Paragonimus heterotremus Chen et Hsia, 1964 (Digenea: Paragonimidae): species identification based on the biological and genetic criteria, and pathology of infection.

As a result of the experimental infection of rats with metacercariae of Paragonimus heterotremus Chen et Hsia, 1964 from crabs (Potamiscus tannanti) caught in Yen Bai province, Vietnam, it was found that worms migrated into the lungs, to the liver and less frequently to the tissue that lines body cavities of the hosts, where they reached the adult stage, but in the muscles, worms stayed at the larval stage. Studies have shown that for P. heterotremus, rats can simultaneously play the role of the final and paratenic host; herewith, an infection with the trematode of this species can lead to the development of three forms of paragonimiasis: pulmonary, hepatic and muscular. Eggs from the adult worms localised in the liver, unlike eggs from the adult worms localised in the lungs, were not excreted into the external environment, but accumulated inside the organ. Histology and description of changes, which take place on the external surface of organs affected with P. heterotremus, are given in this study. Based on the behavioural characteristics of worms during rat infection and molecular genetic data, we established that worms from Vietnam and India should be assigned to different species of Paragonimus. P. heterotremus distribution is limited to the territory of the Southeast China, Northern Vietnam, Laos and Thailand.

Expanded complement of Niemann-Pick type C2-like protein genes in Clonorchis sinensis suggests functions beyond sterol binding and transport.

BACKGROUND: The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles. METHODS: We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues. RESULTS: Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage. CONCLUSIONS: The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.

Digenean Holostephanus (Trematoda: Digenea: Cyathocotylidae) metacercariae in common carp (Cyprinus carpio Linnaeus, 1758) muscle: zoonotic potential and sensitivity to physico-chemical treatments.

Metacercariae of various species within the genus Holostephanus Szidat, 1936 (Trematoda: Digenea: Cyathocotylidae) occur in muscles of both farmed and wild fish, including common carp (Cyprinus carpio Linnaeus, 1758). The life cycle includes a snail as first intermediate host, fish as second intermediate host and birds or mammals as final hosts. We studied the zoonotic potential and the viability of Holostephanus metacercariae from common carp following exposure to various physical and chemical treatments. Muscle tissue samples of common carp specimens from a fish farm in the north-eastern part of Hungary were examined and metacercariae recovered. The zoonotic potential was evaluated experimentally by using small mammals as models (albino mice, n = 2; and Syrian hamsters, n = 4) infected per os with Holostephanus cysts. Parallelly, Metagonimus metacercariae were used as positive controls. We could not confirm the zoonotic potential of Holostephanus metacercariae as they did not survive in the mammalian intestine whereas Metagonimus metacercariae developed to the adult stage. We assessed the viability of metacercariae isolated from common carp specimens during exposure to different physical treatments (temperatures of -18°C, +20°C, +40°C and +60°C) and chemical agents (5% and 10% acetic acid and 10% sodium chloride (NaCl)). Metacercariae lost viability by freezing at -18°C (2 h), heating at 60°C (20 min), incubation in 5% and 10% acetic acid (5 min) and 10% NaCl (2 h). These methods served as models to investigate the effectiveness of food preparation techniques (such as cold and hot smoking, freezing, salting and pickling) on the survival of metacercariae.

Microphallus ochotensis sp. nov. (Digenea, Microphallidae) and relative merits of two-host microphallid life cycles.

