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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2022 Aug 24;46(4):1036–1046. doi: 10.1007/s12639-022-01526-x

Economic losses, morpho-molecular identification, and identity of Fasciola species recovered from Egypt

Omima Ramadan Abdel‑Fatah 1, Waleed M Arafa 2,✉, Ahmed Anwar Wahba 3, Khaled Mohamed El‑Dakhly 2
PMCID: PMC9606198  PMID: 36457773

Abstract

A retrospective study to estimate economic losses caused by livers condemnation, due to fascioliasis, of slaughtered cattle and buffaloes in Egypt during the period of 2016–2020, was done. Moreover, a morpho-molecular identification of collected liver flukes from slaughtered animals in municipal abattoirs was conducted. Livers of naturally infected carcasses were obtained from slaughtered animals in Beni-Suef, Cairo and Tanta provinces, Egypt during 2019–2020 for phenotypic characterization of recovered Fasciola species and molecular identification of collected worms using PCR targeting the ITS-1 region. Findings of the retrospective study revealed that percentages of livers condemnation of cattle and buffaloes ranged from 0.79 to 0.66% during the period from 2016 to 2020. The highest percentages were detected in the south Egypt (2.5–6.0%) with the highest economic losses (261850–616300 USD annually). Morphometrically, collected flukes categorized into Fasciola hepatica and Fasciola gigantica. No intermediate forms (Fasciola sp.-like) were detected. Sequencing analysis of ITS-1 PCR products showed that only Fasciola hepatica (26/34) and F. gigantica (8/34) isolates were found, with no intermediate forms, Fasciola sp.-like, could be identified. Currently, Fasciola hepatica was 100% identical with the Egyptian species (LC076196 and JF294998), French species (JF294999), and Iranian species (MF969009 and MK377150). Moreover, the obtained F. gigantica species showed 100% identity with Egyptian ones (LC076125, LC076108 and KX198619), Iranian (KF982047 and MF372919), and other GenBank specimens from Vietnam, Cameroon and India. In conclusion, South Egypt showed the highest economic losses due to fascioliasis, especially Aswan province. Fasciola hepatica was more common than F. gigantica, while the hybrid form was not detected.

Keywords: Fasciola hepatica, Fasciola gigantica, Ruminants, Morphometry, PCR, Egypt, ITS-1

Introduction

Fascioliasis is a liver parasitic disease caused by trematodes belonging to family Fasciolidae, primarily Fasciola hepatica and Fasciola gigantica, with certain Lymnaeidae snails act as intermediate hosts affecting various livestock as well as humans worldwide (Yavuz et al. 2007; Soliman 2008; Khoramian et al. 2014). In Egypt, liver flukes, Fasciola spp., negatively impacted on Egypt's farming industry and public health, with a potential zoonotic importance of humans infection (Soliman 2008). Fasciola species cause economically important disease in sheep and cattle, with an estimated 250 million sheep and 350 million cattle infected globally (Hillyer and Apt 1997; Bennett and pelaar 2005; Schweizer et al. 2005; Beesley et al. 2017). In Egypt, the annual losses due to Fasciola species infections among the animals were 190 million Egyptian pounds (El-Tahawy et al. 2017). The disease potentially interferes with the food intake and digestion of infected livestock (Batmaz 2013; Irak et al. 2018), together with huge economic losses in endemic areas due to excessive mortality and morbidity in sheep and goats as well as increased sensitivity to secondary infections and greater preventative costs (Karpamar et al. 2012).

Fascioliasis, mainly induced by F. hepatica, is the most frequent vector-borne helminthic disease. The worldwide distribution of such species in various countries might be attributed to the overlapping environments encouraging the life cycle (Mas-Coma 2005; Mas-Coma et al. 2005). Meanwhile, F. gigantica has a lesser extent of distribution, particularly in Africa, Asia and the southern Europe (Mas-Coma and Bargues 1997; Ashrafi et al. 2006). In Asian countries, the distribution of both F. hepatica and F. gigantica is overlapped making it difficult to distinguish the involved species (Ashrafi et al. 2006). Quantification the morphometric differences between allopatric specimens of F. hepatica and F. gigantica by using a computer image analysis system (CIAS) have recently been proposed and applied, with special references to traditional measurements, definitive host influence and geographical distribution of intermediate snail hosts (Periago et al. 2006). The co-existence of the two fasciolids in livestock implies on a complicated picture of possible ways of transmitting the infection (Ashrafi et al. 2004).

In Egypt, the prevalences of fascioliasis were 50.6% and 32.3% in cattle and buffaloes, respectively (Bazh et al. 2012). In bile ducts of definitive hosts, liver flukes produce severe hepatic damage (Amer et al. 2016). Furthermore, in Dakhla Oasis, Egypt about 11.1% of examined cattle livers were harboring F. hepatica (Arafa et al. 2018). Lymnaea species snails are a group of freshwater snails worldwide distributed with Lymnaea natalensis is the most common snail host for those liver flukes (Adediran and Uwalaka 2013; Chontananarth et al. 2017; Lee et al. 2017) Emaciation, diarrhea, various degrees of icterus, decreased milk output with an unpleasant taste, and anestrumanimals were among the clinical findings (Bazh et al. 2012). It was estimated that the global annual economic loss would be $2 billion (Rokni et al. 2010). In Egypt, the annual losses of animals due to fascioliasis estimated to be over 190 million Egyptian pounds, according to a report issued by the Egyptian Academy of Scientific Research and Technology (El-Tahawy et al. 2017; Abdel-Hakeem and Omar 2020).

Molecular biology has been proposed to support the taxonomy of various helminths based on DNA genetic markers of nuclear ribosomal DNA (rDNA) and special mitochondrial DNA, particularly DNA amplification using Polymerase Chain Reaction (PCR) and other sequencing techniques (Prasad et al. 2007). The Internal Transcribed Spacers (ITS-1 and ITS-2) of nuclear rDNA, which are found between the succeeding coding regions (18S, 5.8S, and 28S) in metazoan parasites, were found to be useful for species identification (Nolan and Cribb 2005).

Therefore, the current study aimed for estimation economic losses of fascioliasis in Egypt due to liver condemnations of cattle and buffaloes through a retrospective study. Moreover, morpho-molecular identification of Fasciola species, in the main abattoirs using ITS-1 marker was carried out.

