Wide bovine tick-borne pathogen spectrum: Predominancy of Theileria annulata and the first molecular detection of Ehrlichia minasensis in Turkey.

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them.

Whole genome sequence and diversity in multigene families of Babesia ovis.

Ovine babesiosis, caused by Babesia ovis, is an acute, lethal, and endemic disease worldwide and causes a huge economic loss to animal industry. Pathogen genome sequences can be utilized for selecting diagnostic markers, drug targets, and antigens for vaccine development; however, those for B. ovis have not been available so far. In this study, we obtained a draft genome sequence for B. ovis isolated from an infected sheep in Turkey. The genome size was 7.81 Mbp with 3,419 protein-coding genes. It consisted of 41 contigs, and the N50 was 526 Kbp. There were 259 orthologs identified among eight Babesia spp., Plasmodium falciparum, and Toxoplasma gondii. A phylogeny was estimated on the basis of the orthologs, which showed B. ovis to be closest to B. bovis. There were 43 ves genes identified using hmm model as well. They formed a discriminating cluster to other ves multigene family of Babesia spp. but showed certain similarities to those of B. bovis, B. caballi, and Babesia sp. Xinjiang, which is consistent with the phylogeny. Comparative genomics among B. ovis and B. bovis elucidated uniquely evolved genes in these species, which may account for the adaptation.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72-3.46) and 1.46% (CI 0.87-2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43-14.90) and 0.33% (CI 0.02-1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01-0.51) and 2.08% (CI 1.25-3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey.

Discovery of a new Hepatozoon species namely Hepatozoon viperoi sp. nov. in nose-horned vipers in Türkiye.

Although Hepatozoon spp. remains the most prevalent intracellular protozoa infecting snakes, it was reported only in a few snake species of the Colubridae family in Türkiye. Moreover, studies on these hemoparasites are not available in venomous nose-horned vipers from Türkiye. In this study, we investigated Hepatozoon spp. in three individual Vipera ammodytes using morphological and molecular methods. Our results were positive for intraerythrocytic Hepatozoon spp. gamonts in all three snakes, exhibiting low parasitemia. The microscopic findings were further confirmed through molecular data. A genus-specific PCR assay targeting the 18S rRNA gene region of Hepatozoon spp., was performed using HemoF/HemoR and Hep300/Hep900 primers. The obtained sequences were concatenated and used in phylogenetic analyses in comparison with different Hepatozoon species. Although our (OP377741) isolate was separated into a different branch, it was clustered with the isolates of H. massardi (KC342526), H. cevapii (KC342525), and H. annulatum (ON262426) from Brazilian snakes. Moreover, gene similarity and pair-wise distance between our isolate and other Hepatozoon species infecting snakes were found to be 89.30-98.63% and 0.009-0.077, respectively. Hence, we reported a new species of Hepatozoon, namely Hepatozoon viperoi sp. nov. infecting V. ammodytes. Since the literature does not indicate the existence of such a Hepatozoon species in V. ammodytes in different countries, our data may contribute to the expanding knowledge of Hepatozoon species in snakes, providing new insights into the biodiversity of the haemogregarine protozoan parasite.

Protozoan and Rickettsial Pathogens in Ticks Collected from Infested Cattle from Turkey.

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05−100% nucleotide identity with those deposited in GenBank (query cover range: 89−100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey.

A survey on equine tick-borne diseases: The molecular detection of Babesia ovis DNA in Turkish racehorses.

