Q3 lab-on-chip real-time PCR for the diagnosis of Leishmania infantum infection in dogs.

Leishmaniasis is a vector-borne disease caused by many Leishmania spp. which infect humans and other mammalian hosts. Leishmania infantum is the main agent of canine leishmaniasis (CanL) whose diagnosis is usually confirmed by serological and molecular tests. This study aimed to evaluate the clinical and analytical sensitivities of a lab-on-chip (LOC) real-time PCR applied on the portable Q3-Plus V2 platform (Q3 qPCR) in the detection of L. infantum. The Q3 qPCR performance was assessed by comparing the quantification cycle (Cq) values with those obtained using the qPCR run on a CFX96 Real-Time System (CFX96 qPCR). A total of 173 DNA samples (extracted from bone marrow, lymph node, blood, buffy coat, conjunctival swab, and skin) as well as 93 non-extracted samples (NES) (bone marrow, lymph node, blood, and buffy coat) collected from dogs were tested with both systems. Serial dilutions of each representative DNA and NES sample were used to assess the analytical sensitivity of the Q3 qPCR system. Overlapping Cq values were obtained with the Q3 qPCR and CFX96 qPCR, both using DNA extracted from L. infantum promastigotes (limit of detection, <1 promastigote per milliliter) and from biological samples as well as with NES. However, the Q3 qPCR system showed a higher sensitivity in detecting L. infantum in NES as compared with the CFX96 qPCR. Our data indicate that the Q3 qPCR system could be a reliable tool for detecting L. infantum DNA in biological samples, bypassing the DNA extraction step, which represents an advance in the point-of-care diagnostic of CanL.

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.

Isoenzyme characterization of Leishmania infantum toward checking the antioxidant activity of superoxide dismutase and glutathione peroxidase.

BACKGROUND: Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. METHOD: After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. RESULTS: GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. CONCLUSION: The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

Re-emergence of human leishmaniasis in northern Italy, 2004 to 2022: a retrospective analysis.

BackgroundHuman leishmaniasis is a protozoan disease transmitted by sand flies and endemic in the Mediterranean region. In Italy, leishmaniasis is present in the south and the western coastal regions, with an epidemic peak detected in northern Italy in the early 1970s.AimTo examine temporal trends, and demographic, clinical, geographical and environmental features of human leishmaniasis cases recorded by the local health unit (LHU) of Bologna, northern Italy.MethodsIn this retrospective observational study, we analysed human leishmaniasis cases recorded from 2004 to 2022 within the Bologna LHU. We also conducted serological investigations for canine leishmaniasis in owned dogs living near the place of infection of human cases.ResultsIn total, 173 cases of human leishmaniasis were detected, and 154 cases were considered autochthonous. An increase of human cases was observed since 2004, with incidence peaks above 2 cases/100,000 inhabitants in 2013, 2018 and 2022; epidemic peaks were preceded by dry summers. Most cases lived in the plain and hilly areas less than 400 m above sea level and many resided in isolated housing, in city outskirts, and/or near uncultivated areas, watercourses and railway sections. The incidence of canine leishmaniasis did not increase in the study period.ConclusionAn epidemic of human leishmaniasis with fluctuating annual numbers of cases, probably related to environmental and climatic factors, was identified in the Bologna LHU. Understanding the risk factors and the environmental characteristics related to places of infection is crucial to evaluate the public health implications of leishmaniasis.

Hydrazinated geraniol derivatives as potential broad-spectrum antiprotozoal agents.

Geraniol, a primary component of several essential oils, has been associated with broad-spectrum antiprotozoal activities, although moderate to weak. This study primarily concentrated on the synthesis of hydrazinated geraniol derivatives as potential antiprotozoal agents. The synthesised compounds were tested in vitro against different parasitic protozoans of clinical relevance, including Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania infantum. Compounds 6, 8, 13, 14 and 15 demonstrated low micromolar activity against the different parasites. Compounds 8, 13, 14 and 15 had the highest efficacy against Trypanosoma brucei rhodesiense, as indicated by their respective IC50 values of 0.74, 0.56, 1.26 and 1.00 µM. Compounds 6, 14 and 15 displayed the best activity against Trypanosoma brucei brucei, with IC50 values of 1.49, 1.48 and 1.85 µM, respectively. The activity of compounds 6, 14 and 15 also extended to intracellular Trypanosoma cruzi, with IC50 values of 5.14, 6.30 and 4.90 µM, respectively. Compound 6, with an IC50 value of 11.73 µM, and compound 14, with an IC50 value of 8.14 µM, demonstrated some modest antileishmanial activity.

Alternative hosts of Leishmania infantum: a neglected parasite in Europe.

