INFECTION AND IMMUNITY, June 1994, p. 2562-2567 Vol. 62, No.

6
0019-9567/94/$04.00+0
Copyright C) 1994, American Society for Microbiology

Identification of a Novel Antigen from Corynebacterium

pseudotuberculosis That Protects Sheep
against Caseous Lymphadenitis
J. WALKER,I* H. J. JACKSON,' D. G. EGGLETON,2 E. N. T. MEEUSEN,' M. J. WILSON,'
AND M. R. BRANDON'
Centre for Animal Biotechnology, School of Veterinary Science, The University of Melboume,'
and CSL Ltd.,2 Parkville, Victoria 3052, Australia
Received 13 December 1993/Returned for modification 29 January 1994/Accepted 1 April 1994

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A 40-kDa protein antigen from Corynebacterium pseudotuberculosis has been identified by application of a
strategy that employs locally derived antibody-secreting cells (ASC). ASC probes generated by culture of ASC
obtained from the lymph node draining the site of infection showed a specificity restricted to a 40-kDa antigen.
Analysis of immunoblots with sequential serum samples taken from sheep during the course of experimental
primary infection with C. pseudotuberculosis also revealed the 40-kDa antigen as an early immunodominant
antigen. Sheep vaccinated with two 100-,ug doses of a 40-kDa antigen preparation in aluminium hydroxide
adjuvant were protected against infection with C. pseudotuberculosis, with an 82% reduction in the proportion
of infected sheep and a 98% reduction in lung lesions. Sera from vaccinated sheep exhibited a strong response
only to the 40-kDa antigen on immunoblots. These results strongly suggest that the 40-kDa antigen plays a
major role in immunity to caseous lymphadenitis.

