Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM)
-
Elham Hajialilo
,
Ahmad Hosseini-Safa
,
Adel Spotin
,
Mehrzad Saraei
,
Morteza Ghanbari Johkool
,
Hossein Piri
,
Peyman Heydarian
Abstract
Background: The liver flukes of the Fasciola species and Dicrocoelium spp. are recognised as parasites of domestic and wild herbivores. Both species of F. hepatica and F. gigantica as well as D. dendriticum are distributed in Iran. The present study aimed to identify Fasciola spp. and Dicrocoelium spp. using mitochondrial Cox1 (cytochrome c oxidase I) gene by HRM method.
Methods: Totally, thirty infected liver specimens were collected from the sheep (n:23) and cattle (n:7) at the abattoirs of Qazvin Province, northwest Iran in 2022. DNA extraction and PCR amplification of Cox1 gene were conducted by HRM technique. DnaSP v.5.0 was used for compression of diversity indices of ribosomal 28S rDNA and mitochondrial Cox1 markers of Dicrocoelium spp. The taxonomic status of Dicrocoelium spp. was performed by sequencing and phylogenetic analysis.
Results: Overall, 26 and 4 isolates were identified as F. hepatica and F. gigantica, respectively. D. dendriticum was the sole infecting species of Dicrocoelium revealed by HRM analysis. Genomic analysis showed a moderate (28S rDNA genes: 0.600±0.215) to high (Cox1: 0.733±0.155) haplotype diversity for D. dendriticum.
Conclusion: The parasite-dependent mitochondrial gene (Cox1) could identify a higher genetic diversity of D. dendriticum compared to nuclear 28S rDNA gene. HRM technique in the present study found to be a reliable technique for identification and genetic diversity of liver flukes but more comprehensive and in-depth studies in different parts of the country are needed.
1. Bazsalovicsová E, Králová-Hromadová I, Špakulová M, Reblánová M, Oberhauserová K (2010). Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants. Helminthologia, 47:76-82.
2. Mas-Coma S, Bargues MD, Valero M (2005). Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol, 35:1255-1278.
3. Mas-Coma S, Bargues M (1997). Human liver flukes: a review. Res Rev Parasitol, 57:145-218.
4. Sohrabi A, Amini F )1967(. A report of the liver distomatosis in the northern part of Iran. Report Inst Pub Hlth Res, Tehran. (1617).
5. Ashrafi K, Valero M, Panova M, Periago M, Massoud J, Mas-Coma S (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.
6. Forghanparast K, Ashrafi K (2000). Comparison of the Formalin-Ether and the Katz Kato in The Parasitological Diagnosis of Human Fascioliasis. Journal of Guilan University of Medical Sciences, 9:1-5.
7. Arjona R, Riancho JA, Aguado JM, Salesa R, Gonzalez-Macias J (1995). Fascioliasis in developed countries: a review of classic and aberrant forms of the disease. Medicine, 74:13-23.
8. Mas-Coma S (2005a). Epidemiology of fascioliasis in human endemic areas. J Helminthol, 79:207-216.
9. Pritchard G, Forbes A, Williams DJ, Salimi‐Bejestani M, Daniel R (2005). Emergence of fasciolosis in cattle in East Anglia. Vet Rec, 157:578-582.
10. Otranto D, Traversa D (2003). Dicrocoeliosis of ruminants: a little known fluke disease. Trends Parasitol, 19:12-15.
11. Stein C, Kuchenmüller T, Hendrickx S, et al (2007). The global burden of disease assessments—WHO is responsible? PLoS Negl Trop Dis, 1:e161.
12. 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, 179:266-271.
13. Hosseini-Safa A, Rokni MB, Mosawi SH, et al (2019). High-resolution melting analysis as an appropriate method to differentiate between Fasciola hepatica and F. gigantica. Iran J Public Health, 48:501-507.
14. Magalhães KG, Passos LKJ, Carvalho OdS (2004). Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR. Mem Inst Oswaldo Cruz, 99:421-424.
15. Morozova E, Ryskov A, Semyenova S (2002). RAPD variation in two trematode species (Fasciola hepatica and Dicrocoelium dendriticum) from a single cattle population. Genetika, 38:977-983.
16. Ngui R, Lim YA, Chua KH (2012). Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis. PloS One, 7:e41996.
17. Schweizer G, Meli ML, Torgerson PR, Lutz H, Deplazes P, Braun U (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, 150:164-169.
18. Shahnazi M, Ebadi M, Abbaspoor Z, et al (2020). Molecular characterization of Fasciola and Dicrocoelium species isolated from ruminant livestock in Qazvin, Iran. Infect Disord Drug Targets, 20:737-742.
