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RESEARCH ARTICLE
Multi-Etiological Nature of Tuberculosis-Like
Lesions in Condemned Pigs at the
Slaughterhouse
Fernando Cardoso-Toset
1,2
*, Jaime Gómez-Laguna
2
, Shyrley P. Amarilla
3
, Ana I. Vela
4,5
,
Librado Carrasco
3
, Jose F. Fernández-Garayzábal
4,5
, Rafael J. Astorga
1
,
Inmaculada Luque
1
1Department of Animal Health, Faculty of Veterinary Medicine, ‘International Excellence Agrifood Campus,
CeiA3’, Córdoba, Spain, 2Department of R&D, CICAP - Food Research Centre, Pozoblanco, Córdoba,
Spain, 3Department of Anatomy and Comparative Pathology, Faculty of Veterinary Medicine, ‘International
Excellence Agrifood Campus, CeiA3’, Córdoba, Spain, 4Department of Animal Health, Faculty of Veterinary
Medicine, Complutense University, Madrid, Spain, 5VISAVET Health Surveillance Centre, Faculty of
Veterinary Medicine, Complutense University, Madrid, Spain
*v52catof@uco.es;fcardoso@cicap.es
Abstract
Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other
than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in
pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughter-
house due to generalized TBL were sampled and selected for analysis. Pyogranulomatous
(56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of
the granulomas observed in both lymph nodes and lungs belonged to more advanced
stages of development (stages III and IV) whereas in the liver and the spleen most of lesions
belonged to intermediate stages (stages II and III). Different microorganisms were simulta-
neously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis com-
plex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main
groups of microorganisms detected after bacteriological analysis, with Trueperella pyo-
genes and Streptococcus suis as the most frequently isolated species. Mycobacteria
belonging to MTC were the most frequently detected pathogens in granulomatous and pyo-
granulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria
were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples
(37.2%). These results may provide new insights into the pathogenesis and diagnosis of
this pathology. The importance of coryneform bacteria and streptococci in such processes
must be evaluated in future studies.
Introduction
Tuberculosis-like lesions (TBL) can be an important cause of condemnation in swine at abat-
toir inspection representing significant important economic losses [1]. In pigs these lesions are
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 1/12
OPEN ACCESS
Citation: Cardoso-Toset F, Gómez-Laguna J,
Amarilla SP, Vela AI, Carrasco L, Fernández-
Garayzábal JF, et al. (2015) Multi-Etiological Nature
of Tuberculosis-Like Lesions in Condemned Pigs at
the Slaughterhouse. PLoS ONE 10(9): e0139130.
doi:10.1371/journal.pone.0139130
Editor: Pere-Joan Cardona, Fundació Institut
d’Investigació en Ciències de la Salut Germans Trias
i Pujol, Universitat Autònoma de Barcelona, SPAIN
Received: May 27, 2015
Accepted: September 8, 2015
Published: September 29, 2015
Copyright: © 2015 Cardoso-Toset et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was financially supported by the
Council of Economy, Science, Innovation and
Employment of the Andalusian Government (AGR-
2685-2012) and by the Centre for the Development of
Industrial Technology (CDTI) of Spain (project
reference IDI-20111632/IDI-20111633). Cardoso-
Toset F was funded by a grant of the Agrifood
Campus of International Excellence Programme
(ceiA3) from the Ministry of Education, Culture and
described as necrotic-calcified, proliferative or purulent gross lesions compatible with tubercu-
losis (TB) [2,3]. Although TBL in pigs are frequently limited to head lymph nodes, different
body locations such as other lymph nodes and thoracic or abdominal organs can be also
affected [3,4].
Granulomatous and pyogranulomatous lesions can be identified in TBL according to the
cellular components [5]. Granulomas, as the main lesions associated with TB, have been widely
classified within different stages of development that may help in the interpretation of disease
progression [4,6,7,8]. More advanced stages of granulomas have been associated with primary
sites of infection [3,4], but also with a lower bacterial load [3,6].
