Duration: 10/01/2011 to 09/30/2016

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, 2000), the second most common cause of nursing dairy calf mortality, and the leading cause of weaned dairy heifer mortality (USDA, 2002).

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 hosts 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-1027 has improved our understanding of some aspects of the problem. For example, it has been shown that vaccination for respiratory pathogens and administration of antimicrobials prior to shipment or on feedlot arrival can decrease respiratory disease in calves at high risk for the problem (Frank et al, 2002). Other research has shown that, in contrast to long established dogma, calves with circulating levels of maternal antibodies can be successfully vaccinated against respiratory pathogens, leading to improved resistance to disease when maternal antibodies have waned (Zimmerman et al, 2006). Studies have revealed a predominance of new patterns of microbial infection in BRD (Fulton et al, 2009b); evidence that some pathogens can modulate the immune response to other organisms, leading to disease of enhanced severity (Gershwin et al, 2005); and the discovery that a key bacterial pathogen can exploit a critical receptor on host white blood cells to induce cell suicide and a destructive inflammatory cascade (Jeyaseelan et al, 2002). See more examples in the section Related, Curent 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-1027 project.

The importance of the work described is that the combined effort of members of NC-1027 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-1027 can be improved by interaction with researchers outside NC-1027, including those in government laboratories and in the pharmacologics/biologics industry, and with veterinary practitioners, cattle producers, and other stakeholders. Members of NC-1027 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 industrys 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-1027 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

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). In most cases, primary viral infection is followed by secondary bacterial infection, with viral pathogens typically modulating the host immune response or damaging airway epithelial cells to predispose the host to the secondary bacterial infection. Bacterial pathogens also produce virulence factors that contribute to respiratory pathology and counteract the host immune response, enhancing their persistence. While viral and bacterial respiratory pathogens have numerous strategies by which they establish infection and cause respiratory disease, factors other than respiratory infection are also necessary to induce BRD. The additional necessary factors are related to 1) the bovine hosts specific ability to resist disease following respiratory infection and 2) the way cattle are managed (Fulton, 2009a; Taylor et al., in press).

NC-1027 research activities, past 5 years:

Research carried out by members of NC-1027 in the past 5 years has revealed much about how infectious agents, host resistance, and management practices work together to induce BRDC, and about how some practices can decrease BRD (Fulton, 2009b). Some highlights of recent work which has elucidated the means by which bacterial and viral pathogens interact with the host immune response to cause BRD: it was shown that the latency related gene of BHV-1 impairs the induction of apoptosis (Jones et al, 2006), that BHV-1 gE is necessary for anterograde transport in neurons (Brum et al, 2008), and that the BHV-1 protein CIPO1 interferes with host cell interferon synthesis (Saira et al, 2009), with this protein being upregulated in cattle treated with dexamethasone (Workman et al, 2009). The role of BVDV nonstructural proteins in viral assembly and impairment of host immune responsiveness was shown (Henningson et al, 2009; Liang et al, 2009). Through a proteomics approach, it was shown that cytopathic BVDV downregulates several kinases in bovine macrophages, likely explaining at least in part its cytopathic effect (Pinchuk et al, 2008; Lee et al, 2008). BVDV was also shown to affect molecules associated with function of professional antigen presenting cells (Lee et al, 2009). M. haemolytica leukotoxin was shown to associate with lipid rafts in bovine cell membranes, and is internalized via clathrin and dynamin-2 dependent mechanisms (Attapatu et al, 2007); mechanisms by which H. somni induces thrombosis, through disruption of endothelial cells and platelet aggregation, were also discovered (Behling-Kelly et al, 2007; Kuckleberg et al, 2007). Research revealed that microbes can manipulate the immune response to the hosts detriment, such as the induction of disease-enhancing immunoglobulin E (IgE) by BRSV and H. somni (Gershwin et al., 2005), or the suppression of immune cell functions such as oxygen radical production by M. bovis (Wiggins et al, 2010). Adhesins of M. haemolytica that are involved in airway epithelial cell binding were characterized (Kisiela et al, 2009). It was shown that white tail deer fawns (Ridpath et al, 2007; Ridpath et al 2008) or alpacas (Topliff et al, 2009) can be acutely or persistently infected with BVDV, making these hosts a possible source of BVDV infection for cattle. Feedlot BRD was shown to cluster into 7 different patterns based on the timing of disease from feedlot arrival, and the pattern was associated with decreased performance by affected cattle (Babcock et al, 2010). It was shown that the pattern of pathology and microbial infection in feedlot BRD has changed over several decades, with chronic lung lesions associated with Mycoplasma bovis being relatively more common than acute lesions associated with M. haemolytica (Fulton et al, 2009b).

