Mortality in Syrian hamsters infected with Paragonimus kellicotti.

In an attempt to find a small animal model for paragonimiasis, Syrian hamsters were infected with between 1 and 16 metacercariae of Paragonimus kellicotti. A definitive mortality dose-response was observed with 90% of all hamsters given 3 or more parasites succumbing to the infection within 35 days. Hamsters demonstrated acute pleuritis, reactive mesothelial hyperplasia, subpleural accumulations of reactive and mature plasma cells, neovascularization, fibrohistiocytic thickening with and without giant cells, raised fibroconnective tissue lesions, and granulomatous inflammation with hemorrhage. Perivascular plasmacytic (lymphocytic) infiltrate, multifocal bronchopneumonia, and parenchymal necrotizing suppurative granulomatous inflammation, hemorrhagic pneumonia, and diffuse sprinkling of eosinophils, neutrophils, and intraalveolar macrophages also were observed. The response observed here may represent a new small animal mortality model useful in the search for new compounds to treat early trematode infections.     

Purification and properties of a thiol protease from lung fluke adult Paragonimus ohirai

The thiol protease was purified from adult Paragonimus ohirai by alpha 1-antitrypsin-Sepharose, Sephadex G-75 and CM-cellulose, measuring its activities to hydrolyze hemoglobin and tosyl-L-lysine alpha-naphthyl-ester. The purified protease showed a single band on polyacrylamide disc gel isoelectrophoresis as zymogram with Tos-Lys-NE and also by protein staining, and its pI was found to be 6.4. The molecular weight was calculated to be 29,000 by gel filtration and 27,000 by SDS-polyacrylamide gel electrophoresis as a single polypeptide. The protease hydrolyzed hemoglobin and Tos-Lys-NE optimally at pH 4.0 and 5.0, respectively. The both hydrolyzing activities were inhibited by alpha 1-AT and soybean trypsin inhibitor as well as thiol protease inhibitors such as antipain, E-64 and p-hydroxymercuriphenylsulfonate. These results indicate that this enzyme is a new type thiol protease.     

Electron microscopic study of spermatogenesis in the lung fluke (Paragonimus miyazakii)

Summary The characteristics of spermatogenesis in a type of pulmonary parasite, Paragonimus miyazakii have been observed using the electron microscope. Groups of several spermatocytes revealed mutual cytoplasmic connection. That degree of this fusion increased as spermatogenesis progressed, and finally developed into a so-called cytophore. Then, this cytophore remained joined with a spermatid by a short stalk until the spermatid changed into a sperm. The nucleus of the spermatid became elongated with a string-like arrangement of the chromatin, which, in turn, showed increased electron density. At the pole of the spermatid, linearly arranged microtubules developed just below the plasma membrane. Close to an elongated portion at the pole, two separate flagella start growing and later fuse with the sperm itself. In the sperm tail a couple of tail filament complexes, longitudinally oriented slender mitochondria, and a tubular structure were present.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

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.     

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.     

An endogenous inhibitor of thiol protease from adult lung fluke Paragonimus miyazakii

An endogenous inhibitor of thiol protease from adult Paragonimus miyazakii was found to occur during the gel filtration on Sephacryl S-300. The partially purified inhibitor was specific for thiol proteases such as ficin and papain. The inhibitor also suppressed tosyl-L-lysine alpha-naphthylester hydrolytic activity of Paragonimus thiol protease. The molecular weight of the inhibitor was found to be 430,000 by the gel filtration. This inhibitor was thermostable and resistant to trypsin and glycosidase digestions.     

Ultrastructure of Mehlis' gland in the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae)

Mehlis' gland of a digenetic trematode, Paragonimus ohirai, is composed of two types of secretory cells, DB and CB. The less abundant type (DB) produces dense bodies, with the cytoplasm characterized by greatly distended cisternae of rough endoplasmic reticulum. The other type (CB) synthesizes clear, vesicular bodies. Its cytoplasm contains numerous mitochondria, rough endoplasmic reticulum with narrow cisternae, and abundant Golgi complexes. Processes of the two cell types converge on the ootype-proximal uterine wall, pass through the epithelium, and finally open into the lumen. These proximal processes contain longitudinally arranged microtubules whose luminal ends are anchored to the epithelium by ring-form septate desmosomes. According to the distribution of the two types of processes, three different zones (DB, mixed, and CB) can be recognized within the epithelia. As the CB processes enter the lumen predominantly beyond the uterine valve region, this cell may produce secretions required for egg shell maturation or hardening. The role of DB cells (which enter the lumen more commonly in the ootype near the oviduct) remains unknown.     

