The

Veterinary Journal
The Veterinary Journal 172 (2006) 526–531
www.elsevier.com/locate/tvjl

Comparative efficacy of two single-dose bacterins in the control

of Mycoplasma hyopneumoniae in swine raised under
commercial conditions in Brazil
Maria Regina Baccaro a, Flavio Hirose b, Ossamuro Umehara b,
Lineu C.B. Gonçalves c, Daniela Sabatini Doto a, Renata Paixão a,
Luciane Tieko Shinya a, Andrea Micke Moreno a,*
a
Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Av. Dr. Orlando Marques de Paiva,n. 87,
CEP 05508-000, São Paulo, SP, Brazil
b
Laboratórios Pfizer Ltda, Av. Alexandre Dumas, n 1711, 8 andar, CEP 04717-004, São Paulo, SP, Brazil
c
Rua Dr. Antonio Roberto Neto, n 214, CEP 05366-160, São Paulo, SP, Brazil

Abstract

A field study was conducted in Brazil to evaluate the efficacy of single vaccination of pigs with two bacterins to prevent Myco-
plasma hyopneumoniae lung lesions. The first (T1) treatment group (174 pigs) was injected with 2 mL of saline solution; group T2
(177 pigs) with 2 mL of bacterin A, and group T3 (174 pigs) with 2 mL of bacterin B. On days-on-test (DOT) 0, 35, 66, 97 and 125,
blood samples and tonsil swabs were collected from selected pigs for antibody determination (indirect ELISA) and PCR assay for
the presence of M. hyopneumoniae. Pigs were slaughtered on DOT 126–129 and lung lesions were scored blindly.
Bacterin A vaccinated pigs had significantly (P 6 0.05) lower lung lesion scores (0.2%) than bacterin B (0.4%) or saline-treated
pigs (1.2%); there was also a significantly lower (P 6 0.05) number of pigs with lung lesions (27.1%), than bacterin B (38.2%) or
saline-treated (55.4%) pigs. The two vaccines had similar (P > 0.05) results in terms of mean weight gain, average daily weight gain,
feed efficiency, frequency of PCR positives, and there was similar antibody conversion (ELISA). It was concluded that although the
productivity parameters and antibody conversions were similar, bacterin A was more effective in preventing and reducing the sever-
ity of lung lesions than bacterin B.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Enzootic pneumonia; Mycoplasma hyopneumoniae; Lung lesion; Swine; Vaccine

1. Introduction common pathogens detected in pigs with respiratory dis-

Porcine respiratory disease complex (PRDC) is a
serious health problem in growing and finishing pigs typ-
ically around 16–22 weeks of age. PRDC is characterised
by slow growth, decreased feed efficiency, lethargy, anor-
exia, fever, cough and dyspnoea (Ross, 1999; Kim et al.,
2003). Mycoplasma hyopneumoniae is one of the most
*
Corresponding author. Tel.: +55 11 30911294; fax: +55 11
30917928.
E-mail address: morenoam@uol.com.br (A.M. Moreno).
ease. In Brazil, a serological survey involving 2808 serum
samples from seven States showed that 27.7% of pigs were
serologically positive, and that 59.09% of herds contained
pigs that were serologically positive to M. hyopneumoniae
(Moreno et al., 1999).
Commercial bacterins requiring two intramuscular
injections have been available in Brazil for several years
and are routinely used within the swine industry. When
compared to non-vaccinated control pigs, one such bac-
terin has been shown in a field study to demonstrate good
efficacy in reducing lung lesions leading to better produc-

