Potential Hybridization of Fasciola hepatica and F. gigantica in Africa—A Scoping Review

The occurrence of Fasciola gigantica and F. hepatica in Africa is well documented; however, unlike in Asia, there is a paucity of information on the existence of hybrids or parthenogenetic species on the continent. Nonetheless, these hybrid species may have beneficial characteristics, such as increased host range and pathogenicity. This study provides evidence of the potential existence of Fasciola hybrids in Africa. A literature search of articles published between 1980 and 2022 was conducted in PubMed, Google Scholar, and Science Direct using a combination of search terms and Boolean operators. Fasciola species were documented in 26 African countries with F. hepatica being restricted to 12 countries, whilst F. gigantica occurred in 24 countries, identified based on morphological features of adult Fasciola specimens or eggs and molecular techniques. The co-occurrence of both species was reported in 11 countries. However, the occurrence of potential Fasciola hybrids was only confirmed in Egypt and Chad but is suspected in South Africa and Zimbabwe. These were identified based on liver fluke morphometrics, assessment of the sperms in the seminal vesicle, and molecular techniques. The occurrence of intermediate host snails Galba truncatula and Radix natalensis was reported in Ethiopia, Egypt, South Africa, Tanzania, and Uganda, where F. hepatica and F. gigantica co-occurrences were reported. The invasive Pseudosuccinea columella snails naturally infected with F. gigantica were documented in South Africa and Egypt. In Zimbabwe, P. columella was infected with a presumed parthenogenetic Fasciola. This suggests that the invasive species might also be contributing to the overlapping distributions of the two Fasciola species since it can transmit both species. Notwithstanding the limited studies in Africa, the potential existence of Fasciola hybrids in Africa is real and might mimic scenarios in Asia, where parthenogenetic Fasciola exist in most Asian countries. In South Africa, aspermic F. hepatica and Fasciola sp. have been reported already, and Fasciola hybrids have been reported? in Chad and Egypt. Thus, the authors recommend future surveys using molecular markers recommended to identify Fasciola spp. and their snail intermediate hosts to demarcate areas of overlapping distribution where Fasciola hybrids and/or parthenogenetic Fasciola may occur. Further studies should also be conducted to determine the presence and role of P. columella in the transmission of Fasciola spp. in these geographical overlaps to help prevent parasite spillbacks.


