In vitro feeding of Hyalomma excavatum and Hyalomma marginatum tick species.

The rearing of ticks is an important technique for studies aiming to elucidate the course and pathogenesis of tick-borne diseases (TBDs). TBDs caused by protozoans (Theileria, Babesia) and bacteria (Anaplasma/Ehrlichia) impose a serious constraint upon livestock health and production in tropical and sub-tropical regions where the distributions of host, pathogen, and vector overlap. This study focuses on Hyalomma marginatum, one of the most important Hyalomma species in the Mediterranean region, being a vector of the virus that causes Crimean-Congo haemorrhagic fever in humans, together with H. excavatum, a vector of Theileria annulata, an important protozoan of cattle. The adaptation of ticks to feeding on artificial membranes allows the creation of model systems that can be put to use examining the underlying mechanisms of pathogen transmission by ticks. Silicone membranes, in particular, offer researchers the flexibility to adjust membrane thickness and content during artificial feeding. The aim of the present study was to develop an artificial feeding technique using silicone-based membranes for all developmental stages of H. excavatum and H. marginatum ticks. Attachment rates after feeding on silicone membranes for females H. marginatum and H. excavatum were 8.33% (8/96) and 7.95% (7/88), respectively. The use of cow hair as a stimulant increased the attachment rate of H. marginatum adults in comparison to other stimulants. The engorgement of H. marginatum and H. excavatum females took 20.5 and 23 days with average weights of 307.85 and 260.64 mg, respectively. Although both tick species could complete egg-laying, and this was followed by hatching of larvae; their larvae and nymphs could not be fed artificially. Taken together, the results of the present study clearly indicate that silicone membranes are suitable for feeding of H. excavatum and H. marginatum adult ticks, supporting engorgement, laying of eggs, and hatching of the larvae. They thus represent a versatile tool for studying transmission mechanisms of tick-borne pathogens. Further studies are warranted to examine attachment and feeding behaviours in order to increase the success of artificial feeding of larvae and nymphal stages.

Effects of flumethrin and eprinomectin in naturally infected mice with Myocoptes musculinus.

Mite infestations can occur in laboratory mice and cause an undesirable immune system response and adversely affect study results. Myocoptes musculinus is a mite species that can occasionally parasitized in laboratory-housed and breeding mice. In this study, the efficacy of flumethrin and eprinomectin were compared in 30 male Balb/c mice naturally infested with M. musculinus. Balb/c mice were divided into three different groups comprising eprinomectin treated, flumethrin treated and untreated control. Eprinomectin and flumethrin applied at doses of 5 mg/kg and 1 mg/kg body weights, respectively. An equal volume of mineral oil was also applied to untreated control group. Clinical scoring was done in all groups before drug administration and on days 7, 14 and 21. Scrap samples were collected and evaluated before the study (day 0) and on days 7, 14 and 21 during the study. Each colony was also videotaped for 15 min every day to calculate the Pruritic Index (PI: scratching and gnawing acts/mouse/minute). Obtained results showed that both eprinomectin and flumethrin pour-on applications significantly decreased the number of different life stages of the parasite in the skin scrapings. Both eprinomectin and flumethrin pour-on applications were found to be effective in the treatment of M. musculinus infestation in mice. However, compared to eprinomectin applied group, slightly higher number of eggs, adult and damaged adults (P < 0.01) were observed in group treated with flumethrin at day 14 after treatment. A significant reduction in PI in eprinomectin treated group 1 day after treatment, while reduction in PI was 3 days after treatment in flumethrin treated group. Clinical scoring data indicated complete recovery in the eprinomectin treated group 21 days after treatment, while partial recovery was observed in the flumethrin treated group. As a result, it can be concluded that pour-on application of eprinomectin is more effective compared to flumethrin against natural M. musculinus infestation in mice.

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.

In vitro feeding of all life stages of two-host Hyalomma excavatum and Hyalomma scupense and three-host Hyalomma dromedarii ticks.

Ticks are blood-sucking ectoparasites and can transmit various pathogens of medical and veterinary relevance. The life cycle of ticks can be completed under laboratory conditions on experimental animals, but the artificial feeding of ticks has attracted increased interest as an alternative method. This study represents the first report on the successful in vitro feeding of all life stages of two-host tick species, Hyalomma scupense and Hyalomma excavatum, and the three-host tick Hyalomma dromedarii. The attachment and engorgement rates of adults were 84% (21/25) and 76% (19/25) for H. scupense females. For adult H. excavatum and H. dromedarii, 70% (21/30) and 34.4% (11/32) of the females attached and all attached females successfully fed to repletion. The oviposition rates of the artificially fed females were 36.4%, 57.1% and 63.1% for H. dromedarii, H. excavatum and H. scupense, respectively, with a reproductive efficiency index varying between 44.3 and 60.7%. For the larvae, the attachment and engorgement rates were 44.2% (313/708) and 42.8% (303/708) for H. dromedarii, 70.5% (129/183) and 56.8% (104/183) for H. excavatum and 92.6% (113/122) and 55.7% (68/122) for H. scupense. The attachment and engorgement rates for the nymphs were 90.2% (129/143) and 47.6% (68/143) for H. dromedarii, 66.7% (34/51) and 41.2% (21/51) for H. excavatum, and 44.1% (30/68) and 36.8% (25/68) for H. scupense. Molting rates of the immature stages varied between 71.3% (216/303) and 100% (68/68) for the larvae and between 61.9% (13/21) and 96% (24/25) for the nymphs. The successful in vitro feeding of all stages of the three Hyalomma species makes this method a valuable tool for tick research, with potential applications in studies on the pathogens transmitted by these tick species such as Theileria annulata.

