NC_old1202: Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[02/28/2013] [02/12/2014] [01/09/2015] [02/03/2016] [01/30/2017]

Date of Annual Report: 02/28/2013

Report Information

Annual Meeting Dates: 12/01/2012 - 12/02/2012
Period the Report Covers: 10/01/2011 - 09/01/2012

Participants

" Bert Stromberg, University of Minnesota
" Qijing Zhang, Iowa State University
" Phil Hardwidge, Kansas State University
" David Francis, South Dakota State University
" Amin Fadl, University of Wisconsin-Madison
" Jun Lin, University of Tennessee
" Weiping Zhang, South Dakota State University
" Mo Saif, The Ohio State University
" Gireesh Rajashekara, The Ohio State University
" Alexandra Armstrong , University of Arizona
" Chang Won Lee, The Ohio State University
" Prageeth Wijemanne, University of Nebraska-Lincoln
" Zach Stromberg, University of Nebraska-Lincoln
" Rodney Moxley, University of Nebraska-Lincoln
" Margaret Khaitsa, North Dakota State University
" Devendra Shah, Washington State University
" Tom Besser, Washington State University
" Richard Isaacson, University of Minnesota
" Dave Benfield, The Ohio State University
" David Renter, Kansas State University
" T.G. Nagaraja, Kansas State University
" Linda Mansfield, Michigan State University

Brief Summary of Minutes

1. Qijing Zhang, Chair of NC-1202, gave an update on the renewed proposal. The proposal is accessible on the web site for NC-1202. Anybody interested in the proposal can print out a copy directly from the web page.
2. Administrative Advisor, Dr. Stromberg, congratulated everyone for the successful renewal of the group. The group was given a new NC number; NC1202. He made a presentation on overview of the expectation for the groups and submitting annual report and encouraged new memberships.
3. Appendix E uploading. Members of NC-1202 are required to submit the Appendix E form via the NIMSS system. Members dont have to be state representatives. Investigators from medical schools are allowed and encouraged to become member of NC-1202.
4. Gireesh Rajashekara was elected as the secretary and will become the Chair of NC1202, replacing Lynn Joens on the organizing committee.
5. New members were introduced.
6. Student awards. NC-1202 offers awards for students to compete in the Gastroenteric Disease Section of CRWAD. This year two awards will be offered, one for oral presentation and another for poster presentation. Radhey Kaushik serves as Chair for the student award selection committee. He will continue to serve as Chair for the next year. Selection of a co-chair working with him was accomplished. Historically, the student awards were funded by annual registration fee. How to increase the number of abstracts submitted to the Gastroenteric Disease Section was discussed. One suggestion was to offer student travel awards or increase the dollar amount of the awards. This requires industry sponsors and contact with industries for possible support of NC-1202 activities was proposed. There was also a discussion on changing the registration fee to annual dues, which will ensure a steady source of income for the committee.
7. There was discussion on naming the student awards. The group unanimously agreed to name the awards for Lynn Jeons and David Francis, respectively, in honor of their scientific accomplishments and their dedication to NC1202.
8. Proposed title change for the Gastroenteric Disease Section of CRWAD. There was a motion to change the section name from Gastroenteric Disease to a new name. Dr. Rod Moxley will discuss with CRWAD executive committee to finalize the name for the section in the future CRWAD meetings. The rationale for the change is that many members of NC-1202 work with zoonosis and food safety and this change will attract more submission of abstracts to the section. The attendee unanimously voted for the title change. Rodney Moxley will take this issue to the CRWAD Council for approval.
9. Annual report. Annual state reports are due to Qijing Zhang and those who have not submitted are asked to do so. Bert Stromberg emphasized the importance of outcomes and impact. These should be clearly stated in the report.
10. To reduce registration fee, the committee voted in favor of eliminating food items for future meetings.

Accomplishments

Objective 1. Focus on emerging issue- identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine A. Campylobacter jejuni Michigan The LOS of C. jejuni can mimic gangliosides enriched on peripheral nerves leading to autoimmunity. Because up to 37.7% of dairy cattle have been found to shed C. jejuni, we sought to determine whether the calves were the source of the family infections. Preliminary typing results suggest that some calf and human C. jejuni isolates have similar characteristics. Draft Genome Sequences of 2 C. jejuni Clinical Isolates, NW and 2600. NW was isolated from a patient returning from Kenya with acute gastroenteritis. Strain D2600 was also isolated from a patient with gastroenteritis. These clinical isolates colonized C57BL/6 interleukin 10 knockout (IL-10-/-) mice without inducing a robust inflammatory response. Genome sequences of NW and D2600 will facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model. Iowa We compared the clinical isolates of C. jejuni clone (clone SA) from sheep abortions with the human isolates using PFGE, MLST, and serotyping. The results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. Comparative genomics analysis of C. jejuni isolates associated with sheep abortion in the U.S. and the U.K. and non-abortifacient isolates have been performed. Our findings indicated the presence of several genes/gene clusters that were uniquely/predominantly associated with the clone SA isolates. Using one of these genes, we have developed a specific and sensitive rapid screening/identification technique for clone SA (loop-mediated isothermal amplification method). Additionally adaptive changes in transcriptome of clone SA upon exposure to host specific factors are being determined. Ohio Here, we determined the prevalence, genotypic, and phenotypic properties of Campylobacter isolated from fecal samples of dairy cattle and starlings our results highlight starlings as potential reservoirs for C. jejuni and may play important role in the epidemiology of clinically important C. jejuni in dairy population. B. Shiga toxin-producing E. coli (STEC) Kansas Our objective was to determine the prevalence of seven STEC O groups (O26, O45, O103, O111, O121, O145, and O157) and four major STEC virulence genes (stx1, stx2, eae and ehxA) in cohorts of commercial feeder cattle using selective culture methods, four types of IMS beads (O26, O103, O111 and O157), and an 11-gene multiplex PCR (mPCR). Within-cohort prevalence significantly (P < 0.05) varied for certain O-specific serogroup genes. Because cattle are known reservoirs of STEC, we investigated fecal carriage of E. coli O104 in US cattle. Our results suggest that E. coli O104 is present in cattle feces, but the strains do not appear to carry genes characteristic of the virulent hybrid strain. Fresh steam-flaked corn (SFC) is a hotspot for aggregation of house flies (HF) that likely deposit /acquire fecal bacteria, including E. coli O157, on/from this substrate. SFC was screened by a culturing approach for fecal coliforms before and after exposure to HF under the natural conditions in a cattle feedlot in Kansas. Our data show that HF likely play a role in contamination of SFC with E. coli and other enteric bacteria. Washington State We have developed a 48-plex chromosomal backbone SNP allele assay to efficiently type E. coli O157 isolates into the eight lineages as well as 4 sub-lineages. These SNP genotypes are strongly associated with the Stx-encoding bacteriophage chromosomal insertion site genotypes that we have used previously identify and differentiate clinical and bovine biased genotypes of this pathogen. Using the SNP assay, we have genotyped approximately 800 isolates of known sources, with the goal of evaluating seasons, production systems, hosts of origin, interventions, etc., for effects on clinical and bovine-biased genotypes. The most pronounced effect seen is that a high proportion (>90%) of isolates from feedlots belong to a single, clinical genotype that alone accounts for >2/3 of human infection. We are currently evaluating whether this genotype is favoured by feedlot (high grain) rations. We have investigated seasonal variation of O157 shedding by cattle using experimental inoculations to determine whether the variation seen is due to cattle factors, gastrointestinal factors, or extrinsic factors. Results indicate no significant seasonal variation in the amount or duration of shedding, consistent with seasonal variation being due to factors extrinsic to cattle. We are testing the hypothesis that high summertime seasonal shedding of O157 results from introduction of new season forage crops, by sampling pens of cattle just prior to and just after introduction of the new forage crops. The initial trials have showed consistent and sometimes dramatic increases in O157 shedding following these introductions. C. Caliciviruses Ohio We compared Porcine Sapovirus SaV resistance to physicochemical treatments to that of HuNoVs (by reverse transcription-PCR), FCV and MNV (by infectivity assays). The higher stability of SaV than FCV to heat and acid, and its higher resistance to UV light than FCV and MNV make this enteropathogenic virus a promising surrogate for HuNoVs in both in vivo and in vitro studies. We found that NoV VLPs bind to lettuce using multiple carbohydrates, suggesting that simple water washes may be insufficient to remove NoV contamination. We found that SaV RNA persisted with higher titers on outer leaves that were damaged vs. control non-damaged leaves for at least 21 days. However, the virus was not transferred from outer to inner leaves. We screened 1,874 fecal samples from clinically healthy finisher pigs in nine North Carolina swine farms during 2009 by RT-PCR-coupled hybridization assays using primers and probes specific for porcine NoVs or using RT-PCR and calicivirus universal primers. The overall prevalence of porcine NoVs was 18.9%, and we detected GIX SaVs for the first time in the US. We also determined the full-length genome sequence of porcine GII.18 NoV. Our study provides the first GII.18 strain full-genome sequence data which can be used for genomic comparisons and primer design and molecular manipulation (infectious clone) for future pathogenesis studies. D. Turkey and Chicken Enteric viruses Southeast Poultry Research Laboratory, ARS, USDA Using metagenomic approaches we identified a novel parvovirus from enteric content of chickens and turkeys that were affected by enteric diseases. Comparative sequence analysis showed that the chicken parvovirus (ChPV) and turkey parvovirus (TuPV) represented a new member in the Parvovirus family. Following experimental infection, two-day-old broiler chickens showed characteristic signs of enteric disease. Our data indicate that ChPV alone induces RSS in broilers and an important determinant in the complex etiology of enteric diseases of poultry. A comparative metagenome comparing the RNA viral metagenomes from a healthy flock and a sister flock affected by enteric disease was further analyzed. This analysis revealed numerous picornaviruses that were determined to be homologous to the avian turdiviruses and the turkey and duck hepatitis viruses. Further, molecular diagnostic assays targeting the picornavirus capsid protein gene were designed and tested using archived and field enteric samples. E. Rotavirus Ohio Fecal samples collected in different seasons in 2004 and 2011 from diarrheic and healthy nursing piglets from 5 selected swine farms in the US were screened for group (Gp) A, B and C rotavirus using RT-PCR. Our results indicate high genetic heterogeneity in RVC genes and the concurrent co-circulation of different genotypes. Our preliminary data indicate that there are multiple genotypes of RVA (not only G4 and G5 as in current vaccine) currently circulating in US swine. In conclusion, our study demonstrates that infection with distinct RVC is more frequent among nursing piglets than RVA and RVB infections. These findings are of utmost importance to design updated RV vaccines. F. Antimicrobial resistance Washington State The CTX-M families of resistance gene determinants have spread globally in human hospitals, mainly plasmid-borne in E coli and Klebsiella infections. Recently their incidence in companion animals and livestock has reportedly increased. We have detected a high prevalence of CTX-M positive fecal E. coli in dairy calves in Washington State and have determined that their emergence in Washington dairy animals probably occurred between 2008 and 2011. We are currently studying risk factors for their presence on 30 Washington dairy farms. An individual based model was developed to simulate spread of antibiotic resistant pathogens in a veterinary teaching hospital. Preliminary simulations indicated that the probability of transmission depends on the nature of each hospital site, patient length of stay and the frequency of decontamination. Tennessee Bacterial pathogens have co-evolved with host to counteract the effect of endogenous AMPs. However, molecular mechanisms of AMP resistance in Campylobacter are still largely unknown. Our study identified four genetic loci in Campylobacter that will be useful for characterizing molecular basis of Campylobacter resistance to AMPs, a significant knowledge gap in Campylobacter pathogenesis. Despite prevalent beta-lactam resistance in C. jejuni, the molecular basis of beta-lactam resistance in C. jejuni is still largely unknown. We performed an in vivo random transposon mutagenesis to identify genes required for beta-lactam resistance in C. jejuni 81-176. We identified a novel mechanism of beta-lactam resistance in C. jejuni, which will help us better understand the development and regulation of ²-lactam resistance, a significant issue in many bacterial pathogens. We hypothesize that gut microbiome of food animals also contain diverse and novel AR genes which may contribute to the emergence of AR in foodborne human pathogens. Our study showed the effectiveness of functional cloning approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in gut microbiome of chickens from different production systems, and demonstrated functional compatibility of the novel AR genes in enteric pathogens. Iowa We identified a predicted arsenic efflux protein (ArsB) involved in inorganic arsenic resistance and a novel membrane transporter (named ArsP) that contributes to organic arsenic resistance in Campylobacter. The arsenic resistance profiles in C. jejuni isolates from different animal hosts including human, sheep, chicken, and turkey were found to be significantly different with all tested arsenic compounds. Specifically, the sheep isolates had the lowest level of arsenic resistance, while the isolates from turkey had the highest level of arsenic resistance. In another study, genetic mechanisms for differential expression of the CmeABC Efflux Pump in naturally occurring isolates of C. jejuni have been determined. Isolates from turkeys, broilers and humans were analyzed for CmeA, CmeB, and CmeC expression by immunoblotting and cmeB expression by real time RT-PCR. The adaptive mechanisms employed by C. jejuni to erythromycin treatment were studied. The transcriptomes of C. jejuni NCTC 11168 was determined after 30 min treatment with lethal or sub-lethal doses of Ery by competitive microarray hybridizations. Following the treatment with Ery, 139 genes were up-regulated and 119 were down-regulated. Interestingly, several gene operons with putative involvements in drug efflux were up-regulated after exposure to the lethal dose of Ery. Detailed functional analyses of these operons indicated that outer membrane/periplasmic proteins have a role in both oxygen tolerance and chicken colonization in C. jejuni. G. Lawsonia intracellularis Minnesota The aim of this study was to describe an alternative protocol for cultivation of L. intracellularis in cell monolayers. McCoy cell monolayers were heavily infected by L. intracellularis demonstrating the success of the alternative bag incubation protocol. Incubation in the bag provided environmental conditions for L. intracellularis to infect the cells, similar to the conventional protocol. Based on these preliminary results, we believe this approach can be used for static cultivation of bacteria without requiring a Tri-gas incubator. Objective 2. Focus on effective intervention- develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and foodborne disease agents. A. C. jejuni Michigan We hypothesized that in the absence of the regulatory cytokine IL-10, uncontrolled induction of proinflammatory cytokines and infiltration of inflammatory cells is responsible for tissue obliteration in colitis. The work shows for the first time the adaptive immune response (Type 1 and 17 response) that leads to inflammatory bowel disease in this genetically susceptible mouse model. These results can be used to understand this process in humans. Future work is directed towards understanding the relative importance of Type 1 and 17 pathways and innate and adaptive lymphocytes in C. jejuni induced colitis. Lack of good, tractable small animal models has limited understanding of GBS pathogenesis and new therapies. Our results showed NOD, NOD B7-2-/-, and NOD IL-10-/- mice develop neurological signs including decreased activity, failure to rear, splayed feet, shortened stride length, foot drag, and hind limb paralysis among other signs. Sciatic nerve lesions were consistent with acute inflammatory demyelinating polyradiculoneuropathy seen in GBS. Ohio Whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejunis adaptation. Our studies suggest that the RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogens remarkable potential for adaptation. The methylmenaquinol:fumarate reductase, Mfr of C.jejuni is a Tat-dependent periplasmic redox protein that contributes to the metabolism of fumarate. Since chemically oxidized redox-enzymes, contribute to the generation of oxidative stress in E. coli, we assessed the role of Mfr in C. jejunis resistance to hydrogen peroxide (H2O2). Our results suggested that Mfr contributes to H2O2 resistance in C. jejuni. The role of exopolyphosphatases (PPX/GPPA) in poly-P homeostasis and C. jejuni pathobiology remains unexplored. Here we characterized for the first time the role of PPX/GPPA proteins of C. jejuni. Our results expand our knowledge about the multifactorial regulation of poly-P and ppGpp metabolism, two critical molecules that modulate environmental stress responses and virulence in C. jejuni. Here we investigated the effect of nanoparticle encapsulated outer membrane proteins (OMPs) on C. jejuni colonization in chickens. Chicken were vaccinated nanoparticle encapsulated OMPs by different routes (subcutaneous (s. c) or oral) and doses of candidate vaccine. In conclusion, the s.c route of vaccination with nanoparticle encapsulated OMP of C. jejuni may serve as a candidate vaccine in control of C. jejuni colonization. Arizona We are continuing with our efforts to develop an efficacious vaccine using an attenuated Salmonella vector to express C. jejuni proteins shown to be involved in the colonization of broilers. The Salmonella expressing the CjLAJ3 protein was evaluated as vaccine. There was not a significant reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressed CjLAJ3 protein following challenge. We plan conduct two and three component vaccine studies to determine if a combination of multiple expressed proteins will further reduce the Campylobacter load in broilers. Tennessee We have characterized two Ferric enterobactin (FeEnt) receptors (CfrA and CfrB) in C. jejuni and C. coli, the enteric human pathogens that do not produce any siderophores. Together, our study identified and characterized a novel periplasmic trilactone esterase and suggested a new model of FeEnt acquisition. We also established that Campylobacter could utilize high-affinity salmochelin for iron acquisition, and provided insights into the delicate interaction between Campylobacter and host during infection. The USDA licensed live attenuated S. Typhimurium vaccine strain Ç8914 and vector pYA3493 were used for construction of recombinant Salmonella vaccine expressing CmeC or CfrA. In conclusion, we have successfully developed CfrA- and CmeC-based subunit vaccines, which provides us a solid foundation to evaluate different vaccination regimens for effective mitigation of Campylobacter in poultry in the future. The growth promoting effect of Antibiotic Growth Promoters has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Our study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals. Washington State We have evaluated chicken egg-yolk derived antibodies against several C. jejuni chicken colonization associated proteins has opened opportunities for their use as novel passive immunotherapeutic agents to reduce C. jejuni loads in broiler chickens. B. Enterotoxigenic E. coli (ETEC) South Dakota In this study, we applied a genetic fusion strategy to construct a single antigen consisting of representative antigenic peptides from K88, F18, and LT, examined the fusion antigen vaccine development against ETEC associated diarrhea. Immunized piglets were protected when challenged with virulent ETEC strain. Results indicated that live E. coli strain expressing the K88-F18-LTA2:B fusion antigen can be developed as a broadly protective vaccine against porcine post-weaning diarrhea. We described a reproducible gnotobiotic piglet model of post-weaning ETEC diarrhea and efficacy tests of subunit vaccines consisting of K88 (F4) fimbriae and/or heat labile enterotoxin (LT) delivered by the intranasal route. The current study provides a system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies for the development of vaccines for ETEC. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin. Significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains. Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea. Flagella of F18+ ETEC strains play a role in adherence and infection. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro Nebraska Here we determined whether ETEC exposure to glucose will affect its ability to adhere to intestinal epithelial cells via its effects on LT production. This is the first study demonstrating that glucose (at a specific concentration) in the culture medium, through its effects on LT production, significantly affects bacterial adherence, lending support to the hypothesis that the composition of dietary nutrients in the intestine of the host may directly influence the severity of ETEC infection. Kansas We investigated intestinal epithelial cell signal transduction pathways activated by ETEC and quantified the relative importance of these host pathways to enterotoxin (LT) -induced ETEC adherence. We show that ETEC activates both NF-kB and mitogen-activated protein kinase signalling pathways through mechanisms that are primarily dependent upon LT. Our findings provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence. The extent to which ETEC subverts innate immune responses by directly targeting the NF-kB pathway is an understudied topic. We found that ETEC secretes a heat-stable, proteinaceous factor that blocks NF-kB signaling normally induced by TNF, interleukin-1â, and flagellin. These data suggest that ETEC evades the host innate immune response by directly modulating NF-kB signaling. C. Cryptosporidium parvum Illinois We hypothesized that L-PUFFA block a conserved apicomplexan microneme secretion pathway that regulates parasite motility. Our results suggest microneme secretion is a potential target that could be exploited for development of new drugs for the treatment of not only cryptosporidiosis but also other serious apicomplexan diseases such as toxoplasmosis. To probe this hypothesis I have leveraged NC1041 funding by partnering with Dr. David Sibley, Washington University School of Medicine on an NIH grant entitled, Designing Selective Inhibitors of Calcium-Dependent Kinases in Parasites. D. Shiga toxin producing E. coli (STEC) Nebraska Mucosal explants from the colons of three slaughtered steerswere inoculated with 13 representative strains divided among STEC serogroups O26, O45, O103, O104, O111, O121, and O145. Adherence studies with the same strains were also done with human Caco-2 cells. To our knowledge, this is the first report of this organism being able to colonize bovine colonic epithelium. Also, based on the work reported herein, some STEC also have virulence mechanisms in place allowing them to invade colonic epithelial cells. Kansas Our objective was to determine whether commercially available pre-harvest interventions that were implemented to control fecal shedding of E. coli O157 in commercial feedlot cattle had an effect on fecal shedding of non-O157 STEC. By studying cattle that received one, both, or neither of two interventions a siderophore receptor and porin proteins-based vaccine (VAC) and a direct-fed microbial product (DFM) we found no significant treatment effects on fecal shedding of non-O157 STEC serogroups. Our objective was to determine whether the addition of starch to a dried distillers grains DDG-supplemented diet negates the effects of DG on fecal shedding of E. coli O157:H7. Our study confirms the positive association between feeding DG and fecal shedding of E. coli O157:H7 and starch addition to the DDG diet did not have effect on fecal shedding of E. coli O157:H7. We found that phosphorylation of RPS3 Ser209 by the kinase IKKb was crucial for nuclear localization of RPS3 in response to activating stimuli. Moreover, virulence protein NleH1 of E. coli O157:H7 specifically inhibited phosphorylation of RPS3 Ser209 and blocked RPS3 function, thereby promoting bacterial colonization and diarrhea but resulting in less mortality in a gnotobiotic piglet-infection model. Functional differences and interactions between the E. coli type III secretion system effectors NleH1 and NleH2. We found functional differences between NleH1 and NleH2 in their ability to regulate the host NF-kB pathway. Our data show that NleH1 and NleH2 have pronounced functional differences in their ability to alter host transcriptional responses to bacterial infection. E. Norovirus Ohio We tested ways to increase HuNoV infectivity in Gn pigs to create a more robust animal model for HuNoV. We also investigated the impact of an innate immunity mediator, IFN-± as an antiviral for oral treatment of HuNoV-infected Gn pigs. Simvastatin induced significantly earlier onset and longer duration of fecal virus shedding in treated pigs, frequently with higher fecal viral titers. Finally, in contrast to the increased HuNoV shedding that simvastatin induced, curtailed viral shedding was observed in HuNoV-infected pigs pre-treated with recombinant human IFN-± (rhIFN-±). This finding indicates that IFN-± has potential as an antiviral against HuNoV. Kansas The conserved key sites of 3Cpro or 3CLpro in Picorna, Noro, and Corona viruses (Supercluster) may serve as attractive targets for the design of broad-spectrum antivirals. We reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. Furthermore we reported the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., aldehyde (GC373), a bisulfite adduct (GC376), and a ±-ketoamide (GC375), against viruses that belong to this supercluster. F. L. intracellularis Minnesota Non-pathogenic L. intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. The goal of this study was to determine if feces from rabbits experimentally infected with L. intracellularis could be the source of infection for naïve weanling foals. The results support the role of rabbits as asymptomatic amplifier of L. intracellularis and serve as source of infection for susceptible foals. The objective of this study was to determine the efficacy of an avirulent L. intracellularis vaccine in preventing proliferative enteropathy in weanling foals. Intra-rectal administration of a commercial avirulentporcine vaccine against L intracellularis resulted in complete protection against proliferative enteropathy in the foals in this study This pilot study aimed to demonstrate the susceptibility of rabbits to L. intracellularis obtained from a clinical equine proliferative enteropathy case, and thus provide a potential animal model for investigations on pathogenesis and therapy in horses. This initial study for the development of an EPE in a rabbit model simulates natural infection, as typical lesions, immune response, and fecal shedding were present. F. Rotavirus Kansas In this study, we examined the roles of the key enzymes for cellular triglyceride TG synthesis (lipogenesis) in the replication of rotaviruses by using inhibitors of fatty acid synthase, long chain fatty acid acyl-CoA synthetase (ACSL). Triacsin C, a natural ACSL inhibitor from Streptomyces aureofaciens, was found to be highly effective against rotavirus replication. Many novel triacsin C analogs significantly reduced rotavirus replication. Our results suggest triacsin C and/or its analogs as potential therapeutic options for rotavirus infections. Ohio We investigated the impact of lactic acid bacteria colonization (Lactobacillus rhamnosus strain GG and Bifidobacterium lactis, probiotics) with/without mothers milk/cols on B cell antibody responses to an attenuated oral rotavirus (RV) vaccine in a gnotobiotic (Gn) pig model. Milk/cols did not affect probiotic colonization in RV vaccinated pigs. However milk/cols fed unvaccinated pigs shed higher probiotic bacterial titers than unvaccinated controls. In RV vaccinated pigs, milk/cols and probiotics enhanced mean serum IgA RV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to milk fed, non-probiotic supplemented pigs. We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate and antibody responses and rotavirus (RV) vaccine efficacy in Gn pigs and challenged with virulent RV (VirRV) with or without vitamin A supplementation. Our results suggest that prenatal VAD causes an imbalance in innate immune responses and exacerbates VirRV infection. Vitamin A supplementation failed to alleviate the VAD-associated imbalance in innate immune responses or the exacerbated VirRV infection. Additionally, our results suggest that vitamin A metabolism may be affected in VAD piglets, leading to altered immune function. Illinois A sialyllactose-containing neoglycolipid was synthesized and assayed for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoyl- phosphatidylethanolamine by reductive amination. Results of in vivo animal challenge studies using newborn pigs, demonstrated SLPE afforded complete protection from rotavirus disease. In collaboration with Dr. Sharon Donovan, we also have demonstrated selected natural human milk oligosaccharides, the third most abundant component in human milk, reduce the duration of diarrhea in piglets, possibly in part by promoting immunoglobulin response to rotavirus infection and modulating the gut microbiota. South East Poultry, USDA, ARS, Georgia In order to validate an institutional effluent decontamination system (EDS) in use at our facility, we determined the time and temperature necessary for the complete inactivation of common poultry enteric viruses. This investigation determined the thermal inactivation curve for rotavirus, reovirus and astrovirus in a buffered solution, as well as the endpoint thermal inactivation of these non-enveloped viruses in the EDS, which decontaminates effluent prior to disposal in the municipal sewer. This study confirmed the full inactivation of these viruses by the EDS. G. Salmonella Washington State We generated and screened >4000 mutants of a highly pathogenic S. Enteritidis strain in vitro for their virulence characteristics and identified several novel virulence associated genes. In addition, we are also evaluating the potential usefulness of the virulence-attenuated mutants as a live attenuated vaccine. We characterized more than 100 S. Enteritidis strains for their virulence characteristics and identified several strains that differ in their virulence and survival within egg albumen. We have performed global comparative transcriptome analysis using a next generation sequencing on multiple high and low-pathogenic strains of S. Enteritidis. This led to identification of several previously uncharacterized genes whose expression is upregulated in high-pathogenic strains. We screened a library of >20,000 mutants of a highly pathogenic strain of S. Enteritidis using oral infection in one-day old chickens as a model system and identified several genes that play a role in intestinal invasion and/or systemic dissemination of S. Enteritidis in chickens. Objective 3. Focus on disseminating knowledge Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories Arizona Dr. Joens organized the University of Arizona Food Safety Conference on October 12, 2012, held at the Omni Tucson National Resort. Approximate attendance was 108 attendees, with 20% of attendees coming from food safety industries or government agencies. The Food Safety Conference consisted of 10 hours of contact time over one day, with seven invited speakers followed by a poster session and a dinner to facilitate dialogue and collaborative work. Minnesota The Principle Investigators and Students involved in the project have given presentations and updates on both swine and equine proliferative enteropathy at various scientific, veterinary, and diagnostic meetings in the previous year. These include the Leman Swine Conference, the Rushmore Symposium, the American Association of Equine Practitioners, the American College of Veterinary Internal Medicine and the American Association of Swine Veterinarians. Kansas Through several venues we have provided educational opportunities to professional and graduate students as well as continuing education to veterinarians on enteric pathogens in livestock production systems. The majority of the shared information has focused on the impacts of Shiga toxin-producing Escherichia coli and Salmonella in the beef industry, the conclusions that can be reached based on recent research, and the potential opportunities to reduce these pathogens in beef production systems. Michigan Dr. Mansfield organized a seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University on October 19, 2012. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. Dr. Mansfield helped to organize and attended the USDA Enteric Diseases Meeting NC1041 in Chicago, Illinois on December 1st and 2nd. Dr. Mansfield gave an invited talk entitled Murine models of the autoimmune neuropathy Guillain Barré Syndrome for the Small Animal Models of Enteric Diseases, NIAID, NIH, September 13-14, 2012. She also gave an invited talk entitled C. jejuni induces mixed Type 1 and 17 responses in acute and chronic disease for the Enterics Research Investigational Network, Cooperative Research Centers Annual Meeting, NIH, Seattle, Washington, May 22-23, 2012. North Dakota North Dakota State University (NDSU) and Makerere University (MAK) in Kampala, Uganda offered the joint course International Animal Production, Disease Surveillance and Public Health again in summer 2012 with 23 student attendees 5 from US and 18 from 6 higher education institutions in East Africa. Additionally, 6 students from MAK enrolled in the MS degree in International Infectious Disease Management attended the summer course. Also in July, 2012 an International conference was held in Kampala Uganda funded by USAID initiative on Africa-US Capacity building in Higher Education for Developmentthe project is Capacity building in Integrated Management of Transboundary Animal Diseases & Zoonoses in East & Central Africa (ECA). This project is currently implemented using One Health approach through AFRUS-IDM (Africa-US Integrated Disease Management), a network of higher education institutions in East & Central Africa (University of Nairobi, Kenya; Sokoine University of Agriculture, Tanzania; Mekelle University, Ethiopia; Umutara Polytechnic, Rwanda; Sheik Technical Veterinary School, Somalia) and the US and Canada (Columbus State University, Kansas State University, Michigan State University, University of Minnesota, Washington State University, and University of Saskatchewan, Canada), respectively. Nebraska Knowledge pertinent to NC-1041 activities was disseminated to undergraduate students, graduate students, professional veterinary students, veterinarians, physicians, food processors, researchers, cattle producers and other decision makers regarding pre-harvest food safety of cattle food projects. A listing of specific presentations and dates is provided in Section VI, below.