Materials and methods

Estimating economic losses due to livers contamination through a retrospective study

The study was a five-year retrospective abattoir-based assessment comprising slaughtered cattle and buffaloes during the period from January 2016 to December 2020 in almost Egyptian provinces. Daily records for those slaughtered livestock, were recovered from archival documents of the general organization of veterinary services in Egypt. The total number of slaughtered animals, and the number of livers condemned due to the liver flukes infection were recorded for each province. The prevalence of Fasciola species was annually estimated. Egyptian provinces were divided into north (15 provinces), middle (6 provinces) and south (6 provinces) to facilitate reading the data. Estimated liver cost was calculated by multiplying number of condemned livers × average liver weight (5 kg) × average price of each Kg (10 USD = 150-160LE based on the currency rate exchange) (Yatswako and Alhaji 2017).

Collection of adult flukes and morpho-molecular study of Fasciola species

Sixteen Liver of naturally infected cattle, buffaloes and sheep were obtained from slaughtered animals in the main abattoir in Cairo (Basatin abattoir; 8 cattle and 2 sheep), Beni-Suef (5 cattle), Tanta (one cattle) during 2019–2020. Livers were visually examined on both parietal and visceral surfaces, then palpated and incised ventrally so that the incision crossed most of bile ducts. In addition to the bile ducts, gall bladders were all incised and examined. Flukes were collected from infected livers and roughly identified into F. hepatica and F. gigantica.

As early as possible, specimens were transported to the laboratory of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University for parasitological investigations.

Morphometric characterization of Fasciola species

Intact adult flukes were frequently cleaned in saline, gently compressed between glass slides, fixed in 10% neutral buffered formalin and stained with acetocarmine and mounted in Canada balsam (Muller et al. 2002). Identification of the recovered fasciolids based on morphological features (Urquhart et al. 1996) was conducted. Morphometric measurements of 55 adult flukes F. hepatica representing all forms of approximately 1500 flukes collected from 12 livers were applied. Moreover, morphometric measurements of 23 adult flukes F. gigantica representing 300 flukes collected from 4 livers were conducted. Data of both fasciolids were compared with those of adult cattle from Egypt (Periago et al. 2008) and Iran (Ashrafi et al. 2006). Criteria of traditional measurements of the recovered adult fasciolids were done according the key given by Valero et al. (2005), Ashrafi et al. (2006) and Periago et al. (2006, 2008). The measurements were made with a calibrated stereomicroscope and microscope. All measurements were in millimeters. Measurements comprised lineal biometric characters [body length (BL), maximum body width (BW), cone length (CL), cone width (CW),maximum diameter of the oral sucker (OSmax), minimum diameter of the oral sucker (OSmin), maximum diameter of the ventral sucker (VSmax), minimum diameter of the ventral sucker (VSmin), distance between the anterior end of the body and the ventral sucker (A-VS), distance between the oral sucker and the ventral sucker (OS-VS), distance between the ventral sucker and the union of the vitelline glands (VS-Vit), distance between the union of the vitelline glands and the posterior end of the body (Vit-P), distance between the ventral sucker and the posterior end of the body (VS-P), pharynx length (PhL) and pharynx width (PhW)], areas[body area (BA), oral sucker area (OSA) and ventral sucker area (VSA)] and ratios [body length over body width (BL/BW) and oral sucker area over ventral sucker area (OSA/VSA)].

Molecular identification of Fasciola species

DNA extraction

DNA was extracted from the cone-shaped projection of 34 intact adult flukes preserved in 70% ethanol representing all measurements of the morphologically identified flukes using QIAamp DNA Mini Kit (Cat. No. 51304) (Qiagen, Gmbh, Germany) according to the manufacturer’s instructions at the Faculty of Veterinary Medicine, Beni-Suef, Egypt. DNA extracts were stored at − 20 °C until use.

DNA amplification

PCR targeting ITS1 region of adult fasciolids using the specific oligonucleotide primer forward ITS-1-F 5′-TTGCGCTGATTACGTCCCTG-3′ and reverse ITS-1-R 5′-TTGGCTGCGCTCTTCATCGAC-3′ (Metabion, Germany) with an expected amplicon size of 680 bp (Dar et al. 2012). Briefly, each 25 µL volume consisted of 12.5 µL 2X Emerald Amp GT PCRmaster mix (Code No. RR310A, Takara, Japan), 1 µL of the forward primer (20 pmol/µL), 1 µL of the reverse primer (20 pmol/µL), 6 µL DNA (20 ng/µL) DNA template sample and 4.5 µL PCR grade water. PCR cycling started with an initial denaturation for 5 min at 94 °C followed by 35 cycles of denaturation for 30 s at 94 °C, annealing for 40 s at 53 °C and extension for 45 s at 72 °C. The final extension at 72 °C was allowed to proceed for 10 min. PCR amplicons were separated in a 1.5% agarose gel in tris–acetate EDTA buffer and photographed under UV transillumination following staining with ethidium bromide (Dar et al. 2012).

Sequencing and phylogenetic analysis

Amplified PCR products of Fasciola species samples were purified using Thermo Scientific GeneJET PCR Purification Kit (#K0701, Lithuania). The purified products were sequenced on an Applied Biosystems 310 automated DNA sequencer using cycle sequencing ABI prism Big Dye terminator chemistry (Perkin-Elmer/Applied Biosystems, Foster City, CA).The obtained sequences were aligned using Clustal W method (Altschul et al. 1990) Sequences were compared using the algorithm BLASTn with the available data of National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov). Six variable sites throughout the obtained sequences were compared to identify the precise Fasciola species (Dar et al. 2012). Neighbor-joining (NJ) method (Saitou and Nei 1987) was used to conduct the phylogenetic analyses of the obtained data using MEGA 10 (Kumar et al. 2018). Paragonimus westermani was used as the outgroup to root trees for both F. hepatica and F. gigantica. Neighbor-joining (NJ) trees were tested using 1000 bootstrap (Felsenstein 1985).

Statistical analysis

Morphometric findings of the recovered fasciolids were expressed as mean ± stander error (SE) using IBM SPSS for Windows, v.22 (IBM, Armonk, NY, USA). Moreover, data of liver condemnation between every two successive years was compared using Chi square. P value considered significant at P < 0.05.

