Hasan Eren

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle

Graphical abstract Highlights ► 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.

Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals

Bovine tropical theileriosis, caused by the tick-borne protozoan Theileria annulata, imposes a serious constraint upon breed improvement programmes and livestock production in tropical and sub-tropical regions of the world. Animals that recover from primary infection serve as carriers and play a critical role in the epidemiology of the disease, acting as reservoirs of infection. However, conclusive identification of carrier animals can be problematic. This study describes assessment of candidate target genes for PCR assay-based detection of T. annulata infected carrier animals. Following in silico screening and rejection of three major multi-copy gene families, an assay based on PCR amplification of a 312 bp segment of the T. annulata gene for cytochrome b (Cytob1 assay) was established. Sensitivity was evaluated using serial dilutions of blood obtained from experimentally infected calves, while specificity was confirmed by testing DNA representing twelve different T. annulata stocks and other Theileria and Babesia species. Direct comparison with other target genes and published data indicated that Cytob1 PCR-based assays provide the greatest level of sensitivity, combined with a high level of specificity and the ability to detect different T. annulata genotypes. It can be concluded that the cytochrome b gene is the optimal target for PCR amplification and its incorporation in a Reverse Line Blot Assay offers the most sensitive method yet devised to detect the parasite in carrier animals. The use of this assay will increase the accuracy of epidemiological studies aimed at improving disease control in endemically unstable regions.

A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata.

In this comparative study unfed nymphs of four Hyalomma tick species (Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum) were allowed to engorge on calves experimentally infected with Theileria annulata. The infection prevalence in the salivary glands of the adult female and male ticks of each Hyalomma species used in the study were assessed. The infection prevalence with T. annulata was high and did not vary markedly in the four Hyalomma tick species. The mean number of infected acini per tick in female and male ticks was different with female ticks having higher numbers of infected acini than the male ticks. The sex difference was more significant between H.a. anatolicum and H.a. excavatum than between H. detritum and H.m. marginatum. This study clarifies the roles of four Hyalomma tick species, and their sex, in the development of T. annulata.

Studies on the epidemiology of tropical theileriosis (Theileria annulata infection) in cattle in Central Anatolia, Turkey.

An epidemiological survey for Theileria annulata infection was conducted in 12 selected villages around Ankara in Central Anatolia, Turkey, during the period April 1990 to January 1993. During the survey, 198 cattle of 30 local breeds, 84 Holstein-Friesian×local breeds and 84 Holstein-Friesian breed were examined for antibodies to T. annulata and the presence of the vector ticks. Four species of Hyalomma ticks were identified: Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavtum, Hyalomma detritum and Hyalomma marginatum marginatum. Salivary gland staining indicated that infected adult ticks of all four species were present and, therefore, were implicated in the transmission of tropical theileriosis in the field. Generally, the Hyalomma infestation rate was low, with the heaviest infestations occurring on the older animals. Young adults and calves had very low infestation rates. Most ticks seen on cattle were adults, very few nymphs were found. The blood smear and serological examination of the 198 cattle conducted in March, before the start of the first disease season, showed that the prevalence of piroplasmosis was 11.1% (22 out of 198) and the seroprevalence of T. annulata was 10.6% (21 out of 198). Forty-three animals were then excluded from the study because they were seropositive and/or harboured piroplasms. Ninety-two seronegative animals showed piroplasmosis (92 out of 155) and 34 seronegative animals became seropositive for T. annulata (34 out of 155) during the three disease seasons. One animal became clinically ill with tropical theileriosis and required treatment. The incidence of cattle showing piroplasmosis and disease in the total study sample was 50.7% and 0.5% per disease season, respectively. The seroconversion rate of new infection with T. annulata in the total study was 14.3% per animal season. The number of cattle showing piroplasmosis was much greater than the number of seropositive cattle, which may indicate the presence of another species of Theileria. The two different management systems encountered in the study were considered to have influenced the tick infestation levels.

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

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.

