Marinus Infection Research Articles

Perkinsus marinus tissue distribution and seasonal variation in oysters Crassostrea virginica from Florida, Virginia and New York.

Perkinsus marinus infection intensity was measured in eastern oysters Crassostrea virginica collected in October and December 1993, and March, May, and July 1994 from 3 U.S. sites: Apalachicola Bay (FL), Chesapeake Bay (VA), and Oyster Bay (NY). Gill, mantle, digestive gland, adductor muscle, hemolymph, and remaining tissue (including gonadal material and rectum) were dissected from 20 oysters from each site at each collection time. Samples were separately diagnosed for P. marinus infections by incubation in Ray's Fluid Thioglycollate Medium (RFTM) and subsequent microscopic quantification of purified enlarged hypnospores. At all sampling times and sites, average P. marinus infection intensity (g wet wt tissue-1 or ml hemolymph-1) was lowest in hemolymph samples, and generally highest in the digestive gland. Perkinsus marinus prevalence was 100% at both FL and NY sites for each of the 5 collection times, and, for the VA site, was less than 100% in only 1 month (May 1994). Seasonal intensity patterns and mean total body burdens differed among the sites. Average body burden was highest in VA during October and progressively declined to a minimum in May. This decline was probably due to mortality of heavily infected oysters and diminution of parasite activity associated with colder temperatures and reduced salinities. Intensities varied little during the months of October and December at both the FL and NY sites. Minimum average intensities were observed in March in FL oysters and May in NY oysters. Relatively high P. marinus infection levels that persisted throughout the winter in NY oysters compared with VA oysters could reflect constant high salinity in Long Island Sound which favors parasite activity, and also rapid decline in temperature in the fall that may have prevented epizootic oyster mortalities.

The Eastern oyster: Crassostrea virginica

A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using I g of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 g of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot- blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as dilution endpoint PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification ofP. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat. The severe disease caused by the endoparasitic protozoan Perkinsus marinus (Apicomplexa: class Perkinsea; Levine, 1978) is a major cause of mortality in the eastern oyster (Crassostrea virginica) along the Gulf of Mexico and Atlantic coast, U.S.A. A review on the epizootiology of the disease has been published by Andrews (1988). In its vegetative form as a trophozoite, P. marinus can proliferate throughout the tissues of the oyster by multiple fission, budding, or both. Eventually, mature tropho- zoites enlarge and then undergo rapid reductive divisions to form a hypnospore that enters the water column and sporulates to release large numbers ofbiflagellated zoospores (1,000-2,000 per hypnospore; Perkins, 1966). These motile zoospores pre- sumably give rise to trophozoites once they infect an oyster, but neither the mechanism of infection is known, nor the oyster life stage that is the most susceptible to parasite entry. Although significant progress has been made in understanding this disease over the last 25 yr (Perkins, 1988), new methodologies need to be developed to address these gaps in our knowledge before coherent treatment and management strategies can be formu- lated. tions in oyster seed stocks, latent infections in overwintering oyster populations, the onset of infection in oyster larvae and spat, the presence of P. marinus in other marine organisms that may serve as secondary vectors or reservoirs, and the genetic structure of parasite field populations. In response to these needs, the present paper describes a semiquantitative PCR-based di- agnostic assay for P. marinus DNA that provides both a rapid and sensitive in vivo assay for the detection of P. marinus in oyster hemolymph and gill tissues.

Disease processes of the parasite Perkinsus marinus in eastern oyster Crassostrea virginica:minimum dose for infection initiation, and interaction of temperature, salinity and infective cell dose