Mycobacterium avium complex (MAC), Mycobacterium tuberculosis complex (MTC) and
Rhodococcus equi have been reported as the species most frequently associated with TBL, and
these infections typically result in indistinguishable gross lesions in pigs [5,9,10,11,12,13].
Other genera such as Corynebacterium spp., Streptococcus spp. or Staphylococcus spp., have
also been isolated in caseous lymphadenitis in pigs, highlighting the potential diversity of path-
ogens that might be associated with TBL in this species [1,14,15]. This diversity of microor-
ganisms together with the zoonotic nature of several of them, are factors that should be
considered by public health authorities [1].
Detailed studies evaluating the relative importance of microorganisms other than Mycobac-
terium spp. identified from TBL in pigs are scarce [1,10]. In this work a histopathological and
microbiological evaluation of TBL in pigs is shown. Results of this study can help to better
understand the interaction among microorganisms in pigs affected by TBL to improve the
knowledge on the pathogenesis and diagnosis of this pathology.
Material and Methods
Ethics statement
This study did not involve purposeful killing of animals. Samples were collected from pigs after
routine slaughter and meat inspection procedures. No ethical approval was deemed necessary.
Study design and sampling
A total of 171 pigs where the carcasses were totally condemned due to the identification of gen-
eralized disease according to the European regulation for meat inspection (Regulation 2004/
854/EC) were sampled at two different slaughterhouses between January 2011 and June 2014.
All animals passed antemortem clinical inspection were apparently healthy free-range pigs
over 14 month-old raised in extensive systems from 56 farms located in South West Iberian
Peninsula (Andalusia and Extremadura regions in Spain). After meat inspection procedures
selected organs affected by TBL were sampled according to previous reports [2,3] including
submandibular lymph nodes, lungs, liver and spleen to evaluate disseminated lesions [3,4,9]
(Fig 1A and 1B). From these animals, a total of 352 samples were removed at the slaughter-
house and transported to the laboratory for analysis. To avoid cross contamination, different
sets of sterile instruments and vials were used to collect and transport samples from each ani-
mal. Whenever possible, one well-defined lesion was selected in each organ which was divided
into two portions: one portion was subjected to histopathological analysis and the other was
immediately submitted to bacterial culture and frozen at -20°C to perform qPCR assays [12].
However, when small-sized disseminated lesions were observed, lesions that were similar in
appearance and concentrated in one locality were selected and submitted to each analysis.
Multi-Etiological Nature of TBL in Pigs
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 2/12
Sport and by the Santander Universities Global
Division.
Competing Interests: The authors have declared
that no competing interests exist.
Histopathological analysis
Submandibular lymph nodes, lungs, liver and spleen tissue samples were fixed in 10% neutral
buffered formalin and 4 μm sections were stained with haematoxylin and eosin for histopatho-
logical examination and by the Ziehl-Neelsen (ZN) method to detect acid-fast bacilli (AFB) by
light microscopy [2,8]. Each sample was classified according to the identification of specific
Fig 1. A-G. A) TBL in the submandibular lymph node of an affected pig. Bar, 1cm. B) TBL in the spleen of an affected pig. Bar, 1cm. C) Microscopic image of
a TBL lesions in the lymph node of an affected animal showing a profuse infiltrate of degenerated neutrophils. HE. Bar, 200μm. D) Clustered epithelioid
macrophages surrounded by lymphocytes and erythrocytes in a stage I granuloma in the liver. HE. Bar, 50μm. E) Coalescent stage II granulomas in the
lymph node of a pig showing epithelioid macrophages completely enclosed by a thin capsule, with peripheral infiltration of scattered lymphocytes. HE. Bar,
100μm. F) Stage III granuloma with a central necrotic core, partially mineralized, surrounded by a dense connective tissue capsule infiltrated by lymphocytes
and scattered neutrophils. HE. Bar, 100μm. G) Thickly encapsulated, large, irregular, multicentric granulomas with prominent caseous necrosis and
multifocal islands of mineralization (stage IV granulomas). HE. Bar, 500μm.
