Molecular Characterization of Human and Animal Isolates of Echinococcus granulosus in the Thrace Region, Turkey

Year 2012, , 261 - 267, 01.03.2012

Canan Eryıldız Nermin Sakru

https://doi.org/10.5152/balkanmedj.2012.072

Abstract

Objective: Echinococcus granulosus is the causative agent of cystic echinococcosis in humans and many domestic animals, and remains an important global health problem. The aim of this study was to genotype E. granulosus isolates obtained from humans and animals in the Thrace Region of Turkey. Material and Methods: A total of 58 isolates were obtained from patients who underwent surgery at several hospitals and from animals at a slaughterhouse in the province of Edirne. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal internal transcribed spacer 1 fragments, and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) of the partial mitochondrial NADH dehydrogenase subunit 1 (ND1) gene, was used to characterize human and animal E. granulosus isolates. To investigate the genetic characteristics of isolates, deoxyribonucleic acid (DNA) sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and ND1 genes was performed. Results: Fifty-eight E. granulosus isolates, including 42 from human, 13 from cattle and 3 from sheep were, analyzed. The results indicated two distinct genotypes: the G1 (sheep strain) and G7 (pig strain) genotypes. The sheep strain was shown to be the most common genotype of E. granulosus affecting humans, sheep and cattle. Among the concatenated partial CO1 and ND1 sequence data, eight haplotypes of Echinococcus species were identified in the present study. Conclusion: This is the first report indicating that the E. granulosus pig strain is present in humans in this region. We suggest that new strategies be designed for E. granulosus control programs in Turkey. Turkish Anahtar Kelimeler: Echinococcus granulosus, genotip, PCR-RFLP, PCR-SSCP, CO1, ND1, Edirne, Trakya bölgesi, Türkiye Amaç: Echinococcus granulosus insanlarda ve birçok evcil hayvanda kistik ekinokokkozisin etkenidir ve dünya çapında önemli bir halk sağlığı sorunudur. Bu çalışmanın amacı Türkiye'nin Trakya bölgesinde insanlardan ve hayvanlardan elde edilen E. granulosus izolatlarının genotipini belirlemektir. Gereç ve Yöntemler: Edirne ilinde çeşitli hastanelerde opere edilen insanlardan ve kesimhanedeki hayvanlardan toplam 58 izolat elde edildi. E. granulosus'un insan ve hayvan izolatlarını karakterize etmek için; ribozomal birinci internal transcribed spacer fragmanının analizi için PCR-RFLP (polimeraz zincir reaksiyonu - restriksiyon fragman uzunluğu polimorfizmi) yöntemi ve parsiyel mitokondriyal NADH dehidrogenaz subunit 1 geninin analizi için PCR-SSCP (polimeraz zincir reaksiyonu - tek sarmal konformasyon polimorfizmi) yöntemi kullanıldı. İzolatların genetik özelliklerinin ileri incelemeleri için mitokondriyal sitokrom oksidaz c subunit 1 (CO1) ve NADH dehidrogenaz subunit 1 (ND1) gen bölgelerinin DNA (deoksiribonükleik asit) dizi analizi yapıldı. Bulgular: Kırk iki insan, 13 sığır ve 3 koyun kökenli olan 58 E. granulosus izolatı analiz edildi. Sonuçlar G1 (evcil koyun suşu) ve G7 (domuz suşu) olmak üzere iki farklı genotip bulunduğunu gösterdi. İnsan, koyun ve sığırları etkileyen E. granulosus'un en yaygın genotipinin koyun suşu olduğu gösterildi. Parsiyel mitokondriyal CO1 ve ND1 verileri birlikte değerlendirildiğinde mevcut çalışmadaki E. granulosus izolatlarında 8 haplotip tanımlandı. Sonuç: Bu çalışma; Trakya bölgesindeki insanlarda E. granulosus'un domuz suşunun varlığını gösteren ilk rapordur. Türkiye'de E. granulosus'un kontrol programları için yeni stratejiler oluşturulmasını önermekteyiz.

