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Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs’ lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae . A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis , whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.
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Tonnietal. Veterinary Research (2022) 53:41
https://doi.org/10.1186/s13567-022-01061-w
RESEARCH ARTICLE
The role ofco-infections inM.
hyopneumoniae outbreaks amongheavy
fattening pigs: aeld study
Matteo Tonni1* , Nicoletta Formenti1, M. Beatrice Boniotti1, Flavia Guarneri1, Federico Scali1, Claudia Romeo1,
Paolo Pasquali2, Maria Pieters3, Dominiek Maes4 and Giovanni L. Alborali1
Abstract
Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fat-
tening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence
on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial
swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and
1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A,
and Group C was located in a different building. Pigs’ lungs were scored at the slaughterhouse. TBS were tested for
the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped
by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence
and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma
hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher.
Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections)
increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory
syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae
and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections
suggest that increased biosecurity might be beneficial for lung lesion sequelae.
Keywords: Multiple-locus variable-number tandem repeat analysis, Mycoplasma hyopneumoniae, swine, variable-
number tandem repeat type, fattening pigs
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Introduction
Mycoplasma hyopneumoniae is one of the primary agents
involved in porcine respiratory disease complex (PRDC)
[1] along with a combination of other infectious viral and
bacterial pathogens [14]. Multiple patterns of co-infec-
tion are possible [5] depending on the timing of infection
and the pathogens involved. e immunomodulatory
effect that viruses play in virus-bacteria superinfections is
sometimes altered by M. hyopneumoniae that promotes
both viral and bacterial infections. An example is the
potentiation effect of M. hyopneumoniae during PRRSV
co-infections that also leads to an increase in viral shed-
ding [5]. e outcomes of co-infections vary depending
on the type of interactions; i.e., synergy, neutrality, or
antagonism among the microorganisms [5]. e clinical
signs and lung lesions of M. hyopneumoniae infections
depend on many factors [2], such as viral and bacterial
superinfections [1] or the presence of different circulat-
ing variable-number tandem repeat (VNTR) types. e
Open Access
*Correspondence: matteo.tonni@izsler.it
1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna,
Via Bianchi, 9, 25124 Brescia, Italy
Full list of author information is available at the end of the article
Page 2 of 10
Tonnietal. Veterinary Research (2022) 53:41
effects of lung microbiota on lung health [6] and possi-
ble lung lesion sequelae are still unclear, although some
studies are starting to shed light on this topic [7, 8]. M.
hyopneumoniae is one the most common agents associ-
ated with pneumonia in the slaughterhouse setting [5,
9]. e impact of other co-infecting microorganisms on
lung lesions has been hypothesized [10] and investigated
by experimental infection [11, 12], but studies performed
under field conditions are lacking. e genomic variabil-
ity of M. hyopneumoniae [1317] and the resulting lung
lesions [18] have been widely studied. However, little is
known about the role of co-infections on M. hyopneumo-
niae genetic diversity and their differential effects on the
resulting lung sequelae.
is study was performed to assess the dynamics of
M. hyopneumoniae infection during clinical respira-
tory outbreaks in heavy fattening pigs. In particular, the
main purpose was to clarify the role of co-infections in
M. hyopneumoniae outbreaks and their impact on conse-
quent lung lesions. An additional aim was to investigate
how co-infection affects the presence of M. hyopneumo-
niae genotypes and whether infections of multiple VNTR
types might influence the magnitude of lung lesions.
In this study, M. hyopneumoniae seemed to play a cen-
tral role in respiratory outbreaks in fattening pigs, with
a direct effect on lung lesion scores. Moreover, the pres-
ence of M. hyopneumoniae infections of multiple VNTR
types led to more severe lung lesions. Otherwise, co-
infections were found to have marginal effects with no
relevant influence on the severity of lung lesions.
Materials andmethods
Study design
is study was performed from May 2016 to April 2018
in 10 fattening farms. All the enrolled farms belonged to
multisite production flows with a history of M. hyopneu-
monaie. All pigs enter the fattening units at the approxi-
mate age of 60days. Veterinary practitioners belonging
to two major practices were asked to immediately contact
Istituto Zooprofilattico Sperimentale della Lombardia e
dell’Emilia Romagna (IZSLER) if they suspected an enzo-
otic pneumonia (EP) outbreak in finishing pigs. Herds in
which fattening pigs showed continuative clinical signs
of sneezing and a dry non-productive cough [19] were
enrolled in the present study. Herds in which pigs showed
signs such as fever, anorexia, and laboured breathing
were excluded because these signs suppose the presence
of other bacterial and/or viral agent outbreaks [19].
In every farm, 3 groups (Groups A, B, and C) of 10 pigs
each were defined in relation to the animals that mani-
fested symptoms first. Pigs within each group were ran-
domly selected. Group A comprised pigs that first showed
acute clinical signs. Group B comprised asymptomatic
pigs housed in the pen next to Group A. Group C com-
prised asymptomatic pigs housed in another barn of the
farm. e pigs enrolled in the study were individually
identified with ear tags. e housing conditions were the
same for Groups A, B, and C. Tracheobronchial swabs
(TBS) were simultaneously collected from all 30 pigs on
each farm at two time points: at the onset of clinical signs
in Group A (T0) and 32 ± 6days later, a reasonable time
interval for M. hyopneumoniae infection to turn into the
recovery phase (T1) [1]. Lungs of the selected pigs were
scored for lesions at the slaughterhouse (T2).
