Proteomic approach for characterization of immunodominant membrane-associated 30- to 36-kiloDalton fraction antigens of Leishmania infantum promastigotes, reacting with sera from Mediterranean visceral leishmaniasis patients

The aim of the present study was to identify and characterize proteins of a 30- to 36-kDa fraction of Leishmania infantum promastigote membranes previously shown to be an immunodominant antigen(s) in Mediterranean visceral leishmaniasis (MVL) and a consistent and reliable serological marker of this disease. By the first approach, Coomassie-stained protein bands (32- and 33-kDa fractions) that specifically reacted by immunoblotting with sera from MVL patients were excised from the gel and submitted to enzymatic digestion to generate peptides. Four peptides were sequenced, three of which were shown to be definitely associated with MVL-reactive antigens and ascribed to a mitochondrial integral ADP-ATP carrier protein from L. major, a putative NADH cytochrome b(5) reductase, and a putative mitochondrial carrier protein, respectively. The second approach combined two-dimensional gel electrophoresis of membrane antigens and mass spectrometry (liquid chromatography-mass spectrometry/mass spectrometry) by using a quadrupole time-of-flight analysis. Six immunoreactive spots that resolved within a molecular mass range of 30 to 36 kDa and a pH range of 6.7 to 7.4 corresponded to four Leishmania products. The sequences derived from two spots were ascribed to a beta subunit-like guanine nucleotide binding protein, known as the activated protein kinase C receptor homolog antigen LACK, and to a probable member of the aldehyde reductase family. One spot was identified as a probable ubiquinol-cytochrome c reductase (EC 1.10.2.2) Rieske iron-sulfur protein precursor. The remaining three spots were identified as truncated forms of elongation factor 1alpha. These antigens correspond to conserved proteins ubiquitously expressed in eukaryotic cells and represent potential candidates for the design of a reliable tool for the diagnosis of this disease.     

Purification and characterization of an 80-kilodalton membrane protein from Leishmania donovani.

Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. We previously described the development of 16 monoclonal antibodies specific for L. donovani. The epitope recognized by one of these monoclonal antibodies, D13, is present at high density on nearly all isolates of L. donovani and, along with two other monoclonal antibodies, has been used to develop a sensitive and specific competitive assay for serodiagnosis of visceral leishmaniasis. In this report, we characterize the antigens recognized by D13 by immunoprecipitation of [35S]methionine-labeled promastigotes as two proteins (apparent molecular mass, 72 and 80 kilodaltons). Pulse-chase studies showed no evidence of a precursor-product relationship for the two proteins. We purified the 80-kilodalton protein (p80) to homogeneity by detergent solubilization of promastigote membranes, immunoaffinity chromatography, and ion-exchange chromatography. The epitope on p80 recognized by D13 was completely destroyed by proteolysis but was not affected by periodic acid treatment. P80 did not bind to the radioiodinated lectins concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Its apparent molecular mass was not affected by tunicamycin. Thus, it does not appear to be glycosylated. This protein is highly immunogenic and may prove useful for immunoprophylaxis and serodiagnosis of visceral leishmaniasis.     

Identification of an immunodominant antigenically conserved 32-kilodalton protein from Cowdria ruminantium.

Western blotting (immunoblotting) of Cowdria ruminantium antigens with goat or mouse antiserum identified a periodate-resistant, proteinase K-sensitive immunodominant antigen of 32,000 daltons. This protein, designated Cr32, could be demonstrated in goat choroid plexus infected with one of two different Cowdria stocks. Antisera against nine different Cowdria stocks from Africa and the Caribbean region recognized Cr32, which indicates that this protein contains conserved antigenic determinants.     

Immunoblot analysis of the humoral immune response to Leishmania donovani infantum polypeptides in human visceral leishmaniasis.

Using the immunoblot technique, we have compared the reactions of Leishmania donovani infantum polypeptides with the immunoglobulin G of human sera from patients with parasitologically proven L. d. infantum infection, with suspected visceral leishmaniasis, and with other leishmaniases, protozoiases, helminthiases, and fungal or bacterial diseases. A 94-kDa component reacted with all L. d. infantum-infected sera and with 75% of sera from patients with clinical and serological but no parasitological diagnoses. No reaction was observed with sera from patients in the other disease groups or with control sera. Studies of eight different isolates, subspecies, and species of the genus Leishmania demonstrated that the 94-kDa component was expressed in all strains examined.     

Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis

Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.     

