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SID 5
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Research Project Final Report
Note
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SID 5 (Rev. 3/06)
Project identification
SE3024
Low dose infection in cattle: disease dynamics and
diagnostic strategies
Contractor
organisation(s)
Veterinary Laboratories Agency
Woodham Lane
New Haw, ADDLESTONE
Surrey
KT15 3NB
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 27
£
466,621
start date ................
01/10/2002
end date .................
30/09/2006
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
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The balance of these data are being written up for publication in scientific journals at the moment.
Publication of this form by Defra could be viewed as disclosure of the data, and this could exclude
these data from publications in such peer-reviewed journals. However, we intend to submit these
papers in the next 3-6 months, and I am therefore aksing Defra not to publish the report before March
2007.
Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Bovine TB may spread via cattle-to-cattle transmission and also through the involvement of environmental
reservoirs. In the majority of cattle (ca. 60 %) presenting with bovine tuberculosis in GB, pathology is
restricted to the lower respiratory tract (lung and/or lymph nodes draining the lung) and the most
appropriate experimental model best reproducing this pathology presentation is challenge causing
disease in the lower respiratory tract, ie the intratracheal challenge model, which is being used in these
studies. An additional model of experimental infection via the aerosol challenge route was also
established, which also targets the lower respiratory tract. Applying both models side-by-side
synergistically allowed us to define factors relevant to transmission, pathogenesis, and the performance of
immuno-diagnostic tests, with particular emphasis on studying the disease in animals that presented with
low or very low bacillary loads as well as latently infected animals. The specific aims of this project were
as follows:
 To determine the minimum infective dose of M. bovis in cattle (objs. 1, 2).
 To generate a “memory cow” model of protective immunity, re-infection/re-exposure and potential
latency of M. bovis in cattle (Obj. 3).
 To establish an aerosol challenge model for M. bovis infection in cattle (Obj. 4).
In objecties 1 and 2, we determined the minimum infective dose of Mycobacterium bovis necessary to
stimulate specific immune responses and generate pathology in cattle. Calves were infected by the
intratracheal route with 1,000, 100, 10 and 1 colony-forming units (CFU) of M. bovis. Results showed that
half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine
tuberculosis, whereas the other half of the animals infected with 1 CFU presented without signs of
disease. No signficiant difference in the severity of pathology was observed after intratracheal challenge
after infection with the 1-1000 CFU dose range, although higher infective doses (>10,000 CFU) resulted in
more severe disease and pathology (data not shown), in line with the more severe disease observed after
aerosol infection with a similar high dose. All animals that developed pathology were skin test positive and
produced specific IFN- and IL-4. There was no difference in the size of the skin test reaction, the time
taken to achieve a positive IFN- result, or in the levels of IFN- and IL-4 between animals infected with
the different doses of M. bovis, suggesting that current diagnostic tests (skin test and IFN-test) can
detect cattle soon after M .bovis infection regardless of dose. In objective 4, an aerosol challenge model
for cattle was established by titrating infective M. bovis doses, and the results demonstrated that even
high doses delivered by aerosols (ca. >104 CFU) resulted in visible pathology confined to the lower
respiratory tract although bacilli could also be detected in some animals in the upper respiratory tract.
Therefore, the following conclusions can be made from the results of objs. 1,2 and 4 (see also detailed
SID 5 (Rev. 3/06)
Page 2 of 27
discussion of objective 4 below) can be summarised as follows:
 Intratracheal and aerosol routes of experimental challenge routes are the most appropriate routes
because they best reflect the lesion most commonly associated with natural infection (in GB).
 The lower respiratory tract is highly susceptible, infective doses of 1 CFU will result in pathology and
disease in the lung and/or associated lymph nodes.
 Predominant disease phenotype seen in GB is likely to be caused by aerosol infection of small numbers
of bacilli delivered by small aerosol particles (3-5 organisms) to the lung.
 At these low infection doses, it is possible to produce, apart from VL/Cu+ animals, also animals that are
NVL/cu+ or NVL/cu- yet positive in skin test and IFN- test: Such animals are therefore not ‘falsepositives’ but infected sub-clinically and could pose future infection risks to herds.
Objective 3. In order to study immune responses in cattle containing the infection with low, or
undetectable bacillary loads, we generated a “memory cow” model that we adapted from a drug-assisted
model of protective immunity developed originally for mice. We aimed to address the following specific
questions:
 How do diagnostic tests work in animals with very low bacillary loads, or in a state of latency?
 Does a primary infection protect against cattle against M.bovis re-infection?
 Identify possible correlates of protection/markers of disease severity.
 Identify potential protective antigens.
We could show that cattle infected with M. bovis (spoligotype 9) and then treated with isoniazid (INH)
harbour minimal or no pathology compared to untreated animals, yet still presented with strong cellular
immune responses (IFN-, DTH). In addition, INH-treated cattle were protected against rechallenge with
M. bovis, and we used these animals to explore immunological correlates of protection as well as to
define potential protective antigens. Therefore, this objective had cross-cutting relevance also for Defra’s
cattle TB vaccine development programme. For example, we could show that IL-4 splice variants have
been shown to antagonise/down-regulate IL4-mediated cellular responses, furthermore, that one splice
variant, IL43, was elevated in this model of protective immunity against M. bovis. In relation to the
expected outputs from Obj. 3, the following conclusions can be made:
 BOVIGAM responses contract with treatment/reduction in bacillary load, but animals present with
positive results at most time points, i.e. would still be detected by BOVIGAM assay even at low or
undetectable bacillary loads (see also comments below to objective 4).
 Primary infection confers significant degree of protection against re-infection.
 This protection was against heterologous M. bovis strain (cross-protection), which is encouraging for
vaccine development using BCG or one particular attenuated M. bovis strain.
 Successful chemotherapy requires active immunity.
 IFN- has been confirmed as a marker of bacterial load/pathology.
 The IL43 splice variant constitutes a surrogate of protection.
Objective 4. Development of an aerosol delivery model.
A model was developed to accurately deliver M. bovis to calves via the aerosol route. This model was then
used to deliver decreasing infective doses (104, 102, 101 CFU). The results indicated a decrease in
pathology with aerosol delivery resulting between the high dose and the two lower doses. However,
even with the highest dose almost exclusively in pathology restricted to the lower respiratory tract (lung
and lung associated lymph nodes). These data, together with results from obj. 1, support the
conclusions discussed above in the context of obj. 1. The value of the aerosol infection system lies with
the combination of the natural host, the natural route of infection and the use of a low M. bovis dose to
infect calves. This approach has given further insight into the relationship between infectious dose, the
immunology of infection and the type of pathological picture that follows. The lower infectious doses in
particular, induced an immune state more akin to that seen often in the field, where the relationship
between disease status and immune response is complex. In particular, animals infected with the lowest
infection dose not only presented as NVL/culture-negative but were also skin test negative yet still
detectable using the BOVGIAM test. Results of the aerosol challenge model will be discussed in relation
to the intratracheal challenge model.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
SID 5 (Rev. 3/06)
Page 3 of 27







the scientific objectives as set out in the contract;
the extent to which the objectives set out in the contract have been met;
details of methods used and the results obtained, including statistical analysis (if appropriate);
a discussion of the results and their reliability;
the main implications of the findings;
possible future work; and
any action resulting from the research (e.g. IP, Knowledge Transfer).
Objectives 1: To define the relationship between low infectious doses of M. bovis and immunological
parameters, diagnostic tests and severity of pathology in cattle; and objective 2: End-point titration of
infective dose in the intratracheal challenge model.
Abstract. The aim of this work was to determine the minimum infectious dose of M. bovis necessary to stimulate
specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by
the intra-tracheal route with 1000, 100, 10 or 1 CFU of M. bovis. Specific immune responses (IFN- and IL-4) to
mycobacterial antigens were monitored throughout the study, and responses to the tuberculin skin test assessed
[1] at two time points. Detailed post mortem examinations [2] were performed to determine the presence of
pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. In
the group infected with the lowest dose (1 CFU), one half of the animals developed pulmonary typical of bovine
tuberculosis: The minimal infective dose using the intratracheal infection route to infect cattle with M.bovis is
therefore 1 CFU. The large majority of the animals infected with the higher doses also developed pathology
typical of bovine tuberculosis, and M. bovis could be cultured from tissues. No difference in the severity of
pathology were observed for the different M. bovis doses. All animals that developed pathology were skin testpositive and produced specific IFN- and IL-4 responses. No differences in the size of the skin test reactions, the
times taken to achieve a positive IFN- result, or the levels of IFN-and IL-4 responses were observed for the
different M. bovis doses, suggesting that current diagnostic assays (tuberculin skin test and IFN- test) can detect
cattle soon after M. bovis infection regardless of the dose. This information should be useful in modelling the
dynamics of bovine TB in cattle and in assessing the risk of transmission.
Results.