doi:10.1371/journal.pone.0139130.g001
Multi-Etiological Nature of TBL in Pigs
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 3/12
structures, namely, epithelioid cell, multinucleated giant (MNG) cell, lymphocyte and/or neu-
trophil infiltration, connective tissue capsule formation, antigen-antibody deposits, necrosis
and mineralization. The presence of granulomas with epithelioid cells and MNG cells in the
absence of foreign bodies or fungal structures was considered compatible with a diagnosis of
TB. Granulomas were classified into four stages (I–IV) based on the pathological characteriza-
tion of TB granulomas previously described [7,8](Fig 1D–1G). Lesions characterized by a
necrotic core with an abundant neutrophil infiltration surrounded by epithelioid cells and a
rim of connective tissue with infiltrate of mononuclear cells were described as pyogranulomas
(Fig 1C).
TB diagnosis
The presence of MTC and MAC in the lesions was tested using a duplex real time PCR (qPCR)
previously validated by our research group [16]. Fat and connective tissue were removed from
affected organs and lesions were subsequently minced into small pieces with sterile scissors.
For every sample, up to 2 g of tissue were homogenized in a stomacher with 10 mL of sterile
distilled water for a duration of 2 min. The obtained solution was centrifuged for 10 min at
1,400 gresulting in a pellet for each sample. Genomic DNA was extracted from 25 mg of tissue
homogenate using NucleoSpin
1
Tissue DNA isolation kit (Macherey-Nagel GmbH, Düren,
Germany) according to the manufacturer’s instructions. DNA yields and quality were deter-
mined using a NanoDrop 3300 spectrophotometer (Thermo scientific).
All reactions were run in duplicate in a Agilent Technologies Mx3000P thermocycler under
the following conditions: initial denaturation at 95°C for 10 min, 40 cycles of amplification
consisting of denaturation at 95°C for 30 sec, primer annealing at 65°C for 30 sec, and exten-
sion at 72°C for 30 sec. To check the specificity of the amplified products, DNA from M.bovis
and M.avium isolates and non template controls were included in each assay and used as posi-
tive and negative controls, respectively.
ZN staining was performed in tissue samples negative to qPCR and examined for AFB as
described by Santos et al. [2].
Bacterial isolation
A swab from each sample submitted to bacterial culture was plated on Blood Agar Base and
Columbia Blood Agar Base with nalidixic acid and colistin sulfate (Oxoid ltd., Hampshire,
UK), supplemented with 5% sterile defibrinated sheep blood and incubated both in aerobic
and microaerophilic (5% CO
2
) conditions at 37°C for 48 h. One representative colony of the
most abundant morphologically distinct colonies were selected, subcultured and grown in the
same conditions for further biochemical identification. Gram staining, bacterial morphology
and production of catalase and cytochrome oxidase were performed as preliminary identifica-
tion tests according to standard procedures [17]. Further biochemical identification was per-
formed using commercial identification galleries (API
1
Coryne, API
1
20Strep, API
1
20E and
API
1
20NE, bioMérieux, Marcy-l’Etoile, France) according to manufacturer’s instructions. Iso-
lates were identified as a particular species only if identification scores in the multi-substrate
identification systems were excellent, very good or good (99.9–99.0% ID); otherwise, identifica-
tion was made only at the genus level (spp.). Latex agglutination test for the identification of
streptococcal groups (Streptococcal grouping kit, Oxoid ltd, Hampshire, UK), and Christie
Atkins Munch-Petersen test (CAMP test) were used for identification if necessary according to
previous reports [18,19,20]. Pure cultures of each isolate were stored at -70°C.
Multi-Etiological Nature of TBL in Pigs
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 4/12
16S rRNA gene sequencing
Coryneform bacteria isolates (Gram-positive, catalase variable and oxidase negative irregularly
shaped rods) were identified applying 16S rRNA gene sequencing due to the limited capacity
of biochemical methods to discriminate between species [21]. The 16S rRNA gene of each iso-
late was amplified by PCR and further sequenced to determine genotypic identity [22]. The
determined sequences consisted of about 1,400 nucleotides and were compared with the
sequences of other Gram-positive species available in the GenBank database, by using the
FASTA program (http://www.ebi.ac.uk/fasta33).
