Soil factors influencing the distribution of Oncomelania quadrasi, the intermediate host of Schistosoma japonicum, on Bohol Island, Philippines.

Soil conditions essential to the survival of Oncomelania quadrasi on Bohol Island in the Philippines were examined to clarify the factors limiting distribution of the snail and to develop a method for breeding large numbers of the snail in the laboratory. Soil samples in and around snail habitats were analysed and used for breeding experiments in the laboratory. Experiments using paddy soil derived from different parent materials revealed that the numbers of juvenile snails hatched varied widely between several soil samples. The best soils for reproduction generally had a pH of 5.6-7.9 and > 200 mg of available CaO/100 g. These soil factors, in addition to shade and moisture, determine the optimum conditions for the breeding of O. quadrasi in the field as well as in the laboratory. The determination of the optimum conditions for laboratory breeding of O. quadrasi and other intermediate snail hosts should facilitate detailed study of the hosts and the development of better methods to control or eradicate schistosomiasis and other snail-transmitted diseases.

Ultrasonographic and serologic abnormalities in Schistosoma japonicum infection in Leyte, the Philippines.

We have identified specific ultrasonographic changes in Schistosoma japonicum-infected patients associated with serologic indicators of general liver function. An ultrasonographic examination concomitant with hematologic and biochemical serum analyses was performed on 102 patients at the Schistosomiasis Hospital in Leyte, The Philippines. The ultrasonographic liver images were classified into four patterns, according to the development of periportal fibrosis and the patterns of echogenic bands. Eleven cases with a long-term infection showed typical septal formation (network pattern). Other ultrasonographic changes in the portal system, such as the severity of splenomegaly, did not correlate with the age of the study patients or the duration of their infection; however, the production of collateral vessels was clear in the group of older patients. Among various hematologic and biochemical serum indicators of liver damage, the serum levels of total bile acid (TBA) and procollagen-III-peptide (P-III-P) strongly correlated with the development of hepatic fibrosis and protal hypertension. These findings suggest that the ultrasonographic liver patterns classified here, along with the changes in serum levels of TBA and P-III-P, provide useful indicators for field monitoring of S. japonicum infection.

Improvement of ultrasonographic and serologic changes in Schistosoma japonicum-infected patients after treatment with praziquantel.

We previously reported ultrasonographic and serologic abnormalities in 102 patients infected with Schistosoma japonicum in Leyte, The Philippines. These patients were subsequently treated with praziquantel (3 x 20 mg/kg), and changes in ultrasonographic images and the serum levels of liver function markers in 52 patients were followed up every three months for a period of 17 months. Improvement in the thickening of the portal vein wall and the intensity of echogenic bands was detected six months after treatment with praziquantel. The level of splenomegaly was also reduced in 42 patients who originally did not show the production of collateral vessels. A significant decrease in the serum total bile acid (TBA) level was detected in all patients six months after treatment with praziquantel. However, significant ultrasonographic changes could not be detected in the patients classified as type 3, with severe hepatic fibrosis caused by the long-term infection. These results clearly show that ultrasonographic examination, along with data on the serum TBA level, provides a sensitive tool to monitor the severity of hepatic fibrosis and portal hypertension caused by S. japonicum infection, as well as the improvement resulting from praziquantel treatment.

Serologic and ultrasonographic parameters of praziquantel treatment of hepatic fibrosis in Schistosoma japonicum infection.

We describe the parameters useful in evaluating the development of hepatic fibrosis in Schistosoma japonicum infection, as well as its improvement after treatment with praziquantel (PZQ). Various serologic parameters and ultrasonographic images were examined, and their changes were monitored using rabbits infected with 200 or 300 cercariae of S. japonicum. Infected rabbits were administered one oral treatment of PZQ at a dosage of 100 mg/kg at 6, 12, or 24 weeks after infection. Histopathologic examinations revealed that PZQ had a strong and rapid effect, even on damage that developed long after the infection. The improvement of moderate hepatic fibrosis that developed over 24 weeks after infection was also detected by histopathologic examinations. The serum level of total bile acid was the most sensitive parameter in evaluating the severity of hepatic fibrosis and its improvement after treatment with PZQ. The level of serum procollagen-III-peptide was also useful in evaluating the development of hepatic fibrosis, but not in its improvement. Ultrasonography revealed specific echogenic bands and nodules according to the progress of granuloma formation and fibrosis, and the reversal of these changes could also be observed after treatment with PZQ.

Existence of host-related DNA sequences in the schistosome genome.

DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

Dynamic changes of DNA sequences in Schistosoma mansoni in the course of development.

