Induction of resistance in Oncomelania hupensis nosophora against Schistosoma japonicum, but not against Paragonimus ohirai, using irradiated miracidia

Oncomelania hupensis nosophora snails sensitized with X-irradiated Schistosoma japonicum miracidia demonstrated resistance against a following challenge infection with non-irradiated homologous miracidia. The resistance in O. h. nosophora against S. japonicum was acquired within 1 day of sensitization, and it was strongest in a group challenged at an interval of 3 days. The resistance persisted for at least 4 weeks. Histological examinations revealed amoebocyte accumulation around the challenged S. japonicum sporocysts. On the other hand, when O. h. nosophora sensitized by exposure to X-irradiated P. ohirai or S. japonicum miracidia were subsequently challenged with normal P. ohirai miracidia, no resistance was observed, although they expressed the resistance against heterologous S. japonicum infection.     

Comparative studies of the defense mechanism against Schistosoma japonicum of schistosome-susceptible and -resistant Oncomelania nosophora

The amphibious snail Oncomelania nosophora is an intermediate host of Schistosoma japonicum. Previously we reported that there are two strains of the snail, one resistant and one susceptible to a Mindoro, the Philippines, strain of S. japonicum. The resistant snails were collected from Nirasaki and susceptible snails from Kisarazu, Japan. To determine early cellular responses in the two snail strains, we examined histologic alterations in the snails for up to 18 h after the initial exposure to miracidia. In both strains, the penetrating miracidia were distributed in the foot, mantle, gills, heart, stomach, and kidney, and the mean number of penetrating miracidia was similar in both strains. After penetration, snail hemocytes migrated toward the larvae, and by 12 h after exposure, substantial numbers of penetrated larvae were surrounded and encapsulated by hemocytes. The percentage of larvae encapsulated by hemocytes during 12-18 h after the exposure was significantly higher in the resistant Nirasaki strain (60.9+/-19.8%) than in the susceptible Kisarazu strain (42.3+/-15.0%). In a few snails of the Nirasaki strain, all the larvae found were encapsulated by hemocytes. The differences in hemocyte responses between the two strains may explain the susceptibility of the snails to schistosome larvae.     

In vitro cultivation of Paragonimus miyazakii and P. ohirai

Excysted metacercariae of Paragonimus miyazakii and P. ohirai were cultured in various media at 37.5 C in a 5% CO2 atmosphere. Paragonimus miyazakii grew rapidly and showed a well-developed ovary, uterus, and testes at 172 days in NCTC 109 supplemented with 30% rabbit serum, 50% egg yolk-109, and rabbit red blood cells (RBC's). However, none of the worms formed yolk or eggs in these cultures. On the other hand, P. ohirai grew to the adult stage, in which vitellaria and imperfect ova were formed, in NCTC 109 supplemented with 30% dog serum, 10% yeast extract Earle's solution (YLE), and dog RBC's at 252 days. The maximum body length of these worms measured 7.0 mm (mean 5.5 mm) at 252 days. The dog RBC's were an essential ingredient of the culture medium for the development of P. ohirai. Additions of liver concentrate, chick embryo extract (CEE), and egg yolk-109 in the medium did not provide any additional benefits for the development of worms. Using this supplemented medium, adult worms of P. ohirai removed from rats were maintained in vitro to examine their ability to lay eggs. Egg laying occurred during the first 10-13 days for worms that survived more than 60 days. The number of eggs deposited in this medium was about 2 times that found when Hanks' BSS and NCTC 109 were used.     

Transplantation of Schistosoma japonicum daughter sporocysts in Oncomelania hupensis

Schistosoma japonicum daughter sporocysts obtained from infected Oncomelania hupensis hupensis were successfully transplanted to parasite-free O. hupensis hupensis. Survival and infection rates of recipient snails were 80% and 75% respectively. Intramolluscan development of transplanted daughter sporocysts in recipient snails appears to proceed in a similar manner as those reported for transplanted S. mansoni and S. haematobium in their respective snail intermediate hosts. Complete colonization of the digestive gland of recipient snails by sporocysts was observed 80 days after transplantation. Cercarial production during a 10-day observation from recipient snails was characterized by periods of high and low and irregular daily emissions. The average daily cercarial production was 150 per snail. Cercariae produced by recipient snails were infective to mice. Of those cercariae exposed to mice, approximately 30% developed to adult schistosomes. These results have definitive utility in the maintenance of S. japonicum in the laboratory.     