Common vector-borne diseases of horses include equine piroplasmosis (EP) caused by Babesia caballi and Theileria equi, and equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum. Equine piroplasmosis leads to severe health issues in horses and restrictions on the movement of horses internationally. Anaplasma phagocytophilum causes an acute febrile illness in horses and is also of zoonotic importance. In the present study, blood samples were collected from 152 Turkish racehorses from three different provinces (Izmir, Gaziantep, and Konya) of Turkey to investigate the prevalence of EP and EGA. Standard and nested polymerase chain reactions were performed to identify equine piroplasms and A. phagocytophilum, respectively. PCR primers targeting Babesia spp. 18S rRNA, B. caballi BC48, T. equi EMA-1, and A. phagocytophilum 16S rRNA genes were used for molecular diagnosis. Following the cloning and subsequent sequencing of PCR-positive samples, a total of 15 (9.9%) horses were found to be infected with at least one pathogen. Theileria equi and A. phagocytophilum were found in 3.3% (5/152) and 6.6% (10/152) of the samples, respectively. Although B. caballi specimens were not detected in any of the samples, a positive signal was detected for the Babesia genus-specific 18S rRNA PCR. Subsequent sequencing of this signal revealed 100% identity to Babesia ovis. This is the first detection of B. ovis DNA in racehorses in Turkey to the best of our knowledge. Additionally, this study also reports the first molecular identification of A. phagocytophilum in Turkish racehorses. Based on this report, it is recommended that future epidemiological studies on horses also take B. ovis, a parasite usually found in sheep, into consideration and that further detailed studies be conducted to unravel the transmission pathways and potential clinical effects of B. ovis in horses.

Molecular detection of selected tick-borne pathogens infecting cattle at the wildlife-livestock interface of Queen Elizabeth National Park in Kasese District, Uganda.

In Uganda, ticks and tick-borne diseases (TBDs) pose a big challenge to farmers. They reduce cattle productivity and cause severe economic damage. Several studies have documented the prevalence of tick-borne pathogens in cattle; however, their genetic characteristics and the role of wildlife-livestock interaction in the epidemiology of the TBDs are not well documented. This study assessed the prevalence and genetic diversity of various tick-borne pathogens (TBPs) as well as the risk factors associated with the occurrence of TBPs in blood samples of 208 randomly selected cattle from 16 farms located around Queen Elizabeth National Park (QENP) in Kasese District in western Uganda. Farming practices, disease challenges, and the level of wildlife-livestock interactions were assessed by a questionnaire survey amongst farm owners. Polymerase chain reaction (PCR) assays revealed that 62.9% (131/208) cattle samples were positive for one or more pathogens. Using specific PCR assays, we detected Theileria spp., Theileria parva, Anaplasma marginale, Anaplasma platys-like, and Babesia bigemina at 50.5%, 27.9%, 19.2%, 11.5% and 8.7%, respectively. We also confirmed the infection of samples by Theileria velifera and Theileria mutans after sequencing the Theileria spp. 18S rRNA gene. The risk factors associated with the occurrence of TBPs included communal grazing, herd size, age, and proximity to QENP. Phylogenetic analysis of the T. parva p104 gene showed a high identity to the previous isolates from Uganda and other East African countries and clustered closer to the buffalo (Syncerus caffer) isolates, suggesting a possible cross-species transmission. The sequences of A. marginale groEL and B. bigemina RAP-1a formed well-supported clades with high identities to the previous isolates identified from central and eastern Uganda. The isolates obtained from A. phagocytophilum 16S rRNA gene sequences showed relationship with A. platys-like, Anaplasma sp., uncultured Anaplasma species and A. phagocytophilum isolates from Africa, Asia, Europe, and the USA. The findings of the present study showed that TBDs are still a burden to farmers and that management practices in this area may increase the transmission of pathogens between livestock and wildlife.

Primary Tick-Borne Protozoan and Rickettsial Infections of Animals in Turkey.