1. Skin test and pathological findings.
Table 1 shows the skin test results at 12 weeks post infection with M. bovis (Table 1). Of the 20 animals infected,
14 became skin test-positive (applying the standard interpretation of the skin test). The 6 skin test-negative
animals were distributed among the 3 groups as follows; 3 calves receiving 1CFU (animal no.s 2805, 2871,
2877), 1 calf receiving 10 CFU (animal no. 2866), 1 calf receiving 100 CFU (animal no. 2859), 1 calf receiving
1000 CFU (animal no. 2924). These results were confirmed at the second skin test at weeks 23-25 post-infection
(data not shown). There was no significant difference (Chi-squared p=0.6267) in the distribution of skin testpositive and negative animals between the different groups. All skin test-positive animals had visible pathology in
the respiratory lymph nodes and most also contained lesions in the upper lung. Mycobacterial culture on modified
Middlebrook 7H11 agar and acid fast staining confirmed the presence of M. bovis in lesioned tissues. The degree
of pathology (see Table 2. Pathology Scores) of all animals was comparable, there was no significant difference
(Kruskal-Wallis p=0.3896) between groups that had received infective doses between 1 and 100 CFU M. bovis.
All skin test-negative animals presented without gross pathology and were M. bovis culture-negative.
SID 5 (Rev. 3/06)
Page 4 of 27
2. Immunological responses.
All skin test-positive calves (14 animals) developed positive specific IFN- responses (24-hour whole blood assay
and BOVIGAM ELISA) between 3-5 weeks post-infection (p.i.) (see Table 3. for Summary of IFN- and IL-4
responses). The time to positivity (number of weeks p.i. until a positive response was observed) and the intensity
of the response did not vary significantly between groups that received different doses of M. bovis. There was no
significant difference in the cytokine response to the immunodominant proteins ESAT6 and CFP10 relative to M.
bovis dose (data not shown). 6-day whole blood supernatants from 13/14 calves produced specific IL-4 (B-cell
bioassay). IL-4 responses were transient and initially observed between 5-7 weeks p.i. for most animals. There
was no difference in the strength of the IL-4 response relative to M. bovis dose.
SID 5 (Rev. 3/06)
Page 5 of 27
Concluding remarks.
In summary, data from Objectives 1 and 2 showed that 1CFU of M. bovis is sufficient to cause established
tuberculous pathology in cattle. This pathology is identical to that resulting from significantly higher experimental
doses (up to 1000 CFU in this study) and reflects the pathology seen in naturally infected field reactor cattle.
Cattle infected with 1CFU that developed pathology exhibited strong positive responses to the diagnostic
tuberculin skin test. Furthermore, the infectious dose of M. bovis had no bearing upon the time taken to achieve a
positive IFN- response in those animals that developed pathology. These data are in accord with very low
numbers of bacilli transmitted aerogenously between cattle. Comfortingly, the animals that do go on to develop
pathology and therefore become a likely source of contamination within a herd can be detected at an early stage
with the IFN- and tuberculin skin tests. Furthermore, the Intratracheal route of experimental challenge is appropriate
because it best reflect the lesion most commonly associated with natural infection (in GB), t he lower respiratory tract is
highly susceptible, infective doses of 1 CFU will result in pathology and disease in the lung and/or associated
lymph nodes, and the predominant disease phenotype seen in GB is caused by aerosol infection of small numbers of bacilli
delivered by small aerosol particles (3-5 organisms) to the lung.
Orignial Objective 2. To define the degree of protection conferred to cattle by low doses of M. bovis
against sub-sequent high-dose M. bovis re-challenge and what impact this outcome has on the sensitivity
of diagnostic tests.
This was removed after approval by Defra (17 November 2003) to allow lower titration of M. bovis (Obj. 1) to
proceed down to 1CFU/animal.
Objective 3. (VLA) To define how diagnostically useful immune parameters (e.g. skin test and IFNresponses) develop over time in animals that recover from primary M. bovis infection and to test
whether such animals are protected against sub-sequent re-infection (cattle memory model).
Experiment 3A (VLA). Isoniazid-attenuated primary infection with virulent M. bovis: assessment of drug
effectiveness and safety.
Abstract. The aim of this preliminary experiment was to assess the potential usefulness of a drug-attenuated
acute M. bovis infection of cattle as a means of generating cattle presenting with low or very low bacillary load
and to assess the dynamics of the immune response in such animals during the course of treatment. 16 calves
were infected with a dose of M. bovis known to result in pathology (350 CFU). At 4 weeks p.i. (when a stable
specific immune response was observed) 8 of these were treated with the TB drug isoniazid (INH; 25mg/kg/day)
for a period of 10 weeks. INH treatment was stopped 5 days prior to necropsy. Skin tests were performed 1-2
weeks prior to necropsy. INH-treated cattle were compared with the untreated M. bovis challenge controls in
terms of their specific IFN- responses and pathology. Serum samples were taken to measure liver function
markers (known side effects of INH include liver damage), and plasma INH monitored to ensure drug clearance.
Our results demonstrated the effectiveness of INH-treatment to reduce disease burden (ie bacillary loads and
SID 5 (Rev. 3/06)
Page 6 of 27
severity of pathology), and that this system is appropriate for use in objective 3B. In addition, we could
demonstrate that no adverse hepato-toxcicity was induced by INH treatment.
Results.
1. Skin test and pathological findings. All animals in both groups were skin test positive at 13 weeks post
infection. Mean pathology scores at necropsy were significantly lower in the INH treated animals than in the
control group (table 4). Histopathology (FIG.1) confirmed that lesions in the INH treated group were less severe
than in the control group
FIG.1. Histopathology after INH treatment
CW3196.
Infected Isoniazid treated
CW3199. Infected and untreated
Left tracheobronchial lymph node. Granuloma with
central mineralization. Numerous associated giant
cells surround mineralized debris (arrows). Note
lack of necrosis and scant peripheral fibrosis. H&E
100x
Caudal mediastinal lymph node. Central
mineralization and necrosis (arrowhead) with
scattered lymphocytes and macrophages at the
periphery. Giant cells present rimming central
mineralization (arrow).
H&E 100x
TABLE 4. Pathology scores
Animal
M. bovis + INH
CW 3195
CW 3196
CW 3197
CW 3198
101721
201708
601726
601740
Mean
M. bovis
CW 3199
CW 3200
CW 3201
CW 3202
101714
401724
501718
701734
Mean
*, p-values
(Mann-W hitney)
Lymph Node Lung Sub total Culture Score
0
3
0
2
0
0
2
0
0.9
2
0
0
3
0
0
3
2
1.2
2
3
0
5
0
0
5
2
2.1
1
2
0
2
0
2
7
4
2
9
2
3
3
3
6
15
7
6.0
7
3
4
5
4
3
4
3
4.1
16
5
7
8
7
9
19
10
10.1
16
4
11
7
3
17
18
22
12.3
0.0032
0.0027
0.0014
0.0039
Semi-quantitative scoring of pathology taken from Vordermeier et al (2002), Infect. Immun. 70(6):3026-3032
Semi-quantitative scoring of CFU from cultured tissues courtesy of A.O.W helan.
2. IFN- responses There was no difference in mean antigen specific interferon- production between the INH
treated group and the control group (FIG.2). However, individual results indicated that there was a tendency in
some INH treated animals towards lower levels of specific IFN-production
SID 5 (Rev. 3/06)
Page 7 of 27
FIG. 2. Mean IFN-gamma responses of INH-treated and untreated animals. Data expressed as the mean OD450 responses
(+ SEM), with OD values of medium control wells subtracted from OD values from wells stimulated with the indicated antigens.
Mean IFN responses INH treated animals
1.0
OD 450nm
Mean IFN responses untreated animals
CFP10/ESAT6
PPDA
PPDB
0.5
0.0
1
-0.5
2
3
4
5
6
7
8
9
1.5
CFP10/ESAT6
PPDA
PPDB
1.0
OD 450nm
1.5
0.5
0.0
10
1
Weeks post infection
2
3
4
5
6
7
8
9
10
Weeks post infection
-0.5
3. Liver function markers. The key liver function markers aspartate aminotransferase, gamma glutamyl
transferase and total bilirubin were measured using kits available from Olympus UK Ltd (London, UK). It was
found that in all the animals, levels of these markers remained constant and within normal limits, showing that
none of the animals were experiencing hepatotoxic side effects of INH treatment (data not shown).
4. INH clearance. HPLC measurement of plasma INH levels during treatment and 3 days prior to post mortem
showed that the daily dosing regime maintained INH in the circulation during treatment, and that all INH was
cleared from the system within 1 week of treatment cessation (FIG.3). Consequently, a 4-week resting period
between treatment cessation and re-infection was sufficient to ensure that no residual INH affected the bacilli
used to re-infect the calves in Obj. 3B (see below).
FIG. 3. Pharmacokinetic of INH-treatment of cows. Serum INH levels were measured at the beginning of treatment (week 5)
during treatment (week 8) and one after cessation of therapy. Data from representative animal is shown.
sample data from one animal
circulating INH g per
ml
5
4
3
2
1
14
w
ee
k
8
ee
k
w
w
ee
k
5
0
Experiment 3B (VLA/IAH). INH-assisted protective immunity against re-infection with M. bovis.
