Dynamic transcriptome landscape of Paragonimus proliferus developmental stages in the rat lungs.

Paragonimus proliferus, a lung fluke of the genus Paragonimus, was first reported in Yunnan province, China. P. proliferus can infect Sprague-Dawley (SD) rats and cause lung damage, but there is still no direct evidence of human infection. Until now, there has been a lack of studies on P. proliferus parasitism and development in mammalian lung tissue. The aim of this study was to perform transcriptomic profiling of P. proliferus at different developmental stages. SD rats were infected with P. proliferus metacercariae obtained from crabs; worms isolated from the lungs at different time points as well as metacercariae were subjected to whole transcriptome sequencing. Overall, 34,403 transcripts with the total length of 33,223,828 bp, average length of 965 bp, and N50 of 1833 bp were assembled. Comparative analysis indicated that P. proliferus, similar to other Paragonimus spp., expressed genes related to catabolism, whereas P. proliferus-specific transcripts were related to the maintenance of cellular redox homeostasis, sensitivity to bacteria, and immune response. Transcriptional dynamics analysis revealed that genes involved in the regulation of catabolism and apoptosis had stable expression over the P. proliferus life cycle, whereas those involved in development and immune response showed time-dependent changes. High expression of genes associated with immune response corresponded to that of genes regulating the sensitivity to bacteria and immune protection. We constructed a P. proliferus developmental model, including the development of the body, suckers, blood cells, reproductive and tracheal systems, lymph, skin, cartilage, and other tissues and organs, and an immune response model, which mainly involved T cells and macrophages. Our study provides a foundation for further research into the molecular biology and infection mechanism of P. proliferus.

Molecular identification of morphologically similar microcercous cercariae of two trematode families, Paragonimidae and Troglotrematidae, concurrently found in the same snail species of the subfamily Triculinae.

Microcercous cercariae possess a very short tail and are produced by digenean species of several families including medically important species, such as members of the genera Paragonimus Braun, 1899, Nanophyetus Chapin, 1927 and Troglotrema Odhner, 1914. During our survey of cercariae of Paragonimus spp. in Vietnam, we found microcercous cercariae from ten (0.29%) out of 3,400 snails of Triculinae gen. sp. 2. They were morphologically and molecularly analysed for species identification. The molecular analysis, based on ITS2 sequences, revealed two distinct species: four specimens were identical to Paragonimus proliferus Hsia et Chen, 1964 (Paragonimidae Dollfus, 1939), and the other six specimens were closest to members of the family Troglotrematidae Odhner, 1914 and were temporarily named Troglotrematidae gen. sp. Morphologically, cercariae of the two species found in this study are similar to each other in their gross characteristics but can be distinguished from one another by subtle morphological details. The cercaria of P. proliferus has an I-shaped excretory bladder and does not have mucous gland cells. In contrast, that of Troglotrematidae gen. sp. has a Y-shaped excretory bladder and mucous gland cells. Besides, the redia of P. proliferus is elongate with a short intestine and contains 5-6 cercariae whereas that of Troglotrematidae gen. sp. is more round with a longer intestine and harbours 3-4 cercariae. Our results have shown the importance of the shape of the excretory bladder and the presence/absence of mucous gland cells of the cercaria as well as the shape and size of the redia, and its intestinal length as valuable taxonomic characters of intramolluscan trematode larvae. In addition, the finding of similar microcercous cercariae of different species in the same snail species suggests that careful attention to morphological details is required in the differentiation of Paragonimus cercariae and those of closely related species.

Morphological and molecular characterization of the metacercaria of Paragonimus caliensis, as a separate species from P. mexicanus in Costa Rica.

The trematode Paragonimus mexicanus is the etiological agent of paragonimiasis, a food-borne zoonotic disease in Latin America. This species, as well as Paragonimus caliensis, have been reported from Costa Rica, but it is not known if the two are synonymous. Two types of Paragonimus metacercariae from freshwater pseudothelphusid crabs from several localities in Costa Rica were recognized by light microscopy. Morphologically, these corresponded to descriptions of P. mexicanus and P. caliensis. Metacercariae of the former species lacked a membrane or cyst and their bodies were yellow in color. Those of P. caliensis were contained in a transparent thin cyst and were pink in color. Morphotypes of metacercariae were determined using scanning electron microscopy (SEM). Based on the number and distribution of papillae in the ventral sucker, three morphotypes were found for P. mexicanus and two for P. caliensis. Analysis of DNA sequences (nuclear ribosomal 28S and ITS2 genes, and partial mitochondrial cox1 gene) confirmed the presence of P. mexicanus and provided the first molecular data for P. caliensis. The two species are phylogenetically distinct from each other and distant from the Asian species. The confirmation of P. caliensis as a separate species from P. mexicanus raises several questions about the ecology, biological diversity, and epidemiology of the genus Paragonimus in Costa Rica.

