A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments.

During genetic engineering, DNA is inserted into a plant's genome, and such insertions are often accompanied by the insertion of additional DNA, deletions and/or rearrangements. These genetic changes are collectively known as insertional effects, and they have the potential to give rise to unintended traits in plants. In addition, there are many other genetic changes that occur in plants both spontaneously and as a result of conventional breeding practices. Genetic changes similar to insertional effects occur in plants, namely as a result of the movement of transposable elements, the repair of double-strand breaks by non-homologous end-joining, and the intracellular transfer of organelle DNA. Based on this similarity, insertional effects should present a similar level of risk as these other genetic changes in plants, and it is within the context of these genetic changes that insertional effects must be considered. Increased familiarity with genetic engineering techniques and advances in molecular analysis techniques have provided us with a greater understanding of the nature and impact of genetic changes in plants, and this can be used to refine pre-market assessments of genetically engineered plants and food and feeds derived from genetically engineered plants.

Norovirus GI and GII and hepatitis A virus in berries and pomegranate arils in Canada.

Between 2016 and 2021, the Canadian Food Inspection Agency (CFIA) collected 4218 samples of fresh and frozen berries (blackberries, blueberries, raspberries, strawberries and mixed berries) and pomegranate arils at retail across 11 major cities in Canada and tested these samples for the presence of norovirus GI, norovirus GII and hepatitis A virus RNA. The purpose of this testing was to provide information on the prevalence of these viruses in berries and pomegranate arils on the Canadian marketplace. Of the 926 fresh fruit samples tested, norovirus GI RNA was detected in one raspberry sample and norovirus GII RNA was detected in one strawberry sample. Of the 3292 frozen fruit samples tested, norovirus GI RNA was detected in one blackberry sample, one raspberry sample and one strawberry sample, and norovirus GII RNA was detected in one blueberry sample, three raspberry samples, four strawberry samples, one pomegranate arils sample and one mixed berry sample. None of the fresh or frozen fruit samples tested positive for hepatitis A virus RNA. No statistically significant associations were observed between the prevalence of viral RNA in samples of fresh and frozen fruit, between the prevalence of viral RNA in samples of domestic and imported fruit or between the prevalence of viral RNA in samples of specific fruit types. Overall, the prevalence of norovirus GI and GII RNA together in fresh and frozen fruit samples in Canada was 0.36 %. The results of this study may be used to refine surveillance programs for norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils, e.g. by adapting the commodities tested and/or the numbers of planned samples to better target these hazards. This information may also be used to inform other Government of Canada approaches to better understand the controls associated norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils.

Microbial safety of cheese in Canada.

A profile of the microbial safety of cheese in Canada was established based on the analysis of 2955 pasteurized and raw-milk cheeses tested under Canada's National Microbiological Monitoring Program (NMMP) and 2009 raw-milk cheeses tested under the Targeted Survey Program. 97.8% of NMMP and 99.6% of Targeted Survey cheese samples were assessed as being of satisfactory microbiological safety. Under the NMMP, Salmonella spp. was detected in 2 samples, Listeria monocytogenes was detected in 15 samples and no Escherichia coli O157/H7:NM (non-motile) was detected. Cheese samples assessed as having unsatisfactory levels of S. aureus and generic E. coli were found in 18 and 41 samples, respectively. Under the Targeted Survey, L. monocytogenes was detected in 2 samples, while no Salmonella spp. or E. coli O157/H7:NM were detected. Cheese samples assessed as having investigative and unsatisfactory levels of S. aureus were found in 4 and 2 samples respectively. No samples were found to have investigative or unsatisfactory levels of generic E. coli. For cheese samples collected under the NMMP, logistic regression models indicated that contamination was more frequent in raw-milk cheeses compared to pasteurized-milk cheeses (OR = 5.0, 95% CI (3.0, 8.3)), and in imported cheeses compared to domestic cheeses (OR = 8.2, 95% CI (4.1, 16.1)). A statistically significant association was found between cheese samples assessed as having unsatisfactory levels of generic E. coli and detection of L. monocytogenes, Salmonella spp. or levels of S. aureus that were assessed as unsatisfactory (p < .001). These test results will help support risk analysis and inform food safety decisions.

