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Abstracts
Workgroup: Frontiers in Disease Research
Comparative examination of biochemical correlates of disease resistance in selectively bred Crassostrea virginica and Crassostrea ariakensis
Principal Investigator(s):
Dr. Gustavo Calvo, Virginia Institute of Marine Science, calvo@vims.edu
Co-Investigator(s):
Dr. Stephen L. Kaattari, Virginia Institute of Marine Science
Funding Period: 01/01/00-12/31/01
Objective Ia. Assess Perkinsus marinus resistance in putatively resistant and susceptible stocks of oysters. Results: The final cumulative mortality of the selected groups of Crassostrea virginica oysters was Delaware bay (DB) < Crossbreed (XB) = Mobjack bay (MB) < Rappahannock River (RR). Concurrent MSX infection accounted for some of the early losses in the RR and MB groups. The RR oysters died within the first year of the experiment, most likely due to low resistance to MSX. The P. marinus body burden of the XB oysters was higher than in the DB and MB oysters; however, this was likely due to their more rapid attainment of market size and, thus, their higher rate of filtration. XB oysters reached Virginia market size within the first year of the experiment, and well before the onset of second year mortalities. In laboratory challenge of susceptible and resistant C. virginica and C. ariakensis oysters, P. marinus infections were found to be lower in the XB and DB groups than in the RR or C. ariakensis groups, as assessed by total parasite body burden. This is in contradiction to previous findings that the prevalence and intensity of P. marinus infections in C. ariakensis deployed in the field were lower than that found in C. virginica. The unexpectedly elevated levels of acute P. marinus infections in the C. ariakensis oysters may have been an artifactual result of mantle cavity challenge with large numbers of P. marinus cells. This result may have implications as the mechanism by which oysters of differential susceptibility are able to handle and clear the steady stream of infectious cells encountered during normal feeding in contrast to the large pulses of infective cells administered during artificial infection.
Objective Ib. Assess the potential correlation between endogenous oyster plasma protease and low molecular weight protease inhibitor activities and P. marinus resistance. Results: The assessment of the correlation of protease and protease inhibitor activity was not possible due to the presence of parasite in all field samples, and the inability to extract adequate hemolymph from the small oysters held in quarantine.
Objective Ic. Assess the potential correlation between inducible oyster plasma protease and protease inhibitor activities and P. marinus resistance. Results: The oyster plasma total protein content in all oyster groups underwent a seasonal variation, with progressively lower plasma protein content as the summer progressed. The initial decrease in protein concentration coincided with the occurrence of MSX disease, and the magnitude of the decline concentration corresponded with the degree of strain susceptibility. There was no apparent correlation between the level of oyster plasma inhibitory activity against P. marinus proteases and the parasite body burden. The protease inhibitory activity was similar between all groups at all time points. This contradicts previous research (Oliver et al, 2000), but this difference may be due to optimization of the hide powder azure technique to render it more sensitive and decrease the variability between replicates. In laboratory challenges of susceptible and resistant C. virginica as well as C. ariakensis, there were no differences in the level of protease inhibitory activity between oysters; however, there was an apparent upregulation in the protease inhibitor activity in all oyster groups following experimental infection with P. marinus. While the ubiquity of this response precludes its use as a screening tool for potentially disease resistant oysters, it does open a potential avenue in the study of the oyster innate immune repertoire.
Objective IIa: Assess P. marinus resistance in putatively resistant and susceptible stocks of oysters by assessment of inhibition of plasma degradation by P. marinus protease mediated by low molecular weight constituents of resistant oyster plasma. Results: This experiment was expanded into a large matrix, where the susceptibility of all plasma from all oyster groups to degradation by P. marinus proteases was monitored in the presence or absence of the low molecular weight fraction of all oyster groups. Plasma proteins from all oysters groups were susceptible to degradation by P. marinus proteases, as determined by the appearance of numerous protein degradation fragments and disappearance of some parent bands. This degradation was not eliminated by the low molecular weight fraction of any of the oyster strains, indicating that the ability to prevent proteolytic degradation of plasma proteins may not be crucial to the progression or final outcome of P. marinus infection.
