Production of monoclonal antibodies to the cuticle of advanced third-stage larva of Gnathostoma spinigerum.

This study has demonstrated that sera from Balb/c mice infected with live advanced third-stage larvae (aL3), but not those immunized with crude larval extract, immunoprecipitated the 25-kDa protein from surface-iodinated extract of aL3. Hybridoma cell lines derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissue of aL3 including the esophagus, intestine, muscle and cuticle by immunofluorescence assay. However, none of the cuticle-positive hybridoma cell lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. Western blot analysis showed that monoclonal antibodies (MAbs) secreted by clones derived from one of the cuticle-positive hybridoma lines recognized proteins of molecular weights ranging from 55-96 kDa. The MAbs most likely reacted with the collagenous component of the cuticle.

Lack of efficacy of quinine and artemether against advanced third-stage larvae of Gnathostoma spinigerum in vitro.

The efficacy of quinine and artemether--the effective blood schizontocide in malarial treatment--has been in vitro tested with the advanced third-stage larvae of Gnathostoma spinigerum. All larvae were collected from freshwater eel (Fluta alba) and exposed to the culture medium, each containing either quinine dihydrochloride or artemether at a final concentration of 20 microg/ml and 0.5 microg/ml, respectively for 21 consecutive days. Larval motility was assessed daily and the topographical changes were assessed using scanning electron microscope after 21-days of drug exposure. All worms moved actively for 21 days of study period and no change in surface ultrastructure was observed. Quinine and artemether at these concentrations have no effect on movement and topographical changes on the advanced third-stage larvae of this parasite.

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.

Gnathostoma spinigerum: analysis of protein patterns by two dimensional gel electrophoresis.

The protein extracts from male (MS) and female (FS) adults and advanced third-stage larvae (LS) of Gnathostoma spinigerum were separated by high resolution two-dimensional gel electrophoresis (2-DE). The polypeptide spots, as detected by silver staining, were subsequently identified. The spot patterns of LS, MS and FS were highly complex and consisted of more than 75, 44, 52 prominent spots, respectively. In addition, the stage-specific protein patterns were identified. This 2-DE database should provide an important reference for future biological and biochemical studies of G. spinigerum.

Movability of advanced third-stage larva of Gnathostoma spinigerum exposed to albendazole sulphoxide in vitro.

Movability of advanced third-stage larvae of Gnathostoma spinigerum exposed to albendazole sulphoxide (AlbSO), the active metabolite of albendazole, was determined in vitro. Larvae in control groups moved actively with the whole body for all 21 days of the study period. In larvae treated with AlbSO 1 microg/ml, the movement was significantly reduced after 11 days exposed to the drug and to be only a part of body on the 15th-21st days. In larvae treated with AlbSO 2 microg/ml, the movement was initiated in decreasing after 9th days and to be only a part of body on the 12th-17th days. Finally, worms were immobile but not dead on the 20th-21st days. Although there was no larvae died at 21st days exposed to AlbSO in both concentrations; but all worms were sluggish and may die later. These lethargic worms may not be able to migrate in patients and leading to cure. Albendazole may not be benefit for acute symptom clearance; however, it can prevent the recurrent migratory swelling after the treatment of 21 day-course.

A dot-ELISA test using monoclonal antibody-purified antigens for the diagnosis of paragonimiasis caused by Paragonimus heterotremus.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody-affinity chromatography was developed for detecting antibodies to Paragonimus heterotremus in four groups of subjects. They consisted of 30 patients with P. heterotremus infection, 93 patients with other parasitic infections, 18 patients with pulmonary tuberculosis and 30 normal, healthy controls. Sensitivity, specificity, as well as positive and negative predictive values of the test were 100, 97, 88, and 100%, respectively.

Partially purified antigens of Paragonimus heterotremus for serodiagnosis of human paragonimiasis.

The preparative crude extract of Paragonimus heterotremus was fractionated by isoelectric focusing. Fractions at pH 5 which contained a specific antigen with a relative molecular weight of 31.5 kDa were pooled and used in an indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis for diagnosis of human paragonimiasis. The sensitivity and specificity of ELISA were found to be 100% and 99% respectively. The band of 31.5 kDa antigenic component was found to give consistent reaction with paragonimiasis sera. The sensitivity, specificity and predictive value (positive and negative) of immunoblot analysis for the 31.5 kDa band were all 100%.

Comparison of cysticercus extract, cyst fluid and Taenia saginata extract for use in ELISA for serodiagnosis of neurocysticercosis.

Cysticercus cellulosae extract (CS), cyst fluid (CF), and an extract of Taenia saginata adult worm (TS) were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human neurocysticercosis in Thai patients. ELISA sensitivity was found to be 78.13%, 81.25% and 62.50%, respectively. False positivity was 6.66% with CS and 0% with other antigens. CF gave positivity with a pooled visceral gnathostomiasis serum and 3 of 10 (30%) of angiostrongyliasis sera. CS produced weakly positive ELISA with pooled opisthorchiasis and visceral gnathostomiasis sera. TS gave weak positive ELISA with a pooled opisthorchiasis serum. It was concluded that CF was the best antigen for use in ELISA for serodiagnosis of human neurocysticercosis.

Specificity of antibodies in cerebrospinal fluid of human cerebral gnathostomiasis cases.

