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.

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.

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.

Molecular identification of Theileria and Babesia in ticks collected from sheep and goats in the Black Sea region of Turkey.

A molecular survey was undertaken in the Black Sea region of Turkey to determine the presence of Theileria and Babesia species of medical and veterinary importance. The ticks were removed from sheep and goats, pooled according to species and locations, and analyzed by PCR-based reverse line blot (RLB) and sequencing. A total of 2241 ixodid ticks belonging to 5 genus and 12 species were collected and divided into 310 pools. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 310 pools tested, 46 (14.83%) were found to be infected with Theileria or Babesia species, and the overall MLE of the infection rate was calculated as 2.27% (CI 1.67-2.99). The MLE of the infection rates were calculated as 0.691% (CI 0.171-1.78) in Haemaphysalis parva, 1.47% (CI 0.081-6.37) in Rhipicephalus sanguineus, 1.84% (CI 0.101-7.87) in Ixodes ricinus, 2.86% (CI 1.68-4.48) in Rhipicephalus turanicus, 5.57% (CI 0.941-16.3) in Hyalomma marginatum, and 6.2% (CI 4.02-9.02) in Rhipicephalus bursa. Pathogens identified in ticks included Theileria ovis, Babesia ovis, Babesia bigemina, and Babesia microti. Most tick pools were infected with a single pathogen. However, five pools displayed mixed infections with T. ovis and B. ovis. This study provides the first molecular evidence for the presence of B. microti in ticks in Turkey.

Molecular identification of Theileria and Babesia in sheep and goats in the Black Sea Region in Turkey.

This study was carried out to investigate presence and distribution of Theileria and Babesia species via microscopic examination and reverse line blotting (RLB) techniques in sheep and goats in the Black Sea region of Turkey. For this purpose, 1,128 blood samples (869 sheep and 259 goats) were collected by active surveillance from sheep and goats in different provinces of various cities in the region in the years 2010 and 2011. Smears were prepared from the blood samples, stained with Giemsa, and examined under the light microscope for Theileria and Babesia piroplasms. The genomic DNAs were extracted from blood samples. The length of 360-430-bp fragment in the variable V4 region of 18S SSU rRNA gene of Theileria and Babesia species was amplified using the gDNAs. The polymerase chain reaction products were hybridized to the membrane-connected species-specific probes. A total of 38 animals (3.37%) including 34 sheep (3.91%) and 4 goats (1.54%) were found to be positive for Theileria spp. piroplasms in microscopic examination of smears while Babesia spp. piroplasm could not detected. Infection rates were 34.64% in sheep, 10.04% in goats, and totally 28.99% for Theileria ovis while 0.58% in sheep and totally 0.44% for Babesia ovis. However, Theileria sp. OT3 was detected in 2.65% of sheep and 2.04% of all animals; besides Theileria sp., MK had 0.58% prevalence in sheep and 0.77% in goats, with a total 0.62% with RLB. Although T. ovis and Theileria sp. MK were determined in both sheep and goats, B. ovis and Theileria sp. OT3 were observed only in the sheep. These results provide the first detailed molecular data for sheep and goat theileriosis and babesiosis in the region.

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.

[Tick (Ixodoidea) and flea (Siphonaptera) species on three red foxes (Vulpes vulpes) in Erzurum province]. / Erzurum Ilinde Üç Kirmizi Tilkide (Vulpes vulpes) Kene (Ixodoidea) ve Pire (Siphonaptera) Türleri.

In this study, three red foxes (Vulpes vulpes) which died after traffic accidents were examined in terms of ectoparasites, in the province of Erzurum in January of 2009. 13 ticks and 74 fleas were collected from the foxes and taken to the laboratory in separate glasses containing 70% alcohol. Ticks were taken directly from 70% alcohol and have been identified under the stereo microscope. Fleas were cleared in 10% KOH solution during 4-13 days and washed in distilled water 3-4 times and were identified under the light microscope. Two tick species Ixodes hexagonus Leach, 1815 (5 males, 7 females) and Haemaphysalis numidiana Neumann, 1905 (1 male) and four flea species Pulex irritans Linne, 1758 (23 males, 37 females), Chaetopsylla globiceps Tacshenberg, 1880 (11 female), Ctenocephalides canis Curtis, 1826 (2 female) and Ctenocephalides felis felis Bouche, 1835 (1 female) were identified. In this study H. numidiana have been identified in the fox for the first time.