Parasitic diseases caused by ticks constitute a barrier on global animal production, mainly in tropical and subtropical regions. As a country with a temperate and subtropical climate, Turkey has topography, climate, and pasture resources, and these resources are suitable for animal breeding and parasite-host-vector relationships throughout the country. This geography restricts the regulations on animal movements in the southeastern and eastern Anatolia because of the close contact with the neighboring states. The livestock resources in Turkey are regulated by strong foundations. Almost 30% of the agriculture-based gross domestic product is provided by the livestock industry. Parasitic diseases arising from ticks are endemic in Turkey, and they have a significant impact on the economy and animal health, particularly for ruminants. The main and economically-important tick-borne diseases (TBDs) suffered by animals include theileriosis, babesiosis, hepatozoonosis, and cytauxzoonosis caused by protozoa, and anaplasmosis and ehrlichiosis caused by rickettsiae. The most common hemoprotozoan and rickettsial agents are Anaplasma marginale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma platys, Babesia bigemina, Babesia caballi, Babesia ovis, Cytauxzoon felis, Ehrlichia canis, Hepatozoon canis, Theileria annulata and Theileria equi. These diseases are basically controlled through treatment and measures for tick control. Vaccination can be performed for only tropical theileriosis caused in Turkey. We reviewed the studies published in domestic and international journals to gather epidemiological data regarding the major TBDs suffered by animals in Turkey.

Tick-Borne Hemoparasites of Sheep: A Molecular Research in Turkey.

Tick-borne diseases (TBDs) indulge in severe economic losses in the livestock industry by adversely affecting the small ruminant breeding in tropical and subtropical zone countries, including Turkey. Turkey encompasses a wide land area representing diverse climatic conditions. The present study explored the presence and distribution of Babesia ovis, Theileria ovis, Theileria lestoquardi, Anaplasma ovis, Anaplasma phagocytophilum and the co-occurrence status of these pathogens. A total of 299 sheep blood samples were collected from fifteen provinces located in six different geographical regions in Turkey. PCR analyses were executed using species-specific primers based on Babesia ovis BoSSU rRNA, Theileria ovis ToSSU rRNA, Theileria lestoquardi 18S rRNA, Anaplasma ovis Major Surface Protein (AoMSP4), and Anaplasma phagocytophilum 16S rRNA genes. Overall, 219 (73.24%) sheep were found to be infected with at least one of the following protozoan and rickettsial pathogens; B. ovis, A. ovis,T. ovis, and A. phagocytophilum. Theileria lestoquardi was not detected in any blood sample. The global prevalence of B. ovis, A. ovis, T. ovis, and A. phagocytophilum was estimated to be 2.68%, 16.05%, 41.47%, and 57.19%, respectively. Besides this, dual (24.41%), triple (9.03%), and quadruple (0.67%) co-infections were detected in the study. Phylogenetic analysis revealed significant nucleotide sequence identities between the sequences obtained in this study and the sequences registered in the GenBank. This study provides relevant data regarding the predominance of ovine tick-borne protozoan and rickettsial agents in Turkey. A high molecular prevalence of tick-borne pathogens (TBPs) was identified in the study. This situation indicates that TBPs should be screened continuously, and necessary control measures should be taken to prevent diseases caused by tick-borne protozoan and rickettsial agents.

Molecular detection of genotypes and subtypes of Cryptosporidium infection in diarrheic calves, lambs, and goat kids from Turkey.

