Brief Summary of Minutes of Annual Meeting

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.

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. 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. 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. 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 Produced numerous reports abstracts and scientific manuscripts that were published in various journals Developed a course International Animal Production, Disease Surveillance and Public Health 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. 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. 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. Our findings address a public health concern regarding environmental quality surrounding swine production units. 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. 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. 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. 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. 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. 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. 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. 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. 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.