Results

Economic losses of fascioliasis due to liver condemnation

All over the Egyptian provinces, it has been found that the total of percentages of liver condemnation among slaughtered cattle and buffaloes were 0.79, 0.97, 0.98, 0.89 and 0.66% in 2016, 2017, 2018, 2019 and 2020, respectively. Liver condemnations significantly varied (P < 0.05) among north, middle and south districts of Egypt, and varied from year to another. In the south of Egypt, the largest percentages (2.5–6.0%) of liver condemnation due to fascioliasis were observed. Meanwhile, liver condemnations due to fasciolosis were 0.19%-0.98% in the north, and 0.20% -0.37% in the middle of Egypt. Concomitantly, in the south of Egypt, estimated economic losses induced by liver condemnation due to fascioliasis ranged from 261850 to 616300 USD annually. Furthermore, it ranged from 58750 to 261850USD and 78550 to 128950 USD in the north and middle of Egypt, respectively (Table 1). In the north of Egypt, percentages of condemned livers /slaughtered animals were 0.99, 0.71, 0.26, 0.20 and 0.19% during 2016–2020. In the middle Egypt, condemned livers /slaughtered animals percentages were 0.21, 0.23, 0.20, 0.37 and 0.36%, during the five years period from 2016 to 2020, respectively. Lastly, percentages of infections in the south Egypt were 2.54, 4.46, 6.00, 5.01 and 3.42%, during the five years period from 2016 to 2020, respectively (Fig. 1 and Table 1). Among all, Aswan province had the highest percentage of fascioliasis, ranged from 26 to 60% according the year.

Table 1.

Liver condemnations and estimated economic losses due to fasciolosis of slaughtered cattle and buffaloes in Egypt during 2016–2020

Year Slaughtered animals Condemned livers Chi-square P value
Number Percentage Estimated economic losses (USD $)
2016
North Egypt 530117 5237 0.99 261850 220.0358 < 0.00001*
Middle Egypt 808081 1660 0.21 83000 10.4623 0.001218*
South Egypt 207941 5274 2.54 263700 1080.8156 < 0.00001*
Total 1546139 12171 0.79 608550 277.4935 < 0.00001
2017
North Egypt 475281 3395 0.71 169750 1188.26 < 0.00001*
Middle Egypt 697364 1605 0.23 80250 14.8215 0.000118 *
South Egypt 182643 8140 4.46 407000 463.3074 < 0.00001*
Total 1355288 13140 0.97 657000 0.9301) 0.334833 (NS)
2018
North Egypt 580970 1491 0.26 74550 46.628 < 0.00001*
Middle Egypt 782824 1571 0.20 78550 410.2222 < 0.00001*
South Egypt 205257 12326 6.00 616300 185.3668 < 0.00001*
Total 1569051 15388 0.98 769400 72.2192 < 0.00001*
2019
North Egypt 698928 1391 0.20 69550 0.5673 0.451343 (NS)
Middle Egypt 864288 3204 0.37 160200 0.4017 0.526209 (NS)
South Egypt 224221 11336 5.10 566800 649.8331 < 0.00001*
Total 1787437 15931 0.89 796550 544.3042 < 0.00001*
2020
North Egypt 608739 1175 0.19 58750 NA- NA
Middle Egypt 707681 2579 0.36 128950 -NA NA-
South Egypt 180575 6168 3.41 308400 -NA NA-
Total 1496995 9922 0.66 496100 NA NA
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P value considered significant at P < 0.05. Estimated liver cost was calculated by multiplying average liver weight (5 kg) * price of each kg (10 USD)

NS non-significant, NA not applicable

Fig. 1.

Fig. 1

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A map denoting the distribution pattern of fascioliasis in slaughtered cattle and buffaloes in Egyptian provinces during 2016–2020. The mark filled circle denoted provinces with low prevalences, filled rectangle indicated provinces with moderate prevalences and filled triangle denoted provinces with high prevalences. Among All provinces, Aswan filled star had the highest infection rate. The dark buffaloes symbol referred to the locations of abattoirs

Morphological features of the recovered flukes

Morphometrically, and according the criteria of differentiation, the revealed specimens categorized into Fasciola hepatica and Fasciola gigantica (Fig. 2). No intermediate forms (Fasciola sp.-like) could not be found. Among the examined livers, about 12/16 had F. hepatica, and 4/16 had F. gigantica. Measurements of specimens of F. hepatica are correlated to those of Periago et al., 2008 in Egypt in the following criteria: BL, BA, CL, VS-Vit, VS-P, OSmax, VS min, VSmax, OSA,PhL, PhW and they correlated to those of Ashrafi et al., 2006.in Iran in the followings: BW, BL/BW, CW, A-VS, OS-VS, Vit-P,OS min, VSA, OSA/VSA (Fig. 2, Table 2). Furthermore, Measurements of specimens of F. gigantica are correlated to those of Periago et al., 2008 in Egypt in the following criteria:BA, CL, A-VS, OS-VS, VS-Vit, Vit-P, VS-P, OSmax, PhL,PhWand they correlated to those of Ashrafi et al. (2006) in Iran in the followings: BL, BW, BL/BW, CW, OS min, VS min, VSmax, OSA, VSA, OSA/VSA (Table 3).

Fig. 2.

Fig. 2

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Some macroscopic and microscopic features of the collected flukes. a Liver from cattle in beni-suef abattoir showing adult Fasciola species. b Numerous Fasciola species from slaughtered animal. c A ventral sucker from adult Fasciola gigantica. d Lateral aspect of adult Fasciola gigantica showing vittiline glands. e The anterior end of adult Fasciola hepatica. f Dimensions of a ventral sucker of adult Fasciola hepatica. Scale bar = 500 µm

Table 2.

Comparative morphometric parameters of the liver fluke, Fasciola hepatica, obtained from bovines in Egypt during 2016–2020

