Assessment of human enteric viruses in shellfish from the northern Adriatic sea

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.     

Round-robin comparison of methods for the detection of human enteric viruses in lettuce

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calicivirus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety.     

Coliphage as pressure surrogates for enteric viruses in foods

In this study the potential of using selected bacteriophages as pressure surrogates for hepatitis A virus (HAV) and Aichi virus (AiV) was investigated. The coliphages included, T₄, MS2, Qβ, λ imm 434, λ cI 857 and λ cI 857A. T₄ displayed similar pressure responses as HAV and was chosen for further study. The most pressure-resistant phage, MS2, was selected as a possible surrogate to estimate AiV inactivation by high pressure processing (HPP). HAV, AiV and their selected bacteriophage surrogates were treated at a range of pressures and times in three different media. All four were treated in phosphate-buffered saline (PBS), artificial seawater (ASW) or oyster slurry (OS) at 250, 400 or 500 MPa for 1, 5 or 10 min at 20 °C. While T₄ had similar pressure resistance to HAV under conditions of high (500 MPa) and lower pressure (250 MPa), inactivation trends were very different following treatment at 400 MPa and when the viruses were suspended in OS. MS2 showed similar resistance as AiV but at ambient treatment temperatures only. The highest levels of inactivation of MS2 were achieved at 60 °C and 500 MPa. AiV was eliminated at 60 °C for 5 min at ambient pressure, but > 3 log survived exposure to 60 °C at 500 MPa. This degree of protection by pressure may be important in determining the mechanisms of pressure and heat resistances in other viruses.Industrial relevanceGreater knowledge of the responses of viruses and their surrogates to high pressure will aid in the validation of new high pressure-processed food that may be at risk to contamination from HAV or other enteric viruses.     

Tracing enteric viruses in the European berry fruit supply chain

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses.     

Inactivation of internalized and surface contaminated enteric viruses in green onions

With increasing outbreaks of gastroenteritis associated with produce, it is important to assess interventions to reduce the risk of illness. UV, ozone and high pressure are non-thermal processing technologies that have potential to inactivate human pathogens on produce and allow the retention of fresh-like organoleptic properties. The objective of this study was to determine if UV, ozone, and high pressure are effective technologies compared to traditional chlorine spray on green onions to reduce enteric viral pathogens and to determine the effect of location of the virus (surface or internalized) on the efficacy of these processes. Mature green onion plants were inoculated with murine norovirus (MNV), hepatitis A virus (HAV) and human adenovirus type 41 (Ad41) either on the surface through spot inoculation or through inoculating contaminated hydroponic solution allowing for uptake of the virus into the internal tissues. Inoculated green onions were treated with UV (240 mJ s/cm²), ozone (6.25 ppm for 10 min), pressure (500 MPa, for 5 min at 20 °C), or sprayed with calcium hypochlorite (150 ppm, 4 °C). Viral inactivation was determined by comparing treated and untreated inoculated plants using cell culture infectivity assays. Processing treatments were observed to greatly affect viral inactivation. Viral inactivation for all three viruses was greatest after pressure treatment and the lowest inactivation was observed after chlorine and UV treatment. Both surface inoculated viruses and viruses internalized in green onions were inactivated to some extent by these post-harvest processing treatments. These results suggest that ozone and high pressure processes aimed to reduce the level of microbial contamination of produce have the ability to inactivate viruses if they become localized in the interior portions of produce.     

Prevalence of human pathogenic enteric viruses in bivalve molluscan shellfish and cultured shrimp in south west coast of India

The prevalence of human enteric viruses in bivalve molluscan shellfish and shrimp collected off the south west coast of India was studied to assess the extent of fecal pollution of coastal environment. Out of 194 samples analyzed, 37% of oyster, 46% of clam and 15% of shrimp samples were positive for enteroviruses (EV). Adenoviruses (ADV) were detected in 17% of oyster and 27% of clam samples. However, other enteric viruses such as noroviruses (NoV) and hepatitis A virus (HAV) were not detected in any of the samples. High prevalence of EV and ADV was noticed between May to December. Thirty four percent of oyster and 49% of clam samples showed fecal coliform values higher than the limit. MS-2 phage was detected in 57% of oyster and 73% of clam samples. The presence of MS-2 phage and human enteric viruses showed association while fecal coliforms and enteric viruses showed no association. However, 17 samples, which were positive for enteric viruses (EV and ADV), were negative for MS-2 phage.     

