Brief Summary of Minutes of Annual Meeting

SEPTEMBER 19: Meeting began 8:30 am. Brief discussion of applying for CAP when next opportunity arises, per discussions between Chris Chase and Amelia Woolums with Peter Johnson over e-mail. CAP award for BRD is conceivable; time frame within which we could apply depends on when next award is open. The Johnes CAP has been renewed for 4 years ($1.2 million/year); the avian flu CAP is applying for a renewal, and the PRRS CAP is submitting a revised renewal proposal. If either PRRS or AI arent renewed, funds will be freed up to begin a new CAP; if they are both renewed, a new CAP wont be possible in FY2009 (or longer). Station reports 8:45 am - 12:20 pm with break 10:30-10:45. Lunch (sponsored by Novartis Animal Health) began at 12:20. 1:00 pm: Teleconference with Gary Sherman. Discussed points in our letter to him of August 17th. Has talked with people at CSREES about reports from other multistate projects. Some are feeling less successful, as are we; others doing better. Some talk at CSREES re that, if many projects are having problems like us, changes may be made so that projects can function more effectively (as multistate projects are vital to CSREES mission). Wont be fast, but hes going to try to have gatherings at CSREES, maybe a conference on revitalizing multistate committee process, to find out what can be done to improve situation. Principles are the Ag Expt Station directorsCSREES is just in an overview position, as the law provides. Law very broad, so lots of discretion left to Ag Expt Station directors to use Hatch money as at they see fit. Not enough money to go around as with everywhere elsestate support for universities dropping so much has contributed a lot to this. Thus Ag Expt station directors re-direct Hatch funds to do whatever they feel is most necessary. This is allowable under law. Law does state that 25% of funds need to go to multistate projectsthis is audited to ensure compliance. But many different activities can fit under this 25%. Should talk to our Ag Expt Station director re how this is broken down. Bottom line: Expt Station directors are using their authority in difficult times to do the best they can with the funds they have. Not to say we cant make a case re BRD being an area that requires particular support&but need to keep this in mind when we visit Expt Station Directors. Also remember that BRD is still well supported by NRI, as compared to other animal diseases (tho he notes that problem with getting funds from NRI to study more applied problems is valid). --remember that other researchers are far worse off than we are, in that BRD is at least still supported by NRI. Money going in to Hatch has been steady, but in real dollars has declined. State funding for Expt Station activities has also declined. Every group needs to be known to their Ag Expt. Station directorsmake your case whenever necessary. More than in old days, need to make our mission more clearneed to do a bit more marketing of what we are trying to do than used to be necessary. Have had similar discussions with APHIS and ARSthey all need more money, but they cant lobby! One can argue that we are in perfect storm AI (avian flu), BSE, etc. do make animal health on the radar of the Secretary of Homeland Security. Gary was at meeting with aide of Sec of HS: how high on list is animal health on radar of Sec HS answer was that animal health was high --may need to make marriage of convenience with bioterrorism/agroterrorism link and BRD --we are in a time where the best chance for seeking extra funding is hereBUT the federal budget right now not in a good place due to war on terrorism. Comment from Ricardo Rosenbusch: article from Sciencesome think industry will pick up slack in human health research funding that government doesnt supplybut a study shows that industry follows lead of federal fundingso we might expect similar outcomes for animal health. GS agreed that, from his experience, this is true. Gary will finish going through our 6 points; answers probably wont satisfy us, but he hopes to have more information back to us in next year re challenges multistate projects are facing. Old model clearly is not workingthey cant lobby, but they can talk about how programs can best be run in current climate: how to write laws, make programs that are more supportive. Hopefully in form of conferences, etc. Especially good to try to figure out what is different between multistate projects doing well vs those not doing wellwhat lessons can we learn from successful projects? Specific comments re points in our letter to him: 1. Hatch funds havent declined relatively, but have declined in terms of real dollars. Bigger problem: Ag Expt Station directors have made necessary redirection of funds to preserve faculty lines. He had this experience personally. University administrators must do creative financing to keep faculty positions in place. Some faculty may not realize how thinly funds are spread, and how creative administrators must be, to keep faculty lines in place. This type of use of funds is within discretion of Ag Expt Station directors. 2. Re disproportionate funding/redirection of funding: again, related to discretion of Ag Expt Station director at each institution. Gary thinks disproportionate funding is probably exactly a problem, and we can only find out reasons via frank discussions with Ag Expt Station directors. He thinks in most cases while, decisions may seem unfair/capricious, these people are actually doing the best they can with a bad situation. 3. Re question of whether funding available from our Experiment Stations merits participation in NC1027: he hopes NC1027 never goes away, but he also realizes we can only be so altruistic about where we spend our time. He hears from lots of land grant colleges who say that 75% of their funding comes from NIH, so thats what they have to work on. Related to our later point that BRD not a big model for human disease, so realizes that we cant tap into NIH funds as well as other veterinary researchers. This is probably one of the stronger arguments to make to veterinary school administrators and Ag Expt Station directors. Redistribution of Hatch funds needs to be done on a transparent basis. If we have good logic re why were not in good shape for competing for NIH money, but relative importance of field were in is high (and this is true for BRD)constantly make comparison between losses due to BRD and level of funding for BRD researchthis needs to keep being drummed on til it hits the right set of ears. Is participation in NC1027 worth it? He leaves that decision to us, but the fact that we are thinking this way gives him some incentive to move forward to get CSREES administrators to look at whether multistate projects need to be run under a different model. Some multistate projects are in worse shape than us. 4. Re this [instances when scientists at historically participating stations have wanted to participate but cant get support due to funds going to other departments/colleges]: he will look into this. Could be that Expt Station Director cant transfer funds to the dept. this person is in? We told him to call Jeff Lakritz at Ohio State for more details. R. Rosenbusch asked: can you address as 2 separate issueswhether the person can even get travel funds, vs whether the person can get research funds? We told him Jeff would probably appreciate even travel funds. 