Assessment of Vibrio parahaemolyticus levels in oysters (Crassostrea virginica) and seawater in Delaware Bay in relation to environmental conditions and the prevalence of molecular markers to identify pathogenic Vibrio parahaemolyticus strains.

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

Assessment of chemical, biological and immunological properties of "Damiana de California" Turnera diffusa Willd extracts in Longfin yellowtail (Seriola rivoliana) leukocytes.

In Mexican herbal medicines or natural remedies, Turnera diffusa (Turneraceae) known as "Damiana de California", has ethnopharmacological relevance, including aphrodisiac, diuretic, and antimicrobial activities. To explore the immunological effect of infusion and methanolic extracts from Damiana de California, this study investigated its chemical, biological, antimicrobial and immunological properties in Longfin yellowtail Seriola rivoliana leukocytes. The analysis of chemical compounds revealed a considerable level of total phenolic and flavonoid contents in the infusion compared with methanolic extract. Furthermore, the antioxidant activity showed high hydroxyl radical scavenging activity in infusion extract compared with BHT positive control. Superoxide radical scavenging activity and ion chelation were higher in methanolic extract followed by infusion treatment. Interestingly, notable antimicrobial activity was observed in both extracts of T. diffusa against Vibrio parahaemolyticus. An in vitro study was performed using leukocytes of S. rivoliana treated with infusion or methanolic extracts at 12.5, 25 and 50 µg/mL for 24 h. Remarkably, infusion extract induced proliferation at any concentration but not the methanolic extract, which was diminished in a dose-dependent fashion. The immunostimulation study demonstrated that the phagocytosis activity increased in those leukocytes stimulated with methanolic extract but diminished the respiratory burst activity, in contrast to the activity observed in those leukocytes stimulated with infusion treatment. Finally, leukocytes incubated with the extracts and confronted with V.parahaemolyticus up-regulated the transcription of proinflammatory cytokine IL-1ß gene in a dose response relationship. These findings suggest that the infusion treatment has potential therapeutic properties, promoting the antioxidant capacity and enhancing immune parameters in Longfin yellowtail S. rivoliana.

Vibrio parahaemolyticus risk assessment in the Pacific Northwest: it's not what's in the water.

The Gram-negative bacterium Vibrio parahaemolyticus (Vp) is a major cause of illness associated with the consumption of raw or undercooked seafood, primarily oysters. This species is a natural member of the bacterial community in brackish waters and is bioaccumulated by oysters through filter feeding. Only a subset of strains is thought to be pathogenic. Currently known virulence markers include the gene for the thermostable direct hemolysin (tdh). In this work we analyzed water and oysters for total Vp and strains encoding tdh from 26 oyster-growing areas of the Puget Sound and Pacific coast of Washington state in 2007 and 2008. In addition, possible plankton-associated Vp were assessed from net tow samples. The density of both total and tdh+ Vp in the water column were considerably higher in 2008 than 2007. However, the concentrations of both total and tdh+ Vp in the oyster tissue was similar for both years. A high proportion of Vp strains in the water column was found to be tdh+ in both 2007 and 2008; however, tdh+ strains were detected at much lower levels in oysters. The data show that analysis of Vp density in the oysters is a better risk assessment tool than density in the overlying water column.

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Estimating the burden of acute gastroenteritis and foodborne illness caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus by using population-based telephone survey data, Miyagi Prefecture, Japan, 2005 to 2006.

Most cases of acute gastroenteritis and foodborne disease are not ascertained by public health surveillance because the ill person does not always seek medical care and submit a stool sample for testing, and the laboratory does not always test for or identify the causative organism. We estimated the total burden of acute gastroenteritis in Miyagi Prefecture, Japan, using data from two 2-week cross-sectional, population-based telephone surveys conducted in 2006 and 2007. To estimate the number of acute gastroenteritis illnesses caused by Campylobacter, Salmonella, and Vibrio parahaemolyticus in Miyagi Prefecture, we determined the number of cases for each pathogen from active laboratory-based surveillance during 2005 to 2006 and adjusted for seeking of medical care and submission of stool specimens by using data from the population-based telephone surveys. Monte Carlo simulation was used to incorporate uncertainty. The prevalence of acute gastroenteritis in the preceding 4 weeks was 3.3% (70 of 2,126) and 3.5% (74 of 2,121) in the winter and summer months, yielding an estimated 44,200 episodes of acute gastroenteritis each year in this region. Among people with acute gastroenteritis, the physician consultation rate was 32.0%, and 10.9% of persons who sought care submitted a stool sample. The estimated numbers of Campylobacter-, Salmonella-, and V. parahaemolyticus -associated episodes of acute gastroenteritis were 1,512, 209, and 100 per 100,000 population per year, respectively, in this region. These estimates are significantly higher than the number of reported cases in surveillance in this region. Cases ascertained from active surveillance were also underrepresented in the present passive surveillance, suggesting that complementary surveillance systems, such as laboratory-based active surveillance in sentinel sites, are needed to monitor food safety in Japan.