1090-0233/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2005.07.012
 M.R. Baccaro et al. / The Veterinary Journal 172 (2006) 526–531
tivity performance (Baccaro et al., 2002). Recently, two
new products have been introduced, both recommending
single-dose vaccination. Morris et al. (2001) found no
significant differences under field conditions between
the single-dose and the two-dose bacterins in reducing
lung lesions and in the performance parameters of pigs.
Roof et al. (2001) in a vaccination/challenge study com-
pared the efficacy of one-dose and two-dose bacterins;
the products tested were Ingelvac M. hyo (one dose)
and Ingelvac M. hyo (two doses), both from Boehringer
Ingelheim, and RespiSure One (one dose) and RespiSure
(two doses) from Pfizer Animal Health. Vaccinations
were carried out on days 0 and 14 (for the two-dose vac-
cines) and challenge was performed on Day 28 by intra-
tracheal administration of a virulent M. hyopneumoniae.
Pigs were slaughtered on Day 57. The two one-dose bac-
terins had similar lung lesions with Ingelvac M. hyo hav-
ing had significantly higher average daily weight gains
than the controls, while RespiSure One was similar to
Ingelvac M. hyo or to the controls.
The use of single dose vaccines has the advantage of
reducing labour, causing less stress to the animals and
yielding better meat quality due to fewer injection sites.
The present study was designed to evaluate under com-
mercial conditions the efficacy of two single-dose bacte-
rins in the control of M. hyopneumoniae as measured by
the lung lesion scores at slaughter, and the productivity
performance of growing/finishing pigs, as measured by
weight gain, average daily weight gain and feed
efficiency.
2. Materials and methods
2.1. Experimental design and treatments
The study was conducted in Brazil between November
2001 and March 2002. Five hundred and twenty-five pigs
on a commercial farm with a known history of enzootic
pneumonia, were individually identified by numbered
ear tags and randomly allocated to one of three groups:
Treatment 1 (T1 – 174 pigs), Treatment 2 (T2 – 177 pigs)
or Treatment 3 (T3 – 174 pigs). At weaning, when they
were between 25 and 31 days of age, the pigs were weighed
and vaccinated. The animals in the T1 group received 2
mL of sterile saline solution, the pigs in T2 group received
2 mL of bacterin A (RespiSure1 One, Pfizer Animal
Health) and the pigs in T3 group received 2 mL of bacterin
B (Ingelvac M. hyo, Boehringer Ingelheim). The date of
injection was day-on-test 0 (DOT 0).
All pigs were observed for 30 min post-treatment for
evidence of abnormal clinical signs. The animals in each
treatment group were assigned by weight to pens (with
9–10 pigs per pen during the nursery phase). The 9–10
heaviest pigs in each treatment group were randomly as-
signed to three pens (the 9–10 heaviest pigs from the T1
527
group were placed in one pen; the 9–10 heaviest pigs
from T2 in another pen, and the 9–10 heaviest pigs of
T3 in a third pen); the next 9–10 heaviest pigs from each
treatment group were randomly assigned to three other
pens and so on until all pigs had been assigned (18 pens
per treatment).
On DOT 35, the pigs were weighed and moved to the
grower/finisher unit. The two heaviest pens from each
treatment in the nursery phase were randomly assigned
to one pen in the grower/finisher unit (20 pigs per
pen). The second two heaviest pens from each treatment
in the nursery phase were moved to another pen and
thus successively until all 18 pens/treatment were moved
to nine pens in the grower/finisher unit.
The piglets were all housed in similar conditions, re-
ceived the same feed and were subjected to the same
management regime. The animals did not receive any
antibiotic treatment and were individually monitored
during the entire experimental period.
2.2. Lung lesion scores
All pigs were slaughtered by pen on DOT 126–129.
At slaughter, the lung lesions were scored according to
the system described by Madec et al. (1982). The exam-
iner was unaware of the identity of the treatment group.
2.3. Mortality
Mortality was recorded for all pigs that died after
weaning and throughout the grower/finisher phase to
slaughter and a necropsy was performed to determine
the cause of death when possible.
2.4. Blood and tonsil swab samples
Three pigs from each of the two nursery pens per
treatment that had been transferred to one pen in the
grower/finisher unit were randomly selected prior to
the start of the study (totalling 27 pigs per treatment
in 9 pens). These animals underwent blood and tonsil
swab sampling at the time of injection (DOT 0) and
on DOT 35, 66, 97 and 125. Serum samples were
assayed by indirect ELISA test (Idexx Laboratories)
for detection of antibodies against M. hyopneumoniae.
Tonsil swabs were evaluated for presence of M. hyo-
pneumoniae by the nested-PCR technique as described
by Casalmiglia et al. (1999).
2.5. Body weight
To calculate the weight gain during each growth
phase, all pigs were weighed individually at weaning
(DOT 0), on the day they were moved to grower/finisher
facilities (DOT 35) and on the day before slaughter
(DOT 125).
528 M.R. Baccaro et al. / The Veterinary Journal 172 (2006) 526–531