A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables

Fresh-cut vegetables are prone to be contaminated with foodborne pathogens during growth, harvest, transport and further processing and handling. As most of these products are generally eaten raw or mildly treated, there is an increase in the number of outbreaks caused by viruses and bacteria associated with fresh vegetables. Foodborne pathogens are usually present at very low levels and have to be concentrated (i.e. viruses) or enriched (i.e. bacteria) to enhance their detection. With this aim, a rapid concentration method has been developed for the simultaneous recovery of hepatitis A virus (HAV), norovirus (NV), murine norovirus (MNV) as a surrogate for NV, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enterica. Initial experiments focused on evaluating the elution conditions suitable for virus release from vegetables. Finally, elution with buffered peptone water (BPW), using a Pulsifier, and concentration by polyethylene glycol (PEG) precipitation were the methods selected for the elution and concentration of both, enteric viruses and bacteria, from three different types of fresh-cut vegetables by quantitative PCR (qPCR) using specific primers. The average recoveries from inoculated parsley, spinach and salad, were ca. 9.2%, 43.5%, and 20.7% for NV, MNV, and HAV, respectively. Detection limits were 132 RT-PCR units (PCRU), 1.5 50% tissue culture infectious dose (TCID₅₀), and 6.6 TCID₅₀ for NV, MNV, and HAV, respectively. This protocol resulted in average recoveries of 57.4%, 64.5% and 64.6% in three vegetables for E. coli O157:H7, L. monocytogenes and Salmonella with corresponding detection limits of 10³, 10² and 10³ CFU/g, respectively.Based on these results, it can be concluded that the procedure herein is suitable to recover, detect and quantify enteric viruses and foodborne pathogenic bacteria within 5 h and can be applied for the simultaneous detection of both types of foodborne pathogens in fresh-cut vegetables.     

蛤类滚筒式分级工艺参数优化

对蛤类滚筒式分级工艺参数进行研究。采用单因素试验和正交试验研究处理量、网板孔径增量、滚筒转速和滚筒直径对分级准确率的影响。结果表明:网板孔径增量是影响分级准确率的主要因素,其次是滚筒直径,再次是处理量,影响最小的是滚筒转速。最佳工艺参数为处理量6t/h,网板孔径增量2mm,滚筒转速12r/min,滚筒直径600mm。     

Diarrhetic shellfish poisoning toxin esters in Danish blue mussels and surf clams

Until recently, little focus was given to the presence of diarrhetic shellfish poisoning (DSP) toxin esters in seafood products. However, during the last few years, the occurrence of a high percentage of esters of the total amount of DSP toxins present in some seafood products has been observed. Samples of Danish surf clams (Spisola spp.) and blue mussels (Mytilus edulis) from 1999-2004 were analysed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the presence of DSP toxin esters. The samples contained only okadaic acid and esters of okadaic acid. The level of total okadaic acid equivalents ranged from 224 to 2516 µg kg^-1 in surf clams. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 83 to 98%, mean 95%. The level of total okadaic acid equivalents ranged from 43 to 1631 µg kg^-1 in blue mussels. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 21 to 86%, mean 59%. The probability of a high percentage of okadaic acid esters seems to increase with higher amounts of total okadaic acid equivalents in the bivalves. The large prevalence of DSP toxin esters are of particular importance because of the increased use of chemical methods instead of mouse bioassay for the detection of DSP toxicity.     

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 onsite 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 × 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.     

The effect of cranberry juice and cranberry proanthocyanidins on the infectivity of human enteric viral surrogates

The effect of cranberry juice (CJ) and cranberry proanthocyanidins (PAC) on the infectivity of human enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), MS2(ssRNA) bacteriophage, and phiX-174(ssDNA) bacteriophage was studied. Viruses at high (∼7 log₁₀ PFU/ml) or low (∼5 log₁₀ PFU/ml) titers were mixed with equal volumes of CJ, 0.30, 0.60, and 1.20 mg/ml final PAC concentration, or water and incubated for 1 h at room temperature. Viral infectivity after treatments was evaluated using standardized plaque assays. At low viral titers, FCV-F9 was undetectable after exposure to CJ or the three tested PAC solutions. MNV-1 was reduced by 2.06 log₁₀ PFU/ml with CJ, and 2.63, 2.75, and 2.95 log₁₀ PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. MS2 titers were reduced by 1.14 log₁₀ PFU/ml with CJ, and 0.55, 0.80, and 0.96 log₁₀ PFU/ml with 0.15, 0.30, and 0.60 mg/ml PAC, respectively. ?-X174 titers were reduced by 1.79 log₁₀ PFU/ml with CJ, and 1.95, 3.67, and 4.98 log₁₀ PFU/ml with PAC at 0.15, 0.30, and 0.60 mg/ml, respectively. Experiments using high titers showed similar trends but with decreased effects. CJ and PAC show promise as natural anti-virals that could potentially be exploited for foodborne viral illness treatment and prevention.     

Preservation and shelf-life extension of shrimps and clams by high hydrostatic pressure

Clam ( Venus gallina ) and shrimp ( Parapenaeus longirostris ) samples were high hydrostatic pressure (HHP) treated at 200, 220 and 250 MPa at 25, 30, 40 and 50 °C for 10 and 20 min. Based on the results of microbial reduction, the best combinations of HHP treatments were determined as 250 MPa, 50 °C, 10 min for shrimps and 220 MPa, 50 °C, 10 min for clams. HHP-treated samples stored at 25 °C (room tempertur) and 4 °C (refrigeration temperature) were analysed. According to the results evaluated, shelf-life of shrimps was found to be 12 and 16 days for storage at room and refrigeration temperatures, respectively, as compared with 4 days for non-HHP-treated samples at 4 °C. Similarly shelf-life for the clam samples was found to be 12 days for storage at room temperature and 18 days for storage at refrigeration temperature as compared with 4 days for non-HHP-treated samples at 4 °C.