Prevalence of enterovirus and hepatitis A virus in bivalve molluscs from Galicia (NW Spain): inadequacy of the EU standards of microbiological quality

A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.     

Cryptosporidium contamination in harvesting areas of bivalve molluscs

Cryptosporidium contamination was evaluated in areas in Galicia (northwestern Spain) where bivalve molluscs are harvested. Galicia is the main mussel-producing region in Europe. Data were collected on water contamination of effluents that are discharged into these areas. Cryptosporidium spp. were detected by immunofluorescence microscopy and molecular methods in 71% of the river water samples (n = 7), 64% of raw sewage samples (n = 11), 50% of effluents from wastewater treatment plants (n = 16), and 29.3% of the mussel samples (Mytilus galloprovincialis, n = 184). Cryptosporidium parvum was identified in all samples of contaminated mussels, Cryptosporidium muris was found in three samples of effluent from wastewater treatment plants, and Cryptosporidium baileyi was found in a sample of raw sewage. Further studies are needed to determine the parasitological quality of water in these shellfish harvesting and recreational areas. Cryptosporidium could be a public health risk from consumption of raw or undercooked contaminated molluscs and use of contaminated waters for recreational purposes.     

Occurrence of toxigenic Clostridium difficile in edible bivalve molluscs

Clostridium difficile is an anaerobic bacterium commonly considered to be responsible for antibiotic-associated gastrointestinal diseases, ranging from diarrhea of varying severity to pseudomembranous colitis. The aim of this study was to assess the occurrence of C. difficile in marine edible bivalve molluscs, which, as filter feeding organisms, are able to accumulate particles suspended in water, including microorganisms. Samples of Mytilus galloprovincialis, Tapes philippinarum, and Venus verrucosa were collected from mussel farms and fishmongers in the province of Naples (Southern Italy). C. difficile was found in 49% of the 53 samples investigated. Sixteen isolates were grouped in 12 known different PCR ribotypes (001, 002, 003, 010, 012, 014/020, 018, 045, 070, 078, 106, and 126), whereas 10 additional isolates were grouped in 8 new PCR riboprofiles. Two toxinotypes (0 and V) were found. Fifty eight percent of the isolates were toxigenic. These findings indicate that toxigenic C. difficile strains can be isolated in bivalve molluscs. Marine filter feeding organisms, therefore, may be considered as reservoir of toxigenic strains of C. difficile. The ingestion of raw or poorly cooked contaminated seafood and the high temperature resistance of the spore-forming C. difficile could represent an important source of exposure and pose human health concern.     

Consumption of bivalve molluscs in Italy: estimated intake of cadmium and lead.

Concentrations of cadmium and lead were determined in nine different species of mollusc bivalves (Modiolus barbatus, Venus verrucosa, Scapharca inaequivalvis, Tapes decussatus, Callista chione, Pecten jacobeus, Ensis siliqua, Venus gallina, Cardium tubercolata) collected from different coastal areas of the Adriatic Sea (north, middle and south Adriatic). The levels of cadmium and lead found in bivalves from the north Adriatic Sea were significantly (P < 0.001) higher than those detected in those from the middle and south Adriatic Sea. In some species of molluscan bivalves (M. barbatus and T. decussatus) from the north Adriatic Sea, concentrations of cadmium and lead exceeded the maximum limit (2 mg/kg w.w.) established by the Italian legislation. Weekly intakes were estimated and compared with the Provisional Tolerable Weekly Intake (PTWI) recommended by the FAO/WHO Expert Committee on Food Additives.     

Detection of norovirus RNA in bivalve molluscs by using bacteria-culture-employed method (A3T method)

Norovirus (NV) RNA has rarely been detected in foods despite the use of highly sensitive methods such as RT-PCR and real-time RT-PCR. In the modified method (A3T method) reported previously, a bacterial culture process was introduced into the standard protocol for NV detection to remove some inhibitor(s) present in food ingredients. To confirm the efficiency of the A3T method and to examine NV contamination in bivalve molluscs, we tried to detect NV RNA in bivalve molluscs on the market and in oyster samples associated with foodborne outbreaks by using the standard method and the A3T method. NV RNAs were detected in 20 samples (18.0%) of 111 bivalve molluscs, including oysters, on the market by use of the A3T method, while only one sample (0.9%) was positive according to the standard method. NV RNA was also detected in 10 of 35 oyster samples related to foodborne outbreaks by the A3T method. Those results show that the A3T method is suitable for the detection of NV in bivalve molluscs in general laboratories.     

