INFECTION AND IMMUNITY,
Copyright © 1998, American Society for Microbiology
Feb. 1998, p. 474–479Vol. 66, No. 2
Vaccine Potential of Attenuated Mutants of Corynebacterium
pseudotuberculosis in Sheep
CAMERON P. SIMMONS,1,2* SARAH J. DUNSTAN,2MARY TACHEDJIAN,1,3JOLANTA KRYWULT,3
ADRIAN L. M. HODGSON,4AND RICHARD A. STRUGNELL1,2
CRC for Vaccine Technology1and Department of Microbiology and Immunology,2The University of Melbourne,
and CSIRO Division of Animal Health,3Parkville, Victoria, Australia 3052, and CSIRO Division of
Animal Health, Australian Animal Health Laboratory, Geelong, Victoria, Australia 32204
Received 26 June 1997/Returned for modification 22 September 1997/Accepted 3 November 1997
Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etio-
logical agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats.
Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as
veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C.
pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge.
The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node
persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable
specific gamma interferon (IFN-?)-secreting lymphocytes and induced low levels of antibodies to C. pseudotu-
berculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by
aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the
clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain
expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective
immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction
of IFN-?-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The
results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines
or as vaccine vectors.
Corynebacterium pseudotuberculosis is a gram-positive, my-
colic acid-containing facultative intracellular pathogen which is
phylogenetically related to Mycobacterium tuberculosis (16). C.
pseudotuberculosis is the etiological agent of caseous lymphad-
enitis (CLA) in both sheep and goats. CLA is a chronic disease
characterized by the formation of necrotic lesions that in sheep
are typically located in superficial lymph nodes and the lungs
(1). Transmission of disease is though to occur via contamina-
tion of shearing wounds with viable bacteria originating from
the discharging lung abscesses of infected sheep (6, 19). In
Australia, CLA is one of the most prevalent diseases of sheep
and, as a consequence, has an economic impact due to reduced
wool production by infected animals and condemnation of
carcasses and skins in abattoirs (17, 18). C. pseudotuberculosis
infection of humans has also been reported (20).
While the pathogenic process employed by C. pseudotuber-
culosis in causing CLA in sheep and goats is not well defined,
at least two major virulence determinants have been identified.
One of these is the toxic lipid cell wall, which may mediate the
bacterium’s resistance to killing by phagocytic cells (7, 8). The
other identified virulence determinant is a sphingomyelin-de-
grading phospholipase D (PLD) exotoxin (12). PLD is thought
to mediate dissemination of the pathogen within the host by
increasing local vascular permeability (1). CLA vaccines for-
mulated from concentrated, formalin-inactivated C. pseudotu-
berculosis culture supernatants containing PLD have consider-
able efficacy (3–5). A role for PLD in the virulence of C.
pseudotuberculosis was confirmed when two independently
constructed pld mutants were shown to be attenuated in
sheep (10) and goats (15), respectively. One of these mu-
tants (Toxminus), when used as a vaccine against CLA in
sheep, elicited a protective immune response (10). Such live
attenuated mutants of C. pseudotuberculosis hold promise as
veterinary vaccine vectors, since immune responses to coex-
pressed antigens can be elicited in vaccinated sheep (11). Im-
portantly, the immune response to an antigen delivered by a
live vector can potentially be long lasting, thus circumventing
the requirement for multiple vaccinations.
There is, however, evidence from studies of attenuated Sal-
monella typhimurium mutants to suggest that the type of atten-
uating mutation used to construct a vaccine vector can criti-
cally affect the immunogenicity of the strain. This has been
attributed to the different in vivo growth rates or levels of host
persistence of the mutants and concomitant altered interaction
with the host immune system (13). Toward the development of
new attenuated strains of C. pseudotuberculosis for use as vac-
cine vectors, we have previously constructed and assessed the
vaccine potential of an attenuated aroQ mutant in a mouse
model (24). The aroQ gene encodes a type II 3-dehydroquinase
enzyme likely to be involved in the biosynthesis of aromatic
amino acids in the bacterium. This mutant, when used as a
vaccine in mice, elicited an immune response which protected
vaccinees from wild-type C. pseudotuberculosis challenge (24).
