Objective—To identify the Moraxella bovis cytotoxin
Procedure—Hemolytic and nonhemolytic strains of
M bovis were compared by use of western blotting to
identify proteins unique to hemolytic strains.
Oligonucleotide primers, designed on the basis of
amino acid sequences of 2 tryptic peptides derived
from 1 such protein and conserved regions of the C
and B genes from members of the repeats in the
structural toxin (RTX) family of bacterial toxins, were
used to amplify cytotoxin-specific genes from M
bovis genomic DNA. Recombinant proteins were
expressed, and antisera against these proteins were
produced in rabbits.
Results—Several proteins ranging in molecular mass
from 55 to 75 kd were unique to the hemolytic strain.
An open reading frame encoding a 927-amino acid protein
with a predicted molecular mass of 98.8 kd was
amplified from M bovis genomic DNA. The deduced
amino acid sequence encoded by this open reading
frame was homologous to RTX toxins. Antisera against
the recombinant carboxy terminus encoded by this
open reading frame neutralized hemolytic and cytolytic
activities of native M bovis cytotoxin.
Conclusions and Clinical Relevance—A gene was
identified in M bovis that encodes a protein with
sequence homology to other RTX toxins. Results of
cytotoxin neutralization assays support the hypothesis
that M bovis cytotoxin is encoded by this gene and
belongs in the RTX family of bacterial exoproteins.
Identification of this gene and expression of recombinant
cytotoxin could facilitate the development of
improved vaccines against infectious bovine keratoconjunctivitis.
(Am J Vet Res 2001;62:1222–1228)
Objective—To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK).
Animals—107 beef steers.
Procedures—2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin–M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined.
Results—No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non–IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves.
Conclusions and Clinical Relevance—The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.
OBJECTIVE To evaluate changes in systemic and ocular antibody responses of steers following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin (MbxA).
ANIMALS 13 Angus steers with ages ranging from 318 to 389 days and weights ranging from 352 to 437 kg.
PROCEDURES Steers were assigned to receive 500 μg of a precipitated (MbxA-P; n = 5) or partially solubilized (MbxA-S; 5) recombinant MbxA subunit adjuvanted with polyacrylic acid. A control group (n = 3) received the adjuvant alone. Each steer received the assigned treatment (1 mL/nostril) on days 0 and 28. Serum and tear samples were collected on days 0 (before vaccination), 14, 28, 42, and 55. Changes in MbxA-neutralizing antibody titers and MbxA-specific IgG concentrations in serum and tears and changes in MbxA-specific IgA concentrations in tears were measured.
RESULTS Mean fold changes in MbxA-specific IgG concentration in serum and tears and MbxA-neutralizing antibody titer in tears for the MbxA-P group were significantly greater than those for the MbxA-S and control groups. Mean serum MbxA-neutralizing antibody titer did not differ among the 3 groups. Although the mean fold change in tear MbxA-specific IgA concentration differed significantly among the groups in the overall analysis, post hoc comparisons failed to identify any significant pairwise differences.
CONCLUSIONS AND CLINICAL RELEVANCE Systemic and ocular immune responses induced by intranasal administration of the MbxA-P vaccine were superior to those induced by the MbxA-S vaccine. Additional research is necessary to determine whether the MbxA-P vaccine can prevent naturally occurring infectious bovine keratoconjunctivitis.