Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.

Fasciola gigantica: studies of the tegument as a basis for the developments of immunodiagnosis and vaccine.

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.

Scanning electron microscopic study of microfilaria and the third stage larva of Wuchereria bancrofti.

The surface structures of microfilaria and of the third stage larva of Wuchereria bancrofti were studied by scanning electron microscopy. Distinct features were observed that could be used for differentiating species of this parasite. Specifically, the sheath of microfilariae of W. bancrofti projected beyond the head. The head region of the microfilaria was composed of a cephalic cap with hook, mouth and amphidial opening, and its cuticle showed annulation. Spines were absent at the first transverse annulation, and the tail end showed a slight constriction. In the infective stage larva, characters which are used for differentiating species, such as the two bubble-like ventro-lateral papillae and one dorso-terminal papilla were rather similar to each other in size, but the grooves seen around the base were absent. A previously unreported feature of the third stage larva of W. bancrofti that was discovered in this study is a papilliform process on the left side of the posterior region, between the anus and the tail end.

Diagnosis of cattle fasciolosis by the detection of a circulating antigen using a monoclonal antibody.

A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.

Clonning and sequence analysis of the 26 kDa glutathiones-transferase gene of Schistosoma mekongi.

The number of genomic DNA or cDNA sequences of Schistosoma mekongi accessible in Genbank or EMBL is very limited up to now. Recently, two reports have appeared on the molecular phylogeny of Schistosoma species inferred from partial sequence data of rRNA genes; no further sequence data of S. mekongi is available yet. Knowledge of the molecular structure of protein coding genes of S. mekongi will provide a better understanding of gene function in the genus Schistosoma. A cDNA library of S. mekongi adult male was constructed and a cDNA encoding the 26 kDa glutathione S-transferase protein of this species was cloned. Sequence analysis of this cDNA confirmed the close phylogenetic relationship of S. mekongi to S. japonicum.

Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.

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 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.

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.

Monoclonal antibodies against Schistosoma mekongi surface tegumental antigens.

Monoclonal antibodies were produced from naturally infected BALB/c mice. Thirteen hybridomas which were found to produce monoclonal antibodies against surface tegumental antigens of Schistosoma mekongi by ELISA assay were used in this study. The antigen specificities of hybridomas reactive with surface tegumental antigens were characterized and localized by immunoblotting analysis and Avidin-Biotin method. Of the 13 hybridomas, only three produced monoclonal antibodies to the single epitopes in the surface tegumental antigens. These epitopes (125 kDa, 97 kDa and 38 kDa) have been found to be the major antigenic components of the surface tegument of S. mekongi. The 38 kDa antigen was found to associate with the surface tegumental layers, the muscular layers lying just beneath the tegument, as well as in the gut surface. The 97 and 125 kDa antigens were detectable only in the surface tegumental area. The biochemical identity of these proteins or glycoproteins is unknown. However, these antigens have also been described in S. japonicum and S. mansoni.

Characterization of two monoclonal antibodies against teguments of adult and schistosomula of Schistosoma japonicum.

Two monoclonal antibodies (McAbs) were produced from BALB/c mice hyperimmunized with tegumental extract of Schistosoma japonicum (Chinese strain). The two McAbs were characterized with regard to antibody isotype, antigen binding specificity and parasite stage specificity. One McAb, 8G9-5, was identified as IgM, whereas the other McAb, 9E7, was determined to be IgG2a. Immunoblotting assay indicated that McAb 8G9-5 binds strongly to the band of tegumental antigens of Mr 64 kDa but also binds weakly to other bands at 116, 105, 97, 54, 50, 47 and 45 kDa, whereas 9E7 McAb reacts specifically at Mr 54 kDa. Anatomical localization of the antigens in the adult worm by indirect immunofluorescence assay indicated that the target epitopes of McAb 8G9-5 are in the intra-tegumental structure, whereas the McAb 9E7 epitope is on the surface membrane. The two McAbs also react at similar sites within the teguments of schistosomula and lung worms.

