Susceptibility of wallabies to Fusobacterium necrophorum

Wallabies (Macropus rufogriseus) were not appreciably more susceptible than rabbits or mice to Fusobacterium necrophorum, a fact established by the subcutaneous injection of a series of graded doses into animals of each species. The strikingly frequent occurrence of necrobacillosis in captive macropods is therefore not due to a uniquely high susceptibility. A vaccine containing inactivated F necrophorum culture emulsified with Freund's complete adjuvant failed to increase the resistance of wallabies to subcutaneous challenge with a moderate dose of the homologous strain. The control of necrobacillosis in captive wallabies must therefore depend on managemental measures aimed at minimising faecal contamination of the environment and damage to the buccal mucous membrane and skin.     

坏死梭杆菌研究进展

坏死梭杆菌(Fusobacterium necrophorum,Fn)是一种严格厌氧的革兰阴性多形态杆菌,其致病因子包括内毒素、白细胞毒素、血小板凝集因子、血凝素和溶血素等,其中主要的致病因子是白细胞毒素。由坏死梭杆菌引起的疾病在不同国家和地区均有发生,鹿、羊等反刍动物感染坏死梭杆菌时表现为跛行,关节肿大,肝脓肿和腐蹄病,猪、马等动物感染坏死梭杆菌时主要表现为跛行,严重时继发其他细菌感染,对养殖业造成巨大损失;人类感染该细菌时表现为急性咽炎综合征(Lemierres syndrome)。论文通过对国内外坏死梭杆菌及其疫苗研究和新型佐剂的开发进行综述,以期为该细菌疫苗的研制提供参考。     

坏死梭杆菌白细胞毒素研究进展

坏死梭杆菌是动物和人的各种坏死化脓感染的条件性致病菌.坏死梭杆菌的白细胞毒素是一种高度不稳定性分泌蛋白,被认为是主要的毒力因子.坏死梭杆菌白细胞毒素基因的开放阅读框(lktAORF)包括9 726 bp,编码3 241个氨基酸,总分子质量为336 ku的蛋白,且与其他细菌的细胞毒素没有任何相似的序列.覆盖在整个坏死梭杆菌lktA ORF上的5个短的重叠的多肽分别是BSBSE,SX,GAS,SH和FINAL,将它们在大肠埃希菌中表达,所有的多肽都有免疫原性,但GAS引起最小的抗体反应,BSBSE和SH对坏死梭杆菌攻击诱导产生了很强的保护力,比坏死梭杆菌的培养上清内全长活性lkt或无活性上清的保护性要好得多.     

坏死梭杆菌白细胞毒素研究进展

坏死梭杆菌白细胞毒素(Lkt)是一组对反刍动物白细胞特别是多形性白细胞(PMNs)有特异性毒性作用的细胞外毒素,被认为是坏死梭杆菌感染动物的主要毒力因子。白细胞毒素的物理稳定性较低,高温或极端pH环境中都能使白细胞毒素活性丧失。研究发现,白细胞毒素开放阅读框(ORF)全长9 726bp,由3个基因(lktB、A和C)组成,结构基因是第2个基因(lktA)。白细胞毒素对白细胞的毒性作用有剂量依赖性,并且溶血活性较低,不能在豚鼠猪皮肤上形成皮肤坏死症状。     

Pathogenicity of Fusobacterium necrophorum biovar B.

Previous studies showed that the high minimum infective dose (more than 10(6) organisms) of biovar A strains of Fusobacterium necrophorum for mice by subcutaneous inoculation could be greatly reduced, often to less than 10 organisms, by suspending the fusobacteria in sublethal doses of broth cultures of certain other bacterial species, such as Staphylococcus aureus. The present study showed that no such enhancement of infectivity occurred with two biovar B strains. This observation, together with the low pathogenicity of pure cultures of these strains for mice, suggests that biovar B plays no more than a minor role in the aetiology of necrobacillosis.     

Pathogenicity of Fusobacterium necrophorum biovar C in mice by intraperitoneal and intraportal injections

Three strains of Fusobacterium necrophorum biovar C were injected into mice intraperitoneally and intraportally. All the mice survived. In one mouse out of 15 mice injected intraperitoneally, a few focal abscesses were formed in the liver. The microorganisms were recovered from the liver abscess and the tissue of liver with abscess. No changes were observed in the organs of other 14 mice and no bacteria were recovered from them. In the 15 mice injected intraportally, no liver abscesses and no macroscopic changes in the organs were formed. However, the inoculated bacteria were recovered from the liver of four mice. The pathogenicity of F. necrophorum biovar C was weaker than that of other two biovars.     

坏死梭杆菌检测方法研究进展

坏死梭杆菌是一种严格厌氧的革兰阴性(G-)杆菌,可以依据坏死梭杆菌的菌体形态、菌落特征、生化试验、耐药性试验、酶特性试验等进行检测。根据坏死梭杆菌的16 S rRNA基因序列、16 S～23 SrRNA基因间序列以及rpoB基因序列,可以对其进行菌种水平上的鉴定;根据坏死梭杆菌的gyrB基因序列和白细胞毒素操纵子启动子区序列,可以对坏死梭杆菌进行亚种水平的鉴别。环介导的等温扩增(LAMP)技术的发展也为坏死杆菌病的快速诊断提供了检测方法。     

Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leucocidin

The capacity of extracts from toxigenic and non-toxigenic ruminant strains of Fusobacterium necrophorum to protect against challenge with homologous and heterologous bacteria was examined in mice. The numbers of F. necrophorum which were infective or lethal for mice increased 5- to 8-fold in animals which had been previously inoculated with complete Freund's adjuvant (FCA). Although preparations containing lipopolysaccharide (LPS) and outer membrane proteins (OMP) from several strains gave protection against a non-toxigenic strain (FnB-3), they did not significantly immunize mice against a challenge infection with a toxigenic bovine strain, FnB-1. Only material which had been prepared by gel filtration of 18-h liquid culture supernates of toxigenic F. necrophorum elicited significant immunity against homologous challenge with FnB-1. This preparation contained LPS and the majority of the leucotoxic activity. However, passive protection was not afforded to mice inoculated with bovine or rabbit sera which possessed high neutralization titres against the leucocidin.     

