Abstract

INTRODUCTION

The larval stage (metacestode) of Echinococcus granulosus, the causative agent of cystic echinococcosis (CE), is the source of a globally distributed zoonotic parasitic disease that causes major medical, veterinary and economic losses in endemic countries, including Iran¹1. McManus DP, Zhang W, Li J, Bartley PB. Echinococcosis. Lancet. 2003;362:1295-304.^,22. Moro P, Schantz PM. Echinococcosis: a review. Int J Infect Dis. 2009;13:125-33..

The adult stage of the cestode resides in the small intestine of carnivores, being the domestic and wild canids, the definitive hosts. The intermediate host that harbours metacestodes (hydatid cysts) in liver, lungs and other organs can be one of the numerous species of domestic and wild ungulates, including sheep, goats, cattle, buffalos, camels. Humans may be infected through the accidental intake of parasite eggs in contaminated water or vegetables, or by direct contact with dogs³3. Eckert J, Gemmell MA, Meslin FX, Pawlowski ZS, editors. WHO/OIE manual on echinococcosis in humans and animals: a public health problem of global concern. Paris: WHO; 2001.. CE imposes a considerable economic impact in Iran⁴4. Fasihi Harandi M, Budke CM, Rostami S. The monetary burden of cystic echinococcosis in Iran. PLoS Negl Trop Dis. 2012;6:e1915.. A number of studies have estimated the prevalence of CE in Iranian livestock to be between 5.1% and 74.4% in sheep; 3.5% and 38.3% in cattle; 2% and 20% in goats; 11.9% and 70% in buffalos; and between 25.7% and 59.3% in camels⁵5. Dalimi A, Motamedi G, Hosseini M, Mohammadian B, Malaki H, Ghamari Z, et al. Echinococcosis/hydatidosis in western Iran. Vet Parasitol. 2002;105:161-71.^,66. Rokni M. Echinococcosis/hydatidosis in Iran. Iran J Parasitol. 2009;4:1-16.. Human cases of CE are widespread in Iran and are routinely reported from medical centres and hospitals across the country, including approximately 1% of all surgical cases⁶6. Rokni M. Echinococcosis/hydatidosis in Iran. Iran J Parasitol. 2009;4:1-16.^,77. Sadjjadi SM. Present situation of echinococcosis in the Middle East and Arabic North Africa. Parasitol Int. 2006;55 Suppl:S197-202..

In the past 50 years, significant phenotypic and genetic variation has been revealed among E. granulosus isolates from different intermediate host species in several geographical areas. This led to the establishment of new species and genotypes. The understanding the E. granulosus species and genotypes has had a significant impact on the epidemiology and control strategies for the disease, as well as for future vaccine and drug design⁸8. McManus DP. Current status of the genetics and molecular taxonomy of Echinococcus species. Parasitology. 2013;140:1617-23.^,99. Thompson RC. The taxonomy, phylogeny and transmission of Echinococcus. Exp Parasitol. 2008;119:439-46.. Based mainly on the E. granulosus mitochondrial DNA-based studies, it has been shown that E. granulosus comprises 10 genotypes (G1-G10), which have been characterized as distinct species, comprising E. granulosus sensu stricto (G1, G2 and G3); E. equinus (G4); E. ortleppi (G5); and the controversial group formed by G6-G10 species that according to some authors should be regarded as one species, and to others such as the three species namely E. canadensis, E. borealis and E. intermedius. The validity of the G9 genotype is not clear⁹9. Thompson RC. The taxonomy, phylogeny and transmission of Echinococcus. Exp Parasitol. 2008;119:439-46.

10. 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.

11. Lavikainen A, Haukisalmi V, Lehtinen MJ, Henttonen H, Oksanen A, Meri S. A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data. Parasitology. 2008;135:1457-67.

12. Saarma U, Jõgisalu I, Moks E, Varcasia A, Lavikainen A, Oksanen A, et al. A novel phylogeny for the genus Echinococcus, based on nuclear data, challenges relationships based on mitochondrial evidence. Parasitology. 2009;136:317-28.

