Detection of salivary antibodies to crude antigens of Opisthorchis viverrini in opisthorchiasis and cholangiocarcinoma patients.

Opisthorchis viverrini (O. viverrini; known as human liver fluke) is a major health problem in the northeastern region of Thailand. Infection with O. viverrini is the cause of hepatobiliary disease and cholangiocarcinoma (CCA). Previous studies demonstrated specific antibodies to crude O. viverrini antigens in serum from O. viverrini-infected patients. However, no studies have measured specific antibodies to O. viverrini antigens in saliva from patients with opisthorchiasis and CCA. The objective of the study was to detect specific antibodies to crude O. viverrini antigens in saliva from patients with opisthorchiasis and CCA, and to evaluate their use for diagnosis of O. viverrini infection. Saliva samples from 23 control subjects, 30 opisthorchiasis patients, and 38 CCA patients were collected. ELISA was established for detection of salivary IgA and IgG to crude O. viverrini antigens. ANOVA was used to compare salivary IgA and IgG levels among groups. Salivary IgA to crude O. viverrini antigens in CCA patients was significantly higher than controls (p = 0.007). Salivary IgG in CCA patients was significantly higher than opisthorchiasis patients and controls (p = 0.010 and p < 0.001, respectively). The cut-off value from salivary IgG test demonstrated higher accuracy for positivity of O. viverrini infection than salivary IgA. In conclusion, specific antibodies to crude O. viverrini antigens were detected in saliva of patients with opisthorchiasis and CCA. Salivary antibodies reflect serum immune response to O. viverrini infection, and salivary IgG tends to be a good candidate for diagnosis of O. viverrini infection.

Oral fluids for the immunodiagnosis of Schistosoma mansoni infection.

Saliva and oral transudate were evaluated for their potential as human specimens in the detection of IgG antibodies against soluble Schistosoma mansoni egg antigen (SEA). Preliminary laboratory testing of 49 subjects, 37 with parasitological proven infection and 12 negative controls, displayed 100% sensitivity in ELISA using serum and oral transudate and 94.6% using saliva. The specificity of the ELISA with serum was 100% versus 91.7% with both oral fluids. Significant Spearman rank correlations of anti-SEA IgG levels with egg counts were observed for serum, oral transudate and saliva (P < 0.05). The sensitivity of dot-ELISA was 100% for serum, 89% for transudate and 81% for saliva, and specificity was 100% for all 3 samples. The immunodiagnostic value of ELISA for the detection of anti-SEA IgG antibodies in oral transudate was further evaluated in 197 individuals from an endemic area of Brazil. The ELISA using serum and oral transudate showed sensitivities of 98.8% and 100% respectively and specificities of 67.8% and 64.3% respectively. Use of oral fluids for the diagnosis of S. mansoni infection was equivalent to sera with respect to test efficacy, offering an alternative to blood collection.

Immunoepidemiology of intestinal helminthic infections. 2. Immunological correlates with patterns of Trichuris infection.

The significance of parasite-specific serum and secretory immunoglobulin (Ig) isotype responses as determinants of Trichuris trichiura infection intensity in endemic communities is discussed. Comparison of age-dependent isotype responses and the age profiles of infection intensity in 2 endemic communities with markedly different levels of T. trichiura transmission suggest that serum IgA responses may reflect the accumulated past experience of infection and thus may be relevant in acquired immunity to T. trichiura and contribute to the age-convexity of infection intensity in areas of intense transmission. Preliminary analysis of data from a second community-based study shows that parasite-specific secretory IgA in saliva increases with age and correlates negatively with infection intensity, suggesting that secretory IgA may also be implicated in acquired immunity to this gut-dwelling nematode.

Blocking of nonspecific IgG in indirect radioimmunoassay for detecting Ascaris suum antigens.

