Molecular Characterization of Fasciola hepatica Isolates by RAPD-PCR and Ribosomal ITS1 Sequencing

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Group: 2010
Subgroup: Volume 12, Issue 1
Date: January 2010
Type: Original Article
Start Page: 27
End Page: 32

Authors:

  • MB Rokni
  • Department of Parasitology and Mycology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran ,
  • H Mirhendi
  • Deptartment of Parasitology and Mycology, School of Public Health, Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Tehran, Iran
  • M Behnia
  • Department of Parasitology and Mycology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran ,
  • M Fasihi Harandi
  • Department of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
  • N Jalalizand
  • Laboratory of Molecular Biology, Isfahan Research Center of Public Health, Isfahan, Iran

      Correspondence:

      Affiliation: Deptartment of Parasitology and Mycology, School of Public Health, Institute of Public Health Research, Tehran University of Medical Sciences
      City, Province: Tehran, Tehran
      Country: Iran
      Tel: +98-21-88950184
      Fax: +98-21-88950184
      E-mail: mirhendi@tums.ac.ir

Abstract:


Background: Understanding genetic structure and status of genetic variation of the Fasciola hepatica populations has important implications for epidemiology and effective control of fasciolosis. The aim of the present study was to genetically characterize F. hepatica isolates from different hosts, using sequence analysis of ribosomal ITS1 and RAPD-PCR.

 

Methods: Fifty three adult F. hepaticas were isolated from naturally infected cattle, sheep, buffalo and goat from two regions in Iran. Genomic DNA was extracted from 70% ethanol preserved flukes. RAPD-PCR with a set of arbitrary primers (UBC90 and R151) was used to estimate genetic variation within the species. Ribosomal ITS1 region of the isolates was amplified, using primers specifically designed for this study. Ten samples (4 sheep, 2 cattle, 3 buffaloes and one goat isolate) were sequenced at ITS1 and analyzed, using DNASIS and ClustalW softwares.

 

Results: F. hepatica ITS1 region was amplified successfully for all samples and a band of 470 bp was shown in all cases. Different isolates did not show any significant genetic variations in rDNA-ITS1 as all the sequences showed to be 100% identical. RAPD results of 52 samples, in particular those with UBC90, showed different patterns within F. hepatica isolates of each host. RAPD data for this primer showed three different patterns for each of sheep and cattle isolates and two patterns in buffalo isolates. All the 14 cattle isolates came up with an identical pattern, using primer R151.

 

Conclusion: The study showed the variability of F. hepatica isolates in Iran, using RAPD markers. No intraspecies variation was seen in the Iranian F. hepatica isolates at ITS1 rRNA gene, indicating highly conserved nature of this region.

  

Keywords: Fasciola hepatica; RAPD-PCR; Ruminant; Iran

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Introduction

 

Fasciolosis, caused by the digenetic trematodes of the genus Fasciola, is one of the most important plant-borne helminth infections of human and livestock in many parts of the world.1 Annual economic loss caused by the disease is estimated to be US$2 billion mainly due to condemned livers, reduced milk yield, fertility disorders and reduced meat production.2

During the past two decades, the disease has emerged as a serious medical and veterinary problem in Iran. At least 17000 human infections were reported in Northern Province of Gilan since 1989.3 The prevalence of animal fasciolosis for cattle and sheep has been reported in different parts of Iran to be 1.07 % and 0.59%;4 25.9% and 5.3%,5 and 4.6% and 5.7%,6 respectively.

Two species of F. hepatica and F. gigantica were recognized in humans and domestic farm animals. F. hepatica is the dominant species in both human and livestock in Iran.7 Based on morphological and morphometric criteria, F. hepatica was reported from several species of ruminants including cattle, sheep, goats and buffaloes.

Understanding genetic structure and status of genetic variation of the parasite populations has important implications for epidemiology and effective control of fasciolosis. The degree of genetic variability in F. hepatica in Iran is not clear and the genetic characterization of F. hepatica from different livestock has not been performed before.

