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Marine Organisms as Disease Vectors
Project Goal
This project will assess the impact of physiological and immunological changes on the abundance and pathogenicity of specific bacterial pathogens in oysters. Further, in cooperation with the Functional Genomics project, this project will investigate the relationship between genetic profiles, physiological status and the immune status of oysters under laboratory challenges. Learn more about the background information for this project »
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Abstract
In this project researchers are testing whether changes in the coastal marine environment, such as low oxygen (hypoxia), metals, and organic pollutants can increase the risk that oysters, or other marine fish and shellfish, will pass disease-causing microorganisms (pathogens) to humans. Like other filter-feeding bivalves such as clams and mollusks, oysters harbor and concentrate pathogens that occur in their marine habitat. Through direct contact with the oysters and their surrounding water or consumption of oysters humans can be exposed to these pathogens. Previous studies conducted in several laboratories have shown that poor water quality, such as low oxygen levels, low pH, metal and organic pollutants, can impair some of the specific mechanisms that oysters use to kill or inactivate these bacterial pathogens.
At the end of Year Two, MODV personnel have developed and implemented a method for quantifying the ability of oysters to eliminate bacteria that penetrate their tissues. Studies using this method suggest that exposure to low oxygen and less so to cadmium suppresses the ability of oysters to eliminate culturable bacteria from their tissues. This assay has been translated into a field health assessment, showing that the ability of oysters to eliminate invading bacteria varies as a function of season and of tidal creek source. Furthermore, preliminary data suggest that when the ability of an oyster to eliminate bacteria is compromised by environmental or seasonal stressors, oyster tissue burdens of human disease pathogens, such as Enterococci, increases. These data will contribute to the development of a mathematical model that predicts the increased risk of exposure to pathogens when humans consume or come in contact with oysters and the coastal waters they inhabit. These data have been presented at one regional and one national meeting, and abstracts have been submitted for three additional national/international meetings in early 2007.
Short-Term Objectives
Project objectives include:
- Design protocols for physiological⁄immunological assessments of individual oysters in the metals challenge experiments for the Functional Genomics project;
- Assess the load of total bacteria in oyster hemolymph;
- Conduct an in-house workshop to identify PAHs to be tested, and design experiments that will be used to measure host⁄pathogen responses to these compounds;
- Assist the Marine Genomics Core and Functional Genomics project with developing and validating oyster microarrays in field studies;
- Participate in a variety of formal and informal education and outreach activities and assist in the development of an interactive website to communicate the results of our studies to environmental and public health managers and the public at large;
- Assist in the ongoing process of identifying and promoting interactions with potential user groups, including the public health community and shellfish biologists, as well as other national and international scientific endeavors that will enhance the value of the data produced as part of this project.
Expected Results
Evaluate multiple stressors to the physiological and immunological function of oysters.
Accomplishments:
- In collaboration with other members of the OHH Marine Genomics Core and the Environmental Monitoring and Assessment group, research assistant Kolo Rathburn assisted in tidal creek field assessment and collected 25 oysters from each OHH sampling site. Gill and hepatopancreas from each animal at each site was preserved for transcript profiling, to be conducted in 2006-2007.
