From the greenhouse to poultry facilities, from the classroom to 4-H camps, current Salmonella research is devoted to plants, animals, and youth food safety education.
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Explore This IssueApril/May 2018
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Focus on Tomatoes
While most salmonellosis cases occur due to the consumption of contaminated poultry products, the power of fresh produce, especially tomatoes, to cause salmonellosis cannot be overlooked, says Gireesh Rajashekara, DVM, PhD, a professor in the Food Animal Health Research Program at the Ohio Agricultural Research and Development Center in the Department of Veterinary Preventive Medicine at the Ohio State University, Wooster, Ohio.
“Tomatoes have been frequently associated with wide-scale salmonellosis outbreaks worldwide over the past decades,” Dr. Rajashekara points out. “More than dozen outbreaks have been reported since 2001 in the U.S.”
The sources of these outbreaks in many instances remain unknown, Dr. Rajashekara notes. “The Salmonella infections of tomato plants and fruits do not cause any symptoms,” he relates. “The common practice of washing does not remove Salmonella, as it can internalize and avoid exposure to commonly used surface sanitizers.”
Courtesy of a $500,000 grant provided by the USDA National Institute for Food and Agriculture (NIFA), Dr. Rajashekara is in the final year of a research project focused on Salmonella contamination of tomatoes.
“Our overall goal is to understand plant-Salmonella interactions, identify environmental and biological factors that influence Salmonella persistence in tomato plants and fruits, and identify novel effective strategies to control Salmonella in produce,” he explains. “The long-term goal is to significantly improve the safety and sustainability of the tomato industry by reducing the spread of high-risk human pathogens.”
The specific objectives of Dr. Rajashekara and his research team are: 1) to illuminate both how contamination of the tomato plant with Salmonella occurs and how pathogens survive or proliferate in plant tissues and fruits, and 2) to develop novel effective control methods to prevent Salmonella contamination of tomato plants and fruits.
“We are specifically interested in understanding the environmental factors and phytopathogen infections on the survival of Salmonella in tomato plants and fruits,” Dr. Rajashekara says.
By growing Salmonella-contaminated tomato plants in green house conditions, Dr. Rajashekara and his colleagues, which include plant pathologist Sally Miller, PhD, and doctoral student Loïc Deblais, MS, observed that specific environmental temperature and relative humidity conditions have significant impact on Salmonella persistence in contaminated tomato plants.
“High environmental temperature, greater than 77 degrees Fahrenheit, significantly reduces Salmonella abundance and persistence over time on the surface of the tomato plants, however environmental temperatures did not affect internalized Salmonella,” Dr. Rajashekara explains. “On the other hand, low relative humidity levels, less than 40 percent, increased the probability of dissemination of Salmonella in the plant. Similarly, when tomato plants were experimentally infected with plant pathogens, we observed that the plant pathogens could increase the abundance and persistence of Salmonella in tomato plant tissues, which is most likely due to competition for available nutrients in the plant tissues. Salmonella once infected seems to compete successfully with plant pathogens to survive in tomato plants.”
These tomato plant studies are facilitated by the use of highly sensitive real-time bioluminescent imaging, real-time in planta imaging, Dr. Rajashekara says. “This technique allows visualizing, in a non-destructive way, the exact location of Salmonella in tomato plant and fruit tissues,” he notes, “thus providing a greater understanding of Salmonella survival and persistence in different plant tissues. This technique also allows rapid assessment of the effect of sanitizers or disinfectants on Salmonella survival and persistence in tomato plant tissues and fruits.”
Big Work with Small Molecules
Another goal in Dr. Rajashekara’s lab is to develop novel antimicrobials to control Salmonella using new generation small molecules. “Previous studies have shown that new generation small molecules are effective even against multi-drug resistant pathogens,” Dr. Rajashekara relates. “We identified several novel anti-Salmonella compounds that are effective against even the internalized Salmonella in tomato plants and fruits. These small molecules are compatible with the use of alternative control methods such as beneficial plant associated microorganisms, so thereby can be combined with bio-control approaches to enhance Salmonella control in production systems.”
Since poultry obviously is a major target in Salmonella control efforts, Dr. Rajashekara’s team also tested their novel small molecules in chickens and found that these small molecules are effective in reducing Salmonella in infected chickens.
