Molecular Identification of Fasciola and Dicrocoelium species isolates in ruminants live stock from Kashan and Arak in center of Iran

doi:10.21203/rs.3.rs-4956663/v1

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Molecular Identification of Fasciola and Dicrocoelium species isolates in ruminants live stock from Kashan and Arak in center of Iran

https://doi.org/10.21203/rs.3.rs-4956663/v1

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Fascioliasis and Dicrocoeliasis are important trematode infections that affect humans and ruminants worldwide. Molecular techniques have a conclusive role in detection liver flukes. The purpose of the current study was to find out the genotypic diversity of Fasciola and Dicrocoelium spp. isolated from different hosts in Iran. Totally, 160 and 200 adult Fasciola and Dicrocoelium spp. isolates collected from infected cattle, sheep, and goats from two abattoirs in the center of Iran. PCR-RFLP, and DNA sequences nuclear markers (18S, 28S, ITS) and the mitochondrial marker (ND1, CO1) were applied. PCR products of Dicrocoelium and Fasciola samples, were subjected to digestion by Bfa1, TruiI, BsrB1, ECO881, and Hind III enzymes. DNA from 60 isolates of Fasciola and Dicrocoelium of different hosts were sequenced and evaluated. The PCR reaction showed the length of 18S, 28S, ND1, CO1 of Fasciola at 260bp, 618bp, 700bp, and 500bp, and the length of the ITs2 and 28S of Dicrocoelium was 236bp and 963bp respectively. D. dendriticum has an RFLP pattern of 110, and 126bp (ITS2), and 116, 293, 409bp (28s) using, Bfa1 and Tru1I restriction enzymes. F. gigantica has a profile of 333, and 285bp (28s) using Bsrb1 enzyme. The RFLP pattern of genotype F. hepatica was 73, 120, and 507bp (ND1) and 119 and 381bp (CO1) in size using Hind III and ECO881 enzymes. Using the PCR-RFLP, three species of F. hepatica, F. gigantica, and D.dendriticum were identified. To uncover the genetic population structure of liver flukes across the country, future studies are still required.

