香芪口服液对大肠杆菌耐药抑制作用

通过香芪口服液对耐药大肠杆菌作用前后环丙沙星和庆大霉素最小抑菌浓度测定、质粒图谱分析以及外膜蛋白图谱分析,研究香芪口服液对耐药大肠杆菌的体外耐药性的抑制作用.结果表明:中药作用后,大肠杆菌对环丙沙星和庆大霉素最小抑菌浓度无变化;中药作用后的大肠杆菌的质粒图谱部分质粒消失,1号菌株ompA恢复表达,2号菌株ompF恢复表达;此中药与大肠杆菌质粒拷贝、恢复外膜通道蛋白的正常表达有密切关系,为今后耐药抑制剂的研制奠定理论基础.     

鸭疫里默氏杆菌外膜蛋白A克隆表达及纯化

为了制备鸭疫里默氏杆菌(Riemerella anatipestifer,RA)外膜蛋白A(Outer membrane protein-A,OmpA)。本研究将RA云南流行株外膜蛋白OmpA基因PCR克隆至质粒pET32a,构建重组表达质粒pET32a-OmpA,将其转化大肠杆菌TransB感受态细胞,用IPTG诱导表达,并将表达产物进行初步纯化。结果表明OmpA基因在大肠杆菌原核表达系统中获得了高效表达,表达的OmpA重组蛋白分子量为61 kDa,表达量占菌体蛋白的40%。OmpA重组蛋白经纯化后纯度达90%。本研究为RA的ELISA抗原的制备奠定了基础。     

鸡源大肠杆菌耐药性分析及中药对大肠杆菌耐药性消除作用的研究

研究鸡源大肠杆菌耐药性及中药对大肠杆菌耐药性消除作用。选择22种抗生素采用Kirby-Bauer法对山西省部分地区分离的20株致病性鸡源大肠杆菌进行耐药性检测,对分离菌株进行质粒分析和耐药质粒转化试验,并分析多种中药对大肠杆菌耐药性的消除作用。结果显示,20株试验菌对测试抗生素均存在不同程度的耐药性,其中5耐及5耐以上的菌株占分离菌株的90%,试验菌对青霉素耐药率最高(90%),其次为恩诺沙星(85%),而对黏杆菌素和呋喃妥因的耐药性最低;分离出12种质粒谱型,同一地区、同一鸡场菌株的质粒图谱相同或相似,不同地区菌株的质粒图谱不同,仅有部分相同或相近的流行质粒共存;对其中5株菌株进行耐药质粒的转化试验,发现同一质粒可编码1个至数个耐药性基因,不同质粒可以携带相同的耐药性基因;中药单剂或合剂对大肠杆菌的耐药性有一定的消除作用,其中黄芩的消除效果最好,中药处理后部分菌株质粒图谱无变化,只有黄连和双黄连造成2条质粒带丢失;对中药提取物作用后的消除子进行药敏试验,结果发现大肠杆菌对环丙沙星等多种抗生素恢复了敏感性。研究结果提示联合使用中药治疗鸡源大肠杆菌病有显著效果。     

临床分离食品动物源多重耐药大肠杆菌耐药分子特征研究

本研究从主动外排机制、膜孔蛋白缺失及氟喹诺酮类药物作用靶位改变等几个方面探讨,临床分离的20株动物源性多重耐药大肠杆菌的耐药分子特征。实验结果表明,20株临床分离大肠杆菌gyrA83、gyrA87、parC80的突变率分别为95％、85％、55％。gyrA和parC共同突变的有11株,突变率为55％;20株多重耐药菌大肠杆菌普遍存在主动外排机制,主要介导对部分氨基糖苷类、四环素、氟苯尼考和氟喹诺酮类药物耐药,当添加外排泵抑制剂PAβN后多数菌株庆大霉素、新霉素、四环素、氟苯尼考及氟喹诺酮类药物的MIC都降低了2倍～256倍。利用建立的ELISA方法检测外排泵AcrA蛋白的表达水平,结果证实所有,临床分离菌外排泵表达都增高;20株分离大肠杆菌中,部分菌株缺失OmpC或OmpF蛋白,同时缺失这两个蛋白的只有3株。部分菌株OmpF蛋白条带附近存在有多重耐药相关蛋白（Mar）。本研究结果揭示多重耐药大肠杆菌对常用抗菌药物高水平的耐药表型是主动外排机制、药物作用靶位的改变、外膜通透性的改变及其它机制共同作用的结果。     