Publications

1. Sahin O, Fitzgerald C, Stroika S, Zhao S, Sippy RJ, Kwan P, Plummer PJ, Han J, Yaeger MJ, Zhang Q. 2012. Molecular Evidence for Zoonotic Transmission of an Emergent Highly Pathogenic Campylobacter jejuni Clone in the United States. J. Clin. Microbiol. 50:680-687. PMID: 22189122. PMCID: PMC3295108. 2. Plummer P, Sahin O, Burrough E, Sippy R, Mou K, Rabenold J, Yaeger M, Zhang Q. 2012. Critical Role of LuxS in the Virulence of Campylobacter jejuni in a Guinea Pig Model of Abortion. Infect. Immun. 80:585-93. PMID: 22144479. PMCID: PMC3264297. 3. Luo Y, Sahin O, Dai L, Sippy R, Wu Z, Zhang Q. 2012. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive and Specific Detection of a Campylobacter jejuni Clone. J. Vet. Med. Sci. 74:591596. PMID: 22188995. 4. Burrough ER, Zuowei Wu, Orhan Sahin, Zhang Qijing, Mike J. Yaeger. 2012. Spatial Distribution of Putative Growth Factors in the Guinea Pig Placenta and the Effects of These Factors, Plasma, and Bile on the Growth and Chemotaxis of C. jejuni. Vet. Pathol. 49:470-481. PMID: 22081135. 5. Luangtongkum T, Shen Z, Seng VW, Sahin O, Jeon B, Liu P, Zhang Q. 2012. Impaired fitness and transmission of macrolide-resistant Campylobacter jejuni in its natural host. Antimicrob. Agents Chemother. 56:1300-1308. PMID: 22183170. PMCID: PMC3294946. 6. Han J, Wang Y, Sahin O, Shen Z, Guo B, Shen J, and Zhang Q. 2012. A fluoroquinolone resistance associated mutation in gyrA affects DNA supercoiling in Campylobacter jejuni. Front. Microbiol. 2:21. PMID: 22919613. PMCID: PMC3417464. 7. Sippy R, Sandoval-Green CM, Sahin O, Plummer P, Fairbanks WS, Zhang Q, Blanchong JA. 2012. Occurrence and molecular analysis of Campylobacter in wildlife on livestock farms. Vet. Micro. 157:369-75. PMID: 22266157. 8. Hwang S, Zhang Q, Ryu S, Jeon B. 2012. Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni. J Bacteriol. 2012 Oct 12. [Epub ahead of print]. PMID: 23065977. 9. Shen Z, Pu XY, and Zhang Q. 2011. Salicylate functions as an efflux pump inducer and promotes the emergence of fluoroquinolone-resistant Campylobacter jejuni mutants. Appl Environ Microbiol. 77:7128-33. PMID: 21821741. PMCID: PMC3194847. 10. Kassem II, Sanad YM, Stonerock R, Rajashekara G. 2012. An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers. Poult Sci. Apr;91(4):1032-7. 11. Yasser M. Sanad, Gary Closs Jr, Anand Kumar, Jeffrey T. LeJeune, Gireesh Rajashekara. 2012. Molecular Epidemiology and Public Health Relevance of Campylobacter Isolated from Dairy Cattle and European Starlings in Ohio, USA. Foodborne pathogens and diseases, (In press) 12. Issmat I. Kassem1, Mahesh Khatri, Malak A. Esseili, Yasser M. Sanad, Yehia M. Saif, Jonathan W. Olson2, Gireesh Rajashekara. 2012. Respiratory proteins contribute differentially to Campylobacter jejunis survival and in vitro interaction with hosts intestinal cells. BMC Microbiology, Nov 13;12(1):258. 13. Sanad, Y.M., I.I. Kassem, Z. Liu, J. Lin, J. T. Lejune, and G. Rajashekara. 2012. Occurrence of the invasion associated marker (iam) in C. jejuni isolated from cattle. BMC Research Notes. 4:570 14. Jun Lin J, KS Mateo, M Zhao, AK Erickson, N Garcia, D He, RA Moxley and DH Francis. 2012. Protection of Piglets against Enteric Colibacillosis by Intranasal Immunization with K88ac (F4ac) Fimbriae and Heat Labile Enterotoxin of Escherichia coli. Vet Microbiol. Epub ahead of press. 15. Zeng, X., F. Xu, Y. Mo, and J. Lin. 2012. Identification and characterization of a periplasmic trilactone esterase, Cee, revealed novel features of ferric enterobactin acquisition in Campylobacter. Molecular Microbiology (Accepted). 16. Theoret JR, Cooper KK, Zekarias B, Roland KL, Law BF, Curtiss R 3rd, Joens LA. The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination. Clin Vaccine Immunol. 2012 ;19(9):1426-31. 17. Cooper KK, Cooper MA, Zuccolo A, Joens LA. Re-sequencing of a virulent strain of Campylobacter jejuni NCTC11168 reveals potential virulence factors. Res Microbiol. 2012 Oct 6. doi:pii: S0923-2508(12)00136-2. 10.1016/j.resmic.2012.10.002. [Epub ahead of print] 18. Bell JA, Jerome JP, Plovanich-Jones AE, Smith EJ, Gettings JR, Kim HY, Landgraf JR, Lefébure T, Kopper JJ, Rathinam VA, St Charles JL, Buffa BA, Brooks AP, Poe SA, Eaton KA, Stanhope MJ, Mansfield LS. Outcome of infection of C57BL/6 IL-10(-/-) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up- and down-regulated in vivo. Microbial Pathogenesis 2012 Aug 31. pii: S0882-4010(12)00151-9. doi: 10.1016/j.micpath.2012.08.001. 19. Jerome J.P., Klahn B.D., Bell J.A., Barrick J.E., Brown C.T., and Mansfield L.S. 2012 Draft Genome Sequences of Two C. jejuni Clinical Isolates, NW and D2600, Journal of Bacteriology, in press. 20. Kim JS, Artymovich KA, Hall DF, Smith EJ, Fulton R, Bell JA, Dybas L, Mansfield LS, Tempelman R, Wilson DL, Linz JE. 2012. Passage of Campylobacter jejuni through the chicken reservoir or mice promotes phase variation in contingency genes Cj0045 and Cj0170 that strongly associates with 21. colonization and disease in a mouse model. Microbiology February 16, 2012 as doi:10.1099/mic.0.057158-0. 22. Vannucci FA, Beckler D, Pusterla N, Mapes SM, Gebhart CJ. 2012. Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy. Vet. Micro. (In press) 23. Sampieri F, Vannucci FA, Allen AL, Pusterla N, Antonopoulos A.J., Ball K.R., Thompson J., Dowling P.M., Hamilton D.L., Gebhart C.J. 2012. Species-specificity of equine and porcine L. intracellularis isolates in laboratory animals. Can J Vet Res. (In press) 24. Chander Y, Primus A, Oliveira S, Gebhart CJ. 2012. Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated Brachyspira hampsonii J. Vet. Diag. Investi. 24:903-910. 25. Pusterla N, Mapes S, Gebhart C. 2012. L. intracellularis-specific interferon c gene expression by peripheral blood mononuclear cells in vaccinated and naturally infected foals. Vet. J. 192:249-251. 26. Sampieri F, Allen AL, Pusterla N, Vannucci FA, Anatonopoulos AJ, Hamilton, DL, and Gebhart CJ. 2012. The rabbit as an infection model for equine proliferative enteropathy. Can J. Vet. Res. (In press ) 27. Viott AM, Franca SA, Vannucci FA, Cruz ECC, Costa MC, Gebhart CJ and Guedes MC. 2012. Infection of sparrows (Passer domesticus) and different mice strains with Lawsonia intracellularis. Pesquisa Veterinaria Brazileira. (In press) 28. Vannucci, FA, Foster DN and Gebhart CJ. 2012. Comparative transcriptional analysis of homologous pathogenic and non-pathogenic Lawsonia intracellularis isolates in infected porcine cells. PloS ONE 7(10): e46708. doi:10.1371/journal.pone.0046708 29. Nogradi N, Slovis NM, Gebhart CJ, Wolfsdorf KE, McCrcken JL, Scoggin CF, Kass PH, Mapes SM, Toth B, Lundquist ML, Pusterla N. 2012. Evaluation of the field efficacy of an avirulent live Lawsonia intracellularis vaccine in foals. Vet. J. 192:511-513. 30. Pusterla N, Vannucci FA, Guzman DSM, Mapes S, White A, DiFrancesco M, Gebhart C. 2012. Transmission of Lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits. Vet. Journal. (In press). 31. Vannucci FA, Pusterla N, Mapes SM, Kelley M, Gebhart CJ. 2012. Evidence of host adaptation in Lawsonia intracellularis infections. Vet Res. 43:53-65. 32. Pusterla N., Mapes S. and Gebhart C. 2012. Further investigation of exposure to Lawsonia intracellularis in wild and feral animals captured on horse properties with equine proliferative enteropathy. Vet. J. 194:253-255. 33. Vannucci F.A., Wattanaphansak S., Gebhart C.J. 2012. An alternative method for cultivation of Lawsonia intracellularis. J. Clin. Microbiol. 50:1070-1072. 34. Pusterla N., Vannucci, F.A., Mapes S.M., Nogradi, N., Collier J.R., Hill J.A., DiFrancesco M., White A.M., Akana N.K., Simonek G., Gebhart C.J. 2012. Efficacy of an avirulent live vaccine against Lawsonia intracellularis in the prevention of proliferative enteropathy in experimentally infected weanling foals. Am. Jr. Vet. Res. 73:741-746. 35. Paradis M., Gebhart C.J., Toole D., Vessie G., Winkelman N.L., Bauer S.A., Wilson J.F., McClure C.A. 2012. Subclinical Ileitis: Diagnostic and performance parameters in a multi-dose mucosal homogenate challenge model. J. Swine Health and Prod. 20:137-141. 36. Cull CA, Paddock ZD, Nagaraja TG, Bello NB, Babcock AH, Renter DG. Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle. Vaccine 2012; 30(43): 6210-6205. 37. Jacob ME, Nagaraja TG. Use of direct-fed microbials as a preharvest food safety intervention in cattle. p 189-202. In: Direct-Fed Microbials and Probiotics for Animals: Science and Mechanisms of Action. T. R. Callaway and S. C. Ricke (eds.) 2012. Springer Publ., NY. 38. Bai J, Paddock ZD, Shi X, Li S, An B, Nagaraja TG.. Applicability of a Multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Path Dis. 2012, 9:541-548. 39. Paddock ZD, Shi X, Bai J, Nagaraja TG. Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces. Vet Microbiol 2012, 156:381-388. 40. Jacob ME, Shi X, An B, Nagaraja TG, Bai J. Evaluation of a multiplex real-time PCR for the quantification of Escherichia coli O157 in cattle feces. Foodborne Path Dis 2012, 9:79-85. 41. Eberhart LJ, Deringer JR, Brayton KA, Sawant AA, Besser TE, Call DR. Characterization of a novel microcin that kills enterohemorrhagic Escherichia coli O157:H7 and O26. Appl Environ Microbiol. 2012 Sep;78(18):6592-9. doi: 10.1128/AEM.01067-12. PMID: 22773653; PMCID: PMC3426703. 42. Irshad H, Cookson AL, Hotter G, Besser TE, On SL, French NP. Epidemiology of Shiga toxin-producing Escherichia coli O157 in very young calves in the North Island of New Zealand. N Z Vet J. 2012 Jan;60(1):21-6. PubMed PMID: 22175425. 43. Kudva IT, Davis MA, Griffin RW, Garren J, Murray M, John M, Hovde CJ, Calderwood SB. Polymorphic Amplified Typing Sequences and Pulsed-Field GelElectrophoresis Yield Comparable Results in the Strain Typing of a Diverse Set of Bovine Escherichia coli O157:H7 Isolates. Int J Microbiol. 2012;2012:140105. doi: 10.1155/2012/140105. Epub 2012 Aug 7. PubMed PMID: 23049559; PubMed CentralPMCID: PMC3461292. 44. Bono JL, Smith TP, Keen JE, Harhay GP, McDaneld TG, Mandrell RE, Jung WK, Besser TE, Gerner-Smidt P, Bielaszewska M, Karch H, Clawson ML. Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans. Mol Biol Evol. 2012 Aug;29(8):2047-62. Epub 2012 Feb 21. PubMed PMID: 22355013; PubMed Central PMCID: PMC3408066. 45. Shringi S, García A, Lahmers KK, Potter KA, Muthupalani S, Swennes AG, Hovde CJ, Call DR, Fox JG, Besser TE. Differential virulence of clinical and bovine-biased enterohemorrhagic Escherichia coli O157:H7 genotypes in piglet and Dutch belted rabbit models. Infect Immun. 2012 Jan;80(1):369-80. PMID: 22025512; PMCID: PMC3255674. 46. Pham T, Gao X, Tsai K, Olsen R, Wan F, Hardwidge PR. Functional differences and interactions between the E. coli type III secretion system effectors NleH1 and NleH2, Infection & Immunity, 2012 Jun;80(6):2133-40. PMID: 22451523 47. Wang X, Hardwidge, PR. Enterotoxigenic Escherichia coli (ETEC) prevents host NF-kB activation by targeting IkBa polyubiquitination, Infection & Immunity, 2012, Oct.1 [Epub ahead of print] PMID: 23027537 48. Wang X, Gao X, Hardwidge PR. Heat-labile enterotoxin-induced activation of NF-kappaB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence, Cellular Microbiology, 2012 Aug;14(8):1231-41. PMID: 22452361. 49. Zhang, W. & D. A. Sack. 2012. Progress and hurdles in the development of vaccines against enterotoxigenic Escherichia coli in humans. Expert Review of Vaccines. 11(6):677-94. 50. Ruan, X., Crupper S.C., Schultz B.D., Robertson D.C., and W. Zhang. 2012. Escherichia coli expressing enteroaggregative heat-stable toxin 1 (EAST1) did not cause an increase of cAMP or cGMP levels in cells, and no diarrhea in 5-day old gnotobiotic pigs. PLoS ONE. 7(8):e43203. 51. Duan, Q, M. Zhou, X. Zhu, Y. Yang, J. Zhu, W. Bao, S. Wu, X. Ruan, W. Zhang, and Zhu G. 2012. Flagella from F18+ Escherichia coli play a role in adhesion to pig epithelial cell lines. Mcrobiol Pathogen. In press. 52. Duan, Q., Zhou M., Zhu X., Bao W., Wu S., Ruan X., Zhang W., Yang Y., Zhu J., and Zhu G. 2012. The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in an IPEC-J2 cell model in vitro. Vet. Microbiol. 160(2012):132-140. 53. Santiago-Mateo K, M Zhao, J Lin, W. Zhang, DH Francis. 2012. Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic E. coli strain that expresses the same adhesion and enterotoxins. Vet Microbiol.159:337-342. 54. Erume J, P Wijemanne, EM Berberov SD. Kachman, DJ Oestmann, DH. Francis, RA. Moxley. 2012. Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine. Vet Microbiol 161:315-324. 55. Kim Y, Lovell S, Tiew K, Mandadapu SR, Alliston KR, Battaile KP, Groutas WC, Chang K. Novel Broad-Spectrum Antivirals against 3C or 3C-like proteases of Picornaviruses, Noroviruses and Coronaviruses. Journal of Virology, 2012, 86(21):11754-62. 56. Kim Y, George D, Prior AM, Prasain K, Hao S, Le DD, Hua DH, Chang K. Novel triacsin C analogs as potential antivirals against rotavirus infections. Eur J Med Chem, 2012, 50:311-8. 57. Esseili MA, Wang Q, Zhang Z, Saif LJ. Internalization of sapovirus, a surrogate for norovirus, in romaine lettuce and the effect of lettuce latex on virus infectivity. Appl Environ Microbiol. 2012. 78:6271-9 58. Esseili MA, Wang Q, Saif LJ. 2012. Binding of human GII.4 norovirus virus-like particles to carbohydrates of romaine lettuce leaf cell wall materials. Appl Environ Microbiol. 78:786-94. 59. Jung, K., Q. Wang, Y. Kim, K. Scheuer, Z. Zhang, Q. Shen, K-O. Chang, and L. J. Saif. 2012. The effects of simvastatin or interferon-± on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals. PLoS One 7:e41619. 60. Wang, Q., K. Scheuer, Z. Ahang, W. A. Gebreyes, B. Z. Molla, A. E. Hoet, and L. J. Saif. 2011. Characterization and prevalence of a new porcine Calicivirus in Swine, United States. Emerging infectious diseases 17:1103-1106. 61. Wang Q., Z. Zhang and L.J. Saif. 2012. A cultivable porcine sapovirus surrogate for human caliciviruses: stability and attachment to lettuce. Appl Environ Microbiol. 78:3932-40. 62. Harada S., Oka T., Tokuoka E., Kiyota N., Nishimura K., Shimada Y., Ueno T., Ikezawa S., Wakita T., Wang Q. Saif L. J., Katayama K. 2012. A confirmation of sapovirus re-infection gastroenteritis caseswith different genogroups and genetic shifts in the evolving sapovirus genotypes, 2002-2011. Arch Virol 157:19992003. 63. Amimo, J.O., Vlasova, A.N., Saif, L.J. 2013. Prevalence, genetic heterogeneity and identification of a potential new VP4 genotype of porcine group c rotaviruses in nursing and weaned piglets in Ohio, USA. Submitted to Veterinary Microbiology. 64. Khaitsa, M.L. and Dawn Doetkott. 2012 (Accepted in August 2011). Antimicrobial Drug Resistance and Molecular Characterization of Salmonella Isolated from Domestic Animals, Humans and Meat Products, In: Salmonella - A Dangerous Foodborne Pathogen, Barakat S. M. Mahmoud (Ed.), ISBN: 978-953-307-782-6. 65. Michael Muleme, Robert Barigye, Margaret L. Khaitsa, Eugene Berry, Anthony W. Wamono, Chrisostom Ayebazibwe. 2012. Effectiveness of vaccines and vaccination programs for the control of foot-and-mouth disease in Uganda, 20012010. Trop. Ani. Health and Prod. September 2012. 66. Stella Opendi Sasanya, Susan Olet, Robert Littlefield, Charles L. Stoltenow and Margaret L. Khaitsa. 2012. Spatial-temporal Distribution of the 2007 Melamine Associated Nephrotoxic Renal failure among Pets and Factors Associated with Pet Survival. Journal of Food Protection Trends. In Press. 67. George S, K Circle, S Lindblom, S Vilain, AJM Rosa, D Francis, V Brözel1, and RS Kaushik. 2012. Assessment of Toll-like Receptors in the Ileum of Weanling Pigs- Responses to Feed Antibiotic Chlortetracycline and Gnotobiotic Conditions. Clin Cellular Immunol. Epub ahead of print. 68. Wang, Z., X. Zeng, Y. Mo, K. Smith, J. Lin. 2012. Identification and characterization of a bile salt hydrolase for developing novel alternatives to antibiotic growth promoters. Applied and Environmental Microbiology 78:8795-8802 69. Lin, J., A.A. Hunkapillar, A.C. Layton, Y. Chang, K.R. Robbins. 2012. Response of intestinal microbiota to antibiotic growth promoters in chickens. Foodborne Pathogens and Diseases (In Press) 70. Hoang, K., Y. Wang, and J. Lin. 2012. Identification of genetic loci required for Campylobacter resistance to fowlicidin-1, a chicken host defense peptide. Frontiers in Cellular and Infection Microbiology 2:32. Doi:10.3389/fcimb.2012.00032 71. Zhou, W., Y. Wang, and J. Lin. 2012. Functional cloning and characterization of antibiotic resistance genes from chicken gut microbiome. Applied and Environmental Microbiology. 78:3028-3032 72. Cummings KJ*, Warnick LD, Davis MA, Eckmann K, Gröhn YT, Hoelzer K, MacDonald JK, Root TP, Siler JD, McGuire SM, Wiedmann M, Wright EM, Zansky SM, Besser TE. Farm animal contact is a risk factor for salmonellosis caused by bovine-associated subtypes. (Accepted, Emerg Inf Dis). 73. Morales CA, Guard J, Sanchez-Ingunza R, Shah DH, Harrison M. Virulence and metabolic characteristics of Salmonella enterica serovar enteritidis strains with different sefD variants in hens. Appl Environ Microbiol. 2012 Sep;78(18):6405-12. doi: 10.1128/AEM.00852-12. Epub 2012 Jun 22. PubMed PMID: 22729535; PubMed Central PMCID: PMC3426706. 74. Neal-McKinney JM, Lu X, Duong T, Larson CL, Call DR, Shah DH, Konkel ME. Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLoS One. 2012;7(9):e43928. doi: 10.1371/journal.pone.0043928. Epub 2012 Sep 4. PMID: 22962594; PMCID: PMC3433458. 75. Shah DH, Casavant C, Hawley Q, Addwebi T, Call DR, Guard J. Salmonella Enteritidis strains from poultry exhibit differential responses to acid stress, oxidative stress, and survival in the egg albumen. Foodborne Pathog Dis. 2012 Mar;9(3):258-64. PMID: 22304629; PMCID: PMC3326446. 76. Shah DH, Zhou X, Kim HY, Call DR, Guard J. Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen. Infect Immun. 2012 Dec;80(12):4203-15. PMID: 22988017; PMCID: PMC3497420. 77. Subbiah M, Shah DH, Besser TE, Ullman JL, Call DR. Urine from Treated Cattle Drives Selection for Cephalosporin Resistant Escherichia coli in Soil. PLoS One. 2012;7(11):e48919. doi: 10.1371/journal.pone.0048919. PMID: 23145021; PMCID: PMC3492140. 78. Morrison CM, Dial SM, Day WA Jr, Joens LA. Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis. Appl Environ Microbiol. 2012 Apr;78(8):2867-73. Epub 2012 Feb 3. 79. Li M, Bauer LL, Chen X, Wang M, Kuhlenschmidt TB, Kuhlenschmidt MS, Fahey GC, Jr, and Donovan SM (2012) Microbial Composition and In Vitro Oligosaccharides and Prebiotics Differ between Formula-Fed and Sow-Reared Piglets. J. Nutrition 142:1-10. 80. Fuller KL, Kuhlenschmidt TB, Kuhlenschmidt MS, Jimenez-Flores R, and Donovan SM. (2012) Milk Fat Globule Membrane Isolated from Buttermilk or Cheese Whey and their Lipid Component Inhibit Infectivity of Rotavirus In Vitro. J. Dairy Science (in press). 81. Li, M, Monaco MH, Wang, M, Comstock SS, Kuhlenschmidt TB, Kuhlenschmidt MS, Donovan SM, (2013) Human milk oligosaccharides and prebiotics reduce the duration of rotavirus-induced diarrhea in piglets. JPGN (in press). 82. Lin, J., K. S. Mateo, M. Zhao, A. K. Erickson, N. Garcia, D. He, R. A. Moxley, and D. H. Francis. 2012. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli. Vet. Microbiol. (in press, VETMIC-5932) 83. Shippy, D. C. and A. A. Fadl. 2012. Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine. BMC Microbiology (Accepted for publication).