The aim of the present study was to compare the vaccine
efficacies of aroQ and pld mutants of C. pseudotuberculosis with
regard to induction of immune responses which are protective
against ovine CLA. The capacity of these mutants to elicit
protective immune responses may correlate with their poten-
tial as vaccine vectors for the delivery of heterologous antigens
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Melbourne, Parkville, Victoria, Australia 3052.
Phone: 61 3 9344 5712. Fax: 61 3 9347 1540. E-mail: r.strugnell
MATERIALS AND METHODS
Bacterial strains and culture. C. pseudotuberculosis C231 is a sheep-patho-
genic wild-type strain (2). C. pseudotuberculosis TB521 is a pld mutant obtained
through allelic exchange of the native pld gene with a cloned pld sequence
specifically mutated at the position encoding the active site of the exotoxin.
Previous studies have established that the histidine at position 20 of the mature
PLD protein is part of the enzyme active site (9, 26). Through site-directed
mutagenesis of the pld gene sequence, substitution of the histidine residue to
serine at position 20 of the mature PLD protein rendered the molecule enzy-
matically inactive (26). By homologous recombination, this mutated pld sequence
was recombined with the C231 chromosome so as to replace the native sequence.
The resultant mutant, TB521, expressed enzymatically inactive PLD at levels
approximately equivalent to that of the wild-type parent (data not shown). The
technique of achieving site-specific allelic exchange at the pld locus and the
screening of mutants has been previously described (10). To verify allelic ex-
change, the DNA sequence encompassing the mutation was amplified by PCR,
and the PCR product was sequenced to verify replacement of the native pld
sequence with the mutated sequence. TB521 has previously been shown to be
attenuated in both mice (24). Strains CS100 and CS200 are aroQ mutants of
C231 and TB521, respectively, and were constructed by allelic exchange (24).
TB111 is a ?pld mutant of C. pseudotuberculosis harboring the entire tran-
scriptional unit of the pld gene on the Escherichia coli-C. pseudotuberculosis
shuttle vector pEP-2 and has been described previously (26). Supernatants from
TB111 cultures were used to partially purify PLD for serological assays. C.
pseudotuberculosis strains were grown at 37°C in brain heart infusion (BHI;
Oxoid, Basingstoke, England) broth or agar supplemented with erythromycin (30
?g ml?1) when appropriate. As previously described (10), BHI plates containing
5% sheep erythrocytes and 10% filtered Rhodococcus equi supernatant were
used to identify strains which expressed PLD. For vaccine preparation, C.
pseudotuberculosis strains were grown in 500 ml of BHI broth at 37°C for 20 h
with shaking and then pelleted. Cells were then washed once with phosphate-
buffered saline (PBS), pelleted, and resuspended in 10 ml of PBS prior to
Serological assays. The serological responses of sheep following vaccination to
C. pseudotuberculosis antigens were determined in an enzyme-linked immunosor-
bent assay (ELISA). To assess responses to secreted proteins, supernatants from
TB111 were first precipitated by 45% ammonium sulfate precipitation of filter-
sterilized culture supernatants. The precipitated proteins were resuspended in
PBS (pH 7.4), and salt was removed by dialysis against four changes of PBS (4°C
for 24 h). The composition of the precipitated proteins was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining.
The results indicated PLD comprised at least 90% of the precipitated protein.
