Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia

A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs.     

A Minchinia-like haplosporidan parasitizing blue crabs,Callinectes sapidus

A haplosporidan parasite was found infecting two moribund blue crabs, Callinectes sapidus, from Virginia and North Carolina. Spore stages were not found in either crab. Because of the presence of haplosporosomes in the cytoplasm, the parasites are thought to be a species of Minchinia or Urosporidium.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status

Several Vibrio species are known to be pathogenic to the Pacific oyster Crassostrea gigas. Survival varies according to pathogen exposure and high mortality events usually occur in summer during gametogenesis. In order to study the effects of gametogenetic status and ploidy (a factor known to affect reproduction allocation in oysters) on vibriosis survival, we conducted two successive experiments. Our results demonstrate that a common bath challenge with pathogenic Vibriosplendidus and Vibrio aestuarianus on a mixture of mature, spawning and non-mature oysters can lead to significant mortality. Previous bath challenges, which were done using only non-mature oysters, had not produced mortality. Immunohistochemical analyses showed the affinity of Vibrio for gonadic tissues, highlighting the importance of sexual maturity for vibriosis infection processes in oysters. Mortality rate results showed poor repeatability between tanks, however, in this bath challenge. We then tested a standardized and repeatable injection protocol using two different doses of the same combination of two Vibrio species on related diploid and triploid oysters at four different times over a year. Statistical analyses of mortality kinetics over a 6-day period after injection revealed that active gametogenesis periods correspond to higher susceptibility to vibriosis and that there is a significant interaction of this seasonal effect with ploidy. However, no significant advantage of triploidy was observed. Triploid oysters even showed lower survival than diploid counterparts in winter. Results are discussed in relation to differing energy allocation patterns between diploid and triploid Pacific oysters.     

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota)

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

A new Perkinsus species (Apicomplexa,Perkinsea) from the abalone Haliotis ruber

A new protozoan of the genus Perkinsus is described from the muscle and hemolymph of the blacklip abalone, Haliotis ruber, from South Australia. It occurs in the muscle of the adductor and mantle, and free and in brownish masses in the hemolymph. Cells cultured in thioglycollate medium produced a prezoosporangium which stained blue-black with iodine. The parasite differs from Perkinsus marinus, the only other member of the class, in having a much larger trophozoite, an eosinophilic vacuoplast when present, a short discharge tube, and appears to be uninfective to oysters.     

Cryptosporidium: New developments in cell culture

Studies on Cryptosporidium species have been hampered by the limited amount of parasitic stages available for research. One of the major objectives of many laboratories is to develop a reproducible culture model for this important parasite. Recent research has resulted in long-term culturing of Cryptosporidium in cell culture using pH modification, sub-culturing and gamma irradiation. Further advances in the in vitro culturing of Cryptosporidium revealed that this parasite can complete its life cycle in culture medium overcoming the problem of using the host cells, as host cell overgrowth and aging resulted in the termination of the Cryptosporidium life cycle prior to its completion. Improved methods for visualizing life cycle stages in cell-free culture have also been developed. This review will discuss factors that can influence the success of Cryptosporidium culture in vitro and propose new ideas for the future optimization of the cell-free culture system.     

In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle

Fat body, ovaries without their surrounding connective tissue, and ovarian connective tissue of the ovoviviparous cockroach Leucophaea maderae were incubated in vitro. Incorporation of ¹⁴C-labelled amino acids into proteins by all three tissues was investigated quantitatively and qualitatively during oöcyte maturation (corpora allata active), immediately after ovulation, and during the gestation period (corpora allata inactive). Proteins in the tissues and in the incubation media were processed separately. The qualitative analysis involving disk electrophoretic separation of Ringer soluble tissue proteins or of proteins in the incubation media and subsequent autoradiography of the dried gels showed that all three tissues synthesize the same 26 proteins in all stages of the sexual cycle. An additional fraction is produced by the ovarian connective tissue only. All three tissues synthesize proteins at a higher rate during oöcyte maturation than during gestation.     

