First report on histology and ultrastructure of an intrahemocytic paramyxean parasite (IPP) from tunicate Halocynthia roretzi in Korea

In 2004, epizootiological studies were conducted on mass mortalities of tunicates Halocynthia roretzi in Goje, Korea. The clinical characteristics of infected H. roretzi were weakness of the tunic, loss of elasticity, and finally death involving a rupture of the tunic. Histological studies revealed severe hemocyte infiltration in the connective tissue surrounding the intestine and mantle of infected H. roretzi. Hypertrophied eosinophilic hemocytes containing several cytoplasmic vacuoles were observed in the connective tissue surrounding the intestine, gill and mantle. Ultrastructural examination revealed the presence of a parasite in the cytoplasm of hemocytes. Secondary cells were observed in the primary cell of the parasite. Spore formation within primary cells suggests that the parasite may be an intrahemocytic paramyxean parasite (IPP) and may cause mass mortality of H. roretzi.     

Apoptosis in the pathogenesis of infectious diseases of the eastern oyster Crassostrea virginica

Apoptosis, or programmed cell death, has been reported as being pivotal in infectious diseases of different organisms. The effects of apoptosis on the progression and transmission of the protistan parasites Perkinsus marinus and Haplosporidium nelsoni in the eastern oyster Crassostrea virginica were studied. Oysters were diagnosed for their respective infections by standard methods, and apoptosis was detected using in situ hybridization to detect DNA fragments by end labeling on paraffin sections. A digoxigenin nucleotide probe was used to label the 200 bp fragment produced by apoptosis and detected immunohistochemically using an antidigoxigenin peroxidase conjugate. The probe/DNA fragment complex was stained with a peroxidase substrate and tissues were counterstained with methyl green. Uninfected oysters had large numbers of apoptotic hemocytes present in the connective tissue underlying the stomach, gill, and mantle epithelia, whereas oysters infected with P. marinus had a reduced number of apoptotic hemocytes. The parasite may prevent hemocyte apoptosis in order to yield a greater number of hemocytes in which to house itself. Large numbers of P. marinus cells in some infected oysters were eliminated via apoptosis in the stomach epithelia, disabling the spread of infectious particles through seawater. The oysters infected with H. nelsoni also had reduced numbers of apoptotic hemocytes, while part of the vesicular connective tissue cells were apoptotic. H. nelsoni plasmodia were eliminated via apoptosis in some oysters. Apoptosis may enhance progression and prevent transmission of infectious oyster diseases.     

Perkinsus mediterraneus n. sp., a protistan parasite of the European flat oyster Ostrea edulis from the Balearic Islands, Mediterranean Sea

A new species, Perkinsus mediterraneus, a protistan parasite of the European oyster Ostrea edulis (L.), farmed along the coast of the Balearic Islands, Mediterranean Sea, is described. Morphological examinations with light and transmission electron microscopy, DNA sequence-analysis and enlargement in Ray's fluid thioglycollate medium (RFTM) confirmed that this parasite belongs to the genus Perkinsus. Specific morphological and genetic characteristics indicated that it should be considered a new species in the genus. Sequencing of the small subunit ribosomal (ssu rRNA) gene confirmed that the parasite belongs to the genus Perkinsus, and sequences of the internal transcribed spacer (ITS) were distinct from any Perkinsus ITS sequences previously published and/or deposited in the GenBank. Phylogenetic analysis revealed that the ITS sequences of the new species formed a monophyletic group comprising a sister clade to the P. atlanticus/olseni group. In addition, morphological differences were observed between the new species and the other described Perkinsus spp.. After incubation in RFTM for 1 wk, the prezoosporangium had reached an extremely large size (97.4 +/- 1.99 microm) (mean +/- SE), and after 2 wk incubation had again almost doubled in size (167.1 +/- 8.09 microm). The discharge-tube length was one sixth the diameter of the zoosporangium, i.e. a ratio of 17.36:97.38, the lowest ratio observed for any Perkinsus species. At the ultrastructural level, zoosporangia and zoospores exhibited some differences compared to other Perkinsus species.     

