Veterinary Immunology and Immunopathology 231 (2021) 110161

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Strong antibody responses to Mycobacterium bovis infection in domestic pigs

and potential for reliable serodiagnostics
Archana A. Sridhara a, Ashley Johnathan-Lee a, Rubyat Elahi a, Maria A. Risalde b, c,
Christian Gortazar b, W. Ray Waters d, Konstantin P. Lyashchenko a, Michele A. Miller e, *
a
Chembio Diagnostic Systems, Inc., 3661 Horseblock Road, Medford, NY 11763, USA
b
SaBio Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo 12, 13005 Ciudad Real, Spain
c
Dpto. de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba, Agrifood Excellence International Campus (ceiA3), 14071,
Córdoba, Spain
d
National Animal Disease Center, 1920 Dayton Avenue, Ames, IA 50010, USA
e
Department of Science and Innovation-National Research Foundation Centre of Excellence for Biomedical TB Research, South African Medical Research Council Centre
for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town
8000, South Africa

A R T I C L E I N F O A B S T R A C T

Keywords: Mycobacterium bovis (M. bovis), the main cause of animal tuberculosis (TB), can infect a wide variety of domestic
DPP and wild animal species, including suids. Suids may serve as reservoir hosts or disease sentinels in different
MAPIA scenarios. Accurate detection of M. bovis infection in pigs is important for TB control programs. Although pre­
MPB83
vious studies have shown the value of serological assays for screening animal populations, the diagnostic ac­
Serological assays
Suids
curacy was considered suboptimal. In this study, we used Dual Path Platform (DPP) technology and multi-
antigen print immunoassay (MAPIA) to characterize antigen recognition profiles and temporal antibody re­
sponses. Four M. bovis experimentally infected pigs developed an early antibody response to antigen MPB83,
with a peak in IgG levels starting around 4–6 weeks post-inoculation, although none of the pigs developed an­
tibodies to fusion protein CFP10/ESAT6 within 16 weeks of the experiment. Three of four experimentally
infected pigs developed antibody responses before detectable antigen-specific interferon gamma responses.
Naturally infected pigs with gross lesions containing viable M. bovis showed IgM (19/40 infected animals) and
IgG (39/40) antibody responses to both MPB70/MPB83 (39/40) and CFP10/ESAT6 (34/40). Using MPB70/
MPB83 antigen alone to measure IgG antibody levels by DPP assay, an estimated test sensitivity was 97.5 % (95
% CI: 85.3− 99.9 %). None of the 57 negative control samples had detectable IgM or IgG antibodies to either of
the two test antigens in DPP assay, suggesting an estimated specificity of 100 % (95 % CI: 92.1–100.0 %) in pigs.
MAPIA showed robust IgG reactivity to multiple protein antigens of M. bovis in the naturally infected pigs. The
results demonstrate that serological assays which detect IgG antibodies to MPB83 have high sensitivity and
specificity for accurate detection of M. bovis infection in pigs. Further investigations should be done to validate
anti-MPB70/MPB83 antibodies as a reliable serodiagnostic biomarker for TB diagnosis in pigs.

Abbreviations: A-PPD, Mycobacterium avium purified protein derivative; B-PPD, Mycobacterium bovis purified protein derivative; CFP10, culture filtrate protein 10
kD; CI, confidence interval; CFU, colony forming units; DPP, dual path platform; ELISA, enzyme-linked immunosorbent assay; ESAT6, early secretory antigen target 6
kD; IFNγ, interferon gamma; IgM, immunoglobulin M; IgG, immunoglobulin G; IGRA, interferon gamma release assay; M. bovis, Mycobacterium bovis; MAPIA, multi-
antigen print immunoassay; MTC, Mycobacterium tuberculosis complex; PBS, phosphate buffered saline; PCR, polymerase chain reaction; RLU, relative light units; TB,
tuberculosis; TBL, TB compatible lesions.
* Corresponding author at: DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis
Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town 8000, South
Africa.
E-mail address: miller@sun.ac.za (M.A. Miller).