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 28:61-68 (1997) - doi:10.3354/dao028061 Disease processes of the parasite Perkinsus marinus in eastern oyster Crassostrea virginica: minimum dose for infection initiation, and interaction of temperature, salinity and infective cell dose Chu FLE, Volety AK Experiments were conducted to: (1) test the response of oysters to different doses of the oyster parasite Perkinsus marinus and to 2 stages, meronts or prezoosporangia; and (2) investigate the synergistic effects of temperature, salinity and infective cell concentration on P. marinus infection in oysters. A dose-dependent response of P. marinus infection was found in oysters inoculated with 0, 10, 102, 104, and 105 meronts or prezoosporangia per oyster and maintained at 22 to 25°C and 14 to 21 ppt for 8 to 12 wk. The minimum dose required to infect oysters was 102 meronts or prezoosporangia per oyster through shell cavity inoculation. Interactive effects between temperature, salinity, and infective cell dose on P. marinus prevalence was insignificant in the experiment, in which oysters were challenged by 0, 2.5 × 103 or 2.5 × 104 meronts per oyster and held at 9 temperature-salinity regimes (10, 15 and 25°C at 3, 10 and 20 ppt). However, there was a significant positive interaction relevant to infection intensity between temperature and salinity, and between temperature and meront dose. Temperature was the most important factor followed, respectively, by the infective cell dose and salinity in determining the susceptibility to P. marinus in oysters. Reduced condition index was observed in moderately to heavily infected oysters and in oysters at 25°C. Oyster · Crassostrea virginica · Oyster parasite · Oyster disease · Perkinsus marinus · Temperature · Salinity Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 28, No. 1. Publication date: January 16, 1997 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1997 Inter-Research.

Enhancement of Perkinsus marinus disease progression in TBT-exposed oysters ( Crassostrea virginica)

In vitro exposure of Crassostrea virginica hemocytes to tributyltin (TBT) can inhibit antimicrobial defense mechanisms dependent on reactive oxygen intermediates, as measured by chemiluminescence (CL). However, tissue TBT concentrations attained in this in vivo study (~ 7500 ppb, dry weight) did not inhibit hemocyte recruitment, phagocytosis, CL activity or serum lysozyme. This TBT level was nevertheless effective in enhancing progression and lethality of Perkinsus marinus infections. Immune stimulation was found to be associated with advanced P. marinus infections, as indicated by increased hemocyte recruitment into the circulation and increased CL activity. This played no apparent role in controlling the infection and was insensitive to TBT. Using this battery of standard assays of defense-related functions, the exacerbation of P. marinus infections by TBT could not be attributed to its putative immunosuppresive activity.

A Semiquantitative PCR Assay for Assessing Perkinsus marinus Infections in the Eastern Oyster, Crassostrea virginica

A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using 1 microgram of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 microgram of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot-blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a dilution series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as "dilution endpoint" PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification of P. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat.

Defense Responses of Hemocytes Withdrawn from Crassostrea virginica Infected with Perkinsus marinus

Hatchery-produced, unparasitized oysters ( Crassostrea virginica) were transplanted to high and low salinity sites in the Chesapeake Bay in May 1992. These locations included Mobjack Bay, Virginia (MB, ∼20%), the Choptank River, Maryland (CR, ∼14%), and the Wye River, Maryland (WR, ∼8%); the salinities subsequently varied with rainfall in the region of each site. The protozoan parasite Perkinsus marinas rapidly infected the oysters at each site. Oysters were periodically sampled and individually assayed for intensity of P. marinus infection, total circulating hemocyte count (THC) and zymosan-iuduced chemiluminescence (CL) activity of the hemocytes. The CL assay measures the production of reactive oxygen intermediates (ROIs) thought to mediate antimicrobial activity of the hemocytes. The MB oysters were 100% infected and showed a high intensity of infection by October, 1992; during the following year, high levels of infection were reached rapidly and the population was completely decimated by mid-July, 1993. Disease intensity and progression were slower in oysters from the low salinity sites (CR and WR). The THC and zymosan-stimulated CL responses of hemocytes from moderately or heavily infected oysters were significantly higher than those of uninfected or lightly infected oysters. The increased number of hemocytes from oysters with advanced infections probably represents a general protective response to the presence of infectious agents in tbe hemolymph. Hemocytes withdrawn from oysters with heavy P. marinus infections have markedly increased CL activity after phagocytosis of zymosan particles. Statistical analysis of the data showed that this activated condition was strongly associated with the level of P. marinus infection of the host, but not influenced by the ambient salinity of the oysters' habitat. However, phagocytosis of P. marinus merozoites by hemocytes fails to trigger CL, regardless of the infection intensity of the oysters from which the hemocytes were withdrawn. These data suggest that ingestion of microorganisms may nonspecifically enhance hemocytic defense responsiveness, as measured by their increased production of ROIs upon subsequent phagocytic stimulation by zymosan. However, not all microbes can initiate responses from these activated hemocytes; this may partially explain the virtual inability of C. virginica hemocytes to control P. marinus infections by intracellular cidal mechanisms.

