Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Project N° FP6 – 003713
EPIGENEVAC
Epidemiology and new generation vaccines for Ehrlichia
and Anaplasma infections of ruminants
Instrument : STREP
Thematic priority : Specific Measures in Support
of International Cooperation ( INCO) – Developing Countries (DEV)
FINAL ACTIVITY REPORT
Period covered :
1st July 2005 to 30 June 2009
Date of preparation :
September 2009
Start date of project :
1st July 2005
Project coordinator :
Dominique MARTINEZ
Project organisation :
CIRAD
Duration : 48 months
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
EXECUTIVE SUMMARY
Objectives and approaches
The general objective of the project was to contribute to increased productivity of livestock
by controlling ticks and tick-borne diseases in a context of sustainable production
systems and environmental safety. To do so, integrated control is necessary where
specific diagnostic and efficient vaccines are essential to reduce the use of acaricides
which are costly, induce resistance of vectors, are harmful for the environment and raise
food safety issues. The aim must be to limit their use to specific periods of high
infestations, identified by monitoring vector population dynamics, where the direct effect of
ticks on animals is detrimental, and to follow an application schedule that maintains natural
or vaccine induced enzootic stability.
More specifically, the project dealt with Ehrlichia ruminantium (cowdriosis or heartwater)
and Anaplasma marginale (anaplasmosis) infections of ruminants with the ultimate goal of

developing improved multi-component vaccines

developing or improving molecular diagnostic tests (multi-parasite detection) for
extensive use in epidemiological studies aimed at giving a regional description of
the ticks and tick-borne diseases situation

contributing to evaluate the efficacy, impact and cost-effectiveness of the control
methods and more specifically of the new vaccines in well characterized farming
systems.
The project was divided in two related headings.

A programme of laboratory and experimental-oriented work making extensive use
of genomics applied to the complete E. ruminantium and A. marginale genomes
and its exploitation for the identification of protective antigens that can be used to
design improved vaccines. Genome analysis is also used as a rapid and
straightforward way to improve or complement molecular tests for detection and
typing of pathogens.

A field-oriented programme of work on the epidemiology of cowdriosis and
anaplasmosis using the diagnostic tools previously developed, linked to parallel
monitoring of vector populations and host densities. These studies will be
conducted on a sufficiently large scale to provide regional maps of diseases with
quantitative indicators to help decision-making in animal health interventions. This
will enable the efficacy and epidemiological impact of new vaccines to be evaluated
in an optimal manner in well-described epidemiological situations.
Research Consortium
The research network comprised 11 Institutions from 5 European countries including one
associated state, and 6 Developing countries. Two of the African participating Institutions
(CIRDES and ITC) have a regional mandate in West and Central Africa which widen the
impact of the project to countries and Institutions that are not formal members of the
consortium. Overall, the members of the EPIGENEVAC consortium had the technical
potential and a large access to various field situations, to properly achieve the specific
objectives of the project.
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Contractors of EPIGENEVAC consortium
Country
CIRAD, Campus International de Baillarguet,
Montpellier
France
University of Utrecht, Faculty of veterinary
medicine, 3508 TD
University of Edimburgh, Easter Bush
Veterinary Centre
University of Berne, Institute for veterinary
virology
IBET, Oeiras,
Netherlands
ISRA, Dakar,
CIRDES, Bobodioulasso,
ITC, Banjul
University of Makerere, Kampala
OVI,
INTA, Buenos Aires
Senegal
Burkina Faso
Gambia
Uganda
South Africa
Argentina
Scientific
in
charge
Dominique
Martinez
(coordinator)
Frans Jongejan
United
Kingdom
Switzerland
Ivan Morrison
Portugal
Manuel
JT
Carrondo
Arona Gueye
Hassane Adakal
Bonto Faburay
Margaret Saimo
Mirinda Van Kleef
Marisa Farber
Giuseppe Bertoni
Results achieved
Genomics
After 4 years of activity, the complete genome sequences of 3 strains of E. ruminantium have
been obtained and annotated In silico. Comparative genomic with other Anaplasmataceae
revealed that the gene organization is highly conserved between the 3 E. ruminantium
strains and an important co-linearity is also observed with the Anaplasma marginale genome
reflecting a close genome organisation between these pathogens. A striking feature of E.
ruminantium genome is the presence of long intergenic spaces, which leads to a very low
proportion of coding genome (around 63-64%) unusual in bacteria. Genome-wide
polymorphism has been described with identification of a unique mechanism of genome
plasticity. This mechanism is based on multiple tandem repeats of a 150 bp period present in
the intergenic spaces and responsible for an active process of expansion/contraction due to
the addition or loss of complete repeat sequences. This genomic information is intended to
be further used in comparative studies with 3 new E. ruminantium strains of virulent and
attenuated phenotypes which have been sequenced in a parallel project and are under
annotation at the end of the project.
The extended information obtained on the E. ruminantium genome generated mainly by
CIRAD and OVI contractors has been the basis for setting up subsequent functional genomic
studies and to develop improved molecular diagnostic and genetic diversity studies for use in
epidemiology.
Genome scale genomic information opens the way for multiple investigations. However, it
does not lead to direct and simple identification of efficient vaccine antigens or virulence
mechanisms. Sequence information has thus been completed with functional studies at the
transcription as well as the protein levels with the assumption that combining the various and
complementary approaches to better understand the mechanisms of host-vector-pathogen
interactions would help identifying key genes/proteins for immunization.
 The dialogue between E. ruminantium and the host cells which was explored at a limited
level until recently by looking essentially at outer membrane protein (OMP) gene
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
expression in tick and ruminant endothelial cells has been extended at the full genome
scale. From the genomic sequence data, a bank of oligo-probes was designed by bioinformatics, the oligo-chips produced and their performance validated by hybridization
using genomic ER DNA. Since E. ruminantium is an obligate intracellular bacterium,
studying bacterial mRNA is not straightforward because of major contamination by cell
RNA. For this reason, a SCOTS technique (selective capture of transcribed sequences)
was adapted to E. ruminantium. Transcripts amplified by SCOTS were generated along
the intra-cellular (endothelial cells) lifecycle of virulent and attenuated E. ruminantium
Gardel and Senegal isolates. Slides hybridizations done in time-course experiments
comparing attenuated and virulent E. ruminantium Gardel strains led to the identification
of 34 genes with expression significantly modulated between both phenotypes among
which 19 have unknown function, 6 are involved in metabolism and 4 are orthologs of
virulent factors in other bacteria. The study of their function, role in virulence and potential
as vaccine candidates could only be initiated within the timeframe of the project, but not
fully completed.
The developments necessary to conduct transcriptomic studies mainly done at CIRAD,
were very cumbersome but at the end the approach was successful. In addition to the
identification of 34 genes of interest, it is now possible to extend the approach to the
comparison of E. ruminantium transcriptome in various cell environments (tick versus
ruminant) and possibly strain comparison for further characterization of pathogen-cells
interactions.

To investigate the presence of proteins in a given biological compartment at a specific
time and in a defined environment, proteome studies were required in a further step to
genomic and transcriptomic studies. Two-dimensional polyacrylamide gel electrophoresis
combined with MALDI-TOF mass spectrometry was performed in time course
experiments encompassing the lifecycle of E. ruminantium (Reticulate bodies RB and
elementary bodies EB stages) in bovine endothelial cells (BAE), the comparative analysis
of gels showed that when comparing three types of samples BAE, RB and EB, 60
proteins spots were identified to be exclusively for EB while 10 protein spots were found
to be exclusively from RB. Protein spots have been collected and the identity of these
proteins when available will shed light on molecules which are critical during the
development cycle.
Although the proteome study (mainly done at IBET), was concentrated on timedependent expression, the strategy can be applied to other critical issues such as the
effect of different cells lines (ticks versus endothelial cell lines) on E. ruminantium protein
expression. Identification of immunoreactive proteins with 2D immunoblots probed with
animal sera will also be helpful to characterize antigenic proteins to develop more
effective vaccines.
Overall, nucleotide sequence data were widely used for the development of diagnostics and
the identification of possible candidate vaccine antigens (described below). However, the
functional genomic and proteomic studies could be undertaken and successfully developed
only in the second half of the project. A first set of very promising results (differentially
expressed genes and proteins) has been obtained but could not be fully exploited within the
timeframe of the project. Nevertheless, these results open major avenues for the future of E.
ruminantium research.
