Vaccine 28 (2010) 6265–6272
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Oral vaccination of badgers (Meles meles) with BCG and protective immunity
against endobronchial challenge with Mycobacterium bovis
Leigh A.L. Corner a , Eamon Costello b , Damien O’Meara b , Sandrine Lesellier a,e , Frank E. Aldwell c ,
Mahavir Singh d , R. Glyn Hewinson e , Mark A. Chambers e , Eamonn Gormley a,∗
a
School of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Dublin 4, Ireland
Central Veterinary Research Laboratory, Backweston, Celbridge, Co. Kildare, Ireland
Immune Solutions Ltd., Centre for Innovation, The University of Otago, Dunedin, New Zealand
d
LIONEX GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany
e
TB Research Group, Department of Statutory and Exotic Bacteria, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK
b
c
a r t i c l e
i n f o
Article history:
Received 25 May 2010
Received in revised form 18 June 2010
Accepted 30 June 2010
Available online 15 July 2010
Keywords:
Oral BCG
Badgers
Tuberculosis
Vaccine
a b s t r a c t
Eurasian badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the
transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use
in badgers is considered a key element of any long-term sustainable campaign to eradicate the disease
from livestock in both countries. The aim of this study was to investigate the protective response of
badgers vaccinated orally with Bacille Calmette–Guérin (BCG) encapsulated in a lipid formulation, followed by experimental challenge with M. bovis. A group of badgers was vaccinated by inoculating the
BCG–lipid mixture containing approximately 108 colony forming units (cfu) of BCG into the oesophagus.
The control group was sham inoculated with the lipid formulation only. Thirteen weeks after vaccination
all the badgers were challenged with approximately 104 cfu of M. bovis delivered by endobronchial inoculation. Blood samples were taken throughout the study and the cell mediated immune (CMI) responses
in peripheral blood were monitored by the IFN-␥ ELISA and ELISPOT assay. At 17 weeks after infection
all the badgers were examined post-mortem to assess the pathological and bacteriological responses to
challenge. All badgers in both groups were found to be infected. However, a significant protective effect of
BCG vaccination was measured as a decrease in the number and severity of gross lesions, lower bacterial
load in the lungs, and fewer sites of infection. The analysis of immune responses showed that vaccination with BCG did not generate any detectable CMI immunological responses, however the levels of the
responses increased in both groups following M. bovis infection. The results of the study showed that
vaccination with oral BCG in the lipid formulation generated a protective effect in the badgers.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Bovine tuberculosis, caused by Mycobacterium bovis infection,
is present in the Eurasian badger (Meles meles) populations in Ireland and Great Britain [1,2]. The badger is a significant reservoir
of infection for domestic animals and continued infection in the
badger population has significant economic effects on the cattle
industry. That infection in the badger population is a source of infection for cattle in Ireland and GB was demonstrated by the reduction
in the incidence of tuberculosis in cattle herds following removal
of infected badgers [3,4]. Vaccination of badgers is seen as a key
element in any long-term strategy to eradicate the disease from
cattle. The BCG (Bacille Calmette–Guérin) vaccine, an avirulent live
∗ Corresponding author. Tel.: +353 1 716 6073; fax: +353 1 716 6091.
E-mail address: egormley@ucd.ie (E. Gormley).
0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2010.06.120
strain of M. bovis, is the only vaccine currently available for use in
domestic and wild animals. Several studies have shown that BCG is
protective in cattle [5], deer [6], brushtail possums [7], ferrets [8],
wild boar [9] and badgers [10,11]. In wild brushtail possums BCG
was found to prevent infection and had high protective efficacy
(69–95%) [12,13]. Badgers have also been successfully vaccinated
by administering BCG by a variety of different routes: intradermal
[10] and subcutaneous and mucosal [11].
For the BCG vaccine to be used to control tuberculosis in wild
badgers on a wide scale, and in a cost-effective manner, it is
likely that oral delivery would be the method of choice. As has
been shown in the control of wildlife rabies in Europe and North
America, a vaccination program based on the delivery of vaccine
in an oral bait to a wild animal population can be successfully
undertaken [14,15]. Vaccination studies including the oral route
of delivery have shown that it is necessary for BCG to be delivered live in order to generate immunity [16]. In studies where
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L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
unprotected BCG did not induce protection it was suggested that
this might be due to killing of the BCG in the stomach, as intragastric vaccination was ineffective compared with intraduodenal
vaccination [17]. In other studies, protection has been achieved by
encapsulating BCG in a lipid matrix formulation [18]. The matrix
is composed of triglycerides of fatty acids with varying concentrations of myristic acid, palmitic acid, stearic acid, oleic acid and
linoleic acid that provides a stable storage and delivery vehicle
for BCG [18,19]. When lipid-encapsulated BCG was delivered to
mice by mouth, the bacilli established replicating populations in
the lymph nodes of the alimentary tract, principally in the mesenteric lymph nodes (MLNs) draining the gastro-intestinal tract [20].
It also appears that encapsulation prolongs the in vivo survival
of the BCG and helps to confer long lasting protective immune
responses [21]. Studies have shown that the oral vaccine can induce
protective immune responses in mice [22], guinea pigs [23,24]
brushtail possums [25] and cattle [26]. Oral BCG has also been
shown to prevent M. bovis infection in wild possum populations
following natural exposure to M. bovis [13,27]. The aim of the study
reported here was to determine if live BCG, encapsulated in the
lipid matrix, would induce a protective response when administered by the by intra-oesophageal route to badgers. Vaccinated
badgers were challenged by the endobronchial route with virulent
M. bovis and the response to infection determined by post-mortem
examination and culture, and measurement of immunological
responses.