Keywords

Echinococcus granulosus, genotype, PCR-RFLP, PCR-SSCP, CO1, ND1, Edirne, Thrace region, Turkey

Molecular Characterization of Human and Animal Isolates of Echinococcus granulosus in the Thrace Region, Turkey

Abstract

References

• Soulsby EJL. Helminths, arthropods and of domesticated animals. 7th ed. London: Bailliere Tindall; 1982. p.92-136.
• Eckert J, Deplazes P. Biological, epidemiological, and clinical aspects of Echinococcosis, a zoonosis of increasing concern. Clin Microbiol Rev 2004;17:107-35. [CrossRef]
• Eckert J, Schantz PM, Gasser RB, Torgerson PR, Bessonov AS, Movsessian SO, et al. Geographic distribution and prevalence. In: Eckert J, Gemmell MA, Meslin F-X, Pawlowski ZS, editors. WHO/OIE manual on Echinococcosis in humans and animals: a public health problem of global concern. Paris: World organisation for animal health; 2001. p.101-43.
• Yazar S, Ozkan AT, Hökelek M, Polat E, Yilmaz H, Ozbilge H, et al. Cystic echinococcosis in Turkey from 2001-2005. Turkiye Parazitol Derg 2008;32:208-20.
• Thompson RCA. The taxonomy, phylogeny and transmission of Echinococcus. Exp Parasitol 2008;119:439-46. [CrossRef]
• Nakao M, McManus DP, Schantz PM, Craig PS, Ito A. A molecular phylogeny of the genus Echinococcus inferred from complete mitochondrial genomes. Parasitology 2007;134:713-22. [CrossRef]
• Thompson RCA, McManus DP. Aetiology: parasites and lifecycles. In: Eckert J, Gemmell MA, Meslin F.-X, Pawlowski ZS, editors. WHO/OIE manual on Echinococcosis in humans and animals: a public health problem of global concern. Paris: World organisation for animal health; 2001. p.1-19.
• Thompson RCA, Lymbery AJ. The nature, extent and significance of variation within the genus Echinococcus. Adv Parasitol 1988;27:209-58. [CrossRef]
• Thompson RCA. Biology and systematics of Echinococcus. In: Thompson RCA, Lymbery AJ, editors. Echinococcus and hydatid disease. Wallingford: CAB International; 1995. p.1-50.
• Zhang L, Eslami A, Hosseini SH, McManus DP. Indication of the presence of two distinct strains of Echinococcus granulosus in Iran by mitochondrial DNA markers. Am J Trop Med Hyg 1998;59:171-4.
• McManus DP. Molecular discrimination of taeniid cestodes. Parasitol Int 2006;55 Suppl:S31-7. [CrossRef]
• Vural G, Baca AU, Gauci CG, Bagci O, Gicik Y, Lightowlers MW. Variability in the Echinococcus granulosus cytochrome c oxidase 1 mitochondrial gene sequence from livestock in Turkey and a re-appraisal of the G1-3 genotype cluster. Vet Parasitol 2008;154:347-50. [CrossRef]
• Snabel V, Altintas N, D’Amelio S, Nakao M, Romig T, Yolasigmaz A, et al. Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country. Parasitol Res 2009;105:145-54. [CrossRef]
• Beyhan YE, Umur S. Molecular characterization and prevalence of cystic echinococcosis in slaughtered water buffaloes in Turkey. Vet Parasitol 2011;181:174-9. [CrossRef]
• Utuk AE, Simsek S, Koroglu E, McManus DP. Molecular genetic characterization of different isolates of Echinococcus granulosus in east and southeast regions of Turkey. Acta Trop 2008;107:192-4. [CrossRef]
• Simsek S, Balkaya I, Koroglu E. Epidemiological survey and molecular characterization of Echinococcus granulosus in cattle in an endemic area of eastern Turkey. Vet Parasitol 2010;172:347-9. [CrossRef]
• Simsek S, Balkaya I, Ciftci AT, Utuk AE. Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey. Vet Parasitol 2011;178:367-9. [CrossRef]
