Mutlu Sevinc

A comparative study on the prevalence of Theileria equi and Babesia caballi infections in horse sub-populations in Turkey.

Blood and serum samples were taken from 481 horses, from a stud farm or a racecourse, and tested by microscopic examination of blood smears and cELISA for Theileria equi (T. equi) and Babesia caballi (B. caballi) infections. At the time of sampling, animals were also examined for tick infestations and clinical disease, which were not observed in any of the sampled horses. During the microscopic examination of thin blood smears, parasites were detected in the three horses from the racecourse. Overall seroprevalence of infection was detected as 18.50% (89 of 481 horses) by cELISA, with T. equi being significantly more prevalent than B. caballi. Of the 481 blood samples, 78 (16.21%) were serologically positive for T. equi and 4 (0.83%) were serologically positive for B. caballi. In addition, 7 (1.46%) samples were positive for both T. equi and B. caballi antibodies. Seropositivity rates in the racecourse horses were higher than those determined in the stud farm horses. The rates for T. equi, B. caballi and both species were 13.39, 0.52 and 0% in the horses from the stud farm and 27, 2 and 7% in the racecourse horses, respectively. These results indicate that equine piroplasmosis is more common in racehorses than studhorses and therefore it might be a serious concern in horses that participate to international races.

Babesia ovis infections: Detailed clinical and laboratory observations in the pre- and post-treatment periods of 97 field cases

Ovine babesiosis, caused by Babesia ovis, is of major economic importance in Turkey. The changes in the blood profile of infected animals are informative about the course of infection. The aim of the present study was to evaluate the hematological and biochemical changes in the pre- and post-treatment periods of the natural B. ovis infections. The presence of the parasites was confirmed by microscopy and polymerase chain reaction (PCR) analysis. On the basis of the clinical and laboratory findings, the infections were categorized into different groups according to the degree of anemia and the level of parasitemia. All infected sheep were treated with imidocarb dipropionate (IMDP). The blood pictures in the pre- and post-treatment periods were compared. Pancytopenia occurred in animals with severe anemia and very high parasitemia, and bicytopenia in the other groups. The platelet count (PLT), plateletcrit (PCT) and mean platelet volume (MPV) returned to the normal ranges after treatment, except those in the group with severe anemia. In the biochemical profile, B. ovis infection caused an increase in blood urea nitrogen and total bilirubin, and these parameters returned to normal levels after treatment. The indirect fluorescein antibody test (IFAT) results showed that 38.1% of the cases raised specific antibodies during the period of infection, with titers ranging from 1/160 to 1/640. All of 45 animals re-examined after treatment were seropositive, with high titers that rose up to 1/5120.

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.

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.

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.

Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

ABSTRACT In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis.

Determination of immunoreactive proteins of Babesia ovis

Babesia ovis, an intraerythrocytic protozoan parasite transmitted by ticks, causes severe infections in sheep in tropical and subtropical regions of the world. Parasite-specific immunoreactive proteins have been used as antigen in the serological diagnosis of babesiosis. There is no study about determination of B. ovis-specific proteins in sheep. This study was planned to determine the immunoreactive proteins of B. ovis. In this study, two splenectomized lambs, and twelve seropositive sheep and five seronegative lambs for anti-B. ovis antibodies were used as materials. Infected blood samples at 5% of parasitemia from the two splenectomized lambs experimentally infected with a virulent B. ovis field strain were analyzed for B. ovis-specific proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB). B. ovis-specific five major proteins were recognized by anti-B. ovis serum but not by healthy sheep serum. They were of approximate molecular weights 154, 109, 77, 58, and 38 kDa. As the control samples, protein profiles of the blood extracts of two lambs before splenectomy operation were also blotted with the immune sera, but none of the five proteins was detected. These proteins were also immunoblotted with heterologous positive and negative sheep sera. All of twelve positive sera recognized the 109 kDa protein with 100 percent sensitivity. The 77 kDa protein reacted in 11 of 12 sera (91.6%). The sensitivities of the other 3 proteins ranged between 83.3% and 25%. The five protein bands immunoblotted with sera of the 5 negative lambs did not give any positive reaction. The results of this study revealed the presence of proteins recognized by the serum antibodies of experimentally and naturally infected sheep with B. ovis. Additional studies on the purification of these proteins and on subsequently their utilization in a serodiagnostic method are required to improve the serological diagnosis of ovine babesiosis.

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.

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.

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.