Progress 02/07/02 to 01/31/07
Outputs
Progress Report Objectives (from AD-416) The objectives of the research are to reduce losses to respiratory tract disease in ruminants by developing effective mucosal and parenteral vaccines containing a) clean knockout mutants of Mannheimia and Pasteurella species, b) iron-regulated outer membrane proteins of M. haemolytica, and c) bovine defensin and inactivated M. haemolytica leukotoxin A. Classical and molecular techniques will also be developed to characterize and diagnose ruminant adenoviruses to assess their importance in respiratory Approach (from AD-416) Immunogenic preparations of iron-regulated outer membrane proteins and inactivated leukotoxin A will be prepared from M. haemolytica. Bovine beta defensins will be purified from bovine neutrophils. In frame gene- deletion mutants for five candidate target genes will be made from Mannheimia, Pasteurella, and Haemophilus. Then cattle will be immunized with each of these preparations and their resultant immune response determined and characterized. New adenovirus isolates will be identified and characterized by restriction endonuclease analysis, hexon PCR, restriction fragment length polymorphism analysis, and hexon DNA sequence analysis. Prevalence and importance of these isolates will be determined by seroepidemiology and inoculation of virus into ruminants. Significant Activities that Support Special Target Populations High-quality reagents to characterize adenoviruses from ruminants have been lacking in the U.S. Plaque-purified clones and antibodies for prototype cattle, sheep, and goat adenoviruses were produced and tested for purity. The adenoviruses and antiserum are being used as reference reagents to serotype newly-isolated adenoviruses from ruminants and to determine the prevalence of this group of viruses in the ruminant population. Two new species of adenovirus were isolated and characterized from goats, and one was isolated from deer. Two species of bovine adenovirus were isolated and characterized for the first time from U.S. cattle. Bovine adenovirus 10 has been shown to produce clinical disease and lesions in calves. Knowledge gained will allow for the determination of importance of specific serotypes of adenovirus in the respiratory disease complex so that methods of control and diagnosis can be developed. A method was developed for isolating ovine lung dendritic cells. A real- time PCR assay for the nonstructural protein 2 (NS2) gene was devised which showed that BRSV can productively infect neonatal ovine lung dendritic cells and alveolar macrophages. Additional real-time PCR assays were developed for proinflammatory and immunomodulatory ovine cytokines. The data indicate that BRSV induces immunomodulatory cytokines (IL-4 and IL-10) during the early stages of infection (days 3 or 5 PI) which would result in a failure to clear BRSV and leave the host susceptible to secondary bacterial infections. These findings will enable us to design more effective countermeasures for control of viral respiratory disease. 378 head of beef feeder calves were studied in 4 separate trials over a 30 day period at 3 feedlots in Southwestern Iowa. The calves were sourced from consignors located in the states of Georgia, Iowa, and Missouri. 99 Paired sera were collected from representative calves from each consigner, and nasal swabs were collected from all calves on arrival and after approximately 30 days on feed. The samples will be used to determine the seroprevalance of adenovirus, the incidence of both Mannheimia haemolytica and Pasteurella multocida, to culture for Mannheimia haemolytica and Pasteurella multocida, and to create a collection of bovine P. mutocida for international study of genetic diversity. Accomplishments Histophilus somni (formerly Haemophilus somnus) is an important and costly cause of respiratory disease in feedlot cattle. A new modified- live vaccine strain was constructed for inclusion in multivalent vaccine formulations. The strain can be utilized together with our previous Mannheimia haemolytica and Pasteurella multocida modified-live vaccine strains to impart additional disease resistance in at-risk cattle populations of use to the pharmceutic industry and cattleman. This accomplishment supports Component 4: Countermeasures to Prevent and Control Respiratory Diseases; Problem Statement 4A: Ruminant Respiratory Diseases, of the National Program 103 Action Plan. A problem facing respiratory and enteric disease investigators is the determination of mucosal bacterial colonization characteristics in preface to disease or immunity induction. Marker bacteria can be instrumental to such study and the development of effective immunogens. We used a promoter-trap vector to identify and characterize several effective Pasteurella promoters which are expected to be useful for expression of protein in several Pasteurellaceae hosts. These genetic elements will enable us to evaluate mucosal (oral, intranasal, drinking water) vaccines against several important etiologic agents and to better target the construction of new more effective vaccines. This accomplishment supports Component 4: Countermeasures to Prevent and Control Respiratory Diseases; Problem Statement 4A: Ruminant Respiratory Diseases, of the National Program 103 Action Plan. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 10 Number of Invention Disclosures submitted: 1 Number of Patent Applications filed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 6 Number of Newspaper Articles,Presentations for NonScience Audiences: 1
Impacts
(N/A)
Publications
- Briggs, R.E., Tatum, F.M. 2005. Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae. Applied and Environmental Microbiology. 71(11):7187-7195.
- Thomas, C.J., Hoet, A.E., Sreevatsan, S., Wittum, T.E., Briggs, R.E., Duff, G.C., Saif, L.J. 2006. Transmission of bovine coronavirus and serologic responses in feedlot calves under field conditions. American Journal of Veterinary Research. 67(8):1412-1420.
- Meyerholz, D.K., Gallup, J.M., Tatjana, L., De Macedo, M.M., Lehmkuhl, H.D. , Ackermann, M.R. 2006. Pretreatment with Recombinant Human Vascular Endothelial Growth Factor Virus Replication and Inflammation in a Perinatal Lamb Model of RSV Infection. Viral Immunology. 20(1):188-196.
- Kawashima, K., Meyerholz, D.K., Gallup, J.M., Grubor, B., Lazic, T., Lehmkuhl, H.D., Ackermann, M.R. Differential Expression of Ovine Innate immune Genes by Preterm and Neonatal Lung Epithelia Infected with Respiratory Syncytial Virus. Viral Immunology. 19(2):316-323.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is relevant to the Agricultural Research Service's National Program 103 Animal Health Action Plan. Infectious respiratory tract diseases are the leading cause of loss from disease in the cattle, sheep, and goat industries. Annual loss in the U.S. is estimated to exceed three billion dollars for cattle alone. Losses are from mortality, reduced feed efficiency, and slaughter condemnations, as well as prevention and treatment measures. Death losses to the cattle industry from respiratory disease are currently about 1% of low-risk cattle and about 4% of high-risk cattle. Animals which contract disease but survive, about 10% and 40% of the respective risk groups but reaching 90% of some populations, do not grow well and result in a lower quality product. Wild populations of
bighorn sheep can suffer total die-off by respiratory disease and a number of these populations are in decline due to persistent respiratory disease problems. Means to protect these populations are needed to preserve genetic diversity essential to wildlife species. Viral agents are well recognized as primary pathogens and even uncomplicated infections can cause substantial economic loss. Infectious Bovine Rhinotracheitis Virus (IBRV), Respiratory Syncytial Virus (RSV), Parainfluenza Type 3 Virus (PI3V), and Bovine Viral Diarrhea Virus (BVDV) are generally considered the most economically important viruses and therefore are the focus of most research efforts; yet the magnitude and complexity of the problem still exists. A portion of this problem is attributable to respiratory coronavirus and adenoviruses, which have not been extensively evaluated. Respiratory viruses also predispose animals to costly bacterial pneumonia caused by Mannheimia haemolytica, Pasteurella multocida, and
Haemophilus somnus, which commonly affect beef cattle. P. multocida and M. haemolytica commonly affect dairy cattle. These bacteria remain the major cause of loss from mortality, reduced feed efficiency, treatment costs, and reduced product quality. Control of respiratory disease of cattle, sheep, and goats is currently based on vaccines, some of which are not particularly effective, and on antibiotics, which are both costly and potentially dangerous to long-term public health. Commercial vaccines are available for bacterial respiratory disease, but their efficacy in the field is questionable. An underlying problem is the current beef cattle marketing system, which does not allow timely vaccination before the peak period of disease occurring soon after shipping. Antibiotic usage at this time is prevalent, costly, but beneficial. Vaccination in sheep and goats is complicated by the broad number of M. haemolytica serovars against which protection is necessary. Viral vaccines are
commercially available which provide reasonable levels of control for the well characterized viruses. The impact and control of adenovirus and coronavirus needs investigation. The role of adenovirus in causing or contributing to respiratory disease is being evaluated using serologic and virus isolation methods. An understanding of the molecular basis of pathogenesis is essential to the development of effective intervention strategies to control bacterial respiratory disease. With the identification of key microbial and host molecules involved in disease progression, the design of effective vaccines and therapeutics should be possible. The goal in the design of these products is for a rapid response to allow effective use in feeder beef cattle already in marketing channels, for reduced adverse local reactions to control carcass damage, and to reduce our dependence on costly antibiotic treatments. The molecular mechanisms of respiratory tract colonization and disease progression are
being elucidated by construction and testing of non-polar bacterial mutants. New modified- live vaccine candidates for rapid natural induction of disease resistance are being constructed and evaluated. Mucosal colonization in preface to disease, and adaptive mucosal immune response is being studied to enable new avenues of disease intervention. Products of this research are utilized by commercial biologics firms, regulatory agencies, field veterinarians, cattle producers, wildlife managers, state diagnostic laboratories, and other scientists. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2002) Construct Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate temperature-conditional plasmid vectors for construction of Pasteurellaceae mutants. Analyze convalescent and field sera for antibody to IROMPs. Characterize adenovirus isolates using classical and molecular methods. Design and evaluate adenovirus
family and genus specific PCR primers. Year 2 (FY 2003) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate Pasteurellaceae mutants in vitro and/or in vivo. Continue to characterize adenovirus isolates, sequence hexon genes, and prepare polyclonal antisera to new isolates. Prepare iron-regulated outer membrane proteins (IROMPs) from M. haemolytica. Year 3 (FY 2004) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Assess mutant pathogenicity. Construct modified-live vaccine strains of M. haemolytica, P. multocida and/or H. somnus. Continue to characterize adenovirus isolates, sequence hexon genes. Evaluate serologic relationships among the ruminant adenoviruses and construct phylogenetic trees. Year 4 (FY 2005) Establish colonization models and assess colonization of bacterial mutants. Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest.