Multi-host pathogens that infect various animal species and humans are considered of great importance for public and animal health. Leishmania spp. parasites are a characteristic example of such pathogens. Although leishmaniosis in humans is endemic for about 100 countries around the world it is classified as a neglected tropical disease. There are three main forms of leishmaniosis in humans: cutaneous (CL), visceral (VL) and mucocutaneous leishmaniosis (MCL). Each year, about 30,000 new cases of VL and more than 1 million new cases of CL are recorded. In Europe L. infantum is the dominant species with dogs being reservoir hosts. Apart from dogs, infection has been recorded in various animals, which suggests that other species could play a role in the maintenance of the parasite in nature. Herein we provide an in-depth review of the literature with respect to studies that deal with Leishmania infantum infections in domestic and wild animal species in Europe. Given the fact that domesticated and wild animals could contribute to the incidences of leishmaniosis in humans, the aim of this paper is to provide a comprehensive review which could potentially be used for the development of measures when it comes to the control of the Leishmania infantum parasite.

Cutaneous Leishmaniasis Caused by Leishmania infantum, Israel, 2018-2021.

Cutaneous leishmaniasis (CL) is endemic to Israel. Previously, CL caused by Leishmania infantum had been reported in Israel only once (in 2016). We report 8 L. infantum CL cases; 7 occurred during 2020-2021. None of the patients had systemic disease. L. infantum CL may be an emerging infection in Israel.

Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

Matrix metalloproteinases -2 and -9 expression in dogs with visceral leishmaniasis: A systematic review.

The matrix metalloproteinases (MMPs) are engaged in the degradation and remodeling of the extracellular matrix and vessels, allowing the progression of pathological processes. Recent studies pointed that MMP -2 and -9 are promising visceral leishmaniasis biomarkers. Thus, the present studystudy aimed to review published scientific literature related to MMP-2 and -9 activity on canine visceral leishmaniasis (CVL). The review followed the PRISMA method, searching for articles in ScienceDirect, PubMed, Scopus, Lilacs, Medline and Google Scholar from inception until 20 March 2022 by employing the following terms: "dog", "matrix metalloproteinases" and "Visceral Leishmaniasis" or "Kala Azar". The selected articles were read in full and only those consistent with the eligibility criteria were included in the review. Of 238 articles from the initial search, only five were deemed eligible, which were conducted between 2010 and 2018. All studies were performed in Brazil. It was observed that there was a higher expression of proMMP-2 in cerebrospinal (CS) fluid and serum and active MMP-2 in different skin areas, mainly in high parasite load areas. As for MMP-9, the pro and active forms were both expressed in CS fluid, serum and different skin areas. The MMP-2 can be considered a biomarker of bad prognostic as it plays an inflammatory role with a greater release in the initial phase of the disease, where MMP-9 is perceived in the chronic phase of CVL. Future research on the subject with greater methodological rigor and bigger sample sizes are mandatory to clarify the role of MMPs on disease progression.

Enhanced apoptotic index in hepatocytes, Kupffer cells, and inflammatory infiltrate showed positive correlation with hepatic lesion intensity, parasite load, and clinical status in naturally Leishmania-infected dogs.

It is unknown if Leishmania amastigote infections affect hepatocytes and Kupffer cell apoptosis, and the role played by apoptosis in liver lesions in leishmaniasis is still unclear. Clinically affected and subclinically infected dogs with leishmaniosis and uninfected controls were assessed. Parasite load, biochemical markers for evaluation of liver damage, morphometry (area, perimeter, number of inflammatory focus, major and minor diameters), apoptosis in hepatic tissue (hepatocytes, Kupffer cells, and inflammatory infiltrates) and cellularity in inflammatory foci were quantified. The parasite load in clinically affected dogs proved to be higher than in the other groups. All morphometric parameters (area, perimeter, number of inflammatory focus, major and minor diameters) from clinically affected were higher than the values found in the subclinically infected and uninfected control dogs. Only clinically affected dogs presented high levels of ALT, FA, GGT and cholesterol in serum. Strong positive correlation was observed between biochemical markers for evaluation of liver damage (ALT, FA, GGT and cholesterol) and hepatic apoptosis (hepatocytes, Kupffer cells, and inflammation). Clinically affected dogs showed a more intense hepatic lesion. Hepatocytes showed a higher rate of apoptosis in Leishmania-infected dogs than in uninfected control dogs. The Kupffer cell apoptotic index and apoptosis within the inflammatory infiltrates were higher in clinically affected dogs. The apoptotic index evaluated in hepatocytes, Kupffer cells, and inflammatory infiltrates showed a positive correlation with the intensity of the hepatic lesion, parasite load, and clinical status. Apoptotic cells also showed positive immunostaining for TUNEL, Bcl2, and Bax. Our data showed that hepatic apoptosis was related to the severity of liver damage, the progression of infection, and the parasite load in leishmaniasis. Apoptotic regulated cell recruitment modulated the inflammatory response and favored the survival and dissemination of parasites, depending on the clinical status of the Leishmania-infected dogs.

