Progress 10/01/12 to 10/12/16
Outputs
Target Audience:The NC-1202 group includesbacteriologists, virologists, molecular biologists, pathologists, and immunologists with a history of collaboration, that update the group during the annual meeting in December, in conjunction with the Conference of Research Workers in Animal Diseases (CRWAD) annual meeting in Chicago, IL.Target audiences reached by our efforts include students, scientists, academia, and government officials in the animal and food safety fields as well asanimal health companies in industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Hands-on training, independent study projects, and graduate projects provided training to students at the University of Arizona in various aspects including the culture of Campylobacter, poultry husbandry and vaccination, quantification of Campylobacter from vaccination studies and subsequent data analysis, as well as protocol development for the study of immune responses in vaccine studies. As well, Dr. Armstrong, as chair of the University of Arizona Food Safety Consortium, organized numerous annual Food Safety conferences to members of the academic community, regulatory agencies and the food safety industries to update on research, and to facilitate dialogue and collaborative work. How have the results been disseminated to communities of interest?Presentations and posters were presented at multiple scientific conferences (nationalConference of Research Workers in Animal Diseases, international Campylobacter, Helicobacter and Related Organisms Conference, international Vaccine Technologyconference), agencies (Maricopa County Environmental Services, AZ Governor's Economic Development conference, Bill and Melinda Gates Foundation)as well as presentations to numerous animal health companies, to disseminate knowledge regarding this research. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Recombinant Attenuated Salmonella Vaccine (RASV) constructs expressing Campylobacter geneswere developed in conjunction with Dr. Curtiss' and Dr. Roland's labs at Arizona State University andtested in multiple broiler chicken vaccine trials for the reduction of C. jejuni colonization.Single and multiple-component vaccines were tested, using homologous and heterologous challenge and various vaccination methods including spray and water vaccination. Results indicate that RASVs are effective and compatible with industry methods and can be developedcommercially to reduce Campylobacter in poultry, with the end goal of reducing transmission of Campylobacter to humans, therefore reducing human illnesses associated with the handling and consumption of poultry. Presentations and posters were presented at multiple scientific conferences as well as presentations to animal health companies to disseminate knowledge regarding this research.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Ahmad, M. ELISA Development for Identification of Antibody Response to Poultry Vaccination. (Honor's thesis and poster). University of Arizona, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Law B. Update on Campylobacter. Presentation at 2016 NC1202 Meeting, December 3, 2016. Chicago, IL.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The NC-1202 group consists of bacteriologists, virologists, molecular biologists, pathologists, and immunologists with a history of collaboration, that update the group during the annual meeting in December, in collaboration with the CRWAD annual meeting in Chicago, IL. Target audiences reached by our efforts include students, scientists, industry and government officials in the food safety and food safety vaccine segments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Armstrong, as chair of the University of Arizona Food Safety Consortium, organized the 6th annual University of Arizona Food Safety Conference on September 18, 2015. The keynote address was provided by Bonnie Fernandez-Fenaroli, Executive Director for the Center for Produce Safety and outcomes from University of Arizona food safety research programs were presented. In attendance were members of the academic community, regulatory agencies and the food production and processing industries. How have the results been disseminated to communities of interest?Dr. Armstronggave an oral presentation, "Use of a Recombinant Attenuated Salmonella Typhimurium Vaccine Vector for the Reduction of Campylobacter jejuni in Broiler Chickens" at the Conference of Research Workers in Animal Disease (CRWAD) in Chicago, IL in December, 2014. Dr. Law updated regarding the progress of the vaccine at the annual NC-1202 technical committee meeting (Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety), held in conjunction with the CRWAD conference in Chicago, IL in December, 2014. A presentation entitled "Goals and Impacts of A4161 Program: Prevention and Control of Poultry Flocks and Poultry Products, Including Eggs", was given by Dr. Law and Dr. Godwin on the associated projects with the A4161 program for the reduction of Campylobacter and Salmonella at the USDA NIFA Food Safety Project Directors Meeting on July 24, 2015 in Portland, OR. Numerous meetings have been held throughout the year with industry for discussions regarding the vaccine technology, industry assessments and application, and possible future collaborations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Campylobacter jejuni Recombinant attenuated Salmonella vaccines (RASV) that have previously been shown to produce reduction of C. jejuni colonization of broiler chickens are being utilized in industry compatible conditions for assessment of utility in production and refined to produce greater and more consistent reductions. Studies utilizing methods that increase feasibility of application of the vaccine to industry have contributed to the refinement of a potential product for administration to poultry which may be efficacious in commercial production environments to reduce Campylobacter loads. Ultimately this application may translate to reduced human illnesses in populations handling/consuming meat from vaccinated poultry. The testing of additional potential antigens has further refined the vaccine construct under development. Two to three replicates of vaccine dose studies utilizing initial and booster doses of the cjLAJ2 RASV at 106, 107, or 108/chick (a cost effective dose range) were conducted. Results indicate the potential for a reduced dose to maintain efficacy with regard to reduction of colonization by C. jejuni NCTC11168 following challenge. Studies investigating the efficacy of non-gavage-based, industry compatible delivery methods were also performed. A commercial spray cabinet was used to administer vaccine, as was addition of the vaccine to drinking water. Initial and booster doses of the vaccine were administered via either spray/spray, spray/water or water/water. Results of these studies indicate that these dosing methods may be viable options for effective delivery of this vaccine to poultry, though further development is required.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Armstrong A and Law B. Use of a Recombinant Attenuated Salmonella Typhimurium Vaccine Vector for the Reduction of Campylobacter jejuni in Broiler Chickens. Presentation at Conference of Research Workers in Animal Disease, December 8, 2014. Chicago, IL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Law B. Update on Campylobacter. Presentation at 2014 NC1202 Meeting, December 6, 2014. Chicago, IL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Law B, Zhang Q, Godwin S, Hassan H. Goals and Impacts of A4161 Program: Prevention and Control of Poultry Flocks and Poultry Products, Including Eggs. Presentation at USDA NIFA Food Safety Project Directors Meeting, July 24, 2015. Portland, OR.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The NC-1202 group consists of bacteriologists, virologists, molecular biologists, pathologists, and immunologists with a history of collaboration, that update the group during the annual meeting in December, in collaboration with the CRWAD annual meeting in Chicago, IL. Target audiences reached by our efforts include students, scientists, industry and government officials in the food safety and food safety vaccine segments. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Armstrong, as chair of the University of Arizona Food Safety Consortium, organized the 5th annual University of Arizona Food Safety Conference on October 10, 2014, held at the Westward Look Wyndham Grand Resort. Approximate attendance was 90 participants, with 30% of attendees coming from food safety industries or government agencies. The Food Safety Conference consisted of 9 hours of contact time over one day, with invited speakers, breakfast and lunch, a poster session, and breakout sessions to facilitate dialogue and collaborative work. How have the results been disseminated to communities of interest? A presentation entitled "Campylobacter prevention and control methods" was given by Dr. Law at an international technical vaccine conference, the Vaccine Technology V Conference, in Playa del Carmen, Mexico on June 11, 2014 regarding the results and impacts of Campylobacter on the public health burden. Dr. Armstrong spoke on the topic, "Salmonella, Campylobacter and the Future of Food Safety Vaccination" at a meeting of the Maricopa County Environmental Services Department on July 31st, 2014. She also gave an oral presentation, "Use of a Recombinant Attenuated Salmonella Typhimurium Vaccine Vector for the Reduction of Campylobacter jejuni in Broiler Chickens" at the Conference of Research Workers in Animal Disease (CRWAD) in Chicago, IL in December, 2014. Dr. Law presented vaccine development work at the University of Arizona Food Safety Consortium Annual Conference in Tucson, AZ in October, 2014 and gave an oral presentation regarding the progress of the vaccine at the annual NC-1202 technical committee meeting (Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety), held in conjunction with the CRWAD conference in Chicago, IL in December, 2014. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Campylobacter jejuni Efforts to develop an efficacious vaccine using a recombinant attenuated Salmonella vaccine in collaboration with Dr. Ken Roland at Dr. Curtiss' lab at Arizona State University to express C. jejuni proteins to reduce the colonization of C. jejuni in broiler chickens were continued. The knowledge from these chicken vaccination studies contributes to the development of a vaccine which can be eventually made commercially available to reduce Campylobacter in poultry, with the end goal of reducing transmission of Campylobacter to humans, therefore reducing human illnesses associated with the handling and consumption of poultry and its associated sequelae. Two component vaccine studies reveal that 2 or 3 way component RASV vaccines may provide one such efficacious and feasible vaccination strategy. Heterologous vaccine challenge studies and subsequent strain colonization studies allow refinement of the chicken vaccination model in use, allowing further work to be targeted to the challenges of producing an industry compatible vaccine candidate. Two replicates of heterologous challenge vaccination studies were conducted. Chickens were vaccinated with 1ml at ~1x1010 of vaccine expressing CjLAJ2 and challenged with C. jejuni poultry isolate RM1221, with both studies resulting in a 1 log reduction. Further colonization studies were conducted for various strains to assess optimal challenge doses for heterologous challenges. A third replicate vaccine study testing dual doses (1ml each at ~1x1010 CFU) of vaccines expressing CjLAJ1 and CjLAJ2 and challenged with the homologous strain C. jejuni NCTC 11168 resulted in a 2-log reduction, in addition to the first two replicates demonstrating a near total 7-log reduction and the other vaccinate group demonstrating a modest 1-log reduction of C. jejuni compared to the positive controls.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2014
