INFECTION AND IMMUNITY, JUlY 1992, p. 2900-2905 Vol. 60, No. 70019-9567/92/072900-06$02.00/0Copyright © 1992, American Society for Microbiology

Rational Attenuation of Corynebacterium pseudotuberculosis:Potential Cheesy Gland Vaccine and Live Delivery VehicleADRIAN L. M. HODGSON,* JOLANTA KRYWULT, LEIGH A. CORNER, JAMES S. ROTHEL,

AND ANTHONY J. RADFORDCSIRO Division ofAnimal Health, Animal Health Research Laboratory, Private Bag No. 1, Parkville,

Victoria 3052, Australia

Received 23 December 1991/Accepted 23 April 1992

The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosischromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C.pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at dosestwo logs higher than that at which the wild-type strain produces the disease. This clearly establishes PLD as amajor C. pseudotuberculosis virulence factor. Vaccination of sheep with live Toxminus C. pseudotuberculosiselicits strong humoral and cell-mediated immune responses and protects the animals from wild-type challenge.

Corynebacterium pseudotuberculosis is the gram-positivebacterium that causes caseous lymphadenitis (CLA orcheesy gland) in sheep and goats. CLA in sheep is charac-terized by abscessation of the superficial lymph nodes, andthe infection can result in reduced wool production and meatlosses due to carcass condemnation (16). For some years thesecreted phospholipase D (PLD) exotoxin of C. pseudotu-berculosis has been implicated as a major virulence factorfor this bacterium (2, 22). It has been suggested that thephospholipase activity of PLD facilitates the disseminationand infiltration of the bacteria into host tissue (2). There isclear empirical evidence that PLD is a major virulence factorsince sheep are protected from CLA by vaccination with acrude, formalin-treated PLD preparation derived from pre-cipitating C. pseudotuberculosis culture supernatant (3).

Phospholipases are common among bacterial pathogensand play a key role in the virulence of bacteria from severalgenera: Listeria (13), Bacillus (5, 9), Pseudomonas (14), andClostridium (12, 24, 25). One strategy employed to elucidatethe role of phospholipases in bacterial infections has been tointroduce mutations within the structural genes and examinethe effect on bacterial pathogenicity. Thus the listeriolysinand phospholipase C genes of Listeria monocytogenes weremutated individually, yielding avirulent variants (11) andbacteria with reduced virulence (13), respectively. Similarly,deletion of a phospholipase C gene from Pseudomonasaeruginosa resulted in less-virulent bacteria (15). Such ap-plications of site-specific mutagenesis establish a clear rolefor the targeted phospholipase proteins in virulence anddemonstrate that it is possible to attenuate bacterial patho-gens by deleting a single virulence gene. The recent cloningand sequencing of the C. pseudotuberculosis PLD gene (6)have now made it possible to apply this gene deletionapproach to investigate more directly the role of PLD in C.pseudotuberculosis infection.

Here we describe site-specific mutagenesis of the PLDgene in C. pseudotuberculosis and results of sheep vaccina-tion and challenge trials using the PLD-negative bacterium(designated Toxminus). The potential of the Toxminus mu-

* Corresponding author.

tant as a vaccine against CLA and a live vector for thedelivery of recombinant veterinary vaccines will be dis-cussed.

MATERIALS AND METHODS

Bacterial strains and plasmids. Escherichia coli DH5-ot(BRL) was host for a pUC118 clone of a Corynebacteriumerythromycin resistance gene (7, 20) and the E. coli-Myco-bacterium-Corynebacterium shuttle plasmids pEP2 andpEP3 (17). The C. pseudotuberculosis strain 231 (wild typefor PLD production) was obtained from Doug Burrell(CSIRO Division of Animal Health, Glebe, New SouthWales, Australia). Rhodococcus equi CC50 was a gift fromKeith Hughes (Melbourne University, Department of Veter-inary Science, Werribee, Victoria, Australia).

