Evaluation of the immunogenicity of the P97R1
adhesin of Mycoplasma hyopneumoniae as a
mucosal vaccine in mice
Austen Y. Chen,1Scott R. Fry,1Judy Forbes-Faulkner,2Grant Daggard1
and T. K. S. Mukkur1
T. K. S. Mukkur
1Department of Biological and Physical Sciences, University of Southern Queensland,
Toowoomba, Queensland, Australia
2Oonoonba Veterinary Laboratory, DPI, Townsville, Queensland, Australia
Received 14 March 2005
Accepted 24 February 2006
The immunogenicity of P97 adhesin repeat region R1 (P97R1) of Mycoplasma hyopneumoniae,
an important pathogenesis-associated region of P97, was evaluated in mice as a mucosal vaccine.
Mice were immunized orally with attenuated Salmonella typhimurium aroA strain CS332
harbouring a eukaryotic or prokaryotic expression vector encoding P97R1. Local and systemic
immune responses were analysed by ELISA on mouse sera, lung washes and splenocyte
supernatants following splenocyte stimulation with specific antigens in vitro. Although no
P97R1-specific antibody responses were detected in serum and lung washes, significant gamma
interferon was produced by P97R1-stimulated splenocytes from mice immunized orally with
S. typhimurium aroA harbouring either expression system, indicating induction of a cell-mediated
immune response. These results suggested that live bacterial vectors carrying DNA vaccines or
immunized with the vaccine carrier S. typhimurium aroA CS332 induced serum IgG, but not
mucosal IgA, against P97R1 or S. typhimurium aroA CS332 whole-cell lysate, emphasizing the
importance of assessing the suitability of attenuated S. typhimurium antigen-carrier delivery
vectors in the mouse model prior to their evaluation as potential vaccines in the target species,
which in this instance was pigs.
Porcine enzootic pneumonia (PEP) caused by Mycoplasma
hyopneumoniae is a chronic, non-fatal respiratory disease. It
affects pigs of all ages, resulting in slow growth and reduced
feed efficiency, and causes significant economical losses in
the pig industry worldwide (Sheldrake et al., 1991; Thacker
et al., 1998). PEP is often followed by secondary infections
by Porcine reproductive and respiratory syndrome virus and
opportunistic bacterial pathogens (Calsamiglia et al., 1999;
Sheldrake et al., 1991; Thacker et al., 1998, 1999). As current
market vaccines (bacterins prepared by chemical inactiva-
tion of whole-cell M. hyopneumoniae) do not protect pigs
completely from establishment of M. hyopneumoniae infec-
tion and/or secondary infection (Kristensen et al., 1981;
Maes et al., 1999; Murphy et al., 1993; Pallares et al., 2000;
P97 adhesin has probably been the most-studied and best-
defined potential protective antigen of M. hyopneumoniae
since it was identified as an important adhesin responsible
for the adherence of M. hyopneumoniae to respiratory
ciliated epithelial cells in swine (Zhang et al., 1995).
Furthermore, a repeat region of P97 called R1 (P97R1)
bindingsites (Hsu &Minion, 1998; Hsu etal.,1997) andhas
been reported to function independently from other P97
regions (Minion et al., 2000). P97R1 has been shown to
induce significant P97R1-specific serum IgG titres in both
mice and pigs (Chen et al., 2001), indicating that it is
immunogenic. However, the immunogenicity of P97R1 has
yet to be evaluated as a DNA vaccine. Evaluation of this
immunization strategy is important, as DNA vaccines have
been reported to induce both humoral and cell-mediated
an adhesin, generation of anti-P97R1 immune responses at
the mucosal site may facilitate the prevention of coloniza-
tion of M. hyopneumoniae on respiratory epithelial cells
be achieved by using live attenuated pathogens as antigen
carriers, such as aromatic-dependent Salmonella and
Abbreviations: AP, alkaline phosphatase; IFN-c, gamma interferon;
P97R1, P97 repeat region R1; PEP, porcine enzootic pneumonia.
The GenBank/EMBL/DDBJ accession number for the P97 sequence
of M. hyopneumoniae is AY957500.
46088 G 2006 SGM Printed in Great Britain 923
Journal of Medical Microbiology (2006), 55, 923–929
Shigella species, Mycobacterium bovis Bacille Calmette–
Gue ´rin and Listeria monocytogenes, to deliver DNA vaccine
plasmids with a view to generating mucosal antibodies
(Darji et al., 1997; Dietrich et al., 1998; Fennelly et al., 1999;
Paglia et al., 1998; Pasetti et al., 1999; Sizemore et al., 1995,
1997). The aim of this study was to evaluate humoral, local
and cell-mediated immune responses against P97R1 as a
bacterial DNA vaccine delivered orally via an attenuated
S. typhimurium aroA in a mouse model. As a comparison,
the immunogenicity of S. typhimurium aroA expressing
P97R1 as a recombinant protein was also investigated.
