Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell-derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti-HuNoV efficacy of two alcohol-based commercial hand sanitizers and 60% ethanol by suspension assay using RNase-RT-qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase-RT-qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2-3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

Efficacy of a disinfectant containing silver dihydrogen citrate against GI.6 and GII.4 human norovirus.

AIMS: Human norovirus is a major public health burden and is resistant to numerous sanitizers and disinfectants. In this study, we tested the efficacy of an antimicrobial product containing a blend of silver ions and citric acid (silver dihydrogen citrate; SDC) against GI.6 and GII.4 HuNoV. METHODS AND RESULTS: Pure® hard surface disinfectant (Pure Bioscience, El Cajon, CA) was evaluated using ASTM International virucidal suspension and stainless steel carrier assays. The effect of SDC (or citrate alone) exposure on viral integrity was evaluated using RT-qPCR, transmission electron microscopy, sodium dodecyl sulphate-polyacrylamide gel electrophoresis/Western blot analysis and a receptor-binding assay. Suspension assays showed a 4·0 log10 reduction in RNA copy number within 5 min, while carrier assays showed a 2·0-3·0 log10 reduction in 30 min. Incorporating a simulated soil load into the sample matrix significantly reduced product efficacy. Treated particles displayed deformation and aggregation, a 50% reduction in viral capsid protein band intensity, and an 80% reduction in histo-blood group antigen receptor-binding ability. CONCLUSIONS: Our results suggest that SDC acts exclusively on the viral capsid, and shows efficacy against HuNoV when used on precleaned surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study sheds light on the mechanisms and efficacy of a novel antimicrobial against HuNoV. Our results suggest: (i) silver ions exclusively target the viral capsid, and not the RNA genome; (ii) citrate is not crucial for HuNoV capsid deformation.

Destruction of the Capsid and Genome of GII.4 Human Norovirus Occurs during Exposure to Metal Alloys Containing Copper.

Human norovirus (HuNoV) represents a significant public health burden worldwide and can be environmentally transmitted. Copper surfaces have been shown to inactivate the cultivable surrogate murine norovirus, but no such data exist for HuNoV. The purpose of this study was to characterize the destruction of GII.4 HuNoV and virus-like particles (VLPs) during exposure to copper alloy surfaces. Fecal suspensions positive for a GII.4 HuNoV outbreak strain or GII.4 VLPs were exposed to copper alloys or stainless steel for 0 to 240 min and recovered by elution. HuNoV genome integrity was assessed by reverse transcription-quantitative PCR (RT-qPCR) (without RNase treatment), and capsid integrity was assessed by RT-qPCR (with RNase treatment), transmission electron microscopy (TEM), SDS-PAGE/Western blot analysis, and a histo-blood group antigen (HBGA) binding assay. Exposure of fecal suspensions to pure copper for 60 min reduced the GII.4 HuNoV RNA copy number by ∼3 log10 units when analyzed by RT-qPCR without RNase treatment and by 4 log10 units when a prior RNase treatment was used. The rate of reduction of the HuNoV RNA copy number was approximately proportional to the percentage of copper in each alloy. Exposure of GII.4 HuNoV VLPs to pure-copper surfaces resulted in noticeable aggregation and destruction within 240 min, an 80% reduction in the VP1 major capsid protein band intensity in 15 min, and a near-complete loss of HBGA receptor binding within 8 min. In all experiments, HuNoV remained stable on stainless steel. These results suggest that copper surfaces destroy HuNoV and may be useful in preventing environmental transmission of the virus in at-risk settings.

Molecular methods used to estimate thermal inactivation of a prototype human norovirus: more heat resistant than previously believed?

Two molecular-based methods for estimating capsid integrity as a proxy for virus infectivity were used to produce thermal inactivation profiles of Snow Mountain virus (SMV), a prototype human norovirus (HuNoV). Monodispersed virus suspensions were exposed to 77, 80, 82 and 85 °C for various times, pre-treated with either propidium monoazide (PMA) or RNase, and subjected to RNA isolation followed by RT-qPCR amplification. D-values were 25.6 ± 2.8, 3.1 ± 0.1, 0.7 ± 0.04 and 0.2 ± 0.07 min at 77, 80, 82 and 85 °C, respectively for PMA-treated SMV; and 16.4 ± 0.4, 3.9 ± 0.2 0.9 ± 0.3 and 0.12 ± 0.00 min at 77, 80, 82 and 85 °C, respectively for RNase-treated SMV. Corresponding zD values were 3.80 °C and 3.71 °C for PMA and RNase-treated virus, respectively. Electron microscopy data applied to heat-treated virus-like particles supported this relatively high degree of thermal resistance. The data suggest that SMV is more heat resistant than common cultivable HuNoV surrogates. Standardized thermal inactivation methods (such as milk pasteurization) may not be stringent enough to eliminate this virus and perhaps other HuNoV.

