High Salinity Relaying to Reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay Oysters (Crassostrea virginica)

Cases of Vibrio infections in the United States have tripled from 1996 to 2009 and these infections are most often associated with the consumption of seafood, particularly oysters (Crassostrea virginica). Information is needed on how to reduce numbers of Vibrio parahaemolyticus and Vibrio vulnificus in bi‐valve molluscan shellfish (for example, oysters). The purpose of this study was to evaluate the effectiveness of high salinity relaying or treatment in recirculating aquaculture systems (RASs) as methods to reduce the abundance of V. parahaemolyticus and V. vulnificus in oysters. For relaying field trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 h, and then transferred to high (29 to 33 ppt.) and moderate (12 to 19 ppt.) salinities. For RAS treatment trial, oysters were transferred to 32 to 34 ppt. salinity at 15 °C. After 7, 14, 21, and in some instances 28 d, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real‐time PCR. Initial levels of V. parahaemolyticus and V. vulnificus ranged from 3.70 to 5.64 log₁₀ MPN/g, and were reduced by 2 to 5 logs after 21 to 28 d in high salinity water (29 to 34 ppt.). Oyster mortalities averaged 4% or less, and did not exceed 7%. Relaying of oysters to high salinity field sites or transfer to high salinity RAS tanks was more effective in reducing V. vulnificus compared with V. parahaemolyticus. These results suggest that high salinity relaying of oysters is more effective in reducing V. vulnificus than V. parahaemolyticus in the oyster species used in this study.     

低温贮藏条件下创伤弧菌和副溶血性弧菌失活模型的建立

为掌握对虾中创伤弧菌和副溶血性弧菌在5℃和一18~C低温贮藏条件下失活动力学特征,分别采用线性模型、Weibull模型、Logistic模型对创伤弧菌和副溶血性弧菌的失活曲线进行拟合。研究结果表明,在5~C条件下,创伤弧茵VvHB09的耐冷力较强;．18℃条件下,副溶血性弧菌ATCC17802和创伤弧菌VvSH09的耐冷力较强。线性模型比weibull模型、Logistic模型更适合拟合创伤弧菌和副溶血性弧菌的失活特征。     

Occurrence of Vibrio parahaemolyticus in Two Oregon Oyster-growing Bays

ABSTRACT: Occurrence of Vibrio parahaemolyticus in 2 Oregon oyster-growing areas (Yaquina andTillamook Bays) was studied from November 2002 to October 2003. Vibrio parahaemolyticus was detected in 15.0% of oyster, 20.0% of seawater, and 47.5% of sediment samples with very low levels of pathogenic strains being detected in oysters (≤3.6 most probable number [MPN] /g). The densities of total and pathogenic V. parahaemolyticus were higher in sediment (≤1100 and ≤43 MPN/g) than in seawater (≤15 and ≤3.6 MPN/100 mL) or oyster (≤43 and ≤3.6 MPN/ g). Densities of V. parahaemolyticus in both bays were positively correlated to water temperatures ( P < 0.01), with higher densities in samples being detected in summer, especially July and August. There was no correlation between the densities of V. parahaemolyticus and water salinity or the densities of V. parahaemolyticus and bacterial populations in seawater. Freshly harvested oysters should be kept at refrigeration temperatures to prevent rapid growth of pathogenic V. parahaemolyticus in contaminated oysters.     

同时检测海产品中副溶血弧菌和霍乱弧菌的合检方法评价研究

目的建立水产品中副溶血弧菌和霍乱弧菌合检方法,并对合检方法对比进行评价。方法利用副溶血弧菌和霍乱弧菌阳性菌株,制备纯菌液、不同浓度梯度的人工污染样品。通过对30份纯菌液、30份人工污染样品和250份实际样品的检测,将建立的合检方法与行业标准(SN/T 1022-2010进出口食品中霍乱弧菌检验方法和SN/T 0173-2010进出口食品中副溶血性弧菌检验方法)进行比较,对合检方法进行效果评价。结果实验结果表明副溶血弧菌和霍乱弧菌合检方法,与行业标准检测结果完全一致。结论该方法可靠,对实验仪器和操作人员的要求低,具有良好的实用性,适合基础检测实验室。     

Effectiveness of depuration of Pacific Oyster (Crassostrea gigas): removal of bioaccumulated Vibrio vulnificus by UV-treatment

Food Science and Biotechnology - The present study aimed to evaluate the efficacy of a depuration system equipped with UV-irradiation to control Vibrio vulnificus infection such as septicemia (or...     