Methods to prevent or control BRD, such as metaphylactic use of antimicrobials and vaccination, have been evaluated by NC-1027 researchers. The addition of M. haemolytica outer membrane proteins was shown to improve efficacy of commercial vaccines (Confer et al, 2006). Intranasal vaccination with an M. haemolytica vaccine containing cholera toxin provided effective protection against disease due to M. haemolytica (Confer et al, 2009; Ayalew et al, 2009). The efficacy of vaccination to prevent infection transmitted by BVDV PI calves (Fulton et al, 2006a), or to minimize disease in calves vaccinated in the presence of maternal antibody (Zimmerman et al, 2006; Zimmerman et al, 2009) was confirmed. The relative value of two commonly used antimicrobials in stocker cattle at high risk for BRD was evaluated (Nickell et al, 2008). It was shown that single vaccination of at-risk cattle can be as effective as vaccination with booster in controlling BRD in at least some cases (Step et al, 2009).

Diagnostic tests were refined through NC-1027 research. The relative value of various tests for diagnosis of BVDV PI cattle was established (Fulton et al, 2006), as was the impact of sample handling on test results (Funk et al, 2008a; Funk et al, 2008b). It was shown that different tests of cell mediated immunity (CMI) following viral respiratory vaccination do not agree, indicating that multiple tests should be used when evaluating CMI to vaccination (Reber et al, 2006). Preliminary characterization of biosensors that are in development for chute-side BVDV testing was accomplished (Muhammad-Tahir et al, 2007; Jin et al, 2008).

NC-1027 outreach, past 5 years:

In addition to completing impactful basic and applied BRD research, in 2009 NC-1027 hosted the 2009 Bovine Respiratory Disease Symposium in conjunction with the Summer Academy of Veterinary Consultants meeting. Over 200 attendees from around the world and representing private practice, academia, government, and industry attended. The Symposium included both presentations from speakers who are recognized authorities on various subjects relevant to BRD, as well as breakout sessions where participants met in small groups to discuss issues and opportunities for further development related to the control and prevention of BRD. The schedule of presentations at the Symposium was listed in the CRIS annual report in 2009; the presentations included subjects related to field management of BRD, the impact of cattle marketing on BRD, microbial pathogenesis and immunity in BRD, and summaries of past research and future research needs. In addition to lectures, 3 breakout sessions occurred which allowed participants to gather in small groups to discuss and brainstorm ways to address issues and problems related to BRD control. The titles of the breakout sessions were: 1) What 21st century management practices influence the development of BRD, and how can management be modified to limit BRD?; 2) How does a growing non-farm public influence animal health delivery and research, and how can animal health researchers most effectively interact with such a public; and 3) Where should BRD research be focused in the next 10-20 years, and how can scientists be encouraged to undertake a career in bovine health research? 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 volume 10, issue 2 (December 2009) of Animal Health Research Reviews. As Animal Health Research Reviews is searchable via PubMed and other biomedical research databases, the Symposium proceedings are thus accessible world wide. 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.

