Brief Summary of Minutes of Annual Meeting

Agenda: Saturday, November 13 8:00 AM Registration and Continental Breakfast 8:30 AM Meeting Call to Order and Introduction of Members and Guests 8:45 AM Opening Remarks by Dr. Bert Stromberg, NC 1007 Administrative Advisor 9:00 AM CSREES Report by Dr. Mary Torrence 9:15 AM Interdisciplinary Project Report and Planning Session, led by Dr. Isaacson 10:00 AM Break 10:30 AM Rushmore Conference Update and Planning Session, led by Dr. Francis Progress Reports and Principal Accomplishments 11:00 AM Coronavirus, Rotavirus, Other Viral Enteric Pathogens Moderator: Dr. Kapil 11:30 AM: Campylobacter, Lawsonia Moderator: Dr. Joens 12:00 PM: Lunch 1:00 PM: Brachyspira Moderator: Dr. Gebhart 1:20 PM: Salmonella Moderator: Dr. Isaacson 1:40 PM: Escherichia coli Moderator: Dr. Moxley 2:10 PM Break 2:40 PM Cryptosporidium Moderator: Dr. Gebhart 3:00PM Discussion of Strategic Concerns 5:00PM Adjourn Saturday, November 14 8:00 AM Business meeting of Technical Committee Members 9:00AM Announcements 9:30 AM Discussion of Cooperative Research for Next Year 12:00 PM Adjourn Summary of the Meeting: Saturday November 13, 2004 The meeting was called to order by Dr. Gebhart at 8:30 AM. The list of NC1007Station Representatives and Collaborators was circulated for updates. The minutes from last years meeting were distributed and approved. The Fall 2004 NCRA Regional Update Newsletter was distributed. Meeting dues were briefly discussed. It was decided to keep the dues at $50.00. Under the heading old business, there was discussion on the need for industry support and the fact that this issue should be re-addressed. Dr. Stromberg presented his opening remarks. Two years from now we have a project rewrite. He stressed that project review has been changed. It will be very important to show that there is true collaboration between stations, not merely sharing of reagents. The expectation will be that real sharing is evident in the research, for example by multi-institutional publications or multi-state proposals. Another important consideration will be meeting attendance. Participants should focus on presenting rationale and impact of their work. Through discussion, we should define our longer term goal and ways to effectively interact. He suggested submission of a multi-state proposal to NRI. Realizing this agencys restricted funding, it would be important to know which entities they want to focus on. Dr. Mary Torrence, CSREES representative, stated that the focus of her agency is food safety. The NRI budget has increased slightly and the number of priority areas has been reduced from 8 to 3-4. This will have a positive impact on the size of the awards. She reiterated her suggestion to submit a large collaborative research proposal to either NRI or NIH. Another possibility would be to apply for a CAP award. The focus of the CAP or network awards is currently also on food safety, at the expense of the epidemiology program. Flexibility towards emerging issues is important. She pointed to the success of the PRRS group in obtaining a CAP award and said that this committee had the same potential for obtaining this type of award. What is important to be successful in these endeavors is to think differently, to de-emphasize individual grants and to focus on impact and outcome. Dr. Isaacson led a discussion aimed at generating ideas for a group project. He emphasized that it will be necessary to think big and think different. He mentioned that he sent out an e-mail message to solicit input for a pre-proposal, but that the response from the group was very limited. Despite this, he said that he did submit a proposal on changes in the microflora of the gut in response to antibiotics. The proposal was not funded, but will be resubmitted with a revised epidemiological component. It was pointed out that important elements for the success of the PRRS group in obtaining a grant were the formation of partnerships leading to real collaboration, a multistate approach, looking at outcomes and bringing in the Pork Producers organization. Dr. Isaacson pointed out that it is important to come up with things that unite us. Dr. Gebhart suggested to focus on a few areas of expertise, with 1-2 investigators per area. Dr. Torrence mentioned that some groups use a piece-by- piece approach, rather than submitting an all-encompassing