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 Aydin 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.

Evaluation of Cases with Suspected Canine Leishmaniasis History: A Five-year Retrospective Study (2016-2021)

Objective: This study aimed to evaluate the polymerase chain reaction (PCR) and immunofluorescence antibody test (IFAT) results of suspected samples with canine leishmaniasis (CanL) that were sent to the Parasitology Department Laboratories of the Veterinary Faculty in Aydin Adnan Menderes University. Methods: The age, gender, and breed of the dogs to be evaluated for CanL were recorded, and IFAT was performed using 80 blood serum samples collected from them. Additionally, after the isolation of genomic DNA of 27 blood samples, PCR of these samples was performed using primers that amplify the 145 bp kDNA region of Leishmania species. Results: Thirty-seven (46.25%) of the serum samples were seropositive in at least one dilution (1/64 or 1/128) according to IFAT. Five (18.5%) of the twenty-seven samples were positive for Leishmania DNA according to PCR. According to IFAT, 38.7% of male dogs and 59% of female dogs were positive. The highest number of seropositive samples were detected in dogs aged 3-5 years (11/27). Conclusion: Considering the zoonotic potential of leishmaniasis, which is considered endemic in the region, and the high positivity of the IFAT/PCR results, veterinarians should use advanced diagnostic methods, especially serological and molecular tests, in dogs with suspected CanL. The data obtained show that the risk of infection caused by Leishmania spp. is high in the region. Therefore, it is important to routinely ensure the control of CanL to protect both human and animal health.

Prevalence of Theileria/Babesia Species in Ruminants in Burdur Province of Turkey.

PURPOSE: Theileriosis and babesiosis, two tick-borne haemoparasitic diseases (TBHDs) of ruminants, are caused by protozoan parasites of the genus Theileria and Babesia, respectively. Among them, some species are considered to be highly pathogenic causing serious economic losses to livestock holders especially in tropic and subtropic regions. Local and/or general control measures are needed to be applied to reduce economic impact of TBHDs. Prevalence studies are essential for the implementation and/or design of effective prevention and control measures based on true epidemiological data. Therefore, this study aimed to investigate the presence, prevalence and possible cross infections of Theileria/Babesia species between sheep, goat and cattle herds in Burdur province in Turkey. METHODS: A total of 964 blood samples were collected from sheep (n = 330), goat (n = 300) and cattle (n = 334) from five different districts of Burdur province. The samples were investigated for ovine and bovine Theileria/Babesia species using reverse line blot (RLB) hybridization assay. RESULTS: In small ruminants, T. ovis was the most abundant Theileria species detected in sheep with a rate of 79.69%. Among Babesia species, B. ovis and B. crassa were detected only in blood of goats (0.66%) and sheep (1.12%) as single and mixed infections, respectively. In cattle, T. annulata, B. bovis, Babesia spp. were detected in rates of 0.59%, 3.29%, 3.59%, respectively. CONCLUSION: Obtained results clearly indicated that no cross infections with Theileria/Babesia species occurred in small ruminant and cattle herds that use the same grazing area.

Seroprevalence and molecular detection of Leishmania spp. in cats of West Aegean Region, Turkey.

Zoonotic visceral leishmaniasis is caused by protozoa of the genus Leishmania. Although dogs are considered to be primary reservoir hosts of Leishmania spp., several mammals, such as foxes, jackals and small rodents may also be hosts of different Leishmania spp. Previously, cats were considered as asymptomatic hosts of the parasite without acting as a reservoir. In recent years, there has been an increasing number of leishmaniasis cases in cats, especially in regions where the infection endemically occurs. This data indicate that cats are more likely to be one of the main reservoirs of Leishmania spp. rather than being a non-reservoir host. The aim of this study is to determine the prevalence of Leishmania spp. by molecular and serological techniques among owned and stray cats in four different cities located in western part of Turkey. A total of 386 blood and 301 serum samples were collected from cats in Western Turkey where leishmaniasis is endemic. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania spp. antigens. Blood samples collected from 386 cats were examined by PCR for the presence of Leishmania spp. According to PCR results using RV1/RV2 primers, nine (2.3%) out of 386 samples were positive for Leishmania spp. Further PCR analysis using MC1/MC2 primers showed that one cat in Izmir was found to be infected with L. infantum/donovani complex. Sequence analysis revealed the presence of L. infantum, L. major and L. tropica among sampled cats in western part of Turkey. On the other hand, IFAT results indicated that an overall of 47 out of 301 (15.6%) cats that examined by PCR were found to have anti-Leishmania antibodies. Also, six of the seropositive cats were owned animals. The present study demonstrated that both owned and stray cats can be infected with Leishmania spp. and might be potential reservoirs for other animals and humans. Therefore, all communities living in or nearby endemic regions should be made aware of the role of cats as potential reservoirs of infection. In endemic regions, all animals should be protected against infection with insecticides and monitored routinely to control the spread of zoonotic visceral leishmaniasis.