In the case of A. marginale the full genome sequence was not generated by the consortium
but acquired from collaborations and international data-bases as it was released. Genomic
studies were limited to excretion/secretion products with the assumption that such products
have a very high chance to be virulence factors, as well as some outer-membrane proteins
(OMPs). The work concentrated on a set of 12 CDS identified by subtractive hybridization or
an Alkaline Phosphatase gene (Pho-A) fusion system and considered putative exported
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
virulence factors. Among them a Patatin-like protein (PLP) and a Twin Arginine Translocation
pathway (Tat) concentrated the effort for characterisation using several approaches of
comparative and evolutionary analysis, structural models, and complementation using E coli
deficient mutants. Extensive functional analysis of the Tat pathway known to be important for
the export of virulence factors in many pathogenic bacteria has been conclusively conducted.
The final results show that this pathway is functional in A. marginale and that obligate
intracellular pathogens of mammals and arthopods (Anaplasma, Ehrlichia, Wolbachia and
Rickettsia) seem to have maintained the Tat system functioning with only one substrate
(Ubiquinol-cytochrome c reductase). In addition, 3 putative exported proteins of unknown
function but comprising predicted domains of potential interest for immunisation were
identified and evaluated for their ability to trigger the immune response of ruminants. One of
them AM1108 appeared very promising since it induced significant cattle PBMC proliferation,
up-regulation of TNF-α and INF-, and was recognized by sera from naturally infected
animals.
The approaches selected to identify virulence factors of A. marginale (done at INTA) were
successful. They effectively conducted to the identification of a set of excreted/products
amongst which several were classified as unknown but those with characterized orthologs in
other bacteria (Tat pathway) proved to be effectively involved in virulence. The other CDS
identified are thus major candidates to explore in order to characterize the virulence
mechanisms of A. marginale with a potential of developing improved control methods.
One important objective of the project was the identification of tick genes involved in
development and transmission of pathogens for a possible development of transmission
blocking vaccines.
A significant part of the work dedicated to the interactions between tick cells and pathogens
was made possible by the availability of a unique collection of 25 cell lines from different
species of ticks developed and maintained by CTVM. At the end of the project a panel of
thirteen tick cell lines from vector (A. variegatum) and non-vector (B. decoloratus, B.
microplus, I. ricinus, I. scapularis and R. appendiculatus) species that support growth of E.
ruminantium were available.
In A marginale transmission by Boophilus microplus, suppression subtractive hybridization
(SSH) approach identified 10 genes differentially expressed with confirmation by quantitative
Real-time PCR. Silencing of gene expression by RNA interference was confirmed for 8 out of
9 sequences analyzed. Of them 2 genes encoding putative flageliform silk protein and
subolesin produced significantly lower A. marginale msp4 levels with respect to untreated
control after RNAi which suggests that they might be involved in initial stage of cell infection.
For the rest of the genes, although silencing was confirmed, change of infection level of A.
marginale in cells was not statistically significant. After the initial work conducted on A.
marginale, the effect of silencing the tick subolesin gene by RNAi on the growth of E.
ruminantium in tick cells (IDE8 cells) was also tested. It resulted in reductions of E.
ruminantium map1-1 transcripts of 83% (CTVM Gardel strain) and 73% (attenuated Gardel
strain) as compared to levels in control, non-silenced cultures. Subolesine gene silencing
was afterward successfully achieved on A. variegatum vector cells but the effect on E.
ruminantium growth in these cells has not been done yet.
A panel of tick cell lines has been developed by CTVM and is available to investigate tickpathogen interactions. RNAi silencing of tick genes proved to be a useful tool to determine
the effect of tick genes on the development of the pathogens (mainly done at Utrecht with
CTVM collaborations). Associated with the high-throughput studies of pathogens
transcriptome or proteome (described above), and possibly similar approaches applied to the
host cells, more integrative investigations on tick-pathogens interactions are now possible to
envisage.
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Vaccines
One major objective of the project was to identify new targets for future improved E.
ruminantium vaccines taking advantage of the technologies now available to generate highthroughput genomic data. The associated important challenge was to improve immunological
screening in vitro since genomic information is not sufficient to give the value of a candidate
immunogen and testing all vaccine candidates on animals is impossible.

Vaccine candidates
From the E. ruminantium annotation data-base, a group of around 100 vaccine candidate
antigens were selected for cloning and expression (mostly by OVI and to a lesser extent
by CIRAD) in order to evaluate their potential by immuno-screening of recombinant
products. 72 E. ruminantium genes were cloned from which 59 expressed in E. coli.
Among them, 57 were tested in vitro using PBMCs of which 7 induced both significant
cell proliferation and INF- production indicating a Th1 like response (OVI) considered to
be the basis for immune protection although the fine protective mechanisms are afr from
being fully understood. These 7 most promising ORFs were cloned in the mammalian
expression vector (pVCMiUBs) and the gene products were expressed in E. coli for
boosting. Groups of sheep were immunised with different cocktails of 3 or 4 of these
candidate ORFs by DNA prime-recombinant protein boost. Clear humoral and T cell
immune responses were elicited but no significant protection to a virulent challenge was
conferred.
The absence of protection could be due to the absence of protective capacity of these
genes/antigens and/or inappropriate delivery route. This confirmed the major gap still
existing in the understanding of protective immune responses, the influence of the
delivery route and the poor predictive capacity of in vitro immuno-screening as it was
currently practiced.

Immunological screening of antigens
The need for a better understanding of immune responses leading to protection was
foreseen in the project with a special focus on the ability of ruminant dendritic cells (DC)
to properly initiate and drive the immune response towards protection. These cells were
expected to be used as antigen presenting cells to develop improved immunological
probes (T cell lines and clones) for the screening of candidates antigens generated by
the genomics approach. The work on DC was dedicated to CTVM.
Deriving dendritic cells from the circulation to study the immune response and develop an
antigen screening system was not successful and collecting these DC by lymph duct
cannulation was not carried out since the competent surgeon left and CTVM could not
get a licence for carrying out animal experiments with E. ruminantium. The focus was
then changed from DCs to alternative sources of antigen presenting cells (Theileria
infected macrophage cell lines). These cells were successfully superinfected with E.
ruminantium to test the hypothesis that they may function as presenters of bacterial
antigens and therefore as substitutes for DCs. This system was used successfully to
generate E. ruminantium specific T cell populations from live cattle previously vaccinated
by infection and treatment.
Enriched specific CD4 and CD8 T cell populations were generated by the Theileria
infected macrophage cell lines superinfected with E. ruminantium used as APC. However
this was not achieved on time for use to screen antigens that were finally selected only
using PBMC activation and INF- secretion along the project as described previously.
Delivery systems
Besides the intrinsic potential of antigen to induce an appropriate immune response, the way
the molecules are presented to the immune system is crucial for efficacy. Cloning the most
promising candidate genes in various delivery systems such as plasmid, Pox-virus or Virus-
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Like-Particle (VLP) together with improved immunisation schedule, was planned in the
project.
 DNA vaccination in pCMViUB was considered the best approach by OVI for testing
candidate vaccines in animals since this type of vaccination gave significant protection
when using a cocktail of 4 ORF (1H12 locus) and the cpg1 gene of E. ruminantium in
controlled experimental conditions before the start of the project. However, as mentioned
above, the 7 new E. ruminantium vaccine targets identified from in silico annotation and
further selected by immunoscreening did not confer protection to sheep when delivered in
this vector.

The poxvirus delivery was not undertaken since it was planned only in the case where a
very promising antigen was discovered. Indeed, making constructions of a recombinant
virus is a complex and long process that cannot be applied to numerous antigens. Such a
promising antigen was not identified in the project. In addition, recombinant capripox virus
constructed with a E. ruminantium model antigen (MAP1) by CIRAD did not elicited any
visible immune response to the transgene in small ruminants even when the map1 codon
usage was modified for a better expression in eukaryotic cells. The same construct done
by CIRAD in another project using a morbillivirus gene (Peste des petits ruminants)
conferred a very strong protection to goats. This highlights the problem of expressing
bacterial genes in an eukaryotic expression system. The problem remains unsolved for
Ehrlichia.

The VLP strategy for E. ruminantium was quickly stopped. Indeed, rotaviral VLP
containing the MAP1 (model) antigen fused with VP2 were successfully produced by
CIRAD (in another project with INRA), but did not induce any T or B cell response against
the transgene. The same result was obtained when MAP1 was replaced by ovalbumin
and the immune response investigated in mice with very fine tools such as T cell clones
specific for T cell epitopes of ovalbumin. VLP were of normal size and structure but the
foreign antigen was clearly not seen by the immune system, posing basic problem of
antigen processing and presentation that were out of the scope of the present project.
