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The aim of this study was to evaluate the relationship between naturally occurring Leishmania spp. infections in dogs (Canis familiaris) and the practical implications of the use of serological and molecular methods to confirm diagnoses. The study population consisted of 96 domestic dogs in southeastern Brazil. Serum samples were tested for the presence of anti-Leishmania immunoglobulin G (IgG) antibodies using four commercial canine visceral leishmaniasis kits. Dogs confirmed positive by immunofluorescence antibody test (IFAT) were culled and samples from mesenteric lymph nodes, spleen border, bone marrow and ear skin were taken and submitted to DNA extraction. PCR reactions were performed using primers that amplify a 300–350 bp fragment of the Leishmania ribosomal internal transcribed spacer 1 (ITS1) region. The ITS1 amplified products were analyzed by PCR-RFLP using Hae III restriction endonuclease. To confirm the Leishmania species detected by PCR, each purified sample was sequenced in duplicate. Of the 96 serum samples submitted to serological assays, 8 (8.3%) tested positive for Leishmania by IFAT, 4 (4.1%) by ELISA, 2 (2.1%) by rK39 RDT and 7 (7.3%) by DPP. Four of these infected dogs (50%) were found to be infected only by Leishmania braziliensis or Leishmania amazonensis, and their serum samples tested positive by IFAT and DPP. These findings demonstrate for the first time that cross-reactivity of L. braziliensis and L. amazonensis infection in dogs can be found using the DPP serum test. This is the first record of Leishmania (Leishmania) amazonensis confirmed by a specific molecular marker in dogs (Canis familiaris) from Belo Horizonte, Brazil.

BACKGROUND Leishmaniases are a serious health problem in southeast Brazil, including the city of Belo Horizonte (BH), Minas Gerais state (MG), where there are high rates of incidence and mortality due to visceral leishmaniases. BH is divided into nine sanitary districts (SD) of which one, the Venda Nova SD, was selected for this study because it has high rates of positivity for canine leishmaniasis and high incidence of human leishmaniasis. OBJECTIVES This study aimed to survey the sand fly fauna in Venda Nova SD from August 2011 to July 2013 and perform a descriptive analysis of the vector population. METHODS The sampling was carried out using automatic HP light traps at all covered areas of the Venda Nova SD, in a total of eighteen light traps. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A simple environmental description was done for it area and Kernel estimation was used to infer vector density for each study site. FINDINGS A total of 2,427 sand fly specimens belonging to eight species and five genera were collected of which 95.3% were Lutzomyia longipalpis. The seasonal variation curve was delineated by this species. Lu. longipalpis was the most abundant at all collection points and in all months of the study, and exhibited a natural infection rate of 1.01% for Leishmania infantum and 1.77% for Leishmania braziliensis. MAIN CONCLUSIONS The results show the presence and adaptation of Lu. longipalpis to the anthropic environment of BH and reinforces its role as the main vector of L. infantum in the region.

International audience; Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1β, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

The extracellular vesicles (EVs) released by Leishmania can contribute to the establishment of infection and host immunomodulation. In this study, we characterized the shedding of EVs from Leishmania (Leishmania) amazonensis promastigotes. This species is the causative agent of cutaneous leishmaniasis, and its role during interactions with bone marrow-derived macrophages (BMDMs) and peritoneal B-1 cells was evaluated. Leishmania amazonensis promastigotes cultivated in vitro at different times and temperatures spontaneously released EVs. EVs were purified using size-exclusion chromatography (SEC) and quantitated by nanoparticle tracking analysis (NTA). NTA revealed that the average size of the EVs was approximately 180 nm, with concentrations ranging from 1.8 × 108 to 2.4 × 109 vesicles/mL. In addition, the presence of LPG and GP63 were detected in EVs obtained at different temperatures. Naïve BMDMs stimulated with EVs exhibited increased IL-10 and IL-6 expression. However, incubating B-1 cells with parasite EVs did not stimulate IL-10 expression but led to an increase in the expression of IL-6 and TNFα. After 7 weeks post-infection, animals infected with L. amazonensis promastigotes in the presence of parasite EVs had significant higher parasite load and a polarization to Th2 response, as compared to the group infected with the parasite alone. This work demonstrated that EVs isolated from L. amazonensis promastigotes were able to stimulate macrophages and B-1 cells to express different types of cytokines. Moreover, the immunomodulatory properties of EVs probably contributed to an increase in parasite burden in mice. These findings suggest that the functionality of L. amazonensis EVs on immune system favor of parasite survival and disease progression.

