Two new species of Unikaryon (Microsporidia) hyperparasitic in microphallid metacercariae (Digenea) from Florida intertidal crabs.

The genus Unikaryon (Microsporidia) holds exclusively hyperparasites of Platyhelminthes. Four species of Unikaryon are presently known from trematodes infecting mollusks and fish, and one from a cestode infecting a fish. Here we report two species of Unikaryon from microphallid trematode metacercariae parasitizing the brachyuran crabs, Panopeus herbstii and Pachygrapsus transversus, collected from intertidal habitats in Florida. The first microsporidium, which we assign here to a new species, Unikaryon panopei sp. n., was isolated from Microphallus sp. encysted in Panopeus herbstii from Tampa Bay. The specific designation for the second Unikaryon sp. (Unikaryon sp. 2), which occurred in metacercaria of Diacetabulum sp. found in P. transversus from the Florida Keys, is pending due to the lack of SSrDNA sequence data. Light and electron microscopy demonstrates that both species display characteristics of the genus Unikaryon including the arrangement of spores in sets of two, large posterior vacuole, and eccentric position of the polar filament. Spores of Unikaryon panopei sp. n., unlike those of Unikaryon sp. 2, assemble in large membrane-bound masses containing hundreds of organisms, and display a larger number of polar filament coils - 7-8, compared to 4-5 in Unikaryon sp. 2 The SSUrDNA-inferred phylogenetic analysis places Unikaryon panopei in one clade with Unikaryon legeri, the only other molecularly characterized member of the genus, with 94% of SSUrDNA similarity. These findings increase the number of species parasitizing trematodes and broaden the host range of Unikaryon spp.

Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50µg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries.

Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni.

BACKGROUND: Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. METHODS: Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. RESULTS: The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5-7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. CONCLUSION: NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Experimental infection with Paragonimus heterotremus metacercariae in laboratory animals in Manipur, India.

This study was aimed to find out the host-parasite relationship between Paragonimus heterotremus isolated as metacercariae from mountain crabs, Indochinamon manipurensis, in Manipur, India and laboratory animals such as puppies, albino rats, Swiss mice, guinea pigs, and rabbits, as experimental animals. The animals were fed with the metacercariae. Infected animals were sacrificed 35 to 430 days after feeding to recover worms, which were used to determine the developmental stages. Adult worms (n = 14) were recovered from 3 puppies > or = 70 days after feeding and immature worms (n = 25) were recovered from 2 other puppies 35 or 43 days after infection. The infection rate in puppies was 100%. Juvenile worms were recovered from 3 of 13 rats: 1 of 11 rats whose viscera and cavities were examined and both of two rats whose muscles were examined. Rats were not a suitable animal model for pulmonary infection with P. heterotremus. Mice, guinea pigs, and rabbits were also found to be insusceptible to pulmonary infection with P. heterotremus.

Infection dynamics and molecular identification of metacercariae in cyprinoids from Chiang Mai and Sakon Nakhon Provinces.

The infection dynamics of metacercariae were assessed in cyprinoid fish (cyprinoids) from Chiang Mai and Sakon Nakhon Provinces, Thailand, during October 2008 to September 2009. The samples were collected during 3 seasons from rivers and local markets. Metacercarial infection was determined by acid-pepsin digestion and confirmed using HAT-RAPD PCR method. Thirteen and 16 species of cyprinoids were collected from Chiang Mai and Sakon Nakhon with overall prevalences of metacercarial infection of 84.5 and 47.6%, respectively. Haplorchis taichui, Haplorchoides sp, and Centrocestus caninus were found in Chiang Mai and 4 species of metacercariae: H. taichui, Haplorchoides sp, O. viverrini and an unknown trematode species in Sakon Nakhon. H. taichui and Haplorchoides sp metacercariae in 3 species of cyprinoids (Henicorhynchus siamensis, Cyclocheilichthys armatus, Amblyrhynchichthy truncatus) had the highest prevalence (100%) in Chiang Mai, while the highest prevalence (100%) of metacercaria in Sakon Nakhon was Haplorchoides sp in 1 species of cyprinoids (Cyclocheilichthys armatus). The overall prevalence from Chiang Mai Province was highest in the rainy season (95.6%), lower in the hot-dry season (88.1%) and lowest in the cool season (72.5%). In Sakon Nakhon Province the highest prevalence was in the hot-dry season (52.7%), and lower in the rainy and cool season, 44.4% and 43.5%, respectively. The HAT-RAPD profiles confirmed the identity of metacercariae and adult stage of H. taichui, Haplorchoides sp, C. caninus and O. viverrini.

Seasonal variation of metacercariae in cyprinoid fish from Kwae Noi Bamroongdan Dam, Phitsanulok Province, northern Thailand.

Abstract. A seasonal investigation of the occurrence of metacercariae in cyprinoid fish was conducted at the Kwae Noi Bamroongdan Dam, Phitsanulok Province, northern Thailand during December 2008-August 2009. A total 361 cyprinoid fish from 10 species were examined by artificial digestion method evaluating for metacercariae. The metacercariae determined were Haplorchis taichui, Haplorchis pumilio, Haplorchoides sp and Centrocestus caninus. The prevalence of metacercariae ranged from 76.5% to 82.6%, with an intensity of infection of 23.4 to 36.6. H. taichui, Haplorchoides sp and C. caninus had the highest prevalence in the cool season, while H. pumilio was more prevalent during the hot season. The highest infection prevalence (100%) was seen in Rasbora metallicus, Barbodes gonionotus and R. metallicus. No significant differences in the type of metacercariae were found between seasons, indicating year-round infestation of cyprinoid fish.

Occurrence of Clinostomum complanatum in Aphanius dispar (Actinoptrygii: Cyprinodontidae) collected from Mehran River, Hormuzgan Province, South of Iran.

OBJECTIVE: To investigate the possibility of Aphanius dispar (A.dispar) acting as intermediate host for Clinostomum complanatum (C. complanatum), from Mehran River, Hormuzgan Province of Iran. METHODS: During a biological study of A. dispar in Mehran River, Hormuzgan Province, South of Iran, a total of 97 fish specimens were collected in 24 January 2006. RESULTS: 4 specimens (4.12%) including 1 male and 3 female were found infected with C. complanatum metacercaria. These metacercaria were coiled in the epiderm on the body surface of infected fishes. The infection is known as yellow spot disease. The parasite abundance, intensity and prevalence were 0.05%, 1.25% and 4.12%, respectively. The infection was higher in females than males. CONCLUSIONS: To the best of our knowledge, this is the first report on occurrence of C. complanatum metacercaria in A. dispar in Iran.