Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica.

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.

Predisposition to reinfection by intestinal helminths after chemotherapy in south Thailand.

Rates of reinfection by the intestinal helminths hookworm, Ascaris lumbricoides and Trichuris trichiura after chemotherapy were studied in two villages in Phang-Nga Province, southern Thailand. It was found that intensity of infection levels attained after reinfection correlated positively with pretreatment intensities of infection for all parasites. This implies that certain persons in the community are predisposed to receiving high numbers of worms, due either to environmental or personal risk factors. Therefore, it would be advantageous to identify such persons and treat them preferentially. Targeted chemotherapy, however, should be combined with efforts to identify the risk factors that vary within the community and direct educational efforts or environmental intervention towards the section of the community most affected by the parasites.

Prevalence, incidence, intensity and associated morbidity of intestinal helminths in south Thailand.

Intensive surveys for Ascaris lumbricoides, Trichuris trichiura and hookworm were carried out in two villages in Phang Nga Province, southern Thailand, in order to measure prevalence, estimate incidence and determine the relation between intensity of infection and morbidity before and after chemotherapy. The study populations were a small upland village community (Nai Tone) and a grade school in a small coastal village (Boh Saen). About half of the Nai Tone villagers were given a broad spectrum antihelminthic (albendazole), and the Boh Saen students were all treated successively with three drugs: piperazine citrate to treat for Ascaris, pyrantel pamoate to treat for hookworm, and mebendazole to treat for Trichuris. Stool examinations were made using the quick Kato smear technique, questionnaires were administered concerning a variety of possible symptoms, and anthropometric and blood biochemical parameters were measured both before and after treatment. The prevalence of Ascaris was 31.0 and 22.6%, hookworm was 89.1 and 88.0% and Trichuris was 59.7 and 77.8% in the Nai Tone and Boh Saen study populations, respectively. Average intensity of Ascaris was highest in the 0-9 year age class (greater than 32,000 epg) in Nai Tone Village. Hookworm intensity of infection was higher in males than in females in all age classes, and in Nai Tone Village at least 25% of males and 20% of females had 8000 or more epg of faeces. Trichuris intensity of infection was highest between 5 and 10 years of age in both populations. The only signs or symptoms showing a significant (P less than 0.05) difference between high and low classes of intensity of infection and a significant improvement (P less than 0.01) after drug treatment, were headache and flatulance in the case of hookworm infection in Boh Saen School. The presence of multiple infections made testing of hypotheses concerning particular parasite species difficult.

Genetic variation and differentiation of three Schistosoma species from the Philippines, Laos, and Peninsular Malaysia.

Electrophoretically-detected allozyme variation is described in strains of Schistosoma japonicum (4 Philippine strains), S. mekongi (Laos), and an undescribed anthropophilic S. japonicum-like schistosome from Peninsular Malaysia. Result, together with those reported previously for 8 other strains (S. japonicum, China, Formosa, Japan, Philippines; S. mekongi, 2 substrains; Malaysian schistosome, 2 strains) permit a composite genetic characterization of 15 strains of Asian schistosomes at 9-18 presumptive loci. The proportion of polymorphic loci (P) and the mean heterozygosity per locus (H) were zero in all strains. Although this was expected for strains that had been in laboratory culture for up to 50 years, we expected to detect variation in strains based on 10-50 recently field-collected infected snails. We expected S. japonicum to be as variable as S. mansoni (P = 0.13 (0-0.33), H = 0.04, 18 loci, 22 strains) as it is believed to reproduce sexually, has an evolutionary history of several million years, inhabits a wide geographic range, coevolved with a genetically variable intermediate snail host, and has a diversity of mammalian hosts. No differences were detected between the 5 S. japonicum strains from Leyte and Luzon (Philippines), between the 3 S. mekongi strains, or between the 3 Malaysian schistosome strains; these groups and the remaining S. japonicum strains representing Mindoro (Philippines), China, Formosa, and Japan each have distinctive multilocus electromorphic patterns. Nei's genetic distances (D) were calculated to estimate interstrain and interspecific divergence. Interstrain genetic distances in S. japonicum averaged greater than 0.3; much higher than those reported previously for S. mansoni (D = 0.06, D(max) = 0.24). S. japonicum (Mindoro) was moderately differentiated from the Leyte-Luzon strains (D = 0.29, 12 loci). Estimates of the S. japonicum China-Philippine distance (D greater than 0.4, 11 loci) are high for conspecific populations and further studies of the still poorly characterized Chinese parasite may reveal that these are, in fact, separate species. S. japonicum is shown to be only distantly related to S. mekongi and the Malaysian schistosome (D greater than 1); the latter is closely related to, but genetically quite distinct from, S. mekongi (D = 0.61 +/- 0.275, 11 loci) and warrants recognition as a new species. The medical significance of the isogenic nature of the Asian schistosome strains and their evolutionary divergence are discussed.

Incidence of endemic Opisthorchis viverrini infection in a village in northeast Thailand.