High throughput analysis of amnesic shellfish poisoning toxins in bivalve molluscs by dispersive solid-phase extraction and high-performance liquid chromatography using a monolithic column

Domoic acid and its isomers are algal neurotoxins responsible for the human intoxication syndrome known as amnesic shellfish poisoning. A rapid and simple HPLC–UV/DAD method based on the use of a monolithic silica column is proposed for the determination of ASP toxins in shellfish samples. To the best of our knowledge, a monolithic column is applied for the first time to the analysis of marine toxins.     

超高效液相色谱-串联质谱法测定双壳贝类中9种腹泻性贝类毒素

目的建立超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法,测定双壳贝类样品中9种腹泻性贝类毒素(DSP)。方法样品经100%甲醇提取过滤后,经ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)色谱柱分离,乙腈-0.01%氨水为流动相,流速为0.2 ml/min,柱温为30℃,采用电喷雾电离和正负离子分段扫描,在多反应监测(MRM)模式下测定游离态的毒素(原多甲藻酸1、原多甲藻酸2、原多甲藻酸3、大环内酯类扇贝毒素2、软海绵酸、鳍藻毒素1、鳍藻毒素2、虾夷扇贝毒素和类虾夷扇贝毒素)。当软海绵酸、鳍藻毒素1或鳍藻毒素2任一种或多种毒素呈阳性时,须采用碱水解将酯化的软海绵酸、鳍藻毒素1和鳍藻毒素2转化为游离态形式后,用UPLCMS/MS测定其水解态毒素含量,外标法定量。结果 9种DSP在3个添加水平的平均回收率为85.9%~95.3%,相对标准偏差(RSD)为2.76%~4.18%,检出限(LOD)为4.4~9.9μg/kg,定量限(LOQ)为14.7~33.0μg/kg。结论该方法操作简便、灵敏度高,适用于DSP的日常检测。     

Detection of hepatitis A virus in oysters

A digoxigenin-labelled RNA probe with a sensitivity of 800 50% tissue culture infectious dose (TCID₅₀) was used to detect Hepatitis A Virus (HAV) in oysters. We studied the influence of extraction methodology on riboprobe detection. Oyster samples obtained by four methods of extraction and extraction-concentration were spiked with HAV (CF53 strain). There was no correlation between protein concentration and turbidity of samples, and anti-digoxigenin antibodies showed a non specific reaction. Background noise was independent of protein concentration and disappeared when HAV RNA isolation by phenol/chloroform extraction was introduced, but HAV RNA could not be detected by this technique. In the presence of Acid Guanidinium Thiocyanate (AGT), RNA from HAV suspension was detected following phenolic extraction with a detection threshold of 8.10⁴ TCID₅₀ of spotted virus. HAV detection in oyster extract by a digoxigenin-labelled riboprobe appeared useful in shellfish virology, at least for a primary screening of samples.     

Identification of chemical detected in kiwi fruit during analysis for residual pesticide

An unknown peak was detected in a GC chromatogram of many kiwi fruit extracts during analysis for pesticide residues. It was identified by GC/MS as diphenyl 2-ethylhexyl phosphate (DPEHP), used as a plasticizer and flame retardant. The concentration of DPEHP was investigated in 15 samples of kiwi fruit, and it was detected at between 0.02 and 0.14 microgram/g in 10 of the samples. It might be due to migration of DPEHP into the fruit from the printed portion of the polyethylene terephthalate (PET) package.     