Begin 3-fold DNA sequencing coverage of the M. haemolytica serotype 2 genome. Reconstruct ruminant adenovirus hexon gene phylogenetic trees. Continue to characterize adenovirus and sequence hexon genes. Year 5 (FY 2006) Construct one or more marker strains of M. haemolytica for studies of colonization and spread in cattle and/or bighorn sheep. Tools for assessment of colonization will enable improved means to control this key step in pathogenesis of disease. Construct new modified-live vaccine strain of H. somnus. A live vaccine may offer improved control of this costly disease agent. Develop improved diagnostic and epidemiologic tools for the detection and better understanding of the significance of adenoviral disease in ruminants and characterize the host response to challenge viruses grown in transformed endothelial cell lines. Complete studies to determine whether there is a modulation of cytokine secretion by ruminant alveolar macrophages or dendritic cells infected with RSV;
and f) continue to characterize the bovine lymphocyte surface molecule recognized by monoclonal antibody BAQ155A. 4a List the single most significant research accomplishment during FY 2006. Identification of gene promoters which promise more effective expression of proteins in Pasteurella. A problem facing respiratory and enteric disease investigators is the determination of mucosal bacterial colonization characteristics in preface to disease or immunity induction. Marker bacteria can be instrumental to such study and the development of effective immunogens. We used a promoter-trap vector to identify and characterize several effective Pasteurella promoters which are expected to be useful for expression of protein in several Pasteurellaceae hosts. These genetic elements will enable us to evaluate mucosal (oral, intranasal, drinking water) vaccines against several important etiologic agents and to better target the construction of new more effective vaccines. This accomplishment
supports the Mechanism of Disease component of the National Program 103 Action Plan. 4b List other significant research accomplishment(s), if any. BRSV productively infects neonatal ovine lung dendritic cells with enhanced IL-4 and IL-10 gene transcripts. We first sought to determine whether bovine respiratory syncytial virus (BRSV) can productively infect neonatal ovine lung dendritic cells (DCs). Secondly, we determined whether there was a modulation of proinflammatory or immunomodulatory cytokines during the early stages of BRSV infection (day 3 or day 5 PI). A method was developed for isolating ovine lung dendritic cells. A real-time PCR assay for the nonstructural protein 2 (NS2) gene was devised for determining whether BRSV can replicate in lung DCs. Finally, real-time PCR assays were developed for ovine cytokines. The data indicate that BRSV induces immunomodulatory cytokines (IL-4 and IL-10) which would result in a failure to clear BRSV and leave the host susceptible to
secondary bacterial infections. These findings will enable us to design more effective countermeasures for control of viral respiratory disease. This accomplishment supports the Animal Immunology component of the National Program 103 Action Plan. Construction of a modified-live Histophilus somni vaccine strain. Histophilus somni (formerly Haemophilus somnus) is an important and costly cause of respiratory disease in feedlot cattle. A new modified- live vaccine strain was constructed for inclusion in multivalent vaccine formulations. The strain can be utilized together with our previous Mannheimia haemolytica and Pasteurella multocida modified-live vaccine strains to impart additional disease resistance in at-risk cattle populations. This accomplishment supports the Strategies to Control Infectious and Non-Infectious Disease component of the National Program 103 Action Plan. 5. Describe the major accomplishments to date and their predicted or actual impact. Stress or respiratory
viral infection was found to predispose calf nasal passages to massive colonization by M. haemolytica. M. haemolytica, typically rare on nasal surfaces, was found to reside for long periods of time in the palatine tonsils, a location which can seed the nasal passages with the organism. Strains spread rapidly through herds of cattle whether or not they are stressed. As the bacterium colonizes the nasal passages or tonsils, the cattle respond immunologically and resist nasal colonization at a later time. Certain vaccines also similarly elicit serotype-specific resistance to nasal colonization. Because massive nasal colonization greatly predisposes calves to bacterial pneumonia, resistance to such colonization is a desirable goal for disease control methods. To better understand disease progression and to produce better vaccines, techniques were developed to genetically manipulate M. haemolytica, P. multocida, and H. somnus. Included in these techniques are a series of
temperature-sensitive plasmid vectors which allow alteration or deletion of genes in Pasteurellaceae bacteria without the introduction of foreign DNA into their chromosome. Many of these techniques now are patented, and six organisms so produced are currently licensed for use in veterinary vaccines. A combination M. haemolytica/P. multocida single-dose oral vaccine was formulated to address the need for rapid immunity and reduction of injection-site reactions. Evaluations in laboratory and field experiments show that the vaccine reduces nasal colonization by M. haemolytica, pneumonic lung lesions, and mortality from respiratory disease. Vaccination with the new vaccine significantly improves the performance of calves in the feedlot, a benefit not reported for any currently-available respiratory disease vaccine. Live vaccine strains were constructed for protection of wild Bighorn sheep, which may prove effective following application on hay in the winter. These studies involve the
Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. High-quality reagents to characterize adenoviruses from ruminants have been lacking in the U.S. Plaque-purified clones and antibodies for prototype cattle, sheep, and goat adenoviruses were produced and tested for purity. The adenoviruses and antiserum are being used as reference reagents to serotype newly-isolated adenoviruses from ruminants and to determine the prevalence of this group of viruses in the ruminant population. Two new species of adenovirus were isolated and characterized from goats, and one was isolated from deer. Two species of bovine adenovirus were isolated and characterized for the first time from U.S. cattle. Bovine adenovirus 10 has been shown to produce clinical disease and lesions in calves. Knowledge gained will allow for the determination of importance of specific
serotypes of adenovirus in the respiratory disease complex so that methods of control and diagnosis can be developed. These studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Maintained a CRADA with Biotechnology Research and Development Corporation (BRDC). Technical assistance was provided to commercial biologics firms on design, construction, and evaluation of bacterial respiratory disease agent vaccine candidates. Technical assistance on deer adenovirus hemorrhagic disease was provided to the California Department of Fish and Game, Oregon
Department of Fish and Wildlife, and Idaho Department of Fish and Game. Technical assistance was provided on parainfluenza virus type 3 and bovine respiratory syncytial virus to scientists at Iowa State University to study immune reaction to virus infection in the lung of small ruminants. Provided technical expertise in immunology to a university. Adenovirus genetic sequences were deposited in GenBank, making them available to the global research community. Provided Mannheimia diagnostic assistance, reagents, and bacterial strains to scientists in domestic and foreign laboratories. Adenovirus diagnostic assistance (isolation of the virus, serotyping, serologic testing, and antiserum to the prototype ovine and bovine adenoviruses) was provided to scientists in domestic and foreign diagnostic laboratories. Served as reviewers for manuscripts submitted to refereed national and international scientific publications. Served on graduate student programs of study committees for students
enrolled in Immunobiology and in Veterinary Pathology at a university. Attended the joint meetings of the American Association of Veterinary Laboratory Diagnosticians and United States Animal Health Association and participated as a member of the Diagnostic Virology Committee (AAVLD) and the Committee on Infectious Diseases of Cattle, Bison and Lama (Vice Chair of Committee) and Committee on Sheep and Goats (USAHA). Attended NC-107 Committee on bovine respiratory disease and gave a presentation. Attended the Biotechnology Research and Development Corporation (BRDC) annual Scientific Advisory Committee meeting and presented research results on a BRDC Grant. Consulted by Gregory Grey, University of Iowa Medical School, organizer of the National Surveillance for Emerging Human Adenovirus Study Group to provide technical assistance on adenovirus typing and animal adenovirus cultures to use in test development to detect adenoviruses in sewage. Represented NADC at the 40th Meeting of the
UJNR at the National Institute of Animal Health, Tsukuba, Japan and presented 4 papers on research at NADC.
Impacts
(N/A)
Publications
- Benko, M., Harrach, B., Both, G.W., Russell, W.C., Adair, B.M., Adam, E., deJong, J.C., Hess, M., Johnson, M., Kajon, A., Kidd, A.H., Lehmkuhl, H.D., Li, Q., Mautner, V., Pring-Akerblom, P., Wadell, G. 2005. Adenoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., editors. Virus Taxonomy. Eighth Report of the International Committee on the Taxonomy of Viruses. San Diego, California: Elsevier Academic Press. p. 213-228.
- Fulton, R.W., Briggs, R.E., Ridpath, J.F., Saliki, J.T., Confer, A.W., Payton, M.E., Duff, G.C., Step, D.L., Walker, D. 2005. Transmission of bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves. Canadian Journal of Veterinary Research. 69:161-169.
- Lehmkuhl, H.D. 2005. Overview of Research at the National Animal Disease Center[abstract]. United States Japan Natural Resources Animal and Avian Health Panel. p. 4-5.
- Arbetman, A.E., Lochrie, M., Shangzhen, Z., Wellman, J., Scallan, C., Doroudchi, M.M., Lehmkuhl, H.D., Hobbs, L.A., Pierce, G.F., Colosi, P. 2005. Novel caprine adeno-associated virus (AAV) capsid (AAV-GO.1) is closely related to the primate AAV-5 and has unique tropism and neutralization properties. Journal of Virology. 79(24):15238-15245.
- Fach, S.J., Brockmeier, S., Hobbs, L.A., Lehmkuhl, H.D., Sacco, R.E. 2006. Pulmonary dendritic cells isolated from neonatal and adult ovine lung tissue. Veterinary Immunology and Immunopathology. 112(3-4):171-82.
- Sacco, R.E., Waters, W.R., Rudolph, K.M., Drew, M.L. 2006. Comparative nitric oxide production of LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries. Comparative Immunology Microbiology and Infectious Diseases. 29(1):1-11.
- Briggs, R.E., Tatum, F.M. 2005. Generation and Molecular Characterization of New Temperature-Sensitive Plasmids for the Pasteurellaceae. Applied and Environmental Microbiology. 71(11):7187-7195.
- Tatum, F.M., Briggs, R.E. 2005. Construction of in-frame aroa deletion mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus using a new temperature-sensitive plasmid. Applied and Environmental Microbiology. 71(11):7196-7202.