Deletion and/or amplification of DNA sequences in Schistosoma mansoni were demonstrated by Southern blot analysis. Total cellular DNAs and genomic clones derived from S. mansoni miracidia, adult males and females were used as probes. Endonuclease BamHI-restricted DNAs from miracidia, adult males and females of both S. mansoni and S. japonicum were reacted to each probe. Hybridization with a total cellular DNA from S. mansoni miracidia as a probe showed elimination of signals in S. mansoni adults. On the other hand, blot analysis using a total cellular DNA from S. mansoni adult males as a probe revealed elimination of hybridization signals in S. mansoni miracidia. Hybridization with a clone SmE15 DNA from S. mansoni miracidia as a probe showed no signal in the DNAs from S. mansoni adults, indicating these sequences deleted in adults. Hybridization experiments using the probes SmF25 and SmM51 which are 1.3 and 2.2 kb fragments cloned from S. mansoni adult females and males respectively, demonstrated no signal to DNA from S. mansoni miracidia. Our data suggested the existence of stage-specific DNA sequences in S. mansoni. We propose a model for multiple-step rearrangement of DNA sequences in S. mansoni during the course of development.

Oral itraconazole and topical miconazole with débridement for Acanthamoeba keratitis.

We treated three patients who had Acanthamoeba keratitis with oral itraconazole, a new antifungal agent, topical miconazole, and surgical débridement of the lesion. In these patients, healing and regression of the keratitis began six or seven days after initiation of oral itraconazole and miconazole 0.1% eyedrops (every hour during the day). The clinical signs of corneal infection disappeared after nine weeks in Patient 1, after five weeks in Patient 2, and after eight weeks in Patient 3. Visual acuities improved markedly from hand motions to 20/30 in Patient 1, from counting fingers to 20/16 in Patient 2, and from hand motions to 20/40 in Patient 3. In these patients, no systemic or topical signs of toxicity or adverse reactions were noted during the course of treatment.

Growth and fecundity of Schistosoma japonicum in mice maintained at different environmental temperatures.

On weeks 7 and 9 post infection (PI), the number of Schistosoma japonicum in mice maintained at low (5 degrees C) and high (32 degrees C and 35 degrees C) temperatures did not differ from those in controls (25 degrees C), but shorter males were observed in hosts at 5 degrees C. Further, both male and female worms recovered on week 7 PI from mice kept at 35 degrees C were shorter than those recovered from controls. Staining analysis revealed that worm maturation was not affected in mice kept at 5 degrees C and 32 degrees C. However, some female worms from mice kept at 35 degrees C had no eggs in the uterus. The day of patency of worms in the temperature-stressed mice did not differ from that in controls. EPG in mice kept at 32 degrees C rose to a high level on weeks 7-8 PI which was about 10 times as many as those in control mice, while EPG in mice kept at 5 degrees C appeared to be lower than in controls. The 7-week tissue egg count revealed that the hot- and cold-stressed mice supported significantly less egg productivity per female worm than control mice. The environmental temperature could not alter the migration pattern of schistosomula from the skin to lungs in mice.

Schistosoma japonicum: the effect of serum fractions precipitated by ammonium sulfate on viability of schistosomula.

The effect of serum fractions precipitated by a saturated ammonium sulfate solution on viability of Schistosoma japonicum schistosomula was investigated. Axenized cercariae were incubated for 10 days at 37 degrees C in NCTC 109 supplemented with the 34%, 40%, 50%, 62% and 68% fractions of human or rabbit serum in an equivalent concentration to 10% serum. Only larvae cultured in media with the 68% fraction of human or rabbit serum retained the transparent feature and active motility, while the larvae cultured in other media became opaque and immotile. This was also true in media with human serum albumin or fatty acid-free human serum albumin at concentrations of 1.0 and 10.0 mg/ml of NCTC 109. On 10-day culture a part of larvae in the medium with the 68% fraction of human serum began to feed on red blood cells and the intestine was nearly closed by 21 days. These results suggest that serum fraction of 68% ammonium sulfate consisting of albumin is an important constituent in supporting the further growth of schistosomula in vitro.

Ultrastructural observations on damage to schistosomula of Schistosoma japonicum by mouse neutrophils in vitro.

Ultrastructure of schistosomula of Schistosoma japonicum was studied in relation to the effect of mouse neutrophils in vitro. In the presence of antibody and complement, damage to the schistosomular tegument covered with many neutrophils began to appear within 1 h incubation. Exfoliation of the granular cytoplasm of the tegument, disorganization of the muscle layers and vacuolation in the inner tissues were seen in a part of neutrophil-attached parasites by 2 h incubation. However, the majority of schistosomula with fewer cells retained their integrity till 16 h. The cytochemical examination of schistosomula incubated with mouse neutrophils for 1 and 2 h demonstrated clearly the localization of the peroxidase activity on the surfaces of the cells and the parasites.

