Vaccination and Protection of Pigs against Pleuropneumonia with aVaccine Strain of Actinobacillus pleuropneumoniae Produced
by Site-Specific Mutagenesis of the ApxII OperonC. T. PRIDEAUX,* C. LENGHAUS, J. KRYWULT, AND A. L. M. HODGSON
Animal Health Laboratory, CSIRO Division of Animal Health,Geelong, Victoria 3120, Australia
Received 16 April 1998/Returned for modification 6 July 1998/Accepted 26 January 1999
The production of toxin (Apx)-neutralizing antibodies during infection plays a major role in the inductionof protective immunity to Actinobacillus pleuropneumoniae reinfection. In the present study, the gene encodingthe ApxII-activating protein, apxIIC, was insertionally inactivated on the chromosome of a serovar 7 strain,HS93. Expression of the structural toxin, ApxIIA, and of the two genes required for its secretion, apxIB andapxID, still occurs in this strain. The resulting mutant strain, HS93C2 Ampr, was found to secrete theunactivated toxin. Pigs vaccinated with live HS93C2 Ampr via the intranasal route were protected against across-serovar challenge with a virulent serovar 1 strain of A. pleuropneumoniae. This is the first reportedvaccine strain of A. pleuropneumoniae which can be delivered live to pigs and offers cross-serovar protectionagainst porcine pleuropneumonia.
Actinobacillus pleuropneumoniae is a member of the familyPasteurellaceae and is the etiological agent of porcine pleuro-pneumonia, an acute or chronic infection affecting pigs of allages. The disease, characterized by hemorrhagic, fibrinous, andnecrotic lung lesions, is highly contagious and causes majorlosses to the swine industry (25). To date, 12 serovars havebeen identified worldwide (serovars 1 to 12). Within a geo-graphical region a small number of serovars predominate; forexample, in Australia serovars 1, 7, and 12 make up approxi-mately 90% of isolates.
A number of potential virulence factors have been identifiedfor A. pleuropneumoniae, including a family of secreted toxins(3, 5, 26, 29). These secreted toxins, or Apx toxins, are mem-bers of the RTX toxin family (11–13). The role of Apx toxinsin A. pleuropneumoniae virulence was first demonstrated withspontaneous and chemically induced nonhemolytic mutantswhich were found to be completely or partially avirulent; thisrole was later confirmed by using transposon mutagenesis (1,15, 17, 29, 30, 33, 34). At least three different Apx toxins areproduced by A. pleuropneumoniae, designated ApxI, ApxII,and ApxIII. ApxI shows strong hemolytic activity, and ApxIIshows relatively low hemolytic activity. Both are cytotoxic andactive against a broad range of cells of different types andspecies (9, 19, 28). ApxIII is nonhemolytic but strongly cyto-toxic, with a host range including porcine alveolar macro-phages and neutrophils (19, 29). Currently, no identified sero-var of A. pleuropneumoniae produces all three Apx toxins, withthe majority producing only two, while a small number produceonly one (8, 10–12, 19, 29).
Production and secretion of active RTX toxins requires theactivity of at least four genes, apxC, -A, -B, and -D. The apxAgene encodes the structural toxin, and the apxC gene encodesa posttranslational activator which is involved in the transfer ofa fatty acyl group from an acyl carrier protein to the structuraltoxin (18). Activation of ApxA is required for target cell bind-
ing. The apxB and apxD genes encode proteins that are re-quired for secretion of the activated toxin (7, 36). ApxI andApxIII are encoded by operons that consist of the four con-tiguous genes (-C, -A, -B, -D) expressed from a single promoterlocated 59 of the apxC gene. The ApxII operon contains onlythe apxA and apxC genes expressed as a single RNA transcript.Secretion of ApxII is dependent on the activity of the apxIBand apxID gene products (13).
