Brief Summary of Minutes of Annual Meeting

The meeting was held during Dec. 4-5, in Chicago, IL, in conjunction with the annual CRWAD meeting. 1. Proposal rewrite a. A writing team was assembled and team members include Linda Mansfield, Richard Isaacson, and others. b. Rewrite and submission of the proposal will be completed in 2011. c. Along with the resubmission, the committee discussed potential expansion of the scope to include poultry and possible name change for NC1041. The main contact for poultry experts would be Mo Saif, Laslo and Zack (SE Poultry Research Center). d. Mark Robinson can keep us abreast of the NIFA priorities. 2. Rushmore Conference a. The committee decided to hold another Rushmore conference on enteric diseases. b. The proposed meeting will be in conjunction with the CRWAD meeting next year (2011) in Chicago and will run from the Friday Evening through Sunday noon immediately preceding CRWAD. c. David Francis, Rod Moxley, and Richard Isaacson will lead the organizing effort. Richard Isaacson has agreed to write a USDA Conference Grant which will focus on expenses incurred through inviting speakers for the meeting. d. Urgent business with regard to the meeting includes identification of an over-all theme and session themes likely to stir interest in the meeting and a willingness of members of the medical research community to join us for the meeting. e. In concert with the identification of timely conference themes, we need to identify speakers to anchor sessions and who have fresh ideas sufficient to draw attendance and also to foster collaborations with NC-1041 participants. 3. Appendix E Form - Everyone was reminded to fill out the Appendix E form. This can be done through each universitys AES office. 4. USDA NIFA - Mark Robinson gave an update on USDA NIFA and state representatives presented progress reports.

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 Iowa Ecology and pathogenesis of a newly emerged and highly pathogenic Campylobacter jejuni clone. We recently identified a highly pathogenic Campylobacter jejuni clone (SA) as the major cause of sheep abortion in the United States. To delineate the pathogenesis of this unique clone, we determined the complete genome sequence of a representative clone SA isolate (IA3902) and initiated functional genomics analysis. To determine if the SA clone was also present in healthy sheep, we tested a total of 103 intestinal content and 124 bile samples from a lamb slaughterhouse, and isolated Campylobacter from 45 and 14 of these samples, respectively. Molecular fingerprinting of 48 C. jejuni isolates with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) showed the existence of multiple PFGE profiles and MLST types (14 STs), of which some were more predominant than others. These results indicate a high genetic diversity of C. jejuni isolates from healthy sheep and that multiple genotypes including the highly abortifacient C. jejuni clone SA colonize sheep without causing clinical diseases. These findings suggest that clone SA is well adapted in the sheep production system and is present in both intestinal contents and gall bladder. Michigan Monitoring Campylobacter jejuni using Fluorescent in situ Hybridization. FISH was used following pre-bleaching to detect C. jejuni in paraffin-embedded tissues. A scoring system was created to assess the specificity of the FISH staining. When scored in a blinded fashion, the total score of the positive slides was 97 of a possible 220 points, while the total score of the negative slides was 25 of 220. The average score of a positive slide was 19.4 of a possible 44 points, while negative slides averaged a score of 5 of 44. Prebleaching of autofluorescent epitopes in tissues significantly enhanced the ability to visualize this bacterium in tissues after challenge infection. Since all slides were blinded to the scorer, the results showed that FISH can be a reliable method of tracking C. jejuni in paraffin-embedded tissue. Arizona Feedlot Sampling for the Presence of Campylobacter jejuni. Sampling for the presence of Campylobacter jejuni was accomplished for year two for a cohort of 36 cattle. Fecal swabs were taken from these cattle at the range, at arrival at the feedlot, and monthly for 6 months at the feedlot, and at day of slaughter. Carcass swipes were taken with both the USDA-FSIS method, and the ventral midline method at dehiding, and evisceration. Environmental samples at the feedlot including fecal material from the birds, were conducted prior to arrival, and at 3 months and 6 months. Swipes of feed bunks, watering units, drag swabs of pens, and flies at the feedlot were sampled beginning at prior to arrival of cattle and monthly after for six months. Only pigeons were positive for C. jejuni at pre-arrival of the calves. Other sources for acquiring C. jejuni started