Genetic variation of Taenia saginata cyst isolates from Iraq based on mitochondrial COX1 sequences

Cysticercus bovis, the larval stage of T. saginata, causes bovine cysticercosis. Cattle get infected with T. saginata by ingesting the eggs excreted from humans infected with T. saginata. Humans infected by consuming beef infected with larval cysticercus of T. saginata (C. bovis) (Abusier et al., 2007; Ogunremi &Benjamin, 2010). A bovine carcass infected with C. bovis might contaminate 8 – 20 individuals (Sato et al., 2018). T. saginata is found worldwide and affects developing and industrialized countries (Dorny et al., 2000; Silva & Costa-Cruz, 2010). Cysticercosis usually results in few clinical signs or is asymptomatic, especially if the infection is mild. On the other hand, cases are accountable for significant economic losses in the meat industry (WHO, 2005; Torgerson, 2013). Typically, cysticercosis is diagnosed through macroscopic examination throughout carcasses’ post-mortem inspections; yet, the approach was criticized for its low sensitivity in the detection of cysticercosis (Geysen et al., 2007) and diagnostic competence (Abuseir et al., 2006). Molecular methods, like PCR, have excellent specificity and sensitivity, allowing for accurate differentiation and identification of Taenia species while overcoming various drawbacks of traditional approaches (Yamasaki et al., 2004; Gonzalez et al., 2004; Sato et al., 2018).

Advanced tools for detecting and researching the relation between taeniid species have been developed thanks to the introduction of molecular genetic approaches. Mitochondrial DNA sequencing has proven helpful in identifying and genetically characterizing such parasites (Bowles & McManus, 1994). Mitochondrial genes, particularly COX1, are widely used markers for the identification of the helminth parasite (Gasser et al., 1999). In addition, the parasites’ genetic population structure can help with epidemiological investigations and focus on their evolutionary history (Campbell et al., 2006; Anantaphruti et al., 2013). Knowing the parasite’s population variations aids in the analysis of transmission patterns (Pajuelo et al., 2017).

Several studies have looked into human taeniid tapeworm genetic variations, but most studies focused on T. solium and human cysticercosis (Rostami et al., 2015). There’s a scarcity of knowledge on genetic variation in T. saginata from various world regions. In 2007, the whole mitochondrial genome of T. saginata was reported (Jeons et al., 2007). T. saginata was genetically characterized in Ethiopia and Thailand (Okamoto et al., 2010; Hailemariam et al., 2014). In surrounding countries, Jahed Khaniki et al. (2009) recorded a low rate of C. bovis infection in Iran (0.25 %), as well as Kus et al. (2014) reported that the prevalence of infection in Turkey ranged from 0.3 to 30 %.In Iraq, thorough molecular investigations are necessary to improve our understanding of such species’ genetic diversity and to develop an efficient parasite vaccine. In Iraq, epidemiological data are scarce, and the available pieces of literature are few; the prevalence of bovine cysticercosis in cattle, buffaloes, and taeniasis in humans were recorded in Iraqi province (San & Zana, 2017; Al-Jadar & Hayatee 1988; Kadir & Salman, 1999; Musa, 2017; Al-Saqur et al., 2020).Because genetic information on Iraqi T. saginata isolates is scarce, this study was performed using COX1 sequences to analyze the intra-specific variation of T. saginata isolates acquired from cattle in the Sulaymaniyah province of Iraq.

From December 2020 until May 2021, 37 T. saginata cysts were obtained from cattle in the Modern Sulaimani Slaughterhouse in the Sulaymaniyah district of Iraq. The specimens were disinfected and preserved using 70 % ethyl alcohol. Following the manufacturer’s instructions, genomic DNA was extracted from each scolex using the EasyPure^TM Genomic DNA kit (Trans Gen Biotech Co., China) and stored at -20°C. JB3 (forward): 5′-TTTTTTGGG-CATCCTGAGGTTTAT-3′ and JB4.5 (reverse):5′-TAAAGAAAGAA-CATAATGAAAATG-3′ were employed for amplifying a 400-bp-long mitochondrial COX1 gene fragment (Bowles et al., 1992). The amplification was carried out with f-Pfu DNA polymerase (SBS Genetech Co., China) under the same circumstances as Rostami et al. (2015).

SiMax PCR Products/Agarose Gel Purification Kit was used to purify the purified DNA fragments from agarose gel (SBS Genetech Co.). An ABI -3730XL capillary machine was used to sequence the purified DNA (Macrogen Inc., South Korea). In BioEdit software, sequences were aligned with ClustalW multiple sequence alignments (Hall, 1999). Also, the representative COX1 nucleotide sequences have been submitted to the NCBI and are accessible in the Gen-Bank database under the accession numbers OK036447 – OK036451.Using the maximum likelihood method, a phylogenic tree was generated based on the reference sequences of T. saginata and related species (Supplementary Table 1). Genetic distances were estimated using Kimura’s 2-parameter model, and the tree topology’s robustness was assessed using a bootstrap value of 1000 repetitions using datasets available in MEGA 7 version 7. (Kumar et al., 2016). The maximal composite possibility model has been utilized to calculate the evolutionary divergence regarding nucleotide sequences of T. saginata from the present and previously published studies for further analysis (Kumar et al., 2016).

