New Insights into Pathogenesis and Immunity of Leishmania Parasites with Emphasis on Vaccines

En diferentes regiones de Latinoamérica la infección por T. cruzi y Leishmania se superponen, por lo cual se reportan infecciones mixtas circulantes, debido a esto; deben realizarse pruebas diagnósticas específicas para evitar reacciones cruzadas entre estas dos patologías. Objetivo: Determinar patrones de fluorescencia que permitan la diferenciación entre Leishmaniasis, enfermedad de Chagas e infección mixta empleando epimastigotes de T. cruzi. Metodología: Se empleó la técnica de Inmunofluorescencia Indirecta utilizando epimastigotes de T. cruzi (TcV autóctono) como antígeno figurado frente a un panel de muestras de suero codificados como A, B, C y D correspondientes a pacientes con infección por: Leishmaniasis (A), Infección mixta por Leishmania y Chagas(B), Enfermedad de Chagas (C) y sin ninguna de las dos infecciones (D). Resultados: En los cuatro paneles de muestras se observaron diferentes patrones de intensidad de fluorescencia a nivel de membrana y núcleo de los epimastigotes de T. cruzi (TcV autóctono). Conclusiones: La técnica de Inmunofluorescencia (IFI) con antígenos de epimastigotes de T. cruzi a demostrado utilidad en la diferenciación entre enfermedad de Chagas, Leishmaniasis y/o infecciones mixtas por ambos parásitos en aquellas zonas donde la coexistencia de ambas es habitual.

O presente trabalho realizou estudos in silico para predição das atividades farmacocinéticas, físico-químicas, toxicológicas e biológicas de metabólitos isolados de Libidibia ferrea Martius, planta utilizada popularmente para o tratamento de inflamações e lesões, como antidiabética, antimicrobiana, antifúngica e antileishmaniose. As comparações foram realizadas através dos programas: chemsketch, mcule property calculator, preadmet, protox e pass online. Os resultados revelaram que apenas as moléculas lupenona e o trans-farnesol apresentaram absorção intestinal acima de 90%. Os metabólitos ácido gálico, catequina, metil-galato, quercetina e trans-farnesol seguem a regra de lipinsk em todos os seus parâmetros pré-condicionados. No que se refere a inibição enzimática, cada uma das moléculas do estudo demonstrou inibição direcionada a pelo menos duas enzimas da CYP. A lupenona foi a única molécula que se apresentou ativa contra o protozoário leishmania, além de ser promissora contra neoplasias e inflamação. A catequina e quercetina demonstraram resultados positivos no que tange a atividade antioxidante, e a maioria das substâncias do estudo em questão apresentaram capacidade de proteção mucomembranosa. Nenhuma substância demonstrou embriotoxicidade ou teratogênese, mas todas as moléculas demonstraram possuir certo nível de toxicidade em outros parâmetros de análise. Em termos farmacocinético, toxicológico e biológico, as moléculas pauferrol A, B e C foram as menos promissoras, enquanto que a lupenona, catequina e quercetina apresentaram os melhores resultados.

