Molecular investigation of Anaplasma phagocytophilum and related strains among sheep flocks from different parts of Türkiye; with a note of phylogenetic analyses of Anaplasma phagocytophilum- like 1.

Anaplasma phagocytophilum is a vector-borne zoonotic pathogen and can infect various vertebrate hosts, especially cattle, sheep, goats, horses, and dogs. Molecular-based studies have revealed that the agent has a high genetic diversity and closely related strains circulate in hosts. In this study, 618 sheep blood samples obtained from different geographic regions of Türkiye were researched for A.phagocytophilum and related strains with PCR, RFLP, and DNA sequence analyses. The DNA of these pathogens was detected in 110 (17.79%) samples. RFLP assay showed that all positive samples were infected with A.phagocytophilum-like 1, whereas A.phagocytophilum-like 2 and A.phagocytophilum were not detected. Partial parts of 16 S rRNA gene of seven randomly selected positive samples were sequenced. The phylogenetic analyses of these isolates revealed that at least two A.phagocytophilum-like 1 isolates circulate among hosts in Türkiye and around the world. A.phagocytophilum-related strains have been reported in molecular-based studies over the last few years, but there is a lack of data on the vector competence, epidemiology, clinical symptoms, and genetic diversity of these pathogens. Therefore, large-scale molecular studies are still needed to obtain detailed data on the above-mentioned topics.

Anaplasma capra: a new emerging tick-borne zoonotic pathogen.

The genus Anaplasma includes A. marginale, A. centrale, A. bovis, A. ovis, A. platys, and A. phagocytophilum transmitted by ticks, some of which are zoonotic and cause anaplasmosis in humans and animals. In 2012, a new species was discovered in goats in China. In 2015, the same agent was detected in humans in China, and it was provisionally named Anaplasma capra, referring to 2012. The studies conducted to date have revealed the existence of A. capra in humans, domestic animals, wild animals, and ticks from three different continents (Asia, Europe, and Africa). Phylogenetic analyses based on gltA and groEL sequences show that A. capra clearly includes two different genotypes (A. capra genotype-1 and A. capra genotype-2). Although A. capra human isolates are in the genotype-2 group, goat, sheep, and cattle isolates are in both groups, making it difficult to establish a host genotype-relationship. According to current data, it can be thought that human isolates are genotype-2 and while only genotype-1 is found in Europe, both genotypes are found in Asia. Anaplasma capra causes clinical disease in humans, but the situation is not yet sufficient to understand the zoonotic importance and pathogenicity in animals. In the present review, the history, hosts (vertebrates and ticks), molecular prevalence, pathogenic properties, and genetic diversity of A. capra were evaluated from a broad perspective.

First Molecular Evidence of Babesia vogeli, Babesia vulpes, and Theileria ovis in Dogs from Kyrgyzstan.

Tick-borne parasitic diseases cause mild to severe infections among vertebrate hosts, including dogs. Species in the genus Babesia are important tick-borne pathogens and have worldwide distributions. Although there are data on the prevalence and distribution of Babesia species among dogs around the world, there is no information available in Kyrgyzstan, according to a literature review. In this study, 337 dogs were screened by nested PCR for the presence of the 18S small subunit ribosomal RNA (18S SSU rRNA) gene of piroplasm species. Overall prevalence was 6.23% (21/337) for Babesia/Theileria spp. DNA sequencing of positively tested samples revealed that eighteen samples were infected with Babesia vogeli (B. vogeli) (5.34%), two samples with B. vulpes (0.59%), and one sample with Theileria ovis (T. ovis) (0.29%). The phylogenetic analyses and nucleotide sequences in contrast with those present in GenBank revealed that two nucleotide substitutions (594th and 627th) were found between B. vogeli isolates, including ours, indicating that the mutation is relatively rare. The sequences of other pathogens obtained in this study confirmed 100% nucleotide identity with B. vulpes and T. ovis sequences in GenBank. To the best of our knowledge, B. vogeli, B. vulpes, and T. ovis were detected for the first time in dogs from Kyrgyzstan, and it is thought that results will contribute to the understanding of the epidemiology of canine tick-borne pathogens in the country.