Citation:
McCloskey, A. Development of Recombinant Attenuated Salmonella Vaccines expressing Campylobacter jejuni genes Cj0113, Cj0982c, Cj1656c or Cj0404 for reduction of colonization by C. jejuni. (Master's thesis). University of Arizona, 2014.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2014
Citation:
Johnson, S. Development of Salmonella vector vaccines targeting Campylobacter jejuni dps and ctsr proteins for the reduction of colonization in broiler chickens. (Master's thesis). University of Arizona, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Law B, Armstrong A, Curtiss R, Roland K. Making Food Safer: Campylobacter Prevention and Control Measures. In Abstracts of posters and oral presentations from Vaccine Technology V, 8-13th June 2014. Paradisus Playa del Carmen, Mexico. Engineering Conferences International.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The NC1202 group consists of bacteriologists, virologists, molecular biologists, pathologists, and immunologists with a history of collaboration, that update the group during the annual meeting in December in collaboration with the CRWAD annual meeting in Chicago, IL. Regarding this project,a Campylobacter symposium organized by Randall Singer’s group (University of Minnesota) in conjunction with Charles Hofacre (University of Georgia) in furtherance of the outreach goals of this Campylobacter vaccine project targeted veterinarians and researchers with interest in Campylobacter.The symposium was offered in July 2013 at the annual AVMA/AAAP (American Veterinary Medical Association/American Association of Avian Pathologists) meeting in Chicago, Illinois.The symposium was recorded in digital HD and will be available for distribution. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? TheCampylobacter symposium offered in July 2013 at the annual AVMA/AAAP (American Veterinary Medical Association/American Association of Avian Pathologists) meeting in Chicago, Illinois provided professional development to veterinarians, researchers, and students.Since the symposium was recorded in digital HD and will be available for distribution, it allows future veterinarians, researchers, and students access to gain a background in Campylobacter. How have the results been disseminated to communities of interest? A poster waspresented at a dedication to the new School of Animal and Comparative Biomedical Sciences at the University of Arizona March 29, 2013 to enhance public understanding and increase interest in learning invaccine development and sciencein the new school. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) Efforts to develop an efficacious vaccine using arecombinant attenuated Salmonella vaccinein collaboration with Dr. Roy Curtiss’ lab at Arizona State University to express C. jejuni proteins to reduce the colonization of C. jejuni in broilers are being continued. Preliminary poultry vaccine studies testing an initial format 2-way vaccine have been conducted. Chickens were vaccinated with dual doses of vaccines expressing CjLAJ1 and CjLAJ2 and challenged with the homologous strain C. jejuni NCTC 11168. Results differed for the two groups, with one vaccinate group demonstrating a near total 7-log reduction and the other vaccinate group demonstrating a modest 1-log reduction of C. jejuni compared to the positive controls.The CjLAJ3 protein was found not to be effective as a vaccine candidate in reducing the numbers of C. jejuni in chickens, and will not be included in component vaccines. 2) A Campylobacter symposium was organized by Randall Singer’s group (University of Minnesota) in conjunction with Charles Hofacre (University of Georgia) in furtherance of the outreach goals of the vaccine project. The symposium was offered in July 2013 at the annual AVMA/AAAP (American Veterinary Medical Association/American Association of Avian Pathologists) meeting in Chicago, Illinois. This symposium targeted veterinarians and researchers with interest in Campylobacter. The symposium featured talks ranging from Campylobacter ecology within the broiler environment to the human burden of illness associated with Campylobacter. Talks were given by globally recognized experts in the field of Campylobacter research, including Drs. Robert Tauxe, Jaap Wagenaar, and Nigel French. The symposium was recorded in digital HD and will be available for distribution.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Armstrong, A, Curtiss R, Roland K, Law B. Use of a Recombinant Attenuated Salmonella Typhimurium Vector Vaccine for the Reduction of Campylobacter jejuni in Broiler Chickens. In Abstracts of posters and oral presentations from the 17th International Workshop on Campylobacter, Helicobacter and Related Organisms,15-19th September 2013. University of Aberdeen, 2013. J Med Microbiol. (http://jmm.sgmjournals.org/content/62/Pt_9/suppl/DC1)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Berghaus RD, Thayer SG, Law BF, Mild RM, Hofacre CL, Singer RS. Enumeration of Salmonella and Campylobacter spp. in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks. Appl Environ Microbiol. 2013 Jul;79(13):4106-14. doi: 10.1128/AEM.00836-13. Epub 2013 Apr 26.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Campylobacter jejuni 1. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ3 protein. We are continuing with our efforts to develop an efficacious vaccine using an attenuated Salmonella vector to express C. jejuni proteins shown to be involved in the colonization of broilers. The Salmonella strain used as the vector was developed by Curtiss and has three different mutations which results in its attenuation. Gene CjLAJ3 was inserted into an expression plasmid, cloned and amplified in an E. coli shuttle vector, extracted, and electroporated into the Salmonella vector. Chicks were orally vaccinated with the vector vaccine or empty vector at 10 and 16 days. Chicks were orally challenged with the homologous C. jejuni strain NCTC11168 at 10 days after the final vaccination. Ten days post challenge the chickens were necropsied, the ceca were removed, and the cecal contents serially diluted and direct plated for enumeration of C. jejuni on Campy Cefex plates. 2. Challenge studies. Challenge studies were conducted in chickens with several strains of C. jejuni. 3. Focus on disseminating knowledge. Dr. Joens organized the University of Arizona Food Safety Conference on October 12, 2012, held at the Omni Tucson National Resort. Approximate attendance was 108 attendees, with 20% of attendees coming from food safety industries or government agencies. The Food Safety Conference consisted of 10 hours of contact time over one day, with seven invited speakers followed by a poster session and a dinner to facilitate dialogue and collaborative work. PARTICIPANTS: Lynn Joens, Professor; Bibiana Law, Associate Research Professor; Alexandra Armstrong, Assistant Research Scientist; Alix McCloskey, Doctoral Student; Shivanna Johnson, Master's student TARGET AUDIENCES: The research provided laboratory and field training for students. The Food Safety Conference targeted food safety researchers, students, industry professionals, public health members and government agency representatives. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Campylobacter jejuni 1. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ3 protein. There was not a significant reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressed CjLAJ3 protein following challenge. Although another trial needs to be conducted, these initial results demonstrate that the CjLAJ3 protein is not effective as a vaccine candidate in reducing the numbers of C. jejuni in chickens. 2. Challenge studies. Generally, poultry derived isolates colonize at consistently high levels with relatively low challenge doses (<10^5), while human clinical isolates colonize poorly or variably even at relatively high challenge doses (10^6-10^7). These challenge studies will assist us in selecting dosages for future challenges of chicks in vaccine studies. 3. Focus on disseminating knowledge. In the post-conference evaluations, attendees found the ability to network with food safety researchers, students, industry professionals, public health members and government agency representatives to be the most valuable part of attending the Food Safety Conference. Researchers who attended the workshop had the opportunity to interact with stakeholders and learn of current food and water safety concerns and research needs; food safety professionals and funding agencies came away from the conference with a greater understanding of what food safety research is being done by University of Arizona Food Safety Consortium members and who they could collaborate with on future projects; students discovered different areas of food and water safety through the presentations as well as in discussions with fellow attendees which they found beneficial in assisting with determining their future educational goals and career paths.
