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1.
Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells. 相似文献
2.
Comparative biological features of a rat liver abscess model induced with three Fusobacterium necrophorum strains 总被引：3，自引：0，他引：3
Several biological features were compared in a rat liver abscess model, using intraportal inoculations with 3 bovine strains of Fusobacterium necrophorum which varied in virulence. Serum alanine aminotransferase activities were increased significantly (P less than 0.05) in rats inoculated with F necrophorum 2101 by postinoculation hours 6, 12, and 24. Thereafter, alanine aminotransferase values returned to base line for the remainder of the experiment. Also, rats inoculated with F necrophorum 2101 had a significantly greater (P less than 0.05) weight loss than did the control rats during the first 5 postinoculation days and developed leukocytosis characterized by a neutrophilia with a left shift. The duration of the bacteremia was related directly to the virulence of the F necrophorum strain. Fusobacterium necrophorum 2101, a biotype A which was the most virulent, induced the most persistent bacteremia; F necrophorum 2035, a biotype B which was the least virulent, produced the shortest bacteremia; and F necrophorum 2030, a biotype AB which was of intermediate virulence, led to bacteremia of intermediate duration. Plasma endotoxin was demonstrated intermittently during the first 24 hours, but did not correlate with the bacteremia. 相似文献
3.
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. 相似文献
4.
K F Lechtenberg T G Nagaraja H W Leipold M M Chengappa 《American journal of veterinary research》1988,49(1):58-62
Twenty-eight abscessed livers were collected from feedlot cattle at an abattoir; specimens were obtained from 49 abscesses for bacteriologic culture and for histologic examination. Cultural procedures included techniques to enumerate and isolate facultative and obligate anaerobic bacteria. Anaerobic bacteria were isolated from all 49 abscesses, whereas facultative bacteria were isolated from only 22. Mean bacterial counts for anaerobic and facultative bacteria were 3 X 10(8) and 8 X 10(8) bacteria/g of purulent material, respectively. Fusobacterium necrophorum, the only anaerobe isolated, was detected in 100% of the abscesses. Fusobacterium necrophorum biotype A was isolated from 57% of the abscesses (in pure culture from 75%), and F necrophorum biotype B was isolated from 47% of the abscesses (from 96% with mixed infections). Corynebacterium pyogenes was the predominant facultative bacterium isolated. Histologic changes in abscesses were qualitatively similar; abscesses were pyogranulomatous, with a necrotic center surrounded by zones of inflammatory tissue. However, the severity of lesions varied, depending on the F necrophorum biotype involved. Portal triad fibrosis and bile-duct proliferation were most severe in biotype A and mixed biotype B infections and less severe in abscesses from which biotype B was isolated in pure culture. 相似文献
5.
Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice 总被引：4，自引：0，他引：4
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. 相似文献
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7.
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. 相似文献
8.
Ribosome-rich extracts (RRE) were prepared by differential and ultracentrifugation from 25 bovine and 6 ovine isolates of Fusobacterium necrophorum (FN) including both biotypes A and B. A pooled rabbit antiserum was prepared against whole-cell and sonicated whole-cell bacterins of F necrophorum isolate FN 3080, and a 2nd pooled rabbit antiserum was prepared against a RRE of FN 3080. The RRE of the 25 bovine isolates were tested against the FN 3080 whole-cell antiserum, using Ouchterlony double-immunodiffusion procedures. One to 3 precipitin lines were observed with the 25 isolates. The individual bovine isolates were found to have lines of identity with 5 to 21 of the other 24 isolates. The 25 bovine isolates and the 6 ovine isolates were then compared, using the FN 3080 RRE antiserum. One to 3 precipitin lines were observed for the 31 isolates with the RRE antiserum, and lines of identity were observed between all 31 of the isolates. These results indicated that common antigens are present in the RRE from a wide variety of F necrophorum isolates including both A and B biotypes. 相似文献
9.
Tadepalli S Stewart GC Nagaraja TG Jang SS Narayanan SK 《Veterinary microbiology》2008,127(1-2):89-96
Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections. 相似文献
10.