Molecular detection and phylogenetic analysis of Toxocara vitulorum in feces and milk samples from naturally infected water buffaloes.

Toxocara vitulorum infects cattle and water buffalo, leading to mild to severe infection in calves and has wide geographic distributions, especially in tropical and subtropical countries. This work aimed to assess the prevalence, distributions, and phylogeny of T.vitulorum in water buffaloes in different parts of Sivas, one of the essential buffalo-breeding areas in Türkiye. T.vitulorum was found in 42 (8.23%) and 54 (10.58%) fecal (n:510) samples using microscopic and molecular techniques, respectively. T.vitulorum was higher in animals aged 0-6 months compared to other groups. Furthermore, when animals aged 0-6 months were grouped within themselves, the prevalence of T.vitulorum in 1-3 month-old-animals was higher than in both younger than one month and older than three months. T.vitulorum was detected in fecal samples obtained from animals older than six months. In colostrum/milk samples (n:100), T.vitulorum-larvae were found in 4% and 10% with microscopic and molecular techniques, respectively. The larvae were detected in colostrum/milk samples in the mother between the 2nd and 28th days postpartum-period. The ITS-1-gene of 11 PCR-positive samples was sequenced. The 98.99-100% nucleotide identity was determined between our T.vitulorum isolates and those present in GenBank. In conclusion, this is the first molecular survey and phylogenetic analysis of T.vitulorum in fecal and colostrum/milk samples from naturally infected water buffaloes. Data obtained in this study will help to understand the life cycle and epidemiology of the nematode. Data also revealed that veterinarians should consider older animals as well as young animals in their control program of nematode infections in farms.

Molecular survey of Anaplasma phagocytophilum and related variants in water buffaloes: The first detection of Anaplasma phagocytophilum-like 1.

Anaplasma phagocytophilum infects various hosts and lead to mild to severe infection. Currently, two A.phagocytophilum-related variants have been documented in different countries. Although limited, there are studies revealing the presence of A.phagocytophilum in water buffaloes, but no study investigating A.phagocytophilum-like 1 and -like 2. A.phagocytophilum and related variants were investigated using PCR, PCR-RFLP, and DNA sequence analysis in water buffaloes in Türkiye. 364 buffalo blood samples were examined for A.phagocytophilum and related strains. Seven buffaloes were determined to be positive with PCR and PCR-RFLP revealed that all samples were A.phagocytophilum-like 1. According to the partial sequence of 16 S rRNA gene, A.phagocytophilum like-1 may split into two different variants. This work supplies the first molecular report of A.phagocytophilum-like 1 in water buffaloes. However, a lack of information is present on the pathogen's clinical manifestations and vector species. There is still a need to investigate vectors and clinical signs of the pathogen.

Molecular prevalence of bovine hemoplasmosis in Turkey with first detection of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos in cattle and water buffalo.

Hemoplasma species can cause infection varying from mild to severe in a wide range of hosts, including cattle and water buffalo. Two hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been reported in cattle and water buffalo from different parts of the world to date. There was a lack of information on the presence and distribution of these pathogens in Turkey despite the negative economic impact on livestock production. This study aimed to develop a duplex PCR assay amplifying the 16S rRNA gene, in order to analyze DNA samples obtained from 297 cattle and 360 water buffaloes, and to determine the molecular prevalence of bovine hemoplasma species in Sivas province. Bovine hemoplasma species were found in 94 of 297 (31.64%) cattle and in 17 of 360 (4.72%) water buffaloes in this study. Randomly selected six positives PCR products (three samples each species) obtained from cattle and water buffaloes were sequenced, and the consensus sequences were uploaded to GenBank. Nucleotide similarity of 96.97-100% was determined between M. wenyonii isolates obtained in this study and those of M. wenyonii isolates present in the GenBank database, whereas C. Mycoplasma haemobos isolates from this study shared 99.04-100% homology with the C. Mycoplasma haemobos isolates uploaded to the GenBank. With the current study, the molecular presence of M. wenyonii and C. Mycoplasma haemobos were documented for the first time in cattle and water buffaloes in Turkey. Considering the rate of prevalence, veterinarians should take precautions against bovine hemoplasma species to protect animal health.

