Prevalence, Molecular Characterization and Phylogenetic Analysis of Fasciola spp. Based on ITS2 Gene Sequence from Slaughtered Animals in Abattoirs and its public Health in Qualyobia Province, Egypt

Fascioliasis is a parasitic infection with a significant negative impact on animal’s productivity and causing widespread human zoonotic disease in several countries including Egypt, Algeria, Tunisia, Botswana, Niger, Vietnam, Mauritania, Korea, and Japan (Robertson and Mochankana, 2018).

Egypt is regarded as one of the Fascioliasis endemic countries in the world and of high parasitic burden in infected animals and humans (Arafa et al., 2018). Fascioliasis caused by F. hepatica and F. gigantica both utilize animals and humans as final hosts (Amer et al., 2016). Identification of these parasites based on the morphological and morphometric criteria; however, these traditional methods are still unreliable in definite differentiation (Liu et al., 2014). For that, molecular methods must be used to overcome the challenges of identifying species based on their phenotypic features (Shoriki et al., 2016). When Fasciola hepatica flukes mature, they take on the appearance of brown, flatworms in the shape of leaves that are typically 20 to 30 mm in length and 13 mm in diameter. They have two suckers anteriorly; the larger, or acetabulum, located on the ventral side and allows the fluke to adhere to the bile duct wall and stay there, while the smaller, anterior sucker feeds on the bile. Fasciola gigantica flatworms can reach lengths of up to 5 to75 mm in length and 12 mm in diameter but are look similar to their smaller cousins. (Mas-Coma, et al., 2014).

In Egypt, F. gigantica is common among ruminants along the Nile Valley, while F. hepatica is reported in imported sheep and cattle whereas; both species co-exist alongside native livestock animals in Egypt (Mas-Coma et al., 2019). Fascioliasis decreases animal productivity, weight gain, and the output of meat and milk. Due to decreased immunity brought on by chronic Fascioliasis and liver condemnation during postmortem examination in slaughterhouses, it also contributes to moderate icterus, metabolic problems, and secondary infections, while acute Fascioliasis may result in fatalities (Eman et al., 2016).

Fasciola has a complicated life cycle that involves intermediate snail hosts like the common snail and final hosts such as mammals like humans. One miracidium is produced when eggs lost in the stool of the last host and embryonate in fresh water. To spread infection, the miracidium pierces the snail’s (Lymnaeidae family) tegument. The majority of Fasciola transmission worldwide is carried out by snails of the species Galba and Radix, particularly Galba truncatula. The distribution of the intermediate host, which is required to maintain the parasite’s viability in endemic areas, is reflected in the distribution of Fasciola risk and infection (Vázquez et al., 2018).

The grazing habits of animals on grassland are thought to have caused a variation in Fascioliasis. In general, cattle like to graze close to springs and streams where snails are prevalent. In contrast, compared to sheep, goats often have the lowest infection rate. Goats graze differently than sheep because they naturally prefer to eat leaves and health at elevated areas, opposite to sheep which frequently graze on the ground. The risk of infection in goats may be reduced by this type of grazing because there is less exposure to contagious metacercaria (Manoochehr et al., 2017).

Molecular studies have confirmed that both species can be differentiated by mitochondrial DNA sequencing of NDI and COI or nuclear ribosomal of internal transcribed spacers (ITS) 1 and 2 (Amer et al., 2011). Studies in Thailand have also verified that Fasciola hybrid form present in the livestock liver that has been identified as hybrids based on the sequences of ITS1 and ITS2 (Siribat et al., 2018). For molecular and genetic characterization of F. gigantica, F. hepatica, and Fasciola hybrid form, ITS1, and ITS2 have been renowned and more efficient genetic markers.