Bacterial strains. M. hyopneumoniae strain A518/1 was provided
by Ms J. Forbes-Faulkner (Oonoonba Veterinary Laboratory,
Queensland, Australia). r2m+S. typhimurium strain P9121 was
provided by Dr Bruce Stocker (Stanford University, California,
USA) and S. typhimurium aroA strain CS332 was obtained from
McMaster Laboratory, Sydney University Campus, CSIRO Division
of Animal Health, Australia.
Growth and isolation of genomic DNA of M. hyopneumo-
niae. M. hyopneumoniae was grown in modified Friis mycoplasma
broth as described by Friis (1975). The culture was grown at 37uC
at 100 r.p.m. until acidity (yellow–brown colour indicating a pH
change) was observed. Genomic DNA was extracted using a
Quantum Prep AquaPure Genomic DNA kit (Bio-Rad).
Construction of P97R1 expression plasmids. Based on the pub-
lished P97 gene sequence (GenBank accession no. U50901), the P97
gene was obtained by PCR from strain A518/1 genomic DNA using
The P97 PCR product was purified using the High Pure PCR
Product Purification kit (Roche) and cloned into the prokaryotic
expression vector pTrcHis2 (Invitrogen) according to the manufac-
turer’s instructions. The pTrcHis2-P97 plasmid was purified using
a QIAprep Spin Miniprep kit (Qiagen) and sequenced. The P97
sequence has been submitted to GenBank under accession no.
AY957500. P97R1 was PCR amplified from pTrcHis2-P97 and cloned
into pTrcHis2 using forward primer 59-GAAGGTAAAAGAGA-
TGTAAGTGAAAAGCCAGTATTAGTAGCAACTG-39, and into the
eukaryotic expression vector pcDNA3.1 (Invitrogen) using the same
primers except for the addition of a Kozak sequence (ATTATGG) to
the 59 end of the forward primer. The resultant plasmids, pTrcHis2-
P97R1 and pcDNA3.1-P97R1, were then purified and sequenced.
Expression of the P97R1 fusion protein in S. typhimurium
aroA and purification. The plasmid pTrcHis2-P97R1 purified
from Escherichia coli TOPO 10 cells was first electrotransformed
into r2m+S. typhimurium P9121; the purified plasmid was sub-
sequently electrotransformed into S. typhimurium aroA CS332 using
a 1 mm cuvette at 129 V, 25 uFD and 1?7 kV (Electro Cell
Manipulator ECM600 Electroporation System). SDS-PAGE was
performed with a 12% gel using the Bio-Rad Mini Protein II Cell
apparatus according to the manufacturer’s instructions. Western
blot analysis was carried out according to the protocol for the use
of anti-His(C-terminal)–HRP antibody (Invitrogen). The P97R1
fusion protein was visualized using 5 ml TMB stabilized substrate
(Promega). P97R1 was purified under denaturing conditions using
a QIAexpress kit for high-level expression and purification of
66His-tagged proteins (Qiagen). Protein concentration was deter-
mined using the Coomassie Plus Protein Assay (Pierce) according to
the manufacturer’s instructions.
Transient expression of the P97R1 fusion protein in mam-
malian cells. Transient transfection of COS-7 cells with pcDNA3.1-
P97R1 was performed using Lipofectamine 2000 (Life Technologies)
according to the manufacturer’s instructions. Detection of the P97R1
fusion protein was performed using a WesternBreeze Novex Chromo-
genic Western Blot Immunodetection kit (Invitrogen) following the
manufacturer’s instructions. The antibody used to detect P97R1 was
alkaline phosphatase (AP)-conjugated antibody anti-V5 (Invitrogen),
as P97R1 was fused to the C-terminal V5 epitope on the vector.
In vitro invasion assay for S. typhimurium aroA CS332. An
in vitro invasion assay for S. typhimurium aroA CS332 into the
human embryonic intestinal cell line HI-407 was carried out as
described by Walker et al. (1992). Both S. typhimurium aroA and
S. typhimurium aroA carrying heterologous plasmids were tested
for their invasiveness.
