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1.
Vet Parasitol Reg Stud Reports ; 32: 100740, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35725103

RESUMO

Bats are parasitized by a wide spectrum of ecto and endoparasites, but their role as a reservoir for some zoonoses is not fully understood. The objective of this work was to evaluate the presence of Leishmania DNA in the blood of bats from 30 municipalities in the state of Minas Gerais, Brazil. We analyzed samples of 120 bats, covering 29 species. The blood samples were used for DNA extraction and submitted to conventional PCR analysis with primers directed to the Leishmania ITS-1 region of the rRNA. In total, 1.67% (2/120 samples) were positive for Leishmania spp., detected in animals from the metropolitan region of Belo Horizonte, the state capital. Sequencing of the positive samples revealed that both bats were infected with Leishmania (Leishmania) infantum. Considering the adaptability of some bats species to synanthropic environments, the results of the present work can contribute to a better comprehension of the leishmaniasis cycle and epidemiology.


Assuntos
Quirópteros , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Animais , Brasil/epidemiologia , Leishmania infantum/genética , Leishmaniose/epidemiologia , Leishmaniose/veterinária , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária
2.
Parasit Vectors ; 15(1): 211, 2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35710435

RESUMO

BACKGROUND: Classically, dogs have been considered to be the only reservoir of leishmaniasis in urban areas. However, in a previous study, we found a 33.3% prevalence of Leishmania infantum in the spleens of Norway rats (Rattus norvegicus) sampled in the underground sewer system of the city of Barcelona (Spain). The aim of the present study was to verify, using molecular methods, the potential reservoir role of these rats in the same sewer system. METHODS: A sensitive real-time PCR (qPCR) assay, DNA sequencing and phylogenetic analysis were carried out to identify and quantify the presence of L. infantum DNA in sand fly individuals captured in the same underground sewer system of Barcelona as in our previous study and in the spleens and ears of rats captured in the same sewer system. RESULTS: Leishmania infantum DNA was found in 14 of the 27 (51.9%) sand flies identified as Phlebotomus perniciosus, and 10 of the 24 (41.7%) rats studied were infected. Leishmania infantum was found in the spleens (70%) and in the ears (40%) of the infected rats. Quantitative results revealed the presence of high loads of L. infantum in the rats studied (> 3 × 106 parasites/g ear tissue) and among the sand flies (> 34 × 106 parasites in 1 individual). CONCLUSIONS: The molecular methods used in this study demonstrated a high prevalence of L. infantum in the underground sewer populations of both R. norvegicus and P. perniciosus. These results suggest that sewer rats, in addition to dogs, are likely to act as reservoirs of leishmaniasis in cities, where sewer systems seem to offer the ideal scenario for the transmission of leishmaniasis. Therefore, to achieve the WHO 2030 target on the elimination of leishmaniasis as a public health problem successfully, an efficient control strategy against leishmaniasis in rats and sand flies should be implemented, particularly in the sewer systems of urban areas of endemic countries.


Assuntos
Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Phlebotomus , Psychodidae , Animais , DNA , Cães , Leishmania infantum/genética , Leishmaniose/veterinária , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Phlebotomus/parasitologia , Filogenia , Psychodidae/parasitologia , Ratos , Espanha/epidemiologia
3.
Acta Trop ; 232: 106512, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35568069

RESUMO

The genus Leishmania comprises a wide range of species, some of which are pathogenic to humans and each of which has a different tissue preference, resulting in one of the three clinical forms of human leishmaniasis: visceral, cutaneous, or mucocutaneous. Although, all pathogenic species are deposited intradermally in the mammalian host upon an infectious sand fly bite, only the viscerotropic strains can leave the skin and reach the internal organs. We assume that Leishmania tissue tropism is not only the result of Leishmania genetic determinism but is also governed by the interaction of the parasite with different vectorial and human host elements. To shed light on these elements and key steps determining the course of the infection, we describe throughout this review the disease's progression from the early stages of infection taking place in the skin to the late stages succeeding in the parasite's visceral dissemination. Hence, we address the question of Leishmania tropism, through providing relevant hypotheses and answers gathered from the literature.


Assuntos
Leishmania , Leishmaniose Cutânea , Leishmaniose Visceral , Animais , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Mamíferos , Pele/patologia , Tropismo
4.
Parasitol Res ; 121(7): 2129-2140, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35614147

RESUMO

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.