Publications
- 1. Morrison CM, Dial SM, Day WA Jr, Joens LA. Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis. Appl Environ Microbiol. 2012 Apr;78(8):2867-73. Epub 2012 Feb 3.
- 2. Theoret JR, Cooper KK, Zekarias B, Roland KL, Law BF, Curtiss R 3rd, Joens LA. The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination. Clin Vaccine Immunol. 2012 Sep;19(9):1426-31. Epub 2012 Jul 11.
- 3. Cooper KK, Cooper MA, Zuccolo A, Joens LA. Re-sequencing of a virulent strain of Campylobacter jejuni NCTC11168 reveals potential virulence factors. Res Microbiol. 2012 Oct 6. doi:pii: S0923-2508(12)00136-2. 10.1016/j.resmic.2012.10.002. [Epub ahead of print]
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Campylobacter jejuni 1. Identification of C. jejuni proteins involved in host colonization. A major thrust of our laboratory is examining C. jejuni proteins for their role in the colonization of chicks. Genes expressing proteins that are secreted and have a surface orientation, or are up-regulated, or express known colonization factors are being mutated and examined for their role in gut colonization. One of those proteins is gene CjLAJ3. The gene encoding the protein was mutated and examined for its role in poultry colonization. The ∆CjLAJ3 mutant and C. jejuni parent strain were examined for colonization traits in poultry. 2. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ2 protein. The Salmonella strain used as the vector was developed by Curtiss and has three different mutations which results in its attenuation. Gene CjLAJ2 was inserted into an expression plasmid, cloned and amplified in an E. coli shuttle vector, extracted, and electroporated into the Salmonella vector. Chicks were fasted and orally vaccinated with the vector vaccine or empty vector at 10 and 16 days. Trial 1 chicks were orally challenged with a homologous C. jejuni strain and trial 2 chicks were orally challenged with a heterologous C. jejuni strain at 10 days after the final vaccination. Ten days post challenge the chickens were necropsied, the ceca were removed, and the cecal contents serially diluted and plated for enumeration. PARTICIPANTS: Lynn Joens, Principal Investigator, Professor; Bibiana Law, Assistant Research Professor; Alexandra Armstrong, Doctoral Candidate; Rita Mild, Doctoral Candidate; Lisbeth Echevarria, Doctoral Candidate. TARGET AUDIENCES: Laboratory and field training for graduate students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Campylobacter jejuni 1. Identification of C. jejuni proteins involved in host colonization. All the birds inoculated with the wild-type strain were colonized at an average of 7.42E+07 CFU/g, whereas, 3 of 30 birds (<10 CFUs) were colonized at an average of 7.33E+01 CFU/g with the C. jejuni mutant strain ∆CjLAJ3. These results indicate that the CjLAJ3 protein maybe an excellent vaccine candidate to reduce the numbers of C. jejuni in chickens. 2. Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ2 protein. In trial 1, we had a 4-log reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressed CjLAJ2 protein following challenge with the homologous strain, as compared to chicks receiving the empty vector (EV) vaccine. There was a 2-log reduction in C. jejuni when the CjLAJ2 vaccinates were compared to the normal controls. In trial 2, we had an overall reduction of 1.0 log of C. jejuni in cecal contents of chicks vaccinated with the CjLAJ2 protein following challenge with a heterologous strain, as compared to chicks receiving the EV vaccine. These trials demonstrated a significant reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressing the CjLAJ2 protein and challenged with the homologous strain. In addition, a 1-log reduction was present in chicks challenged with a heterologous strain. These results demonstrate that cecal numbers of C. jejuni can be significantly reduced using a Salmonella vaccine expressing gene CjLAJ2.
Publications
- Cooper, K.K., Cooper, M.A., Zuccolo, A., Law, B., L.A. Joens. 2011. Complete Genome Sequence of Campylobacter jejuni Strain S3. J Bacteriol. 193(6):1491-2.
- Mild, R.M., L.A. Joens, Friedman, M., Olsen, C.W., McHugh, T.H., Law, B., Ravishankar S. 2011. Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast. J Food Sci. 76(3):M163-8.
- Theoret J.R., Cooper K.K., Glock RD, L.A. Joens. 2011. A Campylobacter jejuni Dps Homolog Has a Role in Intracellular Survival and in the Development of Campylobacterosis in Neonate Piglets. Foodborne Pathog Dis. 8(12): 1263-1268.
- Morrison, C.M., Armstrong, A.E., Evans, S., Mild, R.M., Langdon, C.J., L.A. Joens. 2011. Survival of Salmonella Newport in oysters. Int J Food Microbiol. 2011 Aug 2;148(2):93-8. Epub 2011 May 13.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: A. Salmonella Newport a) Mutation of genes in SPI1 and SPI2. Well characterized genes in Salmonella Pathogenicity Island 1 (invA) and Pathogenicity Island 2 (ssaV) were mutated in Salmonella Newport, which resulted in invasion and phagocyte survival deficiencies, respectively. These mutants along with the parent strain were allowed to colonize oysters and were maintained in aquaria. b) Identification of colonization/invasion genes. Analysis of the microarrays from the transposon site hybridization (TraSH) assay was completed. These analyses indicated over 90 genes involved in the missing transposon gene pool that was recovered from the challenged oysters. The missing genes included structural proteins such as those needed for the formation of flagella and the type III secretion system as well as proteins involved in metabolic functions and protein transport. Precisely, the genes were pliC a lysozyme inhibitor, flgE the flagellar hook protein, ydiM an inner membrane transport protein, and the protein A3103 that is homologous to the secretin outer membrane pore protein of the type III secretion system. To test whether the genes were truly necessary for survival, the four genes were mutated, and the mutants along with the wildtype strain were used to infect oysters. Results showed that the elimination of motility by mutating the flagella apparatus significantly reduced Salmonella's ability to colonize oysters. By 30 days post-infection, the flagellar mutant (flgE gene) was unable to be detected in oysters, whereas, the wildtype was present at an average of 10^2 CFU/g. There was no significant difference between the levels of wildtype and the other 3 mutant strains. B. Campylobacter jejuni a) Feedlot Sampling for the Presence of Campylobacter jejuni. Sampling for the presence of Campylobacter jejuni was accomplished for year two for a cohort of 36 cattle. Fecal swabs were taken from these cattle at the range, at arrival at the feedlot, and monthly for 6 months at the feedlot, and at day of slaughter. Carcass swipes were taken with both the USDA-FSIS method, and the ventral midline method at dehiding, and evisceration. Environmental samples at the feedlot including fecal material from the birds, were conducted prior to arrival, and at 3 months and 6 months. Swipes of feed bunks, watering units, drag swabs of pens, and flies at the feedlot were sampled beginning at prior to arrival of cattle and monthly after for six months. b) Pulsed-field gel analysis of C. jejuni isolates from various species at the University of Arizona feedlot prior to and upon the arrival of calves from the range were conducted. PARTICIPANTS: Principal Investigator: Lynn A. Joens; Assistant Research Professor: Bibiana Law, Ph.D.; Post-doctorates: Kerry Cooper, Ph.D., Margarethe Cooper, Ph.D.; Graduate Students: Chris Morrison-Ph.D candidate, Alex Armstrong-Ph.D candidate, Crystal Brillhart-Ph.D candidate, Rita Mild-Ph.D candidate, and Kelsey Shaner-M.S. candidate. TARGET AUDIENCES: Laboratory and field training for graduate students and post-doctorates. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Salmonella Newport - Survival studies in the oysters revealed that neither of the type-three secretion systems (TTSS) were critical for Salmonella's invasion and survival in oysters. However, the TraSH studies did show the importance of flagella genes in the colonization of the oyster with S. Newport and also with the survival of the bacterium in oyster tissue. B. Campylobacter jejuni - Only pigeons were positive for C. jejuni at pre-arrival of the calves. Other sources for acquiring C. jejuni started to emerge between the months of October through December, including pen floors, feed bunks, and the watering units. Only 1 of 36 cattle were positive for C. jejuni at arrival at the feedlot, but by the end of month one, 28/36 cattle were already positive for C. jejuni. The six months after arrival of the cattle showed a dramatic increase in the presence of C. jejuni in all samples except feed and flies. These sources remained positive until the day of processing in which 30 of 36 cattle fecal samples were positive. During processing, a significant number of carcasses were positive for C. jejuni after evisceration with the highest number of positives coming from swipes from the ventral midline sites. Surprisingly, unlike year 1, carcass and ground beef samples from the carcasses were positive upon testing for C. jejuni. Clearly, C. jejuni is present all the way through the feedlot, during processing and aging of the beef carcass, and in the resulting hamburger. This should raise the spectra of hamburger as a definite risk factor in acquiring campylobacteriosis. In addition, we have isolates that are involved in transmission dynamics, bacterial fitness, and host specificity as shown in the pulsed-field gels from year one. At three months, one isolate from flies and birds had spread to a calf in pen 1 and in pen 2. Only a few calves were colonized at three months in the feedlot with C. jejuni, but at six months there was a major change in isolates colonizing the calves. In addition, there was one calf which became colonized with three different C. jejuni isolates at the same sampling time point. Sequencing of these isolates should identify genes that can be grouped into these categories and may lead to virulence factors that can be targeted in vaccine preparations.
