Fusobacterium necrophorum determined as abortifacient in sheep by laser capture microdissection and fluorescence in situ hybridization.

Fluorescent in situ hybridization (FISH) has been extensively used for identification of individual microbial cells within their natural environment. The present work describes the identification of Fusobacterium necrophorum in formalin-fixed tissue samples from three sets of ovine twins aborted at late pregnancy by a technique that combines laser capture microdissection (LCM) and fluorescent in situ hybridization (LCM-FISH). Cultural bacteriological examination had failed to identify an infectious agent but by histological examination, large colonies of bacteria associated with tissue inflammation were seen. In situ hybridization visualized the bacteria in the tissue samples and micro-colonies closely associated with lesions were isolated by LCM. PCR-amplification and sequencing of 16S rRNA gene from the microdissected bacteria identified the organisms as Fusobacterium necrophorum. A rRNA-targeting oligonucleotide probe specific for F. necrophorum was used in a FISH assay. In situ hybridization showed a high density of F. necrophorum in all examined tissue sections. Simultaneous probing with a general bacterial probe EUB338 and the specific probe for F. necrophorum showed that no other bacteria could be detected in the tissue sections. We therefore conclude that F. necrophorum was the likely cause of abortion in these sheep.

Immunohistochemical and ultrastructural identification of Fusobacterium necrophorum subsp. necrophorum in bovine fatal necrotizing glossitis.

A 37-day-old male Japanese black calf showing marked salivation and leucocytosis died and was examined the tissues histologically. Histological lesions were characterized by severe focal necrotic glossitis on the ventral side of the root of the tongue. Immunohistochemically, Fusobacterium necrophorum subsp. necrophorum antigen was detected in the necrotic tissues and its distribution corresponded to that of the gram-negative, nonsporeforming, long filamentous organisms. Ultrastructural similarities between the organism and F. necrophorum subsp. necrophorum, but not subsp. funduliforme were observed. These findings clearly demonstrated that the fatal necrotic glossitis was caused by F. necrophorum subsp. necrophorum. This is the first report of bovine fatal necrotizing glossitis with leucocytosis caused by F. necrophorum subsp. necrophorum infection, and this organism may be an important fatal pathogen in calves with glossal lesions.

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.

Ultrastructure and molecular characterization of Fusobacterium necrophorum biovars.

The ultrastructural features and molecular components of 18 strains of Fusobacterium necrophorum biovars A, AB and B, isolated from animal and human infections, were examined by electron microscopy, multilocus enzyme electrophoresis (MEE) and by sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-PAGE). High resolution scanning electron microscopy revealed that the strains possessed a convoluted surface pattern. Transmission electron microscopy showed that all strains possessed a cell wall structure typical of gram-negative bacteria. Bleb formation was not uncommon. Numerous extracellular materials, resembling lipopolysaccharide (LPS) fragments, surrounded cells of both human strains and biovar B animal strains. Biovar A field strains revealed capsules as stained by ruthenium red whereas a stock culture strain showed the capsule only when immunostabilized with hyperimmune serum. Starch gel electrophoresis showed all strains to possess adenyl kinase, glutamate dehydrogenases and lactate dehydrogenase; each enzyme migrated uniformly (monomorphic) among the strains and represented an electrotype. However, SDS-PAGE indicated differences in the protein profiles between all of the strains; the most distinctly different was a human isolate (FN 606). Silver staining to detect LPS showed extensive "ladder" patterns among the majority of biovar A strains but not in the animal biovar B strains. Immunoblotting of LPS with a rabbit antiserum prepared against phenol extracted LPS from a biovar A animal isolate (LA 19) suggested marked variability in the LPS antigens among the isolates studied.

Adherence of Fusobacterium necrophorum to bovine ruminal cells.

The adherence of Fusobacterium necrophorum to the surface of bovine ruminal epithelial cells was paralleled by the organism's haemagglutinating ability. Treatment of the bacterial cells with haemagglutinin antiserum caused a reduction in the degree of attachment. The purified haemagglutinin became bound to the membranes of ruminal epithelial cells but lost its adherence when pre-incubated with haemagglutinin antiserum. These findings suggest that the adherence of F. necrophorum to the membrane of the ruminal cells is mediated by haemagglutinin.