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Cooperative Oxford Laboratory
Contamination of Atlantic coast commercial shellfish Abstrast: Shellfish (oysters and/or clams) were obtained from 37 commercial harvesting sites in 13 Atlantic coast states from Maine to Florida and one site in New Brunswick, Canada. Gill washings from each of 25 shellfish at each site were examined by immunofluorescence microscopy (IF A) for oocysts of Cryptosporidium. Gill washings from another 25 shellfish at each site were grouped into five pools of five shellfish each. DNA from each pool was utilized for PCR and genotyping. Oocysts were found in 3.7% of 925 oysters and clams examined by IFA in shellfish from New Brunswick and 11 of 13 states. Cryptosporidium DNA was detected by PCR in 35.2% of 185 pools. Cryptosporidium parvum genotypes 1 and 2, and Cryptosporidium meleagridis, all of which have been identified in infected humans, were identified at 37.8% of the sites. Gill washings from every site were tested for the presence of infectious oocysts by biological assay in neonatal BALB/c mice but no mice were found infected, suggesting that either the oocysts were no longer infectious or infections in mice were below the level of detection. Collectively, these findings indicate that Cryptosporidium species, indicative of pollution from human and animal feces and potentially infectious for humans, were found in commercial shellfish from 64.9% of sites examined along the Atlantic coast by either microscopy or molecular testing. Previous reports link periods of high rainfall with the elevated numbers of pathogen contaminated shellfish. Because shellfish in the present study were examined during a period of exceptionally low precipitation, the data are thought to underestimate the number of Cryptosporidium contaminated shellfish likely to be found during periods of normal or above normal precipitation.
Serological affinities of the oyster pathogen Perkinsus marinus (Apicomplexa) with some dinoflagellates (Dinophyceae) Abstrast: The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular-genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflage11ates. To test for evidence of serological epitopes common to P. marinus and dinoflage11ates, we probed 19 free-living and 8 parasitic dinoflage11ate, or dinoflage11ate- like, species for cross-reactivity with polyclonal antibodies to P. marinus. Three of 19 free-living dinoflage11ates (16%), and 7 of 8 parasitic dinoflage11ates (88%) were labeled by anti-F. marinus antibodies. In reciprocal immunoassays using poly clonal antibodies to the Hematodinium sp. dinoflage11ate parasite of Norway lobsters, Nephraps norvegicus, P. marinus and the same 7 parasitic dinofla- ge11ates labeled by anti-F. marinus antibodies, were again labeled. The dinoflage11ate-like parasite of prawns Pandalus platyceras was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody-binding epitopes on ce11s of P. marinus and several dinoflage11ates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflage11ates suggests a closer phylogenetic link to the syndinean dinoflage11ate lineage. The consistent failure of the dinoflage11ate-like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Hematodinium-species parasitic dinoflage11ates.
Two epizootic diseases in Chesapeake Bay commercial clams, Mya arenaria and Tagelus plebeius
Temporal variability of Cryptosporidium in the
Oyster biomass, abundance, and harvest in northern Chesapeake Bay: trends and forecasts
Remote acoustic habitat assessment techniques used to characterize the quality and extent of oyster bottom in the Chesapeake Bay
The location, composition, and origin of oyster bars in Mesohaline Chesapeake Bay
Juvenile oyster disease (JOD) and management strategies:
No evidence of accommodation in the eyes of the bottlenose dolphin, Tursiops truncatus
A digital presentation of the Maryland oyster habitat and associated bottom types in the Chesapeake Bay (1974-1983) Abstract:Between 1975 and 1983, the Maryland Department of Natural Resources conducted a survey of Maryland's portion of the Chesapeake Bay to reassess the extent and condition of oyster bottom (bars) that were initially surveyed in 1912. A variety of methods were employed to assess bottom condition, including bottom grabs, sounding poles, and dragged microphones. The survey used six categories to describe substrate type. Three of these represented oyster bottom: cultch (exposed oyster shell), cultch with sand, and cultch with mud. The other three categories were non-oyster bottom: sand, mud, and consolidated hard sediment. Bottom characterizations were drawn on 37 transparent Mylar sheets. Although the resulting charts were used to generate new legal oyster bar boundaries, the original bottom characterizations were never published. We present here the digitized Mylars in a Geographic Information System (GIS) polygon format, which is now available as a digital file. A comparison of results with more recent habitat assessments indicate the survey was a generally valid representation of the true bottom condition. The digitized survey results, however, could be misapplied if used as an exact description of current bottom conditions, because in many areas, cultch categories are actually shell covered by varying depths of sediment. This work provides a demonstration of how GIS-based analysis can be employed to interpret historical surveys of oyster habitats and provide a useful management product to resource managers.
Ultrastructural characteristics of the in vitro cell cycle of the protozoan oyster pathogen Perkinsus marinus Abstract:Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marin us, a pathogen of the eastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell culture was propagated from infected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts, were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles with vacuoplasts. There were micropores on the inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles, and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggesting a function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and may specialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infected oyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose, hypha-like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangial stages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.
Sudden appearance of cysts and ellobiopsid parasites on zooplankton in a Michigan lake: a potential explanation of tumor-like anomalies Abstract:Cysts on calanoid and cyclopoid copepods, previously reported as tumor-like anomalies (TLAs) in Lake Michigan and Europe, appeared briefly in Patterson Lake, a small Michigan inland lake. Cysts were rare (4% maxi- mum) in samples collected on September 11, 1999, but appeared with high frequency on calanoid adults (49%) and cyclopoid nauplii (73%) in samples collected on October 16. By October 30, cysts were again rare (0.4% maximum).Cysts most commonly appeared on the lateral surface of the animal at the articulation of the 151 and 2nd prosomal seg- ments and often consisted of herniated copepod tissues. Transparent, pyriform cysts co-occurred in low frequency with other types of cysts and are believed to be the trophomeres and gonomeres of ellobiopsid parasites. Histologic manifes- tations of cysts were diverse; herniations consisted of acellular yolk-like material and apparent host tissue, while cysts thought to be Ellobiopsis contained cells with different degrees of nuclear staining and unusual spherical bodies. Her- nias were experimentally induced on live calanoid copepods by piercing the carapace with a fine needle, suggesting that ellobiopsid parasites may cause the hernias by puncturing the carapace of their hosts. Ellobiopsid parasites are common on marine crustacean zooplankton but have been recorded only once before in freshwater.
Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms Abstract:The prevalence of Cryptosporidium, Giardia and Eimeria, in healthy, asymptomatic, post-weaned and mature cattle was investigated on three Maryland farms. One farm, a dairy research facility, had 150 multiparous Holstein milking cows; 24 were examined and Cryptosporidium andersoni was detected in three (12.5%) but neither Giardia nor Eimeria was detected. The second farm, a commercial dairy, had 57 multiparous Holstein milking cows and an equal number of heifers. Of 19 cows examined, C. parvum, Giardia duodenalis, and Eimeria bovis and/or E. ellipsoidalis were detected in two (10.5%), two (10.5%) and one (5.26%) cow, respectively. Of 23 heifers examined, C. parvum, Giardia, and E. bovis and E. ellipsoidalis, was detected in two (8.7%), four (17.4%), and five (21.7%), heifers, respectively. The third farm, a beef cattle breeding and genetics research facility, had 1807- to 9-month old purebred black Angus. Of 118 examined for C. parvum and Giardia, 34 (28.8%) and 44 (37.3%) were positive, respectively, of 97 examined for E. bovis and/or E. ellipsoidalis 32 (33.0%) were positive. These findings, based on a method with a minimum detection level of 100 oocysts of C. parvum/g of feces, which underestimates the number of infected cattle, clearly demonstrate the presence of low level, asymptomatic infections in post-weaned and adult cattle in the United States and indicate the potential role of such cattle as reservoirs of infectious parasites.
An Index of Ecosystem Integrity for Northern Abstract:We propose a theoretical approach to evaluating the quality of large ecosystems under anthropogenic stress, and present a partial application of these ideas. We synthesized data from a variety of Chesapeake Bay monitoring programs into composite variables expressing ecosystem integrity. The analysis spanned most of the larger Maryland tributaries to the Bay (tidal fresh to mesohaline areas) for nine years (1986-1994). An index modeled on the Index of Biotic Integrity (IBI) and a multivariate ordination and ranking method combined information on aquatic plant, plankton, benthic and fish assemblages with water quality-based habitat goals. The multivariate method proved superior to the IBI approach in discriminating ecosystem integrity. A logistic regression model with watershed land use characteristics and water depth as independent variables explained a significant amount of variation in the multivariate rank index. Urban land use had the most negative effect on the index, whereas forested land had the most positive effect.
Life history and habitat observations of softshell clams
Abstract:Population densities, survival, factors associated with mortalities, and growth of softshell clams, Mya arenaria, in two northeastern New Jersey estuaries were studied from 1993 through 1997. The study areas were near shore where low-tide water depths ranged from 15 to 90 em. Juvenile densities were high only in 1993. Light sets of juveniles from 1994 to 1997 disappeared by the end of their first summer. The longest living softshells were the abundant 1993 year class, which survived for 26 mo in the Shrewsbury River. This contrasts with life spans of 7-12 years for softshells in New England. Mortalities of softshells were correlated with: (1) predation by the striped killifish, Fundulus majali.\', and mummichog, F. heteroclitus; (2) mats of sea lettuce, Viva lactusa; and (3) high temperatures (30-32 QC). Softshell sarcoma was also present and may have contributed to mortalities. The effects of the mortality agents varied among locations and years. The softshells of the Shrewsbury River averaged about 23 mm and 40 mm long at the end of their first and second growing seasons, respectively.
Analysis of extracellular proteins of two Perkinsus spp. isolated from the softshell clam Mya arenaria in vitro Abstract:Biochemical characterization of the extracellular proteins (ECP) of two softshell clam Perkinsus spp. cloned isolates, Perkinsus chesapeaki isolate G-117 and Perkinsus marinus H-49, was performed and compared to that of the oyster-derived P. marinus isolate P-l. G-1l7 and H-49 demonstrated distinct differences in enzyme activities; however, all three isolates shared common bands. Substrate-impregnated gels showed H-49 to possess proteolytic activities while G-117 did not. Inhibition studies revealed that H-49 ECP contain serine proteases similar to those described for P-l. The G-1l7 ECP lacked proteolytic activity but showed a higher production of lipolytic enzymes than H-49 or P-l. Optimal in vitro growth temperatures for the two clam isolates were generally lower than those for P-l. G-117 showed faster growth at lower salinities than either H-49 or P-l. Clam Perkinsus spp, isolates appear to be better adapted to lower salinities and temperatures than the P. marinus isolate of the eastern oyster.
Prevalence of Perkinsus spp. in Chesapeake Bay soft-shell clams,Mya arenaria Linnaeus, 1758 during 1990-1998 Abstract:Prevalence and intensity of Perkinsus spp. infections were determined in soft-shell clams Mya arenaria during 1990 to 1998 based upon incubation of rectal tissues in Ray's fluid thioglycollate medium. During the study, soft-shell clams were collected from 18 sites in the upper Chesapeake Bay in Maryland. Enlarged hypnospores were found in -7% (114/1,705) of the soft-shell clams. Peak prevalences occurred in the fall of 1992 with -53% (16/30) at Piney Point and 50% (15/30) at Eastern Neck, and in August 1995 with -64% (18/28) and -37% (11/30) at Cedar Point and Piney Point, respectively. This investigation provides evidence that Perkinsus spp. infections in soft-shell clams are more common than previously thought.
