Investigation of Anti-Toxoplasma gondii Antibodies in Cats Using Sabin- Feldman Dye Test in Ankara in 2016

Amaç: Toksoplasmozis, zorunlu hücre içi protozoon olan Toxoplasma gondii’nin (T.gondii) etken olduğu, tüm dünyada yaygın görülebilen ve tüm vertebralıları tutabilen multisistemik bir hastalıktır. T. gondii için bilinen tek kesin konak Felidae ailesinin üyeleridir. Çalışmamızda Ankara’da kedilerde Sabin-Feldman Dye Testi (SFDT) ile anti-Toxoplasma gondii antikorlarının tespiti amaçlanmıştır. Aynı bölgede daha önceden yapılan çalışmalar ile karşılaştırmalar yapılarak Toxoplasmozun yayılımı açısından günümüzdeki durumun değerlendirilmesi hedeflenmiştir. Yöntemler: Çalışmamızda kullanılan Toxoplasma Rh suşunun idamesi laboratuvarımızda sağlanmaktadır. T.gondii tanımlanmasında kullanılan SFDT serolojik bir testtir ve altın standart olarak kabul edilmektedir. Çalışmanın materyali Mart 2016 - Ekim 2016 tarihleri arasında Ankara’da kliniklere müracaat eden kedilerden kan örnekleri alınarak sağlanmıştır. Kedilerden alınan kan örnekleri inaktive edilerek SFDT 1/4, 1/16, 1/64, 1/256, 1/1024 titrelerde çalışılmıştır. Bulgular: Toxoplasma gondii araştırması yapılan kedilerin 56’sında (%43,4) SFDT 1/16 titrede, 7’sinde (%5,4) 1/64 titrede, 23’ünde (%17,8) 1/256 titrede pozitif saptanırken, 43’ünde (%33,3) negatif çıkmıştır. Demografik bilgiler ile SFDT sonuçlarının karşılaştırılmasında; pozitif test sonuçlarının cinsiyet ve yaş ile ilişki göstermediği bulunmuştur (Sırasıyla P=0,803 ve P=0,991). Sokak kedilerinde ev kedilerine göre seropozitiflik fazladır (P<0,001). Sadece ticari kuru mama ile beslenen kedilerde test sonuçları negatiftir (P<0,001). Avlanan kedilerde pozitiflik avlanmayanlara göre fazladır (P<0,001). Sonuç: Bu çalışma ile kedilerin %66,6’sında seropozitiflik tespit edilmiştir ki halen oldukça yüksek bir orandır. Sonuçta, avlanan ve doğal beslenen sokak kedilerinde Toxoplasma açısından önlemlerin alınması halk sağlığı için bir zorunluluktur.Objective: Toxoplasmosis, in which obligate intracellular protozoa Toxoplasma gondii (T.gondii) is the causative organism, is a multisystemic disease that can be seen all over the world and can impair all vertebrates. the only hosts known for T.gondii are members of Felidae family. Our study aimed to determine anti-Toxoplasma gondii antibodies with Sabin-Feldman Dye Test (SFDT) in cats in Ankara. It’s aimed to evaluate the current situation in terms of Toxoplasmosis spread by comparing our findings with previous studies in the same region. Methods: Rh strain of Toxoplasma used in our study is maintained in our laboratory. SFDT is still accepted as the gold standard. Material of the study was obtained by taking blood samples from cats who were admitted to the clinics between March 2016 and October 2016 in Ankara. Blood samples were inactivated and measurements were done with SFDT 1/4, 1/16, 1/64, 1/256, 1/1024 titers. Results: SFDT resulted positive in 56 (43.4%) cats at a dilution of 1/16, in 7 (5.4%) cats at a dilution of 1/64, in 23 (17.8%) cats at a dilution of 1/256 and negative in 43 (33.3%) cats. Comparison of demographic data with SFDT results showed that positive test results did not differ according to gender and age (P=0.803 and P=0.991, respectively). Seropositivity was higher in stray cats than house cats (P<0.001). Test results were negative in the cats that fed only by commercial dry food (P<0.001). Positivity in hunter cats was more than in non-hunters (P<0.001). Conclusion: Seropositivity was detected in 66.6% of the cats, which was quite a high rate. As a result, taking precautions in terms of Toxoplasma for stray cats that are hunting and feeding naturally is a necessity for public health

