Genetic diversity and haplotypes of Cysticercus tenuicollis isolates from slaughtered sheep and goats in Elazig and Bingol provinces of Turkey.

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 (π: 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.

Sequence and Haplotype Analyses of Ligula intestinalis in Acanthobrama marmid (Cyprinidae) in Turkey.

PURPOSE: Ligulosis caused by Ligula intestinalis adversely affects the fisheries carried out in the lakes and ponds, causing economic losses in the fish industry. In this study, it was aimed to reveal the molecular characterization of L. intestinalis isolates obtained from woodfish (Acanthobrama marmid) in Keban Dam Lake in Elazig province of Turkey by using mt-CO1 gene sequences and to determine the genetic differences and haplotypes between the isolates. METHODS: In the examination made in terms of L. intestinalis, the intestine of the fish was opened with the help of fine-tipped scissors, the contents were allowed to come out, and the parasites were taken into a petri dish containing phosphate buffered saline (PBS). Then, L. intestinalis plerocercoids were taken into 15 ml falcon tubes containing 70% ethanol and stored at - 20 °C until further analysis. From each isolate, total gDNA was extracted from the plerocercoids. A partial (480 bp) mt-CO1 gene was amplified by PCR and sequenced unidirectionally. The final size of the trimmed sequences was 392 bp for 43 sequences. Sequence and haplotype analyses were performed, followed by phylogenetic analyses. RESULTS: All isolates were confirmed as L. intestinalis by BLAST analysis. In addition, 87 nucleotide mutation positions were determined among 43 CO1 gene sequences. As a result of the haplotype network performed for the mt-CO1 gene region of L. intestinalis isolates; arranged in a star-like configuration with the main haplotype (Hap05), separated from other haplotypes by 1-6 mutation steps, and 29 haplotypes were identified, covering 13.9% (6/43) of the total isolates. Also, 75 variable (polymorphic) sites were determined, 52 of which were parsimony informative sites. CONCLUSIONS: The molecular characterization of L. intestinalis in woodfish (A. marmid) was identified for the first time in Turkey.

Cloning and expression of Fasciola hepatica enolase gene and efficacy of recombinant protein in the serodiagnosis of sheep fasciolosis.

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.

In Silico Evaluation of the Haplotype Diversity, Phylogenetic Variation and Population Structure of Human E. granulosus sensu stricto (G1 Genotype) Sequences.

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.

A demographic survey on the prevalence of gastrointestinal parasites based on socioeconomic determinants in Pakistan.

INTRODUCTION: The present study was conducted to investigate prevalence of intestinal parasites and the risk factors related to socio-demographic characteristics of patients admitted in pathology ward, General Hospital, Gujranwala. METHODOLOGY: 318 stool samples were collected from patients and examined under light microscope by using wet mount technique. While socio-demographic information was collected in the form of a questionnaire. RESULTS: The results showed seven (n = 7) species of intestinal parasites were prevalent in stool samples of patients. Among them, four (n = 4) were helminth and three (n = 3) were protozoan parasites causing single and mixed infections. Overall prevalence of intestinal parasites was 78.3% (n = 249/318) considering both male and female patients. Highest prevalence was recorded for A. lumbricoides (n = 125, 39.3%) followed by H. nana (n = 10, 3.1%), S. stercoralis and T. saginata (n = 6, 1.9%). Among protozoan parasites, higher prevalence was recorded in G. lamblia (n = 23, 7.2%) followed by E. histolytica (n = 21, 6.6%). Among single infections, the most prevalent parasite was A. lumbricoides and less prevalent parasites were S. stercoralis and T. saginata. The factors that had significant effect (p < 0.05) on prevalence of parasitic species were contaminated water, food, soil, and surrounding environment. CONCLUSIONS: The present study determined that the parasite helminth (A. lumbricoides, H. nana, S. stercoralis, T. saginata) and protozoan (G. lamblia and E. histolytica) are common that pose an important public health concern in Pakistan.

Cloning, expression and serodiagnostic potential of HSP70 of Taenia multiceps in sheep.