• Simsek S, Kaplan M, Ozercan IH. A comprehensive molecular survey of Echinococcus granulosus in formalin-fixed paraffinembedded tissues in human isolates in Turkey. Parasitol Res 2011;109:411-6. [CrossRef]
• Bowles J, McManus DP. Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method. Mol Biochem Parasitol 1993;57:231-40. [CrossRef]
• Simsek S, Eroksuz Y. Occurrence and molecular characterization of Echinococcus granulosus in Turkish mouflon (Ovis gmelinii anatolica). Acta Trop 2009;109:167-9. [CrossRef]
• Gasser RB, Zhu X, McManus DP. Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism. Acta Trop 1998;71:107-15. [CrossRef]
• Sharbatkhori M, Mirhendi H, Jex AR, Pangasa A, Campbell BE, Kia EB, et al. Genetic categorization of Echinococcus granulosus from humans and herbivorous hosts in Iran using an integrated mutation scanning-phylogenetic approach. Electrophoresis 2009;30:2648-55. [CrossRef]
• Byun SO, Fang Q, Zhou H, Hickford JG. An effective method for silver-staining DNA in large numbers of polyacrylamide gels. Anal Biochem 2009;385:174-5. [CrossRef]
• Bowles J, Blair D, McManus DP. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol Biochem Parasitol 1992;54:165-73. [CrossRef]
• Okamoto M, Bessho Y, Kmiya M, Kurosawa T, Horii T. Phylogenetic relationships within Taenia taeniaeformis variants and other taeniid cestodes inferred from the nucleotide sequence of the cytochrome c oxidase subunit I gene. Parasitol Res 1995;81:451-8. [CrossRef]
• Bowles J, McManus DP. NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus. Int J Parasitol 1993;23:969-72. [CrossRef]
• Bowles J, Blair D, McManus DP. Molecular genetic characterization of the cervid strain (‘northern form’) of Echinococcus granulosus. Parasitology 1994;109:215-21. [CrossRef]
• Lavikainen A, Lehtinen MJ, Meri T, Hirvela-Koski V, Meri S. Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus. Parasitology 2003;127:207-15. [CrossRef]
• Hüttner M, Nakao M, Wassermann T, Siefert L, Boomker JD, Dinkel A, et al. Genetic characterization and phylogenetic position of Echinococcus felidis (Cestoda: Taeniidae) from the African lion. Int J Parasitol 2008;38:861-8. [CrossRef]
• Okamoto M, Nakao M, Blair D, Anantaphruti MT, Waikagul J, Ito A. Evidence of hybridization of between Taenia saginata and Taenia asiatica. Parasitol Int 2010;59:70-4. [CrossRef]
• Gasser RB, Zhu X, McManus DP. NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 1 sequences compared for members of the genus Taenia (Cestoda). Int J Parasitol 1999;29:1965-70. [CrossRef]
• Harandi MF, Hobbs RP, Adams PJ, Mobedi I, Morgan-Ryan UM, Thompson RCA. Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran. Parasitology 2002;125:367-73.
• Wachira TM, Bowles J, Zeyhle E, McManus DP. Molecular examination of the sympatry and distribution of sheep and camel strains of Echinococcus granulosus in Kenya. Am J Trop Med Hyg 1993;48:473-9.
• Bowles J, van Knapen F, McManus D. Cattle strain of Echinococcus granulosus and human infection. The Lancet 1992;339:1358. [CrossRef]
• Scott JC, Stefaniak J, Pawlowski ZS, McManus DP. Molecular genetic analysis of human cystic hydatid cases from Poland: identification of a new genotypic group (G9) of Echinococcus granulosus. Parasitology 1997;114:37-43. [CrossRef]
• Sharbatkhori M, Mirhendi H, Harandi MF, Rezaeian M, Mohebali M, Eshraghian M, et al. Echinococcus granulosus genotypes in livestock in Iran indicating high frequency of G1 genotype in camels. Exp Parasitol 2010;124:373-9. [CrossRef]