Immunization of mice with S. typhimurium aroA CS332 live
vector vaccines. Preparation of live Salmonella carrier vaccines
and mouse immunization were carried out as described by Fagan
et al. (1997). The vaccine groups comprised: (i) S. typhimurium
aroA CS332 carrying pTrcHis2-P97R1 [Salmonella(pTrcP97R1)]; (ii)
Salmonella(pcDNA3.1-P97R1); (iii) Salmonella(pTrcHis2 vector);
(iv) Salmonella(pcDNA3.1 vector); and (v) PBS (pH 7?2). Each
group contained five mice. On day 0, 6–8-week-old female BALB/c
mice were each immunized orally with 0?25 ml Salmonella suspen-
sion (26108c.f.u.) for groups 1–4. On day 28, mice were given a
second dose of 0?25 ml Salmonella suspension (36108c.f.u.) orally.
For the PBS group, each mouse was given 0?25 ml PBS orally on
days 0 and 28. On day 56, all mice were sacrificed. The molecular
microbiological manipulations carried out in this investigation were
approved by the Institutional Biosafety Committee and the experi-
ments carried out in mice were approved by the Animal Ethics
Committee of the University of Southern Queensland.
Estimation of antibody responses. Sera and lung washes col-
lected as described elsewhere (Fagan et al., 1997) were stored at
220uC. S. typhimurium CS332 whole-cell lysate was prepared by
adding formalin to the cell culture to a final concentration of 1%,
incubating at 37uC for 2–4 h and checking for sterility. The culture
was pelleted by centrifuging for 10 min at 2000 g and resuspended
in bicarbonate buffer (30 mM sodium carbonate, 20 mM sodium
hydrogen carbonate, pH 9?6). The suspension was sonicated
(5620 s on/off cycles) at a 60% duty cycle, adjusted to an OD600of
0?4 and stored at 220uC. ELISA was performed according to
Desolme et al. (2000) with the following modifications. Microtitre
plates were coated with either 50 ml purified P97R1 (5 mg ml21) or
50 ml S. typhimurium whole-cell lysate. The secondary antibody
used was horseradish peroxidase-conjugated goat anti-mouse IgG or
IgA (Sigma). The variation between plates was normalized against
positive control sera by multiplying the test sample titre by the ratio
of the mean positive control titre to the positive control titre for a
particular plate (Mukkur et al., 1995). The antigen-specific antibody
titres were determined as the reciprocal of the dilution at a cut-off
point of 2?5 times the absorbance of the lowest dilution of the sera
from non-immunized mice.
Determination of cytokine production. The spleens from each
vaccine group were removed aseptically and pooled into 5 ml
Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing
100 U penicillin/streptomycin ml21and 50 mM 2-mercaptoethanol
on ice. Splenocytes were isolated using a cell constrainer (Becton
Dickinson) and centrifuged at 800 g for 10 min at room tempera-
ture. The supernatant was removed, the cell pellet was resuspended
924Journal of Medical Microbiology 55
A. Y. Chen and others
in 10 ml of the above medium and a 10 ml aliquot was removed for
cell counting before the solution was centrifuged again as described
above. The washed cell pellet was resuspended in 10 ml DMEM con-
taining 100 U penicillin/streptomycin ml21, 10% fetal bovine serum
and 50 mM 2-mercaptoethanol and diluted to 56106cells ml21.
Cells (2 ml) were added to each well of a 24-well plate. The antigen
stimulant (purified P97R1 fusion protein) was then added at a
concentration of 1 mg ml21to wells in duplicate. The plate was
incubated at 37uC in 5% CO2 for 72 h. The splenocyte culture
supernatants were collected and stored at 220 or at 270uC for
long-term storage. Cytokine production in the splenocyte super-
natants was measured using a Mouse Cytokine ELISA kit (Pierce)
according to the manufacturer’s instructions.
Statistical analysis. Values for antibody titres and cytokine
production were compared using one-way ANOVA. Results were
considered significant for values of P ¡0?05.
Analysis of the P97R1 sequence
DNA sequencing analysis showed that the P97R1 fragment
from M. hyopneumoniae strain A518/1 was 285 bp and
contained 11 (A/T)KP(E/V)(A/T) amino acid repeats,
which are characteristic of P97 (Wilton et al., 1998), and
one VKPVA sequence (Fig. 1).
Expression of P97R1 in S. typhimurium aroA
and protein purification
Expression of P97R1 in S. typhimurium aroA CS332 was
observed by Western blotting following IPTG induction or
simply by growing the bacteria overnight in Terrific broth
containing 100 mg ampicillin ml21. The P97R1 fusion
protein was detected by anti-His(C-terminal)–HRP anti-
body in the Western blot. The size of P97R1 was estimated
to be ~17 kDa including the 3–4 kDa C-terminal tag from
the vector (Fig. 2).
