A new type of advanced third-stage larvae of the genus Gnathostoma in freshwater eels, Fluta alba, from Nakhon Nayok, central Thailand.

Five advanced third-stage larvae of a newly identified type of genus Gnathostoma were collected from freshwater eels, Fluta alba, which were purchased at a market in Nakhon Nayok, central Thailand. The most remarkable characteristic of the newly identified larvae was the larger body size compared with any other larva of Gnathostoma spp. They were also distinguishable from other species by the shape of their hooklets, which branched in a complex manner at the base: this had not been previously observed in any other larval Gnathostoma. The newly described larvae had an average number of 44.5, 45.0, 49.0 and 55.1 hooklets on the head-bulb from the first to the fourth rows, respectively, which were comparable to those of larval G. spinigerum. However, the average number of nuclei in each intestinal cell was 2.21 and fewer than those of the larvae of G. spinigerum. These results suggest that the new type of larvae belong to either G. vietnamicum, G. malaysiae, or constitute a new species of the genus Gnathostoma.

Immunodiagnosis of human trichinellosis and identification of specific antigen for Trichinella spiralis.

Crude antigens obtained from the infective stage larvae of Trichinella spiralis were used in an ELISA for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 96.8% when performed on sera of groups 2 and 3. Cross-reaction was observed with the sera of patients with capillariasis, gnathostomiasis, opisthorchiasis, and strongyloidiasis and opisthorchiasis with hookworm infection. The sensitivity of the test was 100% when performed on sera of group 1, which were collected 57 days after infection. Western blot analysis revealed that a specific antigen for T. spiralis was a component of M(r) 109.

Towards a suitable antigen for diagnosis of Gnathostoma spinigerum infection.

Advanced third-stage larvae of G. spinigerum were obtained from two separate sources, namely from cysts in the livers of naturally infected eels (L3E) and from experimentally infected mice (L3M). Morphology of the L3E was studied microscopically. The larvae were homogenized in distilled water, 1% Triton X-100 or 1% sodium deoxycholate containing protease inhibitors. Protein compositions of the three crude extracts were compared, on the same weight basis, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining while their antigenicities were studied by Western blot analysis using serum of a patient with parasitologically confirmed gnathostomiasis. Distilled water was found to be the best extraction solution in solubilizing proteins especially the diagnostic antigen, namely the 24,000 (24 kDa) mol. wt component from the larvae. The L3E and L3M contained relatively equal amounts of the 24 kDa antigen. This diagnostic component was anatomically located in the body fluid, oesophagus and intestine of the larva.

Immunodiagnosis of human trichinellosis using excretory-secretory (ES) antigen.

Infective first stage larvae of Trichinella spiralis were recovered from muscles of laboratory infected mice by digesting the muscles with 1% HC1-1% pepsin and collecting the larvae by modified Baerman's method. The larvae were cultivated in a serum-free medium for 18 h. The ES antigen obtained from the culture medium was used in an enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 100%. The sensitivity of the test was also 100% when performed on sera of group 1 collected at days 57 and 120 after infection. Sera collected earlier (day 23) and those collected 700 days after infection had negligible reactivity. Thus IgG-ELISA using ES antigen of the L1 was useful not only for diagnosis but also in evaluation of cure. Western blot analysis revealed that specific antigens of T. spiralis were 94, 67, 63, and 39 kilodalton components.

Gnathostomiasis in Thailand: a survey on intermediate hosts of Gnathostoma spp. with special reference to a new type of larvae found in Fluta alba.

To clarify current status of gnathostomiasis in Thailand, a survey on intermediate hosts has been carried out at various localities since 1987. It was found that Fluta alba (Fresh water eel) as well as Channa striata (snake-headed fish) might be important in playing a role of transmitting the infection either among humans or reservoir animals. During the three years from 1987 to 1989, larvae of Gnathostoma spinigerum were found in 80-100% of F. alba obtained from markets in Nakhon Nayok, with a maximum recovery of 2,582 larvae per eel. Among larvae found in these eels, five were peculiar in possessing four rows of hooklets with complicated branches at the base. Epithelial cells of the intestine of these larvae contained 1-2 nuclei. These observations indicate that the larvae are different from those of reported species of Gnathostoma from Thailand including G. spinigerum, suggesting a possibility of the advanced third-stage larvae of G. malaysiae.

Purification of Gnathostoma spinigerum specific antigen and immunodiagnosis of human gnathostomiasis.

Specific antigen of G. spinigerum which has been shown to be a protein with a relative mol. wt of 24,000 (24K) was prepared from the advanced third-stage larvae (L3) obtained from the livers of naturally infected eels. The L3 were ground and extracted with water. Purification procedures involved gel filtration, chromatofocussing and anion exchange column chromatographies, while characterization of the specific antigen was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining, Western blot analysis and isoelectric focussing. The specific antigen which has a pI of 8.5 was used as antigen in the indirect enzyme-linked immunosorbent assay (ELISA) to detect specific antibody in four groups of individuals, namely five parasitologically diagnosed gnathostomiasis patients (group 1); 15 clinically diagnosed gnathostomiasis patients (group 2); 136 patients with other parasitic infections (group 3); and 25 normal healthy parasite-free controls. Sensitivity, specificity and predictive values (positive and negative) of the assay were 100%.

Monoclonal antibody to a diagnostic Mr 24,000 antigen of Gnathostoma spinigerum.

