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Infection and Immunity, December 1998, p. 5613-5619, Vol. 66, No. 12
Department of Anatomy, Pathology, and
Pharmacology, College of Veterinary Medicine, Oklahoma State
University, Stillwater, Oklahoma 74078
Received 12 May 1998/Returned for modification 25 June
1998/Accepted 8 September 1998
Pasteurella haemolytica serotype 1 is the bacterium
most commonly associated with bovine shipping fever. The presence of
antibodies against P. haemolytica outer membrane proteins
(OMPs) correlates statistically with resistance to experimental
P. haemolytica challenge in cattle. Until now, specific
P. haemolytica OMPs which elicit antibodies that function
in host defense mechanisms have not been identified. In this study, we
have cloned and sequenced the gene encoding one such protein, PlpE.
Analysis of the deduced amino acid sequence revealed that PlpE is a
lipoprotein and that it is similar to an Actinobacillus
pleuropneumoniae lipoprotein, OmlA. Affinity-purified, anti-PlpE
antibodies recognize a protein in all serotypes of P. haemolytica except serotype 11. We found that intact P. haemolytica and recombinant E. coli expressing PlpE
are capable of absorbing anti-PlpE antibodies from bovine immune serum,
indicating that PlpE is surface exposed in P. haemolytica and assumes a similar surface-exposed conformation in E. coli. In complement-mediated killing assays, we observed a
significant reduction in killing of P. haemolytica when
bovine immune serum that was depleted of anti-PlpE antibodies was used
as the source of antibody. Our data suggest that PlpE is surface
exposed and immunogenic in cattle and that antibodies against PlpE
contribute to host defense against P. haemolytica.
Pasteurella haemolytica
serotype 1 (S1) is the organism most commonly associated with shipping
fever, a disease of beef cattle characterized by fibrinous
pleuropneumonia (reviewed in references 9 and
15). The disease is of significant economic
importance to the beef industry in the United States, accounting for
annual losses approaching 1 billion dollars (18). Shipping
fever pneumonia is precipitated by stress-inducing conditions such as
shipping, viral infections, inhalation of diesel fumes, overcrowding,
and weaning (9, 15). P. haemolytica S1 resides in
small numbers in the upper respiratory tracts of cattle, and the tonsil
has been shown to be a reservoir (16, 46). Cells proliferate
under stressful conditions and are aerosolized in large numbers into lung alveoli, where they cause the disease (16).
Numerous surface and secreted molecules of P. haemolytica S1
have been studied to evaluate their roles in immunity to P. haemolytica infection (reviewed in reference
6). A secreted cytolytic toxin, leukotoxin (Lkt)
(44), is a significant P. haemolytica virulence factor. In one study, a vaccine consisting of recombinant Lkt (rLkt)
did not provide protection against experimental P. haemolytica challenge (11). However, inclusion of that
rLkt in a commercial vaccine resulted in enhanced resistance to
challenge (11). Those results are in agreement with data
from a prior study (45), which suggested that antibodies
against Lkt and surface antigens are necessary for protective immunity
to P. haemolytica.
The P. haemolytica surface antigens likely to be most
important in contributing to protective immunity are outer membrane proteins (OMPs). Vaccination of cattle with an OMP-enriched fraction of
P. haemolytica cell envelopes significantly reduces lung
damage following experimental challenge with a P. haemolytica strain of the homologous serotype (29).
Bovine antibody responses to proteins present in P. haemolytica surface extracts correlate statistically with
resistance to pneumonia (10, 47). Our group and others have
analyzed the bovine antibody response to PomA, a protein belonging to
the OmpA family (28), to a 94-kDa P. haemolytica
OMP (34), and to several membrane lipoproteins (12-14, 37). These studies suggest a role for outer membrane antigens in
eliciting protective immunity. However, the capacity for P. haemolytica OMP-specific antibodies to function in host defense mechanisms remains uncharacterized. For the development of
more-effective vaccines, it will be important to characterize
individual OMPs and identify those that elicit host antibodies which
enhance resistance to P. haemolytica infection.
