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Improvement of current and development of new vaccines for theileriosis and babesios of small ruminants

Final Report Summary - PIROVAC (Improvement of current and development of new vaccines for theileriosis and babesios of small ruminants)

Executive Summary:
PiroVac is a major international research project designed to develop control measures to combat two major tick-borne diseases of small ruminants, namely theileriosis and babesiosis. This EU-funded research programme aims at improving existing vaccines, designing new vaccines and on capacity building in partner laboratories both in Europe and in endemic areas.
Small ruminant piroplasmosis is a major threat to livestock production in many areas of the developing world. Theilerosis and babesiosis, caused by the protozoan parasites Theileria lestoquardi, T. uilenbergi and Babesia ovis, infect sheep and goats causing disease, production loss and sometimes death. Consequently, these diseases have a major impact on animal welfare and stock-holder prosperity throughout the world.
By developing effective measures to control these important diseases, the PiroVac project represents a major contribution to achieving the United Nation's Millennium Development Goals of food security, food safety, poverty alleviation, animal welfare and environmental sustainability.

PiroVac is a collaborative effort among a number of established research groups working on theileriosis and babesiosis. The consortium also encompasses laboratories involved in malaria research in order that scientific and technological knowledge in that field can be translated into tools and reagents for small ruminant piroplasms. Industrial expertise in vaccine development and delivery systems has also been incorporated in order to maximise the potential for translational application.

The PiroVac project was developed as an integrated approach, encompassing immunology, molecular biology, bioinformatics and genetic engineering together with pathogen genomics and host genetics which is directed at addressing two broad aims:

(1) Development of effective and reliable vaccines for use in disease control campaigns for sustainable livestock development.
(2) Capacity-building for the sustainable implementation of integrated control measures required for disease control and/or eradication through increasing scientific knowledge, training and improvement of infrastructure.

The specific goals of the project are:

(1) Improvement and development of live attenuated vaccines for the control of small ruminant theileriosis and babesiosis through determining the effectiveness of attenuation using:

a) in vivo assessment of attenuation, analysing clinical and immunological criteria (both humoral and cellular responses) of immunised and challenged animals
b) subtractive libraries and microarray analysis for the identification of attenuation markers

(2) Sub-unit vaccine design through:

a) Identification of suitable antigens using a combination of genomics, bioinformatics and gene expression analysis coupled with experimental confirmation of antigen localisation and presentation. To facilitate antigen discovery, parasite molecules involved in host cell invasion, activation of cytokine-producing CD4+ T cells and NK cells and activation of cytotoxic T-lymphocytes involved in killing of T. lestoquardi-infected leucocytes will be identified.
b) Immunological characterisation of the identified antigens as potential vaccine candidates.

(3) Vaccination trials using:

a) live attenuated vaccines
b) recombinant protein and DNA vaccines

The PiroVac project contributed to the understanding of the immunological and molecular mechanisms involved in host-pathogen interaction. Tools for the detection and identification of the tackled parasites were developed, reagents required for the characterisation of the innate and adaptive immunity of small ruminants were generated and genomes of the parasites were sequenced. The attenuation of T. lestoquardi was successful an attenuated strain of T. lestoquardi was evaluated as to its potential as a vaccine candidate.
PiroVac held a number of project meetings and was co-organising an international scientific conference “Emerging and Re-Emerging Epidemics Affecting Global Health”, held 2012 in Orvieto, Italy. Knowledge dissemination was performed via scientific exchange visits, giving young researchers from endemic regions the chance to visit a partner laboratory of the consortium. Links have been established to scientists outside the project, e.g. via joint conferences.

