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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

2018 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, Subobjective 1.1c, over the past year the collection of Mycoplasma bovis isolates has grown to include 98 from bison and over 200 from cattle. These were primarily obtained from animals with mycoplasmosis but nasal cavity isolates from healthy carriers were also acquired. All isolates have been typed by multilocus sequence typing. Isolates from cattle appear to be rapidly evolving and diversifying since we continue to discover many novel MLST sequence types among this group. In contrast, only two novel sequence types have been identified among recent bison isolates, including one that was previously found only in cattle. This could signal the introduction into the bison population of M. bovis clones that were previously restricted to cattle. M. bovis and M. bovirhinis isolates to be sequenced by the ARS have been selected. The scope of our genomics-based studies has recently been expanded through newly formed collaborations with the University of Saskatchewan, South Dakota State University, Kimron Veterinary Institute (Israel) and the government of New Zealand, with whom ARS researchers in Ames, Iowa plan to engage in a worldwide, genome-based comparison of several hundred isolates of M. bovis. In support of Objective 1, Subobjective 1.2b, ELISA testing of 172 sera collected between 2012 and 2015 from bison in six herds managed by the U.S. Fish and Wildlife Service has been completed and it was found that only 3 tested positively. While M. bovirhinis was recovered from the nasal cavity of 44 percent of bison sampled from these same herds, M. bovis was not recovered from any animal. ELISA testing has been initiated on an additional group of several hundred sera obtained from bison in the U.S. and Canada and are continuing to acquire both sera and nasal swabs from additional bison, including those in the U.S. Fish and Wildlife herds. In support of Objective 2, Subobjective 2.2, relevant bovine genes, such as boToll-like receptor (boTLR)2 boTLR4, MD2, and CD14 have been cloned. A human cell line, which does not normally express these proteins has been transfected with the respective vectors containing the genes to generate boTLR4/MD2/CD14-expressing cells. We are currently working to generate TLR2 expression cells. Once all stable cell lines are established, relevant studies utilizing these cell lines will be conducted. In support of Objective 2, Subobjective 2.3, calves were co-infected with Mycoplasma bovis and/or Bovine Viral Diarrhea Virus (BVDV) in two experiments that were conducted to characterize host functional genomic responses to infection. Samples collected at necropsy included serum, retropharyngeal lymph node, palatine tonsil, liver, mesenteric lymph node, tracheal-bronchial lymph node, spleen and thymus. To date, different ribonucleic acids have been extracted from all of these tissues at necropsy and RNA sequencing and data analyses are in progress. In support of Objective 3, Subobjective 3.1a, codon-optimized DNA fragments encoding putative protective sequences of Mycoplasma bovis, Mycoplasma ovipneumoniae, BVDV, Mycobacterium bovis, Histophilus somni, have been designed, cloned and expressed in Mannheimia haemolytica by ARS researchers at Ames, Iowa. A Mycoplasma bovis construct of this work is currently undergoing evaluation in a cattle efficacy trial. In support of Objective 3, Subobjective 3.1b, though we have been unable to generate a double mutant of Histophilus, we were able to generate a different double mutant that is suitable for vaccine efficacy evaluation. In addition, a Mannheimia strain which expresses an immunogenic fragment of a protein, which is also suitable for evaluation as a vaccine has been generated.

1. Killing of respiratory bacteria by host white blood cell-derived small proteins. Previous studies indicated that white blood cell-derived small proteins can kill respiratory bacteria. The question to be investigated this year was whether these small proteins can kill Mycoplasma bovis, an important respiratory bacterium in cattle. ARS researchers at Ames, Iowa, have compared the ability of certain small proteins produced by bovine white blood cells to kill bacteria associated with bovine respiratory disease complex. Of the four small proteins examined, two were found to be very effective in killing M. bovis. Using several techniques, it was shown that this protein was able to kill by causing structural damage to the membrane. It will be important to evaluate the biological activity of this protein in cattle to determine whether it reduces the levels of respiratory pathogens.

