A survey of infective larvae of Gnathostoma in eels sold in Ho Chi Minh City.

To investigate the distribution of Gnathostoma spp in Ho Chi Minh City (HCM city), 1,081 eels were purchased from a local market twice a month from March 1998 to February 1999. Infective larvae of Gnathostoma spp detected from the flesh and liver of eels by the press preparation technique were examined and identified. Three hundred and fifty advanced third-stage larvae were recovered from liver, none from the flesh. The average rate of infection was 0.11; a high rate of infection was found from August to November and a low rate of infection from February to May. The average number of larvae/eel was 2.9; the greatest number of larvae/eel was in January whereas the lowest was in March and April. There was a marked decrease in both prevalence and intensity of infection from February to May, followed by a rise from June. The finding suggests that in HCM city, the infection rate abruptly decreases soon after the end of the rainy season and starts to rise when the rain comes and reaches its peak at the end of the rainy season. All recovered larvae were identified as G. spinigerum.

Toxocara and Gnathostoma among stray canines in Bangkok.

Stomachs and intestines of 88 adult and 112 young stray dogs were obtained from the Rabies Control Subdivision, Bangkok, and examined especially for the presence of Gnathostoma spinigerum and Toxocara canis. Forty-five dogs were found positive for T. canis (overall prevalence 22.5%) but none were found infected with G. spinigerum. The prevalence of T. canis in young dogs was 37.5% (42 of 112) whereas in adult dogs was only 3.4% (3 of 88). The total number of T. canis recovered from the 45 positive dogs was 272 (averaging 6.0 worms/dog). This includes 268 worms from 42 young dogs (averaging 6.4 worms/dog) and four worms from three adult dogs (averaging 1.3 worms/dog). The average number of worms, according to sex and stage, per young dog were as follows: male worms 2.4 +/- 3.5 (range 0-15), female worms 2.8 +/- 3.5 (0-16), immature worms 1.2 +/- 2.5 (0-9), and all worms 6.4 +/- 8.2 (1-34). The maximum number of worms per young dog was 34 while the minimum was one, and 35.7% (15/42) of these young dogs harbored only one worm. The body length of the recovered T. canis were as follows: males measuring 3.0-12.0 cm (averaging 7.1 +/- 2.1 cm), females 4.1-18.2 cm (11.0 +/- 4.1 cm), and immature worms 0.7-3.7 cm (2.1 +/- 0.8 cm).

Seasonal variation in the intensity of Gnathostoma larvae in swamp eels (Fluta alba) sold in a local market in Bangkok.

The viscera of swamp eels were obtained from a local market in Bangkok twice a month from June 1996 to May 1997. The livers were separated, weighed and counted. Gnathostome larvae were recovered from the livers by the digestion technic, examined, identified, and counted. A total of 12,278 Gnathostoma larvae were obtained from 18,561.1 g (15,264 pieces) of eel livers. The overall average number of larvae/g liver and the overall average number of larvae/liver are 0.91 and 0.94, respectively. The greatest number of larvae/g liver (on average) was in December (high levels of infection during the months of October to December) whereas the lowest was in April (lowest levels of infection during the months of March to April). Thus there was a marked decrease in the average number of larvae/g liver during January to April, which then started to rise in May. This finding suggests that the level of infection abruptly decreases soon after the completion of the rainy season, starts to rise when the rain has come, and reaches its peak when the amount of rainfall is highest. More than 99% of the total gnathostome larvae recovered were identified to be G. spinigerum, and 25.4% of the entire larvae recovered bore variant or abnormal cephalic hooklets. The most common unusual feature was that there were extra rudimentary hooklets above row one, below row four and in between the four rows of hooklets which comprised 21.4%. In addition, the body size and the number of cephalic hooklets of G. spinigerum are also discussed.

Morphological variation and abnormality of cephalic hooklets of Gnathostoma spinigerum hepatic stage larvae from laboratory infected mice.

One thousand advanced third-stage larvae of Gnathostoma spinigerum from laboratory infected mice, two to five weeks after being fed with infected cyclops, were examined specifically for the morphology of their cephalic hooklets. Among these, only the 15-day old (early hepatic-stage) larvae and the 30-day old (late hepatic-stage) larvae were measured for the size of their body and hooklets. The average body size of the 15-day old and 30-day old larvae were 3.4 +/- 0.4 x 0.4 +/- 0.04 mm and 4.9 +/- 0.4 x 0.5 +/- 0.04 mm, respectively. The average size of the hooklets from rows one to four of the 15-day old larvae was 14.6 +/- 1.7 x 6.8 +/- 0.6 microm, 15.6 +/- 2.0 x 7.2 +/- 0.5 microm, 16.0 +/- 1.8 x 7.4 +/- 0.6 microm and 15.9 +/- 1.9 x 7.3 +/- 0.6 microm, respectively. Those of the 30-day old larvae were 15.1 +/- 1.7 x 7.1 +/- 0.6 microm, 16.3 +/- 1.6 x 7.5 +/- 0.7 microm, 16.5 +/- 1.7 x 7.8 +/- 0.6 microm and 16.3 +/- 1.7 x 7.6 +/- 0.8 microm, respectively. The average number of cephalic hooklets from rows one to four of the two- to five-week old larvae were 42.8 +/- 2.6, 45.3 +/- 2.8, 46.9 +/- 2.8 and 50.2 +/- 2.9, respectively. Several types of morphological variation and abnormality of the cephalic hooklets were observed. The most common ones were extra rudimentary hooklets below row four or within the four rows of hooklets (10.8%), the present of a fifth row of hooklets (1.9%), abnormal hooklets in only row four (1.2%), lobed or branched hooklets (0.5%), spiral arrangement of the four rows of hooklets (0.4%), and fragmented hooklets (0.4%).