Haemocytic and humoral activities of eastern and Pacific oysters following challenge by the protozoan Perkinsus marinus

Eastern oysters ( Crassostrea virginica) and Pacific oysters ( Crassostrea gigas) were exposed to the protozoan parasite Perkinsus marinus. Pacific oysters were less susceptible to P. marinus infection than eastern oysters. The concentration of circulating haemocytes, percentage of granulocytes and plasma haemagglutinin titre increased in Pacific oysters exposed to P. marinus as compared to unexposed Pacific oysters. Similar increases were not observed in eastern oysters exposed to P. marinus. The percentage of granulocytes, percentage of phagocytic haemocytes, number of zymosan per phagocytic haemocytes and plasma haemagglutinin titre were greater in Pacific oysters than in eastern oysters heavily infected with P. marinus. However, the concentration of circulating haemocytes and the concentrations of plasma protein and lysozyme were lower in Pacific oysters. The data suggest that Pacific oysters may offer a less favourable environment for the development of P. marinus as compared to eastern oysters for at least two possible reasons; i.e. the elevated cellular and humoral activities may degrade the parasite more effectively, and/or the lower plasma protein levels may limit parasite growth.

Levels of intracellular free amino acids used for salinity tolerance by oysters (Crassostrea virginica) are altered by protozoan (Perkinsus marinus) parasitism

Free amino acid (FAA) levels were measured from May through October 1991 in gill tissues of two groups of juvenile oysters (Crassostrea virginica Gmelin), one transferred from a low salinity field site (8‰) to a field site of high salinity (20‰) and high Perkinsus marinus (Mackin, Owen, and Collier) prevalence, the other kept at the low salinity field site. Within 24 h, glycine levels in the oysters transferred to high salinity increased 8-fold, taurine concentrations doubled and the total FAA pool rose from 150 μmol g−1 dry wt to 400 μmol g−1 dry wt. Taurine levels reached a plateau within 20 d after transfer to high salinity and remained at that level until P. marinus infections were detected 85 d after transfer. Taurine and glycine levels declined by ∼40% in the high salinity population as infection intensity increased between 70 and 105 d. Total FAA declined by approximately 33% over this period. The oysters kept at low salinity were not infected and continued to grow while the infected high salinity oysters showed no increase in shell length after Day 85. FAA levels in the low salinity group remained relatively constant throughout the experiment except for an initial rise triggered by an increase in ambient salinity from 8 to 12‰. The results suggest that salinity tolerance mechanisms in C. virginica may be impaired by P. marinus infection.

Reproductive capacity of the eastern oyster Crassostrea virginica infected with the parasite Perkinsus marinus

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 23:135-144 (1995) - doi:10.3354/dao023135 Reproductive capacity of the eastern oyster Crassostrea virginica infected with the parasite Perkinsus marinus Kennedy VS, Newell RIE, Krantz GE, Otto S We studied the influence of the protozoan parasite Perkinsus marinus (Mackin, Owen &AMP Collier) on reproduction in the eastern oyster Crassostrea virginica (Gmelin) from 2 sub-estuaries of central Chesapeake Bay, Maryland, in 1990 and 1991. One hypothesis was that increased levels of infection by P. marinus would result in smaller egg size and diminished lipid reserves within individual eggs. Our results did not reveal any consistent deleterious effects of P. marinus on these gametogenic characteristics, although some of our sample sizes were small and the average disease intensities were low. Our second hypothesis, that there would be a decline in reproductive output as a result of high levels of P. marinus infection, was supported for one estuary when those oysters were near their peak of reproductive condition (July). We found no evidence to support our third hypothesis that there would be a negative influence of infections during the 1990 reproductive season on subsequent reproductive output (summer 1991). Based on published evidence that P. marinus can inhibit oyster growth, we speculate that oysters infected at low intensities by P. marinus can divert energy from growth to reproduction. This differential energy partitioning allows infected oysters to produce fewer eggs than uninfected oysters but with no change in the amount of lipid reserves stored in each egg. Oyster . Disease . Reproduction . Crassostrea virginica . Perkinsus marinus Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 23, No. 2. Publication date: October 26, 1995 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1995 Inter-Research.