One of the major difficulties limiting the development of
new defined-subunit vaccines is the identification or selection
of protective antigens. Bacteria and parasites present the
researcher and the host with a complex array of immunogenic
molecules. To date, candidate vaccine antigens have been
selected on the basis of their known or putative role in
infection, e.g., bacterial toxins or essential enzymes of parasites
such as glutathione-S-transferase (19), or on the basis of their
identification with antibodies from immune sera. However, the
complexity of the immune response to the pathogen is usually
indicated by the very diverse antibody response in immune
sera, and reliance on selection of molecules judged to be
immunodominant on the basis of their reactivity with serum
antibody has not always been successful in identifying protec-
tive antigens. For example, immunodominant membrane pro-
teins of Mycoplasma hyopneumoniae (21) and Plasmodium
falciparum (20) have not proved to be protective.
Caseous lymphadenitis (CLA) is a common and economi-
cally important disease of sheep and goats, caused by infection
with Corynebacterium pseudotuberculosis. The disease is char-
acterized by the formation of encapsulated necrotic or caseous
lesions that in sheep are located primarily in peripheral lymph
nodes and the lungs. C. pseudotuberculosis is a facultative
intracellular pathogen that multiplies within macrophages. It is
readily phagocytosed by nonimmune macrophages, and follow-
ing phagocytosis with ovine (11) and caprine (22) macro-
phages, phagosomes fuse with lysosomes but bacteria continue
to multiply within phagolysosomes, leading to cell death,
release of bacteria, and ultimately a necrotic lesion. The
response of the host is to contain the pyogranuloma by the
formation of a strong collagen capsule (15). The immune
response to natural infection has been shown to include a
strong humoral component, with circulating antibodies de-
tected to a large number of antigens (9). Cell-mediated
immunity has also been implicated in control of CLA lesion
*
Corresponding author. Phone: 61 3 344 7348. Fax: 61 3 347 4083.
2562
development, with lymphocytes observed inside the collagen
capsule in intimate contact with necrotic tissue (23). The
intracapsular lymphocytes have a defined architecture of three
layers, with high proportions of CD8 and of -yi T-cell subpopu-
lations being striking features of the observed cellular response
to C. pseudotuberculosis infection.
Two major virulence determinants of C. pseudotuberculosis
are currently recognized-a potent phospholipase D (PLD)
exotoxin (14) of 31 kDa, believed important in facilitating
spread of the disease, and a toxic lipid associated with the cell
wall (4, 12). Production of both of these factors by strains of C.
pseudotuberculosis has been correlated with virulence (17).
Vaccination against CLA in sheep has been attempted with
a variety of antigens, with variable success. Jolly (14) showed
the involvement of toxoid in protection. More recently, puri-
fied toxoid has been shown to provide a similar level of
protection to a complex array of culture supernatant-derived
antigens (5). Comparison of protection against CLA achieved
with whole-culture, culture supernatant, and whole-cell anti-
gens has led others (2, 3) to conclude that cell-associated
antigens were required as well as toxoid for optimal protection.
The ability of cell-associated antigens to protect lambs against
CLA has been recently confirmed by using bacterial cell walls
with muramyl dipeptide as an adjuvant (1). Hodgson et al. (13)
have shown that infection of sheep with an avirulent toxin-
minus strain of C. pseudotuberculosis with a deletion of the
PLD toxin gene induces strong immunity to CLA, providing
incontrovertible evidence that antigens other than PLD con-
tribute to protection.
Despite many studies demonstrating the involvement of an
antigen(s) other than toxin in protection, no such antigen has
been described. We report here the identification of an antigen
from C. pseudotuberculosis by application of a strategy that
utilizes the local immune response. Others have used B cells
from a site of infection to derive monoclonal antibodies for
antigen identification (24) but did not analyze directly the
spectrum of specificities of antibodies produced by the B cells.
Studies in our laboratories of the local immune response in
VOL. 62, 1994 SUBUNIT VACCINE AGAINST OVINE CASEOUS LYMPHADENITIS
tissue at the time of infection, or rejection of infection, have
shown that antibody-secreting cells (ASC) obtained locally
produce antibodies of highly restricted specificity, especially
compared with the serum response (16). Such ASC probes
have been applied to the identification of a novel antigen, and
furthermore, we demonstrate that vaccination of sheep with
the native antigen provides a high level of protection against
experimentally induced CLA.
MATERIALS AND METHODS
Bacterial strains and culture. C. pseudotuberculosis WA1030
was grown at 37°C in brain heart infusion broth (Difco) or on
the same medium solidified with 1.5% Bacto Agar (Difco). A
number of field isolates of C. pseudotuberculosis designated
A01 to A05 were also used in this study.
Sheep. All animals were from a CLA-free flock of Romney-
Marsh sheep bred at the CSL Field-station, Woodend, Victo-
ria, Australia. Adult sheep, used for immune response studies,
were housed inside and fed a diet of chaff and pellets. Lambs,
used for the vaccine trial, remained in paddocks.
Preparation of ASC probes. To study the local immune
response to infectious challenge with C. pseudotuberculosis,
ASC probes were prepared by culture of ASC following
infection. Naive adult sheep that had not previously been
vaccinated against CLA or infected with this bacterium were
used. Sheep were infected by subcutaneous injection of 1 ml of
dilute suspension of caseous material obtained from a CLA