Prevalence of Gastrointestinal Parasites in Stray Cats of İzmir

Gastrointestinal parasites of cats can affect animal health and welfare, as well as human health because of some zoonotic parasites including Toxoplasma gondii, Cryptosporidium spp., Isospora spp., Blastocystis sp., and Toxocara spp. Therefore, it is fairly important to investigate the presence of gastrointestinal parasites in stray cats in order to reveal the frequency of parasite diseases and to prevent the spread of parasitic diseases. A total of 465 feces samples were collected from Veterinary Clinics located in 5 different districts of İzmir. For microscopic examination, all feces samples were processed by centrifugation-sucrose flotation. In addition, cat feces with diarrhea were stained by the by Kinyoun acid-fast staining for the diagnosis of Cryptosporidium spp. As a result, 73 of 465 (15.6%) cats were found to be infected with at least one of the following parasites: Blastocystis sp., Isospora spp., Cryptosporidium spp., Toxoplasma gondii-like oocyte, Toxocara spp., Hymenolepis spp. and Dipylidium caninum. Among the studied stray cats, Blastocystis sp. was detected as the most prevalent protozoon parasite (10.5%) in stray cats. Overall, the results show that stray cats are a significant source for distribution of various parasite diseases to humans and animals in İzmir, Turkey

In silico discovery of antigenic proteins and epitopes of SARS-CoV-2 for the development of a vaccine or a diagnostic approach for COVID-19

In the genome of SARS-CoV-2, the 5′-terminus encodes a polyprotein, which is further cleaved into 15 non-structural proteins whereas the 3′ terminus encodes four structural proteins and eight accessory proteins. Among these 27 proteins, the present study aimed to discover likely antigenic proteins and epitopes to be used for the development of a vaccine or serodiagnostic assay using an in silico approach. For this purpose, after the full genome analysis of SARS-CoV-2 Wuhan isolate and variant proteins that are detected frequently, surface proteins including spike, envelope, and membrane proteins as well as proteins with signal peptide were determined as probable vaccine candidates whereas the remaining were considered as possible antigens to be used during the development of serodiagnostic assays. According to results obtained, among 27 proteins, 26 of them were predicted as probable antigen. In 26 proteins, spike protein was selected as the best vaccine candidate because of having a signal peptide, negative GRAVY value, one transmembrane helix, moderate aliphatic index, a big molecular weight, a long-estimated half-life, beta wrap motifs as well as having stable, soluble and non-allergic features. In addition, orf7a, orf8, and nsp-10 proteins with signal peptide were considered as potential vaccine candidates. Nucleocapsid protein and a highly antigenic GGDGKMKD epitope were identified as ideal antigens to be used in the development of serodiagnostic assays. Moreover, considering MHC-I alleles, highly antigenic KLNDLCFTNV and ITLCFTLKRK epitopes can be used to develop an epitope-based peptide vaccine

Discovery of new Toxoplasma gondii antigenic proteins using a high throughput protein microarray approach screening sera of murine model infected orally with oocysts and tissue cysts

Abstract Background Toxoplasma gondii is an obligate intracellular protozoan parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations in immune compromised humans. The parasite has also been recently linked to behavioral diseases in humans and other mammalian hosts. New antigens are being evaluated to develop a diagnostic kit for the diagnosis of acute infection or a protective vaccine. Methods In this study, we have focused on the discovery of new antigenic proteins from T. gondii genomic data using a high throughput protein microarray screening. To date, microarrays containing > 2870 candidate exon products of T. gondii have been probed with sera collected from patients with toxoplasmosis. Here, the protein microarrays are probed with well-characterized serum samples from animal models administered orally with oocysts or tissue cysts. The aim was to discover the antigens that overlap in the mouse profile with human antibody profiles published previously. For this, a reactive antigen list of 240 antigens recognized by murine IgG and IgM was identified using pooled sera from orally infected mice. Results Analyses of screening data have identified plenty of antigens and showed strong immunogenicity in both mouse and human antibody profiles. Among them, ROP1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA14, MIC1, MIC2 and MAG1 have shown strong immunogenicity and used as antigen in development of vaccines or serological diagnostic assays in previous studies. Conclusion In addition to the above findings, ROP6, MIC12, SRS29A and SRS13 have shown strong immunogenicity but have not been tested in development of a diagnostic assay or a vaccine model yet

