Location: Ruminant Diseases and Immunology Research
2024 Annual Report
Objectives
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.
Approach
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 Objective 1, new contemporary bovine viral diarrhea virus (BVDV) clinical tissue and serum samples dating from 2020 to 2023 were collected from numerous diagnostic and state laboratories in North America. To date, a total of 78 samples from Florida, California, Ohio, and Ontario have been obtained. Sequencing and assembly of BVDV genomic sequences from these contemporary samples are underway. Novel isolates and strain prevalence are being analyzed. Additionally, serum samples from these new batches are being utilized to evaluate animal exposure and circulating antibodies to BVDV strains. Bioinformatic pipelines and protocols were established for downstream analyses of isolates to characterize molecular determinants, especially the generation and nature of strain specific deletion viral genomes. These pipelines and protocols will be used for analyses of these newly acquired contemporary strains.
In support of Objective 2, tandem in vitro and in vivo studies were conducted to analyze the cross-protective ability of 15 different BVDV2a strains. In vivo studies generated serum antibodies against each strain that were assessed for cross-reactivity by in vitro virus neutralization assay. From these initial studies, strains of interest with high cross-reactivity are being chosen for further analysis for characterizing protective immune responses and unique attributes of the potentially immunodominant isolates. Additionally, new in vivo studies have been approved for completion in FY25 to better understand the implications and dynamics of BVDV infection on immune cell depletion. Similar experiments are planned for new isolates acquired as part of objective 1.
In support of Objective 2, 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. RNA was extracted from 2,196 samples (three samples from 732 animals). Sequencing has been done in all samples. Bioinformatics analysis is being initiated using samples at the three interval points.
In support of Objective 2, we have obtained synthetic bovine respiratory syncytial virus (BRSV) attachment protein (G protein) peptides. To better understand the immune response to BRSV infection we will assess the white blood cell (WBC) responses in cows with a history of annual BRSV vaccinations. Following stimulation with synthetic peptides derived from the BRSV G protein sequence, WBCs from cows with known major histocompatibility complex class II alleles will be examined for their response to peptides, with the objective to identify novel WBC epitopes. An epitope is the part of a pathogen that the immune system recognizes. This information will be of interest to researchers, veterinarians, and producers interested in vaccine development.
In support of Objective 2, small non-coding ribonucleic acids (sncRNAs) and messenger RNA (mRNA) are being analyzed to establish their association and determine their relationship. Animals were assigned to three groups: Control, Mycoplasma bovis (MB), or co-infection (Dual). The greatest number of differentially expressed sncRNAs and mRNAs are being identified in thymus. The protein tyrosine phosphatase receptor type O (PTPRO) genes was consistently differentially expressed when comparing expression among the different groups. Additional analyses will take place to establish pathways in which the virus affects the host.
In support of Objective 3, a novel vaccine adjuvant approach to improve long lasting immunity and retain the highest levels of safety is under development. An in vivo trial to compare the experimental vaccination strategy against commercial options is approved and planned for FY25. In addition to comparing markers of humoral and cellular immunity, which are previously validated, the vaccine effectiveness will be compared to the commercial options in a heterologous BVDV challenge. Pending an initial assessment of the vaccine efficacy and function, a long-term protection study of 6 months post immunization is planned and has been approved by the IACUC.
In support of Objective 3, an adenoviral vector encoding an antiviral biotherapeutic protein from our collaborators at the Plum Island Animal Disease Center was received. Using an in vitro cell culture system, we previously demonstrated that this antiviral biotherapeutic protein can effectively neutralize bovine viral diarrhea virus (BVDV). Since in vitro cell culture studies were completed ahead of schedule, we conducted an animal trial to test the effectiveness of this adenoviral vector expressing biotherapeutic protein against BVDV infection in calves. BVDV typically infects lymphoid tissues and causes severe damage to thymus compromising morphology and immunosuppression in calves. The gross weight and morphology of the thymuses of the calves were significantly preserved when this vector, which expresses biotherapeutic protein, was administered one day before and one day after BVDV challenge compared calves infected with BVDV only. We are currently in the process of further optimizing this vector system to ensure the complete protection of thymus and reduce viral shedding in calves.
Accomplishments
1. Identification of differentially expressed transfer ribonucleic acid fragments in experimental bovine respiratory disease. Understanding the molecular mechanisms underlying immune responses can allow informed decisions in drug or vaccine development and assist in the identification of biomarkers to predict exposure or evaluate treatment efficacy. ARS scientists in Ames, Iowa conducted a study to identify differentially expressed transfer ribonucleic acid (RNA)-derived fragments (tRFs) in calves challenged with Mycoplasma bovis (M. bovis) or co-infected with M. bovis and bovine viral diarrhea virus (BVDV). Serum, white blood cells (WBC), liver, mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), spleen, and thymus were collected. The expression of tRFs in MLN, TBLN, thymus, and spleen were highly correlated with each other, but not with serum and WBC. The highest number of differentially expressed tRFs were found in thymus and MLN, consistent with lymphoid depletion previously observed in BVDV. tRFs were increased in coinfected compared to control and MB groups in liver, MLN, TBLN, thymus and WBC. Identifying the targets of differentially expressed tRFs induced by infection will aid our understanding of the molecular mechanisms underlying host immune responses to respiratory disease in cattle and will be of interest to scientists studying bovine respiratory diseases.