Expression of P97R1 in mammalian cells
Plasmid pcDNA3.1-P97R1 purified from S. typhimurium
aroA CS332 was transfected into COS-7 cells. Expression of
the P97R1 fusion protein (fused to the C-terminal V5
epitope) was examined by Western blotting using anti-V5–
AP antibody (Fig. 3). The size of the P97R1 fusion protein
expressed in COS-7 cells was similar to that expressed in
In vitro invasiveness of S. typhimurium aroA
An investigation was carried out to test the effect of the
presence of recombinant plasmids on the in vitro invasive-
ness of S. typhimurium aroA CS332. The results showed that
Fig. 1. DNA and amino acid sequences of the P97R1 frag-
ment from M. hyopneumoniae strain A518/1.
Fig. 2. Expression of the P97R1 fusion protein in S. typhimur-
2, pTrcHis2 vector; 3, induction of P97R1 expression by IPTG;
4, expression of P97R1 in S. typhimurium aroA CS332 in
Terrific broth containing 100 mg ampicillin ml”1after overnight
incubation. P97R1 was detected using anti-His(C-terminal)–
HRP antibody in the Western blot.
BenchMark Protein Ladder;
Fig. 3. Expression of P97R1 fusion protein in COS-7 cells by
Western blot. Lanes: 1, BenchMark Protein Ladder; 2, COS-7
cells transfected with pcDNA3.1-P97R1; 3, COS-7 cells trans-
fected with pcDNA3.1 vector. Detection of the P97R1 fusion
protein was carried out using a WesternBreeze Novex Chromo-
genic Western Blot Immunodetection kit (Invitrogen) using anti-
V5–AP antibody (Invitrogen).
M. hyopneumoniae P97R1 as a mucosal vaccine
the ability of S. typhimurium aroA CS332 to invade the
human intestinal cell line HI-407 was not affected by the
presence of either plasmid vector or recombinant plasmid
Analysis of antibody response by ELISA
Sera and lung washes were analysed for antigen-specific
antibody responses. No serum or mucosal antibody (IgG or
IgA) responses against the P97R1 fusion protein were
detected in mice immunized with Salmonella(pTrcP97R1)
or Salmonella(pcDNA3.1-P97R1) when compared with
control groups vaccinated with PBS, Salmonella(pTrcHis2
vector) or Salmonella(pcDNA3.1 vector). All of the groups,
except for the PBS control group, induced serum IgG
(Table 2). Although each group was immunized with the
same amount of S. typhimurium, the magnitude of the IgG
responses among these groups varied. However, no mucosal
IgA against S. typhimurium aroA whole-cell lysate was
detected in the lung washes of any of these groups.
Measurement of cytokine levels by ELISA
Splenocytes isolated from pooled spleens from each group
of five mice were stimulated with the P97R1 fusion protein
and cultured in duplicate in vitro for 3 days. The super-
natants were removed and used for determination of
cytokine production using a mouse cytokine ELISA. Both
the Salmonella(pTrcP97R1) and Salmonella(pcDNA3.1-
gamma interferon (IFN-c) but not interleukin-4 when
compared with the control groups (Fig. 4).
Studies of P97R1 in different strains have shown that they
contain different numbers of repeats of the amino acid
sequence (A/T)KP(E/V)(A/T) arranged in tandem (Wilton
et al., 1998). In our study, analysis of M. hyopneumoniae
strain A518/1 revealed that P97R1 contains 11 (A/T)KP(E/
V)(A/T) repeats and one VKPVA sequence. The presence of
VKPVA is not common, but has also been found in strain
Sue (Wilton et al., 1998). Our results confirmed the vari-
ability of both the number and the composition of P97R1
repeats in M. hyopneumoniae.
Table 1. Evaluation of the in vitro invasiveness of S. typhi-
murium aroA CS332
Results are given as the mean number of viable bacteria (c.f.u.)
per well±SEM from duplicate wells and counted by plating in
S. typhimurium aroA CS332
Table 2. Estimated antibody levels against P97R1 and the antigen carrier S. typhimurium aroA CS332 in vaccinated versus
Results are given as the mean titre±SD from the five mice in each group.