Leukoreduction as a control measure in transfusion transmission of visceral leishmaniasis.

BACKGROUND: Asymptomatic visceral leishmaniasis (VL) infection is a risk for transfusion safety. Leukoreduction has been an alternative for the prevention of some blood-borne diseases, including VL. This study aimed to evaluate the role of leukoreduction of cellular blood components as a control measure for transfusional VL transmission. RESEARCH DESIGN AND METHODS: A total of 161 polytransfused patients with non-leukoreduced blood components (HNL), 95 polytransfused with leukoreduced blood components (LH), and 202 non-transfused (NT) from endemic regions for VL and with a similar epidemiological profile. The detection of antibodies against VL was performed by ELISA and the presence of the parasite was investigated by real-time PCR. Statistical significance was defined as p < .05. RESULTS: When comparing three groups, ELISA results were statistically significant (p = .0065). The residual analysis of ELISA showed statistically significant for the HNL group compared to the general group (p = .002; OR: 5.6; CI: 1.7-25.8), demonstrating that individuals who received non-leukoreduced transfusions are five times more likely to acquire Leishmania infantum infection than the general. DISCUSSION: Higher prevalence in the group with HNL and low prevalence in those who received LH, similar to NT patients, highlight the risk of transfusional VL transmission and reinforce the role of leukoreduction in its prevention.

Dogs with canine visceral leishmaniasis have a boost of extracellular vesicles and miR-21-5p up-expression.

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 1010 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 1010 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.

Role of antigen-presenting cells in non-ulcerated skin lesions caused by Leishmania (Leishmania) infantum chagasi.

In Central America, infection by Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis (NUCL). This work aimed to evaluate the participation of subpopulations of antigen-presenting cells in skin lesions of patients affected by NUCL through double-staining immunohistochemistry using cellular and intracellular markers. Twenty-three skin biopsies from patients affected by NUCL were used. Histological sections stained by HE were used for histopathological study. Immunohistochemical studies were performed using primary antibodies against Langerhans cells, dermal dendritic cells, T lymphocytes, and the cytokines IL-12, IFN-γ, TNF-α, iNOS, and IL-10. The histopathological lesions were characterized by an inflammatory infiltrate, predominantly lymphohistiocytic, of variable intensity, with a diffuse arrangement associated with epithelioid granulomas and discreet parasitism. Double-staining immunohistochemistry showed higher participation of dendritic cells producing the proinflammatory cytokine IL-12 in relation to the other evaluated cytokines. Activation of the cellular immune response was marked by a higher density of CD8 Tc1-lymphocytes followed by CD4 Th1-lymphocytes producing mainly IFN-γ. The data obtained in the present study suggest that antigen-presenting cells play an important role in the in situ immune response through the production of proinflammatory cytokines, directing the cellular immune response preferentially to the Th1 and Tc1 types in NUCL caused by L. (L.) infantum chagasi.

Synthesis and Evaluation of Marine-Inspired Compounds Result in Hybrids with Antitrypanosomal and Antileishmanial Activities.

Natural products are a very rich source for obtaining new compounds with therapeutic potential. In the search for new antiparasitic and antimicrobial agents, molecular hybrids were designed based on the structures of antimicrobial marine quinazolinones and eugenol, a natural phenolic compound. Following reports of the therapeutic potential of quinazolinones and eugenol derivatives, it was expected that the union of these pharmacophores could generate biologically relevant substances. The designed compounds were obtained by classical synthetic procedures and were characterized by routine spectrometric techniques. Nine intermediates and final products were then evaluated in vitro against Trypanosoma brucei and Leishmania infantum. Antifungal and antibacterial activity were also evaluated. Six compounds (9b, 9c, 9d, 10b, 10c, and 14) showed mild activity against T. brucei with IC50 in the range of 11.17-31.68 µM. Additionally, intermediate 9c showed anti-Leishmania activity (IC50 7.54 µM) and was six times less cytotoxic against THP-1 cells. In conclusion, novel derivatives with a simple quinazolinone scaffold showing selectivity against parasites without antibacterial and antifungal activities were disclosed, paving the way for new antitrypanosomal agents.

Seroprevalence of canine leishmaniosis in asymptomatic dogs in Kosovo.