Identification and Characterization of a Novel, 37-Kilodalton Leishmania donovani antigen for diagnosis of Indian visceral leishmaniasis

The biggest challenge in the serological diagnosis of visceral leishmaniasis (VL) is to find a biomarker with a high specificity. This study was undertaken to identify novel Leishmania donovani antigens to solve the existing problem. The soluble L. donovani promastigote antigen was separated by SDS-PAGE, and a Western blot was probed with pooled sera of five subjects with confirmed VL before (n = 9 pools) and after (n = 9 pools) treatment and at the 6-month follow-up visit (n = 9 pools), healthy controls not from an area of endemicity (n = 9 pools), and healthy controls from an area of endemicity. The antibody response to the identified partially purified antigen was ascertained by an enzyme-linked immunosorbent assay (ELISA) with 70 sera from patients with parasitologically confirmed VL, 48 sera from healthy controls from an area where the disease is not endemic, 60 sera from healthy controls from an area of endemicity, and 42 sera from patients in different disease groups. The eluted protein was subjected to two-dimensional (2D) gel electrophoresis, Western blotted, and probed with sera from patients with confirmed VL and from healthy controls not from an area of endemicity. The antigenic protein was further characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The identified protein (BHUP2) corresponds to a cytochrome c-like synthesis protein of 37 kDa. ELISA results were 94% sensitive, whereas specificities with sera from healthy controls from an area of endemicity, healthy controls not from an area of endemicity, and disease controls were 98%, 100%, and 97%, respectively. The antigen identified via a proteomics-based approach has a strong potential for further development as a diagnostic tool for VL.     

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis.

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral leishmaniasis.     

Purification of two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis

Two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis were purified using species-specific monoclonal antibodies and characterized. The molecular weights of the proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were approximately 70,000 and approximately 72,000, respectively. The 70 kDa protein, which appears as a diffuse band on silver staining, was resolved into a doublet by Western blotting with monoclonal antibody. Though of similar molecular weight and amino acid composition, the two proteins were shown to be distinct by peptide mapping and Western blotting of the purified material. The two proteins are recognized specifically by human visceral leishmaniasis serum and not by serum from cutaneous leishmaniasis or Chagas' disease. These proteins will be useful in developing a direct serodiagnostic assay for visceral leishmaniasis.     

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.     

Molecular fingerprinting of Leishmania infantum strains following an outbreak of visceral leishmaniasis in central Israel

Human and canine visceral leishmaniasis caused by Leishmania infantum emerged in central Israel after an absence of over 30 years. The origin of this outbreak was investigated by examining genetic polymorphisms in 37 strains isolated from dogs and patients with visceral leishmaniasis in the continuously active northern Israeli and West Bank foci and in a new Israeli focus using DNA fingerprinting with the human multilocus minisatellite probe 33.15. Analysis of the patterns obtained by DNA fingerprinting separated the strains geographically into northern (clade B) and central (clades A and C) genotypic groups. These results suggest that the emergence of visceral leishmaniasis in central Israel is due not to parasite spread from northern Israel to the new focus but rather to increased parasite transmission in central Israel and the West Bank coupled with changes in the ecoepidemiology of this region.     

Eco-epidemiology of visceral and cutaneous leishmaniasis in the Yemen Arab Republic. I. Presence, in sympatric condition, of Leishmania infantum and Leishmania donovani complexes

In complement to a previous survey, the authors proceed to the analysis of strains isolated from visceral human and canine leishmaniasis. Finally, among eight human strains isolated and identified with an enzymatic method, seven belong to the Leishmania donovani complex and one to the L. infantum complex. The L. donovani complex is represented by the MON-31 and MON-83 zymodem. The first one is also present in Saudi Arabia and Ethiopia. The second one, corresponding to a small variant, pleads for an intrafocal polymorphism phenomenon which was until now unknown in the L. donovani complex. The L. infantum complex is observed: 1) in sympatria with L. donovani in mountainous areas; 2) alone in the Tihama coastal plain. As for human cutaneous leishmaniasis present in the same focuses it is caused by L. tropica MON-71 and not by the above mentioned complexes.     

Identification and characterization of an immunodominant 28-kilodalton Coxiella burnetii outer membrane protein specific to isolates associated with acute disease

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an approximately 28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a approximately 28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.     

Antibodies against a Leishmania infantum peroxiredoxin as a possible marker for diagnosis of visceral leishmaniasis and for monitoring the efficacy of treatment

Diagnosis of leishmaniasis is frequently based on serological methods, such as direct agglutination, immunofluorescence tests and ELISA assays with Leishmania total extracts, as antigen, however due to highly inconclusive results, more reliable tests are needed. In the present study, the prevalence of antibodies to a number of recombinant proteins (LmSIR2, LmS3a, LimTXNPx, LicTXNPx and LiTXN1) highly conserved among Leishmania species, were evaluated by ELISA in Leishmania infantum infected children from an endemic area of Portugal. We found that sera from children patients had antibodies against the different recombinant proteins, LicTXNPx presented the highest immuno-reactivity compared to the other and the most often recognized in the case of acute visceral leishmaniasis (VL). Moreover, in children treated with meglumine antimoniate or amphotericin B, antibodies against some of the recombinant proteins declined, whereas conventional serology using crude extracts showed little or no difference between the pre- and post-treatment values. The highest reduction was observed in the case of antibodies against the LicTXNPx protein. These results suggest that the antibodies against LicTXNPx might be a useful constituent of a defined serological test for the diagnosis and the monitoring of the therapeutic response in VL. The monitoring and follow-up in a large-scale field trials of such marker in areas where leishmaniasis is endemic will lend support to this.     