Vaccination against porcine pleuropneumonia has utilized,to date, bacterins or subunit vaccines based on various com-ponents of A. pleuropneumoniae. Results obtained with bac-terin vaccines have offered, at best, homologous protectionagainst the serovar used to prepare the vaccine material. Incontrast, natural infection of pigs with any one serovar servesto prevent natural reinfection with any serovar (24). Apx’s arethought to be of particular importance for the induction ofprotective immunity; nonhemolytic mutants cannot induceprotective immunity in animals (17), and commercial bacterinvaccines that lack Apx do not provide adequate protection (16).Previously we (26) demonstrated the ability of an A. pleuro-pneumoniae mutant deficient in chromosomal apxA and apxCgenes to express and secrete an unactivated form of ApxI froma plasmid-encoded apxIA gene. This engineered strain wasfound to be attenuated in a mouse model and, when admin-istered as a live vaccine, offered protection against homologousand heterologous challenge.
The use of a plasmid-borne protective antigen in a livevaccine strain is limited due to the potential of the plasmid tobe lost during in vivo replication of the vaccine. Here wedescribe the construction of an A. pleuropneumoniae vaccinestrain by using site-specific mutagenesis of the apxIIC gene onthe chromosome. The resulting strain produces and secretes anunactivated ApxIIA by using chromosomally encoded genes,thus ensuring that the protective antigen is maintained withinthe vaccine strain, unlike in previous experiments, in whichApxIA was expressed from a plasmid and could therefore belost from replicating bacteria. The potential of this modifiedstrain to protect pigs from cross-serovar challenge with virulentA. pleuropneumoniae was investigated.
* Corresponding author. CSIRO, Division of Animal Health, Ani-mal Health Laboratory, Private Bag No. 24, Geelong, Victoria 3120,Australia. Phone: 61-3-5227 5000. Fax: 61-3-5227 5531. E-mail:[email protected].
Bacterial strains and growth conditions. The A. pleuropneumoniae bacterialstrains used in this study (serovar 1, HS25; serovar 7, HS93) were isolated frompigs with pleuropneumonia and kindly supplied by Pat Blackall (Animal Re-search Institute, Yeerongpilly, Queensland, Australia). Strains of A. pleuropneu-moniae were grown in brain heart infusion broth (BHI), supplemented withnicotinamide adenine dinucleotide (NAD; Sigma Chemical Co., St. Louis, Mo.)to a final concentration of 10 mg/ml. Blood agar was prepared by adding 5%sterile defibrinated horse erythrocytes to the BHI agar. The antibiotics used andtheir final concentrations were as follows: kanamycin, 25 mg/ml; streptomycin, 50mg/ml; and ampicillin, 5 mg/ml, unless stated otherwise. Escherichia coli DH5awas used throughout this study, by standard techniques (31).
Isolation, amplification, and Southern blot analysis of A. pleuropneumoniaegenomic DNA. Isolation of A. pleuropneumoniae genomic DNA was performedas described by Prideaux et al. (26), using lysozyme and proteinase K digestionfollowed by phenol-chloroform-isoamyl alcohol extraction. Amplification of spe-cific regions of the A. pleuropneumoniae genome was achieved by PCR, using thebuffer and cycle conditions described previously (26) and a Perkin-Elmer CetusDNA thermal cycler.
Southern blot analysis of PCR products was performed by standard techniques(31), with a final washing stringency of 0.1% sodium dodecyl sulfate (SDS)–0.13SSPE (13 SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7])at 65°C for 10 min.
Construction of recombination plasmids. The plasmid pEP-CAmpr was con-structed for use in site-specific mutagenesis of the apxIIC gene. A 3.4-kb frag-ment containing the apxIIC gene was isolated by PCR. Specific oligonucleotidesfor use in PCR were synthesized with a Pharmacia Gene Assembler Plus DNAsynthesizer (59, CGCACCATGGTCGGGC; 39, CTAACAGCTAGTGCA)based on published sequence (32). The fragment contained 2.0 kb of DNA 59 and900 bp 39 of the apxIIC gene (480 bp). The PCR fragment was cloned into theMycobacterium-E. coli shuttle vector pEP2 (27). The resulting plasmid, pEP2-CA, contained a unique XbaI restriction site located 180 bp downstream of theapxIIC translational start site. The Ampr gene from the 4.2-kb plasmid of Pas-teurella haemolytica A1 (23) was cloned into the unique XbaI site of pEP-CA togenerate the recombination plasmid pEP-CAmpr.