为分离纯化奶牛腐蹄病坏死杆菌,分析其与其他菌株的亲缘关系,本研究利用坏死杆菌白细胞毒素特异性引物,对奶牛腐蹄病病牛蹄部拭子样品进行了PCR检测,利用厌氧培养基对PCR检测阳性样品进行了坏死杆菌的分离培养,以分离的坏死杆菌基因组DNA为模板,对白细胞毒素基因进行了克隆和序列分析。结果显示,9份奶牛腐蹄病病牛蹄部拭子样品PCR检测结果均为阳性,对其中一份样品中的坏死杆菌进行分离培养,获得了纯培养物,命名为bFR13-1。坏死杆菌bFR13-1菌株白细胞毒素基因测序结果显示,与GenBank已发表的H05、A25和B35菌株的白细胞毒素基因在核苷酸水平的同源性分别为98.40%、98.35%和90.79%,推导氨基酸的同源性分别为97.7%、97.6%和89.0%。进化树分析结果显示,坏死杆菌bFR13-1菌株白细胞毒素与H05菌株的同源性最高,bFR13-1菌株与H05菌株和A25菌株呈较近亲缘关系。结果表明,不同坏死杆菌分离株的白细胞毒素呈现一定的变异性,这种变化是否与坏死杆菌致病性相关,值得深入研究。 相似文献
11.
GAO Jing LI Chun-qiu YAO Shuang ZHANG Li-chun WANG Xin-yu WANG Zhi-hui ZHAO Xi-wen WEI Shan WANG En-yu SUN Dong-bo WU Rui GUO Dong-hua 《中国畜牧兽医》2015,42(9):2308-2312
For separation and purification of Fusobacterium necrophorum of cow footrot, and analysis of genetic relationship with other strains, the hoof ministry swab samples were detected by PCR based on specific primers of leukotoxin gene, and genomic DNA were isolated from PCR positive samples of Fusobacterium necrophorum culturing in anaerobic medium.The genes of leukotoxin were cloned and sequenced.The results showed that nine of hoof ministry swab samples were all PCR positive samples, and we obtained Fusobacterium necrophorum pure culture from one of the samples which named bFR13-1.The gene sequencing results indicated that the homologies of leukotoxin gene nucleotide sequence of bFR13-1 strain compared with H05, A25 and B35 strains from GenBank were 98.40%, 98.35% and 90.79%, respectively, and the homologies of deduced amino acid sequence were 97.7%, 97.6% and 89.0%, respectively.Phylogenetic tree analysis results showed that leukotoxin gene of Fusobacterium necrophorum bFR13-1 and H05 had high homology and bFR13-1, H05 and A25 showed a close genetic relationship.The result indicated that leukotoxin showed variability between different Fusobacterium necrophorum isolated strains, and it was worth to study whether this change and pathogenicity of Fusobacterium necrophorum were related. 相似文献
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13.
Fusobacterium necrophorum, a Gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically, and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses, ruminant foot abscesses and human oral infections. The pathogenic mechanism of F. necrophorum is complex and not well defined. Several toxins, such as leukotoxin, endotoxin, haemolysin, haemagglutinin and adhesin, have been implicated as virulence factors. Among these, leukotoxin and endotoxin are believed to be more important than other toxins in overcoming the host's defence mechanisms to establish the infection. F. necrophorum is encountered frequently in mixed infections and, therefore, synergisms between F. necrophorum and other pathogens may play an important role in infection. Several investigators have attempted to induce protective immunity against F. necrophorum using bacterins, toxoids, and other cytoplasmic components. Generally, none of the immunogens has afforded statisfactory protection against Fusobacterium infections. Because of the unavailability of suitable immunoprophylaxis, the control of F. necrophorum infection has depended mainly on the use of antimicrobial compounds.Abbreviations CFU
colony-forming units
- DNA
deoxyribonucleic acid
- DNase
deoxyribonuclease
- Eh
redox potential
- ELISA
enzyme-linked immunosorbance assay
- LPS
lipopolysaccharide
- MPN
most-probable number
- PMN
polymorphonuclear cells
- rRNA
ribosoma ribonucleic acid
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- subsp.
subspecies 相似文献
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15.