lesion; the suspension was prepared by homogenizing approx-
imately 0.1 g of exudate with 1 ml of phosphate-buffered saline
and contained 6.8 x 106 CFU of C. pseudotuberculosis per ml.
The subcutaneous injection was given on the outer aspect of
the right hock, resulting in a local infection, detected as a
swelling within 7 days. Following infection, sequential samples
of blood were taken and sheep were killed by intravenous
injection of 30 ml of sodium pentabarbitone (Lethabarb)
either 7 or 14 days after infection. The popliteal lymph node
has been shown by previous tracer studies with Evans blue dye
to drain the area of infection. The left and right popliteal
lymph nodes were dissected out, and mononuclear leukocyte
suspensions were prepared on Ficoll-Isopaque gradients. The
mononuclear cells, containing ASC, were cultured at 2 x 106
cells per ml in Dulbecco's modified essential medium (Difco)
containing 10% (vol/vol) heat-inactivated fetal calf serum, 2 x
10-' M 2-mercaptoethanol, and the antibiotics penicillin (100
U/ml) and streptomycin (100 ,ug/ml). Mitogens were added to
ASC cultures at the following concentrations: pokeweed mito-
gen (Gibco), 25 ,ug/ml, final concentration; lipopolysaccharide
(LPS; Sigma), 50 ,ug/ml, final concentration; and dextran
sulfate (D.SO4; BDH) 20 ,ug/ml, final concentration. After 7
days of culture at 37°C in a 5% CO2 incubator, cells were
removed by centrifugation (1,000 x g, 10 min), and the
supernatants were retained. These supernatants obtained by
culture of ASC are referred to as ASC probes.
Antigen identification. ASC probes were used to screen
whole-cell antigens of C. pseudotuberculosis by immunoblots.
Washed whole cells (10 ,ul of cells per lane at an optical density
at 600 nm [OD600] of 1.0) were solubilized with loading buffer,
run on 12.5% (wt/vol) sodium dodecyl sulfate (SDS)-polyacryl-
amide gels (Mini-Gels; Bio-Rad) under reducing conditions
using a discontinuous buffer system, and then electroblotted
onto nitrocellulose (BA35; Schleicher & Schuell). After block-
ing overnight in Tris-buffered saline (pH 7.4) with 0.05%
(vol/vol) Tween 20 (TBS), blots were incubated with either
serum (diluted 1/100 in TBS) or ASC probe (undiluted) for 1
h at room temperature. After four washes in TBS, bound sheep
2563
antibodies were detected by incubation with an anti-sheep
horseradish peroxidase conjugate (Dako) diluted 1/600 in TBS
followed by washing and incubation in diaminobenzidine sub-
strate (Sigma; 2 mg/ml) and H202 (20 mM) in citrate buffer,
pH 5.5.
Antigen purification. The 40-kDa antigen identified by ASC
probes was prepared from 50 liters of culture supernatant that
had been concentrated by pressure dialysis. Ammonium sul-
fate was added to 50% saturation, and precipitated proteins
were removed by centrifugation. The supernatant was applied
to a phenyl-Sepharose hydrophobic chromatography column
(Pharmacia) and eluted with a decreasing (NH4)2SO4 gradient.
ASC probes were used to monitor fractionation of the 40-kDa
antigen preparation.
Vaccination trial. Sixty-four wether lambs, approximately 6
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months old, were randomly allocated to one of three groups
and were identified by ear tags. Sheep in group 1 received 100
jig of 40-kDa antigen preparation in aluminum hydroxide,
group 2 animals received 5 ,ug of 40-kDa antigen preparation
per dose given in aluminum hydroxide, and group 3 sheep
received a placebo injection of aluminum hydroxide alone.
Each animal was given two subcutaneous injections 4 weeks
apart. Three weeks following the second vaccination, sheep
were bled and challenged by subcutaneous injection of a
washed suspension of 2 x 106 in vitro-grown C. pseudotuber-
culosis cells in the lower right back leg. Three months after
infectious challenge, the sheep were slaughtered at a commer-
cial abattoir. As ears and tags were removed before inspection,
sheep were identified by consecutive numbering of the gam-
brels or hangers, and these numbers were coded to ear tag
numbers for later analysis. Carcasses were examined by De-
partment of Primary Industry inspectors. Lungs were removed
and placed in plastic bags for subsequent scoring of lesions.
Lesions were detected by palpation, and any enlarged lymph
node or swelling in tissue was examined by serial incisions.
After all tissues had been examined and results had been
recorded, gambrel numbers were decoded and group lesion
scores were collated.
Assay of serum antibodies. Antibody responses in sera were
assayed by enzyme-linked immunosorbent assay (ELISA).
Whole cells diluted to an OD600 of 0.01 or culture supernatant
or 40-kDa antigen preparation diluted to approximately 5 ,ug
of protein per ml in bicarbonate buffer, pH 9.6, were dispensed
in 100-,lI aliquots into 96-well microtiter plates (Nunc Immu-
noplate). After coating overnight at 4°C, wells were washed
and blocked with TBS for 1 h. Dilutions of sera were incubated
for 1 h at 37°C, wells were washed, and 100-tLI aliquots of
anti-sheep peroxidase conjugate (Dako) diluted 1/5,000 in TBS
were added. Conjugate was incubated for 1 h, wells were
washed with TBS, and bound conjugate was detected with
100-,u aliquots of tetramethylbenzidine (100 ,ug/ml; Aldrich)
substrate with 20 mM H202. ELISA reactions were stopped
with 50 p,l of 2 M H2SO4.
Statistical methods. Results of the vaccination trial were
analyzed by standard statistical methods (10).
RESULTS
Antigen identification. Sequential serum samples taken
from naive (CLA-free) sheep during the course of a primary
infection were screened by immunoblots against whole-cell
antigens. Serum antibody responses are shown in Fig. la. Little
antibody response was detected during the first 4 to 5 days
(Fig. la, panels i and ii). A band at approximately 28 kDa
observed with preinfection sera from naive sheep increased in
intensity with sera from day 4 of infection onwards. This
2564 WALKER ET AL. INFECT. IMMUN.