Culture supernatant proteins isolated in this manner were solubilized at 10 ?g/ml
in bicarbonate buffer (pH 9.6) and used to coat 96-well microtiter plates (Nunc
Immunoplates). For preparation of whole-cell lysate antigens, washed overnight
cultures of C. pseudotuberculosis were passed through a French press three times
at a pressure of 1,000 lb/in2. Insoluble material was removed by centrifugation,
and the supernatant containing soluble cellular proteins was removed and freeze
dried. Freeze-dried antigens were solubilized at 10 ?g/ml in carbonate buffer and
used to coat 96-well microtiter plates, which were then incubated at 4°C over-
night. The protein concentration in solutions of coating buffer were determined
by specific protein determination assay (Bradford assay), using a dye reagent
concentrate (Bio-Rad, Hercules, Calif.). Following antigen coating, ELISA
plates were washed and blocked with 3% bovine serum albumin in PBS for 1 h
prior to addition of serum. Serum was diluted in PBS containing 0.3% bovine
serum albumin and 0.05% Tween 20 and, following serial dilution, incubated in
wells for 2 h at 37°C. Wells were washed with PBS containing 0.05% Tween 20,
100 ?l of the appropriate mouse anti-sheep immunoglobulin G (IgG) subclass-
specific antibody (kind gifts from Ken Beh, CSIRO Division of Animal Health,
Sydney, Australia) was added, and the mixture was incubated for 2 h at 37°C.
Following washing, a sheep anti-mouse horseradish peroxidase-conjugated anti-
body (Silenus) was added for 2 h at 37°C. Wells were then washed, and bound
conjugate was detected by using Immunopure o-phenylene diamine (Pierce) with
H2O2the as substrate. Reactions were stopped with 20 ?l of 2.5 M H2SO4, and
optical densities (ODs) were read at 492 nm. Titers were expressed as the
reciprocal of the dilution which gave an OD threefold above the OD of preim-
mune serum analyzed on the same plate. Titers obtained from preimmune serum
of individual animals were always below 300.
Assay for cellular immune responses. Cellular responses to C. pseudotubercu-
losis antigens were quantified by detection of gamma interferon (IFN-?) in
plasma of 1-ml whole-blood cultures stimulated with 5 ?g of a C. pseudotuber-
culosis soluble whole-cell lysate. Whole blood was incubated in duplicate with
and without antigen for 18 h at 37°C in 5% CO2, when plasma was collected and
stored at ?20°C until analyzed. Levels of IFN-? in plasma were determined by
using a commercial capture ELISA for the detection of ovine and bovine IFN-?
(CSL Ltd., Parkville, Victoria, Australia) according to the manufacturer’s in-
structions. Levels of IFN-? were expressed as stimulation indices. These were
calculated by dividing the mean ELISA OD attained with plasma derived from
antigen-stimulated blood by the mean OD obtained when blood was cultured
Sheep vaccination and challenge. Nine-month-old merino wethers were se-
lected from a flock with no history of vaccination or CLA. Prescreening of sheep
involved analysis of serum antibody for reactivity with C. pseudotuberculosis
whole-cell lysate antigens in an ELISA. The use of whole-cell lysates as antigens
in ELISAs has previously been applied to identify sheep with CLA (25). Of 63
sheep screened, 30 with the lowest C. pseudotuberculosis-specific serum antibody
levels were randomly divided into six groups of five. Sheep were vaccinated
subcutaneously above the left hind lateral claw, a site drained by the left popliteal
lymph node. Groups of sheep received either 106or 108CFU of the aroQ
mutants CS100 and CS200; sheep given the pld mutant TB521 received either 106
or 104CFU. Sheep in one group were vaccinated with 106CFU of the wild-type
strain C231. The rationale for including in this study sheep vaccinated with
wild-type strain C231 came from studies by Pepin et al. (21) and Hodgson et al.
(10) which demonstrated that sheep experimentally infected with virulent C.
pseudotuberculosis developed concomitant, acquired immunity to reinfection.
Consequently, these sheep could serve as positive controls for protective immu-
nity. All sheep were allowed to graze freely and were bled fortnightly for isolation
of serum and for blood cultures. Observation of vaccine-induced reactogenicity
at the vaccination site were made weekly. Thirty-eight days postvaccination, all
sheep, including naive controls, were challenged subcutaneously in the right hind
leg with 106CFU of C231. All sheep were sacrificed 38 days postchallenge, and
subjected to a full necropsy. At necropsy, the left and right hind popliteal lymph
nodes were individually collected for bacterial culture. The iliofemoral, medial
iliac, superficial cervical, and superficial inguinal lymph nodes were dissected in
situ for evidence of abscessation. The lungs, kidneys, and intestines were re-
moved from the carcass and similarly analyzed for evidence of abscessation.