Molecular Survey of Anaplasma phagocytophilum and Related Strains in Sheep and Goats from Sivas; with a High Prevalence of Anaplasma phagocytophilum-like 1

Objective: This study aimed to investigate Anaplasma phagocytophilum and related strains (A. phagocytophilum-like 1 and like 2) in sheep and goats for the first time in Sivas province with molecular techniques. Methods: The study material was composed of 247 animal (159 sheep and 88 goats) blood samples from four districts of Sivas province (Sivas City Center, Kangal, Koyulhisar, and Yildizeli). A. phagocytophilum and related strains were screened with polymerase chain reaction (PCR), PCR-RFLP, and DNA sequence analysis. Results: A. phagocytophilum related strains were found in 44.93% (111/247) of the small ruminants using PCR. The infection rate was 45.91% (73/159) in sheep and 43.18% (38/88) in goats. In this study, 110 samples were positive for only A. phagocytophilum-like 1, while A. phagocytophilum-like 1 and like 2 were mix-infection in one sample. A. phagocytophilum was not detected in sheep or goats. Two randomly selected PCR products were sequenced in both directions, and the consensus sequences were deposited on the GenBank under accession numbers: ON598644 and ON598645. Nucleotide similarity of 99.34-100% was determined between A. phagocytophilum-like 1 isolates obtained in this study and those of A. phagocytophilum-like 1 isolates present in the GenBank database. Conclusion: This study provides the first molecular data on A. phagocytophilum-like 1 and like 2 in Sivas province. Considering the high positive rate of the A. phagocytophilum-like 1 in sheep and goats, there is a paucity of data on clinical symptoms and vector species of the pathogen. Therefore, further studies are needed to investigate the vector tick species and clinical symptoms of the pathogen in the host.

The detection and phylogenetic analysis of Anaplasma phagocytophilum-like 1, A. ovis and A. capra in sheep: A. capra divides into two genogroups.

In this study, the presence, prevalence, and genotypes of Anaplasma phagocytophilum, A. ovis, and A. capra in sheep were investigated based on 16 S SSU rRNA, groEL, and gtlA gene-specific polymerase chain reaction (PCR), respectively. The sequences of the genes were used for detection of the phylogenetic position of the species. Additionally, a restriction fragment length polymorphism (RFLP) were carried out for discrimination of A. phagocytophilum and related variants (A. phagocytophilum-like 1 and 2). The prevalence of Anaplasma spp. was found as 25.8% (101/391), while it was found that A. ovis, A. phagocytophilum-like 1, and A. capra are circulating in the sheep herds in Kyrgyzstan, according to the PCRs, RFLP and the partial DNA sequencing results. The positivity rates of A. phagocytophilum-like 1, A. ovis, and A. capra genotype-1 were 6.9, 22.5, and 5.3%, respectively. A total of 32 (8.2%) sheep were found to be mix infected. Moreover, phylogenetic analyses and sequence comparison with those available in the GenBank showed that A. capra formed two distinct genetic groups (A. capra genotype-1 and A. capra genotype-2). Considering the zoonotic potential of these species, it may be necessary to make changes in the interpretation of anaplasmosis cases in animals and there is a need for further studies to determine the pathogenicity of the species/genotypes circulating in animals.

Buffaloes as new hosts for Anaplasma capra: Molecular prevalence and phylogeny based on gtlA, groEL, and 16S rRNA genes.