Assuntos
Antiprotozoários , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Quinolinas , Animais , Antiprotozoários/uso terapêutico , Leishmaniose/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Nitritos/uso terapêutico , Polímeros/uso terapêutico , Quinolinas/uso terapêutico
5.
Acta Trop ; 232: 106522, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35597263

RESUMO

Most microorganisms including Leishmania parasites compete with the innate immune defenses of the infected hosts to acquire iron, an essential nutrient necessary for their growth and replication. In mammals, iron is predominantly bound to protein carriers such as transferrin and ferritin and the strategies adopted by the infected host to restrict its uptake by pathogens are still not elucidated. We compared herein the development of anti-transferrin and anti-ferritin antibodies in hosts that differs by their susceptibility to Leishmania infection. Results showed that Leishmania infantum naturally-infected dogs which have developed canine leishmaniasis (CanL) demonstrated higher titers of IgG antibodies anti-leishmanial antigens and anti-iron binding proteins than those infected without clinical signs. In the experimental mouse model, C57BL/6 mice resisted L. major infection, developed lower titers of Leishmania-specific IgG antibodies than BALB/c susceptible mice but demonstrated also the production of anti-transferrin and anti-ferritin IgG antibodies. Overall, results are in favor that mechanisms, other than the polyclonal activation of B cells associated-hypergammaglobulinemia, a characteristic of susceptible animals, are likely involved and require a replicating parasite for the limitation of iron uptake.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Leishmaniose , Parasitos , Animais , Doenças do Cão/parasitologia , Cães , Ferritinas , Imunoglobulina G , Ferro , Proteínas de Ligação ao Ferro , Leishmaniose/veterinária , Leishmaniose Visceral/parasitologia , Mamíferos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transferrina
6.
Emerg Infect Dis ; 28(6): 1211-1223, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35608628

RESUMO

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.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Parasitos , Animais , Doenças do Cão/parasitologia , Cães , Transmissão Vertical de Doenças Infecciosas , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Estados Unidos/epidemiologia
7.
Microbiol Spectr ; 10(2): e0067922, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35384718

RESUMO

Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and hematological parameters in both preclinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFNγ signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection, respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1ß and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1,939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFNγ signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses, and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and ∼10% of host DEGs vary in infections with either parasite species. IMPORTANCE Visceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.


Assuntos
Leishmania donovani , Leishmania infantum , Leishmaniose Visceral , Parasitos , Animais , Leishmania donovani/genética , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Mamíferos/genética , Camundongos , Camundongos Endogâmicos BALB C , Parasitos/genética , RNA-Seq , Esplenomegalia
8.
BMC Vet Res ; 18(1): 135, 2022 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-35410221

RESUMO

BACKGROUND: Responsible companion animal guardianship (RCAG) comprises a set of concepts involving activities, behavior and care that guardians must provide to ensure the welfare of their animals. When such principles are disregarded, the risk of animals developing zoonotic diseases, such as canine visceral leishmaniasis (CVL), increases. This disease is a public health problem in many urban settings in Brazil because dogs are the main reservoirs of Leishmania and are involved in the transmission of the parasites to humans. Our analytical cross-sectional epidemiological survey aimed to investigate the prevalence of CVL in a city in southeastern Brazil and to establish the association between the disease and a number of predictor variables including dog traits, socioeconomic status of guardians, ecological features of the domicile and RCAG. RESULTS: Our study showed that the global prevalence of CVL in the sample canine population was 6.7% (47/704). All variables related to better dog care were associated with lower chances of infection. Multiple regression analysis revealed that the chances of animals being seropositive for CVL were significantly (p < 0.05) higher when guardians had no formal education or possessed a university degree (vs. those with complete primary or secondary schooling) and when dogs were sheltered outside the house and had free access to the streets. An additional novel finding was that dogs that were acquired as puppies presented half of the chance of developing the disease in comparison with those acquired at the adult stage. Geographically weighted logistic regression coefficients showed that the strengths of the predictor/CVL associations varied depending on the studied geographical space. Both models demonstrated that the associations were always in the same directions. CONCLUSIONS: Our findings indicate that regardless of age and mode of acquisition, adult dogs should be submitted to clinical evaluation and tests for CVL. RCAG can exert positive effects on the control of CVL.


