Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatmen...Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.展开更多
Objective:To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum(L infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dr...Objective:To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum(L infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination teals(FD-DAT) that use freezedried antigen.Methods:Glycerol-preserved DAT antigen was prepared and stored at different temperatures.We tested antigen stored at 4℃,22-37℃and 50℃over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009,and 80 of these samples were pooled to produce sera.Each pooled serum contained 10 sera.All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT,FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens;tests were performed on both human and dog sera over a period of 12 months. Results:There was strong agreement between the results obtained using GP-DAT and FDDAT antigens stored at 22-37℃for 12 months for both human(100%) and dog(100%) pooled sera.The direct agglutination test results were highly reproducible(weighted kappa:GP=0.833, FD=0.979 and FF=0.917).Conclusions:Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field,this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.展开更多
Objective:To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband,Fars Province,South of Iran.Methods:Sticky papers and Sherman trap were used for collection of sand flies ...Objective:To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband,Fars Province,South of Iran.Methods:Sticky papers and Sherman trap were used for collection of sand flies and rodents,respectively.Polymerase chain reaction(PCR)of kDNA,ITS1-rDNA were used for identification of Leishmania parasite in sand flies as well as rodents.Results:Totally 2010 sand flies were collected and the species of Phlebotomus papatasi Scopoli was the common specimen in outdoors and indoors places.PCR technique was employed on 130females of Phlebotomus papatasi.One of them(0.76%)was positive to parasite Leishmania major(L.major)and one specimen(0.76%)was positive to Leishmania infantum.Microscopic investigation on blood smear of the animal reservoirs for amastigote parasites revealed 16(44%)infected Tatera indica.Infection of them to L.major was confirmed by PCR against kDNA loci of the parasite.Conclusions:The results indicated that Phlebotomus papatasi was the dominant species circulating two species of parasites including L.major and Leishmania infantum among human and reservoirs.Furthermore,Tatera indica is the only main host reservoir for maintenance of the parasite source in the area.展开更多
Although Indirect Immuno-Fluorescent Antibody Test (IFAT), performed employing “in house” prepared antigen, is considered by several authors as the golden standard for the quantisation of anti-leishmania antibodies ...Although Indirect Immuno-Fluorescent Antibody Test (IFAT), performed employing “in house” prepared antigen, is considered by several authors as the golden standard for the quantisation of anti-leishmania antibodies in dogs, there is a lack of papers reporting a description of the different patterns of fluorescence that can be observed. An incorrect identification of patterns of fluorescence may be an important source of bias in the interpretation of results. Previous papers report different criteria to define as “positive” a specific pattern of fluorescence, namely: membrane fluorescence, homogeneous fluorescence of the body, or homogeneous fluorescence of the body plus flagellum. In this paper, we report a detailed description of preparation of slides and of the patterns of fluorescence that can be obtained employing “in house” prepared antigen. At least six main patterns of fluorescence may be observed: 1): homogeneous cytoplasmatic green fluorescence;2): membrane pattern, in which the fluorescence is mainly localized along the entire perimeter of the parasites;3): coarse-speckled cytoplasmatic fluorescence;4): flagellar pattern, in which the fluorescence is localized exclusively onto the flagellum;5): punctiform pattern, in which the fluorescence is localized exclusively at the basis of the flagellum;6): nuclear pattern, in which only the nucleus of the parasite shows a homogeneous green fluorescent. The significance of each pattern is discussed.展开更多
Objective: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infanturn proliferation, morphology, and the driving mechanism in leishmanicidal activ...Objective: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infanturn proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. Methods: I., infantum promastigotes were treated with 50 and 100 μg/mL AEPa for 72 h and then antipro- mastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. Results: Treatment with AEPa reduced the numbers ofL infantum promastigotes (50% inhibitory concen- tration (ICso) = 65.9μg/mL; selectivity index (SI)= 22.1) and amastigotes (ICso = 37.9 μg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2μg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 μg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration 〉1000μg/mL). Conclusion: AEPa presented antileishmanial activity against the promastigotes and amastigotes of I.. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.展开更多