Introduction
Fasciolosis is an important food and water-borne zoonotic infection of human, domestic, and wild animals mainly caused by the two liver flukes, Fasciola hepatica (Linnaeus, 1758) and F. gigantica (Cobbold, 1856) [1][2][3]. Of the two species, F. hepatica has been shown to have a cosmopolitan distribution occurring in five continents except for Antarctica [4,5], whilst F. gigantica is restricted to the tropical and subtropical regions of Asia and Africa [6]. According to [7], the distribution of both Fasciola species is associated with the availability and dispersal of viable snail vectors that act as -intermediate hosts (IHs) as well as climatic and ecological conditions suitable for the survival of these snails [8][9][10].
Previous data have suggested that F. hepatica originated from Eurasian ovicaprines, especially Ovis species [11]. This concept has been generally accepted due to the dispersion of its preferred IH snail, Galba truncatula (Müller, 1774), which is associated with areas with mild and cold climates [12], and very high altitudes [11]. Recent data elucidated that the ancestral fasciolids of F. hepatica and F. gigantica are speculated to have emerged in the lowlands of East Africa [13], followed by speciation of F. hepatica in the Eurasian Near East and F. gigantica in Africa [11]. Fasciola hepatica then spread from Eurasia to other parts of the world [11] where its main IH is G. truncatula [14,15]. In Africa, the cryptic species Galba mweruensis (Connolly, 1929) has been indicated as an IH of both Fasciola species [16,17] but has only been proven to transmit F. hepatica in Lesotho and Ethiopia [18,19].
Based on [11], the assumption that the emergence of Fasciolinae species and secondary colonization by F. gigantica and other Fasciola species in Africa was favored by a switch of IHs from planorbid to lymnaeid snails as stated by [20] does not fit the current knowledge. Fasciola gigantica is distributed throughout western, sub-Saharan, and eastern Africa following the wide distribution of the snail intermediate host species Radix natalensis (Krauss, 1848) that it adapted to [11]. Furthermore, this fasciolid species has adapted to ruminants from families Giraffidae, Reduncinae, and Alcelaphinae in sub-Saharan Africa as definitive hosts [11]. Following the movement of animals facilitated by humans and the subsequent introduction of intermediate host snail species into new areas, F. gigantica spread to other regions. In these regions, its main IHs are the Radix species of the "auricularia super-species" (Hubendick, 1951) R. rubiginosa (Minchelin, 1831) in Asia, and R. natalensis in Africa [9,21].
The spread of both Fasciola species led to overlapping distributions in areas where climatic conditions allow the IHs of both species to survive and co-exist, particularly in tropical regions of Africa and Asia [11,22]. In South Africa, the invasive snail Pseudosuccinea columella (Say, 1817) has been suspected to be the vector snail responsible for possibly transmitting both Fasciola species, thus contributing towards the overlap [23], following an observed increase in infection rates of both Fasciola species coinciding with the introduction of the invasive snail species in the country [24]. Pseudosuccinea columella has been reported to be responsible for the secondary transmission of F. hepatica in South America and the Caribbean region [9,25], and it has been proven to naturally transmit F. gigantica in Africa [23,26]. Therefore, it can be hypothesized that P. columella may potentially be transmitting F. hepatica in Africa, supported by successful experimental infections of the Egyptian P. columella population with F. hepatica [27] and natural infections with an unknown yet suspected Fasciola hybrid in South Africa [23] and Zimbabwe [28], thus facilitating the overlapping geographical distribution of Fasciola species in some African countries as reported in Egypt [29] and South Africa [30].
In areas of geographical overlap, hybridization between F. hepatica and F. gigantica has been reported, resulting in Fasciola hybrid species consisting of admixed/introgressive genotypes due to interspecific mating [31,32]. According to [13], this hybridization may have occurred about 2000 years ago in China, resulting in parthenogenetic populations which were then spread across multiple Asian countries where they co-exist with F. gigantica. However, due to a lack of standardized methods for identifying these populations, the parthenogenetic Fasciola in Japanese populations was initially misidentified as F. hepatica [33]. Moreover, parthenogenetic Fasciola was previously called aspermic Fasciola but the discovery of few aspermic triploid individuals that produced and stored mature sperms in their seminal vesicles led to this term being disregarded. Thus, parthenogenetic Fasciola can be distinguished from other Fasciola hybrids by its ability to reproduce and maintain successive generations [13].
Although [13] suggests that the parthenogenetic Fasciola populations have not been reported outside of Asia, other forms of Fasciola hybrid populations have been suspected in some African countries, as evidenced by the recent reports of aspermic F. hepatica and unidentified Fasciola sp. [23,28,30]. However, they could have been missed outside Asia due to the paucity of research on the occurrence of Fasciola hybrids in the areas of geographical overlap [30,34]. Moreover, African studies have not applied nuclear phosphoenolpyruvate carboxykinase (PEPCK) and DNA polymerase delta (POLD) markers which are recommended as suitable markers to identify parthenogenetic Fasciola [13]. Nonetheless, knowledge of the occurrence of Fasciola hybrids is significant since hybrid forms may have increased in their geographic and host expansion [2]. Hence, this review aimed to assess the possible hybridization of F. hepatica and F. gigantica resulting in parthenogenetic Fasciola in Africa as presented in published literature, and the role of P. columella in driving the convergence of the two Fasciola spp., allowing hybridization in the process.

Materials and Methods
The scoping review was conceptualized to address the following review questions: (1) What is the distribution of Fasciola species in Africa? (2) Which African countries have co-occurrences/overlapping geographical distribution of F. hepatica and F. gigantica? (3) Which intermediate snail hosts are implicated in these overlapping distributions? (4) Has the hybridization of F. hepatica and F. gigantica occurred in Africa? (5) If no, are there signs that hybridization is in the process or potentially occurring in Africa but underdocumented? (6) If yes, is it occurring in the same way as in China, where two pure parental Fasciola species are hybridizing or other Asian countries where pure maternal F. gigantica is hybridizing with parthenogenetic species? To answer these questions, articles published in peer-reviewed journals reporting on the distribution of F. hepatica, F. gigantica, snail IHs, and the occurrence of Fasciola hybrids/parthenogenetic Fasciola populations in Africa were retrieved and appraised. Guidelines from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) were followed using the approaches for scoping review described by [35].