Measurements of gravid adult fasciolids F. hepatica (Periago et al. 2008; n = 82) F. hepatica (Ashrafi et al. 2006; n = 99) F. hepatica (the current study; n = 55)
BL 15.48–28.71 (23.73 ± 0.33) 11.74–28.72 (19.57 ± 0.34) 10.9–30.0 (21.75 ± 4.83)
BW 8.21–14.27 (10.54 ± 0.15) 5.37–12.90 (8.63 ± 0.15) 5.10–12.50 (8.49 ± 1.70)
L/W 1.65–2.76 (2.27 ± 0.03) 1.58–3.08 (2.28 ± 0.03) 1.46–3.98 (2.61 ± 0.64)
BA 92.73–303.90 (180.90 ± 4.70) 71.17– 299.54 (171.68 ± 5.12) 75.2–331.2 (187.80 ± 65.81)
CL 1.36–2.98 (2.23 ± 0.04) 1.52–2.86 (2.18 ± 0.03) 1.10–3.10 (2.20 ± 0.51)
CW 2.05–3.99 (3.18 ± 0.04) 2.06–3.72 (2.88 ± 0.04) 1.0–3.10 (2.08 ± 0.39)
A-VS 2.01–3.52 (2.78 ± 0.03) 1.63–2.99 (2.33 ± 0.03) 1.20–3.50 (2.25 ± 0.46)
OS-VS 1.44–2.62 (2.07 ± 0.03) 1.06–2.32 (1.69 ± 0.03) 0.90–2.60 (1.73 ± 0.41)
VS-Vit 8.07–19.0 (14.24 ± 0.25) 4.98–17.03 (10.55 ± 0.22) 6.00–21.0 (13.25 ± 3.34)
Vit-P 3.30–10.4 (6.55 ± 0.16) 2.71–8.80 (5.64 ± 0.13) 2.50–9.1 (5.73 ± 1.53)
VS-P 12.4–25.08 (20.79 ± 0.31) 8.88–24.92 (16.24 ± 0.32) 9.0–28.0 (18.87 ± 4.57)
OS min 0.45–0.89 (0.70 ± 0.01) 0.44–0.85 (0.64 ± 0.01) 0.26–0.85 (0.59 ± 0.14)
OS max 0.69–1.01 (0.86 ± 0.01) 0.26–0.95 (0.70 ± 0.01) 0.45–1.36 (0.89 ± 0.23)
VS min 0.82–1.37 (1.04 ± 0.01) 0.80–1.19 (1.00 ± 0.01) 0.70–1.60 (1.11 ± 0.23)
VS max 0.97–1.49 (1.14 ± 0.01) 0.83–1.24 (1.05 ± 0.01) 0.84– 1.70 (1.27 ± 0.24)
OSA 0.27–0.69 (0.49 ± 0.01) 0.14–0.69 (0.45 ± 0.01) 0.14–1.22 (0.57 ± 0.25)
VSA 0.69–1.61 (0.94 ± 0.02) 0.66–1.47 (1.05 ± 0.02) 0.57–2.66 (1.44 ± 0.58)
OSA/VSA 0.25–0.72 (0.53 ± 0.01) 0.15–0.85 (0.44 ± 0.01) 0.16–0.83 (0.43 ± 0.17)
PhL 0.58–1.02 (0.79 ± 0.01) 0.57–0.83 (0.70 ± 0.01) 0.44–1.01 (0.74 ± 0.15)
PhW 0.32–0.55 (0.42 ± 0.01) 0.21–0.52 (0.36 ± 0.01) 0.26–0.80 (0.53 ± 0.13)
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Table 3.

Comparative morphometric parameters of the liver fluke, Fasciola gigantica, obtained from bovines in Egypt during 2016–2020

Measurements of gravid adult fasciolids F. gigantica (Ashrafi et al. 2006; n = 48) F. gigantica (Periago et al. 2008; n = 12) Fasciola gigantica (the current study; n = 23
BL 30.70–52.12 (39.51 ± 0.84) 35.25–48.71 (44.65 ± 1.15) 17.8–53.0 (42.63 ± 10.78)
BW 6.45–11.38 (8.87 ± 0.16) 8.23–13.6 (10.36 ± 0.46) 6.80–12.0 (9.10 ± 1.49)
L/W 3.20–5.86 (4.48 ± 0.08) 3.43–5.50 (4.37 ± 0.17) 3.00–6.41 (4.95 ± 1.0)
BA 217.5–585.92 (354.09 ± 12.76) 226.16–475.9 (359.20 ± 19.05) 243.0–583.1 (402.80 ± 93.80)
CL 2.12–2.99 (2.55 ± 0.03) 2.61–3.68 (3.16 ± 0.11) 1.90–4.0 (2.86 ± 0.47)
CW 2.84–4.39 (3.46 ± 0.05) 3.25–4.34 (3.81 ± 0.10) 2.00–3.90 (2.63 ± 0.47)
A-VS 1.70–2.97 (2.39 ± 0.03) 2.44–3.39 (2.98 ± 0.08) 1.90–4.00 (2.76 ± 0.55)
OS-VS 1.06–2.01 (1.62 ± 0.03) 1.63–2.52 (2.18 ± 0.08) 1.10–3.80 (2.13 ± 0.70)
VS-Vit 16.05–31.61 (21.87 ± 0.55) 21.15–30.76 (26.04 ± 0.82) 19.0–32.0 (26.34 ± 3.58)
Vit-P 9.03–19.43 (13.79 ± 0.38) 9.86–19.72 (14.98 ± 0.81) 8.00–21.0 (14.89 ± 3.39)
VS-P 26.73–47.76 (35.58 ± 0.81) 31.01–45.39 (41.02 ± 1.21) 31.00–49.0 (40.84 ± 5.76)
OS min 0.57–0.95 (0.76 ± 0.01) 0.72–0.88 (0.79 ± 0.01) 0.40–0.90 (0.66 ± 0.14)
OS max 0.83–1.14 (0.93 ± 0.01) 0.84–1.05 (0.95 ± 0.02) 0.70–1.14 (0.95 ± 0.16)
VS min 1.37–1.83 (1.57 ± 0.01) 1.26–1.52 (1.43 ± 0.02) 1.30–1.85 (1.59 ± 0.14)
VS max 1.44–1.96 (1.63 ± 0.02) 1.35 – 1.67 (1.53 ± 0.03) 1.18–2.00 (1.68 ± 0.23)
OSA 0.51– 1.05 (0.71 ± 0.01) 0.51–0.71 (0.60 ± 0.02) 0.32–1.02 (0.66 ± 0.21)
VSA 1.98–3.49 (2.56 ± 0.05) 1.34–2.02 (1.74 ± 0.06) 1.80–3.70 (2.68 ± 0.56)
OSA/VSA 0.22–0.37 (0.28 ± 0.00) 0.27–0.53 (0.35 ± 0.02) 0.08–0.39 (0.25 ± 0.08)
PhL 0.75–1.03 (0.88 ± 0.01) 0.75–0.94 (0.84 ± 0.02) 0.10–1.04 (0.72 ± 0.24)
PhW 0.36–0.65 (0.48 ± 0.01) 0.45–0.59 (0.50 ± 0.01) 0.30–0.75 (0.58 ± 0.13)
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PCR amplification and phylogenetic analysis

PCR amplification of the DNA of the collected Fasciola species using of ITS-1 resulted in an amplicon size of 680 bp (Fig. 3). Sequence analysis showed that only Fasciola hepatica (26/34) and F. gigantica (8/34) could be detected. Distinctly, no intermediate forms were elucidated among the examined sequences (Fig. 4). The obtained sequences showed 99–100% identity to each other and the previously GenBank isolates. There were 6 nucleotide positions varies among the different isolates (Fig. 4). Fasciola hepatica isolates of the current study were 100% identical with Fasciola Egyptian isolates (LC076196 and JF294998), French isolate (JF294999), and Iranian isolates (MF969009 and MK377150). Approximately 17 sequences deposited in GenBank as F. hepatica under accession numbers OL635617–OL635633 (Fig. 5). Moreover, 4 sequences had accession numbers OL635645–OL635648 showed 100% identity with F. gigantica GenBank isolates. The obtained F. gigantica isolates showed 100% identity with Egyptian isolates (LC076125, LC076108 and KX198619), Iranian isolates (KF982047 and MF372919) and other GenBank F. gigantica isolates from Vietnam. Cameroon, and India (Fig. 6).

