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This study was undertaken in two different climatic areas of Turkey to determine the presence of tick-borne pathogens of medical and veterinary importance. The ticks were removed from humans, pooled according to species and developmental stages, and analyzed by PCR, reverse line blot (RLB) and sequencing. Of the 2333 removed ticks from 10 species, 1238 (53.06%) were obtained from the arid cold zone, and the remaining 1095 (46.93%) were obtained from the humid zone. The removed ticks were identified as Hyalomma marginatum, Hyalomma detritum, Hyalomma excavatum, Rhipicephalus bursa, Rhipicephalus turanicus, Rhipicephalus sanguineus, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis sulcata, Ixodes ricinus, Haemaphysalis and Ixodes spp. nymphs. The dominant species was L ricinus (61.27%) in the humid zone, whereas the Haemaphysalis spp. nymph dominated (30.29%) in the arid zone. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 169 pools tested, 49 (28.99%) were found to be infected with the pathogens, and the overall MLE of the infection rate was calculated as 2.44% (CI 1.88-3.17). The MLE of the infection varied among tick species, ranging from 0.85% (CI 0.23-2.34) in Haemaphysalis spp. nymph to 17.93% (CI 6.94-37.91) in D. marginatus. Pathogens identified in ticks included Theileria annulata, Babesia ovis, Babesia crassa, Anaplasma/Ehrlichia spp., Anaplasma ovis, Ehrlichia canis, Anaplasma phagocytophilum, Hepatozoon canis and Hepatozoon fells. Most tick pools were infected with a single pathogen. However, four pools infected with H. canis displayed infections with B. crassa, A. phagocytophilum and E. canis. The sequencing indicated that Anaplasma/Ehrlichia spp. was 100% identical to the sequence of Ehrlichia sp. Firat 2 and 3 previously identified from Hyalomma anatolicum. (C) 2013 Elsevier B.V. All rights reserved.

In this study, a novel Babesia sp. infecting goats was detected and its phylogenetic relationship to related species was determined. A total of 200 blood samples collected from sheep (n=78) and goats (n=122) were examined in the study. The V4 hypervariable region of the 18S rRNA gene of the novel Babesia sp. was amplified by PCR and analysed using a reverse line blot hybridization assay adapted for small ruminants. Samples from seven goats hybridized to Theileria/Babesia catch-all and Babesia catch-all probes and did not hybridize to any species-specific probe tested, suggesting the presence of an unrecognized Babesia species or genotype. Sequencing results showed the isolate to clearly differ from ovine Babesia species and genotypes currently available in the GenBank database. The isolate showed 90.9%, 93.5%, and 93.4% identity to B. ovis, B. motasi, and B. crassa, respectively and 91-93% similarity to Babesia genotypes recently described in small ruminants. The highest homology (∼96-97%) observed was with Babesia odocoilei, Babesia sp. EU1, and Babesia divergens. The new isolate was provisionally designated Babesia sp. The study contributes to better insight into the distribution and phylogenetic diversity of piroplasms in small ruminants. The survey indicated a high prevalence of piroplasms in small ruminants (21.5%). Of those detected, T. ovis was the most prevalent (17%), followed by Babesia sp. (3.5%), and B. ovis (2%).

A total of 1064 adult ticks identified as Hyalomma marginatum ( n = 965), Rhipicephalus turanicus ( n = 86), Haemaphysalis inermis ( n = 9) and Rhipicephalus bursa ( n = 4) along with 20 egg masses derived from H. marginatum ( n = 18) and R. bursa were screened for Babesia occultans . The ticks were combined into 328 pools and infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of these pools, 23 (7%) were positive for B. occultans , and the overall MLE was calculated as 2.27% (CI 1.48–3.35). Infection prevalence in tick species was 7.09% for H. marginatum (22/310, MLE 2.41, CI 1.55–3.59) and 7.14% for R. turanicus (1/14, MLE 1.11, CI 0.07–5.18). None of H. inermis and R. bursa was infected with the parasite. Host-seeking H. marginatum females displayed an infection prevalence of 1.68% (2/119, MLE 0.63, CI 0.11–2.06), while no parasite DNA was detected in the males. The prevalence of infection in feeding H. marginatum varied, ranging from 27.27% (9/33, MLE 4.61, CI 2.26–8.52) in males to 25.92% (7/27, MLE 8.22, CI 3.72–15.75) in females. Of the egg masses examined, 4 (22.22%) of H. marginatum and 1 (50%) of R. turanicus were infected with B. occultans . To the best of our knowledge, this is the first time that B. occultans has been detected in R. turanicus.