Impact Statements

" Identification of C. jejuni clone SA as a cause of recent foodborne outbreaks and sporadic cases provided strong evidence for zoonotic transmission of this emergent clone from ruminants to humans, indicating that clone SA is an important threat to public health.
" Identification of several clone SA-associated genes provides potential targets for controlling this zoonotic pathogen.
" Analysis of clone SAs adaptive responses opens new avenues for developing diagnostic tools and potential vaccines against C. jejuni sheep abortion.
" Studies on antibiotic resistance mechanisms have the potentials for developing strategies to control antimicrobial resistance in C. jejuni.
" Molecular epidemiological studies of Campylobacter in food animals species would help to determine the potential risk of certain clonal types for human infections thus facilitate effective control and preventive measures, and limit the public health impact.
" The studies focus on the development of innovative strategies to control Campylobacter infection in humans and in animal reservoirs, consequently reducing the occurrence of foodborne illness.
" Understanding the role of TAT system and Tat-dependent proteins in C. jejuni pathobiology would provide novel ways to develop anti campylobacter strategies. For example, identification of small molecule chemical inhibitors that specifically inhibit TAT translocation.
" Development of vaccines to control Campylobacter colonization of poultry and other food animals is a critical step in the multifaceted approach needed to control this bacteria in the food animals.
" Development of chicken egg-yolk derived antibodies against several C. jejuni chicken colonization associated proteins has opened opportunities for their use as novel passive immunotherapeutic agents to reduce C. jejuni loads in broiler chickens.
" This study demonstrates for the first time that calves colonized with C. jejuni without disease can carry strains that have the genetic characteristics associated with those from Guillain Barré patients. This implicates calves as a source for the strains of campylobacter that induce autoimmune diseases like this peripheral neuropathy. These are the most dangerous forms of this food borne bacterium.
" The work shows for the first time the adaptive immune response that leads to inflammatory bowel disease in this genetically susceptible mouse model. These results can be used to understand this process in humans.
" New models for GBS can be used to test treatment and prevention modalities and therapies.
" Development of an alternative protocol for cultivation of L. intracellularis in cell monolayers will opens a new field for in vitro studies and gives an opportunity to engage more research institutes in this area. In addition, studies using various conditions of growth and incubation in the bags can now be conducted.
" Demonstration of attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro is valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.
" Identification of feces from rabbits experimentally infected with L. intracellularis as a source of infection for naïve weanling foals support the role of rabbits as asymptomatic amplifier of L. intracellularis and their role as source of infection for susceptible weanling foals.
" We demonstrated the efficacy of an avirulent L. intracellularis vaccine in preventing proliferative enteropathy in weanling foals and may also reduce environmental contamination with the organism on endemic farms.
" We demonstrated the susceptibility of rabbits to L. intracellularis obtained from a clinical equine proliferative enteropathy case, and thus provide a potential animal model for investigations on pathogenesis and therapy in horses.
" The close association between Stx-encoding bacteriophage insertion sites and chromosomal backbone SNP allele patterns shows that the clinical and bovine-biased genotypes are the result of evolutionary divergence, and not an artifact of bacteriophage movements.
" The strong association between the most frequent clinical genotype and feedlot cattle suggests that feedlot rations preferentially support this genotype. If confirmed experimentally, this could open the door to new interventions with potential to specifically reduce prevalence of clinical genotypes in the cattle reservoir, indirectly promoting public health.
" The extrinsic origin of seasonal variation suggests that control efforts should focus much more strongly on environmental non-cattle reservoirs of this pathogen.
" The association of increased prevalence of O157 immediately following introduction of fresh forages also provides a new avenue for research to reduce the prevalence of this agent in the cattle reservoir.
" Baseline prevalence estimates for STEC O groups and virulence genes in feces of commercial feeder cattle are critical to populate risk assessments and identify the distribution and potential risk of human illness due to STEC infections.
" The STEC serogroup O104 recovered from the feces of US feedlot cattle do not appear to carry genes characteristic of the virulent hybrid strain of STEC O104 that caused the large foodborne outbreak in Germany and France in 2011.
" Steam-flaked corn in cattle feedlots is a hotspot for deposition and acquisition of Escherichia coli and other coliforms by house flies; methods to reduce fly access to SFC should be evaluated in order to reduce bacterial contamination.
" Our data demonstrate that the two copies of E. coli O157:H7 non-locus of enterocyte effacement (LEE)-encoded protein H, designated NleH1 and NleH2, have pronounced functional differences in their ability to alter host transcriptional responses to bacterial infection.
" We found that IKKb-dependent modification of a specific amino acid in RPS3 promoted specific NF-kB functions that underlie the molecular pathogenetic mechanisms of E. coli O157:H7.
" Our findings on the intestinal epithelial cell signal transduction pathways activated by ETEC and the relative importance of these host pathways to enterotoxin (LT) -induced ETEC adherence provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence.
" Research results show that enterotoxigenic Escherichia coli (ETEC) evades the host innate immune response by directly modulating NF-kB signaling.
" Construction of a tripartite antigen using genetic fusion strategy, and application of this fusion antigen for developing a live vaccine strain broadly protective against ETEC associated with porcine postweaning diarrhea.
" Demonstration of ETEC fimbria and enterotoxin antigens and intranasal vaccination route for vaccine development against ETEC.
" Development of a live attenuated vaccine that carries an LT toxoid antigen for vaccine development to protect against post-weaning diarrhea.
" Determination of virulence significance of EAST1 toxin leads to better understand of ETEC pathogenesis in diarrhea disease, and provides important information for effective vaccine development against this pathogen.
" Determination of the role played by flagella in F18+ ETEC strain adherence improves our understanding ETEC pathogenesis, and guides us to develop more effective vaccine against F18+ ETEC associated diarrhea and edema disease in young pigs.
" Our study addressing the effects of glucose on adherence of porcine-origin enterotoxigenic E. coli (ETEC) to porcine jejunal epithelium (IPEC-J2 cells) is the first to demonstrate that glucose (at a specific concentration) in the culture medium, through its effects on LT production, significantly affects bacterial adherence, lending support to the hypothesis that the composition of dietary nutrients in the intestine of the host may directly influence the severity of ETEC infection.
" We have identified compounds that have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster, which includes picornaviruses, caliciviruses and, coronaviruses, by targeting 3Cpro or 3CLpro.
" Studies of HuNoV transmission, inactivation and screening for anti-viral drugs can be performed first in vitro using validated calicivirus surrogates to obtain preliminary data prior to in vivo human volunteer studies for final evaluation.
" An understanding of the mechanism of NoV contamination and the calicivirus mode of transmission in vegetables will enhance efforts for enhancing food safety and decreasing infections in consumers, thereby promoting public health.
" Continuous monitoring of NoVs and SaVs in swine is crucial since porcine NoVs are most closely related to HuNoVs and there is a zoonotic potential for swine NoVs or for transmission of HuNoVs to pigs. Our results will help to improve public health and swine health.
" The prophylaxis or treatment of rotavirus diarrheal disease by nutritional intervention, through the use of easily deliverable receptor mimetics and nutriceuticals, is potentially of great significance for the control of this disease in both agricultural and human medicine arenas.
" Our results suggest a crucial role of lipid metabolism in rotavirus replication, and triacsin C and/or its analogs as potential therapeutic options for rotavirus infections.
" Studies looking at the impact of probiotic colonization and maternal col/milk feeding on RV vaccine efficacy neonatal Gn pig model that mimics breast fed children will provide crucial information for the development of optimal oral vaccination strategies and use of targeted probiotics in breast-fed and formula-fed infants to efficiently protect them against enteric pathogens.
" Use of inexpensive vitamin A as a supplement in children and their mothers may improve mucosal immune responses and oral vaccine efficacy to enhance the overall health of children with micronutrient deficiencies that are wide-spread in developing countries. This strategy may be applicable to rotavirus and also other important mucosal infections in neonatal humans and animals.
" The effectiveness of future RV vaccines may differ depending on the predominant genogroups (A, B, C) and genotypes circulating; hence, the most common RV genogroups and genotypes should be the prime targets for vaccine development.
" Identification of several previously uncharacterized genes whose expression is upregulated in high-pathogenic S. Enteritidis strains or genes that play a role in intestinal invasion and/or systemic dissemination has opened up opportunities to study their roles in infection and egg contamination and investigate their potential usefulness as targets for development of newer vaccines and therapeutics.
" Natural products (e.g. L-UFFA), that interfere with parasite-host cell interactions required for infection could be utilized as nutriceutical feed additives to inhibit parasite infectivity and thus prevent or reduce cryptosporidium load environmental contamination.
" Emergence of bacterial antibiotic resistance has become a serious problem worldwide. Our studies may open new avenues for treatment and prevention of resistant foodborne pathogens important in animal health and food safety.
" The detection of the recent emergence of CTX-M E. coli in northwest cattle populations presents a unique opportunity to determine causes of widespread changes in bacterial resistance.
" If plasmid types are stably associated with CTX-M genes we should be able to determine whether the bacterial host or the plasmid is the epidemiological unit of importance in the dissemination of resistance.
" Veterinary teaching hospitals report outbreaks of antibiotic resistant pathogens but to date there are no published models to explain or predict occurrence of transmission.
" The Master of Science (M.S.) degree as well as graduate certificate in International Infectious Disease Management and Biosecurity (2008-2011) program at NDSU provides further opportunities for expanding student careers and joint research & educational opportunities for students and faculty from the US and East Africa.

Date of Annual Report: 02/12/2014

Report Information

Annual Meeting Dates: 12/07/2013 - 12/08/2013
Period the Report Covers: 10/01/2012 - 09/01/2013

Participants

" Bert Stromberg, University of Minnesota
" Qijing Zhang, Iowa State University
" Phil Hardwidge, Kansas State University
" David Francis, South Dakota State University
" Amin Fadl, University of Wisconsin-Madison
" Jun Lin, University of Tennessee
" Weiping Zhang, South Dakota State University
" Mo Saif, The Ohio State University
" Gireesh Rajashekara, The Ohio State University
" Alexandra Armstrong , University of Arizona
" Rodney Moxley, University of Nebraska-Lincoln
" Margaret Khaitsa, North Dakota State University
" Devendra Shah, Washington State University
" Richard Isaacson, University of Minnesota
" Dave Benfield, The Ohio State University
" David Renter, Kansas State University
" T.G. Nagaraja, Kansas State University
" Linda Mansfield, Michigan State University
" K. J. Yoon, Iowa State University
" Dick Hesse, Kansas State University
" Qiuhong Wang, The Ohio State University
" Douglas Marthaler, University of Minnesota
" Connie Gebhart, University of Minnesota
" Bledar Bisha, University of Wyoming
" Bibiana Law, University of Arizona
" John David Kabasa, Makerere University, Uganda

Brief Summary of Minutes

Brief Summary of Minutes of Annual Meeting

1. Qijing Zhang, Chair of NC-1202, gave an update on the overall goals of the NC1202 group. The proposal is accessible on the web site for NC-1202. Anybody interested in the proposal can print out a copy directly from the web page.
2. Administrative Advisor, Dr. Stromberg, made a presentation on overview of the expectation for the groups and submitting annual report and encouraged new memberships. Also emphasized the importance of how our work impacts industry.
3. Appendix E uploading. Members of NC-1202 are required to submit the Appendix E form. This must be done by Jan. 15, 2014 via the NIMSS system. Members dont have to be state representatives. Investigators from medical schools are allowed and encouraged to become member of NC-1202.
4. Drs. Qijing Zhang and Gireesh Rajashekara are the Chair and Chair-Elect, respectively, at the 2013 annual meeting. The term will be for 2 years. Jun Lin will be the secretary.
5. New members were introduced.
6. Student awards. NC-1202 offers awards for students to compete in the Pathobiology of Enteric and Foodborne Pathogens Section of CRWAD. This year three awards were offered, two for oral presentation and one for poster presentation. Dr. Radhey Kaushik serves as Chair for the student award selection committee. He will continue to serve as Chair for the next year. Selection of a co-chair working with him was suggested. Historically, the student awards were funded by annual registration fee. How to increase the number of abstracts submitted to the Pathobiology of Enteric and Foodborne Pathogens Section was discussed. One suggestion was to offer student travel awards or increase the dollar amount of the awards. This requires industry sponsors and contact with industries for possible support of NC-1202 activities was proposed. All the members were encourage bring their students to meeting.
7. Dr. Rod Moxley discussed with CRWAD executive committee to finalize the new name Pathobiology of Enteric and Foodborne Pathogens for the Gastroenteric Diseases section in the future CRWAD meetings following 2012 annual meeting. The rationale for the change is that many members of NC-1202 work with zoonosis and food safety and this change will attract more submission of abstracts to the section. The new name was approved for the 2013 meeting and for the future meeting.
8. Annual report. Annual station reports were due to Qijing Zhang and those who have not submitted are asked to do so. Bert Stromberg emphasized the importance of outcomes and impact. These should be clearly stated in the report.
9. Dr. Rod Moxley suggested that the station reports not be sent to everyone as it has lot of un-published data. Maybe using two e-mail server lists would be better, one to send out the station report to the chair and the secretary of the NC1202 group and other one for general communication. Also, it was encouraged that the station representative would communicate with other researchers on the contents of the station reports if necessary.
10. Dr. Radhey Koushik, indicated that no official representative from South Dakota has been nominated since Dr. Francis retired. It was suggested that the administrative advisor would contact the South Dakota station director regarding appointing a new member. NC1202 group also suggested to encourage other stations (For example, Indiana, Purdue, Missouri, North Carolina, California etc.,) to join the group
11. There was discussion on bringing together the researchers working on microbiome of livestock. Dr. Richard Isaacson is trying to co-ordinate this and possibly draft a proposal to facilitate getting some funding from USDA or other federal sources. He also suggested that we organize an Animal Microbiome mini symposium and he will take the lead to co-ordinate and organize the meeting.
12. There was discussion on including the PEDV research into this group as it appropriately fits in the enteric diseases category which is a main focus of this group. All the station representatives were encouraged to ask their colleagues working on PEDV to participate in the NC1202 meeting and also include the PEDV work in their station report.

Accomplishments

Objective 1. Focus on emerging issue- identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine A. Campylobacter jejuni Michigan Emerging Campylobacter Species: Multiple molecular typing schemes applied to define genetic relationships among C. jejuni isolates that share the same LOS classification. Data demonstrated that C. jejuni populations associated with enteritis are highly diverse based on several genotyping systems. Furthermore, several clonal complexes had associations with particular LOS classes. C. jejuni isolates from calves have A, B, and C LOS biosynthetic locus classes similar to human GBS associated strains, thereby providing direct support for zoonotic transmission. Iowa Campylobacter-associated ruminant abortion: 42 C. jejuni isolates representing the disease occurred in the U.K. during 2002-2008 were examined using MLST, PFGE and array-based CGH. In contrast to the late U.S. isolates, a high genetic diversity was revealed among the sheep abortion isolates within the U.K. Clone, and there was only minimal overlap among other genotypes between the two countries. C. jejuni isolates from the early-U.S abortions (pre-2000) somewhat resembled the U.K. population. Aclinical isolate (IA3902) of clone SA is most closely related to non-abortifacient reference strain C. jejuni NCTC11168. Notably, the variable genes in the capsular polysaccharides biosynthesis and O-linked glycosylation loci of IA3902 are highly homogenous to their counterparts in C. jejuni subsp. doylei and C. jejuni G1, which are known to be frequently associated with bacteremia. Transcriptomic and proteomic profiles revealed that the pathways of energy generation, motility, and serine utilization were significantly up-regulated in IA3902, whereas the pathways of iron uptake and proline, glutamate, aspartate, and lactate utilization were significantly down-regulated. Immunoproteomic analyses identified 26 immunogenic proteins, some of which were previously shown to induce protective immunity. Washington State Campylobacteriosis epidemiology and molecular epidemiology: In Washington State, the two counties with the highest concentrations of dairy cattle also report the highest incidences of campylobacteriosis. Conditional logistic regression analysis of case-control data from both counties found living or working on a dairy farm and Hispanic ethnicity to have the strongest positive associations with campylobacteriosis. Sequence types found commonly in human isolates were also commonly found in bovine isolates suggesting that in areas with high concentrations of dairy cattle, exposure to dairy cattle may be an important risk for campylobacteriosis. Ohio A Longitudinal Study on Campylobacter spp. in Commercial Turkey Flocks. Eight hundred and ten samples were collected from birds aged from one-week to slaughter. Overall Campylobacter prevalence was 55.8%. flaA-RFLP and MLST subtyping detected isolates mostly constituting Farm homogenous groups. C. coli isolates displayed greater resistance than C. jejuni to most antimicrobials. Pathogenesis of C. jejuni Induced Abortion in Pregnant Ewes. C. jejuni clone SA from both ovine and bovine abortion cases were evaluated for their ability to cause abortion and associated symptoms using a pregnant ewe model, a natural host. Our results data indicate that abortion-associated C. jejuni induce abortion or stillbirth in sheep and down- or up-regulation of specific genes in the uterus of infected host might be implicated in the disease progression. B. Salmonella Washington State Development of a multiplex qPCR for the serotype-specific detection of S.Enteritidis: We optimized TaqMan assay targeting genes invA, sdfI and prot6E. The assay was 100% specific, distinguishing from non-S. Enteritidis strains and discriminating plasmid from non-plasmid bearing strains. Detection limit of the assay was as little as 1 CFU in clean drag-swab samples. In environmental drag swabs, the assay displayed 100% sensitivity, specificity, accuracy, positive predictive value and negative predictive value. Kansas Prevalence of Salmonella in feces and subiliac lymph nodes of commercial feedlot cattle. The prevalence and concentration of Salmonella in feedlot cattle feces, as well as within the peripheral lymph nodes of corresponding beef carcasses, were high and may represent a significant food safety risk. C. Shiga toxin-producing E. coli (STEC) Nebraska Development of ELISAs and monoclonal antibodies against O antigens of adulterant strains of non-O157 of STEC; O26, O45, O103, O111, O121. Preliminary ELISAs using these antigens were developed and will be used to screen for anti-O antigen specific MAb. Evaluation of agar plating media for isolation of FSIS-adulterant STEC. Our results indicated that: (1) none are specific for these organisms; (2) mPossé yields a less subjective and less complicated colony phenotype compared to mRainbow and USMARC, (3) mRainbow is less selective than either USMARC or mPossé, (4) USMARC allowed for the greatest growth of control organisms with STEC-like phenotype and STEC organisms; (5) lack of specificity is a concern with each of these media, and (6) crowding on plates prevented accurate detection of colony phenotypes with all three media. Prevalence of FSIS-adulterant STEC in hide sponge samples from beef cattle at harvest. A total of 576 hide samples were subjected to enrichment culture, IMS, agar plating, multiplex PCR, and other tests. The prevalence distribution of targeted O-antigen genes in the samples was O26, 26%; O45, 6%; O103, 41%; O111, 3%; O121, 3%; O145, 2%, and O157, 41%. A total of 884 isolates of the targeted O-groups were detected, and distributed as follows: O26, 10.4%; O45, 2.4%; O103, 40.7%; O111, 1.4%; O121, 0.6%; O145, 0.6%; O157, 44.0%. Only 39 of these 884 isolates had one or more of the four virulence factors used to screen for pathogenicity (stx1, stx2, eae, ehxA), and only 8 (0.9%) were adulterants based on the FSIS definition. Kansas Prevalence of Shiga toxin-producing STEC serogroups and associated virulence genes (stx1, stx2, eae and ehxA) in feces of commercial feedlot cattle. Our results indicate that STEC serogroups (O26, O45, O103, O121, O145 and O157) of potential public health importance were identified in feces from this commercial feedlot cattle population with O103, O157 and O26 being the most prevalent serogroups. Prevalence of STEC-8 in muscoid flies in the confined cattle environment. House flies (HF, n=180) and stable flies (SF, n=180) were collected on a weekly in cattle feedlot in Nebraska and Kansas and screened for STEC-8. The concentration of enterics per SF was significantly lower than that of HF. The most common serotype was O104 with a prevalence of 10%, followed by O103 (5%), O45 (1.7%), O121 (1.1%) and O26 and O157 (0.5% each). Interestingly, three HF were shown to carry multiple serogroups while none of the flies were positive for O145 and O111. Real-time PCR to detect and quantify STEC-7 in cattle feces. Our data suggest that multiplex real-time PCR and conventional-PCR are most useful in identifying high-shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces. A four plex qPCR assay for the quantification of E. coli O157 in cattle feces based on serogroup specific O157 antigen (rfbE O157), Shiga toxins 1 and 2 (stx1 and stx2) and intimin (eae). The detection limit of the mqPCR assay for E. coli O157 with DNA extracted directly from cattle feces was 7.8x104 CFU/g. However, after six-hour enrichment, sensitivity increased to 3.3x100 CFU/g. The assay targeting the four genes has the potential to be a high-throughput method for detecting and quantifying E. coli O157 in cattle feces. Development of multiplex qPCR assay for the quantification of the six major non-O157 STEC (O26, O103, O111, O45, O121 and O145) in cattle feces. Primers were designed targeting the O-antigen genes. The assays were specific for all the target genes. The detection limits of the assays were 103 CFU/ml, 104 CFU/g, and 102 CFU/g for pooled pure cultures, before and after enrichment of spiked fecal samples, respectively. Washington State Over 600 isolates were genotyped using Shiga toxin encoding bacteriophage insertion sites (SBI), lineage specific polymorphisms (LSPA-6), multi-locus VNTR analysis and the tir 255T>A polymorphism. The results clearly showed phylo-geographic structuring of this bacterium, and divergent evolution of this serotype on these US and Australia continents. As a follow-up, we are conducting a comparative study involving those two countries plus Argentina, using SNP and SBI genotyping. Preliminary results further support for phylo-geographic structuring, and in addition, strong correlation between the existence of strains carrying the Stx2a-encoding bacteriophage and the incidence of human O157-related disease. We participated in a second collaborative study of STEC in New Zealand, a case-control study to identify sources of human infection in that country. Results of which 1) implicated environmental and animal contact as the major source of human infection (rather than foods), and 2) showed phylo-geographical structuring of the pathogen between the North and South Islands (PMID: 24079470). We have developed a 48-plex chromosomal backbone SNP allele assay for STEC to efficiently type STEC isolates into the eight lineages defined by Bono et al., as well as 4 sub-lineages. Using the SNP assay, we have genotyped over 2000 isolates of known sources, with the goal of evaluating seasons, production systems, hosts of origin, interventions, etc., for effects on clinical and bovine-biased genotypes. A previously observed effect, >90% of feedlot cattle isolates, was clarified to be the result of a single over-represented feedlot. We conducted a switch-back cattle challenge trial to test whether grain diets selected for clinical vs bovine-biased genotypes of O157. The results did not show any effect of grain vs forage rations on the shedding of clinical genotypes of O157, but did show that grain rations were associated with lower shedding and more rapid clearance of O157 infection than were forage-based rations. We completed an experimental challenge study on seasonal variation of O157 shedding by cattle. Our results indicate that seasonal variation is principally due to seasonal variation in oral exposure of cattle to this pathogen. We are also sampling pens of dairy and feedlot cattle just prior to and just after introduction of the new forage crops. Trials to date have shown sometimes dramatic increases in O157 shedding following these introductions. North Dakota A study investigated prevalence of STEC in (feces and hides) beef cattle in North Dakota at different production stages from post-weaning to slaughter. Over all 15.6% of adult cattle and 8.75% of steer calves shed STEC O157:H7; over the study period (September 2008 to May 2009). We investigated occurrence of STEC and Salmonella in Feedlot runoff from 2 feedlots in Fargo, and Cogswell, North Dakota. Salmonella spp. prevalence was 81.1 %. Twenty of the 37 samples tested positive with at least one of the (STEC) serotypes. Five of the 37 samples were positive for O157:H7. For the non- O 157 isolates; O103 37.8%, O121 32.4%, 0111 18.9%, O26 13.5%, O113 5.4%, and O145 8.1%). D. Brachyspira hampsonii Minnesota Molecular characterization and epidemiology of Brachyspira hyodysenteriae in U.S. swine herds. Fifty-eight B.hyodysenteriae isolates from nine states across the U.S., were genotyped and analyzed on three levels (intra-site, intra-system and international) using MLST based on seven housekeeping genes. This is the first study to characterize the strains of B.hyodysenteriae currently circulating in swine herds across the U.S. and to elucidate their diversity, distribution and epidemiology. E. Caliciviruses Ohio Pathogenesis of GIII.2 bovine norovirus, CV186-OH/00/US strain in gnotobiotic calves. We demonstrated persisting diarrhea and prolonged fecal shedding, but with a lack of significant intestinal lesions in gnotobiotic calves infected with GIII.2 BoNoV, CV186-OH/00/US strain. Most infected calves exhibited two clinical signs: i) acute but persisting diarrhea; and ii) acute moderate to severe lethargy. The prolonged fecal shedding of GIII.2 BoNoV might partially explain how this virus is maintained as endemic infections in cattle. Prevalence and Molecular Characterization of sapoviruses (SaVs) and noroviruses (NoVs) in pigs. Fecal samples (n=139) collected on pig farms in Ohio were screened for caliciviruses by RT-PCR. No NoVs were detected from nursing piglets. Different SaV genogroups (G), including a newly emerging genotype within GVII (DO19 Korea-like), were detected in 10.1% of piglets. The SaV prevalence was 0-36.7% at farm level at one time point. To our knowledge, this is the first report of porcine DO19 Korea-like SaVs in the US. We tested 413 pooled fecal samples from healthy finisher pigs in North Carolina swine farms collected in 2009. RT-PCR assays revealed a PoNoV prevalence of 18.9%. All NoVs belonged to typical PoNoV genotypes and no human-like NoVs were detected from these pigs. Porcine NoV-negative samples (n=343) were subsequently screened using universal calicivirus primers, and 17 SaV strains were confirmed by sequencing. Detection of Porcine Kobuviruses in US Swine. Kobuvirus, an emerging member of the Picornaviridae family, was detected by primers designed for SaV for the first time from Ohio nursing piglets. F. Rotavirus Minnesota Detection of RVC in porcine samples including lung tissue. Group C rotavirus (RVC) was detected in 4,590 samples tested between December 2009 and October 2011. These porcine intestinal tissues samples also had RV-like lesions. A majority of single RVC infections were found in d3 day old piglets. RV analyses of 635 porcine lung tissues revealed RVC (10%). Based on VP7 gene segment sequencing, 65 RVC samples belonged to 9 G genotypes. Identification of swine RVB in the United States. RVB was detected in 81 of 173 samples tested. Nine RVB positive samples were negative for all other known pathogens. The majority of RVB samples contained a mixed infection with RVA and/or RVC. Interestingly, in the <21 day age group, RVC prevalence was higher than the prevalence of RVA. Swine RVA/RVB/RVC co-infections were higher than expected. Phylogenetic analysis revealed 20 genotypes. G. Porcine Epizootic Diarrhea Virus (PEDV) Minnesota Complete genome sequencing of the first PEDV strain from the United States. PEDV is emerged in the United States in April 2013. A PEDV strain (CO/13) was obtained from a 7 day-old piglet, and Next Generation Sequencing characterized the complete genome. The complete PEDV genome of CO/13 had the highest nucleotide percent identity (99.5%) to Chinese strain AH2012 Ohio Detection of porcine epidemic diarrhea virus (PEDV) in US swine, pathogenesis in gnotobiotic (Gn) pigs and isolation in cell culture. Gnotobiotic pigs exhibited acute severe diarrhea/vomiting post-inoculation at 24-48 hr, followed by dehydration and collapse. Pathologic lesions included severe atrophic enteritis with vacuolation of superficial epithelial cells in cecum and colon. Our data suggest that the PC21A strain of PEDV is highly enteropathogenic and acutely affects the entire intestine, although principally the jejunum and ileum, leading to severe atrophic enteritis and death. We have screened 30 field pig fecal samples from OH and collaborating NC 1202 states (MN, KS). To, only 1 OH strain has been confirmed to serially replicate in Vero cells. H. Antimicrobial Resistance Washington State CTX-M families of resistance gene determinants: We visited 30 Washington State Dairy farms and found high prevalence of cefotaxime, cefepime resistant E. coli on 28/30 farms. Cefepime resistance was highly correlated with the presence of blaCTX-M. Risk factors for high prevalence of blaCTX-M-bearing E. coli included recent animal movements on the farm, region of Washington, use of residual fly sprays, direction of feeding calves, frequency of adding new bedding to calf and use of florfenicol on calves. Any ceftiofur use in calves was not associated with a higher risk. Development of a genotyping method for E. coli similar to multilocus sequence: In order to evaluate the less costly and time-consuming 2-locus method on a set of field cattle E. coli isolates, we chose 75 blaCTX-M-bearing E. coli from our study and typed them using the established 7-locus method and compared those results to the 2-locus method. Our analysis showed the 2-locus genotyping method may be of use epidemiologically in the case of limited resources. Tennessee Regulation of beta-lactam resistance in C. jejuni. We have demonstrated that Cj0843c, a putative lytic transglycosylase involved in cell wall metabolism, was required for induction of ²-lactamase-mediated ²-lactam resistance in Campylobacter. The purified Cj0843c has been successfully crystalized for structural analysis and subsequent molecular docking to identify specific inhibitors. In addition, we observed a single nucleotide mutation (G?T transversion) in the upstream of Cj0299 (a ²-lactamase gene) plays a critical role in acquired ²-lactam resistance. Development of novel alternatives to antibiotic growth promoters for enhanced animal health and food safety. Bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. We also successfully completed a pilot HTS and identified several BSH inhibitors with potential as alternatives to AGP. Iowa PNAs function as antisense agents by binding specifically to complementary sequences in DNA and RNA and inhibiting gene expression and/or translation. PNAs targeting the three components of CmeABC were evaluated for inhibition of CmeABC expression. Compared with the individual PNAs, the combination of CmeA and CmeB PNAs produced stronger inhibition of CmeABC expression. Both CmeA and CmeB PNAs, individually or in combination, reduced the MICs for wild-type and antibiotic-resistant Campylobacter strains significantly. We determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. The arsB and acr3 are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. Objective 2. Focus on effective intervention- develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and foodborne disease agents. A. Campylobacter jejuni Michigan Characterize definitively the neurological signs and disease lesions associated with GBS and MFS in murine models using single blind methods. Mice were gavaged orally with C. jejuni strains HB93-13 and 260.94 from patients with GBS and assessed for clinical neurological signs, anti-ganglioside antibodies, cellular infiltrates, and lesions in peripheral nervous tissue. Our results strongly supported that NOD WT mice are the best mouse model of natural onset GBS. Determine whether innate responses and adaptive responses mediate GBS and MFS in murine models We demonstrate that C57BL/6 IL-10-/- mice infected with a C. jejuni colitogenic human isolate had significantly upregulated Type1 and 17 but not Type2 cytokines in the colon coincident with infiltration of phagocytes, T cells and Innate Lymphoid Cells (ILCs). In contrast, C. jejuni GBS patient strains induced mild colitis associated with blunted Type 1/17 but enhanced Type 2 responses. Moreover, Type2 but not Type1/17 antibodies cross-reacted with peripheral nerve gangliosides demonstrating autoimmunity. Determine effects of Humicrobiota on murine host responses in the presence and absence of three pathotypes of Campylobacter jejuni. C57BL/6 mice with human microbiota had significant elevations in autoimmune responses after infection with C. jejuni compared to congenic infected mice with mouse microbiota. Thus, the humicrobiota, but not the Momicrobiota conferred a GBS immune response mediated by a Th2 dependent mechanism after infection with an enteric C. jejuni strain. This suggests that the pathogenesis of GBS includes factors mediated by the microbiome. Ohio Transducer Like Proteins of C. jejuni mediate substrate specific chemotaxis and virulence. Chemotaxis assays revealed novel ligands for some of the Tlps. Chemotaxis towards aspartate was affected in tlp2, 6 and 10. The tlp3 displayed a decreased chemotaxis towards TCA cycle intermediates like pyruvate and tlp6 towards isocitrate and succinate. The tlp2 deletion mutant was defective in chemotaxis towards inorganic phosphate and ferrous sulfate. These findings reveal that more than one Tlp is responsible for sensing a nutrient in C. jejuni. In vitro virulence studies revealed that tlp8 and 9 had a significant defect in both invasion and intracellular survival in INT 407 cells; however, only tlp10 mutant was defective in colonization of the chicken cecum. Functional Characterization of Exopolyphosphatase/ Guanosine Pentaphosphate Phosphohydrolase (PPX/GPPA) Enzymes of C. jejuni. ppx mutants exhibited increased capacity to accumulate poly-P, however only ppx1 and dkppx mutants showed decreased accumulation of ppGpp. The lack of ppx genes resulted in defects in motility, biofilm formation, nutrient stress survival, invasion and intracellular survival indicating a critical role for ppx genes in C. jejuni pathophysiology. Both ppx1 and ppx2 mutants were resistant to human complement-mediated killing; however, the dkppx mutant was sensitive. Interestingly, the chicken serum did not have any effect on the ppx mutants survival. Arizona C. jejuni vaccines. Recombinant attenuated Salmonella vaccines (RASV) have been adapted to stably express protective antigens at high levels. The CjLAJ3 protein was found not to be effective as a vaccine candidate in reducing the numbers of C. jejuni in chickens. Chickens were vaccinated with dual doses (1ml each at ~1x1010 CFU) of vaccines expressing CjLAJ1 and CjLAJ2 and challenged with the homologous strain C. jejuni NCTC 11168. Results differed for the two groups, with one vaccinate group demonstrating a near total 7-log reduction and the other vaccinate group demonstrating a modest 1-log reduction of C. jejuni compared to the positive controls. Vaccination followed by heterologous challenge showed C. jejuni colonization reduction was not significant, likely due to variable doses for challenge strains. Three additional genes, Cj1475c, Cj0113 and Cj1534c, have been cloned into the Salmonella vaccine strain and will be tested in triplicate. Tennessee Ferric enterobactin acquisition systems in Campylobacter. We have characterized two FeEnt receptors (CfrA and CfrB) in Campylobacter. Recently, we identified and characterized a novel periplasmic trilactone esterase (Cee), and revealed specific TonB-ExbB-ExbD energy transduction systems required for ferric enterobactin acquisition in Campylobacter. We also firmly established that Campylobacter could utilize high-affinity salmochelin by using FeEnt acquisition system. Development and evaluation of Campylobacter vaccine. The live Salmonella-vectored vaccines did not trigger potent immune response and confer protection against Campylobacter in broilers. The conditions for preparing chitosan encapsulated CfrA- and CmeC-based mucosal vaccines have been optimized. To construct DNA vaccine for in ovo and intranasal immunization, the cmeC and cfrA genes were cloned into the eukaryotic expression vector, pCAGGS, with significant modifications. Specifically, a eukaryotic ribosomal binding site sequence was attached immediately upstream of the start codon for enhanced translation. Novel prebiotics and probiotics to control Campylobacter and Salmonella in Pre-harvest poultry. Experiments were conducted evaluating the efficacy of several probiotic bacterial strains (Bacillus, Lactobacillus, and Pedioccocus) and three feed additives (Grape seed extract, thymol and yerba mate) to reduce Salmonella and Camylobacter carriage in the poultry gastrointestinal tract. The probiotics and grape extracts were found to be effective against vertical transmission of Salmonella. None of the probiotics and feed additives reduced Campylobacter colonization. The combination did not enhance the efficacy of either treatment alone. B. STEC and ETEC Nebraska Adhesion to and invasion of bovine and human colonic epithelial cells by non-O157 STEC. Non-O157 STEC strains of varying intimin and flagellar composition were compared for their ability to adhere, cause attaching-effacing (A/E) lesions, and invade bovine and human colonic mucosal epithelial cells. Our studies suggest that most non-O157 STEC adulterant strains, and potentially STEC O104:H4, have the capacity to colonize bovine and human intestinal epithelium. Bioinformatics analysis of type II secretion system (T2SS ) genes of porcine-origin ETEC. The T2SS of 2534-86 and 3030-2 shared high amino acid identity with H10407, and low amino acid identity with TRH7000 even though they both had similar nucleotide identity with H10407 and V. cholerae. Compared to this, the WT LT non-producer G58-1 shared similar levels of nucleotide identity with H10407 and TRH7000 while having a lower level of amino acid identity of with H10407 and TRH7000, respectively. Effects of glucose on expression of LT and STb gene in porcine ETEC. Exposure of the bacteria to glucose at certain concentrations in the media results in maximal production of LT and significantly decreased production of STb. We constructed luminescence fusions to evaluate expression of of LT (eltAB) or STb (estB) of porcine ETEC strain 2534-86. Our results supported that estB is subject to catabolite repression, but did not support the hypothesis that eltAB is subject to catabolite de-repression. Kansas Escherichia coli O26 in feedlot cattle. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle that were enrolled in a study to evaluate an STEC siderophore receptor and porin (SRP®) vaccine (VAC) and a direct-fed microbial (DFM; L. acidophilus and P. freudenreichii). The overall prevalence E. coli O26 was higher by the culture-based method compared to the PCR assay. The interventions (VAC and or DFM) had no impact on fecal shedding of O26. The majority of the O26 recovered from feedlot cattle feces was atypical EPEC and not STEC. Modulation of NF-ºB-dependent responses is critical to the success of attaching/effacing (A/E) human EPEC and EHEC. Our data identified GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by A/E pathogens to inhibit NF-ºB-dependent host innate immune responses. Type III secretion system (T3SS) effector proteins. The NleF, contributes to E. coli and C. rodentium colonization of piglets and mice, respectively. Using a yeast two-hybrid screen, we identified Tmp21, a type-I integral membrane protein and COPI-vesicle receptor involved in trans-Golgi network function, as an NleF-binding partner. Effects of T3SS effector proteins on the innate immune function. We screened ~9,000 human proteins to identify NleH1 kinase substrates and identified the v-crk sarcoma virus CT10 oncogene-like protein (CRKL), a substrate of the BCR/ABL kinase. Knockdown of CRKL abundance prevented NleH1 from inhibiting RPS3 nuclear translocation and NF-ºB activity. We propose that the CRKL interaction with IKKâ recruits NleH1 to the IKKâ complex where NleH1 then inhibits the RPS3/NF-êB pathway. The role of flagella in the pathogenesis of F4ac+ ETEC. Isogenic fliC, motA, and faeG mutants were construced. Both the fliC and faeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. However, there was no difference in adhesion between the motA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro. The effect of the Quorum Sensing (QS)I on the expression of virulence factors in STEC and VTEC. Recombinant E. coli transformed with yenI produced acyl-homoserine lactone synthase (AHL). However, the AI-1 positive recombinant F18ab E. coli exhibited impaired expression of flagella, decreased motility, reduced biofilm formation and AI-2 production, as well as attenuated adherence and invasion on IPEC-J2 cells. This study provides new insights to the crucial function of AI-1 in regulating STEC virulence. The role of flagella in the F18ab E. coli invasion process. Escherichia coli F18ab variant strains are associated with edema disease in pigs worldwide. Flagellated but non-motile bacteria invade piglet epithelial cells even more efficiently than the parent wild-type (WT) strain in vitro. By contrast, the non-flagellated bacteria have significantly reduced invasion as compared with the parent strain. C. Salmonella Wisconsin Examine the relative contribution of GidA and the MnmE in modulation of Salmonella virulence. Both in vitro and in vivo data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis. Our data showed the GidA and MnmE proteins are cytoplasmic proteins associated with the membrane of the Salmonella cell. Washington State Virulence characterization of S. Enteritidis mutants in orally inoculated day-old chickens: Four S. Enteritidis Tn5 mutants (SEN0034, fliH, SEN1393 and spvR) were indistinguishable from the isogenic wild-type strain when orally inoculated in one-day-old chickens whereas two mutants (CsgB and PegD) were defective for intestinal colonization and eight mutants (hilA, SEN3503, SEN0803, SEN2278, fljB, rfbM, rfbN and pipA) showed significant in vivo attenuation in more than one organ. Virulence and phenotypic characterization of dimethyl adenosine transferase (KsgA) mutant of S. Enteritidis: The ksgA mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens when compared to the wild-type. Phenotype Microarray showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur and peptide nitrogen nutrients in the ksgA at 42 C with no major differences observed at 28°C. Identification and characterization of the immunomodulatory activity of CpG motifs of Salmonella: We identified a total of 256 CpG motifs (6-mers) in the Salmonella pan-genome. Interestingly 4 CpG motifs induced production of IL-1² in avian macrophages, a marker for immune-stimulation, similar to LPS, suggesting that these CpG motifs can significantly stimulate innate responses in the chickens. Screening of the remaining 245 CpG motifs is currently underway. D. L. intracellularis and Brachyspira Minnesota Transcriptional profiling of pathogenic and non-pathogenic Lawsonia intracellularis isolates. A total of 401 genes were exclusively expressed by the pathogenic variant. Of the 319 genes which were commonly expressed in both pathogenic and non-pathogenic variants, no significant difference was observed by comparing their normalized transcription levels. Unexpectedly, these genes demonstrated a positive correlation (r2=0.81; p<0.05), indicating the involvement of gene silencing (switching off) mechanisms to attenuate virulence properties of the pathogenic variant during multiple cell passages. Experimental reproduction of brachyspiral colitis in pigs infected with B. hampsonii. This study evaluated the susceptibility of growing pigs to a new Brachyspira species, B.hampsonii (two different clades), compared with B. murdochii and B. hyodysenteriae infection. Our pig study demonstrated the experimental reproduction of brachyspiral colitis induced by a novel strongly hemolytic Brachyspira species (B. hampsonii). The course of disease and the pathological changes were similar between the two clades. However, the severity of the disease in animals infected with B. hyodysenteriae was lower than expected. Validation of a broth microdilution method and investigation of in vitro antimicrobial susceptibilities of porcine Brachyspira species. Using 40 isolates (10 B.hyodysenteriae, 10 B.hampsonii, 10 B.pilosicoli and 10 B.murdochii), the MICs for all antimicrobials obtained by the broth dilution method were generally comparable with those obtained by the agar dilution method. E. Calicivirus Ohio The effects of simvastatin or interferon-± on infectivity of human norovirus in the gnotobiotic pig model for the study of antivirals. This NC collaboration and studies of new NoV antivirals were done with Dr. K. Chang of KSU. Simvastatin induced significantly earlier onset and longer duration of fecal virus shedding in treated pigs. In contrast to the increased HuNoV shedding that simvastatin induced, curtailed viral shedding was observed in HuNoV-infected pigs pre-treated with recombinant human IFN-± (rhIFN-±). This finding indicates that IFN-± has potential as an antiviral against HuNoV. Kansas Structural and Inhibitor Studies of Norovirus 3C-like Proteases. We performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonanceenergy transfer assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murinenorovirus-1 (MNV-1, genogroup V) were compared for the inhibitory activities of a synthetic protease inhibitor (GC376). Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease. We investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Peptidyl compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system. F. Rotavirus Illinois In collaboration with Dr. Sharon Donovan, we also have demonstrated selected natural human milk oligosaccharides (HMO), the third most abundant component in human milk, reduce the duration of diarrhea in piglets, possibly in part by promoting immunoglobulin response to rotavirus infection and modulating the gut microbiota. Ohio Probiotics: effects on neonatal innate immune responses, immune homeostasis, RV infections and vaccine efficacy. Effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on attenuated (Att) HRV vaccination and virulent HRV (VirHRV) challenge were assessed in Gn pigs. Diarrhea and HRV shedding were significantly reduced in colonized pigs following VirHRV challenge. By enhancing Th1 responses to oral AttHRV vaccine, while inducing Treg responses to VirHRV, the selected probiotics were beneficial in improving responses to an enteric vaccine and alleviating the severity of an enteric infection in neonates. Both LA and LGG modulated common gut transcriptome responses related to host metabolism, mucosal integrity and immunity as well as responses unique to each strain. These findings imply that identification of probiotic strain specific responses could facilitate the design of probiotic-based interventions to moderate specific enteric conditions. Recombinant monovalent llama-derived nanoantibody fragments (VHH) as a passive treatment for rotavirus-induced diarrhea. Supplementation of the milk diet with 3B2 VHH clone nanoAbs for 9 days conferred full protection against RV diarrhea and significantly reduced virus shedding. Administration of comparable levels of porcine IgG Abs protected 4 of 6 piglets from HRV diarrhea, but significantly reduced virus shedding in all piglets. G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against HRV diarrhea or virus shedding when administered in similar quantities. Objective 3. Focus on disseminating knowledge Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories Arizona A Campylobacter symposium was organized by Randall Singers group (University of Minnesota) in conjunction with Charles Hofacre (University of Georgia) in furtherance of the outreach goals of the vaccine project. The symposium was offered in July 2013 at the annual AVMA/AAAP meeting in Chicago, Illinois. This symposium targeted veterinarians and researchers with interest in Campylobacter. The symposium featured talks ranging from Campylobacter ecology within the broiler environment to the human burden of illness associated with Campylobacter. Talks were given by globally recognnized experts in the field of Campylobacter research, including Drs. Robert Tauxe, Jaap Wagenaar, and Nigel French. The symposium was recorded in digital HD and will be available for distribution. Minnesota The Principle Investigators and Graduate Students involved in the project have given presentations and updates on both swine and equine enteric diseases at various scientific, veterinary, and diagnostic meetings in the previous year. These include the Conference of Research Workers in Animal Disease, the Leman Swine Conference, the Chinese Leman Swine Conference, the American Association of Veterinary Laboratory Diagnosticians, the American Association of Equine Practitioners, the American College of Veterinary Internal Medicine and the American Association of Swine Veterinarians. They have disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians (both swine and equine). Kansas Through several venues we have provided educational opportunities to professional and graduate students as well as continuing education to veterinarians on enteric pathogens in livestock production systems. The majority of the shared information has focused on the impacts of Shiga toxin-producing Escherichia coli and Salmonella in the beef industry, the conclusions that can be reached based on recent research, and the potential opportunities to reduce these pathogens in beef production systems. Michigan Dr. Mansfield organized a Fall seminar in enteric research. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Cathy Robinson, PhD, Ankit Malik, MS, PhD candidate, and Brian Nohomovich, DO, PhD candidate. Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health in Washington, D.C. on Enteric Diseases. Dr. Mansfield also assisted in organizing the Merial-NIH Veterinary Scholars Symposium: Comparative Medicine: Meeting Global Needs that was held in East Lansing, MI in August 2013 where she organized a session devoted to food borne pathogens. North Dakota North Dakota State University (NDSU) and Makerere University (MAK) in Kampala, Uganda offered the joint course International Animal Production, Disease Surveillance and Public Health again in summer 2013 with 9 student attendees 3 from US and 6 from MAK in East Africa. The three students from the US came from NDSU (1), Jamestown College (1) and University of Minnesota (1). Additionally, 6 students from MAK enrolled in the MS degree in International Infectious Disease Management attended the summer course. Also in July, 2013 at the end of the summer course, an International conference was held in Kampala, Uganda. The nine students who participated in the summer course attended the conference. Other students from MAK also attended and shared their experiences in One Health outreach activities that they had conducted in Uganda. Other attendees of the conference came from several universities in East Africa and US (University of Minnesota and Tufts University). Nebraska Knowledge pertinent to NC-2012 activities was disseminated to undergraduate students, graduate students, professional veterinary students, veterinarians, physicians, food processors, researchers, cattle producers and other decision makers regarding pre-harvest food safety of cattle food projects.