Specificity of antibodies in cerebrospinal fluid (CSF) of human cerebral gnathostomiasis cases were examined by indirect fluorescent antibody technique against paraffin sections of Gnathostoma spinigerum larva. Specific greenish fluorescence was observed at cuticle, esophagus, muscle cells, intestinal cell cytoplasm and microvilli. CSF of confirmed cerebral cysticercosis cases gave fluorescence mostly at the cuticle. It is suggested that parasite-specific antigen may be present on intestinal cell microvilli and CSF would be a good source of antibodies in studying specificity of antibodies to gnathostome infections.

Scanning electron microscopy of cuticular surface of the adults Paragonimus heterotremus in Phitsanulok, Thailand.

Scanning electron microscope observation of surface structure was made on adult Paragonimus heterotremus (Phitsanulok, Thailand) and the surface topography of the anterior end, suckers, body cuticle and papillae was described. The numerous thorn-like tegumentary spines were distributed all over the surface. The spines were well-developed and branched. The papillae were divided into two types: dome-shaped and ciliated. Two pairs of papillae were seen around the lip of the oral sucker, while those around the ventral sucker were not evident.

Intestinal capillariasis: indigenous cases from Chiang Mai and Phayao provinces, Thailand.

Two cases of intestinal capillariasis were presented from new locations in northern Thailand, i.e., Phayao and Chiang Mai provinces. Both of them had chronic voluminous diarrhea and malabsorption. It was believed that they acquired the infection indigenously. Both adult worms and their eggs, presented in the feces, were identified as Capillaria philippinensis but with morphological variation. The infection was treated effectively with a prolonged administration of mebendazole.

Serodiagnosis of human paragonimiasis caused by Paragonimus heterotremus.

Enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination tests (IHA) were evaluated for serodiagnosis of human paragonimiasis caused by Paragonimus heterotremus using homologous adult worm extract as antigen. IgG-ELISA was the most sensitive, being positive in all paragonimiasis sera tested while IHA and IgA-ELISA gave 88% and 59% positive rates respectively. Cross reactivity in IgG-ELISA was detected with fascioliasis sera, producing overall assay specificity of 97%. It is suggested that IgG-ELISA is a reliable serodiagnostic test for human paragonimiasis caused by Paragonimus heterotremus.

Anatomical localization of Gnathostoma spinigerum larval antigens by an indirect fulorescent antibody test.

Indirect fluorescent antibody test (IFAT) was performed on sections of Gnathostoma spinigerum advanced third-stage larva with gnathostomiasis, angiostrongyliasis, trichinosis, strongyloidiasis and cysticercosis sera. Positive fluorescence was observed with the first three sera. Fluorescence was associated with the anterior part of the esophagus, surface of the cuticle and cytoplasmic granules of the intestine. Absorption of sera with gnathostome antigen did not elicit fluorescence. The results suggest that substances secreted from the esophagus and intestine constitute antigens in excretory-secretory products of the larva.

Prevalence of toxoplasma antibodies in blood donors and pregnant women in Khon Kaen Province.

Serum samples from blood donors and pregnant women in Khon Kaen were examined for antibodies to Toxoplasma by an indirect hemagglutination and indirect fluorescent antibody techniques. It was found that 6.4 per cent of the blood donors were positive by the indirect hemagglutination and 6.2 per cent by indirect fluorescent antibody tests. The seroprevalence in pregnant women were 12.0 per cent by indirect hemagglutination and 4.7 per cent by indirect fluorescent antibody tests. The frequency distribution curves of indirect hemagglutination titers were unimodal in both the groups studied. From the basis of these findings, it was concluded that toxoplasmosis is not endemic in Khon Kaen and the transmission occurs at a very low level.

Scanning electron microscopy of the early third-stage larvae of Gnathostoma spinigerum.

Scanning electron microscopic observations were made on the early third stage (eL3) larvae of Gnathostoma spinigerum (Sakolnakhon, northeast Thailand) from 3-week-old infected cyclops (Mesocyclops leuckarti). The morphological surfaces of the anterior end, head spine, body cuticle, amphid, papillae, posterior end of larvae were described and compared with the advance third-stage (aL3) larvae.

Serodiagnosis of human gnathostomiasis.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for serodiagnosis of human ocular and visceral gnathostomiasis in comparison to an indirect haemagglutination (IHA) and precipitin (PPT) tests. The ELISA antibody titers were found to range from 1:400 to 1:51,200 against somatic and 1:200 to 1:25,600 against excretory-secretory (ES) antigens. When sera were tested at single dilutions, the ELISA was positive in 7 of 8 gnathostomiasis cases while only 5 and 3 were positive by IHA and PPT respectively. The overall specificity of the ELISA was 96.7% and 97.4% with somatic and ES antigens respectively. Since somatic and ES antigens produced similar ELISA results, either can be used for diagnostic purpose. It was suggested that the ELISA was a reliable serodiagnostic test for human gnathostomiasis.

Filariasis in Tak Province, northwest Thailand: the presence of subperiodic variant Wuchereria bancrofti.

The microfilariae found in carriers at Tak Province, Northwestern Thailand were morphologically and morphometrically studied. It was found that the parasites conformed to that of W. bancrofti microfilaria. The microfilarial periodicity as determined from four carriers was found to be nocturnally (early evening) subperiodic type showing a distinct peak at 1800 hours.