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Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Cross-reactions with Fusobacterium necrophorum were found in a PCR designed for detection of a wide range of mycoplasma species. Twenty-five strains of Fusobacterium were examined; all 14 F. necrophorum strains reacted positively, whereas all 7 Fusobacterium nucleatum strains reacted negatively. Two strains that were not F. necrophorum yielded variable results.

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.