Despite these poorly encouraging results, INTA initiated the production of VP2/6 rotaviral
msp1-VLP for A. marginale. In this case, only characterized T and B cell epitopes were
fused to VP2 and VP6 respectively, in contrast to the full MAP1 antigen which was fused
with VP2 in the case of E. ruminantium. The work was not achieved before the end of the
project.

A proof of principle was also provided by the University of Bern that a CAEV gag peptide
may be used as a carrier for goat vaccines. The rationale of the immunology studies
done by BERN in the project was to improve basic knowledge on goat (and more widely
ruminant) immunology for an application to heartwater to which goats are particularly
susceptible. The delivery of an E. ruminantium peptide identified from a MAP vaccine
candidate antigen using the carrier peptide developed by BERN is envisaged for the
future.

The most efficient experimental vaccine remains the inactivated vaccine formulation
already developed by the consortium (CIRAD). Its advantage on live attenuated E.
ruminantium immunisation which also confers a very strong immune protection is that it is
far more stable, can be injected subcutaneously, and can be formulated with any E.
ruminantium strain to overcome antigenic diversity. The mass production process in
bioreactor has been fully developed and optimised by IBET with the objective of industrial
production. The last step consisting of the industrial formulation of the antigen in oil
adjuvant was successfully performed with optimum parameters determined. The highspeed homogenizer appeared the most rapid and convenient method amongst the
techniques compared for preparing W/O emulsions, producing emulsions that were
stable upon storage at 4°C for at least 1 month. However, E. ruminantium emulsification
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
in ISA 50 adjuvant using the homogenizer was found to be particularly difficult. The use
of ISA 70 adjuvant resulted in stable and homogenous emulsions of particles ranging
approx 1µm diameter with the lowest degradation profile of emulsified E. ruminantium
antigens (both at 4°C and 38°C). ISA 70M was also shown of particular interest due to its
excellent results in terms of emulsion stability and E. ruminantium antigen integrity
overtime at 4ºC. These two adjuvants should undoubtedly be considered as an
alternative to ISA 50 for testing in future vaccination trial.
Despite the various sophisticated delivery systems tested with either model antigens (E.
ruminantium MAP1) or vaccine candidate antigens (7 E. ruminantium putative candidates),
no immune response (VLP and recombinant pox) or protection (DNA / recombinant protein
prime-boost vaccination) could be significantly achieved. Inactivated formulation of E.
ruminantium in ISA50 oil adjuvant remains the most efficient method of immunization with an
industrial production process fully validated. Interestingly two new promising oil adjuvants
were evaluated which nevertheless remain to be tested in vaccination trials.
Diagnostic and epidemiology
Vaccines remain essential tools for the control of vector borne diseases, but appropriate
integrated control strategies cannot be adequately achieved without an appropriate
knowledge of disease epidemiology and dynamics of tick-vector-host interactions. In the
case of tick-borne Rickettsiales which are obligate intracellular parasites difficult to cultivate,
PCR-based methods for the detection and genetic characterization represent the methods of
choice. These molecular methods were extensively developed in the project in order to
improve the sensitivity of detection and to characterize the genetic diversity of E.
ruminantium and to a lower extent of A. marginale.

Pathogen detection and isolation
The specific detection of E. ruminantium based on the amplification of a fragment of the
pCS20 ORF was improved at CIRAD by developing a nested PCR showing a 2 log
sensitivity improvement. This nPCR was extensively validated and used in field studies.
Quantitative Real-time PCRs (QPCR) were also developed to quantify the number of
bacteria in cultures, tissues or vectors. QPCR targeting the pCS20 (CIRAD, OVI) or the
map1.1 genes (CTVM) were developed and used in animal experiments, whereas a
map1-QPCR was developed and standardized to follow the mass production process in
culture flasks or bioreactors (IBET). Nested-PCR based on msp5 for A. marginale
detection was already available and was therefore used. However, in the field, hosts and
vectors can be co-infected by several parasites. Thus, in addition to species-specific PCR
methods, reverse line blot hybridization method developed by the consortium in a
previous project was used to simultaneously detect several parasites in pathological
specimen and to detect new species using Anaplasma-Ehrlichia “catch-all probes”. The
RLB was improved by adding improved probes generated from the results of field
studies. New pathogens genotypes were identified and the prevalence of infections
including co-infections was determined in various places (INTA, Makerere).
New E. ruminantium and A. marginale strains were isolated as stabilates or in culture as
a continuous process along the project. Indeed, beside the genetic characterization that
can be done directly in pathological samples, characterizing the phenotype of pathogens
is crucial, in particular cross-protection between isolates for appropriate vaccine
formulations.

Pathogen Genotyping
The diversity of pathogens is a key problem recognized since a long time, which applies
to A. marginale and even more E. ruminantium. Insufficient information was available at
the beginning of the project regarding the extent and the dynamic of this diversity in order
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
to formulate appropriate vaccines. This was particularly true for E. ruminantium where
antigenic diversity strongly hampers vaccine efficacy in the field. A main objective of the
genetic characterization was to try to find correlates between phylogenetic clusters and
phenotypic characters such as cross-protection. The full genome information generated
in the project on several strains of E. ruminantium and available on A. marginale from
collaborations (USA) were extensively used for this purpose (see above). The
polymorphism of E. ruminantium was characterized at the genome scale from the full
genome comparison of the various strains available. Polymorphism consists in SNPs, a
small number of strain-specific ORFs, truncated ORFs and highly polymorphic genes
such as the OMP genes. All of these polymorphisms were used to develop a panel of
typing methods for answering the various questions.
The first method to characterize the extent of E. ruminantium diversity and its
geographical dispersal was based on PCR or nPCR map1 amplification followed by
RFLP analysis or sequencing (CIRAD, CIRDES, ITC). Unexpected large number of
genotypes circulating at the village level was revealed by extensive studies conducted
mainly in Burkina Faso (CIRDES). More than 10 to 12 different map1 genotypes with
various degrees of cross-protection were shown to circulate in a restricted area. It was
subsequently confirmed in the field that good protection using an inactive vaccine can be
achieved only if an appropriate strain is included. However, defining the appropriate
cocktail with such diversity is not straightforward: not all strains combinations can be
tested by cross-protection and the frequency of the various genotypes in an E.
ruminantium population can evolve in a given area. This large diversity of map1
genotypes was confirmed in all countries where studies were conducted: Africa (OVI,
CIRDES, ISRA, Makerere), Caribbean (CIRAD), Madagascar. On the other hand, similar
genotypes can be found in very distant regions suggesting that their number is not
unlimited. However, although map1 is a good marker of diversity, it is not a geographical
or a cross-immunity predictor.
Phylogenetic trees constructed from various individual gene sequences by OVI were also
unable to give any useful correlates with E. ruminantium phenotypes.
Multi-Locus Sequence Typing (MLST) was thus developed since it was shown to be
informative in phylogeography studies in other bacterial models. Optimization of nested
PCRs targeting 6 housekeeping genes was done (among a set of 16 initially tested) and
their use validated on a set of E. ruminantium reference strains for MLST. Similarly,
Multilocus VNTR analysis (MLVA) was also developed since it gives good correlates in
some bacteria with phenotypic characters such as host spectrum or virulence. 8 E.
ruminantium VNTR were fully validated amongst a set of 27 initially identified for use in
MLVA. Both methods tested on a limited number of selected E. ruminantium strains
proved to be appropriate to carry out phylogenetic analysis. Extensive use on a large
number of well characterized ER samples remains necessary to try to find correlates with
various phenotypes. These extensive studies could not be achieved during the project.
A battery of performing species-specific detection methods have been made available along
the project for epidemiological or experimental studies. In parallel, various complementary
typing methods were also developed, validated and made available for epidemiology. The
prevalence of the studied parasites, their genotype in most instances, their phenotype when
isolates could be obtained, have been determined in various field conditions in Africa, the
Caribbean and Argentina. Data-bases have been built and a first set of partial distribution
maps generated. Correlates between genetic clusters and phenotypes (cross-protection,
virulence, origin…) of pathogens have not been obtained yet but the panel of typing methods
developed is intended to be extensively used from field sampling to achieve this goal which
remains a major challenge for the future.
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
LIST OF DELIVERABLES
Deliv
erabl
e
No
Deliverable title
WP
N°
Date
due
Actual /
Forecast
delivery
date
D1.1
Computerized annotation data bases (full
E. ruminantium genomes, A. marginale
OMP)
1
6
6
12
1, 6
D1.2
Polymorphic genes of ER through
comparison of Gardel and Welgevonden
isolates
1
6
6
2
1
D1.3
Genes
potentially
involved
in
pathogenicity, environmental adaptation,
attenuation and virulence from annotation
in silico
1
6
6
10
1, 6
D1.4
Genes for vaccine studies from annotation
in silico (E. ruminantium & A. marginale)
1
6
6
9
1, 11
D1.5
DNA
micro-array
chips
ruminantium (transcriptome)
E.