Experimental groups Antibody titres against P97R1Antibody titres against Salmonella
Serum IgGSecretory IgA Serum IgGSecretory IgA
Salmonella(pTrcHis2 vector) (prokaryotic plasmid control)
Salmonella(pcDNA3.1 vector) (eukaryotic plasmid control)
Fig. 4. In vitro production of IFN-c by P97R1-stimulated sple-
nocytes. Pbs, Mice vaccinated with PBS; Trv, mice vaccinated
with Salmonella(pTrcHis2 vector); Pcv, mice vaccinated with
with Salmonella(pTrcP97R1); PcP97R1, mice vaccinated with
mean±SEM from duplicate culture wells pooled from five mice.
*, Significant difference in IFN-c production (P <0?05) com-
pared with the control groups (Pbs, Trv and Pcv).
Results areshown as the
926Journal of Medical Microbiology 55
A. Y. Chen and others
In this present study, the immunogenicity of S. typhimur-
ium harbouring P97R1 under the control of a eukaryotic
or prokaryotic expression system was investigated in mice.
Serum and local immune responses were compared between
Salmonella(pTrcP97R1) and Salmonella(pcDNA3.1-P97R1).
Serum ELISA results showed that neither Salmonella-
(pTrcP97R1) nor Salmonella(pcDNA3.1-P97R1) induced
measurable P97R1-specific IgG, IgM or IgA antibodies
compared with the control groups. Similarly, no IgG or IgA
against P97R1 was detected in the lung washes of mice
vaccinated with either Salmonella(pTrcHis2-P97R1) or
Salmonella(pcDNA3.1-P97R1). In contrast, a previous
study (Fagan et al., 1997) reported that mice immunized
orally with S. typhimurium aroA strain SL3261 expressing
the M. hyopneumoniae NrdF fragment produced significant
NrdF-specific IgA in the lungs, but failed to elicit significant
levels of IgG, IgM or IgA in the serum. The failure to induce
serum antigen-specific antibody responses in both studies is
unclear, but possibly is due to the fact that the nature of the
antigen may have had an impact on the type of immune
response, as suggested by Boyle et al. (1997). However, the
failure to induce P97R1-specific mucosal IgA in our study
may have been due to the use of S. typhimurium aroA strain
CS332 rather than strain SL3261. In all of the groups
against S. typhimurium was detected and no lung IgA was
found. This observation is supported indirectly by previous
studies (Mukkur & Walker, 1992; Mukkur et al., 1995) in
which mice and sheep vaccinated orally with strain CS332
did not induce detectable mucosal IgA in intestinal washes,
despite the fact that strain CS332 was invasive in vivo, as
judged by the production of acute enteritis of jejunum and
ileum accompanied by multiple focal hepatitis, albeit low
grade, although the latter was not observed in sheep (Begg,
1990). To ensure that S. typhimurium aroA CS332 was
invasive in this study, an in vitro invasion assay was
performed, which showed that S. typhimurium invaded
intestinal HI-407 cells relatively efficiently and that there
was no significant difference in invasiveness between S.
typhimurium and S. typhimurium carrying heterologous
plasmids. Thus it was apparent that other factors may have
contributed tothevariability in immunogenicity inmucosal
vaccination models. The animal and the strain used have
been shown to affect the extent and quality of the immune
response (Begg, 1990; Dunstan et al., 1998; Soo et al., 1998;
Valentine et al., 1998). For example, it has been shown that
Ityrmice vaccinated orally with S. typhimurium expressing
Leishmania gp63 antigen induce Th1 responses, whereas Itys
mice induce Th2 responses when immunized with the
same vaccine (Soo et al., 1998). The gene selected for
insertional mutagenesis of the bacterial live vector has also
been shown to influence the extent and quality of the
immune response. Studies comparing three S. typhimurium
MudJ lacZ-inactivated mutants (CL288, CL401 and CL554)
and a prototype aroA mutant showed that the former
mutant strains elicited higher serum IgG and/or mucosal
IgA titres than the prototype aroA mutant strain (Valentine
et al., 1998), presumably because they were able to persist in
the murine tissues for longer periods than the aroA mutant
Another possible explanation for the poor induction of
local mucosal IgA by mucosal immunization could be the
observation that cells presenting the encoded antigen after
DNA transfer or vaccine carrier have been found to persist
mainly in spleen and lymph nodes for a considerable
length of time. This may be one reason for the immune
response being Th1-oriented, with very little local antibody
production (Darji et al., 2000; Dunstan et al., 1998;
Reinhardt et al., 2001; Roberts et al., 1998; Woo et al.,
2001; Zhang et al., 2001). Similarly, in our study, although
Salmonella(pTrcHis2-P97R1) and Salmonella(pcDNA3.1-
P97R1) did not induce P97R1-specific serum IgG or
mucosal IgA, both groups induced significant P97R1-
specific IFN-c production compared with the control
groups. This finding is supported by other studies in
which Salmonella-based DNA vaccines or Salmonella
expressing heterologous antigens were reported to induce
a predominately Th1 response (Darji et al., 2000; Dunstan
et al., 1998; Reinhardt et al., 2001; Roberts et al., 1998; Woo
et al., 2001; Zheng et al., 2001). M. hyopneumoniae potential
antigens NrdF and P97 used as vaccines have also been
shown to induce a presumed antigen-specific Th1 response,
although only in pigs, considered to be responsible for
higher mean daily weight gains and reduced lung lesion
scores (Fagan et al., 2001; Shimoji et al., 2003). In contrast,
pigs vaccinated intramuscularly with the P97 fusion protein
induced a strong P97-specific serum antibody response, but
failed to protect pigs against M. hyopneumoniae challenge
(King et al., 1997). In fact, serum antibodies induced by
commercial M. hyopneumoniae vaccines do not correlate
with protection against M. hyopneumoniae challenge (Maes
et al., 1999; Pallares et al., 2000; Thacker et al., 1998). Thus
it has been suggested that serum antibodies may not play
a protective role in PEP; instead cell-mediated immune
responses may be important in mediating protection
against PEP (Darji et al., 1997; Shimoji et al., 2003;
Thacker et al., 2000).
