Leishmania parasites are a group of kinetoplastid pathogens that cause a variety of clinical disorders while maintaining cell communication by secreting extracellular vesicles. Emerging technologies have been adapted for the study of Leishmania-host cell interactions, to enable the broad-scale analysis of the extracellular vesicles of this parasite. Leishmania extracellular vesicles (LEVs) are spheroidal nanoparticles of polydispersed suspensions surrounded by a layer of lipid membrane. Although LEVs have attracted increasing attention from researchers, many aspects of their biology remain unclear, including their bioavailability and function in the complex molecular mechanisms of pathogenesis. Given the importance of LEVs in the parasite-host interaction, and in the parasite-parasite relationships that have emerged during the evolutionary history of these organisms, the present review provides an overview of the available data on Leishmania, and formulates guidelines for LEV research. We conclude by reporting direct methods for the isolation of specific LEVs from the culture supernatant of the promastigotes and amastigotes that are suitable for a range of different downstream applications, which increases the compatibility and reproducibility of the approach for the establishment of optimal and comparable isolation conditions and the complete characterization of the LEV, as well as the critical immunomodulatory events triggered by this important group of parasites.

Extracellular vesicles released by Leishmania spp. (LEVs) are increasingly recognized as key mediators of parasite communication and host immune modulation. Lipids, although central to LEV biogenesis and function, remain understudied in the context of Leishmania pathogenicity. Here, we investigated the presence and distribution of lipid-rich structures in Leishmania (L.) amazonensis promastigotes and intracellular amastigotes using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and fluorescence microscopy with Bodipy staining. Promastigotes at the stationary phase exhibited abundant vesicle accumulation in the flagellar pocket, Golgi-associated autophagic structures, and lipid body–like inclusions. Infected BALB/c peritoneal macrophages contained amastigotes within parasitophorous vacuoles, also displaying lipid-rich compartments. Bodipy staining confirmed the presence of neutral lipid bodies in promastigotes, supporting their involvement in LEV formation. These findings suggest that lipid-enriched LEVs contribute to membrane remodeling, intracellular survival, and host cell modulation. Our study provides experimental evidence supporting lipid-centered mechanisms in Leishmania LEV biology and highlights potential targets for therapeutic intervention.

Leishmaniases are zoonotic vector-borne diseases caused by a wide variety of Leishmania species with complex transmission cycles involving different reservoirs, potential new hosts and vectors. Similarly, to other eukaryotes, Leishmania use released extracellular vesicles (LEVs) to play important initial interactions that are crucial to modulate the subsequent systemic immune response on the establishment of infection in humans and other important hosts like dogs. Recent studies in endemic areas of Brazil concluded that canine infections were predominantly due to L. amazonensis and not restricted to L. infantum (syn. Leishmania chagasi). Under these premises, the diagnosis of canine leishmaniasis needs to be improved, including the identification of current etiological agent, the clinical differential diagnosis and the histopathologic features. In this way, the dual aim of that study is to register collected observations for the diagnosis of natural canine infections and to insert in vitro results in the field of LEVs that still research gaps to be filled to understand the mechanisms and biological aspects involving the parasite-host interactions. Therefore, the future studies of Parasitology research for both of these fields are very important for the interventions for the prevention, control, elimination and eradication worldwide.

Lipid-rich structures are frequently observed in Leishmania spp., yet their morphological diversity and stage-specific distribution remain insufficiently described. Here, we performed an ultrastructural and fluorescence-based characterization of Leishmania (L.) amazonensis promastigotes and intracellular amastigotes using transmission electron microscopy (TEM), scanning electron microscopy (SEM), BODIPY® lipid staining, and Nanoparticle Tracking Analysis (NTA). Promastigotes exhibited vesicle-like and membrane-derived compartments concentrated in the flagellar pocket, as well as electron-dense lipid bodies adjacent to the Golgi complex. Intracellular amastigotes displayed rounded morphology within parasitophorous vacuoles and contained distinct lipid-rich inclusions. BODIPY staining confirmed neutral lipid pools in promastigotes. NTA detected submicron particles in culture supernatants, with amastigotes showing higher particle abundance (3.1–5.9 × 10⁸ particles/mL) and smaller mean sizes (132–171 nm) compared with promastigotes. These observations provide a descriptive account of lipid-rich and vesicle-like structures in L. amazonensis and offer morphological context for future investigations into membrane dynamics, lipid metabolism, and parasite–host interactions. No inference of vesicle identity, biogenesis, or function is made, in accordance with MISEV2023 recommendations.