Fusobacterium necrophorum hemolysin in bovine hepatic abscess

An immunofluorescence study was made on bovine hepatic abscess containing Fusobacterium necrophorum predominantly. The abscess section stained with anti F. necrophorum hemolysin serum demonstrated fluorescence which formed irregular and granular shapes. Actinomyces pyogenes isolates from the abscess were not stained with the serum. These findings suggest that the bacterial hemolysin contributes to the formation of the hepatic abscess during an infection with F. necrophorum.     

牛源坏死梭杆菌43K外膜蛋白的黏附特性研究

旨在明确牛源坏死梭杆菌43K OMP的黏附特性。将43K OMP基因克隆连接至pET-32a载体,转化入大肠杆菌BL21 DE3中,通过IPTG诱导进行原核表达,应用黏附试验、天然蛋白竞争试验、抗体抑制试验和蛋白酶水解试验,明确牛源坏死梭杆菌43K OMP的黏附性,同时将纯化的重组蛋白和提取的天然43K OMP与牛子宫内膜细胞和牛乳腺上皮细胞共孵育,观察43K OMP对细胞的黏附作用。结果显示：43K OMP基因克隆到pET-32a载体中,随后在大肠杆菌BL21 DE3以包涵体形式成功表达;携带重组质粒(H2019)的大肠杆菌经IPTG诱导后,与空载体对照相比,与宿主细胞的结合显著增强(P<0.05);天然43K OMP与细胞共孵育后,黏附细胞的细菌数量显著降低(P<0.05);H2019与43K OMP多抗或单抗预孵育后,显著降低黏附宿主细胞的细菌数量(P<0.05);经不同浓度蛋白酶K处理H2019后,黏附细胞的细菌数量显著降低(P<0.05),且与蛋白酶K浓度呈现剂量依赖关系。同时,43K OMP天然蛋白和重组蛋白能黏附于牛子宫内膜细胞和乳腺上皮细胞表面。...     

Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice

Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.     

Immunization of mice against Fusobacterium necrophorum infection by perenteral or oral administration of vaccine

Immunization of mice against Fusobacterium necrophorum infection was attempted by using 3 vaccination procedures: (1) intraperitoneal (IP) injection of F necrophorum cells in saline solution, (2) IP injection of cells with added aluminum hydroxide adjuvant, and (3) feeding of a powdered mouse diet containing lyophilized cells. One or 2 weekly IP injections of the bacteria cells (in saline solution) for 3, 6, or 12 weeks resulted in protection of 48.7% to 64.5% of the mice against challenge exposure. Of the 2 control groups (given saline solution only), 100% and 97.4% became infected. Weekly IP injections of bacterial cells in an aluminum hydroxide adjuvant for 3, 6, or 12 weeks resulted in protectivity of 54.1% to 77.5%. Of the control mice (given adjuvant only), 97.5% became infected. Bacterial cells fed to mice at a dose level of 1.5 mg (dry weight)/g of powdered diet for 30 days (4 or 5g of diet each day) resulted in only a delay in the mean time of death as compared with the rapid death of the control mice. The feeding dose of 0.15 mg of cells/g of diet did not delay the mean time of death.     

坏死梭杆菌白细胞毒素基因的BSBSE片段的原核表达及抗血清制备

本研究以国内分离的牛源坏死梭杆菌F4基因组DNA为模板,应用PCR方法扩增BSBSE片段,克隆到pMD18-T载体上,鉴定并测序正确后,构建pET32a-BSBSE表达质粒,转化E．coli BL21（DE3）经IPTG诱导、SDS—PAGE检测重组蛋白表达、Western blot检测重组蛋白反应原性。以该重组蛋白免疫兔制备抗血清,经间接ELISA检测抗血清效价。结果表明,PCR扩增得到1100bp的BSBSE片段,以此片段构建了pET32a—BSBSE表达质粒,经诱导后获得了目的蛋白表达,以Western blot检测该重组蛋白证明为本研究的目的蛋白,并且与抗体具有反应原性。以间接ELISA检测该重组蛋白免疫兔制备的抗血清效价达10^5。研究结果将为坏死梭杆菌毒力因子（1kt）的深入研究奠定了物质基础。     

Aggregation of bovine platelets by Fusobacterium necrophorum.

Washed cell suspensions of biovar A strains of Fusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca(2+)-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide of F. necrophorum biovar A strains.     

Phylogenetic analysis of Fusobacterium necrophorum, Fusobacterium varium and Fusobacterium nucleatum based on gyrB gene sequences

The nucleotide sequences of the DNA gyrase B subunit gene (gyrB) of Fusobacterium necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. varium were determined and analyzed together with those of F. nucleatum subsp. nucleatum and F. nucleatum subsp. vincentii. On the phylogenetic tree constructed, the strains of each fusobacterial species formed distinct clusters with deep sublines. The degree of sequence similarity within each cluster was 93.2% or more, whereas similarities between clusters ranged from 70.1 to 72.7%. These clusters were recovered with 100% bootstrap probabilities and are in very good agreement with the species of Fusobacterium. These data suggest that gyrB is an accurate genealogical marker for the classification of the fusobacterial taxa considered in this study.