Biology and Life Sciences

Abstract: Toxoplasmosis is a prevalent zoonotic disease worldwide, affecting approximately one-third of the global human population. Primary infection with Toxoplasma gondii during pregnancy can induce miscarriage or congenital infection, leading to irreversible damage to the foetus. Moreover, reactivation of T. gondii infection in immunosuppressed individuals can result in fatal outcomes. No vaccine exists to prevent human disease caused by this parasite. Thus, a vaccine that could induce complete and lasting protection against human toxoplasmosis is an unmet need. In this work, a subunit vaccine, consisting of T. gondii membrane proteins (TGMP) from the T. gondii Me49 strain plus CpG-oligodeoxynucleotide adjuvant (CpG), was tested using BALB/cByJ mice. Intranasal immunisation with TGMP plus CpG (TGMP+CpG) raised TGMP-specific serum IgG and intestinal IgA antibody levels, and parasite-specific IFN-γ-producing CD4+ and CD8+ memory T cells. Dense granule proteins (GRA) 2 and 7, surface antigen (SAG)-related sequences 25, 29B, and 34A, microneme protein (MIC) 10, toxofilin, nascent polypeptide-associated complex (NAC) domain-containing protein and NAC subunit beta were identified as immunogenic proteins in the TGMP. Mice immunised with TGMP+CpG were challenged with T. gondii tachyzoites and showed a significant reduction in the parasitic burden in the peritoneal exudate, spleen, and lungs, compared to mice sham-immunised with CpG alone. Altogether, these results indicate that mucosal immunisation with TGMP plus CpG adjuvant is worth exploring as a vaccination approach to prevent toxoplasmosis.

Abstract: The gastrointestinal and pulmonary systems are severely affected in both homozygous and heterozygous cystic fibrosis (CF) patients. Cystic fibrosis (CF) is a genetic disorder affecting epithelial ion transport and multiple organ systems. While bacterial and fungal comorbidities are well studied, parasitic infections remain underexplored. This narrative review addresses an unexplored area, inspired by the common gastrointestinal symptoms observed in both CF and certain intestinal parasites. It aims to explore potential links between CF and intestinal parasitic diseases, based on clinical case reports, literature reviews, and research articles. In this study, we used various search engines, including PubMed, ScienceDirect, Elsevier, Wiley, ResearchGate, and Google Scholar. This was constructed by employing keywords such as Cystic fibrosis, CFTR, cystic fibrosis associated with pathogens, intestinal inflammation, bowel disease, intestinal protozoa, and helminthic infections. Once similar symptoms and molecular/immune mechanisms were detected, our keywords extended to include specific parasites associated with CF and/or CFTR, as well as chronic lung inflammation, pancreatitis, or diabetes. This review also examines immunomodulation in CF, with or without parasitic infection, and the influence of CF on predisposition to parasitic infection, diagnosis, or treatment. In a nutshell, we found only a few case reports on CF-protozoan infection comorbidities in non-Middle Eastern countries, and even those are dated two decades ago. This indicates the underestimation of the possibility of CF-comorbidity, which might be life-threatening. Hence, CF diagnosis in patients with intestinal parasitic infections, family pedigree reviews before treatment, and in vivo/ex vivo research studies are recommended.

Abstract: Background/Objectives: Protozoan parasites of the genus Leishmania are causative agents of a group of devastating human diseases, known as leishmaniasis. These microorganisms possess very unusual mechanisms of gene expression that are poorly understood. This study was aimed at analyzing the tRNA repertoire encoded in the Leishmania infantum genome, a species responsible for the most severe form of disease, visceral leishmaniasis. tRNAs are adaptor molecules aimed at decoding mRNAs into proteins. Results: A total of 92 tRNA genes, dispersed on 38 loci were identified; often located in regions where unidirectional gene arrays converge. Putative intronic sequences were inferred for three tRNA genes, and, remarkably, 9 tRNAs were identified within the protein-coding sequences of annotated genes. According to structural predictions, the L. infantum tRNA repertoire covers 49 of the 61 possible anticodons, but because of the well-documented wobble phenomenon, these are sufficient to decode all codons in the 8532 protein-coding genes currently annotated in its genome. As illustrated in this study, codon usage is a well-conserved trait among different Leishmania species but differs substantially regarding the codon usage of its human host. Finally, we analyzed tRNA adaptation index (tAI) parameters, codon usage metrics, and relative protein expression levels. Conclusions: Apart from providing the tRNA gene repertoire and its genome distribution, we have shown the existence of a statistically significant, positive correlation between the tAI scores and protein expression levels in L. infantum promastigotes.

