Brief Summary of Minutes of Annual Meeting

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.

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.

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.