Abstract. The aim of this experiment was to assess whether INH-treatment of acute M. bovis infection as a
regime for generating protective immunity to re-infection, and to investigate possible immunological correlates of
protection as well as determining the performance of the BOVIGAM assay in animals presenting with no or minor
gross pathology and low bacillary loads. 24 calves (3 groups of 8) were used in this experiment. 2 groups of 8
calves were infected with 350 CFU M. bovis (spoligotype 9, sp. 9) at week 0. From 3 weeks p.i. all 16 M. bovis
(sp.9)-infected calves received treatment with INH (25mg/kg/day) which lasted for 14 weeks (i.e. until week 17 of
the experiment). Animals were then taken off INH treatment and rested for 4 weeks. At week 22 of the
experiment, one group of M. bovis (sp.9)-infected/INH-treated calves, and the third group of 8 naïve calves were
challenged with 1000 CFU M. bovis (sp.35). All 24 animals were skin tested 1 week prior to necropsy at week 34.
Immunological responses (IFN-, IL-4, IL-10, TNF-) were monitored. In addition PBMC were collected for mRNA
extraction to investigate IL4 splice variants, recently described in cattle, and correlated with immune protection in
humans infected with M. tuberculosis [3-5]. Serum and plasma samples were also collected as above. Detailed
post mortem examinations, microbiological culture of tissues and histopathological analyses were carried out on
all animals. We could show that BOVIGAM responses contract with treatment/reduction in bacillary load, but
animals present with positive results at most time points, i.e. would still be detected by BOVIGAM assay even at
low or undetectable bacillary loads, that primary infection confers significant degree of protection against reinfection, that FN- is a marker of bacterial load/pathology, and that the IL43 splice variant constitutes a
surrogate of protection.
SID 5 (Rev. 3/06)
Page 8 of 27
Results.
1. Skin test, pathological findings and M. bovis recovery from PM tissues
All 16 INH treated animals were skin test positive at week 32, immediately prior to post mortem, as were the 8
type 35 challenge controls. Animals treated with INH and rested (Group A) showed significantly less pathology
than the challenge controls (Group C, table 5). Group B animals (INH-treated animals re-challenged with sp. 35)
also presented with substantially reduced gross pathology compared to the sp. 35 infected control group C (table
5). Thus INH-treatment of M.bovis infected cattle resulted in the development of protective immunity. M. bovis
was cultured from tissue collected and the spoligotypes of the recovered organism established: Visibly lesioned
animals in group B were either positive for type 9 M.bovis only (ie were fully protected against sp 35 rechallenge), for type 35 alone (ie chemotherapy had cleared the sp. 9 primary inoculum but animals were not fully
protected against sp. 35 re-challenge), or harboured both type 9 and type 35 M. bovis .
Table 5. Gross pathology
Pathology Scores
Treatment
Calf
Lymph Nodes
Lung
Subtotal
Gp.A. M.bovis type 9 + INH alone
3375
3376
3377
3378
101233
201946
600672
400770
Total
1
1
0
1
1
0
0
2
6
0
0
0
0
1
0
0
5
6
1
1
0
1
2
0
0
7
12
Gp.B. M.bovis type 9 + INH type 35 challenge
3371
3372
3373
3374
500177
701232
700946
701633
Total
1
0
0 (culture +ve)
0
1
0
1
3
4
6
0
0
2
0
3
0
12
9
p value (Mann-Whitney) Gp.A-Gp.B = 0.597, nsd
1
0
0
4
10
0
2
3
20
Gp.C. M.bovis challenge control, type 35 alone
3421
3422
3423
3424
101938
401139
601625
SID 5 (Rev. 3/06)
5
0
0
6
6
3
0
3
1
0
9
5
0
3
Page 9 of 27
8
1
0
15
11
3
3
701612
5
12
17
Total
25
33
58
p value (Mann-Whitney) Gp.A-Gp.C = 0.04 statistically significantly different
p value (Mann-W hitney) Gp.B-Gp.C = 0.126 nsd
( ) indicates the numbers of areas of the lung affected
2. INH clearance
HPLC analysis for both INH and its metabolite N-acetyl INH (revealed through treatment of the plasma sample
with HCl at 800 C prior to derivatization) showed INH to have been completely cleared from the circulation prior to
infectious rechallenge at week 22 (FIG.4).
FIG.4. Pharmacokinetics of INH in serum. Results from all 8 calves that were subsequently re-challenged with M. bovis
artweek 22 are shown.
total circulating INH + N acetyl INH g per ml
INH g per ml
20
3371
3372
3373
3374
3375
3376
3377
3378
15
10
5
0
0
5
10
15
20
weeks post infection
3. Cytokines
a) IFNCytokine assays for IFNshowed a similar pattern of fluctuation with time to that seen in previous
experiments. In all four groups, specific responses developed within three weeks of infection (FIG.5.). These
responses gradually cionytracted during INH treatment until week 17 when treatment was stopped. Subsequent
to treatment cessation, a sharp increase was observed in some animals in all groups, suggesting that cessation
of treatment was allowing mycobacteria to resume growth (homologous rechallenge). Interestingly, INH-treated
animals that were not re-challenged but presented without gross pathology (NVL) exhibited a peak of IFN-
responses both to PPD-B and ESAT-6/CFP-10 shortly after treatment cessation (Fig. 5, upper right panel) that
was not as evident in the corresponding animals that showed gross pathology (VL, Fig. 5, upper left panel). This
suggests that an active memory immune response post-treatment cessation - generated during the INH-treatment
- is required for effective chemotherapy.
INH-treated calves that were re-challenged with the heterologous M.bovis strain (sp. 35) at week 22 could also
be grouped into animals with and without gross pathology (VL and NVL animals, respectively). In both groups of
animals we could observe the brief response peak following treatment cessation (ie prior to re-challenge),
although the peak was less pronounced in the VL group (Fig. 5 lower left panel) than for the NVL animals (Fig. 5,
lower right panel). Interestingly, following sp. 35 re-challenge only the NVL animals developed a further IFN-
response peak, mainly to ESAT-6 and CFP-10 stimulation followed by a generally downward trend over the postrechallenge period (Fig. 5, lower right). In contrast, the ESAT-6/CFP-10 peak following re-challenge in the VL
animals was less pronounced and closely followed the PPD-B responses. Further, both responses did not
decrease over time but remained stable or followed an upward trend (Fig. 5, lower left). As ESAT-6 and CFP-10induced IFN- responses correlate with bacterial load and the severity of pathology we propose that these
response peaks after INH-treatment cessation, and following re-challenge in the ‘fully protected’ (ie NVL) animals
represent the reduction of bacterial loads as consequence of anamnestic and protective immune responses [3].
FIG.5. Kinetics of in vitro IFN- responses as measured by BOVIGAM assay. Results are presented as mean OD450 (+ SEM)
after stimulation with PPD-B or ESAT-6/CFP-10 with medium background levels subtracted. Upper panels: Group A animals
(INH-treatment, but no re-challenge). VL animals, left panel; NVL animals, right panel. Lower panels: Group B animals (INHtreated, then sp. 35 re-challenged). Left panel, VL animals; right panel, NVL animals.
SID 5 (Rev. 3/06)
Page 10 of 27
Optical Density 450nm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
CFP10/ESAT6
PPDB
10
20
30
40
weeks PI
20
30
Optical Density 450nm
Optical Density 450nm
10
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
10
20
30
40
weeks PI
INH
mean Bovigam data type 35 challenge NVL (n=2)
INH
mean Bovigam data type 35 challenge VL (n=6)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
optical density 450nm
Mean Bovigam data NVL no challenge (n=3)
Mean Bovigam data VL no challenge (n=5)
40
weeks after first infection
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
10
20
30
40
weeks after first infection
INH
INH
Challenge
week 22
Challenge
week 22
b) IL10 cytokine assays using five day whole blood culture supernatants showed that a peak of specific IL10
activity was always detected within two weeks of commencement of INH treatment (FIG.6). Interestingly, following
M. bovis sp. 35 challenge of un-treated animals we did not observe a corresponding IL-10 response (data not
shown), nor was an IL-10 response induced in the INH-treated animals following M. bovis sp. 35 re-challenge
(data not shown). This observation can be interpreted in several ways. Firstly, the presence of a specific IL-10
response in protected animals before re-challenge could suggest that IL-10 constitutes a potential marker
(surrogate/correlate) of protection, for example T regulatory cell produced IL-10 could be involved in the formation
of solid T cell memory. Equally feasible is the interpretation that T regulatory cell-produced IL-10 is acting a antiinflammatory agent counteracting the effects of IFN- that would otherwise contribute to immunopathology [7].
Only further experiments will allow us to assign a function to these IL-10 responses.
FIG.6. Mean specific IL10 (n=16) responses. IL-10 in culture supernatants was determined by ELISA, IL-10 production from
cultures stimulated with either PPD-B or a peptide cocktail of ESAT-6 and CFP-10, with medium background values
subtracted, is expressed as mean units (+ SEM). Shown are results of all 16 INH-treated animals up to the time of rechallenge (week 22). No further IL-10 production was observed post-rechallenge (not shown).
25
PPDB
ESAT6/CFP10
Units of IL10
20
15
10
5
0
2
4
-5
6
8 10 12 14 16 18 20 22
weeks post infection
start INH
treatment
INH
stopped
c)TNFand IL-4cytokine ELISA assays did not yield any data that could usefully be correlated with either INH
treatment, pathology or culture data.
d) IL4 splice variants
The presence of 2 IL4 splice variants IL42 and IL43 were assessed in bovine ex vivo un-stimulated PBMC.