In addition to the 2009 BRD Symposium, NC-1027 members also organized the 4th BVDV Symposium: BVDV Variability: Impact on Virulence, Host Range and Control, Phoenix, AZ, January 26, 2009 in conjunction with National Cattlemens Beef Association meeting. This international meeting had both a scientific and producer component with 180 attendees for the scientific program and 150 attendees for the producer program. NC-1027 also continued productive interactions with veterinarians in the field through annual meetings at the American Association of Bovine Practitioners (AABP) Annual Convention. Starting in 2006, NC-1027 Technical Committee members held meetings which were open to all attendees, for the purposes of facilitating discussion between veterinarians in the field and BRD researchers. These meetings were well attended, and their success contributed to the decision by the AABP Board of Governors to establish a BRD Committee, for the purposes of improving education of members regarding issues related to BRD. In 2009 the open meetings sponsored by NC-1027 were replaced by open meetings of the BRD Committee. Two members of NC-1027 currently sit on the BRD committee, and others participated in the open meetings in 2009 and 2010.

Knowledge gaps to be filled by activities in the proposed project (2011-2016):

While members of NC-1027 have completed research that provides foundational knowledge regarding the mechanisms by which bacteria, viruses, and the host response interact to cause BRD, that is leading to the development of more accurate and effective diagnostic tests, and that is providing evidence-based guidance for the development of management practices and therapeutic modalities to limit the impact of BRD, important knowledge gaps remain and will be addressed in the upcoming project. Knowledge gaps to be addressed by NC-1027 in 2011-2016:

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.
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.
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.
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.
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.
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.
7. The concept of regional voluntary programs to eliminate specific BRDC pathogens will tested be tested in the Michigan Upper Peninsula BVDV Project.

Objectives

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.
   
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.
   
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.
   
4. To develop management practices, including rationally applied therapeutic and preventative interventions, that minimize the impact of BRD on cattle health, welfare and productivity.
   
5. To promote open scientific exchange and dialogue among scientists, veterinarians, allied industry professionals and cattlemen to advance BRD research initiatives.
   
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.
   

