Final Report Summary - CSFV_GODIVA (Improve tools and strategies for the prevention and control of classical swine fever)
Executive summary:
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the World organization for animal health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability for oral application for wild boar and backyard pigs. The CSFV_GODIVA project aimed at improving the tools and strategies for the prevention and control of CSF for domestic pigs and wild boar. A new modified live CSF marker vaccine allowing differentiation between infected and vaccinated animals (DIVA) was developed. Immunization and challenge trials showed that the antibody titers after a single intramuscular or oral vaccination were stable for a minimum of 6 month and full protection from lethal challenge infection was observed.
Project context and objectives:
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the world organization for animal health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability to be applied orally for wild boar vaccination. Project CSFV_GODIVA aimed at improving the tools and strategies for the prevention and control of CSF, including the development of a new modified live CSF marker vaccine for domestic pigs and wild boar. The work performed under the CSFV_GODIVA was divided into 5 major tasks. The main objectives within each task are summarized below.
Task A: Recent developments in new generation live marker vaccines and molecular diagnostic tools
In several countries worldwide, CSF has been eradicated from the domestic pig population. Despite intensive efforts on the national and international level, complete eradication from the European Union (EU) has proven to be elusive, especially since wild boar populations in several European countries are endemically infected. The current control strategy within the EU is a strict stamping out strategy without prophylactic vaccination. Nevertheless, community legislation also foresees the possible use of emergency vaccination in domestic pigs as well as in wild boar. For vaccination of wild boar, the live C-strain vaccine has historically been used. The disadvantage of this vaccine is that no differentiation between CSF vaccinated and infected animals can be made. This causes serious complications for disease surveillance. To ease trade restrictions for domestic pigs, potent marker vaccines are required that allow reliable differentiation of infected from vaccinated animals. The currently available E2 subunit vaccines allow this differentiation but lack some properties that live vaccines exhibit e.g. fast and sound protection after single application, prevention of trans-placental infection, suitability for use via the oral application route. The aim of Task A was the final development of a live marker vaccine candidate, orally applicable to wild boar and intramuscularly applicable to domestic pigs. The availability of a safe and efficacious marker vaccine and its accompanying DIVA diagnostic assays provides a highly valuable tool that can be applied in control and eradication strategies and can accommodate for the use of marker vaccines as stipulated in EU directives.
Task B: Epidemiology
Although the experience with the implementation of emergency control measures during CSF outbreaks in the domestic pig population is increasing, the excessive killing of pigs and destruction of animal products occurring during such outbreaks remains a critical and ethical concern. Emergency vaccination to control CSF ('vaccination-to-live' strategy) could dramatically improve the situation, however requires an in-depth evaluation of its efficacy and potential further consequences before being considered to be a valid alternative. Due to the limited availability of data on the application of emergency disease control in livestock populations, epidemiological modelling provides a useful tool to aid strategy evaluation.
Task C: Improve detection and diagnosis and validation (including DIVA diagnostic tools)
It is self-explanatory that the potential application of a marker vaccine for emergency vaccination is dependent on the availability of a DIVA diagnostic assay (or test protocol) with acceptable test characteristics. Although preliminary data were available on the DIVA potential of a number of commercially available tests, an in depth evaluation and validation of a DIVA test for use in conjunction with the new marker vaccine was still lacking. In parallel, it was recommended to identify new antigens and to develop new serological DIVA assays specifically to be used with the new marker vaccine. As laboratory capacity was shown to be a limiting factor during the management of CSF outbreaks in the past, the development of new cost-effective mass-diagnostic techniques and the development of low-tech, on-site techniques for use under field conditions were believed to be beneficial tools to increase laboratory capacity during outbreaks. The development and validation of DIVA diagnostic tools for CSF was the main objective of Task C.
Work developed under this task consisted of the following parts:
- Evaluation of existing commercially available antibody ELISA's for use as a DIVA test with the new marker vaccine;
- Identification of new antigens and development of new antibody ELISA's for use with the new marker vaccine;
- Development of suspension microarray DIVA assays based on Luminex technology for high throughput detection of viral nucleic acids and/or antibodies;
- Development of magnetic bead array or other assay for DIVA detection of viral nucleic acids;
- The evaluation of a portable PCR assay for use under field conditions (on-site detection);
- Evaluation of the general applicability and fitness for purpose of infra-red thermography for the early detection of suspicious animals.
Task D: Improve knowledge on immunological reactions directly related to vaccine improvement
The numerous animal experiments carried out for the development of the new marker vaccine provided an ideal opportunity for the simultaneous study of the immunological mechanisms involved in the protection against CSF. As the new marker vaccine could also be used in CSFV endemic populations in the future, where young piglets have maternal antibodies due to wild type infection or vaccination of the sow, an assessment of the duration of this maternal immunity and its potential interference with vaccine efficacy was necessary.
Work developed under this task was focused on the following objectives:
- Study of the humoral immune responses after vaccination and infection with CSF and comparison of immunological responses after vaccination with the C-strain and the new marker vaccine;
- Study of the local virus replication in tonsils and early immune responses in tonsils after oral vaccination and challenge;
- Comparison of virus replication and responses in immune cells from wild boar versus domestic pigs;
- Study of the maternal immunity against CSF and its potential interference with vaccine efficacy.
Task E: Alternative methods of suppression of viral replication
Despite antiviral treatment being commonly used in human medicine for the treatment of a number of viral diseases, the use of antiviral drugs in veterinary medicine is rare. The rapid onset of the effect induced by antiviral treatment could however be a major benefit in the control of highly infectious diseases such as CSF. In vitro activity of a certain class of antiviral molecules against CSFV had already been demonstrated. Further confirmation of the potential of this molecule was needed in vivo in addition to the initial exploration of a number of alternative methods for the suppression of viral replication for example the use of small interfering RNAs.
Work developed under this task was focused on the following objectives:
- Demonstration of the potential of antiviral treatment to reduce CSFV transmission from treated to untreated pigs in an animal experiment;
- Study of the potential emergence of antiviral resistance by means of quasispecies analysis;
- Evaluation of the in vitro interference capacity of modified live CSFV and of selected small interfering RNAs.
Project results:
TASK A: Recent developments in new generation live marker vaccines and molecular diagnostic tools (Task leader: FLI)
The work of Task A was developed under five work packages as described further:
Work package 1: Selection of a final live vaccine candidate (WP leader: VAR)
Objectives
The main objective of WP1 was to select a final vaccine candidate based on pre-existing data and comparative animal trials. The choice was to be made between marker vaccine candidates Flc11 and CP7_E2alf.
Summary of results of the WP
In a first step pre-existing performance data were collected from publications, project reports and other communications. In total, 14 studies (seven published articles) on CP7_E2alf and three studies (two published articles) on Flc 11 were collected and the data were categorized (e.g. safety, efficacy, DIVA potential).
Thereafter, comparative animal trials were performed for both intramuscular and oral vaccination. All assessments were done in comparison with the 'gold standard' C-strain vaccine. In detail, the two chimeric Pestiviruses CP7_E2alf and Flc11 were directly compared in three separate animal trials using domestic pigs. The studies were carried out by partners 9 (IVI), 7 (FLI) and 5 (CVI) respectively. In the first study, the immunity induced by single oral vaccination was assessed by challenge infection 14 days after vaccination. The second study also included a challenge infection 21 days after single oral vaccination in addition to a challenge infection carried out 14 days after vaccination as in the previous experiment. The third study assessed the immunity induced by single intramuscular vaccination at 7 and 14 days post vaccination. In all studies, a group of animals vaccinated with the C-strain was included as a positive control. Challenge infection was carried out by oro-nasal administration of the highly virulent CSFV strain Koslov in all studies. In each study, a thorough assessment of safety, efficacy and DIVA potential of each vaccine candidate was made. Accompanying the comparative challenge trials, multiple vaccinations with both candidates was performed by partner 7 (FLI). All studies were published in peer reviewed journals (Blome et al., 2012; Eble et al. 2012).
Objectives
The objectives of WP2 were the following:
1) The establishment of Master Cell Stock and Master Seed Virus for the final candidate;
2) To define the parameters for the scale-up of production;
3) The evaluation of a pilot vaccine in target animals.
Summary of results of the WP
Master cell stock (MCS) and working cell stock (WCS) of swine kidney cells were prepared and validated according to European Pharmacopoeia (Ph. Eur.) guidelines.
Master seed virus (MSV) and working seed virus (WSV) of the recombinant CP7_E2alf virus were validated following Ph. Eur. Guidelines. The virus strain was confirmed free of Mycobacterium, Mycoplasma, bacteria, fungi, yeasts and bovine and porcine pathogens, and Brucella.
Infection parameters of CP7_E2alf [multiplicity of infection (MOI), time of infection (TOI) and harvesting time] were optimized providing useful recommendations to increase yield. Moreover, a production method at pilot scale on roller bottles and bioreactors was established.
Objectives
The main objective of WP3 was to assure the efficacy of the CP7_E2alf vaccine under laboratory and field conditions in naïve piglets and in piglets with maternally derived antibodies (MDA) against CSF. This work package was divided into three subtasks.
Summary of results of the WP
Subtask 3.1 Experimental infections assuring efficacy
Subtask 3.1 aimed at testing the efficacy of the CP7_E2alf vaccine by means of experimental infections.
The efficacy of CP7_E2alf after intramuscular and oral administration in 6-10 week old piglets free of Pestivirus antibodies was tested at CAO-DVMP. The vaccine proved to be effective in piglets challenged with highly virulent CSFV with the exception of one orally vaccinated piglet (out of 15) which succumbed at 7 days post-challenge (7 dpc). When the same experimental design was repeated in piglets with MDA against CSF (induced by CP7_E2alf vaccination of the sows), in the orally vaccinated piglets partial protection was observed as seven out of 15 piglets succumbed to the challenge infection. A study conducted by the same partner tested the ability of the CP7_E2alf vaccine to prevent transplacental infection in pregnant sows at 40-50 days of gestation. The vaccine showed a reduction but not full prevention of transplacental infection.
Subtask 3.2 Field trials in domestic pigs with the final vaccine candidate
Field trials with the CP7_E2alf vaccine were initially planned to be carried out in commercial farms in China in comparison to the widely used C-strain vaccine. It proved to be very difficult to obtain the permissions for the importation of the vaccine into China and for use in the field as not all safety data were available at the time of the application. In order to search for clarification and to facilitate the process, the Coordinator Dr Frank Koenen, Deputy Coordinator Dr Ase Uttenthal and the Deputy Task leader of task A Dr Sandra Blome visited Dr Rong Gao in Chengdu, China in July 2012. A comparative study was planned to be performed in a high containment unit. Despite all the efforts made to perform a trial in China, it was not possible that this trial was initiated within the timeframe of the project. A 'field-type' study was conducted at FLI and was successfully completed. The study confirmed the safety of CP7_E2alf vaccination in domestic pigs under field-like conditions in Germany. Two field studies were also conducted in wild boar in Italy to evaluate the effectiveness of CP7_E2alf as an oral vaccine for wild boar. Due to the absence of a positive marker in the baits it was not possible to confirm that the analyzed animals have eaten the baits. Nevertheless, virus neutralizing antibodies were detected in 36.3% of the wild boars that were shot after a single vaccination campaign. This study also showed that there is no loss of the original viral titer within the baits after 24 hours.
Subtask 3.3 Field and laboratory trials of vaccination in CSFV endemic populations
A study of vaccine efficacy in piglets from C-strain vaccinated sows was performed at DTU-Vet. CP7_E2alf provided protection against severe clinical signs and mortality in those piglets positive for C-strain MDA. The DIVA potential of the vaccine in these piglets was proven as well.
Work package 4: New methods for the easy oral application of the vaccine (WP leader: FLI)
Objectives
The development of a new generation marker vaccine for oral application has to be accompanied by the optimization of a suitable bait formulation. For this reason, different options for oral vaccination of wild boar and emergency vaccination of domestic pigs were under investigation in the framework of WP4. The studies included uptake and bait design studies. Final goal was the selection of a baiting system for the final marker vaccine candidate.
