Affinity purified oval antigen for diagnosis of Opisthorchiasis viverrini.

Monoclonal antibodies (MAb) were raised against an oval antigen of the liver fluke Opisthorchis viverrini which is the causative agent of a parasitosis, i.e. opisthorchiasis in Thailand. The antibodies were used in an affinity column to purify the O. viverrini oval antigen from a crude extract of adult parasites by chromatography. The oval antigen was then used in a membrane (dot) ELISA for detecting antibodies in serum samples of parasitologically confirmed Opisthorchis viverrini infected individuals (adult parasites were found in stools after praziquantel treatment and salt purgation), as well as of individuals infected with other parasites and parasite-free controls. The MAb-based dot-ELISA using the affinity purified O. viverrini oval antigen revealed 100% sensitivity, specificity and accuracy for detecting O. viverrini infection. The test is simple, rapid and highly reproducible. Several samples can be tested at the same time without the requirement for special equipment or much increase in testing time; thus it is suitable for mass screening for O. viverrini exposure, especially in new endemic areas. Furthermore using serum specimens could increase patient and community compliance compared to the conventional parasitological survey which uses stool samples for the detection of O. viverrini ova, without treatment and subsequent salt purgation, this conventional method shows a low sensitivity and is also unpleasant to both the sample donors and the laboratory technicians which has historically shown a further negative impact on the final outcome.

Comparative horizontal starch gel isoenzyme electrophoresis of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Foot muscle tissue extracts from six lymnaeid species of the Indo-Pacific region [Lymnaea (Bullastra) cumingiana and L. (Radix) quadrasi from the Philippines, L. (R.) rubiginosa from Indonesia and Thailand, and L. (R.) viridis from Guam and Hong Kong] were subjected to horizontal starch gel isoenzyme electrophoresis and assayed for seven isoenzymes (AcP, AlP, CA, EST, LAP, CAT and GOT) to elucidate their taxonomic relationships. L. cumingiana exhibited banding patterns for EST, LAP and CAT uniquely different from the rest, thus supporting the hypothesis that it is a distinct species. Zymogram patterns for AlP, CA, EST and LAP attest to the close affinity between L. quadrasi and L. rubiginosa (Indonesia and Thailand). Minor differences suggest a closer relationship between the two geographical strains of L. rubiginosa than with L. quadrasi, lending support to the hypothesis that L. quadrasi is inseparable as a race or variety from the typical L. swinhoei Adams, which in turn is but a race of L. auricularia, which also encompasses L. rubiginosa. The two geographical strains of L. viridis from Guam and Hong Kong showed the greatest consistency with regards to similarity and congruence in banding patterns. Non-specific esterases (EST) were the most useful in distinguishing the six species from each other.

Comparative radular morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

The radular morphology of Lymnaea (Bullastra) cumingiana was compared to that of five other Indo-Pacific lymnaeid "species", namely: L. (Radix) quadrasi (Philippines), L. (R.) rubiginosa (Indonesia and Thailand) and L. (R.) viridix (Guam and Hong Kong) in order to investigate the taxonomic relationship among the six species. Although all six species uniformly exhibited a unicuspid, slightly asymmetrical central (rachidian) tooth and tricuspid laterals, interesting differences were noted among the outer marginals. These were observed to be uniquely bicuspid in L. cumingiana, predominantly tricuspid in L. quadrasi, tetracuspid in L. rubiginosa (Indonesia and Thailand) and multicuspid in L. viridis (Guam and Hong Kong). Thus, the results support the hypotheses that L. cumingiana is a unique species compared to the rest, that L. quadrasi is closely related to L. rubiginosa (Indonesia and Thailand) and that the two geographical isolates of L. viridis have not diverged. Radular morphology was therefore found to have a limited significance in elucidating the taxonomic relationship between the six groups of lymnaeids studied.

Ecological observations on Lymnaea (Bullastra) cumingiana.

Field surveys conducted at Echague, Isabela and San Pablo, Laguna revealed that Lymnaea (Bullastra) cumingiana, the natural second snail intermediate host of Echinostoma malayanum in the Philippines, exhibits a moderate degree of diversity in its choice of habitats. Rice fields of all stages of development, stagnant shallow streams and springs are the main areas where the snail can be collected from at Echague, Isabela. However, they were absent in rice fields that had been extensively sprayed with molluscicides to control the "golden apple snail" (Ampullarius canaliculatus). In contrast, they were also very abundant in the highly eutrophic waters of Sampaloc lake, San Pablo, Laguna. L. cumingiana co-exists with various species of insects, snails, fish and plants in these habitats. Information on ecological characteristics affecting its distribution will be useful for those who wish to collect and study this species in the future.

