Liver Fluke Species Identification Isolated From Humans and Animals Using PCR-RFLP and DNA Sequencing

1. INTRODUCTION

The Fasciola species is an extremely pathogenic parasite that affects humans and animals and this disease has a dual impact on human health and economic losses [1], [2]. The parasite habitat is the livers and bile ducts of humans and animals. In livestock, it causes numerous financial losses, such as a decrease in milk, meat, and wool production [3]. This fluke directly affects livestock productivity through host mortality and partial or complete liver condemnation. Furthermore, it has an indirect effect on the host growth and feeding, which negatively reflect on the white and milk yield [4]. The effect of animal Fasciolasis on weight will be varied depending on the age group and poorer carcass quality indicators [5]. The human Fascioilasis which is known as a neglected disease is occurring in the area where the peoples eat raw water plants that may be contaminated with metacercaria [6]. Due to the epidemiological traits of Fasciola hepatica and Fasciola gigantica infections in humans, accurate and precise diagnoses are crucial for early uncomplicated treatment. The species of liver fluke have differential characteristics and aspects, also the different type of lymnaeid snails serve as the intermediate host of the liver flukes [7]. The clinical, pathological, and immunological diagnosis approaches are not efficient methods to differentiate between the species of liver fluke [7], [8]. Furthermore, the morphometric methods faced many limitations, the differentiation of F. hepatica and F. gigantica is currently based on implementing numerous molecular methods using various DNA markers [7]. Various molecular diagnostic tools, including polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP), and rDNA sequencing, have been used to find the genetic variation and species identification of Fasciola spp. [9], [10], [11], [12]. Furthermore, due to the shortcomings of the morphometric approach, many molecular strategies have been applied for genotyping and classifying liver flukes throughout the world. These strategies use various molecular markers [13]. A quick and straightforward PCR-RFLP experiment using the common restriction enzyme Dra II is presented to differentiate between species of liver flukes. Based on the 28S, rDNA gene sequence recently discovered the polymorphism among liver fluke populations [9], [14]. The rDNA region has been applied to establish the genetic polymorphism of Fasciola spp. in Spain. This revealed that heterozygous specimens had nucleotide variants [15]. In a new study, a genetically varied type of liver fluke was discovered, named Fasciola intermediate, it is morphologically similar to F. gigantica and F. hepatica [16]. The present study is an attempt to find the species of the liver flukes isolated from humans and animals using PCR-RFLP and DNA sequencing.

2. MATERIALS AND METHODS

2.2. DNA Extraction and PCR

The DNA was extracted from the tissue of the isolated worm (whole body of the worm), the liver flukes were ground individually in an Eppendorf tube using micro-pistil, and the commercial DNA extraction kit (ADD prep, Tissue Genomic DNA extraction kit) was used. The extracted DNAs were stored at −20°C.

In the present study, two molecular approaches were followed RFLP and DNA sequencing, and two sets of primers were used Table 1. The 30 μL PCR reactions prepared to amplify the target region of the liver fluke DNA contained 2 μL of the target DNA (sample), 15 μL of master mix 2× (Amplicon, Skovlunde, Denmark), 1 μL of forwarding primer, 1 μL of reverse primer, and 11 μL deionized distilled water (ddH₂O), the conventional PCR was used (Thermocycler BIO-RAD iQ TM), the program of the thermo-cycler was set for both primers as follow; initial denaturation 5 min at 95°C followed by 35 cycles at 95°C for 50 s, 55°C for 40 s, and 72°C for 1 min with a final extension of 7 min at 72°C.

Table 1: The nucleotide sequences of primers used in the present study

3. RESULTS

Out of 48 liver flukes isolated from humans and animals, all PCR runs successfully amplified the suspected band (618 bp) for 28S rDNA primer and (836 bp) for the FCOX1 primer (Figs. 1 and 2). Furthermore, for genetic characterization and species identification of liver flukes, DNA sequencing for FCOX1 and RFLP were done.

Fig. 1. Gel electrophoresis of the PCR product of the primer 28S rDNA. M= DNA ladder (50 bp), N = Negative control, 1–8 = The samples.

Fig. 2. Gel electrophoresis of the PCR product of the primer FCOX1. M = DNA ladder (50 bp), N = Negative control, 1–9 = The samples.