Mutagenesis of the apxIIC gene. Site-specific mutagenesis of the apxIIC geneutilized the recombination plasmid pEP-CAmpr. Cesium chloride-purified pEP-CAmpr DNA was isolated from E. coli and linearized with ClaI. Followingdigestion, the DNA was purified by phenol-chloroform extraction and ethanolprecipitated. A total of 3 mg of linearized DNA was electroporated (0.2-cm-diameter cuvettes; 400 V; 1.25 kV) into A. pleuropneumoniae HS93 (serovar 7,ApxII) by using the protocol described previously by Frey (7). Products of theelectroporation were plated onto (BHI-NAD) blood agar plates containing am-picillin at a concentration of 1 mg/ml. Colonies that were ampicillin resistant anddid not produce a zone of hemolysis on blood agar plates were selected forfurther characterization.
PCR was used to examine the region of the A. pleuropneumoniae chromosomecontaining the apxIIC gene, employing oligonucleotides that bound to the apxIIoperon (59TACAGAACGTTGGTA, 39CTAACAGCTAGTCCA) at the com-mencement of the apxIIC open reading frame and 900 bp after its closing.
Western blot analysis. Western blot (immunoblot) analysis was performed asdescribed previously by Sambrook et al. (31). Rabbit sera were produced againstculture supernatants of A. pleuropneumoniae HS25 (serovar 1) by the methoddescribed previously (5). Rabbit sera were preabsorbed with an isolate of HS93that lacks both the apxIIA and apxIIC genes, and therefore does not produce Apx(26), before use at a 1 in 50 dilution. The conjugate, used at a 1:1,000 dilution,was sheep anti-rabbit immunoglobulin affinity-isolated horseradish peroxidase-conjugated antiserum (Silenus), with tetramethylbenzidine (22) as the substrate.Bacterial samples for Western blot analysis were prepared by diluting overnightcultures 1:20 and incubating at 37°C with shaking until an optical density at 600nm (OD600) of 0.8 was reached. At this time, culture supernatant (12,000 3 g for5 min) and cell pellet (12,000 3 g for 5 min; washed in an equal volume ofphosphate-buffered saline and lysed in loading buffer) samples were taken(equivalent to 20 ml of total culture) and analyzed by SDS-polyacrylamide gelelectrophoresis by using the discontinuous buffer system (21). Separated proteinswere transferred to nitrocellulose with a Bio-Rad transblot cell by using theprotocols outlined by the manufacturer.
Attenuation of HS93C2 Ampr in mice. Overnight cultures of A. pleuropneu-moniae were grown with vigorous shaking at 37°C in BHI broth supplementedwith NAD. The following day a 1 in 20 dilution was made, and the new cultureswere incubated until an OD600 of 0.8 was reached, at which point the count ofviable A. pleuropneumoniae was found to be 109 CFU per ml. Various dilutionsof A. pleuropneumoniae cultures were prepared so that the desired number ofbacteria were contained in 200 ml of BHI broth. Six-week-old female BALB/cmice (Walter and Elisa Hall Institute of Medical Research, Parkville, Australia)were maintained in PC1 facilities with water and food ad libitum. Mice wereinjected intraperitoneally (i.p.) with 200 ml of A. pleuropneumoniae preparation.Control mice received 200 ml of BHI broth i.p. The number of surviving mice at24 h postchallenge was recorded; and these mice were considered to havereceived a sublethal dose.
