Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Three graduate students and multiple undergraduate and veterinary studentsreceived bench-top laboratory research trainingon this pre-harvest food safety project during its 5 year period. These students learned how to work with pathogenic Salmonella strains and the bacteriophages that infect them, as well as how to develop an animal model of salmonellosis in dairy calves, and how to use the modelto test hypotheses. The target audience also includes bovine veterinary clinicians and their clients, dairy and beef producers, who will benefit directly from this project because it initiated work designed toreplace antibiotics with bacteriophages to decrease Salmonella in food animals. Changes/Problems:A major challenge occurred in the final year of this project when the biocontainment facility which houses our calves was closed for repair. We were unable to pursue our calf studies during this time, but will pursue them within the objectives of the next Hatch project. What opportunities for training and professional development has the project provided?Three Master's level graduate students, eight veterinary student workers, and four undergraduate student workers were trained over the course of this project. The graduate students presented the results of their work, in either poster or platform format, in local, regional, and national venues, including Phi Zeta Research Emphasis Day (local), Research Week (local), Southeastern Branch of the American Society for Microbiology (regional), and the AmericanCollege of Veterinary Pathologists annual meeting (national). How have the results been disseminated to communities of interest?The gradaute, veterinary,and undergraduate students supported by this Hatch project all were trained in the field of pre-harvest food safety, an area in which none of them had previous experience.The three graduate students gavemultiple presentations to diverse audiences, including veterinarians and microbiologists. Thus, they educated individuals about the expanding field of food safety. Also, as antibiotic use in animals comes under increasing oversight, the use of bacteriophages as novel "antimicrobials" to treat diseased animals opens up a new field of investigation inthe increasingly antibiotic-free world of food animal production. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT The overall results from this project indicate that a novel treatment approach utilizing specialized viruses called bacteriophages, which 'feed' on bacteria, reduces the number of the bacterial pathogen Salmonella in experimentally infected calves.Salmonellosissometimes is fatal in calves and is a significant problem in both beef and dairy herds.Also, cattle products contaminated with Salmonellacause foodborne diseases in people. In this project, two strains of Salmonella were used to cause disease in 6-8 week old orally infected dairy calves. Mixtures of 5-7 different bacteriophages, isolated from samples of naturally infected animals, were amplified in the lab and administered to calves showing signs of disease. We used standardmicrobiology techniques to count the number of Salmonella in fecal samples from phage-treated and non-treated calves, and observed that the treated calves shed >1,000-foldless Salmonellathan untreated calves. These findings show that bacteriophages can be used to treat salmonellosis in calves.Phage trreatment is an alternative to antibiotic treatment, and thus potentially reduces the occurrence of multi-drug resistant strains of Salmonella. And, as the number of Salmonella being shed by cattle is reduced byphage treatment, cattle products consumed by people will have less Salmonella contamination, and the number of people infected by contaminated cattle products will decrease. RESULTS Objective 1: Isolate and characterize lytic bacteriophages that target food-borne bacterial pathogens, including E. coli O157:H7 and Salmonella serotypes. Approximately 30 Salmonella-targeted bacteriophages (phages) were isolated and characterized during the project. Included in their characterization was testingfor the ability to lyse16 serotypes of Salmonella, which told us each phage's host range. Because we developed experimental disease models in calves using Salmonella serotypes Newport and Enteritidis (see Objective 2 results, below), we chose 5 Newport-targeted phages, and 7 Enteritidis-targeted phages, to examine the efficacy of phage treatment of bovine salmonellosis