Duration: 10/01/2016 to 09/30/2021

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Bovine respiratory disease (BRD) is the leading cause of mortality for all classes of cattle and calves in the United States (USDA, 2006). In 2006, respiratory disease accounted for 28.7% of all deaths, with losses due to animal death alone costing producers over $692 million annually (USDA, 2006). This estimate does not include cost of medication, labor, and production losses associated with respiratory disease; those costs, which have not been well estimated, likely increase all BRD-related losses to over $1 billion per year (Ishmael, 2001; Kapil and Basaraba, 1997). Thus BRD has a significant impact on profitability of U.S. cattle operations, as well as a significant impact on well-being of U.S. cattle. Recent NAHMS surveys also confirm that BRD is the leading cause of morbidity and mortality in U.S. feedlots (USDA, 2011), the most common cause of nursing dairy calf mortality, and the leading cause of weaned dairy heifer mortality (USDA, 2014). BRD is the result of multiple factors acting in concert. Infection by viral and bacterial respiratory pathogens causes the pathologic lesions characteristic of BRD (Woolums et al., 2009). Viral infection is nearly always the primary infectious insult, with subsequent infection by opportunistic bacterial organisms exacerbating the resulting lung pathology. The viral agents most commonly involved in BRDC include bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza type 3 virus (PI-3V), and bovine respiratory coronavirus (BRCV); the predominant bacterial agents include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis (Fulton et al., 2009a; Gagea et al., 2006). Other agents may be found in isolated cases. When BRD affects a group of cattle, various combinations of multiple viral and bacterial agents are typically involved. However, the viral pathogens of BRD can also be found to circulate in cattle showing few or no signs of disease, and the bacterial pathogens can be identified as part of the normal flora of the nasopharynx in healthy cattle. Since the microbial pathogens of BRD may cause little or no disease, factors other than respiratory infection must be necessary to induce BRD. The additional necessary factors are related to 1) the bovine host’s specific ability to resist disease following respiratory infection and 2) the way cattle are managed, including factors such as mixing animals from multiple sources, transportation, and weaning, castration, and/or dehorning immediately prior to shipment and mixing (Fulton, 2009a; Taylor et al., in press). Thus, BRD needs to be considered a disease complex and must be approached by attacking the multiple etiologies involved in its occurrence. Although BRD is an important cause of cattle morbidity and mortality, research carried out by members of NC-1192 has improved our understanding of some aspects of the problem. Genetic factors associated with BRD susceptibility have been explored with promising results (Zanella et al., 2011; Neibergs et al., 2015). Significant efforts have also been directed at improving clinical BRD diagnosis. NC-1192 members have developed enhance scoring systems to improve BRD diagnosis in dairy calves (Aly et al., 2014; Love et al., 2014). Efforts by many NC-1192 members to characterize virulence factors are ongoing. See more examples in the section “Related, Current and Previous Work”. Although these discoveries have improved foundational knowledge regarding the causes of BRD, and have revealed pathways amenable to mitigating the disease in at least some groups of cattle, gaps in our knowledge of factors that contribute to BRD persist. New pathogens have recently been identified to predominate in BRD in some cattle (Fulton et al, 2009b; Gagea et al, 2006), but it is not known whether these agents have gained a foothold because of particular pathogenic mechanisms or because of changes in management that predispose cattle to infection by them. Rapid identification of pathogens, ideally at “chute-side”, could help manage cattle with BRD in ways to better limit spread of disease, but rapid tests for BRD pathogens are as yet not available. Management of co-infections, such as gastrointestinal parasitism, may impact the ability of cattle to respond to respiratory infection, but little research has yet evaluated the impact of parasitism or other non-respiratory infections on BRD. While vaccines that improve the immune response to BRD are widely used and can be efficacious, factors which limit their impact need to be addressed; among these include the fact that vaccination is sometimes but not always effective in calves with circulating maternal antibody, and for some infectious agents, vaccines are not available, or have not been confirmed to be effective in the field. These and other knowledge gaps will be addressed by research in the proposed NC-1192 project. The importance of the work described is that the combined effort of members of NC-1192 is the most broad, collaborative, and multidisciplinary effort to ameliorate BRD in North America. If the described research is not undertaken, it will not be possible for veterinarians and producers to develop science-based approaches to minimize or prevent BRD in cattle managed under modern U.S. husbandry practices. While the work described is technically and logistically challenging, scientists at the participating stations have the necessary experience and skills to carry out the proposed research. The group will consist of a team of researchers with a broad range of experience with the techniques necessary to undertake the research. Participating researchers possess the necessary contemporary skills in molecular biology, immunology, virology, bacteriology, and animal management to carry out research to test relevant hypotheses and to then develop science-based integrated diagnostic and preventative strategies. Ongoing progress of useful research by NC-1192 can be improved by interaction with researchers outside NC-1192, including those in government laboratories and in the pharmacologics/biologics industry, and with veterinary practitioners, cattle producers, and other stakeholders. Members of NC-1192 have worked hard to develop relationships with national organizations to allow for substantial dialog to occur regarding the unanswered questions and persistent problems related to BRD. This “two way street” has helped researchers understand ongoing and new challenges that the industry faces while at the same time allowing us to articulate challenges that researchers face in conducting relevant research that meets the industry’s needs. This open dialog has in part been responsible for increased funding being made available to fund much needed BRD research. Ultimately, the value of the research proposed here is most significant only if it is translated from research discovery to field application. NC-1192 prides itself in being a primary source of information about BRD for veterinarians and producers. Its research effort helps to provide better surveillance of the causes of BRD, improves understanding of the complex molecular events involved in BRD polymicrobial infections, visualizes and tests new management strategies, and articulates a cutting-edge team approach that coordinates new knowledge with validated practices. Through integrated efforts, we will facilitate the dissemination of this information, as well as that developed by other BRD initiatives, to the beef and dairy industry where is can be applied. Impacts of the proposed research will include: 1. Veterinarians and cattle producers will have access to science-based recommendations for the control and prevention of BRD in cattle managed in modern U.S. production systems; 2. Researchers in academic, government, and industry laboratories will be provided with basic foundational and applied information regarding BRD that will be necessary for their their ongoing work to advance scientific discovery in the fields of vaccinology, immunology, microbiology, pharmacology, and animal husbandry; 3. Scientists, veterinarians, and policy makers working to minimize unnecessary use of antimicrobials will be provided with basic and applied information regarding methods to enhance resistance to BRD by maximizing the use of vaccines and management strategies that will minimize the need for antimicrobials; 4. Scientists, educators, and policy makers will be provided with cutting-edge information regarding the mechanisms by which cattle develop BRD, and regarding science-based methods to minimize or prevent the impacts of BRD; 5. Veterinarians and cattle producers will be regularly educated regarding both new developments in the science of BRD, and in rational and practical methods to limit the impact of BRD in U.S. cattle.