Methods

Objective 1: MI and OK will develop improved methods for BVDV testing. MI will develop strategies to screen herds for infection with BVDV based on the use of sentinel animals (Pillars et al., 2002), a rapid and cost effective strategy. Detection of virus neutralizing (VN) antibodies in young cattle is highly associated with circulating BVDV in dairy herds but has not been validated in beef herds. MI will compare the use of sentinel animals to screen beef herds for BVDV infection to the results to individual animal testing. A subset of unvaccinated 6 month old calves (sentinels) will be tested for VN antibodies to both genotype 1 and 2 BVDV. At the same time, testing of all individual cattle on the farm will be conducted using pooled rtPCR on skin samples. The presence of VN titers in sentinel animals will be compared to test results from all individual animals. OK will compare genomics based testing for BVDV to traditional virus isolation (VI). OK will also validate genomics based testing for BHV-1 and BCV, using samples from experimentally and naturally infected cattle. NADC will develop PCR tests for multiple bovine adenovirus serotypes; these will be validated using known strains in-house and then tested against field isolates from samples from live cattle and necropsy specimens. MI and VA will develop biosensors for the rapid identification of BRD agents. MI will continue ongoing work to use biosensors to identify BVDV PI cattle (Muhammed-Tahir et al., 2005), which will refine and optimize biosensors for real time and multiplex BVDV detection systems. Testing will continue to emphasize the development of biosensors that can work with a variety of different test substances (whole blood, serum, nasal swabs, and skin samples). VA will develop and test optical biosensors for multiple agents, focusing particularly on making DNA-identifying biosensors optimally sensitive without prior PCR. Biosensor diagnosis is field compatible and rapid (minutes), so these methods will address demands of veterinarians for field-expedient patient-side diagnosis. WI and SD will continue to work in developing new state-of-the-art diagnostic tools for BRD agents; this effort will be headed by participants in the Wisconsin and South Dakota Veterinary Diagnostic Laboratories. Objective 2: CA, CO, VA, OK, NADC, and SD will evaluate the action of bacterial pathogens in BRD, the interaction among multiple bacterial pathogens, and the interaction among bacterial pathogens, viral pathogens, and environmental factors. CA has determined that Histophilus somni IbpA has two toxic Fic motifs that cause bovine alveolar epithelial cells to retract; they will now investigate the interaction of these two motifs with bovine brain microvascular endothelial cells to investigate interaction of H. somni with the host. Protection of host cells with antibody to IbpA DR2/Fic is under study as a way of manipulating the dynamic interaction. CO and VA will collaborate on research of biofilms, microbial communities supported by a pathogen-derived extracellular matrix, in BRD. They will work to clarify how host-derived protein molecules substitute for or enhance biofilm formation by BRD pathogens, using an in vitro culture system that mimics this microenvironment in vivo. It is likely that expression of genes and proteins by bacteria attached to host macromolecules differs significantly from bacteria grown using conventional broth culture techniques, and this culture system has in fact revealed proteins that are expressed only under conditions that favor attachment to host macromolecules. This induced antigen technology will be used to identify unique antigens that are essential for in vivo survival and persistence or that may contribute to in vivo pathogenesis and antimicrobial resistance. NADC and SD will collaborate on a project to establish the dynamics of Mannheimia/Pasteurella colonization/shed/spread in bighorn sheep, using attenuated and marked M. haemolytica and P. multocida, and nasopharyngeal challenge to evaluate colonization in the pharyngeal region and fecal shedding. NADC will also evaluate the effect of cell-surface sialylation of P. multocida and its role in pathogenesis and immune evasion. OK will determine important immunogens of M. haemolytica, characterizing genes, mapping epitopes, and confirming immunogenicity. CA, GA, and KS will evaluate how coinfections and environmental factors impact the immune response in BRD. CA will characterize the IgE response to BRSV infection coinfection with H. somni, and the impact of environmental pollens, dusts, and molds on IgE production in infectious BRD. GA will evaluate the effect of gastrointestinal (GI) parasitism on immune response to viral BRD vaccines, specifically testing the hypothesis that GI parasitism induces an unfavorable T helper type 2 (TH2) immune response to viral respiratory vaccination, which is expected to be associated with an ineffective or potentially disease enhancing immune response in viral BRD. KS will focus on developing a better understanding of the risk factors (both environmental and host factors) that influence the onset and progression of bovine respiratory disease (BRD); they will work to understand various aspects of BRD including behavioral changes, physiological changes, early pathological changes, and tangential risk factors. MI, NADC, and NE will evaluate the role of viral pathogens in BRD; MI will study the effect that BVDV PI calves have on non-PI cattle in feedlot settings after different intervention strategies are used. Variables