Effects of High Hydrostatic Pressure on the Physical,Microbial, and Chemical Attributes of Oysters (Crassostrea virginica)

The change in the quality attributes (physical, microbial, and chemical) of oysters (Crassostrea virginica) after high hydrostatic pressure (HHP) treatment at 300 MPa at room temperature (RT, 25 °C) 300, 450, and 500 MPa at 0 °C for 2 min and control oysters without treatment were evaluated over 3 wk. The texture and tissue yield percentages of oysters HHP treated at 300 MPa, RT increased significantly (P < 0.05) compared to control. Aerobic and psychrotrophic bacteria in control oysters reached the spoilage point of 7 log CFU/g after 15 d. Coliform counts (log MPN/g) were low during storage with total and fecal coliforms less than 3.5 and 1.0. High pressure treated oysters at 500 MPa at 0 °C were significantly higher (P < 0.05) than oysters HHP treated at 300 MPa at 0 °C in lipid oxidation values. The highest pressure (500 MPa) treatment in this study, significantly (P < 0.05) decreased unsaturated fatty acid percentage compared to control. The glycogen content of control oysters at 3 wk was significantly higher (P < 0.05) when compared to HHP treated oysters [300 MPa, (RT); 450 MPa (0 °C); and 500 MPa (0 °C)]. HHP treatments of oysters were not significantly different in pH, percent salt extractable protein (SEP), and total lipid values compared to control. Based on our results, HHP prolongs the physical, microbial, and chemical quality of oysters.     

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae,V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872–1:2007 and TS 21872–2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.     

A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnificus

A microbiological survey was conducted to determine the levels of total and pathogenic Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) in Pacific oysters (Crassostrea gigas) collected from commercial growing areas in the North Island, New Zealand.The survey was intended to be geographically representative of commercial growing areas of Pacific oysters in New Zealand, while selecting the time frame most likely to coincide with the increased abundance of pathogenic vibrio species. Vp was detected in 94.8% of oyster samples examined (n = 58) with a geometric mean concentration of 99.3 MPN/g, while Vv was detected in 17.2% of oyster samples examined with a geometric mean concentration of 7.4 MPN/g. The frequency of Vp positive samples was 1.7 fold greater than reported in a study conducted three decades ago in New Zealand. Potentially virulent (tdh positive) Vp was detected in two samples (3.4%, n = 58) while no trh (another virulence marker) positive samples were detected. 16 S rRNA genotype could be assigned only to 58.8% of Vv isolates (8:1:1 A:B:AB ratio, n = 10). There was a good agreement [98.2% of Vp (n = 280) and 94.4% of Vv (n = 18) isolates] between molecular tests and cultivation based techniques used to identify Vibrio isolates and there was a significant (R² = 0.95, P < 0.001, n = 18) linear relationship between the MPN estimates by real-time PCR and cultivation. There was no significant correlation between any of the environmental parameters tested and Vp or Vv concentrations.     

Reductions of Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas) by depuration at various temperatures

Consumption of raw oysters has been linked to several outbreaks of Vibrio parahaemolyticus infection in the United States. This study investigated effects of ice storage and UV-sterilized seawater depuration at various temperatures on reducing V. parahaemolyticus in oysters. Raw Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10290, 10292, 10293, BE 98-2029 and 027-1c1) at levels of 10⁴⁻⁶ MPN/g. Inoculated oysters were either stored in ice or depurated in recirculating artificial seawater at 2, 3, 7, 10, 12.5, and 15 °C for 4–6 days. Holding oysters in ice or depuration of oysters in recirculating seawater at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration at temperatures between 7 and 15 °C reduced V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days with no loss of oysters. Depuration at refrigerated temperatures (7–15 °C) can be applied as a post-harvest treatment for reducing V. parahaemolyticus in Pacific oysters.     

姜汁对太平洋牡蛎（Crassostrea gigas）酶解液风味改善的研究

通过感官评价、顶空固相微萃取与气相色谱-质谱联用技术（solid phase micro-extraction gas chromatographmassspectrometer,SPME-GC/MS）以及计算相对气味活度值（ralative odour active value,ROAV）的方法分析太平洋牡蛎（Crassostrea gigas）酶解前后挥发性成分的变化情况,探讨姜汁对牡蛎脂质氧化的抑制作用和改善牡蛎酶解产物风味的效果。结果表明：与新鲜牡蛎相比,酶解液中脂质氧化产物,如异戊醛、壬醛、2-壬烯醛和3,5-辛二烯酮含量分别增加至5.09%、1.6%、1.3%、0.5%,使牡蛎酶解液呈现出较重的腥味与哈喇味,说明牡蛎风味的劣变与酶解过程中发生的脂质氧化有关。牡蛎酶解过程中添加姜汁,脂肪氧化产生的不愉快成分如（E）-2-戊烯醛、（Z）-4-庚烯醛、壬醛、2,3-辛二酮等未被检测到;异戊醛、己醛、（E）-2-己烯醛等物质的相对含量分别减少了81.02%、33.28%、54.39%。姜汁中挥发性成分如香叶醇、香茅醇、对伞花烃和芳樟醇等成为其新的主体呈味成分,并贡献出花香、柠檬香等愉快气味。因此,姜汁对改善牡蛎酶解液的风味有显著效果。     