Canine leishmaniosis (CanL) is a neglected zoonotic disease caused by Leishmania spp. Leishmania infantum is the species responsible for the zoonotic form of the disease where dogs are reservoir hosts. This study aimed to determine the seroprevalence of CanL in asymptomatic dogs in Kosovo. Blood samples were collected from 285 dogs in all seven regions in Kosovo (35-50 samples per region) from summer 2021 to spring 2022. Sera were tested using enzyme-linked immunosorbent assay (ELISA), and the presence of anti-Leishmania IgG was confirmed by an indirect fluorescent antibody test (IFAT). The true overall seroprevalence of CanL of asymptomatic dogs in Kosovo with ELISA was 4.21% (95% CI: 2.42-7.21) while with IFAT was 3.51% (95% CI: 1.92-6.34). The highest rates were found in the Prishtina region to be 8.0% (4/50) by ELISA and 6.0% (3/50) by IFAT, and in the Mitrovica region, the prevalence was 0% (0/40). There were no significant differences among the different regions, gender, age, health status, and breed. These findings highlight the presence of CanL in most regions of Kosovo and underline the veterinary relevance of clinically asymptomatic dogs infected with Leishmania.

Characterization and Proteomic Analysis of Plasma EVs Recovered from Healthy and Diseased Dogs with Canine Leishmaniosis.

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Inhibition of Leishmania infantum Trypanothione Reductase by New Aminopropanone Derivatives Interacting with the NADPH Binding Site.

BACKGROUND: As a result of the paucity of treatment, Leishmaniasis continues to provoke about 60,000 deaths every year worldwide. New molecules are needed, and drug discovery research is oriented toward targeting proteins crucial for parasite survival. Among them, trypanothione reductase (TR) is of remarkable interest owing to its vital role in Leishmania species protozoan parasite life. Our previously identified compound 1 is a novel chemotype endowed with a unique mode of TR inhibition thanks to its binding to a formerly unknown but druggable site at the entrance of the NADPH binding cavity, absent in human glutathione reductase (hGR). METHODS: We designed and synthesized new 3-amino-1-arylpropan-1-one derivatives structurally related to compound 1 and evaluated their potential inhibition activity on TR from Leishmania infantum (LiTR). Cluster docking was performed to assess the binding poses of the compounds. RESULTS: The newly synthesized compounds were screened at a concentration of 100 µM in in vitro assays and all of them proved to be active with residual activity percentages lower than 75%. CONCLUSIONS: Compounds 2a and 2b were the most potent inhibitors found, suggesting that an additional aromatic ring might be promising for enzymatic inhibition. Further structure-activity relationships are needed to optimize our compounds activity.

Geographic Origin and Vertical Transmission of Leishmania infantum Parasites in Hunting Hounds, United States.

Vertical transmission of leishmaniasis is common but is difficult to study against the background of pervasive vector transmission. We present genomic data from dogs in the United States infected with Leishmania infantum parasites; these infections have persisted in the apparent absence of vector transmission. We demonstrate that these parasites were introduced from the Old World separately and more recently than L. infantum from South America. The parasite population shows unusual genetics consistent with a lack of meiosis: a high level of heterozygous sites shared across all isolates and no decrease in linkage with genomic distance between variants. Our data confirm that this parasite population has been evolving with little or no sexual reproduction. This demonstration of vertical transmission has profound implications for the population genetics of Leishmania parasites. When investigating transmission in complex natural settings, considering vertical transmission alongside vector transmission is vital.

Autochthonous Leishmania infantum in Dogs, Zambia, 2021.

Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.

Protective immune response mediated by neutrophils in experimental visceral leishmaniasis is enhanced by IL-32γ.

Neutrophils are important cells in protection against microbial infections including visceral leishmaniasis (VL). It is well known that IL-32γ increases the protective T helper 17 cell mediated immune response against Leishmania infantum. Thus, in this study we evaluated whether IL-32 γ can increase the protective role of neutrophils against VL. In comparison with wild type (WT) mice, transgenic mice for human IL-32 γ (IL-32 γ Tg) presented a higher frequency and absolute number of neutrophils in both spleen and liver after the establishment of L. infantum infection. The IL-32 concentrations correlated with neutrophil numbers in the infected tissues. The IL-32 γ -induced recruitment of neutrophils was dependent on IL-17, since inhibition of Th17 T cells generation and IL-17 production with digoxin treatment reversed the effects of IL-32 γ. In murine neutrophils, the presence of IL-32 γ enhanced the phagocytosis of L. infantum via CR3. In addition, murine IL-32 γ Tg neutrophils were able to kill L. infantum due to the increased production of ROS when compared with WT neutrophils. In fact, IL-32 γ Tg mice lost their ability to control infection by L. infantum when neutrophils were depleted. In parallel, treatment of human neutrophils with recombinant IL-32 γ increased phagocytosis and ROS-dependent killing of L. infantum, similarly to murine IL-32 γ Tg neutrophils. The data show that IL-32 γ induces neutrophil recruitment to organs affected by VL and increases phagocytosis and killing of L. infantum by neutrophils. Together, data indicate the pivotal axis IL-32 γ -Th17-neutrophils to control VL.