Prevalence of tick-borne haemoparasites in small ruminants in Turkey and diagnostic sensitivity of single-PCR and RLB

Background: Tick-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. Results: Overall, 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. Conclusion: The 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

Infection dynamics of Theileria annulata over a disease season following cell line vaccination

Tropical theileriosis is a tick-borne haemoparasitic disease of cattle caused by the protozoan parasite Theileria annulata. Globally, the economic impact of the disease is immense and enhanced control measures would improve livestock production in endemic regions. Immunisation with a live attenuated vaccine is an effective and widely used control method, however, the repeated use of live vaccines may have an impact on the field parasite population at a genetic level. Additionally, there has been an increasing number of reports of vaccine breakthrough cases in recent years. Thus, the present study was designed to evaluate the genetic composition of a parasite population over a disease season in a locality where live cell line vaccination is practised. A diverse range of parasite genotypes was identified and every T. annulata positive cattle blood sample harboured multiple parasite genotypes. An alteration in the major genotype and an increasing multiplicity of infection in individual animals was observed over the course of the disease season. Vaccination status was found not to effect within-host multiplicity of infection, while a significantly higher number of genotypes was detected in grazed cattle compared to non-grazed ones. A degree of genetic isolation was evident between parasite populations on a micro-geographic scale, which has not been reported previously for T. annulata. Analysis of parasite genotypes in vaccinated animals suggested only a transient effect of the vaccine genotype on the genetic diversity of the T. annulata population. The vaccine genotype was not detected among clones of two vaccine ‘breakthrough’ isolates and there is no suggestion that it was responsible for disease. The obtained data indicated that in the system studied there is no apparent risk of introducing the vaccine genotype into the population with only a transient effect on the genetic diversity of the parasite population during the disease season

High genetic diversity and differentiation of the babesia ovis population in Turkey

Babesia ovis is a tick‐transmitted protozoan haemoparasite causing ovine babesiosis in sheep and goats leading to considerable economic loss in Turkey and neighbouring countries. There are no vaccines available, therapeutic drugs leave toxic residues in meat and milk, and tick vector control entails environmental risks. A panel of eight mini‐ and micro‐satellite marker loci was developed and applied to study genetic diversity and substructuring of B. ovis from western, central and eastern Turkey. A high genetic diversity (He = 0.799) was found for the sample of overall B. ovis population (n = 107) analyzed. Principle component analysis (PCoA) revealed the existence of three parasite subpopulations: (a) a small subpopulation of isolates from Aydin, western Turkey; (b) a second cluster predominantly generated by isolates from western Turkey; and (c) a third cluster predominantly formed by isolates from central and eastern Turkey. Two B. ovis isolates from Israel included in the analysis clustered with isolates from central and eastern Turkey. This finding strongly suggests substructuring of a major Turkish population into western versus central–eastern subpopulations, while the additional smaller B. ovis population found in Aydin could have been introduced, more recently, to Turkey. STRUCTURE analysis suggests a limited exchange of parasite strains between the western and the central–eastern regions and vice versa, possibly due to limited trading of sheep. Importantly, evidence for recombinant genotypes was obtained in regionally interchanged parasite isolates. Important climatic differences between the western and the central/eastern region, with average yearly temperatures of 21°C versus 15°C, correspond with the identified geographical substructuring. We hypothesize that the different climatic conditions may result in variation in the activity of subpopulations of Rhipicephalus spp. tick vectors, which, in turn, could selectively maintain and transmit different parasite populations. These findings may have important implications for vaccine development and the spread of drug resistance.Instituto de PatobiologíaFil: Mira, Anabela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Unlu, Ahmet Hakan. Van Yuzuncu Yil University. Vocational School of Gevas; TurquíaFil: Bilgic, Huseyin Bilgin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Bakirci, Serkan. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Hacilarlioglu, Selin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Karagenc, Tulin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Carletti, Tamara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Weir, William. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shiels, Brian. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino UnidoFil: Shkap, Varda. Kimron Veterinary Institute. Division of Parasitology; IsraelFil: Aktas, Munir. Firat University. Faculty of Veterinary Medicine. Department of Parasitology; TurquíaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The secreted Theileria annulata Ta9 protein contributes to activation of the AP-1 transcription factor.