Theileria spp. infect wild and domestic ruminants in the tropical and subtropical regions of the world. Two species, T. lestoquardi and T. ovis, are suspected to cause ovine theileriosis in Iran. The epidemiological aspects of ovine theileriosis in Iran are poorly understood, and further investigations by sensitive and precise techniques are required. In this study, the use of a nested PCR for amplification of a fragment of the 18S ribosomal DNA from virtually all species of Theileria is described. For differentiation of various Theileria spp. a RFLP assay was developed as a diagnostic tool enabling direct, concurrent, highly specific and sensitive identification of Theileria spp. The sensitivity of the nested PCR for Theileria species was 10−5% parasitemia. Restriction fragment length polymorphism (RFLP) of the PCR products allowed differentiation between three different Theileria species (T. annulata, T. lestoquardi and T. ovis) and seems to be useful for differentiation of other species such as T. separata and Theileria spp. china. From 100 field blood samples obtained from sheep in East and South-East Iran, 56% were positive for Theileria spp. by nested-PCR compared with 21% by microscopic examination. Out of 56 positive samples, 12.5% (7/56) were positive for T. ovis and 87/5% (49/56) were positive for T. lestoquardi. This is the first report in which T. ovis has been detected in Iran using molecular identification techniques.

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T annulata and 7: buffelilorientatis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was 7: buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina. (C) 2008 Elsevier B.V. All rights reserved.

Detection and identification of Theileria and Babesia species in 920 apparently healthy small ruminants in eastern Turkey, as well as parasite genetic diversity, was investigated using a specifically designed reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified and hybridized to a membrane onto which catchall and species-specific oligonucleotide probes were covalently linked. Three Theileria and one Babesia genotype were identified. Comparison of the Theileria genotypes revealed 93.6-96.2% similarity among their 18S rRNA genes. Two Theileria shared 100% and 99.7% similarity with the previously described sequences of T. ovis and Theileria sp. OT3, respectively. A third Theileria genotype was found to be clearly different from previously described Theileria species. The genotype was provisionally designated as Theileria sp. MK. The Babesia genotype shared 100% similarity with Babesia ovis. The survey indicated a high prevalence of piroplasm infections in small ruminants (38.36%). Theileria spp. prevalence was 36.08%. Prevalence of B. ovis was 5.43%. The most abundant Theileria species identified was T. ovis (34.56%) followed by Theileia sp. MK (1.30%) and Theileria sp. OT3 (0.43%).

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.

This study was carried out to determine the prevalence and distribution of tropical theileriosis in cattle in eastern Turkey by microscopical, serological and molecular methods. A total of 1561 whole blood, 1505 serum and 1483 blood smear samples were collected from cattle of various breeds and ages in 11 towns of Eastern Turkey. Theileria annulata piroplasm DNA extracted from cattle blood was amplified by polymerase chain reaction (PCR) using species-specific primers. Serum antibodies against T. annulata were investigated by indirect fluorescence antibody test (IFAT). Blood smears were examined for Theileria piroplasms by microscopical examination (ME). In the examination of DNA extracted from 1561 blood samples, an amplicon with the size of 721bp was obtained in 37.8% (590/1561) of these samples. Serum antibodies against T. annulata and piroplasm of Theileria spp. were detected in 34.9% (526/1505) and 19.7% (293/1483) of the samples, respectively. The differences between ME and PCR results and between ME and IFAT results were statistically significant (P < 0.05). In contrast, there was no significant difference between the PCR and IFAT results. A total of 179 ticks (136 female; 43 male) belonging to Hyalomma spp. were collected from cattle from three towns. Ticks were identified to be Hyalomma anatolicum anatolicum on the basis of morphological features.

Aktas, Munir/0000-0002-3188-8757 WOS: 000188537100001 PubMed: 15036571 A total of 2388 cattle and 442 shelters, from two provinces (Elazig and Malatya) endemic for tropical theileriosis in the east of Turkey, were studied for Hyalomma tick populations from July 1993 to July 1995 in Elazig and from May 1998 to January 1999 in Malatya. Four thousand five hundred and eighty one of 7455 Hyalomma ticks were collected from cattle, the other ticks (2874) were collected from shelters. All of the ticks collected from shelters were Hyalomma anatolicum anatolicum. Two thousand eight hundred and ninety five (63.1%) of 4581 Hyalomma ticks collected from cattle were H.a. anatolicum. 23.8% (1047/4581), 11.7% (536/4581) and 0.6% (3/4581). of Hyalomma ticks were Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum, respectively. A total of 5909 Hyalomma adult ticks collected from cattle (3362/5909) and shelters (2447/5909) were dissected and salivary glands were stained with Methyl-green/Pyronin method. Thousand one hundred and fifty (46.9%) of 2447 H.a. anatolicum collected from shelters and 412 (19.1%) of 2147 H.a. anatolicum collected from cattle were positive for Theileria infection. Twenty (2.4%) of 820 H.a. excavatum and 23 (4.6%) of 495. H. detritum collected from cattle were positive. The mean number of infected acini per infected male and female ticks collected from cattle were 11.3 and 22.4 in H.a. anatolicum, 4 and 6.8 in H.a. excavatum, 17.9 and 18.3 in H. detritum, respectively. In H.a. anatolicum collected from shelters, the above rates were 11.8 and 17.6 in male and female ticks, respectively. The prevalence and intensity of Theileria infection was greater in female ticks than in males. (C) 2003 Elsevier B.V. All rights reserved.

