Final Report Summary - WILDTBVAC (Integrated solutions for Tuberculosis control in animals combining vaccination and multi-species diagnostics)
Executive Summary:
WildTBVac is a small or medium-scale focused research project targeted to SMEs of FP7-KBBE-2013-7-single-stage type in the area 2.1.4 Socio-economic research and support policies. The project is a grant to transfer research developed in the previously EU funded project TB-STEP (#212414) to the production sector by building a pre-commercial set of TB control solutions. Specific objectives are the following: a) To standardize and optimize the production of an inactivated TB vaccine; b) to evaluate vaccine-compatible multi-species diagnostic strategies; c) to assess the field efficacy of integrated TB control strategies for wild boar; d) to evaluate the field efficacy of oral vaccination for TB control in domestic and feral pigs, and e) to define the exploitation and commercialization strategy.
Project management has taken care of receiving and distributing the funding, coordinating the research, demonstration and other activities and collecting the individual technical and financial reports in a timely and consistent manner.
The project has produced a bank of seeds from a selected isolate of M. bovis for medium-large scale production according to an established quality control protocol. Costs of production have been determined. Oral delivery of the inactivated M. bovis bacteria did not compromise diagnostic skin and IGRA tests using defined antigens, but intramuscular did so. The protein target MPB83 has been successfully produced and both a LFD (lateral flow device) prototype and a DR (double recognition) ELISA have been developed. A first validation using experimental and field sera has been carried out showing that: a) the LFA resulted in a sensitivity value of 86.6 % and an specificity of 96% in wild boar and pigs; b) the DR-ELISA had a sensitivity of 99.1%, and specificity of 99.2%; c) DR-ELISA on sera samples from different zoo species and pet animals have been tested with good results; d) it yielded a sensitivity of 86% and an specificity of 87%; e) deer samples yielded an estimate of a 94% of SP and 50% of SN. Use of the vaccine as a bait in wildlife is compatible with diagnostic in cattle by the existing or novel tuberculin skin testing formats as well as with the use of IGRA tests utilizing tuberculin or defined antigens and with the potential use of MPB83-DR-ELISA.
TB control strategies at the wildlife-livestock interface depends on many factors including (a) the single or multi-host nature and other characteristics of the pathogen, (b) the availability of suitable diagnostic tools, (c) the characteristics of the wildlife host(s), (d) the geographical range of the pathogen/reservoir (improved control in isolated versus continuous populations) and the scale of the control effort (large-scale longitudinal programs are better), (e) the attitude of the stakeholders involved (highly dependent on their education and communication provided to them
The domestic pig field test has been delayed but already confirmed that vaccine parenterally administered is well tolerated and immunogenic. Currently, the proof of concept that vaccination in domestic suids protects as efficiently as in wild suids and that was the main goal of this task has not been demonstrated in these settings and requires further histopathological and microbiological studies that should clarify the pathogenetic aspects of the vaccination-infection interaction.
Vaccine doses requirements for European and American expected demand through a six years projection sum up over 4 million doses. Maximum production needs per year were estimated at 1.45 million vaccine doses.
The project has generated 11 SCI papers and 9 congress presentations in addition to a divulgation article and information made available at a website (www.wildtbvac.eu).
Project Context and Objectives:
Recent studies have shown estimations of worldwide 50 million cattle infections with M. bovis, at a cost to the agricultural community of 2-3 billion € per year. In developing countries, M. bovis infection is still widespread, in both cattle and humans. Even in developed countries, with heavy and longtime investments in eradication of disease from livestock which seemed to have reached success, complete eradication appears to be hampered by the reiteration of outbreaks related to the presence of wildlife reservoirs of M. bovis. Under these circumstances, a single case of a reportable disease in livestock can result in serious economic consequences for the producer, public, and governments. Within this scenario, there’s room and clear business opportunities for integrated control products or solutions that aim at addressing what is considered a global problem, animal tuberculosis. Our overall objective is therefore to take the results of the TB-STEP project - “Strategies for the eradication of bovine tuberculosis”, funded under FP7-KBBE-2007 with project number 212414, where most of the WildTBVac partners were involved, and build a pre-commercial set of TB control solutions by means of testing the technical and economic viability of the aforementioned integrated approach. WildTBVac specific objectives are the following:
To standardize and optimize the production of a TB Vaccine (prototype resulting of the TB-STEP project)
To evaluate several vaccine-compatible diagnostic strategies (also resulting from the TB-STEP project) that will include multi-species DIVA tests (wild boar, domestic pigs, cattle)
To assess the field efficacy of integrated TB control strategies including oral vaccination for TB control in wild boar
To evaluate the field efficacy of integrated TB control strategies including oral vaccination for TB control in domestic and feral pigs
To define the exploitation and commercialization strategy including clear technology transfer approaches and development of a market oriented business.
Project Results:
WP1:Management and coordination (NEIKER)
Consortium management tasks have been focused on coordinating exchanges of information and materials between the partners, setting up meetings and communications between the task leaders and supervising deadline compliance for deliverables and milestones regarding the technical aspects of the project. The coordinator has taken care also of distributing the funding and requiring the partners to make their financial reports on time and according to the Commission specifications.
There have been no relevant problems. Most of the efforts have been devoted to clarify administrative concepts in order to make the reports. The most important problem has been a delay in the initiation of the experimental vaccination in Italy that was caused by the long time consumed by the obtainment of the administrative permits.
There has been no consortium change.
There have been 5 project meetings summarized in the following table:
Meeting
Date
Time
Venue
Attendees
1
Kick-off
14-1-2014
9:30 13:30
NEIKER, Derio, Spain
17
YFE, IZI, RAJ, JGA, ISE, LDO, JBE, CGO, ACH, RHA, PRU, BLA, PPA, BCH, FBU, MVO, GJO
2
18-6-2014
18:00 18:50
Town Hall, Cardiff, UK
8
RAJ, JGA, CGO, LDE, JBE, MVO, GJO, ARA
3
9-12-2014
15:30 16:30
Adobe Connect, Videoconference (https://neikertecnalia.adobeconnect.com/rajustenkrd/)
9
JGA, RAJ, CGO, JBE, ARA, BLA, PRU, FBU, MGA
4
3-3-2015
15:00 15:40
Adobe Connect, Videoconference (https://neikertecnalia.adobeconnect.com/rajustenkrd/)
11
RAJ, PPA, JBE, ARA, BLA, PRU, RLA
Off-line: MVO, GJO, CGO. MGA
5
24-6-2015
9:30 15:30
NEIKER, Derio, Spain
12
RAJ, JGA, ISE, LDE, JBE, MVO, GJO, CGO, MGA, BLA, PRU, AVE
Table 1. Meetings table
Project planning has been carried out as expected and is currently completed but for the experiment on pigs in Italy. This delay has not allowed to verify the use of the vaccine in domestic pigs at the end of the project, but has provided material for the setting up of diagnostic tests. The work is underway, so it will generate the planned information that will be shared by the partners and submitted as an addendum when ready.
The website was deployed in the early months (www.wildtbvac.eu) of the project and is still available and functional.
WP2: Vaccine optimization and scale up (NEIKER)
Summary of progress towards objectives
A bank of strains seeds from a selected isolate of M. bovis has been produced.
The medium-large scale production of the selected vaccine strain in terms of shortest time, maximum amount and optimal and stable genetic and antigenic features has been produced and a quality control protocol has been established and is being used for the current batches.
The composition of the vaccine product according to the route of administration in terms of dose and adjuvants for maximal protection and minimal diagnostic interference has been fixed according to the currently available information.
Costs of production have been determined and updated according to real experience in producing and shipping to partners and out of the EU collaborators.
Significant results
A method for medium-large scale production has been set up with the necessary safety and quality controls applied and the product has been delivered to partners and third parties for testing as needed.
Deviations
Only limited changes according to the budgetary shortcuts have been deemed necessary in terms of production systems and equipments to fulfil current and future needs.
Task 2.1 Equipment set-up
Task Leader: NEIKER (Acquisition and setting-up the equipment needed).
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support).
Task Objectives
The main objective of this task is to acquire and set-up the equipment needed to produce the vaccine in a medium-scale pre-industrial fashion.
Results
The equipment to produce and harvest bacteria has been set up in the BSL3 laboratory and its performance checked. In addition, a high capacity home-made inactivation system has been manufactured, transferred to BSL3 laboratory facilities and validated
Deviations from the work programme, and corrective actions taken/suggested
The final budget did not allow the purchase of a freeze-drier and a bio-reactor as initially planned and for which most of this task was designed. Anyway, the work has been performed following the current protocols and with the existing equipment but with some modifications for an increased productive capacity and biosafety.
Task 2.2 Large scale mycobacterial growth
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (diagnostic validation research).
Task Objectives
Task 2.2 goals were to produce seeds for 1030 vaccine batches and vaccine for other WPs under current conditions. Mycobacterial cells grown in supplemented 7H9 medium were harvested by centrifugation or filtration and then aliquots were freeze-dried to produce the seeds stock. The aim is to produce enough vaccine doses under current conditions and to provide them to other partners in order to be used in diagnostic validation research activities.
Results
All the planned 1030 seeds have been produced. They are kept in 50% glycerol at -80ºC. Vaccine has been produced and partners have been provided with sufficient doses to be used in other WPs. Additionally, in order to take advantage of resources available from third parties interested in our product, and therefore in line with this project broad objectives in terms of dissemination and assessment of product properties, vaccine doses have been made available for both additional uses by this consortium partners (IREC, VISAVET, AHVLA) and for research groups. The latter include: Celine Richomme & Maria Laura Boschiroli (ANSES), Suelee Robbe Austerman & Pauline Nol (USDA-APHIS-VS-STAS), Jose Antonio Ortiz (Las Lomas deer farm, Cádiz), Natasha Rudenko (Academy of Sciences of the Czech Republic), Quim Segalés (CRESA) and Eamonn Gormley (UCD Dublin).
Deviations from the work programme, and corrective actions taken/suggested
Instead of using freeze-drying to lyophilize the seeds, these are kept at -80ºC in vials containing 0.5 ml 50% glycerol and 5% 7H9 broth.
Task 2.3 Definition of optimal production conditions
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (effects of vaccine characteristics on interference with diagnostic performance).
Task Objectives
This task was aimed at defining the optimal production conditions in terms of time, growth and bacterial harvesting. In order to achieve this, optimum bacterial production and time parameters were assessed in culture. Slight medium formulation differences, static or shaking conditions, use of a small volume pre-culture, air exchange or airtight conditions and clump formation are the anylised variables. To harvest cells centrifugation and filtering are the methods compared.
Results
The results obtained for the comparison between shaking and static culture conditions indicate that shaking promotes cell clumping if no additional dispersant is included in the medium. But the effects of dispersants on the antigenicity of bacteria cultured in dispersant-enriched medium are unknown. The use of a starter pre-culture improves production in terms of bacterial amount obtained, shorter time and lower clump formation. Under current conditions, clump formation is directly related to culture age. Culturing bacteria for periods longer than 2-3 weeks promotes tough clump formation. Air exchange conditions can only be tested in small flasks because the bio-reactor could not be purchased. Centrifugation must be the harvesting method since no modern filtering system could be obtained.
Deviations from the work programme, and corrective actions taken/suggested
Comparison between culturing bacteria under airtight or air-exchange conditions were only tested in a small scale (flasks containing 10 ml of medium) instead of using a bio-reactor. Filtration has been excluded for cell harvesting.
Task 2.4 Stability of vaccine batches
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (effects of vaccine genetic and stability characteristics on interference with diagnostic performance).
Task Objectives
This task 2.4 is focused on assessing genetic and antigenic stability of vaccine batches. To grow and check thirty seeds/batches at different post-production times. Spoligotyping method has been used to make sure the original genetic profile has not changed. Two dimensions electrophoresis, western-blot and inoculation in guinea pigs were used in order to assess the antigenic stability of seeds. To test changes in the material of models described in WP3 require that for improved diagnostic performance.
Results
Vaccine products derived from the thirty seeds prepared for this purpose were analysed at different post-production times using spoligotyping, 2D/WB and guinea pig inoculation. Spoligotype of tested vaccine products has not changed in comparison with the original spoligotype (original low-passage strain).
Figure 1. Genetic stability as observed in the spoligotyping
Figure 2. Batch stability
Figure 3. Skin test reactivity induced by the vaccine in guinea pigs.
The vaccine strain grows better in static and air-tight cultures for 2-3 weeks. Both seeds and vaccine batches remain genetically and antigenically stable for, at least, three years in standard refrigeration conditions
Deviations from the work programme, and corrective actions taken/suggested
No deviations to be reported.
Task 2.5 Final optimal formulation
Task Leader: NEIKER
Other Participants: UCLM (formulae testing in wild boar), VISAVET (formulae testing in pigmy pig), ISS (formulae testing in pig), VACUNEK (technology follow-up for industrial and commercial feasibility and support).
Task Objectives
The aim of this task was to define the final formulation as well as the quality control procedure. To do the former, four formulations were assayed in-vivo in order to choose the best one according to the protection it provides and to their cross-effects over diagnostic tools. This experiment was performed to define the indications for use of the vaccine. The quality control procedure to ensure the purity, the genetic identity, the antigenic ability, the inactivation and the innocuousness (no negative cross effects) of the final product were defined as well.
Results
Design of experimental groups and settings. Four formulations were assayed in-vivo. These combine 2 antigen concentrations (high-dose or 107 CFU & low-dose or 103 CFU) and 2 administration routes (oral without adjuvant and intramuscular with Montanide adjuvant).
Deviations from the work programme, and corrective actions taken/suggested
Pigmy pigs will not be used for in-vivo testing in VISAVET due to animal availability problems. Instead, goats benormal young pigs were finally used.
WP3: Evaluation of vaccine-associated diagnostic strategies including the use of defined diagnostic antigens (AHVLA/DEFRA)
Task 3.1 Tuberculin skin test and IGRA reactivity
Task Leader: DEFRA
Other Participants: NEIKER will use the reagents in a set of cattle, VISAVET will evaluate a define set of animals by both diagnostic protocols (skin and IFN-g tests).
Task Objectives
The main objective of this task was to perform a vaccination experiment in cattle and for Defra to supply defined DIVA antigens to other partners. The underlying hypothesis investigated in this work package is that oral uptake of the wildlife vaccine by bystander domestic animals (e.g. cattle or domestic, free-ranging pigs) will not induce systemic immune responses and therefore will not compromise the use of tuberculin or of defined DIVA antigens in diagnostic assays probing cellular immunity (skin test or blood-based IGRA).
NEIKER also used the reagents in a set of cattle and VISAVET evaluated a defined set of animals by both diagnostic protocols (skin and IFN-g tests)
Results
APHA experiment
We assessed the suitability of tuberculin as a diagnostic reagent for the skin test and IGRA in cattle vaccinated with inactivated M. bovis strain 1403 (Neiker), also called mdR below. To this end, 6 calves (aged 5-7 months) were vaccinated with 106-107 CFU of killed bacteria which is the standard dose (also called Full Dose, FD) by either the oral or intramuscular route according to the supplier’s guidelines (Neiker). Six weeks post vaccination, these animals received intradermal injections (0.1 ml volume) in the neck with avian tuberculin (PPD-A; 2,500 IU [Prionics]) and bovine tuberculin (PPD-B; 3,000 IU [Prionics]). Skin induration was measured at the injection sites prior to and 72 hours after the skin test, and the results are expressed as the difference in skin thickness between the two readings. In animals vaccinated by the oral route, no positive skin test reactions were observed when using either the SIT (PPD-B only) or SICCT (PPD-B – PPD-A) readouts (
). In contrast, all animals vaccinated by the intramuscular route showed positive skin test reactions in both tests (
).
Table 2. Skin test results using tuberculin reagents in vaccinated cattle. Values represent the change in skin thickness (differences in mm). SIT: response considered positive if PPD-B ≥ 4mm. SICCT: response considered positive if the difference between PPD-B and PPD-A is > 4mm. Test positive results are highlighted in red.
The whole blood IGRA test using tuberculin reagents was also performed at time points pre- and post-vaccination. IFN-γ levels in the plasma supernatant were quantified using the Bovigam ELISA kit (Prionics), and samples were considered test positive if the O.D. for PPD-B stimulated cultures minus the O.D. for PPD-A stimulated cultures was > 0.1. All 12 animals demonstrated IFN-γ responses to the positive control PWM (data not shown). None of the animals vaccinated by the oral route tested positive in the IGRA assay at any time point. In contrast, all six animals vaccinated by the intramuscular route showed positive IGRA test results 2 weeks post vaccination, which persisted for the duration of the experiment (
).
Table 3. IFN-γ responses in vaccinated cattle using the tuberculin readout (PPDB – PPDA). Values represent ΔO.D. values (PPDB - PPDA). Test positive results are highlighted in red.
In summary, the results of Task 3.1 indicate that in contrast to intramuscular delivery, oral delivery of the inactivated M. bovis strain 1403 vaccine did not compromise diagnostic tests for bovine TB that ulitise tuberculin reagents.
Additional experiment conducted at Neiker:
We had the opportunity to incorporate data from an independent experiment conducted at Neiker that confirmed our findings that intramuscular injection of the standard full dose induced both skin test and IFN-γ responses. In this experiment, calves were either vaccinated with 107 CFU (MdR-FD, n = 10) or a reduced dose (MdR-RD, n = 10) of 103 CFU, or left unvaccinated (n=5). Blood samples for IFN-γ testing were collected at the time of vaccination (day 0) and at days 15, 43, 78, 86 and 186 post-vaccination. Blood samples were stimulated with PPDA and PPDB; IFN-γ responses were determined by Bovigam ELISA. The same interpretation criteria were applied as described above. Two tuberculin skin tests were performed at days 43 and 78 post-vaccination using PPDB (single intradermal test, SIT) with the standard test interpretation being applied.
The results are summarised in Table 4 and demonstrate that animals vaccinated with the full dose (MdR-FD = 107 CFU) developed positive IFN-γ responses as early as 15 days post-vaccination and these responses remained positive in between 88 and 100 % of animals until the last blood sampling was performed (day 186). In contrast, 90% of the animals vaccinated with the reduced dose (MdR-RD = 103 CFU) were IFN-γ test-negative 15 days post-vaccination, and the majority remained test negative over the course of the experiment. (Table 3.3). Moreover, one animal that tested positive in this test, was test positive before the vaccine was applied (false-positive). Similar observations were made when skin test responses were considered: 40 and 50 % of the MdR-FD vaccinated animals tested positive at the two skin tests, respectively, whilst only 1/10 (10% of the reduced dose vaccinated cattle tested positive (Table 4). As expected, none of the unvaccinated control animals tested positive to either test (Table 4). In conclusion, this experiment confirmed the observation that intramuscular vaccination with the full dose of this vaccine will compromise the specificities of skin and IFN-γ tests. Further, it demonstrates that a reduced vaccine dose will only lead to limited test positivity in these tests probing cell-mediated immunity.
