Modeling the importance of fish condition, overall health, and disease on the fecundity of White Perch in the Choptank River.

OBJECTIVE: Modeling of fecundity with allometric, nutritional, and environmental covariates has increased sensitivity of reproductive metrics in many fish species. In estuaries with heavy anthropogenic influence, resident species often experience sublethal health impacts because of increased stress, which can include increases in gonadal pathology, intersex, or potential reproductive failure. This study models the fecundity of the estuarine species White Perch Morone americana in response to health parameters identified as signals of habitat stress, including gross pathology presentation, nutritional condition, and disease presence. METHODS: Subpopulation fecundity in the Choptank River (Maryland) of the Chesapeake Bay was estimated using stereological fecundity sampling methods and modeled using information-theoretic approaches of model selection. Nutritional and health parameters identified through health assessment techniques, specific somatic indices, and disease presence were selected as covariates. RESULT: Nutrition demonstrated limited influence on model fit as compared to models with only conventional allometric variables such as weight and length. Of the health variables, gross pathology and somatic indices showed minimal influence on selection, but mycobacterial infection, a chronic condition in the Chesapeake Bay among temperate basses, showed measurable influence. Models with mycobacteriosis included were 40 times more likely the best fit when compared to models with only allometric parameters. CONCLUSION: Whether this has a region-wide influence on all subpopulations will require further research and sampling of the magnitude of mycobacteriosis infection.

Growth Rates of Vibrio parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium.

The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Vibrio parahaemolyticus in the Chesapeake Bay: Operational In Situ Prediction and Forecast Models Can Benefit from Inclusion of Lagged Water Quality Measurements.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis. Given its natural presence in brackish waters, there is a need to develop operational forecast models that can sufficiently predict the bacterium's spatial and temporal variation. This work attempted to develop V. parahaemolyticus prediction models using frequently measured time-indexed and -lagged water quality measures. Models were built using a large data set (n = 1,043) of surface water samples from 2007 to 2010 previously analyzed for V. parahaemolyticus in the Chesapeake Bay. Water quality variables were classified as time indexed, 1-month lag, and 2-month lag. Tobit regression models were used to account for V. parahaemolyticus measures below the limit of quantification and to simultaneously estimate the presence and abundance of the bacterium. Models were evaluated using cross-validation and metrics that quantify prediction bias and uncertainty. Presence classification models containing only one type of water quality parameter (e.g., temperature) performed poorly, while models with additional water quality parameters (i.e., salinity, clarity, and dissolved oxygen) performed well. Lagged variable models performed similarly to time-indexed models, and lagged variables occasionally contained a predictive power that was independent of or superior to that of time-indexed variables. Abundance estimation models were less effective, primarily due to a restricted number of samples with abundances above the limit of quantification. These findings indicate that an operational in situ prediction model is attainable but will require a variety of water quality measurements and that lagged measurements will be particularly useful for forecasting. Future work will expand variable selection for prediction models and extend the spatial-temporal extent of predictions by using geostatistical interpolation techniques.IMPORTANCEVibrio parahaemolyticus is one of the leading causes of seafood-borne illness in the United States and across the globe. Exposure often occurs from the consumption of raw shellfish. Despite public health concerns, there have been only sporadic efforts to develop environmental prediction and forecast models for the bacterium preharvest. This analysis used commonly sampled water quality measurements of temperature, salinity, dissolved oxygen, and clarity to develop models for V. parahaemolyticus in surface water. Predictors also included measurements taken months before water was tested for the bacterium. Results revealed that the use of multiple water quality measurements is necessary for satisfactory prediction performance, challenging current efforts to manage the risk of infection based upon water temperature alone. The results also highlight the potential advantage of including historical water quality measurements. This analysis shows promise and lays the groundwork for future operational prediction and forecast models.

Environmental Determinants of Vibrio parahaemolyticus in the Chesapeake Bay.