- Fulton, R.W., Johnson, B.J., Briggs, R.E., Ridpath, J.F., Saliki, J.T., Confer, A.W., Burge, L.J., Step, D.L., Walker, D.A., Payton, M.E. 2006. Challenge with bovine viral diarrhea virus by exposure to persistently infected calves: protection by vaccination and negative results of antigen testing in nonvaccinated acutely infected calves. Canadian Journal of Veterinary Research. 70(2):121-127.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Infectious respiratory tract diseases are the leading cause of loss from disease in the cattle, sheep, and goat industries. Annual loss in the U. S. is estimated to exceed three billion dollars for cattle alone. Losses are from mortality, reduced feed efficiency, and slaughter condemnations, as well as prevention and treatment measures. Death losses to the cattle industry from respiratory disease are currently about 1% of low-risk cattle and about 4% of high-risk cattle. Animals which contract disease but survive, about 10% and 40% of the respective risk groups but reaching 90% of some populations, do not grow well and result in a lower quality product. Wild populations of bighorn sheep can suffer total die-off by respiratory disease. Means to protect these populations are needed to preserve
genetic diversity essential to wildlife species. Viral agents are well recognized as primary pathogens and even uncomplicated infections can cause substantial economic loss. Infectious Bovine Rhinotracheitis Virus (IBRV), Respiratory Syncytial Virus (RSV), Parainfluenza Type 3 Virus (PI3V), and Bovine Viral Diarrhea Virus (BVDV) are generally considered the most economically important viruses and therefore are the focus of most research efforts; yet the magnitude and complexity of the problem still exists. A portion of this problem is attributable to respiratory coronavirus and adenoviruses, which have not been extensively evaluated. Respiratory viruses also predispose animals to costly bacterial pneumonia caused by Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus, which commonly affect beef cattle. P. multocida and M. haemolytica commonly affect dairy cattle. These bacteria remain the major cause of loss from mortality, reduced feed efficiency, treatment
costs, and reduced product quality. Control of respiratory disease of cattle, sheep, and goats is currently based on vaccines, some of which are not particularly effective, and on antibiotics, which are both costly and potentially dangerous to long-term public health. Commercial vaccines are available for bacterial respiratory disease, but their efficacy in the field is questionable. An underlying problem is the current beef cattle marketing system, which does not allow timely vaccination before the peak period of disease occurring soon after shipping. Antibiotic usage at this time is prevalent, costly, but beneficial. Vaccination in sheep and goats is complicated by the broad number of M. haemolytica serovars against which protection is necessary. Viral vaccines are commercially available which provide reasonable levels of control for the well characterized viruses. The impact and control of adenovirus and coronavirus needs investigation. The role of adenovirus in causing or
contributing to respiratory disease is being evaluated using serologic and virus isolation methods. An understanding of the molecular basis of pathogenesis is essential to the development of effective intervention strategies to control bacterial respiratory disease. With the identification of key microbial and host molecules involved in disease progression, the design of effective vaccines and therapeutics should be possible. The goal in the design of these products is for a rapid response to allow effective use in feeder beef cattle already in marketing channels, for reduced adverse local reactions to control carcass damage, and to reduce our dependence on costly antibiotic treatments. The molecular mechanisms of respiratory tract colonization and disease progression are being elucidated by construction and testing of non-polar bacterial mutants. New modified- live vaccine candidates for rapid natural induction of disease resistance are being constructed and evaluated. 2. List the
milestones (indicators of progress) from your Project Plan. Year 1 (FY 2001) Construct Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate temperature-conditional plasmid vectors for construction of Pasteurellaceae mutants. Analyze convalescent and field sera for antibody to IROMPs. Characterize adenovirus isolates using classical and molecular methods. Design and evaluate adenovirus family and genus specific PCR primers. Year 2 (FY 2002) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate Pasteurellaceae mutants in vitro and/or in vivo. Continue to characterize adenovirus isolates, sequence hexon genes, and prepare polyclonal antisera to new isolates. Prepare iron-regulated outer membrane proteins (IROMPs) from M. haemolytica. Year 3 (FY 2003) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Assess mutant pathogenicity. Construct modified-live
vaccine strains of M. haemolytica, P. multocida and/or H. somnus. Continue to characterize adenovirus isolates, sequence hexon genes. Evaluate serologic relationships among the ruminant adenoviruses and construct phylogenetic trees. Year 4 (FY 2004) Establish colonization models and assess colonization of bacterial mutants. Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Begin 3-fold DNA sequencing coverage of the M. haemolytica serotype 2 genome. Reconstruct ruminant adenovirus hexon gene phylogenetic trees. Continue to characterize adenovirus and sequence hexon genes. Year 5 (FY 2005) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Continue to evaluate colonization of bacterial mutants. Continue to obtain genome sequence of M. haemolytica serotype 2. Refine adenovirus species-specific PCR primers and develop real time PCR test. Continue to characterize adenovirus and sequence hexon
genes. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Milestone Fully Met 2. Continue to evaluate colonization of bacterial mutants. Milestone Substantially Met 3. Refine adenovirus species-specific PCR primers and develop real time PCR test. Milestone Substantially Met 4. Continue to characterize adenovirus and sequence hexon genes. Milestone Substantially Met 5. Continue to obtain genome sequence of M. haemolytica serotype 2. Milestone Not Met Critical SY Vacancy 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The project is scheduled to be included in the NP 103 Animal Health Panel Review, conducted by the Office of
Scientific Quality Review, to begin September 2005 with the new project plan expected to be certified and implemented July 2006. FY 2006: Construct one or more marker strains of M. haemolytica for studies of colonization and spread in cattle and/or bighorn sheep. Tools for assessment of colonization will enable improved means to control this key step in pathogenesis of disease. Construct new modified-live vaccine strain of H. somnus. A live vaccine may offer improved control of this costly disease agent. Develop improved diagnostic and epidemiologic tools for the detection and better understanding of the significance of adenoviral disease in ruminants and characterize the host response to challenge viruses grown in transformed endothelial cell lines. Complete studies to determine whether there is a modulation of cytokine secretion by ruminant alveolar macrophages or dendritic cells infected with RSV; and f) continue to characterize the bovine lymphocyte surface molecule recognized
by monoclonal antibody BAQ155A. FY 2007: Evaluate respiratory tract colonization by marker strains of M. haemolytica. Tools for assessment of colonization will enable improved means to control this key step in pathogenesis of disease. Construct new M. haemolytica serotype 2 vaccine strain. A live vaccine may offer improved control of this costly disease agent. Continue to develop and test adenovirus vaccine strategies in the host species using an established vaccine/challenge model. Determine whether the T cell cytokine response to RSV infection in ruminants is skewed toward a Th2 response by evaluating transcriptional factors such as T-bet and GATA-3 as well as IL-12R?2. FY 2008: Evaluate M. haemolytica serotype 2 vaccine strain for efficacy in reduction of respiratory tract colonization. The live vaccine may offer improved control of this costly disease. Evaluate shed spread of M. haemolytica among bighorn sheep using marker strain. Disease control in bighorn sheep depends
greatly on epidemiologic aspects of M. haemolytica. Study the interaction between RSV-infected epithelial cells and leukocytes in the lung of ruminants. 4a What was the single most significant accomplishment this past year? Modification of M. haemolytica vaccine strain to increase immunogenicity and reduce virulence in bighorn sheep. We've found that modified-live vaccine strains can be very effective in controlling pneumonic pasteurellosis in sheep and cattle, but bighorn sheep are sensitive to adverse reactions. Modifications of a vaccine strain were incorporated which both increase expression of an important immunogen and reduce the possibility of adverse reactions. The new strain is useful for formulation of new commercial vaccines for domestic ruminants and may lead to a useful vaccine for bighorn sheep. 4b List other significant accomplishments, if any. ARS scientists working on this project have developed a method for isolation of respiratory tract dendritic cells from
ruminant lung tissue. Respiratory tract dendritic cells were isolated using a magnetic- activated cell sorter (MACS). Following MACS purification, the cells were examined by electron microscopy and by flow cytometry for dendritic cell- specific surface molecules and tracer endocytosis. During this past year, dendritic cells have been isolated from neonatal lambs as well as adult sheep. This is the first documented isolation of dendritic cells from neonatal animals. It has been shown that the pulmonary dendritic cells from neonates and adults are similar phenotypically and functionally. This is in contrast to the current dogma that suggests that neonatal dendritic cells are less mature than those of adults. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Stress or respiratory viral infection was found to predispose calf nasal passages to massive colonization by M. haemolytica. M. haemolytica, typically rare on nasal
surfaces, was found to reside for long periods of time in the palatine tonsils, a location which can seed the nasal passages with the organism. Strains spread rapidly through herds of cattle whether or not they are stressed. As the bacterium colonizes the nasal passages or tonsils, the cattle respond immunologically and resist nasal colonization at a later time. Certain vaccines also similarly elicit serotype-specific resistance to nasal colonization. Because massive nasal colonization greatly predisposes calves to bacterial pneumonia, resistance to such colonization is a desirable goal for disease control methods. To better understand disease progression and to produce better vaccines, techniques were developed to genetically manipulate M. haemolytica, P. multocida, and H. somnus. Included in these techniques are a series of temperature-sensitive plasmid vectors which allow alteration or deletion of genes in Pasteurellaceae bacteria without the introduction of foreign DNA into
their chromosome. Many of these techniques now are patented, and six organisms so produced are currently licensed for use in veterinary vaccines. A combination M. haemolytica/P. multocida single-dose oral vaccine was formulated to address the need for rapid immunity and reduction of injection-site reactions. Evaluation in laboratory and field experiments show that the vaccine reduces nasal colonization by M. haemolytica, pneumonic lung lesions, and mortality from respiratory disease. Vaccination with the new vaccine significantly improves the performance of calves in the feedolot, a benefit not reported for any currently-available respiratory disease vaccine. Live vaccine strains were constructed for protection of wild Bighorn sheep, which may prove effective following application on hay in the winter. These studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease
Components of the Animal Health Program. High-quality reagents to characterize adenoviruses from ruminants have been lacking in the U.S. Plaque-purified clones and antibodies for prototype cattle, sheep, and goat adenoviruses were produced and tested for purity. The adenoviruses and antiserum are being used as reference reagents to serotype newly-isolated adenoviruses from ruminants and to determine the prevalence of this group of viruses in the ruminant population. Two new species of adenovirus were isolated and characterized from goats, and one was isolated from deer. Two species of bovine adenovirus were isolated and characterized for the first time from U.S. cattle. Bovine adenovirus 10 has been shown to produce clinical disease and lesions in calves. Knowledge gained will allow for the determination of importance of specific serotypes of adenovirus in the respiratory disease complex so that methods of control and diagnosis can be developed. These studies involve the
Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A license agreement was established with a commercial veterinary biologics firm to transfer vaccine strains. Maintained a CRADA. Technical assistance was provided to commercial biologics firms on design, construction, and evaluation of bacterial respiratory disease agent vaccine candidates. Technical assistance on deer adenovirus hemorrhagic disease was provided to the California Department of Fish and Game, Oregon Department of Fish and Wildlife, and Idaho Department of Fish and Game. Technical assistance was
provided on parainfluenza virus type 3 and bovine respiratory syncytial virus to scientists at Iowa State University to study immune reaction to virus infection in the lung of small ruminants. Provided technical expertise in immunology to a university. Adenovirus genetic sequences were deposited in GenBank, making them available to the global research community. Provided Mannheimia diagnostic assistance, reagents, and bacterial strains to scientists in domestic and foreign laboratories. Adenovirus diagnostic assistance (isolation of the virus, serotyping, serologic testing, and antiserum to the prototype ovine and bovine adenoviruses) was provided to scientists in domestic and foreign diagnostic laboratories. Attended NC-107 Committee on bovine respiratory disease and gave a presentation. Attended the Biotechnology Research and Development Corporation (BRDC) annual Scientific Advisory Committee meeting and presented research results on a BRDC Grant. 7. List your most important
publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Served as reviewers for manuscripts submitted to refereed national and international scientific publications. Served on graduate student programs of study committees for students enrolled in Immunobiology and in Veterinary Pathology at a university. Attended the joint meetings of the American Association of Veterinary Laboratory Diagnosticians and United State Animal Health Association and participated as a member of the Diagnostic Virology Committee (AAVLD) and the Committee on Infectious Diseases of Cattle, Bison and Llama (Vice Chair of Committee) and Committee on Sheep and Goats (USAHA).
Impacts
(N/A)
Publications
- Briggs, R.E., Tatum, F.E. 2004. Construction of Pasteurella haemolytica vaccines. U.S. Patent 6,793,927.
- Meyerholz, D.K., Grubor, B., Fach, S.J., Sacco, R.E., Lehmkuhl, H.D., Gallup, J.M., Ackermann, M.R. 2004. Reduced clearance of respiratory syncytial virus in a preterm lamb model. Microbes and Infection. 6(14) :1312-1319.