维氏气单胞菌JLL-1株外膜蛋白的原核表达及其免疫原性检测

为研究维氏气单胞菌(Aeromonas veronii)外膜蛋白(outer membrane protein,OMP)的免疫原性,本试验对A.veronii的OmpAⅠ基因进行克隆,构建了原核表达质粒pET-30a(+)-OmpAⅠ,并转入大肠杆菌BL21(DE3),将其表达后纯化重组蛋白,利用SDS-PAGE和Western blot分析鉴定。将灭活苗、弗氏佐剂(OmpAⅠ)、纯化蛋白及PBS缓冲液分别免疫鲤鱼后,检测每周各试验组鱼体血清非特异性免疫指标(酸性磷酸酶、碱性磷酸酶、超氧化物歧化酶活力)及特异性IgM抗体水平变化情况,并于加强免疫2周后对鲤鱼进行攻毒保护性试验。结果显示:重组蛋白免疫鲤鱼后能产生明显的免疫反应,保护率在77%以上;受免鲤鱼血清中IgM抗体水平、酸性磷酸酶、碱性磷酸酶及超氧化物歧化酶活力活力较对照组有显著提高。结果表明,重组蛋白OmpAⅠ具有较好的免疫原性和保护作用,为进一步研究外膜蛋白基因工程疫苗的开发奠定基础。     

新城疫病毒F蛋白和禽致病性大肠杆菌OmpA嵌合蛋白的表达及双特异性抗体制备

为获得Ⅶ型新城疫病毒(Newcastle disease virus, NDV)融合蛋白(fusion protein, F蛋白)和O18型禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)外膜基因A(outer-membrane proteases, OmpA)嵌合蛋白和双特异性抗体，试验首先采用基因合成技术获得Ⅶ型NDV F基因和O18型APEC的OmpA基因片段，使用同源重组技术将F基因插入到OmpA基因中，再将重组基因片段插入到原核表达载体pGEX-4T-1中构建嵌合表达质粒，对重组质粒中的插入基因序列进行测序鉴定；将序列正确的嵌合表达质粒转入大肠杆菌BL21(Rosetta)感受态细胞中，利用IPTG诱导嵌合蛋白表达，使用SDS-PAGE和Western-blot检验嵌合蛋白的表达情况；纯化嵌合蛋白后免疫兔获得抗血清，抗血清再经抗原亲和纯化层析柱纯化获得多克隆抗体，采用间接酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)测定抗体效价。结果表明：成功合成了Fo和OmpA1、OmpA...     

加减三黄汤对耐药大肠杆菌acrA mRNA表达水平的影响

为了解加减三黄汤对大肠杆菌耐药性的影响,采用实时荧光定量方法检测了外输泵acrA mRNA表达水平的变化。将100只昆明系小鼠分为阳性组(感染耐药性大肠杆菌2 751株和2 952株),阴性组,攻毒治疗组(2 751株+加减三黄汤,2 952株+加减三黄汤),8d后将鼠随机剖杀,取肝组织抹片培养大肠杆菌,做药敏试验,同时提取质粒,检测大肠杆菌外输泵基因acrA的mRNA表达量。结果显示,经加减三黄汤在体治疗后的大肠杆菌耐药性下降了,每μL中mRNA拷贝数为2 751株最高达1016,2 952株1015;用药后各菌株均有下降;相差极显著(P≤0.01),耐药性越大的拷贝数越多。表明加减三黄汤对耐药大肠杆菌外输泵基因acrA的mRNA表达水平影响与耐药性有一定的相关性。     