Farm management
All farms were located in the northern region of Italy and
followed an all-in/all-out protocol within a multisite pro-
duction flow system. Pigs were housed in confined barns
with natural ventilation systems. Vaccination protocols
included Aujeszky disease virus, porcine circovirus type
2 (PCV2), and M. hyopneumoniae. On all farms, vaccina-
tion against Aujeszky disease was performed three times
(the last during the fattening phase), whereas PCV2 vac-
cination involved a single shot in the farrowing site. e
M. hyopneumoniae vaccination strategies are shown in
Table1. None of the considered fattening farms adopted
the anthelmintic treatment regime.
None of the selected pigs had undergone any phar-
macological treatment with the exception of individual
therapy with tetracyclines or macrolides administered to
animals not under study in all farms following the onset
of respiratory symptoms.
Sampling procedures
Tracheobronchial swabs (TBS) were collected through a
60-cm catheter (Portex® Catheter; Smiths Medical, Min-
neapolis, MN, USA). As the pig inspired, the catheter was
inserted into the trachea with the aid of a laryngoscope
and mouth speculum. e catheter was gently inserted
deeply into the trachea until the first branching of the
bronchial tree in accordance with a standard procedure
[20]. Immediately after sampling, the tip of the catheter
was inserted into a sterile tube with 2mL of phosphate-
buffered saline (PBS) and transferred under refrigerated
conditions (5°C ± 3°C) to the IZSLER laboratory.
Lungs were scored after slaughter following the
method proposed by Madec and Kobisch [21] based
on the magnitude of greyish to purplish consolidated
pneumonic areas on each lung lobe surface: 0% = no
lesion, <25% = score of 1, 25%–49% = score of 2, 50%–
74% = score of 3, and >75% = score of 4. Based on the
total lung score (range of 0 to 28), the severity of lung
lesions was classified as suggested by Hillen etal. [22]:
no lesions (score of 0), mild lesions (score of 1–4), severe
Page 3 of 10
Tonnietal. Veterinary Research (2022) 53:41
lesions (score of 5–9), or very severe lesions (score
of 10).
Pathogen detection
Pathogen investigation focused on M. hyopneumoniae
and the main PRDC agents known to occur in the consid-
ered geographic area [4]: Actinobacillus pleuropneumo-
niae, Mycoplasma hyorhinis (M. hyorhinis), Glässerella
parasuis, Bordetella bronchiseptica, Trueperella pyo-
genes, porcine reproductive and respiratory syndrome
virus (PRRSV), PCV2, swine influenza A virus (swIAV),
and Pasteurella multocida [4]. Samples positive for M.
hyopneumoniae were also genotyped by multiple-locus
variable-number tandem repeat analysis (MLVA).
Bacterial isolation
Because of its ease and reliability of execution [1], bac-
terial culture was adopted to detect P. multocida, B.
bronchiseptica, and T. pyogenes. A drop of the tracheo-
bronchial secretions contained in the sterile tube with
TBS and PBS was seeded onto a blood agar culture
plate through a sterile loop. All plates were incubated at
37°C in an aerobic atmosphere for 24 ± 3h. e bacte-
rial growth was evaluated by examining the morphology
of the colony, performing Gram staining, and performing
biochemical tests.
In the case of a mucous colony, P. multocida was sus-
pected and specific biochemical tests (API NE; bioMer-
iéux) were carried out to confirm the pathogen identity
[23]. No colony suspected to be B. bronchiseptica or T.
pyogenes was detected; therefore, these two pathogens
were excluded from the study.
Tracheobronchial swab samples were considered
positive when at least one colony of P. multocida was
detected.
Molecular biology
e biomolecular assays used in the present study were
chosen to meet the requirements of the quality manage-
ment system of IZSLER based on UNI CEI EN ISO/IEC
17025 [24].
DNA and RNA were extracted from TBS and lung
homogenates using an automated extraction platform
(KingFisher; ermo Fisher Scientific, Waltham, MA,
USA). e eluted nucleic acids then underwent molecu-
lar tests. Real-time polymerase chain reaction (PCR)
for M. hyopneumoniae, M. hyorhinis, A. pleuropneumo-
niae, PRRSV, PCV2, and swIAV were carried out using
a C1000 thermal cycler (Bio-Rad Laboratories, Hercules,
CA, USA), while conventional PCR (G. parasuis) was
performed on an Applied Biosystems GeneAmp® PCR
System 9700 (ermo Fisher Scientific).
e real-time PCR adopted to detect M. hyopneumo-
niae followed the protocol proposed by Marois etal. [25]
with the detection limit set at a 37-cycle threshold (Ct)
value. Positive samples were further submitted to MLVA
genotypic analysis.
Another real-time PCR assay based on the gene encod-
ing M. hyorhinis protein p37 [26] was developed to detect
this mycoplasma, setting a 38-Ct limit. PRRSV RNA was
detected using the Applied Biosystems LSI VetMAX
PRRSV EU/NA real-time PCR kit (ermo Fisher Scien-
tific) as specified by the manufacturer, with a threshold
detection limit of 37-Ct. e presence and quantification
of PCV2 was assessed through real-time PCR accord-
ing to the protocol published by Olvera etal. [27]; the
IZSLER laboratory threshold limit was set at 40-Ct.
