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The Veterinary Journal xxx (2008) xxx–xxx
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Reduced lung lesions in pigs challenged 25 weeks after

the administration of a single dose of Mycoplasma
hyopneumoniae vaccine at approximately 1 week of age
S.C. Reynolds a,*, L.B. St Aubin b, L.G. Sabbadini b, J. Kula c, J. Vogelaar d,
P. Runnels b, A.R. Peters e
a
Veterinary Medicine Research and Development, Pfizer Animal Health, Sandwich CT13 9NJ, UK
b
Veterinary Medicine Research and Development, Pfizer Animal Health, Kalamazoo, MI, USA
c
Assured Research Services Inc., French Village, MO 63036, USA
d
Barton’s West End Facilities, Oxford, NJ 07863, USA
e
ARPEXAS Ltd., Folkestone, UK

Accepted 19 March 2008

Abstract

Two independent studies assessed the duration of immunity of an inactivated adjuvanted Mycoplasma hyopneumoniae vaccine against
mycoplasmal pneumonia in seronegative (study A, n = 52) and seropositive (study B, n = 52) pigs. The pigs were allocated randomly to
treatment and were then injected with a single dose of either the vaccine or a placebo at approximately 1 week of age. Twenty-five weeks
after treatment administration, the pigs were challenged with a virulent strain (LI 36, Strain 232) of M. hyopneumoniae and the extent of
lung lesions consistent with mycoplasmal pneumonia was assessed 4 weeks later.
In study A, the geometric mean lung lesion score (expressed as least squares mean percentages of lung lesions) was significantly
(P = 0.0001) lower in vaccinated (0.3%, n = 20) than in control pigs (5.9%, n = 24) seronegative to M. hyopneumoniae at enrolment; sim-
ilarly, in study B, the extent of lung lesions was significantly reduced (P = 0.0385) in seropositive vaccinated pigs (2.0%, n = 22) com-
pared to controls (4.5%, n = 26). At the end of the investigation period, 4 weeks after challenge, mean antibody sample-to-positive
(S/P) ratios were significantly higher both in seronegative (P = 0.0012) and seropositive (P = 0.0001) vaccinated pigs (mean values = 0.77
and 0.81, respectively) than in controls (mean values = 0.51 and 0.38, respectively).
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Vaccine; Mycoplasma hyopneumoniae; Pig; Lung lesion; Maternal antibody; Duration of immunity

Introduction Actinobacillus pleuropneumoniae, Haemophilus parasuis,

Mycoplasma hyopneumoniae is a mucosal respiratory
pathogen which causes significant economic losses in pig
production world-wide. M. hyopneumoniae is the aetiolog-
ical agent of mycoplasmal pneumonia, and the primary
causative agent of enzootic pneumonia where the interac-
tions between bacteria such as Bordetella bronchiseptica,
*
Corresponding author. Tel.: +44 1304 641250; fax: +44 1304 653158.
E-mail address: sandwich-cr.ahpdinfotemp@pfizer.com (S.C. Rey-
nolds).
Pasteurella multocida or Streptococcus suis results in a
more severe and a longer duration of disease (Maes
et al., 1996; Buddle and O’Hara, 2005). M. hyopneumoniae
has also been considered to be one of the three most com-
mon pathogens (with porcine reproductive and respiratory
syndrome virus and swine influenza virus) detected in por-
cine respiratory disease complex (PRDC) seen in growing
and fattening pigs (Thacker et al., 1999; Thacker, 2001).
Infection is initiated when M. hyopneumoniae adheres
intimately to the surface of ciliated epithelial cells which
induces extensive loss of cilia, destruction and exfoliation

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doi:10.1016/j.tvjl.2008.03.012

Please cite this article in press as: Reynolds, S.C., et al., Reduced lung lesions in pigs challenged 25 weeks after ..., The Veterinary
Journal (2008), doi:10.1016/j.tvjl.2008.03.012
 ARTICLE IN PRESS
2 S.C. Reynolds et al. / The Veterinary Journal xxx (2008) xxx–xxx
of epithelial cells, and results in reduced or loss of function
of the mucociliary apparatus (Zhang et al., 1995; Hsu and
Minion, 1998; Kwon et al., 2002; Minion, 2002). At nec-
ropsy, well demarcated tan-grey to dark red-purple areas
of consolidations, typical of M. hyopneumoniae infections,
are present in the cranio-ventral parts of the lung 2-4 weeks
post infection (Kwon et al., 2002).
