Molecular and immunological characterization of encoding gene and 14-3-3 protein 1 in Fasciola gigantica.

A cDNA encoding Fg14-3-3 protein 1 was cloned by immunoscreening of an adult-stage Fasciola gigantica cDNA library using a rabbit antiserum against tegumental antigens of the parasite. The protein has a deduced amino acid sequence of 252 residues and a calculated molecular weight of 28.7 kDa. It shows sequence identity values between 57.6 and 58.1% to the human 14-3-3 beta, zeta, theta, and eta proteins and is in a phylogenetic cluster with the 14-3-3 protein 1 of Schistosoma spp. Nucleic acid analyses indicate that the Fg14-3-3 protein 1 is encoded by a single copy gene and that this gene is expressed as a transcript of 1250 nucleotides. In adult and 4-week-old parasites the gene's transcriptional and translational products were localized in the gut epithelium, parenchyma, tegument cells, and in the reproductive organs. An antiserum against recombinant Fg14-3-3 protein 1 detected a slightly smaller 14-3-3 protein in the parasite's excretion/secretion material and showed cross-reactivity with 14-3-3 proteins in extracts of other trematodes and mouse. Antibodies against Fg14-3-3 protein were detected in the sera of rabbits as early as 2 weeks after infection with metacercariae of F. gigantica and the antibody titre increased continuously over a 10-week observation period.

Histopathological alterations in the edible snail, Babylonia areolata (spotted babylon), in acute and subchronic cadmium poisoning.

Histopathological alterations in 6- to 8-month-old juvenile spotted babylon, Babylonia areolata, from acute and subchronic cadmium exposure were studied by light microscopy. The 96-h LC(50) value of cadmium for B. areolata was found to be 3.35 mg/L, and the maximum acceptable toxicant concentration (MATC) was 1.6 mg/L. Snails were exposed to 3.35 and 0.08 mg/L (5% of MATC) of cadmium for 96 h and 90 days, respectively. After exposure the gill, the organs of the digestive system (proboscis, esophagus, stomach, digestive gland, and rectum), and the foot were analyzed for cadmium accumulation. The results showed that most digestive organs had a high affinity for cadmium. The main target organ was the stomach, which could accumulate on average 1192.18 microg/g dry weight of cadmium. Cadmium was shown to accumulate to a lesser extent in the digestive gland, gill, rectum, esophagus, proboscis, and foot. Histopathological alterations were observed in the gill and digestive organs (proboscis, esophagus, stomach, and rectum). The study showed that the stomach and gill were the primary target organs of both acute and subchronic exposure. Gill alterations included increased size of mucous vacuoles, reduced length of cilia, dilation and pyknosis of nuclei, thickening of basal lamina, and accumulation of hemocytes. The epithelial lining of the digestive tract showed similar alterations such as increased size of mucous vacuoles, reduced length of cilia, and dilation of nuclei. In addition, fragmentation of the muscle sheath was observed.

Ultrastructural changes in various organs of the fish Puntius gonionotus fed cadmium-enriched cyanobacteria.

The accumulation and toxicity of cadmium in Puntius gonionotus fish that consumed the cyanobacterium Spirulina platensis contaminated with cadmium were studied. Fish were fed cadmium-contaminated cells for 4 weeks, after which cadmium accumulation in various organs was determined. The highest cadmium content was found in the kidney (56.0 microg Cd/g wet weight). Cadmium was not detected in the gill during the entire 4 weeks of cadmium feeding. Histopathological alteration of cells was observed in the gill, kidney, and liver. The results showed that dietary cadmium caused hypertrophy and edema of gill filaments. Coagulative necrosis and karyolysis of the nucleus were observed in the kidney. Vacuoles and hyaline droplets had accumulated in the epithelial cells of the proximal tubule. In the liver vacuolation of the cytoplasm, infiltration of macrophages, and focal necrosis were found. The ultrastructural changes that occurred in the cells of different organs were similar. These included a proliferation of vacuoles and lysosomes, formation of myelin bodies, degranulation, vesiculation, and dilation of rough endoplasmic reticulum, as well as swelling of mitochondria with loss of cristae.