2.2. Vaccination with BCG
2. Methods
The BCG Pasteur strain used in this study has frequently been
used in animal studies and was recommended by a joint WHO,
FAO/OIE consultative group in 1994 for use in vaccine trials in
animals [28]. The BCG was formulated in a lipid matrix, as previously described [19]. This preparation is liquid at 37 ◦ C (which
enables dispersal of viable bacilli), but solidifies below 30 ◦ C. The
lipid was an animal derived fractionated complex lipid which contained triglycerides of the following fatty acids: 1% myristic acid,
25% palmitic acid, 15% stearic acid, 50% oleic acid and 6% linoleic
acid, and has been used in previous oral BCG vaccination studies
in mice and possums [18,19]. For formulation, broth-cultured BCG
was pelleted and resuspended in the lipid medium that had been
warmed to 37 ◦ C to achieve a final concentration of 1 × 108 colony
forming units (cfu)/ml of lipid [18]. BCG (and control) formulations
were transferred to 20 ml Luer-Lock syringes and allowed to solidify
at 4 ◦ C while being gently mixed. Before use, the syringes containing the lipid mixtures were warmed to room temperature, then a
1.5 mm plastic cannula was attached and, to ensure accurate dosing, the cannula was filled with lipid. Badgers were vaccinated by
passing the cannula approximately 10 cm down the oesophagus
and inoculating 1 ml of the BCG–lipid mixture. The control group
(N = 7) was sham inoculated with the lipid formulation only. The
vaccinated badgers (N = 7) received approximately 108 cfu of BCG
as determined by retrospective counts following extraction of BCG
from representative samples of the lipid-formulated vaccine [18].
To detect excretion of BCG, fresh faeces were collected from pens
on days 1, 2, 3 4, 9 and 17 post-vaccination.
2.1. Handling of captive badgers
2.3. M. bovis suspension and experimental infection
All work with badgers was carried out under licences issued by
National Parks and Wildlife Service and the Department of Health
and Children, and ethical approval was obtained from the UCD animal research ethics committee. Fourteen badgers were used in the
study, all obtained from wild populations that were free of tuberculosis (as assessed by historical record and immunological assays
at least twice prior to vaccination). The badgers were maintained
in captivity in groups of 2–4, in outdoor pens each with an area
of ∼200 m2 , with earthen floors covered in grass and containing
some shrubs. In each pen were two or three wooden setts that each
contained an inner nest box. The badgers were fed proprietary dog
biscuits, raw peanuts and fresh chicken pieces. Fresh water was
available ad libitum. The badgers were acclimatised to captivity for
at least 6 weeks prior to the commencement of the experiment.
The badgers were allocated to either a vaccine group (seven badgers) or a non-vaccinated control group (seven badgers). Badgers
in the vaccine group were housed separately from the control badgers and the groups contained similar numbers of male and female
badgers.
For handling, the badgers were anaesthetised with ketamine
hydrochloride (10 mg/kg) and medetomidine hydrochloride
(0.1 mg/kg, Domitor® , Pfizer) co-administered by intramuscular
injection. The badgers were examined 2 weeks before vaccination
and at 0, 2, 4, 7, 10 and 13 weeks post-vaccination and 2, 4, 6, 9, 12
and 16 weeks after infection. Badgers were challenged at week 13
post-vaccination. On each occasion the badgers were weighed and
examined for signs of disease or injury, and blood was collected by
jugular venopuncture. A tracheal aspirate was collected by passing
a 1.5 mm (outside diameter, OD) catheter down the trachea to the
bifurcation and as the catheter was withdrawn mucus was drawn
into the lumen of the catheter. The mucus was flushed from the
lumen of the catheter and washed from the outside of the catheter
with phosphate buffered saline containing 0.05% Tween-80 (PBST)
and collected for culture.
M. bovis strain M2137, originally isolated from a tuberculous badger, was grown to stationary phase in Middlebrook 7H9
broth (Difco, Becton Dickinson and Company, Sparks, MD, USA)
supplemented with 0.1% Tween-80, centrifuged, and the pellet resuspended and washed in PBS containing 0.05% Tween-80
(PBST). The suspension, containing approximately 1 × 108 cfu/ml,
was stored at −70 ◦ C in 2.5 ml aliquots [29]. For inoculation the
suspension was thawed and adjusted to the required concentration with PBST. The badgers were anaesthetised and a small
animal endoscope (3.7 mm OD fibre optic endoscope, Veterinary
Endoscopy Systems, UK) was introduced into the bronchus of the
right middle lobe (RMD). When in place a 1 mm OD cannula with
a void volume of 0.8 ml was inserted deep into the bronchus and
1.0 ml of the M. bovis suspension instilled. The cannula was flushed
with 1.0 ml of sterile PBS. The badgers were immediately placed
in right lateral recumbency and allowed to recover. The inoculum
stock was then re-cultured to determine the precise dose delivered to the badgers. The actual suspension used for inoculation
contained 8 × 103 cfu/ml.
2.4. Post-mortem examination
At 17 weeks post-infection (pi) all the badgers were anaesthetised and then euthanased with an intravenous overdose of
sodium pentabarbitone. The euthanased badgers were subjected
to a detailed necropsy. The severity of the gross lesions in lymph
nodes and organs were scored according to the scheme described
previously and individual scores of lesions were added to generate
an overall pathology score [29]. The anatomical sites examined for
gross pathology and by histopathology, and the samples collected
for bacteriological examination are listed in Table 1. All the lymph
nodes (LN) visible at each site were collected and a small portion
of each was fixed for histological examination and the remainder
retained for bacteriology. Tissues for bacteriology were collected
L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
Table 1
Vaccination of badgers with BCG by the oral route and response to experimental
infection with Mycobacterium bovis: distribution of infection.