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.

Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis.

Babesiosis is a disease complex caused by unicellular Babesia parasites and among them, malignant ovine babesiosis caused by B. ovis has a devastating economical impact on the small ruminant industry. The control of disease is mainly based on chemotherapy and preventing animals from tick infestation and to date no vaccine is available against ovine babesiosis. The requirement for vaccination against B. ovis infection in endemically unstable regions is necessary for implementation of effective disease control measures. The aim of the present study was to evaluate the effectiveness of different immunisation protocols against disease in sheep experimentally vaccinated with recombinant B. ovis apical membrane antigen-1 (rBoAMA-1) and/or live, a B. ovis-infected cell line. Sheep were divided into four experimental groups, plus a control group. Animals were immunised either with the B. ovis stabilate, or with rBoAMA-1, or with both rBoAMA-1 and the B. ovis stabilate. Western blots and ELISAs indicated that immunisation with rBoAMA-1 resulted in generation of a specific response against the recombinant protein, but the degree of antibody response did not correlate with the level of induced protection against challenge. The strongest immune response was induced in animals co-immunised with the live B. ovis stabilate plus rBoAMA-1. Both the hematological and parasitological findings indicated that this co-immunisation regimen has vaccine potential to limit losses incurred by ovine babesiosis in endemic countries.

Tick Bites on Humans in Southwestern Region of Turkey: Species Diversity

Objective: The aim of the present study was to determine tick species found on humans who suffered from tick bite in the Southwestern Anatolia Region, Turkey. Methods: Between January and October 2007, ticks were collected from people admitted to the city and/or town hospitals with complaints of tick bites in nine different provinces of Turkey. Genus and/or species of the ticks in adult, larva and nymph stages were identified microscopically. Identification was done using related taxonomic keys. Results: A total of 2.610 ticks were collected from humans who were admitted to the hospitals with complaints of tick bites in the Southwestern Anatolia Region in the present study. Of these, 1.858 samples were collected from the Aegean Region and the remaining 752 from the Mediterranean Region of the country. The ticks were identified as Hyalomma spp. (78.58%), Rhipicehalus spp. (18.89%), Ixodes spp. (0.88%), Dermacentor spp. (0.77%), Haemaphysalis spp. (0.61%), Argas spp. (0.23%), and Ornithodoros spp. (0.04%). Results indicated that the majority of the ticks were nymphs of Hyalomma spp. (n=1.582). Nymphal stage was most commonly encountered from the Aegean Region and the Mediterranean Region with a prevalence of 46.13% (n=1.204) and 14.48% (n=378) respectively. Within the collected adult ticks (n=913), the majority of the samples were identified as H. marginatum (n=233, 26.09%). Conclusion: The results indicate the high diversity of tick species infesting humans in the Southwestern Anatolia Region, Turkey. So, it is crucial to publish information on tick bite prevention, which would play an important role in reducing the incidence of tick-borne diseases.

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.

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.

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 Gevas, 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.

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.

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.

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.

A parasitological, molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey.

Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.

Determination the Role of Rhipicephalus sanguineus for Transmission of Leishmania major to Reservoir Animals.

OBJECTIVE: This study aimed to explore the role of Rhipicephalus sanguineus in the transmission of Leishmania major, the etiological agent of zoonotic cutaneous leishmaniasis. METHODS: Ten gerbils (Meriones unguiculatus) were infected with promastigotes of L. major, and 10 gerbils were maintained as controls. In a controlled environment, 2000 R. sanguineus larvae were fed to two gerbils. Following feeding to gerbils, 65 tick pools were prepared from the engorged larvae and molted unfed nymphs. These pools were tested for the presence of L. major using polymerase chain reaction and real time (RT) PCR. RESULTS: One of the infected gerbil was anesthetized and necropsied following the dropping of all fed larvae. Following the examination, amastigotes were detected in all organs and tissues. PCR and RT-PCR were performed to test whether the engorged R. sanguineus larvae successfully took the parasite while feeding and was able to transmit it to the next nymphal stage; however, none of the tick pools were found to be positive for L. major. CONCLUSION: Although L.major was not detected in ticks that fed on gerbils, using dogs in experimental studies related to leishmaniasis will give clearer results in terms of detecting the potential role of insects and acars.

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.