Metacercarial polymorphism and genetic variation of Paragonimus heterotremus (Digenea: Paragonimidae), and a re-appraisal of the taxonomic status of Paragonimus pseudoheterotremus.

Paragonimus heterotremus, which is an important pathogen for human paragonimiasis in Asia, is recognized as having the smallest metacercariae (maximum diameter < 300 µm) of any previously reported Paragonimus species. Recently, P. pseudoheterotremus has been described from Thailand as a new species having metacercariae (about 200 µm) slightly smaller than those of Thai P. heterotremus. In fact, the small size of P. pseudoheterotremus metacercariae is compatible with those of P. heterotremus from India and China. In this study in Vietnam, we found variably sized small metacercariae which are expected to consist of both P. heterotremus and P. pseudoheterotremus. Contrary to expectation, the adult flukes obtained by separate infection of experimental cats with different sized metacercariae were all identified as P. heterotremus, using both morphological and molecular characteristics. The molecular analyses of an extensive collection of P. heterotremus/P. pseudoheterotremus isolates from Asian countries also indicated that genetic distances between different populations of P. heterotremus are even larger than that between P. pseudoheterotremus and P. heterotremus. The haplotype network showed that all P. heterotremus and P. pseudoheterotremus isolates formed a P. heterotremus complex consisting of three groups with strong geographical origins. In addition, the Indian P. heterotremus group is the root of the other P. heterotremus and P. pseudoheterotremus populations. Based on the observed metacercarial polymorphisms and genetic variation in P. heterotremus, P. pseudoheterotremus should be considered a geographically isolated population of the P. heterotremus complex.

Systems biology studies of adult paragonimus lung flukes facilitate the identification of immunodominant parasite antigens.

BACKGROUND: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. CONCLUSIONS: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future.

Pleuropulmonary paragonimiasis: mimicker of tuberculosis.

Infection caused by the lung fluke is endemic in north eastern parts of India. Paragonimus westermani and Paragonimus heterotremus are known to be endemic in eastern Indian states of Manipur and Nagaland. The infection is related to eating habits of the locals and is acquired by ingestion of raw, inadequately cooked crabs or crayfish containing encysted metacercariae which act as second intermediate hosts during the life cycle of the lung fluke. Diagnosis is generally delayed due to lack of suspicion and presentation similar to tuberculosis which is endemic in the population. We report pleuropulmonary paragonimiasis in a soldier from eastern India who presented with chest pain, haemoptysis, and eosinophilia. He gave history of consumption of raw crabs while on leave at his native village in Nagaland. Ova morphologically resembling Paragonimus heterotremus were detected in sputum and bronchoalveolar lavage specimen. Symptoms resolved with praziquantel treatment.

Ectopic (subcutaneous) Paragonimus miyazakii infection in a dog.

Ectopic infection with Paragonimus miyazakii was determined to be the cause of a subcutaneous inguinal mass in a 15-month-old, male, boar-hunting dog. On histologic examination, the mass comprised granulomatous panniculitis, intralesional adult trematodes and eggs, and lymphadenitis. Extrapulmonary paragonimosis in animals is rare. This appears to be the first report in a dog of ectopic P. miyazakii infection with mature trematodes and eggs that involved the inguinofemoral lymphocenter and surrounding subcutis.

Description of a new lung fluke species, Paragonimus vietnamensis sp. nov. (Trematoda, Paragonimidae), found in northern Vietnam.

