Elucidating the role played by bone marrow in visceral leishmaniasis.

Leishmaniasis is a widespread group of infectious diseases that significantly impact global health. Despite high prevalence, leishmaniasis often receives inadequate attention in the prioritization of measures targeting tropical diseases. The causative agents of leishmaniasis are protozoan parasites of the Leishmania genus, which give rise to a diverse range of clinical manifestations, including cutaneous and visceral forms. Visceral leishmaniasis (VL), the most severe form, can be life-threatening if left untreated. Parasites can spread systemically within the body, infecting a range of organs, such as the liver, spleen, bone marrow and lymph nodes. Natural reservoirs for these protozoa include rodents, dogs, foxes, jackals, and wolves, with dogs serving as the primary urban reservoir for Leishmania infantum. Dogs exhibit clinical and pathological similarities to human VL and are valuable models for studying disease progression. Both human and canine VL provoke clinical symptoms, such as organ enlargement, fever, weight loss and abnormal gamma globulin levels. Hematologic abnormalities have also been observed, including anemia, leukopenia with lymphocytosis, neutropenia, and thrombocytopenia. Studies in dogs have linked these hematologic changes in peripheral blood to alterations in the bone marrow. Mouse models of VL have also contributed significantly to our understanding of the mechanisms underlying these hematologic and bone marrow abnormalities. This review consolidates information on hematological and immunological changes in the bone marrow of humans, dogs, and mice infected with Leishmania species causing VL. It includes findings on the role of bone marrow as a source of parasite persistence in internal organs and VL development. Highlighting gaps in current knowledge, the review emphasizes the need for future research to enhance our understanding of VL and identify potential targets for novel diagnostic and therapeutic approaches.

Leishmania exposure in dogs from two endemic countries from New and Old Worlds (Brazil and Portugal): evaluation of three serological tests using Bayesian Latent Class Models.

BACKGROUND: Zoonotic leishmaniosis caused by Leishmania infantum is endemic in several countries of the Mediterranean Basin, Latin America, and Asia. Dogs are the main hosts and reservoirs of human infection. Thus, from a One Health perspective, early diagnosis of Leishmania infection in dogs is essential to control the dissemination of the parasite among other dogs and to humans. The aim of this study was to estimate the diagnosis accuracy of three serological tests to detect antibodies to Leishmania in dogs from two endemic settings using Bayesian latent class models (BLCM). METHODS: A total of 378 dogs from two Portuguese and Brazilian endemic areas of leishmaniosis (194 animals from Portugal and 184 from Brazil) were screened. Detection of anti-Leishmania antibodies was performed using two commercial ELISA (L. infantum IgG-ELISA® and EIE-LVC®) and a rapid immunochromatographic test (DPP-LVC®). Bayesian latent class models were used to estimate Leishmania infection prevalence, together with sensitivities and specificities of the three diagnostic tests, in the two dog populations simultaneously. Predictive values were also calculated. Credibility intervals (CI) were obtained, considering different types of prior information. RESULTS: A posterior median Leishmania seroprevalence of 13.4% (95% CI 9.0-18.7) and of 21.6% (15.0-28.3) was estimated to the Portuguese and Brazilian dog subpopulations, respectively. The Bayesian analysis indicated that all tests were highly specific (specificity above 90%), and that the DPP-LVC® was more sensitive (96.6%; 83.1-99.9) than both ELISAs in the Portuguese subpopulation, while in the Brazilian subpopulation, EIE-LVC® and L. infantum IgG-ELISA®, had the highest sensitivity (88.2%; 73.7-97.0) and specificity (98.7%; 95.1-99.9), respectively. CONCLUSIONS: In general, the levels of diagnosis accuracy of the three serological tests to detect Leishmania antibodies assessed by BLCM indicate their utility in canine epidemiological studies. The same approach should be used to assess the performance of these techniques in the clinical management of infected and sick dogs using representative samples from the wide spectrum of clinical situations, namely from subclinical infection to manifest disease. The low positive predictive value of the serological tests used in the current protocol of the Brazilian Ministry of Health suggests that they should not be used individually and may not be sufficient to target reservoir-based control interventions.