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.

Evaluation of a PCR and comparison with RLB for detection and differentiation of Theileria sp. MK and other Theileria and Babesia species of small ruminants.

Theileria sp. MK in sheep and goats were detected first time by polymerase chain reaction (PCR) and detection limit of PCR and reverse line blotting (RLB) were compared. A part of 18S ssu rRNA gene was amplified from blood samples that were taken from sheep and goats naturally infected with Theileria sp. MK by PCR. Detection limit of both PCR and RLB methods was one infected cell in 10(7) sheep erythrocytes. Nine hundred twenty field samples that had been tested previously by RLB were evaluated by the PCR assay. As found by RLB previously, 12 of 920 (1.30%) samples were detected as positive by PCR. Two positive PCR products, one of which was from sheep and the other from goat, were sequenced. These sequences were identical to the reported nucleotide sequence of Theileria sp. MK. It is concluded that the PCR described in this study will be useful for epidemiological studies and for discrimination between Theileria sp. MK and other Theileria species. In addition, PCR has superiority over RLB because of its ease of use and time period required.

[Use of multiplex PCR for the diagnosis of Theileria annulata and Theileria buffeli]. / Theileria annulata ve Theileria buffeli'nin Teshisinde Multiplex PCR'in Kullanilmasi.

The species causing theileriosis in cattle in Turkey are Theileria annulata and T. buffeli. While T. buffeli is low in pathogenicity or non-pathogenic , T. annulata is very pathogenic and causes tropical theileriosis with high morbidity and mortality in cattle. In this study, a multiplex PCR was used for a simultaneous diagnosis of these species. Genes for the merozoite surface antigen (Tams 1) and the major piroplasm surface protein (MPSP) were amplified with PCR for T. annulata and T. buffeli, respectively. It was found that both single and mixed infection with T. annulata and T. buffeli could be diagnosed with multiplex PCR.

Seroprevalence of hypodermosis in cattle in some provinces of Turkey.

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in the east and southeast of Turkey. For this purpose, a total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. The highest percentage of seropositivity were detected at Elazig province (26.3%) followed by Malatya (22.3%) and Diyarbakir provinces (22.1%). The seropositivity rate was higher in female (31%) than male (14.1%). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3-4 ages and 5 and up ages, respectively.

[PCR-RFLP analysis of the Tams1 gene of Theileria annulata]. / Theileria annulata Tams1 geninin PCR-RFLP analizi.

Tams1 is a merozoite surface antigen of Theileria annulata. Genetical variations of Tams1 make studies of vaccine and diagnostic tests like ELISA difficult. In this study, Tams1 genes of 89 T. annulata isolates obtained from natural infected cattle in Elazig and Bingöl regions were tested with PCR-RFLP. Six different restriction profiles (a, b, c, d, e, f) were detected. The number of restriction profiles of 89 samples was found to be as follows: 78(a), 2(b), 2(c), 5(d), 1(e), and 1(f).

[Survey of T. annulata and T. buffeli/orientalis in cattle in the region of Erzincan using reverse line blotting]. / Erzincan yöresinde sigirlarda Theileria annulata ve Theileria buffeli/orientalis'in reverse line blotting yöntemi ile arastirilmasi.

This study was carried out to investigate Theileria annulata and T. buffeli/orientalis in cattle in the region of Erzincan using reverse line blotting (RLB) and microscopical examination. A total of 123 blood samples and thin blood smears were collected from cattle in distinct locations. Thin blood smears were microscopically examined for Theileria piroplasms. The 18S SSU rRNA gene in the DNA of Theileria spp extracted from blood was amplified and used in RLB. For this purpose, PCR products were hybridized with specific probes for over-all Theileria spp., T. annulata and T. buffeli/orientalis as well as Babesia spp. While Theileria spp. were observed in 14 out of 123 cattle, (11.38 %) during microscopical examination, T. annulata was detected in 19 (15.45%) cattle and T. buffeli/orientalis, in 12 (9.76%) by RLB, respectively. Mixed infection was also detected in three samples.

Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species.

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.