Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.

Isolation, characterization, molecular analysis and application of bacteriophage DW-EC to control Enterotoxigenic Escherichia coli on various foods.

Among food preservation methods, bacteriophage treatment can be a viable alternative method to overcome the drawbacks of traditional approaches. Bacteriophages are naturally occurring viruses that are highly specific to their hosts and have the capability to lyse bacterial cells, making them useful as biopreservation agents. This study aims to characterize and determine the application of bacteriophage isolated from Indonesian traditional Ready-to-Eat (RTE) food to control Enterotoxigenic Escherichia coli (ETEC) population in various foods. Phage DW-EC isolated from Indonesian traditional RTE food called dawet with ETEC as its host showed a positive result by the formation of plaques (clear zone) in the bacterial host lawn. Transmission electron microscopy (TEM) results also showed that DW-EC can be suspected to belong to the Myoviridae family. Molecular characterization and bioinformatic analysis showed that DW-EC exhibited characteristics as promising biocontrol agents in food samples. Genes related to the lytic cycle, such as lysozyme and tail fiber assembly protein, were annotated. There were also no signs of lysogenic genes among the annotation results. The resulting PHACTS data also indicated that DW-EC was leaning toward being exclusively lytic. DW-EC significantly reduced the ETEC population (P ≤ 0.05) in various food samples after two different incubation times (1 day and 6 days) in chicken meat (80.93%; 87.29%), fish meat (63.78%; 87.89%), cucumber (61.42%; 71.88%), tomato (56.24%; 74.51%), and lettuce (46.88%; 43.38%).

Physicochemical Parameters Affecting Norovirus Adhesion to Ready-To-Eat Foods.

The adhesion of noroviruses to strawberry, turkey slices, ham, and cheddar cheese was studied using murine norovirus 1 (MNV-1) as a surrogate for human norovirus (NoV). Based on plaque assay, the recovery and adhesion of MNV-1 depended on the food type (turkey versus strawberry), pH of the initial suspension buffer (pH 4 versus pH 7), and food fat composition (C8 versus C18). Recovery of infectious particles from turkey was 68% compared to 9.4% from strawberry. On turkey, adhesion of MNV-1 was lower at pH 7 (pH of fecal matter), and virus particles adhered to this pH were recovered more easily (33,875 PFU) than at pH 4 (pH of vomitus). The presence of fat and the composition of fatty acids seemed to increase MNV-1 recovery and adherent viral particles recovered but did not affect adhesion (68% on fat-free turkey and regular turkey). Adherent MNV-1 particles recovered from stainless steel coated with saturated fatty acid (C8, C14, C18) increased significantly with chain length (P < 0.05), but adhesion did not seem to change. Using liquid droplet contact angle to measure surface energy, it was deduced that hydrophobic interactions contribute considerably to the adhesion of MNV-1 to stainless steel, polyvinyl chloride, and high-density polyethylene. IMPORTANCE Ready-to-eat (RTE) foods are major vehicles of transmission of foodborne viral pathogens, including NoV. The high incidence of gastroenteritis caused by viruses is due largely to their persistence in the environment and adhesion to different kinds of surfaces in the food industry, including the foods themselves. Compared with bacteria, adhesion of viruses to surfaces is poorly understood. Better knowledge of the physicochemical parameters involved in the adhesion of NoV to ready-to-eat foods is essential to devising effective strategies for reducing the persistence and, thus, the transmission of this virus.

Screening of Ready-to-Eat Meat Products for Hepatitis E Virus in Switzerland.

Seroprevalence data for pig herds suggested that there must be a relevant reservoir for hepatitis E virus (HEV) in Switzerland. To know more about the viral presence in ready-to-eat meat products, we screened pork liver sausages and raw meat sausages from the Swiss retail market for the presence of HEV. Testing was performed with a detection method where the virus extraction step was optimized. As for the performance of the improved method, the mean recovery rate for the mengovirus process control was 24.4%, whereas for HEV-inoculated sample matrices between 10.4 and 100% were achieved. The limit of detection was about 1.56 × 103 and 1.56 × 102 genome copies per gram for liver sausages and raw meat sausages, respectively. In the screening programme, HEV-RNA was detected in 10 of total 90 (11.1%) meat products, 7 of 37 (18.9%) liver sausages, and 3 of 53 (5.7%) raw meat sausages. Virus loads of up to 5.54 log10 HEV genome copies per gram were measured. All sequences retrieved from positive samples belonged to HEV genotype 3. The significance of the presented work was a current overview of the HEV prevalence in ready-to-eat meat products on the Swiss retail marked and an improvement of the extraction efficiency of the HEV detection method.