Media. Transformed E. coli was grown in Luria broth (19)containing 50 ,ug of ampicillin (Sigma Chemical Co.), 50 ,ugof kanamycin sulfate, 100 ,ug of erythromycin (BoehringerGmbH, Mannheim, Germany) or 150 ,ug of hygromycin B(Sigma) per ml as required. C. pseudotuberculosis wascultured at 37°C in brain heart infusion broth (Difco Labo-ratories) containing 0.1% Tween 80 (BHI) at 37°C. Trans-formed cells were selected on BHI agar supplemented with50 p,g of kanamycin, 150 ,ug of hygromycin B or 100 ng oferythromycin per ml. Putative C. pseudotuberculosis mu-tants were selected on BHI agar containing 100 ng oferythromycin and 150 ,ug of hygromycin B (Sigma) per ml.To detect PLD activity, C. pseudotuberculosis was culturedon sheep blood plates (Luria broth agar containing 5%defibrinated whole sheep blood and 10% filtered (0.2-,umpore size) culture supernatant from Rhodococcus equibroth). Factors present in the culture supernatant of R. equiact in synergy with PLD to enhance hemolysis.DNA techniques. All DNA manipulations were conducted

by using standard protocols (19). Genomic DNA was iso-lated from C. pseudotuberculosis as described previously (6,17).

Construction of a recombination plasmid. A recombinationplasmid was constructed by cloning a 1.5-kb SacI fragmentcarrying the PLD gene (6) into the PstI site of pEP2. A1.7-kb HindIII fragment containing an erythromycin resis-tance gene (7, 20) was cloned into the PLD gene deleted with

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PstI (pBTB58). The orientation of the PLD and erythromy-cin genes was determined by Sall restriction analysis.

Mutagenesis of the C. pseudotuberculosis PLD gene. Therecombination plasmid was electroporated into wild-type C.pseudotuberculosis 231 (21), and transformants were se-lected on BHI agar containing erythromycin and kanamycin.C. pseudotuberculosis harboring the recombination plasmidwas then electroporated with pEP3, and the transformantswere selected on BHI agar plates supplemented with eryth-romycin, kanamycin, and hygromycin B. The presence ofplasmid pEP3 was confirmed by using Southern blot analy-sis. A transformant containing both plasmids was grownovernight with shaking in BHI containing all three drugs.The culture was subcultured 1:100 in 10 ml of BHI supple-mented with only hygromycin B and shaken overnight. Thesubculturing regimen was repeated a total of three times.The final broth culture was dilution plated onto sheep bloodplates supplemented with erythromycin and hygromycin B.Single colonies were patched to BHI agar plates containingboth erythromycin and hygromycin B and also onto platessupplemented only with kanamycin.

Examination of mutated C. pseudotuberculosis for PLDactivity. Three mutated C. pseudotuberculosis strains weregrown for 2 days in BHI, pelleted, resuspended in 100 ,ul ofphosphate-buffered saline (PBS), and then sonicated torelease cellular protein. Samples (10 ,ul) were spotted ontosheep blood plates and held at 37°C overnight and comparedwith samples obtained from the wild-type strain.DNA analysis of C. pseudotuberculosis mutants. Total ge-

nomic DNA was isolated from three Toxminus C. pseudo-tuberculosis colonies. DNA was digested with SacI, electro-phoresed, and Southern blotted to Zeta-Probe nylon filters(Bio-Rad). Filters were hybridized in 50% formamide-1.0%sodium dodecyl sulfate (SDS)-0.5% skim milk powder-0.5%SSPE (lx SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mMEDTA [pH 7.7]) (19) overnight at 37°C with a PLD gene-specific probe labelled with 3 P by using random primers (4)and then were washed at increasing stringency as necessary(up to 65°C in 0.2x SSC) (lx SSC is 0.15 M NaCl plus 0.015M sodium citrate) and exposed to X-ray film (Fuji RX).Wild-type C. pseudotuberculosis and C. pseudotuberculosistransformed with pBTB58 were included as controls. Toremove the first probe, filters were held in 0.4 M NaOH at45°C for 30 min, washed in 0.1x SSC-0.1% SDS-0.2 M TrisHCI (pH 7.5) at 45°C for 30 min before being hybridized withan erythromycin gene-specific probe, and then washed andexposed to X-ray film as described above.

