢镰刀菌(Fusarium oxysporum)、马铃薯晚疫疫霉菌、辣椒炭疽菌(Colletotrichum capsici)均有一定的抑制作用,尤其对马铃薯晚疫疫霉菌的抑菌效果显著,抑菌率达89%。采用PCR方法对菌株HC5进行多种抗生素合成基因的检测,扩增到786 bp的硝吡咯菌素片段和587 bp的氢氰酸片段,表明菌株HC5能够代谢产生这两种抗生素,单一或协同发挥拮抗作用。研究表明,菌株HC5是一株具有开发潜力的生防细菌。     

2016 — 2020年西北部分地区马铃薯致病疫霉交配型分析

为合理选用抗性品种防治马铃薯晚疫病，采用对峙培养的方法对 2016 — 2020年分离自甘肃、青海和宁夏等3省(区)25个县(区)的694株马铃薯致病疫霉[Phytophthora infestans(Mont.) de Bary]进行了交配型测定。结果供试菌株中只检测到了A1交配型和A2交配型，未发现A1A2和SF(自育型)，其中A2交配型菌株为优势菌株，2016 — 2020年占所测菌株的比例分别为86.4%、96.3%、86.4%、80.9%、96.5%。5年间A2交配型菌株占比呈先升后降再升趋势。A2交配型比例的增大给马铃薯晚疫病的防控和抗病育种带来新的挑战。     

地黄强致病型病原菌的分离及其专化型鉴定

为探索地黄连作栽培中真菌病害的大规模爆发机制, 明确自毒物质所介导的土壤病原菌增多的根际生物学过程, 本研究利用PDA平板法, 经过显微鉴定和真菌ITS鉴定分析, 从地黄连作土壤和发病植株中分离到镰刀菌菌株31株, 并进行了致病性检测。结果表明, 串珠镰刀(菌株编号RPP009)、茄病镰刀(菌株编号CCS013)、禾谷镰刀(菌株编号CCS024)、雪霉叶枯病菌(菌株编号CCS038)和尖孢镰刀(菌株编号CCS043)在接种3 d后就表现出明显的致病能力, 严重影响地黄幼苗的株高(为对照的23.40%~30.20%)和植株鲜重(为对照的23.58%~38.94%), 并引起地黄植株变黄畸形, 根毛和须根减少, 在接种2周后致使全部植株枯萎死亡, 具有很强的致病性。专化型鉴定表明, 菌株Fusarium oxysporum (菌株编号CCS043)和Monographella nivale (菌株编号CCS038)只侵染地黄, 不侵染其他试验材料, 植株感病初期在中午时分下部叶片缺水萎蔫, 早晚又有所恢复, 如此反复, 至3~5 d, 叶片则不能恢复正常直立, 枯萎死亡。镜检发现, 感病植株茎部维管束变褐色至暗褐色, 后逐渐导致周围海绵组织破裂, 进而致使地下块根逐渐变黑腐烂, 初步判定两株菌株为地黄专化型病原菌。     

禾谷镰刀菌拮抗菌株的筛选及鉴定

禾谷镰刀菌引起的小麦赤霉病能够导致小麦产量的大幅降低和品质的严重损失。为探索赤霉病生物防治方法,本试验从扬花期被禾谷镰刀菌侵染的麦穗上分离得到28株芽孢杆菌,利用平板对峙法对其进行筛选,其中20株菌对禾谷镰刀菌表现出明显的拮抗作用。根据生理生化特性和16S rDNA序列分析,这20株拮抗菌分别被鉴定为甲基营养型芽孢杆菌(11株)、解淀粉芽孢杆菌(2株)、枯草芽孢杆菌(2株)、暹罗芽孢杆菌(3株)和特基拉芽孢杆菌(2株)。这些菌株能够有效降低小麦赤霉病的发病率、发病程度和病情指数,其中菌株JS62N、JS15E和JS29I能够将赤霉病发病率降低80%以上;菌株JS29I、JS62N、JS39C和JS39D能够将赤霉病发病程度降低80%以上。16株菌能将病情指数降低80%以上;所有菌株均能极显著增加小麦的百粒重,JS12Q效果最好(+101.4%)。本研究结果为小麦赤霉病的防治提供了生物防治材料。     

Identification of Escherichia coli from shellfish and related environments by automicrobic system

A total of 463 fecal coliform positive isolates obtained from shellfish and related samples gave typical Escherichia coli IMViC reactions. E. coli identifications for 458 (99%) of these isolates were confirmed using a combination of the Automicrobic System (AMS) and the API 20E system (reference system). The AMS (test system) identified 433 isolates as E. coli; the remaining 25 (5%) isolates were identified as E. hermanii by the test system and as E. coli by the reference system. Additional tests performed on the isolates identified as E. hermanii confirmed those AMS identifications to be incorrect.     