a(i) a(ii) a(iii) (a) (b) (C)

97kD
67kD
H]
59kD -
97kD -
67kD -
59kD -
37kD -

A
K'1'
4.-

IY&
I4- 37kD -
m

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0247 0 2 4 7 11 14
Days Days

b(i) b(ii) b(iii) b(iv) FIG. 2. (a) SDS-polyacrylamide gel protein profiles of antigen
preparations of C. pseudotuberculosis. Lanes: 1, whole cells; 2, concen-
_k, _ I.'kE
1 97kD-.
67kD-
lI trated culture supernatant (9 ,ug of protein loaded); 3, 40-kDa antigen
preparation (3 p.g of protein loaded). (b) Immunoblot of supernatant
59kD- I. w antigens (as shown in panel a, lane 2), reacted with ASC probes
. -D derived from a challenged node. ASC probe cultures were stimulated
97kD- 00 :^ 4
with either phytohemagglutinin (PHA) or LPS-D.SO4 (LPS). (c)
37kD- M.
Immunoblot of 40-kDa antigen probed with representative pre- and
67kD-
59kD- postimmunization sera from a sheep vaccinated with the 40-kDa
8 antigen preparation.
37kD- k, '^ * Li=
4

DA._.N
_ 4_#3#s g
{

{, K ASC probes prepared from lymph nodes taken from sheep

i .-s
cI C*lj
s CT s V)
;> X > 4 CL4
0-.
g from sheep killed 14 days after a primary infection showed
k R C6
FIG. 1. Analysis of sera (a) and ASC probes (b) from naive sheep
with a primary infection of C. pseudotuberculosis against whole-cell
antigens. (a) Sequential serum samples from three sheep between days
0 and 7 of infection (i) or days 0 and 14 of infection or serum taken at
day 14 of infection (iii). Arrows indicate 40-kDa antigen. (b) ASC
probes derived from four sheep 7 days (i) and 14 days (ii, iii, and iv)
after a primary infection. Arrows indicate 40-kDa antigen. ASC probes
were prepared by culturing mononuclear leukocytes obtained from
popliteal lymph nodes draining infected or contralateral uninfected
sites in the presence of either pokeweed mitogen (PWM) or LPS-
D.SO4 (LPS).
antigen was identified as a major cell protein of 28 kDa on
SDS-polyacrylamide gel electrophoresis (PAGE) of whole
cells (Fig. 2a, lane 1) that gave a background reaction on
immunoblots. Other minor bands did not increase in intensity
from days 2 to 7 of infection (Fig. la, panel i). Infections of C.
pseudotuberculosis that were allowed to proceed for 14 days
stimulated antibodies to a wide range of antigens, the immu-
noblot patterns of sera becoming more complex from day 7
onwards, as the infection progressed.
killed at day 7 of infection showed little reactivity on immu-
noblots (Fig. lb, panel i). The background band at 28 kDa
observed on immunoblots with preinfection sera was also seen
with ASC probes prepared from lymph nodes draining infected
cn cn
0.4 a4 sites of sheep killed at day 7 (Fig. lb, panel i) and from lymph
04 04 nodes draining uninfected sites (Fig. lb, panel ii). ASC probes
strong and restricted reactivity on immunoblots to whole-cell
antigens (Fig. lb), especially compared with serum antibody.
Probes from ASC draining the infected site were strongly
reactive, while those from the contralateral uninfected site
showed background reactivity to the 28-kDa protein (Fig. lb).
The spectrum of reactivity of ASC probes at day 14 to
antigens of C. pseudotuberculosis showed some variation be-
tween sheep, with a number of bands highlighted by some
sheep (Fig. lb, panels iii and iv). However, a 40-kDa antigen
was consistently and strongly recognized on immunoblots and
in two sheep was the major antigen recognized (Fig. lb, panels
ii and iii). Analysis of immunoblots of sera showed that
infected sheep also had circulating antibody to the 40-kDa
antigen, and sequential serum samples showed this was one of
the earliest antigens recognized during the course of infection
(Fig. la, panel ii). ASC probes also strongly recognized the
40-kDa antigen in concentrated culture supernatant (Fig. 2b),
with a minor band at 38 kDa also visible, which we believe to
be a smaller form of the same antigen. Polyclonal sera raised to
the 40-kDa antigen preparation also recognized the 38-kDa
band, seen in Fig. 2c as the lower band of a doublet.
ELISA of antibodies in scrum taken at day 14 and in ASC
probes from the same sheep to whole-cell and supernatant
antigens of C. pseudotuberculosis confirmed the results ob-
tained from the immunoblots (Tables 1 and 2). Antibody
VOL. 62, 1994 SUBUNIT VACCINE AGAINST OVINE CASEOUS LYMPHADENITIS 2565

