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Molecular characterization of Latin American and Mediterranean Babesia bovis and B. bigemina strains and its application for the development of improved control strategies

Final Report Summary - MEDLABAB (Molecular characterisation of Latin American and Mediterranean Babesia bovis and B. bigemina strains and its application for ... improved control strategies)

The EU-funded project MEDLABAB aimed at controlling two very important cattle diseases. It was an INCO Specific Targeted Research Project (STREP No. 003691), funded under the Sixth Framework Programme, within the Thematic Priority of 'Improved control strategies for parasitic protozoa from cattle'. It dealt with bovine babesiosis, a serious cattle disease that limits the production of meat and milk and is caused by Apicomplexan parasites of the genus Babesia sp. It affects vast tropical and subtropical regions of the world, where the tick vector thrives. The goal of the project has been to generate new knowledge and biotechnological tools that can lead to a better control of the disease. The specific aims included the identification of molecular markers for the typification of Babesia bovis and B. bigemina strains present in Latin America and Mediterranean Europe, the study of genetic and antigenic polymorphisms in vaccine candidate antigens among different field isolates, and the detection of alternative carriers or reservoirs for these parasites. The information gathered can be useful for vaccine design and for decision-making about adequate control measures.

In addition, the project aims included developing new diagnostic methods for bovine babesiosis. An additional important goal of the project has been to maximise a fruitful interaction between scientists and students from the countries involved, leading to strengthened research capabilities; and to increase awareness and available information about this disease.

To accomplish the mentioned objectives, a consortium was gathered composed of research institutions of seven countries:
(1) University of Alcala de Henares, Spain;
(2) Center of Research in Veterinary and Agricultural Sciences (CICVyA), National Institute of Agricultural Technology (INTA), Argentina;
(3) Istituto Zooprofillatico della Sicilia, Italy;
(4) Erciyes University, Turkey;
(5) the Brazilian Agricultural Research Corporation (EMBRAPA), Brazil;
(6) the National Institute for Investigation in Forestry, Agriculture and Animal Production (INIFAP), Mexico; and
(7) the Institute for Biological and Experimental Technology (IBET), Portugal.

WP 1 was aimed at the collection of B. bovis and B. bigemina DNA samples from geographical isolates of different bovine babesiosis-endemic regions; the collection of a set of merozoite antigen preparations from the same isolates; the assembly of a bank of B. bovis and / or B. bigemina-infected serum samples; and the collection of infected blood samples to be used as inoculum to start in vitro cultures. For DNA extraction, two types of samples were obtained. On one hand, bovine whole blood samples were randomly collected in different regions of high endemicity for the vector ticks that transmit B. bovis and B. bigemina parasites. The presence of parasite DNA was confirmed by PCR, nested PCR or RLB. In this way, from a total of 2473 samples collected in Argentina, Italy, Turkey and Portugal, 82 B. bovis-positive and 60 B. bigemina-positive DNA samples could be gathered and conform a virtual DNA bank available for further studies within the consortium. On the other hand, blood samples from babesiosis clinical cases, with higher parasitemia, were also collected, and whenever possible, parasitemia was amplified in splenectomised naïve bovines. These samples, together with reference strains regularly used in some of the participating countries, also form part of the MEDLABAB DNA bank, and include 27 B. bovis samples and 20 B. bigemina samples. In addition blood samples with high parasitemia were stored in liquid nitrogen in the presence of cryopreservatives, as inocula to start in vitro cultures; and for antigen preparation. With respect to serum samples, a virtual bank of over 5 000 bovine samples from tick-endemic regions was gathered. Samples are maintained frozen in the different participating laboratories. In most cases, the presence of antibodies against B. bovis and / or B. bigemina was assayed by immunofluorescence or ELISA based on merozoite extracts or recombinant antigens. In addition to these samples, experimental infections of cattle were performed with attenuated or pathogenic B. bovis and B. bigemina strains, and serum samples were collected from these animals at different times post-infection.

WP 2 was aimed at the production of in vitro cultures of B. bovis and B. bigemina and the isolation of parasite biological clones. Methods for in vitro cultivation of these parasites have long been established, and although they are relatively simple, they require expertise, fully dedicated personnel, available animal facilities for the periodical provision of bovine erythrocytes and serum, and sterile culture facilities. In addition, some parasite lines show intrinsic limitations to grow in culture. In spite of these limitations, cultures of different pathogenic and attenuated strains could be successfully established in three participating laboratories. Also, other two groups got intensive training in Babesia sp. culturing techniques that can be applied for future projects. In addition, as a contribution to the simplification of B. bovis culturing techniques, a culturing method that reduces the amount of serum needed was developed.

