Research Article

Molecular Sequencing Analysis of Fasciola Spp. in Sheep

Israa M. Essa*, Ghazi Y. Azzal, Alaa Tariq Abdulwahid

Department of Parasitology, College of Veterinary Medicine, University of Basrah, Basra, Iraq.

Received | June 14, 2024; Accepted | July 26, 2024; Published | August 22, 2024

*Correspondence | Israa M. Essa, Department of Parasitology, College of Veterinary Medicine, University of Basrah, Basra, Iraq; Email: israa.essa@uobasrah.edu.iq

Citation | Essa IM, Azzal GY, Abdulwahid AT (2024). Molecular sequencing analysis of fasciola spp. in sheep. Adv. Anim. Vet. Sci. 12(10): 1846-1852.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.10.1846.1852

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Fasciola, commonly known as liver fluke, is a parasitic flatworm which classify under the Plagiorchiida Order of the Trematoda Class in the Platyhelminthes Phylum. The genus of Fasciola includes mainly F. hepatica Linnaeus, 1758, and F. gigantica Cobbold, 1855 in addition to a number of invalid species such as F. indica, F. californica, F. halli and F. nyanzae (Itagaki et al., 2022; Madsen et al., 2022). Wide range of animal species and humans can infect with Fasciola resulting in a public health concern and severe economic losses in livestock industry (Beesley et al., 2018; Regasa et al., 2021; Zerna et al., 2021). Lifecycle of Fasciola comprises five phases; egg (pass through feces of mammalian host), miracidia (free-swimming and infect the intermediate host), cercariae (erupt from snail host and attached to aquatic plants), metacercarial cysts (in aquatic plants) and adult fluke (mammalian host). After ingestion, young flukes were excyst in duodenum, penetrate intestinal wall, enter the peritoneal cavity, and migrate to liver penetrating the capsule and tunnel of parenchyma. After growing and destroying tissues, flukes enter the bile ducts and occasionally the gallbladder where they mature and begin produce eggs (Phalee et al., 2015; Moazeni et al., 2016; Lalor et al., 2021; Stuen et al., 2022).

According to amount of ingested metacercariae, clinical symptoms might range from mild to severe devastating illness. Acute form of infection is manifested by sudden death, anemia, and painful-distended abdomen could occur seasonally in sheep. While the chronic form of disease might occur at any season particularly winter causing a submandibular edema, unthriftiness, anemia and decreased milk yield (Constable et al., 2016; Forbes, 2017). Based on stage of disease, diagnosis can be done traditionally using the fecal sedimentation test especially in chronic and subacute infections (Kajugu et al., 2015). However, serological enzyme-linked immunosorbent assays (ELISAs) and molecular techniques such as polymerase chain reaction (PCR) have provided reliable tools in last decades (Webb et al., 2018; Caravedo et al., 2020). Furthermore, genetic research has determined the origin, toxicity, evolution, development of parasite and resistance to various anthelmintics (Cwiklinski et al., 2015; Beesley et al., 2017).

In Iraq, molecular prevalence of Fasciola have been studied in Wasit (Abdulwahed et al., 2019), Mosul (Hamoo et al., 2019), Sulaymaniyah (Raoof et al., 2020), and Erbil (Muhammad and Hassan, 2021) provinces. Therefore, this study aims to investigate the prevalence rate of Fasciola in livers of 361 slaughtered sheep at Basra province (Iraq) with molecular testing and phylogenetic analysis of obtained flukes in NCBI.

Table 1: Total results for gross examination of livers of 361 sheep.

Animal	Total No.	Positives [No. (%)]
		Total	Juvenile worms	Adult worms
Sheep	361	29 (8.03%)	23 (79.31%) *	6 (20.69%)
p-value			0.0263 S

Significance (S) *; Non-significance (NS)

MATERIALS AND METHODS

Samples

During November (2023) to February (2024), livers of totally 361 slaughtered sheep at the official and private abattoirs in Basra province (Iraq) were examined grossly to detect the presence of flukes that collected initially in plastic petridishes contained phosphate buffered saline (PBS).

Morphology

At laboratory, the collected flukes were examined directly based on the external characteristics as described by Rokni et al. (2010).

