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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Research Project #432020

Research Project: Identification of Disease Mechanisms and Control Strategies for Bacterial Respiratory Pathogens in Ruminants

Location: Ruminant Diseases and Immunology Research

2019 Annual Report

Objective 1. Define the virulence determinants and mechanisms involved with the principal bacteria associated with bovine respiratory disease, including identifying microbial mechanisms used by commensal bacteria to become pathogens, and identifying the mechanisms of bacterial colonization in target hosts. Subobjective 1.1: Identify microbial mechanisms used by commensal bacteria to become pathogens. Subobjective 1.2: Identify the mechanisms of bacterial colonization of the host. Objective 2. Determine the host-pathogen interactions associated with respiratory infections, including developing animal disease models to study respiratory disease complex, identifying the host factors that drive the early innate immune response to bacterial respiratory infections, and characterizing functional genomics of the host associated with respiratory infection. Subobjective 2.1. Continue the development of animal disease models to study respiratory disease complex. Subobjective 2.2. Identify the host factors that drive the early innate immune response to bacterial infection. Subobjective 2.3. Characterize functional genomics of the host associated with respiratory infection. Objective 3: Develop intervention strategies to reduce antibiotic use, including developing vaccines that will induce early mucosal immunity in young animals, and developing and evaluating immune-modulators to prevent and/or treat respiratory disease. Subobjective 3.1. Develop vaccines that induce early mucosal immunity in young animals. Subobjective 3.2. Develop and evaluate immune modulators to prevent and/or treat respiratory disease.

Binding of bacteria to mucosal surfaces, and evasion of host innate and adaptive immunity are critical to successful colonization and maintenance of infection. Identification of key molecular players in these interactions should enable potentially effective intervention strategies. We will utilize a coordinated and multipronged approach to characterize molecular mechanisms promoting respiratory bacterial pathogen colonization, adherence, and persistence in cattle. We plan to use experimental ruminant models and specific mutants to describe molecular mechanisms enabling bacteria to colonize, adhere and grow in the respiratory tract, and to examine the influences of primary viral infection on secondary bacterial infections. While much knowledge has been gained regarding the individual pathogens involved in BRDC, less is known concerning co-infections involving bacterial and viral respiratory pathogens. Given the expertise of our research team, and specific etiologic agent prevalence in the field, we will focus on BVDV and BRSV as the viral pathogens, and Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis as the bacterial agents. We plan to continue the development of reproducible models of viral predisposition to bacterial disease and to characterize the host and infectious agents’ response using a comprehensive approach. Bacterial genes or gene products identified in these studies will be used, based on their importance in colonization, for developing and testing novel vaccines. Furthermore, we will examine the potential of immunomodulators to enhance the host response to infection with respiratory pathogens. The overriding goal of this plan is to develop preventative measures that aid in the reduction or elimination of BRDC in beef and dairy cattle. Reductions in BRDC will be of substantial economic benefit to cattle producers. However, as specific bacterial pathogens involved in BRDC are significant causes of morbidity and mortality in wild ruminant populations, there are aspects of this research plan that include those species. For example, bighorn sheep suffer severe die-offs as a result of respiratory disease and it is considered the major factor impacting the long-term sustainability of bighorn sheep populations. Moreover, M. bovis has additionally emerged in North American bison, causing substantial economic losses to producers and threatening the stability of heritage herds. Therefore, strategies to reduce respiratory disease in wild ruminant populations will be of substantial value to the public interest in sustaining wildlife populations, as well as reduce economic losses to bison producers.

