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. 2015 Jan-Mar;10(1):46-55.

Morphological and molecular discrimination of fasciola species isolated from domestic ruminants of urmia city, iran

Mohammad Yakhchali  1 , Reza Malekzadeh-Viayeh  2 , Abbas Imani-Baran  3 , Karim Mardani  4
Affiliations

Morphological and molecular discrimination of fasciola species isolated from domestic ruminants of urmia city, iran

Mohammad Yakhchali et al. Iran J Parasitol. 2015 Jan-Mar.

Abstract

Background: The trematodes of the genus Fasciola (the liver flukes) are among the well-known instances of food-borne parasites worldwide. Differentiation of Fasciola species is important because of their different transmission and epidemiological characteristics. The current study was undertaken to discriminate Fasciola species in the domestic ruminants of Urmia city, Iran.

Methods: Adult flukes were isolated from the naturally infected livers of the slaughtered water buffaloes and sheep. The flukes were initially identified based on morphological and morphometric parameters. A 618-bp-long fragment of the 28SrRNA gene of Fasciola was amplified by polymerase chain reaction (PCR). The amplified fragment was digested by DraII or AvaII enzymes for a restriction fragment length polymorphism (RFLP) analysis and sequenced for the phylogenetic tree construction.

Results: Based on the morphometric examination, the flukes belonged to F. hepatica, F. gigantica and an intermediate Fasciola form. The PCR-RFLP analysis was able to differentiate F. hepatica from F. gigantica. While the phylogenetic reconstruction justified, to some extent, the morphological diagnosis, it failed to segregate F. hepatica from F. gigantica identified in this and the previous studies.

Conclusion: To resolve fully the problem of taxonomy and evolution in Fasciola species, employing a broad range of molecular and morphological approaches is necessary. This is crucial for epidemiological surveys and successful clinical management of their infection.

Keywords: 28SrRNA gene; Liver flukes; Morphology; PCR-sequencing; RFLP.

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Figures

Fig.1:
Fig.1:
Agarose gel electrophoresis of 28SrRNA gene of the liver flukes. (a) Lanes 1–7, F. hepatica and lanes 8–14, F. gigantica from water buffaloes. (b) Lanes 1–2, F. hepatica and lanes 3–7, F. gigantica from sheep. Lane P, positive control; Lane N, negative control; Lane M, 250bp DNA size marker
Fig. 2:
Fig. 2:
Restriction fragment length polymorphism (RFLP) pattern of the PCR products of the liver flukes after digestion with AvaII restriction enzyme. Lanes 1–6, F. hepatica; Lanes 8–15, F. gigantica. Lanes 7 and 16, 618-bp-long PCR products of F. hepatica and F. gigantica, respectively; Lane M, 250bp DNA size marker
Fig. 3:
Fig. 3:
Restriction fragment length polymorphism (RFLP) pattern of the PCR products of the liver flukes after digestion with DraII restriction enzyme. Lanes 1–7, PCR products of F. hepatica; Lanes 9–16, PCR products of F. gigantica. Lane P: 618-bp-long PCR product of F. gigantica; Lane M, 250bp DNA size marker
Fig. 4:
Fig. 4:
Phylogenetic tree constructed by maximum likelihood (ML) method and based on the Kimura 2-parameter model using 16 nucleotide sequences of Fasciola hepatica and F. gigantica. The tree with the highest log likelihood (−1475.6886) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site
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