- Fach, S.J., Meyerholz, D.K., Gallup, J.M., Ackermann, M.R., Lehmkuhl, H.D., Sacco, R.E. 2005. Neonatal lamb respiratory tract dendritic cells and the effects of RSV infection [abstract]. Federation of American Societies for Experimental Biology Conference. 19:A403.
- Lehmkuhl, H.D. 2004. Adenovirus infection in sheep and goats. Proceedings of the United States Animal Health Association. Available: http://www. usaha.org/committees/sg/sg.shtml.
- Meyerholz, D.K., Grubor, B., Gallup, J.M., Lehmkuhl, H.D., Anderson, R.D., Lazic, T., Ackermann, M.R. 2004. Adenovirus-mediated gene therapy enhances parainfluenza virus 3 infection in neonatal lambs. Journal of Clinical Microbiology. 42(10):4780-4787.
- Ackermann, M.R., Meyerholz, D.K., Grubor, B., Lehmkuhl, H.D., Sacco, R.E., Gallup, J.M., Derscheid, R., Lazic, T., Fach, S., Kawashima, K. 2004. Innate immunity of respiratory epithelia: effects of preterm birth and respiratory syncytial virus infection [abstract]. Conference of Research Workers in Animal Diseases. p. 136.
- Delhon, G., Tulman, E.R., Afonso, C.L., Lu, Z., Concha-Bermejillo, A., Lehmkuhl, H.D., Piccone, M.E., Kutish, G.F., Rock, D.L. 2004. Genomes of the parapoxviruses of ORF virus and bovine papular stomatitis virus. Journal of Virology. 78(1):168-177.
- Fach, S.J., Meyerholz, D.K., Gallup, J.M., Ackermann, M.R., Lehmkuhl, H.D., Sacco, R.E. 2004. Effect of BRSV infection in neonatal lambs on respiratory tract dendritic cells [abstract]. Autumn Immunology Conference. p. 52.
- Meyerholz, D.K., Grubor, B., Fach, S.J., Sacco, R.E., Lehmkuhl, H.D., Gallup, J.M., Ackermann, M.R. 2004. Increased severity of bovine respiratory syncytial virus infection in preterm lambs [abstract]. Bi- Annual All-Iowa Virology Symposium. Paper No. 12.
|
Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Infectious respiratory tract diseases are the leading cause of loss from disease in the cattle, sheep, and goat industries. Annual loss in the U. S. is estimated to exceed three billion dollars for cattle alone. Losses are from mortality, reduced feed efficiency, and slaughter condemnations, as well as prevention and treatment measures. Death losses to the cattle industry from respiratory disease are currently about 1% of low-risk cattle and about 4% of high-risk cattle. Animals which contract disease but survive, about 10% and 40% of the respective risk groups but reaching 90% of some populations, do not grow well and result in a lower quality product. Wild populations of bighorn sheep can suffer total die-off by respiratory disease. Means to protect these populations are needed to preserve
genetic diversity essential to wildlife species. Viral agents are well recognized as primary pathogens and even uncomplicated infections can cause substantial economic loss. Infectious Bovine Rhinotracheitis Virus (IBRV), Respiratory Syncytial Virus (RSV), Parainfluenza Type 3 Virus (PI3V), and Bovine Viral Diarrhea Virus (BVDV) are generally considered the most economically important viruses and therefore are the focus of most research efforts; yet the magnitude and complexity of the problem still exists. A portion of this problem is attributable to respiratory coronavirus and adenoviruses, which have not been extensively evaluated. Respiratory viruses also predispose animals to costly bacterial pneumonia caused by Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus, which commonly affect beef cattle. P. multocida and M. haemolytica commonly affect dairy cattle. These bacteria remain the major cause of loss from mortality, reduced feed efficiency, treatment
costs, and reduced product quality. Control of respiratory disease of cattle, sheep, and goats is currently based on vaccines, some of which are not particularly effective, and on antibiotics, which are both costly and potentially dangerous to long-term public health. Commercial vaccines are available for bacterial respiratory disease, but their efficacy in the field is questionable. An underlying problem is the current beef cattle marketing system, which does not allow timely vaccination before the peak period of disease occurring soon after shipping. Antibiotic usage at this time is prevalent, costly, but beneficial. Vaccination in sheep and goats is complicated by the broad number of M. haemolytica serovars against which protection is necessary. Viral vaccines are commercially available which provide reasonable levels of control for the well characterized viruses. The impact and control of adenovirus and coronavirus needs investigation. The role of adenovirus in causing or
contributing to respiratory disease is being evaluated using serologic and virus isolation methods. An understanding of the molecular basis of pathogenesis is essential to the development of effective intervention strategies to control bacterial respiratory disease. With the identification of key microbial and host molecules involved in disease progression, the design of effective vaccines and therapeutics should be possible. The goal in the design of these products is for a rapid response to allow effective use in feeder beef cattle already in marketing channels, for reduced adverse local reactions to control carcass damage, and to reduce our dependence on costly antibiotic treatments. The molecular mechanisms of respiratory tract colonization and disease progression are being elucidated by construction and testing of non-polar bacterial mutants. New modified- live vaccine candidates for rapid natural induction of disease resistance are being constructed and evaluated. 2. List the
milestones (indicators of progress) from your Project Plan. Year 1 (FY 2001) Construct Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate temperature-conditional plasmid vectors for construction of Pasteurellaceae mutants. Analyze convalescent and field sera for antibody to IROMPs. Characterize adenovirus isolates using classical and molecular methods. Design and evaluate adenovirus family and genus specific PCR primers. Year 2 (FY 2002) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Evaluate Pasteurellaceae mutants in vitro and/or in vivo. Continue to characterize adenovirus isolates, sequence hexon genes, and prepare polyclonal antisera to new isolates. Prepare iron-regulated outer membrane proteins (IROMPs) from M. haemolytica. Year 3 (FY 2003) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Assess mutant pathogenicity. Construct modified-live
vaccine strains of M. haemolytica, P. multocida and/or H. somnus. Continue to characterize adenovirus isolates, sequence hexon genes. Evaluate serologic relationships among the ruminant adenoviruses and construct phylogenetic trees. Year 4 (FY 2004) Establish colonization models and assess colonization of bacterial mutants. Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Begin 3-fold DNA sequencing coverage of the M. haemolytica serotype 2 genome. Reconstruct ruminant adenovirus hexon gene phylogenetic trees. Continue to characterize adenovirus and sequence hexon genes. Year 5 (FY 2005) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Continue to evaluate colonization of bacterial mutants. Continue to obtain genome sequence of M. haemolytica serotype 2. Refine adenovirus species-specific PCR primers and develop real time PCR test. Continue to characterize adenovirus and sequence hexon
genes. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled to be completed under Year 4 and were fully or substantially completed. Establish colonization models and assess colonization of bacterial mutants. Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Reconstruct ruminant adenovirus hexon gene phylogenetic trees. Continue to characterize adenovirus and sequence hexon genes. The following milestone was not fully or substantially met because of the enormity of the project. Initiate 3-fold DNA sequencing coverage of the M. haemolytica serotype 2 genome. A plasmid library of genomic DNA was constructed and genome sequencing is now approximately 20% complete. B. List the milestones that
you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The Year 5 milestones are listed below with a description of the anticipated outcomes. The project is scheduled to be included in the NP 103 Animal Health Panel Review, conducted by the Office of Scientific Quality Review, to begin September 2005 with the new project plan expected to be certified and implemented July 2006. Year 5 (FY 2005) Construct additional Pasteurellaceae mutants lacking possible virulence determinants of interest. Continue to evaluate colonization of bacterial mutants. Continue to obtain genome sequence of M. haemolytica serotype 2. Refine adenovirus species-specific PCR primers and develop real time PCR test. Continue to characterize adenovirus and sequence hexon genes. Year 6 (FY 2006) Evaluate the vaccine potential of new M. haemolytica vaccine strains in cattle and bighorn sheep. Develop improved
diagnostic and epidemiologic tools for the detection and better understanding of the significance of adenoviral disease in ruminants and characterize the host response to challenge viruses grown in transformed endothelial cell lines. Complete analysis of M. haemolytica genome data and compare to data from M. haemolytica serotype 1 genome project. Determine whether the T cell cytokine response to RSV infection in ruminants is skewed toward a Th2 response and characterize lymphocyte subpopulations in bovine tonsil and peripheral lymph nodes. Year 7 (FY 2007) Target specific genes with probable relevance to virulence in the Pasteurellaceae for inactivation based on comparative genomics of M. haemolytica. These will be useful for elucidation of the genes' role in disease pathogenesis. Continue to develop and test adenovirus vaccine strategies in the host species using an established vaccine/challenge model. Study the interaction between RSV-infected epithelial cells and leukocytes in the
lung of ruminants. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004 year: Heavy nasopharyngeal colonization with M. haemolytica in stressed calves is a prerequisite to disease losses from pneumonia in beef and dairy calves. A combination M. haemolytica serotype 1 / P. multocida serotype A:3 single-dose orally-administered vaccine previously constructed by ARS scientists working at USDA-ARS National Animal Disease Center, Ames, IA, on this project, was evaluated in a field trial in cooperation with New Mexico State University and Oklahoma State University. The number of calves shedding (and therefore colonized with) virulent M. haemolytica field strains of serotype 1 was greatly reduced among vaccinates (P<0.001) throughout the 35 day feeding period. Field strains of serotype 6 (a somewhat less common pathogen similar in virulence to serotype 1), however, appeared to have filled the niche opened up by
effective vaccination against serotype 1 as more vaccinates than non-vaccinates shed these virulent strains. The findings underscore the desirability of vaccination against both serotypes 1 and 6 for effective control of pneumonic pasteurellosis B. Other Significant Accomplishment(s), if any: 1) In accordance with CRIS objectives during this year we characterized new adenovirus isolates from cattle, sheep and goats using classical and molecular techniques. Characterization of goat adenovirus isolates from Minnesota and Canada indicate that there may be across species antigenic relationships among the ruminant Mastadenovirus although they differ at the genetic level. Consensus primers for dependovirus and autonomous parvoviruses were developed to help characterize a recent goat adenovirus/dependovirus to explore their roles in pathogenicity. 2) The M. haemolytica vaccine modified-live strain which has proven effective when administered orally or by injection in calves is also
effective in bighorn sheep, but, unlike in calves, the strain is still somewhat virulent in bighorn sheep. A new modified-live strain was constructed which is expected to be effective when administered orally, but have reduced virulence in bighorn sheepre. Vaccine strains of serotype 2 and 10 based on this new genetic modification may finally give us practical means to protect bighorn sheep from devastating outbreaks of pneumonic pasteurellosis. 3) ARS scientists working on this project have developed a method for isolation of respiratory tract dendritic cells from ruminant lung tissue. Respiratory tract dendritic cells were isolated using a magnetic- activated cell sorter (MACS). Following MACS purification, the cells were examined by electron microscopy and by flow cytometry for dendritic cell- specific surface molecules and tracer endocytosis. It will be determined if respiratory tract dendritic cells or alveolar macrophages can be infected by respiratory syncytial virus (RSV).