Because of the potentially low sensitivity of TBS for
PRRSV and PCV2, serology data was collected at both
T0 and T1, to better assess the farm status, resulting in
seroprevalences > 90% for both cases and at both time
Table 1 Description of the ten fattening pig herds included in the study
* Vaccination strategies were resumed in 1 (single shot at 20–28days) or 2 (double shots before and after weaning).
T0: onset of clinical signs during the EP outbreak.
Farm Herd Size (Nb of pigs) Enrolled pigs M. hyopneumoniae vaccination
schemes* Age of pigs at T0 (days) Age at
slaughtering
1 4000 30 1 131 245
2 4800 30 1 153 250
3 10 000 30 1 176 270
4 1500 30 1 204 280
5 3200 30 1 158 255
6 2300 30 2 238 265
7 3900 30 2 143 280
8 5300 30 2 232 275
9 1500 30 1 206 265
10 4500 30 2 124 255
Page 4 of 10
Tonnietal. Veterinary Research (2022) 53:41
points. SwIAV was detected through real-time reverse-
transcriptase PCR with amplification of the M gene of
influenza A virus following the protocol described in the
Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals [28]. Detection of G. parasuis was performed
using the qualitative PCR protocol standardized by
Oliveira etal. [29]; the PCR product was loaded on a 2.0%
agarose gel, run at 100V for 40min, and visualized under
ultraviolet light.
Mycoplasma hyopneumoniae MLVA genotyping
For the samples positive for M. hyopneumoniae by real-
time PCR, the number of copies was elaborated based
on a standard reference curve. ese samples were gen-
otyped after with MLVA. Briefly, the method consisted
of four conventional PCR for the amplification of Locus
1, Locus 2, P97-RR1, and P97-RR2 genes in accord-
ance with the scheme proposed by Charlebois etal. [13]
and Tonni etal. [17]. e PCR were performed with an
Applied Biosystems thermocycler (ermo Fisher Scien-
tific), and the PCR products were examined by capillary
electrophoresis (QIAxcel; Qiagen, Hilden, Germany). In
case of unclear results, further evaluation was performed
with a 2.0% high-resolution agarose gel run at 100V for
2h and visualized under ultraviolet light. For each VNTR
locus, the estimated number of tandem repeats was cal-
culated according to the allele calling table (Additional
file1). Each VNTR type was defined based on the num-
ber of repeats per locus. e VNTR type nomenclature
was assigned following the chronological order of identi-
fication at the IZSLER laboratory.
e presence of one or more M. hyopneumoniae VNTR
types per sample was defined as a single (SN) or mixed
(MX) infection, respectively.
Statistical analysis
For all 300 pigs, the probability of being infected by M.
hyopneumoniae during the outbreak and before slaugh-
tering was examined through a mixed logistic regres-
sion, including time (T0 or T1), experimental group (A,
B, or C), time × group interaction, and infection status
(infected or not infected by other pathogens) as explana-
tory variables. In the subset of pigs infected by M. hyo-
pneumoniae (n = 229), we also explored the variation in
the ln-transformed number of copies through a general
linear mixed model, including the same set of explana-
tory variables. Finally, the effect of these same variables
on the probability of having an MX infection was ana-
lysed through a second mixed logistic regression. In all
these models, pig and farm ID were included as random
factors to account for repeated measures on the same
individual and for within-farm variability, respectively.
In all cases, models in which the farm was included as a
random intercept were significantly different from simple
models (all p < 0.0001).
Finally, we explored variation in the aggregated lung
lesion scores of pigs (i.e., no lesions, mild lesions, severe
lesions, or very severe lesions) observed at T2 through
two different mixed ordinal logistic regressions. In the
first model, we included all pigs (n = 300) and examined
the effect of the group and M. hyopneumoniae infec-
tion status (i.e., infected at least once in time or never
infected). In this model, we also included the infec-
tion status by M. hyorhinis, A. pleuropneumoniae and P.
multocida, because they are known to potentially cause
severe lung lesions. In the second model, we included
only animals that had been infected at least once by M.
hyopneumoniae (n = 280) and considered as explanatory
variables the experimental group, the total number of co-
infections, and MLVA history (i.e., MX genotype infec-
tion at least once in time or always SN infection). In both
models, the farm ID was included as a random intercept.
In all analyses, we started from full models and
obtained minimal models through backward elimination
of non-significant variables (partial p-value for removal
set at 0.15). For the linear model, comparisons of signifi-
cant variables with more than two levels were analysed
through t-tests on differences in least squares means,
applying Holm correction for multiple comparisons. For
logistic regressions, odds ratio (OR) estimates and their
95% confidence intervals (CIs) are reported. For the lin-
ear mixed model, normality of residuals was assessed
visually. For logistic regressions, overdispersion was
checked through the generalized chi-square/degree of
freedom ratio, and the areas under the receiver operating
characteristic curves (AUC) are reported below.
All analyses were carried out through PROC GLIM-
MIX in SAS/STAT 9.4 software (SAS Institute Inc., Cary,
NC, USA).
