Molecular characterization and phylogenetic studies of Echinococcus granulosus and Taenia multiceps coenurus cysts in slaughtered sheep in Saudi Arabia

Abstract Taeniids, consisting of two genera Echinococcus and Taenia, are obligatory tapeworms of mammals, and their pathogenicity was due to infection with larval stages. Hydatid (the larval stage of Echinococcus granulosus) and coenurus (the larval stage of Taenia multiceps) cysts are prevalent in domestic, wild ruminants, livestock, swine, and dogs, and accidentally they could also be found in humans. They lead to different clinical manifestations that cause economic loss in livestock and human morbidity. In Saudi Arabia, few studies were performed on hydatid and coenurus cyst genetic variations. The main goal of the present study was to identify E. granulosus and T. multiceps cyst isolates collected from slaughtered Harri sheep in Saudi Arabia by partial sequencing with PCR amplification of the cytochrome C oxidase 1 (COX1) gene. Molecular and phylogenetic evaluation based on COX1 sequences indicated that cyst isolates belong to E. granulosus and T. multiceps, respectively, successfully submitted in NCBI Genbank. Molecular characterization showed a low nucleotide diversity with two submitted isolates of coenurus with related isolates of Genbank. Conversely, E. granulosus isolates showed higher nucleotide diversity. The reported data could serve as a foundation for future molecular epidemiological and biological studies.


Introduction
Taeniid tapeworms belong to subclass Eucestoda, order Cyclophyllidea, and family Taeniidae, and they represent zoonotic parasites of mammals and livestock. Family Taeniidae consists of two genera, Echinococcus and Taenia, and they are mainly focused on for their socioeconomic impact by causing human morbidity and loss of livestock [1,2].
Echinococcus granulosus causes hydatidosis disease due to its larval stage infection. Hydatidosis (cystic echinococcosis) is widely distributed, and its prevalence varies according to climate and their contact with livestock [3]. Hydatid cysts have been reported in different livestock, including sheep, camels, cattle, and goats, with various incidence rates, resulting in significant economic losses [4][5][6]. Recently, it was recorded that the monetary burden of surgically treated human cystic echinococcosis increased that differs from one country to another [7]. In addition, it was reported that about 2.5% of humans infected with hydatidosis died after resorting to surgery [8]. There are three possibilities for cystic echinococcosis management: medical treatment with albendazole, percutaneous procedures, and surgery that depends on cyst stage, state, and organ location [9].
Coenurus represents the infective larval stage of Taenia multiceps; they are also widely distributed mainly in the tropical countries in Africa, Asia, and the Middle East [10]. T. multiceps are found as an obligate intestinal tapeworm of dogs, also commonly found in sheep and goats, as an intermediate host for the parasite, but the infection was extended to cattle and humans [11]. Mainly, coenurus cysts present in the brain in either acute or chronic form also could be frequently found in intramuscular and subcutaneous tissues [12][13][14]. Coenurosis leads to different clinical manifestations according to the location and extent of the coenuri. Coenurus cysts in the brain cause fever, muscle tremors, hemorrhagic retinal lesions, paralysis, ataxia, blindness, nystagmus, dysmetria, and scoliosis [15,16]. However, coenuri cysts in the subcutaneous and intramuscular tissues damage the functioning of normal organs and lead to muscular pain [11].
Due to the veterinary and medical significance of both E. granulosus and T. multiceps, there has been an intensive focus on their ecological, epidemiological, and taxonomic studies. Molecular characterization among many species found within Taeniidae, especially for the genus Echinococcus, has been well documented, but this was scarce with coenurus. However, there are very few documented studies on genetic variability regarding Taeniids in Saudi Arabia.
The present study aimed to (i) genetically characterize hydatid (larval stage of E. granulosus) and coenurus (larval stage of T. multiceps) cysts isolated from slaughtered Harri sheep in Taif, Saudi Arabia, by using mitochondrial cytochrome C oxidase 1 (COX1) and NADH dehydrogenase subunit 1 (NAD1) genes. Also, the study aimed to (ii) report the genetic relationship between selected genes loci of Taif isolates and other isolates present in other countries in Genbank.

Sample collection
The present study was carried out on Harri sheep from an animal slaughterhouse in Taif governorate (21.2819°N, 40.3841°E), Mecca Province, Saudi Arabia. During postmortem veterinary examination, meat was systematically inspected for hydatid and coenurus cysts occurrence by applying procedures of the routine meat inspection (sampling period from January to October 2020). Hydatid cysts were found in muscles, liver, and viscera, but coenurus cysts were found only in muscles. Therefore, we have collected samples from 20 sheep (gender, female; age, 1-2 years), about 200-300 g of muscles from each animal, which contains either hydatid or coenurus cysts from slaughtered sheep under permission from the Ministry of Environment, Water and Agriculture (KSA). Each cyst from a different animal was labeled and handled as a different isolate before carrying out the examination.

