Preservation of Leishmania donovani promastigotes in blood agar slants

Long-term cultivation of Leishmania promastigotes by weekly passage to fresh medium was reported to be disadvantageous because needs labor, risk of contamination, lowering in infectivity and virulence pattern. Cryopreservation and Lyophilization require expensive facilities which could be a burden and unaffordable to most laboratories of developing countries where the disease is endemic. These problems could be minimized by simple preservation of Leishmania donovani promastigotes in blood agar slants at 7-8 degrees C for 6-7 months. The preserved promastigotes were examined for viability up to one year at a regular interval of one month. Viable promastigotes were found and revived successfully from all the slants stored up to 7 months after that, the viability of promastigotes was found to be decreased in the slants of 8-9 month storage. No viable promastigotes were recovered from the slants stored up to 11-12 months. By this method, the promastigotes can easily be stored up to 7 months without loss of biological activity. The number of passage of promastigotes to fresh medium has been greatly reduced by this method from 30 times to 01 when compared with weekly passage in liquid medium. This simple and economical method can be recommended for short storage of Leishmania culture without loss of any activity.     

Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis

During a 20-month period, more than 500 splenic aspirations were performed in 89 patients with suspected or proven visceral leishmaniasis. The two complications which occurred (intra-abdominal bleeding and penetration of the intestine in one patient each) both resolved with conservative management. Parasite density in splenic aspirate smears was graded on a logarithmic scale from 0 (no parasites in 1,000 microscopic fields) to 6+ (greater than 100 parasites per microscopic field). Among 46 newly diagnosed and 17 relapsed or drug-resistant patients with visceral leishmaniasis, the average initial parasite grade was 4.35 +/- 0.92 (mean +/- SD) and 4.15 +/- 1.37, respectively. The grading system was useful in measuring the speed of response to treatment, and in distinguishing slow responders from nonresponders. This was especially valuable for managing patients with drug-resistant visceral leishmaniasis. The system also provided a means of comparing the efficacy of different treatment regimens, and for calculating the optimum duration of treatment.     

Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors

Human visceral leishmaniasis (kala-azar) transmitted by blood transfusion has been described in previous reports. Seroprevalence of antibodies to Leishmania donovani was shown to be related to prior blood transfusions in multiply transfused hemodialysis patients in Natal, Rio Grande do Norte, Brazil. In this study, a possible correlation between seroreactivity and the presence of L. donovani DNA was investigated in asymptomatic healthy blood donors. Sera were tested using the fucose mannose ligand (FML) ELISA, which was shown to have a sensitivity of 100%, a specificity of 96-100%, reliability, and diagnostic and prognostic potential for the detection of human and canine kala-azar, respectively. Leishmanial DNA was assessed by the polymerase chain reaction (PCR) and dot-blot hybridization techniques in blood and bone marrow samples. Among 21 FML-seroreactive asymptomatic blood donors, 5 (24%) were positive by the PCR and 9 (43%) were positive in a dot-blot assay of blood samples, showing a significant correlation (chi2 = 14.24, P < 0.01). No Leishmania DNA was detected in 20 FML non-reactive blood donors. Our results point to the need for control of transmission of kala-azar by blood transfusion in areas endemic for this disease.     

Plasma membrane of Leishmania donovani.

Plasma membrane of Leishmania donovani promastigotes was isolated by disrupting the cells in Dounce homogenizer and found to be having two fractions M1 and M2. Chemical analysis of the two membrane fractions revealed that M1 had less RNA content and high sterol-phospholipid molar ratio than M2. M1 was also rich in membrane marker enzymes, e.g., 5' nucleotidase and acid phosphatase. Glucose-6-phosphatase, the marker enzyme of endoplasmic reticulum was higher in M2 fraction. The electron micrograph also revealed the presence of plasma membrane vesicles in M1 fraction.     

Concurrent human infection with Leishmania donovani and Leishmania braziliensis braziliensis

In the suburban district of Campo Grande, Municipality of Rio de Janeiro (Brazil) cases of both Leishmania domovani and L. b. braziliensis infections occur. In March 1982 we examined a white male child, five years old, with fever, weight loss and distended abdomen, symptoms said to have started three months before. After the first month a papular lesion appeared on his forehead, rapidly increasing in size and becoming ulcerated: Parasites were isolated from both the ulcer and the bone marrow by culturing in BHI-agar with rabbit blood and a liquid overlay of modified LIT medium. Using the techniques of zymodeme analysis, schizodeme analysis and serodeme analysis using monoclonal antibodies, the parasite from the ulcer was identified as L. b. braziliensis and that from the bone marrow as L. donovani. This seems to be the first described case of concurrent infection of man with a visceral and cutaneous Leishmania. It indicates that, at least in humans, a previous infection with L. donovani does not protect against L. b. braziliensis. This result has important implications for the development of vaccines against leishmaniases.     