Summary of results of the WP
As it was demonstrated in previous experimental uptake studies that spherical baits could show an enhanced uptake in young wild boar, spherical baits were produced and distributed in small field trials conducted in France. These studies were designed to comparatively assess different bait types including the traditional baits currently in use for oral C-strain vaccination of wild boar. It could be shown that bait uptake by young wild boar in the field was almost independent from bait size and shape. After all, it was concluded that for the time being, the traditional C-strain baits present the best option. For this type of baits, all production methods and baiting systems are in place and are thus easily transferred to CP7_E2alf. Optimization might be possible in future both in terms of flavoring and baiting scheme.
Work package 5: Field trial on wild boars, including baiting. Data collection/evaluation on wild boar vaccination (WP leader: IZS-UM)
Objectives
The aim of this study was to evaluate the effectiveness of a new generation of a live marker vaccine developed under this project by application of oral vaccination on wild boar in field trials.
Summary of results of the WP
In order to quantify vaccination efficacy in the field, initial experiments were carried out. The first experiment covered field evaluation of consumption of different types of baits by camera trapping. The second was a preliminary study in captivity in order to validate incorporation of biomarkers (iophenoxic acids [IPA]). The studies using camera trapping showed that irrespective of study site and season, non-target species such as Eurasian badgers, Red foxes, mustelids and birds were frequently observed consuming baits. For the wild boar itself, it was seen that neither small spherical baits, nor traditional vaccine baits were consumed by young wild boar less than 10 months, while adult and sub-adult animals ate them occasionally. The third bait type (Spanish formulation) was not consumed by either piglets or adults. These results possibly arise from the high availability of natural and artificial food resources and suggest that a specific solution should be explored for each eco-region depending on wild boar local food availability and habits.
TASK B: Epidemiology (Task leader: ONCFS)
The work of Task B was developed under five work packages as described further:
Work package 6: Comparative evaluation of classic and alternative control strategies in domestic pigs based on economic analysis of CSF-spread model (WP leader: UFZ)
Objectives
The objectives of WP6 were the following:
1) The conceptualisation and implementation of an expert-agreed evaluation model;
2) The statistical analysis of available data preparing agreed and uncertain parameterisation;
3) The validation of the epidemiological model (data-driven and pattern-oriented);
4) The reference simulation for EU strategies by strategic efficiency, costs and associated risks;
5) The comparative simulation experiment for alternative strategies defined in the project;
6) The quantitative analysis of cost-benefit and efficiency of strategies for comparison.
Summary of results of the WP
From simulations of the CSF-spread model the following conclusions could be drawn:
(1) emergency vaccination with a live vaccine in a zone of 3.000 m around detected outbreaks, as control measure is
(i) (at least) as effective as pre-emptive culling of the 1.000 m zones,
(ii) can be used to replace the usual pre-emptive cull of 1.000 m zones, and
(iii) as safe as pre-emptive culling after applying the standard protocol to lift restrictions;
(2) emergency vaccination applied by oral administration via feed would increase efficacy and safety of measures due to greater performance in mass-application;
(3) emergency vaccination is a practical control alternative to prevent mass-destruction of (non-infected) livestock;
(4) emergency vaccination should be equipped with rapid testing procedures that allow demonstration of absence of virus in order to
(i) resolve issues with growing animals within restriction zones (e.g. test to slaughter) and
(ii) enable strategy amendments that increase efficacy (e.g. prolonged restriction zones) and hence increase safety of restricted areas prior to end-screening measures;
(5) emergency vaccination combined with pre-emptive culling is a possible but futile concept;
(6) emergency vaccination will require commercialisation of a limited number of pigs that are seropositive to vaccine virus;
(7) emergency vaccination with vaccine following the DIVA principle would allow the demonstration of success of the control measure during and after the final diagnostic screening to regain the CSFV free status. Follow-up of the epidemiological situation during the epidemic, however logically, will be based on rapid testing for virus presence.
Work package 7: Model based analysis of field data on CSFV transmission and persistence in the wild (WP leader: ONCFS)
Objectives
The objectives of WP7 were the following:
1) To define the critical (key) parameters responsible for CSF persistence in the wild and to explore in particular the effect of hunting pressure and seasonality, landscape structure, population size and wild boar demography;
2) To provide an operational tool to predict CSF spread and persistence in a given wild boar population and to evaluate the efficacy of oral vaccination of wild boar using the C-strain or the new marker vaccine in different designs of baiting;
3) To theoretically evaluate the relative performance of complex strategies combining vaccination and human induced modifications of the demography of wild boar (i.e. hunting and contraception).
Summary of results of the WP
During the first project period, an analysis of the correlation between the 'pro-active', 'contemporaneous', 'post-active' vaccination treatment and time horizon with circulating infection was performed. The key-parameters of circulation and persistence of CSF in wild boar based on data and literature were reviewed and the spatially-explicit individual-based model was validated.
Work package 8: Descriptive epidemiology in backyard pigs of classical swine fever (WP leader: UCM)
Objectives
The main objectives of this work package were, firstly, to predict the population and geographical distribution of backyard pigs (BYP) in the EU and, secondly, to evaluate the epidemiological role that BYP have in the CSF spread.
Summary of results of the WP
The first objective was accomplished by using a two-step modelling approach based on generalized linear mixed models using up to 52 socio-economic and cultural factors to estimate the BYP density at NUT3 level in the whole EU. This approach allowed us to produce a BYP density map at NUT3 level for the whole EU (which is already available). Moreover, the model revealed that BYP are concentrated in areas with traditional agricultural practices, particularly in regions in which agricultural holdings have mixed livestock and a low standard gross margin, farm owners are old (i.e. age 55-66) and labour force is familiar, among other factors. Results of the BYP density map produced under this WP also agreed with areas identified by experts to have high abundance of BYP in an expert opinion elicitation conducted during 2012.
The evaluation of the epidemiological role of BYP for CSF spread was performed only for Bulgaria, as it was the only country with detailed information available, and it was approached in two ways. Firstly, we evaluated whether or not BYP is a risk factor for CSF occurrence by using the Bayesian logistic regression model. Results of the statistical model revealed that BYP is a significant risk factor for CSF occurrence in Bulgaria, but not the most important one. In fact, high risk areas for CSF occurrence were those concentrating a high number of other low biosecurity farms, particularly family farms type B and East Balkan pig herds, as well as those farms with high number of outgoing pig shipments and low levels of personal consumption (i.e. economically deprived areas).
Work package 9: Early warning systems of CSF and surveillance in domestic pigs and wild boar (WP leader: FLI)
Objectives
Major goal of WP9 was the development of an optimized surveillance strategy focusing on the situation in the industrial sector, backyard pig populations and wild boar based on model-based simulations. As a first step, the currently applied surveillance strategies and early warning systems regarding CSF in domestic pigs and wild boar were reviewed and statistically described. The review included both CSF affected and non-affected countries and was combined with the development of a new statistical methodology to calculate corrected sample sizes for finite populations.
Summary of results of the WP
Surveillance strategies and legal provisions regarding CSF in domestic pigs were reviewed in the framework of subtask 9.1 using records and surveys from different regions of the EU. Thereby, also the statistical properties of the employed systems were assessed.
One point with optimization potential was found to be the sample collection: sampling schemes that are laid down in EU legislation do not correct for test properties such as sensitivity and specificity of diagnostic tests. To point out possible ways of improving the system and thus increasing reliability, the current system was compared to a corrected sample size that takes into account test characteristics. The corrected sample was optimized in such a way that it provided at least one true positive test result if the true prevalence was higher than the design prevalence. Furthermore, a simulation study was conducted to demonstrate the effect of different herd size distributions and various prevalences at animal and herd level on the design prevalence. It could be concluded that the currently used virological and serological tests applied in combination with the current sampling strategy is suitable to attest freedom from disease for the design prevalences and confidence levels specified in the CSF diagnostic manual of the European Union. Targeted sampling on clinically diseased animals (e.g. febrile pigs) would allow minimizing the number of virological tests. However, in order to correct for the properties of the combined test the number of examined animals must be increased. In countries with a high number of backyard pigs (low average herd size, high number of herds) the sample size, false and true test results increase significantly when the same monitoring and test parameters are used. Therefore, attesting freedom from CSF in these areas becomes difficult by solely relying on laboratory testing given non-perfect test properties.
Work package 10: Explorative molecular epidemiology (distribution, clustering, species specificity) (WP leader: TiHo)
Objectives
The aim of WP10 was to provide information on the epidemiological situation of CSF in countries with a high prevalence of backyard pig farming such as most countries of the Balkan Peninsula in order to facilitate epidemiological investigations and risk analyses. This included the characterization and phylogenetic analysis of recent CSFV strains from the Balkan region as well as a complete genome sequencing of selected strains using conventional as well as high capacity sequencing techniques. To make the obtained information available for use in subsequent studies sequences and epidemiological data were entered in existing databases. Moreover, this WP was aimed at the characterization of a quasispecies structure of CSFV by the analysis of the NS5B region of the genome in order to study the impact of a potential quasispecies structure on epidemiology as well as on in vitro results.
Summary of results of the WP
With the intention to shed light on the overall epidemiological situation in countries with a high proportion of backyard pig farming such as countries of the Balkan Peninsula, molecular epidemiology was performed with a total of 97 CSFV isolates originating from outbreaks that occurred in South-Eastern European countries between 1994 and 2007. After having obtained these isolates from collaborating National Reference laboratories, they were subjected to partial sequencing (fragments of the 5'NTR and E2 gene) and subsequent phylogenetic analysis. The genetic typing showed that almost all isolates belonged to genotype 2.3. On the basis of these sequences and additional sequences from outbreaks in Eastern and Western European countries obtained from the EU and OIE Reference Laboratory for CSF in Hannover (EURL) CSF virus database, two clusters were distinguished within subtype 2.3. These were tentatively named 2.3.1 and 2.3.2. Beyond the molecular studies, also an attempt was made to elucidate the overall epidemiological situation by compiling all data presented in the framework of different TAIEX (technical assistance and information exchange instrument of the institution building unit of directorate-general enlargement of the European Commission) events as well as data available from EU and OIE workshops. This information together with data obtained through molecular epidemiology was made available through publication (Blome et al., 2010). Moreover, the used isolates were integrated in the virus collection of the EURL, and sequence data as well as basic epidemiological data was uploaded into the CSF virus database.
TASK C: Improve detection and diagnosis and validation (including diva diagnostic tools) (Task leader: SVA)
The work of Task C was developed under three work packages as described further:
Work package 11: Evaluation of currently available antibody ELISAs for DIVA purposes. Characterisation of monoclonal antibodies and antigens for potential use in ELISAs (WP leader: TiHo)
Objectives
In the framework of WP11 an evaluation study of commercially available CSFV antibody ELISAs was implemented to decide which ELISA can be used for DIVA purposes in combination with 3rd generation modified life CSFV marker vaccines. Additionally, for the improvement of existing ELISAs or for the development of new tests monoclonal antibodies as well as novel antigens were characterized. Based on the outcome of these studies, suitable ELISAs were selected for an evaluation within an inter-laboratory comparison test. This was aimed at the determination of statistical measurements in order to draw conclusions about the feasibility of implementation of the selected 3rd generation marker vaccine and DIVA diagnostic tools for the control of CSF.
Summary of results of the WP
To determine the DIVA potential of commercially available test systems, seven CSFV E2 and Erns antibody ELISAs, respectively, were assessed in a preliminary inter-laboratory comparison test. The E2-ELISAs CHEKIT* CSF Sero (IDEXX CSF Sero Ab Test) and HerdChek* CSFV Ab (IDEXX CSFV Ab Test) performed best with regard to sensitivity. Furthermore, both tests showed a good practicability and reproducibility, making them the most promising DIVA ELISA candidates for a DIVA strategy with marker vaccines containing the E2 protein of Bovine viral diarrhea virus (BVDV) (e.g. Flc9). Additionally, both E2-ELISAs achieved best results in terms of sensitivity with animals vaccinated with CP7_E2alf and could therefore be used for the detection of CP7_E2alf marker vaccinated animals. The Erns-ELISA CHEKIT* CSF Marker (IDEXX CSFV Marker Test) achieved also a good sensitivity and reproducibility. However, as this test cannot distinguish between pestiviruses, it was ruled out for a DIVA strategy with chimeric vaccines that contain the Erns of ruminant pestiviruses (e.g. CP7_E2alf and Flc11). The PrioCHECK® CSFV Erns ELISA (Prionics) was the only test suitable for this purpose, even though some improvements were needed with regard to its sensitivity, selectivity, and especially robustness. Since the marker vaccine CP7_E2alf was selected as final vaccine candidate, great efforts were made to improve the PrioCHECK® CSFV Erns ELISA.
