Parasite-specific Antigens Research Articles

Expression and characterization of a parasite-specific antigen on macrophages after infection with Leishmania donovani.

A rabbit polyclonal antibody to crude soluble antigen of Leishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infected in 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 type of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.

Age-dependency of serum isotype responses and antigen recognition in human whipworm (Trichuris trichiura) infection.

The present study examines the age-dependency of parasite-specific isotype responses and antigen recognition profiles of individuals within a Trichuris trichiura endemic community, in order to evaluate the significance of serum antibodies as determinants of observed age-related patterns of infection intensity. A high degree of individual heterogeneity is observed in isotype responses to separated T. trichiura antigens by Western blot. Recognition by IgG1 antibodies exhibits marked age-dependency. The age-profiles of IgG1 responses to selected antigens of 16-17 kDa and 90 kDa molecular weight reflect the age-related changes in current infection intensity at the population level. Similarly, mean age patterns of IgG2 responses to a 90 kDa antigen, and mean IgG4 responses to a 16-17 kDa antigen reflect mean infection levels. IgG3 responses are negligible, and for methodological reasons, both IgE and IgM specificities are not presented. IgA responses to separated antigens of 16-17 kDa and 90 kDa, exhibit age-profiles which may suggest the development of an IgA-mediated acquired resistance to T. trichiura with age. IgA levels remain elevated throughout early adulthood, when infection intensity levels markedly decrease, supporting the hypothesis that IgA antibodies may be significant in generating the convex nature of the age-infection profile of T. trichiura.

Dirofilaria immitis: Detection of parasite-specific antigen by monoclonal antibodies in glomerulonephritis in infected dogs

For the identification of circulating parasite antigens associated with immune-complex glomerulonephritis in dogs infected with Dirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of cross-reactivity with Toxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-1, an IgG2a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100 degrees C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected with D. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted.

Efficacy of milbemycin oxime in chemoprophylaxis of dirofilariasis in cats

Summary Although cats are less susceptible to infection with Dirofilaria immitis than are dogs, the possibility of severe consequences from infection or adulticidal treatment renders preventive treatment a desirable alternative in endemic areas. To evaluate the efficacy of milbemycin oxime as a chemoprophylactic agent in cats, 48 cats were inoculated with infective D immitis larvae. Single oral treatment with 2.3 mg of milbemycin oxime (0.5 to 0.9 mg/kg of body weight) at 30 or 60 days after inoculation with infective larvae gave strong but incomplete protection. Treatment at 60, as well as 90, days after inoculation with infective larvae was completely effective in preventing development of infection. A control group of inoculated, but untreated, cats was monitored biweekly for hematologic changes and for changes in parasite-specific serum antigen and antibody concentrations. Pronounced increases in total leukocyte counts and eosinophil numbers were associated with the estimated time of in vivo molting from fourth- to fifth-stage larvae. Antibody reactivity correlated with infection status, but serum antigen concentrations through 161 days after inoculation were undetectable.

An hypothesis to explain why cell-mediated immunity alone can contain infections by certain intracellular parasites and how immune class regulation of the response against such parasites can be subverted.

Cells with a low density of parasite-specific antigens on their surface are postulated to be susceptible to a cell-mediated attack but not to effector mechanisms normally activated following the binding of specific antibody to the infected cell. It is further postulated that such infected cells normally induce a cell-mediated response, and that cells infected with slow-growing intracellular parasites have a low density of parasite-specific antigens on their surface. Despite these general postulates, cell-mediated immunity is not invariably induced following natural infection by certain slow-growing parasites, such as those responsible for leprosy, tuberculosis, and the leishmaniases, and antibody can be induced that is exclusive of a strong, cell-mediated response. It is proposed that certain events in such cases subvert the normal regulatory processes that control the class of immunity induced. In these cases, the parasite-infected cells, bearing a low representation of parasite antigens, induce antibody even though they are not susceptible to antibody-dependent effector mechanisms, and so they are not eliminated. In this case, chronic infection and uncontrolled growth of the parasite occurs, often with fatal consequences.