TABLE 1. Titers in serum' assayed by ELISA using whole-cell TABLE 3. Field trial lesion scores of sheep 3 months after
and supernatant antigens infectious challenge with C. pseuidotuibercuilosis
ELISA OD459 No. of infected No. of sheep with Mean no. of lung
Treatment group sheep/no. in popliteal lymph lesions + SD
Day 7-Day infection 14-Day infection group node lesions (range)
postinfection
Whole Supernatant Whole Supernatant 40-kDa antigen
cells antigens cells antigens 100 ,ug 2/21" 0 0.1 0.5 (1-2)"
0 0.580 0.207 0.400 0.162 5 ,ug 9/22 0 1.4 2.6 (1-12)"
2 0.545 0.189 0.361 0.160 Unvaccinated 12/22 5 6.7 + 12.6 (1-46)
4 0.544 0.204 0.362 0.165 controls
7 0.620 0.225 0.380 0.517 " Significantly different from control group, see text for details.
1l - 0.593 1.355
14 0.744 1.523
All sera were assayed at a 1/100 dilution.

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responses developed in sera from day 7 onwards. Antibody
reactivity of the probe from the challenged site reacted
strongly with antigens at day 14, but the ASC probe from 7-day
infections was negative, as were probes prepared from lymph
nodes draining uninfected sites. Other experiments have
shown that inclusion of mitogens in cultures leads to a higher
level of antibody, as measured by ELISA, but does not affect
the limited spectrum of antigens recognized by ASC probes on
immunoblots.
Antigen preparation. The 40-kDa antigen was fractionated
from 50 liters of culture supernatant by hydrophobic chroma-
tography with a decreasing gradient of 50 to 0% saturated
ammonium sulfate. The 40-kDa antigen eluted between 25 and
15% saturated (NH4)2SO4 and was approximately 50% pure as
judged by scanning densitometry of Coomassie-stained SDS-
PAGE gels (Fig. 2a, lane 3); this is defined as the 40-kDa
antigen preparation. The major contaminating moieties were
peptides of less than 20 kDa derived from the culture medium.
The large scale precluded some techniques, such as gel chro-
matography, for further purification. Silver staining highlighted
a red-to-rust brown diffuse band at the top of the gel. The
31-kDa PLD toxin was the major protein present in bacterial
culture supernatants (Fig. 2a, lane 2) but could not be detected
either on stained gels of the 40-kDa antigen preparation or on
immunoblots of sera from sheep immunized with the 40-kDa
antigen preparation; such blots showed that only the 40-kDa
antigen was immunogenic (Fig. 2c). The contaminating bands
of <20 kDa visible in Fig. 2a, lane 3, were not immunogenic
when reacted with ASC probes (Fig. 2b) or with the sheep sera
(Fig. 2c). Six field isolates of C. pseudotuberculosis were shown
by immunoblotting experiments using ASC probes to also
contain the 40-kDa antigen (not shown).
Immune response to vaccination. Antibody titers of vacci-
TABLE 2. Titers in ASC probes" assayed by ELISA using whole-
cell and supernatant antigens
ELISA OD450
Origin of prohe Mitogen" 7-Day infection 14-Day infection
Whole Supernatant Whole Supernatant
cells antigens cells antigens
Challenged site PWM 0.079 0.034 0.092
LPS-D.SO4 0.151 0.065 t).105
Unchallenged PWM 0.140 0.048 0.067
site LPS-D.SO4 0.225 0.105 0.092
aASC probes were assayed at a 1/2 dilution.
"PWM, pokeweed mitogen.
nated sheep were measured by ELISA prior to infectious
challenge. Following primary and secondary vaccinations,
there was no difference in the group mean titers to the 40-kDa
antigen of sheep vaccinated with 5 ,ug of antigen (mean OD450
+ standard deviation = 0.318 + 0.166) compared with those of
sheep vaccinated with 100 ,ug of antigen (0.294 + 0.177).
Unvaccinated control sheep had significantly lower titers
(OD450 ± standard deviation = 0.075 + 0.122). Immunoblots
of sera from vaccinated sheep showed a strong response only
to the 40-kDa antigen (Fig. 2c).
Lesion scores. Infectious challenge with C. pseudotuberculo-
sis in the right hock 3 months prior to slaughter resulted in a
satisfactory level of infection, with 55% of control animals
developing lesions (Table 3).
Vaccination with 100-[wg doses of the 40-kDa antigen prep-
aration had a major effect on development of disease and
significantly reduced the infection rate from 55 to 5% (z test
value = 3.58, P < 0.002). Vaccination with 5-pLg doses of