Immune response dynamics and Lutzomyia longipalpis exposure characterize a biosignature of visceral leishmaniasis susceptibility in a canine cohort.

BACKGROUND: Reports have shown correlations between the immune response to vector saliva and Leishmaniasis outcome. We followed dogs in an endemic area for two years characterizing resistance or susceptibility to canine visceral leishmaniasis (CVL) according to Leishmania infantum diagnosis and clinical development criteria. Then, we aimed to identify a biosignature based on parasite load, serum biological mediators' interactions, and vector exposure intensity associated with CVL resistance and susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: A prospective two-year study was conducted in an area endemic for CVL. Dogs were evaluated at 6-month intervals to determine infection, clinical manifestations, immune profile, and sandfly exposure. CVL resistance or susceptibility was determined upon the conclusion of the study. After two years, 78% of the dogs were infected with L. infantum (53% susceptible and 47% resistant to CVL). Susceptible dogs presented higher splenic parasite load as well as persistence of the parasite during the follow-up, compared to resistant ones. Susceptible dogs also displayed a higher number of correlations among the investigated biological mediators, before and after infection diagnosis. At baseline, anti-saliva antibodies, indicative of exposure to the vector, were detected in 62% of the dogs, reaching 100% in one year. Higher sandfly exposure increased the risk of susceptibility to CVL by 1.6 times (CI: 1.11-2.41). We identified a discriminatory biosignature between the resistant and susceptible dogs assessing splenic parasite load, interaction of biological mediators, PGE2 serum levels and intensity of exposure to sandfly. All these parameters were elevated in susceptible dogs compared to resistant animals. CONCLUSIONS/SIGNIFICANCE: The biosignature identified in our study reinforces the idea that CVL is a complex multifactorial disease that is affected by a set of factors which are correlated and, for a better understanding of CVL, should not be evaluated in an isolated way.

Effects of larval rearing substrates on some life-table parameters of Lutzomyia longipalpis sand flies.

Sand flies are the insects responsible for transmitting Leishmania parasites, the causative agents of leishmaniasis in humans. However, the effects of sand fly breeding sites on their biology and ecology remain poorly understood. Herein, we studied how larval nutrition associated with putative breeding sites of the sand fly Lutzomyia longipalpis affects their oviposition, development, microbiome, and susceptibility to Leishmania by rearing L. longipalpis on substrates collected from an endemic area for leishmaniasis in Brazil. The results showed that female L. longipalpis select the oviposition site based on its potential to promote larval maturation and while composting cashew leaf litter hindered the development, larvae reared on chicken feces developed rapidly. Typical gut microbial profiles were found in larvae reared upon cashew leaf litter. Adult females from larvae reared on substrate collected in chicken coops were infected with Leishmania infantum, indicating that they were highly susceptible to the parasite. In conclusion, the larval breeding sites can exert an important role in the epidemiology of leishmaniasis.

Solid lipid nanoparticles as a novel formulation approach for tanespimycin (17-AAG) against leishmania infections: Preparation, characterization and macrophage uptake.

17-N-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) is an inhibitor of heat shock protein 90 (Hsp90), which has been studied in the treatment of cancer such as leukemia or solid tumors. Alternatively, 17-AAG may represent a promising therapeutic agent against leishmaniasis. However, the delivery of 17-AAG is difficult due to its poor aqueous solubility. For exploring the therapeutic value of 17-AAG, we developed solid lipid nanoparticles (SLN) by double emulsion method. SLN exhibited ~100 nm, PDI < 0.2 and zeta potential ~20 mV. In addition, SLN were morphologically spherical with negligible aggregation. The entrapment efficiency of 17-AAG into the lipid matrix reached at nearly 80%. In a separate set of experiments, fluorescent SLN (FITC-labeled) showed a remarkable macrophage uptake, peaking within 2 h of incubation by confocal microscopy. Regarding the drug internalization as critical step for elimination of intracellular Leishmania, this finding demonstrates an important feature of the developed SLN. Collectively, these data indicate the feasibility of developing SLN as potential delivery systems for 17-AAG in leishmaniasis chemotherapy.