to emerge between the months of October through December, including pen floors, feed bunks, and the watering units. Only 1 of 36 cattle was positive for C. jejuni at arrival at the feedlot, but by the end of month one, 28/36 cattle were already positive for C. jejuni. During processing, a significant number of carcasses were positive for C. jejuni after evisceration with the highest number of positives coming from swipes from the ventral midline sites. Pulsed-field gel analysis of C. jejuni isolates from various species at the University of Arizona feedlot. Various species were sampled for the presence of C. jejuni at the University of Arizona feedlot prior to and upon the arrival of calves from the range. A number of these samples were positive and at three months, one isolate from flies and birds had spread to a calf in pen 1 and in pen 2. Only a few calves were colonized at three months in the feedlot with C. jejuni, but at six months there was a major change in isolates colonizing the calves. In addition, there was one calf which became colonized with three different C. jejuni isolates at the same sampling time point. Identification of C. jejuni proteins involved in host colonization. Genes expressing proteins that are secreted and have a surface orientation, or are up-regulated, or express known colonization factors are being mutated and examined for their role in gut colonization. One of those proteins is TlyA which is secreted as a possible hemolysin. The gene secreting this protein has been mutated in our laboratory in C. jejuni strain M129 and examined for its role in the colonization of 14 day-old chicks. Chicks challenged with the tlyA mutant were not colonized. Ohio Campylobacter epidemiology in cattle. In this study, we determined the genetic relatedness of C. jejuni and C. coli isolated form cattle across different geographical locations in the U.S. and investigated their potential for invasion and persistence in human cell lines as well as their antibiotic resistance properties. Campylobacter spp were detected in 181 (19.2%) out of a total of 944 fecal samples. Our results showed that the prevalence of C. jejuni in cattle feces was lower than that of C. coli. The PFGE analysis suggested that the C. jejuni and C. coli isolates were genetically diverse and geographically constrained, possibly reflecting regional prevalence and adaptation. The cattle isolates of C. jejuni showed high invasion and intracellular survival capacity in INT 407 intestinal cells. On the other hand, C. coli strains showed lower invasion and intracellular survivability. Further, bovine Campylobacter isolates showed high resistance to several antimicrobial agents including ciprofloxacin, erythromycin, gentamicin, and nalidixic acid. Furthermore, MLST analysis of 19 selected C. jejuni isolates identified 7 isolates that belonged to ST-21 clonal complex, 2 isolates to ST-42 clonal complex, and one isolate to ST-61 clonal complex which are the main three common STs from human C. jejuni. Occurrence of the invasion associated marker (iam) in Campylobacter jejuni isolated from cattle. We investigated the occurrence of the iam in C. jejuni isolated from cattle, an increasingly important source of Campylobacter infections. To assess the contribution of the iam to C. jejunis host adaptation, we also characterized the potential of iam-containing cattle isolates for chicken colonization and human cell invasion. We show that the prevalence of iam in cattle C. jejuni is relatively lower as compared to isolates occurring in humans and chickens, suggesting the potential for this marker to differentiate C. jejuni from disparate hosts. In addition, iam was polymorphic and certain alleles occur in cattle isolates that were capable of colonizing and invading chickens and human intestinal cells, respectively, which might be important in C. jejunis host(s) association B. Salmonella Arizona Salmonella Newport - emerging pathogen in oysters. Well characterized genes in Salmonella Pathogenicity Island 1 (invA) and Pathogenicity Island 2 (ssaV) were mutated in Salmonella Newport, which resulted in invasion and phagocyte survival deficiencies, respectively. Survival studies revealed that neither of these type-three secretion systems (TTSS) were critical for Salmonella's invasion and survival in oysters. Analysis of the microarrays from the transposon site hybridization (TraSH) assay was completed. These analyses indicated over 90 genes involved in the missing transposon gene pool that was recovered from the challenged oysters. The missing genes included structural proteins such as those needed for the formation of flagella and the type III secretion system as well as proteins involved in metabolic functions and protein transport. The results showed that the elimination of motility by mutating the flagella apparatus significantly