Zoosporulation of a new Perkinsus species isolated from
Abstract:A gill-associated Perkins us sp. isolated from the softshell clam Mya arenarial is described as a new species, P. chesapeaki sp. novo Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia. Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including an open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned. Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gill-associated Perkins us from the softshell clam as a new species.
Epizootiology of the parasitic dinoflagellate Hematodinium sp. in the American blue crab Callinectes sapidus Abstract: Hematodinium sp. is a parasitic dinoflagellate that infects and kills blue crabs Calli- nectes sapidus. Periodic outbreaks of dinoflagellate infections with subsequent high host mortalities prompted a study of the epizootiology and distribution of the crab pathogen. Hemolymph samples from over 13000 crabs were assessed for infections over 8 yr. Moderate to high prevalences were found at several locations along the Atlantic and Gulf coasts of the United States. In the coastal bays .of Maryland and Virginia, prevalence followed a seasonal pattern, with a sharp peak in late autumn. Infections were significantly more prevalent in crabs measuring less than 30 mm carapace width; host sex did not influence prevalence. Prevalences were highest in crabs collected from salinities of 26 to 30 %0; no infected crabs were found in salinities below 11 %0. Intensity of infection did not vary among crab sizes, molt stages, or sexes. Naturally and experimentally infected crabs died over 35 and 55 d in captivity, with a mean time to death of approximately 13 and 42 d, respectively. Several other crus- taceans, including gammaridean amphipods, xanthid (mud) crabs, and the green crab Carcinus maenus, were found with Hematodinium-like infections. Considering its widespread distribution and high pathogenicity, we suggest that Hematodinium sp. represents a significant threat to blue crab populations in high salinity estuaries along the Atlantic and Gulf coasts of the USA.
Protease inhibitors in plasma of the softshell clam Mya arenaria: Identification and effects of disseminated sarcoma Abstract:Despite the ecologic and economic significance of the softshell clam (Mya arenaria), little is known about the humoral factors involved in its host defense mechanisms. Protease inhibitors, a group of proteins believed to playa role in host defense mechanisms against infections and proliferative diseases, have recently been identified in bivalve molluscs. In the present study we provide evidence for the presence of protease inhibitors in softshell clam plasma. Levels of protease inhibitory activities against the enzymes tested varied greatly, e.g. 1 J.lg of plasma protein inhibited 35.3:t 9.69 ng pepsin (aspartic protease), 4.9:t 1.45 ng papain (cysteine protease) and 3.1:t 0.88 ng trypsin (serine protease). On the contrary, the level of anti-metalloprotease (thermo lysin) activities was much lower. The sensitivity to methylamine and the ability to protect trypsin from active site trypsin inhibitors provided evidence for the presence of an Ci2-macroglobulin-like molecule in softshell clam plasma. In the Chesapeake Bay widespread epizootics of disseminated sarcoma have been described in M. arenaria populations. The impact of this lethal proliferative disorder on clam defense responses has received little attention. In this study the effects of sarcoma progression on plasma protease inhibitory activities were, therefore, assessed. Clams with early stages of sarcoma showed a non-significant decrease in protease inhibitor levels. Clams with advanced stages of sarcoma showed a significant decrease in the ability to inhibit trypsin and papain, while the protease inhibitory activity levels against aspartic and metalloprotease were completely exhausted.
Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay Abstract:Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.
Giardia duodenalis cycts of genotype a recovered from clams in theChesapeake Bay subestuary, Rhode River Abstract:Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodena/is Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.
Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay. Abstract:Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1 % recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts.
Characterization of two Perkinsus spp. from the softshell clam, Mya arenaria, using the small subunit ribosomal RNA genes Abstract:Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morpho- logically different Perkinsus species isolates from the gill (G 117) and the hemolymph (H49) of the softshell clam, Mya arena ria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G 117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of> 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G 117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.
Discrimination between two Perkinsus spp. isolated from the softshell clam Mya arenaria by sequence analysis of two internal transcribed spacer regions and 5.8S ribosomal RNA genes Abstract:The internal transcribed spacer (ITS-l and ITS-2) regions and the 5.8S ribosomal RNA gene of 2 Perkinsus spp. (G 117 and H49) originating from the softshell clam, Mya arenaria, of the Chesapeake Bay were cloned and sequenced to obtain evidence for their genetic divergence. A high level of heterogeneity in both regions, probably resulting from deletions, insertions, and base substitutions, was evident from alignments of the sequences of the 2 isolates with published sequences of other Perkinsus spp. The isolate G 117 and other Perkinsus spp. were highly divergent (13-26 % and 19-20 % sequence divergence in ITS-l and ITS-2, respectively). These regions in the isolate H49 and Perkinsus marinus were similar (99.07 % and 99 % for ITS-l and ITS-2, respectively). Evidence obtained from a phylogenetic analysis using the aligned sequences suggests that G 117 and H49 belong to 2 distinct species of Perkinsus. The isolate G 117 possibly belongs to an as yet undescribed species of Perkinsus, and H49 belongs to the species P. marinus. The conclusions drawn from the genetic analysis of H 49 and G 117 are supported by previously reported morphological characteristics (McLaughlin & Faisal, 1998b). Isolates H49 and G 117 originated from the same molluscan species demonstrating that at least 2 different species of Perkinsus can co-exist in 1 host.
Salinity and temperature effects on Hematodinium sp.