proposal. Dr. Gebhart suggested looking at antibiotics in the feed versus the problem of taking antibiotics out of the feed. After some further discussion, Dr. Isaacson focused on what appeared to be the most productive idea: definition of the normal gut flora in the presence or absence of antibiotics. Dr. Torrence mentioned that CDC involvement might be feasible for this type of project. Dr. Isaacson then gave a brief overview of the approaches he had taken to gather preliminary data for this type of project. Dr. Stromberg asked how we would all fit in. Dr. Isaacson suggested to use the remainder of the day for further brainstorming. Dr. David Francis announced that the Third International Rushmore Conference will be held on September 30 and October 1, 2005 at the Rushmore Plaza Hotel in Rapid City, SD. The suggested conference title is: Strategies in the Prevention of Enteric Disease and Dissemination of Food-Borne Pathogens Currently secured funds total $15,000. This meeting is to be viewed as an important part of the outreach component of the NC-1007 group. The focus of this meeting will be less on mechanisms of disease than what are we going to do about it. Four sessions are proposed. Each session will be composed of a keynote address and one or more internal and external invited presentations. The following points were made on meeting format and proceedings: -Not every speaker has to be interested in enteric diseases. -The focus of the meeting should not be limited to veterinary medicine. -Oral presentations should primarily be given by the invited speakers. These will be of the overview type. Most other presentations, except for 4-5, which will be converted to oral presentations, will be presented as posters. -Dr. David Francis will put the conference grant together. -Does the series Advances in Experimental Medicine still exist? -A draft of the meeting format was distributed. -Nominations are needed for speakers. The names of potential speakers should be submitted to Dr. Francis via e-mail. -Dr. Francis suggested inviting people from major granting agencies to come talk to us in conjunction with this meeting. -A website would be very useful. From 11:00 AM on participants from the stations represented at the meeting discussed overview presentations of progress and principal accomplishments made during the last year on the project(s) they were associated with. Starting at 3 PM, the discussion of strategic concerns was continued. Dr. Isaacson recapped the morning discussion and acted as a facilitator. He proposed to brainstorm ideas and get them on the table. He stressed that responsibilities needed to be defined and an action plan had to be formulated. Various ideas were put forward, the two main themes being the effects of antibiotics on colonization and the alteration of the gut microflora in response to antibiotics. General comments All bacterial pathogens spend most of their life cycle as commensals. What do we know about their relationship with the host? All our efforts are focused on disease aspects, not on the bacterial-host interaction. Do we have to focus our efforts at the whole animal level? Growth promoters produce changes in the microflora and enhance/optimize metabolic activity. This enhances the health of an animal in a general sense. A consortium grant could focus on host factors and individual grants on virulence factors. Colonization-related ideas Dr. Joens said that pathogenesis and colonization are the strengths of this group. Dr. Joens commented on the host-dependent effect of colonization with Campylobacter. Dr. Moxley indicated that E.coli O:157 receptors are present only on certain cell types in cattle, explaining why cattle do not show disease. Dr. Joens mentioned that the host response should not be overlooked and that we have the advantage of animal models of foodborne disease within the group. Dr. Francis said that biofilm formation, resulting in resistance to antibiotics, is a form of colonization. Dr. Moxley mentioned quorum sensing in the gut. Dr. Gebhart proposed colonization as the central theme. Different environmental conditions (presence or absence of antibiotics in the food) and differences between pathogens could be tested within this framework. Dr. Joens mentioned several angles that could be explored within the colonization framework: -Receptors versus no receptors. -Bacterial