Accurate Identification of Leishmania Parasites in Sandflies by Polymorphism Analysis of COII Gene Using PCR and qPCR-HRM Techniques in Iranian Border with Iraq
Identifying Leishmania by COII and qPCR
Abstract
Background: Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sandflies are important for treatment and local control.
Methods: Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sandflies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concentration was measured at 260 and 280 nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Results: Among about 3000 collected sandflies, 89 female P. papatasi were identified and two with L. major. In amplified fragment of COII gene among 611 bp, 452 bp had no genetic variations with low polymorphic sites (P=0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). L. major was discriminated in P. papatasi with 0.84 °C Tm and unique curve based on thermodynamic differences was an important criteria using HRM technique.
Conclusion: Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accurate diagnostic procedures for leishmaniasis control.
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2. Ghafari SM, Fotouhi-Ardakani R, Parvizi P (2020a) Designing and developing a high- resolution melting technique for accu- rate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real- time PCR and in silico genetic evalua- tion. Acta Trop. 211: e105626.
3. Ghafari SM, Ebrahimi S, Nateghi-Rostami M, Bordbar A, Parvizi P (2020b) Com- parative evaluation of salivary glands pro- teomes from wild Phlebotomus papatasi proven vector of zoonotic cutaneous leishmaniasis. Vet Med Sci. 00: 1–8.
4. Dostálová A, Volf P (2012) Leishmania de- velopment in sand flies: parasite-vector interactions overview. Parasit Vectors. 5: 276.
5. Parvizi P, Ready PD (2008) Nested PCRs and sequencing of nuclear ITS-rDNA frag-
ments detect three Leishmania species of gerbils in sand flies from Iranian foci of
zoonotic cutaneous leishmaniasis. Trop Med Int Health. 13(9): 1159–1171.
6. Müller KE, Zampieri RA, Aoki JI, MuxelSM, Nerland AH, Floeter-Winter LM (2018) Amino acid permease 3 (aap3) Coding sequence as a target for Leish- mania identification and diagnosis of leish- maniases using high resolution Melting analysis. Parasit Vectors. 11: 421.
7. Asfaram S, Fakhar M, Mirani N, De- rakhshani niya M, Valadan R, Ziaei Hez- arjaribi H, Emadi SN (2019) HRM–PCR is an accurate and sensitive technique for the diagnosis of cutaneous leishmani- asis as compared with conventional PCR. Acta Parasitol. 65(2): 310–316.
8. Fotouhi-Ardakani R, Dabiri S, Ajdari S, Alimohammadian MH, AlaeeNovin E, Taleshi N, Parvizi P (2016) Assessment of nuclear and mitochondrial genes in precise identification and analysis of ge- netic polymorphisms for the evaluation of Leishmania parasites. Infect Genet Evol. 46: 33–41.
9. Karaku ŞM, Pekag Irba ŞM, Demir S, Eren H, Toz S, Özbel Y (2017) Molecular screening of Leishmania spp. Infection and blood meals in sand flies from a leish- maniasis focus in southwestern Turkey. Med Vet Entomol. 31: 224–229.
10. Antonia AL, Wang L, Ko DC (2018) A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis. Peer J. 6: e5905.
11. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M (2018) Real time PCR applications for diagnosis of leishmaniasis. Parasit Vectors. 11: 273.
12. Rojas-Jaimes J, Rojas-Palomino N, Pence J, Lescano AG (2019) Leishmania spe- cies in biopsies of patients with different clinical manifestations identified by high resolution melting and nested PCR in an Endemic district in Peru. Parasite Epi- demiol Control. 3: e 00095.
13. Ahuja K, Vats A, Beg MA, Kariyawasam K, Chaudhury A, Chatterjee M, Karun- aweera ND, Selvapandiyan A (2020) High resolution melting based method for rap- id discriminatory diagnosis of Frotorco- infecting Leptomonas seymouri in Leish- mania donovani-induced leishmaniasis. Parasitol Int. 75: e102047.
14. Schönian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, Jaffe CL (2003) PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Mi- crobiol Infect Dis. 47(1): 349 ̶ 358.
15. Parvizi P, Benlarbi M, Ready PD (2003) Mitochondrial and Wolbachia markers for the sand fly Phlebotomus papatasi: little population differentiation between peridomestic sites and gerbil burrows in Isfahan Province, Iran. Med Vet Entomol. 17: 351–362.
