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Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.
Paragonimus westermani, the lung fluke, is known to migrate to the pulmonary tissue of mammalian hosts and causes pathological changes in the lungs. An acidic thiol-dependent proteinase with a molecular weight of approximately 20,000 daltons was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. On SDS-PAGE, the molecular weight of the enzyme was 17,500 daltons. Isoelectric point was 6.45. The enzyme was similar to the acidic cysteine proteinase of vertebrates in the properties of pH 5.5 for at least two days when stored at 4 degrees C. The cysteine proteinase was capable of degrading collagen and hemoglobin. Sera of patients with paragonimiasis and mice infected with P. westermani reacted in immunoblots with the partially purified proteinase. This result suggested that the cysteine proteinase of P. westermani may play a role in migration in tissues, and in acquisition of nutrients by parasites from the host. It is also potentially an antigen for the serodiagnosis of paragonimiasis.
Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.
Paragonimus westermani is a medically important foodborne trematode occurring throughout southeast Asia. We have used molecular techniques to test the hypothesis that the parthenogenetic triploid form of P. westermani has arisen only once. Sources of data for comparison were: (a) restriction fragment length polymorphisms (RFLPs) of ribosomal internal transcribed spacers (ITS); and (b) 'fingerprint' patterns observed when genomic digests were probed with simple sequence repeats (ATT)10 and (ATGT)7. In all cases there were distinct differences among triploid isolates from southwest Japan, northeast China and Korea. These findings are considered in the context of previous cytogenetic, allozyme, mitochondrial-RFLP and partial cytochrome c-oxidase subunit I (COI) sequence studies and indicate that triploid lineages may have arisen independently on more than one occasion. We favour this view. An alternative explanation is that the triploids did have a single origin, but that different clonal lineages have undergone subsequent mutations.
The presence of cross-reacting antigens between Paragonimus westermani, Schistosoma mansoni, and Fasciola hepatica adult worms was demonstrated by Ouchterlony immunodiffusion and enzyme-linked immunosorbent assay (ELISA). A serum bank was developed against the three trematode genera to serve as probes to determine the presence of cross-reacting antibodies to P. westermani worm extracts. In this manner, it was possible to demonstrate that antigens common to F. hepatica and S. mansoni tegument were also present in P. westermani worm extracts. Likewise, it was possible to demonstrate that the F. hepatica antigens which bind to Concanavalin A, as well as the subfraction which in isoelectric focusing has a pI of 4.2, were also found in P. westermani worms. Also, a monospecific polyclonal serum to a Fasciola/Schistosoma cross-reacting antigen and the anti-P. westermani serum both reacted in Ouchterlony immunodiffusion with the P. westermani antigenic extract, each producing a line which linked with each other indicating common antigenic determinants and suggesting a common antigen among the digenetic trematodes. Finally, the P. westermani antigenic extracts induced in mice the production of antibodies which reacted with S. mansoni adult worm antigens by ELISA. As all of the Fasciola and Schistosoma sera were prepared against antigenic preparations which induced in mice protection to challenge infection with S. mansoni, this suggested that the P. westermani worms also contain protective antigens against S. mansoni. Immunity to Schistosoma mansoni infection was induced in mice by vaccination with Paragonimus westermani whole worm extracts (PwWWE). Immunized mice showed as high as a 67% worm burden reduction over controls. High doses of PwWWE did not confer protection to S. mansoni infection. Thus, in this study, immunity in heterologous systems was demonstrated and the existence of a common protective antigen shared by the digenetic trematodes was suggested.
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Paragonimus westermani is an endemic disease and has had scattered distribution in Northern Taiwan for the long period of 108 years. In order to know the current status of the prevalence of this disease in Taipei County, an investigation by means of examination of snail, crab and stool for identification of cercariae, metacercariae and ova of Paragonimus westermani was carried out in five small highly endemic foci-Alilao, Shengkeng, Shuangchi, Shihjen and Taiping from July 1984 to June 1985. The results obtained are summarized as follows: The infection rate for 17, 412 fresh-water snails, Semisulcospira libertina, the first intermediate host and for 180 fresh-water crabs, Eriocheir japoniucs, the second intermediate host was both negative in the first four places. On the contrary, the rate for 879 fresh-water snails, Semisulcospira libertina, for 298 fresh-water crabs, Potamon miyazaki and Potamon dehaani and for 45 school-children was zero, 26.8% and zero respectively, in the last one. Based on the above findings, it is concluded that in the last 20 years, Paragonimus westermani might probably be under control in the former areas which are now developed districts. This development has probably influenced the prevalence and distribution of the disease. But it is still prevalent in the latter area located in remote undeveloped mountain.
