Comparing listeriosis risks in at-risk populations using a user-friendly quantitative microbial risk assessment tool and epidemiological data.

Although infection by the pathogenic bacterium Listeria monocytogenes is relatively rare, consequences can be severe, with a high case-fatality rate in vulnerable populations. A quantitative, probabilistic risk assessment tool was developed to compare estimates of the number of invasive listeriosis cases in vulnerable Canadian subpopulations given consumption of contaminated ready-to-eat delicatessen meats and hot dogs, under various user-defined scenarios. The model incorporates variability and uncertainty through Monte Carlo simulation. Processes considered within the model include cross-contamination, growth, risk factor prevalence, subpopulation susceptibilities, and thermal inactivation. Hypothetical contamination events were simulated. Results demonstrated varying risk depending on the consumer risk factors and implicated product (turkey delicatessen meat without growth inhibitors ranked highest for this scenario). The majority (80%) of listeriosis cases were predicted in at-risk subpopulations comprising only 20% of the total Canadian population, with the greatest number of predicted cases in the subpopulation with dialysis and/or liver disease. This tool can be used to simulate conditions and outcomes under different scenarios, such as a contamination event and/or outbreak, to inform public health interventions.

Evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the "sweating" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

Campylobacter spp. in Icelandic poultry operations and human disease.

We describe the observed relationship of campylobacter in poultry operations to human cases in a closed environment. During 1999 in Iceland, domestic cases of campylobacteriosis reached peak levels at 116/100,000 and in 2000 dropped to 33/100,000. Approximately 62% of broiler carcass rinses were contaminated with Campylobacter spp. in 1999. During 2000, only 15% of the broiler flocks tested Campylobacter spp. positive. In 2000, carcasses from flocks which tested positive on the farms at 4 weeks of age were subsequently frozen prior to distribution. We suggest that public education, enhanced on-farm biological security measures, carcass freezing and other unidentified factors, such as variations in weather, contributed to the large reduction in poultry-borne campylobacteriosis. There is no immediate basis for assigning credit to any specific intervention. We continue to seek additional information to understand the decline in campylobacteriosis and to create a risk assessment model for Campylobacter spp. transmission through this well defined system.

A descriptive analysis of giardiasis cases reported in Ontario, 1990-1998.

Cases of giardiasis in Ontario were described using notifiable disease data from the Ontario Ministry of Health for the years 1990-1998 inclusive. The mean annual age- and sex-adjusted incidence rate was 25.77 cases per 100,000 population for the 25,289 cases reported. Children under five years of age had the highest incidence of disease. Males had a higher mean annual incidence in all age groups. Four deaths occurred among cases. The most frequently reported symptoms were loose stools or watery diarrhea (50.1%). A seasonal pattern was noted, peaking in late summer and early autumn. The most frequently reported probable risk settings were the home (40.1%) and travel (39.1%). The study findings suggest that a high proportion of cases occur in urban areas and spatial analysis showed the highest incidence around Lake Huron and Georgian Bay. Unfiltered water and person-to-person contact are believed to be important sources of infection.

Crohn's disease and Mycobacterium avium subsp. paratuberculosis: current issues.

Crohn's disease is a chronic debilitating inflammatory bowel disease of unknown etiology. Proposed causes include bacterial or viral infection, diet or exposure to tobacco smoke, genetic abnormality, and immune dysfunction. The bacterium Mycobacterium avium subsp. paratuberculosis (Map) has received much research attention as a potential cause of the disease. Map causes Johne's disease in ruminants. The pathology of Johne's disease superficially resembles that of Crohn's disease in humans. Some researchers have shown evidence of Map in intestinal tissues of Crohn's disease patients. Studies are in progress to investigate the possibility that Map exists in milk from infected cows and survives pasteurization. This is a controversial subject with the potential for media attention and public outcry. We examined the current literature and concluded that insufficient evidence exists at this time to implicate any one factor, including Map in milk, as the definitive cause of Crohn's disease. The high degree of uncertainty in this issue requires regulators to recognize the need for effective risk communication as ongoing research provides additional information about the disease.