Incidence, measured as the proportion of persons whose stools become positive within one year, was studied in endemic Opisthorchis viverrini, the human liver fluke, in a northeastern Thai village over a two-year period. Incidence was higher in males than in females, especially in children under five years of age. It was at least 47% overall in the first year of the study, but declined to below 20% per year in the second. This is attributed to drying of a local water reservoir and decline in availability of infective stages in fish. The fluctuation of incidence is probably due to the large variations in rainfall from year to year. The rate of reversion from positive to negative varied from 2% to 6% per year.

Consistency of fecal egg output in patients with Opisthochiasis viverrini.

Counts of Opisthorchis eggs in two samples of feces arbitrarily taken 0-21 days apart from each of 209 patients from northeastern Thailand were carried out by Stoll's dilution technique. Based on the number of eggs/mg feces, each patient was classified as uninfected, lightly infected (less than 1 egg/mg), moderately infected (1-10/mg), heavily infected (greater than 10-50/mg), or very heavily infected (greater than 50/mg). The intensity of infection for each of the 209 individuals as shown by first and second counts was compared. Of the 209 individuals, 150 (71.8%) remained within the same egg excretion category, with all but five (2.4%) of the remainder being in an adjacent category. Statistical analysis showed no effect of variation in interval between stool counts on the accuracy or consistency of replicate counts.

Morbidity in relation to intensity of infection in Opisthorchiasis viverrini: study of a community in Khon Kaen, Thailand.

A study of the prevalence and intensity of opisthorchiasis viverrini in relation to morbidity as determined by standard medical examination was carried out in Nong Ranya, a small village containing 309 people in northeastern Thailand. Opisthorchis viverrini infection as determined by Stoll's quantitative egg count method had an overall prevalence of 94% and reached 100% prevalence in most age groups above the age of 10 years. With respect to intensity, 6% were uninfected, 26% had light (less than or equal to 1 eggs per mg [epmg] of feces), 37% moderate (greater than 1-10 epmg), 25% heavy (greater than 10-50 epmg), and 5% very heavy (greater than 50 epmg) infections. Peak intensity in both males and females occurred at age 40 and above. A history of eating "koipla" (a sauce consisting of ground up raw fish), of feeling weak, and of having right upper quadrant abdominal pains occurred most frequently in the infected groups and was correlated with intensity of infection. Regardless of intensity of infection, only a small proportion of the population were unable to carry out their routine activities. Anorexia, nausea or vomiting bore little relationship to the presence or intensity of infection. Hepatomegaly at the mid-clavicular line occurred in 14% of the population, mainly in the more heavily infected groups. Neither jaundice nor splenomegaly was observed in the population.

Measurement of incidence of the human liver fluke, Opisthorchis viverrini, in northeast Thailand.

A survey of a community in northeast Thailand where Opisthorchis viverrini is endemic permitted comparison of two methods of measuring incidence: direct determination of the rate at which an uninfected group became infected between two surveys a year apart, and estimation of incidence from age-specific prevalence data using a logarithmic regression method. Both methods revealed that incidence increased with age in young children, and estimation from age-prevalence data showed that the increase was roughly linear from near birth to about age 5 years, beyond which no clear trend in incidence was evident. A catalytic infection model incorporating an infection rate increasing from birth to age 5, and remaining constant thereafter, gave an excellent fit to age-prevalence profiles. Both methods of determining incidence are sensitive to errors in diagnosis, but the direct determination method is more sensitive to the presence of false negatives. A method for correcting this bias is given. The regression method, which was less sensitive to yearly variation in incidence and is easier to use, is recommended for preliminary surveys to identify villages with high transmission intensity.

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.

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.

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.

Isoenzyme analyses of Malaysian Schistosoma, S. mekongi and S. japonicum by isoelectric focusing in polyacrylamide gel.

Isoenzyme patterns of adult Malaysian Schistosoma, S. mekongi and S. japonicum strains were analysed by isoelectric focusing (IEF) in polyacrylamide gel. Enzyme patterns obtained from Malaysian Schistosoma homogenates differed from those of S. mekongi and S. japonicum strains. Malaysian Schistosoma was found to differ from S. japonicum by 8 enzymes, namely phosphoglucomutase, phosphoglucoisomerase, malate dehydrogenase, acid phosphatase, hydroxy-butyrate dehydrogenase, hexokinase and alkaline phosphatase, and from S. mekongi by phosphoglucomutase, malate dehydrogenase, aldolase and alkaline phosphatase. These results and the distinct biology of the parasite suggest that Malaysian Schistosoma is a new species in the S. japonicum complex.

Initial characterization of Trypanosoma lewisi surface membrane antigen.

The purified T. lewisi were subjected to hypotonic lysis plus freezing and thawing in acetone dry ice bath. The trypanosome ghosts were obtained after repeated washing and centrifugation. The homogenized ghost suspension was assayed for enzyme Na++K+ ATPase activity to ratify the presence of the trypanosome surface membrane. Membrane solubilized in sodium dodecyl sulfate were fractionated by gel filtration on Sephadex columns equilibrated with the detergent and electrophoresis in polyacrylamide gel. Crude trypanosome surface membrane antigens were tested for their immunogenicity, administered to rats in Fraund's complete adjuvant. The results of these experiments indicated that the protective immunogen is tightly bound to the membrane since the use of strong anionic detergent is necessary in its extraction.