Project Context and Objectives:
In the context of global warming and globalisation, vector-borne diseases, including tick-borne diseases, are expected to emerge, with an increasing risk of pathogen transmission to animals and humans worldwide. Ticks transmit a greater variety of protozoan, bacterial, rickettsial and viral pathogens than any other arthropod vector group.
Moreover, ticks and tick-borne diseases (TTBD) pose a major constraint for the development and improvement of the livestock industry. They cause economical losses by decreasing milk production, effecting weight loss, and increasing risk for bacterial, viral and fungal infections. Hence, 80% of 1200 million cattle are at risk for TTBDs causing a global annual loss of US$ 7000 million. Although exact figures for individual diseases are not available, the economical loss alone due to T.parva-infection has been calculated to be about US$ 200 million and for T.annulata-infections to be about US$ 500 million.
Theilerosis and babesiosis, caused by the protozoan parasites Theileria lestoquardi, T. uilenbergi and Babesia ovis, are considered as a major threat to small ruminants in many areas of the developing world as they have a major impact on animal welfare and stock-holder prosperity throughout the world. Accordingly, they are ranked high in terms of their impact on the livelihood of resource poor farming communities.
Generally, management of TTBDs is primarily through control of the tick vector using acaricides, although this is unsustainable due to increasing acaricide resistance and food safety concerns. In a limited area of the endemic regions, attenuated live vaccines have been applied for bovine piroplasmosis (Theileria and Babesia), but not at the same level for small ruminant piroplasmosis. It is, therefore, imperative to work out integrated control measures including the development of efficient and appropriate vaccines. Nevertheless, this question was in the focus of the Pirovac project. The phylosophy behind this project was based on the integration of pathogen and host genomics in vaccine research, which may revolutionize the way scientists approach the challenge of discovering and developing safe and effective vaccines. Thus, the scientific challenge was to use the genomic data obtained in this and other projects to design tools and combine them with the huge progress in immunology, molecular biology, bioinformatics, genetic engineering and ultimately translate this progress into policy innovations and strategies, contributing to animal welfare through developing effective measures to control these important diseases.

The general objective of the project is to ensure food security and to improve food safety by improving existing and developing new vaccines against small ruminant piroplasmosis caused by T. lestoquardi, T. uilenbergi, and B. ovis.

Specific objectives
1. Improvement and development of live attenuated vaccines for the control of small ruminant theileriosis (T. lestoquardi and T. uilenbergi) and Babesiosis (Babesia ovis) through determining the effectiveness of attenuation using:

a. In vivo assessment of attenuation by analyzing clinical and immunological criteria (humoral and cellular responses) of immunized and challenged animals
b. Subtractive libraries and microarrays for the identification of attenuation markers

2. Design of subunit vaccine through:
a. Identification of suitable antigens using a combination of genomics, bioinformatics and gene expression analysis coupled with experimental confirmation of antigen localisation and presentation.
b. Immunological characterization of the identified antigens as potential vaccine candidates.

3. Vaccination trials using:
a. Live attenuated vaccines
b. Recombinant protein and DNA vaccines

Project Results:
To achieve the above mentioned goals, it was important to first focus on the creation of the required infrastructre. Thus, during the first 18 months several actions corresponding to the workpackages described in the proposal have been developed. For an efficient coordination of the consortium, two meetings were held during the first period to setup priorities. In the first line this included preparation of infective material, cell culture systems, antibodies, establishment of immunological assays for the evaluation of the host immune response against the parasites, gene sequencing, bioinformatics and preparation of DNA, RNA and cDNA. Indeed many of these requirment were successfully fulfilled. Briefly, the following major results were achieved:

• Tick colonies have been established in Germany, Sudan, Turkey and UK
• Infective material (Theileria, Babesia) has been distributed to different partners of the project. In addition, cell cultures have been successfully established in several laboratories of the consortium.
• DNA, RNA, cDNA and number of recombinant proteins have been prepared and used for the preparation of polyclonal antisera.
• Several genes have been identified, cloned, sequenced and recombinantly expressed. A few of these recombinant proteins have been used for the production of polyclonal antisera in rabbit.
• Sequencing of T. lestoquardi, T. uilenbergi and B. ovis has been initiated and the generated results were provided to the partners of the project in the second and third periods of the project for further characterisation and use.
• Database and bioinformatic analysis have been conducted on known genes of T. annulata, T. lestoquardi, T. parva and Plasmodium and Babesia parasites. Using a comparative approach to identify orthologes of proteins of the above mentioned parasites known to be involved in host-pathogen interaction have been cloned. For example AMAI-1, MyoA, GAP45, GAP50, MTIP, ICP, SIR2.
• A comparison of gene expression profiles was initiated and further experiments should put more light on key-differences that determine the virulence of the pathogens.
• NKp46 marker is considered as a reliable marker of NK6 cells. Given the role NK cells in the immune response to the infection with Theileria parasites experiments were successfully conducted to clone this molecule in sheep and a monoclonal antibody recognising this molecule has been generated in mice and were used for further characterisation of NK ovine cell.
• Experiments were performed to identify the role of cytotoxic T-lymphocytes in the protective immune response against T. lestoquadri infection. Based on the measurement of IFN-gamma by peripheral blood mononuclear cells of infected animals after in vitro stimulation with T. lestoquardi-infected cell lines. This system was used for studying the immune response of sheep to infection with T. lestoquardi and to vaccination in the second and third periods.