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Myospora metanephrops (n. g., n. sp.) from marine lobsters and a proposal for erection of a new order and family (Crustaceacida; Myosporidae) in the Class Marinosporidia (Phylum Microsporidia)

In this study we describe, the first microsporidian parasite from nephropid lobsters. Metanephrops challengeri were captured from an important marine fishery situated off the south coast of New Zealand. Infected lobsters displayed an unusual external appearance and were lethargic. Histology was used to demonstrate replacement of skeletal and other muscles by merogonic and sporogonic stages of the parasite, while transmission electron microscopy revealed the presence of diplokaryotic meronts, sporonts, sporoblasts and spore stages, all in direct contact with the host sarcoplasm. Analysis of the ssrDNA gene sequence from the lobster microsporidian suggested a close affinity with Thelohania butleri, a morphologically dissimilar microsporidian from marine shrimps. Whilst morphological features of the lobster parasite are consistent with members of the family Nosematidae, molecular data place the parasite closer to members of the family Thelohanidae. Due to the contradiction between morphological and molecular taxonomic data, we propose the erection of a new genus in which the lobster parasite is the type species (Myospora metanephrops). Furthermore, we recommend the erection of a new family (Myosporidae) and a new order (Crustaceacida) to contain this genus. The taxonomic framework presented could be further applied to the re-classification of existing members of the Phylum Microsporidia.     

Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.     

Tissue damage and leukocytic infiltration following attachment of the mite Unionicola intermedia to the gills of the bivalve mollusc Anodonta anatina

Unionicola intermedia lives as a parasite in the gill region of Anodonta anatina. It attaches to the gills of its host by means of the pedipalps which cause displacement, rupture, and erosion of the gill epithelium. The pedipalps are sunk deeply into the underlying connective tissue of the gills and produce a leukocytic infiltration into the damaged area and a consequent edema of the gill filaments.     

Host-parasite relationship of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica in the Argentinean Patagonian coast

The association of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica is described. The identity of the green alga was confirmed by molecular studies; the alga was found within the hemocytes that infiltrate the connective tissue of the geoduck siphons. Cytological characteristics of hemocytes were not altered by algal infection; very often the algae were seen enveloped by a digestive vacuole within the hemocyte cytoplasm, evidencing diverse degrees of resorption. Connective cells of siphons were rarely infected by C. parasitica. The mean prevalence of C. parasitica was higher (82%) in San Matías Gulf (42°00′S, 65°05′W) than in San José Gulf (45%) (40°32′S, 64°02′W); except for spring, when the two locations showed no differences in prevalences (80%). Independently of location, season and host size, infected geoducks showed lower condition index values than uninfected ones. Regarding other bivalve species, only one specimen of the razor clam Ensis macha was found infected, and none of the oysters Ostrea puelchana and Pododesmus rudis and scallop Aequipecten tehuelchus was parasitized by the green alga.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

Echinococcus multilocularis: Two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.     

The immunopathogenesis of Entamoeba histolytica

Amebiasis is the disease caused by the enteric dwelling protozoan parasite Entamoeba histolytica. The WHO considers amebiasis as one of the major health problems in developing countries; it is surpassed by only malaria and schistosomiasis for death caused by parasitic infection. E. histolytica primarily lives in the colon as a harmless commensal, but is capable of causing devastating dysentery, colitis and liver abscess. What triggers the switch to a pathogenic phenotype and the onset of disease is unknown. We are becoming increasingly aware of the complexity of the host-parasite interaction. During chronic stages of amebiasis, the host develops an immune response that is incapable of eliminating tissue resident parasites, while the parasite actively immunosuppresses the host. However, most individuals with symptomatic infections succumb only to an episode of dysentery. Why most halt invasion and a minority progress to chronic disease remains poorly understood. This review presents a current understanding of the immune processes that shape the outcome of E. histolytica infections during its different stages.