Genotypes Identification of Echinococcus granulosus Isolated from Iranian Dogs and Camels using Three Polymerase Chain Reaction‑Based Methods of cox1 Gene

Document Type : Original Article

Authors

1 Department of Medical Parasitology and Mycology, School Medicine, Kashan University of Medical Sciences, Kashan,

2 Department of Medical Parasitology and Mycology, School Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

10.4103/iahs.iahs_91_20

Abstract

Aims: Hydatidosis is an important zoonotic disease that is caused by a tiny tapeworm, namely Echinococcus granulosus. In this study, three
polymerase chain reaction (PCR)‑based methods, including, high resolution melting (HRM) analysis, DNA sequencing, and PCR‑restriction
fragment length polymorphism (RFLP) have been used for genotype the identification of E. granulosus isolates from dogs and camels in
Zarinshahr and Najafabad, Isfahan province, Iran. Materials and Methods: A total of 200 adult worms of 40 dogs and 51 samples of camel
hydatid cysts were examined. Molecular characterization of isolates was performed using HRM assay, sequencing of DNA, and digestion Rsa1
pattern coding for the mitochondrial cox1 gene. For analysis of the HRM melting curve, we used the Tm within the range of 77.50°C–79.23°C.
Results: HRM analysis revealed that 72.5%, 15%, and 12.5% dog’s genotypes and 41.17%, 21.56%, and 35.29% camel genotypes were
G1, G3, and G6, respectively. PCR‑RFLP analysis, spare parts 310 bp and 138 bp of cox1 that shows the G1 genotype in all of the isolates.
Sequence analysis as well as HRM assay was confirmed genotypes of G1, G3, and G6 in camels and dogs. Based on three methods of the
cox1 gene the dominant genotype was G1. Conclusion: The PCR‑RFLP only identified the G1 genotype, whereas the HRM analysis, as well
as DNA sequencing, were detected three genotypes G1, G3, G6, therefore, these two methods have enough accuracy for the determination of
genotypes of E. granulosus. This information leads to a better understanding of the biological characteristics of E. granulosus genotypes in
Iran and shows the camel as a source of human hydatidosis.

