Ecological setting of phlebotomine sand flies in the Republic of Kosovo.

Sand flies (Diptera, Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis, as well as phleboviruses. In the Balkans, the endemicity and spreading of sand fly-borne diseases are evident, particularly in the Republic of Kosovo, a country with a predominantly humid continental climate. To date, understanding the drivers behind the spatial structure and diversity patterns of sand fly communities in humid continental regions remains limited. Therefore, elucidating the geographical and ecological factors contributing to the presence of potential vector species in the country is crucial. We aimed to enhance our understanding of factors influencing sand fly occurrence in cool and wet wintering humid continental areas, which could serve as a model for other countries with similar climatic conditions. Therefore, we assessed the currently known sand fly fauna through detailed environmental analyses, including Voronoi tessellation patterns, entropy calculations, Principal Coordinate and Component Analyses, Hierarchical Clustering, Random Trees, and climatic suitability patterns. Notable differences in the ecological tolerance of the species were detected, and the most important climatic features limiting sand fly presence were wind speed and temperature seasonality. Sand flies were observed to prefer topographical environments with little roughness, and the modelled climatic suitability values indicated that, dominantly, the western plain regions of Kosovo harbour the most diverse sand fly fauna; and are the most threatened by sand fly-borne diseases. Phlebotomus neglectus and P. perfiliewi, both confirmed vectors for L. infantum and phleboviruses, were identified as two main species with vast distribution in Kosovo. Contrary to this, most other present species are relatively sparse and restricted to temperate rather than humid continental regions. Our findings reveal a diverse potential sand fly fauna in Kosovo, indicating the need for tailored strategies to address varying risks across the country's western and eastern regions in relation to leishmaniasis control amidst changing environmental conditions.

Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Emergence of imported cutaneous leishmaniasis caused by Leishmania major: a case series from Kerala, India.

Cutaneous leishmaniasis (CL) is often considered a 'great imitator' and is the most common form of leishmaniasis. The Leishmania species responsible for CL varies among countries, as these species exhibit specific distribution patterns. The increased mobility of people across countries has resulted in the imported incidences of leishmaniasis caused by non-endemic species of Leishmania. During 2023, we confirmed three CL cases caused by L. major from Kerala, India, and upon detailed investigation, these were identified to be imported from the Middle East and Kazakhstan regions. This is the first report of CL caused by L. major from Kerala. The lesion morphology, detection of anti-rK 39 antibody and Leishmania parasite DNA from the blood samples were the unique observations of these cases. Kerala, being an emerging endemic zone of visceral leishmaniasis (VL) and CL, the imported incidences of leishmaniasis by non-endemic species can pose a significant threat, potentially initiating new transmission cycles of leishmaniasis caused by non-endemic species.

Enhancing the efficacy of fluconazole against Leishmania major: Formulation and evaluation of FLZ-nanoemulsions for topical delivery.

BACKGROUND: Cutaneous Leishmaniasis (CL) remains a significant public health concern, particularly in the tropical and subtropical regions. Present treatment options for CL such as Fluconazole (FLZ) face limitations, including low solubility and bioavailability. This study aimed to address these challenges by investigating the use of nano-emulsions (NEs) to enhance the efficacy of FLZ against Leishmania major(L.major). MATERIALS AND METHODS: FLZ-NEs were formulated with oleic acid, Tween-20, and ethanol using low-energy emulsification at various surfactant/co-surfactant ratios. Subsequently, a comprehensive analysis was conducted to assess the physicochemical characteristics of the samples. This analysis encompassed stability, zeta potential, pH, viscosity, refractive index, and droplet size. We then studied the anti-parasitic properties of these optimized FLZ-NEs both in vitro and in vivo. RESULTS: The selected nano-emulsion (NE) formulation (2 % oleic acid, 20 % Tween 20, 10 % ethyl alcohol) showcased desirable properties like small droplet size (10.51 ± 0.24 nm), low dispersity (0.19 ± 0.03), and zeta potential value (- 0.41 ± 0.17 mV), key for stability and targeted drug delivery. This optimal formulation translated into remarkable efficacy. In vitro, FLZ-NEs demonstrated a threefold increase in their ability to combat promastigotes and a remarkable thirtyfold increase in their ability to combat amastigotes. Additionally, they demonstrated a ninefold advantage in their ability to specifically target parasites within infected macrophages, thereby attacking the infection site. These promising in vitro results translated into improved outcomes in vivo. Compared to other chemicals studied, FLZ-NE-treated mice showed decreased disease severity, weight growth, and quicker ulcer healing. It was further supported by histopathological research, which showed reduced tissue damage linked to Leishmania infection. CONCLUSION: These findings show the potential of nanotechnology-based drug delivery in improving anti-leishmanial treatment.