as an alternative to antibiotic treatment. Each of these 12 phages were further characterized by examining their (1)ability to lyse Salmonella in liquid culture, (2) morphology using electron microscopy, and (3)ability to bind their hostby performing standard absorption curves. Objective 2: Develop bacteriophage delivery approaches for chickens and cattle. Two Salmonella infection models in calveswere utilized for examining phage delivery. The S. Newport modelhad been previously developed inthe Pricelab in anticipation for usein this Hatch Project. Then, in 2012, a human outbreak of S. Enteritidis disease traced to contaminated ground beef prompted us to develop a second infection model in calves, using S. Enteritidis. To date, six dairy calves have been infected with S. Enteritidis; two of these calves also received the 7-hage cocktail discussed in Objective 1. We chose to administer the phage (and the Salmonella) orally usinga dosing syringe, as oral transmission is the naturalroute for Salmonella and the best route to administer phage into the gut where Salmonella colonizes and causes disease. However, these calves were housed in an indoor biocontainment stall, and not in a pasture, so dosing syringe administration is not feasible for phage treating a herd of cattle. A more logical approach to herd phage treatment is by administering the phage intotheir drinking water, which is normally placed in water troughs which are refilled with tap water regularly. Therefore, we also examined each of the cocktail phages' survivability in tap water by adding a knowntiter of phage to sterile tap water and incubating overnight, after which the phage were re-titered. We learned that several of our phage are sensitive to the chlorine in tap water; adding skim milk powder to the water reversed this sensitivity, most likely because the casein in milk binds the chlorine. These results indicate that we need to use skim milk-treated tap water to administer phage to cattle from a water trough. Objective 3: Determine the effect of bacteriophage treatment on pathogen shedding and severity of animal disease. Bacteriophage cocktails were used to treat calves experimentally infected with S. Newport and S. Entertitidis. Bacteria and phages were enumerated in fecal samples to monitor shedding. In addition, calf temperature (fever) and fecal consistency (formed -> watery diarrhea) were measured. Calves infected with 5 x 109 colony forming units ofS. Newport showed disease signs of diarrhea and fever on day 2 or 3 post-inoculation. Fecal samples showed that large numbers of salmonellaeare shed during the acute phase of the disease, with numbers slowly decreasing for 7-10 days. When the 5-phage S. Newport cocktail was administered to infected calves when they first showed a fever spike, the fever rapidly decreased, as did the number of salmonellae in the feces. Relatively low numbers of phage were shed in the feces within 24 h of phage treatment initiation. A similar approach was followed with the S. Entertitidis infection model; however, the results were quite different. Of the six calves infected, four received no phage treatment and two were treated with the 7-phage S. Entertitidis cocktail.Three of the control calves shed moderate amounts of salmonella in their feces,and one control calf shed high amounts of salmonella in its feces. One of the treated calves fecally shed low amounts of salmonella; the second shed high amounts of salmonella in its feces. Septicemia developed in three of the calves with the most severe disease, and salmonella was recovered from the peripheral lymph nodes of two of these calves. Interestingly,the peripheral lymph nodes of one of the treated calves yielded phages, too. More work needs to be performed to develop a reproducible S. Entertitidis infection model in calves before we can examine the efficacy of phage treatment. Objectives 4 & 5. These objectives were carryovers from previous Hatch projects which we decided not to pursue when work with the Salmonella-infected calf models and bacteriophage treatment showed promising results.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Kitchens, S.R., Wright, J.C., Duran S.H., and Price, S.B., Factors influencing environmental Salmonella incidence and proximal movement in a multi-species animal facility . 