Neodiplostomum leei n. sp. (Digenea: Neodiplostomidae) from chicks infected with metacercariae from the grass snake Rhabdophis tigrina

Neodiplostomula from the European grass snake Rhabdophis tigrina, in the Republic of Korea, were thought to represent 2 species: Pharyngostomum cordatum (large-sized neodiplostomula), an intestinal trematode of cats, and Neodiplostomum seoulense (small-sized neodiplostomula), an intestinal trematode of humans and rodents. The present study describes N. leei n. sp. (Digenea: Neodiplostomidae) on the basis of adult flukes recovered from the small intestines of chicks experimentally infected with small-sized neodiplostomula from the grass snake. The new species differs from N. seoulense in terms of the more extensive distribution of vitellaria, the more severe bilobation of 2 testes (each having a dumbbell shape), the absence of a genital cone, and the smaller size of the tribocytic organ and eggs. Neodiplostomula of the new species and N. seoulense are almost indistinguishable. The new species develops into an adult in chicks, whereas N. seoulense matures in mice and rats. Neodiplostomula of the new species migrate to the liver in mice and rats, without maturing. Results show that there are at least 3 species of neodiplostomid trematodes in the grass snake in the Republic of Korea, i.e.. P. cordatum, N. seoulense and N. leei n. sp.     

Phylogenetic Relationships of 3 Korean Neodiplostomum Species (Digenea: Neodiplostomidae) Based on Partial CO1 Gene

The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.     

Neodiplostomum spratti n. sp. (Digenea: Diplostomidae) from Antechinus spp. (Marsupialia: Dasyuridae) in Australia,with notes on other diplostomids from Australian mammals

Neodiplostomum spratti n. sp. is described from Antechinus stuartii and A. swainsonii (Marsupialia: Dasyuridae). The status of Neodiplostomum minor, N. flabelliformis, N. intermedius, N. sarcophilus, N. diaboli and Pharyngostomoides dasyuri is reviewed. New hosts records for N. intermedius are Rattus lutreolus, R. leucopus, R. rattus (all Rodentia: Muridae) and Dasyurus maculatus (Dasyuridae). The following undescribed species are recorded: Neodiplostomum spp. from Dasyurus maculatus from Victoria, Dasyurus viverrinus from Tasmania, Perameles nasuta (Marsupialia: Peramelidae) from New South Wales, Sminthopsis leucopus (Dasyuridae) from New South Wales, and a diplostomulum from the lung of Perameles nasuta from New South Wales. The validity of Fibricola is reconsidered and it is proposed that it be considered a synonym of Neodiplostomum. The new combinations Neodiplostomum minor (Dubois, 1936), N. sarcophilus (Sandars, 1957) and N. flabelliformis (Pearson & Dubois, 1985) are proposed: all three were previously attributed to Fibricola.     

Experimental human infection with Fibricola cratera (Trematoda: Neodiplostomidae)

Fibricola cratera is a strigeoid trematode indigenous to North America that, heretofore, was known only to infect wild mammals. Herein, it is reported that an experimental inoculation of a human volunteer produced a patent infection that lasted 40 months. Symptoms of epigastric discomfort, loose stools and flatulence occurred over the first year of infection and ameliorated thereafter. Eggs per gram of stool were low (less than or equal to 2) throughout the course of infection and were not detected by the standard technique of formalin-ether concentration. To monitor infection, the entire stool sample was examined each month after sieving through No. 10 (pore size 2 mm) and 100 (pore size 145 microns) sieves and collecting eggs on a No. 325 (pore size 45 microns) sieve. This is the first report of a North American strigeoid trematode capable of maturing in a human and is only the second species of strigeoid known to do so. The other species is F. seoulensis which has been implicated in 26 human infections in Korea.     

Introduction of a Brachylaima species (Digenea: Brachylaimidae) to Australia

A Brachylaima species recorded previously from the house mouse (Mus domesticus) in South Australia is shown to use the introduced European snails Cernuella virgata and Cochlicella barbara as first intermediate hosts, and the same two species and Theba pisana as second intermediate hosts. The life cycle is typical of Brachylaima species. The parasite is capable of infecting at least two species of native Australian rodent, Rattus fuscipes and Leporillus conditor. Because of the intermediate hosts used, it is hypothesized that this trematode species has been introduced from Europe. The parasite may be conspecific with one of a number of described European species but difficulties with the literature concerning these species have required that the present form be designated only as Brachylaima sp.     