Quantitative risk assessment of Vibrio parahaemolyticus in finfish: a model of raw horse mackerel consumption in Japan.

The aim of this study was to evaluate the effects of implemented control measures to reduce illness induced by Vibrio parahaemolyticus (V. parahaemolyticus) in horse mackerel (Trachurus japonicus), seafood that is commonly consumed raw in Japan. On the basis of currently available experimental and survey data, we constructed a quantitative risk model of V. parahaemolyticus in horse mackerel from harvest to consumption. In particular, the following factors were evaluated: bacterial growth at all stages, effects of washing the fish body and storage water, and bacterial transfer from the fish surface, gills, and intestine to fillets during preparation. New parameters of the beta-Poisson dose-response model were determined from all human feeding trials, some of which have been used for risk assessment by the U.S. Food and Drug Administration (USFDA). The probability of illness caused by V. parahaemolyticus was estimated using both the USFDA dose-response parameters and our parameters for each selected pathway of scenario alternatives: washing whole fish at landing, storage in contaminated water, high temperature during transportation, and washing fish during preparation. The last scenario (washing fish during preparation) was the most effective for reducing the risk of illness by about a factor of 10 compared to no washing at this stage. Risk of illness increased by 50% by exposure to increased temperature during transportation, according to our assumptions of duration and temperature. The other two scenarios did not significantly affect risk. The choice of dose-response parameters was not critical for evaluation of control measures.

Quantitative modeling for risk assessment of Vibrio parahaemolyticus in bloody clams in southern Thailand.

A risk assessment of Vibrio parahaemolyticus in bloody clams (Anadara granosa) consumed in southern Thailand was conducted. This study estimated the prevalence and concentration of pathogenic V. parahaemolyticus in bloody clams at harvest and retail stages; and during this process, methods to detect the total and pathogenic V. parahaemolyticus were investigated. Consumption of bloody clams and cooking efficiency were studied using interviews and on-site observation of consumers. A beta-Poisson dose-response model was used to estimate probability of illness applying estimation methods for the most likely parameter values presented by USFDA. Microbial and behavioral data were analyzed by developing a stochastic model and the simulation gave a mean number of times a person would get ill with V. parahaemolyticus by consuming bloody clams at 5.6 x 10(-4)/person/year. Sensitivity analysis demonstrated the fraction of people who did not boil the clams properly was the primary factor in increasing risk. This study serves as an example of how a microbiological risk assessment with limited data collection and international cooperation leads to valuable local insight.

Avaliação quantitativa do risco de doença, causada por Vibrio arahaemolyticus, associado ao consumo de ostras (Crassostrea brasiliana) cruas cultivadas e comercializadas no Estado de São Paulo / Quantitative risk assessment of illness, caused by Vibrio parahaemolyticus, associated with the consumption of raw oysters (Crassostrea brasiliana) farmed and commercialized in the State of São Paulo

Vibrio parahaemolyticus (Vp) é uma bactéria naturalmente presente em regiões estuarinas, sendo a principal causa de gastrenterite de origem bacteriana associada a pescados, principalmente ostras cruas. Nesta pesquisa, foi desenvolvida uma avaliação quantitativa de risco para avaliar a probabilidade de Vp causar doença após o consumo de ostra crua, produzida e comercializada no Estado de São Paulo. O estudo incluiu a identificação e caracterização do perigo, a avaliação da exposição e a caracterização do risco. Um modelo matemático foi desenvolvido. Este modelo leva em consideração o comportamento de Vp em ostras na cadeia produtiva, em cada estação do ano, além da relação entre a dose de Vp ingerida e a probabilidade de desenvolver a doença. A avaliação da exposição foi desenvolvida em três etapas: cultivo, pós-coleta e consumo. Na etapa de cultivo foram considerados os fatores que influenciam a prevalência e o número de Vp em ostras no momento da coleta. Na etapa pós-coleta, foram descritas as práticas da indústria e foram considerados os fatores associados ao processamento, transporte e manipulação. Já na etapa de consumo foram considerados os fatores como a quantidade de ostras consumidas por porção, o peso médio por ostra consumida e a população de Vp patogênico no momento do consumo. O resultado do modelo quantitativo da avaliação da exposição foi, então, integrado ao modelo dose-resposta, Beta-Poisson, para se obter uma estimativa do risco. Esta estimativa expressa o impacto da exposição humana a Vp, sobre a saúde pública, associada ao consumo de ostras. A simulação de Monte Carlo foi utilizada para avaliar o efeito da variabilidade e incerteza das variáveis do modelo sobre a estimativa do risco. O modelo prediz uma probabilidade de ocorrência de doença de 4,6x10-4, por porção de ostra, consumida ao longo do ano. As variáveis que possuem maior influência sobre o risco de ocorrência de doença são a população de Vp em ostras no cultivo, a temperatura de transporte das ostras até o varejo e a porcentagem de Vp patogênico em ostra, no momento do seu consumo. O modelo evidencia que uma das maneiras de reduzir o risco de ocorrência de doença seria intervir nas condições de transporte de ostras até o varejo por meio da sua refrigeração. Com o modelo é possível identificar fatores e simular cenários para avaliar o comportamento de V. parahaemolytícus como um perigo microbiológico, ao longo da cadeia produtiva de ostra até o momento do seu consumo. Também é possível avaliar o impacto de medidas de intervenção na cadeia produtiva. As suposições adotadas limitam a aplicabilidade do modelo. Portanto, é necessário que o modelo seja validado, particularmente com relação ao número de casos de doença causados por Vp, cujos dados de vigilância epidemiológica inexistem no Brasil