Results
Histopathological analysis
A total of 352 samples from 171 slaughtered pigs with TBL were evaluated. Pyogranuloma was
the lesion most frequently detected in all the examined organs (198/352; 56.2%) (Table 1)in
approximately 60% of the animals (104/171; 60.8%).
Granulomatous lesions were observed in 71/352 (20.2%) of samples, being described in 38/
171 of animals (22.2%). The presence of concomitant pyogranulomatous and granulomatous
lesions in different organs was observed in 14 out of 171 animals (8.2%). Necrotic foci or
lesions showing intense mineralization and fibrosis, with absence of epithelioid cells or
MNGCs (13/352; 3.7%) were separately considered as these lesions could not clearly be classi-
fied as either pyogranulomas or granulomas. Other lesions such as interstitial pneumonia,
catarrhal-purulent bronchitis, periportal fibrosis, periesplenitis and interstitial and multifocal
hepatitis were also detected in the absence of granulomatous or pyogranulomatous lesions (71/
352, 20.2% samples; and 1/171, 0.6% animals). Finally, in a reduced number of cases, lesions
could not be detected (38/352, 10.8% samples; and 9/171, 5.3% animals).
Granulomas were mostly observed in samples from submandibular lymph nodes and to a
lesser extent in liver, lungs and spleen (Table 1). Regarding the stage of granulomas, 31% of
granulomas belonged to the initial stages (I and II), whereas 69% of the granulomas were
included within the stages III and IV. This pattern was confirmed for lymph node and lung
samples, whereas in the spleen and the liver most of the granulomas belonged to the stages II
and III (Table 2).
TB diagnosis
The genome of MTC was amplified on samples from 65 animals (65/171 animals; 38%). In 44
out of these 65 animals generalized TBL affecting submandibular lymph nodes and other
organs (lungs, liver or spleen) were detected. In 25 out of these 44 animals (56.8%) MTC was
Table 1. Type of lesions identified from samples.
Total SLN
a
Lungs Liver Spleen
N° (%) N° (%) N° (%) N° (%) N° (%)
Pyogranuloma 198 (56.2) 94 (60.3) 42 (49.4) 33 (48.5) 29 (67.4)
Granuloma 71 (20.2) 38 (24.4) 14 (16.5) 14 (20.6) 5 (11.6)
Necrosis or calcification 12 (3.4) 8 (5.1) 0 (0) 4 (5.9) 0 (0)
Other lesions 33 (9.4) 2 (1.3) 23 (27.1) 7 (10.3) 1 (2.3)
No lesions 38 (10.8) 14 (8.9) 6 (7.1) 10 (14.7) 8 (18.6)
Total 352 (100) 156 (100) 85 (100) 68 (100) 43 (100)
a
SLN: submandibular lymph nodes.
doi:10.1371/journal.pone.0139130.t001
Multi-Etiological Nature of TBL in Pigs
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 5/12
detected only in submandibular lymph nodes; in 6/44 (13.6%) mycobacteria were detected in
submandibular lymph nodes and lungs, liver and/or spleen; whereas in 13/44 animals (29.5%)
mycobacteria were detected only in lungs, liver or spleen (data not shown). MAC was detected
only in one case associated with pyogranulomatous lesions in a liver.
AFB were recorded in 15/269 (5.6%) of qPCR negative samples and in 9/105 (8.6%) of
qPCR negative animals by ZN staining.