Moreover, in Egypt, researchers found the hybrid form that has a morphometric character between both species. The modern techniques of PCR and DNA sequencing ease the species identification, strain clarification (Sumruayphol et al., 2020). The selected gene or sequence needs to be common, extraordinarily moderated within, and sufficiently diverse between taxa. In a perfect world, the variable areas ought to have neighboring preserved areas so that „global” oligonucleotide primers may be chosen (Yuan et al., 2016).

Egypt is considered as one of the Fascioliasis-endemic areas in the world (Amer et al., 2016) and the disease burden is high in the Nile Delta region. Both species, Fasciola hepatica and Fasciola gigantica, are commonly seen present in Egyptian sheep, and the occurrence of the hybrid form has been reported (Amer et al., 2011). This disease is severely affecting the sheep farming industry and causing severe economic loss, especially in the reduction of wool production, meat, and total body weight (Amer et al., 2016).

The objectives of this study were to determine the prevalence of Fascioliasis in slaughtered animals, molecular characterization, and phylogenetic analysis of Fasciola spp. worm and its public health in Qualyobia Province, Egypt.

The current study was performed in different slaughtered houses of the Qualyobia region, located in Lower Egypt. It is located in the Nile Delta region, north of Cairo. This study was approved by the Department of Zoonoses, Faculty of Veterinary Medicine, Benha University, Egypt. The weather is typically dry, with scorching or extremely hot summer days and warm or mild winter days.

At the abattoir, physical examination (antemortem) was performed shortly prior to slaughter of 286 animals to determine sex, age, body condition, grazing system and region. Following animal slaughter, the liver was extensively checked post-mortem for the presence of liver flukes. Multiple cuts and subcuts about 1 cm thick were done to check for the existence of Fasciola parasites, which made scratchy sounds, exerted brownish fluid, and immature Fasciola. Identification of the Fasciola species based on the morphological characters of the agent and classify into F. gigantic and F. hepatica (Urquhart et al., 1996).

The liver was collected from (286) slaughtered animals (88 sheep, 26 goats, 68 cattle, 25 buffaloes, and 79 camels) from June 2018 until March 2021 from El-Ramla abattoir, Toukh abattoir, Sendyoon abattoir, mini-abattoirs in El-Shamut, and Shebeen Al-Qanater abattoir at Qualyobia province, Egypt and were examined for Fasciola flukes during the regular postmortem inspection. The obtained worms were washed separately using saline water (0.9%) and repeated washing at least thrice to eliminate the debris, all samples were transported in ice box to laboratory of Zoonoses, Faculty of veterinary medicine, Benha University, then were maintained in ethanol (70%), and kept at – 20°C for the extraction of genomic DNA.

Liver flukes were taken out of the 70% ethanol and dyed with Carmine staining method according to (Gibbons et al., 1996). The flukes were washed in tap water to eliminate any remaining ethanol from preservation, and then they were rehydrated using a graduated range of alcohol (70%, 50%, 30%, 10%, and distilled water). The next step was to regressively stain the flukes by soaking them in Aceto Alum Carmine stain for an entire night. The excess stain was then rinsed off with distilled water. To remove excess staining without losing the coloration, they were then immersed in alcoholic acid (2 ml of concentrated HCl in 100 ml of 70% ethanol) for 2-4 hours. The samples were subsequently dehydrated in escalating series of graded alcohols (50% - 70% - 80% - 90% - 100%) and then cleaned with clove oil for 24hours. The specimens were then mounted using Canada balsam and internal organs examined by light microscope.