2.6. Feed conversion ratio
All pigs in a pen were fed ad libitum from a single fee-
der. The weight of all feed added to each feeder and the
date of each addition was recorded throughout the
grower/finisher phase. There was no feed remaining
when pigs were sent to slaughter.
2.7. Statistical analysis
Frequency distributions of lung lesion scores were
calculated for each lobe and treatment. The arc sin
square root transformation was applied to the percent
of lung affected from each animal. The transformed per-
centage of lung damage was analysed using a general lin-
ear mixed model analysis of variance including the fixed
effect of treatment and the random effects of block,
block by treatment interaction as the error term for test-
ing treatment and residual. Least-squared means were
back-transformed for presentation.
Body weight was analysed using a general linear re-
peated measures mixed model analysis of variance with
a model including the fixed effects of treatment, day of
study, treatment by day of study interaction and ran-
dom effects of block, block by treatment interaction as
the error term for testing treatment, animal (block by
treatment), block by treatment by day of study as the er-
ror term to test day of study and treatment by day of
study and residual.
Average daily weight gains between weaning and
moving to grower/finisher, between weaning and slaugh-
ter and between moving to the grower/finisher and
slaughter were calculated using estimate statements.
Contrasts were used to compare the average daily
weight gains between treatments.
The feed conversion ratio was analysed using a gen-
eral linear mixed model analysis of variance including
the fixed effect of treatment and the random effects of
block and residual.
Frequency distributions of PCR and ELISA results
were calculated for each treatment at each day when
samples were collected. Differences between treatments
were tested on each day of data collected using the
CMH test of general association. The statistical signifi-
cance was set at a 0.05.
3. Results
A total of 525 pigs were enrolled in the study: 174 in
treatment 1 (T1), 177 in treatment 2 (T2) and 174 in
treatment 3 (T3) at study initiation (Table 1). During
the study period, 6, 6 and 5 pigs died in Groups T1,
T2 and T3, respectively. Many pigs lost their ear tags
(62 pigs) during the transportation from the farm to
the slaughterhouse and could not be identified at slaugh-
ter for lung lesion scoring, and thus the total number of
pigs examined for lung lesions were: 139 in T1, 155 in T2
and 152 in T3 groups. One pig pre-selected for sampling
in T2 and one in T3 died before DOT 35 and one pig in
T2 died before DOT 66 sampling. There was no adverse
reaction in any of the M. hyopneumoniae bacterins vac-
cinated or saline-treated pigs. Of seventeen dead pigs, all
had ileitis confirmed by PCR detection and histopathol-
ogical examination.
3.1. Lung lesion scores
Vaccination with bacterin A or bacterin B signifi-
cantly reduced (P 6 0.05) the number of pigs with lung

Table 1
Number of pigs enrolled in the study comparing saline solution, bacterin A and bacterin B
Treatment groups Number of pigs
Initial Mortality DOT 125 (weighed) Examined at slaughter Lost ear tagb
T1 – saline solution 174 6 167a 139 29
T2 – bacterin A 177 6 171 155 16
T3 – bacterin B 174 5 169 152 17
Total 525 17 507a 446 62
DOT, day-on-test.
a
One pig was not weighed on DOT 125 because it was not found.
b
Pigs that lost ear tags during transportation from the farm to the slaughterhouse.