13. Moks E, Jõgisalu I, Valdmann H, Saarma U. First report of Echinococcus granulosus G8 in Eurasia and a reappraisal of the phylogenetic relationships of 'genotypes' G5-G10. Parasitology. 2008;135:647-54.^-1414. Lymbery AJ, Jenkins EJ, Schurer JM, Thompson RC. Echinococcus canadensis, E. borealis, and E. intermedius. What's in a name. Trends Parasitol. 2015;31:23-9.. Recently, the lion strain has been proposed as another new species and E. felidis was settled as a sister taxon of E. granulosus sensu stricto¹⁵15. 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..

Several molecular studies performed in Iran have shown the occurrence of E. granulosus sensu stricto (G1-G3) and E. canadensis (G6)¹⁶16. Harandi MF, Hobbs RP, Adams PJ, Mobedi I, Morgan-Ryan UM, Thompson RC. Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran. Parasitology. 2002;125:367-73.

17. Sharbatkhori M, Fasihi Harandi M, Mirhendi H, Hajialilo E, Kia E. Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran. Parasitol Res. 2011;108:521-27.

18. 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.

19. Sharbatkhori M, Kia EB, Fasihi Harandi M, Jalalizand N, Zahabiun F, Mirhendi H. Comparison of five simple methods for DNA extraction from Echinococcus granulosus protoscoleces for PCR amplification of ribosomal DNA. Iran J Parasitol. 2009;4:54-60.

20. Hajialilo E, Fasihi Harandi M, 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.

21. Pezeshki A, Akhlaghi L, Sharbatkhori M, Razmjou E, Oormazdi H, Mohebali M, et al. Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran. J Helminthol. 2013;87:387-91.^-2222. Pour A, Hosseini S, Shayan P. Comparative genotyping of Echinococcus granulosus infecting buffalo in Iran using cox1 gene. Parasitol Res. 2011;108:1229-34.. Due to the lack of more accurate data from this region, the present study has investigated the population genetic pattern of E. granulosus isolated from livestock and humans by sequencing and the phylogenetic analysis of cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes.

MATERIALS AND METHODS

Collection of samples

The present cross-sectional study was performed from April 2011 to July 2012. Hydatid cysts of liver and lung tissues were collected from sheep, goats, cattle, buffalos and camels, in different slaughterhouses, in Golestan province of northern Iran, southeast of the Caspian sea (Table 1). Cysts that did not contain parasites or calcified cysts were excluded from the study. Molecular techniques were conducted on isolates using clean cyst fluid samples and some whitish germinal layer. Additionally, formalin-fixed paraffin-embedded tissues (FFPT) from patients with histologically confirmed hydatid cysts coming from a private hospital of Gorgan were also evaluated in this study (Table 1). Protoscoleces from individual cysts were aspirated and washed three times with normal saline and preserved at -20 °C until used for the molecular analysis.

DNA Extraction

Isolates underwent four freeze and thaw cycles in liquid nitrogen alternated with a passage in a water bath at 95 °C. Samples were then suspended in 200 µL of tissue lysis buffer and 80 µL of proteinase K and incubated at 56 °C overnight²³23. Kamenetzky L, Canova SG, Guarnera EA, Rosenzvit MC. Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts. Exp Parasitol. 2000;95:122-7.. Subsequently, genomic DNA was isolated from the homogenised suspension using a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) according to the manufacturer's recommended protocol.

Extracting DNA from FFPE tissues of human CE was performed using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

A spectrophotometer (NanoDrop(r) ND-1000, Thermo Scientific, Massachusetts, USA) was used to ensure the quality of DNA extraction. The genomic DNA was kept at -20 °C until amplification.

Individual genomic DNA samples were analysed using amplification of two mitochondrial DNA fragments within cox1 and nad1 genes, separately. JB3 (TTTTTTGGGCATCCTGAGGTTTAT) and JB4.5 (TAAAGAAAGAACATAATGAAAAT G) sequences were used as cox1 forward and reverse primers, and MS1 (AGATTCGTAAGGGGCCTAATA) and MS2 (ACCACTAACTAATTCACTTTC) sequences were used as nad1 forward and reverse primers, respectively¹⁸18. 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.. PCR was carried out in a final volume of 50 µL, including 4 µL (50-100 ng) of genomic DNA, 3.5 mM MgCl₂, 250 µM of dNTPs, 25 pmol of each primer and 2 U of Taq polymerase, under the following conditions: 35 cycles of 94 °C for 30 s, 50 °C for 45 s, 72 °C for 35 s, followed by a final extension of 72 °C for 10 min Negative (no added DNA) and positive controls were included in each PCR experiment. PCR products were analysed by electrophoresis in ethidium bromide-stained 1% agarose gels prepared in TBE buffer (65 mM Tris-HCl, 22.5 mM boric acid, 1.25 mM EDTA, pH 9). The gels were visualized using an UV transilluminator (UVitec, Cambridge, UK).

