Host infectiousness to insect vectors is a crucial parameter for understanding the transmission dynamics of insect-borne infectious diseases such as leishmaniases. Despite their importance, critical factors influencing the outwards transmission of Leishmania major, including parasite distribution within the host body and the minimum number of skin amastigotes required for vector infection, remain poorly characterized. To address these gaps, we studied these parameters in the natural North African reservoir host Meriones shawi and in BALB/c mice infected with a low parasite dose. Using qPCR, we quantified Leishmania loads in different zones (regions) of infected ear pinnae, whereas microscale infectiousness was evaluated via microbiopsies and fluorescence microscopy. The amastigote distribution within infected ears was heterogeneous, with pronounced differences between the lesion center, lesion margin, and visually unaffected surrounding skin. Phlebotomus papatasi females that fed in areas where no amastigotes were detected via microscopy did not become infected. In M. shawi, lesion margins have emerged as the most effective source of infection. The number of amastigotes at bite sites where sand fly females became infected ranged from 4--500, with as few as 2--10 amastigotes sufficient to initiate vector infection. This low infection threshold was confirmed by experiments in which P. papatasi was fed through a chick-skin membrane. In contrast, the BALB/c mouse model showed only minor differences in infectiousness between lesion centers and margins. The minimum infectious dose in BALB/c mice was approximately 100 times greater than that in M. shawi, with successful infections occurring at sites containing 1,500-10,000 amastigotes. These findings advance our understanding of Leishmania transmission by addressing critical knowledge gaps and enabling more accurate modelling of cutaneous leishmaniasis epidemiology. Moreover, this study highlights the importance of incorporating natural host models in research, as the dynamics of disease progression and transmission parameters can differ significantly between natural hosts and standard laboratory models.

Arboviruses and Emergent viruses Institut Pasteur d'Algérie Algiers Algeria

Department of Parasitology Charles University Prague Czech Republic

Department of Zoology Charles University Prague Czech Republic

UMR BioCIS CNRS Université Paris Saclay Orsay France

UR ESCAPE USC Anses Faculty of Pharmacy Université de Reims Champagne Ardenne Reims France

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Abadías-Granado I, Diago A, Cerro PA, Palma-Ruiz AM, Gilaberte Y. Cutaneous and mucocutaneous leishmaniasis. Actas Dermosifiliogr. 2021;22:491–502. PubMed

Ruiz-Postigo JA, Jain S, Madjou S, Al E. Global leishmaniasis surveillance, 2022: assessing trends over the past 10 years. 2023. p. 471–87. Available from: https://www.who.int/publications/i/item/who-wer9840-471-487

Alcover MM, Rocamora V, Ribas A, Fisa R, Riera C. Underestimation of human cutaneous leishmaniasis caused by Leishmania infantum in an endemic area of the Mediterranean basin (Balearic Islands). Microorganisms. 2023;11(1):126. doi: 10.3390/microorganisms11010126 PubMed DOI PMC

Copeland HW, Arana BA, Navin TR. Comparison of active and passive case detection of cutaneous leishmaniasis in Guatemala. Am J Trop Med Hyg. 1990;43(3):257–9. doi: 10.4269/ajtmh.1990.43.257 PubMed DOI

Ashford RW. The leishmaniases as emerging and reemerging zoonoses. Int J Parasitol. 2000;30(12–13):1269–81. doi: 10.1016/s0020-7519(00)00136-3 PubMed DOI

Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27(2):123–47. doi: 10.1111/j.1365-2915.2012.01034.x PubMed DOI

Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 2006;22(9):439–45. PubMed

Kamhawi S, Serafim TD. Patchy parasitized skin governs Leishmania donovani transmission to sand flies. Trends Parasitol. 2017;33(10):748–50. doi: 10.1016/j.pt.2017.08.004 PubMed DOI PMC

Doehl JSP, Bright Z, Dey S, Davies H, Magson J, Brown N, et al. Skin parasite landscape determines host infectiousness in visceral leishmaniasis. Nat Commun. 2017;8(1):57. doi: 10.1038/s41467-017-00103-8 PubMed DOI PMC

Sadlova J, Vojtkova B, Lestinova T, Becvar T, Frynta D, Benallal KE, et al. Infectiousness of asymptomatic Meriones shawi, reservoir host of Leishmania major. Pathogens. 2023;12(4):614. doi: 10.3390/pathogens12040614 PubMed DOI PMC

Volf P, Volfova V. Establishment and maintenance of sand fly colonies. J Vector Ecol. 2011;36:1–9. PubMed

Dean S, Sunter J, Wheeler RJ, Hodkinson I, Gluenz E, Gull K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5(1):140197. doi: 10.1098/rsob.140197 PubMed DOI PMC

Ashwin H, Sadlova J, Vojtkova B, Becvar T, Lypaczewski P, Schwartz E, et al. Characterization of a new Leishmania major strain for use in a controlled human infection model. Nat Commun. 2021;12(1):215. doi: 10.1038/s41467-020-20569-3 PubMed DOI PMC

Kirstein OD, Abbasi I, Horwitz BZ, Skrip L, Hailu A, Jaffe C, et al. Minimally invasive microbiopsies: a novel sampling method for identifying asymptomatic, potentially infectious carriers of Leishmania donovani. Int J Parasitol. 2017;47(10–11):609–16. doi: 10.1016/j.ijpara.2017.02.005 PubMed DOI PMC