19. Shokouhi S, Mirzaei A, Naserifar R, Abdi J (2018). Dicrocoelium dendriticum Infection Among Livestock In Western Iran. Southeast Asian J Trop Med Public Health, 49:755-760.
20. Hussein EM, Al-Mohammed HI, Hussein AM (2009). Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis. Parasitol Res, 105:1053-1060.
21. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem, 50:1156-1164.
22. Pangasa A, Jex AR, Campbell BE, et al (2009). High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. Mol Cell Probes, 23:10-15.
23. Radvansky J, Resko P, Surovy M, Minarik G, Ficek A, Kadasi L (2010). High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism. Anal Biochem, 398:126-128.
24. Talmi-Frank D, Nasereddin A, Schnur LF, et al (2010). Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis, 12;4(1):e581.
25. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem, 49:853-860.
26. Moghadamizad Z, Hosseini-Safa A, Mohebali M, Heydarian P, Aryaeipour M, Rokni MB (2020). Specific detection of Fasciola hepatica and F. gigantica in infected domesticated animals using high-resolution melting analysis (HRM). Iran J Public Health, 49:521-529.
27. Arbabi M, Hooshyar H, Lotfinia M, Bakhshi MA (2020). Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences. Mol Biochem Parasitol, 236:111260.
28. Aryaeipour M, Rouhani S, Bandehpour M, Mirahmadi H, Kazemi B, Rokni MB (2014). Genotyping and phylogenetic analysis of Fasciola spp. isolated from sheep and cattle using PCR-RFLP in Ardabil province, northwestern Iran. Iran J Public Health, 43:1364-71.
29. Bozorgomid A, Rouhani S, Harandi MF, Ichikawa-Seki M, Raeghi S (2020). Genetic diversity and distribution of Fasciola hepatica haplotypes in Iran: Molecular and phylogenetic studies. Vet Parasitol Reg Stud Reports, 19:100359.
30. Reaghi S, Haghighi A, Harandi MF, Spotin A, Arzamani K, Rouhani S (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, 9:1034-38.
31. Vilas R, Criscione C, Blouin M (2005). A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites. Parasitology, 131:839-46.
32. Mitchell G, Cuthill G, Haine A, et al (2017). Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum. Vet Parasitol, 235:100-105.
33. Farjallah S, Slimane BB, Piras CM, Amor N, Garippa G, Merella P (2013). Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers. Exp Parasitol, 135:471-478.
34. Ashrafi K, Valero MA, Peixoto RV, Artigas P, Panova M, Mas-Coma S (2015). Distribution of Fasciola hepatica and F. gigantica in the endemic area of Guilan, Iran: Relationships between zonal overlap and phenotypic traits. Infect Genet Evol, 31:95-109.
35. Bozorgomid A, Nazari N, Rahimi H, et al (2016). Molecular characterization of animal Fasciola spp. isolates from Kermanshah, Western Iran. Iran J Public Health, 45:1315-21.
36. Yakhchali M, Malekzadeh-Viayeh R, Imani-Baran A, Mardani K (2015). Morphological and molecular discrimination of Fasciola species isolated from domestic ruminants of Urmia city, Iran. Iran J Parasitol, 10:46-55.
37. Peng M, Ichinomiya M, Ohtori M, Ichikawa M, Shibahara T, Itagaki T (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.
38. Rouhani S, Raeghi S, Spotin A (2017). Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran. Pak J Biol Sci, 20:204-209.
39. Sharifiyazdi H, Moazeni M, Rabbani F (2012). Molecular characterization of human Fasciola samples in Gilan province, Northern Iran on the basis of DNA sequences of ribosomal and mitochondrial DNA genes. Comp Clin Path, 21:889-894.
40. Gasser RB (2006). Molecular tools—advances, opportunities and prospects. Vet Parasitol, 136:69-89.
41. Hu M, Gasser RB (2006). Mitochondrial genomes of parasitic nematodes–progress and perspectives. Trends Parasitol, 22:78-84.
42. Jex AR, Littlewood DTJ, Gasser RB (2010). Toward next-generation sequencing of mitochondrial genomes—focus on parasitic worms of animals and biotechnological implications. Biotechnol Adv, 28:151-159.
43. Zarowiecki M, Huyse T, Littlewood D (2007). Making the most of mitochondrial genomes–markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea). Int J Parasitol, 37:1401-1418.
44. Le T, Blair D, McManus D (2001). Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L.(Platyhelminthes; Trematoda). Parasitology, 123:609-621.
45. Liu GH, Yan HB, Otranto D, et al (2014). Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences. Mol Phylogenet Evol, 79:325-331.
46. Wang XY, Zhao GH, Liu GH, et al (2013). Characterization of Dicrocoelium chinensis from domestic yaks in Gansu and Sichuan provinces, China, using genetic markers in two mitochondrial genes. Mitochondrial DNA, 24:263-266.