by-products. -Tissue environment. -Expression of bacterial proteins. -Colonization factors have been studied for more than 30 years. -What would be new in colonization? To be relevant, what is being proposed has to lead to an intervention strategy. Alteration of the gut microflora -Changes in microbial flora are the result of changes in glycosylation. Dr. Isaacson stated that there are 500-1000 bacterial species in the gut, with tremendous concentration variation between them. Entire population measurement is important -Healthy animals grow better. Is this a microflora shift? -Ohio said that a population shift may be difficult to predict, but that the effect of a population shift can definitely be studied -Because of changes in antibiotic use regulations in Europe, more antibiotics have to be used there now to treat sick pigs. -Are the tools available to focus the proposal around microflora changes? Sunday, November 14, 2004 The business meeting of technical committee members was started at 8AM. The committee decided that the next NC-1007 meeting will take place in Rapid City, SD, after the conclusion of the Rushmore Conference. The Conference is fulfilling our obligation to disseminate information. There was discussion on ways to involve industry and commodity groups. One possibility would be to invite representatives from industry and relevant commodity groups to the conference and have an open evening session with them. Dr. Francis will send out a list of potential invitees. An attempt will be made to get Dr. Gerberding from CDC as a speaker. The general meeting was started at 9:00AM. New business Dr. Gebhart announced the meeting date/place for the next NC-1007 meeting. Judges were selected for the poster session of the enteric disease section of the CRWAD meeting. Dr. Joens announced that a new chair will need to be appointed for the Enteric Disease Session of CRWAD. Nominations should be submitted by e-mail by May, 2005. A keynote speaker is needed for next years Enteric Disease Session. Dr. Linda Saif was mentioned as a potential speaker. Proposal(s) 1. Microflora Dr. Gebhart suggested to pursue the microflora approach as our main proposal, since it has already been submitted once by Dr.Isaacson. The objectives would be to look at the effect of growth promoters on gut microflora and the relationship to public health. Dr. Isaacson has contacted another group for a swine model. This group would be specifically mentioned in the proposal. Other Producer groups (beef, dairy, poultry) should be focused upon. Dr. Isaacson said that the existing proposal can only be changed in the area of epidemiology. Dr. Francis said that it is unlikely that everyone from the group is a participant in this proposal. 2. Colonization Dr. Joens proposed seeking seed money for the colonization approach related to antibiotics (~$50,000) from producer groups. Data obtained with this money could be used to write an NIH proposal. Dr. Gebhart indicated that she is willing to help in getting industry support for this approach. Dr. Francis thought that USDA is not likely to fund this type of proposal. He was in favor of seeking industry support and said that we could take advantage of the presence of industry people at next years Rushmore conference. Dr. Gebhart concurred. Dr. Isaacson said that it would be OK to have a stakeholders meeting during the Rushmore Conference. Dr. Joens said that the concept was to get seed money so that more people would be involved. Subgroups within the committee could then submit grants, either to NIH or USDA. 3. Conclusions -Dr. Isaacson will resubmit the microflora proposal. -Small group collaboratives are planned for next year. -The group will seek seed money for research, both from industry and producer groups. -A stakeholders meeting will be held in conjunction with the Rushmore Conference. Publications Dr. Stromberg re-emphasized the need for shared publications. The meeting was adjourned at approximately 12:00 PM. Next meeting Information: Location: Rapid City, SD Date: October 2, 2005 Respectfully submitted, Roger Maes NC-1007 Secretary

