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.

Sex ratio and susceptibility of the golden apple snail, Pomacea canaliculata.

Golden apple snails, Pomacea canaliculata, were collected at two localities having different ecological environments. In both canal and pond, P. canaliculata males were found more than females during the dry season (summer and winter). In the canal, the male snails were highest in number (86.67%) in May. When rain started, they began decreasing and were lowest at 33.33% in August. Of 575 snails collected, 30.6% were infected by one or more of the three groups of amphistome, distome and echinostome metacercariae. There were two high peaks of infection in April and October, as 60.7% and 68.4%, respectively, during which there were more males than females. The average number of parasites per snail which was highest at 54 was found in the medium-sized males (25 out of 35 males) in October. The number of parasites per snail was significantly correlated with the collected males (p < 0.01), but such relationship was not occurred with the females. Of the females, only the large-sized individuals were infected. In the pond, the female snails were present in much greater numbers than the males during the reproductive time (June-September). The females were highest (94.23%) in August. Only 24 (4.0%) of 605 snails were infected; most of the infected snails were large.

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.

Natural parasitic infection of the golden apple snail, Pomacea canaliculata.

Golden apple snails, Pomacea canaliculata, were collected once a month during a year to search for their natural parasites. The collections were made at two localities having different ecological environments. Of 576 collected snails from a canal, 176 individuals (30.6%) were infected by three groups of metacercariae. These parasites were amphistome, distome and echinostome metacercariae, which had prevalences of 23.5, 19.5 and 0.5%, respectively. The incidence of infection was highest (68.4% in October) when the snail population was composed of the old, juvenile and young Pomacea. Amphistome metacercariae were found most frequently and echinostome metacercariae the least frequently; both parasites were localized in the foot muscle of the snails and had a Shannon index of zero. The range of amphistomes was 1 to 115 with the mean +/- SD of 1 +/- 2 and 95% CL of 1, 2. Distome metacercariae were found primarily in the heart (range: 1-13), and also in the foot muscle (range: 1-5) and kidney (range: 1-14), with a Shannon index of 0.4. The means +/- SD (with 95% CL) were 3 +/- 4 (95% CL = 1, 5), 3 +/- 4 (95% CL = 2, 4) and 2 +/- 1 (95% CL = 1, 2) for the foot muscle, heart and kidney, respectively. The snails from a pond, another locality, had a low proportion of infected individuals. Of 605 snails, only 24 individuals (4.0%) were infected, with the prevalence of amphistomes, distomes and echinostomes being 0.8, 1.8 and 2.1%, respectively. The incidence of infection for each month was zero or less than 10%, except in May when it was 30.2%.

Mosquito larvae and associated macroorganisms occurring in gem pits in southern Tha Mai District, Chanthaburi Province, Thailand.

Aquatic field studies were conducted in Tha Mai District, Chanthaburi Province, Thailand. Larval habitats of Anopheles dirus were examined from November 1986 through June 1988 in 42 manmade gem pits. Larvae were found in pits containing clear water under full or partial shade. The abundance of different kinds of mosquito larvae were related to seasonal changes in these aquatic habitats. Variations in An. dirus density and occurrence were related to predators populations, ie Notonectidae and fish.

Anopheles dirus size and fecundity: relationship to larval density and protein accumulation.

Anopheles dirus were reared at two different larval densities (100/pan and 400/pan) to produce two different size classes of adults. Both the wing length and fecundity of females from the two densities were significantly different (p less than 0.001). Adult size was related to larval density and protein accumulation during immature life. Egg production was also related to adult size. Consequently, larger field adults have the potential to live longer and produce more eggs. The smaller adult size associated with crowded larval development was apparently caused by space associated effect on feeding rather than by a shortage of food per se.

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.

Long residual activity of Bacillus sphaericus 1593 against Culex quinquefasciatus larvae in artificial pools.

The microbial larvicide, Bacillus sphaericus 1593, was evaluated against Culex quinquefasciatus larvae in artificial pools at Huay Kwang area in Bangkok between March and October 1985. This study was aimed at determining the residual activity of B. sphaericus 1593 in waste water under field conditions. The larvicidal activity of B. sphaericus 1593 was found to persist for at least 5 months in artificial pools containing waste water. The populations of B. sphaericus 1593 in the test water fluctuated, decreasing by approximately 4.25 and 3.47 log10 colony forming unit (cfu)/ml from the original concentrations in 60 and 80 days after application and then increasing approximately 2.92 and 2.77 log10 cfu/ml in 92 and 72 days for pools No. 1 and No. 2, respectively. This evidence indicates that B. sphaericus 1593 can recycle in such conditions.

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.

Scanning electron microscopic study of the tegumental surface of the hybrid schistosome between Schistosoma mekongi and S. japonicum-like (Malaysian).

Hybridization experiments between the two non-sibling species of schistosomes, Schistosoma mekongi in man and S. japonicum-like (Malaysian) in rodents, were carried out. Two laboratory-bred snail species, Tricula aperta (beta race), the snail host of S. mekongi and Robertsiella kaporensis, the snail host of S. japonicum-like (Malaysian), were used for the production of cercariae. Cross mating between S. mekongi and S. japonicum-like (Malaysian) were achieved in the laboratory by the usual procedure of exposing snails to single miracidia of each species, then exposing mice to cercariae emanating from two snails only, each infected with a different species. Hybrid eggs and miracidia were used to infect snails of both species. The resultant F1 cercariae were used to infect mice. It was shown in this study that the attempt to cross these two species of schistosomes could be achieved in the laboratory, but the results provided very low yield of hybrid worms and eggs. F1 hybrid adult worms from S. mekongi male and S. japonicum-like (Malaysian) female were obtained and examined for the microtopography of the tegument by scanning electron microscopy. The tegumental surface of the hybrid male schistosome resembled the male parent, S. mekongi, with a few characters which resembled the male, S. japonicum-like (Malaysian). The surface tegument of the hybrid male worm was characterized by the presence of highly-branched and perforated ridges interspersed with a large number of papillae all over the body surface with the heaviest concentration on the middle portion of the body. There were four types of papillae present; the pleomorphic papillae; the cratered papillae, with or without cilia; the hemispherical sensory papillae with cilia; and the fungiform papillae. Spines were absent on the body surface except in the oral and ventral suckers and in the gynecophoral canal. The tegument lining the gynecophoral canal was characterized by the presence of low ridges with scattered papillae with small number of short spines in the posterior portion of the canal. In contrast to the male, the female hybrid worm had numerous spines all over the body surface with the most concentration in the posterior region. Among the spines were low perforated ridges. Two types of papillae were present in the female hybrid; the cratered papillae, with or without cilia, and the hemispherical papillae.

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.