antigen resulted in a small and insignificant reduction in the
infection rate to 41% (z test value = 1.25, P < 0.20).
Lung lesion scores provided the best measure of protection
achieved by vaccination with the 40-kDa antigen preparation.
More control sheep developed lung lesions than developed
lesions in lymph nodes, and those infected often had multiple
lesions in the lung (Table 3). Of the nine control sheep with
lung lesions, five did not have lesions in popliteal or other
lymph nodes.
Vaccination with 100-pLg doses of the 40-kDa antigen prep-
aration gave a significant 98% reduction in the incidence of
lung lesions (unpaired t test, t = 2.046, 41 df, P < 0.025; Table
3). The 5-pLg dose sheep showed a 79% reduction (t = 1.932, 42
df, P < 0.05). Vaccination also tended to reduce the lesion size,
with control group lesions having an average diameter of 67
mm and lesions in the 5- and 100-p.g dose groups having
average diameters of 37 and 26 mm, respectively. These data
show that in addition to preventing actual infection as reflected
in the number of lung lesions, vaccination with the 40-kDa
antigen preparation also limited the development of estab-
lished foci of infection.
The right popliteal lymph node drains the site of infectious
challenge; however, only 5 of the 22 right popliteal lymph
nodes from control sheep showed lesion development. Vacci-
nation of sheep with the 40-kDa antigen preparation at 5- and
0.408 100-,ug dose levels prevented infection in the right popliteal
0.333 lymph node (Table 3). The only other lymph nodes with lesion
0.040 development were the prescapular nodes. Three control sheep
0.072 and one sheep in the 5-p.g dose group had lesions in the
prescapular lymph nodes. No sheep in the 100-,ug dose group
had detectable lesions in peripheral or visceral lymph nodes.
2566 WALKER ET AL.
DISCUSSION
Antibody probes produced by ASC taken from the lymph
nodes draining the site of infection with C. pseudotuberculosis
showed restricted antigenic reactivity on immunoblots, partic-
ularly compared with the diverse spectrum of antigens identi-
fied by antibodies from serum present in the sheep at the time
of sampling of lymph nodes. This restricted response identified
a novel 40-kDa antigen from C. pseudotuberculosis. The kinet-
ics of the serum immune response of naive sheep also high-
lighted the 40-kDa antigen as one of the predominant antigens
recognized (Fig. la) at day 7 postinfection. By day 14, when
ASC from lymph nodes were obtained, the serum response was
heterogeneous and the 40-kDa antigen response was no longer
obvious over other antigens on immunoblots. Taken together,
the restricted response of ASC probes and the more hetero-
geneous response in serum show that the greater part of serum
antibody to an infectious challenge is not being synthesized in
the draining lymph nodes. The pool of antibodies in serum
specific for antigens other than the 40-kDa antigen are pre-
sumably being synthesized in other lymphoid tissues, like the
spleen.
Vaccination of sheep with 100 ,ug of the 40-kDa antigen
preparation gave up to 82% reduction in the proportion of
infected sheep. This level of protection compares favorably
with the levels of protection achieved with currently available
commercial vaccines (5, 7). The lower dose level of 5 ,ug of
40-kDa antigen preparation did not provide a significant level
of protection in terms of infected sheep but did significantly
reduce the incidence of lung lesions. This lower level of
protection was not reflected in lower group mean titers of
antibody, with group means of the 5- and 100-,ug dose groups
not being significantly different. In addition, there was no
discernible relationship between lung lesion scores and anti-
body titers. These data strongly implicate cellular immunity as
the primary mechanism of immune rejection of infection.
Immunoblot analysis of sera from sheep vaccinated with the
40-kDa antigen preparation showed a strong antibody re-
sponse restricted to the 40-kDa protein (Fig. 2c). The lower-
molecular-mass components of approximately 20 and less than
15 kDa in the 40-kDa antigen preparation were not immuno-
genic. These were assumed to be derived from medium
components of bovine origin. There was no response detected