Based on morphology of metacercariae and adult worms together with molecular data from our previous study, we describe herein a new lung fluke species named Paragonimus vietnamensis sp. nov. Metacercariae of this new species is round and extremely large in size (nearly 800 mum in diameter) having a fragile outer cyst of variable thickness and a thin fragile inner cyst. There are little or no space between metacercaria and the inner cyst wall. These and other morphological features of metacercariae of P. vietnamensis sp. nov. are not completely identical with but have some similarities with those of P. microrchis, P. proliferus, or P. menglaensis. On the other hand, adult worms obtained by experimental infection of these large metacercariae are oval in shape, having a ventral sucker slightly larger than the oral one, and having singly arranged relatively short cuticular spines. These morphological features of adult worms are partly similar to but not identical with those of P. microrchis, P. skrjabini, P. yunnanenis, P. xiangshanensis, or P. harinasutai. Taken these morphological data and our previous molecular phylogenetic analyses of ITS2 and CO1 sequences of metacercariae and adult worms together, P. vietnamensis sp. nov. is different from any other known Paragonimus spp.

Paragonimiasis and its intermediate hosts in a transmission focus in Lao People's Democratic Republic.

Human cases of paragonimiasis have been reported in Lao People's Democratic Republic from several provinces, however, detailed descriptions of a focus of transmission including parasites species and intermediate hosts are lacking. Field investigations were carried out between February and July 2004 in three neighboring villages of the province of Vientiane. Persons with chronic cough (more than three weeks) were identified during a door-to-door survey and three sputum samples were examined for Paragonimus spp. and tuberculosis on two consecutive days. Potential intermediate snail and crustacean hosts were sampled among the populations' water-contact sites and examined for metacercarial infections or cercarial shedding. Four dogs were infected with metacercaria and the species of the subsequently retrieved adult parasites were identified. 12.7% (15 cases) of all chronic cough patients (0.6% of the total population) suffered from paragonimiasis. Samples of five species of freshwater snails, two species of crabs and one species of prawn were collected from the Nam Sêt River. None of the snails were infected. Metacercariae of P. harinasutai, P. bangkokensis and P. heterotremus were found in Potamon lipkei crabs while an infection with P. westermani was observed in a Chulathelphusa brandti crab. No paragonimid metacercaria was found in the Macrobrachium dienbienphuense prawns Dang and Nguyen 1972. This study demonstrates the importance of Paragonimus spp. as an etiological agent in patients with chronic cough and suggests that similar transmission might be widespread in Lao People's Democratic Republic. We document herein the transmission of several Paragonimus species indicating that paragonimiasis is an active zoonosis in Lao People's Democratic Republic.

Otitis media and a neck lump--current diagnostic challenges for Paragonimus-like trematode infections.

A 29 year-old Nigerian studying in the UK presented with a neck lump and otitis media. Paragonimus-like trematode eggs were found in the neck lump aspirate. Morphologically these eggs resembled Paragonimus uterobilateralis or Achillurbainia congolensis. We favoured the diagnosis of achillurbainiasis over extrapulmonary paragonimiasis on the basis of clinical features and because we could not amplify DNA sequences using PCR primers specific for Paragonimus species. We discuss current diagnostic challenges for this rare parasitic infection.

Effects of L-type Ca2+ channel antagonists on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate.

The inhibitory effects of L-type Ca2+ channel antagonists on Na cholate-induced in vitro excystment (CIIE) of Paragonimus ohirai metacercariae were studied. At concentrations of 10 microM, nicardipine and nimodipine inhibited CIIE completely and by approximately 92%, respectively. Nitrendipine and (+/-)-verapamil inhibited CIIE by about one half and one third, respectively. Nifedipine and diltiazem did not inhibit CIIE significantly. At higher concentrations, nitrendipine at 20 microM completely inhibited CIIE, and (+/-)-verapamil at 40 microM inhibited CIIE by 93%. Nifedipine and diltiazem inhibited CIIE only slightly and little, respectively, even at 40 microM. Complete inhibition by nicardipine at 10 microM required preincubation of metacercariae with the antagonist for 15 min. The inhibitory effects of nicardipine and nimodipine were reversible, and most of the nimodipine-treated metacercariae could excyst within 1 h after being washed, but the nicardipine-treated ones started to excyst 1 h after washing. Nicardipine suppressed the active movement of encysted juveniles evoked by Na cholate, whereas nimodipine did not suppress this significantly. These results suggested that L-type Ca2+ channels appeared to be involved in CIIE of P. ohirai metacercariae and that the inhibitory effect of the channels was due primarily to factors other than the inhibition of muscular activity, probably involving the secretion and release of enzymes lytic against the metacercarial cyst wall.

[Pleuro-pulmonary paragonimiasis]. / La paragonimose pleuropulmonaire.

