Progress 10/01/07 to 09/30/14
Outputs
Target Audience: Researchers and industry personnel interested in pre and post-harvest contamination anddisinfectionof fresh produce. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate andundergraduate students and visiting scientists. How have the results been disseminated to communities of interest? Results have been published in various reports and manuscvripts. The last manuscript is currently being reviewed. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Pathogen inactivation: The first processes evaluated were various chemical sanitizers commonly used by the food industry. Cryptosporidium parvum, E. intestinalis, 5-strain pool of Salmonella and 5-strain pool of E. coli O157:H7 were used to compare their responses to chemical treatments. A 3 and 6 log reduction was obtained with E. coli O157:H7 when treated with 0.5% hydrogen peroxide for 1 min and 5 min respectively. Treatment with Pro-San yielded a 3 log reduction when incubated for 1 or 5 min. A 4 log reduction in viability was observed when E. intestinalis spores were treated with 0.5% hydrogen peroxide while Pro-San had no effect. Neither treatment was effective against C. parvum. Different concentrations and combinations of levulinic acid (LA)/SDS were tested for their ability to reduce infectivity of C. parvum, E. intestinalis, and E. coli O157:H7. Pathogens were treated at 20 ± 2oC. When microsporidia spores and Cryptosporidium oocysts were treated for 30 and 60 min, none of the combinations proved to be effective at inactivating the spores or oocysts. At 3% LA/2%SDS and 2% LA/1%SDS for 30 min, 1 hr and 2 hr no effect on parasite viability was observed. E. coli O157:H7 was highly susceptible at various concentrations and exposure times tested, but SDS or levulinic acid alone had very limited effect on E. coli viability. Thermal inactivation. Common freezing methodologies used with strawberries and raspberries (whole or cut, with or without syrup) and blanching of spinach leaves before freezing were used to determine inactivation and survival of foodborne pathogens. Strawberries and raspberries (whole or cut, with or without syrup, and sugar) were inoculated with a five-serotype mixture of Salmonella, five isolate mixture of E. coli O157:H7, E. intestinalis, and C. parvum. E. coli O157:H7 and Salmonella were not affected by any of the freezing processes whereas partial inactivation was achieved with Cryptosporidium and microsporidia. Less than 1 log reduction was observed with Salmonella or STEC (raspberries, whole and sliced strawberries) with a non-significant but larger reduction when using water instead of syrup or sugar. At least a 4 log reduction was noted with Cryptosporidium in all treatments. With microsporidia in raspberries, 3, 1, and 0.8 log reductions were observed in water, sugar, and syrup respectively. When inoculated onto whole strawberries, 1, 0.47 and 0.70 log reductions were achieved with water, syrup and sugar respectively. Similar results were observed when using sliced strawberries. Blanching inactivated bacteria significantly, but did not eliminate them completely (detectable by enrichment) and parasites were not infectious after blanching. Basil, broccoli, bean sprouts, and green peas were experimentally inoculated with E. coli O157:H7, Salmonella, microsporidia, and C. parvum as described previously and blanched at various temperatures (71oC-100oC) for various periods of time (0-60 sec). Three other sanitizing vegetable washes marketed for prewash of fresh produce were studied.. The vegetable washes evaluated included citric acid and a combination of ozone, or ultrasonic-ozone-nanosilver. Method A used ozone exclusively and Method B used ozone, silver and ultrasonic technologies. No significant bacterial inactivation or removal was observed using any of the systems tested in green onions and herbs. Similarly, Cryptosporidium was not affected by any of these systems but viable microsporidia was not recovered from the ozone-only based method. Cross contamination of fresh produce can occur during harvest. The coring process during lettuce harvesting and cross contamination of coring blades and lettuce heads was examined. Coring tools were sanitized using diluted chlorine or 3% LA/3% SDS solution (LA/SDS). Contaminated blades transferred bacterial pathogens from head to head when using all concentrations of E. coli, Salmonella, and