Vaccination and challenge of pigs. Six-week-old pigs were prebled to screenfor existing antibodies against A. pleuropneumoniae HS93 (serovar 7) and ApxI.Pigs found to be negative in these tests were randomly assigned to experimentalgroups. Nine 6-week-old pigs received 109 CFU of the A. pleuropneumoniaevaccine strain in 1 ml of growth medium, via intranasal inoculation on day 0,while nine control pigs received 1 ml of BHI. The vaccine was prepared byinoculating 10 ml of BHI-NAD (10 mg/ml) with a single colony of the vaccinestrain and growing with shaking at 37°C until an OD600 of 0.8 was reached. Thevaccination schedule was repeated on day 14. On day 28, the nine vaccinated andnine control pigs were divided into groups of six and three. The two groups of sixpigs (i.e., vaccinated and unvaccinated) were challenged with 2 3 109 A. pleu-ropneumoniae HS25 (serovar 1) in 2 ml of growth medium via the intranasalroute, while the groups of three were given 2 ml of BHI broth in a similarmanner. The challenge strain was prepared by inoculating a single colony ofHS25 into BHI-NAD (10 mg/ml) and growing until an OD600 of 0.8 was reached.At this time the viable count was 109 CFU/ml. At 5 days postchallenge, pigs wereeuthanized, and the number and severity of lung lesions were recorded.
RESULTS
Characterization of the apxIIC mutant. Site-specific muta-genesis of the apxIIC gene utilized the recombination plasmidpEP-CAmpr. This plasmid contains the apxIIC open readingframe insertionally inactivated by the introduction of an Ampr
gene into the unique XbaI site. The inactivated apxIIC gene isflanked by 2.0 kb of genomic DNA upstream (59) and 900 bpdownstream (39). pEP-CAmpr was linearized and electroporat-ed into A. pleuropneumoniae HS93, and the products of ho-mologous recombination were selected by plating on bloodagar plates containing ampicillin.
Genomic DNA was extracted from the nonzoning, ampicil-lin-resistant mutants designated HS93C2 Ampr and the par-ent strain, HS93. PCR was used to examine the region of theA. pleuropneumoniae chromosome containing the apxIIC gene.The PCR product (Fig. 1) obtained by using HS93C2 Ampr
genomic DNA (3.5 kb) was approximately 1.8 kb larger in sizethan that obtained with the parent strain, HS93. This increasein size corresponded to the size of the Ampr gene. Products ofthe PCRs were further characterized by Southern blot hybrid-ization with the isolated Ampr or apxC gene as a probe (Fig. 1).Hybridization of the apxC gene probe to the PCR productsfrom HS93 and HS93C2 Ampr confirmed that the region ofthe chromosome containing the apxIIC gene had been ampli-fied. The Ampr gene probe hybridized to the PCR productobtained from HS93C2 Ampr, confirming that this strain con-tained the Ampr gene associated with the apxC gene. The PCRproduct obtained when HS93 genomic DNA was used as tem-plate did not hybridize to the Ampr gene probe.
Characterization of Apx expression by HS93C2 Ampr. Log-arithmic cultures of HS932 Ampr and HS93 were examined byWestern blotting with antisera raised in rabbits against thesecreted proteins of A. pleuropneumoniae HS25 (i.e., serovar 1,ApxI and ApxII). A toxin-deficient strain of HS93 resultingfrom deletion of the apxIIC and apxIIA genes (26) was used asa negative control. The Apx-deficient mutant (HS93 Tox2) didnot react specifically with the anti-Apx antisera in the regioncorresponding to the ApxII molecular weight. Supernatant andcellular material from both the HS93C2 Ampr mutant and theparent strain, HS93, produced a single polypeptide, correspond-ing in size to ApxII, that reacted with the anti-APX rabbit sera(Fig. 2). A potential high-molecular-weight HS93 Tox2 poly-peptide may have reacted with the antisera. The preabsorptionof the sera with HS93 Tox2 prior to use and the position of theband suggest that it corresponds to nonspecific cross-reactionwith material remaining in the loading well.