Keywords


1.     Yanagida T, Mohammadzadeh T, Kamhawi S, Nakao M, Sadjjadi SM, Hijjawi N, et al. Genetic polymorphisms of Echinococcus granulosus sensu stricto in the Middle East. Parasitol Int 2012;61:599-603.
2.     Ito A. Review of Echinococcus and Echinococcosis, Part A. in: Andrew Thompson RC, Lymbery AJ, Deplazes P.Ito Parasites & Vectors 2017;10:408.
3.     Chaâbane-Banaoues R, Oudni-M’rad M, Cabaret J, M’rad S, Mezhoud H, Babba H. Infection of dogs with Echinococcus granulosus: Causes and consequences in an hyperendemic area. Parasit Vectors 2015;8:1-9.
4.     Budke CM, Deplazes P, Torgerson PR. Global socioeconomic impact of cystic echinococcosis. Emerg Infect Dis 2006;12:296-303.
5.     WHO. Echinococcosis. Available from: https://www.who.int/ news-room/fact-sheets/detail/echinococcosis.[Last accessed on 23 March 2020].
6.     Bagheri R, Haghi SZ, Amini M, Fattahi AS, Noorshafiee S. Pulmonary hydatid cyst: Analysis of 1024 cases. Gen Thorac Cardiovasc Surg 2011;59:105-9.
7.     Khalkhali HR, Foroutan M, Khademvatan S, Majidiani H, Aryamand S, Khezri P, et al. Prevalence of cystic echinococcosis in Iran: A systematic review and meta-analysis. J Helminthol 2018;92:260-8.
8.     Harandi MF, Budke CM, Rostami S. The monetary burden of cystic echinococcosis in Iran. PLoS Negl Trop Dis 2012;6:e1915.
9.     McManus DP, Gray DJ, Zhang W, Yang Y. Diagnosis, treatment, and management of echinococcosis. BMJ 2012;344:e3866.
10.     Thompson RC. The taxonomy, phylogeny and transmission of Echinococcus. Exp Parasitol 2008;119:439-46.
11.     Nakao M, Lavikainen A, Yanagida T, Ito A. Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae). Int J Parasitol 2013;43:1017-29.
12.     NakaoM,LiT,HanX,MaX,XiaoN,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.
13.     McManus DP. Current status of the genetics and molecular taxonomy of Echinococcus species. Parasitology 2013;140:1617-23.
14.     Cucher MA, Macchiaroli N, Baldi G, Camicia F, Prada L, Maldonado L, et al. Cystic echinococcosis in South America: Systematic review of species and genotypes of Echinococcus granulosus sensu lato in humansand natural domestic hosts. Trop Med Int Health 2016;21:166-75.
15.     Amin Pour A, Hosseini SH, Shayan P. Comparative genotyping of Echinococcus granulosus infecting buffalo in Iran using Cox1 J Microbiol Methods gene. Parasitol Res 2011;108:1229-34.
16.     Youssefi MR, Tabaripour R, Omrani VF, Spotin A, Esfandiari B. Genotypic characterization of Echinococcus granulosus in Iranian goats. Asian Pac J Trop Dis 2013;3:362-6.
17.     Mahami-Oskouei M, Ghabouli Mehrabani N, Miahipour A, Fallah E. Molecular characterization and sequence analysis of Echinococcus granulosus from sheep isolates in East Azerbaijan province, northwest of Iran. J Parasit Dis 2016;40:785-90.
18.     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.
19.     Hajialilo E, Harandi MF, Sharbatkhori M, Mirhendi H, Rostami S. Genetic characterization of Echinococcus granulosus in camels, cattle and sheep from the south-east of Iran indicates the presence of the G3 genotype. J Helminthol 2012;86:263-70.
20.     Ebrahimipour M, Sadjjadi SM, Yousofi Darani H, Najjari M. Molecular studies on cystic echinococcosis of camel Camelus dromedarius and report of Echinococcus ortleppi in Iran. Iran J Parasitol 2017;12:323-31.
21.     Cardona GA, Carmena DA. Review of the global prevalence, molecular epidemiology and economics of cystic echinococcosis in production animals. Vet Parasitol 2013;192:10-32.
22.     Spotin A, Gholami S, Nasab AN, Fallah E, Oskouei MM, Semnani V, et al. Designing and conducting in silico analysis for identifying of Echinococcus spp. with discrimination of novel haplotypes: An approach to better understanding of parasite taxonomic. Parasitol Res 2015;114:1503-9.
23.     Shariatzadeh SA, Spotin A, Gholami S, Fallah E, Hazratian T, Mahami-Oskouei M, et al. The first morphometric and phylogenetic perspective on molecular epidemiology of Echinococcus granulosus sensu lato in stray dogs in a hyperendemic Middle East focus, northwestern Iran. Parasit Vectors 2015;8:409.