- In 2004-2005 a prototype assay was developed to monitor the distribution and fate of live bacterial pathogens injected in C. virginica. Oysters are injected with a known amount (usually 10 5) colony-forming units (CFU) of a strain of Vibrio campbellii that has been transfected with a multi-copy plasmid encoding resistance to the antibiotics chloramphenicol and kanamycin. At specified time points after injection of bacteria, the oyster is opened, its tissues homogenized and a measured amount of the homogenate is layered onto microbial culture plates containing drug-selective media. Bacterial colonies that grow on the culture plate after 24 hours are counted as CFU. A change in CFU per gram oyster tissue indicates a change in the ability of the oyster to render bacteria non-culturable, also called the bacteriostatic or killing activity. Those bacteria that cannot be detected by the CFU assay may have been removed from tissues completely (cleared); alternatively those bacteria may be still be present in tissues but unable to grow out on culture media. In order to distinguish these two possibilities, we conduct a second assay on the tissue homogenate of bacteria-injected oysters to quantify the number of bacteria that are still present in tissues, but have not been substantially degraded. This assay for “intact” bacteria is based on real-time PCR quantification of the plasmid that can be retained only by bacteria that have not been substantially degraded by the oyster’s immune system. A change in the number of intact bacteria reflects a change in the ability of oysters to degrade bacteria. Thus, the pool of intact bacteria includes both culturable bacteria CFU as well non-culturable bacteria that are sufficiently intact to retain the plasmid. This prototype assay incorporates measurements of two functional components of the oyster’s immune system: (1) mechanisms that render bacteria non-culturable and (2) mechanisms that degrade bacteria. It is important to distinguish between these two mechanisms, since it is possible that, under favorable conditions, non-culturable bacteria may become culturable pathogens again. In addition, non-culturable bacteria may still have pathogen effects on a host species.
- In 2005-2006 studies using the bacterial challenge assay, as detailed above, provided a striking picture of seasonal and environmental impacts on the ability of oysters to eliminate bacterial pathogens. We currently assess safety of oysters for consumption by CFU or other quantification of targeted bacteria (such as enterococci and fecal coliforms). The data we present below suggests that the abundance and persistence of a pathogen within an oyster varies in a predictable pattern with environmental and seasonal conditions. End-users of these data include the OHH Pathogen Source Tracking Project, as well as public health groups such as DHEC.
Hypoxia as a natural stressor (laboratory study). Postdoctoral research associate Brett Macey showed that oysters held at sub-lethal levels of hypoxia (20% of air saturation) with high levels of CO 2 and low pH (hypercapnic hypoxia, HH) retained higher levels of culturable bacteria in their tissues than did animals maintained in air-saturated water over 60 min after injection of bacteria (Figure 1; 2-way ANOVA, p=0.003; *significant pairwise differences between treatments, Holm-Sidak method).
- High numbers of intact bacteria could still be detected by real-time PCR in both normoxia- and HH-exposed animals throughout the 60 min timecourse. By 60 min after injection, the percentage of intact bacteria that remained culturable (CFU/intact X 100%) was significantly higher in oysters exposed to HH (Figure 2). Taken together these data suggested that exposure to HH impairs mechanisms of bacteriostasis in oyster tissues. This laboratory experiment also demonstrated that the bacterial clearance assay can be used quantify the increased burden of bacterial pathogens in oysters as a function of a natural stressor, such as environmental hypoxia accompanied by naturally co-occurring hypercapnic hypoxia and acidosis. These data will be important in developing quantitative models of human exposure to pathogens in hypoxic environments.
- Metal contaminant effects (laboratory). Master’s degree candidate Heidi Williams exposed oysters to an environmentally-relevant dose of cadmium (50 ppb) at 25 oC in 25ppt seawater for 30 days, then used the bacterial clearance assay to examine bacteriostatic activity of cadmium-exposed as compared to unexposed oysters. There was a significant effect of time and but no significant effect of cadmium-exposure (two way ANOVA), on the ability of oysters to render bacteria non-culturable (Figure 3), although there was a significantly higher number of culturable bacteria in the tissues of cadmium-exposed animals at 30 min after injection, compared to control, unexposed animals. Interestingly, cadmium-exposed animals did have significantly lower total hemocyte counts mL -1 hemolymph than did animals held for 30 days without cadmium exposure (Figure 4; one-way ANOVA; p = 0.038).