reduced Salmonella's ability to colonize oysters. By 30 days post-infection, the flagellar mutant (flgE gene) was unable to be detected in oysters, whereas, the wildtype was present at an average of 102 CFU/g. There was no significant difference between the levels of wildtype and the other 3 mutant strains. Kansas Prevalence and persistence of Salmonella in cohorts of feedlot cattle. Our objective was to determine factors associated with fecal prevalence of Salmonella at feedlot entry and within 24 hrs of harvest (preharvest), and to assess potential persistence of Salmonella strains within cattle populations. We demonstrated (II.1.1) that the fecal prevalence of Salmonella immediately prior to harvest may be higher in subsets of the feedlot population and that specific Salmonella strains appear to persist within and among feedlot cohorts throughout the feeding period. These findings are unique and may facilitate the development of approaches to controlling Salmonella in commercial feedlots. C. Non-O157 Shiga toxin-producing E. coli (STEC) Nebraska Preliminary studies were conducted to validate a published protocol for the concurrent detection of STEC O26, O103, O111, O145 and O157, and to further test its utility for detection of STEC O45 and O121. With only a limited number of studies, it was evident that a screening step in the published protocol may result in a significant number of misclassified organisms or false negative results. A multiplex PCR was developed for the concurrent detection O26, O103, O111, O157, and will be further tested for detection of O45, O121, and O145. Washington State University We 1) applied multiple genotyping systems to a diverse set of E. coli O157:H7 isolates, 2) performed and analyzed genomic sequencing (454 Titanium) to pooled DNA from 30 isolates of bovine biased genotypes (SBI genotypes 3 and 4) under-represented in previous SNP discovery efforts, and 3) compared Stx converting bacteriophage insertion site genotyping to SNP genotyping in a large (N=400 isolates) set of E. coli O157:H7 isolates of diverse (human clinical, environmental, bovine, and retail meat) isolates. 1) Strong concordance was detected between genotypes defined by Stx-converting bacteriophage insertion sites, Lineage Specific Polymorphism Assays, the presence the Q933 bacteriophage allele, and the presence of a specific single non-synonymous nucleotide polymorphism in the tir, all of which detected the same groups of isolates as belonging to genotypes frequently identified in human disease (clinical genotypes) and genotypes common in cattle but rarely found associated with human disease. 2) Over 200 novel, unique SNPs were detected in the SBI genotype 3 and 4 pooled DNA. A subset of 96 of these SNPs were selected for inclusion in a 96-plex SNP allele assay, using the Illumina GoldenGate system. 3) Very strong clustering of SBI genotypes was observed in a phylogenic tree of >400 diverse E. coli O157:H7 isolates generated by SNP polymorphisms by Jim Bono and colleagues at the USDA Meat Animal Research Center. On-going collaborative efforts with that group are expected to result in an efficient SNP genotyping multiplexed test system that will unambiguously classify O157:H7 isolates into groups with strong or very weak human infection potential. D. Caliciviruses Ohio Validation of tissue culture-adapted porcine sapovirus (SaV) as an improved surrogate for HuNoVs to assess viral stability and decontamination methods. Our lab has successfully adapted this strain to cell culture, including serial passage in readily available pig kidney cell lines. Recent improvements to maintain the in vitro propagation of TC-Po/SaV include the use of commercially available bile acids in the culture medium to substitute for the restricted availability of germfree pig intestinal contents. Also serial passage of our original TC-Po/SaV strain has produced consistently higher viral titers in cell culture (range 106-107 TCID50/mL). These improvements also enable other labs to more readily utilize the TC-Po/SaV as a surrogate for the study of decontamination of human NoVs. Characterization and prevalence of a new porcine calicivirus in US swine. New porcine caliciviruses, designated St-Valerien-like viruses, were detected from Canadian swine in 2009. We detected a similar virus, NC-WGP93C strain, from swine fecal samples from NC. We characterized the genome of this strain, designed a real-time RT-PCR assay for virus detection and performed a prevalence study of apparently healthy finisher pigs. The NC-WGP93C strain is genetically similar to the Canadian strains, sharing 89.3-89.7% overall genomic nucleotide identity, and 95.9-96.3% amino acid identity for the predicted capsid protein VP1. The new caliciviruses were detected in all nine North Carolina farms tested (prevalence of 23.8%; range 2.6 to 80%). Our