https://doi.org/10.1016/j.vetimm.2020.110161
Received 17 January 2020; Received in revised form 11 November 2020; Accepted 14 November 2020
Available online 19 November 2020
0165-2427/© 2020 Elsevier B.V. All rights reserved.
A.A. Sridhara et al.
1. Introduction
Mycobacterium bovis (M. bovis), the primary etiologic agent of animal
tuberculosis (TB), can infect a wide variety of domestic and wild animal
species, including suids. Suids have been previously considered to be
dead-end hosts of M. bovis, however, more recent studies suggest that in
certain scenarios, they can serve as maintenance hosts. Wild boar (Sus
scrofa) are a TB reservoir host in Spain (Naranjo et al., 2008) and
extensively reared black pigs in Italy (Di Marco et al., 2012) and Spain
(Cano-Terriza et al., 2018). It has been shown that inter-species in­
teractions with infected suids are significant risk factors for disease
transmission (Barasona et al., 2014). Even in situations where it does not
appear that pigs play a role in transmission, they may serve as useful
sentinels for TB in livestock and wildlife (Bailey et al., 2013; Nugent
et al., 2015). Therefore, accurate detection of M. bovis infection in pigs is
important for TB control programs.
Previous studies report that serological assays can be useful for bTB
detection in suid populations (Cardoso-Toset et al., 2017; Roos et al.,
2018; Thomas et al., 2019). However, in order to optimize performance
of serological tests, it is essential to understand the host-determined
characteristics of antibody responses to M. bovis, as well as identify
immunodominant antigens for the target species. Studies in experi­
mentally infected cattle and white-tailed deer (Odocoileus virginianus)
have shown that the kinetics of the humoral response vary between
individuals as well as antigen recognition profiles (Lyashchenko et al.,
1998; Waters et al., 2004).
The humoral response to MPB70 and MPB83 proteins is reportedly
consistent in M. bovis-infected livestock and wildlife species (Waters
et al., 2004, 2006; Lyashchenko et al., 2008; Cardoso-Toset et al., 2017;
Infantes-Lorenzo et al., 2017). However, there are variable antibody
responses to other antigens, such as ESAT6 or CFP10. Serological assays
which include complex antigens, such as bovine purified protein de­
rivative (B-PPD), may increase diagnostic sensitivity, although assays
that use combinations of specific antigens may have higher test sensi­
tivity and specificity (Cardoso-Toset et al., 2017). Since IgM responses
usually precede IgG, detection of both antibody classes may improve
identification of infected animals. For example, assays that measured
both IgM and IgG antibodies to MPB83 in M. bovis experimentally
infected cattle had increased sensitivity as compared to measurement of
responses by either isotype alone (Waters et al., 2006). In wild boar, a
MPB83-based lateral flow assay (INgezim TB CROM Ab; Ingenasa,
Madrid, Spain) yielded sensitivity and specificity values of 83 % and 97
%, respectively (Fresco-Taboada et al., 2019). Therefore, aims of this
study were to investigate temporal development of antibody responses
in experimentally infected pigs and characterize IgM and IgG responses
to CFP10/ESAT6 and MPB70/MPB83 in naturally infected pigs.
2. Materials and methods
2.1. Animals
To analyze antibody responses in pigs with M. bovis infection, two
sets of serum samples were retrospectively tested in this study. The first
set of specimens was serially collected from experimentally infected
pigs, whereas the second set was obtained from M. bovis-infected and
uninfected pigs identified in slaughterhouses via the TB surveillance
program in Spain.
The experimentally infected cohort consisted of four domestic white
pigs inoculated with 2 mL of a suspension containing 105 CFU of a field
strain of M. bovis (spoligotype SB0295), administered by the oropha­
ryngeal route, following the protocol described by Ballesteros et al.
(2009). A control group of four pigs was sham inoculated. Blood samples
in lithium heparin and without additives were collected biweekly over
the 16-week study period for interferon-gamma release assay (IGRA)
and serological studies, respectively. Animals were anesthetized by
intramuscular injection of Zoletil® (SBC Virbac Biotech Co., Ltd.,
2
Veterinary Immunology and Immunopathology 231 (2021) 110161
Carros, France) and euthanized by captive bolt, followed by a
post-mortem examination in order to assess the presence or absence of
TB compatible lesions (TBL) and perform microbiological culture.