Results
Mycoplasma hyopneumoniae infection andVNTR types
Overall, 442 of the 586 examined TBS (corresponding
to 300 different pigs) were positive for M. hyopneumo-
niae (75.4%; 95% CI 71.9–78.9). e prevalence of posi-
tive swabs in farms ranged from 8.5% to 100%. A detailed
breakdown of the prevalence of M. hyopneumoniae and
all other infections by time and by experimental group is
provided in Table2. From T0 to T1, 11 pigs of Group C
that developed non-respiratory disease and 3 pigs (1 in
each group) that died were excluded from the study.
e probability of a pig being infected by M. hyopneu-
moniae varied significantly with groups, by the M. hyor-
hinis infection status, and by the P. multocida infection
status (all p < 0.05) (mixed logistic regression, N = 600
samples, AUC = 0.93; Table3). In detail, pigs from Group
Page 5 of 10
Tonnietal. Veterinary Research (2022) 53:41
A had higher odds of being infected by M. hyopneumo-
niae than pigs from both Group B (OR, 2.7; 95% CI 1.1–
6.2) and Group C (OR, 11.7; 95% CI 4.9–27.9), whereas
pigs from Group B were more likely to be infected than
pigs from Group C (OR, 4.4; 95% CI 2.1–9.3). Addition-
ally, co-infection by M. hyorhinis was positively associ-
ated with the M. hyopneumoniae infection status (OR,
1.8; 95% CI 1.1–3.0), whereas P. multocida infection
was instead negatively associated with it (OR, 0.5; 95%
CI 0.6–0.9). Overall, the prevalence of M. hyorhinis was
indeed higher in pigs co-infected by M. hyopneumoniae,
whereas the prevalence of P. multocida was conversely
higher in pigs free from M. hyopneumoniae infection
(Figure1).
e number of copies of M. hyopneumoniae in positive
pigs ranged from 7.1 to 21.8 ln-copies [mean ± stand-
ard error (SE), 14.8 ± 0.2]. It increased with both M.
hyorhinis (parameter estimate ± SE, 1.11 ± 0.33 ln cop-
ies) and A. pleuropneumoniae co-infection (0.65 ± 0.40
ln copies) and varied significantly in time depending
on the group (all p < 0.05) (linear mixed model, N = 442
samples; Table4). At T0, there was no difference in the
number of copies between positive animals from Groups
A and B (padj = 0.055), but they both had a significantly
higher number of copies than positive pigs from Group
C (padj < 0.0001 and p = 0.003, respectively). However,
the number of copies in Groups A and B decreased sig-
nificantly from T0 to T1 (padj = 0.002 and padj = 0.01),
while the average number of copies in positive pigs from
Group C did not change over time (padj = 0.95). As a con-
sequence, at T1 there was no longer a difference in the
number of copies among any of the groups (all padj > 0.05).
Detailed results of the DSLM post-hoc comparisons are
shown in Table4.
With respect to the VNTR types, the overall preva-
lence of MX infections in the examined positive sam-
ples was 12.3% (95% CI 9.0–15.5). A breakdown of
prevalence by time and experimental group is shown
in Table3. e probability of positive pigs having an
MX infection increased significantly with PRRSV co-
infection (OR, 3.1; 95% CI 1.1–9.3) and varied in time
depending on the group (both p < 0.05) (mi xed logistic
Table 2 Respiratory infections in heavy-fattening pigs: prevalence (% of infected animals/examined animals) at T0 and
T1 and incidence (% of infected animals/susceptible animals) at T1 in Group A (NT0 = 100, NT1 = 99), Group B (NT0 = 100,
NT1 = 99) and Group C (NT0 = 100, NT1 = 88)
Infection Group T0 T1
P (%) 95% CI (%) P (%) 95% CI (%) I (%) 95% CI (%)
M. hyopneumoniae A 85.0 77.9–92.1 86.9 80.1–93.6 26.7 4.3–49.0
B 82.0 74.3–89.7 73.7 64.9–82.6 27.8 7.1–48.5
C 62.0 52.3–71.7 61.4 51.0–71.7 29.7 15.0–44.5
A. pleuropneumoniae A 27.0 18.1–35.8 24.2 15.6–32.8 23.3 13.6–33.0
B 19.0 11.2–26.8 20.2 12.1–28.2 22.2 13.2–31.3
C 22.0 13.7–30.3 14.8 7.2–22.3 12.3 4.8–19.9
G. parasuis A 40.0 30.2–49.8 55.6 45.6–65.5 50.8 38.1–63.6
B 28.0 19.0–36.9 56.6 46.6–66.5 52.1 40.5–63.7
C 31.0 21.8–40.2 40.9 30.4–51.4 31.7 19.9–43.4
M. hyorhinis A 67.0 57.6–76.4 64.6 55.1–74.2 59.4 42.4–76.4
B 64.0 54.4–73.6 59.6 49.7–69.4 54.3 37.8–70.8
C 55.0 45.1–64.9 45.4 34.8–56.1 35.7 21.2–50.2
P. multocida A 52.0 42.0–62.0 8.1 2.6–13.5 4.2 0–9.8
B 7.0 1.9–12.1 36.4 26.7–46.0 35.9 26.1–45.7
C 5.0 0.6–9.3 22.7 13.8–31.6 22.9 13.8–31.9
PCV2 A 11.0 4.8–17.2 8.1 2.6–13.5 0
B 9.0 3.3–14.7 7.1 1.9–12.2 0
C 8.0 2.6–13.4 10.2 3.8–16.7 1.7 0–5.9
PRRSV A 11.0 4.8–17.2 2.0 0–4.8 1.1 0–3.3
B 6.0 1.3–10.7 1.0 0–3.0 1.0 0–3.2
C 3.0 0–6.4 0 0 0
SwIAV A 0 0 0
B 0 0 0
C 2.0 0–4.8 0 0
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Tonnietal. Veterinary Research (2022) 53:41
regression, N = 400 samples; AUC = 0.84; Table 3). In
detail, in Groups A and B there was no variation in the
occurrence of MX infection from T0 to T1, whereas in
pigs from Group C there was a significant increase in
the probability of showing an MX at T1 (OR, 11.1; 95%
CI 3.5–36.5; padj = 0.0008) (see also Figure2).
