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





- Dassanayake, Rohana
- Casas, Eduardo
- Ma, Hao
- Kaplan, Bryan
- Nielsen, Daniel
- Sacco, Randy
- Tatum, Fred
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Host-pathogen Interactions and Control Strategies for Bacterial Respiratory Pathogens in Cattle **Location:
                            Ruminant Diseases and Immunology Research** 2022 Annual Report **Objectives**
                                

Objective 1: Define the virulence determinants and mechanisms used by Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Mycoplasma mycoides cluster agents to cause disease in ruminant species.
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 infection with Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Mycoplasma mycoides cluster agents, including development of animal models.
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 prevent or treat respiratory infections that minimizes the development of antibiotic resistant bacteria. This includes the development of easily administered vaccines and developing and evaluating immune-modulators to prevent and/or treat respiratory disease.
Subobjective 3.1: Develop and test vaccines that induce early immunity in young animals.
Subobjective 3.2: Develop and test vaccines that induce early mucosal immunity in young animals.
Objective 4: Following identification of virulence determinants, utilize synthetic genome and other approaches to engineer Mycoplasma mycoides cluster agents for enhancing the understanding of disease pathogenesis and for use as potential vaccines.
Objective 5: Determine the role of surface lipoproteins for vaccine enhancement of disease in Mycoplasma mycoides subsp. mycoides small colony. **Approach**
                                

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 plan to utilize a coordinated, multipronged approach to characterize molecular mechanisms promoting respiratory bacterial colonization, adherence, and persistence in cattle. While much knowledge has been gained from studying individual pathogens, less is known concerning co-infections involving bacterial and viral pathogens. Given the expertise of our research team, we will focus on BHV-1 and BRSV as the viral pathogens, and Mannheimia haemolytica, Pasteurella multocida, and Mycoplasma bovis as the bacterial agents. Mycoplasma mycoides was added to this project by the USDA Animal Health NPLs in response to congressional appropriations. We will continue the development of experimental animal models and specific mutants to describe molecular mechanisms enabling bacteria to colonize the respiratory tract, and examine influences of primary viral infection on secondary bacterial infections. Bacterial genes or gene products so identified, will be used for developing and testing novel vaccines and/or immunomodulators. The overriding goal is to reduce or eliminate BRDC, which will substantially benefit producers. However, specific pathogens involved in BRDC can cause significant disease in wild ruminants, there are aspects of this plan that include isolates from those species. For example, M. bovis has emerged in bison, causing substantial economic losses and threatening stability of heritage herds. Therefore, strategies to reduce respiratory disease in wildlife will be valuable to the public interest in sustaining these populations, as well as reduce economic losses to producers. **Progress Report**
                                

In support of Objective 1, we utilized our proprietary bacterial-vector system to generate putative Mannheimia haemolytica (M. haemolytica) adhesin mutants. Previously, we generated M. haemolytica and Pasteurella multocida (P. multocida) filamentous hemagglutinin, sialic acid, and capsular mutants. The identity of most mutants has been confirmed by polymerase chain reaction, Western blot, and/or sialic acid uptake assays. These mutants will be tested in future animal studies to assess their role in colonization in the upper respiratory tract.

In support of Objective 2, we successfully purified lipopolysaccharides (LPS) from wildtype M. haemolytica which is currently being tested for purity and concentration. Pro- and anti-inflammatory cytokine and housekeeping gene-specific primers were designed and the specificity of primers were tested on cellular ribonucleic acid (RNA) prepared from bovine peripheral blood mononuclear leukocytes. Quantitative polymerase chain reaction assay was performed and specific primer pairs without any non-specific signals were identified. These specific primers will be used to identify the pro- and anti-inflammatory cytokine gene expression patterns of extracted cellular RNA from bovine leukocytes which were stimulated with purified lipopolysaccharides and lipoproteins. Once the protocol is optimized to extract LPS from Pasteurella species, LPS from a M. haemolytica sialic acid mutant and P. multocida wild-type and sialic acid mutants will be prepared. These studies will provide important information on bacterial molecules that stimulate pro- and/or anti-inflammatory cytokine expression in bovine leukocytes.

