Induction of the Viable-but-Nonculturable State in Salmonella Contaminating Dried Fruit.

Salmonella can become viable but nonculturable (VBNC) in response to environmental stressors, but the induction of the VBNC state in Salmonella contaminating ready-to-eat dried fruit is poorly characterized. Dried apples, strawberries, and raisins were mixed with a five-strain cocktail of Salmonella at 4% volume per weight of dried fruit at 109 CFU/g. The inoculated dried fruit were then dried in desiccators at 25°C until the water activity (aw) approximated that of the uninoculated dried fruit. However, Salmonella could not be recovered after drying, not even after enrichment, suggesting a population reduction of approximately 8 log CFU/g. To assess the potential impact of storage temperature on survival, dried apples were spot-inoculated with the Salmonella cocktail, dried under ambient atmosphere at 25°C, and stored at 4 and 25°C. Spot inoculation permitted recovery of Salmonella on dried apple after drying, with the population of Salmonella decreasing progressively on dried apples stored at 25°C until it was undetectable after about 46 days, even following enrichment. The population decline was noticeably slower at 4°C, with Salmonella being detected until 82 days. However, fluorescence microscopy and laser scanning confocal microscopy with the LIVE/DEAD BacLight bacterial viability system at time points at which no Salmonella could be recovered on growth media even following enrichment showed that a large proportion (56 to 85%) of the Salmonella cells on the dried fruit were viable. The data suggest that the unique combination of stressors in dried fruit can induce large numbers of VBNC cells of Salmonella. IMPORTANCE Salmonella is a leading foodborne pathogen globally causing numerous outbreaks of foodborne illnesses and remains the leading contributor to deaths attributed to foodborne disease in the United States and other industrialized nations. Therefore, efficient detection methods for Salmonella contaminating food are critical for public health and food safety. Culture-based microbiological methods are considered the gold standard for the detection and enumeration of Salmonella in food. Findings from this study suggest that unique stressors on dried fruit can induce the VBNC state in Salmonella, thus rendering it undetectable with culture-based methods even though the bacteria remain viable. Therefore, strong consideration should be given to using, in addition to culture-based methods, microscopic and molecular methods for the accurate detection of all viable and/or culturable cells of Salmonella contaminating dried fruit, as all of these cells have the potential to cause human illness.

A comprehensive perspective of food nanomaterials.

Nanotechnology is a rapidly developing toolbox that provides solutions to numerous challenges in the food industry and meet public demands for healthier and safer food products. The diversity of nanostructures and their vast, tunable functionality drives their inclusion in food products and packaging materials to improve their nutritional quality through bioactive fortification and probiotics encapsulation, enhance their safety due to their antimicrobial and sensing capabilities and confer novel sensorial properties. In this food nanotechnology state-of-the-art communication, matrix materials with particular focus on food-grade components, existing and novel production techniques, and current and potential applications in the fields of food quality, safety and preservation, nutrient bioaccessibility and digestibility will be detailed. Additionally, a thorough analysis of potential strategies to assess the safety of these novel nanostructures is presented.

Foodborne outbreaks in Canada linked to produce: 2001 through 2009.

Foodborne disease outbreaks associated with fresh fruits and vegetables have been increasing in occurrence worldwide. Canada has one of the highest per capita consumption rates of fresh fruits and vegetables in the world. In this article, we review the foodborne disease outbreaks linked to produce consumption in Canada from 2001 through 2009. The 27 produce-related outbreaks included an estimated 1,549 cases of illness. Bacterial infection outbreaks represented 66% of the total. Among these, Salmonella was the most frequent agent (50% of outbreaks) followed by Escherichia coli (33%) and Shigella (17%). Cyclospora cayetanensis was the only parasite detected and was associated with seven outbreaks. Among the foodborne viruses, only hepatitis A was implicated in two outbreaks. The food vehicles most commonly implicated in outbreaks were leafy greens and herbs (26% of outbreaks), followed by seed sprouts (11%). Contamination sources and issues related to the future control of fresh produce-related foodborne disease outbreaks also are discussed.

Listeria monocytogenes in two different poultry facilities: Manual and automatic evisceration.

Listeriosis is a serious foodborne disease caused by Listeria monocytogenes, a pathogen often found in food processing plants. Poultry meat and its derivatives may harbor L. monocytogenes even if good manufacturing practices are implanted in abattoirs. Little information exists in Brazil on the frequency of L. monocytogenes contamination, even though the country is considered the top poultry meat exporter in the world. This study attempted to compare 2 exporters poultry facilities following same the standards but differing only in manual (plant M) or automatic (plant A) evisceration. Eight hundred fifty-one samples from food, food contact and non-food contact surfaces, water, and workers' hands were collected from cage to finished products over a 1-yr period. In plant A, 20.1% of the samples were positive for L. monocytogenes, whereas in plant M, 16.4% was found. The greatest incidence of contamination with the pathogen in plant A was found in non-food contact surfaces (27.3%), while in plant M, it was found in products (19.4%). The most prevalent serovars were 1/2a or 3a (plant M) and 4b, 4d, or 4e (plant A). Despite having proper hygiene and good manufacturing practices, controlling the entry and persistence of L. monocytogenes in processing facilities remains a formidable task.

