Purification and partial characterization of Fusobacterium necrophorum haemolysin

Abstract Extracellular haemolysin of Fusobacterium necrophorum was partially purified by diethylaminoethyl-cellulose column chromatography and Sephadex G-200 gel filtration. The purified preparation was shown to be homogenous by polyacrylamide gel electrophoresis. In sodiumdodecylsulfate-polyacrylamide gel electrophoresis, the haemolysin was divided into two bands. Their M _rs were approximately 54000 and 48000. It was heat-sensitive and oxygen-labile. Inactivated haemolysin in air could be reactivated by the dialysis with ammonium sulfate solution containing cysteine monohydrochloride.     

Effect of Fusobacterium necrophorum haemolysin on peripheral leukocytes in vitro

Abstract Fusobacterium necrophorum haemolysin was cytotoxic for animal leukocytes. The most sensitive cells to the haemolysin were rabbit leukocytes, of which disruption of the cells and protoplastic extrusion were induced. The response was dose- and time-dependent, and was neutralised by antiserum. Scanning electron microscopy revealed that the haemolysin induced destruction of rabbit leukocytes, leaving membrane fragments.     

Adherence of Fusobacterium necrophorum to bovine hepatic cells

Abstract The adherence of Fusobacterium necrophorum to the surface of bovine hepatic cells was investigated. Hemagglutinating strain VPI2891 adhered well to the cell membranes, whereas non-hemagglutinating strain S-45 adhered less well. The adherence of the former strain was strongly inhibited by the homologous anti-hemagglutinin serum. Immunofluorescence study revealed that the purified hemagglutinin bound to the membranes of the hepatic cells. These findings suggest that the adherence of F. necrophorum to the bovine hepatic cells is mediated by the bacterial hemagglutinin and has pathogenic importance in bovine necrobacillosis.     

Biochemical and biological characterization of ruminal Fusobacterium necrophorum

Abstract Biochemical characteristics, biological activities, and antimicrobial susceptibilities of ruminal Fusobacterium necrophorum (eight subsp. necrophorum and eight subsp. funduliforme ) and of isolates (three of each subsp.) obtained from bovine hepatic abscesses were determined. F. necrophorum subsp. necrophorum strains had higher phosphatase and DNase activities, produced more leukotoxin, and were more pathogenic to mice than subsp. funduliforme strains. The leukotoxin titer for culture supernatants of ruminal subsp. necrophorum strains was approximately 15 times lower than that of hepatic subsp. necrophorum strains. Hemagglutination activity was present in all hepatic, but only in some ruminal, strains of subsp. necrophorum . The antimicrobial sensitivity profile of the ruminal isolates was similar to that of hepatic isolates.     

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe

Abstract A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemology of ovine foot abscess and other diseases caused by F. necrophorum .     

Detection of antibodies against Fusobacterium necrophorum and Porphyromonas levii‐like species in dairy cattle with papillomatous digital dermatitis

Bovine digital epidermitis involves different pathologies, including PDD, interdigital dermatitis, and foot rot. Bacteriological and molecular biological studies suggest that these are multimicrobial infections. During our study on the isolation of treponemes from biopsies of PDD, colonies producing black pigment were isolated frequently from the primary cultures, suggesting that Porphyromonas species were present. Moreover, 16S rRNA genes of Fusobacterium necrophorum and Porphyromonas levii‐like species were detected in the lesions. We therefore determined whether an immunological response could be elicited by a P. levii‐like organism isolated from a PDD lesion, as well as two subspecies of F. necrophorum in the sera from cows with and without PDD. A total of 151 serum samples were collected from 85 cows with PDD lesions and 33 cows without lesions on 12 PDD‐positive farms and from 33 cows on two PDD‐free farms. ELISA data showed that IgG antibody levels against antigens of P. levii‐like species and F. necrophorum subsp. necrophorum were significantly higher in cows on PDD‐positive farms than in cows on PDD‐free farms, regardless of the presence of PDD lesions in the cows on the PDD‐positive farms. However, F. necrophorum subsp. funduliforme was present at low levels in both groups. The ELISA results were confirmed by western blot analysis. Furthermore, antigens of these bacteria were detected in PDD‐biopsy sections examined by immunohistochemical staining. F. necrophorum subsp. necrophorum and P. levii‐like species may be involved in the pathogenesis of PDD.     

Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.     

Partial characterization of Fusobacterium necrophorum protease

Partial characterization of Fusobacterium necrophorum protease was investigated. The protease was partially purified by gel filtration with Toyopearl HW 55. The final preparation was inactivated completely by heating at 60 degrees C for 30 min and inhibited by ascorbic acid, sodium thioglycollate and p-hydromercuribenzoate. Antibody response to the protease was demonstrated in mice receiving 10(4) CFU of F. necrophorum.     

Necrobacillosis: A case report of complicated Fusobacterium necrophorum septicemia

Necrobacillosis due to Fusobacterium necrophorum is an uncommon anaerobic infection. It has a wide range of presentations and commonly presents as Lemierre's syndrome. We present a case of necrobacillosis defined by F. necrophorum bacteremia with epidural and pararectal fluid collection without evidence of internal jugular vein thrombophlebitis.     