The group of pigs with naturally acquired M. bovis infection was
comprised of 40 Iberian pigs that had gross TBL, from which M. bovis
was isolated as previously described (Thomas et al., 2019). The negative
control group included 57 white pigs in which infection was ruled out
based on the absence of gross TBL and negative culture results for
members of the M. tuberculosis complex (MTC).
2.2. Mycobacterial culture and speciation
All tissues with present or suspicious TBL were cultured for MTC.
When TBL were not observed, a pool of mandibular, tracheobronchial
and mediastinal lymph nodes (LNs) from each animal was also cultured.
The tissue samples were placed in a sterile container and stored at − 80
◦
C until bacteriological culture. The samples were inoculated into
Coletsos and 0.2 % (w/v) pyruvate-enriched Löwenstein-Jensen media
(Difco, Madrid, Spain) after decontamination with a final concentration
of 0.37 % hexadecylpyridinium chloride (Corner and Trajstman, 1988).
Isolates were identified by means of PCR (Wilton and Cousins, 1992) and
spoligotyping (Kamerbeek et al., 1997).
2.3. Interferon-gamma release assay (IGRA)
Blood samples were collected from experimental pigs into tubes with
lithium heparin and transferred to the laboratory in the same facility at
room temperature. Stimulation of whole blood with PBS (nil control),
and avian and bovine tuberculin purified protein derivative (avian PPD
and bovine PPD, respectively; 20 μg/mL; CZ Veterinaria, Porriño, Spain)
was performed within 8 h of collection. Pokeweed mitogen (PWM; 20
μg/mL; Sigma-Aldrich, Spain) was used as a positive control. Blood
cultures were incubated for 24 h at 37 ◦ C in a humidified incubator (5 %
CO2). Plasma was harvested after 24 h and stored at − 20 ◦ C until
assayed. Detection of IFNγ in the supernatant was performed using a
quantitative ELISA (PIERCE Endogen, Rockford, IL, USA), following
manufacturer’s recommendations. An animal was considered IGRA
positive when the optical density (OD) of the sample stimulated with
bovine PPD minus the OD of the nil was ≥ 0.05 and greater than the OD
of the sample stimulated with avian PPD minus the nil.
2.4. Multi-antigen print immunoassay (MAPIA)
Serum IgG antibodies to a panel of selected M. bovis proteins were
detected by MAPIA, performed as previously described (Lyashchenko
et al., 2017). The antigen panel included four recombinant proteins
(ESAT6, CFP10, MPB70, and MPB83), three hybrid proteins
(CFP10/ESAT6, MPB70/MPB83, and CFP10/MPB83), and one native
antigen (B-PPD). Swine IgG antibodies were detected in serum samples
by incubation on nitrocellulose strips impregnated with antigens, fol­
lowed by immunodetection with alkaline phosphatase-conjugated Pro­
tein G (Rockland Immunochemicals Inc., Limerick, PA, USA). MAPIA
bands were developed with 5-bromo-4-chloro-3-indoyl-phosphate/ni­
troblue tetrazolium (Seracare Life Sciences, Milford, MA, USA).
MAPIA results were evaluated visually, with a band of any intensity
considered as an antibody positive reaction.
2.5. Dual path platform (DPP)
Serum antibody levels were measured by DPP assay employing re­
combinant fusion proteins MPB70/MPB83 and CFP10/ESAT6 immobi­
lized onto a strip membrane as two separate test lines. For independent
IgM and IgG antibody detection, colloidal gold nanoparticles were
functionalized with goat anti-pig IgM (Bio-Rad, Hercules, CA, USA) and
Protein A/G (BioVision, Milpitas, CA, USA), respectively. The assay
required 5 μL sample and 20 min until results were read. Signal intensity
A.A. Sridhara et al.
of test lines was measured by an optical reader in relative light units
(RLU) as described (Lyashchenko et al., 2017).