Assuntos
Doenças do Cão , Leishmaniose Visceral , Animais , Brasil/epidemiologia , Estudos Transversais , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Humanos , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Animais de Estimação
9.
Exp Parasitol ; 236-237: 108250, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35390313

RESUMO

Visceral leishmaniasis (VL) is the deadliest form of leishmaniasis without a safer treatment option. This study implies drug repurposing to find a novel antileishmanial compound, namely febrifugine dihydrochloride (FFG) targeting Leishmania antioxidant system. Starting with virtual screening revealed the high binding affinity and lead likeness of FFG against the trypanothione reductase (TR) enzyme of Leishmania donovani, followed by experimental validation. The promastigotes inhibition assay gave the IC50 concentration of FFG and Miltefosine (positive control) as 7.16 ± 1.39 nM and 11.41 ± 0.29 µM, respectively. Their CC50 was found as 451 ± 12.73 nM and 135.9 ± 5.94 µM, respectively. FFG has been shown to increase the reactive oxygen species (ROS), leading to apoptosis-like cell death among L. donovani promastigotes. Spleen touch biopsy resulted in 62% and 55% decreased parasite load with FFG and miltefosine treatment, respectively. Cytokine profiling has shown an increased proinflammatory cytokine response post-FFG treatment. Moreover, FFG is safe on the liver toxicity parameter in mice post-treatment.


Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Animais , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Citocinas/metabolismo , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas , Quinazolinas
10.
Vet Parasitol ; 305: 109700, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35397378

RESUMO

Leishmaniosis is a zoonotic disease with a very complex pathogenesis modulated by the interaction between the parasite, the vector and the host. Although the pathological characteristics have been extensively studied in the typically affected organs, some locations such as muscles and reproductive organs have been less studied. The objective of this study was to evaluate the presence of lesions in the temporal muscle and the male reproductive organs (testicle and epididymis) and correlate their characteristics with the presence of the parasite and with the clinical status of the dogs. The temporal muscle was studied in 25 infected beagle dogs (nine females and 16 males) and five uninfected control dogs (two females and three males) and the testicle and epididymis in the 19 males. Dogs were euthanized one year after infection and clinical signs, anti-Leishmania serum antibodies, and lymph node parasite load were assessed. Muscular and reproductive lesions were characterized by H&E and immunohistochemistry (IHC). The presence of the parasite in the lesions was evaluated using IHC and molecular techniques. Myositis was observed in 72% (18/25) of the dogs and was characterized by lymphoplasmacytic or histiocytic lesions. Mild and severe lesions were detected, the latter being statistically associated with the presence of the parasite and with the clinical status of the dogs. Orchitis was observed in 50% (8/16) of the dogs and was mainly mild and lymphoplasmacytic. No statistical relationship was found between testicular lesions and the presence of the parasite or the clinical status. Epididymitis was observed in 87.5% (14/16) of the dogs, and the lesions were often infiltrated by numerous histiocytes and neutrophils. Epididymal lesions were statistically associated with the clinical status of the dogs and with the presence of the parasite in the lesions. IgG and IgM immunoglobulins were found in all lesions, suggesting a local immune response with reactivation of the infection. Leishmania was more frequently detected in severe and histiocytic lesions, although some lesions had no detectable parasites. These results have shown that lesions in the temporal muscle, epididymis, and testicles are common in dogs infected by Leishmania infantum and that dogs may show a different response to infection. This response is characterized by varying degrees of cellular and immune responses associated with a variable presence of the parasite.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Doenças do Cão/parasitologia , Cães , Epididimo , Feminino , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Músculo Temporal/patologia , Testículo
11.
Turkiye Parazitol Derg ; 46(1): 28-33, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35232702

