Brief Summary of Minutes of Annual Meeting

Minutes Research and outreach activities were reported by voting members in attendance and by guests from the BRD CAP (Dr. Terry Lehenbauer), USDA ARS (Drs. Carol Chitko-McKown, Larry Kuehn, and Richard Leach), Iowa State University (Dr. J.R. Tait), and WTAMU (Dr. John Richeson). Dr. Deb Hamernik (AA) advised the project members to continue collaborating and reporting any funding leveraged from the project. She also provided a handout with information regarding various organizations working to increase funding for animal health research, with contact information (see attached handout). Dr. Margo Holland (NIFA) provided the group with information regarding NIFA funding opportunities. She encouraged the group to apply for trainee fellowships through the NIFA Fellowship program. Some discussion ensued regarding the difficulty in funding trainees with the current limitations of the program (e.g., postdoc trainees cannot be funded until they have advanced to candidacy, by which time, at many institutions, they are nearly done with their programs). Members pointed out the difficulty in funding grad students in first 2 years of their programs&would be better if a program to do this was available. Dr. Holland encouraged members to send input, as stakeholders, to USDA. Also some discussion regarding USDA/NIH Dual Purpose Dual Benefit RFA: several felt the RFA would be more impactful if it funded research using ag animal diseases as models for human diseases, and not just research on zoonotic diseases. She encouraged the group to send feedback to Mark Mirando. She pointed out input directly from stakeholders is more impactful than her comments regarding the program. Dr. Holland also said that Foundational RFA will probably be released in late Sept., and this will have funding for FY12 and FY13 combined. She advised group to lean toward submitting if they are inclined to, as there will not be another opportunity until the FY14 RFA. She also provided other information regarding funding opportunities, FY11 funding rates, and personnel changes at NIFA (see attached handout). Discussion of potential BRD Symposium 2014. Dr. Amelia Woolums led discussion of whether group should hold a 2014 BRD Symposium, which would be 5 years after 2009 Symposium (BRDS 2009). Dr. Woolums gave group a short summary of 2009 meeting, noted that BRDS 2009 website is still active (www.brdsyomposium.org), and that people can still access papers and powerpoints from the meeting on the site. Also, the meeting made $19,000 in profit; the money has been used to provide an additional $1000 for each of 3 student research awards at AABP, and $10,000 is available for seed money for next Symposium if group decides to hold one. Consensus was that BRDS 2014 was a good idea. Should be held in conjunction with BRD CAP if they are willing; Dr. Woolums will contact Dr. Alison Van Eenennaam (extension leader for CAP) to see if her group is willing to work together on this. Dr. Fulton suggested we try to get more involvement from AAVLD and from producer organizations (NCBA, Heifer Rearers Organization, etc.). Volunteers to serve on BRDS 2014 Organizing Committee: Robert Fulton, Christine Navarre, Carol Chitko-McKown, Laurel Gershwin, Amelia Woolums, Chris Chase. Plan to add Dr. Van Eenennaam; Dr. Dale Grotelueschen (Pfizer) was also recommended as a good candidate to join committee as rep from industry. Proposed site: immediately before 2014 AVC Summer meeting (location TBA); consensus was that AVC was easy to work with and very helpful in terms of local logistics. Amelia Woolums or Chris Chase will contact AVC leadership to ask if we can hold BRDS 2014 in conjunction with 2014 AVC summer meeting. Business meeting: election of new Chair and Secretary. Dr. Chase (Chair) and Dr. Woolums (Secretary) have been serving for several consecutive years. They asked group if the group wished to elect new officers; they are willing to continue serving but also would be very happy for new people to step in. Group felt they were doing a good job and asked that they continue serving. Vote was taken with unanimous agreement for Dr. Chase to stay on as Chair and Dr. Woolums to stay on as secretary. Discussion of 2013 meeting: decided to meet in conjunction with AABP, since tentative plan for 2014 will be to meet in conjunction with Summer AVC meeting. Date for 2013 NC1192 Technical Committee meeting: September 18, 2013, Milwaukee WI (in conjunction with AABP). More details and info re hotel will be sent out in early summer 2013.