Table 4. IFN-γ and skin test responses in vaccinated cattle using the tuberculin readout (PPDB – PPDA) for the interpretation of the IFN-γ and the interpretation of the single intradermal skin tests (PPDB) to classify animals as positive or negative (interpretation criteria as described in
and
).
Deviations from the work programme, and corrective actions taken/suggested
Some unscheduled work has been done in cattle that support the conclusions of the partner DEFRA experiment. This adds value to the project, no corrective action required. .
Task 3.2. Antigenically defined skin test and IGRA reactivity.
Task Leader: PRIONICS (provide peptide cocktails PC-EC and PC-HP for stimulation of blood and the IGRA Bovigam)
Other Participants: NEIKER applied the test in a set of cattle, DEFRA (vaccinate cattle and test in skin test and IGRA) and VISAVET carried out a field trial to define the sensitivity and specificity of these specific antigens under specific conditions.
Task Objectives
As tuberculin has inherent problems of production, quality control, lack of specificity, we also evaluated whether defined antigens such as ESAT-6, CFP-10, Rv3615c, and Rv3020c can be used in assays of cellular immune responses such as skin test and IGRA (cattle, pigs).
Results
We assessed the suitability of defined antigens as diagnostic reagents for the skin test and IGRA in cattle vaccinated with inactivated M. bovis strain 1403 (Neiker) as described for Task 3.1. For the skin test, two protein cocktails were formulated containing either: (i) the proteins ESAT-6, CFP-10 and Rv3615c (APHA-1); or (ii) the proteins ESAT-6, CFP-10, Rv3615c and Rv3020c (APHA-2). Previous data generated at APHA has shown that these defined antigen skin test reagents induce positive skin test reactions in TB-reactor cattle with confirmed infection (APHA-1 [n=62]: test sensitivity of 85.48% [95%CI of 74.22% to 93.14]; APHA-2 [n=16]: test sensitivity of 93.75% [95%CI of 69.77% to 99.8%], data not shown). At the time of skin testing, the cattle detailed in Task 3.1 (i.e. cattle 1 to 12) were also skin tested with APHA-1 and APHA-2. In both cases, the protein cocktails were delivered by intradermal injection (0.1 ml volume) at a concentration of 10μg/protein/injection. Skin induration was measured at the injection sites prior to and 72 hours after the skin test, and the results are expressed as the difference in skin thickness between the two readings. As previously shown for tuberculin reagents (Task 3.1) no positive skin test reactions to either APHA-1 or APHA-2 defined antigen cocktails occurred in animals vaccinated via the oral route (Table 3.4). However, in contrast to that seen for tuberculin reagents (Task 3.1) limited skin test reactions were also observed in animals vaccinated via the intramuscular route, with only a single animal testing positive to the APHA-1 reagent (Table 5).
Table 5. Skin test results using defined antigens in vaccinated cattle. Values represent the change in skin thickness (Δmm). APHA-1 and -2: response considered positive if ΔΔ mm is ≥ 2mm.
Simultaneous with carrying out whole blood IGRA using tuberculin reagents, the performance of defined antigen reagents was also assessed in the vaccinated cattle detailed in Task 3.1. Whole blood samples taken from animals at time points pre- and post-vaccination were stimulated for 24 hours with peptide cocktails containing the following defined antigens: (i) E/C (ESAT-6 and CFP-10 [APHA]); (ii) Rv3615c (APHA); (iii) PC-EC (ESAT-6 and CFP-10 [Prionics]); and (iv) PC-HP (ESAT-6, CFP-10 and 4 additional mycobacterial gene products [Prionics]). All peptides were used at a concentration of 5μg/peptide/ml. IFN-γ levels in the plasma supernatant were quantified using the Bovigam ELISA kit (Prionics), and samples were considered test positive if the O.D. for peptide stimulated cultures minus the O.D. for unstimulated cultures was > 0.1. All 12 animals demonstrated IFN-γ responses to the positive control PWM (data not shown). Oral vaccination with the inactivated M. bovis strain 1403 did not induce IFN-γ responses to any of the defined antigen peptide cocktails during the course of the experiment, with the exception of one animal that tested positive to the PC-HP peptide cocktail at week 7 post vaccination (Table 6). In contrast, more frequent responses to the defined antigens were observed in animals vaccinated via the intramuscular route, where all six animals tested positive to E/C, PC-EC and PC-HP at least on one occasion post vaccination (Table 6). Responses to the E/C peptide cocktail were first seen 2 weeks post vaccination, which persisted throughout the course of the experiment in all animals. Similar results were seen with the PC-EC and PC-HP peptide cocktails, although the frequencies of response were lower at some time points when compared to the E/C cocktail. In contrast, only one animal tested positive to the Rv3615c peptide cocktail (Table 6).
Table 6. IFN-γ responses in vaccinated cattle using defined antigens
In summary, the results of Task 3.2 indicate that oral delivery of the inactivated M. bovis strain 1403 did not compromise diagnostic skin tests using defined antigens. Intramuscular vaccination resulted only in low responses in 1/6 animals. Although oral immunization also did not compromise IGRA diagnostic tests that utilize defined antigens E/C, Rv3615c and PC-EC), these tests were compromised when the vaccine was given via the intramuscular route.
Deviations from the work programme, and corrective actions taken/suggested
There are no deviations from the work programme.
Task 3.3. Serological tests in pigs using LFD
Task Leader: ISS (evaluate the feasibility of sero-dominant antigen MPB83 in IGRA test)
Other Participants: NEIKER will provided the vaccine, PRIONICS and INGENASA provided serological tests using MPB70 and MPB83 as antigens on the LFD-Comb platform for evaluation in pigs, ISS evaluated the feasibility of sero-dominant antigen MPB83 in IGRA test and VACUNEK analyzed and compare results with its own proprietary basic ELISA kit).
Overall summary of work progress and achievements
One of the aims of this task was to develop new one-step assays able to detect antibodies in the field based on the Lateral-Flow Technology. This assays works with low sample volumes, can be performed rapid (the entire test procedure is completed in 12 minutes) and easily without special equipment directly on the field and can provide a useful tool in situations where laboratory support and skilled personnel are limited.
During this reporting period, INGENASA has worked in the development and evaluation of the LF assay based on the recombinant protein MPB83, to be used in the field with serum/blood samples from pigs and wild board. The assay has been validated in the laboratory with samples from Partners 5 and 7. Additionally, INGENASA has already produced a big batch that will be distributed to the partners for further validation.
Summary of significant results
The protein target MPB83, necessary for the subsequent assay development, has been successfully expressed and purified.
A LFD (lateral flow device) prototype for specific anti-TB antibodies detection has been designed and developed.
A protocol for performing the rapid assays has been established.
A first validation of the rapid assays, using experimental and field sera has been carried out.
A big batch has been produced.
Introduction
The LFD for TB antibody detection is based on the recombinant MPB83 protein of Mycobacterium bovis. The first step was the expression and purification of these targets in order to use them as capture reagent on the nitrocellulose and labelled with latex particles to use as signal generator. Once the proteins were expressed and characterised, the lateral flow device was designed using a double recognition (DR) approach, where the antibody present in the sample is bound twice to the same antigen, the MPB83 protein in this case. If the tested sample contains specific antibodies, they will be captured firstly by the latex particles labelled with MPB83 and secondly by the MPB83 immobilized on the nitrocellulose membrane giving as a result the appearance of a coloured line on the membrane. In addition, green latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test.
Description of work
MPB83 has been expressed in the E.coli system. The protein was assayed to found the optimal conditions for the assay, that is, the optimal concentration of MPB83 protein adsorbed on the nitrocellulose membrane and the best conditions to conjugate the proteins to the latex particles. Moreover, in order to establish the best conditions to perform the assay different parameters such as sera dilutions, running buffers, kind and sizes of membranes, latex... were tested to determine the accuracy of the assay. It is important to remark that the present test is a qualitative assay, resulting in a positive or negative decision.
In order to test the new LFD with blood, a spike in test was performed by adding positive and negative serum samples into blood from pig.
The schematic diagram of the LFD is showed in Figure 4. It is based on a direct immunoassay called Double Recognition antibody (DR) with two differentiated areas:
1. Sample window: it contains the latex particles dried on the sample pad: red microspheres covalently linked to MPB83 protein and green microspheres are used as test control.
2. Result window: it contains a nitrocellulose membrane with two lines printed: the test line formed by the MPB83 protein and the control formed by a specific MAb for the control protein.
Figure 4. Schematic diagram of the LFD developed for the task 3.3.
To carry out the test, 150 μl of 1/50 dilution of sample (serum or blood) were added to the sample pad. To carry out the test, 150 μl of 1/50 dilution of sample (serum or blood) were added to the sample pad followed by 4 drops of running buffer. The specific antibodies present in sample react with the latex particles coated with MPB83. This complex flows through the membrane and will react with the immobilized MPB83 protein on the membrane, making the test line visible. Interpretation of results should be done 12 minutes after the addition of the sample. A positive result for TB antibodies is recorded for samples with red and green lines visible. In the case of negative samples, only a green line will be show up. If no green line appears, the test will be invalid. No results must be considered after 12 minutes.
Figure 5. Interpretation of results after adding the porcine serum sample to the LFD developed.
Results
In order to determine the diagnostic sensitivity and specificity in pigs and wild boards, two panels of sera had been evaluated. On the one hand, 30 serum samples of negrody pigs from Partner 6, and 181 negative pig sera from farms free of TB. The results of the evaluation of these serum samples on the LF devices are shown in the next table:
TB-LFD
Positive pig sera (negrody pig)
30
26
Negative pig sera
181
174
Table 7. Results to determine the diagnostic sensibity and specity
Of the 30 TB positive samples, there were 4 false negatives from the LFA resulted in a sensitivity value of 86.6 %. Among the negative samples, 7 were tested as false positives by using LFA, resulted a 96% of specificity.
The second panel of sera consists of 113 wild board positive and 132 negative sera for TB. All these sera were provided by Partner 5. All these sera were also tested by the DR-ELISA developed during the project, and this technique was considered the reference technique. One hundred twelve of 113 positive sera were positive for the TB line resulted a sensitive value of 99.1%, and 132 of the 133 negative sera were negative, resulted a specificity value of 99.2%.
ELISA DR
TB-DR
Positive
113
112
Negative
132
133
Table 8.
Even though more samples need to be tested, these preliminary results suggest that the developed LFD provides a reliable method for rapid detection of TB antibodies in pig and wild board. INGENASA has currently available a big batch of LFDs that could be sent to others Partners.
Perspectives
Given the obtained results so far with this LFD are very promising, the next step will be the evaluation of the test by the other project partners using samples from real or experimental infections.
Deviations from Annex I
The development and validation of the LFD for TB antibodies detection in pigs, has been a hard and long work. For that reason, the evaluation of the test for other Project Partners is delayed. However, currently a batch is available for evaluation and the collaboration among partners will continue.
Task 3.4. Multi-species serological assay
Task Leader: INGENASA. MPB83-DR-ELISA Validation and Serological studies.
Other Participants: NEIKER provide the sera of TB status-defined cattle, DEFRA (supply sera from M. bovis infected cattle), PRIONICS will evaluate the suitability of a novel serologic test using MPB70/MPB83 antigens on the LFD-Comb platform for detection of TB specific immune response in different hosts. The LFD-Comb platform combines 8 single LFD tests in a user-friendly comb shape format to allow efficient testing of a large number of samples with a very rapid turn-around time. In addition with the use of a proteinA/G conjugate, the test is capable of detecting antibody classes from multiple species. UCLM will supply sera from wild boar of known infection status, VACUNEK will analyze and compare results with its own proprietary basic ELISA kit and VISAVET will supply sera samples to INGENASA from different hosts (domestic, wildlife, zoo animals, peridomestic) for validation of the serological test.
Task Objectives
The major aim of this task was to conduct the MPB83-DR-ELISA validation and to perform Serological studies. A double recognition ELISA (DR-ELISA) based on the recombinant MPB-83 antigen expressed in the baculovirus system, was used to perform serological studies in the animal species studied (wild board, pigs, feral pigs, cattle...) in order to establish and validate a DIVA assay that allows the differentiation between vaccinated and infected animals. The MPB83-DR-ELISA, developed during the EU project TB-STEP, presents the capability of detecting antibodies against the MPB83 protein with high sensitivity and specificity in several species, therefore, the same assay was used in all cases.
In parallel, using the same recombinant antigen and the same strategy of double recognition, a new approach has been developed based on Lateral-flow devices (LFDs) technology. This represents a well-established technology appropriate for use in a wide variety of point-of-care (POC) or field-use applications. The benefits of LFDs include: 1) User-friendly format. 2) Very short time to generate a test result. 3) Long-term stability over a wide range of climates. 4) Relatively inexpensive to make. These features make strip tests ideal for applications such as home testing, rapid point of care testing, and testing in the field.
In the second period of the project, Partner 7 (INGENASA) has continued working in the evaluation of the multi-specie MPB83-DR-ELISA using samples from different species. As an example, a panel of 47 fallow deer serum samples provided by Partner 5 (UCLM) was tested for the first time with the DR-ELISA prototype. Out of the 47 animals, 13 presented clinical signs and were slaughtered and necropsied, detecting lesions in 10 of them. Of the 10 samples considered positives, there were 2 false negative from the ELISA, resulted in a sensitivity of 80%. Among the 37 negative samples, one was tested as false positive by using the ELISA, resulting in a specificity of 97.3%. These results indicate that the MPB83-DR ELISA is also suitable for this species and therefore it could be a useful tool for the control of the disease in fallow deer.
Results
A double recognition ELISA (DR-ELISA) based on the MPB83 was developed under the TB-STEP project (FP7-KBBE-2007). This assay uses the recombinant protein MPB83 both as antigen coated in the ELISA plate and as conjugated to recognize the antibodies bounded to the coated MPB83. This assay doesn’t use any anti-specie antibody and for that reason could be used with sera from any specie. The assay is the same in all cases with some slight modifications in the sample or in the conjugate dilutions.
To validate the DR-ELISA with sera from different species, UCM has provided INGENASA with samples from zoo animals. A total of 170 sera samples from, Capuchin’s monkey, Yellow armadillo, Meerkat, Patagonian’s sea lion, Californian’s sea lion, Asiatic’s elephant, Watussi, Porcupine, Seal, Oystercatcher, Chimpanzee etc, have been analysed by DR-ELISA.
Most of them have been tested twice and in duplicate by DR-ELISA to study the presence of antibodies against the MPB83, the others have been analysed only once due to low volume available. Positive results were only obtained in 13 out of 170 sera. Thirty seven out of these 170 sera have been checked by TB culture isolation (Ctbc). The comparison between both assays (DR-ELISA and culture isolation) is summarized in the next table. The qualitative results (positive/ negative) showed a 75% of agrement between them (κ=0.4064).
Table 9. Contingency table for bacterial isolation and DR ELISA results with Zoo Animals
Moreover, sera from pets (dogs, cats and guinea pigs) and farm animals (goats and pigs) have been also studied. Seven sera from cats and 9 sera from dogs were collected and tested. Only two dogs were postitive by the DR-ELISA. In the case of the guinea pigs, 21 samples out of 58 were positives. All these results, have to be checked by other techniques.
Additionally, Partner 8 (UCM) has collected sera from 505 goats. All of them were analysed by the DR-ELISA, detecting 300 positives. In order to study the accuracy of these results, it has been carried out their comparison with other studies (INF, TB culture isolation...) which have been carried out by Partner 8. When the results of TB culture isolation from 168 studied samples, were compared with the DR-ELISA, a level of SN and SP of 86% and 87% respectively were observed. If TB culture isolation and/or lesion were compare with the DR-ELISA, the SN increase up to 90% (data not shown).
Table 10. TB Ab response by DR-ELISA versus TB culture isolation in goats.
The validation of the DR-ELISA was also carried out with serum samples from badgers. Sixty one samples were provided by Partner 2 (APHA):
• 36 sera from experimentally TB-infected badgers
• 14 sera from known TB-negative badgers
• 11 samples from a heat-inactivated M.bovis vaccine trial (6 from vaccinated animals)
The results showed that all infected animals presented Ab against the MPB83 protein that were detected in the DR-ELISA. Besides, it is important to mention that the vaccinated animals did not have specific antibodies against TB. The results are shown in the following Figure.
Figure 6. TB Ab response in badgers by DR-ELISA
Cattle serum samples from the vaccination experiment carried out by Partner 4 (APHA) described in Task 3.1 were also analysed by DR-ELISA. Briefly, 6 calves (aged 5-7 months) were vaccinated with 106-107 CFU of bacteria by either oral or intramuscular route according to the supplier’s guidelines (Neiker). Similarly to the SIT or SICCT results obtained by Partner 4,in animals vaccinated by the oral route, no positive Ab response were observed by DR-ELISA. By the contrary, all six animals vaccinated by intramuscular route showed positive response after 2 weeks post-vaccination. (Figure 7).
Figure 7. TB Ab response in cattle by DR-ELISA. Vaccinated animals by oral route (blue) or intramuscular route (red)
These last results, with serum from badger and cattle, are based on experimental animals, therefore, field samples will have to be tested for a better validation of the assay.
Finally, a panel of 198 serum samples from deer provided by Partner 5 (UCLM) has also been tested by the DR-ELISA. In contrast with the results obtained with samples from other animal species, in this case, the level of SN it is not as high. Preliminary results show a 94% of SP but only a 50% of SN. The same happens when field sera from cattle are used. During the next period of the project, INGENASA will work on the improvement of the assay with these kind of samples.
Overall summary of work progress.
Work progress on this deliverable has led to the recommendation that the vaccine could be used to vaccinate wildlife in baited form in conjunction with the following in cattle: (i) continuation of existing tuberculin skin testing or novel skin test formats based on defined antigens; (ii) the use of IGRA tests utilizing tuberculin or defined antigens; (iii) the use of the serology MPB83-DR-ELISA. However, we applied only single doses of this vaccine to cattle, and we therefore recommend further studies sensitising cattle with multiple doses and by repeat administration to confirm our findings. We do not recommend using this vaccine in cattle via the systemic route in control scenarios combining vaccination with test and slaughter. We therefore recommend to evaluate the effectiveness of this vaccine in cattle when given via the oral route should its application to cattle be considered.