Molecular Assay

Firstly, the samples of liver fluke were thawed in water path, washed extensively in PBS, and subjected to steps of the DNAs extraction as described in the Protocol B of G-SpinTM Total DNA Extraction Kit (Intron Biotechnology, Korea). After estimation of purity and concentration of extracted DNAs using the Nanodrop System (Thermo-Scientific, UK), the MasterMix tubes were prepared following the manufacturer instructions of the AccuPowerTM PCR PreMix (Bioneer, Korea) using one set of designed primers: ISR-F (5´-ATC ACT GAT GGG GTG CCT AC-3´) and ISR-R (5´-CCG GAT ACA TTA GGG AAA CG-3´) by targeting the 18S rRNA gene of Fasciola spp. in the NCBI-GenBank (MW620063.1) isolate at a final volume of 20 μl. Then, the MasterMix tubes were amplified in the Thermal Cycler (Bio-Rad, USA) at the following conditions: 1 cycle for initial denaturation (95°C / 5 minutes), 35 cycles for denaturation (95°C / 30 seconds), annealing (56°C / 30 seconds) and extension (72°C / 1 minute), and 1 cycle for final extension (72°C / 7 minutes). Electrophoresis of PCR products were performed in agarose-gel (1.5%) stained with Ethidium Bromide at 100 Volt and 80 Am for 1 hour. According to size of ladder marker, positive samples were identified under UV transilluminator at ≈592 bp.

For phylogeny, DNAs of five positive local Fasciola isolates were sequenced by the Macrogen Company (Korea), and the data were submitted in the NCBI-GenBank, and analysed using the MEGA Software (version x) to detect the percentage of identity between the local study isolates and global NCBI-BLAST isolates.

Statistical Analysis

The t-test, Odd ratio and risk were served in the GraphPad Prism Software (version 6.0.1) to detect significant differences at a level of P<0.05 (*), P<0.01 (**), P<0.001 (***) and P<0.0001 (****), (Gharban, 2023).

RESULTS AND DISCUSSION

In the present study, 8.03% of sheep were infected with liver flukes with significant prevalence (P<0.0263) of juvenile flukes (79.31%) when compared to adults (20.69%). Based on morphology, all adult flukes were diagnosed as Fasciola (Table 1). In comparison to national studies, there was 0.28% in Kirkuk (Abass et al., 2018), 55.71% in Sulaimani (Ali et al., 2021), 1.76% in Duhok (Nerway et al., 2021), 5.5% in Salah al-Din (Sulieman et al., 2021), 10% in Mosul (Al-Lahaibi et al., 2023), and 2% in Erbil (Koyee et al., 2023); whereas globally, there was 13.2% in Ethiopia (Jittapalapong, 2007), 3.09% in India (Shende et al., 2007), 6.6% in Iran (Khanjari et al., 2014), 14.7% in Egypt (Amer et al., 2016), 100% in Indonesia (Kusumarini et al., 2020) and 26.2% in Turkey (Ayvazoğlu et al., 2023). This variation might attribute to number of tested samples, topographical properties, management system, climatic conditions, degree of exposure to intermediate host, and therapeutic / prevention schemes applied for controlling infection.

 

Table 2: Distribution of liver flukes according to animal risk factor (age and sex).

Factor	Category	Total No.	Positive		Risk	Odds Ratio
			No.	%
Age (Year)	< 1	117	0	0	0	0
	1-2	148	4	2.7	0.1	0.079
	>2-3	62	14	22.58	2.756	3.252
	>3	34	11	32.35 *	3.781 ****	5.096 ****
p-value			0.0426 S		0.0001 S	0.0001 S
Sex	Female	266	24	9.02	1.711 ****	1.781 ****
	Male	95	5	5.26	0.284	0.556
p-value			0.0621 NS		0.0001 S	0.0001 S

 

Significance (S) *; Non-significance (NS).