多杀性巴氏杆菌OmpA基因的原核表达及生物信息学分析

本试验旨在探究羊源多杀性巴氏杆菌OmpA基因的原核表达及其生物信息学特征。以羊源多杀性巴氏杆菌HN-01株基因组为模板,设计特异性引物扩增OmpA基因;构建pET-28a （+）-OmpA重组质粒后转化大肠杆菌BL21（DE3）感受态细胞,将鉴定正确的重组菌经IPTG诱导表达;通过SDS-PAGE及Western blotting分析表达蛋白的特征,并运用生物信息学工具对OmpA基因序列进行分析。结果显示,羊源多杀性巴氏杆菌OmpA基因大小约为1 044 bp,该基因序列与HN-06株的同源性达89.72%。通过诱导后发现,pET-28a （+）-OmpA重组菌最佳诱导条件为1 mmol/L IPTG 37℃诱导6 h,表达的重组蛋白大小约为40 ku,以包涵体的形式存在。Western blotting结果显示,约40 ku的重组蛋白携带His标签。经生物信息学分析,OmpA分子式为C₁₆₈₄H₂₆₁₉N₄₅₇O₅₀₅S₃,属碱性疏水蛋白,其多肽链的1-21位氨基酸为信号肽区域,并具有多种结构。综上所述,OmpA可能具有特殊结构,与众多外膜蛋白结构特点相似。本研究构建了多杀性巴氏杆菌OmpA基因原核表达系统,优化诱导条件后能稳定获得OmpA重组蛋白,为进一步探究巴氏杆菌的致病机理提供理论依据。     

多杀性巴氏杆菌OmpA基因的原核表达及生物信息学分析

本试验旨在探究羊源多杀性巴氏杆菌OmpA基因的原核表达及其生物信息学特征。以羊源多杀性巴氏杆菌HN-01株基因组为模板,设计特异性引物扩增OmpA基因;构建pET-28a(+)-OmpA重组质粒后转化大肠杆菌BL21(DE3)感受态细胞,将鉴定正确的重组菌经IPTG诱导表达;通过SDS-PAGE及Western blotting分析表达蛋白的特征,并运用生物信息学工具对OmpA基因序列进行分析。结果显示,羊源多杀性巴氏杆菌OmpA基因大小约为1 044 bp,该基因序列与HN-06株的同源性达89.72%。通过诱导后发现,pET-28a(+)-OmpA重组菌最佳诱导条件为1 mmol/L IPTG 37℃诱导6 h,表达的重组蛋白大小约为40 ku,以包涵体的形式存在。Western blotting结果显示,约40 ku的重组蛋白携带His标签。经生物信息学分析,OmpA分子式为C_(1684)H_(2619)N_(457)O_(505)S_3,属碱性疏水蛋白,其多肽链的1-21位氨基酸为信号肽区域,并具有多种结构。综上所述,OmpA可能具有特殊结构,与众多外膜蛋白结构特点相似。本研究构建了多杀性巴氏杆菌OmpA基因原核表达系统,优化诱导条件后能稳定获得OmpA重组蛋白,为进一步探究巴氏杆菌的致病机理提供理论依据。     

猪源大肠杆菌O157:H7体外氟苯尼考耐药诱导及对抗菌药物敏感性变化研究

为初步研究猪源大肠杆菌O157:H7 (E.coli O157:H7)对氟苯尼考耐药性的产生和消除机制,本研究采用亚抑菌浓度体外耐药诱导的方法将两株猪源大肠杆菌O157:H7诱导成氟苯尼考高度耐药菌株,采用无氟苯尼考压力下连续传代培养的方法将获得的氟苯尼考耐药菌株的氟苯尼考耐药性消除,检测耐药诱导菌和耐药消除菌对抗菌药物的敏感性,并检测菌株质粒携带的耐药基因。结果显示,经氟苯尼考耐药诱导,猪源大肠杆菌O157:H7对氟苯尼考、阿莫西林、头孢唑啉、头孢拉定和头孢噻吩由敏感变为耐药,对头孢噻肟的敏感性由敏感变为中介,对氧氟沙星、环丙沙星和阿奇霉素由中介变为耐药;而经耐药消除后,菌株恢复对上述药物的敏感性;在菌株的质粒中检测到氟苯尼考耐药基因、喹诺酮类耐药基因和β-内酰胺酶基因,与耐药表型相符。结果表明,在氟苯尼考压力的长期存在下,猪源大肠杆菌O157:H7对氟苯尼考产生耐药,且对青霉素类、头孢类和喹诺酮类药物产生交叉耐药,在去除氟苯尼考压力下连续培养,可消除菌株的部分耐药性。     