Persistence and transferability of noroviruses on and between common surfaces and foods.

Human noroviruses (HuNoV) are the leading cause of foodborne disease, and poor personal hygiene practices of infected workers are the most common mode of contamination. The purpose of this study was to characterize the persistence and transferability of representative noroviruses Norwalk virus (NV), Snow Mountain virus (SMV), and murine norovirus 1 (MNV-1) on and between solid surfaces and foods. Changes in virus concentration on artificially inoculated solid surfaces (stainless steel, ceramic, and Formica) or lettuce were monitored over a period of 14 to 42 days. Virus transfer was evaluated from donor (solid surface) to recipient (food, e.g., lettuce and sliced turkey deli meat) for up to 2 h postinoculation. Viruses were recovered by elution and titered with reverse transcription quantitative PCR (RT-qPCR) and/or infectivity assay, as appropriate. Based on RTqPCR, the concentration of NV and SMV on surfaces dropped gradually over time, with an average reduction of 1.5 to 2.0 and 1.8 to 2.3 log, respectively, after 42 days, with no statistically significant differences by surface. When inoculated onto lettuce stored for 2 weeks at 4°C and room temperature, the titers of NV and SMV dropped by approximately 1.0 and 1.2 to 1.8 log, respectively. Comparatively, the RT-qPCR signal associated with purified HuNoV RNA placed on the same surfaces was more rapidly lost to degradation. Transfer efficiency ranged from 0 to 26 % for lettuce and from 55 to 95 % for sliced turkey deli meat, with statistically significant differences (P ≤ 0.05) in transferability as a function of contact pressure (100 and 1,000 g/9 cm(2)) and inoculum drying time. When similar experiments were done with MNV-1, infectious virus failed to be detected on solid surfaces after storage day 21, although the virus did persist on lettuce. This study provides much needed quantitative data for use in risk assessment efforts intended to characterize the transmission of HuNoV during food preparation and handling.

Impact of storage temperature and product pH on the survival of Listeria monocytogenes in vacuum-packaged souse.

Souse is a fully cooked, ready-to-eat gelled pork product. There is a zero-tolerance policy for Listeria monocytogenes in ready-to-eat meat products. The survival and/or growth of L. monocytogenes in souse is unknown. The effectiveness of three different souse formulations (pH 4.3, 4.7, and 5.1) for controlling the growth of L. monocytogenes at two refrigerated storage temperatures (5 and 10 degrees C) was evaluated. All products were vacuum packaged. Uninoculated product was prepared as the control, and other products were artificially surface contaminated with a three-strain cocktail of L. monocytogenes (10(6) CFU/ cm2). Microbial counts were obtained on selective and nonselective media twice weekly through 8 weeks of storage. Souse did not support the growth of L. monocytogenes regardless of product formulation or storage temperature. At 5 degrees C, D-values for products with pH values of 4.7 and 5.1 were not different, but survival of L. monocytogenes in product with a lower pH (4.3) was decreased compared with survival in products with higher pH values (P < 0.05). Survival of L. monocytogenes was not impacted by storage temperatures (P > 0.05). Consumer acceptability (n = 75 souse consumers) of pH 4.3 products was not different from that for (typical) pH 4.7 products (P > 0.05). These results indicate that conventionally produced souse does not support the growth of L. monocytogenes and that inactivation of the organism is more likely in products formulated at a lower pH (< or = 4.3) without affecting consumer acceptance.

PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores.

This paper describes a molecular-based method which is able to discriminate between viable and inactivated Bacillus subtilis spores by utilizing the DNA-intercalating dye propidium monoazide. The approach should be valuable in our attempt to employ molecular methods to streamline the evaluation of process validation using bacterial endospores.

Development of a risk-ranking framework to evaluate potential high-threat microorganisms, toxins, and chemicals in food.