Phylogeny and classification of the Digenea (Platyhelminthes: Trematoda)

Complete small subunit ribosomal RNA gene (ssrDNA) and partial (D1-D3) large subunit ribosomal RNA gene (lsrDNA) sequences were used to estimate the phylogeny of the Digenea via maximum parsimony and Bayesian inference. Here we contribute 80 new ssrDNA and 124 new lsrDNA sequences. Fully complementary data sets of the two genes were assembled from newly generated and previously published sequences and comprised 163 digenean taxa representing 77 nominal families and seven aspidogastrean outgroup taxa representing three families. Analyses were conducted on the genes independently as well as combined and separate analyses including only the higher plagiorchiidan taxa were performed using a reduced-taxon alignment including additional characters that could not be otherwise unambiguously aligned. The combined data analyses yielded the most strongly supported results and differences between the two methods of analysis were primarily in their degree of resolution. The Bayesian analysis including all taxa and characters, and incorporating a model of nucleotide substitution (general-time-reversible with among-site rate heterogeneity), was considered the best estimate of the phylogeny and was used to evaluate their classification and evolution. In broad terms, the Digenea forms a dichotomy that is split between a lineage leading to the Brachylaimoidea, Diplostomoidea and Schistosomatoidea (collectively the Diplostomida nomen novum (nom. nov.)) and the remainder of the Digenea (the Plagiorchiida), in which the Bivesiculata nom. nov. and Transversotremata nom. nov. form the two most basal lineages, followed by the Hemiurata. The remainder of the Plagiorchiida forms a large number of independent lineages leading to the crown clade Xiphidiata nom. nov. that comprises the Allocreadioidea, Gorgoderoidea, Microphalloidea and Plagiorchioidea, which are united by the presence of a penetrating stylet in their cercariae. Although a majority of families and to a lesser degree, superfamilies are supported as currently defined, the traditional divisions of the Echinostomida, Plagiorchiida and Strigeida were found to comprise non-natural assemblages. Therefore, the membership of established higher taxa are emended, new taxa erected and a revised, phylogenetically based classification proposed and discussed in light of ontogeny, morphology and taxonomic history.     

Metabolic response of perfused livers to various oxygenation conditions

Isolated liver perfusion systems have been used to characterize intrinsic metabolic changes in liver as a result of various perturbations, including systemic injury, hepatotoxin exposure, and warm ischemia. Most of these studies were done using hyperoxic conditions (95% O(2)) but without the use of oxygen carriers in the perfusate. Prior literature data do not clearly establish the impact of oxygenation, and in particular that of adding oxygen carriers to the perfusate, on the metabolic functions of the liver. Therefore, herein the effects of oxygen delivery in the perfusion system on liver metabolism were investigated by comparing three modes of oxygenation. Rat livers were perfused via the portal and hepatic veins at a constant flow rate of 3 mL/min/g liver in a recirculating perfusion system. In the first group, the perfusate was equilibrated in a membrane oxygenator with room air (21% O(2)) before entering the liver. In the second group, the perfusate was equilibrated with a 95% O(2)/5% CO(2) gas mixture. In the third group, the perfusate was supplemented with washed bovine red blood cells (RBCs) at 10% hematocrit and also equilibrated with the 95% O(2)/5% CO(2) gas mixture. Oxygen and CO(2) gradients across the liver were measured periodically with a blood gas analyzer. The rate of change in the concentration of major metabolites in the perfusate was measured over time. Net extracellular fluxes were calculated from these measurements and applied to a stoichiometric-based optimization problem to determine the intracellular fluxes and active pathways in the perfused livers. Livers perfused with RBCs consumed oxygen at twice the rate observed using hyperoxic (95% O(2)) perfusate without RBCs, and also produced more urea and ketone bodies. At the flow rate used, the oxygen supply in perfusate without RBCs was just sufficient to meet the average oxygen demand of the liver but would be insufficient if it increased above baseline, as is often the case in response to environmental perturbations. Metabolic pathway analysis suggests that significant anaerobic glycolysis occurred in the absence of RBCs even using hyperoxic perfusate. Conversely, when RBCs were used, glucose production from lactate and glutamate, as well as pathways related to energy metabolism were upregulated. RBCs also reversed an increase in PPP fluxes induced by the use of hyperoxic perfusate alone. In conclusion, the use of oxygen carriers is required to investigate the effect of various perturbations on liver metabolism.     