Progress Report
In support of Objective 1, ARS researchers at Ames, Iowa, conducted a calf trial designed to document colonization and shedding of Mannheimia haemolytica (M. haemolytica) mutants in conjunction with bovine herpesvirus-1. The BHV-1 infection was found to enhance shedding of both serotypes 1 and 6 M. haemolytica, as well as a Mannheimia varigena (M. varigena) which was part of the normal flora of some of the experimental calves upon arrival at the NADC. Genome sequence of the M. varigena isolate confirmed its identity and showed an intact leukotoxin gene consistent with its hemolytic phenotype. Lung challenge trials are required to confirm whether this M. varigena may be an emerging pathogen in cattle. Whole genome libraries have been prepared for 40 bison isolates, 14 cattle isolates and 2 deer isolates of Mycoplasma bovis, and for 4 isolates of Mycoplasma bovirhinis. Mycoplasma bovis isolates from cattle appear to be rapidly evolving and diversifying since we continue to discover many novel genetic groups known as multilocus sequence types among this bacterial species. Although bison isolates appear to be evolving far less rapidly, we have identified a few novel sequence types among recent isolates, including some that were previously found only in cattle. This could signal the introduction into the bison population of Mycoplasma bovis clones that were previously restricted to cattle. Conversely, we have now identified in cattle sequence types previously found only in bison. In the U.S., sequence types appear to only rarely be shared between bison and cattle, while in Canada there is much more extensive sharing of genotypes between these hosts. The scope of our genomics-based studies continues to expand through collaborations with the University of Saskatchewan, Canada, South Dakota State University, Kimron Veterinary Institute (Israel) and the Ministry of Primary Industries (New Zealand), with whom we are engaged in a worldwide, genome-based comparison of several hundred isolates of Mycoplasma bovis. Related to this effort, during FY19 we implemented a new MTA/CA with the New Zealand Ministry for Primary Industries. Enzyme-linked immunosorbent assay testing has been completed for over 3000 sera from healthy bison in the U.S. and Canada, collected between 1984 and 2019. These samples represent 31 different bison farms, herds or locations, some public and some private, including Yellowstone and other federally managed herds in the U.S. and several federally or provincially managed herds in Canada. Data collation has just begun, but it appears only about 5 percent of bison have antibodies indicative of prior exposure to M. bovis. Positive samples include sera collected as early as 1988, many years prior to recognizing the emergence of M. bovis as a disease threat in bison. Nasal swabs were collected at the time of bleeding for several hundred U.S. bison sampled between 2012 and 2019. Only one was culture- or PCR-positive for M. bovis, from a herd that had experienced an outbreak roughly 6 months prior. While preliminary, these data suggest the prevalence of M. bovis in healthy bison may be relatively low. Supporting Objective 2, ARS researchers at Ames, Iowa, have cloned several bovine Toll-like receptors (TLRs), boTLR2, boTLR4, MD2, and CD14. In order to pinpoint the role of each of these molecules in inducing proinflammatory cytokine expression, a cell is needed which does not already contain these molecules. A human cell line, which does not normally express TLRs and associated proteins has been transfected with the respective vectors containing the genes to generate boTLR2/MD2/CD14- and boTLR4/MD2/CD14-expressing cells. Human cell lines expressing boTLRs were generated, however, the cell lines failed to maintain stable expression of respective genes. ARS researchers at Ames, Iowa have also cloned, expressed and purified lipoproteins from both Pasteurella multocida and Mycoplasma bovis. Therefore, effects of bacterial lipopolysaccharides and lipoproteins on proinflammatory cytokine expression will be performed using cattle peripheral blood mononuclear cells. These studies will provide important information on bacterial molecules that stimulate proinflammatory cytokine expression in bovine cells. Such information could be used to development treatment modalities which reduce inflammation that occurs during bovine respiratory disease. Initial efforts were made to identify differentially expressed genes associated with bovine respiratory disease complex in different tissues. An experiment was conducted with the objective to establish whether differences in gene expression in palatine tonsil and retropharyngeal lymph node occur when calves were colonized by Mannheimia haemolitica (MH) or Pasteurella multocida (PM). There were 32 differentially expressed genes detected between the MH and control calves, and 70 differentially expressed genes observed between the PM and controls. There were 69 differentially expressed genes seen when the comparison was made between the combined MH and PM groups, with the control group. There were 14 genes that were differentially expressed in all contrasts. Genes identified as differentially expressed in these tissues with these pathogens, need to be replicated, to establish if these genes are potential candidate markers to develop intervention strategies with the objective to minimize the negative effects of bovine respiratory disease. In support of Objective 3, an experimental vaccine for Mycoplasma bovis was previously constructed by ARS researchers at Ames, Iowa. The vaccine was evaluated for safety and efficacy, using a single intranasal dose, in both cattle and bison. Following virulent challenge, vaccinated animals exhibited reduced lung lesion, middle ear infection, and joint infection. Infectious Mycoplasma lung load was reduced among vaccinates regardless of lung lesion status. A mouse model of infection was used for preliminary evaluation of several small antimicrobial proteins against Histophilus somni. The proteins were found to elicit up to 100 percent control of infection in this model. The most effective protein is currently being evaluated in a calf pneumonia model of infection. A potential new vaccine for bovine respiratory syncytial virus and human respiratory syncytial virus has been tested by ARS researchers at Ames, Iowa, in collaboration with researchers at Rutgers University and Mount Sinai Medical School. The vaccine utilizes an attenuated Newcastle disease virus containing a gene from bovine respiratory syncytial virus. Studies published using the vaccine in mice have shown the vaccine to provide protection. Preliminary vaccination and challenge trials in calves have now been conducted and the vaccine was found to provide incomplete protection. Thus, results of these studies in cattle have provided researchers with data that indicates that dosage of the vaccine needs to be re-evaluated for larger animal species. ARS researchers at Ames, Iowa, conducted new trials using a 5-fold higher dose of the vaccine and administered two doses three weeks apart. Vaccinated calves were found to have reduced lung lesions following virulent challenge compared to non-vaccinated calves. In addition, viral load in the lungs was reduced in vaccinates. Recently, ARS researchers have determined that vaccinated calves have reduced gene expression levels of inflammatory mediators. Additional experiments were undertaken this year repeat the studies, with similar protection observed. Nasal washes collected from these studies are being evaluated for mucosal antibody induction by the vaccine.