Depuration of Oysters (Crassostrea gigas) Contaminated with Vibrio parahaemolyticus and Vibrio vulnificus with UV Light and Chlorinated Seawater

The efficacy of depuration using UV light and chlorinated seawater for decontaminating Vibrio parahaemolyticus and Vibrio vulnificus from oysters was investigated. Oysters were contaminated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10(4) to 10(5) CFU ml(-1) for bioaccumulation. The depuration was conducted in a closed system in which 350 liters of seawater was recirculated at a rate of 7 liters/min for 48 h at room temperature. Counts of V. parahaemolyticus or V. vulnificus were determined at 0, 6, 18, 24, and 48 h. Three treatments were conducted: T1, control treatment; T2, UV treatment; and T3, UV plus chlorine treatment. After 48 h of depuration of V. parahaemolyticus, T3 reduced the count by 3.1 log most probable number (MPN) g(-1) and T2 reduced the count by 2.4 log MPN g(-1), while T1 reduced the count by only 2.0 log MPN g(-1). After 48 h of depuration of V. vulnificus, T2 and T3 were efficient, reducing the counts by 2.5 and 2.4 log MPN g(-1), respectively, while T1 reduced the count by only 1.4 log MPN g(-1). The UV light plus chlorine treatment was more efficient for controlling V. parahaemolyticus in oysters. Both UV light and UV light plus chlorine were efficient for V. vulnificus. The present study is the first report showing the efficacy of depuration systems for decontaminating V. parahaemolyticus and V. vulnificus in oysters cultivated on the Brazilian coast. This study provides information on processes that can contribute to controlling and preventing such microorganisms in oysters and could be used for effective postharvest treatment by restaurants and small producers of oysters on the coast of Brazil.     

Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas): accumulation from water and survival during cold storage and thermal process

This study investigated accumulation of Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas) exposed to contaminated water and survival of V. parahaemolyticus in the clams during cold storage and heating processes. Vibrio parahaemolyticus could be accumulated in clams to a level similar to that of contaminated water within 12 h of exposure of clams to contaminated water at temperatures between 9 and 33 °C. Keeping clams stored at 5 and 0 °C for 10 days resulted in 1.98 and 2.32 log MPN g^?1 reductions of V. parahaemolyticus, respectively, in the clams. Frozen storage at ?18 °C for 15 days or at ?30 °C for 30 days were capable of reducing V. parahaemolyticus from 4.05 log MPN g^?1 to non‐detectable levels (< 3 MPN g^?1). A heating process in hot water at 80 °C or higher for 1 min also reduced V. parahaemolyticus in the clams to non‐detectable levels.     

食品中副溶血性弧菌的危害分析、检测与预防控制

副溶血性弧菌是一种广泛分布于近岸海水、海底沉积物和海产品中的嗜盐性细菌，也是引起我国特别是沿海地区细菌性食物中毒危害的首要食源性致病菌。为了提高人们对副溶血性弧菌的重视程度，降低食源性疾病的风险，本文就副溶血性弧菌的生物特性、毒力因子、污染途径、食物中毒特点、检测方法以及预防措施作简要综述。     

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX^® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+ V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+ V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX^® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX^® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+ V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+ V. parahaemolyticus.     