Objective 1  Define mechanisms of pathogen-host-environmental interactions in enteric and food borne diseases: Brachyspira The biochemical properties of membrane-associated proteases of B. pilosicoli were investigated by NE. Campylobacter The cytolethal distending toxin (CDT) of Campylobacter species was characterized by NE. Although cdtB gene sequences are present in both C. jejuni and C. coli, production of CDT correlated positively only with C. jejuni. AZ is assesing the pathogenicity of C. jejuni in broilers. Their studies indicate that isolates from poultry exhibit a wide range of virulence traits and suggest that only certain clusters of isolates, a minority, from poultry may be virulent and capable of causing disease in humans. AZ has also isolated novel virulence genes from C. jejuni using a green fluorescence protein promoter library. They continue to explore the environmental factors associated with C. jejuni biofilm development on abiotic surfaces, focusing on the early stages of biofilm development and attachment to surfaces. Lawsonia MN has performed a metabolic reconstruction of Lawsonia based on information derived from the whole genomic sequence. This information may help define necessary nutritional components for cell-free growth of this obligately intracellular bacterium. Also, they have cloned, expressed, and purified a flagellar protein of Lawsonia, FliC. Escherichia coli NE, as a result of preliminary experiments conducted at SD, has constructed a single knockout STb- strain of E. coli using the lambda red recombinase-based linear transformation technique. This will be used to construct double and triple-knockout mutants in porcine origin enterotoxigenic E. coli in order to delete the antibiotic resistance markers following the inactivation of each gene. SD has investigated the factors influencing F18+ E. coli receptor expression in weanling pigs. Their findings suggest that diet and microbial flora may be at least partially responsible for inducing F18+ receptor expression in these pigs. They continue to characterize the receptor specificity of D88ac fimbriae. To date, observations of these studies suggest that several regions of the FaeG genes are likely involved in receptor recognition, with the region from aa133 to 156 imparting variant receptor specificity. WA is studying E. coli O157:H7 excretion over 12 months in dairy heifers with the aim of identifying high versus low shedders, concentrations, and persistence of excretion. They are also examining the role of RAJ colonization in influencing feedlot E. coli epidemiology. This work aims to identify E. coli O157:H7 super-shedders and examine how their presence within a feedlot pen influences the E. coli excretion of in-contact cattle in the same pen. Samples from retail beef and raw milk within WA State for sampled for non-O157:H7 STEC. Isolates obtained have been characterized on the basis of virulence marker possession and serogroup. These food non-O157:H7 isolates are being compared to local human isolates based on genotype, serogroup, and virulence marker profiles. Salmonella Research examining maintenance and transmission of multi-drug resistant S. Newport in dairy herds has been completed by WA. Data indicate that separation of hospital pens and maternity pens is an effective intervention for reducing the prevalence and persistence of these organisms. Also, a new DNA microarray approach has been used for molecular fingerprinting of Salmonella R- plasmids. Porcine Enteric Calicivirus OH identified bile acids as the active factor in intestinal content fluid required for PEC growth. They propose a novel mechanism for enteric calicivirus growth dependent on bile acids, ubiquitous molecules present in the intestine at the site of the virus replication that involves the PKA cell-signaling pathway and a possible down-regulation of innate immunity. Porcine Group A Rotavirus IL pursued a receptor therapeutic approach for porcine rotavirus disease, based on previous studies showing that gangliosides are required by sialic acid-dependent strains for enterocyte infectivity. Since infection of enterocytes is preceded by attachment of virus to the cell surface, it follows that an understanding of the biology of this interaction is seminal to prophylactic or therapeutic intervention. Bovine Coronavirus Several methods were used to detect bovine coronavirus in soil samples by KS and they concluded that this virus was inactivated in the soil environment. They also assessed the effectiveness of several disinfectants on emerging and enteric viruses in the environment, concluding that RNA was relatively more stable than