The studies on Cryptosporidium infections of animals in Turkey mostly rely on microscopic observation. Few data are available regarding the prevalence of Cryptosporidium genotypes and subtypes infection. The aim of this study is to analyse the detection of Cryptosporidium genotypes and subtypes from young ruminants. A total of 415 diarrheic fecal specimens from young ruminants were examined for the Cryptosporidium detection by use of nested PCR of the small subunit ribosomal RNA (SSU rRNA) gene and the highly polymorphic 60 kDa glycoprotein (gp60) gene followed by sequence analyses. The results of this study revealed that 25.6% (106 of 415) of the specimens were positive for Cryptosporidium spp. infection. We identified 27.4% (91/333), 19.4% (13/67), and 13.4% (2/15) of positivity in calves, lambs and goat kids, respectively. Genotyping of the SSU rRNA indicated that almost all positive specimens were of C. parvum, except for one calf which was of C. bovis. Sequence analysis of the gp60 gene revealed the most common zoonotic subtypes (IIa and IId) of C. parvum. We detected 11 subtypes (IIaA11G2R1, IIaA11G3R1, IIaA12G3R1, IIaA13G2R1, IIaA13G4R1, IIaA14G1R1, IIaA14G3R1, IIaA15G2R1, IIdA16G1, IIdA18G1, IIdA22G1); three of them (IIaA12G3R1, IIaA11G3R1 and IIaA13G4R1) was novel subtypes found in calves and lambs. Additionally, three subtypes (IIaA11G2R1, IIaA14G3R1, and IIdA16G1) were detected in young ruminants for the first time in Turkey. These results indicate the high infection of Cryptosporidium in Turkey and propose that young ruminants are likely a major reservoir of C. parvum and a potential source of zoonotic transmission.

Molecular Detection and Assessment of Risk Factors for Tick-Borne Diseases in Sheep and Goats from Turkey.

BACKGROUND: Tick-borne diseases mainly, theileriosis, babesiosis and anaplasmosis cause significant economic losses in livestock globally, including Turkey. The tick-borne pathogens of small ruminants in Turkey have been studied widely but information on molecular characterization and disease occurrence is still limited. METHODS: In this study, both microscopy and molecular detection and characterization for Theileria spp. Babesia ovis, Anaplasma ovis and Anaplasma phagocytophilum was conducted. A total of 133 blood samples of tick-infested small ruminants (105 sheep and 28 goats) were collected from Turkey: half of the animals had clinical signs of tick-borne disease infections. RESULTS: Using PCR assays and microscopy, 90.2% and 45.1% of the samples were positive for at least one pathogen, respectively. Overall, the infection rates of A. phagocytophilum, B. ovis, A. ovis, Theileria spp. were 66.7%, 62.4%, 46.6% and 7.0%, respectively. Fifty-nine of the 133 (44.4%) samples were co-infected with two or more pathogens. Sex, season and B. ovis positivity were significant risk factors for occurrence of clinical disease. Sequence and phylogenetic analysis based on B. ovis 18S small subunit rRNA, A. ovis major surface protein 4, Theileria spp. 18S rRNA and A. phagocytophilum 16S rRNA genes showed that the isolates in this study clustered together in well-supported clades with those previously collected from Turkey and other countries. CONCLUSIONS: The study shows B. ovis as the most significant pathogen associated with clinical and fatal cases in small ruminants from Turkey. Female sex and summer season are associated with increased risk of the disease. This study shows high infection rates with the pathogens among small ruminants including A. phagocytophilum which has veterinary and public health importance.

Endemic instability of ovine babesiosis in Turkey: A country-wide sero-epidemiological study.

Ovine babesiosis is an endemic tick-borne disease of small ruminants in the Middle East, European, and some African and Asian countries, including Turkey. This study assessed whether the endemic status of this disease was stable or instable, which is important for disease control efforts. For this aim, 4115 sheep blood samples were collected from 81 cities in the seven geographical regions of Turkey. The diagnosis of Babesia ovis was made using microscopic and serological techniques. Thin blood smears were prepared from anticoagulated venous blood. Serum samples were screened for specific antibodies using an enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). Recombinant Babesia ovis secreted antigen 1 (rBoSA1) was used in the ELISA. The antigen slides used in the IFAT were prepared from the B. ovis-infected blood at a high level of parasitemia (5 %). The animals were divided into three groups according to their age: group I (one to six months), group II (6-12 months), and group III (older than one year). The endemic status of B. ovis was determined according to the inoculation rate (h value) calculations. Babesia spp. merozoites were observed in 40 (0.97 %) of the slides. Seropositivity rates were 29.89 % (1230/4115) and 49.16 % (2023/4115) by the ELISA and IFAT, respectively. According to the IFAT results, 31.7 %, 33.6 %, and 52.8 % of the animals were seropositive in groups I, II, and III, respectively. The inoculation rates of the animals indicated that the endemic status of ovine babesiosis was mostly instable throughout the country. Endemic stability was found only in group I from four regions (Central Anatolia, Eastern Anatolia, Aegean, and Mediterranean). Based on these results, the risk of clinical infection due to tick infestation was high when the maternal immunity and non-specific age resistance weakens or disappears. Thus, vaccination is needed to protect sheep against B. ovis infections in Turkey.