First record of Leishmania spp. in birds and bats from Sudan

Objective Based on the (phosphoenolpyruvate carboxykinase, PEPCK) dominant epitopes gene of Leishmania infantum that has been analyzed and selected, we construct the corresponding recombinant prokaryotic expression vector to obtain the recombinant protein, construct the corresponding recombinant eukaryotic expression vector and verify the expression of eukaryotic vector in NIH3T3 cells, which laid the foundation for the subsequent vaccination and infection of animal experiment. Methods Based on the PEPCK dominant epitopes gene sequence, the recombinant prokaryotic and eukaryotic expression plasmid pET32a- PEPCK and pVAX1- EPEPCK were constructed by PCR reaction and enzyme reaction and were respectively transfected into E. coli and NIH3T3 cells of mice for expression. The expression of prokaryotic recombinant plasmid and the prokaryotic expressed protein purified by HisTrap affinity column were detected through SDS-PAGE and Western-Blot, and the expression of pVAX1-E PEPCK was verified by immunofluorescence. Results The correct sequencing results of the recombinant prokaryotic and eukaryotic vectors showed that the construction of recombinant vectors was successful. The positive results of 48.08 kD of recombinant protein were detected in SDS-PAGE and Western Blot, and immunofluorescence results of NIH3T3 cells transfected with eukaryotic recombinant vectors showed positive results. Conclusion We successfully constructed the recombinant prokaryotic and eukaryotic expression vectors: pET32a- PEPCK and pVAX1-E PEPCK , successfully got the recombinant proteins that followed by proteinic purification and confirmed the expression of pVAX1-E PEPCK in NIH3T3 cells, which is the basis for experiments where animals are vaccinated with a DNA prime-protein boost vaccines. 摘要：目的 基于前期分析并选取的婴儿利什曼原虫 PEPCK 的优势表位基因, 构建相应重组原核表达载体以获得 重组蛋白, 构建相应重组真核表达载体并验证真核载体在 NIH3T3 细胞中的表达, 为后续动物的免疫和感染实验备下 基础。 方法 根据 PEPCK 优势表位基因序列, 经 PCR 反应及酶切连接构建重组原核与真核表达质粒 pET32a- PEPCK 和 pVAX1-E PEPCK 并分别转染至 E. coli 和 NIH3T3 细胞中进行表达。采用 SDS-PAGE 电泳和 Western Blot 鉴定原核重 组质粒在大肠杆菌中的表达和原核表达蛋白被镍柱纯化后的情况, 采用免疫荧光实验验证真核重组质粒在细胞中的 表达。 结果 重组原核与真核载体的正确测序结果表明重组载体构建成功。SDS-PAGE 电泳和 Western Blot 的结果 显示, 大肠杆菌中表达的原核重组蛋白以及纯化后的蛋白在 48.08 kD 处存在条带, 转染了真核重组载体的 NIH3T3 细胞 的免疫荧光结果呈阳性。 结论 成功构建了 PEPCK 优势表位基因的重组原核和真核表达载体: pET32a- PEPCK 和 pVAX1-E PEPCK , 成功表达相应的重组蛋白并纯化并验证了 pVAX1-E PEPCK 在 NIH3T3 细胞中的表达, 为后续 DNA 疫苗初次免疫和蛋白疫苗加强免疫的动物实验备下基础。

Vorinostat is a drug used to treat cutaneous T cell lymphoma whose action mechanism is based on Histone Deacetylase inhibition. Histone Deacetylases are a family of enzymes that remove acetyl groups from histone and non-histone proteins that control many crucial processes, such as gene regulation, cell cycle progression, differentiation, and apoptosis. Histone Deacetylase homologues are also expressed in parasites of the genus Plasmodium, Leishmania, Cryptosporidium, Schistosoma, Entamoeba, and others. In this way, antiparasitic properties of Vorinostat have been explored. The aim of this review is to report the current state knowledge of Vorinostat as antiparasitic drug against Plasmodium, Leishmania, Cryptosporidium, Schistosoma and Entamoeba in order to support future investigation in this field. The authors revised the recent and relevant literature concerning the topic and discussed advances and limitations of studies on Vorinostat as potential drug to treat human parasitic diseases. Vorinostat has been efficient in vitro and, in some cases, in vivo, against parasites that cause parasitic diseases, such as malaria, leishmaniasis, cryptosporidiosis, amoebiasis, and schistosomiasis. In vitro and in vivo models have demonstrated the antiparasitic activity of Vorinostat, however, the challenge is to assay its activity in animal models and to evaluate if Vorinostat is safe for humans as new alternative to treat human parasitic infections.

