Effects of a collagenolytic cell wall component from Fusobacterium necrophorum subsp. necrophorum on rabbit tissue-culture cells

The effects on rabbit tissue-cultured cells of collagenolytic cell wall component (CCWC) from Fusobacterium necrophorum subsp. necrophorum were investigated. Scanning electron microscopy demonstrated that CCWC damaged the cell surfaces of the rabbit granulocytes and hepatocytes but the effects of the cells differed from each other. Granulocytes appeared smooth and morphologically irregular whereas hepatocytes looked rough and had tiny holes in the cell membranes. Differences in cell viability were observed in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt) assay. The findings suggest that cytotoxic activity in vivo may well contribute to the establishment of an initial injury in visceral tissues, and the action of CCWC could increase the chances of survival for an invading F. necrophorum subsp. necrophorum at the first stages of infection.     

Immunohistochemical and ultrastructural identification of Fusobacterium necrophorum subsp. necrophorum in bovine fatal necrotizing glossitis

A 37-day-old male Japanese black calf showing marked salivation and leucocytosis died and was examined the tissues histologically. Histological lesions were characterized by severe focal necrotic glossitis on the ventral side of the root of the tongue. Immunohistochemically, Fusobacterium necrophorum subsp. necrophorum antigen was detected in the necrotic tissues and its distribution corresponded to that of the gram-negative, nonsporeforming, long filamentous organisms. Ultrastructural similarities between the organism and F. necrophorum subsp. necrophorum, but not subsp. funduliforme were observed. These findings clearly demonstrated that the fatal necrotic glossitis was caused by F. necrophorum subsp. necrophorum. This is the first report of bovine fatal necrotizing glossitis with leucocytosis caused by F. necrophorum subsp. necrophorum infection, and this organism may be an important fatal pathogen in calves with glossal lesions.     

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。...     

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.     

Endotoxin from Fusobacterium necrophorum of bovine hepatic abscess origin.

The endotoxic activity of Fusobacterium necrophorum bov 5 was investigated. The supernatant (S) fluid and cell wall (CW) preparation, obtained after differential centrifugation of the ruptured cell mass, were lethal for mice. The toxicity of the S fluid was stable during prolonged storage, treatment with formalin, and heating for 15 minutes at 80, 100, and 121 C, but was destroyed by alkaline hydrolysis with 0.25 N NaOH. The toxic factor was found in a high molecular weight (MW) fraction after gel filtration. The properties exhibited by the toxic S fluid resembled those of endotoxic lipopolysaccharide (LPS). Extracted and partially purified LPS (endotoxin) from F necrophorum bov 5 demonstrated a mouse median lethal dose (mouse LD50) of 16.8 mg/kg of body weight. The toxic LPS material, a high molecular weight moiety as estimated by gel filtration, was resistant to ribonuclease (RNase), deoxyribonuclease (DNase), and pronase treatment. A positive Shwartzman reaction (median skin lesion dose (SLD50) equal to 3.32 mug/kg of body weight) and biphasic fever response (minimal dose required to produce a fever index of 40 sq cm which falls on the linear portion of dose-response curve (FL40) equal to 0.41 mug/kg of body weight) further indicated the toxin was endotoxin in nature. The LPS from F necrophorum bov 5 was less toxic than Salmonella typhimurium LPS; but had considerable toxicity for experimental animals. The toxic activity of the partially purified F necrophorum bov 5 endotoxin was separated into 2 fraction regions by diethylaminoethyl (DEAE)-cellulose chromatography. The data provide evidence for the production of a potent endotoxin, possibly composed of more than one toxic component, which may be released upon cell disruption.     

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.     

Biochemical characterization of the leukotoxins of three bovine strains of Fusobacterium necrophorum

The biochemical characteristics of the leukotoxins of 3 bovine isolates of Fusobacterium necrophorum which represent biotypes A, AB, and B were compared. Two methods were used for the production of the leukotoxins: medium M-1 continuous dialysis sac cultures and brain-heart infusion agar plate cultures. The supernatant cultural fluids were fractionated sequentially by membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. The ultrafiltrates (less than 500 mol wt) were fractionated by gel-permeation chromatography, using G-10 Sephadex. The leukotoxins of the 3 F necrophorum strains were estimated to have a molecular weight between 350 and 450. The leukotoxins in the ultrafiltrates (less than 500 mol wt) were stable at 60 C for 4 hours and at 100 C for 30 minutes, stable to extremes of pH (3 to 11), and stable to degradative enzymes including trypsin, protease, alpha-amylase, lipase, deoxyribonuclease, and ribonuclease. Significant differences were not observed in the biochemical characteristics of the leukotoxins produced in vitro by the 3 F necrophorum biotypes. These assays were done, using monolayers of mouse peritoneal macrophages. The monolayers were exposed to the 4 ultrafiltrates of both the continuous dialysis sac and brain-heart infusion agar cultures (pH 7.2) for 4 hours at 4 C, 25 C, and 37 C. Maximal cytotoxic activity in the assays was at 37 C.     