Comparative shell morphology of Lymnaea (Bullastra) cumingiana (Pulmonata: Lymnaeidae) and related taxa in the Indo-Pacific region.

Comparative shell morphology using both quantitative and qualitative parameters was employed to investigate the taxonomic relationship between the endemic Philippine species, Lymnaea (Bullastra) cumingiana and five other lymnaeid "species" in the Indo-Pacific region, namely: L. (Radix) quadrasi (Philippines). L. (Radix) rubiginosa (Indonesia), L. (Radix) rubiginosa (Thailand), L. (Radix) viridis (Guam) and L. (Radix) viridis (Hong Kong). Fifty randomly chosen adult specimens of each species were studied and compared, although only field-collected specimens were studied for the first four groups and laboratory-raised specimens for the last two group. Results strongly suggested that L. cumingiana is a distinct species among the rest. L. quadrasi, L. rubiginosa (Indonesia) and L. rubiginosa (Thailand) exhibited great affinity towards each other. Likewise, the two geographical isolates of L. viridis were practically identical to each other except for some minor size differences.

Comparison of the protective efficacy on mekongi schistosomiasis in mice induced by antigens derived from cercariae, schistosomulae and adult worms.

Protective efficacy of the extracts of cercariae, schistosomulae and adult worms of S. mekongi was studied in mice receiving immunizations with these extracts emulsified with Freund's complete adjuvant initially and incomplete adjuvant subsequently, and compared with mice receiving physiological saline with or without adjuvants as controls. After challenge with cercariae, the animals were sacrificed and the larvae or adult worms harvested by lung recovery and perfusion techniques on day 5 and weeks 6-8, respectively. Worm reduction rates were significantly higher in mice receiving extracts of schistosomula (59%) and adult worms (51%) than in those receiving the cercarial extracts (31%). Similar findings were obtained with the perfusion technique showing worm reduction rates of 57%, 53% and 30% in mice receiving extracts of schistosomulae, adult worms and cercariae, respectively. ELISA antibody titers were correspondingly increased in mice receiving extracts of schistosomulae and adult worms, but not in those receiving cercariae. This apparent association may be inadequate to suggest that the increase in ELISA titer be used as an indicator for resistance in mekongi schistosomiasis.

Factors affecting laboratory acclimatization of field collected Lymnaea (Bullastra) cumingiana Pfeiffer (Pulmonata: Lymnaeidae).

Lymnaea (Bullastra) cumingiana, the newly discovered natural second intermediate host of Echinostoma malayanum in the Philippines, is a sensitive and delicate lymnaeid species which requires certain conditions for successful transport from the field and cultivation in the laboratory. Field collected specimens were found to be best transported in styrofoam containers lined with wet filter paper or containing natural substrate and vegetation instead of Sphagnum moss. The method is convenient and produces a survival rate of 73-86%. However, transport time is crucial and mortality increases the longer the snails are in transit. For optimal results in laboratory acclimatization, snails are best raised in wide-mouthed containers providing a large exposed water surface area. Adequate aeration is advised but vigorous bubbling of the water should be avoided. Water should be replaced with filtered dechlorinated water every 2 to 3 days, depending on water quality. A combination of fresh lettuce leaves and a few flakes of fish food was found to be ideal. Lastly, population density was the most significant factor affecting survival and so overcrowding should be avoided.

Lymnaea (Bullastra) cumingiana Pfeiffer (Pulmonata: Lymnaeidae): second intermediate host of Echinostoma malayanum in the Philippines.