1. Role of disulfide bonds in host white blood cell-derived small proteins in the killing of respiratory bacteria. Previous studies indicated that small host white blood cell-derived proteins can effectively kill most bovine respiratory bacteria. The goal of one study this year was to identify whether these small proteins form intrachain disulfide bonds or whether such bonds were important for antimicrobial activities. ARS researchers at Ames, Iowa have compared a small protein with two cysteine residues, where two cysteines were replaced with other amino acid residues. Using several techniques, it was shown that the small proteins do not form intra-chain disulfide bonds and disulfide bonds appeared not to be essential for potent antimicrobial activity. The small protein showed improved protection in a mouse model of bovine respiratory disease. Therefore, this protein may prove useful as a treatment to reduce the severity of bovine respiratory disease complex. Pharmaceutical companies looking to develop alternatives to antibiotics for the treatment of bovine respiratory disease will benefit from these findings.

2. Novel nanoparticle vaccine for bovine respiratory syncytial virus fails to protect vitamin A deficient calves. Nanoparticle-based vaccines have shown promise as vaccine delivery vehicles and to potentiate the immune response due to their ability to provide sustained release of pathogen molecules and to induce both antibody- and cell-mediated immune responses. ARS researchers at Ames, Iowa, in collaboration with researchers at Kansas State University and Iowa State University, have developed a novel nanovaccine delivered via the intranasal route. Calves receiving this vaccine exhibited reduced lung lesions, reduced viral burden, and decreased virus shedding compared to unvaccinated calves. However, it was found that vitamin A deficient calves were not protected by the nanovaccine. These data show that in producing vaccine-ready calves, proper nutritional balance is critical to a successful vaccination program. These findings will be used by ruminant nutritionists, veterinarians, farmers and vaccine manufacturers in reducing the negative effects of bovine respiratory disease.

Review Publications
Falkenberg, S.M., Dassanayake, R.P., Walz, P., Casas, E., Neill, J.D., Ridpath, J.F. 2019. Frequency of bovine viral diarrhea virus detected in subpopulations of peripheral blood mononuclear cells in persistently infected animals and health outcome. Veterinary Immunology and Immunopathology. 207:46-52.
Guerra-Maupome, M., Palmer, M.V., McGill, J.L., Sacco, R.E. 2019. Utility of the neonatal calf model for testing vaccines and intervention strategies for use against human RSV infection. Vaccines. 7(1):7.
Lippolis, J.D., Powell, E.J., Reinhardt, T.A., Thacker, T.C., Casas, E. 2019. Symposium review: omics in dairy and animal science-promise, potential, and pitfalls. Journal of Dairy Science. 102(5):4741-4754.
Bennett, G.L., Tait, R.G., Shackelford, S.D., Wheeler, T.L., King, D.A., Casas, E., Smith, T.P.L. 2019. Enhanced estimates of carcass and meat quality effects for polymorphisms in myostatin and mu-calpain genes. Journal of Animal Science. 97(2):569-577.