Objective IIb. Determine if resistant oyster plasma is capable of digesting P. marinus ECP. Results: Coincubation of oyster plasma with ECP did not result in degradation of the ECP proteins. Conversely, substantial degradation of hemolymph proteins occurred. In order to track the degradation of the oyster proteins, the proteins were labeled with biotin molecules prior to co-incubation with P. marinus ECP. This allowed an enzyme-coupled streptavidin molecule to be used as a probe, rather than a polyclonal antiserum. This had the advantage of ensuring that all proteins were tracked, regardless of their immunogenicity, and that the degradation products of those proteins could also be tracked, even if they were no longer recognized by the antiserum. The experiment demonstrated that the degradation of certain plasma proteins coincided with the appearance of apparent degradation products, some of which were of a similar molecular weight between three different oyster species. It was noted in a separate experiment that the pattern of plasma proteins visible by SDS-PAGE is relatively simple in young, uninfected oysters prior to co-incubation with P. marinus ECP. Comparison of protein patterns either by one or two dimensional electrophoresis or by mass spectroscopy may provide an early indicator of infection by tracking the products of exposure to specific P. marinus proteases.
Objective IIc: Determine if co-incubation of Dermo resistant Crassostrea plasma with P. marinus cells inhibits their replication or viability. Results: Rather than the simple retardation of growth anticipated, oyster tissue extracts and hemolymph actually induce the production of in vivo type forms of the parasite (e.g. tomonts, prezoosporangia) not commonly seen in in vitro culture. This demonstrates that the inclusion of oyster extracts into the cell culture can be used to study the mechanisms of activation and differentiation of P. marinus cells. Furthermore, the inclusion of these supplements caused an upregulation of a set of low molecular weight proteases to be secreted by P. marinus into the cell culture supernatant. This upregulation did not occur in the presence of homogenates of P. marinus-tolerant oyster species. It was reasoned that this "reversion" to a form more closely resembling the in vivo state would be associated with an increase in virulence of the P. marinus cell line.
Objective IId. Determine the in vivo capacity of purified proteases or inhibitors to retard or protect against P. marinus infection. Results: Inhibition of proteolytic activity did not occur in the oyster samples, as demonstrated in objective IIa; therefore, it was considered more appropriate not to simply co-inject P. marinus cells with protease or potential protease inhibitors, but rather to determine if supplements with homogenates or plasma would induce a more virulent cell. Thus we used P. marinus cells cultured in the presence or absence of C. virginica hemolymph or tissue homogenate to experimentally infect oysters. The body burden at four weeks demonstrated a significant increase in the infection level in the oysters administered P. marinus cells grown in the presence of oyster products. These results have led to a new grant which aims to determine the mechanisms of regulation of P. marinus proteases and other extracellular products by exposure to oyster products, and the potential association of upregulation of these products with enhanced parasite virulence.
IMPACTS and/or BENEFITS:
The results of this grant have enhanced the understanding of the potential mechanisms of P. marinus differentiation and virulence in both susceptible and tolerant hosts. The availability of media supplements that enhance differentiation and alter the expression of potential protease and other virulence factors has created the ability to use subtractive techniques of immunization and hybridization to attempt isolation and characterization. Furthermore, it has enabled the search for the molecular cues present in susceptible oysters that are responsible for this phenotypic change.
PROJECT PUBLICATIONS:
Earnhart, C. and Kaattari, S. Optimization of Protease Inhibitor Assays for Eastern Oyster (Crassostrea virginica) Immune Assessments. Presentation to the Eastern Fish Health Workshop, 2000.
MacIntyre, E. and Kaattari, S. Altered Perkinsus protease profiles upon exposure to selected oyster tissue homogenates. Presentation to the Annual Meeting of the National Shellfisheries Association, 2001, Orlando, FL.
Kaattari, S., MacIntyre, E. and Earnhart, C. Modulation of Perkinsus marinus functions by host-derived products. Presentation to the Annual Meeting of the National Shellfisheries Association, 2002, Mystic, CT.
MacIntyre, E., Earnhart, C. and Kaattari, S. In press. Host oyster tissue extracts modulate in vitro protease expression and cellular differentiation in the protozoan parasite, Perkinsus marinus. Parasitology
Calvo, G., Earnhart, C. and Kaattari, S. Disease resistance and potential biochemical correlates in a selectively bred oyster strain. Presentation to the Annual Meeting of the National Shellfisheries Association, 2001, Orlando, FL.
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