Bacterial isolation
A total of 235 isolates were obtained after bacteriological culture (Table 3). Due to the high
number of bacterial species detected in low percentages the analysis was focused on coryne-
form bacteria and streptococci as the main groups of microorganisms detected in this study
besides MTC. Coryneform bacteria were identified in 100 out of 235 isolates (42.5%) and were
recovered from 69 animals (40.3%). Most coryneform microorganisms were identified as True-
perella pyogenes (formerly Arcanobacterium pyogenes) (72%), which was isolated from a signif-
icant number (69.6%) of examined animals (Table 3). Streptococci were also identified in a
notable number of isolates (65/235; 27.7%) and animals (48/171; 28.1%). Streptococcus suis was
the species most frequently identified within this group (40% of isolates and 47.9% of animals
in which streptococci were isolated) followed by Streptococcus porcinus and Streptococcus dys-
galactiae spp. equisimilis (Table 3).
Organic distribution of identified microorganisms
The frequency of detection of microorganisms in TBL from the examined organs is shown in
Table 4. MTC, coryneform bacteria and streptococci were detected in all analysed organs.
MTC was more frequently detected in submandibular lymph nodes (32.7%), whereas coryne-
form bacteria were more frequently isolated from lungs (25.9%) and spleen (37.2%). T.pyo-
genes was the species identified in over 60% of cases associated with coryneform bacteria in all
examined organs (data not shown). Streptococci were equally isolated from lesions of all exam-
ined organs.
Microorganisms and type of lesions at individuals
The bacteria identified from different type of lesions are summarized in Table 5. Twenty-three
animals (13.4%) yielded negative results both by microbiological and qPCR studies despite
having presented microscopic lesions. In 75 (43.9%) of the pigs, a single microorganism was
identified, whereas in 73 (42.7%) of the animals two or more microorganisms were detected.
The isolation of coryneform bacteria from MTC positive pigs was frequent (10.5%), with T.
pyogenes being identified in 77.8% (14/18) of these cases. Moreover, different species of
Table 2. Distribution of granulomas per examined organ and stage of development.
Total SLN
a
Lung Liver Spleen
N° (%) N° (%) N° (%) N° (%) N° (%)
Stage I 2 (2.8) 0 (0) 1 (7.7) 1 (7.1) 0 (0)
Stage II 20 (28.2) 10 (25.6) 3 (23.1) 5 (35.7) 2 (40)
Stage III 27 (38) 16 (41) 3 (23.1) 6 (42.9) 2 (40)
Stage IV 22 (31) 13 (33.3) 6 (46.1) 2 (14.3) 1 (20)
Total 71 (100) 39 (100) 13 (100) 14 (100) 5 (100)
a
SLN: submandibular lymph nodes.
doi:10.1371/journal.pone.0139130.t002
Multi-Etiological Nature of TBL in Pigs
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coryneform bacteria were isolated from MTC negative pigs (16.9%), with T.pyogenes as the
main species identified (17/29; 58.6%).
Mycobacteria belonging to MTC were identified in 19/38 (50%) of pigs in which granuloma
was the unique detected lesion (Table 5). Bacteria other than mycobacteria were also detected
from granulomatous lesions in 13/38 of animals (34.2%), whereas no microorganisms were
identified in 6/38 (15.8%). However, when pyogranuloma was considered as a sole lesion, most
animals were negative to MTC (70/104; 67.3%) (Table 5). Coryneform bacteria (50/104; 48.1%
animals) and less frequently streptococci (26/104; 25% animals) were the main microbial
agents isolated from these lesions.
Table 3. Microorganisms isolated from lesions.
Isolates Positive animals
N° % N° %
Coryneform bacteria 100 42.5 69 40.3
Trueperella pyogenes 72/100 72 48/69 69.6
Corynebacterium suicordis 12/100 12 10/69 14.5
Rhodococcus equi 3/100 3 3/69 4.3
Corynebacterium xerosis 4/100 4 2/69 2.9
Corynebacterium spp. 2/100 2 1/69 1.4
Corynebacterium ulcerans 1/100 1 1/69 1.4
Corynebacterium urealyticum 1/100 1 1/69 1.4
Other coryneform bacteria
a
5/100 5 5/69 7.2
Streptococci 65 27.7 48 28.1
Streptococcus suis 26/65 40 23/48 47.9
Streptococcus porcinus 12/65 18.5 7/48 14.6
Streptococcus dysgalactiae spp. equisimilis 6/65 9.2 5/48 10.4
Streptococcus equi spp. zooepidemicus 5/65 7.7 4/48 8.3
Streptococcus agalactiae 4/65 6.1 4/48 8.3
Streptococcus alactolyticus 4/65 6.1 2/48 4.2
Streptococcus uberis 2/65 3.1 2/48 4.2
Other streptococci
b
6/65 9.2 6/48 12.5
Enterococcus spp.