5. Re Hatch funds being last vestige of support for veterinary translational/applied researchthis is the case we need to make with our Ag Expt Station directors. Point out this gapits real. Worse for some diseases than others. Make this point to amplify our other point that there may be too few people left in this field to provide critical mass to support animal health the way it needs to. We need to continue to make this case that the gap is real between what is fundable at NRI vs what we need so that it doesnt dead-end at basic research level. 6. Re brain drain from BRD researchhe asked how many grad students are here at our meeting? We told him none. This should be a major arrow in our quiver why we need our share of funds from our respective Expt Stations. Reality is that we are in a time when we all need to market better what we need for what we are doing. Other comments from Gary Sherman: Strategies for revitalizing multistate projects: some have done well via meeting at CRWAD. They see rationale for going to AABP, but in the sense of trying to get more research money, and seeing blend of basic and applied research, CRWAD is good venue. PRRS had a conference and had great turnout; brucellosis group did too. A BRD symposium at CRWAD might be a good idea. Robert Fulton: how much should we emphasize clinically applied research with Hatch/1433 money? They are encouraged at their station to use Hatch/1433 to serve as springboard for another grant to an outside agency. Which do you think Hatch/1433 funds are supposed to be used for? G. Sherman says he hears people above him say that Hatch/1433 should be used to solve practical problems, but reality is probably that your institution needs you to use the funds as springboard to get more extramural funds. However, they do say these funds are supposed to be for local/applied problems, so realizes they are speaking out of both sides of their mouth. Again, problem is that there is just not enough money to do both. Hatch/1433 funds are not enough to support a research program any morethus reality is that people are using these funds to get more extramural funds. Ricardo Rosenbusch: is there any merit in bringing these attentions to AABP, AVC, producer groups? Although as a USDA employee G. Sherman cannot be involved with lobbying, he acknowledged that it could be helpful for any group in our position to get organized, and then ask to meet with Secretary Johanns [who resigned the morning after this discussion] and others. Stakeholder groups important from political standpoint; CRWAD important from a scientific standpoint. We need to be focused on both arms of this: pushing for political attention, and pushing to show scientific strengths of our group. Some groups that are effective have a governmental relations committee of their own, focused like a laser, on bringing these problems to the attention of people in government. Gale Buchanan (former head of Dept. of Animal and Crop Sciences at University of Georgia) is our undersecretary. Christopher Chase comment: we dont have grassroots support like CRWAD, so this is why we are meeting at AABPuntil we get the political mandate that the PRRS group does, meeting at CRWAD may not do us as much good. G. Sherman concedes that if we dont have enough manpower to do both, we need to get constituent groups organized to help us bring attention to the problem first. 2:20 - 3:15 pm: Discussion of possible symposium on animal health research funding. A. Woolums presented proposed agenda, list of invitees, objectives/outcomes. Also presented 2 other options: a) BRD symposium to show current research, and have discussions, or b) just write white paper and take it out to groups. We planned to submit conference grant to NRI to support this, but P. Johnson said NRI cannot support conference if it is explicitly aimed at lobbying. Could support a national/international conference on BRD research, which could be helpful to showcase state of research and needs for future research. Group discussion: BRD symposium would be more positiveeasy to selleasier to get funding. This is sounding like better outcome. We were successful in 2001 getting support for AVC symposium. Comment: meeting at Amarillo in 1983 had better stature. Proceedings were referenced for years. Comment: Ray Loan and Texas A&M really got behind that meeting. AVC supported with attendees. Texas Cattle Feeders supported banquet, got behind meeting politically. If we can get Texas Cattle Feeders behind us, good year-to-year support. Consider American Farm Bureau, too. Two years from now: AABP in Omahamight be good site. Or Albuquerque (both AVC and AABP meeting there, but in 3 years). AVC more diversemore cow-calf, some dairynot just feedlot, like is thought. Consensus was that would be ideal to have symposium a day or two before AVC. If we apply for NRI conference grant and are funded, would get money fall 08; need to spend by fall 09. Committee to write grant and put together plan for BRD Research Symposium: A. Woolums (chair), C. Chase, R. Fulton, R. Rosenbusch. Will model on Amarillo 2003 BRD meeting; tentatively plan to hold in conjunction with AVC at a centrally located U.S. site. Comment: Animal Health Research Reviews might be good site for proceedings to be published. Can get electronically. Short break 3:15-3:20. 