Complement-mediated lysis is an important host defense mechanism
against microbial infection and is believed to play a role in
controlling P. haemolytica pneumonia. Serum complement
concentrations were found to be lower in stressed cattle after
transport to a feedlot (40). Lower complement concentrations
were associated with higher morbidity in the feedlot, and morbid calves
had significantly lower complement levels than did healthy calves in
the same feedlots (40). These data suggest that a decrease
in serum complement levels might facilitate P. haemolytica
infection. However, complement-mediated killing of P. haemolytica requires sensitization with antibodies (27). Antibodies against surface-exposed epitopes of OMPs
are likely to play an important role in complement-mediated lysis of
P. haemolytica.
Cattle that are resistant to P. haemolytica-induced
pneumonia develop antibodies to a surface-exposed, ~45-kDa OMP
(36). The purpose of this study was to determine, through
genetic cloning and DNA sequencing, the specific identity of the
immunogenic 45-kDa protein and to evaluate the contribution of
antibodies against this protein to complement-mediated killing of
P. haemolytica. We found that the 45-kDa protein is a
lipoprotein, designated PlpE, and that antibodies against PlpE, present
in bovine immune sera, contribute to complement-mediated killing of
P. haemolytica.
Bacteria, bacteriophage, culture media, and genomic library.
P. haemolytica (89010807N) S1 was grown in BHI broth or on
BHI agar (Difco Laboratories, Detroit, Mich.) as previously described (32). Escherichia coli BB4 and XL1-Blue and
bacteriophages Bovine immune sera and purification of antibodies.
Two
bovine immune sera were used, one from a calf hyperimmunized with live
P. haemolytica (25) and one from a calf that was
vaccinated with P. haemolytica OMPs and was resistant to
experimental P. haemolytica challenge (7).
Briefly, the OMP-vaccinated calf was vaccinated subcutaneously on day 0 and day 21 with P. haemolytica S1 OMPs (2 mg in 1 ml of
phosphate buffered saline [PBS] and 1 ml of an aluminum
hydroxide-DDA-bromide adjuvant which has been described elsewhere in
more detail [8]). On day 36, the calf was
experimentally challenged transthoracically with 5 ml of a mixture
containing 109 CFU of P. haemolytica S1/ml in
each caudal lung lobe. Lung damage was evaluated upon necropsy 4 days
after challenge, by using a previously described lung lesion score
system (35). The serum used in this experiment was collected
on the day of experimental challenge.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetic and Immunologic Analyses of PlpE, a
Lipoprotein Important in Complement-Mediated Killing of
Pasteurella haemolytica Serotype 1
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ZAPII and R408 were supplied with a P. haemolytica genomic DNA library (Clontech Laboratories, Palo Alto,
Calif.) (37) and were grown according to the manufacturer's
instructions. Recombinant E. coli strains were grown in the
presence of ampicillin (50 µg/ml).
Gene cloning, DNA sequencing, site-directed mutagenesis, and sequence analysis. The plpE gene was cloned by screening a P. haemolytica expression library (described above), according to the manufacturer's instructions, with affinity-purified anti-45-kDa antibodies. Additional DNA cloning was performed as described previously (37). Plasmid inserts were progressively deleted from both ends by using the Erase A Base kit (Promega Corp., Madison, Wis.), and progressively smaller plasmid inserts were sequenced by using the universal and reverse primers. Both DNA strands were sequenced. DNA sequencing was performed at the Oklahoma State University Recombinant DNA/Protein Resource Facility, on an Applied Biosystems (Foster City, Calif.) model 373A automated DNA sequencer. Site-directed mutagenesis was performed by using the Gene Editor in vitro Mutagenesis System (Promega Corp.). Mutations were confirmed by DNA sequence analysis. Sequences were analyzed with MacVector/Assemblylign software (Oxford Molecular Group, Inc., Campbell, Calif.). The deduced amino acid sequence of PlpE was compared with other sequences in GenBank by using BLAST 2.0 (1), and alignments were generated with CLUSTALW 1.7 at the Baylor College of Medicine Search Launcher.
