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Öğe Afrika Kıtasına Seyahat Edenlere Bulaşabilecek Paraziter Hastalıklar(2018) Karakavuk, Muhammet; Aykur, Mehmet; Unver, Aysegul; Döşkaya, MertHastalıkların yayılmasında seyahat önemli bir yer tutmakta olup seyahat edenlerin sayısı her geçen gün artmaktadır. Buna bağlı olarak seyahat sırasında ya da sonrasında meydana gelen hastalıkların önemi artmaktadır. Paraziter hastalıklar özellikle az gelişmiş ülkelerde epidemik veya endemik olup yüksek sayıda ölümlere neden olmaktadır. Gelişmiş ülkelerden az gelişmiş ülkelere seyahat eden insanlarda seyahat sırasında paraziter hastalıkların bulaşma riski daha yüksektir. Dünya nüfusunun %15’i Afrika kıtasında yaşamaktadır. Kıta coğrafik, ekonomik ve gelişmişlik açısından Doğu Afrika, Güney Afrika, Kuzey Afrika ve Batı Afrika olmak üzere dört bölgeye ayrılmaktadır. Son yıllarda Afrika kıtasına yapılan uluslararası seyahatler artarak devam etmektedir. Bu seyahatler sırasında sıtma, schistosomiasis, trypanosomiasis (Afrika Uyku Hastalığı), onchocerciasis, lenfatik filariasis ve leishmaniasis gibi paraziter hastalıkların bulaşma riski bulunmaktadır. Afrika kıtasındaki ülkelere seyahat etmeden önce bölgede bulunan hastalıklara karşı önlem almak hayati önem arz etmektedirÖğe Amerika Kıtasına Seyahat Edenlerde Risk Oluşturabilecek Paraziter Enfeksiyonlar ve Alınacak Önlemler(2018) Aykur, Mehmet; Karakavuk, Muhammet; Unver, Aysegul; Dağcı, HandeSon 10 yılda uluslararası seyahat eden kişi sayısı giderek artmaya devam etmektedir. Çeşitli amaçlarla yapılan bu seyahatlerle birlikte paraziter hastalıkların bulaşma riski de artmaktadır. Risk olturan enfeksiyonlardan biri sıtma olup, bulaşabilecek türler Plasmodium vivax ve Plasmodium falciparum türleridir. Leishmaniasis vakalarının dağılımı ABD’nin güneyinden Arjantin’in kuzeyine kadar olan bölgelerde bildirilmiştir. Yılda ortalama 57.000 kutanöz ve mukokutanöz leishmaniasis vakasına rastlanırken, yaklaşık 4.000 visseral leishmaniasis vakası görülmektedir. Chagas hastalığı, 21 ülkede endemik ve her yıl yaklaşık altı milyon insanın etkilendiği bildirilmektedir. Bu kıtada 25 milyon insan schistosomiasis riski altında olup, bunların %90’ı Brezilya’da yaşamaktadır. Dünya Sağlık Örgütüne (DSÖ) göre Ekvator, Kolombiya, Brezilya, Guatemala, Meksika ve Venezuela’da seyahat edenlerin onchocerciasis ile bunun yanı sıra yaklaşık olarak 12,6 milyon insan lenfatik filariasis (%80’i Haiti’de) enfeksiyonuyla karşılaşma riski bulunduğu bildirilmektedir. Bu bölgelere yapılan seyahatlerde gerekli önlemler alınmadığı ve uygun profilkatik ilaçlar uygulanmadığı durumlarada önemli mortalite ve morbiteler görülebilmektedir.Öğe Aşı adayı toxoplasma gondii GRA8 proteininin oluşturduğu immun yanıt ve korunmanın araştırılması(Ege Üniversitesi, Sağlık Bilimleri Enstitüsü, 2019) Karakavuk, Muhammet; Döşkaya, MertToxoplasma gondii insan dâhil tüm sıcakkanlı hayvanları enfekte edebilen, insan ve hayvanlarda ciddi klinik tablolara yol açan zoonotik bir protozoondur. Dünya nüfusunun ortalama %30‘u T. gondii ile enfektedir. Toksoplazmozis çiftlik hayvanlarında da büyük ekonomik kayıplara neden olmaktadır. Bu yüzden T. gondii‘ye karşı aşı geliştirmek oldukça önemlidir. Bu amaçla daha önceki çalışmamızda kuvvetli immun yanıt uyaran GRA8 geni öncelikle biyoinformatik olarak incelenmiş, proteinin hücre içi ve transmembran kısımları atılıp, sinyal peptidi eklenmiştir. Protein pcDNA.3.3 vektörü içine klonlanarak sadece GRA8 plazmid içeren ve CpG+Escort ile takviye edilmiş GRA8 plazmid DNA aşıları geliştirilmiştir. Daha sonra Swiss Webster fareler üç hafta ara ile iki kez SC olarak aşılanmış ve uyarılan sıvısal immun yanıt Western Blot ve ELISA ile hücresel immun ise flow sitometri ve MTT testleri ile belirlenmiştir. DNA aşılarının koruyuculuğu, 7-8 tane T. gondii PRU suşuna ait doku kistlerinin oral yoldan uygulanması ile tespit edilmiştir. Dokularda oluşan kistler faz kontrast mikroskobu ile sayılmıştır. Elde edilen sonuçlara göre, aşılama sonrası sadece GRA8 plazmid ve Cpg+Escort+GRA8 plazmid içeren aşı grupları kontrollere göre kuvvetli IgG yanıtı uyarmıştır (P<0,001). IgG1 ve IgG2a yanıtı incelendiğinde Th1-Th2 kutuplaşmasının