Source: OHIO STATE UNIVERSITY submitted to
COMBATING THE THREAT OF ZOONOTIC DISEASES IN ANIMAL AGRICULTURE: IMPLICATION TO PUBLIC HEALTH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
1001041
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Rakashekara, GI.
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Food Animal Health Research Program
Non Technical Summary
Foodborne and waterborne diseases constitute a significant burden for public health. More than two-thirds of infectious diseases affecting humans are zoonotic and food producing animals including poultry serve as reservoir for these pathogens. Furthermore, for many of zoonotic diseases there is are effective vaccine or control measures and the use of antibiotics is complicated either by the emergence of resistant bacteria. Together, these public health and safety concerns underscore the critical need for integrated research efforts to better understand the ecology and epidemiology of zoonoses, pathogenesis and immune response to these zoonotic pathogens so that effective strategies can be developed to prevent the dissemination of these organisms in the environment and limit the spread of these zoonotic agents to humans. The focus of this project will be to: 1) understand the ecology and epidemiology of the zoonotic pathogens in animal reservoirs and their environment, 2) investigate the occurrence, dissemination and ecological fitness of antibiotic resistant pathogens and 3) develop and evaluate efficient methods/strategies and models for the detection and prevention of zoonotic pathogens in the farm to table continuum.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124030110120%
7124030116020%
7124030117020%
7223599110010%
7223399110010%
7223299110110%
7223299110010%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
3299 - Poultry, general/other; 4030 - Viruses; 3599 - Swine, general/other; 3399 - Beef cattle, general/other;

Field Of Science
1101 - Virology; 1160 - Pathology; 1100 - Bacteriology; 1170 - Epidemiology;
Goals / Objectives
The overall goal of this research is to combine the research expertise of the Food Animal Health Research Program to enhance the understanding of the microbiology, epidemiology and pathogenesis of zoonotic pathogens and develop effective control strategies for these infectious agents of livestock that have potential for zoonotic transmission. Towards this end, we propose to investigate the following aspects of several zoonotic pathogens for which food-producing animals serve as reservoirs. Understand the mechanisms of microbial pathogenesis of zoonotic pathogens of food safety and public health concerns. Understand immunity to these pathogens using animal models. Understand the epidemiology and ecology of these foodborne zoonotic pathogens in the food producing animals, fresh produce and their production environments. Understand the routes of interspecies transmission of zoonotic pathogens Develop and evaluate vaccines, therapeutics, adjuvants, and diagnostics for foodborne zoonotic pathogens.
Project Methods
Methods & Analysis Due to the wide variety of zoonotic pathogens that may emerge as potential public health threats, the specific methods will vary from individual project to project. However, our underlying strategy is to use a hypothesis-driven research approach to provide sound scientific data concerning the research problems investigated. Research may include development and/or utilization of animal models of infection to characterize mechanisms of pathogenesis and pathogen/host interactions. These include studies of pathogenesis and comparative pathogenesis, interspecies transmission of pathogens, understanding the molecular basis of pathogenesis, studies of pathogen and host gene expression and characterization of host genetic risk factors for disease susceptibility. We will exploit current advances in the detection of gene and protein expression patterns to assess disease progression, and to seek unique host cellular signatures indicative of infection and/or protective immune responses. Studies of the natural rates of horizontal gene transfer, especially of plasmid-encoded toxins and antibiotic resistance factors, in the microbial ecosystems likely to be most exposed to foodborne pathogens. For the studies on epidemiology of infections in natural non-human and human hosts, we will use extensive in-field sampling strategies coupled with epidemiological practices targeted at identifying factors that influence the distribution, acquisition, course and severity of human disease. This will also include studies on pathogens in the environment and non-food-producing vectors, when those studies may contribute to the understanding of factors bearing on emergence, transmission, persistence, development of virulence, host switching, and/or epidemic/epizootiologic thresholds as they directly pertain to human disease. The unique gnotobiotic and specific pathogen free animal facilities (piglets and calves) available at the FAHRP will be used to study the microbial community population structure, metatranscriptomics, metabolomics and impact of probiotic or prebiotic or symbiotic interventions for infectious diseases using these animals as models. This approach has provided researchers with the unparalleled opportunity to discover how diet and bacterial populations in newborn animals modulate and influence the animals' response to infection. For food safety studies, targeting each specific group of individuals (producers) with tailored food safety messages that address their biases and pre-existing beliefs will enhance adoption and assist them in making more informed decisions about their health. This mental model approach to message development and delivery can be applied to a variety of topics and will enhance the effectiveness of other outreach and engagement activities.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:Livestock and vegetable producers, Food Processors, Scientists, Veterinarians, Policy Makers, Consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several graduate and undergraduate students and postdoctoral fellows were trained in the area of food safety, epidemiology, diagnostics, antimicrobial resistance, vaccine development, pre- and post-harvest control strategies, omics approaches, aswell as in basic research relating to mechanisms of disease pathogenesis and immunity. These trainings resulted in the completion of Masters and Graduate students' dissertation and endured them with skills to conduct independent research. Similarly, researchers were trained in extension and education such that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest? The knowledge gained was communicated to different stakeholders and is expected to enhance food safety, reduce risk of zoonoses, aid in the development of vaccines and therapeutic against human infections and preparedness against emerging and re-emerging infectious disease of livestock as well as public health significance. These reseach activities have generated publications that will benefit scientists, farmers, producers, consumers, policy makers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Interspecies transmission of influenza viruses using turkey as a model We will continue to characterize swine and human influenza isolates in turkeys to assess their potential for interspecies transmission and reverse zoonosis. We will also apply virus histochemistry technique as a practical approach to predict the in vivo pathogenicity of influenza viruses. Heterologous and heterosubtypic protection by live and inactivated vaccines againstlosand high pathogenicity avian influenza in chickens We will continue to optimize and test different prime & boost or combined vaccination approach usinginactivatedvaccine, subunit vaccine, and live attenuated influenza vaccines to increase the breadth of the immunity. Development and evaluation of broadly cross-reactive swine influenza virus vaccine To Elucidate cross-protective efficacy of combined NanoFlu and Nano-11-Flu vaccines in pigs. Determine the efficacy of Nano-11-Flu vaccine in influenza maternal antibody positive pigs Development and evaluation of a potent oral Salmonella vaccine for poultry To study the efficacy of oral Salmonella vaccine in homologous and heterologous bacterial challenge trials in poultry. Coronaviruses We will stusy the contribution of individual mutation sites of spike protein and non-structural protein (nsp) 16 and the deletion of ORF3 accessory gene in PEDV attenuation. In 2019, we plan to introduce combined mutations in one PEDV genome to generate efficacious attenuated PEDV vaccine candidates." We will investigate how the two swine enteric coronaviruses with similar tissue tropisms, PEDV and PDCoV, do in vivo interactions. Deltacoronaviruses (PDCoV and ADCoV). We will next evaluate the phylogenetic relationships between the current avian and porcine DCoVs to investigate their potential for transmission between avian and livestock species. We will also inoculate Gn piglets with terrestrial bird/porcine-like ADCoV to see if productive infection can be established. Wewill investigatethe effect of vitamin A (VA) supplementation in VA deficient pregnant sows, compared with VA sufficient sows on:i) upregulation of homing molecules and trafficking of immune cells via the gut-mammary gland (MG)-secretory IgA axis and modulation of gut/MG immune responses in uninfected pregnant sows; ii) enhancing lactogenic immunity after primary porcine epidemic diarrhea virus (PEDV) infection of pregnant sows and piglet passive protection to PEDV challenge (model epidemic virus infection); and iii) maintenance and reactivation of anamnestic rotavirus (RV)-specific IgA memory B cell responses in RV re-exposed pregnant sows and piglet passive protection to RV challenge (model zoonotic/endemic virus infection). Rotavirus -We will conduct metabolomics study to identify effect HRV infection and rotavirus (RV) vaccination on host metabolome under malnourished conditions. Identification of HRV-associated metabolic biomarkers will delineate host pathways and molecular targets, which can be used to develop RV therapies targeting pathways perturbed by HRV. We will obtain fecal transplants form Kenyan healthy and stunted (malnourished) infants to evaluate the combined effects of intestinal dysbiosis and protein-calorie malnutrition on the immune function and HRV vaccine efficacy in neonatal Gn pigs. We will evaluate if the generated Rotavirus C-VLPs can be used to boost the pre-existing RVC-specific immunity in gilts. We will also conduct comparative evaluation of the pathogenesis of the current and historic (Cowden) strains of porcine RVC in Gn pigs and porcine small intestinal enteroids. Campylobacter We will test 9 RASVs expressing Campylobacter antigens to protect chickens against Campylobacter colonization. APEC We will test the efficacy of GI-7 (growth inhibitor) and QSI-7 (quorum sensing inhibitor), in field simulated conditions and identify the mechanisms of action of these GI's. We will also test the efficacy of probiotics and peptides to control APEC in chickens. We plan to further characterizethe organic acids and small peptides produced by the probiotics using chromatography methods (LC-MS and CE-MS). Probiotic encapsulation and drug delivery efficiency studies will also be performed prior to in vivo studies.