1
12
36
15
1, 6
D1.6
E. ruminantium gene expression profiles
in different host cells and environmental
conditions
1
36
48
96
1, 6
D2.1
Recombinant E.
marginale proteins
A.
2
3-20
6 / 36
33
1, 11
D2.2
Antibodies
proteins
recombinant
2
6-24
12/ 48
6
1, 11
D2.3
Dendritic cell and monocyte presentation
systems
2
12
12 / 48
36
2
D2.4
T cell lines or PBMC of immune animals
for antigen screening
2
36
12 / 48
66
2
D2.5
Recombinant proteins inducing specific
Th1
cellular
responses
(vaccine
candidates)
2
6-24
6 / 48
14
1, 6
D3.1
Genes cloned in an eukaryotic expression
plasmid (selected from T-cell screening of
recombinant proteins)
3
1224
/ 36
4
1, 6
D3.2
Protective genes/proteins identified after
ruminant immunisation & challenge
3
30
12 / 48
31
1, 6
D3.3
Recombinant capripox vaccine
3
42
/ 42
22
1
D3.4
Recombinant rotavirus VLP vaccine
3
42
24 / 42
10
1, 5
D3.5
Most efficient
system
3
48
/ 48
41
7
D3.6
Most efficient immunisation schedule
3
48
/ 48
47
7
D3.7
VLP product for assessment and efficacy
testing (VLP-map model)
3
24
/ 48
20
5
D3.8
Formulation for long term storage of
vaccines
3
48
12 / 48
22
5
to
from
ruminantium
specific
immunisation
&
delivery
10
Estimate
d
indicativ
e
Person
month
Used
indicativ
e
Person
month
Lead
Parti
cipan
t
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
D4.1
New oligo-probes (Universal reverse line
blot assay) & PCR primers for
uncharacterised parasite species
4
12
12 / 36
210
3
D4.2
PCR primers and oligo probes developed
for E. ruminantium strain genotyping
4
18
12 / 36
34
1
Dat
e
due
Actual /
Forecast
Delivery
date2
Delivera
ble
No1
Deliverable title
WP
N°
Estimate
d
indicativ
e Person
Used
indicati
ve
Person
month
month
Lead
Particip
ant
D4.3
Micro array biochips for multi
pathogen detection or genotyping
4
18
/ 36
56
3, 1
D5.1
Field sites for epidemiological
studies selected with in depth
description
of
agro-ecological
conditions as well as parasitic
environment (from published and
grey
literature,
reports…).
Epidemiological surveys designed
5
6
6
16
7, 10
D5.2
New isolates of E. ruminantium and
A. marginale as well as other
parasite species characterised
5
1248
12 / 48
74
7, 11
D5.3
E. ruminantium genetic clusters
correlated with cross-protection
clusters for subsequent prediction of
cross-protection
directly
from
genetic typing
5
24
12 / 48
72
7, 8
D5.4
Bank and database of pathogens
5
12
12
39
7, 10
D5.5
GIS database developed
5
12
12 / 24
106
7
D5.6
Distribution maps of tick and
pathogens including E. ruminantium
strain genotypes
5
2448
24 / 48
106
7
D5.7
Efficacy of inactivated vaccine (gold
standard, extensive trials available)
as compared to new generation
vaccines (limited field trials)
5
3648
12 / 48
84
7, 8
D6.1
Strategic plan for public awareness
6
3
3
3
1
D6.2.
Daily coordination. Proceedings and
minutes of coordination meetings
and technical workshops
6
12243648
12 / 48
48
1
D6.3
Final
Plan
for
using
disseminating knowledge
6
42
12 / 48
3
1, 3
and
11
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
D1.1 to D1.4. Delivered as planned in the project. However annotation is never fully
completed and new genes or polymorphisms will potentially be explored as new
information is generated in the future. The usefulness of this annotation is already
evidenced by utilisation of the data to compare with 3 new strains including attenuated
phenotypes which are being sequenced besides this project.
D1.5. The in silico data base of oligonucleotides is completed. Oligos have been
synthesized and slides comprising 40 000 spots in each chamber have been produced
(Agilent) and validated by genomic DNA hybridization. They are available for E.
ruminantium transcriptome studies.
D1.6. Expression has been demonstrated for several sets of genes (OMPs, unique or
truncated genes) as necessary for the experiments conducted.
As far as genome-scale E. ruminantium transcriptome is concerned, all SCOTS samples
for Gardel (virulent and attenuated) as well as Senegal virulent E. ruminantium samples
have been generated and their quality validated. Hybridizations have been done to
compare attenuated versus virulent Gardel in time course experiments. 34 genes with
expression significantly modulated between virulent and attenuated strains have been
identified.
D2.1. 72 E. ruminantium genes have been cloned from which 59 expressed in E. coli. 11
genes of A. marginale have been successfully expressed.
D2.2. Sera specific to recombinant proteins have been raised in goats and rabbits against 5
polymorphic E. Ruminantium MAP antigens and tested in Western-blot. No new immune
sera to recombinant proteins have been raised in addition to those produced during year 1.
The effort has concentrated on the production of recombinant proteins and their evaluation
as immunogens.
D2.3. Dendritic cell generation from blood was poorly successful and lymph duct
cannulation could not be undertaken since the competent surgeon left and CTVM could not
get a licence for carrying out animal experiments with E. ruminantium.
As an alternative source of antigen presenting cells Theileria annulata schizont-infected
lymphoblastoid cell lines were successfully superinfected by E. Ruminantium. They have
been used as APC for PBMC of E. Ruminantium immunised cattle in year 4 to generate
specific T cell lines.
D2.4. Antigen screening has been done using PBMC but no long term T cell lines have
been generated. T cell lines have been generated (D2.3) but not yet used for screening
recombinant antigens.
D2.5. Fifty seven recombinant proteins have been produced (see D2.1) and tested in vitro
using PBMCs among which 7 induced both significant cell proliferation and INF-
production indicating a Th1 like response (i.e. candidate vaccine antigens).
D3.1. The 7 genes mentioned in D2.5 have been inserted in the pCMVUbi for testing as
immunogens
D3.2. Goat protection (50%) was demonstrated using a mixture of 4 recombinant MAP
proteins in year 1. The 7 vaccine candidates described in D2.5 and D3.1 were tested on
sheep using a DNA prime-recombinant protein boost immunisation protocol. Immune
responses were elicited but no significant survival to virulent challenge was conferred.
D3.3 In absence of clear protection conferred by of the most promising candidate antigens
identified by immuno-screening (D2.5 to 3.2), poxvirus delivery was not performed. In
12
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
addition this system used with a model map1 gene did not elicited an immune response
against the transgene in small ruminants questioning about the ability of the system to
properly express bacterial genes (the system has been fully validated on foreign viral
genes as a positive control.
D3.4. The production of VP2/6 rotaviral msp1-VLP for A. marginale has been initiated.
Cloning of VP2-MSP1 T cell epitopes chimera has been done in pFASTBAC and the
Cloning of VP6-MSP1 B cell epitopes chimera is in progress. Expression in baculovirus in
for the autoassembling of VLPs has not been completed and should be continued.
This strategy was abandoned for E. ruminantium since MAP1-VLP although successfully
produced did not induce any T or B cell response against the transgene (see D3.7). In this
case the full antigen was fused with VP2 whereas for A. marginale only relevant epitopes
are fused. Reactivation of this strategy for E. ruminantium would depend on results
obtained with MSP1.
D3.5, D3.6. Only feasible after the production of a candidate recombinant vaccine and
results of immunisation of D3.2. No significant protection results have been achieved with
the new candidate antigens and subsequently optimization of the immunisation schedule
was not possible.
D3.7. Approach originally planned only with the most promising antigen as for recombinant
poxvirus. However, currently in stand-by since it was shown by CIRAD outside this project
that chimaeric MAP1-rotaviral pseudo-particles successfully produced with an appropriate
conformation where unable to induce any protection against the transgene (map1) whereas
responses were achieved against the rotavirus antigens. The absence of response was
confirmed in another experimental system consisting in Ovalbumine-rotaviral VLP
immunisation of mice. Again no B or T cell responses were observed against the
ovalbumine transgene. The system could be reconsidered pending the results obtained
with Anaplasma msp1 (D3.4)
D3.8. Long-term storage influence on efficacy has been completed for the ISA50
inactivated vaccine. The process of E. ruminantium formulation in adjuvant for an industrial
production has been developed and validated. It appears that ISA70 and ISA70M have
better stability performances with less antigen degradation than ISA50 when preserved at
4°C and 38°C. These new adjuvants have to be considered for future commercial vaccines
but their protective capacity has to be tested before as compared to that of ISA50.