The control of multi-factorial swine respiratory diseases
is substantially more difficult than it would be with myco-
plasmal pneumonia or any of the other respiratory disease
individually. It requires meticulous historical, clinical, and
laboratory investigations of the herd to understand the
infection pattern: the timing of infections and identification
of the pathogens involved, the host–pathogen and patho-
gen–pathogen relationship, or the spread of infection
among animals. Because M. hyopneumoniae is very effective
at evading and also at modulating the natural immune
response, the exposure of this organism to the immune sys-
tem is minimal and the immune response is complex.
Vaccination has become an important tool in the con-
trol of respiratory diseases of swine; however immunisation
efficacy varies from herd to herd and depends also on the
environmental factors and the management practices. In
addition, within a herd the serological status varies consid-
erably between individuals with age, the presence of mater-
nally-derived antibodies, or exposure to infection, and at
the time of vaccination there is commonly a mixture of pigs
seronegative or seropositive to M. hyopneumoniae or to
other pathogens. The influence of maternal antibodies on
the induction of a protective immunity after early vaccina-
tion is still not well understood. Recent studies have shown
that M. hyopneumoniae bacterin administered to pigs in the
presence of maternal antibodies has little or no detectable
effect on the development of an antibody response (Daw-
son et al., 2002; Martelli et al., 2006).
In the two laboratory studies presented in this paper we
assessed the duration of immunity in seronegative and
seropositive pigs following administration of an inactivated
adjuvanted M. hyopneumoniae vaccine (Stellamune One,
Pfizer)1 at approximately 1 week of age. Twenty-five weeks
after vaccination, the pigs were infected with a virulent
strain of M. hyopneumoniae. The induction of an antibody
response and the reduction of lung lesions typical of myco-
plasmal pneumonia were evaluated 4 weeks after challenge.
Materials and methods
Duration of immunity was assessed in 52 pigs, seronegative at enrolled,
in a study (study A) conducted at Assured Research Services (A.R.S. Inc.,
French Village, USA), and in 52 pigs, seropositive at enrolment, in study B
conducted at Barton’s West End (Oxford, USA). For both studies, the
experimental protocol was ethically reviewed and the studies conducted in
accordance with the Code of Federal Regulations for animal welfare (9
CFR, 1999a), and product evaluation (9 CFR, 1999b), local legislation
and Good Clinical Practices. The animals were under observation from
1
Also known RespiSure One in the USA.
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Journal (2008), doi:10.1016/j.tvjl.2008.03.012
the day before treatment administration through to the end of the study
(day 202 in study A and day 208 in study B).
All personnel participating in the studies including the examining
veterinarian, individuals responsible for scoring lungs, and for laboratory
investigations, were unaware of the allocation of animals to treatment and
were not involved in the administration of treatment. The dispenser, who
administered all treatments, was not involved in any other aspect of the
studies.
Animals
Pigs had not received any vaccine or other immunologically active
drugs active against M. hyopneumoniae prior to enrolment. Routine pro-
cedures (such as iron injection, teeth clipping, and castration of males)
were performed according to the normal procedures at each source farm.
For pigs in study A, routine procedures were performed within the first
24 h after birth and prior to dosing, whereas in study B, routine proce-
dures were performed 3 days after dosing.
On the day of treatment administration (day 0), animals were assigned
to one of two treatment groups, either vaccine (n = 26 both in study A and
study B) or placebo (n = 26 both in study A and study B), according to a
generalised random block design with blocks based on litter (two control
and two vaccinated pigs per litter) and post-weaning pen.
Study A, seronegative animals
The animals selected for the study were sourced from a US genetic
multiplier herd which had been monitored regularly both serologically and
clinically. In this herd there was no serological or clinical evidence of M.
hyopneumoniae infection. Although it is not possible to prove that the
piglets were free of the organism, the herd was considered to be free of M.
hyopneumoniae.
Pigs included in the study were from 13 sows seronegative to M.
hyopneumoniae (sample-to-positive [S/P] ratios range: 0.000–0.026). A
total of 52 male pigs, 7–8 days of age, were individually identified with ear
tags and were enrolled into the study. All pigs were seronegative to M.
hyopneumoniae on the day before treatment administration (S/P ratios
range: 0.000–0.457). A blood sample was collected and pigs were dosed
with either placebo or with Stellamune One while on the source farm.