Work package 12: Development of alternative new methods for DIVA detection of nucleic acids and antibodies for laboratory use and for on-site testing of domestic pigs and wild boar (WP leader: SVA)
Objectives
The aim of WP 12 was to develop and test novel methods for the detection of nucleic acids and antibodies in pigs and wild boar infected with CSFV or vaccinated with Cp7E2alf. The work package was divided in several subtasks. The first subtask was dealing with sample preparation and enrichment methods, the second with suspension microarray based DIVA assays, the third with basic methods for detection and differentiation of viral nucleic acids, the fourth with evaluation of a portable PCR assay for on-site detection and the fifth with standardization of the methods.
Summary of results of the WP
The work with the first subtask demonstrated that methods only requiring basic technology can be used for successful extraction of CSFV RNA in the field or in basic laboratories. It was demonstrated that the use of filter paper, like FTA paper (GE healthcare), is a very convenient, simple and economical method for collecting; storing and transporting samples as well as extracting RNA from samples of potentially CSFV infected pigs or wild boars. Because of its robustness and simplicity this method is very suitable for use in the field and because it is an economical method, larger number of animals can be sampled. This sampling procedure is convenient to use in combination with field based PCR assays. Elution of RNA from the filter papers can be done by submerging filter paper punches in a simple elution buffer and following a very simple protocol. Our results demonstrated that there is an upper limit to the number of filter paper punches that can be eluted together without causing inhibition in subsequent RT-PCR steps. DMPK B and FTA-Elute are superior to FTA Classic cards in this setting. The use of handheld magnet in combination with reagents from Nordiag and magnetic beads for extraction of nucleic acids was tested as an alternative to filter papers (Dynal MPC-S, Dynal Biotech, Norway). It was concluded that the handheld magnet gives a faster and more reproducible result under different environmental conditions but it involves more steps than the filter paper method and is therefore more cumbersome. The method takes around 90 min to complete a preparation of 10 samples. These collection and extraction methods simplify sampling and submission of samples to the laboratory and enable analysis directly on site, thereby providing a rapid result to support further actions. Even though the field sample preparation methods yielded a lower amount of viral nucleic acids, they are useful for speeding up the diagnostic process.
For the second subtask, the Luminex xMAP technology, which has become more and more widely used for multiplexing detection of agents, was used. This task started with the development of a suspension microarray assay for the detection and differentiation of both wild type and CP7_E2alf vaccine RNAs simultaneously. The assay had similar sensitivity for the detection of wild type viruses and 10-fold lower sensitivity for the marker vaccine, compared to real-time RT-PCR assays. When evaluated in a duplex format, the sensitivity was reduced a 10-fold further. Unlike wild type viruses, the marker vaccine candidate CP7_E2alf replicates only transiently in pigs upon vaccination, yielding very low levels of viral nucleic acids in a very short time period, close to the detection limit of the highly sensitive real-time RT-PCR. Based on these results and the fact that the Luminex assay is also more labor intensive and more expensive, it was concluded that the Luminex assay for the detection of nucleic acids is not a suitable alternative DIVA method. However, a serological DIVA assay based on the Luminex technology could be beneficial for the mass screening of serum samples.
Therefore, the focus was shifted to a serological assay. To develop a serological DIVA assay, three viral proteins, namely BVDV Erns, CSFV Erns and E2 were produced in insect cells using the baculovirus expression system, and subsequently purified by affinity chromatography. The assay was evaluated by testing 365 samples and compared to ELISA. Out of the 365 samples, 360 samples were correctly diagnosed by the E2 antibody detection, resulting in a sensitivity of 98.5% and a specificity of 98.9%. For the CSFV Erns antibody detection, 155 out of the 187 samples reached the same results by both the microsphere immunoassay and ELISA. Some samples had different results between the Luminex assay and ELISA that was still under further improvement. BVDV Erns antibody detection had low MFI values (between 27 and 6720 with a median value of 762). As the MFI values from vaccinated pigs were slightly higher than those from naïve pigs, a very low antibody response to BVDV Erns antigen in pigs upon vaccination with CP7_E2alf was suggested. The DIVA potential of these assays was confirmed by testing well defined samples. The multiplex immunoassay offers a new format for high-throughput DIVA diagnosis of classical swine fever and marker vaccine CP7_E2alf. It could be used in reference laboratories as a confirmatory tool.
In the third subtask the loop-mediated isothermal amplification (LAMP) assay for basic laboratories was tested. For field application, both magnetic bead array (MBA) and LAMP are suitable, as they don't required advanced instruments. Further comparison of both methods found LAMP was better suitable for field condition as it required fewer steps and should give quicker results compared to MBA. The results showed that the sensitivity of the RT-LAMP was 10-fold less than real-time RT-PCR for the detection of marker vaccine CP7_E2alf. However, this is a very useful assay for use in laboratories equipped with basic instruments only. The only instrument needed to perform this assay is a heating block or even a simple stove and results can be read by naked eye. Combined with sample collection and extraction on filter paper, the LAMP assay is a very economical of detecting infected animals that can be used under very simple conditions in the field or elsewhere.
For a similar purpose the fourth subtask was devoted to the evaluation of a portable PCR instrument for use in the field. Initially an instrument from Smiths detection was the focus of attention. This was a very complete and safe, but very expensive instrument. This instrument was subsequently withdrawn from the market. Therefore the smartcykler from Cepheid was tested. This instrument gave excellent results in our trials. The instrument can be used in the field but needs high voltage and therefore a generator must accompany the instrument. Subsequently a truly field based PCR instrument from Tetracore was chosen. This instrument, T-Core 4, is portable, runs on batteries or connected to the grid and has four cells for performing PCR. Results are calculated and displayed in a connected lap top computer. Tetracore supplies wet and dry RT-PCR kits for use with the T-Core 4 and both were tested in our experiments. Initial experiments gave poor results due to technical problems with the PCR instrument. These problems were later resolved and the instrument and the accompanying wet and dry kits were tested with the Tetracore instrument in parallel with other PCR instruments (Rotorgene and Smartcykler). Both the wet and dry kits contain an inhibition control labelled with Cy5. The portable PCR was tested using a serial RNA dilution of the EU CSFV reference sera 1 and 2 and serial dilution of CSFV Koslov. Standardization was integrated in each subtask.
Work package 13: Development and validation of new methods for the easy selection of suspicious animals (infrared thermography) and sample reduction (pooling) (WP leader: TiHo)
Objectives
In the framework of WP13, the evaluation of infrared thermography as a non-invasive tool for a risk based sampling scheme in case of animal disease outbreaks in pigs (CSF) was in the foreground. In an overall evaluation the assessment of practicability and reliability for detection of febrile animals in pig groups or herds, the application and validation under defined conditions, and terminal tests in the field were included. Moreover, WP13 was aimed at the evaluation of pooling of samples as a method to reduce the vast number of samples that have to be analyzed during an outbreak of CSF.
Summary of results of the WP
Various infectious diseases are accompanied by fever as an early and reliable symptom. For this reason, risk based sampling schemes for animal disease outbreaks like CSF are based on this clinical sign. In practice, external factors like ambient temperature and stress reactions related to rectal temperature measurement hamper the implementation. In order to overcome this problem, as well as to simplify the selection of suspicious animals, the non-invasive infrared thermography technique for the measurement of skin temperature was tested and evaluated.
With the analysis of infrared thermography pictures of CSFV infected pigs in experimental settings, as well as of pigs in commercial fattening and breeding piggeries, a significant correlation between skin temperature of several body regions and rectal temperature was found. However, it was ascertained that several technical-physical factors (sharpness of the images, correct angle between camera and object), as well as environmental parameters (ambient temperature, relative humidity, air movement, solar radiation, other sources of warmth, skin contamination) have to be considered to gain significant results. Especially on weaner pigs the ambient temperature had a significant effect. This indicated that also individual parameters (age, sex, biological status, psychological stress, feeding, skin irritation, local inflammation, topography of the measuring point) influenced the infrared measurement considerably. The lack of robustness of this method became also apparent by measurement of pig groups. Since the movement of the pigs was fast and the space for movement large, it was not possible to take pictures in groups of small pigs and groups with more than 15 animals. These results underlined the complexity of the infrared thermography technique and the need for experience and background information to obtain reliable results.
Together with a lack of sensitivity, selectivity, and robustness, it is doubted that infrared thermography is applicable as a diagnostic tool to select diseased animals in CSF outbreaks under field conditions. Fitness for purpose cannot be testified without reservation.
Outbreaks of CSF in domestic pigs or wild boar populations are accompanied not only by a vast number of pigs that have to be clinically investigated but also by vast numbers of samples that have to be investigated for CSF virus, antigen or genome, and antibodies. In this context, diagnostic capacities available at the laboratories can be bottle necks and may hamper rapid analyses of samples. Sample pooling, especially for highly sensitive real-time RT-PCR assays, can greatly augment the number of samples that can be analyzed without having to increase the capacity of the laboratory and without an increase in associated costs. To assess the diagnostic reliability an evaluation of serum sample pooling with regard to overall performance and influence of sample quality was performed. Especially for the pooling of wild boar samples from monitoring and surveillance programs, sample quality might be a critical factor as these samples often arrive in bad to almost non-existent quality.
TASK D: Improve knowledge on immunological reaction directly related to vaccine improvement (Task leader: ANSES)
The work of Task D was developed under one single work package as described further:
WP14: Improve knowledge on immunological reactions directly related to vaccine improvement (WP leader: Anses)
Objectives
The CP7_E2alf was compared to the C-strain for its immune responses modulation when applied intramuscularly or orally at different times before challenge. Further objectives were to understand the early mechanisms induced at the tonsil level after oral immunization and their implication in reducing the virus replication, to identify possible differences between immune cells derived from wild boars or domestic pigs, and to evaluate the possible interference of maternal antibodies against vaccination efficacy.
Summary of results of the WP
The C-strain has been used for years to vaccinate wild boars by means of distribution of baits. When orally applied the C-strain is able to protect piglets as soon as three days after vaccination. Here, CP7_E2Alf has been evaluated for its potency to induce a very early immune response and for the mechanisms behind this protection. Other samples obtained from the WP3 on efficacy trials permitted to investigate later challenge protocols.
TASK E: Alternative methods of suppression of viral replication(Task leader: VAR)
The work of Task E was developed under one single work package as described further:
WP15: Exploration of alternative methods of suppression of viral replication (WP leader: VAR)
Objectives
This WP was aimed at exploring a number of alternatives to stamping and vaccination, as potential aids in the control of CSF outbreaks. These included the evaluation of the potential of antiviral treatment to reduce CSF virus transmission as well as the assessment of the risk for the development of antiviral resistance against this molecule. Further objectives were the evaluation of the capability of live attenuated vaccine strains to interfere with wild type CSFV replication and the assessment of the potential of RNA interference by testing selected small interfering RNAs (siRNAs).
Summary of results of the WP
The molecule 5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is an antiviral molecule belonging to the class of the imidazopyridines and had previously been demonstrated to be an active inhibitor of the replication of CSFV in vitro. In WP15, an animal experiment was performed to evaluate the efficacy of BPIP in preventing CSF virus transmission from treated to untreated pigs. The study showed that a 15-day treatment with BPIP administered via the feed was able to reduce, but not completely prevent, virus transmission from CSFV infected to sentinel animals. Treatment with BPIP reduced the duration of viraemia in CSFV infected pigs resulting in a lower occurrence of virus transmission to sentinel animals and a lower genome load measured by RT-PCR in the tonsils from these sentinel animals. The results of this study demonstrated the potential of antiviral treatment as an early emergency measure in the case of a CSF outbreak.