The above mentioned achievements and the generated knowledge were applied to address the tasks of the different workpackages.

Attenuation markers
Taking use of the progress achieved in T.annulata modell, experiments we conducted to look for the upregulation or downregulation of genes involved in the attenuation of T.lestoquardi such as MMP9 using RT-PCR. MMP9 is a recgnoized attenuation marker in T.annulata-infected cells and plays a crucial role in the metastasis of Theileria-infected cells into different organas of the infected hosts. It could be confirmed that in T.lestoquardi-transformed ovine cells, MMP9 is significantly down regulated, indicating that the attenuated cells due to long term cultivation loss substantially their capacity to infilterate hosts organs.
A part of MMP9, TNF-alpha is also downregulated in long term cultures of both T.annulata and T.lestquardi-infected cells.
Taking these data together, it seems that these parasites share many similar mechanisms regarding their virulence and metastasis capacity.
The role of sialic acid in the dissemination of cells infected with T. lestoquardi was also investigated. It was found that there are clear differences between attenuated and non- attenuated infected cells. These results showed changes in the amount of CD44 following long passages of the cell lines. In acomparative study, differences were also observed at the level of CD44 between T. annulata infected clonal cell lines that were sensitive or resistant to the anti-theilericidal drug (buparvaquone) treatment. The level of CD44 was higher in the resistant than in the sensitive cell lines.

Analysis of genetic diversity of T. lestoquardi
In the third period other isolates of T. lestoquardi were analysed as to their genetic diversity. The validated ten microsatellite primer sets were used to genotype parasite DNA samples derived from 92 Omani infected sheep. A DNA sequencer was used to determine the size of each amplicon. Using this sample set all of the microsatellites were found to be polymorphic with the number of alleles for each marker ranging from 2 (for micro-satellite TL25) to 15 (for micro-satellite TL16). The average number of alleles per microsatellite was 10.33.

Two broad patterns of diversity were observed;
• alleles differing by a unit repeat motif length, with no intermediate sizes (TL05, TL07, TL13, TL16 and TL19) and
• a continuous spectrum of size (TL04, TL18, TL280 and TL281) for mini-satellite alleles.

The former pattern has been postulated to occur via replication slippage or unequal crossing over during meiosis, resulting in changes in copy number of repeat motifs; while the latter pattern has been attributed to step-wise mutation. An infinite allele model has been proposed to describe the resulting allelic variation, which can explain the patterns of diversity exhibited by these types of markers. Sequencing a number of alleles from each locus would be necessary for confirmation of the mutational mechanism, by identifying that mutation in the first group of markers occurs in a step-wise fashion, whereas for the second group, a more complex mutation mechanism would be predicted to operate.

Further characterisation of allelic diversity of T. lestoquardi from three sheep populations in three regions of Oman (in Ash Sharqiyah, Ad Dakhiliyah, Al Batinah) was performed.

The overall number of alleles per locus ranged from 5 (for locus TL07) to 13 (for locus TL281). Allelic diversity at each locus, summarized as unbiased heterozygosity (H) from the distribution of allele frequencies, revealed higher levels of genetic diversity among parasites in Ash Sharqiyah (H = 0.6256) and Ad Dakhiliyah (H = 0.6171) than from Al Batinah (H = 0.5161).

Moreover, the quality of the data allowed construction of multi-locus haplotypes for each sample, generated using the designated predominant allele at all the examined loci. A total of 89 isolates were genotyped and demonstrated that they differ from each other at most of the examined loci. Thus, most of the T. lestoquardi populations derived from infected animals in Oman are represented by a unique haplotype. In summary, excellent progress has been made in the development of a set of specific markers for future analysis of the population genetics of T. lestoquardi. Initial work shows evidence of significant genetic diversity in the parasite population, also within Omani isolates.