WP4: Integrated field TB control in wild boar (IREC/UCLM)
Summary of progress towards objectives
The focus of the task is on understanding single versus integrated control. We assess the combined effect of vaccination and culling strategies in the context of integrated disease control, considering all available tools.
This task is completed. The main field studies have already been described in the deliverables on vaccination (4.1) and on test and cull (4.2). Some additional field studies have been carried out, including a test and cull and vaccination trial on a wild boar farm. This report focuses on evaluating and discussing integrated TB control strategies in wild boar and at the wild boar – cattle interface.
Regarding vaccination, laboratory evidence and field data available to date indicate a significant reduction of lesion scores and TB prevalence in vaccinated wild boar (see deliverable 4.1). An additional experiment carried out in a wild boar farm and hunting estate showed that vaccination reduces the prevalence of TB-compatible lesions by 75% in vaccinated wild boar as compared to non-vaccinated controls.
The selective culling attempt carried out in WildTBvac, despite of huge investments in terms of facilities and effort, failed to achieve a significant reduction in TB prevalence (see D.4.2). Hence, there is no obvious advantage in combining selective culling with vaccination.
By contrast, it is known that random (i.e. non-targeted) wild boar culling reduced TB prevalence between 21-48% (Boadella et al. 2012). Reducing the population size will reduce densities and lower total costs for TB control. Moreover, random culling may help to achieve an initial reduction in prevalence because of the density-dependent effect on infection transmission and because of the lower mean age of the surviving population. This might facilitate vaccination strategies (Boadella et al.2012; Anderson et al. 2013).
Task 4.1 Controlled field assessment of oral vaccination in wild boar
Task Leader: UCLM (bait production and deployment, field sampling and gene expression response).
Other Participants: NEIKER and VACUNEK (vaccine production and adaptation for field testing), DEFRA (supplies consultency and reagents), INGENASA (serology to assess TB and significant co-infections), VISAVET (collaborate in the field studies mainly in laboratory diagnosis (culture and IGRA)), PRIONICS supply peptide cocktails PC-EC and PC-HP based on the antigens ESAT6 and CFP10 for stimulation of blood for IGRA.
Task Objectives
This task was bait production and deployment, field sampling and gene expression response. A controlled and replicated design allowed (i) describing the pathology and culture score and (ii) measuring the serum antibody and gene expression response of field vaccinated wild boar piglets as compared to unvaccinated controls. This yielded an assessment of the % change in these indicators as compared to data obtained in captivity (Ballesteros et al. 2009, Garrido et al. 2011). This experiment was be performed on a 10,000 ha study area split into two parts.
Results
Two vaccine candidates weredeployed, on about 5,000 ha each: BCG (live vaccine) and heat-inactivated M. bovis (inactivated vaccine, IV). Both BCG and IV have shown protection against challenge with M. bovis under laboratory conditions (Gortazar et al. 2014, Beltrán-Beck et al. 2014a).
Safe and specific vaccine deployment is a key concern of oral vaccination strategies. In addition, confirmation of bait uptake (i.e. use of marked baits) is needed to generate sound scientific data. In the wild boar field vaccination experiments, safety and specificity were confirmed through camera trap surveys and analyses of target and non-target host tissues for BCG (Beltrán-Beck et al. 2014b). Furthermore, daily bait deployment at dusk and collection of non-consumed baits immediately after dawn improved bait specificity and limited BCG inactivation due to high environmental temperatures. However, this procedure is labor-intensive and could be avoided by deploying only the IV.
Assessing vaccination efficacy under field conditions is challenging. In the ongoing wild boar vaccination field trials, vaccination sites were purposefully selected away from cattle farms, since risks of cattle contamination with live BCG could not be excluded a priori. Hence, no results will become available in terms of reductions in cattle TB breakdowns. This will require further experiments on and around TB-positive cattle farms. However, effects of wild boar vaccination are measured in the wild boar target host. This can be tested at the individual level (piglets with and without biomarker) and at the population scale (treatment sites before and after; treatment sites vs. controls) and preliminary information regarding lesion and culture scores in piglets is encouraging for the IV vaccine (Díez-Delgado et al., in prep.).
Figure 8 Summary of field results
Partner 7 (INGENASA) has evaluated the Ab response of 22 wild board after vaccination in experimental condition with inactivated vaccine by the oral route. All sera were tested by I-ELISA and DR-ELISA. The results are shown in the following Figure. As may be seen, the results are very similar by using both assays. No positive Ab response were observed after vaccination by oral route. By the contrary, all animals showed positive response after challenge. (Figure 9)
Figure 9 Results of sera tested by I-ELISA and DR-ELISA
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
Next steps in the next period
No accurate cost estimations are currently available for this field experiment. Also, results are currently available for only two bait deployment years, and this experiment is aimed at lasting at least four years. Finally, the newly developed vaccine and selective baiting tools deserve knowledge transfer from the lab to the market, and attainment of regulatory approval for distribution in the field.
Task 4.2 Test and cull strategies in wild boar captured under field conditions
Task Leader: UCLM (wild boar capturing, handling and field testing, and field sampling).
Other Participants: INGENASA (serology to assess TB and significant co-infections), PRIONICS (to supply its serologic LFD-Comb assay for laboratory diagnosis) and VISAVET (to perform laboratory diagnosis of culled animals (microbiological culture and IGRA)).
Task Objectives
To make use of existing and new plate and lateral-flow antibody tests (Aurtenetxe et al. 2008, Boadella et al. 2011a) for test and cull strategies in wild boar captured under field conditions at high TB prevalence sites. Test-negative wild boar were tested, tagged and released at the site of capture if negative. Test-positive animals were culled and submitted to further analysis. TB prevalence and wild boar abundance was continuously monitored to assess the impact of selective culling (Boadella et al. 2011b).
Results
We assessed the suitability of targeted culling as a means for tuberculosis (TB) control in intensely managed wild boar hunting estates. The 6,000 ha large study area included one capture site, one control site, and two release sites. Each site was fenced. In summer 2012, 2013 and 2014, a total of 929 wild boar were live-captured by means of cage traps and corral traps. All wild boar were micro-chipped and tested immediately after capture for antibodies against the Mycobacterium tuberculosis complex (MTC) with an animal side lateral flow ELISA. Wild boar were released according to their TB status: Seropositive individuals into the release sites (hunted after summer), and seronegative individuals back into the capture area. The annual summer seroprevalence of antibodies against the MTC declined significantly in live-captured wild boar piglets from the capture site, from 43.86% in 2012 to 26.74% in 2013 (39% reduction in seroprevalence). Autumn-winter MTC infection prevalence was calculated based on culture results of hunter-harvested wild boar. No significant changes between hunting seasons were recorded in the capture site and in the control site, and prevalence trends through time were similar in both sites. However, in the release sites the seasonal autumn-winter MTC infection prevalence increased significantly in non-yearling hunter-harvested wild boar from 40.54% in 2011-2012 to 64.29% in 2012-2013 (59% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted culling as a single tool, failed to reduce TB prevalence as compared to the control site. The knowledge generated will help to improve wild boar and TB management in semi-intensive settings.
Figure 10 TB infection prevalence under a targeted culling strategy troughout time
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
Task 4.3 Assessment of the combined effect of previous tasks
Task Leader: UCLM
Other Participants: VISAVET
Task Objectives
The focus of the task is on modelling of single versus integrated control. We will use modelling to assess the combined effect of both strategies, based on the rationale that reducing the population will reduce densities and lower total costs for TB control (Boadella et al.2012). Specifically, VISAVET will participate in the statistical analysis of the data obtained in tasks 3.1 and 3.2.
Progress towards task objectives for the reporting period
This task is in good progress. Raw datasets on culling and on selective culling are available, while data on field vaccination are already available for two years. Data for the third year of vaccination will become available in May 2015 (when the cultures are deemed to be finished).
Results
Figure 11. Flow chart of the available disease control options and results assessment in diseases shared with wildlife
Vaccination
Results of the field vaccination experiment (oral vaccination) after two consecutive years are detailed in D.4.1. Briefly, while the heat-inactivated M. bovis conferred some protection, BCG apparently failed to achieve protection.
In the experiment with parenteral heat-inactivated M. bovis, promising results were obtained. Piglet vaccination with heat-inactivated M. bovis started in 2012. A total of 362 wild boar piglets were vaccinated in the farm in 2012 and 2013. No adverse local or systemic reactions were detected after vaccination by visual inspection.
From November 2014 to December 2015, 173 three to four year old wild boar, which had been vaccinated as piglets, were hunted. In the same period, 144 additional hunted wild boar had not been vaccinated.
Parenteral vaccine efficacy was measured by the presence of TB compatible lesions (TBL). The TBL prevalence was 2.7% and 10.9% for vaccinated and control wild boar, respectively (Chi square= 6.69 1 d.f. p= 0.009; Figure 2). Thus, parenteral vaccination reduced lesion prevalence by 74.7%. Among those animals with TBL, no difference in the lesion score was found between vaccinated and non-vaccinated ones. No adverse reactions to the vaccine were observed at postmortem examination.
Figure 12. .- Prevalence of TB-compatible lesions (TBL) in control and parenterally vaccinated wild boar.
Culling and test & cull strategies
Regarding vaccination, laboratory evidence and field data available to date indicate a significant reduction of lesion scores and TB prevalence in vaccinated wild boar (see deliverable 4.1). An additional experiment carried out in a wild boar farm and hunting estate showed that vaccination reduces the prevalence of TB-compatible lesions by 75% in vaccinated wild boar as compared to non-vaccinated controls.
The selective culling attempt carried out in WildTBvac, despite huge investments in terms of facilities and effort, failed to achieve a significant reduction in TB prevalence (see D.4.2). Hence, there is no obvious advantage in combining selective culling with vaccination.
By contrast, it is known that random (i.e. non-targeted) wild boar culling reduced TB prevalence between 21-48% (Boadella et al. 2012). Reducing the population size will reduce densities and lower total costs for TB control. Moreover, random culling may help to achieve an initial reduction in prevalence because of the density-dependent effect on infection transmission and because of the lower mean age of the surviving population. This might facilitate vaccination strategies (Boadella et al.2012; Anderson et al. 2013). Another interesting observation is that the highest TB prevalence ever recorded (close to 90%) occurs in a protected area (Doñana National Park, Huelva, Spain), where no hunting takes place and population control is usually limited to disease surveillance purposes.
Integrated disease control strategies and their practicality
Table 10 presents the top twenty management interventions, shown in order of perceived effectiveness, as determined by the expert panel.
The most effective intervention identified based on expert opinion was banning supplemental feeding of game species. However, this option was not considered practical by stakeholders. The most effective and practical interventions were the separation of wildlife and livestock access to waterholes, testing cattle every 3 months on farms with a recent positive TB case and removing gut-piles from the land after hunting events. Although all three of these options were well supported, each stakeholder group supported different approaches more strongly, suggesting that it might be effective to promote different disease management contributions in different stakeholder communities. This integrated approach contributes to the identification of the optimum combination of management tools that can be delivered effectively.
Table 11. The 20 management interventions regarding TB control at the wildlife – livestock interface, shown in order of perceived effectiveness as determined by the expert panel (Cowie et al. 2015).
Discussion and conclusions
Assessing the efficacy of single disease control tools, such as vaccination alone or targeted culling alone, remains challenging under natural conditions. Assessing the combined effect of these two tools is even more difficult. However, wildTBvac allowed moving several steps ahead in our understanding of several disease control tools regarding the wild boar – M. bovis binomium.
Regarding vaccination, while the outcome of the four-year field oral vaccination experiment is still incomplete (last animals being sampled in 2016), some positive indications exist regarding the heat-inactivated M. bovis vaccine, as reported in D.4.1. Additionally, the smaller experiment on parenteral vaccination of farmed wild boar piglets with the same product indicated a promising 75% protection against TBL (Figure 2).
Regarding culling, while it is known that random culling reduces TB prevalence in wild boar (Boadella et al. 2012), the selective culling experiment carried out in wildTBvac failed to achieve a significant effect at the population level (see deliverable 4.2). However, under farm conditions, the targeted culling of all adult wild boar testing positive to MTC antibodies by ELISA probably contributed to keep the breeding facilities TB-free.
Given the abovementioned results, we suggest that in more or less natural field settings, culling (random culling) may contribute to reduce wild boar TB prevalence prior to vaccination. We also suggest that, while selective culling failed under field conditions, it might be an interesting tool in wild boar farms, were access and testing of every single individual is granted. In conclusion, culling might be combined to improve vaccination efficacy, but the specific tool (random or targeted) will depend on the setting.
Various general inferences can be made. First, all options for disease control at the wildlife-livestock interface, including those of no intervention, need to be considered, either individually or combined. Second, combining several disease control tools in integrated strategies is likely to reduce the cost and effort required for disease control. Integrated strategies are also preferred since no single control measure is universally applicable. Third, the success of disease control in wildlife depends on many factors, including (a) the single or multi-host nature and other characteristics of the pathogen, (b) the availability of suitable diagnostic tools, (c) the characteristics of the wildlife host(s), (d) the geographical range of the pathogen/reservoir (improved control in isolated versus continuous populations) and the scale of the control effort (large-scale longitudinal programs are better), (e) the attitude of the stakeholders involved (highly dependent on their education and communication provided to them)(Cowie et al. 2015, Gortázar et al. 2015).
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
WP5: Evaluation of efficacy of heat-inactivated Mycobacterium bovis as oral vaccine in domestic pigs against tuberculosis (ISS)
Summary of progress towards objectives
This task is in an advanced stage., but is not completed. Pigs have been vaccinated in 7 groups in order to detect infection by M. bovis and to differenciate it from other closely related mycobacterial infections. Also pigs in two farms with a history of natural TB infection have been vaccinated. Even though the whole follow-up has not been completed due to a delay in obtaining the permits for the experiment, the works are progressing and data will be available in about 12 weeks after the official end of the project.
Task 5.1 Assessment of immune status of vaccinated piglets.
Task Leader: ISS coordinates the activities, chooses the sows and monitors the animals.
Other Participants: VISAVET set up the protocols for detection in faeces and saliva in samples obtained from experimentally infected pigs; DEFRA supplids reagents for skin test and IGRA assays and INGENASA will supplied the diagnostic test prototypes to the participating partners.
Task Objectives
Farms with semi free-roaming system were selected in Sicily on the light of a high degree of prevalence for tuberculosis due to Mycobacterium bovis. Two of them were chosen and successively, 8-10 tuberculosis-free sows were selected in each, according to the results of IFN-gamma test as described by Pesciaroli (Pesciaroli et al., 2012). 20-25 Piglets from those sows were vaccinated as reported elsewhere (Garrido et al., 2011), just after weaning and a similar number of age-matched piglets were kept as unvaccinated controls. Before vaccination and at 2-month intervals, from each animal, blood was collected and skin test was performed to assess the immune response.
Overall summary of work progress
During this reporting period, Partner 7 (INGENASA) has continued working on the evaluation of the pig sera samples from the vaccination/challenge experiments carried out at VISAVET (Partner 8) in the previous period. Briefly, in the experiment 1, Seven groups of 4 pigs each were vaccinated, infected or left as controls (see table). The vaccine consisted of heat-inactivated M. bovis by oral route.
Group 1
Control group: No vaccination/No infection
Group 2
Machuquillo vaccinated: D0 Vaccination/No infection
Group 3
BCG Vaccinated: D0 Vaccination/No infection
Group 4
MAH Infected: No vaccination/D0 Infection MAH
Group 5
MAA Infected: No vaccination/D0 Infection MAA
Group 6
Mbovis/MAA Infected :No vaccination/D0 Infection Mbovis-MAA
Group 7
Mbovis infected: No vaccination/D0 Infection Mbovis
Table 12. Animal groups in experiment 1.
Initially, the antibody response against M. bovis was analyzed by INGEZIM TB DR ELISA (DR-ELISA) and INGEZIM TB indirect ELISA (i-ELISA). As it has been reported in WP3, INGENASA has worked on the final development and validation of a lateral-flow assay (LFA) to detect Ab against M.bovis in serum/blood samples from pigs and wild board. This new LFA has been used to evaluate those sera. The results were compared with those obtained by ELISA.
The results showed that the correlation of the LFA with the two ELISAs is very high. There are only two differences in samples of the 30 days post vaccination/infection: One sample from the group of animals infected with M. bovis that was negative by LFA, negative by indirect ELISA, and positive by DR-ELISA. In this case, the positive results was very close to the cut-off of the assay. By the contrary, there was one sample positive by LFA and DR-ELISA, but negative by i-ELISA. The next tables show the summary of the LFA results compared with ELISAs.
Table 13. Summary of the LFA results compared with ELISAs.
These results correspond to a sensitivity value of 92.9 and 92.3 % for DR-ELISA and i-ELISA respectively and a specificity value of 98.3 for both assays.
In the Experiment 2, two groups of nine animals were studied. The first was the control and it did not receive the vaccine and the other was vaccinated day 0 and 30. All animals were infected with M. bovis at day 60 and finally were bled at day 90 and 120.
Group 1
Control group: d0 No Vaccine /d30 No boost/d60 Infection
Group 2
Machuquillo vaccinated: d0 Vaccination/ d30 boost/d60 infection
Table 14. Animal Groups in experiment 2
The correlation between the DR-ELISA and LFA in this experiment was also high. The summary is showed in the next table:
Table 15. Correlation between the DR-ELISA and LFA in experiment 2
The differences found have been in samples close to the cut-off of the ELISA assay. This data resulted in a sensitivity value of 90.6 % and a specificity value of 93.6 %.
As conclusion, the data obtained with the LFA showed a high sensitivity and specificity, although a little bit lower that the ELISAs. However this pen-side test offers advantages that should be taken into consideration: it is rapid, economic and simple-to use diagnostic tool, since it does not require any kind of instrumentation and the results is interpreted visually. Further validation in different scenarios will be carried out in the next future.
Task 5.2 M. bovis and corresponding lesions assessment
Task Leader: ISS vaccination of animals, monitoring and post mortem analysis
Other Participants: NEIKER and VACUNEK provide ready-to-use- vaccine; UCLM (provides expertise in
assessing the response to vaccination),
Task Objectives
Vaccinated and unvaccinated piglets will be maintained in their natural environment to guarantee a natural
exposure to the pathogens. Pigs will be slaughtered at 15 months of age and a complete post mortem analysis
will be performed, collecting oral, thoracic and abdominal lymph nodes, along with liver, spleen, lungs and
intestines to assess the presence and the severity of lesions. According to standard microbiological tests, and
the presence of M. bovis will be assessed in the different organs.