Occurrence and survival of coliform bacteria, Escherichia coli and Salmonella in various manure and compost

pp. 865–874
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 10² to 10⁶ cfu g¹ dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria ( E. coli , E. vulneria , Pantoea sp. and Buttiauxella agrestis ) were identified whereas isolates of Serratia marcescens , not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost.     

Comparison of the Transport of Tetracycline-Resistant and Tetracycline-Susceptible Escherichia coli Isolated from Lake Michigan

It was recently reported that tetracycline could enhance the mobility of manure-derived Escherichia coli within saturated porous media (Walczak et al. (Water Research 45:1681-1690, 2011)). It was also shown, however, that E. coli from various sources could display marked variation in their mobility (Bolster et al. (Journal of Environmental Quality 35:1018-1025, 2009)). The focus of this research was to examine if the observed difference in the mobility of manure-derived tetracycline-resistant (tet(R)) and tetracycline-susceptible (tet(S)) E. coli strains was source-dependent. Specifically, E. coli were isolated from Lake Michigan, and the influence of tetracycline resistance on Lake Michigan-derived E. coli was investigated through column transport experiments. Additionally, a variety of cell morphology and surface properties were determined and related to the observed bacterial transport behavior. Our experimental results showed that, consistent with previous observations, the deposition rate coefficients of the tet(R)E. coli strain was ~20-100% higher than those of the tet(S)E. coli strain. The zeta potential of the tet(R)E. coli cells was ~25 mV more negative than the tet(S)E. coli cells. Because the surfaces of the E. coli cells and the quartz sands were negatively charged, the repulsive electrostatic double-layer interaction between the tet(R)E. coli cells and the quartz sands was stronger, and the mobility of the tet(R)E. coli cells in the sand packs was thus higher. The tet(R)E. coli cells were also more hydrophilic than the tet(S)E. coli cells. Results from migration to hydrocarbon phase (MATH) tests showed that about ~35% more tet(S)E. coli cells partitioned to the hydrocarbon phase. As it was previously shown that cell hydrophobicity could enhance the attachment of bacterial cells to quartz sand, the difference in cell hydrophobicity could also have contributed to the observed higher mobility of the tet(R)E. coli cells. The size of the tet(R) and tet(S)E. coli cells were similar, suggesting that the observed difference in their mobility was not size-related. Characterization of cell surface properties also showed that tet(R) and tetS E. coli cells differed slightly in cell-bound lipopolysaccharide contents and had distinct outer membrane protein profiles. Such difference could alter cell surface properties which in turn led to changes in cell mobility.     

Multitest system for biochemical identification of Salmonella, Escherichia coli, and other Enterobacteriaceae isolated from foods: collaborative study

Nine laboratories evaluated the ability of the MICRO-ID test system to correctly identify members of the Enterobacteriaceae. A total of 78 isolates representing 11 genera of enterics that had been previously isolated from foods were used in the collaborative study. The collaborators streaked each isolate on plate count agar and incubated the plates overnight at 35 degrees C to check purity of the isolates. Then they proceeded with the method in which an isolated colony is transferred to a plate count agar slant and is incubated 18-24 h at 35 degrees C. Growth from the slant is emulsified in 3.5 mL physiological saline to a density comparable to a McFarland No. 2 tube and is then used to inoculate the test (MICRO-ID) strip. The strip is incubated 4 h at 35 degrees C and the reactions are read and recorded. Isolates are identified by using an octal code and the test kit manual. The system correctly identified 98.8% of the Salmonella isolates, 97.7% of the E. coli isolates, and 84.6% of the other 9 enteric genera tested. The system has been approved interim official first action as an alternative to conventional biochemical tests (1) for presumptive generic identification of food-borne Salmonella and for screening and elimination of non-Salmonella isolates; (2) for identification of E. coli from foods; and (3) for presumptive generic identification of other Enterobacteriaceae isolated from foods.     