Identification and analysis of immunodominant antigens for ELISA-based detection of Theileria annulata

Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals.

[Detection of Anaplasma / Ehrlichia Species of Cattle and Ticks in Aydın Region].

OBJECTIVE The aim of this study is to detect the Anaplasma/Ehrlichia species of cattle and ticks and to provide knowledge on the prevalence of these species during sampling periods. METHODS A total of 679 blood and 186 tick samples were collected from the Osmanbükü, Akçaova, Dalama, and Söke districts of Aydın. The samples were screened with genus polymerase chain reaction (PCR) for Anaplasma/Ehrlichia spp., species-specific polymerase chain reaction for Anaplasma marginale and A. centrale, and nested PCR for A. bovis and A. phagocytophilum. RESULTS A. centrale was detected in Söke during September and in Dalama and Akçaova during March, June, September, and December. A. marginale was detected in Osmanbükü during June; in Söke during March and December; in Akçaova during June, September, and March; and in Dalama during the entire sampling period. A. phagocytophilum was detected in all regions during the entire sampling period. None of the samples were positive for A. bovis. Mixed infections were detected in 50 blood samples. A. marginale and A. phagocytophilum were detected in the tick samples. CONCLUSION In this study, A. phagocytophilum was abundantly detected compared with A. marginale and A. centrale. A. phagocytophilum and A. centrale were extensively found in Akçaova and A. marginale was mostly seen in Dalama. Parasites were extensively detected in September and March. The analysis indicated that collected ticks were infected with different Anaplasma/Ehrlichia species.

Prevalence of Larval-Stage Dicrocoeliidae (Digenea) Trematodes in Helix lucorum (Mollusca: Pulmonata) in Van Province

OBJECTIVE The aim of this study was to investigate the prevalence of larval-stage Dicrocoeliidae trematodes in Helix lucorum, a land snail found in Van Province. METHODS Helix lucorum snails were collected in April, May, and June 2017 from Edremit and Gevaş, the central districts of Van Province, especially from natural areas where ruminants predominate. The snails were anesthetized with magnesium chloride, were removed from their shells, and their digestive glands were disrupted. The disrupted parts were examined under a microscope. RESULTS In Van Province, H. lucorum snails were found to be intermediate hosts for Dicrocoelium trematodes with a prevalence of 22%. The larval stages detected in the microscope are photographed and shown in detail. The number of infection with larval stages of the parasite was found to be highest in May. CONCLUSION Helix lucorum the land snail, serves as an intermediate host for some developmental stages of the Dicrocoeliid trematodes, is also consumed as nutrients by humans in some countries. Based on the obtained results in this study, it can be concluded that this snail would have important effects on animal health in the Van region which has a hard climate and a border with Iran.

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.

Selection of Genetic Markers to Determine Diversity in Theileria annulata Populations after Recombination.

OBJECTIVE Selecting polymorphic mini- and microsatellite markers to determine genetic diversity and chromosomal regions exhibiting elevated rates of recombination in Theileria annulata populations after recombination. METHODS The Unipro UGENE software was used to select markers. A score at which 10 times more tandem repeats (TRs) were identified in the real DNA sequence than those in the scrambled sequences of T. annulata was used as the cutoff. TRs containing minimum three nucleotides in length for microsatellite and six nucleotides for minisatellite regions and having a repeat motif identity between 96%-100% with the unit size repeated minimum three times were screened through the whole genome using the suffix array algorithm. RESULTS A total of 359 minisatellites and 8973 microsatellites were identified. TRs were screened one by one through the whole genome; mini- and microsatellites representing a single region and having suitable regions for primer design were selected based on their localization on T. annulata chromosomes, their repeat motif identity (>96%), and their repeat length (<1500 bp). The primers used to amplify selected candidates were designed, and each primer was used to check 27 different isolates of T. annulata. CONCLUSION In the present study, a total of 13 polymorphic mini- and microsatellite markers located on the different chromosomes were selected to determine the population diversity of T. annulata.