DNA sequencing and phylogenetic analysis

A panel of 74 PCR amplicons for each cox1 and nad1 gene was purified and subjected to sequencing in two directions, using the same forward and reverse PCR primers.

The electropherogram of each sequence was visually checked and the sequences were compared to each other and with reference sequences using the BioEdit²⁴24. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-8. and the BLAST softwares available at http://www.ncbi.nlm.nih.gov/. The representative sequences for both cox1 and nad1 genes were submitted to GenBank.

Three separate phylogenetic analyses of sequencing data were conducted (i) using: pcox1 data for sequences determined in the present study only, and pcox1 data for T. saginata as the outgroup; (ii) pnad1 data for sequences determined in the present study only, and pnad1 data for T. saginata as the outgroup; (iii) concatenated pcox1+pnad1 data representing all genetic variations detected in the present study, previously described E. granulosus genotypes (G1-G10), Echinococcus species along with T. saginata as the outgroup. The character-based Bayesian inference method (BI) was used for the analyses. BI was executed using the software MrBayes v.3.1.2 available at http://mrbayes.csit.fsu.edu/index.php. Posterior probabilities (pp) were designed for 2,000,000 generations (ngen: 2,000,000; burnin: 20 000) by means of the Monte Carlo Markov Chain method and four simultaneous tree-building chains (nchains:4), with each 100^th tree saved (samplefreq:100). The evolutionary distance was determined using the General Time Reversible evolutionary model (nset: 6), allowing for a gamma-shaped variation in mutation rates between codons (rates: g). The TreeviewXv.0.5.0 program²⁵25. Page RD. TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996;12:357-8. was used to indicate the consequence tree.

RESULTS

Seventy-four CE isolate fragments of approximately 450 bp and 400 bp long were successfully amplified within cox1 and nad1 genes, respectively. The obtained consensus sequences of cox1 and nad1 genes were 366 bp and 378 bp, respectively. Fifty-eight (78.3%), 2 (2.7%), 11 (15%) and 3 (4%) isolates belonged to the G1, G2, G3 and G6 genotypes, respectively. All four human CE isolates belonged to the G1 genotype (Table 1). The sequence alignments of the isolates displayed eight characteristic profiles in cox1 sequences and 5 characteristic profiles in nad1 sequences. Sequence profiles for cox1 (designated as Golc1-8) and nad1 (designated as Goln1-5) were submitted to GenBank, accession numbers KM513626- KM513633 and KM513634- KM513638, respectively. As some E. granulosus sequences were the same in different hosts, the equal sequence profile in different hosts was named as sub-numbers and submitted to GenBank with the related accession numbers KT074941- KT074949 and KT074936- KT074940 for cox1 and nad1 genes, respectively. For example Golc6 (AN: KM513631), and Golc6-1 (AN: KT074949), have the same cox1 sequence in cattle and camel hosts, respectively (Table 2).

Separate phylogenetic analyses of pcox1 and pnad1 data sets were conducted, and all the combinations of cox1 and nad1 sequence types, representing all the 74 isolates in the present study were determined. These analyses revealed a concordance between the genotypic classification of pcox1 and pnad1, inferring the utility of combined pcox1 and pnad1 data to access the haplotypic variation among E. granulosus. Hence, each pair of pcox1 and pnad1 sequence types (e.g. Golc1- Golc8 and Goln1- Goln5) was used to define the ''working'' haplotypes (see Table 2 and Fig. 1). In all the cases, concatenated reference sequences represented the same isolate (i.e. Golc1 and Goln3 sequences were derived from the same isolate representing the haplotype 2 (H2) in the Table 2). A data set representing the concatenated pcox1+pnad1 sequences for all the 15 haplotypes (H1-H15) detected in this study was employed, along with key reference data (comprising concatenated pcox1+pnad1 sequences from previous studies representing all the recognized Echinococcus species and E. granulosus ''genotypes'', with T. saginata as the outgroup; see Table 2 and Fig. 1).