Lung lesions
e raw total lung lesion scores in pigs examined at T2
(n = 287) ranged from 0 to 22 (mean ± SE, 4.9 ± 0.3).
Table 3 Minimal models exploring factors aecting variation in M. hyopneumoniae infection status, ln-transformed no. of
copies and VNTR types in pigs
In all the models, pig IDs and farm IDs were included as random intercepts.
mixed logistic regression; * linear mixed model; a only positive animals; b Group C held as reference; c T0 held as reference; d not-infected held as reference.
Response variable Explanatory variable Parameter
estimate ± SE Statistic p-value
M. hyopneumoniae infection statusGroupbA 2.67 ± 0.55 χ22 = 32.8 <0.0001
B 1.98 ± 0.47
TimecT10.37 ± 0.33 χ21 = 0.25 0.62
M. hyorhinis
infection statusdInfected 0.58 ± 0.27 χ21 = 4.59 0.032
P. multocida infection statusdInfected 0.71 ± 0.33 χ21 = 4.49 0.034
Group:Timeb, c Group A: T10.42 ± 0.61 χ22 = 4.01 0.13
Group B: T11.01 ± 0.50
M. hyopneumoniae no. of copies (ln-
transformed)*, a GroupbA 3.45 ± 0.54 F2, 425 = 19.7 <0.0001
B 2.04 ± 0.55
TimecT10.03 ± 0.59 F1, 425 = 14.7 0.0001
M. hyorhinis
infection statusdInfected 1.11 ± 0.33 F1, 425 = 11.2 0.0009
A. pleuropneumoniae infection statusdInfected 0.65 ± 0.40 F1, 425 = 2.70 0.10
Group:Timeb, c Group A: T11.84 ± 0.75 F2, 425 = 3.48 0.032
Group B: T11.74 ± 0.77
M. hyopneumoniae MLVA†, a GroupbA1.73 ± 0.51 χ22 = 1.02 0.60
B1.18 ± 0.49
TimecT12.41 ± 0.59 χ21 = 9.20 0.0024
PRRSV
infection statusdInfected 1.13 ± 0.56 χ21 = 4.09 0.043
Group:Timeb, c Group A: T12.54 ± 0.70 χ22 = 13.42 0.0012
Group B: T12.13 ± 0.71
Figure1 M. hyopneumoniae co-infections in fattening pigs.
Prevalence of other respiratory infections in pigs infected (dark bars,
n = 442) and not infected (white bars, n = 144) by M. hyopneumoniae.
Bars indicate 95% Confidence Intervals. Asterisks indicate significance
level at p < 0.05 in the mixed logistic regression model.
Table 4 Dierences of least square means by time and
group in M. hyopneumoniae no. of copies (ln-transformed) in
samples (N = 442) from infected pigs
Time Group Estimate ± SE t425 padj
T0A vs B 1.41 ± 0.47 2.83 0.055
A vs C 3.45 ± 0.54 6.37 <0.0001
B vs C 2.04 ± 0.55 3.72 0.0031
T1A vs B 1.31 ± 0.51 2.54 0.11
A vs C 1.61 ± 0.57 2.82 0.056
B vs C 0.30 ± 0.58 0.51 0.99
T1 vs T0A1.81 ± 0.47 3.84 0.0020
B1.71 ± 0.50 3.40 0.010
C 0.03 ± 0.59 0.06 0.95
Page 7 of 10
Tonnietal. Veterinary Research (2022) 53:41
Ninety-two pigs showed no lesions, 76 pigs showed
mild lesions, 67 pigs showed severe lesions, and 52 pigs
showed very severe lesions as shown in Table5. Over-
all, the probability of observing severe lesions was sig-
nificantly affected only by a history of M. hyopneumoniae
infection (χ21 = 20.15; p < 0.0001), with pigs infected
at least once having 40 times the odds of showing very
severe lesions. Indeed, of the 19 animals that were never
infected by M. hyopneumoniae, 17 showed no lesions
and 2 showed mild lesions; none showed severe or very
severe scores. Neither the experimental group nor any
of the other respiratory infections included in the model
had a significant effect on lung lesions (all p > 0.05). Con-
sidering only pigs with a history of M. hyopneumoniae
infection, lung lesions were affected only by M. hyopneu-
moniae21 = 12.1; p < 0.0001). Pigs with MX infections
had significantly higher odds of showing severe lesion
scores than pigs with SN infections (OR, 3.0; 95% CI
1.6–5.6). Once again, neither the experimental group nor
the number of co-infections had any effect on the sever-
ity of lung lesions (p = 0.36 and p = 0.76, respectively).
