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    Cloning and expression of Fasciola hepatica enolase gene and efficacy of recombinant protein in the serodiagnosis of sheep fasciolosis
    (Elsevier, 2023) Celik, Figen; Simsek, Sami; Selcuk, Muhammed Ahmed; Kesik, Harun Kaya; Kilinc, Seyma Gunyakti; Celik, Burcak Aslan
    Fasciolosis caused by Fasciola hepatica is a disease of zoonotic importance that is common worldwide and can cause serious problems in farm animals, some wild animals and humans. The development of diagnostic kits for the correct diagnosis of fasciolosis in sheep is important in terms of preventing yield losses. With this study, it is aimed to clone and express the enolase gene to be isolated from adult F. hepatica and to determine the effectiveness of the recombinant antigen in the serodiagnosis of sheep fasciolosis. For this aim, primers were designed to amplify the enolase gene from the F. hepatica enolase sequence, mRNA was isolated from F. hepatica adult fluke obtained from an infected sheep followed by cDNA was obtained. Enolase gene was amplified by PCR and the product was cloned and then expressed. The efficiency of the purified recombinant protein was displayed by Western blot (WB) and ELISA using positive and negative sheep sera. As a result, the sensitivity and specificity rates of the recombinant FhENO antigen were 85% and %82.8 by WB while the rates were 90% and 97.14% by ELISA, respectively. At the same time, in sheep blood sera samples collected from the Elazig and Siirt provinces of Turkey, 100 (50%) of 200 sera were found to be positive by WB and 46 (23%) were found to be positive by ELISA. The most important problem in ELISA was the high cross-reaction rate of the recombinant antigen used, as in WB. In order to prevent the cross-reactions, it will be useful to compare the genes encoding the enolase protein of parasites from the closely related parasite family, and select the regions where there are no common epitopes, and clone them and test the purified protein.
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    Co-infection of Echinococcus equinus and Echinococcus canadensis (G6/7) in a gray wolf in Turkey: First report and genetic variability of the isolates
    (Elsevier, 2023) Kilinc, Seyma Gunyakti; Celik, Figen; Kesik, Harun Kaya; Selcuk, Muhammed Ahmed; Ahmed, Haroon; Simsek, Sami
    Cystic echinococcosis (CE) is an important zoonotic diseases caused by larval form of Echinococcus granulosus sensu lato. The material of this study was the gray wolf (Canis lupus), which was found dead in the rural area of Bingol province of Turkey. The animal was brought to Veterinary Faculty for necropsy and many of adult Echinococcus spp. obtained. A total of 9 whole adult worms were morphologically examined under the micro-scope, gDNA was isolated from individual samples, a partial mt-CO1 gene fragment (875 bp) was amplified with PCR and sequenced. According to the phylogenetic analysis, six worms were characterized as E. equinus, while three were reported as E. canadensis (G6/7). It was found that the haplotypes of both species were similar to previously published haplotypes. This is the first report in which E. equinus and E. canadensis (G6/7) adult parasites were detected together in a gray wolf's intestine. The findings are important in that it draws attention to the importance of wild cycle in the spread of CE.
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    Genetic diversity and haplotypes of Cysticercus tenuicollis isolates from slaughtered sheep and goats in Elazig and Bingol provinces of Turkey
    (Wiley, 2024) Karakoc, Sirvan; Kesik, Harun Kaya; Celik, Figen; Selcuk, Muhammed Ahmed; Kilinc, Seyma Gunyakti; Simsek, Sami
    Background: The cestode Taenia hydatigena uses canids, primarily dogs, as definitive hosts, whereas the metacestode larval stage cysticercus infects a range of intermediate hosts, including domestic animals such as goats, sheep and pigs. Objectives: In this study, we aimed to determine the genetic differences and haplotypes of Cysticercus tenuicollis isolates obtained from sheep and goats slaughtered in the Bingol and Elazig provinces of Turkey. Methods: C. tenuicollis isolates were collected from 44 sheep and 26 goats slaughtered in slaughterhouses in Bingol and Elazig. After the isolation of total genomic DNA from C. tenuicollis isolates, the genetic characterization of the partial mitochondrial cytochrome oxidase 1 (CO1) gene region (866 bp) was amplified using specific primers by polymerase chain reaction (PCR), the products were then sequenced, and haplotype and genetic diversity analyses were carried out. Results: As a result of the haplotype network analyses, 34 different haplotypes were detected around the main haplotype (Hap02) arranged in a star-like configuration and separated from other haplotypes by 1-28 mutation steps and covering 22.85% (16/70) of all isolates. Twenty-seven polymorphic fields were detected, 77.77% (21/27) of which were parsimony-informative, and secondary haplotype and nucleotide diversity were observed. Additionally, we detected high intraspecies haplotype diversity (hd: 0.933) and high nucleotide diversity (pi: 0.00383), with 27 different nucleotide variation positions among the haplotypes of the isolates. Tajima's D value was negative, indicating population expansion and/or selection purification. The significantly negative Fu's Fs values indicated recent population expansion or the presence of expected rare haplotypes. Conclusion: The results of this study confirmed that C. tenuicollis isolates clustered in one lineage and were closely related to the relevant reference sequences in different countries, confirming the circulation of C. tenuicollis in different geographical regions.