Isolation and degranulation of mucosal mast cells from the small intestine of parasitized sheep

The isolation of mucosal mast cells and globule leucocytes from the small intestine of sheep immunized with Trichostrongylus colubriformis is described. Sheep mast cell protease was released from these cells in a dose-dependent fashion after incubation with soluble protein from T. colubriformis larvae. Release also occurred with other T. colubriformis antigens whereas non-parasite antigens at comparable protein concentrations evinced only a minimal response. Mucosal mast cells prepared from worm-free sheep also produced a similar minimal response. This is the first report describing the release of sheep mast cell protease from isolated sheep intestinal mucosal mast cells after addition of specific parasite antigens.

Analysis of host components in hydatid cyst fluid and immunoblot diagnosis of human Echinococcus granulosus infection.

To improve serodiagnosis of cystic hydatidosis, immunoblotting studies were performed to look for a highly specific parasite antigen(s). First, commercially available hydatid cyst fluid antigen preparations were characterized by SDS-PAGE and by immunoblotting with sera specific for parasite and host animal proteins. One preparation, designed for use in complement fixation tests, did not appear to be suitable for immunoblotting because of the low concentrations of parasite antigens. Several host proteins, including serum albumin and IgG, were detected in the cyst fluid. Sera from patients with Echinococcus granulosus infections and other parasitic diseases were examined by immunoblotting for antibodies against specific cyst fluid parasite antigens. Several parasite antigens were variably recognized. Only one antigen, a 40 kDa protein, was recognized by all E. granulosus-infected patients. Reactivity against this antigen was also observed in all sera from E. multilocularis, cysticercosis, and schistosomiasis patients as well as in some filariasis cases. Two E. granulosus antigens, molecules of 12.5 and approximately 17 kDa, were only recognized by antibodies from some E. granulosus patients.

Macrophage regulation of immune responses of spleen cells from mice infected with Trypanosoma cruzi

Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.

Changes in Antibody Profile after Treatment of Human Onchocerciasis

To define the changes in antibody response to Onchocerca volvulus antigens after treatment of patients with onchocerciasis, IgG and IgE antibodies were examined quantitatively and qualitatively in 21 patients and 3 control individuals before and sequentially for 14 days after treatment with diethylcarbamazine. The quantitative levels of IgE and IgG responses (both polyclonal and O. volvulus-specific) remained essentially unchanged for all patients, but 9 of the 21 patients showed intensified responses to one or more parasite-specific antigens, and 8 of 21 developed antibodies to previously undetected antigens. There was a significant correlation between the intensities of infection and the development of newly recognized anti-O. volvulus antibodies. These studies demonstrate that O. volvulus-specific IgE and IgG antibody responses are, at least transiently, enhanced by treatment with diethylcarbamazine and that after treatment, parasites possibly release antigens previously hidden from the host's immune response.

Characterization and in vitro translation of Toxoplasma gondii ribonucleic acid

RNA was extracted from purified tachyzoites of Toxoplasma gondii (RH strain) by sequential centrifugation in guanidine hydrochloride, urea, and lithium chloride. The subunits of the RNA were characterized by denaturing and non-denaturing electrophoresis in agarose gels. Poly(A) +-RNA, purified by oligo(dT)-cellulose affinity chromatography, was translated in a rabbit reticulocyte lysate assay and the products were immunoprecipitated with an experimentally infected mouse serum and a naturally infected human serum. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, fluorography of the polypeptides confirmed that the mRNA translated specific parasite antigens.

Characterization of parasite antigens from human hydatid cyst fluid by SDS-PAGE and IEF

Combining high resolution power of SDS-PAGE and IEF with the specific immunological recognition of a human antiserum directed against Echinococcus granulosus antigens, we could identify, in 4 hydatid cystic fluids of human origin, 4 antigens with a molecular weight in the range 32-13 KD, and an antigen of 200 KD which, in reducing conditions, gave 2 bands of 67 and 52 KD. In addition, mainly in one of the cystic fluids, there were at least another 4 specific non-reducible bands with a molecular weight ranging from 80 to 40 KD. Specific parasite antigens, which constitute not more than 3% of total protein content of the cystic fluid, migrate, in isoelectric focusing, from a pH of less than 5 to more than 8.