A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.     

Occurrence of Perkinsus sp. in undulated surf clams Paphia undulata from the Gulf of Thailand

The undulated surf clam Paphia undulata supports Thailand's largest shellfishery in the Gulf of Thailand, with landings in 1999 recorded at 70000 t (metric tonnes) yr(-1). We report, for the first time, the prevalence of Perkinsus sp. in clams in the Gulf. A monthly survey from January to December 2001 utilizing the fluid thioglycollate medium (FTM) method showed that average monthly prevalence was 84.7% (n = 360). The monthly percentage of infected clams was generally 100%, with low prevalence in May (66.7%) and no infection in September. The monthly mean infection intensity in terms of Perkinsus sp. cells g(-1) tissue varied from 0 in September to 187 759 +/- 18970 (x +/- SE) in October. No obvious annual variation in intensity and prevalence was observed. Prezoosporangia that developed in FTM were 25 to 75 pm in diameter. A few days after incubation in aerated seawater, the prezoosporangia underwent successive binary cell division and formed motile zoospores (2 to 5 microm long). The zoospores were released into the seawater through a discharge tube formed during the 2- and 4-cell stages. Serial semi-thin sections (1 to 4 pm thickness) of clam tissue (n = 120 clams) showed developing trophozoites 3 to 6 pm in diameter within gills, connective tissue, gonads and, especially, the digestive glands. Microscopic features of different life stages indicated that Perkinsus sp. in Thailand closely resembled P. olseni (= P. atlanticus) reported in Australia, New Zealand, Korea, Japan, Spain and Portugal.     

A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus

Invertebrates display effective innate immune responses for defense against microbial infection. However, the protozoan parasite Perkinsus marinus causes Dermo disease in the eastern oyster Crassostrea virginica and is responsible for catastrophic damage to shellfisheries and the estuarine environment in North America. The infection mechanisms remain unclear, but it is likely that, while filter feeding, the healthy oysters ingest P. marinus trophozoites released to the water column by the infected neighboring individuals. Inside oyster hemocytes, trophozoites resist oxidative killing, proliferate, and spread throughout the host. However, the mechanism(s) for parasite entry into the hemocyte are unknown. In this study, we show that oyster hemocytes recognize P. marinus via a novel galectin (C. virginica galectin (CvGal)) of unique structure. The biological roles of galectins have only been partly elucidated, mostly encompassing embryogenesis and indirect roles in innate and adaptive immunity mediated by the binding to endogenous ligands. CvGal recognized a variety of potential microbial pathogens and unicellular algae, and preferentially, Perkinsus spp. trophozoites. Attachment and spreading of hemocytes to foreign surfaces induced localization of CvGal to the cell periphery, its secretion and binding to the plasma membrane. Exposure of hemocytes to Perkinsus spp. trophozoites enhanced this process further, and their phagocytosis could be partially inhibited by pretreatment of the hemocytes with anti-CvGal Abs. The evidence presented indicates that CvGal facilitates recognition of selected microbes and algae, thereby promoting phagocytosis of both potential infectious challenges and phytoplankton components, and that P. marinus subverts the host's immune/feeding recognition mechanism to passively gain entry into the hemocytes.     

Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells,tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.     

Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus,a protistan parasite of the clam Tapes decussatus

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.     

