ABSTRACT
Objective: Echinococcus granulosus contains a complex of different strains that represent diversity in the pattern of the life cycle and also their host types. So far 10 genotypes of this parasite have been identified, using molecular methods. The current study aimed to evaluate and compare the genotypic diversity of E. granulosus metacestodes from livestock of Turkey and Iran. Methods: A total of 90 livestock liver and lung organs infected with hydatid cyst from industrial slaughterhouses of Bonab Province in the East Azerbaijan Province in Iran (60 samples, including 30 sheep and 30 cattle) and Van Province in Turkey (30 samples, including 15 sheep and 15 cattle) were collected. DNA was extracted from the protoscolices or germinal layers and polymerase chain reaction (PCR) were utilized, targeting the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) genes. PCR products were isolated from the electrophoresis gels and sequenced. The sequences were compared with each other, as well as with those related available sequences in the GenBank, using the BioEdit software and the BLAST algorithm. Finally, the phylogenetic trees were constructed by comparing sequences of cox1 and nad1 fragments, using the MEGA7 software and the maximum likelihood method. Results: All samples sequenced from Iran corresponded to the genotype G1 (100%). Among the samples from Turkey, 15 samples (78.9%) were identified as G1 while only one sample (5.3%) corresponded to the genotype G3 and 3 isolates (15.8%) were defined as genotypes G1/G3. Five distinct haplotypes were determined within the examined isolates from sheep and cattle in both countries and all isolates clustered in one group. Phylogenetic analysis revealed that the intra-species genetic variations were 0.0-0.6% and 0.0-1.4% for cox1 and nad1, respectively. Conclusion: The dominant genotype of E. granulosus sensu stricto of livestock in both countries was the G1 (sheep strain) genotype. Our findings indicate that the sheep-dog cycle is the leading cycle of E. granulosus in these two areas. Hence, adopting regional common policies and bilateral cooperation helps to control the disease in livestock as well as in human in these two regions. Further study is required to compare the genetic diversity of human isolates of E. granulosus in these two countries.
Subject(s)
Cattle Diseases/parasitology , Echinococcosis/veterinary , Echinococcus granulosus/classification , Genetic Variation , Livestock , Sheep Diseases/parasitology , Abattoirs , Animals , Base Sequence , Cattle , Echinococcosis/parasitology , Echinococcus , Echinococcus granulosus/genetics , Echinococcus granulosus/isolation & purification , Electron Transport Complex IV/genetics , Genotype , Haplotypes , Humans , Iran , NADH Dehydrogenase/genetics , Phylogeny , Polymerase Chain Reaction , Sheep , TurkeyABSTRACT
AIM: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples. MATERIALS AND METHODS: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel. RESULTS: The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination. CONCLUSION: Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.