Regarding Babesia, a total of 20 Rap-1 genes were characterised (genes' sequences, presence of different rap-1 gene types (rap-1a, rap-1b and rap-1c), polymorphism of the different gene copies, and variability of the intergenic regions), for 5 pathogens of sheep from China: Babesia sp. Xinjiang and 4 isolates of the B. motasi-like phylogenetic group. The data were then compared with the already published sequences of B. ovis rap-1 multicopy genes. Peptites, recombinant proteins and polyclonal antisera was produced.

Genome analysis
A great emphasis was given to the generation of genomic data for Theileria lestoquardi. Draft assemblies have been generated for both parasite genomes and the bulk of the T. lestoquardi genome has been annotated using T. annulata as a model and then manually curated
The genome of T. lestoquardi was sequenced using the 454 platform followed by denovo assembly and annotation was then transferred from the closely related T. annulata genome using RATT (via PAGIT package). This generated 3199 annotated genes. Subsequently the entire annotation of the genome was manually curated allowing correction of predicted open reading frames, start and stop codons, and identification of intron/exon splice junctions. This considerably improved the quality of the genome annoatation. In addition, genes previously missing from the annotation that were predicted from T. annulata were identified and annotated to provide predicted protein coding sequences leading to a total of 3,371 annotated genes. This assembly comprises 363 scaffolds with 1,225 contigs with a genome size of 7.3Mb indicating that some of the genome sequence has not been captured in the assembly, or the genome is smaller than that of T.annulata.
A major problem encountered with the genome assembly was the inability to assemble and validate annotation of large repeated gene families encoding secretome proteins. These gene families are of considerable interest as they are likely to be involved in host parasite interaction, including manipulation of the immune response, and are candidates for events that allowed speciation of T. lestoquardi from the closely related T. annulata. To capture these gene families, obtain missing regions of the genome and improve genome annotation, it was decided to re-sequence the T. lestoquardi genome using long range Pac Bio sequencing technology (http://www.gatc-biotech.com/en/gatc/sequencing-technologies/pacbio-rs-ii.html) to allow assembly of contigs encompassing repeated sequence. This project was commenced in March 2014, using the infected cell line (TlShiraz) employed to generate the previous genome sequence assembly. To enrich for parasite DNA, large-scale cultures were generated and subjected to methodology for enrichment of parasite nuclei. DNA was then isolated, subjected to clean up and quality control procedures required by GATC biotech. The DNA was then sequenced under commercial contract by GATC; sequence was retrieved in September 2013. Initial analysis indicates 37% of the sequence reads to be T. lestoquardi with X18 fold genome coverage, and an average read length of 6 kb with up to individual 20 kb sequence reads. This is a considerable improvement on sequence length previously obtained. Required work will be to clean up errors and assemble the sequence and then investigate assembly of the TashAT locus as a model for a repeat gene family. The assembled sequence will be compared to the existing genome sequence for final annotation and deposited in to the public repository site and publication of the genome sequence.
The activities during period 3 considerably improved the quality of the T. lewenshuni genome,which was sequenced using Solexatechnology to yield some 5 million reads. Seventeen K-meranalysis showed that the genome size is surprisingly about 18Mb (more than double size of the genomes of other sequenced Theileria genomes). Genome assembly generated 317 contigs, which comprised 157 scaffolds. In total 7134 genes were predicted of which 6867 (96.26%) were annotated. Analysis of the predicted genes found that many of them are present as single copies in T. annulata and T. parva, were duplicated in this genome, indicating potential duplication of the whole genome sequence. To confirm this, a BAC library has been constructed, from which large fragments are going to be sequenced and will be used to curate the assembly of the genome.
A considerable improvement on sequence length previously obtained was generated. The assembled sequence will be compared to the existing genome sequence for final annotation and deposited to the public repository site and publication of the genome sequence.

B. ovis genome: The genomic DNA of genomic of B.ovis was initially sequenced using 454 technology and the genome assembled de novo. Although the sequence data obtained were suitable for initial microsatellite prediction, it was decided that further sequencing and greater read depth was required to perform annotation. Additional sequence was generated using the Illumina platformand a genome assembly performed de novo using the combined 454 and Illumina data. Contigs were scaffolded and aligned against B. bovis using ABACAS software. The assembly comprised 774 contig scaffolds (>500bp)
Furthermore, a homologous protein to B. bovis bovipain-2 (ACV41421) and P. falciparum falcipain-2 (XP_001347832.1) was identified in the sequenced genome of B. ovis, and named ovipain-2. A papain-typical pre-pro-peptide organization (53 kDa) and an extracellular active site were in silico predicted. The identification of the ovipain-2 gene in the B. ovis sequenced genome was carried out.

