Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolated from bovine ruminal contents and liver abscesses.

Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.     

Endotoxin-triggered haematological interactions in Fusobacterium necrophorum infections

The haematological mechanisms in the course of liver abscess formation were evaluated. They were examined by employing viable cells of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in comparison with their endotoxins. Whole cell infection with F.n. necrophorum led to neutrophilia and to a concomitant monocytosis in parallel with those responses induced by the in vivo injection of its endotoxin. Viable infection with F.n. funduliforme was characterized by a sustained endotoxin-related monocytosis against neutropenia. The stimulatory impact of endotoxin on monocytes when released from a viable F.n. funduliforme infection suggested an inherently peculiar mechanism which differed from the induction of both neutrophilia and monocytosis when F.n. funduliforme endotoxin was administered alone. The neutrophilic inducing capacity of the F.n. necrophorum endotoxin was equally illustrated by its positive chemotactic effect on polymorphonuclear neutrophils in vitro. The data presented here emphasize the virulence of F.n. necrophorum viewed in reference to changes in leucocyte trafficking and as complemented by a relatively high endotoxin content.     

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.     

Proposal of two subspecies of Fusobacterium necrophorum (Flügge) Moore and Holdeman: Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898)

The biological and biochemical properties, DNA base compositions, and levels of DNA-DNA homology of two biovars of Fusobacterium necrophorum were examined. Some differences were found between the two biovars in biological and biochemical properties. The G + C contents of DNAs from biovar A strains VPI 2891T (T = type strain), NCTC 10576, N167, Fn47, and Fn43, were 32, 30, 29, 28, and 31 mol%, respectively. The G + C contents of DNAs from biovar B strains Fn524T, 606, Fn49, Fn45, and 1260 were 30, 31, 27, 31, and 30 mol%, respectively. Labeled DNA from biovar A strain VPI 2891T exhibited 100 to 80% relatedness to DNAs from biovar A strains and 59 to 51% relatedness to DNAs from biovar B strains. Labeled DNA from biovar B strain Fn524T exhibited 100 to 81% relatedness to DNAs from biovar B strains and 71 to 60% relatedness to DNAs from biovar A strains. Therefore, the names Fusobacterium necrophorum subsp. necrophorum subsp. nov., nom. rev. (ex Flügge 1886), and Fusobacterium necrophorum subsp. funduliforme subsp. nov., nom. rev. (ex Hallé 1898), are proposed for Fusobacterium necrophorum biovars A and B, respectively. The type strain of F. necrophorum subsp. necrophorum is strain VPI 2891 (= JCM 3718 = ATCC 25286), and the type strain of F. necrophorum subsp. funduliforme is strain Fn524 (= JCM 3724).     

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.     

Fusobacterium necrophorum infections in animals: pathogenesis and pathogenic mechanisms

Fusobacterium necrophorum, a Gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), have been recognized, that differ morphologically, biochemically, and biologically. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis), either specific or non-specific infections, in a variety of animals. Of these, bovine liver abscesses and foot rot are of significant concern to the cattle industry. Liver abscesses arise with the organisms that inhabit the rumen gaining entry into the portal circulation, and are often secondary to ruminal acidosis and rumenitis complex in grain-fed cattle. Foot rot is the major cause of lameness in dairy and beef cattle. The pathogenic mechanism of F. necrophorum is complex and not well defined. Several toxins or secreted products, such as leukotoxin, endotoxin, hemolysin, hemagglutinin, proteases, and adhesin, etc., have been implicated as virulence factors. The major virulence factor appears to be leukotoxin, a secreted protein of high molecular weight, active specifically against leukocytes from ruminants. The complete nucleotide sequence of the leukotoxin operon of F. necrophorum has been determined. The operon consists of three genes (lktBAC) of which the second gene (lktA) is the leukotoxin structural gene. The leukotoxin appears to be a novel protein and does not share sequence similarity with any other leukotoxin. F. necrophorum is also a human pathogen and the human strains appear to be different from the strains involved in animal infections.     

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.     

Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and an aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. Several toxins or secreted products have been implicated as virulence factors. The major factors contributing to ruminal colonization and invasion into the liver are hemagglutinin, endotoxin and leukotoxin, of which leukotoxin is the protective antigen. In some conditions, the organism synergistically interacts with Arcanobacterium pyogenes, a facultative anaerobic organism and a secondary etiologic agent, to cause liver abscesses.     