Various DNA-based techniques can be applied to the genetic study of Fasciola spp. Nucleotide sequence comparison of Internal Transcribed Spacers (ITS1 and ITS2) of ribosomal DNA is a reliable approach to systematic molecular study of most helminth parasites.8 Random Amplified Polymorphic DNA PCR (RAPD-PCR) is a simple, reliable, and fast technique for estimating the extent of genetic variability within and between species.1,9

The aim of the present study was to characterize genetically adult F. hepatica isolates from cattle, sheep, buffalos and goats from different parts of Iran, using sequence analysis of ribosomal ITS1 and RAPD-PCR.

 

 

Materials and Methods

 

Livers from sheep, cattle, buffaloes and goat were collected at local slaughterhouses from two geographical regions (Table 1). Hepatic bile ducts were macroscopically examined for the presence of adult Fasciola worms. The worms were rinsed with normal saline solution and morphologically identified to the species level according to standard taxonomic keys.10,11 The confirmed F. hepatica specimens were transferred to vials containing 70% ethanol until used for DNA extraction.

 

Table 1: Number of Fasciola hepatica specimens isolated from farm animals and their geographical origins 

Host

Number of F.
hepatica
isolates

Location of
origin

Sheep

30

Tehran

Cattle

14

Tehran

Buffalo

  8

Urmia (NW Iran)

Goat

  1

Urmia (NW Iran)

Total

53

-

 

 

We used a simple and efficient method for isolating DNA. Briefly, 20 µL lysis buffer (100 mM Tris-HCl, pH=8, 10 mM EDTA, 100 mM, NaCl and 2% Triton X-100) was added on 5 mm2 cuts of each worm and they were placed on a microscope slide. The worm was rigorously crushed between two slides for about 30 seconds. More lysis buffer was added on the slide when appropriate. Finally, the homogenate was transferred to a 1.5 ml tube and lysis buffer was added to a final volume of 400 µL. Total genomic DNA was extracted, using standard phenol-chloroform procedures.12 DNA samples were stored at -20oC until their use. Care was taken to ensure that there was no contamination with host tissues.

PCR was used to amplify nuclear ITS1 of the ribosomal DNA (rDNA) repeats. A primer pair, FascF (5’-ACC GGC GCT GAG AAG ACG- 3’) as forward and FascR (5’-CGA CGT ACG TGC AGT CCA- 3’) as reverse, was designed which amplified a ~ 470 bp fragment in ITS1 region. The primers were designed, using available genetic sequence data of ITS1 for F. hepatica in GenBank. PCR reaction volumes of 50 µl contained 1 µl (about 10 ng) DNA template, 400 µM dNTP’s, 25 pmol of each primer, 1.25 units Taq DNA polymerase and 1.5 mM MgCl2 in reaction buffer. PCR amplification was carried out in a thermocycler (Corbett Research, Australia) under the following temperature conditions: an initiation of 95oC for 5 min, followed by 30 cycles of 94oC for 30 s (denaturation), 58oC for 60 s (annealing), 72oC for 45 s (extension) and a final extension of 72oC for 6 min.

ITS1 sequences of 10 F. hepatica isolates (4 sheep, 2 cattle, 3 buffaloes and one goat isolate) were obtained in an automatic DNA sequencer (ABI Prism 377, Perkin Elmer). Sequence comparisons with published sequences of ITS1 were made in GenBank (www.ncbi.nlm.nih.gov/blast). Multiple sequence alignment was carried out, using ClustalW with default settings and DNASIS software (Hittachi 2003, Japan).

For RAPD analysis, four primers were initially tested of which 2 (UBC90: GGG GGT TAG G and R151: GCT GTA GTG T) were selected. Reaction volumes of 25 µl contained 1 µl DNA template, 400 µM dNTP’s, 25 pmol of each primer and 1.25 units Taq DNA polymerase. The temperature profile was as follows: an initiation of 95oC for 5 min, followed by 40 cycles of 94oC for 40 s (denaturation), 32oC for 60 s (annealing), 72oC for 60 s (extension) and a final extension of 72oC for 6 min. PCR products were electrophoresed on either 1.5% (for ITS1 PCR) or 1.2% (for RAPD-PCR) agarose gel and visualized by UV illumination after ethidium bromide staining.