c
19 8.1 14 8.2
Carnobacterium spp.
d
17 7.2 14 8.2
Aerococcus spp.
e
13 5.5 11 6.4
Staphylococcus spp.
f
7 3 7 4.1
Pasteurella multocida 4 1.7 4 2.3
Others
g
10 4.2 7 4.1
Total 235 100 171 100
a
Rhodococcus boritolerans,Dietzia timorensis,Pseudoclavibacter spp, Brevibacterium spp and
Actinomyces masicol (1 isolate/each)
b
Streptococcus spp. (3 isolates), S.mitis,S.rattus and S.bovis (1 isolate/each)
c
E.faecium (8 isolates) E.durans (3 isolates), E.faecalis (6 isolates), E.gallinarum and E.avium (1 isolate/
each)
d
C.maltaromaticum (16 isolates) and C.divergens (1 isolate)
e
A.urinae (7 isolates), A.viridans (4 isolates) and A nurinaequi (2 isolates)
f
S.sciuri,S.xylosus (2 isolates/each), Staphylococcus spp., S.aureus and S.haemolyticus (1 isolate/each)
g
Leuconostoc spp. (4 isolates), Escherichia coli (2 isolates) Mezorhizobium spp., Halospirulina spp.,
Glanulicatella spp. and Lactococcus lactis (1 isolate/each)
doi:10.1371/journal.pone.0139130.t003
Multi-Etiological Nature of TBL in Pigs
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Discussion
Tuberculosis-like lesions include a wide range of lesions grossly compatible with TB [2,3].
However, previous studies have shown that pathogens other than mycobacteria may cause
indistinguishable lesions in pigs [10]. The involvement of different pathogens in the develop-
ment of these lesions needs to be evaluated to assess an accurate diagnosis of TBL in pigs and
to establish effective control measures against this disease [1].
In this study, pyogranuloma was the predominant lesional pattern (104/171 animals;
60.8%) with granuloma being detected only in 38 out of 171 animals (22.2%). The high number
of pyogranulomatous lesions detected in the present study (198/352 samples; 56.2%) suggests
the importance of pyogenic bacteria in the etiology of TBL in pigs. In fact, a wide spectrum of
bacteria belonging to twenty different genera was detected. MTC was detected in 65 (38%) of
Table 4. Frequency of detection of microorganisms from TBL within the examined organs.
Total SLN
a
Lungs Liver Spleen
N° (%) N° (%) N° (%) N° (%) N° (%)
MTC 82 (23.3) 51 (32.7) 12 (14.1) 14 (20.6) 5 (11.6)
Coryneform bacteria 98 (27.8) 46 (29.5) 22 (25.9) 14 (20.6) 16 (37.2)
Streptococci 62 (17.6) 30 (19.2) 16 (18.8) 10 (14.7) 6 (13.9)
Others 63 (17.9) 28 (17.9) 15 (17.6) 13 (19.1) 7 (16.3)
a
SLN: submandibular lymph nodes.
doi:10.1371/journal.pone.0139130.t004
Table 5. Frequency of detected microorganisms and type of lesions identified at individual level.