The aim of this study was to determine the efficacy of the recombinant protein expressed by the T. multiceps HSP70 gene in the immunodiagnosis of sheep coenurosis. Specific primers were designed to amplify the gene encoding HSP70 from the whole genome sequence of T. multiceps, mRNA was isolated from C. cerebralis cysts obtained from an infected sheep's brain, and then, cDNA was isolated. The gene region related to the designed primers was amplified by PCR, and the obtained product was cloned and expressed. The specificity of the recombinant protein purified afterwards was demonstrated by Western Blot using known positive and negative sheep sera. Additionally, the efficiency of the recombinant protein in ELISA was determined with sera collected from 1207 sheep. Finally, the sensitivity and specificity values ​of the recombinant TmHSP70 antigen in the Western Blot were 100 %, while the sensitivity and specificity rates of the ELISA were 66.6 % and 52.6 %, respectively. At the same time, 567 (46.97 %) of the 1207 analyzed serum samples were found to be positive by ELISA. In conclusion, the Western Blot technique had a higher quality than ELISA in detecting the TmHSP70 antigen for the serodiagnosis of sheep coenurosis.

Genetic diversity and haplotypes of paediatric hydatid cyst isolates and first occurrence of E. canadensis (G6/G7) in paediatric cases in Turkey.

Cystic echinococcosis (CE) is a neglected zoonotic tropical disease caused by Echinococcus granulosus sensu lato. The aim of this study was to investigate the genetic variation of hydatid cyst isolates obtained from surgically confirmed paediatric cases originating from two different regions in eastern Turkey. Seventeen paediatric cases aged between 6 and 16 were operated by open surgery, and the germinal layers of their cysts were obtained for further molecular analyses. After genomic DNA isolation, 875 bp mt-CO1 gene fragments were amplified in all samples by PCR. Then, the unidirectional sequence analyses of the PCR products were carried out. According to the BLAST analyses of 17 sequences, 16 of these sequences were matched with E. granulosus sensu stricto, while one sequence was identified as E. canadensis (G6/G7) for the first time in paediatric cases in Turkey. High haplotype diversity and low nucleotide diversity were observed in the E. granulosus s.s. sequences.

Molecular Characterization of Hydatid Cysts Cases in a Wild Boar and Mule in Turkey

Objective: Cystic echinococcosis (CE) is a zoonotic infection that affects humans, livestock and wild animals through the larval form of Echinococcus granulosus sensu lato (s.l.). Molecular and taxonomic studies carried out in the recent years accept that Echinococcus granulosus s.l., a complex of 5 cryptic species, causes CE. In this study, we performed morphological and molecular characterisation of cyst isolates obtained from a wild boar and mule naturally infected with hydatid cyst. Methods: After gDNA isolation, the mitochondrial cytochrome oxidase subunit 1 (mt-CO1) gene region was amplified by polymerase chain reaction (PCR) with specific primers. The amplified mt-CO1 PCR products were purified and one-way DNA sequence analysis was performed. Results: Comparison of the partial sequences of mt-CO1 gene from the hydatid cyst isolates with that of reference sequences in GenBank revealed 100% similarity with E. granulosus sensu stricto (G1-G3) sequences. Conclusion: To the best of our knowledge, this is the first study to determine the molecular characterisation of Echinococcus species in a wild boar in Turkey.

In Silico Analysis of the Biodiversity and Conservation Status of Mitochondrial Cytochrome C Oxidase Subunit 1 (CO1) Gene of Taenia multiceps.

PURPOSE: Taenia multiceps resides in the small intestine of carnivores such as dogs, foxes, woles, jackals, while Coenurus cerebralis which is the larval form usually settle in the central nervous system and spinal cord of intermediate hosts like sheep, cattle and goats. The aim of this study was to analyze the haplotype diversity, genetic variation and population structure of the mt-CO1 gene sequences of sheep isolates of T. multiceps had been submitted to GenBank from different countries. METHODS: A total of 102 sequences from the mt-CO1 gene fragment belong to T. multiceps sheep isolates were used for bioinformatic analyses. Haplotype analysis, phylogenetic analysis and diversity, neutrality, fixation and gene flow analyses were applied to the sequences. RESULTS: As a result, 20 haplotypes together with different multiple nucleotide changes were determined after the sequence analysis. Trimmed fragment length was 337 bp hereby 19 polymorphic areas, 12 of which were parsimony informative, were identified, and any insertion-deletion was found. The number of mutations between major haplotypes and the others range from one to nine. The highest (0.72) genetic differentiation (Fst value) was observed between Turkey and Egypt populations while the lowest (- 0.22) was reported from Greece. These findings are important in terms of showing the diversity of nucleotide variation in T. multiceps sheep isolates. CONCLUSIONS: This study serves as the basis for future large-scale studies on T. multiceps worldwide epidemiology, bioecology, geographic distribution and population structure.