 

 

Results

 

Fifty-three F. hepatica isolates were studied. The list of isolates according to their host and geographical origin is listed in Table 1. DNA was successfully extracted from all the isolates by the appropriate techniques. ITS1 PCR amplified all of the isolates, producing a single band of ~470 bp (Figure 1). The size was exactly expected according to the data derived and analyzed from GenBank.  ITS1 sequence analysis of 10 F. hepatica isolates showed exactly 100% similarity between the isolates and also with all available ITS-sequence data of F. hepatica in GenBank (GenBank accession nos. AB207145, AB207141, AB207140, AB207139, AJ243016, AM900370, AM709649,  AM709648, AM709647, AM709646,  AM709645,  AM709644, AM709643, AM709622, AM709621, AM709620,  AM709619,  AM709618, AM709617,  AM709616, AM709615, AM709614,  AM709613,  AM709612, AM709611, AM709610,  AM709609,  AM709500,  AM709499,  AM709498, AM707030, AJ628432, AJ628431).

 

 

 

 

Fig. 1: Agarose gel electrophoresis of ITS1 PCR product of representative Fasciola hepatica isolates from sheep and cattle in Iran. Lanes 18-22: sheep isolates, Lanes 23-25: cattle isolates, Lane 26: negative control, M: 100bp DNA size marker

 

No nucleotide variations were observed in the data in GenBank and in our sequences, among the same host and between different hosts Figure 2.

 

 

 

Fig. 2: Agarose gel electrophoresis of RAPD-PCR products of Fasciola hepatica isolates from Iran. A: RAPD-PCR pattern-I produced by primer UBC90, all sheep isolates. M, DNA size marker. B:  RAPD-PCR pattern-II produced by primer UBC90, all sheep isolates. M, DNA size marker

 

Potential intraspecific variation within F. hepatica was investigated by RAPD-PCR. Both oligonucleotide primers provided distinct patterns of amplified genomic DNA. Isolates with similar banding pattern were classified as a single type. Results of two RAPD profiles, using two oligonucleotide primers (UBC90 and R151), are shown in Table 2 and Figure 2. RAPD results showed different patterns within F. hepatica isolates of each host. This was especially evident in RAPD-PCR amplifications. Using primer UBC90, RAPD data for this primer revealed three different patterns for each of sheep and cattle isolates and 2 patterns in buffalo isolates (Table 2). Although some minor variations were detected in the relative intensity of some bands of the same size, pattern-I and pattern-II were readily distinguishable (Figure 3). Pattern-I amplified by primer UBC90 was produced in more than 64% of all of the isolates (Table 2) while all other isolates (35.3%) produced different patterns (II or III).

 

 

Table 2: Results of RAPD-PCR study of 52 F. hepatica isolates from sheep, cattle and buffaloes, showing three banding patterns using primers UBC90 and R151

F. hepatica

host origin

Pattern I

No (%)

Pattern II

No (%)

Pattern III

No (%)

 

Primer UBC90

 

 

Sheep

19 (63.4)

  4 (13.3)

7 (23.3)

Cattle

  9 (64.3)

  4 (28.6)

1 (7.1)

Buffalo

  5 (71.4)

  2 (28.6)

0 (0)

Total

33 (64.7)

10 (19.6)

8 (15.7)

 

Primer R151

 

 

Sheep

27 (90.0)

  2 (6.7)

1 (3.3)

Cattle

14 (100)

  0 (0)

0 (0)

Buffalo

  3 (60.0)

  2 (40.0)

0 (0)

Total

44 (89.8)

  4 (8.2)

1 (2.0)

 

 

 

Fig. 3: Agarose gel electrophoresis of RAPD-PCR products of Fasciola hepatica isolates from Iran. A: RAPD-PCR pattern-I produced by primer UBC90, all cattle isolates. M, DNA size marker. B: RAPD-PCR pattern-I produced by primer R151, all cattle isolates. M, DNA size marker. C: RAPD-PCR patterns produced by primer UBC90, Lanes 35-36 and 39 are buffalo isolates presenting RAPD pattern-I. Lanes 40 and 42-43 are buffalo isolates showing RAPD pattern-II. Lane 37 is the only single goat isolate. Lanes 38 and 41 are poorly amplified. M, DNA size marker.