Infection with Eimeria tenella: modulation of lymphocyte blastogenesis by specific antigen, and evidence for immunodepression.

The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.

Specific and Non-Specific Lymphocyte Blastogenic Responses in Individuals Infected with Trypanosoma Cruzi *

Lymphocyte blastogenic response to various parasite-specific and non-parasite antigens was measured in 31 Trypanosoma cruzi-infected individuals representing the indeterminate, megadisease and cardiomyopathy forms of Chagas' disease. The non-parasite antigens and mitogens stimulated responses in the peripheral blood mononuclear cell (PBMC) cultures of the infected individuals to levels not different than the stimulation seen in PBMC of the normal control group (n = 10). The cell culture antigen (CC-Ag) derived from trypomastigote-amastigote forms of T. cruzi and the epimastigote antigen (EPI-Ag) elicited a strong response among all infected individuals in contrast to virtually no response among the normal controls. Comparisons among the individuals with T. cruzi infection stimulated with EPI-Ag showed no significant difference between the clinical groups. However, stimulus with CC-Ag showed the megadisease group response to be greater than the response of the indeterminate group, but not different than than of the cardiomyopathy group. The possible biological implications of these findings are discussed.

The shedding of the outer glycocalyx of juvenile Fasciola hepatica

Freshly excysted Fasciola hepatica possess an outer glycocalyx which on incubation at 37° C is rapidly shed. Using an ELISA technique the release of this parasite antigen was shown to be temperature-dependent and to occur in both normal bovine serum as well as in serum free conditions. The ELISA failed to detect the antibody—antigen complexes that occurred when flukes were incubated in immune serum. Release of specific parasite antigen fell slowly with in vitro cultured flukes, but increased with in vivo cultured flukes. Using a fluorescence inhibition assay, antigens with high ELISA titres inhibited surface fluorescence of the parasite suggesting that the ELISA was detecting surface antigens as well as other parasite metabolic products. Several metabolic blocking agents and commercial anthelminthics were titrated against juvenile F. hepatica to screen for inhibition of the surface — tegument shedding: there was little selective inhibition of surface shedding without a significant loss of motility.

Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells.

We have developed a procedure for the dispersion of mast cells from the intestinal lamina propria (LP) and epithelium of rats infected with the intestinal nematode, Nippostrongylus brasiliensis. The dispersed cells are morphologically and histochemically similar to intestinal mucosal mast cells (MMC) in situ and are distinguishable from peritoneal mast cells (PMC). MMC derived from the LP or epithelium of parasitized animals secrete histamine in response to the specific parasite antigens as well as anti-IgE. Unlike PMC, these cells are unresponsive to the basic secretagogues 48/80 and bee venom peptide 401. Similarly, bee venom peptide 401 conjugated with dansyl chloride binds to PMC and mast cells in the thymus and intestinal serosa, but not to mast cells in or derived from the intestinal LP and epithelium. Studies on PMC treated by the intestinal cell isolation procedure show that the functional characteristics of the MMC cannot be solely attributed to the isolation procedure. Thus, MMC have been isolated and shown to be morphologically, histochemically, and functionally different from PMC, as suggested by previous in vivo studies of the normal intestine.

Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Highly purified Plasmodium knowlesi schizonts were used to produce a hyperimmune anti-parasite serum in a rhesus monkey. Proteins of membranes from normal and P. knowlesi-infected erythrocytes, as well as purified schizonts, were solubilized in 1% Triton X-100 and analyzed by bidimensional electrophoretic techniques. Of seven parasite-specific antigens identified in membranes of parasitized erythrocytes by crossed immune electrophoresis against monkey anti-parasite serum, only three could be detected in the purified schizonts. Bidimensional focusing-dodecyl sulfate/polyacrylamide gel electrophoresis of membranes from parasitized cells revealed three proteins, in the 55,000-90,000 molecular weight region, with isoelectric points between pH 4.5 and pH 5.2, that could not be detected in normal membranes or purified schizonts. Membranes of normal erythrocytes and uninfected erythrocytes that had been incubated with sera from monkeys with 25-50% parasitemia did not react with the monkey anti-parasite serum.