Publications
- Ravishankar S, Zhu L, Reyna-Granados J, Law B, Joens L, Friedman M. Carvacrol and cinnamaldehyde inactivate antibiotic-resistant Salmonella enterica in buffer and on celery and oysters. J Food Prot. 2010 Feb;73(2):234-40.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: A. Salmonella Newport - Sampling oysters for Salmonella from the Yaquina river basin in Oregon. Twenty oysters were collected from the Yaquina river each month starting in January 2009 to examine the role of climate change on the presence of Salmonella. Oysters were collected by the Hatfield Marine Science Center of Oregon State University and sent to the University of Arizona for culture analysis. A total of 880 oysters were examined from January to November with 26 positive for Salmonella giving us a 2.9% prevalence rate. More importantly, oysters were only positive for Salmonella during the spring and summer months (April-July). All Salmonella isolates were tested with antisera specific to Salmonella serovar Newport, with 34% (9/26) being positive for S. Newport. B. Campylobacter jejuni - 1) Sequencing and annotation of C. jejuni strain S3 (poultry). We have hypothesized that non-invasive C. jejuni strains express or harbor genes that are responsible for a fluid exudate phenotype in infected piglets which is absent in piglets infected with invasive strains. The fluid producing C. jejuni strain RM1221 has been sequenced but is resistant to genetic manipulation and would be of limited use in the current project. Therefore, C. jejuni strain S3 was sequenced since this strain is easily transformable and produces intestinal lesions and a watery exudate similar to RM1221 in infected piglets. Sequencing of S3 was accomplished by the Genomic Analysis and Technology Core (GATC) Facility at the University of Arizona using 454 technology. Sequencing of the C. jejuni S3 genome resulted in 14 large contigs at 42x coverage with a genome size of 1.7 Mb. This sequence is being finished and annotated at the Arizona Genomics Institute (core facility), then it will be submitted to GenBank. 2) Identification of C. jejuni proteins in intestinal fluid exudate of infected piglets. One ml of filtered pooled intestinal fluid from pigs infected with RM1221 or filtered pooled fecal material from normal pigs was added to a set of Centricon Centrifugal Devices, subjected to low-speed centrifugation and the filtrate recovered. Equal amounts of normal and infected piglet intestinal fluid proteins were mixed together, concentrated, and labeled with Cy2 to serve as the internal standard. Normal and infected piglet intestinal fluid proteins were labeled with Cy3 and Cy5, respectively. After labeling, the Cy2, Cy3, and Cy5 labeled proteins were pooled and subjected to 2D gel electrophoresis. Protein spots of interest were picked and digested. All spectra were searched against the bacterial protein database from NCBI. Genes encoding four proteins from the DIGE separation were detected in the genome of C. jejuni at a homology of >98%; reciprocal BLASTp searches revealed no orthologs with similarly high homology indicating that the proteins are encoded by the C. jejuni genome. PARTICIPANTS: Principal Investigator: Lynn A. Joens; Assistant Research Professor: Bibiana Law, Ph.D.; Post-doctorates: Kerry Cooper, Ph.D., Margarethe Cooper, Ph.D.; Graduate Students: James Theoret-Ph.D candidate, Chris Morrison-Ph.D candidate, Alex Armstrong-Ph.D candidate, Crystal Brillhart-Ph.D candidate, Rita Mild-Ph.D candidate. TARGET AUDIENCES: Laboratory training for graduate students and post-doctorates PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Salmonella Newport - Salmonella Newport is a pathogen colonizing bivalves and in doing so presents a food-safety risk to individuals that consume raw oysters. Sampling oysters for Salmonella from the Yaquina river basin in Oregon. These data imply that Salmonella spp. and especially S. Newport are still being detected in oysters consumed by the public but the risk is greater in the warmer months. However, the prevalence numbers of S. Newport are lower than those from oysters examined in 2002-2003 (7.4%). TraSH studies should detect genes needed for the Salmonella JJPX01.0014 clone to colonize and survive in oysters. B. Campylobacter jejuni - Infection of humans with C. jejuni most often results in a watery diarrhea; however, pathogenic factors associated with this common disease manifestation are unknown. Several factors may have complicated the study of these factor(s) and the resulting pathology. Our data indicates that C. jejuni strains obtained from clinical cases are generally invasive and that these strains induce a hemorrhagic disease, not watery diarrhea. We speculate that invasive, non-fluid producing strains may be overrepresented in clinical cases as people infected with fluid producing strains, though debilitated, may be less likely to seek medical attention. Use of clinical strains most often in research may have led to the understudy of underrepresented fluid producing strains. Our analysis of the virulence phenotypes of C. jejuni poultry isolates in the piglet model has identified non-clinical, non-invasive strains that induce a watery diarrhea. It is anticipated that these studies, designed to identify novel C. jejuni fluid producing factor(s), will significantly increase the understanding of this common pathology resulting from C. jejuni infection. Moreover, insights provided by these studies will provide reagents for new diagnostic tests and targets for new anti-campylobacter therapeutics and vaccines. 1) Sequencing and annotation of C. jejuni strain S3 (poultry). Sequencing and finishing S3 (non-invasive, fluid producing) of C. jejuni should allow us to make comparisons of genes being up-regulated between a non-invasive, fluid producing strain (S3) and sequenced invasive, clinical strains in the infected piglet model and possibly identify the gene(s) responsible for this phenotype of watery diarrhea. 2) Identification of C. jejuni proteins in intestinal fluid exudate of infected piglets. The DIGE separation analysis of fluid exudate from pigs infected with S3 will be combined with the up-regulation analysis to identify genes involved in intestinal fluid production in the piglet model. Mutation of these genes will allow us to test this hypothesis of fluid production (watery diarrhea) in the piglet model.
Publications
- Law, B.F., S. M. Adriance, L.A. Joens. Comparison of in vitro Virulence Factors of Campylobacter jejuni to in vivo Lesion Production. Foodborne Pathogens and Disease. 6(3):377-85. 2009.
