Introduction
Cystic echinococcosis (CE), an important zoonotic disease which is widespread around the world, is caused by the larval stages of Echinococcus granulosus, a helminth from the family Taeniidae. Carnivores, particularly dogs and other canines, serve as definitive hosts. Intermediate hosts, which harbour metacestodes, include a wide range of wild and domestic herbivores. Humans may serve as accidental hosts and the consumption of food contaminated with parasite eggs or the direct transmission of eggs from dogs can lead to infection (Craig & Larrieu, Reference Craig and Larrieu2006). Past taxonomical and molecular studies on E. granulosus demonstrated that this species is composed of several strains that are genetically different from one another (Farhadi et al., Reference Farhadi, Fazaeli and Haniloo2015). Until now, ten distinct genotypes (G1–G10) have been detected, including the species E. granulosus (genotypes G1, G2, G3; sheep and buffalo strains), E. equinus (G4; horse strain), E. ortleppi (G5; cattle strain) and E. canadensis (G6–G10) (Thompson, Reference Thompson2008; Alvarez Rojas et al., Reference Alvarez Rojas, Romig and Lightowlers2014). Later investigation by Lymbery et al. (Reference Lymbery, Jenkins, Schurer and Thompson2015) demonstrated that the G6 (camel strain) and G7 (pig strain) genotypes are single species which are different from the G8 and G10 genotypes (cervid strains). Hence, the G6/G7 genotypes are named E. intermedius, G8 is named E. borealis and G10 is named E. canadensis (Lymbery et al., Reference Lymbery, Jenkins, Schurer and Thompson2015).
CE is a cosmopolitan zoonotic disease, with a high prevalence rate in livestock and humans in some parts of America (especially South America), Australia, northern and eastern Africa, southern and central parts of Russia, central Asia, China and parts of the Mediterranean region (Grosso et al., Reference Grosso, Gruttadauria, Biondi, Marventano and Mistretta2012). In Iran, hydatidosis is an important public-health problem and the disease is observed in different parts of the country. In livestock, the total prevalence of CE is estimated to be 5.9% in sheep, 8.8% in cattle, 6.4% in goats, 16.5% in buffalos and 32.7% in camels (Khalkhali et al., Reference Khalkhali, Foroutan, Khademvatan, Majidiani, Aryamand, Khezri and Aminpour2017). The data on operated cases revealed that, in this country, 1.18–3 cases per 100,000 surgeries in hospitals are related to hydatidosis (Ghaffari, Reference Ghaffari1999; Fasihi Harandi et al., Reference Fasihi Harandi, Budke and Rostami2012). The total prevalence of cystic echinococcosis in humans is calculated to be 4.2% in Iran (Khalkhali et al., Reference Khalkhali, Foroutan, Khademvatan, Majidiani, Aryamand, Khezri and Aminpour2017).
Molecular data and the sequencing of different isolates from livestock have proved the presence of various genotypes (G1–G10) of E. granulosus around the world (Grosso et al., Reference Grosso, Gruttadauria, Biondi, Marventano and Mistretta2012). G1 is the most commonly identified genotype (72.9%) in humans and animals throughout the world (McManus, Reference McManus2013). In Iran, restriction fragment length polymorphism (RFLP) and sequence analysis have detected different genotypes, including G1–G3, G6 and G7, in animal and human isolates. To date, no study has been conducted regarding the sequencing of different genotypes of E. granulosus and the genetic characterization of CE in the central part of Mazandaran province, northern Iran. Hence, the present study was designed to identify E. granulosus genotypes in different host species using polymerase chain reaction (PCR)-RFLP of internal transcribed spacer-1 (ITS1) ribosomal DNA and the sequencing of the cytochrome c oxidase subunit 1 (Cox1) mitochondrial gene for the first time in this region.