47. Khan MA, Afshan K, Nazar M, Firasat S, Chaudhry U, Sargison ND (2020). Genetic analysis confirms the presence of Dicrocoelium dendriticum in the Himalaya ranges of Pakistan. Parasitol Int, 81:102276.
48. Zhao GH, Bian QQ, Ren WX, et al (2013). Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes. Mitochondrial DNA, 24:683-688.
49. Spotin A, Mahami-Oskouei M, Harandi MF, et al (2017). Genetic variability of Echinococcus granulosus complex in various geographical populations of Iran inferred by mitochondrial DNA sequences. Acta Trop, 165:10-16.
2. Mas-Coma S, Bargues MD, Valero M (2005). Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol, 35:1255-1278.
3. Mas-Coma S, Bargues M (1997). Human liver flukes: a review. Res Rev Parasitol, 57:145-218.
4. Sohrabi A, Amini F )1967(. A report of the liver distomatosis in the northern part of Iran. Report Inst Pub Hlth Res, Tehran. (1617).
5. Ashrafi K, Valero M, Panova M, Periago M, Massoud J, Mas-Coma S (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.
6. Forghanparast K, Ashrafi K (2000). Comparison of the Formalin-Ether and the Katz Kato in The Parasitological Diagnosis of Human Fascioliasis. Journal of Guilan University of Medical Sciences, 9:1-5.
7. Arjona R, Riancho JA, Aguado JM, Salesa R, Gonzalez-Macias J (1995). Fascioliasis in developed countries: a review of classic and aberrant forms of the disease. Medicine, 74:13-23.
8. Mas-Coma S (2005a). Epidemiology of fascioliasis in human endemic areas. J Helminthol, 79:207-216.
9. Pritchard G, Forbes A, Williams DJ, Salimi‐Bejestani M, Daniel R (2005). Emergence of fasciolosis in cattle in East Anglia. Vet Rec, 157:578-582.
10. Otranto D, Traversa D (2003). Dicrocoeliosis of ruminants: a little known fluke disease. Trends Parasitol, 19:12-15.
11. Stein C, Kuchenmüller T, Hendrickx S, et al (2007). The global burden of disease assessments—WHO is responsible? PLoS Negl Trop Dis, 1:e161.
12. 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, 179:266-271.
13. Hosseini-Safa A, Rokni MB, Mosawi SH, et al (2019). High-resolution melting analysis as an appropriate method to differentiate between Fasciola hepatica and F. gigantica. Iran J Public Health, 48:501-507.
14. Magalhães KG, Passos LKJ, Carvalho OdS (2004). Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR. Mem Inst Oswaldo Cruz, 99:421-424.
15. Morozova E, Ryskov A, Semyenova S (2002). RAPD variation in two trematode species (Fasciola hepatica and Dicrocoelium dendriticum) from a single cattle population. Genetika, 38:977-983.
16. Ngui R, Lim YA, Chua KH (2012). Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis. PloS One, 7:e41996.
17. Schweizer G, Meli ML, Torgerson PR, Lutz H, Deplazes P, Braun U (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, 150:164-169.
18. Shahnazi M, Ebadi M, Abbaspoor Z, et al (2020). Molecular characterization of Fasciola and Dicrocoelium species isolated from ruminant livestock in Qazvin, Iran. Infect Disord Drug Targets, 20:737-742.
19. Shokouhi S, Mirzaei A, Naserifar R, Abdi J (2018). Dicrocoelium dendriticum Infection Among Livestock In Western Iran. Southeast Asian J Trop Med Public Health, 49:755-760.
20. Hussein EM, Al-Mohammed HI, Hussein AM (2009). Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis. Parasitol Res, 105:1053-1060.
21. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem, 50:1156-1164.
22. Pangasa A, Jex AR, Campbell BE, et al (2009). High resolution melting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans. Mol Cell Probes, 23:10-15.
23. Radvansky J, Resko P, Surovy M, Minarik G, Ficek A, Kadasi L (2010). High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism. Anal Biochem, 398:126-128.
24. Talmi-Frank D, Nasereddin A, Schnur LF, et al (2010). Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis, 12;4(1):e581.
25. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem, 49:853-860.
26. Moghadamizad Z, Hosseini-Safa A, Mohebali M, Heydarian P, Aryaeipour M, Rokni MB (2020). Specific detection of Fasciola hepatica and F. gigantica in infected domesticated animals using high-resolution melting analysis (HRM). Iran J Public Health, 49:521-529.
27. Arbabi M, Hooshyar H, Lotfinia M, Bakhshi MA (2020). Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences. Mol Biochem Parasitol, 236:111260.