D4.1. RLB was extensively used on field samples mainly by INTA, ITC and Makerere
partners. INTA has improved a set of probes. New Anaplasma genotypes were
characterized.
D4.2. New primers and nPCR targeting 12 polymorphic genes (unique or truncated genes)
as well as 8 housekeeping genes have been developed, validated and used to genotype
field E. ruminantium strains by MLST. A MLVA method has also been developed : 17 out of
23 VNTR identified have been developed for genotyping, 10 were finally selected and 8
nPCR were fully validated on reference E. ruminantium strains for extensive use on field
samples.
D4.3. Diagnostic DNA chips have not been developed since the RLB assay (membranebased format) for pathogen detection appeared more adapted for tropical conditions. More
emphasis has been made on PCR followed by several typing methods such as MLST,
MLVA (see D4.2)
D5.1. In all countries field studies have been conducted since the beginning of the project
with collection of a significant number of samples.
13
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
D5.2. New E. ruminantium or A. marginale have been identified by molecular methods
completed by isolation in vitro for some. These strains have been used in phylogenetic
studies. New strain characterization is considered an on-going process to complete genetic
analysis and pathogen mapping.
D5.3. Clustering based on the polymorphic or housekeeping genes (MLST) studied so far
has not shown correlation with cross-protection groups. The MLVA method showed a good
discriminatory index but has not yet been used on a set of samples large enough to
conclude on its predictive capacity of cross-protection. This approach is continued as new
information is generated.
D5.4. Bank of pathogens and their products has been incremented from field studies and
used for genetic characterization and epidemiology (linked to D5.2)
D5.5 & 5.6. Map generation and data-base (serology, PCR & RLB results) has been
initiated by some partners and up-dating must be a continuous process.
D5.7. Efficacy of the cowdriosis inactivated vaccine has been evaluated in the field in
Burkina Faso during a previous project and has highlighted the crucial impact of strain
diversity on efficacy. A better characterization of E. ruminantium population structure and
dynamic in the field has been undertaken in Burkina Faso where the field trials have been
conducted, and extensive diversity studies conducted in the various Africa partners (map1,
MSLA, MLVA genotyping). This must be continued with the aim of developing appropriate
cocktail vaccine formulation.
D6.1. Achieved in year 1
D6.2. Progress reports have been provided each year. A launching (April 2005, Senegal)
and a Mid-term (June 2008, Montpellier) coordination meetings have been organized.
Results were reviewed and planning was done during these meetings.
D6.3. Final plan for disseminating the knowledge has been finalised.
14
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
PLAN FOR USING AND DISSEMINATING THE KNOWLEDGE
Exploitable knowledge and its use
Overview table
Exploitable
knowledge
Exploitable
products or
measure
Sector
of
application
Timetable
for
commerci
al use
Patents
or
other
IPR
protection
Owner &
other
partner(s)
E.
ruminantium
Genome
sequence and
Annotation
Annotation
data-bases for
E. ruminantium
(3 strains)
Medical
(veterinary)
Not
commercial
as such
Published
in
the
public
domain
(see
references)
CIRAD & OVI
Genomic
polymorphism
of
E.
ruminantium
DNA
fragments for
diagnostic,
polymorphic
genes
for
genotyping
Medical
(veterinary)
> 2010
2 patents :
CIRAD
Frutos
R.,
Ferraz
C.,
Demaille
J.,
Martinez
D.
2006.
Sequences for
differential
diagnostic of
Ehrlichia
ruminantium
and
use
thereof
=
Séquences de
diagnostic
différentiel
d'Ehrlichia
ruminantium et
leur utilisation.
Genève :
WIPO, 70 p.
PCT/EP2004/0
13853,
2004//1/0/.
Industry
15
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Frutos, R., N.
Vachiery,
T.
Lefrançois, C.
Ferraz,
J.
Demaille and
D.
Martinez
(2008). Target
genes
for
strain-specific
diagnostic of
Ehrlichia
ruminantium
and
use
thereof. Brevet
PCT/IB2006/0
03870
publications
(see
references)
Cloning and
expression of
in
silico
annotated
genes
Recombinant
proteins of E.
ruminantium
and
A.
marginale
Medical
(veterinary)
Mass
production
process,
downstream
processing
and storage
conditions for
a heartwater
inactivated
vaccine
Inactivated
heartwater
vaccine
Medical
(veterinary)
> 2012
Industry
> 2010
(strain
diversity)
Industry
Patent to be
considered
depending on
vaccine
protection
results
OVI
and
CIRAD
(E.
ruminantium)
Process
published
in
the
public
domain.
IBET
with
CIRAD and
OVI
Protection
is
based on the
diffusion of the
necessary
biological
resources and
the control of
the technology
is
not
straightforward
16
INTA
(A.
marginale)
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Bioinformatics
services:
Pipeline
for
automatically
processing
sequence
data
and
performing
polymorphism
and
phylogenetic
analysis
of
multilocus
sequence
typing
scheme
(MLST). This
method
is
applied
for
bacterial
taxonomy and
bacterial
population
studies. The
pipeline has a
module-based
structure and
the whole tool
will
be
available for
downloading
from
INTA
server and for
local
installation
MLST pipeline
Research,
Clinical
Microbiology
2010-2011
The system is
developed
under
Linux
(source code
freelydistributed and
available to the
general public)
INTA
Anaplasma
marginale
gene products
for using as
vaccine
targets
(virulence
factors). The
antigens are
expected
to
be delivered
in
Virus-like
particles
Baculovirus
expressing A.
marginale
antigens
Research,
Veterinary field
> 2012
The
baculovirus
expressing A.
marginale
antigens
will
be tested in
vaccine trials,
supported by
institutional
grants (INTA)
during
forthcoming
period
INTA
Patents to be
considered
depending on
vaccine
protection
results
17
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Dissemination of knowledge
Publications
1. Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber,
E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, F., Zweygarth, E.,
Jongejan, F., Maillard, J-C., Berthier, D., Botha, M., Joubert, F., Thomson, N.R., Allsopp, M.T.,
and Allsopp, B.A. 2005. The genome of the heartwater agent, Ehrlichia ruminantium, contains
multiple tandem repeats of actively variable copy number. Proceedings of the National
Academy of Sciences of the United States of America. 102:838-843.
2. Isabel Marcelino, Célia Veríssimo, Marcos F.Q. Sousa, Manuel J.T. Carrondo, Paula M. Alves
(2005). Characterization of Ehrlichia ruminantium replication and release kinetics in
endothelial cell cultures. Vet Microbiol, 110(1-2):87-96.
3. Peixoto C, Marcelino I, Vachiery N, Bensaid A, Martinez D, Carrondo MJT, Alves P,. 2005.
Quantification of Ehrlichia ruminantium by Real Time PCR. Vet. Microbiol., 107 : 273-278
4. Bekker C.P.J., Postigo M, Taoufik A, Bell-Sakyi L, Ferraz C, Martinez D, Jongejan F. 2005.
Transcription analysis of the major antigenic protein 1 multigene family of three in vitro
cultures Ehrlichia ruminantium isolates. J. Bacteriol., 187 (14) : 4782-4791.
5. Frutos R, Alain Viari, Conchita Ferraz, Sophie Eychenié, Yane Kandassamy, Isabelle Chantal,
Albert Bensaid, Anne Morgat, Eric Coissac, Nathalie Vachiery, Jacques Demaille, and
Dominique Martinez. 2006. Comparative genomic analysis and genome evolution of three
phenotypically distinct strains of Ehrlichia ruminantium. J. Bacteriol., 188 (7) : 2533-2542.
6. Frutos R, Alain Viari, Conchita Ferraz, Albert Bensaid, Anne Morgat, Frederic Boyer, Eric
Coissac, Nathalie Vachiery, Jacques Demaille, and Dominique Martinez. 2006. Comparative
genomics of three strains of Ehrlichia ruminantium : A review. Ann. N. Y. Acad. Sci., 1081:
417-433
7. Fluri, A., Nenci, C., Zahno, M. L., Vogt, H. R., Charan, S., Busato, A., Pancino, G., Peterhans,
E., Obexer-Ruff, G., and Bertoni, G. (2006). The MHC-haplotype influences primary, but not
memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes
of the caprine arthritis encephalitis virus Gag protein. Vaccine 24, 597-606.