viral proteins and needed longer and higher concentration treatments for complete inactivation. Cryptosporidium parvum IL found that mucins, plasma membrane vesicles and fractioned cell membranes inhibited binding of sporozoites to host cells. They have purified and partially characterized this membrane inhibitory activity which has led to the discovery of a bovine intestinal-derived lipid fraction that inhibits sporozoite infectivity of host cells. Using suppressive subtractive hybridization, they identified a small number of candidate genes that appear to be differentially expressed. These are being cloned and sequenced. Finally, IL has determined the Cryptosporidium oocysts are effectively retarded from overland transport by vegetative filter strips 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. Objective 2  Develop and improve diagnostics, treatment, and preventative measures for enteric and food-borne diseases: Escherichia coli NE is performing clinical trials to test the effect of an enterohemorrhagic E. coli (EHEC) type III secreted protein vaccine on prevalence of E. coli O157:H7 in a research beef feedyard. The data suggest that vaccinating against EHEC type III secreted proteins has a significant effect on E. coli O157:H7 colonization of the terminal rectum of cattle. NE is also performing a clinical trial testing the effect of a direct-fed microbial product on prevalence of E. coli O157:H7 in a research beef feedyard. This study shows that feeding a combination of Lactobacillus crispatus and Propionibacterium freudenreichii direct-fed microbial product was not effective in reducing the probability of E. coli O157:H7 fecal shedding or colonization of the terminal rectum by this organism. A large-scale clinical trial that tested the effect of EHEC type III secreted protein vaccine and direct-fed microbials on the prevalence of E. coli O157:H7 in commercial beef feedyards was conducted. Vaccination of cattle within commercial feedlots was effective for reducing the probability of detecting E. coli O157:H7 from ROPES and might be beneficial as a pre-harvest intervention strategy against E. coli O157:H7. The effect of the EHEC type III secreted protein vaccine in weanling calves on the ranch was tested for reduction of the prevalence of E. coli O157:H7 in feedlot cattle. They concluded that the prevalence of E. coli O157:H7 during fall weaning was minimal on the Montana ranches tested, and the low level of shedding persisted throughout the feeding period. Therefore, the effect of vaccination as a pre-harvest food safety intervention strategy for backgrounders and feeders could not be determined. SD has developed an optimized multiplex PCR to detect virulence genes in Enterotoxigenic E. coli (ETEC). This test appears to eliminate or substantially reduce incidence of false positive test results. Salmonella AZ examined oysters from U.S. coastal waters for the prevalence of fecal coliforms and Salmonella. S. Newport was the major pathogen isolated at a prevalence rate of 7.8%. Virtually all isolates were resistant to ampicillin and tetracycline. Campylobacter WA performed a survey of thermophilic Campylobacter prevalence on Pacific NW cattle farms of various types. Overall detected prevalence of C. jejuni was about 40% and of C. coli less than 10%. C. coli expressed antimicrobial resistance and multiple antimicrobial resistance much more frequently than did C. jejuni. Lawsonia MN evaluated the effectiveness of hyper-immunized chicken eggs for controlling Lawsonia infection in growing swine and found that these antibodies significantly affected the reduction in average daily gain during a Lawsonia infection. MN also developed and tested a method for molecular epidemiologic typing of Lawsonia. This Variable Number Tandem Repeat typing method was shown to be stable and conserved in individual Lawsonia isolates under various conditions. MN developed and compared two methods for measuring the viability of Lawsonia and found that there was no significant difference between flow cytometry or a direct count method for determining Lawsonia viability. Finally, MN compared the agreement of two Lawsonia serology tests, an indirect fluorescence antibody test and an immunoperoxidase monolayer assay, for sensitivity and specificity in diagnosis of field cases of ileitis. There was an excellent level of agreement between the