Relationship between pollution and susceptibility to infectious disease in the eastern oyster, Crassostrea virginica

Infectious disease epizootics seriously affect many populations of marine organisms. Pollution has been hypothesized to contribute to some aquatic epizootics, although this link has not been adequately examined. To further evaluate this hypothesis, the effect of a complex mixture of sediment derived pollutants on the susceptibility of the eastern oyster, Crassostrea virginica, to an infectious disease was investigated. The disease studied is caused by the protozoan parasite Perkinsus marinus (Dermo) and results in significant oyster mortalities in the mid-Atlantic region of the United States. Oysters were exposed to 0.15 and 30% dilutions of water soluble fractions, generated from sediments collected from the Elizabeth River, a heavily polluted subestuary of the Chesapeake Bay, USA. Oysters were then challenged with P. marinus meronts. Pollutant exposure enhanced preexisting P. marinus infections and increased the oysters' susceptibility to experimentally induced infection, in a dose-dependent manner.

Evaluation of methods using ray's fluid thioglycollate medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica

Accurate detection and quantification of parasite body burden are critical for understanding many aspects of host-parasite interactions. The standard assay for diagnosing Perkinsus marinus infections in Crassostrea virginica involves incubation of oyster tissue in Ray's fluid thioglycollate medium (RFTM), followed by iodine staining and microscopic examination for parasites. The original RFTM tissue assay is destructive and provides only a ranked level of infection intensity. A recent modification provides a technique to enumerate P. marinus after incubation in RFTM and determines total body burden. Application of this technique to hemolymph samples has provided a nondestructive assay. We provide a critical evaluation of these three assays. Data from previous studies were not adequate to critically evaluate performance of the tissue and hemolymph assays. Sensitivity and accuracy of these assays were therefore compared against weight-standardized body burden in Delaware Bay oysters over the course of a year. Determination of total body burden was significantly more sensitive than the other assays. Neither tissue nor hemolymph assays provided accurate estimates of individual infection intensities and were insensitive at low infection levels. The tissue assay was easier to use and slightly more accurate than the hemolymph assay, and both provided reasonable estimates of average infection level in the population. In summary, total body burden assessment is recommended when highly accurate measures of infection intensity or prevalence are necessary, the tissue assay is recommended for monitoring epizootics because of its simplicity and accuracy at the population level, and the hemolymph assay is only recommended when oysters must be kept alive.

Perkinsus marinus Infection and Potential Defense-Related Activities in Eastern Oysters, Crassostrea virginica: Salinity Effects

Infection by Perkinsus marinus and potential defenserelated activities were determined in eastern oysters ( Crassostrea virginica) maintained under different salinity treatments. Oysters were inoculated with trophozoites (10 6 trophozoites per oyster) of P. marinus and incubated in a closed system for 5 weeks in water of 3, 10, and 20 Opt at 22.0 ± 1.0°C. Oysters were then sacrificed and assessed for P. marinus infection. Simultaneously, potential defenserelated activities of individual oysters from each treatment group including controls were examined. Prevalence and intensity of P. marinus infections were found to be positively correlated with salinity. Infections were detected in all salinity groups, but heavy infections were found only at 10 and 20 ppt. No significant difference in total hemocyte counts and percentage granulocytes existed between control and P. marinus-challenged or between infected and uninfected oysters at any salinity. Total hemocyte counts in oysters maintained at 10 ppt were significantly higher than at 3 ppt but were not significantly different from oysters at 20 ppt. Total hemocyte counts were positively correlated with percentage of granulocytes in oysters. Lysozyme concentrations were significantly lower in the oysters maintained in 10 and 20 ppt water than in those maintained in 3 ppt water. Lysozyme concentration was positively correlated with protein concentration and condition index. Hemolymph protein and condition index appeared to be lower in infected than in uninfected oysters, however, these differences were statistically insignificant.