Sheep vaccination and challenge trials. Nine-month-oldewes were used in the sheep vaccination and challenge trials.To minimize the possibility of C. pseudotuberculosis infec-tion prior to use, the lambs received no vaccination and wereneither shorn nor tail docked. In the first trial, three sheepwere each vaccinated just above the coronet of the left hindlateral claw with 2 ml of PBS containing either 108 or 1010CFU of one of the Toxminus C. pseudotuberculosis strains.Three control sheep were injected with 2 ml of PBS, and afurther three were injected with 2 ml of PBS containing 106CFU of wild-type C. pseudotuberculosis. All sheep werenecropsied at week 8, and the major lymph nodes and organswere examined for abscesses. The left popliteal lymph nodewas collected for microbiological examination. In a secondexperiment, five sheep each were injected subcutaneouslyinto the cleft between the first phalanges on the left hind clawwith 2 ml of PBS containing either 2 x 107 or 2 x 105 CFUof Toxminus C. pseudotuberculosis. Similarly, four sheepreceived a dose of 4 x 105 CFU of wild-type C. pseudotu-

ScS1I

/\/\/ \

/\/\

PLD gene

500 bp

ICHR ERM PLD Recombination

Pv Sm Sm c

FIG. 1. Construction of a PLD gene-specific recombination cas-sette. The PLD gene was cloned into plasmid pEP2. The erythro-mycin resistance gene was then cloned into the PLD gene, thereindeleting 286 bp. CHR, chromosomal DNA sequences flanking PLD;S, signal sequence; PLD, PLD gene; V, pEP2 vector; ERM,erythromycin resistance gene; Sc, Sacl; SI, Sall, Pv, PvuII; P, PstI;Sm, SmaI.

berculosis and two controls were injected with 2 ml of PBS.At week 9, all the sheep, including five new unvaccinatedcontrols, were challenged by injecting the right hind hoofwith 4 x 106 CFU of wild-type C. pseudotuberculosis.The sheep were necropsied 18 weeks postvaccination, and

the major lymph nodes and organs were examined forabscessation. The left, or right popliteal lymph node or bothwere collected for microbiological examination.

Immunological assays. Antibody response to Toxminusvaccination and virulent C. pseudotuberculosis challengewas measured by an indirect enzyme-linked immunosorbentassay (ELISA) using 0.5 jig of sonicated C. pseudotubercu-losis Toxminus per well as antigen. Titers were determinedas the reciprocal of the dilution that gave half the maximumoptical density obtained. Mean titers are expressed as geo-metric means. T-cell response was measured by quantifyingthe release ofgamma interferon in a whole-blood culture (18)incubated with 5 pug of C. pseudotuberculosis Toxminussonicate per ml. Levels of interferon in plasma were assayedby using a commercial capture-tag ELISA for the detectionof ovine and bovine gamma interferon (CSL Ltd, Parkville,Victoria, Australia). Interferon levels are expressed as stim-ulation indices which were calculated by dividing the ELISAoptical density attained with plasma derived from bloodcultured with antigen by that obtained when the blood wascultured without antigen.

RESULTS

Mutagenesis of the C. pseudotuberculosis PLD gene. Tomake a vector capable of site-specific recombination withthe C. pseudotuberculosis chromosome, an erythromycinresistance gene was cloned into a deletion derivative of thePLD gene (Fig. 1). Wild-type C. pseudotuberculosis wastransformed with the recombination plasmid (pBTB58) andsubsequently with the hygromycin resistance shuttle plasmidpEP3. Plasmid pEP3 retains the same origin of replication aspEP2 but carries a hygromycin instead of a kanamycinresistance gene. Cells transformed with both plasmids wereselected on media containing kanamycin, erythromycin, andhygromycin. To maintain plasmid pEP3 and promote theloss of plasmid pBTB58 through plasmid segregation, cellscontaining both plasmids were subcultured to media contain-ing only hygromycin. To detect those cells in which recom-bination into the chromosomal PLD gene had occurred,cultures were plated onto blood plates containing bothhygromycin and erythromycin. Greater than 90% of thehygromycin- and erythromycin-resistant colonies failed toproduce zones of lysis on blood plates. Furthermore, all of

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A BA B C D E F G

M.O

mutated PLD gerne(C,D.E ) ..ANN

A B C D E F G

'P4 up' -

FIG. 2. Southern blot analyses of C. pseudotuberculosis mutants. (A) SacI-digested genomic DNA hybridized with a PLD-specific probe.The probe was made by labelling a 1.5-kb SacI fragment containing the PLD gene. Lanes: A, wild-type C. pseudotuberculosis 231; B, strain231 containing recombination plasmid pBTB58; C, mutant 1; D, mutant 2; E, mutant 3; F, same as lane B but DNA undigested; G, undigestedDNA from C. pseudotuberculosis retaining PLD activity after mutagenesis procedure. Arrow at PLD gene shows position of 2.0-kb molecularweight marker; arrow at mutated PLD gene is 3.4-kb mark. Higher-molecular-weight and ghost bands are probably partially digested material.Note: there are no Sacl sites in pBTB58. (B) Same as Panel A, except that the filter was washed and then hybridized with an erythromycingene-specific probe. The probe was made by labelling a 1.7-kb HindIll fragment containing the erythromycin resistance gene. Note theabsence of the 2.0 kb PLD gene Sacl fragment in lanes A and B.

