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.     

Identification of immune complex antigens in sera of Indian kala-azar patients

Level of circulating immune complex (IC) in visceral leishmaniasis is much higher than that in control sera. In immunoblot experiment, treatment of kala-azar IC with patient sera showed at least 6 bands of which the band around 55 kDa region was most prominent. The band at 55 kDa is primarily due to the presence of an antigen recognized by its corresponding antibody present in the patient sera. This was confirmed by using radiolabelled antibody from kala-azar patient serum and antipromastigote serum. The heavy chain of IgG originating from IC is also present in the same region which was detected by its recognition with antihuman IgG. The IC gave a band at 55 kDa region with sea-urchin antitubulin. Kala-azar sera also reacted with purified rat brain tubulin. The present results suggest that a tubulin like protein is present at 55 kDa region along with the heavy chain of IgG.     

Variation of antigenicity and serological reaction to Pneumocystis carinii in Korea.

The present study observed the variation of antigenicity of Pneumocystis carinii and serum IgG antibody reaction to the antigens from different localities in Korea. Antigens of rat P. carinii and sera of inhabitants were collected at Chunchon. Chungju, Kwangju, and Seoul during 1995-1996. Enzyme-linked Immunosorbent Assay and immunoblot were used for immune reaction. Absorbance of 1,294 human sera ranged between 0.01 and 0.93. Sera from Chunchon showed higher absorbances than those from other areas. Immunoblotting revealed IgG antibody reactions to 116, 100, and 45-55 kDa antigenic bands of rat P. carinii, but the frequencies of positive reaction to individual bands were variable by localities. Total 62.6% of the sera showed the reaction to 116 kDa band while 37.7% reacted to 100 kDa band and 32.0% to 45-55 kDa bands. For the reaction to 116 kDa, the reaction rate was 60.0% to 82.6% by localities. It is found that the reaction rates of the human sera to rat P. carinii antigen are variable according to the localities. Also, the high molecular antigen of 116 kDa of rat P. carinii is the most frequent antigenic band reacting to human sera.     

Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation

Aspergillus fumigatus is a highly pathogenic fungus causing a wide spectrum of diseases in immunocompromised as well as immunocompetent hosts. The present work was undertaken to evaluate the cytotoxic nature of fractionated antigens of A. fumigatus against the mammalian cell lines (J774, RAW, CHO and L929). An enriched protein antigenic fraction of A. fumigatus was subjected to con A Sepharose and phenyl Sepharose chromatography. Antigenic fractions, ConAub (conA unbound) and PSC III (fraction III of phenyl Sepharose column) containing low mw antigens showed higher cytotoxicity as compared to other antigenic fractions. PSC III was further purified on HPLC resulting in an 18 kDa homogeneous protein. The purified protein showed high ELISA absorbance values for specific IgG and IgE antibodies in sera of ABPA patients. Monoclonal antibody raised against Asp fl, a major allergen/antigen of A. fumigatus recognised the purified 18 kDa by ELISA and western blot. The 18 kDa allergen/antigen or Asp fl showed similar toxicity towards all the four cell lines (macrophage and fibroblast) with an IC50 of 75 ng/ml or 4.16 nM. Reduction in toxicity of 18 kDa at low temperatures and potentiation in presence of ammonium chloride and monensin indicates mechanism of internalisation of 18 kDa in eukaryotic cells is similar to -sarcin. The present work shows that the 18 kDa allergen/antigen (Asp fl) is a major cytotoxin secreted by A. fumigatus which may play multiple roles in the pathogenesis of Aspergillosis through allergenicity, antigenicity and cytotoxicity. (Mol Cell Biochem 167: 89-97, 1997)     

A monoclonal antibody monitoring band 3 modifications in human red blood cells

Morphologic and methabolic erythrocyte modifications are thought to be the basis of cell removal from circulating blood. A significant role has been ascribed to the immunological network which may remove aged or misshapen erythrocytes through the binding of specific autoantibodies. Along this line recent observations indicate that a senescence antigen appears in consequence of postsynthetic modifications of band 3, one of the most important erythrocyte membrane proteins, which accounts for many functional activities of the red cells. On this basis, we raised a mouse hybridoma anti-band 3 monoclonal antibody (B6 MoAb) of the IgG2a class which monitors band 3 differences among normal red blood cells separated by Percoll density gradient. These differences are outlined by the decrease of B6 MoAb binding to band 3 monomer, the appearance of an 80–90 kDa new band, lighter than band 3, and the increase of low molecular weight fragments in the 4.5 region. The B6 MoAb appears to be very useful in detecting modifications of band 3 since it bind to a 19 kDa Chy-Try fragment estimated to be sensitive to aging.Abbreviations PBS Phosphate Buffer Saline - MoAb Monoclonal Antibody - RBCs Red Blood Cells - PMSF Phenylmethylsulphonyl Fluoride - PVC Polyvinyl Chloride - ACD Acid Citrate Dextrose - HMWP High Molecular Weight Polymers - Chy-Try Chymotrypsin-Trypsin Digested - i.p. intraperitoneum - ELISA Enzyme Linked Immuno Sorbent Assay - Hepes 4-(2-Hydroxyethyl)-piperazine-1-ethane-sulfonic acid. Enzymes: trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), neuraminidase (EC 3.2.1.18)     