Diagnosis of opisthorchiasis by enzyme-linked immunosorbent assay using partially purified antigens.

Opisthorchis viverrini antigens were partially purified from adult worms collected from liver and extrahepatic biliary system of infected hamsters. Tegument fraction was obtained by chemical extraction, whereas other fractions were purified by Sephadex G-200 gel filtration chromatography. Five fractions of O. viverrini antigens were obtained, namely tegument extract, somatic extract, fraction 1 (P1), fraction 2 (P2) and fraction 3 (P3), respectively. The enzyme-linked immunosorbent assay technique was used to compare the reactivity of the five partially purified antigens. The sensitivity and specificity of all five antigens were compared by testing against the sera of 78 O. viverrini-infected individuals from O. viverrini endemic areas and 70 individuals from non-endemic areas infected with hookworm, Trichuris and Ascaris including 49 individuals with negative stool examination. The assays performed with tegument extract, somatic extract and P1 fraction were found to have 100% sensitivity, whereas the sensitivities of those with P2 and P3 were 96.1% and 83.3%, respectively. The tegument extract had the highest specificity as demonstrated by the lowest cross-reactivity with other parasites. Our results indicated that surface tegument is the most suitable antigen for use in immunological diagnosis of opisthorchiasis.

Bionomics of Anopheles minimus and its role in malaria transmission in Thailand.

The bionomics of Anopheles minimus, one of the main malaria vectors in Thailand, were conducted in Pakchong district, Nakhon Ratchasima province, from January 1984 to June 1985. The prevalence of An. minimus was influenced by monthly rainfall, relative humidity, temperature and wind velocity, with a major peak of density from September to November. An. minimus preferred to feed on animal rather than on human, tended to bite human more outdoors than indoors, and thus exhibiting zoophilic and exophilic behaviour. The biting activity of the mosquitoes on animal exhibited high densities throughout the night in all seasons, whereas on human they tended to be an early evening biter in the dry cool season, and early morning biter in the wet season, and thus increasing the chance of man-vector contact. The life expectancy of An. minimus varied from month to month, ranging from 2.7 to 11.5 days, with the longest longevity during the dry cool season. The natural malaria infection rate of this species was very low. Out of 1,518 dissected guts, only 0.4% were found infected with oocysts. There were no sporozoites detected in the 1,560 dissected salivary glands.

Bionomics of Anopheles maculatus complex and their role in malaria transmission in Thailand.

The bionomics of Anopheles maculatus complex and its role in malaria transmission were conducted in Pakchong and Sadao districts, Nakhon Ratchasima and Songkhla provinces, respectively, from January 1984 to July 1985. In Pakchong, An. maculatus species A was the most dominant species, followed by species B form F and species C which was rare. The densities of species A and species B form F were high between July and November, with their peaks in October. Biting activities of both species occurred through out the night, with a major peak during the first quarter of the night on all seasons. In Sadao, only An. maculatus species B form E was detected with peak densities between February and June. Biting activities of this species varied according to seasons. The prevalence of mosquitoes was influenced by monthly rainfall, relative humidity and air-temperature. All species of female An. maculatus complex studied prefered to feed on animal rather than on human, and tended to bit human more outdoors than indoors, and thus exhibiting a zoophilic and exophagic behaviour. Life expectancies of An. maculatus species A ranged from 1.6 to 6.6 days, species B form F from 1.1 to 8.1 days, and species B form E from 0.7 to 21.2 days. The natural malaria infection rate was very low. Out of 4,430 guts dissected, only 0.23% were found infected with oocysts. There were no sporozoites detected in the 4,472 dissected salivary glands.

The host-parasite relationship between the Saudi Arabian Schistosoma mansoni and its intermediate and definitive hosts. 3. Effects of duration of exposure and cercarial concentration on the infection of mice by S. mansoni cercariae.