Myxobolus cuneus n. sp. (Myxosporea) infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) in Brazil: histopathology and ultrastructure

The characteristics of Myxobolus cuneus n. sp. and its relationship to the host Piaractus mesopotamicus are described based on light and electron microscopy and histological observations. Polysporic plasmodia measuring 20 microm to 2.1 mm in size were found in 63.3 % of the P. mesopotamicus examined. The parasite was found in the gall bladder, urinary bladder, gills, spleen, fins, head surface, liver and heart. Generative cells and disporoblastic pansporoblasts occurred along the periphery of the plasmodia, and mature spores were found in the internal region. The mature spores had a pear shaped body in frontal view, with a total length of 10.0 +/- 0.6 microm and a width of 5.1 +/- 0.3 microm (mean +/- SD). The spore wall was smooth with sutural folds. The polar capsules were elongated, were pear shaped, and equal in size (length 5.7 +/- 03 microm; width 1.7 +/- 0.2 microm), with the anterior ends close to each other. The polar filaments were tightly coiled in 8-9 turns perpendicular to the axis of the capsule. The plasmodia were always found in connective tissue (wall of the arterioles of the gill filaments, serous capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). The parasite caused important damage in the gills, where development occurred in the wall of gill filament arterioles; a mild macrophage infiltrate was also observed. In advanced developmental stages, the plasmodia caused deformation of the arteriole structure, with a reduction and, in some cases, obstruction of the lumen. The parasite was found throughout the period studied and its prevalence was unaffected by host size, season or water properties.     

Perkinsus olseni detected in Vietnamese aquacultured reef clams Tridacna crocea imported to the USA, following a mortality event

Morbidity and mortality were observed in a group of 30 reef clams Tridacna crocea that were imported to Florida, USA, from a Vietnamese culture facility and held in research facility aquaria. Clinical signs included an incompletely extended mantle, slow mantle responses to stimuli, and sloughing of byssal tissue beginning 2 to 5 d prior to death. Necropsy findings included emaciation, visceral mass edema, and rare multifocal 1 mm off-white to light-tan gill nodules. Histopathology revealed marked inflammation and necrosis within the visceral mass and gills, with interstitial edema and atrophy of glandular, gonadal, and muscular tissues. Inflamed tissues contained large numbers of 10 to 15 microm extracellular round organisms consistent with Perkinsus sp. trophozoites. The organisms often formed clusters of 1 to 4 cells and were surrounded by a 1 to 3 microm rim of eosinophilic material variably forming a radiating corona pattern and by 3 to 4 host hemocytes with dense round nuclei. Polymerase chain reaction assays indicated the presence of Perkinsus sp. DNA in these animals, and species-specific assays indicated the presence of P. olseni, and possibly other Perkinsus spp., but not P. marinus. Identification of Perkinsus spp. other than P. marinus in T. crocea imported from Vietnam confirms that importation of untested and unquarantined ornamental reef clams has possibly allowed incursion of P. olseni into the USA.     

Spatio-temporal patterns of perkinsosis in the Manila clam Ruditapes philippinarum from Arcachon Bay (SW France)

Pathogens belonging to the genus Perkinsus infect many bivalve molluscan species around the world, including the Manila clam Ruditapes philippinarum. We investigated the spatial distribution of this parasite at 34 stations throughout Arcachon Bay (SW France). Prevalence of perkinsosis was 93% and mean infection abundance was 96 x 10(3) cells g(-1) wet gill. Lowest mean abundances were found close to the Leyre River mouth and a significant negative correlation was observed between mean abundance and salinity. Perkinsosis was rare at the oceanic site where salinities and other environmental parameters were stable. A second aim of this study was to survey perkinsosis during annual cycles at 4 sites within Arcachon Bay. Prevalence and intensities (+/- SE) of the disease were high, on average between 70 and 100%, and 130 x 10(3) +/- 6.7 x 10(3) cells g(-1) wet gill. No seasonal cycle was evident. Clams were infected at 9 mm shell length and infection increased with clam size. The third objective was to determine the disinfection and infection kinetics through a 21 mo reciprocal transplantation between a nearly Perkinsus sp.-free area and a highly affected site. Disinfection appeared to be a very slow process and was similar at the site with favorable conditions for Perkinsus sp. as at the site with unfavorable conditions. Conversely, infection acquisition appeared to be episodic with spatially defined areas. Consequently, the overall lack of a clear seasonal infection pattern is interpreted as the combination of episodic infection events and slow disinfection kinetics.     

