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Background Snails of the Lymnaeidae family are the intermediate hosts of Fasciola species, the causative agents of fascioliasis. The purpose of this study was to determine the prevalence of Fasciola species in lymnaeid snails and to investigate the association of geoclimatic factors and Fasciola species distribution in northwestern provinces of Iran using geographical information system (GIS) data. Methods A total of 2000 lymnaeid snails were collected from 33 permanent and seasonal habitats in northwestern Iran during the period from June to November 2021. After identification by standard morphological keys, they were subjected to shedding and crushing methods. Different stages of Fasciola obtained from these snails were subjected to the ITS1 polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method for species identification. The associations of weather temperature, rainfall, humidity, evaporation, air pressure, wind speed, elevation, and land cover with the distribution of Fasciola species were investigated. Geographical and statistical analysis was performed using ArcMap and SPSS software, respectively, to determine factors related to Fasciola species distribution. Results Of the 2000 snails collected, 19 were infected with Fasciola hepatica (0.09%), six with F. gigantica (0.03%), and 13 with other trematodes. Among geoclimatic and environmental factors, mean humidity, maximum humidity, and wind speed were significantly higher in areas where F. hepatica was more common than F. gigantica . The altitude of F. hepatica -prevalent areas was generally lower than F. gigantica areas. No significant relationship was observed between other investigated geoclimatic factors and the distribution of infected snails. Conclusions The present study showed the relationship of humidity and wind speed with the distribution of snails infected with F. hepatica or F. gigantica in the northwestern regions of Iran. In contrast to F. gigantica , F. hepatica was more prevalent in low-altitude areas. Further research is recommended to elucidate the relationship between geoclimatic factors and the presence of intermediate hosts of the two Fasciola species. Graphical Abstract

Fasciolosis is a parasitic disease affecting humans and livestock, caused by digenean trematodes of the genus Fasciola , primarily F. hepatica and F. gigantica . This study investigates the coexistence of these species and their hybrids in yaks from Tibet, China. We analyzed the nuclear rDNA internal transcribed spacer (ITS) regions, including ITS1 and ITS2, through Sanger sequencing and Next-Generation Sequencing (NGS) to assess single-nucleotide polymorphisms (SNPs). Our results reveal that one specimen (NM008B) is identical to pure F. hepatica , while another (NM008A) contains genetic information from both F. hepatica and F. gigantica , indicating potential hybridization or introgression. The morphological analysis reveals that the collected adult F. hepatica specimens exhibit distinct characteristics, while the hybrid specimens display “intermediate” features of F. hepatica and F. gigantica . This study is the first to document the coexistence of F. hepatica and hybrid Fasciola forms in a single yak. The findings underscore the complexities of hybridization dynamics and the necessity for advanced molecular techniques in accurately identifying Fasciola species. Future research should focus on mitochondrial DNA and other nuclear gene analysis to further elucidate the nature of these hybrids and their ecological implications.

In the present study, 16 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from sheep and cattle from seven geographical locations in Niger were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361/362 bp, respectively, for all liver fluke samples sequenced. Comparison of the ITS sequences of the Niger Fasciola samples examined in the present study with that of Fasciola hepatica, Fasciola gigantica, and the “intermediate Fasciola” from elsewhere revealed that the Niger Fasciola samples examined represent two species, namely F. hepatica and F. gigantica. This is the first demonstration of the existence of both F. hepatica and F. gigantica in Niger by a genetic approach, which provides foundation for further studies on F. hepatica and F. gigantica in Niger and has implications for studying the population genetic structure of the Niger Fasciola and for the diagnosis and control of the disease they cause.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica , is a globally significant parasitic disease affecting livestock and humans. Both species exist in South Africa, however, information on their geographical distribution and genetic diversity is limited. This study investigated the genetic diversity and phylogenetic relationships of Fasciola species in South Africa using Pepck, COI and ITS markers. Two hundred and seven Fasciola spp. specimens analysed in this study showed that 74.4% of isolates identified as F. hepatica and 25.6% as F. gigantica based on the Pepck gene. The geographical distribution revealed that F. hepatica occurred in all surveyed provinces but was dominant in the temperate zones of South Africa. Fasciola gigantica was confined to subtropical zones, with co-occurrence observed in Limpopo, Mpumalanga, and KwaZulu-Natal provinces. The COI and ITS genes in both Fasciola species generated fourteen and eight haplotypes respectively, with low to moderate haplotype diversity (Hd = 0.287 – 0.509 for COI and 0.056 – 0.239 for ITS) and low nucleotide diversity (Pi = 0.00085 – 0.00284 for COI and 0.00024 – 0.00034 for ITS), suggesting recent population diversification between the two species. The study samples yielded six haplotypes based on COI, with three haplotypes comprising most samples i.e. Hap-1 ( F. gigantica) and Hap-5 and Hap-6 ( F. hepatica) . With ITS, two haplotypes represented most samples, i.e. Hap-1 ( F. gigantica ) and Hap-3 ( F. hepatica) . There was no observed unique distribution of haplotypes based on provinces or agro-ecological climatic zones for both markers. Phylogenetic analysis of both COI and ITS genes confirmed the grouping of the haplotypes and reiterated their relatedness. However, one isolate identified as F. gigantica in COI and with F. hepatica in ITS based on BLAST and further formed haplotypes with F. gigantica and F. hepatica haplogroups for COI and ITS, respectively. This suspected hybrid Fasciola form was from an animal with a mixed infection. These findings highlight the complex genetic structure of Fasciola spp. populations in South Africa and provide new insights into their epidemiology, with implications for targeted control strategies and further investigation of cryptic/hybrid lineages.

