Progress 07/15/12 to 07/14/15
Outputs
Target Audience:Target audiences would include professionals, researchers and scientists in the area of food microbiology, food technology, biotechnology, microbiology, food safety; undergraduate students; producers, supply chain and retailers from the food industry; consumers and the general public. Blueberries are of economic importance to Maine. Growers in our state can directly apply successful technology developed from this study to their blueberry crops. The comprehensive overview of intervention techniques will expand upon industry knowledge on the effectiveness of their pre-established processing interventions are against T. gondii. Changes in intervention behavior can be established to lower risks of T. gondii contamination. This expansion on intervention technology knowledge will allow for the increase safety of those most affected by T. gondii pregnant women and the immunocompromised. An efficient and easy to interrupt detection method can also be utilized by the food industry to address T. gondii contamination concerns. The continuing evolution of this optical DNA sensing method will allow the mainstreaming of routine T. gondii testing in the American food system. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Throughout the conduct of this project, it has provided research opportunities for both graduate and undergraduate students to finish their master's and honors thesis and capstone projects. These training opportunities allowed the students to participate in exploring the science behind developing a novel detection method for significant foodborne pathogens. Through mentoring, training and independent laboratory activities, the students were able to learn the necessary techniques to carry out the objectives of their individual projects and develop meaningful protocols based on validated data and standards. Their projects abstracts have been accepted and were presented to international conference (Institute of Food Technologists - IFT Annual Conferences 2013, 2014 and 2015). How have the results been disseminated to communities of interest?The results of this project have been presented in various meetings within and outside the university. This project has expanded the number of scientists conducting research on T. gondii through training by collaborators in the USDA. It has led to the creation of facilities and protocols in order to handle working with a protozoan. New standard operating procedures have been developed both at the University of Maine and the ERRC at Wyndmoor. Protozoans are historically more difficult to work with as compared to most bacteria. Opening doors to T. gondii research at the University of Maine as well as at ERRC Wyndmoor has led to an increased awareness of protozoan work and potential new areas of research for scientists at both research centers. More public awareness of T. gondii has also been accomplished through presentations at the University of Maine's GradExpo, the IFT and IAFP Annual Meeting. By disseminating the purpose and the progress of this study, members of the community are becoming aware of the relevance and usefulness of this project. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. We determined the effectiveness of using peroxyacetic acid, lactic acid, chlorine dioxide and chlorine washes to remove T. gondii oocysts inoculated on the surface of blueberries. Blueberry samples (10g, n=5) were spot inoculated with 5-log T. gondii oocysts. After drying for 30 minutes, the samples were placed in peroxyacetic acid (1%), lactic acid (2%), ClO2 (1, 3, 5, 10, and 15ppm), NaClO (100, 150, and 200 ppm), or water [berries:solution = 1:1 (w/v)] and vortexed (Fisher ) for 2 minutes to simulate washing process. To determine log reduction, oocysts removed from the blueberries surface were enumerated using a compound microscope at 400x. by examining the counts of oocysts in the washing solutions. The water wash removed 4.5 log oocysts from the surface of blueberries. The peroxyacetic acid and lactic acid washes removed significantly (p<0.05) more oocysts, 4.9 and 4.8 log, respectively, than the water wash, whereas the ClO2 and NaClO washes removed similar levels of oocysts as the water wash. All the chemical wash treatments did not alter the appearance of blueberries. Results indicate that peroxyacetic and lactic acid washes are more effective in removing T. gondii oocysts from blueberry surface than ClO2, NaClO and water washes. Peroxyacetic acid removed just over 5 log of the 5.2 log inoculated oocysts while water washes only remove 4.7 log. Interventions targeting the oocyst stage of T. gondii are imperative for controlling T. gondii contamination of fruits