EVALUATION OF MICRONUTRIENT-DEPENDENT METABOLIC SUPPORT OF LEISHMANIADONOVANI PROMASTIGOTES IN VITRO
DOI:
https://doi.org/10.22159/ijap.2026v18i3.57368Keywords:
Novy-McNeil-Nicol medium, Leishmania species, Vitamin A, Vitamin D₃, ZincAbstract
Objective: Leishmaniadonovani is a type of protozoan parasite that causes visceral leishmaniasis. The present study was conducted to assess how specific micronutrients influence the metabolism, growth, and viability of Leishmania donovani promastigotes in vitro.
Methods: Leishmania donovani promastigotes were cultured in Novy-McNeil-Nicol (NNN) medium and allocated into six groups: control (no supplements), vitamin A (G1), zinc (G2), vitamin D₃ (G3), combined vitamin A + zinc + vitamin D₃ (G4), and G4 with additional supplementation in the liquid phase (G5). Stock solutions of vitamins and zinc were aseptically prepared, filtered, and added to solid or liquid media. Parasite density and viability were monitored every two days using a hemocytometer and trypan blue staining.
Results: Parasite proliferation in the vitamin-supplemented media was comparable to that observed in the control group. The parasites continued to proliferate for more than 20 days without subculture. Cell counts were recorded on days 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, with the highest proliferation rate observed on day 8.
A marked increase in parasite numbers was detected in media supplemented with all vitamins, in both solid and liquid forms. The maximum cell density reached (107.25 ± 3.10) × 10⁶ cells/ml, compared to (73.50 ± 1.92) × 10⁶ cells/ml in the NNN control medium.
After day 8, parasite density gradually declined over time. By day 20, the cell count decreased to (23.50 ± 3.11) × 10⁶ cells/ml in the vitamin-supplemented media, whereas the NNN medium showed a lower count of (4.50 ± 0.58) × 10⁶ cells/ml. Statistical analysis demonstrated significant differences between all tested media (p < 0.05).
Conclusion: The study’s findings demonstrate that Leishmaniadonovanipromastigotes exhibit a marked dependence on zinc, vitamin A, and vitamin D₃ for metabolic maintenance in vitro, highlighting exploitable nutritional vulnerabilities rather than therapeutic benefit.
References
1. Wamai RG, Kahn J, McGloin J, Ziaggi G. Visceral leishmaniasis: a global overview. J Glob Health Sci. 2020;2(1). doi:10.35500/jghs.2020.2.e3
2. Ghazi HF, Mohammed ST, Zabbon AA, Shaaban MT. The effect of contraceptive pills on the severity of visceral leishmaniasis by measuring some physiological parameters. Al-Mustansiriyah J Sci. 2024;35(4):64–71. doi:10.23851/mjs.v35i4.1547
3. Sharief AH, GAsim Khalil EA, Suliaman SM. In vitro cultivation of Leishmaniadonovanipromastigotes: growth potential of human urine as replacement of fetal calf serum. Ann Syst Biol. 2021;4(1):001–004. doi:10.17352/asb.000010
4. Nayak A, Akpunarlieva S, Barrett M, Burchmore R. A defined medium for Leishmania culture allows definition of essential amino acids. ExpParasitol. 2018;185:39–52. doi:10.1016/j.exppara.2018.01.009
5. Feleke BE, Feleke TE. Micronutrient levels and their effects on the prognosis of visceral leishmaniasis treatment, a prospective cohort study. BMC Infect Dis. 2020;20(1):867. doi:10.1186/s12879-020-05615-1
6. Paul UK, Chakraborty PK, Hoque MR, Jafrin W, Sarkar S, Akhter S, Roy AS. Serum iron status among visceral leishmaniasis patients. Mymensingh Med J. 2014;23(4):654–7. doi:https://europepmc.org/article/med/25481581 PMID:25481581
7. Rajakumari R, Oluwafemi OS, Thomas S, Kalarikkal N. Dietary supplements containing vitamins and minerals: formulation, optimization and evaluation. Powder Technol. 2018;336:481–92. doi:10.1016/j.powtec.2018.06.026