A new digenean species, Microphallus ochotensis sp. nov., was described from the intestine of Pacific eiders (Somateria mollissima v-nigrum) from the north of the Sea of Okhotsk. It differs from other microphallids in the structure of the metraterm, which consists of two distinct parts: a sac with spicule-like structures and a short muscular duct opening into the genital atrium. Mi. ochotensis forms a monophyletic clade together with other congeneric species in phylograms derived from the 28S and ITS2 rRNA gene. Its dixenous life cycle was elucidated with the use of the same molecular markers. Encysted metacercariae infective for birds develop inside sporocysts in the first intermediate host, an intertidal mollusc Falsicingula kurilensis. The morphology of metacercariae and adults was described with an emphasis on the structure of terminal genitalia. Considering that Falsicingula occurs at the Pacific coast of North America and that the Pacific eider is capable of trans-continental flights, the distribution of Mi. ochotensis might span the Pacific coast of Alaska and Canada. The range of its final hosts may presumably include other benthos-feeding marine ducks as well as shorebirds. We suggest that a broad occurrence of two-host life cycles in microphallids is associated with parasitism in birds migrating along sea coasts. The chances that migrating birds would stop at a site where both first and second intermediate hosts occur are relatively low. The presence of a single molluscan host in the life cycle increases the probability of transmission.

Immune response of rats vaccinated orally with various plant-expressed recombinant cysteine proteinase constructs when challenged with Fasciola hepatica metacercariae.

BACKGROUND: Cysteine proteinases of Fasciola hepatica are important candidates for vaccine antigens because of their role in fluke biology and host-parasite relationships. In our previous experiments, we found that a recombinant cysteine proteinase cloned from adult F. hepatica (CPFhW) can protect rats against liver fluke infections when it is administered intramuscularly or intranasally in the form of cDNA. We also observed considerable protection upon challenge following mucosal vaccination with inclusion bodies containing recombinant CPFhW produced in Escherichia coli. In this study, we explore oral vaccination, which may be the desired method of delivery and is potentially capable of preventing infections at the site of helminth entry. To provide antigen encapsulation and to protect the vaccine antigen from degradation in the intestinal tract, transgenic plant-based systems are used. METHODOLOGY: In the present study, we aimed to evaluate the protective ability of mucosal vaccinations of 12-week-old rats with CPFhW produced in a transgenic-plant-based system. To avoid inducing tolerance and to maximise the immune response induced by oral immunisation, we used the hepatitis B virus (HBV) core protein (HBcAg) as a carrier. Animals were immunised with two doses of the antigen and challenged with 25 or 30 metacercariae of F. hepatica. CONCLUSIONS: We obtained substantial protection after oral administration of the plant-produced hybrids of CPFhW and HBcAg. The highest level of protection (65.4%) was observed in animals immunised with transgenic plants expressing the mature CPFhW enzyme flanked by Gly-rich linkers and inserted into c/e1 epitope of truncated HBcAg. The immunised rats showed clear IgG1 and IgM responses to CPFhW for 4 consecutive weeks after the challenge.

Infectivity and development of Opisthorchis viverrini metacercariae in immunosuppressed Barbonymus gonionotus fingerlings (Cyprinidae).

Opisthorchiasis caused by Opisthorchis viverrini infection, is a serious public health problem in Lao PDR, Cambodia, Vietnam and Thailand. Cyprinid fish are required for the transmission of O. viverrini metacercariae to fish eating-mammal hosts including humans. The transmission of infective metacerariae is an important event, potentially resulting in the development of cholangiocarcinoma in cases of chronic opisthorchiasis. For a better understanding of the development of O. viverrini metacercariae in the second intermediate host, Barbonymus gonionotus (Cyprinidae), the immune response of infected B. gonionotus through fibrous tissue formation and superoxide dismutase (SOD) activity were investigated. The immunosuppressant (prednisolone) was administered to B. gonionotus infected with O. viverrini cercariae to study infectivity and development of the metacercariae. Additionally, the effects of prednisolone were studied by the investigation of fibrous tissue formation around the metacercariae, as well as SOD activity. In our study, prednisolone administered prior to infection in B. gonionotus had higher metacercarial recovery when compared to the non-administered control group. On the other hand, the administration of prednisolone after the O. viverrini infection allowed metacercariae to develop to the infective stage. Energy consumption from encystation to the infective stage required 443.9-463.7degree-days. The results show that immunosuppression decreases fibrous tissue and SOD activity in infected fish, and also affects the ability of O. viverrini metacercariae to develop to the infective stage. The immune response to metacercarial development of O. viverrini in cyprinid fish should be further studied to gain a greater understanding of this process, and hence, a better understanding of the mechanism(s) involved in metacercarial wall formation.