The presence of capsule in Cryptococcus neoformans influences the gene expression profile in dendritic cells during interaction with the fungus.

The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1alpha, IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.

Survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus.

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

Norovirus cross-contamination during food handling and interruption of virus transfer by hand antisepsis: experiments with feline calicivirus as a surrogate.

While there is good epidemiological evidence for foods as vehicles for norovirus transmission, the precise means of spread and its control remain unknown. The feline calicivirus was used as a surrogate for noroviruses to study infectious virus transfer between hands and selected types of foods and environmental surfaces. Assessment of the potential of selected topicals in interrupting such virus transfer was also made. Ten microliters of inoculum of feline calicivirus deposited onto each fingerpad of adult subjects was allowed to air dry and the contaminated area on individual fingerpads was pressed (10 s at a pressure of 0.2 to 0.4 kg/cm2) onto 1-cm-diameter disks of ham, lettuce, or brushed stainless steel. The virus remaining on the donor and that transferred to the recipient surfaces was eluted and plaque assayed. Virus transfer to clean hands from experimentally contaminated disks of ham, lettuce, and stainless steel was also tested. Nearly 46 +/- 20.3, 18 +/- 5.7, and 13 +/- 3.6% of infectious virus was transferred from contaminated fingerpads to ham, lettuce, and metal disks, respectively. In contrast, approximately 6 +/- 1.8, 14 +/- 3.5, and 7 +/- 1.9% virus transfer occurred, respectively, from ham, lettuce, and metal disks to hands. One-way analysis of variance test showed that pretreatment (washing) of the fingerpads either with water or with both topical agent and water significantly (P < 0.05) reduced virus transfer to < or = 0.9%, as compared with < or = 2.3 and < or = 3.4% transfer following treatments with either 75% (vol/vol) ethanol or a commercial hand gel containing 62% ethanol, respectively. Despite wide variations in virus transfer among the targeted items used, intervention agents tested reduced virus transfer significantly (P < 0.05) when compared with that without such treatments (71 +/- 8.9%). These findings should help in a better assessment of the potential for cross-contamination of foods during handling and also assist in developing more effective approaches to foodborne spread of norovirus infections.

A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus.

Feline calicivirus (FCV) has been used by researchers as a surrogate for Norwalk virus (NV), since they share a similar genomic organization, physicochemical characteristics, and are grouped in the same family, Caliciviridae. Unlike NV, however, FCV can grow in established cell lines and produce a syncytial form of cytopathic effect. In this report, we describe the development and standardization of a plaque assay for FCV using monolayers of an established line of feline kidney (CrFK) cells in 12-well cell culture plates. The assay method has demonstrated reproducibility, ease of performance and resulted in clear plaque zones, readable in 24 h after virus inoculation. The infectivity titre of the virus by this plaque assay agreed well with tissue culture infectious dose(50) (TCID(50)) determinations. The described plaque assay would be a valuable tool in conducting various quantitative investigations using FCV as a model for NV and Norwalk-like viruses (NLV).

A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters.

DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.

Foodborne outbreaks in Canada linked to produce.

Examples of foodborne outbreaks traced to fresh fruits and vegetables can be found worldwide. The quantity of produce eaten per capita has been increasing steadily over the past two decades, creating a heightened potential for produce-related foodborne disease. A number of outbreaks identified during this time period were reviewed, with particular emphasis placed on incidents that have occurred in Canada. The collective information highlights the diversity of infectious agents and produce items involved, with a view to the prevention of fresh produce-related foodborne disease in the future.

Patterns of chemokine expression in models of Schistosoma mansoni inflammation and infection reveal relationships between type 1 and type 2 responses and chemokines in vivo.