Adult flukes were obtained from laboratory rats and hamsters fed with metacercariae isolated from Lymnaea (Bullastra) cumingiana Pfeiffer and were identified as Echinostoma malayanum Leiper based on the presence of a circumoral collar of 43-45 spines and the highly lobed testes. This discovery is of public health significance because L. (Bullastra) cumingiana in eaten raw by certain local populations in northern Luzon. The proposed new species, Cathaemasia cabrerai (Jueco and Monzon, 1984), is probably invalid because it apparently was based on E. malayanum flukes that had lost their circumoral collar of spines after treatment with praziquantel and bithionol. This is the first case where L. (Bullastra) cumingiana has been proven to be of medical importance in the Philippines. i.e., as the natural second intermediate host of E. malayanum. The first intermediate host and reservoir host of this parasite in the country are still unknown. Previously, only E. ilocanum was reported to be present in the Philippines.

Investigation on immunity induced by Schistosoma spindale against S. mekongi in experimental mice.

An investigation on immunity induced by Schistosoma spindale cercariae (cattle and swamp buffalo schistosome) against S. mekongi (human schistosome) was conducted in Swiss albino mice. The studies comprised the development patterns of homologous immunity of S. spindale and heterologous immunity induced by S. spindale against S. mekongi. The development pattern of homologous immunity was studied in mice with an immunization of 100 S. spindale cercariae. At one week intervals, between 2 to 16 weeks after immunization, they were each challenged with 500 S. spindale cercariae. Significant homologous immunity, as judged by lung recovery assay five days after challenge, occurred from week 5 to week 16 with week 8 giving the highest homologous immunity (68.1% of schistosomular reduction). Using the above information mice, with an eight-week immunization period of 100 S. spindale cercariae, were tested for resistance to heterologous S. mekongi infection. The criteria used to evaluate their immune status was schistosomular lung recovery, daily egg output, worm recovery and tissue egg count. The results showed that mice immunized with S. spindale cercariae could develop heterologous immunity against S. mekongi infection. Manifestation of immunity was demonstrated by significant reduction in mean schistosomular recovery (31.4%), in mean daily egg output per female worm (16.7%), in mean worm recovery (64.2%) and in mean egg deposition in the liver tissue and intestines per female worm (37.05%).

Lung-migration patterns of Schistosoma mekongi and S. spindale in mouse.

Two groups of laboratory-bred Swiss albino mice were used to study the lung-migration patterns of Schistosoma mekongi and S. spindale. The first group was individually infected with 100 S. mekongi cercariae by hair-looping application on shaved abdomen. The latter group was individually exposed to 500 S. spindale cercariae by tail immersion. Each group of these infected mice was then divided into subgroups. The number of schistosomulae was determined using a lung recovery assay starting from the second day after infection and continuing for 15 consecutive days. The results revealed a sharp peak of both S. mekongi and S. spindale on the fifth day post cercarial infection.

Scanning electron microscope studies of anesthetized Tricula aperta, vector of mekong Schistosoma.

Nembutal, menthol, Ketaset, and Curarine were used to anesthetize beta race of Tricula aperta snails in preparation for study by scanning electron microscopy. The snails remained sensitive to the glutaraldehyde fixative in low level anesthesia of nembutal, menthol and Ketaset. Snails were also sensitive to Curarine and retracted into their shells. The best relaxations were obtained by initial anesthesia with nembutal or menthol followed by curarine. Snails relaxed by this procedure showed relatively little reaction to the fixative solution and remained extended outside their shells. The surface ultrastructure of the snails was well preserved, showing microvillate, secretory, and ciliated sensory cells. The mucoid secretions remained on the surface of cilia on the body and foot of the snails as spherical granules. There were two types of ciliated cells: (1) Those with few short cilia showing 1-3 microns above the microvilli of surrounding cells and (2) Those with numerous cilia up to 10 microns long. Results of transmission electron microscopic studies of these snails show no disruption of the ultrastructure of the surface cells.

Mekong schistosomiasis. 4. A parasitological survey of wild rodents, domestic pigs and cattle on Khong Island, Laos.

No evidence of infection with the Mekong Schistosoma was found in 12 Rattus exulans, 81 R. r. molliculus, and 10 Bandicota savilei caught in the vicinity of and downstream from the schistosomiasis transmission focus on Khong Island, South Laos, and examined by dissection and portal perfusion. Likewise, no eggs of the Mekong Schistosoma were detected in faeces of 15 domestic pigs or 43 domestic cattle examined on Khong Island both by merthiolate-iodine-formalin concentration and by the hatching technique. These results suggested that the wild rodents listed above, as well as pigs and cattle in the vicinity of Khong Town, may not contribute significantly to the transmission of the Mekong Schistosoma under present conditions.