Made available in DSpace on 2021-06-25T10:30:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) The Mayaro virus (MAYV) is an arbovirus that circulates mainly in tropical forests or rural areas in Latin America and is transmitted mainly by Haemagogus mosquitoes. The objective of this study was to evaluate the vector competence, microbiome, and the presence of Wolbachia in three Aedes albopictus populations infected with MAYV. The vector competence was assessed based on viral infection and transmission by RT-qPCR. In addition, the microbiome was evaluated by amplification of the 16S rRNA V4 region and PCR to detect the presence of Wolbachia (strain wAlbA/wAlbB). Our results show that all three populations were susceptible to MAYV infection. The potential transmission of the MAYV was consistent in all populations of naïve mosquitoes injected (more than 50%). The microbiome analysis revealed 118 OTUs (operational taxonomic unit) from the three populations, 8 phyla, 15 classes, 26 orders, 35 families, 65 genera, and 53 species. All populations had Pseudomonas and Wolbachia as predominant genera. There was no difference between the variables for MAYV and Wolbachia (wAlbA or wAlbB) in the abdomen. However, in the head + thorax samples at 14 dpi, there was a difference between the two populations, indicating a possible correlation between the presence of Wolbachia (wAlbB) and infection. Overall, we show evidence that Ae. albopictus displays significant infection and transmission competence for the MAYV in the laboratory, and its bacterial microbiota play an important role in the host, mainly the strains of Wolbachia. The influence of the intestinal microbiota of Ae. albopictus is poorly known, and a better understanding of these interactions would open new perspectives for disease control through the manipulation of microbial communities. The exact contribution of this mosquito species to the transmission of the MAYV in the field remains to be confirmed. Grupo Mosquitos Vetores Endossimbiontes e Interação Patógeno-Vetor Instituto René Rachou-Fiocruz Grupo Taxonomia de Flebotomíneos e Epidemiologia das Leishmanioses Instituto René Rachou-Fiocruz Department of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP) School of Agricultural Sciences Central Multiuser Laboratory São Paulo State University (UNESP) Institute of Biotechnology São Paulo State University (UNESP) Department of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP) School of Agricultural Sciences Central Multiuser Laboratory São Paulo State University (UNESP) Institute of Biotechnology São Paulo State University (UNESP) CAPES: 001 FAPESP: 2013/11343-6 FAPESP: 2016/16952-9 FAPEMIG: APQ-00913-16 FAPEMIG: APQ-02760-17 FAPERJ: E-16/2014

Leishmania (Viannia) braziliensis is the most common etiological agent of cutaneous and mucocutaneous leishmaniasis (MCL) in Latin America. An interesting aspect of the disease outcome caused by this species is the appearance of non-ulcerated atypical cutaneous leishmaniasis. Atypical (AT) lesions are often associated with therapeutic failure when treated with antimony(Sb)-based drugs. Refractory cases are not necessarily due to intrinsic parasite drug resistance. The status of in vitro drug susceptibility from L. braziliensis field isolates is less assessed than patient treatment outcome. In this work, L. braziliensis isolated from typical CL (6), MCL (1) and AT (3) lesions and vector (1) were tested for their susceptibility to amphotericin B (AmB), miltefosine (MIL), glucantime (GLU) and non-comercial meglumine antimoniate (MA). Overall, intracellular amastigotes of all isolates were sensitive to the tested antileishmanial drugs except AT lesions-derived strains 316, 330 and 340 that presented in vitro resistance against SbV-based drugs. Although susceptible to miltefosine - based on phenotypic screening - intramacrophagic quiescent amastigotes could restore infection. L. braziliensis promastigotes isolated from AT lesions also displayed 29% reduced capacity to infect human monocyte-derived macrophages when compared with parasites obtained from patients with typical lesions, MCL or from sand-fly. These data indicate differences in drug susceptibility and infectiveness among L. braziliensis isolated from patients exhibiting different types of lesions and highlight the importance of its characterization for drug response prediction outcome in clinical practice.

SUMMARY Activation of the NLRP3 inflammasome by Leishmania parasites is critical for the outcome of leishmaniasis, a disease that affects millions of people worldwide. We investigate the mechanisms involved in NLRP3 activation and demonstrate that caspase-11 (CASP11) is activated in response to infection by Leishmania species and triggers the non-canonical activation of NLRP3. This process accounts for host resistance to infection in macrophages and in vivo. We identify the parasite membrane glycoconjugate lipophosphoglycan (LPG) as the molecule involved in CASP11 activation. Cytosolic delivery of LPG in macrophages triggers CASP11 activation, and infections performed with Lpg1−/− parasites reduce CASP11/NLRP3 activation. Unlike bacterial LPS, purified LPG does not activate mouse CASP11 (or human Casp4) in vitro, suggesting the participation of additional molecules for LPG-mediated CASP11 activation. Our data identify a parasite molecule involved in CASP11 activation, thereby establishing the mechanisms underlying inflammasome activation in response to Leishmania species., Graphical Abstract, In Brief Activation of the NLRP3 inflammasome is critical for the outcome of leishmaniasis. De Carvalho et al. show that Leishmania lipophosphoglycan (LPG) triggers caspase-11 activation in macrophage cytoplasm and the non-canonical activation of NLRP3, thereby establishing the mechanisms underlying NLRP3 activation in response to Leishmania.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

Background Little is known about the genetic diversity of Leishmania infantum isolates in the state of Minas Gerais, Brazil. Methods Molecular characterization of hsp70, mpi and ITS1 was carried out for 29 isolates of L. infantum from the bone marrow of naturally infected dogs from the cities of Divinopolis, Para de Minas and Brumadinho, located in the central-west, central and central regions of the state of Minas Gerais, Brazil, respectively. Results Analysis of the parasite nucleotide sequences demonstrated very high homogeneity of the studied samples. Conclusions In the endemic regions studied, parasites are genotypically indistinguishable.

