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. 2020 Feb 18;44(2):441–446. doi: 10.1007/s12639-020-01201-z

Morphologic and molecular analysis of Dicrocoelium dendriticum species from Iranian native sheep

Laya Shamsi 1, Mousa Tavassoli 1,✉, Soraya Naem 1, Alireza Mahmoudian 2, Elham Ahmadi 3
PMCID: PMC7244644  PMID: 32508420

Abstract

Dicrocoeliosis is a worldwide parasitic disease of ruminants which affects the liver. In this current study, the phylogenic pattern of Dicrocoelium species in Iranian native sheep from three different geographical regions was investigated by investigating a 520 bp fragment of mitochondirial NAD1 gene. The analysis of the NAD1 oligo nucleotide sequences from 10 D. dendriticum (GenBank accession numbers: MG889399 to MG889408) revealed few non-significant differences, suggesting limited application for NAD1 gene as a biomarker for study of genetic variation in Dicrocoelium. The morphometrical study also showed a significant relationship for the several morphometric indices among the Dicrocoelium spp. isolates from different regions of Iran.

Keywords: Dicrocoelium, Sheep, Iran, NADH

Introduction

Dicrocoelium dendriticum, the lancet fluke, can be found in the bile ducts and gallbladder of definitive hosts causing Dicrocoeliosis. Domestic and wild ruminants are common hosts, but horses, rabbits, dogs, pigs and humans can also be infected (Otranto and Traversa 2003). A wide range of land snails and ants species (as the first and the second intermediate hosts, respectively) are involved in a complex life cycle of the parasite (Otranto and Traversa 2003). Ruminants and occasionally humans usually become infected following ingestion of ants carrying metacercariae (Karadag et al. 2005), causing weight loss, growth delay, anemia, digestive disorders, edema, reduced milk production and cost related to anthelminthic treatment in ruminants (Manga-González et al. 2001; Otranto and Traversa 2003; Ferreras-Estrada et al. 2007). Spurious infections may result from the ingestion of undercooked or raw infected liver (Di 2010).

Fields with dry, chalky and alkaline soils are suitable areas for the intermediate hosts of Dicrocoelium (Manga-González et al. 2001).

A high rate of D. dendriticum incidence has been reported in Iranian native ruminants (Ansari-Lari and Moazzeni 2006; Daryani et al. 2006; Ghazani et al. 2008; Ahmadi and Meshkehkar 2010). Epidemiological studies have shown that D. dendriticum has low parasitic specificity in definitive and intermediate hosts (Rosick and Groschaft 1982). Conventional diagnosis is based on the detection of eggs in the feces or isolation of adult parasite from the liver during post mortem examination (Manga-Gonzalez et al. 1991). Despite the importance of Dicrocoeliasis, little information is available regarding the morphometric and genomic properties of the parasite in Iran.

Therefore, to understand the intras- and inter-specific variations, the mitochondrial NADH gene variations were investigated among the D. dendriticum species isolated in sheep from geographically distinct locations in Iran.

Materials and methods

Morphometric analysis

Totally, 245 adult Dicrocoelium parasites were collected from livers and gall bladders from sheep slaughtered in three distinct mountainous, lowlands and coastal geographical areas, including Sabzevar (Khorasan Razavi), Qom, Ilam, Qazvin, Hamadan, Sanandaj, Kermanshah, Urmia, Noshahr and Ahvaz cities (Fig. 1). The indices of body length and width, the inner and outer diameter of the oral sucker, the inner and outer diameter of the Acetabulum, the distance between the suckers, the ratio of oral sucker to Acetabulum, the width and the length of the testicle, the ovary length and width, the length of yolk and the distance from the beginning and the end of the yolk were determined using a light microscope, as explained somewhere else (Taira et al. 2006).

Fig. 1.

Fig. 1

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The map showing the sampling areas in Iran, marked with triangles

Statistical analysis

Analysis of variance (ANOVA) method was used to investigate the differences between different morphometric indices of D. dendriticum (SPSS v. 21) and the P values < 0.05 were statistically considered significant.

Molecular analysis

Out of 245 adult worms, 10 parasites were carefully washed using physiological buffer saline (PBS), stored in 70% ethanol solution and froze at − 20 °C.

