Subscribe Now Subscribe Today
Research Article
 

Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran



Soheila Rouhani, Saber Raeghi and Adel Spotin
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Background and Objective: Fascioliasis is economically important to the livestock industry that caused with Fasciola hepatica and Fasciola gigantica. The objective of this study was to identify these two species F. hepatica and F. gigantica by using nuclear and mitochondrial markers (ITS1, ND1 and CO1) and have been employed to analyze intraspecific phylogenetic relations of Fasciola spp. Materials and Methods: Approximately 150 Fasciola specimens were collected, then stained with haematoxylin-carmine dye and observed under an optical microscope to examine for the existence of sperm. The ITS1 marker was used to identify different Fasciola and phylogenetic analysis based on ND1 and CO1 sequence data were conducted by maximum likelihood algorithm. Results: Fasciola samples were separated into 2 groups. Almost all specimens had many sperms in the seminal vesicle (spermic fluke) and one fluke did not contain any sperm in the seminal vesicle. The aspermic sample had F. gigantica RFLP pattern with ITS1 gene. Phylogenetic analysis based on NDI and COI sequence data were conducted by maximum likelihood showed a similar topology of the trees obtained particularly for F. hepatica and F. gigantica. Conclusion: This study demonstrated that aspermic Fasciola found in this region of Iran has same genetic structures through the spermic F. gigantica populations in accordance to phylogenetic tree.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Soheila Rouhani, Saber Raeghi and Adel Spotin, 2017. Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran. Pakistan Journal of Biological Sciences, 20: 204-209.

DOI: 10.3923/pjbs.2017.204.209

URL: https://scialert.net/abstract/?doi=pjbs.2017.204.209
 
Received: December 01, 2016; Accepted: February 14, 2017; Published: March 15, 2017


Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

The Fasciola species are found in domestic ruminants and may occasionally affect humans. Fascioliasis is economically important to the livestock industry. It is estimated that economic losses is over two billion US dollars annually. Human infection has been reported worldwide and is serious health problem1. About 180 million people are at risk of infection worldwide in endemic areas2.

The causative species of the disease are Fasciola hepatica and Fasciola gigantica. Fasciola hepatica is distributed worldwide, whereas F. gigantica is restricted to tropical regions and has been found in Africa as well as South and Southeast Asia3.

Fasciola hepatica and F. gigantica can be differentiated based on morphological criteria such as body size, ratio of body length to width and shape; however, these criteria are not always reliable because of the morphological diversity within the species4.

These species are meiotically functional diploid and therefore they have abundant mature sperm (spermic fluke) in the seminal vesicles5. This male reproductive organ is the common predominant characteristic of both species6. On the other hand intermediate Fasciola flukes, which have morphological characteristic intermediate between F. hepatica and F. gigantica and no sperm (aspermic fluke) in their seminal vesicles have been found in Asian countries7. Fasciola flukes reproduce bisexually and aspermic Fasciola probably reproduce parthenogenetically. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the F. hepatica/F. gigantica type, which includes DNA fragments of both F. hepatica and F. gigantica.

These two species can be identified using nucleotide sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS1) and 2 (ITS2)8,9. Additionally, DNA sequences of mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (CO1) genes have been employed to analyze intraspecific phylogenetic relations of Fasciola spp10,11.

There are some reports from Iran ruminant’s fascioliasis particularly in cattle and buffaloes based on geography and climate variability12,13. Also, there have been no valuable reports on molecular and spermatogenic characterization of Fasciola flukes. The objective of this study was to characterized the Fasciola flukes from North West of Iran for first time based on spermatogenic status to identify of probably hybrid of Fasciola sp. in Iran and PCR-RFLP method in ribosomal ITS1 and mitochondrial ND1 and CO1 sequences for phylogenetic analysis.

MATERIALS AND METHODS

Sample collections and spermatogenic status assessment: One hundred and fifty Fasciola specimens were collected from the bile ducts of 4 buffaloes, 38 cattle at slaughter house located in 7 different geographical locations in the North West of Iran (Azerbaijan) from January-September, 2015 (Table 1). This region of Iran borders Iraq, Turkey and Armenia with different mountain climate and biosphere reserve. Fasciola flukes were collected, washed in 0.9% saline solution, fixed in 70% ethanol between two glass slides and transported to the laboratory for further studies. The whole body of Fasciola including seminal vesicle in the anterior part of worm was stained with haematoxylin-carmine dye and observed under an optical microscope to examine for the existence of sperm14. Prior to staining, a small posterior part of fluke was used for DNA extraction.

