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that the mean FEC in a group is strictly related to its variance (Taylor's power law) using published data and our own unpublished data on horses from France, Poland, and Mexico.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.

Abstract:

Autophagy is a conserved cellular degradation process involving ATG proteins, with ATG4 proteases essential for processing ATG8 family proteins during autophagosome formation. In Trichomonas vaginalis, the role of autophagin proteases in processing autophagy markers TvAtg8a and TvAtg8b has not been fully characterized. In this study, we expressed and purified recombinant TvAtg4.4 and demonstrated its cysteine protease activity in vitro. TvAtg4.4 rapidly processed TvAtg8aGST and, to a lesser extent, TvAtg8bGST. Enzymatic assays confirmed substrate specificity and inhibition by cysteine protease inhibitors. TvAtg4.4 mRNA expression increased under glucose restriction, and immunolocalization showed its presence in autophagic vesicles, cytoplasm, endoplasmic reticulum, Golgi, lysosomes, hydrogenosomes, and nucleus. Colocalization with TvAtg8a and TvAtg8b supports its functional role in autophagy. The localization of TvAtg4.4 in T. vaginalis autophagosomes and ER suggests its involvement in the cleavage of TvAtg8a and TvAtg8b after synthesis and in the delipidation or deconjugation of these proteins from the autophagosome outer membrane before autophagosome-lysosome fusion. These findings clarify the enzymatic function and cellular localization of TvAtg4.4, provide insight into autophagy mechanisms in T. vaginalis, and suggest potential novel roles for this protease in parasite biology.

Abstract: The absence of molecular tools for manipulation of gene expression in the pathogenic free-living amoeba Naegleria fowleri has historically limited our understanding of gene function in the organism and has coincidently impacted the identification of potential druggable pathways and proteins. Here, we describe the development of approaches for the generation of transgenic amoebae using polyethyleneimine nanoparticles to deliver plasmids designed to confer antibiotic resistance and fluorescence to the cells. Through a series of optimization steps, we found that transfection of plasmids encoding the fluorescent protein mCherry fused by a T2A self-cleaving peptide to a codon-optimized puromycin acetyltransferase selectable marker yielded fluorescent cells that were resistant up to 100 µg/mL puromycin. Transfected trophozoites harbored between 45 and 65 copies of the transgene per cell and both fluorescence and resistance were persistent in the presence of selector through continued passages. The development of these approaches is anticipated to enable application of an array of genetic manipulation techniques including forward and reverse genetics to the study of this important pathogen.

Abstract:

Gastrointestinal nematodes are among the most significant parasites affecting liverstock health and productivity, leading to major economic losses and contributing to the global increase in resistance to anthelmintics. Biological control using fungi with ovicidal and nematophagous activity offers an environmentally friendly alternative. This study investigated, for the first time, the interactive effects between the nematophagous/larvicidal fungus Duddingtonia flagrans and the ovicidal fungus Pochonia chlamydosporia under natural infection conditions. Eighteen Holstein × Zebu males (12–15 months old) were divided into three groups (n = 6): T1 (D. flagrans), T2 (D. flagrans + P. chlamydosporia), and control. Treatments were administered orally daily (6 g/100 kg BW of each fungus; 10⁶ chlamydospores/g) for nine months. Faecal egg counts (EPG) and infective larvae in pasture (L3) were monitored. Groups T1 and T2 showed significantly lower EPG values than the control during most of the experimental period. Haemonchus spp. was identified as the predominant nematode, confirming its epidemiological relevance. The combined fungal treatment exhibited synergistic activity, enhancing parasite suppression through complementary ovicidal and larvicidal mechanisms. This approach proposes a sustainable and reproducible alternative to the excessive use of chemical compounds, contributing innovative and applicable solutions to national livestock production and integrated animal health.