2.6. Data analysis
Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) was used for
statistical analyses. Species-specific cut-off values were calculated using
receiver operating characteristic curves to determine seroreactivity rates
for each test antigen and antibody isotype. Relative antibody levels were
expressed as the ratios of test line intensity in RLU to the respective cut-
off value. A signal-to-cut-off (s/co) ratio at ≥ 1.0 was used to distinguish
reactive and non-reactive results. Diagnostic sensitivity of the DPP assay
was assessed by calculating the proportion of samples containing IgM
and/or IgG antibodies against either test antigen in the total number of
samples from M. bovis-infected animals. Diagnostic specificity was
determined by calculating the proportion of non-reactive samples in the
control groups. Diagnostic accuracy was estimated by calculating the
proportion of all true results in the number of all tested samples. The test
performance characteristics and seroreactivity rates found for each in­
dividual antigen used in the DPP assay were assessed at 95 % confidence
interval (CI).
3. Results
Progressive disease in all experimental M. bovis-inoculated pigs was
confirmed at necropsy by the presence of visible gross TBL where
tuberculous granulomas were observed by histopathological evaluation
(hematoxylin and eosin staining), as well as by isolation of the micro­
organism through MTC culture. All isolates belonged to the same spo­
ligotype as the M. bovis strain used for challenge. None of the control
pigs had evidence of infection.
Ante-mortem monitoring of the cell-mediated immune response in
experimental M. bovis-inoculated pigs was based on PPD IGRA results.
All pigs tested negative in the bovine PPD IGRA on day of inoculation, as
well as 2 weeks post-M. bovis inoculation, with 2 animals (A and D)
becoming bovine PPD IGRA positive at 4 weeks post-inoculation. All 4
pigs were bovine PPD IGRA positive from 6 weeks post-inoculation
onwards (Table 1). The stimulation of whole-blood cultures with PWM
led to responses of IFNγ in plasma that exceeded an OD of 0.5 at every
time point tested.
Pigs that were naturally M. bovis-infected were confirmed by the
presence of gross TBL and the isolation of the microorganism through
MTC culture. Animals found to be positive to either TBL only or culture
only were omitted from the study. Uninfected pigs from which samples
were collected at the abattoir did not present any gross lesions consistent
with TB, and the pool of mandibular, tracheobronchial and mediastinal
LNs from these animals assessed by mycobacterial culture was also MTC
negative.
Antibody responses to fusion protein MPB70/MPB83, but not to
CFP10/ESAT6, were detected by DPP assay in all four pigs inoculated
with M. bovis. As shown in Fig. 1, three experimentally infected pigs (A,
B, C) developed both IgM and IgG antibody responses, whereas one
Table 1
Initial detection of cell-mediated and humoral immune responses in pigs
experimentally infected with M. bovis.
1
Test conversion time
Animal
IFN-gamma IgM antibody IgG antibody
A 4 2 6
B 6 4 4
C 6 2 4
D 4 ND 2 6 mentally infected pigs, detection of IgM in naturally infected pigs did not
1
Results are shown as time (weeks) post-inoculation with M. bovis when first
test positive results were obtained.
2
Not detected.
3
Veterinary Immunology and Immunopathology 231 (2021) 110161
animal (D) produced only an IgG response. IgM seroconversion was
detectable as early as 2 weeks after M. bovis inoculation, followed by a
peak of IgM antibody at week 4 and rapid decline, although low levels of
IgM remained detectable for 16 weeks (pigs A, B, C). In contrast, more
robust and sustainable IgG responses developed in all infected pigs
starting at 4 weeks (animals B, C) or 6 weeks (animals A, D) after M. bovis
inoculation (Table 1, Fig. 1). No antibody response to CFP10/ESAT6
antigen was detected by the DPP assay in the experimentally infected
group over the period of 16 weeks (data not shown).
In agreement with DPP results, MAPIA results using an extended
panel of mycobacterial antigens demonstrated predominant IgG reac­
tivity to MPB83 and MPB70/MPB83, with a later weak response to
CPF10/MPB83 in three of the four M. bovis-infected pigs (Fig. 2). None
of the four uninfected pigs in the negative control group showed
detectable antibodies to any of the antigens in MAPIA or DPP assay (data
not shown).