Related, Current and Previous Work

Members of NC-1192 identified several knowledge gaps related to the objectives set forth in the current multistate project. Information has been generated to address these knowledge gaps and is presented here. 1. The role of biofilm formation in disease due to bacterial respiratory pathogens will be determined; this will form the basis for improved methods to counteract chronic respiratory disease, which is becoming more prevalent in feedlot BRD and is an ongoing problem in calf BRD. VA showed that Histophilus somni forms biofilm in vitro and also in the bovine host; this was associated with upregulation of genes related to exopolysaccharide production. Importantly, after cattle were challenged with H. somni, biofilm including P. multocida was identified. This indicates that these two BRD pathogens collaborate to establish infection in the host. Some isolates of P multocida were better able to form biofilm than others; H. somi enabled poor biofilm forming strains of P. multocida to co-establish in the biofilm. This work revealed new mechanisms by which bacterial pathogens can establish in the bovine host and evade immunity. Therapeutic targets aimed at blocking the ability of bacteria to form biofilms could help the bovine host resist infection by these bacteria (55, 56, 57, 61). 2. Biosensors for field or chute-side diagnosis of BVDV and other pathogens will be further refined, addressing a repeatedly stated need by producers for improved ways to rapidly identify BRD pathogens. MI evaluated patient-side diagnostic kits for human respiratory syncytial virus (HRSV) detection for use in detecting bovine respiratory syncytial virus (BRSV), using BRSV isolates obtained from GA and other sites. Kits were shown to identify BRSV, with the Tru RSV assay showing the best agreement in preliminary testing. On field samples, the Tru RSV assay was in agreement with RT-PCR 40 of 52 times (k=0.490) (71). VA is working 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. 3. The use of clinical and laboratory tests to more quickly and accurately diagnose BRD and to characterize the prognosis of affected cattle will be refined, to allow improved diagnosis and informed guidance for decision making regarding the best management of BRD affected cattle. MI completed work indicating that testing of blood collected from cattle at slaughter by the bovine TB gamma interferon ELISA was feasible and provided accurate information regarding the TB exposure status of cattle at the plant, a major point of cattle concentration. This has the possibility of improving control and eradication of bovine TB (53, 54). CA completed and published an enhanced BRD scoring system based on dichotomous variables that is designed to improve feasibility for on-farm use (46). VA developed an ELISA to detect bovine antibodies to H. somni exopolysaccharide (55). MO has demonstrated an association between clinically diagnosed field cases of BRD and increases in lipopolysaccharide binding protein and haptoglobin in feedlot cattle. Transferrin concentrations were not significantly associated with BRD status (43). 4. The methods by which viral and bacterial pathogens circumvent the host immune response will be elucidated. While it has been well established that BRD pathogens can circumvent host immunity, the details of the mechanisms are not very clear, impairing the possibility of applying new developments in treatment and prevention of immune dysfunction to be applied to BRD. NE in collaboration with LA completed research indicating that the BHV1 ORF2 product promotes BHV1 latency by promoting survival of infected neurons and through DNA binding, which increases the half-life of the ORF2 product. Work was also completed showing that the viral VIP 16 and bICP0 are activated in a dexamethasone model of stress-induced latency reactivation. 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-? 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-? gene promoters (IFN-?1 and IFN-?3) in a dose dependent fashion. These studies provided evidence that bICP27 inhibited IFN-?1 and IFN- ?3 promoter activity, thus interfering with the host response to BHV-1 infection (26, 28, 29, 58, 64, 65, 75, 82). OK and NADC identified bovine coronavirus (BoCV) from outbreaks of BRD, and sequenced a region of the envelope Spike protein genome sequenced. This showed that BoCV from respiratory disease outbreaks were from a newly identified clade, clade 2; this is in contrast to BoCV in the licensed BoCV MLV vaccine and the historical enteric isolates, which were BoCV clade 1. New clade isolates may be causing disease by escaping host immunity established to clade 1 isolates. SD completed research showing that cytopathic (CP) and noncytopathic (NCP) BVDV induce autophagy in infected cells, and that suppression of autophagy decreased viral replication. Work also showed that BVDV strains vary in their effect on NK cell activation, with more virulent BVDV strains inducing different profiles of cell surface marker expression than less virulent strains. MS, SD, and TX evaluated the impact of BVDV on antigen cell presentation. MS used comparative protein profiling approach to elucidate the mechanisms involved in BVDV interference of monocyte and dendritic cell antigen presentation, which allows the virus to avoid effective recognition and elimination by innate and adaptive immune responses. This research confirms that low doses of BVDV significantly affect early apoptotic and oxidative stress mechanisms in cp and ncp BVDV-infected monocytes compared to control cells. A culture system for producing mature dendritic cells in culture has been optimized, allowing evaluation of antigen presentation. This model system will allow assessment of differences in autophagosome trafficking by cp and ncp BVDV, as well as other pathogens. The impact on dendritic cell activation means that immunity at the earliest stages of the host response are impaired by BVDV infection . WI continued research to determine how neutrophil extracellular traps are induced by H. somni and M. hemolytica. WI also showed that BHV-1 infection of respiratory epithelial cells led to release of factors that impaired formation of extracellular traps in some but not all circumstances, and that macrophages can remodel extracellular traps by use of DNAse II (5, 41). WI also evaluated the synergistic effects of BHV-1 and M. haemolytica infection in bovine bronchial epithelial cells through gene microarray analysis. 