Abstract:

Bumped kinase inhibitor 1708 (BKI-1708), previously demonstrated to target apicomplexan kinases and CDPK1 and MAPKL1, exhibits remarkable activity against Toxoplasma gondii infection both in vitro and in vivo. Notably, BKI-1708 does not affect the viability of mammalian cells. Upon exposure to BKI-1708 exposure, T. gondii tachyzoites form large multinucleated complexes named baryzoites and remain trapped in within host cells. In this study, additional potential molecular targets for BKI-1708 were identified in soluble extracts of T. gondii ME49 tachyzoites and human foreskin fibroblasts (HFF) using differential affinity chromatography coupled to mass spectrometry (DAC-MS). Beyond kinases, secondary interactions in T. gondii involved the binding of proteins associated with cell division, cytoskeleton, vesicular trafficking, secretory organelles, and transcriptional and translational regulators. In non-infected HFFs, BKI-1708 interactors included cytoskeletal regulators along with multiple RNA/DNA-binding proteins. Upon infection, this profile shifted, with cytoskeletal components no longer detected, while nucleic acid–binding proteins remained present, consistent with infection-induced chromatin and transcriptional remodeling. This multi-target interference could contribute to the impaired cytokinesis and the formation of multinucleated baryzoites, aligning with the concept that antiprotozoal drugs exert efficacy through coordinated perturbation of multiple cellular processes rather than a single dominant target.

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Toxoplasma gondii, the causative agent of toxoplamosis, a disease widely distributed, is an intracellular parasite that invades host cells of different tissues using specialized endocytic activity. Recent studies suggest that tunneling nanotubes (TNTs), thin cell surface projections, may participate in the parasite-host cell interaction process. We report results on the involvement of host cells TNTs in the adhesion and internalization of T. gondii tachyzoites to epithelial LLC-MK² cells. Microscopy analysis showed that incubating cells in 0.45 M sucrose induces reversible assembly of TNTs without affecting cell viability. The presence of extended TNTs correlated with increase on parasite adhesion and reduction of parasite entry, suggesting a structural or signaling role in mediating adhesion. TNTs assembled following sucrose incubation contain both actin and tubulin components. These results highlight the functional relevance of TNTs in T. gondii host cell interaction, especially in parasite adhesion, opening new perspectives for understanding T. gondii-host cell interaction.

Abstract: A wide range of Leishmania species, transmitted by phlebotomine, cause a group of diseases known as leishmaniasis, which present diverse clinical manifestations. Leishmaniasis has a high impact on the most vulnerable communities, primarily affecting people suffering from malnutrition or living in poor housing conditions. Due to leishmaniasis are strongly associated with poverty the access to treatment is often limited. Besides, high drug toxicity and therapeutic failure, related to the emergence of drug resistance, remain major challenges associated with currently available treatments. Therefore, there is a need for developing new therapeutic approaches safer and more effective. Drug combinations and drug repurposing are two strategies currently used in the development of new treatments targeting multiple diseases. The combination of drugs with different mechanisms of action can minimize the emergence of resistance and allow drug dosage reduction, increasing the likelihood of successful drug repurposing. The research evaluated the leishmanicidal effects of combining antitumoral agents (paclitaxel and docetaxel) with standard drugs (miltefosine and paromomycin). Results demonstrated synergistic effects at higher doses. Furthermore, the antitumoral compounds boosted the host immune response by promoting macrophage polarization toward the M1 phenotype, which is essential for parasite control. These findings highlight a promising therapeutic approach that could improve treatment efficacy and reduce resistance.