Four pathogenic and two attenuated biological clones of B. bovis were obtained during the project. Their parental strains were S2P, a pathogenic isolate collected in the NW of Argentina, and R1A, originally a pathogenic isolate collected in the centre of this country that was later attenuated by serial passages in splenectomised bovines. Parasites were multiplied in continuous in vitro culture, and clones were obtained by the limited dilution method. In a first round of cloning, 11 primary biological clones were obtained from the S2P strain and three from the R1A strain. Clones showing the highest in vitro growth rates (four pathogenic and two attenuated) were chosen and their phenotypes, in vivo evaluated by experimental inoculation of calves. Different clinical parameters were assessed, such as the increase of body temperature, the maximum hematocrit decrease, the levels of parasitemia measured in peripheral blood and the occurrence of nervous symptoms. This experiment showed that all clones kept their parental phenotypes.

WP 3 was focused on the study of polymorphism in genes that encode for B. bovis and B. bigemina antigens that can be used for future recombinant vaccines and new diagnostic methods; as well as the detection of conserved B-cell epitopes in these antigens. In this work package, already defined antigens and previously uncharacterised ones were studied. The first included, for B. bovis, the genes encoding for the merozoite surface antigen family (msa-1, msa-2a1, msa-2b, msa-2a/b and msa-2c) as well as rap-1, ama, acs-1 and trap genes; and for B. bigemina, rap-1a, 1b, 1c and GP45. These genes were amplified from DNA of different isolates, sequenced and deposited in the GenBank. Conserved and polymorphic regions were determined in sequence alignments, and B-cell epitopes were predicted using bioinformatic algorithms. In addition to the studies carried out on already described antigens, some other were described during this project and constitute promising candidates for the development of new control strategies. These include: B. bovis serine and a cystein protease; B. bovis MIC1-like protein; B. bovis PFS230-A; a highly conserved ribosomal phosphoprotein in B. bovis; B. bigemina spherical body protein 1 (SBP-1) and ama-1, ortologues of the antigens of same name in B. bovis.

WP 4 was aimed at the development of molecular markers for strain typification. These markers can be useful in epidemiological and population genetic studies, as well as to understand vaccine failures. Several approaches to define molecular markers were used in the case of B. bovis.

The objectives of WP 5 were the search for alternative reservoirs of B. bovis and B. bigemina parasites. To this aim, whole blood was collected from different mammals that live in tick-endemic regions, and the presence of parasite DNA was analysed in extracted samples by PCR, nested PCR, Real Time PCR or RLB. Additionally, in some cases, the presence of specific antibodies was analyzed in collected serum samples. The conclusions that can be drawn from the results obtained are the following:
(i) significant reservoir hosts for B. bovis and B. bigemina include: wild ruminants, buffaloes and equines;
(ii) hosts with little or no significance as reservoir include wild and domestic carnivores, wild boar and small wild mammals.
Our results suggest that it would be convenient to include equines and water buffaloes in control campaigns against babesiosis, especially in places where they share grazing lands.

WP 6 was aimed at the development of new diagnostic methods for bovine babesiosis. Significant progress was achieved in the development of both direct and indirect methods.

WP 7 concerned internal communications within the consortium. A fruitful interaction among members was generated by the organisation of three meetings, the first in Buenos Aires, Argentina, in December 2005, the second in Palermo, Italy, in May 2007 and the third, in Buenos Aires, in September 2008. These meetings were organised immediately after or before congresses where the project results could be shared within the participants and with other members of the scientific community. In this way, the Babesia World Summits I and II followed the first and second meetings; and the VI International Conference on Ticks and Tick-borne Pathogens (TTP-6) preceded the third MEDLABAB consortium meeting. Communication among members was also maintained through exchange of information and methodologies via e-mail, visits and training periods, joint collaborative research, and a mailing list, containing relevant information on the project topics, such as recent publications and news.

WP 8 was aimed at the dissemination of information, which was successfully achieved by the following efforts:
(i) creation of a website (please see online) that presented information on the project and project members, as well as generalities on bovine babesiosis and the epidemiological situation in each of the participating countries;
(ii) a fruitful production of scientific reports that includes 31 full papers published in scientific journals, 21 short communications in a special issue of the Parassitologia journal, 6 manuscripts in preparation or submitted, 112 abstracts contributed to scientific meetings;
(iii) 3 PhD theses, 7 MSc theses and 7 undergraduate theses, already defended; as well as 5 ongoing PhD theses, in all cases related to the project objectives;
(iv) 3 articles in diffusion magazines and 38 conferences dictated (with an estimated audience of over 2300).

WP 9 concerned the financial management of this project, which was carried out by the financial coordinator.

It is important to mention that the interactions between MEDLABAB partners are bound to continue beyond the timeframe of this project. Samples have been exchanged between partners for the testing of molecular markers and diagnostic methods, which will be carried out in the near future. Additionally, collaborative proposals between two or more partners have been presented to funding organisms to allow that the research lines that started with this project can continue in the future.