Accomplishments Objective 1: To aid the rapid identification and subsequent management of BRD by developing, validating and guiding the application of new state-of-the-art diagnostic tools. KS, WI, and SD reported results from state diagnostic lab testing for BRD pathogens. MI continued work to adapt a human RSV diagnostic test for rapid patient-side diagnosis of BRSV, with testing of clinical samples and comparison of results of the HRSV test with RT-PCR. The HRSV diagnostic assay was in agreement with RT-PCR 50 of 66 times (k=0.484). There appears to be a problem with false positive results (assuming the rtPCR is correct). This could be a matrix problem; MI is currently working to understand this phenomenon. NE is undertaking studies to examine the viruses that are present in calves suffering from BRDC versus healthy calves, based on the hypothesis that there novel viruses present in the respiratory tract of healthy cattle versus those suffering from BRDC. Nasal swabs from health calves will be compared to those with BRD. PCR and the ViroChip will be used to identify known viruses associated with BRDC. Deep sequencing will also be performed to identify novel viruses that are present. If new viruses are discovered, long-term studies will focus on growing these viruses and testing whether they cause disease in cattle. Sampling of herds with a history of BRD is underway. OK in collaboration with NADC isolated BVDV from 1264 PI cattle entering a feedlot between 2004  2008. Of the 1264 PI isolates there were 12.0% BVDV 1a, 78.3% BVDV1b, and 9.7% BVDV2a. The BVDV1b was the predominant subtype (P value <0.05). VA continued work to develop a highly sensitive and mobile detection device that will identify the presence of H. somni among livestock. The detector under study uses nanoparticle-based optical fiber biosensors (NOFS) to identify H. somni in materials such as nasal secretions from infected cattle. The sensor uses oligonucleotide sequences specific to different H. somni isolates coupled with an ionic self-assembled multilayer (ISAM) films onto the surface of the optical fiber cladding of the biosensor. The ISAM/probe duplex hybridized with the target DNA, and was detected and quantified based on the alteration of optical power transmitting through the fiber. Hybridization of the probe with DNA derived from the sample results in a significant decrease in the optical power transmitted through the fiber. The resulting portable sensing method would be useful for field applications where compact equipment (smaller than a laptop) can be combined to simply include a light source, optical fiber, detector, and computer. During specimen testing, DNA hybridization will alter the optical properties of the attached thin film, which will immediately modify the transmission characteristics of the fiber and produce an observable output indicating the presence and concentration of each target antigen. Specificity is provided by the careful selection of DNA probes. Sensitivity is obtained by tailoring the optical fiber and thin-film fabrication process and refining the signal processing algorithm. Objective 2: To elucidate key steps in the dynamic interactions between pathogens, host immunity and the environment, and to determine how manipulation of these factors can reduce the risk of BRD CA has collaborated with WA, SD, OK, and the BRD CAP to carry out challenge studies using individual BRD pathogens, for the purposes of characterizing host gene expression in response to infection with individual pathogens. NC1192 input to CAP during 2011 meeting led CAP to increase the number of cattle per group (from n = 2 to n = 6), which all agreed would improve the strength of the research findings. KS evaluated risk factors associated with feedlot BRD incidence and found that factors related to weather, as well as characteristics related to the cohort (month and year of arrival, gender, days on feed, risk designation, and other factors) and distance transported were significantly associated with feedlot BRD. WI continued to investigate extracellular trap formation by bovine leukocytes exposed to M. haemolytica or its leukotoxin (LKT). Bovine monocyte-derived macrophages and alveolar macrophages, but not freshly isolated peripheral blood monocytes, produce extracellular traps (which we term METs) upon exposure to M. haemolytica or its LKT in vitro. Some of the M. haemolytica cells are ensnared within METs and a portion of those cells are killed within METs. Histophilus somnii cells also elicit trap formation by bovine leukocytes in vitro in a dose- and time-dependent manner. WI performed gene microarray analysis of bovine bronchial epithelial cells exposed in vitro to BHV-1, M. haemolytica, or both agents. Preliminary analysis revealed differential regulation (>2 fold, P<0.05) of 978 transcripts by BHV-1 alone, 2040 transcripts by M. haemolytica alone, and 3500 genes by BHV-1 and M. haemolytica in combination. Functional analysis of the microarray data revealed alterations in genes involved in biological processes of