tests, with a Kappa value of 0.87. Enteric Calicivirus OH conducted a cross-sectional study in normal adult pigs to investigate the prevalence of porcine sapovirus Cowden and porcine noroviruses. The prevalence of porcine Norovirus was 23% and that of porcine Sapovirus was 30%. The high prevalence of these Noroviruses raises the question of whether pigs may be reservoirs for certain strains of human Noroviruses. To determine risk assessments for occurrence of human and animal caliciviruses in U.S. market oysters and to develop strategies to reduce viral gastroenteritis associated with their consumption, OH surveyed regional oyster harvests for caliciviruses. The results confirm the presence of human caliciviruses in raw oysters and suggest that they should be considered a risk factor in outbreak inveastigations. OH also determined both antigenic and genetic relationships of bovine noroviruses to human noroviruses and found that they do not share significant antigenic and genetic relationships. Bovine Coronovirus OH compared bovine coronovirus shedding patterns and antibody titers in two groups of calves after shipment to a feedlot and commingled at arrival. Based on the findings, they suggest vaccination of feedlot calves against bovine coronovirus at least 3 weeks prior to shipping to feedlots. KS recommends using the monoclonal (8F2), which has been fully developed and characterized, for immunohistochemical staining of intestinal sections for bovine coronaviruses. Objective 3  Provide training and continuing education opportunities and dissemination of information to students, producers, consumers, veterinarians and diagnostic laboratories: Information on E. coli O157:H7 research, which included the results of clinical trials with the vaccine and direct-fed microbial was disseminated to the public by NE in the form of extension publications; extension meetings with producers, youth groups, and veterinarians; a release to the Associated Press which was covered by 51 newspapers and magazines in the U.S. and Canada; lectures to students in animal science and microbiology classes at UNL (two different courses). Groups in which extension presentations were given included: the Beef Quality Workshop (Mitchell, NE); Nebraska Beef Youth (Lincoln, NE); Northeast Equipment and Farm Show (Madison, NE); Nebraska Veterinary Medical Association (Lincoln, NE); Nebraska Beef Council (Lincoln, NE); and Nebraska Cattlemens Animal Health and Nutrition Committee (Lincoln, NE). The Principle Investigators, Graduate and Post-Graduate Students, and Veterinary Students involved in the MN project have given presentations and updates on ileitis 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 and the American Association of Swine Veterinarians. They have disseminated new information, reagents, and procedures to producers, industries, veterinary diagnostic laboratories and veterinarians. In addition, they have presented two international workshops, including theoretical lectures and practical laboratories, on the diagnosis and control of ileitis in pigs.

Arizona Konkel, M.E. and L.A. Joens: Campylobacter : Molecular and Cellular Biology. Chapter 22. Horizon Press. 2004. Konkel, M.E., Monteville, M.R., Klena, J.D., Joens, L.A.: Microbial Foiod Safety in Animal Agriculture: Current Topics by M.E. Torrence and R.E. Isaacson. Chapter 21, Iowa Atate Press. June, 2003. Lee, M.K., S.J. Billington, L.A. Joens. Potential Virulence and Antimicrobial Resistance in Campylobacter jejuni Isolates Obtained from Food and Companion Animals. In Press. Foodborne Path. Dis. 2004. Raphael, B.H. and L.A. Joens, 2004. Ferrous Iron Uptake is independent of FeoB in Campylobacter jejuni. Can. J. of Micro. In Press. Brands, D.A. A. Inman, C.H. Gerba, J.Mare, S.J. Billington, L.Saif, J.Levine, and L.A. Joens. 2004. The Prevalence of Salmonella spp. In United States oysters. In Press. App. Envir. Micro 2005. Brands, D.A., S.J. Billington, J.F. Levine, and L.A. Joens. Genotypes and antibiotic Resistance of Salmonella Newport Isolates from U.S. Market Oysters. In Press Foodborne Path. Dis. 2005 Illinois J Trask, S. McLaughlin, MS Kuhlenschmidt and P. Kalita 2004 Overland and Near-surface Transport of Cryptosporidium parvum. Environ. Qual. 33:984-983. Johnson, JK, Schmidt, Gelberg, HB, and Kuhlenschmidt MS. 2004. Microbial Adhesion of Cryptosporidium parvum sporozoites: Purification of an Inhibitory Lipid from Bovine Mucosa. J. Parasitology 90:980-990. Kansas Barkey, T.I., Dritz, S.S., Nietfeld, J.C., Johnson, B.J., Minton, J.E. 2004. Effect of dietary nannoligosaccharide and sodium chlorate on the growth performance, acute-phase response, and shedding of weaned pigs challenged with Salmonella enteric serotype Typhimurium. J Anim. Sci. 2004;82:397-404. Minnesota Gebhart, C.J. and R.M.C Guedes. 