In order to evaluate the risk and the genetic mechanisms for the potential development of antiviral resistance against this molecule, the quasispecies structure of the NS5B genome region, the area targeted by the antiviral molecule, was determined by cloning and sequencing and was compared between the wild type CSFV strain Wingene and its BPIP resistant variant. The antiviral resistant variant was generated in-vitro by subsequent passaging in the presence of increasing concentrations of BPIP. The study showed that intra-host variability was present in the wild type Wingene population and increased significantly when antiviral pressure was applied. Mutations found in the BPIP-resistant variant suggested that more than one mutation was responsible for the development of the resistant phenotype. Although no clear evidence was found for the presence of published resistance associated mutations in the wild type Wingene population, the finding of one particular mutation (R3407H) in the wild type and BPIP-resistant Wingene population drew our attention as this mutation was mapped to a resistance associated region in NS5B. These findings clearly highlight the usefulness of quasi-species analysis for the research in future antiviral therapy.
Also in WP15, the effect of RNA interference (RNAi) on CSFV infection was studied in vitro. Small interfering double-stranded RNA molecules (siRNAs) targeting the non-structural protein NS2-3 and 3´ non-translated region (3´NTR) encoding genome region of the recent German CSFV field isolate 'Roesrath' were used to assess RNA interference with homologous as well as heterologous CSFV strains. It was demonstrated that the selected siRNAs were able to inhibit viral replication after homologous infection using current CSFV strains of genotype 2.3. No complete inhibition but a rather diverse repression of CSFV replication could be attained in vitro. Due to the requirement for a 100% sequence homology the effect of in vivo application of siRNAs as antiviral drugs seems to be limited. In addition it has been reported that the development of successful in vivo siRNA delivery systems pose significant challenges, also for other viruses.
In a final part of WP15, the potential role of virus interference in early protection against CSFV vaccination was assessed. An in vitro study was carried out using the CP7_E2alf vaccine virus for super-infection of persistently CSFV infected as well as uninfected porcine kidney cell lines. The results indicated a powerful viral interference capacity of CSFV infection.
Management of the project and other activities (WP16 and WP17)
WP16: Management of the project (WP leader: VAR)
Objectives
The objective of this WP was the overall management of the project including the signing of Consortium and Grant Agreements and the monitoring of compliance herewith, the regular communication with the EC scientific officer, the maintenance of the project website and the EC webportal via ECAS, the preparation of Amendments to the Grant Agreement and of the periodic and final activity reports, the organisation of 6-montly project meetings and of the dissemination meeting, the management of the financial aspects of the project and disbursements of funds to the partners, the general responsibility and accountability for progress of the project against milestones and deliverables as specified in the grant agreement.
WP17: Other activities (WP leader: VAR)
Objectives
The objectives of WP17 were the development of the project website, the inclusion of a potential new beneficiary or invitation of scientists from Romania, Bulgaria or other relevant third countries, and the dissemination of the final results of the project.
Summary of results of the WP
Collaboration with scientists from Romania was attempted for the work concerning the epidemiology of CSF in backyard pigs but proved to be unsuccessful in the end. Collaboration with the Bulgarian authorities was established instead and the necessary data to perform the work on backyard pigs were obtained from Bulgaria.
The development of the project website and the dissemination of the final results of the project are described in the next section of the report.
Potential impact:
Socio-economic impact and wider societal implications
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the World Organization for Animal Health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability to be applied orally for wild boar vaccination. Project CSFV_GODIVA was dedicated to improving the tools and strategies for the prevention and control of CSF, including the development of a new modified live CSF marker vaccine for domestic pigs and wild boar.
In order to improve tools and strategies for the prevention and control of CSF, the work developed under CSFV_GODIVA project aimed to fulfil the following objectives:
- the development of a new live CSF marker vaccine with DIVA (Differentiation of Infected from Vaccinated Animals) diagnostic assay for domestic pigs and wild boar;
- the evaluation of the benefits of CSF emergency vaccination of domestic pigs and the vaccination of wild boar;
- the improvement of wild boar baiting systems;
- the study of the epidemiology of CSFV in backyard pigs;
- the evaluation of CSF surveillance systems in domestic pigs, wild boar and backyard pigs;
- the assessment of the potential use of anti-viral treatment to reduce CSFV transmission;
- the study of the immunological reactions after CSFV infection and vaccination.
These objectives were organised in five tasks and were outlined in the workplan providing a logical process for improving of knowledge on critical aspects of CSF and the development of a live marker vaccine allowing the DIVA principle.
By including several National reference laboratories (NRL) and the European community reference laboratory (CRL) for CSF and a Chinese partner, the project reinforced the European network of experts for European research in epidemiology and diagnostics in CSF.
Several outcomes of the project emphasis its successful contribution to improve tools and strategies for the prevention and control of CSF:
A new marker vaccine has been submitted for registration and will allow improving CSF eradication in the future:
Vaccination as an alternative to pre-emptive culling will reduce the number of healthy pigs that are destroyed. The ultimate deliverable of the project was to present a marketable live marker vaccine for intramuscular administration to domestic pigs and for oral application in wild boar. A few months before the finalization of the project the dossier for marketing of the injection vaccine was submitted for EMA. Several studies also target the use of the oral vaccine but the initial application is on injection. The dossiers were produced through a very constructive collaboration between the industrial partner and all research partners. The new DIVA vaccine has been analysed in comparison to the C-strain vaccine both for safety and efficacy. Humoral and cellular immunological parameters have been investigated both after vaccination and after subsequent challenge of vaccinated pigs in various age groups. The emergency use of the vaccine has been analysed by reducing the time from vaccination till the highly virulent challenge. The impact of maternal immunity and effect on pregnant animals has also been analysed.
The vaccine has been tested as a bait vaccine for wild boar or for oral application in domestic backyard pigs. The baits have been evaluated in field trials in France and Italy. The main obstacle for the bait uptake seems to be the availability of other food sources. With plenty of usual food the baits will have too much competition.
Improved CSF diagnosis:
The DIVA vaccine is only of use if accompanying DIVA diagnostics is available. The available serological DIVA diagnostic systems were validated and compared in a review. Within the consortium and in collaboration with external partners new improved diagnostic tests were produced and the available tests improved. As the vaccine is a live vaccine further DIVA tests that can differentiate between the wild-type virus and the vaccine virus was needed, a genetic DIVA was developed and the test was further implemented for a field portable PCR assay.
Development of epidemiological tools for a better understanding of CSF and to support decision makers:
A comprehensive study on the epidemiology of CSFV in backyard pigs and wild boar has been an impressive support for the cost/benefit decisions on when to vaccinate and to decide which groups are at risk. Based on the model an evaluation of CSF surveillance in domestic pigs, wild boar and backyard pigs could be tested with highlights of strengths/weaknesses. The model will assist the European decision makers and be a valuable tool in emergency situations when enormous socio-economic decisions are taken.
From simulations of the CSF-spread model it can be concluded that emergency vaccination is a practical control alternative to prevent mass-destruction of (non-infected) livestock if accompanied with rapid testing procedures that allow demonstration of absence of virus. Only a limited number of pigs that are seropositive to vaccine virus will be commercialized. In addition emergency vaccination with the novel DIVA vaccine would allow the demonstration of success of the control measure during and after the final diagnostic screening to regain the CSFV free status. Follow-up of the epidemiological situation during the epidemic, however, will be mainly based on rapid testing for virus presence. For this purpose, PCR strategies were developed within the project that is able to differentiate field CSFV and vaccine virus. For domestic pigs the currently used virological and serological tests applied in combination with the current sampling strategy are suitable to attest freedom of disease as specified in the CSF diagnostic manual of the EU.
Alternative methods were studied:
An antiviral molecule belonging to the class of the imidiazopyridines administered via the feed was able to reduce but not completely prevent CSFV transmission from treated CSFV infected to sentinel animals. Proof of principle for the efficacy of antiviral treatment against CSF transmission was shown.
Transfer of knowledge on relevant aspects of CSF and transfer of diagnostic and epidemic tools through training and traineeships to a large number of professionals, decision makers and scientist.
The project provided employment, education and training for young post-graduate and post-doctoral researchers. They gained intra and inter-disciplinary experience and training working within an international, multidisciplinary team including working with industry and in a high level biosecurity environment. In addition young scientists were trained in presentation opf results and the compilation of manuscripts for publication.
The outcomes of the project will benefit EU consumer driven concerns with regard to animal welfare, food safety and food quality and will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of CSF and therefore reducing the socio-economic consequences of potential incursions of the disease from infected regions. In addition, assistance in the preservation of small to medium sized family farms, as viable economic units within a rural environment will contribute to maintaining rural infrastructure of EU regions and member states.
Main dissemination activities and exploitation of results
Exploitation and dissemination of the results was considered by the consortium to be vital. Dissemination of the results of the project was fulfilled through a series of internal and external dissemination activities.
Internal dissemination
The consortium was composed by a multidisciplinary scientific team from 17 partners based in 11 different countries. The ability of the consortium to successfully complete the project was attributed to the scientific expertise in different fields including vaccine development, virology, diagnostics, molecular biology including sequencing, immunology, epidemiology and disease surveillance/control, conduct of animal experiments, wild boar baiting and field trial experience. The presence of an industrial partner in the project led to the development of a CSF marker vaccine that can be produced at an industrial scale and that could become commercialised after registration.
Internal dissemination between partners was achieved through the organisation of 6-montly project meetings, teleconferences, communication on the project website via Mid Monthly Messages, training of scientific staff and exchanges of personnel between institutes; transfer of laboratory tests and test protocols between partners, detailed reporting including full materials and methods in the periodic reports to the EC.
Training of scientific staff and exchanges of personnel
Ilse vangeel (VAR) visited the Animal Health Department of the complutense university of Madrid (UCM) for two weeks in February 2011. The purpose of the visit was to learn a number of epidemiological methods that are used to study the epidemiology of epizootic diseases and for disease surveillance, control and risk analysis. Ilse Vangeel was trained by Dr. Beatriz Martinez-Lopez into the following methods: social network analysis, cluster analysis, geographic information systems (GIS) and Bayesian modelling for risk factor and risk analysis.
Desislava Yordanova Rangelova (DTU-Vet) visited Anses, Ploufragran for 2 weeks in 2012. Her travel expenses were funded by an EPIZONE Short Term Mission. She was trained by Patricia Renson to perform isotype specific antibody ELISAs in the laboratory. After her training, the test protocols were transferred from Anses to DTU-Vet. As part of the collaboration, a joint publication is being prepared for submission to Veterinary Research.
External dissemination
External dissemination of results was achieved through the following series of activities:
- Publications in scientific peer reviewed journals;
- Oral and poster presentations at conferences and scientific meetings;
- PhD theses;
- Organisation of the project's dissemination meeting;
- Project website (see http://www.csfvaccine.org online);
- Reports to the EC.
The CSFV_GODIVA dissemination meeting
The dissemination meeting organised by the coordinator was held in Brussels on the 31st of January 2013. A total of 68 participants attended the meeting: 31 members of the CSFV_GODIVA project and 37 guests from international institutions EU DG Research and DG Sanco and OIE, and national institutions for food safety, public health, nature and forestry; ministries, farmers unions, universities and other research institutions. Participants included representatives from 12 different EU countries and from Switzerland and Russia. Major achievements of the project were presented by several partners of the consortium. Presentations were given about the development of the new marker vaccine, the development of improved and new DIVA diagnostic tools, the comparative evaluation of CSF emergency control strategies in domestic pigs, field studies for vaccination of wild boar, the risk of backyard pigs for the transmission of CSF to EU countries, immunology of CSF, and the potential of antiviral treatment to reduce CSFV transmission. The meeting ended with a round table discussion with active participation from the audience.
Development of the project website
The website for the CSFV_GODIVA project (see http://www.csfvaccine.org online) was launched during the first project meeting in Brussels in May 2009. The website contains an area which can be accessed by the general public with general information about the project such as background, objectives, partners, description of work packages; and a restricted access area which can be accessed only by members of the consortium. The latter part of the website was used to share documents such as meeting presentations, meeting summaries, milestones and deliverables documents, periodic reports etc.
List of websites:
http://www.csfvaccine.org.