The effect of controlled release copper sulfate and tributyltin fluoride on the mortality and infectivity of Schistosoma mansoni miracidia.

The experiments were conducted to assess the effect of sublethal concentration of controlled release copper sulfate (EC-8) and tribultyltin fluoride (CBL-9B) on the mortality and infectivity of Schistosoma mansoni miracidia. It was found that LC50 and LC90 of EC-8 to the miracidia were 8.5 mg/l per hr and 15.75 mg/l per hr respectively. For CBL-9B, LC50 was 19.75 microgram/l per hr and LC90 was 32.0 microgram/l per hr. Exposing of the miracidia to 0.5 mg/l of EC-8 or 4.0 microgram/l of CBL-9B respectively, for only one hour can prevent their transmission to the snail host. The results indicate that immobilization of the free living stages of the schistosome parasite was not required to interrupt their transmission to the next hosts.

Enzyme-linked immunosorbent assay for detection of Opisthorchis viverrini infection.

Antibodies to O. viverrini in the sera of people from endemic and non-endemic areas were investigated using indirect ELISA technique. For the patients from the endemic area, 92.8% who passed eggs in the stool were found to be positive for O. viverrini antibody. In addition, 46.5% of the people who did not pass eggs in the stool were also found to have low titer of O. viverrini antibody. On the other hand only 2.4% of the people from the non-endemic area with other intestinal parasite infections were found to have O. viverrini antibody in their sera. It was concluded that positive reaction of O. viverrini antibody is not cause by cross-reaction with other parasites but low liter of antibody is probably due to low-level or past infection. There is a positive correlation between the titer of O. viverrini antibody and intensity of infection as indicated by number of eggs excreted per milligram of feces. Patients with a few O. viverrini eggs in feces, but biopsy-proved-cholangiocarcinoma had very high titer of antibody.

Opisthorchis viverrini : intensity and rates of infection in cyprinoid fish from an endemic focus in Northeast Thailand.

The infection rates and monthly variations in intensities of Opisthorchis viverrini infection in cyprinoid fish from an endemic focus in Northeast Thailand were investigated between April 1980 and March 1981. Out of six species of cyprinoid fish examined, four were found to harbour O. viverrini metacercariae. All four species of infected fish showed high rates of infection. Among the four species of O. viverrini infected fish, Cycloccheilicthys apagon and Puntius leiacanthus were found in abundance. The results indicate very little variation in the rates of infection throughout the year. On the other hand, intensities of infection varied from month to month, ranging from 8 to 88 metacercariae per fish for C. apagon and from 8 to 32 metacercariae per fish for P. leiacanthus. O. viverrini metacercariae were found in the fish muscle throughout the body; however, the body muscle harboured the highest density of metacercariae, followed by the dorsal fin, pectoral fin, pelvic fin and anal fin muscles respectively. In lieu of all these findings, it is therefore invalid to assume as indicated by the former workers that any potential intermediate fish host that does not have the O. viverrini metacercariae in its pectoral fin muscle in negative with opisthorchiasis.

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.

Biology of Robertsiella kaporensis snails and Malaysian Schistosoma.

Studies on the bionomics and host-parasite relationship of Robertsiella kaporensis and Malaysian Schistosoma were investigated. The study was divided into 4 parts: cultivation of snails, R. kaporensis, and maintenance of Malaysian Schistosoma life cycle, daily cercarial shedding cycle in R. kaporensis, miracidial load and cercarial shedding pattern and the infectivity of Malaysian Schistosoma cercariae. R. kaporensis were cultured in the laboratory with the use of plastic container provided with fine sand. The snails were fed with diatoms and Saraca leaves. The development period for the snail eggs was about 20-30 days, the young grew to maturity in 14-15 weeks, and the average growth rate of snails was 0.23 mm per week. The daily cercarial shedding cycle of snails had shown that the peak emergence of cercariae of Malaysian Schistosoma occurred at night, between 6-10 pm. The miracidial load which yielded the best results in terms of percentage infection rates of snails and cercarial output was the miracidial concentration of 8 miracidia per snail. The study on infectivity of Malaysian Schistosoma cercariae has shown that there was a decrease in infectivity of the cercariae to mammalian hosts as the cercariae increased in age. The percentage infection rate of mice and numbers of worms recovered were highest in mice infected with cercariae of 0-1/2 hr. old. Infection of cercariae fell rapidly after the cercariae were 16 hr. old.

Comparative methods of infecting mice with Schistosoma mekongi.

Experiments were carried out to investigate the most suitable cercarial concentration and method of exposure for the experimental mice infected with cercariae of Schistosoma mekongi. Swiss albino mice of 2 months old were exposed individually by three methods exposure : abdominal skin exposure, tail immersion and subcutaneous injection. Cercarial concentrations of 10 to 20 cercariae per mouse were used for each method of exposure. In general, it was observed that both the infection rate and total worm recovery rose with an increase in cercarial concentration. The highest percentage infection rate and total worm recovery was found in mice infected with 20 cercariae per mouse by the abdominal skin exposure method.

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.