Results from cattle from SE3024 Objective 3B (INH-assisted protection model) were compared with results from
BCG-vaccinated cattle, naïve controls and naturally infected field reactors. mRNA was quantified using paired
primers and taqman probes specific for IL42 and IL43. GAPDH was used as the house-keeping gene to which
cytokines were normalised in order to remove differences in the amounts of mRNA within each sample (see
Technical Annexe for complete methods). Firstly, we investigated field reactors versus naïve controls for the
presence of the splice variants in naturally infected populations. Field reactors were obtained from multi-reactor
breakdowns with culture-confirmed bovine tuberculosis. Dividing the IL43-positive field reactors (which were also
ESAT-6 and/or CFP-10 as well as PPD-B IFN- positive) into animals with and without gross visible pathology
(VL, visible lesion and NVL, non-visible lesion, respectively) (Fig.7) revealed that the highest expression of IL43
was found in the NVL group. IL43 was significantly higher in this group of compared to the VL group (Mann
SID 5 (Rev. 3/06)
Page 11 of 27
Whitney p=0.0091). Thus a high IL43 mRNA expression appeared to correlate with the absence of pathology in
cattle naturally infected with M. bovis.
FIG. 7. IL43 expression in NVL and VL field rectors. Results are expressed as mean IL43 expression/unit GAPDH (+ SEM).
IL43-positive field reactors
*
IL43/unit GAPDH
20
15
10
5
0
NVL
VL
pathology status
We next investigated IL4 splice variants in cattle protected from virulent M. bovis challenge by the INH-treatment
described above. Increased IL43 was observed following M. bovis (sp.9) infection and INH treatment from week
13, and this reached statistical significance on weeks 21 and 25 (Mann Whitney p=0.0159 and p=0.0317
respectively) (Fig. 8). As expected, increased IL43 was also observed in the group B animals before rechallenge (Fig. 9), compared to group A animals (that were INH-treated but not re-challenged). After re-challenge,
IL43 responses remained higher in group B comparEd to group A animals, although this difference in responses
was not significant (Fig. 9). Similar increases in IL43 expression were also observed in BCG vaccinated animals (not
shown, as not part of SE3024). In conclusion, therefore, ex vivo expression of IL43 constitutes a potential surrogate for
protection.
FIG.8. IL43 expression during INH-attenuated M. bovis infection in group A animals. Results of individual animals are
presented with horizontal bars indication median responses.
IL43/unit GAPDH
100
* *
10
1
0.1
0.01
0.001
INH
0
0
3
7
13
21
25
30
34
Mb. sp.9
Fig. 9. IL43 expression during INH-attenuated M. bovis infection in animals from all three groups. For clarity and ease of
making comparisons between groups, only median values are shown. Group A, SP9/INH (blue); group B, SP9/NIH/Sp35
(red), group C, Sp35 infection only (green).
SID 5 (Rev. 3/06)
Page 12 of 27
Interestingly as discussed above, it was IL43, and not IL42 that appeared to be the dominant splice variant in
these investigations with un-stimulated PBMC. We did note however that stimulation of PBMC with antigen
(PPDB) resulted in different patterns of expression, often with strong IL4 and IL42 responses but weak IL43.
Figure 10 below shows the mean PPDB-specific responses in M. bovis-infected, BCG-vaccinated and naïve
cattle.
FIG. 10. PPDB-specific cytokine induction in cultured PBMC after in vitro stimulation with bovine tuberculin( PPD-B).
Compared are responses of M. bovis sp. 9 infected animals, BCG vaccinated calves as well as uninfected/unvaccinated
controls (naïve).
fold increase mRNA
10
IL4
IL42
IL43
8
6
4
2
0
INFECTED
BCG
NAIVE
Differences in the kinetics of bovine splice variants have been noted previously [8], and so taken together the
data suggest differential activation and possibly also different functions for IL42 and IL43 in bovine tuberculosis.
We are now able to pursue this hypothesis of differential splice variant activity in cattle.
4. Potential protective antigens.
INH-treatment conferred a significant degree of protection against re-challenge, which provided us with an
opportunity to compare antigen profiles between treated and sp. 35 re-challenged cows (protected animals) and
animals that were only infected with sp. 35 (unprotected controls). In previous experiments we had shown that
BCG vaccinated animals that were subsequently challenged with M. bovis developed rapid, anamnestic
responses about 2 weeks post-challenge, at time points where responses in unvaccinated control animals had
not developed [6]. We hypothesised that antigens recognised at this early timepoint post-infection would therefore
constitute protective antigens. As discussed above, we observed similar response peaks after re-challenge.
Therefore, we evaluated a panel of 20 antigens for their capacity to induce IFN- responses in INH-treated and
re-challenged cattle and compared their responses to untreated control animals only infected with sp. 35. These
experiments were undertaken 2, 3, and 4 weeks post-challenge with sp. 35 (ie at weeks 24, 25, 26). The results
are presented in Fig. 11, and demonstrate that 2 weeks post-infection very few antigens are recognised in the
control animals, whereas at least 3 antigens induced IFN- responses in the treated animals (antigens indicated
by red boxes in Fig. 11), whilst responses against these and other antigens could be demonstrated in both groups
at the later time points. These three antigens are now being evaluated for their ability to act as subunit vaccines in
protecting cattle against bovine TB (as part of project SE3224).
Fig. 11. IFN- response after stimulation with a panel of antigens. Responses are expressed as mean IFN- production in pg +
SEM. Potential subunit vaccine candidates are indicated by red boxes. Definition of a potential protective antigen: responses 2
weeks post-challenge in protected cattle (M. bovis type 9 infectino, INH-treatment, re-challenge with sp. 9, bottom panel), no
responses as 2 weeks post-challenge in control animals (M.bovis sp. 35 infection only, top panel). Please note that the identity
of the tested antigens is being withheld to protect IP.
SID 5 (Rev. 3/06)
Page 13 of 27
Objective 4. To establish and validate an M. bovis aerosol challenge model for cattle (VSD component,
report prepared by Dr Jim McNair and edited into the body of the complete report by the project co-ordinator, Dr
Martin Vordermeier).
Bovine tuberculosis is primarily a disease of the respiratory tract and the main route of transmission, is
aerogenous. The typical patterns of disease, as seen in field cases, are predominantly within the lower respiratory
tract, and to a lesser extent in the upper tract, with a smaller number of cases observed through disclosure of
lesions in the mesenteric lymph nodes. The dose of M. bovis considered to be infectious is thought to be very
small although this has not been defined experimentally. The prime objective of this experimental series was to
combine the aerogenous route of infection with a defined number of colony forming units to clarify the infectious
doses required to initiate infection. Objective 4 comprised three distinct phases. The first phase was based on
the preparation of unicellular cultures of M. bovis with defined colony forming units. The second phase was based
on developing expertise with the modified Madison aerosol chamber and to define particle size in aerosol
suspensions and M. bovis survival curves. The third phase was the application of the chamber for the delivery of
defined aerosolised doses to naïve cattle followed by measurement of immune responses and definition of the
pathological picture. As the procedures used in this objective are direct outputs of this project, and integral to the
success of these experiments, we will describe them in the context of the individual experiments in detail rather
than listing them in the methodological annexe.
1. Development of the aerosol model to deliver M. bovis to cattle, and determination of particle sizes.
a) Preparation of unicellular M. bovis stocks. Our approach is based on the growth of M. bovis with disruption of
multi cellular clumps and cell enumeration. M. bovis strain AF2122/97 was used throughout all experiments.
100ml volumes of 7H9 broth (0.22um filter sterilised) were seeded with 1ml stock culture and incubated for 7 days
at 37oC. 10ml of the 7H9 broth were transferred to a 7H11 agar flat and allowed to rest for one hour before the
addition of 100 ml 7H9 broth. At 14 days post inoculation, cultures were rocked gently to remove the surface
bacterial growth after which supernatants were pooled. 40ml aliquots of live cultures were transferred to 100ml
vaccine bottles complete with a sealed septum and passed through a 23-guage needle a total of ten times. The
cell suspension was then filtered through a 5µm PVDF filter and stored (frozen at -70oC) in 2ml aliquots.