In contrast to the experimentally infected pigs, animals with natu­
rally acquired M. bovis infection developed strong humoral immune
responses to both MPB70/MPB83 and CFP10/ESAT6 antigens in the
DPP assay (Fig. 3). The antibody responses included IgM and IgG pro­
duced in 19/40 and 39/40 infected animals, respectively (Table 2). The
IgG responses appeared more pronounced, while IgM responses were
relatively infrequent and of lower magnitude. The MPB70/MPB83
protein elicited IgM and IgG antibody responses more frequently (39/
40) than did CFP10/ESAT6 protein (34/40). The latter antigen, despite
the high seroreactivity rate, showed no added value in serological
identification of M. bovis-infected pigs, as all serum samples reactive to
CFP10/ESAT6 also recognized MPB70/MPB83 (Table 2). Combinatorial
analyses revealed that the detection of IgG antibodies to MPB70/MPB83
antigen was sufficient for optimal diagnostic accuracy. Using MPB70/
MPB83 antigen alone to measure only IgG responses, estimated test
sensitivity was 97.5 % (95 % CI: 85.3− 99.9 %). None of the 57 control
samples had detectable IgM or IgG antibodies to any of the two test
antigens in DPP assay, suggesting an estimated specificity of 100 % (95
% CI: 92.1–100.0 %) in pigs.
MAPIA analyses of the serum samples collected from pigs with
naturally acquired M. bovis infection (Fig. 4) revealed the following
differences from the antibody responses found in the experimentally
infected pigs (Fig. 2): 1) significantly more seroreactive antigens; 2)
generally stronger band intensity suggesting higher antibody levels; and
3) evident seroreactivity of MPB70 protein (among additional antigens
other than MPB83), which was not observed during the experimental
infection. These results suggest that early recognition of MPB83 by IgG
antibodies in the experimentally infected pigs continued over the course
of disease in the naturally infected animals. In addition, the MAPIA data
shown in Fig. 4 clearly demonstrated the advantage of using recombi­
nant multi-epitope polyproteins over B-PPD for more sensitive antibody
detection in M. bovis-infected pigs.
4. Discussion
Results from the current study demonstrate that serological assays
which detect antibodies to MPB83 have high sensitivity and specificity
for accurate detection of M. bovis infection in pigs that have been
experimentally infected or have naturally acquired infection. The ki­
netics of the humoral response suggests that pigs will have detectable
antibodies to MPB70/MPB83 early in infection and maintain high levels
through progression to disease. This is similar to results in M. bovis
experimentally infected cattle in which the earliest antibody responses
were detected to MPB83, with a peak in IgG response starting around 6
weeks (Waters et al., 2006). Although IgM responses to MPB70/MPB83
in the DPP assay peaked earlier than IgG in the majority of experi­
add to test sensitivity.
The IgM/IgG responses were detected earlier than the IFN-gamma
responses in 3 of the 4 experimentally infected pigs. In other species,
A.A. Sridhara et al.
Fig. 1. Antibody responses during experimental M. bovis infection in pigs. Results are shown as reader-generated values for IgM (broken line) and IgG (solid line)
antibodies to MPB70/MPB83 detected by DPP assay in serum samples serially collected from M. bovis-inoculated animals A, B, C, and D.
the cell-mediated immune response tends to occur earlier than antibody
detection. In experimentally infected cattle, a robust IFN-gamma
response was detected by 2 weeks post-inoculation with antibody re­
sponses only starting to be detected at 3 weeks (Waters et al., 2010). In a
separate study, although a weak MPB83-specific IgM was detected by 4
weeks, the response was boosted after injection of PPDs for the skin test
(Waters et al., 2006). In contrast, the infected pigs in the present study
had an early robust antibody response, supporting use of serological
tests for M. bovis diagnosis in suids.
The MAPIA results in the naturally infected pigs showed heteroge­
neity in the IgG antibody responses to M. bovis infection in terms of
antigen recognition patterns and confirmed the seroreactivity of the two
antigens, MPB70/MPB83 and CFP10/ESAT6, utilized in the DPP assay.
Interestingly, recognition of MPB70 protein (among additional antigens
other than MPB83) was not observed in the experimentally infected pigs
4
Veterinary Immunology and Immunopathology 231 (2021) 110161
Fig. 2. Antibody responses in pigs experimen­
tally infected with M. bovis. MAPIA was per­
formed as described in Materials and Methods.
Antigens printed onto nitrocellulose membrane
are shown on the right. Each strip represents
antibody result obtained from individual serum
sample collected at specified time over the
course of infection. Visible bands on the strips
indicate the presence of IgG antibody to corre­
sponding antigen(s), with band intensity
generally reflecting the antibody level.
despite the existence of largely cross-reactive epitopes on these two
M. bovis proteins (Lyashchenko et al., 2001). Collectively, the DPP and
MAPIA findings suggest more advanced stages of disease in the group of
naturally infected pigs than that achieved in the experimentally infected
animals. The above differences in the antibody responses between these
two groups of M. bovis-infected pigs may be attributed to the short
duration of the experimental study (16 weeks), differences in the
M. bovis dose received between groups, or possible repeated exposure in
the naturally infected pigs.
Evaluation of rapid methods for TB detection in slaughtered pigs
showed that, unlike PCR and histopathology, antibody detection can be
used for monitoring herds (Cardoso-Toset et al., 2015). In a recent study
using serum samples of 217 pigs with confirmed disease status from
known infected and uninfected free-ranging pig farms in southern Spain
to compare five serological assays (four ELISAs and one lateral-flow
A.A. Sridhara et al. Veterinary Immunology and Immunopathology 231 (2021) 110161

species. However, it also depends upon the antigens selected and

immunoassay format utilized. A recent evaluation of serologic tests in
domestic pigs and wild boar with confirmed TB diagnosis (n = 227) and
disease-free status (n = 366) found a higher sensitivity for P22 ELISA (84
%) than for bPPD ELISA (77 %), and 100 % specificity for both assays,
suggesting the diagnostic potential of antibody detection in identifying
infected pigs (Thomas et al., 2019). Our earlier study using the DPP
VetTB assay to characterize IgG responses in M. bovis-infected animals of
three suid species demonstrated the highest serodiagnostic accuracy was
found in pigs (sensitivity 95 %, specificity 100 %), followed by wild boar
(sensitivity 80 %, specificity 97 %) and warthogs (sensitivity 83 %,
specificity 91 %), with the predominant seroreactivity to MPB83 protein
in all three species (Miller et al., 2019).
In Spain, Iberian pigs are raised using extensive management systems
where the free-ranging animals can share their habitat with other do­
mestic and wildlife species, such as wild boar, which serve as reservoirs
Fig. 3. Levels of serum antibodies detected by DPP assay in pigs with naturally
of M. bovis infection. In a recent epidemiological surveillance study,
acquired M. bovis infection. Results are shown as DPP reader-generated values
(in RLU) obtained for IgM (triangles) and IgG (circles) antibodies to MPB70/ seroprevalence estimated by P22 ELISA testing of 3622 Iberian pigs was
MPB83 and CFP10/ESAT6 antigens detected in 40 culture-positive (solid 2.3 %, whereas herd prevalence assessed in 129 herds was 24.8 %
symbols) and 57 control animals (open symbols). Horizontal broken lines (Cano-Terriza et al., 2018). These findings in combination with isolation
indicate threshold values discriminating antibody negative and antibody posi­ of 25 different spoligotypes identified in the same study have revealed
tive results. Violin plot (Graphpad Prism 8) was applied to analyze data dis­ extensive circulation of M. bovis among pig farms under extensive
tribution and highlight differences between the animal groups in antigen management.
recognition. The presence of M. bovis-infected pigs, especially in open systems,
increases the risk of spillover and spillback. Therefore, in order to
manage these risks through detection of infected animals, a highly
Table 2
sensitive yet specific test is needed that is cost-effective, simple to use
Seroreactivity rates (% and 95 % CI) obtained in DPP assays for detection of IgM
and can be performed with a single easily obtained sample (i.e. blood).
and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 antigens in pigs with
acquired M. bovis infection. The high sensitivity and specificity of the DPP assay, using the MPB70/
MPB83 IgG response, suggests that it can be used for individual live
Test antigen IgM IgG IgM and/or IgG
animal testing. In addition, post-mortem serum samples could be used to
MPB70/MPB83 35.0 % 97.5 % 97.5 % rapidly diagnose TB in abattoir pigs.
(21.1− 51.7) (85.3− 99.9) (85.3− 99.9)
Although the results strongly suggest that MPB83 is an immunodo­
CFP10/ESAT6 30.0 % 85.0 % 85.0 %
(17.1− 46.7) (69.5− 93.8) (69.5− 93.8) minant antigen for humoral responses in M. bovis-infected pigs, the
MPB70/MPB83 and/or 47.5 % 97.5 % 97.5 % number of antigens evaluated in the present study was limited. In
CFP10/ESAT6 (31.63.7) (85.3− 99.9) (85.3− 99.9) addition, testing of larger sample sizes of both known infected and un­
infected animals would increase confidence in the test performance.
In conclusion, results from both M. bovis experimentally infected and
naturally infected domestic pigs show that anti-MPB83 antibody
detection offers a potentially useful diagnostic tool for TB diagnosis in
pigs. Further investigation should be performed to confirm the accuracy
and utility of this assay.

Funding

Fig. 4. Antibody reactivity patterns characterized by MAPIA in pigs with ac­
quired M. bovis infection. MAPIA was performed as described in Materials and
Methods. Antigens printed onto nitrocellulose membrane are shown on the
right. Each strip represents individual antibody profile obtained with serum
sample collected from control or infected animal. Visible bands on the strips
indicate the presence of IgG antibody to corresponding antigen(s), with band
intensity generally reflecting the antibody level.
test), diagnostic sensitivity and specificity estimates ranged from 66 %–
78 % and 99 %–100 %, respectively (Cardoso-Toset et al., 2017).
Therefore, due to insufficient test sensitivity, serodiagnosis has been
proposed as a useful screening tool for pig herds but not for individual
animals.
The diagnostic value of serology in TB detection varies among host
species, as antibody test sensitivity varies dramatically between host
The present work has benefited from the financial aid of research
funded by ‘Plan Nacional’ grant WildDriver CGL2017-89866 (MINECO,
Spain and EU FEDER) and Castilla-La Mancha and EU FEDER grant
MYCOTRAINING (SBPLY/19/180501/000174). Prof. Miller was funded
partially by the South African government through the South African
Medical Research Council and the National Research Foundation [grant
#86949]. The content is the sole responsibility of the authors and does
not necessarily represent the official views of the funders.
Ethical approval
The protocol followed for the handling and sampling frequency
procedures in the experimental study was designed to reduce stress and
health risks for the subjects, according to European (Directive 86/609/
ECC) and Spanish legislation (R.D. 223/1988 and R.D. 1021/2005). This
protocol was approved by the Committee on the Ethics of Animal Ex­
periments of the Regional Agriculture Authority (Comunidad de Madrid,
permit CM180112-01).
In the case of pigs naturally infected with M. bovis, these were not
intentionally killed for this study but were routinely slaughtered during
the period 2015− 16 from the southern and central Spain. No ethical

5
A.A. Sridhara et al.
approval was, therefore, deemed necessary for this part of the study.
Protocols, amendments and other resources were used according to the
guidelines approved by each autonomous government following the R.
D. 1201/2005 of the Spanish Ministry of Presidency. Blood and tissue
samples were collected by veterinarians in compliance with the Ethical
Principles in Animal Research.
Declaration of Competing Interest
All authors declare no conflict of intertest.
Acknowledgements
We are grateful to many colleagues at the Institute for Game and
Wildlife Research (IREC), Health Surveillance Centre (VISAVET) and
University of Cordoba (UCO) for technical support and their help in the
collection of samples.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.vetimm.2020.110161.
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