Abstract:The parasitic dinoflagellate Hematodinium sp. infects and causes mortalities in blue crabs Callinectes sapidus Rathbun 1896 from high salinity coastal embayments. The seasonal infection cycle and apparent salinity and temperature requirements for infections reported from wild crab populations indicate that environmental factors influence the parasite's ability to proliferate within crab hemolymph. A series of experiments held crabs at various water temperatures and salinities to assay infection intensity and crab survival. There was a significant increase in mean intensity in infected crabs held in ambient 15-9 °C seawater for 32 days; at temperatures below 9 °C, mean intensity diminished. Mean intensity decreased significantly in infected crabs held in 10%0 or 29%0 artificial seawater at 9 °C for 73 days; the decrease was significantly greater at 10%0 than at 29%0. Mean intensity increased in infected crabs held in 22%0 seawater at either 12 or 16 °c. Presumably uninfected crabs held at 22 °C presented infections after 14 days. No infections were transmitted by exposure of uninfected crabs to infected crabs after 85 days. Low water temperature and salinity appear to limit the proliferation of Hematodinium sp. in blue crab hemolymph. Apparently uninfected crabs from endemic areas can carry pre-patent or latent infections.
Survival of infectious Cryptosporidium parvum oocysts in seawater and Eastern oysters (Crassostrea virginica) in the Chesapeake Bay Abstract:Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at lOoC and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.
Detection of Cryptosporidium oocysts and Giardia cysts in the tissues of eastern oysters (Crassostrea virginica) carrying principal oyster infectious diseases Abstract:The potential cross-reactivity of the combined Cryptosporidium/Giardia direct immunofluorescence antibodies (IFA) of MERIFLUORTM and HYDROFLUORTM_COMBO tests was examined against tissues containing known developmental stages of 12 pathogens causing the principal infectious diseases in oysters. Spores of Haplosporidium nelsoni and Haplosporidium costale produced positive acid-fast stain (AFS) reactions similar in intensity to Cryptosporidium parvum oocysts, Hexamita nelsoni trophozoites produced positive IFA reactions in both IFA tests; however, the intensity of fluorescence was considerably lower and the fluorescein-staining pattern different than those of Giardia cysts, The applicability of AFS for screening oysters for Cryptosporidium oocysts is low, and positive identification of Cryptosporidium oocysts cannot be accomplished based on the AFS, Presumptive IFA identification of the Cryptosporidium oocysts or Giardia cysts in the oyster tissue should fulfill 3 criteria, i.e., bright-green fluorescence of the same intensity as C. parvum oocysts and Giardia cysts in the positive control, correct size and shape of the fluorescein-stained objects, and oocyst or cyst shell clearly visible.
Giardia sp. cysts and infectious Cryptosporidium parvum oocysts in the feces of migratory Canada geese (Branta canadensis) Abstract:Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment.
In vitro propagation of two Perkinsus species from the softshell clam, Mya arenaria Two continuous, axenic cultures of Perkinsus spp. (H49 and G 117) were obtained from the softshell clam Mya arenariacollected from Swan Point in the Chester River, Chesapeake Bay (Maryland). Isolate H49 was obtained from the hemolymph of a heavily infected clam. Except for their larger size, H49 trophozoites and schizonts exhibited the characteristic morphology of Perkinsus marinus and divided by schizogony. Isolate G 117 was obtained from a combined gill/palp sample of a moderately infected clam. Unlike H49, vegetative forms (trophozoites and schizontsl of G 117 were present along with prezoosporulation stages in the same culture. In culture, G 117 cells multiplied by both schizogony and successive bi-partition. Both H49 and G 117 cells reacted positively with anti-Perkinsus marinus polyclonal serum and formed hypnospores upon incubation in Ray's fluid thioglycolate medium that stained black with Lugol's iodine. This is the first isolation of Perkinsus species from the softshell clam. Studies to determine the infectivity, pathogenicity, enzyme activities, and genotyping of both softshell clam Perkinsus spp. are ongoing.
Histopathological alterations associated with Perkinsus spp. infection in the softshell clam, Mya arenaria Abstract: Softshell clams (Mya arenaria) collected from the Chester River in the upper Chesapeake Bay showed the presence of Perkinsus spp. in - 12 % (28/240) of clams examined. The infection seems to run a mild course with the host prevailing in encapsulating invading parasites The gills appear to be the major site of infection; however, the parasite was also found in the digestive gland, gonads, and kidneys and occasionally in the tissue and sinuses of adductor muscles. Typically, clusters of protozoal cells were embedded in an amorphous PAS-positive substrate and were surrounded by one or more layers of granulocytes. In heavy infections, both free and encapsulated Perkinsusspp cells were observed in affected tissue forming aggregations of different sizes. Within the tissues of M. arenaria, the parasite propagated by schizogony. The presence of large encapsulations in vital organs such as the gills and gonads may adversely affect growth and fertility of affected clams.
Diseases, parasites, and symbionts of blue crabs, Callinectes sapidus, dredged from Maryland portions of Chesapeake Bay Abstract: A 3-year histological study of disease prevalence in 657 blue crabs, Callinectes sapidus, dredged from 31 sites within Maryland portions of Chesapeake Bay during autumn and winter revealed the presence of many diverse parasites and symbionts. A large number of crabs exhibited hemocytic infiltration and encapsulation. Parasites and symbionts identified included viruses (Baculo- Band RLV-RhVA), a rickettsia-like microorganism (RLM), an unusual strandlike organism, unidentified microsporidians and gregarines, both parasitic (Mesanophrys chesapeakensis) and symbiotic (Lagenophrys callinectes and Epistylis sp.) cilates, the nemertean Carcinonernertes carcinophila, and trematode metacercariae, some hyperparasitized by the haplosporidian Urosporidiurn crescens. Signifi- cant differences in disease and parasite frequencies were observed among survey periods. The prevalence of some tissue responses and parasites exhibited seasonal patterns of infection and infestation.
Shellfish benthic habitat assessment in the Chesapeake Bay; progress towards integrated technologies for mapping and analysis Abstract:Combinations of mechanical and remotely sensed technologies were applied to the task of developing techniques to assess and chart the physical condition of oyster habitat in the mesohaline Maryland Chesapeake Bay. Acoustic sub-bottom profiling equipment, side-scan sonar, underwater video. sediment analysis, and seafloor classification systems were tested for their capability in characterizing important components or conditions of the benthic environment. as well as their ability to integrate and display this information via linked Global Positioning System (GPS) and Geographical Information System format (GIS). Acoustic seafloor classification fully integrated with shipboard and land-based GIS, linked to bottom validation techniques. seems to offer the most effective solution to oyster habitat assessment at virtually any scale of interest.