Tissue or samplea
Left mandibular LN
Right mandibular LN
Left parotid LN
Right parotid LN
Left deep cervical LN
Right deep cervical LN
Left caudal cervical LN
Right caudal cervical LN
Left axillary LN
Right axillary LN
Left inguinal LN
Right inguinal LN
Left popliteal LN
Right popliteal LN
Left bronchial LN
Right bronchial LN
Anterior mediastinal LN
Posterior mediastinal LN
Left cranial lobe
Left caudal lobe
Right cranial lobe
Right middle lobe
Right caudal lobe
Accessory lobe
Pleura
Mesenteric LN
Hepatic LN
Liver
Spleen
Left kidney
Right kidney
Faecesc
Urinec
Group
Vaccinate
N=7
Control
N=7
0
0
0
0
3 (1–3)
3 (2–3)
0
0
0
0
0
0
0
1 (2)
3 (1–2)
7 (3–5)
2 (2)
5 (3–4)
0
0
0
7 (2–5)
3 (1–3)
2 (2)
3 (1–5)
4 (2–3)
5 (2–4)
0
0
0
0
1
0
0
0
1 (1–2)b
0
0
2 (1–2)
0
0
1(1)
3 (1–2)
0
0
0
1 (1)
4 (1–3)
7 (4–6)
1 (3)
7 (2–6)
2 (1–2)
1 (2)
3 (1–4)
5 (2–5)
2 (1–6)
4 (2–5)
3 (4)
2 (1)
6 (1–4)
0
2 (1–2)
1 (1)
1 (3)
0
1
6267
in the sample could be calculated (cfu/g). Tissue samples were cultured without decontamination except for lungs; these along with
urine and tracheal aspirates were decontaminated with 0.075% w/v
cetylpyridinium chloride (CPC) prior to culture. Faeces were decontaminated with 0.75% w/v CPC prior to culture. For each specimen
two plates of modified Middlebrook 7H11 medium (Difco, Becton
Dickinson and Company, Sparks, MD, USA) containing a mixture of antibiotics (polymyxin B 200,000 unit/l, ticarcillin 100 mg/l,
amphotericin B 10 mg/l and trimethoprim 10 mg/l; Mycobacteria
selectatab, Mast Diagnostics, Derby Road, Bootle, UK), two slopes
of Lowenstein–Jensen with pyruvate (LJP) medium, two slopes of
Stonebrink’s medium with pyruvate (SB) and one tube of BACTEC
12B medium were inoculated. When primary cultures of tissue
samples were contaminated, a stored portion of the macerated
sample was decontaminated with 0.075% w/v CPC and media reinoculated. All inoculated media were incubated at 37 ◦ C for 12
weeks. BACTEC 12B media were checked for growth using the
BACTEC 460 radiometric system (Becton Dickinson and Company,
Sparks, MD). The mean number of colonies on the 7H11 plates was
used to calculate the concentration of M. bovis in the sample. Where
a specimen was positive only in BACTEC 12B, and for urine, faeces
and tracheal aspirates, results were recorded as only positive or
negative.
Preliminary isolate identification was based on colony morphology, growth rate, pigmentation and cording characteristics [31].
Spoligotyping of selected isolates, which confirmed isolates as M.
bovis and differentiates between BCG and the challenge strain, was
performed according to the method of Kamerbeek et al. [32]. All
of the isolates from clinical and post-mortem samples had either
the wild type M. bovis or BCG profile on spoligotyping and those
from the faeces collected in the same pens, a BCG profile (data not
shown).
2.6. Mycobacterial antigens and immunological assays
a
M. bovis was not isolated from the adrenal glands nor organs of the reproductive
tract (testicles and seminal vesicles, or uterus and mammary glands).
b
The number of badgers from which M. bovis was isolated and in parentheses the
concentration of bacilli in the tissue, expressed as log10 colony forming units per
gram of tissue cultured.
c
The culture of urine and faeces was recorded only as positive or negative.
using an aseptic technique to minimise contamination (including
cross-contamination with M. bovis) and so that culturing could be
done without decontamination or with a low concentration of the
decontaminant. Also cultured were urine aspirated from the bladder, rectal faeces, and samples of liver (∼2 g), spleen (∼2 g), half
of each kidney and 2–5 g taken from the centre of each lung lobe.
Where macroscopic lesions were observed on the surface of a lung
lobe or a lesion was palpated within the parenchyma, these were
collected separately for culture. After the samples for culture had
been collected, the lungs were fixed in buffered formalin. After fixation for a minimum of 14 days, the fixed lungs were sliced at 2 mm
intervals and the cut surfaces examined for macroscopic lesions.
After fixation, histopathology samples were embedded in paraffin, sectioned at 3 ␮m and stained with haematoxylin and eosin,
and by the Ziehl–Neelsen method for acid-fast bacteria. The
histopathological examination consisted of the detection of lesions
of tuberculosis, i.e., granulomas containing acid-fast bacteria [30].
Only gross lesions confirmed as tuberculosis by histology and/or
bacteriology are listed in Table 2.
2.5. Bacteriology
Specimens for culture were processed as previously described
[29] and each specimen was cultured separately. Tissue samples
were weighed before culture so that the concentration of M. bovis
PBMC were isolated from whole blood as described previously [33]. The levels of badger IFN-␥ in PBMC cultures stimulated
with bovine tuberculin and CFP10 was measured by ELISA [34].
Briefly, up to 10 ml of heparinised blood was collected from
each animal and 0.75 ml aliquots were dispensed into individual
wells of 24-well tissue culture plates (Costar, UK). In addition,
wells contained 0.75 ml of each of the following: 30 ␮g/ml purified protein derivative of M. bovis (PPD-Bov, Weybridge VLA),
5 ␮g/ml CFP10 (kind gift of Lionex GmbH, Germany), a negative control or 5 ␮g/ml pokeweed mitogen (PWM, Sigma) as a
positive control, diluted in RPMI (Invitrogen Life Technologies)
supplemented with 25 UI/ml of heparin (Roche Diagnostics). The
endotoxin levels of CFP10 were measured at <0.4 EU/␮g antigen.
The cultures were incubated for 16 h at 37 ◦ C in a humidified
atmosphere with 5% CO2 before harvesting of plasma supernatants
after centrifugation. IFN-␥ production was measured for each antigen in duplicate, in a sandwich ELISA using mouse anti-badger
IFN-␥ monoclonal antibody mAb 11b9 (VLA, Weybridge UK) for
capture (diluted 1/200) and rabbit anti-badger IFN-␥ polyclonal
antibody Rb300 for detection (diluted 1/200) [34]. The mean optical density (OD) values were calculated for each antigen–blood
combination and the results are expressed as mean OD of stimulated PBMC minus OD of PBMC cultured in the absence of
antigen.