Antigen preparation and Western immunoblots. Whole-cell lysates were prepared and Western immunoblots were performed as described previously (14, 37). Primary antibodies used for each Western immunoblot experiment are described in Results. Alkaline phosphatase-conjugated mouse monoclonal, anti-bovine immunoglobulin G antibody (Sigma Immunochemicals, St. Louis, Mo.) (1:20,000 in TSGT) was used as the secondary antibody in Western immunoblots.
For Western immunoblots, bovine immune serum was absorbed with intact P. haemolytica by using a modification of a previously described method (19). Logarithmic-phase P. haemolytica cells, from 1 liter of culture (A600 of 0.5), were pelleted by centrifugation, washed once in PBS, and resuspended in immune serum diluted 1:100 in Tris-saline-nonfat dry milk (TSM) (10 mM Tris [pH 7.4], 0.9% [wt/vol] NaCl, 1% nonfat dried milk). Cells resuspended in serum were incubated at 4°C for 3 h on a rocking platform. Following incubation, cells were pelleted by centrifugation at 11,000 × g. The supernatant was carefully removed and stored at
20°C
after the addition of sodium azide at 0.02%. Unabsorbed immune serum was used as a control and was diluted 1:100 in TSM before use. Serum
absorptions with recombinant E. coli(pB4522) and
nonrecombinant E. coli(pBluescript SK) were performed
similarly except that stationary-phase organisms were used.
[3H]palmitic acid labeling of bacterial lipoproteins. Labeling and analysis of P. haemolytica, recombinant E. coli expressing PlpE, and nonrecombinant E. coli were performed by using [9,10-3H]palmitic acid (Dupont, NEN, Boston, Mass.) as described previously (14).
Complement-mediated killing assay. Serum from a calf with a low antibody titer against P. haemolytica as determined by an enzyme-linked immunosorbent assay was used as a complement source. The complement source serum was depleted of any existing antibodies against P. haemolytica by incubation with excess stationary-phase P. haemolytica at 4°C on a rocking platform for 1 h. Before incubation with complement source serum, P. haemolytica cells were washed once with cold PBS (4°C).
Serum from an OMP-vaccinated calf (described above) was used as the source of antibodies. The antibody source serum was heat inactivated at 56°C for 30 min and was used in two different forms. For the first form, anti-PlpE antibodies were removed from the antibody source serum by absorption of the serum with recombinant E. coli expressing PlpE. This process would also remove any anti-E. coli antibodies that were present in the serum. For the second form, control serum from which only anti-E. coli antibodies were removed was prepared by absorption of the antibody source serum with nonrecombinant E. coli(pBluescript SK). For absorbing
sera to be used in complement killing assays, recombinant or
nonrecombinant E. coli cells were grown overnight in 100 ml
of Luria broth (Life Technologies Inc., Grand Island, N.Y.) and
harvested by centrifugation (11,000 × g) at 4°C.
Cells were washed once with PBS and resuspended in 2 ml of antibody
source serum that had been diluted 1:1 with PBS. Cells and serum were
incubated on a rocking platform for 3 h at 4°C. After
incubation, cells were removed from serum by centrifugation. The
process was repeated until the serum absorbed with recombinant E. coli no longer recognized PlpE in a Western immunoblot (data not shown).
The complement-mediated killing assay was developed by modifying the
techniques described by Chae et al. (5) and Murphy et al.
(33). To ensure that the assay was capable of detecting a
change in the amount of bactericidal antibody, numbers of bacteria and
the concentration of complement were evaluated in preliminary experiments (33). For complement-mediated killing assays,
bacteria were grown in BHI broth for 18 h at 37°C, on a rotary
shaker (200 rpm). Cells were washed once with PBS and resuspended in
PBS to an A600 of 0.5. Complement source serum
(50 µl) and antibody source serum (form 1 or 2 described above) (50 µl) were added to 150 µl of PBS. P. haemolytica cells
(~9,000 to 18,000 CFU in 40 µl of PBS) were then added. Immediately
after the addition of P. haemolytica (t = 0)
and after incubation for 30 min in a 37°C water bath (t = 30), 100-µl samples were removed and diluted 1:100 in PBS.