microsporidia. In blades sanitized with LA/SDS cross-contamination was significantly lower than when blades were sanitized with chlorine. Microsporidia was not inactivated using LA/SDS. Improved detection of parasites from produce. The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash/elution solutions and removing them from suspect samples. Sterile E-Pure water, 3% levulinic acid-3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate buffered saline, 0.1% Alconox®, and 1% HCl-pepsin were evaluated for their effectiveness in removing C. cayetanensis, C. parvum, and T. gondii from basil. One hundred and 1,000 oocysts of these parasites were inoculated on 25 g of basil leaves and stored for 1 h at 21oC and 24 h at 4oC. Oocysts inoculated at a concentration of 1,000 oocysts/25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts/25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl-pepsin wash solution. Oocyst recovery rates were higher at 1 h compared to 24 h post-inoculation. The humoral response to Cryptosporidium infections in infected individuals was determined. Using differential in gel electrophoresis (DIGE), specific spots common and unique to two C. parvum isolates were identified. Selected proteins were examined for their use as potential markers to recognize individuals with active cryptosporidiosis for potential use in food and waterborne outbreaks. The select proteins were analyzed and identified by mass spectrometry. In one-dimensional analysis a C. parvum protein of 57 kDa reacted more strongly with acute human sera (p < 0.05) whereas the results of two-dimensional analysis suggest that various C. parvum antigens may be used as markers of early Cryptosporidium infection. C. parvum antigens of 14, 16, 21 or 26 kDa detected in one-dimensional analysis, or 17.5 detected in two-dimensional analysis, reported previously in the literature, did not distinguish recent infection (p < 0.05). A Cryptosporidium serine/threonine phosphatase, Cryptosporidium actin protein, a dynein heavy chain, phosphoglycerate kinase, a chaperone-related protein, and three Cryptosporidium hypothetical proteins were detected. Impact: Disinfectants that can inactivate foodborne parasites and bacteria aid consumers by enabling them to have access to safer foods. Various chemical treatments were evaluated. Hydrogen peroxide and Pro-San were effective in inactivating E. coliO157:H7, but had limited or no effect against parasite viability. Cryptosporidium and microsporidia were not inactivated even when treated for up to 2 hrs with 2% LA/1%SDS or 3%LA/2%SDS at room temperature, whereas E. coli O157:H7 was highly susceptible. Freezing does not kill and minimally affects the viability of Salmonella, STEC O157:H7, and microsporidia but Cryptosporidium is significantly reduced, but not eliminated under the tested conditions. Sanitation systems tested in this study were not effective at killing or removing all foodborne parasites from the surface of vegetables, particularly herbs that are consumed raw, thus there is a need to find effective methods for produce sanitation.Pathogen transfer and cross-contamination can occur when contaminated water, or contaminated coring tools are used during lettuce harvesting. Use of LA/SDS during coring will result in lettuce heads with less bacterial contaminants than with chlorine rinses. Improvements in detection of parasites was addressed by comparing efficient elution methods to isolate oocysts from fresh produce. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. Finally, proteins and peptides that might be useful to detect early Cryptosporidium infections and to determine human immune response associated to a specific Cryptosporidium species have been identified.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2011
Citation:
1. Chandra, V., Torres, M.P. and Ortega Y.R. 2013. Efficacy of wash solutions on Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii recovery from basil. Journal for Food Protection. Submitted.
2. Ortega, Y.R., Torres, M.P., and Tatum, J.M. 2011. Efficacy of levulinic acid/SDS antibacterial against Encephalitozoon intestinalis, Cryptosporidium parvum, and E. coli O157:H7. Journal for Food Protection. 74(1): 140-144.
3. Ortega, Y.R., Torres, M.P., Van Excel, S., Moss, L. and Cama, V. 2007. Efficacy of a sanitizer and disinfectants to inactivate Encephalitozoon intestinalis spores. Journal of Food Protection. 70:681-684.