DNA was extracted using a commercial kit (MBST Co., Tehran, Iran), according to the manufacturer’s instructions and stored at − 20 °C. The polymerase chain reaction (PCR) amplifications were carried out in a 50 µl total volume containing 10 mM Tris–HCl, 50 mM KCl, 4 µl of DNA template, 1.5 mM MgCl2, 200 mM deoxy nucleoside triphosphates (dNTPs), 20 pM of each forward and reverse primers and 2U of Taq DNA polymerase (Fermentas, Hanover, Maryland, USA). The forward and reverse primers (Fig. 2) were designed to amplify a 520 bp fragment of the NADH gene as explained somewhere else (Herwerden et al. 2000). PCR reactions were carried out under the following conditions: one cycle of 94 °C for 5 min as early denaturation followed by 35 cycles of 94 °C for 45 s (denaturation), 60 °C for 1 min (annealing), and 72 °C for 45 s (extension). The last final extension step was applied at 72 °C for 5 min. The PCR products were analyzed using electrophoresis in 1.5% (w/v) agarose gel and visualized using Sybr green (0.001%) under ultraviolet illumination.

Fig. 2.

Fig. 2

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PCR products for NAD1 gene of D. dendriticum in 1.5% agarose gel. Lane M: Gene Ruler TM 100 bp DNA ladder; Lane 1: negative control; Lanes 2: positive control, 3–12: samples from different locations of Iran

For sequence analysis, the PCR products were submitted for sequencing (Sinaclon Co., Tehran, Iran).

Data analysis

The Multiple sequence alignments and the construction of a phylogenetic tree for the nucleotide sequences were generated using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) and MEGA4 software for Maximum-likelihood analysis.

Results

Morphometric

In the present study, the morphometric indices of Dicrocoelium isolated in sheep in different regions of Iran were also compared, and no significant relationship was found (P > 0.05) between the inner diameter of the Acetabulum, the inner diameter of the oral sucker and Acetabulum to oral sucker ratio variables for the samples from different regions (Table 1).

Table 1.

Morphometric indices of sheep Dicrocoelium spp. collected from different regions of Iran (mean ± S.E)

Variables (µm)* ***Regions examined
Region 1 Region 2 Region 3 Region 4
Body length 3480.73 ± 84.45 3853.17 ± 66.62b 4262.54 ± 127.68a 4289.20 ± 134.27a
Body width 1175 ± 74.48a 1087.56 ± 20.10ab 977.23 ± 46.04b 1008.11 ± 65.72b
Length to width ratio 3.26 ± 0.20b 3.64 ± 0.09b 4.41 ± 0.27a 4.54 ± 0.41a
The inner diameter of the oral sucker** 61.45 ± 6.02a 53.26 ± 2.06a 51.17 ± 4.83a 60.74 ± 4.17a
The outer diameter of the oral sucker 75.02 ± 4.61b 89.83 ± 2.52ab 76.51 ± 6.77b 98.35 ± 4.95a
The inner diameter of the Acetabulum** 72.78 ± 6.03a 64.91 ± 1.96a 65.28 ± 7.33a 72.86 ± 5.48a
The outer diameter of the Acetabulum 92.97 ± 4.85b 103.55 ± 2.44ab 97.89 ± 8.24b 119.70 ± 7.35a
Distance between the suckers 349.19 ± 28.40b 410.31 ± 12.87ab 439.22 ± 38.24a 453.37 ± 37.44a
Oral sucker to Acetabulum ratio** 1.26 ± 0.06a 1.16 ± 0.01a 1.28 ± 0.06a 1.22 ± 0.07a
The length of the testicle 256.73 ± 17.79b 298.38 ± 9.10b 297.02 ± 21.69b 375.66 ± 20.85a
The width of the testicle 370.90 ± 34.04ab 369.30 ± 10.71ab 338.23 ± 24.99b 420.95 ± 25.47a
Ovary length 114.90 ± 6.55c 125.03 ± 3.97bc 144.64 ± 17.08ab 167.57 ± 9.93a
Ovary width 190.06 ± 13.12b 207 ± 4.64ab 192.81 ± 14.28a 228 ± 21.29a
Length of yolk 899.20 ± 46.50b 986.02 ± 28.85b 1168.79 ± 80.45a 972.53 ± 49.06b
The distance from the yolk to the end 1530.02 ± 54.71b 1556.83 ± 3.98b 1731.76 ± 108.99ab 1813.00 ± 66.01a
The distance from the yolk to the beginning 1128.41 ± 62.33b 1321.21 ± 26.84a 1435.75 ± 108.48a 1468.36 ± 77.97a
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*Indicating non-significant difference (P < 0.05)