 

 

With primer R151, all the 14 cattle isolates came up with an identical pattern. Most sheep (27/30, 90%) and buffalo (60%) isolates presented the same pattern as cattle isolates (pattern I). Nevertheless, different RAPD patterns were seen in 14.3% of sheep and buffalo isolates with primer R151 (Data not shown). Each RAPD pattern corresponding with each individual primer was identical in sheep, cattle and buffalo isolates.

 

 

Discussion

 

Studies on genetic variability within and between populations of Fasciola have important implications for epidemiology, control and diagnosis of fasciolosis. Different DNA-based molecular techniques have

been applied to these flukes.1 There have been no data regarding genetic characterization of F. hepatica in Iran using defined DNA sequences. This study was carried out to characterize F. hepatica isolates of different livestock from different geographical locations in Iran, using ITS1 sequencing and RAPD-PCR.

There are not many sequence data on rDNA ITS1 as most nuclear DNA studies on Fasciola have focused on ITS2. ITS1 PCR in this study amplified a 463 bp fragment which was composed of 85 bp in 18SrDNA and 378 bp in ITS1 (the whole size of ITS1 in Fasciola is 433 bp, GenBank accession no. AJ243016).

In the present study, 10 F. hepatica isolates from four host species in Iran were sequenced. Different isolates of F. hepatica from different animal hosts showed a uniform sequence pattern and no sequence variation was detected in ITS1, indicating highly conserved nature of rDNA internal transcribed spacers in F. hepatica. This has been shown in several studies in which F. hepatica populations demonstrated very little or no intraspecific variation in ribosomal ITS regions.1,13,14 Although we did not find ITS1 as a proper region for investigating intra-species differences, this highly conserved region could be an ideal target for diagnostic purposes including detection of the DNA in clinical samples or for identification of the worm in PCR-based formats such as sequencing or PCR-RFLP.

Analysis of rDNA is a single locus method demonstrating gene polymorphisms rather than actual polymorphism within a species. This shortcoming is resolved by RAPD-PCR method, which is based on the amplification of total genomic DNA with single arbitrary oligonucleotide primers. This technique has provided us with a suitable means of detecting genetic polymorphisms in parasite populations. RAPD polymorphism has been used for estimating genetic variability within and between parasite species and for evaluating genetic relationships between populations.

In the present study, 52 F. hepatica isolates were analyzed by RAPD, using 2 primers. An estimate of intraspecific variability of F. hepatica from two regions in Iran was achieved, using RAPD-PCR. This study demonstrates clearly that genetic heterogeneity exists within F. hepatica in Iran. Such variability has been reported for other platyhelminthes, Schistosoma mansoni,15 Schistosoma haematobium,16 Clonorchis sinensis,17 and Echinococcus granulosus.18,19 Variability was shown to be present within isolates from the same hosts (sheep, cattle and buffalo) as well as between isolates of different host origin. F. hepatica of sheep and cattle origin seems to show greater variability than that of buffaloes (Table 2, Figure 3).  This may be the result of the gene flow due to more intensive transmission rate of the parasite in sheep and cattle than the transmission rate in buffaloes and more extensive transfer of the animals within the endemic areas through livestock trade.

The limitations of the study, i.e., the use of one goat and sampling from only two regions can be overcome in future studies by using more goats and sampling from more regions. This will help to obtain more reliable and valid data

In conclusion, the results of this study suggest the variability of F. hepatica isolates in Iran, using RAPD markers. Looking at ribosomal ITS regions of F. hepatica, no intraspecies variations were seen in the Iranian F. hepatica isolates; however, the study suggests that F. hepatica ITS1 is an invaluable species marker for diagnostic purposes. In addition, the findings of the present study on the variability of F. hepatica isolates in Iran may have implications in the epidemiology and control programs of fasciolosis as a major helminth zoonosis of medical and veterinary importance. However, further studies with more isolates from different regions are needed to elucidate the nature and extent of the variability documented in this study.

 

 

Acknowledgements

 

The study was financially supported by a grant from Institute of Public Health Research, Tehran University of Medical Sciences, Iran.  The authors declare that there is no conflict of interests.

 

Conflict of interest: None declared.

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