If dendritic cells can be infected by RSV, it is likely that RSV infection would modify the dendritic cell maturation process and bias the cytokine profile. Furthermore, dendritic cells are known to activate naive T cells in the draining lymph nodes. Thus RSV infection of respiratory tract dendritic cells trafficking to the draining lymph node would likely bias the T cell response in the lymph node. 4) Bighorn sheep are more susceptible to infection with Mannheimia haemolytica than are domestic sheep. In collaboration with scientists at the Idaho Department of Fish and Game, ARS scientists working on this project have begun to examine differences in the host inflammatory response to Mannheimia infection. As macrophages play a critical role in the host response to infection, these cells have been derived from peripheral blood of bighorn sheep and domestic sheep. The cultured cells were determined to be macrophages based on morphology, immunohistochemical staining and flow cytometric
analysis. Following a two-week culture period, the macrophages were stimulated with M. haemolytica lipopolysaccharide (LPS) and examined for their ability to secrete nitric oxide. In agreement with previous research, domestic sheep macrophages produce little or no nitric oxide in response to LPS. In contrast, bighorn sheep macrophages stimulated with LPS produced significantly (P<.05) higher levels of nitric oxide compared to controls. Nitric oxide production in response to LPS may play a role in the pathogenesis of M. haemolytica infections. C. Significant Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. a) Stress or respiratory viral infection was found to predispose calf nasal passages to massive colonization by M. haemolytica. M. haemolytica, typically rare on nasal surfaces, was found to reside for long periods of time in the palatine tonsils, a location
which can seed the nasal passages with the organism. Strains spread rapidly through herds of cattle whether or not they are stressed. As the bacterium colonizes the nasal passages or tonsils, the cattle respond immunologically and resist nasal colonization at a later time. Certain vaccines also similarly elicit serotype-specific resistance to nasal colonization. Because massive nasal colonization greatly predisposes calves to bacterial pneumonia, resistance to such colonization is a desirable goal for disease control methods. To better understand disease progression and to produce better vaccines, techniques were developed to genetically manipulate M. haemolytica, P. multocida, and H. somnus. Included in these techniques are a series of temperature-sensitive plasmid vectors which allow alteration or deletion of genes in Pasteurellaceae bacteria without the introduction of foreign DNA into their chromosome. Many of these techniques now are patented, and four organisms so produced
are currently licensed for use in veterinary vaccines. A combination M. haemolytica/P. multocida single-dose oral vaccine was formulated to address the need for rapid immunity and reduction of injection-site reactions. Evaluation in laboratory and field experiments show that the vaccine reduces nasal colonization by M. haemolytica, pneumonic lung lesions, and mortality from respiratory disease. Vaccination with the new vaccine significantly improves the performance of calves in the feedolot, a benefit not reported for any currently-available respiratory disease vaccine. Live vaccine strains were constructed for protection of wild Bighorn sheep, which may prove effective following application on hay in the winter. These studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. b) High-quality reagents to characterize adenoviruses
from ruminants have been lacking in the U.S. Plaque-purified clones and antibodies for prototype cattle, sheep, and goat adenoviruses were produced and tested for purity. The adenoviruses and antiserum are being used as reference reagents to serotype newly-isolated adenoviruses from ruminants and to determine the prevalence of this group of viruses in the ruminant population. Two new species of adenovirus were isolated and characterized from goats, and one was isolated from deer. Two species of bovine adenovirus were isolated and characterized for the first time from U.S. cattle. Bovine adenovirus 10 has been shown to produce clinical disease and lesions in calves. Knowledge gained will allow for the determination of importance of specific serotypes of adenovirus in the respiratory disease complex so that methods of control and diagnosis can be developed. These studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies
to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. c) Not all animals exposed to M. haemolytica develop pneumonia, and these differences may be related to innate pulmonary defense mechanisms. Three antimicrobial anionic peptides were isolated from pulmonary surfactant of cattle and sheep. These peptides were small (721.6 ' 823.7 Da), hydrophilic, and contained repeats of aspartic acid residues. Minimal inhibitory concentrations of anionic peptides and similar analogs were comparable to those of other vertebrate antimicrobial peptides. Other antimicrobial peptides have also been found in ruminants that are also efficacious. These include proline-rich antimicrobial peptides from sheep and goat leukocytes and beta defensins. Further study of these innate immune mechanisms may allow the elucidation of new potential points for disease intervention. These studies involve the Animal Immunology, Genetic Resistance to Disease, and Strategies to
Control Infectious and Non-Infectious Disease Components of the Animal Health Program. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? a) Technical assistance was provided to commercial biologics firms on design, construction, and evaluation of bacterial respiratory disease agent vaccine candidates. b) Technical assistance on deer adenovirus hemorrhagic disease was provided to the California Department of Fish and Game, Oregon Department of Fish and Wildlife, and Idaho Department of Fish and Game. c) Technical assistance was provided on parainfluenza virus type 3 and bovine respiratory syncytial virus to scientists at Iowa State University to study immune reaction to virus infection in the lung of small ruminants. d) Provided technical expertise in
immunology to a university. e) Adenovirus genetic sequences were deposited in GenBank, making them available to the global research community. f) Provided Mannheimia diagnostic assistance, reagents, and bacterial strains to scientists in domestic and foreign laboratories. g) Adenovirus diagnostic assistance (isolation of the virus, serotyping, serologic testing, and antiserum to the prototype ovine and bovine adenoviruses) was provided to scientists in domestic and foreign diagnostic laboratories. h) Served as reviewers for manuscripts submitted to refereed national and international scientific publications. i) Served on graduate student programs of study committees for students enrolled in Immunobiology and in Veterinary Pathology at a university. j) Attended the joint meetings of the American Association of Veterinary Laboratory Diagnosticians and United States Animal Health Association and participated as a member of the Diagnostic Virology Committee (AAVLD) and the Committee on
Infectious Diseases of Cattle, Bison and Lama (Vice Chair of Committee) and Committee on Sheep and Goats (USAHA). k) Attended NC-107 Committee on bovine respiratory disease and gave a presentation. l) Attended the the Biotechnology Research and Development Corporation (BRDC) annual Scientific Advisory Committee meeting and presented research results on a BRDC Grant. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Fach, S.J., W.R. Waters, M.V. Palmer, W.C. Davis, and R.E. Sacco. 2003. Analysis of lymphocytes isolated from white-tailed deer (Odocoileus virginianus) fawns. ARS Immunology Workshop, Beltsville, MD. Meyerholz, D.K., B. Grubor, S.J. Fach, R.E. Sacco, D. Endsley, H.D. Lehmkuhl, J.M. Gallup, M.R. Ackermann. 2004. Respiratory syncytial virus infection in preterm lambs. Federation of American Societies for Experimental Biology, American Society for Investigative Pathology section, Washingtion,
DC, abstract no. 1074.
Impacts
(N/A)
Publications
- TATUM, F.M., BRIGGS, R.E. LKTA DELETION MUTANT OF P. HAEMOLYTICA. 2002. U. S. PATENT 6,495,145.
- TATUM, F.M., BRIGGS, R.E. TEMPERATURE SENSITIVE PLASMIDS OF P. HAEMOLYTICA. 2003. U.S. PATENT 6,573,093.
- BROGDEN, K.A., ACKERMANN, M.R., MCCRAY, P.B., TACK, B.F. ANTIMICROBIAL PEPTIDES IN ANIMALS AND THEIR ROLE IN HOST DEFENSES. INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS. 2003. Vol. 22, p. 465-478.
- MEYERHOLZ, D.K., ANDERSON, R.D., HASKELL, R.E., LEHMKUHL, H.D., GRUBOR, B. M., GALLUP, J.M., ACKERMANN, M.R. CLINICAL AND PATHOLOGICAL PARAMETERS OF PARAINFLUENZA VIRUS-3 INFECTION IN NEONATAL LAMBS WITH CONCURRENT ADENOVIRAL-MEDIATED BETA-DEFENSIN GENE THERAPY. AMERICAN COLLEGE OF VETERINARY PATHOLOGISTS ABSTRACTS. Vet. Pathol. 40:5, 603, Abstract #42.
- GRUBOR, B., MEYERHOLZ, D.K., GALLUP, J.M., BROGDEN, K.A., LEHMKUHL, H.D., ACKERMANN, M.R. THE EFFECTS OF BACTERIAL AND VIRAL PNEUMONIA ON SURFACTANT PROTEIN D MRNA EXPRESSION. AMERICAN COLLEGE OF VETERINARY PATHOLOGISTS ABSTRACTS. 2003. E4.
- Grubor, B., Gallup, J.M., Meyerholz, D.K., Crouch, E.C., Evans, R.B., Brogden, K.A., Lehmkuhl, H.D., Ackermann, M.R. 2004. Enhanced surfactant protein and defensin mrna levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs. Clinical and Diagnostic Laboratory Immunology. 11(3):599-607.
- Fulton, R.W., Briggs, R.E., Payton, M.E., Confer, A.W., Saliki, J.T., Ridpath, J.F., Burge, L.J., Duff, G.C. 2004. Maternally derived humoral immunity to bovine viral diarrhea virus (BVDV)1a, BVDV1b, BVDV2, bovine herpesvirus-1, parainfluenza-3 virus bovine respiratory syncytial virus, Mannheimia haemolytica and Pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination. Vaccine. 22(5-6):643-649.
- Meyerholz, D.K., Grubor, B., Fach, S.J., Sacco, R.E., Ensley, D., Lehmkuhl, H.D., Gallup, J.M., Ackermann, M.R. Respiratory syncytial virus infection in preterm lambs. 2004. Presented at FASEB Meeting. Abstract No. 1074.
- Fajt, V.R., Apley, M.D., Brogden, K.A., Skogerboe, T.L., Karle, V.K., Chin, Y. 2004. The effect of danofloxacin and tilmicosin on body temperatures in beef calves with induced pasteurella (mannheimia) haemolytica pneumonia. American Journal of Veterinary Research. 65(5):610-615.
- Arbetman, A., Lochrie, M., Randlev, B., Surosky, R., Zhou, S., Wellman, J., Pater, C., Lehmkuhl, H.D., Hobbs, L.A., Pierce, G., Colosi, P. 2004. Isolation of a close AAAV5 relative from goat tissue: evidence of host promiscuity. Gene Therapy. Abstract #89.
- Olson, E.J., Haskell, S.R., Frank, R.K., Lehmkuhl, H.D., Hobbs, L.A., Warg, J.V., Landgraf, J., Wunschmann, A. 2004. Isolation of an adenovirus and an adeno-associated virus from goat kids with enteritis. Journal of Veterinary Diagnostic Investigation. 16:461-464.