Establishment of an appropriate inoculum dose of Leishmania donovani promastigotes required to establish a visceral infection in laboratory animal rodent models

Syrian hamsters and BALB/c mice were inoculated intraperitoneally with various doses of stationary phase Leishmania donovani promastigotes derived from primary, secondary and tertiary cultures. Axenic derived amastigotes from a tertiary culture and mass-culture derived promastigotes from primary, secondary, and tertiary cultures were also used. Animals were sacrificed after 30 days incubation period and parasite-loads quantified from Giemsa stained spleen smears. A primary inoculum dose of 1 x10(8) was found to be the most appropriate in effecting a visceral infection. This parasite dose resulted in a spleen parasite-load of 10-20 amastigotes per field of microscope view at x1,000 magnification. Those involved in candidate vaccine molecules or experimental drugs against kala-azar will find these results useful.     

Leishmania major-like antigen for specific and sensitive serodiagnosis of human and canine visceral leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.     

Asymptomatic domestic dogs are carriers of Leishmania infantum: possible reservoirs host for human visceral leishmaniasis in southern Iran

In the past few years, the incidence of human visceral leishmaniasis (HVL) has increased in many districts of Fars Province, southwest of Iran, particularly, among communities of nomadic tribes. Recent epidemiological reports in Leishmania infantum endemic regions of Iran indicate that more than 50–70% of seropositive dogs are asymptomatic for Leishmania infection. Between 2004 and 2006, blood samples were collected from 110 domestic dogs from nomadic and rural areas. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DATs), and for L. infantum kinetoplast deoxyribonucleic acid (kDNA), in polymerase chain reaction (PCR)-based assays. Of the 110 dogs, 5.5% (6/110) were found seropositive and 23% (25/110) PCR-positive. Four of the six seropositive (67%) and 22 of the 25 PCR-positive (88%) were asymptomatic. The rate of infection in dogs from nomadic communities was higher (27%) than dogs from rural areas (18%). Since positivity in the PCR-based assay indicated the presence of L. infantum amastigotes in the peripheral blood of 23% of the subjects, it is clear that these asymptomatic dogs (88%) are quite common in the study areas and probably act as reservoirs in the transmission of Leishmania parasites, to humans and to other dogs, by sandflies. Moreover, our study showed that application of PCR to buffy coat samples gave a better estimate of the real rate of infection in asymptomatic dogs than DAT.     

Evidence for distinct 5'- and 3'-nucleotidase activities in the surface membrane fraction of Leishmania donovani promastigotes

A surface membrane fraction isolated from Leishmania donovani promastigotes contained distinct 5'- and 3'-nucleotidase activities. These were distinguished from each other, and from a previously described surface membrane nonspecific acid phosphomonoesterase, on the basis of several properties. The 5'- and 3'-nucleotidases had p' optima of 6.5 and 8.5, respectively. In contrast to the 3'-nucleotidase, the 5'-nucleotidase was inhibited by both ammonium molybdate and fluoride ions; the latter inducing a biphasic response. Neither divalent cations nor chelators affected the 5'-enzyme activity whereas the 3'-enzyme was inactivated by EDTA. This inactivation was fully reversed following removal of the chelator, either by resuspension of the membranes in EDTA free medium or by addition of certain divalent cations in excess; Co2+ being the most effective. The 5'-nucleotidase had activity with both ribo- and deoxyribonucleotide substrates, whereas the 3'-nucleotidase did not hydrolyse deoxyribonucleotides.     

Mediterranean visceral leishmaniasis in immunocompetent children. Report of two cases relapsed after specific therapy

Visceral leishmaniasis (VL) is endemic in areas bordering the Mediterranean Sea (Spain, Italy, France, Greece, Morocco, Tunisia) where it is caused by Leishmania infantum and is transmitted by the bite of a hematophagous sandfly belonging to Phlebotomus spp.; the dog constitutes the main reservoir of infection. Two cases of VL in immunocompetent children are described. Both patients lived in endemic areas for leishmaniasis (Sicily) and at admission were febrile, pale and had splenomegaly. In both patients anti-leishmania antibodies were present and a definitive diagnosis was confirmed by demonstration of leishmania parasites by microscopy or polymerase chain reaction (PCR) in the bone marrow aspirates. The use of PCR performed on peripheral blood has been reported to be highly sensitive for the diagnosis and follow-up of children with VL. One patient was treated with N-dimethylglucamine, Glucantim, the other one with liposomal Amphotericin B (AmBisome). Both had symptomatic relapses 3 months later, and recovered following re-treatment with AmBisome administered intravenously at a dosage of 3 mg/Kg for ten consecutive days. The patients were monitored for one year after treatment was completed.