3:20- 3:25. Short presentation by LA rep; Dr. Navarre here for Dr. Kousoulas, and couldnt attend this morning 3:25- Discussion with members of biologics/pharmaceutical industry on BRD research funding and ways to optimize interaction between industry/researchers outside industry, chaired by Dan Grooms. Attendees: Dale Groeteleuchen from Pfizer, Bruce Nosky, Merial; Richard Harlan, Novartis; Gerry Mechor, Elanco. Highlights: remember industry needs to see return on their investment. Even basic research done by companies has to be product driven. Issues of interest: why do some cattle get BRD, others dont; animal welfare impact of BRD; BRD/vaccination in beef calves < 4 months old; concern re lack of scientist to do basic research on BRD in the future; interaction of animal behavior/animal husbandry/animal health; better ways to diagnose BRD, especially chute-side. Had short discussion with feedlot practitioner (Dr. Wade Taylor) who joined us. Adjourned at 4:45 PM. SEPTEMBER 20, 2007 Meeting re-convened at 8:35 am with open forum. Seven attendees split about evenly between private practitioners and reps from industry (in addition to NC1027 members). Highlights: discussion of BRD in adult dairy cattle. Discussion of pros/cons of SRP vaccines. Comment: would be good if university done in production setting had a technician on site. Lots of good research opportunities are missed. Should also consider sending grad students to busy practices. Open forum ended at 9:45 pm. Break until 10:00 am. Business meeting: Proposal that 2008 meeting be in conjunction with AABP in Charlotte next year (September 24-25) so the NC-1027 will meet September 24-25, 2008. Bigger question: how do we get more people to come to this meeting? Suggestion: more science would have been interesting (tho it was noted that meeting used to be mostly scientific discussion, and attendance waned in face of that). Proposal by L. Corbeil: also could have a symposium at CRWAD. Ask Bob Ellis to give us half day in Resp section for BRD. Could say sponsored by NC1027wouldnt cost anything. Could build up critical mass for scientific discussion again there. Comment by R. Fulton: meeting at AABP might be more fruitful (in terms of better interaction with practitioners and improving our ability to gain support from stakeholders) if we get on the AABP program. Could have a session on BRD sponsored by NC1027. Other discussion: Debate about how much money was the problem with decreasing activity in NC1027. Point made that CSREES cant expect much activity by group when many stations dont get any funding. Consider using electronic means (listserve +/- website) for interaction between group, annual station report submission; and no longer having a formal business meeting. Instead have symposia/presentations involving NC1027 in conjunction with both CRWAD and AABP. Next Meeting Information: Christopher Chase will organize meeting for next year (September 24-25, 2008). Will ask L. Corbeil to organize half day NC1027-sponsored CRWAD symposium for 2008, Dan Grooms to organize half day NC1027-sponsored AABP symposium in 2008 [they have both since agreed to work on this]. Plan to have business meeting interaction (station report submission) electronically. Adjourned 10:45 AM.

Objective 1: To evaluate the prevalence of viral and bacterial agents of respiratory disease by developing, validating and disseminating new state-of-the-art molecular diagnostics for rapid identification of these agents. South Dakota reported that respiratory pathogens were similar to preceding years with Pasteurella multicida, Mannheimia haemolytica, and noncytopathic bovine viral virus diarrhea (BVDV) most frequently isolated. Georgia, Iowa and Michigan, with Iowa as the lead institution, submitted a proposal to USDA NRICGP to evaluate the prevalence of M. bovis shedding in calves in cow-calf operations in Iowa, Georgia, and Michigan, and to determine whether shedding of M. bovis at the cow-calf operation predicted occurrence of CPPS in the same calves when they are subsequently placed in feedlots in Iowa. Georgia reported a sensitive and accurate method of identification of BRSV nasal shedding, calves were challenged with virulent BRSV by aerosol. Preliminary information suggests that the qRT-PCR was more sensitive than either VI or FA in identifying infected calves. Results of viral shedding will also be compared to results of BRSV IHC of lung tissue and qRT-PCR of RNA extracted from lung tissue to evaluate agreement between each method of identifying virus in nasal swabs and lung. Michigan reported on additional progress on the porous silicon (PS) - based DNA sensor for the detection of the BVDV and compared to the conductometric platform previously developed. A US patent for the direct electropolymerization used in this application has been applied for. The very rough surface of the nano PS layer prepared the conditions for strong adsorption of polypyrrole (PPy) film on the PS layer without metallic an under layer. Because PS-based platform developed in this work was label-free and metal-free, it is easier to fabricate compared with a metal thin-film electrode and does not need any synthesis procedure for molecular labeling. This amperometric sensitivity detection is more reproducible than conventional optoelectronic or potentiometric methods. Generally, PPy film carries one positive charge for every three monomers. If a receptor molecule specific to BVDV has negative surface charge, there will be an electrostatic attraction between the receptor and PPy film. Even though PPy is intrinsically conductive, the conductivity will be varied with the amount of BVDV coupled with the immobilized receptor molecules. Work on further understanding and refining the conductometric biosensor continues. To date, BVDV positive samples show lower resistance than the negative samples. Optimization of antibody concentrations used in the biosensor has greatly reduced variation, but not to an acceptable level. High variability in sensing signal seems to be the most serious problem in this work. To resolve this problem, a screen-printing system was built. Screen-printed electrodes (SPEs) have the same size and thickness and the same annealing procedure will be used for drying the SPEs. NADC performed a large incidence study. 