Schistosomiasis on Bohol Island, Philippines, with special emphasis on the successful discovery of new habitats of the vector snail, Oncomelania quadrasi, and area-wide mollusciciding.

In the endemic area of schistosomiasis on Bohol Island, we succeeded in locating seven new colonies of the vector snail, Oncomelania quadrasi, four in Trinidad and three in Talibon, in addition to six locations from which the snails were previously recorded. We were led by the proximity of the snail habitat to the residence of the infected individuals. In northern Bohol the continuous wet season would appear to make the major differences between the places that do and those that do not support O. quadrasi. Twice-yearly weed clearance and applying chemical molluscicides (niclosamide and phebrol) for the control of O. quadrasi have been extensively in effect over the snail colonies, since July 1986. Although it has not yet yielded satisfactory results in certain colonies, snails have been eliminated from six habitats in a period of two and a half years.

A comparison of the antischistosomal effect of levo- and dextro-praziquantel on Schistosoma japonicum and S. mansoni in mice.

Praziquantel (PZQ) is a racemic compound composed of equal proportions of its optical isomers, levo- and dextro-PZQ. The efficacy of these compounds was compared with that of PZQ in mice infected with Schistosoma japonicum or S. mansoni. Mice were given 50, 2 x 50 (on consecutive days), 500, or 2 x 250 mg/kg of each compound orally 5 weeks after infection. Significant reduction of worm recovery was observed in S. japonicum infection 30 days after treatment with 2 x 50, 500, or 2 x 250 mg/kg of levo-PZQ, whereas no therapeutic effect was demonstrated with dextro-PZQ. Percent reduction in worm burden in mice treated with levo-PZQ was significantly higher than in those with PZQ at a dosage of 2 x 50 mg/kg (67.9% vs. 34.5%). Neither eggs in feces nor miracidial hatching of eggs from the livers and intestines were observed in mice treated with levo-PZQ. In S. mansoni infection, levo-PZQ showed no significant schistosomicidal effect compared with PZQ and dextro-PZQ, although there was reduction in egg counts.

Schistosoma japonicum and S. mansoni: ultrastructural damage in the tegument and reproductive organs after treatment with levo- and dextro-praziquantel.

Ultrastructural observations were made of changes in the tegument and reproductive organs of Schistosoma japonicum and S. mansoni from ICR mice after treatment with praziquantel (PZQ), levo-PZQ, and dextro-PZQ at a single oral dose of 500 mg/kg body weight. No marked difference in types and extent of lesions of the tegument of S. japonicum was found between the compounds regardless of the time of worm recovery after treatment. This was equally true of S. mansoni. Degeneration of the testis, ovary, and vitelline gland of S. japonicum was more prominent in worms administered PZQ and levo-PZQ than in those receiving dextro-PZQ. In S. mansoni, extensive regression of the reproductive organs was observed in male and female worms treated with PZQ and dextro-PZQ, while no serious damage was seen in worms treated with levo-PZQ.

Integration and expression of murine retrovirus-related sequences in schistosomes.

Antibodies against the retrovirus envelope glycoprotein (gp70) of mouse xenotropic retrovirus, BALB virus 2 (Bv2) reacted with the adult worms of Schistosoma japonicum and S. mansoni. This reaction was completely inhibited after adsorption of the antibodies with virions of retrovirus. The reactive schistosome antigen was located in the subtegumental layer of the adult male fluke and in the vitelline gland of the adult female of S. japonicum and S. mansoni. Proteins extracted from both parasites were examined by immunoblot analysis. Anti-Bv2 gp70 antiserum reacted with those proteins from both schistosomes and the band patterns were different among sexes and species. Southern hybridization of the DNA extracted from adults of S. japonicum and S. mansoni demonstrated the presence of sequences homologous to the env gene of mouse ecotropic and xenotropic retroviruses. DNA sequences homologous to the gag and pol regions of the ecotropic murine leukaemia virus were also detected in the DNAs of schistosomes.

Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro.

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

The killing effect of subclasses of mouse IgG antibodies on schistosomula of Schistosoma japonicum.

The killing effect of IgG antibodies in sera from S. japonicum infected mice on schistosomula of S. japonicum was studied in vitro in the presence of complement. Immune mouse sera were fractionated over Protein A-Sepharose and IgG1, IgG2a, IgG2b and IgG3 were purified by affinity chromatography. The killing activity of each subclass of IgG antibodies against schistosomula was observed only in the presence of complement. The activity of IgG1 was higher than that of the other subclasses of IgG. In combination of the two or three subclasses of IgG, the additive mortality rate was given in all combinations of each subclass of IgG. These results support the view that the combination of various subclasses of IgG antibodies potentiates the resultant lethal effect on schistosomula.