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.

We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.

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Fusobacterium necrophorum is the main pathogen that causes numerous necrobacilloses. Hemolysin is one of the major virulence factors involved in fusobacterial infections. In order to investigate the genetic basis of hemolytic activity and the regulation mechanism of the hemolysin expression, a genomic library was constructed from F. necrophorum DNA by ligating DNA fragments generated by partial HindIII digestion with pUC18 vector. The screening of the genomic library with polymerase chain reaction, DNA sequencing and sequence assembly led to a 7.45 kb sequence which includes the putative hly gene and upstream sequence. Clustered putative genes encoding short chain acyl-CoA dehydrogenase (Scad) and electron transfer flavoprotein (Etf) alpha and beta subunits locate upstream of hly. A 535 bp non-coding sequence, possibly with some cis-regulatory elements involved in the regulation of the hemolysin expression in F. necrophorum, locates between etf-beta and hly. The nucleotide sequence of the hly gene indicates it encodes hemolysin. It is the first characterized hemolysin coding gene in F. necrophorum.

To analyze immunodominant regions of leukotoxin protein of Fusobacterium necrophorum strain H05, a series of truncated forms of leukotoxin gene were expressed in Escherichia coli using the vector pGEX-6p-1 or pPROEX HTa. The results of SDS-PAGE showed the truncated forms PL1, PL2, PL4, and PL5 were expressed in Escherichia coli using the vector pGEX-6p-1, and the truncated forms PL3 was expressed in Escherichia coli using the vector pPROEX HTa. These recombinant proteins were able to react with antisera against Fusobacterium necrophorum strain A25. In five recombinant proteins, the recombinant proteins PL1, PL3 and PL4 as vaccine were able to elicit formation of the better protective effects on mice against infection of Fusobacterium necrophorum strain A25.

Leukotoxin is a well-known virulence factor of animal isolates of Fusobacterium necrophorum subspecies necrophorum, and is also expressed by animal isolates of subspecies funduliforme, whereas its presence in isolates from humans has not been fully established. In this study we found that the leukotoxin gene was present in all tested F. necrophorum isolates from humans. Three sequence variants were found, two of which have not been described previously. The sequence types correlated to source of infection. Further studies are needed to examine the role of the leukotoxin in human infections.

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 epidemiology of ovine foot abscess and other diseases caused by F. necrophorum.

Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.