Search Strategy
A literature search was conducted on Google Scholar, PubMed, and Science Direct electronic databases. The search was completed using a combination of Boolean operators (OR, AND) and the following search terms: F. hepatica AND F. gigantica AND co-occurrence,

Inclusion and Exclusion Criteria
Articles were included in the study if they were published in peer-reviewed journals and they: (1) precisely reported on the occurrences and co-occurrences of F. hepatica and F. gigantica and their snail intermediate hosts in Africa; (2) reported on the occurrences of Fasciola hybrids or parthenogenetic Fasciola in Africa; (3) identified eggs or adult flukes up to species level using molecular techniques or morphological features; and (4) were conducted in Africa and published between January 1980 and February 2022.
The review excluded articles that (1) did not identify Fasciola species and their intermediate hosts up to species level; (2) reported on experimental infections; (3) did not contribute towards answering the research questions; and (4) were not conducted in Africa or were published outside the years indicated above and were not written in English.

Charting, Collating, and Summarising Data
Data were extracted from articles that met the inclusion criteria after appraisal. Data concerning the details of the authors and year of publication, the aim or objectives of the study, the country where the study was conducted, the outcomes of the study, and any information relevant to the main objectives of this review were extracted and recorded.

Results
The searches using Google Scholar, PubMed, and Science Direct electronic databases yielded 6721 records, and 23 additional records were identified through reference list screening ( Figure 1). A total of 1023 duplicates were identified and removed. The titles and abstracts of 5698 records were screened for relevance and 5296 records were deemed irrelevant and excluded. Full-text articles of 402 records were extracted and assessed for eligibility, and 161 records were deemed ineligible and excluded because they did not contribute towards answering the review questions. A total of 241 records from 26 African countries were included in the scoping review (Tables S1 and 2). relevant information were identified by screening titles and abstracts to obtain relevant articles. Full texts of relevant articles were retrieved and managed using EndNote reference manager version X9 (Clarivate Analytics, Philadelphia, PA, USA).

Inclusion and Exclusion Criteria
Articles were included in the study if they were published in peer-reviewed journals and they: (1) precisely reported on the occurrences and co-occurrences of F. hepatica and F. gigantica and their snail intermediate hosts in Africa; (2) reported on the occurrences of Fasciola hybrids or parthenogenetic Fasciola in Africa; (3) identified eggs or adult flukes up to species level using molecular techniques or morphological features; and (4) were conducted in Africa and published between January 1980 and February 2022.
The review excluded articles that (1) did not identify Fasciola species and their intermediate hosts up to species level; (2) reported on experimental infections; (3) did not contribute towards answering the research questions; and (4) were not conducted in Africa or were published outside the years indicated above and were not written in English.

Charting, Collating, and Summarising Data
Data were extracted from articles that met the inclusion criteria after appraisal. Data concerning the details of the authors and year of publication, the aim or objectives of the study, the country where the study was conducted, the outcomes of the study, and any information relevant to the main objectives of this review were extracted and recorded.
Most studies identified Fasciola specimens based on morphological features (n = 147), whilst 23 (n = 23) studies identified the parasites using molecular techniques. Other studies used egg morphology from coproscopy (n = 1), serology (n = 1), and 20 (n = 20) studies used a combination of more than one diagnostic technique (Table S1 and Figure 2).

Geographical Distribution and Occurrence of F. hepatica and F. gigantica in Africa
Results show that Fasciola species occur in all five African subregions ( Figure 2). In North Africa, both F. hepatica and F. gigantica have been reported in Egypt and Algeria,  (Table S2).