The optimal production of P. haemolytica leukotoxin in the culture supernatant of a fluid medium is dependent on a number of factors. The leukotoxin has to be produced by using a strain that is known for its ability to produce high quantities of leukotoxin, inoculated into the most suitable type of medium at the correct culture density containing the necessary supplements and harvested after a certain growth period. The volume in which it is produced may also have an influence. Two different procedures are described to produce the leukotoxin in 5 to 15-l quantities in RPMI 1640 medium. The first method used to produce leukotoxin is one that has been repeatedly described since the presence of the leukotoxin was first established in 1978. Using this method seven batches of leukotoxin were produced in litre quantities with leukotoxin activity ranging from 23-67 u/ml. The seed culture inoculum is prepared in brain heart infusion broth, which is centrifuged before the organisms are inoculated into RPMI 1640 medium containing 3.5% foetal calf serum and incubated for only 1 h in a fermenter, after, which the leukotoxin is harvested. An improved alternative method was devised which yielded higher levels of leukotoxin activity by utilising the ability of the P. haemolytica organisms to grow and produce leukotoxin during the logarithmic growth phase in a fermenter. A seed culture harvested in the log phase was prepared in brain heart infusion broth by means of a series of cultures and inoculated into RPMI 1640 containing 3.5% foetal calf serum. Three hours of active growth were allowed during which the leukotoxin was measured by its biological activity and an ELISA assay, and the increase in cell mass by means of the optical density every 30 min. The average leukotoxin biological activity measured 260 u/ml and by means of the ELISA test the leukotoxin concentration measured 315 u/l which is a substantial increase in leukotoxin production. In comparison the average optical density only measured 0.469 at 650 nm. Previous findings were substantiated that the highest cell density was not reflected in the highest leukotoxin activity. It is possible to induce high levels of leukotoxin secretion in submerged cultures with RPMI 1640 medium containing foetal calf serum in the controlled environment of a fermenter in large enough quantities for use as a vaccine by the improved preparation of the seed culture inoculum. 相似文献
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17.
Species of anaerobic bacteria isolated from clinical veterinary specimens were tested for susceptibility to trimethoprim-sulfonamides by the broth-disk elution technique. Three different media were used for each organism: prereduced anaerobically sterilized (PRAS) brain-heart infusion broth (BHI), thioglycollate broth, and a semidefined PRAS medium. Susceptibility results from these media were compared with those determined by interpreting the minimal inhibitory concentration obtained using an agar dilution technique. Results from broth-disk testing in semidefined medium agreed in 68.7% of the cases, in 53.7% for thioglycollate broth, and in 36.9% for BHI. The greatest deviation between techniques occurred with isolates belonging to the genus Bacteroides, followed by those of the genus Clostridium and those of the genus Fusobacterium. This deviation was directly proportional to increasing concentrations of thymidine in the BHI and thioglycollate broths but not with the semidefined medium. We conclude that the broth-disk elution method for measuring susceptibility of obligate anaerobes to trimethoprim-sulfonamides is unsuitable. 相似文献
18.
Cultural characteristics and virulence of strains of Fusobacterium necrophorum isolated from the feet of cattle and sheep 总被引：1，自引：0，他引：1
D. L. EMERY J. A. VAUGHAN B. L. CLARK J. H. DUFTY D. J. STEWART 《Australian veterinary journal》1985,62(2):43-46
Sixty-one isolates of Fusobacterium necrophorum were recovered for study. Thirty-one were obtained from lesions of foot abscess in cattle (25) and sheep (6), 28 were from interdigital lesions in cattle and 2 were from the normal interdigital skin of cattle. The majority of isolates from lesions of foot abscess were virulent, belonged to biotype AB (Fievez 1963), produced flat, irregular shaped, greyish colonies and haemolysis on blood agar, and grew as turbid filamentous suspensions in liquid media. They produced a soluble exotoxin, a leucocidin, and were pathogenic for cattle and mice. Virulent isolates also produced a haemolysin which most readily lysed bovine, equine and chicken erythrocytes; those from sheep were less susceptible while those of rabbit and pig were the most resistant. Isolates recovered from lesions of the feet not classified as foot abscess and from clinically normal feet were predominantly of the B biotype and caused few experimental lesions, produced convex, round, yellow colonies, flocculated and sedimented while growing in liquid medium and produced little or no haemolysin or leucocidin. Routine differentiation between virulent and non-virulent bovine isolates of F. necrophorum could be achieved by assessing the colour, morphology, and degree of haemolytic activity of colonies grown on blood agar. 相似文献
19.
Results listing the identification of brucella isolates received by the National Brucellosis Reference Centre, National Biological Standards Laboratory, Canberra from 1981 to 1985 are presented. The distribution of brucella species and biotypes is shown on a host and state basis. Cultures isolated in Australia were identified as Brucella abortus biotypes 1, 2 and 4, and Strain 19; B. suis biotype 1, and B. ovis. B. melitensis biotype 3 was recovered from man infected in the Mediterranean area. B. abortus biotype 1 was the most frequent isolate. Atypical cultures isolated from cattle included B. suis biotype 1, and erythritol-utilising mutants of Strain 19. The epizootiological implications of these findings are discussed in relation to their impact on the national campaign to eradicate bovine brucellosis. 相似文献
20.
M M Garcia B W Brooks G M Ruckerbauer C E Rigby L B Forbes 《Canadian journal of veterinary research》1988,52(3):338-342
Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4. 相似文献