Bacterial culture from lymph nodes. The bacterial load in popliteal lymph
nodes was determined by fine dissection of the lymph nodes with scissors fol-
lowed by homogenization in 5 ml of saline, using a medical Stomacher 80
(Seward, London, England). Where abscesses were noted in other lymph nodes,
pus was collected and cultured on BHI agar. Identification of C. pseudotubercu-
losis aroQ mutants was made on BHI plates containing erythromycin. Identifi-
cation of the wild-type strain was based on culture morphology and capacity to
cause hemolysis on BHI plates containing sheep erythrocytes and R. equi super-
natant. Conversely, identification of the pld mutant, TB521, was based on lack of
hemolysis on blood plates.
Statistical analysis. Bacterial counts in popliteal lymph nodes from vaccinees
were compared to counts from unimmunized animals by using the nonparametric
Mann-Whitney test. Total IgG1 and IgG2 antibody responses in vaccinated
sheep were compared by the student t test.
aroQ mutants of C. pseudotuberculosis elicit less severe site
reactions. The purpose of this study was to compare the levels
of virulence of aroQ and pld mutants of C. pseudotuberculosis
and assess their efficacy as live vaccines against CLA in sheep.
An ovine model of C. pseudotuberculosis infection, established
previously (10), facilitated this comparison. This model allows
determinations of a strain’s virulence, based on (i) lymph node
colonization/abscessation and immunization site reactogenicity
and (ii) its capacity to elicit a protective immune response,
based on clearance of wild-type challenge bacteria from a
distal draining lymph node.
Semiquantitative observations of reactogenicity at the vac-
cination site on day 9 postvaccination indicated that site reac-
tions in sheep vaccinated with 106CFU of CS100 or CS200
were less severe than those in sheep administered 106CFU
of C231 or TB521, respectively (Fig. 1). Sheep administered
TB521 at a comparable dose had site reactions which were
marginally less severe, but resolved more quickly, than those
observed in sheep administered C231. Site reactions typically
resolved by day 21 postinjection in all sheep except those
administered C231, which persisted till day 28 in some animals.
Humoral immune responses following primary C. pseudotu-
berculosis vaccination. The humoral immune response follow-
ing vaccination with C. pseudotuberculosis strains was assessed
at day 38 postvaccination. The results indicated that following
vaccination, all sheep developed IgG1 and IgG2 antibody re-
sponses specific for C. pseudotuberculosis cell-associated anti-
gens (Fig. 2A). The magnitude of the antibody response to
cell-associated antigens was, however, lower than the response
to antigens isolated from C. pseudotuberculosis culture super-
natants, of which a major protein component is PLD (Fig. 2B).
VOL. 66, 1998 ATTENUATED MUTANTS OF C. PSEUDOTUBERCULOSIS IN SHEEP475
All vaccine strains elicited antibodies to culture supernatant
antigens, with a bias toward the detection of IgG2 over IgG1
(Fig. 2B). Since the ratio of IgG2 to IgG1 did not change
significantly between vaccination groups, we believe the differ-
ences observed are attributable to the different binding affin-
ities of the IgG1 and IgG2 antibody conjugates. The magnitude
of the antibody response to C. pseudotuberculosis antigens was
vaccine dependent, however, with some vaccine strains induc-
ing significantly higher total antibody titers. The sum of the
IgG1 and IgG2 antibody titers to culture supernatant antigens
was significantly lower (P ? 0.05) in sheep vaccinated with 106
CFU of CS100 than in sheep vaccinated with an equivalent
number of the parental strain, C231 (Fig. 2B). Similarly, the
sum of the antibody titer from sheep vaccinated with 106CFU
of CS200 was significantly lower (P ? 0.05) than that observed
from sheep vaccinated with 106CFU of its parental strain,
TB521 (Fig. 2B). Despite these vaccine-dependent differences
in antibody levels, at the time of challenge, all vaccinated sheep
had serum antibodies to C. pseudotuberculosis culture super-
natant and cell-associated antigens.