Abstract: Malaria, caused by Plasmodium parasites, remains a global health crisis, necessitating novel therapeutic strategies targeting host-parasite interactions. During liver stage in-fection, parasites exploit host vesicular trafficking machinery, particularly SNARE pro-teins that mediate membrane fusion. Using a CRISPR/Cas9 knockout system in HeLa cells combined with advanced microscopy of Plasmodium berghei-infected HeLa cells, we identified specific endolysosomal SNAREs VAMP7, VAMP8, Vti1B, and Stx7 to be re-cruited to the parasitophorous vacuole membrane (PVM) with distinct temporal profiles. This demonstrates the parasite’s precise manipulation of host endolysosomal trafficking pathways. VAMP7 and Vti1B localized to the PVM within 30 minutes post-infection suggesting potential roles during invasion, while VAMP8 and Stx7 appeared later toward 24 hpi, coinciding with increased nutrient acquisition. Single gene deletions showed minimal impact, but combinatorial knockouts revealed critical redundancy. VAMP7-VAMP8 as well as VAMP7–Vti1B double KO significantly reduced parasite in-fection and growth, with Vti1B playing a dominant role. Triple KO phenotypes mirrored VAMP7-Vti1B disruption, underscoring Vti1B’s dominant role. SNARE depletion also impaired lysosome-PVM association and LAMP1 positive vesicle recruitment. Our findings indicate Plasmodium hijacks a coordinated host SNARE network to fuse lysosomes with the PVM for nutrient uptake. Targeting Vti1B-containing complexes disrupts this pathway without host cell toxicity, offering a promising host-directed antimalarial ap-proach.

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Between 1993 and 2024, 274 hard ticks (Ixodidae) were collected from 138 underground localities in Croatia. This study represents the most extensive survey of hard tick fauna in Croatian underground habitats to date. The collected hard ticks were classified into three genera and seven species. Ixodes was the most abundant genus, with five species, while Haemaphysalis, and Hyalomma were each represented by one species. The highest number of hard tick species was collected in underground habitats in Dalmatia, followed by north-western Croatia and Slavonia. Ixodes vespertilionis Koch, 1844 was the most abundant species in the collected sample, comprising 81.02%, and was recorded in all studied regions. Only I. vespertilionis was recorded throughout the year, while I. hexagonus was found in nine months, I. frontalis in four months, and the other species in shorter periods. The largest number of I. vespertilionis specimens was collected during the spring months (33.17%), while the fewest were collected in winter (16.58%). The discovery of Ixodes lividus Koch, 1844 in north-western Croatia represents a new country record, increasing the number of hard tick species currently known in Croatia to 28.

Abstract: Toxoplasmosis is a cosmopolitan zoonosis that particularly threatens pregnant women and immunocompromised individuals. Among people living with HIV, Toxoplasma gondii may invade the central nervous system, producing neuropathological effects associated with mental and psychiatric disorders. We assessed the seroprevalence of anti–T. gondii IgG in HIV-infected and HIV-uninfected residents of Iquitos, Peru, and evaluated an in-house ELISA based on total lysed antigen (TLA) against a commercial kit. In this observational cross-sectional study, 151 participants were enrolled: 92 HIV-positive and 59 HIV-negative. Overall IgG seroprevalence was 88.08% (133/151), reaching 91.30% (84/92) in the HIV-positive group and 83.05% (49/59) in the HIV-negative group. Seroprevalence was highest among adults aged 36–60 years (89.33%; 67/75) and among males (89.77%; 79/88) versus females (85.71%; 54/63). Mean optical density values were greater in HIV-positive participants (1.37±0.393; 95% CI 1.28–1.45) than in HIV-negative participants (0.565±0.200; 95% CI 0.510–0.620), and were higher in men overall (1.18±0.502; 95% CI 1.07–1.30). These findings indicate very high exposure to T. gondii in Iquitos, with higher IgG prevalence and antibody levels in HIV-positive individuals. The marked OD difference suggests stronger IgG responses among HIV-positive participants, highlighting potential risk for toxoplasmic encephalitis. Serological screening and preventive counseling may be warranted in high-burden Amazonian communities.

Abstract: 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.