嗜温气单胞菌的鉴定及其外膜蛋白型研究

从病鳖、鳗鱼体内分离到2株嗜温气单胞菌(TAh-3、EAs-1),经形态学观察、生理生化特性等鉴定,确定TAh-3为嗜水气单胞菌,EAs-1为温和气单胞菌。动物感染试验结果表明,TAh-3株为致病性嗜水气单胞菌,对鲫鱼的致死率100%,EAs-1株为非致病性温和气单胞菌。SDS-PAGE电泳结果显示,TAh-3株外膜蛋白(OMPs)型由7条蛋白带组成,其中1条主带大小为40.0kDa;EAs-1株OMPs型由8条蛋白带组成,其中2条主带大小为38.0kDa、45.0kDa。     

Immune response of cattle to Haemophilus somnus lipid A-protein conjugate vaccine and efficacy in a mouse abortion model.

Immunogenicity of the lipid A component of Haemophilus somnus lipooligosaccharide in cattle and mice was examined after purification, detoxification, and covalent conjugation to a protein carrier. After 2 inoculations, a substantial antibody response was induced in most cattle to lipid A and the protein carrier. To determine whether antibodies to lipid A would be protective, 5 x 10(7) colony-forming units of H somnus strain 649 were administered IV to endotoxin-responsive (C3H/HeN) mice. In one study, 8 of 13 C3H/HeN mice aborted when inoculated. In contrast, abortion did not result when mice were inoculated with the same dose of an isolate of H somnus normally found in the prepuce or with the rough mutant Escherichia coli J5. In addition, endotoxin-nonresponsive (C3H/HeJ) mice were significantly (P = 0.03) more resistant to abortion by strain 649 than were C3H/HeN mice, but inoculated C3H/HeN mice were only slightly more resistant to H somnus abortion, compared with control mice. Although a large antibody response to lipid A was detected, there was no significant difference in the immunized group between mice that aborted and mice that delivered normally. Thus, lipooligosaccharide and other properties of virulent H somnus strains may contribute to abortion in mice.     

Characterization and classification of Actinobacillus (Haemophilus) pleuropneumoniae plasmids.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHM0, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: H1(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The H1 and H2 plasmids belonged to different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.     

Killing of Streptococcus uberis by bovine neutrophils following growth in chemically defined media

Following growth in a chemically defined medium (CDM), five strains of Streptococcus uberis were tested for their ability to survive killing by bovine neutrophils. Strains 0140J, ST10, EF20 and C221 were easily killed, whereas strain C197C was highly resistant. The ability of strain 0140J to resist phagocytosis and killing was increased by supplementation of the growth medium with milk whey, casaminoacids, casein, or, to a lesser extent, bovine serum. Supplementation of the growth medium with yeast extract or bovine serum albumin did not affect the resistance of this strain. Following growth in CDM supplemented with casein, strains ST10 and C221, like strain 0140J, were significantly more resistant to killing by neutrophils. The resistance of strains EF20 and C197C was unaffected by the addition of casein to the medium; strain EF20 remained susceptible and strain C197C highly resistant to killing. The effect of supplementing the growth media with components other than casein was only studied for strain 0140J. Decapsulation of strains C197C, ST10 and 0140J, grown in CDM + casein, with type-X hyaluronidase did not significantly affect their ability to survive in the presence of bovine neutrophils.     

The problem of escalating resistance of Haemonchus contortus to the modern anthelmintics in South Africa

During the past decade in South Africa there has been a continual increase in sheep of strains of gastrointestinal helminths resistant to the modern anthelmintics. Five strains of Haemonchus contortus are described in this paper. Despite the fact that 2 of the 5 strains were tested for susceptibility only to ivermectin, a total of 10 instances of resistance were found. Four of the 5 strains were resistant to ivermectin, 2 to closantel, 2 to rafoxanide and 2 to the benzimidazoles. One of these strains was concurrently resistant to 3 different anthelmintic groups, namely, the ivermectins, the benzimidazoles and the salicylanilides. Resistance to ivermectin developed in 2 strains of H. contortus after a history of only 3 treatments with this compound in one instance and 11 treatments in the other. In the latter case drenching with ivermectin was well interspersed with that of other anthelmintics. This rapid development of resistance suggests that there may be cross-resistance between ivermectin and another anthelmintic group. Two of the ivermectin resistant strains were recovered from separate properties in the south-western Cape Province, where Ostertagia circumcincta, which is usually the dominant parasite in this region, was virtually eliminated by the anthelmintic treatment. On each of these properties it was apparently replaced by a resistant strain of H. contortus. A serious threat to control is the dissemination of worm strains with multiple resistance to anthelmintics. The strain of H. contortus resistant to 3 anthelmintic groups has already been widely dispersed, as the farmer concerned suddenly decided to give up farming with sheep and to sell his flock.     

