Using the fatty acid binding protein type I gene as a unique DNA marker, Fasciola flukes can be differentiated by species.

 

Abstract

Background

Fasciola hepatica, F. gigantica, and hybrid Fasciola flukes have been distinguished using multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) for nucleus phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively. However, both approaches have been reported to exhibit discrimination flaws. The objective of this study was to create a multiplex PCR based on the FABP type I gene, a novel nuclear marker.

Methods

Using DNA samples of hybrid Fasciola flukes, F. hepatica, and F. gigantica collected from 11 countries in Europe, Latin America, Africa, and Asia, nucleotide sequence variants of FABP type I were examined. For multiplex PCR, two distinct reverse primers for F. hepatica and F. gigantica as well as a common forward primer were created.

Results

Using multiplex PCR, specific segments of F. hepatica (290 bp) and F. gigantica (190 bp) were amplified satisfactorily. The hybrid flukes, however, included parts from both species. The 1312 Fasciola samples utilised in this investigation could be distinguished with pinpoint accuracy using the multiplex PCR for FABP type I. Notably, this innovative technique showed no discrimination errors.

Conclusions

Instead of pepck and pold, multiplex PCR for FABP type I can be used to distinguish between different species. In the future, a greater variety of Fasciola flukes should be used to continuously test the robustness of the species-specific primer because nucleotide changes in the primer regions may lead to amplification errors.

Graphical abstract

Background

Fasciolosis causes huge economic losses to the livestock industry in endemic areas [1, 2]. Fasciola hepatica and F. gigantica are well-known causative agents of this disease. Both species have normal spermatogenic abilities and reproduce bisexually by fertilization. In contrast, the hybrid Fasciola flukes of the two species have been reported in many Asian countries [3]. Both diploids and triploids have been reported in hybrid Fasciola flukes [4, 5]. Because hybrid flukes harbor a meiotic disorder that affects spermatogenesis, they probably reproduce parthenogenetically [5]. Therefore, it is important to precisely discriminate hybrid flukes from F. hepatica and F. gigantica because they are speculated to have stronger viability than the two species [6].

For nuclear phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively, multiplex polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (RFLP) can distinguish between Fasciola spp. by the fragment patterns of F. hepatica (Fh), F. gigantica (Fg), and the hybrid (both Fh and Fg: Fh/Fg) [7]. The two nuclear markers' detection of the Fh/Fg type suggests that hybrid Fasciola flukes are offspring of the cross-pollination of F. hepatica and F. gigantica [3, 6].

Even though F. hepatica isolates from Afghanistan, Algeria, Ecuador, and Spain revealed discrimination errors in the multiplex PCR for pepck, subsequent nucleotide sequencing of the DNA fragment of pepck allowed accurate species identification. Discrimination mistakes were seen in F. gigantica isolates from Nigeria with regard to pold [12]. The mistake in PCR-RFLP was traced to a single nucleotide alteration at the restriction enzyme's recognition site [12].

The nuclear DNA-encoded fatty acid binding protein (FABP) type I of Fasciola flukes performs a variety of functions, including immunological regulation and anthelmintic sequestration [13]. Additionally, the DNA databank has the messenger RNA (mRNA) sequence for FABP type I [13]. The nucleotide sequence variations of FABP type I in F. hepatica, F. gigantica, and hybrid Fasciola flukes were examined in this work. Then, 1312 Fasciola species from 11 countries in Asia, Africa, Europe, the Near and Middle East, and Latin America were subjected to a multiplex PCR for FABP type I. In place of pepck and pold, the novel multiplex PCR for FABP type I was found to be an effective marker for exact species differentiation of Fasciola spp.

Methods

Fasciola samples

The current study included 1312 Fasciola flukes, including 470 F. hepatica, 609 F. gigantica, and 233 hybrid Fasciola, from 11 different nations, including Afghanistan, Algeria, Peru, Spain, Indonesia, Malaysia, Nigeria, Pakistan, Uganda, Japan, and Bangladesh [8, 9, 11, 12, 14,15,16,17,18,19,20]. Previous research [8, 9, 11, 12, 14, 15, 16, 17, 18, 19] have sequenced the mitochondrial nad1 gene's nucleotides and performed fragment analyses on nuclear pepck and pold. 7, 19, 6, 27, and 15 Fasciola isolates from Afghanistan, Algeria, Peru, Spain, and Nigeria, respectively, showed differences between pepck and pold. Table 1 provides a summary of all information that is known about the Fasciola samples.

  Table 1 Nuclear marker profiles of Fasciola flukes used in this study