Examination of these cultures by Ziel-Nielsen staining revealed a predominance of single cells, with only a few,
small clumps present.
b) Quantificaton of aerosol delivery. M. bovis aerosolised by nebulisation and induced into the airstream of the
Madison chamber was sampled using an all-glass-impinger (AGI) system to trap bacteria and to allow
enumeration. This system also allowed definition of the M. bovis curve generated by the aerosol stream. With the
Madison chamber operating normally and delivering M. bovis to the airstream, air samples were taken at various
SID 5 (Rev. 3/06)
Page 14 of 27
time points for culture to estimate CFU delivered. Titration studies based on M. bovis cultures with defined CFU
indicated a strict relationship between the numbers of M. bovis added to the nebuliser, the generation of a stream
containing aerosolised bacteria and cell survival over a defined period. In addition, the decrease in cell viability
pre and post nebulisation was minimal. Furthermore, our studies indicated that the particle size generated by the
nebuliser was in the range 0.8 to 15µm, a range which is compatible with M. bovis in single cell suspensions.
c) Delivery of non-infectious aerosols, estimation of particle sizes. In order to gain confidence in the operation of
the Madison chamber while delivering an aerosol stream to calves, a number of experiments were carried out
using the chamber, to deliver a non-infectious aerosol to calves. Originally, it was envisaged that calves would
require sedation using Rompum in order to apply the mask over the muzzle for the length of time required to
deliver an infectious dose by aerosol. However, in a series of preliminary experiments, it was evident that calves,
when treated quietly and gently, would accept the mask for up to five minutes. Recordings taken from a
pneumotachometer also indicated that there was no respiratory distress associated with the mask during this time
period. Pneumotachometer data gave extremely accurate and consistent volumes of inspired and expired air over
a given time period. Coupled with the data generated by M. bovis nebulisation experiments, we were confident
that we were able to accurately deliver defined infective doses of M. bovis to individual calves.
Objective 4, milestones 04/04, 04/05, 04/06: Aerosol delivery of M. bovis to calves, dose titrations
Exp. 1. Aerosol model tested with low dose (10 4 CFU)
Exp. 2. Aerosol model tested with low dose (10 2 CFU)
Exp. 3. Aerosol model tested with low dose (101 CFU)
These experiments were designed to assess the effect of delivering aerosolised M. bovis at various doses to the
respiratory tract of cattle, and to assess the effect of dose on immunology and pathogenesis while using the
natural route of infection. This experimental series was based on groups of five calves each experiment, all of
which were purchased from farms with a TB-free history over the previous five years. All calves were Friesian
cross bred, castrated males aged between 4 and 6 months. Each infection was carried out according to the same
protocol. Calves were restrained individually in a cattle crush and a member of staff placed and held the mask in
position. After a short period of familiarisation, the aerosol stream carrying M. bovis was directed to the mask for a
period during which 100 litres of air containing the infectious dose was inhaled. The mask was kept in position for
a further period of two minutes to allow M. bovis exhaled from the lungs to be expelled and trapped within the
HEPA filters linked to the aerosol chamber. All calves were treated similarly within a thirty minute period, following
which, the chamber was removed from the animal accommodation for fumigation and decontamination.
Over a period of nine months, animals were kept and were blood sampled on a regular basis. Immunological tests
were carried out either immediately or, for antibody analysis, samples were stored at -20oC until assayed. One
week before post mortem examination, calves were skin tested using conventional PPDA and PPDB reagents
and interpretation of results. A detailed post mortem examination was carried out when all the tissues and lymph
nodes associated with the upper and lower respiratory tract were examined and sampled for mycobacterial
culture.
a) Infectious dose delivered by aerosol chamber. Prior to each experimental infection, the infectious dose was
calculated from stock cultures of known CFU and titration curves derived from aerosol experiments. Table 6
summarises the mean values calculated for each experiment. When airstream samples taken during the 10 1 CFU
infection and cultured, no bacterial growth was recorded on any plates. This failure to support bacterial growth
meant that the infectious dose given to this group of calves could not be verified.
Table 6: Mean values of infectious dose and inhalation time, calculated for each group of calves (5 calves per
group) infected by aerosolised M. bovis using the modified Madison chamber.
Experiment
Intended dose
Inhalation time
Actual dose
Range
(Secs)
1
104 CFU
170
7.68 (0.29) x 104
7.2– 7.9x104
2
2
10 CFU
147
226 (21.9)
101 - 341
3
101 CFU
169
Unknown*
NA
* Failure to culture M. bovis on this batch of agar plates meant that this infectious dose could not be calculated
There was a high degree of uniformity, within each experimental infection, of the dose delivered to the respiratory
tract of calves. This uniformity was due to a combination of accurate colony forming units defined in stock M.
bovis cultures and the use of a pneumotachometer to measure the volume of air inspired.
b) In vitro cell mediated responses following infection with M. bovis. Before and after experimental infection,
all calves were blood sampled and tested for IFN- release using whole blood cultures stimulated with various
complex and recombinant antigens. Supernatant fluids were harvested and tested for the presence of IFN- using
the Bovigam test kit. Pre-infection, all cattle were found to have negligible IFN- responses. When tested post
SID 5 (Rev. 3/06)
Page 15 of 27
infection using PPDB, there was no difference in the onset of the mean IFN- response in each of the three
groups. However, in general terms, the larger the dose of M. bovis delivered, the greater the IFN- response seen
post infection (Figs. 12-14). Within the 104 and 102 CFU groups all animals remained positive after three weeks
post infection. However, in the group with the lowest infectious dose (10 1 CFU) there were inconsistent responses
throughout the post infection period. All animals were positive at various time points but only two animals (2042-1
and 2275-5) were consistently positive post infection.
IFN- responses to ESAT-6 were also greater with the larger infectious doses, when judged by the mean group
response. However, in both the 102 CFU and 101 CFU groups there was a large degree of variation observed. In
the 102 CFU infection group, two animals (2237-2 and 2242-7) were consistently negative to ESAT-6 and in the
101 CFU group most animals on most occasions remained unresponsive to ESAT-6. Immune responses to
CFP10 were very similar to ESAT-6 but not identical. In the 104 CFU group, the mean response post infection,
was positive throughout the post infection period with the exception of four individual time points. On three of
these occasions, if an animal was negative to CFP10, it was positive to ESAT-6. The IFN- response to CFP10 in
the 102 CFU group was also consistent, with four calves responding to this antigen post infection while one
animal (2239-4), did not respond at any time post infection. Generally, all animals were unresponsive to CFP10 in
the 101 CFU group with the exception of three animals on only one occasion. This M. bovis titration series has
shown that as the infectious dose is lowered, the more inconsistent the immune response to the two recombinant
antigens becomes.
c) Skin testing with conventional PPDA and PPDB. Two animals infected with 104 CFU became ill during the
experiment and were euthanized on animal welfare grounds during weeks 13 and 19 post infection and were not
skin tested. All of the other cattle were skin tested using PPDA and PPDB by intra dermal injection, with skin
thickness measured at 0 and 72 hours post inoculation (table 7). All calves in the 10 4 CFU and 102 CFU groups
that were skin tested were positive using test interpretation criteria as applied under field conditions. The mean
increase in skin thickness for each group was very similar, 17.2 and 13.8 mm respectively. In contrast, from the
five cattle tested in the 101 CFU group, only one (2275-5) was skin test positive. Coincidently, this animal was
also M. bovis culture positive (cranial mediastinal lymph node) and had the greatest responses to PPDB by IFN-
release. This animal had also intermittent but inconsistent IFN- responses to ESAT-6 and CFP10.
Table 7. Summary of the skin test results from each experimental infection (10 4, 102 and 101 CFU), based on the
increase in skin thickness (PPDB – PPDA) at 72 hours post intradermal injection with PPDA and PPDB.
Experiment
1
2
3
Infectious
dose
7.68 x 104
226 (21.9)
Unknown*
Number
tested
3
5
5
Number
positive
3
5
1
Mean increase (mm)
17.2
13.8
1.3
Range
(mm)
9-26.5
7-27
-1 to 5
d) Post mortem examinations. All calves were given a detailed examination post mortem, to identify and record
the presence of tuberculous lesions typical of M. bovis infection. The presence and size of lesions were recorded
and used to calculate a post mortem score for each tissue or lymph node examined and which was used to
compare individual animals or groups (table 8). Samples of tissue, irrespective of the presence of lesions, were
taken for bacteriological culture. For each infectious dose given, there was a very different pathological picture as
defined by the distribution of lesions. All calves given 10 4 CFU were found to have tuberculous lesions that were
confined to the lower respiratory tract with the exception of calf 1952-2 where a single lesion was found in the left
palatine tonsil. All lung lobes were found to have lesions with the exception of calf 2246-2, were the middle and
accessory lobes were free from lesions. Lesions were found in the cranial and caudal mediastinal lymph nodes of
all calves, however, only two calves (1952-2 and 1953-3) were found to have lesions in the cervical lymph node.
The post mortem score in this group ranged between 16 and 48, with the mean value calculated at 33.8 (SD =
12.2).
Table 8. Comparison of the post mortem scores calculated after each experimental infection (10 4, 102 and 101
CFU).
Experiment
Infectious
Number
Number
Mean PM score
Range
dose
examined
lesion
(mm)
positive
1
7.68 x 104
5
5
33.8 (12.2)
16-48
2
226 (21.9)
5
3
3.6 (3.9)
0-9
3
Unknown*
5
0
0
0
By reducing the infectious dose to 10 2 CFU, a much more restricted pattern of lesion development was observed.