 

According to animal risk factors, values of positive rate, Odd ratio and risk were elevated significantly in sheep of >3 years old (32.35%, 3.781 and 5.096, respectively) in comparison to sheep aged >2-3 years (22.58%, 2.756 and 3.252, respectively), 1-2 years (2.7%, 0.1 and 0.079, respectively) and < 1 year (0%, 0 and 0, respectively), (Table 2). Worldwide, several researchers demonstrated that Fasciola infection and the risk of parasite have increased significantly with advancing age of study sheep and being higher at > 3 years (Ahmed et al., 2007; Rinaldi et al., 2015; Denizhan and Bİçek, 2018). This could explain because that either older animals might expose largely and frequently to source of infection or lowering resistance to infection at advanced ages. In contrast, the findings of another study found the lack of significant differences between the study age groups including <1 (8.33%), 1-2 (6.66%), and >2 (12.5%) years (Al-Lahaibi et al., 2023).

Concerning the sex of study sheep, no significant variation (P<0.0621) was seen between female (9.02%) and male (5.26%) sheep; however, females were appeared at higher risk of infection than males. These findings were similar with other researchers (Asadian et al., 2013; Denizhan and Bİçek, 2018; Al-Lahaibi et al., 2023). In contrast, other researchers found that females have higher rate of fasciolosis than males (Isah, 2019; Muhammad and Hassan, 2021). The increasing risk of fasciolosis in females might be attributed to either study female are older in age or immunity of shhep is attenuated due to stressful conditions of breeding and milk production.

Molecular examination using the conventional PCR assay indicated 93.1% (27 / 29) positive samples to Fasciola spp. including 91.3% (21/23) juvenile flukes and 100% (6/6) adult flukes (Table 3, Figure 1). Although, Fasciola species can be distinguished based on their morphological criteria, precise differentiation of two flukes is often difficult due to morphological variations among different isolates (Rokni et al., 2010; Yakhchali et al., 2015). In addition, Marcilla et al. (2002) thought that neither parasitological plus clinical tests nor immunological assays can differentiate between Fasciola species. Therefore, DNA-based diagnostic techniques can confirm morphometric tools and facilitate the task of species differentiation. This could because genotyping characters are not influenced by ecological and geographical factors. In addition, molecular methods can be used to understand the nature and extent of inter- and intra-specific variations in Fasciola species (Qureshi et al., 2019; Das et al., 2022).

 

Table 3: Total results of molecular examination of 29 suspected samples as Fasciola spp. by PCR.

Animal	Total No.	Positives [No. (%)]
		Total	Juvenile worms (Total no. 23)	Adult worms (Total no. 6)
Sheep	29	27 (93.1%)	21 (91.3%) *	6 (100%)
p-value			0.0482 S

 

Significance (S) *; Non-significance (NS).

 

 

In sheep, different genes have been used to identify Fasciola and confirming its species such as Cox1 (Muhammad and Hassan, 2021; Wang et al., 2022), ITS (Kobra et al., 2018; Alsulami et al., 2023), and 18S rRNA (Saadatnia et al., 2022; Sultana et al., 2022) genes. Sequencing data of five local Fasciola isolates were submitted in the NCBI-GenBank under the names of Fasciola hepatica IQ-1 Sheep isolate, Fasciola hepatica IQ-2 Sheep isolate, Fasciola hepatica IQ-3 Sheep isolate, Fasciola hepatica IQ-4 Sheep isolate, and Fasciola hepatica IQ-5 Sheep isolate. Phylogenetic tree analysis detected the presence of significant identity between the study Fasciola isolates and the NCBI-GenBank

 

Saudi Arabian (MN559388.1) F. hepatica at 99.72-99.78% (Figure 2) and a homology sequence identity at 99.76-99.78% (Table 4, Figure 3). These results demonstrated that F. hepatica is the most prevalent Fasciola species in sheep of Basra province as reported in other Iraqi (Koyee et al., 2023; Othman et al., 2023; Gharban et al., 2024) and global (Alsulami et al., 2023; Celik et al., 2023; Gröning et al., 2023) studies.

 

Table 4: Homology sequence identity between the local Fasciola isolates and the NCBI-GenBank Fasciola isolates.