Molecular identification. PCR-RFLP. Fasciola. Dicrocoelium

Iran

Fascioliasis and Dicrocoeliasis are liver fluke infections caused by the three zoonotic worm species Fasciola hepatica, F.gigantica and Dicrocoelium dendriticum and are distributed globally affecting ruminants and occasionally humans that, while treatable, is difficult to diagnose (Shahnazi et al. 2020; Evack et al. 2022).They are heeding for animal farm industry and are considered the result in economic losses due to a decrease in herd productivity as a result of decreased fertility, decreased wool and milk production, and an increase in mortality (Shrestha et al. 2020). Fascioliasis (F. hepatica and F. gigantica) impact on human has also become an emerging public health concern in recent years. Estimates suggest that fascioliasis is an important Foodborne trematodes disease with 2.4 million people are currently infected and 180 million people are at risk in over 70 countries (Chai and Jung 2020). The disease's importance to human health has been highlighted by recent research, and it has been included on the World Health Organization's guideline of neglected tropical diseases (NTPs) for 2030 (Garcia-Corredor et al. 2023). In human fascioliasis, adult worms are commonly found in the bile duct and gallbladder. Although ectopic migration in human is rarely fatal, but sometimes fasciolid worms isolated in ectopic foci, i.e., eye, brain, spinal cord, subcutaneous tissues, cecum, lymph nodes and Mesocolon. It can cause pain, liver and gallbladder damage, and other organ injuries (Kim et al. 2015). Due to the complexity of the transmission cycle, purely human de-worming programs may not be the most cost-effective approach to sustain prevention of fascioliasis. Furthermore, the increasing number of reports of triclabendazole's ineffectiveness in treating fascioliasis due to drug resistance suggests the need for a multidisciplinary approach (Kelley et al. 2016). According to the transmission cycle, it is clear that valid options for controlling Fasciola requires not only human deworming, but also snail control and livestock treatment. A single intervention may not be enough to ensure long-term control of fasciola due to the multidimensional nature of the disease. It is necessary to have a single healthy approach that brings together different disciplines and sectors (Mas-Coma et al. 2018; Quang et al. 2022). Fasciola does not have a comprehensive transmission model yet, even though there are clear advantages and the requirement for more evidence-based guidance in control programs. The current models only provide a limited understanding of the transmission cycle, with no mention of humans as a potential final host (Turner et al. 2016), or they make predictions based on climate factors (Valencia-lópez et al. 2012), consequently, neglecting the underlying transmission mechanisms. In the case of hybrids between the two species, the traditional morphological methods used to distinguish between them can be inconclusive. Fasciolosis and Dicrocoeliasis pose significant economic challenges in communities. Rapid and accurate detection of the parasite is essential for effectively preventing and controlling the diseases. Identification of liver flukes Fasciola and Dicrocoelium species is routinely talented by morphometry assay. Molecular approaches have been used recently to identify these parasites with greater accuracy due to the complexity of characterization through morphological examination. The current molecular access allows for precise identification of helminths at the species level, enabling the identification of worms in definitive hosts ( Afshan et al. 2014; Nukeri et al. 2022; Garcia-Corredor et al. 2023). Various genetic markers, including mitochondrial genes (CO1 and ND1), nuclear gene 28S rRNA, and ribosomal internal transcribed spacers (ITS1 and ITS2), have been beneficial in identifying liver flukes, Fasciola and Dicrocoelium. Molecular approaches have accelerated the identification of morphologically similar parasites, but there is a lack of standardization, and the detection of certain parasitic species remains unknown. Results from studies clearly indicate that coding mitochondrial genes is more practical compared to non-coding ribosomal gene sequences, and mitochondrial DNA sequences allow for faster identification of nucleotide replacement in platyhelminths compared to ITS. Therefore, mitochondrial DNA is more effective at revealing variations among different isolates in comparison to ITS (Shafiei et al. 2014; Chougar et al. 2019; Shahnazi et al. 2020; Calvani and Šlapeta 2021; Kasahara et al. 2021; Hajialilo et al. 2023). Nonetheless, information about the parasite’s genetic divergence and intra-population construction is finite worldwide. The study aimed to assess the genetic variation of samples identified as Fasciola spp. and Dicrocoelium spp. based on morphology, utilizing the PCR-RFLP technique. Furthermore, the genetic diversity, sequencing, and phylogenetic analysis of liver flukes from sheep, goats, and cattle were conducted using 18S, 28S, ITS (rDNA), ND1, and CO1 (mtDNA) genetic markers.

Collection of fluke specimen

A total of 160 and 200 adult samples of Fasciola and Dicrocoelium were randomly collected from the bile duct of naturally infected cross-breed 50 cattle, 55 goats, and 55 sheep during post-mortem inspection at two abattoirs situated in center Iran (Arak abattoir in Markazi province and Kashan in Isfahan province). Macroscopic examination were performed to assess infection caused by Fasciola and Dicrocoelium. Following collection, individual flukes were carefully washed three times, with double distilled water to eliminate bile residues and blood residues adhering to the parasite. The samples were then examined to identify morphometric characteristics using standardized measurements and taxonomic keys recognized for differentiating both Fasciolid species and Dicrocoelium (Shahnazi et al. 2020; Hajialilo et al. 2023). All samples were preserved in 75% ethanol, and stored at -80°C until they were used for molecular examination.