Background:Leishmania infantum is the causative agent of human visceral leishmaniasis(VL)and sporadic human cutaneous leishmaniasis(CL)in the Mediterranean region.The genetic variation of the Leishmania parasites may ...Background:Leishmania infantum is the causative agent of human visceral leishmaniasis(VL)and sporadic human cutaneous leishmaniasis(CL)in the Mediterranean region.The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations.The main objective of this study was to explore the genetic polymorphism in L.infantum isolates from human and animal hosts in different regions of Morocco.Methods:The intraspecific genetic variability of 40 Moroccan L.infantum MON-1 strains isolated from patients with VL(n=31)and CL(n=2)and from dogs(n=7)was evaluated by PCR-RFLP of nagt,a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase.For a more complete analysis of L.infantum polymorphism,we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database.Results:Moroccan L.infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified.Three of the six genotypes were exclusively identified in the Moroccan population of L.infantum:variant M1(15%),variant M2(7.5%),and variant M3(2.5%).The most common genotype(65%),variant 2(2.5%),and variant 4(7.5%),were previously described in several countries with endemic leishmaniasis.Phylogenetic analysis segregated our L.infantum population into two distinct clusters,whereas variant M2 was clearly distinguished from both cluster I and cluster II.This distribution highlights the degree of genetic variability among the Moroccan L.infantum population.Conclusion:The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L.infantum strains isolated from human and canine reservoirs with 6 genotypes identified.Three of the six Moroccan nagt genotypes,have not been previously described and support the particular genetic diversity of the Moroccan L.infantum population reported in other studies.展开更多
基金supported by grants from the Brazilian Agencies:Coordenação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES-Financial code 001)Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico(FUNCAP).
文摘Objective:To evaluate the immunological response elicited by an inactivated bacterial vector carrying the K39 antigen of Leishmania infantum,and a purified antigen.Methods:Mice were subjected to the following treatments:(1)Purified recombinant K39(rK39)protein at a 20μg dose with complete Freund’s adjuvant;(2)Inactivated Escherichia coli(BL21 DE3)carrying the K39 protein at an equivalent total protein content of 200μg;(3)Inactivated bacteria lacking the K39 protein;(4)Non-immunized control animals.Serological monitoring was performed.All groups were challenged by intraperitoneal injection of 10^(7) Leishmania infantum promastigotes.After euthanasia,the liver and spleen were collected to analyze the levels of TNF,IFN-γ,IL-12,IL-4,and IL-10.Results:Mice immunized with purified rK39 or the inactivated bacterial vector carrying the K39 antigen of Leishmania infantum showed a long-lasting immune response with high levels of polyclonal antibodies specifically recognizing the recombinant proteins.The IgG1 subclass was the predominant immunoglobulin;however,the induction of IgG2a and the profile of cytokines produced were indicative of the induction of a mixed-type response.Conclusions:The inactivated bacterial vector carrying the K39 antigen,as well as the purified antigen can induce a long-lasting immune response in immunized mice,predominantly favouring a Th2 profile response.
基金funded by Tehran University of Medical Sciences (Project No:88-01-27-9353)National Institute of Health Research,Islamic Republic of Iran(Project No:241/1441)
文摘Objective:To prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum(L infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination teals(FD-DAT) that use freezedried antigen.Methods:Glycerol-preserved DAT antigen was prepared and stored at different temperatures.We tested antigen stored at 4℃,22-37℃and 50℃over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009,and 80 of these samples were pooled to produce sera.Each pooled serum contained 10 sera.All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT,FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens;tests were performed on both human and dog sera over a period of 12 months. Results:There was strong agreement between the results obtained using GP-DAT and FDDAT antigens stored at 22-37℃for 12 months for both human(100%) and dog(100%) pooled sera.The direct agglutination test results were highly reproducible(weighted kappa:GP=0.833, FD=0.979 and FF=0.917).Conclusions:Because GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field,this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.
基金Supported by the School of Public Health.Tehran University of Medical Sciences.(Project No.13946)
文摘Objective:To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband,Fars Province,South of Iran.Methods:Sticky papers and Sherman trap were used for collection of sand flies and rodents,respectively.Polymerase chain reaction(PCR)of kDNA,ITS1-rDNA were used for identification of Leishmania parasite in sand flies as well as rodents.Results:Totally 2010 sand flies were collected and the species of Phlebotomus papatasi Scopoli was the common specimen in outdoors and indoors places.PCR technique was employed on 130females of Phlebotomus papatasi.One of them(0.76%)was positive to parasite Leishmania major(L.major)and one specimen(0.76%)was positive to Leishmania infantum.Microscopic investigation on blood smear of the animal reservoirs for amastigote parasites revealed 16(44%)infected Tatera indica.Infection of them to L.major was confirmed by PCR against kDNA loci of the parasite.Conclusions:The results indicated that Phlebotomus papatasi was the dominant species circulating two species of parasites including L.major and Leishmania infantum among human and reservoirs.Furthermore,Tatera indica is the only main host reservoir for maintenance of the parasite source in the area.