Table 1: Profiles of Fasciola haplotypes and their accession no. in North West of Iran
Fg: Fasciola gigantica, Fh: Fasciola hepatica

DNA extraction and amplification: Total DNA was extracted from each fl uke with a high pure PCR template preparation Kit (Dynabio®, Takapouzist, Iran), according to the manufacturer's instructions and stored at -20°C until use. DNA fragments of each target regions (nuclear ITS1 region and mitochondrial ND1 and CO1 regions) were amplified by Polymerase Chain Reaction (PCR) using a pair primer. Total volume of reaction was 15 μL containing 1.5 μL DNA template, 5 μL distilled water, 10 p mol of each primers (Forward and Reverse) and 7.5 μL master mix (Amplicon®). The primer sets used were ITS1-F (5-TTGCGCTGATTACGTCCCTG-3) and ITS1-R (5-TTGGCTGCGCTCTTCATCGAC-3) for ITS1 region and Ita 10 (5-AAGGATGTTGCTTTGTCGTGG-3) and Ita 2 (5-GGAGTAC GGTTACATTCACA-3) for ND1 region and Ita 8 (5-ACGTTG GATC ATAAGCGTGT-3) and Ita 9 (5-CCTCATCCAAC ATAACC TCT-3) for CO1. Reaction cycles consisted of an initial denaturing step at 94°C for 90 sec, followed by 35 cycles at 94°C for 90 sec, 53°C (ITS1) or 55°C (ND1 and CO1) for 90 sec and 72°C for 120 sec, with a final extension at 72°C for 10 min using a Eppendorf Mastercycler gradient thermocycler. The DNA fragments were analyzed by 1.5% agarose gel electrophoresis.

Restriction fragment length polymorphism of amplified DNA (PCR-RFLP): The ITS1 marker was used to identify different Fasciola. Briefly, the reaction volume of 10 μL contained 5 μL of PCR products with approximately 680 bp fragments, 1 U of the RsaI restriction enzyme and 1 μL of manufacturer-supplied reaction buffer (Cinagen®, Iran). After incubation at 37°C for 1 h and heat inactivation of RsaI at 65°C for 20 min, the digestions were exposed to 1.5% agarose gel electrophoresis and visualized by with ethidium bromide. The fragment data were recorded with UV illumination (UVITEC).

Sequencing and phylogenetic analysis: The products of ND1 and CO1 were directly sequenced by Bioneer Company using the same primers which were used in the PCR. The sequences were aligned and compared with those of existing sequences from the region, related to Fasciola spp. Available in the GenBank, using the chromas and multiple alignments were performed with data related to Fasciola spp. From Iran and other countries deposited in GenBank. Phylogenetic analysis based on ND1 and CO1 sequence data were conducted by Maximum Likelihood (ML) algorithm using MEGA6 software. All characters were run unordered and equally weighted. Alignment gaps were treated as missing data. Bootstrap analysis were conducted using 1000 replicates.

RESULTS

Spermatogenesis: Fasciola samples were separated into 2 groups consisting of flukes with spermatogenic ability based on the presence of sperm in the seminal vesicles (Table 1). Almost all specimens had many sperms in the seminal vesicle (spermic fluke) and one fluke did not contain any sperm in the seminal vesicle (aspermic fluke).

ITS1 types and species identification: The RFLP pattern of 149 spermic Fasciola isolates showed RFLP pattern of F. hepatica and F. gigantica. The aspermic sample had F. gigantica RFLP pattern.

ND1 and CO1 haplotypes and phylogeny: The ND1 fragments (approximately 535 bp) and CO1 fragments (approximately 438 bp) were amplified for all specimens. Partial sequences of NDI and COI showed 45 and 36 variable sites and also yielded 6 and 8 haplotypes respectively, which were represented by Accession No. KX021266 to KX021279 and deposited in the GeneBank (Table 1). Phylogenetic analysis based on NDI and COI sequence data were conducted by maximum likelihood using MEGA6 with lung fluke, Paragonimus westermani (Accession no. AF219379) designated as an out group branch showed in Fig. 1 and 2.

DISCUSSION

This is the first study on the basis of spermatogenesis characterization of Fasciola sp. in Iran. This study has shown that both spermic and aspermic Fasciola existed in the North West of Iran on the basis of the staining (haematoxylin-carmine dye).

The differentiation of Fasciola species is crucial due to their epidemiological patterns. All of the specimens in this study obtained from cattle (Bus taurus) that traditionally were nurtured. Cattle are the host for both Fasciola species in this region of Iran. The ratio of body length and width (BL/BW) has been considered to be one of the useful criteria for discrimination of both species in Fasciola15. There is only one aspermic sample in this region of Iran and the size of this fluke is similar in range with F. gigantica.