dengeli olduğu tespit edilmiştir. Aşı gruplarında, IFN-γ salgılayan CD4 T lenfositleri ile CD8 T lenfosit oranlarının kontrollere göre sırasıyla 1,31-1,75 ve 1,17-1,70 kat arasında arttığı saptanmıştır. Stimülasyon indeksleri aşı gruplarında kontrol gruplarına göre %7-%34 arasında yüksek olduğu tespit edilmiştir. GRA8 plazmid aşısı uygulanan farelerde doku kisti sayısı 440,74±343,35 olup PBS ve Cpg+Escort+Boş plazmid uygulanan farelere göre belirgin olarak azalmıştır (P<0,0001, ****). Cpg+Escort+GRA8 plazmid aşısı uygulanan farelerde ise doku kisti sayısı ise 804,76±715,87 olup PBS (P=0,0086, **) ve Cpg+Escort+Boş plazmid (P=0,0007, ***) uygulanan farelere göre belirgin şekilde düşmüştür. GRA8 plazmid aşı grubunda oluşan doku kisti sayısı Cpg+Escort+GRA8 plazmid grubuna göre belirgin bir şekilde azalmıştır (P<0,05, *). Sonuç olarak bu çalışmada protein mikroarray taraması ile seçilen aşı adayı GRA8 antijeninden geliştirilen DNA aşılarının kuvvetli sıvısal ve hücresel immun yanıtı uyarabildiği ve toksoplazmozise karşı kısmi korunma sağladığı tespit edilmiştir. Ayrıca ileride yapılacak çalışmalarda GRA8 antijeni ve diğer T. gondii antijenlerinin kullanıldığı multi-antijenik DNA aşılarının geliştirilmesiyle toksoplazmozise karşı tam bir korunmanın olasılığı öngörülmektedir.Öğe Comparison of an in house and a commercial real-time polymerase chain reaction targeting Toxoplasma gondii RE gene using various samples collected from patients in Turkey(Bmc, 2019) Doskaya, Mert; Pullukcu, Husnu; Karakavuk, Muhammet; Sahar, Esra Atalay; Tasbakan, Mehmet Sezai; Tasbakan, Meltem Isikgoz; Guruz, Adnan YukselBackground: Toxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. in this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions. Methods: A total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples. Results: the compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content. Conclusions: Both the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. in house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.Öğe Construction of a multiepitope vaccine candidate against Fasciola hepatica: an in silico design using various immunogenic excretory/secretory antigens(Taylor & Francis Ltd, 2022) Akil, Mesut; Aykur, Mehmet; Karakavuk, Muhammet; Can, Huseyin; Doskaya, MertBackground Fasciola hepatica is an important pathogen that causes liver fluke disease in definitive hosts such as livestock animals and humans. Various excretory/secretory products have been used in serological diagnosis and vaccination studies targeting fasciolosis. There are no commercial vaccines against fasciolosis yet. Bioinformatic analysis based on computational methods have lower cost and provide faster output compared to conventional vaccine antigen discovery techniques. The aim of this study was to predict B- and T-cell specific epitopes of four excretory/secretory antigens (Kunitz-type serine protease inhibitor, cathepsin L1, helminth defense molecule, and glutathione S-transferase) of Fasciola hepatica and to construct a multiepitope vaccine candidate against fasciolosis. Methods and Results Initially, nonallergic and the highest antigenic B- and T- cell epitopes were selected and then, physico-chemical parameters, secondary and tertiary structures of designed multiepitope vaccine candidate were predicted. Tertiary structure was refined and validated using online bioinformatic tools. Linear and discontinuous B-cell epitopes and disulfide bonds were determined. Finally, molecular docking analysis for MHC-I and MHC-II receptors was performed. Conclusion This multi-epitope vaccine candidate antigen, with high immunological properties, can be considered as a promising vaccine candidate for animal experiments and wet lab studies.