Recombinant Proteins
A number of Theileria lestoquardi recombinant proteins were prepared like Tlsp, Clone 5 and T. uilenbergi recombinant protein TuIP. In addition, ten putative surface proteins were cloned into a modified pmaxCloning vector for expression in mammalian cells. Plasmids were sequenced, and expression in mammalian cells was confirmed by transfection of COS7 cells followed by immunofluorescence analysis.
A considerable amount of sequence data were made available to the PIROVAC consortium members. Thus, MyoA, MTIP, GAP50, GAP45 & AMAI1, Actin, Aldolase, MLC, TRAP, RAP-1a61-1, RAP-1a61-2, RAP-1a67 from B. ovis were cloned and expressed. The relevant N-terminal fragment of MyoA and a fragment of GAP45 were successfully cloned. The full sequences of these genes were resolved. The putative PKA sites in MTIP, MyoA and GAP45 were mutated (S>A) and recombinant proteins corresponding to wild type and S>A mutants of MTIP, MyoA and GAP45 were expressed in bacteria and purified. The wild type proteins corresponding to MTIP and MyoA were used to generate specific antibodies.
A recombinant form of ovipain-2 was produced in E. coli, using the pET Champion system (Invitrogen). The expression of ovipain-2 in B. ovis was evaluated in in vitro cultured merozoites by immunoblot and immunofluorescence. Additionally, the neutralizing effect of anti-ovipain-2 antibodies on B. ovis invasion of red blood cells was analyzed. Finally, the effect of anti-ovipain-2 antibodies on B. ovis merozoite growth was evaluated.
For fuctional analysis of these molecules some of these specific antibodies were used in in vitro inhibition assay of host cell invasion by the parasites.
For the identification of GPI-anchored protein genes in Babesia ovis and Theileria annulata a two-step approach was followed. First, GPI-anchored proteins were identified in the predicted proteomes of the piroplasmids B. bovis and Theileria annulata, and second, BLAST searches were carried out in the B. ovis genome using the identified sequences. In the case of B. bovis, 17 GPI-anchored proteins were predicted. Five of the identified proteins were members of the Merozoite Surface Antigen family, for which the presence of GPI anchors had been previously demonstrated by metabolic labelling. The appearance of these proteins in the list of predicted GPI-anchored proteins serves to validate the robustness of the predictive tools applied.

Genetic background of the control of the infection
In third period, the project addressed the question of the genetic background concerning the ability of the host to control a T.lestquardi-infection. For this purpose, two sheep breeds were used: a local Italian breed, the Altamurana, and a Northern European breed, the Suffolk. These animals were infected by inoculating them with attenuated T. lestoquardi-cells. Interstingly, the course of the infection in both breeds was extremely mild. Nevertheless, differences in levels of Haptoglobin (Hp) were observed between breeds before the initiation of the infection. It is obvious that the Altamurana had a generally higher level of Hp prior to infection while the Suffolk had a low initial HP level, which increased in 2 individual at 35 days post infection, although, the levels of Hp measured were highly variable.
The in vitro culture systems established in the first period were used to investigate the susceptibility of the Comisana (S Italian local bred) and Suffolk (exogenous breed) plus the Altamurana (another Local Italian bred) to infection with Babesia parasites. It was found that the red blood cells of the Altamurana sheep were completely refractory to the in vitro development of both Babesia species tested. Cells of two of the Suffolk breed were susceptible to in vitro infection, one being highly susceptible, and cells of one of the Comisana breed was also susceptible.

Host cell invasion
Assessement of the role of parasite molecules in the inavasion of host cells was investigated using an antgiserum generated against BoAMA1. For this purpose an in vitro growth inhibition assay for Babesia ovis was established using cell cultures harbouring sheep erythrocytes. It was found that the Anti-AMA 1 antibodies could inhibit the in vitro growth of B. ovis by 50% (parasite growing below 1% instead of the 2% obtained in the absence of AMA1), indicating that this molecule might be involved in host erythrocyte invasion. Therefore, BoAMAI was considered as a potential vaccine candidate.