16. Parvizi P, Mauricio I, Aransaya AM, Miles MA, Ready PD (2005) First de- tection of Leishmania major in perido- mestic Phlebotomus papatasi from Isfa- han Province, Iran: comparison of nest- ed PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle ki- netoplast DNA. Acta Trop. 93: 75–83.
17. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA.
18. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 30(12): 2725–2729.
19. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformat- ics. 25(11): 1451–1452.
20. Tajima F (1996) The amount of DNA polymorphism maintained in a finite pop- ulation when the neutral mutation rate var- ies among sites. Genetics. 143: 1457–1465.
21. Ibrahim ME, Barker DC (2001) The origin and evolution of the Leishmania donovani complex as inferred from a mitochondri- al cytochrome oxidase II gene sequence.
Infect Genet Evol. 1(1): 61–68.
22. Yatawara L, Le TH, Wickramasinghe S, Agatsuma T (2008) Maxicircle (mitochon- drial) genome sequence (partial) of Leish- mania major: gene content, arrangement and composition compared with Leish- mania tarentolae. Gene. 424(1–2): 80– 86.
23. Babiker AM, Ravagnan S, Fusaro A, Has- san MM, Bakheit SM, Mukhtar MM, Cattoli G, Capelli G (2014) Concomi- tant infection with Leishmania donovani and L. major in single ulcers of Cutane- ous Leishmaniasis patients from Sudan. J Trop Med. e170859.
24. Nebohacova M, Kim CE, Maslov DA (2009) RNA editing and mitochondrial activity in promastigotes and amastigotes of Leish- mania donovani. Int J Parasitol. 39: 635– 644.
25. Shaw JM, Campbell D, Simpson L (1989) Internal frameshifts within the mito- chondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA. Proc Natl Acad Sci USA. 86(16): 6220–6224.
26. Cao DP, Guo XG, Chen DL, Chen JP (2011) Species delimitation and phylo- genetic relationships of Chinese Leish- mania isolates reexamined using kineto- plast cytochrome oxidase II gene sequenc- es. Parasitol Res. 109: 163–173.
27. Mirzaei A, Schweynoch C, Rouhani S, Parvizi P, Scho ̈nian G (2014) Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leish- maniasis in Iran. Trans R Soc Trop Med Hyg. 108: 502–512.
28. Haydon DT, Cleaveland S, Taylor LH, Lau- renson MK (2002) Identifying reservoirs of infection: A conceptual and practical challenge. Emerg Infect Dis. 8(12): 1468– 1473.
29. Yaghoobi-Ershadi MR (2012) Phlebotom- ine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania trans- mission. J Arthropod Borne Dis. 6(1): 1.
30. Boite MC, Mauricio IL, Miles MA, Cu- polillo E (2012) New insights on taxon- omy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis. 6: e1888.
31. Hernandez C, Alvarez C, Gonzalez C, Aya- la MS, León CM, Ramírez JD (2014) Identification of six New World Leish- mania species through the implementa- tion of a High-Resolution Melting (HRM) genotyping assay. Parasite Vectors. 7: 501–508.
32. Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floe- ter-Winter LM (2016) High resolution melting analysis targeting hsp 70 as a fast and efficient method for the discrim- ination of Leishmania species. PLoS Negl Trop Dis. 10(2): e0004485.
33. Słomka M, Sobalska-Kwapis M, Wachulec M, Bartosz G, Strapagiel D (2017) High Resolution Melting (HRM) for High- Throughput Genotyping-Limitations and Caveats in Practical Case Studies. J Clin Child Adolesc Psychol. 18: 2316–2337.
34. Bezerra-Vasconcelos DR, Melo LM, Al- buquerque ÉS, Luciano MCS, Bevilaqua CML (2011) Real-time PCR to assess the Leishmania load in Lutzomyia long- ipalpis sand flies: screening of target genes and assessment of quantitative methods. Exp Parasitol. 129: 234–239.
35. González E, Álvarez A, Ruiz S, Molina R, Jiménez M (2017) Detection of high Leishmania infantum loads in Phleboto- mus perniciosus captured in the leish- maniasis focus of southwestern Madrid region (Spain) by real time PCR. Acta Trop. 171: 68–73.
36. Strelkova MV, Eliseev LN, Ponirovsky EN, Dergacheva TI, Evans DA (2001) Mixed leishmanial infections in Rhom- bomys optimus: a key to the persistence of Leishmania major from one transmis- sion season to the next. Ann Trop Med Parasitol. 95: 811–819.