In our investigation, we showed that P97R1 adhesin pre-
sented to the mouse as either a bacterium-based DNA
vaccine or a bacterium expressing the heterologous P97R1
antigen induced P97R1-specific Th1 responses in mice but
without any anti-P97R1 mucosal antibody responses.
Whether the failure of S. typhimurium aroA CS332 to
induce mucosal antibody responses in this investigation
was associated with the characteristics of the insertional
mutation in the antigen-carrier S. typhimurium strain
warrants further investigation.
We thank Scott Kershaw, Tracy Doan and David Park for technical
assistance and Dr Ashley Plank for advice on statistical analysis of the
data. This work was supported by Project Team Research Program
Grant 179535 from the University of Southern Queensland and Delta
M. hyopneumoniae P97R1 as a mucosal vaccine
Begg, A. P. (1990). Studies on aromatic-dependent Salmonella species
as vaccines in animals. PhD thesis, University of Sydney, Australia.
Boyle, J. S., Koniaras, C. & Lew, A. M. (1997). Influence of cellular
location of expressed antigen on the efficacy of DNA vaccination:
cytotoxic T lymphocyte and antibody responses are suboptimal when
antigen is cytoplasmic after intramuscular DNA immunization. Int
Immunol 9, 1897–1906.
Calsamiglia, M., Pijoan, C. & Trigo, A. (1999). Application of a nested
polymerase chain reaction assay to detect Mycoplasma hyopneumo-
niae from nasal swabs. J Vet Diagn Invest 11, 246–251.
Chen, J.-R., Liao, C.-W., Mao, S. J. & Weng, C.-N. (2001). A recom-
binant chimera composed of repeat region RR1 of Mycoplasma
hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evalua-
tion of specific IgG response in mice and pigs. Vet Microbiol 80,
Darji, A., Guzma ´n, C. A., Gerstel, B., Wachholz, P., Timmis, K. N.,
Wehland, J., Chakraborty, T. & Weiss, S. (1997). Oral somatic
transgene vaccination using attenuated S. typhimurium. Cell 91,
Darji, A., zur Lage, S., Garbe, A. I., Chakraborty, T. & Weiss, S.
(2000). Oral delivery of DNA vaccines using attenuated Salmonella
typhimurium as carrier. FEMS Immunol Med Microbiol 27, 341–349.
Desolme, B., Me ´ve ´lec, M. N., Buzoni-Gatel, D. & Bout, D. (2000).
Induction of protective immunity against toxoplasmosis in mice by
DNA immunization with a plasmid encoding Toxoplasma gondii
GRA4 gene. Vaccine 18, 2512–2521.
Dietrich, G., Bubert, A., Gentschev, I. & 7 other authors (1998).
Delivery of antigen-encoding plasmid DNA into the cytosol of
macrophages by attenuated suicide Listeria monocytogenes. Nat
Biotechnol 16, 181–185.
Dunstan, S. J., Simmons, C. P. & Strugnell, R. A. (1998). Com-
parison of the abilities of different attenuated Salmonella typhimurium
strains to elicit humoral immune responses against a heterologous
antigen. Infect Immun 66, 732–740.
Fagan, P. K., Djordjevic, S. P., Chin, J., Eamens, G. J. & Walker, M. J.