“Small molecules are also compatible with the use of probiotics and they enhance the antimicrobial activity of certain antibiotics that are currently used to control Salmonella in poultry production systems,” Dr. Rajashekara says. “Our study highlights the importance of abiotic (environmental) and biotic (plant pathogens) factors in the survival and persistence of Salmonella in fresh produce. And we are very excited about the potential effectiveness of novel new generation small molecules against Salmonella. We believe our findings will allow the development of new strategies to prevent contamination of human bacterial foodborne pathogens at pre-harvest stages and to implement effective disinfection procedures post-harvest.”
Success with Steam Pasteurization
Vacuum steam pasteurization is proving to be effective for killing Salmonella on several low moisture foods, according to Teresa Bergholz, PhD, a food scientist with North Dakota State University, Fargo.
“Recently, a number of outbreaks of foodborne illness have been attributed to Salmonella on low-moisture foods,” Dr. Bergholz points out. “These foods were previously thought to be relatively low-risk for transmitting foodborne illness, as they typically do not support growth of microbes. With these latest developments, it is clear that effective preventative controls are needed to reduce the risk of human illness associated with low-moisture foods. To that end, we are finding that the use of vacuum steam pasteurization is expected to have greater efficacy against pathogens, as moist heat is more effective at inactivating microbes compared to dry heat.”
In Dr. Bergholz’s recent research funded by the North Dakota Agricultural Products Utilization Commission, she and her colleagues applied steam at lower temperatures to several low moisture foods, including flaxseed, quinoa, and sunflower kernels. “The lower temperatures are relative to greater than 212 degrees Fahrenheit, which is the temperature that would be required to produce steam if not under a vacuum,” she explains. “The results show that vacuum steam pasteurization for 2 to 5 minutes at temperatures ranging from 167 degrees Fahrenheit to 185 degrees Fahrenheit can effectively kill 5 logs of Salmonella.
“Now that we know vacuum steam pasteurization can be effective, using a USDA NIFA grant, we are currently evaluating if Salmonella serovars Agona, Enteriditis, Montevideo, and Tennessee differ in their susceptibility to vacuum steam pasteurization when inoculated onto whole flaxseeds,” Dr. Bergholz continues. “Our initial results indicate that the serovars have similar levels of inactivation. We are also interested in determining if the length of time the pathogen is present on the low moisture food prior to vacuum steam pasteurization impacts the ability of Salmonella to be killed by the treatment.”
Dr. Bergholz says many studies have shown that Salmonella can survive for months or even years on low moisture foods. “Now we want to know if that long-term survival could make Salmonella more resistant or more susceptible to the thermal treatment,” she mentions. “This work is still in progress.”
Another part of this project was to evaluate if vacuum steam pasteurization impacted the quality of the foods that were pasteurized, Dr. Bergholz adds. “In collaboration with food scientist Clifford Hall, PhD, who coordinates the NDSU pulse crops quality program, we measured chemical and microbial changes in whole and milled flaxseed over 28 to 36 weeks after pasteurization,” she relates. “Overall, we saw a reduction in the number of aerobic microbes, yeasts, and molds, and negligible changes in chemical parameters. Thus, we learned that vacuum steam pasteurization can be used to effectively inactivate pathogens and has minimal effects on chemical shelf-life parameters of flaxseed.”
Since poultry products have been frequently implicated in reported cases of salmonellosis, and since there has been little information available about the chicken’s intestinal microbiota and its role in resistance to disease causing pathogens, especially Salmonella, Hosni Hassan, PhD, a professor of microbiology in the Prestage Department of Poultry Science at North Carolina State University (NCSU), Raleigh, spearheaded a research project to tackle these issues.
“Our goal was to develop a novel Salmonella control strategy that promotes the establishment of an intestinal microbiota in the chicken that prevents colonization by pathogenic bacteria and enhances the mucosal immune response to Salmonella vaccination and challenges,” Dr. Hassan relates. “To that end, our objectives were to characterize the changes in the chicken’s gut microbiome during Salmonella infections and identify members of the microbiota associated with decreased Salmonella colonization; and to develop an optimized control program that promotes resistance to colonization and the development of strong mucosal immunity virulent strains of Salmonella enterica.”