Gene Expression Profile in the Liver of BALB/c Mice Infected with Fasciola hepatica.

BACKGROUND: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with F. hepatica metacercariae using a microarray-based methodology. METHODOLOGY: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). RESULTS: We found that F. hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. CONCLUSION: The present study provides us insights at the molecular level about the underlying mechanisms used by F. hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis.

Effect of pond water depth on snail populations and fish-borne zoonotic trematode transmission in juvenile giant gourami (Osphronemus goramy) aquaculture nurseries.

Infection with fish-borne zoonotic trematodes (FZT) is an important public health problem in many parts of Southeast Asia. People become infected with FZT when eating raw or undercooked fish that contain the infective stage (metacercariae) of FZT. The parasites require specific freshwater snails as first intermediate host and a variety of fish species, both wild caught and cultured, as second intermediate host. Aquaculture production has grown almost exponentially in SE Asia and in order to produce fish free from FZT metacercariae, it is important to mitigate factors promoting transmission to fish. Here we report results from a cross-sectional study to look at the association between pond depth and infection with FZT in giant gourami nursery ponds. Density of intermediate host snails was positively associated with pond depth (count ratio associated with a 1m increase in pond depth was 10.4 (95% C.L.: 1.61-67.1, p<0.5)) and this may partly explain the higher prevalence and intensity of FZT infection in juvenile fish. High fry stocking density (>200 fry m(-3)) was associated with lower host snail density (count ratio=0.15) than low stocking density (<100 fry m(3)). Ponds stocked with 100-200 fry m(-3) had snail counts 0.76 (95% C.L.: 0.33-1.75, p n.s.) of those in ponds stocked with fry density of <100 fry m(-3). Since density of intermediate snail hosts was associated with FZT transmission to fish, effort should be taken to reduce snail density prior to stocking the fry, but focus should also be on habitats surrounding ponds as transmission may occur through cercariae produced outside ponds and carried into ponds with water pumped into ponds.

Acanthoparyphium sp. and other metazoan symbionts of the American oyster, Crassostrea virginica, from South Texas.

In this study, we investigated oysters, Crassostrea virginica , from Laguna Madre in South Texas, where a 45-yr old study recorded metacercarial infections of the echinostomatid trematode, Acanthoparyphium spinulosum , an Asian relative of which, Acanthoparyphium tyosenense, has been associated with human infections via the ingestion of raw mollusks. In an effort to examine the base-line infection parameters of Acanthoparyphium sp. in oysters, we examined the effect of distance from the shoreline, which is the habitat of the first intermediate host snail, Cerithidea pliculosa, as well as temporal changes in oyster infection levels, by conducting quarterly collections of oysters during a year. We found that almost all oysters (prevalence = 97.8-100%) were infected regardless of distance to the shoreline and season. However, the abundance of metacercariae was significantly higher close to the shoreline, while no significant temporal changes could be detected. In addition to the echinostomatid, we found a high abundance of the metacestode Tylocephalum sp. and the presence of 4 other metazoan parasites. None of the infections seemed to incur significant tissue damage to the oysters. Our study shows that at least locally, recreational harvesters of oysters may be exposed to Acanthoparyphium sp. Future studies should examine oysters from snail host habitats in the Gulf of Mexico, and the potential zoonotic risk of Acanthoparyphium sp. should be evaluated using experimental infections of animal models.

Echinostoma revolutum: freshwater snails as the second intermediate hosts in Chiang Mai, Thailand.

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice.

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.

Seasonal features of Heterophyopsis continua metacercariae in perches, Lateolabrax japonicus, and infectivity to the final host.

As the second intermediate host, the perch Lateolabrax japonicus provides metacercariae of Heterophyopsis continua to man. Fry of the perch arrive at and grow to mid-sized fish in the brackish water of Jinju Bay, South Korea. The H. continua metacercariae occur in perch as small and mid-sized forms in July and grow to the large form by August. The size distribution of metacercariae shifts dominantly to the large form in September and then remains as it was. Adult H. continua recovery was high from experimental chicks infected with the medium and large metacercariae collected in September and reduced to half from the chicks infected with those of October. Collectively, the H. continua metacercariae grew to the large form in about 20 days and achieved infectivity to chicks after 2 months in the perches.