Oyster shell disarticulation in three Chesapeake Bay tributaries Abstract:We examined the effects of site location, season of deployment, substrate, size class, and salinity and temperature regimen on the time-since-death (TSD) required for the disarticulation of shells of Crassostrea virginica (Gmelin, 1791) at sites located in three Chesapeake Bay tributaries, The mean TSD required for disarticulation was greatest at Chestertown (815.5 days), intermediate at Oxford (718.9 days), and least at Deal Island (630.0 days), which corresponded to progressively increasing mean salinities. The mean TSD for all sites combined was 739.4 days, ranging from 21 to 1,427 days. Within sites, oyster shell size class (i.e., market-sized> 76 mm, small <76 mm) had significant intrasite effects on mean TSD at Chestertown and Oxford, although among sites, a significant interaction existed between size class and sites. Overall, shell length had a strong positive correlation with TSD and accounted for 10.0% of the variability in TSD. Mean annual salinity had a strong negative correlation with TSD, accounting for 18.1 % of the variability in mean TSD. The season of deployment of oyster shells had a significant effect on the mean TSD at Chestertown and Oxford, although not in a consistent manner from one site to another. However, overall, the cumulative percentage disarticulation was greatest in summer (47.6%) and least in winter (6.3%). Among sites, the substrate on which the trays were deployed (i.e., reef or sediment) did not significantly affect mean TSD.
Potential role of the Eastern oyster, Crassostrea viginica, in the epidemiology of Cryptosporidium parvum Abstract:Oysters were placed in an aquarium containing artificial seawater, and Cryptosporidium parvum oocysts were added. Oocysts were later found in the gill washings, hemocytes, and gut contents of the oysters. Hemocytes containing oocysts were intubated into four mice. C. parvum stages developed in the ileal epithelia of all of the mice, indicating that the oocysts in the hemocytes remained infective.
In vitro interactions between hemocytes of the Eastern oyster, Crassostrea virginica Gmelin, 1791 and Cryptosporidium parvum oocysts Abstract:It was demonstrated by an in vitro slide phagocytosis assay that hemocytes of the Eastern oyster, Crassostrea virginica Gmelin, 1791 are capable of rapid recognition and internalization of infectious Cryptosporidium parvum (AUCP-1 strain) oocysts. The incubation of hemocyte monolayers (8.5 X 104 cells) that had received 6.8 X 105 or 3.4 X 105 oocysts was arrested at 5, 15,30,60,90, and 120 min and the oocysts detected by acid-fast stain and immunofluorescent antibody (IFAT). An average of 20.5, 38.3, 50.2, 58.9, 69.0, and 75.0% oocysts were phagocytosed after 5, 15, 30, 60, 90, and 120 min, respectively. The intensity of fluorescence of phagocytosed oocysts significantly de- creased over time (P < 0.01), and their round shape was altered. The number of cells containing oocysts and the mean number of ingested oocysts (range: 1.2-4.5 per cell) increased significantly over time (P < 0.01), whereas the numbers of non phagocytosed oocysts that were adherent to the glass slides significantly decreased (P <0.05). By extrapolation, the results indicate that Cr. virginica is capable of internalizing up to 6.4 X 106 Cryptosporidium oocysts per ml of its hemolymph.
Recombinant expression of the antimicrobial peptide polyphemusin, and its activity against the protozoan oyster pathogen Perkinsus marinus Abstract:Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from natural materials or obtained by chem- ical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptibleE. coli host, weusedacarboxy- terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLiml was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marin us, complete inhibition was observed at 12 ug/ ml, and partial inhibition at 8 ug/ml.
Protozoans isolated from Louisiana shelf sediments subject to hypoxia/anoxia with emphasis on freshwater amoebae and marine flagellates Abstract:Sediments offshore from the Louisiana coast west of the Mississippi Delta Bight were cultured for marine and freshwater protozoans during a period of seasonal benthic hypoxia. The episodes of hypoxia occurring in this area from late spring through early fall follow periods of high volume discharge from the Mississippi River, and coincide with a strong pycnocline between low salinity surface water and deeper more saline water. The study site was located off the Terrebonne/Timbaliet Bay system at depths of 14-22 m where there are recurrent episodes of dissolved oxygen readings approaching 0.0 mg VI, and bottom water and sediments may have an odor of hydrogen sulfide. Sewage contamination at the site has been considered since there are numerous point and non-point sources for pollutants that enter the ecosystem from nearby rivers and bays. We tested bottom sediments to determine whether or not freshwater, soil, or sewage associated protozoans were present as indicators of land or riverine runoff. Two of seven sediment samples yielded freshwater or soil cyst-forming amoebae belonging to the genus Acanthamoeba (A. polyphaga and A. hatchetti); one yielded a cyst-forming freshwater flagellate, Heteromita globosa. All stations were positive for one or more of 15 genera of marine flagellates, four yielded unidentified ciliates and five free-living marine amoebae. Physical features of the seabottom and observations on the benthic biota observed by divers and Remotely Operated Vehicles (ROVs) showed that surficial sediments were anoxic and covered by bacterial mats.