Some reports from the North West of Iran were detected Fasciola spp., with only ITS-RFLP method and constructed phylogenetic trees using nuclear rDNA but this method is not sufficient for Fasciola spp. taxonomic characterization16-18.

Fig. 1: A phylogenetic tree of Fasciola spp. based on ND1 gene in North West of Iran

Fig. 2: A phylogenetic tree of Fasciola spp. based on CO1 gene in North West of Iran

The existence of an endemic intermediate Fasciola form in Northern Iran based on the phylogenetic analysis of the nuclear rDNA19. Base on morphological study and phenotypic analysis of adults Fasciola flukes, there are F. hepatica, F. gigantica and intermediate forms in the endemic region of Gilan, Iran4. Although both species can generally be distinguished on the basis of their morphology but the use of molecular methods and markers are often necessary for species confirmation and to identification of the intermediate forms15.

Previous studies have shown that molecular phylogeny with mtDNA, including ND1 and CO1 can be effectively used for proper differentiation haplotypes and intermediate forms Fasciola sp10,20. Molecular analysis of the intermediate forms are mainly performed in the Far East Asian countries such as China, Japan, Korea and Vietnam14. Existence of several haplotype in North West of Iran demonstrated the effect of ecology and climate which indicates further need for studies in other regions of Iran. These differences showed in North East of Iran before12.

Iran is a vast country with numerous climate regions which may indicate variable Fasciola haplotypes. The phylogenetic analysis using Neighbor Joining as well as maximum likelihood methods showed separate position on both trees (Fig. 1, 2), which supported with different bootstrap values (%) from 1000 replicates probably reflecting the prominent nucleotide that may be attributed to host range and genetic migration. Also P. westermani (AF219379) was used as outgroup. Host specificity, drug susceptibility or resistance and differences in virulence may be influences molecular variation21.

CONCLUSION

It can be concluded that F. hepatica found in this region of Iran is closely related to F. hepatica found throughout of Iran and Asian countries like India and Bangladesh. Also, in this region of Iran, new aspermic haplotypes of F. gigantica are similar to African countries such as Egypt and Zambia and this aspermic Fasciola belonged to F. gigantica clades. Further genetic studies of Fasciola parasite obtained from hosts in different regions are necessary to show the diversity of Fasciola spp. and help the miserable hybrid of Fasciola sp. This is the first report of existence spermic and aspermic Fasciola in the North West of Iran.

ACKNOWLEDGMENT

The authors would like to thank Maragheh University of Medical Sciences for providing funding to this study (Project No: 3139-2017).

SIGNIFICANCE STATEMENTS

Fasciola hepatica and Fasciola gigantica possess abundant mature sperms in their seminal vesicles and thus, they reproduce bisexually. On the other hand, aspermic Fasciola flukes reported from Asian countries, which have no sperm in their seminal vesicles, probably reproduce parthenogenetically. The hybrid origin of aspermic Fasciola flukes was strongly suggested by the presence of the F. hepatica/F. gigantica type, which includes DNA fragments of both F. hepatica and F. gigantica.

REFERENCES
1:  McManus, D.P. and J.P. Dalton, 2006. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology, 133: S43-S61.
CrossRef  |  PubMed  |  Direct Link  |  

2:  Mas-Coma, S., M.D. Bargues and M.A. Valero, 2005. Fascioliasis and other plant-borne trematode zoonoses. Int. J. Parasitol., 35: 1255-1278.
CrossRef  |  Direct Link  |  

3:  Rokni, M.B., W.M. Lotfy, K. Ashrafi and K.D. Murrell, 2014. Fasciolosis in the MENA Region. In: Neglected Tropical Diseases-Middle East and North Africa, McDowell, M.A. and S. Rafati (Eds.). Springer, New York, ISBN: 9783709116135, pp: 59-90.

4:  Ashrafi, K., M.A. Valero, M. Panova, M.V. Periago, J. Massoud and S. Mas-Coma, 2006. Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitol. Int., 55: 249-260.
CrossRef  |  Direct Link  |  

5:  Peng, M., M. Ichinomiya, M. Ohtori, M. Ichikawa, T. Shibahara and T. Itagaki, 2009. Molecular characterization of Fasciola hepatica, Fasciola gigantica and aspermic Fasciola sp. in China based on nuclear and mitochondrial DNA. Parasitol. Res., 105: 809-815.
CrossRef  |  PubMed  |  Direct Link  |  