Publications

A. Publications " Jung, K., Scheuer, K., Zhang, Z., Wang, Q.H., Saif, L.J. Pathogenesis of GIII.2 bovine norovirus, CV186-OH/00/US strain in gnotobiotic calves. Veterinary Microbiology (In press). 2013. " Zufan Sisay, Qiuhong Wang, Tomoichiro Oka and Linda Saif. 2013. Prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in US Swine. Arch Virol 158:1583-8. " Kelly A. Scheuer, Tomoichiro Oka, Armando E. Hoet, Wondwossen A. Gebreyes, Bayleyegn Z. Molla, Linda J. Saif, and Qiuhong Wang. 2013. Prevalence of Porcine Noroviruses, Molecular Characterization of Emerging Porcine Sapoviruses from Finisher Swine in the United States, and Unified Classification Scheme for Sapoviruses. J Clin Microbiol. 51:2344-53. " Tomoichiro Oka, Linda J. Saif, Qiuhong Wang. 2013. Complete Genome Sequence of the first genogroup II genotype 18 porcine norovirus strain QW125. Genome Announcement 1: e00344-13. " Sayaka Takanashi, Linda J. Saif, John H. Hughes, Tea Meulia, Kwonil Jung, Kelly A. Scheuer, Qiuhong Wang. 2013. Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures. Arch Virol [published online Aug. 23, 2013]. " Chattha, K.S., Kandasamy, S., Vlasova, A.N., Saif, L.J. Vitamin A deficiency impairs adaptive B and T cell responses to a monovalent attenuated human rotavirus vaccine and virulent human rotavirus challenge in a gnotobiotic piglet model. 2013. Plos One (in press). " Vlasova, A.N., Chattha, K.S., Kandasamy, S., Rajashekara, G., Saif, L.J. 2013. Lactobacillus rhamnosus (GG) and Bifidobacterium lactis (Bb12) colonization promotes intestinal homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs; Plos One, 8(10):e76962. " Chattha, K.S., Vlasova, A.N., Kandasamy, S., Liu, Z., Rajashekara, G., Saif, L.J. 2013. Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model. J Immunol.; 191(5):2446-56. " Chattha KS, Vlasova AN, Kandasamy S, Esseili MA, Siegismund C, Rajashekara G, Saif LJ.. 2013. Probiotics and colostrum/milk differentially affect neonatal B cell responses to oral rotavirus vaccine. Vaccine. 2013 Apr 8;31(15):1916-23. PMID: 23453730 " Vlasova, A.N., Chattha, K.S., Siegismund, C.S., Kandasamy, S., Saif, L.J. 2013. Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in gnotobiotic pigs. J Immunol.; 190(9):4742-53. " Vega, C.G, Bok, M., Vlasova, A.N., Chattha, K.S., Gómez-Sebastián, S., Nuñez, C., Alvarado, C., Lasa, R., Escribano, J.M., Garaicoechea, L.L., Fernández, F.M., Wigdorovitz, A., Saif, L.J., Parreño, V.G. 2013. Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to rotavirus VP6 Protect Neonatal Gnotobiotic Piglets Against Human Rotavirus Induced Diarrhea. Plos Pathogens, 9(5): e1003334. " Kassem II, Chandrashekhar K, Rajashekara G. 2013. Of energy and survival incognito: A relationship between viable but non-culturable cells (VBNC) formation and inorganic polyphosphate and formate metabolism in Campylobacter jejuni. Frontiers in Microbiology. Vol 4, No. 00183, PMID:23847606; PMCID: PMC3705167 " Annamalai T, Pina-Mimbela R, Kumar A, Binjawadagi B, Liu Z, Renukaradhya GJ, Rajashekara G. 2013. Evaluation of nanoparticle encapsulated OMPs for the control of Campylobacter jejuni colonization in chickens. Poult Sci. Aug;92 (8):2201-11. PMID: 23873570 " Drozd M, Merrick NN, Sanad YM, Dick LK, Dick WA, Rajashekara G. 2013. Evaluating the occurrence of host-specific , general fecal indicators, and bacterial pathogens in a mixed-use watershed. J Environ Qual. May-Jun;42(3):713-25. PMID: 23673938 " Sanad YM, Closs G Jr, Kumar A, LeJeune JT, Rajashekara G.. 2013. Molecular Epidemiology and Public Health Relevance of Campylobacter Isolated from Dairy Cattle and European Starlings in Ohio, USA. Foodborne Pathogens and Disease. Mar;10(3):229-36. doi: 10.1089/fpd.2012.1293. Epub 2012 Dec 21. PMID: 23259503. Dec 21. " Wu Z, Sahin O, Shen Z, Liu P, Miller WG, and Zhang Q. 2013. Multi-omics Approaches to Deciphering a Hypervirulent Strain of Campylobacter jejuni. Genome Biol Evol. 5(11):2217-30. doi: 10.1093/gbe/evt172. PMID: 24201373. " Shen Z, Han J, Wang Y, Sahin O, and Zhang Q. 2013. The contribution of ArsB to arsenic resistance in Campylobacter jejuni. PLoS ONE. 8 (3):e58894. PMID: 23554953. " Oh E, Zhang Q, and Jeon B. 2013. Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni. J Antimicrob Chemother; doi:10.1093/jac/dkt381. PMID: 24084637. " Mu Y, Shen Z, Jeon B, Dai L, and Zhang Q. 2013. Synergistic effects of anti-CmeA and anti-CmeB peptide nucleic acids on sensitizing Campylobacter jejuni to antibiotics. Antimicrob Agents Chemother. 57:4575-7. PMID: 23817373. " Xia Q, Muraoka WT, Shen Z, Sahin O, Wang H, Wu Z, Liu P, and Zhang Q. 2013. Adaptive mechanisms of Campylobacter jejuni to erythromycin treatment. BMC Microbiol. 13:133. doi: 10.1186/1471-2180-13-133. PMID: 23767761. " Burrough, Eric, Samantha Terhorst, Orhan Sahin, Qijing Zhang. 2013. Prevalence of Campylobacter spp. relative to other enteric pathogens in grow-finish pigs with diarrhea. Anaerobe. pii: S1075-9964(13)00094-2. doi: 10.1016/j.anaerobe.2013.06.004. " Hao H, Yuan Z, Shen Z, Han J, Sahin O, Liu P, Zhang Q. 2013. Mutational and transcriptomic changes involved in the development of macrolide resistance in Campylobacter jejuni. Antimicrob Agents Chemother. ;57(3):1369-78. PMID: 23274667. " Qin, Shangshang; Wang, Yang; Zhang, Qijing; Zhang, Maojun; Deng, Fengru; Shen, Zhangqi; Wu, Congming; Wang, Shaolin; Zhang, Jianzhong; Shen, Jianzhong. 2013. Report of ribosomal RNA methylase gene erm(B) in multidrug resistant Campylobacter coli. J Antimicrob. Chemother. (In press). " John P. Jerome, Jeffrey E. Barrick, Hayhung Y. Kim, Brian D. Klahn, C. Titus Brown, Linda S. Mansfield. Reversible motility mutations and parallel Ã54 loss during experimental evolution of Campylobacter jejuni. In review at Molecular Biology and Evolution. " Jerome, J.P. and Mansfield, LS. 2013 Within-host evolution of Campylobacter jejuni, In "Campylobacter Ecology and Evolution", Sheppard, S.K. ed, Sanger Centre, England, pp. 1-25. " Samuelson D.R., Eucker T.P., Bell J.A., Dybas L.A., Mansfield L.S., Konkel M.E. 2013. The Campylobacter jejuni CiaD effector protein activates MAP 1 kinase signaling pathways and is required for the development of acute disease. Cell Communication and Signaling 2013, 11:79, doi:10.1186/1478-811X-11-79. " Malik A., Sharma D., St. Charles J.L., Dybas L.A., Mansfield L.S. 2013. Contrasting immune responses mediate Campylobacter jejuni induced colitis and autoimmunity, Nature Mucosal immunology, In press. " Zeng, X., F. Xu, and J. Lin. 2013. Specific TonB-ExbB-ExbD energy transduction systems required for ferric enterobactin acquisition in Campylobacter. FEMS Microbiology Letter. 347(1):83-91 " Zeng, X., F. Xu, Y. Mo, and J. Lin. 2013. Identification and characterization of a periplasmic trilactone esterase, Cee, revealed a unique pathway of ferric enterobactin acquisition in Campylobacter. Molecular Microbiology. 87(3): 594-608. " Díaz-Sánchez, S., S. Sánchez, S. Herrera-León, C. Porrero, J. Blanco, G. Dahbi, J. E. Blanco, A. Mora, R. Mateo, I, Hanning and D. Vidal. 2013. Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. in large game animals intended for consumption: relationship with management practices and livestock influence. Vet. Micro. 163(3-4):274-81. " Kumar, G., I. Hanning and M. Slavik. 2013. Influence of acid-adaptation on adhesion and invasion of INT 407 cells by Campylobacter jejuni. Foodborne Pathogens and Disease. ahead of print. doi:10.1089/fpd.2013.1544. " " Pendleton, S., D. Biswas, S.C. Ricke and I. Hanning. 2013. Evaluation of Whole Genome Sequencing as a Genotyping Tool for Campylobacter jejuni by Comparison with Pulsed-Field Gel Electrophoresis and flaA typing. Poultry Sci. 92:573-58 " Zeng, X., and J. Lin. 2013. Beta-lactamase induction and cell wall metabolism in Gram-negative bacteria. Frontiers in Antimicrobials, Resistance and Chemotherapy. (Review) Vol. 4. Article 128. doi:10.3389/fmicb.2013.00128 " Pu, Xiao-Ying, Qijing Zhang, Jing-Cao Pan, Zhangqi Shen, Wei Zhang. 2013. Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress. World J Microbiol Biotechnol. 29(2):365-71. " Liu, Yang, Yang Wang, Stefan Schwarz, Yun Li, Zhangqi Shen, Qijing Zhang, Congming Wu, and Jianzhong Shen. 2013. Transferable Multiresistance Plasmids Carrying cfr in Enterococcus spp. from Swine and Farm Environment. Antimicrob. Agents Chemother. 57(1):42-8. " Gehring, Barnett, DebRoy, D'Souza, Eaker, Fratamico, Gillespie, Hegde, Jones, J. Lin, Oliver, Paoli, Perera, Uknalis. 2013. A High-Throughput Antibody-Based Microarray Typing Platform. Sensors. 13:5737-5748. " Xu, F., C. Wu, and J. Lin. 2013. Microbial endocrinology: revealing the mechanisms of signaling language during the interactions between microorganism and host. (Review) Acta Microbiologica Sinica. 53(9):901-907. " Lin, J., A.A. Hunkapillar, A.C. Layton, Y. Chang, K.R. Robbins. 2013. Response of intestinal microbiota to antibiotic growth promoters in chickens. Foodborne Pathogens and Diseases. 10(4): 331-337. " Ricke, S. and I. Hanning. 2013. Food Safety Applications of Nanoparticles. In Nanotechnology Safety. 115-125. " Silfrany, RO., R.E. Caba, F. Solís de los Santos, and I. Hanning. 2013. Detection of Quinolones in Poultry Meat Obtained from Retail Centers in the Santiago Province, the Dominican Republic. Journal of Food Protection. 76:190-369 " Park SH, I. Hanning, W. Gilbert, M. Munro, L. Devareddy and S. Ricke. 2013. Feeding Mice Aged and Fresh Blackberries Powder Supplements Result in Shifts in the Gastrointestinal Microflora. Food Bioscience 1:66-72. " Diaz, S., S. Pendleton, I. Hanning, and D. DSouza. 2013. Next-Generation sequencing: applications for poultry production and food safety. Poultry Science. 92:562-572 " Park, SH., I. Hanning, A. Perrota, BJ Bench, E. Alm, and SC Ricke. 2013. Modifying the gastrointestinal ecology in alternatively raised poultry and the potential for molecular and metabolomic assessment. Poult Sci 92(2):546-61. " Hardy, B., N. Crilly, S. Pendleton, A. Andino, A. Wallis, N. Zhang, and I. Hanning. 2013. Impact of Rearing Conditions on the Microbiological Quality of Retail Poultry Meat. J Food Sci. 78:M1232-1235. " Andino, A., S. Pendleton, N. Zhang, W. Chen, F. Critzer, and I. Hanning. 2013. Survival and virulence of Salmonella spp. in poultry feed in strain and serovar dependent. Poult Sci. Accepted for publication " Gonzalez-Gil, F., S. Diaz-Sanchez, S. Pendleton, A. Andino, N. Zhang, C. Yard, N. Crilly, F. Harte, and I. Hanning. 2013. Yerba Mate Enhances Probiotic Bacteria Growth In Vitro but as a Feed Additive does not Reduce Salmonella Enteritidis Colonization In Vivo. Poult Sci. Accepted for publication. " Lin, J., K. S. Mateo, M. Zhao, A. K. Erickson, N. Garcia, D. He, R. A. Moxley, and D. H. Francis. 2013. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli. Vet. Microbiol. 162:731-739. " Berghaus RD, Thayer SG, Law BF, Mild RM, Hofacre CL, Singer RS. Enumeration of Salmonella and Campylobacter spp. in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks. Appl Environ Microbiol. 2013 Jul;79(13):4106-14. doi: 10.1128/AEM.00836-13. Epub 2013 Apr 26. " Armstrong, A, Curtiss R, Roland K, Law B. Use of a Recombinant Attenuated Salmonella Typhimurium Vector Vaccine for the Reduction of Campylobacter jejuni in Broiler Chickens. In Abstracts of posters and oral presentations from the 17th International Workshop on Campylobacter, Helicobacter and Related Organisms,15-19th September 2013. University of Aberdeen, 2013. J Med Microbiol. " Chiok KL, Addwebi T, Guard J, Shah DH. Dimethyl adenosine transferase (KsgA)deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens. Appl Environ Microbiol. 2013 Oct 11. [Epub ahead of print] PubMed PMID: 24123731. " Crespo R, Garner MM, Hopkins SG, Shah DH. Outbreak of Listeria monocytogenes in an urban poultry flock. BMC Vet Res. 2013 Oct 11;9(1):204. [Epub ahead of print] PubMed PMID: 24119838. " Al-Adwani SR, Crespo R, Shah DH. Production and evaluation of chicken egg-yolk-derived antibodies against Campylobacter jejuni colonization-associated proteins. Foodborne Pathog Dis. 2013 Jul;10(7):624-31. " Campioni F, Davis M, Medeiros MI, Falcão JP, Shah DH. MLVA typing reveals higher genetic homogeneity among S. Enteritidis strains isolated from food,humans and chickens in Brazil in comparison to the North American strains. Int J Food Microbiol. 2013 Mar 15;162(2):174-81. " Jaros P, Cookson AL, Campbell DM, Besser TE, Shringi S, Mackereth GF, Lim E, Lopez L, Dufour M, Marshall JC, Baker MG, Hathaway S, Prattley DJ, French NP. A prospective case-control and molecular epidemiological study of human cases of Shiga toxin-producing Escherichia coli in New Zealand. BMC Infect Dis. 2013 Sep 30;13:450. doi: 10.1186/1471-2334-13-450. PubMed PMID: 24079470. " Jung WK, Bono JL, Clawson ML, Leopold SR, Shringi S, Besser TE. Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7. Appl Environ Microbiol. 2013 Nov;79(22):7036-41. doi: 10.1128/AEM.02173-13. Epub 2013 Sep 6. PubMed PMID: 24014531; PubMed Central PMCID: PMC3811523. " Mellor GE, Besser TE, Davis MA, Beavis B, Jung W, Smith HV, Jennison AV, Doyle CJ, Chandry PS, Gobius KS, Fegan N. Multilocus genotype analysis of Escherichia coli O157 isolates from Australia and the United States provides evidence of geographic divergence. Appl Environ Microbiol. 2013 Aug;79(16):5050-8. doi: 10.1128/AEM.01525-13. Epub 2013 Jun 14. PubMed PMID: 23770913; PubMed Central PMCID: PMC3754714. " Shringi S, Schmidt C, Katherine K, Brayton KA, Hancock DD, Besser TE. Carriage of stx2a differentiates clinical and bovine-biased strains of Escherichia coli O157. PLoS One. 2012;7(12):e51572. doi: 10.1371/journal.pone.0051572. Epub 2012 Dec 11. PubMed PMID: 23240045; PubMed Central PMCID: PMC3519850. " Davis MA, Moore DL, Baker KN, French NP, Patnode M, Hensley J, Macdonald K, Besser TE. Risk factors for campylobacteriosis in two Washington State counties with high numbers of dairy farms. J Clin Microbiol. 2013 Dec;51(12):3921-7. doi: 10.1128/JCM.01433-13. Epub 2013 Sep 11. PubMed PMID: 24025908. " Marthaler D, Rossow K, Culhane M, Collins J, Goyal S, Ciarlet M, Matthijnssens J. 2013. Identification, phylogenetic analysis and classification of porcine group C rotavirus VP7 sequences from the United States and Canada. Virology. 2013:446(1-2):189-98. doi: 10.1016/j.virol.2013.08.001. PMID: 24074581 " Marthaler D, Jiang Y, Otterson T, Goyal S, Rossow K, Collins J. 2013. Complete Genome Sequence of Porcine Epidemic Diarrhea Virus Strain USA/Colorado/2013 from the United States. Genome Announc. 2013:1(4). doi:pii: e00555-13. 10.1128/genomeA.00555-13. PMID: 23929470 " Vannucci FA, Kelley, MR, Gebhart, CJ. 2013. Comparative genome sequencing identifies a prophage-associated genomic isolated linked to host adaptation of Lawsonia intracellularis infections. Vet. Res. 44:49-58. " Fuller KL, Kuhlenschmidt TB, Kuhlenschmidt MS, Jimenez-Flores R, and Donovan SM. (2013) Milk Fat Globule Membrane Isolated from Buttermilk or Cheese Whey and their Lipid Component Inhibit Infectivity of Rotavirus In Vitro. J. Dairy Science 2013 96(6):3488-97 " Hester S, Chen X, Li M, Monaco M, Comstock S, Kuhlenschmidt T, Kuhlenschmidt, MS and Donovan S. (2013) Human milk oligosaccharides inhibit rotavirus infectivity in vitro and in acutely infected piglets. British Journal of Nutrition 110:1233-1242 " Liu Y, Zhang C, Hu D, Kuhlenschmidt, MS, Kuhlenschmidt TB, Mylon SE, Kong R, Bhargava R, Nguyen T H (2013) Role of collector alternating charged patches on transport of Cryptosporidium parvum oocysts in a patchwise charged heterogeneous micromodel. Environ Sci Technol 47 2670-8. " Paul C. Davidson, Theresa B. Kuhlenschmidt, Rabin Bhattarai, Prasanta K. Kalita, Mark S. Kuhlenschmidt (2013) Investigation of Rotavirus Survival in Different Soil Fractions and Temperature Conditions. Journal of Environmental Protection 4: 1-9 " McLaughlin, S. J., Kalita, P. K. and Kuhlenschmidt, M. S. (2013) Fate of Cryptosporidium parvum oocysts within soil, water, and Plant environment. Journal of Environmental Management 131: 121-128 " Shippy, D. C., N. M. Eakley, C. T. Lauhon, P. N. Bochsler and A. A. Fadl. 2013. Virulence characteristics of Salmonella following deletion of genes encoding for the tRNA modification enzymes GidA and MnmE. Microbial Pathogenesis. 57: 1-9. " Erume, J., P. Wijemanne, E. M. Berberov, S. D. Kachman, D. J. Oestmann, D. H. Francis, and R. A. Moxley. 2013. Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine. Vet. Microbiol. 161:315-324. " Fekete, P. J., K. S. Mateo, W. Zhang, R. A. Moxley, R. S. Kaushik, and D. H. Francis. 2013. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells. Veterinary Microbiology 164:330-335. " Vogstad, A. R., R. A. Moxley, G. E. Erickson, T. J. Klopfenstein, and D. R. Smith. 2013. Stochastic simulation model comparing distributions of STEC O157 fecal shedding prevalence between cattle vaccinated with type III secreted vaccines and non-vaccinated cattle. Zoonoses Public Health (epub Jul 5. doi:10.1111/zph.12069. PMID:23826923) " Vogstad, A. R., R. A. Moxley, G. E. Erickson, T. J. Klopfenstein, and D. R. Smith. 2013. Assessment of heterogeneity of efficacy of a three-dose regimen of a type III secreted protein vaccine for reducing STEC O157 in feces of feedlot cattle. Foodborne Pathog. Dis. 10:678-683. " Paddock ZD, Renter DG, Cull CA, Paddock ZD, Bai J, Nagaraja TG. Escherichia coli O26 in feedlot cattle: Fecal prevalence, isolation, characterization and effects of an E. coli O157 vaccine and a direct-fed microbial. Foodborne Pathog Dis. in press. " Smith R, Sanderson MW, Jones R, Renter DG, Larson RL. Economic risk analysis model for Bovine Viral Diarrhea Virus biosecurity in cow-calf herds. Prev Vet Med. in press. " Jacob M, Bai J, Renter DG, Rogers A, Shi X, Nagaraja TG. Comparing real-time and conventional PCR to culture-based methods for detecting and quantifying Escherichia coli O157 in cattle feces. J Food Prot. in press. " Cernicchiaro N, Cull CA, Paddock ZD, Shi X, Bai J, Nagaraja TG, Renter DG. Prevalence of Shiga toxin-producing Escherichia coli and associated virulence genes in feces of commercial feedlot cattle. Foodborne Pathog Dis. 2013; 10(10): 835-841. " Paddock ZD, Bai J, Shi X, Renter DG, Nagaraja TG. Detection of Escherichia coli O104 in the feces of feedlot cattle by a multiplex PCR assay designed to target major genetic traits of the virulent hybrid strain responsible for the 2011 German outbreak. Appl Environ Microbiol. 2013; 79(11): 3522-3525. " Paddock ZD, Renter DG, Shi X, Krehbiel C, DeBey B, Nagaraja TG. Effects of feeding dried distillers grains with supplemental starch on fecal shedding of Escherichia coli O157:H7 in experimentally-inoculated steers. J Anim Sci. 2013; 91(3): 1362-1370. " Daisuke Takahashi, Yunjeong Kim, Scott Lovell, Om Prakash, William C Groutas and Kyeong-Ok Chang, Structural and Inhibitor Studies of Norovirus 3C-like Proteases. 2013, Virus Research Sep 17. [Epub ahead of print] " Yunjeong Kim, William C.Groutas, and Kyeong-Ok Chang. 2013. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease. Antiviral Res. 97(2):161-8. " Gao X, Wang X, Pham TH, Feuerbacher LA, Lubos ML, Huang M, Olsen R, Mushegian A, Slawson C, Hardwidge PR. NleB, a bacterial effector with glycosyltransferase activity, targets GAPDH function to inhibit NF-kB activation, Cell Host & Microbe, 2013, Jan;13(1):87-99. PMID: 23332158 " Zhou M, Duan Q, Zhu X, Guo Z, Li Y, Hardwidge PR, Zhu G. Both flagella and F4 fimbriae contribute to F4ac+ Enterotoxigenic Escherichia coli adherence to IPEC-J2 cells, Veterinary Research, 2013 May 13;44(1):30. PMID: 23668601 " Olsen RL, Echtenkamp F, Cheranova D, Deng W, Finlay BB, Hardwidge PR. The enterohemorrhagic Escherichia coli effector protein NleF binds mammalian Tmp21, Veterinary Microbiology, 2013 May 31;164(1-2):164-70. PMID: 23434013 " Yang Y, Yao F, Zhou M, Zhu J, Zhang X, Bao W, Wu S, Hardwidge PR, Zhu G. F18ab Escherichia coli flagella expression is regulated by acyl-homoserine lactone and contributes to bacterial virulence, Veterinary Microbiology, 2013 Aug 30: 165(3-4):378-83. PMID: 23693029 " Duan Q, Zhou M, Liang H, Zhu X, Guo Z, Li Y, Hardwidge PR, Zhu G. Contribution of flagellin subunit FliC to piglet epithelial cell invasion by F18ab E. coli, Veterinary Microbiology, 2013 Sep 27; 166(1-2):220-4. PMID: 23746569 " Meng X, Meng X, Zhu C, Wang H, Wang J, Nie J, Hardwidge PR, Zhu G. The RNA chaperone Hfq regulates expression of fimbrial related genes and virulence of Salmonella enteritidis, FEMS Microbiology Letters, 2013 Sep;346(2):90-6. PMID: 23808344 " Pham T, Gao X, Singh G, Hardwidge PR. Escherichia coli virulence protein NleH1 Interaction with the v-crk Sarcoma Virus CT10 Oncogene-Like Protein (CRKL) Governs NleH1 Inhibition of the Ribosomal Protein S3 (RPS3)/NF-kappaB Pathway, Journal of Biological Chemistry, 2013, in press " Zhou M, Guo Z, Yang Y, Duan Q, Zhang Q, Yao F, Zhang X, Hardwidge PR, Zhu G, Flagellin and F4 fimbriae have Opposite Effects on Biofilm Formation and Quorum Sensing in F4ac+ Enterotoxigenic Escherichia coli, Veterinary Microbiology, 2013, in press " Rüter C, Hardwidge PR. Drugs from Bugs: Bacterial effector proteins as promising biological (immune-) therapeutics, FEMS Microbiology Letters, 2013, in press Lay press " Mansfield, LS, 2013. Gut microbes may provide targets for food-borne diseases. AgBioResearch Legislative Report on Food Safety and Security, MSU AgBioresearch & MSU Extension Legislative Report 2012-13, p. 19. Book Chapters " Zeng, X., and J. Lin. 2013. Siderophore-mediated iron acquisition for Campylobacter infection. Chapter 10. Pp111- 124 In S. Sheppard and G. Meric (eds), Campylobacter Ecology and Evolution. Horizon Scientific Press, Hethersett, U.K. " Moxley, R. A. 2013. Chapter 6, Enterobacteriaceae, pp. 53-61. In, McVey, D. S., M. Kennedy, and M. M. Chengappa (editors), Veterinary Microbiology, 3rd Ed., Wiley-Blackwell, Hoboken, NJ. " Moxley, R. A. 2013. Chapter 7, Enterobacteriaceae: Escherichia, pp. 62-74. In, McVey, D. S., M. Kennedy, and M. M. Chengappa (editors), Veterinary Microbiology, 3rd Ed., Wiley-Blackwell, Hoboken, NJ. " Moxley, R. A. 2013. Chapter 8, Enterobacteriaceae: Salmonella, pp. 75-84. In, McVey, D. S., M. Kennedy, and M. M. Chengappa (editors), Veterinary Microbiology, 3rd Ed., Wiley-Blackwell, Hoboken, NJ. " Moxley, R. A. 2013. Chapter 9, Enterobacteriaceae: Yersinia, pp. 85-94. In, McVey, D. S., M. Kennedy, and M. M. Chengappa (editors), Veterinary Microbiology, 3rd Ed., Wiley-Blackwell, Hoboken, NJ. " Moxley, R. A. 2013. Chapter 10, Enterobacteriaceae: Shigella, 95-100. In, McVey, D. S., M. Kennedy, and M. M. Chengappa (editors), Veterinary Microbiology, 3rd Ed., Wiley-Blackwell, Hoboken, NJ.

