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 | July 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.

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