The effect of sperm number on fertility in blue fox vixens (Alopex lagopus ) artificially inseminated with frozen silver fox (Vulpes vulpes ) semen

During the breeding seasons of 1989 and 1990, a total of 617 blue fox vixens aged 1 to 6 years (mean +/- SEM, 2.6 +/- 0.1) were inseminated with frozen silver fox semen with either 150 million (n = 213, 1989 + 1990), 100 million (n=172, 1990), 75 million (n = 119, 1989) or 37.5 million (n = 113, 1989) spermatozoa per insemination. Two intrauterine inseminations, each with an insemination volume of 1.0 ml, were performed at 24-hour intervals on the first and second days after maximum vaginal electrical resistance was measured. Conception rates were 87% (186 of 213) with 150 million spermatozoa per insemination, 85% (146 of 172) with 100 million, 77% (91 of 119) with 75 million and 68% (77 of 113) with 37.5 million. The mean numbers of cubs per litter +/- SEM for the four groups were 7.6 +/- 0.2 (168 registered litters), 7.5 +/- 0.3 (115 litters), 6.4 +/- 0.4 (86 litters) and 6.4 +/- 0.4 (75 litters). A negative effect on both the conception rate and mean litter size at whelping was observed with decreasing sperm numbers (conception rate percentage: p = 0.0001, Chi-square, litter size: p = 0.02, Kruskal-Wallis Test). Only the two larger numbers of spermatozoa gave litter sizes comparable to those obtained by artificial insemination (AI) with fresh semen.     

Increased chylomicron triglyceride hydrolysis by connective tissue flow in perfused rat hindlimb. Implications for lipid storage

Skeletal muscle has two circulatory routes, nutritive (in contact with muscle) and non-nutritive (part of which is located in the connective tissue), and the balance of flow between the two is controlled by neural input and circulating vasomodulators. The purpose of this study was to assess muscle triglyceride hydrolysis given that the two circuits may have a differing vascular distribution of hydrolytic activity. The isolated rat hindlimb was perfused with 6% Ficoll((R)) and a radiolabeled chylomicron;-lipid emulsion containing apolipoprotein C-II. Serotonin (0.5-1 micrometer), a model vasoconstrictor previously shown to preferentially increase connective tissue flow, inhibited hindlimb oxygen uptake (from 16.7 +/- 0.6 to 10.2 +/- 1.0, mean +/- SE, n = 7 (P <0.001)) and stimulated [(14)C]-labeled fatty acid uptake into muscles (from 184 +/- 28 to 602 +/- 132, mean +/- SE, n = 7 (P = 0.009)). These effects were reversed by the vasodilator carbamyl choline. Vasopressin resulted in increased oxygen consumption but no change in triglyceride hydrolysis. Cholesteryl oleate uptake (an indicator of endocytosis of the chylomicron or remnant particle) was unaltered by serotonin. It is concluded that chylomicron triglyceride hydrolysis is enhanced by vasoconstrictors that increase connective tissue flow in the perfused rat hindlimb. Increased hydrolysis appears to be primarily due to an increased access of triglyceride to hydrolytic enzymes, presumably lipoprotein lipase associated with the fat cells commonly observed interlaced amongst bundles of muscle fibers.     

Effects of Bucephalus sp. (Trematoda: Bucephalidae) on Perna perna mussels from a culture station in Ratones Grande Island,Brazil

This study reports the prevalence of Bucephalus sp. in Perna perna populations from a culture station of southern Brazil and its effect on the mussel reproductive tissue and immune system. The prevalence of Bucephalus sp. in P. perna (n = 1871) was considered low (3.1%) and did not seasonally vary. Histological sections of the mantle of infected mussels revealed a marked (80%) reduction of the reproductive tissue that was severe even in mussels exhibiting a moderate infection degree. The total (THC) and differential (DHC) hemocyte counts were lower in infected mussels (3.9 x 10(6) hem/ml; granular hemocytes = 33%) as compared with non-infected animals (5.5 x 10(6) hem/ml; granular hemocytes = 40%). The plasma protein concentration did not vary upon infection. Hemocyte infiltration was significantly higher only in mussels with a very heavy infection degree. The parasite sporocysts were never seen encapsulated by the host hemocytes. Our results indicate that Bucephalus sp. promotes a severe castration in its host and apparently evades the mussel immune system.     

