Annual/Termination Reports:

[01/04/2008] [03/31/2009] [02/04/2010] [03/31/2011] [03/09/2012]

Report Information

Participants

Besser, Thomas - Washington State University;
Robertson, Don - Kansas State University;
Joens, Lynn - University of Arizona;
Saif, Linda - Ohio State University;
Kuhlenschmidt, Mark - University of Illinois;
Frances, David - South Dakota State University;
Kaushik, Radhey - South Dakota State University;
Isaacson, Richard - University of Minnesota;
Gebhart, Connie - University of Minnesota;
Khaitsa, Margaret - North Dakota State University;
Mansfield, Linda - Michigan State University;
Stromberg, Bert - AA University of Minnesota;
Robinson, Mark - USDA, CSREES

Brief Summary of Minutes

1. Mark Robinson USDA CSREES representative gave the committee an overview of the organizational and funding situation. Suggested that committee members ensure trhat they properly report extension and outreach activities. In committee reports, he suggested that committee reports ensure that accomplishments be phrased or summarized in non-technical language. Two or three concise sentences describing outcomes are much more useful and will assist properly crediting committee accomplishments.

2. Bert Stromberg, committee advisor, added three specific points: 1) Advised that each station properly report extension and outreach activities, 2) complete SAES422 fully and carefully as these reports will be critical to the mid-term reviews of the NC1041 activities and 3) Ensure that all membership ensure that their Appendix E forms properly acknowledge extension/outreach activies, i.e. all those activities that are not in the other categories.

3. Don Robertson, incoming chair announced his upcoming retirement and the need for NC1041 to identify an alternative secretary/incoming chair.

4. Emerging diseases discussions and collaborations

a. Linda Saif discussed the evaluation of several alternative modern swine waste treatment systems falling under the category cEST (conventional Environmental Systems Technologies) for their ability to inactivate important viral agents porcine norovirus, porcine sapovirus, and rotaviruses of groups A, B, and C. The anaerobic digester systems had the best effect at reducing viruses detectable by PCR. All systems produced virus negative products when tested by either cell cultures or by gnotobiotic pig assay.

b. Linda Saif reported development of specific diagnostic serologic assays for SARS coronavirus. This was a major advance since previous assays cross-reacted with animal group 1 coronavirus nucleoprotein. The 3 region of the nucleoprotein molecule was found to represent a SARS specific region.

c. Linda Saif reported the isolation and characterization of a bovine-like coronavirus from a giraffe. The isolate was a group 2e (bovine-like) CoV. The virus was used to infect calves and the inoculum and the post-calf infection strains were subjected to genomic sequencing. The isolate was also adapted to cell culture, and the post adaptation virus was also subjected to genomic sequencing. The virus was highly pathogenic in the inoculated calves.

d. Tom Besser reported on a pilot project to look for the agent Escherichia albertii. E. albertii is an emerging agent of disease in humans (best characterized in children in Bangladesh), rabbits (shown by experimental infection), wild birds (shown by natural outbreaks in finch species birds in Alaska and previously in Scotland). The challenges of accurate identification were discussed. The API 20E codes typical of this species will be circulated among the NC1041 group. A study of WADDL accessions identified 4 cases among 300 submissions, including 2 calves, 1pig and 1 chicken.

e. Tom Besser reported on the emerging strain of S. Typhimurium TYP035/187 in Washington state. The strain has diverse resistance phenotypes, and has increased from its first identification in 1999 to account for over 50% of bovine isolates and between 5 and 10% of human isolates in 2006. MLVA analysis shows several branches including at least four which apparently independently acquired plamids encoding cmy2 cephalosporinase.

f. Don Robertson reported on the EAST enterotoxin, showing data that this gene is present in diverse E. coli pathotypes including a very common occurrence in porcine E. coli isolates from both pre- and post-weaning ages groups. Don also reported on the great difficulties encountered in the attempt to produce a recombinant that would produce and secrete functional or intact EAST.

g. Lynn Joens reported on Salmonella in oysters, first summarizing the publications from his laboratory reporting on the common isolation of multi-drug resistant S. Newport from oysters on three coasts. He reported on the lesions produced in oyster intestinal tissues of salmonlla colonized oysters. He reported on the prolonged survival of Newport compared to E. coli in depurated oysters. He reported on the isolation of Salmonella from oysters purchased in restaurants. Discussion followed on the significance of the salmonella contamination and the number of committee members who did or did not appreciate raw oysters.

h. Tom Besser reported on his collaborative part of the Joens oyster project. In approximately 1000 oyster cultures from oyster beds in WA state only three salmonella isolates were obtained, none of which were multi-drug resistant. Therefore the frequent Salmonella contamination of oysters with MDR Newport, at least in the Pacific Northwest, seems to no longer be occurring. The cultures will continue to result in twelve sequential monthly samplings of 3 dozen oysters each from multiple bays impacted by livestock farms, by urban runoff and in pristine bays.

5. Interventions:

a. Linda Saif reported on porcine respiratory coronavirus, the SARS-like pneumonia virus of pigs, evaluating the effects of dexamethazone, a drug commonly used in the treatment of SARS. Dexamethasone seemed to have beneficial effects initially, but resulted in impaired viral clearance suggesting that this treatment may have adverse effects in SARS patients.

b. Linda Saif also reported on the effects of Lactobacillus colonization of gnotobiotic pigs infected with rotavirus in a project designed to determine the effects of the probiotic agents on the B-cell responses to the viral infection. Lactobacillus was not found to augment B-cell responses. Discussion followed on several areas including other possible beneficial effects of the probiotic.

c. Lynn Joens reported on a Campylobacter jejuni pilA mutant. The mutant expressed fewer fibers when EM imaged in biofilms, decreased colonization of chicks, and decreased inflammation in a piglet model. Therefore, this protein represents a possible vaccine antigen.

d. Lynn Joens reported on a C. jejuni cytotoxin. Diverse strains exhibited diversity on their invasive phenotype and their secretion of cytotoxin. Cytotoxin in supernatants was analyzed on 2D gels, exhibiting about 20 spots which are currently being evaluated for aa sequence to identify.

e. Rod Moxley described a series of experiments on F4 producing ETEC isolated from pigs, which produced several different toxins including LT and STb. In young (5-9 day) pigs, tested in collaboration with David Frances, deletion of the LT gene abrogated pathogenicity whereas deletion of the STb gene had no detectable effect. LT had no apparent effect on colonization in this experiment. However, in intestinal loops in 6 to 8 weeks old pigs, the opposite effect was seen and LT had no apparent effect. The hypothesis that fimbrial adhesions are required for LT effects on pathogenicity was proposed.

f. Rod Moxley reported a summary of the effects of the E. coli O157:H7 vaccine. Numerous studies showed a consistent trend for decreased fecal shedding, decreased hide contamination, and decreased rectal mucosal colonization in vaccinated cattle.

g. Rod Moxley also reported on experiments evaluating the effects of transformants bearing plasmids encoding ETEC LT on subsequent colonization by wild type F4 ETEC in in vitro experiments. A plasmid bearing LT decreased adherence of the wild type strain. A plasmid bearing a mutant LT which lacked a cAMP effect also decreased subsequent adherence of the wt strain. Similarly, a plasmid bearing the LT b subunit only decreased/abolished subsequent wt strain adherence.

h. Margaret Khaitsa reported on the effects of direct fed microbial product on colonization of cattle with E. coli O157:H7 and with Salmonella following natural exposure. A significant effect decreasing colonization of cattle with O157 was apparent at weeks 3, 6 and 9. No effect of the product on Salmonella contamination was apparent.

i. Mark Kuhlenschmidt reported on investigations on the interaction of Cryptosporidium sporozooites and cells. An effort to identify cellular products that mediate sporozoite internalization identified oleic acid. Oleic and related cis unsaturated fatty acids markedly decreased sporozoite internalization in cells in a dose-dependent manner. Cool movies of the motility of sporozoites and the rapid internalization of sporozoites were shown.

j. Connie Gebhardt gave a fascinating summary of recent progress and work on Lawsonia. A syndrome of ill thrift in foals was described which is attracting a lot of attention in the equine circles was described.

6. Outreach and other activities (Sunday, December 2)

a. The leadership of NC1041 was discussed. Dick Isaacson agreed to take the chair position for the coming term. Linda Mansfield agreed to take the secretary position for the coming term. The committee agreed that Linda would not be expected to lead the renewal effort that would occur during her term but that instead she would be able to form a committee to take responsibility for the renewal.

b. A discussion of the efforts to add new members to NC1041 took place. Candidate institutions to be contacted include Iowa State, NADC, Purdue, and the Nesbitt ARU. In addition, additional participants from KS, AZ (Songer), and OH (LeJeune) were discussed. Several NC-1041 members will follow up with these institutions to pursue this possibility.

c. Linda Mansfield gave the committee an overview of her research areas and interests, including the ILL-10 knockout mouse model of C. jejuni infection and the immunomodulatory effects of ESPs of Trichuris suis.

d. Margaret Khaitsa gave a summary of a program sponsored by ND for study abroad in Uganda. The program focuses on animal production, disease surveillance, and public health activities. The program requires advance training for preparation and the committee discussed how this training could be accomplished in distant sites through internet or DVD formats. The program included 4 students in its first year, and is expected to include more than ten students in this coming summer program.

The meeting adjourned at 11 am, Sunday December 2.

Accomplishments

Interaction of Salmonella Newport with market oysters: To determine the nature of the interactions between Salmonella Newport and oysters or the aquatic environment, five parameters were examined: 1) vertical transmission between generations of the Pacific Oyster Crassostrea gigas, 2) survival of Salmonella Newport and E. coli in seawater containing plankton, 3) survival of Salmonella Newport in seeded oysters, 4) depuration studies in oysters infected with S. Newport or E. coli, and 5) histopathological effects of oyster colonization with S. Newport. Results indicate that vertical transmission is not responsible for infection of multiple oyster generations. S. Newport was found to survive at significantly higher numbers (90 days) in oysters than E. coli (45 days) following a 72-h depuration period. S. Newport is capable of surviving in oysters after exposure to a variety of contamination levels and can do so at significantly higher levels and for longer periods of time than E. coli. S. Newport survived longer in seawater containing plankton than an environmental E. coli strain, but both S. Newport and E. coli were non-viable after 12 days. Finally, immunohistochemical analysis of oyster exposed tissues show that Salmonella can breach the oysters epithelial barriers and reside within the deeper connective tissues of the oyster. <br /> <br /> Reduction of host colonization with Campylobacter jejuni: Poultry colonization studies of the pilus deficient mutant and parent strain of C. jejuni have been completed in triplicate. The pilus mutant was found to colonize with an average of 2.98X105 CFU/g fecal contents, compared to the wild type of 3.17X108 CFU/g fecal contents. In addition, only 10 of 40 chicks were colonized with the pilus mutant, whereas, 38 of 40 chicks were colonized with the parent strain.<br /> The newborn piglet serves as a model system for C. jejuni. Piglets were infected with a parent and pilus mutant strain of C. jejuni and examined for shedding and lesions typical of campylobacteriosis. Shedding was detected only in piglets inoculated with the parent strain of C. jejuni. Lesions of edema with fluid production, catarrhal inflammation, inflammatory cell infiltrate and epithelial cell erosion were present in 3 of 5 piglets inoculated with the parent strain of C. jejuni. Piglets inoculated with the pilus mutant remained normal during the study.<br /> <br /> Toxin production in the piglet model by Campylobacter jejuni: Isolated poultry strains demonstrate different degrees of virulence during invasion and survival in cultured epithelial cells/macrophages. However, the majority of these isolates were pathogenic when tested in the piglet model. In contrast, only isolates which failed to invade epithelial cells in vitro were able to produce a fluid exudate in the piglet model <br /> <br /> Produced numerous reports abstracts and scientific manuscripts that were published in various journals<br /> <br /> Developed a course International Animal Production, Disease Surveillance and Public Health<br /> <br /> Escherichia coli. Studies conducted with isogenic enterotoxigenic E. coli (ETEC) strains indicated that heat-labile enterotoxin (LT) contributes more to virulence in the causation of severe diarrheal disease in pigs less than two weeks old than heat-stable enterotoxin-b (STb). By contrast, STb appear to potentially play a more important role in post-weaned pigs over six weeks old. A clinical trial with an E. coli O157:H7 type III secreted protein vaccine found that three doses of vaccine significantly reduced fecal shedding of this organism in feedlot cattle, further confirming the potential for vaccination to be useful and effective pre-harvest intervention. <br /> <br /> Pathogenic Brachyspira spp. Attachment of B. pilosicoli, but not B. aalborgi, to cultured human intestinal epithelial cells in vitro elicits the release of interleukin 8 that might play a role in the pathogenesis of colitis in human and animals. A high-throughput nucleotide sequencing and bioinformatic analysis method was developed for allelic profiling by multilocus sequence typing of pathogenic B. pilosicoli isolated from humans and animals with colonic spirochetosis. Re-emergence of swine dysentery in swine production units in the mid-west and North Carolina, require renewed efforts into (i) development of improved methods for specific identification and tracking of B. hyodysenteriae in clinical specimens, (ii) monitoring development of antimicrobial resistance, and (iii) understand basic mechanisms of disease pathogenesis. <br /> <br /> Enteric Viruses in Swine Manure: 1) The high prevalence of PoSaV, RV-A and RV-C in pre-treatment manure samples demonstrates that these viruses are widely distributed and stable in the swine population. 2) The results differed among the technologies evaluated and between viruses. 3) The fact that Gn pigs were not infected by post-treatment samples suggests that infectious virus would not be present in the field after treatment. 5) Our results suggest that all the technologies tested were effective in reducing virus infectivity. <br /> Our findings address a public health concern regarding environmental quality surrounding swine production units.<br /> <br /> Antigenic cross-reactivity between SARS-CoV and group 1 animal CoVs and development of improved diagnostics: We confirmed that two-way antigenic cross-reactivity exists between SARS-CoV and porcine group 1 CoVs and identified the cross-reactive region within the N Protein (aa 1-213) and the SARS-CoV specific region (aa 360-412). Use of such paired N protein fragments in ELISA provides new discriminatory diagnostic reagents to detect SARS-specific CoV antibodies in humans and animals. <br /> <br /> Characterization of new animal CoVs to clarify mechanisms of CoV interspecies transmission: We identified a new bovine-like CoV from giraffes and confirmed its experimental interspecies transmission to cattle suggesting that wild ruminants could harbor CoVs transmissible to cattle or vise versa.<br /> <br /> Altered pathogenesis of and immune responses to PRCV due to corticosteroid treatments: One to two doses of corticosteroid given early in PRCV infection initially reduced the pulmonary lesions. However, continued high doses through 6 days led to more extensive viral replication and accentuated lung lesions. Presumably corticosteroid-induced immunosuppression reflected by decreased innate and early cytokine responses followed by further dampening of local and systemic Th1 IFN-³ responses precipitated a failure to curtail viral replication, resulting in increased shedding and lung lesions.<br /> <br /> Immune responses to Lactic acid bacteria and their influence on intestinal infection by enteric viral pathogens: Significant findings include: 1) RV infection alone recruited monocytes/macrophages to the intestine. 2) LAB colonization down-regulated monocyte/macrophages and DCs in systemic tissues after RV infection suggesting an immunomodulatory effect. 3) LAB or RV promoted innate immune maturation in neonates.<br /> <br /> The committee researchers established the critical role of STx2 enterotoxin in the virulence of E. coli O157: H7. Collaborations between South Dakota and Nebraska have shown the critical importance of LT in colonization of ETEC on piglet epithelium.<br /> <br /> Development of novel methods for detection of waterborne pathogens (Cryptosporidium and rotavirus): We are developing new technology for the sensitive detection of environmental microbial pathogens using label-free photonic crystal biosensor technology. Using this new sensor technology, we have demonstrated that porcine rotavirus can be specifically detected in aqueous samples as sensitively as conventional ELISA but without the need for secondary label processing. <br /> <br /> Rotavirus: We have synthesized a sialyllactosylphosphatidylethanolamine (SLPE) neoglycolipid that displays a potent ability to inhibit both virus binding and infectivity in vitro. In field trials this inhibitor blocked infection, virus shedding and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. In collaborative studies with Dr. Sharon Donovan, Department of Food Science and Human Nutrition, we have shown that genistin, inhibits the infectivity of porcine rotavirus in vitro. We are currently examining the effect of this soy isoflavone on rotavirus infection in vivo as well as the possible synergistic effect of SLPE and specific porcine and human milk oligosaccharides. <br /> <br /> Cryptosporidium parvum: Using a cell-suspension sporozoite adhesion assay we have purified a lipid fraction that inhibits sporozoite-host cell invasion. Preliminary data suggest this lipid is inhibiting sporozoite microneme secretion and gliding motility. <br /> <br /> Contamination of Agriculture Watersheds: We have determined the cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil that occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil. <br /> <br /> <br />

Publications

Impact Statements

1. Interaction of Salmonella Newport with market oysters: Impact: These data imply that Salmonella Newport may not be just a transient resident of the oysters gut, along with other wastewater indicators, and that merely testing growing waters, or even the oysters themselves, for coliforms is likely inadequate for preventing oyster-borne salmonellosis.
2. Reduction of host colonization with Campylobacter jejuni Impact: These studies demonstrated the importance of the pilus protein in both colonization of the host but also in lesion production. This indicates that the pilus antigen would be a very effective immunogen for poultry as well as man.
3. Toxin production in the piglet model by Campylobacter jejuni Impact: This study indicates that isolates that have the ability to invade cells lack the gene (s) or failed to express the toxin gene in vivo. The toxin may be an added virulence factor for C. jejuni isolates.
4. Contributed data on risk assessment of antimicrobial resistance in Salmonella species isolated from ND cattle and humans
5. Validated a possible control strategy (Feeding DFM to feedlot cattle) pre-harvest for the control of E. coli O157:H7 shedding.
6. Provided an opportunity for international training and exposure of 3 students through the course International Animal Production, Disease Surveillance and Public Health.
7. Added an international perspective to our research and training thus providing opportunities for faculty and student collaborations.
8. Escherichia coli: The determination that heat-labile enterotoxin is the most important enterotoxin in the causation of diarrheal disease in F4ac+ enterotoxigenic E. coli (ETEC) infections in pigs less than two weeks old sheds new light on the pathogenesis of this organism, and should help in the development of new intervention and therapeutic strategies. The finding that three doses of a vaccine containing type III secreted proteins of E. coli O157:H7 significantly reduces shedding of this organism in the feces of cattle further confirms that vaccination is a useful and effective pre-harvest intervention for this organism.
9. Helicobacter hepaticus: CDT-induced inhibition of antigen-specific local and systemic immune functions might be involved in persistence of bacterial pathogens in susceptible hosts. Identification of a cell receptor for CDT in susceptible hosts will provide a basis for implementation of genetic selection of livestock resistant to major food-borne bacterial diseases, and basic understanding of mechanisms for important bacterial diseases of humans and animals.
10. Pathogenic Brachyspira spp.: Results from current studies extend our previous observations on B. pilosicoli spirochete attachment onto host epithelial cells and further indicate that this interaction elicits the release of cytokine associated with host inflammatory response. The multilocus sequence analysis method will support ongoing efforts to track transmission of pathogenic intestinal spirochetes between animal and environmental sources and humans. With the current re-emergence of B. hyodysenteriae in the US swine industry, close monitoring of clinical isolates against commonly-used antimicrobial agents is an important priority to prevent the spread of the disease.
11. Enteric Caliciviruses in Animal Wastes: We performed the first evaluation of the impact of different candidate ESTs on virus detection in swine manure, and suggests that although only one candidate EST (ATAD) can reduce virus concentrations to undetectable levels as evaluated by molecular techniques, virus infectivity assay (CCIF) and Gn pig inoculation.
12. Studies of the antigenic cross-reactivity between SARS-CoV and group 1 animal CoVs to develop and improve diagnostics, and preventive measures: We found that fragments aa1-213 and aa360-412 of the SARS-CoV N-protein when used together can be valuable discriminatory diagnostic reagents with the latter reflecting only SARS-CoV specificity.
13. Characterization of new animal CoVs: We demonstrated that wild ruminant CoVs are biologically, antigenically and genetically similar to bovine CoVs from domestic cattle suggesting the possibility of interspecies transmission and adaptation of CoVs to new hosts among the ruminant species.
14. Influence of Lactic acid bacterial colonization and rotavirus infection on distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs. Our study demonstrated that either intestinal probiotic bacterial colonization or pathogenic enteric viral infection effectively promoted the development of innate immunity in neonatal pigs. Thus, probiotic bacteria can be used to stimulate early maturation of the neonatal innate immune system.
15. Enterotoxigenic E. coli (ETEC) is a major cause of postweaning diarrhea in pigs and is responsible for substantial economic loss. An efficacious vaccine for the disease is greatly needed and of high priority for the industry. During the past year, we have made substantial progress towards understanding the pathogenesis of ETEC and its virulence determinants that must be considered in the development of an efficacious vaccine.

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Report Information

Participants

Rodney A. Moxley, University of Nebraska-Lincoln;
Margarethe Cooper, University of Arizona;
Kerry Cooper University of Arizona;
Linda Saif, The Ohio State University;
Bill Sischo, Washington State University;
Margaret Khaitsa, North Dakota State University;
Radhey Kaushik, South Dakota State University;
David H. Francis, South Dakota State University;
Mark S. Kuhlenschmidt, University of Illinois;
Richard Isaacson, University of Minnesota;
Gireesh Rajashekara, The Ohio State University;
Vijay A. K. Rathinam, Michigan State University;
David Renter Kansas State University;