Total Granuloma Pyogranuloma Concomitant
lesions
a
Other
lesions
b
N° % N° % N° % N° % N° %
MTC positive animals 65 38 19 50 34 32.7 7 50 5 33.3
Coryneforms 18 10.5 2 5.2 16 15.4 0 0 0 0
Streptococci 6 3.5 1 2.6 2 1.9 0 0 3 20
Others 9 5.3 3 7.9 4 3.8 1 7.1 1 6.7
Coryneforms + streptococci 10 5.8 4 10.5 4 3.8 2 14.3 0 0
Coryneforms + others 4 2.3 1 2.6 2 1.9 1 7.1 0 0
Streptococci+ others 3 1.7 2 5.3 1 1 0 0 0 0
No isolation 15 8.8 6 15.8 5 4.8 3 21.4 1 6.7
MTC negative animals 106 62 19 50 70 67.3 7 50 10 66.7
Coryneforms 29 16.9 1 2.6 24 23.1 1 7.1 3 20
Streptococci 17 9.9 1 2.6 10 9.6 2 14.3 4 26.7
Others 22 12.9 6 15.8 12 11.5 1 7.1 3 20
Coryneforms + streptococci 4 2.3 1 2.6 3 2.9 0 0 0 0
Coryneforms +streptococci + others 1 0.6 0 0 1 1 0 0 0 0
Coryneforms + others 3 1.7 2 5.3 0 0 1 7.1 0 0
Streptococci + others 7 4.1 2 5.3 5 4.8 0 0 0 0
No isolation 23 13.4 6 15.8 15 14.4 2 14.3 0 0
Total 171 100 38 100 104 100 14 100 15 100
a
Granulomatous and pyogranulomatous lesions detected in the same animal
b
Necrotic or calcified foci (5/15) and other lesions or no lesions (10/15)
doi:10.1371/journal.pone.0139130.t005
Multi-Etiological Nature of TBL in Pigs
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the animals, together with an important participation of coryneform bacteria and streptococci
(40.35% and 28.1% positive animals respectively). These findings reinforce the multi-etiologi-
cal nature of TBL.
The detection of multiple microbial agents was frequent (42.7% of analysed animals)
highlighting the importance of performing a thorough microbiological examination of TBL for
disease surveillance [1]. MTC, coryneform bacteria (including T.pyogenes,Corynebacterium
spp., and related genera) and streptococci were the pathogens more frequently detected from
TBL. Other microorganisms also identified but with a lower frequency (Staphylococcus spp.,
Pasteurella multocida,Enterococcus spp., Carnobacterium spp., Aerococcus spp.) can be isolated
from the environment, faeces, skin and mucous membranes of pigs [23], but their importance
in this process is unknown. Therefore, our analysis was focused on the most representative
groups of pathogens identified in the study.
MTC and MAC may play different roles in TBL according to the prevalence of bovine TB.
In this sense, M.avium is the main mycobacteria recovered from TBL in officially bovine TB
free countries [24] as well as in fattening pigs reared in intensive systems [25]. However, in
countries where TB is still prevalent in cattle and wildlife MTC is frequently detected from
TBL in free-range pigs [2,9,13]. Pigs of this study were bred in a free-ranged system sharing
natural resources with other wild and domestic animals in a geographical area in which a high
prevalence of TB infected wild boars has been described [26]. Contact and cross infection
between these populations may occur as has been reported in other areas of Spain [9]. Accord-
ingly, MTC was predominately detected from TBL in our study.
The frequency of isolation of Rhodococcus equi in our study (4/171, 2.3% animals) was
much lower than previously reported in pigs, negative to mycobacteria, and reared in intensive
farms in the Netherlands [10]. Despite further studies being deemed necessary to elucidate
these differences, several factors such as breed susceptibility, herd management and the ecology
of the bacteria may play a role. Corynebacteria other than Rhodococcus equi have been sporadi-
cally related with severe caseous lymphadenitis in pigs, including Corynebacterium ulcerans
and Corynebacterium pseudotuberculosis [14,15]. In the present study, Corynebacterium sui-
cordis was the main species isolated within this genus, but its relative importance was low. T.
pyogenes was the predominant species within this group. This pathogen is involved in miscella-
neous pyogenic infections in pigs and ruminants, including metritis, udder lesions, abscesses,
pneumonia, arthritis, endocarditis, lymphadenitis and osteomyelitis [27,28,29]. Although T.
pyogenes has previously been associated with caseous lymphadenitis in pigs [1], its relative
importance was low in comparison with our study.