Haemoparasitic agents associated with ovine babesiosis: A possible negative interaction between Babesia ovis and Theileria ovis.

Babesiosis, theileriosis, and anaplasmosis are the most common tick-borne diseases in sheep. The majority of anaplasmosis and theileriosis are subclinical; however, babesiosis causes severe infections in small ruminants. Although there are many reports of co-infections with the agents of these diseases, their clinical severity compared with either of the infections alone is unknown. Within the host, interactions between co-infecting species may cause variations in clinical presentation and response to therapy. The aim of this study was to determine the tick-borne agents in sheep located at sites where fatal disease outbreaks caused by babesiosis have commonly been reported. Two hundred and nine sheep with clinical signs suggestive of ovine babesiosis were included in the study. The initial diagnosis of haemoparasites was based on clinical symptoms and microscopy and was confirmed using PCR assays. The blood samples were examined for the presence of Babesia ovis (B. ovis), Anaplasma ovis (A. ovis), A. phagocytophilum, and Theileria ovis (T. ovis). The results showed 86.12% of the animals were infected with one or more pathogens. B. ovis was the dominant pathogen. Overall, the infection rate of B. ovis, A. ovis, T. ovis, and A. phagocytophilum was 70.81%, 56.94%, 21.05%, and 2.39%, respectively. The infection rate of B. ovis alone (31.11%) was higher than A. ovis (9.44%) or T. ovis (1.67%) alone. Co-infections were found at a higher percentage (57.78%) than single infections (42.22%). A. ovis was detected in the blood of a high percentage (98.07%) of co-infected animals. Coexistence of B. ovis and A. ovis (34.45%) was more common than other combinations of species. There was a noticeably low level of co-occurrence between B. ovis and T. ovis (1.11%). During the study, 11 sick animals did not survive despite treatment. Seven were infected with B. ovis alone, three had a dual infection with B. ovis and A. ovis, and one had B. ovis, A. ovis, and T. ovis.

A PCR survey of vector-borne pathogens in different dog populations from Turkey.

In the present study, a total of 192 blood samples were collected from pet dogs, kennel dogs and shepherd dogs in Konya district, Turkey, and tested by specific PCR for the presence of vector-borne pathogens. Several pathogens were identified, most of which can cause substantial morbidity in dogs. PCR results revealed that 54 (28.1%) dogs were infected with one or more pathogens. Positive results were obtained for Babesia spp. in 4 dogs (2.1%), Hepatozoon spp. in 8 dogs (4.2%) and Mycoplasma spp. in 46 dogs (24%). Three dogs (1.6%) were infected with two or three pathogens. The sequence analysis of the positive DNA samples revealed the presence of Babesia canis vogeli, Hepatozoon canis, Hepatozoon sp. MF, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. Ehrlichia canis and Anaplasma platys were not detected. Regardless of ownership status, vector-borne diseases were common in these dog populations. There was significant difference of pathogen prevalence among the different dog populations. Mycoplasma spp. was more frequent in the kennel dogs (31.9%) than in the pet (21.4%) and shepherd dogs (13.8%). Additionally, the frequency of Babesia spp. and Hepatozoon spp. was higher in the shepherd dogs which account for three quarters and half of the total number of Babesia spp. and Hepatozoon spp., respectively. To our knowledge, this is the first report of Mycoplasma infection in dogs in Turkey. The results of the present study provide a foundation for understanding the epidemiology of canine vector-borne diseases (CVBDs), and for strategies to control these diseases in Turkey.