Impact Statements

" Results from comparative and functional genomics studies of C. jejuni will reveal genetic changes contributed to the hyper-virulence in abortion induction, which ultimately will help to design targeted control measures.
" The genetic and phenotypic differences between the abortion-associated C. jejuni isolates from the U.K. and the U.S. further suggest that the dominance of clone SA in the U.S. may be the result of US-specific selection pressures that facilitated the emergence of this clone.
" The in vivo studies in a natural host (sheep) are critical to better characterize the virulence potential and pathogenesis of the abortion-associated C. jejuni. These studies would aid in the development of effective control or prevention strategies.
" Findings from immunoproteomics studies reveal immunogens that may potentially elicit protective immune responses and provide a foundation for developing vaccines against C. jejuni induced sheep abortion.
" Data on the occurrence and genetic diversity of Campylobacter in turkey production chain will aid in the development of informed prevention and control strategies.
" Elucidating the role of exopolyphosphatases in C. jejuni virulence, stress responses and colonization underscores the importance of PPX/GPPA proteins in poly-P and ppGpp homeostasis, two critical molecules that modulate environmental stress responses and virulence in C. jejuni and would enable us to design approaches to control this pathogen in food chain.
" The ability of C. jejuni to sense external environment through chemical gradients is a key to its diverse lifestyles in different hosts. A better understanding of the mechanisms by which C. jejuni specifically colonizes its host can provide useful insights into strategies for control of Campylobacter and its associated diseases in humans.
" Findings on PNAs further demonstrate the feasibility of using PNAs to potentiate antibiotics against antibiotic-resistant Campylobacter.
" Understanding arsenic resistance (ArsB) in C. jejuni provides new insights into the ecology and adaptive mechanisms of Campylobacter in animal food production environments.
" Identification of mouse model for studying GBS induced by C. jejuni, is critical to understand the molecular mechanisms of pathogenesis of GBS. This will aid in developing effective interventions. Also, the availability of mouse model will also allow screening of C. jejuni strains from livestock for potential to cause GBS.
" The knowledge from the chicken vaccination studies contribute to the development of a vaccine which can be eventually made commercially available to reduce Campylobacter in poultry, with the end goal of reducing human illness associated with the handling and consumption of poultry and its associated sequelae.
" Two component vaccine studies reveal that 2 or 3 way component RASV vaccines may provide one such efficacious and feasible vaccination strategy.
" Heterologous vaccine challenge studies and subsequent strain colonization studies allow refinement of the chicken vaccination model in use, allowing further work to be targeted to the challenges of producing an industry compatible vaccine candidate.
" We found that direct contact with cattle and living or working on a dairy farm were significant contributors to the campylobacteriosis disease burden, but in our data Hispanic ethnicity was one of the strongest risk factors. In order to design public health interventions the effect of ethnicity, above and beyond the effects of food consumption, contact with livestock, and age, need to be explored further.
" Emergence of bacterial antibiotic resistance has become a serious problem worldwide. Our studies may open new avenues for treatment and prevention of resistant foodborne pathogens important in animal health and food safety.
" Research on the development of alternatives to antibiotic growth promoters will lead to novel One Health measures for enhanced animal production, food safety, and human health.
" Our model supported findings from observational studies of nosocomial risks in veterinary teaching hospitals; primarily, length of hospital was the strongest risk factor for acquiring resistant and/or non-resistant strains. Other findings included that more frequent cleaning, particularly of locations with high patients traffic, would be beneficial. In addition, improved health care worker hygiene and protective wear, would also be beneficial.
" We elucidated several biologically plausible risk factors for introduction and maintenance of a novel resistance gene on commensal E. coli in dairy cattle (blaCTX-M-bearing E. coli). Recent animal movements on the farm, region of Washington, use of residual fly sprays, direction of feeding calves (youngest to oldest having a lower risk), frequency of adding new bedding to calf hutches, and use of florfenicol on calves.
" The 2-locus genotyping method for E. coli may have better congruence with 7-locus MLST in human clinical E. coli isolates than in field-collected commensal E. coli; so the 2-locus genotyping method may be of use epidemiologically in the case of limited resources.
" Virulence characterization of several S. Enteritidis mutants led to identification of several novel genes that contribute to the intestinal colonization and systemic dissemination of S. Enteritidis in poultry. This information will be valuable for developing live attenuated vaccines to prevent S. Enteritidis infection in poultry.
" Characterizing the immune-modulatory activities of all possible CpG motifs in Salmonella genome will improve our understanding of immunomodulatory mechanisms utilized by Salmonella for successful propagation in the host. Furthermore, the identification of CpG motifs could also serve as targets for development of oligonucleotides that can improve the efficacy of currently used vaccines.
" The multiplex real-time PCR developed for serotype-specific detection of S. Enteritidis will be highly useful for detection of SE in eggs, meat and environmental drag swabs and will significantly reduce time and costs associated with the microbiological procedures.
" The detection of Rotavirus C in lung tissue may be associated with viremia, which occurs with Rotavirus A infections. In addition to influenza A and Mycoplasma hyopneumoniae, RV may be an important cause of respiratory disease.
" We describe a higher prevalence of Rotavirus B in the United States than previously reported, and RVB infections as an important cause of diarrhea in pigs. RVB has not been previously reported as an important cause of swine diarrhea. Since there is no available vaccine for RVB, other methods of prevention and treatment must be researched.
" Probiotic colonization positively affected immune homeostasis/immune responses to an oral AttHRV vaccine and VirHRV infection in a biologically relevant neonatal Gn pig disease model. Ultimately, our results will provide crucial information for the development of innovative oral treatment strategies using targeted probiotics in neonates to efficiently protect them against enteric pathogens and improve vaccine-induced immunity.
" Nanoantibody production can be scaled-up to develop passive treatments or supplemented functional foods such as milk replacement formulas for infants and neonatal animals to treat RV or other enteric pathogen- associated diarrheas.
" The complete PEDV genome sequencing from the US illustrates CO/13 most recent common ancestor, suggesting a possible source. Once more complete genomes become available, sequence analysis will conclude highly variable regions, which can be used to track PEDV strains.
" Information on the pathogenesis of newly emerged US PEDV strains will help improve management and biosecurity. Further, cell culture adapted PEDV strains will serve for serologic diagnosis and candidate vaccines to promote new measures for control and prevention of PEDV infections in the US swine industry.
" Enteric infection of Gn calves with GIII.2 BoNoV, CV186-OH strain is a useful model for comparative pathogenesis studies of other enteric NoV infections, such as human NoV, due to the pathogenic similarities (prolonged shedding) between GIII.2 BoNoV and human NoVs. This model may also help to elucidate the role of antiviral treatments and innate and adaptive immunity in the clearance of NoV from the infected intestine.
" Continuous monitoring of NoVs and SaVs in swine is crucial since porcine NoVs are most closely related to HuNoVs and there is a zoonotic potential for swine NoVs or for transmission of HuNoVs to pigs. Our results will help to improve public health and swine health.
" We successfully demonstrated the experimental reproduction of the brachyspiral colitis caused by B. hampsonii clades I and II. This experimental model may be valuable for evaluating the efficacy of different interventions to prevent and control this emerging pathogen.
" The results on transcriptional profile of L. intracellularis open a new research field for studying target genes involved in the pathogenesis of proliferative enteropathy.
" Development of methods for molecular characterization of B.hyodysenteriae strains will now allow us to investigate their distribution, phylogeny and epidemiology.
" We highlighted the importance of surveillance to detect the potential emergence of Brachyspiral isolates with decreased susceptibility towards commonly used antimicrobials. In addition, we validated the use of a broth dilution technique for routine antimicrobial susceptibility testing of Brachyspira species in the U.S.
" Monoclonal antibodies developed against the O-antigens of FSIS-adulterant non-O157 Shiga toxin-producing Escherichia coli (STEC) strains will be highly useful reagents for implementation in new diagnostic tests, including those involving rapid detection of these organisms in food and other matrices.
" Studies detecting the prevalence of FSIS-adulterant non-O157 and O157 strains in hide samples of beef cattle at harvest will help in the development of a quantitative microbial risk assessment model for these organisms in beef.
" Studies evaluating chromogenic agar plating media for FSIS-adulterant STEC have validated their use for this purpose, but have also revealed the need for their continued improvement.
" Studies evaluating the capacity of FSIS-adulterant non-O157 STEC strains to adhere to bovine and human colonic epithelial cells will increase our knowledge of how these organisms colonize cattle and cause disease in human patients, and may form the basis for future preventive strategies.
" Baseline prevalence estimates for Shiga toxin-producing Escherichia coli O groups and virulence genes in feces and flies, as well as Salmonella in feces and subiliac lymph nodes, of commercial feeder cattle are critical to populate risk assessments and identify the distribution, intervention opportunities and potential risks of human illness.
" Close association between Stx-encoding bacteriophage insertion sites and chromosomal backbone SNP allele patterns shows that the clinical and bovine-biased genotypes are the result of evolutionary divergence, and not an artifact of bacteriophage movements.
" Phylogeographic structuring of O157 explains much or all of international variation in the incidence of O157-associated human disease.
" The extrinsic origin of seasonal variation suggests that control efforts should focus much more strongly on environmental non-cattle reservoirs of this pathogen.
" The association of increased prevalence of O157 immediately following introduction of fresh forages also provides a new avenue for research to reduce the prevalence of this agent in the cattle reservoir.
" Studies of the secretion systems and regulatory mechanisms of enterotoxins in E. coli will increase our knowledge of how they cause disease, and potentially lead to new preventive and therapeutic approaches that reduce our use of antibiotics, e.g. through dietary means.
" We identified GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by attaching/effacing pathogens to inhibit NF-kB-dependent host innate immune responses.
" We determined that the E. coli virulence protein NleF binds the human Tmp21 protein to disrupt host protein trafficking.
" We identified the kinase substrate of the E. coli virulence protein NleH1 as the v-crk sarcoma virus CT10 oncogene-like protein (CRKL) and proposed that the NleH1-CRKL interaction contributes to the ability of NleH1 to inhibit host NF-kappaB signaling.
" We determined that both flagella and F4 fimbriae are required for efficient F4ac+ enterotoxigenic E. coli (ETEC) adhesion in vitro.
" We determined that flagella function as adhesins to enhance the ability of F18ab E. coli to target to piglet epithelial cells.
" We determined that the RNA chaperone protein Hfq contributes to Salmonella enteritidis virulence by regulating fimbrial gene expression.

Date of Annual Report: 01/09/2015

Report Information

Annual Meeting Dates: 12/06/2014 - 12/07/2014
Period the Report Covers: 10/01/2013 - 09/01/2014

Participants

Peter Johnson, USDA;
Margo Holland, USDA;
Roy Curtis, Arizona State University;
Qijing Zhang, Iowa State University;
Frank Blecha, Kansas State University;
Weiping Zhang, Kansas State University;
Phil Hardwidge, Kansas State University;
T.G. Nagaraja, Kansas State University;
Mo Saif, The Ohio State University;
Linda Saif, The Ohio State University;
Gireesh Rajashekara, The Ohio State University;
Issmat Kassem, The Ohio State University;
Qiuhong Wang, The Ohio State University;
Linda Mansfield, Michigan State University;
Dongwan Yoo, University of Illinois at Urbana-Champaign;
Bibiana Law, University of Arizona;
Alexandra Armstrong , University of Arizona;
Richard Isaacson, University of Minnesota;
Douglas Marthaler, University of Minnesota;
Connie Gebhart, University of Minnesota;
Rodney Moxley, University of Nebraska-Lincoln;
Zach Stromberg, University of Nebraska-Lincoln;
Jun Lin, University of Tennessee;
Amin Fadl, University of Wisconsin-Madison;
Bledar Bisha, University of Wyoming;
Sheela Ramamamoorthy, North Dakota State University;
Radhey Kaushik, South Dakota State University;
Joy Scaria, South Dakoda State University;
Devendra Shah, Washington State University;
Kim Chrok, Washington State University;
Jake Elder, Washington State University;
Aruna Ambagala, CFIA-Canada

Brief Summary of Minutes

1. The meeting was called to order by the Chair of NC-1202, Dr. Gireesh Rajashekara at 8:00 am on Dec 6. Dr. Rajashekara introduced Dr. Frank Blecha, the new NC-1202 Administrative Advisor from Kansas State University.

2. Administrative Advisor, Dr. Frank Blecha, gave a short presentation on specifics related to the completion of Midterm Review of NC-1202 multistate project, which was due on Dec 15th, 2014.

3. Nineteen (19) Progress Reports were presented from 9:00 am to 5:30 pm on Dec 6.

4. From 8:30 am to 10:00 am on Dec 7, Drs. Peter Johnson and Margo Holland provided an update on NIFA budget and funding, with introduction of two new programs (CARE and Exploratory) that began in FY2014 and may be included again in FY2015. The NC-1202 group expressed concerns and also provided suggestions for current NIFA funding opportunities, which include; 1) challenge and rationale to include three obligated components (Research, Extension, Education) for large integrated grants. Current policy presented a hurdle to package an effective and successful multi-state proposal; 2) possibility to include a new program/category for junior scientists and provide junior faculty greater opportunity. NIFA may adopt a NIH-like policy to provide incentives for New Investigators. Regarding support for new investigators, Drs. Peter Johnson and Margo Holland suggested NC1202 committee draft and send a recommendation to Dr. Sonny Ramaswamy (NIFA Director).

5. From 10:15 am to 12:00 pm on Dec 7, NC-1202 group members had vivid discussion on the following issues:

• Progress Report. Midterm Review was reminded to be completed by 12/15/2014. Due to confidentiality concern for the date described in the station reports, it was suggested that the progress report will be only sent to the station representatives.

• Budget. $75 registration fee was charged for 2014 NC-1202 annual meeting, which should cover all costs in 2014 with some leftover for 2015. It is expected that a lower registration fee will be charged for 2015 annual meeting.

• Feasibility of organizing a symposium by NC-1202 group. The symposium (focused on enteric disease) will be submitted through CRWAD and is expected to attract more people and increase attendee numbers for CRWAD. Dr. Peter Johnson raised the issue of possible conflict with the symposium organized by other multistate project (e.g. PEDV focus). To address this concern, Dr. Frank Blecha suggested different groups should cooperate with each other. The symposium abstracts will be submitted through CRWAD. To select Keynote Speakers for the symposium, a 5-member committee (Linda Mansfield, Linda Saif, Jun Lin, Gireesh Rajashekara, Radhey Koushik) was established.

• Student awards. NC-1202 offers awards for students to compete in the Pathobiology of Enteric and Foodborne Pathogens Section of CRWAD. This year three awards were offered, two for oral presentation and one for poster presentation. Drs. Radhey Kaushik (Chair) and Weiping Zhang (Vice-Chair) are in charge of the student award selection committee. Dr. Weiping Zhang will serve as Chair for the Pathobiology of Enteric and Foodborne Pathogens Section of CRWAD for 2015. The student awards were funded by the annual registration fee. To increase the number of abstracts submitted to the Pathobiology of Enteric and Foodborne Pathogens Section, the award size will be increased from 2015 with only one award ($200) for each category (Oral or Poster Presentation).

• Collaboration and funding opportunities. Dr. Linda Saif suggested that NC-1202 submit a collaborative, integrated application to the 2015 NIFA Food Security Grant; the new RFA is expected to be released in early 2015. Specific topics suitable for this grant were discussed by the participants; ‘Microbiome & Gut Health’ was selected as the general theme for this application to address a significant and timely food security issue. Upon release of the 2015 RFA, a steering committee will be established to move the project forward. Dr. Mo Saif nominated Dr. Gireesh Rajashekara to lead the steering committee for this collaborative project in the near future.

Accomplishments

Objective 1. Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. A. Campylobacter jejuni Iowa Bacterial factors involved in an emerging SA clone that cause ruminant abortion: Comparative analysis and genetic work of C. jejuni strains showed capsule locus (CPS) and CJSA_0039 (putative tyrosine phosphorylated regulator) were absolutely required for induction of systemic infection and abortion in pregnant guinea pigs. The genes contributing to bacteremia were identified by Tn-seq analysis. A novel transformation strategy was utilized to determine the specific genetic determinants required for abortion. Immunogenicity of membrane-associated proteins of Campylobacter jejuni-associated with sheep abortion. We observed that CgpA, MetK, FabG had the strongest antigenicity, while HtrA, FlgL and Peb4 were less antigenic, and CJSA_0852 had only little reactivity with the sera tested. CgpA, HtrA and FlgL were chosen as subunit vaccine candidates to evaluate the protective immunity against bacterial challenge in our mice model of systemic infection and bacteremia. Results suggest that CgpA may be a potential subunit vaccine candidate against sheep abortion caused by C. jejuni. Prevalence of C. jejuni clone SA in feedlot cattle. A total of 3,184 fecal samples were collected from 66 different feedlot cattle herds in Iowa, Texas, Colorado, Missouri and Kansas, and cultured directly for Campylobacter. The overall prevalence rate of Campylobacter in cattle feces was 72% (2293/3184). Our findings indicate that Campylobacter is commonly prevalent in the U.S. feedlot cattle, and that clone SA constitutes a substantial portion of the commensal cattle C. jejuni population. Ohio Pathogenesis mechanisms associated with Campylobacter jejuni -induced abortion in ewes: We demonstrated that effectiveness of using sheep (the natural host) to study the pathogenesis of sheep abortion associated C. jejuni. We also highlighted the responses of the host and the progression of symptoms associated with infections in pregnant sheep. 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. 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: This study highlighted the high prevalence, genotypic diversity, and antimicrobial resistance of Campylobacter spp in commercial turkey from farm to slaughter. Michigan In 2013-14, we completed 13 experiments infecting mice with C. jejuni strains from patients with Guillain Barré Syndrome (GBS) and patients with colitis. We have developed several mouse models that will now permit study of C. jejuni strains with selected gene knockouts. We have developed a mouse model of autoimmune diseases arising after C. jejuni infection including Inflammatory Bowel Disease and Guillain Barré Syndrome that has yielded important new knowledge for the medical field and that has been published in a Nature Journal. We now know that the evolution of C. jejuni in the host can lead to changes in its surface coat that can trigger autoimmune disease in genetically susceptible hosts. Top accomplishments include 1) identifying at least three mouse models for studying GBS induced by C. jejuni, 2) Identifying neurological phenotypes and lesions in mouse models of GBS using a new digital gait analysis video camera and software, 3) demonstrating that colitis and GBS disease outcomes after C. jejuni infection have alternative T cell regulatory pathways in the C57BL/6 mouse model, and 4) showing effects of a Type III secretion system of C .jejuni on the outcome of infection with C. jejuni in an animal model. B. Salmonella Washington State Comparing antibiotic resistance in temporally related Salmonella from cattle and humans: We analyzed 5,124 human clinical isolates from Washington State Department of Health, 391 cattle clinical isolates from the Washington Animal Disease Diagnostic Laboratory, and 1864 nonclinical isolates from foodborne disease research on dairies in the Pacific Northwest. Salmonella Typhimurium (ST), Salmonella Newport (SN) and Salmonella Montevideo (SM) were the most common serovars in both humans and cattle. The ST and SN from cattle had greater probability of resistance to multiple classes of antimicrobials than ST and SN from humans (p<0.0001). However, occurrence of profiles unique to cattle and not observed in temporally related human isolates, indicates these profiles are circulating in cattle only. Further AMR diversity assessment using different measures and new techniques (e.g. bootstrapping) showed that although there were shared profiles between humans and cattle, the expected and observed number of profiles was different, suggesting Salmonella and associated resistance from humans and cattle may not be wholly derived from a common population. Most prevalent poultry-associated Salmonella serotypes (MPPSTs) differ in their susceptibility to widely used carcass sanitizer, chlorine: Significant proportion of marketed poultry meat in the US remains contaminated with few Most Prevalent Poultry-associated Salmonella seroTypes (MPPSTs). A simulation model-derived data suggest that MPPSTs differ in their susceptibility to chlorine and that the level of CME in immersion chilling is an important contributing factor in Salmonella survival. C. Shiga toxin-producing E. coli (STEC) Nebraska Detection of enterohemorrhagic Escherichia coli (EHEC) in samples of feces, hides, and carcasses of culled dairy cows at harvest. We determined the proportion of fecal, hide, and de-hided carcass surface samples positive for seven EHEC serotypes (O26, O45, O103, O111, O121, O145, and O157) in culled dairy cattle at slaughter using a convenience sampling approach. One hundred culled dairy cows from the western U.S. were sampled over 5 weeks at a commercial abattoir (June to July 2014). Our results suggest that adulterant EHEC may be highly prevalent in culled dairy cattle at slaughter, further emphasizing the importance of effective pre- and post-harvest interventions. Erosive colitis in a yearling feedlot heifer caused by enterohemorrhagic Escherichia coli O165:H25. An EHEC strain that was identified as serotype O165:H25 and positive for stx2c, eae-?, and ehxA was isolated from the tissue of erosive colitis in a yearling feedlot heifer. This is the first clinical case of EHEC O165:H25 infection identified in cattle, and also represents one of the oldest bovine animals known to be clinically affected with any EHEC serotype. Wyoming Development of MALDI-TOF MS protocols for diagnosis of antimicrobial resistant bacteria. A total of 1,177 presumptive AMR Escherichia coli (ciprofloxacin and cefotaxime resistant), Staphylococcus spp. (cefoxitin resistant), and Enterococcus spp. (erythromycin resistant) isolates from wildlife, feed, and water associated with concentrated animal feeding operations were analyzed using developed MALD-TOF MS method. Detailed analyses of mass spectral profiles identified numerous spectral peaks, which may be useful for subtyping applications, including differentiation of AMR phenotypes. Kansas Multiple STEC projects have been successfully completed, which include 1) summer and winter prevalence of STEC in feces of feedlot cattle. This study indicated that non-O157 STEC are rare, but generally follow the same seasonal pattern as O157 STEC. However, further research is needed to determine if this trend holds true across other cohorts of cattle, feedlots and regions of the country. 2) feedlot- and pen-level prevalence of 7 STEC serotypes in feces of commercial feedlot cattle. Results provided insight on the feedlot- and pen-level fecal prevalence of E. coli serogroups in pre-harvest commercial feedlot cattle. 3) Develop culture- and PCR-based methods to detect 7 major serogroups of STEC. This study refined culture-based methods and compare with PCR method for detection of six non-O157 STEC serogroups in cattle feces (n = 576). 3) Comparison of a four-plex real-time PCR assay, conventional PCR and culture-based method for detection of STEC in feces of feedlot cattle. Our results indicated the multiplex quantitative PCR (mqPCR) assay detected more samples as positive compared to conventional PCR or culture-based method; 4) Pooling of immunomagnetic separation beads does not affect sensitivity of detection of seven serogroups of STEC in cattle feces. Pooling of IMS beads is advantageous because of reduced time, labor and expense required to detect and isolate STEC-7.; 5) Application of multiplex real-time PCR for the detection of six major non-O157 E. coli serogroups in feces of feedlot cattle. The higher relative sensitivity coupled with reduction in time and labor make mqPCR assays a good alternative to conventional PCR and culture-based methods for detection of the six non-O157 E. coli serogroups in cattle feces; 6) Escherichia coli O104 in feedlot cattle feces: Prevalence, isolation and characterization. Cattle shed serogroup O104 in feces, but only a few strains (11.2 %) carried stx1 gene and none of the isolated strains carried genes characteristic of the hybrid pathotype; 7) Quantification of six non-O157 E. coli serogroups in cattle feces by spiral plating method. This study indicated that the spiral plating technique combined with PCR confirmation has the potential to be a useful technique to quantify non-O157 E. coli serogroups in cattle feces. D. Brachyspira hampsonii Minnesota Molecular characterization of emerging “Brachyspira hampsonii” isolates. The novel pathogen, “Brachyspira hampsonii”, consists of two distinct genetic clades (I and II) and the pathogenicity of each clade has been confirmed experimentally in pigs. With one exception, all evaluated swine systems were infected with a single specific clade. Sites and systems that were positive for “B. hampsonii” repeatedly over several years were found to be temporally infected with the same ST or two clonally related STs. E. Coronavirus Ohio Detection of porcine deltacoronavirus (PDCoV) in US swine, pathogenesis of OH strains in gnotobiotic (Gn) pigs. The PDCoV, a new coronavirus, has been simultaneously and frequently detected in diarrheic fecal samples from pigs. The original and Gn pig-passaged OH-FD22 and OH-FD100 samples were confirmed virulent and free of other viral pathogens. These PDCoV pools will provide a useful challenge strain for pathogenesis and vaccine studies. Our study, for the first time, confirmed that PDCoV induces clinical disease and pathologic lesions in pigs. Minnesota Rapid detection and phylogenetic analysis of porcine deltacoronavirus. A real time RT-PCR was developed to detect and characterize PDCoV. A majority of the positive PDCoV samples (n=69, 78%) were co-infected with at least one of the previously described pathogens while 20 (22%) were negative for previously described pathogens. The results indicate that PDCoV is a common pathogen in the Midwest. F. Rotavirus Minnesota Widespread rotavirus H (RVH) in commercially raised pigs. The swine RVH VP6 nucleotide percent identity had a higher similarity to the novel human RVH than swine RVB strains. In the United States, 30 positive RVH samples were detected form 10 different states. Sequencing analysis suggested RVH has been circulating in the United States swine population for at least one decade but probably longer. Detection and high occurrence of porcine rotavirus A, B, and C by real time RT-PCR in diagnostic samples. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). Although RVA was detected at the highest overall percentage (62%), 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1–20 days of age), while RVA and RVB were most predominant in ?21 day old pigs. G. Antimicrobial Resistance Tennessee Regulation of beta-lactam resistance in Campylobacter jejuni. Our molecular and biochemical studies demonstrated that Cj0843c, a putative lytic transglycosylase involved in cell wall metabolism, was required for induction of ?-lactamase-mediated ?-lactam resistance in Campylobacter. The crystals of Cj0843c have been significantly improved (the crystal diffracted to 2.57 Å resolution) and will be used for structural analysis and subsequent molecular docking to identify specific inhibitors. Molecular basis of conjugation in Campylobacter jejuni. Conjugation is an important mechanism for horizontal gene transfer. Using C. jejuni as a model recipient strain, we have made significant progresses in the mechanisms of conjugation. First, we observed that heat shock treatment could dramatically increase conjugation efficiency of C. jejuni. Second, using a unique co-transformation strategy in conjunction with whole genome sequencing and comparative genomics analysis, we have identified factors required for high frequency conjugation in C. jejuni. Development of novel alternatives to antibiotic growth promoters (AGP) for enhanced animal health and food safety. Bile salt hydrolase (BSH), a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Our studies suggested that BSH inhibitors are promising alternatives to AGP for enhanced animal growth performance and food safety. The BSH from L. salivarius is an ideal candidate for screening desired BSH inhibitors. Ohio 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. We highlighted 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. Notably, antibiotic resistance was detected even in isolates collected from farms under organic management. Kansas Effects of in-feed copper and tylosin supplementations on copper and antibiotics resistance in fecal enterococci of feedlot cattle. The objective was to investigate whether feed supplementation of copper, at elevated level, co-selects for macrolide resistance in fecal enterococci in the absence of tylosin. A longitudinal study indicated that due to the selection pressure feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B) mediated copper and tylosin resistant fecal enterococci in feedlot cattle. Effects of in-feed copper, chlortetracycline, and tylosin on the prevalence of transferable copper resistance gene, tcrB, among fecal enterococci of weaned piglets. Longitudinal study found that the supplementation of antibiotics in combination with copper increased the prevalence of copper-resistant enterococci. Objective 2. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens. A. Campylobacter jejuni Iowa Molecular genetic analysis of C. jejuni strains. Toxin-antitoxin (TA) systems are pairs of genes that encode a stable toxin and an unstable antitoxin and their primary function is to maintain the plasmid by eliminating plasmid-free daughter cells through a post segregation killing mechanism. Our recent study first demonstrated that the existence of the type I and type II TA systems in pVir plasmid of C. jejuni. In this study, we report the identification and characterization of the type II toxin-antitoxin system in the pVir plasmid of C. jejuni clone SA strain IA3902, a highly abortifacient isolate from sheep. Our results establish that CJSA_pVir0046 and CJSA_pVir0046 encode a functional TA system in C. jejuni. Control of Campylobacter in poultry flocks. We conducted a longitudinal epidemiological study to identify the risk factors associated with variation in Campylobacter prevalence in U.S. commercial broiler production. Cecal contents (n=5521) and boot swabs (n=1029) were collected from 369 broiler flocks on 15 different farms with repeated sampling. In order to identify the risk factors associated with Campylobacter prevalence at the farm and house/flock levels, farms were divided into 2 categories based on the overall number of Campylobacter positive flocks reared during the entire study period (i.e., low prevalence vs high prevalence). Arizona C. jejuni vaccine development. Recombinant attenuated Salmonella vaccines (RASV) have been adapted to stably express protective antigens at high levels. Two replicates of heterologous challenge vaccination studies were conducted with both studies resulting in a 1 log reduction. A third replicate vaccine study testing dual doses (1ml each at ~1x1010 CFU) of vaccines expressing CjLAJ1 and CjLAJ2 and challenged with the homologous strain C. jejuni NCTC 11168 resulted in a 2-log reduction. Construction of a single vaccine expressing dual genes and optimization of current constructs is underway. Tennessee Development and evaluation of Campylobacter vaccine. The CmeC- and CfrA-based DNA vaccines have been modified for in ovo immunization. Two independent in ovo vaccination trials have been performed. The in ovo injection of DNA vaccines at 18th day of embryonation failed to trigger significant immune response in broilers and to confer protection against C. jejuni colonization. The chitosan encapsulated subunit mucosal vaccines have been prepared and will be evaluated in broilers in the future. Ohio The role of respiratory proteins in C. jejuni’s pathobiology: The respiratory protein methylmenaquinol:fumarate reductase (Mfr) contributed to the oxidative stress response. Mfr also affected the interaction of the bacterium with macrophages and chicken colonization. Formate dehydrogenase activity and localization are dependent on a functional twin arginine translocation system in the highly invasive strain C. jejuni 81-176. The role of inorganic polyphosphate (poly P) metabolism in C. jejuni’s pathobiology: Exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) also contributed to poly P and ppGpp homeostasis. The PPX/GPPA also modulated environmental stress responses and virulence in C. jejuni. In a separate study, we showed that poly P mediated the modulation of biofilm growth and stability in C. jejuni. Our findings further highlighted the importance of these molecules as potential targets for anti-C. jejuni therapeutics Michigan Janus-faced immunity to an enteric pathogen: How Campylobacter mediates colitis and autoimmunity. C. jejuni is a leading cause of foodborne enteritis and has also been linked to the autoimmune neuropathy, Guillain Barré syndrome (GBS). This study established the first mouse model of an autoimmune disease induced directly by an enteric bacterium. Innate immunity is insufficient to mediate severe C. jejuni induced colitis. Rag1-/- mice on the C57BL/6 background were challenged with C. jejuni alongside C57BL/6 wt mice that serve as a known positive colonization control. Our data showed that T cells are necessary for severe C. jejuni-induced colitis. C. jejuni colonization correlated with colitis induction in as much as the mouse genetic background that supports inflammation (IL-10-/-) had higher colonization extent by the end of the experiment than the mouse genotypes/treatments that did not (BL/6wt or Rag1-/- or Rag1-/- IL-10R blocked mice). Washington Evaluation of Passive Immunotherapeutic Efficacy of Hyperimmunized Egg-Yolk Powder (HEYP) Against Intestinal Colonization of Campylobacter jejuni in Chickens. The results showed that there were no differences in the cecal colonization of C. jejuni between HEYP treated and non-treated control chickens. The use of HEYP at the dose and the regimes used in current study is not efficacious in reducing C. jejuni colonization in chickens. B. STEC and ETEC Nebraska Study of the effects of glucose on heat-labile enterotoxin (LT) and heat-stable enterotoxin-b (STb) gene expression in porcine-origin enterotoxigenic E. coli (ETEC) using lux reporters. This study is critical to demonstrate regulation of the two enterotoxins (LT and STb) concurrently in a given strain and determine whether the effects occur in vivo in inoculated animals. In the past year, we constructed ?crp derivatives of Lux reporter strains, and also made CRP-complemented constructs. The expected effects of catabolite repression on the STb reporter were again seen in the CRP-complemented mutant strain and a trend toward catabolite de-repression was seen in the CRP-complemented LT reporter. Kansas Multiple ETEC projects have been successfully completed, which include 1) ETEC biofilm formation. Mutagenesis study demonstrated that biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain; 2) ETEC and intestinal cell autophagy. ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC; 3) Role of methionine on intestinal cell autophagy during ETEC infection. Methionine deprivation resulted in enhanced ETEC adhesion and increased both the cytotoxicity and apoptotic responses of IPEC-1 cells infected with ETEC. 3) E. coli quorum sensing. The luxS deletion mutant exhibited changed flagella-related phenotypes. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence; 4) E. coli genome sequencing. The objective of this study was to report the draft genome sequence of enterotoxigenic Escherichia coli (ETEC) strain W25K, which causes diarrhea in piglets; 5) Bacterial virulence factors and host inflammation. The Escherichia coli NleH1 and NleH2 virulence proteins differentially regulate host transcription of innate immunity genes. NleH expression was detrimental to C. rodentium in C57BL/10ScNJ mice. C. rodentium that expressed both E. coli NleH1 and NleH2 was hypervirulent in C3H/HeJ mice; 6) ETEC and intestinal immunity. We found that ETEC infection promoted the production of pro-inflammatory cytokines. Meanwhile, ETEC infection affected sIgA transport and Paneth cell function. 7) Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 and corresponding impacts on cattle performance in a commercial feedlot. A siderophore receptor and porin proteins-based vaccine (VAC) and a Lactobacillus acidophilus-based direct-fed microbial (DFM) were evaluated. We observed that the application of these two treatments differentially impacted both fecal shedding of E. coli O157:H7 and cattle performance outcomes, demonstrating need to consider potential food safety impacts as well as cattle and carcass performance, when evaluating potential costs and benefits of interventions; 8) prevalence of STEC-8 in muscoid flies in the confined cattle environment. Prevalence of STEC-8 in house flies has been assessed on the temporal and spatial basis. Washington We completed a comparative study involving US, Australia and Argentina strains of E. coli O157:H7 isolated from cattle, using SNP and SBI genotyping. Results strongly support for phylogeographic structuring. Countries with high incidence of human O157-related disease have much higher frequency of strains encoding Stx2a. Novel SNP lineages of E. coli O157:H7 comprised substantial proportions of the populations in countries outside of the US, where the SNP typing panel was developed. We participated in a second comparative study of geographic divergence of E. coli O157:H7 between the US and New Zealand. We conducted an oral immunization trial of cattle with E. coli O157:H7, using a pool of three spontaneously Stx-negative E. coli from the EHEC1 lineage We are continuing to test the hypothesis that high summertime seasonal shedding of O157 results from introduction of new season forage crops. Most of the data to date have focused on adult dairy cattle and shown relatively minor impact on STEC shedding including O157. By comparing STEC shedding in adjacent pens with shared water troughs with shedding in adjacent pens lacking shared water troughs, the specific role of common water sources in STEC epidemiology was evaluated. The project is ongoing but the preliminary analysis, a general linear model of absolute STEC prevalence difference conditional on shared water trough or shared fence line, found no effect of shared water through on absolute prevalence difference. C. Salmonella Tennessee Evaluation of the use of probiotics and botanicals as feed additives to reduce Salmonella colonization in a chick model. Botanical treatments supported the growth of the probiotics and were biocidal in vitro but had limited effectiveness in vivo. Feed inclusion concentrations are being adjusted for optimization. Kansas A randomized trial to assess whether enrofloxacin metaphylaxis for bovine respiratory disease affects fecal shedding of Salmonella and Campylobacter in feedlot cattle. Preliminary results indicate that metaphylactic administration of enrofloxacin in feedlot cattle did not significantly affect fecal prevalence of Salmonella and Campylobacter. Additionally, metaphylactic administration of enrofloxacin did not impact naladixic acid or ciprofloxacin susceptibilities of Salmonella isolated from feeder cattle feces. Washington Transcriptional profiling of Salmonella Enteritidis strains using RNA-Seq identifies genes consistently highly expressed in biologically relevant microenvironments: A core of 23 genes are consistently highly expressed in three highly pathogenic S. Enteritidis strains, regardless of growth microenvironments representing the avian host. The role of these genes in bacterial physiology and virulence are being investigated for development of new vaccines or antimicrobial agents Overexpression of catalytically inactive dimethyl adenosine transferase (KsgA) unveils contribution of KsgA to Salmonella Enteritidis physiology and virulence: The results support KsgA as a potential candidate for drug and/or vaccine development. Identification and characterization of the immunomodulatory activity of CpG motifs of Salmonella: Cystine-guanine (CpG) motifs are DNA sequences (6 bp) that can have strong immune-modulatory activities. We identified 256 unique CpG motifs within the Salmonella pan-genome. We then synthesized 256 oligodinucleotides (ODNs) each containing a single CpG motif repeated 3 times. This study comprehensively characterized the immune-modulatory activities of all the possible CpG motifs found within Salmonella pan-genome and identified a number of immunostimulatory motifs. These newly identified CpG motifs could serve as targets for the development of ODNs that stimulate immune responses against Salmonella in mammals and chickens and also to improve the efficacy of currently used vaccines. D. Brachyspira Minnesota Association of Brachyspira antibiograms with species and source. In general, the in vitro MICs of Brachyspira species were low for tiamulin and valnemulin; and high for lincomycin and tylosin. In contrast to B. hyodysenteriae and “B. hampsonii”, several B. pilosicoli and B. murdochii isolates showed high tiamulin MICs. The odds of an isolate having a low MIC if originating from a sow site were significantly higher than those originating from a finisher site. Most isolates with high MIC profiles originated from three swine production systems. Analysis of virulence-associated genes in known and novel Brachyspira species to develop pathogen-specific diagnostic assays. Based on the most frequently detected virulence genes and usefulness of sequences to discriminate pathogenic Brachyspira species, the nox gene was determined to be a useful target for design of diagnostic PCR assays. Species-specific PCR assays for B. hyodysenteriae, “B.hampsonii”, and the Brachyspira genus were developed and evaluated. Further, the B. hyodysenteriae and “B. hampsonii” primer sets were evaluated for their utility as a duplex PCR. E. Streptococcus suis Kansas Streptococcus suis virulence. The SSU0587 gene encodes a protein of 1,491 amino acids that possesses beta-galactosidase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both beta-galactosidase activity and adherence to microvascular endothelial cells and reduced concentration in the brains of infected mice. F. Calicivirus Kansas Cathepsin L plays a crucial role in the replication of caliciviruses. This conclusion was supported by various evidence using recombinant cathepsin, diverse calicivirus, and cathepsin L inhibitors. Cathepsin L could be a therapeutic target as demonstrated in severe acute respiratory syndrome coronavirus and Ebola virus. Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors. Among the lead compounds, a class of tripeptidyl transition state inhibitors were further characterized and validated. Two tripeptidyl compounds possess a potential as a broad range antiviral agents G. Rotavirus Ohio Probiotics: effects on neonatal innate immune responses, immune homeostasis, RV infections and vaccine efficacy. We demonstrated that in the neonatal Gn pig disease model, selected G+ and G-probiotics exert their beneficial effects by promoting immunomaturation and immune responses and regulating immune homeostasis, thereby modulating responses to virulent HRV and alleviating diarrhea. Our findings will contribute to alternative low cost probiotic treatments applicable to neonates (or mothers) to moderate RV disease, enhance oral vaccine efficacy, and reduce neonatal morbidity and mortality. Comparative in vivo and in vitro studies of porcine rotavirus G9P and human rotavirus Wa (G1P) in gnotobiotic pigs: Our comparative studies suggest that PRV G9P may replicate in immune swine cells which may facilitate its more efficient dissemination and persistence in swine. These findings contribute basic knowledge about RV replication and the virus associated immunopathology. Wyoming An improved method to capture and detect viruses from bioaerosols has been developed. H. Cryptosporidium parvum Illinois Our results suggest microneme secretion is a potential target that could be exploited for development of new drugs for the treatment of not only cryptosporidiosis but also other serious apicomplexan diseases such as toxoplasmosis. Objective 3. Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures. The PIs and Graduate Students involved in the project have been continuing to give presentations and updates enteric diseases and food safety at various scientific, veterinary, and diagnostic meetings in the previous year. They have effectively disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians. They also have generated many high impact peer-reviewed journal articles. The members have actively organized various outreach/education activities, such as 1) Dr. Armstrong organized the 5th annual University of Arizona Food Safety Conference on October 10, 2014; 2) Dr. Irene Hanning presented Webinar on Food Safety in Organic Poultry via eXtension Archived at http://www.extension.org/pages/25242 (National, 2014); 3) two half day symposia were organized by Dr. Linda Mansfield (MSU) to discuss scientific progress. Fall seminar series in Large Animal Clinical Sciences at the College of Veterinary Medicine at MSU where preharvest food safety was prominently featured. People attending came from the Agricultural, Veterinary Medicine, Microbiology and Food Science, and Human Nutrition departments. Objective 4. Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings. • NC-1202 members and their students presented their work in numerous national and international meetings. We held annual NC1202 meetings in Dec of 2014, and also sponsored 3 student awards for best oral and poster presentations at the Conference of Research Workers in Animal Disease meeting. • NC-1202 members have established active collaborations, partly reflected by joint grant/publications. Following are three active integrated food safety projects in which our NC1202 members are project directors and involve several NC1202 members at the participating institutions as co-project directors: 1) Zhang, Q. (PD, Iowa State Univ.) and other Co-PDs who are NC1202 members: Jun Lin (Univ. of Tennessee), Gireesh Rajashekara (Ohio State Univ). USDA NIFA Food Safety Challenge Grant, Novel Approaches for Mitigation of Campylobacter in Poultry. $2,500,000. Award period: 07/01/2012 – 06/30/2017. 2) Law, B. (PD, Univ. of Arizona) and other Co-PIs who are NC1202 members: Roy Curtiss III is a scientific advisor, and Ken Roland from the Curtiss lab is the Co-PI. USDA NIFA Food Safety Challenge Grant, The Development of an Efficacious Vaccine to Reduce Campylobacter in Chickens. $2,500,000. Award period: 8/1/2012 – 7/31/2017. 3) Moxley, R. (PD, Univ. of Nebraska) and T.G. Nagaraja and David Renter (Co-PDs, Kansas State Univ.) USDA NIFA Coordinated Agricultural Program Award, Shiga-toxigenic Escherichia coli (STEC) in the Beef Chain: Assessing and Mitigating the Risk by Translational Science, Education and Outreach. Total funding: $25,000,000 for 16 institutions, 51 collaborators. Award period: 1/1/2012 – 12/31/2016

Publications

Peer-reviewed journal articles • Zuowei Wu, Rachel Sippy, Orhan Sahin, Paul Plummer, Ana Vidal, Diane Newell, and Qijing Zhang. 2014. Genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolates associated with sheep abortion in the United States and the Great Britain. J Clin Micro, 52:1853-61. PMID: 24648552. • Zuowei Wu, Orhan Sahin, Fei Wang, Qijing Zhang. 2014. Proteomic identification of immunodominant membrane-related antigens in Campylobacter jejuni associated with sheep abortion. Journal of Proteomics, 99:111-122. PMID: 24487037. • Orhan Sahin, Eric R. Burrough1, Nada Pavlovic, Tim S. Frana, Darin M. Madson, and Qijing Zhang. 2014. Campylobacter jejuni as the cause of canine abortions in the Midwest United States. J Vet Diagn Invest, 26:699-704. PMID: 25085872. • Ma L, Wang Y, Shen J, Zhang Q, Wu C. 2014.Tracking Campylobacter contamination along a broiler chicken production chain from the farm level to retail in China. Int J Food Microbiol. 181:77-84. PMID: 24831929. • Wang Y, M. Zhang, F. Deng, Z. Shen, C. Wu, J. Zhang, Zhang Q, and J. Shen. 2014. Emergence of multidrug-resistant Campylobacter with a horizontally acquired ribosomal RNA methylase. Antimicrob. Agents Chemother. 58(9):5405-5412. • Ma, L., Z. Shen, G.W. Naren, H. Li, X. Xia, C. Wu, J. Shen, Q. Zhang, and Y. Wang. Identification of a novel G2073A mutation in 23S rRNA in amphenicol-selected Campylobacter jejuni. PLoS One. 9(4):e94503. doi: 10.1371/journal.pone.0094503 • Su, C.C., A. Radhakrishnan, N. Kumar, F. Long, J. R. Bolla, H.T. Lei, J. A. Delmar, S. V. Do, T.H. Chou, K. R. Rajashankar, Q. Zhang and E. W. Yu. 2014. Crystal structure of the Campylobacter jejuni CmeC outer membrane channel. Protein Sci. 23:954-961. • Shen, Z., T. Luangtongkum, Z. Qiang, B. Jeon, L. Wang, and Q. Zhang. 2014. Identification of a novel membrane transporter mediating resistance to organic arsenic in Campylobacter jejuni. Antimicrob Agents Chemother. 58(4):2021-2029. • Oh, E., Q. 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Antibiotic growth promoters enhance animal production by targeting bile salt hydrolase and its producers. Frontiers in Microbiology (section Antimicrobials, Resistance and Chemotherapy). 5:33. doi: 10.3389/fmicb.2014.00033 • Wu, J.E, J. Lin, and Q. Zhong. 2014. Physical and antimicrobial characteristics of thyme oil emulsified with soluble soybean polysaccharide. Food Hydrocolloids. 39:144-150. • Smith, K., X. Zeng, J. Lin. 2014. Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system. PLOS One 9(1):e85344. DOI: 10.1371/journal.pone.0085344 • Zhang, H.W., X. Zeng, Q.Qi, K.L. Sun, C.J.Ma, X.J. Hu, J. Lin. 2014. The interaction of CmeA and CmeC is independent of substrate and CmeB in Campylobacter jejuni CmeABC system. Journal of Chinese Science Bulletin (In Press) • Zeng, X., S. Brown, B. Gillespie, J. Lin. 2014. A single nucleotide in promoter modulates the expression of the ?-lactamase OXA-61 in Campylobacter jejuni. 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Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeq®) in gnotobiotic pig model. Vaccine 32:816-24. • Lee,C.S., C. Lee, J. Marion, Q. Wang, L.J. Saif and J. Lee. 2014. Occurrence of human enteric viruses at freshwater beaches during swimming season and its link to water inflow. Science of the Total Environment 472:757-766. • Jung, K, K. Scheuer, Z. Zhang, Q. Wang, L. J. Saif. 2014. Pathogenesis of GIII.2 bovine norovirus, CV186-OH/00/US strain in gnotobiotic calves. Vet Microbiol. 168:202-207. • Takanashi, S., L.J. Saif, J. H. Hughes, T. Meulia, K. Jung, K. A. Scheuer, and Q. Wang. 2014. Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures. Arch. Virol. 159:257-266. • Amimo, J.O, Okoth, E, Junga, J O, Ogara, W.O, Njahira, M.N, Wang, Q ,Vlasova, A.N, Saif, L.J, and Djikeng, A. 2014. 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Impact Statements

Studies on C. jejuni clone SA will provide insights into how this bacterium causes disease and how protective immunity is generated, which will be critical for us to develop vaccines and other interventions to control the disease in pregnant animals.
The data generated from prevalence studies in cattle feces and milk will significantly enhance our knowledge of the ecology and epidemiology of Campylobacter in ruminant reservoirs and provide important information for the development of preharvest and postharvest strategies to control Campylobacter transmission to humans.
The studies on Campylobacter in poultry flocks will generate important information that can be utilized to control Campylobacter in poultry, which is a significant source of foodborne illnesses. The outcomes will improve food safety at multiple levels of production and consumption, and will benefit the U.S. poultry industry by helping them meet the new performance standards set by USDA-FSIS.
Development of effective Campylobacter vaccine for poultry would reduce Campylobacter load in poultry, consequently reducing human Campylobacteriosis.
Litter management studies have shed light on potentially easy and producer-friendly approaches to reduce Campylobacter contamination in broilers and cognate production practices.
In 2013-14, we (Michigan) completed 13 experiments infecting mice with C. jejuni strains from patients with Guillain Barré Syndrome (GBS) and patients with colitis. We identified mouse models for studying GBS induced by C. jejuni, and used the model system to elucidate C. jejuni-host interaction mechanisms.
We have demonstrated that passive immunotherapy using antibodies against C. jejuni colonization associated proteins is not effective in reducing C. jejuni colonization in chickens. This study raises the need to develop better understanding of colonization factors of C. jejuni in chickens so that improved methods can be developed for control of this public health pathogen in the reservoir host (poultry).
The important risk factors for campylobacteriosis vary according to the demographic characteristics of the population, including g age, occupation and ethnicity. We found that direct contact with cattle and living or working on a dairy farm were significant contributors to the campylobacteriosis disease burden, but in our data Hispanic ethnicity was one of the strongest risk factors. In order to design public health interventions the effect of ethnicity, above and beyond the effects of food consumption, contact with livestock, and age, need to be explored further.
Antimicrobial resistance studies may open new avenues for treatment and prevention of resistant foodborne pathogens important in animal health and food safety.
Research on the development of alternatives to antibiotic growth promoters will lead to novel ?One Health? measures for enhanced animal production, food safety, and human health.
The detection of two strains of B. hyodysenteriae with high MIC antibiograms will enable tracking the potential spread of such strains.
Our model supported findings from observational studies of nosocomial risks in veterinary teaching hospitals; primarily, length of hospital was the strongest risk factor for acquiring resistant and/or non-resistant strains. Other findings included that more frequent cleaning, particularly of locations with high patients traffic, would be beneficial. In addition, decreasing the rate of transmission given contact, i.e. improved health care worked hygiene and protective wear, would also be beneficial.
We elucidated several biologically plausible risk factors for introduction and maintenance of a novel resistance gene on commensal E. coli in dairy cattle (blaCTX-M-bearing E. coli).
We have evidence showing that blaCTX-M may confer a selective advantage over blaCMY-2 depending on the environmental concentration of ceftiofur.
We have evaluated and compared methods for a set of cattle-origin blaCTX-M-bearing E. coli. We also compared combinations of loci and measured diversity and correspondence to the standard 7-locus MLST scheme for E. coli. A six-locus method including 5 of the standard loci plus fimH was as or more discriminatory than MLST.
The research on whether enrofloxacin metaphylaxis affected the prevalence and fluoroquinolone susceptibility profiles of Salmonella and Campylobacter recovered from feces of feedlot cattle will enhance risk management plans for antimicrobial use in cattle.
Virulence characterization of several S. Enteritidis mutants led to identification of several novel intervention targets. CpG study will improve our understanding of immunomodulatory mechanisms utilized by Salmonella for successful propagation in the host. Furthermore, the CpG motifs identified in this study could also serve as targets for development of oligonucleotides that can improve the efficacy of currently used vaccines.
We have demonstrated that multi-drug resistance is common among Salmonella isolates belonging to 8 major serotypes that are commonly isolated from poultry in the US. We have also demonstrated that majority of MDR isolates are biofilm formers suggesting possible association between these two phenotypes. This study will contribute toward understanding population dynamics of Salmonella in poultry and aid in developing strategies to control these Salmonella in a serotype-independent manner.
?B. hampsonii? genotypes were found to be epidemiologically related to their site and system of origin. The detection of the same or clonally related genotypes in a site temporally, despite a history of apparently successful ?B. hampsonii? elimination or control, suggests the possibility of a re-infection from a source within the site and/or system.
Swine dysentery in swine farms incurs huge economic loss. Our VDL assay has been and continues to be a preferred reference laboratory for Brachyspira diagnostic testing and research trials.
Data from studies on USDA, FSIS-adulterant (EHEC) will be used to populate a microbial risk assessment model with the goal of reducing the occurrence and public health risks of these organisms in beef. Studies of the regulatory mechanisms of enterotoxin expression in ETEC swine will increase our knowledge of how these organisms cause disease, and potentially lead to new preventive and therapeutic approaches that reduce our use of antibiotics, e.g. through dietary means.
Risk factors and prevalence estimates for Shiga toxin-producing Escherichia coli O groups and virulence genes in feces, as well as Salmonella in feces and subiliac lymph nodes, of commercial feeder cattle are critical to populate risk assessments and identify the distribution, intervention opportunities and potential risks of human illness.
The research on bacterial virulence mechanisms will enhance the development of vaccines and antimicrobial products.
Our preliminary data show that house flies in confined cattle facilities carry STEC-8 and likely play a role in the ecology and dissemination of this food-borne pathogen. Integrated management of house flies should be incorporated into pre-harvest food safety strategies.
Phylogeographic structuring of O157 explains much or all of international variation in the incidence of O157-associated human disease.
The extrinsic origin of seasonal variation suggests that control efforts should focus much more strongly on environmental (non-cattle) reservoirs of this pathogen.
The results from pathogenesis studies of OH strains of PDCoV in Gn pigs will provide new reagents and assays for PDCoV and contribute to the comprehensive understanding of the pathogenesis of US PDCoV strains to promote new measures for control and prevention of PDCoV infections in US swine.
Knowledge of basic aspects of homologous and heterologous RV replication in the swine host will aid in: a) development of more efficacious preventive or therapeutic tools; and b) understanding of the mechanisms of RV interspecies transmission
Studies on the effect of probiotics on neonatal immune responses and rotavirus infection will provide crucial information for the development of innovative oral treatment strategies using targeted probiotics in neonates to efficiently protect them against enteric pathogens and improve vaccine-induced immunity.
While the pathogenesis of RVH in pigs is unknown, the detection of rotavirus H in pigs identifies another possible cause of diarrhea in pigs. The identification of rotavirus H in humans and pigs may suggest a possible interspecies transmission, which needs further investigating.
We describe the occurrence of PDCoV in diagnostic samples and the phylogenetic relationships of the global PDCoV strains. Since PDCoV was recently identified in the United States, the pathogenesis and emergence of PDCoV is still unknown. Serological assays are needed to determine the prevalence of this pathogen.
Our study highlights the different etiology for swine rotavirus A, B, and C within four pig age groups (1-3, 4-20, 21-55, and >55 days) within the United States. However, the cause for the differences between RV infections still needs further investigating, as well as whether global swine rotavirus etiology is the same as the United States.
Cryptosporidium parvum causes a debilitating diarrhea of livestock either alone or in concert with other enteropathogens. Our studies have great potential to benefit not only animal health but will reduce the likelihood of zoonotic spread of Cryptosporidium parvum through contamination of the water supply from domestic livestock operations.

Date of Annual Report: 02/03/2016

Report Information

Annual Meeting Dates: 12/05/2015 - 12/06/2015
Period the Report Covers: 10/01/2014 - 09/30/2015

Participants

Peter Johnson, USDA
Margo Holland, USDA
Roy Curtis, Arizona State University
Qijing Zhang, Iowa State University
Frank Blecha, Kansas State University
Weiping Zhang, Kansas State University
Phil Hardwidge, Kansas State University
M.M. Chengappa, Kansas State University
T.G. Nagaraja, Kansas State University
Linda Saif, The Ohio State University
Gireesh Rajashekara, The Ohio State University
Hua Wang, The Ohio State University
Qiuhong Wang, The Ohio State University
Dongwan Yoo, University of Illinois at Urbana-Champaign
Richard Isaacson, University of Minnesota
Fernando Leite, University of Minnesota
Connie Gebhart, University of Minnesota
Rodney Moxley, University of Nebraska-Lincoln
Jun Lin, University of Tennessee
Bledar Bisha, University of Wyoming
Sheela Ramamamoorthy, North Dakota State University
Radhey Kaushik, South Dakota State University
Joy Scaria, South Dakoda State University
Devendra Shah, Washington State University
Tom Besser, Washington State University
Kaori Mohr, Zoetis
Jenny Welch, Zoetis
Teshome Yehualaeshet, Tuskegee University
Suja Aarattuthodiyil, University of Arkansas at Pine Bluff
Deland J Myers Sr, Prairie View A&M University
Tumen Wuliji, Lincoln University in Missouri

Brief Summary of Minutes

Minutes Attachment:

Summary of Meeting Minutes 2015.docx

Accomplishments

Objective 1. Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals.  A. Campylobacter jejuni   Iowa Pathogenesis of Campylobacter jejuni clone SA. We identified a highly virulent, tetracycline-resistant C. jejuni clone (named clone SA) that has recently emerged in ruminant reservoirs and become the predominant cause of sheep abortion in the United States. To understand the virulent mechanisms of clone SA, we took advantage of natural competence of C. jejuni, positive selection in animal model, and next generation sequencing to directly identify the genetic determinant causing abortion. We proved that sequence polymorphisms in the outer membrane protein are both necessary and sufficient for inducing abortion in pregnant animals. These findings identify a key virulence determinant responsible for abortion induction by Campylobacter and illustrate the powerfulness and necessity of combining experimental evolution with genomic approaches for understanding bacterial pathogenesis.    B. Salmonella   Washington State Antimicrobial resistance profiling of > 600 isolates belonging to twelve most prevalent poultry associated Salmonella serotypes (MPPSTs) recovered from the US poultry sources. Most of the isolates were pan-susceptible (52%) or displayed resistance to 1 (24.5%) or 2 (20.5%) antibiotic classes, whereas few isolates (3%) were resistant to ≥3 antibiotic classes. Multi-drug resistance (≥3 antibiotic classes) including resistance to third generation cephalosporin (2.4%) was primarily limited to Heidelberg and Kentucky. All MPPSTs except Kentucky were recently identified by CDC among the top 30 clinically relevant serotypes isolated from human illnesses in the US. The prevalence of Enteritidis (R2 = 0.650) and Heidelberg (R2 = 0.899) significantly (P < 0.01) correlated with the annual incidence of human illness due to these serotypes.    We assessed the antimicrobial activity of chlorine against twelve MPPSTs using a chicken-meat-based model to simulate the processing environment within the immersion chilling tank. We observed that the level of chicken meat extract contamination is an important contributing factor for survival of Salmonella against chlorine treatment.    Genetic characterization of S. Kentucky isolates recovered from the US poultry (n=140) and human (n=29). Based on PFGE patterns, two major clusters (A and B) were identified at a similarity level of 50%. Our data suggests that the clinical isolates within cluster B are likely of domestic origin and clinical isolates within cluster A are likely of international origin and indicate a possible international spread of multi-drug resistant S. Kentucky.     C. Shiga toxin-producing E. coli (STEC)     Nebraska Comparison of enrichment broths for supporting growth of Shiga toxin-producing Escherichia coli. We observed that STEC grew significantly better when enriched in EC (E. coli broth) compared to TSB. Modification of TSB by the addition of bile salts improved the growth and detection of STEC compared to TSB alone.   Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and pre-intervention carcass surfaces of feedlot cattle at harvest. This study indicates that “top 6” and O157:H7 EHEC are present on hides, and to a lesser extent, pre-intervention carcasses of feedlot cattle at harvest. However, continued improvement in non-O157 detection methods is needed for accurate estimation of prevalence given the discordant results across protocols.   Prevalence and concentration of enterohemorrhagic Escherichia coli in culled dairy cows at harvest.   Matched samples (n = 300) were collected from 100 cows at harvest and tested by a culture-based method and 2 molecular-based methods, NeoSEEK STEC (NS), and Atlas STEC EG2 Combo (Atlas). This study provides evidence that non-O157 EHEC are ubiquitous on hides of culled dairy cattle, and feces are an important source of non-O157 EHEC hide contamination.   Hemorrhagic colitis associated with enterohemorrhagic Escherichia coli O165:H25 infection in a yearling feedlot heifer. A 1-year-old feedlot beef heifer was moribund with neurological signs and bloody diarrhea.   An E. coli O165:H25 strain was isolated from the colonic mucosal tissue, and by microarray analysis contained diverse typical virulence genes. To our knowledge: this is the first report of disease in cattle associated with EHEC O165:H25 infection; the oldest bovine EHEC disease case with isolation of the pathogen; and the first bovine case to demonstrate grossly-evident, hemorrhagic, colonic mucosal erosions associated with EHEC infection.     Kansas Seasonal aspects of prevalence of non-O157 STEC in feces of feedlot cattle. We conducted a study to determine the prevalence of seven STEC-7 and their virulence genes in feces of commercial feedlot cattle during summer and winter months. We did identify seasonal differences and fecal shedding was highly variable between pens of cattle, which needs to be explored further.   Regional, feedlot, and pen-level variability in prevalence of STEC. We conducted a study looking at regional, feedlot, and pen-level variability in prevalence of non-O157 STEC in feces of feedlot cattle. The pre-harvest fecal prevalence estimates, and their distribution across pens, feedlots, and states obtained in this study provide necessary data to integrate into the quantitative microbial risk assessment.   Quantification of Microbial Transfer from Hides to Carcasses during Commercial Beef Slaughter Operations. We are collecting hide and carcass samples from each of 4 large commercial slaughter plants in the north and south regions. We are evaluating matched samples through the beef slaughter process at five sampling points: 1) hides (hide-on) on the kill floor; 2) pre-wash carcass; 3) post-wash/pre-evisceration carcass; 4) post-evisceration carcass; 5) post-evisceration/post-final intervention carcass.   Prevalence and concentration of STEC-7 on hides of fed and culled cattle harvested within the same plant and time. Our group and others have generated limited data on STEC prevalence and concentration for each of these cattle types. However, there is a challenge when synthesizing data/results from multiple independent studies that may differ in several important factors (e.g., time/season, geography, and diagnostic methods) other than simply cattle type. We have established a collaboration to address this issue.   Basic Reproduction Number and Transmission Dynamics of Shiga toxin-producing E. coli. We developed a Markov chain model to simulate the dynamics of 7 serogroups of Shiga toxin-producing Escherichia coli (O26, O45, O103, O111, O121, O145, and O157) in cattle production environments based on a set of cross-sectional data in two years in two states. We demonstrated that different detection sensitivity levels of the pathogen change the dynamics of prevalence, thus better detection techniques and estimates of their performance are critical for risk assessment and future studies.   Role of stable flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) in the confined cattle environment. Stable flies were collected over three summer months and processed individually for STEC. Our data indicated that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.   Role of house flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) in the confined cattle environment. The results from this study indicate that house flies likely play a role as vectors and/or reservoirs of non-O157 STEC in confined cattle environments.   D. Brachyspira hampsonii   Minnesota Identify the molecular epidemiology of pathogenic Brachyspira species circulating in U.S. swine herds. We characterized isolates from diverse epidemiological sources using MLST and compared the genotypes from multiple sites and systems within the U.S. Further, we compared genotypes from the U.S. with those identified globally. Though the species is genetically diverse, with 5 genetic groups identified to date, it represents a single species, genetically closest to the commensal Brachyspira species.    E. Coronavirus     Ohio Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets versus 26-day-old weaned pigs. Our study confirmed that the US PEDV PC21A is highly enteropathogenic in conventional, seronegative nursing piglets. Our study was the first to explore differences in the the innate immune responses of nursing and weaned pigs to PEDV infection.   Isolation and characterization of porcine deltacoronavirus (PDCoV) from pigs with diarrhea in the United States. PDCoV OH-FD22 strain collected from intestinal contents of a diarrheic pig from Ohio was successfully isolated in ST and LLC-PK cell cultures. The results of this study are critical for the development of new serologic tests for PDCoV and expansion of our knowledge of the biology and epidemiology of PDCoV in swine.     Illinois In our study, the role of IFN responses for PEDV infections was examined. Our data suggest that type I IFNs seem to restrict PEDV, and in turn PEDV seems to code for IFN antagonizing proteins. There is a need to conduct such studies to understand the pathogenesis of PEDV, which will provides a new insights into the design of a new vaccine candidate for control of the disease.     Minnesota Genomic and evolutionary inferences between American and global strains of PEDV. We determined the complete genomes of 93 PEDV field samples from US swine and analyzed the data in conjunction with complete genome sequences available from GenBank (n=126) to determine the most variable genomic areas. Our findings suggest that significant genetic events outside of the spike region have contributed to the evolution of PEDV.   Characterization and evolution of porcine deltacoronavirus in the United States. We investigated the presence of PDCoV in diagnostic samples, which were further categorized by case identification (ID), and the association between occurrence, age, specimen and location between March and September 2014. Approximately, 7% of the case IDs submitted from the US were positive for PDCoV. Our results indicate that oral fluids continue to be a valuable specimen to monitor swine herd health, and PDCoV has been circulating in the US prior to 2014.   Identification and complete genome of Seneca Valley Virus in vesicular fluid and sera of pigs affected with ediopathic vesicular cisease, Brazil. Numerous ongoing outbreaks in Brazilian swine herds have been characterized by vesicular lesions in sows and acute losses of neonatal piglets. The complete genome of Seneca Valley virus was identified in vesicular fluid and sera of sows, providing evidence of association between SVV and vesicular disease and viremia in affected animals.     South Dakota We developed and validated new method for whole genome sequencing of PEDV directly from fecal samples. A total of 56 samples were tested using our method.   We also developed an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of PEDV. All assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.     F. Calicivirus       Ohio The genetic mechanism of cell culture adaptation of an enteric calicivirus, porcine sapovirus (PoSaV) Cowden strain was investigated. By using the reverse genetics system, we identified that four (178, 289, 324, and 328) amino acid substitutions in VP1, but not the substitutions in the RdRp region, were critical for the cell culture adaptation of PoSaV Cowden strain. Our findings have revealed the molecular basis for PoSaV adaptation to cell culture.   Elucidation of the mechanisms of NoV (and infectious SaV) entry through the roots, their interaction with components of the vascular system, and their internal transport inside lettuce plants. Our preliminary results showed that following RNase treatment of the samples, HuNoV particles could not be detected in lettuce or spinach leaves when the virus was introduced through the petiole or the roots. In contrast, porcine SaV particles were detected under both settings.   Identification of the lettuce cell wall carbohydrate(s) involved in NoV binding and the inhibition of such binding by plant-derived lectins. Our results suggest that H type HBGA-like carbohydrates exist in lettuce tissues, and GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall materials.     G. Rotavirus     Minnesota Widespread rotavirus H (RVH) in commercially raised pigs. In the United States, 30 positive RVH samples were detected form 10 different states. Sequencing analysis suggested RVH has been circulating in the United States swine population for at least one decade but probably longer.   Detection and high occurrence of porcine rotavirus A, B, and C by real time RT-PCR in diagnostic samples. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). Although RVA was detected at the highest overall percentage (62%), 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1–20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs.        H. Antimicrobial Resistance     Iowa Mutation and antibiotic resistance. Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. We performed experiments to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance. The mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni. The mutY point mutation also led to &sim; 700-fold increase in the emergence of ampicillin-resistant mutants. The G595 → T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter.   Function and regulation of multidrug transporters. The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. Among the 64 C. jejuni isolates examined in this study, We observed that overexpression of cmeABC did not affect the susceptibility of C. jejuni to most tested antimicrobials except for chloramphenicol, but promoted the emergence of ciprofloxacin-resistant mutants under antibiotic selection. Differential expression of CmeABC may facilitate Campylobacter adaptation to antibiotic treatments.   Rising fluoroquinolone resistance in Campylobacter of bovine origin. Here, we analyzed the antimicrobial susceptibilities of C. jejuni and C. coli obtained from 65 different feedlot cattle in different states with total 320 C. jejuni and 115 C. coli isolates. Our findings reveal the drastic increase in the prevalence of fluoroquinolone-resistant Campylobacter in feedlot cattle in the United States and highlight the need for enhanced effort to understand the ecology of antibiotic resistant Campylobacter in the ruminant reservoir.     Tennessee Molecular basis of conjugation in Campylobacter jejuni. Conjugation is an important mechanism for horizontal gene transfer. Using a unique co-transformation strategy in conjunction with comparative genomics analysis, Cj1501c was identified as a key restriction-modification enzyme involved in high frequency conjugation in C. jejuni.   Development of bile salt hydrolase (BSH) inhibitors, novel alternative to antibiotic growth promoters.   Our previous studies suggested that BSH inhibitors are promising alternatives to AGP for enhanced animal growth performance and food safety. The in vivo efficacy of riboflavin, one BSH inhibitor identified from high-throughput screening, was evaluated in a chicken study. At 21 days of age, average body weight per bird in riboflavin-treated group is significantly higher than that in control group, supporting our hypothesis. The BSH from L. salivarius was successfully crystalized (1.8 Å resolution) and the motifs and amino acids critical for BSH activity were identified.     Ohio Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania: Campylobacter occurred in 9.5% of the beef carcasses and 13.4% of the raw milk samples, respectively. Resistance to various antibiotics was observed. In addition, 46.3% isolates were classified as multi drug resistant Campylobacter.   Antimicrobial Resistance and Genotypic Diversity of Campylobacter Isolated from Pigs, Dairy, and Beef Cattle in Tanzania: Campylobacter occurred in 32.5, 35.4, and 19.6% of the pig, dairy, and beef cattle samples, respectively. Resistance to important antimicrobials and multi-drug resistance was also observed; with the highest resistance noted for ampicillin (75.7%) and the lowest for chloramphenicol (4.5%), MLST typing revealed seven novel sequence types (six C. jejuni and one C. coli).       Wyoming We have developed MALDI-TOF MS protocols for identification and typing of antimicrobial resistant bacteria. Approximately 3,000 presumptive cephalosporin and fluoroquinolone resistant E. coli, macrolide resistant Enterococcus spp., and methicillin resistant Staphylococcus spp. isolates collected from 40 cattle facilities and associated wildlife were initially identified by MALDI-TOF MS.       Objective 2. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.     Campylobacter jejuni     Arizona jejuni vaccine development. Recombinant attenuated Salmonella vaccines (RASV) are being utilized in industry compatible conditions for assessment of utility in production and refined to produce greater and more consistent reductions. Results indicate the potential for a reduced dose to maintain efficacy with regard to reduction of colonization by C. jejuni NCTC11168 following challenge. Studies investigating the efficacy of non-gavage-based, industry compatible delivery methods were also performed. A commercial spray cabinet was used to administer vaccine, as was addition of the vaccine to drinking water. Results of these studies indicate that these dosing methods may be viable options for effective delivery of this vaccine to poultry, though further development is required.   Tennessee Development and evaluation of Campylobacter vaccine. Two large chicken trials were performed to immune response and protective efficacy of intranasal vaccination of prepared subunit vaccines.   Intranasal vaccination with encapsulated protein significantly elicited serum IgG and mucosal IgA response. However, the encapsulated DNA vaccine did not trigger significant immune response   Ohio Control of broiler litter contamination with C. jejuni: Our approach evaluated the impact of different chemical treatment of litter with [aluminum sulfate (Alu), sodium bisulfate (Sob), and magnesium sulfate (Mgs)] and the combination of three chemicals (Alu+Sob+Mgs) on the C. jejuni survival. The litter treatment with combination of three compounds can significantly reduce the number of C. jejuni in ceca and in litter under specific experimental conditions. Metagenomics analysis also confirmed this finding.   The role of chemotaxis in C. jejuni’s pathobiology: Our findings highlighted important roles for chemotaxis proteins in the pathobiology of C. jejuni. Our study also revealed that certain chemotaxis proteins (Tlps) can serve as possible targets for controlling C. jejuni in hosts.   Transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants:   Our study highlighted a novel regulatory role for the Ppk enzymes, which advanced our understanding of the biology of this organism. Our findings further highlighted the importance of these enzymes as potential targets for anti-C. jejuni therapeutics      B. STEC and ETEC     Nebraska Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. Molecular and biochemical studies demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.     Kansas ETEC vaccinology. We used quantitative proteomics to identify 24 proteins that differed in abundance in membrane protein preparations derived from wild-type vs. a type II secretion system mutant of ETEC. A subset of these proteins and identified antigens were generated and evaluated in mice in terms of immunogenicity and protective efficacy. Immunization with either Skp or MipA provided an intermediate degree of protection, 68 and 64 %, respectively.   Role of lipid rafts in bacterial infection. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity.   Washington We conducted an oral immunization trial of cattle with E. coli O157:H7, using a pool of three spontaneously Stx-negative E. coli from the EHEC1 lineage (beta-glucuronidase negative, sorbitol non-fermenting, eae positive, O157 antigen positive, fliC-H7 positive), administered in repeated frequent (2/wk for six weeks), high (10e9/dose) oral gavage challenges. Taking into account both O157 vaccine strains and challenge strains, the O157 immunized group did not exhibit reduced RAJ colonization compared to the sham-immunized group. We are continuing to test the hypothesis that high summertime seasonal shedding of O157 results from introduction of new season forage crops. We are sampling pens of dairy and feedlot cattle just prior to and just after introduction of the new forage crops as well as transition feeding. Most of the data to date have focused on adult dairy cattle and shown relatively minor impact on STEC shedding including O157. By comparing STEC shedding in adjacent pens with shared water troughs with shedding in adjacent pens lacking shared water troughs, the specific role of common water sources in STEC epidemiology was evaluated. The preliminary analysis, a general linear model of absolute STEC prevalence difference conditional on shared water through or shared fence line, found no effect of shared water through on absolute prevalence difference.     C. Salmonella     Washington We completed virulence testing and metabolic profiling of a Salmonella pathogenicity island 13 (SPI-13) mutant of S. Enteritidis and demonstrated that SPI-13 contributes to (i) pathogenesis in streptomycin pre-treated mice but not in day-old chickens and (ii) the metabolic fitness of Salmonella Enteritidis through glucuronic acid and tyramine metabolism.   We tested the virulence of 90 wild-type Salmonella isolates belonging to twelve most prevalent poultry-associated Salmonella serotypes (MPPSTs) using avian macrophages as a surrogate model for intracellular survival. We found that there are significant inter- and intra-serotype differences in the invasion and survival of MPPSTs in avian macrophages and induction of nitric oxide. Most notably S. Schwarzengrund, a serotype associated with invasive Salmonella infection in human, showed higher invasion (3.35%) and higher intracellular survival (8.1%) whereas Kentucky, a serotype rarely associated with human disease, showed low invasion (1.7%) and low intracellular survival (1.15%) in avian macrophages.     D. Brachyspira     Minnesota Identify the epidemiological associations of antibiograms of Brachyspira hyodysenteriae and “Brachyspira hampsonii” circulating in U.S. swine herds. General antimicrobial susceptibility trends for U.S. Brachyspira species showed high susceptibility to tiamulin, valnemulin, and carbadox and low susceptibility to tylosin and lincomycin. Very few U.S. B. hyodysenteriae strains showed low susceptibility to multiple drugs (little tiamulin resistance). The general patterns of antimicrobial susceptibility profiles varied by the species of Brachyspira, by type of site, and by system of origin (suggesting system-specific susceptibility).   Identify candidate virulence-associated genes of Brachyspira hyodysenteriae and “Brachyspira hampsonii”. To date, we have sequenced the genomes of 14 B. hyodysenteriae and 2 “B. hamsponii” isolates. Genomes of reference high and low virulence strains, one each, are available publically. After genome assembly (either de novo sequencing or by reference mapping), comparisons of high and low virulence B. hyodysenteriae strains have been performed using four different methods.    E.  Calicivirus and Norovirus   Kansas Entry of caliciviruses. We reported that bile acids facilitate virus escape from the endosomes into the cytoplasm for successful replication of porcine enteric calicivirus (PEC). This is through the ceramide formation by the activation of sphingomyelinase (ASM). Inhibition of ASM resulted in the retention of PEC, feline calicivirus, or murine norovirus in the endosomes in correlation with reduced viral replication, suggesting the importance of viral escape from the endosomes for the replication of various caliciviruses.   Norovirus antivirals. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of anti-norovirus therapeutics. We have reported the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrated in vivo efficacy of an inhibitor using the murine model of norovirus infection.     F. Rotavirus   Ohio Broad-spectrum antibiotic and probiotic treatments: Effects on VirHRV disease severity and immune responses in Gn pigs colonized with/without the defined commensal microflora (DMF). We have demonstrated that in Gn pigs colonized with defined microflora, broad-spectrum antibiotic treatment resulted in increased HRV infection and disease severity, likely, altering innate and adaptive immune responses and bacterial composition. Additionally, we confirmed that EcN probiotic treatment moderated the above parameters, thus, ameliorating HRV disease.   Comparative in vivo and in vitro studies of porcine rotavirus G9P and human rotavirus Wa (G1P) in gnotobiotic pigs: The changing epidemiology of porcine and human group A rotaviruses (RV), including emerging G9 strains, may compromise the efficacy of current vaccines. Understanding the genetic features of the new emerging RV strains will contribute to development of new surveillance and safe and efficacious vaccines.   Wyoming Develop improved methods to capture and detect viruses from bioaerosols. Modification of SKC BioSamplers for capture of viruses from bioaerosols improved detection sensitivity by 8.5x and 2x for the virus surrogates MS2 and Φ6 bacteriophages, respectively. The modifications to the SKC BioSampler are straight forward and add minimum cost, allowing for simplification of sampling procedures and addition of Sample buffer incorporated protectants.     G. Cryptosporidium parvum   Illinois We have developed a battery of complementary in vitro and in vivo assays that allow us to quantitfy Cryptosporidium, Plasmoddium, and Toxoplasma microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity or growth of these parasites is inhibited by CpV and CDPK inhibitors.     H. Microbiome-Host Interactions   Ohio Establishment of a human microbiota transplanted neonatal pig model for future research to assess interactions among enteric pathogens, diet and host factors (commensal bacteria and immunity). We have recently established a human microbiota transplanted neonatal Gn pig model. This model will allow increased knowledge of the interactions among the commensal microbiota, enteric pathogens, immunity and diet and to test future potential interventions to restore the affected immune function and microbial structure.   Minnesota Characterizing the functional microbiome of the swine gut in response to treatment with growth promotor tylosin. The microbiome of animals treated with tylosin, an antimicrobial growth promoter, were analyzed with PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) to predict their metagenome and allow for metabolic analyses. The swine used in this study were housed in two independent commercial farms (10 swine per group) and were fed normal diet (Control) or diet containing tylosin (40 ppm). Large scale changes in function of gut microbiome were not observed between the two farms or tylosin treatment. The major functions found in the microbiome of animals were carbohydrate metabolism (10.6%), amino acid metabolism (9.4%), membrane transport (13.1%) and DNA replication and repair (9.4%). Other studies have also found that differences in gene function of a microbiome remain largely the same despite perturbation of microbial populations within the gut.     I. Clostridium difficile and Clostridium sordellii.   South Dakota Genomic and phenomic characterization of Clostridium difficile and Clostridium sordellii. Despite strong similarities between the two Clostridial species, differences in their host tissue preference place C. difficile infections in the gastrointestinal tract and C. sordellii infections in soft tissues. To improve our understanding of C. sordellii and C. difficile virulence and pathogenesis, we performed a comparative genomic and phenomic analysis of the two. The proteins involved in the metabolic differences between C. sordellii and C. difficile should be targets for further studies.       Objective 3. Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures.   The PIs and Graduate Students involved in the project have been continuing to give presentations and updates enteric diseases and food safety at various scientific, veterinary, and diagnostic meetings in the previous year. They have effectively disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians. They also have generated many high impact peer-reviewed journal articles. The members have actively organized various outreach/education activities, such as 1) Dr. Armstrong, as chair of the University of Arizona Food Safety Consortium, organized the 6th annual University of Arizona Food Safety Conference on September 18, 2015. The keynote address was provided by Bonnie Fernandez-Fenaroli, Executive Director for the Center for Produce Safety and outcomes from University of Arizona food safety research programs were presented. In attendance were members of the academic community, regulatory agencies and the food production and processing industries; and 2) Through several venue Kansas State have provided educational opportunities to professional and graduate students as well as continuing education to veterinarians on enteric pathogens in livestock production systems. The majority of the shared information has focused on the impacts of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter, and antimicrobial use/resistance in the beef industry, the conclusions that can be reached based on recent research, and the potential opportunities to reduce these pathogens in beef production systems.     Objective 4. Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings.   <ul> <li>NC-1202 members and their students presented their work in numerous national and international meetings. We held annual NC1202 meetings in Dec of 2015, and also sponsored 3 student awards for best oral and poster presentations at the Conference of Research Workers in Animal Disease meeting. </li> <li>NC 1202 organized a Mini-Symposium focused on Microbiome and Animal Health on Dec 7, 2015. Seven keynote speakers were invited to present their research work. NC 1202 members discussed the theme for 2016 symposium and unanimously selected the topic of antimicrobial resistance.</li> <li>NC-1202 members have established active collaborations, partly reflected by joint grant/publications. Following are three active integrated food safety projects in which our NC1202 members are project directors and involve several NC1202 members at the participating institutions as co-project directors: </li> </ul> <ol> <li>Zhang, Q. (PD, Iowa State Univ.) and other Co-PDs who are NC1202 members: Jun Lin (Univ. of Tennessee), Gireesh Rajashekara (Ohio State Univ). USDA NIFA Food Safety Challenge Grant, Novel Approaches for Mitigation of Campylobacter in Poultry. $2,500,000. Award period: 07/01/2012 – 06/30/2017 </li> <li>Law, B. (PD, Univ. of Arizona) and other Co-PIs who are NC1202 members: Roy Curtiss III is a scientific advisor, and Ken Roland from the Curtiss lab is the Co-PI. USDA NIFA Food Safety Challenge Grant, The Development of an Efficacious Vaccine to Reduce Campylobacter in Chickens. $2,500,000. Award period: 8/1/2012 – 7/31/2017.</li> <li>Moxley, R. (PD, Univ. of Nebraska) and T.G. Nagaraja and David Renter (Co-PDs, Kansas State Univ.) USDA NIFA Coordinated Agricultural Program Award, Shiga-toxigenic Escherichia coli (STEC) in the Beef Chain: Assessing and Mitigating the Risk by Translational Science, Education and Outreach.  Total funding: $25,000,000 for 16 institutions, 51 collaborators. Award period: 1/1/2012 – 12/31/2016  </li> </ol> <ul> <li>Dr. Rajashekara led NC-1202 team for submission of a CAP project to NIFA Food Security Program in 2015, which was not awarded but received “High Priority” score.</li> </ul>