Fig. 3.

Fig. 3

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Amplified ITS-1 ribosomal region products on agarose gel. Lane N is a sample treated with saline (negative control). Lane P is a positive control. Lanes 1–23 represented PCR products of amplified Fasciola species specimens (680 bp). Lane 7 represented a negative specimen. Lanes M represented 100 bp ladder

Fig. 4.

Fig. 4

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Nucleotide sequence alignment of ITS-1 of Fasciola species (intermediate form of Vietnam AB514870, and Japan AB514866), F. hepatica Egypt (previous study, LC076196), F. hepaticaof the current study, F. gigantica Egypt (previous study, LC076127), and F. giganticaof the current study

Fig. 5.

Fig. 5

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Phylogenetic relationships between Fasciola hepatica ITS-1 sequences of the current study with other F. hepatica and Fasciola species intermediate form GB isolates using neighbour joining tree. Fasciola hepatica isolates of the current study (OL635617–OL635633), violet square labelled, were closely to each other and previously deposited Egyptian, French and Iranian F. hepatica isolates. The intermediate forms of Fasciola species of previous studies from Egypt, Saudi-Arabia, Vietnam, South-Korea, and Japan were away from isolates of the current study. Paragonimus westermani was used to root the current tree

Fig. 6.

Fig. 6

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Phylogenetic relationships between Fasciola gigantica ITS-1 sequences of the current study with other F. gigantica and Fasciola species intermediate form GB isolates using neighbour joining tree. Fasciola gigantica isolates of the current study (OL635645–OL635648), violet square labelled, were closely to each other and previously deposited Egyptian, Iranian and Nigerian F. gigantica isolates. The intermediate forms of Fasciola species of previous studies from Egypt, Saudi-Arabia, Vietnam, South-Korea, and Japan were away from isolates of the current study. Paragonimus westermani was used to root the current tree

Discussion

Fascioliasis caused by the common liver flukes, Fasciola hepatica and Fasciola gigantica, is common in a wide range of animals with significant negative economic losses, particularly in both large and small ruminants due to the high opportunity of the infection associated with expected morbidity and mortality (Amer et al. 2016; Dar et al. 2012; Olsen et al. 2015). Dramatically, economic losses due to the infection range from sudden deaths, in acute infections, to reduced body conditions, infertility and decreased yield, in chronic infections (Calvani and Šlapeta 2021).

The current investigation revealed that the percentages of liver condemnation form slaughtered cattle and buffaloes nationwide ranged from 0.66 to 0.79% per year (2016–2020). Meanwhile, the highest percentages of annual liver condemnation were in the south of Egypt (2.5–6.0%), associated with the highest economic losses (261850–616300 USD). Furthermore, Aswan recorded the highest liver condemnation percentage (26–60%) all over the Egyptian provinces. Similarly, Mohammed and Maky (2020) recorded that 4529010 Egyptian pound (383063 USD) economic loss for two years (2017–2018) due to meat condemnation in three abattoirs (Alexandria, Qena and Aswan) including 4290 (2.55%) livers were condemned. Moreover, Rassol et al. (2020) determined 7553 (9.3%) condemned livers in Abu Simbel Abattoir, Aswan Governorate. This percentage was resulted from (86.3%) fascioliasis, (8.7%) cirrhosis, (2.3%) abscesses, (1.5%) calcified cyst, (0.6%) hepatitis and (0.6%) cysticercus bovis. Also, they reported the economic losses were 152718 USD caused by fascioliasis. Abd El-aziz et al. (2021) reported 11712 LE economic losses annually due to fascioliasis along (2017 and 2018). Furthermore, Jaja et al. (2017) assessed the prevalence and monetary losses associated with Fasciola infection at three abattoirs in Eastern Cape Province from 2010–2012 and founded that annual prevalence of Fasciola at abattoirs AB1, were (3.2%, 2.2% and 2.0%), AB2 (6.4%, 4.6% and 3.5%), AB3 (14.4%, 6.9% and 9.5%) for year 2010, 2011 and 2012 respectively. The total financial loss due to Fasciola infection during the active survey of the three abattoirs was ZAR 44, 930 (3456.2 USD). In addition, Opio et al. (2021) detected that monetary loss due to liver damage was 9900 UGX (2.67 USD)/infected animal.at the Lira Municipal abattoir in Northern Uganda.

On the other hand, Elshraway and Mahmoud (2017) found that out of 2251 slaughtered cattle and buffaloes of both sexes, 695 (30.88%) were infected with Fasciola species, in El-Kharga, Egypt. Moreover, 51/ 458 (11.1%) of examined cattle livers were harboring F. hepatica in Dakhla Oasis, Egypt (Arafa et al. 2018). In similar African countries, F. hepatica and F. gigantica were reported in 20–26% of slaughtered sheep in Ethiopia (Sissay et al. 2007). Furthermore, the prevalences of Fasciola spp. in trade cattle slaughtered at Minna, Suleja, Bida, Kontagora and New-Bussa municipal abattoirs in Nigeria during the period of 2005–2014 were 1.31, 1.70, 1.11, 1.05, and 1.24%, respectively with an overall 10-years prevalence of bovine fasciolosis was 1.46%, and livers of slaughtered animals condemned as a result of Fasciola spp.-induced pathological lesions Yatswako and Alhaji (2017) In Iran, an Arabian and Asian country with similar geographical conditions, Kordshooli et al. (2017) mentioned that 3.44% of cattle, goats, and sheep slaughtered at Jahrom main abattoir during 2011–2015, were infected with Fasciola spp. The mean prevalence of fasciolosis for cattle, sheep, and goat was 11.15, 5.22, and 2.15%, respectively. The lower prevalence in the current study might be attributed to the regular deworming of livestock with the proper anthelmintics, combating of the vector intermediate hosts via the elimination of lymnaeid snails with molluscicides and removal of ponds of stagnant water.