Brief Summary of Minutes

Accomplishments

Progress of Work and Principal Accomplishments<br /> <br /> Objective 1. Focus on emerging diseases- 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.<br /> <br /> A. Salmonella enterica<br /> We sequenced the genotype JJPX01.0014 found in oysters and are currently annotating the sequence. We have also produced a S. Newport mutant library for use in transposon site hybridization studies. The culturing of cells and genomic extraction were repeated using a non-mutagenized Salmonella Newport JJPX 01.0014 control. PCR to the T7 promoter and linker sites in the mutant library was performed with a positive conformation that the transposon had been randomly inserted. We conducted a survey of oysters purchased from restaurants in Tucson from 2007-2008. Samples were tested for Salmonella using a modified version of the FDA BAM manual. A total of 2281 oysters were tested for the presence of Salmonella. Twenty-eight oysters were found to be positive for Salmonella giving us a 1.2% prevalence rate. In addition, 43% of the oysters positive for Salmonella were of the S. Newport serovar.<br /> <br /> The genetic diversity of E. coli O157:H7 and Salmonella isolated at the various fecal sampling stages of feedlot steers fed with direct-fed microbial (DFM; Lactobacillus acidophilus; BT1386) and those not fed DFM during finishing was evaluated. A total of 123 of 454 (27.1 %) and 112 of 454 (24.7 %) E. coli O157:H7 were recovered from FG and the FS respectively (P < 0.05). For DFM treated steers, 48 of 66 (72.7 %) and 34 of 66 (51.5 %) tested positive for E. coli O157:H7 in FG and the FS, respectively (P < 0.05). The control steers tested E. coli O157:H7 positive in 57 of 71 (76 %; FG) and 41 of 71 (57.7 %; FS). (P < 0.05). Pulsed field gel electrophoresis (PFGE) analysis was performed on E. coli O157:H7 (n =194) and on fecal Salmonella (n = 58) isolated from the FS and FG. Of the 194 E. coli O157:H7 isolates tested, PFGE revealed 21 distinguishable genotypes. New genotypes were isolated from 1 of 39 (2.6 %) treated and 19 of 55 (34.5 %) control steers (P < 0.05). For Salmonella, 9 distinguishable genotypes were identified by PFGE. New Salmonella genotypes were observed among 1 of 29 (3.5 %) treated and 2 of 29 (6.9 %) control steers (P < 0.05). Based on data from this study, DFM protected cattle from recurrent E. coli O157:H7 infections and not Salmonella.<br /> <br /> Salmonella Typhimurium serovars isolated from naturally infected feedlot cattle (n = 138) in North Dakota were characterized for antimicrobial resistance (AMR), presence of integrons and genotypic relatedness using PFGE assays. All 58 Salmonella isolates tested were resistant to e1 of the antimicrobials with 56/58 (96.6%) showing multi-drug resistance (resistant to e2 antimicrobials). Twenty-nine were Salmonella serovars Typhimurium var Copenhagen and were positive for class I integron, two of which also tested positive for integron 2. The 58 Salmonella isolates belonged to 9 distinguishable PFGE profiles, with the most prevalent genotype accounting for 46.6% (27/58) of the isolates. These data indicate that Salmonella Typhimurium serovars isolated from naturally infected feedlot cattle in ND showed widespread AMR with presence of class 1 integron and a wide variety of distinguishable PFGE profiles. <br /> <br /> The rate of new introduction of multidrug resistant Salmonella into Northwestern dairy farms averaged 0.91 new introductions per herd-year but was quite variable among study farms. The main variable that seemed to explain the farm-farm difference was the use of commercial, off-farm heifer raising at calf ranches where animals from many farms are mixed. Contamination of feeds with MDR salmonella was found to be very infrequent and very low concentration, and did not account for any new MDR salmonella introductions. A single strain of MDR Typhimurium was observed to spread to many study herds and a genetic analysis, using multilocus VNTR analysis has been completed that demonstrated multiple acquisitions of Cmy2 bearing plasmids within this strain<br /> <br /> To test the hypothesis that resistance genes undergo mutations that are selectively neutral, we hybridized 57 E. coli isolates onto our resistance gene microarray. We expected that mutations preventing gene expression would occur more frequently in a low antimicrobial use setting like a cow-calf operation than in an intense antimicrobial use setting like a dairy calf raiser. The results of these hybridizations did not support our hypothesis. Plasmid profiles and transformations onto selective media indicated that the unknown ²-lactam resistance gene or genes are likely to be on the chromosome. One of four isolates with ampicillin resistance and no ²-lactamase genes had no plasmids and the plasmids from the others failed to transform onto ampicillin plates. Plasmid profiles from the other 6 isolates showed plasmids of diverse sizes, including one or two very large (ca. 200 kb) plasmids. The effort to isolate plasmids carrying the targeted unknown resistance genes has met with mixed success.<br /> <br /> B. Campylobacter jejuni<br /> <br /> We hypothesized that particular genetic backgrounds enhance susceptibility of IL-10-/- mice to C. jejuni. C. jejuni stably colonized C57BL/6 IL-10-/-, C57BL/6 IL-10+/+, NOD IL-10-/-, NOD IL-10+/+, C3Bir IL-10-/-, and C3H/HeJ IL-10+/+ mice, while only IL-10-/- mice had typhlocolitis that mimicked human campylobacteriosis. C. jejuni was detected in blood and spleens of many infected C3Bir IL-10-/-, NOD IL-10-/-, and NOD IL-10+/+ but not C57BL/6 mice indicating that genetic background influenced extraintestinal spread. In C3H/HeJ IL-10+/+ mice, lack of TLR4 and the Cdcs1 allele were not sufficient for enteritis, but in C3Bir IL-10-/- mice these combined deficiencies enhanced both extraintestinal spread of C. jejuni and sensitivity to colon microbiota. All infected IL-10-/- and IL-10+/+ mice had anti-C. jejuni specific IgG antibody that failed to protect IL-10-/- mice against enteritis. These data demonstrate that lack of IL-10 had a greater effect on C. jejuni induced GI disease than other immune elements such as TLR4 (C3H/HeJ) and MHC H-2g7, diabetogenic genes, and CTLA-4 (NOD) and that host genetic background is in part responsible for disease phenotype. C3Bir IL-10-/- mice where Cdcs1 impairs gut barrier function provide a new murine model of C. jejuni and can serve as surrogates for immunocompromised patients with extraintestinal spread. <br /> <br /> C. Lawsonia intracelluaris<br /> <br /> Weve continued to add to our database of L. intracellularis genotypic types (based on VNTR typing) from various proliferative enteropathy outbreaks of pigs, horses, and other animal species. We collected and analyzed samples from each of 42 proliferative enteropathy outbreaks in pig herds, from 14 horse herds, and from outbreaks in 5 other animal species. From the data analyzed so far, no genetic variation was found within clinical types of proliferative enteropathy within a pig outbreak.<br /> <br /> <br /> There are no detectable differences between the PFGE patterns of three pig (North American or European) and a horse isolate using the restriction enzyme Pme I. The Sse8387 I restriction enzyme resulted in an inconsistent PFGE pattern in some isolates because of the presence of variable concentrations of the largest (194 kb) plasmid of L. intracellularis, which is not cut by this enzyme.<br /> <br /> On the day of sampling, all foals appeared in good health and none were showing clinical signs compatible with EPE. With the exception of one previously diagnosed case of EPE from farm 1, all remaining foals had normal total solid serum concentrations. Exposure rate for the study farms ranged from 29.7% to 45.5% and all fecal samples tested negative by PCR for the presence of L. intracellularis. The present study documents the high exposure rate to L. intracellularis in resident foals from farms with documented cases of EPE.<br /> <br /> Fresh feces from jack rabbits (100), striped skunks (22), feral cats (14), Brewers blackbirds (10), Virginian opossums (9), raccoons (4), ground squirrels (3) and coyotes (2) were collected either from the ground or after trapping the animals using live traps. Feces from jack rabbits, striped skunks, Virginian opossums and coyotes tested PCR positive for L. intracellularis, while all feces from feral cats, Brewers blackbirds, raccoons and ground squirrels tested PCR negative for L. intracellularis. PCR testing on DNA extracted directly from feces was positive for L. intracellularis in 6 out of 164 feces. The largest number of PCR positive L. intracellularis fecal samples was observed in striped skunks, followed by Virginian opossums, jack rabbits and coyotes. Since the fecal samples were collected at equine farms with confirmed cases of EPE, striped skunks, Virginian opossums, jack rabbits and coyotes may act as potential sources of infection to susceptible weanlings.<br /> <br /> D. Escherichia coli<br /> <br /> The objective of this study was to compare the accuracy of the QIAamp DNA Stool Mini Kit (QIAamp Kit) in detecting E. coli O157:H7 in enrichment and non-enrichment cattle feces. Using conventional culture methods (gold standard), 456 fecal samples were analyzed for detectable levels of E. coli O157:H7. QIAamp Kit was used to purify fecal DNA (from enrichment and non-enrichment feces) and E. coli O157:H7 genes (Stx1 and Stx 2) were targeted and amplified by PCR. A total of 199/456 (43.6%) fecal samples were positive for E. coli O157:H7 by culture. Of the 456 enrichment fecal samples, E. coli O157:H7 Shiga toxin-like genes were detected from 159/456 (34.6%) enrichment fecal samples compared to 43/456 (9.4%) targeted from non-enrichment samples. <br /> <br /> We demonstrated that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding ability to promote subsequent bacterial adherence. Increased adherence was not due to change in the surface expression of the host receptor for the K88ac adhesion.<br /> <br /> We constructed and tested a mutant LT toxin containing an amino acid substitution at position 192, and prepared a chimeric LT/STb gene by linkage of STb gene to the LTb subunit gene segment. These genes were transformed into an E. coli strain that produced K88ac fimbriae to form a live vaccine. Purified antigens from constructs expressing K88ac or LT were employed in a subunit vaccine delivered by the intranasal route. Both vaccines stimulated IgA antibody production in young pigs. Pigs receiving the living vaccine were protected from virulent challenge.<br /> <br /> To develop a piglet model for studying diarrhea disease and developing vaccines, we challenged gnotobiotic piglets with constructed isogenic E. coli strains expressing porcine 987P (F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal disease when inoculated with constructs expressing human or porcine enterotoxins.<br /> <br /> In this study, we evaluated extracts of the fermented soy product, Dochi, for its ability to block K88 ETEC binding to enterocytes. We found that Dochi extracts were able to significantly inhibit binding of K88 ETEC to brush borders isolated from porcine enterocytes (50% inhibition, P < 0.05) and cultured porcine intestinal epithelial (IPEC-J2) cells (60% inhibition, P < 0.05). The inhibitory compound in the Dochi extract was found to elute during fractionation at an approximate molecular weight of 1300 Da.<br /> <br /> E. Calicivirus<br /> <br /> About 50% of foodborne outbreaks of known etiology are caused by HuNoVs. Specific attachment of the tissue culture adapted porcine sapovirus Cowden strain (TC-Po/SaV) to the vegetable surface will be analyzed next by blocking attachment with specific antibodies, synthetic oligosaccharides, etc. For post-harvest processing, the efficacy of different treatments on reduction of infectious enteric caliciviruses in vegetables will be evaluated using the TC-Po/SaV as a HuNoV surrogate. We tested if TC-Po/SaV attaches to lettuce leaves at different pHs. At pH 5.0 and pH 6.0, 82% and 76% of virus bound to the lettuce leaves, respectively. At pH 3.0, 4.0 and 8.0, viral titers were below 10, the detection limit of our infectivity assay.<br /> <br /> F. Coronaviruses (CoVs<br /> <br /> We sequenced and analyzed the full-length genomes of four coronaviruses (CoVs) each from a distinct wild ruminant species in Ohio: sambar deer, a waterbuck, a sable antelope and a white-tailed deer. For two of the CoVs (sambar deer and waterbuck), complete genomes from both the cell culture-adapted and gnotobiotic calf-passaged strains were also sequenced and analyzed. Phylogenetically, wild ruminant CoVs belong to group 2a CoVs with the closest relatedness to the most recent BCoV strains. High nucleotide identities (99.4-99.6%) among the wild ruminant strains and the most recent BCoV strains (BCoV-LUN and BCoV-ENT, 1998) further comfirm the close relatedness. Comparative genetic analysis of captive wild ruminant CoVs and BCoV strains from various NC states suggests that no specific genomic markers are present that allow discrimination between the bovine strains and bovine-like CoVs from captive wild ruminants; furthermore, no specific genetic markers were identified that defined cell culture or calf-passaged strains, or host origin of strains. Our findings suggest that cattle may be reservoirs for CoVs that infect captive wild ruminants or vice.<br /> <br /> G. Cryptosporidiumparvum and Rotavirus<br /> <br /> We are developing and optimizing a new method for the sensitive detection of environmental microbial pathogens, especially waterborne pathogens, using label-free photonic crystal biosensor technology. The optical biosensor operates by measuring changes in the reflected resonant wavelength. Using this new sensor technology, we have demonstrated in preliminary experiments that porcine rotavirus can be specifically detected in aqueous samples as sensitively as conventional ELISA but without the need for secondary label processing.<br /> <br /> Objective 2. Focus on effective interventions- Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents<br /> <br /> A. Campylobacter jejuni<br /> <br /> In our current studies we demonstrated a reduction in colonization and invasion of epithelial cells in vitro by a Cj1534 mutant. Essentially, a 10-fold reduction in adherence was noted in the Cj1534 mutant strain when compared to the adherence of the parent strain. The invasion results were surprising, with average invasion numbers of 1.8 x 103 for the parent M129 strain, but no invasion of the INT 407 cells with the mutant strain. Therefore, the mutation of the Cj1534 gene had a reduction affect on the colonization of the host cells with C. jejuni, but the mutation also dramatically reduced the invasion. We showed that infected C57BL/6 and congenic interleukin (IL)-10-/- mice serve as models of C. jejuni colonization and enteritis, respectively. Thus, C57BL/6 are resistant to C. jejuni-induced disease. We investigated the interaction of C. jejuni with murine bone marrow-derived DCs (BM-DCs) to assess bacterial killing, DC activation, and ability of C. jejuni-infected BM-DCs to stimulate Campylobacter-specific T cell responses in vitro. BM-DCs challenged with C. jejuni efficiently took up and killed C. jejuni 11168 and significantly up-regulated surface MHC-II, CD40, CD80 and CD86 demonstrating a mature phenotype. Infected BM-DCs secreted significant amounts of tumor necrosis factor-alpha, IL-6 and IL-12p70. Formalin-killed C. jejuni also induced maturation of BM-DCs with similar cytokine production but at a significantly lower magnitude than live bacteria. Maximal activation of murine BM-DCs required internalization of C. jejuni; attachment alone was not sufficient to elicit significant responses. Also, various strains of C. jejuni elicited different magnitudes of cytokine production from BM-DCs. Finally, using a coculture system, C. jejuni-infected BM-DCs induced high-level interferon-³ production from CD4+ T cells indicating Th1 polarization.<br /> <br /> The twin-arginine translocation (TAT) pathway represents an important virulence mechanism in many bacterial pathogens. Our recent study with IA of the C. jejuni TAT system has indicated that the deletion in a TAT system functional gene significantly compromises the bacterial ability to tolerate stress responses including biofilm formation and cause persistent infection in chickens. C. jejuni has a limited number of genes that contribute to stress responses; however, the high prevalence of C. jejuni infections in domestic livestock including poultry suggests that the TAT system may allow this fastidious pathogen to successfully overcome a number of environmental stresses during transmission.<br /> <br /> B. Salmonella enterica<br /> <br /> The Cj1534 gene was cloned into a plasmid vector pYA4495-1 and expressed in a lac operon under a PTRC promoter. Hatchlings were purchased and placed in isolation. Birds were immunized orally with ~109 viable cells of Salmonella Typhimurium x9088 (pYA3493-empty vector control; pYA4495-1-vector with Cj1534) on days 3, 10, and 16. The vaccinated birds were challenged on day 26 with ~105 viable cells of C. jejuni strain NCTC11168 and necropsied 10 days later. Birds vaccinated with the Cj1534 protein expressed by S. Typhimurium demonstrated a C. jejuni reduction of 3.5 logs after challenge exposure.<br /> <br /> C. Escherichia coli<br /> <br /> A study was completed to compare the importance of heat-labile enterotoxin (LT) and heat-stable enterotoxin-b in piglets <2 weeks old. Based on the PI percent weight change per h and serum bicarbonate concentrations, the virulence of the STb- mutant (?estB) did not significantly differ from that of the parent. However, deletion of the LT genes (?eltAB) in the STb- mutant resulted in complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. However, in contrast to previous studies in gnotobiotic piglets, there was no evidence that expression of LT enhanced the ability of the F4+ ETEC strain to colonize the small intestine.<br /> <br /> Recently, we reported the occurrence of E. coli O157:H7 infection of the urinary bladders of gnotobiotic piglets following oral inoculation and causation of attaching-effacing enterocolitis. In the present study, we conducted transmission electron microscopy on formalin-fixed tissues from these pigs and found that the infecting E. coli O157:H7 organisms had caused pedestal formation at the attachment sites to the transitional epithelial cells, similar to the lesions typically caused in the intestinal tract.<br /> <br /> We began testing the efficacy of a galactooligosaccharide (GOS) for reducing adherence and colonization of the gnotobiotic piglet intestine by enteropathogenic E. coli (EPEC) and E. coli O157:H7. A 2X2 factorial design with four treatment groups was used: GOS vs control X 2 bacterial strains (EPEC strain E2348/69 or E. coli O157:H7 Shigatoxin-negative ATCC strain 43888). All inoculated piglets (both those treated with GOS and the non-GOS controls) developed gross and microscopic intestinal lesions consistent with EPEC and E. coli O157:H7 infection. However, there was a numerical reduction of the respective inoculum strains isolated from the ileum and spiral colon in CFU/g of PBS-rinsed intestine obtained at necropsy. At the concentration of GOS used, piglets developed mild osmotic diarrhea.<br /> <br /> A trial was conducted to test the efficacy of 2- and 3-dose regimens of an Escherichia coli O157 vaccine product on cattle shedding E. coli O157:H7 in feces, or being colonized at the terminal rectal mucosa (TRM), and having a humoral response, compared to placebo-treated controls. Vaccine efficacy was 33% and 65% for 2- and 3- dose regimens, respectively. Cattle receiving two doses of vaccine had an increase in EspB antibody titers between days 0 and 63 (p=.0.04). Cattle receiving two or three doses of vaccine had an increase in O157 LPS antibody titers between days 0 and 63 (p<0.0001), and between days 0 and 91 (p<0.0001). A dose-response for fecal shedding efficacy, and humoral antibody responses to EspB and O157 LPS following vaccination were detected.<br /> <br /> A trial was conducted to test vaccinating feedlot steers against type III secreted proteins of E. coli O157:H7 on the proportion of steers shedding E. coli O157:H7 in feces when fed diets with or without corn wet distillers grains plus solubles (WDGS). Steers were vaccinated three times at four-week intervals beginning at trial initiation. Rectal fecal samples were collected 3, 6, 9, and 12 wks post-vaccination. There was no interaction (P=0.97) between diet and vaccination for E. coli O157:H7 shedding. There was no interaction between treatment and sampling time (P > 0.40) and no effect of time (P = 0.17) on E. coli O157:H7 shedding. Vaccinated steers were 43% less likely to shed E. coli O157: than non-vaccinated steers. Steers fed WDGS were 2.1 times more likely to shed E. coli O157:H7 than cattle fed the control diet.<br /> <br /> Our initial objective was to determine the fitness cost associated with carriage of peH4H. Growth curve experiments and direct competition studies demonstrated that under most conditions the wild-type strain (H4H) is able to out-compete the plasmid cured strain (NC5). This finding suggests that peH4H confers a direct selective advantage on its host. The critical test occurred when we added peH4H plasmid back into strain NC5 by electroporation to make strain NC5p. However, growth curve and competition studies showed that the fitness advantage was not recovered. At this point we assume that NC5 was altered in some manner during the curing/electroporation process.<br /> <br /> Because plasmid curing is not a viable approach to conduct these studies, we moved peH4H into a lab strain of E. coli, DH10B (designated GH hereafter). We also included a second plasmid, Cp, from strain H4H that harbors resistance to trimethoprim (see below). Cp was included because of the possibility that there was an important interaction between peH4H and Cp. Introduction of these plasmids into GH produced strain H4H-GH, Cp-GH, and H4H-GH-Cp. We then competed these strains against the parental stock (GH) using a serial passage experiment over 8 days. In all cases, addition of either H4H or Cp increased the fitness of GH with a possible additive effect when both plasmids were introduced into GH. We have passaged H4H in LB media over 1,450 generations and strains H4HGH and CpGH over 500 generations and so far we have found no evidence of any plasmid-free strains in these cultures. Thus, both peH4H and Cp appear to convey some kind of fitness advantage to strain GH.<br /> <br /> This study assessed the effect(s) of ceftiofur on blaCMY-2 transfer and dissemination by: 1) an in vivo experimental study in which calves were inoculated with competent blaCMY-2  bearing plasmid donors and susceptible recipients and subjected to ceftiofur selection, and 2) an observational study to determine if ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed blaCMY-2 plasmid transfer in both ceftiofur-treated and untreated calves, but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P=0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of blaCMY-2 bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.<br /> <br /> The proportion of cephalosporin resistant E. coli returned to starting levels by the end of the sampling period (15 days). 86% of the E. coli isolated from calf feces on the first day of sampling (before any antibiotic treatment was administered) were resistant to cephalosporin compared to 23% of E. coli isolated from cow feces on the first day of sampling. Treatment with antibiotics reduced the total number of E. coli in the feces of fresh cows and in the feces of calves. While the trends for cows and calves was similar, the baseline and magnitude of effects were dramatically different.<br /> <br /> D. Lawsonia intracellularis<br /> <br /> The disinfectants Synergize®, Roccal®-D plus, Virkon-S®, Tek-Trol®, Nolvasan S®, DC&R®, and Certi-Dine® were tested for activity against two isolates from affected pigs. Both isolates were completely inactivated with Synergize® (1:256), Roccal®-D (1:256) and DC&R® (1:128). Inactivation was e 99% with Virkon-S® (1%) and 90-99% with Tek-Trol® (1:256) and Nolvasan S® (1:128) and <90% with Certi-Dine® (1:128) within 10 minutes. <br /> <br /> The objectives of this study were to evaluate the humoral immune response and onset and duration of fecal shedding in pregnant broodmares after administration of a modified-live vaccine of L. intracellularis and to determine if specific maternal antibodies against L. intracellularis are passively transferred to foals. All vaccinated mares remained healthy and no adverse vaccine reactions were observed. Fecal shedding was found in 3 mares and lasted from 1 to 3 days. Serologic responses were detected in all vaccinated mares with titers ranging from 60 to 240. Antibodies against L. intracellularis persisted for up to 10 weeks. Colostral antibodies were detected in 2 out of the 8 seropositive mares. Maternally derived antibodies were detected in 6 foals and persisted for up to 8 weeks.<br /> <br /> E. Porcine Group A Rotavirus<br /> <br /> We recently synthesized a sialyl-lactosylphosphatidyl-ethanolamine (SLPE) neoglycolipid. In field trials this inhibitor blocked infection, virus shedding and diarrhea using a twice a day dosage administered to newborn pigs at the time of virus inoculation. Results from these studies suggest that specific oligosaccharide/isoflavone mixtures or profiles may be engineered to provide a deliverable nutriceutical for the treatment and prevention of rotavirus disease across susceptible animal species and humans.<br /> <br /> F. Cornavirus<br /> <br /> 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 potentially relevant to SARS. To establish a persisting PRRSV infection, weaned PRRSV and PRCV seronegative pigs were initially inoculated intranasally (IN) and intramuscularly with PRRSV SD23983 strain at about 3 wks of age. At 10 days after PRRSV infection, anesthetized pigs were inoculated IN and intratracheally with the PRCV ISU-1 strain. The PRRSV+PRCV pigs had greater reductions in weight gains and more severe gross or histological pneumonic lesions compared to either single infection at PRCV PID 2 to 21. The enhanced disease coincided with higher Th1 (IFN-³, IL-12) but lower Th2 (IL-4) cytokine responses in serum for the dually infected pigs at PRCV PID 6 to 21. Greater amounts of PRCV antigen and RNA were observed in the lungs of the dually-infected pigs at PRCV PID 2 compared to PRCV single infection, possibly due to suppression of innate immune responses (IFN± and NK cell activity) by the ongoing PRRSV infection; however, the opposite trend (decreased PRCV antigen and RNA) was noted at PRCV PID 4 to 21. In contrast, PRRSV RNA levels were higher in the dually-infected pigs compared to PRRSV single infection at PRCV PID 4 to 14, consistent with more severe PRRSV-related alveolar macrophage apoptosis in the lungs detected by in situ Tunnel assay. Antibody (Ab) titers to PRRSV were higher for dually-infected compared to single infected pigs at PRCV PID 4 to 21 (PRRSV PID 14 to 31), reflecting increased PRRSV replication. In contrast, lower virus neutralization Abs to PRCV were observed in the dual-infected pigs compared to single PRCV infection at PRCV PID 21, which coincides with lower PRCV RNA levels in dual-infected pigs (PRCV PID 4 to 21).<br /> <br /> G. Cryptosporidium parvum<br /> <br /> Suppressive subtractive hybridization experiments aimed at identifying specific sporozoite genes expressed in response to host cell attachment or exposure to the inhibitory lipid indicate these processes occur without the necessity for attachment- or lipid-induced differential gene expression. This lipid, introduced as part of different dietary regimens, also is being examined for its ability to reduce or eliminate bovine cryptosporidiosis in newborn calves. We determined that cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips (VFS) and that the mechanism of this retardation is specific adhesion to the clay particles of the soil that occurs as a consequence of reduced flow over a vegetated surface as compared to bare soil.<br /> <br /> H. Lactic acid bacteria and probiotics<br /> <br /> We evaluated the patterns of TLR2- (recognizes peptidoglycan), TLR3- (recognizes dsRNA) and TLR9- (recognizes CpG) expressing antigen presenting cells (APC) (defined by CD14, SWC3, CD11R1 markers) in spleen and blood of gnotobiotic (Gn) pigs after colonization with a mixture of two strains of lactic acid bacteria (LAB), L. acidophilus and L. reuteri or infection with the virulent human rotavirus (HRV) Wa strain. We demonstrated that LAB induced strong TLR2-expressing APC responses in blood and spleen; HRV induced a TLR3 response in spleen; and TLR9 responses were induced by either HRV (in spleen) or LAB (in blood). LAB and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of LAB. Overall, the frequencies of TLR-expressing CD14+ APCs were higher than CD14- APCs. LAB enhanced the IFN-³ (Th1) and IL-4 (Th2) responses in serum, but they had a suppressive effect on the TLR3- and TLR9-expressing CD14- APC responses in spleen and the serum IFN-± response induced by HRV.<br /> <br /> We examined rotavirus-specific IFN-³ producing CD4+, CD8+ and CD4+CD8+ T cell responses in gnotobiotic pigs infected with virulent human rotavirus (HRV) or vaccinated with an attenuated HRV vaccine (three or two doses) or an attenuated oral rotavirus vaccine priming and VP 2/6-virus-like particle (VLP) vaccine intranasal boosting regimen. In virulent HRV infected pigs, HRV-specific IFN-³ producing T cells resided primarily in ileum. Attenuated-HRV+2/6VLP induced similar frequencies of intestinal IFN-³ producing T cells as the virulent HRV, whereas 3x or 2x doses of attenuated HRV vaccine were less effective. Protection rates against rotavirus diarrhea upon virulent HRV challenge were significantly correlated with frequencies of intestinal IFN-³ producing T cells, suggesting their role in protective immunity.<br /> Objective 3. Focus on disseminating knowledge- Provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians and diagnostic laboratories. <br /> NDSU in conjunction with the Department of Veterinary Public Health and Preventive Medicine, Makerere University (Mak), Kampala Uganda, developed a 3-credits short term course (4 weeks) International Animal Production Disease Surveillance and Public Health. The course was designed to facilitate diversity in student training and exposure. In order to produce a broadly inclusive, open minded, and globally engaged science workforce.<br />

Publications

Impact Statements

1. Oral vaccination of chicks with a Salmonella vector expressing the Cj1534 protein significantly reduced numbers of C. jejuni in the ceca of challenged birds.
2. Porcine rotavirus can be specifically detected in aqueous samples as sensitively as conventional ELISA but without the need for secondary label processing
3. We have determined the Cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips
4. We showed that dendritic cells from resistant mice process C. jejuni and present its antigens in the context of MHC-II leading to activation of T cells that can mediate. These findings provide a cost effective animal model that can be used to study the genetic basis of virulence of the enteric pathogen C. jejuni protective immune responses
5. VNTR typing continues to be the most sensitive differentiation tool for typing of Lawsonia isolates.
6. Demonstrated the high exposure rate to L. intracellularis in resident foals from farms with documented cases of the disease
7. The side by side comparison of the in vitro activities of disinfectants against Lawsonia in this study could serve as a guide for disinfectant selection for control of Lawsonia in the environment
8. The finding that heat-labile enterotoxin (LT), as opposed to heat-stable enterotoxin-b (STb), was responsible for most of the diarrheagenic effects in F4ac+ enterotoxigenic E. coli (ETEC) infections in pigs less than two weeks old indicates that intervention and prevention efforts need to include targeting this enterotoxin.
9. The finding that E. coli O157:H7 infects the urinary bladder and causes cystitis in the gnotobiotic piglet following intestinal infection provides supportive evidence for the hypothesis that this organism can cause cystitis in human patients.
10. Characterized Salmonella Typhimurium serovars isolated from naturally infected feedlot cattle in ND and showed widespread AMR with presence of class 1 integron and a wide variety of distinguishable PFGE profiles
11. Provided an opportunity for international training and exposure of students from several US institutions through the course International Animal Production, Disease Surveillance and Public Health.
12. Use of the only cultivable enteropathogenic calicivirus, the TC-Po/SaV as a surrogate for HuNoVs to study virus attachment revealed that TC-Po/SaVs are stable at acid pH and attach to lettuce leaves. Thus our research will help to reduce the incidence of foodborne illnesses due to consumption of unprocessed fruits and vegetables contaminated with enteric caliciviruses (human or animal origin)
13. Our findings suggest that wild ruminants may harbor CoVs transmissible to cattle or vise versa and that these CoVs may represent host range variants of an ancestral CoV
14. The adjuvant effect of LAB colonization during rotavirus infection or vaccination seems to be through the activation of the TRL2 and TLR9 pathways. LAB enhanced B cell responses to a live oral rotavirus vaccine
15. South Dakota in collaboration with Nebraska and Kansas have explored the roles of E. coli enterotoxins LT, STa and STb in microbial adhesion and diarrheal disease in pigs. Each toxin significantly contributed to diarrhea, and with regard to that activity there appears to be no difference in the behavior of toxins variants associated with human or porcine ETEC. LT enhances bacterial colonization, apparently by preconditioning epithelial cells for bacterial adhesion.