Materials and methods
Collection and examination of hydatid cysts
In the current study, 63 hydatid cyst samples were collected from December 2016 to March 2017. Fifty-seven livestock samples (40 sheep and 17 cattle) were obtained from a slaughterhouse in Babol, the central part of Mazandaran province. Six archived formalin-fixed paraffin-embedded tissues (FFPE) were obtained from human cases with a history of hydatid cyst surgery from Shahid Beheshti Hospital, Babol, Iran. Regarding the animal samples, the whole content of the cysts from the lung and liver were examined under a light microscope for the presence of protoscoleces. The protoscoleces were washed by double-distilled water and stored at −20°C until used.
DNA extraction and molecular assays
Genomic DNA from protoscoleces was extracted using a Tissue DNA extraction kit (Bio Basic, Markham, Ontario, Canada) according to the manufacturer's instructions. The quality and concentration of extracted DNA was measured with a spectrophotometer (NanoDrop® 2000C, Thermo Scientific, Waltham, Massachusetts, USA). The genomic DNA was stored at −20°C until PCR amplification.
For DNA extraction from FFPE samples, serial sections of 7-mm thickness were obtained from the FFPE blocks. The deparaffinization of sections was performed according to the procedure of Kalantari et al. (Reference Kalantari, Bayani and Ghaffari2016). Deparaffinized tissues were kept at −20°C until used. The DNA extraction of tissue samples was performed using a PCRBio rapid extract kit according to the manufacturer's protocol (PCR BioSystems, London, UK). After evaluating the quality of extracted DNA, the samples were stored at −20°C until used.
PCR-RFLP of the ITS1 gene
The primer pairs used in this study for the amplification of the ITS1 gene were EgF (5′-AGAGCACTTTTGTATGCA-3′) and EgR (5′-ATGGTTGTTATCGCTGCGA-3′), which produce a 460-bp fragment (described by Moghaddas et al., Reference Moghaddas, Borji, Naghibi, Shayan and Razmi2015). The DNA amplification was performed in a total volume of 25 μl, under the following temperature conditions: an initial denaturation step at 95°C for 5 min; 35 cycles at 94°C for 45 s (denaturation), 50°C for 45 s (annealing) and 72°C for 45 s (extension); with a final extension step at 72°C for 10 min. After the determination of PCR products in 2% agarose gels, the products were digested with the restriction enzyme Bsh1236I according to Moghaddas et al. (Reference Moghaddas, Borji, Naghibi, Shayan and Razmi2015). The products were electrophoresed in 3% agarose gel, inspected under UV light and photographed using a gel documentation system (Vilbert, Lourmat, France).
PCR amplification of the Cox1 gene
A fragment of the cytochrome c oxidase subunit 1 (Cox1) gene was amplified. The forward and reverse primers of the Cox1 gene were JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′), which were selected according to Bowles et al. (Reference Bowles, Blair and McManus1992) for the amplification of 450-bp fragments. The PCR amplification was performed in a total volume of 25 μl with the thermal program as follows: an initial denaturation step at 95°C for 5 min; 40 cycles at 94°C for 55 s (denaturation), 54.7°C for 55 s (annealing) and 72°C for 1 min (extension); and a final extension step at 72°C for 10 min. The PCR products were electrophoresed on a 2% agarose gel, and the bands were visualized under UV light and photographed using a gel documentation system (Vilbert).
PCR amplification of the bcl-2 gene
In order to confirm the extracted DNA from the FFPE samples, a fragment of the bcl-2 gene (cell death regulatory gene) was amplified using primer pairs bcl-2 forward (5′-TGGCCAGGGTCAGAGTTAAA-3′ and bcl-2 reverse (5′-TGGCCTCTCTTGCGGAGTA-3′). All stages of PCR amplification were carried out based on the PCR protocols described by Liu et al. (Reference Liu, Wang, Ren, Qu, Sun, Zhang, Qiu and Wang2014).
Sequencing
The PCR products of the Cox1 mitochondrial gene were purified and subjected to sequencing with both forward and reverse primers by Bioneer Company (South Korea). The sequencing results were analysed using Chromas (v.2.6.4) software and compared with each other and the reference sequence in the GenBank database by BLAST analysis (www.ncbi.nlm.nih.gov).