Hazard identification and exposure assessment for microbial food safety risk assessment.

The four cornerstones of microbial food safety risk assessment are hazard identification, exposure assessment, hazard characterization, and risk characterization. These steps represent a systematic process for identifying adverse consequences and their associated probabilities arising from consumption of foods that may be contaminated with microbial pathogens and/or microbial toxins. This paper presents a discussion of the first two steps: hazard identification and exposure assessment, and considerations for different approaches that can be used to analyze the relevant information.

Survival of listeria innocua in salmon following cold-smoke application.

The ability of Listeria innocua to survive on salmon fillets during cold smoking in a commercial processing plant was investigated using a central composite rotatable response surface design to examine smoking temperatures in the range of 18 to 30 degrees C and a smoke time from 2 to 14 h. Smoke temperature did not significantly (P < 0.05) reduce counts of L. innocua on the salmon. However, the smoking time had a significant effect on L. innocua. The smoking time was directly related to the reduction in count (R2 = 0.831), and a 3-log cycle reduction in count was observed when the smoking time was 12 h. The reduction in L. innocua levels on the fish was unaffected by the pH, water activity, and salt concentration of the fillet.

A simulation model for studying the role of pre-slaughter factors on the exposure of beef carcasses to human microbial hazards.

A Monte Carlo simulation model was constructed for assessing the quantity of microbial hazards deposited on cattle carcasses under different pre-slaughter management regimens. The model permits comparison of industry-wide and abattoir-based mitigation strategies and is suitable for studying pathogens such as Escherichia coli O157:H7 and Salmonella spp. Simulations are based on a hierarchical model structure that mimics important aspects of the cattle population prior to slaughter. Stochastic inputs were included so that uncertainty about important input assumptions (such as prevalence of a human pathogen in the live cattle-population) would be reflected in model output. Control options were built into the model to assess the benefit of having prior knowledge of animal or herd-of-origin pathogen status (obtained from the use of a diagnostic test). Similarly, a facility was included for assessing the benefit of re-ordering the slaughter sequence based on the extent of external faecal contamination. Model outputs were designed to evaluate the performance of an abattoir in a 1-day period and included outcomes such as the proportion of carcasses contaminated with a pathogen, the daily mean and selected percentiles of pathogen counts per carcass, and the position of the first infected animal in the slaughter run. A measure of the time rate of introduction of pathogen into the abattoir was provided by assessing the median, 5th percentile, and 95th percentile cumulative pathogen counts at 10 equidistant points within the slaughter run. Outputs can be graphically displayed as frequency distributions, probability densities, cumulative distributions or x-y plots. The model shows promise as an inexpensive method for evaluating pathogen control strategies such as those forming part of a Hazard Analysis and Critical Control Point (HACCP) system.

Pre-slaughter control of Escherichia coli O157 in beef cattle: a simulation study.

A stochastic simulation model was used to assess the benefit of measures implemented in the pre-slaughter period that are aimed at reducing the contamination of beef carcasses with Shiga-like-toxin-producing Escherichia coli O157. The scenario studied was based on an abattoir processing approximately 1000 head of lot-fed cattle per day. Input assumptions were described using probability distributions to reflect uncertainty in their true values. Control measures that were assessed were based on either a reduction in herd prevalence of infection, reduction in opportunity for cross-contamination in the processing plant by re-ordering of the slaughter queue, reduction of concentration of E. coli O157 in fresh faeces, or a reduction in the amount of faeces, mud and bedding ('tag') transferred from the hide to the carcass. Some control measures evaluated were hypothetical in nature and were included to assist with the planning of research priorities. Simulations suggested that the greatest potential impact is associated with vaccination and with an agent that reduces shedding E. coli O157 in faeces. Knowledge of herd-test results obtained by testing a sample of animals from the herd provides only a minor advantage in control programmes, although application of a rapid test to all animals in all lots might be of some benefit. Under most scenarios, there is ample opportunity for cross-contamination to occur within the slaughter plant as a result of early entry of cattle contaminated with E. coli O157. An industry-wide reduction in the amount of tag attached to hides and addition of a source of cattle having a prolonged average fasting time were not predicted to have a large impact on mean amount of carcass contamination with E. coli O157.