24.     Arbabi M, Pirestani M, Delavari M, Hooshyar H, Abdoli A, Sarvi S. Molecular and Morphological Characterizations of Echinococcus granulosus from Human and Animal Isolates in Kashan, Markazi Province, Iran. Iran J Parasitol 2017;12:177-87.
25.     Lymbery AJ, Thompson RC. The molecular epidemiology of parasite infections: Tools and applications. Mol Biochem Parasitol 2012;181:102-16.
26.     Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 2007;8:597-608.
27.     Tong SY, Giffard PM. Microbiological applications of high-resolution melting analysis. J Clin Microbiol 2012;50:3418-21.
28.     Heritier L, Verneau O, Breuil G, MeistertzheimaAL. The high resolution melting analysis (HRM) as a molecular tool for monitoring parasites of the wildlife. Parasitology 2017;144:563-70.
29.     Hernández C, Alvarez C, González C, Ayala MS, León CM, Ramírez JD. Identification of six new world Leishmania species through the implementation of a high-resolution melting (HRM) genotyping assay. Parasit Vectors 2014;7:501.
30.     Higuera SL, Guhl F, Ramírez JD. Identification of trypanosoma cruzi discrete typing units (DTUs) through the implementation of a highresolution melting (HRM) genotyping assay. Parasit Vectors 2013;6:112.
31.     Peña VH, Fernández GJ, Gómez-Palacio AM, Mejía-Jaramillo AM, Cantillo O, Triana-Chávez O. High-resolution melting (HRM) of the cytochrome B gene: A powerful approach to identify blood-meal sources in Chagas disease Vectors. PLoS Negl Trop Dis 2012;6:e1530.
32.     Dehghani M, Mohammadi MA,Rostami S, Shamsaddini S, Mirbadie SR, Harandi MF. High-resolution melting analysis(HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto. Parasitol Res 2016;115:2715-20.
33.     Ma J, Wang H, Lin G, Craig PS , Ito A, Cai Z, et al. Molecular identification of Echinococcus species from eastern and southern Qinghai, China, based on the mitochondrial cox1 gene. Parasitol Res 2012;111:179-84.34.     Santos GB, Espinola SM, Ferreira HB, Margis R, Zaha A. Rapid detection of Echinococcus species by a high-resolution melting (HRM)
approach. Parasit Vectors 2013;6:327.
35.     Addy F, Alakonya A, Wamae N, Magambo J, Mbae C, Mulinge E, et al. Prevalence and diversity of cystic echinococcosis in livestock in Maasailand, Kenya. Parasitol Res 2012;111:2289-94.
36.     Rostami Nejad M, Roshani M, Lahmi F, Nazemalhosseini Mojarad E. Evaluation of four DNA extraction methods for the detection of Echinococcus granulosus genotype 1. Gastroenterol Hepatol Bed Bench 2011;4:91-4.
37.     Rostami S, Talebi S, Babaei Z, Sharbatkhori M, Ziaali N, Rostami H, et al. High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: Differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato. Parasitol Res 2013;112:3441-7.
38.     Avila HG, Santos GB, Cucher MA, Macchiaroli N, Pérez MG, Baldi G, et al. Implementation of new tools in molecular epidemiology studies of Echinococcus granulosus sensu lato in South America. Parasitol Int 2017;66:250-7.
39.     Eskandari F, Mohaghegh MA, Mirzaei F, Ghomashlooyan M, Hejazi SH. Molecular characteristics of echinococcus granulosus strains isolated from iranian camel using high resolution melting analysis of atp6 and co×1 genes. Avicenna J Clin Microbiol Infect 2018;5:14-9.
40.     Wongkamchai S, Monkong N, Mahannol P, Taweethavonsawat P, Loymak S, Foongladda S. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay. Vector Borne Zoonotic Dis 2013;13:31-6.
41.     Li J, Zhao GH, Lin R, Blair D, Sugiyama H, Zhu XQ. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis. Parasitol Res 2015;114:4225-32.
42.     Costa JM, Cabaret O, Moukoury S, Bretagne S. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Methods 2011;86:357-63.
43.     Pangasa A, Jex RA, Campbell BE, Bott NJ, Whipp M, Hogg G, et al. High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. Mol Cell Probes 2009;23:10-5.
44.     Zhang P, Liu Y, Alsarakibi M, Li J, Liu T, Li Y, et al. Application of HRM assays with Eva Green dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 2012;111:2157-63.
45.     Cai XQ, Yu HQ, Li R, Yue QY, Liu GH, Bai JS, et al. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis. ScientificWorldJournal 2014;2014:e893981.