DNA Extraction, Amplification and Alignment

For the extraction of total genomic DNA, segments from the apical and lateral regions of fresh Fasciola and Dicrocoelium were excised and subsequently rinsed with distilled water prior to the DNA isolation process. After allowing the samples to dry, they were placed in a lysis solution containing 50 mM Tris, 1% SDS, and proteinase K, followed by incubation at 56°C for a duration of 12 to 14 hours. The DNA was then extracted from the crushed samples using Takaposi column kits (South Korea), following the manufacturer's instructions, with the exception of modifications made to the volumes of lysis solution and proteinase K. The concentration of the extracted DNA was examined using a NanoDrop spectrophotometer, while quality assessments were performed using electrophoresis with a 1.5% agarose gel. DNA fragments of ITS1 + 2 and 28s regions of Dicrocoelium as well as 28s, ND1 and CO1, 160 genes of Fasciola were amplified by polymerase chain reaction (PCR). Each PCR reactions (25 𝜇L) was performed containing a mixture of4𝜇L of DNA solution, 1.25 units of Taq DNA polymerase, 2.5 𝜇L of 10x PCR buffer, 2 µl MgCl2, 1µl of each primer, 0.4 µl of dNTPs, and 12.85𝜇L of sterile water. The sequences of the primers are shown in Table 1. PCR amplification was performed for one cycle of 90 s at 94∘ C, followed by 30 cycles of 90 s at 94∘ C, 90 s at 55∘ C, 120 s at 72∘ C, and a final extension step of 72∘ C for 10 min followed by cooling at 4∘ C. The PCR products were loaded onto 1.5% agarose gels with 1× TAE and run at 50 V for 25 min. multiple bands were produced, and the bands of the correct length were excised from the gel for sequencing. Finally, on PCR products two-directional Sanger sequencing using the forward and reverse primers was done. The sequence was compared to sequences deposited in Gen Bank using the Basic Local Alignment Search Tool (BLAST) (National Center for Biotechnology Information) and matched other species of Dicrocoelium as well as F. gigantica ITS2, 18s, 28s, CO1 and ND1 sequences. The PCR reaction showed the length of 18S, 28S, ND1, CO1 of fasciola at 260bp, 618bp, 700bp, and 500bprespectively, and the length of the ITs2 and 28S of Dicrocoelium was 236bp and 963bp respectively.

Table 1

Sequences of the primers used in PCR reaction
Trematode	Gene	Forward	length	Reverse	length
Fasciola	CO1	5'-ACGTTGCATCATAAGCGTGT-'3	20	5'-CCTCATCCAACATTACCTCT-'3	20
	ND1	5'-AAGGATGTTGCTTTGTCGTGG-'3	21	5'-GGAGTACGGTTACATTCACA-'3	20
Dicrocoelium	CO1	5'-TGTGTTCGTTCTGCTTTGGG-'3	20	5'-ACTCACACAACACGCCAATC-'3	20
Dicrocoelium	ITS2	5'-CTTACAAACTATCACGACGC-'3	20	5'-GATTAGAAGGCCGTATTTCGGA-'3	22
Dicrocoelium	28S	5'-GTGGCCAGTTGGTCATTAGG-'3	20	5'-ACCTCAGTCTGGACAAGCCA-'3	20
Dicrocoelium	ITS1	5'- GTAAGCCTGTCCTGTGTGCTCGTGC-'3	25	5'- GCCACGACTCCGTAGACACATCTCC-'3	25

PCR-RFLP Analysis

A PCR-RFLP method was done to specifically detection F. hepatica from F. gigantica in ND1 with HinIII enzyme, in CO1 with ECO881 enzyme and 28s with BrsrB1 enzyme. About Dicrocoelium we used to distinguish D.dendriticum from other species belong dicrocoelidae family in ITS2 and 28s genes, Bfa1 and Tru1I restriction enzymes, respectively.

Sequencing and Phylogenetic Analysis

The PCR products of 18s, 28s, COI, and NDI from ten Fasciola isolates and fifteen Dicrocoelium isolates obtained from each host species (cattle, sheep, and goat) were sequenced in both directions using the Sanger method. The same primers utilized in the PCR were employed for sequencing. The resulting sequences were aligned and compared with existing Fasciola spp. sequences available in GenBank, utilizing the BLAST program. A multiple alignment was conducted with data pertaining to Fasciola spp. from Iran and other countries that are also deposited in GenBank. Phylogenetic analysis was executed using the Maximum Likelihood method with MEGA 5.0 software, and bootstrap analyses with 1,000 replicates were performed to assess the reliability of the results (Kumar et al. 2018).

A total of 160 Fasciola and 200 Dicrocelium fresh adult liver trematode were chosen for molecular analysis from two geographic regions following the confirmation of the parasite's identities through diagnostic keys and morphological characteristics among the isolated goats, sheep and cows. The frequency distribution of Fasciola and Dicrocoelium examined in various hosts across the two areas of Kashan and Arak cities is presented in Table 2.