WOS: 000353602800017, PubMed ID: 25771934, In this study, asymptomatic dogs in nine provinces of Turkey were surveyed to investigate the prevalence and intensity of Hepatozoon canis infection. DNA obtained from blood samples collected from 694 domestic dogs (243 stray, 288 shelter, and 163 pets) of both genders and varying ages were evaluated by polymerase chain reaction (PCR). In addition, 285 thin blood smears prepared from these blood samples were also evaluated for microscopic examination. Direct microscopy revealed Hepatozoon gamonts in the peripheral blood of three of 285(1.0%; 95% confidence interval (CI): 0.21-3.04) tested. Using PCR, 155 of the 694(22.3%; 95% CI: 19.28-25.61) were found to be positive for the presence of H. canis DNA. The prevalence of infection was higher in adult dogs (26.2%; 95% CI: 22.1-30.7) than young animals (16.4%; 95% CI: 12.2-21.3). Although the prevalence determined by PCR was higher in male dogs (24.5%; 95% CI: 19.6-29.9) than in female dogs (20.8%; 95% CI: 16.9-25.1), gender differences were not significant. Pet dogs had a lower prevalence of infection (10.4%; 95% CI: 6.2-16.2) compared to stray (26.3%; 95% CI: 20.9-32.3) and shelter dogs (25.7%; 95% CI: 20.7-31.1), but no significant association between stray and shelter dogs was found for the presence of the parasite. Partial sequences of the 18S ribosomal RNA (rRNA) gene shared 99-100% similarity with the corresponding H. canis isolates. This epidemiological survey revealed a high prevalence of H. canis in dogs from several provinces in Turkey, and it suggests that the age and origin are associated with the parasite. (C) 2015 Elsevier B.V. All rights reserved., Scientific and Technical Research Council of Turkey (TUBITAK) [110 O 870], This work was supported financially by a grant (110 O 870) from the Scientific and Technical Research Council of Turkey (TUBITAK). We thank all veterinarians, technicians, and animal breeders in the region for their assistance in sample collection.

The genomic region spanning the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was cloned and sequenced from sixteen Theileria isolates. Each Theileria species possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among ruminant Theileria species. The spacers were most polymorphic in the agent of tropical theileriosis, Theileria annulata, and were more conserved in two benign species, Theileria buffeli and Theileria sergenti Chitose. Phylogenetic analysis of the rDNA ITS1-5.8S rRNA gene-ITS2 region clearly separated each taxon, placing them in three clusters. One held T. annulata, Theileria parva, and Theileria mutans, with the latter two most closely related. The second held T. sergenti Ikeda, T. sergenti Chitose, and T. buffeli, with the latter two most closely related. The third cluster held the Theileria ovis isolates.

A survey of Theileria parasites in cattle in eastern Turkey was carried out using specific polymerase chain reaction. A total of 252 blood samples were collected from clinically healthy cattle between June and July 2004. Of 252 blood samples examined, 41 (16%) were positive for piroplasms by microscopy, whereas 114 (45%) were positive for the presence of at least one species of Theileria by PCR. The percentages of positive animals for Theileria annulata and benign Theileria species (Theileria sergenti/buffeli/orientalis) were 39% (99/252) and 7% (18/252), respectively. By allele-specific PCR examination of 18 field isolates which were positive for benign Theileria parasites, 8 samples were only amplified by B-type specific primers and 10 samples were amplified by both of the B and C-type specific primers, indicating a mixed infection with B and C-type of the parasite. None of the field isolates was amplified by I-type specific primers. Three samples were co-infected with T. annulata and benign Theileria parasites. Two of them which were infected with B-type parasite were also infected with T. annulata, the other sample which was infected both of B and C-type parasites was also infected with T. annulata. A total of 724 ixodid ticks were collected from the cattle. Hyalomma anatolicum anatolicum was the dominant species with 32% (230/724) in the region. H. a. excavatum, Boophylus annulatus and Rhipicephalus bursa represented 25% (183/724), 19% (140/724) and 15% (112/724) of the total number of ticks, respectively. R. sanguineus was the minor species and represented 8% (59/724) of the tick population.

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.

Tick-borne diseases in ruminants are common in tropical and subtropical regions and lead to meat and milk production losses. In this study, polymerase chain reaction (PCR) was used to assess the presence of Theileria ovis in Rhipicephalus bursa ticks. We have demonstrated that the PCR enabled detection of T. ovis in field isolates of R. bursa collected from naturally infested sheep and goats in eastern Turkey. The sampling was done in spring season (between May and June 2004). A total of 420 R. bursa were collected and randomly selected 192 number of them (97 female and 95 male) were dissected. Primers specific for 520 bp fragments small subunit ribosomal RNA (ssu rRNA) gene of T. ovis amplified products from 37 of the 192 (19.27%) samples. The parasite was detected in 17 (17.52%) female and in 20 (21.05%) male ticks. Two T. ovis amplicons from the tick samples were purified and sequenced. The resulting sequences were identical to the nucleotide sequence of the Turkish sheep strain of T. ovis. These results showed that R. bursa might play an important role in the field as a natural vector of T. ovis.

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 107 sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.