In support of Objective 2, blood samples were collected from animals challenged with a vaccine platform based on Mannheimia haemolytica, expressing Mycoplasma mycoides antigens. Animals were allocated in a control group, a group vaccinated with an inactive form of M. haemolytica, and a group vaccinated with M. haemolytica expressing Mycoplasma mycoides antigens. RNA will be extracted and sequenced from the blood of each animal. Messenger and small non-coding RNA will be sent to sequencing. The goal is to understand, at the molecular level, how this vaccine is protecting cattle against pathogens producing respiratory disease. 

In support of Objective 2, Ribonucleic acid (RNA) was extracted from animals challenged with bovine viral diarrhea virus and Mycoplasma bovis. The objective was to establish a relationship between expression of non-coding RNAs and messenger RNA. Animals were allocated to a control group, a group challenged with Mycoplasma bovis, and a group first challenged with bovine viral diarrhea virus, and then with Mycoplasma bovis. After euthanasia, blood, liver, spleen, thymus, and retropharyngeal, mesenteric, and tracheobronchial lymph nodes were collected. RNA was extracted and sequenced from each tissue and animal. Messenger and non-coding RNA sequence was obtained. The bioinformatic analysis of non-coding and messenger RNA will be initiated soon.
 
In support of Objective 3, we previously generated and evaluated a modified-live M. haemolytica mucosal vaccine expressing (and secreting) selected gene fragments of Mycoplasma bovis in animals. Calves vaccinated with M. haemolytica expressing M. bovis vaccine payload showed vaccine-induced highly significant protection against M. bovis lung challenge associated with reduced clinical signs, lung lesions, lung bacterial loading, and mortality. Protection was associated with significant systemic and local (mucosal) humoral immune response. The same regions of M. bovis protective gene fragments were codon-optimized to express in a prokaryotic expression system (Escherichia coli), and purified as recombinant proteins. Recombinant proteins will be formulated in a commercially available adjuvant to prepare an injectable vaccine which will be expected to induce rapid and long-lasting immune responses in cattle. Based on this approach,  we have developed a modified-live M. haemolytica mucosal vaccine candidate against Mycoplasma mycoides subspecies mycoides small colony (MmmSC), the causative agent of contagious bovine pleuropneumonia. This modified-live M. haemolytica mucosal vaccine expressing MmmSC immunogenic antigens will be evaluated to determine its protective immune responses against MmmSC challenge.

In support of Objective 3, we produced a new two-way mucosal vaccine to control Mannheimia haemolytica and Mycoplasma ovipneumoniae in bighorn sheep. The live vaccine was transferred to the Colorado Department of Wildlife and it was administered to captive bighorn sheep on feed. Results indicate that the vaccine induced humoral responses to both M. haemolytica and M. ovipneumoniae antigens. Additional testing of this vaccine is planned for later in 2022. **Accomplishments**
                                

