Introduction

Trematodes of the genus Fasciola cause fasciolosis which is often known as liver fluke infection. The parasite F. hepatica infects animals with one of the widest regional distributions and causes significant losses to the global livestock industry (Charlier et al. 2020). It is listed as one of the most important neglected zoonotic diseases which affect both humans and domesticated animals (Mas-Coma et al. 2014). Fasciola hepatica causes a common foodborne illness in individuals that are linked to eating raw vegetables such as aquatic plants or alfalfa and drinking water that has been tainted with infective metacercaria (Cabada et al. 2018; Sabourin et al. 2018). Similar to humans, animals can get fasciolosis by consuming polluted food and water, which has a negative impact on their health and reduces animal output and, consequently, farm economic growth (Beesley et al. 2018).

There are many zoonotic trematodiasis that have been estimated in the past to cause 665,352 disability-adjusted life years (DALYs) throughout the world (Fürst et al. 2012). Fasciolosis, a type of trematode infection that spreads through contaminated food, is found all around the world. Records show that Fasciola infection has been identified in 81 different countries (Caravedo and Cabada 2020). Fasciola hepatica is commonly present in tropical and subtropical regions such as those in the Middle East (Egypt and Iran), South America (Bolivia, Ecuador, and Peru), and Asia (Mas-Coma et al. 2009). Infection with Fasciola spp. increases the risk of co-infections, decreases production and fertility, and causes considerable morbidity and mortality in livestock. These factors add up to approximately €2.5 billion in annual economic losses worldwide (Mazeri et al. 2017). When animals consume infected encysted metacercariae that have erupted from the intermediate host and settled on the grass, they become infected. Following excystment in the gut, the parasite pierces the intestinal wall and migrates to the liver, where it causes two clinical phases of the disease to develop (Corrales et al. 2021). Acute fasciolosis sets in due to tissue destruction and hemorrhages brought on by the parasites tunneling and feeding activity in the liver tissue, as well as immunopathology brought on by the host’s immune responses. In the chronic phase, the flukes move into the bile ducts, where they mature and lay eggs. Generally, the severity of infection is more in sheep than in cattle because they are less resistant to parasites. The most prevalent type of fasciolosis in sheep farms is subclinical and has minimal liver fluke loads, but it can still affect the wool quality and weight gain (Hayward et al. 2021) and make sheep more vulnerable to other diseases (Munita et al. 2019).

In addition to serving as a source of protein, livestock is seen as a direct source of revenue and employment (Mehmood et al. 2017). Thus, the food security and finance of the affected countries are threatened by this parasitosis (Webb and Cabada 2018). It is considered the primary contributor to financial losses in the cattle sector, particularly through liver damage, high morbidity and mortality, decreased output of meat and milk, and increased costs for anthelmintics and veterinary care (El-Tahawy et al. 2017; Fanke et al. 2017).

The occurrence and spread of the disease are also being impacted by climate change (Mas-Coma et al. 2019) and the trading in live animals is accelerating the spread of novel species to new areas as well as encouraging the development of hybrid forms (Agatsuma et al. 2000). Fasciola distribution causes controversy over species identification in several countries where morphological criteria are applied in research investigations. Presently, the GenBank database has a significant number of incomplete nad1 and cox1 gene sequences of Fasciola species isolated from different host species (Reaghi et al. 2016). Indicators like genetic diversity and population enable us to identify parasite adaptation and fitness in specific habitats (Rouhani et al. 2017).

Molecular studies play a significant role in the detection of dissemination of Fasciola hepatica due to host spectrum and ecological diversity (Thang et al. 2019). Diversity indices advocate that using the main population in defined ecosystems assists in better comprehending the biological perspectives and forecasting antigenic and phenotypic differences (Ai et al. 2011).

The genetic diversity of the F. hepatica population is also influenced by the intermediate hosts where clonal expansion occurs (Beesley et al. 2017). The potential of the intermediate host to spread metacercariae was confirmed by Vilas et al. (2012). Furthermore, the same multilocus genotype was found to be shared among cattle and sheep. The non-significant difference in genetic diversity of F. hepatica isolates from domesticated animals was observed by Beesley et al. (2017); however, a wide spectrum of wild animals being infected by F. hepatica is supposed to be responsible for maintaining genetic diversity. The available literature indicates a high genetic diversity level in F. hepatica populations, which could facilitate adaptation to environmental selection pressure through clonal reproduction in snails, allowing for the rapid spread of resistant populations (Elliott et al. 2014).