Paragonimiasis is a food borne zoonosis due to a trematode belonging to the genus Paragonimus. Although present throughout the world, about 90% of the cases occur in Asia where around 20 million people are infected. The parasitic cycle is complex with two different intermediate hosts. Man is infected by ingesting the raw or undercooked flesh of the second host - a freshwater crab or prawn - or possibly of a paratenic mammal host (wild boar), which contains the infective larval stage metacercariae that reaches the lung which is the main target organ. Epidemiological, pathological, and clinical aspects are reviewed. The main symptoms are protracted cough, and recurrent "benign" hemoptysis. Abnormal pleuro-pulmonary imaging features are constant, but protean and non-specific, leading to frequent confusion with tuberculosis. Diagnosis is easily achieved by ova search in the sputum or pleural fluid, or by serology. Evolution is usually considered benign, although not well known. Finally, praziquantel is the effective first choice treatment. Some paradoxical aspects of this disease are underlined such as: underdiagnosis despite a very simple diagnostic procedure, or opposite tendencies according to location, either extinction or re-emergence.

Effects of blockers of Ca2+ channels and other ion channels on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate.

The inhibitory effects of various ion channel blockers were examined on in vitro excystment of Paragonimus ohirai metacercariae induced by a bile salt, sodium cholate. At a concentration of 10 microM, bepridil, a non-selective Ca(2+) channel blocker, completely inhibited in vitro excystment, whereas TEA, lidocaine, and R(+)-IAA-94, channel blockers against K(+), Na(+) and Cl(-) ions, respectively, benzamil, an Na(+)/H(+) and Na(+)/Ca(2+) ion exchanger blocker, and R(+)-DIOA, a [K(+), Cl(-)] cotransporter inhibitor, did not. Considering the previous result that Ca(2+) ionophores are also efficient inducing factors for in vitro excystment of P. ohirai metacercariae and the present result, bile salts appear to induce the excystment of P. ohirai metacercariae through evoking the Ca(2+) channels of target cells within the metacercarial juveniles.

Morphological description and life cycle of Paragonimus sp. (Trematoda: Troglotrematidae): causal agent of human paragonimiasis in Colombia.

The first morphological description is made of all stages of the life cycle of a Paragonimus species infecting humans in Colombia. Larval stages were obtained both in vitro and from field collections. Adult Paragonimus spp. are described. The aquatic snail Aroapyrgus sp. serves as an intermediate host of this species, both naturally and experimentally, yielding rediae and cercariae. Crabs (Hypolobocera bouvieri monticola and H. emberarum) were found to be the natural second intermediate hosts, and individuals of another crab species (Strengeria sp.) were also infected in the laboratory.

[Development of Paragonimus westermani ichunensis in a reservoir host]. / Razvitie Paragonimus westermani ichunensis v rezervuarnom khoziaine.

Experimental observations showed, that the muscle larva stage of the trematodes Paragonimus westermani ichunensis located in a reservoir host can enlarge its body size. A rate of growth in trematode individuals varied both among different host animals and within one host specimens. When rodents are infected with such muscle larvae, the individuals with the size exceeding 1.15 x 0.33 mm invade into the lungs oh host, while smaller individuals secondarily migrate to muscles.

In vitro excystation of Paragonimus heterotremus metacercariae.

In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39-40 C, and osmolarity of 250-350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5-4 microg/ml), L-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1-8 microM), N-tosyl-L-phenylalanine chloromethyl ketone (0.1-0.4 mM), N alpha-p-tosyl-L-lysine chloromethyl ketone (25-200 microg/ml), iodoacetic acid (0.5-4 mM), and phenylmethylsulfonyl fluoride (1-4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.

Effect of ionophores on in vitro excystment of Paragonimus ohirai metacercariae.

Ca2+ ionophores (A23187 and ionomycin) induced in vitro excystment of Paragonimus ohirai metacercariae, whereas Na+ ionophore (monensin) and K+ ionophores (nigericin and valinomycin) did not. The effect of A23187 on in vitro excystment was dependent on the concentration of the ionophore. A high concentration of 1 microM or more induced rapid excystment, but resulted in the death of metacercarial juveniles. The appropriate concentration of 0.2 microM induced in vitro excystment by 5 h after incubation with little or no damage to the juveniles.

Structural and immunological characteristics of a 28-kilodalton cruzipain-like cysteine protease of Paragonimus westermani expressed in the definitive host stage.

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.