Piglets were initially with their sows in farrowing crates at the source
farm. To prevent swine dysentery they were given access to medicated
creep feed (with 35 g/ton tiamulin and 400 g/ton chlortetracycline) for 4
days prior to their transfer to ARS. At approximately 2 weeks of age, the
animals were transferred to the ARS research facilities where they
remained for the duration of the study. Upon arrival, the piglets were
housed in a grower barn in nursery pens until they were approximately 6
weeks of age, and were then moved to grower pens. At approximately 15
weeks of age, the pigs were transferred to a finisher barn. Pigs were
maintained as original groups throughout their time at the research
institute.
For the first few days after arrival at the research institute, the pigs
were fed a medicated transition feed with 35 g/ton tiamulin and 400 g/ton
chlortetracycline, again to prevent swine dysentery. They were then offered
a commercial pre-starter, and a starter diet with 50 g/ton carbadox. From
approximately 2 months of age, pigs were given grower and finisher diets
that contained no antimicrobials. Feed and water were provided ad libi-
tum throughout the study. In the nursery unit, grower and finisher barns,
temperature and ventilation were controlled.
Study B, seropositive animals
Because the animals were sourced from a commercial herd with a
history of M. hyopneumoniae associated disease, it must be assumed that
the pigs selected for the study were from sows that could have been
exposed to M. hyopneumoniae organisms. In this herd, M. hyopneumoniae
vaccination was used as a means of disease control. It is likely that
colostral transfer of antibodies of vaccinal or natural origin, and early
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S.C. Reynolds et al. / The Veterinary Journal xxx (2008) xxx–xxx
weaning at approximately 2–3 weeks of age reduced transmission of M.
hyopneumoniae from dam to piglet, however the piglets could have been
exposed to the organism. Pigs from this herd are however more likely to be
representative of commercially reared pigs.
Pigs included in the study were from seven sows seropositive to M.
hyopneumoniae (S/P ratios range: 0.688–2.607). A total of 52 pigs of either
sex, 4–8 days of age, were individually identified with ear tags and were
enrolled into the study. All pigs were seropositive (S/P ratios range: 0.983–
3.940) to M. hyopneumoniae on the day before treatment administration.
The treatment vaccine or placebo was administered and a blood sample
collected. Piglets were with the sows in farrowing crates, then at approx-
imately 2 weeks of age the animals were transferred to Barton’s West End
research facilities where they remained for the duration of the study. Upon
arrival, piglets were housed by litter in 13 pens of four pigs per pen until
the end of the study. Pigs were fed medicated feed (with 35 g/ton tiamulin
and 400 g/ton chlortetracycline) for the prevention of swine dysentery
through the weaning period, and then were offered a commercial grower
non-medicated diet through to the end of the study. Feed and water were
given as per routine site practices.
Vaccination
The vaccine, Stellamune One, consists of a M. hyopneumoniae bacterin,
strain P-5722-3 (NL 1042), a field strain isolated from a pig in the USA,
inactivated with binary ethylamine and coupled with Amphigen (Pfizer), a
mineral oil adjuvant (Charlier et al., 2000). Animals were administered a
2-mL commercial dose of either placebo (sterile liquid adjuvant) or vac-
cine (between 4.57 and 5.20RP [Relative Potency] in study A, and between
3.96 and 5.49RP in study B) by the IM route into the neck. Day 0 was the
day of vaccination in both studies.
Challenge
The challenge inoculum was from a stock of frozen lung homogenate
(LI 36) from pigs exposed to strain 232, and was prepared by and pur-
chased from Iowa State University (USA) in 1999. The supply of LI 36
had previously been tested and was shown to produce lung lesions char-
acteristic of mycoplasmal pneumoniae at the titre and volumes used in the
present studies. Fresh challenge inoculum was prepared each day and was
tested for bacterial contamination and concentration.
Study A, seronegative animals
Twenty-five weeks after treatment administration, 44 animals were
challenged intranasally with a 5 mL suspension (1:25 dilution) of virulent
M. hyopneumoniae lung homogenate at a concentration of approximately
106 CCU/mL (Colour Changing Units) on three consecutive days (days
173, 174, and 175 of study). Pigs were anaesthetised and the challenge
suspension was instilled in roughly equal volumes into the nasal passage
via the two nostrils.
Study B, seropositive animals
Twenty-five weeks after treatment administration, 48 animals were
challenged intranasally with a 10 mL suspension (1:25 dilution) of virulent
inoculum at a concentration of approximately 107 CCU/mL on three
consecutive days (days 178, 179, and 180 of study). Pigs were anaesthetised
and the challenge suspension was split between the two nostrils. The
10 mL volume was used in this study based on prior experience with the
challenge stock (LI 36) and its ability to reproduce lesions in seropositive
pigs.