Dilutions were prepared and plated on BHI agar plates for determination
of CFU. To monitor the killing activity of the complement source serum
alone, a control with only complement source serum and no antibody
source serum was evaluated in an identical manner. We also determined
that the heat-killed antibody source serum alone had no killing
activity (data not shown). Percent killing was calculated by the
following formula: {(CFUt=0 CFUt=30)/CFUt=0} × 100. Percent survival was calculated as 100 (percent killing).
Statistical analysis. Within each experiment, complement-mediated killing assays were done in triplicate for each antibody source serum and the complement source serum control. Additionally, three separate experiments were done on different days. Statistically significant differences between percent killing by different sera within experiments were determined by Student's t test (2).
Nucleotide sequence accession number. The nucleotide sequence of P. haemolytica plpE has been deposited in GenBank under accession no. AF059036.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning of plpE.
To isolate a clone expressing the
immunogenic 45-kDa protein, we screened a genomic library of P. haemolytica S1 with anti-45-kDa antibodies that were affinity
purified from bovine immune serum. We isolated recombinant ZAPII
phage that reacted with the affinity-purified antibodies. A recombinant
plasmid containing a 4.5-kbp insert was excised from one phage clone
and transformed into E. coli XL1-Blue. We subcloned a
2.2-kbp fragment from this insert into pBluescript SK() and named
this plasmid pB4522. E. coli(pB4522) expressed a 45-kDa
protein that was recognized by the affinity-purified antibodies and
bovine immune serum (Fig. 1a). We named
this protein PlpE.
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Surface exposure of PlpE. As mentioned earlier, we previously demonstrated the presence of an immunogenic 45-kDa P. haemolytica protein that is surface exposed (36). To determine if PlpE expressed by the recombinant E. coli strain corresponds to the strongly immunogenic P. haemolytica surface protein, we examined surface exposure of the protein on intact P. haemolytica. Absorption of bovine immune serum with intact P. haemolytica resulted in a loss of antibody reactivity on Western immunoblots with rPlpE and a protein of the same Mr in P. haemolytica whole-cell lysates (Fig. 1a and b), suggesting that PlpE is surface exposed in P. haemolytica. Similarly, absorption of the same bovine immune sera with intact recombinant E. coli(pB4522) expressing PlpE resulted in a loss of reactivity to rPlpE and to a 45-kDa protein in P. haemolytica (Fig. 1c and d). These data suggest that PlpE is also exposed on the surface of recombinant E. coli and that PlpE is the primary 45-kDa surface-exposed immunogen of P. haemolytica.
DNA sequence analysis of PlpE. DNA sequencing of the cloned insert in pB4522 revealed an open reading frame of 1,068 nucleotides that begins with a GTG codon and encodes a protein with a calculated molecular mass of 39.1 kDa (Fig. 2). The deduced amino acid sequence contains a putative hydrophobic signal peptide followed by a consensus lipoprotein processing site (LSAC) (Fig. 2). The calculated molecular mass of the putative mature form of PlpE is 37.03 kDa. The N-terminal region of the mature PlpE contains eight imperfect copies of a repeated hexapeptide (Fig. 2) that are encoded by 18-nucleotide repeats (Fig. 3). The hexapeptide repeat is predicted to form a large hydrophilic domain in PlpE (Fig. 4). PlpE also has numerous other hydrophilic domains that correspond to regions with a high probability of being surface exposed (Fig. 4). A search of GenBank sequences and subsequent sequence alignments revealed that the deduced amino acid sequence of PlpE has 18% identity and 32% similarity to an outer membrane lipoprotein (OmlA) produced by Actinobacillus pleuropneumoniae serotype 1 and 20% identity and 35% similarity to the OmlA protein from A. pleuropneumoniae serotype 5 (data not shown). Although both of those proteins lack the hexapeptide repeat mentioned above, they contain regularly spaced PK and PQ repeats in the same region (4, 17, 23).