Spatial distribution and intensity of Perkinsus marinus infections in Oyster Recovery Areas in Maryland Abstract:The project described here reports on the status and spatial variability of Perkinsus marinus in three oyster recovery areas (ORAs) during 1994. The objectives of the study were to examine spatial variability in P. marin us infections among oyster bars along single tributaries and within single oyster bars. In addition, the study was conducted to compare estimates of prevalence based on intensive and spatially accurate patent-tong sampling with estimates of prevalence based on traditional dredge sampling of 30 oysters. For comparative purposes, patent-tong and dredge sampling were both conducted in each of four oyster bars, within 35 days, during September-October 1994. In June 1994, a pilot study was conducted to test the use of long range navigation (LORAN) and global positioning system (GPS) for the accurate deployment of patent-tongs and to compare hemolymph and tissue Ray fluid thioglycollate medium assays. In summary, results from this investigation support three conclusions: (I) During fall 1994, the variation of P. marinus prevalence, among and within oyster bars in sampled ORA tributaries, was small «30%) but statistically significant; (2) traditional dredge samples of 30 oysters per bar provided estimates of prevalence remarkably similar (within 5%) to the ones obtained from patent-tong samples of an average of 340 oysters per bar; (3) RFTM assays conducted in the spring showed that hemolymph, rectum, and combined gill and palp samples gave equivalent determinations of prevalence.
Management alternatives for protecting Crassostrea virginica fisheries in Perkinsus marinus enzootic and epizootic areas Abstract:We review management of oyster stocks infected by Perkinsus marinus, comparing previously published recommendations with current practices, with emphasis on the public fishery in the Maryland portion of Chesapeake Bay. The epizootiology of perkinsiasis is described, particularly the spread of the disease into low salinity areas. We also describe recent attempts to develop a policy and management framework for restoration of oyster populations that have been depleted by P. marinus and Haplosporidium nelsoni. It is apparent from experiences in Gulf of Mexico estuaries, Long Island Sound, and Maryland that strong recruitment can, to some extent, offset the impacts of P. marinus on oyster fisheries. Although improved management practices so far have had very limited success in maintaining harvestable stocks in the Chesapeake, it is clear that the recruitment potential of oyster populations has not been diminished to a critical point. Strategies designed to enhance and supplement natural recruitment, along with maintaining growing areas as free from P. marinus infections as possible, currently offer the most promise for maintaining harvestable stocks. In combination, new developments in research, management, monitoring, and policy are cause for guarded optimism, both for larger, sustainable harvests and for restoration of some of the ecological functions of healthy oyster populations.
A synopsis of juvenile oyster disease (JOD) experimental studies in Crassostrea virginica Abstract:In the late 1980's juvenile oysters, Crassostrea virginica, spawned and cultured in New England and New York, began experiencing up to nearly 100% mortalities in some batches of juveniles. The cause of these mortalities was not ascertained immediately, but examination of dead and dying oysters did not reveal a previously recognized disease syndrome. Early studies showed that it was not an environmental or genetic problem, thus we hypothesized that a new, transmissible disease agent caused the observed mortality. This was verified under laboratory conditions where the disease was readily transmissible. Transmission was enhanced by warm water temperatures, 22-26°C, and salinities of 18-30 ppt. Also, the infectious agent was filterable and sensitive to erythromycin, supporting the hypothesis that the causative agent may be a protistan parasite in the 2-6 µm size range. No evidence to support a viral or bacterial etiology was found.
Mesanophrys chesapeakensis n. sp., a histophagous ciliate in the blue crab, Callinectes sapidus, and associated histopathology Abstract:Histophagous ciliate infections were infrequently detected in blue crabs, Callinectes sapidus, from Chesapeake Bay, Delaware Bay, and a coastal bay of Maryland. Associated pathology included hemocyte aggregations in various tissues and nodule formation. Protargol silver staining of cultured ciliates revealed an oral apparatus morphologically similar to that of other hemolymph-infecting ciliates of the genus Mesanophrys. The oral apparatus included three distinct oral polykinetids and an oral dikinetid consisting predominantly of a b segment and a smaller c segment. Morphologic comparison to other histophagous ciliates infecting crustaceans showed that the ciliate in blue crabs is a distinct species, Mesanophrys chesapeakensis n. sp., family Orchitophryidae.
Population and disease dynamics of Maryland oyster bars: a multivariate
classification analysis
Use of a tetrazolium-based cell proliferation assay to measure effects of in vitro conditions on Perkinsus marinus (Apicomplexa)proliferation Abstract:Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus, at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10-40° C permitted growth, which was maximized at 35° C. pH 6.0-8.5 permitted growth, which was maximized at 7.0-7.5. Osmolalities of 340-1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1-10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0,1:1 DME/ Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.
Acanthamoeba pearci, n.sp. (Protozoa: Amoebida) from sewage contaminated sediments Abstract:Seabottom sediments from a discontinued Philadelphia-Camden 40-Mile ocean sewage disposal site were cultured for cyst-forming free-living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga. Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well-described species within the family Acanthamoebidae Sawyer & Griffin, 1975.
Hematodinium perezi infections in adult and juvenile blue crabs, Callinectes sapidus from coastal bays of Maryland and Virginia, USA Abstract: In coastal bays of Maryland and Virginia, USA, adult and juvenile blue crabs Callinectes sapidus were severely infected with the parasitic dinoflagellate Hemato- dinium perezi. Dinoflagellates were observed in the hemocoel of all infected crabs; associated histopathological changes were evident in some tissues. Dinoflagellates could be observed with an inverted microscope through the 5th pleopod of heavily infected juvenile crabs (5 to 29 mm) without invasion. This note documents a high prevalence of H. perezi infections in juvenile blue crabs from coastal bays in Maryland and Virginia. The seasonal infection prevalence cycle reported by previous authors is consistent with observations made during this study.