Import and efflux of flubendazole in Haemonchus contortus strains susceptible and resistant to anthelmintics

Drug entry into the body of a helminth is a key factor in the efficacy of anthelmintics. The present project was designed to study the ex vivo uptake and efflux of the benzimidazole anthelmintic flubendazole (FLU) in four strains of H. contortus: the ISE strain (fully susceptible to anthelmintics), the ISE-S strain (resistant to ivermectin), the BR strain (resistant to benzimidazoles) and the WR strain (multi-resistant). The transport of FLU between dead and living nematodes was also compared as well as the effect of verapamil, an inhibitor of the main efflux ABCB1 transporter (P-glycoprotein), on FLU accumulation in nematodes. The obtained results showed that FLU is able to effectively enter H. contortus adults due to high FLU lipophilicity. Passive diffusion is probably the only mechanism in both FLU import and efflux from nematodes. No differences in FLU transport were found among four H. contortus strains with different sensitivity to anthelmintics. No active FLU efflux from H. contortus and no effect of verapamil were observed, indicating that H. contortus cannot protect itself against FLU by the active removal of this anthelmintic from its body.     

Bacterial adherence in mastitis caused by Escherichia coli.

The possible role of bacterial adherence in the pathogenesis of experimental mastitis in the mouse was examined with four strains of Escherichia coli. Two of these strains had a known adhesion antigen (K88) and two did not. The K88 antigen did not play a significant role in the virulence or infectivity of E. coli either in the murine or bovine mammary gland. Two E. coli strains, W1 (K88+) and J2 (K88-) were virulent in the mouse but did not adhere to epithelial cells. Both these strains produced clinical mastitis in the cow. A third strain, D282 (K88-), produced mild disease in the mouse but was avirulent in the cow. The fourth strain, 233/ID (K88+), was avirulent in both the mouse and the cow. Strains D282 and 233/1D were killed rapidly by bovine serum whilst J2 and W1 were more resistant. All strains were more sensitive than the control resistant strain E. coli P4, which is known to be highly virulent for the lactating udder.     

鸡柔嫩艾美耳球虫不同抗药性虫株的种内多态性研究

应用RAPD技术进行柔嫩艾美耳球虫4个单一抗药性虫株与1个敏感株的基因组DNA多态性分析，发现4个抗药性虫株及敏感株之间的相似值均大于99%；OPAO3引物对抗盐霉素株扩增出一条约500bp的特异条带，OPH02引物对抗克球粉株和抗盐霉素株均扩增出了约1kb的第三条主带，这些特异条带的出现很可能与球虫抗药性基因有关，有可能用于抗药性虫株的诊断与鉴定。     

Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model

The recently completed genome sequence of Haemophilus parasuis strain SH0165 allowed us to screen putative OMPs for the development of recombinant vaccines. The objective of this study was to evaluate the immunogenicity and protective efficacy of three OMPs of H. parasuis. Three putative OMPs (SmpA, YgiW and FOG) were cloned, expressed and purified by Ni affinity chromatography using nitriloacetic acid resin. Mice were immunized either individually (individual protein, IP) or synergistically (synergistic protein, SP) with the recombinant proteins. A significant increase in IgG titer was detected in all protein-immunized mice. Isotyping studies revealed that the antibodies produced were predominantly IgG2a-type, indicating a predominant Th1 response. A significant increase was observed in IL-2, IL-4 and IFN-γ levels in the culture supernatants of splenocytes isolated from immunized mice. Furthermore, mice were challenged intraperitoneally with 6×10(9)CFU (5×LD(50)) of highly virulent homologous serovar 5 strain (SH0165) or 7.0×10(9) CFU (5×LD(50)) of the heterologous serovar 4 strain (MD0322) at fourteen days after the last immunization. All of the recombinant proteins enhanced survival and reduced histopathological lesions. Our results indicated that the three OMPs showed protection both individually and synergistically against infection with the highly virulent H. parasuis in mice.