Abnormal health and clinical observations
In both studies, observation of general health was performed daily by a
stockperson from the day before the start of the study through to the end
Please cite this article in press as: Reynolds, S.C., et al., Reduced lung lesions in pigs challenged 25 weeks after ..., The Veterinary
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3
of the study to ensure overall animal comfort and to quickly detect
abnormalities; any animal showing signs of abnormal health which
required veterinary attention was examined by a veterinarian, and con-
current medication was given at the discretion of the veterinarian and
according to normal practices.
Lung lesions
In both studies, efficacy was evaluated by assessing the extent of lung
lesions typical of M. hyopneumoniae infection. Based on experimental
infection models, where lungs are usually examined from approximately
14 days up to 40 days after infection corresponding to increased severity of
the lesions (Whittlestone, 1972; Feenstra et al., 1994), a necropsy was
performed approximately 28 days after challenge. Lungs were removed
from the carcasses and each of the seven pulmonary lobes was evaluated
both visually and by palpation. Chronic classic purple/grey lesions were
recorded by sketching the approximate area(s) of the lesions on a dia-
grammatic outline of the lungs and the areas primarily associated with M.
hyopneumoniae infections were identified. The scoring method described
by Pointon et al. (1992) was used to assess the extent of tissue consoli-
dation in each lobe and an estimation of the percent gross involvement for
each lobe was given.
Polymerase chain reaction (PCR) for M. hyopneumoniae
The purpose of conducting the PCR testing from samples collected at
the end of the study was to assess whether the vaccination could eliminate
M. hyopneumoniae organisms from the respiratory tract of challenged
pigs. Lung tissue samples were collected aseptically from the right cardiac
lobe at necropsy, regardless of the presence of lung lesions, using clean,
sterile instruments for each pig to avoid cross contamination. Samples
were tested using a M. hyopneumoniae specific PCR assay in accordance
with the standard procedures of the testing laboratory (South Dakota
Animal Disease Research and Diagnostic Laboratory, USA).
Serology
Blood samples were collected on study days – 1, 28, 56, 84, 113, 140,
166, 172 and 202 from seronegative pigs (study A) and on study days – 1,
34, 75, 103, 131, 166 and 208 from seropositive pigs (study B). Measure-
ment of serum antibody to M. hyopneumoniae was performed in accor-
dance with the M. hyopneumoniae standard indirect ELISA procedures of
the testing laboratory (Tween 20-ELISA, Veterinary Diagnostic Labora-
tory of Iowa State University, USA) (Bereiter et al., 1990). For each
sample, ELISA titres (optical density [OD] readings) were used to calcu-
late S/P ratios where S was defined as sample OD - negative control OD,
and P was defined as positive control OD - negative control OD. In both
studies, S/P ratios <0.5 were considered negative and all other values were
positive.
Statistical analysis
The primary parameter of efficacy was evaluated by a significant
reduction in the extent of lung lesions typical of M. hyopneumoniae. In
addition serological data were analysed. For both clinical and serological
data two-sided tests were used, and the statistical differences between
treatments were assessed at the 5% level of significance. All data were
analysed using SAS programme version 6.12.
Per cent gross involvement per each lung lobe was weighted using the
following ratios of individual lung lobes to total lung mass: left cranial
10%, left middle 10%, left caudal 25%, right cranial 10%, right middle
10%, right caudal 25%, and accessory 10%. The weighted lung lobe values
were then summed across lobes to yield the percentage of total lung with
lesions prior to analysis (Pointon et al., 1992). Percentages of total lung
with lesions were transformed using the arcsin square root and then
analysed using a mixed linear model that included the fixed effect of
treatment and random effect of block. Linear combinations of the
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4 S.C. Reynolds et al. / The Veterinary Journal xxx (2008) xxx–xxx

parameter estimates were used in a priori contrasts after testing for a
significant (P 6 0.05) treatment effect. Comparisons were made between
treatment groups.
Geometric mean S/P ratios at each sampling day were calculated from
least squares means of the (ln[S/P ratio + 1]). The S/P ratios were ana-
lysed using a linear mixed model with repeated measures that included the
fixed effects of treatment, sampling day, and interaction between treat-
ment and sampling day and the random effects of block, animal, and
interaction between treatment and block. Linear combinations of the
parameter estimates were used in a priori contrasts after testing for sig-
nificant (P 6 0.05) treatment or treatment by sampling day interaction
effects. Comparisons were made between treatment groups at each sam-
pling day.
Results
At enrolment, all animals included in the studies were in
good health with no clinical signs of respiratory disease.