results indicate that St-Valerien-like caliciviruses are prevalent in certain US swine farms. E. Coronaviruses Ohio Full genomes of mink Coronaviruses (CoVs) from fecal samples from diarrheic mink in fur farms in Wisconsin and Minnesota were sequenced and analyzed. Full-genomic sequencing and phylogenetic analysis demonstrated that the CoVs from mink belonged to a potentially new Subgroup, 1c of Group 1 CoVs. The MCoVs clustered together with the recently identified ferret enteric CoV (FECoV), but were distinct from swine CoVs in Group 1 (TGEV, PRCV, PEDV). Also MCoVs possessed high genomic variability and relatively low overall nucleotide sequence identities (91.7%) between contemporary strains suggesting that they are under strong selective pressure. F. BVDV Kansas Onset and duration of transient infections among antibody-diverse beef calves exposed to a Bovine Viral Diarrhea Virus persistently infected calf. Study objectives consisted of 1) estimating the onset and duration of TI based on serum VI and rRT-PCR and 2) determination of the potential of TI cattle to shed BVDV. Two 21 day studies were performed where one PI calf was commingled with a confirmed non-PI cattle population with heterogeneous BVDV antibody status (n=12 and n=15, respectively). After PI exposure, virus isolation on serum and nasal swabs failed to detect BVDV among non-PI cattle. The results demonstrated (II.1.2) that BVDV transmission from persistently infected (PI) cattle to non-PI cattle can result in transient infections that may not lead to overt clinical disease, but could lead to further transmission of BVDV. The speed with which exposed cattle become transiently infected and their potential ability to shed the virus may impact design and implementation of BVDV control programs. G. Enterococci Kansas We demonstrated that the Red Flour Beetle, Tribolium castaneum, can acquire antibiotic-resistant enterococci from animal feed and transfer them to sterile feed. We show that management of T. castaneum may be important to prevent the spread of antibiotic-resistant and virulent enterococci in animal feed and feed manufacturing environments. Our findings also indicate that stored-product insects carry antibiotic-resistant and potentially virulent enterococci. We also have shown that their dispersal behavior and capacity to transport antibiotic-resistant bacteria make house flies a potential threat to public health; thus, area wide management may be needed to mitigate health risks. H. E. coli O157 Kansas We have developed a multiplex real-time PCR (II.1.6) for quantification of E. coli O157 and a multiplex PCR (II.1.7) for detection of seven serotypes of Shiga-toxin producing E. coli (STEC) in bovine feces. These assays could prove to be extremely useful for identifying, quantifying and/or characterizing these important enteric bacteria. I. Brachyspira species Minnesota We have evaluated a diagnostic test that can be used to speciate otherwise non-typable Brachyspira isolates from clinical swine samples. This test involves amplifying a highly variable region of the Brachyspira NADH Oxidase (nox) gene with PCR and then sequencing this PCR product for species identification. In addition, we have used this test to identify 79 non-typable Brachyspira isolates derived from recent swine clinical samples. All of these isolates tested negative for B. hyodysenteriae and B. pilosicoli by PCR. Five Brachyspira species commonly isolated from swine including B. pilosicoli, B. hyodysenteriae, B. intermedia, B. murdochii, and B. innocens were obtained from ATCC and used as controls for the new typing system. A PCR targeting a highly discriminatory and species-specific region of the Brachyspira nox gene was develope. J. Lawsonia intracellularis Minnesota The MLST system, PFGE and Variable number tandem repeat (VNTR) were established molecular typing of L. intracellularis pure culture isolates of porcine, equine, and rodent origin. Porcine and equine models were developed for immune response, pathogenesis, and diagnostic studies of L. intracellularis infection. We also developed improved diagnostics, a quantitative polymerase chain reaction (qPCR) assay, for detecting and monitoring proliferative enteropathy in various species. In addition, we used serial serological and fecal PCR testing for targeted treatment of weanling foals on a farm with endemic L. intracellularis. Furthermore, an alternative protocol for cultivation of L. intracellularis was developed. 