- Seasonal effects (field assessment). Brett Macey, Heidi Williams, and Research Assistant Kolo Rathburn recently completed a year-long study to examine seasonal differences in the ability of oysters to render bacteria non-culturable and to degrade these injected bacteria. Oysters were collected from a relatively pristine site, Schooners Creek, SC, every 6 – 8 weeks from November 2005 to late September 2006. After three days in the laboratory, animals were injected with V. campbellii and CFU mL -1 tissue quantified at 60 min after injection of bacteria. Oysters displayed significant differences in bacterial clearance as a function of season and/or water temperature (Figure 5; one-way ANOVA, p<0.001; Holm-Sidak method). Animals collected in January 2006 were much less able to inactivate bacteria than animals collected in other seasons from the same field site.
- Water quality effects (field assessment). Oysters collected from OHH tidal creek sites by the monitoring and assessment group were tested in the bacterial clearance assays by Brett Macey, Heidi Williams, research assistant Kolo Rathburn and summer student Thomas Miller. Oysters collected from Guerin Creek, a forested reference site, retained significantly lower numbers of culturable bacteria at 60 min after injection of bacteria than oysters from any other creek (Figure 6; one-way ANOVA, p<0.001). The density of hemocytes in hemolymph among oysters from the various tidal creeks did not differ significantly (Figure 7; one-way ANOVA, p=0.991). Notably, average total hemocyte count (THC) mL -1 hemolymph of oysters from tested tidal creeks was negatively correlated with the average number of culturable Vibrio campbellii g -1 tissue recovered at 60 min after injection of 10 5 bacteria per animal. The observed negative correlation (Figure 8; linear regression, r 2 = -0.831) is statistically significant (p<0.001). There was a strong positive correlation (r=0.740; p=0.02) between Enterococci contamination of whole oyster tissues (but not water concentrations of Enterococci) and average CFU V. campbellii g−1 tissue recovered at 60 min after injection of bacteria (Figure 9). In other words, high pathogen burden in oyster tissues correlated with reduced ability the oyster to eliminate pathogens.
- Neither THC mL -1 hemolymph nor CFU g -1 oyster tissue were significantly correlated with pathogen loads in the surrounding water (Pathogen Source Tracking Project). Correlations with other environmental assessments, including water and oyster tissue pathogens will be assessed as data become available.
- Distribution of invasive bacterial pathogens in oyster tissues. In 2004-2005 CofC graduate student Heidi Williams developed a prototype assay to monitor tissue distribution and fate of live bacterial pathogens in C. virginia. In 2005-2006, she used this assay used to show that oyster tissues play distinct roles in the elimination of invading pathogens. While injected bacteria are distributed to and rapidly eliminated from all tissues of the oyster (data not shown), the mantle retains a larger percentage of the total culturable bacteria that can be recovered from all tissues at 60 and 120 min after injection (Figure 9). The results of these experiments will be critical to understanding the processes that alter the persistence of bacterial pathogens in oysters and other marine species harvested for human consumption. End-users of these data include the OHH Pathogen Source Tracking Project, as well as public health groups such as DHEC.
Transfer of Results
As project research nears specific endpoints, the group will conduct at least one planning⁄evaluation meeting with representatives of the public health community and other identified end user groups to improving study designs and enhance the value of the data obtained from this study, as well as determine avenues for information transfer.
Publications/Presentations:
- Macey, B.M., Burnett, L.E., Burnett, K.G. 2006. Effects of hypercapnic hypoxia on the clearance of Vibrio campbellii in the Eastern oyster, Crassostrea virginica. 31st Annual Eastern Fish Health Workshop. Charleston, SC. March 2006.
- Macey, B.M., Burnett, L.E. Burnett, K.G. 2006. Effects of hypercapnic hypoxia on the clearance of Vibrio campbellii in the Eastern oyster, Crassostrea virginica. Tenth Congress of the International Society of Developmental and Comparative Immunology. Charleston, SC. July 2006.
Public Information and Outreach:
For More Information
Contact: Karen Burnett, (843) 762.8933
Email: BurnettK@cofc.edu