The badger IFN-␥ Elispot was carried out as described previously
[35]. Briefly, 96-well ELISPOT plates (Millipore multi-screen, Millipore Ltd., UK) were coated with capture mAb 11b9 at 10 ␮g/ml
in carbonate/bicarbonate buffer, pH 9.6 overnight at 4 ◦ C. Lymphocytes were isolated from heparinised whole blood and 100 ␮l of
cells (final concentration 2 × 106 cells/ml) resuspended in RPMI,
supplemented with 5% fetal calf serum, (Invitrogen Life Technolo-
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L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
Table 2
Vaccination of badgers with BCG by the oral route and response to experimental infection with Mycobacterium bovis: distribution of gross lesions subsequently confirmed as
tuberculosis.
PM groupa
Vaccine
Control
Badger
BR4-30
BR4-50
BR4-53
BR4-55
BR4-62
BR4-89
BR4-91
BR4-17
BR4-23
BR4-56
BR4-66
BR4-79
BR4-94
BR4-97
Tissueb
Total score
BrL
BrR
MedA
MedP
1c
0
0
0
0
0
0
0
0
0
0
0
1
1
3
3
1
2
1
3
4
1
3
4
3
4
4
1
0
0
0
0
0
0
0
0
0
0
0
0
4
0
3
0
0
0
0
0
0
2
1
2
4
1
1
1
Lung lobe
LCR
LCD
RCR
RMD
RCD
Acc
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
0
0
0
3
0
0
0
1
0
2
3
2
2
3
4
4
0
3
4
0
4
4
0
2
0
0
0
0
0
1
1
1
0
4
1
0
0
1
0
0
0
0
0
0
4
0
0
0
0
0
4
Pleura
Hep
Kid
0
0
1
1
0
1
1
0
0
1
0
0
1
2
0
0
0
0
1
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
3
0
0
0
12
7
4
5
6
8
10
8
11
11
14
10
18
9
a
Badgers were examined post-mortem at 17 weeks after infection. No macroscopic lesions were observed in the reproductive tract (testicles and seminal vesicles, or
uterus and mammary glands), the lymph nodes (LN) of the head (mandibular, parotid, deep cervical), the tonsils, superficial body LNs (caudal cervical, axillary, inguinal or
popliteal), nor in the spleen, liver, adrenal glands, or mesenteric LN.
b
BrL, left tracheobronchial LN; BrR, right tracheobronchial LN; MedA, anterior mediastinal LN; MedP, posterior mediastinal LN; LCR, left cranial lobe; LCD, left caudal lobe;
RCR, right cranial lobe; RMD, right middle lobe; RCD, right caudal lobe; Acc, right accessory lobe; Hep, hepatic LN and Kid, kidney.
c
Lesions severity score.
gies), non-essential amino acids, 5 × 10−5 M ␤-mercaptoethanol
(Invitrogen Life Technologies) and 100 U penicillin/100 ␮g streptomycin per ml (Invitrogen Life Technologies) was diluted in
100 ␮l with one of each of the following: PPD-Bov (30 ␮g/ml,
Institute of Animal Science and Health, Lelystad, Netherlands),
CFP10 (5 ␮g/ml), Con-A (5 ␮g/ml) diluted in supplemented RPMI.
Negative controls were incubated in supplemented RPMI without antigen. The plates were incubated for 16 h at 37 ◦ C and 5%
CO2 . The liquid was discarded, the plates washed three times in
distilled water, followed by three washes with buffer (PBS, 0.05%
Tween-20, v/v). 100 ␮l of mAb Rb300 diluted 1/400 in PBS, 0.05%
Tween-20, 0.1% albumin, was added to each well and the plates
incubated at 37 ◦ C for 1 h. The plates were then washed three
times with wash buffer. Biotinylated mouse rabbit anti-mouse
IgG (Sigma, Ireland) was diluted 1/1000 in PBS, 0.05% Tween-20,
0.1% albumin, and added to the plates, which were incubated at
37 ◦ C for 1 h. The plates were washed and 100 ␮l of streptavidinalkaline-phosphatase (Mabtech AB, Hamburg, Germany) diluted
1/4000 in PBS, 0.05% Tween-20, 0.1% albumin, and incubated at
37 ◦ C for 1 h. Again the plates were washed with wash buffer and
100 ␮l of BCIP/NBT (bromo-4-chloro-3-indolyl phosphate – Nitro
blue tetrazolium, Sigma–Aldrich) substrate added to each well and
the reaction was stopped after 10–20 min by liberal washing with
water. The plates were allowed to dry and spots counted using an
automated AID ELISPOT reader (Autoimmun Diagnostika GmbH,
Strasberg, Germany) and the net spot forming units (SFU) were
calculated.
2.7. Statistical analysis
The data were tested for normality using GraphPad
Prism version 4.00 for Windows (GraphPad Software, USA,
www.graphpad.com). Differences between groups were analysed
using Mann–Whitney test or t-test using GraphPad and SPSS
version 12.0.1 for Windows (Apache Software Foundation, USA,
www.spss.com).
3. Results
3.1. Post-mortem examination of badgers
Thirteen weeks after vaccination all the badgers in both groups
were challenged with 8 × 103 cfu of M. bovis by endobronchial
inoculation. At 17 weeks post-infection all the badgers were examined post-mortem to assess the pathological and bacteriological
responses to challenge. Infection was found to be established in
all of the badgers as judged by the presence of gross lesions and
culture for M. bovis.
The lesions in the vaccine group were restricted to the thoracic cavity in all except one badger, BR4-62 (Table 2). Lesions in
the thoracic lymph nodes were only present in the right tracheobronchial LN (BrR) in 6/7 badgers, but were seen in the BrR, left
tracheobronchial (BrL) LN and posterior mediastinal LN (Med P) in
the seventh badger (BR4-30). The most severe lung lesions were
associated with the RMD (right middle lobe) in vaccinated badgers.
Lesions were observed in a second lobe in three badgers and in
two additional lobes in BR4-30. Other gross lesions observed were
pleurisy in four badgers and lesions in the hepatic LN (Hep) of one
badger (BR4-62).
When compared with the control sham-vaccinated group, the
vaccine group had significantly fewer (MW test, p = 0.036) lesions
than the control group (Fig. 1A). Gross lesions in the control group
were observed in two thoracic lymph nodes (BrR and MedP LN) in
all badgers and also in the BrL in BR4-97, and the BrL and anterior
mediastinal (MedA) LN in BR4-94 (Table 2). The most severe lung
lesions in the control group were associated with the RMD in four
badgers, the accessory lobe (Acc) in two badgers and the right caudal lobe (RCD) in another badger, BR4-66. Lesions were also seen in
two or more lobes in four badgers. Other gross changes observed
were pleurisy in three badgers, lesions in an HEP of BR4-94 and a
kidney lesion in BR4-66. The kidney lesion was in the papilla of the
right kidney and there was exudate containing AFB present in the
renal pelvis. In addition to the number of gross lesions observed, the
severity of the lesions in the vaccine group was significantly lower
than in the control group (t-test, p = 0.015, Fig. 1B). Following histological examination the vaccine group had marginally fewer sites
L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
6269
Fig. 1. Vaccination of badgers with BCG by the oral route and response to experimental endobronchial infection with Mycobacterium bovis. (A) Number of confirmed gross
lesions of tuberculosis. The vaccine group had fewer lesions (median 3) than the control group (median 4) and the difference was significant (MW test, p = 0.036). Line
indicates median value. (B) Severity of disease based on severity score for each tissue. The vaccine group had significantly lower severity scores than the control group (t-test,
p = 0.015). Line indicates mean value. (C) Bacterial load in the most severely affected lung lobe. The vaccine group had a lower bacterial load than the control group and the
difference was significant (t-test, p = 0.036). Line indicates mean value. (D) Number of sites of infection. There were fewer sites in the vaccine group (mean 7.1) than in the
control group (mean 8.7) and the difference was significant (t-test, p = 0.031). Line indicates mean value.
with histological lesions (median 5, range 2–6) than the control
group (median 5, range 4–6) but the difference was not significant.
3.2. Bacteriological examination of badgers
The distribution of samples from which M. bovis was isolated
and the bacterial load in each sample is shown in Table 1. In the
vaccine group the bacterial load in the most heavily infected lung
lobes was significantly less (t-test, p = 0.036) than in the control
group (Fig. 1C). There were fewer sites of infection in the vaccine
group (mean 7.1) than the control group (mean 8.7) and the difference was significant (t-test, p = 0.031, Fig. 1D). In the vaccine group
the bacterial load in the most severely infected thoracic LNs (mean
4.43 ± 0.30) was less than in the control group (mean 5.00 ± 0.31)
however, the difference was non-significant (t-test, p = 0.104, data
not shown). There were some differences between groups in the
extra-thoracic sites of infection. M. bovis was isolated from the
spleen and both kidneys of one control and the spleen of another,
but not from these organs in the vaccine group. M. bovis was isolated
from the urine of one control badger (3 log10 cfu/ml) that had renal
lesions (BR4-66), and from the faeces of vaccinated badger BR4-55.
Only one tracheal aspirate was culture positive and that was collected from a control badger, BR4-66, 3 weeks after challenge. BCG
was also isolated from tissues of four vaccinated badgers (Table 3).
In three of these badgers BCG was isolated along with the M. bovis
challenge strain.
Seventeen faecal samples were collected from pens containing
vaccinated badgers over a period of 17 days post-vaccination. These
included multiple samples collected on a single day from one pen.
BCG was isolated on only two occasions and from the same pen, one
that contained three vaccinated badgers. In addition, BCG was only
found in low numbers: at a concentration of 20 cfu/g faeces on day
3 post-vaccination and at 1 cfu/g faeces on day 17 post-vaccination.
3.3. Clinical examination
Throughout the study, the general health of all badgers was
monitored routinely to detect evidence of any adverse effects
resulting from vaccination and/or challenge. The body weights of
the badgers in both treatment groups increased from the time
of vaccination in June (mean 10.39 kg ± sd 1.07) to challenge in
September (mean 13.02 kg ± sd 1.57), were maximum in November at 9 weeks pi (mean 14.21 kg ± sd 1.59), and declined towards
the end of the study in January (mean 12.29 kg ± sd 1.62). There
was no difference between groups in the way the body weights
changed. In addition, the body weights of the badgers were higher
at the time of post-mortem than at the time of vaccination (mean
increase 2.28 kg ± sd 1.22). No clinical signs related to vaccination
or the experimental infections were seen in any badger.
The body weights of badgers in both treatment groups increased
from the time of vaccination (mean overall body weight 10.39 ± sd
1.07) to”. There was no difference between groups in the way the
body weights changed.
Table 3
Vaccination of badgers with BCG by the oral route and response to experimental
infection with Mycobacterium bovis: distribution of isolated strains in cervical lymph
nodes.
Badger
BR4-30
BR4-50
BR4-53
BR4-55
BR4-62
BR4-89
BR4-91
a
Tissuea
RDC
LDC
–
BCG + M. bovis
–
BCG
BCG + M. bovis
M. bovis
–
–
M. bovis
–
M. bovis
–
BCG + M. bovis
–
LDC, left deep cervical lymph node, RDC, right deep cervical lymph node.
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L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
Fig. 2. Badger IFN-␥ release from PBMC stimulated with PPD-Bov (A and C) and CFP10 (B and D) as measured by ELISA (A and B) and ELISPOT (C and D). Arrow indicates time
of infection with M. bovis at week 13. Time points are weeks post-vaccination (T0). Results are presented as mean values ± SD.
3.4. IFN- responses of badgers following BCG vaccination and
infection with M. bovis
The IFN-␥ responses in PBMC of all badgers were monitored
in vitro by the badger IFN-␥ ELISA and ELISPOT assay using PPDBov and CFP10 as stimulating antigen. The response profiles of
both vaccinated and control groups were similar using either assay
(Fig. 2A–D) and were consistent with the observed severity of
pathology at the end of the study in both groups. Antigen specific
responses were not observed following vaccination with BCG but
were detected as early as 2 weeks post-challenge in both groups.