Phylogenetic analyses of concatenated data, for the haplotypes 1-15, were performed by using BI, including representative sequence data for all the recognized species of Echinococcus and genotypes of E. granulosus, as well as T. saginata as an outgroup (Table 2 and Fig. 1). A consensus tree has been built and is shown in Figure 1.

Most of the isolates (78.3%) were identified as G1 and were clustered in the G1 reference genotype (accession nos: cox1, U50464; nad1, AJ237632). Isolates that were identified as G2 (2.7%) were clustered in the G2 reference sequences (accession nos: cox1, M64662; nad1. AJ237633). Isolates identified as G3 (14.86%) clustered in the G3 reference sequences (accession nos: cox1, M64663; nad1. AJ237634) and isolates identified as G6 (13.1%) were clustered in the G6 reference sequences (accession nos: cox1, M84666; nad1, AJ237637) (Fig. 1).

H1-H9 represents the G1 genotype, H10 belongs to the G2 genotype, H11-H14 represents the G3 genotype, and H15 belongs to the G6 genotype. A consensus tree based on the phylogenetic analyses of concatenated cox1 and nad1 sequences revealed two distinct clusters. One cluster contained a G1-G3 complex (pp = 1.00) and the other cluster contained the G6-G10 complex (pp = 1.00). Fourteen haplotypes (H1-H14) were found in the G1-G3 cluster, and one haplotype was placed in the G6-G10 complex, the E. canadensis (Fig. 1).

DISCUSSION

The results of this study showed that the G1 genotype (E. granulosus sensu stricto) was the most prevalent identified genotype among the 74 CE isolates from the Golestan province, northern Iran. The G1 genotype was identified in all of the sheep, goat, buffalo and human isolates. Furthermore, 72.5% of the cattle and approximately half of the camel (44.44%) isolates also belonged to the G1 genotype. Two (5%) and nine (22.5%) of the cattle isolates had the G2 and G3 genotype, respectively. Also, two (22.2%) and three (33.3%) camel isolates showed the G3 and G6 genotypes, respectively.

A recent study performed in this region using the ITS1-RFLP method, has reported the G1 genotype in all the human, sheep and cattle isolates and both, G1 and G6 genotypes, in camel isolates³¹31. Gholami S, Sosari M, Fakhar M, Sharif M, Daryani A, Hashemi M, et al. Molecular characterization of Echinococcus granulosus from hydatid cysts isolated from human and animals in Golestan province, north of Iran. Iran J Parasitol. 2012;7:8-16.. However, the G1-G3 genotypes cannot be differentiated by the ITS1-RFLP. In another study, all the five cattle and 10 sheep isolates from the Golestan province had the G1 genotype¹⁸18. 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.. The finding of the G1 genotype as the most prevalent one suggests that the sheep-dog cycle is dominant in CE from the Golestan province. G1 is the most frequent genotype found in humans and livestock throughout the world³²32. Casulli A, Manfredi MT, La Rosa G, Cerbo ARD, Genchi C, Pozio E. Echinococcus ortleppi and E. granulosus G1, G2 and G3 genotypes in Italian bovines. Vet Parasitol. 2008;155:168-72.^,3333. Yan N, Nie HM, Jiang ZR, Yang AG, Deng SJ, Guo L, et al. Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences. Vet Parasitol. 2013;196:179-83.; however, in some north African countries, including Mauritania and Sudan, G6 is the most common genotype in sheep, cattle, camels and humans³⁴34. Bardonnet K, Piarroux R, Dia L, Schneegans F, Beurdeley A, Godot V, et al. Combined eco-epidemiological and molecular biology approaches to assess Echinococcus granulosus transmission to humans in Mauritania: occurrence of the camel strain and human cystic echinococcosis. Trans R Soc Trop Med Hyg. 2002;96:383-6.^,3535. Omer RA, Dinkel A, Romig T, Mackenstedt U, Elnahas AA, Aradaib IE, et al. A molecular survey of cystic echinococcosis in Sudan. Vet Parasitol. 2010;169:340-6..