In mature Paragonimus westermani, specific activity of parasitic cysteine protease declines. To clarify which of the known 17-, 27-, and 28-kDa enzyme activities is decreased, the cysteine proteases were purified from the crude extracts of metacercariae, 4- and 7-wk juveniles, and 16-wk adults by gel filtration, ion-exchange, and affinity matrix chromatographies; the enzyme activity was monitored with the fluorogenic substrate, Cbz-phe-arg-AMC. In addition to 3 known enzymes, 2 other cysteine proteases at 15 and 53 kDa were identified in juveniles and adults and were purified. The 2 novel enzymes were most active in 0.1 M ionic strength and pH 5-6 and were inhibited by N-(N-[L-3-transcarboxyrane-2-carbonyl]-L-leucyl)agamatine, iodoacetamide, and leupeptin. Of the 5 enzymes, specific activities of metacercarial 27- and 28-kDa enzymes were lowered from metacercaria to 16 wk. Between 4 and 16 wk, activities of 3 cysteine proteases of juveniles and adults were additionally exhibited. The activity changes of 5 different cysteine proteases may be associated with migration and immune evasion during the maturation stage of P. westermani when the parasite environment is changing.
Paragonimus westermani is a common fluke in Korea. The present study aimed to observe the cell mediated immune response in experimental paragonimiasis of mice. The mouse (BALB/c) was orally inoculated with 40 metacercariae of P. westermani from Cambaroides similis. During the infection (1, 2, 4, 6 weeks) of mouse, blastogenic response of splenic lymphocytes to P. westermani adult antigen, metacercaria antigen, and PHA were observed. Sera from infected and noninfected mice added to normal mouse splenic lymphocytes with or without PHA. The blastogenic response of splenic lymphocytes to PHA was reduced after 1 week of infection. However after 6 weeks of infection, the response was restored to the control level. The blastogenic response of splenic lymphocytes to P. westermani adult or metacercaria antigen increased significantly on 1 week after infection, and maintained up to 6 weeks after infection. The response of non-infected mice was suppressed by addition of the infected mouse serum. The present results suggested that cellular immunity was involved in P. westermani infected mice and that P. westermani anti-serum inhibited proliferation of T lymphocytes.
The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.
Paragonimus westermani causes inflammatory lung disease in humans. The parasite excretes a host of biologically active molecules, which are thought to be involved in pathophysiological and immunological events during infection. Analyses of the 2-DE protein profiles of the excretory-secretory products (ESP) of adult P. westermani revealed approximately 147 protein spots, at least 15 of which were identified as cysteine proteases (CPs), at pHs between 4.5 and 8.5, and molecular weights (MWs) between 27 and 35 kDa. An additional three CPs (designated as PwCP-3, -8 and -11) were newly recognized by TOF/TOF MS. Their molecular biological information, which shared a high level sequence homology, was elucidated. The majority of the CPs reacted strongly with sera from paragonimiasis patients. When we observed the chronological changes in the antibody responses of the respective CPs against canine sera collected serially at 1, 3, 5, 7, 11 and 14 wk after experimental infection, these molecules exhibited a multiplicity of distinct immune recognition patterns. Our results clearly showed that P. westermani adult ESP were principally composed of excretory-secretory CPs, and that these CPs may exert effects not only on host tissue degradation and nutrient uptake, but also on the immune-regulating cells via synergistic and independent interactions.
Paragonimus westermani is a trematode parasite, which causes pulmonary and/or extrapulmonary granulomatous disease in humans. Successful invasion of the host tissue is critical for the survival of this tissue-invasive parasite. The enzymatic hydrolysis of host proteins is clearly a prerequisite of this process. In this study, we have investigated the functional roles of the excretory-secretory cysteine proteases of P. westermani newly excysted metacercariae (PwNEM) in tissue invasion. The 27 and 28 kDa enzymes (PwMc27 and PwMc28) purified from PwNEM excretory-secretory products (ESP), preferentially degraded fibrillar proteins, but not globular proteins. PwMc28 significantly facilitated the invasion of PwNEM into mouse peritoneum, whereas a diffusible cysteine protease inhibitor, trans-epoxysuccinyl-L-leuciloamido-(4-guanidino) butane (E-64) inhibited this process dose-dependently. Two distinct isoforms of PwMc28 (PwMc28a and PwMc28b), which exhibited two amino acid differences in their mature domains, were identified by tandem mass spectrometry and sequence analysis. Both enzymes were localized at the tegument on the anterior border and on the oral sucker, which suggests excretion-secretion via exocytosis or via the excretory canal network. The mRNA transcripts of PwMc28a and b were expressed abundantly during the active invasion/migration through the host's tissues, suggesting their relevant function to tissue invasion/migration in the definitive host.