Öğe Cryptosporidium spp. during chemotherapy: a cross-sectional study of 94 patients with malignant solid tumor(K Faisal Spec Hosp Res Centre, 2021) Karabey, Mehmet; Can, Huseyin; Oner, Tulay Oncu; Doskaya, Mert; Alak, Sedef Erkunt; Doskaya, Aysu Degirmenci; Karakavuk, MuhammetBACKGROUND: Cryptosporidium spp. is a protozoan parasite that infects many vertebrate animals, including humans. Since Cryptosporidium spp. can cause chronic life-threatening diarrhea and severe malabsorption in immunocompromised patients, we investigated the prevalence of this parasite among patients undergoing chemotherapy for malignant solid tumors. OBJECTIVE: Investigate the prevalence of Cryptosporidium spp. in stool samples. DESIGN: Cross-sectional. SETTING: Tertiary care. PATIENTS AND METHODS: Stool samples were collected from adult patients with malignant solid tumors receiving chemotherapy and diarrhea. Cryptosporidium spp. prevalence was determined using Ziehl-Neelsen staining, ELISA, and real-time PCR targeting of the COWP gene. MAIN OUTCOME MEASURE: The prevalence of Cryptosporidium spp. in patients undergoing chemotherapy for malignant solid tumors. SAMPLE SIZE: 94 RESULTS: The prevalence was 2.1% (2/94), 5.3% (5/94), and 5.3% (5/94) as detected by Ziehl-Neelsen staining, real-time PCR and ELISA, respectively. The prevalence reached 8.5% (8/94) using all results obtained from the three methods. Among eight positive stool samples, four were positive by at least two different methods (Ziehl-Neelsen staining-ELISA or ELISA-real-time PCR) whereas the remaining four were positive by either ELISA or real-time PCR. CONCLUSION: These findings show the risk of cryptosporidiosis in cancer patients and the necessity to use at least two diagnostic methods during the diagnosis of cryptosporidiosis to reach more accurate and trustworthy results. LIMITATIONS: Further studies with a larger sample size are recommended. CONFLICT OF INTEREST: None.Öğe Detection of Echinococcus granulosus and Echinococcus multilocularis in Cyst Samples Using a Novel Single Tube Multiplex Real-Time Polymerase Chain Reaction(Ankara Microbiology Soc, 2016) Can, Huseyin; Inceboz, Tonay; Caner, Ayse; Atalay Sahar, Esra; Karakavuk, Muhammet; Doskaya, Mert; Celebi, Fehmi; Degirmenci Doskaya, Aysu; Gulce Iz, Sultan; Guruz, Yuksel; Korkmaz, MetinCystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RTPCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes;. Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTIGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specificity by distilled water at 10(6)-10(5)-10(4)-10(3)-10(2)-10(1)-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/mu l reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specificity of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specificity of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specific, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples.Öğe Detection of Toxoplasma gondii in a Eurasian Badger (Meles meles) Living in Wildlife Areas of Izmir, Turkey(2018) Karakavuk, Muhammet; Aldemir, Duygu; Şahar, Esra Atalay; Can, Hüseyin; Özdemir, Hüseyin Gökhan; Döşkaya, Aysu Değirmenci; Döşkaya, MertToxoplasma gondii is an obligatory intracellular protozoon parasite that causes toxoplasmosis in humans and all warm-blooded animals. in this study, we aimed to investigate the presence of T. gondii DNA in a Eurasian badger (Meles meles) that was found dead in the wildlife area of Izmir. According to the results of real time polymerase chain reaction, T. gondii REP gene was found to be positive in the Eurasian badger brain homogenate. in conclusion, Eurasian badger, a known carnivore, can be a potential source of toxoplasmosis in the natural settings of İzmir, Turkey.Öğe Determining the analytical sensitivity of polymerase chain reaction targeting Ehrlichia spp. disulfide oxidoreductase gene: Molecular diagnosis of ehrlichiosis in a dog clinically suspected with leishmaniasis(2022) Karakavuk, Muhammet; Aykur, Mehmet; Can, Hüseyin; Döşkaya, Aysu Değirmenci; Dağcı, Hande; Gürüz, Adnan Yüksel; Döşkaya, MertEhrlichia spp. is tick-borne zoonotic pathogen that can infect humans and animals. Nowadays, among the tests used in the diagnosis of ehrlichiosis, the importance of molecular methods is increasing steadily due to their high sensitivity and specificity. The aim of this study was to determine the analytical sensitivity of a conventional polymerase chain reaction (PCR) targeting Ehrlichia spp. disulfide oxidoreductase (DSB) gene. Ehrlichia spp. DSB gene was cloned into the TOPO vector. After TOPO plasmid containing DSB gene were