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Report Information

Participants

Alali, Walid (walali@uga.edu)-University of Georgia; Besser, Thomas (tbesser@vetmed.wsu.edu)-Washington State University;Francis, David (david.francis@sdstate.edu)-South Dakota State University; Isaacson, Richard (isaac015@umn.edu)-University of Minnesota; Joens, Lynn, (joens@ag.arizona.edu)-University of Arizona; Khaitsa, Margaret (Margaret.Khaitsa@ndsu.edu)-North Dakota State University; Kuhlenschmidt, Mark (kuhlensc@illinois.edu)-University of Illinois; Kaushik, Radhey (Radhey.kaushik@sdstate.edu)-South Dakota State University; Mansfield, Linda (mansfie4@cvm.msu.edu)-Michigan State University; Moore, Dale (damoore@vetmed.wsu.edu)-Washington State University; Moxley, Rodney (rmoxley1@unl.edu)-University of Nebraska; Rajashekara, Gireesh (Rajashekara.g@osu.edu)-Ohio State University;Robinson, Mark (mrobinson@nifa.usda.gov)-USDA/NIFA; Saif, Linda (saif.2@osu.edu)-Ohio State University; Sischo, William (wmsischo@vetmed.wsu.edu)-Washington State University; Stromberg, Bert (b-stro@umn.edu)-University of Minnesota; Zhang, Qijing (zhang123@iastate.edu)-Iowa State University

Brief Summary of Minutes

Notes from NC1041 Meeting, Chicago, IL; December 56, 2009
(Submitted by Linda S. Mansfield,)
Sunday Meeting
1. NC1041 Meeting Planning 2010
a. Leadership for next year
i. Organizer serves a 2 year term as secretary and a 2 year term as chair
ii. Linda Mansfield will write report and organize the meeting in 2010 and 2011.
b. Tasks
i. Prepare and agenda and assign section leaders
1. Rewrite should be on the agenda for the next year
2. Need to prepare outline for the proposal during the 2010 meeting
3. Set up an email list
ii. Compile reports for meeting
iii. Assemble writing team for beginning proposal&due date.
c. The Next Proposal
i. Impact documentation
1. short term and long term goals
2. It is useful to get our work into the popular press and to be able to document the public perception
3. Everyone in the group should fill out an appendix E at their AES unit offices

ii. Timeline - by the next year put together a timeline
1. Rewrite should be on the agenda for the next year
2. We will by next year have feedback from the midyear review

iii. Outline
1. We have some progress on the outline.
2. This task must be done during the next meeting
3. Bert Stromberg will bring format for the proposal rewrite to the next meetings

iv. Define stakeholders and start getting input and support from them
1. Stakeholders reports will be very important in the rewrite
2. We must show that stakeholders have been involved
3. Who are our stakeholders? We need to know and demonstrate this.
4. MIMs system setup - Set up in NIMS system operated out of Maryland
a. *Send information and ideas for renewal outline NIMS system
b. We have to do a comparative study of what others are doing in this work.
i. NC(North Central)1041
ii. Food Safety but not the same NCES1033 as Clemson Univ.
5. Stakeholders
a. The Public
b. Consumer groups
c. Industry groups
d. Pork Producers
e. State Medical Epidemiologists
f. WE NEED TO BE INTERESTED IN WHAT WE ARE DOING DESPITE THE FLUCTUATIONS OF THE STAKEHOLDERS

v. Topic Discussions
1. Focus Areas
a. Annual Priorities
b. Intersections between agriculture and public health
c. Veterinary medicine has a disconnect with Public Health
d. Move to Food Safety thus public health animal models become important to us as a group
e. We must distinguish ourselves from the NIFA group. We are not sure what will happen with this


2. Training the next generation of scientists
a. Margarets work to train the next generation of agricultural scientists
3. International Work
a. Why are you going over to Africa and South East Asia?
i. This is done due to biosecurity and food safety that is an international issue
ii. Name the diseases that can be moved around
iii. Microbes that are translocated across international boundaries can be identified
iv. Must justify to US institutions what the benefits are to the US partners for international actions
b. Locations
i. ILRE-international livestock institution
ii. Walter Reed built a facility at Makerere University, Uganda for examining Avian Influenza
iii. USAID in East Africa
4. Organisms on which to focus
a. Interact with public health groups to use their lists of microorganisms of interest e.g. A, B and C list
b. This task will help us in terms of identifying stakeholders
c. Need to get basic info in this area

2. NC 1041 should have a joint meeting
a. US Japan Malnutrition Conference
i. Richard Guemant Past Chair:
ii. John Clemens-Korea
b. USDA National lab-Qijang had this for an idea for joint collaborations
c. 4th Rushmore Conference  we said we would have a meeting

3. Funding Issues
a. How Hatch funds are designed to work
i. Hatch ==> divided into regional associations ==> there is a dollar allocation by a formula ==> 25% must be spent on multistate projects ==> institutions divide it up ==> no formula for these divisions ==> very little accountability
ii. Very few of us get funding from the AES for research
iii. Very little of the funds are actually going for research
iv. This is an administrative issue
v. All institutions do this differently
1. Univ. Wisconsin has a competition
vi. Formula funds and multistate funds
1. Each has different uses.
2. Multistate is more competitive
vii. Financial data for group is backing in database. This needs to be updated. Use it for lobbying
viii. In 2008 there was a bump in Hatch funds. 150 million more into Hatch
1. 245,000
2. 559,000 multidata
3. Reported through 547,000 by NIH
4. AES Stations 3X10^6 total state and
5. Hatch and Multistate funding records are managed through the Cris system
6. There is supposed to be in 1 to 1 relationship between money allocated and the work done; the reality doesnt match this.
ix. Institute of Food and Agriculture.
1. Growth in scale of projects
2. AFRI funding was bumped up
3. Food Safety, international food security
4. Next Steps
a. Compile the meeting notes and send to Dick
b. Send a copy of last proposal to members.
i. Tom Besser has this
ii. NIMS has this
c. Get a list of participants in 2009
i. Dick will add new people into the system
ii. We have to fill out appendix E first
d. Dave Francis is organizing the poster-talk judging for students. Does this transition?
i. Piggyback with them
e. We want to get graduate students involved
i. Make list of trained grad students and what they are doing now
1. Grad students and postdocs should be invited
a. Possibly one per station.
b. We can forgo the registration fee for them.
c. Money can be achieved through USDA as a separate grant
ii. Junior faculty members can be encouraged to join.
1. Benefit is establishing a network of collaborators
2. Junior faculty need to have some incentive for joining
3. Exchange of reagents and collaborations are the benefits
4. Holding this meeting in conjunction with CRWAD made the cost reasonable. This is not likely to change
f. Meeting Ideas
i. NRSP8-genomic support project ½ day invited presentations
ii. 2nd ½ day-station reports
iii. All room  mostly plant and animal genomics
iv. Symposia
1. could be organized on common topics and start, then-have discussions by breakouts
2. Symposium session becomes part of the meeting
3. Speakers
a. Tim Johnson
b. Syed Hashsham  microbial ecology
4. Topics for a symposium on Sunday morning
a. *Decision-to think about topics and send soon
b. Then we will solicit some speaker names
c. Gut microbiome
d. Metagenomics - Andy Banson
i. Pyrosequencing
ii. Illumina sequencing
5. Funding
a. Try to get money from Bob
b. Raise dues slightly to find this
c. Corporate sponsors might pay for this
i. Can we get a corporate sponsor?

g. Midterm Progress Report is due soon
i. Seek collaborations that gain funding
ii. Technology transfer
iii. Funding levels are always in debate
iv. Rewrite is due in 2011
v. What is the status and future of the Rushmore Conference?
vi. SAES-funding report.
vii. NIFA opportunities
1. Mark Robinson is the NIFA liaison
2. Need participation of group members
viii. Nar Agriculture+ &. Org. funding issues NC1041

Accomplishments

Objective 1. Focus on emerging diseases- 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.<br /> <br /> A. S. enterica<br /> <br /> Arizona<br /> 1) Sampling oysters for Salmonella from the Yaquina river. Oysters were examined from January to November with 26 positive for Salmonella giving us a 2.9% prevalence. Oysters were only positive for Salmonella during the spring and summer months. Of the Salmonella isolates collected, 34% (9/26) were S. Newport.<br /> <br /> 2) Identification of colonizing/invasion genes. The whole genome of S. Newport genotype JJPX01.0014 was sequenced. The contigs aligned to NC_011080 with greater than 99% homology. One large difference was the presence of a 30,000 bp insertion in NC_011080 that is not present in S. Newport JJPX01.0014.<br /> <br /> Kansas<br /> 1) Norepinephrine Increases Horizontal Gene Transfer Rates Between Enteric Bacteria. The effect of catecholamines on the rate of conjugative transfer between enteric pathogens was assessed. There was a significant increase in the rate of horizontal gene transfer of a conjugative plasmid between Salmonella and E. coli in the presence of norepinephrine.<br /> <br /> Minnesota<br /> Cultivation medium affects the accuracy of S. enterica surveillance. Four different Salmonella strains were competed in broth or bovine feces at varied combinations and concentrations. In all experiments, S. Newport was the most competitive, regardless of the starting concentration and cultivation protocol. One strain of S. Typhimurium was rarely detected in competition, even when it was the only strain present in bovine feces. Overall, the probability of detecting a specific Salmonella strain had little to do with its starting concentration in the sample.<br /> <br /> North Dakota<br /> 1) Shedding of Salmonella in beef cattle at different production stages. Prevalence of Salmonella in calves remained constant (~50%) September & November 2008, peaked in December 2008 (100%) and then was dropped (27.7%) in February 2009. It rose again in June to 48% at slaughter. AMR patterns remained consistent throughout the study for cattle with resistance observed against the same six antimicrobials. During February 2009 some calves had isolates resistant to cefoxitin. A high association of the presence of the integrase gene was observed in cattle and calves. A negative association was seen towards Cefoxitin and the presence of the integrase.<br /> <br /> 2) Characterization of clinical Salmonella isolates from cattle and humans in the US and Uganda: AMR and class 1 integron. Pan susceptible isolates were 14.3% in cattle and 19.3% in humans. For cattle and human isolates 64% and 25%, respectively, were resistant to 2 or more antibiotics while 48% and 11%, respectively, had resistance to e5 antimicrobials. Among isolates from US 25% of the cattle samples were positive for for class 1 integron. Among the 74 isolates from Uganda all 100% were resistant to e5 antimicrobials. Relatively high resistance to ciprofloxacin was seen in 66.7% cattle and 64% humans. A total of 45.8% of human and 46.2% of cattle isolates tested positive for presence of integron 1.<br /> <br /> B. C. jejuni<br /> <br /> Arizona<br /> 1) Feedlot Sampling for the Presence of C. jejuni. Half of the cohort (18) received a natural diet and the other half a similar diet with rumensin, tylosin and implants of estradiol and progesterone. Flies and pigeons were positive for C. jejuni at arrival of the calves. Later, pen floors, feed bunks, and the watering units were positive. During the first three months 2 of 36 calves were positive for C. jejuni. However, the next six months all samples showed increases in the presence of C. jejuni except feed and flies. On the day of processing all cattle fecal samples were positive. An increase in the percentage of carcasses was positive for C. jejuni after evisceration with the highest number of positives coming from the ventral midline. Carcass samples and ground beef samples from the carcasses were negative.<br /> <br /> 2) Sequencing and annotation of C. jejuni strains S3 (poultry) and M129 (clinical). C. jejuni M129 genome resulted in 11 large contigs at 42x coverage and a genome size of 1.61Mb. Previous studies have demonstrated the presence of four genomic islands (CJIE1, 790bp; CJIE2, 1,109bp; CJIE3, 644bp; and CJIE4, 953bp) encoded by the fluid producing strain RM1221 that are present in or have limited distribution in other C. jejuni strains. Complete RM1221 islands 1, 2 and 4 are present in the genomes of fluid producing strains S3; none of these islands are present in the invasive non-fluid producing strain M129.<br /> <br /> 3) Identification of C. jejuni proteins in intestinal fluid exudate of infected piglets. One ml of filtered pooled intestinal fluid from pigs infected with RM1221 or filtered pooled fecal material were pooled and subjected to 2D gel electrophoresis. Four unique proteins were detected: CJE0556, CJE0595, CJE1447, and CJE1464. These proteins were found to be encoded within genomic islands CJIE2 (CJE0556, CJE0595) and CJIE4 (CJE1447, CJE1464).<br /> <br /> Michigan<br /> C57BL/6 interleukin-10-/- mice were infected with seven genetically distinct C. jejuni strains. Four strains colonized the mice and caused disease; one colonized with no disease; two did not colonize. A microarray comparison of the strain that colonized mice without disease to C. jejuni 11168 that caused disease revealed that putative virulence determinants, including loci encoding surface structures known to be involved in C. jejuni pathogenesis and differed from or were absent in the strain that did not cause disease.<br /> <br /> Ohio<br /> Nearly 60% of cattle populations carry Campylobacter. Occupational exposure to raw meat, consuming ground beef, drinking unpasteurized milk and contact with cattle are implicated in Campylobacter infections. Campylobacter spp were detected in 24% of fecal samples. 71 C. jejuni isolates, 130 C. coli isolates, and 63 other spp of Campylobacter were isolated and confirmed by multiplex.<br /> <br /> C. L. intracelluaris<br /> <br /> Minnesota<br /> 1) Involvement of cell-mediated immune response and specific local mucosal IgA production in L. intracellularis experimental infection. Five-week-old pigs were inoculated with L. intracellularis, intestinal mucosa homogenate from proliferative enteropathy diseased pigs, or mock infection solution. Weak IFN-³ production was detected in one pig of the pure culture group and two pigs of the mucosal homogenate group 14 days pi and in two animals of each group 20 days pi. All pigs in both inoculated groups were seropositive for Lawsonia (IgG) on day 20. Inoculated pigs showed very weak dose dependent DTH reactions. Eight pigs from the pure culture group and seven from the mucosa homogenate group had detectable IgA titers in the intestinal lavage 22 days pi.<br /> <br /> Ohio<br /> Detection and quantification of L. intracellularis. A SYBR green quantitative PCR assay targeting a unique hypothetical protein was developed for detecting and quantifying L. intracellularis. The method detects as few as 3 copies per PCR reaction of the bacterium growing in IEC-18 rat epithelial cells. The qPCR assay was successful in detecting L. intracellularis in fecal samples collected from pigs.<br /> <br /> D. E. coli<br /> <br /> Kansas<br /> 1) A Multiplex PCR procedure for Detection of Six Major Virulence Genes in E.coli O157:H7. A multiplex PCR procedure that detected six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in E. coli O157:H7 was developed. The procedure was validated with a total of 222 isolates. Fecal samples spiked with E. coli O157 amplified all six genes if the concentration of E. coli O157 was 104 CFU/g of feces. However, if PCR was carried out after 6 h enrichment, the detection limit was 10 CFU/g of feces.<br /> <br /> 2) Genetic Relatedness of E. coli O157 Isolates from Cattle Feces and Beef Carcasses. We used PFGE to characterize E. coli O157 isolates from pre-evisceration carcasses and feces that were recovered from 37 E. coli O157-positive truckloads at a commercial abattoir. Among all isolates, there were 17 PFGE types (95% homology) and 37 subtypes (100% homology). Specific subtypes were detected on multiple occasions and from different sample types within loads, among loads, and among days. Within truckload, the percentages of carcass isolates that were identical to high-shedder or low-shedder fecal isolates, as determined by PFGE, were 69.2% and 46.0%, respectively. The percentages of carcass isolates that were the same subtype as high-shedder or low-shedder fecal isolates were 35.3% and 58.8%, respectively.<br /> <br /> 3) Genetic Variations in Shiga Toxin-producing Abilities of Bovine and Human E. coli O157:H7. Reverse passive latex agglutination test was used to evaluate 107 isolates (50 human, 57 bovine) for Stx1 and Stx2 production. Stx2 production of e1:8 was found in 86.0% of human isolates compared to 26.3% of bovine isolates. Bovine isolates with the presence of the TNP regions were associated with significantly lower Stx2 production, while the Q933 gene was associated with higher Stx2 production. Q933 was a better indicator of high Stx2 production by human and bovine isolates and may be a useful screening method to assess their potential to cause human disease.<br /> <br /> 4) Effects of Mucin and its Carbohydrate constituents on E. coli O157 Growth in Batch Culture Fermentations with Ruminal or Fecal Microbial Inoculums. Our objective was to test the effects of mucin and its carbohydrate constituents on in vitro growth of E. coli O157 in ruminal or fecal microbial fermentations. In ruminal fermentations, fucose, mannose, glucuronic acid, galacturonic acid, glucosamine, galactosamine, and mucin had no effect on E. coli O157 concentration compared to the control fermentation. At 24 h, the concentration of E. coli O157 in fermentations with galactose was lower than the control. However, including gluconic acid as substrate increased E. coli O157 concentration at 24 h. In fecal fermentations, mannose, galactose, gluconic acid, glucuronic acid, galacturonic acid, glucosamine, and mucin increased E. coli O157 growth compared to control at 24 h, while galactosamine and fucose did not. Gluconic acid was the most stimulatory substrate.<br /> <br /> South Dakota<br /> 1) Expression of heat-labile enterotoxin increases E. coli adhesion to intestinal epithelial cells. We used the K88ac adhesin producing E. coli strain 1836-2: 8035 containing the intact LT operon cloned into pBR322; 8221 containing the LT operon a mutation in the A subunit; and 8589 containing only the cloned LTb subunit. We found that the strains expressing the intact toxin, a non-toxigenic mutant of LT or the toxins B subunit (8035, 8221, 8589) each were able to induce significantly higher adhesion when compared to a non-toxigenic, adhesin expressing, or non-ETEC strains (8017, G58-1 respectively). <br /> <br /> 2) Avirulent enterotoxigenic E. coli strains act as probiotics against pathogenic K88+ E. coli. We utilized a piglet ETEC challenge model to test the ability of non-pathogenic ETEC constructed strains to colonize the intestine and competitively exclude virulent ETEC. All K88ac receptor-positive piglets in the placebo group developed diarrhea and became dehydrated after 12 hours. Piglets inoculated with either strain containing the modified LT (8221 or 8488) did not exhibit clinical signs of disease following ETEC challenge while piglets inoculated with 8017 showed mild to no diarrhea. This study suggests that pre-inoculation with avirulent strains expressing adhesive fimbriae and a non-toxic form of LT provides significant protection from ETEC challenge when provided 24 hr before that challenge.<br /> <br /> 3) Assessment of Efficacy of oral E. coli constructs for protecting weaned pigs from enterotoxigenic E. coli. Piglets suckled their dams for 5 days then weaned to milk replacer. they were inoculated orally with a placebo or with isogenic E.coli strains at 7 days of age and again at days 14. At 21 days, piglets were challenged with the wild type virulent ETEC strain 3030-2 (K88+/LT/STb). Following challenge, all receptor positive pigs in the placebo control group developed diarrhea and became dehydrated. None of the piglets inoculated with any of the isogenic strains exhibited any clinical signs of disease following ETEC challenge.<br /> <br /> E. Calicivirus<br /> <br /> Ohio<br /> 1) Attachment and inactivation of foodborne enteric caliciviruses in lettuce. We assessed attachment of enteric caliciviruses to lettuce by immunofluorescence, infectivity assays and real time RT-PCR. For post-harvest processing, the efficacy of different treatments on reduction of infectious enteric caliciviruses in vegetables was evaluated. We conclude that at the virus pI, virions may precipitate to form aggregates that may facilitate virus binding to lettuce. The overall virus titers did not change after incubation at RT for 1 hr (pH 4-8); < 1.0 log10 reduction occurred at pH 3. Three different elution buffers were tried to elute TC-Po/SaV from experimentally contaminated lettuce. Only minimum essential medium (MEM) plus 2% fetal bovine serum (FBS) (pH7.4 - 7.7) were positive. Thus TC-Po/SaV remains infectious on lettuce leaves after storage at 4°C for 1 wk, which is the general shelf life of lettuce.<br /> <br /> 2) Detect and characterize NoVs and SaVs from swine and determine their genetic and antigenic relationships to the human strains. We initiated a study to determine the relationship between NoV prevalence in barns, farms and production systems. To date, porcine NoVs were detected from 33% pooled fecal samples. No human-like NoVs have been found by using separate RT-PCR assays with human NoV-specific primers or calicivirus universal primers followed. Recently, we detected a new calicivirus (WGP93C strain) by using RT-PCR with the calicivirus universal primer set followed by sequencing analysis of the RT-PCR products. Nucleotide BLAST search based on virus-specific 265 nucleotides revealed 88% identity with the new calicivirus, St-Valerien-like virus.<br /> <br /> Objective 2. Focus on effective interventions- Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents<br /> <br /> A. C. jejuni<br /> <br /> Arizona<br /> Effect of the Cj1534 mutation on the ability of C. jejuni to adhere to and invade epithelial cells. Cj1534, a putative bacterioferritin gene of C. jejuni, is up-regulated greater than 5-fold in studies conducted in chicks and in piglets. Mutation of the gene demonstrated a significant reduction in the colonization of chicks by the bacterium. Our current studies also demonstrated a reduction in colonization and invasion of epithelial cells in vitro by a Cj1534 mutant.<br /> <br /> Michigan<br /> C. jejuni is linked to the development of autoimmune diseases. We investigated the role of TLR2, TLR4, MyD88, and TRIF signaling in C. jejuni-induced inflammatory activation of DCs. DC upregulation of MHC-II and costimulatory molecules after C. jejuni challenge was profoundly impaired by TLR2, TLR4, MyD88, and TRIF deficiencies. Similarly, C. jejuni-induced secretion of IL-12, IL-6, and TNF-± was significantly inhibited in TLR2-/-, TLR4-/-, MyD88-/-, and TRIF-/- DCs compared to wild-type DCs; however, the magnitude of inhibition was greater in MyD88-/-, TRIF-/-, and TLR4-/- DCs than in TLR2-/- DCs. C. jejuni induced interferon regulatory factor-3 (IRF-3) phosphorylation and IFN-² secretion by DCs in a TLR4-TRIF-dependent fashion, further demonstrating activation of this pathway. TLR2, TLR4, MyD88, and TRIF deficiencies all impaired Th1-priming ability of C. jejuni-infected DCs. Thus, our results show that cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes represents a novel mechanism mediating C. jejuni-induced inflammatory responses of DCs.<br /> <br /> Ohio<br /> <br /> 1) C. jejuni colonization factors. The twin-arginine translocation (TAT) pathway represents an important virulence mechanism in many bacterial pathogens. The deletion of TAT significantly compromised the bacterial ability to tolerate stress responses including biofilm formation and cause persistent infection in chickens.<br /> <br /> 2) PolyPhosphate Kinases. Understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Polyphosphate kinase 1 is a key enzyme mediating the synthesis of polyphosphate, an essential molecule for survival, mediating stress responses, host colonization and virulence in many bacteria. Our findings demonstrate that C. jejuni ppk1 mutant was deficient in poly P accumulation and was associated with decreased ability to form viable but non-culturable cells 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.<br /> <br /> B. S. enterica<br /> <br /> Arizona<br /> 1) Vaccination of chicks with a S. Typhimurium strain (x9088) containing a plasmid vector expressing the Cj1534 gene. Hatchlings were immunized orally with viable cells of S. Typhimurium x9088 (pYA3493-empty vector; pYA4495-1-vector with Cj1534) on days 3, 10, and 16. The vaccinated birds were challenged on day 26 with viable cells of C. jejuni strain NCTC11168. Birds vaccinated with the Cj1534 protein demonstrated a C. jejuni reduction of 3.5 logs after challenge exposure.<br /> <br /> Kansas<br /> Efficacy of the S. Newport SRP® Vaccine in Feedlot Cattle. Our objectives were to determine the effects of a commercially available S. Newport siderophore receptor and porin protein (SRP®). There were no differences between SRP® vaccinated and control cattle in fecal prevalence of Salmonella or any cattle health or performance parameters. Salmonella were recovered from all ten replicates (pairs) of pens, with cumulative prevalence ranging from 1.5% to 22%. Cumulative prevalence of fecal shedding was 10.2% and 10.9% for SRP® vaccinated and control cattle, respectively.<br /> <br /> Washington<br /> 1) Sources and transmission of multidrug resistant Salmonella that cause infection in humans. Analysis of PFGE and MLVA genotype data of human- and bovine strains suggests that S. Typhimurium isolates are more genetically diverse than Newport isolates and that multidrug resistance within that serotype is polyclonal.<br /> <br /> 2) Characterization of bacterial antimicrobial resistance using a validated DNA microarray. We developed a 203-probe oligonucleotide array targeting resistance and virulence genes of Gram-negative pathogens. 59 dairy herds were sampled to estimate the rate of introduction of new multidrug-resistant (MDR) S. enterica strains onto commercial dairy herds. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year. The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. 56% had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. 13 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains.<br /> <br /> Recently, a new clade of S. Typhimurium, WA-TYP035/187, was reported in cattle and humans in the Pacific Northwest. The objective of this study was to describe a possible mechanism of acquisition of third generation cephalosporin resistance in this clade. Ceftazidime resistance increased steadily among WA-TYP035/187 isolates from 0% in 1999 to77.8% in 2006. 59 commercial dairy farms were sampled 7 times over 15 to 21 mo to determine the role of animal movement, including off-farm rearing of heifers, in the inter-herd transmission of MDR Salmonella spp. Logistic regression models indicated that off-farm heifer raising, including contract heifer raising where heifers commingle with cattle from other farms, and herd size per 100-animal increment were significantly associated with the introduction of new MDR Salmonella strains. The negative binomial regression similarly revealed that commingled, herd size per 100 animals, and a history of clinical salmonellosis diagnosed before the study were significantly associated with the number of new MDR Salmonella strains that were introduced.<br /> <br /> C. E. coli<br /> <br /> Kansas<br /> 1) Efficacy of E. coli O157:H7 SRP-based Vaccine in Feedlot Cattle. We evaluated the efficacy of an anti-E. coli O157 SRP-based vaccine in feedlot. 60 cattle were randomly allotted to 3 treatment groups: control, vaccinated with 2 ml or with 3 ml with vaccine. The vaccine at the 3 ml dose reduced the prevalence of E. coli O157 compared to the control (17.7% vs 33.7%). A similar numerical trend was observed with the 2 ml dose (29.1%), but differences were not statistically significant. Additionally, the 3 ml dose of SRP vaccine reduced the number of days cattle tested culture positive for E. coli O157, and the number of days cattle were identified as high-shedders compared to control.<br /> <br /> 2) Associations between E. coli O157:H7 in Feces and on Hides at Harvest and Contamination of Pre-evisceration Beef Carcasses. We found 38.5% of hides and 10.5% of carcasses positive for E. coli O157:H7. All truckload-level predictors significantly affected the probability of an E. coli O157:H7-positive carcass, including presence of a high shedder within the truckload, high (> 25%) within-truckload fecal prevalence, and high (> 50%) within-truckload hide prevalence; the only significant animal-level predictor was having a positive hide.<br /> <br /> 3) Prevalence of E. coli O157:H7 in Gut Contents of Beef Cattle at Slaughter. The prevalence of E. coli O157:H7 in the rumen, cecum, colon, and rectum were determined. Overall prevalence of E. coli O157:H7 in cattle sampled was 20.3%. E. coli O157:H7 in rectal contents was positively associated with presence in the rumen or colon but not in the cecum. The majority (79 to 90%) of isolates obtained within the same animal shared a common PFGE type. There was no significant difference between gut locations in reduction following acid challenge.<br /> <br /> 4) Feeding Dried or Wet Distillers Grains at Varying Inclusion Levels to Feedlot Cattle Affects the Fecal Prevalence of E. coli O157:H7. The inclusion of DG increased the prevalence of E. coli O157:H7 compared to the control cattle. The type of DG did not impact prevalence. Prevalence was not different between cattle fed diets with 0 or 20% DG, however, cattle fed 40% DG had significantly higher prevalence than either 0 or 20% DG. Similarly, the prevalence of super shedders was not different between cattle fed diets with WDG or DDG. Prevalence of super shedders in cattle fed 0 or 20% DG were not different, however, cattle fed 40% DG diets had a higher prevalence of high shedders than cattle fed either 0 or 20% DG.<br /> <br /> Nebraska<br /> Efficacy of a prebiotic galactooligosaccharide for reducing intestinal colonization by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7. Two litters of gnotobiotic piglets were used in a 2 X 2 factorial design with four treatment groups: GOS vs control with 2 challenge strains (EPEC strain E2348/69 or E. coli O157:H7 Shigatoxin-negative ATCC strain 43888). All inoculated piglets developed gross and microscopic intestinal lesions consistent with EPEC and E. coli O157:H7 infection. GOS reduced adherence of EPEC and EHEC in both the ileum and colon. However, the only statistically significant effect was a reduction of EHEC in the colons of piglets.<br /> <br /> Washington <br /> Bovine-biased lineages of EHEC O157 were non-virulent in the neonatal piglet model. Bovine-biased lineages were discovered to uniformly lack stx2, but rather carried stx1 and/or stx2c. These results suggest that approximately half of the isolates of O157 from the bovine reservoir have significantly less virulence than the rest.<br /> <br /> D. L. intracellularis<br /> <br /> Minnesota <br /> 1) In vitro antimicrobial activity against 10 North American and European L. intracellularis isolates. Our in vitro results showed that each L. intracellularis isolate had a different antimicrobial sensitivity pattern and these data can be utilized as an in vitro guideline for the further antimicrobial evaluation of field L. intracellularis isolates.<br /> <br /> 2) In vitro assessment of the effectiveness of powder disinfectant (Stalosan® F) against L. intracellularis using two different assays. Our results indicate that Stalosan F in both powder and suspension forms is able to inactivate over 99% of L. intracellularis after 30min of exposure.<br /> <br /> 3) Evaluation of in vitro bactericidal activity of commercial disinfectants against L. intracellularis. Results demonstrate that QAC, combinations of QACs with aldehydes, and oxidizing agents would perform well for inactivation of L intracellularis under field conditions.<br /> <br /> E. Antimicrobial resistance<br /> <br /> Washington<br /> <br /> Direct competition of plasmid bearing strains demonstrates that blaCMY-2 plasmids incur a measureable fitness cost in the face of plasmid-free competitors (3-12% loss per generation). Using a oligonucleotide microarray we showed that most genes on the plasmid backbone are quiescent in broth culture (log and stationary phases). In addition, all of the known antimicrobial resistance genes are expressed constitutively.<br /> <br /> Ceftiofur was not associated with any clear change in the number of blaCMY-2 expressing bacteria and conjugation rates between donor and recipient strains were not different from control groups.<br /> <br /> The objective of a clinical field trial was to determine the effect of colostrum supplementation of the milk replacer ration on morbidity, mortality, feed intake, and weight gain of preweaned calves. Calves receiving supplemental colostrum had less diarrhea and received fewer antimicrobial treatments than control and placebo calves. The results indicated that calf diarrhea was associated with low serum IgG levels and low-weight calves. Grain consumption and weight gain over the first 28 d of life were significantly greater in colostrum-supplemented calves compared with control calves.<br /> <br /> We determined the effect of raising pre-weaned dairy calves without antimicrobials in the milk. Newborn calves were allocated to one of 4 groups. Calves in the conventional therapy (CT) group were treated with sulfamethoxazole/trimethoprim, spectinomycin, penicillin and bismuth-pectin for diarrhea. The targeted therapy (TT) group included bismuth-pectin for diarrhea and antimicrobial treatment only in cases of fever or depressed attitude. Conventionally treated calves had 70% more diarrhea than TT calves and AB-milk calves had 31% more diarrhea than the NoAB-milk calves. The TT calves have a higher average daily gain at 28 days and consumed more grain compared to CT calves.<br /> <br /> We described the geographic, farm type and animal type factors associated with multiple antimicrobial resistance in bovine fecal E. coli. MAR (multiple antimicrobial resistance) was higher in isolates obtained from California cattle compared to Washington and Oregon cattle. MAR progressively decreased in isolates from calves on calf ranches, feedlot steers, dairy cattle and beef cow-calf cattle. MAR was higher in isolates obtained from calves than from adult cattle, in isolates from conventional farms than from organic farms, and in isolates from beef cow-calf farms in proximity to intensive dairy farm regions than in remote locations.<br /> <br /> F. Porcine Group A Rotavirus<br /> <br /> Ohio<br /> Rotavirus-like particle vaccines with/without attenuated rotavirus priming. We evaluated numbers of rotavirus-specific antibody-secreting cells (ASCs) and virus neutralizing (VN) antibody titers against Wa RV. The highest numbers of RV-specific IgA and IgG ASCs pre-challenge were induced in ileum by the AttMRV+2/6VLP vaccine, whereas the 2/6/7VLP+2/6/4VLP vaccine induced IgA and IgG ASCs in ileum only post-WaRV challenge. Both vaccines induced statistically similar levels of serum IgG antibodies pre-challenge, but the AttMRV+2/6VLP vaccine stimulated significantly higher serum IgA pre-challenge and serum IgG and IgA antibodies post-challenge. VN titers to the Wa strain were statistically higher pre-challenge for the AttMRV+2/6VLP vaccine than the 2/6/7VLP+2/6/4VLP vaccine but statistically similar post-challenge. Both vaccines induced similar low levels of heterotypic protection against diarrhea (36-37%) compared to 71% for the homotypic G1 AttWaRV+2/6VLP vaccine.<br /> <br /> G. Cornavirus<br /> <br /> Ohio<br /> Clarify the effects of experimental dual-infection by porcine reproductive and respiratory syndrome virus and porcine respiratory coronavirus on respiratory disease in pigs as a model for respiratory viral co-infections potentially relevant to the Severe Acute Respiratory Disease Syndrome. We investigated the effects of a prior and ongoing PRRSV infection, on PRCV co-infection and clinical disease and the pathologic and immunologic interactions of the two viruses, as a model for respiratory viral co-infections potentially relevant to SARS. The PRRSV/PRCV pigs had greater reductions in weight gains and more severe gross or histological pneumonic lesions compared to either single infection at PRCV PID 2 to 21. The PRRSV/PRCV dual virus-infected pigs had significantly suppressed innate immune responses, as evident by reduced IFN-± level in lung and blood. In addition, systemic NK cell-mediated cytotoxicity was significantly reduced in PRRSV only infected pigs. Upon co-infection with PRCV, there was a synergistic suppression of NK cell-mediated cytotoxicity. Co-infection by PRRSV and PRCV led to enhanced PRRSV replication in lung, a trend towards increased serum Th1 (IFN³ and IL-12), but decreased Th2 (IL-4) cytokine responses, and clinically exacerbated PRRSV pneumonia.<br /> <br /> H. C. parvum<br /> <br /> Illinois<br /> Experiments are under way to characterize the mechanism of inhibition of sporozoite adhesion. Suppressive subtractive hybridization experiments aimed at identifying specific sporozoite genes expressed in response to host cell attachment or exposure to the inhibitory lipid indicate these processes occur without the necessity for attachment- or lipid-induced differential gene expression. This lipid, introduced as part of different dietary regimens, also is being examined for its ability to reduce or eliminate bovine cryptosporidiosis in newborn calves.<br /> <br /> I. Microbiome<br /> <br /> Minnesota<br /> Alteration of the Pig Distal Gut Microbiota by Tylosin as an Antibiotic Growth Promoter. We hypothesized that the effects of AGPs are mediated by compositional changes of the pig gut microbiota. Fecal samples from tylosin treated and tylosin-non-treated pigs were collected 5 times at 3-week intervals. The sequences of the V3 hypervariable region of 16S rRNA were determined. The pig distal gut bacterial communities of both groups were dominated by Firmicutes and Bacterioidetes accounting for > 80% of total sequences, and showed highly diverse community structure (Shannon > 4.3, and Simpson 1-D > 0.9). Most of the sequences (> 95% of the total sequences) were shared by pigs in the two treatment groups. Components of the classes Actinobacteria, Clostridia, Fibrobacter, and Erysipelotrichi were different between the groups.<br /> <br /> Objective 3. Focus on disseminating knowledge- Provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians and diagnostic laboratories.<br /> <br /> North Dakota<br /> North Dakota State University and Makerere University Uganda offered the joint course International Animal Production, Disease Surveillance and Public Health in summer 2009. Eight students from the US and two students from MAK attended the course. In 2009, a planning grant from USAID was obtained. As a result of that grant, three planning meetings were held in East Africa and in Fargo, ND to develop a 10-year strategic plan on capacity building in Integrated Management of Transboundary Animal Diseases & Zoonoses in East & Central Africa.<br /> <br /> Washington<br /> WSU Developed an extension program centered around calf health and calf science.<br /> The issue of antimicrobial resistance in food animal agriculture was addressed by conducting clinical trials to assess alternatives to antimicrobials in dairy calf-raising and developing outreach to three different audiences.<br />