A total of 12103 snails examined in Central Kashmir for determining the population dynamics of cercariae revealed overall prevalence to be 4.03%. Gymnocephalus (0.13%), furcocercous (0.28%), echinostome (0.34%) and xiphido-cercaria (3.26%) were recorded. The prevalence of cercaria was recorded highest in summer (4.28%) followed by spring (4.05%) and autumn (3.32%). None of the cercaria was recorded during winter season. Morphologically identified cercariae of Veterinary importance were subjected to molecular analysis using genus (28S rDNA and ITS-2) specific primers. The isolates of gymnocephalus cercaria (FA28, FC28, FZ28, FC2) were identified as cercarial stages of Fasciola spp. The phylogenetic trees revealed that the isolates FA28 and FC28 belonged to Fasciola gigantica and FZ28 and FC2 isolate to F. hepatica . The isolate of Fasciola gigantica (FA28, FC28, FZ28) showed 2, 4 and 13 nucleotide polymorphisms. There was addition and deletion of 1 and 8 nucleotides at various positions in case of Fasciola isolates respectively. Besides this, there were nucleotide substitutions at 4 positions along with presence of nucleotide T at 475 position which confirmed it be Fasciola hepatica . The isolates of echinostome cercaria (B1, BD13 and GY) were identified as cercarial stages of Moliniella anceps , Echinoparyphium recurvatum and family Echinostomatidae respectively. The Moliniella anceps isolate showed prominent differences at 8 positions with respect to other Echinostomatidae spp. The insertion of C at position 612 confirmed it to be Moliniella anceps , while as other two isolates showed 2 nucleotide polymorphisms each after 28S gene amplification. On ITS-2 rDNA analysis, the isolate B1 showed 7 nucleotide polymorphisms and phylogenetic tree revealed that the isolate B1, also belonged to Echinoparyphium recurvatum . The study made it very clear that molecular characterization employing internal transcribed spacer (ITS-2) and 28S ribosomal DNA sequences are reliable approach for genetic differentiation of cercarial stages of trematodes. The phylogenetic taxonomy of echinostomes is still unclear and molecular diversity found in this study is perhaps the first study from India as well as in Indian subcontinent. So, focus should be made more on echinostomes for understanding their morphological, biological and molecular diversity for clarifying their taxonomic position.

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis , with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.

In the present study, a polymerase chain reaction-linked single-strand conformation polymorphism (PCR-SSCP) approach combined with DNA sequencing was used to characterise samples of Fasciola spp. from different host species and geographical locations in mainland China. The first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by PCR from individual Fasciola and analysed by SSCP. SSCP analyses displayed three different banding profiles that allowed the identification of all Fasciola samples examined into three groups: Fasciola hepatica, F. gigantica and the “intermediate” Fasciola. Then, the ITS-1 rDNA was sequenced from representative Fasciola samples, and analysis of the complete ITS-1 sequences supported the identification of all Fasciola samples by SSCP approach. The length of the ITS-1 sequences was 422 bp for all Fasciola samples sequenced. Although there was no variation in length or composition of the ITS-1 sequences among multiple specimens within each of the taxa, F. hepatica and F. gigantica differed by 1.2% in their ITS-1 sequences, whereas the “intermediate” Fasciola was unique, in which two different ITS-1 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is identical to that of F. gigantica. This study demonstrated that PCR-SSCP analysis of the ITS-1 rDNA followed by selective sequencing provides a reliable approach for the accurate identification of Fasciola spp., and also supports the existence of the “intermediate” Fasciola between F. hepatica and F. gigantica in mainland China.