Cellular immune responses to primary C. pseudotuberculosis
infections. Cellular immune responses to C. pseudotuberculosis
were assessed in groups of sheep which received the highest
dose of each vaccine strain. The detection of IFN-? in plasma
of antigen-stimulated whole-blood cultures was used as an
indicator of antigen-specific cellular immune responses. On
day 14 postvaccination, only sheep vaccinated with 106CFU of
C231 or 106CFU of TB521 had circulating lymphocytes which
produced IFN-? upon antigen stimulation in vitro (Fig. 3).
IFN-? was not detected in the plasma of stimulated blood
cultures on day 7 postvaccination (data not shown). Stimulated
blood cultures from sheep vaccinated with either CS100 or
CS200 did not produce detectable IFN-? at any time point
Clinical findings at necropsy. In situ dissection and qualita-
tive observation of different lymph nodes and organs in each
animal indicated there were vaccine-dependent differences in
the degree of abscessation resulting from C. pseudotuberculosis
challenge (Table 1). Four of five unvaccinated sheep chal-
lenged with C231 displayed clinical signs of CLA. Indeed, in
three of these animals, abscesses extended to lymph nodes
other than the right popliteal. Despite evidence of infection by
the challenge strain in sheep vaccinated with 108CFU of
CS100 or CS200, these animals displayed less severe clinical
symptoms of CLA compared to unvaccinated controls. Thus,
while there was abscessation in the right popliteal lymph nodes
of some vaccinated animals, lower numbers of other lymph
nodes were affected than in unvaccinated sheep. Conversely,
with regard to popliteal lymph node abscessation, sheep vac-
cinated with 106CFU of CS100 or CS200 appeared as suscep-
tible as unimmunized animals. Without exception, sheep vac-
cinated with 106CFU of TB521 were free from CLA caused by
the wild-type challenge strain. One animal in this group did,
however, have an abscess in the left popliteal lymph node that
was attributed to colonization of the vaccine strain. Two of five
sheep vaccinated with 104CFU of TB521 displayed abscessa-
tion which was restricted to the right popliteal lymph node.
Sheep vaccinated and challenged with C231 had more suppu-
rative lesions in the left popliteal lymph node (three of five
sheep) than the right popliteal lymph node (two of five sheep).
Correspondingly, there were more sheep with abscesses in the
left iliofemoral lymph node (four of five sheep) than in the
right iliofemoral node (none of five sheep).
Persistence of C. pseudotuberculosis vaccine strains. At nec-
ropsy, the left (draining vaccination site) and right (draining
FIG. 1. Clinical scores of adverse reactions occurring 9 days postvaccination
at the injection site of sheep vaccinated with C. pseudotuberculosis C231, the pld
mutant TB521, or the aroQ mutant CS100 or CS200. Site reactions were scored
semiquantitatively, using the following criteria: 0, no significant adverse reaction;
1, ?0.5-cm-diameter defined nodule but ?1.5 cm with no pustulance evident; 2,
?1.5-cm-diameter defined nodule but ?3 cm with no pustulance evident; 3,
?3-cm-diameter defined nodule with surrounding swelling and/or soft pustulant
FIG. 2. Mean (plus standard deviation) IgG1 and IgG2 antibody titers spe-
cific for C. pseudotuberculosis soluble whole-cell lysate antigens (A) and culture
supernatant antigens (B) at day 38 postvaccination. There was a significant
difference (P ? 0.05) in the sum of the antibody responses to secreted proteins
in sheep vaccinated with 106CFU of C231 compared to sheep vaccinated with
106CFU of CS100 (denoted by A and A*, respectively). Similarly, there was a
significant difference (P ? 0.05) in the sum of the antibody responses in sheep
vaccinated with 106CFU of TB521 compared to sheep vaccinated with 106CFU
of CS200 (denoted by B and B*, respectively). The dashed line represents the
limit of antibody detection.
