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Öğe Babesia ovis infections: Detailed clinical and laboratory observations in the pre- and post-treatment periods of 97 field cases(ELSEVIER SCIENCE BV, 2013) Sevinc, Ferda; Sevinc, Mutlu; Ekici, Ozlem Derinbay; Yildiz, Ramazan; Isik, Nermin; Aydogdu, UgurOvine 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. (C) 2012 Elsevier B.V. All rights reserved.Öğe Determination of immunoreactive proteins of Babesia ovis(ELSEVIER, 2013) Sevinc, Ferda; Guler, Leyla; Sevinc, Mutlu; Ekici, Ozlem Derinbay; Isik, NerminBabesia 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. (C) 2013 Elsevier B.V. All rights reserved.Öğe The effect of 12 successive blood passages on the virulence of Babesia ovis in splenectomized lambs: A preliminary study(ELSEVIER, 2014) Sevinc, Ferda; Sevinc, Mutlu; Koc, Yilmaz; Alkan, Fahrettin; Ekici, Ozlem Derinbay; Yildiz, Ramazan; Isik, NerminBabesia ovis, the primary causative agent of ovine babesiosis, is a tick-borne hemoprotozoan parasite and causes serious losses in small ruminant livestock in tropical and subtropical regions. The current control of ovine babesiosis has been base on chemotherapy and limited tick control measures. Attenuated parasites which are valuable sources of live vaccines have been used to prevent babesiosis in cattle. There is a necessity to the studies about prevention of the disease by means of immunoprophylaxis in sheep. For this purpose, this study was planned to obtain the attenuated derivatives of a virulent B. ovis strain by successive blood passages in splenectomized lambs. A total of seventeen, six-month-old lambs, 13 of which splenectomized, 4 of which spleen intact lambs, known to be negative for the presence of hemoparasites and anti-B. ovis antibodies were used. The splenectomized lambs were used for passaging the virulent B. ovis field strain. Four intact lambs divided into two groups were used to evaluate the pathogenicity of the virulent strain and twelfth passage strain of B. ovis. Blood collected from a clinically evident case of babesiosis in the field was inoculated intravenously into the first splenectomized lamb, and then the acute clinical disease was occurred. The same process was successively applied to the rest of 12 splenectomized lambs to have the attenuated B. ovis strain. The clinical, microscopic and hematological changes after inoculation were recorded in each passage of the lambs. All of the passage lambs were exhibited the severe symptoms of the disease from the 2nd day of inoculation. The same treatment protocol including vitamin B12 and the anti-babesial drug imidocarb dipropionate (IMDP) was applied to all splenectomized lambs on the fourth or fifth day of the inoculation. Although the symptoms of the disease were less severe than those of splenectomized lambs; two intact lambs infected with the virulent strain and two intact lambs infected with the twelfth passage blood were also showed the clinical and hematological findings of the disease on the 2nd and 3rd day after inoculation. Infection-related symptoms in all intact lambs disappeared spontaneously from the 6th day of inoculation without the application of any drug. The results of the study showed that there was no change on the virulence of B. ovis through the 12 successive blood passages. Therefore, the further passages or alternative attenuation methods may be necessary to attenuate the virulent strain of B. ovis. (C) 2013 Elsevier B.V. All rights reserved.Öğe Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey(JAPAN SOC VET SCI, 2016) Zhou, Mo; Cao, Shinuo; Sevinc, Ferda; Sevinc, Mutlu; Ceylan, Onur; Liu, Mingming; Wang, GuanboConsidering 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.Öğe Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay(AMER SOC MICROBIOLOGY, 2015) Sevinc, Ferda; Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, OnurIn 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 DH5 alpha 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.Öğe Molecular detection and characterization of Hepatozoon spp. in dogs from the Central part of Turkey(ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2015) Aydin, Mehmet Fatih; Sevinc, Ferda; Sevinc, MutluCanine 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 666 bp 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. (C) 2015 Elsevier GmbH. All rights reserved.