Survival of Porcine teschovirus as a surrogate virus on pork chops during storage at 2°C.

Due to the lack of an efficient cultivation system, little is known about the stability and inactivation of hepatitis E virus (HEV). In addition, there is a lack of information on which cultivable virus(es) are suitable as model or surrogate viruses for HEV. Murine norovirus (MNV) and F-RNA coliphage MS2 are potential surrogates and F-RNA coliphages are a potential indicator for enteric viruses. However, the numbers of F-RNA coliphages excreted by swine are relatively low. In contrast, Porcine teschovirus (PTV) is cultivable and is excreted abundantly. PTV is readily detected on swine carcasses and the potential of PTV as a viral indicator of fecal contamination on hog carcasses is currently being explored, however, there is no information on the environmental stability of PTV. The survival of PTV was determined on vacuum packaged pork chops during storage at 2°C using cultivation and molecular techniques and compared to published data on the survival of MNV and MS2 under similar conditions. Viable PTV was reduced by ≥1.8log units compared to a reduction of 0.6 log genomic copies after 7weeks. The viability data indicates that PTV is less stable than MS2 and MNV during storage at 2°C whereas similar reductions in genomic copies were observed for all 3 viruses. This study provides data on the survival of PTV on pork and insight on the potential of PTV as a surrogate for HEV in the pork processing chain.

Comparison of ZetaPlus 60S and nitrocellulose membrane filters for the simultaneous concentration of F-RNA coliphages, porcine teschovirus and porcine adenovirus from river water.

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C-E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×10(6) PFU/100 mL, 1×10(5) gc/100 mL and 3×10(5) gc/100 mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5-154 PFU/100 mL), PTV in 12/12 samples (397-10,951 gc/100 mL), PAdV in 1/12 samples (15 gc/100 mL), and F-RNA GIII in 1/12 samples (750 gc/100 mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR.

Numbers of coliforms, Escherichia coli, F-RNA phage, rotavirus, bovine enteric calicivirus and presence of non-O157 STEC on commercial vacuum packaged beef.

The numbers of coliforms, Escherichia coli, F-RNA coliphages, bovine enteric calicivirus (BEC) and rotavirus (RV) and presence of non-O157 shiga toxigenic E. coli (STEC) were determined on commercial vacuum packaged beef subprimals at the retail level from swabs obtained from the entire surfaces of 150 cuts that originated from federally and provincially registered plants. The prevalence and log mean numbers of E. coli were higher in provincially registered plants than in federally registered plants; 64% vs 20%, respectively, and -0.3 vs -1.22 log cfu/100 cm(2), respectively. In contrast, the prevalence and mean log numbers of F-RNA coliphages were lower for the provincially registered plants than for the federally registered plants; 31% vs 68% and -0.86 vs -0.13 log cfu/100 cm(2), respectively. One E. coli sample tested positive for stx2 and eae. F-RNA coliphages associated with human origin (GII/GIII) were detected in 12% and 30% of samples that originated from provincially and federally registered plants, respectively. RV RNA was detected in 4% of samples while BEC RNA was not detected. Although the infectivity of RV is unknown, the presence of viable F-RNA coliphages suggests that consumers could potentially be at risk when consuming undercooked meat that is contaminated with RV.

Effect of handling and storage conditions and stabilizing agent on the recovery of viral RNA from oral fluid of pigs.

There is an increasing interest in using oral fluid to determine herd health and documenting the circulation of viruses in commercial swine populations but little is known about the stability of viruses in oral fluid. Hepatitis E virus (HEV) is a zoonotic virus which is widespread in swine herds. Information on optimal handling methods such as heat treatments, freezing and RNA stabilization agents is needed to prevent or minimize degradation of viral RNA by degradative enzymes. The objectives of the study were to determine optimum handling conditions of the oral fluid before RNA extraction and to compare the performance of the RNeasy Protect Saliva Mini kit, which contains a stabilizing agent, with that of the QIAamp Viral RNA Mini kit, which does not contain a stabilizing agent. Preliminary studies with oral fluid inoculated with HEV indicated that a heat treatment of 60°C for 15min was detrimental to HEV RNA. HEV was recovered from 25/25 and 24/25 samples of oral fluid when samples were incubated for ≤24h at 4°C and 30days at -20°C, respectively, without a stabilizing agent and extracted with the QiaAMP kit. In contrast, HEV RNA was detected in 16/25 and 11/25 samples when samples were incubated with a stabilizing agent for 24h at 37°C and 30days at -20°C, respectively, and extracted with the RNeasy Protect Saliva kit. Moreover, the mean number of genome copies/ml of HEV recovered from oral fluid stored at -20°C without the stabilizing agent was 2.9 log units higher than oral fluid stored at -20°C in the presence of the stabilizing agent. The recovery of RNA from HEV, F-RNA coliphage MS2 and murine norovirus (MNV), which are surrogates for norovirus, was significantly greater when oral fluid was incubated for 24h at 4°C than when oral fluid was stabilized with RNAprotect Saliva Reagent for 24h at 37°C, where the relative differences between the two processes were 1.4, 1.8, and 2.7 log genome copies/ml for MS2, MNV, and HEV, respectively. The findings suggest that it is unnecessary to stabilize oral fluid from swine for the detection of viral RNA, provided the samples are stored at 4°C or frozen at -20°C, and that the RNeasy Protect Saliva Mini kit did not perform well for the detection of viral RNA.