Local isolate			NCBI-BLAST isolate
No.	Access No.	Band size	Country	Access No.	Identity
IQ. No. 1	PP779808.1	511 bp	Saudi Arabia	MN559388.1	99.72%
IQ. No. 2	PP779809.1	504 bp	Saudi Arabia	MN559388.1	99.78%
IQ. No. 3	PP779810.1	499 bp	Saudi Arabia	MN559388.1	99.72%
IQ. No. 4	PP779811.1	514 bp	Saudi Arabia	MN559388.1	99.72%
IQ. No. 5	PP779812.1	510 bp	Saudi Arabia	MN559388.1	99.78%

 

CONCLUSIONS AND RECOMMENDATIONS

Molecular and phylogenetic analysis of ovine liver flukes indicated worthily that F. hepatica was the more prevalent Fasciola species in sheep of Basra province. Also, our results provided an initial basic data for monitoring this potentially important parasite in field. However, the main limitations of the present study include the low number of examined animals, short period of study, and disapproving of some owners to examine their slaughtered animals. Therefore, it is necessary to develop the suitable parasite control measures (e.g. controlling the snails, judicious annual using of flukecide, frequent examination of fecal samples of field animals, and destroying of infected livers) and evaluation the local epidemiology of fasciolosis. Also, wider surveys including different areas and animals with using the advanced diagnostic assay in combination with morphology should be conducted.

 

ACKNOWLEDGMENTS

Authors thank the Head and Staff of the Department of Parasitology (College of Veterinary Medicine, University of Basrah) and all veterinarians who contributed in collection of liver flukes.

NOVELTY STATEMENT

The current study provided a new recent data about the molecular prevalence of ovine fasciolosis in Basra province (Iraq), and analysed phylogenetically the local isolates to detect their identity with the global isolate for first time in study areas.

AUTHOR’S CONTRIBUTIONS

IME: Gross examination of slaughtered sheep, collection of liver flukes, and phylogenetic analysis of local Fasciola spp. isolates. GYAA: Morphological examination of collected flukes, and statistical analysis of obtained results. ATA: Molecular analysis of collected flukes.

All authors have approved the final copy of the manuscript.

Funding

This study didn’t receive any funding support

Ethics Approve and Participant Consent

The current study was licensed by the Scientific Committee of the Department of Parasitology in the College of Veterinary Medicine (University of Basrah, Basra, Iraq).

Avaialability of Data and Materials

All obtained data were included in this manuscript.

Conflict of Interest

The authors have declared no conflict of interest.

REFERENCES

Abass KS, Ibrahim EK, Khalaf RN, Esmail RH (2018). Prevalence of liver fluke infections in slaughtered animals in Kirkuk Province. Iraq. J. Anim. Sci. Livest. Prod., 2 (2): 1-6. https://doi.org/10.21767/2577-0594.10005.

Abdulwahed TK, Al-Amery AM (2019). Morphological and molecular study of Fasciola spp. in sheep in Alkut city. Int. J. Biosci., 14 (1): 121-130. https://doi.org/10.12692/ijb/14.1.121-130.

Ahmed EF, Markvichitr K, Tumwasorn S, Koonawootrittriron S, Choothesa A, Jittapalapong S (2007). Prevalence of Fasciola spp infections of sheep in the Middle awash River Basin, Ethiopia. Southeast Asian J. Trop. Med. Public Health, 38 (1): 51-56.

Ali KN, Marif HF, Kakarash NA, Mohammed HO (2021). Liverfluke Coprological Cross-Sectional Survey in Cattle, Sheep and Goats in Sharazur District Kurdistan-Iraq. Al-Anbar J. Vet. Sci., 14 (1): 29-40. https://doi.org/10.37940/AJVS.2021.14.1.3.

Al-Lahaibi BY, Al-Taee AF, Suleiman EG (2023). Evaluation of Fasciola spp infection in cattle and sheep in Mosul city. Assiut Vet. Med. J., 69 (177): 27-33. https://doi.org/10.21608/avmj.2022.173875.1099.

Alsulami MN, Mohamed K, Wakid MH, Abdel-Gaber R, Timsah AG, Al-Megrin WAI, El-Kady AM (2023). Molecular Characterization of Fasciola hepatica in Sheep Based on DNA Sequences of Ribosomal ITS-1. Inf. Drug Resist., 2023: 6661-6671. https://doi.org/10.2147/IDR.S421206.

Amer S, ElKhatam A, Zidan S, Feng Y, Xiao L (2016). Identity of Fasciola spp. in sheep in Egypt. Parasit. Vectors, 9: 1-8. https://doi.org/10.1186/s13071-016-1898-2.