Although the E. granulosus G2 genotype was primarily introduced as a Tasmanian sheep strain, it was later found in other hosts, including goats, cattle, buffalos, camels and humans, from different countries. In Iran, this genotype has been previously reported and dogs are the definitive hosts. To the best of our knowledge, the present study reports the first occurrence of E. granulosus G2 genotype in intermediate hosts in this country.

The E. granulosus G3 genotype has been previously reported in humans, sheep, goats, cattle and camels in different countries, including Iran²⁰20. Hajialilo E, Fasihi Harandi M, 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.^,2121. Pezeshki A, Akhlaghi L, Sharbatkhori M, Razmjou E, Oormazdi H, Mohebali M, et al. Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran. J Helminthol. 2013;87:387-91.^,3636. 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.

37. Espinoza S, Salas AM, Vargas A, Freire V, Diaz E, Sánchez G, et al. Detection of the G3 genotype of Echinococcus granulosus from hydatid cysts of Chilean cattle using cox1 and nd1 mitochondrial markers. Parasitol Res. 2014;113:139-47.

38. Umhang G, Richomme C, Boucher JM, Hormaz V, Boué F. Prevalence survey and first molecular characterization of Echinococcus granulosus in France. Parasitol Res. 2013;112:1809-12.

39. Piccoli L, Bazzocchi C, Brunetti E, Mihailescu P, Bandi C, Mastalier B, et al. Molecular characterization of Echinococcus granulosus in south-eastern Romania: evidence of G1-G3 and G6-G10 complexes in humans. Clin Microbiol Infect. 2013;19:578-82.

40. Singh BB, Sharma JK, Ghatak S, Sharma R, Bal MS, Tuli A, et al. Molecular epidemiology of Echinococcosis from food producing animals in north India. Vet Parasitol. 2012;186:503-6.

41. Busi M, Snabel V, De Liberato C, D'Amelio S. Molecular genotyping of Echinococcus granulosus hydatid cysts in Italy reveals the presence of three distinct genotypes. Parassitologia. 2004;46 Suppl 1:164.^-4242. M'rad S, Oudni-M'rad M, Filisetti D, Mekki M, Nouri A, Sayadi T, et al. Molecular identification of Echinococcus granulosus in Tunisia: first record of the Buffalo strain (G3) in human and bovine in the country. Open Vet Sci J. 2010;4:27-30.. The present study found this genotype in nine of 40 cattle (22.5%) and two of nine camels (22.2%). However, no sheep, goat, buffalo, or human isolates harboured the G3 genotype in the present study.