2. Ghafari SM, Fotouhi-Ardakani R, Parvizi P (2020a) Designing and developing a high- resolution melting technique for accu- rate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real- time PCR and in silico genetic evalua- tion. Acta Trop. 211: e105626.
3. Ghafari SM, Ebrahimi S, Nateghi-Rostami M, Bordbar A, Parvizi P (2020b) Com- parative evaluation of salivary glands pro- teomes from wild Phlebotomus papatasi proven vector of zoonotic cutaneous leishmaniasis. Vet Med Sci. 00: 1–8.
4. Dostálová A, Volf P (2012) Leishmania de- velopment in sand flies: parasite-vector interactions overview. Parasit Vectors. 5: 276.
5. Parvizi P, Ready PD (2008) Nested PCRs and sequencing of nuclear ITS-rDNA frag-
ments detect three Leishmania species of gerbils in sand flies from Iranian foci of
zoonotic cutaneous leishmaniasis. Trop Med Int Health. 13(9): 1159–1171.
6. Müller KE, Zampieri RA, Aoki JI, MuxelSM, Nerland AH, Floeter-Winter LM (2018) Amino acid permease 3 (aap3) Coding sequence as a target for Leish- mania identification and diagnosis of leish- maniases using high resolution Melting analysis. Parasit Vectors. 11: 421.
7. Asfaram S, Fakhar M, Mirani N, De- rakhshani niya M, Valadan R, Ziaei Hez- arjaribi H, Emadi SN (2019) HRM–PCR is an accurate and sensitive technique for the diagnosis of cutaneous leishmani- asis as compared with conventional PCR. Acta Parasitol. 65(2): 310–316.
8. Fotouhi-Ardakani R, Dabiri S, Ajdari S, Alimohammadian MH, AlaeeNovin E, Taleshi N, Parvizi P (2016) Assessment of nuclear and mitochondrial genes in precise identification and analysis of ge- netic polymorphisms for the evaluation of Leishmania parasites. Infect Genet Evol. 46: 33–41.
9. Karaku ŞM, Pekag Irba ŞM, Demir S, Eren H, Toz S, Özbel Y (2017) Molecular screening of Leishmania spp. Infection and blood meals in sand flies from a leish- maniasis focus in southwestern Turkey. Med Vet Entomol. 31: 224–229.
10. Antonia AL, Wang L, Ko DC (2018) A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis. Peer J. 6: e5905.
11. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M (2018) Real time PCR applications for diagnosis of leishmaniasis. Parasit Vectors. 11: 273.
12. Rojas-Jaimes J, Rojas-Palomino N, Pence J, Lescano AG (2019) Leishmania spe- cies in biopsies of patients with different clinical manifestations identified by high resolution melting and nested PCR in an Endemic district in Peru. Parasite Epi- demiol Control. 3: e 00095.
13. Ahuja K, Vats A, Beg MA, Kariyawasam K, Chaudhury A, Chatterjee M, Karun- aweera ND, Selvapandiyan A (2020) High resolution melting based method for rap- id discriminatory diagnosis of Frotorco- infecting Leptomonas seymouri in Leish- mania donovani-induced leishmaniasis. Parasitol Int. 75: e102047.
14. Schönian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, Jaffe CL (2003) PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Mi- crobiol Infect Dis. 47(1): 349 ̶ 358.
15. Parvizi P, Benlarbi M, Ready PD (2003) Mitochondrial and Wolbachia markers for the sand fly Phlebotomus papatasi: little population differentiation between peridomestic sites and gerbil burrows in Isfahan Province, Iran. Med Vet Entomol. 17: 351–362.
16. Parvizi P, Mauricio I, Aransaya AM, Miles MA, Ready PD (2005) First de- tection of Leishmania major in perido- mestic Phlebotomus papatasi from Isfa- han Province, Iran: comparison of nest- ed PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle ki- netoplast DNA. Acta Trop. 93: 75–83.
17. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA.
18. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 30(12): 2725–2729.
19. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformat- ics. 25(11): 1451–1452.
20. Tajima F (1996) The amount of DNA polymorphism maintained in a finite pop- ulation when the neutral mutation rate var- ies among sites. Genetics. 143: 1457–1465.
21. Ibrahim ME, Barker DC (2001) The origin and evolution of the Leishmania donovani complex as inferred from a mitochondri- al cytochrome oxidase II gene sequence.
Infect Genet Evol. 1(1): 61–68.