1. Comparative study of antibacterial activity and stability of a new antimicrobial peptide. The increasing prevalence of antibiotic resistance among pathogenic microbes highlights the urgent need for the identification and development of reagents for an alternative to antibiotics. Antimicrobial peptides (AMPs) are highly effective against microbial pathogens causing diseases in humans and animals. However, AMPs are sensitive to proteases and kidney clearance. Therefore, ARS researchers in Ames, Iowa, developed a protease-resistant small peptide and tested it for trypsin (a common protease) resistance and serum stability, toxicity, and in vitro and in vivo anti-bacterial activities against Histophilus somni, one of the bacterial organisms causing respiratory diseases in cattle. The peptide was able to kill H. somni very efficiently, showed minimal toxicity, and was resistant to trypsin. These results demonstrate the possible utility of an alternative treatment for the control of bacteria causing respiratory diseases. 2. Development of new mucosal vaccine to control pneumonic Pasteurellosis in cattle. Mannheimia haemolytica remains the largest single cause of disease losses to the cattle industry, and there is strong interest in the cattle industry for improved vaccines against pneumonic Pasteurellosis and mucosal vaccine delivery. A novel vaccine against bovine pneumonic Pasteurellosis caused by Mannheimia haemolytica was developed and tested by ARS researchers in Ames, Iowa. In a calf vaccination/challenge study it was demonstrated that a single intranasal dose of the vaccine confers protection against pneumonic disease following challenge with virulent Mannheimia haemolytica. The protection was associated with major reductions in the amount of the disease agent in lung tissue following challenge, as well as reduced lung damage associated with pneumonic disease. The cattle industry has shown interest in commercialization of this new mucosal vaccine. 3. Inhibition of Interleukin (IL)-17A reduces the inflammatory response to Mannheimia haemolytica lung infection of bovine calves. Mannheimia haemolytica infections are often characterized by white blood cell infiltration and dysregulated inflammatory responses in the lungs. IL-17A is thought to play a key role in this inflammatory response by attracting white blood cells, activating innate and adaptive immune cells, and further exacerbating lung pathology. Iowa State University and ARS researchers in Ames, Iowa, used a small molecule inhibitor, ursolic acid (UA), to suppress IL-17A production and determine the downstream impact on the immune response and disease severity following Mannheimia infection in calves, The results support the hypothesis that IL-17A signaling may contribute to lung immunopathology following Mannheimia infections in calves. Further understanding of this inflammatory pathway could expand therapeutic intervention strategies for managing respiratory disease of beef and dairy cattle. **Review Publications**
                            *Dassanayake, R.P., Porter, T.J., Samorodnitsky, D., Falkenberg, S.M., Nicholson, E.M., Tatum, F.M., Briggs, R.E., Palmer, M.V., Casas, E. 2022. Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A. Biochemical and Biophysical Research Communications. 595:76-81. https://doi.org/10.1016/j.bbrc.2022.01.071.*
                                *Briggs, R.E., Billing, S.R., Boatwright Jr, W.D., Chriswell, B.O., Casas, E., Dassanayake, R.P., Palmer, M.V., Register, K.B., Tatum, F.M. 2021. Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens. Microbial Pathogenesis. 161. Article 105159. https://doi.org/10.1016/j.micpath.2021.105159.*
                                *Cushman, R.A., Bennett, G.L., Tait, R.G., McNeel, A.K., Casas, E., Smith, T.P.L., Freetly, H.C. 2021. Relationship of molecular breeding value for beef tenderness with heifer traits through weaning of their first calf. Theriogenology. 173:128-132. https://doi.org/10.1016/j.theriogenology.2021.07.020.*
                                *Eder, J.M., Sacco, R.E. 2022. Ex vivo activated CD4+ T cells from young calves exhibit Th2-biased effector function with distinct metabolic reprogramming compared to adult cows. Veterinary Immunology and Immunopathology. 248. Article 110418. https://doi.org/10.1016/j.vetimm.2022.110418.*
                                *Falkenberg, S.M., Bauermann, F., Scoles, G.A., Bonilla, D., Dassanayake, R.P. 2022. A serosurvey for ruminant pestivirus exposure conducted using sera from stray Mexico origin cattle captured crossing into Southern Texas. Frontiers in Veterinary Science. 9. Article 821247. https://doi.org/10.3389/fvets.2022.821247.*
                                *Lippolis, J.D., Putz, E.J., Reinhardt, T.A., Casas, E., Weber, W.J., Crooker, B.A. 2022. Effect of Holstein genotype on immune response to an intramammary Escherichia coli challenge. Journal of Dairy Science. 105(6):5435-5448. https://doi.org/10.3168/jds.2021-21166.*
                                *Mosena, A.S., Falkenberg, S.M., Ma, H., Casas, E., Dassanayake, R.P., Booth, R., De Mia, G., Schweizer, M., Canal, C., Neill, J.D. 2021. Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates. Journal of Virological Methods. 299. Article 114328. https://doi.org/10.1016/j.jviromet.2021.114328.*


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