Binding specificities of mono- and polyclonal antibodies to the protozoan oyster pathogen Perkinsus marinus Abstract:In an effort to develop direct detection methods for Perkins us marinus cells in host tissue and environmental samples, the production of both mono- and polyclonal antibodies specific for this apicomplexan oyster pathogen was attempted. A particulate hypnospore immunogen was isolated from infected oyster hemolymph following incubation in Ray's fluid thioglycollate medium. Polyclonal antisera produced in both mice and rabbits following immunization with this preparation exhibited high antibody titers for pathogen cell epitopes, but not for host oyster tissue epitopes. At least some antibodies to hypnospore antigenic determinants recognized common epitopes on trophozoite cells in infected oyster tissues, as well as on zoospores and other proliferative cells produced by incubation of infected oyster hemolymph in sterile seawater. Polyclonal antibodies also recognized a diffusable, noncellular substance present in host oyster tissues in areas surrounding focal lesions. Rabbit polyclonal antibodies did not recognize 2 Dermocystidium species infecting salmonid fishes or Haplosporidium nelsoni infecting eastern oysters Crassostrea virginica. These antibodies did recognize many, but not all, Perkins us species infecting selected molluscan hosts worldwide. Monoclonal antibodies produced to date apparently recognize epitopes unique to the hypnospore immunogen.
Acanthamoeba stephensoni, n.sp. (Protozoa:Amoebida) from sewage contaminated shellfish beds in Raritan Bay, New York Abstract:Marine sediments from 12 shallow water stations in Raritan Bay, New York were tested forthe presence of Acanthamoeba. Eight stations were positive for one or more species of Acanthamoeba, A. castellanii, A. comandoni, A. hatchetti, A. lenticulata, A. polyphaga, A. rhysodes, and Acanthamoeba spp. Isolates that grew at 38-40° C were found at four stations (A. comandoni, A. lenticulata, and two unidentified strains). The two unknown strains were characterized on the basis of morphological features, isoenzyme profiles, and mouse pathogenicity tests. One of the two strains was determined to be a new species and is designated herein as Acanthamoeba stevensoni n. sp., A TCC 50388. Mature cysts were most similar to those of morphological Group II of Pussard & Pons (1977). Acanthamoeba stevensoni n. sp. was isolated from inshore coastal sediments where seawater ranged from 20-300/00 (ppt.). The sediments supported commercially valuable populations of hard clams, Mercenaria mercenaria, that required depuration prior to sale because of contamination by sewage-associated bacteria.
Lethal parasites in oysters in from coastal Georgia with discussion of disease and management implications Abstract:Extensive mortalities of oysters, Crassostrea virginica, occurred from 1985 through 1987 in coastal waters of Georgia. Fluid thioglycolate cultures of oysters collected from 16 of 17 locations revealed infections by the apicomplexan parasite Perkinsus marinus. An ascetosporan parasite, Haplosporidium nelsoni, was also observed in histopathological examination of oysters from 4 of the locations. While the range of H. nelsoni currently is recognized as the east coast of the United States from Maine to Florida, this is the first report of the parasite in Georgia waters. This paper documents the occurrence of these two lethal parasites in oysters from coastal waters of Georgia, along with potential disease and management implications. Results of an earlier in- dependent and previously unpublished survey are also discussed which document the presence of P. marinus in Georgia as early as 1966.
Transmission studies of sarcomas in the softshell clam, Abstract:Transmission experiments with adult soft-shell clams (Mya arenaria) demonstrated that clam sarcomas are transmissible with hemolymph from neoplastic animals but not with cell-free ultrafiltrates. Non-neoplastic clams were injected with either hemolymph from neoplastic clams or a cell-free ultrafiltrate prepared from a subsample of the same hemolymph. Injected clams were held in separate flow-through aquaria and examined for sarcomas by histocytology and histology. Data at 17 weeks showed a 44% prevalence of sarcomas in clams injected with neoplastic inoculum. No sarcomas were observed either in clams injected with a cell-free ultrafiltrate or in the control animals. The lack of sarcomas in clams injected with the ultrafiltrate argues against a viral etiology for the disease.
Different proteins in the hemolymph sera from sarcomatous and healthy softshell clams Mya arenaria Abstract:1. Serum proteins showed quantitative and qualitative differences between sarcomatous and healthy soft shell clams, Mya arenaria L. 2. Total protein concentration was significantly higher in the serum of sarcomatous clams than of healthy clams. 3. According to SDS-PAGE, more serum proteins with more variability distinguished sarcomatous clams from healthy ones. 4. Sarcomatous clams had unique serum proteins of Mr 23,000, 45,000 and 54,000. Healthy clams had unique serum proteins of Mr 24,000, 103,000 and 105,000. 5. During disease progression, sarcoma-specific proteins appeared while normal proteins disappeared. 6. We propose that some sarcoma-associated proteins may have tumor promoting and/or cytotoxic functions and that some normal proteins which disappear during disease progression may be involved in the humoral defense system.
Bifurcation of gill filaments in winter flounder (Pleuronectes americanus Walbaum) from Long Island Sound Abstract:Morphological and histological examinations of gills excised from adult winter flounder, Pleuronectes americanus Walbaum, collected at clean and contaminated areas of Long Island Sound were undertaken to assess possible biological consequences of contamination. On the basis of previous studies, three collection sites were chosen: Shoreham, New York, a relatively clean, unindustrialized area, and New Haven Harbor, Connecticut and Hempstead Harbor, New York, both industrialized and heavily populated. Gill samples were taken monthly at all three sites for light and scanning electron microscopy (SEM) examination. Results from both techniques suggest a relationship between contamination of the sediments and the prevalence of bifurcated gill filaments, the condition being most severe at New Haven Harbor. The bifurcations were not consistently associated with any parasitological or pathological conditions when examined by light microscopy. Gill samples were subsequently taken from juvenile winter flounder caught in New Haven Harbor to determine how early in the life cycle gill bifurcations develop. The data show that the majority of these anomalies begin in juvenile flounder rather than in embryos, larvae, or adults. Gill bifurcations were found in 27% of the 2-year-old flounder, compared to 12% of the I-year-old fish and 15% ofthe adults from the same area.
