الملخص
ObjectiveTo determine the virulence of desert-type Leishmania donovani strains through animal infection experiments and to explore preservation methods for maintaining their pathogenicity.Methods The isolated strain was cultured in vitro for 7, 30, 36, 44, 60, 90, and 150 days, respectively， and inoculated into Lagurus lagurus (L.lagurus) with the dose of 2.6×105 per animal by intraperitoneal injection. The spleen coefficient, infection rate, and antibody positive rate of the inoculated animals were detected at day 60 after infection. The desert-type Leishmania donovani strain was further inoculated with Cricetulus migratorius (C.migratorius) and L. lagurus, respectively, for passaging and preservation. The survival time of two kinds of animals andpathogenicity change of the stain in their bodies were compared.ResultsAfter inoculation of desert-type Leishmania donovani strains cultured in vitro for 7-150 days, the spleen coefficient of inoculated L.lagurus gradually increased from 1% on day 7 to 2.2% on day 30, which was more than 10 times of the normal spleen coefficient. Additionally, on day 60, the spleen coefficient remained 3 times higher than the normal value. The infection rate and antibody positive rate decreased from 80% on day 7 to 0% on day 60. At 90 days, there were no significant differences between the infected groups and the control group, and all the observed indexes were within the normal range. The survival time of L.lagurus infected with the in vivo passage strain ranged from 1 to 13 months, and half of the infected individuals died within 4 months. In contrast, C.migratorius had a survival time ranging from 5 to 31 months, and half of the infected individuals died within an average of 13.7 months. There was a significant difference in the average time of death between the two groups (t=0.000 1, P<0.001), but no significant difference in spleen coefficient (t=0.990, P>0.05). This strain exhibited equal virulence in both animals and remained virulent for up to 4 years after continuous passage.ConclusionWith the prolonged culture time, the virulence of the strain decreases gradually. At 90 d, it has no pathogenicity to L. lagurus. Long-term in vitro culture fails to preserve it's pathogenicity to L.lagurus. Only in vivo inoculation can maintain the virulence of this strain.
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Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of ～65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN-γ, TNF-α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.
الملخص
Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.
الملخص
Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of 65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN-γ, TNF-α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.
الملخص
Leishmania donovani is one of the causative agents of visceral leishmaniasis. The immune response against Leishmania depends on CD4+ T helper type 1 cells. The immune system is unable to combat Leishmania because the parasite can exert several immune suppressive mechanisms that facilitate escaping the immune responses. One of these mechanisms is the up-regulation of programmed death-1/programmed death ligand-1 pathway which causes T cells to undergo exhaustion. Autophagy is strongly linked to the immune response, with some research indicating that activating autophagy reduces the immune response to some intracellular pathogens, while others indicate that activating autophagy limits the growth of intracellular pathogens. Leishmania was found to subvert the host defense mechanisms for its own persistence, such as Leishmania-induced autophagy modulation. Leishmania was reported to activate autophagy in different studies, thus getting a dual benefit by evading the immune system and simultaneously utilizing the autophagy byproducts as nutrients. In this review, we introduced different immune evasion/suppressive mechanisms used by Leishmania, and different immunotherapies which were developed accordingly. We focused on the programmed death-1/programmed death ligand-1 pathway as well as autophagy with the potential interplay of both mechanisms.
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Background: Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. Aim: The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. Methods: We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Results: Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1–99.1) and 98.1% (95% confidence interval: 90.1–99.9), and 98.5% (95% CI: 92.4–99.9) and 98.1% (95% CI: 90.1–99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. Limitations: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Conclusion: Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.
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Objective To analyze the epidemiological and clinical data of an imported case of visceral leishmaniasis in Henan Province, and explore the method of laboratory diagnosis of kala-azar. Methods The epidemiological and clinical data of an imported visceral leishmaniasis patient were analyzed. Leishmania donovani bodies in bone marrow smears were observed microscopically. The antibody was detected by using rK39 dipstick test strips. Two pairs of specific primers, K13A-K13B and LITSR-L5.8S, were used to amplify kinetoplast DNA and internal transcribed spacer of rDNA of the parasite, respectively. Results The patient had been in the epidemic area of visceral leishmaniasis, and had symptoms such as irregular fever, splenomegaly, pancytopenia, and inversed ratio of albumin and globulin. The amastigotes of L. donovani were found in the bone marrow smears, and rK39 test strip was positive, and the PCR products of K13A-K13B and LITSR-L5.8S were 87 bp and 285 bp respectively. The similarities of the two fragment sequences to the corresponding sequences of L. donovani were 94% and 100%, respectively. Conclusion The case is diagnosed as visceral leishmaniasis according to the epidemiological data, clinical manifestations and laboratory test results of the patient, and the pathogen is L. donovani.