2004. Lawsonia intracellularis. In C.L. Gyles, et al, (ed.), Pathogenesis of Bacterial Infections in Animals, 3rd ed. Blackwell Publishing, Ames, IA. Walter, D., C.J. Gebhart, J. Kroll, J.T. Holck, and W. Chittick. 2004. Serologic profiling and proper vaccination timing. J. Swine Health Production. (in press). Guedes, R.M.C. and C. J. Gebhart. 2003. Polyclonal and monoclonal antibodies for diagnosis of proliferative enteropathy. J. Vet. Diag. Investi. 15:438-446. Nebraska Berberov, E. M., Y. Zhou, D. H. Francis, M. A. Scott, S. D. Kachman, and R. A. Moxley. 2004. Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of Escherichia coli that produces multiple enterotoxins. Infect. Immun. 72:3914-3924. Dassanayake, R. P., N. E. Caceres, G. Sarath, and G. E. Duhamel. 2004. Biochemical properties of membrane-associated proteases of Brachyspira pilosicoli isolated from humans with intestinal disorders. J. Med. Microbiol. 53:319-323. Dassanayake, R. P., Y. Zhou, S. Hinkley, C. J. Stryker, G. Plauche, J. T. Borda, K. Sestak, and G. E. Duhamel. Characterization of cytolethal distending toxin of Campylobacter species isolated from captive macaque monkeys. J. Clin. Microbiol., in press. De Cock, H. E. V., S. L. Marks, B. A. Stacy, T. Zabka, J. Burkitt, G. Lu, D. J. Steffen, and G. E. Duhamel. 2004. Ileocolitis associated with Anaerobiospirillum in cats. J. Clin. Microbiol. 42:2752-2758. Folmer, J. D., C. N. Macken, R. A. Moxley, D. R. Smith, S. Hinkley, G. E. Erickson, A. A. Potter, B. B. Finlay, and T. J. Klopfenstein. 2004. Vaccination and direct-fed microbials as intervention strategies for reduction of E. coli O157:H7 in feedlot steers. 2004 Nebraska Beef Cattle Report, University of Nebraska Cooperative Extension MP 80-A, pp. 68-71. Johansson, K. E., G. E. Duhamel, B. Bergsjö, E. O. Engvall, M. Persson, B. Pettersson, and C. Fellström. 2004. Identification of three clusters of canine intestinal spirochaetes by biochemical and 16S rDNA sequence analysis. J. Med. Microbiol. 53:345-350. Moxley, R. A. 2004. Escherichia coli O157:H7: an update on intestinal colonization and virulence mechanisms. Anim. Health Res. Rev. 5:15-33. Potter, A. A., S. Klashinsky, Y. Li, E. Frey, H. Townsend, D. Rogan, G. Erickson, S. Hinkley, T. Klopfenstein, R. A. Moxley, D. R. Smith, and B. B. Finlay. 2004. Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine 22:362-369. Smith, D. R., J. T. Gray, R. A. Moxley, S. M Younts-Dahl, M. P. Blackford, S. Hinkley, L. Hungerford, C. T. Milton, and T. J. Klopfenstein. 2004. A diagnostic strategy to determine the Shiga toxin-producing Escherichia coli status of pens of feedlot cattle. Epidemiol. Infect. 132:297-302. Yang, Z., J. Kovar, J. Kim, J. Nietfeldt, D. R. Smith, R. A. Moxley, M. E. Olson, P. D. Fey, and A. K. Benson. 2004. Identification of common subpopulations of non-sorbitol-fermenting, $-glucuronidase-negative Escherichia coli O157:H7 from bovine production environments and human clinical samples. Applied Environ. Microbiol. 70:6846-6854. Ohio Chang, K.O., S.V. Sosnovtsev, G. Belliot, Y. Kim, L.J. Saif, K.Y. Green. 2004. Bile acids are essential for porcine enteric calicivirus replication in association with down-regulation of signal transducer and activator of transcription 1. Proc Natl Acad Sci USA. Jun 8; 101(23):8733-8. Han, M.G., J.R. Smiley, C. Thomas, L.J. Saif. 2004. Genetic Recombination between Two Genotypes of Genogroup III Bovine Noroviruses (BoNVs) and Capsid Sequence Diversity among BoNVs and Nebraska-Like Bovine Enteric Caliciviruses. J. Clin. Microbiol. 