8. Bezerra IP, Braga DS, De Mello MF, Pratti JE, Pereira JC, Marcia A, Ramos TD, Vale AM, Gomes DC, Leonel H. Dietary vitamin D₃ deficiency increases resistance to Leishmania (Leishmania) amazonensis infection in mice. Front Cell Infect Microbiol. 2019;9:448329. doi:10.3389/fcimb.2019.00088
9. Schmid A, Walther B. Natural vitamin D content in animal products. AdvNutr. 2013;4(4):453–62. doi:10.3945/an.113.003780
10. Hewison M. Vitamin D and the immune system: new perspectives on an old theme. EndocrinolMetabClin North Am. 2010;39(2):365–79. https://www.endo.theclinics.com/article/S0889-8529(10)00012-5/abstract
11. Pawłowska M, Mila-Kierzenkowska C, Szczegielniak J, Woźniak A. Oxidative stress in parasitic diseases—reactive oxygen species as mediators of interactions between the host and the parasites. Antioxidants. 2024;13(1):38. doi:10.3390/antiox13010038
12. Ross AC. Vitamin A. In: Bioactive compounds and cancer. Totowa, NJ: Humana Press; 2010. p. 335–56. doi:10.1007/978-1-60761-627-6_16
13. Stephensen CB. Vitamin A, immunity, and infection. In: Nutrition, immunity, and infection. 2017. p. 181–96. https://www.taylorfrancis.com/chapters/edit/10.1201/9781315118901-11/vitamin-immunity-infection-charles-stephensen
14. Kumar VU, Kt MF, Sharma A, Bisht P, Dhingra S, Ravichandiran V, Ramesh M, Murti K. The possible role of selected vitamins and minerals in the therapeutic outcomes of leishmaniasis. Biol Trace Elem Res. 2023;201(4):1672–88. doi:10.1007/s12011-022-03311-6
15. Maret W. Zinc in cellular regulation: the nature and significance of “zinc signals”. Int J Mol Sci. 2017;18(11):2285. doi:10.3390/ijms18112285
16. Kumari A, Singh KP, Mandal A, Paswan RK, Sinha P, Das P, Ali V, Bimal S, Lal CS. Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmaniadonovani. PLoS One. 2017;12(6):e0178800. doi:10.1371/journal.pone.0178800
17. Saini S, Bharati K, Shaha C, Mukhopadhyay CK. Zinc depletion promotes apoptosis-like death in drug-sensitive and antimony-resistant Leishmaniadonovani. Sci Rep. 2017;7(1):10488. doi:10.1038/s41598-017-10041-6
18. Carvalho S, Barreira da Silva R, Shawki A, Castro H, Lamy M, Eide D, Costa V, Mackenzie B, Tomas AM. LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmaniainfantum parasites. MolMicrobiol. 2015;96(3):581–95. doi:10.1111/mmi.12957
19. Mohammed ST, Ghazi HF, Ahmed HY, Majeed HA, Ajah HA, Ali EN, Ali LQ. Preparation a new culture medium by using Gonadermalucidum for cultivation of Leishmaniadonovanipromastigote parasite. Microbes Infect Dis. 2025. doi:10.21608/MID.2025.377492.2724
20. Ali EN. Measurement of protein and albumin/globulin ratio in patients infected with visceral leishmaniasis. Am J Polit Sci. 2013;13(1):170–4. doi:10.32947/ajps.v13i1.212
21. Stanić M. Trypan blue staining does not interfere with the emission spectra of fluorescent dyes in confocal microscopy. Genet Appl. 2020;4(2):50–2. doi:10.31383/ga.vol4iss2pp50-52
22. Alsaad RKA, Kawan MH. In vitro cultivation of Leishmania spp. using blood from human and dog in Novy-MacNeal-Nicolle (NNN) modified medium. Ann RSCB. 2021;25(6):17753–62. https://systems.uomisan.edu.iq/cv/uploads/files/dwo8qa6g4nex_i3.pdf
23. Pucadyil TJ, Chattopadhyay A. Cholesterol: a potential therapeutic target in Leishmania infection? Trends Parasitol. 2007;23(2):49–53. doi:https://www.cell.com/trends/parasitology/abstract/S1471-4922(06)00295-9
24. Rodahl K, Moore T. The vitamin A content and toxicity of bear and seal liver. Biochem J. 1943;37(2):166. doi:10.1042/bj0370166. PMID:16747610; PMCID: PMC1257872
25. Mohamed ST, Sulaiman HHNM, Kamal SB, Aja HA. Effect of Fusariumgraminarum silver-nanoparticles on IL-10 and INF-γ cytokines levels in the mice by Leishmaniadonovani in vivo. Iraqi J Biotechnol. 2019;18(2). https://www.jige.uobaghdad.edu.iq/index.php/IJB/article/view/340