Molecular identification of Paragonimus ohirai and P. westermani from Anhui Province, China

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) region were determined from seven adults of species Paragonimus collected from Jinde and Xiuning Counties, Anhui Province, China. Among these, the nucleotide sequence obtained from one Paragonimus adult (Jinde County) was identical to the ITS2 sequence of P. ohirai previously reported. In order to confirm the result, partial regions of mitochondrial cytochrome C oxidase I (COI) and NADH dehydrogenase 1 (ND1) from the putative P. ohirai sample were further sequenced. They showed a high level of similarity with those of P. ohirai, COI (99.7%) and ND1 (99.5%), supporting the result obtained from the ITS2. In addition to this, we designed P. ohirai- and P. westermani-specific primers (BDW and BD2OH) from ITS2 to identify P. westermani and P. ohirai easily and rapidly. After testing utility of the primers, they were applied to identify seven unidentified Paragonimus samples collected from Jinde and Xiuning Counties, China. All the examined samples showed P. westermani band pattern, and it was reconfirmed by sequencing their ITS2 regions that they are P. westermani. This result indicates that the two newly designed specific primers could be quite helpful for easily identifying P. westermani and P. ohirai, that most of Paragonimus in Jinde and Xiuning Counties consist of P. westermani, and that P. ohirai exists in Jinde County with minority.     

Evidence for metacercarial polymorphism in lung flukes, Paragonimus ohirai and Paragonimus iloktsuenensis

Cross breeding experiments between 2 species of lung fluke, Paragonimus ohirai and Paragonimus iloktsuenensis, were carried out using metacercarial characteristics as distinguishing markers. All metacercariae of F1 obtained were identical to those of P. ohirai. In the F2 and BF1 of F1 X P. iloktsuenensis, both the P. ohirai and P. iloktsuenensis types of metacercariae appeared. In the BF1 of F1 X P. ohirai, however, only the P. ohirai type metacercariae were produced. No intermediate type between the 2 species appeared. The results obtained demonstrate that the differences in the metacercariae, which were previously regarded as the most important characteristic for specific discrimination between these 2 flukes, are only a hereditary phenomenon within a single species. The metacercarial form, number of cyst layers, and body size seem to be controlled by a couple of alleles or very closely linked genes following simple Mendelian inheritance. Furthermore, we confirmed that reproduction in the lung flukes depends on cross-fertilization.     

Malic dehydrogenase patterns in different strains of Schistosoma japonicum and subspecies of Oncomelania hupensis.

Malic dehydrogenase (MDH) of the Formosan Philippine and Indonesian Strains of Schistosoma japonicum and 3 subspecies of snails, Oncomelania hupensis formosana, O. h. quadrasi, and O. h. lindoensis were identified by disc electrophoresis with 10.5% acrylamide gel columns using tris-malic buffer. Male worm extracts of S. japonicum demonstrated 3, 2, and 2 clear MDH patterns and female worm extracts showed patterns of 2, 1 and 1 for the Formosan, Philippine and Indonesian strains of the parasite, respectively. MDH patterns were the same for all 3 subspecies of snails; one major and one minor band being found in all.     

Isozyme patterns of Schistosoma japonicum and S. mansoni

Isozyme patterns of six enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, hexokinase, malate dehydrogenase, 6-phosphate dehydrogenase and phosphoglucomutase were examined in electrophoresed homogenates of adult male worms of Schistosoma japonicum and S. mansoni. In general, enzyme patterns obtained from the parasite homogenates differed from that of host (mouse) blood and muscle, indicating that electrophoretic patterns from parasite extracts are most probably of parasite origin. Adult male and female S. mansoni worms yielded identical patterns. However, all six enzyme patterns showed distinct differences between S. japonicum and S. mansoni. These results suggest that S. japonicum is clearly distinguishable from S. mansoni at the molecular level.     

Novel C-banding patterns in Paragonimus ohirai (Trematoda: Platyhelminthes)

The C-banding pattern for the karyotype of Paragonimus ohirai representing individuals in a new population is reported. The short arm of chromosome 4 consisted of a large pericentromeric proximal C-band block and euchromatic tip. This pattern has not been observed previously and is designated as type E. Other new observations were: chromosome 5 was composed of pericentromeric heterochromatin, a lightly stained intercalary band at the middle portion of the short arm, and a lightly stained interstitial band at the terminal region of the long arm. Chromosome 7 consisted of pericentromeric heterochromatin and a lightly stained telomeric band at the short arm.