Abstract: Oropouche fever (OF), caused by Oropouche virus (OROV), has expanded beyond its Amazonian range into Minas Gerais (MG), Brazil, raising concern about transmission in extra‑Amazonian Atlantic Forest landscapes. Critical gaps persist regarding Culicoides vector communities, anthropophily, and climate-sensitive transmission risk in these newly affected regions. We conducted targeted entomological surveys in five MG outbreak communities using CDC light traps and Protected Human Attraction (PHA) to characterize Culicoides composition. Females underwent RT-qPCR for OROV (n = 819) and physiological assessment (n=312). We developed an entomological alert framework that integrates blood-fed abundance, minimum infection rate (MIR) upper confidence bounds, and environmental drivers via generalized additive mixed models, which explained 68% of the variability in Culicoides abundance and the alert index across communities. We collected 1,171 Culicoides representing five species, C. leopoldoi (79.1%) and the primary vector C. paraensis (20.3%) predominated. C. paraensis was documented for the first time in all five outbreak areas and dominated PHA captures (90%), confirming anthropophily. Although no specimens tested OROV-positive (consistent with expected field infection rates of 0.01–1%), MIR upper bounds reached 132/1,000 in low-sample settings and humidity and temperature strongly modulated abundance. This operational baseline and alert index transform virologically negative, sparse surveillance data into prioritized targets for in-tensified sampling and vector control during early, low‑prevalence phases, when con-tainment of OROV’s extra‑Amazonian spread is still achievable.

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Background: While Plasmodium falciparum (Pf) genomes are constantly evolving to counter new antimalarial drugs, Pf parasites currently allow ancient Pf-malaria-combating red blood cell (RBC) genetic variants to markedly protect humans against onset of severe Pf disease and death. The prevalences of sickle-trait hemoglobin (HbAS) RBCs and “dual-gene protection” type-O HbAS RBCs are substantial in Pf-endemic regions thousands of years after the “sickle” HbS hemoglobin allele (HBB gene variant) and the type-O ABO blood group first emerged. Do Pf-human coevolution data and growing interest in transfusion services in Africa suggest rescue exchanges of “evolution-engineered” RBCs warrant evaluation? Methods: We reviewed transfusion-related publications and data regarding Pf-combating RBC genetic variants and a worrisome Pf genotype (Pfsa+++) that completely eliminates HbAS survival-promotion. Results: Clinicians in Africa are eager to advance transfusion therapies and exploit automated continuous-flow apheresis machines for RBC exchange. There is no evidence the low prevalence of Pfsa+++ is increasing or the combination of the survival-promoting effects of HbAS hemoglobin and type-O blood group provides less than an additive increase in protection. Conclusions: Geneticists can support evaluating therapeutic use of HbAS RBCs by explaining how the prevalence of the worrisome Pfsa+++ genotype might be low and unchanging due to an equilibrium between competing selection pressures and “fitness costs.” Since HbAS hemoglobin alone provides 90% protection against death, conceivably, no human with type-O HbAS RBCs has ever died from Pf malaria. So, it seems prudent to evaluate converting children with life-threatening Pf infections into type-O HbAS patients via exchange transfusion – now.

Abstract: Outbreaks of emerging and re-emerging diseases of both animals and plants are increasing due to climate change, globalization, land-use change, and agricultural intensification. While most pathogen monitoring programs focus on zoonotic threats, wildlife and other organisms in natural habitats can also serve as reservoirs and sentinels for pathogens of agricultural and ecological concern. Plant communities and the pathogens circulating within them are underrepresented in integrated disease monitoring frameworks. This study demonstrates how biodiversity and zoonosis monitoring programs conducted in protected habitats (tallgrass prairies and woodlands) across Illinois, together with insect specimens preserved in biorepositories, can be leveraged to improve knowledge of the identities and ecological associations of a wide range of potential pathogens. We developed an integrative workflow combining taxonomic identification, molecular screening, and epidemiological inference to detect vector-borne plant pathogens from archived insect material. Focusing on Hemiptera (Auchenorrhyncha), we screened specimens for phytoplasmas (Mollicutes), uncultured bacterial plant pathogens transmitted by sap-feeding insects, and characterized host-pathogen associations. At least three distinct phytoplasma strains were detected: ‘Candidatus Phytoplasma asteris’ (16SrI-B), ‘Candidatus Phytoplasma pruni’–related strains (16SrIII), and ‘Candidatus Phytoplasma sacchari’–related strains (16SrXI-H). The latter represents the first documented occurrence of a 16SrXI-H phytoplasma subgroup in Illinois. Overall, we identified five insect specimens harboring phytoplasmas across four preserved sites, all of them were previously unreported insect-phytoplasma associations. These findings demonstrate the value of existing biodiversity infrastructures for proactive surveillance of plant pathogens and extend the One Health paradigm to explicitly include natural ecosystems.

