Comparison of in vitro-cultured and wild-type Perkinsus marinus. II. Dosing methods and host response.
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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. (+info)
Comparison of in vitro-cultured and wild-type Perkinsus marinus. III. Fecal elimination and its role in transmission.
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Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite may be voided from live oysters and subsequently dispersed in the water column. The experiments described here were designed to quantify P. marinus shed in the feces and pseudofeces of experimentally infected oysters. The results indicated that parasites were shed in 2 phases. A 'decreasing' phase occurred within 2 wk of challenge and before net parasite proliferation began in the host. An 'increasing' phase occurred after P. marinus had begun replicating. The quantity of P. marinus recovered in the feces and pseudofeces of exposed oysters was only about 5 % of the dose administered. In vitro-cultured P. marinus were eliminated at a greater rate than wild-type P. marinus and the fraction discharged was not associated with culture phase. Oysters that were continuously dosed with P. marinus in their food gradually lost the ability to discard the parasite in pseudofeces. The quantity of P. marinus shed in feces of infected oysters was correlated with both the P. marinus body burden and subsequent survival time, suggesting that noninvasive fecal counts could predict infection intensity and survival. The results indicate that in an epizootic, shedding of P. marinus via feces is relatively small compared to the potential number released by cadavers of heavily infected oysters, but that fecal discharge may be important in transmission before infections become lethal. (+info)
Hsp70 expression in thermally stressed Ostrea edulis, a commercially important oyster in Europe.
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Synthesis of heat shock proteins (Hsps) in response to elevated temperatures and other denaturing agents is a common feature of prokaryotic and eukaryotic cells. The heat-induced expression of Hsp70 family members in the gills and mantle of Ostrea edulis, a highly valued fisheries resource inhabiting primarily estuarine environments, has been studied. O edulis is exposed to a variety of natural and anthropogenic stresses in the environment. Two isoforms of about 72 kDa and 77 kDa were constitutively present in unstressed organisms, reflecting the housekeeping function performed by these proteins under normal circumstances. Their expression in animals undergoing thermal stress was highly variable, and on the average, little change occurred under different experimental conditions. A third isoform of about 69 kDa was induced in both tissues after exposure to > or = 32 degrees C; its synthesis was detected within 4 hours of poststress recovery at 15 degrees C, reaching the maximum expression after 24 hours in the gills and after 48 hours in the mantle and declining thereafter. Hsp69 expression was low at 38 degrees C, a temperature lethal for about 50% of the individuals tested. Densitometric analysis of Western blots revealed that Hsp69 was mostly responsible for the significant heat-induced overexpression of Hsp70s in O edulis. Comparison with heat shock responses in tissues of Crassostrea gigas indicated a similar pattern of Hsp70 expression. In this organism, however, Hsp69 was induced after exposure to > or = 38 degrees C. We conclude that tissue expression of Hsp69 in O edulis, and possibly other bivalves, is an early sign of thermal stress; determining whether these changes also correlate with other major environmental stresses is the goal of ongoing studies. (+info)
Geographic structure in the European flat oyster (Ostrea edulis L.) as revealed by Microsatellite polymorphism.
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Genetic differentiation of the flat oyster (Ostrea edulis) was studied along the European coast, from Norway to the Black Sea, by means of variation at five microsatellite loci. The results show a mild but significant isolation-by-distance profile, a noticeable between-sample variance in expected heterozygosity, and a tendency for Atlantic populations to be less variable than Mediterranean ones. This does not provide support for the existence of a single large panmictic population for this larvae-broadcasting species, but rather for the relative independence of local stocks. Comparison with data on allozyme variation from the literature confirms this view. It also leads us to suggest that the behavior of some sampled protein loci may depart from the average, so caution should be used when inferring neutral gene flow. (+info)
Ovacystis-like condition in the eastern oyster Crassostrea virginica from the northeastern Gulf of Mexico.
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Histological examination of the eastern oyster Crassostrea virginica from a study in Pensacola Bay, Florida, revealed 2 cases of abnormally large, basophilic ova that resembled cells characteristic of ovacystis disease previously reported in oysters from Maine and Long Island. The hypertrophied gametes measured up to 250 pm in diameter, had scant cytoplasm and contained granular nuclear-masses of Feulgen-positive material. Electron microscopy of reclaimed tissue revealed these masses to consist of virus-like particles (average 46 nm) similar to those reported in cases of ovacystis. (+info)
Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters.
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Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g(-1). Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh(+) V. parahaemolyticus than previously reported. (+info)
Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.
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The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked. (+info)
Virus-contaminated oysters: a three-month monitoring of oysters imported to Switzerland.
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Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs. (+info)