Microscopic examination
Ten samples of hydatid cysts (of E. granulosus) and the other ten samples of coenurus cysts (of T. multiceps) were collected from animal meat and carefully opened with the help of a sterile scalpel. First, protoscolices of hydatid and coenurus cysts were scraped off the inner wall of their cysts. Then, protoscolices were loaded on glass slides, covered, and examined under a light microscope without staining.

DNA extraction of protoscolices
About 300 µL of lysis buffer TNES (10 mM Tris, 400 mM NaCl, 100 mM Na 2 EDTA, 0.6% SDS, pH 7.5) was added to hydatid protoscolices in a microtube; then samples were frozen (in liquid nitrogen) and thawed (5×). To facilitate sample digestion, samples were ground before proteinase K (8 µL, 20 mg/mL) and incubated overnight at 37°C. Next, phenol/chloroform (100 µL) was added to the sample and centrifuged (16,500 g) for 5 min, and then, the clear upper phase was transferred into a new 1.5 mL Eppendorf. Next, for DNA precipitation, the same volume of absolute ethyl alcohol and sodium acetate (1%, 3 mol) was added to the sample and stored overnight (−20°C). Finally, samples were centrifuged (16,500 g) for 5 min, the supernatant was discarded, the pellet containing DNA was left to dry, and the pellet was finally dissolved in 100 µL sterile deionized water [17]. DNA was extracted from coenurus protoscolices with the same previous procedure using TNES lysis buffer and phenol/chloroform, but there was no need for freezing, thawing, or grinding.

Sequencing and phylogenetic evaluation
Different PCR products of COX1 and NAD1 of E. granulosus and T. multiceps cysts were randomly selected and subjected to sequencing using an ABI Prism 3730 Genetic Analyzer automated sequencer. Four sequences of COX1 regions (two of each hydatid and coenurus cysts) were directly submitted in NCBI Genbank to have accession numbers. Sequences of COX1 and NAD1 were aligned with different sequences in Genbank using online NCBI BLAST, phylogenetically estimated, and finally viewed as rectangular cladogram in the phylogenetic Tree View [19].

Morphological and microscopic examination of cysts
Hydatid and coenurus cysts appeared as fluid-filled round/ oval sacs with different sizes embedded in the muscles of sheep. Protoscolices of both cysts were carefully scraped off the inner wall of their cyst rather than collecting them from cysts' inner fluid. E. granulosus protoscolices appeared as an oval body with an invaginated scolex provided with several hooks (Figure 1). Visual examination showed numerous scolices in the inner wall of coenurus cysts. Microscopically, the isolated protoscolex showed both large and small rostellar taeniid hooks and four scup-shaped suckers ( Figure 2).

PCR, sequencing, and phylogenetic analysis
Mitochondrial DNA was used to amplify two separate gene loci, COX1 and NAD1, for both E. granulosus and T. multiceps cysts to yield amplicons of 446 bp and 520 bp, respectively. The present results report the success of PCR amplification for both selected portions (Figures 3 and 4). However, NAD1 amplification shows other lower nonspecific bands.
Randomly selected PCR products from the two loci were sequenced by using their forward/reverse primers. Sequences of COX1 were deposited in Genbank and have been assigned different accession numbers: E. granulosus (COX1) MZ345697. 1