Survival of murine norovirus and F-RNA coliphage MS2 on pork during storage and retail display.

The existence of animal strains of norovirus (NV) that are closely related to human strains raises concerns about interspecies recombination and the potential for zoonotic transmission through undercooked meat products. Contamination of meat with NV can occur both via meat processing operations and poor food handler hygiene. There is a lack of knowledge on the survival of NV on raw meat because NV cannot be effectively cultivated and its detection is limited to molecular methods. The survival of murine norovirus (MNV) and MS2, as surrogates for NV, was determined on pork chops by plaque assay and real time RT-PCR. Both MNV and MS2 displayed very high survival rates on vacuum packaged raw pork chops that were stored at 2°C for up to 7 weeks and numbers declined little during subsequent retail display for 7 days. Maximum reductions for MNV and MS2 were 1.0 log PFU/cm(2) and 0.6 log genome copies(GC)/cm(2) or 1.1 log PFU/cm(2) and 1.2 log GC/cm(2), respectively. The viability of MS2 and MNV was not affected by the proteolytic enzymes present in the meat nor by numbers of bacteria that increased with time during storage in vacuum packs and while on retail display. MNV and MS2 can be considered as good surrogates for NV on raw meat. The findings of this study indicate that potentially pathogenic NV will likely survive extremely well on fresh meat and consumers could potentially be at risk when consuming undercooked meat that is contaminated with NV.

Development of an optimized method for the recovery of infectious F-RNA coliphage MS2 from meat.

F-RNA coliphages, part of the gut flora and likely to be deposited on meat along with other enteric organisms during carcass dressing and processing, may be regarded as an indicator and/or surrogate for potential zoonotic enteric viruses. There is no recommended sampling method for viruses on meats and there is a lack of information on the attachment of enteric viruses or F-RNA coliphages to gauze swabs, cellulose sponges and muscle and fat tissue. The objective of this work was to optimize the recovery of MS2 from muscle and fat tissue of meat by comparing phosphate buffered saline (PBS), 10% beef extract pH 7.2, and tryptose phosphate (2.9%) glycine (6%) broth pH 9.5 as eluants. The sampling techniques of excision, swabbing with gauze or cellulose sponges were compared with homogenizing the inoculated entire muscle or fat surface area. The recovery of MS2 from cellulose sponges using beef extract was significantly higher (P=0.001) than tryptose phosphate glycine broth which was significantly higher (P=0.0001) than PBS. There was no significant difference in the recovery between tryptose phosphate glycine broth and beef extract (P=0.92) and there was no significant difference between PBS and beef extract (P=0.10) when MS2 was recovered from gauze. No significant differences were observed between the different eluants with muscle tissue (P=0.91). When MS2 was recovered from muscle tissue with beef extract significant differences were observed (P=0.002); the sampling techniques of homogenizing the entire sample (56%) was equal to excision (43%) (P=0.23) and swabbing with a cellulose sponge (38%) (P=0.06) which were significantly higher than swabbing with gauze (28%), a second grouping of means indicated that homogenizing the entire sample was significantly higher than the other three sampling techniques. When MS2 was recovered from fat, significant differences were observed (P=0.000); homogenizing the entire sample (78%) was equal to excision (74%), which were significantly higher than swabbing with gauze (49%) or cellulose sponge (29%). The recovery of MS2 from meat is affected by the sampling technique. When choosing a nondestructive sampling method such as a cellulose sponge, a higher recovery can be obtained with beef extract as an eluant.