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the World organization for animal health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability for oral application for wild boar and backyard pigs. The CSFV_GODIVA project aimed at improving the tools and strategies for the prevention and control of CSF for domestic pigs and wild boar. A new modified live CSF marker vaccine allowing differentiation between infected and vaccinated animals (DIVA) was developed. Immunization and challenge trials showed that the antibody titers after a single intramuscular or oral vaccination were stable for a minimum of 6 month and full protection from lethal challenge infection was observed.
Project context and objectives:
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the world organization for animal health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability to be applied orally for wild boar vaccination. Project CSFV_GODIVA aimed at improving the tools and strategies for the prevention and control of CSF, including the development of a new modified live CSF marker vaccine for domestic pigs and wild boar. The work performed under the CSFV_GODIVA was divided into 5 major tasks. The main objectives within each task are summarized below.
Task A: Recent developments in new generation live marker vaccines and molecular diagnostic tools
In several countries worldwide, CSF has been eradicated from the domestic pig population. Despite intensive efforts on the national and international level, complete eradication from the European Union (EU) has proven to be elusive, especially since wild boar populations in several European countries are endemically infected. The current control strategy within the EU is a strict stamping out strategy without prophylactic vaccination. Nevertheless, community legislation also foresees the possible use of emergency vaccination in domestic pigs as well as in wild boar. For vaccination of wild boar, the live C-strain vaccine has historically been used. The disadvantage of this vaccine is that no differentiation between CSF vaccinated and infected animals can be made. This causes serious complications for disease surveillance. To ease trade restrictions for domestic pigs, potent marker vaccines are required that allow reliable differentiation of infected from vaccinated animals. The currently available E2 subunit vaccines allow this differentiation but lack some properties that live vaccines exhibit e.g. fast and sound protection after single application, prevention of trans-placental infection, suitability for use via the oral application route. The aim of Task A was the final development of a live marker vaccine candidate, orally applicable to wild boar and intramuscularly applicable to domestic pigs. The availability of a safe and efficacious marker vaccine and its accompanying DIVA diagnostic assays provides a highly valuable tool that can be applied in control and eradication strategies and can accommodate for the use of marker vaccines as stipulated in EU directives.
Task B: Epidemiology
Although the experience with the implementation of emergency control measures during CSF outbreaks in the domestic pig population is increasing, the excessive killing of pigs and destruction of animal products occurring during such outbreaks remains a critical and ethical concern. Emergency vaccination to control CSF ('vaccination-to-live' strategy) could dramatically improve the situation, however requires an in-depth evaluation of its efficacy and potential further consequences before being considered to be a valid alternative. Due to the limited availability of data on the application of emergency disease control in livestock populations, epidemiological modelling provides a useful tool to aid strategy evaluation.
Task C: Improve detection and diagnosis and validation (including DIVA diagnostic tools)
It is self-explanatory that the potential application of a marker vaccine for emergency vaccination is dependent on the availability of a DIVA diagnostic assay (or test protocol) with acceptable test characteristics. Although preliminary data were available on the DIVA potential of a number of commercially available tests, an in depth evaluation and validation of a DIVA test for use in conjunction with the new marker vaccine was still lacking. In parallel, it was recommended to identify new antigens and to develop new serological DIVA assays specifically to be used with the new marker vaccine. As laboratory capacity was shown to be a limiting factor during the management of CSF outbreaks in the past, the development of new cost-effective mass-diagnostic techniques and the development of low-tech, on-site techniques for use under field conditions were believed to be beneficial tools to increase laboratory capacity during outbreaks. The development and validation of DIVA diagnostic tools for CSF was the main objective of Task C.
Work developed under this task consisted of the following parts:
- Evaluation of existing commercially available antibody ELISA's for use as a DIVA test with the new marker vaccine;
- Identification of new antigens and development of new antibody ELISA's for use with the new marker vaccine;
- Development of suspension microarray DIVA assays based on Luminex technology for high throughput detection of viral nucleic acids and/or antibodies;
- Development of magnetic bead array or other assay for DIVA detection of viral nucleic acids;
- The evaluation of a portable PCR assay for use under field conditions (on-site detection);
- Evaluation of the general applicability and fitness for purpose of infra-red thermography for the early detection of suspicious animals.
Task D: Improve knowledge on immunological reactions directly related to vaccine improvement
The numerous animal experiments carried out for the development of the new marker vaccine provided an ideal opportunity for the simultaneous study of the immunological mechanisms involved in the protection against CSF. As the new marker vaccine could also be used in CSFV endemic populations in the future, where young piglets have maternal antibodies due to wild type infection or vaccination of the sow, an assessment of the duration of this maternal immunity and its potential interference with vaccine efficacy was necessary.
Work developed under this task was focused on the following objectives:
- Study of the humoral immune responses after vaccination and infection with CSF and comparison of immunological responses after vaccination with the C-strain and the new marker vaccine;
- Study of the local virus replication in tonsils and early immune responses in tonsils after oral vaccination and challenge;
- Comparison of virus replication and responses in immune cells from wild boar versus domestic pigs;
- Study of the maternal immunity against CSF and its potential interference with vaccine efficacy.
Task E: Alternative methods of suppression of viral replication
Despite antiviral treatment being commonly used in human medicine for the treatment of a number of viral diseases, the use of antiviral drugs in veterinary medicine is rare. The rapid onset of the effect induced by antiviral treatment could however be a major benefit in the control of highly infectious diseases such as CSF. In vitro activity of a certain class of antiviral molecules against CSFV had already been demonstrated. Further confirmation of the potential of this molecule was needed in vivo in addition to the initial exploration of a number of alternative methods for the suppression of viral replication for example the use of small interfering RNAs.
Work developed under this task was focused on the following objectives:
- Demonstration of the potential of antiviral treatment to reduce CSFV transmission from treated to untreated pigs in an animal experiment;
- Study of the potential emergence of antiviral resistance by means of quasispecies analysis;
- Evaluation of the in vitro interference capacity of modified live CSFV and of selected small interfering RNAs.
Project results:
TASK A: Recent developments in new generation live marker vaccines and molecular diagnostic tools (Task leader: FLI)
The work of Task A was developed under five work packages as described further:
Work package 1: Selection of a final live vaccine candidate (WP leader: VAR)
Objectives
The main objective of WP1 was to select a final vaccine candidate based on pre-existing data and comparative animal trials. The choice was to be made between marker vaccine candidates Flc11 and CP7_E2alf.
Summary of results of the WP
In a first step pre-existing performance data were collected from publications, project reports and other communications. In total, 14 studies (seven published articles) on CP7_E2alf and three studies (two published articles) on Flc 11 were collected and the data were categorized (e.g. safety, efficacy, DIVA potential).
Thereafter, comparative animal trials were performed for both intramuscular and oral vaccination. All assessments were done in comparison with the 'gold standard' C-strain vaccine. In detail, the two chimeric Pestiviruses CP7_E2alf and Flc11 were directly compared in three separate animal trials using domestic pigs. The studies were carried out by partners 9 (IVI), 7 (FLI) and 5 (CVI) respectively. In the first study, the immunity induced by single oral vaccination was assessed by challenge infection 14 days after vaccination. The second study also included a challenge infection 21 days after single oral vaccination in addition to a challenge infection carried out 14 days after vaccination as in the previous experiment. The third study assessed the immunity induced by single intramuscular vaccination at 7 and 14 days post vaccination. In all studies, a group of animals vaccinated with the C-strain was included as a positive control. Challenge infection was carried out by oro-nasal administration of the highly virulent CSFV strain Koslov in all studies. In each study, a thorough assessment of safety, efficacy and DIVA potential of each vaccine candidate was made. Accompanying the comparative challenge trials, multiple vaccinations with both candidates was performed by partner 7 (FLI). All studies were published in peer reviewed journals (Blome et al., 2012; Eble et al. 2012).
Objectives
The objectives of WP2 were the following:
1) The establishment of Master Cell Stock and Master Seed Virus for the final candidate;
2) To define the parameters for the scale-up of production;
3) The evaluation of a pilot vaccine in target animals.
Summary of results of the WP
Master cell stock (MCS) and working cell stock (WCS) of swine kidney cells were prepared and validated according to European Pharmacopoeia (Ph. Eur.) guidelines.
Master seed virus (MSV) and working seed virus (WSV) of the recombinant CP7_E2alf virus were validated following Ph. Eur. Guidelines. The virus strain was confirmed free of Mycobacterium, Mycoplasma, bacteria, fungi, yeasts and bovine and porcine pathogens, and Brucella.
Infection parameters of CP7_E2alf [multiplicity of infection (MOI), time of infection (TOI) and harvesting time] were optimized providing useful recommendations to increase yield. Moreover, a production method at pilot scale on roller bottles and bioreactors was established.
Objectives
The main objective of WP3 was to assure the efficacy of the CP7_E2alf vaccine under laboratory and field conditions in naïve piglets and in piglets with maternally derived antibodies (MDA) against CSF. This work package was divided into three subtasks.
Summary of results of the WP
Subtask 3.1 Experimental infections assuring efficacy
Subtask 3.1 aimed at testing the efficacy of the CP7_E2alf vaccine by means of experimental infections.
The efficacy of CP7_E2alf after intramuscular and oral administration in 6-10 week old piglets free of Pestivirus antibodies was tested at CAO-DVMP. The vaccine proved to be effective in piglets challenged with highly virulent CSFV with the exception of one orally vaccinated piglet (out of 15) which succumbed at 7 days post-challenge (7 dpc). When the same experimental design was repeated in piglets with MDA against CSF (induced by CP7_E2alf vaccination of the sows), in the orally vaccinated piglets partial protection was observed as seven out of 15 piglets succumbed to the challenge infection. A study conducted by the same partner tested the ability of the CP7_E2alf vaccine to prevent transplacental infection in pregnant sows at 40-50 days of gestation. The vaccine showed a reduction but not full prevention of transplacental infection.
Subtask 3.2 Field trials in domestic pigs with the final vaccine candidate
Field trials with the CP7_E2alf vaccine were initially planned to be carried out in commercial farms in China in comparison to the widely used C-strain vaccine. It proved to be very difficult to obtain the permissions for the importation of the vaccine into China and for use in the field as not all safety data were available at the time of the application. In order to search for clarification and to facilitate the process, the Coordinator Dr Frank Koenen, Deputy Coordinator Dr Ase Uttenthal and the Deputy Task leader of task A Dr Sandra Blome visited Dr Rong Gao in Chengdu, China in July 2012. A comparative study was planned to be performed in a high containment unit. Despite all the efforts made to perform a trial in China, it was not possible that this trial was initiated within the timeframe of the project. A 'field-type' study was conducted at FLI and was successfully completed. The study confirmed the safety of CP7_E2alf vaccination in domestic pigs under field-like conditions in Germany. Two field studies were also conducted in wild boar in Italy to evaluate the effectiveness of CP7_E2alf as an oral vaccine for wild boar. Due to the absence of a positive marker in the baits it was not possible to confirm that the analyzed animals have eaten the baits. Nevertheless, virus neutralizing antibodies were detected in 36.3% of the wild boars that were shot after a single vaccination campaign. This study also showed that there is no loss of the original viral titer within the baits after 24 hours.
Subtask 3.3 Field and laboratory trials of vaccination in CSFV endemic populations
A study of vaccine efficacy in piglets from C-strain vaccinated sows was performed at DTU-Vet. CP7_E2alf provided protection against severe clinical signs and mortality in those piglets positive for C-strain MDA. The DIVA potential of the vaccine in these piglets was proven as well.
Work package 4: New methods for the easy oral application of the vaccine (WP leader: FLI)
Objectives
The development of a new generation marker vaccine for oral application has to be accompanied by the optimization of a suitable bait formulation. For this reason, different options for oral vaccination of wild boar and emergency vaccination of domestic pigs were under investigation in the framework of WP4. The studies included uptake and bait design studies. Final goal was the selection of a baiting system for the final marker vaccine candidate.
Summary of results of the WP
As it was demonstrated in previous experimental uptake studies that spherical baits could show an enhanced uptake in young wild boar, spherical baits were produced and distributed in small field trials conducted in France. These studies were designed to comparatively assess different bait types including the traditional baits currently in use for oral C-strain vaccination of wild boar. It could be shown that bait uptake by young wild boar in the field was almost independent from bait size and shape. After all, it was concluded that for the time being, the traditional C-strain baits present the best option. For this type of baits, all production methods and baiting systems are in place and are thus easily transferred to CP7_E2alf. Optimization might be possible in future both in terms of flavoring and baiting scheme.