Anaplasma capra is a tick-borne pathogen that was discovered for the first time in goats in China in 2012. The studies carried out from the first detection in China to the present have revealed the presence of this species in eight countries including Angola, France, Iranian, South Korea, Kyrgyzstan, Malaysia, Spain, and Türkiye in three continents (Africa, Asia, and Europe). It has also been determined that humans, sheep, cattle, dog, and wild animals are the hosts of A. capra. It was investigated whether water buffaloes were the host of A. capra using nested-PCR and DNA sequencing in this study. The prevalence of A. capra in Turkish water buffalo herds was investigated and phylogenetic analyzes were performed on the basis of gltA, groEL, and 16S rRNA genes. A total of 364 water buffalo blood samples were examined in terms of A. capra using gltA gene species-specific nested-PCR. A. capra were detected in 52 of 364 (14.28%) blood samples. There was no statistically significant difference between the prevalence, gender, and age parameters. The gltA, groEL, and 16S rRNA genes in randomly selected three positive samples were sequenced. A. capra isolates obtained from water buffaloes in this study shared 85.20-100%(gltA), 89.84-100%(groEL), and 99.82-100%(16S rRNA) nucleotide similarity with A.capra isolates present in GeneBank. Phylogenetic analyses of gtlA and groEL genes revealed that A. capra divided in two different genogroups. In conclusion, this study revealed that water buffalo is a new host of A. capra. However, comprehensive studies are needed to determine the pathogenicity, vectors, and biological properties of A. capra in this new host.

The first molecular detection of Anaplasma capra in domestic ruminants in the central part of Turkey, with genetic diversity and genotyping of Anaplasma capra.

Tick-borne diseases have been an increasing threat to human and animal health all over the world. Anaplasmosis is one of the emerging tick-borne diseases and has zoonotic potential. A new novel species, which was detected in China in 2010-2012 and provisionally named Anaplasma capra in 2015, causes zoonotic infections and infects many different animal species. In this study, we investigated the presence of A. capra in domestic ruminants from Turkey. A total of 468 blood samples (cattle, sheep, and goat) were examined by the gltA gene-specific nested polymerase chain reaction, revealing the presence of A. capra in six samples (1.28%): one of them from cattle (0.41%) and the other five from sheep (3.22%). According to DNA sequences results of the gltA gene, A. capra isolates identified in the present study were shown high nucleotide similarity with A. capra isolates detected from different hosts. However, the nucleotide differences were detected in the same nucleotide positions between A. capra isolates. For this reason, we thought that at least two different A. capra genotypes could be circulating in the world. As a result, it is seen that A. capra, which was determined to be a new species with zoonotic potential, was revealed in European and Asian countries and in different hosts. In order to raise awareness about human anaplasmosis infections, it is important to reveal the prevalence of the species in the world. The emergence of A. capra in Turkey reveals the need for a re-evaluation of the human and animal health risk analysis in terms of anaplasmosis.

First molecular detection of Anaplasma species in cattle from Kyrgyzstan; molecular identification of human pathogenic novel genotype Anaplasma capra and Anaplasma phagocytophilum related strain.

Anaplasmosis is a rickettsial infection with significant effects on human and animal health, and the discovery of new species or genotypes with zoonotic potential in recent years has increased this importance. The aim of this study was to provide the first assessment of the molecular etiology and prevalence of bovine anaplasmosis in Kyrgyzstan (specifically in the Chuy, Talas, Djalal-Abad, Naryn, and Issyk-Kul regions). The prevalence of bovine anaplasmosis was determined as 1.7% (6/358). PCR and partial DNA sequencing results of the 16S ribosomal RNA (rRNA) gene revealed that Anaplasma centrale, A. phagocytophilum like-1, and the human pathogenic novel genotype A. capra are circulating in cattle herds in Kyrgyzstan. Six DNA nucleotide sequences obtained in this study were deposited in GenBank under the following accession numbers: A. centrale (MW672117, MW672118, MW672119, MW672120), A. phagocytophilum (MW672121), and A. capra (MW672115).