26. Vallochi AL, Teixeira L, Oliveira KDS, Maya-Monteiro CM, Bozza PT. Lipid droplet, a key player in host-parasite interactions. Front Immunol. 2018;9:1022. doi:10.3389/fimmu.2018.01022
27. Prakash S, Rai AK. Retinoic acid shows direct parasiticidal activity by targeting ergosterol pathway in Leishmaniadonovani: a potential therapeutic advancement. J BiomolStructDyn. 2023;41(23):14473–83. doi:10.1080/07391102.2023.2193983
28. Vellozo NS, Pereira-Marques ST, Cabral-Piccin MP, Filardy AA, Ribeiro-Gomes FL, Rigoni TS, DosReis GA, Lopes MF. All-trans retinoic acid promotes an M1-to-M2 phenotype shift and inhibits macrophage-mediated immunity to Leishmania major. Front Immunol. 2017;8:1560. doi:10.3389/fimmu.2017.01560
29. Kinoshita, T., Koike, K., Mwamtemi, H. H., Ito, S., Ishida, S., Nakazawa, Y., Kurokawa, Y., Sakashita, K., Higuchi, T., Takeuchi, K., Sawai, N., Shiohara, M., Kamijo, T., Kawa, S., Yamashita, T. & Komiyama, A. (2000). Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors. Blood, The Journal of the American Society of Hematology, 95(9), 2821-2828.doi: https://doi.org/10.1182/blood.V95.9.2821.009k11_2821_2828
30. Martori C, Velez R, Gállego M, Mesa I, Ferreira R, Alberola J, Rodríguez-Cortés A. Vitamin D and leishmaniasis: neither seasonal nor risk factor in canine host but potential adjuvant treatment through CBD103 expression. PLoSNegl Trop Dis. 2021;15(8):e0009681. doi:10.1371/journal.pntd.0009681
31. Rodríguez-Cortes A, Martori C, Martinez-Florez A, Clop A, Amills M, Kubejko J, Llull J, Nadal JM, Alberola J. Canine leishmaniasis progression is associated with vitamin D deficiency. Sci Rep. 2017;7(1):3346. doi:10.1038/s41598-017-03662-4
32. de Almeida Machado P, Escrivani DO, Gomes DCO, Rossi-Bergmann B, Chaves SP, Coimbra ES, de Matos Guedes HL. Vitamin D increases killing of intracellular Leishmaniaamazonensis in vitro independently of macrophage oxidative mechanisms. Parasitology. 2020;147(14):1792–800. doi:10.1017/S0031182020001791
33. Koshy KT, Beyer WF. Vitamin D₃ (cholecalciferol). In: Analytical profiles of drug substances. Vol. 13. Academic Press; 1984. p. 655–715. doi:10.1016/S0099-5428(08)60205-1
34. Falih BT, Mohammed ST, Mohammed NJ. Effects of the silver nanoparticle synthesis from the leaves of the Capparisspinosa plant on the liver of mice infected with visceral leishmaniasis. Casp J Environ Sci. 2022;20(4):785–91. doi:https://cjes.guilan.ac.ir/article_5765_859ca9b87631a31f76abb88e7e037ed6.pdf
35. Ramos-Martinez E, Gutierrez-Kobeh L, Villaseñor-Cardoso MI. The role of vitamin D in the control of Leishmania infection. Can J PhysiolPharmacol. 2015;93(5):369–76. doi:10.1139/cjpp-2014-0372
36. Aghaei M, Aghaei S, Kouhiyan M, Shahmoradi Z, Hejazi SH. The interaction of zinc as an essential trace element with Leishmania parasites: a systematic review. Adv Biomed Res. 2024;13:73. doi:10.4103/abr.abr_187_23
37. Bafghi AF, Noorbala M, Noorbala MT, Aghabagheri M. Anti-leishmanial effect of zinc sulphate on the viability of Leishmaniatropica and L. major promastigotes. Jundishapur J Microbiol. 2014;7(9):e11192. doi:10.5812/jjm.11192
38. Sundar S, Singh J, Singh VK, Agrawal N, Kumar R. Current and emerging therapies for the treatment of leishmaniasis. Expert Opin Orphan Drugs. 2024;12(1):19–32. doi:10.1080/21678707.2024.2335248
39. Siripattanapipong S, Boontanom P, Leelayoova S, Mungthin M, Tan-Ariya P. In vitro growth characteristics and morphological differentiation of Leishmaniamartiniquensispromastigotes in different culture media. Acta Trop. 2019;197:105039. doi:10.1016/j.actatropica.2019.05.03.
Published
How to Cite
Issue
Section
Copyright (c) 2026 HANAN FAISAL GHAZI, SHAWQ RAAFAT ALNAQQASH, SABAA TAHER MOHAMMED, ALI MURTATHA HASAN, HAMZIA ALI AJAH, EKHLASS N. ALI, NASSREEN N. MZHR
This work is licensed under a Creative Commons Attribution 4.0 International License.