Work package 5: Field trial on wild boars, including baiting. Data collection/evaluation on wild boar vaccination (WP leader: IZS-UM)
Objectives
The aim of this study was to evaluate the effectiveness of a new generation of a live marker vaccine developed under this project by application of oral vaccination on wild boar in field trials.
Summary of results of the WP
In order to quantify vaccination efficacy in the field, initial experiments were carried out. The first experiment covered field evaluation of consumption of different types of baits by camera trapping. The second was a preliminary study in captivity in order to validate incorporation of biomarkers (iophenoxic acids [IPA]). The studies using camera trapping showed that irrespective of study site and season, non-target species such as Eurasian badgers, Red foxes, mustelids and birds were frequently observed consuming baits. For the wild boar itself, it was seen that neither small spherical baits, nor traditional vaccine baits were consumed by young wild boar less than 10 months, while adult and sub-adult animals ate them occasionally. The third bait type (Spanish formulation) was not consumed by either piglets or adults. These results possibly arise from the high availability of natural and artificial food resources and suggest that a specific solution should be explored for each eco-region depending on wild boar local food availability and habits.
TASK B: Epidemiology (Task leader: ONCFS)
The work of Task B was developed under five work packages as described further:
Work package 6: Comparative evaluation of classic and alternative control strategies in domestic pigs based on economic analysis of CSF-spread model (WP leader: UFZ)
Objectives
The objectives of WP6 were the following:
1) The conceptualisation and implementation of an expert-agreed evaluation model;
2) The statistical analysis of available data preparing agreed and uncertain parameterisation;
3) The validation of the epidemiological model (data-driven and pattern-oriented);
4) The reference simulation for EU strategies by strategic efficiency, costs and associated risks;
5) The comparative simulation experiment for alternative strategies defined in the project;
6) The quantitative analysis of cost-benefit and efficiency of strategies for comparison.
Summary of results of the WP
From simulations of the CSF-spread model the following conclusions could be drawn:
(1) emergency vaccination with a live vaccine in a zone of 3.000 m around detected outbreaks, as control measure is
(i) (at least) as effective as pre-emptive culling of the 1.000 m zones,
(ii) can be used to replace the usual pre-emptive cull of 1.000 m zones, and
(iii) as safe as pre-emptive culling after applying the standard protocol to lift restrictions;
(2) emergency vaccination applied by oral administration via feed would increase efficacy and safety of measures due to greater performance in mass-application;
(3) emergency vaccination is a practical control alternative to prevent mass-destruction of (non-infected) livestock;
(4) emergency vaccination should be equipped with rapid testing procedures that allow demonstration of absence of virus in order to
(i) resolve issues with growing animals within restriction zones (e.g. test to slaughter) and
(ii) enable strategy amendments that increase efficacy (e.g. prolonged restriction zones) and hence increase safety of restricted areas prior to end-screening measures;
(5) emergency vaccination combined with pre-emptive culling is a possible but futile concept;
(6) emergency vaccination will require commercialisation of a limited number of pigs that are seropositive to vaccine virus;
(7) emergency vaccination with vaccine following the DIVA principle would allow the demonstration of success of the control measure during and after the final diagnostic screening to regain the CSFV free status. Follow-up of the epidemiological situation during the epidemic, however logically, will be based on rapid testing for virus presence.
Work package 7: Model based analysis of field data on CSFV transmission and persistence in the wild (WP leader: ONCFS)
Objectives
The objectives of WP7 were the following:
1) To define the critical (key) parameters responsible for CSF persistence in the wild and to explore in particular the effect of hunting pressure and seasonality, landscape structure, population size and wild boar demography;
2) To provide an operational tool to predict CSF spread and persistence in a given wild boar population and to evaluate the efficacy of oral vaccination of wild boar using the C-strain or the new marker vaccine in different designs of baiting;
3) To theoretically evaluate the relative performance of complex strategies combining vaccination and human induced modifications of the demography of wild boar (i.e. hunting and contraception).
Summary of results of the WP
During the first project period, an analysis of the correlation between the 'pro-active', 'contemporaneous', 'post-active' vaccination treatment and time horizon with circulating infection was performed. The key-parameters of circulation and persistence of CSF in wild boar based on data and literature were reviewed and the spatially-explicit individual-based model was validated.
Work package 8: Descriptive epidemiology in backyard pigs of classical swine fever (WP leader: UCM)
Objectives
The main objectives of this work package were, firstly, to predict the population and geographical distribution of backyard pigs (BYP) in the EU and, secondly, to evaluate the epidemiological role that BYP have in the CSF spread.
Summary of results of the WP
The first objective was accomplished by using a two-step modelling approach based on generalized linear mixed models using up to 52 socio-economic and cultural factors to estimate the BYP density at NUT3 level in the whole EU. This approach allowed us to produce a BYP density map at NUT3 level for the whole EU (which is already available). Moreover, the model revealed that BYP are concentrated in areas with traditional agricultural practices, particularly in regions in which agricultural holdings have mixed livestock and a low standard gross margin, farm owners are old (i.e. age 55-66) and labour force is familiar, among other factors. Results of the BYP density map produced under this WP also agreed with areas identified by experts to have high abundance of BYP in an expert opinion elicitation conducted during 2012.
The evaluation of the epidemiological role of BYP for CSF spread was performed only for Bulgaria, as it was the only country with detailed information available, and it was approached in two ways. Firstly, we evaluated whether or not BYP is a risk factor for CSF occurrence by using the Bayesian logistic regression model. Results of the statistical model revealed that BYP is a significant risk factor for CSF occurrence in Bulgaria, but not the most important one. In fact, high risk areas for CSF occurrence were those concentrating a high number of other low biosecurity farms, particularly family farms type B and East Balkan pig herds, as well as those farms with high number of outgoing pig shipments and low levels of personal consumption (i.e. economically deprived areas).
Work package 9: Early warning systems of CSF and surveillance in domestic pigs and wild boar (WP leader: FLI)
Objectives
Major goal of WP9 was the development of an optimized surveillance strategy focusing on the situation in the industrial sector, backyard pig populations and wild boar based on model-based simulations. As a first step, the currently applied surveillance strategies and early warning systems regarding CSF in domestic pigs and wild boar were reviewed and statistically described. The review included both CSF affected and non-affected countries and was combined with the development of a new statistical methodology to calculate corrected sample sizes for finite populations.
Summary of results of the WP
Surveillance strategies and legal provisions regarding CSF in domestic pigs were reviewed in the framework of subtask 9.1 using records and surveys from different regions of the EU. Thereby, also the statistical properties of the employed systems were assessed.
One point with optimization potential was found to be the sample collection: sampling schemes that are laid down in EU legislation do not correct for test properties such as sensitivity and specificity of diagnostic tests. To point out possible ways of improving the system and thus increasing reliability, the current system was compared to a corrected sample size that takes into account test characteristics. The corrected sample was optimized in such a way that it provided at least one true positive test result if the true prevalence was higher than the design prevalence. Furthermore, a simulation study was conducted to demonstrate the effect of different herd size distributions and various prevalences at animal and herd level on the design prevalence. It could be concluded that the currently used virological and serological tests applied in combination with the current sampling strategy is suitable to attest freedom from disease for the design prevalences and confidence levels specified in the CSF diagnostic manual of the European Union. Targeted sampling on clinically diseased animals (e.g. febrile pigs) would allow minimizing the number of virological tests. However, in order to correct for the properties of the combined test the number of examined animals must be increased. In countries with a high number of backyard pigs (low average herd size, high number of herds) the sample size, false and true test results increase significantly when the same monitoring and test parameters are used. Therefore, attesting freedom from CSF in these areas becomes difficult by solely relying on laboratory testing given non-perfect test properties.
Work package 10: Explorative molecular epidemiology (distribution, clustering, species specificity) (WP leader: TiHo)
Objectives
The aim of WP10 was to provide information on the epidemiological situation of CSF in countries with a high prevalence of backyard pig farming such as most countries of the Balkan Peninsula in order to facilitate epidemiological investigations and risk analyses. This included the characterization and phylogenetic analysis of recent CSFV strains from the Balkan region as well as a complete genome sequencing of selected strains using conventional as well as high capacity sequencing techniques. To make the obtained information available for use in subsequent studies sequences and epidemiological data were entered in existing databases. Moreover, this WP was aimed at the characterization of a quasispecies structure of CSFV by the analysis of the NS5B region of the genome in order to study the impact of a potential quasispecies structure on epidemiology as well as on in vitro results.
Summary of results of the WP
With the intention to shed light on the overall epidemiological situation in countries with a high proportion of backyard pig farming such as countries of the Balkan Peninsula, molecular epidemiology was performed with a total of 97 CSFV isolates originating from outbreaks that occurred in South-Eastern European countries between 1994 and 2007. After having obtained these isolates from collaborating National Reference laboratories, they were subjected to partial sequencing (fragments of the 5'NTR and E2 gene) and subsequent phylogenetic analysis. The genetic typing showed that almost all isolates belonged to genotype 2.3. On the basis of these sequences and additional sequences from outbreaks in Eastern and Western European countries obtained from the EU and OIE Reference Laboratory for CSF in Hannover (EURL) CSF virus database, two clusters were distinguished within subtype 2.3. These were tentatively named 2.3.1 and 2.3.2. Beyond the molecular studies, also an attempt was made to elucidate the overall epidemiological situation by compiling all data presented in the framework of different TAIEX (technical assistance and information exchange instrument of the institution building unit of directorate-general enlargement of the European Commission) events as well as data available from EU and OIE workshops. This information together with data obtained through molecular epidemiology was made available through publication (Blome et al., 2010). Moreover, the used isolates were integrated in the virus collection of the EURL, and sequence data as well as basic epidemiological data was uploaded into the CSF virus database.
TASK C: Improve detection and diagnosis and validation (including diva diagnostic tools) (Task leader: SVA)
The work of Task C was developed under three work packages as described further:
Work package 11: Evaluation of currently available antibody ELISAs for DIVA purposes. Characterisation of monoclonal antibodies and antigens for potential use in ELISAs (WP leader: TiHo)
Objectives
In the framework of WP11 an evaluation study of commercially available CSFV antibody ELISAs was implemented to decide which ELISA can be used for DIVA purposes in combination with 3rd generation modified life CSFV marker vaccines. Additionally, for the improvement of existing ELISAs or for the development of new tests monoclonal antibodies as well as novel antigens were characterized. Based on the outcome of these studies, suitable ELISAs were selected for an evaluation within an inter-laboratory comparison test. This was aimed at the determination of statistical measurements in order to draw conclusions about the feasibility of implementation of the selected 3rd generation marker vaccine and DIVA diagnostic tools for the control of CSF.
Summary of results of the WP
To determine the DIVA potential of commercially available test systems, seven CSFV E2 and Erns antibody ELISAs, respectively, were assessed in a preliminary inter-laboratory comparison test. The E2-ELISAs CHEKIT* CSF Sero (IDEXX CSF Sero Ab Test) and HerdChek* CSFV Ab (IDEXX CSFV Ab Test) performed best with regard to sensitivity. Furthermore, both tests showed a good practicability and reproducibility, making them the most promising DIVA ELISA candidates for a DIVA strategy with marker vaccines containing the E2 protein of Bovine viral diarrhea virus (BVDV) (e.g. Flc9). Additionally, both E2-ELISAs achieved best results in terms of sensitivity with animals vaccinated with CP7_E2alf and could therefore be used for the detection of CP7_E2alf marker vaccinated animals. The Erns-ELISA CHEKIT* CSF Marker (IDEXX CSFV Marker Test) achieved also a good sensitivity and reproducibility. However, as this test cannot distinguish between pestiviruses, it was ruled out for a DIVA strategy with chimeric vaccines that contain the Erns of ruminant pestiviruses (e.g. CP7_E2alf and Flc11). The PrioCHECK® CSFV Erns ELISA (Prionics) was the only test suitable for this purpose, even though some improvements were needed with regard to its sensitivity, selectivity, and especially robustness. Since the marker vaccine CP7_E2alf was selected as final vaccine candidate, great efforts were made to improve the PrioCHECK® CSFV Erns ELISA.