Detection of Echinococcusgranulosus and Echinococcusmultilocularis in Cyst Samples Using a Novel Single Tube Multiplex Real-Time Polymerase Chain Reaction

Dünyada ve ülkemizde Echinococcus granulosus ve Echinococcus multilocularis'in neden olduğu sırasıyla kistik ekinokokkoz (KE) ve alveolar ekinokokkoz (AE) önemli helmint hastalıkları arasındadır. Türkiye'de yapılan epidemiyolojik çalışmalar KE prevalansının 291-585/100.000 arasında olduğunu göstermiştir. AE seroprevalansının da %3.5 oranında olduğu bildirilmiştir. KE ve AE tanısında klinik bulgular yanında genelde radyoloji (ultrason, bilgisayarlı tomografi , manyetik rezonans) ve serolojik yöntemler kullanılmaktadır. Kesin tanı patolojik incelemeye dayalıdır. Hidatik kistlerin steril olması ya da protoskoleks içermemesi durumunda ise, patolojik tekniklerle E.granulosus ve E.multilocularis tür ayrımında zorluklar yaşanmaktadır. Bu çalışmada, aynı test içerisinde AE ve KE tanısının yapılmasına imkan verebilecek Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') ve Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primerleri ve üç farklı prob; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fl oresan-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-fosfat-3') ve Multilocularis (5'-LC705CTGTGATCTTGGTGTAGTAGTTGAGATT-fosfat-3') kullanılarak, E.granulosus ve E.multilocularis mitokondriyal 12S rRNA genini hedefl eyen yeni bir multipleks gerçek zamanlı polimeraz zincir reaksiyonu (M-RTPCR) yönteminin geliştirilmesi amaçlanmıştır. M-RT-PCR sırasında, E.granulosus (GenBank: AF297617.1) ve E.multilocularis (GenBank: NC_000928.2) mitokondriyal 12S rRNA gen bölgelerini içeren plazmidler pozitif kontrol olarak kullanılmıştır. M-RT-PCR yönteminin geliştirilmesinde ayrıca, patolojik olarak KE (n: 10) ve AE (n: 15) olduğu doğrulanmış hastalara ait kist örneklerinin yanı sıra, negatif kontrol olarak sağlıklı insan DNA örnekleri (n: 25) ve 12 farklı parazite (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) ait DNA örnekleri de çalışılmıştır. Testin analitik duyarlılığının saptanması için TOPO klonlama ile E.granulosus ve E.multilocularis pozitif kontrol plazmidleri oluşturulmuştur. Pozitif kontrol plazmidi, analitik duyarlılık ve özgüllüğün belirlenmesi amacıyla her reaksiyonda 106-105-104-103-102-101-1 plazmid kopya olacak şekilde distile su ile sulandırılmıştır. Çalışmamızın sonuçları, testin pozitif kontrol plazmidi kullanılarak elde edilen analitik duyarlılığının, E.granulosus ve E.multilocularis için 1 plazmid kopya/?l örnek olduğunu göstermiştir. Testin 12 farklı parazite ait DNA örneği ile çapraz reaksiyon vermemesi, analitik özgüllüğünü ortaya koymuştur. Yirmibeş hastaya ait kist örneğinde Echinococcus DNA varlığının gösterilmesi ve tür ayrımının yapılabilmesinin yanında, sağlıklı (negatif kontrol) insan DNA örnekleri ile çapraz reaksiyon vermemesi, testin klinik duyarlılık ve özgüllüğünün %100 olduğunu göstermiştir. Sonuç olarak, bu çalışmada geliştirilen M-RT-PCR yönteminin, kist örneklerinde ekinokokkoz tanısının konulmasına ek olarak, E.granulosus ve E.multilocularis'in ayırt edilmesinde duyarlı, özgül, hızlı ve güvenilir bir yöntem olduğu tespit edilmiştirCystic 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 fi ndings, are being used. The defi nitive 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-fl uoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-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 confi rmed 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 specifi city by distilled water at 106-105-104-103-102-101-1 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/?l reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specifi city 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 specifi city of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specifi c, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst sample

Development of a novel recombinant ELISA for the detection of Crimean-Congo hemorrhagic fever virus IgG antibodies

Crimean-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 (P0.05) 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

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

International audienceAbstract Background 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

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey

International audienceToxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation