ABSTRACT Foodborne viruses such as norovirus and hepatitis A virus (HAV) are highly transmissible, persistent in the environment, and resistant to many conventional inactivation methods. Foods can become contaminated with these viruses either at the source of harvest or during food handling and processing. Multiple lines of evidence suggest that foodborne viruses can survive desiccation and dry conditions. Several foodborne virus outbreaks have been linked to low-moisture foods (LMFs), indicating that these foods can be vehicles of virus transmission. However, the efficiencies of common virus extraction methodologies have not been examined with LMFs. We adapted the International Organization for Standardization (ISO) 15216-1:2017 method for virus recovery for use with chocolate, pistachios, and cornflakes. We also developed a magnetic bead assay for the recovery of HAV from LMFs and used the porcine gastric mucin–coated magnetic beads (PGM-MBs) to extract norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV) from the same LMFs. The efficiency of virus recovery using the bead-based assay was then compared with that of the ISO 15216-1:2017 method. In chocolate and pistachios, the recovery rates with the PGM-MB method were 5.6- and 21.3-fold higher, respectively, for FCV and 1.65- and 18-fold higher, respectively, for MNV than those with the ISO 15216-1:2017 method. However, the PGM-MB method failed to recover MNV and FCV from cornflakes. The recovery rates for HAV in chocolate, pistachios, and corn flakes with the magnetic bead method were 11.5-, 3-, and 5.6-fold higher, respectively, than those with the ISO 15216-1:2017 method. Thus, depending upon the food matrix and the target virus, the bead-based assays can be used to efficiently and rapidly extract viruses from LMFs. HIGHLIGHTS Anti-HAV–coated magnetic beads can be used to efficiently extract HAV from LMFs. Extraction efficiency of the PGM-MB method depends on the tested matrix. Bead-based assays are faster and have fewer steps than the ISO 15216-1:2017 method.
Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)–mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.
Enterobacter sakazakii has been implicated as the causal organism in a severe form of neonatal meningitis, with reported mortality rates of 40 to 80%. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. In this study, clinical and foodborne isolates of E. sakazakii were evaluated for enterotoxin production by the suckling mouse assay. In addition, suckling mice were challenged both orally and by intraperitoneal injection. Of 18 E. sakazakii strains evaluated, four were found to test positive for enterotoxin production. All strains of E. sakazakii were lethal to suckling mice at 10 8 CFU per mouse by intraperitoneal injection, while two strains caused death by the peroral route. In in vitro assays, CHO, Vero, and Y-1 cells demonstrated both cell lysis and rounding when exposed to E. sakazakii strain LA filtrates. This is the first report describing any putative virulence factors of E. sakazakii .