According to the manufacturer’s instructions, the genomic DNA was extracted from each adult worm (10 Fasciola flukes) using a QIAamp® DNA Mini Kit from Qiagen in Hilden, Germany. Genomic DNA was eluted in 50 µl elution buffer and stored at −20°C until used for molecular identification. The primer pair DSJF included a forward primer (5’ –ATA TTG CGG CCA TGG GTT AG-3’) and a reverse primer known as DSJ3 (5’- CCA ATG ACA AAG TGA CAG CG-3’) that was unique for F. hepatica was employed. Another species-specific reverse primer DSJ4 (5’- CCA ATG ACA AAG TAA CAG CA-3’) specific for F. gigantica was also used with DSJF forward primer. These Fasciola specific primers were used at a concentration of 50 ρmol for the amplification of approximately 300 bp fragment for Fasciola species (Ai et al., 2010). All PCR amplification reactions, including control negative samples, were all based on a 25-ml reaction mixture containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 200 mM of each deoxyribonucleotide triphosphate, 1.25 U Taq polymerase and 1 ml DNA template. The thermocycler that was set to give 5 min at 94°C, followed by 30 cycles, each of 30 s at 94°C, 30 s at 60°C and 30 s at 72°C, before a final extension for 5 min at 72°C. The amplicons (4 ml) from each PCR were separated by electrophoresis in 1.0% (w/v) agarose gel, stained with ethidium bromide, and photographed using a gel documentation system (UVI tec, Cambridge, U.K.).

The sequencing of random samples of the amplicons occurs using DSJf/DSJ3 from F. hepatica samples and DSJf/DSJ4 from F. gigantica samples and findings matching the appropriate ITS-2 sequences of F. hepatica (GenBank accession MZ396929.1) and F. gigantica (MN970008.1). The produced sequences were manually adjusted, then they were compared to equivalent sequences of the F. hepatica and F. gigantica accessible in GenBank database by the Basic Local Alignment Search Tool (BLAST) application (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_ TYPE=Blast Search). The ITS2 rDNA gene sequences of various Fasciola isolates from various geographical locations were obtained from GenBank (www.ncbi.nlm.nih.gov/genbank), and the completed protein sequences were aligned using the MEGA6.0 programme (Tamura et al.,, 2013), using default alignment parameters and manual adjustment. The maximum likelihood approach was used to conclude the phylogenetic analysis. For genetic sequence and correlation analysis of the ITS2 gene in diverse Fasciola isolates belonging to different locations, MEGA 6.0 was employed. This programme allowed for the creation of multiple alignments of the ITS2 gene for all Fasciola isolates (Li, 2003).

Chi square (X2) test was used to determine association between variables (prevalence, locality, season, sex, age, and rearing system) and their degree of significance. A P value <0.05 was considered significant.

The infection rate of Fascioliasis in examined slaughtered animals in different abattoirs in Qualyobia was 17.8% (51/286). The specific prevalence of Fascioliasis was 7(28%), 23(26.1%), 16(23.5%), 5(19.2%), and 0 (0%) in buffaloes, sheep, cattle, goats, and camels, respectively (Table-1). So, the higher infection rate was found in buffaloes and sheep then cattle and goats, but this difference was not significant with P value >0.05 (Table-1).

Table 1: Shows the prevalence of Fasciola species among slaughtered animals

Table 2: Shows the frequence distribution of Fasciola species among positive slaughtered animals:

Table 3: Shows the infection rate of Fasciola spp. among sheep and goats in relation to associated risk factors:

Table 4: Shows the infection rate of Fasciola spp. among cattle, buffaloes, and camels in relation to associated risk factors

F. hepatica was the predominant species 52.9% (27/51), F. gigantica was 35.3% (18/51), mixed infection was 5.9% (3/51) and larval stage was 5.9% (3/51) (Table-2). Based on the animals species; sheep has 12 F. hepatica, 8 F. gigantica and 3 larval stages, goats have 4 F. hepatica and 1 F. gigantica, cattle has 9 F. hepatica, 4 F. gigantica and 3 mixed infection while buffaloes have 2 F. hepatica and 5 F. gigantica (Table-2). Based on PCR product, 10 Fasciola isolates were corresponding to Fasciola hybrid form as they given positive result when used with primer of F. hepatica, and F. gigantica (Figure-2,3).