Effectiveness of icing as a postharvest treatment for control of Vibrio vulnificus and Vibrio parahaemolyticus in the eastern oyster (Crassostrea virginica)

The focus of this research was to investigate the efficacy of icing as a postharvest treatment for reduction of the levels of Vibrio vulnificus and Vibrio parahaemolyticus in commercial quantities of shellstock oysters. The experiments were conducted in June and August of 2006 and consisted of the following treatments: (i) on-board icing immediately after harvest; (ii) dockside icing approximately 1 to 2 h prior to shipment; and (iii) no icing (control). Changes in the levels of pathogenic Vibrio spp. during wholesale and retail handling for 2 weeks postharvest were also monitored. On-board icing achieved temperature reductions in all sacks in accordance with the National Shellfish Sanitation Program standard, but dockside icing did not meet this standard. Based on one-way analysis of variance, the only statistically significant relationship between Vibrio levels and treatment occurred for samples harvested in August; in this case, the levels of V. vulnificus in the noniced oysters were significantly higher (P < 0.05) than were the levels in the samples iced on-board. When analyzing counts over the 14-day storage period, using factorial analysis, there were statistically significant differences in V. vulnificus and V. parahaemolyticus levels by sample date and/or treatment (P < 0.05), but these relationships were not consistent. Treated (iced) oysters had significantly higher gaping (approximately 20%) after 1 week in cold storage than did noniced oysters (approximately 10%) and gaping increased significantly by day 14 of commercial storage. On-board and dockside icing did not predictably reduce the levels of V. vulnificus or V. parahaemolyticus in oysters, and icing negatively impacted oyster survival during subsequent cold storage.     

环介导等温扩增技术检测创伤弧菌方法的建立与应用

根据创伤弧菌vvhA基因设计4条特异性引物,采用环介导等温扩增方法(LAMP)建立创伤弧菌快速检测方法。结果表明,建立的LAMP方法具有高度特异性,经对13株细菌进行扩增,创伤弧菌为LAMP阳性,其他菌株均为LAMP阴性。该方法对培养的创伤弧菌的检测下限为51CFU/mL,可在60min内完成扩增反应。用建立的LAMP方法对120份海产品进行检测,共检出37份LAMP阳性样本,与传统培养方法结果一致。该方法与传统方法相比,可节省大量时间,且对实验仪器和操作人员的要求低,具有良好的实用性。     

波动温度模拟蟹肉副溶血性弧菌生长模型

研究模拟蟹肉在4℃~28℃范围内副溶血性弧菌的生长动力学曲线,采用Belehradek方程描述温度对副溶血性弧菌的生长动力学影响。通过在设计的波动温度下模拟蟹肉中副溶血性弧菌的生长动态模型而得到预测值,并与在此波动温度下副溶血性弧菌的实测值进行比较,本文同时采用了偏差因子和准确因子来验证模型对波动温度的预测适用性。     

风险分级矩阵在贝类海产品中副溶血性弧菌风险评估的应用研究

目的 研究风险分级矩阵方法学应用以及我国沿海地区居民贝类海产品中副溶血性弧菌污染的健康风险等级.方法 利用贝类海产品消费量以及副溶血性弧菌污染等数据,计算危害严重性(5分制)和疾病发生可能性(5分制)参数,导入风险分级模型矩阵,对我国沿海地区不同人群副溶血性弧菌健康风险进行赋值和等级评价.结果 我国沿海地区全人群和贝类...     

水产品中副溶血性弧菌PMA-ddPCR活菌定量检测方法的研究

本研究旨在针对水产品中活的副溶血性弧菌建立一种快速定量的PCR检测方法。基于叠氮溴化丙锭(PMA)在一定光照条件下能抑制死亡菌DNA扩增,以及微滴式数字PCR技术能将检测精度扩展至单分子目标基因,并实现绝对定量的特点,以副溶血性弧菌tlh基因为目的片段设计及筛选适合的特异性引物与探针,优化反应体系,通过对PMA浓度及曝光条件等优化,建立了一种联用PMA-dd PCR技术快速检测水产品中活的副溶血性弧菌的定量检测方法。研究结果显示:选择16μg/m L作为PMA工作浓度,曝光时间为8 min,此条件下能够完全抑制副溶血性弧菌死菌的DNA扩增并对活菌扩增无影响。通过对比PMA-dd PCR和PMA-q PCR的检测低限分别为2×10~1 cfu/mL、2×10~2 cfu/mL,PMA-dd PCR法的灵敏度比PMA-q PCR法的高。应用PMA-dd PCR定量方法检测人工污染的基围虾和小帆立贝这两种海产品,在基围虾中最低可检出1.9×10~1 cfu/g的副溶血性弧菌、在小帆立贝中最低可检出8.9 cfu/g的副溶血性弧菌。该研究为将PCR技术实际应用于水产品中低量污染、活的副溶血性弧菌的定量检测奠定了基础。     

Optimization of resuscitation-promoting broths for the revival of Vibrio parahaemolyticus from a viable but nonculturable state

Food Science and Biotechnology - This study was conducted to examine the effect of formulated resuscitation-promoting broths on the revival of viable but nonculturable Vibrio parahaemolyticus...