Tracking and modeling norovirus transmission during mechanical slicing of globe tomatoes.

Recent epidemiological evidence indicates that preparation of fresh produce for use as ingredients in ready-to-eat food in commercial settings has been a significant source of the norovirus (NoV) infections in the U.S. This research investigated the dissemination of NoV from a single tomato to many others via the use of an 11-horizontal blade slicer commonly found in restaurants or sandwich shops. A total of eight trials were conducted. The source of contamination in each trial was a soak-inoculated, air-dried globe tomato containing ~8log10 murine norovirus (MNV). Each trial began by slicing a single un-inoculated tomato in the slicer, followed by slicing an inoculated tomato. This was then followed by slicing 9 to 27 un-inoculated tomatoes. A similar and constant hand pressure on the slicer was used in every trial. Three slices from each tomato were collected for virus elution, concentration, and extraction before RT-PCR detection of MNV. The change in MNV per sliced tomato was averaged over all eight trials, and two mathematical models were fit to the average data using a logarithmic model or a power model. Regression analysis determined that the equation that best fit the data was y=-0.903∗ln(x)+7.945, where y=log10 MNV per slicing and x=tomato slicing number. An acceptable fit (R(2)=0.913) was indicated. The MNV levels transferred (y) generally decreased as the number of tomatoes sliced (x) increased, with some exceptions. Infrequent but erratic transfers, where the MNV level of a subsequent tomato was higher than that of a preceding tomato, occurred in later transfer of some trials. In contrast, the first and second transfers of each trial were always shown to have sharply decreased levels of MNV from the inoculum. The MNV log10 reduction per slicing event changes throughout the process: with a predicted 0.63log10 reduction from tomato 1 to tomato 2 (76% reduction); a 0.07log10 reduction predicted from tomato 13 to tomato 14 (a 14% reduction); and 0.03log10 reduction predicted from tomato 27 to tomato 28 (a 7% reduction). Virus transfer is clearly variable even given the consistent slicing procedure used throughout each trial. This study illustrates the complex nature of risk prediction associated with NoV cross-contamination during food preparation in commercial establishments.

Comparison of RNA extraction methods for the detection of a norovirus surrogate in ready-to-eat foods.

Four nucleic acid extraction methods were evaluated for the purpose of quantifying a norovirus surrogate (murine norovirus [MNV-1]) concentrated from different food samples. Simple (strawberries and lettuce) and complex (sliced turkey breast, soft-shell clams, and potato salad) food matrices were inoculated with a viral suspension containing high (4×10(5) PFU) or low (4×10(3) PFU) numbers of viral particles. MNV-1 was eluted using either the Pulsifier™ or repetitive pipetting. The four methods were based on using magnetic silica (MiniMAG), non-magnetic silica (bioMérieux Basic kit), silica membrane (Qiagen kit), and phenol (TriReagent) for RNA extraction. The greatest recovery of viral RNA from simple matrices was obtained using magnetic silica for both inoculation levels. For strawberries, the addition of pectinase during the elution step improved RNA recovery when the Pulsifier was used with silica membrane extraction and when repetitive pipetting was used with magnetic silica extraction. In the case of complex matrices, the extraction of high or low numbers of MNV-1 was highest overall using magnetic silica. The exception was soft-shell clams with a high viral load, in which the greatest recovery was obtained with the phenol-based method. In general, magnetic silica was the most effective for extracting both high and low numbers of MNV-1 particles from a wide range of foods.

Potential risk of norovirus infection due to the consumption of "ready to eat" food.

In this study, we investigated the presence of enteric viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and adenovirus (HAdV), in vegetables available on the Italian markets. For this aim, 110 national and international "ready to eat" samples were collected and analyzed by biomolecular tests and positive samples were confirmed by sequencing. All samples (100 %) were negative for HAV, HEV, and HAdV, while 13.6 % (15/110) were positive for NoV. Actually there is not a formal surveillance system for NoV infections in Italy but we clearly demonstrated a potential risk associated with the consumption of "ready to eat" vegetables. This study confirmed for the first time in Italy the presence of norovirus in semi-dried tomatoes by PCR technique.

Investigations on the frequency of norovirus contamination of ready-to-eat food items in Istanbul, Turkey, by using real-time reverse transcription PCR.

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.