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Epidemiology and new generation vaccines for Ehrlichia and anaplasma infections of ruminants

Final Report Summary - EPIGENEVAC (Epidemiology and new generation vaccines for Ehrlichia and anaplasma infections of ruminants)

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 characterised 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.

Results achieved

After four years of activity, the complete genome sequences of three strains of E. ruminantium have been obtained and annotated In silico. Comparative genomic with other Anaplasmataceae revealed that the gene organisation is highly conserved between the three 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 three 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 immunisation.

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 high-throughput 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.

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-like particle (VLP) together with improved immunisation schedule, was planned in the project.

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 characterisation represent the methods of choice. These molecular methods were extensively developed in the project in order to improve the sensitivity of detection and to characterise the genetic diversity of E. ruminantium and to a lower extent of A. marginale.