Reliability of an ordinal rating system for assessing the amount of mud and feces (tag) on cattle hides at slaughter.

A study was conducted to provide a quantitative description of the amount of tag (mud, soil, and bedding) adhered to the hides of feedlot beef cattle and to appraise the statistical reliability of a subjective rating system for assessing this trait. Initially, a single rater obtained baseline data by assessing 2,417 cattle for 1 month at an Ontario beef processing plant. Analysis revealed that there was a strong tendency for animals within sale-lots to have a similar total tag score (intralot correlation = 0.42). Baseline data were summarized by fitting a linear model describing an individual's total tag score as the sum of their lot mean tag score (LMTS) plus an amount representing normal variation within the lot. LMTSs predicted by the linear model were adequately described by a beta distribution with parameters nu = 3.12 and omega = 5.82 scaled to fit on the 0-to-9 interval. Five raters, trained in use of the tag scoring system, made 1,334 tag score observations in a commercial abattoir, allowing reliability to be assessed at the individual level and at the lot level. High values for reliability were obtained for individual total tag score (0.84) and lot total tag score (0.83); these values suggest that the tag scoring system could be used in the marketing and slaughter of Ontario beef cattle to improve the cleanliness of animals presented for slaughter in an effort to control the entry of microbial contamination into abattoirs. Implications for the use of the tag scoring system in research are discussed.

Simulation modeling for microbial risk assessment.

Quantitative microbial risk assessment implies an estimation of the probability and impact of adverse health outcomes due to microbial hazards. In the case of food safety, the probability of human illness is a complex function of the variability of many parameters that influence the microbial environment, from the production to the consumption of a food. The analytical integration required to estimate the probability of foodborne illness is intractable in all but the simplest of models. Monte Carlo simulation is an alternative to computing analytical solutions. In some cases, a risk assessment may be commissioned to serve a larger purpose than simply the estimation of risk. A Monte Carlo simulation can provide insights into complex processes that are invaluable, and otherwise unavailable, to those charged with the task of risk management. Using examples from a farm-to-fork model of the fate of Escherichia coli O157:H7 in ground beef hamburgers, this paper describes specifically how such goals as research prioritization, risk-based characterization of control points, and risk-based comparison of intervention strategies can be objectively achieved using Monte Carlo simulation.

Quantitative risk assessment for Escherichia coli O157:H7 in ground beef hamburgers.

Quantitative Risk Assessment (QRA) is a methodology used to organize and analyze scientific information to estimate the probability and severity of an adverse event. Applied to microbial food safety, the methodology can also help to identify those stages in the manufacture, distribution, handling, and consumption of foods that contribute to an increased risk of foodborne illness, and help focus resources and efforts to most effectively reduce the risk of foodborne pathogens. The term Process Risk Model (PRM) is introduced in this paper to describe the integration and application of QRA methodology with scenario analysis and predictive microbiology to provide an objective assessment of the hygienic characteristics of a manufacturing process. The methodology was applied to model the human health risk associated with Escherichia coli O157:H7 in ground beef hamburgers. The PRM incorporated two mathematical submodels; the first was intended to described the behaviour of the pathogen from the production of the food through processing, handling, and consumption to predict human exposure. The exposure estimate was then used as input to a dose-response model to estimate the health risk associated with consuming food from the process. Monte Carlo simulation was used to assess the effect of the uncertainty and variability in the model parameters on the predicted human health risk. The model predicted a probability of Hemolytic Uremic Syndrome of 3.7 x 10(-6) and a probability of mortality of 1.9 x 10(-7) per meal for the very young. These estimates are likely high for all hamburger meals, but may be reasonable for the home-prepared hamburgers described by this model. The efficacy of three risk mitigation strategies were evaluated by modifying the values of the predictive factors and comparing the new predicted risk. The average probability of illness was predicted to be reduced by 80% under a hypothetical mitigation strategy directed at reducing microbial growth during retail storage through a reduction in storage temperature. This strategy was predicted to be more effective than a hypothetical intervention which estimated a plausible reduction in the concentration of E. coli O157:H7 in the feces of cattle shedding the pathogen and one aimed at convincing consumers to cook hamburgers more thoroughly. The conclusions of this approach are only accurate to the extent that the model accurately represents the process. Currently, uncertainty and ignorance about the hygienic effects of the individual operations during production, processing, and handling limit the applicability of a PRM to specify HACCP criteria in a quantitative manner. However, with continuous improvement through stimulated research, a PRM should encompass all available information about the process, food, and pathogen and should be the most appropriate decision-support tool since it represents current knowledge.