Through a cooperative agreement with the U.S. Food and Drug Administration, the Institute of Food Technologists developed a risk-ranking framework prototype to enable comparison of microbiological and chemical hazards in foods and to assist policy makers, risk managers, risk analysts, and others in determining the relative public health impact of specific hazard-food combinations. The prototype is a bottom-up system based on assumptions that incorporate expert opinion/insight with a number of exposure and hazard-related risk criteria variables, which are propagated forward with food intake data to produce risk-ranking determinations. The prototype produces a semi-quantitative comparative assessment of food safety hazards and the impacts of hazard control measures. For a specific hazard-food combination the prototype can produce a single metric: a final risk value expressed as annual pseudo-disability adjusted life years (pDALY). The pDALY is a harmonization of the very different dose-response relationships observed for chemicals and microbes. The prototype was developed on 2 platforms, a web-based user interface and an Analytica(R) model (Lumina Decision Systems, Los Gatos, Calif., U.S.A.). Comprising visual basic language, the web-based platform facilitates data input and allows use concurrently from multiple locations. The Analytica model facilitates visualization of the logic flow, interrelationship of input and output variables, and calculations/algorithms comprising the prototype. A variety of sortable risk-ranking reports and summary information can be generated for hazard-food pairs, showing hazard and dose-response assumptions and data, per capita consumption by population group, and annual p-DALY.

Inactivation of bacterial pathogens in human milk by high-pressure processing.

Low-temperature, long-time (LTLT) pasteurization assures the safety of banked human milk; however, heat can destroy important nutritional biomolecules. High-pressure processing (HPP) shows promise as an alternative for pasteurization of breast milk. The purpose of this study was to investigate the efficacy of HPP for inactivation of selected bacterial pathogens in human milk. Human milk was inoculated with one of five pathogens (10(8) to 10(9) CFU/ml), while 0.1% peptone solution solutions with the same levels of each organism were used as controls. The samples were subjected to 400 MPa at 21 to 31 degrees C for 0 to 50 min or to 62.5 degrees C for 0 to 30 min (capillary tube method) to simulate LTLT pasteurization. Tryptic soy agar and selective media were used for enumeration. Traditional thermal pasteurization resulted in inactivation (> 7 log) of all pathogens within 10 min. In human milk and in peptone solution, a 6-log reduction was achieved after 30 min of HPP for Staphylococcus aureus ATCC 6538. After 30 min, S. aureus ATCC 25923 was reduced by 8 log and 6 log in human milk and peptone solution, respectively. Treatments of 4 and 7 min resulted in an 8-log inactivation of Streptococcus agalactiae ATCC 12927 in human milk and peptone solution, respectively, while Listeria monocytogenes ATCC 19115 required 2 min for an 8-log inactivation in human milk. Escherichia coli ATCC 25922 was inactivated by 8 log after 10 min in peptone solution and by 6 log after 30 min in human milk. These data suggest that HPP may be a promising alternative for pasteurization of human milk. Further research should evaluate the efficacy of HPP in the inactivation of relevant viral pathogens.

Feasibility of utilizing bioindicators for testing microbial inactivation in sweetpotato purees processed with a continuous-flow microwave system.

Continuous-flow microwave heating has potential in aseptic processing of various food products, including purees from sweetpotatoes and other vegetables. Establishing the feasibility of a new processing technology for achieving commercial sterility requires evaluating microbial inactivation. This study aimed to assess the feasibility of using commercially available plastic pouches of bioindicators containing spores of Geobacillius stearothermophilus ATCC 7953 and Bacillus subtilis ATCC 35021 for evaluating the degree of microbial inactivation achieved in vegetable purees processed in a continuous-flow microwave heating unit. Sweetpotato puree seeded with the bioindicators was subjected to 3 levels of processing based on the fastest particles: undertarget process (F(0) approximately 0.65), target process (F(0) approximately 2.8), and overtarget process (F(0) approximately 10.10). After initial experiments, we found it was necessary to engineer a setup with 2 removable tubes connected to the continuous-flow microwave system to facilitate the injection of indicators into the unit without interrupting the puree flow. Using this approach, 60% of the indicators injected into the system could be recovered postprocess. Spore survival after processing, as evaluated by use of growth indicator dyes and standard plating methods, verified inactivation of the spores in sweetpotato puree. The log reduction results for B. subtilis were equivalent to the predesigned degrees of sterilization (F(0)). This study presents the first report suggesting that bioindicators such as the flexible, food-grade plastic pouches can be used for microbial validation of commercial sterilization in aseptic processing of foods using a continuous-flow microwave system.

Recovery and detection of Vibrio vulnificus during cold storage.