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Site-directed mutagenesis of the GTG start codon. To verify that GTG functions as a translational start codon for PlpE, we performed site-directed mutagenesis and converted the codon to GGG. Western immunoblots of whole-cell lysates from the wild-type and mutant E. coli(pB4522) strains, probed with anti-45-kDa antibodies, revealed that the mutant no longer produced a 45-kDa immunoreactive protein, suggesting that GTG functions as the translational start codon (Fig. 5).
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Lipid modification of PlpE.
Because the deduced amino acid
sequence contained a consensus lipoprotein processing site, we examined
P. haemolytica and E. coli(pB4522) for the
presence of 45-kDa lipid-modified proteins. P. haemolytica,
E. coli(pB4522), and nonrecombinant E. coli(pBluescript SK) were grown in the presence of
[3H]palmitic acid. A 45-kDa, 3H-labeled
lipoprotein is present in whole-cell lysates of P. haemolytica and E. coli(pB4522) but absent from the
nonrecombinant E. coli strain (Fig.
6).
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Conservation of PlpE among serotypes of P. haemolytica. To determine if PlpE is expressed by other P. haemolytica serotypes, we examined whole-cell lysates of P. haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13, and 14 and an untypeable strain of P. haemolytica, by Western immunoblot analysis, for reactivity with the anti-45-kDa antibodies (Fig. 7). The antibodies reacted strongly with a 45-kDa protein in serotypes 1, 5, 6, 7, 8, 12, and 14; a 38-kDa protein in serotype 2; a 36-kDa protein in serotype 13; and an ~80-kDa protein in serotype 9 and the untypeable strain. The antibodies reacted weakly with a unique band at 43 kDa in serotype 9. The antibodies did not react strongly with a protein in the serotype 11 strain.
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Role of anti-PlpE antibodies in complement-mediated killing of P. haemolytica. We sought to determine if anti-PlpE antibodies contribute to complement-mediated killing of P. haemolytica. Since we had observed that E. coli(pB4522) has the capacity to remove anti-PlpE antibodies from bovine serum (Fig. 1d), we used the recombinant strain to remove those antibodies from immune serum of a calf that was vaccinated with P. haemolytica OMPs. We then compared rates of complement-mediated killing of P. haemolytica using, as an antibody source, bovine immune serum or immune serum depleted of anti-PlpE antibodies. As shown in Fig. 8, immune serum that was depleted of anti-PlpE antibodies caused less killing of P. haemolytica than did immune serum that was not depleted of those antibodies. For each of three separate experiments, the difference in killing activity between the two sera was statistically significant (P < 0.003).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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As mentioned earlier, numerous studies have indicated that P. haemolytica OMPs are important in eliciting protective immunity in cattle and that antibody responses of cattle to P. haemolytica OMPs correlate with resistance to experimental P. haemolytica challenge. However, not all antibody responses to individual antigens contribute significantly to host defense, and some may actually be detrimental to certain defense mechanisms (30, 38, 49). Therefore, one of our goals is to identify and characterize individual P. haemolytica OMPs that elicit antibodies which function in host immune mechanisms.
In our previous work, we observed an immunoreactive band at 45 kDa on Western immunoblots of P. haemolytica OMPs that were probed with three different sera from cattle resistant to P. haemolytica infection (36). Those studies also revealed the 45-kDa antigen to be a surface-exposed protein. Here, we present genetic and immunologic characterization of a P. haemolytica 45-kDa surface-exposed lipoprotein (PlpE) and demonstrate that bovine antibodies against PlpE contribute to complement-mediated killing of P. haemolytica. The results of our study demonstrate that PlpE is the major P. haemolytica antigen responsible for the significant, immunoreactive band at 45 kDa in Western immunoblots probed with immune sera from cattle.