4. Survival of Pathogens in Frozen Fruits. At-A-Glance 22(1). Center for Food Safety. UGA. March 2013.
5. Sanitation of Lettuce Coring Tools. At-a-Glance. 20(2). Center for Food Safety. UGA. August 2011.
6. Potential Targets for Detecting Cryptosporidium. At-a-Glance. 19(2). Center for Food Safety. UGA. July 2010.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: This year we addressed two issues on pathogens in fruits and herbs. The first is survival of foodborne pathogens when frozen and the second to determine better methods to recover/remove parasites from basil. OBJECTIVE 1. To determine if foodborne pathogens could survive freezing when stored in water, syrup, or sugar. Foodborne outbreaks have been associated with consumption of contaminated berries. This contamination can occur during pre or post-harvest practices, or even during handling of food at the homes. Freezing is a common practice to preserve berries at home and by the food industry. Raspberries and sliced or whole strawberries were experimentally inoculated with Salmonella spp (pool of five isolates), STEC O157:H7 (pool of 5 isolates), Cryptosporidium parvum oocysts, and Encephalitozoon intestinalis (microsporidia) spores. Log reduction due to freezing was determined by comparing the survival at 4 C minus the log survival at -23 C. Less than 1 log reduction was observed with Salmonella or STEC (raspberries, whole and sliced strawberries) with a non-significant but larger reduction when using water instead of syrup or sugar. At least a 4 log reduction was noted with Cryptosporidium in all treatments. With microsporidia in raspberries, 3, 1, and 0.8 log reductions were observed in water, sugar, and syrup respectively. When inoculated onto whole strawberries, 1, 0.47 and 0.70 log reductions were achieved with water, syrup and sugar respectively. Similar results were observed when using sliced strawberries. OBJECTIVE 2. Removal of Cyclospora and Cryptosporidium from basil using different wash solutions. 25 gram basil leaves were spot inoculated individually with 100 to 1000 oocysts of Cyclospora and Cryptosporidium and incubated at RT for 1 hour and 4 C for 24 hours. The inoculated samples were then hand washed for 1 minute with the following solutions: E-pure H2O, 3% levulinic acid/3% sodium dodecyl sulfate, PBS, 1M glycine, 0.1% Alconox, and 1% HCl/pepsin. DNA was extracted from the washed samples and amplified using nested PCR for the detection of both parasites. From the six wash solutions that were tested initially on the recovery of Cyclospora, only four (E-pure H2O, 1M glycine, 0.1% Alconox, and 1% HCl/pepsin) were kept for the following trials since they were most effective. All four wash solutions could detect both parasites' oocysts at the concentration of 1000 oocysts/25 g basil. However, 0.1% Alconox and 1% HCl/pepsin worked better at identifying 100 oocysts/25 g basil. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Freezing does not kill and minimally affects the viability of Salmonella, STEC O157:H7, and microsporidia. On the contrary, viability of Cryptosporidium is significantly reduced but not eliminated by freezing in all of the tested conditions. With the emerging parasitic foodborne illnesses related to fresh produce, an efficient elution method to isolate oocysts from the suspected food is crucial. Since Cyclospora and Cryptosporidium have a low infectious dose, it is very important to maximize the recovery. The findings of this study can be used to improve the sensitivity of foodborne parasite detection.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Two objectives of this project were examined: Objective 1. To determine the survival of foodborne pathogens (five isolate mixture of E. coli O157:H7 and Salmonella) after blanching. Basil, broccoli, bean sprouts, and green peas were experimentally inoculated with E. coli O157:H7, Salmonella, microsporidia, and Cryptosporidium parvum and blanched at various temperatures (71oC-100oC) for various periods of time (0-60 sec.). After blanching, bacterial contaminants were removed from the vegetables and grown in their respective selective media. Freezing of contaminated produce was also done to determine if this step would be a critical control point. E. coli O157:H7 and Salmonella were not affected by the freezing processes. Blanching could reduce the contaminant loads in produce but did not eliminate them. Inactivation varied based on the exposure time and blanching temperatures. Cryptosporidium and microsporidia were susceptible at high temperature treatments. Pathogens could not be detected if produce was rinsed with 3%levulinic acid/3%SDS prior to the blanching process. Objective 2. To examine the coring process during lettuce harvesting and identify the contamination sources and cross contamination of coring blades and lettuce heads. The effects of sanitizing the coring tools with diluted chlorine or 3% levulinic acid/3% SDS solution (LA/SDS) was also studied. Five-serotype mixture of Salmonella and five-isolate mixture of E. coli O157:H7 were used to experimentally inoculate soil, water, and coring blades before the coring process. Iceberg lettuce heads were used in all experiments. Experimental inoculations were examined using various concentrations of contaminants (3 log to 7 log). Contaminated blades can transfer bacterial pathogens from head to head when using all concentrations of E. coli, Salmonella, and microsporidia. If