Table 2 The frequency distribution of Dicrocelium and Fasciola for molecular investigation from different hosts studied in Arak and Kashan regions

Total

Kashan Region

Arak Region

Present

Number

parasite

Present

Number

parasite

Host

Fasciola

Dicrocoelium

33.3

Fasciola

Dicrocoelium

Cattle

Fasciola

Dicrocoelium

33.3

Fasciola

Dicrocoelium

Sheep

Fasciola

Dicrocoelium

33.3

Fasciola

Dicrocoelium

Goat

160

200

100

Fasciola

Dicrocoelium

100

Fasciola

Dicrocoelium

Total

Microscopic examination showed that hepatic samples were infected with the both Fasciola species (F. hepatica, and F. gigantica) and only D. dendritium, according to the standard measurements and morphometric characteristics.

PCR of Fasciola CO1, ND1, 18s and 28s regions

Polymerase chain reaction product showed the presence of specified bands of Fasciola species by using the recommended primers as 500bp (COI), 700bp (NDI), 260bp (18s) and 681bp (28s) after optimizing the thermal gradient PCR done. Fig. 1(a,b) and Fig. 2(a,b) show the electrophoresis results of the PCR product of the CO1, ND1, 18s and 28sgenes of Fasciola liver trematode collected from Arak and Kashan regions in sheep, goat and cattle.

PCR-RFLP of Fasciola CO1, ND1 and 28s genes

To determine the enzymatic digestion pattern of the CO1, ND1 and 28sregions of Fasciola liver flukes, the RFLP method of the ECO881, Hind III and BsrBI restriction enzymes was used. The ECO881 and Hind III enzymes have no effect on F.gigantica, but they cut F.hepatica into a fragment of 119,381bp and bp73, bp120 and bp507 fragments respectively (Fig. 1d, 1e). The BsrBI restriction enzyme had no effect on genotype F. hepatica, but it cuts genotype of F.gigantica into a fragment of 285bp and 233bp (Fig. 2c).

PCR of Dicrocoelium ITS2 and 28s genes

The PCR reaction product showed the presence of specified bands of Dicrocoelium species by using the recommended primers as 236 bp (ITS2) and 963 bp (28s) after optimizing the thermal gradient PCR done. Fig. 3(a,b) and Fig. 4(a,b) show the electrophoresis results of the PCR product of the ITS2 and 28s genes of Dicrocoelium liver trematode collected from Arak and Kashan regions in sheep, goat and cattle.

PCR-RFLP of Dicrocoelium ITS2, and 28s genes

To determine the enzymatic digestion pattern of the ITS2, 28s regions of Dicrocoelium liver flukes, the RFLP method of the Bfa1, and Tru1Irestriction enzymes was used. The Bfa1 and Tru1Ienzymes cuts D.dendriticu into a fragment of 110bp and 126bp and 116bp, 145bp, 293bp and 409bp, respectively. Fig. 3(c,d) and Fig. 4(c,d) clearly present the only predominant genotype of Dicroceolium in various hosts in Iran is D.dendriticum