Abstract: Malaria remains one of the most pressing public health challenges in Africa, a continent that bears a significant percentage of the global malaria morbidity and mortality. From the earliest microscopic discoveries of Plasmodium to the era of genomics and vaccine innovations, Africa has stood both as the epicentre of the disease, and the focal point of global research and control efforts. The continent’s unique ecological, genetic, and socio-political contexts have also shaped the evolution of the parasite, the host, and the mosquito vector. African scientists, institutions, and communities have progressively transitioned from being subjects of investigations to active contributors in malaria research; advancing studies in epidemiology, molecular biology, pharmacogenomics, and vaccine development. This review traces the journey of malaria science from its microscopic origins to genomic breakthroughs; emphasising how Africa’s contributions, challenges, and innovations have redefined global understanding. It also highlights the importance of locally-driven research, surveillance, and policy frameworks to translate genomic data into practical solutions, aiming towards equitable and sustainable malaria elimination on the continent.

Abstract: Haemonchus contortus (H. contortus) is a gastrointestinal parasite that affects small ruminants, causing anemia, edema, and, in severe cases, death, posing a significant threat to livestock production. This study focused on analyzing the parasite’s sexual differentiation to identify potential molecular targets for the development of control strategies. The genes daf-12, fem-1, and sdc-2 were evaluated based on their orthologs in Caenorhabditis elegans. Specific primers were designed, and nucleic acids were extracted from L3 larvae and adult male and female H. contortus. Gene presence and expression were analyzed using PCR and RT-PCR, along with protein structure modeling and phylogenetic analysis. Results showed differential gene expression depending on life stage and sex: daf-12 was highly expressed in L3 larvae, indicating its involvement in early development; fem-1 showed higher expression in males, suggesting a role in male sexual differentiation; while sdc-2 was more expressed in females, implying a function in regulating female characteristics. These findings provide key insights into the molecular mechanisms underlying sexual differentiation in H. contortus, which could contribute to the development of novel control tools and help mitigate the economic losses caused by this parasite in livestock production.

Abstract: This study was conducted to assess the involvement of two lizard species: the sand lizard (L. agilis) and the common lizard (Zootoca vivipara), and their Ixodes ricinus ticks, in the circulation spirochetes of the Borrelia burgdorferi s.l. complex. Lizards were captured at three study sites in suburban areas of western Poland. Common lizards were less abundant and occurred only at one site. A total of 1,129 ticks were collected from 167 sand lizards and 164 individuals from 42 common lizards. Biopsies of the distal part of the lizard tail were taken from 172 animals. All samples that tested positive by real-time PCR underwent subsequent nested PCR targeting the flaB gene, followed by sequencing. At least 6.3% of I. ricinus ticks (MIR) from L. agilis, and 6.1% from Z. vivipara, were infected. Borrelia lusitaniae was the most prevalent species in L. agilis-derived ticks, accounting for 73.2% of all infected samples, followed by B. burgdorferi s.s. (23.0%). Conversely, this latter species prevailed (90%) over B. lusitaniae (10%) in tick samples from Z. vivipara. Therefore, we believe that sand lizards are competent reservoir hosts for B. lusitaniae, while the role of Z. vivipara for this species is unclear. The high prevalence of B. burgdorferi s.s. was also found in infected larval samples (40.7%) and biopsies (60%) of L. agilis. Thus, in our opinion, these two lizard species could be another group of reservoir hosts for this human pathogen, along with birds and rodents.

Abstract:

Toxoplasma gondii, the causative agent of toxoplasmosis widespread in animals and humans, is an intracellular apicomplexan protozoan parasite infecting a variety of host cells. Gene editing using CRISPR-Cas9 has become a standard tool to investigate the molecular genetics of this interaction. With respect to gene knock-out (KO) studies, the general paradigm implies that the gene of interest is expressed in the wildtype and that only the gene of interest is affected by the knock-out. Consequently, the observed phenotype depends on presence or absence of genes of interest. To challenge this paradigm, we knocked out two open reading frames (ORFs) constitutively expressed in T. gondii ShSp1 tachyzoites, but not essential, namely ORF 297720 encoding a trehalose-6-phosphatase homolog and ORF 319730 encoding a You2 C2C2 zinc finger homolog. We analyzed the proteomes of tachyzoites isolated at a late stage of infection, of intracellular tachyzoites and of host cells at an early stage of infection. The intended KO proteins were present in the T. gondii Sp1 wildtype but absent in the KO clones. Moreover, besides differentially expressed (DE) proteins specific to each KO, 17 DE proteins common to both KOs were identified in isolated and 39 in intracellular tachyzoites. Moreover, 76 common DE proteins were identified in host cells. Network and enrichment analyses showed that these proteins were functionally related to antiviral defense mechanisms. These results indicate that the KO of a gene of interest may not only affect the expression of other genes of the target organism, in our case T. gondii, but also the gene expression of its host cells. Therefore, phenotypes of KO strains may not be causally related to the KO of a given gene.