No lesions were found in two calves (2022-2 and 2247-4) at gross post mortem examination, while three calves
were found to have lesions in the left and right bronchial, cranial and caudal mediastinal lymph nodes. Only two
SID 5 (Rev. 3/06)
Page 16 of 27
lung lobes were found to be lesioned, the left cranial lung and the right caudal lung. All other lung lobes were
found to be lesion free. The mean post mortem score for this group was 3.6 (SD = 3.9) At the lowest infectious
dose (101 CFU) no lesions were disclosed in any of the calves exposed, in any of the upper or lower respiratory
tract tissues examined. All tissues examined were of a normal appearance and size. The only exception was the
palatine tonsils which were seen to have discreet, pus filled areas which were culture negative for M. bovis. The
post mortem score for these calves, individually, and for the group was 0. At the time of writing, one tissue sample
(the cranial mediastinal lymph node from calf 2275-5) was found to be culture positive for acid fact bacilli and later
confirmed as M. bovis AF2122/97 by spoligotyping and VNTR analysis. The presence of this isolate, albeit from
only one animal, validates the infectious dose given to this group and helps to explain the immune responses
generated following exposure.
There is a close association between the higher infectious dose (104 CFU), the pattern of lesion development and
the immune responses measured. All five caves were lesioned, all were skin test positive and all had strong,
persistent in vitro IFN- responses to PPDB, ESAT-6 and CFP10. In contrast, infections with either 102 or 101
CFU were characterised by a reduction or absence of lesions, and, inconsistent in vitro immune responses. When
102 CFU was given, the two calves without lesions were skin test positive, had strong in vitro IFN- responses to
PPDB and although seen at a much lower level, IFN- responses to ESAT-6 and CFP10 were also present. In
contrast, the IFN- response to CFP10 was absent in an animal that was lesioned.
The greatest degree of variation was seen in the group given 10 1 CFU were only one animal was skin test
positive. This calf was also culture positive for M. bovis. All animals in this group were positive to PPDB at a
number of time points post infection, however, most animals were negative to both ESAT-6 and CFP10 for most
of the post infection period and were only positive on an intermittent basis. There is an implication that at this
infectious dose, animals are more likely to be positive by PPDB than by recombinant antigens. This pattern was
also seen in the animal which was culture positive, when PPDB induced responses to a greater extent than
responses provoked by either ESAT-6 or CFP10.
e) Bacteriological culture. There was evidence of spread from the lower to the upper respiratory tract following
culture of tissue samples taken from calves infected with 104 CFU. In two calves, M. bovis was isolated from the
upper trachea. In a third animal, samples from the lower trachea and the left medial retropharyngeal were culture
positive. In a fourth animal the right and left palatine tonsil, the left pharyngeal tonsil and the nasopharynx were
culture positive. There was no evidence of disease spread from the lower tract in calves infected with 10 2 CFU. In
this group, M. bovis was confirmed in lesioned tissue samples in the three calves where lesions were recorded.
However, M. bovis was cultured from one animal (2022-2, caudal mediastinal lymph node) where no lesions were
recorded) but not isolated from any tissue samples taken from animal 2247-4, which was not lesioned. At the time
of writing, only one M. bovis culture positive tissue (caudal mediastinal lymph node taken from animal 2275-5)
has been recorded in the group infected with 101 CFU. All cultured samples taken from this group will be kept for
a total of 86 days before recording the final result.
Conclusions
This work highlights the value of using the natural host for M. bovis infection models and the importance of using
a natural route of infection to interrogate the effects of dose on infection establishment, pathogenesis, host
immunity and diagnosis. This approach, intended to mimic tield type infections, will give invaluable insight into
naturally acquired M. bovis infection by using a natural route of infection in cattle. In particular, the lower
infectious doses induced an immune state more akin to frequently observed in the field, where the relationship
between disease status and immune response can be complex. This model can be exploited to explore these
complexities and to improve our diagnostic capabilities.
Discussion of data in objective 4 in relation to results from objective 1.
Interestingly, and seemingly in contrast to the intratracheal infection model, a reduction in pathology was
observed with decreasing infective doses (i.e. 10,000 CFU compared to the two lower doses). However, not the
same dose titrations were performed in both infection models and we could show in previous studies with the
intratracheal challenge model that more severe disease and pathology resulted after infection with of > 10,000
CFU than with the lower doses applied here (< 1000 CFU). Therefore, the results between high and low doses
(>10,000 CFU and below 1000 CFU) are comparable between the intratracheal and the aerosol challenge routes.
The dose range between 100 and 1000 CFU need to be evaluated in the aerosol model to allow full comparison
with the intratracheal model. Further, aerosol infection with 10 CFU resulted in no visible pathology, although the
exact infective dose could not be determined in this experiment. However, at face value, these findings seem to
be in contrast to the intratracheal model where doses between 1 and 10 CFU resulted in visible pathology.
However, even intratracheal infection with 1 and 10 CFU did not result in visible pathology in all animals (for
example only 3/6 animals infected with 1 CFU presented as VL animals, or 5/6 infected with 10 CFU), furthermore
the severity of pathology induced after intratracheal challengewith 10 or 100 CFU compared to that induced after
aerosol infection with 100 CFU was of similar range and not significantly different (1-12 with intratracheal infection
at these doses compared to 0-9 with 102 CFU delivered by aerosol). Thus, and taking into accout the relatively
small numbers of animals that we were able to use in these experiments, it is difficult to conclude unambiguously
that fundamental differences exist between these two models at these low and limited infection doses. Further
studies will be required to determine this without ambiguity. One obvious theoretical difference between the two
SID 5 (Rev. 3/06)
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models is that the intratracheal route delivers the infective inoculum concentrated to the same site in the lung
(right cranial lobe), which from previous studies we deem to be highly susceptible. Therefore such aninfection
results in a more uniform disease pattern which is a signficiant advantage when performing, for example
vaccination studies, where financial constraints necessitate that all animals develop disease. The aerosol route
will deliver the dose dispersed over the whole lung, i.e. it is possible that areas of the lungs are targeted that are
not equally susceptible to infection, therefore resulting potentially resulting in less uniform disease development.
However, more studies will be needed to address this hypothesis.
SID 5 (Rev. 3/06)
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Figure 12. Mean IFN- responses to PPDB, ESAT-6 and CFP10 following infection with 104 CFU M. bovis.
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Figure 13. Mean IFN- responses to PPDB, ESAT-6 and CFP10 following infection with 102 CFU M. bovis.
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Figure 14. Mean IFN- responses to PPDB, ESAT-6 and CFP10 following infection with 101 CFU M. bovis.
SID 5 (Rev. 3/06)
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Project SE3024: Summary of main findings and suggestions for further research
A. Main results:
 Intratracheal and aerosol routes of experimental challenge routes are the most appropriate routes
because they best reflect the lesions most commonly associated with natural infection (in GB).
 The lower respiratory tract is highly susceptible, infective doses of 1 CFU delivered directly to the
lung by the intratracheal route will result in pathology and disease in the lung and/or associated
lymph nodes.
 Taking results of objectives 1 and 4 together, one conclude that the predominant disease phenotype
seen in GB is caused by aerosol infection of small numbers of bacilli delivered by small aerosol
particles (3-5 organisms) to the lung.
 The observations above therefore will allow a more informed and precise analysis of
transmission risks, and of the virulence of M. bovis infection of cattle.
 BOVIGAM responses contract with reduction in bacillary load, but animals present with
positive results at most time points, although results using the lowest infective dose delivered by
aerosol showed a more variable response with the defined antigens (ESAT-6/CFP-10) not
performing consistently as well as tuberculin. Animals infected with this dose and route also at
certain time points would have escaped detection by the BOVIGAM test.
 Using low infective doses or chemotherapy, it is possible to produce, apart from VL/Cu+ animals,
also animals that are NVL/cu+ or NVL/cu- yet positive in skin test and IFN- test: Such animals are
therefore not ‘false-positives’ but infected sub-clinically and could pose future infection risks to
herds. This is particularly relevant in the case of new outbreaks in otherwise non-endemic regions of
GB, where the rapid removal of any infected animal is a priority. Furthermore, it emphasises also the
relevance of using alternative diagnostic tests like the BOVGIAM IFN-test in this scenario to
maximise the identification and removal of the number of infected cattle. This conclusion is also
supported by results from objective 4 where animals infected with the lowest infection dose not only
presented as NVL/culture-negative (ie could be considered as latently infected) but were also skin
test negative yet detectable using the BOVGIAM test.
 Primary infection confers significant degree of protection against re-infection.
 This protection was against a heterologous M. bovis strain (cross-protection), which is encouraging
for vaccine development using BCG or one particular attenuated M. bovis strain.
 Successful chemotherapy requires active immunity.
 IFN- has been confirmed as a marker of bacterial load/pathology.
 The IL43 splice variant constitutes a surrogate of protection.
 Memory cow model can also be used to prioritise potential protective antigen for subunit vaccine
development, and 3 candidates have been identified in this experiment.
 Aerosol infection was intended to mimic field type infections, and gave invaluable insight into
naturally acquired M. bovis infection by using a natural route of infection. In particular, the lower
infectious doses induced an immune state more akin to frequently observed in the field, where the
relationship between disease status and immune response can be complex. This model can be
exploited to explore these complexities and to improve our diagnostic capabilities.
 Results from project SE3024 have also cross-cutting relevance for Defra’s cattle TB vaccine
development programme. These cross-cutting research results are indicated in this summary by
italics.
B. Possible future work:
 Investigations into the nature and degree of latency of TB in cattle, both by field and experimental
studies.
 To investigate the functions of IL-4 splice variants in the regulation of immunity in cattle.
