High-Resolution Melting Analysis as an Appropriate Method to Differentiate between Fasciola hepatica and F. gigantica
-
Ahmad HOSSEINI-SAFA
,
Mohammad Bagher ROKNI
,
Sayed Hussain MOSAWI
,
Peyman HEYDARIAN
,
Hakim AZIZI
,
Afshin DAVARI
,
Mojgan ARYAIEPOUR
Abstract
Background: Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-Resolution Melting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively.
Methods: Ninety-three Fasciola spp. samples were collected from infected slaughtered animals in different regions of Iran, including North West (Ardebil Province) and South East (Zahedan Province) during 2016. Genomic DNA from the samples was extracted using a DNA extraction kit and then after Real-Time PCR amplification, HRM was done.
Results: Overall, 59 and 34 isolates were identified as F. hepatica and F. gigantica, respectively. The percentages of each species from animals were as follows: sheep (F. hepatica, 80.39% and F. gigantica, 19.61%), cattle (F. hepatica, 42.85% and F. gigantica, 57.15%).
Conclusion: HRM technique developed in the present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.
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2. Rokni MB, Lotfy WM, Ashrafi K, Murrell KD (2014). Neglected Tropical Diseases-Middle East and North Africa. Springer, 59-90.
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5. Marcilla A, Bargues MD, Mas-Coma S (2002). A PCR-RFLP assay for the dis-tinction between Fasciola hepatica and Fasciola gigantica. Mol Cell Probes, 16(5):327-333.
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7. Alasaad S, Soriguer RC, Abu-Madi M et al (2011). A TaqMan real-time PCR-based assay for the identification of Fasciola spp. Vet Parasitol, 179(1-3), 266-271.
8. Huang W, He B, Wang C, Zhu X( 2004). Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet Parasitol,120(1-2), 75-83.
9. Magalhães KG, Passos LKJ, Carvalho OdS (2004). Detection of Lymnaea columella in-fection by Fasciola hepatica through Multi-plex-PCR. Mem Inst Oswaldo Cruz, 99(4): 421-424.
10. Schweizer G, Meli M, Torgerson PR et al (2007). Prevalence of Fasciola hepatica in the intermediate host Lymnaea truncatula detected by real time TaqMan PCR in populations from 70 Swiss farms with cattle husbandry. Vet Parasitol, 150(1-2): 164-169.
11. Ngui R, Lim YA, Chua KH ( 2012). Rapid detection and identification of human hookworm infections through high reso-lution melting (HRM) analysis. PloS One ,7(7):e 41996.
12. Chua KH, Lim SC, Ng CC et al (2015). De-velopment of high resolution melting analysis for the diagnosis of human ma-laria. Scientific Reports, 5:15671.
13. Rojas A, Segev G, Markovics A, Aroch I, Baneth G (2017). Detection and quantifi-cation of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocerco-sis. Parasites Vectors, 10: 435
14. Tun S, Ithoi I, Mahmud R et al (2015). De-tection of helminth eggs and identifica-tion of hookworm species in stray cats, dogs and soil from Klang Valley, Malay-sia. PloS One, 10(12): e0142231.
15. Rokni MB, Mirhendi H, Mizani A et al (2010). Identification and differentiation of Fasciola hepatica and Fasciola gigantica us-ing a simple PCR-restriction enzyme method. Exp Parasitol,124(2): 209-213.
16. Aryaeipour M, Rouhani S, Bandehpour M et al (2014). Genotyping and phylogenetic analysis of Fasciola spp. isolated from sheep and cattle using PCR-RFLP in Ar-dabil province, northwestern Iran. Iran J Public Health, 43( 10): 1364–71.
17. Aryaeipour M, Bozorgomid A, Kazemi B et al (2017). Molecular and Morphometrical Characterization of Fasciola Species Isolat-ed from Domestic Ruminants in Ardabil Province, Northwestern Iran. Iran J Public Health, 46(3):318-325.
18. Higuera SL, Guhl F, Ramírez JD (2013). Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the im-plementation of a High-Resolution Melt-ing (HRM) genotyping assay. Parasites Vectors, 6:112.
19. Rokni MB, Bozorgomid A, Heydarian P, Aryaeipour M (2018). Molecular Evidence of Human Fasciolosis Due to Fasciola gi-gantica in Iran: A Case Report. Iran J Public Health, 47(5):750-754.