Results
According to the results obtained in the first period of the project (M18) we chose 2 free roaming pig farms considered endemic for tuberculosis due to M.bovis. Those two farms have around 200 adults and are farrow-to-finish.
We selected pigs within 5 months of age to be enrolled in the trial. In the first farm we included 68 pigs, in the second farm 63 with an age distribution reported in the following figure.
Figure 13 Age distribution per farm
Safety of the vaccine.
Safety was previously verified in mice. Mice were inoculated either by parenteral or oral route and were monitored for 15 days to exclude any deleterious effect. Following vaccine administration mice did not show any sign and the vaccine (data not shown).
Selected pigs were individually identified, bled and then vaccinated. In the first farm, 31 out 68 pigs were intramuscularly vaccinated and the remaining were kept unvaccinated. In the second farm 43 out 63 pigs were vaccinated and the remaining were kept unvaccinated. Following vaccination, pigs did not show any adverse reaction corroborating the preliminary results previously obtained in mice.
Blood samples taken before vaccination was then used to verify the immune status of pigs enrolled in the trial. IGRA results are reported in the following tables.
IGRA
negative
positive
unvaccinated
25
6
vaccinated
13
24
Table 16. IGRA results in FARM 1
IGRA
negative
positive
unvaccinated
20
0
vaccinated
43
0
Table 17. IGRA results in FARM 2
These results showed that in farm 1 the circulation of M.bovis is likely to involve pigs in the first stage of their life and they become infected very early. It is our opinion that the use of vaccine also in IGRA positive animals could give the project an added value because we could be able to assess the effect of vaccination in conditions that can naturally occur in field conditions.
Ninety days after vaccination, blood samples were collected in vaccinated and unvaccinated pigs. Blood samples were then stimulated with bovine and avium PPDs. Interferon gamma was then quantified by using a commercial ELISA kit (R&D Systems) according to Pesciaroli et al., 2012.
Results are depicted in the following tables.
In the farm 1 32 pigs were randomly selected (they are free ranging therefore the capture is not controlled). Of those 15 were unvaccinated and 17 vaccinated. The 15 unvaccinated pigs resulted all negative except one that gave positive results to the second IGRA. Nine out 17 vaccinated pigs resulted negative at IGRA performed before vaccination. Six of those resulted still negative at the second IGRA while three of those became positive. The remaining vaccinated pigs (8 out 17) that were already positive at IGRA performed before vaccination, resulted still positive at IGRA performed after vaccination.
Unvaccinated pigs (15)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
14
1
positive
0
0
Vaccinated pigs (17)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
6
3
positive
0
8
Table 18. IGRA results after vaccination in FARM 1
In the farm 2 57 pigs were randomly selected (they are free ranging therefore the capture is not controlled). Of those 15 were unvaccinated and 42 vaccinated. The 15 unvaccinated pigs resulted all negative except one that gave positive results to the second IGRA. All vaccinated pigs were negative at IGRA performed before vaccination Twenty-one of those resulted negative at IGRA performed after vaccination while 21 of those became positive.
Unvaccinated pigs (15)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
14
1
positive
0
0
Vaccinated pigs (42)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
21
21
positive
0
0
Table 19. IGRA results after vaccination in FARM 2
Overall, from this field test, it is possible to infer important even not conclusive insights. First of all, we have confirmed that vaccine parenterally administered is well tolerated without any sign of suffer or distress in the recipient animals.
Secondarily, we ascertained that piglets that become positive to the second IGRA except in one case in farm 1 and 1 case in farm 2 were those previously vaccinated. This is unlikely correlated to a sensitisation to wild M. bovis rather to the induction of an immune response operated by the vaccine. This means that the vaccine is immunogenic and also that infection was not disseminated enough among the animals enrolled in the experiment. This is the reason why we could not kill the animals as planned because likely they were still uninfected. To overcome this problem, we decided to keep the animals alive to give more time to get the infection. At the endo of the follow up, twenty-four pigs were randomly selected from the two herds enrolled in the field test. At slaughter, a complete post-mortem analysis was performed.
Fourteen out 24 selected pigs were vaccinated while 10 out 24 selected pigs were unvaccinated control animals. As reported above, those pigs were previously bled at 90 days after vaccination and blood samples were collected and stimulated with bovine or avium PPDs. Interferon gamma was quantified by using a commercial ELISA kit (R&D Systems) according to Pesciaroli et al., 2012.
Previous IGRA for the slaughtered pigs:
IGRA
negative
positive
groups
vaccinated
7
7
controls
9
1
IGRA performed the day before the slaughtering:
IGRA
negative
positive
groups
vaccinated
3
11
controls
9
1
At post-mortem a complete analysis of organs and draining lymph nodes was done and results are depicted as follows:
Post mortem analysis
negative
positive
groups
vaccinated
3
11
controls
8
2
Slaughter analysis with the IGRA
IGRA
negative
Positive
Post mortem
positive
0
12(1)
negative
12
0
Three out 12 pigs resulted positive to IGRA for M.avium
Overall, the animals which resulted positive at slaughter showed classical signs of tuberculosis in one or more lymph nodes with, in some cases, also lesions in parenchimatous organs, such as liver, lung and kidneys, although bacterial isolatio and identification has not been done at this time. These results clearly suggest that the vaccination is not able to exert any significant effect in improving the pathological status of infected animals.
In order to ascertain that lesions are associated to the infection we correlated the immune status previously observed with the presence of lesions, irrespectively to the vaccination status, with the following results:
Post mortem analysis
negative
positive
IGRA
positive
1
7
negative
11
5
As expected, there is a high correlation between the IGRA negative animals and those without lesions, suggesting that, presumably, those animals are still uninfected. There is, in addition, a good degree of correlation between IGRA positive animals and those with lesions (infected).
At slaughter, the rate of animals with lesions was 78% among vaccinated animals and 20% among unvaccinated. These results are counter-intuitive and far from expected according to preliminary experiences, but were consistent with IGRA results and indicate that, at the time of submitting this report, vaccination did not just fail to protect, but seemed to favour the detection of infection in terms of gross lesions. Currently, the proof of concept that vaccination in domestic suids protects as efficiently as in wild suids and that was the main goal of this task has not been demonstrated in these settings and requires further histopathological and microbiological studies that should clarify the pathogenetic aspects of the vaccination-infection interaction.
Pesciaroli M, Russo M, Mazzone P, Aronica V, Fiasconaro M, Boniotti MB, Corneli S, Cagiola M, Pacciarini M, Di Marco V, Pasquali P. Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs. Vet Immunol Immunopathol. 2012 Aug 15;148(3-4):369-72.
WP6: Dissemination, exploitation and market preparation (INGENASA)
During the course of the project, the Consortium has evaluated and validated two different complementary products:
An oral TB vaccine for wild life animals that has been tested in pigs, wild boards and cattle.
A double recognition ELISA test, associated to the vaccine, based on the recombinant MPB83 (MPB83-DR-ELISA) that was developed during the EU project TB-STEP. This assay permits to detect antibodies (Ab) against M. bovis in serum and it is able to distinguish infected from vaccinated animals. The assay has been tested in swine, including wild and domestic pigs, and it is suitable for wild (badgers, camelids, zoo animals...) and domestic animals species. Moreover, an indirect ELISA for TB Ab detection in pigs has also been evaluated.
According to The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA Journal 2015;13(12):4329, 191 pp. doi:10.2903/j.efsa.2015.4329 the overall proportion of cattle herds infected with, or positive for, M. bovis remained very low in the EU (0.8% of the existing herds in the EU), although there is a heterogeneous distribution of M. bovis in Europe. The prevalence ranges from absence of infected/positive animals in many EU officially tuberculosis free status (OTF) regions, to a prevalence of 11.6% in the non-OTF regions of the United Kingdom (England, Northern-Ireland and Wales).
In the OTF regions, the proportion of herds infected with M. bovis decreased further to 0.011% in 2014. This can be partly explained by the inclusion of Hungary, in 2014, in the list of OTF European Countries Members States (MS). Hungary had 16,419 cattle herds and reported 3 infected ones in 2014. Also, in OTF countries, the number of herds infected with M. bovis reported was on average lower than in 2013.
Figure 14. Status of countries regarding bovine tuberculosis due to M. bovis, 2014
In the non-OTF regions, in 2013, the reported number of herds positive for M. bovis was, for most MS, was similar or lower than in 2014. However, an increase was noteworthy in Spain (1,526 in 2013 and 1,867 in 2014) and Ireland (4,640 in 2013 and 6,623 in 2014) while in the United Kingdom a decrease was reported (10,956 in 2013 to 10,172 in 2014). Overall, the reported proportion of herds positive for M. bovis in the non-OTF regions has slowly increased during the last years
Bovine tuberculosis is distributed worldwide, but control programs have eliminated or nearly eliminated this disease from domesticated animals in many countries. Nations currently classified as tuberculosis-free include Australia, Iceland, Denmark, Sweden, Norway, Finland, Austria, Switzerland, Luxembourg, Latvia, Slovakia, Lithuania, Estonia, the Czech Republic, Canada, Singapore, Jamaica, Barbados and Israel. Eradication programs are in progress in other European countries, Japan, New Zealand, the United States, Mexico, and some countries of Central and South America.
Figure 15. Number of bovine TB cases reported by country to OIE in 2011
Although cattle are the primary hosts for M. bovis, other domesticated and wild mammals can also be infected and isolations have been reported from sheep, goats, equines, camels, pig, wild board, dog, cats, felines including lions and tigers, badgers...
In 2014, 14 MS and two non-MS investigated animal species other than cattle for M. bovis. M. bovis was reported in 816 animals other than cattle: alpacas (34), badgers (218), bison (3), cat (24), deer (106), dog (1), goat (29), guinea pig (1), lamas (3), pet animal (1), pig (153), sheep (1), wild boar (219), wild animal (1) and zoo animal (2). Seventeen MS and two non-MS investigated animals for Mycobacterium species other than M. bovis. M. tuberculosis was reported in two pigs and 38 cattle and M. caprae was reported in 126 animals by four MS (Austria, Germany, Hungary and Spain): cattle (68), deer (10), goats (6), sheep (1) and wild boar (41).
Because the course of disease is slow, taking months or years to kill an infected animal, an animal can spread the disease to many others before the first clinical signs appears. Therefore, movement of undetected infected domestic animals and contact with infected wild animals are the major ways of spreading the disease.
Each year, the experts confer more epidemiological relevance to the wild reservoirs as a difficulty in the TB eradication Therefore, the new developed tools would help to create new and more effective plans to control and finally eradicate TB from domestic animals and consequently human TB.
Animal TB control and eradication programs management, are mainly designed and implemented by International Institutions (OIE) and National Animal Health Institutions. We have the advantage that important Institutions in this field are members of WildTBVac Consortium and, therefore, the developed products have been already tested and validated by them. This represents a great opportunity for innovative businesses [...]to find a niche on the market. Having the advisement and collaboration from the Partners of the Consortium, Partners 2 (VACUNEK) and 7 (INGENASA) have designed a business plan for a vaccine production plant and for the commercialization of the new diagnostic assays, respectively.
The TB diagnostic techniques most frequently used nowadays are intradermal tuberculin test or gamma interferon assay. This requires handling the wild animals, well-equipped laboratories and skilled personnel. Detection of specific antibodies is an important alternative tool to control the disease.
Partner 7 (INGENASA), produces and commercializes diagnostic kits under rigorous quality control rules (ISO 14001:2004 and ISO 9001:2008), for a big number of diseases of different species including, pigs, cattle, horses, dogs or cats. Partner 7 already has a broad distribution network across the word, with presence in more than 63 different countries and, therefore, products validated within this project are expected to be easily introduced into the market using resources and networks of the company. We sell our own products to end users, institutions, private companies Public Health Agencies, etc. We can also provide reagents in bulk to other kit manufacturers, or finished products to distributors.
The objectives of the plan will cover:
1.- Evaluation and quantification of the real weight of the wild animals in the epidemiology of TB in some key areas for each country, specially, where eradication plans have been less effective.
To do this, according to the Health Authorities:
The guidelines to make the sampling, number of specimens... will be provided.
The new serological assay will be used to know the prevalence of the infection within the wild population.
The convenience to go ahead with the following steps, will be evaluated according to the guidelines of the Health Authorities. When the estimated prevalence was considered significant enough to interfere with the bovine eradication programs, different solutions could be suggested:
a.- Establishment of a vaccination program, using the edible vaccine developed within the project.
b.- Monitoring of the prevalence of TB in wild animals in the controlled areas.
2.- After evaluation of the results of this plan on the bovine TB incidence, the establishment of the program in other areas will be considered.
The selection of markets will be done by following the priorities below:
Priority 1 (During the first 2 years). We would like to work in
Countries with long history and experience applying bovine TB eradication programs, without obtaining a final eradication + those countries in which the Partners of the Consortium could have interest and could support the advantage of using the ELISA test (Spain, Italy, England ...)
Priority 2 (From the third year and thereafter):
Countries with high bovine population + countries with bovine TB eradication programs going on (New Zealand, the United States , some countries in Europe, Central and South America...)
Income Projections
Although it is hard to estimate the provisional diagnostic market, trying to be very cautious, we have set our objectives of sales of diagnostics for the following 5 years. To prepare them, we established the following premises:
1.- Sales price per individual test will be 1,0 €
2.- On each area in which the system will be introduced, we will estimate a minimum number of 200 samples and a maximum of 1.000
3.- Once the program is implanted in one area, the monitoring will remain (with the same number of test) for at least 5 years.
In order to show the projected annual profit, an optimistic and a cautious estimation has been performed. A summary is showed in the following Table:
Year 1
Year 2
Year 3
Option 1
Option 2 (conservative)
Option 1
Option 2 (conservative)
Option 1
Option 2 (conservative)
Nº of countries
6
3
10
6
12
8
Nº areas/country
4
2
6
3
8
4
Nº test/year
1000
200
1000
200
1000
200
Objetive sales in €
24.000,00 €
1.200,00 €
60.000,00 €
3.600,00 €
96.000,00 €
6.400,00 €
Cumulative sales
24.000,00 €
1.200,00 €
84.000,00 €
4.800,00 €
180.000,00 €
11.200,00 €
Table 20. Projected annual profit
Regarding vaccine perspectives, a detailed business plan is provided in deliverable D6.64. Production projections are as follows:
Table 21. Quantification of number of doses and their derived production costs at Iberian market.
Vaccine doses requirements for Iberian demand through the six years of projections sum up over half million doses. Highest production needs per year reach 210.000 vaccine doses.
Table 22. Quantification of number of doses and their derived production costs at International market.
Vaccine doses requirements for European and American demand through the six years of projections sum up over 4 million doses. Maximum production needs per year reach 1.45 million vaccine doses.
Authorities’ successful plan for TB eradication could affect to the projections previously cited, so half million doses facilities will cover the highest demand for Iberian territory through the 6 year period as well as projections up to the third year of the project.
Potential Impact:
The problem of wild boar as TB maintenance hosts is particularly relevant in Spain and Portugal, but extends to several other EU countries (France has recently observed an association between wild boar TB seroprevalence and bovine TB outbreaks) and to Hawaii in the USA. Moreover, free ranging domestic pigs can also act as M. bovis maintenance hosts as was recently described in Italy (Di Marco et al. 2012). Therefore, initially we aim at two potential differentiated markets: 1) Wild boar or feral pigs, who include game producers and animal health or environment authorities in Europe and South America; 2) Domestic pigs, from both an industrial production and animal health authorities perspective in countries with a significant proportion of open-air bred pigs such as the Mediterranean countries and Hungary.
The potential economic benefits of this are quite high as such a wildlife vaccine/domestic diagnosis strategy that would not lead to false-positivity in domestic animals would avoid costs to the taxpayers of compensation payments and costs of increased testing and testing frequencies, as well as the direct economic losses to farmers due to decreased production, veterinary care, movement and trade restrictions and loss of product quality image. Further, it would enhance stakeholder acceptance and confidence in such a strategy, which are necessary requirements for the implication of any such strategy. Potential customers are in principle governments, public administrations and large pig farming companies (including public and private hunting grounds, national parks, etc.). Therefore, suitable customers are to be found both in A) government agencies, and B) in the private sector.
Vaccines developed in WILDTBVAC would be of interest for animal health authorities interested in livestock TB control. Interest towards vaccine and diagnostics research has already been expressed by colleagues from the US Department of Agriculture and from several agencies of EU Member States (MS) including France, Austria and Germany, and non-member states such as Switzerland. Vaccines and diagnostic tools would also be of interest for wildlife conservation agencies. Farmers, mainly those focused on high quality free-range production might greatly benefit from a tool that can greatly simplify the only expensive test-and-cull strategy applied to bovine TB that is currently being questioned by some medical researchers. The porcine sector is particularly relevant in Mediterranean EU MS, but other MS do also find pig TB and would probably be interested in diagnostics. The hunting sector, which is quite significant in some EU MS such as Spain and France, wild boar farmers/producers would be most interested in products developed by WILDTBVAC.
The three veterinary companies included in the partnership are in excellent position to spread the technology refined in this project and to take advantage from the market studies set as a specific outcome of this project.
The overall expected impact of WildTBVac outcomes are:
At an Activity level: a) Sustainable production and management of biological resources. Focusing on a targeted activity that addresses specific animal pest or disease that in addition has a direct relation with food security as well; b) Helping to implementation of the Action Plan against the rising threats from Anti Microbial Resistance (AMR). Although there is no direct relationship with AMR, as a general strategy, vaccination is aimed at reducing prevalence of infection without directly killing the pathogens, but by increasing resistance to infection. In this case doing this in susceptible animal species should decrease human exposure and therefore, the need for antibiotic treatment in human patients.
At an Area level: a) Socio-economic research and support policies. Providing tools needed by policy makers and other actors to support implementation of relevant strategies to avoid direct economic losses to farmers due to movement and trade restrictions.
At a Topic level: KBBE.2013.1.4-07 by bringing about results on three main issues addressed in the Call: a) Opportunity for innovative applications; b) Production of a dedicated business plan; c) Development of new patents.