Work package 12: Development of alternative new methods for DIVA detection of nucleic acids and antibodies for laboratory use and for on-site testing of domestic pigs and wild boar (WP leader: SVA)
Objectives
The aim of WP 12 was to develop and test novel methods for the detection of nucleic acids and antibodies in pigs and wild boar infected with CSFV or vaccinated with Cp7E2alf. The work package was divided in several subtasks. The first subtask was dealing with sample preparation and enrichment methods, the second with suspension microarray based DIVA assays, the third with basic methods for detection and differentiation of viral nucleic acids, the fourth with evaluation of a portable PCR assay for on-site detection and the fifth with standardization of the methods.
Summary of results of the WP
The work with the first subtask demonstrated that methods only requiring basic technology can be used for successful extraction of CSFV RNA in the field or in basic laboratories. It was demonstrated that the use of filter paper, like FTA paper (GE healthcare), is a very convenient, simple and economical method for collecting; storing and transporting samples as well as extracting RNA from samples of potentially CSFV infected pigs or wild boars. Because of its robustness and simplicity this method is very suitable for use in the field and because it is an economical method, larger number of animals can be sampled. This sampling procedure is convenient to use in combination with field based PCR assays. Elution of RNA from the filter papers can be done by submerging filter paper punches in a simple elution buffer and following a very simple protocol. Our results demonstrated that there is an upper limit to the number of filter paper punches that can be eluted together without causing inhibition in subsequent RT-PCR steps. DMPK B and FTA-Elute are superior to FTA Classic cards in this setting. The use of handheld magnet in combination with reagents from Nordiag and magnetic beads for extraction of nucleic acids was tested as an alternative to filter papers (Dynal MPC-S, Dynal Biotech, Norway). It was concluded that the handheld magnet gives a faster and more reproducible result under different environmental conditions but it involves more steps than the filter paper method and is therefore more cumbersome. The method takes around 90 min to complete a preparation of 10 samples. These collection and extraction methods simplify sampling and submission of samples to the laboratory and enable analysis directly on site, thereby providing a rapid result to support further actions. Even though the field sample preparation methods yielded a lower amount of viral nucleic acids, they are useful for speeding up the diagnostic process.
For the second subtask, the Luminex xMAP technology, which has become more and more widely used for multiplexing detection of agents, was used. This task started with the development of a suspension microarray assay for the detection and differentiation of both wild type and CP7_E2alf vaccine RNAs simultaneously. The assay had similar sensitivity for the detection of wild type viruses and 10-fold lower sensitivity for the marker vaccine, compared to real-time RT-PCR assays. When evaluated in a duplex format, the sensitivity was reduced a 10-fold further. Unlike wild type viruses, the marker vaccine candidate CP7_E2alf replicates only transiently in pigs upon vaccination, yielding very low levels of viral nucleic acids in a very short time period, close to the detection limit of the highly sensitive real-time RT-PCR. Based on these results and the fact that the Luminex assay is also more labor intensive and more expensive, it was concluded that the Luminex assay for the detection of nucleic acids is not a suitable alternative DIVA method. However, a serological DIVA assay based on the Luminex technology could be beneficial for the mass screening of serum samples.
Therefore, the focus was shifted to a serological assay. To develop a serological DIVA assay, three viral proteins, namely BVDV Erns, CSFV Erns and E2 were produced in insect cells using the baculovirus expression system, and subsequently purified by affinity chromatography. The assay was evaluated by testing 365 samples and compared to ELISA. Out of the 365 samples, 360 samples were correctly diagnosed by the E2 antibody detection, resulting in a sensitivity of 98.5% and a specificity of 98.9%. For the CSFV Erns antibody detection, 155 out of the 187 samples reached the same results by both the microsphere immunoassay and ELISA. Some samples had different results between the Luminex assay and ELISA that was still under further improvement. BVDV Erns antibody detection had low MFI values (between 27 and 6720 with a median value of 762). As the MFI values from vaccinated pigs were slightly higher than those from naïve pigs, a very low antibody response to BVDV Erns antigen in pigs upon vaccination with CP7_E2alf was suggested. The DIVA potential of these assays was confirmed by testing well defined samples. The multiplex immunoassay offers a new format for high-throughput DIVA diagnosis of classical swine fever and marker vaccine CP7_E2alf. It could be used in reference laboratories as a confirmatory tool.
In the third subtask the loop-mediated isothermal amplification (LAMP) assay for basic laboratories was tested. For field application, both magnetic bead array (MBA) and LAMP are suitable, as they don't required advanced instruments. Further comparison of both methods found LAMP was better suitable for field condition as it required fewer steps and should give quicker results compared to MBA. The results showed that the sensitivity of the RT-LAMP was 10-fold less than real-time RT-PCR for the detection of marker vaccine CP7_E2alf. However, this is a very useful assay for use in laboratories equipped with basic instruments only. The only instrument needed to perform this assay is a heating block or even a simple stove and results can be read by naked eye. Combined with sample collection and extraction on filter paper, the LAMP assay is a very economical of detecting infected animals that can be used under very simple conditions in the field or elsewhere.
For a similar purpose the fourth subtask was devoted to the evaluation of a portable PCR instrument for use in the field. Initially an instrument from Smiths detection was the focus of attention. This was a very complete and safe, but very expensive instrument. This instrument was subsequently withdrawn from the market. Therefore the smartcykler from Cepheid was tested. This instrument gave excellent results in our trials. The instrument can be used in the field but needs high voltage and therefore a generator must accompany the instrument. Subsequently a truly field based PCR instrument from Tetracore was chosen. This instrument, T-Core 4, is portable, runs on batteries or connected to the grid and has four cells for performing PCR. Results are calculated and displayed in a connected lap top computer. Tetracore supplies wet and dry RT-PCR kits for use with the T-Core 4 and both were tested in our experiments. Initial experiments gave poor results due to technical problems with the PCR instrument. These problems were later resolved and the instrument and the accompanying wet and dry kits were tested with the Tetracore instrument in parallel with other PCR instruments (Rotorgene and Smartcykler). Both the wet and dry kits contain an inhibition control labelled with Cy5. The portable PCR was tested using a serial RNA dilution of the EU CSFV reference sera 1 and 2 and serial dilution of CSFV Koslov. Standardization was integrated in each subtask.
Work package 13: Development and validation of new methods for the easy selection of suspicious animals (infrared thermography) and sample reduction (pooling) (WP leader: TiHo)
Objectives
In the framework of WP13, the evaluation of infrared thermography as a non-invasive tool for a risk based sampling scheme in case of animal disease outbreaks in pigs (CSF) was in the foreground. In an overall evaluation the assessment of practicability and reliability for detection of febrile animals in pig groups or herds, the application and validation under defined conditions, and terminal tests in the field were included. Moreover, WP13 was aimed at the evaluation of pooling of samples as a method to reduce the vast number of samples that have to be analyzed during an outbreak of CSF.
Summary of results of the WP
Various infectious diseases are accompanied by fever as an early and reliable symptom. For this reason, risk based sampling schemes for animal disease outbreaks like CSF are based on this clinical sign. In practice, external factors like ambient temperature and stress reactions related to rectal temperature measurement hamper the implementation. In order to overcome this problem, as well as to simplify the selection of suspicious animals, the non-invasive infrared thermography technique for the measurement of skin temperature was tested and evaluated.
With the analysis of infrared thermography pictures of CSFV infected pigs in experimental settings, as well as of pigs in commercial fattening and breeding piggeries, a significant correlation between skin temperature of several body regions and rectal temperature was found. However, it was ascertained that several technical-physical factors (sharpness of the images, correct angle between camera and object), as well as environmental parameters (ambient temperature, relative humidity, air movement, solar radiation, other sources of warmth, skin contamination) have to be considered to gain significant results. Especially on weaner pigs the ambient temperature had a significant effect. This indicated that also individual parameters (age, sex, biological status, psychological stress, feeding, skin irritation, local inflammation, topography of the measuring point) influenced the infrared measurement considerably. The lack of robustness of this method became also apparent by measurement of pig groups. Since the movement of the pigs was fast and the space for movement large, it was not possible to take pictures in groups of small pigs and groups with more than 15 animals. These results underlined the complexity of the infrared thermography technique and the need for experience and background information to obtain reliable results.
Together with a lack of sensitivity, selectivity, and robustness, it is doubted that infrared thermography is applicable as a diagnostic tool to select diseased animals in CSF outbreaks under field conditions. Fitness for purpose cannot be testified without reservation.
Outbreaks of CSF in domestic pigs or wild boar populations are accompanied not only by a vast number of pigs that have to be clinically investigated but also by vast numbers of samples that have to be investigated for CSF virus, antigen or genome, and antibodies. In this context, diagnostic capacities available at the laboratories can be bottle necks and may hamper rapid analyses of samples. Sample pooling, especially for highly sensitive real-time RT-PCR assays, can greatly augment the number of samples that can be analyzed without having to increase the capacity of the laboratory and without an increase in associated costs. To assess the diagnostic reliability an evaluation of serum sample pooling with regard to overall performance and influence of sample quality was performed. Especially for the pooling of wild boar samples from monitoring and surveillance programs, sample quality might be a critical factor as these samples often arrive in bad to almost non-existent quality.
TASK D: Improve knowledge on immunological reaction directly related to vaccine improvement (Task leader: ANSES)
The work of Task D was developed under one single work package as described further:
WP14: Improve knowledge on immunological reactions directly related to vaccine improvement (WP leader: Anses)
Objectives
The CP7_E2alf was compared to the C-strain for its immune responses modulation when applied intramuscularly or orally at different times before challenge. Further objectives were to understand the early mechanisms induced at the tonsil level after oral immunization and their implication in reducing the virus replication, to identify possible differences between immune cells derived from wild boars or domestic pigs, and to evaluate the possible interference of maternal antibodies against vaccination efficacy.
Summary of results of the WP
The C-strain has been used for years to vaccinate wild boars by means of distribution of baits. When orally applied the C-strain is able to protect piglets as soon as three days after vaccination. Here, CP7_E2Alf has been evaluated for its potency to induce a very early immune response and for the mechanisms behind this protection. Other samples obtained from the WP3 on efficacy trials permitted to investigate later challenge protocols.
TASK E: Alternative methods of suppression of viral replication(Task leader: VAR)
The work of Task E was developed under one single work package as described further:
WP15: Exploration of alternative methods of suppression of viral replication (WP leader: VAR)
Objectives
This WP was aimed at exploring a number of alternatives to stamping and vaccination, as potential aids in the control of CSF outbreaks. These included the evaluation of the potential of antiviral treatment to reduce CSF virus transmission as well as the assessment of the risk for the development of antiviral resistance against this molecule. Further objectives were the evaluation of the capability of live attenuated vaccine strains to interfere with wild type CSFV replication and the assessment of the potential of RNA interference by testing selected small interfering RNAs (siRNAs).
Summary of results of the WP
The molecule 5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is an antiviral molecule belonging to the class of the imidazopyridines and had previously been demonstrated to be an active inhibitor of the replication of CSFV in vitro. In WP15, an animal experiment was performed to evaluate the efficacy of BPIP in preventing CSF virus transmission from treated to untreated pigs. The study showed that a 15-day treatment with BPIP administered via the feed was able to reduce, but not completely prevent, virus transmission from CSFV infected to sentinel animals. Treatment with BPIP reduced the duration of viraemia in CSFV infected pigs resulting in a lower occurrence of virus transmission to sentinel animals and a lower genome load measured by RT-PCR in the tonsils from these sentinel animals. The results of this study demonstrated the potential of antiviral treatment as an early emergency measure in the case of a CSF outbreak.