الملخص
Objective To analyze the epidemiological and clinical data of an imported case of visceral leishmaniasis in Henan Province, and explore the method of laboratory diagnosis of kala-azar. Methods The epidemiological and clinical data of an imported visceral leishmaniasis patient were analyzed. Leishmania donovani bodies in bone marrow smears were observed microscopically. The antibody was detected by using rK39 dipstick test strips. Two pairs of specific primers, K13A-K13B and LITSR-L5.8S, were used to amplify kinetoplast DNA and internal transcribed spacer of rDNA of the parasite, respectively. Results The patient had been in the epidemic area of visceral leishmaniasis, and had symptoms such as irregular fever, splenomegaly, pancytopenia, and inversed ratio of albumin and globulin. The amastigotes of L. donovani were found in the bone marrow smears, and rK39 test strip was positive, and the PCR products of K13A-K13B and LITSR-L5.8S were 87 bp and 285 bp respectively. The similarities of the two fragment sequences to the corresponding sequences of L. donovani were 94% and 100%, respectively. Conclusion The case is diagnosed as visceral leishmaniasis according to the epidemiological data, clinical manifestations and laboratory test results of the patient, and the pathogen is L. donovani.
الملخص
Aims: This study investigates the activity of tetracyclic iridoid compounds against Leishmania spp. and the mechanism(s) of action. Study Design: An experimental study. Place and Duration: Department of Parasitology, Noguchi Memorial Institute for Medical Research, between September 2017 and July 2018. Methodology: The 50 % inhibitory concentration (IC50) of compounds against Leishmania donovani and L. major promastigotes were determined after 48 hours of incubation using the Alamar blue. Cytotoxicity of compounds was determined against cell lines using MTT assay. The anti-amastigote activity of compounds was further assessed by DAPI (4′,6-diamidino-2-phenylindole) staining. The mechanism of cell death induced by compounds was determined using nexin assay. Mitosis, cytokinesis and morphometry were monitored by DAPI and Kinetoplastid Membrane Protein (KMP) staining. Cell cycle arrest induced by compounds was analyzed by FACS. Results: Molucidin and ML-F52 inhibited the growth of promastigote in L. donovani (Molucidin; IC50 = 2.94±0.60 µM, ML-F52; IC50 = 0.91±0.50 µM) and L. major (Molucidin; IC50 = 1.85± 0.20 µM, ML-F52; IC50 = 1.77± 0.20 µM). ML-F52 had a 10-fold cytotoxic effect on parasites relative to normal cell lines. Against intracellular forms, Molucidin and ML-F52 inhibited intracellular amastigote replication and infectivity. Amphotericin B, Molucidin and ML-F52, induced a dose-dependent apoptotic effect on promastigotes. Although no change in KMP-11 expression was observed, iridoids inhibited cell division and morphological changes in promastigote cultures. Molucidin and ML-F52 induced apoptotic mechanism of cell death, inhibited cytokinesis and induced phenotypic changes in promastigotes. Molucidin further induced ‘’nectomonad-like’’ forms and loss of kDNA, ML-F52 induced ‘cell-rounding’ with loss of flagellum. Molucidin also induced cell growth arrest at G2-M phase (54.5 %). A significant induction of apoptosis (P = .05) was shown by an enhanced peak in the sub-G1 confirming the apoptotic inducing properties of iridoids. Conclusion: This study shows the anti-leishmania activity of tetracyclic iridoids which could be further investigated for the development of new chemotherapy against Leishmaniasis.
الملخص
Cutaneous leishmaniasis (CL) caused by Leishmania donovani is an endemic vector-borne disease in Sri Lanka. Over 2,500 cases have been reported since 2000 and the number of CL cases has dramatically increased annually. Total 57 clinically suspected CL patients attending the dermatology clinic in Anuradhapura Teaching Hospital were recruited from January to June 2015. Slit skin smears and skin biopsies were taken from each of the subjects. Clinical and epidemiological data were obtained using interviewer administered questionnaire. Forty-three (75.4%) patients among 57 were confirmed positive for L. donovani. The majority of infected patients was males (P=0.005), and the most affected age group was 21–40 years. Soldiers in security forces, farmers, and housewives were identified as high risk groups. The presence of scrub jungles around the residence or places of occupation (P=0.003), the presence of sandflies (P=0.021), and working outsides more than 6 hr per day (P=0.001) were significantly associated with CL. The number of lesions ranged from 1–3, and the majority (76%) of the patients had a single lesion. Upper and lower extremities were the prominent places of lesions, while the wet type of lesions were more prevalent in females (P=0.022). A nodular-ulcerative type lesion was common in both sexes. The presence of sandflies, scrub jungles, and outdoor activities contributed to spread of Leishmania parasites in an endemic pattern. Implementation of vector control programs together with health education with regard to transmission and prevention of CL are necessary to control the spread of this infection.