Schistosomes in the Xe Kong river of Cambodia: the detection of Schistosoma mekongi in a natural population of snails and observations on the intermediate host's distribution.

A natural population of Neotricula aperta (Gastropoda: Pomatiopsidae) in the Xe Kong river of Cambodia was found to be infected with Schistosoma mekongi--apparently the first time this parasite has been found in snails outside the Mekong river. Only 0.14% of the N. aperta collected were found infected. Potential habitats for N. aperta were examined in selected rivers of central and southern Laos and Cambodia, so that the density of N. aperta at each site could be estimated. At survey sites where sufficient snails were collected, the population was also screened for infection with S. mekongi. The geographical distribution of N. aperta outside the Mekong river is shown to be much greater than previously documented; this is in agreement with predictions based on palaeo-geographical models and the evolutionary history and historical biogeography (phylogeography) of the species. The Xe Kong river is identified as a potential source of N. aperta colonists entering Cambodia from Laos. The findings, which indicate that there is a risk of human infection with S. mekongi in areas distant from the Mekong river, have important implications for schistosomiasis-control strategies in the region.

Investigation of Nematopsis spp. oocysts in 7 species of bivalves from Chonburi province, Gulf of Thailand.

This is the first detailed report of Nematopsis spp. in Thai bivalves. A monthly survey was conducted on 7 species of commercial bivalves from Chonburi province, on the eastern seaboard of Thailand, from November 2000 to November 2001 to investigate the prevalence of the apicomplexan parasite Nematopsis Schneider, 1892. Nematopsis spp. sporozoites were found in the cultivated bivalves Arcuatula arcuatula, Anadara granosa and Perna viridis as well as the locally harvested Paphia undulata. They were not found in Donax faba, Meretrix meretrix or Saccostrea cucullata. Using light microscopy, we were able to identiby 4 oocyst morphotypes of the gregarine Nematopsis spp. Prevalence of Nematopsis spp. during the 13 mo sampling period was highest in A. arcuatula (91.8%; n = 110) and lowest in A. granosa (59.2%; n = 130). The morphology of the oocysts differed between hosts, with an average (x +/- SD) length/width of 16.28 +/- 0.64/12.01 +/- 0.35 microm (n = 50) for A. arcuatula, 16.90 +/- 0.71/12.69 +/- 0.33 microm (n = 50) for A. granosa, 17.61 +/- 0.69/12.72 +/- 0.36 microm (n = 50) for P. viridis, and 11.21 +/- 0.62/8.55 +/- 0.52 microm (n = 50) for P. undulata. Identification of oocysts of these apicomplexan gregarines to species was not attempted. The prevalence of infection in relation to habitat and time of sampling is discussed.

Cadmium biosorption by cells of Spirulina platensis TISTR 8217 immobilized in alginate and silica gel.

The biosorption of cadmium by immobilized Spirulina platensis on alginate gel and silica gel was studied. The maximum biosorption capacities for alginate immobilized cells and silica immobilized cells were 70.92 and 36.63 mg Cd/g biomass, respectively. Temperature did not have an influence on metal sorption, whereas an initial pH solution did. Sorption occurred in a wide pH range (pH 3-8). The highest adsorption of alginate immobilized cells was at pH 6, while silica immobilized cell adsorption was not affected at pH between 4 and 7. The immobilized cells were reused in consecutive adsorption-desorption. The results showed that immobilized cells could be repeatedly used in the sorption process up to five times.

Opisthorchis viverrini and opisthorchiasis: a historical review and future perspective.

Opisthorchiasis is caused by the liver fluke, Opisthorchis viverrini. The fluke afflicts approximately seven million inhabitants in northeastern and northern Thailand. The fluke utilizes, respectively, freshwater snails and cyprinoid fish as its first and second intermediate hosts. Man is the accidental definitive host who acquires infection through the consumption of improperly cooked fish. Information pertaining to the history, the life-cycle, the epidemiology and transmission dynamics, and diagnosis of the fluke is compiled and presented.

Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus).

In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C(3)H(8)NO(5)P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p

Kinetics of basic dye (methylene blue) biosorption by giant duckweed (Spirodela polyrrhiza).

Wastewater containing pigments and/or dyes can cause serious water pollution problems in the form of reduced light penetration and photosynthesis, and the toxicity from heavy metals associated with pigments and/or dyes. Laboratory investigations, of the potential use of dried Spirodela polyrrhiza biomass as an adsorbent for the removal of the basic dye methylene blue from aqueous solution were conducted. A series of experiments were undertaken in an agitated batch adsorber to assess the effect of the system variables, i.e. sorbent dosage, pH, and contact time. The results showed that as the amount of the dried S. polyrrhiza increased, the percentage of dye sorption increased accordingly. At pH 2.0 the sorption of dye was not favorable, while the sorption at other pHs (3.0-11.0) was remarkable. There was no significant difference in the dye concentration remaining when the pH was increased from 3.0 to 11.0. The dye removal time was influenced by the initial dye concentration, and the process followed the first-order rate kinetics. The rate constants for intraparticle diffusion were 1.00 and 3.27 mg/g/min1/2 for 300 and 500 mg/l of dye, respectively.

Histopathological alterations of white seabass, Lates calcarifer, in acute and subchronic cadmium exposure.

Histopathological alterations to white seabass, Lates calcarifer aged 3 months in acute and subchronic cadmium exposure were studied by light and scanning electron microscopy. The 96-h LC50 values of cadmium to L. calcarifer was found to be 20.12 +/- 0.61 mg/l and the maximum acceptable toxicant concentration (MATC) was 7.79 mg/l. Fish were exposed to 10 and 0.8 mg/l of Cd (as CdCl,H2O) for 96 h and 90 days, respectively. The study showed that gill lamellae and kidney tubules were the primary target organs for the acute toxic effect of cadmium while in the subchronic exposure, the toxic effect to gills was less than that of kidney and liver. Gill alterations included edema of the epithelial cells with the breakdown of pillar cell system, aneurisms with some ruptures, hypertrophy and hyperplasia of epithelial and chloride cells. The liver showed blood congestion in sinusoids and hydropic swelling of hepatocytes, vacuolation and dark granule accumulation. Lipid droplets and glycogen content were observed in hepatocytes at the second and third month of subchronic exposure. The kidney showed hydropic swelling of tubular cell vacuolation and numerous dark granule accumulation in many tubules. Tubular degeneration and necrosis were seen in some areas.

The phylogeography of Asian Schistosoma (Trematoda: Schistosomatidae).

Partial (DNA) sequences are presented for 2 nuclear (18S and 28S rRNA genes) and 2 mitochondrial (12S rRNA and ND1 genes) loci for 5 species belonging to the Schistosoma japonicum, S. sinensium and S. indicum groups of Asian Schistosoma. Fresh field isolates were collected and cultured for the following taxa: S. incognitum (S. indicum group, central Thailand), S. mekongi (S. japonicum group, southern Laos), S. ovuncatum (S. sinensium group, northern Thailand), S. spindale (S. indicum group, northeast Thailand and central Thailand isolates) and S. sinensium (S. sinensium group, Sichuan Province, China). This represents the first published DNA sequence data for S. ovuncatum and for S. sinensium s.s. from the type locality in China. The paper also presents the first sequence data at the above loci for S. incognitum (except for the 28S sequences) and S. sinensium. Congruence was observed between the phylogenies estimated for each locus, although the relationships of S. incognitum were not so well resolved. Fitch-Margoliash, maximum likelihood (M/L) and maximum parsimony methods were used to estimate the phylogenies and the agreement between them was similar to that observed between loci. The ML tree was considered to best represent the data and additional 28S sequences (taken from the GenBank), for S. haematobium, S. japonicum, S. mansoni and Orientobilharzia turkestanicum, were used to construct an overall phylogeny. The S. indicum group taxa showed considerable divergence from the other Asian species and closest affinity with the African group. S. ovuncatum and S. sinensium appeared as sister taxa but their status as sibling species remained supported. The findings are discussed in the context of phylogeographical hypotheses for the origin of Schistosoma. An Asian origin for Schistosoma is also considered.