Vibrio parahaemolyticus (Vp) is naturally present in estuarine regions and is the main cause of gastroenteritis associated with the consumption of bivalve molluscan shellfish, specially raw oysters. In this research, a quantitative risk assessment was developed to evaluate the probability of Vp causing disease after consumption of raw oyster, produced and commercialized in the state of Sao Paulo. The study included the identification and characterization of the hazard, exposure assessment and risk characterization. A mathematical model was developed. This model takes into account the behavior of Vp in oysters in the productive chain, for each season of the year, besides the relationship between the number of cells of Vp ingested and the probability of developing the disease. The exposure assessment was done in three steps: farming, after harvesting and consumption. At the farming step, the factors that influence the prevalence and the population of Vp at the time of harvesting were considered. At the after harvesting step, the factors associated with transportation, handling and processing were considered. At the consumption step, factors related to the amount of oysters and the average weight per oyster consumed and the density of pathogenic Vp at the time of consumption were considered. Then, the quantitative model of exposure assessment was integrated to the dose-response model, BetaPoisson, in order to obtain a risk estimate. This calculation expresses the impact of the human exposure to Vp associated with the consumption of oysters on public health. The Monte Carlo simulation was used to evaluate the effect of variability and uncertainty of variables of the model in the risk estimation. The model predicts a probability of occurrence of the disease of 4,6x10-4 per serving of oyster consumed during one year. The variables showing the greatest influence on the risk of occurrence of disease are the density of Vp in oyster in the farming step, the temperature during transportation of oysters to the retail market and the percentage of pathogenic Vp strains in oysters,' at the moment of consumption. The model indicates that the use of refrigeration during transportation of oysters to retail could reduce the risk of disease. The model allows the identification of factors and the simulation of scenarios in order to evaluate the behavior of V. parahaemolyticus, as a microbiologícal hazard, in the productive chain of oyster to the consumption. It is also possible to evaluate the impact of intervention measures in the productive chain. The assumptions Iimit the application of the model. Therefore, it is necessary to validate the model, particularly in relation to the number of cases of dísease caused by V. parahaemolyticus of which the data on epidemiologic surveillance do not exist in Brazil

Comparison of sensitivity analysis methods based on applications to a food safety risk assessment model.

Sensitivity analysis (SA) methods are a valuable tool for identifying critical control points (CCPs), which is one of the important steps in the hazard analysis and CCP approach that is used to ensure safe food. There are many SA methods used across various disciplines. Furthermore, food safety process risk models pose challenges because they often are highly nonlinear, contain thresholds, and have discrete inputs. Therefore, it is useful to compare and evaluate SA methods based upon applications to an example food safety risk model. Ten SA methods were applied to a draft Vibrio parahaemolyticus (Vp) risk assessment model developed by the Food and Drug Administration. The model was modified so that all inputs were independent. Rankings of key inputs from different methods were compared. Inputs such as water temperature, number of oysters per meal, and the distributional assumption for the unrefrigerated time were the most important inputs, whereas time on water, fraction of pathogenic Vp, and the distributional assumption for the weight of oysters were the least important inputs. Most of the methods gave a similar ranking of key inputs even though the methods differed in terms of being graphical, mathematical, or statistical, accounting for individual effects or joint effect of inputs, and being model dependent or model independent. A key recommendation is that methods be further compared by application on different and more complex food safety models. Model independent methods, such as ANOVA, mutual information index, and scatter plots, are expected to be more robust than others evaluated.