378 head of beef feeder calves were studied in 4 separate trials over a 30 day period at 3 commercial feedlots in southwestern Iowa. The calves were sourced from consignors located in the states of Georgia, Iowa, and Missouri. All nine serotypes of adenovirus were found to actively infected calves during the 30 days. All calves suffered active infections, many suffered infections with multiple serotypes, and infection with some serotypes was correlated with M. haemolytica infection. Adenovirus may be causing considerable losses to the U.S. beef industry and warrant a commercially-available vaccine. Nasal swabs were analyzed by culture for the presence of P. multocida and M. haemolytica. The former was recovered from a high percentage of calves on arrival at the feedyard (approximately 40%) and became more prevalent during the approximately 30 day study period. M. haemolytica recovery was variable, from 10% to nearly 70% of each group indicating a moderate to high level of stress and/or concurrent viral infection. Shedding of Mannheimia haemolytica was associated with seroconversion to adenovirus. Oklahoma repoted on the prevalence of bovine viral diarrhea virus (BVDV) persistently infected (PI) cattle in beef breeding herds in 30 herds with 4530 calves. The samples collected by ear notches were tested for BVDV antigen using immunohistochemistry (IHC) and antigen capture ELISA (ACE). Animals with initial positives on both IHC and ACE were sampled again for both tests, and serums were collected for viral propagation and sequencing of a viral genomic region, 5-untranslated region (5-UTR) for viral subtyping. Samples were also collected from the dams of PI calves. There were 25 PI calves from 4530 samples (0.55%) and these PI calves were from 5/30 herds (16.7%). Two herds had multiple PI calves and three herds had only one PI calf. Only 1/25 PI calves dam was PI (4.0%). The subtype of all the PI isolates was BVDV1b. Histories of the ranches indicated 23/30 had herd additions of untested breeding females. Twenty-four of 30 herds had adult cowherd vaccinations against BVDV using primarily killed BVDV vaccines at pregnancy examination. Oklahoma reported on BVDV incidence in a cow study. The prevalence of bovine viral diarrhea virus (BVDV) persistently infected (PI) cattle in beef breeding herds was determined in 30 herds with 4530 calves. The samples collected by ear notches were tested for BVDV antigen using immunohistochemistry (IHC) and antigen capture ELISA (ACE). Animals with initial positives on both IHC and ACE were sampled again for both tests, and serums were collected for viral propagation and sequencing of a viral genomic region, 5-untranslated region (5-UTR) for viral subtyping. Samples were also collected from the dams of PI calves. There were 25 PI calves from 4530 samples (0.55%) and these PI calves were from 5/30 herds (16.7%). Two herds had multiple PI calves and three herds had only one PI calf. Only 1/25 PI calves dam was PI (4.0%). The subtype of all the PI isolates was BVDV1b. Histories of the ranches indicated 23/30 had herd additions of untested breeding females. Oklahoma also reported on testing for BVDV in the feedlot where they tested 3023 cattle with 10 cattle determined to be PI (0.33%) using the IHC test on fixed ear notch samples. These include cattle in 32 pens with 7 pens with PI cattle. Two pens have more than one PI calf. Oklahoma also reported on the levels of BVDV virus from PI animals over an 11-month interval. These levels of infectious virus in nasal swabs demonstrate and confirm the high potential of spread of BVDV from PI cattle to susceptible cattle. They monitored viral infectivity and used various tests for PI calves, including the antigen capture ELISA (ACE) on fresh notches and serums, IHC on formalin notches, viral titration (VT) on serum and nasal swabs (NS), and PCR of nasal swab materials, serums, and ear notches. Virus was detected in nasal swabs and serums of PI cattle throughout the study. PI calves do not change test status for ACE and IHC using ear notches and remain positive over time. Selected PI calves were inconsistent for PCR detection of viral genomic material in fresh ear notches. The ACE and IHC remain positive in all of the calves tested from day 0 and at monthly intervals through eleven months of continual testing. Oklahoma also reported on a bovine herpesvirus 1 study. They have obtained a group of almost 50 BHV-1 isolates including current MLV strains, and reference strains such as Cooper (respiratory 1.1 strain) and K-22 (genital 1.2 strain) from respiratory cases in feedlots, genital infections in dairy cattle, neonatal calf disease, and aborted fetuses from heifers/cows recently receiving MLV vaccines. They are currently performing genomic studies to detect differences among the strains. To date several have been examined by restriction endonuclease enzyme fragment polymorphism (RFP). RFP testing using traditional enzymes to date has detected differences among selected field strains and vaccines. Wisconsin reported their BVDV testing rate. BVDV positive testing rate of 0.17% in serum (8 of 4,678), 3.3% in buffy coat (20 of 601), 0.3% in ear notch ELISA (48 of 14,287) and 2.3% in PCR ear notch pools (40 of 1,726). Objective 2: To investigate the basic biology, molecular pathogenesis, and immunopathogenesis of polymicrobial infections including important viral and bacterial agents. South Dakota reported that BVDV PI microarray expression profiling study. Lung leukocytes from PI and non-PI calves were used to determine differences in gene expression profile due to BVDV infection. upregulated includes pro-apoptotic genes like p53 were upregulated while, anti-apoptotic genes and pro-inflammatory genes were down-regulated. California reported on a vaccine trial with a formalin killed alum adjuvanted vaccine and with a H. somni bacterin with a challenge with combined or single pathogens. The BRSV vaccine did not shift the immune response to a Th2 cytokine mediated response and the induction of IgG2 by the bacterin resulted in solid protection from H. somni infection. Cytokine analysis using TaqMan RT-PCR revealed a mixed profile with neither Th1 nor Th2 dominance. California also reported on in vitro development of primary ciliated bovine bronchial epithelial (BE) cell line for use in co-infection studies. Additional studies with bovine turbinate cells to evaluate binding of H. somni to infected and uninfected cells in the presence and absence of inhibitors for binding (such as dextran sulfate and heparin sulfate) are ongoing, including examining the evaluation of BT and BE cells for their ability to alter H.somni binding after various times of infection. California also reported on a study with calves were either immunomodulated with BCG vaccination or mock vaccination potentially to down-regulate Th2 responses, then dually infected with BRSV and H. somni. Necropsy results, H. somni isolation, and clinical signs have been evaluated. Other parameters such as interferon gamma production by PBMC and bronchial lymph node cells are still