Cadmium biosorption by Sphingomonas paucimobilis biomass.

Among microorganisms isolated in Bangkok, the gram-negative bacterium Sphingomonas paucimobilis exhibited the greatest cadmium tolerance. It was able to survive in the medium containing cadmium as high as 200 mg/l. However, concentrations of cadmium at 25-200 mg/l inhibited its growth. The biosorption properties for cadmium of this bacterial biomass and the effects of environmental factors (i.e., biosorbent type, initial pH and biosorbent concentration) on the cadmium biosorption were explored. The results showed that the cadmium removal capacity of living cells was markedly higher than that of nonliving cells. Cadmium biosorption by S. paucimobilis biomass was also affected by the initial pH and biosorbent concentration.

Phytoremediation potential of Spirulina (Arthrospira) platensis: biosorption and toxicity studies of cadmium.

This study examines the possibility of using Spirulina (Arthrospira) platensis TISTR 8217 to remove low concentrations of cadmium (less than 100 mg/l) from wastewater. The cyanobacteria were exposed to six different cadmium concentrations for 96 h, and the growth rate was determined using an optical density at 560 nm. The inhibiting concentration (IC50) was estimated using probit analysis. The IC50 at 24, 48, 72, and 96 h were 13.15, 16.68, 17.28, and 18.35 mg/l Cd, respectively. Cellular damage was studied under a light microscope and a transmission electron microscope. Swollen cells and fragmented filaments were observed. Cell injury increased with increasing concentrations of cadmium. Ultrastructural changes were observed in the algae exposed to cadmium concentrations both close to IC50 (14.68 mg/l) and at IC50 (18.35 mg/l). The alterations induced by cadmium were disintegration and disorganization of thylakoid membranes, presence of large intrathylakoidal space, increase of polyphosphate bodies, and cell lysis. In addition, the cadmium adsorption by algal cells was studied. Environmental factors were found to have an effect on biosorption. The uptake of cadmium was not affected by the temperature of the solution, but the sorption was pH dependent. The optimum pH for biosorption of algal cells was 7. The cadmium uptake process was rapid, with 78% of metal sorption completed within 5 min. The sorption data fit well to the Langmuir isotherm. The maximum adsorption capacity for S. platensis was 98.04 mg Cd per g biomass.

Production and characterization of a monoclonal antibody against recombinant fatty acid binding protein of Fasciola gigantica.

In Fasciola parasites fatty acid binding proteins (FABPs) are the carrier proteins that help in the uptake of fatty acids from the hosts' fluids. Attempts have been made to utilize both native and recombinant FABP (rFABP) for immunodiagnosis and vaccine development for fasciolosis. In this study, we have produced a number of monoclonal antibodies (MoAbs) against rFABP of Fasciola gigantica. These MoAbs were initially screened against rFABP by ELISA and then tested for their specificities by immunoblotting. Five stable clones were selected and characterized further: four of them were of the isotype IgG(1) while one clone was IgG(2a). All the MoAbs reacted with rFABP which has a molecular weight (MW) of 20 kD and with at least two isoforms of native proteins at MW 14.5 kD that were present in the tegumental antigen (TA) and crude worm extracts, and the excretion-secretion materials. Immunoperoxidase staining of frozen sections of adult parasites by using these MoAbs as primary antibodies indicated that FABP were present in high concentration in the parenchymal cells and reproductive tissues, in low concentration in the tegument and caecal epithelium. All MoAbs cross-reacted with a 14.5 kD antigen present in the whole body (WB) extract of Schistosoma mansoni, while no cross-reactivities were detected with antigens from Eurytrema pancreaticum and Paramphistomum spp.