476SIMMONS ET AL.INFECT. IMMUN.
challenge site) hind popliteal lymph nodes were aseptically
removed from all sheep and processed for quantitative bacte-
rial culture of the vaccine strain (left) and the challenge strain
(right) (Fig. 4). Left popliteal lymph nodes from sheep infected
with CS100 and CS200 were sterile and contained no ab-
scesses. This result indicated that at the doses given, CS100
and CS200 were either unable to colonize or unable to persist
in the left popliteal lymph nodes for 76 days. Conversely, four
of five sheep vaccinated with 106CFU of TB521 harbored
between 102and 104bacteria in their left popliteal lymph
nodes (Fig. 4). Indeed, there was evidence of abscessation in
one of these lymph nodes from which TB521 was isolated
(Table 1). All randomly selected colonies isolated from this
node were nonhemolytic on blood plates, suggesting that ab-
scessation resulted from TB521 colonization. Four of five left
popliteal lymph nodes isolated from sheep vaccinated with
C231 harbored significant numbers of bacteria (range, 102to
107CFU) (Fig. 4). Three of these nodes contained abscesses
Colonization by the wild-type challenge strain. The quanti-
tative observations made regarding the number of challenge
bacteria in the right popliteal lymph node of each animal
partially reflected the number of abscesses found at necropsy.
Enumeration of the number of wild-type challenge bacteria
isolated from the right popliteal lymph nodes of unvaccinated
sheep indicated that these animals were highly susceptible to
infection (Fig. 4). Low numbers of C231 were isolated from the
left popliteal lymph node of one unvaccinated animal, which
also had the highest bacterial load in the right lymph node. The
presence of challenge bacteria in the left popliteal lymph node
may have arisen as a result of systemic spread of the organism,
since the right iliofemoral lymph node of this animal was also
severely abscessed. Importantly, the isolation of C231 in all
unimmunized sheep confirmed the infectious nature of the
challenge inoculum. Sheep vaccinated with CS100 or CS200,
irrespective of the dose, also harbored high numbers of wild-
type bacteria in the right popliteal lymph nodes (Fig. 4). This
result indicated that CS100 and CS200, when used as vaccines,
were unable to elicit an immune response that prevented in-
fection. One animal in the group vaccinated with 108CFU of
CS200 also carried C231 challenge bacteria in the left popliteal
lymph node. This animal also had the highest bacterial counts
in the right popliteal lymph node, which was correspondingly
severely abscessed. Given that no other lymph nodes were
visibly abscessed in this animal, contamination during lymph
node collection cannot be excluded.
In sheep vaccinated with 106CFU of TB521, there was a
significant difference (P ? 0.01) in the median number of
challenge bacteria recovered from the right popliteal lymph
node compared to unimmunized control animals (Fig. 4).
There was no evidence of abscessation in the right popliteal
lymph nodes of TB521-vaccinated animals (Table 1). Sheep
vaccinated with 106CFU of C231 and subsequently challenged
with the same strain also harbored significantly fewer (P ?
0.05) challenge bacteria (between 10 and 104CFU) in the right
popliteal lymph nodes compared to unimmunized control an-
imals (Fig. 4). Despite the significant reduction in the number
of challenge bacteria in these nodes, two of five lymph nodes
contained abscesses (Table 1).
We have previously reported that aroQ mutants of C.
pseudotuberculosis are highly attenuated in a BALB/c mouse
model of infection yet, at the appropriate dose, could elicit an
immune response that protected mice from homologous chal-
lenge (24). Our results for sheep, a natural host for C. pseudo-
FIG. 3. Stimulation indices representing C. pseudotuberculosis antigen-spe-
cific IFN-? release from stimulated whole-blood isolated from individual sheep
14 days postvaccination. Blood from uninfected control sheep did not produce
IFN-? following antigen stimulation.
TABLE 1. Number of sheep in each vaccine group (n ? 5) with abscesses in specific lymph nodes or organs at necropsy (day 76)
from which C. pseudotuberculosis could be recovereda
Lymph node or
No. of sheep in each vaccine group with abscesses (by location)
CS100 (108b) CS200 (108) TB521 (106) C231 (106) CS100 (106) CS200 (106) TB521 (104) Naive
Right medial iliac
Left superficial cervical
Right superficial inguinal
aWith one exception, wild-type C. pseudotuberculosis was recovered from all abscesses.
cAbscess from which TB521 was recovered.