(1997). Oral immunization of mice with attenuated Salmonella
typhimurium aroA expressing a recombinant Mycoplasma hyopneu-
moniae antigen (NrdF). Infect Immun 65, 2502–2507.
Fagan, P. K., Walker, M. J., Chin, J., Eamens, G. J. & Djordjevic, S. P.
(2001). Oral immunization of swine with attenuated Salmonella
typhimurium aro A SL3261 expressing a recombinant antigen of
Mycoplasma hyopneumoniae (NrdF) primes the immune system for a
NrdF specific secretory IgA response in the lungs. Microb Pathog
Fennelly, G. J., Khan, S. A., Abadi, M. A., Wild, T. F. & Bloom, B. R.
(1999). Mucosal DNA vaccine immunization against measles with a
highly attenuated Shigella flexneri vector. J Immunol 162, 1603–1610.
Friis, N. F. (1975). Mycoplasma of the swine – a review. Nord Vet
Med 27, 329–336.
Gurunathan, S., Klinman, D. M. & Seder, R. A. (2000). DNA
vaccines: immunology, application, and optimization. Annu Rev
Immunol 18, 927–974.
Hsu, T. & Minion, F. C. (1998). Identification of the cilium binding
epitope of the Mycoplasma hyopneumoniae P97 adhesin. Infect
Immun 66, 4762–4766.
Hsu, T., Artiushin, S. & Minion, F. C. (1997). Cloning and functional
analysis of the P97 swine cilium adhesin gene of Mycoplasma
hyopneumoniae. J Bacteriol 179, 1317–1323.
King, K. W., Faulds, D. H., Rosey, E. L. & Yancey, R. J., Jr (1997).
Characterization of the gene encoding Mhp1 from Mycoplasma
hyopneumoniae and examination of Mhp1’s vaccine potential.
Vaccine 15, 25–35.
Kristensen, B., Paroz, P., Nicolet, J., Wanner, M. & de Weck, A. L.
(1981). Cell-mediated and humoral immune response in swine after
vaccination and natural infection with Mycoplasma hyopneumoniae.
Am J Vet Res 42, 784–788.
Maes, D., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens,
B., Verbeke, W., Viaene, J. & de Kruif, A. (1999). Effect of vaccination
against Mycoplasma hyopneumoniae in pig herds with an all-in/all-
out production system. Vaccine 17, 1024–1034.
Minion, F. C., Adams, C. & Hsu, T. (2000). R1 region of P97 mediates
adherence of Mycoplasma hyopneumoniae to swine cilia. Infect
Immun 68, 3056–3060.
Mukkur, T. K. & Walker, K. H. (1992). Development and duration of
protection against salmonellosis in mice and sheep immunised with
live aromatic-dependent Salmonella typhimurium. Res Vet Sci 52,
Mukkur, T. K., Walker, K. H., Baker, P. & Jones, D. (1995). Systemic
and mucosal intestinal antibody response of sheep immunized with
aromatic-dependent live or killed Salmonella typhimurium. Comp
Immunol Microbiol Infect Dis 18, 27–39.
Murphy, D., Van Alstine, W. G., Clark, L. K., Albregts, S. & Knox, K.
(1993). Aerosol vaccination of pigs against Mycoplasma hyopneu-
moniae infection. Am J Vet Res 54, 1874–1880.
Paglia, P., Medina, E., Arioli, I., Guzman, C. A. & Colombo, M. P.
(1998). Gene transfer in dendritic cells, induced by oral DNA
vaccination with Salmonella typhimurium, results in protective
immunity against a murine fibrosarcoma. Blood 92, 3172–3176.
Pallares, F. J., Berrocal, F., Sanchez, A., Oliva, J. E., Martinez, J. S. &
Munoz, A. (2000). Comparison of two different treatments against
swine enzootic pneumonia in three sites production system. In
Proceedings of the 16th International Pig Veterinary Society Congress,
p. 502. Melbourne, Australia.
Pasetti, M. F., Anderson, R. J., Noriega, F. R., Levine, M. M. & Sztein,
M. B. (1999). Attenuated DguaBA Salmonella typhi vaccine strain
CVD 915 as a live vector utilizing prokaryotic or eukaryotic
expression systems to deliver foreign antigens and elicit immune
responses. Clin Immunol 92, 76–89.
Reinhardt, R. L., Khoruts, A., Merica, R., Zell, T. & Jenkins, M. K.
(2001). Visualizing the generation of memory CD4 T cells in the
whole body. Nature 410, 101–105.
Roberts, M., Li, J., Bacon, A. & Chatfield, S. (1998). Oral vaccination
against tetanus: comparison of the immunogenicities of Salmonella
strains expressing fragment C from the nirB and htrA promoters.