Correction: High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil-A Phase II study.

[This corrects the article DOI: 10.1371/journal.pntd.0006871.].

Deciphering the Role Played by Autophagy in Leishmania Infection.

In recent decades, studies have shown that, depending on parasite species and host background, autophagy can either favor infection or promote parasite clearance. To date, relatively few studies have attempted to assess the role played by autophagy in Leishmania infection. While it has been consistently shown that Leishmania spp. induce autophagy in a variety of cell types, published results regarding the effects of autophagic modulation on Leishmania survival are contradictory. The present review, after a short overview of the general aspects of autophagy, aims to summarize the current body of knowledge surrounding how Leishmania spp. adaptively interact with macrophages, the host cells mainly involved in controlling leishmaniasis. We then explore the scarce studies that have investigated interactions between these parasite species and the autophagic pathway, and finally present a critical perspective on how autophagy influences infection outcome.

A docking-based structural analysis of geldanamycin-derived inhibitor binding to human or Leishmania Hsp90.

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis.

An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4-rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4-rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4-rLci1A and E4-rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Corrigendum: Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

[This corrects the article DOI: 10.3389/fimmu.2018.02558.].

Natural infection by Leishmania infantum in the Lutzomyia longipalpis population of an endemic coastal area to visceral leishmaniasis in Brazil is not associated with bioclimatic factors.

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoan Leishmania infantum and in Brazil is transmitted mainly by the bite of Lutzomuyia longipalpis sand flies. Data about the presence, distribution, natural infection rate, seasonal and monthly dynamics of the vector population are important for optimizing the measures to control VL in endemic areas. This study aimed to identify sand fly fauna in an endemic area for VL to detect the prevalence of L. infantum infection in the Lu. longipalpis population and to elucidate the influence of bioclimatic factors on the monthly fluctuations of this vector. HP light traps were monthly set in the intradomicile and peridomicile of residences located in the central and beachfront areas of Camaçari, a VL endemic area. The sand fly collection was conducted in two periods: i) period 1-between December 2011 and November 2012 and ii) period 2-August 2014 and July 2015. Sand fly species were identified and detection of L. infantum infection by qPCR was performed in pools of female Lu. longipalpis. For the first time, the parasite load of positive pools was correlated with the number of Lu. longipalpis captured per month in both periods. Correlation analyses between the monthly fluctuation of the sand fly population and bioclimatic indices of the municipality in both collection periods were also performed. In both evaluated periods, more than 98% of the collected sand flies were Lu. longipalpis, confirming the predominance of this species in the region. It was captured mostly in the beachfront area in all months evaluated (99%). For the period 1, Leishmania DNA was detected in 81% of tested pools representing a minimal infection rate of 9.6%. In the period 2, 40% of the pools were positive with a minimal infection rate of 10.2%. Infected sand flies were only detected in the beachfront area in both periods. The parasite load was low and did not vary in the evaluated months despite the number of collected sand flies. No correlation was observed for climatic factors in both areas of Camaçari. These findings emphasize the high risk of Leishmania transmission in Camaçari regardless of the season and that other factors, aside from bioclimatic elements, are influencing the sand fly population monthly fluctuation.

Proteomic Analysis Reveals a Predominant NFE2L2 (NRF2) Signature in Canonical Pathway and Upstream Regulator Analysis of Leishmania-Infected Macrophages.