Haplosporidium nelsoni(MSX) in Eastern Oyster Populations of North Carolina

Abstract Populations of the eastern oyster Crassostrea virginica from 36 locations off the coast of North Carolina were examined cytologically in the fall of 1989 for Haplosporidium nelsoni (MSX). The pathogen was found in oysters from 11 (31%) of the sites. Systemic Perkinsus marinus infections were noted in oysters from 26 (72%) of the locations.

Seasonal infection intensity cycle of the parasite Perkinsus marinus (and an absence of Haplosporidium spp.) in oysters from a South Carolina salt marsh

Investigation of the prevalence of the protozoan parasite Perkinsus marinus in populations of intertidal oysters (Crassostrea vircjnica) from a South Carolina salt marsh led to the finding of a distinct, 4 phase cycle of infection intensity. The cycle consisted of characteristic quiescent, pre-virulent, virulent and remission stages of infection. For the first time, P. marinus infection intensity in South Carolina oysters was found to have a statistically significant positive correlation with temperature and salinity, and a negative correlation with oyster condition index. Our data also indicated that P. marinus infection in adult oysters from South Carolina is not size dependent. Based on a limited, qualitative h~stopathological of oyster tissue samples, we found no evidence to indicate the presence of Haplosporidium spp. in the North Inlet salt marsh/estuary system. These results strongly suggest that highly localized mass mortalities of oysters in North Inlet have been due to virulent levels of P. marinus mfecbons.

Effect of temperature and salinity on in vitro culture of the oyster pathogen, Perkinsus marinus (Apicomplexa: Perkinsea)

Perkinsus marinus is one the oyster diseases which caused severe oyster mortalities in Virginia particularly in the 1950s and during 1986 and 1987. It is believed that temperature and salinity control the incidence and intensity of P. marinus infection in oysters. Experiments were conducted to examine the effect of salinity and temperature on in vitro culture of P. marinus. The results indicate that a temperature of 28°C and salinities between 10 and 34‰ favor the development of P. marinus presporangia. Sporulation (release of free swimming biflagellated zoospores) occurred within 2 to 4 days. Presporangia can withstand temperatures as low as 4°C for a time period ≤4 days. Sporulation was observed in presporangia cultures previously incubated at 4°C after they were transferred to 28°C for 1–6 days. Sporulation also took place in presporangia cultured in water of 6‰ at 28°C. Zoospores can survive from 3 to 28 days in water of 4–32‰ at 20°–28°C. Zoospores died in 1 day when transferred from 28° to 4°C.

Enhancement of protozoan pathogen Perkinsus marinus infections in American oysters Crassostrea virginica exposed to the chemical carcinogen n-nitrosodiethylamine (DENA)

Enhancement of protozoan pathogen Perkinsus marinus infections in American oysters Crassostrea virginica exposed to the chemical carcinogen n-nitrosodiethylamine (DENA)

Metabolic changes induced in oysters ( Crassostrea virginica) by the parasitism of Boonea impressa (Gastropoda: Pyramidellidae)

1. 1. The ectoparasitic snail Boonea (= Odostomia) impressa is an important parasite of oysters. Although results varied among tissues and levels of parasitism, all oysters parasitized by B. impressa exhibited alterations in biochemical composition after 1 month. 2. 2. Few changes were observed in small (2–4 cm) or large (6–8 cm) oysters parasitized by 5 or 10 snails, respectively. In contrast, carbohydrate concentration decreased in small oysters parasitized by 15 or 25 snails and increased in large oysters parasitized by 30 snails. 3. 3. Decreased carbohydrate content probably resulted from direct removal of assimiliated carbon by B. impressa. Increased carbohydrate concentration may be due to the combined impact of B. impressa with infections by the protozoan Perkinsus (= Dermocystidium) marinus. P. marinus infection increased in intensity with snail parasitism. Increased lipid content in the mantle tissue of large oysters and increased taurine content in most oysters and oyster tissues could be similarly explained. 4. 4. Overall, mantle tissue was the most severely affected, possibly because snail parasitism produces direct injury to this tissue as well as the systemic impact caused by removal of assimilated carbon.