10 selected colonies producing zones were resistant tokanamycin whereas 50 zoneless mutants examined were allkanamycin sensitive. These results suggested that plasmidpBTB58 was lost from the host cell and that a double-crossover recombination event had introduced the erythro-mycin resistance gene into the chromosome thus inactivatingthe PLD gene. To confirm that the mutated bacteria were notproducing PLD, cell lysates were spotted onto blood plates.Only cellular protein obtained from the parental C. pseudo-tuberculosis produced a zone of erythrocyte lysis. Thisresult suggested that the mutants were incapable of produc-ing zones of lysis on sheep blood plates because they did notproduce PLD and not because they produced a nonsecretedmutant PLD protein.DNA analysis of C. pseudotuberculosis mutants. To confirm

that the predicted recombination event with the C. pseudo-tuberculosis chromosome had occurred, total DNA purifiedfrom three Toxminus isolates, one variant capable of pro-ducing zones after mutagenesis, the wild-type bacteriumharboring pBTB58, and the wild-type strain was examinedby using Southern blot analysis (Fig. 2). When the wild-typeC. pseudotuberculosis genome cut with Sacl was probed byusing the PLD gene, a single 2.0-kb band was observed (Fig.2A). Genomic DNA isolated from three Toxminus bacteriaanalyzed in the same way showed only a single band of 3.4kb (Fig. 2A). When the same filter was subsequently probedwith an erythromycin resistance gene-specific probe, onlythe 3.4-kb Sacl fragment and pBTB58 DNA within nonmu-tated isolates hybridized (Fig. 2B). This confirmed that theerythromycin gene had been incorporated into the PLD geneand is consistent with the occurrence of a double-crossoverevent resulting in the replacement of the chromosomal PLDgene with the deleted PLD-erythromycin resistance gene

cassette.When uncut genomic DNA isolated from C. pseudotuber-

culosis that could still lyse sheep blood plates, even after themutagenesis procedure, was probed by using the PLD gene

probe, a band pattern identical to that produced by the

pBTB58 control was observed (Fig. 2A, lanes G and F). Thisresult suggested that the ability to lyse sheep blood was

retained because pBTB58 had not been lost from the cell andthe chromosomal copy of the PLD gene had not beenmutated.

Necropsy examination of sheep. A preliminary trial involv-ing three replicates per treatment was set up to determine theclinical response of sheep to vaccination with Toxminus C.pseudotuberculosis. No abscesses were detected in the threesheep vaccinated with 108 CFU of Toxminus bacteria,whereas all of the animals injected with 106 CFU of thewild-type strain showed extensive abscessation of the pop-liteal lymph node. This result suggested that deletion of thePLD gene had attenuated C. pseudotuberculosis. Two of thesheep vaccinated with 1010 CFU of Toxminus had smalldisseminated abscesses within the draining popliteal lymphnode, but Toxminus bacteria could be cultured from onlyone of these lymph nodes.A larger dose-response and challenge trial was then un-

dertaken both to confirm the attenuation of Toxminus and toascertain whether Toxminus was capable of stimulating a

protective immune response. Sheep were vaccinated in theleft hind hoof with various doses of Toxminus and subse-quently challenged by injecting the wild-type strain into theright hoof. At necropsy the major organs and lymph nodeswere examined for abscessation; however, abscesses were

confined to either the site of injection, the draining lymphaticduct, within the popliteal lymph node, or a combination of allthree sites. The number, size, and distribution of abscesseswere related to the challenge dose or level of protectionelicited. Unvaccinated sheep receiving 106 CFU of wild-typebacteria at the commencement of the experiment displayedthe most severe clinical signs: multiple abscesses up to 2.5cm in diameter. Toxminus even at the high dose of 107 CFUbacteria produced no abscesses (Tables 1 and 2). In contrast,sheep injected with 105 or 106 CFU of wild-type C. pseudo-tuberculosis formed abscesses from which the injected bac-