Publications

Publications:<br /> <br /> Arizona <br /> <br /> Law, B.F., S. M. Adriance, L.A. Joens. Comparison of in vitro Virulence Factors of Campylobacter jejuni to in vivo Lesion Production. Foodborne Pathogens and Disease. 6(3):377-85. 2009.<br /> <br /> Wilson, M. K., A. B. Lane, B. F. Law, W. G. Miller, L. A. Joens, M. E. Konkel, and B. A. White. Analysis of the Pan Genome of Campylobacter jejuni Isolates Recovered from Poultry by Pulsed-Field Gel Electrophoresis, Multilocus Sequence Typing (MLST), and Repetitive Sequence Polymerase Chain Reaction (rep-PCR) Reveals Different Discriminatory Capabilities. Microbial Ecology 58:843855. 2009. DOI 10.1007/s00248-009-9571-3.<br /> <br /> Illinois<br /> <br /> Pineda, MF, L. L. Chan, Theresa B. Kuhlenschmidt, Mark S. Kuhlenschmidt, and Brian. T. Cunningham (2009) Rapid Label-Free Selective Detection ofPorcine Rotavirus using Photonic Crystal Biosensors for Groundwater Monitoring. IEEE Sensors Journal 9(4):470-477.<br /> <br /> Andres, A., Donovan, S.M. and Kuhlenschmidt, M.S. 2009. Isoflavones and virus infections (Invited Review), J. Nutritional Biochemistry (20(8):563-9).<br /> <br /> Liu, Y, Dao Janjaroen, Mark S. Kuhlenschmidt, Theresa B.Kuhlenschmidt, Thanh H Nguyen. 2009. Deposition of Cryptosporidium Parvum Oocysts on Natural Organic Matter Surfaces: Microscopic Evidence for Secondary Minimum Deposition in a Radial Stagnation Point Flow Cell. Langmuir (in press)<br /> <br /> Donovan, S.M., Andres, A., Mathai, R.A., Kuhlenschmidt, T.B. and Kuhlenschmidt, M.S. 2009. Soy formula and isoflavones and the developing intestine. Nutritional Reviews (In Press).<br /> <br /> McLaughlin, S.J., Kuhlenschmidt, T.B., Kalita, P.K. and Kuhlenschmidt, M.S. 2009. Interaction of cryptosporidium parvum oocysts with soil particles andvegetated filter strips (Submitted).<br /> <br /> Kansas<br /> <br /> Alam M.J., Renter D.G., Taylor E.V., Mina D.M., Moxley R.A., Smith D.R. Antimicrobial susceptibility profiles of Salmonella enterica serotypes recovered from pens of commercial feedlot cattle using different sampling approaches. Current Microbiology 2009; 58(4): 354-359.<br /> <br /> Rao S., Van Donkersgoed, J., Bohaychuk V., Besser T., Song X., Wagner B., Hancock D., Renter D.G., Dargatz D, Morley P.S. Antimicrobial drug use and antimicrobial resistance in enteric bacteria among cattle from Alberta feedlots. Foodborne Pathogens and Disease, in press.<br /> <br /> Van Donkersgoed, J., Bohaychuk V., Besser T., Song X., Wagner B., Hancock D., Renter D.G., Dargatz D. Occurrence of foodborne bacteria in Alberta feedlots. Canadian Veterinary Journal 2009; 50(2): 166172.<br /> <br /> B. Other Publications.<br /> Journal Articles:<br /> <br /> Coolon J.D., Jones K.L., Narayanan S., Wisely S.M. Microbial Ecology of the Intestinal Flora of P. maniculatus and P. leucopus of the Tri-State Mining District. Molecular Ecology, in press.<br /> <br /> Dodd C., Renter D.G., Fox J.T., Shi X., Sanderson M.W., Nagaraja T.G. Genetic relatedness of Escherichia coli O157 isolates from cattle feces and pre-intervention beef carcasses. Foodborne Pathogens and Disease, in press.<br /> <br /> Taylor E.V., Kastner J.J., Renter D.G. Challenges involved in the Salmonella Saintpaul outbreak and lessons learned. Journal of Public Health Management and Practice, in press.<br /> <br /> Walker, C., Shi X., Sanderson M. J., Sargeant J. S., Nagaraja T. G.. Prevalence of Escherichia coli O157:H7 in gut contents of beef cattle at slaughter. Foodborne Pathogens and Disease, in press.<br /> <br /> Reinstein S., Fox J.T., Shi X., Alam M.J., Renter D.G., Nagaraja T.G. Prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle. Applied and Environmental Microbiology 2009; 75(16): 5421-5423.<br /> <br /> Alam M.J., Renter D.G., Ives S.E., Thomson D.U., Hollis L.C., Sanderson M.W., Nagaraja T.G. Potential associations between fecal shedding of Salmonella in feedlot cattle treated for apparent respiratory disease and subsequent adverse health outcomes. Veterinary Research 2009; 40:02.<br /> <br /> Jacob M.E., Fox J.T., Drouillard J.S., Renter D.G., Nagaraja T.G. Evaluation of feeding dried distillers grains with solubles and dry-rolled corn on the fecal prevalence of Escherichia coli O157:H7 and Salmonella spp. in cattle. Foodborne Pathogens and Disease 2009; 6(2): 145-153.<br /> <br /> Peterson G., Bai J., Narayanan S. A co-printed oligomer to enhance reliability of spotted microarrays. J Microbiol Methods 2009; 77(3): 261-266.<br /> <br /> Fox, J. T., Drouillard, J. S., and Nagaraja T. G. Competitive exclusion Escherichia coli cultures on E. coli O157 batch culture ruminal or fecal microbial fermentation. Foodborne Pathogens and Disease 2009; 6:193-199.<br /> <br /> Thomson D. U., Loneragan, G. H., Thornton A. B., Lechtenberg K. F., Emery D. A., Burkhardt D. T., Nagaraja T. G. .Use of a siderophore receptor and porin proteins-based vaccine to control the burden of Escherichia coli O157:H7 in feedlot cattle. Foodborne Pathogens and Disease 2009; 6:871-877.<br /> <br /> Fox, J. T., Thomson, D. U., Drouillard J. S., Thornton, A. B., Burkhardt D. T., Emery D. A., Nagaraja T. G. Efficacy of Escherichia coli O157:H7 siderophore receptor/porin proteins-based vaccine in feedlot cattle naturally shedding E. coli O157. Foodborne Pathogens and Disease 2009; 6:893-899.<br /> <br /> Fox, J. T., Drouillard J. S., Nagaraja T. G. 2009. Effects of mucin and its carbohydrate constituents on Escherichia coli O157 in batch culture fermentations with ruminal or fecal microbial inoculums. Journal of Animal Science 2009; 87:1304-1313.<br /> <br /> Thornton, A. B., Thomson D. U., Fox J. T., Loneragan G. H., Burkhardt D. T., Emery D. A., Nagaraja T G. .Siderophore receptor/porin proteins-based vaccination reduces prevalence and shedding of Escherichia coli O157:H7 in experimentally inoculated cattle. Journal of Food Protection 2009; 72:866-869.<br /> <br /> Jacob, M. E., Callaway T. R., Nagaraja T. G. Dietary interactions and interventions affecting Escherichia coli O157 colonization and shedding in cattle. Foodborne Pathogens and Disease 2009; 6:785-792.<br /> <br /> Theses and Dissertations:<br /> <br /> Taylor, Ethel. 2009. Foodborne disease in the United States: from surveillance to policy. MPH, Report. (Advisor, D.G. Renter)<br /> <br /> Lubbers, Brian. 2009. The Impact of Oxytetracycline Dosing on Bacterial Populations and Transfer of Resistance Elements between Salmonella and E. coli in Vitro and in Vivo. Ph.D. Dissertation. (Minor Advisor: S. Narayanan).<br /> <br /> Other Presentations and Published Abstracts:<br /> <br /> Renter, David G. (invited presentation). E. coli O157:H7  Dissemination and Persistence in Bovine Production Environments. The Third Governors Conference on Ensuring Food Safety: E. coli O157:H7  progress and challenges. University of Nebraska-Lincoln. 2009.<br /> <br /> Nagaraja, T. G. (invited presentation). Feeding Distillers Grains to Ruminants: What do we know so far? The Third Governors Conference on Ensuring Food Safety: E. coli O157:H7  progress and challenges. University of Nebraska-Lincoln. 2009.<br /> <br /> Renter D.G., Dodd, C.C., Thomson D.U., Nagaraja T.G. Salmonella in beef cattle production systems. BIFSCO - Beef Industry Safety Summit, San Diego CA. 2009.<br /> <br /> Dodd, C.C., Renter D.G., Thomson D.U., Nagaraja T.G. A randomized trial assessing effects of a commercially available Salmonella vaccine in commercial feedlot cattle. Conference for Research Workers in Animal Disease, Chicago, IL. 2009. #64.<br /> <br /> Dodd, C.C., Renter D.G., Nagaraja T.G., Shi X. Prevalence and persistence of Salmonella within pens of feedlot cattle. Conference for Research Workers in Animal Disease, Chicago, IL. 2009. #44.<br /> <br /> Peterson, G, Bai, J., Nagaraja, T.G., Patton, J., and Narayanan.S. Diagnostic Microarray for Human and Animal Bacterial Diseases. Conference for Research Workers in Animal Disease, Chicago, IL. 2009.<br /> <br /> Peterson, G., and Narayanan, S. Norepinephrine Increases Horizontal Gene Transfer Rates Between Enteric Bacteria. Conference for Research Workers in Animal Disease, Chicago, IL. 2009.<br /> <br /> Gart, E., and Narayanan, S. Influence of variable concentration of catecholamines on the growth of Citrobacter rodentium and expression of virulence genes. Conference for Research Workers in Animal Disease, Chicago, IL. 2009.<br /> <br /> Nagaraja, T. G. Pathogen concerns for organically-grown beef. Symposium on organic poultry and red meats-microbiology considerations: What do we know, what more do we need to know and how do we get there? Institute of Food Technology Annual Meeting, Anaheim, CA, 2009.<br /> <br /> Michigan<br /> <br /> Bell JA, St. Charles JL, Murphy AJ, Rathinam VA, Plovanich-Jones AE, Stanley EL, Wolf JE, Gettings JR, Whittam TS, Mansfield LS. 2009. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10(-/-) mice, BMC Microbiology, 2009 Mar 18;9:57, PMID: 19296832.<br /> <br /> Rathinam VA, Appledorn DM, Hoag KA, Amalfitano A, Mansfield LS. 2009. Campylobacter jejuni-induced activation of dendritic cells involves cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes, Infection and Immunity, 2009 Mar 30, PMID: 19332531.<br /> <br /> Rathinam VA, Hoag KA, and Mansfield LS. 2008. Dendritic cells from C57BL/6 mice undergo activation and induce Th1-effector cell responses against Campylobacter jejuni. Microbes and Infection 2008 Oct;10(12-13):1316-24. Epub 2008 Aug 5, PMID: 18725315.<br /> <br /> Mansfield LS, Patterson JS, Fierro BR, Murphy AJ, Rathinam VA, Kopper JJ, Barbu NI, Onifade TJ, and JA Bell. 2008. Genetic background of IL-10-/- mice alters host-pathogen interactions with Campylobacter jejuni and influences disease phenotype, Microbial Pathogenesis 2008 Oct;45(4):241-57 Epub2008 Jun 11.<br /> <br /> Parthasarathy G and Mansfield LS. 2008. Recombinant IL-4 (rIL-4) enhances Campylobacter jejuni invasion of Intestinal Pig Epithelial Cells (IPEC-1), Microbial Pathogenesis, Accepted April 22, 2009. ISSN 0882-4010.<br /> <br /> Abstracts<br /> V.A.K. Rathinam, Daniel M. Appledorn, Kathleen A. Hoag, Andrea Amalfitano and Linda S. Mansfield. 2009. TLR4?MyD88 and TLR4?TRIF signaling axes cooperate in activating dendritic cells in response to an intracellular enteric pathogen. Pattern Recognition Molecules and Immune Sensors, Keystone Meetings, Banff, Alberta CA, March 2009. <br /> <br /> J.A. Bell, J.L. St. Charles, A.J. Murphy, V.A. Rathinam, A.E. Plovanich-Jones, E.L. Stanley, J.E. Wolf, J.R. Gettings, T.S. Whittam, Tristan Lefebure, Michael Stanhope, L.S. Mansfield. 2009. Pathogenicity of Campylobacter jejuni strains in C57BL/6 IL-10-/- mice varies with strain genetic background, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.<br /> <br /> J.P. Jerome, A.E. Plovanich-Jones, J.A. Bell, Linda S. Mansfield. 2009. Genetic changes during serial passage of Campylobacter jejuni in C57BL/6 IL-10-/- mice are associated with enhanced virulence, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.<br /> <br /> J.D. Olmstead, V.A.K. Rathinam, L.S. Mansfield. 2009. Determining organ specific recovery of Campylobacter jejuni in a murine disease model, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.<br /> <br /> JL St. Charles1,3, JA Bell1, and LS Mansfield. 2009. Development of murine models for study of Guillain-Barré Syndrome, FWDIRN Annual Meeting, Stevenson, WA. March 30-April 1, 2009.<br /> <br /> Minnesota<br /> <br /> BOOK CHAPTER<br /> Gebhart, C.J. and R.M.C. Guedes. (In press). Lawsonia intracellularis and the proliferative enteropathies. In C.L. Gyles, et al., (ed.), Pathogenesis of Bacterial Infections in Animals, 4th ed. Wiley-Blackwell Publishing, Hoboken, NJ. <br /> <br /> JOURNAL ARTICLES<br /> Wattanaphansak, S., R.S. Singer and C.J. Gebhart. 2009. Evaluation of in vitro bactericidal activity of commercial disinfectants against Lawsonia intracellularis. J. Swine Health and Production (accepted).<br /> <br /> Pusterla, N., R. Jackson, S. M. Mapes, J. Noland, R.M. Stenbom and C. Gebhart. 2009. Lawsonia intracellularis: Humoral immune response and fecal shedding in weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live vaccine. Vet. J. (in press). <br /> <br /> Guedes, R.M.C. and C.J. Gebhart. 2009. Evidence of involvement of cell-mediated immune response and specific local mucosal IgA production in Lawsonia intracellularis experimental infection. 2009. Can. J. Vet. Res. (in press).<br /> <br /> Pusterla, N. and C. Gebhart. 2009. Equine proliferative enteropathy caused by Lawsonia intracellularis. Equine Vet. J. (in press).<br /> Pusterla, N., J.R. Collier, S.M. Mapes, S. Wattanaphansak, C. Gebhart. 2009. Effect of administration of a virulent live vaccine of Lawsonia intracellularis on mares and foals. Vet Rec. 164:783-785. <br /> <br /> Wattanaphansak, S., R.S. Singer, R.E. Isaacson, J. Deen, B.R. Gramm and C.J. Gebhart. 2009. In vitro assessment of the effectiveness of powder disinfectant (Stalosan F) against Lawsonia intracellularis using two different assays. Vet. Microbiol. 136:403-407.<br /> <br /> Pusterla, N., R. Jackson, R. Wilson, J. Collier, S. Mapes and C. Gebhart. 2009. Temporal detection of Lawsonia intracellularis using serology and PCR in Thoroughbred horses residing on a farm endemic for equine proliferative enteropathy. Vet. Microbiol.136:173-176.<br /> <br /> Wattanaphansak, S., R.S. Singer and C.J. Gebhart. 2009. In vitro antimicrobial activity against 10 North American and European Lawsonia intracellularis isolates. Vet. Microbiol. 134:305-310.<br /> <br /> Wattanaphansak, S., Singer, R.S., Isaacson, R.E., Deen, J., Gramm, B.R., and Gebhart, C.J. In vitro assessment of the effectiveness of powder disinfectant (Stalosan F) against Lawsonia intracellularis using two different assays. Veterinary Microbiology 136: 403 (2009).<br /> <br /> Singer, R.S., Mayer, A.E., Hanson, T.E., and Isaacson, R.E. Do microbial interactions and cultivation media decrease the accuracy of Salmonella surveillance systems and outbreak investigations? Journal of Food Protection 72:707 (2009).<br /> <br /> Nebraska<br /> Refereed Journal Articles:<br /> <br /> Alam, M. J., D. Renter, E. Taylor, D. Mina, R. Moxley, and D. Smith. 2009. Antimicrobial susceptibility profiles of Salmonella enterica serotypes recovered from pens of commercial feedlot cattle using different types of composite samples. Current Microbiol. 58:354-359.<br /> <br /> B. Other Publications.<br /> <br /> Refereed Journal Articles:<br /> <br /> Smith, D. R., R. A. Moxley, R. E. Peterson, T. J. Klopfenstein, G. E. Erickson, G. Bretschneider, E. M. Berberov, and S. Clowser. 2009. A two-dose regimen of a vaccine against type III secreted proteins reduced Escherichia coli O157:H7 colonization of the terminal rectum in beef cattle in commercial feedlots. Foodborne Pathog. Dis. 6 (2):155-161.<br /> <br /> Moxley, R.A., D.R. Smith, M. Luebbe, G. E. Erickson, T. J. Klopfenstein, and D. Rogan. 2009. Escherichia coli O157:H7 vaccine dose-effect in feedlot cattle. Foodborne Pathog. Dis. 6 (7):879-884.<br /> <br /> Smith, D. R., R. A. Moxley, T. J. Klopfenstein, and G. E. Erickson. 2009. A randomized longitudinal trial to test the effect of regional vaccination within a cattle feedyard on Escherichia coli O157:H7 rectal colonization, fecal shedding, and hide contamination. Foodborne Pathog. Dis. 6 (7):885-892.<br /> <br /> Book Chapters:<br /> <br /> Moxley, R. A., D. R. Smith. 2010. Attaching-effacing Escherichia coli infections in cattle. Veterinary Clinics of North America: Food Animal Practice. (accepted)<br /> <br /> Extension Reports:<br /> <br /> Rich, A. R., A. N. Jepson, M. K. Luebbe, G. E. Erickson, T. K. Klopfenstein, D. R. Smith, and R. A. Moxley. 2009. Vaccination to reduce the prevalence of Escherichia coli O157:H7 in feedlot cattle fed wet distillers grains plus solubles. J. Anim. Sci. Vol. 87, E-Suppl. 3:268<br /> <br /> Research Presentations and Published Abstracts:<br /> <br /> Klopfenstein, T. J., D. R. Smith, G. E. Erickson, and R. A. Moxley. Feeding distillers grains and E. coli O157:H7. 2009 Nebraska Beef Report. Agricultural Research Division, University of Nebraska Cooperative Extension, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, MP92:47-49.<br /> <br /> Extension and Teaching Presentations:<br /> <br /> Smith, DR. Pre-harvest interventions for food safety. Universidad Autonoma Chapingo, Chapingo, Mexico, Mexico Date: 10/20/2009 <br /> <br /> Smith, DR. The ecology of food safety pathogens in live cattle: with emphasis on Escherichia coli O157:H7 and Salmonella. 1er Simposium Internacional de Inocuidad de alimentaria. Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional Autonoma d Mexico. Mexico City, DF, Mexico. Date: 10/21/2009 <br /> <br /> Smith, DR. On-farm practices that improve the safety of food: the example of Escherichia coli O157:H7 in cattle feedlots. 1er Simposium Internacional de Inocuidad de alimentaria. Facultad de Medicina Veterinaria y Zootecnia. Universidad Nacional Autonoma d Mexico. Mexico City, DF, Mexico. Date: 10/22/2009 <br /> <br /> Smith DR,Rich AR, Jepson AN, Luebbe MK, Erickson GE, Moxley RA, Klopfenstein TJ. A randomized trial to test the effect of vaccination and feeding high levels of wet distillers grains plus solubles on the probability for feedlot cattle to shed Escherichia coli O157:H7 in feces. International Society for Veterinary Epidemiology and Economics. Durban, South Africa. Date: 08/12/2009 <br /> <br /> Smith DR. Can what we feed cattle affect the safety of food? The effect of distillers grains on E. coli O157:H7 <br /> Date: 05/05/2009 <br /> <br /> DR Smith, AN Jepson, AR Rich, MK Luebbe, RA Moxley, GE Erickson, TJ Klopfenstein. A randomized trial to test the effect of vaccination and feeding high levels of wet distillers grains plus solubles on the probability for feedlot cattle to shed Escherichia coli O157:H7 in feces. Beef Industry Safety Summit. BIFSCO Date: 03/04/2009 <br /> <br /> Smith DR. An update on pre-harvest control of E. coli O157:H7. Nance County Cattlemen Assoc. Date: 02/16/2009 <br /> <br /> Smith DR. E. coli O157:H7 interventions and distillers grains. Nebraska Beef Feedlot Roundtable. Norfolk, Lexington, Bridgeport. Date: 02/10/2009 <br /> <br /> Smith DR. Beef cattle diseases: Can practitioners get paid for population thinking. 18th Annual Alabama Conference for Food Animal Veterinarians <br /> Date: 02/07/2009 <br /> <br /> Smith DR. Applying population thinking to neonatal calf diarrhea. 18th Annual Alabama Conference for Food Animal Veterinarians. Birmingham, AL Date: 02/07/2009 <br /> <br /> Smith DR. Population thinking in medicine. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> Smith DR. The art and science of interpreting diagnostic tests. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> Smith DR. The art and science of population diagnostics. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> Smith DR. Applying population thinking to neonatal calf diarrhea. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> Smith DR. Applying population diagnostics to the control of BVDV. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> Smith DR. Applying population diagnostics to the control of Johnes disease. North American Veterinary Conference. Orlando, FL Date: 01/19/2009 <br /> <br /> AN Jepson, RA Moxley, DR Smith, GE Erickson, TJ Klopfenstein, D Rogan. Vaccine dose and serological response of feedlot cattle vaccinated against Escherichia coli O157:H7. Conference of Research Workers in Animal Disease. Chicago, IL <br /> Date: 12/07/2008 <br /> <br /> A. R. Rich, A. N. Jepson, M. K. Luebbe, G. E. Erickson, T. J. Klopfenstein, D. R. Smith, R. A. Moxley. E.coli O157:H7 -incidence, control, and distillers grain <br /> <br /> North Dakota<br /> <br /> Oloya, D. Doetkott, M.L. Khaitsa. 2009. Antimicrobial drug resistance and molecular characterization of Salmonella isolated from domestic animals, humans and meat products. J. Foodborne Pathogens & Disease, 2009, 6(3) 273-284. <br /> <br /> Luke C. Heider, M.L. Khaitsa, et al. 2009. Genetic and phenotypic characterization of the blaCMY gene from Escherichia coli and Salmonella enterica isolated from food-producing animals, humans, the environment and retail meat. J. Foodborne Pathogens & Disease, 2009, 6(3) 273-284.<br /> <br /> Papers submitted and currently under review: <br /> Ebot S.Tabe, James Oloya, Dawn K. Doetkott, Margaret L. Khaitsa. 2009. Characterization of multidrug-resistant Salmonella Typhimurium serovar Copenhagen isolated from feedlot cattle. Journal of Food Protection Trends, Manuscript ID FPT 09-29, Submitted September 29, 2009.<br /> <br /> Abstracts submitted to Conferences:<br /> Tabe ES, Oloya J, Doetkott DK, Bauer ML, Mahero MW, Khaitsa ML. 2009. Comparative effect of direct-fed-microbials on fecal shedding and genotypic diversity of Escherichia coli O157:H7 and Salmonella in Feedlot cattle. In Proceedings of the 12th International Symposium on Veterinary Epidemiology and Economics (ISVEE), August 10-14, 2009 Durban, South Africa.<br /> <br /> Michael Mahero, Susan Olet, Dawn Doetkott, Margaret Khaitsa. Characterisation of Antimicrobial Resistance (AMR) and Presence of Class 1 Integrons In Salmonella Serovars Isolated From Clinical Cases of Animals And Humans In North Dakota. North Dakota Academy of Science, April 30, 2009 Fargo ND, p.58.<br /> <br /> M.W.Mahero, D. K. Doetkott, S. Olet, D.K. Byarugaba, M. L.Khaitsa. Characterisation of Antimicrobial Resistance and Presence of Class 1 Integrons in Salmonella Isolated from Animals and Humans In North Dakota, USA and Kampala Uganda. North Dakota EPSCOR Conference, September 24, 2009 at North Dakota State University, Fargo ND. Poster #70.<br /> <br /> Nesemeier, B., Doetkott, D., Olet, S and Khaitsa, ML. Characterization of Salmonella spp. isolated from beef cattle from post weaning to slaughter. North Dakota EPSCOR Conference, September 24, 2009 at North Dakota State University, Fargo ND. Poster #71.<br /> <br /> Michael Mahero, D. K. Doetkott, S. Olet, D.K. Byarugaba, M. L.Khaitsa. Characterization of Antimicrobial Resistance and Presence of Class 1 Integrons in Salmonella Isolated from Animals and Humans In North Dakota, USA and Kampala Uganda. North Dakota. In Proceedings of the 69th annual meeting of the North Central Branch of the American Society for Microbiology, October 9-10, at La Crosse, WI.<br /> <br /> Ohio<br /> Azevedo, M.S.P., L. Yuan, A.M. Gonzalez, K. Jeong, T. Nguyen, C. Iosef, K. Lovgren-Bengtsson, B. Morein, and L.J. Saif. 2009. Protection and antibody secreting cell responses induced by a combined vaccine of attenuated human rotavirus oral or intranasal priming and intranasal 2/6 VP rotavirus-like particle boosting with ISCOM in gnotobiotic pigs. Clin. Vaccine. Immun. (submitted).<br /> <br /> Azevedo, M.S.P., A.M. Gonzalez, L. Yuan, K. Jeong, C. Iosef, T. van Nguyen, K. Lovgren-Bengtsson, B. Morein and L.J. Saif. 2009. Oral versus intranasal prime/boost regimen using attenuated HRV or 2/6 VLP with ISCOM influences protection and antibody secreting cell responses to rotavirus in neonatal gnotobio. Vaccine Immun. (in revision).<br /> <br /> D. Gangaiah, Issmat I. Kassem, Zhe Liu, and Gireesh Rajashekara. 2009. Importance of Polyphosphate Kinase 1 for Campylobacter jejuni: Viable-but-Nonculturable Cell Formation, Natural Transformation, and Antimicrobial Resistance. Applied Environ. Microbiol. (In Press)<br /> <br /> Drozd M., II. Kassem, W. Gebreyes, G. Rajashekara. 2010. A quantitative polymerase chain reaction assay for detection and quantification of Lawsonia intracellularis. J. Vet. Diagnos. Investigation (In Press)<br /> <br /> Rajashekara, G, Drozd, M., Gangaiah, D., Jeon, B., Liu, Z., and Zhang, Q., 2009, Functional Characterization of The Twin-arginine Translocation System in Campylobacter jejuni. Foodborne pathogens and Dis. 6: 935-945.<br /> <br /> Abstracts<br /> <br /> Saif, L.J. Gut reactions to rotavirus vaccines in a gnotobiotic piglet disease model: a priming lesson for neonatal mucosal immunity. The 5th Intl. Veterinary Vaccines and Diagnostics Conference, Madison, WI, July 19-23, 2009.<br /> <br /> Vlasova, A.N., M.S.P. Azevedo, H.R. Resque, A.M. Hartzler, and L.J. Saif. The effect of virulent human rotavirus challenge on TLR3 and 4 responses and plasmacytoid dendritic cell distribution in gnotobiotic pigs vaccinated with rotavirus-like particles (VLPs). Proc. 14th Intl. Congress of Mucosal Immunology (ICMI), Abstract, Boston, MA. July 5-9, 2009.<br /> <br /> Resque, H.R., A.N. Vlasova, M.S.P. Azevedo, A.M. Hartzler, and L.J. Saif. Rotavirus-like particle (CLP) vaccines with/without attenuated rotavirus priming induce heterotypic rotavirus antibody responses but low protection rates to heterotypic rotavirus challenge. Proc.14th Intl. Congress of Mucosal Immunology (ICMI), Abstract, Boston, MA. July 5-9, 2009.<br /> <br /> Wang, Q., Z. Zhang, and L.J. Saif. Impact of pH on attachment of the enteropathogenic calicivirus porcine sapovirus to lettuce leaves. Amer. Soc. Virol. 28th Annual Mtg, Juniv. Of British Columbia, Vancouver, BC, Canada, July 11-15, 2009.<br /> <br /> Renukaradhya, G., K. Alekseev, K. Jung, and L. J. Saif. Distorted immune responses in pigs to porcine respiratory coronavirus previously infected with porcine reproductive and respiratory syndrome virus: a respiratory viral co-infection model. CRWAD Meeting, Chicago, IL, December 6- 8, 2009.<br /> <br /> Saif, L.J., Jung, K., G.J. Renukaradhya and Zhang, X. Respiratory viral co-infections and corticosteroid treatments influence disease, pathology and immunologic responses to a porcine respiratory coronavirus of pigs. CRWAD Meeting, Chicago, IL, December 6- 8, 2009.<br /> <br /> Y. M. Sanad*, Z. Liu, I. Kassem, D. Gangaiah, J. T. LeJeune, G. Rajashekara. Molecular Epidemiological studies on Campylobacter species in cattle. CRWAD Meeting, Chicago, IL, December 6- 8, 2009.<br /> <br /> D. Gangaiah*, I. Kassem, J. Torrelles, Z. Liu, Y. Sanad and G. Rajashekara. Role of Polyphosphate Kinase 2 in Campylobacter jejuni stress responses, host colonization and pathogenesis. CRWAD Meeting, Chicago, IL, December 6- 8, 2009.<br /> <br /> D. Gangaiah , Issmat I. Kassem, Byeonghwa Jeon, Zhe Liu, Qijing Zhang, and Gireesh Rajashekara. 2009. "Polyphosphate Kinase 1 is Important for VBNC Formation, Natural Transformation and Antimicrobial Resistance in Campylobacter jejuni." In: 15th International workshop on Campylobacter, Helicobacter and Related Organisms. September 2Toki Messe, Niigata, Japan.<br /> <br /> D. Gangaiah, B. Adhikari, B. Jeon, Q. Zhang , Z. Liu, Y. Sanad, M. Drozd, and G. Rajashekara. 2009. "Deciphering the role of Polyphosphate kinases in Campylobacter jejuni colonization/ pathogenesis." In: 146th Annual AVMA Convention. July 11,2009, Seattle, Washington, USA.<br /> <br /> Rajashekara, Gireesh. 2009. "Campylobacter jejuni stress responses and in vivo colonization: Relevance to food safety." In: International conference on Food, Environment, and Health. June 20,2009, Xian, China. <br /> <br /> South Dakota<br /> <br /> Glenn GM, DH Francis and EM Danielsen. 2009. ToxinMediated Effects on the Innate Mucosal Defenses: Implications for Enteric Vaccines. Infect Immun 77: 5206-5215. Minireview.<br /> <br /> Johnson AM, RS Kaushik, PR Hardwidge. 2009. Disruption of transepithelial resistance by enterotoxigenic Escherichia coli. Vet Microbiol. In press. (accepted on Aug 7, 09).<br /> <br /> Johnson, AM, RS Kaushik, DH Francis, JM Fleckenstein, and PR Hardwidge. 2009. The heat-labile enterotoxin promotes enterotoxigenic Escherichia coli adherence to intestinal epithelial cells. J Bacteriol. 191: 178-186.<br /> <br /> Lin J, T. Wolff, A Erickson and D Francis. 2009. Effect of bacitracin tetracycline resistance in Escherichia coli and Salmonella. Vet Microbiol 138: 353-360.<br /> <br /> Rettedal E, S Vilain, S Lindblom, K Lehnert,C Scofield,S George,S Clay, RS Kaushik, AJM Rosa, DFrancis, and VS Brozel. 2009. Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline. Appl Environ Microbiol 75: 54895495.<br /> <br /> Zhang, W, C Zhang, DH Francis, Y Fang, D Knudsen, JP Nataro, and DC Robertson. 2010. Genetic fusions of LTAB and STa toxoids of porcine enterotoxigenic Escherichia coli (ETEC) elicited neutralizing anti-LT and anti-STa antibodies. Infect Immun. In Press.<br /> <br /> Zhang, W., Y. Fang, and D. H. Francis. 2009. Characterization of the binding specificity of K88ac and K88ad fimbriae of enterotoxigenic Escherichia coli by constructing K88ac/K88ad chimeric FaeG major subunits. Infect Immun. 77: 699-706. <br /> <br /> Washington<br /> <br /> Adhikari B, Besser TE, Gay JM, Fox LK, Davis MA, Cobbold RN, Berge ACB, McClanahan R, Hancock DD. 2009. Introduction of New Multidrug Resistant Salmonella enterica Strains into Commercial Dairy Herds. J Dairy Science. 2009; 92:4218-4228.<br /> <br /> Adhikari B, Besser TE, Gay JM, Fox LK, Davis MA, Cobbold RN, Berge ACB, McClanahan R, Hancock DD. 2009. The Role of Animal Movement, Including Offfarm Rearing of Heifers, in the Inter-Herd Transmission of MDR Salmonella. J Dairy Science. 2009; 92:4229-4238.<br /> <br /> Adhikari B, Besser TE, Gay JM, Fox LK, Hancock DD, Davis MA. Multilocus variable number of tandem repeats analysis and plasmid profiling to study the occurrence of blaCMY-2 within a PFGE-defined clade of Salmonella enterica serovar Typhimurium isolated from cattle and humans. Applied and Environmental Microbiology. 2009 Oct 30. [Epub ahead of print] PubMed PMID: 19880639.<br /> <br /> BERGE A.C.B., BESSER T.E., MOORE D.A., SISCHO, W.M. 2009. EVALUATION OF THE EFFECTS OF ORAL COLOSTRUM SUPPLEMENTATION DURING THE FIRST FOURTEEN DAYS ON THE HEALTH AND PERFORMANCE OF PREWEANED CALVES. JOURNAL OF DAIRY SCIENCE 92:286-295.<br /> <br /> BERGE A.C., HANCOCK D.D., SISCHO W.M., BESSER T.E. 2009. GEOGRAPHIC, FARM AND ANIMAL FACTORS ASSOCIATED WITH MULTIPLE ANTIMICROBIAL RESISTANCE IN BOVINE FECAL ESCHERICHIA COLI IN THE WESTERN UNITED STATES. JOURNAL OF AMERICAN VETERINARY MEDICAL ASSOCIATION (IN PRESS).<br /> <br /> BERGE A.C., MOORE D.A., BESSER T.E., SISCHO W.M. 2009. TARGETING THERAPY TO MINIMIZE ANTIMICROBIAL USE IN PRE-WEANED CALVES: EFFECTS ON HEALTH, GROWTH, AND TREATMENT COSTS. JOURNAL OF DAIRY SCIENCE 92:4707-14.<br /> <br /> Call, DR, MA Davis, and AA Sawant. 2008. Antimicrobial resistance in beef and dairy cattle production. Animal Health Research Reviews 9:159-167. PMID: 18983724.<br /> <br /> Call, DR, RS Singer, D Meng, SL Broschat, LH Orfe, JM Anderson, DR Herndon, LS Kappmeyer, JB Daniels, and TE Besser. BlaCMY-2 positive Inc A/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. In press, Antimicrobial Agents and Chemotherapy.<br /> <br /> Daniels JB, Call DR, Hancock DD, Sischo WM, Baker K, Besser TE. The role of ceftiofur in the selection and dissemination of blaCMY-2  mediated cephalosporin resistance in Salmonella enterica and commensal Escherichia coli isolated from cattle. Applied and Environmental Microbiology 75:3648-3655.<br /> <br /> Davis, MA, KNK Baker, DR Call, LD Warnick, Y Soyer, M Wiedmann, Y Gröhn, PL McDonough, DD Hancock, and TE Besser. 2009. Multiple locus variable number of tandem repeats typing method for Salmonella enterica serovar Newport. Journal of Clinical Microbiology 47:1934-1938.<br /> <br /> Leopold SR, Magrini V, Holt NJ, Shaikh N, Mardi ER, Cagno J, Orgura Y, Iguchi A, Hayashi T, Mellmann A, Karch H, Besser TE, Sawyer SA, Whittam TS, Tarr PI. 2009. A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis. Proc National Acad Sciences. 106(21):8713-8. ZW011<br /> <br /> Meng, D, SL Broschat, and DR Call. 2008. A Java-based tool for the design of classification microarrays. BMC Bioinformatics 9:328. PMID: 18680597.<br /> <br /> Shah DH, Shringi S, Besser TE, Call DR. Escherichia coli O157:H7. In: Liu, D, Editor. Molecular Detection of Foodborne Pathogens, CRC Press, Chapter 27. 2009.<br /> <br /> Soyer Y, A Moreno Switt, MA Davis, J Maurer, PL McDonough, D J Schoonmaker-Bopp, NB Dumas, T Root, LD Warnick, YT Gröhn, M Wiedmann. 2009. Salmonella 4,5,12:i:-: an emerging Salmonella serotype that represents multiple distinct clones. Journal of Clinical Microbiology . In press.<br /> <br /> Vanaja SK, Springman AC, Besser TE, Whittam TS, Manning SD. 2009. Differential Expression of Virulence and Stress Fitness Genes between Clinical and Bovinebiased Genotypes of Escherichia coli O157:H7. In press, Applied and Environmental Microbiology.<br /> <br /> Whitworth JH, Fegan N, Keller J, Gobius KS, Bono JL, Call DR, Hovde CJ, Hancock DD, Besser TE. At an international scale, E. coli O157:H7 Stx bacteriophage insertion genotypes have a variable distribution between human and cattle hosts. Applied and Environmental Microbiology. 2008; 74:7447-7450.<br /> <br /> Whitworth JH, et al. Diverse genetic markers concordantly identify bovine-origin Escherichia coli O157:H7 under-represented in human disease. In press, Applied and Environmental Microbiology.<br /> <br /> Zhou X, Konkel ME, Call DR. Type III secretion system 1 of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines. Microbiology. March 2009. 155(Pt3):837-51.<br /> <br /> Zhou, X, DH Shah, ME Konkel, and DR Call. 2008. Type III secretion system 1 genes in Vibrio parahaemolyticus are positively regulated by ExsA and negatively regulated by ExsD. Molecular Microbiology 69:747-764. PMID: 18554322.<br />