Crude water extract (CA) was prepared from the advanced third-stage larvae of Gnathostoma spinigerum collected from livers of naturally infected eels. The extract was partially purified by chromatofocussing column chromatography and the fraction which contained specific antigen of G. spinigerum which was an Mr 24,000 glycoprotein was used to immunize five Balb/c mice for preparing immune splenocytes. Spleen cells were collected from one mouse which showed high serum titre by indirect enzyme-linked immunosorbent assay and contained specific antibody to the Mr 24,000 antigen as checked by Western blot analysis. The spleen cells were fused with myeloma Sp2/0 cells at a ratio of 10 spleen cells per one myeloma cell using polyethylene glycol 3350 as a fusogen. Thirteen out of 174 growing polyclones (7.5%) produced antibodies to the partially purified CA fraction. Among them, two polyclones produced antibody directed to the Mr 24,000 protein. These two polyclones were subjected to monocloning by limiting dilution and a monoclone GN6/24 which produced monoclonal antibody to the specific Mr 24,000 protein of G. spinigerum was obtained.

Studies on immunodiagnosis of human paragonimiasis and specific antigen of Paragonimus heterotremus.

Adult Paragonimus heterotremus were recovered from the lungs and pleural cavity of cats orally infected with metacercariae. The worms were ground and extracted with distilled water. The soluble crude antigen (CA) contained about 40% proteins which could be fractionated by gel filtration on Sephadex G-200 into three profiles namely the F1, F2 and F3. The CA and its Sephadex profiles were used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to P. heterotremus in three groups of patients, i.e. patients whose sputum and/or faeces revealed P. heterotremus eggs (group 1), patients with other parasitic infections (group 2), bacterial proven tuberculosis patients (group 3) and healthy, parasite-free controls (group 4). The sensitivity and specificity of the assay when the F1 was used as the antigen were 100%. Western blot analysis revealed that specific antigen of P. heterotremus was a non-protein component of Mr35 kDa.

Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis.

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.

Effect of single-dose albendazole and single-dose mebendazole on Necator americanus.

One hundred two children (43 males and 59 females) aged 6 to 14 years with positive stool examination by Kato-Katz and/or Harada-Mori culture techniques for N. americanus, were randomly divided into two groups. Group I with 48 children were treated with a single dose albendazole, 400 mg. Group II, 54 children, received a single dose mebendazole, 600 mg. After treatment, repeated stool examination was performed on Day 14, Day 21 and Day 28. The children were considered cured when stool examination was negative on all three occasions by both methods. The cure rate was 64% in Group I and 11% in Group II. The difference was statistically significant (p less than 0.05). The eggs reduction rate was 98% in Group I and 95% in Group II. Mild and transient side effects such as nausea, dizziness and headache were observed in both groups. Albendazole, 400 mg, as a single dose treatment was shown to be superior to mebendazole, 600 mg, single dose for the mass treatment of hookworm infection, especially that of Necator americanus, in an endemic area.

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.

Transmammary transmission of Necator americanus larva in the human host.

The prevalence of Necator americanus in the 128 nursing mothers at Saraburi hospital was 61%. The examination of milk from these mothers revealed the presence of N. americanus in one case. The finding suggested that milk could be a potential source of hookworm infection in man.

Experimental cross-breeding of Ancylostoma tubaeforme (Zeder, 1800) and Ancylostoma caninum (Ercolani, 1859).

In experimental crosses between A. tubaeform and A caninum the worms failed to produce progeny in dual-strain combinations. Even though these two strains did not fail to copulate. Egg production was observed only in identical single-strain combinations. This supports the assumption that the two species are genetically separate and valid.

Prevalence and zoonotic potential of Ancylostoma ceylanicum in cats in Thailand.

Ancylostoma ceylanicum and Ancylostoma caninum were found in cats from Prachin Buri, Thailand, with infection rates of 92% and 23%, respectively. In this survey, 75% of cats were infected with A. ceylanicum alone, the rest had mixed infections of A. ceylanicum and A. caninum. The worm burden range in 26 cats for A. ceylanicum and A. caninum were 1 to 83 and 1 to 10, respectively. For A. ceylanicum, both males and females were found in the gut from the first part of duodenum to rectum. In the case of A. caninum the distribution was not constant. The sex ratio between male and female A. ceylanicum was 1:1.4. The egg count for A. ceylanicum was in the range 31-150 per gram of faeces (mean 70). The zoonotic potential of these parasites was discussed.

Light microscopy and scanning electron microscopy of Bathmostomum sangeri Cobbold, 1879, of elephants.

Bathmostomum sangeri is an intestinal parasite of the elephant. Males measured 12.15-14.25 mm in length; females measured 14.98-17.68 mm in length. Buccal capsule is well-developed and funnel-shaped. There is a raised and transverse fissure ridge around the oral margin. The internal wall of the buccal capsule is raised into a series of circular ridges or lamellae. Teeth or cutting plates could not be seen. Spicules are stout, wing-like structures. The telamon is pear-shaped, but a gibernaculum is not present. There are two pairs of papillae on the either side of the cloacal opening. The female tail is gradually tepering.

Hypodontus macropi, Mönnig, 1929 from a Bennetts' wallaby Macropus rufogrisea.

Hypodontus macropi was found in Macropus rufogrisea. Males measured 12.73--13.93 mm in length; one female was 19.07 mm in length. The buccal capsule was funnel-shaped. The mouth opening was directed antero-ventrally. There was a pair of large cutting plates on the dorsal margin of the buccal capsule. The brusal rays were well-developed. Spicules were equal and each bore a cuticular wing. A gubernaculum and a telamon were present. The vulva was situated near the anus. The female tail, 0.163 mm in length, was suddenly tapering.