being evaluated. Georgia establish long term cultures of primary bovine bronchial respiratory epithelial cells (BREC) from samples collected from bronchial brushings of living calves. In preliminary studies, the cells have grown well from bronchial brushings, establishing a differentiated monolayer. Preliminary studies indicate that the cells can be infected with BRSV. Georgia also reported on a leukocyte reactivation from neonatal calves. Proliferative responses against bovine viral diarrhea virus (BVDV) and mycobacterial purified protein derivatives (PPDs) were evaluated. Prior to parturition dams of the calves received a vaccine containing inactivated BVDV, but were not vaccinated against mycobacterial antigens. The results showed that all calves had essentially no IgG in circulation at birth, but comparable and significant levels by day 1. Calves receiving whole colostrum had enhanced leukocyte responses to BVDV antigen 1 and 2 days after ingestion of colostrum. In contrast, calves receiving frozen colostrum, or cell-free colostrum did not respond to BVDV. These results indicated that transfer of live maternal cells from colostrum to neonatal calves enhanced responses to antigens against which the cow had previously responded (BVDV) in the first days of life, but not to antigens for which the cow was naïve (mycobacterial PPD). Kansas reported on work in collaboration with Nebraska that tested the reactivation property and immune response against a gE cytoplasmic tail truncated and a Us9 acidic domain deleted BHV-1 mutants. Several calf and rabbit studies show that both the mutant viruses replicated efficiently in the nasal and ocular epithelium during primary infection. Upon reactivation from latency, both the mutant viruses were not shed and only the respective rescued wild-type viruses were present in the nasal and ocular shedding. Primary immune responses against both the mutant viruses, based on serum neutralization titers were higher than the entire gE and Us9 ORF-deleted viruses (reported last year). Upon dexamethasone induced reactivation, seroconversion was detected only for the respective rescued virus-infected calves. Mississippi is conducting experimental annotation of the three bovine respiratory disease (BRD) pathogens Mannheimia haemolytica, Histophilus somni, and Pasteurella multocida by proteogenomic mapping to improve their ongoing structural annotations and provide functional annotation. In 2007, proteogenomic maps of Mannheimia haemolytica strain PHL213 and P. multocida strain 3480 were produced. Proteins were isolated from each strain in triplicate and analyzed by multi-dimensional protein identification technology (MuDPIT) using two-dimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The resulting mass spectra were searched against their respective protein databases using SEQUEST (Bioworks 3.2 cluster; ThermoElectron). The lists of peptides identified from the genome sequences were compared with the lists identified from protein databases. NADC conducted insertional mutational studies of P. multocida enzymes involved in sialic acid metabolism. These mutants are significantly attenuated in a mouse septicemia model. Also sialic acid mutants may be useful as live vaccines. They constructed sialic acid uptake mutants of bovine P. multocida. Filamentous hemagglutinin (FHA) of B. pertussis plays a critical role in bacterial adherence to host cells and invasion of respiratory epithelial cells. NADC constructed FHA mutants of bovine P. multocida. NADC also developed for isolating ovine lung dendritic cells. A real-time PCR assay for the nonstructural protein 2 (NS2) gene was devised which showed that BRSV can productively infect neonatal ovine lung dendritic cells and alveolar macrophages. Additional real-time PCR assays were developed for proinflammatory and immunomodulatory ovine cytokines. The data indicate that BRSV induces immunomodulatory cytokines (IL-4 and IL-10) during the early stages of infection (days 3 or 5 PI) which would result in a failure to clear BRSV and leave the host susceptible to secondary bacterial infections. Oklahoma has been working on the development of chimeric Mannheimia haemolytica vaccines containing highly immunogenic epitopes from leukotoxin A (LKT) and the outer membrane protein (OMP), PlpE. The five chimeric proteins used as vaccines differed in the number of copies of the LKT and PlpE epitopes they contained and the presence or absence of GST leader sequences or glycine-serine spacers. Mice were intraperitoneally vaccinated with 25, 50 and 75 ¼g quantities of each chimeric protein. Western blot analysis and enzyme-linked immunosorbent assays (ELISA) demonstrated the immune response to the chimeric proteins was strong and specific to PlpE and LKT. Complement-mediated killing assays established that the sera were bactericidal to M. haemolytica in the presence of complement. MTT assays of the hyper-immune murine sera showed that active LKT is neutralized by anti-leukotoxin antibodies formed after vaccination. While all of the chimeric proteins did induce some level of immune response, SAC88 and SAC89 were most immunogenic in that anti-SAC88 and anti-SAC89 sera exhibited the highest complement meditated killing activity and some of the highest levels of LKT neutralizing antibodies. Wisconsin reported on intracellular pathways that result in bovine leukocyte death following exposure to M. haemolytica LKT. LKT internalization can occur by both clathrin-dependent coated pits and lipid rafts. Both of these pathways are inhibited by reducing levels of the scission protein dynamin-3, using siRNA. LKT internalization required actin filament formation, and involves relocation of dynamin from mitochondria to the cell membrane. LKT can be visualized as bound to the outer mitochondrial membrane suing scanning electron microscopy. LKT binding to mitochondria is associated with a drop in mitochondrial membrane potential. These results were obtained using LKT from which LPS was removed, were not observed using LKT produced by an lktC mutant (provided by Dr. S. Highlander, Baylor), and were blocked by addition of an anti-LKT Mab (provided by Dr. Srikumaran, Washington State). The above results were obtained using the BL3 bovine lymphoblastoid cell line, but are being confirmed using peripheral blood neutrophils. A student in the lab is designing an LKT construct to track internalization and transport of LKT in leukocytes. This effort has been aided by Dr. Maheswaran at Minnesota. Wisconsin also reported on the