PCR-RFLP of Fasciola 28s gene in Kashan region

PCR of DicrocoeliumITS2 and 28s genes inKashan and Arak

Among gastrointestinal parasitic infections, Fascioliasis and Dicrocoeliasis represent the most significant helminth diseases affecting domestic livestock, with occasional occurrences in humans, particularly in tropical and subtropical regions such as Iran. The parasitizing trematodes responsible for these infections lead to substantial economic losses within the livestock sector, manifested through reductions in milk production, body weight, and the culling of affected animals and their organs. Fascioliasis is classified as a zoonotic "neglected tropical disease," which poses considerable public health risks and can, in certain instances, be fatal. The differentiation of Fasciola and Dicrocoelium species through morphometric techniques presents challenges, including morphological variations influenced by the parasite's age, host type, or infection intensity (Aryaeipour et al. 2017; Kumar et al. 2018; Shahnazi et al. 2020; Kruchynenko et al. 2022). Consequently, molecular methods are recommended as effective approaches for identifying the predominant species in a given area. Molecular techniques based on DNA are both accurate and dependable for identifying various species of Fasciola and Dicrocoelium in endemic regions (Ngoc Anh et al. 2018; Javanmard et al. 2022). Among these techniques, PCR-RFLP stands out as one of the most effective methods for differentiating these trematodes (Shahnazi et al. 2020). The PCR-RFLP assay serves as a robust tool for distinguishing between F. hepatica and F. gigantica. In prior research, a PCR-RFLP approach utilizing the partial rDNA of ITS1 along with the RsaI restriction enzyme was employed for the differentiation and identification of Fasciola species (Dar et al 2012). These techniques have been applied to distinguish between Fasciola species by analyzing the profiles produced by the action of endonucleases on the ITS genes of these parasites. In previous researches the RsaI restriction enzyme targeting the ITS1 region to effectively differentiate between F. hepatica and F. gigantic have been employed (Ichikawa et al. 2011; Aryaeipour et al. 2014; Ngoc Anh et al. 2018). The successful management of these diseases has led to the accurate identification of the parasite and its prevalent species. In various regions worldwide, including Iran, different genetic markers such as ITS1, ITS2, ND1, and Cox1 are utilized to identify the dominant species of Fasciola. Additionally, a range of restriction enzymes is applied for the precise diagnosis of Fasciola species (Bozorgomid et al 2016; Sarkari et al. 2017; Piri et al 2018; Shokouhi et al. 2019). The research conducted by Saadatnia et al. (2022) utilized the Rsa1 enzyme for specific identifications of Fasciola species. Additionally, investigations by Aryanpour et al. (2014) and Sarkari et al. (2017) focused on Fasciola obtained from domestic animals in various regions of Iran, employing ITS1 and the Rsa1 enzyme for their analyses. The findings indicated that the populations of both species were equivalent; however, Piri et al. (2018) conducted a study on Fasciola in Hamedan, utilizing ITS1 and the enzyme Rsa1. Their results revealed that F. hepatica was the predominant Fasciola species in that area, based on the analysis of all samples. Additionally, Mir et al. (2019) examined Fasciola in Zabol County, employing ITS1 and the enzyme Rsa1. They reported that out of 70 samples, 63 were identified as F. hepatica, while only 7 were diagnosed as F. gigantica, establishing F. gigantica as the dominant Fasciola species in that region. The findings of this study revealed that the population of F. hepatica surpassed that of F. gigantica, demonstrating the prevalence of F. hepatica, consistent with observations made in central Iran. Due to endemic nature of fascioliasis and dicrocoeliasis in Iran, along with the favorable conditions for the spread of these diseases, liver fluke infections have emerged as a significant concern within the country's public health priorities (Arbabi et al. 2018). The effective combat against this disease necessitates the precise identification of the parasite and its prevalent species. Consequently, numerous studies have been conducted across various regions of the country to determine the dominant species of Fasciola. Similar to other investigations in Iran, our research employed restriction enzymes to conduct RFLP-PCR. The findings of our study demonstrate that the PCR-RFLP technique serves as a powerful method for identifying both genotypes of F. hepatica and F. gigantica in diverse hosts throughout Iran. Additionally, sequencing ITS fragments proves to be an appropriate genetic marker for distinguishing the genotype of Fasciola, as well as for examining interspecies variation and phylogenetic traits (Levy et al. 2023). The molecular approach utilizing DNA analysis is regarded as a more reliable means of identifying Fasciola species compared to alternative methods. Consequently, the DNA markers necessary for the identification of Fasciola species encompass the Mitochondrial COI gene, ITS2, 5.8s, ITS1, and ND1. Among these, the internal transcribed spacer (ITS-2) region of ribosomal DNA (rDNA) has emerged as the most commonly employed and adaptable genetic marker for phylogenetic analysis, demonstrating successful application in various regions globally (Ngoc Anh et al. 2018; Mitchell et al. 2021; Javanmard et al. 2022; Alsulami et al. 2022). In recent years, there has been a notable enhancement in the sensitivity and specificity of molecular markers used for the identification of Dicrocoeliid liver flukes (Nezami et al. 2019; Khan et al. 2021; Javanmard et al. 2022). Conventional PCR and DNA sequencing techniques have been employed to amplify segments of nuclear ribosomal genes, including their internal transcribed spacers, along with mitochondrial DNA loci, which have been established for the detection of dicrocoeliid parasites (Liu et al. 2014; Bian et al. 2015; Shahnazi et al. 2020; Javanmard et al. 2022). Molecular analysis of the NADH dehydrogenase (NAD1) gene indicated that this genetic marker is unsuitable for the molecular characterization of D. dendriticum (Shamsi et al. 2020). Conversely, while ribosomal cistron DNA and cytochrome oxidase subunit 1 (cox1) have been proposed as suitable for investigating intra-species variations within D. dendriticum, the limited number of reference sequences for these genes hampers the interpretation of potential correlations between genetic variations, hosts, and the origin of Dicrocoelium (Khan et al. 2021). Consequently, numerous studies have utilized ITS fragments for the genetic examination of this trematode. Additionally, the ITS 2 fragment of the rRNA gene is recognized as a reliable genetic marker for molecular studies involving flatworms (Javanmard et al. 2022). Furthermore, phylogenetic analysis indicated that all our sequences clustered within a single clade alongside the majority of the previously submitted sequences. Consistent with our results, a study conducted by Liu et al. amplified and sequenced the ITS and mitochondrial genome of D. dendriticum and D. chinensis, demonstrating that both fragments were sufficiently distinct to differentiate the two species, while exhibiting limited variation within each species (Liu et al. 2014). Furthermore, research conducted on sheep and goats in China revealed that phylogenetic analysis of the ITS 2 fragment was insufficient to distinguish D. dendriticum isolates according to their host species and geographical origin (Bian et al. 2015). Similarly, a study in Iran failed to identify significant intra-species variations in D. dendriticum through molecular examination of the ITS fragment (Gorjipoor et al. 2015). These findings are corroborated by additional studies that demonstrate a high degree of similarity in the rRNA gene of D. dendriticum isolates from various global locations (Dar et al. 2020). The rDNA ITS-2 of Dicrocoelium serves as an effective genetic target owing to its widespread genomic presence and the abundance of species-specific variable sequences that are bordered by highly conserved sequences. This characteristic facilitates the binding of universal primers and allows for the differentiation among various trematode species (Khan et al. 2024).