To explore the roles of chemokines in type 1 and type 2 responses in vivo, we examined mRNA expression for a panel of up to 17 chemokines in experimental mouse models using Schistosoma mansoni. These studies revealed that Mig (monokine induced by gamma interferon), cytokine-responsive gene 2/10-kDa interferon-inducible protein, RANTES, lymphotactin, macrophage inflammatory protein 1beta (MIP-1beta), JE/monocyte chemoattractant protein 1, and MIP-2 are associated with type 1 egg-induced responses and that thymus-derived chemotactic agent 3 (TCA3), eotaxin, MIP-1alpha, and MIP-1gamma are associated with type 2 egg-induced responses. After cercarial infection, both type 1-associated and type 2-associated chemokines were elevated in the livers of infected mice presensitized with eggs and recombinant interleukin-12 (rIL-12), a regimen that diminishes pathology. Neutralization of IL-12 or gamma interferon during egg deposition reversed the effects of prior treatment with rIL-12, leading to a return to larger granulomas; persistently elevated expression of TCA3, eotaxin, and MIP-1alpha; and a marked reduction in the expression of type 1-associated chemokines despite the maintenance of a dominant type 1 cytokine response in the draining lymph nodes. Our findings suggest that there are patterns of coordinate chemokine expression characteristic of type 1 and type 2 responses in vivo; that the cells recruited by a given pattern of chemokines may differ, depending on the composition of peripheral populations; and that patterns of tissue expression of chemokines may determine the character of an inflammatory response independently of the dominant pattern of differentiation of antigen-specific T cells. Our data reveal new relationships between chemokines and polarized immune responses and suggest that end organ inflammation might be altered by chemokine blockade without necessitating reversal of the phenotype of the majority of differentiated T cells.

Adsorption, attachment and biofilm formation among isolates of Listeria monocytogenes using model conditions.

AIMS: To determine whether isolates of Listeria monocytogenes differ in their ability to adsorb and form biofilms on a food-grade stainless steel surface. METHODS AND RESULTS: Strains were assessed for their ability to adsorb to a test surface over a short time period. Although some differences in numbers of bound cells were found among the strains, there were no correlations between the degree of adsorption and either the serotype or source of the strain. The ability of each strain to form a biofilm when grown with the test surface was also assessed. With the exception of a single strain, all strains adhered as single cells and did not form biofilms. Significant differences in adherence levels were found among strains. Strains demonstrating enhanced attachment produced extracellular fibrils, whereas those which adhered poorly did not. A single strain formed a biofilm consisting of adhered single cells and aggregates of cells. CONCLUSIONS: Significant differences were found in the ability of various L. monocytogenes strains to attach to a test surface. In monoculture, the majority of strains did not form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in attachment and biofilm formation among strains provide a basis to study these characteristics in L. monocytogenes.

Standardization of a method to determine the efficacy of sanitizers in inactivating human pathogenic microorganisms on raw fruits and vegetables.

The efficacy of sanitizers in killing human pathogenic microorganisms on a wide range of whole and fresh-cut fruits and vegetables has been studied extensively. Numerous challenge studies to determine the effects of storage conditions on survival and growth of pathogens on raw produce have also been reported. Results of these studies are often difficult to assess because of the lack of sufficient reporting of methods or, comparatively, because of variations in procedures for preparing and applying inocula to produce, conditions for treatment and storage, and procedures for enumerating pathogens. There is a need for a standard method to accurately determine the presence and populations of pathogenic microorganisms on produce. The adoption of standard, well-characterized reference strains would benefit a comparative assessment of a basic method among laboratories. A single protocol will not be suitable for all fruits and vegetables. Modifications of a basic method will be necessary to achieve maximum recovery of pathogens on various types of produce subjected to different sanitizer or storage treatments. This article discusses parameters that must be considered in the course of developing a basic standard method against which these modifications could be made.

Cytokine-responsive gene-2/IFN-inducible protein-10 expression in multiple models of liver and bile duct injury suggests a role in tissue regeneration.

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl(4) treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl(4), D-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl(4) injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.

A small outbreak of listeriosis potentially linked to the consumption of imitation crab meat.

A small outbreak of listeriosis involving two previously healthy adults occurred in Ontario. Food samples obtained from the refrigerator of the patients included imitation crab meat, canned black olives, macaroni and vegetable salad, spaghetti sauce with meatballs, mayonnaise and water. All of the samples except the water contained Listeria monocytogenes. The three most heavily contaminated samples were the imitation crab meat, the olives and the salad which contained 2.1 x 109, 1.1 x 107 and 1.3 x 106 cfu g-1, respectively. L. monocytogenes serotype 1/2b was isolated from the patients, as well as from the opened and unopened imitation crab meat. Molecular typing of the isolates by both randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) typing demonstrated the imitation crab meat and clinical strains to be indistinguishable. Challenge studies performed with a pool of L. monocytogenes strains showed that imitation crab meat, but not olives, supported growth of the organism. In this study we have shown for the first time the potential involvement of imitation crab meat in a small outbreak of listeriosis. In terms of disease prevention, temperature control is critical to prevent or reduce the growth of this foodborne pathogen. In addition, with refrigerated products having a long (> 30 d) shelf life, additional safety factors must be used to prevent the growth of foodborne pathogens such as L. monocytogenes.

Rapid concentration and detection of hepatitis A virus from lettuce and strawberries.

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.

Contamination of foods by food handlers: experiments on hepatitis A virus transfer to food and its interruption.

Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm(2) resulted in transfer of 9.2% +/- 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% +/- 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.