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Respiratory tract diseases are the leading cause of loss from disease in the cattle, sheep, and goat industries. Annual loss in the U.S. is estimated to exceed one billion dollars for cattle alone. Viral agents are well recognized as primary pathogens and even uncomplicated infections can cause substantial economic loss. Infectious Bovine Rhinotracheitis Virus (IBRV), Respiratory Syncytial Virus (RSV), Parainfluenza Type 3 Virus (PI3V), and Bovine Viral Diarrhea Virus (BVDV) are generally considered the most economically important viruses, yet the magnitude and complexity of the problem still exists. A portion of this problem is attributable to Respiratory Coronavirus and Adenoviruses, which have not been extensively evaluated. Respiratory viruses also predispose animals to costly bacterial pneumonia caused by Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus, which
commonly affect beef cattle. P. multocida and M. haemolytica commonly affect dairy cattle. These bacteria remain the major cause of loss from mortality, reduced feed efficiency, treatment costs, and reduced product quality. Commercial vaccines are available for bacterial respiratory disease, but their efficacy in the field is questionable. An underlying problem is the current beef cattle marketing system, which does not allow timely vaccination before the peak period of disease occurring soon after shipping. Antibiotic usage at this time is prevalent, costly, but beneficial. Vaccination in sheep and goats is complicated by the broad number of M. haemolytica serovars against which protection is necessary. The role of Adenovirus in causing or contributing to respiratory disease is being evaluated using serologic and virus isolation methods. The molecular mechanisms of respiratory tract colonization and disease progression are being elucidated by construction and testing of
non-polar bacterial mutants. New modified-live vaccine candidates for rapid natural induction of disease resistance are being constructed and evaluated. Finally, innate mechanisms of respiratory tract resistance to bacterial infection are being characterized as a new potential point for disease intervention. 2. How serious is the problem? Why does it matter? Annual loss in the U.S. to respiratory disease is estimated to exceed one billion dollars. Losses are from mortality, reduced feed efficiency, and slaughter condemnations, as well as prevention and treatment measures. Control of respiratory disease of cattle, sheep, and goats is currently based on vaccines, which are not particularly effective, and on antibiotics, which are both costly and potentially dangerous to long-term public health. Death losses to the cattle industry from respiratory disease are currently about 1% of low-risk cattle and about 4% of high- risk cattle. Animals, which survive, about 10% and 40% of the
respective risk groups, do not grow well and result in a lower quality product. Cattle with old lesions of pneumonia at slaughter have a reduced carcass quality. Wild populations of bighorn sheep can suffer total die-off by respiratory disease. Means to protect these populations are needed to preserve genetic diversity essential to wildlife species. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 103, Animal Health (100%) This research supports the Host/Pathogen Interactions and Disease Control Strategies components of National Program 103, Animal Health. An understanding of the molecular basis of pathogenesis is essential to the development of effective intervention strategies to control bacterial respiratory disease. With the identification of key microbial and host molecules involved in disease progression, the design of effective vaccines and therapeutics should be possible. The goal in the
design of these products is for a rapid response to allow effective use in feeder beef cattle already in marketing channels, for reduced adverse local reactions to control carcass damage, and to reduce our dependence on costly antibiotic treatments. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: Currently available vaccines against bacterial respiratory disease are relatively slow to induce resistance and are often inadequate because current marketing systems do not facilitate timely administration. A new combination M. haemolytica/P. multocida single-dose orally-administered vaccine, constructed by ARS scientists working at USDA-ARS National Animal Disease Center (NADC), Ames, IA, on this project, was evaluated in a field trial in cooperation with New Mexico State University and Oklahoma State University. Weight gain of vaccinates was improved by 14 pounds per head (P<0.02) over that of controls
during the 35 day trial with little evidence of compensatory gain by the controls after the period of peak susceptibility. The new vaccine, if commercially available, would be the first respiratory disease vaccine to show a clear performance advantage to beef producers. B. Other Significant Accomplishment(s), if any: 1) Respiratory tract disease is an important cause of economic loss to the livestock industry. Adenovirus associated respiratory tract disease is under-diagnosed because adenoviruses are difficult to isolate and once isolated most laboratories do not have the ability to characterize them. Adenoviruses for characterization were either isolated in our laboratory from tissues submitted from clinical cases of respiratory tract disease or were provided by diagnostic laboratories. Some of these isolates were characterized as bovine adenovirus type 7 (BAdV-7). BAdV-7 may potentially be a systemic infection and disease, not just confined to the respiratory tract. This
information is important for veterinary diagnosticians. The frequency of occurrence of this virus in cattle in the U.S. suggests the need to develop a vaccine. 2) A patented temperature-conditional plasmid shuttle vector, previously constructed by ARS scientists working on this project, contains 2 mutations within the origin-of-replication of a plasmid isolated from M. haemolytica. By site-directed mutagenesis, ARS scientists working on this project showed that a single nucleotide substitution was sufficient to cause temperature-conditional replication, suggesting that the second substitution is inconsequential. Nucleotide substitutions were introduced at different sites to construct new temperature-sensitive origins-of- replication with different and useful properties compared to the original patented vector. These results will allow scientists to improve the utility of this or related plasmid vectors for use in the construction of new genetically-modified organisms of bacteria for
laboratory study or vaccine usage. C. Significant Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. a) Stress or respiratory viral infection was found to predispose calf nasal passages to massive colonization by M. haemolytica. M. haemolytica, typically rare on nasal surfaces, was found to reside for long periods of time in the palatine tonsils, a location which can seed the nasal passages with the organism. Strains spread rapidly through herds of cattle whether or not they are stressed. As the bacterium colonizes the nasal passages or tonsils, the cattle respond immunologically and resist nasal colonization at a later time. Certain vaccines also similarly elicit serotype-specific resistance to nasal colonization. Because massive nasal colonization greatly predisposes calves to bacterial pneumonia, resistance to such colonization is a desirable goal for disease
control methods. To better understand disease progression and to produce better vaccines, techniques were developed to genetically manipulate M. haemolytica, P. multocida, and H. somnus. Many of these techniques now are patented, and four organisms so produced are currently licensed for use in veterinary vaccines. A combination M. haemolytica/P. multocida single-dose oral vaccine was formulated to address the need for rapid immunity and reduction of injection-site reactions. Evaluation in laboratory and field experiments show that the vaccine reduces nasal colonization by M. haemolytica, pneumonic lung lesions, and mortality from respiratory disease. Vaccination with the new vaccine significantly improves the performance of calves in the feedolot, a benefit not reported for any currently-available respiratory disease vaccine. A live vaccine strain was constructed for protection of wild Bighorn sheep, which may prove effective following application on hay in the winter. These
studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. b) High-quality reagents to characterize adenoviruses from ruminants have been lacking in the U.S. Plaque-purified clones and antibodies for prototype cattle, sheep, and goat adenoviruses were produced and tested for purity. The adenoviruses and antiserum are being used as reference reagents to serotype newly-isolated adenoviruses from ruminants and to determine the prevalence of this group of viruses in the ruminant population. Two new species of adenovirus were isolated and characterized from goats, and one was isolated from deer. Two species of bovine adenovirus were isolated and characterized for the first time from U.S. cattle. Bovine adenovirus 10 has been shown to produce clinical disease and lesions in calves. Knowledge gained will allow for the determination of
importance of specific serotypes of adenovirus in the respiratory disease complex so that methods of control and diagnosis can be developed. These studies involve the Pathogen Detection and Diagnostics, Mechanisms of Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. c) Not all animals exposed to M. haemolytica develop pneumonia, and these differences may be related to innate pulmonary defense mechanisms. Three antimicrobial anionic peptides were isolated from pulmonary surfactant of cattle and sheep. These peptides were small (721.6 ' 823.7 Da), hydrophilic, and contained repeats of aspartic acid residues. Minimal inhibitory concentrations of anionic peptides and similar analogs were comparable to those of other vertebrate antimicrobial peptides. Other antimicrobial peptides have also been found in ruminants that are also efficacious. These include proline-rich antimicrobial peptides from sheep
and goat leukocytes and beta defensins. Further study of these innate immune mechanisms may allow the elucidation of new potential points for disease intervention. These studies involve the Animal Immunology, Genetic Resistance to Disease, and Strategies to Control Infectious and Non-Infectious Disease Components of the Animal Health Program. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: a) Develop sensitive polymerase chain reaction (PCR) assay for detection of M. haemolytica/P. multocida colonization of mucosal surfaces; b) characterize newly isolated untyped adenoviruses from cattle, sheep and goats; c) isolate antimicrobial peptides from sheep tracheal cell extracts by cation-exchange chromatography and purify by High Performance Liquid Chromatography (HPLC), sequence purified peptides by N-terminal Edman degradation, beta-defensins will be used directly, and cathelicidins will be synthesized; and d) perform 3-fold coverage
nucleotide-sequencing of M. haemolytica serotype 2 genome. Year 2: a) Construct M. haemolytica serotypes 1, 2, and 10 which express an immunogenic leukotoxoid only 30% the size of the active protein; b) construct M. haemolytica serotype 1 with small 100 amino acid deletions in the N-terminal region of the encoded LktA product for structure/function analysis of the leukotoxin protein with bovine lymphocytes; c) develop improved PCR primers for the Mastadenovirus genus; d) assess the antimicrobial activity of natural beta-defensins, natural cathelicidins, and synthetic cathelicidins against sheep pathogens P. multocida and M. haemolytica and determine both minimal inhibitory concentrations and minimal bactericidal concentrations; and e) Analyze and begin annotation of sequence data from M. haemolytica serotype 2 genomic sequencing project, use PCR techniques for gap-closure of sequence contigs. Year 3: a) Target specific genes with probable relevance to virulence in the
Pasteurellaceae for inactivation. These will be useful for elucidation of these gene's role in disease pathogenesis; b) develop improved diagnostic and epidemiologic tools for the detection and better understanding of the significance of adenoviral disease in ruminants; and c) determine the ability of ovine beta-defensins to augment and direct adaptive immune responses to co-administered antigens for M. haemolytica, and immunize animals intranasally with mixtures of beta-defensins and M. haemolytica leukotoxin, and determine the humoral and protective response; and d) Complete analysis of M. haemolytica genome data and compare to data from M. haemolytica serotype 1 genome project. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? a) Consulted and
assisted an animal health industry on design, construction, and evaluation of vaccine candidates. b) Attended NC-107 Committee and gave a presentation. c) Attended the the Biotechnology Research and Development Corporation (BRDC) Scientific Advisory Committee and presented research results on a BRDC Grant. d) Provided Mannheimia diagnostic assistance, reagents, and bacterial strains to scientists in domestic and foreign laboratories. e) Provided adenovirus diagnostic assistance (isolation of the virus, serotyping, serologic testing and antiserum to the prototype ovine and bovine adenoviruses) to scientists in domestic and foreign diagnostic laboratories. f) Provided technical assistance on deer adenovirus hemorrhagic disease to several state Departments of Fish and Game. g) Provided technical assistance on parainfluenza virus type 3 and bovine respiratory syncytial virus to scientists at a state university to study immune reaction to virus infection in the lung of small ruminants. h)
Served as reviewers for manuscripts submitted to refereed national and international scientific publications. i) Corresponded with scientists (Veterinary Medical Research Institute, Hungarian Academy of Sciences) in Budapest, Hungary on the Adenovirus Study Group of the International Committee on Taxonomy of Viruses on adenovirus taxonomy and classification. Results from our laboratory have helped to establish a new genus Atadenovirus in the Adenoviridae family. j) Attended the joint meetings of the American Association of Veterinary Laboratory Diagnosticians and United States Animal Health Association and participated as a member of the Diagnostic Virology Committee (AAVLD) and the Committee on Infectious Diseases of Cattle, Bison and Lama (Vice Chair of Committee) and Committee on Sheep and Goats (USAHA). k) Co-organized the 37th Meeting of the United States-Japan Cooperative Program in Natural Resources Panel of Animal and Avian Health Meeting held in Ames, IA, November 2002. l)
Member of the NADC Public Relations Committee. Worked in the NADC Booth at the Iowa Cattlemen's Association Convention. m) Participated in organizing and conducting the meeting on 'Diseases at the Interface Between Domestic Livestock and Wildlife Species' held in Ames, IA, July 17-18, 2003. n) Participated as a member of the Sheep Health Committee of the National Institute for Animal Agriculture. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). LEHMKUHL, H.D., HOBBS, L.A., WOODS, L.W. NEED FOR WILDLIFE SPECIFIC TOOLS: CHARACTERIZATION OF AN ADENOVIRUS THAT CAUSES HEMORRAGIC DISEASE IN DEER. DISEASES AT THE INTERFACE BETWEEN DOMEATIC LIVESTOCK AND WILDLIFE SPECIES. July 17-18, 2003. p. 22.