Funded by a $2.5 million USDA NIFA grant that was awarded in 2012 and runs through June 30, 2018, Dr. Hassan, his NSCU colleagues, and collaborators at the University of North Carolina-Chapel Hill (UNC-CH) have used an attenuated strain of Salmonella, which was developed by his students during 2007 through 2010 and patented in 2012, as vaccine. “The strain is different than the current vaccines and its authenticity and efficacy were first tested in the mice model of salmonellosis,” Dr. Hassan relates, noting that the mechanism of action hasn’t been published, so is yet confidential. “We also sequenced the complete genome of this patented Salmonella vaccine strain.”
The research team used the novel vaccine strain in its poultry studies alone, and in combinations with other reagents, such as prebiotic galacto-oligosaccharides (GOS), for modulating the gut microbiome of egg laying chickens.
“We isolated and characterized several poultry-specific probiotic organisms from the healthy experimental birds and selected three isolates for complete genome sequencing,” Dr. Hassan points out.
The researchers identified the composition and the development of the chicken gut microbiome as a function of age, vaccination, adding prebiotic GOS to the diet, adding poultry-specific probiotic isolates to the diet, and combinations thereof.
Dr. Hassan says these efforts are based on the fact that the gut microbiota plays an important role in the digestion of complex plant fibers and polysaccharides; and the microflora also provides protection against colonization by invasive pathogenic organisms (colonization resistance).
“We also examined the effects of these treatments on the chicken’s immune system,” Dr. Hassan continues. “Based on our results, we conclude that vaccination and modulation of the gut microbiome enhanced the bird’s ability to resist Salmonella infections. We currently have work in progress to identify the effects of these treatments on gut metabolites and how they contribute to mechanism(s) for resistance to pathogens.”
An article about the vaccine was just submitted to a peer reviewed journal in March 2018.
Dr. Hassan says the NCSU Office of Technology Commercialization and New Ventures has signed a confidential disclosure agreement with a commercial vaccine production company to evaluate and discuss the data. “We don’t know when and if our new vaccine will be available commercially,” he notes.
Youth Education and Outreach
An additional major component of this research, Dr. Hassan points out, has been to work with professional educators and extension 4-H faculty at NCSU, who have been co-principal investigators, to develop educational materials for K-12 students and 4-H groups to teach consumers about food safety, the science behind preventing foodborne disease, and their role in preventing salmonellosis.
“For this we set out to develop innovative K-12 curriculum material in subjects related to food safety, disease transmission, and next generation genetic research to engage students in agricultural sciences and communicate their role in food safety,” Dr. Hassan relates. “We aimed to develop instructional materials based on the 4-H national standards to communicate and teach 4-H members the technology and science used to produce safe food and their responsibilities in preventing foodborne disease.”
In collaboration with the Kenan Institute at NCSU, Dr. Hassan’s team selected nine K-12 school teachers from around the state of North Carolina to participate in the program. “These nine ‘Food Safety Kenan Fellows’ spent the summers of 2012 and 2013 working in three different research labs, the microbiology and immunology labs at NCSU, and the Microbiome Core Facility at UNC-CH, learning about the microbiological, immunological, and the molecular biology/genome sequencing aspects of food safety,” Dr. Hassan says. “By 2014, the Food Safety Kenan Fellows developed and field tested three food safety curricula for elementary, middle, and high schools.”
The co-principal investigators and collaborators from the Kenan Institute, the 4-H team and the NCSU IntelliMedia Group (computer engineering), in collaboration with the 4-H field agents from around the state, carried out the curriculum development and dissemination phase of the project, Dr. Hassan points out. “These collaborators piloted the curriculum, including testing the Intellimedia software, and addressing issues encountered with development and implementation of the curricula during this process,” he says.
The IntelliMedia Group developed, refined, and published the Crystal Island game-based learning environment and supporting websites for the project, Dr. Hassan continues. “The development effort has been focused on making the system easy to deploy by improving the distribution of the Crystal Island game through the project website, and management and monitoring of students through the Teacher Portal web application,” he explains. “The program has been evaluated by the national 4-H organization and we expect the program will be adopted and used nationally by 4-H clubs.”
The microbiology/food Safety curricula was modified for 4-H clubs’ activities. “The curricula were delivered and piloted in elementary and middle school enrichment programs and summer 4-H camp activities in some 16 North Carolina counties,” Dr. Hassan relates. “Clearly, we are very excited about and satisfied with the progress made and the learning outcomes. We are hopeful that these young minds will be the future food safety leaders and educators.”