Table 2
Number and percentage of pigs with M. hyopneumoniae lung lesions according to treatment
Treatment group Pigs with lung lesions Pigs without lung lesions Total number of pigs examined
T1 – saline solution 77 (55.4%)a 62 (44.6%) 139
T2 – bacterin A 42 (27.1%)b 113 (72.9%) 155
T3 – bacterin B 58 (38.2%)c 94 (61.8%) 152
a,b,c
Between treatments, different superscripts are significantly different (P 6 0.05).
 M.R. Baccaro et al. / The Veterinary Journal 172 (2006) 526–531 529

lesions compared to saline solution (Table 2). The sal-
ine-treated group had 77/139 (55.4%) of pigs with lung
lesions compared to 42/155 (27.1%) in the bacterin A
vaccinated group and 58/152 (38.2%) in the bacterin
B vaccinated group. The number of pigs with lung le-
sions in the T2 group was significantly lower
(P 6 0.05) than in the T3 group. The severity of M.
hyopneumoniae as determined by the lung lesion scores
(Table 3), were also significantly lower (P 6 0.05) in
the T2 (0.20%) than T3 (0.40%) or saline-treated pigs
(1.20%). Lung lesion score using bacterin B was also
significantly lower (P 6 0.05) than in the saline control
group.
bacterin A had 13/25 (52.0%) and bacterin B had 17/
26 (65.4%) positive PCR on DOT 125.
The numbers of ELISA positive animals (Table 5)
were 0 on DOT 0 in the saline and bacterin A groups
and 1/27 (3.7%) in the bacterin B vaccinated group.
On DOT 35, the proportion of pigs that showed sero-
conversion was 96.2% (25/26) in both vaccinated groups
that continued higher than 96% through to the end of
the study, while the control group had 0% (0/27) on
DOT 35, 7.7% (2/26) on DOT 66, 70.4% on DOT 97
and 92.6% (25/27) on DOT 125.
3.3. Body weights

3.2. PCR and ELISA results
The proportion of pigs with PCR positive results for
M. hyopneumoniae tonsil swabs (Table 4) was similar for
all treatment groups (P > 0.05) on DOT 0, 35, 66 and
97. On DOT 125, the two vaccinated groups had a sig-
nificantly lower (P = 0.0065) number of pigs with posi-
tive results compared to saline-treated pigs. There was
no significant difference between the two vaccinated
groups. The saline-treated group had 25/27 (92.6%),
There was no significant (P > 0.05) difference in mean
body weights between treatment groups at any time
evaluated (Table 6). At the end of the study on DOT
125, the least squares mean body weights were: saline
group 83.19 kg, T2 group 85.83 kg and T3 86.55 kg.
The total mean weight gains during the 125 days study
period were similar in all groups: saline 77.25 kg, T2
79.91 kg and T3 80.81 kg. The difference in average daily
weight gains between treatment groups (Table 7) also
was not significant (P > 0.05).

Table 3
Total severity of lung lesions back transformed least squares means (%) in each treatment group
T1 – saline solution T2 – bacterin A T3 – bacterin B
Back transformed least squares means (%) 1.20a 0.20b 0.40c
a,b,c
Between treatments, lung lesions back transformed least squares means with different superscripts are significantly different (P 6 0.05).

Table 4
Number of PCR positive pigs for M. hyopneumoniae at each sampling day
Treatment groups Number of PCR positive pigs/total (%)
DOT 0 DOT 35 DOT 66 DOT 97 DOT 125
T1 – saline solution 13/26a (50%) 1/27 (3.7%) 16/26a (61.5%) 25/27 (92.6%) 25/27a (92.6%)
T2 – bacterin A 14/27 (51.9%) 7/26b (26.9%) 19/25b (76.0%) 23/25 (92.0%) 13/25b (52.0%)
T3 – bacterin B 8/27 (29.6%) 3/26b (11.5%) 18/26 (69.2%) 24/26 (92.3%) 17/26b (65.4%)
P values 0.1262 0.0657 0.3583 0.6401 0.0065
DOT, day-on-test.
a
Samples from one pig were not suitable for testing.
b
One pig died before sampling.

Table 5
Number ELISA positive pigs for M. hyopneumoniae antibody at each sampling day
Treatment groups Number of ELISA positive pigs/total (%)
DOT 0 DOT 35 DOT 66 DOT 97 DOT 125
A a a,A a
T1 – saline solution 0/26 (0%) 0/27 (0%) 2/26 (7.7%) 19/27 (70.4%) 25/27 (92.6%)
T2 – bacterin A 0/27 (0%) 25/26b,B (96.2%) 24/25b,B (96.0%) 25/25b (100%) 25/25 (100%)
T3 – bacterin B 1/27 (3.7%) 25/26b,B (96.2%) 26/26b (100%) 25/26b (96.2%) 26/26 (100%)
P values Not tested 3 · 10 12 5.4 · 10 11 3.5 · 10 4 0.1063
DOT, day-on-test.
A
Samples from one pig were not suitable for testing.
B
One pig died before sampling.
a,b
Between treatments, different superscripts are significantly different (P 6 0.05).
530 M.R. Baccaro et al. / The Veterinary Journal 172 (2006) 526–531

Table 6 pigs was similar to that reported by Baccaro et al.