28. Aryaeipour M, Rouhani S, Bandehpour M, Mirahmadi H, Kazemi B, Rokni MB (2014). Genotyping and phylogenetic analysis of Fasciola spp. isolated from sheep and cattle using PCR-RFLP in Ardabil province, northwestern Iran. Iran J Public Health, 43:1364-71.
29. Bozorgomid A, Rouhani S, Harandi MF, Ichikawa-Seki M, Raeghi S (2020). Genetic diversity and distribution of Fasciola hepatica haplotypes in Iran: Molecular and phylogenetic studies. Vet Parasitol Reg Stud Reports, 19:100359.
30. Reaghi S, Haghighi A, Harandi MF, Spotin A, Arzamani K, Rouhani S (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, 9:1034-38.
31. Vilas R, Criscione C, Blouin M (2005). A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites. Parasitology, 131:839-46.
32. Mitchell G, Cuthill G, Haine A, et al (2017). Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum. Vet Parasitol, 235:100-105.
33. Farjallah S, Slimane BB, Piras CM, Amor N, Garippa G, Merella P (2013). Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers. Exp Parasitol, 135:471-478.
34. Ashrafi K, Valero MA, Peixoto RV, Artigas P, Panova M, Mas-Coma S (2015). Distribution of Fasciola hepatica and F. gigantica in the endemic area of Guilan, Iran: Relationships between zonal overlap and phenotypic traits. Infect Genet Evol, 31:95-109.
35. Bozorgomid A, Nazari N, Rahimi H, et al (2016). Molecular characterization of animal Fasciola spp. isolates from Kermanshah, Western Iran. Iran J Public Health, 45:1315-21.
36. Yakhchali M, Malekzadeh-Viayeh R, Imani-Baran A, Mardani K (2015). Morphological and molecular discrimination of Fasciola species isolated from domestic ruminants of Urmia city, Iran. Iran J Parasitol, 10:46-55.
37. Peng M, Ichinomiya M, Ohtori M, Ichikawa M, Shibahara T, Itagaki T (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.
38. Rouhani S, Raeghi S, Spotin A (2017). Spermatogenic and Phylo-molecular Characterizations of Isolated Fasciola Spp. From Cattle, North West Iran. Pak J Biol Sci, 20:204-209.
39. Sharifiyazdi H, Moazeni M, Rabbani F (2012). Molecular characterization of human Fasciola samples in Gilan province, Northern Iran on the basis of DNA sequences of ribosomal and mitochondrial DNA genes. Comp Clin Path, 21:889-894.
40. Gasser RB (2006). Molecular tools—advances, opportunities and prospects. Vet Parasitol, 136:69-89.
41. Hu M, Gasser RB (2006). Mitochondrial genomes of parasitic nematodes–progress and perspectives. Trends Parasitol, 22:78-84.
42. Jex AR, Littlewood DTJ, Gasser RB (2010). Toward next-generation sequencing of mitochondrial genomes—focus on parasitic worms of animals and biotechnological implications. Biotechnol Adv, 28:151-159.
43. Zarowiecki M, Huyse T, Littlewood D (2007). Making the most of mitochondrial genomes–markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes: Digenea). Int J Parasitol, 37:1401-1418.
44. Le T, Blair D, McManus D (2001). Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L.(Platyhelminthes; Trematoda). Parasitology, 123:609-621.
45. Liu GH, Yan HB, Otranto D, et al (2014). Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences. Mol Phylogenet Evol, 79:325-331.
46. Wang XY, Zhao GH, Liu GH, et al (2013). Characterization of Dicrocoelium chinensis from domestic yaks in Gansu and Sichuan provinces, China, using genetic markers in two mitochondrial genes. Mitochondrial DNA, 24:263-266.
47. Khan MA, Afshan K, Nazar M, Firasat S, Chaudhry U, Sargison ND (2020). Genetic analysis confirms the presence of Dicrocoelium dendriticum in the Himalaya ranges of Pakistan. Parasitol Int, 81:102276.
48. Zhao GH, Bian QQ, Ren WX, et al (2013). Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes. Mitochondrial DNA, 24:683-688.
49. Spotin A, Mahami-Oskouei M, Harandi MF, et al (2017). Genetic variability of Echinococcus granulosus complex in various geographical populations of Iran inferred by mitochondrial DNA sequences. Acta Trop, 165:10-16.
| Files | ||
| Issue | Vol 52 No 4 (2023) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijph.v52i4.12455 | |
| Keywords | ||
| High-resolutionmelting (HRM) Fasciola hepatica Fasciola gigantica Dicrocoelium dendriticum CoxI | ||
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How to Cite
1.
Hajialilo E, Hosseini-Safa A, Spotin A, Saraei M, Ghanbari Johkool M, Piri H, Heydarian P. Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM). Iran J Public Health. 2023;52(4):818-828.