Abstract:

Throughout history, species within the genus Entamoeba have been described using a set of criteria that have not always been applied consistently, resulting in a taxonomy that is often confusing and controversial. Several factors have influenced the application of these criteria, including the limited number of morphological characters useful for taxonomic studies, overlapping host ranges among many species, the occurrence of mixed infections in some hosts, and a cosmopolitan distribution largely associated with human and animal movements. The incorporation of biochemical and genetic data as diagnostic and differential criteria, following the development of these techniques in the second half of the twentieth century, has enabled the recognition of cryptic species as well as the proposal of new taxa; however, significant taxonomic issues remain unresolved. This review summarises the historical development and main controversies in the taxonomy of the genus Entamoeba, from its origins in the late nineteenth century, when morphology and host association were the only available criteria, to the present day, in which molecular approaches provide a more realistic view of species diversity and interspecific relationships. Based on this analysis, general principles are proposed as a pragmatic synthesis to guide future taxonomic work on Entamoeba.

Abstract: The protozoan pathogen Toxoplasma gondii is responsible for toxoplasmosis a disease that can be deadly in immunocompromised patients and the developing fetus during pregnancy. Current treatments are widely considered to be suboptimal. We have recently reported that 5-fluoropyrimidines have highly promising anti-toxoplasmosis effects and are taken up by the parasite by a high affinity uracil/uridine transporter, TgUUT1. Here, we attempt to identify the gene encoding this transport protein. The only nucleoside or nucleobase family identified in the T. gondii genome was the Equilibrative Nucleoside Transporter (ENT) family, with four members. Of these, TgAT1 is known to be purine-specific, and deletion of the TgENT2 and TgENT3 genes, either separately or jointly, did not affect uridine transport or sensitivity to 5-fluoropyrimidines. In contrast, depletion of TgENT1, an essential gene, resulted in significant reduction in the uptake of both uracil and uridine but not of the amino acid tryptophan. Moreover, expression of TgENT1 in a Leishmania mexicana cell line with low endogenous uracil uptake rates significantly increased uracil uptake for these cells. We conclude that it is highly probable that TgENT1 encodes the T. gondii uracil/uridine transporter.

Abstract: Orthohalarachne attenuata and O. diminuata mites are parasites of the respiratory system of pinnipeds. During hosts dives, mites are challenged to cope with changing conditions of available oxygen in the nasal cavity. Adults and nymphs live inside the host but larvae are active and responsible for colonizing new hosts. Hence, larvae are also exposed to environmental conditions with variable temperatures, dehydration or changes in salinity. In this study, we studied the effect of hypoxia, humidity and salinity on survival of mite larvae in O. attenuata and O. diminuata. We found that both species are highly tolerant to withstand long times under hypoxia. In turn, both species showed low survival when exposed to direct air. Finally, the hyperosmotic solution was highly harmful for O. attenuata compared to O. diminuata. Our results show that humidity rather than oxygen availability is a constraint for survival and a limitation for dispersal when searching for new hosts. The present study expands our knowledge of ecophysiology and adaptations to changing conditions experienced during the dispersal of these marine parasite species.

Abstract: Reindeer in the southern Palearctic remain poorly documented, especially regarding helminths. Limited data exist for their small, isolated, and conservation-important populations. Because helminths affect survival, reproduction, and population stability, and act as biogeographic indicators, knowledge of their diversity in these southern regions is considered essential for research and management purposes. A total of 242 fecal samples were obtained from 2012 to 2025 from reindeer inhabiting China, Kazakhstan, Mongolia, and Russia. These samples were examined by coproscopy. Morphological diagnoses were supplemented with DNA analyses. Trematodes of Paramphistomoidea, cestodes of Moniezia, and nematodes identified as E. rangiferi, O. macrotis, the dimorphic O. gruehneri/O. arctica, as well as Nematodirus, Capillaria, and unidentified small strongylids were revealed. All taxa detected in this study have been reported previously for R. tarandus and for the Palearctic, exception for Nematodirus sp. for the southern area. However, examinations of reindeer from selected regions in Russia, as well as Mongolia and Inner Mongolia (China) were carried out for the first time. Southern range limits were established for E. rangiferi in China and O. macrotis in Russia. Species O. macrotis has been proposed as a biogeographical marker for wild reindeer in the Eastern Siberia, while Capillaria may indicate domestic herds.