blades are sanitized with LA/SDS cross-contamination is significantly low PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The bacterial contaminants are reduced after the blanching process; however, this method should not be considered an exclusive process to render pathogen-free produce. The times and temperatures of effective pathogen destruction tends to be where the product loses its fresh appearance. Parasites were susceptible to the blacking process. Pathogen transfer can occur when contaminated water, or contaminated coring tools are used during lettuce harvesting. Reduction of bacterial contaminants in lettuce and coring tools was achieved when coring tools were rinsed with diluted chlorine and more yet when rinsed with LA/SDS. Use of LA/SDS during coring will result in lettuce heads with less contaminant than with chlorine rinses.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Increased consumer demand for fresh fruits and vegetables and so-called "natural" products has coincided with the heightened circulation of "freeze-killed" fruit ingredient alternatives to traditionally pasteurized or heat-treated products such as fruit pieces and purees. Several national and international outbreaks of foodborne illnesses have been associated with frozen fruits (i.e. norovirus and raspberries, Cyclospora and raspberries and cake filling, Salmonella Typhi and frozen mamey). Food composition, rate of cooling, and temperature may influence the inactivation and survival of foodborne pathogens. We investigated common freezing methodologies on strawberries and raspberries (whole or cut, with or without syrup) and blanching of spinach leaves before freezing to determine inactivation and survival of foodborne pathogens. Strawberries and raspberries (whole or cut, with or without syrup, and sugar) were inoculated with five-serotype mixture of Salmonella, five isolate mixture of E. coli O157:H7, Encephalitozoon intestinalis, and Cryptosporidium parvum. Berries were frozen for 48 hr and pathogens recovered and analyzed for the presence of viable organisms. Blanching experiments were done using spinach leaves. E. coli O157:H7 and Salmonella were not affected by any of the freezing processes whereas partial inactivation was achieved with Cryptosporidium and microsporidia (<2-3 logs MPN or CFU/25 gr). Blanching inactivated bacteria significantly but did not eliminate them completely (detectable by enrichment) and parasites were not infectious after blanching. The second objective in this project was to evaluate three vegetable wash solutions available in the market to determine their effect on pathogen removal from green onions and herbs (basil, cilantro, and parsley). The pathogens tested in the laboratory were Salmonella, E. coli O157:H7, Cryptosporidium, and microsporidia. The number of organisms inoculated that remain in the herbs after wash and in the wash solution was determined as well as their viability. The vegetable washes evaluated included citric acid and a combination of ozone, or ultrasonic-ozone-nanosilver). Method A uses ozone exclusively and Method B uses ozone, silver and ultrasonic technologies. These washes are intended for use in the home, restaurant, cruise ships, or food processors. The objective of this part of the project was to determine the viability of Salmonella, E. coli O157:H7, Microsporidia, and Cryptosporidium parvum when treated with wash solutions on pathogen removal and inactivation from green onions and herbs. We inoculated Cryptosporidium parvum, Encephalitozoon intestinalis, five-serotype mixture of Salmonella and five-isolate mixture of E. coli O157:H7 on green onions, basil, cilantro, and parsley. No significant bacterial inactivation or removal was observed using any of the systems tested in green onions and herbs. Similarly, Cryptosporidium was not affected by any of these systems but viable microsporidia was not recovered from the ozone only based method. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Additional kill steps are necessary when preparing minimally processed and preserved foods (by blanching and freezing) and the survival varies according to the food matrix as well as the pathogen in question. Sanitation systems tested in this study were not effective at killing or removing all foodborne pathogens from the surface of vegetables, particularly herbs that are consumed raw thus there is a need to find effective methods for produce sanitation.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Two objectives of this project were examined: Objective 1. Following the information obtained in 2008, selected proteins from Cryptosporidium parvum were examined for their use as potential markers to recognize individuals with active cryptosporidiosis during a food and waterborne outbreak. Proteins were separated using a 1 and 2-D gel electrophoresis followed by western blot analysis. Human sera from Cryptosporidium-infected individuals was used. The select proteins were analyzed and identified by mass spectrometry. In one-dimensional analysis a C. parvum protein of 57 kDa reacted more strongly with acute human sera (p < 0.05) whereas the results of two-dimensional analysis suggest that C. parvum antigens of 43.4, 50.3, 50.3, 47.6, 64.7, and 50.3 kDa, with pIs of 5.4, 7.0, 7.2, 5.3, 6.6, and 6.7, respectively, may be used as markers of early Cryptosporidium infection. C. parvum antigens of 14, 16, 21 or 26 kDa detected in one-dimensional