Our result revealed that the relation between the incidence of Fascioliasis and gender which revealed that males 40/51 (78.4%) more than females 11/51 (21.6%) animal’s carcasses. In all animals’ species male was infected more than female except in cattle but was not significant except in goat with P value =004 (Table-3). Locality was not affected on Fasciola infection in sheep in contrast to in goats with P value = .02. The incidence of Fascioliasis was higher in age more than 3 years in sheep and goats (Table-3). Fascioliasis was higher in sheep during autumn, spring, winter, and summer 11/23 (47.8%), 4/13 (30.8%), 6/36 (16.7%) and 2/16 (12.5%) respectively and was significantly with P value = .03 (Table-3). In goats, the infection was observed more in winter 4/5 (80%) than spring 1/11 (9.1%) with P value= .003 (Table-3). There was a significant difference in sheep and goats in between different seasons because of the rainy season, presence of fresh green grazing pasturing and spreading of the snail’s host. High Fasciola infection recorded in animals reared in free rang system (outdoors) than housed ones (indoors), 44/51 (86.3%) and 7/51 (13.7%) in all animals respectively with P value = .00, .000 and .01 in sheep, buffaloes, and goats, respectively and these were significantly (Table-3,4). Most of infected slaughtered animals suffered from weakness, rough coat, pasty feces, and pale mucus membrane. Moreover, the higher infection found in animals that slaughtered in shops at villages than that slaughtered in abattoir due to lake of meat inspection, 17/36 (47.2%), 6/52 (11.5%) in sheep, 4/19 (21.1%), 1/7 (14.3%) in goats. 14/29 (48.3%), 2/39 (5.1%) in cattle, 7/11 (63.6%), 0/14 (0%) in buffaloes that slaughtered in shops in villages and abattoirs respectively (Table-3,4).

The incidence of Fascioliasis was higher in age 2-4 years in buffaloes (Table-4) and in age more than 4years in cattle (Table-4), this was not significantly with P value >0.05. Moreover, in rainy season than dry ones due to presence of fresh green grazing pasturing, high population of snails and encysted metacercaria. According to seasons, the infection in cattle and buffaloes were 7/25(28%), 3/10 (30%) in winter, 3/16 (18.8%), 2/5 (40%) in spring, 2/9 (22.2%), 0/2 (0%) in summer and 4/18 (22.2%), 2/8 (25%) in autumn respectively, but this was not significantly (Table-4). The differences among the geographical locations could be attributed mainly due to increase in irrigated land masses in the research area and farmers’ propensity to feed their livestock in these marshy and wet areas due to a lack of feed. The ecological condition is favorable for the survival and development of the snail intermediate host for species of Fasciola.

Grossly regarding Fascioliasis infection during slaughterhouse postmortem inspection revealing the external smooth liver surface declared multiple white or creamy tunnels ranging from few millimeters to nearly 3 cm (Figure-1, A) represented the postmortem liver fibrosis appear from external liver surface. Fasciola tunnels that were observed from undamaged liver surfaces oozing grassy blackish hemorrhagic exudate and declared different took photos of creamy leaf-like Fasciola spp. about 1.5-2.0 cm in length and about 1.0 cm in width (Figure-1, B). These reduced the value of the infected carcass and the liver and led to consumer rejection of the liver.

In this study, 10 isolates of Fasciola were investigated from 51 naturally infected sheep, goats, cattle, and buffaloes. Genomic DNA was productively extracted, and the specific primers (DSJf/DSJ3 for F. hepatica and DSJf/DSJ4 for F. gigantica) were used in this work successfully amplified a fragment of 300 bp of a part of ITS2 gene of Fasciola samples (Figure 2,3). 10 isolates give bands at 300 pb when used primers of Fasciola hepatica and primers of Fasciola gigantica this mean that the ten isolates are intermediate form of Fasciola. Alignment analysis through online blast reveals similarity of 100% (isolate W1 GenBank accession OP245092)) and some variability (SNP, INDEL) in (isolate W7 GenBank accession OP245094) of inspected sequence fragment when compared with GenBank database of F. hepatica and F. gigantica available reference data (GenBank accession MZ396929.1) and (MN970008.1) respectively. Furthermore, it’s clear and interesting that 1 of 2(50 %) of isolates shows SNP (T to C) of the 282nd, (C to T) of the 321st nucleotide of an investigated fragment (Figure 5). Indel found in W7 isolate at 310 pb by deletion or insertion of G nucleotide. SNP may be due to the presence of hybrids in our isolates. The phylogenetic tree revealed the two isolates cluster in two clades (Figure 6).