Vibrio parahaemolyticus naturally occurs in brackish and marine waters and is one of the leading causes of seafood-borne illness. Previous work studying the ecology of V. parahaemolyticus has often been limited in geographic extent and lacked a full range of environmental measures. This study used a unique large data set of surface water samples in the Chesapeake Bay (n = 1,385) collected from 148 monitoring stations from 2007 to 2010. Water was analyzed for more than 20 environmental parameters, with additional meteorological and surrounding land use data. The V. parahaemolyticus-specific genetic markers thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) were assayed using quantitative PCR (qPCR), and interval-censored regression models with nonlinear effects were estimated to account for limits of detection and quantitation. tlh was detected in 19.6% of water samples; tdh or trh markers were not detected. The results confirmed previously reported positive associations for V. parahaemolyticus abundance with temperature and turbidity and negative associations with high salinity (>10 to 23‰). Furthermore, the salinity relationship was determined to be a function of both low temperature and turbidity, with an increase of either nullifying the high salinity effect. Associations with dissolved oxygen and phosphate also appeared stronger when samples were taken near human developments. A renewed focus on the V. parahaemolyticus ecological paradigm is warranted to protect public health.IMPORTANCE Vibrio parahaemolyticus is one of the leading causes of seafood-borne illness in the United States and across the globe. Exposure is often through consuming raw or undercooked shellfish. Given the natural presence of the bacterium in the marine environment, an improved understanding of its environmental determinants is necessary for future preventative measures. This analysis of environmental Vibrio parahaemolyticus is one of only a few that utilize a large data set measured over a wide geographic and temporal range. The analysis also includes a large number of environmental parameters for Vibrio modeling, many of which have previously only been tested sporadically, and some of which have not been considered before. The results of the analysis revealed previously unknown relationships between salinity, turbidity, and temperature that provide significant insight into the abundance and persistence of V. parahaemolyticus bacterium in the environment. This information will be essential for developing environmental forecast models for the bacterium.

Recreational swimmers' exposure to Vibrio vulnificus and Vibrio parahaemolyticus in the Chesapeake Bay, Maryland, USA.

Vibrio vulnificus and Vibrio parahaemolyticus are ubiquitous in the marine-estuarine environment, but the magnitude of human non-ingestion exposure to these waterborne pathogens is largely unknown. We evaluated the magnitude of dermal exposure to V. vulnificus and V. parahaemolyticus among swimmers recreating in Vibrio-populated waters by conducting swim studies at four swimming locations in the Chesapeake Bay in 2009 and 2011. Volunteers (n=31) swam for set time periods, and surface water (n=25) and handwash (n=250) samples were collected. Samples were analyzed for Vibrio concentrations using quantitative PCR. Linear and logistic regressions were used to evaluate factors associated with recreational exposures. Mean surface water V. vulnificus and V. parahaemolyticus concentrations were 1128CFUmL(-1) (95% confidence interval (CI): 665.6, 1591.4) and 18CFUmL(-1) (95% CI: 9.8, 26.1), respectively, across all sampling locations. Mean Vibrio concentrations in handwash samples (V. vulnificus, 180CFUcm(-2) (95% CI: 136.6, 222.5); V. parahaemolyticus, 3CFUcm(-2) (95% CI: 2.4, 3.7)) were significantly associated with Vibrio concentrations in surface water (V. vulnificus, p<0.01; V. parahaemolyticus, p<0.01), but not with salinity or temperature (V. vulnificus, p=0.52, p=0.17; V. parahaemolyticus, p=0.82, p=0.06). Handwashing reduced V. vulnificus and V. parahaemolyticus on subjects' hands by approximately one log (93.9%, 89.4%, respectively). It can be concluded that when Chesapeake Bay surface waters are characterized by elevated concentrations of Vibrio, swimmers and individuals working in those waters could experience significant dermal exposures to V. vulnificus and V. parahaemolyticus, increasing their risk of infection.

Impact of Hurricane Irene on Vibrio vulnificus and Vibrio parahaemolyticus concentrations in surface water, sediment, and cultured oysters in the Chesapeake Bay, MD, USA.