List of Websites:
http://www.wildtbvac.eu
WildTBVac is a small or medium-scale focused research project targeted to SMEs of FP7-KBBE-2013-7-single-stage type in the area 2.1.4 Socio-economic research and support policies. The project is a grant to transfer research developed in the previously EU funded project TB-STEP (#212414) to the production sector by building a pre-commercial set of TB control solutions. Specific objectives are the following: a) To standardize and optimize the production of an inactivated TB vaccine; b) to evaluate vaccine-compatible multi-species diagnostic strategies; c) to assess the field efficacy of integrated TB control strategies for wild boar; d) to evaluate the field efficacy of oral vaccination for TB control in domestic and feral pigs, and e) to define the exploitation and commercialization strategy.
Project management has taken care of receiving and distributing the funding, coordinating the research, demonstration and other activities and collecting the individual technical and financial reports in a timely and consistent manner.
The project has produced a bank of seeds from a selected isolate of M. bovis for medium-large scale production according to an established quality control protocol. Costs of production have been determined. Oral delivery of the inactivated M. bovis bacteria did not compromise diagnostic skin and IGRA tests using defined antigens, but intramuscular did so. The protein target MPB83 has been successfully produced and both a LFD (lateral flow device) prototype and a DR (double recognition) ELISA have been developed. A first validation using experimental and field sera has been carried out showing that: a) the LFA resulted in a sensitivity value of 86.6 % and an specificity of 96% in wild boar and pigs; b) the DR-ELISA had a sensitivity of 99.1%, and specificity of 99.2%; c) DR-ELISA on sera samples from different zoo species and pet animals have been tested with good results; d) it yielded a sensitivity of 86% and an specificity of 87%; e) deer samples yielded an estimate of a 94% of SP and 50% of SN. Use of the vaccine as a bait in wildlife is compatible with diagnostic in cattle by the existing or novel tuberculin skin testing formats as well as with the use of IGRA tests utilizing tuberculin or defined antigens and with the potential use of MPB83-DR-ELISA.
TB control strategies at the wildlife-livestock interface depends on many factors including (a) the single or multi-host nature and other characteristics of the pathogen, (b) the availability of suitable diagnostic tools, (c) the characteristics of the wildlife host(s), (d) the geographical range of the pathogen/reservoir (improved control in isolated versus continuous populations) and the scale of the control effort (large-scale longitudinal programs are better), (e) the attitude of the stakeholders involved (highly dependent on their education and communication provided to them
The domestic pig field test has been delayed but already confirmed that vaccine parenterally administered is well tolerated and immunogenic. Currently, the proof of concept that vaccination in domestic suids protects as efficiently as in wild suids and that was the main goal of this task has not been demonstrated in these settings and requires further histopathological and microbiological studies that should clarify the pathogenetic aspects of the vaccination-infection interaction.
Vaccine doses requirements for European and American expected demand through a six years projection sum up over 4 million doses. Maximum production needs per year were estimated at 1.45 million vaccine doses.
The project has generated 11 SCI papers and 9 congress presentations in addition to a divulgation article and information made available at a website (www.wildtbvac.eu).
Project Context and Objectives:
Recent studies have shown estimations of worldwide 50 million cattle infections with M. bovis, at a cost to the agricultural community of 2-3 billion € per year. In developing countries, M. bovis infection is still widespread, in both cattle and humans. Even in developed countries, with heavy and longtime investments in eradication of disease from livestock which seemed to have reached success, complete eradication appears to be hampered by the reiteration of outbreaks related to the presence of wildlife reservoirs of M. bovis. Under these circumstances, a single case of a reportable disease in livestock can result in serious economic consequences for the producer, public, and governments. Within this scenario, there’s room and clear business opportunities for integrated control products or solutions that aim at addressing what is considered a global problem, animal tuberculosis. Our overall objective is therefore to take the results of the TB-STEP project - “Strategies for the eradication of bovine tuberculosis”, funded under FP7-KBBE-2007 with project number 212414, where most of the WildTBVac partners were involved, and build a pre-commercial set of TB control solutions by means of testing the technical and economic viability of the aforementioned integrated approach. WildTBVac specific objectives are the following:
To standardize and optimize the production of a TB Vaccine (prototype resulting of the TB-STEP project)
To evaluate several vaccine-compatible diagnostic strategies (also resulting from the TB-STEP project) that will include multi-species DIVA tests (wild boar, domestic pigs, cattle)
To assess the field efficacy of integrated TB control strategies including oral vaccination for TB control in wild boar
To evaluate the field efficacy of integrated TB control strategies including oral vaccination for TB control in domestic and feral pigs
To define the exploitation and commercialization strategy including clear technology transfer approaches and development of a market oriented business.
Project Results:
WP1:Management and coordination (NEIKER)
Consortium management tasks have been focused on coordinating exchanges of information and materials between the partners, setting up meetings and communications between the task leaders and supervising deadline compliance for deliverables and milestones regarding the technical aspects of the project. The coordinator has taken care also of distributing the funding and requiring the partners to make their financial reports on time and according to the Commission specifications.
There have been no relevant problems. Most of the efforts have been devoted to clarify administrative concepts in order to make the reports. The most important problem has been a delay in the initiation of the experimental vaccination in Italy that was caused by the long time consumed by the obtainment of the administrative permits.
There has been no consortium change.
There have been 5 project meetings summarized in the following table:
Meeting
Date
Time
Venue
Attendees
1
Kick-off
14-1-2014
9:30 13:30
NEIKER, Derio, Spain
17
YFE, IZI, RAJ, JGA, ISE, LDO, JBE, CGO, ACH, RHA, PRU, BLA, PPA, BCH, FBU, MVO, GJO
2
18-6-2014
18:00 18:50
Town Hall, Cardiff, UK
8
RAJ, JGA, CGO, LDE, JBE, MVO, GJO, ARA
3
9-12-2014
15:30 16:30
Adobe Connect, Videoconference (https://neikertecnalia.adobeconnect.com/rajustenkrd/)
9
JGA, RAJ, CGO, JBE, ARA, BLA, PRU, FBU, MGA
4
3-3-2015
15:00 15:40
Adobe Connect, Videoconference (https://neikertecnalia.adobeconnect.com/rajustenkrd/)
11
RAJ, PPA, JBE, ARA, BLA, PRU, RLA
Off-line: MVO, GJO, CGO. MGA
5
24-6-2015
9:30 15:30
NEIKER, Derio, Spain
12
RAJ, JGA, ISE, LDE, JBE, MVO, GJO, CGO, MGA, BLA, PRU, AVE
Table 1. Meetings table
Project planning has been carried out as expected and is currently completed but for the experiment on pigs in Italy. This delay has not allowed to verify the use of the vaccine in domestic pigs at the end of the project, but has provided material for the setting up of diagnostic tests. The work is underway, so it will generate the planned information that will be shared by the partners and submitted as an addendum when ready.
The website was deployed in the early months (www.wildtbvac.eu) of the project and is still available and functional.
WP2: Vaccine optimization and scale up (NEIKER)
Summary of progress towards objectives
A bank of strains seeds from a selected isolate of M. bovis has been produced.
The medium-large scale production of the selected vaccine strain in terms of shortest time, maximum amount and optimal and stable genetic and antigenic features has been produced and a quality control protocol has been established and is being used for the current batches.
The composition of the vaccine product according to the route of administration in terms of dose and adjuvants for maximal protection and minimal diagnostic interference has been fixed according to the currently available information.
Costs of production have been determined and updated according to real experience in producing and shipping to partners and out of the EU collaborators.
Significant results
A method for medium-large scale production has been set up with the necessary safety and quality controls applied and the product has been delivered to partners and third parties for testing as needed.
Deviations
Only limited changes according to the budgetary shortcuts have been deemed necessary in terms of production systems and equipments to fulfil current and future needs.
Task 2.1 Equipment set-up
Task Leader: NEIKER (Acquisition and setting-up the equipment needed).
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support).
Task Objectives
The main objective of this task is to acquire and set-up the equipment needed to produce the vaccine in a medium-scale pre-industrial fashion.
Results
The equipment to produce and harvest bacteria has been set up in the BSL3 laboratory and its performance checked. In addition, a high capacity home-made inactivation system has been manufactured, transferred to BSL3 laboratory facilities and validated
Deviations from the work programme, and corrective actions taken/suggested
The final budget did not allow the purchase of a freeze-drier and a bio-reactor as initially planned and for which most of this task was designed. Anyway, the work has been performed following the current protocols and with the existing equipment but with some modifications for an increased productive capacity and biosafety.
Task 2.2 Large scale mycobacterial growth
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (diagnostic validation research).
Task Objectives
Task 2.2 goals were to produce seeds for 1030 vaccine batches and vaccine for other WPs under current conditions. Mycobacterial cells grown in supplemented 7H9 medium were harvested by centrifugation or filtration and then aliquots were freeze-dried to produce the seeds stock. The aim is to produce enough vaccine doses under current conditions and to provide them to other partners in order to be used in diagnostic validation research activities.
Results
All the planned 1030 seeds have been produced. They are kept in 50% glycerol at -80ºC. Vaccine has been produced and partners have been provided with sufficient doses to be used in other WPs. Additionally, in order to take advantage of resources available from third parties interested in our product, and therefore in line with this project broad objectives in terms of dissemination and assessment of product properties, vaccine doses have been made available for both additional uses by this consortium partners (IREC, VISAVET, AHVLA) and for research groups. The latter include: Celine Richomme & Maria Laura Boschiroli (ANSES), Suelee Robbe Austerman & Pauline Nol (USDA-APHIS-VS-STAS), Jose Antonio Ortiz (Las Lomas deer farm, Cádiz), Natasha Rudenko (Academy of Sciences of the Czech Republic), Quim Segalés (CRESA) and Eamonn Gormley (UCD Dublin).
Deviations from the work programme, and corrective actions taken/suggested
Instead of using freeze-drying to lyophilize the seeds, these are kept at -80ºC in vials containing 0.5 ml 50% glycerol and 5% 7H9 broth.
Task 2.3 Definition of optimal production conditions
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (effects of vaccine characteristics on interference with diagnostic performance).
Task Objectives
This task was aimed at defining the optimal production conditions in terms of time, growth and bacterial harvesting. In order to achieve this, optimum bacterial production and time parameters were assessed in culture. Slight medium formulation differences, static or shaking conditions, use of a small volume pre-culture, air exchange or airtight conditions and clump formation are the anylised variables. To harvest cells centrifugation and filtering are the methods compared.
Results
The results obtained for the comparison between shaking and static culture conditions indicate that shaking promotes cell clumping if no additional dispersant is included in the medium. But the effects of dispersants on the antigenicity of bacteria cultured in dispersant-enriched medium are unknown. The use of a starter pre-culture improves production in terms of bacterial amount obtained, shorter time and lower clump formation. Under current conditions, clump formation is directly related to culture age. Culturing bacteria for periods longer than 2-3 weeks promotes tough clump formation. Air exchange conditions can only be tested in small flasks because the bio-reactor could not be purchased. Centrifugation must be the harvesting method since no modern filtering system could be obtained.
Deviations from the work programme, and corrective actions taken/suggested
Comparison between culturing bacteria under airtight or air-exchange conditions were only tested in a small scale (flasks containing 10 ml of medium) instead of using a bio-reactor. Filtration has been excluded for cell harvesting.
Task 2.4 Stability of vaccine batches
Task Leader: NEIKER
Other Participants: VACUNEK (technology follow-up for industrial and commercial feasibility and support). PRIONICS and INGENASA (effects of vaccine genetic and stability characteristics on interference with diagnostic performance).
Task Objectives
This task 2.4 is focused on assessing genetic and antigenic stability of vaccine batches. To grow and check thirty seeds/batches at different post-production times. Spoligotyping method has been used to make sure the original genetic profile has not changed. Two dimensions electrophoresis, western-blot and inoculation in guinea pigs were used in order to assess the antigenic stability of seeds. To test changes in the material of models described in WP3 require that for improved diagnostic performance.
Results
Vaccine products derived from the thirty seeds prepared for this purpose were analysed at different post-production times using spoligotyping, 2D/WB and guinea pig inoculation. Spoligotype of tested vaccine products has not changed in comparison with the original spoligotype (original low-passage strain).
Figure 1. Genetic stability as observed in the spoligotyping
Figure 2. Batch stability
Figure 3. Skin test reactivity induced by the vaccine in guinea pigs.
The vaccine strain grows better in static and air-tight cultures for 2-3 weeks. Both seeds and vaccine batches remain genetically and antigenically stable for, at least, three years in standard refrigeration conditions
Deviations from the work programme, and corrective actions taken/suggested
No deviations to be reported.
Task 2.5 Final optimal formulation
Task Leader: NEIKER
Other Participants: UCLM (formulae testing in wild boar), VISAVET (formulae testing in pigmy pig), ISS (formulae testing in pig), VACUNEK (technology follow-up for industrial and commercial feasibility and support).
Task Objectives
The aim of this task was to define the final formulation as well as the quality control procedure. To do the former, four formulations were assayed in-vivo in order to choose the best one according to the protection it provides and to their cross-effects over diagnostic tools. This experiment was performed to define the indications for use of the vaccine. The quality control procedure to ensure the purity, the genetic identity, the antigenic ability, the inactivation and the innocuousness (no negative cross effects) of the final product were defined as well.
Results
Design of experimental groups and settings. Four formulations were assayed in-vivo. These combine 2 antigen concentrations (high-dose or 107 CFU & low-dose or 103 CFU) and 2 administration routes (oral without adjuvant and intramuscular with Montanide adjuvant).
Deviations from the work programme, and corrective actions taken/suggested
Pigmy pigs will not be used for in-vivo testing in VISAVET due to animal availability problems. Instead, goats benormal young pigs were finally used.
WP3: Evaluation of vaccine-associated diagnostic strategies including the use of defined diagnostic antigens (AHVLA/DEFRA)
Task 3.1 Tuberculin skin test and IGRA reactivity
Task Leader: DEFRA
Other Participants: NEIKER will use the reagents in a set of cattle, VISAVET will evaluate a define set of animals by both diagnostic protocols (skin and IFN-g tests).
Task Objectives
The main objective of this task was to perform a vaccination experiment in cattle and for Defra to supply defined DIVA antigens to other partners. The underlying hypothesis investigated in this work package is that oral uptake of the wildlife vaccine by bystander domestic animals (e.g. cattle or domestic, free-ranging pigs) will not induce systemic immune responses and therefore will not compromise the use of tuberculin or of defined DIVA antigens in diagnostic assays probing cellular immunity (skin test or blood-based IGRA).
NEIKER also used the reagents in a set of cattle and VISAVET evaluated a defined set of animals by both diagnostic protocols (skin and IFN-g tests)
Results
APHA experiment
We assessed the suitability of tuberculin as a diagnostic reagent for the skin test and IGRA in cattle vaccinated with inactivated M. bovis strain 1403 (Neiker), also called mdR below. To this end, 6 calves (aged 5-7 months) were vaccinated with 106-107 CFU of killed bacteria which is the standard dose (also called Full Dose, FD) by either the oral or intramuscular route according to the supplier’s guidelines (Neiker). Six weeks post vaccination, these animals received intradermal injections (0.1 ml volume) in the neck with avian tuberculin (PPD-A; 2,500 IU [Prionics]) and bovine tuberculin (PPD-B; 3,000 IU [Prionics]). Skin induration was measured at the injection sites prior to and 72 hours after the skin test, and the results are expressed as the difference in skin thickness between the two readings. In animals vaccinated by the oral route, no positive skin test reactions were observed when using either the SIT (PPD-B only) or SICCT (PPD-B – PPD-A) readouts (
). In contrast, all animals vaccinated by the intramuscular route showed positive skin test reactions in both tests (
).
Table 2. Skin test results using tuberculin reagents in vaccinated cattle. Values represent the change in skin thickness (differences in mm). SIT: response considered positive if PPD-B ≥ 4mm. SICCT: response considered positive if the difference between PPD-B and PPD-A is > 4mm. Test positive results are highlighted in red.
The whole blood IGRA test using tuberculin reagents was also performed at time points pre- and post-vaccination. IFN-γ levels in the plasma supernatant were quantified using the Bovigam ELISA kit (Prionics), and samples were considered test positive if the O.D. for PPD-B stimulated cultures minus the O.D. for PPD-A stimulated cultures was > 0.1. All 12 animals demonstrated IFN-γ responses to the positive control PWM (data not shown). None of the animals vaccinated by the oral route tested positive in the IGRA assay at any time point. In contrast, all six animals vaccinated by the intramuscular route showed positive IGRA test results 2 weeks post vaccination, which persisted for the duration of the experiment (
).
Table 3. IFN-γ responses in vaccinated cattle using the tuberculin readout (PPDB – PPDA). Values represent ΔO.D. values (PPDB - PPDA). Test positive results are highlighted in red.
In summary, the results of Task 3.1 indicate that in contrast to intramuscular delivery, oral delivery of the inactivated M. bovis strain 1403 vaccine did not compromise diagnostic tests for bovine TB that ulitise tuberculin reagents.
Additional experiment conducted at Neiker:
We had the opportunity to incorporate data from an independent experiment conducted at Neiker that confirmed our findings that intramuscular injection of the standard full dose induced both skin test and IFN-γ responses. In this experiment, calves were either vaccinated with 107 CFU (MdR-FD, n = 10) or a reduced dose (MdR-RD, n = 10) of 103 CFU, or left unvaccinated (n=5). Blood samples for IFN-γ testing were collected at the time of vaccination (day 0) and at days 15, 43, 78, 86 and 186 post-vaccination. Blood samples were stimulated with PPDA and PPDB; IFN-γ responses were determined by Bovigam ELISA. The same interpretation criteria were applied as described above. Two tuberculin skin tests were performed at days 43 and 78 post-vaccination using PPDB (single intradermal test, SIT) with the standard test interpretation being applied.
The results are summarised in Table 4 and demonstrate that animals vaccinated with the full dose (MdR-FD = 107 CFU) developed positive IFN-γ responses as early as 15 days post-vaccination and these responses remained positive in between 88 and 100 % of animals until the last blood sampling was performed (day 186). In contrast, 90% of the animals vaccinated with the reduced dose (MdR-RD = 103 CFU) were IFN-γ test-negative 15 days post-vaccination, and the majority remained test negative over the course of the experiment. (Table 3.3). Moreover, one animal that tested positive in this test, was test positive before the vaccine was applied (false-positive). Similar observations were made when skin test responses were considered: 40 and 50 % of the MdR-FD vaccinated animals tested positive at the two skin tests, respectively, whilst only 1/10 (10% of the reduced dose vaccinated cattle tested positive (Table 4). As expected, none of the unvaccinated control animals tested positive to either test (Table 4). In conclusion, this experiment confirmed the observation that intramuscular vaccination with the full dose of this vaccine will compromise the specificities of skin and IFN-γ tests. Further, it demonstrates that a reduced vaccine dose will only lead to limited test positivity in these tests probing cell-mediated immunity.