The multiple alignments of sequences were performed using the ClustalW method of Mega (v.6) software (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011). The phylogenetic analysis of the different haplotypes obtained from this work, together with the reference sequences of E. granulosus genotypes (G1–G10) and other Echinococcus species, was constructed according to the neighbour-joining (NJ) method using Mega (v.6) software. For the comparison of a relevant outgroup sequence, a sequence of Taenia saginata was also selected.
Results and discussion
In total, 63 CE samples, including 40 sheep and 17 cattle along with 6 human isolates, were evaluated. In the animal isolates, 29 samples were obtained from the liver and 28 samples were from the lung. In human samples, 4 samples were obtained from the liver and 1 sample each from the lung and the peritoneal cavity. Amplification of the bcl-2 gene was used as an internal control for human samples, and a 152-bp fragment was amplified in the all extracted DNA from FFPE tissue samples.
In the present study, PCR-RFLP of the ITS1 gene and digestion with Bsh1236I was performed, as previously examined by Moghaddas et al. (Reference Moghaddas, Borji, Naghibi, Shayan and Razmi2015). The application of this method had several advantages, including reduction in PCR program time, reduction of digestion time and cost effectiveness. Here, the ITS1 gene was amplified successfully in 5 human and 37 animal samples (9 cattle, 28 sheep). The RFLP with Bsh1236I showed that 100% of isolates had G1–G3 genotypes. Our findings are supported by the results of previous studies performed in different parts of Iran (Kia et al., Reference Kia, Rahimi, Sharbatkhori, Talebi, Fasihi Harandi and Mirhendi2010; Hajialilo et al., Reference Hajialilo, Sharbatkhori, Mirhendi and Rostami2012; Khademvatan et al., Reference Khademvatan, Yousefi, Rafiei, Rahdar and Saki2012; Pezeshki et al., Reference Pezeshki, Akhlaghi, Sharbatkhori, Razmjou, Oormazdi, Mohebali and Meamar2012; Dousti et al., Reference Dousti, Abdi, Bakhtiyari, Mohebali, Mirhendi and Rokni2013; Hanifian et al., Reference Hanifian, Diba, Tappeh, Mohammadzadeh and Mahmoudlou2013).
All human and animal isolates were further evaluated using the Cox1 gene. A 450-bp fragment was amplified in 2 human, 9 cattle and 20 sheep samples. Two-direction sequencing was carried out for these isolates. BLAST analysis showed 100% identity, 100% coverage and 0.0 E-value with reference sequences in GenBank (accession number KX269858.1). All human isolates had G1 genotypes. In cattle, 8 (88.8%) isolates had the G1 genotype and only 1 (11.1%) had the G3 genotype. G1 and G2 genotypes were identified in 15 (75%) and 5 (25%) of the sheep isolates, respectively. The G2 genotype was also observed in one cattle sample and the overall frequency of the G2 genotype was 16.1% (table 1). These outcomes show that G1 is the most common genotype (80.6%). According to several studies, G1 is the most commonly identified genotype in livestock and humans worldwide (Eryıldız & Şakru, Reference Eryıldız and Şakru2012; McManus, Reference McManus2013; Sharma et al., Reference Sharma, Sehgal, Fomda, Malhotra and Malla2013; Alvarez Rojas et al., Reference Alvarez Rojas, Romig and Lightowlers2014; Hu et al., Reference Hu, Wang and Zhong2015; Sharbatkhori et al., Reference Sharbatkhori, Tanzifi, Rostami, Rostami and Harandi2016). The molecular and sequencing data on CE in Iran shows that the G2 genotype has only been reported in cattle and humans (Rostami et al., Reference Rostami, Torbaghan, Dabiri, Babaei, Mohammadi, Sharbatkhori and Harandi2015; Sharbatkhori et al., Reference Sharbatkhori, Tanzifi, Rostami, Rostami and Harandi2016). The comparison of our results with those of other studies mentioned above shows that this genotype is infrequent in our country and it is limited to the northern part of Iran. The other genotype detected in the present study