First Molecular Detection of Dirofilaria immitis and D. repens in Dogs from Kyrgyzstan.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are the causative agents of cardiopulmonary and subcutaneous dirofilariosis, respectively. This neglected disease mainly seen in dogs, cats and wild carnivores is re-emerging recent years. No study was conducted on dirofilariosis in dogs in Kyrgyzstan. PURPOSE: The goal of this study was to investigate Dirofilaria species using PCR and sequencing in dogs from Kyrgyzstan. METHOD: Dirofilaria spp. infection in dogs was screened via convential PCR and sequencing in 337 dogs from Kyrgyzstan. RESULT: The overall prevalence of Dirofilaria spp. was 0.59% (2/337): DNA of D. immitis was detected in one sample and DNA of D. repens in second positive sample. In second sample, parallel co-infection of D. repens with Wolbachia was also found. While D. immitis sequence showed 98.70-100% similarity with previously reported sequences of D. immitis from dog blood, D. repens shared 100% identity with other sequences of D. repens. CONCLUSION: These results provided first evidence for Dirofilaria spp. in Kyrgyzstan and emphasized the veterinary and medical importance.

Molecular detection of tick-borne rickettsial and protozoan pathogens in domestic dogs from Turkey.

BACKGROUND: Canine tick-borne parasites have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary importance, domestic dogs play an important role in the transmission cycles of some agents by acting as reservoirs and sentinels. This study investigated Babesia, Theileria, Anaplasma, and Ehrlichia species in asymptomatic dogs in ten provinces of Turkey. METHODS: DNA obtained from blood samples collected from 757 domestic dogs (243 stray, 351 shelter, 163 pet) of both sexes and various ages were evaluated using PCR and reverse line blotting (RLB) assays. RESULTS: Of the 757 dogs tested, 41 (5.4%) were found to be infected with one or more parasites. Ehrlichia canis (37/757, 4.9%) was the most common canine tick-borne pathogen, followed by Anaplasma platys (4/757, 0.5%). Babesia canis and Theileria annulata were each detected in 1 (0.13%) sample. Combined infection of E. canis and A. platys was detected in 2 (0.3%) samples. The prevalence of tick-borne pathogens was higher in adult dogs (6.8%) than in those under one year old (3.1%). Difference in infection rate of male and female dogs was not significant. Pet dogs had a lower prevalence of infection (1.2%) compared to stray (7.4%) and shelter dogs (6%) although the difference between stray and shelter dogs was not significant. CONCLUSIONS: Babesia canis, T. annulata, A. platys, and E. canis species were identified at the molecular level in dogs in several provinces of Turkey, with E. canis being the most common species among tick-borne pathogens. Detailed studies should be conducted regarding the existence and prevalence of B. canis and Dermacentor reticulatus in eastern Turkey.

A molecular and parasitological survey of Hepatozoon canis in domestic dogs in Turkey.

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.

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey.

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.

A study on ovine tick-borne hemoprotozoan parasites (Theileria and Babesia) in the East Black Sea Region of Turkey.

In this study, the frequency of Theileria and Babesia species was assessed via reverse line blotting and blood smear-based diagnostic methods in small ruminants. A total of 201 apparently healthy animals from 26 randomly selected herds located in 4 locations (Artvin, Giresun, Gumushane, and Tokat) of East Black Sea Region of Turkey were investigated for the blood protozoans. In a polymerase chain reaction (PCR), the hypervariable V4 region of the 18S ribosomal RNA gene was amplified with a set of general primers specific for all Theileria and Babesia species. The PCR products were hybridized against catchall and species-specific (Theileria spp., Theileria lestoquardi, Theileria ovis, Theileria sp. OT1, Theileria sp., OT3, Theileria sp., MK, Theileria luwenshuni, Theileria uilenbergi, Babesia spp., Babesia ovis, Babesia motasi, and Babesia crassa) probes. Theileria piroplasms were identified in nine (4.47%) samples by microscopic examination. Reverse line blotting (RLB) detected the infection in 19.90% of the samples. The infection rate of sheep (28.90%) was higher than goats (4.10%). T. ovis, Theileria sp., MK, and Theileria sp. OT3 were detected by RLB. The most prevalent Theileria species was T. ovis (18.90%) followed by Theileria sp. MK (0.99%). Theileria sp. OT3 was detected in one sample (0.43%). A single animal was infected as mix with T. ovis and Theileria sp. MK. The other Theileria (T. lestoquardi, Theileria sp. OT1, T. luwenshuni, and T. uilenbergi) and Babesia (B. ovis, B. motasi, and B. crassa) species were not detected. This study is the first molecular survey on ovine tick-borne protozoans in East Black Sea Region of Turkey.