Molecular studies that employ both nuclear and mitochondrial DNA have identified diverse fluke populations across the globe. The nuclear DNA markers, ribosomal internal transcribed spacer (ITS), phosphoenolpyruvate carboxykinase (pepck), DNA polymerase delta (pold), cytochrome c oxidase I (cox1), and NADH dehydrogenase (nad1) genes are some of the markers that can be used for genetic characterization of Fasciola spp., as described by Sarkari et al. (2017) and Chougar et al. (2019).

Mitochondrial DNA (mt-DNA), has been used as a marker for population diversity. Mitochondrial cox1 and nad1 genes are frequently used as DNA markers to investigate the genetic variability of Fasciola spp. These markers are of high importance to study biogeography and population structure of F. hepatica (Itagaki et al. 2005). Furthermore, microsatellite DNA simple sequence repeat markers have also been studied to reveal the population structure of various parasites (Yin et al. 2016; Moendeg et al. 2017). The genetic information of the population and the level of intimate kinship are frequently assessed using analysis of mt-DNA sequences because of the absence of recombination, maternal inheritance, conserved structure, higher mutation rate and a relatively higher evolutionary rate (Jia et al. 2012). To identify genetic variations within Fasciola species, incomplete sequences of cox1 and nad1 genes have been effectively employed (Bowles and McManus 1993). To understand the population dynamics, it is essential to assess the genetic diversity in F. hepatica isolates globally. This study analyzed the cox1 and nad1 gene sequences available in the GenBank repository to describe genetic variation, population structure, and phylogeny of F. hepatica.

Methods

Data Collection

A total of 604 gene sequences of F. hepatica were retrieved and a dataset was produced after selecting the cox1 (n = 247) and nad1 (n = 357) genes, submitted to the National Center for Biotechnology Information, USA (NCBI), database until 9 February 2023.

Alignment and Phylogenetic Analysis

By using Molecular Evolutionary Genetics Analysis Version 11 (MEGA 11), all gene sequences were assembled into it in FASTA format. The reference sequences cox1 (accession no. LC273025) and nad1 (accession no. MK838688) were used to align all of the sequences after they had been cut off at both ends. Short gene sequences were eliminated leaving 604 gene sequences of 387 bp cox1 (n = 247) and 689 bp nad1 (n = 357) for bioinformatic analysis. Phylogenetic trees were created from the sequences of both gene regions using the neighbor-joining (NJ) model and the Jukes–Cantor nucleotide distance measure. The 1000 bootstrap replicates were used to obtain statistical support for the branch specificity. To determine the strength of the relationships, the sequence of Schistosoma japonicum was used as an outgroup.

Haplotype analysis and networking

The sequences in FASTA format were examined during the haplotype analysis by using the tool DnaSP 6 (Rozas et al. 2017). Calculations were made to estimate the genetic composition of both the genes using the haplotype and nucleotide change values, nucleotide and haplotype numbers, and the neutrality indices. For a visual depiction of the associations between haplotypes, the sequences were converted to Nexus format (Maddison et al. 1997) and a haplotype network was created using the PopArt (Population Analysis with Reticulate Trees) application (Leigh and Bryant 2015).

Results

A total of 604 gene sequences from F. hepatica isolates were analyzed in this study, comprising 247 cox1 sequences obtained from 9 countries and 357 nad1 sequences obtained from 11 countries (as shown in Table 1). All of these sequences were sourced from the NCBI database.

Table 1 Accession numbers of cox1 and nad1 gene fragments of F. hepatica isolates used in the study
Full size table

Polymorphism and haplotype analysis

Mutations were detected at 154 and 275 different points within the cox1 and nad1 gene sequences. Forty-six distinctive haplotypes were identified by the analysis of 247 cox1 gene sequences (Table 2). The dominating haplotype among these was Hap01, which had 64 genetic sequences. By evaluating 357 nad1 genetic sequences, 98 haplotypes were recognized (Table 3). Out of these, Hap11 was the dominant haplotype and consisted of 113 gene sequences.

Table 2 Haplotypes of cox1 sequences of F. hepatica and accession numbers of isolates forming groups
Full size table
Table 3 The haplotypes of nad1 sequences of F. hepatica and accession numbers of isolates forming groups
Full size table

Haplotype network

There were 46 haplotypes in the cox1 haplotype network (Fig. 1). The major haplotype in the network was Hap01, which contributed 25.91% (64/247) and was followed by Hap15 with 18.21% (45/247). A single distinct haplotype mainly composed 65.21% (30/46) of the total haplotype network. Individual haplotypes came from Algeria (n = 13), Iran (n = 7), Ecuador (n = 3), Iraq (n = 3), Spain (n = 3), and Italy (n = 1).