20. Aryaeipour M, Kia EB, Heidari Z, Sayyad Talaie Z, Rokni MB (2015). Serological study of Human Fasciolosis in Patients Referring to the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2008-2014. Iran J Parasitol,10(4):517-522
21. Itagaki T, Tsutsumi K, Sakamoto T, Tsu-tsumi Y (1995). Characterization of genet-ic divergence among species within the genus Fasciola by PCR-SSCP. Japanese J Parasitol, 44, 244-247.
22. Krämer F, Schnieder T(1998). Sequence het-erogeneity in a repetitive DNA element of Fasciola. Int J Parasitol ,28: 1923-9.
23. Bozorgomid A, Nazari N, Rahimi H et al (2016). Molecular Characterization of An-imal Fasciola spp. Isolates from Kerman-shah, Western Iran. Iran J Public Health,45(10):1315-1321
24. Le TH, Nguyen KT, Nguyen NT et al (2012). Development and evaluation of a single step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gi-gantica (Fasciolidae; Trematoda; Platyhel-minthes). J Clin Microbiol, 50(8):2720-6.
25. Magalhaes KG, Jannotti-Passos LK, Caldei-ra RL et al (2008). Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR. Vet Parasitol,152(3-4): 333-338.
26. Hosseini-Safa A, Mohag MA et al (2016). First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis. Gastroenterol Hepatol Bed Bench, 9(Suppl1):S70-S74.
27. Safa AH, Harandi MF, Tajaddini M et al (2016). Rapid identification of Echinococcus granulosus and E. canadensis using high-resolution melting (HRM) analysis by fo-cusing on a single nucleotide polymor-phism. Jap J Infect Dis, 69(4):300-305.
28. Pangasa A, Jex AR, Campbell BE et al (2009). High resolution melting-curve (HRM) analysis for the diagnosis of cryp-tosporidiosis in humans. Mol Cell Probes, 23(1): 10-5.
29. Radvánský J, Bazsalovicsová E, Králová-Hromadová I, Minárik G, Kádaši Ľ (2011). Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trema-toda: Fasciolidae), the giant liver fluke of ruminants. Parasitol Res, 108(1): 201-209.
2. Rokni MB, Lotfy WM, Ashrafi K, Murrell KD (2014). Neglected Tropical Diseases-Middle East and North Africa. Springer, 59-90.
3. Simsek S, Utuk AE, Balkaya I (2011). Molec-ular differentiation of Turkey cattle iso-lates of Fasciola hepatica and Fasciola gigantica. Helminthologia, 48, 3-7.
4. Zhang P, Liu Y, Alsarakibi M et al (2012). Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages. Parasi-tol Res, 111(5), 2157-2163.
5. Marcilla A, Bargues MD, Mas-Coma S (2002). A PCR-RFLP assay for the dis-tinction between Fasciola hepatica and Fasciola gigantica. Mol Cell Probes, 16(5):327-333.
6. Cai XQ, Yu HQ, Li R et al (2014). Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis. ScientificWorldJournal, 893981.
7. Alasaad S, Soriguer RC, Abu-Madi M et al (2011). A TaqMan real-time PCR-based assay for the identification of Fasciola spp. Vet Parasitol, 179(1-3), 266-271.
8. Huang W, He B, Wang C, Zhu X( 2004). Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet Parasitol,120(1-2), 75-83.
9. Magalhães KG, Passos LKJ, Carvalho OdS (2004). Detection of Lymnaea columella in-fection by Fasciola hepatica through Multi-plex-PCR. Mem Inst Oswaldo Cruz, 99(4): 421-424.
10. Schweizer G, Meli M, Torgerson PR et al (2007). Prevalence of Fasciola hepatica in the intermediate host Lymnaea truncatula detected by real time TaqMan PCR in populations from 70 Swiss farms with cattle husbandry. Vet Parasitol, 150(1-2): 164-169.
11. Ngui R, Lim YA, Chua KH ( 2012). Rapid detection and identification of human hookworm infections through high reso-lution melting (HRM) analysis. PloS One ,7(7):e 41996.
12. Chua KH, Lim SC, Ng CC et al (2015). De-velopment of high resolution melting analysis for the diagnosis of human ma-laria. Scientific Reports, 5:15671.