To determine if a storm event (i.e., high winds, large volumes of precipitation) could alter concentrations of Vibrio vulnificus and V. parahaemolyticus in aquacultured oysters (Crassostrea virginica) and associated surface water and sediment, this study followed a sampling timeline before and after Hurricane Irene impacted the Chesapeake Bay estuary in late August 2011. Aquacultured oysters were sampled from two levels in the water column: surface (0.3 m) and near-bottom (just above the sediment). Concentrations of each Vibrio spp. and associated virulence genes were measured in oysters with a combination of real-time PCR and most probable number (MPN) enrichment methods, and in sediment and surface water with real-time PCR. While concentration shifts of each Vibrio species were apparent post-storm, statistical tests indicated no significant change in concentration for either Vibrio species by location (surface or near bottom oysters) or date sampled (oyster tissue, surface water, and sediment concentrations). V. vulnificus in oyster tissue was correlated with total suspended solids (r = 0.41, P = 0.04), and V. vulnificus in sediment was correlated with secchi depth (r = -0.93, P <0.01), salinity (r = -0.46, P = 0.02), tidal height (r = -0.45, P = 0.03), and surface water V. vulnificus (r = 0.98, P <0.01). V. parahaemolyticus in oyster tissue did not correlate with environmental measurements, but V. parahaemolyticus in sediment and surface water correlated with several measurements including secchi depth [r = -0.48, P = 0.02 (sediment); r = -0.97, P <0.01 (surface water)] and tidal height [r = -0.96, P <0.01 (sediment), r = -0.59, P <0.01 (surface water)]. The concentrations of Vibrio spp. were higher in oysters relative to other studies (average V. vulnificus 4 × 10(5) MPN g(-1), V. parahaemolyticus 1 × 10(5) MPN g(-1)), and virulence-associated genes were detected in most oyster samples. This study provides a first estimate of storm-related Vibrio density changes in oyster tissues, sediment, and surface water at an aquaculture facility in the Chesapeake Bay.

Antimicrobial susceptibility of Vibrio vulnificus and Vibrio parahaemolyticus recovered from recreational and commercial areas of Chesapeake Bay and Maryland Coastal Bays.

Vibrio vulnificus and V. parahaemolyticus in the estuarine-marine environment are of human health significance and may be increasing in pathogenicity and abundance. Vibrio illness originating from dermal contact with Vibrio laden waters or through ingestion of seafood originating from such waters can cause deleterious health effects, particularly if the strains involved are resistant to clinically important antibiotics. The purpose of this study was to evaluate antimicrobial susceptibility among these pathogens. Surface-water samples were collected from three sites of recreational and commercial importance from July to September 2009. Samples were plated onto species-specific media and resulting V. vulnificus and V. parahaemolyticus strains were confirmed using polymerase chain reaction assays and tested for antimicrobial susceptibility using the Sensititre® microbroth dilution system. Descriptive statistics, Friedman two-way Analysis of Variance (ANOVA) and Kruskal-Wallis one-way ANOVA were used to analyze the data. Vibrio vulnificus (n = 120) and V. parahaemolyticus (n = 77) were isolated from all sampling sites. Most isolates were susceptible to antibiotics recommended for treating Vibrio infections, although the majority of isolates expressed intermediate resistance to chloramphenicol (78% of V. vulnificus, 96% of V. parahaemolyticus). Vibrio parahaemolyticus also demonstrated resistance to penicillin (68%). Sampling location or month did not significantly impact V. parahaemolyticus resistance patterns, but V. vulnificus isolates from St. Martin's River had lower overall intermediate resistance than that of the other two sampling sites during the month of July (p = 0.0166). Antibiotics recommended to treat adult Vibrio infections were effective in suppressing bacterial growth, while some antibiotics recommended for pediatric treatment were not effective against some of the recovered isolates. To our knowledge, these are the first antimicrobial susceptibility data of V. vulnificus and V. parahaemolyticus recovered from the Chesapeake Bay. These data can serve as a baseline against which future studies can be compared to evaluate whether susceptibilities change over time.