Discussion
e present study was performed to investigate the
dynamics of M. hyopneumoniae and its interactions
with other respiratory pathogens in pig farms. e farms
enrolled in the study were therefore specifically selected
because of reported clinical signs such as non-productive
coughing and sneezing, which led to the suspicion of EP
outbreaks. Consequently, the prevalence of M. hyopneu-
moniae in our sampled population was indeed higher
than that of all other respiratory infections.
e three different groups of pigs sampled in each farm
were defined according to farm localization and clinical
conditions, leading to different exposures to M. hyopneu-
moniae among the groups independent of the sampling
time. Group A included pigs in which clinical signs were
first observed, and as expected, this group had the high-
est probability of being infected by M. hyopneumoniae.
Conversely, animals housed near Group A or stalled in
another building (Group B and C, respectively) were less
likely to be positive for M. hyopneumoniae.
On the contrary to the infection status, the M. hyo-
pneumoniae load varied in time but with a different pat-
tern depending on the group. e highest PCR number
of copies was detected at T0 in Groups A and B and at T1
in Group C. Because the highest burden of M. hyopneu-
moniae has been proven to occur at 28days post-infec-
tion [30], it can be assumed that the early stages of the
EP outbreaks occurred at T0 in Groups A and B and at
T1 in Group C. Considering the higher sensitivity of real-
time PCR on TBS samples collected in the early stage of
the outbreak [31], these findings suggest that sampling of
pigs with clinical signs may lead to a more effective and
timely diagnosis of EP outbreaks.
e role of M. hyopneumoniae as a gate opener for sec-
ondary bacterial infections, especially P. multocida and A.
pleuropneumoniae, is well known [2, 19]. In the present
study, the wide panel of analysed pathogens allowed for
the study of several possible co-infections, but the only
positive association was detected between M. hyopneu-
moniae and M. hyorhinis, with the latter more frequently
observed in the presence of the former. Mycoplasma
hyopneumoniae is one of the main pathogens involved
in swine pneumonia [1], whereas M. hyorhinis generally
has slight importance with mild or no clinical respiratory
signs [32]. No positive association was detected between
the presence of M. hyopneumoniae and other respira-
tory pathogens, with P. multocida even showing a slightly
negative association with M. hyopneumoniae infection.
e prevalence of P. multocida was indeed higher in
Figure2 Dynamics of M. hyopneumoniae mixed infections in
fattening pigs. Prevalence of M. hyopneumoniae mixed infections
(i.e. showing multiple VNTR genotypes) in positive pigs at T0 (light
bars) and T1 (dark bars) per experimental group. Error bars indicate
95% Confidence Intervals. Asterisks indicate significance level at
padj < 0.001 in t-tests on differences of least squares means.
Table 5 Severity of lesions based on the lung lesion score
according to the Madec and Kobish method [18]
Data refer to all 10 farms for a total of 287 scored pigs.
a Score = 0; b score 1–4; c score 5–9; d score 10.
Magnitude of lesion P (%) 95% CI (%)
No lesiona32.1 26.6–37.5
Mild lesionsb26.0 21.3–31.6
Severe lesionsc23.3 18.4–8.3
Very severe lesionsd18.1 13.6–22.6
Page 8 of 10
Tonnietal. Veterinary Research (2022) 53:41
pigs free from M. hyopneumoniae, which may suggest
an unfavourable interaction between these two bacte-
rial infections. However, a possible explanation for this
observation is the strict selection criteria of the enrolled
farms, which included pigs with signs of EP but without
the typical signs of a complicated infection (e.g., pyrexia,
anorexia, lethargy, or even death) [1]. In other words, the
rigorous enrolment of farms with pigs showing typical
signs of EP may have promoted the selection of pure and
uncomplicated M. hyopneumoniae infections; therefore,
the presence of P. multocida was presumably not due to
secondary irruption. Furthermore, because P. multocida
was isolated on only a few farms, we cannot exclude the
possibility that this weak negative association was due to
the limited sample size.
Although no association was detected between the
presence of M. hyopneumoniae and A. pleuropneumo-
niae, co-infection by these two bacteria was found to be
more common when M. hyopneumoniae showed a higher
number of copies in the PCR assay. Similarly, infections
with M. hyorhinis were more frequent with a higher M.
hyopneumoniae load. Despite the fact that the exact
mechanisms of co-infections in respiratory diseases are
not well known [5], a possible explanation for this result
is the need for M. hyopneumoniae to be well established
in the respiratory tract to potentiate secondary patho-
gens [5]. Consequently, containing the proliferation of M.
hyopneumoniae could be beneficial to avoid A. pleuro-
pneumoniae and M. hyorhinis co-infections.
e prevalence of several respiratory pathogens seemed
to vary with time and by group, but the often-limited
number of infected pigs coupled with the non-random
selection of farms did not allow for a more detailed analy-
sis of the other infection dynamics. Furthermore, the vac-
cination strategies (including the type of vaccine used)
were not considered as variables in this study and should
be investigated in the future.
e respiratory pathogens considered in this study are
the most common agents detected in pig farms in the
study area [4]; therefore, since no deworming scheme
has been adopted and because Ascaris suum was not
considered, this should be considered a limitation of the
research.
Actinobacillus pleuropneumoniae is a major agent of
chronic pleurisy [1]. However, we rarely found pleural
lesions in our sample, and this prompted us to not con-
sider the pleurisy score.