• M’rad S, Filisetti D, Quidni M, Mekki M, Belguith M, Nouri A, et al. Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination. Vet Parasitol 2005;129:267-72. [CrossRef]
• Zhang L, Gasser RB, Zhu X, McManus DP. Screening for different genotypes of Echinococcus granulosus within China and Argentina by single-strand conformation polymorphism (SSCP) analysis. Trans R Soc Trop Med Hyg 1999;93:329-34. [CrossRef]
• Abushhewa MH, Abushhiwa MHS, Nolan MJ, Jex AR, Campbell BE, Jabbar A, et al. Gentic classification of Echinococcus granulosus cysts from humans, cattle and camels in Libya using mutation scanning-based analysis of mitochpndrial loci. Mol Cell Probes 2010;24:346-51. [CrossRef]
• Jabbar A, Narankhajid M, Nolan MJ, Jex AR, Campbell BE, Gasser RB. A first insight into the genotypes of Echinococcus granulosus from humans in Mongolia. Mol Cell Probes 2011;25:49-54. [CrossRef]
• Nakao M, Li T, Han X, Ma X, Xiao N, Qiu J, et al. Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences. Int J Parasitol 2010;40:379-85. [CrossRef]
• Obwaller A, Schneider R, Walochnik J, Gollackner B, Deutz A, Janitschke K, et al. Echinococcus granulosus strain differentiation based on sequence heterogeneity in mitochondrial genes of cytochrome c oxidase-1 and NADH dehydrogenase-1. Parasitology 2004;128:569-75. [CrossRef]
• Pour AA, Hosseini SH, Shayan P. Comparative genotyping of Echinococcus granulosus infecting buffalo in Iran using cox1 gene. Parasitol Res 2011;108:1229-34. [CrossRef]
• Bart JM, Abdukader M, Zhang YL, Lin RY, Wang YH, Nakao M, et al. Genotyping of human cystic echinococcosis in Xinjiang, PR China. Parasitology 2006;133:571-9. [CrossRef]
• Sharbatkhori M, Fasihi Harandi M, Mirhendi H, Hajialilo E, Kia EB. Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran. Parasitol Res 2011;108:521-7. [CrossRef]
• Varcasia A, Canu S, Kogkos A, Pipia AP, Scala A, Garippa G, et al. Molecular characterization of Echinococcus granulosus in sheep and goats of Peloponnesus, Greece. Parasitol Res 2007;101:1135-9. [CrossRef]
• Bardonnet K, Benchikh-Elfegoun MC, Bart JM, Harraga S, Hannache N, Haddad S, et al. Cystic echinococcosis in Algeria: cattle act as reservoirs of a sheep strain and may contribute to human contamination. Vet Parasitol 2003;116:35-44. [CrossRef]
• Eckert J, Thompson RCA. Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans. Acta Trop 1997;64:19-34. [CrossRef]
• Villabos N, Gonzalez LM, Morales J, de Aluja AS, Jimenez MI, Blanco MA, et al. Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico. Vet Parasitol 2007;147:185-9. [CrossRef]
• Moro PL, Nakao M, Ito A, Schantz PM, Cavero C, Cabrera L. Molecular identification of Echinococcus isolates from Peru. Parasitol Int 2009;58:184-6. [CrossRef]
• Pawlowski Z, Stefaniak J. The pig strain of Echinococcus granulosus in humans: a neglected issue? Trends Parasitol 2003;19:439.
• Breyer I, Georgieva D, Kurdova R, Gottstein B. Echinococcus granulosus strain typing in Bulgaria: the G1 genotype is predominant in intermediate and definitive wild hosts. Parasitol Res 2004;93:127-30. [CrossRef]
• Casulli A, Interisano M, Sreter T, Chitimia L, Kirkova Z, La Rose G, et al. Genetic variability of Echinococcus granulosus sensu stricto in Europe inferred by mitochondrial DNA sequences. Infect Genet Evol 2012;12:377-83. [CrossRef]