The result of RFLP analysis of the PCR product of the 28S rDNA primer (618 bp), which is digested by restriction enzyme (DraII) showed two patterns, the first one has two bands of (529 bp and 89 bp), while the other pattern is not digested and the 618bp band remains. Table 2 and Fig. 3, the first pattern belongs to F. hepatica, and the second is F. gigantica [15]. The DNA sequencing of the PCR product of the FCOX1 primers showed the genetic variation, and the result of the nucleotide analysis indicates the presence of F. hepatica and F. gigantica in the Kurdistan-Iraq, the results were deposited in the Genbank the following Accession numbers; (OP718780 and OP718781) and (OP718782, OP718783, and OP718784).

Table 2: Restriction fragment length polymorphism Patterns of PCR products of 28S rDNA primer digested with DraII enzyme

Fig. 3. Restriction fragment length polymorphism Patterns of PCR products of 28s rDNA primer digested with DraII enzyme: M = DNA Ladder (50 bp), 1–13 = The samples isolated from animals (1–11) and humans (12 and 13).

4. DISCUSSION

The present study is an attempt to evaluate two DNA markers to find the genetic characterization and species identification of liver flukes isolated from humans and animals, the RFLP and DNA sequencing were used for two DNA regions of the liver flukes. There are no adequate studies on the molecular and species identification of the liver fluke isolated from humans in Kurdistan Region – Iraq. In the present study, RFLP techniques were applied to identify the Fasciola spp. Many studies have been published in various countries throughout the world to identify the genetic polymorphism and species identification of this fluke, including Iraq, Iran, and Thailand [12], [13], [15]. In a study on the molecular diagnosis of Fasciola hepatica in livestock utilizing the COX1 gene in Erbil Province, Kurdistan – Iraq, PCR, and sequencing were used to identify the genetic variation among isolated liver fluke from animals they found genetic variation and they conclude that DNA sequencing is a differential technique for finding genetic variation and species identification using COX1 region, this finding in agreement with result [12]. In another study in Duhok governorate – Iraq, specifically used the ribosomal DNA markers; ITS1, and they found the common species that are responsible for animal fascioliasis are F. hepatica and F. gigantica this result agrees with the current finding [15].

Despite earlier studies, recent investigation on the prevalence of fascioliasis and molecular characterization of Fasciola spp. in sheep and goats was done in the Sulaymaniyah province of northern Iraq, using DNA markers and sequencing of the incomplete 28S rRNA gene and codon analysis, they found that F. hepatica represented the majority of the identified, followed by F. gigantica represented the other two field sequences, this study also supports the finding of the present study and they confirmed the Sulaymaniyah is the place to both F. hepatica and F. gigantica. the 28S rDNA is confirmed as a potential biomarker in identifying various Fasciola species, it was also used in the current study [9].

Furthermore, many studies from different areas demonstrate that molecular approaches are applicable to discriminate Fasciola species, the commonly used restriction enzymes Ava II and Dra II, based on a sequence of the 28S rRNA gene, COX1, ITS1, and ITS2 [15], [16], [17], [18]. They concluded that RFLP is rapid and easy for species identification of liver flukes, and they support current results that indicated the incubation period of DNA digestion by the restriction enzymes can be shortened, also different enzymes can be used in future studies to find the most applicable enzyme to discriminate all three species of Fasciola.

Despite the use of genetic markers for Fasciola species identification, the immunological markers were done to find the efficacy and applicability to discriminate the species of liver flukes, a study from Duhok, Kurdistan – Iraq demonstrated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) in infected sheep with Fasciola spp. the direct examination visual inspection of the liver and immunological assay was followed [7], the result shows the infection rate among animals at a high rate while the species of liver flukes cannot be accurately diagnosed immunologically due to morphologically high similarity, this make researches to adapt molecular markers exactly PCR-PFLP which is the dependable approach for the detection of Fasciola species.

5. CONCLUSION

The our finding concluded that all species of Fasciola were found in studied area and F. hepatica is the common species responsible of human infection. Furthermore, the present study concludes the use of molecular markers PCR-RFLP with different restriction enzymes for species identification and genetic variation is rapid, easy, and reliable. Furthermore, nucleotide sequencing can be used for species identification and genotyping while the nucleotide sequence analysis and editing will make mistakes or substation of the nucleotide base pair and the result may be not accurate as in the PCR-RFLP which is depend on the restriction side and it will be highly specific.