Molecular detection and identification of Anaplasma and Ehrlichia species in cattle from Turkey.

Bovine anaplasmosis is a tick-borne rickettsial disease widespread in tropical and subtropical areas. We investigated the presence and distribution of Anaplasma spp. in cattle from 6 provinces in Turkey. For amplification of the segment spanning the V1 region of the 16S ribosomal RNA (rRNA) gene of Anaplasma species, a reverse line blot (RLB) hybridization assay was performed on 389 blood samples. RLB identified Anaplasma infections in 9.0% (35/389) of the samples. The most frequently found species was A. marginale (11/389, 2.8%), followed by A. centrale (4/389, 1.0%) and A. phagocytophilum (4/389, 1.0%). Eighteen of 35 PCR-positive samples gave positive signals to the catch-all probes, but did not show any response to the species-specific probes tested. Sequencing results of 5 representative amplicons randomly selected from these specimens indicated that 3 were 100% identical to the sequence of A. ovis, and the other 2 sequences were 99.5% identical to the sequence of Ehrlichia sp. Omatjenne strain. The results further confirmed that A. ovis and Ehrlichia sp. Omatjenne infection occurs in cattle populations in Turkey.

Molecular evidence for Anaplasma phagocytophilum in Ixodes ricinus from Turkey.

This study investigated the presence of the pathogen Anaplasma phagocytophilum in ixodid ticks removed from humans living in three provinces (Giresun, Trabzon, Rize) in the east of the Black Sea Region of Turkey. A total of 1097 ixodid ticks were examined for the presence of A. phagocytophilum DNA. From the 95 pooled tick samples tested, species-specific fragments of A. phagocytophilum (11/95 samples, 11.6%) were amplified by nested PCR. Adult Ixodes ricinus (9/53 samples, 17.0%) and Ixodes spp. nymphs (2/9 samples, 22.2%) were infected with A. phagocytophilum. None of the remaining tick species gave a positive result for the presence of the pathogen. All nested PCR-positive samples were directly sequenced. The partial sequences (457bp) of the amplicons obtained from the infected tick pools were 100% identical to one another and to previously isolated sequences from human patients. To obtain a longer 16S rRNA gene sequence, one representative sample was reamplified with the universal primer set. The longer representative sequence (1306bp) also shared 99.92% similarity (a single adenine deletion) with the recently reported complete sequence of A. phagocytophilum.

Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks.

A polymerase chain reaction (PCR) assay followed by partial sequencing of the 16S ribosomal RNA gene was performed for the presence of Ehrlichia and/or Anaplasma. A total of 242 ixodid ticks were collected from domestic ruminants and their shelters, as well as humans, and their individual salivary glands were dissected out for DNA. From the 242 ticks analyzed, six (2.47%), comprising three Hyalomma anatolicum anatolicum, one Rhipicephalus bursa, and two Rhipicephalus sanguineus, were positive. Of these sequenced samples directly obtained from the PCR products, three sequences from H. a. anatolicum were identical to that of the gene of Ehrlichia spp. strains. One sequence identified in R. bursa was closely related to Anaplasma platys. The remaining two sequences detected in R. sanguineus were similar to that of the gene of Anaplasma ovis. The study presented here provides preliminary data regarding the presence of rickettsial pathogens in ticks in Turkey.

Molecular detection of Theileria and Babesia infections in cattle.

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 T. buffeli/orientalis) 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 T. 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.