Concerning the dynamics of M. hyopneumoniae
VNTR types, the prevalence of M. hyopneumoniae MX
infections was stable through time in Groups A and
B, whereas it increased significantly from T0 to T1 in
Group C. Considering the aforementioned trend of
the M. hyopneumoniae load that suggested the onset
of the outbreak at T0 in Groups A and B and at T1 in
Group C, the MX infections appeared to occur mainly
in pigs later and farthest from where the outbreak of EP
started.
Since the endemic presence of M. hyopneumoniae com-
plicates the interpretation of these findings, the currently
available data did not allow us to assess the actual mecha-
nism that is occurring. However, a possible interpreta-
tion is that when an EP outbreak occurs, the VNTR types
involved remain the same throughout the pathogenic
evolution within each pig. e subsequent phase of the
outbreak with extensive shedding of M. hyopneumoniae
instead induces the circulation of high loads of different
VNTR types, increasing the risk of pigs being exposed to
multiple VNTR types. e data show a complex dynamic
of different genotype infections with an evolution of the
VNTR types along the different stages of the outbreak.
erefore, the timing of sampling could greatly affect
the likelihood of detecting all genotypes hosted by a pig.
However, these results should be interpreted with cau-
tion because although TBS are considered a very sensi-
tive type of sample for intra vitam diagnosis [20], VNTR
types are individually hosted in each lung lobe [17], and
the ability of TBS to detect all VNTR types in each lung
has never been assessed.
TBS analysis was our primary choice because it is the
most sensitive method for the diagnosis of M. hyopneu-
moniae in live animals [20]. However, its sensitivity for
some of the other investigated respiratory pathogens is
either lower or unknown. For instance, A. pleuropneu-
moniae and G. parasuis are more reliably cultured from
lung tissue [1, 33], and this should be considered a partial
limitation of this procedure. Despite this, TBS were cho-
sen because they would have allowed for the successful
detection of the widest range of pathogens among those
known to circulate in the study area.
Additionally, we found that the probability of M.
hyopneumoniae-positive pigs having an MX infection
increased significantly with PRRSV co-infection. e
presence of active PRRSV circulation is considered an
indirect index of biosecurity [34], and low biosecurity
has also been suspected of being related to infection with
multiple M. hyopneumoniae VNTR types [35]. us,
improvement in external biosecurity could prevent the
entrance of pathogens such as PRRSV [34] and might also
avoid the introduction of several M. hyopneumoniae gen-
otypes. However, these results should be interpreted with
caution because of the very limited presence of PRRSV in
our sample, and further investigations are surely needed
to draw more substantial conclusions.
e high prevalence of M. hyopneumoniae among
lung lesion-associated bacteria is well known [5], and
in the present study we indeed observed significantly
Page 9 of 10
Tonnietal. Veterinary Research (2022) 53:41
more severe lung lesions in pigs with at least one posi-
tive PCR for M. hyopneumoniae. However, the sever-
ity of lesions was not related to the group, suggesting
that the time point at which pigs become infected dur-
ing the outbreak is not important; the critical point
is rather the infection by M. hyopneumoniae itself. A
practical implication of this is that invivo samplings
during an EP outbreak could allow for early identifica-
tion of pigs that will likely develop lung sequelae.
Studying co-infections is very complex and challeng-
ing, and the evolution of co-infections in pigs during
the fattening period is a largely unexplored field [5].
e present study partially sheds light on M. hyopneu-
moniae and other respiratory co-infections during EP
outbreaks. Previous studies have revealed the ability
of M. hyorhinis to exacerbate M. hyopneumoniae lung
lesions [32]. In the present study, the presence and load
of M. hyopneumoniae in TBS were significantly asso-
ciated with the presence of M. hyorhinis, but this co-
infection was not associated with a worsening of lesion
severity. Furthermore, despite the well-known ability
of A. pleuropneumoniae and P. multocida to cause lung
lesions [36], no relationship has been detected between
the presence of these two bacteria and the magnitude
of lesions. In general, the presence of multiple co-infec-
tions was not related to the severity of lung lesions in
our sample, indicating that M. hyopneumoniae has a
dominant role in determining their magnitude.
e longer finishing period adopted in Italy surely
influences the dynamics of infection [1]. Indeed, an
extensive study on Italian pig farms revealed that a his-
tory of A. pleuropneumoniae infection is a risk factor
for pleuritis sequelae [37]. Likewise, M. hyopneumo-
niae, which has the same tendency to become chronic,
could follow a similar evolution. Notably, however,
the clinical evaluation of pigs in the present study was
carried out only at the beginning of the outbreak (T0)
and after slaughtering (T2) by lung lesion scoring. An
additional evaluation of the clinical status at T1 could
indeed help to shed further light on the outbreak
evolution.
e relationship between the detection of specific
strains of M. hyopneumoniae or the presence of multiple
VNTR types and lung lesion scores is controversial [13,
17, 18, 38, 39]. In this study, pigs with a history of MX
infection during the outbreak show more severe lung
lesions. erefore, an improvement in lung lesion scores
could be achieved by limiting the circulation of diverse
genotypes (e.g., by improving farm biosecurity as men-
tioned above), as suggested by Michiels etal. [18].
In conclusion, M. hyopneumoniae was found to play a
central role in respiratory outbreaks in fattening pigs, while
co-infections seemed to play a more marginal role with no
relevant effect on the severity of lung lesions. e presence
of M. hyopneumoniae infections of multiple VNTR types
during the finishing period led to more severe lung lesions
in this study. erefore, improving farm management and
biosecurity and in turn limiting the circulation of multi-
ple genotypes could potentially lead to a better lung lesion
status.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13567- 022- 01061-w.