Most of the human CE isolates from Iran have been reported as belonging to the G1 genotype. This is consistent with the findings of the present study, as all four human FFPE tissues had the G1 genotype. In a study from the Isfahan province of central Iran, using PCR-RFLP, all 30 human CE isolates were reported as belonging to the G1 genotype⁴³43. Kia EB, Rahimi H, Sharbatkhori M, Talebi A, Fasihi Harandi M, Mirhendi H. Genotype identification of human cystic echinococcosis in Isfahan, central Iran. Parasitol Res. 2010;107:757-60.. Another study, using SSCP coupled to partial cox1 and nad1 sequencing, found that all 23 human CE originally from central Iran had the G1 genotype¹⁸18. 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.. A study in the Ardabil province of north western Iran reported seven and two human CE isolates belonging to the G1 and G3 genotypes, respectively²¹21. Pezeshki A, Akhlaghi L, Sharbatkhori M, Razmjou E, Oormazdi H, Mohebali M, et al. Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran. J Helminthol. 2013;87:387-91.. Harandi et al., using a PCR-RFLP method, reported 33 and three human CE isolates as belonging to the G1 and G6 genotypes, respectively¹⁶16. Harandi MF, Hobbs RP, Adams PJ, Mobedi I, Morgan-Ryan UM, Thompson RC. Molecular and morphological characterization of Echinococcus granulosus of human and animal origin in Iran. Parasitology. 2002;125:367-73.. The only human CE isolate from the Kerman province of south western Iran showed a G6 genotype²⁰20. Hajialilo E, Fasihi Harandi M, 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.. A recent study reported the G6 genotype in all eight FFPE tissues from cerebral Echinococcus cysts in patients from a Tehran hospital. The authors also claimed that the G6 genotype has a higher affinity for the human brain than the G1 genotype⁴⁴44. Sadjjadi SM, Mikaeili F, Karamian M, Maraghi S, Sadjjadi FS, Shariat-Torbaghan S, et al. Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans. Int J Parasitol. 2013;43:875-7.. In a study on 125 human isolates conducted by Rostami et al. (2015), G1 and G6 genotypes were shown to be present in 54.4 and 40.8% of the isolates, respectively. The G6 genotype is especially prevalent in south-eastern parts of the Iran where humans were slightly more infected by camel strains (G6) than G1 strains (45.8 vs. 41.7%). These data show that humans are quite susceptible to infections by the G6 genotype (E. canadensis), and also that there is an active camel-dog cycle in many parts of the country with camel and sheep as potential intermediate hosts. The results of the present study indicate the interaction of the camel-dog and the sheep-dog cycles in this Iranian region⁴⁵45. Rostami S, Shariat Torbaghan S, Dabiri S, Babaei Z, Ali Mohammadi M, Sharbatkhori M, et al. Genetic characterization of Echinococcus granulosus from a large number of formalin-fixed, paraffin-embedded tissue samples of human isolates in Iran. Am J Trop Med Hyg. 2015;92:588-94.. In the present study, two of nine (22.2%) camel isolates had the G6 genotype. This finding is almost entirely in accordance with a previous study conducted on 19 camel isolates from central Iran that found the G6 genotype in 31.6% of isolates, with most of the isolates belonging to E. granulosus sensu stricto (68.4%)¹⁷17. Sharbatkhori M, Fasihi Harandi M, Mirhendi H, Hajialilo E, Kia E. Sequence analysis of cox1 and nad1 genes in Echinococcus granulosus G3 genotype in camels (Camelus dromedarius) from central Iran. Parasitol Res. 2011;108:521-27.. Furthermore, the low frequency of the G6 genotype in this study may be the result of low breeding and slaughtering of camels in this region.

In the present study, most isolates designated as haplotypes 1 to14 (H1-H14), formed a strongly supported clade (pp = 1.00) together with reference sequences representing E. granulosus G1-G3 genotypes (E. granulosus sensu stricto), and the exclusion of E. felidis (pp = 1.00). H15 as the only haplotype belonging to the G6 genotype was clustered in the E. granulosus G6-G10 genotypes (known as the E. canadensis G6 genotype) with a maximal statistical support (pp = 1.00); a strong support was also placed in a smaller cluster in the G6 and G7 genotypes (pp = 0.98).

There are still controversies on the nature of E. canadensis (G6, G7, G8 and G10). The G7 (pig strain) is predominantly found in Europe while the G6 genotype has been found in central Asia, the middle east/ north Africa and South America with a very distinct epidemiological and biological context in comparison with G8 and G10 genotypes. According to Lymbery et al. (2015), G6 and G7 are not believed to be sympatric in most parts of the globe and the nomenclature of G6-G10 genotypes warrants more precise explanation. The division of Echinococcus granulosus sensu lato into G-numbers is a relic of the 1990s and should be reconsidered¹⁴14. Lymbery AJ, Jenkins EJ, Schurer JM, Thompson RC. Echinococcus canadensis, E. borealis, and E. intermedius. What's in a name. Trends Parasitol. 2015;31:23-9.^,4646. Nakao M, Lavikainen A, Yanagida T, Ito A. Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae). Int J Parasitol. 2013;43:1017-29.. This is especially true for the micro-variants G1-3 and G6/7, whose biological relevance are largely questionable.

The present study found that the predominant genotype is G1 (78.4%), as in other areas of the country, and describes the first report of the G2 genotype identified in cattle hosts. Also, this study confirms previous reports of the G3 genotype in camels and cattle species in the country. As humans can be infected with G2 and G3 genotypes, the epidemiological implication of cattle and camels in maintaining the transmission cycle of different E. granulosus genotypes warrants more attention.

ACKNOWLEDGMENTS

The authors would like to thank all the veterinary staff of different slaughterhouses that helped the collection of samples for this study. This work was performed as part of a MSc. thesis carried out by A.T. and was equally financially supported by the Vice-Chancellor for Research of Kerman University of Medical Sciences, grant No. 90-435; and the Vice-Chancellor for Research of Golestan University of Medical Sciences, grant No. 35/808.

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