Comparison of in vitro-cultured and wild-type Perkinsus marinus. II. Dosing methods and host response

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally transmit parasites by 'natural' methods. In addition, the host's condition may affect a study's outcome. The experiments reported here examined the effect of dosing method and host metabolic condition on measures of virulence for the oyster parasite Perkinsus marinus. Oysters, Crassostrea virginica, were challenged with wild-type and cultured forms of P. marinus via feeding, shell-cavity injection, gut intubation and adductor-muscle injection. For both parasite types, adductor-muscle injections produced the heaviest infections followed by shell-cavity injection, gut intubation, and feeding. There was no difference in parasite burdens between oysters fed cultured cells by acute vs chronic dosing, and parasite loads stabilized over time, suggesting a dynamic equilibrium between invasion and elimination. P. marinus distribution among tissues of challenged oysters indicated that parasites invaded the mantle and gill, as well as the gut, which has been considered the primary portal of entry. Frequency distributions of P. marinus in oysters challenged with 3 different culture phases indicated an aggregated distribution among hosts and suggested that stationary-phase parasites were easiest for the oyster to control or eliminate and log-phase parasites were the most difficult. Host metabolic condition also affected experimental outcomes, as indicated by increased infection levels in oysters undergoing spawning and/or exposed to low oxygen stress.     

Copper exposure affects hemocyte apoptosis and Perkinsus marinus infection in eastern oysters Crassostrea virginica (Gmelin)

Dermo disease in the eastern oyster (Crassostrea virginica) is caused by an intracellular protistan parasite Perkinsus marinus. The progression and outcome of this disease is determined by a complex interplay between the host's immunity and parasite's escape mechanisms, both of which can be influenced by environmental pollutants including heavy metals such as copper (Cu). The goal of the present study was to determine the effects of Cu on the levels of apoptosis (which can serve as an important host defense mechanism) in oyster immune cells (hemocytes) in?vitro and in?vivo as well as on the establishment of P.?marinus infections in?vivo. Surprisingly, Cu exerted opposing effects on apoptosis levels of hemocytes in?vitro and in?vivo, stimulating apoptosis in isolated hemocytes but suppressing it during Cu exposure of whole oysters. The mechanisms of this effect are presently unknown and may be related to the different bioavailability of the metal in?vitro and in?vivo. As expected, Cu accumulated in oyster soft tissues during in?vitro exposure. Unexpectedly, this metal also strongly accumulated in hemolymph plasma which is classically considered isoionic with the surrounding seawater, likely reflecting the presence of soluble Cu-binding proteins in oyster plasma. Cu reduced growth of P.?marinus in?vitro and greatly reduced infection levels of hemocytes in?vivo, presumably by direct toxic effects on the parasite. As a possible parasitic counterbalance, Cu accumulation in the hemocytes was reduced by P.?marinus infection, although this reduction was not sufficient to prevent the parasiticidal effects of the heavy metal in?vivo. This effect of Cu may be useful as a potential therapeutic against Dermo disease in aquaculture conditions. Overall, this study provides important new insights into the potential role of environmental metals in host-parasite relationships and disease dynamics in C.?virginica.     