RESUMO

Objective: This study aimed to evaluate the polymerase chain reaction (PCR) and immunofluorescence antibody test (IFAT) results of suspected samples with canine leishmaniasis (CanL) that were sent to the Parasitology Department Laboratories of the Veterinary Faculty in Aydin Adnan Menderes University. Methods: The age, gender, and breed of the dogs to be evaluated for CanL were recorded, and IFAT was performed using 80 blood serum samples collected from them. Additionally, after the isolation of genomic DNA of 27 blood samples, PCR of these samples was performed using primers that amplify the 145 bp kDNA region of Leishmania species. Results: Thirty-seven (46.25%) of the serum samples were seropositive in at least one dilution (1/64 or 1/128) according to IFAT. Five (18.5%) of the twenty-seven samples were positive for Leishmania DNA according to PCR. According to IFAT, 38.7% of male dogs and 59% of female dogs were positive. The highest number of seropositive samples were detected in dogs aged 3-5 years (11/27). Conclusion: Considering the zoonotic potential of leishmaniasis, which is considered endemic in the region, and the high positivity of the IFAT/PCR results, veterinarians should use advanced diagnostic methods, especially serological and molecular tests, in dogs with suspected CanL. The data obtained show that the risk of infection caused by Leishmania spp. is high in the region. Therefore, it is important to routinely ensure the control of CanL to protect both human and animal health.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmania , Leishmaniose Visceral , Leishmaniose , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Feminino , Leishmania infantum/genética , Leishmaniose/diagnóstico , Leishmaniose/epidemiologia , Leishmaniose/veterinária , Leishmaniose Visceral/parasitologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Estudos Retrospectivos
12.
PLoS Pathog ; 18(3): e1010375, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35294501

RESUMO

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Proteômica
13.
Acta Trop ; 230: 106385, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35245491

RESUMO

OBJECTIVES: Leishmaniasis is a vector-borne disease and dogs may act as urban reservoirs. Turkey and most of the Mediterranean basin countries are endemic for leishmaniasis. In this study, it is aimed to report the autochthonous leishmaniasis cases, with all the components of the infection cycle (reservoir, vector, and the host) in a region close to Europe. METHODS: Nine human and four canine autochthonous leishmaniasis cases were included in the study. Direct microscopy, culture methods, serological, and molecular tests were applied to the samples obtained from the cases. RESULTS: VL and CL patients consisted of 2 L.infantum, 1 L. donovani, 2 L. tropica, and 2 L. tropica,1 L. major,1 L. infantum infected patients respectively. CanL cases were infected with L. infantum, L. donovani, L. tropica, and L. major. CONCLUSIONS: All the cases were autochthonous cases located in Manisa province. As Greece and all the Mediterranean basin countries in Europe share competent vectors, it is concluded that the detection of all 4 species of Leishmania parasites in such proximity to Europe poses an important public health threat for Europe. This study reports all four species of Leishmania spp., including L. major and L.donovani in close proximity to continental Europe.


Assuntos
Doenças do Cão , Leishmania donovani , Leishmania infantum , Leishmania major , Leishmaniose Cutânea , Leishmaniose Visceral , Animais , Cães , Humanos , Leishmania donovani/genética , Leishmania infantum/genética , Leishmania major/genética , Leishmaniose Cutânea/epidemiologia , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Saúde Pública
14.
Acta Trop ; 230: 106412, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35305943

RESUMO

Vaccination against visceral leishmaniasis (VL) should be considered as a control measure to protect against disease, and amastigote-specific proteins could help to develop such vaccines, since this parasite form is in contact with the host immune system during the active disease. In this study, a Leishmania amastigote-specific protein, LiHyG, was evaluated as recombinant protein (rLiHyG) as vaccine candidate against Leishmania infantum infection in BALB/c mice. The protein was associated with saponin (rLiHyG/Sap) or Poloxamer 407-based polymeric micelles (rLiHyG/Mic) as adjuvants, and animals receiving saline, saponin or micelle as controls. Immunological and parasitological analyses were performed before (n = 8 per group; as primary endpoint) and after (n = 8 per group; as secondary endpoint) infection. Results showed that, in both endpoints, rLiHyG/Sap and rLiHyG/Mic induced higher levels of IFN-γ, IL-12 and GM-CSF in spleen cell cultures from vaccinated animals, besides elevated presence of IgG2a isotype antibodies. Decreased hepatotoxicity and 'positive lymphoproliferative response were also found after challenge. Such findings reflected in significantly lower levels of parasite load found in their spleens, livers, bone marrows and draining lymph nodes. In conclusion, rLiHyG associated with Th1-type adjuvant could be considered for future studies as vaccine candidate to protect against VL.