Checklist of Snail Intermediate Hosts of F. hepatica and F. gigantica in Africa
Evidence from the reviewed studies shows that both G. trancatula and R. natalensis snails occur in North, South, and East African subregions, whilst West and Central regions only documented the occurrence of R. natalensis (Table 1). Additionally [16], documented the presence of a cryptic Galba species, G. mweruensis, in Southern Africa (Lesotho) and East Africa (Ethiopia, Uganda, and Tanzania). In North Africa, both R. natalensis and G. truncatula were documented specifically in Egypt, whilst Algeria, Morocco, and Cameroon documented the presence of G. truncatula only. Reviewed studies further showed that Fasciola species in North Africa can infect other snail species with evidence of natural infections in Biomphalaria alexandrina (Ehrenberg, 1831) and P. columella by Egyptian F. gigantica and Bulinus truncatus (Audouin, 1827) by Tunisian F. hepatica (Table S3). In Central Africa, R. natalensis and P. columella were the only lymnaeid species documented in Cameroon. Southern African region showed the highest species diversity by documenting five lymnaeid species including P. columella, R. natalensis, R. auricularia (Linnaeus, 1758), R. rubiginosa (Michelin, 1831) and G. truncatula. South Africa documented all five lymnaeid species whilst Zimbabwe and Namibia recorded R. natalensis and P. columella. Zambia and Lesotho recorded R. natalensis and G. truncatula, respectively. Botswana recorded R. natalensis and R. auricularia. Evidence of natural infections in P. columella with Fasciola sp. was documented in Zimbabwe and South Africa. In West Africa, R. natalensis was the only lymnaeid species documented in Benin, Côte d'Ivoire, Niger, Nigeria, and Senegal. East African countries where R. natalensis and G. truncatula were documented include Ethiopia, Tanzania, and Uganda, whilst Kenya and Madagascar reported R. natalensis (Table 1). Results further showed evidence of infection of Bio. pfeifferi (Krauss, 1848) and Bio. sudanica (Martens, 1870) by F. gigantica in Kenya (Table S3).

Occurrence and Identification of Fasciola Hybrids/Aspermic and Suspected Parthenogenetic Fasciola in Africa
Results show that Fasciola hybrids were confirmed in cattle from Egypt and Chad, and in buffalo and sheep from Egypt. These Fasciola hybrids were genetically identified by comparing the nucleotides of the ITS-1 and ITS-2 sequences and individuals displayed heterozygosity with the ITS-Fh/Fg genotype thus confirming mixed bases of both Fasciola types at the variable sites [1,2,29]. Furthermore, studies also compared the nuclear ITS gene sequences to the mitochondrial sequences of NADH dehydrogenase subunit I (NDI) and cytochrome c-oxidase subunit I (COI) regions. The hybrid individuals identified as one Fasciola species at ITS-1 and ITS-2, however, displayed sequences of the other species at NDI and/or COI (Table 2). Morphologically, the length/width ratio of Fasciola hybrid adult flukes (1.86-3.37 mm) significantly differed from those of F. gigantica (3.43-5.50 mm) and F. hepatica (1.65-2.76 mm) ( Table 2). Hybrid species also showed variations in the size and position of the oral and ventral suckers, the structure of intestinal caeca, and the position and branches of testes from F. hepatica and F. gigantica (Table 2). Results also showed that some specimens which possessed morphometric characters of F. hepatica displayed a close genetical relation to F. gigantica based on the PCR-linked restriction fragment length polymorphism (PCR-RFLP) using the AvaII restriction enzymes [105]. [2] To molecularly characterize Fasciola flukes using the ITS-1 and 2 nuclear markers to confirm species and any hybrid forms.  Parthenogenetic Fasciola were suspected in the form of aspermic Fasciola species from cattle in South Africa. The aspermic Fasciola sp. (suspected parthenogenetic) were characterized as specimens with no sperms in their seminal vesicles or had "very scanty sperms" and mean length/width ratio measurements that significantly varied from those of F. gigantica and F. hepatica which were 2.02 ± 0.35 mm, 2.79 ± 0.48 mm and 4.41 ± 1.10 mm, respectively [30]. Unidentified Fasciola sp. isolated from P. columella were recorded in Zimbabwe and South Africa (Table S2 and Table 2). In Zimbabwe, the unidentified Fasciola sp. showed a close affinity to F. gigantica and F. hepatica on a phylogenetic tree based on the ITS marker [28]. In South Africa, the unidentified Fasciola sp. Showed a close affinity to F. gigantica on BLAST and genetic distance; however, they formed their own haplotype and clade different from that of F. gigantica and F. hepatica based on the ITS-1 marker [23].