Impacts
What was accomplished under these goals? Interspecies transmission of influenza viruses using turkey as a model Our study demonstrated the susceptibility of turkey hens to SIVs and human influenza viruses. Additionally, virus histochemistry can be a useful in vitro tool to investigate the pattern of influenza virus attachment to different tissues and as predictor for the in vivo pathogenicity of influenza virus in turkeys. Our turkey model will serve as a great tool to assess potential for interspecies transmission and reverse zoonosis of influenza virus. Heterologous and heterosubtypic protection by live and inactivated vaccines against low and high pathogenicity avian influenza in chickens Our study demonstrated that pc4-LAIV can provide good protection in young chickens and prime-boost vaccination with pc4-LAIV and IIV induces a robust protective immune response against heterologous, and partial protection against heterosubtypic HPAI challenges. Continuous fine-tuning of pc4-LAIV, by selecting the most potent interferon-inducing viral subpopulations, along with the use of multivalent H5 and H7 formulation of pc4-LAIV may protect chickens against the two most important subtypes of avian influenza viruses. Development and evaluation of broadly cross-reactive swine influenza virus vaccine: This study demonstrated that Nano-11 nanoparticles based flu vaccine candidate induced moderate mucosal IgA and T cell responses compared to chitosan particles in the respiratory tract and slightly reduced the nasal viral shedding in pigs. Development and evaluation of a potent oral Salmonella vaccine for poultry:This study demonstrated that oral Salmonella subunit vaccine delivered through chitosan and polyanhydride particles enhance mucosal antibody responsed and can reduce colonization and shedding of Salmonella. Coronaviruses: Cytopathic effects (CPE) in deltacoronavirus (PDCoV) infectedcellsconsisted of enlarged and rounded cells followed by cell shrinkage and detachment, were identified by the 3rdviral passage in the IPEC-J2 cells.PDCoV antigen was detected in the cells showing CPE.Bydouble immunofluorescent andTUNELstaining, mostPDCoV antigen-positive IPEC-J2 cells failed to show TUNEL-positive signals, indicating that PDCoV-infected IPEC-J2 cells may not undergo apoptosis, but rather necrosis, similar to necrotic cell death of infected enterocytesin vivo. Pregnant second trimester gilts had significantly higher levels of circulating PEDV IgA and IgG Abs and ASCs and PEDV virus neutralizing (VN) Abs post PEDV infection. Coinciding with the significantly higher PEDV Ab responses in second trimester gilts,the survival rate of their PEDV-challenged piglets was 100%,compared with87.2%, 55.9% and 5.7% for first, third and mock litters, respectively.Additionally, piglet survival positively correlated with PEDV IgA Abs and ASCs and VN Abs in milk and PEDV IgA and IgG Abs in piglet serum. At post-inoculation hour (PIH) 16 or 24,when vomiting was first or frequently observed, respectively, PEDV infection resulted in significantly reduced numbers of serotonin-positive enterochromaffin (EC) cells in duodenum, mid-jejunum, ileum, or colon. Avian Deltacoronavirus: We analyzed 1,236 avian cloacal swabs during 2015-2017 and tested them for γ- and δ-CoV using genus-specific reverse transcription-PCR assays. A total of 61 (4.93%) samples were γ-CoV positive with up to 29 positive samples per state. In contrast, there were only 14 samples positive for δ-CoV, corresponding with a prevalence of 1.13%.Interestingly, the prevalence of δ- and γ-CoVs in aquatic birds was 1.34% and 6.3%, respectively, compared to only 0.6% and 0% in terrestrial birds. Porcine rotavirus C. There was a significantly higher prevalence in litters with diarrhea born to gilts compared with those born to multiparous sows. Of 113 piglet fecal samples tested, 76.1% were RVC positive. RVC RNA was detected in higher quantities and significantly more frequently in piglets with diarrhea compared with healthy ones (81 vs. 67%). Semi-purified VLPs were used as antigens in ELISA assay to detect swine RVC Ab in serum and milk from the sows. Using RVC-VLP ELISA we demonstrated that gilts with diarrheic litters had significantly lower RVC Ab titers in milk compared with those of sows with healthy litters. Campylobacter: We identifiedE. coliNissle 1917 (EcN) with antimicrobial activity againstCampylobacterin vitro. We formulated EcN microcapsules using FDA approved alginate and chitosan nanoparticles. The EcN microcapsules displayed; better survivability under simulated harsh gastrointestinal conditions, sustained EcN release, and enhanced antimicrobial activity againstCampylobacter. Salmonella We identified four novel small molecule inhibitors ofSalmonella serotypes. The antimicrobial efficacy of these compounds was not altered in biofilm-protected Salmonella and also reduced Salmonella burden in broiler chicken's ceca when treated for 5 days. Wecompared the genomic composition ofS.Heidelberg isolated from environmental samples of different breeder farms in the Midwest, US. Whole genome data showed differences in specific metabolic pathways between poultry production system related to horizontal gene transfer. We analyzed theefficacy and antimicrobial characterization of different known probiotic bacteria againstSalmonella infectionsin vitro. Two probiotics were identified as significant inhibitors and early characterization studies helped us to derive potential peptides that also significantly reduced the salmonella infection. Avian pathogenicE. coli (APEC): We identified three novel SM growth inhibitors (GI-6, GI-7 and GI-10) with potent anti-APEC efficacy in chickens. These GI's considerably reduced the APEC induced mortality of chickens, APEC load and APEC lesions severity in chickens. These GI's affected the APEC cell membrane and were effective against multiple APEC serotypes including those are antibiotics resistant and on APEC biofilm. We also identified two probiotics and their derived small peptides (P-1, P-2, and P-3) with antimicrobial activity against APEC. We identified 10 quorum sensing inhibitors (QSI) that inhibited the AI-2 activity of APEC O78. Seven were tested in one-week old broiler chickens for their efficacy against APEC O78.The QSI-5 and QSI-10 resulted in 100% and 75%. reduced the pathological lesion (>85%) in different organs and reduced bacterial load up to 5.2-6.1 logs and3.4-4.3 logs, respectively in all internal organs compared to control.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu, J, R.M. Gallo, M. Khan, G.J. Renukaradhya and R.R. Brutkiewicz (2018). Neurofibromin 1 Impairs Natural Killer T-Cell-Dependent Antitumor Immunity against a T Cell Lymphoma. Front. Immunol., 8:1901. PMID: 29354122
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Li, Y, P. Shang, C. Carrillo, P. Naraghi-Arani, C.J. Jain, G.J. Renukaradhya, A.W. Firth, E.J. Snijder and Y. Fang* (2018). Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses. Virology, 517:164-176. PMID: 29325778
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Renu, S, S. Dhakal, E. Kim, J. Goodman, Y.S. Lakshmanappa, M.J. Wannemuehler, B. Narasimhan, P.B. Boyaka, and G.J. Renukaradhya* (2018). Intranasal delivery of influenza antigen by nanoparticles, but not NKT-cell adjuvant differentially induces the expression of B-cell activation factors in mice and swine. Cellular Immunology (In Press), pii: S0008-8749(18)30075-3. doi: 10.1016/j.cellimm.2018.04.005. PMID: 29665972
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dhakal, S, S. Renu, S. Ghimire, Y.S. Lakshmanappa, B. Hogshead, N.F. Ruiz, F. Lu, H. HogenEsch, S. Krakowka, C.W. Lee and G.J. Renukaradhya* (2018). Mucosal immunity and protective efficacy of intranasal inactivated influenza vaccine is improved by chitosan nanoparticle delivery in pigs. Frontiers in Immunology, 9:934. PMID: 29770135
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dhakal, S, X. Cheng, J. Salcido, S. Renu, K. Bondra, Y.S. Lakshmanappa, C. Misch, S. Ghimire, N.F. Ruiz, B. Hogshead, S. Krakowka, K. Carson, J. McDonough, C.W. Lee and G.J. Renukaradhya* (2018). Liposome nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs. International Journal of Nanomedicine, 13:6699-6715. PMID: 30425484
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhugunissov, K, Y. Bulatov, D. Taranov, Z. Yershebulov, Z. Koshemetov, A. Zhunushov, G.J. Renukaradhya, K. Tabynov and Y. Abduraimov (2018). Beta-propiolactone inactivated bivalent bluetongue virus vaccine containing Montanide ISA-71VG adjuvant induces long-term immune response in sheep against serotypes 4 and 16 even after 3 years of controlled vaccine storage. Veterinary Microbiology, 226:23-30. PMID: 30389040.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Guo R, P. Shang P, C.A. Carrillo, Z. Sun, Y.S. Lakshmanappa, X. Yan, G.J. Renukaradhya, J. McGill, C.J. Jaing, M.C. Niederwerder, R.R.R. Rowland and Y. Fang (2018). Double-stranded viral RNA persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus. Virology, 524:78-89. PMID: 30165309
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Renu, S, A.D. Markazi, S. Dhakal, Y.S. Lakshmanappa, S.R. Gourapura, R. Shanmugasundaram, S. Senapati, B. Narasimhan, R.K. Selvaraj and G.J. Renukaradhya* (2018). Surface engineered polyanhydride based oral Salmonella subunit nanovaccine for poultry. International Journal of Nanomedicine (In Press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Renu, S, A.D. Markazi, S. Dhakal, Y.S. Lakshmanappa, R. Shanmugasundaram, R. Selvaraj and G.J. Renukaradhya. Engineering of Targeted Mucoadhesive Chitosan Based Salmonella Nanovaccine for Oral Delivery in Poultry. Immunology 2018, 105th Annual Meeting of the American Association of Immunologists, May 4 - 8, 2018, Austin, Texas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Dhakal, S, R. Sankar, S. Ghimire, Y.S. Lakshmanappa, B. Hogshead, N.F. Ruiz, S. Krakowka, C.W. Lee and G.J. Renukaradhya. Mucosal immunity and protective efficacy of inactivated influenza virus vaccine is improved by chitosan nanoparticle intranasal delivery in pigs. Immunology 2018, 105th Annual Meeting of the American Association of Immunologists, May 4 - 8, 2018, Austin, Texas.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Renu, S, Y. Han, Y.S. Lakshmanappa, S. Dhakal, N.C. Tardi, N. Feliciano-Ruiz, S. Ghimire, R. Selvaraj and G.J. Renukaradhya. Oral drinking water delivered chitosan-Salmonella nanovaccine for poultry. Abstract #127, AAVI mini-symposium and 99th Annual CWRAD meeting, December 2-4, 2018, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Lunney J.K, M. Bailey, J. Manirarora, G.J. Renukaradhya, S. Kenney, J. Labresh, Y. Sang, O. Francis and L. Wooldridge. US-UK Collaborative Swine Immune Toolkit Initiative: Development and characterization of new immune reagents for swine health, vaccine and disease studies. (i) North American PRRS Symposium and Emerging and Foreign Animal Diseases, December 1-2, 2018, Chicago, IL. (ii) 99th Annual CWRAD meeting, December 2-4, 2018, Chicago, IL.
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Ghorbani A, Ngunjiri JM, Xia M, Elaish M, Jang H, KC M, Abundo MC, Jiang X, Lee CW. Heterosubtypic protection by live attenuated and chimeric norovirus P-particle-M2e vaccines in chickens.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jang H, Ngunjiri JM, Elaish M, Lee CW. Efficacy and Synergy of Live-attenuated and Inactivated Influenza Vaccines in Young Chickens. 98th Annual Conference of Research Workers in Animal Diseases meeting. Chicago, IL. December 3-5, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Lee CW, Jang H, Ngunjiri JM, Elaish M, Ghorbani A, KC M, Abundo M. Prime-boost vaccination using live and inactivated virus vaccines for broader protection against avian influenza. 10th International Symposium on Avian Influenza. Brighton, UK. April 15-18, 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kathayat D, Helmy YA, Deblais L, Rajashekara G. 2018. Novel small molecules affecting cell membrane as potential therapeutics for avian pathogenic Escherichia coli. Scientific Reports 8:15329.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Mawad A, Helmy YA, Shalkami AG, Kathayat D, Rajashekara G. 2018. E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro. Appl Microbiol Biotechnol. 2018 Oct 9. doi: 10.1007/s00253-018-9417-3. [Epub ahead of print]. PMID:30302522
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deblais L, Scaria J, Rajashekara G. 2018. Draft genome sequences of Salmonella entericasubsp. enterica serotype Heidelberg from chicken and turkey farm environments. Microbiol Resour Announc 7:e01204-18. https://doi.org/10.1128/MRA.01204-18.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deblais L, Lorentz B, Scaria J, Nagaraja KV, Nisar M, Lauer D, Voss S, Rajashekara G. 2018. Comparative Genomic Studies of Salmonella Heidelberg Isolated From Chicken- and Turkey-Associated Farm Environmental Samples. Front Microbiol 9.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deblais L, Helmy YA, Kathayat D, Huang H, Miller SA, Rajashekara G. 2018. Novel Imidazole and Methoxybenzylamine Growth Inhibitors Affecting Salmonella Cell Envelope Integrity and its Persistence in Chickens. Sci Rep 8:13381.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Helmy, YA., Deblais, L., Kassem II, Kathayat, D., and Rajashekara, G. (2018). Novel small molecule modulators of quorum sensing in avian pathogenic Escherichia coli (APEC). Virulence. DOI: 10.1080/21505594.2018.1528844.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miyazaki A, Kandasamy S, Michael H, Langel SN, Paim FC, Chepngeno J, Alhamo MA, Fischer DD, Huang HC, Srivastava V, Kathayat D, Deblais L, Rajashekara G, Saif LJ, Vlasova AN. 2018. Protein deficiency reduces efficacy of oral attenuated human rotavirus vaccine in a human infant fecal microbiota transplanted gnotobiotic pig model. Vaccine. 2018 Oct 8;36(42):6270-6281.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jang H, Elaish M, Kc M, Abundo MC, Ghorbani A, Ngunjiri JM, Lee CW. Efficacy and synergy of live-attenuated and inactivated influenza vaccines in young chickens. PLoS One. 13(4):e0195285. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hu H, Jung K, Wang Q, Vlasova AN, Saif LJ. 2018. Development of a one-step RT-PCR assay for detection of pancoronaviruses (?-, ?-, ?-, and ?-coronaviruses) using newly designed degenerate primers for porcine and avian fecal samples. Journal of Virological Methods 256:116-122.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jung, K., Miyazaki, A., Saif, L.J. (2018) Immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (Gn) pigs infected with porcine epidemic diarrhea virus (PEDV) and serum cytokine responses of Gn pigs to acute PEDV infection. Research in Veterinary Science. Jun 12;119:99-108.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jung, K., Miyazaki, A., Hu, H., Saif, L.J. (2018) Susceptibility of porcine IPEC-J2 intestinal epithelial cells to infection with porcine deltacoronavirus (PDCoV) and serum cytokine responses of gnotobiotic pigs to acute infection of the IPEC-J2 cell culture-grown PDCoV. Veterinary Microbiology. 21: 49-58.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Oka T, Stoltzfus GT, Zhu C, Jung K, Wang Q, Saif LJ. 2018. Attempts to grow human noroviruses, a sapovirus, and a bovine norovirus in vitro. PLoS ONE 13(2): e0178157. https://doi.org/10.1371/journal.pone.0178157
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Esseili MA, Meulia T, Saif LJ, Wang Q. 2018. Tissue distribution and visualization of internalized human norovirus in leafy greens. Appl Environ Microbiol 84:e00292-18. doi: 10.1128/AEM.00292-18. [Epub ahead of print] (selected as Spotlight)
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yunfang Su, Yixuan Hou, Melanie Prarat, Yan Zhang, and Qiuhong Wang. 2018. New variants of porcine epidemic diarrhea virus with large deletions in the spike protein in United States, 2016-2017. Archives of Virology. 163(9):2485-2489.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Chun-Ming Lin; Shristi Ghimire; Yixuan Hou; Patricia Boley; Stephanie N. Langel; Anastasia N. Vlasova; Linda J. Saif; Qiuhong Wang 2018. Pathogenicity and immunogenicity of attenuated porcine epidemic diarrhea virus PC22A strain in conventional weaned pigs. BMC Veterinary Research
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Yixuan Hou, Tea Meulia, Xiang Gao, Linda J Saif and Qiuhong Wang. 2018. The deletion of both tyrosine-based endocytosis signal and endoplasmic reticulum-retrieval signal in the cytoplasmic tail of spike protein attenuates PEDV in pigs. J Virol.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Qiuhong Wang, Anastasia N. Vlasova, Scott P. Kenney, Linda J. Saif. Emerging and re-emerging coronaviruses in pigs. Current Opinion in Virology.
