Comparative genomic analysis and phylogenetic position of Theileria equi

Lowell S Kappmeyer 3 Mathangi Thiagarajan 2 7 David R Herndon 3 Joshua D Ramsay 0 Elisabet Caler 2 Appolinaire Djikeng 6 Joseph J Gillespie 4 5 Audrey OT Lau 0 1 Eric H Roalson 8 Joana C Silva 4 Marta G Silva 3 Carlos E Suarez 3 Massaro W Ueti 3 Vishvanath M Nene 6 Robert H Mealey 0 Donald P Knowles 0 3 Kelly A Brayton 0 1 0 Department of Veterinary Microbiology & Pathology, Washington State University , Pullman, WA 99164-7040 , USA 1 Paul G. Allen School for Global Animal Health, Washington State University , Pullman, WA 99164-7040 , USA 2 J. Craig Venter Institute , Rockville, MD 20850 , USA 3 Animal Disease Research Unit, Agricultural Research Service, USDA , Pullman, WA 99164-7030 , USA 4 Institute for Genome Sciences and Department of Microbiology and Immunology, University of Maryland School of Medicine , Baltimore, MD 21201 , USA 5 Virginia Bioinformatics Institute at Virginia Tech , Blacksburg, VA 24061 , USA 6 International Livestock Research Institute , P.O. Box 30709, Nairobi 00100 , Kenya 7 Current address: Frederick National Lab for Cancer Research , Rockville, MD 20852 , USA 8 School of Biological Sciences, Washington State University , Pullman, WA 99164-4236 , USA Background: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. Results: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. Conclusions: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms. - Background Equine piroplasmosis of horses, mules, donkeys and zebras is caused by the tick-borne apicomplexan protozoan parasites Babesia caballi and Theileria equi, transmitted by ixodid ticks such as Dermacentor nitens (B. caballi) and Rhipicephalus microplus (T. equi) [1,2]. Although endemic in most countries [3], the U. S., until recently, has been considered free of infection. Equine infections in Florida with B. caballi and T. equi were diagnosed between 1961 and 1969 leading to an eradication campaign which lasted twenty-five years and cost twelve million dollars [4]. The re-emergence of T. equi in Florida [4] and Texas [5] raised concern of its further spread within the U. S., and indeed, infected horses have been identified in 12 states [6,7]. The cause of the 2008 Florida outbreak was due to iatrogenic transmission, but two tick species, Amblyomma cajennense and D. variabilis, were identified as novel vectors in the 2009 Texas outbreak [5]. The re-emergence of this pathogen in the U.S. impacts global movement and health of horses and affects the multi-billion dollar equine industry. Additional members of the phylum Apicomplexa, important to global human and animal health include the organisms in the genus Plasmodium as well as T. parva and T. annulata, and Babesia bovis causes of malaria, bovine theileriosis and babesiosis, respectively. The phylogenetic position of T. equi has been controversial, and the organism has been renamed several times [8]. Molecular phylogenetic analyses indicate an intermediate position for T. equi between B. bovis and Theileria spp. [9,10] and is supported by the genomic data presented here which provides the deepest phylogenetic analysis to date. Collective data supports the concept that a new genus placement sister to Theileria may be appropriate for T. equi. Similar to bovine theileriosis caused by T. annulata, transmission of T. equi to equids eventually results in lysis of erythrocytes and prolonged anemia. Anemia associated with T. parva occurs later during infection and is comparatively and clinically mild [11]. Infection of B- and T-lymphocytes by T. parva and mononuclear phagocytes and B-lymphocytes by T. annulata lead to reversible cell transformation [12]. Infection of peripheral blood mononuclear cells (PBMCs) by T. equi has been reported [8,13]. However the role of PBMC infection in the pathogenesis of T. equi, unlike T. parva and T. annulata remains unresolved, and PBMC proliferation and/or transformation have not been associated with clinical equine piroplasmosis. The primary clinical outcome of acute T. equi infection is anemia and the associated erythrolysis is independent of parasite-specific immune responses [14]. Resolution of acute disease is followed by apparent lifelong parasite persistence within equids [15]. Persistence is characterized by the continuous presence of 103 to 106 infected peripheral erythrocytes per ml/blood resulting in efficient acquisition and transmission by ticks [16]. A hallmark of pathogens that establish persistent infection and avoid immune elimination is the presence of an immunodominant, variable multigene family responsible for immune evasion, such as VESA1 (Variant Erythrocyte Surface Antigen 1) in B. bovis [17], PfEMP1 (Erythrocyte Membrane Protein 1) in P. falciparum [18] and VSG in T. brucei [19]. An analogous family was not detected in T. equi. A candidate multigene gene family in T. equi encodes Equi Mer (...truncated)