101st Annual Meeting of the Southeastern Branch of the American Society for Microbiology. Kennesaw, GA, November 14, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Shirley, James F., Kitchens, Steven R., Walz, Paul H., Wright, James C., and Price, Stuart B., Factors influencing environmental Salmonella incidence and proximal movement in a multi-species animal facility . 101st Annual Meeting of the Southeastern Branch of the American Society for Microbiology. Kennesaw, GA, November 14, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Kitchens, S.R., Wright, J.C., Duran S.H., and Price, S.B., Factors influencing environmental Salmonella incidence and proximal movement in a multi-species animal facility . Abstracts of 2015 Phi Zeta Research Day Forum, Auburn, AL. November 4, 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Shirley, James F., Kitchens, Steven R., Walz, Paul H., Wright, James C., and Price, Stuart B., Characterization and Simplification of a Bacteriophage Cocktail to Reduce Salmonella Lymph Node Carriage in Calves . Abstracts of 2015 Phi Zeta Research Day Forum, Auburn, AL. November 4, 2015.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: This projecttargets two audiences. One of those audiences would be producers interested in reducing Salmonella in cattlein order to reduce the number of human cases of salmonellosis caused by exposure to contaminated bovine products. A second audience would be veterinarians who treat cattle for salmonellosis. The advantage for either of these audiences ofusing a bacteriophage treament approach to reducing Salmonella is the fact that no antibiotics would be required, thus avoiding the spectre of spreading antibiotic resistancefrom food animals to people. Changes/Problems: The Sugg Calf Isolation Facility, in which we conduct our calf infection experiments, is scheduled for renovation in the next project year. No animals can be housed in the facility during this renovation, which will prevent us from working with calves for approximately 7 months. This will have a significant impact on our ability to pursue the goals of the project. What opportunities for training and professional development has the project provided? Graduate Students: J. Forrest Shirley continued his MS Thesis research in calendar year 2014. He is working on Objective 3, specifically with the development and characterization of the S. Enteritidis experimental infection model in calves. Steven Kitchens began his MS Thesis research during this project period. He is focusing on Objective 1, performing the bacteriophage isolation and characterization. He also is pursuing the environmental Salmonella project. Veterinary Students: Veterinary students Jasmine Morris and Amelie Rivaleau performed work on Objective 3 during the latest project period. They cared for and cultured the calves, and enumerated Salmonella colonies from the calf fecal samples. Undergraduate Student: Undergraduate student Austin Conley enrolled in a Research Problems course in the Fall of 2014. He was assigned to work on Objective 1 with Steven, where he learned how to isolate and characterize bacteriophages, and worked on the environmental study, also with Steven. How have the results been disseminated to communities of interest? The results from both Objectives 1 and 3 were disseminated to broad local, regional and national audiences during calendar year 2014. Graduate students J. Forrest Shirley and Steven Kitchens presented their findings in departmental seminars, to the local Phi Zeta Research Emphasis Day and Research Week 2014, and to the regional Southeastern Branch meeting of the American Society for Microbiology. Principal investigatorStuart Price presented work from Objective 3 at the national General Meeting of the American Society for Microbiology. What do you plan to do during the next reporting period to accomplish the goals? Work will continue on the environmental study (Objective 1) as Steven Kitchens performs a second year of quarterly Salmonella sampling at the vet school. An emphasis will be placed on examining the interaction of Salmonella isolates and bacteriophages isolated alongside them in the environment. Forrest Shirley will use a 7-phage cocktail to treat S. Enteritidis-infected calves to determine if this 'living' antimicrobial treatment can prevent severe disease and invasion of the peripheral lymph nodes (Objective 3). In addition, work on the S. Newport infection model in calves will expand as we try to determine the lowest dose of phages that significantly reduces fecal shedding and abrogates disease (Objective 3).
Impacts
What was accomplished under these goals?