Abstract:

Background: Clinical research lab is a key component of the conduct of clinical studies. Enrolment, follow up of patients is mainly based on the diagnosis skills of lab technicians and automates performance. To overcome this challenge, since 2020, as part of the quality assurance of its lab research activities, GRAS has registered with the College of American Pathologists (CAP) and the Clinical Laboratories Services (CLS) Johannesburg, South Africa for external proficiency testing (EPT) of its laboratory. The performances achieved during these last five years are reported. Methods: PT samples were couriered out three times a year (quadr-month) (hereafter referred to as Q) to GRAS Lab and results were generally due back two to three weeks later. For parasitology, challenges specimen are thick and thin blood films stained with Giemsa and mounted with strip to protected them for multiple use, or photographs also called ̎ whole slide images ̎ (WSI). For biochemistry and haematology, a set of 5 samples were received for processing. All the evaluations were performed according to the GRAS lab internal established procedures. Results: The overall CAP parasitological laboratory performance in term of diagnosis accuracy of malaria parasites and other blood parasites from 2020 to 2024 was 97.3% (ranging from 93,33 to 100%); with 93,33%, 100%, 100%, 93.33% and 100% achieved respectively in 2020, 2021, 2022, 2023 and 2024. Annually, the number of evaluated microscopists varied according to operational staff at the time of evaluation. In total, 31 microscopists were enrolled in CLS PT scheme, with 73.9% graded as “experts” microscopists and 19.2% graded as “reference” microscopists. For haematology, the PT showed 100% accuracy over the 4 years participation indicating the high-performance level of the tested automates and the comparability of data between automates. Same trend was observed for biochemistry PT results with an overall score of 92,12% ranging from 78% to 100%. Conclusion: The Proficiency testing has demonstrated value as an important laboratory quality assurance tool to guaranty the accuracy skill for malaria parasites and other blood parasites diagnosis by lab technicians and results generated by the automates. It has helped laboratories identify issues related to test design and performance.

Abstract: Type-P5 ATPases are the least characterized among the P-type ATPases and this is especially true in the case of the malaria parasite. In this study, Spf1, a subtype-P5A ATPase of yeast, and ATP13A2, a subtype-P5B ATPase of humans, were used as templates to extensively characterize the sequences and structural features of haemosporidian type-P5 ATPases. Malaria parasites have both subtype-P5A and subtype-P5B ATPase genes and the structural features of the proteins recapitulate the known structures of subtype-P5A and subtype-P5B ATPases respectively. Detailed structural analysis detected an addition-al alpha-helix in the P-domain of subtype-P5A ATPases which is not found in subtype-P5B ATPases. This feature may be an additional signature to distinguish subtype-P5A and subtype-P5B ATPases in addition to the differences in the membrane loops of the N-terminal domain, the arm in the P-domain of subtype-P5A, and substrate differences. A notable difference in the type-P5 ATPases from the malaria parasite, as compared to the templates, is the insertion of multiple regions of variable and low-complexity regions that form intrinsically disorganized loops. These loops may form a shroud-like structure that protects the core ATPase structure and/or participates in low-affinity interprotein interactions. Homology modeling did not provide definitive answers about the substrate specificity of the haemosporidian type-P5 ATPases. However, the haemosporidian sub-type-P5A ATPase is likely an ER transmembrane dislocase as are the other subtype-P5A ATPases. In contrast, the subtype-P5B ATPases of the malaria parasite are not likely to be polyamine transporters in lysosomes, as have been described in fungi and metazoans. This suggests that subtype-P5B ATPases have undergone lineage specific divergence in regard to their function(s).