Seroprevalence of zoonotic pathogens in stray cats in an urban area of northeast Spain.

The feline population is extensive in urban areas worldwide, comprising stray and domestic cats. Cats, acting as reservoirs, can transmit various zoonotic organisms to humans, which can cause significant public health issues. We evaluated the seroprevalence of zoonotic pathogens in stray cats in an urban area of northeast Spain (the city of Zaragoza) to assess potential risks to human health. A total of 88 sampled cats (52 females and 36 males) underwent antibody evaluation using the indirect immunofluorescence technique. Seroprevalence rates were determined for IgG antibodies to Bartonella henselae (36.3%), Toxoplasma gondii (31.8%), Rickettsia felis (14.7%), Rickettsia typhi (9%), and Leishmania infantum (10.2%). Our results confirmed the presence in stray cats of antibodies against all those pathogens, indicating that they all circulate in the feline population in Zaragoza. Male cats exhibited a higher predisposition to T. gondii, whereas females showed an increased likelihood of contracting B. henselae. This difference may be attributed to distinct behaviors according to sex. Our findings underscore the importance of maintaining and intensifying surveillance coupled with preventive measures against zoonotic pathogens in cats. They highlight the need for comprehensive control strategies designed to mitigate public health risks associated with feline populations.

Performance of a point-of-care test for the detection of anti-Leishmania infantum antibodies is associated with immunofluorescent antibody titer and clinical stage of leishmaniosis in dogs from endemic regions.

Canine leishmaniosis (CanL) is caused by the protozoal parasite Leishmania infantum, which is transmitted by sand flies in warm climates across the world. Because dogs are considered a primary domestic reservoir for the parasite that causes leishmaniosis in humans, it is important from a One Health perspective that CanL be properly managed. In endemic regions, CanL is a common differential diagnosis in sick dogs because the clinical signs and clinicopathological disorders of the disease are non-specific, variable, and may overlap those of other common conditions. Diagnosis is based on the presence of compatible clinical signs, laboratory abnormalities, and confirmation by serological and parasitological evidence of infection. Here, we describe the performance of a point-of-care (POC) immunoassay that uses recombinant antigens to detect canine anti- L. infantum antibodies in a convenience sample set from a diagnostic laboratory, a group of canine patients with clinical staging, and in apparently healthy dogs from endemic areas. An immunofluorescence antibody test (IFAT) was used as the semiquantitative reference method. In the convenience sample set with high IFAT titers (≥ 1:800), the POC immunoassay demonstrated perfect agreement with IFAT (100%; 90/90). Using samples from dogs staged as either LeishVet Stage 2 or 3 or LeishVet Stage 1, positive agreement of the POC immunoassay with the IFAT was 98.8% (82/83) and 83.8% (31/37), respectively. The negative agreement with IFAT was 98.9% (272/275) in apparently healthy dogs from endemic areas of Greece and Italy. Since the performance of the POC immunoassay was associated with IFAT titer and clinical stage of CanL, the test may help veterinarians when determining if CanL is likely responsible for a patient's clinical picture or when evaluating an apparently healthy patient prior to vaccination.

Encapsulation of Citrus sinensis essential oil and R-limonene in lipid nanocarriers: A potential strategy for the treatment of leishmaniasis.

Leishmaniases, a group of neglected tropical diseases caused by an intracellular parasite of the genus Leishmania, have significant impacts on global health. Current treatment options are limited due to drug resistance, toxicity, and high cost. This study aimed to develop nanostructured lipid carriers (NLCs) for delivering Citrus sinensis essential oil (CSEO) and its main constituent, R-limonene, against leishmaniasis. The influence of surface-modified NLCs using chitosan was also examined. The NLCs were prepared using a warm microemulsion method, and surface modification with chitosan was achieved through electrostatic interaction. These nanocarriers were characterized by differential scanning calorimetry (DSC), X-ray diffraction (XRD), transmission electron microscopy, and dynamic light scattering (DLS). In vitro cytotoxicity was assessed in L929 and RAW 264.7 cells, and leishmanicidal activity was evaluated against promastigote and amastigote forms. The NLCs were spherical, with particle sizes ranging from 97.9 nm to 111.3 nm. Chitosan-coated NLCs had a positive surface charge, with zeta potential values ranging from 45.8 mV to 59.0 mV. Exposure of L929 cells to NLCs resulted in over 70 % cell viability. Conversely, surface modification significantly reduced the viability of promastigotes (93 %) compared to free compounds. Moreover, chitosan-coated NLCs presented a better IC50 against the amastigote forms than uncoated NLCs. Taken together, these findings demonstrate the feasibility of using NLCs to overcome the limitations of current leishmaniasis treatments, warranting further research.