Impact Statements

1. The in vitro antimicrobial activity against 10 North American and European L. intracellularis isolates showed that each L. intracellularis isolate had a different antimicrobial sensitivity pattern and these data can be utilized as an in vitro guideline for the further antimicrobial evaluation of field L. intracellularis isolates.
2. The in vitro assessment of the effectiveness of powder disinfectant (Stalosan® F) against L. intracellularis indicate that Stalosan F in both powder and suspension forms is able to inactivate over 99% of L. intracellularis after 30min of exposure.
3. Bactericidal activity of commercial disinfectants against L. intracellularis was evaluated and these data provide an in vitro guide for disinfectant selection to control L intracellularis.
4. We have identified natural products (e.g. L-UFFA) or synthetic derivatives that interfere with parasite-host cell interactions required for infection. Such molecules, particularly natural products, could be utilized as nutriceutical feed additives to inhibit parasite infectivity and thus prevent or reduce parasite load environmental contamination. Such an approach could benefit not only animal health but would reduce the likelihood of zoonotic spread of Cryptosporidium parvum through contamination of the water supply from domestic livestock operations.
5. South Dakota in collaboration with Nebraska has demonstrated that E. coli enterotoxin LT enhances attachment of ETEC to pig intestinal epithelial cells. microbial adhesion. These results suggest that the use of LT in vaccines would enhance protective responses. worked with Kansas resulted in the development of LT-STa genetic fusions for the production of enterotoxin antigens for vaccine development. Studies conducted in South Dakota have established efficacy of modified live ETEC vaccines in protecting weaned piglets short-term by competitive exclusion and long term by stimulating immunity.
6. Development of An extension program centered around calf health and calf science was developed and implemented. Complex issues, such as resistance in bacteria from the use of antimicrobials in food animal agriculture requires an integrated approach  from the science to its translation to judicious drug use on-farm but also a wide reach, to multiple audiences.
7. Data demonstrated that Salmonella spp. and especially S. Newport are still being detected in oysters consumed by the public but the risk is greater in the warmer months. However, the prevalence numbers of S. Newport are lower than those from oysters examined in 2002-2003 and from oysters obtained from restaurants in 2007-2008. TraSH studies should detect genes needed for the Salmonella JJPX01.0014 clone to colonize and survive in oysters.
8. Birds and possibly flies were shown to be a significant source of C. jejuni at the feedlot.
9. Sequencing and finishing of two strains, M129 (invasive, non-fluid producing) and S3 (non-invasive, fluid producing) of C. jejuni, should allow us to make comparisons of genes being up-regulated between the two strains in the infected piglet model and possibly identify the gene(s) responsible for this phenotype.
10. The DIGE separation analysis of fluid exudate from pigs infected with S3 will be combined with the up-regulation analysis to identify genes involved in intestinal fluid production in the piglet model. Mutation of these genes will allow us to test this hypothesis in the piglet model.
11. Work demonstrated that the older piglet would serve as an excellent model to examine the efficacy of putative C. jejuni vaccines.
12. It was established that Salmonella shedding prevalence in both calves and cattle fluctuated at different sampling times along the beef production points and that multi drug resistance was common throughout the study. Salmonella isolates from Uganda exhibited more multi-drug resistance compared to those from North Dakota. Also, presence of class-1 integron was more associated with AMR in Ugandan isolates compared to ND isolates.
13. The study abroad program provided US students the opportunity to participate in the International Animal Production, Disease Surveillance and Public Health for the third successive year.
14. Results showed that prebiotic galactooligosaccharide (GOS) may potentially be a useful means to reduce E. coli O157:H7 intestinal colonization in the intestines of infants.
15. Research demonstrated that caliciviruses can attach and replicate in lettuce. An understanding of the mechanisms of interaction of enteric caliciviruses with vegetables will permit development of improved prevention and disinfection strategies for foodborne viruses both pre- and post-harvest. Thus our research will help to reduce the incidence of foodborne illnesses due to consumption of unprocessed fruits and vegetables contaminated with enteric caliciviruses (human or animal origin).
16. Our research provided estimated the prevalence of norovirus in swine and identified new (or emerging) NoVs in swine and determined their relationship to human NoVs and built an extensive sequence database for porcine NoVs. Knowing the diversity of swine NoVs and their relatedness to human strains will enable us to pinpoint the possible origin (swine or human) of emerging NoV strains.
17. Identified genetic relatedness will provide an integrated global picture of C. jejuni epidemiology in the cattle population in the US and help to determine the potential risk of certain clonal types for human infections.
18. Our findings demonstrated the importance of controlling concurrent respiratory viral infections of pigs with PRSSV and PCRV to improve swine health. They also have potential relevance to prognosis of SARS in patients with existing or prior respiratory viral co-infections.
19. The common P-type of rotavirus influenced induction of cross-protective VN antibodies and that cross-reactive VN antigens on VP7 may also play a role. This information is critical for vaccine design to protect again multiple serotypes of rotavirus in the field.
20. Identification of small molecule chemical inhibitors that specifically inhibit TAT translocation would lead to novel antibacterials for control of Campylobacter. Such approaches will be more practical for use in chickens because of the risk associated with the emergence of resistant bacteria with the use of antibiotics. Identification of proteins that are translocated through the TAT system will reveal novel virulence or host adaptation proteins, permitting development of novel antimicrobial strategies.
21. Elucidating the role of polyphosphates kinases in C. jejuni virulence, stress responses and colonization would enable us to design approaches to control this pathogen in food chain.
22. We developed a microarray chip and software analysis platform that allows for rapid molecular identification of human and animal bacterial pathogens with high specificity and reliability and is capable of detecting bacterial pathogens in complex sample matrices including soil and feces.
23. A multiplex PCR procedure was developed that detected six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in E. coli O157:H7. This assay has diagnostic value.
24. We showed that Shiga toxin production may serve as useful markers to identify cattle strains of E. coli O157:H7 that are most likely to cause human disease.
25. Our studies showing effects of catecholamine hormones on horizontal gene transfer rates between enteric bacteria are important because the results provide a mechanistic explanation of how host factors may drive the development of highly virulent and multidrug resistant strains of enteric bacteria, and how physiological and psychological stress may influence the duration and severity of disease caused by enteric bacterial pathogens.
26. A relatively new vaccine technology utilizes the iron requirement of pathogenic bacteria by targeting siderophore receptor and porin proteins (SRP®). We found that an anti-Escherichia coli O157 SRP-based vaccine reduced fecal shedding of E. coli O157:H7 in feedlot cattle naturally shedding the organism. Results from this trial and others indicate that a E. coli O157:H7 SRP® vaccine may serve as a preharvest intervention to reduce the burden of E. coli O157:H7 on cattle presented for slaughter and ultimately improve food safety.
27. Although Salmonella may be commonly isolated from feces of feedlot cattle, there are many serotypes and their potential virulence in cattle and/or people can vary. The conditionally licensed and commercially available Salmonella Newport SRP® vaccine could potentially stimulate immunity and provide cross-protection to multiple different Salmonella serotypes, which are important to public health as well as to cattle health and production. However, our study (in one commercial feedlot) showed that the vaccine had no significant effect on Salmonella fecal prevalence, cattle health, or cattle performance.
28. In our recent experimental trial we confirmed the positive association between fecal shedding of E. coli O157:H7 and feeding distiller grains to feedlot cattle and showed that the inclusion level (20 vs. 40%) but not the type of DG (dry vs. wet) affects fecal prevalence of E. coli O157:H7.
29. Murine models are expected to act as surrogates for humans to study C. jejuni strains coming from humans, cattle and swine. We used the models to establish the effect of host passage and diet on disease outcome (pathotype observed). We also screened a panel of C. jejuni strains from human patients in the murine model to observe whether variations in pathotype could be observed based on the genotype of the organism.
30. We showed that dendritic cells from resistant mice process C. jejuni and present its antigens in the context of MHC-II leading to activation of T cells that can mediate protective immune responses. Furthermore, our results show that cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes represents a novel mechanism mediating C. jejuni-induced inflammatory responses of DCs. This was the first demonstration of such a mechanism for an intact bacterium. These findings provide a cost effective animal model that can be used to study the genetic basis of virulence of the enteric pathogen C. jejuni. Furthermore, in this study period we used these murine models to determine several genetic determinants of the host that control susceptibility to enteric disease and the various pathotypes observed in patients due to this bacterium.
31. Overall, the probability of detecting a specific Salmonella strain had little to do with its starting concentration in the sample. The bias introduced by culture could be dramatically biasing Salmonella surveillance systems and hindering traceback investigations during Salmonella outbreaks. Future studies should focus on the microbiological explanations for this Salmonella inter-strain variability, approaches for minimizing the bias, and estimations of the public health significance of this bias.
32. Microbiome analysis of the pig intestinal tract has shown that there are bacterial compositional differences when growth promoters are used and are likely to mediate the process of growth promotion. These results also provide us with a reference of the pig distal gut microbiota profile and fundamental knowledge for later studies to design new strategic approaches to replace AGPs in the future, such as probiotics or prebiotics.

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Report Information

Participants

" Besser, Thomas, Washington State University

" Francis, David, South Dakota State University

" Isaacson, Richard, University of Minnesota

" Francis, David, South Dakota State University

" Gephart, Connie, University of Minnesota

" Joens, Lynn, University of Arizona

" Khaitsa, Margaret, North Dakota State University

" Kaushik, Radhey, South Dakota State University

" Lin, Jun, University of Tennessee

" Mansfield, Linda, Michigan State University

" Moxley, Rodney, University of Nebraska

" Nagaraja, T.G., Kansas State University

" Narayanan, Sanjeev, Kansas State University

" Rajashekara, Gireesh, The Ohio State University

" Renter, David , University of Nebraska

" Robinson, Mark, USDA/NIFA

" Shah, Devendra, Washington State University

" Sischo, William, Washington State University

" Stromberg, Bert, University of Washington

" Zhang, Qijing, Iowa State University

Brief Summary of Minutes

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.

Accomplishments

Objective 1. Focus on emerging issue- identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine<br /> <br /> A. Campylobacter jejuni<br /> <br /> Iowa <br /> <br /> 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.<br /> <br /> Michigan<br /> <br /> 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.<br /> <br /> Arizona<br /> <br /> 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. <br /> <br /> 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. <br /> <br /> 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.<br /> <br /> Ohio<br /> <br /> 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. <br /> <br /> 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<br /> <br /> B. Salmonella<br /> <br /> Arizona<br /> <br /> 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. <br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> C. Non-O157 Shiga toxin-producing E. coli (STEC)<br /> <br /> Nebraska<br /> <br /> 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.<br /> <br /> Washington State University<br /> <br /> 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.<br /> <br /> D. Caliciviruses<br /> <br /> Ohio<br /> <br /> 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.<br /> <br /> 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. <br /> <br /> E. Coronaviruses<br /> <br /> Ohio<br /> <br /> 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.<br /> <br /> F. BVDV<br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> G. Enterococci<br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> H. E. coli O157<br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> I. Brachyspira species<br /> <br /> Minnesota<br /> <br /> 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.<br /> <br /> J. Lawsonia intracellularis<br /> <br /> Minnesota<br /> <br /> 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.<br /> <br /> 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.<br /> <br /> A. C. jejuni<br /> <br /> Michigan<br /> <br /> 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.<br /> <br /> Ohio<br /> <br /> 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.<br /> <br /> 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. <br /> <br /> 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. <br /> <br /> 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.<br /> <br /> B. E. coli<br /> <br /> South Dakota<br /> <br /> 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. <br /> <br /> Nebraska<br /> <br /> 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.<br /> <br /> C. Cryptosporidium parvum<br /> <br /> Illinois<br /> <br /> 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.<br /> <br /> 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. <br /> <br /> D. Coronaviruses<br /> <br /> Ohio<br /> <br /> 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. <br /> <br /> 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. <br /> <br /> E. E. coli O157<br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> F. BVDV<br /> <br /> Kansas<br /> <br /> 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.<br /> <br /> G. Norovirus<br /> <br /> Kansas<br /> <br /> 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. <br /> <br /> H. L. intracellularis<br /> <br /> Minnesota<br /> <br /> 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.<br /> <br /> Objective 3. Focus on disseminating knowledge  Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories<br /> <br /> Michigan<br /> <br /> 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.<br /> <br /> Nebraska<br /> <br /> 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.<br /> <br /> Minnesota<br /> <br /> 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).<br />

Publications

Bell JA, St. Charles JL, Murphy AJ, Rathinam VA, Plovanich-Jones AE, Stanley EL, Wolf JE, Gettings JR, Whittam TS, Mansfield LS. 2008. Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10-/- mice, BMC Microbiology, 2009 Mar 18;9:57, PMID: 19296832.<br /> <br /> Rathinam VA, Appledorn DM, Hoag KA, Amalfitano A, Mansfield LS. 2009. Campylobacter jejuni-induced activation of dendritic cells involves cooperative signaling through TLR4-MyD88 and TLR4-TRIF axes, Infection and Immunity, June 2009, p. 2499-2507, Vol. 77, No. 6, PMID: 19332531.<br /> <br /> Parthasarathy G and Mansfield LS. 2009. Recombinant IL-4 (rIL-4) enhances Campylobacter jejuni invasion of Intestinal Pig Epithelial Cells (IPEC-1), Microbial Pathogenesis, 2009 Jul;47(1):38-46. Epub 2009 May 3, PMID: 19409975.<br /> <br /> Wilson DL, Rathinam VK, Qi W, Wick LM, Landgraf J, Bell JA, Plovanich-Jones A, Parrish J, Finley RL, Mansfield LS and Linz JE. 2010. Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL-10 deficient mice. Microbiology, Papers in Press. Published April 1, 2010 as doi:10.1099/mic.0.035717-0.<br /> <br /> Jerome, J.P., J.A. Bell, A.E. Plovanich-Jones, J.E. Barrick, C.T. Brown, L.S. Mansfield. 2010. Standing genetic variation in contingency genes drives the adaptation of Campylobacter jejuni to a novel host. PloS One, Addressing critiques prior to resubmission after a positive review.<br /> <br /> Bell, J.A., J. R. Gettings, J. P. Jerome, J. J. Kopper, A. Plovanich-Jones, V. A. K. Rathinam, J. L. St. Charles, E.J. Smith, A. G. Staunton, and Mansfield, L.S. Campylobacter jejuni genome content and virulence phenotypes (pathotypes) in C57BL/6 IL-10-/- mice, submitted to Infection and Immunity.<br /> <br /> Page, A., N.M. Slovis, C.J. Gebhart, K. Wolfsdorf, S.M. Mapes And N. Pusterla. 2010. Use of serial serological and fecal PCR testing for targeted treatment of weanling foals on a farm with endemic Lawsonia intracellularis. JAVMA (in press).<br /> <br /> Pusterla, N., S. Mapes, C. Johnson, N. Slovis, A. Page and C. Gebhart. 2010. Comparison of feces versus rectal swabs for the molecular detection of Lawsonia intracellularis in foals with equine proliferative enteropathy. J. Vet. Diag. Invest. 22:741-744.<br /> <br /> Wattanaphansak, S., C.J. Gebhart, J.M. Anderson, and R.S. Singer. 2010. Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis. J. Vet. Diagn. Invest. 22:598-602.<br /> Guedes, R.M., and C.J. Gebhart. 2010. Evidence of cell-mediated immune response and specific local mucosal immunoglobulin (Ig) A production against Lawsonia intracellularis in experimentally infected swine. Can. J. Vet. Res. 74:97-101.<br /> <br /> Pustela, N., S. Wattanaphansak, S. Mapes, J. Collier, J. Hill, M. Difrancesco, and C. Gebhart. 2010. Oral infection of weanling foals with an equine isolate of Lawsonia intracellularis, agent of equine proliferative enteropathy. J. Vet. Intern. Med. 24:622-627.<br /> <br /> Pustela, N., R. Jackson, S.M. Mapes, J. Noland, R.M. Stenbom, and C. Gebhart. 2010. Lawsonia intracellularis: humoral immune response and fecal shedding in weanling foals following intra-rectal administration of frozen- thawed or lyophilized avirulent live vaccine. Vet. J. 186:110-112.<br /> <br /> McOrist, S. and C.J. Gebhart. In press. Porcine proliferative enteropathies. In G. Stevenson, et al. (ed), Diseases of Swine, 10th ed. Iowa State University Press, Ames, IA.<br /> Gebhart, C.J. and R.M.C. Guedes. 2010. Lawsonia intracellularis and the proliferative enteropathies. In C.L. Gyles, et al., (ed.), Pathogenesis of Bacterial Infections in Animals, 4th ed. Wiley-Blackwell Publishing, Hoboken, NJ. <br /> <br /> Burrough, Eric R., Orhan Sahin, Paul J. Plummer, Qijing Zhang, and Michael J. Yaeger. 2009. Pathogenicity of an emergent, ovine abortifacient Campylobacter jejuni clone following oral inoculation in a pregnant guinea pig model. AJVR 70:1269-1276.<br /> <br /> Guo, B., J. Lin, D.L. Reynolds, and Q. Zhang. 2010. Contribution of the Multidrug Efflux Transporter CmeABC to Antibiotic Resistance in Different Campylobacter Species. Foodborne Pathogens and Disease. 7: 77-83.<br /> <br /> Chen, X., N.G. Yun, C.M. Wu, Y. Wang, L. Dai, L.N. Xia, P.J. Luo, Q. Zhang, and J.Z. Shen. 2010. Prevalence and Antimicrobial Resistance of Campylobacter Isolates from Broilers in China. Vet. Microbiol. 144:133-139.<br /> <br /> Mao, G., G. A. Kraus, I. Kim, M. E. Spurlock, T. B. Bailey, P. M. Dixon, Q. Zhang, and Donald C. Beitz. 2010. Mitochondrially targeted vitamin E normalizes hepatic mitochondrial 4 oxidative stress and inhibits fat deposition in mice. J. Nutrition. 140: 1425-1431.<br /> <br /> Burrough, E.R., K. DiVerde, O. Sahin, P. J. Plummer, Q. Zhang, and M. J. Yaeger, DVM. 2010. Expression of Toll-like receptors 2 and 4 in subplacental trophoblasts from guinea pigs aborting following infection with Campylobacter jejuni. Veterinary Pathology. doi: 10.1177/0300985810375809<br /> <br /> Jeon, Byeonghwa, Yang Wang, Haihong Hao, Yi-Wen Barton, Qijing Zhang. 2010. Contribution of CmeG to antibiotic and oxidative stress resistance in Campylobacter jejuni. J. Antimicrob. Chemother. doi:10.1093/jac/dkq418.<br /> <br /> Burrough, Eric R., Orhan Sahin, Paul J. Plummer, Kevin DiVerde, Qijing Zhang, Michael J. Yaeger. 2010. Comparison of two commercial ovine Campylobacter vaccines and an experimental bacterin in a guinea pig model. AJVR. In presss.<br /> <br /> Plummer, Paul, Jinge Zhu, Masato Akiba1, Dehua Pei, and Q. Zhang. 2010. Identification of a Key Amino Acid of LuxS Involved in AI-2 Production in Campylobacter jejuni. PLoS ONE. In press.<br /> <br /> Butler JE. K.M. Lager, I. Splichal, D. Francis, I. Kacskovics, M. Sinkora, N. Wertz, J. Sun, Y. Zhao, W.R. Brown, R. DeWald, S. Dierks, S. Muyldermans, J.K. Lunney, P.B. McCray, C.S. Rogers, M.J. Welsh, P. Navarro, F. Klobasa, F. Habe and J. Ramsoondar. 2009. The piglet as a model for B cell and immune system development. Review. Vet Immunol Immunopathol 128: 147-170 <br /> <br /> Zhang, W, C Zhang, DH Francis, Y Fang, D Knudsen, JP Nataro, and DC Robertson. 2010. Genetic fusions of LTAB and STa toxoids of porcine enterotoxigenic Escherichia coli (ETEC) elicited neutralizing anti-LT and anti-STa antibodies. Infect Immun. 78: 316-325. <br /> <br /> Zhang W and DH Francis. 2010. Genetic fusions of heat-labile toxoid (LT) and heat-stable toxin b (STb) of porcine enterotoxigenic Escherichia coli elicit protective anti-LT and anti-STb antibodies. Clin Vacc Immunol. 17: 1223-123<br /> <br /> Lin J, AK Erickson, KS Mateo, M Zhao, RA Moxley and DH Francis. Protection of piglets from postweaning diarrhea by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli. In revision.<br /> <br /> Fekete, PZ, KS Mateo, RW Zhang, RS Kaushik, PR Hardwidge, RA Moxley and DH Francis. Multiple mechanisms for alteration by heat-labile enterotoxin in the ability of enterotoxigenic Escherichia coli to adhere to IPEC-J2 porcine epithelial cells. In revision.<br /> <br /> George S, K Circle, S Lindblom, S Vilain, A Rosa, D Francis, V Brözel and RS Kaushik. Assessment of toll-like receptors in the ileum of weanling pigs - responses to feed antibiotic chlortetracycline and gnotobiotic conditions. Submitted.<br /> Mateo KS, JH Ayres, M Zhao, JE Butler and DH Francis. Intestinal resection and anastomosis in neonatal gnotobitic piglets. Submitted.<br /> <br /> Ravishankar S, Zhu L, Reyna-Granados J, Law B, Joens L, Friedman M. Carvacrol and cinnamaldehyde inactivate antibiotic-resistant Salmonella enterica in buffer and on celery and oysters. J Food Prot. 2010 Feb;73(2):234-40.<br /> <br /> Kuhlenschmidt MS, Rolsma MD, Kuhlenschmidt T, Gelberg HB. Characterization of a Porcine Enterocyte Receptor for Group A Rotavirus. In: Mechanisms in the Pathogenesis of Enteric Diseases. ed., Paul PS, Francis DH, Benfield DA., pp. 135-143, Plenum Press, New York, N.Y., 1997.<br /> <br /> MD Rolsma, TB Kuhlenschmidt., HB Gelberg and MS Kuhlenschmidt (1998) Structure and Function of a Porcine Enterocyte Ganglioside Receptor for Group A Rotavirus. Journal of Virology 72:9079-9091.<br /> TB Kuhlenschmidt. B Hanafin and MS Kuhlenschmidt (1999) Sialic Acid Dependence and Indpendence of Group A Rotavirus. Adv. In Experimental Biology and Medicine 473:309-317 <br /> <br /> Gelberg HB, Thulin JD, Kuhlenschmidt MS. Application of Intestinal Xenografts to the Study of Enteropathogenic Infectious Disease. In: Mechanisms in the Pathogenesis of Enteric Diseases. ed., Paul PS, Francis DH, Benfield DA., pp. 31-35, Plenum Press, New York, N.Y., 1997.<br /> <br /> Rolsma MD, Kuhlenschmidt TK, Gelberg HB, Kuhlenschmidt MS. 1998 Structure and function of a ganglioside receptor for porcine group A rotavirus. J. Virol. 72:9079-9091 <br /> <br /> Moreno B, Bailey BN, Luo S, Martin MB, Kuhlenschmidt M, Moreno SN, Docampo R, Oldfield E. (2001) (31)P NMR of apicomplexans and the effects of risedronate on Cryptosporidium parvum growth. Biochem Biophys Res Commun. 284:632-637.<br /> <br /> J Trask, S. McLaughlin, MS Kuhlenschmidt and P. Kalita 2004 Overland and Near-surface Transport of Cryptosporidium parvum. Environ. Qual. 33:984-983.<br /> <br /> Johnson, JK, Schmidt, Gelberg, HB, and Kuhlenschmidt MS. 2004. Microbial Adhesion of Cryptosporidium par-vum sporozoites: Purification of an Inhibitory Lipid from Bovine Mucosa. J. Parasitology 90:980-990.<br /> <br /> Wetzel DM., Schmidt J, Kuhlenschmidt MS., Dubey JP, and Sibley LD. 2005.Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infection and Immunity 73:5379-5387. <br /> <br /> Andres, S. M. Donovan, T. B. Kuhlenschmidt, and M. S. Kuhlenschmidt (2007) Isoflavones at Concentrations Present in Soy Infant Formula Inhibit Rotavirus Infection in Vitro. J. Nutr., September 1, 2007; 137(9): 2068 - 2073.<br /> <br /> Johanna S. Salzer, Innocent B. Rwego, Tony L. Goldberg, Mark S. Kuhlenschmidt, and Thomas R. Gillespie. Giardia sp. and Cryptosporidium sp. Infections in primates in distrubed and undisturbed forest in western Uganda. (2007) . J Parasitol. 2007 Apr ;93 (2):439-40.<br /> <br /> Bergner, TB Kuhlenschmidt and MS Kuhlenschmidt 2010. Characterization of a Synthetic Receptor Mimetic for Group A Porcine Rotavirus (manuscript in preparation).<br /> <br /> Johnson JK, Kuhlenschmidt MS, Gelberg HB. Glycoconjugates and plasma membrane vesicles inhibit Cryptosporidium parvum sporozoite-host cell recognition. Vet Pathol 34:509, 1997.<br /> <br /> Williams, M. S., J. L. Withee, E. D. Ebel, N. E. Bauer, W. D. Schlosser, W. T. Disney, D. R. Smith, and R. A. Moxley. 2010. Determining relationships between the seasonal occurrence of Escherichia coli O157:H7 in live cattle, ground beef, and humans. Foodborne Pathog. Dis. 7:1247-1254<br /> <br /> Erume, J., E. M. Berberov, and R. A. Moxley. 2010. Comparison of the effects of different nutrient media on production of heat-stable enterotoxin-b by Escherichia coli. Vet. Microbiol. 144:160-165.<br /> <br /> Moxley, R. A., and D. R. Smith. 2010. Attaching-effacing Escherichia coli Infections in cattle. Vet. Clin. Food Anim. 26:29-56<br /> <br /> Qiuhong Wang, Kelly Scheuer, Zhenwen Zhang, Kwonil Jung, Wondwossen A. Gebreyes, Bayleyegn Z. Molla, Armando E. Hoet,and Linda J. Saif. Characterization and prevalence of a new porcine calicivirus in US swine. Emerging Infectious Diseases (submitted 2010).<br /> <br /> Vlasova, A.N., Halpin, R., Wang, S., Ghedin, E., Spiro, D.J., Saif, L.J. Molecular characterization of a potentially new subgroup 1 coronavirus associated with mink epizootic catarrhal gastroenteritis (ECG). [submitted to J Gen Virol].<br /> <br /> Renukaradhya, G.J., Alekseev, K.P., Jung, K., Fang, Y., Saif, L.J. Porcine reproductive and respiratory syndrome virus induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs. Viral Immunology. 23: 457-466. 2010.<br /> <br /> Jung, K., Gurnani, A., Renukaradhya, G.J., Saif, L.J. Nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus. Veterinary Immunology and Immunopathology. 136:335-9. 2010.<br /> <br /> Jung, K., Renukaradhya, G.J., Alekseev, K.P., Fang, Y., Tang, Y., Saif, L.J. Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections including SARS. Journal of General Virology. 90: 2713-2723. 2009.<br /> <br /> Yasser Sanad, Issmat I. Kassem, Jun Lin, Jeffrey T. LeJeune, Gireesh Rajashekara. Occurrence of the invasion associated marker (iam) in Campylobacter jejuni isolated from cattle (Submitted for publication).<br /> Mary Drozd, Dharanesh Gangaiah, Zhe Liu, and Gireesh Rajashekara. Contribution of PhoX to Twin Arginine Translocation mediated Campylobacter jejuni function and resilience to Environmental Stresses (Submitted for publication).<br /> <br /> Dharanesh Gangaiah, Zhe Liu, Jesús Arcos, Issmat I. Kassem, Yasser Sanad, Jordi B. Torrelles and Gireesh Rajashekara. 2010. Polyphosphate Kinase 2: A Novel Determinant of Stress Responses and Pathogenesis in C. jejuni. PLoS One, Aug 17;5(8). pii: e12142<br /> <br /> Issmat I. Kassem, Yasser Sanad, Dharanesh Gangaiah, Michael Lilburn, Jeffery Lejeune, Gireesh Rajashekara. 2010. Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter. J Appl Microbiol. 2010 Jul 18. [Epub ahead of print}.<br /> <br /> Dharanesh Gangaiah, Issmat I. Kassem, Zhe Liu, and Gireesh Rajashekara. 2009. Importance of polyphosphate kinase 1 for C. jejuni viable-but-nonculturable cell formation, natural transformation, and antimicrobial resistance. Appl Environ Microbiol. 75:7838-49.<br /> <br /> Saif, L.J., M.P. Pensaert, K. Sestak, S.G. Yeo, K. Jung. Coronaviruses. In: Viral Pathogens (J.J. Zimmerman, et al. eds.), 10th Edition of Diseases of Swine, Wiley-Blackwell. In Press.<br /> <br /> Nickell JS, White BJ, Larson RL, Renter DG. A simulation model to quantify the value of implementing whole herd Bovine Viral Diarrhea Virus testing strategies in beef cowcalf herds. J Vet Diagn Invest. 2011; 23(2): in press. <br /> <br /> Sargeant JM, OConnor A, Renter DG, Kelton D, Snedeker K, Wisener L, Leonard E, Guthrie A, Faires M. Reporting of methodological features in observational studies of pre-harvest food safety. Prev Vet Med, in press.<br /> <br /> Nickell JS, White BJ, Larson RL, Renter DG. Onset and duration of transient infections among antibody- diverse beef calves exposed to a Bovine Viral Diarrhea Virus persistently infected calf. Inter J Appl Res Vet Med. 2010; 8(4) in press. <br /> <br /> Taylor E, Renter DG, Nagaraja TG. Genetic variations in Shiga toxin-producing abilities of bovine and human Escherichia coli O157:H7. Zoo and Public Health, in press.<br /> <br /> Dodd C, Renter DG, Thomson D, Nagaraja TG. A randomized field trial to determine the effects of a commercially available vaccine against Salmonella in feedlot cattle. Americ J Vet Res., in press.<br /> <br /> Smith R, Sanderson MW, Renter DG, White B, Larson R. A stochastic risk-analysis model for the spread of Bovine Viral Diarrhea Virus after introduction to naïve cow-calf herds. Prev Vet Med. 2010; 95(1-2): 86-98.<br /> <br /> Jacob M, Paddock Z, Renter DG, Lechtenberg K, Nagaraja TG. Feeding dried or wet distillers grains at various inclusion levels to feedlot cattle affects fecal shedding of Escherichia coli O157:H7. Appl and Environ Microbiol. 2010; 76(21): 7238-7242.<br /> <br /> Jacob M, Renter DG, Nagaraja TG. Animal- and truckload-level associations between E. coli O157:H7 in feces and on hides at harvest and contamination of pre-evisceration beef carcasses. J Food Protec. 2010; 73(6): 1030-1037.<br /> <br /> Dodd C, Renter DG, Fox J, Shi X, Sanderson MW, Nagaraja TG. Genetic relatedness of Escherichia coli O157 isolates from cattle feces and pre-intervention beef carcasses. Foodborne Pathog and Dis. 2010; 7(4): 357-365.<br /> <br /> Taylor E, Kastner J, Renter DG. Challenges involved in the Salmonella Saintpaul outbreak and lessons learned. J Public Health Manag and Pract. 2010; 16(3): 221-231.<br /> <br /> Walker, C. X. Shi, M. J. Sanderson, J. S. Sargeant, and T. G. Nagaraja.. Prevalence of Escherichia coli O157:H7 in gut contents of beef cattle at slaughter. Foodborne Pathog and Dis. 2010; 7:249-255.<br /> <br /> Jacob, M.E., J.T. Fox, T.G. Nagaraja, J.S. Drouillard, R.G. Amachavadi, S.K. Narayanan. Effects of feeding elevated concentrations of supplemental copper and zinc on antimicrobial susceptibilities of fecal bacteria in feedlot cattle. Foodborne Pathog and Dis. 2010; 7:643-648.<br /> <br /> Jacob M. E., D. G. Renter, and T. G. Nagaraja. Animal- and truckload-level associations between E. coli O157:H7 in feces and on hides at harvest and contamination of pre-evisceration beef carcasses. J. Food Prot. 2010. 73:1030-1037.<br /> <br /> Bai J., X. Shi and T. G. Nagaraja. A Multiplex PCR Procedure for detection of six major virulence genes in Escherichia coli O157:H7. J. Microbiol. Meth. 2010. 82:85-89.<br /> <br /> Amachawadi, R. G., N. W. Shelton, M. E. Jacob, X. Shi, S. K. Narayanan, L. Zurek, S. S. Dritz, J. L. Nelssen, M. D. Tokach, and T. G. Nagaraja. Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine. Foodborne Pathog and Dis. 2010; 9:1089-1097.<br /> <br /> Channaiah LH, Subramanyam B, Zurek L. Survival of Enterococcus faecalis OG1RF:pCF10 in poultry and cattle feed: vector competence of the Red Flour Beetle, Tribolium castaneum (Herbst). J Food Protect. 2010; 73(3): 568-573.<br /> <br /> Channaiah LH, Subramanyam B, McKinney, and Zurek L. Stored-product insects carry antibiotic-resistant and potentially virulent enterococci. FEMS Micro Ecol. 2010; 74(2): 464-471.<br /> <br /> Chakrabarti S, Kambhampati S, Zurek L. Assessment of house fly dispersal between rural and urban habitats in Kansas, USA. J Kansas Entomol Soc. 2010; 83(2): 172-188. Walker, C. X. Shi, M. J. Sanderson, J. S. Sargeant, and T. G. Nagaraja 2010.. Prevalence of Escherichia coli O157:H7 in gut contents of beef cattle at slaughter. Foodborne Path. Dis. 7:249-255.<br /> <br /> Y. Kim and K.O. Chang. 2010. Chapter 6. Porcine enteric caliciviruses. Caliciviruses: Molecular and Cellular Virology. Horizon Scientific Press. <br /> K.O. Chang and Y. Kim. 2010. Chapter 9. Reverse Genetics System and Replicon system of Caliciviruses: Molecular and Cellular Virology. Horizon Scientific Press.<br />