Short-term infection of striped bass Morone saxatilis with Mycobacterium marinum.

Striped bass Morone saxatilis were studied in order to characterize their immune responses over the short term following challenge with Mycobacterium marinum. The expression of immunity-related genes (IL-1beta, TNF-alpha, Nramp and TGF-beta) quickly increased following infection with M. marinum, but these genes were subsequently down-regulated despite the fact that bacterial counts remained high. The number of monocytes and neutrophils also initially increased at 1 d postinfection. This confirms the importance of these types of cells in initial inflammation and mycobacterial infection in striped bass. The phagocytic index of splenic leukocytes over these same time frames did not change significantly following infection. The discrete window in which inflammatory mechanisms were stimulated in striped bass may be related to the intracellular nature of this pathogen.

Historical presence (1975-1985) of mycobacteriosis in Chesapeake Bay striped bass Morone saxatilis.

A retrospective analysis of archived tissue blocks has revealed that mycobacteriosis was apparent in Chesapeake Bay striped bass as early as 1984. Of 37 cases available from the years 1975 to 1985, 2 fish were found positive based on histopathology and genus-specific PCR. Multi-gene sequencing places the bacteria from the 2 positive cases (1984 and 1985) within the Mycobacterium tuberculosis clade with closest resemblance to the recently described fish pathogen M. pseudoshottsii. Our data confirms that mycobacteriosis is not a new disease of Chesapeake Bay striped bass and underscores the value of archived tissues in epidemiological examinations.

Influence of nutritional state on the progression and severity of mycobacteriosis in striped bass Morone saxatilis.

Challenge studies with Mycobacterium marinum clearly demonstrate that a poor diet affects the progression and severity of mycobacteriosis in striped bass Morone saxatilis. Fish (n = 512 total, wt = 65 +/- 15 g) were inoculated intraperitoneally with 10(4) colony-forming units (CFU) g(-1) body weigth (BW) or a physiological saline solution (controls) and evaluated for 8 mo. Inoculated fish fed a low-ration diet (0.15% BW d(-1)) developed a severe, systemic infection characterized by a high bacterial load (>10(8) CFU g(-1) spleen) and poor granuloma formation, which commonly progressed to mortality by 6 wk. In contrast, inoculated fish fed an adequate ration diet (1% BW d(-1)) developed classic granulomatous inflammation of reduced severity and total body energy similar to that found in uninoculated controls (p > 0.05). After 4 wk, fish fed adequate rations maintained an equilibrium state throughout the study period, even though 10(6) CFU g(-1) spleen mycobacteria were consistently cultured. In a second study, reactivation of an acute inflammatory state was demonstrated by placing previously infected fish on reducing diets (0.073% BW d(-1)). In both studies, the energetic demand of this disease was only appreciable when associated with active, severe, inflammatory states. To our knowledge, this study is the first to demonstrate the interaction of diet and mycobacteriosis in fish.

Expanded range and new host species of Mycobacterium shottsii and M. pseudoshottsii.

Mycobacterium shottsii and M. pseudoshottsii are recently described mycobacteria commonly isolated from Chesapeake Bay striped bass Morone saxatilis. However, their distribution in striped bass outside of the Chesapeake region and their ability to infect alternative hosts have not been described. Mycobacteria identified as M. shottsii (based on fatty acid methyl ester analysis and multigene sequencing) were isolated from striped bass collected in Albemarle Sound, North Carolina, and white perch Morone americana in the Rhode River, Maryland, and detected in striped bass from the New York Bight off Long Island, New York. Mycobacterium pseudoshottsii were isolated from white perch in the Rhode and Corsica rivers, Maryland, and detected in striped bass in the New York Bight. This work demonstrates that these mycobacteria can be found outside of the Chesapeake Bay as well as in hosts other than striped bass.