VOL. 66, 1998 ATTENUATED MUTANTS OF C. PSEUDOTUBERCULOSIS IN SHEEP 477
tuberculosis infections, support the observation that these aroQ
strains are indeed attenuated. However, at the doses used for
vaccination in the current trial, these strains did not elicit
immune responses which protected sheep from CLA.
The observation that the degree of vaccination site reacto-
genicity in sheep given 106CFU of CS100 or CS200 was less
severe than that found in sheep given 106CFU of either C231
or TB521 is consistent with the hypothesis that aroQ mutants
have a reduced capacity to multiply in vivo. This hypothesis is
supported by the observation that the left popliteal lymph
nodes of sheep immunized with the aroQ mutants were sterile
at necropsy. This finding suggests that aroQ mutants are either
unable to persist in or unable to colonize this draining lymph
node. In direct contrast, in four of five sheep vaccinated with
106CFU of the pld mutant, TB521, nonhemolytic bacteria
could be recovered from the left popliteal lymph node, which
drains the vaccination site. Indeed, the left popliteal lymph
node from one of these sheep displayed abscessation that was
attributed to colonization by TB521. In comparison to sheep
vaccinated with C231, however, animals immunized with TB521
had reduced vaccination site reactogenicity, lower vaccine
strain colonization of the left popliteal lymph node, and a
concomitant reduction in the number of vaccination-induced
abscesses. These observations confirm that mutation of the
gene encoding the PLD exotoxin (His203Ser20) is sufficient
to significantly attenuate the bacterium in its natural host. C.
pseudotuberculosis mutants either with deletions in the pld
gene (10) or with histidine-to-tyrosine substitutions at position
20 of the mature PLD protein (23) have previously been shown
to be attenuated.
While all sheep infected with C. pseudotuberculosis strains
developed IgG antibodies to both cell-associated and culture
supernatant antigens, there were some significant differences
in the magnitude of the responses. Despite different vaccine
doses, sheep infected with 106CFU of C231 or TB521 had
significantly higher antibody titers to culture supernatant anti-
gens compared to sheep vaccinated with 108CFU of CS100 or
CS200, respectively. The detection of IFN-? in the plasma of
stimulated whole blood isolated from sheep vaccinated with
106CFU of TB521 or C231 suggests that these strains also
elicit cellular immune responses. The inability to detect IFN-?
from sheep vaccinated with CS100 or CS200 suggests either
that these strains are relatively poor stimulators of IFN-?-
secreting cells or that the kinetic of the acquired immune
response is different. The capacity of TB521 and C231 to elicit
IFN-?-secreting lymphocytes in whole blood of vaccinees cor-
related with a reduction in the number of challenge bacteria in
the right popliteal lymph node. Thus, the induction of an
adequate Th1-type T-cell response, characterized by IFN-?
production, may be an essential component in the induction of
acquired resistance to C. pseudotuberculosis infection by live
attenuated vaccines. Previous studies of mice (24) and sheep
(27) support this contention. Furthermore, the importance of
Th1-type immune responses in acquired resistance to other
faculative intracellular bacterial pathogens is well character-
While Th1-type cellular immune responses are likely to be
an important component in acquired resistance to C. pseudo-
tuberculosis, humoral immune responses, particularly to anti-
gens found in culture supernatants, have also been suggested
to mediate immunity. In Australia, current commercial vac-
cines against CLA consist of inactivated C. pseudotuberculosis
culture supernatant antigens, of which PLD is a component
(3–5, 11). It has been hypothesized that the presence of anti-
PLD antibodies at the time of C. pseudotuberculosis challenge
could abolish toxin induced vascular permeability and thus
limit dissemination of the pathogen (12). Evidence that im-
mune responses to PLD can mediate immunity has been shown
in sheep studies using chromatographically purified PLD as a
subunit vaccine (4). However, a correlation has not been es-
tablished in individual sheep between the magnitude of the
antibody response to PLD and protection from CLA (3). An-
other protein secreted by C. pseudotuberculosis, a 40-kDa
serine protease, has also been used as a subunit vaccine to elicit
protective immune responses in sheep (28, 29). The presence
of antibodies to the 40-kDa protein did not correlate with pro-
tection from challenge, however, leading the authors to suggest
that cellular immune responses mediated protection (28).