and other produce. The use of peroxyacetic acid and lactic acid washing could be a viable method of controlling T. gondii oocysts contamination on blueberries. Other processing interventions such as freezing and irradiation were also studied. Freezing was shown to be an effective method in controlling T. gondii tissue cysts in contaminated meat products at -20°C for 24h. Low dosages of irradiation 0.2-0.6 kGy were studied in their effectiveness of controlling T. gondii. A self-contained Lockheed Corporation 137CS gamma radiation source at a dose rate of 0.075 kGy/min was utilized. After treatment, oocysts were recovered from berries. Human foreskin fibroblast (HFF) cells were maintained as monolayers in a 96well microplate, exposed to excysted T. gondii recovered from berries, and incubated for 7 days. The viability of HFF cells was evaluated using 3 (4,5dimethylthiazol2yl) 2,5diphenyl tetrazolium (MTT) assay. In addition, blueberries were analyzed for compression firmness, surface color, and total anthocyanins, immediately after each treatment. HFF cells inoculated with oocysts recovered from the 0.6 kGy treated berries retained viability at 93% compared to the control, indicating that treated oocysts were less infectious to HFF cells. The result showed that gamma radiation significantly (P < 0.05) inactivated T. gondii oocysts on blueberries. Quality analysis showed that there was no significant change in texture, anthocyamins, or color in berries after the irradiation treatment. Findings of this study indicated that lowdose irradiation is a potential intervention measure for controlling T. gondii contamination on blueberries without affecting product quality. Objective 2. We developed a rapid and specific optical sensing method for the detection of T. gondii by DNA sandwich hybridization with oligonucleotide-functionalized gold nanoparticles (AuNps). Pairs of thiol-modified oligonucleotides (30-mer) were immobilized onto AuNps forming probes to target the 529-bp region of the ME-49 strain of T. gondii. The 163-bp locus in the 529-bp region found in every strain of T. gondii was amplified by specific primers to be used as a target. The AuNps-oligio probes hybridized with the target DNA under optimal temperature and incubation time (56oC, 30 minutes) forming an AuNps-oligio and target DNA structure. Results showed that after the addition of NaCl salts (3M, 100 μL), the AuNp-oligio probe and target DNA structure remained red, while the samples with non-target DNA changed to purple. The color difference between target and non-target DNA was due to the aggregation of the AuNp-oligo; without the target DNA, the AuNp-oligo probes aggregated which resulted in the change to the purple color. Human eyes can detect the color change without the aid of any additional equipment. This method requires less than an hour for detection after the DNA preparation. The AuNp-DNA hybridization was confirmed through gel electrophoresis and the aggregation was corroborated with spectrophotometric data. The colorimetric properties of this specific detection method of T. gondii offer a significant advantage over the traditional cat and mouse bioassays in terms of time, cost and simplicity.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Lacombe, A., A. Breard, A. C-A. Hwangc, D. Hill, X. Fan, L. Huang, and V.C.H. Wu*. 2015. Inactivation of Toxoplasma gondii on Blueberries using Low-Dose Irradiation. Food Control.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Lacombe, A., A. Breard, A. C-A. Hwangc, D. Hill, X. Fan, L. Huang, and V.C.H. Wu*. 2015. Inactivation of Toxoplasma gondii on Blueberries using Low-Dose Irradiation. International Association for Food Protection (IAFP) Annual Meeting. Portland, OR.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang and V.C.H. Wu. 2014. Efficacy of Peroxyacetic Acid, Lactic Acid, Chlorine Dioxide, and Chlorine Washes in Removing Toxoplasma gondii from the Surface of Blueberries. International Food Technologists Annual Meeting. Food Microbiology Division: Graduate Research Paper Oral Competition Finalist & Food Microbiology Division: Food Safety and Defense Poster Presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang and V.C.H. Wu. 2014. Developing a Rapid Colorimetric Detection Method Utilizing Gold Nanoparticles (AuNPs) for the Detection of Toxoplasma gondii. International Food Technologists Annual Meeting. Food Microbiology Division: Food Safety and Defense Poster Presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang and V.C.H. Wu. 2013. Efficacy of Peroxyacetic Acid and Lactic Acid Washes on Removal of Toxoplasma gondii Oocysts from Blueberries. International Association for Food Protection European Symposium.