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 Outcome of C. jejuni Infection of C57BL/6 IL-10-/- Mice Varies with Strain. Ten individually caged mice were inoculated per os with ~1010 cfu of each single C. jejuni strain. Mice were monitored for clinical signs for 35 days (or until clinical signs necessitated euthanasia) and then euthanized and necropsied. Presence of C. jejuni in tissues and feces was evaluated by culture and PCR; histopathology, in sections of the ileocecocolic junction; and antibody response, by ELISA. Mice inoculated with broth and C. jejuni 11168 served as controls. Variation was observed in colonization proficiency, mouse survival after inoculation, and degree of clinical signs and pathology. Three strains were less proficient colonizers; five strains caused severe disease in the majority of mice. Seven strains caused bloody diarrhea in at least one mouse. One strain produced neurological signs. These results, combined with published data, distinguished five pathotypes of C. jejuni in C57BL/6 IL-10-/- mice: no colonization, colonization with little or no disease, colonization with moderate or severe enteritis, colonization with hemorrhagic enteritis, and colonization with neurological sequelae either with or without enteritis. Preliminary comparisons of genome content (presence or absence of COGS) of the 10 sequenced strains did not reveal genome-wide differences that corresponded to differences in pathogenicity in mice. These results indicate that C. jejuni-infected C57BL/6 IL-10-/- mice display a spectrum of disease outcomes similar to that of humans. Genetic differences related to differences in pathogenicity are likely to be found as point mutations, indels, or changes in gene expression. Ohio Dynamics of Campylobacter jejuni Colonization in poultry. We are using bioluminescent C. jejuni to visualize temporally in real-time the pathogen-host interaction in a greater detail. We have confirmed that bioluminescence can be efficiently expressed in C. jejuni using a plasmid that has the lux genes. We have generated a bioluminescent C. jejuni by integrating bioluminescence reporter, the lux operon, into C. jejuni chromosome through homologous recombination. Over a period of two weeks, we did not observe any bioluminescent signal when the chicks were imaged alive or when the organs were harvested and imaged. However, when the organs were processed and plated onto agar plates a sporadic, weak and inconsistent bioluminescent colonies were noticed suggesting inefficient colonization of bioluminescent C. jejuni or precolonization of chicks with naturally occurring C. jejuni. Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter. Specifically, we constructed bioluminescent Campylobacter strains and used them to monitor the survival of these pathogens in litter (bedding) material. We inserted shuttle plasmids carrying the luminescence genes (luxCDABE) into C. jejuni and C. coli to construct bioluminescent strains of these pathogens. The strains were spiked into microcosms containing samples of litter-washings and dry litter collected from different enclosures that housed broiler chickens. Our results show that C. jejuni and C. coli survived for at least 20 days in reused (old) litter while the growth of these pathogens was inhibited in clean (new) litter. Our study indicated that the Bioluminescence provided a simple, sensitive, and rapid approach for analyzing the growth dynamics of Campylobacter. We investigated the role of PPK1 in C. jejuni pathogenesis, stress survival and adaptation. Our findings demonstrate that C. jejuni ppk1 mutant was deficient in poly P accumulation, which was associated with decreased ability to form viable but non-culturable cells (VBNC) under acid stress. The ppk1 mutant also showed decreased frequency of natural transformation and increased susceptibility to various antimicrobials. Furthermore, the ppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the ppk1 mutant with the wildtype copy of ppk1 restored the deficient phenotypes to levels similar to those of the wildtype. Our results suggest that poly P plays an important role in stress survival and adaptation and might contribute to genome plasticity, and spread and development of antimicrobial resistance in C. jejuni. Polyphosphate Kinase 2. We demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The ppk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. Importantly, the ppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as chicken colonization. Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. B. E. coli South Dakota Development of a Subunit Vaccine for Enterotoxigenic E. coli in pigs. We tested subunit vaccines consisting of K88 fimbriae and/or heat labile enterotoxin (LT) for efficacy against K88+ ETEC in weanling-age piglets. We also analyzed antibody responses to the vaccine antigens. The results suggest that an intranasal vaccine consisting of both antigens is highly protective against a vigorous experimental challenge with K88+ ETEC. Further, this study suggests that the immune response to these antigens occurs rapidly and to such a sufficient titer that pigs are protected at about the age of weaning when post weaning disease is most likely to occur. Finally, the current study provides a model system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies in the development of vaccines for ETEC in humans. Nebraska Inverse relationship between fluid accumulation and adherence of enterotoxigenic Escherichia coli in ligated jejunal loops. The effect of expression of LT and STb by F4ac+ ETEC was studied in ligated jejunal assays in weaned purebred Yorkshire pigs originating from two different farms, each group conducted as an independent experiment. The results of the study involving differential expression of heat-labile enterotoxin (LT) and heat-stable enterotoxin-b (STb) by enterotoxigenic Escherichia coli of swine found that enterotoxin-induced fluid accumulation, which was almost entirely due to STb, flushes progeny organisms into the lumen of the bowel and thereby promotes shedding of the infectious agent in the feces, increasing the likelihood of transmission to new hosts. C. Cryptosporidium parvum Illinois Our laboratory has discovered and characterized a subset of long-chain polyunsaturated fatty acids (L-PUFA), originally isolated from bovine colostrum, which block in vitro Cryptosporidium parvum and Toxoplasma gondii host cell infectivity (Figures 1 and 2) as well as both Toxoplasma gondii and Plasmodium gallinaceum infectivity in vivo (Figures 3 and 4). The results of in vitro host cell invasion assays indicate multiple L-PUFA can inhibit the invasion of MDBK cells by C. parvum sporozoites; however, there is a remarkable structural specificity requirement. To display inhibitory activity the fatty acids must be unsaturated, have a free carboxyl end, and have at least one bend (unsaturation) along the carbon chain. Elaidic acid, the trans isomer of oleic acid, which differs only in conformation (straight versus bended hydrocarbon chain, respectively, showed no inhibitory activity. In addition, L-PUFA must contain less than sixteen carbons and no more than twenty carbons, with maximal activity between eighteen and twenty carbons. Of the fatty acids tested within these specifications, polyunsaturated fatty acids were more effective inhibitors than monounsaturated fatty acids. Preliminary results suggest L-PUFA are inhibiting parasite-host cell invasion by inhibiting sporozoite microneme secretion and gliding motility. Control of Microbial Contamination of Agriculture Watersheds. In this continuing study, rotavirus survival was investigated in three different soil fractions and at three different temperatures (4, 25, and 37C). A rotavirus suspension was mixed with whole soil, sand, and clay and allowed to incubate for up to 30 days. Samples were collected daily to investigate virus survival over time, which was quantified using a tissue-culture infectivity assay. In summary, our preliminary results indicate, in the absence of any soil particles, rotavirus survival is highest at 4C, with survival decreasing as temperature increases. These data also indicate whole soil has some protective effect, allowing rotavirus to survive better in soil for the entire range of temperatures and for more than a week at 37C. The results also show that sand fractions are the most effective media for reducing rotavirus survival at all temperature conditions tested. D. Coronaviruses Ohio We used PRCV infection of pigs as a model to determine the impact of co-infection with PRRSV on the severity of CoV-induced respiratory disease. We investigated the effects of a prior and ongoing PRRSV infection (down-regulator of innate immunity), on PRCV (up-regulator of innate immunity) co-infection and clinical disease and the pathologic and immunologic interactions of the two viruses, as a model for respiratory viral co-infections. The PRRSV/PRCV pigs had greater reductions in weight gains, more frequent fever, and more severe gross or histological pneumonic lesions compared to either single infection at PRCV PID 2 to 21. We evaluated the innate and adaptive immune responses in the 4 groups of pigs. The PRRSV/PRCV dual virus-infected pigs had significantly suppressed innate immune responses, as evident by reduced interferon (IFN)-± levels in lung and blood. These findings imply that innate immune suppression induced by prior infection with an immunomodulating respiratory virus may contribute to more severe disease after a respiratory CoV infection. We examined "NO levels in the lungs of pigs infected with either PRCV or PRRSV, or co-infected with PRRSV and PRCV over the time course of the infection, and analyzed the antiviral effects of "NO on these two viruses in an in vitro system using a "NO donor, S-nitroso-N-acetylpenicillamine (SNAP). A large increase in "NO levels was detected in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. The PRRSV/PRCV dual- and PRCV single-infected pigs had 2.4 to 14 times higher BAL "NO levels at PRCV PID 2 to 10 