Asadian S, Mohebali M, Moudi M, EshratBeigom KIA, Heidari Z, Asgari M, Rokni MB (2013). Seroprevalence of human fascioliasis in Meshkin-Shahr district, Ardabil Province, northwestern Iran in 2012. Iran. J. Parasitol., 8 (4): 516-521.

Ayvazoğlu C, Kızıltepe Ş, Demir PA, Eser G (2023). Prevalence and Economic Importance of Fasciolosis in Slaughtered Sheep in Iğdır Province of Turkey. Euroasia J. Math. Eng. Nat. Med. Sci., 10 (26): 87-95. https://doi.org/10.5281/zenodo.7771735.

Beesley NJ, Williams DJ, Paterson S, Hodgkinson J (2017). Fasciola hepatica demonstrates high levels of genetic diversity, a lack of population structure and high gene flow: possible implications for drug resistance. Int. J. Parasitol., 47 (1): 11-20. https://doi.org/10.1016/j.ijpara.2016.09.007.

Beesley NJ, Caminade C, Charlier J, Flynn RJ, Hodgkinson JE, Martinez-Moreno A, Williams DJL (2018). Fasciola and fasciolosis in ruminants in Europe: Identifying research needs. Transbound. Emerg. Dis., 65: 199-216. https://doi.org/10.1111/tbed.12682.

Caravedo MA, Cabada MM (2020). Human fascioliasis: current epidemiological status and strategies for diagnosis, treatment, and control. Res. Rep. Trop. Med., 9: 149-158. https://doi.org/10.2147/RRTM.S237461.

Celik F, Simsek, S Selcuk MA, Kesik HK, Kilinc SG, Celik BA (2023). Cloning and expression of Fasciola hepatica enolase gene and efficacy of recombinant protein in the serodiagnosis of sheep fasciolosis. Vet. Parasitol., 320: 1-12 https://doi.org/10.1016/j.vetpar.2023.109961.

Constable PD, Hinchcliff KW, Done SH, Grünberg W (2016). Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs and goats. Elsevier Health Sciences.Pp: 641-645. 

Cwiklinski K, Dalton JP, Dufresne PJ, La Course J, Williams DJ, Hodgkinson J, Paterson S (2015). The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution. Genome Boil., 16, 1-13. https://doi.org/10.1186/s13059-015-0632-2.

Das S, Mandal SC, Baidya S, Pandit S, Jas R, Batabyal S, Chaudhury N (2022). First report of intra-species morphological variation of Fasciola gigantica from different host origins (cattle and buffalo). Indian J. Anim. Health, 61 (2): 294-299. https://doi.org/10.36062 /ijah.2022.10322.

Denizhan V, Bİçek K (2018). Seroprevalence of Fasciola hepatica in sheep in the province of Van and some districts. Atatürk Ünivers. Vet. Bilim. Derg., 13 (3): 278-284. https://doi.org/10.17094/ataunivbd.500867.

Forbes A (2017). Liver fluke infections in cattle and sheep. Livestock, 22 (5): 250-256. https://doi.org/10.12968/live.2017.22.5.250.

Gharban HA (2023). Molecular prevalence and phylogenetic confirmation of bovine trichomoniasis in aborted cows in Iraq. Vet. World, 16 (3): 580-587. https://doi.org/10.14202/vetworld.2023.580-587.

Gharban HA, Sray AH, Essa I.M (2024). Serological prevalence of anti-Fasciola hepatica antibodies in sheep. Egypt. J. Vet. Sci., 55 (6): 1583-1590. https://doi.org/10.21608/ejvs.2024.257463.1742.

Gröning H, Oksanen A, Skrzypczak T, Autio T (2023). A survey for antibodies against Fasciola hepatica in cattle and sheep in Finland indicates a low level of exposure. Acta Vet. Scand., 65 (1): 1-9. https://doi.org/10.1186/s13028-023-00688-9.

Hamoo RN, Al-Rubaye FSI, Mustafa NG (2019). Molecular characterization and phylogenetic analysis of Fasciola gigantica in Iraqi sheep using ITS1. Adv. Anim. Vet. Sci., 7: 256-260. https://doi.org/10.17582/journal.aavs/2019/7.4.256.260.

Isah UM (2019). Studies on the prevalence of fascioliasis among ruminant animals in northern Bauchi state, North-Eastern Nigeria. Parasite Epidemiol. Control, 5: 1-7. https://doi.org/10.1016/j.parepi.2019.e00090.