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: S. N. Langel, F. C. Paim, M. A. Alhamo, A. Buckley, A. Van Geelen, K. M. Lager, A. N. Vlasova, L. J. Saif: Stage of gestation at porcine epidemic diarrhea virus infection of pregnant swine impacts maternal immunity and lactogenic immune protection of neonatal suckling piglets. Submitted to Frontiers in Immunology
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: S. N. Langel, F. C. Paim, K. M. Lager, A. N. Vlasova and L. J. Saif: Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): Historical and current concepts. Virus Res, 226, 93-107 (2016) doi:10.1016/j.virusres. 2016.05.016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 1- Kathayat D, Mawad AMM, Closs G Jr., Helmy YA, Deblais L, Srivastava V, Blakeslee JJ, Rajashekara G. 2018. Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12 possesses strong antimicrobial activity against avian pathogenic Escherichia coli. OARDC annual conference 2018, Wooster, OH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 6- Closs G., Helmy Y.H., Howell A., Kathayat D., and Rajashekara G (2018). Efficacy and antimicrobial characterization of different probiotic bacteria against Salmonella infections in-vitro. The 99th annual conference of research workers in animal Diseases (CRWAD). Dec 1st- 4th. Chicago. IL. USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 2- Kathayat D, Helmy YA, Deblais L, Rajashekara G. 2018. Small molecule adjuvants potentiate the antimicrobial activity of colistin against avian pathogenic Escherichia coli. Abstract submitted to 99th annual conference of research workers in animal diseases (CRWAD), Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 3- Yosra A. Helmy, Dipak Kathayat, Mostafa Ghanem, Loic Deblais, Gary Closs, Vishal Srivastava, Mohamed El-Gazzar, and Gireesh Rajashekara (2018). Novel small molecules with antimicrobial activities against Mycoplasma gallisepticum infections in poultry. The 99th annual conference of research workers in animal Diseases (CRWAD). December 1st- 4th. Chicago. IL. USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 4- Yosra A. Helmy, Dipak Kathayat, Loic Deblais, Gary Closs, Vishal Srivastava and Gireesh Rajashekara (2018). Novel quorum sensing inhibitors to control colibacillosis in chickens. CFAES Annual Research Conference. April 27th, Ohio State University, Wooster, Ohio State, USA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 5- Srivastava V, Huang HC, Deblais L, Miyazaki A, Kandasamy S, Langel SN, Paim FC, Chepngeno J, Kathayat D, Vlasova AN, Saif LJ, Rajashekara G (2018). Lower efficacy of human rotavirus vaccine in a microbiota humanized gnotobiotic pig model: A microbiota prospective. The 99th annual conference of research workers in animal diseases (CRWAD) Dec 1st -4th, Chicago. IL. USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 7- Yunfang Su, Yixuan Hou, Melanie Prarat, Yan Zhang, and Qiuhong Wang. Archives o Virology. New PEDV variants with a large deletion in the spike protein in United States, 2016-2017. (poster). Proc. 37th American Society for Virology Annual meeting, Abstract#P10-2, College Park, Maryland. July 14-18, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 9- Yixuan Hou, Tea Meulia, Xiang Gao, Linda J Saif and Qiuhong Wang. A tyrosine-based sorting motif in PEDV spike protein is an endocytosis signal and contributes to viral virulence. (oral) Presentation #260. The 99th Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL. December 2-4, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 10- Qiuhong Wang, Linda J. Saif, Yixuan Hou, Chun-Ming Lin, Xiang Gao, Xinsheng Liu, Thavamathi Annamalai. Molecular attenuation mechanisms of porcine epidemic diarrhea virus (PEDV) in pigs. (oral) Presentation #261. The 99th Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL. December 2-4, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 11- S. N. Langel, A. N. Vlasova, F. C. Paim, M. A. Alhamo, A. Buckley, K. M. Lager, L. J. Saif: Gestational age impacts mucosal immunity and the gut-mammary gland-secretory IgA axis in porcine epidemic diarrhea virus (PEDV)-infected pregnant swine and lactogenic immune protection of their piglets. American Society of Virology Annual Meeting, College Park, MD (2018)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. S. N. Langel, F. C. Paim, M. A. Alhamo, K. M. Lager, Q. Wang, A.N. Vlasova, L.J. Saif: Defining the gut-mammary gland-secretory IgA (sIgA) axis during porcine epidemic diarrhea virus (PEDV) infection in pigs. XIVth International Nidovirus Symposium, Kansas City, MO (2017)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 8- Yixuan Hou, Yunfang Su, Linda J Saif, and Qiuhong Wang. Porcine Epidemic Diarrhea Virus Lacking Ribose 2O-Methyltransferase Activity is Attenuated in Pigs. (Oral). Proc. 37th American Society for Virology Annual meeting, Abstract#W16-6, College Park, Maryland. July 14-18, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 3. J. Chepngeno, A. Diaz, Chimelo, F.C., S. Takanashi, H. Michael, L.J. Saif, A.N. Vlasova. 2018. Rotavirus C prevalence in healthy and diarrheic piglets: effects of maternal immunity. CRWAD 2018  The 99th Annual Conference of Research Workers in Animal Diseases. Dec 2-4, 2018; Chicago, Illinois


Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Livestock and Vegetable Producers, Food Processors, Scientists, Veterinarians, Policy Makers, Consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several graduate and undergraduate students and postdoctoral fellows were trained in the area of food safety, epidemiology, diagnostics, antimicrobial resistance, vaccine development, pre- and post-harvest control strategies, omics approaches, as well as in basic research relating to mechanisms of disease pathogenesis and immunity. These trainings resulted in the completion of Masters and Graduate students' dissertation and endured them with skills to conduct independent research. Similarly, researchers were trained in extension and education such that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest? The knowledge gained was communicated to different stakeholders and is expected to enhance food safety, reduce risk of zoonoses, aid in the development of vaccines and therapeutic against human infections and preparedness against emerging and re-emerging infectious disease of livestock as well as public health significance. These reseach activities have generated publications that will benefit scientists, farmers, producers, consumers, policy makers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Rotavirus We will generate RVC VLPs and establish RVC antibody ELISA We will use the established ELISA to evaluate RVC Ab prevalence in sows in Ohio We will use the qRT-PCR to evaluate RVC prevalence in the nursing piglets. We will comparatively assess the pathogenesis of porcine RVCs of genotypes G3 and G6 in 1-7 and 21 day old germfree piglets. Influenza We will further optimize the newly upgraded real-time RT-PCR protocol for swine influenza viruses. In addition, we will validate the new protocol with experimental tissue and swab samples. We will further optimize the vaccine components and vaccination regimens for broader protection against antigenically different and emerging strains of influenza virus. We will test the efficacy of the optimized vaccine and vaccination protocol against highly pathogenic avian influenza viruses of different lineage and subtypes. We will continue to characterize influenza isolates of different origin (including swine and human influenza viruses) in turkeys to assess their potential for interspecies transmission and reverse zoonosis. Investigate the mechanisms involved in induction of protective response by nanoparticle based influenza virus vaccine delivered intranasally in parallel in mice and pigs. To study the efficacy of oral Salmonella vaccine in homologous and heterologous bacterial challenge trials in poultry. Salmonella and Campylobacter We will test probiotic bacteria Butiricicoccus, Lactobacillus and Bifidobacterium alone or in combination with small molecule growth inhibitor to control Salmonella and Campylobacter in infected broiler and layer chickens We will identify the drug target of the novel growth inhibitors effective against Salmonella to understand the mechanisms of action of small molecules We will test the effect of nanoparticle entrapped small molecule for the control of Campylobacter and Salmonella in chickens. We will evaluate attenuated Salmonella expressing Campylobacter antigens to reduce Campylobacter and Salmonella in chickens. We will test the effect of nanoparticle encapsulated E. coli Nissle probiotic for the control of Campylobacter in chickens.

Impacts
What was accomplished under these goals? Campylobacter: We showed that pre-treatment of HT-29 human colonic cells with the probiotic EcN significantly affect C. jejuni's invasion and no intracellular C. jejuni were recovered. EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity, cell proliferation and cellular immunity. We showed that C. jejuni exhibited enhanced chemoattraction to and respiration of formate in comparison to other organic acids. Formate also significantly increased C. jejuni's growth, motility, and biofilm formation under microaerobic (5% O2) conditions. However, formate reduced oxidase stress under microaerobic conditions as well as aerotolerance and biofilm formation under ambient oxygen conditions. Salmonella We identified four novel small molecule inhibitors of Salmonella and with restricted effects on other beneficial bacteria at 200 μM. The antimicrobial efficacy of these compounds was not altered in biofilm-protected Salmonella and the compounds enhanced the in vitro efficacy of existing antibiotics used to treat Salmonella in poultry and humans. The compounds also reduced Salmonella burden in broiler chicken's ceca with minimal impact on the cecal microbiota. Whole genome comparison of S. Heidelberg isolates from chicken and turkey farm environment showed differences in specific metabolic pathways between poultry production system related to horizontal gene transfer (type IV secretion system, conjugative transfer, and phage proteins). In collaboration with Department of Animals Sciences, University of Georgia, we developed and evaluated the efficacy of oral delivered subunit Salmonella vaccine through stomach pH conditions resistant biodegradable particles in poultry, and found satisfactory results in terms of induction of mucosal IgA response in the intestines. Rotavirus Because most field strains of porcine rotavirus C (RVC) cannot be propagated in a cell culture, virus like particles is the method of choice to generate antigen to evaluate antibody responses to genetically heterogeneous porcine RVCs. Previously identified field porcine rotavirus C (RVC) strains of G3 and G6 G-genotypes and the historic/reference RVC Cowden strain (G1) were selected to generate virus-like particles. VP4 gene (that determines P-genotype) sequencing demonstrated that in addition to the different G-genotypes, they possess different (P8/P9) P-genotype specificity. We are now cloning VP2, VP6, VP7 and VP4 genes from the 3 different strains to generate virus-like particles that can be used as a VLP cocktail or chimeric VLPs in ELISA to evaluate RVC-specific immune responses in the sows of piglets with or without diarrhea. qRT-PCR was validated in the lab using available fecal samples from feral swine (from USDA-APHIS) and from healthy sows and their piglets and 4 (out of 44) samples and 1 (out of 116 samples) were found to be positive. Fecal sample collection was initiated in cooperation with one of the Ohio swine farms. Caliciviruses: We found that human norovirus (HuNoVs) utilizes the same capsid sites to bind to histo-blood group antigens (HBGA) in humans and HBGA-like carbohydrates in lettuce. HuNoVs can be internalized and distributed to edible leaves of lettuce and spinach via contaminated irrigation water from roots. Influenza We designed a new RRT-PCR primer based on the Swine Influenza Virus (SIV) Matrix gene sequences available in the GenBank database. Compared with NVSL method, our newly designed primer was at least 100 times more sensitive for detection of a specific group of SIVs that belong to North American triple reassortant lineage while maintaining similar or better sensitivity for other SIVs when used in combination with existing primers. We investigated heterosubtypic protection conferred by pc4-LAIV with or without the M2eP combined treatment or boost vaccination. This study reveals protective efficacy of LAIV which was enhanced by M2eP boost vaccination early in the course of challenge against heterosubtypic H5N2 avian influenza virus. Prime-boost vaccination of LAIV and M2eP provided a long-lasting level of broadly reactive antibodies against LAIV that reacted with the whole virus particles from different avian influenza virus subtypes (H5N2 and H7N3). We demonstrated that inactivated influenza A virus and conserved T and B cell peptides entrapped in biodegradable poly lactic-co-glycolic acid (PLGA), polyanhydride, liposome, Nano-11 and chitosan nanoparticles delivered to mucosal site by intranasal delivery in the form of mist to pigs induced broadly cross-protective response against a virulent, zoonotic and heterologous swine influenza virus challenge indicated by reduced clinical flu symptoms and substantially reduced lung pathology in pigs.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Elaish M, Ngunjiri JM, Ghorbani A, Jang H, KC M, Abundo MC, Pantin-Jackwood M, Bowman AS, Torchetti MK, Lee CW. Transmission of influenza viruses to turkeys. 10th Annual CEIRS Meeting. Atlanta, GA, July 16th, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Deblais L, Lorentz B, Scaria J, Nagaraja KV, Nisar M, and Rajashekara G. Comparative genomic study of Salmonella Heidelberg from chicken and turkey poultry production systems.�CWARD meeting (Chicago  USA  December 2017)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lee, C.W and G.J. Renukaradhya. Universal Influenza Vaccine. Represented OARDC, The Ohio State University to the US Capital Hill Washington DC meeting attended by US senators and USDA team, April 5, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Renu, S, A.D. Markazi, S. Dhakal, Y.S. Lakshmanappa, R. Shanmugasundaram, R. Selvaraj and G.J. Renukaradhya. Formulation and characterization of bioadhesive polyanhydride nanoparticles based Salmonella vaccine for oral delivery in poultry. OARDC Research Conference, The Ohio State University, Columbus, Ohio. April 20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Deblais L, Helmy YA, Candelero-Rueda R, Kathayat D, Huang HC, Miller SA, and Rajashekara G. Reduction of Salmonella burden in poultry using new generation small molecules. OBASM (Columbus  USA  April 2017).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Ghorbani A, Ngunjiri JM, Jang H, Elaish M, Lee CW. Protective efficacy of NS-1 truncated live attenuated influenza vaccine combined with or without M2e subunit vaccine against heterologous and heterosubtypic challenges. 97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jang H, Elaish M,, Ngunjiri JM, Lee CW. Application of NS1-truncated variant as live attenuated influenza vaccine for early protection and its complementary use with inactivated vaccine in chickens 97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Elaish M, Ngunjiri JM, Jang H, Lee CW. Evaluation of in vitro biological role of ectodomain of the influenza virus matrix protein 2 (M2e) specific antibodies in chickens. 97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Helmy YA, Kassem II, Deblais L, and Rajashekara G (2017). Control of Avian Pathogenic E. coli (APEC) using Quorum Sensing Inhibitor Small Molecules. The American Association of Avian pathologist (AAAP), July 21-25, Indianapolis, IN, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, K. Bondra, D. Shyu, K. Tabynov, C.W. Lee and G.J. Renukaradhya. Intramuscular route of delivery of PLGA-nanoFlu vaccine improves antibody response in pigs. OARDC Research Conference, The Ohio State University, Wooster, Ohio. April 20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, K. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C.W. Lee and G.J. Renukaradhya. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. Abstract # 263, Immunology 2017, AAI meeting, Washington Convention Center, Washington DC, May 12-16, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manirarora, J, M. Bailey, G.J. Renukaradhya, S. Kenney, J. Labresh, Y. Sang, O. Francis, L. Wooldridge, and J.K. Lunney. US-UK Collaborative Swine Immune Toolkit Initiative: Development of new immune reagents for swine health, vaccine and disease studies. Immunology 2017, AAI meeting, Washington Convention Center, Washington DC, May 12-16, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lakshmanappa, Y.S, P. Shang, S. Dhakal, S. Renu, B. Hogshead, P. Bernardo, X. Yan, Y. Fang and G.J. Renukaradhya. Concurrent but consecutive vaccination of modified live type 1 and type 2 PRRSV provides better protection in nursery�pigs. Abstract #44, North American PRRS Symposium Emerging and Foreign Animal Diseases and National Swine Improvement Federation NAPRRS-NSIF Joint Conference, December 1-3, 2017,�Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhakal, S, R. Sankar, S. Ghimire, Y.S. Lakshmanappa, B. Hogshead, N.F. Ruiz, S. Krakowka, C.W. Lee and G.J. Renukaradhya. Chitosan delivery of inactivated influenza vaccine improves heterologous protection by enhancing antibody and cellular immune responses in pigs. Abstract #103, AAVI mini-symposium and 98th Annual CWRAD meeting, December 3-5, 2017,�Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Renu, S, A.D. Markazi, S. Dhakal, Y.S. Lakshmanappa, R. Shanmugasundaram, R. Selvaraj and G.J. Renukaradhya. Mucoadhesive Chitosan Nanovaccine oral delivery against Salmonella in poultry. Abstract #125, AAVI mini-symposium and 98th Annual CWRAD meeting, December 3-5, 2017,�Chicago, IL.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Kassem II, Helmy YA, Kashoma IP, and Rajashekara G (Nov, 2016).�The emergence of antibiotic resistance on poultry farms.�In: Ricke, S., (ed.), Achieving sustainable production of poultry meat: Safety, quality and sustainability. Volume 1. Burleigh Dodds Science Publishing, UK. ISBN: 978-1-78676-064-7
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lakshmanappa, Y.S, P. Shang, S. Dhakal, S. Renu, B. Hogshead, P. Bernardo, X. Yan, Y. Fang and G.J. Renukaradhya. Concurrent but consecutive vaccination of modified live type 1 and type 2 PRRSV provides better protection in nursery�pigs. Abstract #114, AAVI mini-symposium and 98th Annual CWRAD meeting, December 3-5, 2017,�Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lunney J.K, M. Bailey, J. Manirarora, G.J. Renukaradhya, S. Kenney, J. Labresh, Y. Sang, O. Francis and L. Wooldridge. US-UK Collaborative Swine Immune Toolkit Initiative: Development and characterization of immune reagents for swine health, vaccine and disease studies. 98th Annual CWRAD meeting, December 3-5, 2017,�Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Francis, O, M. Bailey, L. Wooldridge, J.K. Lunney, J. Manirarora, G.J. Renukaradhya, S. Kenney, J. Labresh and Y. Sang. US-UK Collaborative Swine Immune Toolkit Initiative: Development of new immune reagents for swine health, vaccine and disease studies. British Society for Immunology Congress, Brighton, UK, December 4, 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ohba M, Oka T, Ando T, Arahata S, Ikegaya A, Takagi H, Ogo N, Zhu C, Owada K, Kawamori F, Wang Q, Saif LJ, Asai A (2017). Antiviral effect of theaflavins against caliciviruses. J Antibiot (Tokyo). 70(4):443-447. doi: 10.1038/ja.2016.128. Epub 2016 Oct 19. PubMed PMID: 27756911.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, K. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C.W. Lee and G.J. Renukaradhya (2017). Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs. J Control Release, 247:194-205. PMID: 28057521
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Deblais L, Lorentz B, Scaria J, Nagaraja KV, Nisar M, and Rajashekara G (2017). Comparative genomic study of Salmonella Heidelberg from chicken and turkey poultry production systems (Submitted to Frontiers in Microbiology).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Helmy YA., Kassem II, Kumar A and Rajashekara G (2017). In vitro evaluation of the impact of the probiotic E. coli Nissle 1917 on Campylobacter jejunis invasion and intracellular survival in human colonic cells. Frontiers in Food Microbiology. doi.org/10.3389/fmicb.2017.01588.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Elaish M, Ngunjiri JM, Ali A, Xia M, Ibrahim M, Jang H, Hiremath J, Dhakal S, Helmy YA, Jiang X, Renukaradhya GJ, Lee CW. Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens. PLoS One. 2017 Feb 2;12(2):e0171174.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Dhakal, S, J. Goodman, K. Bondra, Y.S. Lakshmanappa, J. Hiremath, D. Shyu, K. Oyuang, K. Kang, S. Krakowka, M.J. Wannemuehler, C.W. Lee, B. Narasimhan and G.J. Renukaradhya (2017). Polyanhydride nanovaccine against swine influenza virus in pigs. Vaccine, 35(8): 1124-1131. PMID: 28117173
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mailybayeva, A, B. Yespembetov, S. Ryskeldinova, N. Zinina, A. Sansyzbay, G.J. Renukaradhya, N. Petrovsky, and K. Tabynov, (2017). Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats. Plos One, 12(10): e0186484. PMID: 29023541
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wide but variable distribution of a hypervirulent�Campylobacter jejuni�clone in beef and dairy cattle in the United States.Tang Y, Meinersmann RJ, Sahin O, Wu Z, Dai L, Carlson J, Plumblee J, Genzlinger L, LeJeune JT, Zhang Q. Appl Environ Microbiol. 2017 Sep 29. pii: AEM.01425-17. doi: 10.1128/AEM.01425-17. [Epub ahead of print]
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Complete Genome Sequences of Two Phage-Like Plasmids Carrying the CTX-M-15 Extended-Spectrum ?-Lactamase Gene. Colavecchio A, Jeukens J, Freschi L, Edmond Rheault JG, Kukavica-Ibrulj I, Levesque RC,�LeJeune J, Goodridge L.Genome Announc. 2017 May 11;5(19). pii: e00102-17. doi: 10.1128/genomeA.00102-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Rising fluoroquinolone resistance in Campylobacter isolated from feedlot cattle in the United States. Tang Y, Sahin O, Pavlovic N,�LeJeune J, Carlson J, Wu Z, Dai L, Zhang Q. Sci Rep. 2017 Mar 29;7(1):494. doi: 10.1038/s41598-017-00584-z.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Food Safety Instruction Improves Knowledge and Behavior Risk and Protection Factors for Foodborne Illnesses in Pregnant Populations.Kendall P, Scharff R, Baker S,�LeJeune J, Sofos J, Medeiros L. Matern Child Health J. 2017 Aug;21(8):1686-1698. doi: 10.1007/s10995-017-2291-2.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Food Indwelling Clostridium difficile in Naturally Contaminated Household Meals: Data for Expanded Risk Mathematical Predictions.Rodriguez-Palacios A, Ilic S, LeJeune JT. Infect Control Hosp Epidemiol. 2017 Apr;38(4):509-510. doi: 10.1017/ice.2016.332. Epub 2017 Feb 7. No abstract available.�
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Amadi VA, Matthew-Belmar V, Subbarao C, Kashoma I,�Rajashekara G, Sharma R, Hariharan H, Stone D (2017). Campylobacter Species Isolated from Pigs in Grenada Exhibited Novel Clones: Genotypes and Antimicrobial Resistance Profiles of Sequence Types. Foodborne Pathog Dis. 2017 Jul;14(7):419-425. doi: 10.1089/fpd.2016.2229.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: On-farm starling populations and other environmental and management factors associated with the presence of cefotaxime and ciprofloxacin resistant E. coli among dairy cattle in Ohio. Medhanie GA, Pearl DL, McEwen SA, Guerin MT, Jardine CM, Schrock J, LeJeune JT. Prev Vet Med. 2016 Nov 1;134:122-127. doi: 10.1016/j.prevetmed.2016.10.006. Epub 2016 Oct 11.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Issmat I. Kassem, R. Candelero-Rueda, K. Esseili, and G. Rajashekara. (2017). Formate simultaneously reduces oxidase activity and enhances respiration in Campylobacter jejuni. Scientific Reports. 7:40117.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kassem II., Helmy YA., Kathayat D, Candelero-Rueda R, Kumar A, Deblais L, Huang H, Sahin O, Zhang Q, and Rajashekara G (2017). Nonculturability Might Underestimate the Occurrence of�Campylobacter�in Broiler Litter. Foodborne Pathogens and Disease. doi.org/10.1089/fpd.2017.2279
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Nisar M, Kassem II,�Rajashekara G, Goyal SM, Lauer D, Voss S, Nagaraja KV (2017). Genotypic relatedness and antimicrobial resistance of Salmonella Heidelberg isolated from chickens and turkeys in the midwestern United States. J Vet Diagn Invest. 2017 May;29(3):370-375. doi: 10.1177/1040638717690784.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Esseili, M.A. Tegtmeier S, Saif LJ, Farkas T, and Wang Q. Differential Tissue Distribution of Internalized Human Norovirus, Porcine Sapovirus and Tulane Virus in Lettuce and Spinach Plants. IAFP 2017, July 9-12 in Tampa, Florida. (Oral T7-08)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Deblais L, Helmy YA, Kathayat D, Vrisman C, Candelero R, Huang HC, Miller SA, and Rajashekara G. A combined genomic and in vivo imaging platform to understanding Salmonella-tomato plant host interactions. NIFRA IAFP meeting (Tampa  USA  July 2017).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Elaish M, Ngunjiri JM, Jang H, Ghorbani A, KC M, Abundo MC, Lee CW. Potential mechanism of protection by M2e-based vaccine in chickens. American Society for Virology - 2017 Annual MeetingAmerican Society for Virology - 2017 Annual MeetingAmerican Society for Virology, 36th Annual Meeting, University of Wisconsin-Madison, June 24-28, 2017. American Society for Virology - 2017 Annual Meeting


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Livestock and Vegetable producers, Food processors, Scientists, Veterinarians, Policy makers, Consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate and undergraduate students and postdoctoral fellows were trained in the area of food safety, epidemiology,diagnostics, vaccine development, pre- and post harvest control strategies, as well as in basic research relating tomechanisms of disease pathogenesis and immunity. These trainings resulted in the completion of Masters and Graduate students dissertation andendured them with skills to conduct independent research. Similarly researchers were trained in extension and educationsuch that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest?The knowledge gained was communicated to different stakeholders and is expected to enhance food safety, reduce risk of zoonoses, aid in the development of vaccines and therapeutic against human infections and preparedness against emerging and re-emerging infectious disease of livestock as well as public health significance. These reseach activities have generated publications that will benefit scientists, farmers, producers, consumers, policy makers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Caliciviruses: -It will be interesting to evaluate the potential interspecies transmission of closely related animal and human SaVs, such as GI,GII and GV SaVs, using experimental animals. -Molecules on lettuce that are involved in the direct binding of GI.1/Norwalk to lettuce need to be investigated further. Next, we will look for nontoxic inhibitors to block binding and prevent HuNoV contamination. Campylobacter: - We are currently analyzing the impact of organic farming on the gut microbiome of chickens and on the abundance of antibiotic resistance markers. - We are optimizing chemically-based interventions to control Campylobacter in litter and broiler focks. - We are currently investigating the efficacy of EcN in controlling Campylobacter colonization of chickens. - We are optimizing the small molecules to test them in chickens and evaluate their ability to target specific pathways that are essential for Campylobacter survival. Influenza: - An in-depth analysis will be done to determine which ISGs need to be upregulated to enhance NS1-truncated live attenuated influenza vaccine efficacy. - Different prime & booster or combined vaccination approach using inactivated vaccine, subunit vaccine, and live attenuated vaccines to increase the breadth of the immunity. - Investigate the mechanisms involved in induction of protective response by nanoparticle based influenza virus vaccine delivered intranasally in parallel in mice and pigs. - Analyze the efficacy of influenza peptides and adjuvant based nanoparticle platform to improve cross-protective immunity in pigs. Rotavirus: We will continue to monitor distribution and genetic make-up of RVs in the pig population.

Impacts
What was accomplished under these goals? Caliciviruses: -We sequenced the full length genomes of three new porcine SaVs belonging to three genogroups (GV, GVII and GVIII) and classified human and animal SaVs into 15 genogroups (G) based on the major structural protein VP1 sequences. - We showed that HuNoV GII.4 internalized through the roots of lettuce and spinach plants and viral RNA was detected in their leaves for at least up to 6 days post inoculation (dpi). The RNA titers for both TV and SaV were significantly higher in the central vein than the top of leaves in both plants, suggesting that TV and SaV maybe more restricted than HuNoVf to the central veins (or are more susceptible to degradation inside the leaves). - We demonstrated that H type HBGA-like carbohydrates are present in the cell walls of lettuce, and the virus-like particles (VLPs) of GII.4/HS194 strain bind to α-L-fucose residues in lettuce. GI.1 VLPs showed much stronger binding signal than GII.4 VLPs in ELISA. Also, our data suggested that GII.4 strains generally have a broader HBGA binding pattern than GI.1/Norwalk strain. Rotavirus: A notable advance is the recent development of RT-PCR assays to monitor the epidemiology of group A,B and C RVs by NC-1041 collaborators (Amimo et al., 2013a; Amimo et al., 2013b). An important finding is the emergence of new genotype of group C RV as a major cause of diarrhea in neonatal piglets (Amimo et al., 2013b (78)). Campylobacter: - We showed that the average number of Campylobacter jejuni-positive in organically grown hens was lower in comparison to conventionally grown hens. Isolates from organically grown hens on two farms exhibited significantly lower resistance to ciprofloxacin, erythromycin, and tylosin. - Investigated the efficacy of on-farm practices to control litter as a source of Campylobacter. We selected 4 broiler farms that used the same approach to treat litter. We sampled litter (n = 384) and ceca (n = 192) from 6 houses on each farm. -We showed that the litter treatment reduced the moisture of the litter, but the treatment did not result in reducing the Campylobacter in the flocks in the high prevalence farms. - The pre-treatment of HT-29 human colonic cells with the probiotic EcN showed a significant effect on C. jejuni's invasion (~2.5 log reductions). Furthermore, no intracellular C. jejuni were recovered from EcN treated HT-29 cells after 24 h of incubation. EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity and cell proliferation. - We used high-throughput chemical screens to identify small compounds for control of Campylobacter. Based on rigorous screening, we selected 12 out of 4182 compounds tested. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 μM concentrations. Influenza: -We determined the ability of antibodies induced after M2e-PP vaccination in chickens to bind the native M2e expressed on whole influenza virus particles or on the surface of virus infected cells and their role in inhibiting AI virus replication. While M2e-PP antibodies were able to bind to the purified virus or virus-infected MDCK cells, no difference in binding ability was observed among the 3 virus subtypes tested. IFA results were in agreement with the ELISA data described above. Inhibition of virus replication was dependent on the concentration of M2e-PP antibodies where rapid loss of plaque formation was observed at lower antibody concentrations. However, inhibition of virus replication plateaued at a plaque-neutralization index of ~40% indicating that direct virus neutralization cannot fully explain the mechanism of protection by M2e-PP antibodies. -We determined whether pc4-LAIV can have further advantages, such as early protection and complementary effect on the inactivated vaccine. In our first study, 1-day-year-old birds were vaccinated with pc4-LAIV or IV, monitored for the development of immune responses, and challenged with a heterologous virus at 6 weeks of age. As expected, IV vaccination not only failed to induce seroconversion before 3 weeks of age but also did not efficiently induce IFN-related gene response. In contrast, vaccination with pc4-LAIV induced high transcription level of IFN-related genes and antibody response as early as 2-weeks of age. In a follow up study, we primed the birds with pc4-LAIV at 1 day of age, boosted with IV three weeks later and compared the efficacy with diverse vaccine regimens. The LAIV priming-IV boosting regimen induced the highest antibody titers and provided the greatest total protection from heterologous virus challenge. -We evaluated inactivated influenza A virus entrapped in biodegradable poly lactic-co-glycolic acid (PLGA) and polyanhydride nanoparticles delivered to mucosal site by intranasal delivery in the form of mist to pigs. Our data demonstrated induction of broadly cross-protective response against a virulent, zoonotic and heterologous swine influenza virus challenge, indicated by absence of clinical flu symptoms and substantially reduced lung pathology in candidate nanovaccine received pigs. Immunologically, in nanovaccine administered pigs increased virus specific T cell response associated with reduced challenged virus load in the airways was observed. Usefulness of findings: Caliciviruses: Our results suggest that several animal SaVs have genetic similarities to human SaVs. The characterization of additional complete genomic sequences of animal SaVs may enable the design of more universal SaV primers to detect SaVs from both animals and humans. -Knowledge of the mechanism of HuNoVs translocation inside leafy greens will allow the utilization of better preventive measures. Knowledge about the plant carbohydrates reactive to HuNoVs will allow the identification of HBGA-like carbohydrates in plants for NoV binding Campylobacter: - Our findings suggested that organic farming can play a role in controlling antimicrobial resistant Campylobacter. However, continuous monitoring and adhering to best farming practices are necessary to express this effect. - We showed that the on-farm treatment of broiler litter using standard farming practices was not sufficient for controlling Campylobacter in high-prevalence flocks. Our findings highlight the need for more rigorous approach to control litter as a source of Campylobacter. - We also showed that the use of the probiotic EcN was beneficial for reducing Campylobacter in vitro. This suggests that EcN can potentially be deployed to control Campylobacter in hosts. - We identified small biomolecule that can inhibit Campylobacter. These molecules can be applied to control Campylobacter in hosts. Rotavirus: -Surveys of the dominant strains of RVs circulating in animals of different ages, their diversity and relatedness to human strains and their shedding by RV vaccinated animals is critical to evaluate vaccine efficacy and aid in the design of new vaccines to prevent RV disease across animal species and humans. Influenza: Our data shows that M2e-PP antibodies are capable of recognizing the native M2e epitopes exposed on the surface of influenza virus-infected MDCK cells and whole virus. It also shows that M2e antibodies have a role in blocking viral replication. Future studies are warranted to demonstrate the in vivo mechanism of protection of M2e-PP induced antibodies in chickens. Our study demonstrated that pc4-LAIV can provide protection in young chickens and the live-priming and IV-boosting can enhance antibody response and protection efficacy.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kassem I. I., Y. A. Helmy, I. P. Kashoma, G. Rajashekara. 2016. The emergence of antibiotic resistance on poultry farms. In: Ricke, S., (ed.), Achieving sustainable production of poultry meat: Safety, quality and sustainability. Volume 1. Burleigh Dodds Science Publishing, UK. ISBN: 978-1-78676-064-7.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kassem I. I., O. Kehinde, A. Kumar, and G.Rajashekara. 2016 Antimicrobial-resistant Campylobacter in organically and conventionally raised layer chickens. Foodborne Pathogens and Disease. PMID: 27768387 DOI: 10.1089/fpd.2016.2161
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Kassem I. I., and G. Rajashekara. 2016. Of a Tenacious and Versatile Relic: The Role of Inorganic Polyphosphate (poly-P) Metabolism in the Survival, Adaptation, and Virulence of Campylobacter jejuni. In: de Bruijn, F.J., (ed.), Stress and Environmental Control of Gene Expression in Bacteria. Volume 2. Wiley-Blackwell, USA. pp 1354-1360. ISBN: 978-1-119-00488-2.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Kassem I. I., O. Kehinde, Y. A. Helmy, R. Pina-Mimbela, A. Kumar, K. Chandrashekhar, G. Rajashekara. 2016. Campylobacter in poultry: the conundrums of highly adaptable and ubiquitous foodborne pathogens. In: J. Mei Soon, L. J. Manning, C. A. Wallace (eds.), Foodborne Diseases: Case Studies of Outbreaks in the Agri-Food Industries. CRC Press, Taylor & Francis Group, USA. pp 79112. ISBN: 978-1-4822-0827-6.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kumar A, Gangaiah D, Torrelles JB, Rajashekara G. Polyphosphate and associated enzymes as global regulators of stress response and virulence in Campylobacter jejuni. World J Gastroenterol. 2016 Sep 7;22(33):7402-14.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kumar A, Drozd M, Pina-Mimbela R, Xu X, Helmy YA, Antwi J, Fuchs JR, Nislow C, Templeton J, Blackall PJ, Rajashekara G. Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library. Front Microbiol. 2016 Apr 6;7:405
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Tomoichiro Oka, Zhongyan Lu, Tung Phan, Eric L. Delwart, Linda J. Saif and Qiuhong Wang. Genetic characterization and classification of human and animal sapoviruses. PLoS ONE 11(5): e0156373. doi: 10.1371/journal.pone.0156373(Corresponding authors)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xiang Gao, Malak A. Esseili, Zhongyan Lu, Linda J. Saif, Qiuhong Wang. 2016. Recognizing HBGA-like carbohydrates in lettuce by human GII.4 norovirus. Appl Environ Microbiol. Appl Environ Microbiol. 82(10):2966-74. doi: 10.1128/AEM.04096-15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Qiuhong Wang. Recognition of HBGA-like carbohydrates in lettuce by human GII.4 norovirus. 2016 NoroCORE annual meeting. Crystal city, VA, USA. April 13-15, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Qiuhong Wang, Xiang Gao, Malak Esseili, Linda Saif, Lisa Lindesmith, Ralph Baric, Baijun Kou, Mary Estes, Robert Atmar. Comparison of the binding of GII.4 and GI.1human norovirus strains to lettuce. Oral presentation. The 6th International Calicivirus Conference in Savannah, GA, USA from October 10  October 13.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 1. Jang H, Ngunjiri JM, Lee CW. Association between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens. PLoS One. 11(6):e0156603. 2016
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: 2. Elaish M, Ali A, Xia M, Ibrahim M, Ngunjiri JM, Jang H, Hiremath J, Dhakal S, Helmy YA, Jiang X, Renukaradhya GJ, Lee. Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 1. Jang H, Ngunjiri J, Lee CW. Correlation between Interferon Response and Protective Efficacy of NS1-Truncated Mutants as Influenza Vaccine Candidates in Chickens, 2016 AAAP/AVMA Meeting, San Antonio, TX. August 6-9, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Xiang Gao, Malak A. Esseili, Zhongyan Lu, Linda J. Saif, Qiuhong Wang. Recognizing HBGA-like carbohydrates in lettuce by human GII.4 norovirus. Noro2016. L�beck, Germany. March 17-19, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 2. Elaish M, Ngunjiri JM, Jang H, Lee CW. Evaluation of in vitro biological role of ectodomain of the influenza virus matrix protein 2 (M2e) specific antibodies in chickens97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 3. Jang H, Elaish M,, Ngunjiri JM, Lee CW. Application of NS1-truncated variant as live attenuated influenza vaccine for early protection and its complementary use with inactivated vaccine in chickens 97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 4. Ghorbani A, Ngunjiri JM, Jang H, Elaish M, Lee CW. Protective efficacy of NS-1 truncated live attenuated influenza vaccine combined with or without M2e subunit vaccine against heterologous and heterosubtypic challenges. 97th CRWAD Meeting. Chicago: Dec. 46. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Renukaradhya, G.J. Santosh Dhakal, and Shauna Brummet. Swine Influenza Vaccine, I-Corps@Ohio 2015 Showcase, Boat House at Confluence Park, Columbus, October 23, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal, S., J. Hiremath, J. Goodman, Y.S. Lakshmanappa, D-L. Shyu, K. Oyuang, B. Narasimhan, and G.J. Renukaradhya. Nanoparticle based vaccination strategy against swine influenza virus. Abstract #92, 96th Annual CWRAD meeting, December 6-8, 2015, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bondra, K. Dhakal, S. Krakowka, K. Kang, C.W. Lee and Renukaradhya, G.J. Nanoparticle delivered inactivated swine influenza virus vaccine reduces the lung pathology in heterologous virus challenged pigs. OARDC Research Conference, The Ohio State University, Wooster, Ohio. April 21, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, B. Binjawadagi, K. Kang, J. Goodman, S. Krakowka, B. Narasimhan, C.W. Lee and Renukaradhya, G.J. Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous protection through cell-mediated immunity in pigs. OARDC Research Conference, The Ohio State University, Wooster, Ohio. April 21, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal, S, J. Hiremath, K. Bondra, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, K. Kang, B. Binjawadagi, J. Goodman, K. Tabynov, S. Krakowka, B. Narasimhan, C.W. Lee and Renukaradhya, G.J. Poly-lactic-co-glycolic acid nanovaccine against swine influenza virus in a pig model." Nanovaccine Research Initiative, Iowa State University, Ames, Iowa. May 22  24, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhakal, S, J. Goodman, J. Hiremath, Y.S. Lakshmanappa, D. Shyu, K. Oyuang, C.W. Lee, B. Narasimhan and Renukaradhya, G.J. " Polyanhydride nanovaccine against swine influenza virus in a pig model." Nanovaccine Research Initiative, Iowa State University, Ames, Iowa. May 22  24, 2016.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Livestock and Vegetable producers, Food processors, Scientists, Veterinarians, Policy makers, Consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Secveral graduate and undergraduate students and postdoctoral fellows were trained in the area of food safety, epidemiology, diagnostics, vaccine development, pre- and post harvest control strategies, as well as in basic research relating to mechanisms of disease pathogenesis and immunity. These trainings resulted in the completion of Masters and Graduate students dissertation and endured them with skills to conduct independent research. Similarly researchers were trained in extension and education such that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest?The knowledge gained was communicated to different stakeholders and is expected to enhance food safety, reduce risk of zoonoses, aid in the development of vaccines and therapeutic against human infections and preparedness against emerging and re-emerging infectious disease of livestock as well as public health significance. These reseach activities have generated publications that will benefit scientists, farmers, producers, consumers, policy makers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals?Campylobacer studies: We will expand our studies on Tlps to understand the molecular mechanisms on how these proteins contribute to C. jejuni pathobiology We will study the role of glycosylated proteins, that are affected by ppks mutation, in C. jejuni pathobiology and explore these targets in potential anti-Campylobacter strategies We will test the novel small molecules in for their effect on C. jejuni colonization of chickens and enhance the efficiency of the compounds by downstream modification, target identification and biologically active functional groups. We will continue our molecular epidemiology studies to understand the extent of antibiotic resistance Campylobacter in food animal and develop alternative approaches to mitigate emergence of antibiotic resistance in foodborne pathogens. We will test how infections with tomato pathogens (Clavibacter and Xanthomonas) affect Salmonella survival in tomato plants. In addition, we will study the plant colonization after grafting with infected tools. We further analyze novel anti-Salmonella compounds for their cytotoxicity in tomato seeds and fruits, as well as in human intestinal and macrophage cell lines to facilitate further in vivo testing. Norovirus studies: We will continue to study the exact binding sites on leafy greens for HuNoVs. We will test our findings in cell culture adaptation using other porcine enteric calicivirus first, then in human enteric caliciviruses. Our final goal is to establish a routine cell culture system for HuNoVs. Swine Influenza studies: Investigate the mechanisms involved in induction of protective response by nanoparticle based influenza peptide and killed virus vaccines delivered intrasnally in pigs. Analyze the efficacy of additional influenza peptides nanoparticle platform to improve the cross-protective immunity in pigs. Avian Influenza studies: An in-depth analysis will be done to determine which ISGs need to be upregulated to enhance NS1-truncated live attenuated influenza vaccine efficacy. Assess the prime & booster or combined vaccination approach using recombinant and live attenuated influenza vaccine.

Impacts
What was accomplished under these goals? Campylobacter and Salmonella studies: We showed that Transducer Like Proteins (Tlp) play important role in C. jejuni's adaptation and pathobiology. Our comparative analysis between C. jejuni WT, polyphosphate kinase mutants (?ppk1 and ?ppk2) showed qualitative and quantitative differences in total outer membrane constituents and these differences are related with C. jejuni's capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Our data indicated that the formate dehydrogenase (Fdh) activity and localization in C. jejuni 81-176 were dependent on a functional Tat system. Thus highlights the potential of the Tat system and Fdh as appealing targets for novel anti-C. jejuni therapeutics. Our study highlighted the extent of antimicrobial resistance in Campylobacter occurring in important foods in Tanzania. The potential risks to consumers emphasize the need for adequate control approaches, including the prudent use of antimicrobials to minimize the spread of antimicrobial-resistant Campylobacter. We have identified 12 potential novel small molecule compounds that are effective against C. jejuni and future work on increasing the efficiency of the compounds by downstream modification, target identification and biologically active functional groups must be explored. Our study demonstrated the inability of ST to colonize tomato at high relative humidity and low temperature. Despite the broad range of resistance of ST to antimicrobials, we have identified promising novel anti- ST candidates for future development of anti-Salmonella strategies. Human nrovirus studies. Human norovirus (HuNoV) RNA have been detected from pigs and dogs, raising the concerns of zoonotic potential of HuNoVs. Our lab tried to understand the contamination and survival mechanisms of HuNoVs and their calicivirus surrogates in fresh produce, lettuce and spinach. Because there is no robust cell culture systems for HuNoVs, researches on pathogenesis, virus inactivation, and the development of antivirals and vaccines have been hampered. Therefore, we also tried to study the cell culture adaptation mechanisms of enteric caliciviruses using porcine sapovirus Cowden strain as a model. We hope to provide important information potentially applicable to the establishment of cell culture systems for human enteric caliciviruses. Swine Influenza studies: Development of broadly reactive H1N1 influenza vaccine against swine influenza viruses: Development and evaluation of conserved influenza peptides encased in biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticle delivered to mucosal site by intranasal delivery in the form of mist to pigs. Our data showed induction of broadly protective response against a zoonotic heterologous swine influenza virus challenge in our candidate vaccine received pigs. As immune correlates, in vaccinated pigs increased epitope specific T cell response in the lung lymphocytes was associated with reduced challenged virus load in the airways. To further improve the breadth of immunity against influenza in pigs, killed swine H1N2 virus was encased in PLGA nanoparticles and delivered intranasally and found enhanced antigen specific lymphocyte response in a pre-challenged study. Avian influenza studies: NS1-truncated mutants and IFN to control influenza in chickens. Mutants of influenza virus that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) differ in terms of their immunogenicity and protective efficacy in chickens. The in vivo efficacy was shown to correlate with the level of type I IFN induction in chicken embryonic fibro blast cells. In this study, we sought to establish the relationship between the induction of IFN and IFN-stimulated gene (ISG) responses in vivo and the immunogenicity and protective efficacy of NS1-truncated live attenuated influenza vaccine (LAIV). We have demonstrated that the level of antibody induction and protective efficacy of NS1-truncated LAIV in chickens correlates well with upregulation of ISG expression. An in-depth analysis such as system biology is required to determine which ISGs need to be upregulated to enhance NS1-truncated LAIV efficacy.We clearly demonstrated that accelerated antibody induction and protective efficacy of NS1-truncated LAIV correlates well with upregulation of IFN-stimulated genes. Further, through oral administration of recombinant chicken IFN alpha in drinking water, we provide direct evidence that type I IFN is required for rapid induction of adaptive immune responses and protective efficacy of influenza vaccine in chickens. Our results provide critical information to develop broadly reactive influenza vaccine for poultry.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Jang H, Ngunjiri J, Lee CW. The boosting effect of IFN in immune response to influenza vaccination in chickens. 66th North Central Avian Disease Conference. St. Paul, MN. March 16-17, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ngunjiri JM, Jang H, Elaish M, Lee CW. Potential of endogenously produced and exogenously administered interferon as adjuvant for influenza vaccine. 9th International Symposium on Avian Influenza. Athens GA. April 12-15, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW, Elaish M, Kang KI, Ngunjiri JM, Jang H, Ali A, Xia M, Jiang X. Efficacy of M2e-based vaccine in chickens in comparison to murine model 9th International Symposium on Avian Influenza. Athens GA. April 12-15, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW, Ngunjiri JM, Jang H, Kang KI, Mohamed E. Potential Role of Interferon as Adjuvant for Influenza Vaccine. 152nd AVMA Meeting. Boston MA. July 10-14, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Esseili MA, Gao X, Tegtmeier S, Wang Q. Human norovirus binding to lettuce cell wall components and its internalization through the vascular system. Presented at the USDA NIFA Food Safety Project Directors Meeting, Portland, OR.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Esseili MA, Gao X, Tegtmeier S, Saif LJ, Wang Q. The effect of mechanical damage, heat and flooding on Human Norovirus, Tulane Virus and Murine Norovirus survival on pre-harvest lettuce plants. Presented at the IAFP conference, Portland, OR.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania.Microb Drug Resist. 2015 Jul 8. [Epub ahead of print]. PMID: 26153978
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chandrashekhar K, Gangaiah D, Pina-Mimbela R, Kassem II, Jeon BH, Rajashekara G. Transducer like proteins of Campylobacter jejuni 81-176: role in chemotaxis and colonization of the chicken gastrointestinal tract. Front Cell Infect Microbiol. 2015 May 27;5:46. doi: 10.3389/fcimb.2015.00046. eCollection 2015
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mollenkopf DF, Cenera JK, Bryant EM, King CA, Kashoma I, Kumar A, Funk JA, Rajashekara G, Wittum TE. Organic or antibiotic-free labeling does not impact the recovery of enteric pathogens and antimicrobial-resistant Escherichia coli from fresh retail chicken. Foodborne Pathog Dis. 2014 Dec;11(12):920-9. doi: 10.1089/fpd.2014.1808.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 4. Esseili MA, Saif LJ, Farkas T, Wang Q Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce. Appl Environ Microbiol. 2015 Aug;81(15):5085-92. doi: 10.1128/AEM.00558-15. Epub 2015 May 22. PMID: 26002891
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Elaish M, Ali A, Xia M, Kang Kl, Ibrahim M, Jiang X, Lee CW. Immunogenecity and protective efficacy of M2e-expressing norovirus P particle vaccine in chickens. Vaccine. 33:4901-9. 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ibrahim M, Sultan HA, Alaa GR, Kang KI, Arafa AS, Saif YM, Lee CW. Development of Broadly Reactive H5N1 Vaccine against Different Egyptian H5N1 Viruses. Vaccine. 33(23):2670-7. 2015.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kassem II, Rajashekara G. dehydrogenase localization and activity are dependent on an intact twin arginine translocation system (Tat) in Campylobacter jejuni 81-176. Foodborne Pathog Dis. 2014 Dec;11(12):917-9. doi: 10.1089/fpd.2014.1797. PMID: 25268895
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sahin O, Kassem II, Shen Z, Lin J, Rajashekara G. Zhang Q. 2015. Campylobacter in poultry: ecology and potential interventions. Avian Diseases. 59: 185-200.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 3. Esseili MA, Chin A, Saif L, Miller SA, Qu F, Lewis Ivey ML, Wang Q.Postharvest Survival of Porcine Sapovirus, a Human Norovirus Surrogate, on Phytopathogen-Infected Leafy Greens. J Food Prot. 2015 Aug;78(8):1472-80. doi: 10.4315/0362-028X.JFP-14-518. PMID: 26219360
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 3. John M. Ngunjiri, Prosper Boyaka, Ahmed Ali, Philip I. Marcus, Chang-Won Lee. "A novel SLSYSINWRH motif of the NS1 protein enhances the interferon-inducing particle efficiency and the effectiveness of candidate live-attenuated influenza vaccine. Plos ONE. 10(3):e0118934. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lo�c Deblais, Fulya Gurel1, Sally Miller, and Gireesh Rajashekara. Understanding Salmonella-tomato plant host interactions and development of novel effective control strategies to reduce Salmonella burden in tomato production. IAFP Annual Meeting, July 25th to 28th, Portland, OR 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Issmat I. Kassem, O. O. Kehinde, A. Kumar, K. Chandrashekhar, R. Pina-Mimbela, H. Huang, G. Rajashekara. Litter Chemical Amendments that Reduce pH and Moisture Contribute to the Control of Campylobacter jejuni in Broilers. The 115th General Meeting of the American Society for Microbiology, New Orleans, LA. May 30-June 2, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hiremath, J, M. Elaish, K. Kyung-il, B. Binjawadagi, K. Ouyang, S. Dhakal, C.W. Lee, and G.J. Renukaradhya. PLGA-Nanoparticle entrapped swine influenza virus peptides vaccine induces epitope specific cell-mediated immune response in pigs. Abstract #103, 95th Annual CWRAD meeting, December 7-9, 2014, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Thomas M, Z. Wang, C.C. Sreenivasan, B.M. Hause, G.J. Renukaradhya, F. Li, D.H. Francis, R.S. Kaushik and M. Khatri. Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs. Abstract #177, 95th Annual CWRAD meeting, December 7-9, 2014, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lee, C-W, K. Kang, M. Elaish, J.M. Ngunjiri, H. Jang, A. Ali, J. Hiremath, S. Dhakal, M. Xia, X. Jiang, and G.J. Renukaradhya. Efficacy of M2e-based vaccine in Murine, Avian and Swine Models. Abstract #175, 95th Annual CWRAD meeting, December 7-9, 2014, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2015 Citation: Dhakal, S., J. Hiremath, J. Goodman, Y. S. Lakshmanappa, B. Shyu, K. Oyuang, B. Narasimhan, G.J. Renukaradhya (2015). Nanoparticle based vaccine strategy against swine influenza virus. Nanovaccine Research Initiative, Iowa State University, Ames, Iowa. May 26  28, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Renukaradhya, G.J, Hiremath, J, M. Elaish, K. Kyung-il, B. Binjawadagi, K. Ouyang, S. Dhakal, and C.W. Lee. Nanoparticle entrapped swine influenza virus peptides vaccine induces epitope specific T cell response in pigs. European Veterinary Immunology Workshop, Vienna, Austria, September 2  4, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Lee CW. Approaches Toward the Developemnt of Universal Influenza Vaccines. 2015 World Vaccine Congress, Washing DC. March 2015.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Livestock and vegetable producers, Food processors, Scientists, Veterinarians, policy makers, consumers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Secveral graduate and undergraduate students and postdoctoral fellows were trained in the area of epidemiology, diagnostics, vaccine development, pre- and post harvest control strategies, as well as in basic research relating to mechanisms of disease pathogenesis and immunity. These trainings resulted in the completion of Masters and Graduate students dissertation and endured them with skills to conduct independent research. Similarly researchers were trained in extension and education such that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest? The knowledge gained was communicated to different stakeholders and is expected to enhance food safety, reduce risk of zoonoses, aid in the development of vaccines and therapeutic against human infections and preparedness against emerging and re-emerging infectious disease of livestock as well as public health significance. These reseach activities have generated publications that will benefit scientists, farmers, producers, consumers, policy makers and other stakeholders. What do you plan to do during the next reporting period to accomplish the goals? Food and Environmental Safety: New or Emerging enteric viruses: 1. We will also use a third surrogate virus, Tulane virus whose binding properties mimic HuNoV binding to histo-blood group antigens (HBGA). We will assess the infectivity of the viruses in different sections of the leaves. Will perform similar experiments to assess viral internalization through the roots of the plants. 2. We will test the effect various enzymatic digestions on the CWP isolated through the salt extractants as well as use specific pectinases to asses whether pectins are involved in the binding. Affinity chromatography will be used to capture the specific binding oligosaccharide. Campylobacter molecular pathogenesis and epidemiology 1. Complete screening, identification, and in vivo testing of small bio-molecule inhibitors against Campylobacter in chickens. 2. Characterize the role of formate dehydrogenase and formate metabolism in the ability of Campylobacter to colonize different hosts. 3- Investigate the role of specific methyl-accepting proteins in Campylobacter pathogenesis. 4. Complete studies on Campylobacter’s prevalence and antibiotic resistance in Layer chickens. 5. Apply and analyze a customized litter treatment approach for controlling Campylobacter on broiler farms. 6. Understand mechanisms of polyphosphate regulation of stress responses and virulence Development of broadly reactive H5N1 vaccine against different Egyptian H5N1 viruses In vivo vaccine efficacy study using newly developed Egyptian H5N1 vaccine will be performed in Egypt. Prime & booster or combined vaccination approach using M2e-based vaccine with inactivated vaccine will be further optimized. Efficacy of recombinant M2e vaccine will be further enhanced by incorporation of immuno-stimulatory epitopes or using different adjuvants. Mechanisms involved in protection afforded by recombinant M2e vaccine will be investigated. We will test conserved protein other than M2e (eg. HA2) in norovirus P particle platform in terms of immunogenicity and protective immunity.

Impacts
What was accomplished under these goals? Understand the mechanisms of microbial pathogenesis of zoonotic pathogens of food safety and public health concerns. Elucidation of the mechanisms of norovirus (NoV) and infectious sapovirus (SaV) entry through the roots, their interaction with components of the vascular system, and their internal transport inside lettuce plants: We showed that both murine norovius (MNV) and SaV internalized inside lettuce and spinach leaves. Immunohistochemical staining showed that both SaV and MNV viral particles could be detected inside both spinach and lettuce. The viral particles were aggregated around the vascular bundles and were present in spaces between the cells. Therefore, SaV and MNV internalization through the petiole of spinach and lettuce may occur through a non-specific mechanism. This work further highlighted the need to better understand the contamination of leafy greens with these viruses. Identification of the lettuce cell wall carbohydrate(s) involved in NoV binding and plant-derived lectin inhibitors for binding. Our results indicated that human norovirus viral like particles (HuNoV VLPs) bind the digested cell wall but not the cytosolic contents, confirming our previously published data that HuNoV VLPs bind specifically to lettuce cell wall materials. We showed that a significantly higher HuNoV VLP binding to leaf vein than to leaf lamina. Further analysis suggested that either pectins or the ionically bound cell wall proteins (CWP) mediate the binding, but not hemicelluloses. Our preliminary results suggest that removal of cellulose enhanced NoV VLP binding to this fraction. Taken together, our results suggested that HuNoV VLP does not bind to hemicelluloses or celluloses of lettuce cell wall material (CWM), rather the binding is mediated through either the pectins or the CWP or both. These findings will enhance targeting of specific lettuce cell wall components to reduce norovirus contamination. Campylobacter jejuni survival mechanisms: Respiratory proteins: We recently showed that the respiratory protein methylmenaquinol:fumarate reductase (Mfr) contributes to the oxidative stress response by affecting hydrogen peroxide susceptibility and accumulation in C. jejuni (Kassem et al., 2014). We also showed that formate dehydrogenase activity and localization are dependent on a functional twin arginine translocation system in the highly invasive strain, C. jejuni 81-176 (Kassem and Rajashekara, 2014). These findings highlighted novel roles for the respiratory proteins in C. jejuni’s survival and pathogenesis. Inorganic polyphosphate (poly P) metabolism: Recently, we showed that exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) modulated environmental stress responses and virulence in C. jejuni (Malde et al., 2014). In another study, we showed that poly P mediated the modulation of biofilm growth and stability in C. jejuni (Drozd et al., 2014). Since poly P and associated enzymes have not been detected in eukaryotic cells, our findings further highlighted the importance of these molecules as potential targets for anti-C. jejuni therapeutics Understand immunity to these pathogens using animal models Development of broadly reactive H5N1 vaccine against different Egyptian H5N1 viruses: In this study, a reverse genetically created variant H5N1 virus with five amino acid mutations in the HA gene showed enhanced cross reactivity. This virus showed up to 16 fold increase in reactivity to the classic-lineage H5N1 viruses measured by hemagglutination inhibition (HI) assay while maintaining similar level of reactivity with the variant-lineage viruses compared to wild type virus. In addition, a single amino acid substitution (N165H), of potential glycosylation site at the HA globular head broaden the reactivity to antisera generated against H5N1 viruses from different clusters. The development of influenza vaccine that elicits broad immune responses to different strains and/or subtypes is needed to reduce the costs and labor for reformulation of strain specific inactivated vaccines. Efficacy of Combined Vaccination with Recombinant M2e and inactivated Influenza Vaccine in Chickens: In this study, we tested immune response and protection of a recombinant M2e protein expressed in the surface of norovirus P particle (M2e-PP) in combination with inactivated vaccine in chickens. Chickens in the combined vaccination groups have shown decreased viral shedding titers from tracheal swabs at 1, 3 and 5 days post challenge (DPC) when compared to those birds vaccinated with inactivated vaccine alone or M2e-PP vaccine alone. In addition to additive effect conferred by M2e vaccination, we observed that addition of M2e in inactivated vaccine or prime vaccination of M2e before inactivated vaccine induced significantly higher hemagglutination inhibition antibody titer against inactivated vaccine strain. Our results provide us the baseline data to further optimize the platform to develop universal influenza vaccine for poultry. Understand the epidemiology and ecology of these foodborne zoonotic pathogens in the food producing animals, fresh produce and their production environments. Control of broiler litter contamination with C. jejuni: We tested the impact of different chemical treatments [aluminum sulfate (Alu), sodium bisulfate (Sob), and magnesium sulfate (Mgs)] to reduce C. jejuni contamination of litter and cognate chicken colonization. Using uninoculated chickens, the Alu+Sob+Mgs treatment significantly reduced the number of colonized birds and C. jejuni was not detected in the litter (Kassem et al., 2014; In preparation). Our findings demonstrate a strategy to control C. jejuni in litter, a valuable on-farm resource. Prevalence and antibiotic resistance properties of Campylobacter on Turkey farms: We conducted a recent study to evaluate the prevalence and antibiotic resistance properties of Campylobacter spp on Turkey farms (Kashoma et al., 2014). This study highlighted the high prevalence, genotypic diversity, and antimicrobial resistance of Campylobacter spp in commercial turkey from farm to slaughter. Prevalence and antibiotic resistance properties of Campylobacter on Layer farms: We have recently finalized a study that compared the antibiotic resistance properties of Campylobacter isolated from conventional and organic layer farms (Kehinde et al., 2014; In preparation). Our findings highlight the role of layer farms as potential reservoirs for antibiotic resistance markers, which can pose a risk as potential environmental pollution. Our results also show that the type of farming can affect the emergence of antibiotic resistance in these settings. Understand the routes of interspecies transmission of zoonotic pathogens Fecal contamination of recreational waters: We addressed this research gap by investigating the association between swimming-related illness frequency and water quality determined from multiple bacterial and viral genetic markers. This study provides evidence that a combined measure of recreational water quality that simultaneously considers both bacterial and viral densities, particularly HAdV, may improve prediction of disease risk than a measure of a single agent in a beach environment likely influenced by nonpoint source human fecal contamination. Develop and evaluate vaccines, therapeutics, adjuvants, and diagnostics for foodborne zoonotic pathogens. Novel small molecule inhibitors of Salmonella: In this study, we focused on identifying novel effective control management. A library of 5182 small molecules was tested on S. typhimurium. Among this collection, a total of 21 small molecules having cidal/static effect with a minimal inhibitory concentration between 400 μM to 5 μM were identified. This cluster was screened on 8 other strains of the same genus, and 7 candidates were effective against multiple species.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Different Approaches Toward Universal Influenza Vaccines. Asia Pacific Poultry Conference Proceedings. p.61-63. 2014.adv
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Elaish M, Kang KI, Ibrahim M, Ali A, Awe O, Lee CW. Immunogenicity and protective efficacy of combined vaccination with recombinant M2e and inactivated influenza vaccines in chickens and pigs. Annual Meeting. American Society of Virology. 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ibrahim M, Sultan HA, Arafa AS, Kang KI, Razik AZA, Elaish M, Lee CW. Development of broadly reactive H5n1 vaccine against antigenically diverse Egyptian H5N1 viruses by targeted mutation in the hemagglutinin protein. Annual Meeting. American Society of Virology. 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Elaish M, Kang K, Ali A, Awe O, Ibrahim M, Lee CW. "Immunogenicity and protective efficacy of combined vaccination with norovirus P particle-M2e chimera and inactivated influenza vaccine in chickens". Annual Meeting. St. Paul: 65th North Central Avian Disease Conference. 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Esseili, M.A. Saif L.J, Farkas, T and Wang Q. Survival of Norovirus Surrogates on Lettuce: A Comparison among Feline Calicivirus, Murine Norovirus, Porcine Sapovirus and Tulane Virus. IAFP.P1-88. Indianapolis, IN. Aug. 3-6, 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kassem II, Rajashekara G. Formate Dehydrogenase Localization and Activity Are Dependent on an Intact Twin Arginine Translocation System (Tat) in Campylobacter jejuni 81-176. Foodborne Pathog Dis. 2014 Sep 30. [Epub ahead of print] PubMed PMID: 25268895.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kashoma IP, Kumar A, Sanad YM, Gebreyes W, Kazwala RR, Garabed R, Rajashekara G. Phenotypic and Genotypic Diversity of Thermophilic Campylobacter spp. in Commercial Turkey Flocks: A Longitudinal Study. Foodborne Pathog Dis. 2014 Sep 3.[Epub ahead of print] PubMed PMID: 25184688.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Drozd M, Chandrashekhar K, Rajashekara G. Polyphosphate-mediated modulation of Campylobacter jejuni biofilm growth and stability. Virulence. 2014 Aug 15;5(6):680-90. doi: 10.4161/viru.34348. PubMed PMID: 25127528; PubMed Central PMCID: PMC4139409.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ibrahim M, Sultan H, Arafa A, Kang K, Razik A, Lee CW. "Toward the development of broadly reactive influenza vaccine for the control of highly pathogenic avian influenza H5N1 in Egypt". Annual Meeting. St. Paul: 65th North Central Avian Disease Conference. 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Malde A, Gangaiah D, Chandrashekhar K, Pina-Mimbela R, Torrelles JB, Rajashekara G. Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni. Virulence. 2014 May 15;5(4):521-33. doi: 10.4161/viru.28311. Epub 2014 Feb 25. PubMed PMID: 24569519; PubMed Central PMCID: PMC4063813.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kassem II, Khatri M, Sanad YM, Wolboldt M, Saif YM, Olson JW, Rajashekara G. The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxidesusceptibility and accumulation in Campylobacter jejuni. Microbiologyopen. 2014 Apr;3(2):168-81. doi: 10.1002/mbo3.158. Epub 2014 Feb 7. PubMed PMID: 24515965; PubMed Central PMCID: PMC3996566.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Issmat I. Kassem, K. Gbenga, A. Kumar, K. Chandrashekhar, R. Pina-Mimbela, G. Rajashekara. An evaluation of the impact of litter chemical amendments on reducing Campylobacter jejuni in broilers. Conference of research workers in animal disease (CRWAD), Chicago, IL. December 6- 8, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Issmat I. Kassem, K. Esseili, M. Terribile, G. Rajashekara. Formate decreases oxidase activity and enhances motility, respiration, and biofilm formation in Campylobacter jejuni. The 114th General Meeting of the American Society for Microbiology, Boston, MA. May 17-20, 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Marion JW, Lee C, Lee CS, Wang Q, Lemeshow S, Buckley TJ, Saif LJ, Lee J.Integrating bacterial and viral water quality assessment to predict swimming-associated illness at a freshwater beach: a cohort study.PLoS One. 2014 Nov 19;9(11):e112029. doi: 10.1371/journal.pone.0112029. eCollection 2014. PMID:25409012