DOI: https://doi.org/10.21203/rs.3.rs-1706309/v1
Background: Simultaneous isoenzyme and molecular characterization of Echinococcus granulosus sensu lato have not been investigated in Iran. The present study aims to investigate the isoenzyme pattern of the larval stage of Echinococcus spp. genotypes based on their host types in Iran.
Methods and Results: A total of 32 samples, including 8 human and 24 animal hydatid cyst isolates, were collected from May 2018 to December 2020. The protoscolices were removed from the cyst, and their genotypes were determined by the molecular method followed by isoenzyme electrophoresis. The activities of five enzymatic systems including glucose 6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), malic enzyme (ME), nucleoside hydrolyse 1 (NH1) and isocitrate dehydrogenase (ICD) were evaluated. Sequence analysis of the samples showed the specimens as E. granulosus sensu stricto (G1) 24 (75%), and E. canadensis (G6) 8 (25%) genotypes. The isoenzyme pattern of ICD of both the strains produced a six-band pattern, with different Relative Factors (RF). The G6PD produces two bands with different relative migration in both genotypes. The MDH and NH1 systems revealed two-band pattern in the E. granulosus sensu stricto strain, while it produced only one band in the ME enzyme. Furthermore, in the E. canadensis strain the MDH and NH1 enzymes showed one band and the ME enzyme represented a two-band pattern.
Conclusion: The finding of the present study revealed that the entirely different isoenzyme patterns in the systems of NH1, G6PD, MDH and ME may be used for differentiation of E. granulosus sensu stricto and E. canadensis strains.
Parasitic diseases, especially in developing countries are a major health problem worldwide [1]. Cystic echinococcosis (CE) is a cosmopolitan parasitic zoonosis caused by Echinococcus sensu lato [2]. More than one million people are affected with echinococcosis at any given time in the World Health Organization (WHO) reports [3]. Furthermore, it is ranked second on the list of food-borne parasites globally and is one of the 17 most neglected tropical diseases prioritized by the WHO [3]. While definitive hosts are usually wild dogs and carnivores, a wide range of domestic and wild mammals, even humans, act as intermediate or random hosts [4]. Possible routes of infection for humans are contaminated drinking water, contaminated food, or exposure to a contaminated environment [5]. The disease is widespread worldwide, common in countries especially in the Middle East, India, South America, and Australia [6]. In Iran, extensive infections are observed in dogs and herbivores such as sheep, cattle, camel and goats. Moreover, human patients have been frequently reported from almost all parts of the country, particularly by prominent medical centers [7, 8]. A careful study of various aspects of the disease in Iran is very necessary since it is one of the endemic regions with ~ 1% of its total surgeries related to this disease [9].
There is a lot of heterogeneity in E. granulosus sensu lato due to the presence of intraspecific variants or strains [8, 10]. In addition, better identification of genotypes and their molecular and biochemical differences are of great significance because of the effects of different strains of E. granulosus in designing diagnostic, therapeutic, prevention, and control strategies [11]. Based on the phenotypic and molecular studies, E. granulosus sensu lato has been divided into E. granulosus sensu stricto (including the identified genotypic variants G1-G3), E. felidis (the former ‘lion strain’), E. equinus (the ‘horse strain’, genotype G4), E. ortleppi (the ‘cattle strain’, genotype G5) and E. canadensis [12]. The application of the isoenzyme comparison method is very important due to its very high capabilities in distinguishing species and even different genotypes [13]. The zymogram method is widely implemented for the biochemical classification of several vertebrate parasites, including protozoa and worms [14–18]. This method is an efficient method to study the different species in Echinococcus and particularly to identify different strains of E. granulosus [19].
The aim of the present study was to investigate the isoenzyme pattern of E. granulosus sensu stricto and E. canadensis genotypes of E. granulosus larval stage based on host type in Iran and to obtain data on enzyme pattern changes within each genotype and between these two genotypes of parasites in different hosts. Considering that no phenotypic study based on isoenzyme pattern on Echinococcus granulosus parasite has been performed in Iran so far, the present study will be the first study in this field.
Sample Collection
Animal specimens (sheep, cattle, and camels) from Shiraz, Birjand, and Ilam industrial slaughterhouses were gathered from May 2018 to December 2020. In the meantime, human hydatid cyst isolates were also achievable in different hospitals of Shiraz and Tehran, from where they were transferred to the Parasitology Laboratory at Shiraz University of Medical Sciences (Table 2). The protoscolices were removed from the cysts and rinsed with PBS. Finally, the sediments obtained were kept at -21°C for subsequent experiments.
Enzymes Extraction
Equal volumes of enzyme stabilizers (2mM Dithiothreitol, aminocaproic acid, and EDTA) were added to the sediment of the samples and were thoroughly mixed. Freezing (at -191°C) and thawing (at 30°C) were executed ten times. The supernatant solution containing water-soluble proteins (including enzymes) was transferred into 0.5ml tubes. Next, the samples were centrifuged at 15,000 rpm for 80 minutes at 4°C, and finally, the solution was kept at -70°C until being employed.