Despite the equivocal role of antibodies to proteins secreted
by C. pseudotuberculosis in protective immunity, the magnitude
of the mean antibody response to culture supernatant antigens
by sheep immunized with C231 or TB521, in this trial, corre-
lated with fewer challenge bacteria in the right popliteal lymph
node. Clearly though, the mere presence of these antibodies did
FIG. 4. Isolation of C. pseudotuberculosis mutants and wild-type bacteria from left (draining the vaccination site) and right (draining the challenge site) popliteal
lymph nodes from individual sheep at necropsy (day 76). Bacteria isolated from lymph nodes were identified as described in Materials and Methods. Symbols for mutant
and wild-type bacteria: ?, CS100; ?, CS200; ƒ, TB521; I, C231. There was a significant reduction in the median number of wild-type bacteria (I) recovered from the
right popliteal lymph nodes of sheep vaccinated with 106CFU of C231 (P ? 0.05) or TB521 (P ? 0.01) compared to lymph nodes from naive controls. The dashed
line represents the limit of detection.
478SIMMONS ET AL.INFECT. IMMUN.
not prevent infection of the lymph node draining the challenge Download full-text
site, since CS100- and CS200-vaccinated sheep, while having an-
tibodies to culture supernatant antigens, had significant numbers
of challenge bacteria in their right popliteal lymph nodes.
While most sheep immunized with the aroQ mutant CS100
or CS200 harbored high numbers of challenge bacteria and
also some abscesses in their right popliteal lymph nodes, the
total number of lymph nodes displaying clinical signs of CLA
in these sheep appeared to be lower than in unvaccinated
control animals. Thus, the immune responses induced by the
aroQ mutants used as vaccines did not protect sheep from
infection but did appear to reduce the clinical severity of dis-
ease resulting from wild-type challenge. We hypothesize that
the presence of antibodies to C. pseudotuberculosis antigens at
the time of challenge may help limit the systemic dissemination
of the pathogen to other lymph nodes. In contrast, the immune
response elicited by administration of 106CFU of TB521 pre-
vented colonization with the wild-type challenge strain. Inter-
estingly though, despite the apparent immune status of these
animals, a majority of vaccinees were unable to clear the vac-
cine strain from the lymph node draining the immunization
site. We hypothesize that this is due to the inaccessible location
of the bacteria within granulomas. Indeed, a key process in the
induction of a protective immune response may be the forma-
tion of microscopic pyogenic granulomas, which, by helping to
prevent bacterial dissemination, allow the host to mount an
effective, T-cell-mediated immune response (22). Conversely
however, these granulomas allow persistence of the pathogen
by excluding immunological effectors.
In this study, we have assessed the virulence of aroQ and pld
mutants of C. pseudotuberculosis in sheep and simultaneously
their capacity to act as vaccines against homologous challenge.
The aroQ mutants did not elicit a protective immune response
against CLA. These mutants may be overly attenuated with
respect to in vivo growth to elicit the required response. In
contrast, at the appropriate dose, the pld mutant TB521 elic-
ited a protective immune response, and this was correlated
with persistence of the vaccine strain, the induction of IFN-?-
secreting lymphocytes, and relatively high levels of antibodies
to culture supernatant antigens. Importantly, vaccination with
TB521 did not cause overt CLA in vaccinees. As a result,
TB521, like the previously constructed ?pld mutant (Toxmi-
nus) (10, 11), holds promise as a live vaccine vector for the
induction of cellular and humoral immune responses to heter-
ologous antigens expressed by the bacterium.
We thank Leigh Corner, Noel Collins, and Sandy Matheson for
excellent technical assistance during sheep necropsy. The assistance of
Jan Tennent in reviewing the manuscript was greatly appreciated.
This work was supported by the CRC for Vaccine Technology.
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Editor: R. N. Moore
VOL. 66, 1998ATTENUATED MUTANTS OF C. PSEUDOTUBERCULOSIS IN SHEEP479