Synthesis and activity of benzimidazole N-Acylhydrazones against Trypanosoma cruzi, Leishmania amazonensis and Leishmania infantum.

In this study, we present the design, synthesis, and cytotoxic evaluation of a series of benzimidazole N-acylhydrazones against strains of T. cruzi (Y and Tulahuen) and Leishmania species (L. amazonensis and L. infantum). Compound (E)-N'-((5-Nitrofuran-2-yl)methylene)-1H-benzo[d]imidazole-2-carbohydrazide demonstrated significant activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 0.033 µM and a selectivity index (SI) of 7680. This represents a potency 46 times greater than that of benznidazole (IC50/120 h = 1.520 µM, SI = 1390). Another compound (E)-N'-(2-Hydroxybenzylidene)-1H-benzo[d]imidazole-2-carbohydrazide showed promising activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 3.600 µM and an SI of 14.70. However, its efficacy against L. infantum and L. amazonensis was comparatively lower. These findings provide valuable insights for the development of more effective treatments against Trypanosoma cruzi.

Isolation of two steroidal saponins with antileishmanial activity from Allium giganteum L.

Background and purpose: Alliums are rich sources of steroidal saponins, flavonoids, and sulphoric compounds of which steroidal saponins have recently received more attention due to their important pharmacological activities. Allium giganteum (giant onion) which is named locally "Couria" in the Northeast of Iran, is grown widely in "Kouh-Sorkh" mountains in Khorasan province. Experimental approach: Phytochemical investigation of chloroform-methanol and aqueous extract of the plant resulted in the isolation and identification of two steroidal saponins, using comprehensive spectroscopic methods including 1D and 2D NMR and MS. Findings/Results: The chemical structures of the isolated saponins were determined as (22S)-cholesta-1b,3b,16b,22b-tetraol 5-en, and 3-O-ß-D-glucopyranosyl26-O-ß-D-glucopyranosside and (25R)-26-O-ß-D-glucopyranosyl-5α-furostan-1α,3ß,22α,26-tetraol3-O-{ß-D-galactopyranosyl-(1→2)-O-[ß-D-xylopyranosyl- (1→3)]-O-ß-D-glucopyranosyl-(1→4)-ß-D-galactopyranoside}. Investigation of in vitro antileishmanial activity of the isolated compounds at 10, 50, and 100 µg/mL exhibited significant leishmanicidal against the promastigotes of Leishmania major. Conclusion and implications: The results established a valuable basis for further studies about A. giganteum and the anti-parasitic activity of steroidal saponins.

Mevalonate kinase of Leishmania donovani promotes its survival and plays a pivotal role in pathogenesis.

The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, ∼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.

Molecular Survey of Leishmania Infection of Sand Flies in Karun County, Southwestern Iran.

Background: Zoonotic cutaneous leishmaniasis (ZCL) is widely distributed in Iran and around the world. Also, Khuzestan Province is an endemic focus of ZCL. This study aims to investigate the natural infection of sand flies with the Leishmania parasite in Karun County. Methods: Sand flies were collected from Jangiyeh, Qaleh Chanan, Kut-e-Navaser, and Ghazavieh in the spring and summer in the year of 2019, by installing 60 sticky paper traps each time (30 traps outdoors and 30 traps indoors). Two hundred female sand flies with different abdominal conditions (empty, blood-fed, semi-gravid, and gravid) were examined for infection rate using the Nested-PCR method. Results: In this study, seven species of sand flies including Phlebotomus papatasi, Ph. alexandri, Ph. sergenti, Ph. caucasicus, Sergentomyia tiberiadis, Se. sintoni, and Se. antennata were reported from Karun County, with a frequency of 79.64%, 16.96%, 1.07%, 0.18%, 0.36%, 1.61%, and 0.18%, respectively. Only eleven specimens of Ph. papatasi were found to be positive for Leishmania major, with an overall infection rate of 7.8%. The infection of Ph. papatasi was specifically reported in blood-fed, gravid, and semi-gravid specimens, with infection rates of 17.02%, 4.35%, and 14.29%, respectively. Conclusion: In this study, the infection of L. major from Ph. papatasi was reported. The results can be used in planning the control of ZCL in the study area.

Evaluation of Nanonanoliposomal Curcumin on Cutaneous Leishmaniasis Skin Lesions Caused by Leishmania major in BALB/c Mice.

