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1.
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.  相似文献   

2.
The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.  相似文献   

3.
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.  相似文献   

4.
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   

5.
An 8 year-old cow showing severe dyspnea and nasal mucosal necrosis immediately after parturition was subjected to pathological examination. The principal lesions were fibrinonecrotic rhinitis, necrotic bronchopneumonia and renal infarction. Fusobacterium necrophorum biotype A and Arcanobacterium pyogenes antigens were detected in the nasal and pulmonary lesions. These results suggest that the lesions were caused by a concurrent infection of the detected bacteria and that the pulmonary lesions were caused by the aspiration of infectious materials from the nasal ones. Mucosal coagulative necroses observed as the initial lesions in rhinitis were frequently associated with multiple thrombosis. The findings might suggest that thrombosis played an important role in the development of the nasal lesions.  相似文献   

6.
The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.  相似文献   

7.
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   

8.
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.  相似文献   

9.
At an abattoir, lesion specimens from 140 condemned sheep livers were collected for bacteriologic culture and for pathologic examination. Grossly, 23 lesions were abscesses; from 9 of which, Fusobacterium necrophorum biovar A (3 in pure culture and 6 in mixed culture) was isolated and from 14 of which, biovar B (6 in pure culture and 8 in mixed culture) was isolated. Escherichia coli was the predominant facultative anaerobic bacterium and Clostridium perfringens was the predominant obligate anaerobic bacterium isolated from the 14 lesions with mixed bacterial infection. Histologically, these lesions had a core of coagulation necrosis, encircled by a zone of necrotic phagocytic cells and bacteria with cellular characteristics of F necrophorum biovars A or B, and a connective tissue capsule. Of the 117 lesions without F necrophorum, 49 were culture-positive (for other organisms) and 69 were culture-negative. These 117 lesions were fibrous and were smaller than the 23 abscesses. A variety of gram-positive and gram-negative facultative anaerobic and obligate anaerobic bacteria was isolated from the culture-positive lesions, but always in low numbers. Eleven culture-negative and 18 culture-positive lesions were examined and had histologic characteristics of parasite-induced granulomas, with numerous eosinophils and epithelioid giant cells. Results of the study indicated that the histologic appearance of ovine hepatic lesions with F necrophorum was similar to bovine liver abscesses caused by F necrophorum, but unlike bovine liver abscesses, F necrophorum biovar B was isolated more frequently than was biovar A and often in pure culture. Most of the lesions in the condemned livers were parasite-induced granulomas.  相似文献   

10.
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11.
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   

12.
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   

13.
Transmission experiments were carried out in gnotobiotic pigs to determine whether lesions typical of swine dysentery could be produced by oral inoculation of Treponema hyodysenteriae in combination with Bacteroides vulgatus or Fusobacterium necrophorum, or both. Each of the organisms had been isolated from swine with early lesions of the disease. Lesions were not found in 6 pigs inoculated with T hyodysenteriae alone, in 4 pigs given F necrophorum and T hyodysenteriae, or in 4 pigs given B vulgatus and F necrophorum. Lesions typical of swine dysentery developed in 8 pigs given B vulgatus, F necrophorum, and T hyodysenteriae as well as in 3 of 4 pigs given B vulgatus and T hyodysenteriae. In both of these groups, the inoculated bacteria were recovered from the colon, and T hyodysenteriae was demonstrated in the colonic crypts, epithelium, and lamina propria. The pathogenicity of the T hyodysenteriae was shown by the development of characteristic signs and lesions of swine dysentery in 12 of 14 naturally farrowed pigs inoculated with T hyodysenteriae alone.  相似文献   

14.
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。  相似文献   

15.
The effects of the collagenolytic cell wall component (CCWC) of Fusobacterium necrophorum subsp. necrophorum on bovine hepatic cell and cytoskeletons were investigated. Scanning electron microscopy (SEM) demonstrated that CCWC damaged the cell surfaces, forming tiny holes on the cell membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles revealed that CCWC degraded bovine cytokeratin and vimentin and by indirect fluorescent antibody (IFA) method, it was shown that CCWC caused the deformation of hepatocellular vimentin. This suggested that CCWC contributes to bovine hepatic injury and it may be as important pathogenic factor in the development of bovine hepatic abscesses.  相似文献   

16.
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.  相似文献   

17.
Mice immunised with killed or living Fusobacterium necrophorum, by five different regimens, almost invariably failed to produce antibodies demonstrable by a passive haemagglutination test. An enzyme-linked immunosorbent assay (ELISA), however, usually demonstrated a serum antibody response. This suggested that F necrophorum was not in fact immunosuppressive in mice--a possibility that had been entertained to explain the great difficulty in protecting mice immunologically against challenge with F necrophorum.  相似文献   

18.
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.  相似文献   

19.
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   

20.
Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.  相似文献   

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