In order to estimate the prevalence of Paragonimus westermani among the school children in Ulchin county, Kyungpook Province, five primary schools were selected for sampling. The children were first examined using an intradermal test with Paragonimus antigen, Lot No. 890417, and then examined for Paragonimus eggs from sputa and stools collected from the intradermal-positive cases. In a total of 382 children examined, 53 cases or 13.9 percent revealed positive reactions for Paragonimus westermani. The sex specific rate of infection was a little higher in boys than in girls. The age specific rate of Paragonimus westermani infections varied from age to age. Paragonimus westermani was most prevalent in the 8-year-old group in both sexes and showed about the same proportion among boys and girls of each age group. A comparison of the reliability for the detection of Paragonimus eggs from intradermal positive cases in this study showed that the sputum test was more sensitive than the stool test. Summarizing the results, this study indicates that the prevalence for Paragonimus westermani is still high and the endemic foci of the fluke exists in Ulchin county, Kyungpook Province, Korea.
In order to determine the epidemiological pattern of the Paragonimus westermani, the infestation rates of the cercarial and metacercarial larvae of digenetic trematodes in the snail and crayfish host, and the prevalence of Paragonimus westermani among the residents in the vicinity of the stream Namdae, Wyangpi, and Kwang, in the Ulchin county, Kyungpook province were studied from March to October in 1984. The population density of the snails per square meter of the habitats ranged form 5 to 25, with average of 15. Among the seven habitats, one, Ducheon, had snails infested with the cercariae of Paragonimus westermani, and the proportion of infested snails was very low, the average being 0.152 per thousand. Of six hundred and four crayfish examined, 113 or 18.7 per cent harboured the encysted larvae of Paragonimus westermani. A high infestation rate for the metacercariae in the crayfish was found in two habitats; 39.0 per cent in the Ducheon and 21.6 per cent in the Sokwang. The prevalence of Paragonimus westermani among the residents in Ulchin county was relatively high, 25.8 per cent by the Paragonimus intradermal tests and the difference in the rate of infection between males and females was found to be significant(t>2). The results obtained in this study indicate that endemic foci of Paragonimus westermani exist in Ulchin county and the prevalence of this lung fluke among the residents is relatviely high.
This survey was undertaken to determine the recent prevalence of lung fluke, Paragonimus westermani, in the three localities of Wiseong county, Kyungpook Province, Korea and to compare the prevalence among the residents in the same areas between 1924 and 1980. The three localities, Ku-seong, Sam-chun, and Um-ji villages (dong) have been known as the endemic areas of Paragonimus westermani. The surveys were made on the general population. They were examined using intradermal test with Paragonimus westermani antigen, and the diagnosis of paragonimiasis was made depending on the positive intradermal reaction. The sputum examination could not be performed beacause the residents were inaccessible. The prevalence of Paragonimus westermani among the residents in the three communities varied from 1.4 % to 7.9 per cent, with an average of 4.0 per cent. Whereas, the average reported by Ichinomiya in 1924 was 4.7 %. There are no significant differences in the sex and age specific rates of infection between 1924 and 1980. With the abundant density of Semisulcospira snails and crayfish in Sam-chun stream and the high rate of infection with Paragonimus westermani among the residents in the village, it is found that Sam-chun village, one of the three localitied in Wiseong county, where Ichinomiya (1924) surveyed, was still the endemic focus of Paragonimus westermani.
The cysteine proteases of Paragonimus westermani metacercaria are known to initiate metacercaria excystment. However, their secretory sites, such as the intestine, tegument, and excretory bladder are not well defined. In this study, the metacercariae were labeled with bromodeoxyuridine (BrdU) to distinguish the initial activation sites. BrdU was labeled mainly at the excretory bladder and the excretory granules of the metacercariae and the newly excysted juvenile worms. This result shows that early 'defecation' of the proteases from the excretory bladder may accelerate the excystment of P. westermani metacercariae.