The present work revealed that, morphometrically, and according the criteria of differentiation, the recovered fasciolid specimens were Fasciola hepatica (12/16) and Fasciola gigantica (4/16) of the collected livers. No intermediate forms (Fasciola sp.-like) could be found. Such finding might be due to being that co-infections with both flukes were lacked. On the other hand, the occurrence of F. hepatica / F. gigantica intermediate form (Fasciola-sp.) for the first time in Egypt was recorded (Periago et al. 2008). They referred such finding to the co-existence of both F. hepatica and F. gigantica in the same host. Moreover, in Iran, Ashrafi et al. (2006) revealed the occurrence of F. hepatica and F. gigantica and the intermediate form.

Regarding the molecular tools, it well known that the first nuclear ribosomal internal transcribed spacer (ITS-1) is the most reliable marker to differentiate Fasciola species; F. hepatica, F. gigantica, and Fasciola-sp. like, the intermediate hybrid form (Rokni et al. 2010; Dar et al. 2012; Amer et al. 2011; Ichikawa and Itagaki 2010). Such marker yields an amplicon of 680 bp having 6 variable nucleotide positions based on Fasciola species; F. hepatica, F. gigantica, and their hybrid form (Amer et al. 2016; Dar et al. 2012; Ichikawa and Itagaki 2010). In the current investigation, the obtained sequences were identified as F. hepatica (55/78) and F. gigantica (23/78).Interestingly, it has been found that F. hepatica was more common than F. gigantica. Similarly, Amer et al. (2016) recorded a higher occurrence of F. hepatica (11/17) than F. gigantica (4/17) based on the molecular identification in Egypt. There was no hybrid form detected among the obtained sequences. Currently, it is worthy to mention that each infected liver hada single fasciolidspecies (F. hepatica/F. gigantica) with no animals were co-infected. Meanwhile, the hybrid form was detected in Egypt (Amer et al. 2011) and many Asian countries (Itagaki et al. 2005; Mohanta et al. 2014). The opportunity of the occurrence of the hybrid form depends largely on the co-existence of both fasciolid species in the same definitive host (Amer et al. 2016, 2011; Spithil et al. 1999). The hybrid form occurs as result of mating between adults F.hepatica and F.gigantica carrying nuclear rRNA genes of both parents and mitochondrial genome of the maternal parent (Blair 2005; Le et al. 2008).Meanwhile, introgressed form appears due to crossing of hybrids with one parent species. Consequently, the ribosomal genes of this generation become homozygous toward one Fasciola species as well as mitochondrial genes of the other species. Expectedly, the hybrid forms among the examined sequences were lacked in the current study. This might be attributed to the finding that no co-infections observed among the examined livers. Furthermore, non-overlapping morphological features of the examined isolates showed the occurrence of either F. hepatica or F. gigantica among the examined isolates. As well, sequencing analysis of ITS1 amplicons proved the morphological identification of the obtained isolates. Dar et al. (2012) showed that the intermediate, hybrid, form of Fasciola in Egypt is sporadic and less common. In Egypt and other Africa countries, the identification of Fasciola species based on the morphological features may work efficiently (Amer et al. 2016).Nevertheless, in Japan, Korea, Vietnam, and various Asian countries, the morphological identification of Fasciola species is not reliable due to presence of the intermediate forms (Itagaki et al. 2005).

In the present work, phylogenetic analysis of ITS-1 of F. hepatica isolates (OL635617–OL635633) revealed 100% identity with the previous similar Egyptian isolates (LC076196 and JF294998) (Amer et al. 2016; Dar et al. 2012). Moreover, 100% identity with the French F. hepatica isolate (JF294999) was reported by Dar et al. (2012) and with the Iranian F. hepatica isolate (MF969009) (Mir et al. 2019). Furthermore, our Fasciola gigantica isolates showed 100% identity with the previously recorded Egyptian isolates (LC076125) (Amer et al. 2016). It is worthy to mention that ITS-1 marker was conserved and yielded amplicons with an identity ranging from 99 to 100% with the different F. hepatica and F. gantaica isolates recorded in GenBank. In further studies, other markers like mitochondrial genes, nad1 and cytochrome c oxidase subunit 1, will be included to estimate the genetic variability among the two main fasciolid species in Egypt.

Conclusion

Economic losses caused by fascioliasis in cattle, and buffaloes should be considered by veterinary authorities in Egypt. In the current study, Fasciola hepatica was more predominant than F. gigantica. The co-existence of both F. hepatica and F. gigantica was not existed; consequently, no hybrid form was detected among the examined samples, based on using ITS-1 marker gene sequencing analysis.

Acknowledgements

Authors kindly acknowledge all veterinarians and authorities who facilitated gathering data and specimens collection.

Author contributions

OR; samples collection, lab work, finding analysis, draft manuscript. WMA; designing, drafting manuscript, revising. ANW; drafting manuscript, KME; Designing, revising.

Funding

No funding sources.

Data availability

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

Authors declare that there is no conflict of interest.