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. Three hundred and fifty five genes exhibited comparable expression profiles following virus or bacterial exposure, suggesting that gene regulation by virus exposure is restricted to a small set of specific targets. Co-exposure to BHV-1 and M. haemolytica altered the viral response in a synergistic or antagonistic manner, consistent with our previous findings. BHV-1 induction of expression of IFN-y, IL-1B, IL-1?, IL-8, and Tnf-? was confirmed by RT-PCR. By comparison, M. haemolytica treatment produced significantly greater inductions (>10 fold) of several genes, including CXCL2, PTX3, IL6, IL1A, SERPINB2, and IL8, compared to BHV-1 alone. Surprisingly, co-exposure to BHV-1 and M. haemolytica resulted in a switch from repression to activation for BHV-1 repressed genes such as Cxcl10. 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 (52). 5. The nature of optimally effective and safe vaccines will be better characterized, with particular focus on live vaccines for mucosal (intranasal) administration that are stable and effective. This effort will ensure the progression of new developments in BRD vaccinology based on the most recent knowledge regarding mucosal immunity, providing veterinarians and producers with the most effective tools to prevent BRD. 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 (1). 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 (48). In a pilot study with 20 cattle per treatment group, high risk stocker cattle that received vaccination (MLV 5-way viral and clostridial) at arrival were more likely to be treated for BRD and more likely to die of BRD over an 84-day conditioning period, as compared to cattle that were not vaccinated. Cattle vaccinated at arrival, cattle with high or moderate (103 – 103.9 F) fever at arrival, and cattle treated for BRD gained significantly less weight over an 84-day conditioning period than cattle without these factors. 6. The means by which coinfections among BRD pathogens, or between BRD pathogens and other agents, such as gastrointestinal nematodes, impact the occurrence and outcome of BRD, will be determined. This effort will lead to foundational knowledge regarding the mechanisms of disease and recovery in polymicrobial infection, and to identification of new targets for intervention on the molecular, cellular, whole animal, and herd level. GA 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. In a follow-up to the pilot, MS found that deworming at arrival decreased fecal egg count in treated cattle but was not found to be significantly associated with BRD morbidity, mortality, or weight gain. Presence of a high fever (? 104 F) or gastrointestinal nematode parasitism (as measured by fecal egg count) at arrival were associated with increased risk of treatment for BRD. VA has worked 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. WI conducted research to determine how bovine respiratory epithelial cells respond to infection with BHV-1, M. haemolytica, or both pathogens together, and showed that cells produced a different pattern of inflammatory mediators when they were infected with both pathogens, as compared to either pathogen alone (52). 7. The concept of regional voluntary programs to eliminate specific BRDC pathogens will tested be tested in the Michigan Upper Peninsula BVDV Project. MI continued to work on a bovine viral diarrhea virus (BVDV) eradication program in the Upper Peninsula (UP) of Michigan. Research suggests a minority of herds have PI animals present in them. Elimination of these animals from this low percentage of herds has potential to greatly impact BVDV control in the US. To date (July 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 PI’s have been confirmed in 9 herds (3.9%). Of 17, 917 cattle screened, 24 have been confirmed as PI’s (0.13%). One stakeholder biosecurity practice started has been the mandatory BVDV testing of cattle participating in the UP State Fair. A benchmark goal for the program is to have tested for and controlled BVDV in 80% of the beef and dairy herds and 95% of the cattle in the UP by 2012 (35). In addition to research activities, NC-1192 has participated in significant outreach activities. Members of NC-1192 organized and hosted the 2nd Bovine Respiratory Disease Symposium (July 30-31, 2014) in Denver, Colorado in conjunction with the Academy of Veterinary Consultants summer meeting. Presentations at the symposium covered topics including the current state of BRD in beef and dairy cattle, clinical presentation and diagnosis of BRD, influence of genetics on BRD susceptibility, recent research findings related to pathogenesis, and management of BRD in production systems. Dissemination of information to interested parties occurred by 3 major means: 1) printed proceedings were distributed to all attendees and made available to several interested parties who requested them after the Symposium; 2) after the Symposium the Proceedings were published in Animal Health Research Reviews (December 2014). As Animal Health Research Reviews is searchable via PubMed and other biomedical research databases, the Symposium proceedings are thus accessible worldwide. Other outputs included a website (www.brdsymposium.org) where information regarding the Symposium was posted, and many of the speakers’ presentations were made available free for viewing. While the Symposium itself was the most significant output of the project, the proceedings published in Animal Health Research Reviews may eventually reach even more individuals interested in the prevention and control of bovine respiratory disease.