Objective 1: The 11 bacteriophages isolated in the previous project period were characterized in calendar year 2014. Each of the phages showed unique lysis patterns when tested against the 16 indicator Salmonella serotypes, demonstrating that they are distinct phage species. The bacteriophage discovery effort was expanded in calendar year 2014. Quarterly environmental sampling from sites across the veterinary school campus was initiated in Winter, 2014. Samples (349) were obtained from beef, diary, and equine hospital barns, the dairy parlor, dairy cow pasture, andsites within the Animal Health & Disease Researchunit of the vet school. These samples were enriched for isolation of Salmonella and their bacteriophages. Surprisingly, multiple sites showed contamination with S. Muenster in all fourquarters.A second serotype, S. Cerro, emerged during Spring and Summer quarters.Bacteriophages were isolated from these environmental samples that targeted either S. Muenster or S. Cerro. Objective 3: Work continued on the S. Enteritidis experimental infection model in calves. Three pairs of calves were infected with this serovar; fecal shedding of S. Enteritidis, body temperature, and fecal consistency were measured daily. 3/6 calves showed typical signs of salmonellosis: fever, diarrhea, and anorexia concomitant with significant fecal shedding of the pathogen. However, the other three calves showed a much different disease, characterized by high fever and dissemination of S. Enteritidis to the peripheral lymph nodes. Two of these calves had to be humanely euthanized; the third calf died suddenly following a severe illness.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Price, S.B., Kitchens, S.R., Shirley, J.F., Wright, J.C., and Walz, P.H., Bacteriophage-Resistant Mutant of Salmonella Newport Shows an Unusual Shedding Phenotype in a Calf Model of Infection. 2014. General Meeting Abstracts, American Society of Microbiology 114th Annual meeting, Boston, MA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kitchens, S.R., Wright, J.C., Duran S.H., and Price, S.B. 2014. Preliminary Study of Factors Associated with Environmental Salmonella Prevalence in a Multi-Species Animal Facility. Abstracts of the Southeastern Microbiology Summit - Joint meeting of Southeastern and Florida ASM Branches. Ponte Vedra, FL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Shirley, James F., Kitchens, Steven R., Walz, Paul H., Wright, James C., and Price, Stuart B. 2014. Salmonella Enteritidis contamination of bovine peripheral lymph nodes: inquiry into an outbreak of Salmonella Enteritidis in ground beef. Abstracts of the Southeastern Microbiology Summit - Joint meeting of Southeastern and Florida ASM Branches. Ponte Vedra, FL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Shirley, James F., Kitchens, Steven R., Walz, Paul H., Wright, James C., and Price, Stuart B. 2014. Experimental Infection of Calves with Salmonella Enteritidis: Disease Variability and Peripheral Lymph Node Invasion. Abstracts of 2014 Phi Zeta Research Day Forum, Auburn, AL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kitchens, S.R., Wright, J.C., Duran S.H., and Price, S.B. A one year study of factors associated with environmental Salmonella prevalence in a multi-species animal facility. Abstracts of 2014 Phi Zeta Research Day Forum, Auburn, AL. November 5, 2014.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience for this work is veterinarianswho work with dairy and beef producers, who could use bacteriophage treatment to limit salmonellosis in their client's cattle herds. Bacteriophages could be used by producers in place of antibiotics to treat salmonellosis in cattle, and the ensuing pathogen reduction could decrease salmonellosis in humans. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In calendar year 2013, this project was the source of training for a new Master's degree graduate student . He was trained by the investigators to isolate and cultureSalmonellaand their phages in the laboratory, and to assist the veterinarians in obtainingSalmonella-containinglymph node biopsies from calves. How have the results been disseminated to communities of interest? The results obtained in calendar year 2013 for both Objectives 1 and 3 of this project were presented by the investigators and graduate student to microbiologists at a branch meeting of the American Society for Microbiology, and to an audience of veterinarians at a departmental seminar where the investigators hold faculty appointments. What do you plan to do during the next reporting period to accomplish the goals? Objective1: The 11 bacteriophages will be testedin vitro against 16 strains ofSalmonellato determine their efficiency of killing. Objective 3: A phage mixture will be used to treat calves experimentally infected withS.Enteritidistheto determine if the phages reduce the salmonella in the calves, and thus, reduce the disease signs (fever and diarrhea) and fecal shedding of the pathogen.
Impacts
What was accomplished under these goals?