6:  Terasaki, K., T. Itagaki, T. Shibahara, Y. Noda and N. Moriyama-Gonda, 2001. Comparative study of the reproductive organs of Fasciola groups by optical microscope. J. Vet. Med. Sci., 63: 735-742.
CrossRef  |  Direct Link  |  

7:  Chaichanasak, P., M. Ichikawa, P. Sobhon and T. Itagaki, 2012. Identification of Fasciola flukes in Thailand based on their spermatogenesis and nuclear ribosomal DNA and their intraspecific relationships based on mitochondrial DNA. Parasitol. Int., 61: 545-549.
CrossRef  |  Direct Link  |  

8:  Prasad, P.K., V. Tandon, D. Biswal, L. Goswami and A. Chatterjee, 2008. Molecular identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae) based on the ribosomal internal transcribed spacer regions. Parasitol. Res., 103: 1247-1255.
CrossRef  |  PubMed  |  Direct Link  |  

9:  Periago, M.V., M.A. Valero, M. Panova and S. Mas-Coma, 2006. Phenotypic comparison of allopatric populations of Fasciola hepatica and Fasciola gigantica from European and African bovines using a computer image analysis system (CIAS). Parasitol. Res., 99: 368-378.
CrossRef  |  PubMed  |  Direct Link  |  

10:  Itagaki, T., M. Kikawa, K. Sakaguchi, J. Shimo, K. Terasaki, T. Shibahara and K. Fukuda, 2005. Genetic characterization of parthenogenic Fasciola sp. in Japan on the basis of the sequences of ribosomal and mitochondrial DNA. Parasitology, 131: 679-685.
CrossRef  |  Direct Link  |  

11:  Ichikawa, M., N. Iwata and T. Itagaki, 2010. DNA types of aspermic Fasciola species in Japan. J. Vet. Med. Sci., 72: 1371-1374.
CrossRef  |  Direct Link  |  

12:  Reaghi, S., A. Haghighi, M.F. Harandi, A. Spotin, K. Arzamani and S. Rouhani, 2016. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran. Vet. World, 9: 1034-1038.
CrossRef  |  Direct Link  |  

13:  Ashrafi, K., 2015. The status of human and animal Fascioliasis in Iran: A narrative review article. Iran. J. Parasitol., 10: 306-328.
Direct Link  |  

14:  Mohanta, U.K., M. Ichikawa-Seki, T. Shoriki, K. Katakura and T. Itagaki, 2014. Characteristics and molecular phylogeny of Fasciola flukes from Bangladesh, determined based on spermatogenesis and nuclear and mitochondrial DNA analyses. Parasitol. Res., 113: 2493-2501.
CrossRef  |  Direct Link  |  

15:  Amer, S., A. ElKhatam, S. Zidan, Y. Feng and L. Xiao, 2016. Identity of Fasciola spp. in sheep in Egypt. Parasites Vectors, Vol. 9. 10.1186/s13071-016-1898-2

16:  Aryaeipour, M., R. Soheila, B. Mojgan, M. Hadi, K. Bahram, B.R. Mohammad, 2014. Genotyping and phylogenetic analysis of fasciola spp. isolated from sheep and cattle using pcr-rflp in Ardabil province, Northwestern Iran. Iran. J. Pub. Health, 43: 1364-1371.
PubMed  |  Direct Link  |  

17:  Galavani, H., S. Gholizadeh and K.H. Tappeh, 2016. Genetic characterization of Fasciola isolates from west Azerbaijan province Iran based on ITS1 and ITS2 sequence of ribosomal DNA. Iran J. Parasitol., 11: 52-64.
Direct Link  |  

18:  Yakhchali, M., R. Malekzadeh-Viayeh, A. Imani-Baran and K. Mardani, 2015. Morphological and molecular discrimination of Fasciola species isolated from domestic ruminants of Urmia City, Iran. Iran. J. Parasitol., 10: 46-55.
Direct Link  |  

19:  Amor, N., A. Halajian, S. Farjallah, P. Merella, K. Said and B.B. Slimane, 2011. Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences. Exp. Parasitol., 128: 196-204.
CrossRef  |  Direct Link  |  

20:  Ichikawa-Seki, M., P. Ortiz, M. Cabrera, C. Hoban and T. Itagaki, 2016. Molecular characterization and phylogenetic analysis of Fasciola hepatica from Peru. Parasitol. Int., 65: 171-174.
CrossRef  |  Direct Link  |  

21:  Constantine, C.C., 2003. Importance and pitfalls of molecular analysis to parasite epidemiology. Trends Parasitol., 19: 346-348.
CrossRef  |  Direct Link  |  

©  2021 Science Alert. All Rights Reserved