A search of the CRIS database revealed no other multistate research projects focused on bovine respiratory disease. While other projects focus on respiratory disease in other species, cattle are unique from those species in important anatomic, physiologic, and management aspects; therefore research in other species has very limited value in cattle. The CRIS search did reveal several individual approved research projects that have some degree of overlap with the multistate project proposed here; however, those individual project leaders are also participants in the NC-1027 multistate research effort. Finally, an annual CRIS database search will be conducted to insure that future duplication does not occur between other multistate research projects and the NC-1027 project.

Objectives

1. To elucidate pathways by which host characteristics, pathogen virulence mechanisms, and environmental impacts interact to produce BRD, and to develop strategies to mitigate detrimental factors and enhance protective mechanisms.
   
2. To develop and validate methodologies for accurate BRD diagnosis, objective risk assessment, and surveillance to detect new trends in BRD occurrence.
   
3. To develop and validate management practices and responsibly applied therapeutic and preventative interventions, such as vaccines, antimicrobials, and immunomodulators, to minimize the impact of BRD on cattle, producers, and society.
   
4. To determine how attributes of cattle production systems including epidemiologic, societal, and economic forces contribute to BRD, and to develop ways to catalyze change in those systems to reduce the occurrence of BRD and improve cattle health, welfare, and productivity.
   
5. To promote dialogue and exchange among scientists, veterinarians, allied industry professionals and cattle producers to advance BRD research initiatives, to implement outreach, to disseminate research results, and to facilitate the translation of research findings to practical field applications.
   

