6 research outputs found
Fusobacterium necrophorum leukotoxoid vaccine for prevention of liver abscesses
The efficacy of Fusobacterium
necrophorum crude leukotoxoid vaccine to
immunize and protect steers against experimentally
induced liver abscesses was 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
oil emulsion adjuvant. Vaccine was injected
subcutaneously on days 0 and 21. Blood
samples were collected weekly to monitor
immune response. Three weeks after the
second vaccination, steers were injected
intraportally with F. necrophorum culture to
induce liver abscesses. Three weeks later (day
63), steers were euthanatized and necropsied;
livers were examined, and protection was
assessed. Anti-leukotoxin antibody titers in
the control steers generally did not differ
from the baseline (week 0) titers. The titers
in the vaccinated groups increased, more so
after the second injection, and the increase
was generally dose dependent. At necropsy,
all steers in the control group had liver
abscesses. In the vaccinated groups, two out
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. The difference
suggests a protective effect of antileukotoxin antibodies against experimentally
induced liver abscesses
Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum
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