Assuntos
Leishmania infantum , Vacinas contra Leishmaniose , Leishmaniose Visceral , Leishmaniose , Saponinas , Adjuvantes Imunológicos , Animais , Antígenos de Protozoários/genética , Leishmaniose/prevenção & controle , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética
15.
Acta Trop ; 229: 106371, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35181302

RESUMO

Canine leishmaniosis is a vector-borne disease caused by Leishmania parasites. Serological methods are the most common tests used for the diagnosis. This study aimed to evaluate and compare different serological commercial immunochromatographic rapid tests available in Spain to detect anti-Leishmania canine antibodies. The immunochromatographic tests were evaluated in different groups of dogs (healthy seronegative dogs (n = 21), naturally-sick dogs with moderate anti-Leishmania antibodies (n = 39), naturally-sick dogs with high anti-Leishmania antibodies (n = 37), dogs with the serological result of other pathogens infection (n = 20) and exposed dogs (n = 33)) admitted to the Veterinary Teaching Hospital of the University of Zaragoza (Spain) according to the clinical information sent with the sample to the laboratory for diagnostic purposes. The serology status was also routinely recorded through an in-house enzyme-linked immunosorbent assay (ELISA) and an in-house indirect immunofluorescence test (IFAT). The qualitative commercial serological immunochromatographic tests used were: FASTest LEISH, Uranotest Leishmania, Uranotest Leishmania 2.0, Speed Leish K, Witness Leishmania, and DFV Test Leishmania. Performance measures analyzed for each test were: sensitivity, specificity, and area under the receiver-operating (ROC) curve. The maximum specificity (1.00) was attained for Uranotest Leishmania and DFT Test Leishmania, followed by FASTest LEISH (0.98), Uranotest Leishmania 2.0 (0.98), Speed Leish K (0.98), and Witness Leishmania (0.95). The maximum sensitivity was attained for FASTest LEISH (1.00), followed by Uranotest 2.0 (0.97), Speed Leish K (0.97), Uranotest (0.96), and the lowest results with Witness (0.84) and DFV Test (0.59). Regarding the ROC curve, the maximum value was attained with the FASTest LEISH (0.99), followed by Uranotest (0.98), Uranotest 2.0 (0.97), Speed Leish K (0.97), Witness (0.90), and the lowest result with DFV Test (0.79). Efforts in the field of diagnosis should focus on establishing a commercial immunochromatographic test with high sensitivity and specificity with a reasonable cost-benefit balance.


Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Anticorpos Antiprotozoários , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Hospitais Veterinários , Hospitais de Ensino , Leishmaniose Visceral/parasitologia , Sensibilidade e Especificidade , Testes Sorológicos/veterinária , Espanha
16.
PLoS Negl Trop Dis ; 16(2): e0010170, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35139072

RESUMO

Diseases caused by trypanosomatids are serious public health concerns in low-income endemic countries. Leishmaniasis is presented in two main clinical forms, visceral leishmaniasis-caused by L. infantum and L. donovani-and cutaneous leishmaniasis-caused by many species, including L. major, L. tropica and L. braziliensis. As for certain other trypanosomatids, sexual reproduction has been confirmed in these parasites, and formation of hybrids can contribute to virulence, drug resistance or adaptation to the host immune system. In the present work, the capability of intraclonal and interspecies genetic exchange has been investigated using three parental strains: L. donovani, L. tropica and L. major, which have been engineered to express different fluorescent proteins and antibiotic resistance markers in order to facilitate the phenotypic selection of hybrid parasites after mating events. Stationary and exponential-phase promastigotes of each species were used, in in vitro experiments, some of them containing LULO cells (an embryonic cell line derived from Lutzomyia longipalpis). Several intraclonal hybrids were obtained with L. tropica as crossing progenitor, but not with L. donovani or L. major. In interspecies crossings, three L. donovani x L. major hybrids and two L. donovani x L. tropica hybrids were isolated, thereby demonstrating the feasibility to obtain in vitro hybrids of parental lines causing different tropism of leishmaniasis. Ploidy analysis revealed an increase in DNA content in all hybrids compared to the parental strains, and nuclear analysis showed that interspecies hybrids are complete hybrids, i.e. each of them showing at least one chromosomal set from each parental. Regarding kDNA inheritance, discrepancies were observed between maxi and minicircle heritage. Finally, phenotypic studies showed either intermediate phenotypes in terms of growth profiles, or a decreased in vitro infection capacity compared to the parental cells. To the best of our knowledge, this is the first time that in vitro interspecies outcrossing has been demonstrated between Leishmania species with different tropism, thus contributing to shed light on the mechanisms underlying sexual reproduction in these parasites.