8. Mordasini F., Vogt, H.-R., Zahno, M.-L., Maeschli A., Nenci, C., Zanoni, R. G., Peterhans, E.,
and Bertoni, G. (2006). Analysis of the Antibody Response to an Immunodominant Epitope of
the Envelope Glycoprotein of a Lentivirus and Its Diagnostic Potential. Journal of Clinical
Microbiology 44, 981-991.
9. Ravazzolo, A.-P., Nenci, C., Vogt, H.-R., Waldvogel, A., Obexer-Ruff, G., Peterhans, E., and
Bertoni, G. (2006). Viral load, organ distribution, histopathological lesions, and cytokine mRNA
expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus.
Virology 350(1):116-27.
10. Vachiery N, Thierry Lefrancois, Isabel Esteves, Sophie Molia, Christian Sheikboudou, Yane
Kandassamy, Dominique Martinez. 2006. Optimisation of the inactivated vaccinal dose
against heartwater and in vitro quantification of Ehrlichia ruminantium challenge material.
Vaccine. 24 (22) : 4747-56
11. Isabel Marcelino, Marcos F.Q. Sousa, Célia Veríssimo, António E. Cunha, Manuel J.T.
Carrondo e Paula M. Alves (2006). Process development for the mass production of Ehrlichia
ruminantium. Vaccine, 24(10): 1716-1725.
12. Zweygarth, E., Josemans, A.I., Spickett, A.M., Steyn, H.C., Putterill, J., Troskie, P.C., Mtshali,
M.S., Bell-Sakyi, L., Shkap, V., Fish, L., Kocan, K.M., Blouin, E.F. (2006). In vitro cultivation of
18
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
a South African isolate of an Anaplasma sp. in tick cell cultures. Onderstepoort Journal of
Veterinary Research 73, 251-255.
13. Marcelino, I., Vachiéry N., Amaral, A.I., Roldao, A., Lefrançois, T., Carrondo, M.J.T., Alves, P.,
Martinez, D. 2007. Effect of purification process and the storage conditions on the efficacy of
an inactivated vaccine against hearwater. Vaccine. 4903-4913.
14. Cristina Peixoto, Isabel Marcelino, Ana Isabel Amaral, Manuel JT Carrondo, Paula M Alves,
Purification by membrane technology of an intracellular Ehrlichia ruminantium candidate
vaccine against heartwater, Process Biochemistry 2007; 42(7): 1084-1089
15. R. Frutos, A. Viari, N. Vachiéry, F. Boyer, D. Martinez. 2007. Ehrlichia ruminantium : genomic
and evolutionary features. Trends in Parasitol., 23 (9) : 414-419.
16. Postigo, M., Taoufik. A., Bell-Sakyi, L., de Vries, E., Morrison, I., Jongejan, F. (2007).
Differential transcription of the major antigenic protein 1 multigene family of Ehrlichia
ruminantium in Amblyomma variegatum ticks. Veterinary Microbiology 122, 298-305
17. Faburay, B., Geysen, D., Munstermann, S., Bell-Sakyi, L., Jongejan, F. (2007). Longitudinal
monitoring of Ehrlichia ruminantium infection in lambs and kids by pCS20 PCR and MAP1-B
ELISA in The Gambia. BMC Infectious Diseases 7, 85.
18. Bell-Sakyi, L., Zweygarth, E., Blouin, E.F., Gould, E.A., Jongejan, F. (2007). Tick cell lines:
tools for tick and tick-borne disease research. Trends in Parasitology 23 (9), 23 (9): 450-457.
19. Pisoni G., Bertoni G., Puricelli M., Maccalli M. and Moroni P. (2007). Demonstration of coinfection with and recombination of caprine-arthritis encephalitis virus and maedi-visna virus in
naturally infected goats. J. Virol. 81, 4948-4955
20. Nenci C., Zahno M-L., Vogt H-R., Obexer-Ruff G., Doherr M.G., Zanoni R., Peterhans E. and
Bertoni G. (2007) Vaccination with a T cell-priming Gag peptide of Caprine Arthritis
Encephalitis Virus transiently enhances viral replication in vivo. J. Gen. Virol. 88, 1589-1593
21. B Faburay, D Geysen, S Munstermann, A Taoufik, M Postigo, F Jongejan. 2007. Molecular
detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in the Gambia.
Exp. Appl. Acarol., 42(1):61-74
22. de la Fuente J, Ruybal P, Mtshali MS, Naranjo V, Shuqing L, Mangold AJ, Rodriguez SD,
Jimenez R, Vicente J, Moretta R, Torina A, Almazan C, Mbati PM, de Echaide ST, Farber M,
Rosario-Cruz R, Gortazar C, Kocan KM.. Analysis of world strains of Anaplasma marginale
using major surface protein 1a repeat sequences. Vet Microbiol. 2007 Jan 31;119(2-4):38290. Epub 2006 Nov 2
23. Pisoni G., Moroni P., Turin L. and G. Bertoni (2007). Compartmentalization of small ruminant
lentiviruses between blood and colostrum in naturally infected goats. Virology, 2007 Dec
5;369(1):119-30. Epub 2007 Aug 23
24. N. Vachiéry, G. Maganga, T. Lefrançois, Y. Kandassamy, F. Stachurski, H. Adakal, C. Ferraz,
A. Morgat, A. Bensaid, E. Coissac, F. Boyer, J. Demaille, A. Viari, D. Martinez, R. Frutos.
2007. Differential strain-specific diagnosis of the heartwater agent: Ehrlichia ruminantium.
Infection, Genetics and Evolution. 8 (4): 459-466.
25. De la Fuente J, Blouin EF, Manzano-Roman R, Naranjo V, Almazan C, Perez de la Lastra JM,
Zivkovic Z, Jongejan F, Kocan KM: Functional genomic studies of tick cells in response to
infection with the cattle pathogen, Anaplasma marginale. Genomics 2007, 90(6):712-722.
26. Zivkovic Z, Nijhof AM, Kocan KM, de la Fuente J, Jongejan F: Experimental transmission of
Anaplasma marginale by male Dermacentor reticulatus, BMC Vet Res. 2007 Nov 30;3:32.
27. Bell-Sakyi, L., Zweygarth, E., Blouin, E.F., Gould, E.A., Jongejan, F. (2007). Tick cell lines:
19
Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
tools for tick and tick-borne disease research. Trends in Parasitology 23, 450-457.
28. Faburay, B., Geysen, D., Ceesay, A., Marcelino, I., Alves, P.M., Taoufik, A., Postigo, M., BellSakyi, L., Jongejan, F. (2007). Immunisation of sheep against heartwater in The Gambia using
inactivated and attenuated Ehrlichia ruminantium vaccines. Vaccine 25, 7939-7947.
29. B Faburay, F Jongejan, A Taoufik, A Ceesay, D Geysen. 2008. Genetic diversity of Ehrlichia
ruminantium in Amblyomma variegatum ticks and small ruminants in the Gambia determined
by restriction fragment profile analysis. Vet Microbiol., 126 : 189-199.
30. Postigo, M., Taoufik, A., Bell-Sakyi, L., Bekker, C. P. J., de Vries, E., Morrison W. I., Jongejan,
F. (2008). Host cell-specific protein expression in vitro in Ehrlichia ruminantium. Veterinary
Microbiology 128, 136-147.
31. Cristina Peixoto, Isabel Marcelino, Ana Isabel Amaral, Manuel JT Carrondo, Paula M Alves,
Purification by membrane technology of an intracellular Ehrlichia ruminantium candidate
vaccine against heartwater, Process Biochemistry 2007; 42(7): 1084-1089
32. Isabel Marcelino, Nathalie Vachiéry, Ana I. Amaral, Cristina Peixoto, António Roldão, Thierry
Lefrançois, Manuel J. T. Carrondo, Paula M. Alves, Dominique Martinez. Effect of the
purification process and the storage conditions on the efficacy of an inactivated vaccine
against heartwater, Vaccine 2007; 25(26): 4903-4913.
33. Molia S, Frebling M, Vachiéry N, Pinarello V, Petitclerc M, Rousteau A, Martinez D, Lefrançois
T. 2008. Amblyomma variegatum in cattle in Marie Galante, French Antilles: prevalence,
control measures, and infection by Ehrlichia ruminantium. Vet Parasitol. 153(3-4):338-46.
34. Ruzek, D., Bell-Sakyi, L., Kopecky, J., Grubhoffer, L. (2008). Growth of tick-borne encephalitis
virus (European subtype) in cell lines from vector and non-vector ticks. Virus Research 137,
142-146.
35. Niederhauser, S., Bruegger, D., Zahno, M. L., Vogt, H. R., Peterhans, E., Zanoni, R., Bertoni,
G., 2008. A synthetic peptide encompassing the G5 antigenic region of the rabies virus
induces high avidity but poorly neutralizing antibody in immunized animals. Vaccine 26, 67496753.