There were no signs of abnormal health or evidence of seri-
ous injection site reactions observed in any of the enrolled
animals after treatment administration.
Withdrawals and completions
In study A, 8/52 animals failed to complete the study;
six were vaccinated animals and two were controls. For
welfare reasons, one control animal was euthanased on
study day 19 (chronic septic polyarthritis) and one vacci-
nated animal on study day 95 (non-reducible rectal pro-
lapse). In addition, one vaccinated animal was found
dead on study day 40 (pericardial fluid, haemorrhage on
the epicardium, and congested lungs at necropsy), and
for one vaccinated animal found dead on study day 143
the cause of death was not determined due to advanced
post-mortem change. On the day of challenge inoculation
(day 174), one control and three vaccinated pigs died
because of anaesthetic complications. Forty-four animals
(20 vaccinated and 24 control animals) completed the study
on day 202 (Table 1).
In study B, four vaccinated animals (out of 52 animals
enrolled) failed to complete the study. Two animals were
withdrawn from the study on day 174 because they lost
their identification numbers, were in the same pen, and
could not be conclusively re-identified. Anaesthetic compli-
cations occurred during challenge inoculation and two vac-
cinated pigs were found dead on study days 178 and 179.
Forty-eight animals (22 vaccinated and 26 control animals)
completed the study on day 208 (Table 1).
Abnormal general health observations
In study A, seven vaccinated and three control pigs
showed signs of abnormal health including traumatic inju-
ries, septic arthritis, joint swelling or infection, and enteritis
between approximately 2–4 weeks of age; one vaccinated
pig had a rectal prolapse at approximately 14 weeks of
age, and a chronic abscess and laceration were reported
between approximately 25–27 weeks of age for one vacci-
nated pig and one control pig. One vaccinated pig, found
dead on day of study 40 and suspected to have a gut
oedema, presented at post-mortem examination some tho-
racic lesions with the presence of pericardial fluid, haemor-
rhage on the epicardium, and congested lungs. Coughing
was observed among challenged animals approximately 3
weeks after challenge.
In study B, the signs of abnormal health observed were
mainly caused by traumatic injuries both in vaccinated
(n = 3) and control (n = 6) pigs: between approximately
2-3 weeks of age for testicular trauma, swollen hock, and
then later between approximately 21–30 weeks of age for
deep laceration on the shoulder, left hind leg trauma, and
traumatic loss of hoof. In addition one vaccinated pig pre-
sented with a swelling nodule at approximately 27 weeks of
age and a control pig presented with several abnormal signs
including soft tissue injuries, carbuncle, laminitis, and signs
of cannibalism between approximately 25–29 weeks of age.
Lung lesions
Study A, seronegative animals
The extent of lung lesions was assessed in 44 animals, 20
vaccinated pigs and 24 control pigs (Table 2). Vaccinated
pigs had a significantly (P = 0.0001) lower least squares
(LS) mean percentage of lung lesions than control pigs
(0.3% vs. 5.9%, respectively). The prevalence of lung lesions
at slaughter was lower in vaccinated pigs (n = 9/20, 45%)
than in control pigs (n = 22/24, 92%).

Table 1
Summary of study design in pigs experimentally infected with M. hyopneumoniae 25 weeks after vaccination with Stellamune One
Study A in seronegative pigs Study B in seropositive pigs
Control group Vaccinated group Control group Vaccinated group
Number of pigs at study initiation 26 26 26 26
Number (%) of pigs at study completion 24 20 26 22
(92%) (77%) (100%) (85%)
Number (%) of pigs that failed to complete the studya 2 6 0 4
(8%) (23%) – (15%)
a
In study A, eight pigs failed to complete the study for reasons unrelated to treatment administration. Four pigs were withdrawn and euthanased for
welfare reasons (n = 2) or were found dead (n = 2), and four pigs died due to complications of the anaesthesia on the day of challenge inoculation. In study
B, four pigs failed to complete the study. Two pigs were withdrawn because they lost their identification and two other pigs died due to complications of
the anaesthesia on the day of challenge inoculation.

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Table 2
Extent of lung lesions at slaughter 4 weeks after challenge with M. hyopneumoniae
Study A, seronegative pigs
Control group (n = 24)
Least squares mean percentage of lung lesions in pigs challenged 25 weeks after treatment administration
Lung lesion score 5.9
(Geometric LS mean)
Standard error 1.30
Range 0–36
P value 0.0001
Study B, seropositive animals
The extent of lung lesions was assessed in 48 animals, 22
vaccinated pigs and 26 control pigs (Table 2). Vaccinated
pigs had a significantly (P = 0.0385) lower LS mean per-
centage of lung lesions than control pigs (2.0% vs. 4.5%,
respectively). The number of pigs with lung lesions at
slaughter was lower in the vaccinated treatment group
(n = 19/22, 86%) than in the control treatment group
(n = 25/26, 96%).