Assuntos
Hibridização Genética , Leishmania donovani/genética , Leishmania major/genética , Leishmania tropica/genética , Animais , Linhagem Celular , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Psychodidae
17.
Front Immunol ; 13: 801182, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154115

RESUMO

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2 -/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2 -/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2 -/-) or from protein kinase R (PKR) knock-out mice (pkr -/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr -/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr -/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.


Assuntos
Interferon-alfa/metabolismo , Interferon beta/metabolismo , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos Peritoneais/imunologia , Transdução de Sinais/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , eIF-2 Quinase/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Inativação de Genes , Interferon-alfa/genética , Interferon beta/genética , Leishmaniose Visceral/parasitologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , eIF-2 Quinase/genética
18.
Int J Mol Sci ; 23(3)2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-35163386

RESUMO

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak's isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase-protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts.


Assuntos
Cães/genética , Cães/parasitologia , Leishmania infantum/patogenicidade , Leishmaniose Visceral/genética , Leishmaniose Visceral/veterinária , Estágios do Ciclo de Vida/fisiologia , Macrófagos/parasitologia , Transcriptoma/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Ontologia Genética , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Macrófagos/metabolismo , Proteínas NLR/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Virulência
19.
Parasitol Res ; 121(5): 1389-1406, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35169883

RESUMO

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, being fatal if untreated. In search of a more effective treatment for VL, one of the main strategies is the development and screening of new antileishmanial compounds. Here, we reported the synthesis of seven new acetyl functionalized 1,2,3-triazolium salts, together with four 1,2,3-triazole precursors, and investigated their effect against different strains of L. infantum from dogs and humans. The 1,2,3-triazolium salts exhibited better activity than the 1,2,3-triazole derivatives with IC50 range from 0.12 to 8.66 µM and, among them, compound 5 showed significant activity against promastigotes (IC50 from 4.55 to 5.28 µM) and intracellular amastigotes (IC50 from 5.36 to 7.92 µM), with the best selective index (SI ~ 6-9) and reduced toxicity. Our findings, using biochemical and ultrastructural approaches, demonstrated that compound 5 targets the mitochondrion of L. infantum promastigotes, leading to the formation of reactive oxygen species (ROS), increase of the mitochondrial membrane potential, and mitochondrial alteration. Moreover, quantitative transmission electron microscopy (TEM) revealed that compound 5 induces the reduction of promastigote size and cytoplasmic vacuolization. Interestingly, the effect of compound 5 was not associated with apoptosis or necrosis of the parasites but, instead, seems to be mediated through a pathway involving autophagy, with a clear detection of autophagic vacuoles in the cytoplasm by using both a fluorescent marker and TEM. As for the in vivo studies, compound 5 showed activity in a mouse model of VL at 20 mg/kg, reducing the parasite load in both spleen and liver (59.80% and 26.88%, respectively). Finally, this compound did not induce hepatoxicity or nephrotoxicity and was able to normalize the altered biochemical parameters in the infected mice. Thus, our findings support the use of 1,2,3-triazolium salts as potential agents against visceral leishmaniasis.


Assuntos
Antiprotozoários , Leishmania infantum , Leishmaniose Visceral , Animais , Antiprotozoários/uso terapêutico , Cães , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Sais/farmacologia , Sais/uso terapêutico , Triazóis/farmacologia , Triazóis/uso terapêutico
20.
PLoS Negl Trop Dis ; 16(2): e0010224, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35192633

RESUMO

BACKGROUND: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo. METHODOLOGY/FINDINGS: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo. CONCLUSIONS: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.


Assuntos
Leishmania donovani , Vacinas contra Leishmaniose , Leishmaniose Visceral , Animais , Comunicação Celular , Células Dendríticas , Leishmania donovani/fisiologia , Leishmaniose Visceral/parasitologia , Camundongos , Neutrófilos , Vacinas Atenuadas
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