to the 31-kDa PLD toxin. We have not analyzed the cellular
immune response to vaccination. The significant protection
achieved by vaccination suggests that the 40-kDa antigen plays
a major role in protection against CLA. To date, the only other
recognized protective antigen has been PLD, which is also a
major determinant of virulence (5, 13, 14, 17).
Currently, vaccines against CLA are based on culture super-
natants containing inactivated PLD toxin (5). Protection is
attributed to responses to the PLD toxoid, and vaccination
with purified inactivated PLD has achieved approximately 79%
reduction in infection, similar to the 75 to 80% levels of
protection achieved with the crude complex commercial vac-
cines (5-7). In other studies, we have found that antibody
responses of sheep vaccinated with one particular commer-
cially available vaccine, Glanvac (CSL Ltd), showed low and
inconsistent titers to the 40-kDa antigen. Variability in cellular
or humoral responses to the 40-kDa antigen may provide an
immunological basis for the intermediate levels of protection
achieved with commercial and experimental vaccines (1, 2, 5,
7). As discussed previously, cell-mediated immunity may play a
role in protection against intracellular infection with C.
pseudotuberculosis. The absence of a direct correlation be-
tween antibody titers to the 40-kDa antigen and disease status
INFEc-r. IMMUN.
indicates either that antibody to the 40-kDa antigen is involved
in protection through more indirect mechanisms, for example,
antibody-dependent cell-mediated cytotoxicity, or that anti-
body is not involved in protection. Cytotoxic T-cell responses
to the 40-kDa antigen may mediate protection against CLA, as
has been shown in other intracellular bacterial infections.
There is also the possibility that T-cell responses to antigens
that do not elicit an antibody response mediate protection.
Further studies are required to elucidate the contributions of
humoral and cellular immunity in protection against ovine
CLA.
The function and location of the 40-kDa antigen are not yet
known. The antigen is present at significant levels in culture
supernatant and in detergent extracts of whole cells and is
probably noncovalently attached to the cell or is actively
secreted. The higher reactivity in ELISAs of ASC probes
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specific for the 40-kDa antigen with supernatant antigens than
with cellular antigens (Table 2) supports the concept that the
40-kDa antigen is a secreted protein. Studies are continuing to
clone the gene for and express the 40-kDa antigen and
determine its role in immunity and pathogenesis. The identi-
fication of a major protective antigen of C. pseudotuberculosis
other than PLD should contribute to the development of an
efficacious defined vaccine against CLA.
Studies of serum antibody responses following natural infec-
tion and vaccination have highlighted a wide range of antigens
from C. pseudotuberculosis (8, 9). These previous studies
showed a number of antigens in the 40-kDa region on immu-
noblots, but the strong response to a very wide range of
antigens prevented the selection of any particular antigen as
being important in immunity. Identification of protective anti-
gens by immunoscreening using serum antibodies has yielded
important candidate vaccine antigens, for example, paramyo-
sin of Schistosoma mansoni (18). In previous studies of CLA
(9) and in mycobacterial infections, which like CLA are caused
by facultative intracellular pathogens, effective vaccine anti-
gens have not yet been identified. We have shown here that
ASC probes generated by ASC taken from lymph nodes
draining the site of a challenge infection with C. pseudotuber-
culosis identified a highly protective antigen. This study dem-
onstrates that generation of ASC probes from a site of
infection provides a rational approach to the identification of
candidate vaccine antigens which may have application to a
wide range of diseases of both humans and animals.
ACKNOWLEDGMENTS
This work was supported by the Meat Research Corp. (Australia).
We thank Vern Bowles for critically reviewing the manuscript,
Christine Kerr for typing, and Ken Snibson for photography.
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