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

Molecular surveillance of Theileria parasites of livestock in Oman

Background: Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian Peninsula, and causes high rates of mortality and morbidity in sheep and cattle. However, there is a paucity of information on the distribution of Theileria spp. over the whole region and their impact on different hosts. The present study carried out a country-wide molecular survey for Theileria spp. of livestock in Oman across four governorates. The aim of the survey was to define the prevalence of Theileria spp. in cattle, sheep and goats, highlight risk factors for infection and identify the main tick species involved in parasite transmission. Material and methods: A total of 2020 animals were examined in the survey consisting of sheep [n = 592], goats [n = 981] and cattle [n = 447]. All three species were raised and co-grazed on the same farms. Theileria parasites were detected using PCR-RFLP and RLB of the 18S rRNA gene. Cloning and sequencing of the 18S rRNA was carried out on 11 T. lestoquardi isolates from Ash-Sharqiyah, and Ad-Dhahira governorates, and phylogenetic relationships were inferred using additional sequences of T. lestoquardi, T. annulata and T. ovis available in GenBank. Results: Theileria spp. prevalence was 72.3%, 36.7% and 2.7% among cattle, sheep and goats, respectively. Strong similarity in results was obtained using RLB and PCR-RFLP for detection of Theileria spp. however, RLB detected a higher rate of mixed infection than PCR-RFPL (P < 0.001). Theileria annulata was the only parasite detected in cattle, while sheep and goats carried T. ovis, T. lestoquardi and T. annulata as well as Theileria spp. OT1. Of the four Theileria spp. detected in small ruminants, overall T. ovis was most prevalent (sheep [33.4%], goats [2.0%]), whereas T. lestoquardi was less prevalent (sheep [22.0%], goats [0.5%]). A large proportion of infected sheep (19%) carried mixed infection of T. ovis and T. lestoquardi. However, single T. lestoquardi infections (3.0%) were less prevalent than T. ovis infections (14.5%). Risk of Theileria spp. infection was significantly higher for exotic breeds, relative to native breeds, of cattle (p = 0.00002) and sheep (p = 0.005). Phylogenetic analysis placed T. lestoquardi in Oman in the same clade as other T. lestoquardi strains isolated from the same regional area (Iraq and Iran). The main tick species, identified on the examined animals, Hyalomma anatolicum, was widely distributed and was found in all of the surveyed governorates. Conclusion: Theileria spp. are widespread in Oman with variable prevalence detected in different regions. Two economically important hosts, cattle and sheep are at high risk from virulent T. annulata and T. lestoquardi, respectively. The survey indicates extensive exposure to ticks and transmission of infection that has a significant economic impact. The higher prevalence of T. lestoquardi as mixed rather than single infection requires further investigation

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydın region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal

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

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

Background: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. Results: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Conclusions: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a ‘One Health’ approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans

Identification and characterisation of a Theileria annulata proline-rich microtubule and SH3 domain-interacting protein (TaMISHIP) that forms a complex with CLASP1, EB1, and CD2AP at the schizont surface.

Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria-induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co-immunoprecipitation, we identified a CLASP1/CD2AP/EB1-containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14-3-3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta-p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain-interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1-binding SxIP motif, as well as functional SH3 domain-binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non-infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis

Failure of combination therapy with imidocarb dipropionate and toltrazuril to clear Hepatozoon canis infection in dogs

GAZYAGCI, SERKAL/0000-0002-0043-6942WOS: 000294225700047PubMed: 21472405Current treatments with imidocarb dipropionate for infected dogs with Hepatozoon canis do not always provide parasitological cure. The objective of this study is to determine whether concomitant use of toltrazuril may potentiate the effect of imidocarb dipropionate in the management of H. canis infection (HCI). Twelve dogs were determined to have naturally HCI based on clinical signs, identification of the parasite in blood smears, and serologic assay. The animals were allocated randomly to one of two groups (n = 6 in each group). Dogs in Imi group were given imidocarb dipropionate at a dose of 6 mg/kg body weight subcutaneously in two injections 14 days apart. Imi plus Toltra group was given imidocarb dipropionate as dose mentioned above and toltrazuril at 10 mg/kg/day orally for the first five treatment days. Clinical findings, blood counts and parasitaemia levels in blood before and 14, 28 and 56 days after the initial treatment were performed to evaluate treatment response. The overall clinical efficacy of imidocarb dipropionate with and without toltrazuril was 83.3% and 66.7%, respectively; with a mean recovery time of 21.0 and 25.6 days, respectively. A substantial main effect of time on mean PCV, Hb, WBC, neutrophil and PLT and gradual reduction of parasitaemia were significantly observed in both groups (P < 0.05), whereas no significant difference was noticed between the studied protocols. The parasitologic cure rate at the end of eight weekly observation period was 16.6% and 33.3% in Imi and Imi plus Toltra groups, respectively. Similar clinicopathologic and parasitologic responses were observed in both treated groups; thus, it was concluded that toltrazuril does not reveal additional benefit to imidocarb therapy in dogs with HCI

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