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    In Silico Evaluation of the Haplotype Diversity, Phylogenetic Variation and Population Structure of Human E. granulosus sensu stricto (G1 Genotype) Sequences
    (Mdpi, 2022) Selcuk, Muhammed Ahmed; Celik, Figen; Kesik, Harun Kaya; Kilinc, Seyma Gunyakti; Ahmed, Haroon; Jiang, Nan; Simsek, Sami
    Echinococcus granulosus sensu lato is the causative agent of cystic echinococcosis (CE), which is a neglected zoonotic disease with an important role in human morbidity. In this study, we aimed to investigate the haplotype diversity, genetic variation, population structure and phylogeny of human E. granulosus sensu stricto (s.s.) (G1 genotype) isolates submitted to GenBank from different parts of the world by sequencing the mitochondrial CO1 and ND1 genes. The sequences of the mt-CO1 (401 bp; n = 133) and mt-ND1 (407 bp; n = 140) genes were used to analyze the haplotype, polymorphism and phylogenetic of 273 E. granulosus s.s. (G1 genotype) isolates. Mutations were observed at 31 different points in the mt-CO1 gene sequences and at 100 different points in the mt-ND1 gene sequences. Furthermore, 34 haplotypes of the mt-CO1 sequences and 37 haplotypes of the mt-ND1 sequences were identified. Tajima's D, Fu's Fs, and Fu's LD values showed high negative values in both mt-CO1 and mt-ND1 gene fragments. The haplotype diversities in the sequences retrieved from GenBank in this study indicate that the genetic variation in human isolates of E. granulosus s.s. in western countries is higher than in eastern countries. This may be due to demographic expansions due to animal trades and natural selections.
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    Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker
    (Elsevier, 2024) Celik, Figen; Selcuk, Muhammed Ahmed; Kilinc, Seyma Gunyakti; Kesik, Harun Kaya; Ahmed, Haroon; Wang, Ying; Simsek, Sami
    Cystic echinococcosis (CE) is a common zoonotic disease caused by the larval form of Echinococcus granulosus sensu lato. This study determined the genotype and haplotype differences using the NADH dehydrogenase subunit 5 gene in hydatid cyst samples. Human (n = 12), cattle (n = 28), and sheep (n = 31) hydatid cyst isolates were included. Seventy-one genomic DNA samples were successfully extracted, and a 759 bp mitochondrial NADH dehydrogenase subunit 5 gene fragment was amplified by PCR. Following the sequence analysis, E. granulosus sensu stricto isolates were identified as G1 (n = 61) and G3 (n = 10). A total of 23 haplotypes were obtained from the 71 E. granulosus s.s. G1 and G3 samples. The main haplotype was Hap01 (60.56 %), which consisted of the G1 genotype. The second largest haplotype was Hap04, which consisted entirely of the G3 genotype. Hap14 acted as a bridge between the G1 and G3 genotypes. This study identifies G1 as the dominant genotype in humans and farm animals in Turkey. High haplotype and nucleotide diversity in genotypes were observed. Additionally, this is the first report on the phylogeography and gene flow models of the E. granulosus s. s. population in Turkey using the NADH dehydrogenase subunit 5 gene, the best marker distinguishing between G1 and G3 genotypes.