The responses peaked between 4 and 6 weeks post-challenge in
both vaccine and control groups. Thereafter, the production of
IFN-␥ declined in both groups but remained higher than the preinfection levels. The mean ELISA antigen specific IFN-␥ response
was higher in the control group compared with the vaccine group.
In contrast the response in the vaccine group was higher when the
ELISPOT assay was used. With both assays, however, the differences
were not statistically significant between the groups except for one
time-point 2 weeks post-challenge where the control group was
significantly higher in the response to stimulation with PPD-Bov,
measured by ELISPOT.
4. Discussion
In this study orally delivered lipid-formulated BCG vaccine
induced a protective response in the vaccinated badgers. The protective response was measured as fewer sites with gross lesions, a
decrease in the severity of gross lesions, fewer sites of infection, and
lower bacterial load in the lungs and thoracic lymph nodes compared with the control group. The results are consistent with those
obtained following subcutaneous and mucosal vaccination of badgers with BCG, and are also consistent with the development of a
protective response obtained when lipid-formulated BCG vaccine
was studied in mice [22], possums [18], guinea pigs [23,24] and
cattle [26].
Vaccination with oral BCG did not prevent infection in the badgers, as all badgers of both treatment groups were found infected at
post-mortem. Badgers are very susceptible to endobronchial infection and we have previously shown that they can be experimentally
infected with as few as 10 cfu [29]. However, in order to obtain uniform results, we used a high challenge dose of 104 cfu of M. bovis
[11]. The immune protection demonstrated in the vaccinated group
is therefore likely to be an underestimate of the true vaccine effect,
given the severity of the challenge. The use of an endoscope permitted the delivery of the M. bovis suspension directly to the right
middle and caudal lobes of the lung. The disease produced was
similar to that in naturally infected badgers [36,37]. In the majority
the infection was mild and respiratory excretion of M. bovis after
challenge was rare, detected only once in a tracheal aspirate of
a control group badger. There was dissemination of infection to
extra-thoracic sites in badgers of both treatment groups. Overall,
the distribution of infection was wider than that shown by histology, with the latter generally consistent with the distribution of
gross pathology.
BCG was recovered from faeces and tissues of vaccinated badgers. Faecal excretion of BCG occurred for at least 17 days after
vaccination, although only low concentrations of bacilli were
detected. In similar studies with orally vaccinated possums maximum excretion of BCG in faeces (103 cfu/g of faeces) occurred
on day 2 and excretion was undetectable beyond 7 days postvaccination [25]. In addition, BCG was not cultured from possum
faeces when exposed for 1 week on pasture. It was concluded from
this observation that, due to the loss of viability of BCG in faeces
exposed on pasture, environmental contamination with BCG in faeces would be short-lived and unlikely to pose a problem to cattle
grazing the pasture.
BCG was isolated from one tissue collected at post-mortem from
each of four badgers and in each instance a deep cervical LN (syn.
medial retropharyngeal LN) was infected. In three of these badgers
there was concurrent infection with BCG and the M. bovis challenge strain in the affected tissue. After oral BCG vaccination BCG
has been shown to persist for up to 30 weeks in mice where it
was recovered from the mesenteric LNs [21] and 8 weeks in brushtail possums [25]. Co-infection with BCG and virulent M. bovis in
vaccinated animals has not been reported previously.
L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
As with previous badgers studies there was limited CMI
responses following vaccination with BCG [38]. The BCG dose of
108 cfu was chosen for this study as it conferred significant protection against infection with virulent M. bovis in other animal species
[19,26]. The immunogenicity of BCG in badgers has been investigated in a number of previous studies and low CMI responses
were reported, irrespective of the route of vaccination [35,39,40].
It was suggested that a relatively low T lymphocyte response to
PPD-Bov following BCG vaccination might be a feature characteristic of the mustelid response. The reason for the low response is
unclear though it may be dose-dependent and the response was
too low to be detectable in peripheral blood. In another study carried out with captive badgers, subcutaneous injection of badgers
with high doses (107 –108 cfu) of BCG generated relatively high
IFN-␥ levels when PBMC were stimulated with PPD-Bov and IFN-␥
responses measured by ELISPOT [35]. That study provided evidence of a BCG dependent CMI response in badgers, as has been
found in cattle and mice [5,41]. The generation of elevated immune
responses following BCG vaccination by the oral route has been
associated with increased levels of protection in possums [25].
However, in the current study the lack of response of badgers to
the oral BCG vaccination was not associated with a failure of the
vaccine to protect. It is reasonable to assume that oral-delivered
BCG must have survived passage through the stomach and crossed
the intestinal mucosa to establish infection and induce protective
immunity.
We have previously shown that the production of IFN-␥ by
badger T lymphocytes in response to mycobacterial antigens is
indicative of a cellular response to active infection [38]. The IFN␥ responses of the vaccinated group were significantly higher 2
weeks post-challenge than the non-vaccinated controls when measured by ELISPOT. This apparent anamnestic response in vaccinated
badgers is consistent with observations reported previously for
BCG-vaccinated cattle. These studies showed that BCG vaccinated
calves that were protected against M. bovis challenge developed
cellular immune responses very early after M. bovis challenge at a
time-point when no responses were yet detectable in unvaccinated
controls [42]. From 4 weeks post-challenge, however, the response
of both groups (vaccinates and controls) was similar. Although
some of the pathology scores were significantly lower in the vaccinated badgers than in the controls, the difference in the extent of
lesions was not reflected in distinct immunological profiles, and it
was not possible to correlate post-vaccination peripheral immune
responses induced by oral BCG with the levels of protection that
were measured.
