Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.     

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

, , , , , and 1992. Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting. International Journal for Parasitology 22: 1083–1088. Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75,48, 30, 24 and 22 kDa).     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk

Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 10⁴ colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.     

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.     

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies in horse serum

An enzyme-linked immunosorbent assay (ELISA) was developed and evaluated to detect equine antisperm antibodies (ASA) in horse serum. Six maiden mares between 12 and 18 mo of age were immunized with stallion sperm cells (SC group, N=2), seminal plasma (SP group, N=2), or phosphate-buffered saline (PBS) as a control (C group, N=2). Horses received a second injection of the same antigen 2 wk after the first. Blood was collected weekly for 10 wk after initial immunization and again at Week 15. Serum ASA levels (IgG and IgA) were measured by ELISA using two assay systems, one containing stallion SC as the plate antigen and another containing SP.

In horses immunized with SC, peak IgG levels were detected by ELISA during Wk 2 and 3 after first injection using either plate antigen. The antibody levels persisted through Week 5 and then slowly declined until Week 15. Horses immunized with SP had IgG levels that did not differ from control horses using either ELISA plate antigen. The only significant elevation in serum IgA ASA occured during Week 5 after initial immunization and only in mares immunized with SC as detected by ELISA using SC as the plate antigen. Attachment of ASA to stallion spermatozoa was confirmed by an indirect immunofluorescence assay.     

Monoclonal antibodies as markers of specific cell types and regional antigens in the freshwater planarian Dugesia (G.) tigrina

We have produced monoclonal antibodies (mAb's) against antigens of the fresh-water planarian Dugesia (G.) tigrina (Girard) using standard protocols. Labeling these mAb's with PAP (peroxidase-antiperoxidase) and indirect-immunofluorescence methods, we then determined the distribution of their antigens in the planarian. Out of 112 mAb's that showed some specificity for restricted parts of the planarian, 71 were found to be cell- or tissue-specific — among them 36 for parenchymal cells, 7 for muscle cells, 11 for epidermal cells, 8 for gastrodermis, and 7 to basement membrane. Another 41 showed different, but overlapping, regional specificities, namely to pharynx and parenchyma. So far, we have been unable to isolate specific mAb's against undifferentiated cells (neoblasts). These mAb's should be important tools in study of tissue and cell morphology, regeneration, and growth and degrowth.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.     

An approach to the production of helminth antigens in vitro: The formation of hybrid cells between Fasciola hepatic a and a rat fibroblast cell line

Using polyethylene glycol, hybrid cells were formed between rat fibroblasts lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT, B.C. 2.4.2.8) and cells of the liver fluke Fasciola hepatica. The hybrid cells survived in a medium containing hypoxanthine, aminopterin and thymidine (HAT) indicating that the enzyme deficiency of the parental rat cells had been corrected. Isoelectric focusing in agarose gels showed that the HGPRT activity in the hybrids was of F. hepatica rather than rat origin. F. hepatica chromosomes could not be identified with certainty in hybrids; and fluke antigens, other than HGPRT, could not be detected in them or in culture medium in which they had grown.     

Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay

Rapid and reliable detection of harmful algae in coastal areas and shellfish farms is an important requirement for monitoring programs. Molecular technologies are rapidly improving the detection of phytoplankton and their toxins. Assays are based on the discrimination of genetic differences in the species. A commercially available PCR ELISA Dig Detection Kit in a microtiter plate was adapted for the rapid assessment of specificity of the two probes used in a sandwich hybridization assay. The toxic dinoflagellate Alexandrium minutum was used as the target organism and a capture and signal probe were designed for a species-specific identification of this species. This assay also provided the necessary specificity tests prior to the probes being adapted to an automated biosensor using a sandwich hybridization format. All probes regardless of the detection method must be extensively tested prior to use in the field. Total rRNA was isolated from three different strains of A. minutum and the mean concentration of RNA per cell of was determined to be 0.028 ng ± 0.003. Thus, a standard calibration curve for different RNA concentrations was determined so that cell numbers could be inferred from the assay. The assay and the standard curve were evaluated by using spiked field samples. The results demonstrated that the molecular assay was able to detect A. minutum cells at different cell counts in the presence of a complex background.     

Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

An innovative LC/MS approach applied to the determination of zeralenone in maize: Alternate Isotope-coded Derivatization Assay (AIDA)

Zearalenone is a mycotoxin mainly produced by severalFusarium species, which are known to colonize grains in temperate climates. The purpose of the study is to provide a reliable isotope dilution method for the quantification of this mycotoxin. A derivative of the analyte to be used as standard is obtained by reaction with acetic anhydride, which is available in two pure isotopic forms, a protonated (“light”) and a hexadeuterated (“heavy”). The derivatized standards are added to the matrix split intwo parts. Then, the derivatization procedure is repeated on both matrices derivatizing the part containing the “heavy” labelled standard with the “light” acetic anhydride and the part containing the “light” labelled standard with the “heavy” acetic anhydride. Both extracted mixtures are analyzed by LC/MS, monitoring the “light” and the “heavy” labelled analytes and using the former as standard for the latter in one case and viceversa in the other case. The method allowed to obtain very good results, without the need of IAC purification. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: The Italian Ministry of Health     

Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats

The dinoflagellate, Cochlodinium polykrikoides Margalef, has been responsible for mass mortalities of both wild and farmed fish along the Korean coast on virtually an annual basis since 1982. Economic impacts to the fishing and aquaculture industries are extensive, with a loss of USD $95 million reported in 1995 alone. The use of taxon-specific molecular probes for harmful algal species is recognized as a promising approach for the early detection of bloom formation and as part of an effective mitigation strategy. We have developed and successfully applied large subunit ribosomal RNA (LSU rRNA)-targeted probes in both whole cell and sandwich hybridization assay (SHA) formats for the species-specific detection of C. polykrikoides in Korean coastal waters. Sequences of the D1–D3 variable regions used to design probes were identical between five Korean and one Hong Kong C. polykrikoides isolates, while sequences for several N. American Cochlodinium isolates differed to varying degrees from the former. The automated SHA detected C. polykrikoides at levels as low as 1–3 cells/L in the field, demonstrating its suitability for detecting the target species at pre-bloom concentrations. This method should thus prove valuable to existing monitoring programs aimed at providing aquaculture interests with an early warning of frequently devastating bloom events.