serially diluted, PCR targeting the Ehrlichia spp. DSB gene was performed. While working on this research, blood and skin scraping samples of a stray dog clinically suspected with leishmaniasis as well as treated for leishmaniasis arrived to our laboratory. Thereafter, PCRs targeting Ehrlichia spp. DSB and 16S rRNA and Leishmania kinetoplast DNA (kDNA) genes were performed to identify the pathogen in blood and skin scraping samples of the stray dog. The analytical sensitivity of the PCR assay targeting Ehrlichia spp. DSB gene was 1 ? copy plasmid/reaction using serially diluted TOPO plasmid containing DSB gene. PCR targeting the Ehrlichia spp. DSB gene was positive and PCR targeting Leishmania spp. kDNA was negative in blood and skin samples of the stray dog clinically suspected with leishmaniasis. Using nested PCR targeting Ehrlichia spp. 16S rRNA, E. canis was identified in blood and skin scraping samples of the stray dog. In this study, PCR targeting Ehrlichia spp. DSB gene has been shown to have high sensitivity. Also it was shown molecular methods can help clinicians in differential diagnosis of ehrlichiosis and leishmaniasis to prevent inappropriate treatment.Öğe Development of a new serotyping ELISA for Toxoplasma gondii type II, type III and Africa 1 lineages using in silico peptide discovery methods, well categorized feline and human outbreak serum samples(Bmc, 2022) Can, Huseyin; Aksoy Gokmen, Aysegul; Doskaya, Mert; Erkunt Alak, Sedef; Degirmenci Doskaya, Aysu; Karakavuk, Muhammet; Koseoglu, Ahmet EfeBackground Discovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis. In Turkey, genotypes belonging to type II, type III and Africa 1 lineages were mainly detected. The present study focused on to find out epitopes with high discriminative capacity to serotype these genotypes using well characterized strains isolated from Turkey. Methods To meet this objective, GRA6 and GRA7 genes were sequenced from strains belonging to the type II, III and Africa 1 lineages, and B cell epitopes inside these sequences were predicted by Bcepred and additional docking analysis was performed with B cell receptor. Based on these analyses, 22 peptides harboring lineage specific epitopes were synthesized. Then, the serotyping potency of these peptides was tested using peptide ELISA and well categorized serum samples collected from stray cats infected with genotypes of the different lineages type II (n:9), III (n:1) and Africa 1 (n:1). As a result of peptide-ELISA, a serotyping schema was constructed with peptides that show high discriminative capacity and this assay was validated by sera collected from humans after an outbreak (n:30) and mother/newborn pair sera (n:3). Later, the validated serotyping schema was used to serotype a larger group of human (n:38) and cat (n:24) sera. Results Among 22 peptides, GRA6II/c, GRA7III/d, and GRA6 Africa 1/b epitopes have shown discriminative capacity. During the validation of peptide-ELISA, the serotype of toxoplasmosis outbreak and mother/newborn cases were detected to be serotype II. Moreover, the analyses in a larger group showed that serotype II was prevalent in humans and stray cats. Conclusions Overall, the results showed that the serotyping schema could be successfully used to serotype T. gondii infections caused by type II, III and Africa 1 genotype.Öğe Development of a novel recombinant ELISA for the detection of Crimean-Congo hemorrhagic fever virus IgG antibodies(Nature Research, 2021) Guelce-Iz, Sultan; Elaldi, Nazif; Can, Hueseyin; Sahar, Esra Atalay; Karakavuk, Muhammet; Gul, Aytul; Felgner, Philip LouisCrimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV). Serological screening of CCHF is important and current ELISA use antigens prepared from virus which is expensive due to requirement of high bio-containment facilities. in this study, we aimed to develop a new recombinant ELISA. For this purpose, CCHFV genome were expressed as 13 proteins in E. coli and among them abundantly purified recombinant Nucleocapsid protein (rNP) and Mucin-like variable domain (rMLD) were used as antigen in ELISA (Rec-ELISA). Rec-ELISA using rNP, rMLD and a combination of both (rNP/rMLD) were