**There is no meaningful relation between this area (P < 0.05)

***Region 1: Noshahr; Region 2: Orumiyeh, Sabzevar, Kermanshah, Hamedan, Ilam, Qazvin, Sanandaj; Region 3: Ahvaz; Region 4: Qom

Molecular

Totally, 10 sequences were determined and submitted to GenBank (accession numbers of MG889399, MG889400, MG889401, MG889402, MG889403, MG889404, MG889405, MG889406, MG889407 and MG889408).

No significant correlation was found between the sequences for D. dendriticum isolates.

As shown in Fig. 3, phylogenetic tree for the nucleotide sequences was created using MEGA4 program and based on maximum-likelihood method.

Fig. 3.

Fig. 3

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Phylogenetic analysis of Dicrocoelium spp. based on NAD1 sequences using Maximum-likelihood (ML) method analysis

The partial nucleotide sequences were compared with available sequences for Dicrocoelium species worldwide using Clustal O program (https://www.ebi.ac.uk/Tools/msa/clustalo). All strains isolated in this study were clustered together, indicating that these parasites are identical (Fig. 4).

Fig. 4.

Fig. 4

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Sequences of Dicrocoelium dendriticum isolates in different cities of Iran

Discussion

In the current study, the differences between the D. dendriticum isolates were investigated by morphometric analysis and sequencing. No significant correlations were found between different variables for samples from different regions. Previously, Dicrocoelium species were studied in Italian, Austrian, and German sheep according to the location of testicles within the parasite body (Otranto et al. 2007). In D. dendriticum, the testicles are tandem, but in D. chinensis, the testicles are bilateral (Yamaguti 1971; Taira et al. 2006). The morphology of the testicles in our samples had a tandem pattern, revealing that the spices D. dendriticum is the causing agent of dicrocoeliasis in our study, as shown in several studies (Hinaidy 1983; Taira et al. 2006; Otranto et al. 2007; Bazsalovicsová et al. 2010; Martínez-Ibeas et al. 2011).However, no intraspecific variations have been found within the geographically distant populations of Fasciola hepatica and D. dendriticum (Bazsalovicsová et al. 2010).

The phenotypic and genetic differences in D.dendriticum from Iran have been investigated (Arbabi et al. 2012; Gorjipoor et al. 2015). Gorjipoor et al. (2015) has shown a higher variation in NAD1 gene compared to ITS-2 gene. While, no significant differences have been found between the geographic locations and the host associations (Otranto et al. 2007; Moazeni et al. 2012; Gorjipoor et al. 2015), another study has been shown morphological differences between the D. dendriticum isolates from different locations in Iran (Arbabi et al. 2012).

In another Iranian study (Bian et al. 2015), 3.4–12.3% inter-specific variations in ITS-2 gene of ribosomal DNA (rDNA) has been found among the investigated Dicrocoelium species, while the inter- and intra-specific sequence variations found in another study outside of Iran for the same gene were 0–0.5% and 0–1.3%, respectively (Bazsalovicsová et al. 2010).

The morphological variations in suborder of Plagiorchiata shown in different studies may be caused by the differences in the age and the nutritional status of the host, the maturity status of the parasites, and the climatic and ecological conditions of the areas of study (Tkach et al. 2000). No genetic variations were found in NADH gene our study. Similar findings have also been reported by investigating the variations in 18S and 28S rDNA sequences in Iranian isolates of D. dendriticum (Otranto et al. 2007; Arbabi et al. 2012). However, a Spanish study has shown genetic differences in this parasite using the random amplified polymorphic DNA (RAPD) technique (Sandoval et al. 1999).

Conclusion

In conclusion, investigation of the genetic variations of D. dendriticum in our study suggests that study of NAD1 gene is not a powerful tool to determine the phylogenetic analysis of Dicrocoelium species.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical standards

This article is prepared and presented by the author in accordance with the ethical standards.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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