Background: Curcumin is an extract of rhizome turmeric (diferuloylmethane), with antioxidant, anti-inflammatory, antimicrobial, and anti-parasitic properties, which making it a potential candidate for the treatment of leishmaniasis. The aim of the presented study was to evaluate curcumin as possible candidate for treatment of cutaneous leishmaniasis. Methods: We investigated the physicochemical properties and anti-leishmanial effects of nanoliposomal curcumin (40, 80, and 120 µM) in Leishmania major (MRHO/IR/75/ER) infected BALB/c mice at the faculty of Veterinary Medicinem University of Tehran, Iran. For this aim, L. major promastigotes (MHROM/IR/75/ER) at stationary phase (2×106) were inoculated sub-cutaneously into the upper area of the tail in BALB/c mice (six groups, n= 10 per group). For evaluation of nanoliposomal curcumin, the zeta potential, particle size and stability of nanoliposomal curcumin was determined. Furthermore, the anti-leishmanial effects of nanoliposomal curcumin formulation on the lesion sizes was determined and the parasite burden in the leishmania induced lesion was performed using semi quantitative PCR. Results: Treatment of L. major infected BALB/c mice with nanoliposomal curcumin led to a reduction in the kinetic of the skin lesion size development. The semi quantitative PCR analysis of DNA extracted from the lesions showed reduction of parasite burden. The most effective treatment could be found in 80 µM nanoliposomal curcumin. Treatment with Glucantime, as a positive control, also showed a nearly similar effect compared to the effect of 80 µM nanoliposomal curcumin. Conclusion: Nanoliposomal curcumin could be considered as a potential drug against cutaneous leishmaniasis caused by L. major in susceptible animal models.

Designing a Multiple-Epitope Vaccine Candidate against Leishmania major and Leishmania infantum for Monocyte-Derived Exosome Preparation.

Background: Leishmania is a vector-borne protozoon, which causes visceral, cutaneous and mucocutaneous leishmaniosis in human and animals. Monocyte-derived exosome vaccines can be used as prophylaxis and immunotherapy strategies. The aim of this study was to design a multiple-epitope candidate vaccine using leishmaniolysin (GP63) and rK39 proteins against Leishmania major and L. infantum for monocyte-derived exosome preparation. Methods: This study was carried out in Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, 2023-2024. Effective immunodominant epitopes were selected from two antigenic proteins of GP63 and rK39 using various immunoinformatics and bioinformatics approaches. Vibrio cholerae ß-subunit was used as an adjuvant to stimulate immune responses. Then, appropriate linkers were selected for the fusion of epitopes. The 3D model of candidate vaccine was predicted and validated. Results: This designed candidate vaccine could effectively be used as a prophylaxis strategy against leishmaniosis. Conclusion: A candidate vaccine was designed using bioinformatic and immunoinformatic studies with virtual acceptable quality; however, effectiveness of this vaccine should be verified through further in-vitro and in-vivo studies.

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania.

Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multi-locus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

A breath of fresh air: impact of insect-borne protozoan parasites on the respiratory system.

The protozoan parasites Plasmodium, Leishmania, and Trypanosoma are transmitted by hematophagous insects and cause severe diseases in humans. These infections pose a global threat, particularly in low-resource settings, and are increasingly extending beyond the current endemic regions. Tropism of parasites is crucial for their development, and recent studies have revealed colonization of noncanonical tissues, aiding their survival and immune evasion. Despite receiving limited attention, cumulative evidence discloses the respiratory system as a significant interface for host-pathogen interactions, influencing the course of (co)infection and disease onset. Due to its pathophysiological and clinical implications, we emphasize that further research is needed to better understand the involvement of the respiratory system and its potential to improve prevention, diagnosis, treatment, and interruption of the chain of transmission.

Synthesis, biological evaluation and mechanism of action of benzothiazole derivatives with aromatic hydrazone moiety, a new class of antileishmanial compounds.

Leishmaniasis is a disease caused by protozoa Leishmania spp., considered as a significant and urgent public health problem mainly in developing countries. In the absence of an effective vaccine, the treatment of infected people is one of the most commonly prophylactic measures used to control this disease. However, the therapeutic arsenal is reduced to a few drugs, with serious side effects and variability in efficacy. Attempting to this problem, in this work, a series of benzothiazole derivatives was synthetized and assayed against promastigotes and intracellular amastigotes of L. amazonensis, as well as the toxicity on macrophages. In addition, studies about the mechanism of action were also performed. Among the synthesized molecules, the substitution at position 4 of the aromatic ring appears to be critical for activity. The best compound exhibited IC50 values of 28.86 and 7.70 µM, against promastigotes and amastigotes of L. amazonensis, respectively, being more active than miltefosine, used as reference drug. The in silico analysis of physicochemical and pharmacokinetic (ADMET) properties of this compound suggested a good profile of oral bioavailability and safety. In conclusion, the strategy of using benzothiazole nucleous in the search for new antileishmanial agents was advantageous and preliminar data provide information about the mechanism of action as well as in silico parameters suggest a good profile for preclinical studies.