Fascioliasis caused by the Fasciola hepatica and Fasciola gigantica which is a plant borne trematodes that are hazardous to health and reduce animal productivity and damage economic activities. Consumer rejection of the liver results in a loss for the economy. In addition, an acute infection with Fasciola results in weight loss, stunted growth, and infertility. There was a decrease in productivity while the disease was in its chronic phase Mehmood et al. (2017).

The result of this study indicates that the infection rate of Fascioliasis was (17.8%) among the examined slaughtered animals. The reason for this infection could be due to increase in irrigated land masses in the research area and the propensity of farmers and animal rearers to feed their livestock in marshy and damp areas behind the grain because of a lack of feed. Since the intermediate host favours marshy areas with slow-moving water and small streams, which also provide enough moisture for the infective metacercaria to survive Magaji et al. (2014).

The higher infection was found in buffaloes 28% followed by sheep 26.1%, cattle 23.5%, goats 19.2% and camels 0%. The difference of the incidence among slaughtered animals might be attributed to the grazing style of animals but this difference was not significant with P value >0.05 (Table-1). F. hepatica was the predominant species 27(52.9%) (12 in sheep, 4 in goats, 9 in cattle and 2 in buffaloes), Fasciola gigantica was 18(35.3%) (8 in sheep, 1 in goats, 4 in cattle and 5 in buffaloes), mixed infection was 3(5.9%) in cattle and larval stage was 3 (5.9%) in sheep morphologically (Table-2), our result was lower than that obtained by Usman, (2019) in Nigeria (40.5%) in which higher rate was observed in cattle than sheep and goats, and Ieren et al. (2016) in Nigeria (48%) in which cattle more infected than sheep and goats (males more than females and active rain season than early dry season) and Zemene and Atnaf, (2015) in Ethiopia (21.8%) in which cattle more infected than sheep and goats and in adult animals than young ones, higher infection observed in poor body condition followed by medium body condition. The wide variation in prevalence between various nations could be primarily attributed to variations in climatic conditions, such as rainfall and temperature Fox et al. (2011) as well as other significant factors like grazing in marshy pastures, having access to streams or ponds, having a large herd, and the type of soil Howell et al. (2015). Additionally, human activity (large urbanization, the growth of irrigated regions, and the transformation of natural wetlands into agricultural lands) is linked to the spread of water and food-borne illness Sabourin et al. (2018).

The present study revealed that the occurrence of Fascioliasis in sheep was 23/88 (26.13%), this result was lower than that recorded by Usman, (2019) in Nigeria which was 39.1%, and female sheep recorded higher infection (44.6%) than males (30.7%) in contrast to our result that higher in males (28.57%) than females (20%). Our result was higher than that recorded by Zemene and Atnaf, (2015) in Ethiopia which was 20.14%, and adult sheep more infected than young ones. Also, was higher than that recorded by Jean-Richar et al. (2014) 23% and Ouchene-Khelif et al. (2018) in Algeria 6.5%, the seasonal infection of Fasciola hepatica was highest in winter for sheep.