 To determine the protective potential of subunit candidates identified.
SID 5 (Rev. 3/06)
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Technical Annexe: materials and methods used throughout this project (objectives in brackets, methods
specific to objective 4 are discussed in the context of the results section above).
Mycobacterial antigens. Tuberculin PPD preparations from M. bovis (PPDB) and from M. avium (PPDA) were
produced at the Veterinary Laboratories Agency (VLA) of Weybridge as described previously. Recombinant
ESAT6 and CFP10 were obtained from Dr. Singh, GBF, Braunschweig, Germany, peptide cocktails representing
ESAT-6 and CFP-10 (Vordermeier et al., 2001) were prepared from individual peptides prepared by Pepscan Ltd,
Lelystad, the Netherlands.
Experimental M. bovis infection (objectives 1 and 3). Friesian-cross females or castrated males were obtained
from a herd free of M. bovis infection, as defined by a history of negative skin test results. All animals were also
negative in the IFN- diagnostic test for bovine tuberculosis. Calves were infected intra-tracheally using the GB
field isolate AF2122/97 (spoligotype 9) in all experiments unless otherwise stated. GB field isolate AF 61-1307-01
(spoligotype 35) was used for re-infection of calves in OBJ.3B. During these studies animals were housed in an
high-security isolation unit under negative pressure, and expelled air was filtered. The single comparative
tuberculin skin test (SCITT) was performed on all animals at the time points stated in the text for each objective
above. Euthanasia was carried out by intravenous injection of sodium pentobarbitone, and a detailed post mortem
analysis performed as described below. For the lymph nodes, each of the following types of lymph node (or lymph
node chain) was removed aseptically: right and left lateral retropharyngeal, right and left medial retropharyngeal,
right and left submandibular, right and left cervical superficial, right and left bronchial, crainial mediastinal chain,
caudal mediastinal chain, and mesenteric. These were serially sliced (approx. 2mm slices) with a scalpel and
inspected. Samples from individual nodes and lesions were obtained for mycobacterial culture and
histopathological examination. The remaining superficial and visceral lymph nodes were inspected in situ. For the
lungs, each lobe was serially sliced with a sharp knife. All slices were palpated and inspected. The respiratory
airways were opened as far into the lung parenchyma as possible and inspected.
Isoniazid (INH) treatment of cattle (obj. 3). Isoniazid tablets (isonta/28/100) were obtained from UCB Pharma
Limited (Bedfordshire, U.K.). Tablets were ground up using a conventional coffee bean grinder and the correct
dose/animal (25mg/kg/day, as determined by individual animal weight [9]) suspended in water and fed orally
using a Phillips Cattle Drencher (Cox Surgical, U.K.).
Measurement of Plasma INH using High Performance Liquid Chromatography (HPLC, obj. 3). Plasma INH
was measured following the methodology of Smith et al [10]. Plasma samples were deproteinated by
precipitation with 10% trichloracetic acid prior to derivatization with cinnemaldehyde and neutralization with
ammonium acetate. INH in plasma was then measured. In addition, to determine levels of circulating N acetyl INH
(a metabolite), a second sample was treated with hydrochloric acid prior to derivatization in order to convert N
acetyl INH to INH in the sample. The amounts of INH in each sample was determined by HPLC. Each sample,
and a set of standards treated in the same way, at the same time as the samples, were chromatographed on a
Jupiter C18 5 column (250 x 4.6 mm) using an isocratic solvent system: 0.1 M ammonium acetate, pH
5.6/acetonitrile/methanol (47/23/30 v/v/v) at 1ml/min. The amount in each standard was determined by reading
against a line of peak area against amount of INH plotted for the corresponding set of standards.
N.B. Standard for INH assay kindly provided by Celltech Manufacturing Services Ltd., U.K.).
Liver function assays (ob. 3).
Filter-sterilised (0.2m) serum samples for individual calves were analysed at VLA-Shrewsbury for 3 key liver
function markers; aspartate aminotransferase, gamma glutamyl transferase and total bilirubin, using kits available
from Olympus UK Ltd (London, UK)
Whole blood culture for cytokines (objs. 1-3). Whole blood cultures were performed in 96-well plates in
0.25ml/well aliquots in duplicate by mixing 0.225ml whole blood/well with 0.025ml of antigen-containing solution
(at 10X final concentration required). Supernatants were harvested after 24 hours (for IFN- and TNF-) and 5
days (for IL-4 and IL-10) [2].
IFN- assay (all objectives). 24-hour whole blood supernatants were determined using the BOVIGAM enzyme
immunoassay kit and following the manufacturers instructions (Biocore, USA).
TNF-assay (objective 3B). 24-hour whole blood supernatants were determined using the bovine TNFscreening kit (Endogen).
IL-4 ELISA assay (objective 3B). 3-day whole blood supernatants were determined using the bovine IL-4
screening kit (Endogen).
IL4/B-cell Bioassay (obj. 1-3). 5-day whole blood supernatants were tested in the B cell bioassay as previously
described [11]. Bovine B cells from a naïve skin-test-negative animal were positively sorted from PBMC using the
SID 5 (Rev. 3/06)
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monoclonal antibody ILA58 (IAH, Compton, Berkshire) specific for bovine immunoglobulin (Ig) light chains, which
both labels and preactivates B cells by cross-linking the surface Ig. PBMC were then incubated with rat-antimouse IgG2(a+b)-coated microbeads before positive sorting using the MACS column separation system (Miltenyi
Biotech, Surrey, UK). B cells were eluted, washed and resuspended to 10 6/ml in culture medium (RPMI 1640 with
Glutamax [Gibco] supplemented with 10% foetal bovine serum [Gibco], nonessential amino acids [Gibco],
100U/ml penicillin, 100g/ml streptomycin and 5x10-5M 2-mercaptoethanol), and 0.1ml added/well to 96-well
round-bottomed culture trays together with 50l/well of supernatant to be tested, in duplicate. Plates were
incubated overnight at 37oC and 5%CO2, pulsed with tritiated thymidine, and incubated for a further 24 hours
before harvesting and counting using -scintillation.
IL-10 assay (objective 3B). A bovine IL10 antibody pair obtained from Serotec was used for this assay. 96-well
plates were coated overnight with bovine anti-IL10 prior to adding supernatant samples. Binding of the
biotinylated detection antibody was visualised using horseradish-peroxidase conjugated streptavidin (Sigma) and
Pierce Super Signal ELISA femto maximum sensitivity substrate, the resulting light emissions being read on a
luminometer. IL10 standard was kindly provided by J. Hope, IAH, Compton.
IFN-ELISPOT [12] (objectives 1 and 3). ELISPOT plates (Immobilon polyvinylidene difluoride membranes,
Millipore, France) were coated overnight at 4oC with the bovine IFN- -specific monoclonal antibody clone 5D10
(Biosource International, California, USA). Unbound antibody was removed by washing, and the wells were
blocked with 10% foetal calf serum in RPMI 1640 medium (Life Technologies, Paisley, Scotland, United
Kingdom). PBMC (2 x 105/well suspended in tissue culture medium [RPMI 1640 supplemented with 5% controlled
process serum replacement type 1; Sigma Aldrich; nonessential amino acids; Sigma Aldrich; 5 x 10-5 M 2mercaptoethanol, 100 U of penicillin/ml, and 100 µg of streptomycin sulfate/ml; Sigma Aldrich]) were then added
and cultured at 37°C and 5% CO2 in a humidified incubator for 24 hours. Spots were developed with rabbit serum
specific for IFN- followed by incubation with an alkaline phosphatase-conjugated monoclonal antibody specific for
rabbit immunoglobulin G (Sigma Aldrich). The spots were visualized with 5-bromo-4-chloro-3-indolylphosphatenitroblue tetrazolium substrate (Sigma Aldrich).
RNA extraction, reverse transcription and real-time quantitative PCR (polymerase chain reaction, obj. 3B).
Previous clinical studies have shown that the IL42 splice variant is to be found in un-stimulated PBMC [3-5].
Therefore we used freshly isolated PBMC for all cattle in this report. RNA was prepared from 10 6 PBMC/sample
using the RNeasy mini kit system and following the manufacturers instructions (Qiagen Ltd., West Sussex, UK).
PBMC were isolated by layering over Histopaque 1077 (Sigma) and centrifuging at 800g for 40 minutes at room
temperature, washed twice in Hanks Balanced Salt Solution (HBSS, Life Technologies) and counted. 106 PBMC
were aliquoted into sterile screw top vials, the cells pelleted by centrifugation and the supernatant discarded.
Lysates were prepared by resuspending the pelleted cells in 350l of RLT buffer according to manufacturers
instructions (Qiagen Ltd), and lysates stored at minus 80 oC. Cell lysates were centrifuged through DNA shredder
columns (Qiagen Ltd) prior to ethanol extraction and RNA isolation using the RNeasy column system. RNA
preparations were finally treated with Turbo DNase (Ambion (Europe) Ltd., Huntingdon, UK) for 30 minutes at
37oC. Reverse transcription (RT) was carried out using the Qiagen Reverse iT TM T-primed First Strand Synthesis
Kit and following the manufacturers instructions. RT controls (no RTase enzyme) were included for every sample.