36. Niederhauser, S., Zahno, M. L., Nenci, C., Vogt, H. R., Zanoni, R., Peterhans, E., Bertoni, G.,
2009. A Gag peptide encompassing B- and T-cell epitopes of the caprine arthritis encephalitis
virus functions as modular carrier peptide. J.Immunol.Methods.
37. Kocan KM, Zivkovic Z, Blouin EF, Naranjo V, Almazan C, Mitra R, de la Fuente J. Silencing of
genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental
cycle in Dermacentor variabilis. BMC Dev Biol. 2009Jul 16;9(1):42. PubMed PMID: 19607704.
38. Esteves E, Bastos CV, Zivkovic Z, de La Fuente J, Kocan K, Blouin E, RibeiroMF, Passos LM,
Daffre S. Propagation of a Brazilian isolate of Anaplasma marginale with appendage in a tick
cell line (BME26) derived from Rhipicephalus (Boophilus) microplus. Vet Parasitol. 2009 Apr
6;161(1-2):150-3. Epub 2008 Dec 13. PubMed PMID: 19150177.
39. de la Fuente J, Blouin EF, Manzano-Roman R, Naranjo V, Almazán C, Pérez de la Lastra JM,
Zivkovic Z, Massung RF, Jongejan F, Kocan KM. Differential expression of the tick protective
antigen subolesin in anaplasma marginale- and A. phagocytophilum-infected host cells. Ann N
Y Acad Sci. 2008 Dec;1149:27-35.PubMed PMID: 19120168.
40. Galindo RC, Doncel-Pérez E, Zivkovic Z, Naranjo V, Gortazar C, Mangold AJ, MartínHernando MP, Kocan KM, de la Fuente J. Tick subolesin is an ortholog of the akirins
described in insects and vertebrates. Dev Comp Immunol. 2009Apr;33(4):612-7. Epub 2008
Nov 28. PubMed PMID: 19041667.
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
41. Zivkovic Z, Blouin EF, Manzano-Roman R, Almazán C, Naranjo V, Massung RF, Jongejan F,
Kocan KM, de la Fuente J. Anaplasma phagocytophilum and Anaplasma marginale Elicit
Different Gene Expression Responses in Cultured Tick Cells. Comp Funct Genomics.
2009:705034. Epub 2009 Jul 15. PubMed PMID: 19636428.
42. de la Fuente J, Kocan KM, Blouin EF, Zivkovic Z, Naranjo V, Almazán C, Esteves E, Jongejan
F, Daffre S, Mangold AJ. Functional genomics and evolution of tick-Anaplasma interactions
and vaccine development. Vet Parasitol. 2009 Sep 20. [Epub ahead of print]
43. Ruzek, D., Bell-Sakyi, L., Kopecky, J., Grubhoffer, L. (2008). Growth of tick-borne encephalitis
virus (European subtype) in cell lines from vector and non-vector ticks. Virus Research 137,
142-146.
44. Moretta R., Ruybal P, Mesplet M., Petrigh R, Nuñez P, Gil G, Wilkowsky S, Garbossa G,
Farber M. Flow Cytometry to Evaluate Anaplasma marginale Parasitemia using a Fluorescent
Nucleic Acid Stain. Ann. N.Y. Acad. Sci. 1149: 111–113 (2008).
45. Tomassone L, Nuñez P, Gürtler RE, Ceballos LA, Orozco MM, Kitron UD, Farber M.
Molecular detection of Ehrlichia chaffeensis in Amblyomma parvum ticks, Argentina. Emerg
Infect Dis . 2008 Dec;14(12):1953-5.
46. H.C. Steyn, A. Pretorius, C.M.E. McCrindle, C.M.L. Steinmann, M. Van Kleef A quantitative
real-time PCR assay for Ehrlichia ruminantium using pCS20. Veterinary Microbiology 131
(2008) 258–265
47. Ruybal P, Moretta R, Perez A, Petrigh R, Zimmer P, Alcaraz E, Echaide I, Torioni de Echaide
S, Kocan KM, de la Fuente J, Farber M. Genetic diversity of Anaplasma marginale in
Argentina. Vet Parasitol. 2009 May 26;162(1-2):176-80.
48. Raliniaina M, Meyer D, Pinarello V, Sheikboudou C, Kandassamy Y, Emboulé L, Adakal H,
Stachurski F, Martinez D, Lefrançois T, Vachiéry N. Mining the genetic diversity of Ehrlichia
ruminantium using map gene family: a key for efficient vaccine against heartwater. Veterinary
Parasitology. 2009. In press.
49. Emboulé L, Daigle F, Meyer D, Mari B, Pinarello V, Sheikboudou C, Magnone V, Frutos R,
Viari A, Barbry P, Martinez D, Lefrançois T and Vachiéry N. Innovative approach for
transcriptomic analysis of obligate intracellular pathogen: Selective Capture of Transcribed
Sequences of Ehrlichia ruminantium. BMC Molecular Biology, In press
50. Adakal H, Meyer D, Carasco-Lacombe C, Pinarello V, Allègre F, Huber K, Stachurski F,
Morand S, Martinez D, Lefrançois T, Vachiery N and Frutos R. MLST scheme of Ehrlichia
ruminantium: genomic stasis and recombination in strains from Burkina-Faso. Under review to
Infection Genetic and Evolution. 2009 Aug 25. [Epub ahead of print]
51. Tomassone L., Nuñez P., Ceballos L.A., Gürtler R.E., Kitron U., Farber M.. Detection of
“Candidatus Rickettsia sp. strain Argentina”and R. bellii in Amblyomma ticks (Acari: Ixodidae)
from Northern Argentina”. Submitted to Experimental and Applied Acarology.
52. Isabel Marcelino, Andre Martinho de Almeida, Rita Francisco, Catarina Franco, Ana Varela
Coelho, Manuel J. T. Carrondo, Paula M. Alves. Proteome analysis of E.ruminantium using
two-dimensional gel electrophoresis coupled with mass spectrometry (in preparation).
53. Isabel Marcelino, Catarina Brito, Mónica Barreto, Nathalie Vachiéry, Thierry Lefrançois,
Dominique Martinez, Manuel J. T. Carrondo, Paula M. Alves. Differential expression and
structural analysis of E.ruminantium outer membrane proteins (in preparation).
Poster & communications
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
1. Cristina Peixoto, Isabel Marcelino, Ana I. Amaral, Marcos F.Q. Sousa, Manuel J.T. Carrondo,
Paula. M. Alves (2006). Downstream processing of Ehrlichia ruminantium elementary bodies for
a vaccine against Heartwater. European Downstream Technology Forum, Goettingen,
Germany.
2. Isabel Marcelino, Marcos F.Q. Sousa, Ana I. Amaral, Cristina Peixoto, Nathalie Vachiery,
Thierry Lefrançois, Dominique Martinez, Manuel J. T. Carrondo, Paula M. Alves (2006). Process
development for a veterinary vaccine against Heartwater using stirred tanks.12th International
Congress on Infectious Diseases (ICID), Lisbon, Portugal.
3. Isabel Marcelino, Marcos F.Q. Sousa, Ana I. Amaral, Cristina Peixoto, Nathalie Vachiery,
Thierry Lefrançois, Dominique Martinez, Manuel J. T. Carrondo, Paula M. Alves (2006).
Optimization of a vaccine candidate against Heartwater. Vaccine Technology, Puerto Vallarta,
Mexico.
4. Vachiéry N., Raliniaina M., Stachurski F., Adakal H., Molia S., Lefrançois T. and Martinez D.
Understanding the mechanisms of transmission of Ehrlichia ruminantium and its influence on the
structure of pathogen populations in the field. Ann. N.Y. Acad. Sci. 2006. Fourth Conference on
Rickettsiae and Rickettsial Diseases
5. L. Bell-Sakyi, E.B.M. Koney and O. Dogbey. 2005. “Tick-borne pathogens in Ghanaian cattle”.5th
International Conference on Ticks and Tick-Borne Pathogens, Neuchatel, Switzerland.
6. Participation to the 9th biennal of Society for Tropical Veterinary Medicine: Animal biodiversity
and emerging diseases prediction and prevention. Oral presentation on: “Amblyomma
variegatum ticks and heartwater in three Caribbean islands: tick infection and Ehrlichia
ruminantium genetic diversity in bovine herds”. Publication in New York Academy of Science
dedicated to this conference.