|
Progress 07/15/13 to 07/14/14
Outputs
Target Audience: Blueberries for are used in this study because they are of economic importance to Maine. Growers in our state can directly apply successful technology developed from this study to their blueberry crops. The comprehensive overview of intervention techniques will expand upon industry knowledge on the effectiveness of their pre-established processing interventions are against T. gondii. Changes in intervention behavior can be established to lower risks of T. gondii contamination. This expansion on intervention technology knowledge will allow for the increase safety of those most affected by T. gondii pregnant women and the immunocompromised. An efficient and easy to interrupt detection method can also be utilized by the food industry to address T. gondii contamination concerns. The continuing evolution of this optical DNA sensing method will allow the mainstreaming of routine T. gondii testing in the American food system. Target audiencesalso include professionals, researchers and scientists in the area of food microbiology, food technology, biotechnology, microbiology, food safety; undergraduate students; producers, supply chain and retailers from the food industry; consumers and the general public. Changes/Problems: Along with the originally proposed plaque assay a second assay called MTT is also being utilized in order to gain more insightful results. What opportunities for training and professional development has the project provided? This project provided opportunities for training a M.S. graduate student/research assistant. Training in handling of Toxoplasma gondii Training in cell culture maintenance and assays Training on operation of irradiation and high pressure processing equipment Oral Presentation, University of Maine Graduate Research Expo 2014, 2nd Place Oral Presentation, University of Maine Graduate Research Expo 2013, 3rd Place Oral Presentation, IFT Annual Meeting 2014, Food Microbiology Oral Competition Finalist Poster Presentations, IFT Annual Meeting 2014, Food Microbiology Division How have the results been disseminated to communities of interest? This project has expanded the number of scientists conducting research on T. gondii through training by collaborators in the USDA. It has lead to the creation of facilities and protocols in order to handle working with a protozoan. New standard operating procedures have been developed both at the University of Maine and the ERRC at Wyndmoor. Protozoans are historically more difficult to work with as compared to most bacteria. Opening doors to T. gondii research at the University of Maine as well as at ERRC Wyndmoor has lead to an increased awareness of protozoan work and potential new areas of research for scientists at both research centers. More public awareness of T. gondii has also been accomplished through presentations at the University of Maine’s GradExpo and the IFT Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals? Continue to optimize cell culture assays in order to assess the interventions of irradiation, HPP, freezing and heating on T. gondii oocysts and tissue cysts. Finish objective 2.
Impacts
What was accomplished under these goals?
Objective 1. This study determined the effectiveness of using peroxyacetic acid, lactic acid, chlorine dioxide and chlorine washes to remove T. gondii oocysts inoculated on the surface of blueberries. Blueberry samples (10g, n=5) were spot inoculated with 5-log T. gondii oocysts. After drying for 30 minutes, the samples were placed in peroxyacetic acid (1%), lactic acid (2%), ClO2 (1, 3, 5, 10, and 15ppm), NaClO (100, 150, and 200 ppm), or water [berries:solution = 1:1 (w/v)] and vortexed (Fisher ) for 2 minutes to simulate washing process. To determine log reduction, oocysts removed from the blueberries surface were enumerated using a compound microscope at 400x. by examining the counts of oocysts in the washing solutions. The water wash removed 4.5 log oocysts from the surface of blueberries. The peroxyacetic acid and lactic acid washes removed significantly (p<0.05) more oocysts, 4.9 and 4.8 log, respectively, than the water wash, whereas the ClO2 and NaClO washes removed similar levels of oocysts as the water wash.. All the chemical wash treatments did not alter the appearance of blueberries. This study suggests that peroxyacetic and lactic acid washes are more effective in removing T. gondii oocysts from blueberry surface than ClO2, NaClO and water washes. Interventions targeting the oocyst stage of T. gondii are imperative for controlling T. gondii contamination of fruits and other produce. The use of peroxyacetic acid and lactic acid washing could be a viable method of controlling T. gondii oocysts contamination on blueberries Objective 2. This study developed a rapid and specific optical sensing method for the detection of T. gondii by DNA sandwich hybridization with oligonucleotide-functionalized gold nanoparticles (AuNps). Experiments are being conducted.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang and V.C.H. Wu. 2014. Developing a Rapid Colorimetric Detection Method Utilizing Gold Nanoparticles (AuNPs) for the Detection of Toxoplasma gondii. International Food Technologists Annual Meeting. Food Microbiology Division: Food Safety and Defense Poster Presentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang and V.C.H. Wu. 2013. Efficacy of Peroxyacetic Acid and Lactic Acid Washes on Removal of Toxoplasma gondii Oocysts from Blueberries. International Association for Food Protection European Symposium.