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

First Molecular Characterization of Raphidascaris acus Bloch, 1779 (Nematoda: Anisakidae) from European eels (Anguilla anguilla Linnaeus, 1758) Caught off the Aegean Region Streams, Turkey

In this study, the presence of anisakid nematodes in the European eels (Anguilla anguilla L.) was investigated. A total of 30 specimens of eels were caught by local fishermen from the Buyuk Menderes River, Turkey. Nematoda species of Raphidascaris acus was found from eels. This is the first record of R. acus from A. anguilla from Turkish waters. The ribosomal DNA (rDNA) internal transcribed spacer regions (ITS-1and ITS-2) and 5.8S of this parasitic species was amplified and sequenced. Also, all the nematodes were identified as R. acus based on nucleotide sequence comparisons. Pairwise comparison between the entire ITS regions and 5.8S of the R. acus isolates of A. anguilla (GenBank accession number: KT633862) and other R. acus isolates from Caspian Sea (KM047505), and Vistula Lagoon, Poland (AY603537) showed differences ranging from 0.0 to 1.9% intraspecific nucleotide differences, respectively. With the present study, R. acus infecting A. anguilla caught off the Aegean Region streams were characterized for the first time by sequencing of the rDNA ITS regions and 5.8S

Parasites Detected by Examination of Fecal Samples in Wrestling Camels

First historical findings on camel wrestling, which is now practiced as a festival in Turkey, particularly in certain regions (Marmara, Aegean, Mediterranean) date back to the 15th century. In terms of animal husbandry, parasitic diseases may result in negative outcomes ranging from loss of performance to death for camels. In the present study, annual camel wrestling arenas were visited between December and March (2010-2011), and stool samples were collected from camels from different cities for parasitological analysis. Stool samples of 109 camels from 7 different cities (Aydin, Izmir, Manisa, Denizli, Mugla, Balikesir, and Canakkale) were examined using Baermann-Wetzel stool culture, flotation, and sedimentation techniques for the parasites that live in gastrointestinal tract. The analyses revealed that 74% of the camels (81 of 109) were infected with one or more parasites: Trichostrongylus spp. (47.7%), Ostertagia spp. (27.5%), Dicrocoelium spp. (24.7%), Trichuris spp. (11.9%), Eimeria cameli (11.9%), Capillaria spp. (6.4%), Fasciola spp. (6.4%), Dictyocaulus viviparous (5.5%), Haemonchus spp. (4.5%), Oesophagostomum spp. (4.5%), Cooperia spp. (4.5%), Cooperia oncophora (3.6%), Nematodirus spp. (3.6%), Chabertia ovina (2.7%), Eimeria spp. (1.8%), and Paramphistomum spp. (0.9%). 16 different parasites, at the level of species and genus, were found, of which 14 were helminth (11 nematodes, 3 trematodes), and 2 were protozoans. The present study was the first to report Ostertagia spp., Fasciola spp. Dictyocaulus viviparus, Haemonchus spp., Oesophagostomum spp., Cooperia spp., Cooperia oncophora, Chabertia ovina and Paramphistomum spp. in camels in Turkey. As high as 74 percent of the incidence of parasitic diseases and the wide variety of parasites found in the present study suggest that parasitic infections may be overlooked entity in wrestling camels that are meticulously brought up

Ta9 constructs used for transfections.

<p>Ta9 constructs used for transfections.</p

Western blot of GFP-Ta9 fusion protein detected with immune sera (A) and Ta9-specific affinity purified antibody (B).

<p>A: Lane 1; GFP-Ta9-1-335, 2; GFP-only. B: Lane 1; GFP-Ta9-1-335, 2; GFP-only. M: All Blue Standards (BioRad). Control anti-GFP antibody.</p

The level of Ta9 protein following Bw720c treatment.

<p>A: Hyper-immune sera; Lane 1: BL3 cells non-treated with Bw720c, 2: BL3 cells treated with Bw720c, 3: TBL3 cells non-treated with Bw720c, 4: TBL3 cells treated with Bw720c, 5: GFP-Ta9-1-335 aa. B: Ta9 specific affinity purified ab; Lane 1: BL3 cells non-treated with Bw720c, 2: BL3 cells treated with Bw720c, 3: TBL3 cells non-treated with Bw720c, 4: TBL3 cells treated with Bw720c. M: All Blue Standards (BioRad). Control anti-β-Tubulin.</p