Immunization trials
Protocols were prepared for immunization trials. Three vaccination trials were performed. The first trial was conducted in Turkey. A Turkish stock of Babesia ovis Akçaova, which was cultivated in vitro for several passages, was used to immunise and challenge the sheep. The experiments described here represent the first approach in which a combination of attenuated parasites and a subunit vaccine is applied for the immunization of sheep against B.ovis. Based on the resulrs of the in vitro inhibition of host cell invasion, the immunization was performed by inoculating the animals either with in vitro cultured B.ovis-infected erythrocytes or BoAMA1 recombinant protein or a combination of both. At different intervals of the immunization and challenge, the full range of clinical, haematological and parasitological parameters were evaluated. It was found that the best protection can be achieved by using attenuated and subunit vaccines in one combination.
The second vaccination trials were done in the Sudan by our Sudanes subcontractor Prof. Abdel Rahim El-Hussein. Vaccination trials agaisnt Malignant Theileriosis was performed in the Sudan. It was found that animals, which received both schizonts and recombinant protein, were better protected than animals immunised either with culture vaccines or recombinant protein. All animals showed enlarged superficial LNs, schizonts were detected in three animals starting from day 24, piroplasm detected in three animals from day 28. No deaths among this group and all animals were recovered. Loss of weight varied from + to ++. Interstingly, there was a 10 day delay in the detection of the schizonts compared to animals of group one, which were immunized with recombinant protein only. The clinical signs were much milder in group 2 and none of the animals died.
It was concluded that in future approaches, more emphasis must be put on the optimization of the immunisation protocols in terms of the number of the inoculated cells, concentration of the protein as well as the choice and the concentration of the adjuvant.

The third immunization trial was performed in China by partner 3. A T. uilenbergi recombinant protein (TuIP) was applied to immunize sheep, which consequently were infected with parasitized erythrocytes. Although all the animals developed clinical signs, the immunized group showed a statistically significant reduction in the percentage of the parasitemia. While only 2.24% parasitemia was recorded for the animals of immunized group, this value was 12.8% in the control group (t-test, p<0.05). Moreover, the onset of the parasitemia was substantially delayed in the immunized group.
It is recommended to use a combination of live attenuated parasites and a subunit vaccine in future immunisation trials.

Immune response
In previous studies, the crucial role cytotoxic T-lymphocytes (CTL) in the protective immune response against T. lestoquardi-infection was confirmed. These cells are able to kill their targets in an MHC- class I restricted manner and to produce IFN-gamma after in vitro stimulation with T. lestoquardi-infected cell lines.
Using this in vitro assay, peripheral blood mononuclear cells of infected animals were co-cultured with T. lestoquardi-infected cell lines and their capacity to produce IFN-gamma was assessed. The obtained results confirmed the generation of CTLs in infected and immunised sheep. Accortdingly, this system was used to screen parasite antigens and their derived peptides as to their potential to induce a recall response in these these cells.

In addition to the ten genes already screened in prvious periods, a further three candidate antigenic genes (TQ2, TL16020, TL13245) were cloned during period 3 of the project. These were screened for their ability to induce a CD8 T cell response in cells generated from five sheep, using a IFN-γ assay to TQ2, TL16020, TL13245. Interestingly, no response was seen (n=2). In summary, of the 13 T. lestoquardi genes screened during the entire project lifetime only two (Tl8, Tl9) elicited CTL response from one of five sheep tested. Furthermore different peptide sequence combinations ranging from 9 mer to 12mer were tested for their ability to induce an IFNg response (see Figure below, n=2) to examine the immonodominance a two antigenic epitopes. The 12 mer resulted in highest responses for two experimental repeats, followed by the 9 mer and 10 mer.

Moreover further work on antigen diversity of three of these genes (Tlms1, orthologue of TA17050) the major merozoite surface antigen, Tl-SPAG1 (orthologue of TA03755) the sporozoite surface antigen; and TL-CTL9 (orthologue of TA15705), a major secreted CTL recognised macroschizont antigen) was performed in Period 3.