A review of hepatitis E virus

Hepatitis E virus (HEV) is a major cause of outbreaks and sporadic cases of viral hepatitis in tropical and subtropical countries but is infrequent in industrialized countries. The virus is transmitted by the fecal-oral route with fecally contaminated drinking water being the usual vehicle. Hepatitis resulting from HEV infection is a moderately severe jaundice that is self-limiting in most patients. Young adults, 15 to 30 years of age, are the main targets of infection, and the overall death rate is 0.5 to 3.0%. However, the death rate during pregnancy approaches 15 to 25%. Death of the mother and fetus, abortion, premature delivery, or death of a live-born baby soon after birth are common complications of hepatitis E infection during pregnancy. Hepatitis E virus is found in both wild and domestic animals; thus, HEV is a zoonotic virus. The viruses isolated from swine in the United States or Taiwan are closely related to human HEV found in those areas. The close genetic relationship of the swine and human virus suggests that swine may be a reservoir of HEV. In areas where swine are raised, swine manure could be a source of HEV contamination of irrigation water or coastal waters with concomitant contamination of produce or shellfish. Increasing globalization of food markets by industrialized countries has the potential of introducing HEV into new areas of the world. The purpose of this review is to cover certain aspects of hepatitis E including the causative agent, the disease, diagnosis, viral detection, viral transmission, epidemiology, populations targeted by HEV, and the role of animals as potential vectors of the virus.     

Heat inactivation of hepatitis A virus in dairy foods

Experiments were performed to determine the thermal resistance of hepatitis A virus (HAV) in three types of dairy products containing increased amounts of fat content (skim milk, homogenized milk; 3.5% MFG, and table cream; 18% MFG). HAV-inoculated dairy products were introduced into custom-made U-shaped microcapillary tubes that in turn were simultaneously immersed in a waterbath, using custom-made floating boats and a carrying platform. Following exposure to the desired time and temperature combinations, the contents of each of the tubes was retrieved and was tested by plaque assay to determine the reduction in virus titer. Our data indicated that < 0.5 min at 85 degrees C was sufficient to cause a 5-log reduction in HAV titer in all three dairy products, whereas at 80 degrees C, < or = 0.68 min (for skim and homogenized milk), and 1.24 min (for cream) were needed to cause a similar log reduction. Using a nonlinear two-phase negative exponential model (two-compartment model) to analyze the data, it was found that at temperatures of 65, 67, 69, 71, and 75 degrees C, significantly (P < 0.05) higher exposure times were needed to achieve a 1-log reduction in virus titer in cream, as compared to skim and homogenized milk. For example, at 71 degrees C, a significantly (P < 0.05) higher exposure time of 0.52 min (for cream) was needed as compared to < or = 0.18 min (for skim and homogenized milk) to achieve a 1-log reduction in virus titer. A similar trend of inactivation was observed at 73 and 75 degrees C where significantly (P < 0.05) higher exposure times of 0.29 to 0.36 min for cream were needed to cause a 1-log reduction in HAV in cream, as compared to < or = 0.17 min for skim and homogenized milk. This study has provided information on the heat resistance of HAV in skim milk, homogenized milk, and table cream and demonstrated that an increase in fat content appears to play a protective role and contributes to the heat stability of HAV.     

The survival of hepatitis A virus in fresh produce

Fresh produce has been repeatedly implicated as the source of human viral infections, including infection with hepatitis A virus (HAV). The objective of the present study was to evaluate the HAV adsorption capacity of the surface of various fresh vegetables that are generally eaten raw and the persistence of the HAV. To this end, the authors experimentally contaminated samples of lettuce, fennel, and carrot by immersing them in sterile distilled water supplemented with an HAV suspension until reaching a concentration of 5 log tissue culture infectious dose (TCID50)/ml. After contamination, the samples were stored at 4 degrees C and analysed at 0, 2, 4, 7, and 9 days. To detect the HAV, RT-nested-PCR was used; positive samples were subjected to the quantitative determination using cell cultures. The three vegetables differed in terms of their adsorption capacity. The highest quantity of virus was consistently detected for lettuce, for which only a slight decrease was observed over time (HAV titre = 4.44 +/- 0.22 log TCID50/ml at day 0 vs. 2.46 +/- 0.17 log TCID50/ml at day 9, before washing). The virus remained vital through the last day of storage. For the other two vegetables, a greater decrease was observed, and complete inactivation had occurred at day 4 for carrot and at day 7 for fennel. For all three vegetables, washing does not guarantee a substantial reduction in the viral contamination.     