Methods

Objective 1:To elucidate pathways by which host characteristics, pathogen virulence mechanisms, and environmental impacts interact to produce BRD, and to develop strategies to mitigate detrimental factors and enhance protective mechanisms. MS and SD will characterize host immune responses to infection. MS will collect blood samples for isolation of RNA from white blood cells. Cellular RNA will be subjected to deep sequencing of messenger RNA (RNA seq) to evaluate relative expression of genes related to immune response, inflammatory state, metabolism, and other physiologic functions. Gene expression in cattle that complete a 60-day backgrounding period without being treated for BRD will be compared to that in cattle that are treated for BRD once, and cattle that are treated for BRD three times. Differences in gene expression between these populations will be assessed to determine whether major changes in expression of any gene is related to likelihood of completing the 60-day backgrounding period without developing BRD. SD will confirm findings in previous pilot work. In collaboration with WI they will measure the effect of BVDV strains on the neutrophil, NK and macrophage cytokine expression including TNF-?, interleukin-1? (IL)-1?, interleukin-8 (IL-8) and interleukin-6 (IL-6) through quantitative reverse transcription polymerase chain reaction (q-RT-PCR), and measure the effect of BVDV strains (cp and ncp) on neutrophil and macrophage phagocytic activity. SD will also measure the effect of BVDV strains on the macrophage’s and neutrophils bacterial killing ability and oxidative burst. The effects will be measured using different virulence strains of BVDV. SD will assess the role of dietary fiber in inflammation. The study will consist of 10, 150-200 kg Holstein or Holstein-Jersey steers. The entire experiment will be repeated using 4-5 animals/replicate (4-5 animals X 2 replicates= 8-10 animals). The intestinal-lymph cannulation (ILC) surgery will be performed on all the animals. These animals will be used in three experiments. First, lymph and microbiome profile will be determined in cattle fed a diet based on soy hulls, a rumen-friendly digestible fiber source. Second, lymph and intestinal microbiome profile will be determined in cattle fed conventional corn/soybean based diet. Third, the effect of inclusion of beta-glucans in the conventional corn/soybean based diet on the lymph and microbiome profile will be determined. MS will confirm findings in previous pilot work that indicated that GIN parasitism increased BRD risk in stocker cattle in trials in 2 consecutive years. Sale barn derived stocker cattle (n = 80 per trial) will be procured and assigned to treatment groups: 1) dewormed (fenbendazole and levamisole) and vaccinated (MLV 5-way, clostridial); 2) dewormed only; 3) vaccinated only; 4) not dewormed, not vaccinated. Fecal samples for measurement of FEC will be collected at arrival and every 28 days. Cattle will be monitored daily for 60 days for signs of BRD using a standardized DART (depression, appetite, respiratory, temperature) scoring system. Individuals assessing cattle health will be unaware of the treatment status of cattle. Cattle with signs of BRD will be treated with industry standard practices. Weights will be measured every 28 days and serum neutralizing antibody titers to BHV-1, BRSV, and BVDV1 will be measured at arrival and every 28 days. All cattle will be vaccinated on day 56 in order to determine whether cattle vaccinated at arrival establish a memory response that leads to an anamnestic response at arrival. Outcomes to be assessed will be BRD morbidity and mortality, total morbidity and mortality, ADG, and total pounds gained. MO will investigate the hydrogen peroxide/lactoperoxidase/halide ion system found in the bovine respiratory tract and its impact on common respiratory pathogens. Preliminary findings indicate administration of sodium iodide to calves via orogastric intubation can achieve biologically significant levels of iodine in airway secretions. Further investigation will be directed at evaluating the impact of different methods of oral administration and forms of iodine on achieved concentrations in airway fluid. KS, VA, and WI will investigate virulence factors of BRD pathogens. KS will genotype and produce antimicrobial sensitivity profiles of Mannheimia haemolytica isolates obtained from a variety of sources, including nasal and lung isolates from both normal and pneumonic cattle, using pulsed field gel electrophoresis and standard antimicrobial sensitivity testing, respectively, to determine the genetic and antimicrobial resistance characteristics and variation among virulent and non-virulent isolates. VA will use flow cell cultures to develop biofilms in vitro and analyze them using confocal and electron microscopy. VA will also use allelic mutagenesis to make H. somni mutants that cannot make exopolysaccharide (EPS), necessary for biofilm formation. Virulence of these mutants will be screened in a mouse model and confirmed by bovine respiratory challenge. Bovine isolates of P. multocida will be used to evaluate interactions with H. somni mutants. VA will also investigate the role of small RNA in regulation of EPS and biofilm formation. WI will explore the ability of bacterial BRD pathogens to attach and form a biofilm, in an effort to gain insights into mechanisms by which these organisms colonize the upper respiratory tract in healthy cattle. Using a cell culture method to assess biofilm formation by M. haemolytica and P. multocida on bovine respiratory epithelial cells, WI will investigate how surface components of the bacterial cells affect attachment and biofilm formation and whether viral infection alters biofilm formation on the respiratory epithelium. WI will explore potential interactions among bacterial species (i.e. M. haemolytica and P. multocida) that affect biofilm formation. WI will also assess the interactions among endothelial cells, leukocytes and platelets in response to Histophilus somni. Prior studies indicate that H. somni causes changes in endothelial cells and platelets that contribute to a procoagulant environment. These changes in turn could lead to the thrombus formation commonly seen during H. somni infection in vivo. KS will investigate effect of environmental (eg: ambient temperature) and management (eg: shipping practices) factors on the incidence of BRD will be evaluated using prospective experimental infections using either M. haemolytica or Mycoplasma