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At an abattoir, lesion specimens from 140 condemned sheep livers were collected for bacteriologic culture and for pathologic examination. Grossly, 23 lesions were abscesses; from 9 of which, Fusobacterium necrophorum biovar A (3 in pure culture and 6 in mixed culture) was isolated and from 14 of which, biovar B (6 in pure culture and 8 in mixed culture) was isolated. Escherichia coli was the predominant facultative anaerobic bacterium and Clostridium perfringens was the predominant obligate anaerobic bacterium isolated from the 14 lesions with mixed bacterial infection. Histologically, these lesions had a core of coagulation necrosis, encircled by a zone of necrotic phagocytic cells and bacteria with cellular characteristics of F necrophorum biovars A or B, and a connective tissue capsule. Of the 117 lesions without F necrophorum, 49 were culture-positive (for other organisms) and 69 were culture-negative. These 117 lesions were fibrous and were smaller than the 23 abscesses. A variety of gram-positive and gram-negative facultative anaerobic and obligate anaerobic bacteria was isolated from the culture-positive lesions, but always in low numbers. Eleven culture-negative and 18 culture-positive lesions were examined and had histologic characteristics of parasite-induced granulomas, with numerous eosinophils and epithelioid giant cells. Results of the study indicated that the histologic appearance of ovine hepatic lesions with F necrophorum was similar to bovine liver abscesses caused by F necrophorum, but unlike bovine liver abscesses, F necrophorum biovar B was isolated more frequently than was biovar A and often in pure culture. Most of the lesions in the condemned livers were parasite-induced granulomas.

Isolates of Fusobacterium necrophorum were obtained from 124 bovine hepatic abscesses (in 119 cattle) and from the ruminal contents of 12 cattle. Three biotypes (A, AB, and B) were found. Type A isolates were most common in hepatic abscesses, and type B isolates were most common in ruminal contents. Virulence studies in mice indicated that type A isolates were more pathogenic than types AB or B isolates. The difference in virulences between types A and B isolates was not related to the toxicity of the endotoxin. Results of serotesting (passive hemagglutination) showed that a wide heterogeneity existed between isolates. Minimal inhibitory concentrations of antibiotics for 12 of the isolates (7 type A, 1 type AB, and 4 type B) were as follows: less than or equal to 0.06 IU of penicillin G/ml; less than or equal to 0.5 micrograms of cephaloridine/ml; less than or equal to 2 micrograms of chloramphenicol/ml; less than or equal to 0.06 micrograms of clindamycin/ml; less than or equal to 8 micrograms of erythromycin/ml; less than or equal to 128 micrograms of gentamicin/ml; less than or equal to 0.25 micrograms of oxytetracycline/ml; and less than or equal to 4 micrograms of tylosin/ml.

The in vitro leukotoxic activity of 3 bovine isolates of Fusobacterium necrophorum which varied in pathogenicity were compared. Monolayers of mouse peritoneal macrophages were exposed to culture filtrates from each F necrophorum strain, and cell viability was determined, using the trypan blue dye exclusion test. Two methods were used for production of the leukotoxin: (1) medium M-1 continuous dialysis sac cultures and (2) brain-heart infusion agar plate cultures. Supernatant cultural fluids containing the leukotoxin were subjected to membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. All ultrafiltrates had a cytotoxic effect on the monolayers. Cytotoxic activity was not found in the ultrafilter residues or in the control media ultrafiltrates. Comparative study of leukotoxin production indicated that F necrophorum 2101, type A, produced the most leukotoxin; F necrophorum 2030, type AB, produced slightly less leukotoxin; and F necrophorum 2035, type B, produced small amounts of leukotoxin. Endotoxin activity, as demonstrated by the mouse lethality test, was found in the residues of the XM-100A ultrafilter (100,000 mol wt), but not in the filtrates. Culture supernatant fluids and the XM-100A ultrafiltrates were positive for endotoxin, using the limulus amebocyte lysate assay; however, the other ultrafiltrates with lower mol wt exclusion limits were negative.

The biochemical characteristics of the leukotoxins of 3 bovine isolates of Fusobacterium necrophorum which represent biotypes A, AB, and B were compared. Two methods were used for the production of the leukotoxins: medium M-1 continuous dialysis sac cultures and brain-heart infusion agar plate cultures. The supernatant cultural fluids were fractionated sequentially by membrane-partition chromatography, using ultrafilters with approximate molecular weight (mol wt) exclusion limits of 100,000, 10,000, 2,000, and 500. The ultrafiltrates (less than 500 mol wt) were fractionated by gel-permeation chromatography, using G-10 Sephadex. The leukotoxins of the 3 F necrophorum strains were estimated to have a molecular weight between 350 and 450. The leukotoxins in the ultrafiltrates (less than 500 mol wt) were stable at 60 C for 4 hours and at 100 C for 30 minutes, stable to extremes of pH (3 to 11), and stable to degradative enzymes including trypsin, protease, alpha-amylase, lipase, deoxyribonuclease, and ribonuclease. Significant differences were not observed in the biochemical characteristics of the leukotoxins produced in vitro by the 3 F necrophorum biotypes. These assays were done, using monolayers of mouse peritoneal macrophages. The monolayers were exposed to the 4 ultrafiltrates of both the continuous dialysis sac and brain-heart infusion agar cultures (pH 7.2) for 4 hours at 4 C, 25 C, and 37 C. Maximal cytotoxic activity in the assays was at 37 C.

Several biological features were compared in a rat liver abscess model, using intraportal inoculations with 3 bovine strains of Fusobacterium necrophorum which varied in virulence. Serum alanine aminotransferase activities were increased significantly (P less than 0.05) in rats inoculated with F necrophorum 2101 by postinoculation hours 6, 12, and 24. Thereafter, alanine aminotransferase values returned to base line for the remainder of the experiment. Also, rats inoculated with F necrophorum 2101 had a significantly greater (P less than 0.05) weight loss than did the control rats during the first 5 postinoculation days and developed leukocytosis characterized by a neutrophilia with a left shift. The duration of the bacteremia was related directly to the virulence of the F necrophorum strain. Fusobacterium necrophorum 2101, a biotype A which was the most virulent, induced the most persistent bacteremia; F necrophorum 2035, a biotype B which was the least virulent, produced the shortest bacteremia; and F necrophorum 2030, a biotype AB which was of intermediate virulence, led to bacteremia of intermediate duration. Plasma endotoxin was demonstrated intermittently during the first 24 hours, but did not correlate with the bacteremia.