22. Yatawara L, Le TH, Wickramasinghe S, Agatsuma T (2008) Maxicircle (mitochon- drial) genome sequence (partial) of Leish- mania major: gene content, arrangement and composition compared with Leish- mania tarentolae. Gene. 424(1–2): 80– 86.
23. Babiker AM, Ravagnan S, Fusaro A, Has- san MM, Bakheit SM, Mukhtar MM, Cattoli G, Capelli G (2014) Concomi- tant infection with Leishmania donovani and L. major in single ulcers of Cutane- ous Leishmaniasis patients from Sudan. J Trop Med. e170859.
24. Nebohacova M, Kim CE, Maslov DA (2009) RNA editing and mitochondrial activity in promastigotes and amastigotes of Leish- mania donovani. Int J Parasitol. 39: 635– 644.
25. Shaw JM, Campbell D, Simpson L (1989) Internal frameshifts within the mito- chondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA. Proc Natl Acad Sci USA. 86(16): 6220–6224.
26. Cao DP, Guo XG, Chen DL, Chen JP (2011) Species delimitation and phylo- genetic relationships of Chinese Leish- mania isolates reexamined using kineto- plast cytochrome oxidase II gene sequenc- es. Parasitol Res. 109: 163–173.
27. Mirzaei A, Schweynoch C, Rouhani S, Parvizi P, Scho ̈nian G (2014) Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leish- maniasis in Iran. Trans R Soc Trop Med Hyg. 108: 502–512.
28. Haydon DT, Cleaveland S, Taylor LH, Lau- renson MK (2002) Identifying reservoirs of infection: A conceptual and practical challenge. Emerg Infect Dis. 8(12): 1468– 1473.
29. Yaghoobi-Ershadi MR (2012) Phlebotom- ine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania trans- mission. J Arthropod Borne Dis. 6(1): 1.
30. Boite MC, Mauricio IL, Miles MA, Cu- polillo E (2012) New insights on taxon- omy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis. PLoS Negl Trop Dis. 6: e1888.
31. Hernandez C, Alvarez C, Gonzalez C, Aya- la MS, León CM, Ramírez JD (2014) Identification of six New World Leish- mania species through the implementa- tion of a High-Resolution Melting (HRM) genotyping assay. Parasite Vectors. 7: 501–508.
32. Zampieri RA, Laranjeira-Silva MF, Muxel SM, Stocco de Lima AC, Shaw JJ, Floe- ter-Winter LM (2016) High resolution melting analysis targeting hsp 70 as a fast and efficient method for the discrim- ination of Leishmania species. PLoS Negl Trop Dis. 10(2): e0004485.
33. Słomka M, Sobalska-Kwapis M, Wachulec M, Bartosz G, Strapagiel D (2017) High Resolution Melting (HRM) for High- Throughput Genotyping-Limitations and Caveats in Practical Case Studies. J Clin Child Adolesc Psychol. 18: 2316–2337.
34. Bezerra-Vasconcelos DR, Melo LM, Al- buquerque ÉS, Luciano MCS, Bevilaqua CML (2011) Real-time PCR to assess the Leishmania load in Lutzomyia long- ipalpis sand flies: screening of target genes and assessment of quantitative methods. Exp Parasitol. 129: 234–239.
35. González E, Álvarez A, Ruiz S, Molina R, Jiménez M (2017) Detection of high Leishmania infantum loads in Phleboto- mus perniciosus captured in the leish- maniasis focus of southwestern Madrid region (Spain) by real time PCR. Acta Trop. 171: 68–73.
36. Strelkova MV, Eliseev LN, Ponirovsky EN, Dergacheva TI, Evans DA (2001) Mixed leishmanial infections in Rhom- bomys optimus: a key to the persistence of Leishmania major from one transmis- sion season to the next. Ann Trop Med Parasitol. 95: 811–819.
| Files | ||
| Issue | Vol 16 No 4 (2022) | |
| Section | Original Article | |
| DOI | https://doi.org/10.18502/jad.v16i4.12085 | |
| Keywords | ||
| Cloning, Cytochrome oxidase II, qPCR-HRM | ||
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How to Cite
1.
Ghafari SM, Fotouhi- Ardakani R, Parvizi P. Accurate Identification of Leishmania Parasites in Sandflies by Polymorphism Analysis of COII Gene Using PCR and qPCR-HRM Techniques in Iranian Border with Iraq. J Arthropod Borne Dis. 2023;16(4):301–314.