Ultrastructure of the oviduct of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae)

The oviduct of Paragonimus ohirai is lined by an epithelium composed of cells that rest on a prominent basal lamina and exhibit extensive basal infoldings. Apical surfaces of the epithelial cells are lamellated, and height and abundance of lamellae vary in different regions. Cilia are confined to two separate areas and are variable with respect to their axonemal pattern. There are five principal regions of the oviduct: infundibular (IR), distal ciliary (DCR), preseminal receptacle (PSR), proximal ciliary (PCR), and junctional (JR). The pattern of organization of cytoplasmic organelles such as mitochondria, Golgi complexes, and lysosomes is distinctive within each region. The remainder of the wall consists of circularly arranged myofiber bundles, together with a plexus of nerve fibers. The structure of the oviduct in relation to passage of oocytes, movement of spermatozoa, and fertilization is discussed.     

Comparative proteomics of adult Paragonimus kellicotti excretion/secretion products released in vitro or present in the lung cyst nodule

Paragonimus kellicotti is a zoonotic lung fluke infection, the agent of North American paragonimiasis, and an excellent model for other Paragonimus infections. The excretory/secretory proteins (ESP) released by parasites and presented at the parasite-host interface are frequently proposed to be useful targets for drugs and/or vaccines In vitro culture conditions may alter ESP compared to those produced in vivo. In order to investigate ESPs produced in vivo we took advantage of the fact that adult P. kellicotti reproduce in the lungs of experimentally infected gerbils in tissue cysts. We performed a mass-spectrometric analysis of adult P. kellicotti soluble somatic protein (SSPs) extracts, excreted/secreted proteins (ESPs) produced by adult worms during in vitro culture, and lung cyst fluid proteins (CFPs) from experimentally infected gerbils. We identified 2,137 P. kellicotti proteins that were present in at least two of three biological replicates and supported by at least two peptides. Among those were 1,914 proteins found in SSP, 947 in ESP and 37 in CFP. In silico analysis predicted that only 141 of the total 2,137 proteins were secreted via classical or non-classical pathways. The most abundant functional categories in SSP were storage and oxidative metabolism. The most abundant categories in ESP were proteins related to metabolism and signal transduction. The 37 parasite-related proteins in CFP belonged to 11 functional categories. The largest groups were proteins with unknown function, cytoskeletal proteins and proteasome machinery. 29 of these 37 proteins were shared among all three sample types. To our knowledge, this is the first study that compares in vitro and in vivo ESP for any Paragonimus species. This study has provided new insights into ESPs of food-borne trematodes that are produced and released in vivo. Proteins released at the host-parasite interface may help the parasite evade host immunity and may represent new targets for novel treatments or diagnostic tests for paragonimiasis.     

Ultrastructure of spermatozoa of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae), in the seminal receptacle

The seminal receptacle of Paragonimus ohirai contains not only mature spermatozoa, but also atypical and degenerate ones, suggesting that abnormal spermatozoa are retained in this organ. The spermatozoon is of a parallel biflagellar type with cortical microtubules, consisting of the anterior region, first mitochondrial region, intermediate (amitochondrial) region, second mitochondrial region, posterior nuclear region (PNR) and tail region (TR). The first third of the spermatozoon exhibits typical undulatory movement, while the middle part shows vibratory movement. At the area between head and midsections (H-M area) the peripheral doublets of axonemes are interrupted, and the external ornamentation is distributed widely around this portion. Throughout the immotile PNR and TR, the axonemes lack the dynein arms of their peripheral doublets. H-M, PNR, and TR ultrastructural characteristics are specific in P. ohirai spermatozoon and seem to be closely related to its pattern of movement.