Molecular detection and genetic characterization of Babesia, Theileria and Anaplasma amongst apparently healthy sheep and goats in the central region of Turkey.

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia ovis, Theileria ovis and Anaplasma ovis in 343 small ruminants (249 sheep and 94 goats) from 13 towns in the Central Anatolia region of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. ovis ssu rRNA (BoSSUrRNA), T. ovis ssu rRNA (ToSSUrRNA) and A. ovis major surface protein 4 (AoMSP4) genes, respectively. Fragments of these genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. ovis, T. ovis and B. ovis were 60.0%, 35.9% and 5.2%, respectively. Co-infection of the animals with two or three pathogens was detected in 105/343 (30.6%) of the ovine samples. The results of sequence analysis indicated that AoMSP4 were conserved among the Turkish samples, with 100% sequence identity values. In contrast, the BoSSUrRNA and ToSSUrRNA gene sequences were relatively diverse with identity values of 98.54%-99.82% and 99.23%-99.81%, respectively. Phylograms were inferred based on the BoSSUrRNA, ToSSUrRNA and AoMSP4 sequences obtained in this study and those from previous studies. B. ovis isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, the Turkish T. ovis isolates in the present study formed a monophyletic grouping with the isolates from other countries in a phylogeny based on ToSSUrRNA sequences. Furthermore, phylogenetic analysis using AoMSP4 sequences showed the presence of three genotypes of A. ovis. This study provides important data for understanding the epidemiology of tick-borne diseases in small ruminants and the degree of genetic heterogeneities among these pathogens in Turkey. To our knowledge, this is the first study on the co-infection of Babesia, Theileria and Anaplasma in sheep and goats in Turkey.

Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey.

Considering the scarce information on occurrences of Toxoplasma gondii and Neospora caninum in domestic animals from Turkey, the aim of this study was to investigate the seroprevalence of these parasite infections in cattle, horses, sheep, goats and dogs in Turkey. The specific antibodies against T. gondii and N. caninum were detected by iELISAs based on the recombinant TgSAG2 or NcSAG1 in a total of 2,039 serum samples from eleven provinces. The seroprevalence of T. gondii infections was 46.3%, 4.0%, 20.0%, 12.9% and 19.8%, that of N. caninum infections was 0.3%, 7.4%, 2.1%, 3.2% and 16.6% in the horses, cattle, sheep, goats and dogs, respectively. These results indicated that T. gondii and N. caninum infections are prevalent in Turkish domestic animals.

Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey.

Babesia spp., Theileria spp. and Anaplasma spp. are significant tick-borne pathogens of livestock globally. In this study, we investigated the presence and distribution of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in cattle from 6 provinces of Turkey using species-specific PCR assays. The PCR were conducted using the primers based on the B. bigemina rhoptry-associated protein 1a (BbiRAP-1a), T. annulata merozoite surface antigen-1 (Tams-1), T. orientalis major piroplasm surface protein (ToMPSP) and A. marginale major surface protein 4 (AmMSP4) genes, respectively. Fragments of B. bigemina internal transcribed spacer (BbiITS), T. annulata internal transcribed spacer (TaITS), ToMPSP and AmMSP4 genes were sequenced for phylogenetic analysis. PCR results revealed that the overall infections of A. marginale, T. annulata, B. bigemina and T. orientalis were 29.1%, 18.9%, 11.2% and 5.6%, respectively. The co-infection of two or three pathogens was detected in 29/196 (15.1%) of the cattle samples. The results of sequence analysis indicated that BbiRAP-1a, BbiITS, Tams-1, ToMPSP and AmMSP4 were conserved among the Turkish samples, with 99.76%, 99-99.8%, 99.34-99.78%, 96.9-99.61% and 99.42-99.71% sequence identity values, respectively. In contrast, the Turkish TaITS gene sequences were relatively diverse with 92.3-96.63% identity values. B. bigemina isolates from Turkey were found in the same clade as the isolates from other countries in phylogenetic analysis. On the other hand, phylogenetic analysis based on T. annulata ITS sequences revealed significant differences in the genotypes of T. annulata isolates from Turkey. Additionally, the T. orientalis isolates from Turkish samples were classified as MPSP type 3 genotype. This is the first report of type 3 MPSP in Turkey. Moreover, AmMSP4 isolates from Turkey were found in the same clade as the isolates from other countries. This study provides important data for understanding the epidemiology of tick-borne diseases and it is expected to improve approach for diagnosis and control of tick-borne diseases in Turkey.