Fig. 1
figure 1

Appearance of cox1 (387 bp) haplotypes F. hepatica sequences. The number of mutations that distinguish haplotypes is indicated by screening marks. The geographical distribution of haplotypes is shown in different colors. The size of the circles is related to the haplotype frequency

Full size image

There were 98 haplotypes in the nad1 haplotype network (Fig. 2). The major haplotype in the network was Hap11, which contributed 31.65% (113/357) and was followed by Hap40 with 16.52% (59/357). A single distinct haplotype mainly composed 77.55% (76/98) of the total haplotype network. Individual haplotypes came from Spain (n = 24), Brazil (n = 14), Armenia (n = 12), Ecuador (n = 8), Afghanistan (n = 7), Algeria (n = 7), and Iran (n = 4).

Fig. 2
figure 2

Appearance of nad1 (689 bp) haplotypes F. hepatica sequences. The number of mutations that distinguish haplotypes is indicated by screening marks. The geographical distribution of haplotypes is shown in different colors. The size of the circles is related to the haplotype frequency.

Full size image

Phylogenetic tree

The findings of the phylogenetic analysis were reliable with the haplotype network. The cox1 and nad1 gene sequences were aligned to create the phylogenetic tree as mentioned in Figs. 3 and 4, respectively. Schistosoma japonicum was added as outgroup in both phylogenetic trees. The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei 1987). The optimal trees are shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. This analysis involved 52 and 103 nucleotide sequences for cox1 and nad1 trees, respectively. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA11 (Tamura et al. 2021).

Fig. 3
figure 3

Phylogenetic tree view of F. hepatica sequences using cox1 gene (387 bp)

Full size image
Fig. 4
figure 4

Phylogenetic tree view of F. hepatica sequences using nad1 gene (689 bp)

Full size image

Gene flow, diversity, and neutrality analysis

In Table 4, the diversity and neutrality indices are given for the cox1 and nad1 genes. To ascertain whether populations were under selection pressure, the values of Tajima’s D and Fu’s Fs were determined.

Table 4 Diversity and neutrality indices obtained using nucleotide data of the cox1 (387 bp) and nad1 (689 bp) gene sequences of F. hepatica
Full size table

Diversity and neutrality indices for F. hepatica isolates from various geographical regions

To provide insights into the future population growth in each region, the sequences used in the study were further analyzed to compute diversity and neutrality indices. Diversity and neutrality indices of cox1 and nad1 genes are shown in Tables 5 and 6, respectively.

Table 5 Region-wise diversity and neutrality indices obtained using nucleotide data of the cox1 gene sequences of F. hepatica obtained from the NCBI GenBank database
Full size table
Table 6 Region-wise diversity and neutrality indices obtained using nucleotide data of the nad1 gene sequences of F. hepatica obtained from the NCBI GenBank database
Full size table

For cox1 gene sequences from Ecuador, Iran, Italy, and Spain, the negative but insignificant values of Tajima’s D were observed while this parameter was statistically significant and negative for the Iraqian isolates. This indicated deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. A positively significant value of Tajima’s D was observed for the isolates from Algeria and the value was also positive but insignificant for the isolates from South Africa. Diversity indices were not calculated for the isolates from Australia and Egypt as four or more sequences are needed to compute Tajima’s D value. Fu’s Fs was negative for the entire population indicating an excess number of alleles, as would be expected during genetic hitchhiking or a recent population expansion except for the Algerian and South African isolates which were positive but insignificant.

Regarding nad1 gene sequences, the negatively significant values of Tajima’s D were observed for the isolates from Algeria, Armenia, Brazil, Ecuador, and Spain. Like cox1 gene sequences, this also indicated deviation from neutrality suggesting a recent population expansion. Diversity indices were not calculated for the isolates from Australia and Uruguay as four or more sequences are needed to compute Tajima’s D value. Fu’s Fs was negative for the entire population indicating an excess number of alleles, except for the Argentinian isolates which were positive but insignificant. For nad1 gene analysis, Japan was excluded in indices calculations as only one sequence was included from Japan in this study.

Host-based diversity and neutrality indices for F. hepatica isolates

In order to provide a better comprehensive understanding of the genetic dynamics of the parasite in various hosts and its circulation in the environment, the sequences used in the study were further analyzed to compute diversity and neutrality indices based on their host origin. A total of 12 cox1 and 20 nad1 sequences available in the NCBI GenBank database were without any information about their host. Thus, only 235 cox1 and 337 nad1 gene sequences were included in the host-wise analysis of data. Diversity and neutrality indices of cox1 and nad1 genes are shown in Tables 7 and 8, respectively.