to be evaluated include types of vaccines used (killed vs live or different antigenic make-ups), timing of vaccine administration (pre-arrival vs on-arrival in feedlots), and preconditioning programs. Outcomes to be measured include morbidity, mortality, growth, and economic performance. NADC will evaluate the disease enhancing effect of coinfection between BRSV and M. haemolytica or P. multocida. NE will determine the host cellular genes activated during latency reactivation of BHV-1, using a dexamethasone (DEX) treatment-induced model of stress in calves latently infected with BHV-1. Genes expressed in trigeminal ganglia (TG) of latently infected and noninfected control calves treated with DEX will be compared by gene expression microarray analysis, in situ hybridization, and semi-quantitative RT-PCR. SD will continue to work with NADC and CO in determining the role of white-tailed deer as a reservoir for BVDV infection. This will be done through prevalence studies of BVDV persistently infected deer from regional stratified populations of white-tailed deer. Objective 3. CA and GA will continue cellular and molecular studies on the immunomodulatory effect of BRSV on immune responses to bacterial antigens to which the host is concurrently exposed including Histophilus somni, Mannheimia hemolytica, Pasteurella multicida, and Mycoplasma bovis. CA along with WI will investigate the invasive mechanisms of H. somni at the cellular and molecular levels in both alveolar cells and microvascular endothelial cells in vitro. Biochemical methods, molecular techniques, immuno-confocal microscopy and transwell analysis of invasion are being used for this investigation. Clinical and experimental H. somni disease is being investigated by immunohistochemistry in cattle. GA will evaluate the effect of bovine respiratory syncytial virus (BRSV) infection on cytokines and cell surface markers expressed by airway epithelial cells (AEC) on cell-mediated immune responses. Understanding these early steps in the host immune response to infectious BRD studies will also provide foundational knowledge regarding AEC responses in BRD, which likely have important impact on medium- and long-term responses infectious respiratory disease in cattle. MS, NADC and SD will continue studies on the effect of BVDV infection on innate immunity phagocytic cells. MS will continue on the effect of BVDV infection on bovine monocyte differentiation and function. Bovine monocytes challenged with BVDV and M. haemolytica in vitro will be assessed for phenotypic and functional differentiation parameters and the ability of bovine monocyte ability to uptake and clear M. haemolytica. Correlating results of these functional assays with functional genomics will allow the identification of molecular mechanisms that govern the observed phenotypes. NADC will work with monocyte-derived macrophages with BVDV. SD will examine BVDV strain effect on dendritic cell (DC) function and co-stimulation. DC function will be tested with various strains of BVDV. The DCs will be examined for their functional characteristics to produce Th-1 and/or Th2 cytokines, expression of co-stimulatory molecule and ability to activate T cells. SD in collaboration with NADC will determine the genetic changes in cp BVDV isolates from 11 animals that died from mucosal disease who were persistently infected with the same strain of BVDV. SD will compare the sequences of the original ncp strain to the isolated cp viruses. One of the outcomes will be the determination of genetic alterations that are termed cytopathogenicity markers. SD will then compare the effect these different cp and ncp biotypes on the innate immune system using the macrophage model. SD in collaboration with CO and NADC will evaluate the role of the fetal liver in immune tolerance. The Kupffer cell is a part of a dynamic system in the liver that modulates system-wide immune responses. In the developing fetus, the liver is a lymphoid organ, and even a site of immune education early in immune development. Fetuses were infected at ~75 days of gestation with BVDV and harvested at 1 week, 2 weeks, one month and two months post infection and lymphoid organs and liver harvested. The tissues will be analyzed using immunohistochemistry for BVDV virus and quantitative PCR for cytokine mRNA. Additional sequential samples from the time points above will be sequenced for BVDV genetic changes. NE will continue to identify cellular genes activated by the dexamethasone (DEX) model of stress in the trigeminal ganglion (TG) of BHV-1 latently infected calves. NE has shown that DEX induces cellular factors such as cellular transcription factor, C/EBP-alpha and Notch 1. Cellular gene expression in TG of latently infected calves will be determined at 1½ hour after DEX treatment of latently infected calves versus 3 or 6 hours after DEX treatment using Bovine Genome Microarrays. In situ hybridization and semi-quantitative RT-PCR will be done to confirm that cellular genes identified in microarray analysis are induced or repressed by DEX. NE will also test whether cellular genes stimulated by DEX can regulate productive infection, using a plasmid system that will expressing a specific cellular gene will be co-transfected with genomic BHV-1 DNA and the number of infectious centers compared to cells transfected with an empty expression vector and BHV-1 DNA. Lastly NE will determine if DEX inducible cellular genes regulate viral transcription. OK will determine if the interaction of bovine coronavirus (BCV) and bovine viral diarrhea virus (BVDV) will enhance BRD in calves subsequently challenged with M. haemolytica H. somni or P. multocida. Isolates of M. haemolytica, P. multocida, and H. somni will be compared genotypically and phenotypically with respect to transmissibility and disease outcomes. WI will be evaluating the formation of extracellular traps by bovine leukocytes in response to bacterial pathogens of the BRD complex (M. haemolytica and H. somnii) and how this response is influenced by exposure to respiratory virus or products released from respiratory virus infected cells. These studies will involve a series of in vitro assessments of chromatin and protein release from bovine peripheral blood PMNs, monocytes, and macrophages. As a model system we will use BHV-1 infected bovine bronchial epithelial cells. The results of these in vitro studies will inform subsequent in vivo experiments to determine if extracellular traps are formed during BRD, and whether they play a beneficial or detrimental role in host defense. Objective 4: GA will evaluate mechanisms by which maternal antibody impairs response to vaccination by use of an in vitro model culture system including bovine dendritic cells (DC), B cells, and T helper cells (TH). BRSV-specific antibody production by B cells activated by DC and TH in the presence of BRSV-specific antibody will be evaluated. BRSV specific antibody is expected to suppress B cell activation; it may be that B cell responses can be improved by addition of B cell stimulating factors at the time of antigen exposure. Methods shown to improve B cell responses in the presence of antigen s specific antibody in the in vitro system will then be tested in experimental vaccines given to calves with suppressive levels of maternal antibody Bovine viral diarrhea virus is known to be a part of the bovine respiratory disease complex (BRD), better known as shipping fever. The role that cattle persistently infected with BVDV may play in BRD is unknown. Using a previously developed model, MI will study the effect that PI calves have on non-PI cattle in feedlot settings after different intervention strategies are used (Grooms et al., 2002). Variables that will be studied look include types of vaccines used (killed -vs- live or different antigenic make-ups), timing of vaccine administration (pre-arrival -vs- on-arrival in feedlots), and preconditioning programs. MI is proposing to implement a pilot BVDV eradication program in the Upper Peninsula of Michigan (UP). This will be the first comprehensive eradication program in the US. The program will consist of the following components: Education: The success of the proposed program will be anchored in educating the UP cattle industry on the importance of controlling BVDV. Targeted populations will include producers, veterinarians, and allied industry personnel. Educational efforts will rely heavily on the Michigan State University Extension educators and network. The methods of education will include the following. 1) Producer educational meetings. 2)One-on-one meetings with producers and veterinarians 3) Articles in MSU extension newsletter. 4) BVDV Educational handouts with program specific information.5) A web site will be developed with BVDV and program information. 6) Partnerships with key stakeholder groups including regional breed groups, milk co-ops, Michigan Farm Bureau, Michigan Cattlemen's Association, etc PI Identification and Elimination: At the herd level, a testing strategy will be implemented to first identify herds at high risk for having PI's on them and then to identify individuals that are PI. This protocol will be adapted to each farm depending on the type of farm, farm management and their risk. All samples will be forwarded to the Diagnostic Center for Population and Animal Health via overnight courier. Biosecurity: Biosecurity recommendations for each farm will be made to help reduce the possible reintroduction of BVDV. Each producer will receive a biosecurity plan that is specific for his or her operation. Vaccination: Vaccination will be encouraged to reduce the risk of BVDV transmission on and within herds. Comprehensive vaccine recommendations will be made for each farm based on level of management, risk and producer goals. Vaccination is not required for control and eradication, but is certainly an effective tool to aid in the goal. Surveillance: Once farms have been determined free of BVDV, surveillance programs will be implemented to monitor BVDV status. Sentinels will be introduced every two years to determine if virus is circulating on the farm or not. Other surveillance methods that can be used include necropsy of mortalities and calf screening. Implementation: A regional BVDV control and eradication program will require a long-term commitment. I tis expected that our involvement in this project will last for a minimum of 5 years. NE will develop a vaccine vector that does not express functional domains of three viral proteins that regulate anterograde transport of viral particles in sensory neurons (gE and US9) and inhibit cell-mediated immune-recognition (gN). The BHV-1 gE and US9 genes are required for axonal anterograde transport, and consequently this virus does not reactivate from latency. The BHV-1 envelope protein gN inhibits major histocompatibility class I (MHC-I) presentation in virus-infected cells and targets peptide transport. This virus is expected to replicate like wild-type but will not reactivate from latency in calves and may induce enhanced humoral and cellular immune responses. This vaccine virus would also maintain