Quantitative risk assessment: an emerging tool for emerging foodborne pathogens.

New challenges to the safety of the food supply require new strategies for evaluating and managing food safety risks. Changes in pathogens, food preparation, distribution, and consumption, and population immunity have the potential to adversely affect human health. Risk assessment offers a framework for predicting the impact of changes and trends on the provision of safe food. Risk assessment models facilitate the evaluation of active or passive changes in how foods are produced, processed, distributed, and consumed.

Analysis of whole-cell fatty acid profiles of verotoxigenic Escherichia coli and Salmonella enteritidis with the microbial identification system.

Differentiation of strains within bacterial species, based on gas chromatographic analysis of whole-cell fatty acid profiles, was assessed with 115 strains of verotoxigenic Escherichia coli and 315 strains of Salmonella enteritidis. Fatty acid-based subgroups within each of the two species were generated. Variability of fatty acid profiles observed in repeat preparations from the same strain approached that observed between subgroups, limiting the usefulness of using fatty acid profiles to subgroup verotoxigenic E. coli and S. enteritidis strains.

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.

Evaluation of enrichment procedures for recovering Listeria monocytogenes from dairy products.

Six different enrichment media and five selective plating media were compared for their suitability for the recovery of Listeria monocytogenes from dairy products. These included media used to test milk products by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control (CDC), and media developed by the U.S. Department of Agriculture (USDA) for testing meat and poultry products. Test samples included naturally contaminated goat's milk, cultured milk products and ice cream manufactured with L. monocytogenes, and unpasteurized milk inoculated with heat- and freeze-injured cells of L. monocytogenes. Generally, the media and two-stage enrichment protocol developed by the USDA, with plating of samples after two consecutive 24-h incubation periods, yielded better recoveries than all other enrichment media incubated for 24 h. A modified USDA procedure, incorporating nonselective pre-enrichment of samples by omitting acriflavine and nalidixic acid from the primary USDA enrichment broth, and transfer of a larger volume of the initial culture broth to the secondary enrichment media, significantly increased recoveries of low numbers of sublethally stressed L. monocytogenes. Prolonged incubation of samples in the FDA enrichment broth, for 7 days, did not consistently improve recoveries over the initial 24-h incubation time of the medium. The selective plating medium developed by the USDA, lithium chloride-phenylethanol-moxalactam agar, was the most effective plating agar for isolation of L. monocytogenes following enrichment of samples in any broth culture, and increased recoveries of L. monocytogenes by 19-40% compared with other selective agar media tested.

Microbiological safety of traditional and starter-mediated processes for the manufacture of Italian dry sausage.