DNA Extraction
The DNAs were extracted from all collected samples using a DNA extraction kit (Bioneer Company, South Korea) following the manufacturer's instructions.
PCR and Sequencing
In this study, two different primer pairs were used to amplify genes related to mitochondrial enzymes nad1 and cox1. To synthesize these selected fragments from the mitochondrial genome of the parasite, JB3 (5'-TTTTTTGGGCATCCTGAGGTTTAT-3') and JB4.5 (5'-TAAAGAAAGAACATAATGAAAATG-3') primers were used for the cox1 coding gene and JB11 (5-AGATTCGTAAGGGGCCTAATA-3') and JB12 (5'-ACCACTAACTAATTCACTTTC-3') primers were used for the nad1 coding gene[20]. Total volume of 25 µl was selected for PCR reaction (one unit of Taq DNA polymerase, 2µl of MgCl2, 0.5µl of forward and Reverse primers, 1µl of DNA, 2.5µl of buffer 10X, 2µl of dNTP, and 16.5µl of distilled water). After preparing the PCR mixture, the thermocycler program was set in 35 cycles. Finally, the sequencing of the PCR products and phylogenetic tree were performed.
Isoenzyme Electrophoresis
The Discontinuous Polyacrylamide Gel Electrophoresis Method was applied to perform the isoenzyme electrophoresis by vertical electrophoresis. Electrophoresis was performed using a 4% stacking gel and an 8% separating gel. In the present study, the activities of five enzymatic systems including; Glucose-6-Phosphate Dehydrogenase (G6PD), Malic Enzyme (ME), Isocitrate Dehydrogenase (ICD), Malate Dehydrogenase (MDH), and Nucleoside Hydrolyse 1 (NH1) were assessment. the Relative Factor (RF) or relative migration distance and number and frequency of Isoenzyme Banding Patterns (IBP) or zymodeme of each genotype were determined and compared with each other at the end of the project and staining. RF graphically was calculated from the top of the resolving gel to each band and to the dye front using a ruler for all bands in the isoenzyme pattern. RF was calculated using the following formula.
RF = Migration distance of the protein band/Migration distance of the dye front
RF was defined as the mobility of a protein divided by the mobility of the ion front.
Cocktail staining for visualizing of each enzyme in polyacrylamide gel is shown in table 1.
Table 1. Cocktail staining for visualizing of each enzyme in polyacrylamide gel
|
Enzyme |
Buffer |
Substrate |
Coenzyme |
|
MDH |
Tris-HCl 0.3 mol pH=8 |
Malic acid |
NAD |
|
G6PD |
Tris-HCl 0.3 mol pH=7.4 |
G6P |
NADP |
|
ICD |
Tris-HCl 0.3 mol pH=7 |
Isocitrate |
NADP |
|
NH1 |
Tris-HCl 0.3 mol pH=7.4 |
Inosine |
Not needed |
|
ME |
Tris-HCl 0.3 mol pH=7.4 |
Malic acid |
NADP |
A total of 32 samples, including 8 human and 24 animal (sheep, cattle, and camels) hydatid cyst isolates, were collected. The collected samples by the sequencing of the PCR products were characterized as E. granulosus sensu stricto (24 isolates) and E. canadensis (8 isolates) strains (Table 2).
| Variable | Frequency | E. granulosus sensu stricto (G1) strain | E. canadensis (G6) strain | |
|---|---|---|---|---|
| Tissue of the host | Liver | 22 | 16 | 6 |
| Lung | 10 | 8 | 2 | |
| Location | Shiraz | 16 | 16 | - |
| Tehran | 4 | 4 | - | |
| Ilam | 4 | 4 | - | |
| Birjand | 8 | - | 8 | |
| Host | Human | 8 | 8 | - |
| Sheep | 8 | 8 | - | |
| Cattle | 8 | 8 | - | |
| Camel | 8 | - | 8` | |
Isoenzyme pattern of glucose 6-phosphate dehydrogenase (G6PD)
G6PD system revealed two zymodeme, zymodeme 1 in 24 isolates all of them were E. granulosus sensu stricto strain and zymodeme 2, in 8 isolates, that all of them were the E. canadensis strain (Figure 1).
Isoenzyme pattern of ME
ME revealed two zymodeme, zymodeme 1 in 24 isolates all of them were E. granulosus sensu stricto strain and zymodeme 2, in 8 isolates, that all of them were the E. canadensis strain (Figure 2).
Isoenzyme pattern of MDH
MDH revealed two zymodeme, zymodeme 1 in 24 isolates all of them were E. granulosus sensu stricto strain and zymodeme 2, in 8 isolates, that all of them were the E. canadensis strain (Figure 3).
Isoenzyme pattern of ICD
ICD revealed two zymodeme, zymodeme 1 in 26 isolates, that were included all of E. granulosus sensu stricto strain and 2 E. canadensis isolates, and zymodeme 2, in 6 isolates, that all of them were the E. canadensis strain (Figure 4).