Table 7 Host-wise diversity and neutrality indices obtained using nucleotide data of the cox1 gene sequences of F. hepatica obtained from the NCBI GenBank database
Full size table
Table 8 Host-wise diversity and neutrality indices obtained using nucleotide data of the nad1 gene sequences of F. hepatica obtained from the NCBI GenBank database
Full size table

Regarding the cox1 gene sequences, the values of Tajima’s D were negative but insignificant for the isolates of goat and human origin. Overall, positive and significant Tajima’s D values were observed. A similar pattern of Fu’s Fs values was observed.

Regarding the nad1 gene, the negative and statistically significant value of Tajima’s D indicated events of recent population expansion while this value was positive for the isolates of cattle origin. Fu’s Fs was negative for the isolates of sheep origin while the overall value was insignificant and positive.

The cox1 and nad1 gene sequences were aligned to create the phylogenetic tree (Figs. 5 and 6) to describe host-wise diversity. Schistosoma japonicum was added as an outgroup in both phylogenetic trees. Representative haplotypes were included to construct the phylogenetic trees. For nad1, 52 cattle and 60 sheep isolates were included out of a dataset of 337 sequences while for the cox1 gene, 35 cattle, 15 sheep, 5 goat, and 4 human samples were included out of a dataset of 235 sequences.

Fig. 5
figure 5

Evolutionary analysis of host wise investigation of cox1 gene sequences inferred by maximum likelihood method

Full size image
Fig. 6
figure 6

Evolutionary analysis of host wise investigation of nad1 gene sequences inferred by maximum likelihood method

Full size image

Discussion

Infectious diseases including parasitic infestations are important health problems in both animals and human beings (Mo’awad et al. 2022; Mahmoud et al. 2022). Parasites, in addition to causing diseases in animals, indirectly affect the economics of the farmer by decreasing the productivity potential of animals (Mahmood et al. 2022; Alberfkani et al. 2022). Due to the growing infection risks, fasciolosis, a recurrent ignored tropical disease, is becoming a significant issue for both food quality and human safety. The current study offers significant findings about the genetic analysis and global diversity of F. hepatica.

High levels of rainfall, the optimal combination of temperature and humidity, public eating habits, ecological factors of Fasciola transmission, free ruminant grazing, and the presence of an intermediate host favor the propagation of fasciolosis in an area (Akhlaghi et al. 2017). It is widely known that maximum genetic flow, which increases the actual population size across a range of geographical zones where the features associated with heterogeneity may be prevalent, can frequently result in the genetic variation of parasites. Due to the variety of hosts and ecological circumstances, molecular epidemiology investigations are essential for identifying Fasciola dissemination (Thang et al. 2019).

High variability has also been observed in other research that has used haplotype diversity to investigate F. hepatica. For instance, research employing the nad1 gene identified 24 haplotypes from 79 Brazilian individuals and 37 haplotypes from 130 Iranian individuals (Bozorgomid et al. 2020). Eight haplotypes were found within 90 F. hepatica samples (based on a 535 bp nad1 fragment) and six haplotypes were found within 30 F. hepatica samples (based on a 745 cox1-nad1 concatenated fragment) in previous investigations from Iran and Armenia, respectively (Reaghi et al. 2016; Semyenova et al. 2006). In a Peruvian investigation, the nad1 component from 78 distinct parasites was examined. Eight haplotypes were discovered (Hd = 0.685 and π = 0.00175) (Ichikawa-Seki et al. 2016). Seven cox1 gene haplotypes were found in an Argentine investigation that included 22 participants. When nad4 and nad5, two more mitochondrial genes, were examined, four and three haplotypes were found, correspondingly (Carnevale et al. 2017). Only six cox1 haplotypes (Hd = 0.482 and π = 0.003) and 18 nad1 haplotypes (Hd = 0.832 and π = 0.005) were found in an analysis of 208 samples conducted by Elliott et al. (2014) in Australia. Ichikawa-Seki et al. (2017) discovered that in 211 lamellipodia from different geographic regions of China, 11 haplotypes belonged to an F. hepatica population and 18 haplotypes belonged to an F. gigantica population.

The existence of F. hepatica in Peru, Argentina, Australia, and Southern Brazil with a very limited number of individuals, each from a much larger parental population, producing a Founder’s effect, may be one explanation for both high haplotype diversity and low nucleotide diversity. We propose that F. hepatica was introduced into Brazil in a number of distinct waves of human and cattle immigration in order to more adequately explain the large number of haplotypes that were observed.