Itagaki T, Hayashi K, Ohari Y (2022). The causative agents of fascioliasis in animals and humans: Parthenogenetic Fasciola in Asia and other regions. Infect. Genet. Evol., 99: 1-11. https://doi.org/10.1016/j.meegid.2022.105248.

Jittapalapong S (2007). Prevalence of Fasciola spp infections of sheep in the Middle awash River Basin, Ethiopia. Southeast Asian J. Trop. Med. Public Health, 38 (1): 51-62.

Kajugu PE, Hanna REB, Edgar HW, McMahon C, Cooper M, Gordon A, Fairweather I (2015). Fasciola hepatica: Specificity of a coproantigen ELISA test for diagnosis of fasciolosis in faecal samples from cattle and sheep concurrently infected with gastrointestinal nematodes, coccidians and/or rumen flukes (paramphistomes), under field conditions. Vet. parasitol., 212 (3-4): 181-187. https://doi.org/10.1016/j.vetpar.2015.07.018.

Khanjari A, Bahonar A, Fallah S, Bagheri M, Alizadeh A, Fallah M, Khanjari Z (2014). Prevalence of fasciolosis and dicrocoeliosis in slaughtered sheep and goats in Amol Abattoir, Mazandaran, northern Iran. Asian Pacific J. Trop. Dis., 4 (2): 120-124. https://doi.org/10.1016/S2222-1808(14)60327-3.

Kobra PIRI, Saidijam M, Maghsood A, Matini M, Fallah M (2018). Prevalence of animal fasciolosis and specification of Fasciola spp. isolated from sheep, goats and cattle by molecular method: Hamadan province, west of Iran. Iran. J. parasitol., 13 (4): 524-532.

Koyee QM, Khailany RA, Rahman ML, Nassraldin LN (2023). Histopathologic Changes and Molecular Characterization of Fascioliasis (a Zoonotic Disease) among Slaughtered Livestock in Erbil and Halabja Abattoirs, Kurdistan Region-Iraq. Baghdad Sci. J., 21 (7): 1-19. https://doi.org/10.21123/bsj.2023.9099.

Kusumarini SR, Permata FS, Widyaputri T, Prasetyo D (2020). Prevalence of fasciolosis emphasis on age, origin, body condition and post mortem by geographic information systems on sacrificial examination in Malang District–East Java. J. Phys., 2020 (1): 1-8. https://doi.org/10.1088/1742-6596/1430/1/012025.

Lalor R, Cwiklinski K, Calvani NED, Dorey A, Hamon S, Corrales JL, De Marco Verissimo C (2021). Pathogenicity and virulence of the liver flukes Fasciola hepatica and Fasciola gigantica that cause the zoonosis Fasciolosis. Virulence, 12 (1): 2839-2867. https://doi.org/10.1080/21505594.2021.1996520.

Madsen H, Stauffer JR (2022). Zoonotic trematode infections; their biology, intermediate hosts and control. In Parasitic Helminths and Zoonoses-From Basic to Applied Research. IntechOpen. https://doi.org/10.5772/intechopen.102434.

Marcilla A, Bargues MD, Mas-Coma S (2002). A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica. Mol. Cell. Probes, 16 (5): 327-333. https://doi.org/10.1006/mcpr.2002.0429.

Moazeni M, Ahmadi A (2016). Controversial aspects of the life cycle of Fasciola hepatica. Exp. Parasitol., 169, 81-89. https://doi.org/10.1016/j.exppara.2016.07.010.

Muhammad MJ, Hassan ZI (2021). Molecular Diagnosis of Fasciola hepatica in livestock using cox1 gene in Erbil Provence-Kurdistan Region/Iraq. Zanco J. Pure and Appl. Sci., 33 (4): 36-42. https://doi.org/10.21271/ZJPAS.33.4.4.

Nerway CA, Mero WMS, Mohammed AB (2021). Prevalence of Fasciola spp. among slaughtered livestock in Zakho city, Duhok governorate-Iraq. Acad. J. Nawroz Univer., 10 (2): 199-206. https://doi.org/10.25007/ajnu.v10n2a1034.