Comparative studies on animal models for Opisthorchis viverrini infection: host interaction through susceptibility and pathology.

Syrian hamsters and gerbils are animal models for Opisthorchis viverrini infection. In both models, the parasites develop into adults with different pathologies of the hepatobiliary system. However, no comparative pathological studies have yet been completed. We therefore investigated host interaction through the susceptibility and pathological changes of Syrian hamsters and gerbils infected with 50 O. viverrini metacercariae for 30, 60, and 90 days post-infection. Animals were sacrificed at each time point for comparative study. Susceptibility and infectivity were investigated through worm burden. Parasite morphology and reproductive organs were stained with carmine and observed under light microscopy. Reproductive organs and eggs per worm were counted to confirm worm maturity. Bile acid components of both animal groups were analyzed by thin-layer chromatography. The results showed that infection in gerbils was of greater severity than in Syrian hamsters by observation of bile obstruction, enlargement of the gallbladder and common bile duct, and generation of fibrosis and cirrhosis. The worm burden of infected gerbils was lower than that observed in Syrian hamsters. Infectivity in both Syrian hamsters and gerbils was 100% with infection by 50 metacercariae; whereas with 10 metacercariae, the infectivity in gerbils was zero to very low, but still 100% in Syrian hamsters. The largest body size of worms, and the largest ovary and testes areas, was correlated with eggs per gram of feces and eggs per worm. The bile acid components cholic acid and chenodeoxycholic acid were undetectable in gerbils. The present study suggests that although Syrian hamsters, usually the host selection for an animal model, are susceptible to O. viverrini infection, infected gerbils produce worms that mature more rapidly, have larger body sizes, and more fully developed reproductive organs; this may be caused by the difference in bile acid components.

Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: an implication of high pathogenicity of the European liver fluke.

European liver fluke (Opisthorchis felineus) and Asian liver fluke (Opisthorchis viverrini) are similar in morphology but comparative pathology of the infections has not been described. We therefore did comparative histopathology of both parasites in an experimental animal model. The study was conducted in 3 groups of 105 Syrian golden hamsters; the first and second groups fed with 50 metacercariae of O. felineus (OF) or O. viverrini (OV) and the last group was uninfected controls. Five hamsters in each group were euthanized on weeks 1, 2, 4, 8, 12 and 24 post-infection. The liver tissue was fixed and processed for routine histopathology and immunohistochemistry for proliferation markers (BrdU or PCNA). Overall, the liver histopathology of O. felineus and O. viverrini infection was generally similar. However, various histopathogical features including intense inflammation, fibrosis, biliary and goblet cell hyperplasia and dysplasia occurred earlier in the OF group. In addition, the existence of precancerous lesions such as cholangiofibrosis in a long-term infection was observed only in this group. O. felineus is larger in size than O. viverrini which, together with its excreted and secreted antigens, likely is crucial in the induction of liver fluke induced disease. The differences in nature and timing of the histopathological profile indicate that opisthorchiasis caused by the European liver fluke O. felineus is more pathogenic than its Asian relative O. viverrini.

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP.

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.

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.

Migration, site selection, and development of Ornithodiplostomum sp. metacercariae (Digenea: Strigeoidea) in fathead minnows (Pimephales promelas).