In the present study, the genetic variation and population dynamics of F. hepatica were evaluated. This was accomplished by utilizing sequenced information from the cox1 and nad1 gene sequences that were obtained from GenBank and are frequently utilized for Fasciola species differentiation. As a result of this work, knowledge about global gene flow and population dynamics in F. hepatica infections was gained. For this in silico analysis, we used a total of 247 cox1 (387 bp) and 357 nad1 (689 bp) gene sequences of F. hepatica isolates that were already registered in the NCBI database to assess the genetic diversity and variation of the F. hepatica. The results of the current study show an extremely high global haplotype diversity within both cox1 and nad1 genes. The 604 samples analyzed represented a total of 46 haplotypes for the cox1 and 98 for nad1 genes.

Nucleotide diversity and population expansion were assessed by using neutrality indices including Tajima D, Fu’s Fs, and Fu’s LD (Ramos-Onsins and Rozas 2002). A positive Tajima D value indicates heterozygosity, which is described as having a selective advantage, whereas negative values show that one allele has a selective advantage over the other allele. The Tajima D test measures how much populations deviate from the traditional neutral model. A negative score also denotes a rapid population increase (Vamathevan et al. 2008; Stephens et al. 2001). Both the cox1 and nad1 gene segments in our study had high Tajima’s D values, which suggested a low likelihood of future population growth. The Tajima’s D value of the nad1 gene sequence is lower (2.14910) than that of the cox1 gene sequence (3.40314), which suggests that the former is growing at a slower rate. With a significantly negative value (p < 0.05), Fu’s Fs serves as a marker of population growth sensitivity, showing that the populations share the same gene pool and exhibit similar growth tendencies (Fu 1997; Li et al. 2009). In both the cox1 and nad1 haplotype groupings, our study produced highly positive and statistically non-significant Fu’s Fs values, indicating that these populations have not spread internationally.

The level of polymorphism in the population was found by evaluating nucleotide diversity. We found that the mean nucleotide difference between the nad1 (0.21029) and cox1 (0.17426) gene sequences was larger. In order to determine the uniqueness of haplotypes within the population, haplotype diversity was also evaluated. The values of the nad1 (0.860) and cox1 (0.869) gene sequences were extremely comparable in our investigation.

Our investigation of the cox1 gene sequences resulted in the discovery of 46 haplotypes in total. The network consisted of 30 individual haplotypes, with the dominant haplotype accounting for 25.91% of the whole. In our examination of the nad1 gene sequence, 98 distinct haplotypes were found. There were 76 individual haplotypes, and the predominant haplotype made up 31.65% of the entire network. The major haplotypes all share a common ancestry.

The 387 bp cox1 gene sequences had 154 unique mutations found throughout them, whereas the 689 bp nad1 sequences had 275 unique mutations found. The longer and more complicated evolutionary history of F. hepatica may be reflected in the higher mutation rates. Worldwide, there is a very high level of genetic variation within the F. hepatica species, and the complicated phylogeographic patterns that have been identified by phylogenetic and geographic analyses imply that the intensive animal trade has influenced the species’ current distribution. Several South American, North African, Asian, and Middle Eastern countries (Ecuador, Brazil, Algeria, Iran, Afghanistan, etc.) had a high number of haplotypes detected in this study, suggesting that F. hepatica has been present in these regions for a longer period of time than in some western nations (Italy and Spain).

Region-wise analysis revealed that both the cox1 and nad1 genes showed deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. Furthermore, overall host-wise analysis showed positive and significant Tajima’s D values for both the cox1 and nad1 gene sequences. To the best of our knowledge, this is the first attempt to provide insights into genetic variations and population structure of F. hepatica at a global scale using cox1 and nad1 genes. Our findings suggest the existence of specific variants of F. hepatica in different parts of the world and provide information on the molecular ecology of F. hepatica. In this analysis, 387 bp cox1 and 689 bp nad1 gene consensus sequences were investigated to describe genetic diversity. In the future, we recommend the utilization of longer, rather complete, gene sequences to provide a broader picture of genetic variations among F. hepatica isolates. It is further suggested to study additional mitochondrial markers as the discriminatory power of a set of mitochondrial gene targets is superior to describing genetic diversity.

Conclusion

Communities with a high prevalence of intermediate hosts face a serious threat from F. hepatica. This study is the first bioinformatics investigation to assess the genetic structure F. hepatica gathered globally, despite the fact that numerous molecular studies have been carried out to date. All of the sequencing information from F. hepatica isolates relevant to cattle and humans was presented in this study. We assume that this research can close any knowledge gaps in the area. Our findings also mark a critical development in upcoming epidemiological and bioecological research that may result in effective therapies for specific species or strains.