Abstract:

Background: Leishmaniasis is a zoonotic vector-borne disease and a significant global public health concern worldwide and in Algeria. In This study we have investigated the potential role of ticks and fleas as carriers of Leishmania in endemic regions of Algeria. Methods: Adult ectoparasites were collected from reservoir dogs and cohabiting animals across three provinces: Tizi-Ouzou (northeast), M'Sila (southeast), and Tébessa (extreme east). A subset of 247 ectoparasites was randomly selected for Leishmania DNA screening using ITS1-PCR. Results: Morphological identification revealed two tick species, Rhipicephalus turanicus (378 specimens) and Rhipicephalus sanguineus s.l (127 specimens), and one flea species, Ctenocephalides felis (94 specimens). Dogs were the most heavily infested hosts (74.12%), followed by sheep (9.51%) and cats (9.34%). Leishmania DNA was detected in 36.43% (90/247) of the tested specimens, with higher positivity in ticks (41.32%) compared to fleas (17.64%). Infection rates varied by host species, with dogs harboring the majority of positive ectoparasites (62/90), primarily R. sanguineus s.l (19/30) and R. turanicus (40/115). Leishmania DNA was also detected in ectoparasites collected from cats and sheep, whereas goats and rabbits were free from Leishmania DNA. Conclusions: This investigation highlights the high detection rate of Leishmania DNA in ticks and fleas from animals in Algerian endemic regions, indicating exposure to infected hosts. Together with previous reports, these findings support the view that ticks and fleas may act as incidental hosts or mechanical carriers of the parasite. However, their role in parasite transmission remains unconfirmed and warrant further investigation, particularly through studies assessing vector competence. These results emphasize the need for additional research to clarify the contribution of these ectoparasites to Leishmania transmission and multi-host dynamics.

Abstract: Horse flies (Tabanidae) negatively affect livestock by reducing productivity, compro-mising animal welfare, and serving as mechanical vectors of pathogens. However, the spatial processes shaping their community organization in southern Brazil’s Coastal Plain of Rio Grande do Sul (CPRS) remain poorly understood. To address this, we con-ducted standardized Malaise-trap surveys and combined them with historical–contemporary comparisons to examine distance–decay patterns in community compo-sition. We evaluated both abundance-based (Bray–Curtis) and presence–absence (Jac-card) dissimilarities using candidate models. Across sites, Tabanus triangulum emerged as the dominant species. Dissimilarity in community structure increased monotonically with geographic distance, with no evidence of abrupt thresholds. The square-root model provided the best fit for abundance-based data, whereas a linear model best described presence–absence patterns, reflecting dispersal limitation and environmental filtering across a heterogeneous coastal landscape. Sites within riparian forests and conservation units displayed higher diversity, emphasizing the ecological role of protected habitats and the importance of maintaining connected corridors. Collectively, these findings establish a process-based framework for surveillance and landscape management strategies to mitigate vector, host contact. Future directions include integrating remote sensing and host distribution, applying predictive valida-tion across temporal scales.

Abstract:

Parasites of marine mammals are of significant interest in the fields of evolutionary biology, veterinary medicine, human health, conservation, and ecology. Therefore, every opportunity to study them should be seized. This study aimed to report on specimens of cestodes found in the small intestine of a gray whale (Eschrichtius robustus) in Chukotka, Russia, in 2024. The specimens were examined using light and scanning electron microscopy, histology, and molecular analysis. Based on their morphology and host specificity, the cestodes were identified as Priapocephalus eschrichtii. Analyses of the 18S and 28S ribosomal RNA genes confirmed a close relationship between P. eschrichtii and species of Tetrabothrius, which infect seabirds and whales. Sequences of the 18S, 28S, ITS2 rDNA, and gene CoxI mtDNA are reported here for the first time. More than 50 specimens of P. eschrichtii were detected in 1 meter of intestine of the whale with a maximum length of 50 cm. Chronic lymphoplasmacytic inflammation of the intestinal mucosa was observed in the infected whale. The eggs of the cestodes were clustered within an irregular membrane, which may contribute to multiple infestations of the host and lead to diseases that affect the whale’s health and survival. This study should be continued.

