SummaryAnaplasma species are obligate intracellular rickettsial pathogens transmitted by ticks with an impact on human and animal health. Anaplasma ovis infects sheep and goats in many regions of the world, and it can be diagnosed by different methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR based on the gene coding for major surface protein 4 (MSP-4) was used to examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, were positive. This indicates high prevalence of A. ovis in the countries under investigation, and it can be assumed that the situation in other areas of the world might be similar. Thus, A. ovis should be considered as an important constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.

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Graphical abstractHighlights► Novel multiplex PCR for Theileria annulata, Babesia bovis and Anaplasma marginale. ► Specific and sensitive tool which can be applied to epidemiological studies. ► Simple and efficient assay which has been validated using field samples.

BackgroundTick-borne haemoparasitic diseases (TBHDs), caused by Theileria, Babesia, Anaplasma and Ehrlichia, are common in regions of the world where the distributions of host, pathogen and vector overlap. Many of these diseases threaten livestock production and some also represent a concern to human public health. The primary aim of this study was to determine the prevalence of the above-mentioned pathogens in a large number of blood samples (n = 1979) collected from sheep (n = 1727) and goats (n = 252) in Turkey. A secondary aim was to assess the diagnostic sensitivity of a number of species-specific polymerase chain reaction (PCR) tests and the reverse line blotting (RLB) assay. DNA samples were screened using species-specific PCR for the presence of Theileria ovis, Theileria sp. MK, T. lestoquardi, T. uilenbergi, T. luwenshuni, Babesia ovis, Anaplasma ovis and A. phagocytophilum while RLB was undertaken to test for the presence of all known Theileria, Babesia, Anaplasma and Ehrlichia species. The diagnostic sensitivity of these two approaches was then compared in terms of their ability to detect single species and mixed infections.ResultsOverall, 84 and 74.43% of the small ruminants sampled were identified as hosting one or more pathogen(s) by species-specific PCR and RLB respectively. The presence of Theileria sp. OT1, T. luwenshuni and T. uilenbergi in Turkey was revealed for the first time while the presence of Babesia motasi, B. crassa and T. separata in Turkish small ruminants was confirmed using molecular methods. A high prevalence of mixed infection was evident, with PCR and RLB approaches indicating that 52.24 and 35.42% of animals were co-infected with multiple species, respectively. More than 80% of the mixed infections contained T. ovis and/or A. ovis. The RLB approach was found to be capable of detecting mixed infections with species such as Theileria sp. OT1, Theileria sp. OT3, T. separata, B. crassa and Babesia spp.ConclusionThe results indicated that pathogens causing TBHDs are highly prevalent in sheep and goats in Turkey. The diagnostic sensitivity of species-specific single PCR was generally higher than that of RLB. However, the latter approach was still capable of identifying a high proportion of individuals containing mixed-species infections. The use of species-specific single PCR is recommended to accurately estimate pathogen prevalence and to identify co-infected hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2151-3) contains supplementary material, which is available to authorized users.

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10 Background 11Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian 12 Peninsula, and causes high rates of mortality and morbidity in sheep and cattle.

Theileria annulata is an obligate intracellular protozoan parasite of the phylum Apicomplexa. Theileria sporozoites invade bovine leukocytes and develop into a multinucleate syncytial macroschizont that causes uncontrolled proliferation and dissemination of infected and transformed leukocytes. Activator protein 1 (AP-1) is a transcription factor driving expression of genes involved in proliferation and dissemination and is therefore a key player in Theileria-induced leukocytes transformation. Ta9 possesses a signal peptide allowing it to be secreted into the infected leukocyte cytosol and be presented to CD8 T cells in the context of MHC class I. First, we confirmed that Ta9 is secreted into the infected leukocyte cytosol, and then we generated truncated versions of GFP-tagged Ta9 and tested their ability to activate AP-1 in non-infected HEK293T human kidney embryo cells. The ability to activate AP-1-driven transcription was found to reside in the C-terminal 100 amino acids of Ta9 distant to the N-terminally located epitopes recognised by CD8+ T cells. Secreted Ta9 has therefore, not only the ability to stimulate CD8+ T cells, but also the potential to activate AP-1-driven transcription and contribute to T. annulata-induced leukocyte transformation.