13. Rojas A, Segev G, Markovics A, Aroch I, Baneth G (2017). Detection and quantifi-cation of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocerco-sis. Parasites Vectors, 10: 435
14. Tun S, Ithoi I, Mahmud R et al (2015). De-tection of helminth eggs and identifica-tion of hookworm species in stray cats, dogs and soil from Klang Valley, Malay-sia. PloS One, 10(12): e0142231.
15. Rokni MB, Mirhendi H, Mizani A et al (2010). Identification and differentiation of Fasciola hepatica and Fasciola gigantica us-ing a simple PCR-restriction enzyme method. Exp Parasitol,124(2): 209-213.
16. Aryaeipour M, Rouhani S, Bandehpour M et al (2014). Genotyping and phylogenetic analysis of Fasciola spp. isolated from sheep and cattle using PCR-RFLP in Ar-dabil province, northwestern Iran. Iran J Public Health, 43( 10): 1364–71.
17. Aryaeipour M, Bozorgomid A, Kazemi B et al (2017). Molecular and Morphometrical Characterization of Fasciola Species Isolat-ed from Domestic Ruminants in Ardabil Province, Northwestern Iran. Iran J Public Health, 46(3):318-325.
18. Higuera SL, Guhl F, Ramírez JD (2013). Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the im-plementation of a High-Resolution Melt-ing (HRM) genotyping assay. Parasites Vectors, 6:112.
19. Rokni MB, Bozorgomid A, Heydarian P, Aryaeipour M (2018). Molecular Evidence of Human Fasciolosis Due to Fasciola gi-gantica in Iran: A Case Report. Iran J Public Health, 47(5):750-754.
20. Aryaeipour M, Kia EB, Heidari Z, Sayyad Talaie Z, Rokni MB (2015). Serological study of Human Fasciolosis in Patients Referring to the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2008-2014. Iran J Parasitol,10(4):517-522
21. Itagaki T, Tsutsumi K, Sakamoto T, Tsu-tsumi Y (1995). Characterization of genet-ic divergence among species within the genus Fasciola by PCR-SSCP. Japanese J Parasitol, 44, 244-247.
22. Krämer F, Schnieder T(1998). Sequence het-erogeneity in a repetitive DNA element of Fasciola. Int J Parasitol ,28: 1923-9.
23. Bozorgomid A, Nazari N, Rahimi H et al (2016). Molecular Characterization of An-imal Fasciola spp. Isolates from Kerman-shah, Western Iran. Iran J Public Health,45(10):1315-1321
24. Le TH, Nguyen KT, Nguyen NT et al (2012). Development and evaluation of a single step duplex PCR for simultaneous detection of Fasciola hepatica and Fasciola gi-gantica (Fasciolidae; Trematoda; Platyhel-minthes). J Clin Microbiol, 50(8):2720-6.
25. Magalhaes KG, Jannotti-Passos LK, Caldei-ra RL et al (2008). Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR. Vet Parasitol,152(3-4): 333-338.
26. Hosseini-Safa A, Mohag MA et al (2016). First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis. Gastroenterol Hepatol Bed Bench, 9(Suppl1):S70-S74.
27. Safa AH, Harandi MF, Tajaddini M et al (2016). Rapid identification of Echinococcus granulosus and E. canadensis using high-resolution melting (HRM) analysis by fo-cusing on a single nucleotide polymor-phism. Jap J Infect Dis, 69(4):300-305.
28. Pangasa A, Jex AR, Campbell BE et al (2009). High resolution melting-curve (HRM) analysis for the diagnosis of cryp-tosporidiosis in humans. Mol Cell Probes, 23(1): 10-5.
29. Radvánský J, Bazsalovicsová E, Králová-Hromadová I, Minárik G, Kádaši Ľ (2011). Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trema-toda: Fasciolidae), the giant liver fluke of ruminants. Parasitol Res, 108(1): 201-209.
| Files | ||
| Issue | Vol 48 No 3 (2019) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijph.v48i3.894 | |
| Keywords | ||
| High-resolution melting (HRM) Fasciola hepatica Fasciola gigantica CoxI | ||
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How to Cite
1.
HOSSEINI-SAFA A, ROKNI MB, MOSAWI SH, HEYDARIAN P, AZIZI H, DAVARI A, ARYAIEPOUR M. High-Resolution Melting Analysis as an Appropriate Method to Differentiate between Fasciola hepatica and F. gigantica. Iran J Public Health. 2019;48(3):501-507.
