The PCR amplification of ITS-1 and ITS-2 rDNA yielded fragments of approximately 970 bp in length (Fig. 2). The 59 PCR amplicons of ITS-1 and ITS-2 were subjected to direct sequencing which yielded sequences of 946 bp in length. The sequence was composed of the complete ITS-1 sequence of 422 bp, complete 5.8S sequence of 158 bp, and complete ITS-2 sequence of 366 bp. Out of the 59 Fasciola samples sequenced, 17% were F. hepatica and 83% were F. gigantica, there were no intermediate forms of Fasciola species. Fasciola samples collected from cattle were F. hepatica 2 (9.5%), F. gigantica 19 (90.5%), sheep F. hepatica 8 (36.4%), F. gigantica 14 (63.6%) and all samples from goats were F. gigantica.
The sequences were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers MZ396875, MZ396876, MZ396877, MZ396878, MZ396885, MZ396925, MZ396926, MZ396928, MZ396929, MZ3969332 for Fasciola hepatica and MZ396874, MZ396879- MZ396884, MZ396886- MZ396924, MZ396927, MZ396930, MZ396931 Fasciola gigantica. The sequences were aligned with previously published Fasciola ITS-1, 5.8S and ITS-2 sequences retrieved from GenBank accession numbers JF432073, MK321604, JF496711, AM900371, MW046875, JF496709,KJ789338, HM746787, HQ197358, KF543340, AJ853848, JF432072, KJ789364, HM746785, MK321597, JF432075, GQ231547, AM709647, AM850107 and AM709621 (Alasaad et al. 2007; Ali et al. 2008; Farjallah et al. 2009; Rokni et al. 2010; Amor et al. 2011; Galavani et al. 2016; Evack et al. 2020; Omar et al. 2021).
The sequences of ITS-1 showed that F. hepatica differed form F. gigantica in five variable nucleotides, while the ITS-2 sequences of the examined F. hepatica was different from F. gigantica ITS-2 in seven nucleotides (Table 2). Among the 59 sequenced Fasciola isolates there was no nucleotide variation in the ITS-1 sequences. In ITS-2 sequences there was no nucleotide variation among F. hepatica but nucleotide variation was observed at two nucleotides of F. gigantica at sequence position 842 and 936, two and three Fasciola isolates respectively. This indicates the presence of two genotypes of F. gigantica isolates that were examines. Sequence variation in position 842 was examined only in Narok among sheep and cattle, while in position 936 it was examined in Fasciola samples from the three localities and it occurred in cattle and goat.
Table 2
Comparison of nucleotides at variable sites in ITS-1 and ITS-2 sequences of Fasciola from different hosts and geographical locations
Species | Variable sites in ITS-1 and ITS-2 sequences | Accession numbers | Location |
ITS-1 ITS-2 |
18 | 108 | 202 | 280 | 300 | 815 | 842 | 854 | 860 | 911 | 918 | 936 |
F. gigantica | T | T | T | A | T | C | G | T | T | - | A | G | JF432073 | Iran |
| T | T | T | A | T | C | G | T | T | - | A | G | MK321604 | Chad |
| T | T | T | A | T | C | G | T | T | - | A | G | JF496711 | China |
| T | T | T | A | T | C | G | T | T | - | A | G | AM900371 | Niger |
| T | T | T | A | T | C | G | T | T | - | A | G | MW046875 | Zimbabwe |
| T | T | T | A | T | C | G | T | T | - | A | G | MZ396874 | This study |
| T | T | T | A | T | C | C | T | T | - | A | G | MZ396918 | This study |
| T | T | T | A | T | C | C | T | T | - | A | G | MZ396931 | This study |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396912 | This study |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396916 | This study |
| T | T | T | A | T | C | G | T | T | - | A | T | MZ396919 | This study |
F. hepatica | C | A | C | T | C | T | G | C | C | T | G | G | AM709647 | Spain |
| C | A | C | T | C | T | G | C | C | T | G | G | AM850107 | Niger |
| C | A | C | T | C | T | G | C | C | T | G | G | JF432072 | Iran |
| C | A | C | T | C | T | G | C | C | T | G | G | JF708027 | China |
| C | A | C | T | C | T | G | C | C | T | G | G | MZ396875 | This study |
Phylogenetic tree was constructed for the analysis of phylogenetic diversity of the liver flukes, using ITS1 and ITS2 sequences of F. gigantica and F. hepatica from this study along with available sequences in GenBank from other regions (Fig. 3). Fascioloides magna was used as an outgroup GenBank accession number EF534991 (Khalifa et al. 2016).