Ethics approvals

Not applicable, as no specimens were taken from condemned parts in abattoirs with any experiments done for animals or humans.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Abd El-Aziz OM, Hassanin FS, Shaltout FA, Mohamed OA. Prevalence of some foodborne parasitic affection in slaughtered animals in local Egyptian abattoir. Benha Vet Med J. 2021;40:111–114. doi: 10.21608/bvmj.2021.80316.1432. [DOI] [Google Scholar]
  2. Abdel-Hakeem SS, Omar MAFE. Ovine fascioliasis: environmental epidemiology and meta-analysis of the prevalence, agro-ecological and economic factors in five provinces of the Nile Delta region of Egypt. Damanhour J Vet Sci. 2020;3(1):23–31. doi: 10.5455/djvs.2020.67811. [DOI] [Google Scholar]
  3. Adediran OA, Uwalaka EC. Laboratory breeding of Lymnaea natalensis (Krauss, 1848) intermediate host of Fasciola gigantica (Cobbold, 1856) N Y Sci J. 2013;6(8):55–57. [Google Scholar]
  4. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  5. Amer S, Dar Y, Ichikawa M, Fukuda Y, Tada C, Itagaki T, Nakai Y. Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Parasitol Int. 2011;60:5–12. doi: 10.1016/j.parint.2010.09.003. [DOI] [PubMed] [Google Scholar]
  6. Amer S, El-Khatam A, Zidan S, Feng Y, Xiao L. Identity of Fasciola spp. in sheep in Egypt. Parasit Vectors. 2016;9(1):1–8. doi: 10.1186/s13071-016-1898-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arafa WM, Hassan AI, Snousi SAM, El-Dakhly KM, Holman PJ, Craig TM, Aboelhadid SM. Fasciola hepatica infections in cattle and the freshwater snail Galba truncatula from Dakhla Oasis. Egypt J Helminthol. 2018;92(1):56–63. doi: 10.1017/S0022149X17000086. [DOI] [PubMed] [Google Scholar]
  8. Ashrafi K, Massoud J, Holakouei K, Mahmoodi M, Joafshani MA, Valero MA, Fuentes MV, Khoubbane M, Artigas P, Bargues MD, Mas-Coma S. Evidence suggesting that Fasciola gigantica might be the most prevalent causal agent of fascioliasis in northern Iran. Iran J Public Health. 2004;33:31–37. [Google Scholar]
  9. Ashrafi K, Valero MA, Panova M, Periago MV, Massoud J, Mas-Coma S. Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitol Int. 2006;55:249–260. doi: 10.1016/j.parint.2006.06.003. [DOI] [PubMed] [Google Scholar]
  10. Batmaz H (2013) Koyun ve keçilerin iç hastalıkları: Semptomdan Tanıya Tanıdan Sağaltıma. Nobel Tıp Kitabevi
  11. Bazh EKA, Beder NA, Ayoub M, Sadek K. Fasciola infection among cattle and buffaloes at Behera Governorate. Egypt Zagazig Vet J. 2012;40:125–136. [Google Scholar]
  12. Beesley NJ, Williams DJL, Paterson S, Hodgkinson J. Fasciola hepatica demonstrates high levels of genetic diversity, a lack of population structure and high gene flow: possible implications fordrug resistance. Int J Parasitol. 2017;47(1):11–20. doi: 10.1016/j.ijpara.2016.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bennett R, Pelaar J. Updated estimates of the costs associated with thirty four endemic livestock diseases in Great Britain: a note. J Agric Econ. 2005;56(1):135–144. doi: 10.1111/j.1477-9552.2005.tb00126.x. [DOI] [Google Scholar]
  14. Blair D. Ribosomal DNA, variation in parasitic flatworms. In: Maule AG, Marks NJ, editors. Parasitic flatworms: molecular biology, biochemistry, immunology and control. Wallingford: CABI; 2005. pp. 96–123. [Google Scholar]
  15. Calvani NED, Šlapeta J. Fasciola species introgression: Just a fluke or something more? Trends Parasitol. 2021;37(1):25–34. doi: 10.1016/j.pt.2020.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chontananarth T, Tejangkura T, Wetchasart N, Chimburut C. Morphological characteristics and phylogenetic trends of trematode cercariae in freshwater snails from Nakhon Nayok Province, Thailand. Korean J Parasitol. 2017;55(1):47–54. doi: 10.3347/kjp.2017.55.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dar Y, Amer S, Mercier A, Courtioux B, Dreyfuss G. Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt. Parasite. 2012;19(2):177–182. doi: 10.1051/parasite/2012192177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Elshraway NT, Mahmoud WG. Prevalence of fascioliasis (liver flukes) infection in cattle and buffaloes slaughtered at the municipal abattoir of El-Kharga, Egypt. Vet World. 2017;10(8):914–917. doi: 10.14202/vetworld.2017.914-917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. El-Tahawy AS, Bazh EK, Khalafalla RE. Epidemiology of bovine fascioliasis in the Nile Delta region of Egypt: it's prevalence, evaluation of risk factors and it's economic significance. Vet World. 2017;10(10):1241–1249. doi: 10.14202/vetworld.2017.1241-1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Felsenstein J. Phylogenies and the comparative method. Am Nat. 1985;125:1–15. doi: 10.1086/284325. [DOI] [Google Scholar]
  21. Hillyer GV, Apt W. Food-borne trematode infections in the Americas. Parasitol Today. 1997;13(3):87–88. doi: 10.1016/S0169-4758(97)01000-4. [DOI] [Google Scholar]
  22. Ichikawa M, Itagaki T. Discrimination of the ITS1 types of Fasciola spp. based on a PCR-RFLP method. Parasitol Res. 2010;106:757–761. doi: 10.1007/s00436-010-1724-2. [DOI] [PubMed] [Google Scholar]
  23. Irak K, Celik BA, Karakoc Z, Çelik ÖY, Mert H, Mert N, Kaya MO. Oxidant/antioxidant status, PON1 and ARES activities, trace element levels, and histological alterations in sheep with cystic echinococcosis. Iran J Parasitol. 2018;13(3):448–456. [PMC free article] [PubMed] [Google Scholar]
  24. Itagaki T, Kikawa M, Sakaguchi K, Shimo J, Terasaki K, Shibahara T, Fukuda K. Genetic characterization of parthenogenetic Fasciola sp. in Japan on the basis of the sequences of ribosomal and mitochondrial DNA. Parasitology. 2005;131:679–685. doi: 10.1017/S0031182005008292. [DOI] [PubMed] [Google Scholar]
  25. Jaja IF, Mushonga B, Green E, Muchenje V. Financial loss estimation of bovine fasciolosis in slaughtered cattle in South Africa. Parasite Epidemiol Control. 2017;2(4):27–34. doi: 10.1016/j.parepi.2017.10.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Karpamar A, Yıldırım A, Bişkin Z, Düzlü Ö, İnci A. The investigation of fasciolosis in sheep by copro antigen ELISA and sedimentation-zinc sulphate flotation technique around Zara region (Turkey) Kafkas Üniv Vet Fak Derg. 2012;18:7–12. [Google Scholar]