46.     Sady H, Al-Mekhlafi MM, Ngui R, Atroosh WM, Al-Delaimy AK, Nasr NA, et al. Detection of Schistosoma mansoni and Schistosoma haematobium by real-time PCR with high resolution melting analysis. Int J Mol Sci 2015;16:16085-103.
47.     Bazsalovicsová E, Králová-Hromadová I, Radvánszky J, Beck R. The origin of the giant liver fluke, Fascioloides magna (Trematoda: Fasciolidae) from Croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes. Parasitol Res 2013;112:2661-6.
48.     Maurelli MP, Rinaldi L, Capuano F, Perugini AG, Cringoli G. Development of a real time PCR for the differentiation of the G1 and G2/G3 genotypes of Echinococcus granulosus. Parasitol Res 2009;105:255-9.
49.     Moghaddas E, Borji H, Naghibi A, Shayan P, Razmi GR. Molecular genotyping of Echinococcus granulosus from dromedaries (Camelus dromedarius) in eastern Iran. J Helminthol 2015;89:100-4.50.     Reed GH, Wittwer CT. Sensitivity and specificity of single nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004;50:1748-54.
51.     Gundry CN, Dobrowolski SF, Martin YR, Robbins TC, Nay LM, Boyd N, et al. Base-pair neutral homozygotes can be discriminated by calibrated high resolution melting of small amplicons. Nucleic Acids Res 2008;36:3401-8.
52.     Montgomery J, Wittwer CT, Palais R, Zhou L. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc 2007;2:59-66.
53.     De ML, Takano K, Brochado JF, Costa CV, Soares AG, Yamano K, et al. Infection of humans and animals with Echinococcus granulosus (G1 and G3 strains) and E. ortleppi in Southern Brazil. Vet Parasitol 2011;177:97-103.
54.     Burtis C, Ashwood E, Bruns D. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics 5th Edition. Imprint: Saunders; 2012.
55.     Boubaker G, Macchiaroli N, Prada L, Cucher MA, Rosenzvit MC, Ziadinov I, et al. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex. PLoS Negl Trop Dis 2013;7:e2017.
56.     Adwan G, Adwan K, Bdir S, Abuseir S. Molecular characterization of Echinococcus granulosusisolated from sheep in Palestine. Exp Parasitol 2013;134:195-9.
57.     Sharma M, Sehgal R, Fomda BA, Malhotra A, Malla N. Molecular characterization of Echinococcus granulosus cysts in north Indian patients: Identification of G1, G3, G5 and G6 genotypes. PLoS Negl Trop Dis 2013;7:e2262.
58.     Rostami Nejad M, Taghipour N, Nochi Z, Mojarad EN, Mohebbi SR, Harandi MF, Zali MR. Molecular identification of animal isolates of Echinococcus granulosus from Iran using four mitochondrial genes. J Helminthol 2012;86:485-92.
59.     Mohaghegh MA, Yousefi-Darani H, Azami M, Ghomashlooyan M, Hashemi N, Jabalameli Z, et al. Analysis of the cox1 gene in Echinococcus granulosus from sheep in northeast Iran using PCR high-resolution melting (qPCR-HRM) curve analysis. Trop Biomed 2018;35:91-9.
60.     Ma SM, Maillard S, Zhao HL, Huang X, Wang H, Geng PL, et al. Assessment of Echinococcus granulosus polymorphism in Qinghai province, People’s Republic of China. Parasitol Res 2008;102:1201-6.
61.     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 Tropica 2008;107:192-4.
62.     Moro PL, Nakao M, Ito A, Schantz PM, Cavero CL. Molecular identification of Echinococcus isolates from Peru. Parasitol Int 2009;58:184-6.
63.     Tigre W, Deresa B, HaileA, Gabriel S, Victor B, Pelt JV, et al. Molecular characterization of Echinococcus granulosus s.l. cysts from cattle, camels, goats and pigs in Ethiopia. Vet Parasitol 2016;215:17-21.
64.     Sharbatkhori M, Mirhendi H, Harandi MF, Rezaeian M, Mohebali M, Eshraghian M, et al. Echinococcus granulosus genotypes in livestock of Iran indicating high frequency of G1 genotype in camels. Exp Parasitol 2010;124:373-9.
65.     Pestechian N, Hosseini Safa A, Tajedini M, Rostami-Nejad M, Mousavi M, Yousofi H, et al. Genetic diversity of Echinococcus granulosus in center of Iran. Korean J Parasitol 2014;52:413-8.
66.     Spotin A, Mahami-Oskouei M, Harandi MF, Baratchian M, Bordbar A, Ahmadpour E, et al. Genetic variability of Echinococcus granulosus complex in various geographical populations of Iran inferred by mitochondrial DNA sequences. Acta Tropica 2017;165:10‑6