probed with acute (n=64; collected between days 1 and 7 after onset of symptoms), convalescent (n=35; collected 8 days after onset of symptoms), consecutive sera (n=25) of confirmed CCHF cases and control sera (n=43). The sensitivity and specificity of Rec-ELISA using rNP/rMLD were 73% and 98% in acute cases and 97% and 98% in convalescent cases. The median interquartile absorbance value to discriminate the acute and convalescent phases of CCHF was significantly higher with ELISA using rNP/rMLD (P<0.0001) compared to rNP (P>0.05) and rMLD (P=0.001). These results indicate that the Rec-ELISA using rNP/rMLD may be very useful to diagnose convalescent CCHF cases especially in field studies.Öğe Development of a Rapid-Crypto Colorimetric LAMP Test to Detect Cryptosporidiosis in Feces of Newborns Calves(Springer Int Publ Ag, 2024) Karakavuk, Muhammet; Can, Huseyin; Can, Sengul; Karakavuk, Tugba; Doskaya, Mert; Doskaya, Aysu DegirmenciBackgroundCryptosporidiosis is a disease that causes major intestinal damage in humans and animals. The causative agents of the disease are Cryptosporidium species. In newborn calves, diarrhea can lead to death, resulting in significant economic losses for the farms. Therefore, accurate, rapid, and cost-effective diagnosis of the disease is very important.Material and methodsIn this study, a novel colorimetric loop-mediated isothermal amplification (LAMP) test named Rapid-Crypto Colorimetric LAMP test targeting Cryptosporidium spp. 18S rRNA gene was developed to detect cryptosporidiosis in the feces of newborn calves. The analytical sensitivity of the test was determined by plasmid controls. Clinical sensitivity was determined using the feces of 127 calves collected from farms in Izmir and Manisa provinces. All of the samples were also investigated with Real-Time PCR targeting the Cryptosporidium spp. COWP gene. Cross-reactivity was tested using the DNA of other parasites and bacteria.ResultsAccording to the results, the analytical sensitivity of the Rapid-Crypto Colorimetric LAMP test was found as 1 copy plasmid/reaction. When the results were compared with the Real-Time PCR test, the sensitivity of the Rapid-Crypto Colorimetric LAMP test was 100% and the specificity was 97.4%. The test did not cross-react with other parasites and bacteria. ConclusionThe Rapid-Crypto Colorimetric LAMP test developed in this study provides an advantage in the diagnosis of Cryptosporidium spp. in calf stool samples since it can be applied in basic laboratories or in the field, does not require experienced personnel, and has high sensitivity. Moreover, diagnosis can be made with the naked eye without using any device.Öğe Development of multistage recombinant protein vaccine formulations against toxoplasmosis using a new chitosan and porin based adjuvant system(Elsevier, 2022) Parmaksiz, Selin; Gul, Aytul; Alak, Sedef Erkunt; Karakavuk, Muhammet; Can, Huseyin; Gul, Ceren; Karakavuk, TugbaToxoplasmosis is a global health problem affecting both human and animal populations. The lack of effective treatment makes the development of a vaccine against toxoplasmosis one of the main goals in the management of this disease. In our study, vaccine formulations containing the multistage recombinant antigens, rBAG1 + rGRA1 were developed with a combined adjuvant system consisting of chitosan and Salmonella Typhi porins in micro (MicroAS) and nanoparticulate (NanoAS) forms. BALB/c mice were immunized intraperitoneally with vaccine formulations two times at three-week intervals. Three weeks after the second vaccination, mice were challenged with 7-8 live tissue cysts of the virulent T. gondii PRU strain by oral gavage. Higher cellular uptake by macro-phages and enhanced cellular (IFN-gamma and I-4 in stimulated spleen cells) and humoral (IgG, IgG1, IgG2a) responses were obtained with the adjuvanted formulation, higher with microsystem when compared to that of nanosystem. Microsystem was found to stimulate Th1-polarized immune responses, whereas non-adjuvanted antigens stim-ulated Th2-polarized immune response. The highest survival rate and reduction in cysts numbers and T. gondii DNA were obtained with the adjuvanted antigens. Our study showed that adjuvanted multistage recombinant vaccine systems increase the immune response with strong protection against T. gondii, more profoundly in microparticulate form.