CBA mice macrophages (MØ) control infection by Leishmania major and are susceptive to Leishmania amazonensis, suggesting that both parasite species induce distinct responses that play important roles in infection outcome. To evaluate the MØ responses to infection arising from these two Leishmania species, a proteomic study using a Multidimensional Protein Identification Technology (MudPIT) approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out on CBA mice bone-marrow MØ (BMMØ). Following SEQUEST analysis, which revealed 2,838 proteins detected in BMMØ, data mining approach found six proteins significantly associated with the tested conditions. To investigate their biological significance, enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). A three steps IPA approach revealed 4 Canonical Pathways (CP) and 7 Upstream Transcriptional Factors (UTFs) strongly associated with the infection process. NRF2 signatures were present in both CPs and UTFs pathways. Proteins involved in iron metabolism, such as heme oxigenase 1 (HO-1) and ferritin besides sequestosome (SQSMT1 or p62) were found in the NRF2 CPs and the NRF2 UTFs. Differences in the involvement of iron metabolism pathway in Leishmania infection was revealed by the presence of HO-1 and ferritin. Noteworty, HO-1 was strongly associated with L. amazonensis infection, while ferritin was regulated by both species. As expected, higher HO-1 and p62 expressions were validated in L. amazonensis-infected BMMØ, in addition to decreased expression of ferritin and nitric oxide production. Moreover, BMMØ incubated with L. amazonensis LPG also expressed higher levels of HO-1 in comparison to those stimulated with L. major LPG. In addition, L. amazonensis-induced uptake of holoTf was higher than that induced by L. major in BMMØ, and holoTf was also detected at higher levels in vacuoles induced by L. amazonensis. Taken together, these findings indicate that NRF2 pathway activation and increased HO-1 production, together with higher levels of holoTf uptake, may promote permissiveness to L. amazonensis infection. In this context, differences in protein signatures triggered in the host by L. amazonensis and L. major infection could drive the outcomes in distinct clinical forms of leishmaniasis.

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).

High accuracy of an ELISA test based in a flagella antigen of Leishmania in serodiagnosis of canine visceral leishmaniasis with potential to improve the control measures in Brazil - A Phase II study.

BACKGROUND: Canine Visceral leishmaniasis (CVL) is a serious public health problem, thus for its control, the Ministry of Health in Brazil recommends the rapid diagnosis and euthanasia of seropositive dogs in endemic areas. Therefore, our group had previously selected six recombinant proteins (rLci1, rLci2, rLci4, rLci5, rLci8, and rLci12) due to their high potential for CVL diagnostic testing. The present study aims to produce an immunodiagnostic test using the aforementioned antigens, to improve the performance of the diagnosis of CVL recommended by Brazilian Ministry of Health. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the recombinant proteins in the serological assays, positive and negative samples were selected based on parasitological test (culture) and molecular test (qPCR) of splenic aspirate. Initially, we selected 135 dog serum samples, 73 positives (symptomatic and asymptomatic) and 62 negatives to screen recombinant proteins on ELISA platform. Then, for rLci5 ELISA validation, 361 serum samples collected in a cross-sectional study were selected, being 183 positives (symptomatic and asymptomatic) and 178 negatives. In the screening of the recombinant proteins, rLci5 was the only protein to present a performance statistically higher than the performance presented by EIE-LVC test, presenting 96% (IC 95%; 85-99%) vs. 83% (IC 95%; 69-92%) of sensitivity for symptomatic dogs, 71% (IC 95%; 49-97%) vs. 54% (IC 95%; 33-74%) for asymptomatic dogs and 94% (IC 95%; 83-99%) vs, 88% (IC 95%; 76-95% of specificity. Thus, the rLci5 protein was selected to compose a final ELISA test. Validation of rLci5 ELISA showed 87% (IC 81-91%) of sensitivity, 94% (IC 95%; 90-97%) of specificity and 90% accuracy. Testing the EIE-LVC with the same validation panel, we observed a lower performance when compared to ELISA rLci5 (sensitivity of 67% (IC 95%; 59-74%), specificity of 87% (IC 95%; 81-92%), and accuracy of 77%). Finally, the performance of current CVL diagnostic protocol recommended by Brazilian Ministry of Health, using DPP-LVC as screening test and EIE-LVC as confirmatory test, was compared with a modified protocol, replacing EIE-LVC by rLci5 ELISA. The current protocol presented a sensitivity of 59% (IC 95%; 52-66%), specificity of 98% (IC 95%; 95-99%) and accuracy of 80% (IC 95%; 76-84%), while the modified protocol presented a sensitivity of 71% (IC 95%; 63-77%), specificity of 99% (IC 95%; 97-100%) and accuracy of 86% (IC 95%; 83-89%). CONCLUSION: Thus, we concluded that rLci5 ELISA is a promising test to replace EIE-LVC test and increase the diagnostic performance of CVL in Brazil.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

In Search of Biomarkers for Pathogenesis and Control of Leishmaniasis by Global Analyses of Leishmania-Infected Macrophages.