Canine Leishmaniasis caused by Leishmania infantum is endemic in Mediterranean countries. The disease represents an important public health issue in Kosovo in which the autochthonous cases by humans were reported in the last years. The objective of this study was to survey the serological and clinical data of Canine Leishmaniasis in dogs in southwestern region of Kosovo. The blood samples were collected from stray dogs in four municipalities, respectively Prizren, Gjakova, Rahovec and Deçan. The total 125 samples were collected between April/September 2016. Antibodies of Leishmania infantum were detected by competitive enzyme-linked immunosorbent assay (ELISA IgG). Out of total 125 serum samples, 23 dogs (18.4%) were positive for Leishmania infantum. In three of 23 infected dogs (13%) were shown the clinical signs of Canine Leishmaniasis as skin and ocular lesions, decreased appetite, lameness, diarrhea, lethargy and progressive weight loss. The highest infection rate was detected in Prizren municipality (21.6%), followed by Gjakova (17.6%), Rahovec (17.1%) and Deçan (7.8%). The present study confirmed that the Leishmaniasis is endemic in southwestern part of Kosovo and emphasized the need to establish the surveillance and control of this disease.

In Peru, only 12.7% (n=207) of fish species have had some record as parasitic metazoan hosts. The objective of this investigation was to determine the community of helminth parasites in black cusk-eel Genypterus maculatus (Tschudi, 1846) of north Peru. Fifty specimens of G. maculatus from Puerto Pimentel, province of Chiclayo, department of Lambayeque, Peru were evaluated. For the analysis of the parasitic community, the parasitological ecological indexes of prevalence (P), mean abundance (MA), mean intensity (MI) of infection, the dispersion indices, the correlation between total length (TL) and sex of fish vs parasitological ecological indexes, were calculated. The component community of the parasitic helminths in the evaluated fish was dominated by the presence of endoparasitic flatworms and absence of ectoparasites. The parasitic fauna of G. maculatus included eight endoparasitic species, four trematodes, two cestodes, one acanthocephalan and one nematode species. The species with the highest prevalence and abundance were Helicometra fasciata (Rudolphi, 1819), Scolex pleuronectis Müller, 1788 and Corynosoma australe Johnston, 1937. The prevalence of infection of C. australe was found positively related to the total length of G. maculatus. The sex of G. maculatus was not found associated with the P, MA and MI of any of the helminth parasites. This is the first study of ecological aspect in the parasitic fauna of G. maculatus made in Peru.

Recently, Leishmania infantum has increasingly been detected in stray cats in endemic regions of the world. Cats have been considered playing a role in the epidemiology of visceral leishmaniosis, an endemic zoonosis in Iran. The studies concerning feline leishmaniosis (FeL) allow the hypothesis that cats can be considered as potential reservoirs. The investigations on Leishmania infection in cats are very few in Iran and therefore we aimed to assess the L. infantum infection in stray cats and its possible role in transmission of the disease to human by direct agglutination test (DAT), ELISA, nested-PCR and confirmation via sequencing and phylogenetic analysis in Fars province, Iran. Whole blood samples were obtained from 174 stray cats. Anti-Leishmania antibodies were detected in the sera using DAT and ELISA. DNA was extracted from the buffy coat of each subject and PCR amplified, targeting Leishmania kDNA gene. PCR results were confirmed by sequence analysis. Prevalence of clinical signs in positive cats was 19.0%. Anti-Leishmania antibodies with different titers were detected in 48 (27.59%) and leishmanial DNA in 36 (20.69%) of the cats. The sequencing of PCR-positive cats revealed the parasite as L. infantum. A high seroprevalence of L. infantum was revealed, with higher levels in males, adult cats, and those living in rural districts and southern zones. Despite the reservoir task of cats in nature is still ambiguous, the high serological and molecular detection of L. infantum in stray cats indicates that cats are regularly bitten by infected sand flies in Fars province, southern Iran, and may have a potential reservoir role in the maintenance of L. infantum in the endemic areas of zoonotic visceral leishmaniosis in Iran. Anyway, Leishmania infection must be appraised in the differential diagnosis of cutaneous or systemic clinical signs in cats.