the gE based serological marker. BHV-1 gE cytoplasmic tail-specific polyclonal antibodies specifically react to BHV-1 gE. In summary, this strategy will yield a vaccine vector that can be used to express genes from other pathogens, will not reactivate from latency, and is a DIVA. NE will also a second BHV-1 vaccine vector. This be done by creaing a mutation in the latency-related (LR) gene that will express a FLAG-tagged bICP0 protein (LR-BAC) and will not express gN. A mutation in the first open reading frame of the LR gene does not express proteins encoded by the LR gene, this virus does not reactivate from latency, but the mutation is stable in calves Monoclonal antibodies directed against the FLAG epitope are commercially available making it easy to determine if a virus is derived from the LR-BAC versus a field strain. NE will also construct a series of BHV-1 recombinant viruses expressing 1) secreted BVDV type 1 and type 2 envelope proteins E2.1 and E2.2. 2) BHV-1 vector expressing secreted BCoV (bovine corona virus) envelope spike protein (S) with or without the bovine CD 40 ligand. 3) Construct and analyze a BHV-1 vector expressing chimeric antigen consisting of M. haemolytica neutralizing epitopes of Lkt and immuno-dominant epitope (PlpE-R2) of the outer membrane protein. All the vaccines will contain marker genes. Analysis of humoral and cellular immune response induced by the candidate vaccines described will be carried out via vaccination-challenge studies. OK and KS will develop management practices, including rationally applied therapeutic and preventative interventions that minimize the impact of BRD on cattle health, welfare and productivity. Management of BRD using appropriate antimicrobial therapies will be evaluated to control BRD. High risk calves (light weight calves transported from auction markets) for BRD will be monitored over a 30-45 day interval using approved antimicrobials as well as new products under investigation. These studies will use visual means to detect BRD cases as well as remote sensing procedures. Objective 5: NC-1027 will capitalize on already developed relationships with national organizations (American Association of Bovine Practitioners (AABP) , Academy of Veterinary Consultants (AVC), Conference of Research Workers in Animal Diseases (CRWAD)) by seeking opportunities to integrate and participate in their annual meetings with the specific goal of promoting interactions and dialog between researchers and veterinarians. Specifically, the annual meeting of the NC-1027 will be held in conjunction with the AABP Practitioners Annual Conference (Table 1). Table 1; Future AABP Conferences 2011 St. Louis September 22 - 24 2012 Montreal September 20 - 22 2013 Milwaukee September 19 - 21 2014 Albuquerque September 18 - 20 2015 New Orleans September 17 - 19 Through collaborative efforts with USDA AFRI Program leaders, NC-1027 will seek to integrate the annual meeting of AFRI awardees that have research projects focused on BRD with our annual meeting and the AABP meeting. This meeting will be organized and led by NC-1027 members. Interaction between NC-1027 members, AFRI awardees and conference attendees will occur thru both closed and open scientific exchange (research summaries, poster presentations, etc) and open discussion forums. NC-1027 members will actively engage in committees of national organizations that have relevance to BRD. Specific examples of this would include the AABP BRD and BVDV subcommittees. The specific goal of this interaction will be to provide input into the national organizations direction regarding policy, educational programming, and other activities surrounding BRD Objective 6: Capitalizing on the success of the national BRD Symposium that we organized and presented in 2009, we will organize a similar event during the 5 year project. The specific goal of this symposium is to place a spotlight on BRD while focusing on what has been accomplished and what needs to be done. An organizing committee led by NC-1027 members will be formed to plan the event with the goal to hold the symposium in the 3rd or 4th year of the new project. NC-1027 will organize the annual NC-1027 meeting at the annual American Association of Bovine Practitioners meeting, during which we will invite AABP attendees to participate in discussions of ongoing research on BRD. Specifically, an open round table discussion will be scheduled in which NC-1027 members and AFRI BRD researchers will be present and conference attendees will be invited. NC-1027 will actively engage in the national eXtension initiative. Specifically, members of NC-1027 will be encouraged to become content experts available to answer questions thru the national eXtension Ask and Expert initiative. To facilitate this, MI will take the lead in providing a training session to NC-1027 participants on becoming engaged in the eXtension program. To provide practical up-to-date to date information on BRD, NC-1027 members will collaborate on writing annual review manuscripts focused on different aspects of BRD. These manuscripts will be peer reviewed but applied in nature with the goal providing information to the industry that can be immediately used in reducing the incidence of BRD. NC-1027 will actively look to integrate with other research efforts on collaborative outreach. Specifically, we will work closely with USDA AFRI funded BRD initiatives in helping to integrate their research and outreach activities with ours. By leveraging these efforts, a broader and more effective impact can be achieved.