Impact Statements

1. " Understanding the pathogenesis, epidemiology and ecology of Campylobacter in sheep will enable us to devise effective measures to control this zoonotic pathogen.
2. " FISH was used to definitively localize C. jejuni in host tissues after an oral challenge infection. The scoring protocol provides semiquantitative measures of colonization and invasion by the bacterium. This method can be used on paraffin embedded tissues providing an easy means of assessing tissue distribution of C. jejuni in samples in long term storage.
3. " Virulence phenotypes/pathotypes vary with strain of C. jejuni but the underlying genetic mechanisms controlling these phenotypes is not due to gene content alone.
4. " It is apparent that the TTSS system is not involved in oyster enterocyte uptake of S. Newport. However, the TraSH studies did show the importance of flagella genes in the colonization of the oyster with S. Newport and also with the survival of the bacterium in oyster tissue.
5. " C. jejuni is present all the way through the feedlot, during processing and aging of the beef carcass, and in the resulting hamburger. This should raise the spectra of hamburger as a definite risk factor in acquiring campylobacteriosis. In addition, we have isolates that are involved in transmission dynamics, bacterial fitness, and host specificity as shown in the pulsed-field gels. Sequencing of these isolates should identify genes that can be grouped into these categories and may lead to virulence factors that can be targeted in vaccine preparations.
6. " The TlyA protein maybe an excellent vaccine candidate to reduce the numbers of C. jejuni in chickens.
7. " South Dakota in collaboration with Nebraska has assessed subunit vaccine products for protection against post-weaning ETEC infection in pigs. Results of the study indicate that such a vaccine must contain antigens for both fimbrial adhesins and heat labile enterotoxins to be highly effective
8. " L-PUFA represents a unique opportunity to identify a common mechanism used by the Apicomplexa to infect host cells and thus a potential "Achilles heel" target for the development of new anti-parasitic drugs. Such molecules, particularly natural products, could be utilized as nutriceutical feed additives to inhibit parasite infectivity and thus prevent or reduce parasite load environmental contamination. Such an approach could benefit not only animal health but would reduce the likelihood of zoonotic spread of Cryptosporidium parvum through contamination of the water supply from domestic livestock operations.
9. " The improvement of methods to detect STEC O26, O45, O103, O111, O121, O145, and O157 will help form the basis for future epidemiology studies to monitor prevalence and develop pre-harvest interventions for these important food-borne pathogens.
10. " Results of the study involving differential expression of heat-labile enterotoxin (LT) and heat-stable enterotoxin-b (STb) by enterotoxigenic Escherichia coli of swine increased our understanding of how enterotoxins promote shedding of the infectious agent in the feces and increase the likelihood of transmission to new hosts.
11. " An understanding of the mechanisms of interaction of enteric caliciviruses with vegetables using TC-Po/SaVs as a surrogate for HuNoVs will permit development of improved prevention and disinfection strategies for foodborne viruses both pre- and post-harvest.
12. " Ours is the first report of porcine St-Valerien-like viruses in US swine. Knowing the diversity of swine St-Valerien-like viruses and their relatedness to other caliciviruses will enable us to prepare for a potential emerging infectious disease of swine.
13. " By improving our understanding of CoV evolution, diversity and interspecies transmission, this research contributes new knowledge about CoVs applicable to improving both animal and public health.
14. " Identifying genetic relatedness will provide an integrated global picture of C. jejuni epidemiology in the cattle population in the US and help to determine the potential risk of certain clonal types for human infections
15. " Our findings demonstrate the importance of controlling concurrent respiratory viral infections of pigs to improve swine health
16. " Reduction of both "NO and IFN-± innate immune responses may delay viral clearance and lead to inappropriate adaptive immune responses which hinder clearance and control of PRRSV infections in swine.
17. " Identification of small molecule chemical inhibitors that specifically inhibit TAT translocation would lead to novel antibacterials for control of Campylobacter. Such approaches will be more practical for use in chickens because of the risk associated with the emergence of resistant bacteria with the use of antibiotics. Identification of proteins that are translocated through the TAT system will reveal novel virulence or host adaptation proteins, permitting development of novel antimicrobial strategies.
18. " Elucidating the role of polyphosphates kinases in C. jejuni virulence, stress responses and colonization would enable us to design approaches to control this pathogen in food chain.
19. " This new method for Brachyspira species identification will allow the classification of many of these non-typable isolates. We have also apparently identified a new Brachyspira species that did not key in as any of the previously known swine Brachyspira. These isolates were highly hemolytic and were recovered from affected pigs, which suggest that they are potentially virulent and associated with clinical disease in the field.
20. " An expanded VNTR database will provide bioinformatics data and tools for applying VNTR typing more widely and will further enhance our understanding of the transmission dynamics and epidemiology of proliferative enteropathy in various animal species. This VNTR database can be easily mined for field isolate information based on year of isolation, source, geographic location, VNTR pattern, and other epidemiologic and demographic traits.
21. " Development of reproducible models of L. intracellularis infection will now enable us to evaluate and improve protocols for prevention of the disease in pigs and horses. We further demonstrated that a L. intracellularis isolate from a clinical case of EPE could cause infection in rabbits, with minimal adverse health effects, and thus provide an animal model for investigations on Lawsonia pathogenesis and therapy.
22. " Understanding the cell-mediated and local humoral immune responses to L. intracellularis in pigs has implications for development of immunological detection methods for diagnosing the disease in pigs as well as for evaluating effectiveness of vaccines.
23. " Development of a quantitative PCR assay for L. intracellularis contributes a sensitive, specific, and accurate method for the detection and quantification of L. intracellularis in field samples.
24. " Development of an alternative protocol for cultivation of L. intracellularis in cell culture provides a flexible protocol for testing various environmental conditions for improved L. intracellularis cultivation.
25. " This is the first study to evaluate immune response in vaccinated foals and the first to utilize intra-rectal vaccination for proliferative enteropathy. These data will help determine route, dose, and expected immune response for routine vaccination of foals for L. intracellularis.
26. " Use of serial serological testing, combined with other diagnostic modalities, to identify animals on endemic and non-endemic farms that had been exposed to L. intracellularis will allow targeted placement of intervention or preventative methods of control.
27. " Data support the existence of multiple strain types of E. coli O157:H7 in the animal reservoir and in retail meat contaminants that present greatly differing risk of human infection. The investigation of biological differences associated with these genotypes may identify pre-harvest interventions that strongly affect clinical genotypes.

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Report Information

Participants

" Besser, Thomas, Washington State University
" Debroy, Chobi, Pennsylvania State University
" Francis, David, South Dakota State University
" Gebhart, Connie, University of Minnesota
" Isaacson, Richard, university of Minnesota
" Joens, Lynn, University of Arizona
" Kaushik, Radhey, South Dakota State University
" Kuhlenschmidt, Mark, University of Illinois.
" Lin, Jun, University of Tennessee
" Mansfield, Linda, Michigan State University
" Moxley, Rodney, University of Nebraska
" Rajashekara, Gireesh, The Ohio State University
" Sahin, Orhan, Iowa State University
" Saif, Linda, The Ohio State University
" Saif, Linda, The Ohio State University
" Sellers, Holly, University of Georgia
" Shah, Devendra, Washington State University
" Stromberg, Bert, University of Washington
" Zsak, Laszlo, USDA Southeast Poultry Research Lab
" Zhang, Qijing, Iowa State University

Brief Summary of Minutes

1. Linda Mansfield, Chair of NC-1041, gave an update on the proposal renewal. The proposal has been finished and uploaded onto the web site for NC-1041. Anybody interested in the proposal can print out a copy directly from the web page.
2. Appendix E uploading. Members of NC-1041 are required to submit the Appendix E form. This must be done by Jan. 15, 2012 via the NIMSS system. Members dont have to be state representatives. Investigators from medical schools are allowed and encouraged to become member of NC-1041.
3. Qijing Zhang and Lynn Joens will become the Chair and Chair-Elect, respectively, at the 2012 annual meeting. The term will be for 2 years.
4. New members were introduced.
5. Student awards. NC-1041 offers awards for students to compete in the Gastroenteric Disease Section of CRWAD. This year two awards will be offered, one for oral presentation and another for poster presentation. Radhey Kaushik serves as Chair for the student award selection committee. He will continue to serve as Chair for the next year. Selection of a co-chair working with him was suggested. Historically, the student awards were funded by annual registration fee. How to increase the number of abstracts submitted to the Gastroenteric Disease Section was discussed. One suggestion was to offer student travel awards or increase the dollar amount of the awards. This requires industry sponsors and contact with industries for possible support of NC-1041 activities was proposed. There was also a discussion on changing the registration fee to annual dues, which will ensure a steady source of income for the committee.
6. Proposed title change for the Gastroenteric Disease Section. There was a motion to change the section name from Gastroenteric Disease to Gastroenteric Disease, Zoonosis and Food Safety. The rationale for the change is that many members of NC-1041 work with zoonosis and food safety and this change will attract more submission of abstracts to the section. The attendee unanimously voted for the title change. Rodney Moxley will take this issue to the CRWAD Council for approval.
7. Annual report. Annual state reports are due to Linda Mansfield 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.