The success of oral vaccination of captive badgers significantly
advances the possibility of vaccinating wild badger populations for
the control of M. bovis infection [2]. However, before BCG vaccine
can be utilised to control bovine tuberculosis in wild badger populations it will be essential to study the responses of wild badgers
to oral BCG vaccination. Whereas infection of captive badgers in
a vaccination-challenge experiment can at best reveal the potential of oral BCG vaccination, studies in free-ranging wild animals
under conditions of natural M. bovis transmission will reveal the
full potential of BCG induced protection. Before oral vaccination
is incorporated into the national bovine tuberculosis eradication
program, further studies will be required to determine the presentation and type of bait required for targeting the oral vaccine to
free-ranging badgers and the optimal dose of oral BCG required for
protecting badgers against natural infection with M. bovis.
Acknowledgements
The study described in this paper was funded by the Department of Agriculture, Fisheries and Food (DAFF), Republic of Ireland.
6271
S. Lesellier was also supported through a project funded by the
Department for Environment, Food and Rural Affairs (DEFRA), GB.
Matt Lambeth (Immune Solutions Ltd.) is gratefully acknowledged
for assistance with preparing the oral BCG vaccine. The help of
staff in the Mycobacteriology Laboratory at the Central Veterinary
Research Laboratory (Backweston, Co. Kildare) is appreciated. We
also acknowledge the help and support provided by Ian O’Boyle
(DAFF), Michael Sheridan (DAFF), Margaret Good (DAFF), Paddy
Sleeman (UCC) and Prof. Dan Collins (UCD).
References
[1] Clifton-Hadley RS, Wilesmith JW, Richards MS, Upton P, Johnston S. The
occurrence of Mycobacterium bovis infection in cattle in and around an area subject to extensive badger (Meles meles) control. Epidemiol Infect 1995;114(1):
179–93.
[2] Gormley E, Collins JD. The development of wildlife control strategies for eradication of tuberculosis in cattle in Ireland. Tuber Lung Dis 2000;80(4/5):229–36.
[3] Griffin JM, Williams DH, Kelly GE, Clegg TA, O’Boyle I, Collins JD, et al. The impact
of badger removal on the control of tuberculosis in cattle herds in Ireland. Prev
Vet Med 2005;67(4):237–66.
[4] Donnelly CA, Wei G, Johnston WT, Cox DR, Woodroffe R, Bourne FJ, et al. Impacts
of widespread badger culling on cattle tuberculosis: concluding analyses from
a large-scale field trial. Int J Infect Dis 2007;11(4):300–8.
[5] Buddle BM, de Lisle GW, Pfeffer A, Aldwell FE. Immunological responses and
protection against Mycobacterium bovis in calves vaccinated with a low dose of
BCG. Vaccine 1995;13(12):1123–30.
[6] Griffin JFT, Mackintosh CG, Slobbe L, Thomson AJ, Buchan GS. Vaccine protocols
to optimise the protective eficacy of BCG. Tuber Lung Dis 1999;79(3):135–43.
[7] Aldwell FE, Pfeffer A, DeLisle GW, Jowett G, Heslop J, Keen D, et al. Effectiveness
of BCG vaccination in protecting possums against bovine tuberculosis. Res Vet
Sci 1995;58(1):90–5.
[8] Qureshi T, Labes RE, Cross ML, Griffin JF, Mackintosh CG. Partial protection
against oral challenge with Mycobacterium bovis in ferrets (Mustela furo) following oral vaccination with BCG. Int J Tuberc Lung Dis 1999;3(11):1025–33.
[9] Ballesteros C, Garrido JM, Vicente J, Romero B, Galindo RC, Minguijon E, et al.
First data on Eurasian wild boar response to oral immunization with BCG and
challenge with a Mycobacterium bovis field strain. Vaccine 2009;27(48):6662–8.
[10] Stuart FA, Mahmood KH, Stanford JL, Pritchard DG. Development of diagnostic tests for, and vaccination against, tuberculosis in badgers. Mammal Rev
1988;18:74–5.
[11] Corner LA, Costello E, Lesellier S, O’Meara D, Gormley E. Vaccination of European badgers (Meles meles) with BCG by the subcutaneous and mucosal routes
induces protective immunity against endobronchial challenge with Mycobacterium bovis. Tuberculosis (Edinb) 2008;88(6):601–9.
[12] Corner LA, Buddle BM, Pfeiffer DU, Morris RS. Vaccination of the brushtail possum (Trichosurus vulpecula) against Mycobacterium bovis infection
with Bacille Calmette–Guerin: the response to multiple doses. Vet Microbiol
2002;84(4):327–36.
[13] Tompkins DM, Ramsey DS, Cross ML, Aldwell FE, de Lisle GW, Buddle BM.
Oral vaccination reduces the incidence of tuberculosis in free-living brushtail
possums. Proc Biol Sci 2009;276(1669):2987–95.
[14] Stohr K, Meslin FM. Progress and setbacks in the oral immunisation of foxes
against rabies in Europe. Vet Rec 1996;139(2):32–5.
[15] Brochier B, Aubert MF, Pastoret PP, Masson E, Schon J, Lombard M, et al. Field
use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies
in Europe and North America. Rev Sci Tech 1996;15(3):947–70.
[16] Buddle BM, Aldwell FE, Keen DL, Parlane NA, Hamel KL, de Lisle GW. Oral vaccination of brushtail possums with BCG: investigation into factors that may
influence vaccine efficacy and determination of duration of protection. N Z Vet
J 2006;54(5):224–30.
[17] Buddle BM, Aldwell FE, Keen DL, Parlane NA, Yates G, de Lisle GW. Intraduodenal vaccination of brushtail possums with bacille Calmette–Guerin enhances
immune responses and protection against Mycobacterium bovis infection. Int J
Tuberc Lung Dis 1997;1(4):377–83.
[18] Aldwell FE, Keen DL, Parlane NA, Skinner MA, de Lisle GW, Buddle BM. Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance
to pulmonary tuberculosis in brushtail possums. Vaccine 2003;22(1):70–6.
[19] Aldwell FE, Tucker IG, de Lisle GW, Buddle BM. Oral delivery of Mycobacterium
bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis
in mice. Infect Immun 2003;71(1):101–8.