Additional le1. Allele calling table. Each VNTR type is identified as a
combination of different VNTR locus. The number of repeats of each locus
is determined by the number of base pairs (bp) for that locus.
Acknowledgements
The authors would like to thank Alessandra Pitozzi, Stefania Cippo, Dario
Guerrini, and Daniela Loda for all the help and effort dedicated to the project.
We also thank Angela Morben, DVM, ELS, from Edanz, for editing a draft of this
manuscript.
Author contributions
GLA and DM contributed to the design of the study. Samples were collected
by MT. PCR analyses were conducted and interpreted by FG and MBB. The
statistical analysis was developed by CR, FS and NF. MT, GLA, CR, PP, NF and
DM participated in drafting the manuscript, while all authors participated
in proofreading of the manuscript. All authors read and approved the final
manuscript.
Funding
The study was funded by the IZSLER.
Availability of data and materials
The datasets used and/or analysed during the current study are available from
the corresponding author upon reasonable request. The dataset supporting
the conclusions of this article is included within the article.
Declarations
Ethics approval and consent to participate
All farms enrolled in the study followed their own management practices. No
protocol approval from the ethics committee of Istituto Zooprofilattico Speri-
mentale della Lombardia e dell’Emilia Romagna (IZSLER) was required.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna,
Via Bianchi, 9, 25124 Brescia, Italy. 2 Department of Food Safety, Nutrition
and Veterinary Public Health, Istituto Superiore di Sanità viale Regina Elena
299, 00161 Rome, Italy. 3 Department of Veterinary Population Medicine,
University of Minnesota, 1365 Gortner Ave, St. Paul, MN 55108, USA. 4 Faculty
of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Received: 23 December 2021 Accepted: 20 March 2022
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Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiologic agent of enzootic pneumonia in swine, a prevalent chronic respiratory disease worldwide. Mycoplasma hyopneumoniae is a small, self‐replicating microorganism that possesses several characteristics allowing for limited biosynthetic abilities, resulting in the fastidious, host‐specific growth, and unique pathogenic properties of this microorganism. Variation across several isolates of M. hyopneumoniae has been described at antigenic, proteomic, transcriptomic, pathogenic, and genomic levels. The microorganism possesses a minimal number of genes that regulate the transcription process. Post‐translational modifications (PTM) occur frequently in a wide range of functional proteins. The PTM by which M. hyopneumoniae regulates its surface topography could play key roles in cell adhesion, evasion, and/or modulation of the host immune system. The clinical outcome of M. hyopneumoniae infections is determined by different factors, such as housing conditions, management practices, co‐infections, and also by virulence differences among M. hyopneumoniae isolates. Factors contributing to adherence and colonization as well as the capacity to modulate the inflammatory and immune responses might be crucial. Different components of the cell membrane (i.e. proteins, glycoproteins and lipoproteins) may serve as adhesins and/or be toxic for the respiratory tract cells. Mechanisms leading to virulence are complex and more research is needed to identify markers for virulence. The utilization of typing methods, and complete or partial‐gene sequencing for M. hyopneumoniae characterization has increased in diagnostic laboratories as control and elimination strategies for this microorganism are attempted worldwide. A commonly employed molecular typing method for M. hyopneumoniae is Multiple‐Locus Variable number tandem repeat Analysis (MLVA). The agreement of a shared terminology and classification for the various techniques, specifically MLVA, has not been described, which makes inferences across the literature not suitable. Therefore, molecular trends for M. hyopneumoniae have been outlined and a common terminology and classification based on VNTR types has been proposed. This article is protected by copyright. All rights reserved.
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Objective: To compare pathogen detection from tracheobronchial swabs with lung tissue in diagnostic submissions from pigs with reported respiratory disease. Materials and methods: Individual lung samples (n = 153) from 133 laboratory submissions were included in this study. Inclusion criteria were a lung sample where the tracheal bifurcation or major bronchus was readily identifiable and a clinical report of respiratory disease symptoms. Sterile, nylon-locked swabs were used to sample the largest available airway before the lung tissue was routinely processed for diagnostic testing. Swabs were placed in Amies transport medium and tested in blinded parallel with the lung tissue by bacterial culture and polymerase chain reaction (PCR) for common swine respiratory pathogens. Results: there was excellent agreement between PCR detection from lung and bronchial swab samples for porcine reproductive and respiratory syndrome virus, influenza A virus, Mycoplasma hyopneumoniae, and porcine circovirus 2 (kappa > 0.8, all assays). Agreement between bacterial culture from lung and swabs was substantial for Pasteurella multocida and Salmonella spp. And fair for Streptococcus suis. Lung tissue was culture positive more offen than swabs for Haemophilus parasuis and Actinobacillus spp.; however, in these cases, PCR for the respective pathogen was 100% positive on swab samples regardless of culture status of the swab. Implications: Tracheobronchial swabs are a single, uniform sample that can be easily collected at postmortem and transported to the laboratory for detection of swine respiratory pathogens by culture and PCR. Such swabs may serve as a rapid screening tool for unexpected mortalities in a population. © 2018 American Association of Swine Veterinarians. All rights reserved.