7. Isabel Marcelino, Marcos F.Q. Sousa, Ana I. Amaral, Cristina Peixoto, Nathalie Vachiéry,
Thierry Lefrançois, Dominique Martinez, Manuel J. T. Carrondo, Paula M. Alves (2007).
Development of a vaccine candidate vaccine against heartwater. 20th Meeting of the European
Society for Animal Cell Technology, Dresden, Germany.
8. Cristina Peixoto, Isabel Marcelino, Ana I. Amaral, Marcos F.Q. Sousa, Manuel J.T. Carrondo,
Paula. M. Alves (2006). Downstream processing of Ehrlichia ruminantium elementary bodies for
a vaccine against Heartwater. 14th International Conference on Biopartitioning and Purification,
Lisbon, Portugal.
9. Bell-Sakyi, L., Jongejan, F. (2007). “Genomes, Ticks and Pathogens”. Accompanying Trends in
Parasitology Volume 23, issue 9.
10. Dr. Marisa Farber gave a conference at the VII Congress of the Argentinian Society of
Protozoology, held in Mendoza, Argentina in October 2005. A total of 50 people attended to the
conference where Dr. Farber showed the latest results obtained in her laboratory regarding the
use of BCG as expression system for parasite antigens.
11. Poster : “Flow Cytometry to Evaluate Anaplasma marginale Parasitemia using a Fluorescent
Nucleic Acid Stain” by R. Moretta, P. Ruybal, M Mesplet, R. Petrigh, P. Nuñez, G .Gil, S.
Wilkowsky, G. Garbossa and M. Farber was accepted for publication in the Annals of the New
York Academy of Sciences as an special volume of the works presented at the 9th Biennial
Conference of the Society for Tropical Veterinary Medicine, Mérida, México.
12. 9th biennal of Society for Tropical Veterinary Medicine: Animal biodiversity and emerging
diseases prediction and prevention, June 2007. Oral presentation on: “Amblyomma variegatum
ticks and heartwater in three Caribbean islands: tick infection and Ehrlichia ruminantium genetic
diversity in bovine herds”. In press, 2008, in New York Academy of Science.
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
13. Nathalie Vachiéry gave a conference at the Département de Microbiologie et d’Immunologie de
l’Université de Montréal, 28 September 2007. Title : « Diversité génétique d’ER en Afrique et
dans les Caraïbes : un frein à la vaccination contre la cowdriose »
14. Poster “Tick cell lines at Edinburgh University” (by Lesley Bell-Sakyi) was presented at the
conference held in Edinburgh on 7-8 April 2008, to launch the new Roslin Institute and Easter
Bush Research Consortium.
15. Isabel Marcelino, Catarina Brito, Mónica Barreto, Nathalie Vachiéry, Thierry Lefrançois,
Dominique Martinez, Manuel J. T. Carrondo, Paula M. Alves (2008). Differential expression and
structural analysis of E.ruminantium proteins: identification of potential antigens for a subunit
heartwater vaccine. Vaccine Technology II, Albufeira, Portugal.
16. Measuring antigen-specific Immune responses. Held in La Plagne, France from 30 th January to
3rd February, 2008
17. ICTTD Bioinformatics Workshop: Sequence analysis tools for mining tick and tick-borne
pathogen genomic and EST data . 8th – 15th June , 2008.
18. A poster entitled “Invasion and early development of Ehrlichia ruminantium in tick and Theileria
annulata-infected monocytic cells” (by Lesley Bell-Sakyi, Camila Bastos, Milagros Postigo and
Ivan Morrison) was presented at the TTP6 conference held in Buenos Aires, Argentina on 21-26
September 2008.
19. Oral presentation. TTP6 conference held in Buenos Aires, Argentina on 21-26 September 2008.
Zivkovic Z, Esteves E, Almazan C, Daffre S, Nijhof AM, Kocan C, Jongejan F, De la Fuente J.
Differential expresión and functionnal study of Boophilus microplus male salivary gland genes in
response to Anaplasma marginale.
20. Differential expression and functional study of Boophilus microplus male salivary gland genes in
response to Anaplasma marginale infection (Zivkovic Z, Esteves E , Almazán C , Daffre S,
Nijhof AM, Kocan KM, Jongejan F, de la Fuente J ) – presented by Zorica Zivkovic at the
TTP6 conference held in Buenos Aires, Argentina on 21st – 26th September 2008.
21. Oral presentation :10th biennal of Society for Tropical Veterinary Medicine: One Health, One
Medicine: Building Bridges to Face the Challenge of Emerging and Zoonotic Diseases, June
2009, Germany. “Selective capture of transcribed sequences method of Ehrlichia ruminantium
for transcriptomic study” . Loïc Emboulé, France Daigle, Damien Meyer, Bernard Mari, Valérie
Pinarello, Christian Sheikboudou, Virginie Magnone, Roger Frutos, Alain Viari, Pascal Barbry,
Dominique Martinez, Thierry Lefrançois and Nathalie Vachiéry
22. Poster : 3rd congress of European microbiologist, FEMs Microbiology. June 2009, Sweden :
“MAP proteins of the Rickettsia Ehrlichia ruminantium : A key for efficient vaccine against
heartwater ?” Authors: Meyer D, Lefrançois T, Sheikboudou C, Pinarello V, Giraud-Girard K,
Martinez D and Vachiéry N
23. Poster : 3rd congress of European microbiologist, FEMs Microbiology. June 2009, Sweden :
“High throughput analysis of the transcriptome of ER: Implication for genes involved in the
mechanisms of virulence” : Loïc Emboulé, France Daigle, Roger Frutos, Damien Meyer, Alain
Viari, Valérie Pinarello, Sheikboudou Christian, Virginie Magnone, Bernard Mari, Pascal Barbry,
Dominique Martinez, Thierry Lefrançois and Nathalie Vachiéry
24. A poster entitled “Transformation of bovine monocytes by Theileria annulata renders them
susceptible to infection with Ehrlichia ruminantium” (by Lesley Bell-Sakyi, Niall MacHugh and
Ivan Morrison) was presented at the British Society for Parasitology Spring Meeting in
Edinburgh, UK on 5-8 April 2009.
25. Differential expression and functional study of Boophilus microplus male salivary gland genes in
response to Anaplasma marginale infection (Zivkovic Z, Esteves E , Almazán C , Daffre S,
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Project N° FP6-003713 / EPIGENEVAC / Final report / 2009
Nijhof AM, Kocan KM, Jongejan F, de la Fuente J ) – presented by Zorica Zivkovic at the
STVM conference held in Lubeck, Germany on June 29th – July 3rd 2009.
26. M Van Kleef attended a quantitative real-time PCR Symposium held at the University of Munich
9-13 March 2009.
PhD thesis
1. Milagros Postigo submitted her PhD thesis entitled “Molecular and antigenic characterization of
Ehrlichia ruminantium in Amblyomma variegatum ticks and in vitro cultures” to the University of
Edinburgh at the end of June 2006; her research was supervised by Ivan Morrison and Frans
Jongejan (Utrecht University).
2. Isabel Marcelino submitted her PhD thesis entitled “Development of a vaccine candidate against
heartwater” to the Universidade Nova de Lisboa on 15th of March 2007; her research was
supervised by Dr. Paula M. Alves and Prof. Manuel Carrondo; Dr. Dominique Martinez was a
member of the jury.
3. Bonto Faburay submitted his PhD thesis to the University of Utrecht at the end of June 2007; his
research was supervised by Frans Jongejan (Utrecht University).
4. Rosalia Moretta. Doctoral Thesis at University of Buenos Aires, Argentina. June 2008. Advisor:
Marisa Farber
5. Modestine Raliniaina. PhD thesis University of Montpellier II. « diversité génétique de la
rickettsie Ehrlichia ruminantium en Guadeloupe et à Madagascar : caractérisation moléculaire et
mécanismes de transmission des souches » supervised by CIRAD. Défense in December 2009
6. Hassane Adakal. PhD thesis University of Montpellier II. « Structure génétique des populations
d’Ehrlichia ruminantium au Burkina Faso » supervised by CIRAD. Défense in December 2009
Awards
Attribution of the APDF (Associação Portuguesa de Doutorandos em França) award that
recognized the excellent collaboration between the two institutes CIRAD and IBET during
the PROCORDEL project regarding the “Optimization of a vaccine against Heartwater”
(awarded by the Embassy of France in Portugal).
In addition various courses or conferences, interviews were given by some of the
participants
Organisation of International Conferences
The INTA partner organized and hosted the TTP6 Conference (The VI International
Conference on Ticks and Tick-borne Pathogens in Buenos Aires, Argentina, from
September 21 to 26, 2008.). The scientific responsible for EPIGENEVAC at INTA was the
vice-President of the conference
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