The Pacific Notocotylus atlanticus (Digenea: Notocotylidae)

Recently two unidentified Notocotylus species have been recovered from a mallard in Hokkaido, Japan. Preliminary data indicated that one of them may belong to N. atlanticus—a species found in Europe and on the Atlantic coast of North America. Now we have sequenced partial cox1 for several European N. atlanticus isolates and performed a barcode gap analysis. It has shown the conspecificity of European and Japanese worms, bringing up discussion on distribution, transmission and host specificity in this species.     

Reproduction of the trematode Echinostoma caproni parthenites (Digenea: Echinostomatidae)

Dynamics of the reproduction in the trematode Echinostoma caproni parthenites (Echinostomatidae) was observed. Early laying and maturation of the generative cells are for the first time shown to be characteristic for all parthenogenetic generations. Really the process of reproduction had been finishing to the beginning of the generating of new age by parthenites. Mother sporocysts, as well as redia of different generations, in fact stop producing new generative cells with the beginning of the generating of new age, and assume the function of a brood pouch. This feature was considered previously as peculiar mainly to mother sporocysts. Data on the autotomy of the anterior body end in mother sporocysts are verified. In our opinion, these data are an evidence of an early manifestation of the evolutionary trend to the morpho-functional regress and disintegration of the parasitic stage of mother sporocyst.     

Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae)

Prior exposure of Biomphalaria glabrata to the eggs of an incompatible digenean, Plagiorchis elegans, rendered this snail host less suitable to a compatible species, Schistosoma mansoni. Although P. elegans failed to develop patent infections in B. glabrata, it reduced the production of S. mansoni cercariae by 88%. Concomitantly, host attributes such as reproduction, growth, and survival were compromised. The effect of P. elegans infection was most severe among snails that, in addition, had developed patent schistosome infections. Although few S. mansoni cercariae were produced, egg production by B. glabrata was only 4% of control values. Furthermore, no doubly infected snails survived for more than 3 wk after patency, whereas controls experienced no mortality during the same time period. The above effects were attributable to the establishment and persistence of P. elegans sporocysts in the tissues of the incompatible snail host. Their indirect antagonistic interaction with thelarval stages of S. mansoni may be mediated, in part, through their long-term stimulation of the host's internal defense mechanisms. These findings are discussed with a view to use P. elegans and other plagiorchiid digeneans as agents in the biological control of snails and snail-borne diseases.     

Confirmation of the safety of Lagenidium giganteum (Oomycetes: Lagenidiales) to mammals

Lagenidium giganteum Couch, a fungal parasite of mosquito larvae, is considered a promising agent for operational control. Quantitative reisolation of the fungus from rodent blood and tissue is easily accomplished because the cell walls are much more persistent than those of mammalian cells. The mycelium forms discrete, septate cells very early in its development, in marked contrast to filamentous fungi. The fungus is rapidly cleared from mice following intraperitoneal injection of large quantities of mycelium and oospores. More than 95% of viable cells are cleared within 24 h. Mortality following intravenous injection of mycelium in tail veins of mice is caused solely by embolism of heart and lung tissue. Recently revised U.S. Environmental Protection Agency protocols for testing of microbial pest control agents may have to be altered further to accommodate physically large microbial agents such as L. giganteum.     

Host specificity of Austropeplea ollula (Gastropoda: Lymnaeidae) to miracidial infection with a human intestinal fluke Neodiplostomum seoulense (Trematoda: Diplostomatidae) in Korea

Three species of freshwater pulmonate snails of Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula, and Fossaria truncatula. Of these 3 species, only A. ollula was naturally infected with Neodiplostomum seoulense cercariae (prevalence, 0.3%). In experiments with the laboratory-reared snails, A. ollula was found to be susceptible to the N. seoulense miracidia (prevalence, 10%). This is the first report of A. ollula as the first molluscan intermediate host for N. seoulense in Korea.     

Stictodora lari (Digenea: Heterophyidae): the discovery of the first human infections

Stictodora lari Yamaguti, 1939 (Digenea: Heterophyidae) was originally reported as an avian intestinal parasite and is here reported in humans. The patients were adults, aged 46-69 (2 men and 4 women), who resided in coastal villages in Sachon-gun, Kyongsangnam-do, or Shinan-gun, Chollanam-do. in the Republic of Korea. The worms were recovered after praziquantel treatment and purgation with magnesium salts. A total of 15 S. lari specimens (1-10 per individual) was collected, together with 5 other species of intestinal flukes. The flukes were morphologically characterized by a small body size (0.70-0.86 mm long and 0.27-0.36 mm wide), a ventrogenital sac, and a gonotyl armed with 70-80 spines in the form of a comma or reversed comma lying along their lateral margin. The patients had eaten the raw flesh of mullets and gobies that had been caught in an estuary near their villages. To the best of our knowledge, this is the first record of a human S. lari infection.     