Evaluation of HS93C2 Ampr virulence in mice. To test therelative virulence of A. pleuropneumoniae HS93 and HS93C2
Ampr in mice, various dilutions of each bacteria (2 3 108 to1 3 107 CFU/mouse) were prepared in bacterial growth me-
VOL. 67, 1999 SITE-SPECIFIC MUTAGENESIS OF A. PLEUROPNEUMONIAE 1963
dium (BHI-NAD) and administered to mice i.p. The numberof mice that had received a sublethal dose was determined 24 hpostchallenge. Under our conditions, all mortalities oc-curred within the first 24 h postchallenge. A comparison of thedeaths obtained with each isolate showed that 2 3 108 CFU ofthe parent strain, HS93, was sufficient to kill 100% of mice,while an equivalent challenge with HS93C2 Ampr was suble-thal (Table 1).
Vaccination and challenge of pigs. Two groups of nine6-week-old pigs were vaccinated with either 1 ml of BHI con-taining 109 CFU of HS93C2 Ampr or 1 ml of sterile BHI(unvaccinated) via intranasal inoculation on days 0 and 14.Two weeks after secondary vaccination, six of the HS93C2
Ampr-vaccinated and six of the unvaccinated pigs were chal-lenged intranasally with 2 ml of growth medium containing 2 3109 CFU of HS25. The number and severity of lung lesionspresent in pigs at autopsy 5 days postchallenge were recorded(Table 2). It is our experience that no additional lesions re-sulting from experimental challenge of pigs with this protocoldevelop beyond day 5 postchallenge and that at this time anumber of lesions detected have commenced to resolve. Thethree pigs that were neither vaccinated nor challenged hadno detectable lung lesions present at autopsy. Similarly, pigs(three) that were vaccinated and unchallenged showed no ev-idence of A. pleuropneumoniae infection. The six unvaccinatedpigs that were challenged with HS25 all showed numerous lunglesions at autopsy, characterized as focal abscesses up to 3 cmin diameter, and adhesive pleuritis. Further characterization ofthese lesions showed them to contain high levels of A. pleuro-pneumoniae that had characteristics similar to those of HS25,as judged by colony morphology and zones of hemolysis onblood agar plates. In contrast, of the six pigs that had been
vaccinated with HS93C2 Ampr and challenged with HS25,only one had a lesion at autopsy; this was in the form of a singleadhesion between the lung and the rib cage. Upon closerexamination, this adhesion appeared to be older than the 5days since challenge. Bacteria isolated from this adhesion werenot NAD dependent and are therefore unlikely to have beenA. pleuropneumoniae. Lung samples that were taken from vac-cinated and challenged pigs, homogenized, and plated onblood agar (BHI-NAD) did not yield bacteria.
DISCUSSION
Apx toxins are known to play a major role in both the viru-lence of and induction of protective immunity to A. pleuro-pneumoniae, the causative agent of porcine pleuropneumonia.The principal phagocytic cells of the lung and the first line ofdefense against bacterial invasion are the alveolar macrophages.It is possible that A. pleuropneumoniae colonizes the lungthrough the production of Apx toxins which lyse these cells andthus compromise the primary immune responses of the lung (37).
FIG. 1. Characterization of the apxIIC mutant chromosome. PCR was usedto amplify the region of the A. pleuropneumoniae chromosome containing theapxIIC gene from mutant (HS93C2 Ampr) and wild-type (HS93) strains. (A)PCR products were resolved on gels and stained with ethidium bromide. (B)Southern blot hybridization of the PCR products with the apxIIC gene (Tox2)and the ampicillin resistance gene (Ampr).
FIG. 2. Western blot analysis of ApxII expression. Total cells (cell) and cul-ture supernatant (sup) samples of wild-type HS93, HS93C2 Ampr, and a toxin-deficient mutant (HS93Tox2) were analyzed by Western blotting with Apx-spe-cific antisera.