Stage differentiation
Stage differentiation is crucial for survival and development of a number of parasites. Thus, at a certain period the intracellular parasites have to make a decision to differentiate to the next stage. Of great interest is the observation that there is a link between parasite differentiation and the regulation of heat-shock proteins (HSPs). The expression of these proteins is also increased when the cells are exposed to stress factors such as high temperature, which frequently associates infections with bacteria, viruses and parasites. Fever is one of the most characeristics of acute of bovine and ovine theileriosis which is often associated with the diffentiation of schizonts to merozoites. It was shown that inhibition of HSP90 function by geldanamycin (GA) results in the upregulation of the expression of merozoite markers (TAMR1, TAMS1).

In further experiments it could be demonstrated that stage differentiation is under regulation of epigenetic alterations. Based on bioinformartics analysis, we could show that Theileria possesses HDACs class I, II and III. We further elucidated the role of HDACs parasite in differentiation and attenuation using apicidin as a potent HDACs inhibitor. Treatment of the infected cells with apicidin resulted in a significant upregulation of merozoite markers (TAMR1, TAMS1) indicating the occurrence of differentiation of the schizont to merozoites. Thus, it can be postulated that during passaging of the parasites, epigenetic changes might be responsible for the reduced ability of the schizonts to differentiate to merozoites and to loss of virulence.

Tools for diagnostics and typification
The following diagnostic tools were developed:
• A set of 12 markers (3 micro and 9 minisatellites) for the typification of B. ovis isolates has been developed.
• A multiplex PCR for the detection of Chinese Theileria species was developed and evaluated. Also, a reverse line blot (RLB) assay was developed for the detection and the identification of the different ovine Theileria and Babesia parasites in China.
• ELISA assays were established for the specific detection of parasite specific antibodies and used for the evaluation of the immune response of immunized/infected animals.
• Immunoblotting assays for the detection of parasite antigens were developed

Potential Impact:
In the context of global warming and globalisation, vector-borne diseases, including tick-borne diseases, are expected to emerge as increasingly important, with an increasing risk of pathogen transmission to animals and humans worldwide. Ticks transmit a greater variety of protozoan, bacterial, rickettsial and viral pathogens than any other arthropod vector group. Tick-borne diseases caused by Theileria and Babesia are ranked high in terms of their impact on the livelihood of resource-poor farming communities. This fact has been underlined by an FAO group of Expert Consultants, which stated that “even where it seems that the significance for indigenous breeds in enzootic areas is slight, there may be a great loss of potential production because of the difficulties in promoting up-grading schemes using more productive but susceptible exotic breeds”. Although the same expert group highlighted “the global significance of the small ruminant livestock industry both from the viewpoint of national economists and for individual resource-poor farmers in semi-arid zones”, a number of diseases of small ruminants remain neglected, particularly ticks and tick-borne diseases (TTBDs). Currently, management of TTBDs is primarily through control of the tick vector using acaricides, although this is unsustainable due to increasing acaricide resistance and food safety concerns. It is therefore imperative to work out integrated control measures including the development of efficient and appropriate vaccines.

The following section gives a cumulative overview of the Project’s dissemination activities, such as scientific meetings and knowledge dissemination activities and it introduces information about the contacts established with related projects and organisations. It reports fulfilment of the dissemination work plan and gives some information on future plans related to patents and exploitable foreground generated within the Project.

Target groups
The target groups identified for the PiroVac project are:
• General public
• Academia, scientific experts
• Other relevant research projects
• Farmers
• Industries (Vaccine development)

Instruments and Media
Dissemination activities were performed through various instruments and media. These will were carefully selected for facilitating collaboration among partners and related parties. The main instruments which were used fall within the following areas:
• Project Meetings
• Scientific Events
• Scientific Exchange visits
• Publications
• Project Website
• Flyer/Brochures
• Press Release

Activities targeting the general public
The main dissemination activities targeting the general public included
• a press release on the website of Research Center Borstel introducing the project and its objectives during the start phase of the project,
• the design of a project logo,
• the production of a project flyer containing general information of the project, contact information etc. and
• the creation and the maintenance of a project website

Project logo
The project logo was designed and disseminated to the project partners in order to create a common and recognisable sign referring to the PiroVac project.

Project leaflet
The project leaflet informs on the existence and the purpose of the project. The goal is to make stakeholders aware of the project, of the advantages of multiscale research, of the project home page, the partners involved and the FP7 programme.
The leaflet was distributed during the project's lifetime at conferences, meetings and any events, PiroVac partners participated in or contributed to.
The leaflet is also available for download from the project website.