42: 5214-5224. Han, M.G., Q. Wang, J.R. Smiley, K.O. Chang, L.J.Saif. 2004. Self-assembly of the recombinant capsid protein of a bovine norovirus (BoNV) into virus-like particles and evaluation of cross-reactivity of BoNV with human noroviruses. J. Clin. Microbiol. (in press). Saif, L. J. 2004. Bovine Coronavirus Infections. In Infectious Diseases of Livestock, (Coetzer, J.A.W., Thomson, G.R.) Oxford Univ. Press, Oxford, England. Saif, L.J. 2004. Comparative biology of animal coronaviruses: Lessons for SARS. In: SARS The first new plague of the 21st century (M. Peiris, ed), Blackwell Pub., Oxford, UK (in press). Saif, L.J. 2004. Animal Coronaviruses: Lessons for SARS. In: Learning from SARS: Preparing for the next disease outbreak. SARS workshop sponsored by the Institute of Medicine of the National Academy of Sciences, Washington, DC, Sept. 30-Oct. 1, 2003. (in press). Hodgins, D. C., L. Yuan L., V. Parreno, L.B. Corbeil, and L.J. Saif. Mucosal Veterinary Vaccines. In: Mucosal Immunology. Third Edition. Edited by McGhee J. R. Mestecky, J. et al., Academic Press. 2004 (in press). Yuan, L., G. Stevenson and L.J. Saif. Rotavirus and Reovirus. (2004). In: Diseases of Swine. 9th Edition. Edited by Zimmerman, J. J. et al. Ames, Iowa: Iowa State University Press (in press). Saif. L.J. 2004. Animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? Rev. Sci. Tech. Off. Int. Epiz., 23(2): (in press). Saif, L.J. 2004. Animal coronaviruses vaccines: Lessons for SARS. In: Control of Infectious Animal Diseases by Vaccination (Schudel A., M. Lombard, eds). Dev. Biol. Basel, Karger, vol. 119 (in press). South Dakota Berberov, E.M, Y. Zhou, D.H. Francis, M.A. Scott, S.D. Kachman and R.A. Moxley. 2004. Relative importance of heat labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins. Infect Immun 72: 3914-3924 Washington Bae, W, KN Kaya, DD Hancock, DR Call, YH Park, and TE Besser. Accepted. Prevalence and antimicrobial resistance of thermophilic Campylobacter sp. from cattle farms in the Northwestern United States. Applied and Environmental Microbiology. Borucki, MK, SH Kim, DR Call, S. Smole, and F. Pagotto. Accepted. Discrimination of Listeria monocytogenes epidemic strains using a mixed-genome DNA microarray. Journal of Clinical Microbiology. Cobbold, R. N. and Desmarchelier, P. 2003. In vitro studies on the colonization of bovine colonic mucosa by Shiga-toxigenic Escherichia coli (STEC). Epidemiology and Infection. 132: 87-94. Cobbold, R.N.; Rice, D.H.; Szymanski, M.; Call, D. R.; Hancock, D.D.. 2004. Comparison of Shiga-toxigenic Escherichia coli (STEC) prevalence between dairy, feedlot and cow-calf herds in Washington State. Applied and Environmental Microbiology. 70 (7): 4375-4378. DeFrancesco, K.A.; Cobbold, R.N.; Rice, D.H.; Besser, T.E.; Hancock, D.D.. 2004. Antimicrobial Resistance of Commensal Escherichia coli from Dairy Cattle Associated With Recent Salmonellosis Outbreaks. Veterinary Microbiology. 98 (1): 55-61. González, SF, MJ Krug, ME Nielson, Y. Santos, and DR Call. 2004. Simultaneous detection of marine fish pathogens using multiplexed PCR and DNA microarrays. Journal of Clinical Microbiology 42:1414-1419. Khachatryan, AR, DD Hancock, TE Besser, and DR Call. 2004. The role of calf-adapted Escherichia coli in maintenance of antibiotic resistance in dairy calves. Applied and Environmental Microbiology 70:752-757. Lahmers, S, Y Wu, DR Call, S. Labeit, and H. Granzier. 2004. Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium. Circulation Research 94:505-513. Lane, S., J. Evermann, F. Loge and D.R. Call. 2004. Secondary structure prevents target hybridization to oligonucleotide microarrays. Biosensors and Bioelectronics. ORourke KI, Spraker TR, Hamburg LK, Besser TE, Brayton KA, Knowles DP. 2004. Panicker, G, DR Call, MJ Krug, and AK Bej. Accepted. Detection of pathogenic Vibrio spp. in shellfish and Gulf of Mexico water using multiplex PCR and DNA-array hybridization. Applied and Environmental Microbiology. Straub, TM, Quinoñez Díaz, MD, CO Valdez, DR Call, and DP Chandler. 2004. Using DNA microarrays to detect multiple pathogen threats in water. Water Science and Technology: Water Supply 4:107-114. Warsen, A, MJ Krug, S LaFrentz, DR Stanek, FJ Loge, and DR Call. 2004. Simultaneous discrimination between 15 fish pathogens using 16S rDNA PCR and DNA microarrays. Applied and Environmental Microbiology 70:4216-4221. Willse, A, TM Straub, S. Wunschel, JA Small, DR Call, D Daly, and DP Chandler. 2004. Quantitative oligonucleotide microarray fingerprinting of closely related Salmonella enterica isolates. Nucleic Acid Research 32:1848-1856. Woodford, NL, DR Call, DG Remick, and R Rochford. 2004. A model of angiogenesis in SCID mice xenoengrafted with Epstein-Barr virus transformed B cells. Comparative Medicine 54:209-215.