TABLE 1. Virulence of A. pleuropneumoniae strains in mice
Challenge level(106)a
% of mice dead 24 h postchallenge withindicated strain
HS93 HS93C2 Ampr
200 100 020 15 NT10 0 NT
a Levels below those previously found to give 0% death were not tested (NT).
Targets of the Apx toxins include erythrocytes, the lysis ofwhich leads to an increase in the availability of free iron forbacterial growth. In addition, the Apx toxins contribute tolung damage through the lysis of leukocytes, which leads tolocalized inflammation. Binding of Apx to target cells requiresposttranslational activation of the structural toxin by theApxIIC protein (18). In this study we utilized site-specific mu-tagenesis to inactivate the apxIIC gene on the A. pleuropneu-moniae chromosome and examined the effect of this mutationon virulence, induction of protective immunity following infec-tion, and potential to generate mutants for use as live vaccines.
Site-specific mutagenesis of the apxIIC gene was achievedwith suicide, or nonreplicating, plasmid vectors. This methodhas the advantage of rapid screening of products, as theoreti-cally only those bacteria that have undergone recombinationwith the plasmid vector, resulting in the transfer of the markergene onto the bacterial chromosome, will grow under selectiveconditions. A number of attempts within our laboratory havefailed to transform A. pleuropneumoniae with pEP2, leading tothe conclusion that A. pleuropneumoniae is a nonpermissivehost for this plasmid vector. A serovar 7 strain of A. pleuro-pneumoniae producing ApxII alone was chosen for use in thisstudy, as the genes required for ApxII secretion are not co-transcribed with the structural and activating genes. In addi-tion, serovar 7 is relevant as a vaccine candidate in Australia,as serovar 7 isolates are responsible for large numbers of por-cine pleuropneumonia outbreaks.
Homologous recombination leading to the insertion of theAmpr gene into the apxIIC gene on the chromosome wasconfirmed by PCR and Southern blot hybridization. Insertionof the ampicillin resistance gene into the chromosomal copy ofthe apxIIC gene did not prevent transcription or translation ofthe apxIIA gene, as evidenced by the ability to detect ApxII inWestern blots (Fig. 2). It appears that transcription initiates atthe apxII promoter and continues through the ampicillin resis-tance gene and into the apxIIA gene. Although translation ofan active apxIIC gene product is prevented by the presence ofthe ampicillin resistance gene (Fig. 1), translation of the apxIIAgene must recommence further downstream. Chang et al. (3)have described a potential ribosome binding site, located be-tween the apxIIC and apxIIA genes, which may serve to reini-tiate translation of ApxII. The orientation of the ampicillinresistance gene is opposite that of the apxII operon, and there-fore the ampicillin resistance gene promoter cannot contributeto ApxIIA expression. Insertion of the ampicillin resistancegene into the apxIIC gene appears to have reduced the level ofApxII production, possibly due to polar effects of the ampicil-lin resistance gene promoter on the level of downstreamapxIIA transcription. A potential solution to this possible lim-itation would be to clone the ampicillin resistance gene in thesame orientation as the apxIIA gene, though licensing of thevaccine strain for commercial use would require the removal ofany antibiotic resistance gene from the chromosome. The pres-
ence of ApxIIA in the culture supernatant would also indicatethat activation of ApxIIA is not required for secretion. A sim-ilar observation has been made for both E. coli and P. haemo-lytica, where the RTX toxins produced by these bacteria havebeen shown to be secreted without activation (6, 35).
Inactivation of the apxIIC gene on the chromosome resultedin reduced virulence, as observed in a mouse model in which2 3 108 CFU of the apxIIC-deficient mutant resulted in nomortalities compared to a mortality rate of 100% when micewere inoculated with the same level of the parent strain, HS93(Table 1). This is in agreement with our previous observations(26) with a toxin-deficient strain of A. pleuropneumoniae ex-pressing an unactivated form of ApxIA from a plasmid, whereit was found that unless the toxin was activated, it did notcontribute to bacterial pathogenesis.