PLD genie P

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TABLE 1. No. of sheep that developed abscesses in response toinoculation with wild-type and Toxminus C. pseudotuberculosisa

No. of animals with abscesses in the respectiveleg/no. of animals inoculated

DoseVaccination Challenge (106 WT,(left hoof) right hoof)

107 Tox- 0/5 2/5105 Tox- 0/5 1/5105 WT 2/4 0/4PBS 0/2 2/2106 WT NV 5/5

a Tox- and WT, Toxminus and wild-type C. pseudotuberculosis, respec-tively; PBS, phosphate-buffered saline; NV, not vaccinated.

teria could be isolated (Table 1). Taken together, theseresults confirm that Toxminus is attenuated.When sheep were vaccinated with either 105 or 107 CFU of

Toxminus bacteria and challenged with 106 CFU of thewild-type strain, the proportion of animals with abscesses(Table 1) and the severity of abscessation (Table 2) weregreatly reduced compared with results for the unvaccinatedcontrol animals. These results suggested that Toxminusinfection had stimulated a host protective immune response.None of the animals inoculated with 105 CFU of wild-typebacteria had abscesses in the challenged leg, indicating thatthey were completely protected (Tables 1 and 2). This resultshows that C. pseudotuberculosis infection stimulates a verystrong protective immune response.Immunological responses. Antibody response to the Tox-

minus strain was maximal 2 weeks following vaccination

7000

6000

5000

34000O 3(((

f 2000

1000

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TABLE 2. Severity of abscessation in challenged sheepa

Vaccine dose Sheep no. Clinical finding in challenged leg

10' Tox- (n = 5) 1 1-cm dry abscess at SOI2 1-mm popliteal abscess

105 ToxC (n = 5) 6 1-cm abscess at SOIPBS (n = 2) 11 0.5-cm abscess at SOI

12 1-cm abscess at SOI, two 0.5-cmpopliteal abscesses

Nothing (n = 5) 13 0.5-cm abscess at SOI, four 2.5-cmpopliteal abscesses

14 1-cm abscess at SOI, multiple lymphduct and popliteal abscesses

15 1-cm abscess at SOI, three poplitealabscesses

16 2-cm popliteal abscess17 Abscess in lymph duct, multiple

popliteal abscessesa Tox-, Toxminus C. pseudotuberculosis; PBS, phosphate buffered saline;

Nothing, animals received no inoculant at week 1; SOI, site of inoculation;n = number of sheep in group. All sheep were challenged at week 9 with 106wild-type C. pseudotuberculosis.

(Fig. 3). Since sheep vaccinated with 105 and 107 CFU ofToxminus recorded similar antibody titers with no significantvariation at any time (Fig. 3), it appeared that the responsewas not dose dependent at these inoculation rates. Inocula-tion of sheep with the virulent C. pseudotuberculosis strain231 elicited responses comparable to those resulting fromToxminus vaccination at 2 weeks postinfection (Fig. 3).These peaked at five weeks, being significantly higher thanToxminus vaccination titers (P < 0.05) at 5 and 9 weeks

*- Toxminus 10 5

0 2 5 9 11 14Week after inoculation

FIG. 3. Serological response of sheep to Toxminus C. pseudotuberculosis vaccination. Groups of five sheep were vaccinated with dosesof C. pseudotuberculosis as indicated and bled on designated weeks postinfection. The arrow represents the time at which challenge withvirulent C. pseudotuberculosis (106 CFU) was performed. One sheep in the 105 CFU Toxminus group responded very strongly to challenge,and this biased the average week 14 response. Controls were uninfected sheep which ran with the vaccinated animals.

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U Strain 231

* Toxl

1 Tox2

TTox3

'V .1

3 4 5 6 7

Weeks after InoculationFIG. 4. T-cell response to Toxminus C. pseudotuberculosis vaccination. Individual sheep from the first vaccination trial inoculated with

either wild-type C. pseudotuberculosis 231 (106 CFU) or Toxminus (108 CFU; three replicate sheep called Toxl, Tox2, Tox3) were bledweekly and assayed for antigen-specific sensitized T cells by using a gamma-interferon assay. Uninfected control sheep showed no reactionto the C. pseudotuberculosis antigen.

postinfection. Following challenge at week 9 with the viru-lent strain, Toxminus vaccinates displayed an anamnesticresponse which for most animals only slightly exceededantibody levels resulting from the primary vaccination.Since sheep infected with the wild-type strain producedrelatively high initial antibody responses, challenge had littleeffect on titer after week 9 (Fig. 3).To determine whether Toxminus C. pseudotuberculosis

was capable of eliciting cell-mediated immune responses,T-cell sensitization was measured for individual animalsinoculated with the wild-type and Toxminus bacteria (Fig.4). Toxminus stimulated a strong T-cell response that per-sisted over the 7 weeks of the first sheep inoculation exper-iment.