Western analyses of pregnancy-associated glycoprotein family (PAG) in placental extracts of various mammals

The present study was conducted in order to analyze the immunoreactivity of placental extracts of several animal species and humans against the following three groups of PAG antisera: anti-boPAG-I (R#497), -boPAG-II (R#435), and -caPAG (R#706). Placental proteins were obtained after extraction at neutral pH, followed by ammonium sulfate (A.S.) precipitation, dialysis, and lyophilization. The immunoreactivity of different placental extracts was revealed by the use of monodimensional SDS-PAGE, followed by blotting on nitrocellulose membrane and the identification of immunoreactive proteins after incubation with PAG antisera (Western blot technique). A strong immunoreactivity of proteins from synepitheliochorial placenta (cattle, sheep, goat, bison, buffalo, and deer) was demonstrated in both 20-50% and 50-80% A.S. fractions using the three antisera. Proteins from species with epitheliochorial placenta presented variable profiles of detected PAG-like proteins: in the sow, many immunoreactive forms were revealed by antisera boPAG-I and boPAG-II, whereas in the dromedary, only two forms were revealed by anti-boPAG-II. Concerning other species, our protocols showed for the first time a cross-reaction between PAG antisera with proteins extracted from dog, alpaca, dromedary, sea lion, and human placenta.     

Histopathology and cellular response in Enteromyxum leei (Myxozoa) infections of Diplodus puntazzo (Teleostei)

Enteromyxum leei is an intestinal parasite responsible for serious outbreaks in Mediterranean sharpsnout sea bream Diplodus puntazzo. E. leei infection was experimentally transmitted to healthy D. puntazzo (R) by cohabitation with infected donor fish. Haematological changes and histopathological damage were evaluated in relation to the course of infection. The prevalence of infection in R fish was 100% from day 10 post-exposure (p.e.) onwards, and the infection intensity and histopathological damage increased progressively. Different developmental stages were found in the infected intestines, including proliferative (stages 1–3) and sporogonic (stages 4 and 5) stages. Intestinal damage consisted of vacuolation, necrosis, detachment and sloughing of epithelium, and was correlated with the progression of the infection and with the development of the parasite. Sporogonic stages appeared from day 20 p.e. onwards. Initially, D. puntazzo seems to counteract the infection through the increase in leucocyte numbers, respiratory burst activity, haematopoietic activity and MMC. Two types of eosinophilic granular cells (EGC1 and EGC2) were detected in the intestinal epithelium and lamina propria. EGC1 numbers decreased with the progression of infection, whereas an increase in EGC2 occurred, mainly in the lamina propria. The involvement of the cellular immunity in the response of D. puntazzo to E. leei was demonstrated. The depletion of this response at a certain point of the infection could contribute to the high virulence of this myxozoan in this fish species.     

Life cycle of Coitocoecum plagiorchis (Trematoda: Digenea: Opecoelidae)

The life cycle of Coitocoecum plagiorchis Ozaki, 1926 (Coitocaecum is an incorrect subsequent spelling) was studied in the field and laboratory. The study was conducted at the Futatsu River and the Chikugo River, Kyushu, Japan. Adults of C. plagiorchis were obtained from fishes Coreoperca kawamebari, Rhinogobius spp. and Odontobutis obscura. Cotylomicrocercous cercariae with a two-point stylet (= Cercaria distyloides Faust, 1924) were detected in pleurocerid snails (Semisulcospira spp.). Metacercariae with a cyclocoel were obtained from shrimp (Neocaridina denticulata). The cercariae were experimentally exposed to the shrimp, and subsequently metacercariae with a cyclocoel were recovered from the shrimp within 24 days after exposure. These metacercariae were identified as C. plagiorchis. The sporocysts, cercariae, metacercariae and adults are briefly described.     

Tegumental ultrastructure of adult Gynaecotyla squatarolae (Digenea: Microphallidae)

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. Two ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was seen. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ultrastructure.