Othman VS, Hama AA, Zorab RH, Dalimi A (2023). Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq. Iran. J. Parasitol., 18 (4): 554-562. https://doi.org/10.18502/ijpa.v18i4.14264.

Phalee A, Wongsawad C, Rojanapaibul A, Chai JY (2015). Experimental life history and biological characteristics of Fasciola gigantica (Digenea: Fasciolidae). Korean J. Parasitol., 53 (1): 59-64. https://doi.org/10.3347/kjp.2015.53.1.59.

Qureshi AW, Khan ZU (2019). Molecular Characterization of Fasciola isolates from Sheep and Goat, Based on ITS1 Region amplification, in District Mardan, Pak. Saudi J. Life Sci., 4 (1): 28-32. https://doi.org/10.21276/haya.2019.4.1.3.

Raoof HS, Marif HF, Rahman HS, Omar M, Sheikh B, San Ahmed AM (2020). Molecular characterization and phylogenetic analysis of Fasciola species in sheep and goats in Sulaymaniyah Province, Northern Iraq. J. Zankoy Sulaimani, 22 (1): 297-305.

Regasa A, Seboka M (2021). Review on Fasciolosis, its effect on meat quality/hazards and economical importances. Entomol. Ornithol. Herpetol., 10: 1-9. https://doi.org/10.35248/2161-0983.21.10.247.

Rinaldi L, Biggeri A, Musella V, De Waal T, Hertzberg H, Mavrot F, Catelan D (2015). Sheep and Fasciola hepatica in Europe: the GLOWORM experience. Geospatial Health, 9 (2): 309-317. https://doi.org/10.4081/gh.2015.353.

Rokni MB, Mirhendi H, Mizani A, Mohebali M, Sharbatkhori M, Kia EB, Izadi S (2010). Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method. Exp. Parasitol., 124 (2): 209-213. https://doi.org/10.1016/j.exppara.2009.09.015.

Saadatnia A, Solhjoo K, Davami MH, Raeghi S, Abolghazi A (2022). Molecular identification of Fasciola isolated from the liver of meat animals in Fars Province, Iran. J. Parasitol. Res., 2022: 1-18. https://doi.org/10.1155/2022/4291230.

Shende VH, Maske DK, Jayraw AK, Baviskar BS (2007). Epidemiology of caprine gastrointestinal helminthic infection in central zone of Vidarbha region, Maharashtra state. J. Parasit. Dis., 31 (2): 134-136. https://doi.org/10.1007/s12639-014-0581-9.

Stuen S, Ersdal C (2022). Fasciolosis-an increasing challenge in the sheep industry. Animals, 12 (12): 1491-1507. https://doi.org/10.3390/ani12121491.

Sulieman BM, Mahmood OI, Thamer IK (2021). Diagnosis of infection with hepatic worms in sheep in Salah al-Din governorate. Tikrit J. Pure Sci., 26 (2): 17-21. https://doi.org/10.25130/tjps.26.2021.023.

Sultana N, Pervin M, Sultana S, Mostaree M, Mumu TT, Khan MAHNA (2022). Fascioliasis may promote tuberculous infectivity in small ruminants. Saudi. J. Biol. Sci., 29 (10): 1-11. https://doi.org/10.1016/j.sjbs.2022.103402.

Wang X, Zhang K, Guowu Z, Li Z, Yunxia S, Chengcheng N, Xuepeng C (2022). Molecular characteristics and genetic diversity of Fasciola hepatica from sheep in Xinjiang, China. J. Vet. Res., 66 (2): 199-207. https://doi.org/10.2478/jvetres-2022-0018.

Webb CM, Cabada MM (2018). Recent developments in the epidemiology, diagnosis, and treatment of Fasciola infection. Curr. Opin. Infect. Dis., 31 (5): 409-414. https://doi.org/10.1097/QCO.0000000000000482.

Yakhchali M, Malekzadeh-Viayeh R, Imani-Baran A, Mardani K (2015). Morphological and molecular discrimination of Fasciola species isolated from domestic ruminants of Urmia city, Iran. J. Parasitol., 10 (1): 46-58.

Zerna G, Spithill TW, Beddoe, T (2021). Current status for controlling the overlooked caprine fasciolosis. Animals, 11 (6): 1819-1834. https://doi.org/10.3390/ani11061819.