analysis, or 17.5 detected in two-dimensional analysis, reported previously in the literature, did not distinguish recent infection (p < 0.05). A Cryptosporidium serine/threonine phosphatase, Cryptosporidium actin protein, a dynein heavy chain, phosphoglycerate kinase, a chaperone-related protein, and three Cryptosporidium hypothetical proteins were detected. Antigenic peptides were predicted from these proteins and Cryptosporidium-specific peptides are suggested. Objective 2. Inactivation of foodborne pathogens is a key component of food safety particularly in the ready to eat foods such as fresh produce. The effect of various concentrations of levulinic acid and SDS on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis was determined. Cryptosporidium parvum and Encephalitozoon intestinalis viability was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines respectively. Two E. coli O157:H7 isolates were used in the present study. Different concentrations and combinations of levulinic acid/SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10^5), E. intestinalis spores (10^6), and E. coli O157:H7 (10^7) when in suspension. Pathogens were treated for 30, 60, and 120 min at 20 plus/minus 2C. Spores and oocysts were inoculated onto their respective cell line monolayers. Serial dilutions of treated E. coli were prepared in 0.1% peptone were plated on Sorbitol McConkey Agar with 50 μg/ml nalidixic acid plates and colonies counted 24 hrs later. When microsporidia spores and Cryptosporidium oocysts were treated for 30 and 60 min, none of the combinations proved to be effective at inactivating the spores or oocysts. When the parasites were treated with the higher concentrations, 3% levulinic acid/2%SDS and 2% levulinic acid/1%SDS for 30 min, 1 hr and 2 hr no effect on parasite viability was observed. E. coli O157:H7 was used as a control and was highly susceptible at various concentrations and exposure times tested. SDS alone and levulinic acid had very limited effect on E. coli viability but when in combination they were highly effective at 30 and 60 min incubation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have identified proteins and peptides that might be useful to detect early Cryptosporidium infections and to determine human immune response associated to a specific Cryptosporidium species. The effectiveness of sanitizers against parasites continues to be a challenge. Cryptosporidium and microsporidia are not inactivated even when treated for up to 2 hrs with 2% levulinic acid /1%SDS or 3%levulinic acid/2%SDS at room temperature whereas E. coli O157:H7 was highly susceptible at various concentrations and at shorter exposure times.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Foodborne diseases are being increasingly reported in the U.S. and other countries. It is estimated that every year there are 76 million cases of foodborne illness and 5,000 deaths. Pathogens can contaminate foods, particularly fresh vegetables, during their production, collection, transport, handling or processing stages. Natural reservoirs, such as wild, domestic, and farm animals also play a role either as source or as vectors of pathogens. Significant advances in the detection of these organisms in water were primarily accomplished by the development of molecular detection tools and recovery methods using specific antibodies. These methods have been used with Cryptosporidium in water but they need to be modified to improve the recovery and detection of these pathogens in food items. Two objectives of this project were examined in 2008. Objective 1. Using differential in gel electrophoresis (DIGE), proteins of Cryptosporidium and Cyclospora isolates were identified. Specific spots common and unique in two Cryptosporidium parvum isolates have been identified. These are currently being further characterized by Western blot using sera from convalescent individuals. The protein profile of Cyclospora is currently been examined using isolates from different countries. Objective 2. Inactivation of foodborne pathogens in fruits and vegetables is a key component of food safety .We tested 0.5% hydrogen peroxide and Pro-San on experimentally inoculated lettuce and basil. A 3 log reduction was obtained against Escherichia coli O157:H7 when treated with 0.5% hydrogen peroxide for 1 minute and a 6 log reduction was obtained when incubated for 5 minutes. Treatment with Pro-San yielded a 3 log reduction when incubated for 1 or 5 min. A 4 log reduction in viability was observed when Encephalitozoon intestinalis spores were treated with 0.5% hydrogen peroxide while Pro-San had no effect. Neither treatment was effective against Cryptosporidium parvum. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Protein characterization of foodborne parasites will help to better understand the biology of Cryptosporidium and Cyclospora and to determine specific antigens that could have a potential in diagnostic or vaccine development. Identification of disinfectants that can inactivate Cryptosporidium and microsporidia will provide alternatives for consumers to have safer foods. We have determined that hydrogen peroxide and Pro-San were effective in inactivating E. coliO157:H7, but had limited or no effect against parasite viability. Further testing of additional chemicals against parasites is needed.
Publications
- No publications reported this period
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