Abstract: For eight years prior to Argentina's malaria-free certification in 2019, there were no instances of local transmission. Epidemiological surveillance focused on detecting Plasmodium vivax in areas where the last cases had been reported. During the national malaria surveillance program (2016-2018) in Salvador Mazza (Salta Province, northwestern Argentina), several neighborhoods were randomly selected for the collection of human blood samples to detect any silent circulation of P. vivax. Diagnosis of malaria parasites relied on traditional microscopy and molecular detection using blood collected on filter paper, by amplifying and sequencing a portion of the Plasmodium cytochrome b gene. An autochthonous case of P. vivax was identified in an asymptomatic 64-year-old individual in La Bendición neighborhood, Salvador Mazza. The individual had never traveled to any P. vivax-endemic region. This case is the first detected among 92 samples collected from various localities along Argentina's borders with Bolivia (northwest) and Brazil (northeast). This finding highlights the possibility of silent circulation of P. vivax in areas previously assumed to be malaria-free and raises concerns regarding the timing of the certification, prompting a reevaluation of the current situation. The extent of P. vivax circulation among asymptomatic individuals remains largely unknown. This is the first molecularly confirmed asymptomatic case of P. vivax reported following malaria-free certification in Argentina and the Southern Cone region. Currently, there is no active epidemiological or entomological surveillance in the area where the case was recorded. The permeable nature of the border facilitates disease transmission, and the lack of information about the movement of asymptomatic individuals is particularly concerning. It is imperative that the surveillance system responds effectively to maintain the status as a malaria-free country, prioritizing this within the national health system agenda due to the potential implications of silent malaria circulation and the risk of re-emergence and re-establishment.

Abstract: The aim of this study was to determine the optimal incubation time for accurate assessment of parasitic nematode eggs viability, an important step in improving parasitological diagnostics. The experiment used Ascaris suum eggs were collected from three sources: adult roundworms uteri (U), pig faeces (F) and sewage sludge (S), then incubated at 27°C and monitored weekly. Eggs were classified as dead (with clear deformations), viable (with motile larvae) or of uncertain viability (retaining structural integrity but undeveloped). The results showed that eggs from group U had the highest viability (96%) and developed larvae within 3 weeks. In contrast, group F (52% viability) and S (3% viability) showed delayed development, requiring up to 8-12 weeks for a conclusive viability assessment. These results indicate significant differences in egg viability depending on the sample source and emphasise the need for longer incubation times, particularly for environmental samples such as sewage sludge. The study also highlights the limitations of single time point assessments based solely on egg structure, which can lead to misclassification. In conclusion, prolonged incubation improves diagnostic accuracy by allowing a clearer distinction between viable and non-viable eggs, especially in samples with initially uncertain viability.

Abstract: There is increasing evidence on the occurrence of Crithidia spp. in patients presenting either cutaneous or visceral leishmaniasis, solely or associated with Leishmania. We analyzed the influence of temperature in the growth rate and morphology of two Crithidia fasciculata strains (a reference strain and one isolated from a patient), and the effect of the co-cultivation of Leishmania and Crithidia in parasite isolation, in the infection of macrophages in vitro, and also in infections of hamsters, BALB/c mice and sandflies. In culture, both Crithidia strains could undergo 32oC for 96 h, although major morphological alterations and a decrease in mitochondrial membrane potential were observed. At 34°C, there was an 80% reduction on the number of cells from the patient strain. Mixed cultivation of Crithidia-Leishmania led to the recovery of only Crithidia. In macrophages, C. fasciculata alone was virtually eliminated, and in the co-infection only Leishmania was recovered. The same was observed in vivo. Curiously, C. fasciculata is more resistant to Amphotericin B. Our results indicate that both C. fasciculata strains are unable to reproduce the pathogenic effect in vitro and in vivo models.

Abstract: Sterol biosynthesis is crucial for the function of biological membranes and an important target for anti-protozoan/anti-fungal drugs. In the trypanosomatid parasite Leishmania major, deletion of sterol C14-demethylase (C14DM) results in hypersensitivity to heat, increased plasma membrane fluidity, profound mitochondrial dysfunctions, and reduced virulence in mice. In this study, we show that C14DM-null mutants are defective in their tolerance to membrane disrupting agents and osmotic stress and their ability to form autophagosomes. In addition, C14DM-null mutants exhibit heightened sensitivity to anti-trypanosomatid drugs including antimony, ethidium bromide and pentamidine. Combination of itraconazole (a C14DM antagonist) and pentamidine synergistically inhibit the growth of Leishmania parasites. These findings reveal new insight into the roles of sterol synthesis in protozoan pathogens and highlight the potential of using drug combinations to achieve better treatment outcome.