PCR‐RFLP assay confirms the existence of different mitochondrial lineages of Taenia hydatigena including a possible geographically restricted group

Abstract

Taenia hydatigena is a widespread tapeworm of canids (primarily dogs) that causes cysticercosis in ruminants (domestic and wild) and manifests as depression and weakness secondary to various hepatic damages and sometimes mortality in young animals, although, commonly encountered cases are asymptomatic. In most taeniids, genetic polymorphism has been found to impact host preferences, distribution, disease epidemiology and management. Recently, we identified two main mitochondrial lineages of T. hydatigena in China, and here, we examined the mitochondrial nad4‐nad5 genes of T. hydatigena from China, Nigeria, Pakistan and Sudan to assess the intraspecies variation of isolates from these countries and also the distribution of the distinct mitochondrial groups. In addition to China, haplogroup B variant was found in Pakistan, while haplogroup A demonstrated a widespread distribution. We then designed a PCR‐restriction fragment length polymorphism (PCR‐RFLP) assay using XmiI (AccI) and RsaI (AfaI) restriction enzymes to differentiate members of both haplogroups. This result provides more molecular evidence supporting the existence of distinct mitochondrial variants of T. hydatigena. The epidemiological significance of these different mitochondrial groups remains to be explored further. The current PCR‐RFLP assay offers a useful molecular approach for investigating the genetic population structure of T. hydatigena in enzootic regions and in identifying/discriminating the different mitochondrial groups (haplogroups A and B).

Comparison of mitochondrial genetic variation of Taenia hydatigena cysticerci from China and Mongolia

“…Fragment sizes and pro les of the new haplogroups of T. hydatigena for nad5 gene withen Rsa1 and Acc1 restriction enzymes are showen in (Table 3). Haplogroup A1 was extensively spread than other haplogroupes which is in agreement with the previous study (Ohiolei et al, 2021b). The result as shown in (Fig.…”

“…In a previous report, analyses of the complete mitochondrial genes indicated two major ancestors of T. hydatigena namely A and B. Moreover, a signi cant degree of genetic variation has been found for T. hydatigena especially between Asian and African isolates (China, Nigeria, Pakistan and Sudan) so that different mitochondrial lineages were suggested to exist in these regions (Ohiolei et al, 2021a;Ohiolei et al, 2021b).…”

“…The DNA concentration of each extraction was assessed using a spectrophotometer (Thermo Scienti c Inc. Nano Quant) and kept at -20°C until PCR ampli cation. The nad4 and nad5 genes were ampli ed using the following respective primer pairs, Thn4F (forward): 5'-GTCTATTACTCGTTTTCTAAACAG-3' and Thn4R (reverse): 5'-AATCTCAACCAGACACATAAGTG-3' and Thn5F (forward): 5'-TAGGATTAATTTATGACTAGAGTCTCT-3' and Thn5R (reverse): 5'-CTTCTCTCAATCTACCACTAGAAGAGG-3' (Ohiolei et al, 2021b). In a PCR reaction cocktail containing 12.5 µl premix Ex Taq™ version 2.0 (Takara Bio, Kusatsu, Japan), 10 pmol of each primer, 0.5 µl of genomic DNA extract (concentration (c) ≥ 10 ng), and DNase free water up to the nal volume of 25 µl for nad4.…”

“…Previously described RFLP-PCR assay for T. hydatigena targeting 1,444 bp of the NADH dehydrogenase subunit 5 gene (nad5) (Ohiolei et al, 2021b) was employed. Brie y, the nal PCR products ampli ed with the forward 5'-AGGAGTTACATTGTGTGTGACTGTG-3' and reverse 5'-CCACTTATAAAATTGATTCCTG-3' primers were digested with the following restriction endonuclease enzymes; Rsa1 (Thermo Fisher Scienti c, Vilnius, Lithuania) and Acc1 (Thermo Fisher Scienti c, Vilnius, Lithuania).…”