leukotoxin and Liver-Abscess

The efficacy and the optimum dose of Fusobacterium necrophorum crude leukotoxoid vaccine required to immunize and protect steers against experimentally induced liver abscesses were evaluated. The vaccine consisted of cell-free culture supernatant of a high leukotoxin-producing strain of F. necrophorum, inactivated with formalin and homogenized with an adjuvant. Twenty-five steers were assigned randomly to the following five treatment groups: control; three doses (1.0, 2.0, and 5.0 mL) of the culture supernatant; and 2.25 mL of the concentrated supernatant (equivalent to 5 mL of the original supernatant). Vaccine was injected subcutaneously on d 0 and 21. Blood samples were collected weekly to monitor antileukotoxin antibody titers. Three weeks after the second vaccination (d 42), all steers were injected intraportally with F. necrophorum culture to induce liver abscesses. Three weeks later (d 63), steers were euthanatized and necropsied; livers were examined and protection assessed. Antileukotoxin antibody titers in the control steers generally did not differ from the baseline (wk 0) titers. The titers in the vaccinated groups increased, more so after the second injection, and the increase was generally dose-dependent. Necropsy examination revealed that all steers in the control group had abscesses in the liver. In the vaccinated groups, two of five steers in the 1.0-mL group and one each in the 2.0-, 5.0-, and 2.25-mL (concentrated) groups had liver abscesses. Antileukotoxin antibody titers were higher (P < .05) in steers that did not develop abscesses than in steers that developed abscesses. The difference suggested a protective effect of antileukotoxin antibodies against experimentally induced liver abscesses.

Topics: Animals; Antibodies, Bacterial; Bacterial Vaccines; Cattle; Cattle Diseases; Disease Susceptibility; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Exotoxins; Fusobacterium necrophorum; Immunization; Immunosuppressive Agents; Liver; Liver Abscess; Male

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.

Topics: Animals; Cattle; Cattle Diseases; Cell Line; Cell Survival; Exotoxins; Foot Rot; Fusobacterium Infections; Fusobacterium necrophorum; L-Lactate Dehydrogenase; Liver Abscess; Virulence Factors

The serum antileukotoxin antibody response and protection against subsequent experimental challenge with Fusobacterium necrophorum were investigated in 30 steers vaccinated with crude F. necrophorum leukotoxoid. Culture supernatant of F. necrophorum, strain 25, containing leukotoxoid was concentrated. The steers were assigned randomly to six groups (n = 5): PBS control with Stimulon adjuvant; vaccinated with concentrated supernatant diluted to provide 2.5, 5.0, 10.0, or 20.0 ml with the water-soluble Stimulon adjuvant; and 5.0 ml with the Ribi oil-emulsion adjuvant. The steers were injected subcutaneously on days 0 and 21. Blood samples were collected at weekly intervals to monitor serum antileukotoxin antibody titres. On day 42, all the steers were challenged intraportally with F. necrophorum culture. Three weeks later (day 63), the steers were killed and necropsied for examination of their livers and assessment of protection. Steers vaccinated with crude leukotoxoid tended to have higher antileukotoxin titres than the controls, but the difference was not significant. Also, the antibody titre did not appear to be dose-dependent. In the control group, 3 out of 5 steers developed liver abscesses. The incidence of liver abscesses in steers vaccinated with Stimulon adjuvant was not dose related; however, only 8 of the 25 vaccinated steers developed abscesses. None of the steers vaccinated with the 5.0 ml dose with Ribi had any abscesses. Evidence for a relationship between antileukotoxin antibody and protection was shown by the lower titre in those steers that developed abscesses compared to those that did not. It was concluded that antileukotoxin antibody titres probably provided some degree of protection against experimentally induced liver abscesses, but further dose-titration studies using Ribi or possibly another more effective adjuvant will be needed to confirm this.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bacterial Vaccines; Cattle; Cattle Diseases; Cell Wall Skeleton; Cord Factors; Dose-Response Relationship, Immunologic; Exotoxins; Fusobacterium Infections; Fusobacterium necrophorum; Lipid A; Liver Abscess; Male; Random Allocation; Toxoids; Vaccination; Viral Proteins

The relationship between serum-neutralizing antibody against Fusobacterium necrophorum leukotoxin and hepatic abscesses was investigated in cattle fed diets supplemented with or without tylosin. Sixteen cattle (eight each in tylosin and in control groups) were inoculated intraportally with F. necrophorum. Ultrasonographic scanning showed that all control animals developed hepatic abscesses after inoculation. In the tylosin group, two animals were free of abscess by d 7 and one was free by d 14. Leukotoxin-neutralizing antibody titers were low on d 0, but increased (P < .05) markedly after intraportal inoculation in both groups. In a second study, blood was collected at the time of slaughter from 141 feedlot cattle (36 fed diets with tylosin and 105 fed diets without tylosin), and livers were examined for presence or severity of hepatic abscesses at slaughter. The incidences of hepatic abscesses were 32% in the control group and 6% in the tylosin group. Antibody was detected in all animals; however, antibody titers were greater (P < .05) in cattle with abscessed liver than those without, and greater (P < .01) in the nontylosin than in the tylosin group. Abscess score and antibody titer were correlated (r = .34; P < .0001). We conclude that F. necrophorum leukotoxin is highly antigenic and that anti-leukotoxin antibody titer is related to the severity of hepatic abscesses.

Topics: Animals; Antibodies, Bacterial; Bacterial Toxins; Cattle; Cattle Diseases; Exotoxins; Female; Fusobacterium Infections; Fusobacterium necrophorum; Liver; Liver Abscess; Male; Random Allocation; Tylosin; Ultrasonography