Öğe Molecular detection and genetic characterization of Babesia, Theileria and Anaplasma amongst apparently healthy sheep and goats in the central region of Turkey(ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2017) Zhou, Mo; Cao, Shinuo; Sevinc, Ferda; Sevinc, Mutlu; Ceylan, Onur; Ekici, Sepil; Jirapattharasate, CharoonlukBabesia 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 ofA. 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 Glade 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 ofA 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. (C) 2016 Elsevier GmbH. All rights reserved.Öğe Molecular detection and genetic identification of Babesia bigemina, Theileria annulata, Theileria orientalis and Anaplasma marginale in Turkey(ELSEVIER GMBH, 2016) Zhou, Mo; Cao, Shinuo; Sevinc, Ferda; Sevinc, Mutlu; Ceylan, Onur; Moumouni, Paul Franck Adjou; Jirapattharasate, CharoonlukBabesia 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 la (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. (C) 2015 Elsevier GmbH. All rights reserved.Öğe A new immunoreactive recombinant protein designated as rBoSA2 from Babesia ovis: Its molecular characterization, subcellular localization and antibody recognition by infected sheep(ELSEVIER SCIENCE BV, 2015) Sevinc, Ferda; Cao, Shinuo; Zhou, Mo; Sevinc, Mutlu; Ceylan, Onur; Xuan, XuenanOvine 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 DH5 alpha 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. (C) 2015 Elsevier B.V. All rights reserved.Öğe A PCR survey of vector-borne pathogens in different dog populations from Turkey(SPRINGER INTERNATIONAL PUBLISHING AG, 2017) Guo, Huanping; Sevinc, Ferda; Ceylan, Onur; Sevinc, Mutlu; Ince, Ege; Gao, Yang; Moumouni, Paul Franck AdjouIn 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.Öğe Therapeutic and prophylactic efficacy of imidocarb dipropionate on experimental Babesia ovis infection of lambs(ELSEVIER SCIENCE BV, 2007) Sevinc, Ferda; Turgut, Kursat; Sevinc, Mutlu; Ekici, Ozlem Derinbay; Coskun, Alparslan; Koc, Yilmaz; Erol, MuharremThe objective of this study was to evaluate the therapeutic and prophylactic efficacy of imidocarb dipropionate (IMDP) against babesiosis and to determine specific antibodies against Babesia ovis in experimentally infected lambs. Thirty-six 6-month-old splenectomized lambs were used. The lambs were randomly divided into six groups with six animals each, and were intravenously inoculated with 50 mL B. ovis-infected erythrocytes as follows: group I (therapy group) was treated with IMDP (1.2 mg/kg body weight) starting on the day of onset of clinical signs of babesiosis after the inoculation; group II (untreated control animals) was not treated with any therapeutic treatment after the inoculation; groups III, IV, V and VI (prophylaxis groups) were administered IMDP (2.4 mg/kg body weight) 1, 2, 3 and 4 weeks before the inoculation, respectively. The animals were housed in a tick-proof room with water and food ad libitum up to the 30th day post-inoculation (PI). The lambs were monitored from the first day PI by recording the manifestation of clinical disease, rectal temperature, and the degree of parasitaemia. All the lambs became infected with B. ovis, except five animals from group III, which were treated I week prior to experimental infection. Other animals showed signs of acute clinical babesiosis. The animals treated with IMDP (group I) were able to clear the parasite from the blood circulation after 48 h post-treatment. The recrudescence of B. ovis was observed in two lambs 7 days after treatment, and they were treated with the second similar dose of the drug. Six lambs (1, 1, 2 and 2 lambs in group III, IV, V and VI, respectively) from the prophylaxis groups died within 7-17 days after showing high parasitaemia and clinical symptoms of the disease. Regardless of the clinical symptoms, 83.30% and 66.66% of the lambs which were administered IMDP 1-2 and 3-4 weeks before, were determined to be protected against the virulent field strain of B. ovis. (C) 2007 Elsevier B.V. All rights reserved.