Advances in methodology for immunodiagnosis of parasitic diseases

During recent years, significant progress has been made in the development and improvement of procedures for the immunodiagnosis of parasitic diseases. The purpose of this review is to briefly discuss the inherent advantages and limitations of various immunodiagnostic methods currently in general use, to delineate areas in which further research is needed, and to call attention to new technics that may prove to be valuable additions to the present armamentarium of procedures for the immunodiagnosis of parasitic infections. It is well established that the specificity and sensitivity of an immunodiagnostic procedure depend on the quality of the antigen employed. The introduction of better methods for isolating the serologically active antigen components from crude parasite extracts has significantly improved the pathognomonic value of many immunodiagnostic tests. Since the chemical nature of the major specific antigen(s) differs among the various parasites, no single method is universally suitable for antigen fraetionation. Studies illustrating the variety of physicochemical fractionation procedures that have been used successfully for the isolation of specific parasite antigens are noted. In addition, attention is called to the potential advantages of employing exoantigens (i.e., excretions and secretions of living parasites) in immunodiagnostic tests. Procedures most frequently employed for immunodiagnosis of parasitic diseases are discussed in some detail and include: complement fixation; indirect hemagglutination; indirect immunofluorescence procedures using whole organisms as antigens; flocculation tests employing lecithin-cholesterol crystals, bentonite or latex particles as the antigen matrix; card tests using antigen-sensitized lecithin-cholesterol crystals with added charcoal; agglutination reactions; precipitin tests; and hypersensitivity reactions. The advantages and limitations of these procedures are noted and areas in which their use could be extended are suggested. Certain less commonly employed procedures that have shown considerable promise for serodiagnosis or for basic studies on the immunology of parasitic diseases also are discussed. These include immunoelectrophoresis, the recently developed soluble antigen fluorescent antibody procedure, in vitro histamine release by in vivo-sensitized leukocytes, passive cutaneous anaphylaxis, conglutinating complement absorption procedures, and serological adhesion (immune adherence) reactions. Problems associated with the wide variations of methodology employed in various diagnostic laboratories are noted and the need for improved standardization of antigens and technics is emphasized.

Detection of urinary leishmanial antigen by latex agglutination test (<i>KA</i>tex) in kala-azar patients

Background: A new unique latex agglutination test (KAtex) that detects a stable, nonprotein, disease specific parasite antigen in the freshly voided urine of patients suffering from active kala-azar has been introduced by Kalon Biological Ltd. UK. This is absolutely non-invasive method of diagnosis for visceral leishmaniasis and suitable for implementation as a rapid diagnostic tool at the point of care. Objective: Diagnostic potential of KAtex was evaluated among clinically suspected kala-azar patients in an endemic zone of Bangladesh. Methodology: KAtex was done using freshly voided urine according to the manufacturer’s instructions for sixty (60) clinically suspected patients of kala-azar admitted in Rajshahi Medical College Hospital (RMCH), Bangladesh and forty (40) healthy controls during December 2005 to June 2006. Leishmania nested Polymerase Chain Reaction (Ln-PCR) using peripheral blood buffy coat was performed for all study population (100) and Ln-PCR positive cases were considered as confirmed cases of kalaazar. Results: Out of 60 clinically suspected kala-azar patients, 56 were Ln-PCR positive and 53 of 56 Ln-PCR positive cases were KAtex positive (sensitivity, 94.64%; Mantel-Haenszel Chi sq. 79.66, p= 0.0000, confidence interval [CI], >95 to 100%). None of the healthy controls was found positive by Ln-PCR but 2 of 40 were KAtex positive (specificity, 95%; confidence interval [CI], >95 to 100%). The positive and negative predictive values of KAtex were noted as 98.10% and 92.85% respectively. Conclusion: This limited prospective study suggests that KAtex is an absolutely non-invasive urinebased antigen detection test with high sensitivity and specificity and may be useful for screening active kala-azar patients, particularly suitable for field use. Key words: Visceral leishmaniasis; Kala-azar; KAtex; Ln-PCR; sensitivity; specificity. DOI: 10.3329/bjms.v9i4.6688Bangladesh Journal of Medical Science Vol.09 No.4 July 2010 pp.216-222