cell proliferation, inflammation, cell death, leukocyte migration, and cell surface markers. NE continued research to determine how the BHV-1 LR gene regulates latency and how a viral transcriptional activator (bICP0) stimulates productive while repressing innate immune responses. It was found that the LR gene encodes two micro-RNAs that are expressed during latency, and that both micro-RNAs interact with the RNA sensor, RIG-I, which stimulates the IFN-b signaling pathway. In collaboration with LA, NE showed that bICP27, a viral early protein that shuttles between the nucleus and cytoplasm, inhibits the transcriptional activity of two bovine IFN-² gene promoters (IFN-²1 and IFN-²3) in a dose dependent fashion. These studies provided evidence that bICP27 inhibited IFN-²1 and IFN-b3 promoter activity, thus interfering with the host response to BHV-1 infection. OK and NADC collaborated to evaluate an outbreak of severe BVDV-induced disease occurring in 2 lots of cattle entering a Texas feedlot. Affected animals had severe mucosal lesions in the oral cavity, larynx, and esophagus. Mucosal lesions varied from small (13 mm) infrequent mucosal ulcerations to large (5 mm to 1 cm) and coalescing ulcerations. A calf persistently infected with BVDV arrived with one lot and the isolated virus was genotyped as BVDV1b. Identical BVDV1b strains were isolated from 2 other mortalities. A BVDV2a genotype was also isolated in this outbreak. This genotype was identical to all BVDV2a strains isolated in both lots. OK and NADC assessed the genetic variability of M. haemolytica isolates obtained from fatal BRD cases in the United States (USA) and Australia using REP-PCR and sequencing from the 16s ribosomal sequence. All characterization methods were capable of discriminating between isolates. Modest to moderate diversity was seen amongst the isolates, with as much variation being present within a continent as between the two. This suggests that utilizing samples from diverse origins should permit extrapolation to isolates with distant geographic and temporal relationships. It also suggests that measures effective against the bacterium in one setting can reasonably be expected to be efficacious in another. Further, this information can serve as a baseline in assessing whether M. haemolytica truly is an opportunistic pathogen, or if there are notable features that distinguish commensal isolates from those more likely to be associated with disease. SD and CO collaborated to evaluate the effect of BVDV on innate immunity in the developing fetus. Liver samples were collected at necropsy from fetuses retrieved by cesarean section at gestational day 89, from artificially inseminated heifers. Four of the eight heifers had been inoculated at 75 days in gestation with a type 2 non-cytopathic BVDV strain (96B2222). BVDV antigen was clearly detected in persistently infected tissues in cells primarily located in liver sinusoids and near central veins. Confocal microscopy positively identified the population of cells infected with BVDV in fetal livers as exclusively Kupffer cells. Kupffer cells were not uniformly infected with BVDV at this stage of infection; as uninfected Kupffer cells were also observed in all tissues tested. Kupffer cells are responsible for presenting antigen to lymphocytes that were also present at this critical stage of gestation. In the context of the specific microenvironment of the liver, antigen presentation likely results in systemic tolerance rather than immune activation, contributing to persistent infection. WA serves as the repository for the collection, DNA extraction and storage of 2033 Holstein calf samples (cases/controls) from California and 795 Holstein heifer calf samples from New Mexico as part of the BRD consortium formed from the Agriculture and Food Research Initiative. US Department of Agriculture project 2011-68004-30367 Integrated program for reducing bovine respiratory disease complex in beef and dairy cattle. All of these samples have diagnostic testing for Histophilus somni, Pasturella multicoda, Mannheimia haemolytica, Mycoplasm spp, Arcanobacterium pyrogenes, bovine corona virus, bovine respiratory synctial virus, bovine viral diarrhea virus, and interstitial bovine respiratory virus completed. WA also continues work to identify loci in cattle associated with susceptibility to BRD pathogens in general and also individual pathogens in beef and dairy cattle is ongoing. A feedlot study is beginning this year that will add to 1000 beef cattle to the animal resource populations that the BRD Consortium has been developing. Objective 3: To investigate the mechanisms by which infectious agents work singly or in combination to evade, suppress, or misdirect the host immune response, or to directly induce cellular or molecular pathology, in BRD. CA has evaluated the response of bovine respiratory epithelium to exposure to BRSV and a culture supernatant of H. somni, using microarray analysis of RNA from exposed cells and ELISA. Genes expressed include IL-8, IL-6, prostaglandin synthase, and