Genomic regions conserved in lineage II Escherichia coli O157:H7 strains.

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these lineage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.

Rapid determination of Escherichia coli O157:H7 lineage types and molecular subtypes by using comparative genomic fingerprinting.

In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.

Genotypic characterization and prevalence of virulence factors among Canadian Escherichia coli O157:H7 strains.

In this study, the association between genotypic and selected phenotypic characteristics was examined in a collection of Canadian Escherichia coli O157:H7 strains isolated from humans and cattle in the provinces of Alberta, Ontario, Saskatchewan, and Quebec. In a subset of 69 strains selected on the basis of specific phage types (PTs), a strong correlation between the lineage-specific polymorphism assay (LSPA6) genotype and PT was observed with all strains of PTs 4, 14, 21, 31, 33, and 87 belonging to the LSPA6 lineage I (LSPA6-LI) genotype, while those of PTs 23, 45, 67, and 74 belonged to LSPA6 lineage II (LSPA6-LII) genotypes. This correlation was maintained when additional strains of each PT were tested. E. coli O157:H7 strains with the LSPA6-LI genotype were much more common in the collection than were the LSPA6-LII or lineage I/II (LSPA6-LI/II)-related genotypes (82.6, 11.2, and 5.8%, respectively). Of the strains tested, proportionately more LSPA6-LI than LSPA6-LII genotype strains were isolated from humans (52.7% versus 19.7%) than from cattle (47.8% versus 80.2%). In addition, 96.7% of the LSPA6-LII strains carried the stx(2c) variant gene, while only 50.0% of LSPA6-LI/II and 2.7% of LSPA6-LI strains carried this gene. LSPA6-LII strains were also significantly more likely to possess the colicin D gene, cda (50.8% versus 23.2%), and have combined resistance to streptomycin, sulfisoxazole, and tetracycline (72.1% versus 0.9%) than were LSPA6-LI strains. The LSPA6 genotype- and PT-related characteristics identified may be important markers of specific ecotypes of E. coli O157:H7 that have unique epidemiological and virulence characteristics.

Genome evolution in major Escherichia coli O157:H7 lineages.

BACKGROUND: Genetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E. coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types. RESULTS: A total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15 lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in > or =80% of lineage I and absent from > or = 92% of lineage II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains. CONCLUSION: CGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages.

Microbial assessment of irrigation water used for production of fruit and vegetables in Ontario, Canada.

Five hundred one irrigation water samples were collected from 27 irrigation water sources on 17 farms in southern Ontario, Canada, over a single irrigation season in 2002. The water samples were tested for the presence of the following bacterial water quality indicators: total coliform bacteria, fecal coliforms, Escherichia coli, and fecal streptococci. The median values per 100 ml of these indicators in the irrigation water samples were 3,000, 33, 15, and 1, respectively. Between 70.6 and 98.2% of irrigation water samples contained acceptable levels of fecal coliforms or E. coli, according to published irrigation water quality guidelines. Significant correlations (P < 0.05) were observed between the concentrations of different bacterial indicators and the degree of recent precipitation and concentrations of total coliforms and fecal streptococci. With the exception of fecal streptococci, which increased in number toward the end of the study, none of the indicators displayed a significant trend over the course of the season, as determined by linear regression analysis of indicator concentrations over time (P > 0.05).

Sensitivity of PCR detection of Cyclospora cayetanensis in raspberries, basil, and mesclun lettuce.

The sensitivity of a polymerase chain reaction (PCR) method for detection of Cyclospora cayetanensis in raspberries, basil, and mesclun lettuce was evaluated. The assay could detect 40 or fewer oocysts per 100 g of raspberries or basil, but had a detection limit of around 1000 per 100 g in mesclun lettuce.

Irrigation water as source of foodborne pathogens on fruit and vegetables.