Location of haemagglutinin in bacterial cells of Fusobacterium necrophorum subsp. necrophorum.

The location of haemagglutinin (HA) of Fusobacterium necrophorum subsp. necrophorum VPI 2891 strain was investigated by immunofluorescence, confocal laser scan microscopy and immunoelectron microscopy. The immunofluorescence study demonstrated the fluorescence specific for the HA on the bacterial cells and confocal laser scan microscopy indicated similar fluorescence around the cross section of the bacterial cell. The immunoelectron microscopic study also revealed that the protein A-gold conjugates were located around the bacterial surfaces. These findings suggest that HA is one of the components of the cell surfaces of F. necrophorum subsp, necrophorum.     

Collagenolytic activity of a cell wall preparation from Fusobacterium necrophorum subsp. necrophorum

A collagenolytic preparation of Fusobacterium necrophorum subsp. necrophorum was derived from the bacterial cell. It was further treated for gel permeation with Toyopearl HW 50, followed by Sepharose 4B column chromatography. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the final preparation exhibited one definite band and at least one faint band. It was inactivated completely by adjusting the pH to 4.0 or by heating at 80 degrees C for 30 min.     

Fusobacterium nucleatum and colorectal cancer: A mechanistic overview

Colorectal cancer (CRC) is the third most prevalent cancer in the world. There are many risk factors involved in CRC. According to recent findings, the tumor microenvironment and feces samples of patients with CRC are enriched by Fusobacterium nucleatum. Thus, F. nucleatum is proposed as one of the risk factors in the initiation and progression of CRC. The most important mechanisms of Fusobacterium nucleatum involved in CRC carcinogenesis are immune modulation (such as increasing myeloid-derived suppressor cells and inhibitory receptors of natural killer cells), virulence factors (such as FadA and Fap2), microRNAs (such as miR-21), and bacteria metabolism. The aim of this review was to evaluate the mechanisms underlying the action of F. nucleatum in CRC.     

Chemical composition of endotoxins produced by Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

Abstract The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium .     

The leukotoxin operon of Fusobacterium necrophorum is not present in other species of Fusobacterium

The complete nucleotide sequence of the leukotoxin operon of Fusobacterium necrophorum has been determined. The operon consists of three genes (lktBAC) of which the leukotoxin structural gene is the middle determinant. Southern and western blot analyses and flow cytometry analysis for biological activity of the culture supernatants were carried out to determine if the leukotoxin is present in other species of the genus Fusobacterium. Only the two subspecies of F. necrophorum were found to possess the leukotoxin locus and produce the toxin. The human periodontal pathogen, F. nucleatum does not produce detectable leukotoxin. The F. necrophorum leukotoxin was found to be active against human neutrophils.     

Effects of Fusobacterium necrophorum subspecies necrophorum on extracellular matrix of tissue-cultured bovine kidney cells

The effects of Fusobacterium necrophorum subsp. necrophorum on the extracellular matrix were investigated. The toxic preparation from the culture induced reduction in the number of tissue-cultured bovine kidney cells. The exposed cells often manifested partial loss of cytoplasm and were morphologically irregular. Scanning electron microscopy demonstrated partial loss of the microvilli on the exposed cells and roughness of the cell surfaces. Finally, sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles revealed complete degradation of bovine collagen type 1 after treatment with the toxic preparation. This degradation was inhibited by the addition of homologous antiserum. These findings indicate that the degradation may contribute to the establishment of the infection caused by F.n. subsp. necrophorum.     

Fusobacterium necrophorum septicemia following Epstein-Barr virus infectious mononucleosis

We report herein a case of a 20-year-old previously healthy man who presented, 25 days after the onset of clinically and serologically confirmed Epstein-Barr virus (EBV) infectious mononucleosis, Fusobacterium necrophorum septicemia. The diagnosis of postanginal septicemia was confirmed by repeated demonstration of fusiform, obligate anaerobic, Gram-negative bacilli in anaerobic blood cultures, identified as F. necrophorum 15 days after admission. This case report aims at underlining the need of taking into consideration the possibility of severe Fusobacterium septicemia in previously healthy patients following EBV infectious mononucleosis in order to prevent increased mortality and morbidity.     

Adherence of Fusobacterium necrophorum subsp. necrophorum to ruminal cells derived from bovine rumenitis

Fusobacterium necrophorum subsp. necrophorum strain VPI 2891 was shown to adhere to the surfaces of ruminal cells derived from bovine rumenitis. The strain also attached to bovine type 1 collagen. Treatment of the bacterium with antiserum to bacterial cells reduced attachment. The bacterial attachment was also markedly reduced when the ruminal cells had been pretreated with anticollagen serum. Fluorescence specific for the collagen was demonstrated on the surface of bovine tissue affected with rumenitis. These findings suggest that F. necrophorum subsp. necrophorum strain VPI 2891 adheres to the ruminal cells derived from rumenitis tissue and that the attachment may be mediated by cellular collagen.     

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.