Salmonella is a pathogen transmitted to humans from contaminated food, especially food derived from food animals such as chickens, pigs, and cattle. Treating animals with antibiotics improves growth in food animals, in part because they suffer from fewer diseases, but also results in generating antibiotic resistant pathogens, including Salmonella. We are working to develop treatment alternatives to antibiotics that will reduce the amount ofSalmonella in cattle. One such alternative is the use of tiny viruses, called bacteriophages (phages), which target specific bacteria, in this case, Salmonella. Objective1: In this objective, we proposed to isolate and characterize multiple phage strains which targetSalmonell. In calendar year 2013, we were able to group 11 of our phage strains into specific families using an electron microscope. These 11 phages each fell into one of three families of phages according to their microscopic appearance. This information will help us to design treatment mixtures of phages that contain members of each of the three phage families. Multi-family treatment mixtures of phages, when administered orally, will be more effective at reducing Salmonella in cattle. Objective3: In calendar year 2013, we isolated a mutantSalmonellaNewport that is resistant to 4/5 phages used to treatS.Newport in cattle. This mutant was inoculated orally into calves, and, unlike its parentS.Newport, the mutant did not cause disease. Thus, we may be able to use this mutant strain to vaccinate cattle as a means of preventingSalmonellainfection in calves. Also under this objective we began development of a new experimental infection model in calves using a differentSalmonellastrain,S.Enteritidis. This strain normally moves from chickens into people, but in one recent human outbreak, the source ofS.Enteritidis was shown to be meat from dairy cows. So our goal was to determine if we could reproduceS.Enteritidisdisease in cattle. Indeed, using a large inoculum (1010 cells) ofS.Enteritidis, we showed that this strain ofSalmonelladoes cause disease in cattle. This finding will help us develop bacteriophage treatment protocols to reduceS.Enteritidis in cattle.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
J.F. Shirley, S.R. Kitchens, J.C. Wright, P.H. Walz, and S.B. Price. 2013. Salmonella Enteritidis Isolate Characterization and Bacteriophage Cocktail Selection. Southeastern Branch Meeting, American Society for Microbiology, Conference Abstract, Auburn University, AL
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
S.R. Kitchens, J.C. Wright, P.H. Walz, and S.B. Price. 2013. Bacteriophage-Resistant Mutant of Salmonella Newport and Disease in Experimentally Infected Calves. Southeastern Branch Meeting, American Society for Microbiology, Conference Abstract, Auburn University, AL
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: In the period 1/01/2012 through 12/31/2012, work continued on Aims 1 and 3 of the project. Aim 1: A new set of Salmonella-targeted wild bacteriophages was isolated from bovine fecal cultures and characterized. All of them were examined in vitro for their ability to lyse Salmnella Newport broth cultures. Aim 3: Bacteriophage-resistant Salmonella Newport strains were isolated in the laboratory, and one of them was inoculated into two calves to determine if it could cause disease. The results from this experiment were presented at a professional meeting for the American College of Veterinary Pathologists (ACVP), in the Experimental Pathology poster session. Members of the ACVP are veterinary pathologists, and this work is of interest to them because it describes a novel means for control of an important food animal pathogen. PARTICIPANTS: As in 2011, graduate student/pathology resident Joanna Hyland incorporated portions of this project into her Master's project in 2012. She was taught how to isolate bacteriophage-resistant mutants of Salmonella, and showed the investigators how to obtain lymph node biopsies on infected animals post-mortem. Joanna presented her data at the annual American College of Veterinary Pathology meeting again in 2012, as well as at the local Phi Zeta Research Emphasis Day held at the vet school. Two new veterinary student workers were trained by the investigators in 2012 in calf husbandry, fecal culture collection, and benchtop bacteriology. And a visiting Swiss veterinary student spent two weeks in the PI's lab working with the Salmonella and their bacteriophages. TARGET AUDIENCES: The target audience for this work includes dairy and beef cattle producers, who could use bacteriophage treatment to limit salmonellosis in their cattle herds. Bacteriophages could be used by producers in place of antibiotics to treat salmonellosis in cattle. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Aim 1: One of the new bacteriophages isolated, S55, appeared to be most active in vitro when its Salmonella Newport host was growing in stationary phase. This is in contrast with the other Salmonella-targeted bacteriophages in our treatment mixture, which lyse their host when the bacterium is growing in exponential phase. This finding has important implications for bacteriophage treatment of calves with salmonellosis. Salmonellae probably multiply in different growth phases in the calf, depending on their location in the intestine, and whether they are growing intra-cellularly or extra-cellularly. So finding a bacteriophage that lyses Salmonella Newport during stationary phase growth will strenghthen the therapeutic potential of our bacteriophage treatment mixture, which, with the addition of S55, should lyse the salmonellae regardless of their growth phase inside the calf. Aim 3: The bacteriophage-resistant Salmonella Newport isolated in this project caused less disease signs when inoculated into calves, and was shed in lower numbers in the feces compared to the parent, bacteriophage sensitive, S. Newport. This is an important outcome because, in our animal model, calves inoculated with S. Newport and treated with our bacteriophage cocktail shed bacteriophage-resistant salmonellae, which, in a commercial dairy or beef herd, could be transmitted to other animals. The finding that these bacteriophage-resistant salmonellae are attenuated indicates that infected, treated calves may not be as apt to cause the disease in other animals in the herd.
Publications
- Hyland, J.E., S.B. Price, J.C. Wright, P.H. Walz, and S.R. Kitchens. 2012. Isolation and characterization of bacteriophage-resistant Salmonella enterica serotype Newport. American College of Veterinary Pathologists Program and Abstract Book, 2012 Annual Meeting, Seattle, WA, (#E-30)
- Hyland, J.E., S.R. Kitchens, J.C. Wright, P.H. Walz, and S.B. Price. 2012. Oral bacteriophage treatment to reduce fecal shedding of Salmonella enterica serotype Newport from calves. Abstracts of 2012 Phi Zeta Research Emphasis Day, Auburn, AL (#50).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: In this 12-month period, the investigators focused on the first three primary objectives of the project. (Aim 1) Two new Salmonella Newport-targeted bacteriophages (S49, S50) were isoated and characterized. S50 showed strong lytic activity on S. Newport bacterial lawns and in S. Newport liquid cultures. S49 showed no lytic activity in S. Newport liquid cultures. (Aims 2 & 3) The investigators began to explore the timing of bacteriophage delivery on S. Newport fecal shedding and disease signs in S. Newport experimentally infected calves. Two calves were inoculated with a standard dose of S. Newport (5 x 10^9 cfu's). Bacteriophage treatment (10^10 pfu's of each of 5 bacteriophages) was instituted when each calf's temperature increased 2 C above normal, and treatment continued until their temperature dropped back to normal. Another set of experiments was initiated to develop a multi-strain S. Newport shedding model in calves to determine if bacteriophage treatment could decrease shedding and disease signs from more strains than just the lab strain. Therefore, eighteen strains of S. Newport isolated from clinical samples over a period of years were inoculated concomitantly (3 x 10^8 cfu's of each) into 2 pairs of calves, and fecal shedding was followed daily. One pair of these calves received bacteriophage treatment at the onset of fever. Finally, a competition experiment was carried out in one pair of calves where the parent strain of S. Newport was co-inoculated into two calves with a differentially marked bacteriophage-resistant mutant of the parent S. Newport strain. PARTICIPANTS: Graduate student/pathology resident Joanna Hyland incorporated portions of this project into her Master's project in 2011. As a veterinarian, she had a strong background in working with animals, including calves. New to her professional development was the opportunity to perform benchtop science. During 2011, she learned how to isolate, identify, and culture pathogenic Salmonella species and their bacteriophages. She was shown how to prepare a bacterial/bacteriophage inoculum for administration into calves, and