Accomplishments

Objective 1. Focus on emerging issue- identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine<br /> <br /> A. Campylobacter jejuni<br /> <br /> Michigan<br /> Emerging Campylobacter Species. The best-studied species, C. jejuni and C. coli, exhibit high genomic diversity. However, after sequencing more than forty strains from different hosts, Stanhope and colleagues showed that there are barriers to interspecies recombination in the core genomes of the two species. Additionally, phenotypic differences in disease produced can be discerned among strains of C. jejuni. We delineated five pathotypes of C. jejuni in C57BL/6 IL-10-/- mice: lack of colonization, colonization without disease, colonization with watery or hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. These pathotypes correspond to the spectrum of human disease; however, gene content did not predict pathotype: each strain possessed a unique set of present, absent or divergent virulence factors.<br /> <br /> Tennessee<br /> C. jejun is the leading foodbome human pathogen in the United States and many other industrialized countries. Increasing evidence also indicates that antibiotic use in poultry selects for resistant C. jejuni, posing a significant threat to public health. We have made significant progress to understand the molecular mechanisms of pathogenesis and antibiotic resistance in Campylobacter during the reporting period. The findings have filled a significant gap in antimicrobial resistance development in Campylobacter and provided important information for the development of effective vaccine to control Campylobacter.<br /> <br /> Iowa<br /> A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health. Additionally, we develop a LAMP assay specific for detection of clone SA of Campylobacter jejuni.<br /> <br /> In another study, we determined the direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxS complement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902 luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxS gene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivo model of natural disease.<br /> <br /> B. Non-O157 Shiga toxin-producing E. coli (STEC)<br /> <br /> Nebraska<br /> Validation of Culture Methods for Non-O157 STEC. Non-O157 STEC are enzootic in cattle and constitute an emerging zoonotic threat. A current research goal of the USDA-NIFA is the development of diagnostic tests for STEC O26, O45, O103, O111, O121, O145, and O157:H7. Preliminary studies were conducted to test the validity of a published culture protocol (Possé et al. 2008. FEMS Microbiol. Lett. 282:124-131) for detection of STEC O26, O103, O111, O145, and O157:H7, and to determine whether it can be used for detection of O45 and O121. The results suggest that the differential culture medium described by Possé et al. (FEMS Microbiol. Lett. 2008;282:124-131) can be used to concurrently screen for all seven serogroups (O26, O45, O103, O111, O121, O145, and O157:H7). More research on the development and validation of detection methods for non-O157 STEC is needed. <br /> <br /> North Dakota<br /> A study was conducted to investigate prevalence of Shigatoxin-producing Escherichia coli (STEC) in beef cattle in North Dakota at different production stages from post-weaning to slaughter & testing for presence of Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), Intimin (eaeA) and Enterohemolysin (ehlyA) genes using PCR. The preliminary data indicate presence of several serotypes of Shigatoxin-producing Escherichia coli (STEC) (O157, 0145, 0111, 0103, 026) in feces of beef cattle (both calves and adult cattle) in ND.<br /> <br /> Prevalence of Shiga toxin-producing Escherichia coli serogroup O26 in feces of feedlot cattle. Our preliminary findings are that O26 are common, but with a variable prevalence, in cohorts of feedlot cattle. Furthermore, fecal prevalence of O26 and presence of virulence genes may be less than what was observed for serogroup O157. However, the fecal prevalence of O26 may have a similar temporal distribution in feedlot cattle as observed for O157.<br /> <br /> C. Caliciviruses<br /> <br /> Ohio<br /> The binding mechanisms of HuNoV to leafy greens. Our results indicate that NoV VLPs bind to lettuce CWM specifically by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and thus prevent effective removal by simple washing and disinfection by routine decontamination procedures. These results should help to develop more effective decontamination procedures to control foodborne HuNoV outbreaks. <br /> <br /> Persistence and mode of transmission of sapovirus, a surrogate for norovirus, in leafy greens. Our results indicate that (i) SaV can potentially be transferred from roots to leaves and (ii) that the virus can persist for a prolonged period on the leaf surface once introduced. The persistence of the virus in/on lettuce and the possibility of contamination through roots suggest the need for more rigorous pre-harvest measures to reduce calicivirus contamination.<br /> <br /> Potential interspecies transmission of NoVs between pigs and humans. Although no HuNoVs were detected from swine farms, our data is limited to only one state, North Carolina. Additional studies of porcine NoVs from other states are needed to assess their geographic and age prevalence in pigs and to confirm their relatedness to human strains. <br /> <br /> Detection and characterization of the first feline NoV. Our findings document the discovery of a novel feline calicivirus, different from vesiviruses, and extend the spectrum of NoV host range. Epidemiological studies using feline NoV-specific diagnostic tools and experimental infection of cats are required to understand whether NoVs have a pathogenic role in this species or may be transmitted to humans. Regulations may be needed to control the spread of such new viruses.<br /> <br /> Kansas<br /> Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay. Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer (Fret) substrates. We have established a high throughput assay for screening potential protease inhibitors using the Fret assay. We also demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases were inhibited by commercial protease inhibitors including chymostatin. The interaction of<br /> chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy. The established high throughput assay would be highly useful for screening potential antivirals and study of enzyme-substrate interactions.<br /> <br /> D. Rotavirus<br /> <br /> Illinois<br /> Our findings demonstrate a dramatic reduction in virus shedding and absence of rotavirus disease in SLPE-fed piglets, thus providing proof of concept that a nutriceutical approach of using natural or synthetic receptor mimetics, such as SLPE, hold substantial promise for the prevention of rotavirus disease. This approach may be applicable not only to piglets in a natural field setting, but also as a potential dietary supplement, for example in infant formulae, for the prevention of rotavirus or other gastrointestinal infectious diseases in human infants. <br /> <br /> E. Enterococci<br /> <br /> Kansas<br /> Selection of tcrB gene mediated copper resistant fecal enterococci in pigs fed diets supplemented with copper. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to normal copper suggests that supplementation of copper in swine diets selected for resistance. The existence of a metalassociated co-selection mechanism could potentially be a major issue relative to public health and further studies are needed to determine the magnitude of such an association.<br /> <br /> Characterization of antibiotic resistant and virulent enterococci from animal feed. Our data revealed that feed samples collected from feed mills and swine farms carried antibiotic resistant and potentially virulent enterococci. We have shown that these isolates have the potential to spread the antibiotic resistance by horizontal gene transfer.<br /> <br /> Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. This study shows that house flies and German cockroaches in the confined swine production environment likely serve as vectors and/or reservoirs of antibiotic resistant and potentially virulent enterococci and consequently may play an important role in<br /> animal and public health.<br /> <br /> F. E. coli O157<br /> <br /> Kansas<br /> Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.<br /> <br /> Escherichia coli O157:H7 genetic diversity in bovine fecal samples. A total of 254 E. coli O157:H7 isolates from 27 samples were included in PFGE analysis. Fifteen PFGE subtypes (< 100% Dice similarity) were detected among the 254 isolates and there were no common subtypes between the three locations. Seven of 27 (26%) fecal samples had E. coli O157:H7 isolates with different PFGE subtypes (mean = 2.1) within the same sample. The virulence gene profiles of different isolates from the same sample were always identical, regardless of the number of PFGE types. The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing to other isolates from feces, hides or carcasses as multiple PFGE subtypes are present.<br /> <br /> G. Lawsonia intracellularis<br /> <br /> Minnesota<br /> Subclinical porcine proliferative enteropathy: diagnostic and performance parameters in a multi-dose mucosal homogenate challenge model. The objective of this study was to further define subclinical PE by measuring the impact of varying doses of Lawsonia on clinical signs, Lawsonia shedding and seropositivity, performance, and gross and histopathological intestinal changes in weaned pigs. The results showed that subclinical infection can have a detrimental economical disease impact on swine herds. This study confirms that it is possible to reproduce a spectrum of PE that includes mild and subclinical disease using a mucosal homogenate challenge model which mimics naturally occurring disease in the field.<br /> <br /> Loop mediated isothermal amplification method for detection of L. intracellularis. We have developed a new diagnostic method, based on loop mediated isothermal amplification (LAMP), for the detection of L. intracellularis DNA in fecal samples. Using this method we were able to detect about 10,000 in 1 ml of L. intracellularis. No amplification was obtained with DNA extracts from other swine pathogens, indicating high specificity of this new method. Furthermore, this method was used for detection of L. intracellularis in fecal samples without any extensive DNA extraction process or the use of thermocyclers.<br /> <br /> 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.<br /> <br /> A. C. jejuni<br /> <br /> Michigan<br /> Using a mouse model of campylobacteriosis we have shown that C. jejuni NCTC11168 re-isolated from infected mice (mouse-adapted) is more virulent than the ancestral inoculum. Mouse-adapted C. jejuni also had altered ability to autoagglutinate and interact with epithelial cells. After extensive genetic analysis including Illumina genome re-sequencing, we showed that the only genetic difference between mouse-adapted and ancestral C. jejuni was in homopolymeric guanine tracts of thirteen (out of twenty-nine) contingency loci. This suggests that contingency loci play a role in pathogenesis in the mouse model. We then tested the effect of bottlenecking contingency loci mutational diversity on C. jejuni virulence phenotypes. A variant subjected to a single colony bottleneck could not colonize mice.<br /> <br /> Campylobacter jejuni NCTC 11168 induces a mixed Type1 and Type17 cytokine and cellular response in IL-10 deficient murine host. The inflammatory process was characterized by analyzing for inflammatory cytokines and cell types in the colon and mesenteric lymph nodes, and by C. jejuni reactive antibody induction and cytokine response in serum. Colon homogenate ELISA revealed significant up regulation of IFN-³, IL-17A, IL-22, IL-6 and IL-1² but not IL-4. Mesenteric lymph node analysis revealed increases in IFN-³ positive cells in the CD4+, CD8+ and ³´ T cell and NK cell compartments. IL-17 positive cells were increased in CD4+ T cell and Thy1hiCD3- innate lymphocyte compartments. IL-22 positive cells were increased from CD4+ T cell and NK cell compartments. Time-dependent up regulation in C. jejuni specific IgG2b (Type 17 associated), IgG2c and IgG3 (Type1 associated), but not IgG1 (Type 2 associated) or IgM were also observed in the serum of infected mice. <br /> <br /> Addition of a Thelper-2 (Th2) immunomodulator to antibiotic treatment improves survival and decreases clinical signs of inflammatory bowel disease induced by Campylobacter jejuni infection in a murine model. We have also developed a mouse model of autoimmune diseases arising after C. jejuni infection including Inflammatory Bowel Disease and Guillain Barré Syndrome.<br /> <br /> Ohio<br /> Campylobacter in Beef Cattle. Our studies on cattle associated Campylobacter highlight the importance of cattle as a potential reservoir for clinically important Campylobacter. A better understanding of prevalence, distribution, and molecular epidemiology of C. jejuni strains from beef cattle in the US will constitute a significant preharvest preparedness effort.<br /> <br /> The TAT system and Tat-dependent proteins. Screening for small molecule chemical inhibitors that specifically inhibit TAT translocation would lead to novel antibacterials for control of Campylobacter. Such approaches will be more practical for use in chickens because of the risk associated with the emergence of resistant bacteria with the use of antibiotics. Identification of proteins that are translocated through the TAT system will help elucidate the basis for the TAT system contribution to C. jejuni physiology and virulence. Furthermore, a comprehensive analysis of TAT-substrates may reveal other virulence or host adaptation proteins, permitting development of novel antimicrobial strategies. <br /> <br /> Polyphosphate kinase. No homologs of ppk1 and ppk2 have been identified in genomes of mammals and avian species, and thus both PPK1 and PPK2 can be used as potential anti-C. jejuni targets to reduce the prevalence of C. jejuni in poultry and other livestock species as well as humans. Understanding the role of C. jejuni PPKs on the expression of other genes involved in colonization and persistence is highly valuable to understand many aspects of host-microbe interactions in food producing animals and humans. Further, this work should greatly extend our fundamental knowledge on PPK regulation on various virulence genes in prokaryotes.<br /> <br /> Iowa<br /> To facilitate the control of macrolide-resistant Campylobacter, it is necessary to understand if macrolide resistance affects the fitness and transmission of Campylobacter in its natural host. In this study we conducted pairwise competitions and comingling experiments in chickens using clonally related and isogenic C. jejuni strains, which are either susceptible or resistant to erythromycin (Ery). The findings clearly indicate that acquisition of macrolide resistance impairs the fitness and transmission of Campylobacter in chickens, suggesting that the prevalence of macrolide-resistant C. jejuni will likely decrease in the absence of antibiotic selection pressure.<br /> <br /> Arizona<br /> Identification of C. jejuni proteins involved in host colonization. A major thrust of our laboratory is examining C. jejuni proteins for their role in the colonization of chicks. Genes expressing proteins that are secreted and have a surface orientation, or are up-regulated, or express known colonization factors are being mutated and examined for their role in gut colonization. The results indicate that the CjLAJ3 protein maybe an excellent vaccine candidate to reduce the numbers of C. jejuni in chickens.<br /> <br /> Vaccination of chicks with the attenuated Salmonella vector expressing the CjLAJ2 protein. The Salmonella strain used as the vector was developed by Curtiss (2005) and has three different mutations which results in its attenuation. Gene CjLAJ2, encoding a putative hemolysin protein, was inserted into an expression plasmid, cloned and amplified in a E. coli shuttle vector, extracted, and electroporated into the Salmonella vector. The trials demonstrated a significant reduction of C. jejuni in cecal contents of chicks vaccinated with the vector expressing the CjLAJ2 protein and challenged with the homologous strain. In addition, a 1-log reduction was present in chicks challenged with a heterologous strain.<br /> <br /> B. E. coli<br /> <br /> South Dakota<br /> South Dakota has developed a live vaccine for ETEC that prevents diarrhea in pigs within 24h of the time of vaccination. The vaccine seems to offer both immediate competitive exclusion from the pathogen and longer term protection by immunity.<br /> <br /> Nebraska<br /> Effect of culture media on secretion of heat-labile enterotoxin by porcine-origin enterotoxigenic Escherichia coli strains. This study was done to determine whether porcine WT ETEC isolates and recombinant LT constructs from a porcine isolate secrete LT and if so, to compare their LT secretion levels when grown in different culture media supportive of LT production and secretion. This study demonstrated that porcine WT strains secrete LT, albeit to a lesser level than the high LT-producing prototype human WT strain H10407. The level of LT secretion of porcine WT strains may vary depending on the growth media used. Porcine WT strains and a recombinant LT+ derivative of a porcine WT strain secreted higher levels of LT than did an LT+ clone in DH5±. The apparent reason for the lower level of LT secreted by DH5± is that it has a less efficient type II secretion system, which was been shown in a previous study.<br /> <br /> C. Cryptosporidium parvum<br /> <br /> Illinois<br /> The goal of our research is this area is to define the early mechanisms of Apicomplexa-host interactions and to identify new drug candidates that can block these interactions. We have developed a battery of complementary in vitro and in vivo assays that allow us to quantify Cryptosporidium, Toxoplasma, and Plasmodium microbial adhesion, microneme secretion, gliding motility, in vitro and in vivo infectivity, and to determine the mechanism by which the infectivity of these parasites is inhibited by L-PUFFA. Our results thus far provide hope that small molecule natural products, such as L-PUFFA or CDPK inhibitors will be valuable for the development of new drugs that show broad efficacy for treatment of apicomplexan parasitic diseases. Accordingly, L-PUFFA and CDPK inhibitors represent a unique opportunity to exploit a common mechanism used by the Apicomplexa to invade host cells (microneme secretion) and thus a potential "Achilles heel" target for the development of new anti-parasitic drugs.<br /> <br /> D. E. coli O157<br /> <br /> Kansas<br /> Effects of a vaccine and a direct-fed microbial on fecal shedding of E. coli O157:H7 in pens of commercial feedlot cattle fed a diet supplemented with distillers grains. Our objective was to determine the efficacy of a siderophore receptor and porin (SRP®) proteins-based vaccine (VAC) and a direct-fed microbial (DFM; Bovamine®) for controlling fecal shedding of E. coli O157:H7 in pens of feedlot cattle fed a corn-based diet with 25% distillers grains. We conclude that the SRP vaccine can reduce fecal shedding of E. coli O157:H7 in potentially high risk populations of feedlot cattle.<br /> <br /> Fecal shedding of Escherichia coli O26 in feedlot cattle from a field trial evaluating an Escherichia coli O157:H7 vaccine and a direct-fed microbial. Preliminary analysis demonstrated no significant effects of treatment, or treatment by sampling week interaction, on O26 prevalence. However, a significant (P < 0.01) sampling week effect was observed with higher O26 prevalence in the two weeks prior to harvest compared to the previous two weeks. Preliminary results suggest the treatments had no significant effect on O26 shedding; however, these results are based on pooled colonies and ongoing analysis of individual isolates should provide further detail.<br /> <br /> Modeling the effect of bacterial transfer rates and interventions on the prevalence and concentration of Escherichia coli O157 on beef carcasses. We constructed a risk assessment model using @Risk in Microsoft Excel. Parameter distributions were developed from the existing scientific literature and incorporated into a Monte-Carlo framework. Our results indicate that the percentage of bacteria transferred between hides and carcasses is influential to carcass prevalence and concentration; however, there is little data to inform the distribution of this transfer. As expected, the sensitivity analysis showed successful carcass intervention has the highest impact on final carcass prevalence and concentration.<br /> <br /> Nebraska<br /> Single nucleotide polymorphism analysis for characterization of clinical versus bovine-biased genotypes. Two hundred thirty-one E. coli O157:H7 bovine isolates from a vaccine efficacy study (Moxley et al. 2009. Foodborne Pathog. Dis. 6:879-84) were submitted to the Washington station for single nucleotide polymorphism analysis to determine the proportions that may be classified as clinical versus bovine-biased genotypes. This collaboration is part of a USDA-AFRI-NIFA grant that was awarded to Washington State University and the University of Nebraska-Lincoln, with WSU as the lead institution and Dr. Tom Besser as Principal Investigator<br /> <br /> Efficacy testing of a proprietary compound for protection of gnotobiotic piglets against brain lesions caused by Shiga toxin-producing E. coli O157:H7 infection. Histopathological examinations on sections of formalin-fixed tissues from 20 of 45 gnotobiotic piglets were conducted with results provided to the South Dakota station. Piglets were randomly divided into principals and controls; principals received a proprietary compound for designed to protect against Shiga toxin-induced vascular damage. Controls received a placebo treatment. Dr. Moxley, station representative at Nebraska, served as pathologist, and was blinded to animal assignment to treatment and control groups. This collaboration is part of a grant awarded by the Teijin Corporation to South Dakota State University as lead institution with Dr. David Francis as Principal Investigator.<br /> <br /> E. Non-O157 STEC<br /> <br /> Nebraska<br /> Intimin type characterization of non-O157 STEC isolates. Non-O157 STEC are enzootic in cattle and constitute an emerging zoonotic threat. A current research goal of the USDA-NIFA is the development of pre-harvest interventions for STEC O26, O45, O103, O111, O121, O145, and O157:H7. Preliminary studies were conducted to characterize the STEC isolates from each of these seven serogroups for intimin type since this may influence the site and extent of intestinal colonization, and thereby constitute a suitable target for vaccines or other intervention strategies. <br /> <br /> F. Norovirus<br /> <br /> Ohio<br /> The effects of simvastatin or interferon-± on infectivity of human norovirus in the gnotobiotic pig model for the study of antivirals. Our study demonstrated that use of simvastatin enhanced HuNoV infectivity in the Gn pig model. Thus, its use may also support growth of HuNoV in cell culture. We also verified that the increased infectivity of HuNoV might be associated with the inhibitory effect of statins on innate immunity (IFN-±). This observation might explain the exacerbated HuNoV disease and the related higher mortality in statin-treated humans. In addition, we showed that oral treatment with rhIFN-± can curtail early HuNoV fecal shedding in the Gn pig model. This approach could be applicable to control multiple genogroups and genotypes of HuNoVs, including the GII.4 variants that have emerged each year. Collectively, HuNoV infectivity in Gn pigs can be enhanced by simvastatin treatment or reduced by oral treatment with rhIFN-±. These observations indicate that Gn pigs are a useful model to test the efficacy of antivirals against HuNoV.<br /> <br /> H. L. intracellularis<br /> <br /> Minnesota<br /> Infection of sparrows (Passer domesticus) and different mice strains with L. intracellularis. The susceptibility of sparrows (Passer domesticus) and four strains of mice (Swiss, BALB/c, C-57 and DB-A) to L. intracellularis infection were studied. We found that sparrows do not seem to be relevant in the epidemiology of L. intracellularis. The results showed variations in the establishment of lesions among the four different mice strains used. <br /> <br /> Transcriptional profiling of a pathogenic and an attenuated homologous L.intracellularis isolate during in vitro infection. The current study used high-throughput sequencing technology to characterize the transcriptional profiling of a pathogenic and an attenuated isolate during in vitro infection. We identified distinct genes and pathways between a pathogenic and an attenuated L. intracellularis isolate. This information supports our hypothesis and opens a new research field for studying target genes involved in the ecology, pathogenesis and physiology of L. intracellularis.<br /> <br /> Evidence of host adaptation in L. intracellularis infections. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using porcine and equine isolates. In the pig trial, all animals infected with the porcine isolate demonstrated watery diarrhea and seroconversion from 21 days PI. No clinical signs or seroconversion were observed in equine isolate-infected pigs. Two pigs infected with porcine isolate were euthanized on day 21 PI and showed intestinal proliferative lesions associated with the presence of Lawsonia-specific antigen in the intestinal epithelium identified by IHC. No macroscopic or histologic lesions, including IHC staining, were observed in two equine isolate-infected pigs which were also euthanized 21 days PI. Marked clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates.<br /> <br /> Intracellularis increases Salmonella enterica levels in the intestines of pigs. To determine if there was an association between S. enterica carriage promoted by infection with L. intracellularis, we measured S. enterica loads in 4 intestinal sites of pigs challenged with L. intracellularis (at 5 weeks of age), S. enterica serovar Typhimurium (at 6 weeks of age), or both microbes. Preliminary data demonstrated multiple changes in the intestines of pigs challenged with one or both pathogens compared to non-challenged controls. As well, there were differences in the microbiome compositions between the challenge groups.<br /> <br /> I. Rotavirus<br /> <br /> Kansas<br /> Inhibitory effects of bile acids and synthetic Farnesoid X receptor agonists on rotavirus replication. Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. We examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication, which may serve therapeutic option for rotavirus infection in animals and humans.<br /> <br /> Ohio<br /> Probiotics: Effect on neonatal gut immunity to RV. Prior trials have shown satisfactory protection rates for live oral RV vaccines in developed countries. However, the cost is high and the effectiveness is low for RV vaccines tested in underdeveloped countries where most needed. Thus, alternative low cost treatments to moderate HRV diarrhea or improve vaccine efficacy are urgently needed. An improved understanding of the influence of probiotic bacteria on HRV vaccines and disease in neonatal Gn pigs will permit an extrapolation to interventions for infants to prevent HRV diarrhea.<br /> <br /> Rotavirus Vaccine Efficacy in Neonates: Impact of Vitamin A. Our results suggest that maternal and childhood VAD may decrease HRV vaccine efficacy and exacerbate HRV diarrheal disease. Vitamin A deficiency in children from developing countries is high (11-40%) and may result in reduced efficacy of HRV vaccines and increased severity of HRV disease. A similar scenario is possible in farm animals, especially multiparous animals, but has not been addressed. The association between RV related diarrheal disease and VAD in swine and cattle also should be assessed and vitamin A supplementation may be implemented as a vaccine adjuvant and an intervention strategy.<br /> <br /> <br /> Objective 3. Focus on disseminating knowledge  Provide training or continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories<br /> <br /> Michigan<br /> Several students were mentored for study of enteric diseases of food animals. Jamie Jennifer Kopper (DVM candidate) successfully defended her PhD thesis and will go on to finish two more years of veterinary school. Four other students are pursuing PhD degrees in food safety related to these enteric pathogens including John Paul Jerome, Jessica St. Charles, Ankit Malik and Barbie Gadsden, DVM. Three of these students have passed their preliminary exams, two during 2011. One is preparing for this exam. Four undergraduate students are working in the lab on these projects. One undergraduate was accepted to and started medical school during 2011. <br /> <br /> Dr. Mansfield organized a Spring 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 Christopher Waters, PhD (Biofilms in foodborne bacteria) and John Paul Jerome (Contingency genes in Campylobacter jejuni). Also, Dr. Mansfield attended and presented at a scientific conference held by the National Institutes of Health in Baltimore, Maryland on Enteric Diseases. Dr. Mansfield helped to organize and attended the USDA Rushmore Enteric Diseases Meeting NC1041 in Chicago, Illinois on December 3rd and 4th. Her graduate student John Paul Jerome gave a talk about his work entitled Contingency loci dynamics during Campylobacter jejuni infection. Dr. Mansfield gave a talk for the Department of Large Animal Clinical Sciences at Michigan State University entitled Investigating the role of Campylobacter jejuni in acute and chronic disease: the genetic basis of pathotypes on 02/01/2011. <br /> <br /> North Dakota<br /> The course International Animal Production, Disease Surveillance and Public Health was offered for the fifth time in summer 2011 with 9 US students. The joint Master of Science (M.S.) degree as well as a graduate certificate in International Infectious Disease Management and Biosecurity (2008-2011) program was approved by the ND State Board of Higher Education, The first group of students (8) enrolled in fall, 2011.<br /> <br /> Nebraska<br /> <br /> Knowledge pertinent to NC-1041 activities was disseminated to undergraduate students, graduate students, professional veterinary students, veterinarians, physicians, food processors, researchers, cattle producers and other decision makers regarding pre-harvest food safety of cattle food projects. A listing of specific presentations and dates is provided in Section VI, below.<br /> <br /> Minnesota<br /> The Principle Investigators and Students involved in the project have given presentations and updates on both swine and equine proliferative enteropathy at various scientific, veterinary, and diagnostic meetings in the previous year. These include the Conference of Research Workers in Animal Disease, the Leman Swine Conference, the Rushmore Symposium, the American Association of Equine Practitioners, the American College of Veterinary Internal Medicine and the American Association of Swine Veterinarians. They have disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians (both swine and equine).<br /> <br />

Publications

Wilson DL, Rathinam VK, Qi W, Wick LM, Landgraf J, Bell JA, Plovanich-Jones A, Parrish J, Finley RL, Mansfield LS and Linz JE. 2010. Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL-10 deficient mice. Microbiology. 2010 Jul;156(Pt 7):2046-57. <br /> <br /> Jerome, J.P., J.A. Bell, A.E. Plovanich-Jones, J.E. Barrick, C.T. Brown, L.S. Mansfield. 2010. Standing genetic variation in contingency genes drives the adaptation of Campylobacter jejuni to a novel host. PLoS ONE 6(1): e16399. doi:10.1371/journal.pone.0016399.<br /> <br /> Bell, J.A., J. R. Gettings, J. P. Jerome, J. J. Kopper, A. Plovanich-Jones, V. A. K. Rathinam, J. L. St. Charles, E.J. Smith, A. G. Staunton, and Mansfield, L.S. Outcome of Campylobacter jejuni infection of C57BL/6 IL-10-/- mice varies with strain. In review, Infection and Immunity.<br /> <br /> Dodd C, Sanderson MW, Jacob M, Renter DG. Modeling preharvest and harvest interventions for Escherichia coli O157 contamination on beef cattle carcasses. J Food Prot. 2011; 74(9):1422-1433.<br /> <br /> Nickell JS, White BJ, Larson RL, Renter DG, Sanderson MS, Peck C. Bovine Viral Diarrhea Virus (BVDV) Status in cow-calf herds and associations with biosecurity and production practices among Montana beef producers. Bov Pract. 2011; 45(1): 14-22.<br /> <br /> Dodd C, Renter DG, Shi X, Alam MJ, Nagaraja TG, Sanderson MW. Prevalence and persistence of Salmonella in cohorts of feedlot cattle. Foodborne Pathog Dis. 2011; 8(7): 781-789.<br /> <br /> Nickell JS, White BJ, Larson RL, Renter DG, Sanderson MW. A simulation model to quantify the value of implementing whole-herd Bovine Viral Diarrhea Virus testing strategies in beef cowcalf herds. J Vet Diagn Invest. 2011; 23(2): 194-205.<br /> <br /> Dodd CC, Renter DG, et al. Evaluation of the effects of a commercially available Salmonella Newport siderophore receptor and porin protein vaccine on fecal shedding of Salmonella bacteria and health and performance of feedlot cattle. Am J Vet Res., 2011; 72(2): 239-247.<br /> <br /> Taylor E, Renter DG, Nagaraja TG. Genetic variations in shiga toxin-producing abilities of bovine and human Escherichia coli O157:H7. Zoonoses Public Health, 2011; 58(3): 185-91.<br /> <br /> Sargeant JM, OConnor A, Renter DG, Kelton D, Snedeker K, Wisener L, Leonard E, Guthrie A, Faires M. Reporting of methodological features in observational studies of pre-harvest food safety. Prev Vet Med., 2011; 98(2-3): 88-98.<br /> <br /> Paddock, Z. D., C. E. Walker, J. S. Drouillard, Nagaraja T.G.. Dietary monensin level, supplemental urea, and ractopamine on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. J. Anim. Sci., 2011; 89:2829-2835.<br /> <br /> Amachawadi, R. G., N. W. Shelton, X. Shi, J. Vinasco, S. S. Dritz, M. D. Tokach, J. L. Nelssen, H. M. Scott, T. G. Nagaraja. Selection of tcrB gene mediated copper resistant fecal enterococci in pigs fed diets supplemented with copper. Appl. Environ. Microbiol., 2011; 77:5597-5603.<br /> <br /> Jacob, M. E., K. M. Almes, X. Shi, J. M. Sargeant, and T. G. Nagaraja. Escherichia coli O157 diversity in bovine fecal samples. J. Food Prot., 2011; 74:1186-1188. Yunjeong Kim and Kyeong-Ok Chang. Inhibition of Rotavirus replication by Bile Acids through Farnesoid X Recepter. J Virol., 2011; 85(23):12570-7.<br /> <br /> Aqeel Ahmad, Anuradha Ghosh, Coby Schal, and Ludek Zurek. Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community. BMC Microbiol., 2011; 11(1):23.<br /> <br /> Daisuke Takahashi, Yunjeong Kim, Kyeong-Ok Chang, Asokan Anbanandam, Om Prakash. 2011. 1H, 15N, and 13C resonance assignments of Norwalk virus protease. Biomol NMR Assign. Jun 8. [Epub ahead of print]<br /> <br /> Jacob, M. E., X. Shi, B. An, T. G. Nagaraja, and J. Bai. Evaluation of a multiplex real-time PCR for the quantification of Escherichia coli O157 in cattle feces. Foodborne Path. Dis. In Press.<br /> <br /> Paddock, Z., X. Shi, J. Bai, and T.G. Nagaraja. Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces. Vet. Microbiol. In Press.<br /> <br /> Kyeong-Ok Chang, Daisuke Takahashi, Om Prakash, and Yunjeong Kim. Characterization of Proteases from Norovirus Genogroup I and II with the Fluorescence Resonance Energy Transfer Assay. Virology. In Press.<br /> <br /> Vannucci FA, Wattanaphansak S, Gebhart CJ. 2012. An alternative method for cultivation of Lawsonia intracellularis. J. Clin. Microbiol. (accepted).<br /> <br /> Paradis M, Gebhart CJ, Toole D, Vessie G, Winkelman NL, Bauer SA, Wilson JF, McClure CA. 2011. Subclinical Ileitis: Diagnostic and performance parameters in a multi-dose mucosal homogenate challenge model. 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Impact Statements

1. " These findings provide a cost effective animal model that can be used to study the genetic basis of virulence of the enteric pathogen C. jejuni.
2. " These murine models are useful to determine the genetic determinants of the host that control susceptibility to enteric disease and the various pathotypes observed in patients due to this bacterium.
3. " Useful information is generated on the detection, transmission, dissemination, persistence and survival of enteric bacterial and viral pathogens in food and food animal production environments
4. " We have developed assays to detect major serogroups and virulence genes of STEC (II.1.1) and have demonstrated the diversity of STEC O157 strains in bovine fecal samples. " We have demonstrated that supplementation of copper in swine diets may be associated with antimicrobial resistance in fecal enterococci.
5. " We demonstrated that antimicrobial resistant and potentially virulent enterococci may be present in swine feed at feed mills and farms. " We have quantified the effects of commercial available preharvest interventions targeting STEC O157 on fecal shedding of STEC O157 and O26. " We have shown that bile acids and FXR agonists play important roles in the suppression of rotavirus replication, which may serve a therapeutic option for rotavirus infection in animals and humans.
6. " This information may help to elucidate virulence factor-encoding genes involved in this infection and opens a new research field for studying target genes involved in the ecology, pathogenesis and physiology of L. intracellularis. " This is the first report associating different host susceptibilities to species-specific isolates for L. intracellularis infections.
7. " Efforts to reduce L. intracellularis infections would be expected to be an important adjunct in reducing carriage of S. enterica. " Provided an opportunity for international training and exposure of students from several US institutions through the course International Animal Production, Disease Surveillance and Public Health and the joint Master of Science (M.S.) degree in International Infectious Disease Management and Biosecurity.
8. " A better understanding of calicivirus mode of transmission in vegetables would enhance efforts for maintaining food safety and decreasing infections in consumers. Such results will improve public health. " Continuous monitoring of NoVs in swine is crucial since porcine NoVs are most closely related to HuNoVs and there is a zoonotic potential for swine NoVs or for transmission of HuNoVs to pigs. Our results will help to improve public health and swine health.
9. " Development of HuNoV antivirals is important because HuNoVs cause large-scale epidemics with significant mortality in immunocompromised patients. Thus, the Gn pig model for HuNoV will allow testing of new treatment modalities for HuNoV infection and provide new knowledge on antiviral mechanisms and innate and adaptive immunity.
10. " Identification of small molecule chemical inhibitors that specifically inhibit TAT translocation would lead to novel antibacterials for control of Campylobacter. Such approaches will be more practical for use in chickens because of the risk associated with the emergence of resistant bacteria with the use of antibiotics. Identification of proteins that are translocated through the TAT system will reveal novel virulence or host adaptation proteins, permitting development of novel antimicrobial strategies.
11. " Elucidating the role of polyphosphates kinases in C. jejuni virulence, stress responses and colonization would enable us to design approaches to control this pathogen in food chain. " L-PUFFA and CDPK inhibitors represent a unique opportunity to exploit a common mechanism used by the Apicomplexa to invade host cells (microneme secretion) and thus a potential "Achilles heel" target for the development of new anti-parasitic drugs.
12. A better understanding of the mechanism of NoV contamination of vegetables would enhance efforts for maintaining food safety and decreasing infections in consumers. Prevention of NoV foodborne outbreaks should focus on each stage from pre-harvest to table. Thus, our results will promote public health.
13. " Ours is the first detection and characterization of a feline NoV. Knowing the diversity of feline NoVs and their relatedness to other caliciviruses, including HuNoVs, will enable us to prepare for a potential emerging infectious disease of cats or other species. " Identifying genetic relatedness will provide an integrated global picture of C. jejuni epidemiology in the cattle population in the US and help to determine the potential risk of certain clonal types for human infections.
14. " Identification of the intimin type and subtype in selected non-O157 STEC strains will be used to determine whether this bacterial outer membrane protein mediates adherence to mucosal epithelial cells in different locations in the bovine intestine.
15. " The improvement of methods to detect STEC O26, O45, O103, O111, O121, O145, and O157 will help form the basis for future epidemiology studies to monitor prevalence and develop pre-harvest interventions for these important food-borne pathogens.
16. " The efficacy of SL-DPPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large-scale production capabilities make SLPE a promising candidate for further exploration as a possible prophylactic or therapeutic nutriceutical for combating rotavirus disease in animals.
17. " Loop mediated isothermal amplification may be useful for detection of L. intracellularis in fecal samples when PCR testing is not readily available.
18. " Added an international perspective to our research and training thus providing opportunities for faculty and student collaborations. NDSU and MAK had faculty exchange plus student research conducted at MAK.
19. " Use of inexpensive vitamin A as an adjuvant with current rotavirus oral vaccines will improve their efficacy in children with micronutrient deficiencies and improve the overall health of children in developing countries. This strategy may also be applicable to rotavirus vaccines for animals and to vaccines for other important mucosal infections.
20. " Dissection of the factors involved in gut immune maturation provides a basis for development of cost effective measures to promote and maintain gut integrity and recovery from enteric infections such as RV diarrhea. Administration of probiotics to mothers late in gestation or to infants upon initial breastfeeding may have profound impacts on RV vaccine efficacy. Moreover the probiotics can be tailored to select ones that enhance immune responses to vaccines or to moderate inflammatory responses to infectious agents that provoke tissue damage or more severe disease.

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