Expression of antigens in virulent and avirulent Indian strains ofLeishmania donovani

Indo-Gen mediated surface labelling with¹²⁵I demonstrated differences in surface oriented antigens between virulent and virulent promastigote ofLeishniania donovani, In case of virulent strains, surface polypeptides with molecular masses of 63, 53, 42 and 38 kDa were found to be labelled with¹²⁵I whereas in the case of aviralent stains 68, 55, 50, 46, 42 and 33 Da, components were iodinated. Further studies by immunoblot assay using different subcellular fractions of virulent and avirulent parasites demonstrated that antibody raised against gp63 cross-reacted with the 63 and 60 kDa antigen of the virulent and avirulentLeishmania donovani strains of Indian origin respectively. It indicates that these two polypeptides are antigenically similar. When virulent and avirulent cells were grown in the presence of varying concentration of tunicarnycin and immunoblot with anti gp63, it was observed that with increasing concentration of tunicamycin the 63 kDa polypeptide of the virulent cells shifted to approximately 58–57 kDa and the 60 kDa polypoptide of the aviruleni cells shifted to 57 kDa. This suggests that glycosylation may play an important role in antigenic variation between virulent and avirulent parasites.     

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.     

Seroprevalence of toxocariasis among healthy people with eosinophilia

The aim of this study is to determine the Toxocara seropositive rate among healthy people with eosinophilia. A total of 97 people residing in Seoul who were healthy and whose blood eosinophilia was over 10%, as shown by regular health check-ups in 2004, were subjected to this study. Their sera were tested by immunoblotting and ELISA with the antigen of larval Toxocara canis excretory-secretory (ES) protein. Sixty-five sera were band-positive (67.0%). The seropositve control sera were positive to band sizes of 66 kDa, 56 kDa, 32 kDa, and 13 kDa. In ELISA, 63 sera (65.0%) were positive to T. canis ES protein. There was no significant correlation between the IgG ELISA titer and the level of eosinophilia (r = 0.156, P = 0.156). As there were insufficient data to determine whether there were cross-reactions with other helminthic infections, or whether atopy occurred, further studies are required to verify the cause of the seropositive reactions against T. canis ES antigen. Toxocariasis seropositivity is suggested to be the major cause of eosinophilia, since the Toxocara seroprevalence among Korean rural adults was shown to be approximately 5%.     

C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen

Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the subunit of soybean -conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first -sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.Abbreviations Ara h 1 Arachis hypogaea allergen 1 - Ara h 3 Arachis hypogaea allergen 3 - BCA Bicinchoninic acid - Gly m Bd 28 K Glycine max band 28 kDa allergen - Gly m Bd 30 K Glycine max band 30 kDa allergen - Gly m Bd 68 K Glycine max band 68 kDa allergen - IgE Immunoglobulin E     

Inhibition of the photosynthetic electron transport of isolated thylakoids by hemolyzed rabbit sera. Evidence for the potential involvement of parallel electron transport in photosystem I Mehler reactions

The inhibition patterns of rabbit sera (RS1 & RS2) from two different rabbits on the photosynthetic electron transport of isolated spinach thylakoids were studied. Fifty l of RSI were required for 100% inhibition of a H₂O MV/O₂ reaction, while only 10 l of a 1:10 dilution of RS2 were needed for 100% inhibition. The RS2 serum was greatly hemolyzed. The -globulin fraction from purified rabbit serum (RS1) did not inhibit photosynthetic electron transport, indicating that the antibody fraction of the rabbit serum does not contain the inhibitor. It appears that the inhibitor is from the hemolyzed red blood cells. Rabbit sera added prior to chloroplast illumination caused no inhibition, while addition of rabbit sera during illumination inhibited a H₂O MV/O₂ reaction within 1–3s. Aminotriazole, a catalase inhibitor, did not affect the efficacy of the rabbit sera indicating that the unknown rabbit serum inhibitor is not catalase. Various Hill reactions were employed to determine the site of inhibition. Rabbit sera inhibited the following reactions: DHQ/DCMU MV/O₂, DAD/Asc/DBMIB MV/O₂, and DCIP/Asc/DBMIB MV/O₂. Rabbit sera did not inhibit a H₂O DAD_ox reaction indicating that inhibition is on the reducing side of PSI. However, a H₂O Fd/NADP⁺ reaction was not inhibited by rabbit sera. NADP did not interfere with the ability of RS2 to inhibit a MV-mediated Mehler reaction. In simultaneously measured assays of Fd-mediated O₂ and NADP⁺ reductions, RS2 serum inhibited the reduction of O₂ by ferredoxin without inhibiting the reduction of NADP⁺. These results indicate the potential involvement of parallel (branched) electron transport of the reducing side of PSI in the reduction of oxygen.Abbreviations RS1 and RS2 Rabbit serum 1 and 2 - MV methylviologen - DCMU 3,4-dichlorophenyl-N,N-dimethylurea - KFeCN potassium ferricyanide - DCIP dichlorophenolindolphenol - DAD 2,3,5,6-tetramethyl-p-phenylenediamine - DHQ tetramethyl-p-hydroquinone (durohydroquinone) - MES [2-(N-morpholino)-esthanesulfonic acid] - HEPES [N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid] - DBMIB dibromothymoquinone - PSI and PSII photosystem I and II - Fd ferredoxin - Chl chlorophyll - Asc ascorbate - SOD superoxide dismutase     