Impacts
(N/A)
Publications
- FAJT, V.R., APLEY, M.D., ROTH, J.A., FRANK, D.E., BROGDEN, K.A., SKOGERBOE, T.L., SHOSTROM, V.K., Chin, Y.L. THE EFFECTS OF DANOFLOXACIN AND TILMICOSIN ON NEUTROPHIL FUNCTION AND LUNG CONSOLIDATION IN BEEF HEIFER CALVES WITH INDUCED PASTEURELLA (MANNHEIMIA) HAEMOLYTICA PNEUMONIA. JOURNAL OF VETERINARY PHARMACOLOGY AND THERAPEUTICS. 2003. v. 26(3). p. 173-179.
- BROGDEN, K.A., HEIDARI, M., SACCO, R.E., PALMQUIST, D.E., GUTHMILLER, J.M., JOHNSON, G.K., JIA, H.P., TACK, B.F., MCCRAY, JR., P.B. DEFENSIN-INDUCED ADAPTIVE IMMUNITY IN MICE AND ITS POTENTIAL IN PREVENTING PERIODONTAL DISEASE. ORAL MICROBIOLOGY AND IMMUNOLOGY. 2003. v. 18. p. 95-99.
- HEIDARI, M., HAMIR, A.N., CUTLIP, R.C., BROGDEN, K.A. ANTIMICROBIAL ANIONIC PEPTIDE BINDS IN VIVO TO MANNHEIMIA (PASTEURELLA) HAEMOLYTICA ATTACHED TO OVINE ALVEOLAR EPITHELIUM. INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS. 2002. v. 20. p. 69-72.
- BROGDEN, K.A., ACKERMANN, M.R., WELSH, M.J., ZABNER, J. XYLITOL ACTIVATED INNATE IMMUNITY SUPPRESSES PULMONARY MANNHEIMIA HAEMOLYTICA INFECTIONS IN SHEEP. 103rd GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY. 2003. Abstract p. 250, #E-006.
- GALLUP, J.M., GRUBOR, B., BARUA, A.B., MOHAMMADI, G., BROGDEN, K.A., OLSON, J.A., ACKERMANN, M.R. REPEATED INTRAVENOUS DOSES OF ALL-TRANS-RETINOYL BETA-D-GLUCURONIDE IS NOT EFFECTIVE IN THE TREATMENT OF BACTERIAL BRONCHOPNEUMONIA IN LAMBS BUT IS DEVOID OF GROSS AND ACUTE TOXICITY. MEDICAL SCIENCE MONITOR. 2003. v. 8(9). p. 345-353.
- BROGDEN, K.A., GUTHMILLER, J.M. POLYMICROBIAL DISEASES, A CONCEPT WHOSE TIME HAS COME. ASM NEWS. 2003. v. 69(2). p. 69-73.
- FALES-WILLIAMS, A.J., BROGDEN, K.A., HUFFMAN, E., GALLUP, J.M., ACKERMANN, M.R. CELLULAR DISTRIBUTION OF ANIONIC ANTIMICROBIAL PEPTIDE IN NORMAL LUNG AND DURING ACUTE PULMONARY INFLAMMATION. VETERINARY PATHOLOGY. 2002. v. 39(6). p. 706-711.
- CAVERLY, J.M., DIAMOND, G., GALLUP, J.M., BROGDEN, K.A., DIXON, R.A., ACKERMANN, M.R. COORDINATED EXPRESSION OF TRACHEAL ANTIMICROBIAL PEPTIDE AND INFLAMMATORY-RESPONSE ELEMENTS IN THE LUNGS OF NEONATAL CALVES WITH ACUTE BACTERIAL PNEUMONIA. INFECTION AND IMMUNITY. 2003. INFECTION AND IMMUNITY. v.71(5). p. 2950-2955.
- RADI, Z.A., BROGDEN, K.A., DIXON, R.A., GALLUP, J.M., ACKERMANN, M.R. A SELECTIN INHIBITOR DECREASES NEUTROPHIL INFILTRATION DURING ACUTE MANNHEIMIA HAEMOLYTICA PNEUMONIA. VETERINARY PATHOLOGY. 2002. v. 39(6). p. 697-705.
- FRANK, G.H., BRIGGS, R.E., DUFF, G.C., HURD, H.S. EFFECTS OF INTRANASAL EXPOSURE TO LEUKOTOXIN-DEFICIENT MANNHEIMIA HAEMOLYTICA AT THE TIME OF ARRIVAL AT THE FEEDYARD ON THE SUBSEQUENT ISOLATION OF M. HAEMOLYTICA FROM NASAL SECRETIONS OF CALVES. AMERICAN JOURNAL OF VETERINARY RESEARCH. 2003. v. 64(5). p. 580-585.
- SHILTON, C.M., SMITH, D.A., WOODS, L.W., CRAWSHAW, G.J., LEHMKUHL, H.D. ADENOVIRAL INFECTION IN CAPTIVE MOOSE (ALCES ALCES) IN CANADA. JOURNAL OF ZOO AND WILDLIFE MEDICINE. 2002. v. 33(1). p. 73-79.
- GRUBOR, B., MEYERHOLZ, D.K., GALLUP, J.M., LEHMKUHL, H.D., ACKERMANN, M.R. PARAINFLUENZA VIRUS-3 PULMONARY LESIONS ARE NOT ENHANCED BY BOVINE VIRAL DIARRHEA VIRUS. VETERINARY PATHOLOGY PROCEEDINGS OF THE ANNUAL MEETING OF THE AMERICAN COLL. 2002. v. 39(5). p. 637, #97.
- MEYERHOLZ, D.K., GRUBOR, B., GALLUP, J.M., LEHMKUHL, H.D., EVANS, R.B., ACKERMANN, M.R. ALTERATIONS OF SURFACTANT PROTEIN-A AND TUMOR NECROSIS FACTOR-ALPHA EXPRESSION DURING ACUTE BOVINE RESPIRATORY SYNCYTIAL VIRUS INFECTION. AMERICAN SOCIETY FOR MICROBIOLOGY ANNUAL MEETING. 2003. Abstract p. 675, #Z-004.
- LEHMKUHL, H.D., HOBBS, L.A. PHYLOGENETIC AND SEROLOGIC ANALYSIS OF RUMINANT ATADENOVIRUSES. XIIth INTERNATIONAL CONGRESS OF VIROLOGY. 2002. Abstract p. 311, #V-925.
- BRIGGS, R.E., TATUM, F.M. CONSTRUCTION OF IN-FRAME, NON-POLAR, LKTA MUTANTS OF MANNHEIMIA HAEMOLYTICA USING A NEW TEMPERATURE CONDITIONAL PLASMID AND EFFICACY AS MODIFIED-LIVE MUCOSAL VACCINE CANDIDATES. INTERNATIONAL PASTEURELLACEAE SOCIETY CONFERENCE. 2002. ABSTRACT p. 35, #34.
- TOWNSEND, K.M., GUNAWARDANA, G.A., BRIGGS, R.E. PCR IDENTIFICATION AND TYPING OF PASTEURELLA MULTOCIDA: AN UPDATE. INTERNATIONAL PASTEURELLACEAE SOCIETY CONFERENCE. 2002. Abstract p. 26, #13.
- FULTON, R.W., RIDPATH, J.F., SALIKI, J.T., BRIGGS, R.E., CONFER, A.W., BURGE, L.J., PURDY, C.W., LOAN, R.W., DUFF, G.C., PAYTON, M.E. BOVINE VIRAL DIARRHEA VIRUS (BVDV) 1B: PREDOMINANT BVDV SUBTYPE IN CALVES WITH RESPIRATORY DISEASE. CANADAIAN JOURNAL OF VETERIANRY RESEARCH. 2002. 66(3). p. 181-190.