DISCUSSIONSite-specific mutagenesis is a powerful technique for the

elucidation of gene function in bacterial pathogens and in thepast has been used to determine the role of, for example, thephospholipases C in pathogenesis (13, 15). Recent cloningand sequencing of the first bacterial phospholipase D (6) hasmade it possible to apply this technology to examine the roleof PLD in C. pseudotuberculosis infection. In the studypresented here, deletion of the PLD gene from C. pseudo-tuberculosis yielded a Toxminus variant for which virulencein sheep was reduced by at least two log doses. This resultnow clearly establishes PLD as a major virulence factor forC. pseudotuberculosis and confirms the postulates of previ-

ous workers regarding the importance of this enzyme in thepathology of CLA (2, 3, 22).

Deletion of the C. pseudotuberculosis PLD gene presentsan example of how a single virulence gene deletion can

attenuate a bacterial pathogen to generate a variant that hasthe potential to be used as both a live vaccine and a deliveryvehicle for recombinant antigens, that is, a mutant capable ofstimulating strong immunological responses without elabo-rating clinical signs of disease. Although Toxminus C.pseudotuberculosis stimulated strong humoral (Fig. 3) andcellular (Fig. 4) responses, such responses were generally atlevels lower than those elicited from a wild-type infection(Fig. 3 and 4). The difference in magnitude of the immuneresponses was also reflected by a slightly reduced capacityof the Toxminus vaccination to protect sheep from challengeas compared with protection that resulted as a consequenceof a wild-type infection (Table 1). Wild-type bacteria per-sisted in the host for at least 18 weeks. Toxminus infection,however, was resolved at the site of inoculation within a fewweeks, and Toxminus bacteria could not be cultured fromany of the necropsy tissue sampled from animals in thechallenge trial (week 8 and week 18). These results suggestthat PLD contributes to the in vivo survival of C pseudo-tuberculosis. It is probable that persisting bacteria wouldstimulate the immune system until they became fully isolatedwithin abscesses. Sufficient persistence of bacteria enablingstrong stimulation of the immune system is therefore likelyto be an important attribute of potential live vaccines or

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delivery vehicles. In the case of Toxminus, it should berecognized that in addition to a reduced ability to persistwithin the host, the deletion of PLD removes a majorprotective antigen (3). Nevertheless, greatly reduced clinicalsigns in challenged sheep vaccinated with Toxminus (Tables1 and 2) suggest that this strain could be a useful live CLAvaccine. We can only speculate that delivery of a geneticallytoxoided version of PLD, using live Toxminus, may improveprotection against CLA.The capacity to stimulate cell-mediated immunity (Fig. 4)

in addition to humoral responses (Fig. 3) has been reportedfor other intracellular bacterial pathogens such as Mycobac-terium species (10). Mycobactenum spp. and Corynebacte-rium spp. are both mnembers of the actinomycete and relatedorganisms and each possess an immunoreactive cell surfacestructure containing mycolic acid (2). Progress has beenmade in the past few years toward developing Mycobacte-num bovis BCG as a live vector for vaccine antigens (8).Recently, foreign antigens expressed in recombinant BCGhave been shown to elicit long-lasting humoral and cellularimmune responses in mice (1, 23). Development of plasmidvectors capable of replication in C. pseudotuberculosis (17)together with the Toxminus strain now provides a promisingopportunity to develop Toxminus as a live delivery systemfor recombinant veterinary vaccines. Further work will berequired to determine whether immune responses aremounted against recombinant antigens delivered to sheep inToxminus C. pseudotuberculosis and whether the Toxminusstrain can be used in other animal species.

ACKNOWLEDGMENTS

We thank Lynette Hurst for performing the immunological assaysand Noel Collins, Sandy Matheson, and Alex Cameron for invalu-able assistance with sheep handling. Thanks also to Kim Lund andCatherine Pogson for helping with the necropsy.

This work was supported by the Meat Research Corporation(MRC) and Milstern Health Care (MHC).

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