Microbiological changes occurring during the commercial manufacture of Italian dry sausages (Genoa and salametti) were studied in two urban Canadian centres over a 5 month period. A comparison was made between 6 plants which used bacterial starter cultures and 4 plants where more traditional processes (without starters) were used. A total of 600 samples of raw, fermented and finished products were tested for the presence of coliforms, salmonellae, staphylococci, streptococci, the rate of pH reduction and final water activity (aW). Numbers of total bacteria peaked earlier and were significantly higher in sausages at the fermentation stage produced with starter cultures than in those traditionally manufactured. This corresponded with a more rapid drop in pH of the starter-inoculated products. Staphylococci and streptococci were significantly higher in starter-fermented Genoa sausages at the fermentation stage, but no significant differences were seen in the microbiological content or aW of mature finished sausages manufactured by the two different techniques. Of 128 randomly chosen isolates of coagulase-positive staphylococci, 34.4% were enterotoxin producers and 80% of these produced type A toxin. Enterotoxigenic staphylococci were found in 2 different samples of finished salametti and one sample of finished Genoa made with starter cultures and in one sample of finished Genoa made without added culture. Total numbers of staphylococci in these samples were not greater than 500/g. No correlation between the method of manufacture and presence of enterotoxigenic staphylococci could be made. Five subsamples from one lot of raw Genoa were the only samples positive for Salmonella during this study. Results indicated that low temperature traditional fermentations can yield products which are as safe as those produced by the higher temperature starter-controlled process. One of the most important elements in the traditional process was believed to be the selection and use of raw materials of the highest possible quality.

Occurrence and significance of streptococci in fermented Italian type dry sausage.

Commercial cultures used in Canada for the manufacture of Italian dry sausage were examined to determine their microbial composition and suitability for low temperature (less than or equal to 20 degrees C) meat fermentations. Temperature optima in both laboratory media and commercial meat mixtures were generally too high to allow these cultures to be of substantial advantage in this application. In addition, media used currently for the enumeration of streptococci and related organisms from fermented meat products were found to be inadequately specific and often required confirmatory inspection of colonies by conventional phase contrast microscopy. Streptococci were isolated from Italian dry sausage manufactured commercially with and without added starter cultures. Streptococci persisted in sausages produced by both techniques with slightly higher numbers present in starter-acidulated sausages. About 55.5% of the 312 streptococci studied were enterococci (Lancefield's Group D). Streptococci were found in several samples of commercial starter cultures but it was felt that elevated ripening temperatures used for sausage manufactured by the starter-mediated process and meat handling practices were more important factors influencing streptococci recovery from sausage material.

Prevalence of Salmonella and Thermophilic Campylobacter in Fresh Pork, Beef, Veal and Poultry in Canada 1.

A national surveillance program was undertaken in Canada to establish the prevalence and distribution of Salmonella and thermophilic Campylobacter biotypes in slaughter animals and poultry. During the years 1983 to 1986, samples were collected from federally inspected abbatoirs across Canada and tested at regional laboratories. The laboratory isolation procedure for thermophilic Campylobacter included selective enrichment and isolates were characterized according to Lior's biotyping scheme. Salmonella were isolated from 17.5% pork, 2.6% beef and 4.1% veal carcasses. Thermophilic Campylobacter were isolated from 16.9% pork, 22.6% beef and 43.1% veal carcasses. Salmonella were isolated from 69.1 % turkey and 60.9% chicken carcasses, and thermophilic Campylobacter were isolated from 73.7% and 38.2% turkey and chicken carcasses, respectively. Salmonella typhimurium was the most frequently isolated serotype, and predominant in broiler chickens from 1983 to 1985. Salmonella brandenburg was predominant in pork, and Salmonella schwarzengrund was the primary serotype from turkey carcasses. Campylobacter jejuni biotypes I and II were the most frequently isolated biotypes from beef, veal and poultry. Although Campylobacter coli biotype I was the predominant thermophilic Campylobacter in pork, 41.1% of the biotyped isolates from pork were C. jejuni biotypes I and II.

Isolation and characterization of cephalothin-susceptible Campylobacter coli from slaughter cattle.

In a recent meat survey, 10 of 13 (77%) Campylobacter coli isolates were susceptible to cephalothin. These organisms were isolated from nine slaughter cattle from eight meat packing establishments. All 10 isolates grew at 43 degrees C but not at 25 degrees C, were catalase and oxidase positive, and were susceptible to nalidixic acid (30 micrograms) and cephalothin (30 micrograms). The cultures were subsequently identified as C. coli serogroup 20, biotype I (Lior scheme). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that protein and lipopolysaccharide profiles of whole cell preparations of the 10 cephalothin-susceptible strains and the reference strain for C. coli serogroup 20 were very similar. The plasmid profiles of these 11 strains were identical.