Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

A Case of Post Kala-Azar Dermal Leishmaniasis in India

Post kala-azar dermal leishmaniasis (PKDL) is a rare disease. This is a solitary case report from Orissa, India. We describe a case of PKDL in a 55-year-old male who presented with multiple nodular lesions over face, trunk, and extremities. The patient had been to an endemic area of kala-azar and had a previous history of leishmaniasis. Fine needle aspiration cytology samples from skin nodules revealed Leishmania amastigotes.     

Splenic lesions: FNA findings in 48 cases

OBJECTIVE: To study fine needle aspiration (FNA) cytological findings of splenic lesions and assess the role of FNA in the diagnosis of splenomegaly or splenic tumours. METHODS: This study consisted of 48 cases, 25 males and 23 females. The ages ranged between 3 and 71 years. Most of these cases were aspirated under ultrasonographic guidance and a small number were also aspirated directly by using a 22- to 23-gauge needles. The smears were stained with Wright-Giemsa and Papanicolaou methods. Special stains were used whenever necessary. RESULTS: In this study 14 cases were diagnosed as lymphoma-leukaemia, 7 cases as tuberculosis, 12 cases as kala-azar, 2 cases as hydatid cyst, 3 cases as storage diseases, 3 cases as simple cyst, 2 cases as myeloproliferative disorders, 2 cases as malignant tumours and 3 cases as hamartomas (these were wrongly diagnosed as malignant tumours). CONCLUSION: Splenic aspiration is a safe procedure and is very useful in the diagnosis of parasitic and infectious diseases, especially in endemic countries like Iran.     

Cytokines in the differentiation of Th1/Th2 CD4+ subsets in leishmaniasis

Leishmania major infect only macrophages in the host, where they reside in endolysosomal compartments into which MHC class II molecules co-localize. Experimental infection in mice has provided a useful model for the differentiation of Th1 CD4+ effector lymphocytes that are required for the generation of IFN-γ that activates the macrophage to a microbicidal state. Genetically susceptible BALB/c mice aberrantly activate Th2 CD4+ effector cells that are ineffective in arresting infection. Increasing evidence suggests that, rather than discrete parasite antigens or MHC molecules, cytokines mediate the critical decision in the developmental switch to either the Th1 or Th2 effector phenotype.     

Biochemical and immunological characterization of exometabolites from an Indian strain of Leishmania donovani promastigotes grown in a chemically defined medium

Summary Exometabolites (EXOM) of an Indian strain of Leishmania donovani promastigotes isolated from a chemically defined medium by ultrafiltration consisted of proteins, glycoproteins, lipid and lipophosphopolysaccharide (LPPS). LPPS of Mr 40-28 kDa in SDS-PAGE could be labelled metabolically with [³²P]-phosphate and recovered in the aqueous phase of hot-phenol-water extraction of EXOM (PE-Aq) along with a glycoprotein of Mr 150-130 kDa (GP_150-130) . These two molecules could be eluted from DE-52 column with 200 mM NaCI (D₂). The 300 mM NaCl (D₃) and 400 mM NaCl (D4) eluates from DE-52 column contained one unsaturated polar lipid component. The LPPS had Rf value of 0.65–0.75 in Thin Layer Chromatography (TLC) using saturated phenol water solvent system. EXOM revealed 15 bands in SDS-PAGE of which proteins of Mr 84, 66, 56, 50 and 29 kDa were prominent. When EXOM were fractionated through Con A — Sepharose column, the fraction eluted with -methyl-D-mannoside (Con A-E) had seven bands as revealed by SDS-PAGE of which 25, 16, 13 and 12 kDa glycoproteins were prominent.The antigens present in EXOM can be classified as slower anodic migrating and faster anodic migrating antigens as revealed by immunoelectrophoresis (IEP). The slower anodic migrating antigens, LPPS and GP_150-130 recovered in PE-Aq and D₂ did not cross-react with kala- azar patients' sera but cross-reacted with homologous anti-promastigote sera. Two faster anodic migrating antigens which could be recovered in organic phase of hot phenol extraction of EXOM (PE-O) and eluted in D₃ and D₄ and Con A-E, cross-reacted with kala-azar patients' sera. The antigens of both the classes were sensitive to periodic acid oxidation.     

Leishmania donovani: A Chemically Defined Medium Suitable for Cultivation and Cloning of Promastigotes and Transformation of Amastigotes to Promastigotes

A chemically defined medium using commercially available α-MEM supplemented with HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 × 10⁷/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients’bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16–20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.     

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar,India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.