Study of the content of cadmium,chromium and lead in bivalve molluscs of the Pacific Ocean (Maule Region,Chile)

The concentration of Cd, Cr and Pb in the following species Ameghinomya antiqua, Aulacomya atra and Mytilus chilensis, bivalves from Iloca, Constitución and Pelluhue, coastal towns located in the Maule Region, Chile was determined. Representative tissue samples of each species were analysed by using atomic absorption spectroscopy with flame. Validation of methodology was carried out using a certified reference material (TORT-1). The ranges of concentrations found for Cd, Cr and Pb in the 3 species were, respectively, 0.21–4.32, 0.38–12.62 and 0.43–31.10 mg kg⁻¹ dry weight. In relation to the results obtained and the average of each individual metal in the different species of bivalves, Pb concentrations were found in higher levels rather than those of other metals reported by other authors in a similar work, this element constitutes a potential health hazard for the consumers of shellfish. The results were statistically evaluated for possible correlations in the content of metals in different species and in different sites of origin.     

贝类中甲肝病毒提取方法的比较

目的对贝类甲肝病毒的3种试剂盒提取方法进行比较。方法在贝类样品中添加不同滴度的甲肝减毒疫苗后,采用3种核酸提取法:Roche法、AM1836法、Qiagen74104法。按照其说明书提取模拟样品病毒核酸模板,从抽提效率、抽提核酸的稳定性、抑制剂去除效率3个方面对提取效果进行比较。结果在抽提核酸稳定性、抑制剂去除效率方面,3种方法结果相同,稳定性的Ct值为24.795～28.651,抑制剂去除效率方面提取的核酸10倍稀释核酸Ct值比未稀释核酸Ct值差值均为2.9～3.2左右(F=26.802,P0.05);在提取效率方面,Roche法高于AM1836法和Qiagen74104法,Ct值分别为28.5,28.7和29.0。结论 3种方法中,Roche法在贝类中甲肝病毒提取方面更有优势,可以提高贝类中甲肝病毒检测的灵敏度。     

Pressure inactivation of hepatitis A virus in strawberry puree and sliced green onions

Hepatitis A can be acquired by ingesting contaminated produce. To investigate the potential of high-pressure processing as an intervention strategy for virus in produce, strawberry puree and sliced green onions were inoculated with > 10(6) PFU of hepatitis A virus and treated with pressures ranging from 225 to 375 megapascals (MPa) in 25-MPa increments at ambient temperature. Subsequent virus extraction and plaque assay determined that hepatitis A virus was inactivated in strawberry puree and sliced green onions after 5-min exposures to pressures of 375 MPa with log PFU reductions of 4.32 and 4.75, respectively. Hepatitis A virus was equally sensitive in puree and onions at pressures > or = 350 MPa. For treatments of < 325 MPa, the virus was more sensitive to pressure in strawberry puree than in sliced onions with log reductions of 1.2, 2.06, and 3.13 observed for strawberries and 0.28, 0.72, and 1.42 observed for onions after 5-min treatments at 250, 275, and 300 MPa, respectively. Although high-pressure processing may cause some organoleptic alterations to strawberries and onions, results show high-pressure processing will inactivate hepatitis A virus in these foods.     

女性针织内衣市场调查与分析

对中国女性内衣(主要为文胸和底裤)的市场现状和消费者购买力进行调查,利用SPSS统计软件进行数据处理分析,考察女性内衣市场的消费特点与需求状况。对影响女性内衣消费的因素进行全面调查,如款式风格、面料风格、颜色、价格、消费场所等,并分析创建新的内衣品牌存在的投资风险以及应对策略,为我国内衣品牌的迅速健康发展提供参考。     

Depuration of Mytilus galloprovincialis experimentally contaminated with hepatitis A virus

Mussels (Mytilus galloprovincialis) were contaminated with known amounts of laboratory strains of hepatitis A virus and Poliovirus 1 and the effectiveness of a self-cleansing mechanism was studied using a pilot depuration system. Both viruses were rapidly bioaccumulated by mussels and the maximal concentration of about 10(4) TCID50/ml was reached within 1.5 hours. Depuration was carried out up to 24 h; infectivity titer decreased to 10(2) TCID50/ml and 10(3.2) TCID50/ml within 6 h in hepatitis A virus and Poliovirus 1 contaminated mussels, respectively, but only a very slight further decrease was obtained after 24 h. E. coli was used as a control; within 24 h the concentration decreased from 40 to 2 bacteria/ml of mussel (MPN). The elimination of bacteria is not a reliable parameter to control the effectiveness of viral depuration.