Least squares means of body weights per pig according to treatment (2002) in a study conducted in the same herd that had
Treatment Mean weight per pig (kg)a 23.7% of pigs with lesions in the vaccinated group
groups (two doses Respisure – Pfizer Animal Health) compared
DOT 0 DOT 35 DOT 125 Total weight
gain (Day to 57.4% in the saline-control pigs. Pommier et al. (2000)
0–125) reported a smaller reduction of lung lesions frequency,
T1 – saline solution 5.94 16.62 83.19 77.25 from 73.9% in the control to 53.3% in vaccinated pigs.
T2 – bacterin A 5.92 16.58 85.83 79.91 The challenge to which the pigs were exposed, as
T3 – bacterin B 5.74 16.01 86.55 80.81
demonstrated by the high pneumonia rate in the saline
DOT, day-on-test. group, was corroborated by the ELISA and PCR re-
a
There were no significant differences between treatments on any
sults. ELISA results in the saline group were negative
evaluated day at P 6 0.05.
at the study start until DOT 35. On DOT 66, 7.7% were
positive and this increased to 70.4% by Day 97 and
Table 7
Average daily weight gain (kg/day/pig) during each weighing period
92.6% at the end of the study indicating a progressive
(DOT 0–35; DOT 35–125 and DOT 0–125) in each treatment group exposure to the agent. The percentage of positive PCRs
Treatment groups Mean daily weight gain (kg/day/pig)a
was invariably high in all groups from the beginning of
the study, demonstrating contact with the Mycoplasma
DOT 0–35 DOT 35–125 DOT 0–125
as early as 25–31 days of age.
T1 – saline solution 0.30 0.74 0.62 A single vaccination of pigs at weaning with bacterin
T2 – bacterin A 0.30 0.77 0.64
T3 – bacterin B 0.29 0.78 0.65
A, resulted in a significantly lower lung lesion score
(0.2%) compared to bacterin B (0.4%) or saline-treated
DOT, day-on-test.
a
There were no significant differences between treatments on any
control pigs (1.2%). The mean lung lesion score obtained
evaluated period at P 6 0.05. by Baccaro et al. (2002) using the two-dose vaccine, Res-
ipsure, was also 0.2% compared with 2.5% in the control
group. In the present study the reduction of lung lesion
3.4. Feed conversion rate
scores was of the order of 83% with bacterin A and
66.7% with bacterin B when compared to the control
The feed conversion ratio in the grower/finisher
group. Pommier et al. (2000) reported a 50% reduction
phase, from DOT 35 to DOT 125 (Table 8), was signif-
in lung lesion score severity in vaccinated pigs.
icantly (P 6 0.05) better in both vaccinated groups com-
Reductions in growth rate, weight gain and feed
pared to saline-treated pigs but there was no significant
efficiency associated with enzootic pneumonia have all
difference between the two vaccinated groups. The feed
been reported by Pointon et al. (1985) and Ross (1999).
conversion ratio in the saline-treated group was 3.125,
In the present study, there was no significant (P > 0.05)
in bacterin A was 2.990 and in bacterin B was 2.981.
difference in least square mean body weights or mean
daily weight gains at any time evaluated. Such results
are probably due to the short period of time between peak
4. Discussion
infection (DOT 97) and slaughter (DOT 125) and because
the severity of infection that was not high enough to influ-
The swine herd where the study was conducted was
ence weight and weight gain in the control group (mean
exposed to a high M. hyopneumoniae challenge. Despite
lesion scores of 1.20%). However, it influenced the feed
the elevated pneumonia rate observed at slaughter in the
conversion ratio. This result is in accordance with
saline treated group (77/139–55.4%), the vaccination
Pommier et al. (2000) who did not find any significant dif-
with one dose vaccines reduced this rate significantly.
ferences among average daily weight gains per pig when
The number of pigs with lung lesions was 42/155
they compared vaccinated and non-vaccinated groups.
(27.1%) in the bacterin A group compared to 58/152
The impact of uncomplicated M. hyopneumoniae
(38.2%) in the bacterin B group. The decrease observed