2. Identified and solved problem with current diagnostic laboratory test that leads to false-negative results for Mycoplasma bovis, an important respiratory pathogen. Among the key tests employed to identify isolates as M. bovis is a widely used polymerase chain reaction (PCR) assay developed and reported by clinicians at the Iowa State University Veterinary Diagnostic Laboratory. The intended target of the PCR is a DNA repair gene, known to be highly conserved among isolates. Upon investigating the reason for negative results with some isolates confirmed to be M. bovis on the basis of 16S RNA gene sequence, ARS researchers in Ames, Iowa, discovered that the PCR primers were mistakenly selected from a poorly characterized gene immediately adjacent to the DNA repair gene, predicted to encode a lipoprotein. Further analysis of over 200 isolates revealed the presence of nucleotide changes in one of the primer-binding regions and a gene insertion within the amplified region that leads to a false negative result. These recently published findings are of importance to clinicians using this diagnostic PCR, who should be aware that the amplified fragment lies within a gene that appears to be less well-conserved than the intended target and that occasional false negative results may be obtained.

3. Defined a role for Pasteurella multocida capsule in biofilm formation. Pasteurella multocida is an important animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The capsule of P. multocida, which covers the outer layer of the cell wall, is an essential disease-causing factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals) may be associated with biofilm formation, which has not been characterized in P. multocida. A biofilm is an assemblage of microorganisms that stick to each other and to many different types of surfaces. Biofilm formation by clinical isolates was found to be inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. In collaboration with researchers from Virginia Tech, ARS researchers at Ames, Iowa, created capsule-deficient mutants that were found to form biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. The capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the encasement of large numbers of bacterial cells. This is the first detailed description of biofilm formation by P. multocida, which was found to contain a newly reported sugar. Biofilm formation may make some respiratory infections hard to deal with and by determining factors involved in biofilm formation may lead to more effective methods for their treatment.

Review Publications
Elnaggar, M.M., Abdellrazeq, G.S., Dassanayake, R.P., Fry, L.M., Hulubei, V., Davis, W.C. 2018. Characterization of alpha beta and gamma delta T cell subsets expressing IL-17A in ruminants and swine. Developmental and Comparative Immunology. 85:115-124.
Dassanayake, R.P., Falkenberg, S.M., Register, K.B., Samorodnitsky, D., Nicholson, E.M., Reinhardt, T.A. 2018. Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis. PLoS One. 13(5):e0197677.
Falkenberg, S.M., Dassanayake, R.P., Neill, J.D., Ridpath, J.F. 2018. Evaluation of bovine viral diarrhea virus transmission potential to naïve calves by direct and indirect exposure routes. Veterinary Microbiology. 217:144-148.
Petruzzi, B., Briggs, R.E., Tatum, F.M., Swords, W.E., De Castro, C., Molinaro, A., Inzana, T.J. 2017. Capsular polysaccharide interferes with biofilm formation by Pasteurella multocida Serogroup A. mBio. 8(6):e01843-17.
Silveira, S., Falkenberg, S.M., Elderbrook, M.J., Sondgeroth, K.S., Dassanayake, R.P., Neill, J.D., Ridpath, J.F., Canal, C.W. 2018. Serological survey for antibodies against pestiviruses in Wyoming domestic sheep. Veterinary Microbiology. 219:96-99.
Taxis, T.M., Casas, E. 2017. MicroRNA expression and implications for infectious diseases in livestock. Centre for Agriculture and Biosciences International. 12(26):1-20.
Eberle, K.C., Venn-Watson, S.K., Jensen, E.D., Labresh, J., Sullivan, Y., Kakach, L., Sacco, R.E. 2018. Development and testing of species-specific ELISA assays to measure IFN-gamma and TNF-alpha in bottlenose dolphins (Tursiops truncatus). PLoS One. 13(1):e0190786.
Hofstetter, A.R., Eberle, K.C., Venn-Watson, S.K., Jensen, E.D., Porter, T.J., Waters, T.E., Sacco, R.E. 2017. Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR. PLoS One. 12(12):e0189437.
Register, K.B., Boatwright Jr, W.D., Gesy, K.M., Thacker, T.C., Jelinski, M.D. 2018. Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance. Journal of Veterinary Diagnostic Investigation. 30(4):637-641.
Casas, E., Cai, G., Kuehn, L.A., Register, K.B., McDaneld, T.G., Neill, J.D. 2018. Association of circulating transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle. BioMed Central (BMC) Veterinary Research. 14:89.
McGill, J.L., Kelly, S.M., Kumar, P., Speckhart, S., Haughney, S.L., Henningson, J., Narasimhan, B., Sacco, R.E. 2018. Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf. Scientific Reports. 8:3021.