Autochthonous cases of American cutaneous leishmaniasis (ACL) have been reported since 2001 in the Xakriabá Indigenous Reserve located in the municipality of São João das Missões in northern Minas Gerais state, Brazil. In order to study the presence of Leishmania DNA in phlebotomine sand flies, six entomological collections were carried out from July 2008 through July 2009, using 40 light traps placed in peridomicile areas of 20 randomly selected houses. From October 2011 through August 2012, another six collections were carried out with 20 light traps distributed among four trails (five traps per trail) selected for a previous study of wild and synanthropic hosts of Leishmania. A total of 4,760 phlebotomine specimens were collected belonging to ten genera and twenty-three species. Single female specimens or pools with up to ten specimens of the same locality, species and date, for Leishmania detection by molecular methods. Species identification of parasites was performed with ITS1 PCR-RFLP using HaeIII enzyme and genetic sequencing for SSU rRNA target. The presence of Leishmania DNA was detected in eleven samples from peridomicile areas: Lu. longipalpis (two), Nyssomyia intermedia (four), Lu. renei (two), Lu. ischnacantha, Micropygomyia goiana and Evandromyia lenti (one pool of each specie). The presence of Leishmania DNA was detected in twelve samples from among the trails: Martinsmyia minasensis (six), Ny. intermedia (three), Mi. peresi (two) and Ev. lenti (one). The presence of Leishmania infantum DNA in Lu. longipalpis and Leishmania braziliensis DNA in Ny. intermediasupport the epidemiological importance of these species of sand flies in the cycle of visceral and cutaneous leishmaniasis, respectively. The results also found other species associated with Leishmania DNA, such as Mt. minasensis and Ev. lenti, which may participate in a wild and/or synanthropic cycle of Leishmania transmission in the studied area.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly., Author Summary Leishmania amazonensis, a member of the Leishmania mexicana complex, is the causative agent of localized cutaneous leishmaniasis (LCL) and anergic diffuse cutaneous leishmaniasis (ADCL) [1,2]. It is widely distributed throughout the Amazon basin, where it infects a wide range of terrestrial rodents and, less frequently, marsupials. Its main vector is Lutzomyia flaviscutellata (Diptera: Psychodidae) widely distributed in South America and a recent study has predicted its expansion towards South of Brazil [3]. Moreover, Lutzomyia migonei (França, 1920) can also harbor the infection of this species [4,5]. Although its transmission to man is very uncommon, L. amazonensis triggers an incurable and disseminated form of cutaneous leishmaniasis [2,6]. However, most of the mechanisms involved in L. amazonensis pathogenesis are still unknown, especially those related to surface molecules. Glycoconjugates have been extensively characterized as important for the establishment of infection as they protect the parasite from the early action of the host immune system and therefore acting as invasive/evasive strategies. Consequently, we here present the role of lipophosphoglycan (LPG) of L. amazonensis in the interaction with vertebrate and invertebrate hosts.

Brazil is one of the most important endemic areas for leishmaniasis worldwide. Protected areas that are tourist attractions likely present an important risk of transmission of cutaneous leishmaniasis (CL). Furthermore, with the geographical expansion of visceral leishmaniasis (VL), several studies have recorded the occurrence of its vector, Lutzomyia longipalpis, and cases of human and canine VL in such tourist areas. The Parque Estadual do Sumidouro is an environmentally protected area located in the Brazilian Cerrado biome and in an important area endemic for leishmaniasis in the state of Minas Gerais. The purpose of this study was to monitor the sand fly fauna in areas of tourist activity in the park. Sampling was performed every month, from September 2011 to August 2013, using CDC light traps at six sites of differing environmental characteristics. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A total of 4,675 sand fly specimens of 25 species belonging to nine genera were collected. The most abundant species were Micropygomyia quinquefer, Lutzomyia renei and Pintomyia pessoai, although only Pi. pessoai is implicated in the transmission of Leishmania braziliensis. The species accumulation curve reached saturation on the 16th sampling event. Species richness, diversity and evenness differed among the sampled areas. The seasonal curve was not determined by a single unique species, and no single species was the most abundant in all environments sampled. The main vector of Leishmania (Leishmania) infantum, Lutzomyia longipalpis, accounted for only 5.35% of the specimens collected. Proven or suspected vectors of Leishmania (Viannia) braziliensis were recorded, and one female of the cortellezzii complex tested positive for Le. braziliensis DNA. Even with a low infection rate (0.62%), these data indicate the circulation of the parasite and reinforce the need for entomological and epidemiological surveillance in the park and its surroundings.