Similarly to Lara et al. [1] streptococci were detected in a high percentage of animals. Inter-
estingly, S.suis was the species most frequently identified within this group. This microorgan-
ism has been associated with a wide variety of diseases in pigs such as meningitis, arthritis,
bronchopneumonia, endocarditis, poliserositis and septicaemia and has been considered as an
emerging zoonotic agent in humans secondary to exposition to pigs and pork products [30].
The other two streptococcal species more frequently isolated, Streptococcus porcinus and Strep-
tococcus dysgalactiae spp. equisimilis, are also frequently isolated from pigs with suppurative
infections [27,31].
Negative bacteriological results were observed in accordance with previous reports [1,2,
32]. These results may be attributed to false negative results of bacteriology or animals with
advanced lesions in which viable microorganisms could not be obtained. In this sense, the
21.7% (5/23) of animals that showed microscopic lesions but were negative to both bacterial
culture and qPCR, showed AFB on histopathological examination suggesting possible myco-
bacterial involvement in several of them.
Multi-Etiological Nature of TBL in Pigs
PLOS ONE | DOI:10.1371/journal.pone.0139130 September 29, 2015 9/12
Coryneform bacteria isolation from MTC negative TBL was the pattern most frequently
observed from animals affected by pyogranulomatous lesions (24/104; 23.1%). These results
support the idea that pyogenic bacteria can originate TBL in pigs without the involvement of
mycobacteria. In this sense, although false negative results of the qPCR analysis should be con-
sidered, AFB were only recorded in lesions of two of these animals. Alternatively, simultaneous
detection of coryneform bacteria and MTC was also frequent, suggesting a possible involve-
ment of this microbial association in pigs affected by TBL.
Granulomas were predominately observed in submandibular lymph nodes and to a lesser
extent in other body locations. The submandibular lymph node was the organ in which MTC
was more frequently detected, followed by the liver, lungs and spleen. Interestingly, MTC was
identified in a similar rate from both liver and lung samples, with most of the granulomatous
lesions belonging to stages III and IV. These results support the theory that both the respiratory
and digestive routes of infection play an important role in pigs, as previously suggested [4,9].
Despite several authors have suggested that generalized TB in swine is frequent [3,33], oth-
ers have reported a restriction of TBL to head lymph nodes or less frequently to the respiratory
tract [2,9,34]. Our results are in agreement with this latter statement, since MTC was detected
only in submandibular lymph nodes in more than half of the animals. Interestingly, pyogenic
bacteria, including T.pyogenes,Streptococcus spp., and Corynebacterium spp., were isolated
from TBL observed in other organs from these MTC-positive animals (data not shown). These
findings should be taken into account to avoid misdiagnosis of generalized TB based on gross
inspection and to carry out further studies to determine the true role of these agents, especially
T.pyogenes in pyogranulomatous lesions in pigs
Conclusions
The results of this study show that a wide spectrum of microorganisms different to mycobacte-
ria can be isolated from TBL in pigs, with coryneform bacteria and streptococci as the microor-
ganisms most frequently detected besides MTC. The high frequency of detection of T.pyogenes
in pyogranulomatous lesions is also shown. These results should be considered to prevent mis-
diagnosis of TB based on gross lesions and to establish specific control measures against these
pathogens in pigs.
Author Contributions
Conceived and designed the experiments: FCT JGL LC IL. Performed the experiments: FCT
JGL SPA IL. Analyzed the data: FCT JGL SPA AIV LC JFFG RJA IL. Contributed reagents/
materials/analysis tools: AIV JFFG. Wrote the paper: FCT JGL AIV JFFG IL. Final review of
the manuscript: FCT JGL SPA AIV LC JFFG RJA IL.
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