  27. Khoramian H, Arbabi M, Osqoi MM, Delavari M, Hooshyar H, Asgari M. Prevalence of ruminants fascioliasis and their economic effects in Kashan, center of Iran. Asian Pac J Trop Biomed. 2014;4(11):918–922. doi: 10.12980/APJTB.4.2014APJTB-2014-0157. [DOI] [Google Scholar]
  28. Kordshooli MS, Solhjoo K, Armand B, Dowlatkhah H, Jahromi ME. A reducing trend of fasciolosis in slaughtered animals based on abattoir data in South of Iran. Vet World. 2017;10(4):418–423. doi: 10.14202/vetworld.2017.418-423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Le TH, De NV, Agatsuma NT, Thi T, Nguyen QD, McManus DP, Blair D. Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam. Int J Parasitol. 2008;38(6):725–730. doi: 10.1016/j.ijpara.2007.10.003. [DOI] [PubMed] [Google Scholar]
  31. Lee JH, Quan JH, ChoiI W, Park GM, Cha GH, Kim HJ, Yuk JM, Lee YH. Fasciola hepatica: infection status of freshwater snails collected from Gangwon-do (province), Korea. Korean J Parasitol. 2017;55(1):95–98. doi: 10.3347/kjp.2017.55.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mas-Coma S. Epidemiology of fascioliasis in human endemic areas. J Helminthol. 2005;79:207–216. doi: 10.1079/JOH2005296. [DOI] [PubMed] [Google Scholar]
  33. Mas-Coma S, Bargues MD. Human liver flukes: a review. Res Rev Parasitol. 1997;57:145–218. [Google Scholar]
  34. Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005;35:1255–1278. doi: 10.1016/j.ijpara.2005.07.010. [DOI] [PubMed] [Google Scholar]
  35. Mir S, Dabirzadeh M, Rokni MB, Aryaeipour M, SHahraki M K, Azizi H, Identification and phylogenetic classification of Fasciola species isolated from sheep and cattle by PCR-RFLP in Zabol, in Sistan and Baluchistan province, Southeast Iran. Iran J Public Health. 2019;48:934–942. [PMC free article] [PubMed] [Google Scholar]
  36. Mohammed ES, Maky MA. Meat condemnations and economic importance in the Northern and Southern Egyptian Abattoirs. Ad Anim Vet Sci. 2020;8(1):96–107. [Google Scholar]
  37. Mohanta UK, Ichikawa-Seki M, Shoriki T, Katakura K, Itagaki T. Characteristics and molecular phylogeny of Fasciola flukes from Bangladesh, determined based on spermatogenesis and nuclear and mitochondrial DNA analyses. Parasitol Res. 2014;113:2493–2501. doi: 10.1007/s00436-014-3898-5. [DOI] [PubMed] [Google Scholar]
  38. Muller PY, Janovja kH, Miserez AR, Dobbie Z, Short technical report processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques. 2002;32(6):1372–1379. [PubMed] [Google Scholar]
  39. Nolan MJ, Cribb TH. The use and implications of ribosomal DNA sequencing for the discrimination of Digenean species. Adv Parasitol. 2005;60:101–163. doi: 10.1016/S0065-308X(05)60002-4. [DOI] [PubMed] [Google Scholar]
  40. Olsen A, Frankena K, Bødker R, Toft N, Thamsborg SM, Enemark HL, Halasa T. Prevalence, risk factors and spatial analysis of liver fluke infections in Danish cattle herds. Parasit Vectors. 2015;8:160. doi: 10.1186/s13071-015-0773-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Opio LG, Abdelfattah EM, Terry J, Odongo S, Okello E. Prevalence of fascioliasis and associated economic losses in cattle slaughtered at lira municipality abattoir in northern Uganda. Animals. 2021;11(3):681. doi: 10.3390/ani11030681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Periago MV, Valero MA, Panova M, Mas-Coma S. Phenotypic comparison of all opatric populations of Fasciolahepatica and Fasciolagigantica from European and African bovines using a computer image analysis system (CIAS) Parasitol Res. 2006;99:368–378. doi: 10.1007/s00436-006-0174-3. [DOI] [PubMed] [Google Scholar]
  43. Periago MV, Valero MA, El Sayed M, Ashrafi K, El Wakeel A, Mohamed MY, Desquesnes M, Curtale F, Mas-Coma S. First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta. Egypt Infect Genet Evol. 2008;8:51–58. doi: 10.1016/j.meegid.2007.10.001. [DOI] [PubMed] [Google Scholar]
  44. Prasad PK, Tandon V, Chatterjee A, Bandyopadhyay S. PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA of giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss. Parasitol Res. 2007;101(6):1581–1587. doi: 10.1007/s00436-007-0680-y. [DOI] [PubMed] [Google Scholar]
  45. Rassol AMA, Ahmed AM, Sobhy HM, Abdelgayed SS, Hekal SHA. Effects of fascioliasis on the economic losses of beef liver at Abu Simbel Abattoir, Aswan Governorate. Egypt Adv Anim Vet Sci. 2020;8(11):1175–1179. [Google Scholar]
  46. Rokni MB, Mirhendi H, Behnia M, FasihiHarandi M, Jalalizand N. Molecular characterization of Fasciola hepatica isolates by RAPD-PCR and ribosomal ITS1 sequencing. Iran Red Crescent Med J. 2010;12:27–32. [Google Scholar]
  47. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  48. Schweizer G, Braun U, Deplazes P, Torgerson PR. Estimating the financial losses due to bovine fasciolosis in Switzerland. Vet Rec. 2005;157(7):188–193. doi: 10.1136/vr.157.7.188. [DOI] [PubMed] [Google Scholar]
  49. Sissay MM, Uggla A, Waller PJ. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Trop Anim Health Prod. 2007;39:521–531. doi: 10.1007/s11250-007-9035-z. [DOI] [PubMed] [Google Scholar]
  50. Soliman MFM. Epidemiological review of human and animal fascioliasis in Egypt. J Infect Dev Ctries. 2008;2(03):182–189. doi: 10.3855/jidc.260. [DOI] [PubMed] [Google Scholar]
  51. Spithil TM, Smooker PM, Copeman DB. Fasciola gigantica: epidemiology, control, immunology and molecular biology. In: Dalton JP, editor. Fasciolosis. Oxon: CABI Publishing; 1999. pp. 465–525. [Google Scholar]
  52. Urquhart GM, Armour J, Duncan JL, Dunn AM, Jennings FM. Veterinary parasitology, 2nd edn. Oxford: Blackwell Science; 1996. [Google Scholar]
  53. Valero MA, Panova M, Mas-Coma S. Phenotypic analysis of adults and eggs of Fasciolahepatica by computer image analysis system. J Helminthol. 2005;79:217–225. doi: 10.1079/JOH2005301. [DOI] [PubMed] [Google Scholar]
  54. Yatswako S, Alhaji NB. Survey of bovine fasciolosis burdens in trade cattle slaughtered at abattoirs in North-central Nigeria: the associated predisposing factors and economic implication. Parasite Epidemiol Control. 2017;2:30–39. doi: 10.1016/j.parepi.2017.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yavuz A, İnci A, Yıldırım A, İça A, Düzlü Ö. Distribution of Fasciola hepatica in cattle. Erciyes Üniv Sağlık Bilim Derg. 2007;16:96–102. [Google Scholar]

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Data Availability Statement

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

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