Leishmaniasis is a vector-borne, neglected tropical disease with a worldwide distribution that can present in a variety of clinical forms, depending on the parasite species and host genetic background. The pathogenesis of this disease remains far from being elucidated because the involvement of a complex immune response orchestrated by host cells significantly affects the clinical outcome. Among these cells, macrophages are the main host cells, produce cytokines and chemokines, thereby triggering events that contribute to the mediation of the host immune response and, subsequently, to the establishment of infection or, alternatively, disease control. There has been relatively limited commercial interest in developing new pharmaceutical compounds to treat leishmaniasis. Moreover, advances in the understanding of the underlying biology of Leishmania spp. have not translated into the development of effective new chemotherapeutic compounds. As a result, biomarkers as surrogate disease endpoints present several potential advantages to be used in the identification of targets capable of facilitating therapeutic interventions considered to ameliorate disease outcome. More recently, large-scale genomic and proteomic analyses have allowed the identification and characterization of the pathways involved in the infection process in both parasites and the host, and these analyses have been shown to be more effective than studying individual molecules to elucidate disease pathogenesis. RNA-seq and proteomics are large-scale approaches that characterize genes or proteins in a given cell line, tissue, or organism to provide a global and more integrated view of the myriad biological processes that occur within a cell than focusing on an individual gene or protein. Bioinformatics provides us with the means to computationally analyze and integrate the large volumes of data generated by high-throughput sequencing approaches. The integration of genomic expression and proteomic data offers a rich multi-dimensional analysis, despite the inherent technical and statistical challenges. We propose that these types of global analyses facilitate the identification, among a large number of genes and proteins, those that hold potential as biomarkers. The present review focuses on large-scale studies that have identified and evaluated relevant biomarkers in macrophages in response to Leishmania infection.

The mass use of deltamethrin collars to control and prevent canine visceral leishmaniasis: A field effectiveness study in a highly endemic area.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonosis of great importance. Limitations in current VL control measures compromise efficacy, indicating the need to implement new strategies. The aim of this study was to evaluate the effectiveness of the mass use of deltamethrin-impregnated collars in dogs as a public health measure to control and prevent canine visceral leishmaniasis (CVL). METHODOLOGY: An interventional study was implemented in two endemic areas in the district of Monte Gordo (Bahia-Brazil): an intervention area, in which VL seronegative dogs were collared, and a control area in which only conventional CVL control measures were applied. At baseline, seropositive dogs were removed and seronegative dogs were included. Dogs were then reevaluated every 7-8 months for almost two years. At each time point, dogs in the intervention area that remained seronegative received new collars and newly identified seronegative dogs were included and collared. The local zoonosis control authorities were notified of any dogs that tested seropositive in both areas, which were subsequently marked for euthanasia as mandated by the Brazilian Ministry of Health. PRINCIPAL FINDINGS: In the first serological survey, seroprevalence was similar in both areas. At the second evaluation, significant reductions in seroprevalence were seen in both areas, while seroprevalence in the intervention area reduced to 6.0% during the final evaluation versus an increase of 11.0% in the control area. This significant increase and the estimated relative risk (RR = 0.55) indicated protection against CVL in the intervention area. Although CVL incidence did not differ significantly between the areas, an increased tendency was observed in the control area, which could be due to low seroconversion rates throughout the study or a high loss to follow-up. CONCLUSIONS/SIGNIFICANCE: Although our evaluation of the effectiveness of deltamethrin-impregnated collars as a community-wide public health control measure was inconclusive, this measure likely provides protection over time. In endemic areas of Brazil, this strategy represents an operational challenge for local zoonosis control authorities, indicating the need for adjustments, including improved collar design.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195.

BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis.

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease. In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen. Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61-0.80) very good (0.81-0.99) or perfect (1.00), for most of the parameters analyzed. Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.