Isoenzyme pattern of NH1
Isoenzyme pattern of NH1 revealed two zymodeme, zymodeme 1 in 24 isolates all of them were E. granulosus sensu stricto strain and zymodeme 2, in 8 isolates, that all of them were the E. canadensis strain (Figure 5). The isoenzyme pattern illustration of enzymes used in the present study shown in figure 6, also the number of bands and RF in each enzymatic system is shown in Table 3.
Table 3. Number of bands and RF of each enzyme system in E. granulosus sensu stricto and E. canadensis strains
|
Enzyme system |
E. granulosus sensu stricto strain |
E. canadensis strain |
||||
|
Number of isolates |
Number of bands |
RF |
Number of isolates |
Number of bands |
RF |
|
|
MDH |
24 |
1 |
0.60 |
8 |
2 |
0.51, 0.68 |
|
G6PD |
24 |
2 |
0.6, 0.62 |
8 |
2 |
0.15, 0.2 |
|
ICD |
24 |
6 |
0.58, 0.54, 0.43, 0.34, 0.22, 0.17 |
8 |
6 |
0.65, 0.56, 0.52, 0.35, 0.31, 0.26 |
|
NH1 |
24 |
2 |
0.77, 0.69 |
8 |
1 |
0.73 |
|
ME |
24 |
2 |
0.17, 0.31 |
8 |
1 |
0.26 |
The present study was conducted to investigate simultaneous investigation on the isoenzyme and molecular characterization of E. granulosus sensu lato larval stage in different parts of Iran.
An extensive attention has recently been paid to isoenzyme studies in Iran in the field of parasitology, including studies by Hatam et al. (Leishmania, 2013) [21], Sarkari et al. (Fasciola, 2016) [22], Rayani et al. (Giardia, 2020) [23], and (Trichomonas, 2021) [24]. However, no investigation has been conducted on isoenzyme patterns on E. granulosus parasites in Iran to the best of our knowledge.
The result showed that E. granulosus sensu stricto and E. canadensis genotypes have entirely different isoenzyme patterns in the four enzyme systems used (NH1, G6PD, MDH, and ME), but ICD enzyme system was unable to distinguish all E. granulosus sensu stricto from E. canadensis strains. In E. granulosus sensu stricto strain, MDH and NH1 revealed a two-band pattern whereas, in the ME enzyme it had a single main band. In the E. canadensis strain MDH and NH1 revealed a single band, and in ME enzyme had a two-band pattern. Although, in ICD and G6PD enzymes, both genotypes, had a similar enzymatic pattern, their RF was different.
The provided isoenzyme data represent high degrees of homogeneity within the studied strains (E. granulosus sensu stricto and E. canadensis strains). Marked genetic uniformity with almost identical profiles detected in all isolates with five enzyme systems is also obvious. This highlights the importance of using several different criteria, when attempting to fully characterize an intra-strain differentiation of E. granulosus in any geographical locality and from a particular intermediate (or definitive) host. These criteria include ecological, immunological, morphological, biochemical, isoenzymatic, and in vitro developmental studies [25]. The study by Hogg et al. (1999) [26], with the aim of investigating E. granulosus intra-strain polymorphisms reported limited or no variation within the four studied strains, this was consistent with the isoenzyme pattern reported by McPherson et al. (1982) [25], and Limbery and Thompson (1988) [27].
The results obtained in the current study is consistent with the results reported by other authors in different geographical areas on the ability of the zymodeme technique to differentiate between E. granulosus strains [19, 25, 27]. Turceková et al. (2003) proved that GPI and MDH enzymes are suitable for discriminating G7 and G1 of E. granulosus strains [28].
In a previous electrophoretic study, Le Riche & Sewell (1978) showed that the GPI isoenzyme profiles for E. granulosus of U.K sheep and cattle are very similar. However, these patterns are different from those produced by hydatid material obtained from two infected Nigerian camels [29].
The limitations of our study are the small number of enzyme systems examined, and the lack of E. granulosus larval stage isolates from other intermediate hosts in Iran, including goats.
The finding showed that the E. granulosus sensu stricto and the E. canadensis genotypes have entirely different isoenzyme patterns in the enzymes of NH1, G6PD, MDH, ME. Therefore, these enzyme systems may be implemented to differentiate the two E. granulosus genotypes. The results are important because they state that differentiating between two E. granulosus strains are reachable with a limited number of enzymatic systems.
Ethical approval and consent to participate
Ethical approval was obtained from the Shiraz University of Medical Sciences Ethics Committee (Cod number: IR.SUMS.REC.1399.378)
Consent for publication
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Competing interests
The authors declare that they have no competing interests.
Funding
This article has been derived from a PhD thesis (Cod number: 20037) at Shiraz University of Medical Sciences.
Authors’ contributions
G.R. Hatam, S.M. Sadjjadi and M. Dousti designed the conception of the study; G.R. Hatam and S.M. Sadjjadi technical supports and conceptual advice; M. Dousti, A.H. Radfar, and R. Solgi focus of the molecular study; A. Vafafar focus of the isoenzyme study; All authors contributed to the drafted the manuscript, revised it critically and approved the final version.
Acknowledgements
This article has been derived from a PhD thesis (Cod number: 20037) at Shiraz University of Medical Sciences. We would like to thank the cooperation of all the research centers and units.