- Wilson, M. K., A. B. Lane, B. F. Law, W. G. Miller, L. A. Joens, M. E. Konkel, and B. A. White. Analysis of the Pan Genome of Campylobacter jejuni Isolates Recovered from Poultry by Pulsed-Field Gel Electrophoresis, Multilocus Sequence Typing (MLST), and Repetitive Sequence Polymerase Chain Reaction (rep-PCR) Reveals Different Discriminatory Capabilities. Microbial Ecology 58:843-855. 2009. DOI 10.1007/s00248-009-9571-3.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: A bacterioferritin expressed by C. jejuni upon colonization of abiotic and biotic surfaces during biofilm formation has been discovered in the lab. The gene has been cloned and mutated in C. jejuni by insertion of a chloramphenicol cassette in the center of the gene. The gene has also been expressed in DH5a, tagged with HIS, and purified using a HIS tag column. The gene is conserved among all Campylobacter strains and is highly conserved between C. jejuni sequence strains (NCTC11168, RM1221, and 81-176), with only three nucleotide substitutions. PARTICIPANTS: Lynn Joens: PI, to supervise all aspects of project; James Theoret: Graduate student, to conduct experiments TARGET AUDIENCES: Project provided laboratory training for students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In conjunction with another study, three poultry colonization studies were conducted with the parent and the mutant strain, and a significant reduction was observed in the colonization of birds inoculated with the mutant strain. Thirty-eight of 40 birds were colonized with the parent strain, whereas, only 10 of 40 birds were colonized with the mutant strain of C. jejuni. In the birds that were colonized with the mutant strain, viable numbers averaged over three logs less than in birds colonized with the parent strain. Therefore, we hypothesize that the bacterioferritin protein can serve as an antigen to be used as a vaccine in poultry to reduce the numbers of C. jejuni, therefore reducing the numbers of food-borne illnesses caused by this organism. The data generated from this proposal contributes to knowledge regarding colonization of poultry by C. jejuni, and will serve as preliminary data to obtain funding for vaccination studies to confirm that the bacterioferritin protein would reduce the colonization of C. jejuni in broilers, as well as to examine dosage and delivery methods.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: From previous studies, Yaquina Bay has had oysters contaminated with Salmonella, mostly Salmonella Newport. Salmonella Newport is an emerging pathogen, and is of particular interest to CDC due to its multi-drug resistant nature. Oysters and water from the Yaquina Bay and Tillamook Bay have both been sent every 2nd month to the laboratory at the University of Arizona from Oregon State University. These oysters are homogenized and enriched to isolate for Salmonella Newport. Additionally, oysters from retail outlets such as restaurants in Arizona are being tested every second month for Salmonella. Campylobacter spp. (primarily C. jejuni) cause an estimated 2.4 million cases of gastroenteritis per year in the U. S. yet their pathogenic mechanisms remain largely unknown. A novel pilus expressed by C. jejuni upon colonization of abiotic and biotic surfaces during biofilm formation has been discovered in the lab. This pilin protein has been sequenced and the gene identified in the
genomic sequence of NCTC11168 and a mutant with disrupted pilA has been prepared. The ability of the pilus mutant to adhere and invade epithelial cells was analyzed with HEp2 cells.
PARTICIPANTS: Lynn Joens: PI, to supervise all aspects of project Bibiana Law, Assistant Research Professor, to design protocols, train students, and perform research Crystal Brillhart and Alex Armstrong: Graduate students, to conduct experiments
TARGET AUDIENCES: Project provided laboratory training for students.
Impacts
As we did obtain some Salmonella positives from Tillamook Bay during the study, we are currently processing the samples on a monthly basis. We are analyzing rainfall events and environmental factors to increase our understanding of the contamination. Some oyster samples from the restaurants have also been positive. The Salmonella isolates will be analyzed via Pulsed Field Gel Electrophoresis, and tested for antibiotic resistance. The pilA mutant showed decreased adherence and no invasion in the in vitro assay, indicating that the gene is involved in adherence and invasion.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. We have sampled watering nipples from two more broiler houses and currently have cultured over 250 isolates of bacteria. We are currently performing 16S PCR on each culture and the products will be sent to the sequencing facility. Experiments will be performed to characterize the biofilm bacterial community. We are also currently investigating the conditions required for C. jejuni to form biofilms in well water. Various conditions such as various timepoints, and adding nutrients or organisms such as Pseudomonas will be assessed for their affect on the growth of C. jejuni
biofilms. Ultimately, the goal is to understand how biofilms are formed by C. jejuni and if this is an environmental reservoir involved in the infection of poultry.
Impacts
Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. By using a staining procedure to visualize biofilms, we have determined that 1) C. jejuni can form biofilms on abiotic surfaces such as polyvinyl chloride plastic (PVC), acrylonitrile-butadiene-styrene plastic (ABS), copper, polystyrene, and polypropylene when grown in Mueller-Hinton broth, 2) protein synthesis maybe required for the early stages of biofilm formation as chloramphenicol inhibits biofilm formation, 3) as nutritional and environmental conditions of the medium changes, it can have varying affects on the ability of C. jejuni to form biofilms, and 4) C. jejuni
mutants defective in the putative virulence genes and a quorum sensing gene had varying affects on biofilm formation. Therefore, with the combination of genetic, molecular and microscopy techniques, we have been able to initiate investigations into the mechanisms that control C. jejuni biofilm formation and development. Based on the results, we can see that C. jejuni biofilm formation can be a set of complex actions. To form complex communities, C. jejuni must integrate environmental signals by determining density and nutrient availability. We have sampled a biofilm off a water pipe from a broiler house and over 100 isolates of bacteria were cultured and 16S PCR performed on each culture. The PCR products will be sent to the sequencing facility and experiments to characterize the biofilm bacterial community will be performed. The actions and gene expressions that drive biofilm development will continue to be the point of investigation.
Impacts
Campylobacter jejuni: A microbial community on abiotic surfaces: Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Presence, potential virulence and antibiotic susceptibility of Campylobacter jejuni isolated from food and companion animals: Infection with Campylobacter jejuni is associated with exposure to numerous animal related sources. In this study, we report on the recovery, potential for virulence and antibiotic resistance levels of C. jejuni isolated from food and companion animals. Three hundred seventy-eight fecal samples from food and companion animals and beef carcass swabs were tested for the presence of Campylobacter jejuni. Campylobacter jejuni was isolated from 13.8% (11/80) of dogs, 5% (1/20) of goats, 30.5% (53/174) of cattle and 94.7% (18/19) of beef carcasses. Forty-two of these isolates were tested for susceptibility to four antibiotics, each representing a class of antibiotic approved for use in both humans and animals. No isolates were found to be resistant to erythromycin or gentamicin, 2.4% of isolates were resistant to ciprofloxacin and 28.6% of isolates
were resistant to tetracycline. The virulence of these 42 isolates was assessed using in vitro macrophage survival and epithelial cell association assays. Of these, 34 were able to survive within macrophages through 72 hrs, 39 were able to attach to epithelial cells and 31 were capable of invading epithelial cells. Data from these studies suggests that many of the isolates recovered from the non-poultry animal sources included this study have the capacity to cause disease if transmitted to humans. Campylobacter jejuni: A microbial community on abiotic surfaces: Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries, and a source of public health and economic burden. C. jejuni is consistently present in the intestinal tract of commercial broiler chickens, which are probably the main source of human infections. In fact biofilm formation on watering nipples may play a role in the colonization of broiler chickens. By using a staining procedure to
visualize biofilms, we have determined that 1) C. jejuni can form biofilms on abiotic surfaces such as polyvinyl chloride plastic (PVC), acrylonitrile-butadiene-styrene plastic(ABS), copper, polystyrene, and polypropylene when grown in Mueller-Hinton broth, 2) protein synthesis maybe required for the early stages of biofilm formation as chloramphenicol inhibits biofilm formation, 3) as nutritional and environmental conditions of the medium changes, it can have varying affects on the ability of C. jejuni to form biofilms, and 4) C. jejuni mutants defective in the putative virulence genes and a quorum sensing gene had varying affects on biofilm formation. Therefore, with the combination of genetic, molecular and microscopy techniques, we have been able to initiate investigations into the mechanisms that control C. jejuni biofilm formation and development. Based on the results, we can see that C. jejuni biofilm formation can be a set of complex actions. To form complex communities, C.
jejuni must integrate environmental signals by determining density and nutrient availability. Therefore, these actions and gene expressions that drive biofilm development will continue to be the point of investigation.
Impacts
Presence, potential virulence and antibiotic susceptibility of Campylobacter jejuni isolated from food and companion animals: Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases such as C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options. Campylobacter jejuni: A microbial community on abiotic surfaces: Understanding how biofilms are formed by C. jejuni will contribute to how C. jejuni can be prevented from the formation of C. jejuni biofilms in broiler houses and ultimately result in reducing the colonization of chickens with C. jejuni.
Publications
- Lee, M. K., S A Billington, and L A Joens. 2004. Potential Virulence and Antimicrobial Susceptibility of Campylobacter jejuni Isolates from Food and Companion Animals. Foodborne Pathogens and Disease 1:223-230.