Öğe Ege Üniversitesi Tıp Fakültesi Hastanesinde Toksoplazmozis Sıklığının Real-Time PZR ile Belirlenmesi(2022) Karakavuk, Muhammet; Can, Hüseyin; Döşkaya, Aysu Değirmenci; Gürüz, Adnan Yüksel; Döşkaya, MertToxoplasma gondii insan ve sıcakkanlı hayvanları enfekte edebilen zoonotik protozoon bir parazittir. Bu çalışmada çeşitli hasta gruplarından alınmış klinik örneklerde toksoplazmozis sıklığının araştırılması amaçlanmıştır. Çalışma kapsamında 2009-2019 yılları arasında Ege Üniversitesi Tıp Fakültesi Parazitoloji Anabilim Dalı Moleküler Parazitoloji laboratuvarına gönderilen ağırlıklı olarak amniyon sıvısı, kan ve beyin omurilik sıvısı örneklerinin dahil olduğu toplam 535 klinik örnek değerlendirilmiştir. Bu örneklerde DNA izolasyonu sonrası T. gondii RE geni varlığı Real-Time PZR ile araştırılmıştır. Belirtilen zaman aralığında toksoplazmozis sıklığı %2,61 (14/535) olarak tespit edilmiştir. Hastalığın direkt etkilediği merkezi organlardan alınan örneklerde pozitiflik oranı %5,40 (8/148) iken periferden alınan örneklerde pozitiflik oranı %1,74 (5/286) olarak tespit edilmiş ve bu fark istatistiksel olarak anlamlı bulunmuştur (P0,05). Elde edilen bulgular toksoplazmozis tanısında Real-Time PZR yönteminin oldukça önemli bir yeri olduğunu göstermektedir. Ayrıca, örnek alım yöntemlerinin hastalığın tanısında oldukça önemli olduğu sonucuna varılmıştır.Öğe First time identification of Acanthamoeba genotypes in the cornea samples of wild birds; Is Acanthamoeba keratitis making the predatory birds a target?(Academic Press Inc Elsevier Science, 2017) Karakavuk, Muhammet; Aykur, Mehmet; Sahar, Esra Atalay; Karakus, Mehmet; Aldemir, Duygu; Donduren, Omer; Ozdemir, Huseyin Gokhan; Can, Huseyin; Guruz, Adnan Yuksel; Dagci, Hande; Doskaya, MertAcanthamoeba is a free-living amoeba which can be isolated from environment and among others well known as an opportunist protozoan parasite causing infections in humans and animals. Eyes are extremely important for the wild birds and losing sight ability due to Acanthamoeba can be dangerous. The studies on Acanthamoeba infection in wild birds is very few in world and Turkey therefore we aimed to screen deceased wild birds found in Izmir and Manisa provinces located in western Turkey using PCR and non-nutrition agar (NNA) plate method. Cornea samples were obtained from 18 deceased wild birds. During the external examination, signs of keratitis were observed in two Eurasian sparrowhawks (Accipiter nisus). All of the corneal samples were analyzed by two PCR methods and NNA plate. According to results, the Acanthamoeba positivity in corneal samples was 16.6% and 5.5% by PCR and plate method, respectively. According to sequencing data, two of isolates belonged to genotype T5 and one was genotype T4. In conclusion, Acanthamoeba infection was detected in wild bird cornea samples with/without keratitis for the first time in the world. The result of this study also show that Acanthamoeba can be a cause of keratitis in wild birds of Turkey and thus these predator birds can be a target of other wild animals due to loss of sight ability. In terms of public health, these results show the importance of wild birds as a source of Acanthamoeba infection in nature. (C) 2017 Elsevier Inc. All rights reserved.Öğe Genetic characterization of Toxoplasma gondii strains isolated from humans living in •Izmir, Türkiye(Elsevier, 2024) Karakavuk, Muhammet; Can, Huseyin; Celtik, Aygul; Karakavuk, Tugba; Gul, Ceren; Erdem, Huseyin Aytac; Pullukcu, HusnuPurpose: Toxoplasma gondii is an obligate intracellular zoonotic parasite that can infect all warm-blooded animals, including humans. Currently, clinical findings of toxoplasmosis are being related to T. gondii strains such as Type I genotype may cause high pathogenicity and Type II genotype causes a milder clinical presentation. We have showed in our previous that Type II genotype is the most frequent strain detected in stray cats and wild birds living in natural life of I(center dot)zmir. The aim of this study