Discussion
Previous studies have indicated that F. hepatica has a cosmopolitan distribution [4,5], whereas F. gigantica is restricted to parts of Asia and Africa [6]. Reviewed studies have confirmed that F. gigantica is more widespread in Africa as expected following the distribution of its IHs [100] and was reported in 24 African countries. In contrast, F. hepatica was more restricted in its distribution, corresponding to the restricted distribution of its IHs which occur in cooler parts of Africa [100]. Fasciola hepatica was reported in a few countries including Egypt and the Maghreb countries (Algeria, Morocco, and Tunisia) in North Africa; South Africa, Zimbabwe, and Zambia in Southern Africa; Nigeria and Niger in West Africa; and Tanzania, Uganda, and Ethiopia in East Africa, which corresponded to reports from previous studies [22,[110][111][112]. The results also showed co-occurrences of the two Fasciola species in Algeria, Ghana, Ethiopia, Egypt, Nigeria, South Africa, Niger, Tanzania, Uganda, Zambia, and Zimbabwe.
Fasciola specimens were predominately collected from their "primary domestic definitive hosts" [113], i.e., cattle, sheep, and goats in all subregions. This is not surprising since these mammalian hosts easily consume Fasciola metacercariae from pastures [114] when grazing in areas near water bodies which are habitats of the snail IHs [115]. Results also showed that natural infections of F. hepatica in Africa occurred in cattle, African buffalo, sheep, goat, camel, humans, pig, horse, antelope, duiker, kudu, mule, and donkey, whilst F. gigantica infected cattle, sheep, goat, impala, Kafue lechwe, donkey, African buffalo, humans, antelope, horse, duiker, mule, and eland ( Figure 2). The low number of studies reporting fasciolosis in wildlife supports a suggestion by [116][117][118] that infections might be accidental and a result of shared drinking water between wildlife and cattle since most wildlife animals are browsers and thus less likely to become infected through aquatic vegetation [22]. Moreover, a few reviewed studies reported infections in humans, thus highlighting that human fasciolosis is either occurring at a very low rate or neglected since humans can easily become infected by ingesting watercress or other edible raw plants contaminated with metacercariae, which form part of the regular diet in several countries [119][120][121], or through drinking water contaminated with metacercariae. Studies from North, East, and Southern Africa recorded infections in buffalo, donkey, horse, mule, camel, and pig. A few South, West, and East African studies reported infections in humans and wild animals including antelope, eland, duiker, impala, Kafue lechwe, and kudu.
Reviewed studies show that both G. truncatula and R. natalensis co-occur in Ethiopia, Egypt, South Africa, Tanzania, and Uganda, and these are the countries where both F. hepatica and F. gigantica have been documented. However, other countries such as Algeria, Ghana, Nigeria, Niger, Zambia, and Zimbabwe reported co-occurrence of both F. hepatica and F. gigantica, with only the presence of one IH being documented. According to [111], such outcomes can be attributable to livestock transhumance, or these species use other freshwater snails from other families and not Lymnaeidae or Racidinae and we further add that limited or no effort has been made to look for the IH. However, altitude and topography have been reported to have an influence on the survival of the snail IHs, thus contributing to the occurrence of Fasciola species [22,100,103]. No records on the occurrence of snail IHs of Fasciola species were retrieved for nine African countries (South Sudan, Sudan, Chad, DR Congo, Burkina Faso, Ghana, Mali, Mauritania, and Malawi), thus highlighting the paucity of research on snail IHs-Fasciola spp. in these African countries. Moreover, the presence of lymnaeid snail IHs were reported in Lesotho, Namibia, Benin, Senegal, and Madagascar, where there was no reviewed evidence of Fasciola spp. This raises concern about possible rapid transmission should the trematodes be introduced, and according to [122] it might be harmful to inhabitants should there be an introduction of the parasite by infected livestock near water bodies with IHs and accessed by humans during their anthropogenic activities.
Reviewed studies showed that apart from G. truncatula, F. hepatica has been observed to naturally infect B. truncatus in Tunisia [51]. Additionally, G. mweruensis, based on studies by [16], appears to be well established as a major IH of F. hepatica throughout the Sub-Saharan Africa region and has been reported in Lesotho, Ethiopia, Uganda, and Tanzania. This species is presumed to be the most predominant snail species in the highlands of Ethiopia and Lesotho [18,19] and is believed to be the main IH of both F. hepatica and F. gigantica [17], and potentially other trematode infections [18,19]. Natural infections with F. gigantica were also detected in Bio. alexandrina in Egypt based on molecular techniques [123], and Bio. Pfeifferi and Bio. Sudanica in Kenya [96], and P. columella in South Africa [23] and Egypt [26]. In South Africa [23] and Zimbabwe [28], P. columella has been found naturally infected with a suspected parthenogenetic Fasciola sp. Considering that this invasive snail species transmit F. hepatica in other parts of the world, it can be suggested that P. columella may be responsible for the overlapping distribution of both F. hepatica and F. gigantica and, hence, promote the occurrence of Fasciola hybrids playing a role in the overlap between the two species.
In Asia, the occurrence of Fasciola hybrids has been extensively studied and documented in China [123,124], Vietnam [125][126][127][128], Japan [129,130], Korea [131,132], Bangladesh [133], Nepal [134], and Myanmar [135]. A recent study by [12] highlighted the existence of a parthenogenetic Fasciola population originating from hybridization between "pure" F. gigantica and F. hepatica in China at least 2000 years ago, and these populations have spread to other Asian countries including Vietnam, India, the Philipines, Thailand, Myanmar, Bangladesh, and Nepal where it co-exists with F. gigantica, and these have not been reported to occur outside Asia. Results from this review highlighted that hybridization may have already occurred or is in the process in some African countries including Chad, Egypt, and South Africa. This is supported by the individual flukes which have been identified to have intermediate morphological characters of F. gigantica and F. hepatica in Egypt [1,105,109] and South Africa [30], and those presumed to be F. hepatica with little to no sperm in their seminal vesicle in South Africa [30]. Considering that the Asian parthenogenetic Fasciola which were aspermic and those identified as F. hepatica in Japan were later classified as parthenogenetic hybrids [13], it is possible that the "aspermic" populations found in South Africa might also be parthenogenetic Fasciola. Thus, the presence of parthenogenetic Fasciola in Africa could have been missed due to a lack of specific studies focusing on the detection of hybrid populations, or the use of inappropriate or non-specific markers to identify these populations, especially in areas with overlapping distributions of these two Fasciola species. Itagaki [13] recommended the use of a multiplex PCR based on the PEPCK and POLD markers, but recent studies have suggested that the fatty acid binding protein type I gene markers are more reliable compared to the PEPCK and POLD markers [136].
Fasciola hybrids from Egypt [105] that had morphometric characters of F. hepatica were genetically more related to F. gigantica. The results also showed that similar to other countries where Fasciola hybrids/parthenogenetic populations were reported, these specimens were also characterized by either the ITS-Fh/Fg mixed genotype or the possession of sequences of one Fasciola species at ITS-1 and ITS-2 but sequences of the other species at NDI and/or COI [1,2,29,106,108]. Furthermore, the populations found in P. columella in South Africa [23] and Zimbabwe [28] were shown to be genetically closer to F. gigantica but formed their own clade on the phylogenetic tree based on the ITS-1 marker (genetic distances). Similar observations were reported by [13] that the Asian parthenogenetic Fasciola were genetically closer to F. gigantica than F. hepatica [137][138][139].