文摘Although Indirect Immuno-Fluorescent Antibody Test (IFAT), performed employing “in house” prepared antigen, is considered by several authors as the golden standard for the quantisation of anti-leishmania antibodies in dogs, there is a lack of papers reporting a description of the different patterns of fluorescence that can be observed. An incorrect identification of patterns of fluorescence may be an important source of bias in the interpretation of results. Previous papers report different criteria to define as “positive” a specific pattern of fluorescence, namely: membrane fluorescence, homogeneous fluorescence of the body, or homogeneous fluorescence of the body plus flagellum. In this paper, we report a detailed description of preparation of slides and of the patterns of fluorescence that can be obtained employing “in house” prepared antigen. At least six main patterns of fluorescence may be observed: 1): homogeneous cytoplasmatic green fluorescence;2): membrane pattern, in which the fluorescence is mainly localized along the entire perimeter of the parasites;3): coarse-speckled cytoplasmatic fluorescence;4): flagellar pattern, in which the fluorescence is localized exclusively onto the flagellum;5): punctiform pattern, in which the fluorescence is localized exclusively at the basis of the flagellum;6): nuclear pattern, in which only the nucleus of the parasite shows a homogeneous green fluorescent. The significance of each pattern is discussed.
基金supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico(CNPq-grant number 424820/2016-1)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior(CAPES)+1 种基金Edital PAPQ PROPESP-UFPAthe Instituto Nacional de Biologia Estrutural e Bioimagem-INBEB(CNPq-grant number 465395/2014)
文摘Objective: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infanturn proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. Methods: I., infantum promastigotes were treated with 50 and 100 μg/mL AEPa for 72 h and then antipro- mastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. Results: Treatment with AEPa reduced the numbers ofL infantum promastigotes (50% inhibitory concen- tration (ICso) = 65.9μg/mL; selectivity index (SI)= 22.1) and amastigotes (ICso = 37.9 μg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2μg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 μg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration 〉1000μg/mL). Conclusion: AEPa presented antileishmanial activity against the promastigotes and amastigotes of I.. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.
基金This publication is based on work supported by a grant(GTRX-14-60403-0)from the U.S.Civilian Research&Development Foundation(CRDF Global)with funding from the United States Department of StateThe opinions,findings,and conclusions stated herein are those of the author(s)and do not necessarily reflect those of CRDF Global or the United States Department of State.
文摘Background:Leishmania infantum is the causative agent of human visceral leishmaniasis(VL)and sporadic human cutaneous leishmaniasis(CL)in the Mediterranean region.The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations.The main objective of this study was to explore the genetic polymorphism in L.infantum isolates from human and animal hosts in different regions of Morocco.Methods:The intraspecific genetic variability of 40 Moroccan L.infantum MON-1 strains isolated from patients with VL(n=31)and CL(n=2)and from dogs(n=7)was evaluated by PCR-RFLP of nagt,a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase.For a more complete analysis of L.infantum polymorphism,we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database.Results:Moroccan L.infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified.Three of the six genotypes were exclusively identified in the Moroccan population of L.infantum:variant M1(15%),variant M2(7.5%),and variant M3(2.5%).The most common genotype(65%),variant 2(2.5%),and variant 4(7.5%),were previously described in several countries with endemic leishmaniasis.Phylogenetic analysis segregated our L.infantum population into two distinct clusters,whereas variant M2 was clearly distinguished from both cluster I and cluster II.This distribution highlights the degree of genetic variability among the Moroccan L.infantum population.Conclusion:The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L.infantum strains isolated from human and canine reservoirs with 6 genotypes identified.Three of the six Moroccan nagt genotypes,have not been previously described and support the particular genetic diversity of the Moroccan L.infantum population reported in other studies.