Our analysis of the deduced amino sequence of PlpE revealed several interesting features. Although PlpE migrates at an Mr of 45 kDa on SDS-polyacrylamide gels, the calculated molecular mass of the putative mature form of PlpE is ~37 kDa. This discrepancy may possibly be a result of the high proline content of PlpE (~6%). Proline is a turn-inducing amino acid residue that often causes proteins to migrate slower on SDS-PAGE (39). The deduced amino acid sequence of PlpE also contains a typical signal peptide, followed by a consensus lipoprotein processing site, and in this study we demonstrated lipid modification of rPlpE. Another interesting feature of plpE is that GUG functions as the translational start codon. GUG, AUG, and UUG are in the group of class I initiation codons that support efficient translation (48). In E. coli, the intrinsic activity of GUG is 12 to 15% that of AUG (41). About 8% of known genes from E. coli and other bacteria use GUG as the start codon (43). The aroA gene of Pasteurella multocida appears to use GUG as an initiation codon (21). However, to our knowledge plpE is the first example of a P. haemolytica gene with GUG as a start codon.
Protein sequence similarities are present between the QAQNAP repeats in PlpE and a newly identified peptide repeat (NAP) in some forms of the polymorphic merozoite surface protein 2 (MSP2) from Plasmodium falciparum (22). The NAP repeats, like the QAQNAP repeats in PlpE, occur near the amino terminus of MSP2, and similarities between PlpE and MSP2 include glycines and serines on the amino-terminal side of the repeat regions. Hydrophilicity plots of PlpE and this form of MSP2 are also similar from the amino terminus, extending across the repeated region (data not shown). DNA sequence identity exists as well between the consensus nucleotide sequence encoding the NAP repeat unit of MSP2 (AATGCTCCA) and the consensus nucleotides encoding the corresponding region in the PlpE hexapeptide repeat (AATGCTCCT). In mouse immunization experiments with synthetic peptides and a rMSP2, an immunodominant T-cell determinant was mapped to a region spanning the NAP sequence (42). More detailed immunological studies are required to determine if such a role may exist for the hexapeptide repeat in PlpE.
Within the hexapeptide repeat of PlpE, codon sequences are generally conserved for each amino acid position, and unusual codons for P. haemolytica are also conserved in this region. Seven of the eight codons for the first glutamine of each repeat are CAG, whereas for the second glutamine, six of six codons are CAA. In P. haemolytica, CAA is the preferred codon for glutamine (26) and is reflective of the high moles percent A+T content (~60%) of P. haemolytica chromosomal DNA. For the first alanine in the repeat, six of six codons are GCA, whereas for the second alanine, five of seven codons are GCT. Similarly, all asparagines are encoded by AAT, and all prolines are encoded by CCT. The relative synonymous codon usage in P. haemolytica for CCT was calculated by Lo et al. (26) to be slightly lower than that for CCA. Our recent analyses with a larger number of P. haemolytica genes revealed similar results (31). These data suggest that expansion of the hexapeptide repeat may have occurred by duplication of one or more of the 18 nucleotide repeats.
Additional amino acid sequence alignments revealed that the A. pleuropneumoniae OmlA lipoproteins are similar to PlpE over its entire sequence. A. pleuropneumoniae is a pathogen of pigs that causes a fibrinous pleuropneumonia very similar to that caused by P. haemolytica in cattle. Vaccination of pigs with protein aggregates containing rOmlA, cloned from A. pleuropneumoniae serotype 1, significantly reduced lung damage and death of pigs upon subsequent experimental challenge with a homologous A. pleuropneumoniae serotype (17). Similarly, vaccination of pigs with gel-purified rOmlA, cloned from A. pleuropneumoniae serotype 5a, significantly lowered mortality upon challenge with a serotype 5a strain (4).