infections may be minimal, even in the presence of
in lung lesion frequency in the bacterin A vaccinated
presumed environmental stress (Clark et al., 1993).
Table 8 However, M. hyopneumoniae infections have been
Feed conversion ratio during the grower/finisher phase (from DOT shown to potentiate disease with other infections agents
35–125) such as PRRS virus (Thacker et al., 1999) and bacterial
T1 – saline T2 – T3 – infections (Ross, 1999). The benefits of vaccination may
solution bacterin A bacterin B be greater when such complicating factors are present
Feed conversion ratio 3.125a 2.990b 2.981b (Thacker et al., 2000).
DOT, day-on-test.
The feed conversion ratio was significantly (P 6 0.05)
a,b
Between treatments, feed conversion ratio with different super- lower in both vaccinated groups (bacterin A – 2.990 and
scripts are significantly different (P 6 0.05). bacterin B – 2.981) compared to the saline control group
 M.R. Baccaro et al. / The Veterinary Journal 172 (2006) 526–531
(3.125). These results are in agreement with Baccaro
et al. (2002) who obtained a significantly better feed con-
version ratio in vaccinated pigs (2.527) than in their con-
trol group (2.905).
Whereas the finishing weight and ADG were not sig-
nificant with P values > 0.05, the vaccinated pigs were
heavier on average by 2.5–3 kg. This may be meaningful
from a financial standpoint especially when combined
with the improvement in feed conversion rate. This
financial benefit may increase the incentive to vaccinate,
even in the apparently uncomplicated disease condition
reported in this study.
On DOT 125, the number of M. hyopneumoniae
PCR-positive tonsil swabs was significantly higher
(P 6 0.05) for the saline-treated than either vaccinated
groups. This finding can be related to the higher elimina-
tion of the agent in the control group than in the vacci-
nated groups. Studies associating vaccine efficacy and
M. hyopneumoniae detection by PCR were not found
in the literature. It is expected that vaccination proce-
dures must reduce the presence of the agent in the envi-
ronment and consequently the infection pressure. The
significant reduction of animals eliminating the agent
on DOT 125 in the vaccinated groups, confirms this
supposition.
The number of animals exhibiting ELISA positive re-
sults was >96% by DOT 35 for both vaccinated groups
and was significantly higher than the saline group
through DOT 97. This result is related to the detection
of immune responses to vaccination, demonstrating that
both vaccines induced seroconversion equally (>96%),
in contrast to the results of Roof et al. (2001) who found
a 0% seroconversion with RespiSure One and 80% with
Ingelvac M. hyo.
From the results reported here, it is concluded that
although both vaccines induced a high proportion of
seroconversion and better feed efficiency than the con-
trol, bacterin A (RespiSure One) was more effective than
bacterin B (Ingelvac M. hyo) in preventing and reducing
the severity of lung lesions caused by M. hyopneumoniae
in swine raised under commercial conditions. Greater
weight, better weight gain and improved feed efficiency
are consequences that depend on the severity of disease.
In the present study, although the severity of M. hyo-
pneumoniae infection was not high enough to signifi-
cantly influence weight and weight gain in the control
group, it did influence the feed efficiency. Vaccination
is an important preventative tool in the control of M.
hyopneumoniae infection and the harmful consequences
of the disease. Many variables need to be considered
in the decision to vaccinate and vaccination may not
be appropriate in every case. The decision is best made
in conjunction with a veterinarian familiar with the
herd.
531
Acknowledgements
This study was supported by Pfizer Animal Health.
The authors warmly thank Ms. Vickie King from the
Pfizer VMRD Biometrics, Quality and Technology,
Groton, CT, USA, for her invaluable help in the study
design and for the statistical analysis.
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