Actin degradation concomitant with Fusobacterium necrophorum subsp. necrophorum adhesion to bovine portal cells

The effects of Fusobacterium necrophorum subsp. necrophorum on cellular actin were investigated using tissue-cultured bovine portal cells. Fluorescence studies revealed the appearance of intense fluorescent spots on the cellular actin and the spots increased in a time dependent manner. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles manifested partial or complete degradation of actin preparation after treatment with the bacterial cells. These findings suggest that the bacterial cell wall may contribute to the degradation of the cellular actin during the initial stage of the infection caused by F. necrophorum subsp. necrophorum.     

Ribotyping to compare Fusobacterium necrophorum isolates from bovine liver abscesses, ruminal walls, and ruminal contents.

Restriction fragment length polymorphism analysis of rRNA genes was employed to genetically compare Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolates from multiple abscesses of the same liver and isolates from liver abscesses, the ruminal wall, and ruminal contents from the same animal. Four livers with multiple abscesses and samples of ruminal contents, ruminal walls, and liver abscesses were collected from 11 cattle at slaughter. F. necrophorum was isolated from all liver abscesses, nine ruminal walls, and six ruminal content samples. Chromosomal DNA of the isolates was extracted and single or double digested with restriction endonucleases (EcoRI, EcoRV, SalI, and HaeIII); then restriction fragments were hybridized with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNAs from Escherichia coli. EcoRI alone or in combination with EcoRV yielded the most discriminating ribopatterns for comparison. Within the subspecies multiple isolates from the same liver were indistinguishable based on the ribopattern obtained with EcoRI. The hybridization patterns of liver abscess isolates were concordant with those of the corresponding isolates from ruminal walls in eight of nine sets of samples. None of the six ruminal content isolates matched either the liver abscess isolates or the ruminal wall isolates. The genetic similarity between the isolates from liver abscesses and ruminal walls supports the hypothesis that F. necrophorum isolates of liver abscesses originate from the rumen.     

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.     

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.     

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.     

Conversion of DL-threonine, D-threonine and 2-oxobutyrate into propionate and 2-hydroxybutyrate by Fusobacterium species

The present investigation examined DL -threonine, D- threonine and 2-oxobutyrate conversion into propionate and 2-hydroxybutyrate by various type strains and clinical isolates of Fusobacterium. Except for Fus. naviforme , the type strains were able to produce varying degrees of propionate and/or 2-hydroxybutyrate from DL -threonine. Additionally, D -threonine was converted into an equimolar amount of propionate by Fus. necrophorum subsp. necrophorum , Fus. nucleatum subsp. nucleatum and Fus. varium , and to a lower but significant amount by Fus. mortiferum and Fus. perfoetens. However, the level of propionate remained unchanged for Fus. nucleatum subsp. fusiforme , Fus. nucleatum subsp. vincentii , Fus. naviforme , Fus. necrophorum subsp. funduliforme , Fus. gonidiaforme and Fus. russii. 2-Oxobutyrate was fermented to propionate by all type strains, although Fus. russii reduced it mainly to 2-hydroxybutyrate. Thus, an attempt was made to make use of these features in order to identify clinical isolates.     

Immunobiological properties of lipopolysaccharides isolated from Fusobacterium nucleatum and F. necrophorum

Lipopolysaccharides (LPSs) were isolated from Fusobacterium nucleatum ATCC 10953 and F. necrophorum ATCC 25286 by the hot phenol/water procedure. F. nucleatum LPS was composed of 16% (w/w) carbohydrate, 10% (w/w) hexosamine and 40% (w/w) fatty acid, while F. necrophorum LPS was composed of 26% (w/w) carbohydrate, 12% (w/w) hexosamine and 28% (w/w) fatty acid. These LPS preparations induced mitogenic responses in spleen cells of BALB/c, BALB/c (nu/nu) and C3H/HeN mice, and these responses were suppressed by the addition of polymyxin B. The preparations also induced the polyclonal responses of C3H/HeN spleen cells. In addition, enhanced glucose utilization and interleukin-1 production by murine peritoneal macrophages were demonstrated. Neither spleen cells nor macrophages from the 'LPS-nonresponsive' C3H/HeJ mouse were activated by LPSs from the Fusobacterium species.     

Enzyme-linked immunosorbent assay for the detection of Fusobacterium necrophorum antibody in bovine sera.

An enzyme-linked immunosorbent assay (ELISA) with HCl heat extracted antigen of Fusobacterium necrophorum was developed for the detection of antibody in bovine sera. Optimal conditions for antigen concentration and dilution of bovine serum were established. Pretreatment of positive reference serum with the antigens of different bacteria demonstrated no decrease, whereas the serum pretreated with F. necrophorum antigens revealed a decrease in the ELISA values. The apparent difference in ELISA values was observed between the sera derived from cattle infected and not infected with Fusobacterium necrophorum. These findings indicate that the ELISA detects the antibody to F. necrophorum in bovine sera.     

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 .     

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.     

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.