Measurement of Progress and Results

Outputs

• Biosensors for field or chute-side identification of BRD pathogens will be developed and validated.
• New PCR tests for viral BRD pathogens, including several serotypes of adenovirus, will be developed and validated.
• Use of live animal sentinels for BVDV diagnosis will be tested and validated.
• Information regarding the effect of gastrointestinal parasites on response to BRD vaccination will be available.
• Information regarding the means by which bovine herpesvirus undergoes latency will be available.
• Output 6: BVDV will be eradicated in 80% of the herds in the Upper Penninsula of Michigan, providing a test case for the feasibility of BVDV eradication. Output 7: Information regarding the methods by which viral and bacterial pathogens circumvent the host immune response, including the effect of BVDV on innate immune cell function, will be available. Output 8: Knowledge of methods to improve BRD vaccines, particularly methods to improve the efficacy and stability of mucosal vaccines, will be advanced. Output 9: Improved methods of diagnosing BRD in the field, and of determining the prognosis for cattle with BRD, will be developed. Output 10: Regular educational opportunties for veterinarians will be available through meetings at AABP, AVC, and other conferences, and via regular publication of research summaries via AABP or AVC publications. Output 11: A national BRD Symposium will be held, providing a forum for presentation on the most current knowlege regarding BRD pathogenesis, prevention, and control.

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 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.
• Scientists, educators, and policy makers will be provided with up-to-date 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

(2011): NC-1027/NIFA/AABP BRD presentations Begin assessment of newer generation biosensors Continue BVDV eradication project in Michigan UP Continue development of PCR tests for adenovirus serotypes Continue studies to identify host proteins involved with herpes latency Continue studies to evaluate effect of BVDV on innate immune cells Continue studies on H. somnus on airway cells Continue studies of effect of neutrophil traps on BRD resistance Begin studies to develop improved clinical BRD diagnostic methods Begin studies of optimal use of antimicrobials to control BRD

(2012): NC-1027/NIFA/AABP BRD presentations Complete publication on BVDV PI deer 80% of herds in Michigan UP tested in eradicataion project Continue studies of live animal sentinels for BVDV diagnosis

(2013): NC-1027/NIFA/AABP BRD presentations Begin planning BRD Symposium Validate biosensors for BRD pathogen diagnosis Validate PCR tests for adenovirus serotypes Confirm efficacy of improved M. haemolytica, BHV-1 and BRSV vaccines Validate clinical BRD diagnostic methods

(2014): NC-1027/NIFA/AABP BRD presentations BRD Symposium Disseminate information regarding impact of BVDV on innate immune cells Dissemminate information regarding impact of H. somnus on airway cells Disseminate information regarding value of neutrophil traps in BRD resistance Disseminate information regarding biosensors for pathogen diagosis Disseminate information regarding new PCR diagnostic tests Disseminate information regarding live sentinels for BVDV diagnosis Disseminate information regarding improved vaccines for M. haemolytica, BHV-1, and BRSV Disseminate information regarding improved clinical BRD diagnosis Disseminate information regarding optimal antimicrobial use for BRD control

(2015): NC-1027/NIFA/AABP BRD presentations Publish proceedings of BRD Symposium

Projected Participation

View Appendix E: Participation

Outreach Plan

As described in the project narrative (primarily Objectives 5 and 6), a major component of this project is the continuous distribution of information on BRD to veterinarians and cattle producers coupled with technology transfer of the latest validated diagnostic methods to veterinarians and US veterinary diagnostic laboratory network. This multi-faceted approach is one of the true strengths of this committee.

Basic information generated will be disseminated through peer-reviewed journals. Members of NC-1027 have a tremendous history of publishing their work in peer-reviewed journals, thus helping to advance the science of BRD globally. Te 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-1027. Just as importantly, NC-1027 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-1027 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 2nd National BRD Symposium will be planned and held during the course of the project.

Organization/Governance

The Technical Committee of NC-1027 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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Symposium Proceedings
Proceedings of the 2009 BRD Symposium. 2009. An Health Res Rev 10:99-171.

Attachments

Land Grant Participating States/Institutions

AR, CA, CO, KS, KY, LA, MI, MO, MS, NE, OK, SD, VA, WA, WI

Non Land Grant Participating States/Institutions

Mississippi State University, USDA/NADC