synergyistic effect of active infection with BHV-1 or other bovine respiratory viruses causes more severe pneumonia with M. haemolytica. Infection of primary bovine bronchiolar epithelial cell lines with BHV-1 increases the adhesion of M. haemolytica to these cells. BHV-1 infection increases expression of IL-8, and several inflammatory cytokines. Wisconsin also investigated M. haemolytica outer membrane proteins for their participation in attachment of M. haemolytica cells to bovine bronchial epithelial cells. Recent evidence suggests that one or more lipoproteins are involved in this process. Wisconsin has been assisted in this effort by sera and reagents obtained from Dr. Confer at Oklahoma. Wisconsin also been comparing the responses of bovine pulmonary epithelial and endothelial cells to LPS and LKT. Endothelial cells are more responsive to LPS, exhibiting changes in transepithelial electrical resistance (TEER), expression, of inflammatory cytokines, and morphologic and biochemical changes consistent with apoptosis. Epithelial cells also produced inflammatory cytokines in response to LPS, but did not demonstrate decreased TEER or cytotoxic changes, unless activated neutrophils were included in the incubation mixture. Both epithelial and endothelial cells gave similar signals for TLR-4, suggesting that other differences in signaling are responsible for the dichotomy in response to LPS between the two cell types. Wisconsin has also continued their studies of mechanisms by which Haemophilus somnus causes vasculitis. Using two different isolates of H. somni, that differed in their expression of phosphorylcholine (ChoP) on their LOS, we found that ChoP+ H. somni induced aggregation, while ChoP- H. somni did not. This finding suggested that ChoP on H. somni may interact with platelets, possibly via the PAF receptor, to induce aggregation. However, using an inhibitor of downstream PAF receptor signaling, we found no reduction in platelet aggregation. They inferred that if ChoP binds to the PAF receptor, this event does not induce signaling through this receptor. This observation raises the possibility that platelet aggregation induced by H. somni results from platelet cross-linking of the bacteria via ChoP, rather than initiation of a conventional platelet aggregation pathway. Wisconsin also looked at the effect of H. somni on bovine platelets and bovine pulmonary artery endothelial cells. Endothelial cells were incubated with platelets that had been activated with ADP, H. somni or H. somni LOS. Incubation with activated bovine platelets significantly increased expression of adhesion molecules (ICAM-1, E-selectin) and tissue factor, as measured by flow cytometry, real-time PCR, and western blot analysis. Activated platelets also up-regulated expression of endothelial cell IL-1², MCP-1, and MIP-1± as determined by real-time PCR and an IL-1² ELISA. Bovine platelets, but not H. somni, were internalized by bovine endothelial cells as visualized by transmission electron microscopy. This observation raises the possibility that endocytosis of bovine platelets may contribute, in part, to endothelial cell activation and cell stress. These findings suggest that H. somni activated bovine platelets might play a role in promoting vasculitis by increasing endothelial cell pro-inflammatory responses. The above studies involved collaboration with Dr. Corbeil (UC-San Diego and UC-Davis) and Dr. Inzana (Virg. Tech). Wisconsin also looked at the most severe manifestation of H. somnus infection, thrombomeningoencephalitis (TME). Cerebral microvascular endothelial cells are the primary cellular constituent of the blood brain barrier, which protects the central nervous system from inflammation and infectious agents. We developed an in vitro model and found that binding of H. somnus to bovine brain endothelial cells (BBEC) can be inhibited by high concentrations of heparin, or by pretreating the BBECs with heparinase. Addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no effect. These data suggest a role for sulfated glycosaminoglycans in the adhesion of H. somnus to BBECs. BBECs up-regulated TNF-±, IL-1², tissue factor, and ICAM-1 expression, following exposure to H. somnus. They also down-regulated their expression of occludin, a tight junction protein, and exhibited a decreased ability to activate protein C. Increased permeability of BBECs in response to viable, but not killed H. somnus, was observed as measured by decreased transepithelial electrical resistance (TEER) and by albumin leakage across the monolayers. These changes in monolayer permeability could be blocked by addition of a myosin light chain kinase inhibitor. The observed alterations in the BBECs could play a role in the development of vasculitis, thrombosis and inflammation that results in blood brain barrier dysfunction and bacterial invasion of the brain parenchyma. These studies involved collaboration with Dr. Corbeil and Dr. K.S. Kim (Johns Hopkins). Objective 3:To develop management and prevention strategies that incorporate new vaccines and treatment protocols to combat bovine respiratory disease and reduce economic loss. South Dakota reported on the effect of antigen trafficking following vaccination. Vaccinated with a NCP BVDV vaccine had longer antigen retention than either of the CP BVDV vaccines. Georgia has been working on developing siRNA molecules to inhibit replication of BRSV. Initials results have confirming inhibition of viral replication in vitro. Iowa was the lead station on a collaboration with GA, NADC and IA stations, and included the study of beef calves from GA farms that were sampled on arrival at a transit receiving station in GA, then sampled again at 1 and 2 months after arrival at IA feedlots. A total of 11 farms in GA were sampled. Sampling could detect 100% of calves as positive (one instance). A farm was arbitrarily considered as focally infected if < 10% of calves were infected, and widely infected if the percentage was higher. Only 2 of 11 GA farms were widely infected. Calves from those farms stayed infected at the same or higher proportion upon commingling at feedlot. Calves from the 9 naïve or focally infected farms all became widely infected upon commingling at feedlot. All calves from the second shipment were negative at farm. Overall, the pattern was a shift from naïve or focally infected to widely infected at feedlot. Iowa also tested several antimicrobials with potential usefulness against North American strains of M. bovis. Forty two isolates of M. bovis recovered