A new immunoreactive recombinant protein designated as rBoSA2 from Babesia ovis: Its molecular characterization, subcellular localization and antibody recognition by infected sheep.

Ovine babesiosis, caused by the intra-erythrocytic protozoan parasite Babesia ovis, is an infectious and economically important tick-borne disease of sheep. Diagnostic testing is an essential tool used for the control of the disease. In order to identify and characterize the immunoreactive proteins which are useful in serological diagnosis of the disease, a complementary DNA (cDNA) expression library was constructed from B. ovis merozoite mRNA. A cDNA clone designated as BoSA2 was identified by immunoscreening of a cDNA library using immune sheep serum. The sequence of the BoSA2 cDNA had a partial open reading frame of 1156 nucleotides encoding a polypeptide of 384 amino acid residues. Theoretical molecular mass for the mature protein was 43.5 kDa. The sequence of the BoSA2 was inserted into the expression vector pGEX-4T-1 and then expressed in Escherichia coli DH5α cells as a glutathione S-transferase (GST)-tagged fusion protein. This recombinant fusion protein (rBoSA2) was purified by GST-affinity chromatography. Immunoreactivity of the rBoSA2 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using the sera from the animals naturally and experimentally infected with B. ovis. ELISA results demonstrated that this antigen was useful for the diagnosis of ovine babesiosis. The localization of the BoSA2 protein was shown in and on the parasite and in the cytoplasm of the infected erythrocyte by confocal laser microscope. To our knowledge, rBoSA2 is the second immunoreactive recombinant protein of B. ovis until the present.

Molecular detection and characterization of Hepatozoon spp. in dogs from the central part of Turkey.

Canine hepatozoonosis is a tick-borne protozoal disease caused by Hepatozoon spp. Two species of Hepatozoon are currently known to infect dogs as Hepatozoon canis and H. americanum. Although H. canis generally causes a chronic infection with relatively mild clinical alterations compared to H. americanum, infection by H. canis can be life-threatening. The disease is widespread in USA, Africa, Europe, South America, and Asia. To determine the frequency of infection with Hepatozoon spp. in stray dogs from Central Anatolia Region of Turkey, a total of 221 blood samples collected over a three-year period were evaluated by using genus specific Polymerase Chain Reaction (PCR) designed to amplify a fragment of 666bp located in 18 S rRNA gene of Hepatozoon spp. Eight (3.61%) blood samples were positive for Hepatozoon spp. For the classification of species, all positive PCR products were purified with a PCR purification kit and sequenced. Sequencing results of eight representative amplicons indicated that 6 were 98-99% identical to the sequence of H. canis and the other 2 sequences were 95-97% identical to the sequence of Hepatozoon spp. So it was named Hepatozoon sp. MF. A phylogenetic tree was constructed from the sequences of the tick-borne agents identified previously and in this study using the neighbor-joining method. The nucleotide sequences were compared to the H. canis sequences reported in Turkey using the nucleotide Basic Local Alignment Search Tool (BLAST) program. The results of this study are significant in terms of the presence of a novel canine Hepatozoon genotype.