bovis and retrospective epidemiological studies using large feedlot populations. Objective 2: To develop and validate methodologies for accurate BRD diagnosis, objective risk assessment, and surveillance to detect new trends in BRD occurrence. KS and MO will work to develop tools for diagnosis of clinical BRD. KS will compare clinical, physiological and behavioral characteristics of normal compared to experimentally infected (M. haemolytica and M. bovis) cattle to determine those parameters associated with increased risk or resistance to BRD. These parameters will be evaluated by a clinical illness score (CIS) system, serum biochemical markers, and a combination of pedometer, accelerometers and GPS monitoring. MO is developing a statistical process control model that includes several aspects of feeding and locomotion behavior to objectively identify ill cattle in a production setting. MO is also investigating several biomarkers, including acute phase proteins and blood gas parameters, which may be used to differentiate clinical BRD from other common bovine illnesses. KS and SD will continue to develop and use diagnostic testing methods to survey field case submissions and research specimens for BRD pathogens. 3. To develop and validate management practices and responsibly applied therapeutic and preventative interventions, such as vaccines, antimicrobials, and immunomodulators, to minimize the impact of BRD on cattle, producers, and society. GA will compare the effect of intranasal and subcutaneous modified-live virus booster vaccine on systemic and mucosal immune responses in beef calves that were primed with intranasal vaccine at 1-4 weeks of age. Calves will be assigned to either intranasal or subcutaneous vaccination groups and given modified live BHV1, BRSV, and PI3 vaccines via their assigned route. Subcutaneous MLV BVDV vaccine will be administered to all calves. Calves will be physically separated to prevent transmission of vaccine virus between groups. Blood and nasal secretions will be collected. Blood samples will be analyzed for total IgG concentrations and antibody titers against BHV1 and BRSV. Nasal secretion samples will be analyzed for total IgA and IgG concentrations as well as BHV1-specific IgA and IgG concentrations. Nasal secretion samples will also be analyzed for IFN-? and ? concentrations. GA will assess the impact of the addition of Omnigen-AF to feed on the recall proliferation and cytokine response against BHV-1 and BVDV antigens by peripheral blood mononuclear cells following booster vaccination. Omnigen-AF has been examined for its capacity to enhance antibody responses to vaccines with mixed success. Antibody titers represent the “history” of the response. It is proposed here to see if Omnigen-AF will alter the development of T-helper memory response after vaccination by measurement of T-helper proliferation (relative quantity of circulating memory cells) and the context of helper memory (by measuring the quantity and pattern of recall cytokine production). Memory context can indicate expansion of Th1 (IFN-gamma), Th2 (IL-4), Th17 (IL-17) or Treg (IL-10 and TGF-beta) activity by the vaccine. Omnigen-AF may provide enhanced memory cell development or allow for longer memory activity because of interaction between enhanced innate cells, their products and lymphocytes that provide adaptive function. Differential cytokine quantity and/or pattern may be affected by Omnigen-AF and as a result the level of clonal activation and expansion enhanced. However, up to this point, this hypothesis has not been tested. In this study, animals that have had a priming dose of vaccine will be examined for their booster response to the same vaccine. All animals in the study will be demonstrated to have been primed or will be primed with the vaccine prior to initiation of feeding the Omnigen-AF or control diets. Both humoral (SN titer) and cell-mediate recall responses will be assessed in this study. This study is designed to be a minimal proof of concept study. Heifers will be placed on a standard TMR diet or that diet supplemented with 9gm/100kg of Omnigen-AF for the four months of the experiment. Heifers will receive priming vaccination at about 4-5 weeks prior to the initiation of feeding and sample to determine that all have comparable baseline response to the vaccine antigens. Following a 3.5-month preconditioning period, heifers will receive a booster vaccine, and responses will be monitored for a 2 month feeding period. GA will evaluate the effect of injectable trace minerals on the onset of protection from bovine viral diarrhea virus (BVDV) acute infection induced by a MLV vaccine in newly received stressed beef calves. Calves seronegative to BVDV will be assigned to one of three treatments: MLV BVDV vaccine with a trace mineral injection, MLV BVDV vaccine alone, and unvaccinated control group. All groups will then be challenged 5 days post-vaccination with moderately virulent BVDV-2. Blood samples will be collected to assess leukocyte counts, cortisol and cytokine concentrations, and BVDV antibody titers. Nasal secretions will be sampled to assess viral shedding and IFN-?, ? and ? concentrations. Liver biopsies will be collected to assess mineral status of calves. MS will assess of the impact of on-arrival vaccination on BRD rates in high risk stocker cattle. To extend previous work to assess the impact of on-arrival vaccination on BRD, sale barn derived stocker cattle (n = 80 per trial) will be procured and assigned to treatment groups: 1) dewormed (fenbendazole and levamisole) and vaccinated (MLV 5-way, clostridial); 2) dewormed only; 3) vaccinated only; 4) not dewormed, not vaccinated. Cattle will be monitored daily for 60 days for signs of BRD using a standardized DART (depression, appetite, respiratory, temperature) scoring system. Individuals assessing cattle health will be unaware of the treatment status of cattle. Cattle with signs of BRD will be treated with industry standard practices. Weights will be measured every 28 days and serum neutralizing antibody titers to BHV-1, BRSV, and BVDV1 will be measured at arrival and every 28 days. All cattle will be vaccinated on day 56 in order to determine whether cattle vaccinated at arrival establish a memory response that leads to an anamnestic response at arrival. Outcomes to be assessed will be BRD morbidity and mortality, total morbidity and mortality, ADG, and total pounds gained. MS will assess a parapox vectored BVDV vaccine. To extend previous work done at SD a parapox