Abstract: Faecal egg counts (FEC) are used to assess the intensity of gastrointestinal nematode (GIN) infections in herbivores. FEC distribution is aggregated, meaning that approximately 20% of animals harbour 80% of infections. In times of escalating anthelmintic resistance, it may be necessary to restrict treatment to the animals with the heaviest infections. This strategy is called targeted selective treatment (TST) and is relevant to GIN, for example. The difficulty lies in identifying which animals to treat. One solution is to select potentially at-risk animals based on age (for example, treating the young), or to perform individual faecal egg counts (though this is costly). We propose a solution for determining the suitability of selective treatment based on the level of FEC (200 or 500 eggs per gram of faeces). First, we demonstrate that the mean FEC in a group is strictly related to its variance (Taylor's power law) using published data and our own unpublished data on horses from France, Poland, and Mexico. The study will focus on small and large strongyles in horses. Taylor's power law states that sample variance (Var) and the population mean are related by a simple equation: Var = a Mean^b or log(Var) = log(a) + b log(Mean). We will then evaluate the influence of factors such as age, status (mare, stallion, yearling, etc.), day-to-day variability, and previous anthelmintic treatments on this relationship. Next, to reduce the number of FECs, we estimate the mean FEC on a composite faecal sample. We will then calculate the variability and therefore the number of horses with an FEC above the chosen acceptable level. When the mean is high, the number of horses to be treated is also high and TST is not beneficial. When the FEC is average, TST may be worthwhile, either based on the FEC of individual horses or on the horse class at risk. Based on the percentage of horses with an FEC above the acceptable level, the farmer can decide whether to treat all animals or establish a TST protocol.

Abstract: Neospora caninum, the causative agent of abortion in cattle, has a major economic impact worldwide. This review aims to provide an overview of key advances of the last 5-8 years in understanding host-pathogen interactions, molecular mechanisms, and emerging control strategies. Epidemiological studies have revealed the influence of environmental, genetic, and ecological factors on parasite transmission dynamics, and emphasized the importance of integrated "One Health" strategies. Characteristics of different Neospora strains have been elucidated through animal models and molecular tools such as clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-based gene editing, high-throughput sequencing and advanced proteomics, aiming to shed light on stage-specific gene regulation and virulence factors, contributing to the development of interventions against neosporosis. Insights into immune modulation, immune evasion and parasite persistence contributed to the efforts towards vaccine development. In terms of therapeutics, repurposed drugs but also more targeted inhibitors have shown promising efficacy in reducing parasite burden and mitigating vertical transmission in laboratory models. Here, more recent innovations in nanoparticle-based drug delivery systems and immunomodulatory strategies are prone to enhance therapeutic outcomes. However, a significant challenge remains the integration of molecular and immunological insights into practical applications.

Abstract:

This study presents the complete sequencing and comparative genomic analysis of Leishmania (Viannia) naiffi and Leishmania (Viannia) shawi, species of epidemiological relevance in the Brazilian Amazon. Genome assemblies yielded sizes of 32.13 Mb and 32.51 Mb, with 8,170 and 7,767 annotated genes, respectively. Predicted gene functions were primarily related to catalytic, binding, and ATP-dependent activities. Pangenome analysis revealed a core genome of 6,256 genes alongside notable species-specific differences, including 46 and 25 unique genes in L. naiffi and L. shawi. Functional screening identified pharmacologically promising proteins such as calpains, ABC transporters, and notably, GSK-3. Ploidy analysis indicated tetraploidy on chromosome 8 in L. naiffi and chromosome 2 in L. shawi. Genetic variability assessment detected 34,480 SNPs in L. naiffi and 26,562 in L. shawi, indicating greater genomic diversity in the former. Phylogenetic inference based on the polA1 gene confirmed the placement of both species within the Leishmania (Viannia) subgenus. These findings advance Leishmania genomics knowledge by highlighting unique genetic signatures, regions of high variability, and potential therapeutic targets. This work establishes a foundation for future research on evolution, pathogenicity, and drug development for leishmaniasis.