The metacercarial stage of trematodes is typically considered an encysted, developmentally quiescent, resting stage. Yet the metacercariae of some species of strigeoid trematode undergo extravagant development within specific tissues of their second intermediate host. Our understanding of patterns of migration, site selection and development of these types of metacercariae is known for only a few species. In this study, we characterize the invasion and development of Ornithodiplostomum sp. metacercariae in their second intermediate host, the fathead minnow, Pimephales promelas. Diplostomules completed their migration into the abdominal cavity between 15 min and 48 h p.i. Most diplostomules migrated along muscular and connective tissue then penetrated the peritoneal lining of the abdominal cavity en route to the liver or pancreas. Alternatively, some diplostomules migrated within the host's circulatory system, including the heart and arteries of the hepatic portal system. Metacercarial development in the liver and pancreas involved distinct growth, encystment and consolidation phases. Metacercarial volume increased 15-fold between 48 h and 4 weeks p.i., presumably due to absorptive and/or ingestive feeding activities within host tissues. By 2 weeks p.i., metacercariae were enveloped within a cyst wall and they were found loosely attached to the surfaces of internal tissues or unattached within the body cavity. These results emphasize the complex nature of metacercarial migration and growth and demonstrate that their growth and encystment phases occur within different habitats within their intermediate hosts.

Temporal variation of Meiogymnophallus minutus infections in the first and second intermediate host.

In order to study seasonal patterns of Meiogymnophallus minutus infections in its intermediate hosts, bivalve samples were collected monthly between April 2008 and March 2009 from a high intertidal flat at Courtmacsherry Bay, Ireland. Infection rates in the first intermediate host Scrobicularia plana did not fluctuate significantly with season. Completely developed M. minutus cercariae appeared in daughter sporocysts from June and prevailed from July to October, indicating that transmission of M. minutus from its first to its second intermediate host is confined to this period of the year. All analysed individuals of the second intermediate host Cerastoderma edule were found to be infected with metacercariae. Infection levels significantly increased in September, suggesting recent cercarial invasions. Throughout the year, the majority of metacercariae were hyperinfected by the pathogenic microsporidian Unikaryon legeri. Spreading of hyperinfections was confined to spring and summer. Newly settled metacercariae were not affected by hyperparasitism and presumably retained their infectivity for half a year. Our findings suggest that the spreading of hyperinfections is correlated with higher water temperatures and that the entire metacercarial population has to rebuild every year as a consequence of hyperparasite-induced mortality.

Influence of Calicophoron microbothrium amphistomosis on the biochemical and blood cell counts of cattle.

Sixteen Tuli steers aged 1 year were subdivided into four equal groups (I-IV) and infected with Calicophoron microbothrium metacercariae. Group I received a low dose (LD) of 5000 metacercariae, group II a medium dose (MD) of 15,000 metacercariae, group III a high dose (HD) of 25,000 metacercariae while group IV was the non-infected control (C) group. The experimental animals were monitored daily for clinical signs while ethylene diamine tetraacetic acid (EDTA) blood and serum samples were collected every 7 days until day 28 post-infection, when sample collection was terminated. Samples were processed for full blood count, eosinophils and blood biochemical values for calcium, magnesium, phosphorus, total protein and albumin. Moderate to severe diarrhoea developed in the MD and HD groups at day 21 post-infection. The diarrhoea coincided with a significant decrease (P < 0.05) in total plasma protein, calcium and phosphorus levels, particularly in the MD group. Similarly, a significant decrease (P < 0.05) in the packed cell volume (PCV), the haemoglobin (Hb) and red blood cell (RBC) levels occurred in the MD and HD groups from day 21 post-infection, while a significant increase (P < 0.05) in the circulating eosinophils occurred between 7 and 21 days post-infection in the LD and the HD groups.

On a new lung fluke found in Peru, Paragonimus peruvianus sp. n. (Trematoda: Troglotrematidae)

Paragonimus peruvians sp.n was described from two cats captured in La Asunción and El Guayo, Cajamarca, Peru. The new species is allied to P. mexicanus Miyazaki et Ishii, 1968 in morphology, but they are separated by the branching of the ovary and testes as well as by the character of eggs. It is most likely that human paragonimiasis occurring in Cajamarca area is caused by the new species. The metacercaria belonging probably to P. peruvianus was found in a fresh-water crab, Pseudothelphusa chilensis collected in El Guayo, which is not infrequently eaten uncooked by inhabitants in that area. Ultimately, P. peruvianus seems to be very important in the medical field.