Adult Brugia malayi 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha

We earlier reported a sizeable protection conferred by ‘mitochondria rich’ (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of  34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-α, IFN-γ and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that  34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.     

Linkage mapping of quantitative trait loci controlling seed weight in pea (Pisum sativum L.)

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F₂ progeny of a cross between two cultivated types (Primo and OSU442-15) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (Slow) and a P. sativum ssp. humile accession (JI1794). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the Primo x OSU442-15 cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the JI1794 x Slow cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the Primo x OSU442-15 and JI1794 x Slow crosses.     

IgG and IgE immune response against the surface glycoprotein gp200 of Saccharomyces cerevisiae in patients with atopic dermatitis

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73 %, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis. Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicansallergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn. This revised version was published online in August 2006 with corrections to the Cover Date.     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues

A new polyclonal antibody to the humanerythrocyte urea transporter UT-B detects a broad band between 45 and65 kDa in human erythrocytes and between 37 and 51 kDa in raterythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk)antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band.However, in Jk null erythrocytes [Jk(ab)], only a faint band at55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and ratkidney show UT-B staining in nonfenestrated endothelial cells indescending vasa recta. UT-B protein and mRNA are detected in rat brain,colon, heart, liver, lung, and testis. When kidney medulla or liverproteins are analyzed with the use of a native gel, only a singleprotein band is detected. UT-B protein is detected in cultured bovineendothelial cells. We conclude that UT-B protein is expressed in morerat tissues than previously reported, as well as in erythrocytes.

     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis

Although the existence of O-acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an 2 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9-O-AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients. Published in 2004..     

Chromosome profile ofLeishmania donovani: Interstrain and interspecific variations

The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.     

Expression and characterization of a parasite-specific antigen on macrophages after infection withLeishmania donovani

A rabbit polyclonal antibody to crude soluble antigen ofLeishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infectedin vitro. The determinant was recognized on infected macrophage surface only when F (ab)₂ fragments of anti-leishmanial antiserum was employed in immunofluorescence. F(ab)₂ fragments of human patient sera also could recognize the determinant. The expression of this antigen was not stage-specific for the parasite. Immunochemical analyses revealed this antigen to be of 51 kDa protein. Specific leaching of membrane proteins by trypsin showed three bands of expressed antigens of 26, 11 and 10 kDa, which in all likelihood might be arising from the 51 kDa antigen. The antigen was not expressed until 12 h of post infection, reached a maximum level at 24 h and thereafter attained a steady state level as studied upto 96 h of post infection. This typc of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.     

How Far Are We from Visceral Leishmaniasis Elimination in Bangladesh? An Assessment of Epidemiological Surveillance Data

Introduction

In 2005, Bangladesh, India, and Nepal joined forces to eliminate Visceral Leishmaniasis (or kala-azar) from the region by 2015. In Bangladesh the elimination target is set at less than one new case per 10,000 population per year at upazila (sub-district) level. As the deadline approaches, we review the status of the elimination initiative in this country.

Methods

We collected all available disease surveillance data at the Disease Control Unit of the Directorate General of Health Services, Government of Bangladesh from 1994 to 2013. Additionally, we retrieved data from the Civil Surgeon Office from the Mymensingh district, one of the most heavily affected areas in Bangladesh.

Results

Between 1994 and 2013, 109,266 kala-azar cases causing 329 deaths were reported from 37 endemic districts in Bangladesh. Only 16 districts reported cases every year. The Mymensingh district was the most affected with 53,582 (49.04%) cases. Between 2008 and 2013 only 16 upazilas showed incidence rates above the elimination target in which they ranged from 1.06 to 18.25 per 10,000 people per year.

Discussion

While clear progress has been made towards eliminating VL, 16 upazilas in Bangladesh had not yet reached the target in 2013, based on official notification data that probably suffered from under-reporting bias. The elimination initiative urgently needs to establish methods to ascertain and monitor the elimination target.