Different cultural techniques and molecular methods for the detection of Vibrio vulnificus during cold storage in a model broth system were compared. Two strains of V. vulnificus were grown to stationary phase and inoculated (10(6) CFU/mL) into tryptic soy broth with 2% sodium chloride (TSBN2) or artificial seawater (ASW), both pre-chilled to 5 degrees C. These were stored for 10 days, with sub-sampling conducted at time 0 and every 2 days thereafter. Each subsample was plated, by both pour and spread plate techniques, onto tryptic soy agar 2% sodium chloride (TSAN2) with or without catalase (400 or 600 U) or sodium pyruvate (80 or 160 mg) supplementation. Nucleic acids were extracted from subsamples and subjected to PCR and RT-PCR with hemolysin as the target. Higher recoveries of V. vulnificus were obtained with spread plating compared to pour plating (P<0.05). The addition of sodium pyruvate (80 mg) or catalase (400 U) significantly increased cell recovery (P<0.05). PCR amplification signals were stronger than RT-PCR signals at each timepoint, and results were generally consistent between TSAN2 and ASW for each strain. These results will aid in the design of optimum methods to recover and/or detect V. vulnificus cells subjected to sublethal stress that might be encountered in food processing and storage.

Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters.

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.

Molecular characterization of Listeria monocytogenes of the serotype 4b complex (4b, 4d, 4e) from two turkey processing plants.

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach.

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods.

AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.

Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR.

AIMS: We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria. METHODS AND RESULTS: Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide. The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol. CONCLUSIONS: Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths. SIGNIFICANCE AND IMPACT OF THE STUDY: The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.

Zirconium hydroxide effectively immobilizes and concentrates human enteric viruses.

BACKGROUND: Detection of human enteric viruses in foods and environmental samples requires concentration of viruses from complex matrices before application of molecular or cultural methods. Previous studies have described the use of zirconium hydroxide to concentrate bacteria from clinical, environmental, and food samples. AIMS: Our study describes the application of zirconium hydroxide to concentrate human enteric viruses. METHODS: Poliovirus type 1, hepatitis A virus (HAV) strain HM-175, and Norwalk virus (NV) were used as models. Virus recovery was evaluated both as loss to discarded supernatants and as recovery in the precipitated pellets. RESULTS: Poliovirus type 1, based on the plaque assay recoveries, ranged from 16 to 59% with minimal loss to the supernatant (1-5%). For both HAV and NV, RT-PCR amplicons of appropriate sizes were detected and confirmed in the pellet fraction with no visible amplicons from the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid and inexpensive method shows promise as an alternative means to concentrate enteric viruses.

Human Enteric Viruses as Causes of Foodborne Disease.

Recent epidemiological evidence indicates that enteric viruses are the leading cause of foodborne disease in the U.S.A. and, indeed, worldwide. Certainly, advances in epidemiology and molecular biology have improved the ability to study this previously elusive group of foodborne pathogens. The purpose of this article is to review the agents, transmission routes, epidemiology, persistence, diagnosis, and detection of foodborne viruses and their diseases, with specific reference to the role that contemporary technologies have had in improving our understanding of this important group of emerging foodborne pathogens.

A weighted composite dose-response model for human salmonellosis.

This article describes the development of a weighted composite dose-response model for human salmonellosis. Data from previously reported human challenge studies were categorized into two different groups representing low and moderately virulent/pathogenic Salmonella strains based on a disease end point. Because epidemiological data indicate that some Salmonella strains are particularly pathogenic, and in the absence of human feeding study data for such strains, Shigella dysenteriae was used as a proxy for highly virulent strains. Three single-hit dose-response models were applied to the human feeding study data and evaluated for best fit using maximum likelihood estimation: (1) the exponential (E-1pop), (2) the two-subpopulation exponential (E-2pop), and (3) the Beta-Poisson (BP). Based on the goodness-of-fit test, the E-1pop and BP were the best-fit models for low and moderately virulent/pathogenic Salmonella strains, and the E-2pop and BP models were better for highly virulent/pathogenic strains. Epistemic analysis was conducted by determining the degree of confidence associated with the selected models, which was found to be 50%/50% (E-1pop/BP) for low and moderately pathogenic Salmonella strains, and 9.8%/90.2% (E-2pop/BP) for highly virulent strains. The degree of confidence for each component model and variations in the proportion of strains within each virulence/pathogenicity category were incorporated into the overall composite model. This study describes the influence of variation in strain virulence and host susceptibility on the shape of the population dose-response relationship.