The sensitivity of an indirect radioimmunoassay (IRIA) used for detecting larval body wall (LBW) antigens of Ascaris suum was enhanced by using various blocking agents which prevented nonspecific binding of immunoglobulin G (IgG) or free 125I without preventing binding of specific antibodies to the antigen. The use of blocking agents reduced counts for both positive and negative sera, resulting in an increase in calculated binding ratios (BR) and deltas and, thus, in the sensitivity of the assay. The relative effectiveness of blocking agents, in decreasing order, were turkey serum (TS), rabbit gamma globulin (RGG), rabbit whole serum (RS), bovine serum albumin (BSA), rabbit IgG (RIgG), bovine alpha globulin (BAG), bovine gamma globulin (BGG) and Tween-20.

Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.

Diagnostic values of parasite-specific antibody detections in saliva and urine in comparison with serum in opisthorchiasis.

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P<0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA.

An immunohistochemical investigation of the adult stage of the equine parasite Strongylus vulgaris.

Adult Strongylus vulgaris, collected from the caecum of infected horses and embedded in paraplast using standard methods, were sectioned for immunohistochemistry (IHC) studies. Antibodies were raised in rabbit against the excretory-secretory product (ESP) and against two constituent protein bands (28-30 kDa). The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting indicated the immunogenicity of ESP and of the subunits (28-30 kDa). In ELISA, both rabbit hyperimmune sera recognized the ESP and (28-30 kDa) bands consistently and strongly. Both hyperimmune sera recognized most ESP subunits (80, 60, 54, 42, 35, 30, 20 and 15 kDa) in immunoblots. IHC, using light microscopy, suggested that the following worm tissues reacted strongly and positively with both antisera: amphids, tooth core, intestine, excretory gland and ducts, and hypodermis. Thus, either these are antigen-producing tissues, or antigens sharing common epitopes occur in them. The following tissues reacted weakly: body cuticle, buccal capsule cuticle, oesophagus, and also somatic muscle (non-contractile portion) perhaps due to diffusion of antigen from adjacent tissues. Preimmune serum gave a negative reaction with most worm tissues.

Identification of mouth part antigens of Fasciola gigantica and Toxocara vitulorum and its molecular targets recognized by homologous and heterologous adult anti-sera against adult.

Sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE) fractionation of whole-worm and mouth part antigens of F. gigantica and T. vitulorum showed obvious qualitative and quantitative differences. Three different anti-sera, raised in rabbits against adult extracts of F. gigantica, T. vitulorum and Monieza expansa, were utilized in immunoblotting for identification of mouth part antigens that cross-react with adult worm of the same species or of different species. 7 and 8 poly peptides were recognized in F. gigantica and T. vitulorum mouth parts respectively by their homologous rabbits anti-adult anti-sera. The cross reactive antigens in F. gigantica which recognized by T. vitulorum anti-sera was 234 KD, while two components of M.wt. 113 and 93 KD were detected by M. expansa serum in the same extract. Furthermore, T. vitulorum antigens which cross-reacted with F. gigantica anti-serum 150 and 65 KD and with M. expansa were 65 and 49 KD.

The use of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen.

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

Identification of a laminated layer-associated protein in Echinococcus multilocularis metacestodes.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction lead to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS-PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

Reconstitution in vitro of the motile apparatus from the amoeboid sperm of Ascaris shows that filament assembly and bundling move membranes.

We have developed an in vitro motility system from Ascaris sperm, unique amoeboid cells that use filament arrays composed of major sperm protein (MSP) instead of an actin-based apparatus for locomotion. Addition of ATP to sperm extracts induces formation of fibers approximately 2 microns in diameter. These fibers display the key features of the MSP cytoskeleton in vivo. Each fiber consists of a meshwork of MSP filaments and has at one end a vesicle derived from the plasma membrane at the leading edge of the cell. Fiber growth is due to filament assembly at the vesicle; thus, fiber elongation results in vesicle translocation. This in vitro system demonstrates directly that localized polymerization and bundling of filaments can move membranes and provides a powerful assay for evaluating the molecular mechanism of amoeboid cell motility.