To test the protective efficacy of the vaccine in the targetspecies, we vaccinated pigs with HS93C2 Ampr, a serovar 7strain, via the intranasal route and challenged with HS25 (Ta-ble 2), which belongs to serovar 1 and produces both ApxI andApxII. This combination of Apx production is associated withthe most severe outbreaks of pleuropneumonia (13, 20). Priorto vaccination, pigs were determined to be free of both HS93-and ApxI-specific antibodies, therefore ensuring their naive sta-tus for both vaccination (HS93) and challenge (HS25: ApxI)strains. Vaccination and challenge were both via the intranasalroute. This method of delivery was chosen because it bestmimics the natural route of exposure of pigs to A. pleuropneu-moniae. The three pigs that were vaccinated and not chal-lenged had no lung lesions present at autopsy, indicating thatthe vaccine strain does not cause lesions in pigs that are evi-dent at 3 weeks postvaccination. Previously we had adminis-tered the toxin-deficient strain HS93 Tox2 to pigs at dosessimilar to that of the challenge used in this experiment andautopsied the pigs at day 5 but observed no lesions. Apx-deficient mutants of APP produced by either chemical or trans-poson mutagenesis have previously been shown to have a re-duced ability to induce lung lesions (1, 15, 17, 29, 30, 33, 34).The six unvaccinated pigs challenged with HS25 showed nu-merous lung lesions that were visible on autopsy, indicatingthat the level of challenge used was sufficient to induce lesionsin unprotected animals. In contrast, only one of the six vacci-nated pigs showed any sign of infection, in the form of a singlelung adhesion, which was unlikely to be a result of the chal-lenge. The ability to achieve cross-serovar protection followinglive vaccination, but not after vaccination with bacterin prep-arations, suggests that cross-serovar protection may be depen-dent on the presentation of in vivo-regulated proteins to theimmune system. In addition, the route of vaccination may alsoplay a role in the level of cross-protection obtained. Intranasalvaccination was chosen because it best mimics the naturalroute of A. pleuropneumoniae infection, which is known toinduce an immune response that is cross-protective. In con-trast, bacterin vaccines are delivered by subcutaneous or intra-
TABLE 2. Vaccination and challenge of pigs
Group No. of pigs Extent of lung lesions
Unvaccinated, unchallenged 3 None detected
Vaccinated, unchallenged 3 None detected
Vaccinated, challenged 6 None detected in five animals, but one pig had a small adhesion on the left diaphragmatic lobea
Unvaccinated, challenged 6 Multiple abscesses and adhesive pleuritis
a Not considered to be related to A. pleuropneumoniae challenge (see text for details).
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muscular injection. It has been demonstrated previously that theimmune responses induced by a commercial vaccine are verydifferent from those induced following aerosol exposure of pigs toA. pleuropneumoniae (14). Sera obtained from animals postvac-cination and prior to challenge responded weakly to ApxIIA byenzyme-linked immunosorbent assay (results not shown). Addi-tional work is ongoing to further characterize the immune re-sponses obtained through vaccination with HS93C2 Ampr and tocompare them with those obtained during natural infection.
The findings of this protection study demonstrate the poten-tial of HS93C2 Ampr to be delivered via the nasal route as avaccine to protect pigs against porcine pleuropneumonia. Ac-tivation of ApxIIA was found to be necessary for virulence ina mouse model but not for secretion. The ability of HS93C2
Ampr to protect pigs from virulent A. pleuropneumoniae chal-lenge, together with the central role of Apx immunity in pro-tecting pigs from A. pleuropneumoniae infection, suggests thatactivation of the toxin is not required to induce protectiveimmunity. This is the first report of a live vaccine strain ofA. pleuropneumoniae that is suitable for use in pigs and offerscross-serovar protection.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of the AustralianPig Research and Development Corporation.
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