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Progress 01/01/03 to 12/31/03
Outputs
Cattle and livestock have been shown to shed Campylobacter jejuni indicating that they are potential reservoirs of infection for man. In this study bovine, and other livestock feces or intestinal swabs from carcasses were cultured for C. jejuni using broth enrichment followed by selective plating. Isolates were initially selected based on colony morphology, microscopy, and hippurate hydrolysis and subsequently confirmed as C. jejuni by PCR. To date 213 have been evaluated for C. jejuni, with 21 confirmed positives. The ability of the isolates to survive in macrophages was evaluated in J774A.1 cell line using the gentamicin protection assay. Results indicate the ability of the isolates to survive for 72 hours at levels comparable to C. jejuni M129, a human clinical strain. All isolates were tested against selected antibiotic to test for resistance levels.
Impacts
Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases such as C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options.
Publications
- No publications reported this period
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Progress 10/01/97 to 09/30/02
Outputs
Cattle and livestock have been shown to shed Campylobacter jejuni indicating that they are potential reservoirs of infection for man. In this study bovine, and other livestock feces or intestinal swabs from carcasses were cultured for C. jejuni using broth enrichment followed by selective plating. Isolates were initially selected based on colony morphology, microscopy, and hippurate hydrolysis and subsequently confirmed as C. jejuni by polymerase chain reaction. To date, 212 swabs have been evaluated, yielding 21 confirmed C. jejuni isolates. The ability of isolates to survive in macrophages was evaluated in the J774A.1 cell line using the gentamicin protection assay. Results indicate ability of isolates to survive for 72 hours at levels comparable to C. jejuni M129, a human clinical strain.
Impacts
Campylobacter jejuni has been isolated from many cattle and livestock animals. The presence of such a reservoir in animals contributes to the zoonotic spread of diseases due to C. jejuni to man. Determining the prevalence of C. jejuni in a broad range of species, the virulence of isolates, and antibiotic resistance profiles will add to our understanding of transmission and treatment options.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
To date, 15 adult milk cows and 30 unbred heifer calves aged less than 3 mos. have been sampled. Two fecal swabs were taken directly from each subject and transported to the laboratory in Cary-Blair media on ice. At the laboratory, one swab from each subject was processed for Salmonella and the other processed for Campylobacter. Four cows have tested positive for C. jejuni based on culture, hippurate hydrolysis, and PCR using C. jejuni specific primers. No subjects have tested positive for Salmonella. All C. jejuni isolates obtained in this study will be tested for pathogenicity via cellular invasion and survival assays.
Impacts
Sampling of cattle represent an initial effort to determine the coloniztion of multiple animal sources including chickens. Such efforts will be useful in determining the sources of contamination in livestock and may have an impact in both animal health and food safety.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
1.Campylobacter Intracellular Survival Kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies suggest that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains were constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intraepithelial cell survival as growth curves for the katA mutant and wild type strains generated from long term culture within HEp2 cells are roughly identical. Catalase does
however contribute to intramacrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p less 0.05) relative to the wild type strain after 72 hour incubation with these cells. Additionally, inhibitors of nitric oxide production and the respiratory burst in J774A1 cells increased the ability of the katA mutant to survive within these cells. 2.Bacterial Contamination of Shellfish Recently, there has been an increase in the incidence of bacterial enteric disease from the consumption of raw shellfish. We examined clams and oysters collected from U.S. Coastal waters for the prevalence of fecal coliforms, Campylobacter spp. and Salmonella spp. Shellfish were harvested and shipped to Arizona on ice for bacterial examination. The shellfish meat was collected aseptically, homogenized, and cultured on selective agar following enrichment. DNA was extracted from a portion of each homogenate for PCR analysis. Shellfish samples (n equals
162) from six different bays were examined to date. Coliforms were detected in samples from each site with a prevalence ranging from 3 to 75 percent. Salmonella spp. were also detected in samples from each of the six sites, with a prevalence as high as 74percent. In contrast, Campylobacter spp. were only detected in samples from two different bays. Surprisingly, 21 of 89 samples collected from three of the six sites contained Salmonella spp., only. This result demonstrates that current coliform testing standards alone do not identify other potentially widespread foodborne pathogens.
Impacts
1. C. jejuni is able to survive in macrophages and get to deeper tissue.If it can be determined how C. jejuni can survive in macrophages then it would be possible to find new ways to treat infection before it can invade deeper tissue. 2. Current fecal detection methods include coliforms only. We show that there is no direct correlation between Salmonella and Campylobacter with coliforms. We need to start looking for these other organisms as well as coliforms.
Publications
- Day, W.A., Sajecki, J.L., Pitts, T.M., Joens, L.A., 2000, Role of Catalase in Campylobacter jejuni Intracellular Survival, Infection and Immunity 68(11)6337-6345.
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Progress 01/01/99 to 12/31/99
Outputs
We have continued to look at the kinetics of C. jejuni intracellular survival within macrophages. In addition to previous survival studies performed on the isogenic katA mutant of C. jejuni in mouse peritoneal macrophages, studies in J774 and porcine peritoneal macrophages demonstrated a significant reduction in the number of viable katA mutant cells over a 72 hour period when compared to the wild type control. Invasion studies in epithelial cells with the sodB mutant from Dr. Pickett at the University of Kentucky confirmed her previous results. However, we also found the sodB mutant attached at a significantly lower rate than the wild type control. This finding, in addition to our work shows that this mutant exhibits almost identical survival kinetics to the sodB wild type and M129 strain C. jejuni in macrophages and has led us to believe that disruption of the sodB gene may hinder the organism's ability to adhere to epithelial cells and subsequently invade the cell.
Since the bacteria are actively phagositized in macrophages we did not see reduced numbers of the sodB mutant when infecting J774 macrophages. Inhibitors were used in the above studies involving the katA mutant to determine what is actually mediating the destruction of this mutant in macrophages. The two inhibitors used were NG-L-monomethyl arginine, which competes for nitric oxide synthase production, and apocynin which inhibits NADPH oxidase and the respiratory burst. We were able to recover the katA mutant in numbers analogous to the wild type when one or both of the inhibitors were used in J774 macrophage studies. Further studies are being done utilizing confocal microscopy to determine the intracellular trafficking pattern of C. jejuni within macrophages. We will be looking at phagolysosome development of C. jejuni infected J774 cells in addition to changes in phagolysosomal pH.
Impacts
The pathogenic mechanisms by which Campylobacter jejuni cause disease are currently unclear. Phenotypic traits such as invasion and survival within epithelial and phagocytic cells appear to play an important part in C. jejuni virulence. Therefore, studies dealing with C. jejuni intracellular survival may bring us closer to understanding the mechanisms by which the organism causes disease.
Publications
- Sajecki, Jaime W.A. Day, L.A. Joens. 2000. The role of catalase in Campylobacter jejuni intracellular survival. Infection and Immunity.(in press)
- Konkel, M.E., L.A. Joens, P.F. Mixter, Campylobacter, 2ond Ed. I. Nachamki and M.J. Blaser. "Molecular Characterization of Campylobacter jejuni virulence determinants". P217-240. 1999.
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Progress 01/01/97 to 12/31/97
Outputs
Campylobacterosis: Inhibition of transcription and translation of de novo expressed proteins in C. jejuni blocks its ability to invade host cells in vitro. In order to detect recombinant expressed C. jejuni OMPs, rabbit polyclonal anti-serum to sarkosyl extracted proteins derived from host-cell exposed C. jejuni were produced. Screening of a C. jejuni cosmid library with the anti-serum identified a single clone harboring 34 kb insert DNA. Lysates of this clone did not bind anti-serum specific for plate grown C. jejuni indicating that this epitope was expressed only while cultured with Hep-2 cells. Subclonal analysis identified the serum binding epitope encoded within a 6.6 kb ClaI fragment. Non-reactive subcloned fragments all mapped within a 3 kb portion of the fragment. Sequence and alignment analysis of this DNA identified three open reading frames comprising an operon of high homology to an iron uptake system (ferrichrome uptake operon fhuABCD) in other bacteria.