Ichthyophthirius (Ciliophora): population studies suggest reproduction in host epithelium

Evidence that Ichthyophthirius multifiliis trophonts may reproduce within the epithelium of the host was obtained from experimental infections of channel catfish. Mean number of parasites spontaneously leaving the fish increased from 0 on day 3 postexposure (PE) to 66.5 per fish on day 7 PE. Mean population density in fin, however, increased five-fold from day 3 to day 5 PE in the absence of opportunity for reinfection. At day 3 PE, 100% of parasite loci in fin and gill arches contained solitary trophonts. At day 4 PE, 10% of loci in fin contained clusters of two trophonts; at day 7 PE, 69% contained clusters of two or more trophonts. The first clusters of four trophonts in fin were observed day 5 PE and of eight trophonts, day 8 PE. Trophonts in clusters were flattened against one another.     

Severe apicomplexan infection in the oyster Ostrea chilensis: a possible predisposing factor in bonamiosis

Histological examination of 6455 oysters Ostrea chilensis from Foveaux Strait south of New Zealand over a 5 yr period showed >85% contained apicomplexan zoites, irrespective of season. Zoites occurred around the haemolymph sinuses and the digestive diverticulae at all intensities of infection; occurrence in the sub-epithelium, Leydig tissue and gills/mantle increased with increasing intensity of infection. Many (>35%) oysters were heavily infected, and most of them had severely damaged tissues. Heavy infections affected gametogenesis; 1% of lightly infected oysters had empty gonad follicles lacking germinal epithelium compared with 2% of moderately infected oysters and 9% of heavily infected oysters. Of oysters with empty gonad follicles, 75% were heavily infected with zoites. The parasite spread from the haemolymph sinuses and moved between Leydig cells, causing their dissociation and lysis. Some zoites were intracellular in Leydig cells. Lesions contained many haemocytes phagocytosing zoites, leading to haemocyte lysis and causing a haemocytosis. Fibrosis occurred to repair lesions in a few oysters. The zoites had a typical apical complex with 2 polar rings and 84 sub-pellicular microtubules. Prevalence and intensity of concurrent Bonamia exitiosus infection was related to the intensity of zoite infection, with only 3.8% of B. exitiosus infections occurring in the absence of zoites, 20.0% occurring in light zoite infections, 30.9% in moderate zoite infections, and 45.4% when oysters were heavily infected with zoites. The converse was not the case, as 75.3% of zoite infections occurred in the absence of B. exitiosus infection, including 51.1% of moderate to heavy zoite infections. There was a statistically significant association between intensities of B. exitiosus and of zoites (p < 0.0001). Zoites may increase the susceptibility of oysters to B. exitiosus by occupying and destroying haemocytes, and by destroying connective tissue cells and utilising host glycogen reserves. The parasite may be heteroxenous, with other stages in the terebellid polychaete Pseudopista rostrata.     

Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico.     

Description of Perkinsus beihaiensis n. sp., a new Perkinsus sp. parasite in oysters of Southern China

Oysters were collected from coastal locations in China from 1999-2006 for parasite analyses by molecular, culture, and histological techniques. Polymerase chain reaction-based assays targeting the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex were performed to detect the presence of Perkinsus species. Sequencing and phylogenetic analysis of amplified Perkinsus sp. DNAs indicated that a novel Perkinsus sp. infects Crassostrea hongkongensis, Crassostrea ariakensis, and other bivalve hosts from Fujian to Guangxi provinces in southern China. Prevalence of this Perkinsus sp. reaches as high as 60% in affected oyster populations. Analyses of nucleotide sequences of the rRNA ITS region and of large subunit rRNA and actin genes, consistently confirmed the genus affiliation of this Perkinsus sp., but distinguished it from currently accepted Perkinsus species. Parasite cell types, such as signet ring trophozoites of 2-8 microm diameter, were observed by histology, and application of both genus Perkinsus and Perkinsus species-specific in situ hybridization probes consistently labelled the same Perkinsus sp. cells in histological sections from infected oyster tissues. Combined phylogenetic and histological results support the identity of a new parasite species, Perkinsus beihaiensis n. sp.