vector containing BVDV E2 antigen will be used. Serum neutralizing antibody titers to BVDV1 will be measured at arrival; the cattle will be vaccinated with a single dose of vaccine and antibody response measured every 7 days. At day 56 cattle will be challenged with a heterologous BVDV strain and clinical signs and virus production monitored. MO and CA will work with AL to further assess the efficacy of oral iodine administration as a preventive and/or therapeutic control strategy for BRD. Preliminary in vitro susceptibility assays have been conducted with promising results. Further in vitro studies and field-based clinical trials will be conducted. KS will evaluate the efficacy of various antimicrobial agents for their effectiveness against BRD and to determine their optimal conditions for use. These studies will involve administration of antimicrobials under various conditions in conjunction with experimental infection with M. haemolytica. Outcomes will be determined based on using CIS, physiological and behavirol parameters. MS will assess the effect of feeding tilmicosin without metaphylaxis on BRD in high risk cattle. To extend previous work to determine the impact of tilmicosin feeding in conjunction with ceftiofur metaphylaxis on BRD in high risk stocker cattle, MS investigators will evaluate the impact of tilmicosin feeding without metaphylaxis on BRD occurrence. Four hundred and eighty crossbred beef heifers (approximate arrival weight = 420 lbs) will be assembled by an order buyer in Waynesboro, TN. Following assembly (2-4 d) cattle will be vaccinated against respiratory and clostridial pathogens and assigned a uniquely numbered ear tag. Following processing cattle will be shipped 392 miles to the MAFES White Sand Branch Unit outside of Poplarville, MS. Cattle will be offloaded, weighed in pen scale (groups of 10) and 20 head were randomly placed into receiving pens, where they will have free choice access to a 73% concentrate diet and water. The ration will be formulated to exceed NRC (2000) nutrient requirements for lightweight beef calves. Calves will be monitored daily for symptoms of BRD according the the clinical scoring system described by Perino and Apley (1998), and calves with a score > 1 will be removed from the pen and taken to the working facilities where a rectal temperature (RT) will be determined. If RT is 40oC or greater, the animal was considered morbid. Morbid animals will be treated with enrofloxacin, and if symptoms (clinical score and RT) are still persistent 72 h following treatment the animal will receive florfenicol. Seventy two h after the 2nd treatment, if the animals has not responded based upon clinical score and RT, it will be treated with oxytetracycline. Finally, if the animal is still displaying symptoms 48 h after oxytetracycline treatment, it will be removed from study and classified a chronic. When total group morbidity reached 10%, calves will be individually weighed and randomly assigned to pens (the animals defined as morbid will be randomized across treatments as well). There will be two treatments evaluated, a receiving ration with no added tilmicosin (CON) or a receiving ration with added tilmicosin (TIL) at the level of 568 to 757 g/ton (to be determined once intake is established). Rations will be formulated to be similar in nutrient composition. Calves will be monitored for BRD; a new episode will be defined as an animal displaying signs 21 days since its last antibiotic therapy. Tilmicosin will be fed to the TIL group for 14 per label indications, after which all animals will receive the same diet. On d 28, cattle will be weighed for an interim weight, and on d 56 a final weight will be collected. Cattle will be sold to a commercial feedlot and feedlot performance (daily gain, morbidity and mortality) will be collected from the feedlot. Performance data (DMI, ADG, and feed conversion) as well as health data (morbidity, mortality, treatment success, and new episodes) will be the variables of interest. 4. To determine how attributes of cattle production systems including epidemiologic, societal, and economic forces contribute to BRD, and to develop ways to catalyze change in those systems to reduce the occurrence of BRD and improve cattle health, welfare, and productivity. KS will evaluate management practices within the feedlot industry to determine those factors which are associated with either increased or decreased disease. Parameters to be included in large group statistical analysis include season, shipment time, processing protocols, weight gain and loss, treatment and treatment costs, among others. MS will further refine system dynamics models they have developed to improve the ability of the models to identify points in the cattle marketing system that are amenable to manipulation to improve cattle health. Alternatively, improved models might allow more accurate prediction of when BRD rates are likely to increase, or when certain management changes might best be applied to limit BRD. MO will add to the work proposed by MS by investigating factors that motivate producers to manage cattle. Many different levels of management can be found throughout the beef industry and further work is required to understand why producers ultimately make choices that either prevent or contribute to BRD. If appropriately understood, societal changes may be implemented to reduce management styles that contribute to BRD. 5. To promote dialogue and exchange among scientists, veterinarians, allied industry professionals and cattle producers to advance BRD research initiatives, to implement outreach, to disseminate research results, and to facilitate the translation of research findings to practical field applications. KS will continue to maintain the Beef Cattle Institute (BCI; beefcattleinstitute.org). A major component of the BCI is to address those issues associated with BRD, including prevention, antimicrobial resistance, animal welfare, and sustainability. The BCI increases information access and training opportunities for students and others working in the beef industry through research and practical engagement, in order to provide beef producers with the most current education, research and outreach available. The next BRD Symposium will be held in 2019. Collaborating scientists in NC-1192 will work together to plan the Symposium; it is likely that some investigators from several participating stations will be invited to speak, or will be presenting the results of research at the poster session.