The PCR-RFLP method revealed the presence of three liver fluke species: F. hepatica, F. gigantica, and D. dendriticum in central Iran, which are responsible for infections in cattle, sheep, and goats. This method is known for its simplicity, speed, cost-effectiveness, and accessibility, which allows to distinguish between F. hepatica and F. gigantica. Nevertheless, nucleotide sequencing of the parasite is considered the most effective approach. The findings of this study provide valuable insights that will aid future aid future studies on the distribution of these species in other regions. It seems necessary to develop hygienic policies aimed at preventing and controlling fascioliasis and dicrocoeliasis due to their associated economic impacts.

Funding The Vice Chancellor for Research of Kashan University of Medical Sciences, Kashan, Iran granted this study (projects No. 94142, 97040, 98072)

Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Ethics approval statement. The ethics committee of Kashan University of Medical Sciences approved the current study and all experimental protocols, with the codes IR.KAUMS.REC.1394.142, IR. KAUMS. MEDNT.REC.1397.23 and IR.KAUMS. MEDNET.REC.1398. 060.

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Molecular Identification of Fasciola and Dicrocoelium species isolates in ruminants live stock from Kashan and Arak in center of Iran

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Abstract

Figures

Introduction

Material and methods

Collection of fluke specimen

DNA Extraction, Amplification and Alignment

PCR-RFLP Analysis

Sequencing and Phylogenetic Analysis

Results

PCR of Dicrocoelium ITS2 and 28s genes

PCR-RFLP of Fasciola 28s gene in Kashan region

PCR of DicrocoeliumITS2 and 28s genes inKashan and Arak

Discussion

Conclusion

Declarations

References

Status:

Version 1