was G3, which was observed in one cattle isolate. However, the results of other investigations in Iran revealed that after G1, the G3 genotype is more widespread in our country (Hajialilo et al., Reference Hajialilo, Sharbatkhori, Mirhendi and Rostami2012; Nikmanesh et al., Reference Nikmanesh, Mirhendi, Ghalavand, Alebouyeh, Sharbatkhori, Kia, Mohebali, Eghbali and Rokni2014; Pestechian et al., Reference Pestechian, Hosseini Safa, Tajedini, Rostami-Nejad, Mousavi, Yousofi and Haghjooy Javanmard2014). Another commonly reported genotype in Iran is G6, which was not observed in our work (Sadjjadi et al., Reference Sadjjadi, Mikaeili, Karamian, Maraghi, Sadjjadi, Shariat-Torbaghan and Kia2013; Moghaddas et al., Reference Moghaddas, Borji, Naghibi, Shayan and Razmi2015; Sharbatkhori et al., Reference Sharbatkhori, Tanzifi, Rostami, Rostami and Harandi2016). The absence of the G6 genotype could be due to the lack of camel breeding and consumption of camel meat in this region. These results indicate that the sheep–dog cycle is the most common transmission route of cystic echinococcosis in the study region.
Echinococcus granulosus genotypes and haplotypes obtained from Cox1 gene sequence analysis in different host species, along with reference sequences deposited in GenBank.
In the present study, ten different haplotypes were found among the genotypes by alignment analysis. H1–H2 in the G1 group was the most commonly identified haplotype. The nucleotide sequence of H1–H2 had 100% similarity with haplotypes reported previously from Jordan (G01; AB491414), Europe (EG1; JF513058) and Iran (Golc1; KT074941) (Yanagida et al., Reference Yanagida, Mohammadzadeh and Kamhawi2012; Sharbatkhori et al., Reference Sharbatkhori, Tanzifi, Rostami, Rostami and Harandi2016). The haplotype H3 in our study was similar to the EG4 haplotype detected by Casulli et al. (Reference Casulli, Interisano, Sreter, Chitimia, Kirkova, La Rosa and Pozio2012), which is the common haplotype in Eastern European populations. The H4 haplotype (KM513627, KT074945) is similar to the haplotypes reported in Golestan province, Iran (Casulli et al., Reference Casulli, Interisano, Sreter, Chitimia, Kirkova, La Rosa and Pozio2012; Sharbatkhori et al., Reference Sharbatkhori, Tanzifi, Rostami, Rostami and Harandi2016). Other haplotypes found in the current study were not similar to haplotypes detected in Iran and other parts of the world, which could indicate that these are new haplotypes.
The genetic relationship of all ten haplotypes with reference sequences obtained from GenBank, assessed by the NJ algorithm using Mega (v.6) software, is illustrated in fig. 1.
Genetic relationships between E. granulosus isolates from CE in human and animal samples in the present study and reference sequences of G1–G10 genotypes. The phylogenetic tree was performed by neighbour-joining algorithm, with Taenia saginata as the outgroup. Haplotypes 1–10 were clustered in the G1–G3 genotype complex of E. granulosus.
In conclusion, the molecular analysis of E. granulosus showed that the common sheep strain G1 is predominant in the northern part of Iran, affecting human, sheep and cattle populations. In addition, infected livestock can act as potential reservoirs for human infection. Also, the results of our study showed high genotypic diversity among these isolates. Finally, the results presented here can help improve the implementation of control programmes against cystic echinococcosis and the promotion of public health.
Acknowledgements
The authors would like to thank all the people who helped with this study. We would also like to thank Ms Taraneh Ghaffari for language editing.
Financial support
This study was supported by the Babol University of Medical Science (grant number 9604415).
Conflict of interest
None.
Ethical standards
This study was approved by the Babol University of Medical Science, ethical code number MUBABOL.HRI.REC.1396.154 3943.