Measurement of Progress and Results

Outputs

• Improved basic knowledge of host characteristics, pathogen virulence mechanisms, and environmental factors that contribute to the development of BRD.
• Assays, scoring systems, biometric measurements, and models that accurately detect and diagnose BRD in production settings.
• Prevention strategies, vaccination, and therapeutic technologies that effectively decrease the incidence and impact of BRD.
• Models that describe how and why cattle are managed in ways that either prevent or predispose cattle to developing BRD as well as new strategies to encourage producer adoption of techniques and technologies that reduce BRD incidence and impact.
• Peer-reviewed articles, lay articles, posters, presentations, and symposia to communicate the results of research conducted by members of NC-1192.

Outcomes or Projected Impacts

• Veterinarians and cattle producers will have access to science-based recommendations for the control and prevention of BRD in cattle managed in modern U.S. production systems
• Researchers in academic, government, and industry laboratories will be provided with basic foundational and applied information regarding BRD that will be necessary for their their ongoing work to advance scientific discovery in the fields of vaccinology, immunology, microbiology, pharmacology, and animal husbandry
• Scientists, veterinarians, and policy makers working to maximize judicious use of antimicrobials will be provided with basic and applied information regarding methods to enhance resistance to BRD by maximizing the use of vaccines and management strategies that will minimize the need for antimicrobials
• Scientists, educators, and policy makers will be provided with cutting-edge information regarding the mechanisms by which cattle develop BRD, and regarding science-based methods to minimize or prevent the impacts of BRD
• Veterinarians and cattle producers will be regularly educated regarding both new developments in the science of BRD, and in rational and practical methods to limit the impact of BRD in U.S. cattle

Milestones

(0): Objective 1: MS, MO, and SD have projects planned for 2016, 2017, and 2018. • Objective 2: MO has diagnostics research planned for years 2016-2020. KS has ongoing research planned. • Objective 3: GA, MS, MO, and SD have research related to BRD management planned for 2016, 2017, and 2018. • Objective 4: KS, MS, and MO will have ongoing research related to objective 4. • Objective 5: All stations will report finds through various outlets on a continuous basis. The 3rd BRD symposium will be held in 2019.

Projected Participation

View Appendix E: Participation

Outreach Plan

Basic information generated will be disseminated through peer-reviewed journals. Members of NC-1192 have a tremendous history of publishing their work in peer-reviewed journals, thus helping to advance the science of BRD globally. To extend our impact, an annual contribution to the peer-reviewed literature will be a review of important aspects of BRD co-authored by members of NC-1192. Just as importantly, NC-1192 team members have actively been involved in contributing to the lay literature. Dissemination of information at the applied level will continues through lay publications will continue to be a focus of this project
Annual presentations by team members at annual meetings of the AAVLD-USAHA, CRWAD, AABP and AVC (and others) will aid in the dissemination of information to veterinarians, cattle producers, and researchers, as well as technology transfer of the latest validated diagnostic methods to US veterinary diagnostic laboratory network.
NC-1192 will continue to create dialog surrounding BRD by engaging the industry at various levels. The primary point of engagement will be involvement with national organizations including the USDA, AABP, AVC and NCBA. This will be accomplished by actively participating in and creating open dialog opportunities with these organizations. As a culmination of these efforts, the 3rd “National BRD Symposium” will be planned and held during the course of the project.

Organization/Governance

The Technical Committee will consist of one voting member from each cooperating station as appointed or otherwise designated by that station, with an Administrative Advisor. A President and Secretary of the Technical Committee will be elected by a majority vote of the committee; each will serve a one-year term. Annual meetings will be held at a time and site agreed on by the Technical Committee, with the majority of meetings held in conjunction with a national meeting of an organization related to bovine health (such as the American Association of Bovine Practitioners or the Academy of Veterinary Consultants). At the annual meeting, the Secretary will record the minutes and submit them to the Technical Committee for approval. The President and Secretary will prepare the annual report summarizing material supplied by the voting member from each participating station and, following approval of the report by the Technical Committee, will submit the report to the Administrative Advisor for dissemination to the appropriate parties.

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Attachments

Land Grant Participating States/Institutions

AL, AR, CA, CO, GA, IA, IL, KS, LA, MO, MS, NE, OK, SD, TN, TX, WA, WI

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med, Mississippi State University, USDA-ARS/NE, USDA/NADC, West Texas A&M University