Trematode parasites, or flukes, are a significant economic threat to ruminant production worldwide. Traditional diagnostic methods rely on egg sedimentation from faeces, a time-consuming methodology lacking sensitivity and specificity. This study aimed to develop and validate two detection methods: firstly, qPCR for accurate identification of Fasciola spp., and secondly, a deep amplicon sequencing technique for identifying fluke species using faecal sedimented egg DNA. To detect Fasciola spp., infection, primers targeting mitochondrial DNA were repurposed to develop a SYBR Green qPCR assay. For the identification of fluke species, a deep amplicon sequencing approach was developed. A reference sequence library and taxonomy file were generated for 21 fluke species, potentially enabling species-level sequence read separation for a range of trematodes and extraction of amplicon sequence variants (ASVs). To validate the qPCR and deep amplicon sequencing approach, 402 faecal samples were collected from cattle and sheep across the UK. Fluke eggs were isolated by sedimentation, screened by microscopy and qPCR, Sanger sequencing and deep amplicon sequencing to identify fluke eggs to species level. qPCR demonstrated high analytical sensitivity, detecting Fasciola hepatica DNA down to 19.2 fg and F. gigantica down to 6.4 fg, with no cross-amplification of other flukes. Deep amplicon sequencing was able to detect as few as five F. hepatica and Calicophoron daubneyi eggs and identify mixed infections. High levels of co-infection (14.4%) of F. hepatica and C. daubneyi were observed in faecal samples, followed by single infections with C. daubneyi (12.6%) and F. hepatica (3.2%). Notably, deep amplicon sequencing detected F. hepatica in 20 samples missed by qPCR. Data analysis identified 55 and 32 ASVs for F. hepatica and C. daubneyi, respectively, with phylogenetic clustering within their respective clades. This study developed a qPCR assay for Fasciola spp. detection and validated a deep amplicon sequencing for fluke species differentiation. These approaches are able to identify fluke species in excreta from infected ruminants and provide additional valuable tools for enhancing fasciolosis surveillance and control.

Background In Egypt, liver flukes, Fasciola spp. (Digenea: Fasciolidae), have a serious impact on the farming industry and public health. Both Fasciola hepatica and Fasciola gigantica are known to occur in cattle, providing the opportunity for genetic recombination. Little is known on the identity and genetic variability of Fasciola populations in sheep. Methods This study was performed to determine the prevalence of liver flukes in sheep in Menofia Province as a representative area of the delta region in Egypt, as measured by postmortem examination of slaughtered animals at three abattoirs. The identity and genetic variability of Fasciola spp. in slaughtered animals were determined by PCR-sequence analysis of the nuclear ribosomal internal transcribed spacer 1 (ITS1) and the mitochondrial NADH dehydrogenase subunit 1 ( nad 1) genes. Results Physical inspection of the liver indicated that 302 of 2058 (14.7%) slaughtered sheep were infected with Fasciola spp. Sequence analysis of the ITS1 and nad 1 genes of liver flukes from 17 animals revealed that 11 animals were infected with F. hepatica , four with F. gigantica , and two with both species. Seventy eight of 103 flukes genetically characterized from these animals were F. hepatica , 23 were F. gigantica, and two had ITS1 sequences identical to F. hepatica but nad 1 sequences identical to F. gigantica. nad 1 sequences of Egyptian isolates of F. gigantica showed pronounced differences from those in the GenBank database. Egyptian F. gigantica haplotypes formed haplogroup D, which clustered in a sister clade with haplogroups A, B and C circulating in Asia, indicating the existence of geographic isolation in the species. Conclusions Both F. hepatica and F. gigantica are prevalent in sheep in Egypt and an introgressed form of the two occurs as the result of genetic recombination. In addition, a geographically isolated F. gigantica population is present in the country. The importance of these observations in epidemiology of fascioliasis needs to be examined in future studies.

Fasciola gigantica is a widespread parasite that causes neglected disease in livestock worldwide. Its high transmissibility and dispersion are attributed to its ability to infect intermediate snail hosts and adapt to various mammalian definitive hosts. This study investigated the variation and population dynamics of F. gigantica in cattle, sheep, and goats from three states in Sudan. Mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes were sequenced successfully to examine intra and interspecific differences. ND1 exhibited higher diversity than COI, with 15 haplotypes and 10 haplotypes, respectively. Both genes had high haplotype diversity but low nucleotide diversity, with 21 and 11 polymorphic sites for ND1 and COI, respectively. Mismatch distribution analysis and neutrality tests revealed that F. gigantica from different host species was in a state of population expansion. Maximum likelihood phylogenetic trees and median networks revealed that F. gigantica in Sudan and other African countries had host-specific and country-specific lineages for both genes. The study also indicated that F. gigantica -infected small ruminants were evolutionarily distant, suggesting deep and historical interspecies adaptation.