In order to evaluate the risk and the genetic mechanisms for the potential development of antiviral resistance against this molecule, the quasispecies structure of the NS5B genome region, the area targeted by the antiviral molecule, was determined by cloning and sequencing and was compared between the wild type CSFV strain Wingene and its BPIP resistant variant. The antiviral resistant variant was generated in-vitro by subsequent passaging in the presence of increasing concentrations of BPIP. The study showed that intra-host variability was present in the wild type Wingene population and increased significantly when antiviral pressure was applied. Mutations found in the BPIP-resistant variant suggested that more than one mutation was responsible for the development of the resistant phenotype. Although no clear evidence was found for the presence of published resistance associated mutations in the wild type Wingene population, the finding of one particular mutation (R3407H) in the wild type and BPIP-resistant Wingene population drew our attention as this mutation was mapped to a resistance associated region in NS5B. These findings clearly highlight the usefulness of quasi-species analysis for the research in future antiviral therapy.
Also in WP15, the effect of RNA interference (RNAi) on CSFV infection was studied in vitro. Small interfering double-stranded RNA molecules (siRNAs) targeting the non-structural protein NS2-3 and 3´ non-translated region (3´NTR) encoding genome region of the recent German CSFV field isolate 'Roesrath' were used to assess RNA interference with homologous as well as heterologous CSFV strains. It was demonstrated that the selected siRNAs were able to inhibit viral replication after homologous infection using current CSFV strains of genotype 2.3. No complete inhibition but a rather diverse repression of CSFV replication could be attained in vitro. Due to the requirement for a 100% sequence homology the effect of in vivo application of siRNAs as antiviral drugs seems to be limited. In addition it has been reported that the development of successful in vivo siRNA delivery systems pose significant challenges, also for other viruses.
In a final part of WP15, the potential role of virus interference in early protection against CSFV vaccination was assessed. An in vitro study was carried out using the CP7_E2alf vaccine virus for super-infection of persistently CSFV infected as well as uninfected porcine kidney cell lines. The results indicated a powerful viral interference capacity of CSFV infection.
Management of the project and other activities (WP16 and WP17)
WP16: Management of the project (WP leader: VAR)
Objectives
The objective of this WP was the overall management of the project including the signing of Consortium and Grant Agreements and the monitoring of compliance herewith, the regular communication with the EC scientific officer, the maintenance of the project website and the EC webportal via ECAS, the preparation of Amendments to the Grant Agreement and of the periodic and final activity reports, the organisation of 6-montly project meetings and of the dissemination meeting, the management of the financial aspects of the project and disbursements of funds to the partners, the general responsibility and accountability for progress of the project against milestones and deliverables as specified in the grant agreement.
WP17: Other activities (WP leader: VAR)
Objectives
The objectives of WP17 were the development of the project website, the inclusion of a potential new beneficiary or invitation of scientists from Romania, Bulgaria or other relevant third countries, and the dissemination of the final results of the project.
Summary of results of the WP
Collaboration with scientists from Romania was attempted for the work concerning the epidemiology of CSF in backyard pigs but proved to be unsuccessful in the end. Collaboration with the Bulgarian authorities was established instead and the necessary data to perform the work on backyard pigs were obtained from Bulgaria.
The development of the project website and the dissemination of the final results of the project are described in the next section of the report.
Potential impact:
Socio-economic impact and wider societal implications
Classical swine fever (CSF) is among the most important diseases of domestic pigs worldwide and is notifiable to the World Organization for Animal Health (Office International des Epizooties, OIE). Due to the tremendous socio-economic impact of CSF outbreaks, emergency vaccination scenarios are continuously under discussion within the European Union. Currently available CSF vaccines show restrictions either in terms of marker capacities, immunogenicity or suitability to be applied orally for wild boar vaccination. Project CSFV_GODIVA was dedicated to improving the tools and strategies for the prevention and control of CSF, including the development of a new modified live CSF marker vaccine for domestic pigs and wild boar.
In order to improve tools and strategies for the prevention and control of CSF, the work developed under CSFV_GODIVA project aimed to fulfil the following objectives:
- the development of a new live CSF marker vaccine with DIVA (Differentiation of Infected from Vaccinated Animals) diagnostic assay for domestic pigs and wild boar;
- the evaluation of the benefits of CSF emergency vaccination of domestic pigs and the vaccination of wild boar;
- the improvement of wild boar baiting systems;
- the study of the epidemiology of CSFV in backyard pigs;
- the evaluation of CSF surveillance systems in domestic pigs, wild boar and backyard pigs;
- the assessment of the potential use of anti-viral treatment to reduce CSFV transmission;
- the study of the immunological reactions after CSFV infection and vaccination.
These objectives were organised in five tasks and were outlined in the workplan providing a logical process for improving of knowledge on critical aspects of CSF and the development of a live marker vaccine allowing the DIVA principle.
By including several National reference laboratories (NRL) and the European community reference laboratory (CRL) for CSF and a Chinese partner, the project reinforced the European network of experts for European research in epidemiology and diagnostics in CSF.
Several outcomes of the project emphasis its successful contribution to improve tools and strategies for the prevention and control of CSF:
A new marker vaccine has been submitted for registration and will allow improving CSF eradication in the future:
Vaccination as an alternative to pre-emptive culling will reduce the number of healthy pigs that are destroyed. The ultimate deliverable of the project was to present a marketable live marker vaccine for intramuscular administration to domestic pigs and for oral application in wild boar. A few months before the finalization of the project the dossier for marketing of the injection vaccine was submitted for EMA. Several studies also target the use of the oral vaccine but the initial application is on injection. The dossiers were produced through a very constructive collaboration between the industrial partner and all research partners. The new DIVA vaccine has been analysed in comparison to the C-strain vaccine both for safety and efficacy. Humoral and cellular immunological parameters have been investigated both after vaccination and after subsequent challenge of vaccinated pigs in various age groups. The emergency use of the vaccine has been analysed by reducing the time from vaccination till the highly virulent challenge. The impact of maternal immunity and effect on pregnant animals has also been analysed.
The vaccine has been tested as a bait vaccine for wild boar or for oral application in domestic backyard pigs. The baits have been evaluated in field trials in France and Italy. The main obstacle for the bait uptake seems to be the availability of other food sources. With plenty of usual food the baits will have too much competition.
Improved CSF diagnosis:
The DIVA vaccine is only of use if accompanying DIVA diagnostics is available. The available serological DIVA diagnostic systems were validated and compared in a review. Within the consortium and in collaboration with external partners new improved diagnostic tests were produced and the available tests improved. As the vaccine is a live vaccine further DIVA tests that can differentiate between the wild-type virus and the vaccine virus was needed, a genetic DIVA was developed and the test was further implemented for a field portable PCR assay.
Development of epidemiological tools for a better understanding of CSF and to support decision makers:
A comprehensive study on the epidemiology of CSFV in backyard pigs and wild boar has been an impressive support for the cost/benefit decisions on when to vaccinate and to decide which groups are at risk. Based on the model an evaluation of CSF surveillance in domestic pigs, wild boar and backyard pigs could be tested with highlights of strengths/weaknesses. The model will assist the European decision makers and be a valuable tool in emergency situations when enormous socio-economic decisions are taken.
From simulations of the CSF-spread model it can be concluded that emergency vaccination is a practical control alternative to prevent mass-destruction of (non-infected) livestock if accompanied with rapid testing procedures that allow demonstration of absence of virus. Only a limited number of pigs that are seropositive to vaccine virus will be commercialized. In addition emergency vaccination with the novel DIVA vaccine would allow the demonstration of success of the control measure during and after the final diagnostic screening to regain the CSFV free status. Follow-up of the epidemiological situation during the epidemic, however, will be mainly based on rapid testing for virus presence. For this purpose, PCR strategies were developed within the project that is able to differentiate field CSFV and vaccine virus. For domestic pigs the currently used virological and serological tests applied in combination with the current sampling strategy are suitable to attest freedom of disease as specified in the CSF diagnostic manual of the EU.
Alternative methods were studied:
An antiviral molecule belonging to the class of the imidiazopyridines administered via the feed was able to reduce but not completely prevent CSFV transmission from treated CSFV infected to sentinel animals. Proof of principle for the efficacy of antiviral treatment against CSF transmission was shown.
Transfer of knowledge on relevant aspects of CSF and transfer of diagnostic and epidemic tools through training and traineeships to a large number of professionals, decision makers and scientist.
The project provided employment, education and training for young post-graduate and post-doctoral researchers. They gained intra and inter-disciplinary experience and training working within an international, multidisciplinary team including working with industry and in a high level biosecurity environment. In addition young scientists were trained in presentation opf results and the compilation of manuscripts for publication.
The outcomes of the project will benefit EU consumer driven concerns with regard to animal welfare, food safety and food quality and will undoubtedly contribute to the sustained development of EU agri-food business by improving prevention, early detection and control of CSF and therefore reducing the socio-economic consequences of potential incursions of the disease from infected regions. In addition, assistance in the preservation of small to medium sized family farms, as viable economic units within a rural environment will contribute to maintaining rural infrastructure of EU regions and member states.
Main dissemination activities and exploitation of results
Exploitation and dissemination of the results was considered by the consortium to be vital. Dissemination of the results of the project was fulfilled through a series of internal and external dissemination activities.
Internal dissemination
The consortium was composed by a multidisciplinary scientific team from 17 partners based in 11 different countries. The ability of the consortium to successfully complete the project was attributed to the scientific expertise in different fields including vaccine development, virology, diagnostics, molecular biology including sequencing, immunology, epidemiology and disease surveillance/control, conduct of animal experiments, wild boar baiting and field trial experience. The presence of an industrial partner in the project led to the development of a CSF marker vaccine that can be produced at an industrial scale and that could become commercialised after registration.
Internal dissemination between partners was achieved through the organisation of 6-montly project meetings, teleconferences, communication on the project website via Mid Monthly Messages, training of scientific staff and exchanges of personnel between institutes; transfer of laboratory tests and test protocols between partners, detailed reporting including full materials and methods in the periodic reports to the EC.
Training of scientific staff and exchanges of personnel
Ilse vangeel (VAR) visited the Animal Health Department of the complutense university of Madrid (UCM) for two weeks in February 2011. The purpose of the visit was to learn a number of epidemiological methods that are used to study the epidemiology of epizootic diseases and for disease surveillance, control and risk analysis. Ilse Vangeel was trained by Dr. Beatriz Martinez-Lopez into the following methods: social network analysis, cluster analysis, geographic information systems (GIS) and Bayesian modelling for risk factor and risk analysis.
Desislava Yordanova Rangelova (DTU-Vet) visited Anses, Ploufragran for 2 weeks in 2012. Her travel expenses were funded by an EPIZONE Short Term Mission. She was trained by Patricia Renson to perform isotype specific antibody ELISAs in the laboratory. After her training, the test protocols were transferred from Anses to DTU-Vet. As part of the collaboration, a joint publication is being prepared for submission to Veterinary Research.
External dissemination
External dissemination of results was achieved through the following series of activities:
- Publications in scientific peer reviewed journals;
- Oral and poster presentations at conferences and scientific meetings;
- PhD theses;
- Organisation of the project's dissemination meeting;
- Project website (see http://www.csfvaccine.org online);
- Reports to the EC.
The CSFV_GODIVA dissemination meeting
The dissemination meeting organised by the coordinator was held in Brussels on the 31st of January 2013. A total of 68 participants attended the meeting: 31 members of the CSFV_GODIVA project and 37 guests from international institutions EU DG Research and DG Sanco and OIE, and national institutions for food safety, public health, nature and forestry; ministries, farmers unions, universities and other research institutions. Participants included representatives from 12 different EU countries and from Switzerland and Russia. Major achievements of the project were presented by several partners of the consortium. Presentations were given about the development of the new marker vaccine, the development of improved and new DIVA diagnostic tools, the comparative evaluation of CSF emergency control strategies in domestic pigs, field studies for vaccination of wild boar, the risk of backyard pigs for the transmission of CSF to EU countries, immunology of CSF, and the potential of antiviral treatment to reduce CSFV transmission. The meeting ended with a round table discussion with active participation from the audience.
Development of the project website
The website for the CSFV_GODIVA project (see http://www.csfvaccine.org online) was launched during the first project meeting in Brussels in May 2009. The website contains an area which can be accessed by the general public with general information about the project such as background, objectives, partners, description of work packages; and a restricted access area which can be accessed only by members of the consortium. The latter part of the website was used to share documents such as meeting presentations, meeting summaries, milestones and deliverables documents, periodic reports etc.
List of websites:
http://www.csfvaccine.org.