from Midwest outbreaks in 1999 to 2006 were tested. Of these, 7 were from BRD cases and the rest from mastitis cases, reflecting the shift in case-loads handled by the Iowa Veterinary Diagnostic Lab. Two antimicrobials, ceftiofur and tilmicosin, were previously shown to be ineffective in-vitro against M. bovis in the previous study, and this was confirmed in this study. Enrofloxacin and florfenicol were effective against a majority (over 90%) of strains. Tulathromycin was shown to be ineffective against all strain tested. Michigan has initiated a regional BVDV control program to control and eradicate BVDV. The purpose is to identify benefits and obstacles of such a program and demonstrate a feasible model that may be adopted by other parts of the US. Wisconsin conducted an investigation of the relationships between air quality, a variety of environmental risk factors, and calf respiratory health were studied in 13 naturally ventilated calf barns during winter. A minimum of 12 pre-weaned calves were randomly selected and scored for the presence of respiratory disease in each barn. An air sampling device was used to determine airborne bacteria colony forming units per cubic meter (cfu/m3) of air in calf pens and central alleys within the barns. Temperature and relative humidity were recorded in each calf pen, the barn alley, and outside the barn. Samples of bedding were collected in each pen and dry matter was measured. Pen bedding type and a calf nesting score (degree to which the calves could nestle into the bedding) was assigned to each barn. Calf numbers, barn and pen dimensions, ridge, eave, and curtain openings, and exterior wind speed and direction were determined and used to estimate building ventilation rates. Factors that were significantly associated with a reduction of prevalence of respiratory disease were reduced pen log10 cfu/m3 on BAP, presence of a solid barrier between each calf pen, and increased ability to nest. Individual calf pen log10 cfu/m3 were significantly different from barn alley log10 cfu/m3 on both BAP and EMB. Significant factors associated with reduced calf pen log10 cfu/m3 on BAP were increasing pen area, increasing number of open planes of the calf pen, decreasing pen temperature, and wood-particle bedding. Significant factors associated with reduced alley log10 cfu/m3 on BAP were increased ventilation changes per hour, increased barn volume per kg of calf, reduced pen log10 cfu/m3, and barn type.

Journal Publications: 1. Ridpath JF, CS Mark, CCL Chase, AC Ridpath, and JD Neill. Febrile response and decrease in circulating lymphocytes following acute infection of white tail deer fawns with either a BVDV1 or a BVDV2 strain. J Wildlife Dis. in press. 2. Zimmerman AD, Buterbaugh RE, Herbert JM, Hass JM, Frank NE, Luempert III LG, Chase CCL. Efficacy of bovine herpesvirus-1 inactivated vaccine against abortion and stillbirth in pregnant heifers. JAVMA, 2007; 231:13861389. 3. Corbeil LB, Arnold KF, Kimball R, Berghaus L, Gershwin LJ. Specificity of IgG and IgE antibody responses to Haemophilus somnus infection of calves. Vet Immunol Immunopathol. 2006 Sep 15;113(1-2):191-9. 4. Berghaus LJ, Corbeil LB, Berghaus RD, Kalina WV, Kimball RA, Gershwin LJ. Effects of dual vaccination for bovine respiratory syncytial virus and Haemophilus somnus on immune responses. Vaccine. 2006 Aug 14;24(33-34):6018-27. 5. Donovan DC, Reber AJ, Gabbard JD, Aceves-Avila M, Galland KL, Holbert KA, Ely LO, Hurley DJ. Effect of maternal cells transferred with colostrum on cellular responses to pathogen antigens in neonatal calves. Am J Vet Res, 2007; 668:778-782. 6. Wiggins MC, Woolums AR, Sanchez S, Hurley DJ, Cole DJ, Ensley DT, Pence ME. Prevalence of Mycoplasma bovis in backgrounding and stocker cattle operations. J Am Vet Med Assoc. 2007; 230:1514-1518. 7. Al-Mubarak-A, Simon, J, Coats, C, Okemba, JD, Burton, MD, and Chowdhury, SI. Glycoprotein E(gE) specified by Bovine herpesvirus type5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions. Virology. 2007; 365:398-409. 8. Butchi, NB, Jones, C, Perez, S, Doster, A, and Chowdhury,SI. Role of Envelope protein Us9 in the anterograde transport of BHV-1 following reactivation in the Trigeminal Ganglia . J. Neurovirology. 2007; 13(4):384-388. 9. Muhammad-Tahir, Z, Alocilja, EC, and Grooms, DL. Indium Tin Oxide-Polyaniline Biosensor: Fabrication and Characterization. Sensors. 2007; 7:1123-1140. 10. Lee, S-R, Pharr, GT, Boyd, BL, Pinchuk, LM*. Bovine viral diarrhea viruses modulate toll-likereceptors, cytokines and co-stimulatory molecules genes expression in bovine peripheral blood monocytes.Comparat Immunol Microbiol Infect Dis. 2007; doi:10.1016/j.cimid.2007.06.006S. 11. Thomas, CJ, Hoet, AE, Sreevatsan, S, Wittum, TE, Briggs, RE, Duff, GC, and Saif, LJ. Transmission of bovine coronavirus and serologic responses in feedlot calves under field conditions. Am J Vet Res. 2006; 67:1412-20. 12. Fach, SJ, Brockmeier, SL, Hobbs, LA, Lehmkuhl, HD, and Sacco, RE. Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue. Vet Immunol Immunopathol. 2006; 112:171-82. 13. Kawashima, K, Meyerholz, DK, Gallup, JM, Grubor, B, Lazic, T, Lehmkuhl, HD, and Ackermann MR. Differential expression of ovine innate immune genes by preterm and neonatal lung epithelia infected with respiratory syncytial virus. Viral Immunol. 2006; 19:316-23. 14. Fach, SJ, Meyerholz, DK, Gallup, JM, Ackermann, MR, Lehmkuhl, HD, and Sacco, RE. Neonatal ovine pulmonary dendritic cells support bovine respiratory syncytial virus replication with enhanced interleukin IL-4 and IL-10 gene transcripts. Viral Immunol. 2007; 20:119-30. 15. Meyerholz, DK, Gallup, JM, Lazic, T, de Macedo, MM, Lehmkuhl, HD, and Ackermann MR. Pretreatment with recombinant human vascular endothelial growth factor reduces virus replication and inflammation in a perinatal lamb model of respiratory syncytial virus infection. Viral Immunol. 2007; 20:188-96. 16. Gatto, NT, Estes, DM, Confer, AW, Whitworth, LC, and Murphy, GL. Lung Lesions in SCID-bo and SCID-bg Mice after Intratracheal inoculation with Wild-type or Leucotoxin-deficient Mutant Strains of Mannheimia haemolytica Serotype 1. J Comp Patho. 2006; 134:355-365. 17. Atapattu, DN and Czuprynski CJ. Mannheimia haemolytica leukotoxin binds to lipid rafts in bovine lymphoblastoid cells (BL-3) and is internalized in a dynamin-2 and clathrin-dependent manner. Infect Immun. 2007 Aug 6; [Epub ahead of print] 18. Behling-Kelly, E, Vonderheid, H, Kim, KS, Corbeil, LB, and Czuprynski, CJ. Roles of cellular activation and sulfated glycans in Haemophilus somnus adherence to bovine brain microvascular endothelial cells. Infect. Immun. 