- FULTON, R.W., STEP, D.L., RIDPATH, J.F., SALIKI, J.T., CONFER, A.W., JOHNSON, B.J., BRIGGS, R.E., HAWLEY, R.V., BURGE, L.J., PAYTON, M.E. RESPONSE OF CALVES PERSISTENTLY INFECTED WITH NONCYTOPATHIC BOVINE VIRAL DIARRHEA VIRUS (BVDV) SUBTYPE 1B AFTER VACCINATION WITH HETEROLOGOUS BVDV STRAINS IN MODIFIED LIVE VIRUS VACCINES AND MANNHEIMIA HAEMOLYTICA BACTERIN-TOXOID. VACCINE. 2003. v. 21. p. 2980-2985.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Respiratory tract diseases are the leading cause of loss from disease in the cattle, sheep, and goat industries. Annual loss in the U.S. is estimated to exceed one billion dollars for cattle alone. Viral agents are well recognized as primary pathogens and even uncomplicated infections can cause substantial economic loss. Infectious Bovine Rhinotracheitis Virus (IBRV), Respiratory Syncytial Virus (RSV), Parainfluenza Type 3 Virus (PI3V), and Bovine Viral Diarrhea Virus (BVDV) are generally considered the most economically important viruses. A number of commercial viral vaccines are currently available, yet the magnitude and complexity of the problem still exists. A portion of this problem is attributable to Respiratory Coronavirus and Adenoviruses, which have not been extensively evaluated. Respiratory viruses also predispose animals to costly bacterial pneumonia caused by Mannheimia
haemolytica, Pasteurella multocida, and Haemophilus somnus, which commonly affect beef cattle. P. multocida and M. haemolytica commonly affect dairy cattle. These bacteria remain the major cause of loss from mortality, reduced feed efficiency, treatment costs, and reduced product quality. Commercial vaccines are available for bacterial respiratory disease, but their efficacy in the field is questionable. An underlying problem is the current beef cattle marketing system, which does not allow timely vaccination before the peak period of disease, occurring soon after shipping. Antibiotic usage at this time is prevalent, costly, but beneficial. Vaccination in sheep and goats is complicated by the broad number of M. haemolytica serovars against which protection is necessary. 2. How serious is the problem? Why does it matter? Annual loss in the U.S. to respiratory disease is estimated to exceed one billion dollars from mortality, reduced feed efficiency, slaughter condemnations, and
prevention and treatment measures. Control of respiratory disease of cattle, sheep, and goats is currently based on vaccines, which are not particularly effective, and on antibiotics, which are both costly and potentially dangerous to long-term public health. Death losses to the cattle industry from respiratory disease are currently about 1% of low-risk cattle and about 4% of high-risk cattle. Animals, which survive, about 10% and 40% of the respective risk groups, do not grow well and result in a lower quality product. Cattle with old lesions of pneumonia at slaughter have a reduced carcass quality. Wild populations of bighorn sheep can suffer total die-off by respiratory disease, and means to protect these populations are needed to preserve genetic diversity essential to wildlife species. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 103 - Animal Health (100%). This research supports the
Host/Pathogen Interactions and Disease Control Strategies components. An understanding of the molecular basis of pathogenesis is essential to the development of effective intervention strategies to control bacterial respiratory disease. With the identification of key microbial and host molecules involved in disease progression, the design of effective vaccines and therapeutics should be possible. The goal in the design of these products is for a rapid response to allow effective use in feeder beef cattle already in marketing channels, for reduced adverse local reactions to control carcass damage, and to reduce our dependence on costly antibiotic treatments. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: To better understand disease progression and to produce better vaccines, a temperature-conditional plasmid shuttle vector was previously constructed and used to construct genetically-modified organisms of the
Family Pasteurellaceae. At the National Animal Disease Ccenter (NADC), the mutated origin-of-replication was characterized by genetic sequencing and was found to contain 2 nucleotide substitutions within a 100 bp region compared to the unaltered wild-type origin-of-replication. It is likely a single nucleotide substitution is responsible for the altered characteristics of the temperature-conditional plasmid and the second substitution is inconsequential. A rapidly effective formulation may be developed for oral administration that could replace most or all of the current vaccines which use hypodermic injection. B. Other Significant Accomplishment(s), if any: none. C. Significant Accomplishments/Activities that Support Special Target Populations: none. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? This is a new project based on a Project Plan certified by the Office of Scientific Quality Review, January 18, 2002,
replacing 3625-32000-044-00D. a) Anionic peptides (APs) are small anionic antimicrobial peptides composed of 7 aspartic acid residues and are produced in the lungs of humans, sheep, and cattle. Although expression by epithelial cells of some antimicrobial peptides of humans and ruminants is increased in response to acute infection, AP expression is not increased during acute infection, which suggests that the expression of this peptide is constitutive. The degree of AP expression during the progression (acute, subacute, and chronic) of bronchopneumonia was determined in Mannheimia (Pasteurella) haemolytica inoculated sheep. In the acute group (1 day p. i.), the lungs had lesions typical of bronchopneumonia, and the distribution and intensity of AP immunoreactivity (AP-IR) were similar to those of previous studies. In the subacute group (15 days p.i.), there was prominent hyperplasia of bronchiolar and alveolar epithelial cells, and the chronic group (45 days p.i.) had yet more
pronounced hyperplasia. In the subacute and chronic groups, the intensity and distribution of AP- IR in the cytoplasm of hyperplastic bronchiolar and type II alveolar cells were significantly increased compared to those of saline-inoculated and contralateral (noninoculated) lung lobes. This increased production of AP by hyperplastic cells may protect the lung against further infection until new, fully differentiated epithelial cells are capable of expressing their own inducible array of antimicrobial peptides. b) Surfactant Protein D (SP-D) is a hydrophilic pulmonary surfactant collagenous calcium-dependent lectin which binds to surface glycoconjugates expressed on Gram-negative bacteria, Influenza A virus, and various fungi. Since a hallmark of bronchopneumonia is the initiation of inflammation in the bronchi and bronchoalveolar junction, we chose a model of bronchopneumonia caused by Mannheimia haemolytica to study the expression of SP-D within the bronchioles of infected lambs.
At 15 and 45 days post-inoculation, areas of lung with increased peribronchiolar inflammatory cell infiltrate, epithelial cell hyperplasia, and tortuosity of the airway lumens had decreased intensity of SP-D staining and number of positive cells. In conclusion, cell-associated SP- D protein expression significantly decreases within the hyperplastic epithelium of lungs from the infected animals during chronic pneumonia. c) In the summer and fall of 1993, a newly recognized disease, adenoviral hemorrhagic disease, caused widespread mortality in black-tailed and California deer. Over a thousand deer were estimated to have died. The adenovirus associated with the clinical disease was isolated and characterized, and clinical disease was reproduced experimentally. Observation of the interface between captive deer and free ranging squirrels prompted a preliminary experimental inoculation study to determine if squirrels are capable of carrying and shedding the deer adenovirus. d) Deer
adenovirus produces lesions in blood vessels that can be extremely lethal for mule deer. To characterize the virus, part of the virus genetic material was examined. Analysis of the DNA revealed similarities to several other adenoviruses. The highest similarity was found with the members of the ovine adenovirus isolate 287, bovine adenovirus 4, and duck adenovirus 1. e) Adenoviruses also produce respiratory and enteric disease in cattle. The bovine adenoviruses have been isolated from healthy cattle but more frequently the isolations are associated with some form of clinical disease. Bovine adenovirus 7 was isolated from the blood cells of 8 calves, and antibodies to bovine adenovirus were detected in 38 of 199 (19.1%) calves studied. Results indicate that bovine adenovirus infections can be found in postweaning commingled calves and may be potentially a systemic infection and disease rather than a respiratory mucosal infection alone. 6. What do you expect to accomplish, year by
year, over the next 3 years? Year 1: a) conduct site-directed mutagenesis to determine the relationship of specific nucleotide substitutions to temperature- sensitivity; b) develop sensitive PCR assay for detection of M. haemolytica/P. multocida colonization of mucosal surfaces; c) characterize newly isolated untyped adenoviruses from cattle, sheep and goats; and d) isolate beta defensins from the neutrophils and tracheal epithelium of cattle and sheep, respectively. Year 2: a) construct M. haemolytica serotypes 1, 2, and 10 which express an immunogenic 'leukotoxoid' only 30% the size of the active protein; b) construct M. haemolytica serotype 1 with small 100 amino acid deletions in the N-terminal region of the encoded LktA product for structure/function analysis of the leukotoxin protein with bovine lymphocytes; c) develop improved PCR primers for the Mastadenovirus genus; and d) immunize lambs or calves with defensin and antigens co-mixtures to assess the ability of defensins to
induce and direct adaptive immune responses to select M. haemolytica antigens. Year 3: a) target specific genes with probable relevance to virulence in the Pasteurellaceae for inactivation. These will be useful for elucidation of these genes' role in disease pathogenesis; b) develop improved diagnostic and epidemiologic tools for detection and better understanding of the significance of adenoviral disease in ruminants; and c) assess the immune response of lambs or calves immunized with defensin and antigens co-mixtures to see if defensins induce and direct adaptive immune responses to select M. haemolytica antigens. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? a) Consulted and assisted an animal health industry on design, construction, and evaluation of vaccine candidates. b) Chaired
NC-107 Committee; prepared meeting report. c) Conducted informal cooperative research with industry and with other ARS and university investigators and consulted on Pasteurella and Mannheimia haemolytica classification and typing. d) Collaborated with Hungarian scientists on adenovirus taxonomic classification. e) Provided adenovirus diagnostic assistance (isolation of the virus, serotyping, serologic testing) to scientists in domestic and foreign diagnostic laboratories. f) Provided APHIS, ARS, and university researchers with primary sheep and deer cell cultures. g) Provided technical assistance on deer adenovirus hemorrhagic disease to several state Departments of Fish and Game. h) Provided technical assistance on parainfluenza virus type 3 and bovine respiratory syncytial virus to scientists at a state university to study immune reaction to virus infection in the lung of small ruminants. i) Participated in the Detecting and Controlling BVDV Infections Conference. Discussed
research on BVDV with scientists and industry representatives. j) Attended the XIIth International Congress of Virology in Paris, France. Presented a paper on current ruminant adenovirus research and as a committee member, participated in discussions on adenovirus taxonomy, classification, and diagnosis. k) Served as reviewers for manuscripts submitted to refereed national and international scientific publications.
Impacts
(N/A)
Publications
- Parker, J., Woods, L.W., Lehmkuhl, H.D., Stillian, M.H. Preliminary Investigation of squirrels as a possible reservoir for deer adenovirus. Wildlife Disease Conference. 2002. p. 98. Abstract #74.
- Brogden, K.A. Polymicrobial diseases of animals and humans. Brogden, K.A., Guthmiller J.M., editors. ASM Press, Washington, DC. Polymicrobial Diseases. 2002. p. 3-20.
- Brogden, K.A., Guthmiller, J.M. Polymicrobial diseases: current and future research. Brogden, K.A., Guthmiller J.M., editors. ASM Press, Washington, DC. Polymicrobial Diseases. 2002. p. 403-410.
- Drake, D.R., Brogden, K.A. Continuous culture chemostat systems and flowcells as methods to investigate microbial interactions. Brogden, K.A., Guthmiller J.M., editors. ASM Press, Washington, DC. Polymicrobial Diseases. 2002. p. 21-30.
- Fales-Williams, A.J., Gallup, J.M., Ramirez-Romero, R., Brogden, K.A., Ackermann, M.R. Increased anionic peptide distribution and intensity during progression and resolution of bacterial pneumonia. Clinical Diagnostic Laboratory Immunology. 2002. v. 1. p. 28-32.
- Fent, G.M., Fulton, R.W., Saliki, J.T., Caseltine, S.L., Lehmkuhl, H.D., Confer, A.W., Purdy, D.W., Briggs, R.E., Loan, R.W., Duff, G.C. Bovine Adenovirus serotype 7 infections in postweaning calves. American Journal of Veterinary Research. 2002. v. 63(7). 976-978.
- Grubor, B., Ramirez-Romero, R., Barua, A.B., Gallup, J.M., Crouch, E., Brogden, K.A., Ackermann, M.R. Surfactant protein D expression in normal and pneumonic ovine lung. 102nd American Society for Microbiology. 2002. p. 189. Abstract #E-12.
- Zakhartchouk, A., Bout, A., Woods, L.W., Lehmkuhl, H.D., Havenga, J.E. Odocoileus hemionus deer adenovirus is related to the members of Atadenovirus genus. Archives of Virology. 2002. v. 147(4). p. 841-847.
- Swift, P.K., Woods, L.W., Lehmkuhl, H.D., Jones, K.R. An update of adenoviral hemorrhagic disease in Mule Deer in California. Wildlife Disease Conference. 2002. p. 144. Abstract #115.
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