several matrix metalloproteinases. Results showed good correlation between the results of the microarray and the ELISA, indicating that the microarray accurately predicted expression of the proteins which mediate the inflammatory response to infection. GA recently completed a pilot study to evaluate the impact of gastrointestinal nematode (GIN) parasitism on immune responses to vaccination. In a small study using nursing beef calves, calves with moderate levels of GIN had lower concentrations of SN antibody to BHV-2 at 45 days post weaning and vaccination with a 5-way viral vaccine, as compared to calves with low parasite burdens. Calves with low parasite burdens showed a trend toward increased expression of IFN gamma in response to exposure to BVDV. MI continued work to test a regional BVDV eradication program in the Upper Peninsula (UP) of Michigan. Thru December 31, 2011, 294 (out of an estimated 500 herds in the UP) herds have signed up for the program. In the first five counties, 80% of herds have agreed to participate. Testing has occurred in 232 herds and BVDV PIs have been confirmed in 9 herds (3.9%). Of 17, 917 cattle screened, 24 have been confirmed as PIs (0.13%). One stakeholder biosecurity practice started has been the mandatory BVDV testing of cattle participating in the UP State Fair. SD continued research of the effect of BVDV on dendritic cell antigen presentation. Monocytes were isolated from Holstein Friesians (H.F.) and Brown Swiss (B. Swiss) calves that were 8 months to 1 year of age. Monocytes were differentiated into MDDCs using bovine recombinant IL-4 and GMCSF and confirmed morphologically and phenotypically to be MDDC. The MDDCs had long dendrites and were 5-7 times larger size then monocytes. The cell surface phenotype was CD14-, CD21-, MHCI+, MHCII+, CD86+, DEC205+. 100% of the B. Swiss calves produced MDDCs while only 5.5% of the H.F. were able to generate MDDCs. For MDDCs infection, 4 strains of BVDV were used. No infectious virus production by MDDCs occurred. Interestingly, viral RNA increased in MDDCs through 144 hr after infection. The kinetics of viral RNA production along with the amount of viral RNA was significantly different between viral stains. The study revealed that BVDV replicates in MDDCs but does not produce infectious particles. Accumulation of viral RNA may have significant effects on immune response mounted by MDDCs. VA has continued work to characterize the role of the H. somni biofilm in resistance to host defenses in vitro and in vivo. Following experimental challenge of calves it became clear that Pasteurella multocida, and possibly other organisms, cohabitated with H. somni in biofilms. Therefore, polymicrobial biofilm formation was studied using a drip flow bioreactor (DFR), which can more closely simulate biofilm formation in the lung. A laboratory biofilm was established with H. somni 2336, M. haemolytica A1, and P. multocida A:3. H. somni, M. haemolytica, P. multocida biofilms grown in the DFR resulted in mean viable biofilm log densities of 9.08, 6.9 and 9.56, respectively. When all species were grown together the mean viable biofilm log density was 9.61. In the mixed biofilm, M. haemolytica was not present, and the mean viable biofilm log densities of H. somni and P. multocida were 8.8 and 9.46, respectively. Scanning electron microscopy (SEM) of a mixed biofilm grown in the DFR showed that P. multocida and H. somni co-existed in the same biofilm. Objective 4: To develop management practices, including rationally applied therapeutic and preventative interventions that minimize the impact of BRD on cattle health, welfare and productivity CA is working to develop new vaccine candidates for co-administration to protect against BRSV and H. somni. The selected immunogen for BRSV has been cloned and expressed, and upscale expression for vaccine production is underway. The IbpADR2 from H. somni has been expressed and purified. In a separate study, IbpADR2 has been expressed in microalgae to provide a particulate recombinant antigen for vaccination of calves. GA, KS, NE, and collaborators at 3 other institutions have conducted a survey of cow-calf producers to determine herd-level risk factors for nursing (preweaning) beef calf respiratory disease. Results indicated that certain operation characteristics or management practices were significantly associated either with occurrence of calf BRD or cumulative calf treatment incidence. KS continued work to investigate clinical, behavioral, and pathophysiological data of potential value in detecting the onset and progression of BRD using experimental Mannheimia haemolytica and Mycoplasma bovis pneumonia models. Clinical illness scores (CIS) were assessed in calves challenged with M. bovis and remote triangulation devices recorded the amount of time calves spent in specific areas (near feed bunk, water, and shelter). Calves with more severe disease traveled less distance and spent less time at the hay bunk while spending more time in the shelter. The distance calves traveled was associated with the