Conclusions
Fasciola gigantica and F. hepatica co-occur in Algeria, Ghana, Ethiopia, Egypt, Nigeria, South Africa, Niger, Tanzania, Uganda, Zambia, and Zimbabwe. However, the presence of Fasciola hybrids has only been confirmed in Egypt and Chad, and parthenogenetic populations are suspected in Zimbabwe and South Africa. Galba truncatula and R. natalensis are the main IH of F. hepatica and F. gigantica, respectively, in Africa. However, a cryptic Galba sp. (G. mweruensis) in Sub-Saharan Africa is worth investigating as it is claimed to be the major host of F. hepatica and might also be sustaining both Fasciola species in the continent. The role of P. columella in the transmission of F. gigantica, F. hepatica, and Fasciola sp. is not clear, and the current results suggest that the snail species might be responsible for Fasciola species overlaps as it was proven to transmit both F. hepatica and F. gigantica in other countries. Hence, the authors recommend that surveys should be conducted to assess if this is also the case in Africa and to detect hybrids in areas of geographical overlap using appropriate molecular and morphological techniques. Evidence suggests that Fasciola hybrids recorded in some African countries might be parthenogenetic Fasciola as reported in Asia. This calls for reliable molecular studies such as the concurrent use of ITS-1 and ITS-2 markers with the PEPCK, POLD, and ND1 markers combined with sperm observations for validation and identification of African Fasciola species to avoid misidentifications.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/pathogens11111303/s1. Table S1: Summary of African studies reporting on the presence of Fasciola species recovered from various definitive hosts. Table S2: The distribution and occurrence of Fasciola species in Africa based on studies conducted from 1980-2022. Table S3: Summary of African studies reporting on the occurrence of Fasciola species in their snail intermediate hosts. Table S4: Summary of studies reporting on the occurrence of intermediate hosts of Fasciola spp. in Africa. References