how to follow the course of salmonellosis in calves via fecal culture, temperature assessment, and fecal scoring. She also was mentored for and presented a departmental seminar describing her work and findings. And in December, she presented her work in poster format at a national meeting of her scientific society, the American College of Veterinary Pathology. In addition to providing a rich training ground for a veterinarian/graduate student/resident, the project also hosted two veterinary student workers, who were enrolled in their first or second year of the veterinary curriculum. These students were trained by the faculty investigators both in calf husbandry and care, as well as in benchtop microbiological techniques. A special emphasis on biosafety was placed on their training, as this project was the veterinary students' first exposure to large-scale work with a human bacterial pathogen. TARGET AUDIENCES: The target audience for this project continues to be those working in the food animal production industry, specifically individuals working with beef or dairy cattle. The finding that a calf treated with bacteriophages can transmit those bacteriophages to a co-housed calf has important implications in regards to bacteriophage treatment as an intervention to prevent movement of Salmonella from food animals to humans. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(Aim 1) The discovery and characterization of another S. Newport-targeted bacteriophage,S50, allowed us to incorporate a fifth bacteriophage species into our cocktail for testing in calves experimentally infected with S. Newport. This addition improved the cocktail, since more species of bacteriophage decreases the chance of the S. Newport host from developing bacteriophage resistance. (Aim 2 & 3) One of the two calves inoculated with the single strain of S. Newport developed fever on Day 2 following S. Newport challenge, and was treated with the 5-phage cocktail on Days 3-8. Its temperature returned to normal by Day 5. It shed S. Newport fecally on Days 1-19 post-inoculation (PI), and bacteriophage fecally on Days 3-14 PI. Further work with additional calves needs to be performed to determine if bacteriphage treatment decreases S. Newport fecal shedding and/or disease signs. Interestingly, the second calf, which was co-housed with the first and received the S. Newport challenge concurrently but no bacteriophage treatment, began shedding bacteriophage in its feces on Day 4 PI, and continued through Day 10 PI. This finding indicates that bacteriophages inoculated into one calf can spread via the fecal/oral route to other animals in a herd. The implications of this finding are significant, as it suggests that treating each individual calf or cow in a herd suffering from salmonella contamination would be unnecessary; movement of the bacteriophage via the fecal/oral route to untreated calves would "treat" them, too. The first two calves to receive the 18-strain S. Newport inoculum were controls, used to develop the fecal shedding model. The second pair of calves shed enough S. Newport into their feces to be enumerated by direct plating. Bacteriophage treatment of both calves was initiated on Day 2 PI following a fever spike, and their temperatures returned to normal by Day 5 PI. Although one of the calves stopped shedding countable S. Newport colonies (~10 cfu/g feces) by Day 7, the other calf continued to shed countable S. Newport colonies through Day 11. Additional studies need to be performed to determine if the bacteriophage cocktail is active against multiple strains of S. Newport. In the competition experiment, one of the calves shed the phage-resistant S. Newport strain on Day 1 PI, but not any on the following days, and it did not shed the parent phage sensitive strain at all. The other calf shed both parent and mutant strains of S. Newport on days 1-5 PI. Except for Day 1, this second calf shed a higher percentage of the phage sensitive (parent) strain than the phage resistant strain, suggesting that the spontaneous mutation(s) that conferred phage resistance on the mutant strain may also have made it less fit for survival through the calf gut. Of course, additional calves and competition experiments need to be performed before this conclusion can be proven.
Publications
- Price, S.B., Wright, J.C., and Walz, P.H. 2011. Reduction of Salmonella Newport shedding and disease in experimentally infected calves. American Society for Microbiology Program and Abstract Book, General Meeting, New Orleans, LA (Z-213).