We have also examined the role of catalase in C. jejuni intracellular survival. Initial studies using zymographic native gel analysis identifies a single pattern of catalase activity in C. jejuni lysates indicating the presence of only one catalase gene in this organism. To clone the C. jejuni strain M129 catalase gene a plasmid genomic library was introduced into the double catalase negative E. coli strain UM255. Clones containing the C. jejuni M129 catalase gene were identified using a bubble assay. Sequence and alignment analysis of the insert DNA revealed an ORF encoding catalase with high homology to numerous heme containing catalases present in other proteobacteria. A katA minus strain (JD900) of C. jejuni M129 was constructed via insertion mutagenesis. This strain has no catalase activity verified by spectrophotometric and zymographic analysis and is hyper-sensitive to even low concentrations of hydrogen peroxide (1mM) compared to the wild-type strain. Porcine proliferative
intracellularis: Monoclonal antibodies were produced against L. intracellularis to better characterize the antigens involved in invasion of epithelial cells. Ten Mabs were recently produced using gradient purified L. intracellularis grown in cell culture. These are currently being cloned and characterized for their ability to neutralize growth or invasion of the bacterium. These include clones: 1C2=115 kDa, 2A2=60, 43, 41 kDa, 2B6=60, 43, 41 kDa, 2C1=60 kDa, 3A1=41 kDa, and 3D4= 71 kDa. Non-conserved primers to the L. intracellularis 16s rRNA gene were constructed and examined for their ability to amplify L. intracellularis DNA using standard PCR methods. Essentially, an intense 205 bp product was produced when DNA extracted from PPE tissue or HIE cells infected L. intracellularis was used as template. The 16s rRNA reaction correlated with the results from using C, D primers to insert 78 from L. intracellularis.
Impacts
(N/A)
Publications
- Joens, LA, S Nibbelink, RD Glock. 1997. Induction of gross and microscopic lesions of porcine proliferative enteritis by Lawsonia intracellularis. AJVR, 58:1125-1131.
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Progress 01/01/96 to 12/30/96
Outputs
Lipooligosaccharide (LOS) from isolates B204 (wildtype) and tly- (hemolysin minus mutant) s. Hyodysenteriae was sent for monosaccharide composition analysis. The results of the analysis demonstrated major reductions in the weight percent of carbohydrates in the LOS and also a reduction in the amount of muramic acid in the peptidoglycan layer in the hemolysin minus mutant. A cosmid library of S. Hyodysenteriae isolate B204 was made using a modified pRY107 shuttle vector (containing the SuperCos 1 plasmid) and XL-1 blue MR as the parent E. Coli strain. We have obtained approximately 1000 clones with inserts averaging to cover the genome about 1.5 times. Prelimary screening of the library with monoclonal antibodies specific for LOS carbohydrates have been negative. We are currently screening this expression library with B204 convalescent sera absorbed with S. Inncens or absorbed with a serotype 1 strain of S. Hyodyssenteriae. We are also awaiting the LOS structural data
of S. Hyodysenteriae to initiate complementation studies with strains of Salmonella with mutanted biosynthetic genes for the expression of LPS.
Impacts
(N/A)
Publications
- Kramomtong, I., Neramitmansook, S.C., Joens, L.A., Limamongpranee, S. 1996. Comparison of the ELISA and selective culture in the diagnosis of swine dysentery in Thailand. Vet. Rec. 138(14):332-333.
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Progress 01/01/95 to 12/30/95
Outputs
Further information on S. hyodysenteriae-specific LOS in providing immunity to swine dysentery has been gained. Monoclonal antibodies specific for LOS can neutralize the growth of the spirochete in vitro & may provide some protection in orally immunized mice challenge exposed to S. hyodysenteriae. We have examined field-case sera from pigs with porcine proliferative enteritis (PPE) for antibody to the ISI agent using the Western blot technique. Although experimentally infected pigs often show no serological response to the ISI antigen, pigs with a chronic-type infection do develop an adequate antibody response. We have also demonstrated the presence of the ISI agent in mesenteric lymph nodes, as well as intestinal tissue from experimentally infected pigs. Infected lymph nodes were detected by both PCR and culture. Using zymography (substrate gels) we have demonstrated the presence of a single catalase protein produced by C. jejuni. The gene encoding catalase was cloned
by complementing the double catalase mutant E. coli UM255 with a C. jejuni genomic library in the shuttle vector pRY107. Six catalase positive clones containing identical insert DNA were isolated. The catalase positive recombinant was found to harbor a 4.4 kilobase insert which encodes a catalase protein that co-migrates with the C. jejuni catalase on substrate gels. Subcloning analysis has revealed HincII and EcoRI sites within the catalase gene to be used for cassette mutagenesis to determine if catalase is a virulence factor.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
Growth of the PPE agent in continuous cell lines is extremely hard to monitor. Determination of actual numbers by culture or light microscopy is not possible due to the intracellular nature and size of the organism. The purpose of this work was to develop a competitive PCR assay that could be used to quantitate the organism in cells and in feces. This was accomplished through the development of a competitive PCR involving the internal primers (C/D) and an internal standard (mimic). The mimic (159 bp, was constructed by ligating linkers containing the C/D primer sequences to a 119 bp Xba-I/BssHII fragment from Bluescript (Stratagene). The product was quantitated, 10-fold diluted and the dilutions added to subsequent ileitis DNA templates to quantitate the organism in cell cultures. The product was determined to be 5ng/ul after ligation and amplification. Template DNA from the ileitis agent was co-amplified with the mimic DNA and quantitated by visual comparison. The
age of the pig used in the PPE model and the effect of dexamethasone on lesion production in the pig was examined for future protection studies. Each pig received cells and spent media from a 225 cm2 flask infected for 12 days at 37C under an atmosphere of 5% CO2. Reproduction of PPE (75%) was accomplished in the older animal (7.5 weeks of age) with or without the administeration of dexamethasone. Gross lesions consisted of edema, hemorrhage, and fibrin cast formation in both the small and large intestine. Microscopic lesions demons.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Porcine Proliferative Enteritis:. 1. Cell culture of the ileitis agent has been expanded to a Celligen system which is essentially a cell fermentor system. This system employs beads which provide the epithelial cells an expanded surface area for growth. Oxygen consumption is tracked over a 15 day period and the organism is harvested after consumption levels off. This unit has allowed for the production of a large mass of antigen for vaccine trials which are currently underway. 2. Random primers (10 bp) are currently being used with PCR to screen for the presence of the agent in inoculated cells. Products were obtained with two recently examined primers using low stringency conditions (37(degree)C). Specific amplified products were detected in extracted DNA from cells containing the ileitis agent, but not in noninfected Henle cells. These primers will be used to evaluate various cell lines for the establishment and growth of the organism. In addition, products specific
for the agent will be sequenced, primers developed to the specific sequence and used to detect the agent in fecal preparations from infected pigs in the field.
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Progress 01/01/92 to 12/30/92
Outputs
A "knockout mutant" of the Surpulina hyodysenteriae hemolysin (Tly) was producedfollowing recombination by using a cassette to disrupt the hemolysin gene (tly). Both the mutant strain and parent strain, T42 S. hyodysenteriae, were inoculated into 3 week-old pigs. Of the five three-week old pigs inoculated with the mutant strain, none exhibited symptoms of dysentery during the 30-day study period. Three of the T42 control pigs developed clinical signs of swine dysentery and died during the study period. This study suggests that one of the putative virulence factors of the spirochete may be the Tly hemolysin protein. In an attempt to reproduce porcine proliferative enteritis (PPE) with a definitive agent, enterocytes from PPE affected pigs were enzymatically removed, subjected to antibiotics for 3 hours at 37(degree)C, and lysed with 0.5% deoxycholate. The lysate was inoculated onto Henle cells and the cells examined daily for cytopathic effect. A strict intracellular
organism of 1.4-2.5 (mu)m in length and 0.24-0.29 (mu)m in diameter was identified in infected Henle cells and intragastrically inoculated into 3-week old piglets. The intracellular agent produced clinical signs and proliferative lesions in both the small and large intestine of orally inoculated piglets. Control pigs, which received normal uninfected Henle cells, had no gross or microscopic lesions typical of PPE at necropsy.
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Progress 01/01/91 to 12/30/91
Outputs
The hemolysin from Serpulina hyodysenteriae was further characterized by examining the inhibitory effects on the toxin by different factors including pH, temperature, cations and various blocking agents. The hemolysin was found to be active at temperatures ranging from 20-42(degree)C and from 3.0-9.0 pH. There was no inhibitory effect by calcium, magnesium, BSA, sucrose or 50mM EDTA, but while both zinc and copper were inhibitory. The activity of the hemolysin was partially blocked by the addition of components with a diameter of 1.0-1.1 nm (PEG 1000 or dextran 1500). Neutralization of the hemolysin by newborn calf serum but not fetal calf serum was also observed. The inhibitory effect was shown to be related to the high concentration of high density lipids in the newborn serum. Purification of the hemolysin protein was accomplished using a combined method of HPLC Ion-exchange chromatography and free-flow isoelectric focusing. The purified material was composed of
three isomers having isoelectric points ranging between 5.0-5.5 pl.
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Progress 01/01/90 to 12/30/90
Outputs
The expression of Iak molecules on murine peritoneal macrophages were significantly induced by lipopolysaccharide-like substance (LPSLS) from Treponema hyodysenteriae, but not with LPSLS from Treponema innocens. These results have potentially important implications for the pathogenicity of T. hyodysenteriae, since the lesions of swine dysentery are essentially inflammatory in nature. Hemolysin, extracted from t. hyodysenteriae and partially purified by QAE Ion-exchange chromatography, appears to have enzymatic action. The substrate appears to be a minor lipid present in the cell wall of the erythrocytes. In addition, the lipid contains no phosphate or primary amine groups and does not consist of cholesterol or sphinogomyelin.
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Progress 01/01/89 to 12/30/89
Outputs
Envelope extracts (EE) of T. hyodysenteriae have been examined for immunogenicity in CF-1 mice and pigs. EE which were highly reactive to swine immune sera in the ELISA protected animals against challenge. Treatment with either periodate or proteinase K was shown to alter the immunogenicity of EE. Serum from vaccinated animals identified low molecular weight antigens in the Western blot analysis. In addition, the protection noted in vaccinated pigs appeared to be serotype specific. Lipooligosaccharides (LOS) from T. hyodysenteriae and T. innocens have been analyzed by high performance thin layer chromatography (HPTLC) and gas chromatography (GC). Pipopolysaccharide (LPS) from S. typhimurium was used for comparative purposes. The results indicated that differences in LOS among Treponemal isolates may account for differences in pathogenicity, and that Treponemal LOS is a substantially different compound from LPS. Outer-membrane proteins (OMP) and axial filaments of
T. hyodysenteriae were extracted, separated by SDS-PAGE and transblotted to immobilon for Western blot analysis. All seven proteins in OMP preparations and six proteins in axial filament preparations reacted to swine immune sera. These antigens were shown to be carbohydrate in nature as determined by periodate oxidation.
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Progress 01/01/88 to 12/30/88
Outputs
Two T. hyodysenteriae estrachromosomal DNA libraries have been constructed in E.coli DH5 using the cloning vector pUC 18. From these libraries we have identified two recombinant DNA probes. Hybridization experiments were conducted with cells and DNA from a battery of T. hyodysenteriae and T. innocens isolates, and demonstrated that both probes hybridized to DNA from 11 strains of T. hyodysenteriae, including each of the seven serotypes, but not to any of the 4 T. innocens strains. Western blot analysis of lipooligosaccharide (LOS) from T. hyodysenteriae implies that different epitopes of the LOS are being reorganized when natural infection is contrasted to whole cell immunization. In addition, the LOS from T. hyodysenteriae, when subjected to mild acid hydrolysis, does not resemble the hydrosylate from Salmonella typhimurium (Lipid A). This finding is of key importance as the major biological activity of LOS lies in the Lipid A molecule.
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Progress 01/01/87 to 12/30/87
Outputs
Further work on the development of a DNA probe for the detection of swine dysentery has shown that the probe is as sensitive as standard methods currently used in the diagnosis of swine dysentery. A comparison of lipopolysaccharide extracted from various isolates of T. hyodysenteriae & T. innocens has indicated that an important difference between pathogenic and non-pathogenic isolates may lie in their carbohydrate content. Further work on porcine proliferative enteritis (PPE) has indicated that in addition to Campylobacter spp., at least two viruses are present in the intestine of pigs with PPE.
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Progress 01/01/86 to 12/30/86
Outputs
A method has been developed to test the efficacy of various antibiotics against swine dysentery (SD). Lincomycin and Dimetridazole, incorporated into feed at 1000 ppm, both prevented lesions of SD in orally challenged CF-1 mice. This method will be used to study new antibiotic compounds for anti-treponemal activity. Restriction digests of chromosomal DNA have indicated that there is gene homology within serotypes of T.hyodysenteriae. Using the plasma isolated from T. hyodysenteriae is a cDNA probe, 10 spirchetes/ml were detectable in porcine feces. This method may prove more rapid and sensitive than selective culture.
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Progress 01/01/85 to 12/30/85
Outputs
Further work on the in vitro attachment of T. hyodysenteriae to human embryonic intestinal (HEI) cells has shown that a sialic-acid receptor may be involved and that this receptor is located on the spirochete and not on the host cell. A plasmid has been isolated from T. hyodysenteriae and T. innocens. All plasmids migrated at approximately 8300 Bp. Restriction enzyme testing has shown that the plasmid from T. hyodysenteriae differs from that of T. innocens.
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Progress 01/01/84 to 12/30/84
Outputs
An exotoxin has been detected in culture supernatant fluids from pathogenic isolate B204 T. hyodysenteriae. The exotoxin has been shown to be protein in nature, heat-labile, lethal to mice when inoculated intraperitoneally (IP), and toxic to Vero cells. Mice inoculated IP with the toxin revealed microscopic lesions in the cecum including dilation of the crypts and vacuolation of the goblet cells. Partial purificatin on Sephacryl 200 has yielded one fraction that is lethal for mice. Porcine proliferative ileitis was produced experimentally in conventional pigs five weeks of age. Lesions were present in the ileum of 3 of 5 pigs twenty-two days after oral inoculation with infective porcine ileal scrapings. Campylobacter spp. were found in the epithelial cells lining the crypts of the small intestine of all five inoculated pigs.
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Progress 01/01/83 to 12/30/83
Outputs
LPS from 3 untypable strains of pathogenic Treponema hyodysenteriae was extracted using the Westphal hot phenol-water technique. These were examined serologically in a gel diffusion system against corresponding rabbit antisera. Results indicate that these 3 strains, B8044, B6933, and ACK 300/8, should be considered new serotypes of pathogenic T. hyodysenteriae. Results of attachment studies indicate that pathogentic T. hyodysenteriae probably adheres to intestinal epithelial cells in order to produce disease. Enzymatic treatment of the host cells with periodate and protease reduced attachment of T. hyodysenteriae to the cells; N-acetyl neuraminic acid competitively blocks attachment, while N-acetyl glucoronic acid does not. In vitro assays studying the effects of pig serum on the growth of T. hyodysenteriae and T. innocens indicate that inactivation of nonpathogenic and avirulent strains requires complement alone, while pathogenic T. hyodysenteriae required specific
antibody in addition to complement for killing. Passive protection against swine dysentery was demonstrated in colonic loops instilled in normal pigs. Loops receiving homologous or heterologuous convalescent pig serum with active complement and cells were normal at necropsy, whereas loops which received heat-inactivated immune serum and isolates developed lesions typical of swine dysentery.
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Progress 01/01/81 to 12/30/81
Outputs
Initial phases of a prevalence survey for swine dysentery were planned with other members of the Swine Dysentery Committee of Livestock Conservation Institute. The survey is to be carried out in 1982-83.
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Progress 01/01/80 to 12/30/80
Outputs
Work during the year past has been limited to participation in the preparation of a literature review in conjunction with the Diagnosis of Swine Dysentery Committee of the American Association of Veterinary Laboratory Diagnosticians. The review, entitled, "Diagnosis of Swine Dysentery," is presently under study by committee members.
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Progress 01/01/79 to 12/30/79
Outputs
Contact pigs were exposed directly to feces from Treponema hyodysenteriae-infected mice. Swine dysentery occurred in contact pigs 11 to 13 days after exposure. Contact of pigs with non-swine reservoirs may, therefore, result in transmission of swine dysentery.
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