2. Multivariate analysis reveals that BVDV field isolates do not show a close virus neutralization (VN)-based antigenic relationship across diverse US vaccine strains. Providing protection against bovine viral diarrhea virus (BVDV) is challenging due to the ability of BVDV to infect the fetus, and the increased level of protection needed to protect the fetus. Generally, VN titers indicates how well the antibody response against one virus reacts against other similar viruses, but the relationship between the antibody response and genetic relationships among viruses is not always clear. ARS scientists in Ames, Iowa used two datasets to evaluate bovine viral diarrhea virus (BVDV) vaccination protection by using VN, after combining both data sets to provide a comprehensive analysis. Results from this study supports that genetic assignment of BVDV into subgenotypes, or strains within subgenotypes, based on genome sequence, is not representative of the antibody response to viral molecules that occurs when a virus is used to vaccinate against other related viruses. These results will aid our understanding of host immunity to BVDV infection and provide more informed decisions in the development of BVDV vaccines and will be of interest to scientists, veterinarians, and producers.
3. Development of a novel mucosal RSV vaccine. Bovine RSV (BRSV), causes respiratory disease in young calves, and is a major viral pathogen in the bovine respiratory disease complex in feedlot cattle. In two independent trials, ARS scientists in Ames, Iowa evaluated a nanovaccine which incorporated the BRSV post-fusion (F) and attachment (G) proteins and adjuvant CpG, delivered via intranasal and subcutaneous or intranasal and intranasal prime-boost routes in the neonatal calf model. They compared the nanovaccine performance to a commercial modified live vaccine, and to nonvaccinated control calves. Experimentally challenged calves receiving either nanovaccine treatment exhibited reduced clinical disease scores and less viral replication in nasal samples and lung tissues compared to nonvaccinated calves. The intranasal and subcutaneous or intranasal nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA. The BRSV-F/G CpG nanovaccine has the potential to reduce BRSV disease burden in cattle and these results will be of interest to scientists, veterinarians, and cattle producers.
Review Publications
Sacco, R.E., Jensen, E.D., Sullivan, Y., Labresh, J., Davis, W.C. 2024. An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit. Veterinary Immunology and Immunopathology. Article 110769. https://doi.org/10.1016/j.vetimm.2024.110769.
Ma, H., Alt, D.P., Falkenberg, S.M., Briggs, R.E., Tatum, F.M., Clawson, M.L., Casas, E., Dassanayake, R.P. 2024. Transcriptomic profiles of Mannheimia haemolytica planktonic and biofilm associated cells. PLOS ONE. 19(2). Article e0297692. https://doi.org/10.1371/journal.pone.0297692.
Ma, H., Menghwar, H., Lippolis, J.D., Sarlo Davila, K.M., Casas, E., Dassanayake, R.P. 2024. Draft genome sequence of a multidrug-resistant pseudomonas aeruginosa strain isolated from a dairy cow with chronic mastitis. Microbiology Resource Announcements. Article e0117323. https://doi.org/10.1128/mra.01173-23.
Maina, T.W., Grego, E.A., Broderick, S., Sacco, R.E., Narasimhan, B., Mcgill, J.L. 2023. Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection. Frontiers in Immunology. 14. Article 1186184. https://doi.org/10.3389/fimmu.2023.1186184.
Menghwar, H., Tatum, F.M., Briggs, R.E., Casas, E., Kaplan, B.S., Azadi, P., Dassanayake, R.P. 2023. Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion. Microbiology Spectrum. Article e0294423. https://doi.org/10.1128/spectrum.02944-23.
Ruiz-De La Cruz, G., Sifuentes-Rincon, A.M., Paredes-Sanchez, F.A., Parra-Bracamontes, G., Casas, E., Welsh Jr, T.H., Riley, D., Perry, G., Randel, R.D. 2023. Characterization of intronic SNP located in candidate genes influencing cattle temperament. Brazilian Journal of Animal Science. 52. Article e20220057. https://doi.org/10.37496/rbz5220220057.
Dassanayake, R.P., Ma, H., Casas, E., Lippolis, J.D. 2023. Genome sequence of a multidrug-resistant Pseudomonas aeruginosa strain isolated from a dairy cow that was nonresponsive to antibiotic treatment. Microbiology Resource Announcements. Article e002892. https://doi.org/10.1128/MRA.00289-23.
Ruiz-De La Cruz, G., Sifuentes-Rincon, A.M., Paredes-Sanchez, F.A., Parra-Bracamontes, G.M., Casas, E., Riley, D.G., Perry, G.A., Welsh Jr., T.H., Randel, R.D. 2024. Analysis of nonsynonymous SNPs in candidate genes that influence bovine temperament and evaluation of their effect in Brahman cattle. Molecular Biology Reports. 51(1). Article 285. https://doi.org/10.1007/s11033-024-09264-4.