Table 4. IFN-γ and skin test responses in vaccinated cattle using the tuberculin readout (PPDB – PPDA) for the interpretation of the IFN-γ and the interpretation of the single intradermal skin tests (PPDB) to classify animals as positive or negative (interpretation criteria as described in
and
).
Deviations from the work programme, and corrective actions taken/suggested
Some unscheduled work has been done in cattle that support the conclusions of the partner DEFRA experiment. This adds value to the project, no corrective action required. .
Task 3.2. Antigenically defined skin test and IGRA reactivity.
Task Leader: PRIONICS (provide peptide cocktails PC-EC and PC-HP for stimulation of blood and the IGRA Bovigam)
Other Participants: NEIKER applied the test in a set of cattle, DEFRA (vaccinate cattle and test in skin test and IGRA) and VISAVET carried out a field trial to define the sensitivity and specificity of these specific antigens under specific conditions.
Task Objectives
As tuberculin has inherent problems of production, quality control, lack of specificity, we also evaluated whether defined antigens such as ESAT-6, CFP-10, Rv3615c, and Rv3020c can be used in assays of cellular immune responses such as skin test and IGRA (cattle, pigs).
Results
We assessed the suitability of defined antigens as diagnostic reagents for the skin test and IGRA in cattle vaccinated with inactivated M. bovis strain 1403 (Neiker) as described for Task 3.1. For the skin test, two protein cocktails were formulated containing either: (i) the proteins ESAT-6, CFP-10 and Rv3615c (APHA-1); or (ii) the proteins ESAT-6, CFP-10, Rv3615c and Rv3020c (APHA-2). Previous data generated at APHA has shown that these defined antigen skin test reagents induce positive skin test reactions in TB-reactor cattle with confirmed infection (APHA-1 [n=62]: test sensitivity of 85.48% [95%CI of 74.22% to 93.14]; APHA-2 [n=16]: test sensitivity of 93.75% [95%CI of 69.77% to 99.8%], data not shown). At the time of skin testing, the cattle detailed in Task 3.1 (i.e. cattle 1 to 12) were also skin tested with APHA-1 and APHA-2. In both cases, the protein cocktails were delivered by intradermal injection (0.1 ml volume) at a concentration of 10μg/protein/injection. Skin induration was measured at the injection sites prior to and 72 hours after the skin test, and the results are expressed as the difference in skin thickness between the two readings. As previously shown for tuberculin reagents (Task 3.1) no positive skin test reactions to either APHA-1 or APHA-2 defined antigen cocktails occurred in animals vaccinated via the oral route (Table 3.4). However, in contrast to that seen for tuberculin reagents (Task 3.1) limited skin test reactions were also observed in animals vaccinated via the intramuscular route, with only a single animal testing positive to the APHA-1 reagent (Table 5).
Table 5. Skin test results using defined antigens in vaccinated cattle. Values represent the change in skin thickness (Δmm). APHA-1 and -2: response considered positive if ΔΔ mm is ≥ 2mm.
Simultaneous with carrying out whole blood IGRA using tuberculin reagents, the performance of defined antigen reagents was also assessed in the vaccinated cattle detailed in Task 3.1. Whole blood samples taken from animals at time points pre- and post-vaccination were stimulated for 24 hours with peptide cocktails containing the following defined antigens: (i) E/C (ESAT-6 and CFP-10 [APHA]); (ii) Rv3615c (APHA); (iii) PC-EC (ESAT-6 and CFP-10 [Prionics]); and (iv) PC-HP (ESAT-6, CFP-10 and 4 additional mycobacterial gene products [Prionics]). All peptides were used at a concentration of 5μg/peptide/ml. IFN-γ levels in the plasma supernatant were quantified using the Bovigam ELISA kit (Prionics), and samples were considered test positive if the O.D. for peptide stimulated cultures minus the O.D. for unstimulated cultures was > 0.1. All 12 animals demonstrated IFN-γ responses to the positive control PWM (data not shown). Oral vaccination with the inactivated M. bovis strain 1403 did not induce IFN-γ responses to any of the defined antigen peptide cocktails during the course of the experiment, with the exception of one animal that tested positive to the PC-HP peptide cocktail at week 7 post vaccination (Table 6). In contrast, more frequent responses to the defined antigens were observed in animals vaccinated via the intramuscular route, where all six animals tested positive to E/C, PC-EC and PC-HP at least on one occasion post vaccination (Table 6). Responses to the E/C peptide cocktail were first seen 2 weeks post vaccination, which persisted throughout the course of the experiment in all animals. Similar results were seen with the PC-EC and PC-HP peptide cocktails, although the frequencies of response were lower at some time points when compared to the E/C cocktail. In contrast, only one animal tested positive to the Rv3615c peptide cocktail (Table 6).
Table 6. IFN-γ responses in vaccinated cattle using defined antigens
In summary, the results of Task 3.2 indicate that oral delivery of the inactivated M. bovis strain 1403 did not compromise diagnostic skin tests using defined antigens. Intramuscular vaccination resulted only in low responses in 1/6 animals. Although oral immunization also did not compromise IGRA diagnostic tests that utilize defined antigens E/C, Rv3615c and PC-EC), these tests were compromised when the vaccine was given via the intramuscular route.
Deviations from the work programme, and corrective actions taken/suggested
There are no deviations from the work programme.
Task 3.3. Serological tests in pigs using LFD
Task Leader: ISS (evaluate the feasibility of sero-dominant antigen MPB83 in IGRA test)
Other Participants: NEIKER will provided the vaccine, PRIONICS and INGENASA provided serological tests using MPB70 and MPB83 as antigens on the LFD-Comb platform for evaluation in pigs, ISS evaluated the feasibility of sero-dominant antigen MPB83 in IGRA test and VACUNEK analyzed and compare results with its own proprietary basic ELISA kit).
Overall summary of work progress and achievements
One of the aims of this task was to develop new one-step assays able to detect antibodies in the field based on the Lateral-Flow Technology. This assays works with low sample volumes, can be performed rapid (the entire test procedure is completed in 12 minutes) and easily without special equipment directly on the field and can provide a useful tool in situations where laboratory support and skilled personnel are limited.
During this reporting period, INGENASA has worked in the development and evaluation of the LF assay based on the recombinant protein MPB83, to be used in the field with serum/blood samples from pigs and wild board. The assay has been validated in the laboratory with samples from Partners 5 and 7. Additionally, INGENASA has already produced a big batch that will be distributed to the partners for further validation.
Summary of significant results
The protein target MPB83, necessary for the subsequent assay development, has been successfully expressed and purified.
A LFD (lateral flow device) prototype for specific anti-TB antibodies detection has been designed and developed.
A protocol for performing the rapid assays has been established.
A first validation of the rapid assays, using experimental and field sera has been carried out.
A big batch has been produced.
Introduction
The LFD for TB antibody detection is based on the recombinant MPB83 protein of Mycobacterium bovis. The first step was the expression and purification of these targets in order to use them as capture reagent on the nitrocellulose and labelled with latex particles to use as signal generator. Once the proteins were expressed and characterised, the lateral flow device was designed using a double recognition (DR) approach, where the antibody present in the sample is bound twice to the same antigen, the MPB83 protein in this case. If the tested sample contains specific antibodies, they will be captured firstly by the latex particles labelled with MPB83 and secondly by the MPB83 immobilized on the nitrocellulose membrane giving as a result the appearance of a coloured line on the membrane. In addition, green latex microparticles coated with a control protein and a second line created above the test line, by the immobilization of anti-control protein antibodies, was used as a control of test.
Description of work
MPB83 has been expressed in the E.coli system. The protein was assayed to found the optimal conditions for the assay, that is, the optimal concentration of MPB83 protein adsorbed on the nitrocellulose membrane and the best conditions to conjugate the proteins to the latex particles. Moreover, in order to establish the best conditions to perform the assay different parameters such as sera dilutions, running buffers, kind and sizes of membranes, latex... were tested to determine the accuracy of the assay. It is important to remark that the present test is a qualitative assay, resulting in a positive or negative decision.
In order to test the new LFD with blood, a spike in test was performed by adding positive and negative serum samples into blood from pig.
The schematic diagram of the LFD is showed in Figure 4. It is based on a direct immunoassay called Double Recognition antibody (DR) with two differentiated areas:
1. Sample window: it contains the latex particles dried on the sample pad: red microspheres covalently linked to MPB83 protein and green microspheres are used as test control.
2. Result window: it contains a nitrocellulose membrane with two lines printed: the test line formed by the MPB83 protein and the control formed by a specific MAb for the control protein.
Figure 4. Schematic diagram of the LFD developed for the task 3.3.
To carry out the test, 150 μl of 1/50 dilution of sample (serum or blood) were added to the sample pad. To carry out the test, 150 μl of 1/50 dilution of sample (serum or blood) were added to the sample pad followed by 4 drops of running buffer. The specific antibodies present in sample react with the latex particles coated with MPB83. This complex flows through the membrane and will react with the immobilized MPB83 protein on the membrane, making the test line visible. Interpretation of results should be done 12 minutes after the addition of the sample. A positive result for TB antibodies is recorded for samples with red and green lines visible. In the case of negative samples, only a green line will be show up. If no green line appears, the test will be invalid. No results must be considered after 12 minutes.
Figure 5. Interpretation of results after adding the porcine serum sample to the LFD developed.
Results
In order to determine the diagnostic sensitivity and specificity in pigs and wild boards, two panels of sera had been evaluated. On the one hand, 30 serum samples of negrody pigs from Partner 6, and 181 negative pig sera from farms free of TB. The results of the evaluation of these serum samples on the LF devices are shown in the next table:
TB-LFD
Positive pig sera (negrody pig)
30
26
Negative pig sera
181
174
Table 7. Results to determine the diagnostic sensibity and specity
Of the 30 TB positive samples, there were 4 false negatives from the LFA resulted in a sensitivity value of 86.6 %. Among the negative samples, 7 were tested as false positives by using LFA, resulted a 96% of specificity.
The second panel of sera consists of 113 wild board positive and 132 negative sera for TB. All these sera were provided by Partner 5. All these sera were also tested by the DR-ELISA developed during the project, and this technique was considered the reference technique. One hundred twelve of 113 positive sera were positive for the TB line resulted a sensitive value of 99.1%, and 132 of the 133 negative sera were negative, resulted a specificity value of 99.2%.
ELISA DR
TB-DR
Positive
113
112
Negative
132
133
Table 8.
Even though more samples need to be tested, these preliminary results suggest that the developed LFD provides a reliable method for rapid detection of TB antibodies in pig and wild board. INGENASA has currently available a big batch of LFDs that could be sent to others Partners.
Perspectives
Given the obtained results so far with this LFD are very promising, the next step will be the evaluation of the test by the other project partners using samples from real or experimental infections.
Deviations from Annex I
The development and validation of the LFD for TB antibodies detection in pigs, has been a hard and long work. For that reason, the evaluation of the test for other Project Partners is delayed. However, currently a batch is available for evaluation and the collaboration among partners will continue.
Task 3.4. Multi-species serological assay
Task Leader: INGENASA. MPB83-DR-ELISA Validation and Serological studies.
Other Participants: NEIKER provide the sera of TB status-defined cattle, DEFRA (supply sera from M. bovis infected cattle), PRIONICS will evaluate the suitability of a novel serologic test using MPB70/MPB83 antigens on the LFD-Comb platform for detection of TB specific immune response in different hosts. The LFD-Comb platform combines 8 single LFD tests in a user-friendly comb shape format to allow efficient testing of a large number of samples with a very rapid turn-around time. In addition with the use of a proteinA/G conjugate, the test is capable of detecting antibody classes from multiple species. UCLM will supply sera from wild boar of known infection status, VACUNEK will analyze and compare results with its own proprietary basic ELISA kit and VISAVET will supply sera samples to INGENASA from different hosts (domestic, wildlife, zoo animals, peridomestic) for validation of the serological test.
Task Objectives
The major aim of this task was to conduct the MPB83-DR-ELISA validation and to perform Serological studies. A double recognition ELISA (DR-ELISA) based on the recombinant MPB-83 antigen expressed in the baculovirus system, was used to perform serological studies in the animal species studied (wild board, pigs, feral pigs, cattle...) in order to establish and validate a DIVA assay that allows the differentiation between vaccinated and infected animals. The MPB83-DR-ELISA, developed during the EU project TB-STEP, presents the capability of detecting antibodies against the MPB83 protein with high sensitivity and specificity in several species, therefore, the same assay was used in all cases.
In parallel, using the same recombinant antigen and the same strategy of double recognition, a new approach has been developed based on Lateral-flow devices (LFDs) technology. This represents a well-established technology appropriate for use in a wide variety of point-of-care (POC) or field-use applications. The benefits of LFDs include: 1) User-friendly format. 2) Very short time to generate a test result. 3) Long-term stability over a wide range of climates. 4) Relatively inexpensive to make. These features make strip tests ideal for applications such as home testing, rapid point of care testing, and testing in the field.
In the second period of the project, Partner 7 (INGENASA) has continued working in the evaluation of the multi-specie MPB83-DR-ELISA using samples from different species. As an example, a panel of 47 fallow deer serum samples provided by Partner 5 (UCLM) was tested for the first time with the DR-ELISA prototype. Out of the 47 animals, 13 presented clinical signs and were slaughtered and necropsied, detecting lesions in 10 of them. Of the 10 samples considered positives, there were 2 false negative from the ELISA, resulted in a sensitivity of 80%. Among the 37 negative samples, one was tested as false positive by using the ELISA, resulting in a specificity of 97.3%. These results indicate that the MPB83-DR ELISA is also suitable for this species and therefore it could be a useful tool for the control of the disease in fallow deer.
Results
A double recognition ELISA (DR-ELISA) based on the MPB83 was developed under the TB-STEP project (FP7-KBBE-2007). This assay uses the recombinant protein MPB83 both as antigen coated in the ELISA plate and as conjugated to recognize the antibodies bounded to the coated MPB83. This assay doesn’t use any anti-specie antibody and for that reason could be used with sera from any specie. The assay is the same in all cases with some slight modifications in the sample or in the conjugate dilutions.
To validate the DR-ELISA with sera from different species, UCM has provided INGENASA with samples from zoo animals. A total of 170 sera samples from, Capuchin’s monkey, Yellow armadillo, Meerkat, Patagonian’s sea lion, Californian’s sea lion, Asiatic’s elephant, Watussi, Porcupine, Seal, Oystercatcher, Chimpanzee etc, have been analysed by DR-ELISA.
Most of them have been tested twice and in duplicate by DR-ELISA to study the presence of antibodies against the MPB83, the others have been analysed only once due to low volume available. Positive results were only obtained in 13 out of 170 sera. Thirty seven out of these 170 sera have been checked by TB culture isolation (Ctbc). The comparison between both assays (DR-ELISA and culture isolation) is summarized in the next table. The qualitative results (positive/ negative) showed a 75% of agrement between them (κ=0.4064).
Table 9. Contingency table for bacterial isolation and DR ELISA results with Zoo Animals
Moreover, sera from pets (dogs, cats and guinea pigs) and farm animals (goats and pigs) have been also studied. Seven sera from cats and 9 sera from dogs were collected and tested. Only two dogs were postitive by the DR-ELISA. In the case of the guinea pigs, 21 samples out of 58 were positives. All these results, have to be checked by other techniques.
Additionally, Partner 8 (UCM) has collected sera from 505 goats. All of them were analysed by the DR-ELISA, detecting 300 positives. In order to study the accuracy of these results, it has been carried out their comparison with other studies (INF, TB culture isolation...) which have been carried out by Partner 8. When the results of TB culture isolation from 168 studied samples, were compared with the DR-ELISA, a level of SN and SP of 86% and 87% respectively were observed. If TB culture isolation and/or lesion were compare with the DR-ELISA, the SN increase up to 90% (data not shown).
Table 10. TB Ab response by DR-ELISA versus TB culture isolation in goats.
The validation of the DR-ELISA was also carried out with serum samples from badgers. Sixty one samples were provided by Partner 2 (APHA):
• 36 sera from experimentally TB-infected badgers
• 14 sera from known TB-negative badgers
• 11 samples from a heat-inactivated M.bovis vaccine trial (6 from vaccinated animals)
The results showed that all infected animals presented Ab against the MPB83 protein that were detected in the DR-ELISA. Besides, it is important to mention that the vaccinated animals did not have specific antibodies against TB. The results are shown in the following Figure.
Figure 6. TB Ab response in badgers by DR-ELISA
Cattle serum samples from the vaccination experiment carried out by Partner 4 (APHA) described in Task 3.1 were also analysed by DR-ELISA. Briefly, 6 calves (aged 5-7 months) were vaccinated with 106-107 CFU of bacteria by either oral or intramuscular route according to the supplier’s guidelines (Neiker). Similarly to the SIT or SICCT results obtained by Partner 4,in animals vaccinated by the oral route, no positive Ab response were observed by DR-ELISA. By the contrary, all six animals vaccinated by intramuscular route showed positive response after 2 weeks post-vaccination. (Figure 7).
Figure 7. TB Ab response in cattle by DR-ELISA. Vaccinated animals by oral route (blue) or intramuscular route (red)
These last results, with serum from badger and cattle, are based on experimental animals, therefore, field samples will have to be tested for a better validation of the assay.
Finally, a panel of 198 serum samples from deer provided by Partner 5 (UCLM) has also been tested by the DR-ELISA. In contrast with the results obtained with samples from other animal species, in this case, the level of SN it is not as high. Preliminary results show a 94% of SP but only a 50% of SN. The same happens when field sera from cattle are used. During the next period of the project, INGENASA will work on the improvement of the assay with these kind of samples.
Overall summary of work progress.
Work progress on this deliverable has led to the recommendation that the vaccine could be used to vaccinate wildlife in baited form in conjunction with the following in cattle: (i) continuation of existing tuberculin skin testing or novel skin test formats based on defined antigens; (ii) the use of IGRA tests utilizing tuberculin or defined antigens; (iii) the use of the serology MPB83-DR-ELISA. However, we applied only single doses of this vaccine to cattle, and we therefore recommend further studies sensitising cattle with multiple doses and by repeat administration to confirm our findings. We do not recommend using this vaccine in cattle via the systemic route in control scenarios combining vaccination with test and slaughter. We therefore recommend to evaluate the effectiveness of this vaccine in cattle when given via the oral route should its application to cattle be considered.