The OmlA proteins from different A. pleuropneumoniae serotypes may be more antigenically heterogeneous than are the PlpE proteins from the different P. haemolytica serotypes. A. pleuropneumoniae serotypes 1, 5, and 7 are the most common in North America. Sera from pigs vaccinated with rOmlA (serotype 1) failed to recognize proteins in 6 of 13 A. pleuropneumoniae serotypes and only weakly recognized a protein in 3 of those serotypes (17). Similarly, rabbit antisera against rOmlA from serotype 5a recognized a protein only in serotypes 5a, 5b, and 10 (4, 23). These data suggest that OmlA proteins from a single serotype are unlikely to be cross-protective for heterologous serotypes. Indeed, one A. pleuropneumoniae vaccine currently under evaluation includes, among other antigens, rOmlA proteins from both serotypes 1 and 5 (24).
In contrast, our Western immunoblots revealed that anti-45-kDa antibodies, affinity purified from bovine immune sera, recognize a protein in all but one P. haemolytica serotype (serotype 11). P. haemolytica serotypes 1 and 6 are most frequently isolated from the lungs of pneumonic cattle in the United States. Thus, PlpE may have potential for being a significant cross-protective antigen for those serotypes. Several P. haemolytica serotypes possess immunoreactive antigens with Mrs different from that of PlpE (Fig. 7), including serotypes 2 (~38 kDa) and 9 (~80 kDa) and the untypeable strain (~80 kDa). Although purely speculative at this time, it is possible that variation in the number of hexapeptide repeats could account for these differences in Mrs. Alternatively, as discussed above, different percentages of proline residues in the proteins from different serotypes could alter the mobility of these proteins. Significant differences in P. haemolytica serotype 1 and serotype 9 OMPs were also suggested by the results of a recent vaccine trial. Vaccination of calves with OMPs purified from a serotype 9 strain did not provide significant protection from experimental challenge with a serotype 1 strain (29). Future studies will be necessary to evaluate the capacity of PlpE to enhance protection of cattle against experimental challenge with homologous and heterologous serotypes of P. haemolytica.
Results of the complement-mediated killing assays demonstrate that anti-PlpE antibodies contribute to this mechanism of bovine defense, one that is believed to be important in protection against P. haemolytica pneumonia. Immune serum that was depleted of anti-PlpE antibodies also caused significant amounts of killing, ~75% under these assay conditions. These results suggest that antibodies against other surface antigens also contribute to complement-mediated killing. Absorption of bovine immune serum with intact P. haemolytica results in the loss or reduction of immunoreactivity in Western blots to numerous antigens (Fig. 1a and b), any of which may bind antibodies that effect complement-mediated killing. Identification of these antigens and elucidation of the role of antibodies directed against them in bovine immune mechanisms are currently major focuses of our research.
Control complement-mediated killing assays with only complement source and no antibody source indicated that complement alone failed to kill P. haemolytica, suggesting that complement activation strictly through the alternative pathway does not play a significant role in the killing of the organism. These data are in agreement with those of previous studies which demonstrated that only activation of the complement cascade through the classical pathway is important in killing P. haemolytica (3, 27).
Studies in our laboratory to evaluate the role of anti-PlpE antibodies in other mechanisms of host defense, such as neutrophil phagocytosis and killing, and to identify additional OMPs that may be important in protective immunity against P. haemolytica are under way. The addition of PlpE and other OMPs which elicit protective antibodies to vaccines containing leukotoxin may provide significant protection against pneumonic pasteurellosis.
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ACKNOWLEDGMENTS |
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This work was supported by grant 95-37204-1999 from the National Research Initiative Competitive Grants Program of the USDA, the Oklahoma Agricultural Experiment Station (project OKL02179), and the Oklahoma State University College of Veterinary Medicine. K.P. was supported by an OSU-CVM graduate research assistantship award.
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FOOTNOTES |
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* Corresponding author. Mailing address: Dept. of APP, 209 Vet. Med. Bldg., College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078. Phone: (405) 744-4518. Fax: (405) 744-5275. E-mail: gmurphy{at}okway.okstate.edu.
Editor: T. R. Kozel
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infection and Immunity, December 1998, p. 5613-5619, Vol. 66, No. 12
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