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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Research Project #441143

Research Project: Intervention Strategies to Prevent and Control Viral Respiratory Pathogens of Ruminants

Location: Ruminant Diseases and Immunology Research

2022 Annual Report

Objective 1: Determine the impact of variant and emerging viruses as causative agents of respiratory disease in ruminants, with special emphasis on the role of Bovine Viral Diarrhea virus (BVDV). Subobjective 1A: Conduct whole genome phylogenetic analyses to support molecular epidemiological studies to characterize and determine the significance of ruminant respiratory viruses currently circulating in United States. Subobjective 1B: Identify the molecular determinants that drive strain prevalence, emergence, evolution, virulence, and transmission of bovine respiratory viruses. Subobjective 1C: Conduct metagenomic analyses of respiratory samples from feedlot cattle to determine presence and significance of bovine respiratory viruses. Objective 2: Elucidate the host-pathogen interactions associated with the Bovine Respiratory Disease Complex. Subobjective 2A: Identify host factors associated with viral infection that predispose to respiratory disease. Subobjective 2B: Characterize cellular and humoral responses that drive protective immunity against viral respiratory pathogens. Subobjective 2C: Characterize functional genomics of the host associated with susceptibility to respiratory disease. Objective 3: Develop intervention strategies for controlling viral respiratory infections of ruminants. Subobjective 3A: Develop vaccines and vaccination strategies that provide better cross-protection against emerging and antigenic variant field strains. Subobjective 3B: Improve existing diagnostic tests and testing strategies for the early detection of respiratory viral pathogens of ruminants on relevant farm settings. Subobjective 3C: Develop biotherapeutic platforms for feedlot cattle that induce rapid onset of immunity as a companion to respiratory disease vaccination.

Bovine respiratory disease (BRD) is a major cause of production losses to the cattle industry. The aim of the research in this project plan is to provide scientific information to better understand the viral pathogenesis of BRD. In particular, the disease dynamics of host-pathogen interactions responsible for the BRD will be investigated. Agents of interest include bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1) and bovine respiratory syncytial virus (BRSV). This research will involve a multidisciplinary approach to address the broad and ambitious goal of controlling viral diseases of cattle, with a priority on respiratory viral pathogens. The approach used here is consistent with the multifactorial nature of bovine respiratory disease, as it results from an interplay of infection by multiple viral and bacterial pathogens, stress, immune dysfunction and environmental factors. The first objective of this project addresses the impact of variant and emerging viruses. Screening to determine the incidence of variant and emerging viruses will require the development of surveillance tools and methods to measure impact. This will lead to a greater understanding of viruses that play a role in BRD. A major question here is evaluation of currently marketed vaccines and whether it will be necessary to modify them to protect against emerging/variant viruses. There is a need to identify newly emerging/variant viruses that interact with the host in producing BRD. A second objective examines host/pathogen interactions, specifically to determine how respiratory viral pathogens interact with the host to moderate innate and adaptive immune responses. This includes interaction between BVDV, BRSV, BHV-1 and emerging/variant viruses. It is established that most BRD involves interactions of multiple agents, both viral and bacterial, thus experiments involving multiple agents will be conducted to examine this interplay and how each contributes to BRD. The third objective of this project plan involves defining events that promote the production of a strong, protective immune responses (both innate and acquired immunity). Results from this will reveal targets or points of intervention that can be utilized in the development of robust vaccines and management regimens that reduce the impact of BRD. The knowledge gained here will be used for the design of new vaccines, including subunit vaccines, or for providing greater knowledge for the selection of virus strains to be used in vaccines. This part of the project will evaluate the practical applications of information generated in the form of improved vaccines or vaccination strategies. The ultimate, cumulative goal of this research is to improve control of viral respiratory pathogens that will enhance cattle health and well-being, and reduce production costs for farmers and ranchers.