La Leishmaniosis visceral humana causada por Leishmania donovani es una enfermedad antroponotica endemica en muchos paises de Africa oriental. Sin embargo, se desconoce si animales domesticos o salvajes pueden tener un papel en esta infeccion. El principal objetivo de este estudio fue detectar anticuerpos frente a Leishmania infantum en sueros de perros del noroeste de Uganda mediante ELISA indirecto y evaluar los factores de riesgo. Para ello, se analizaron muestras de suero del ano 2017 y muestras de suero impregnadas en papel de filtro Whatman no3 del 2018. El trabajo de campo se llevo a cabo en los Parques Nacionales de Elizabeth Queen (QENP), Mgahinga, Budongo y en los distritos de Kasese, Kapchorwa, Buliisa, Masindi, Kibingo, Hoima y Kanungu. Las muestras de suero del 2017 fueron todas negativas. La seroprevalencia global obtenida de las muestras impregnadas en papel filtro fue del 50.8%, siendo los distritos de Karusandara y Kilembe los que obtuvieron una mayor proporcion de perros seropositivos. Entre los datos de frecuencia analizados, no se encontro ninguna asociacion estadisticamente significativa entre las variables estudiadas. Si bien, se observaron valores estadisticamente significativos con respecto a porcentajes de positividad registrados por localidad y condicion corporal de los perros muestreados. Los resultados del presente estudio sugieren el posible caracter endemico de la leishmaniosis canina en el noreste de Uganda. No obstante, estudios futuros son necesarios para determinar la prevalencia real de esta infeccion y su correacion con otras tripanosomiasis en todo el pais y evaluar el verdadero rol de los perros como reservorios a la leishmaniosis en esta area geografica.

Chromatographic fractionation of the antileishmanial extract obtained from fermentation of the endophytic fungus Penicillium citrinum, isolated from leaves of Ageratum myriadenia, yielded three diketopiperazine alkaloids; cyclo(L-Pro-L-Leu) (1), cyclo-(L-Pro-L-Phe) (2) and tryprostatin B (3). The structures of these compounds were established on the basis of spectroscopic methods and comparison with the literature. Compounds 1 and 2 were active against both amastigote-like forms of Leishmania (Leishmania) amazonensis and intracelular amastigotes of L. (Leishmania) infantum with approximately 50% of parasite growth inhibition at 100 μM. None of the compounds were considered toxic against human leukemia monocyte cell line (THP-1) at 100 μM. It is the first report about isolation of these diketopiperazines from P. citrinum and their antileishmanial potential against L. (L.) infantum. Key words: Microfungi, natural products, in vitro, Leishmania, amphotericin B, cytotoxic activity.

Alignment of single gene and concatenated sequences of RSP, FAD, STK, A2P, UBE1, MTF, and SF3B1 genes from 41 isolates of the Leishmania donovani complex. All alignment were performed in the software Mega 7.0 using the Muscle algorithm. DNA sequences were obtained by Sanger sequencing.

A Study to Assess the Safety, Efficacy and Immunogenicity of Leishmania Vaccine ChAd63-KH in PKDL

Introduction: Cutaneous leishmaniasis is a mutual parasitic disease, which is endemic in some countries of the world. The use of common drugs has been restricted because of confirmed side effects and drug resistance. There are still serious challenges in the treatment of CL. Developing new therapeutic methods for leishmaniasis, besides obtaining a prompt treatment response that prevents lesions' progression and scarring, is a significant success in the treatment of the disease. Novel approaches such as nanotechnology have been applied as a therapeutic modality. This study aimed at evaluating the influence of three physical approaches combined with nanoparticles on promastigotes, amastigotes, and lesion-induced parasites. Materials and Methods: Leishmania promastigotes were treated with gold nanoparticles combined with different duration of the microwave with 2450 MHz frequency. So, the promastigotes treated with electroporation and ultraviolet irradiation in presence of silver and titanium dioxide nanoparticles respectively. Leishmania amastigotes were preserved by GNP and SNP with different conditions exposure of Microwave irradiation and electroporation. Ultraviolet irradiation in presence of silver nanoparticles used to reduce the size of lesion-induced parasites. Results: Promastigotes survival rate induced a significant decline in different conditions of all three approaches in the presence of nanoparticles in comparison to similar samples without there. The least survival of amastigotes was also recorded in the groups containing nanoparticles received the microwave and electroporation. In addition, the findings on parasite burden and size of lesions showed a significant difference between the treatment groups with SNPs and UV irradiation and control group. Conclusion: The finding emphasized to the utility of new approaches by nanotechnology could be useful for the treatment of cutaneous disease such as leishmaniasis. For validation of this statement need to more research and some studies have been planned for this aim.