- Hyland, J.E., Price, S.B., Wright, J.C., Walz, P.H., and Kitchens, S.R. 2011. Oral bacteriophage treatment to reduce fecal shedding of Salmonella enterica serotype Newport from calves. American College of Veterinary Pathologists Program and Abstract Book Annual Meeting, Nashville, TN (#182)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In previous work, two Salmonella infection models were developed, one in chickens with S. Typhimurium, and a second in cattle with S. Newport. Therefore, in Year 1 of the project, work focused on the first objective: Isolate and characterize lytic bacteriophages (phages) that target Salmonella. Environmental samples and supernatant from Salmonella-positive clinical bacteriology samples were screened for plaque-forming ability on 12 serovars of Salmonella. Plaques were purified and stocked in DMSO at -80 C. To date, 50 phages have been isolated that form plaques on at least one Salmonella serovar. Five S. Typhimurium-targeted phages, and 5 S. Newport-targeted phages, have been selected to comprise "cocktails" for use in their respective animal infection models. The phages in each cocktail was chosen for their unique lysis pattern in the 12 serovar screening assay on solid media and for their lytic characteristics in liquid cultures of their host serovar. PARTICIPANTS: Stuart Price, principal investigator (PI), provided the lab space and personnel to carry out the bacteriological and bacteriophage protocols for the project. He supervised the graduate student, research assistant, lab assistant, and veterinary student workers in the lab, and organized and oversaw the animal work in the isolation facilities. He will submit the progress and final reports for the project, and prepare drafts of manuscripts and extramural grant proposals drawn from the results of the project for review by the co-authors. Jim Wright, co-investigator (CI), will supervise the graduate student and veterinary student workers in handling, inoculating, and taking fecal samples from the calves. He will also perform the statistical analyses of the data and aid PI Stuart Price with experimental design. Paul Walz, co-investigator, will oversee the overall health of the calves, and, as a bovine clinician, provide emergency care for ill animals. He will work with the PI Stuart Price and CI Jim Wright with experimental design. Haroldo Toro, co-investigator, will work with the PI on all aspects of the chicken project, including experimental design, animal husbandry, and chick inoculation. Joanna Hyland, graduate student trainee, will work on the calf project under the supervision of Price, Wright, and Walz. She will perform the in vitro experiments involving Salmonella and bacteriophages, as well as inoculate and culture the calves. She will help the PI prepare manuscripts, and work with the faculty investigators on extramural grant proposals. Steven Kitchens, research assistant II, will provide routine support to the bacteriology and bacteriophage portion of both the calf and chicken projects. He will help with calf and chicken inoculations, and help the Toro group with chicken husbandry and culture. Heather Worley, lab assistant, will perform glassware washing, media preparation, and routine maintenance in the PI's lab. Veterinary student workers will work with the faculty investigators and graduate student with the routine care and culture of the calves. TARGET AUDIENCES: Much of this project involves applied science, and results from it are designed to help the food animal industry. Specifically, poultry producers, dairymen, and beef cattle operations will be the recipients of new knowledge gained about bacteriophage-directed pathogen reduction. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Only environmental samples that contained Salmonella yielded Salmonella-targeted phages. Therefore, we focused our phage-finding efforts on Salmonella-positive cultures obtained from our veterinary bacteriology laboratory, rather than random sampling of multiple environmental sites. Many of the 50 phages in our library lyse S. Typhimurium; far fewer lyse S. Newport. Almost all of the phages in our collection lyse more than one Salmonella serovar. Therefore, to limit the possibility of picking identical species of phage for inclusion in our cocktails, each cocktail was composed of phages that lysed a different set of Salmonella serovars, including S. Typhimurium or S. Newport. In the in vitro liquid culture assays, incorporating a single lytic phage species into a growing culture of its bacterial host results in lysis of almost all of the host cells. However, resistant bacterial survivors multiply despite the presence of high phage titers, and eventually the number of resistant bacteria in the phage-containing culture rivals that of control cultures containing no phage. To prevent selection of resistant phages in our animal infection models, 5 different phages will be mixed and used as a pathogen reduction treatment. If phage treatments reduce Salmonella shedding in food animals, then this novel approach to pathogen reduction could lessen the reliance on antibiotic usage in animal production agriculture.
Publications
- No publications reported this period
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