|
Progress 07/15/12 to 07/14/13
Outputs
Target Audience: Target audiences would include professionals, researchers and scientists in the area of food microbiology, food technology, biotechnology, microbiology, food safety; producers, supply chain and retailers from the food industry; consumers and the general public. The results of this study have already been presented to international meetings, conferences and book of abstracts. It is expected to be published in scientific journals as it progresses and reached its completion. Blueberries for are used in this study because they are of economic importance to Maine. Growers in our state can directly apply successful technology developed from this study to their blueberry crops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided opportunities for training a M.S. graduate student/research assistant. The student was able to present the research results at the 2013 IFT annual meeting in Chicago. Her research results were also presented at the 2013 International Association for Food Protection European Symposium. The student received 3rd in Natural Sciences Oral Presentation category in the GradExpo (2013) from presenting the progress of this project. The project is in collaboration with co-PIs at USDA ARS ERRC Wyndmoor, Pennsylvania. Through this project, the MS graduate was able to work at ERRC to experience operation of irradiation and high pressure processing and conducted experiments between May – Oct, 2013. How have the results been disseminated to communities of interest? The overview of this study has been presented to the local wild blueberry industry. This study has been accepted and presented at the 2013 International Association for Food Protection European Symposium. What do you plan to do during the next reporting period to accomplish the goals? The progress of this study is promising. We will continue on the comprehensive study of intervention practices and their effectiveness in removing or reducing T. gondii contamination and develop an optical DNA sensing method for the rapid and efficient detection of T. gondii
Impacts
What was accomplished under these goals?
In this period, we have focused on using chemical washes to remove potential sources of food contamination concerned with T. gondii oocysts on produce. Blueberry samples (10g) which were spot inoculated with T. gondii oocysts were treated with 1% peroxyacetic acid, 2% lactic acid, Cl2 (100, 150, 200ppm), ClO2 (1, 3, 5, 10, 15ppm) or water [1:1 (w/v) ratio] with 2 minutes of vortexing to simulate washing process. The oocyst removal rate was determined by the number of removed oocysts divided by the number of original inoculated oocysts. Irradiation, high pressure processing, freezing, heating, and microwaving trials are all currently being conducted. The second objective of this study is to design and develop an optical DNA sensing method for the rapid and efficient detection of T. gondii. For the optical DNA sensing method a region of T. gondii's genome must be targeted. There are two common regions targeted by researchers that are well conserved across the different strains of T. gondii – the 529bp region and the B1 gene. The 529bp region was chosen over the B1 gene region. Sandwich hybridization conditions are currently being tested including salt, temperature and S1 enzyme digestion time.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Breard, A., C. Hwang, D. Hill, X. Fan, L. Huang, and V.C.H. Wu. 2013. Efficacy of Peroxyacetic Acid and Lactic Acid Washes on Removal of Toxoplasma gondii Oocysts from Blueberries. International Association for Food Protection European Symposium.
|