Detection of M. hyopneumoniae by PCR
In both studies, the presence of M. hyopneumoniae anti-
gen was detected by PCR of lung tissue samples in all treat-
ment groups: for study A, M. hyopneumoniae was found in
18/20 vaccinated pigs and 23/24 control pigs and for study
B in 14/22 vaccinated pigs and 18/26 control pigs.
Serology
Study A, seronegative animals
Overall there were significant (P = 0.0010) differences in
the serological response between treatment groups. All ani-
mals included in the study were seronegative (S/P ratios
<0.5) the day before treatment administration and there
were no significant (P = 0.3964) differences (mean val-
ues = 0.12 ± 0.02 for vaccinated pigs and 0.13 ± 0.01 for
control pigs) between treatment groups. The serological
response was significantly (P 6 0.0468) higher in vacci-
nated animals on study days 28-84, 166 and 172. Between
12 and 24 weeks after vaccination sporadic incidents
occurred, with S/P ratios >0.5, affecting 8/26 control pigs
on 12 occasions: one pig on study day 84 (value = 0.602,
group mean value = 0.12 ± 0.02), and five pigs on study
day 113 (values = 0.536–0.707, group mean value =
0.20 ± 0.04). Then four pigs on day 140 (values = 0.569–
1.029, group mean value = 0.15 ± 0.04), one pig on day
166 (value = 0.720, group mean value = 0.10 ± 0.03), and
one pig on day 172 (value = 0.614, group mean
value = 0.10 ± 0.03). None of these pigs had S/P ratios
>0.5 for the subsequent blood sample prior to challenge.
After challenge, a marked increase in S/P ratios was
observed in both treatment groups which was significantly
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Journal (2008), doi:10.1016/j.tvjl.2008.03.012
5
Study B, seropositive pigs
Vaccinated group (n = 20) Control group (n = 26) Vaccinated group (n = 22)
0.3 4.5 2.0
0.18 1.46 0.78
0–6 0–36.75 0–13.75
0.0385
higher (P = 0.0012) in vaccinated animals (mean
value = 0.77 ± 0.06) than in control animals (mean
value = 0.51 ± 0.05) on study day 202 (Fig. 1).
Study B, seropositive animals
Overall there were significant (P = 0.0116) differences in
the serological response between treatment groups. On the
day before treatment administration, all enrolled pigs
tested seropositive (mean values = 1.15 ± 0.05 for vacci-
nated pigs and 1.14 ± 0.05 for control pigs) and there were
no differences (P = 0.8302) in mean titres between the
treatment groups. A reduction of geometric mean S/P
ratios was detected 34 days (mean values = 0.40 ± 0.03
for vaccinated pigs and 0.39 ± 0.03 for control pigs) and
75 days (mean values = 0.13 ± 0.017 for vaccinated and
control pigs) after vaccination at approximately 82 days
of age; then mean S/P ratios were low until challenge inoc-
ulation. There were no differences (P P 0.2070) in the
mean S/P ratios between vaccinated and control animals
34, 75, 103, 131, and 166 days after vaccination until chal-
lenge. Although based on the source herd history, piglets in
both treatment groups may have been exposed to M. hyo-
pneumoniae organisms, serological data prior to challenge
indicate that there was no evidence of a significant M. hyo-
pneumoniae infection prior to the challenge administration.
Challenge induced an increase in antibody response
which was significantly higher (P = 0.0001) in vaccinated
animals (mean value = 0.81 ± 0.04) than in control ani-
mals (mean value = 0.38 ± 0.04) on study day 208 (Fig. 2).
Discussion
We report here the results of two independent labora-
tory studies where lung lesions typical of M. hyopneumo-
niae infection were evaluated in seronegative (study A)
and seropositive (study B) pigs vaccinated at approxi-
mately 1 week of age and challenged with a virulent strain
of M. hyopneumoniae 25 weeks later.