was to assess toxoplasmosis seroprevalence in immunocompromised patients, investigate the presence of T. gondii DNA in their blood samples, and genotype the PCR positive ones. Methods: The 42 buffy-coat and serum samples were collected from immunocompromised patients who were from various clinics. Thereafter, Real-Time PCR targeting RE gene of T. gondii was performed with DNA samples obtained from buffy-coat samples. Genotyping was performed by sequencing of GRA6 and GRA7 gene regions of positive DNA samples obtained from tissues of bioassay and PCR positive samples. Results: According to Real-Time PCR results, T. gondii DNA was detected in 23.8% (10/42) samples. Among these 10 samples, two samples were determined as T. gondii Type II genotype. Anti-Toxoplasma IgG antibodies were detected in 28.57% (12/42) samples. Conclusions: Overall, the detection of Type II genotype in humans in I(center dot)zmir province suggested that T. gondii infection in humans, stray cats, and wild animals may be associated to each other in terms of transmission.Öğe Genotyping of Enterocytozoon bieneusi isolates detected in stray cats of Izmir, Turkiye(Springer, 2023) Sügeç, Ecem; Güvendi, Mervenur; Karakavuk, Muhammet; Alak, Sedef Erkunt; Doskaya, Aysu Degirmenci; Un, Cemal; Doskaya, MertThe phylum Microsporidia includes obligate intracellular parasites that can infect humans and various animals. To date, 17 different species within the phylum have been reported to infect humans. Among them, Enterocytozoon bieneusi (E. bieneusi) is one of the most frequently detected species in humans. Identification of E. bieneusi as well as its genotypes in humans and animals is important to reveal their role in transmission to each other. Cats are blamed as the source of E. bieneusi transmission to humans. In this study, we aimed to genotype 170 E. bieneusi positive samples isolated from stool of stray cats living in Izmir province of Turkiye. According to the results, 47 samples were amplified by nested PCR protocol targeting ITS region and successfully sequenced. The phylogenetic analysis showed the presence of zoonotic genotype D and type IV in stray cats, which are also frequently detected in humans. Among the E. bieneusi genotypes detected, the prevalence of type IV (93.6%; 44/47) was very high compared to genotype D. Overall, the identification of zoonotic genotypes of E. bieneusi supports that stray cats can play an important role in the transmission of E. bieneusi to humans in Izmir.Öğe Genotyping ofPneumocystis jiroveciiisolates obtained from clinical samples by multilocus sequencing: a molecular epidemiology study conducted in Turkey(Springer, 2020) Surgec, Ecem; Can, Huseyin; Doskaya, Mert; Karakavuk, Muhammet; Sahar, Esra Atalay; Doskaya, Aysu Degirmenci; Demir, SamiyePneumocystis jiroveciiis an opportunistic respiratory pathogen causing Pneumocystis pneumonia (PcP) in immunocompromised patients. the aim of this study was to investigate the genetic diversity ofP. jiroveciiisolates (n: 84) obtained from PcP patients using multilocus sequencing method based on mt26S, SOD, and CYB loci. Among the 84 clinical samples that were positive forP. jiroveciiDNA, 31 (36.90%) of them were genotyped using at least one locus. of the 31 clinical samples, 26 of them were successfully genotyped using all loci whereas three samples were genotyped using either mt26S/CYB loci or mt26S/SOD loci. Additionally, there were two more clinical samples that were genotyped using CYB or SOD locus. Using mt26S locus, genotypes 2, 3, 7, and 8 were detected. Frequencies of genotype 7 and 8 were higher and both of them were found in 11 (n: 29; 37.93%) clinical samples. Using SOD locus, SOD 1, 2, and 4 genotypes were detected. SOD 1 was the predominant genotype (20/28; 71.42%). During the analyses of CYB locus, CYB 1, 2, 5, 6, and 7 as well as a new CYB genotype were detected. CYB 1 (16/29; 55.17%) and 2 (10/29; 34.48%) were the predominant genotypes. Overall, according to the multilocus sequencing results E, F, M, N, P, and V multilocus genotypes were detected among the PcP patients. in addition, SOD 1 was the predominant genotype and CYB had a more polymorphic locus.