Publications

Peer-reviewed journal articles   Wang, Yang; Dong, Yanni; Deng, Fengru; Liu, Dejun; Yao, Hong; Zhang, Qijing; Shen, Jianzhong; Liu, Zhihai; Gao, Yanan; Wu, Congming; Shen, Zhangqi. 2015. Species shift and multidrug resistance of Campylobacter from chicken and swine, China, 2008-2014. Journal of Antimicrobial Chemotherapy. In press.   Deng, Fengru, Jianzhong Shen, Maojun Zhang, Congming Wu, Qijing Zhang, and Yang Wang. 2015. Constitutive and inducible expression of the ribosomal RNA methylase gene erm(B) in Campylobacter. Antimicrob. Agents Chemother. 59(10):6661-4.   Qin, Shangshang, Hui Qi, Qijing Zhang, Di Zhao, Zhen-Zhen Liu, Hao Tian, Lijuan Xu, Hui Xu, Mengmeng Zhou, Xianju Feng, Hong-Min Liu. 2015. Emergence of extensively drug-resistant Proteus mirabilis harboring a conjugative NDM-1 plasmid and a novel Salmonella genomic island 1 variant (SGI1-Z). Antimicrob. Agents Chemother. 59(10): 6601–6604.   Dai, Lei, Wayne T Muraoka, Zuowei Wu, Orhan Sahin, and Qijing Zhang. 2015. A single nucleotide change in mutY increases the emergence of antibiotic-resistant Campylobacter jejuni mutants. Journal of Antimicrobial Chemotherapy 07/2015; DOI:10.1093/jac/dkv190 Grinnage-Pulley, Tara and Qijing Zhang. 2015. Genetic Basis and Functional Consequences of Differential Expression of the CmeABC Efflux Pump in Campylobacter jejuni Isolates. PLoS ONE 10(7):e0131534.   Sahin, Orhan, Issmat I. Kassem, Zhangqi Shen, Jun Lin, Gireesh Rajashekara, and Qijing Zhang. 2015. Campylobacter in Poultry: Ecology and Potential Interventions. Avian Diseases 59(2):185-200.   Kovač, Jasna, Katarina &Scaron;imunović, Wu Zuowei, Anja Klančnik, Franz Bucar, Qijing Zhang, Sonja Smole Možina. 2015. Antibiotic resistance modulation and modes of action of (-)-[alpha]-pinene in Campylobacter jejuni. PLoS ONE 10(4): e0122871.   Kandasamy, S., Vlasova, A.N., Chattha, K.S., Fisher, D., Shao, L., Rajashekara, G., Saif, L.J. 2015. Escherichia coli Nissle colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model. JVI (Accepted).   Shao, L., Fischer, D.D., Kandasamy, S., Rauf, A., Langel, S.N., Wentworth, D.E., Stucker, K.M., Halpin, R.A., Lam, H.C., Marthaler, D., Saif, L.J., Vlasova, A.N. 2015. Comparative in vitro and in vivo studies of porcine rotavirus G9P[13] and human rotavirus Wa G1P[8]. J Virol. 2015 Oct 14. pii: JVI.02401-15. [Epub ahead of print].   Vlasova, A.N., Kandasamy, S., Chattha, K.S., Rajashekara, G., Saif, L.J. 2015. Comparison of probiotic lactobacilli and bifidobacteria effects, immune responses and rotavirus vaccines and infection in different host species. Veterinary Immunology and Immunopathology. (In press).   Jung, K., Annamalai. T., Lu, Z., Saif, L.J. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing versus 26-day-old weaned pigs. Veterinary Microbiology. July 9;178(1-2): 31-40. 2015.   Jung, K. B. Eyerly, T. Annamalai, Z. Lu and L.J. Saif. 2015. Structural alteration of tight and adherens junctions in villous and crypt epithelium of the small and large intestine of conventional nursing piglets infected with porcine epidemic diarrhea virus. Vet. Microbiol.177(3-4):373-378.   Annamalai, T., L.J. Saif Z. Lu, and K. Jung. 2015. Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs. Vet Immunol and Immunopath doi:10.1016/j.vetimm.2015.09.006.   Hu, H., Jung, K., Vlasova, A.N., Chepngeno, J., Lu, Z., Wang, Q., Saif, L.J. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States. Journal of Clinical Microbiology 53, 1537-1548. 2015.     Esseili MA, Chin A, Saif L, Miller SA, Qu F, Lewis Ivey ML, Wang Q. 2015. Postharvest Survival of Porcine Sapovirus, a Human Norovirus Surrogate, on Phytopathogen-Infected Leafy Greens. J Food Prot 78:1472-1480.   Esseili MA, Saif LJ, Farkas T, Wang Q. 2015. Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce. Appl Environ Microbiol 81:5085-5092.   Esseili MA, Gao X, Tegtmeier S, Saif LJ, Wang Q. 2015. Abiotic stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and its surrogates on Preharvest Leafy Greens. Appl Environ Microbiol doi:10.1128/AEM.02763-15.   Zhongyan Lu, Masaru Yokoyama, Ning Chen, Oka Tomoichiro, Kwonil Jung, Kyeong-Ok Chang, Thavamathi Annamalai, Qiuhong Wang*, and Linda J. Saif*. Molecular mechanism of cell culture adaptation of porcine sapovirus. (J. Virol, Accepted).   Kashoma IP, Kassem II, Kumar A, Kessy BM, Gebreyes W, Kazwala RR and Rajashekara G (2015) Antimicrobial Resistance and Genotypic Diversity ofCampylobacter Isolated from Pigs, Dairy, and Beef Cattle in Tanzania. Front. Microbiol. 6:1240. doi: 10.3389/fmicb.2015.01240   Chandrashekhar K, Kassem II, Nislow C, Gangaiah D, Candelero-Rueda RA, Rajashekara G. Transcriptome analysis of Campylobacter jejuni polyphosphate kinase (ppk1 and ppk2) mutants. Virulence. 2015 Nov 5:1-5. [Epub ahead of print] PubMed PMID: 26537695.   Kashoma IP, Kassem II, John J, Kessy BM, Gebreyes W, Kazwala RR, Rajashekara G. 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] PubMed PMID: 26153978.   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. PubMed PMID: 26075188; PubMed Central PMCID: PMC4444964.   Zeng, X., B. Gillespie, and J. Lin. 2015. Important role of a putative lytic transglycosylase Cj0843 in β-lactam resistance in Campylobacter jejuni. Frontiers in Microbiology (section Antimicrobials, Resistance and Chemotherapy) 6:1292. DOI:10.3389/fmicb.2015.01292   Zhang, Y., J. Lin, Q. Zhong. 2015. S/O/W emulsions prepared with sugar beet pectin to enhance the viability of probiotic Lactobacillus salivarius NRRL B-30514. Food Hydrocolloids (In Press). Xu, F., F. Guo, C. Wu, G. Cui, X. Zeng, B.Yang, H. Zhou, J. Lin. 2015. Transcriptomic analysis of Campylobacter jejuni NCTC 11168 in response to epinephrine and norepinephrine. Frontiers in Antimicrobials, Resistance and Chemotherapy. 6:452. doi: 10.3389/fmicb.2015.00452.   Zeng, X., D. Ardeshna, and J. Lin. 2015. Heat shock enhanced conjugation efficiency in standard Campylobacter jejuni strains. Applied and Environmental Microbiology. 81:4546-4552.   Zhang, Y., J. Lin, Q. Zhong. 2015. The increased viability of probiotic Lactobacillus salivarius NRRL B-30514 encapsulated in emulsions with multiple lipid-protein-pectin layers. Food Research International. 71:9-15.   Lin, J., K. Nishino, M.C. Roberts, M. Tolmasky, R.I. Aminov, and L. Zhang. 2015. Mechanisms of antibiotic resistance. Frontiers in Microbiology (section Antimicrobials, Resistance and Chemotherapy). 6:34. Doi:10.3389/fmicb.2015.00034   Mirajkar NS and Gebhart CG. Comparison of agar dilution and Etest with broth microdilution for susceptibility testing of swine Brachyspira species. J. Vet. Diagn. Invest. 2015 (In press).   Mirajkar, NS, AZ Bekele, YY Chandler, and CJ Gebhart, 2015. Molecular epidemiology of novel pathogen “Brachyspira hampsonii” reveals relationships between diverse genetic groups, regions, host species, and other pathogenic and commensal Brachyspira. J. Clin. Microbiol. PMID:26135863   Mirajkar N.S. and Gebhart C.J. Understanding the molecular epidemiology and global relationships of Brachyspira hyodysenteriae from swine herds in the United States: A Multi-Locus Sequence Typing Approach. PLoS ONE 9(9): e107176. doi: 10.1371/journal.pone.0107176   Vannucci FA, Linhares DC, Barcellos DE, Lam HC, Collins J, Marthaler D. Identification and Complete Genome of Seneca Valley Virus in Vesicular Fluid and Sera of Pigs Affected with Idiopathic Vesicular Disease, Brazil. Transbound Emerg Dis. 2015 Dec;62(6):589-93. doi: 10.1111/tbed.12410. Epub 2015 Sep 7. PMID: 26347296   Borewicz KA, Kim HB, Singer RS, Gebhart CJ, Sreevatsan S, Johnson T, Isaacson RE. Changes in the porcine intestinal microbiome in response to infection with Salmonella enterica and Lawsonia intracellularis. Plos One 2015 Oct 13;10(10):e0139106. doi: 10.1371/journal.pone.0139106. eCollection 2015.   Homwong N, Jarvis MC, Lam HC, Diaz A, Rovira A, Nelson MI, Marthaler D. Characterization and evolution of porcine deltacoronavirus in the United States. Prev Vet Med. 2015 Nov 10. pii: S0167-5877(15)30045-3. doi: 10.1016/j.prevetmed.2015.11.001. [Epub ahead of print] PMID: 26611652.   Jarvis MC, Lam HC, Zhang Y, Wang W, Hesse RA, Hause BM, Vlasova A, Wang Q, Zhang J, Nelson MI, Murtaugh MP, Marthaler D. Genomic and evolutionary inferences between American and global strains of porcine epidemic diarrhea virus. Prev Vet Med. 2015 Nov 10. pii: S0167-5877(15)30041-6. doi: 10.1016/j.prevetmed.2015.10.020. [Epub ahead of print] PMID: 26611651.   Johnson, T., Singer, R., Isaacson, R., Danzeisen, J., Lang, K., Kobluk, K., Rivet, B., Borewicz, K., Frye, J., Englen, M., Anderson, J., and Davies, P. In vivo transmission of an IncA/C plasmid in Escherichia coli depends on tetracycline concentration, and acquisition results in variable cost of fitness. Appl. Environ. Microbiol. 10, 2015.   Pragman, A., Isaacson, R., Wendt, C., and Reilly, C. A method for determining taxonomical contributions to group differences in microbiomic investigations. Journal of Computational Biology, Accepted March 14, 2015.   Kim, H. B. and Isaacson, R. E. The pig gut microbial diversity: understanding the pig gut microbial ecology through the next generation high throughput sequencing. Vet Microbiol. 177:242-251 (2015).   Pérez-Méndez A, Chandler JC, Paar J, Doolittle M, Bisha B, Goodridge LD. 2015. Field-based evaluation of an integrated F-RNA coliphage concentration and detection method. Applied and Environmental Microbiology (submitted).   Chandler JC, Manley WA, Pérez-Méndez A, Bisha B, Adkins JA, Henry CS, Prenni JE, Goodridge LD. 2015. Molecular and phenotypic properties of cantaloupe associated Listeria monocytogenes. Food Microbiology (submitted).   Johnson DC, Bzdek JP, Fahrenbruck CR, Chandler JC, Bisha B, Goodridge LD, Hybertson BM. 2015. An innovative non-thermal plasma reactor to eliminate microorganisms in water. Desalination and Water Treatment. DOI: 10.1080/19443994.2015.1024752.   Bisha B, Brehm-Stecher BF. 2014. Flow cytometry for rapid detection of Salmonella spp. in seed sprouts. ScienceOpen Research. DOI: 10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1.     Wijemanne, P., J. Xing, E. M. Berberov, D. B. Marx, D. H. Francis, and R. A. Moxley. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains. PLoS One 10(3):e0117663.   Stromberg, Z. R., G. L. Lewis, D. B. Marx, and R. A. Moxley. 2015. Comparison of enrichment broths for supporting growth of Shiga toxin-producing Escherichia coli. Curr. Microbiol. 71:214-219.   Stromberg, Z. R., N. Baumann, G. L. Lewis, N. Sevart, N. Cernicchiaro, D. G. Renter, D. B. Marx, R. K. Phebus, and R. A. Moxley. 2015. Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and carcass surfaces of beef feedlot cattle at harvest. Foodborne Pathog. Dis. 12:631-638.   Stromberg, L. R., Z. R. Stromberg, A. Banisadr, S. W. Graves, R. A. Moxley, and H. Mukundan. 2015. Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli. J. Microbiol. Methods 116:1-7.   Moxley, R. A., Z. R. Stromberg, G. L. Lewis, J. D. Loy, B. W. Brodersen, I. R. Patel, and J. Gangiredla. 2015. Hemorrhagic colitis associated with enterohemorrhagic Escherichia coli O165:H25 infection in a yearling feedlot heifer. JMM Case Reports 2:1-6. doi:10.1099/jmmcr.0.005004.   Stromberg, Z. R., G. L. Lewis, S. S. Aly, T. W. Lehenbauer, N. Cernicchiaro, N. and R. A. Moxley. Prevalence and concentration of enterohemorrhagic Escherichia coli in culled dairy cattle at harvest. J. Food Prot. Accepted and in press.   Chopyk, J., R. Moore, Z. R. Stromberg, G. L. Lewis, D. G. Renter, N. Cernicchiaro, R. A. Moxley, and K. E. Wommack. Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides. Microbiome. Submitted.   Stromberg, L. R., N. W. Hengartner, K. L. Swingle, R. A. Moxley, S. W. Graves, G. A. Montaño, H. Mukundan. Membrane insertion for the detection of lipopolysaccharides: exploring the dynamics of amphiphile-in-lipid assays. PLoS Pathog. Submitted.   Stromberg, Z. R., G. L. Lewis, and R. A. Moxley. Comparison of agar media for the detection and quantification of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O121, O145, and O157 in cattle feces. J. Food Prot. Submitted.   Scaria J, Suzuki H, Ptak CP, Chen JW, Zhu Y, Guo XK, Chang YF. Comparative genomic and phenomic analysis of Clostridium difficile and Clostridium sordellii, two related pathogens with differing host tissue preference. BMC Genomics. 2015 Jun 10;16:448.   Okda F, Liu X, Singrey A, Clement T, Nelson J, Christopher-Hennings J, Nelson EA, Lawson S. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus. BMC Vet Res. 2015 Aug 1;11:180.   Zhou M, Duan Q, Li Y, Yang Y, Hardwidge PR, Zhu, G. Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling, Archives of Microbiology, 2015, May 3.   Kumar A, Hays M, Lim F, Foster LJ, Zhou M, Zhu G, Miesner T, Hardwidge PR. Protective enterotoxigenic Escherichia coli antigens in a murine intranasal challenge model, PLoS Neglected Tropical Diseases, 2015, Aug 5;9(8):e0003924.   Zhou M, Yang Y, Chen P, Hu H, Hardwidge PR, Zhu G. More than a locomotive organelle: flagella in Escherichia coli, Applied Microbiology and Biotechnology, 2015.   Zhang Y, Vadlani, PV, Kumar A, Hardwidge PR, Govind R, Tanaka T, Kondo A. Enhanced D-lactic acid production from renewable resources using engineered Lactobacillus plantarum, Applied Microbiology and Biotechnology, 2015.   Cull CA, Renter DG, Bello N.B., Ives S.E., Babcock A.H. Performance and carcass characteristics of commercial feedlot cattle from a study of vaccine and direct-fed microbial effects on E. coli O157:H7 fecal shedding. J An Sci. 2015; 93(6): 3144-3151.   Dewsbury DM, Renter DG, Shridhar PB, Noll LW, Shi X, Nagaraja TG, Cernicchiaro N. Summer and winter prevalence of Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121, O145, and O157 in feces of feedlot cattle. Foodborne Pathog Dis. 2015; (8):726-732.   Noll LW, Baumgartner WC, Shridhar PB, Cull CA, Dewsbury D, Shi,X, Cernicchiaro N, Renter DG, Nagaraja TG. Pooling of immunomagnetic separation beads does not affect sensitivity of detection of six serogroups of Shiga toxin-producing Escherichia coli in cattle feces. Journal of Food Protection (accepted for publication).   Ison S, Delannoy S, Bugarel , Nagaraja TG, Renter DG, den Bakker H, Nightingale K, Fach P, Loneragan G. Targeted amplicon sequencing for SNP genotyping of attaching and effacing Escherichia coli O26:H11 cattle strains using a high-throughput library preparation technique. Appl Enviorn Microbiol (accepted for publication).   Noll LW, Shridhar PB, Dewsbury DM, Shi X, Cernicchiaro N, Renter DG, Nagaraja TG. A comparison of culture- and PCR-based methods to detect six major non-O157 serogroups of Shiga toxin-producing Escherichia coli in cattle feces. PLoS One. 2015; 10(8):e0135446.   Noll LW, Shridhar PB, Shi X, An B, Cernicchiaro N, Renter DG, Nagaraja TG, Bai J. A four-plex real-time PCR assay, based on rfbE, eae, stx1, and stx2 genes, for the detection and quantification of Escherichia coli O157 in cattle feces. Foodborne Pathogens and Disease 12(9):787-794.   Shivanna V, Kim Y, Chang KO. Bile acid-mediated Activation of the Acid Sphingomyelinase Facilitates Endosomal Escape of Porcine Enteric Calicivirus. 2015. Virology. 483:218-28.   Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Kyeong-Ok Chang, William C. Groutas, Recent Advances in the Discovery of Norovirus Therapeutics. J Med Chem. 2015 Aug 17.   Kim Y, Shivanna V, Hua DH, Groutas WC, and Chang KO. Broad-spectrum protease inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.2015. J Virol. 89(9):4942-50.   Kankanamalage AM. Uy WP, Mandadapu SR, Alliston KR, Chang KO, Kim Y, Lovell S, Groutas WC. Structure-Based Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic and Cell-Based Studies. 2015. J Med Chem. 58(7):3144-55.   Toro M, Cao G, Rump L, Nagaraja TG, Meng J, Gonzalez-Escalona N. Genome Sequences of 64 Non-O157:H7 Shiga Toxin-Producing Escherichia coli Strains. Genome Announc. 2015 Oct 1;3(5). pii: e01067-15. doi: 10.1128/genomeA.01067-15.   Amachawadi RG, Scott HM, Vinasco J, Tokach MD, Dritz SS, Nelssen JL, Nagaraja TG. Effects of In-Feed Copper, Chlortetracycline, and Tylosin on the Prevalence of Transferable Copper Resistance Gene, tcrB, Among Fecal Enterococci of Weaned Piglets. Foodborne Pathog Dis. 2015 Aug;12(8):670-8. doi: 10.1089/fpd.2015.1961.   Amachawadi RG, Nagaraja TG. First Report of Anaerobic Isolation of Salmonella enterica from Liver Abscesses of Feedlot Cattle. J Clin Microbiol. 2015 Sep;53(9):3100-1. doi: 10.1128/JCM.01111-15.   Ison SA, Delannoy S, Bugarel M, Nightingale KK, Webb HE, Renter DG, Nagaraja TG, Loneragan GH, Fach P. Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States. Appl Environ Microbiol. 2015 Jun;81(11):3671-8. doi: 10.1128/AEM.00397-15.   Agga GE, Scott HM, Vinasco J, Nagaraja TG, Amachawadi RG, Bai J, Norby B, Renter DG, Dritz SS, Nelssen JL, Tokach MD. Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs. Prev Vet Med. 2015 May 1;119(3-4):179-89.doi: 10.1016/j.prevetmed.2015.02.008.   Amachawadi RG, Scott HM, Aperce C, Vinasco J, Drouillard JS, Nagaraja TG. Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle. J Appl Microbiol. 2015 Jun;118(6):1287-97. doi: 10.1111/jam.12790.   Kumar A, Menon S, Nagaraja TG, Narayanan S. Identification of an outer membrane protein of Fusobacterium necrophorum subsp. necrophorum that binds with high affinity to bovine endothelial cells. Vet Microbiol. 2015 Mar 23;176(1-2):196-201. doi: 10.1016/j.vetmic.2014.12.015.   Amachawadi RG, Scott HM, Nitikanchana S, Vinasco J, Tokach MD, Dritz SS, Nelssen JL, Goodband RD, Nagaraja TG. 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Impact Statements

• Characterization of C. difficile and C. sordellii. These two species are emerging pathogens that infect both humans and animals. Our results based genomic and phenomic analysis of C. difficile and C. sordellii could be useful in designing interventions reduce the incidence of these pathogens in humans, swine and cattle.

Date of Annual Report: 01/30/2017

Report Information

Annual Meeting Dates: 12/03/2016 - 12/04/2016
Period the Report Covers: 01/01/2016 - 01/01/2017