Inhibition of interferon-gamma signaling by Leishmania donovani

Leishmania infection causes marked down-regulation of interferon (IFN)-gamma-induced gene activity in macrophages, but the mechanism of the blockade has not been fully defined. The IFN-gamma signal transduction pathway was analyzed in Leishmania donovani-infected phorbol-differentiated U937 human promonocytic cells. IFN-gamma stimulation induced marked phosphorylation of its own receptor (IFN-gammaR)-alpha chain. Phosphorylation of the receptor subunit was significantly inhibited after 24 h of infection with the parasite, apparently because of decreased amounts of the receptor subunit. Formation of the IFN-gammaR complex, as assessed by tyrosine phosphorylation and association of Jak2, was strongly inhibited in cells infected for 24 h. Inhibition of the IFN-gammaR complex formation correlated with inhibition of STAT1alpha binding to the IFN-gamma response region. Pretreatment with purified parasite lipophosphoglycan before IFN-gamma stimulation had no effect on tyrosine phosphorylation. Thus, inhibition of tyrosine phosphorylation of the IFN-gammaR-alpha chain and subsequent signal transduction are most likely due to the decreased amount of IFN-gammaR-alpha protein after infection.     

Characterization of natural antimony resistance in Leishmania donovani isolates

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of visceral leishmaniasis in India. However, mechanisms of natural resistance are unclear. In this study, we observed that Leishmania donovani clinical isolates not responsive to sodium stibogluconate showed resistance to antimony treatment in both in vitro and in vivo laboratory conditions. The resistant isolates have increased levels of intracellular thiols. This increase in thiol levels was not mediated by the amplification of gamma-glutamylcysteine synthetase, but was accompanied by amplification of trypanothione reductase and an intracellular ATP-binding cassette transporter gene MRPA. The resistance of parasites to antimony could be reversed by the glutathione biosynthesis-specific inhibitor, buthionine sulfoximine, which resulted in increased drug susceptibility. These results suggest the possible role of thiols and MRPA in antimony resistance in field isolates.     

Host immunoglobulin on spleen-derived Leishmania donovani amastigotes

Leishmania donovani, an intracellular pathogen in vertebrates, is found as an amastigote within cells of reticuloendothelial origin. The spleens of experimentally infected Syrian hamsters are frequently used as a source of amastigotes for studies of host-parasite interactions, but amastigotes isolated from hamster spleens have been found to have surface-bound host immunoglobulins, which may confound interpretation of such studies.     

Membrane characterization of amastigote-like forms of Leishmania donovani

During in vitro transformation, Leishmania donovani promastigotes converted into amastigote‐like forms and underwent several changes in membrane parameters. They exhibited significantly increased microviscosity comparable to true amastigotes. Activities of several functionally important membrane bound enzymes were also altered, thereby indicating a change in their orientation. Peanut agglutinin was found to be specific for agglutination of stationary phase promastigotes whereas wheat‐germ agglutinin was specific for the amastigote‐like forms as well as for pure amastigotes, implying the presence of specific glycoconjugates on the parasite surface.     

Adoptive transfer of vaccine-induced resistance to Leishmania donovani

BALB/c mice were immunized with three subcutaneous injections combining killed parasites and glucan, or were untreated. Spleen cells were transferred to syngeneic recipients. Mice which received 5 X 10(8) spleen cells from vaccinated donors demonstrated significant protection against Leishmania donovani challenge as compared to untreated mice receiving immune sera, or mice which received untreated spleen cells.     

pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.     

Screening colonoscopy in asymptomatic average-risk persons with negative fecal occult blood tests.

Colonoscopy was performed on 210 asymptomatic average-risk persons, aged 50-75 years, who had negative fecal occult blood test results. Colonoscopy was complete to the cecum in 209 subjects. Fifty-three subjects (25%) had adenomas and two had cancer. All of the adenomas greater than or equal to 1 cm in size and both cancers occurred in subjects aged greater than or equal to 60 years. Fifty-one percent of subjects with adenomas and one with cancer had no neoplasms distal to the sigmoid-descending colon junction. One subject had a major postpolypectomy hemorrhage that stopped spontaneously. Screening colonoscopy, therefore, has a high yield for detection of neoplasms in asymptomatic average-risk persons aged greater than or equal to 60 years with negative fecal occult blood test results. The yield is low in persons aged 50-54 years and intermediate in persons aged 55-59 years.     

Occurrence of Leishmania donovani parasitemia in plasma of infected hamsters

Intracardiac transfusion of plasma, mononuclear cell fraction and blood of infected hamster donors induced visceral leishmaniasis in normal hamster receptors. At the moment of transfusion, the donors already showed all the typical signs of the disease: ascites, cachexia, as well as splenomegaly and a high parasite load in the spleen and liver. All transfused hamsters developed typical visceral leishmaniasis between 90 and 120 days, indicating that all blood products were infectious. Transfusion of the mononuclear cell fraction induced the highest values of parasitic load (spleen, 766 Leishman Donovan Units (LDU); liver, 2650 LDU), splenomegaly and hepatomegaly (spleen-liver/body relative weight: 1.130 and 6.870, respectively). Animals that received the plasma fraction also developed visceral leishmaniasis, showing similar parasitic load (spleen, 107 LDU; liver, 220 LDU) and spleen-liver/body relative weight (1.005 and 6.35, respectively) than those transfused with whole blood. The finding of typical Leishmania donovani infection in animals transfused with plasma demonstrates the possibility of the extracellular location of parasites, free in this blood fraction deprived of red and white blood cells. Fluorescence-assisted cell sorter analysis (FACS) of plasma showed the presence of particles corresponding in size to amastigotes, which fluoresced strongly with the serum of a patient with Kala-azar (73%), but not with normal serum.