11株鸡新城疫病毒强毒株的分子特性

选取2002～2007年从上海市分离鉴定的11株鸡新城疫病毒（Newcastle disease virus,NDV）强毒株,克隆其融合蛋白（Fusion,F）基因片段,测序后登录GenBank,序列号分别为NDV 022433（EU258661）、022435（EU258662）、0210149（EU258653）、0210154（EU258654）、0210227（EU258656）、0210252（EU258658）、03024（EU258637）、04011（EU258639）、05013（EU258640）、07011（EU258642）和07023（EU258643）。序列分析结果显示：上述分离株的F基因扩增片段为1 654 bp,编码551个氨基酸,其核苷酸序列同源性为94.6%～100%,推导的F蛋白氨基酸序列同源性为93.0%～100%;各分离株F蛋白裂解位点序列均为112R-R-Q-K-R-F117,为基因Ⅶ型NDV强毒株。系统发育分析表明：这11株NDV分离株之间的遗传距离较近,而与传统疫苗株B1、La Sota、V4和F48E9的遗传距离较远。此外,这11株NDV强毒株均具有大多数鹅源NDV毒株特有的973和1 249 nt RsaⅠ位点。     

堆肥中大肠菌群等粪便污染指标菌的残留状况调查

从日本九州地区23处堆肥化设施采集了以牛粪、鸡粪、污泥和餐厨垃圾为原料的堆肥试样29个,调查了大肠菌群等粪便污染指标菌的残留状况。结果表明,使用DESO培养基有11个试样被检出大肠菌群,检出率达38%,菌数在102~106 cfu.g-1的范围。使用API 20E鉴定系统对4个试样中的21个分离纯化菌株进行了菌种鉴定,发现有大肠菌群属的Escherichia coli、Escherichiavulneris、Pantoea sp.和Buttiauxella agrestis,以及非大肠菌群属的Serratia marcescens等肠内细菌科细菌。采用血清凝集试验对5株E.coli的病原性检测结果均为阴性。进一步针对6处堆肥设施的堆肥发酵过程中大肠菌群的消长追踪发现,大肠菌群数从原料到成品出现了逐渐减少直至消失、暂时消失、未完全消失、完全未检出4种走势,显示即使堆肥发酵温度在60℃以上,大肠菌群也有可能通过交叉污染等途径残留在堆肥成品中。     

Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy

The use of Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques for the rapid detection and identification of bacterial contamination in liquids was evaluated. The complex biochemical composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification. Bacterial suspensions (Escherichia coli HB101, E. coli ATCC 43888, E. coli 1224, Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus cereus, and Listeria innocua) were filtered to harvest the cells and eliminate the matrix, which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 6000-4000 cm(-1). The method reproducibly distinguished between different E. coli isolates and conclusively identified the relationship between a new isolate and one of the test species. This methodology may allow for the rapid assessment of potential bacterial contamination in liquids with minimal sample preparation.     

番鸭呼肠孤病毒ZJ99株σC基因的序列分析及其在大肠杆菌中的表达

参考已发表的番鸭呼肠孤病毒(Muscovy duck reovirus, mDRV)的S4序列，自行设计1对扩增σC基因的引物，对mDRV ZJ99株的RNA抽提物进行RT-PCR扩增，成功地获得了843 bp特异性的扩增片段(GenBank: AY619690)。将PCR产物克隆测序，BLASTn比较结果显示, mDRV ZJ99株σC基因与福建MW9710株对应基因的同源性为98％，与法国89026和89330株对应基因的同源性分别为92％和93%； BLASTp比较结果显示, ZJ99株编码的σC蛋白与MW9710、89026和89330株相应蛋白分别具有94％、89％、 89％同一性(identity)和95％、92％、93％相似性(similarity)。进一步将σC基因亚克隆至原核表达载体pET28a， 转化大肠杆菌(Escherichia coli )BL21(DE3)，IPTG诱导阳性重组子，SDS-PAGE电泳检测发现, σC蛋白在大肠杆菌中以包涵体的形式高效表达，目的蛋白的表达量可达到细菌总蛋白的22.9％。