First Evidence of Leishmania infantum Antibodies in Sheep (Ovis aries) from Southern Germany.

In Europe, Leishmania infantum is the most prevalent Leishmania species, and this protozoan is transmitted by phlebotomine sandflies. A recent publication has shown that sheep harbor L. infantum antibodies. This raises questions about the epidemiological role of small ruminants. Therefore, sera from small ruminants located in two southern German federal states, Baden-Wuerttemberg (BW) and Bavaria (BAV), were analyzed with an ELISA to determine the presence of L. infantum antibodies. The species, sex and age (gimmer vs. ewe) were recorded, and a univariate analysis was conducted to determine possible associations. In total, seven sheep flocks (274 sheep/10 goats) from BW and seven sheep flocks (277 sheep/78 goats) from BAV were examined. In BW, four sheep from three flocks tested positive for L. infantum antibodies. In BAV, the same number of positive sheep were detected but in four flocks. The total seropositivity rate in sheep was 1.45%. All goats tested negative. No significant association (p > 0.05) was detected between Leishmania seropositivity and the variables evaluated. Our study reveals the exposure of sheep to L. infantum in a non-endemic area. Further investigation is needed to determine whether sheep can be used as sentinels to identify new phlebotomine habitats and Leishmania risk areas.

Artemisinin: An Anti-Leishmania Drug that Targets the Leishmania Parasite and Activates Apoptosis of Infected Cells.

Leishmaniasis is a relevant disease worldwide due to its presence in many countries and an estimated prevalence of 10 million people. The causative agent of this disease is the obligate intracellular parasite Leishmania which can infect different cell types. Part of its success depends on its ability to evade host defense mechanisms such as apoptosis. Apoptosis is a finely programmed process of cell death in which cells silently dismantle and actively participate in several processes such as immune response, differentiation, and cell growth. Leishmania has the ability to delay its initiation to persist in the cell. It has been well documented that different Leishmania species target different pathways that lead to apoptosis of cells such as macrophages, neutrophils, and dendritic cells. In many cases, the observed anti-apoptotic effect has been associated with a significant reduction in caspase-3 activity. Leishmania has also been shown to target several pathways involved in apoptosis such as MAPK, PI3K/Akt, and the antiapoptotic protein Bcl-xL. Understanding the strategies used by Leishmania to subvert the defense mechanisms of host cells, particularly apoptosis, is very relevant for the development of therapies and vaccines. In recent years, the drug artemisinin has been shown to be effective against several parasitic diseases. Its role against Leishmania may be promising. In this review, we provide important aspects of the disease, the strategies used by the parasite to suppress apoptosis, and the role of artemisinin in Leishmania infection.

Activation of neutrophils excels the therapeutic potential of Mycobacterium indicus pranii and heat-induced promastigotes against antimony-resistant Leishmania donovani infection.

Repurposing drugs and adjuvants is an attractive choice of present therapy that reduces the substantial costs, chances of failure, and systemic toxicity. Mycobacterium indicus pranii was originally developed as a leprosy vaccine but later has been found effective against Leishmania donovani infection. To extend our earlier study, here we reported the immunotherapeutic modulation of the splenic and circulatory neutrophils in favour of hosts as neutrophils actually serve as the pro-parasitic portable shelter to extend the Leishmania infection specifically during the early entry into the hosts' circulation. We targeted to disrupt this early pro-parasitic incidence by the therapeutic combination of M. indicus pranii and heat-induced promastigotes against antimony-resistant L. donovani infection. The combination therapy induced the functional expansion of CD11b+Ly6CintLy6Ghi neutrophils both in the post-infected spleen, and also in the circulation of post-treated animals followed by the immediate Leishmania infection. More importantly, the enhanced expression of MHC-II, phagocytic uptake of the parasites by the circulatory neutrophils as well as the oxidative burst were induced that limited the chances of the very early establishment of the infection. The enhanced expression of pro-inflammatory cytokines, like IL-1α and TNF-α indicated resistance to the parasite-mediated takeover of the neutrophils, as these cytokines are critical for the activation of T cell-mediated immunity and host-protective responses. Additionally, the induction of essential transcription factors and cytokines for early granulocytic lineage commitment suggests that the strategy not only contributed to the peripheral activation of the neutrophils but also promoted granulopoiesis in the bone marrow.