Project website
The PiroVac Home Page is available at the URL http://www.theileria.org/pirovac.

The PiroVac web site is entirely public and available without signing in. It was set up and maintained by partner 2 (UGLA). The web site provides general information on the project and its partners as well as announcements of meetings and other project events. The project leaflet is downloadable via the homepage and the projects scientific publications are listed.

Activities targeting the scientific community
The main dissemination activities targeting the scientific community were
• Project Meetings
• Scientific Conferences
• Scientific exchange visits
• Establishment of links to other projects
• Publications

Links established to other projects and organisations
The project established links and contacts to the following related projects and organisations via joint activities, such as meeting organisation, information exchange, knowledge dissemination, etc:

Organisations:
Society for Tropical Veterinary Medicine (STVM)
The Society for Tropical Veterinary Medicine (STVM) is a non-profit organization whose purpose is the advancement of tropical veterinary medicine, hygiene and related disciplines. The activities of the Society focus scientific interest on strategies to deal with established and changing patterns of diseases affecting animals in the tropics. Tropical Veterinary Medicine has become an area of global importance as world trade continues to expand and the challenge of future research is to develop better diagnosis, treatment and control methods for tropical diseases of livestock species. Significantly, a part of this is to understand the environmental variables which affect the host and disease interactions as well and ultimately to support increased food production in the tropics.

Italian Society for Virology
The Italian Society for Virology (SIV) is a non-profit Association founded in September 2001. The SIV has a multidisciplinary organization and merges the scientific and teaching interests of general, medical, pharmaceutical, veterinary and vegetal virologists.
The aims of the SIV are to promote the knowledge and progress in virological research, to organize meetings and courses, to expand and connect the experience in virology to other related fields, with particular regard to biotechnology. The SIV is a Corporate Member of the European Society for Virology.

Projects:

POSTICK
POSTICK is an Initial Training Network (ITN) funded through the EU Marie Curie actions of FP 7. POSTICK aims to design new effective control strategies for TTBD diseases through understanding the mechanisms of tick-host-pathogen interactions regarding: (a) pathogen diversity, survival and transmission, modulation of host immune response and tick survival and (b) identification of host-pathogen-tick molecules for designing anti-tick vaccine and blocking pathogen transmission.

Arbo-Zoonet
Arbo-Zoonet is the International Network for Capacity Building for the Control of Emerging Viral Vector Borne Zoonotic Diseases. It is a Coordination Action funded by the European Union (EU) under FP7 and was launched in May 2008. The project aims at promoting an international network for capacity building for the control of emerging vector borne zoonotic diseases, in particular West Nile, Rift Valley fever and Crimean-Congo hemorrhagic fever. Specifically, the network addresses integrated control measures including vector control, vaccination programmes and therapy strategies, diagnostic tools and surveillance, public awareness and capacity building in endemic regions.

EDENext
EDENext, Biology and control of vector-borne infections in Europe, is a research project bringing together 46 international partners dedicated to investigating the biological, ecological and epidemiological components of vector-borne disease introduction, emergence and spread, and the creation of new tools to control them.

CCH FEVER
CCH FEVER is a European research network (Collaborative Project) coordinated by the Swedish Institute for Infectious Disease Control (SMI, Prof. Ali Mirazimi, Stockholm – Sweden). The consortium brings together 14 complementary partners, including 2 private companies and 12 laboratories, from 10 Member States and Associated Countries of the EU, as well as USA and South-Africa.
The project is supported by the European Commission under the Health Priority of the 7th Research Framework Programme.

"Molecular epidemiology network for promotion and support of delivery of life vaccines against Theileria parva and Theileria annulata infection in Eastern and Northern Africa"
This network has been founded within the German-African Cooperation Projects in Infectiology, funded by the Deutsche Forschungsgemeinschaft (DFG) and was launched 1st of June 2010.
The general objective of the project is to ensure food security and to improve food safety by developing new and improving existing vaccination against bovine theileriosis caused by T. annulata and T. parva. The anticipated project aims to contribute to poverty alleviation by increasing farmers’ incomes and enhancing the national economies. In addition, the project aims at capacity building and technology transfer to developing countries in Africa.

Furthermore links have been established and maintained to institutions in endemic regions outside the project, such as Sudan, contacts were also established to Ethiopia and Oman.


List of Websites:
www.theileria.org/pirovac