Water-borne transmission of Dermocystidium salmonis
Abstract:Dermocystidium salmonis is a gill pathogen of salmonid fishes in the U.S. Pacific Northwest where it has been associated with mortality of adult and juvenile chinook salmon Oncorhyn- chus tshawytscha. The previously unknown mode of D. salmonis transmission was determined and demonstrated in the laboratory. Uniflagellated zoospores developed within spores obtained from gill cysts and produced infections in pink salmon O. gorbuscha fry. These infections were lethal, and histological examination of infected gill tissue revealed large numbers of D. salmonis cysts in gill epithelia. Electron microscopic examination of immature spores from experimental infections showed that they were identical to immature spores in naturally infected juvenile chinook salmon.
Monoclonal antibodies against Aeromonas salmonicida lipopolysaccharide identify differences among strains Abstract:Monoclonal antibodies (MAbs) directed against Aeromonas salmonidda salmonidda lipopolysaccharide (LPS) were produced and characterized. The specificity of the MAbs (N = 4) to LPS was determined by examination of western blots of proteinase K-treated whole cell preparations of A+ and A- strains, i.e. strains with and without a major surface protein layer, the A layer. The different MAbs identified different epitopes on A. salmonicida LPS. Three of the 4 MAbs (MAb 1, C, and D) reacted with all but a unique group of A. salmonidda strains tested. These MAbs also had binding properties that were insensitive to periodate oxidation of antigen. The fourth MAb (MAb 6) reacted with a more limited collection of bacterial isolates and the binding was sensitive to periodate treatment of antigen. These data demonstrated that heterogeneity is present in the LPS of this species. Lipopolysac- charide from all tested isolates of A. salmonidda salmonicida (N = 10) and A. salmonidda masoudda (N = 2) reacted with each MAb, while isolates of A. salmonicida achromogenes (N = 6) reacted with MAbs 1, C, and D but not with MAb 6. No patterns of similarity were apparent when unclassified isolates of A. salmonidda were examined.
Putative bacterial and viral infections in blue crabs, Callinectes sapidus, held in a flow-through or a recirculating shedding system Abstract:The blue crab (Callinectes sapidus) industry of Chesapeake Bay uses flow-through and recirculation shedding systems to produce soft-shell crabs. Three replicate experiments were performed in summer of 1986 (June, July, August) to compare mortality and crab disease at four different holding densities in the two systems. Replicate sets of crabs (75-80% intermolt) were held for 22 days: in each system, with dead crabs and those appearing moribund removed daily. Selected tissues were removed from moribund crabs and those surviving to the end of the experiments, and were examined histologically. Early mortalities were attributed to bacterial infections, which were first noted between days 2-3 (recirculation) or days 2-20 (flow-through). In five of six instances, infections that we attributed to viruses were first detected from 2 to 18 days later than bacterial infections. As summer progressed, mortalities decreased and fewer crabs had bacterial or viral infections. No statistically significant differences were detected in mortali- ties or in occurrence of bacterial or viral infections between recirculation or flow-through systems, or among the four different crab densities within each system.
Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters Crassostrea gigas by cytophaga-like bacteria Abstract:Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament res ilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20°C. The morphological, distributional, and degradative character- istics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G+C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.
Histopathological and ultrastructural characteristics of bacterial destruction of the hinge ligaments of cultured juvenile Pacific oysters Crassostrea gigas Abstract:Extensive erosion of external ligament surfaces was associated with the presence of dense and homogeneous populations of very long rod-shaped bacteria within the lesions. The distinctive morphological characteristics of these bacteria suggested their probable affinity with one of the marine genera, Cytophaga, Flexibacter or Flavobacterium. The prevalences of bacterial lesions in both hinge ligament elements and in mantle tissues were estimated for a high-mortality population of juvenile oysters from a commercial hatchery. The ligament resilia of 93 % of the oysters were estimated to contain erosive lesions, while only 20% of the ligament tensilia contained lesions. Bacterial infections in mantle tissues were estimated to occur in 47% of the oyster population. Separate morphometric studies using histological material were conducted to test hypotheses con- cerning both the apparent association between bacterial lesions in ligaments and mantle tissues, and the mechanistic basis of the apparent association. The probability of observing a bacterial lesion in mantle tissues was significantly greater (P < 0.05) for oysters observed to have a hinge ligament lesion than it was for all oysters in the sample. The prevalence of erosive perforation of the hinge ligament (7 % ) was insufficient to account for the prevalence of mantle tissue infections (47% ). These findings support the hypothesis that erosive hinge ligament lesions are associated with mantle tissue lesions and mortalities in affected oysters, but do not support the hypothesis that the etiological basis of mantle tissue infection involves direct bacterial invasion following compromise of the mechanical barrier formed by the hinge ligament.
Cytophaga infection in Atlantic salmon Salmo salar in seawater Abstract:A Cytophaga sp. was associated with skin and muscle lesions of moribund Atlantic salmon Salmo salar smolts shortly after they were introduced to seawater in the late winter and spring of 1985, 1986, and 1987. Concurrent with high mortalities, the lesions were most prevalent 1 wk after introduc- tion. Fish introduced to seawater in summer were less severely affected. The lesions initially appeared as white patches on the flanks in the posterior region, and, as the disease progressed, enlarged and extended deep into the underlying muscle. Fish with the lesions exhibited elevated plasma sodium levels, and a probable cause of mortality was osmotic stress. Wet mounts of the lesions from all epizootics revealed numerous filamentous gliding bacteria. Gliding bacteria were isolated on Marine Agar and on seawater Cytophaga Medium from samples obtained during the 1987 epizootics, and were identified as a Cytophaga sp. based on biochemical, morphological, and G + C ratio data. The organism is a marine bacterium requiring at least 10 % seawater for growth in Cytophaga Medium. In addition to differences in growth characteristics, this isolate was serologically distinct from C. psychrophila, Flexibacter columnaris, and F. maritimus. We have experimentally induced the lesions in Atlantic salmon smolts with a pure culture of the organism, thus supporting the hypothesis that the Cytophaga sp. described here is the etiological agent of the disease. |