Further observations on the weak immunogenicity of Fusobacterium necrophorum.

Five virulent strains of Fusobacterium necrophorum resembled a single strain examined earlier by possessing little or no immunogenicity: severe subcutaneous infections cured with metronidazole failed to increase the resistance of mice to subcutaneous challenge 22 days after the cessation of treatment.     

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。     

Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.     

Factors affecting the leukotoxin activity of Fusobacterium necrophorum.

The effect of cultural conditions on the production of leukotoxin by biotypes A and B of F. necrophorum was investigated. Biotypes A and B were grown in prereduced, anaerobically sterilized, brain-heart infusion (BHI) broth. The average leukotoxin titer of culture supernatant was 18 times higher from biotype A strains than from biotype B strains. Leukotoxin activity peaked during the late-log and early-stationary phases of growth, then declined precipitously in both biotypes. F. necrophorum biotype A was grown in different media (BHI, liver infusion, and Eugon broths), at various pH (6.6, 7.3, 7.7, and 8.2), incubation temperatures (30, 35, 39, and 43 degrees C), redox potentials (-352 to +375 mV), and iron concentrations (less than 0.2, 4.2, 42.1, and 361.4 microM). Anaerobic BHI broth with pH from 6.6 to 7.7 at 39 degrees C incubation temperature supported maximal F. necrophorum growth and leukotoxin production. The optimum redox potential for F. necrophorum growth was in the range of -230 to -280 mV. However, the presence of titanium III citrate or dithiothreitol (7.78 mM) in the medium decreased (P less than 0.05) the leukotoxicity of F. necrophorum. Low iron concentration (less than 0.2 microM) decreased (P less than 0.05) growth rate but not leukotoxin activity of F. necrophorum, whereas high iron concentration inhibited the leukotoxin activity.     

The effect of temperature on growth and survival of Fusobacterium necrophorum isolated from bovine liver abscesses.

The ability of Fusobacterium necrophorum to survive or grow in liquid nitrogen or at temperatures between -10 degrees and 59 degrees C was determined. The organism remained viable but did not grow in liquid nitrogen or between -10 degrees and 21 degrees C. It grew between 22 degrees and 43 degrees C. No isolate grew at temperatures above 43 degrees C and all three isolates survived for a minimum of 15 minutes and an average of 25 minutes at 59 degrees C. The optimum temperature for maximum growth was 37 degrees C. The organism survived in ampoules stored in liquid nitrogen for eight years. It survived in liver abscesses stored at -10 degrees C for five years and as cultures in screw capped tubes for three years.     

Ultrastructure and molecular characterization of Fusobacterium necrophorum biovars.

The ultrastructural features and molecular components of 18 strains of Fusobacterium necrophorum biovars A, AB and B, isolated from animal and human infections, were examined by electron microscopy, multilocus enzyme electrophoresis (MEE) and by sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-PAGE). High resolution scanning electron microscopy revealed that the strains possessed a convoluted surface pattern. Transmission electron microscopy showed that all strains possessed a cell wall structure typical of gram-negative bacteria. Bleb formation was not uncommon. Numerous extracellular materials, resembling lipopolysaccharide (LPS) fragments, surrounded cells of both human strains and biovar B animal strains. Biovar A field strains revealed capsules as stained by ruthenium red whereas a stock culture strain showed the capsule only when immunostabilized with hyperimmune serum. Starch gel electrophoresis showed all strains to possess adenyl kinase, glutamate dehydrogenases and lactate dehydrogenase; each enzyme migrated uniformly (monomorphic) among the strains and represented an electrotype. However, SDS-PAGE indicated differences in the protein profiles between all of the strains; the most distinctly different was a human isolate (FN 606). Silver staining to detect LPS showed extensive "ladder" patterns among the majority of biovar A strains but not in the animal biovar B strains. Immunoblotting of LPS with a rabbit antiserum prepared against phenol extracted LPS from a biovar A animal isolate (LA 19) suggested marked variability in the LPS antigens among the isolates studied.     

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.     

PCR法诊断奶牛腐蹄病坏死梭杆菌的研究与应用

试验根据坏死梭杆菌白细胞毒素基因序列的特异性,设计1对引物FLP并建立了PCR检测方法.试验结果表明,引物FLP具有相对较高的特异性和敏感性,其敏感性达到了48.5pg.对现地采集奶牛蹄部病料进行PCR检测,发现引物FLP具有很好的特异性,能够准确诊断出坏死梭杆菌的存在.