Öğe GRA8 DNA vaccine formulations protect against chronic toxoplasmosis(Academic Press Ltd- Elsevier Science Ltd, 2021) Karakavuk, Muhammet; Can, Huseyin; Gul, Aytul; Doskaya, Aysu Degirmenci; Alak, Sedef Erkunt; Un, Cemal; Guruz, Adnan YukselToxoplasma gondii has a very wide host range and infects all warm-blooded animals including humans. The disease causes great economic losses both in animals and humans. Vaccination is the most effective approach to fight against toxoplasmosis however an effective vaccine has not been developed yet. In the present study, GRA8 protein of T. gondii that showed high immunogenicity in our previous microarray screening study was used to develop a DNA vaccine using pcDNA 3.3 vector for the first time. In order to increase the potency of the DNA vaccine, 10 times lower amount of GRA8 DNA vaccine was combined with molecular adjuvant CpG and formulated into a commercial liposome (pcDNA3.3-GRA8+CpG+Escort). Mice were vaccinated intramuscularly two times at three-week intervals and challenged orally with the T. gondii PRU strain tissue cysts. The humoral immune response was determined by Western Blot and ELISA. The cellular immune response was analyzed by flow cytometry, cytokine ELISA and MTT assay. Among the vaccine groups, pcDNA3.3-GRA8 and pcDNA3.3-GRA8+CpG+Escort induced strong IgG response compared to controls (P < 0.001). The IgG1 and IgG2a responses showed a balanced Th1-Th2 polarization. The ratio of CD4(+) and CD8(+) T lymphocytes secreting IFN-gamma increased, and significantly higher extracellular IFN-gamma secretion was achieved compared to the controls (P < 0.01). The amount of tissue cysts in the group of mice vaccinated with pcDNA3.3-GRA8 decreased significantly compared to control groups (P < 0.0001). In the group vaccinated with pcDNA3.3-GRA8+CpG+Escort, the amount of tissue cysts also decreased significantly compared to PBS (P = 0.0086) and Empty plasmid+CpG+Escort (P = 0.0007) groups. This study showed for the first time that pcDNA 3.3. vector encoding GRA8 with or without CpG and Liposome can induce strong cellular and humoral immune responses and confer strong protection against mouse model of chronic toxoplasmosis.Öğe Immunogenicity and protection efficacy of a COVID-19 DNA vaccine encoding spike protein with D614G mutation and optimization of large-scale DNA vaccine production(Nature Portfolio, 2024) Gul, Aytul; Alak, Sedef Erkunt; Can, Huseyin; Karakavuk, Muhammet; Korukluoglu, Gulay; Altas, Ayse Basak; Gul, CerenSevere acute respiratory syndrome coronavirus 2 had devastating consequences for human health. Despite the introduction of several vaccines, COVID-19 continues to pose a serious health risk due to emerging variants of concern. DNA vaccines gained importance during the pandemic due to their advantages such as induction of both arms of immune response, rapid development, stability, and safety profiles. Here, we report the immunogenicity and protective efficacy of a DNA vaccine encoding spike protein with D614G mutation (named pcoSpikeD614G) and define a large-scale production process. According to the in vitro studies, pcoSpikeD614G expressed abundant spike protein in HEK293T cells. After the administration of pcoSpikeD614G to BALB/c mice through intramuscular (IM) route and intradermal route using an electroporation device (ID + EP), it induced high level of anti-S1 IgG and neutralizing antibodies (P < 0.0001), strong Th1-biased immune response as shown by IgG2a polarization (P < 0.01), increase in IFN-gamma levels (P < 0.01), and increment in the ratio of IFN-gamma secreting CD4+ (3.78-10.19%) and CD8+ (5.24-12.51%) T cells. Challenging K18-hACE2 transgenic mice showed that pcoSpikeD614G administered through IM and ID + EP routes conferred 90-100% protection and there was no sign of pneumonia. Subsequently, pcoSpikeD614G was evaluated as a promising DNA vaccine candidate and scale-up studies were performed. Accordingly, a large-scale production process was described, including a 36 h fermentation process of E. coli DH5 alpha cells containing pcoSpikeD614G resulting in a wet cell weight of 242 g/L and a three-step chromatography for purification of the pcoSpikeD614G DNA vaccine.