In both studies, the high prevalence (P92%) of lung
lesions observed in the non vaccinated-control groups gives
evidence that the animals received a severe M. hyopneumo-
niae challenge. The detection of M. hyopneumoniae by PCR
showed that vaccination did not eliminate M. hyopneumo-
niae organisms from the respiratory tract. Not surprisingly
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Fig. 1. Tween 20-ELISA serum antibody (S/P ratio) to M. hyopneumoniae in seronegative vaccinated (—N—) and control (- - - j - - -) pigs following
vaccination (day of study 0) and after M. hyopneumoniae challenge (days of study 173, 174, 175).

Fig. 2. Tween 20-ELISA serum antibody (S/P ratio) to M. hyopneumoniae in seropositive vaccinated (—N—) and control (- - - j - - -) animals following
vaccination (day of study 0) and after M. hyopneumoniae challenge (days of study 178, 179, 180).

some samples collected from pigs in all treatment groups
were free of detectable M. hyopneumoniae; however it is
possible that a negative pig could have been infected. The
samples were collected from roughly the same location at
the right cardiac lobe regardless of the presence of lung
lesions, to avoid bias against evaluating the colonisation
by M. hyopneumoniae based only on the presence of
lesions. Post-mortem examination of lungs for lesions char-
acteristic of M. hyopneumoniae infection showed a signifi-
cant reduction of the extent of lesions in vaccinated pigs
compared to control pigs, in the absence (mean lung lesion
score = 0.3% in vaccinated pigs vs. 5.9% in control pigs,
study A), and in the presence of maternal antibodies (mean
lung lesion score = 2.0% in vaccinated pigs vs. 4.5% in con-
trol pigs, study B) at the time of vaccination. Lung lesions
characteristic of other organisms were not seen.
Similar observations were reported by Dawson et al.
(2002) in two studies conducted under laboratory condi-
tions where seropositive and seronegative pigs adminis-
tered a single dose of M. hyopneumoniae bacterin at

Please cite this article in press as: Reynolds, S.C., et al., Reduced lung lesions in pigs challenged 25 weeks after ..., The Veterinary
Journal (2008), doi:10.1016/j.tvjl.2008.03.012
 ARTICLE IN PRESS
S.C. Reynolds et al. / The Veterinary Journal xxx (2008) xxx–xxx
approximately 24–40 days of age demonstrated no signifi-
cant interaction between serological status and vaccination
on the extent of lung lesions. Pigs were challenged approx-
imately 3 weeks after vaccination and lung lesions were
evaluated 4 weeks later. Thacker et al. (2002) demonstrated
that a single-dose M. hyopneumoniae vaccination and chal-
lenge 14 days later resulted in significantly reduced lung
lesions in vaccinated pigs compared to unvaccinated pigs,
regardless of their serological status when vaccinated.
More recently Martelli et al. (2006) reported no detectable
effect on the antibody response to M. hyopneumoniae in
pigs vaccinated at approximately 1 week of age in the pres-
ence of passively acquired maternal antibodies (from vacci-
nated dams); the study was conducted in a commercial
herd with history of M. hyopneumoniae.
Interestingly, Hodgins et al. (2004) observed that the
levels of maternal antibodies at the time of vaccination
had an effect on the IgG response. The immune mecha-
nisms responsible for protection against M. hyopneumoniae
are complex and not clearly understood. Several authors
demonstrated that there is no direct correlation between
vaccine-induced serum antibody levels and protection
against M. hyopneumoniae (Djordjevic et al., 1997; Thacker
et al., 1998). According to Thacker et al. (2000b), the prim-
ing of both humoral and cellular immunity after vaccina-
tion is probably of significance in the protection against
swine respiratory diseases. A laboratory study showed that
pigs given a two-dose commercial M. hyopneumoniae vac-
cine at approximately 1 week of age, and then challenged
28 days after the second vaccination (day of study 42), pre-
sented significantly more interferon (IFN)-c secreting
blood-borne lymphocytes 6 days after infection, and
increased concentrations of M. hyopneumoniae-specific
IgG and IgA in bronchoalveolar lavage fluid 28 days after
infection, than non-vaccinated animals (challenged and
non-challenged) and vaccinated non-challenged animals.
In the two studies reported here the serological response
was monitored from study initiation and at different time
points through to study termination on day 202 in study
A, and on day 208 in study B. All animals in study A were
seronegative at the time of treatment administration. How-
ever 12 sporadic incidents occurred before challenge among
eight control pigs with S/P ratios >0.5. Since pigs were co-
mingled we would have expected that pig-to-pig transmis-
sion would have resulted in biological amplification of
any individual exposure and a gradual increase in the num-
ber of seropositive pigs and the magnitude of the serologic
responses seen, but this was not observed. These positive
titres would more likely suggest an inconclusive, transient
seropositivity, or assay variation rather than a serological
evidence of an active M. hyopneumoniae infection. After
challenge vaccinated seronegative (study A) and seroposi-
tive (study B) pigs demonstrated an anamnestic response
with antibody levels significantly higher than in control
pigs.