WP4: Integrated field TB control in wild boar (IREC/UCLM)
Summary of progress towards objectives
The focus of the task is on understanding single versus integrated control. We assess the combined effect of vaccination and culling strategies in the context of integrated disease control, considering all available tools.
This task is completed. The main field studies have already been described in the deliverables on vaccination (4.1) and on test and cull (4.2). Some additional field studies have been carried out, including a test and cull and vaccination trial on a wild boar farm. This report focuses on evaluating and discussing integrated TB control strategies in wild boar and at the wild boar – cattle interface.
Regarding vaccination, laboratory evidence and field data available to date indicate a significant reduction of lesion scores and TB prevalence in vaccinated wild boar (see deliverable 4.1). An additional experiment carried out in a wild boar farm and hunting estate showed that vaccination reduces the prevalence of TB-compatible lesions by 75% in vaccinated wild boar as compared to non-vaccinated controls.
The selective culling attempt carried out in WildTBvac, despite of huge investments in terms of facilities and effort, failed to achieve a significant reduction in TB prevalence (see D.4.2). Hence, there is no obvious advantage in combining selective culling with vaccination.
By contrast, it is known that random (i.e. non-targeted) wild boar culling reduced TB prevalence between 21-48% (Boadella et al. 2012). Reducing the population size will reduce densities and lower total costs for TB control. Moreover, random culling may help to achieve an initial reduction in prevalence because of the density-dependent effect on infection transmission and because of the lower mean age of the surviving population. This might facilitate vaccination strategies (Boadella et al.2012; Anderson et al. 2013).
Task 4.1 Controlled field assessment of oral vaccination in wild boar
Task Leader: UCLM (bait production and deployment, field sampling and gene expression response).
Other Participants: NEIKER and VACUNEK (vaccine production and adaptation for field testing), DEFRA (supplies consultency and reagents), INGENASA (serology to assess TB and significant co-infections), VISAVET (collaborate in the field studies mainly in laboratory diagnosis (culture and IGRA)), PRIONICS supply peptide cocktails PC-EC and PC-HP based on the antigens ESAT6 and CFP10 for stimulation of blood for IGRA.
Task Objectives
This task was bait production and deployment, field sampling and gene expression response. A controlled and replicated design allowed (i) describing the pathology and culture score and (ii) measuring the serum antibody and gene expression response of field vaccinated wild boar piglets as compared to unvaccinated controls. This yielded an assessment of the % change in these indicators as compared to data obtained in captivity (Ballesteros et al. 2009, Garrido et al. 2011). This experiment was be performed on a 10,000 ha study area split into two parts.
Results
Two vaccine candidates weredeployed, on about 5,000 ha each: BCG (live vaccine) and heat-inactivated M. bovis (inactivated vaccine, IV). Both BCG and IV have shown protection against challenge with M. bovis under laboratory conditions (Gortazar et al. 2014, Beltrán-Beck et al. 2014a).
Safe and specific vaccine deployment is a key concern of oral vaccination strategies. In addition, confirmation of bait uptake (i.e. use of marked baits) is needed to generate sound scientific data. In the wild boar field vaccination experiments, safety and specificity were confirmed through camera trap surveys and analyses of target and non-target host tissues for BCG (Beltrán-Beck et al. 2014b). Furthermore, daily bait deployment at dusk and collection of non-consumed baits immediately after dawn improved bait specificity and limited BCG inactivation due to high environmental temperatures. However, this procedure is labor-intensive and could be avoided by deploying only the IV.
Assessing vaccination efficacy under field conditions is challenging. In the ongoing wild boar vaccination field trials, vaccination sites were purposefully selected away from cattle farms, since risks of cattle contamination with live BCG could not be excluded a priori. Hence, no results will become available in terms of reductions in cattle TB breakdowns. This will require further experiments on and around TB-positive cattle farms. However, effects of wild boar vaccination are measured in the wild boar target host. This can be tested at the individual level (piglets with and without biomarker) and at the population scale (treatment sites before and after; treatment sites vs. controls) and preliminary information regarding lesion and culture scores in piglets is encouraging for the IV vaccine (Díez-Delgado et al., in prep.).
Figure 8 Summary of field results
Partner 7 (INGENASA) has evaluated the Ab response of 22 wild board after vaccination in experimental condition with inactivated vaccine by the oral route. All sera were tested by I-ELISA and DR-ELISA. The results are shown in the following Figure. As may be seen, the results are very similar by using both assays. No positive Ab response were observed after vaccination by oral route. By the contrary, all animals showed positive response after challenge. (Figure 9)
Figure 9 Results of sera tested by I-ELISA and DR-ELISA
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
Next steps in the next period
No accurate cost estimations are currently available for this field experiment. Also, results are currently available for only two bait deployment years, and this experiment is aimed at lasting at least four years. Finally, the newly developed vaccine and selective baiting tools deserve knowledge transfer from the lab to the market, and attainment of regulatory approval for distribution in the field.
Task 4.2 Test and cull strategies in wild boar captured under field conditions
Task Leader: UCLM (wild boar capturing, handling and field testing, and field sampling).
Other Participants: INGENASA (serology to assess TB and significant co-infections), PRIONICS (to supply its serologic LFD-Comb assay for laboratory diagnosis) and VISAVET (to perform laboratory diagnosis of culled animals (microbiological culture and IGRA)).
Task Objectives
To make use of existing and new plate and lateral-flow antibody tests (Aurtenetxe et al. 2008, Boadella et al. 2011a) for test and cull strategies in wild boar captured under field conditions at high TB prevalence sites. Test-negative wild boar were tested, tagged and released at the site of capture if negative. Test-positive animals were culled and submitted to further analysis. TB prevalence and wild boar abundance was continuously monitored to assess the impact of selective culling (Boadella et al. 2011b).
Results
We assessed the suitability of targeted culling as a means for tuberculosis (TB) control in intensely managed wild boar hunting estates. The 6,000 ha large study area included one capture site, one control site, and two release sites. Each site was fenced. In summer 2012, 2013 and 2014, a total of 929 wild boar were live-captured by means of cage traps and corral traps. All wild boar were micro-chipped and tested immediately after capture for antibodies against the Mycobacterium tuberculosis complex (MTC) with an animal side lateral flow ELISA. Wild boar were released according to their TB status: Seropositive individuals into the release sites (hunted after summer), and seronegative individuals back into the capture area. The annual summer seroprevalence of antibodies against the MTC declined significantly in live-captured wild boar piglets from the capture site, from 43.86% in 2012 to 26.74% in 2013 (39% reduction in seroprevalence). Autumn-winter MTC infection prevalence was calculated based on culture results of hunter-harvested wild boar. No significant changes between hunting seasons were recorded in the capture site and in the control site, and prevalence trends through time were similar in both sites. However, in the release sites the seasonal autumn-winter MTC infection prevalence increased significantly in non-yearling hunter-harvested wild boar from 40.54% in 2011-2012 to 64.29% in 2012-2013 (59% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted culling as a single tool, failed to reduce TB prevalence as compared to the control site. The knowledge generated will help to improve wild boar and TB management in semi-intensive settings.
Figure 10 TB infection prevalence under a targeted culling strategy troughout time
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
Task 4.3 Assessment of the combined effect of previous tasks
Task Leader: UCLM
Other Participants: VISAVET
Task Objectives
The focus of the task is on modelling of single versus integrated control. We will use modelling to assess the combined effect of both strategies, based on the rationale that reducing the population will reduce densities and lower total costs for TB control (Boadella et al.2012). Specifically, VISAVET will participate in the statistical analysis of the data obtained in tasks 3.1 and 3.2.
Progress towards task objectives for the reporting period
This task is in good progress. Raw datasets on culling and on selective culling are available, while data on field vaccination are already available for two years. Data for the third year of vaccination will become available in May 2015 (when the cultures are deemed to be finished).
Results
Figure 11. Flow chart of the available disease control options and results assessment in diseases shared with wildlife
Vaccination
Results of the field vaccination experiment (oral vaccination) after two consecutive years are detailed in D.4.1. Briefly, while the heat-inactivated M. bovis conferred some protection, BCG apparently failed to achieve protection.
In the experiment with parenteral heat-inactivated M. bovis, promising results were obtained. Piglet vaccination with heat-inactivated M. bovis started in 2012. A total of 362 wild boar piglets were vaccinated in the farm in 2012 and 2013. No adverse local or systemic reactions were detected after vaccination by visual inspection.
From November 2014 to December 2015, 173 three to four year old wild boar, which had been vaccinated as piglets, were hunted. In the same period, 144 additional hunted wild boar had not been vaccinated.
Parenteral vaccine efficacy was measured by the presence of TB compatible lesions (TBL). The TBL prevalence was 2.7% and 10.9% for vaccinated and control wild boar, respectively (Chi square= 6.69 1 d.f. p= 0.009; Figure 2). Thus, parenteral vaccination reduced lesion prevalence by 74.7%. Among those animals with TBL, no difference in the lesion score was found between vaccinated and non-vaccinated ones. No adverse reactions to the vaccine were observed at postmortem examination.
Figure 12. .- Prevalence of TB-compatible lesions (TBL) in control and parenterally vaccinated wild boar.
Culling and test & cull strategies
Regarding vaccination, laboratory evidence and field data available to date indicate a significant reduction of lesion scores and TB prevalence in vaccinated wild boar (see deliverable 4.1). An additional experiment carried out in a wild boar farm and hunting estate showed that vaccination reduces the prevalence of TB-compatible lesions by 75% in vaccinated wild boar as compared to non-vaccinated controls.
The selective culling attempt carried out in WildTBvac, despite huge investments in terms of facilities and effort, failed to achieve a significant reduction in TB prevalence (see D.4.2). Hence, there is no obvious advantage in combining selective culling with vaccination.
By contrast, it is known that random (i.e. non-targeted) wild boar culling reduced TB prevalence between 21-48% (Boadella et al. 2012). Reducing the population size will reduce densities and lower total costs for TB control. Moreover, random culling may help to achieve an initial reduction in prevalence because of the density-dependent effect on infection transmission and because of the lower mean age of the surviving population. This might facilitate vaccination strategies (Boadella et al.2012; Anderson et al. 2013). Another interesting observation is that the highest TB prevalence ever recorded (close to 90%) occurs in a protected area (Doñana National Park, Huelva, Spain), where no hunting takes place and population control is usually limited to disease surveillance purposes.
Integrated disease control strategies and their practicality
Table 10 presents the top twenty management interventions, shown in order of perceived effectiveness, as determined by the expert panel.
The most effective intervention identified based on expert opinion was banning supplemental feeding of game species. However, this option was not considered practical by stakeholders. The most effective and practical interventions were the separation of wildlife and livestock access to waterholes, testing cattle every 3 months on farms with a recent positive TB case and removing gut-piles from the land after hunting events. Although all three of these options were well supported, each stakeholder group supported different approaches more strongly, suggesting that it might be effective to promote different disease management contributions in different stakeholder communities. This integrated approach contributes to the identification of the optimum combination of management tools that can be delivered effectively.
Table 11. The 20 management interventions regarding TB control at the wildlife – livestock interface, shown in order of perceived effectiveness as determined by the expert panel (Cowie et al. 2015).
Discussion and conclusions
Assessing the efficacy of single disease control tools, such as vaccination alone or targeted culling alone, remains challenging under natural conditions. Assessing the combined effect of these two tools is even more difficult. However, wildTBvac allowed moving several steps ahead in our understanding of several disease control tools regarding the wild boar – M. bovis binomium.
Regarding vaccination, while the outcome of the four-year field oral vaccination experiment is still incomplete (last animals being sampled in 2016), some positive indications exist regarding the heat-inactivated M. bovis vaccine, as reported in D.4.1. Additionally, the smaller experiment on parenteral vaccination of farmed wild boar piglets with the same product indicated a promising 75% protection against TBL (Figure 2).
Regarding culling, while it is known that random culling reduces TB prevalence in wild boar (Boadella et al. 2012), the selective culling experiment carried out in wildTBvac failed to achieve a significant effect at the population level (see deliverable 4.2). However, under farm conditions, the targeted culling of all adult wild boar testing positive to MTC antibodies by ELISA probably contributed to keep the breeding facilities TB-free.
Given the abovementioned results, we suggest that in more or less natural field settings, culling (random culling) may contribute to reduce wild boar TB prevalence prior to vaccination. We also suggest that, while selective culling failed under field conditions, it might be an interesting tool in wild boar farms, were access and testing of every single individual is granted. In conclusion, culling might be combined to improve vaccination efficacy, but the specific tool (random or targeted) will depend on the setting.
Various general inferences can be made. First, all options for disease control at the wildlife-livestock interface, including those of no intervention, need to be considered, either individually or combined. Second, combining several disease control tools in integrated strategies is likely to reduce the cost and effort required for disease control. Integrated strategies are also preferred since no single control measure is universally applicable. Third, the success of disease control in wildlife depends on many factors, including (a) the single or multi-host nature and other characteristics of the pathogen, (b) the availability of suitable diagnostic tools, (c) the characteristics of the wildlife host(s), (d) the geographical range of the pathogen/reservoir (improved control in isolated versus continuous populations) and the scale of the control effort (large-scale longitudinal programs are better), (e) the attitude of the stakeholders involved (highly dependent on their education and communication provided to them)(Cowie et al. 2015, Gortázar et al. 2015).
Deviations from the work programme, and corrective actions taken/suggested
There are no significant deviations from the work programme.
WP5: Evaluation of efficacy of heat-inactivated Mycobacterium bovis as oral vaccine in domestic pigs against tuberculosis (ISS)
Summary of progress towards objectives
This task is in an advanced stage., but is not completed. Pigs have been vaccinated in 7 groups in order to detect infection by M. bovis and to differenciate it from other closely related mycobacterial infections. Also pigs in two farms with a history of natural TB infection have been vaccinated. Even though the whole follow-up has not been completed due to a delay in obtaining the permits for the experiment, the works are progressing and data will be available in about 12 weeks after the official end of the project.
Task 5.1 Assessment of immune status of vaccinated piglets.
Task Leader: ISS coordinates the activities, chooses the sows and monitors the animals.
Other Participants: VISAVET set up the protocols for detection in faeces and saliva in samples obtained from experimentally infected pigs; DEFRA supplids reagents for skin test and IGRA assays and INGENASA will supplied the diagnostic test prototypes to the participating partners.
Task Objectives
Farms with semi free-roaming system were selected in Sicily on the light of a high degree of prevalence for tuberculosis due to Mycobacterium bovis. Two of them were chosen and successively, 8-10 tuberculosis-free sows were selected in each, according to the results of IFN-gamma test as described by Pesciaroli (Pesciaroli et al., 2012). 20-25 Piglets from those sows were vaccinated as reported elsewhere (Garrido et al., 2011), just after weaning and a similar number of age-matched piglets were kept as unvaccinated controls. Before vaccination and at 2-month intervals, from each animal, blood was collected and skin test was performed to assess the immune response.
Overall summary of work progress
During this reporting period, Partner 7 (INGENASA) has continued working on the evaluation of the pig sera samples from the vaccination/challenge experiments carried out at VISAVET (Partner 8) in the previous period. Briefly, in the experiment 1, Seven groups of 4 pigs each were vaccinated, infected or left as controls (see table). The vaccine consisted of heat-inactivated M. bovis by oral route.
Group 1
Control group: No vaccination/No infection
Group 2
Machuquillo vaccinated: D0 Vaccination/No infection
Group 3
BCG Vaccinated: D0 Vaccination/No infection
Group 4
MAH Infected: No vaccination/D0 Infection MAH
Group 5
MAA Infected: No vaccination/D0 Infection MAA
Group 6
Mbovis/MAA Infected :No vaccination/D0 Infection Mbovis-MAA
Group 7
Mbovis infected: No vaccination/D0 Infection Mbovis
Table 12. Animal groups in experiment 1.
Initially, the antibody response against M. bovis was analyzed by INGEZIM TB DR ELISA (DR-ELISA) and INGEZIM TB indirect ELISA (i-ELISA). As it has been reported in WP3, INGENASA has worked on the final development and validation of a lateral-flow assay (LFA) to detect Ab against M.bovis in serum/blood samples from pigs and wild board. This new LFA has been used to evaluate those sera. The results were compared with those obtained by ELISA.
The results showed that the correlation of the LFA with the two ELISAs is very high. There are only two differences in samples of the 30 days post vaccination/infection: One sample from the group of animals infected with M. bovis that was negative by LFA, negative by indirect ELISA, and positive by DR-ELISA. In this case, the positive results was very close to the cut-off of the assay. By the contrary, there was one sample positive by LFA and DR-ELISA, but negative by i-ELISA. The next tables show the summary of the LFA results compared with ELISAs.
Table 13. Summary of the LFA results compared with ELISAs.
These results correspond to a sensitivity value of 92.9 and 92.3 % for DR-ELISA and i-ELISA respectively and a specificity value of 98.3 for both assays.
In the Experiment 2, two groups of nine animals were studied. The first was the control and it did not receive the vaccine and the other was vaccinated day 0 and 30. All animals were infected with M. bovis at day 60 and finally were bled at day 90 and 120.
Group 1
Control group: d0 No Vaccine /d30 No boost/d60 Infection
Group 2
Machuquillo vaccinated: d0 Vaccination/ d30 boost/d60 infection
Table 14. Animal Groups in experiment 2
The correlation between the DR-ELISA and LFA in this experiment was also high. The summary is showed in the next table:
Table 15. Correlation between the DR-ELISA and LFA in experiment 2
The differences found have been in samples close to the cut-off of the ELISA assay. This data resulted in a sensitivity value of 90.6 % and a specificity value of 93.6 %.
As conclusion, the data obtained with the LFA showed a high sensitivity and specificity, although a little bit lower that the ELISAs. However this pen-side test offers advantages that should be taken into consideration: it is rapid, economic and simple-to use diagnostic tool, since it does not require any kind of instrumentation and the results is interpreted visually. Further validation in different scenarios will be carried out in the next future.
Task 5.2 M. bovis and corresponding lesions assessment
Task Leader: ISS vaccination of animals, monitoring and post mortem analysis
Other Participants: NEIKER and VACUNEK provide ready-to-use- vaccine; UCLM (provides expertise in
assessing the response to vaccination),
Task Objectives
Vaccinated and unvaccinated piglets will be maintained in their natural environment to guarantee a natural
exposure to the pathogens. Pigs will be slaughtered at 15 months of age and a complete post mortem analysis
will be performed, collecting oral, thoracic and abdominal lymph nodes, along with liver, spleen, lungs and
intestines to assess the presence and the severity of lesions. According to standard microbiological tests, and
the presence of M. bovis will be assessed in the different organs.