than the mock control pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with the increased "NO. Our results imply that PRRSV is not susceptible to "NO. E. E. coli O157 Kansas By utilizing modeling techniques to evaluate preharvest interventions for E. coli O157 contamination of beef cattle carcasses, we have shown that combinations of preharvest interventions may be particularly important for supplementing harvest interventions during periods of higher variability in fecal shedding prevalence (i.e., summer). We also have provided useful information on potential effects on E. coli O157 fecal shedding in finishing cattle (and potential intervention opportunities) associated with feeding monensin, urea, ractopamine, or a DFM product. F. BVDV Kansas We used simulation models to quantify the value of implementing whole herd BVDV testing strategies in beef cowcalf herds (II.2.2); the results may be extremely useful for optimizing testing strategies for cowcalf herds. By evaluating data from a state BVDV program (II.2.3), we have generated information that may be useful for animal health professionals since we have indentified potential client education needs for reducing herd exposures to BVDV. G. Norovirus Kansas Our work on norovirus (II.2.4) has demonstrated that AcNg might serve as a good vehicle for oral delivery of IFNs in norovirus infections. In addition, by defining the solution structure of NV Pro (II.2.5), we have provided important information for studying the nature of the protease and protease inhibitors as antivirals against important noroviral infections. H. L. intracellularis Minnesota The humoral immune response and fecal shedding of L. intracellularis was investigated in 20 weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live L. intracellularis vaccine. Foals received either 30 mL frozen-thawed or lyophilized vaccine intra-rectally, given twice, 4 weeks apart. Serum samples from each foal were collected every 4 weeks for 16 weeks following the first vaccination and tested for anti-L. intracellularis specific IgG by IPMA. Rectal swabs were collected every other day following the first vaccination for 4 weeks for detection of L. intracellularis by real-time PCR. Both vaccine formulations administered intra-rectally in weanling foals were safe and led to similar onset and duration of fecal shedding and measurable serum IgG responses against L. intracellularis. A field trial incorporating this intra-rectal vaccine is currently in progress. Objective 3. Focus on disseminating knowledge  Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories Michigan Several students were mentored for study of enteric diseases of food animals. Five students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Jamie Jennifer Kopper (DVM candidate), Ankit Malik and Barbie Gadsden, DVM. Three of these students have passed their preliminary exams. Two are preparing for this exam. Vijay Rathinam, successfully defended his Ph.D. thesis and obtained a postdoctoral position at the University of Massachusetts at Wooster, MA where he is studying the pathogenesis of inflammatory bowel disease due to bacterial pathogens. One undergraduate student working in the lab on these projects was accepted into veterinary schools (Alexander Adrian, University of Minnesota). Dr. Mansfield organized a Fall seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University. People attending have come from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Shannon Manning, PhD, MPH (Shigatoxin producing E. coli) and Robert Britton, PhD (Probiotics for treating enteric diseases). Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health at Cambridge, Maryland on Food and Water borne Pathogens. Dr. Mansfield was an invited speaker for the Norman E. Borlaug International Agricultural Science and Technology Fellowship Program mentor at Polish Food Safety Meeting on 04/23/2010 and gave an invited keynote address at the American Society for Microbiology entitled Examining the genetic basis of pathotypes in Campylobacter jejuni using murine models on 05/28/2010. Dr. Mansfield organized and attended the USDA Multistate Research Project 2009 Meeting NC1041 in Chicago, Illinois on December 4th and 5th. 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 preharvest food safety of cattle food projects. 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; including the Conference of Research Workers in Animal Disease, the Leman Swine Conference, the International Pig Veterinary Society, the American Association of Equine Practitioners, the American College of Veterinary Internal Medicine, the American Association of Swine Veterinarians and the Passion for Pigs Symposium. They have disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians (both swine and equine).

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