[20] Aldwell FE, Baird MA, Fitzpatrick CE, McLellan AD, Cross ML, Lambeth MR, et
al. Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG:
anatomical sites of bacterial replication and immune activity. Immunol Cell
Biol 2005;83(5):549–53.
[21] Aldwell FE, Cross ML, Fitzpatrick CE, Lambeth MR, de Lisle GW, Buddle BM. Oral
delivery of lipid-encapsulated Mycobacterium bovis BCG extends survival of the
bacillus in vivo and induces a long-term protective immune response against
tuberculosis. Vaccine 2006;24(12):2071–8.
[22] Aldwell FE, Brandt L, Fitzpatrick C, Orme IM. Mice fed lipid-encapsulated
Mycobacterium bovis BCG are protected against aerosol challenge with
Mycobacterium tuberculosis. Infect Immun 2005;73(3):1903–5.
6272
L.A.L. Corner et al. / Vaccine 28 (2010) 6265–6272
[23] Clark S, Cross ML, Nadian A, Vipond J, Court P, Williams A, et al. Oral vaccination
of guinea pigs with a Mycobacterium bovis bacillus Calmette–Guerin vaccine in
a lipid matrix protects against aerosol infection with virulent M. bovis. Infect
Immun 2008;76(8):3771–6.
[24] Clark S, Cross ML, Smith A, Court P, Vipond J, Nadian A, et al.
Assessment of different formulations of oral Mycobacterium bovis bacille
Calmette–Guerin (BCG) vaccine in rodent models for immunogenicity and
protection against aerosol challenge with M. bovis. Vaccine 2008;26(46):
5791–7.
[25] Wedlock DN, Aldwell FE, Keen D, Skinner MA, Buddle BM. Oral vaccination
of brushtail possums (Tichosurus vulpecula) with BCG: immune responses,
persistence of BCG in lymphoid organs and excretion in faeces. N Z Vet J
2005;53(5):301–6.
[26] Buddle BM, Aldwell FE, Skinner MA, de Lisle GW, Denis M, Vordermeier
HM, et al. Effect of oral vaccination of cattle with lipid-formulated BCG
on immune responses and protection against bovine tuberculosis. Vaccine
2005;23(27):3581–9.
[27] Corner LA, Norton S, Buddle BM, Morris RS. The efficacy of bacille
Calmette–Guerin vaccine in wild brushtail possums (Trichosurus vulpecula). Res
Vet Sci 2002;73(2):145–52.
[28] Report of a WHO/FAO/OIE Consultation on Animal Tuberculosis Vaccines.
Geneva; 1994.
[29] Corner LA, Costello E, Lesellier S, O’Meara D, Sleeman DP, Gormley E. Experimental tuberculosis in the European badger (Meles meles) after endobronchial
inoculation of Mycobacterium bovis: I. Pathology and bacteriology. Res Vet Sci
2007;83(1):53–62.
[30] Gavier-Widen D, Chambers MA, Palmer N, Newell DG, Hewinson RG. Pathology of natural Mycobacterium bovis infection in European badgers (Meles
meles) and its relationship with bacterial excretion. Vet Rec 2001;148(10):
299–304.
[31] Collins CH, Grange JM, Yates MD. Tuberculosis bacteriology: organisation and
practice. 2nd ed. Oxford; 1997.
[32] Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper
S, et al. Simultaneous detection and strain differentiation of Mycobacterium
tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35(4):
907–14.
[33] Dalley D, Chambers MA, Cockle P, Pressling W, Gavier-Widen D, Hewinson RG.
A lymphocyte transformation assay for the detection of Mycobacterium bovis
infection in the Eurasian badger (Meles meles). Vet Immunol Immunopathol
1999;70(1–2):85–94.
[34] Dalley D, Dave D, Lesellier S, Palmer S, Crawshaw T, Hewinson RG, et al. Development and evaluation of a gamma-interferon assay for tuberculosis in badgers
(Meles meles). Tuberculosis (Edinb) 2008;88(3):235–43.
[35] Lesellier S, Palmer S, Dalley DJ, Dave D, Johnson L, Hewinson RG, et al. The safety
and immunogenicity of bacillus Calmette–Guerin (BCG) vaccine in European
badgers (Meles meles). Vet Immunol Immunopathol 2006;112(1–2):24–37.
[36] Gallagher J, Monies R, Gavier-Widen M, Rule B. Role of infected, nondiseased badgers in the pathogenesis of tuberculosis in the badger. Vet Rec
1998;142(26):710–4.
[37] Gavier-Widen D, Cooke MM, Gallagher J, Chambers MA, Gortazar C. A review
of infection of wildlife hosts with Mycobacterium bovis and the diagnostic difficulties of the ‘no visible lesion’ presentation. N Z Vet J 2009;57(3):122–31.
[38] Lesellier S, Corner L, Costello E, Lyashchenko K, Greenwald R, Esfandiari J,
et al. Immunological responses and protective immunity in BCG vaccinated
badgers following endobronchial infection with Mycobacterium bovis. Vaccine
2009;27(3):402–9.
[39] Lesellier S, Corner L, Costello E, Sleeman P, Lyashchenko KP, Greenwald R, et al.
Immunological responses following experimental endobronchial infection of
badgers (Meles meles) with different doses of Mycobacterium bovis. Vet Immunol
Immunopathol 2009;127(1–2):174–80.
[40] Southey A, Sleeman DP, Lloyd K, Dalley D, Chambers MA, Hewinson RG,
et al. Immunological responses of Eurasian badgers (Meles meles) vaccinated with Mycobacterium bovis BCG (bacillus Calmette Guerin). Vet Immunol
Immunopathol 2001;79(3–4):197–207.
[41] Power CA, Wei G, Bretscher PA. Mycobacterial dose defines the Th1/Th2 nature
of the immune response independently of whether immunization is administered by the intravenous, subcutaneous, or intradermal route. Infect Immun
1998;66(12):5743–50.
[42] Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, Hewinson RG. Correlation of ESAT-6-specific gamma interferon production with
pathology in cattle following Mycobacterium bovis BCG vaccination against
experimental bovine tuberculosis. Infect Immun 2002;70(6):3026–32.