The Ethical Committee approved the study protocol of the University of Sulaimani’s Veterinary Medicine College. All the samples used in this study were taken post-mortem from discarded infected carcasses unfit for human consumption. No animals were killed in the course of this investigation.

The COX1 gene was successfully amplified in all 37 samples. A 384-bp fragment was obtained after the sequences’ trimming and editing. The COX1 gene sequence analysis revealed five distinct haplotypes were identified, designated as IQTS-H1 (n=17), IQTS-H2 (n=8), IQTS-H3 (n=6), IQTS-H4 (n=4), and IQTS-H5 (n=2) (Supplementary Table 2). The pairwise evolutionary divergence between different COX1 haplotypes has been found to range between 0.005 – 0.013, whereas the available nucleotide variation among all five haplotypes is 0.000 – 0.018 (Table 1). In total, nine mutations in the COX1 gene were found in nine segregation sites. The Maximum likelihood approach was used to create a phylogram based on COXI gene sequences. Phylogenetic relationships revealed that all T. saginata haplotypes had been clustered in a single clade, with Korean and Iranian isolates sharing a high degree of closeness (Fig. 1).

Fig.1

Phylogenetic studies of distinct Taenia species have applied various genomic areas, such as 18-S and 28-S ribosomal RNA, in addition to the mitochondrial genes (Hoberg, 2006; Yan et al., 2013). In addition, sequence fragment analysis depending on PCR synthesis of such taeniids’ DNA is one of the molecular methods frequently employed for phylogenetic investigations (Nickish-Rosenegk et al., 1999). Gonzalez et al. (2011) sequenced the appropriate sequences from all taeniid isolates after PCR-amplifying them using particular primers.

This work studied the mitochondrial COX1 gene diversity of 37 T. saginata cattle specimens from Sulaymaniyah, Iraq. COX1 sequence analysis revealed nine nucleotide substitutions, three of which have been nucleotide transversions, in five T. saginata haplotypes. The haplotypes detected eight amino acid alterations attributable to eight nucleotide substitutions (Table 2). There is no indication that cytochrome c oxidase amino acid composition changes impact the parasite adaptability or enzyme’s function. Yet, it was demonstrated in other parasite species that a single amino acid change could influence the biological fitness of an organism (Tachibana et al., 2004; Otsuki et al., 2009). COX1 nucleotide variation was determined to be 0.027 – 0.134 across T. saginata isolates from this investigation and six other Taenia species. Bowles and McManus (1994) and Rostami et al. (2015) calculated the predicted nucleotide variations in COX1 in genus Taenia to be 0.025 – 0.158 and 0.026 – 0.141, respectively. Compared to other haplotypes of such taeniid, COX1 of T. saginata showed a relatively low degree of variation. Similar results were observed by Abuseir et al. (2018) in Germany. In contrast, other studies on the genetic divergence of T. saginata in Asia have revealed considerably higher haplotype diversity in the COX1 gene (Anantaphruti et al.,2013; Sanpool et al., 2017). Furthermore, pairwise comparisons of T. saginata from the experiment with existing mitochondrial sequences from Iranian (Rostami et al., 2015) and Korean (Jeon et al., 2007) cattle revealed 0.000 – 0.018 and 0.000 – 0.008 nucleotide differences in COX1 gene, respectively (Table 1). As a result, the phylogram revealed that the Iraqi T. saginata in this study was equivalent to other T. saginata, sharing 98.18 – 99.74 % identity with the ones from Iran and Korea. The phylogenetic analysis produced a dendrogram clustered all COX1 haplotypes uniformly to a single clade with a T. saginata reference sequence (AY684274 accession number). As distinct sub-clade, other types of the taeniids, including Echinococcus granulosus, T. solium, T. multiceps, T. asiatica, and T. hydatigena, were grouped together.

Understanding the control and epidemiology of parasitic infections requires molecular characterization of veterinary and medical significance parasites. T. saginata can be defined as one of the human and cattle zoonotic parasites with a global range (Rostmai et al., 2015). In the molecular epidemiological surveys of the echinococcosis/taeniasis in various host assemblages and geographical settings, DNA methods are often utilized to identify Echinococcus and Taenia species, strains, and subspecies (McManus, 2006).

The current study added new data about T. saginata mt-DNA cattle haplotypes in Iraq. T. saginata was made up of five haplotypes clustered in a single clade, according to phylogenetic analysis of the COX1 gene calculated using maximum likelihood. Four new strains with new mutations have been discovered in the research area. More thorough research on nuclear genes is needed to fully understand the amount and relevance of genetic variations within populations of the T. saginata.