Progress Report
In support of Subobjective 1A2, samples were received from Cornell University, and from Iowa State University in a collaboration study with the National Animal Disease Center. Eighteen lung samples were received from animals with clinical bovine viral diarrhea virus, 24 nasal swab samples, six feces’ samples, and five bovine viral diarrhea virus isolates. In support of Subobjective 1B1, highly virulent bovine viral diarrhea virus type isolates were analyzed through a bioinformatics pipeline to assess if potential genetic differences between high virulent and typical virulent could be discerned. Information was analyzed using Principal Component Analysis to identify antigen determinants for bovine viral diarrhea virus strains. In support of Subobjective 2A1, we identified two strains of bovine viral diarrhea virus (named Singer and 53637) in vaccines to be immunodominant. An Animal Care and Use Protocol was written to generate monospecific polyclonal antisera against bovine viral vaccine candidates. In support of Subobjective 2B1, we developed in situ hybridization assay to characterize cell mediated immunity responses, as well and including proliferating cell nuclear antigen and program death cell-1 as markers for proliferation and immune suppression. In support of Subobjective 2B2, we conducted a literature search for information about white blood cell (T cells) antigenic determinants (part of a pathogen that an antibody attaches to) from the fusion (F) protein of human respiratory syncytial virus, previously identified in the mouse model or in vitro studies. The sequences of the F protein from human and bovine respiratory syncytial virus were compared by the Basic Local Alignment Tool. This allowed identification of similar bovine respiratory syncytial virus F peptide sequences for each HRSV antigenic determinant. To determine whether these antigenic determinants may be relevant in bovine immunity, peptide sequences including these antigenic determinants were queried against all available bovine leukocyte antigen alleles on the NetMHCpan server ( to identify those that would be predicted to be strong binders (<500 nM). Individual bovine respiratory syncytial virus F peptides were chosen for additional research. In support of Subobjective 2C1, a total of 732 blood samples were received from West Texas A&M University, in a collaboration study with Mississippi State University and the National Animal Disease Center. The objective is to identify transcripts that are differentially expressed in animals vaccinated against bovine viral diarrhea virus (BVDV). The animals were sampled at days 0, 14, and 28, after vaccination. Currently we have extracted RNA from all animals at day 0, and we are on the way of extraction on samples from day 14. Once RNA has been isolated from all samples, they will be sent for sequencing. Subobjective 3C1 focuses on developing intervention strategies for controlling viral respiratory infections of ruminants. In support of subobjective 3C, we developed biotherapeutic platforms for feedlot cattle that induce rapid onset of immunity as a companion to respiratory disease vaccination, serum samples collected from calves inoculated with different bovine viral diarrhea virus (BVDV) isolates were evaluated using viral neutralization assays to identify BVDV isolates that show the broadest cross-neutralization titers. Immunogenic antigens from a selected BVDV isolate have been identified and successfully cloned to produce recombinant BVDV antigens. Several immunostimulants that expect to show the highest immune responses in cattle have been identified and purchased. These immunostimulants along with BVDV antigens will be encapsulated into polyanhydride particles to formulate a BVDV nano-vaccine. We have signed a transmittal memo with ARS researchers at Plum Island Animal Disease Center, Orient Pont, New York to obtain an antiviral protein expression construct to be tested as potential biotherapeutic agent against BVDV. Studies published using this antiviral protein construct in cattle have shown the antiviral activity against foot-and-mouth disease virus and delay in disease development.

1. Serum from stray Mexico-origin cattle captured crossing into southern Texas were positive for bovine viral diarrhea virus (BVDV). Stray cattle along the Texas/Mexico border pose a potential risk for introduction of pathogens. Current methods of control against these pathogens includes capturing stray cattle that are unaccounted for along the border and testing for selected potential pathogens that pose a risk to the cattle industry. ARS researchers in Ames, Iowa, measured titers to BVDV utilizing comparative serology. Titers against all three BVDV species (BVDV-1, BVDV-2, and BVDV-2 (HoBi-like virus) were identified. Fifty percent of the cattle tested had antibodies against BVDV-1, indicating that they had been exposed to it in the past. No sample had an antibody titer higher for HoBi-like virus, as compared to BVDV-1 and BVDV-2. Half the cattle were sero-negative for all three BVDV species which suggests a large number of susceptible animals that are unaccounted for along the border which pose a risk for introduction of new BVDV isolates. This cattle population provides a unique opportunity to evaluate and monitor changes in seroprevalence of economically important cattle diseases affecting the cattle industry.

Review Publications
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.
Dassanayake, R.P., Atkinson, B.M., Mulls, A.S., Falkenberg, S.M., Nicholson, E.M., Casas, E., Narasimhan, B., Bearson, S.M. 2021. Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates. Nature Scientific Reports. 11(1). Article 19276.
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.
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.
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.
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.
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.
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.