Results
According to the results obtained in the first period of the project (M18) we chose 2 free roaming pig farms considered endemic for tuberculosis due to M.bovis. Those two farms have around 200 adults and are farrow-to-finish.
We selected pigs within 5 months of age to be enrolled in the trial. In the first farm we included 68 pigs, in the second farm 63 with an age distribution reported in the following figure.
Figure 13 Age distribution per farm
Safety of the vaccine.
Safety was previously verified in mice. Mice were inoculated either by parenteral or oral route and were monitored for 15 days to exclude any deleterious effect. Following vaccine administration mice did not show any sign and the vaccine (data not shown).
Selected pigs were individually identified, bled and then vaccinated. In the first farm, 31 out 68 pigs were intramuscularly vaccinated and the remaining were kept unvaccinated. In the second farm 43 out 63 pigs were vaccinated and the remaining were kept unvaccinated. Following vaccination, pigs did not show any adverse reaction corroborating the preliminary results previously obtained in mice.
Blood samples taken before vaccination was then used to verify the immune status of pigs enrolled in the trial. IGRA results are reported in the following tables.
IGRA
negative
positive
unvaccinated
25
6
vaccinated
13
24
Table 16. IGRA results in FARM 1
IGRA
negative
positive
unvaccinated
20
0
vaccinated
43
0
Table 17. IGRA results in FARM 2
These results showed that in farm 1 the circulation of M.bovis is likely to involve pigs in the first stage of their life and they become infected very early. It is our opinion that the use of vaccine also in IGRA positive animals could give the project an added value because we could be able to assess the effect of vaccination in conditions that can naturally occur in field conditions.
Ninety days after vaccination, blood samples were collected in vaccinated and unvaccinated pigs. Blood samples were then stimulated with bovine and avium PPDs. Interferon gamma was then quantified by using a commercial ELISA kit (R&D Systems) according to Pesciaroli et al., 2012.
Results are depicted in the following tables.
In the farm 1 32 pigs were randomly selected (they are free ranging therefore the capture is not controlled). Of those 15 were unvaccinated and 17 vaccinated. The 15 unvaccinated pigs resulted all negative except one that gave positive results to the second IGRA. Nine out 17 vaccinated pigs resulted negative at IGRA performed before vaccination. Six of those resulted still negative at the second IGRA while three of those became positive. The remaining vaccinated pigs (8 out 17) that were already positive at IGRA performed before vaccination, resulted still positive at IGRA performed after vaccination.
Unvaccinated pigs (15)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
14
1
positive
0
0
Vaccinated pigs (17)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
6
3
positive
0
8
Table 18. IGRA results after vaccination in FARM 1
In the farm 2 57 pigs were randomly selected (they are free ranging therefore the capture is not controlled). Of those 15 were unvaccinated and 42 vaccinated. The 15 unvaccinated pigs resulted all negative except one that gave positive results to the second IGRA. All vaccinated pigs were negative at IGRA performed before vaccination Twenty-one of those resulted negative at IGRA performed after vaccination while 21 of those became positive.
Unvaccinated pigs (15)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
14
1
positive
0
0
Vaccinated pigs (42)
Post-vaccination IGRA (II)
Negative
Positive
Pre-vaccination IGRA (I)
negative
21
21
positive
0
0
Table 19. IGRA results after vaccination in FARM 2
Overall, from this field test, it is possible to infer important even not conclusive insights. First of all, we have confirmed that vaccine parenterally administered is well tolerated without any sign of suffer or distress in the recipient animals.
Secondarily, we ascertained that piglets that become positive to the second IGRA except in one case in farm 1 and 1 case in farm 2 were those previously vaccinated. This is unlikely correlated to a sensitisation to wild M. bovis rather to the induction of an immune response operated by the vaccine. This means that the vaccine is immunogenic and also that infection was not disseminated enough among the animals enrolled in the experiment. This is the reason why we could not kill the animals as planned because likely they were still uninfected. To overcome this problem, we decided to keep the animals alive to give more time to get the infection. At the endo of the follow up, twenty-four pigs were randomly selected from the two herds enrolled in the field test. At slaughter, a complete post-mortem analysis was performed.
Fourteen out 24 selected pigs were vaccinated while 10 out 24 selected pigs were unvaccinated control animals. As reported above, those pigs were previously bled at 90 days after vaccination and blood samples were collected and stimulated with bovine or avium PPDs. Interferon gamma was quantified by using a commercial ELISA kit (R&D Systems) according to Pesciaroli et al., 2012.
Previous IGRA for the slaughtered pigs:
IGRA
negative
positive
groups
vaccinated
7
7
controls
9
1
IGRA performed the day before the slaughtering:
IGRA
negative
positive
groups
vaccinated
3
11
controls
9
1
At post-mortem a complete analysis of organs and draining lymph nodes was done and results are depicted as follows:
Post mortem analysis
negative
positive
groups
vaccinated
3
11
controls
8
2
Slaughter analysis with the IGRA
IGRA
negative
Positive
Post mortem
positive
0
12(1)
negative
12
0
Three out 12 pigs resulted positive to IGRA for M.avium
Overall, the animals which resulted positive at slaughter showed classical signs of tuberculosis in one or more lymph nodes with, in some cases, also lesions in parenchimatous organs, such as liver, lung and kidneys, although bacterial isolatio and identification has not been done at this time. These results clearly suggest that the vaccination is not able to exert any significant effect in improving the pathological status of infected animals.
In order to ascertain that lesions are associated to the infection we correlated the immune status previously observed with the presence of lesions, irrespectively to the vaccination status, with the following results:
Post mortem analysis
negative
positive
IGRA
positive
1
7
negative
11
5
As expected, there is a high correlation between the IGRA negative animals and those without lesions, suggesting that, presumably, those animals are still uninfected. There is, in addition, a good degree of correlation between IGRA positive animals and those with lesions (infected).
At slaughter, the rate of animals with lesions was 78% among vaccinated animals and 20% among unvaccinated. These results are counter-intuitive and far from expected according to preliminary experiences, but were consistent with IGRA results and indicate that, at the time of submitting this report, vaccination did not just fail to protect, but seemed to favour the detection of infection in terms of gross lesions. Currently, the proof of concept that vaccination in domestic suids protects as efficiently as in wild suids and that was the main goal of this task has not been demonstrated in these settings and requires further histopathological and microbiological studies that should clarify the pathogenetic aspects of the vaccination-infection interaction.
Pesciaroli M, Russo M, Mazzone P, Aronica V, Fiasconaro M, Boniotti MB, Corneli S, Cagiola M, Pacciarini M, Di Marco V, Pasquali P. Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs. Vet Immunol Immunopathol. 2012 Aug 15;148(3-4):369-72.
WP6: Dissemination, exploitation and market preparation (INGENASA)
During the course of the project, the Consortium has evaluated and validated two different complementary products:
An oral TB vaccine for wild life animals that has been tested in pigs, wild boards and cattle.
A double recognition ELISA test, associated to the vaccine, based on the recombinant MPB83 (MPB83-DR-ELISA) that was developed during the EU project TB-STEP. This assay permits to detect antibodies (Ab) against M. bovis in serum and it is able to distinguish infected from vaccinated animals. The assay has been tested in swine, including wild and domestic pigs, and it is suitable for wild (badgers, camelids, zoo animals...) and domestic animals species. Moreover, an indirect ELISA for TB Ab detection in pigs has also been evaluated.
According to The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA Journal 2015;13(12):4329, 191 pp. doi:10.2903/j.efsa.2015.4329 the overall proportion of cattle herds infected with, or positive for, M. bovis remained very low in the EU (0.8% of the existing herds in the EU), although there is a heterogeneous distribution of M. bovis in Europe. The prevalence ranges from absence of infected/positive animals in many EU officially tuberculosis free status (OTF) regions, to a prevalence of 11.6% in the non-OTF regions of the United Kingdom (England, Northern-Ireland and Wales).
In the OTF regions, the proportion of herds infected with M. bovis decreased further to 0.011% in 2014. This can be partly explained by the inclusion of Hungary, in 2014, in the list of OTF European Countries Members States (MS). Hungary had 16,419 cattle herds and reported 3 infected ones in 2014. Also, in OTF countries, the number of herds infected with M. bovis reported was on average lower than in 2013.
Figure 14. Status of countries regarding bovine tuberculosis due to M. bovis, 2014
In the non-OTF regions, in 2013, the reported number of herds positive for M. bovis was, for most MS, was similar or lower than in 2014. However, an increase was noteworthy in Spain (1,526 in 2013 and 1,867 in 2014) and Ireland (4,640 in 2013 and 6,623 in 2014) while in the United Kingdom a decrease was reported (10,956 in 2013 to 10,172 in 2014). Overall, the reported proportion of herds positive for M. bovis in the non-OTF regions has slowly increased during the last years
Bovine tuberculosis is distributed worldwide, but control programs have eliminated or nearly eliminated this disease from domesticated animals in many countries. Nations currently classified as tuberculosis-free include Australia, Iceland, Denmark, Sweden, Norway, Finland, Austria, Switzerland, Luxembourg, Latvia, Slovakia, Lithuania, Estonia, the Czech Republic, Canada, Singapore, Jamaica, Barbados and Israel. Eradication programs are in progress in other European countries, Japan, New Zealand, the United States, Mexico, and some countries of Central and South America.
Figure 15. Number of bovine TB cases reported by country to OIE in 2011
Although cattle are the primary hosts for M. bovis, other domesticated and wild mammals can also be infected and isolations have been reported from sheep, goats, equines, camels, pig, wild board, dog, cats, felines including lions and tigers, badgers...
In 2014, 14 MS and two non-MS investigated animal species other than cattle for M. bovis. M. bovis was reported in 816 animals other than cattle: alpacas (34), badgers (218), bison (3), cat (24), deer (106), dog (1), goat (29), guinea pig (1), lamas (3), pet animal (1), pig (153), sheep (1), wild boar (219), wild animal (1) and zoo animal (2). Seventeen MS and two non-MS investigated animals for Mycobacterium species other than M. bovis. M. tuberculosis was reported in two pigs and 38 cattle and M. caprae was reported in 126 animals by four MS (Austria, Germany, Hungary and Spain): cattle (68), deer (10), goats (6), sheep (1) and wild boar (41).
Because the course of disease is slow, taking months or years to kill an infected animal, an animal can spread the disease to many others before the first clinical signs appears. Therefore, movement of undetected infected domestic animals and contact with infected wild animals are the major ways of spreading the disease.
Each year, the experts confer more epidemiological relevance to the wild reservoirs as a difficulty in the TB eradication Therefore, the new developed tools would help to create new and more effective plans to control and finally eradicate TB from domestic animals and consequently human TB.
Animal TB control and eradication programs management, are mainly designed and implemented by International Institutions (OIE) and National Animal Health Institutions. We have the advantage that important Institutions in this field are members of WildTBVac Consortium and, therefore, the developed products have been already tested and validated by them. This represents a great opportunity for innovative businesses [...]to find a niche on the market. Having the advisement and collaboration from the Partners of the Consortium, Partners 2 (VACUNEK) and 7 (INGENASA) have designed a business plan for a vaccine production plant and for the commercialization of the new diagnostic assays, respectively.
The TB diagnostic techniques most frequently used nowadays are intradermal tuberculin test or gamma interferon assay. This requires handling the wild animals, well-equipped laboratories and skilled personnel. Detection of specific antibodies is an important alternative tool to control the disease.
Partner 7 (INGENASA), produces and commercializes diagnostic kits under rigorous quality control rules (ISO 14001:2004 and ISO 9001:2008), for a big number of diseases of different species including, pigs, cattle, horses, dogs or cats. Partner 7 already has a broad distribution network across the word, with presence in more than 63 different countries and, therefore, products validated within this project are expected to be easily introduced into the market using resources and networks of the company. We sell our own products to end users, institutions, private companies Public Health Agencies, etc. We can also provide reagents in bulk to other kit manufacturers, or finished products to distributors.
The objectives of the plan will cover:
1.- Evaluation and quantification of the real weight of the wild animals in the epidemiology of TB in some key areas for each country, specially, where eradication plans have been less effective.
To do this, according to the Health Authorities:
The guidelines to make the sampling, number of specimens... will be provided.
The new serological assay will be used to know the prevalence of the infection within the wild population.
The convenience to go ahead with the following steps, will be evaluated according to the guidelines of the Health Authorities. When the estimated prevalence was considered significant enough to interfere with the bovine eradication programs, different solutions could be suggested:
a.- Establishment of a vaccination program, using the edible vaccine developed within the project.
b.- Monitoring of the prevalence of TB in wild animals in the controlled areas.
2.- After evaluation of the results of this plan on the bovine TB incidence, the establishment of the program in other areas will be considered.
The selection of markets will be done by following the priorities below:
Priority 1 (During the first 2 years). We would like to work in
Countries with long history and experience applying bovine TB eradication programs, without obtaining a final eradication + those countries in which the Partners of the Consortium could have interest and could support the advantage of using the ELISA test (Spain, Italy, England ...)
Priority 2 (From the third year and thereafter):
Countries with high bovine population + countries with bovine TB eradication programs going on (New Zealand, the United States , some countries in Europe, Central and South America...)
Income Projections
Although it is hard to estimate the provisional diagnostic market, trying to be very cautious, we have set our objectives of sales of diagnostics for the following 5 years. To prepare them, we established the following premises:
1.- Sales price per individual test will be 1,0 €
2.- On each area in which the system will be introduced, we will estimate a minimum number of 200 samples and a maximum of 1.000
3.- Once the program is implanted in one area, the monitoring will remain (with the same number of test) for at least 5 years.
In order to show the projected annual profit, an optimistic and a cautious estimation has been performed. A summary is showed in the following Table:
Year 1
Year 2
Year 3
Option 1
Option 2 (conservative)
Option 1
Option 2 (conservative)
Option 1
Option 2 (conservative)
Nº of countries
6
3
10
6
12
8
Nº areas/country
4
2
6
3
8
4
Nº test/year
1000
200
1000
200
1000
200
Objetive sales in €
24.000,00 €
1.200,00 €
60.000,00 €
3.600,00 €
96.000,00 €
6.400,00 €
Cumulative sales
24.000,00 €
1.200,00 €
84.000,00 €
4.800,00 €
180.000,00 €
11.200,00 €
Table 20. Projected annual profit
Regarding vaccine perspectives, a detailed business plan is provided in deliverable D6.64. Production projections are as follows:
Table 21. Quantification of number of doses and their derived production costs at Iberian market.
Vaccine doses requirements for Iberian demand through the six years of projections sum up over half million doses. Highest production needs per year reach 210.000 vaccine doses.
Table 22. Quantification of number of doses and their derived production costs at International market.
Vaccine doses requirements for European and American demand through the six years of projections sum up over 4 million doses. Maximum production needs per year reach 1.45 million vaccine doses.
Authorities’ successful plan for TB eradication could affect to the projections previously cited, so half million doses facilities will cover the highest demand for Iberian territory through the 6 year period as well as projections up to the third year of the project.
Potential Impact:
The problem of wild boar as TB maintenance hosts is particularly relevant in Spain and Portugal, but extends to several other EU countries (France has recently observed an association between wild boar TB seroprevalence and bovine TB outbreaks) and to Hawaii in the USA. Moreover, free ranging domestic pigs can also act as M. bovis maintenance hosts as was recently described in Italy (Di Marco et al. 2012). Therefore, initially we aim at two potential differentiated markets: 1) Wild boar or feral pigs, who include game producers and animal health or environment authorities in Europe and South America; 2) Domestic pigs, from both an industrial production and animal health authorities perspective in countries with a significant proportion of open-air bred pigs such as the Mediterranean countries and Hungary.
The potential economic benefits of this are quite high as such a wildlife vaccine/domestic diagnosis strategy that would not lead to false-positivity in domestic animals would avoid costs to the taxpayers of compensation payments and costs of increased testing and testing frequencies, as well as the direct economic losses to farmers due to decreased production, veterinary care, movement and trade restrictions and loss of product quality image. Further, it would enhance stakeholder acceptance and confidence in such a strategy, which are necessary requirements for the implication of any such strategy. Potential customers are in principle governments, public administrations and large pig farming companies (including public and private hunting grounds, national parks, etc.). Therefore, suitable customers are to be found both in A) government agencies, and B) in the private sector.
Vaccines developed in WILDTBVAC would be of interest for animal health authorities interested in livestock TB control. Interest towards vaccine and diagnostics research has already been expressed by colleagues from the US Department of Agriculture and from several agencies of EU Member States (MS) including France, Austria and Germany, and non-member states such as Switzerland. Vaccines and diagnostic tools would also be of interest for wildlife conservation agencies. Farmers, mainly those focused on high quality free-range production might greatly benefit from a tool that can greatly simplify the only expensive test-and-cull strategy applied to bovine TB that is currently being questioned by some medical researchers. The porcine sector is particularly relevant in Mediterranean EU MS, but other MS do also find pig TB and would probably be interested in diagnostics. The hunting sector, which is quite significant in some EU MS such as Spain and France, wild boar farmers/producers would be most interested in products developed by WILDTBVAC.
The three veterinary companies included in the partnership are in excellent position to spread the technology refined in this project and to take advantage from the market studies set as a specific outcome of this project.
The overall expected impact of WildTBVac outcomes are:
At an Activity level: a) Sustainable production and management of biological resources. Focusing on a targeted activity that addresses specific animal pest or disease that in addition has a direct relation with food security as well; b) Helping to implementation of the Action Plan against the rising threats from Anti Microbial Resistance (AMR). Although there is no direct relationship with AMR, as a general strategy, vaccination is aimed at reducing prevalence of infection without directly killing the pathogens, but by increasing resistance to infection. In this case doing this in susceptible animal species should decrease human exposure and therefore, the need for antibiotic treatment in human patients.
At an Area level: a) Socio-economic research and support policies. Providing tools needed by policy makers and other actors to support implementation of relevant strategies to avoid direct economic losses to farmers due to movement and trade restrictions.
At a Topic level: KBBE.2013.1.4-07 by bringing about results on three main issues addressed in the Call: a) Opportunity for innovative applications; b) Production of a dedicated business plan; c) Development of new patents.
List of Websites:
http://www.wildtbvac.eu