Awareness is growing that fresh or minimally processed fruit and vegetables can be sources of disease-causing bacteria, viruses, protozoa, and helminths. Irrigation with poor-quality water is one way that fruit and vegetables can become contaminated with foodborne pathogens. Groundwater, surface water, and human wastewater are commonly used for irrigation. The risk of disease transmission from pathogenic microorganisms present in irrigation water is influenced by the level of contamination; the persistence of pathogens in water, in soil, and on crops; and the route of exposure. Groundwater is generally of good microbial quality, unless it is contaminated with surface runoff; human wastewater is usually of very poor microbial quality and requires extensive treatment before it can be used safely to irrigate crops; surface water is of variable microbial quality. Bacteria and protozoa tend to show the poorest survival outside a human host, whereas viruses and helminths can remain infective for months to years. Guidelines governing irrigation water quality and strategies to reduce the risk of disease transmission by foodborne pathogens in irrigation are discussed.

Identification of Escherichia coli O157:H7 genomic regions conserved in strains with a genotype associated with human infection.

Beta-glucuronidase-negative, sorbitol-nonfermenting isolates of Shiga toxin-producing Escherichia coli O157 comprise part of a clone complex of related enterohemorrhagic E. coli isolates. High-resolution genotyping shows that the O157 populations have diverged into two different lineages that appear to have different ecologies. To identify genomic regions unique to the most common human-associated genotype, suppression subtractive hybridization was used to identify DNA sequences present in two clinical strains representing the human lineage I O157:H7 strains but absent from two bovine-derived lineage II strains. PCR assays were then used to test for the presence of these regions in 10 lineage I strains and 20 lineage II strains. Twelve conserved regions of genomic difference for lineage I (CRD(I)) were identified that were each present in at least seven of the lineage I strains but absent in most of the lineage II strains tested. The boundaries of the lineage I conserved regions were further delimited by PCR. Eleven of these CRD(I) were associated with E. coli Sakai S-loops 14, 16, 69, 72, 78, 82, 83, 91 to 93, 153, and 286, and the final CRD(I) was located on the pO157 virulence plasmid. Several potential virulence factors were identified within these regions, including a putative hemolysin-activating protein, an iron transport system, and several possible regulatory genes. Cluster analysis based on lineage I conserved regions showed that the presence/absence of these regions was congruent with the inferred phylogeny of the strains.

Monoclonal antibodies specific for hippurate hydrolase of Campylobacter jejuni.

Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

Survey of Ontario Bulk Tank Raw Milk for Food-Borne Pathogens.

Raw (unpasteurized) milk can be a source of food-borne pathogens. Raw milk consumption results in sporadic disease outbreaks. Pasteurization is designed to destroy all bacterial pathogens common to raw milk, excluding spore-forming bacteria and possibly Mycobacterium paratuberculosis , but some people continue to drink raw milk, believing it to be safe. Current methods for assessing the bacteriological quality of raw milk, such as aerobic plate counts, are not usually designed to detect specific pathogens. The objective of this study was to estimate the proportion of pick-ups (loads of raw milk from a single farm bulk tank) from Ontario farm bulk tanks that contained Listeria monocytogenes . Salmonella spp., Campylobacter spp., and/or verotoxigenic Escherichia coli (VTEC). Samples from 1,720 pick-ups of raw milk were tested for the presence of these pathogens, and 47 L. monocytogenes , three Salmonella spp., eight Campylobacter spp., and 15 VTEC isolates were detected, representing 2.73, 0.17, 0.47, and 0.87% of milk samples, respectively. Estimates of the proportion of theoretical tanker truck loads of pooled raw milk contaminated with pathogens ranged from a low of 0.51 % of tankers containing raw milk from 3 bulk tanks being contaminated with Salmonella spp. to a high of 34.41 % of tankers containing raw milk from 10 bulk tanks being contaminated with at least one of the pathogens. Associations between the presence of pathogens and raw milk sample characteristics were investigated. The mean somatic cell count was higher among VTEC- or L. monocytogenes -positive samples, and the mean aerobic plate count was found to be higher among L. monocytogenes -positive samples. These results confirm the presence of bacterial food pathogens in raw milk and emphasize the importance of continued diligence in the application of hygiene programs within dairies and the separation of raw milk from pasteurized milk and milk products.