Infect Immun 66, 3080–3087.
Sheldrake, R. F., Gardner, I. A., Saunders, M. M. & Romalis, L. F.
(1991). Intraperitoneal vaccination of pigs to control Mycoplasma
hyopneumoniae. Res Vet Sci 51, 285–291.
Sheldrake, R. F., Romalis, L. F. & Saunders, M. M. (1993). Serum
and mucosal antibody responses against Mycoplasma hyopneumoniae
following intraperitoneal vaccination and challenge of pigs with M.
hyopneumoniae. Res Vet Sci 55, 371–376.
Shimoji, Y., Oishi, E., Muneta, Y., Nosaka, H. & Mori, Y. (2003).
Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19
expressing a recombinant protein of Mycoplasma hyopneumoniae P97
Sizemore, D. R., Branstrom, A. A. & Sadoff, J. C. (1995). Attenuated
Shigella as a DNA delivery vehicle for DNA-mediated immunization.
Science 270, 299–302.
Sizemore, D. R., Branstrom, A. A. & Sadoff, J. C. (1997). Attenuated
bacteria as a DNA delivery vehicle for DNA-mediated immunization.
Vaccine 15, 804–807.
928Journal of Medical Microbiology 55
A. Y. Chen and others
Soo, S.-S., Villarreal-Ramos, B., Khan, C. M., Hormaeche, C. E. & Download full-text
Blackwell, J. M. (1998). Genetic control of immune response to
recombinant antigens carried by an attenuated Salmonella typhimur-
ium vaccine strain: Nramp1 influences T-helper subset responses
and protection against leishmanial challenge. Infect Immun 66,
Thacker, E. L., Thacker, B. J., Boettcher, T. B. & Jayappa, H. (1998).
Comparison of antibody production, lymphocyte stimulation and
production induced by four commercial Mycoplasma hyopneumoniae
bacterins. J Swine Health Prod 6, 107–112.
Thacker, E. L., Halbur, P. G., Ross, R. F., Thanawongnuwech, R. &
Thacker, B. J. (1999). Mycoplasma hyopneumoniae potentiation of
porcine reproductive and respiratory syndrome virus-induced
pneumonia. J Clin Microbiol 37, 620–627.
Thacker, E. L., Thacker, B. J., Kuhn, M., Hawkins, P. A. & Waters, W.
R. (2000). Evaluation of local and systemic immune responses
induced by intramuscular injection of a Mycoplasma hyopneumoniae
bacterin to pigs. Am J Vet Res 61, 1384–1389.
Valentine, P. J., Devore, B. P. & Heffron, F. (1998). Identification of
three highly attenuated Salmonella typhimurium mutants that are
more immunogenic and protective in mice than a prototypical aroA
mutant. Infect Immun 66, 3378–3383.
Walker, M. J., Rohde, M., Timmis, K. N. & Guzman, C. A. (1992).
Specific lung and mucosal immune responses after oral immuniza-
tion of mice with Salmonella typhimurium aroA, Salmonella typhi
Ty21a, and invasive Escherichia coli expressing recombinant pertussis
toxin S1 subunit. Infect Immun 60, 4260–4268.
Wilton, J. L., Scarman, A. L., Walker, M. J. & Djordjevic, S. P. (1998).
Reiterated repeat region variability in the ciliary adhesin gene of
Mycoplasma hyopneumoniae. Microbiology 144, 1931–1943.
Woo, P. C. Y., Wong, L.-P., Zheng, B.-J. & Yuen, K.-Y. (2001). Unique
immunogenicity of hepatitis B virus DNA vaccine presented by live-
attenuated Salmonella typhimurium. Vaccine 19, 2945–2954.
Zhang, Q., Young, T. F. & Ross, R. F. (1995). Identification and
characterization of a Mycoplasma hyopneumoniae adhesin. Infect
Immun 63, 1013–1019.
Zhang, Y., Taylor, M. G., Johansen, M. V. & Bickle, Q. D. (2001).
Vaccination of mice with a cocktail DNA vaccine induces a Th1-type
immune response and partial protection against Schistosoma
japonicum infection. Vaccine 20, 724–730.
Zheng, B., Woo, P. C. Y., Ng, M.-H., Tsoi, H.-W., Wong, L.-P. & Yuen,
K.-Y. (2001). A crucial role of macrophages in the immune responses
to oral DNA vaccination against hepatitis B virus in a murine model.
Vaccine 20, 140–147.
M. hyopneumoniae P97R1 as a mucosal vaccine