2006; 74:5311-5318. 19. Behling-Kelly, E, McClenahan, D, Kim, KS, and Czuprynski, CJ. Viable Haemophilus somnus induce myosin light chain kinase-dependent decrease in brain endothelial cell monolayer resistance. Infect Immun. 2007; Jun 25; [Epub ahead of print] 20. Kuckleburg, CJ, Elswaifi, SF, Inzana, TJ, and Czuprynski, CJ. Expression of phosphorylcholine by Histophilus somni induces bovine platelet aggregation. Infect Immun. 2007; 75:1045-1049. 21. Kuckleburg, CJ, McClenahan, DJ, and Czuprynski, CJ. Platelet activation by Histophilus somni and its lipooligosacharride induces endothelial cell proinflammatory responses and platelet internalization. Shock. 2007, Jul 19; [Epub ahead of print] 22. Lago, A, McGuirk, SM, Bennett, TB, Cook, NB, and Nordlund, KV. Calf respiratory disease and pen microenvironments in naturally ventilated calf barns in winter. J. Dairy Sci. 2006; 89:4014-4025. 23. Sylte, M, Inzana, T, and Czuprynski, CJ. Role of tumor necrosis factor in endothelial cell apoptosis caused by Haemophilus somnus lipooligosaccharide. Vet. Immunol. Immunopathol. 2006; 110:303-309. Abstracts and Conference Proceedings: 1. Ridpath JF, CS Mark, CCL Chase and JD Neill. Experimental acute BVDV infection in white tail deer fawn. Proceedings of the 39th annual conference of the American Association of Bovine Practitioners, Saint Paul, MN, September 21-23, 2006, p. 271. 2. Ridpath JF, BE Hessman, JD Neill, RW Fulton, DL Step, A Zimmerman and CCL Chase. Evaluating stability, size requirements, viral load and pooling of ear notch samples in BVDV testing. Proceedings of the 49th annual conference of the American Association of Veterinary Laboratory Diagnosticians, Minneapolis, MN, October 12-18, 2006, p. 213. 3. Tigabu B, A Rosa, G Rosa and CCL Chase. Comparison of immune gene expression in normal and BVDV persistently infected cattle. Abstract 2. 66th annual meeting of the North Central Branch of the American Society of Microbiology, Brookings, SD, October 20-21, 2006, p. 21. 4. Halaweish S, Y Fang, I Halaweish, L Braun and C Chase. Expression of recombinant BVDV-nonstructural proteins to study the humoral immune response against BVDV in cattle. Abstract 1. 66th annual meeting of the North Central Branch of the American Society of Microbiology, Brookings, SD, October 20-21, 2006, p. 21. 5. Tigabu B, A. Rosa, G. Rosa, CCL Chase. Comparison of Immune Gene Expression in Normal and BVDV Persistently Infected Cattle. Abstract, p73. 87th annual meeting of Conference of Research Workers in Animal Disease, Chicago, IL, December 3-5, 2006. 6. Halaweish S, Y Fang, I Halaweish, L Braun, C Chase. Expression of recombinant BVDV-Nonstructural Proteins to Study the Humoral Immune Response against BVDV in Cattle. Abstract, p73. 87th annual meeting of Conference of Research Workers in Animal Disease, Chicago, IL, December 3-5, 2006. 7. Mahmoud MM, LJ Braun, A Hippen, M Thomas, and CC Chase. Effect of source of selenium and vitamin E supplementation on performance and immune response in young dairy calves. Abstract, p84. 87th annual meeting of Conference of Research Workers in Animal Disease, Chicago, IL, December 3-5, 2006. 8. Rosenbusch, R. F. Antimicrobial susceptibility of recent isolates of Mycoplasma bovis. Academy of Veterinary Consultants, Spring 2007 Meeting, Oklahoma City, OK, 2007. 9. S-R Lee1*, G. T. Pharr1, L. M. Pinchuk1. Bovine Viral Diarrhea Viruses Modulate Type I IFN and Pro-Inflammatory Cytokines Production via Toll-Like Receptor-Dependent Mechanisms. S.-R. Lee1*, G. T. Pharr1, L. M. Pinchuk1. Oral presentation at CRWAD, Chicago, IL, Dec.3-5, 2006. 10. Lawrence, ML, SC Burgess, S Bridges, B Nanduri, N Wang, and F McCarthy. Experimental annotation of bovine respiratory disease pathogen genomes by proteogenomic mapping. Plant and Animal Genome XV Conference, San Diego, California, 2007. 11. Confer A. Pathogenesis of pulmonary infections with Pasteurellaceae. Proceedings of the 24th Symposium of the Veterinary Comparative Respiratory Society. Jena, Germany, 2006. 12. Trojan B, Ayalew S, Montelongo M, Confer AW. Intranasal Vaccination of Dairy Calves with Mannheimia haemolytica Chimeric PlpE-LKT (SAC89). ASM abstract for presentation at the annual meeting in Toronto, Canada, May 2007. 13. Ayalew S, Stevens KD, Montelongo M, and Confer AW. Mannheimia haemolytica Chimeric Protein Vaccine Composed of the Major Surface-Exposed Epitope of Outer Membrane Lipoprotein PlpE and the Neutralizing Epitope of Leukotoxin. ASM abstract for presentation at the 107th annual meeting in Toronto, Canada, May 2007. 14. Fulton, RW, Braziel, BC, Kautz, K, Burge, LJ, Weaver, GD, and Hessman, B. Bovine Respiratory Syncytial Virus, Bovine Coronavirus and Bovine Viral Diarrhea Virus Diagnosis by PCR Testing of Nasal Swabs. American Association of Bovine Practitioners Annual Meeting Proceedings. St. Paul, MN, September 21-23, 2006. 15. Ridpath, JF, Hessman, BJ, Neill, J, Fulton, RW, and Step, DL. Parameters of Use of Ear Notch Samples for BVDV Testing: Stability, Size Requirements and Viral Load. American Association of Bovine Practitioners Annual Meeting Proceedings. St. Paul, MN., September 21-23, 2006. 16. Fulton, RW, Whitley, EM, Johnson, BJ, Kapil, S, Ridpath, JF, Burge, LJ, Cook, BJ, and Confer, AW. Bovine Viral Diarrhea Virus Persistent Infectious in Beef Breeding Herds; Utilization of Immunohistochemistry and Antigen Capture ELISA on Ear Notches. 49th Annual Meeting of AAVLD. Minneapolis, MN, October 12-15, 2006. 17. Fulton, RW, Elam, N, Hessman, BE, Johnson, BJ, Kapil, S, Ridpath, JF, Burge, LJ, Braziel, BC, Kautz, K, and Reck, A. Bovine Viral Diarrhea Virus Persistently Infected and Acutely Infected Calves: Assays for Viral Infectivity, Polymerase Chain Reaction Analysis, and Antigen Detection. 49th Annual Meeting of AAVLD. Minneapolis, MN, October 12-15, 2006. 18. Fulton, RW. Detecting Persistently Infected BVDV Cattle in Cow-Calf Herds. Academy of Veterinary Consultants Meeting, Denver, CO, December 1, 2006. Other (Ph.D Dissertations): 1. Tigabu B., PhD. The Effect of Bovine Viral Diarrhea Virus on Immune Gene Expression of Bovine Leukocytes. South Dakota State University, 2007. 2. Chris Kuckleburg, PhD. Bovine platelets activated by Histophilus somni induce endothelial cell activation and apoptosis. University of Wisconsin-Madison, 2007. 3. Erica Behling-Kelly, PhD. Interactions Between Haemophilus somnus and Bovine Brain Endothelial Cells. University of Wisconsin-Madison, 2007.