Evidence Based Insights on Leishmania Replication and Cytokinesis

In order to assess the leishmanian risk related to the presence of sand flies in the Northwest of Morocco, a short entomological survey was realized in Bni Oual locality in May 2015. In total,243 specimens of sand flies were collected. They belong to two genus, four Subgenus and five species. Phlebotomus sergenti is the dominant species of our capture with the proportion of 39.92% followed by Phlebotomus longicuspis (23.87%), Sergentomyia minuta (21,81%), Phlebotomus papatasi (14%) and Phlebotomus ariasi with one species identified. Among these identified species, four of them are considered as vectors of Leishmania that causing cutaneous and viscerals leishmaniasis which shows that the risk of vector-borne leishmaniasis is high in this Moroccan northwest locality.

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. <i>Leishmania</i> exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in <i>Leishmania</i> by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. <i>Δift140</i> cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of <i>Δift140</i> cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, <i>Leishmania</i> promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure; echoing the process that occurs during the promastigote to amastigote differentiation.

New insights with miltefosine unresponsiveness in Brazilian Leishmania infantum isolates

Leishmania parasites as the causative agent of leishmaniasis belong to Trypanosomatidae family.Parasite, vector, vertebrate host and environment are major factors in pathogenesis of Leishmania.Parasite dependent factors are virulence factors which exist in Leishmania species such as LPG, GP63.In recent years, the importance of these factors in the field of vaccine and drug has been considered by researchers.Sand fly biting behavior and salivary gland proteins are vector dependent factors which are effective in the Leishmania pathogenesis.Age, gender, nutrition, immune system, infectious diseases, genetic, occupation, socio-economic characteristics, and habitat are vertebrate host mediated factors.Temperature, rainfall, wind and its speed, soil, and continuous changes in climate are also environmental factors.Therefore the aim of this study was to evaluate the pathogenesis of Leishmania parasites.

- Introduction: Visceral Leishmania represents a challenging diagnosis and treatment because the clinical picture of Visceral Leishmaniasis, so that the treatment outcome is compromised with many factors, thus study try to asses real picture of treatment outcome with determinants. Objective: To assess treatment outcome and determinant factors among patient with visceral Leishmaniasis in Arba Minch General Hospital Leishmania Research and Treatment Center. Methods: Institution-based, Cross-sectional, retrospective study was conducted among Leishmaniasis patients in Arba Minch general hospital. Patient charts that have been treated from January 2011-December 2015 were reviewed. A total of 348 charts were selected by Simple Random Sampling technique and included in the study. The data were entered and cleaned using Epi-info version 7.0 and analysis was carried out using SPSS-20 statistical packages. Multi-variate logistic regression analysis was carried out to identify the independent predictors of treatment outcome. Result: A total of 348 patient's charts were included in the study. The prevalence of poor outcome was found to be 8.6%. The presence of bleeding/Epsitaxis[AOR=5;95% CI:1.3,19.3], tuberculosis[AOR=3.7;95%CI:1.83,6.3], septic syndrome[AOR=4.3;95% CI: 6.1,32], and adverse drug side effect[AOR=4.5;95%CI:2.73,13.6] were the independent predictors of treatment outcome. Conclusion and recommendation: The prevalence of poor treatment outcome among patients with VL in the study area is high. Concomitant infection; bleeding/Epsitaxis, tuberculosis, septic syndrome and adverse side effect were independently associated with poor treatment outcome. The clinical management of VL in co-infected patients is a major challenge that requires new treatment approaches to improve its outcome.