Schistosoma ovuncatum n. sp. (Digenea: Schistosomatidae) from northwest Thailand and the historical biogeography of Southeast Asian Schistosoma Weinland, 1858.

Schistosoma sinensium Bao, 1958 was first isolated from an unidentified snail in Sichuan Province, PR China. This species was apparently rediscovered in Chiang Mai Province, northwest Thailand (Baidikul et al., 1984); the definitive host was the rat Rattus rattus and the intermediate host was the snail Tricula bollingi. In this paper S. sinensium is rediscovered in Sichuan Province and compared with worms recovered from experimentally infected mice, which had been exposed to cercariae shed by T. bollingi from Chiang Mai. Evidence is presented suggesting that the schistosome collected by Baidikul was not S. sinensium and that a new species is involved. The new species, named Schistosoma ovuncatum (etymology: ovum (egg) + uncatus (hooked)), is described and compared with related taxa. All previous papers on the Thai schistosome have used worms recovered from field-collected rodents only; this is the first account in which the life-cycle has been completed in the laboratory, using cercariae shed by T. bollingi, and the resulting worms described. S. ovuncatum differs from S. sinensium in terms of size and shape of body and egg, number of testes, size of ovary, length of vitellarium, intermediate host and biogeographical distribution. The relationships of the two taxa and their position with respect to the Schistosoma indicum- and S. japonicum-groups are discussed. The implications of the findings for the evolution of human schistosomiasis in the region are also commented upon.

Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica.

In this study cDNAs encoding cathepsin L-like proteins of Fasciola gigantica were cloned by the reverse transcription polymerase chain reaction method (RT-PCR) from total RNA of adult specimens. DNA sequence analyses revealed that six different cathepsin L cDNA fragments were isolated, which have DNA sequence identities of 87-99% towards the homologous genes from F. hepatica. Gene expression was studied at the RNA level by Northern and RNA in situ hybridizations. Northern analysis showed the cathepsin L genes to be strongly expressed in adult parasites as a group of 1050 nt sized RNAs. RNA in situ hybridization localized cathepsin L RNA to the cecal epithelial cells. Southern hybridization was used to determine the number of cathepsin L genes and indicated the presence of a family of closely related cathepsin L genes in the genome of F. gigantica.

Fasciola gigantica: surface topography of the adult tegument.

Adult Fasciola gigantica are leaf-shaped with tapered anterior and posterior ends and measure about 35 mm in length and 15 mm in width across the mid section. Under the scanning electron microscope its surface appears rough due to the presence of numerous spines and surface foldings. Both oral and ventral suckers have thick rims covered with transverse folds and appear spineless. On the anterior part of the ventral surface of the body, the spines are small and closely-spaced. Each spine has a serrated edge with 16 to 20 sharp points, and measures about 20 microm in width and 30 microm in height. In the mid-region the spines increase in size (up to 54 microm in width and 58 microm in height) and number, especially towards the lateral aspect of the body. Towards the posterior end the spines progressively decrease in both size and number. The tegumental surface between the spines appears highly corrugated with transverse folds alternating with grooves. At higher magnifications the surface of each fold is further increased with a meshwork of small ridges separated by variable-sized pits or slits. There are three types of sensory papillae on the surface. Types 1 and 2 are bulbous, measuring 4-6 microm in diameter at the base with nipple-like tips, and the type 2 also have short cilia. Type 3 papillae are also bulbous and of similar size but with a smooth surface. These sensory papillae usually occur in clusters, each having between 2 and 15 units depending on the region of the body. Clusters of papillae on the lateral aspect (usually types 1 and 2) and around the suckers (type 3) tend to be more numerous and larger in size. The dorsal side of the body exhibits similar surface features, but the spines and papillae appear less numerous and are smaller. Corrugation and invaginations of the surface are also less extensive than on the ventral side of the body.