The PCR assay was optimized using plasmids containing the constructs for IL4, IL42 and IL43, as well as using
bovine PBMC (ex vivo, antigen-stimulated and mitogen-stimulated). PCR was carried out in triplicates for each
sample using a Universal PCR Mastermix (Applied Biosystems, USA) plus forward and reverse primers and
Taqman probes as described previously [8]. For a positive signal, at least 2 out of the 3 PCR reactions in any
triplicate had to give detectable Ct values. Cytokines were normalized to the housekeeping gene GAPDH [13]. All
primers and probes were manufactured by MWG-Biotech, Ebersberg, Germany. Standard curves for each
cytokine and GAPDH were prepared from the PBMC of two field reactor animals using serial dilutions of cDNA,
extracted and processed as above. The same set of standard curves was used to extract measurements of RNA
from all experiments contained in this report. Variation in GAPDH per samples ranged from 3.5 to 885 arbitrary
units, with 50% samples falling between 17.9 and 304 units GAPDH. A further DNA contamination control
(DNase-treated RNA sample prior to reverse transcription) was included for each sample on each run. Reactions
were carried out using a Rotor-Gene RG300 (Corbett Research, Cambridge, UK). Cycling conditions of an initial
activation step of 10 minutes at 95oC followed by 45 cycles of 95oC for 15 seconds and 60oC for 60 seconds were
used for all primers and probes. A calibration control plasmid was included on each PCR run in duplicate such
that and any minor differences (virtually none) between runs could be normalized and therefore all PCR runs
comparable
SID 5 (Rev. 3/06)
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References cited in text:
1. Anonymous. 2002. European Commission Regulation (EC) NO 1226/2002 of 8 July 2002 amending annexe B
to Council Directive 64/432/EEC. Off. J. Eur. Union (English) L 179: 13.
2. Dean, G.S., S.G. Rhodes, M. Coad, A.O. Whelan, P.J. Cockle, D.J. Clifford, R.G. Hewinson and H.M.
Vordermeier. 2005. Minimum infective dose of Mycobacterium bovis in cattle. Infect. Immun., 73(10): 6467-6471.
3. Dheda, K., J. S. Chang, R. A. M. Breen, A. Jamanda, J. A. Haddock, M. Lipman, L. U. Kim, J. F. Huggett, M. A.
Johnson, G. A. W. Rook and A Zumla. 2005. Expression of a novel cytokine, IL4delta2, in HIV and HIVtuberculosis co-infection. AIDS, 19: 1601-1606.
4. Fletcher, H., P. Owiafe, D. Jeffries, P. Hill, G. A. W. Rook, A. Zumla, T. M. Doherty, R. H. Brookes and the
VACSEL Study Group. 2004. Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL42 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation. Immunology,
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5. Demissie, A., L. Wassie, M. Abebe, A. Aseffa, G. Rook, A. Zumla, P. Andersen, T. M. Doherty and the
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6. Vordermeier, H.M., M.A. Chambers, B.M. Buddle, J.M. Pollock and R.G. Hewinson. 2006. Progress in the
development of vaccines and diagnostic reagents to control tuberculosis in cattle. Vet. J., 171(2): 229-244.
7. O’Garra, A. and P. Vieira. 2004. Regulatory T cells and mechanisms of immune system control. Nat. Med.,
10(8): 801-805.
8. Waldvogel, A. S., M-F. Lepage, A. Zakher, M. P. Reichel, R. Eicher and V. T. Heussler. 2004. Expression of
interleukin-4, interleukin-4 splice variants and interferon-gamma mRNA in calves experimentally infected with
Fasciola hepatica. Vet. Immunol. Immunopathol., 97: 53-63.
9. Leite, R.M.H., R.C. Leite, J.A. Lima, C.B. Foscolo, P.M.P.C. Mota, F.C.F. Lobato and A.P. Lage. 2000. HPLC
identification of isoniazid residues in bovine milk. Arq. Bras. Med. Vet. Zootec., 52(6): 662-668.
10. Smith, P., J. van Dyk and A. Fredericks. 1999. Determination of rifampicin, isoniazid and pyrazinamide by
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11. Rhodes, S.G., N. Palmer, S.P. Graham, A.E. Bianco, R.G. Hewinson and H.M. Vordermeier. 2000. Distinct
responses of gamma interferon and interleukin-4 in bovine tuberculosis. Infect. Immun., 68(9): 5393-5400.
12. Vordermeier, H.M., M. A. Chambers, P.J. Cockle, A.O. Whelan, J. Simmons and R.G. Hewinson. 2002.
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SID 5 (Rev. 3/06)
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References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
SID 5 (Rev. 3/06)
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Scientific journals and books:
Dean, G.S., S.G. Rhodes, M. Coad, A.O. Whelan, P.J. Cockle, D.J. Clifford, R.G. Hewinson and H.M.
Vordermeier. Minimum infective dose of Mycobacterium bovis in cattle. 2005. Infect. Immun., 73(10):
6467-6471.
Rhodes, S.G., J. Sawyer, A.O. Whelan, G.S. Dean, M. Coad, K.J. Ewer, A.S. Waldvogel, A. Zakher, D.J.
Clifford, R.G. Hewinson*, H.M. Vordermeier*. IL-4delta3 splice variant expression in bovine tuberculosis: a
marker of protective immunity? J. Immunol., September 2006, submitted manuscript.
Rhodes, S.G. The cattle model of tuberculosis: studies in the natural host. In; Focus on Tuberculosis
Research. P187-201. Ed. Lucy T. Smithe, Nova Science Publishers Inc., 2005. ISBN I-59454-137-X.
J.D. Rodgers, N.L. Connery, J. McNair, M.D. Welsh, R.A. Skuce, D.G. Bryson, D.N. McMurray & J.M.
Pollock. Experimental exposure of cattle to a precise aerosolised challenge of Mycobacterium bovis: a
novel model for study of tuberculosis. Manuscript in preparation.
J. McNair, J.D. Rodgers, N.L. Connery, , M.D. Welsh, R.A. Skuce, D.G. Bryson, & J.M. Pollock.
Manuscript in preparation.The impact of Mycobacterium bovis aerosol challenge, at different
concentrations, on the immunology and pathology of disease.
Scientific meetings with published abstracts:
Dean, G.S., S.G. Rhodes, M. Coad, A.O. Whelan, P.J. Cockle, D.J. Clifford, R.G. Hewinson and H.M.
Vordermeier. Minimum infective dose of Mycobacterium bovis in cattle. Presentation for the Annual
Congress of the British Society for Immunology, Harrogate, UK, 7-10th Dec. 2004.
Dean, G.S., S.G. Rhodes, M. Coad, A.O. Whelan,B. Villarreal-Ramos, E. Mead, D.J. Clifford, R.G.
Hewinson and H.M. Vordermeier. Isoniazid treatment of bovine tuberculosis: development of a memory
model. Presentation for the 7th International Veterinary Immunology Symposium, Quebec City, Canada,
25-30th July, 2004.
Dean, G.S., S.G. Rhodes, M. Coad, A.O. Whelan,B. Villarreal-Ramos, E. Mead, D.J. Clifford, R.G.
Hewinson and H.M. Vordermeier. Isoniazid treatment of bovine tuberculosis: development of a memory
model. Presentation for the 1st Joint Meeting of European National Societies of Immunology, Paris 6-9th
September, 2006.
Rhodes, S.G., J. Sawyer, A.O. Whelan, G.S. Dean, M. Coad, K.J. Ewer, A.S. Waldvogel, A. Zakher, D.J.
Clifford, R.G. Hewinson*, H.M. Vordermeier*. IL-4delta3 splice variant expression in bovine tuberculosis: a
marker of protective immunity? Presentation for the 1st Joint Meeting of European National Societies of
Immunology, Paris 6-9th September, 2006.
Pollock, J.M., Rodgers, J.D., Welsh, M.D. and McNair, J. (2005). Pathogenesis of bovine tuberculosis: the
role of experimental models of infection. The 4th International Conference on Mycobacterium bovios, 2226 August 2005, Dublin Castle, Dublin, Ireland.
Pollock, J.M., Welsh, M.D. and McNair, J. (2005). Development of new strategies for the control of bovine
tuberculosis in Northern Ireland. Conference Proceedings Symposium Tropical Animal Health, Utrecht,
The Netherlands
Rodgers, J.D., Connery, N., McNair, J., Welsh, M.D., Bryson, T.D.G. and Pollock, J.M. (2005). Aerosol
exposure of cattle to Mycobacterium bovis: A novel method for the study of Tuberculosis. The 6th
International Conference of Mycobacterial pathogenesis, 30 June-3 July 2005, Saltsjobaden, Sweden,
June 2005.
Rodgers, J.D., Connery, N., Mahaffy, H., Breadon, E.L., Milligan, C.R., Colhoun, L., Welsh, M.D., McNair,
J., Bryson, T.D.G. and Pollock, J.M. (2006). Immune responses in cattle using an aerosol infection model
of tuberculosis when challenged with a high dose of Mycobacterium bovis. Poster Presentation at Cellular
mechanisms in host-pathogen interactions, 1-6 June 2006, Elsinore, Denmark.
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