amount of lung lesion. LA developed a subunit vaccine that can elicit strong antibody response against respiratory bovine coronavirus in immunized animals. This method utilized an initial DNA vaccine encoding either the soluble portion of the spike glycoprotein or the soluble portion of the spike glycoprotein fused in-frame to bovine CD154. Animals responded to vaccination, and fusion of CD154 to the soluble portion of the spike glycoprotein resulted in a pronounced increase in circulating and neutralizing serum antibody specific for the BRCoV spike glycoprotein. LA also developed and tested a vaccine from an envelope protein mutant of bovine herpesvirus type 1 (BHV-1). This vaccine induced higher cytotoxic T cell activity and higher serum neutralizing titers than wild type vaccine. Vesicles formation is a mechanism bacteria use for specific secretion and transfer of macromolecules such as proteins and toxins to animals. OK conducted research to determine whether components of vesicles of M. haemolytica were immunogenic in mice and cattle. LC-MS/MS spectrometric analysis of vesicles from this bacterium identified 226 proteins out of which 104 (46%) were cytoplasmic, 5 (2.2%) cytoplasmic membrane, 1 (0.44%) extracellular, 58 (25.6%) OMPs and periplasmic and 58 (25.6%) unknown. Vesicles from M. haemolytica were used to vaccinate dairy calves and Balb/c mice. Analysis of sera from calves and mice by ELISA showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on day 21 and 28 (p < 0.05) compared to day 0. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (p < 0.05) lower than those from non-vaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed that there was 100% serum bactericidal activity in the presence of complement, while there was no killing activity in control mice calves sera. SD along with CO and investigators in Texas, Illinois, Missouri, and New York are involved in the Genetics of Feedlot Health Project which was performed in 2009 and 2010. This study looked at the impact of behavior, genetics, and nutrition, along with microbiology and immunology, as related to respiratory disease and carcass quality. Analysis of the data is continuing, including assessment of cortisol and its relationship to antibody production and proinflammatory responses Objective 5: To promote open scientific exchange and dialogue among scientists, veterinarians, allied industry professionals and cattlemen to advance BRD research initiatives. Members from WA, CA, and SD are participants in the current USDA BRD Coordinated Agricultural Project (CAP), which is currently focused on research to determine the influence of genetics in BRD. Members from other stations (MI and GA) have advised the CAP in their research and outreach efforts. Members from all participating stations presented abstracts and lectures to scientists, veterinarians, allied industry professionals, and cattlemen at various conferences in the past year. Objective 6: To facilitate the translation of research findings to practical field application by developing and integrating BRD educational programming for national veterinary and producer organizations focused on cattle health and management SD, MI, and GA have communicated with the BRD CAP re planning outreach activities related to BRD prevention and control. A presentation was made at the NCBA Cattlemens College on BRD in Nashville TN in February 2012. Also classes specific on bovine respiratory disease were taught to the advanced group of the Southern Great Plains Dairy Consortium-Teaching at Clovis NM in June 2012 KS is the home of the Beef Cattle Institute (BCI). The purpose of the Beef Cattle Institute is to create a collaborative environment to tackle issues facing the beef industry through education, research and outreach. The institute enhances the education and the value of the degrees of students, increases information access and training opportunities for people working in the beef industry, and improves the cultural and intellectual diversity of our student body. The institute sponsors graduate and undergraduate certificate programs, workshops and seminars, and beef cattle performance and health training. Sponsors and partners include AVMA, KVMA, KLA, Kansas Farm Bureau, AVC, AABP, and many industry partners (Bayer, Merial, Pfizer, Novartis, Boehringer Ingelheim). The diagnosis of bovine respiratory diseases (BRD) poses significant challenges to the clinician as there are numerous infectious etiologies, operating singly or most often in combination. OK investigators recently published a review article that describes the traditional tests and several molecular tests and discusses the benefits and limitations of the tests and their interpretation. This article makes the important point that clinicians should consult with their diagnostic laboratory regarding interpretation of test results. The rate of development and use of molecular diagnostic tests have outpaced validation, standardization, and standards for interpretation.

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