Discussion
Cystic echinococcosis and coenurosis are zoonotic diseases that affect humans and livestock due to infection with the larval stages of Echinococcus granulosus and of Taenia multiceps cysts, leading to economic loss that increases annually [4,9,11]. Dogs, especially stray unvaccinated dogs, are definitive hosts for different cestodes of Taeniidae family, including E. granulosus and T. multiceps. Dogs were commonly associated with extensive livestock herding to protect domestic animals against predators that could lead to their infection [20,21]. For the first time, the present study reports the genetic characterization of two different parasite cysts, hydatid (the larval stage of E. granulosus) and coenurus (the larval stage of T. multiceps), in Taif governorate, Makkah Province, Saudi Arabia.
Sheep, goats, cattle, and camels represent the primary livestock species producing red meat in Saudi Arabia, with an estimated total population of 13,444,435 heads [22]. Sheep represent the significant population (72%) of the livestock, and they import large numbers of sheep to satisfy the needs of Saudi citizens. Awassi, Harri, and Najdi are the most available sheep species in Saudi Arabia [23]. Any endemic infection for livestock, such as hydatidosis and coenurosis, could lead to economic setbacks; therefore, there is a need to highlight their study, especially in KSA.
Hydatidosis and coenurosis due to E. granulosus and T. multiceps larval infection, respectively, cause significant damage in livestock that leads to production losses, especially in endemic areas [2,24]. Moreover, these parasites were distributed worldwide in different intermediate hosts, especially in sheep and goats, accidentally humans, reflecting their medical and veterinary importance [25].
The present study aims to identify two Taeniids parasites found in slaughtered Harri sheep meat based on COX1 and NAD1 partial sequencing and phylogenetic relationship. It was reported that the development of molecular techniques rather than traditional morphological criteria has provided improved tools for taeniid species identification and investigating relationships among them. Sequencing of mitochondrial DNA, especially COX1 and NAD1 loci, has been successfully used for molecular characterization and identification of taeniids tapeworm [26,27].
Our choice to use mitochondrial DNA (mtDNA) sequence, especially COX1 and NAD1, for molecular characterization of E. granulosus and T. multiceps larva was based on the previous assessment. mtDNA is widely used in molecular characterization and phylogenetic evaluation studies. COX1 gene represents the most common gene of mtDNA for phylogenetic analysis and evolutionary biology of helminth parasites to determine interspecific and/or intraspecific variation [8,[28][29][30]. The mitochondrial COX1 gene is very suitable for genetic diversity detection and haplotype analysis. Its evolutionary change rate is slow enough for the same species but fast enough for differentiation between different species. Therefore, the mitochondrial COX1 gene has been selected for use in the creation of DNA barcodes and species differentiation of different helminths as our present study [31,32].
Worldwide, there have been well-documented research studies about the genetic variability among many species found within the Taeniidae, especially for the genus Echinococcus. Still, there are a few related species of Taenia, especially for T. multiceps [33]. However, there is a scarcity of studies related to the genetic characterization of E. granulosus and T. multiceps larval stages in KSA and their phylogenetic relationship.
In the current study, a genetically characterized sample based on COX1 isolated from a hydatid cyst of MZ345697.1 showed identity about 94.95% with the other isolate MZ350810.1. For coenurus cyst, isolate1 MZ346598.1 showed 97% homology with the second isolate MZ348363.1. In addition, comparing partial sequences of Taif COX1 isolates from both cysts showed different identities with other isolates from organs present in various hosts found in other countries. The present results are inconsistent with previous studies, in which Al-Hizab et al. [34] and AL-Mutairi et al. [35] have reported E. granulosus species variation based on molecular characterization in infected sheep and camels in the Arabian Peninsula and KSA. AL-Mutairi et al. [35] have reported the phenotypic and genetic characterization of hydatid cysts isolated from sheep and camel meat in Al-Madinah, KSA. Genetic characterization of E. granulosus was based on random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for the whole genome, PCR amplification of COX1 and 12S rRNA genes followed by single-stranded conformation polymorphism (SSCP), and then sequencing. They have investigated that cyst isolates slightly vary from each other and other isolates found in Genbank. They have concluded that there is an intraspecific variation in E. granulosus found in camels and local sheep.
In addition, Christodoulopoulos et al. [36] have investigated COX1 nucleotide diversity of coenurus cyst isolates from sheep and goats in Pakistan, and their phylogenetic analysis shows high related homology with China isolates but highly different from other isolates from different countries. They have reported that coenurus cerebralis commonly found in the brain (cerebral form) could also be found in other non-cerebral tissue such as intramuscular and subcutaneous tissues.
Diversity in the nucleotide sequences of the presently studied isolates was recorded before within the same region, such as in Iran [5,13], China [26], and Italy [37,38]. Most of their phylogenetic analysis depends on one or more mtDNA sequences such as nad1, cox1, and 12S rRNA. They have concluded that there are no monophyletic groups based on the intermediate host species, the organ from which the parasite was isolated, and geographical origin [33,36]. However, reporting parasite characterization and genetic identification will be crucial for controlling and preventing parasitic infections [8].
The limitation of this study is the use of the limited sample size, and only partial sequencing was done for the COX1 gene that could have implications on data interpretation. Thus, we recommend future studies considering a larger sample size, complete sequencing COX1, and other characterization-related genes in a massive epidemiological survey for further evaluation.

Conclusion
The present study reports the molecular characterization of hydatid (the larval stage of E. granulosus) and coenurus (the larval stage of T. multiceps) cysts present in slaughtered sheep meat in Taif, KSA. We have determined sequences differences within isolates of the same species that we submitted in GenBank and other isolates found in Genbank from different host organs in different countries. For a more precise identification and characterization of Taeniids in KSA, additional isolates from other hosts, other geographic areas, different molecular protocols, long-sequenced genes in the mitochondrial and nuclear genome, and further information concerning biological characteristics may be necessary to increase our understanding of the epidemiological distribution in KSA.
Acknowledgments: This work was supported by Taif University Researchers Supporting Project number (TURSP-2020/299), Taif University, Taif, Saudi Arabia. The authors would like to thank the Ministry of Environment, Water and Agriculture in Taif governorate (KSA) for facilitating data and sample collections. Ethics approval: Data and samples were collected from veterinarians as per their periodic examination according to the ministerial recommendation. We have a permission letter from the Ministry of Environment, Water, and Agriculture (KSA) to collect cattle samples from different locations in Taif for our research study with the number 106082/1074/1442, dated 21-02-1442H. Sample collection and all experimental procedures were performed in accordance with a national ethical requirement. National Committee of Bioethics (NCBE) at King Abdulaziz City for Science and Technology (KACST) with number: 10023117, valid till October 1, 2023. , Data availability statement: The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.