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: II. Electron microscopy.

Immunocytochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by immunogold labeling method using seven G. spinigerum specific monoclonal antibodies (MAbs), FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SK-8D4 and SA-9B5. All these MAbs belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate epitopes. The paraformaldehyde-fixed GsAL3 were embedded in Lowicryl K4M medium, and the gold colloidal particles used were 15 nm in size. When the worm sections were probed with FS-3D11, the gold particles appeared to concentrate specifically on the intestinal brush border. When SS-5H5 was applied, the particles were scattered densely over the brush border and in the cytoplasm of epithelial cells. The rest of the MAbs which recognize protein determinants exhibited a lack of labeling. The results suggested that the carbohydrate antigenic determinants recognized by the two MAbs are the most stable and most abundant particularly in the intestine of GsAL3. These results also confirmed the previous finding that the most antigenic site of GsAL3 is the intestine.

Purification of Gnathostoma spinigerum larval antigens by monoclonal antibody affinity chromatography.

An immunoaffinity column was prepared by coupling a partially purified Gnathostoma spinigerum-specific IgG1, MAb SK-6C4 (5 mg/ml) to CNBr-activated Sepharose 4B. Ten milliliters of approximately 0.3 mg/ml of crude soluble G. spinigerum larval antigens (GsAL3) were loaded onto the affinity column at a flow rate of about 5 ml/hour. Elution of the bound antigens was accomplished using 50 mM diethylamine-HCI containing 0.15 M NaCL, pH 11.5. The average amount of eluted antigens obtained by one passage of crude GsAL3 (1-4 mg) through 4 to 8 ml of column matrix was 143 micrograms (range, 67 - 414 micrograms). The minimal amount of purified GsAL3 detectable by ELISA using MAb SK - 6C4 (100 micrograms/ml) was 50 ng/ml. The SK - 6C4 affinity-purified GsAL3 was found to be relatively pure and immunologically specific as determined by SDS-PAGE and Western blotting, respectively.

Immunohistochemical localization of Gnathostoma spinigerum larval antigens by monoclonal antibodies: I. Light microscopy.

Immunohistochemical localization of antigens in advanced third-stage larvae of Gnathostoma spinigerum (GsAL3) was studied by indirect enzyme immunostaining using 7 G. spinigerum specific monoclonal antibodies, FS-3D11, SS-5H5, SK-6C4, SK-4E1, SK-7G6, SD-8D4 and SA-9B5. All these MAb belong to the IgG1 subclass and only FS-3D11 and SS-5H5 recognize carbohydrate determinants. Each MAb exhibited a different reaction pattern and staining intensity in sectioned GsAL3. FS-3D11 bound primarily to the intestinal brush border whereas SS-5H5 reacted with various tissues of the parasite including intestinal epithelium and brush border, lateral cords, muscle, pseudocoel, and cuticle. SK-6C4 predominantly stained muscle, however, SK-4E1 and SK-7G6 exhibited a lack of labeling. SD-8D4 bound to the cuticle and the lateral cords whereas SA-9B5 reacted primarily with the pseudocoel. These results suggest that antigens sharing common epitopes are present in various structures of the larvae with the intestine being the most antigenic site. The present data also suggest that certain GsAL3 antigens recognized by the MAb obtained in this study are sensitive to formalin fixation and/or paraffin embedding since for 2 out of the 7 MAb staining was negative.

Circumoval and larval microprecipitation reactions in experimental and human gnathostomiasis.

Mice, rats and cats were infected either orally or percutaneously with a number of early or advanced third-stage larvae (EL3 or AL3, respectively) of G. spinigerum. Sera obtained from these infected animals and 10 human gnathostomiasis cases were tested against various developmental stages of the parasite which were prepared and used while being alive (fresh) or dead (air-dried) for the circumoval and larval microprecipitation (COP and LMP) reactions. No precipitin reactions were observed in all sera tested against unembryonated eggs, embryonated eggs and first stage larvae neither air-dried nor fresh preparations. Sera were merely reactive giving various degrees of membranous or filamentous precipitates against the air-dried preparation of AL3.

Detection of humoral immune response to Gnathostoma spinigerum in mice.

The humoral immune response to early third stage larvae (EL3) and advanced third stage larvae (AL3) of Gnathostoma spinigerum infection was studied in mice by Ouchterlony gel diffusion technique. The antibodies was detected at week 3 in mice infected with EL3 and remained up to week 10 after infection. Highest positive sample of sera were demonstrated at week 4 to week 7. Similar results were obtained from AL3 infected sera except the antibodies was found and disappeared earlier (week 2 to week 6). G. spinigerum larvae recovery from mice in both groups showed that the number of advanced third stage larvae located in muscle correlated to the peak of positive sera. No cross reaction was observed on positive sera of G. spinigerum and antigens of A. cantonensis, P. siamensis, T. spiralis, O. viverrini and A. ceylanicum. Cross reaction was shown on the G. spinigerum antigen against rat sera with angiostrongyliasis and bandicoot sera with paragonimiasis.