Pruzinova K, Sadlova J, Myskova J, Lestinova T, Janda J, Volf P. Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases. Parasit Vectors. 2018;11(1):37. doi: 10.1186/s13071-018-2613-2 PubMed DOI PMC

Pruzinova K, Sadlova J, Seblova V, Homola M, Votypka J, Volf P. Comparison of bloodmeal digestion and the peritrophic matrix in four sand fly species differing in susceptibility to Leishmania donovani. PLoS One. 2015;10(6):e0128203. doi: 10.1371/journal.pone.0128203 PubMed DOI PMC

Doehl JSP, Sádlová J, Aslan H, Pružinová K, Metangmo S, Votýpka J, et al. Leishmania HASP and SHERP genes are required for in vivo differentiation, parasite transmission and virulence attenuation in the host. PLoS Pathog. 2017;13(1):e1006130. doi: 10.1371/journal.ppat.1006130 PubMed DOI PMC

Myskova J, Votypka J, Volf P. Leishmania in sand flies: comparison of quantitative polymerase chain reaction with other techniques to determine the intensity of infection. J Med Entomol. 2008;45(1):133–8. doi: 10.1603/0022-2585(2008)45[133:lisfco]2.0.co;2 PubMed DOI

Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71(3):267–75. doi: 10.1016/0014-4894(90)90031-7 PubMed DOI

Pinheiro J, Bates D, DebRoy S, Sarkar D, & RCT. nlme: Linear and Nonlinear Mixed Effects Models.e. 2021. Available from: https://cran.r-project.org/package=nlme

Halekoh U, Højsgaard S, Yan J. The R package geepack for generalized estimating equations. J Stat Softw. 2006;15(2):1–11.

Pekár S, Brabec M. Generalized estimating equations: A pragmatic and flexible approach to the marginal GLM modelling of correlated data in the behavioural sciences. Ethology. 2018;124(2):86–93.

R Core T. A language and environment for statistical computing. Available from: http://www.r-project.org/. 2021.

Ghawar W, Bettaieb J, Salem S, Snoussi M-A, Jaouadi K, Yazidi R, et al. Natural infection of Ctenodactylus gundi by Leishmania major in Tunisia. Acta Trop. 2018;177:89–93. doi: 10.1016/j.actatropica.2017.09.022 PubMed DOI

Vojtkova B, Spitzova T, Votypka J, Lestinova T, Kominkova I, Hajkova M, et al. Central Asian rodents as model animals for Leishmania major and Leishmania donovani research. Microorganisms. 2020;8(9):1–20. PubMed PMC

Sadlova J, Seblova V, Votypka J, Warburg A, Volf P. Xenodiagnosis of Leishmania donovani in BALB/c mice using Phlebotomus orientalis: a new laboratory model. Parasit Vectors. 2015;8:158. doi: 10.1186/s13071-015-0765-x PubMed DOI PMC

Vojtkova B, Frynta D, Spitzova T, Lestinova T, Votypka J, Volf P. Repeated sand fly bites of infected BALB/c mice enhance the development of Leishmania lesions. Front Trop Dis. 2021;2(October):1–9.

Miramin-Mohammadi A, Javadi A, Eskandari SE, Mortazavi H, Rostami MN, Khamesipour A. Immune response in cutaneous leishmaniasis patients with healing vs. non-healing lesions. Iran J Microbiol. 2020;12(3):249–55. PubMed PMC

Parkash V, Ashwin H, Dey S, Sadlova J, Vojtkova B, Van Bocxlaer K, et al. Safety and reactogenicity of a controlled human infection model of sand fly-transmitted cutaneous leishmaniasis. Nat Med. 2024;30(11):3150–62. doi: 10.1038/s41591-024-03146-9 PubMed DOI PMC

Seblova V, Volfova V, Dvorak V, Pruzinova K, Votypka J, Kassahun A, et al. Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani. PLoS Negl Trop Dis. 2013;7(4):e2187. doi: 10.1371/journal.pntd.0002187 PubMed DOI PMC

Sadlova J, Myskova J, Lestinova T, Votypka J, Yeo M, Volf P. Leishmania donovani development in Phlebotomus argentipes: comparison of promastigote- and amastigote-initiated infections. Parasitology. 2017;144(4):403–10. doi: 10.1017/S0031182016002067 PubMed DOI PMC

Anjili C, Langat B, Lugalia R, Mwanyumba P, Ngumbi P, Mbati PA. Estimation of the minimum number of Leishmania major amastigotes required for infecting Phlebotomus duboscqi (Diptera: Psychodidae). East Afr Med J. 2006;83(2):68–71. PubMed

Volf P, Hajmova M, Sadlova J, Votypka J. Blocked stomodeal valve of the insect vector: similar mechanism of transmission in two trypanosomatid models. Int J Parasitol. 2004;34(11). PubMed

Elnaiem DA, Ward RD, Young PE. Development of Leishmania chagasi (Kinetoplastida: Trypanosomatidae) in the second blood-meal of its vector Lutzomyia longipalpis (Diptera: Psychodidae). Parasitol Res. 1994;80(5):414–9. doi: 10.1007/BF00932379 PubMed DOI

Serafim TD, Coutinho-Abreu IV, Oliveira F, Meneses C, Kamhawi S, Valenzuela JG. Sequential blood meals promote Leishmania replication and reverse metacyclogenesis augmenting vector infectivity. Nat Microbiol. 2018;3(5):548–55. doi: 10.1038/s41564-018-0125-7 PubMed DOI PMC