The infection rate of Fascioliasis was 5/26(19.2%) in examined goats and was higher between older (2-3, >3years) than young goats, in winter than other seasons, among males than females and in freely reared goats (outdoor) than housed animals (indoor). Our result was higher than that recorded by Hassan et al. (2019) in Giza Governorate 0.89%, IrfanUllah et al. (2016) in Pakistan 6.6%, Çelik and ÇELİK, (2018) in Turkey 14.14%, Abah et al. (2019) in Nigeria 6.2%, Abdelazeem et al. (2020) in Egypt 3.5%, Our result also was higher than that recorded by Ouchene-Khelif et al. (2018) in Algeria 2.5%, the seasonal prevalence of Fasciola hepatica was highest in summer and winter for goat. While our result was lower than that recorded by Usman, (2019) 35% but in those study the infection was higher in females than males. According to age, sex, season, and rearing system, our results agreed with Abdelazeem et al. (2020) in Assiut and Sohag Governorates, Upper Egypt who said that the highest infection found between older than young goats, in winter than other seasons, among males than females and in freely reared goats than housed animals, but disagreed with Abah et al. (2019) who revealed that female goats more infected than male goats.

The infection rate of Fascioliasis in cattle was 16/68 (23.5%) and was higher in female 5/18 (27.7%) than male 11/50 (22%), more at age > 4year 5/17 (29.4%) than in age 2–4year 9/36 (25%) and 1–2year 2/15 (13.33%), in rainy season than dry ones, in winter 7/25 (28%), 4/18 (22.2%) in autumn, 2/9 (22.2%) in summer and 3/16 (18.75%) in spring. In free rang system (outdoor) 13/53 (24.5%) than housed system (indoor) 3/15 (20%), infected animals had pasty feces, pale mucus membrane, rough coat, weakness and 5 infected females not pregnant. Infection more in Shebeen 3/12 (25%) than Benha 9/37 (24.7%) and Toukh 4/19 (21.05%). Infection rate was higher in cattle that slaughtered in shops in village 14/29 (48.3%) than that slaughtered in abattoirs 2/39 (5.1%). Our result was higher than that recorded by Ejeh et al. (2015) in Nigeria 14.6%, Henok and Mekonen, (2011) in Ethiopia 14.6% in which adult recorded higher infection, while was lower than that recorded by Jean-Richar et al. (2014) in the Lake Chad area of Chad 68% in cattle that grazed at lake water which was the source of infection and all infected with F. gigantica, Usman, (2019) in Nigeria 45.7% in which males more infected than females and aged cattle above five years recorded higher infection, Zemene and Atnaf, (2015) in Ethiopia 30.6% in which adult cattle recorded higher infection, Yemisrach and Mekonnen, (2012) in Ethiopia 28.6% but young cattle recorded higher infection than adult ones and Ouchene-Khelif et al. (2018) in Algeria 26.7%, the seasonal infection of Fasciola hepatica was highest in summer and winter for cattle.

The infection rate of Fascioliasis in buffaloes was 7/25 (28%) and was higher in male 6/15 (40%) than female 1/10 (10%), more at age 2–4-year 6/18 (33.3%) than > 4 year 1/7 (14.3%), more in spring 2/5 (40%) than winter 3/10 (30%) and Autumn 2/8 (25%). All infected buffaloes were in free rang system (outdoor) 7/11 (63.6%), infected buffaloes had pasty feces, and some had weakness. Infection more in Benha 5/13 (38.5%) than Toukh 2/6 (33.3%). All infected buffaloes slaughtered in shops in villages. Our result was higher than that recorded by Adane et al. (2019) 20.24% in which the higher infection was recorded in female than male and in old animals than young, Mohammed et al. (2016) 24.4% in which the highest infection found in young than adult animals. Our result in large ruminant (cattle and buffaloes) was 23/93 (24.7%) these was higher than that recorded by Fazly et al. (2015) 6/80 (7.50%) in cattle and buffalo samples and F. gigantica was only identified in the samples.

