Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.     

Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis

Sixteen species-specific monoclonal antibodies were produced against membranes of Leishmania donovani. These antibodies only reacted with determinants present on L. donovani. No cross-reactions were found with any other species of Leishmania or with membranes of Trypanosoma cruzi. An extensive analysis of the binding specificities of selected antibodies was carried out by using whole promastigote homogenates as antigen. Monoclonal antibodies D-1, D-2, D-3, and D-4 correctly identified all 44 L. donovani stocks from a cross-panel of 84 New and Old World Leishmania stocks. Antibodies D-1 and D-2 were also useful for species classification by immunofluorescence. No cross-reactions were observed with any other Leishmania species examined. Based on either Western blot and/or radioimmunoprecipitation analyses, five distinct groups of molecules associated with L. donovani-specific antigenic determinants were identified. These molecules range in m.w. from 18 to 84 kilodaltons. The antigenic molecules recognized by antibodies D-2, D-10, and D-13 are also recognized by antibodies present in sera from patients with visceral leishmaniasis (kala-azar). Kala-azar sera obtained from cases in both the Old and New World specifically compete with these monoclonal antibodies for the appropriate antigenic determinants in Western blot analysis. These monoclonal antibodies and/or the purified protein antigens may be useful in the development of a serologic assay for the clinical diagnosis of visceral leishmaniasis caused by L. donovani and in epidemiologic studies of leishmaniasis.     

Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.     

Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.     

Physical and antigenic heterogeneity in the flagellins of Listeria monocytogenes and L. ivanovii

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 degrees C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 degrees C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.     

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.     

Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp. succinogenes S85 and related fresh isolates.

Polyclonal and monoclonal antibodies to the Cl-stimulated cellobiosidase of Fibrobacter succinogenes subsp. succinogenes S85 reacted with numerous proteins of both higher and lower molecular weights from F. succinogenes subsp. succinogenes S85, but not with Escherichia coli proteins, and only one protein each from Butyrivibrio fibrisolvens and Ruminococcus albus. Different profiles were observed for Western blots (immunoblots) of peptide digests of both the purified enzyme from F. succinogenes and immunoreactive proteins of higher and lower molecular weights, demonstrating that they were different proteins. Therefore, F. succinogenes appeared to produce numerous proteins with one or more common antigenic determinants. However, with the exception of Cl-stimulated cellobiosidase, none were cellulases that have been characterized. An affinity-purified polyclonal antibody to Cl-stimulated cellobiosidase reacted with numerous proteins in cells of each of three fresh isolates of F. succinogenes subsp. succinogenes and one of F. succinogenes subsp. elongata when analyzed by Western blotting. Antibodies to periplasmic cellodextrinase, endoglucanase 2 (EG2), and EG3, when reacted in Western blots with the various cellulases, including Cl-stimulated cellobiosidase, revealed limited antigenic similarity among the different proteins and none with either B. fibrisolvens or R. albus proteins. The periplasmic cellodextrinase antibody reacted with an antigen with a size corresponding to cellodextrinase in each of the three F. succinogenes subsp. succinogenes isolates but not with any antigens from the F. succinogenes subsp. elongata isolate. The anti-EG2 antibody reacted with single antigens in each of the four isolates, while the anti-EG3 antibody reacted with only one of the four isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp. succinogenes S85 and related fresh isolates.

Polyclonal and monoclonal antibodies to the Cl-stimulated cellobiosidase of Fibrobacter succinogenes subsp. succinogenes S85 reacted with numerous proteins of both higher and lower molecular weights from F. succinogenes subsp. succinogenes S85, but not with Escherichia coli proteins, and only one protein each from Butyrivibrio fibrisolvens and Ruminococcus albus. Different profiles were observed for Western blots (immunoblots) of peptide digests of both the purified enzyme from F. succinogenes and immunoreactive proteins of higher and lower molecular weights, demonstrating that they were different proteins. Therefore, F. succinogenes appeared to produce numerous proteins with one or more common antigenic determinants. However, with the exception of Cl-stimulated cellobiosidase, none were cellulases that have been characterized. An affinity-purified polyclonal antibody to Cl-stimulated cellobiosidase reacted with numerous proteins in cells of each of three fresh isolates of F. succinogenes subsp. succinogenes and one of F. succinogenes subsp. elongata when analyzed by Western blotting. Antibodies to periplasmic cellodextrinase, endoglucanase 2 (EG2), and EG3, when reacted in Western blots with the various cellulases, including Cl-stimulated cellobiosidase, revealed limited antigenic similarity among the different proteins and none with either B. fibrisolvens or R. albus proteins. The periplasmic cellodextrinase antibody reacted with an antigen with a size corresponding to cellodextrinase in each of the three F. succinogenes subsp. succinogenes isolates but not with any antigens from the F. succinogenes subsp. elongata isolate. The anti-EG2 antibody reacted with single antigens in each of the four isolates, while the anti-EG3 antibody reacted with only one of the four isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1.

We characterized the structural forms of the human immunodeficiency virus env-encoded proteins with a panel of monoclonal and polyclonal antibodies. Western blot (immunoblot) assays with antibodies specific for gp41 invariably recognized a major component of 160 kilodaltons and a less intense component of 120 kilodaltons in viral lysates. We demonstrated that these species are noncovalently associated tetramers and trimers of gp41 which represent the native form of this protein in virions. These complexes were stable when boiled in the presence of low concentrations of sodium dodecyl sulfate but were dissociated to gp41 monomers at high sodium dodecyl sulfate concentrations. Moreover, two human monoclonal antibodies preferentially recognized the oligomeric complexes over monomeric gp41 in Western blots, indicating the presence of epitopes recognized by the human immune system on the gp41 multimers which are not efficiently expressed by the dissociated monomers. The demonstration of the existence of multimeric env complexes and the enhanced and altered antigenicity of such multimers may be relevant to the design of subunit and recombinant human immunodeficiency virus env vaccines.     

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.     

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.     

Leishmanin skin test in guinea pig with a single purified protein of Leishmania major

A series of hybridomas was produced by fusion of SP2/0 myeloma cells with spleen cells of mice immunized with Leishmania major (L. major). The reactivity of secreted monoclonal antibodies (mAbs) was evaluated against available leishmanin antigen by enzyme linked immunosorbent assay. Only one hybridoma designated as 7F9 secreted IgG1 mAb which was shown to be reactive with leishmanin. This mAb was further tested against four species of Leishmania (L. donovani, L. tropica, L. infantum, L. major) and a recombinant gp63. Among the four species tested it was shown to be only reactive with promastigotes of L. major. The antigen recognized by this mAb was purified and analyzed from both sonicated and supernatant cultures of L. major by immunoaffinity chromatography and reverse phase high performance liquid chromatography. The purified antigen, which gave a single band of 56kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis elicited a strong delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized with L. major. It was almost of the same degree as that produced by leishmanin. These results suggest that an L. major-specific antigen is an alternative as a specific diagnostic skin test reagent, which could lead to a better understanding of the mechanism of DTH in L. major.     

Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry

The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and phosphorylation.     

Species and stage specificity of Leishmania donovani antigens.

Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.