The signs of abnormal health, traumatic injuries, arthri-
tis, enteritis, rectal prolapse or joint swelling, reported in
Please cite this article in press as: Reynolds, S.C., et al., Reduced lung lesions in pigs challenged 25 weeks after ..., The Veterinary
Journal (2008), doi:10.1016/j.tvjl.2008.03.012
7
the two studies are commonly observed in commercial
herds. In study A, cough was observed approximately 3
weeks after challenge. Coughing is commonly observed in
M. hyopneumoniae infections and in other respiratory dis-
eases (Maes et al., 1996), although no correlation with
the severity of infection has been reported (Straw et al.,
1990). The design of the duration of immunity studies pre-
sented in this report did not intend to correlate coughing
with disease after challenge.
The control of multi-factorial swine respiratory diseases
requires strong preventive measures, where an appropriate
choice of vaccination regimen is key to the protection of
the herd: in reducing the infection pressure, the prevalence
and severity of lung lesions, by improving growth perfor-
mances and feed conversion ratios, and by reducing costs
associated antimicrobial treatments (Ross et al., 1984; Pal-
larés et al., 2000; Thacker et al., 2000a; Pallarés et al., 2001;
Smith et al., 2003a, b, c; Haesebrouck et al., 2004). An
early vaccination regimen with immunisation at approxi-
mately 1 week of age should confer seroconversion and
immune protection at an earlier time in life particularly
at around the time of weaning, when pigs from different lit-
ters are mixed together and are likely to face their first
infectious challenge and when the risk of developing
multi-factorial severe pneumonia is exacerbated.
The data reported here complement the wealth of infor-
mation already available from studies conducted in com-
mercial farms across Europe where pigs given one-shot
vaccination at approximately 1 month of age (Smith
et al., 2003a,b), or at approximately 1 week of age (Smith
et al., 2003c) presented significantly reduced lung lesions
(mean lung lesion score = 8.6% vs. 2.4% vs. 5.9%, respec-
tively) compared to control pigs (mean lung lesion
score = 14.9% vs. 7.7% vs. 9.1% respectively).
A laboratory study conducted recently demonstrated an
onset of protective immunity from 2 weeks after vaccina-
tion, as expressed by significantly reduced lung lesions in
vaccinated pigs compared to control pigs (mean lung lesion
score = 12% in vaccinated pigs and 38% in control pigs);
pigs were administered a single dose of commercial vaccine
at approximately 1 week of age, and then were challenged
with a virulent M. hyopneumoniae suspension 2 weeks later
(Horst et al., 2006; Reynolds et al., 2006).
The studies presented in this manuscript demonstrate
that the serological status of pigs at vaccination did not
prevent significant protection from lung lesions or develop-
ment of significant antibody response after challenge 25
weeks after vaccination. With a duration of immunity of
at least 25 weeks, Stellamune One vaccinated pigs will be
protected during longer growing and finishing periods.
Conclusions
The administration of a single dose of M. hyopneumo-
niae bacterin to pigs at approximately 1 week of age
induced a long-lasting protective immunity, and a signifi-
cant reduction in the extent of lung lesions after challenge
 ARTICLE IN PRESS
8 S.C. Reynolds et al. / The Veterinary Journal xxx (2008) xxx–xxx
with a virulent strain of M. hyopneumoniae 25 weeks post-
vaccination in both seronegative and seropositive pigs.
Conflict of interest
None of the authors of this paper has a financial or per-
sonal relationship with other people or organisations that
could inappropriately influence or bias the content of the
paper.
Acknowledgements
The authors are most grateful to Dr. B. Scalzo and Mrs.
Anne Lloyd for their assistance in preparing this paper,
and Dr. T. Meinert for his statistical advice and analysis.
We would like to thank Dr. Eileen Thacker and her labo-
ratory at Iowa State University for preparation and initial
characterisation of the supply of challenge material used in
these studies. We also acknowledge the contribution made
by the staff at Assured Research Services Inc. and at Bar-
ton’s West End Facilities for their scientific and technical
support.
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Please cite this article in press as: Reynolds, S.C., et al., Reduced lung lesions in pigs challenged 25 weeks after ..., The Veterinary
Journal (2008), doi:10.1016/j.tvjl.2008.03.012