The percentage infection rate, worm burden and worm recovery rate in mice increased with an increase in the duration of exposure to cercariae. However, mice exposed to cercariae for 4 min had the same worm burden and worm recovery rates as those exposed for 16 min. Mice exposed to 80 and 160 cercariae each exhibited the highest percentage infection rates. The worm burden was highest in mice exposed to 160 cercariae each, while the worm recovery rate was highest in those exposed to 80 cercariae per mouse.

The host-parasite relationship between the Saudi Arabian Schistosoma mansoni and its intermediate and definitive hosts. 2. Effects of temperature, salinity and pH on the infection of mice by S. mansoni cercariae.

The infectivity of cercariae of the Saudi Arabian isolate of S. mansoni was found to be influenced by factors such as water temperature, salinity and pH. The optimum exposure temperature which resulted into the highest worm burdens and worm recovery rates in mice was 28 degrees C. However, the percentage infection rate was highest at a temperature range of 10 degrees to 34 degrees C. Mice were successfully infected with cercariae of S. mansoni at salinities of 0.5 to 6,400 mg/l. The highest worm burden and worm recovery rate occurred in mice infected by cercariae at a salinity of 100 mg/l, while the percentage infection rate was highest at a salinity range of 0.5 to 1,600 mg/l. Mice exposed to cercariae at the pH of 4.4 and 9.4 did not develop any infection. The percentage infection rate was highest in mice exposed to cercariae at a pH range of 6.4 to 8.4. However, both the worm burden and worm recovery rates were highest in mice at pH 5.4.

The host-parasite relationship between the Saudi Arabian Schistosoma mansoni and its intermediate and definitive hosts. 1. S. mansoni and its local snail host Biomphalaria arabica.

The infectivity of miracidia of the Saudi Arabian isolate of S. mansoni in Bi. arabica was found to be influenced by such factors as miracidial dose, water temperature and salinity. The pre-patent period of S. mansoni in Bi. arabica was 30 to 33 days. Miracidial dose had no effect on the mortality of snails during the pre-patent period. The infection rate increased as the miracidial dose was increased. However, cercarial production was highest in snails exposed to 1 miracidium each and decreased as the miracidial dose was increased. Water temperature during exposure had an influence on the mortality, infection rate and cercarial production in Bi. arabica exposed to S. mansoni miracidia. The infection rate was highest in snails exposed at 28 degrees and 34 degrees C. No infection of Bi. arabica occurred at the temperature of 10 degrees C. The number of cercariae per snail per day was highest in snails exposed to miracidia at 34 degrees C. It was demonstrated that salinity had an influence on the infection of Bi. arabica with miracidia of S. mansoni. The infection rate in snails decreased as the salinity increased up to 4,500 mg/l, above which no infection occurred. The daily pattern of cercarial emergence was rhythmic, whereby 94.7% of the total daily production was released within 6 h from infected Bi. arabica, with a peak between 11 a.m. and 1 p.m.

Clinical field trial of praziquantel in opisthorchiasis in Nong Rangya Village, Khon Kaen Province, Thailand.

A clinical field trial of praziquantel was carried out in Nong Ranya Village, Amphoe Ban Phai, Khon Kaen Province, with a population of 309 individuals, and 94% prevalence rate of opisthorchiasis. A mass treatment was carried out using a single dose of praziquantel at 40 mg per kg body weight. Acceptance for treatment was 91%. Follow-up stool examinations performed on days 14 and 60 gave prevalence rates of 20.5% and 22.2% respectively. Side effects including dizziness, headache, abdominal discomfort, nausea, vomiting, diarrhoea, lassitude, arthralgia, sleepiness, cramps and hot sensation were the complaints from 80% of adults and 40% of children. All of these were mild and transient except in one adult female who had severe diarrhoea and required intravenous fluid infusion.