The current study revealed the Fasciola species that infect sheep, goats, cattle, and buffaloes from Qualyobia, Egypt was Fasciola hybrid form (Figures 2 and 3). This agreed with Mosaab et al. (2021) who found Fasciola hybrid form in sheep and buffaloes in Aswan, Egypt, but disagreed with the results of Amer et al. (2011) who found that F. hepatica was predominant in sheep compared to other hosts, also disagreed with Salihu et al. (2022) who siad that the genotype of Fasciola infecting cattle and other ruminant species in Nigeria was F. gigantica. Moreover, our result disagreed with Abdelazeem et al. (2020) who recorded that the Fasciola species that affected cattle and goats was Fasciola hepatica and gigantica based on the sequencing of (ITS-2) of nuclear ribosomal DNA (rDNA). While our result in camels was 0% and this agreed with Ouchene-Khelifi et al. (2018) in Algeria who not found Fascioliasis in camels, and this may be attributed to those camels not grazed as in small and large ruminant.

Molecular characterization of the Fasciola species were detected based on partial sequences of ITS2 rDNA and the study revealed that the Fasciola spp. sequences from diverse hosts are almost matching to those of earlier available sequences. It has been shown that Fasciola hybrid form is existing in Qualyobia, Egypt, the hybrid form was recorded for the first time at south valley university, Egypt. These outcomes meet the full agreement with those found by Amer et al. (2011) and Mosaab et al. (2021) who recorded the presence of the three Fasciola species in Egypt including the hybrid form, while the present result differed from that recorded by Arafa et al. (2018) Egypt, Cairo who reported pure F. gigantica (isolate from cow and buffalo). However, El-Tahawy et al. (2018) from Nile Delta, Egypt recorded that the Fasciola in Egypt concerning the application of PCR for differentiation of two species of Fasciola by using certain primers of F. hepatica, they recorded that eight samples were found to have positive bands associated to F. hepatica and 2 negative bands representing F. gigantica. Alignment revealed that our result in W7 isolate (GenBank accession OP245094) was typical identical to found in Egypt (MT423006), (MW600275), (AJ853848), Kenya (MN970008), Nigeria (MN608173), Turkey (KY613944), Iran (KF866247), Germany (KF425321), South Africa (MW793533), Belgium (MW046876), Switzerland (MK321643) and Japan (AB010976) while W1 isolate (GenBank accession OP245092) was typical identical to found in Egypt (MT423007), Kenya (MZ396929), Libya (MT025519), Iran (JF432071), India (MZ413927), Viet Nam (MT429176), Saudi Arabia (MN559388), Spain (MG569979) and China (MH385388).

Alignment analysis confirmed our results and revealed similarity between one isolate (W1) with Fasciola hepatica isolate on GenBank when used primer of F. hepatica and W7 revealed similarity with some nucleotide differences (SNP, INDEL) with F. gigantica on GenBank when used primer of F. gigantica and may be due to the presence of hybrids in our isolates, and this outcome may be due to that our isolate is relatively recently introduced to Qualyobia Province as this the first time reported the Fasciola hybrid.

Fascioliasis is a zoonotic disease caused by parasites of the genus Fasciola spp. can result in significant losses for the animal’s industry and have an impact on public health because there is a chance that an illness will spread to people. In present study the infection rate of Fascioliasis in slaughtered animals was 17.8% and the hybrid form was common in the study area. This calls for a policy strategy for the prevention and control of this neglected tropical illness. The suggestions below could be useful. The population needs to be made acutely aware of the threat posed by the spread of Fascioliasis in ruminant animals. More effort should be put into meat control because the area has become a popular selling and buying location for animals from crisis zones. It is necessary to use environmentally acceptable molluscides to reduce the intermediate hosts of the parasitic snails. Lack of sanitation facilities at the local abattoirs studied calls for an urgent concern.

The authors have declared no conflict of interest.

The authors wish to express their gratitude to Faculty of Veterinary Medicine, Benha University for supporting this work.

Lobna M. A. Salem, Nashwa O. Khalifa, Marwa O. Abd El-Halim: Planned the study design, collected, and examined samples, drafted and revised the manuscript. Both authors read and approved the final manuscript.