Location: Animal Disease Research Unit
2025 Annual Report
Objectives
Objective 1: Characterize the immune response that correlates with protection from infection and/or disease.
Sub-objective 1A: Identify the functional antibody profile that predicts protection against bovine anaplasmosis.
Sub-objective 1B: Identify the vaccine candidates against which the protective, functional antibody response is directed.
Objective 2: Develop a vaccine platform for A. marginale antigen expression.
Sub-objective 2A: Determine if A. marginale vaccine candidates expressed by C. burnetii Nine Mile phase II induce protective immunity.
Sub-objective 2B: Develop media that supports A. marginale replication in the absence of host cells.
Objective 3: Devise functional genomics strategies, including gene editing, for developing traits in ruminant livestock to address to enhance disease resistance, enhanced reproductive efficiency, and improve resiliency to ticks, tickborne diseases and other diseases made more prevalent through changing environmental conditions.
Approach
Goal 1A1: Characterize the functional Fc-mediated antibody response produced by immunization of cattle using A. marginale OMPs. Specifically, we will measure antibody dependent cellular phagocytosis by bovine monocytes and neutrophils, antibody dependent complement activation, antibody dependent activation of NK cells and WC1+ 'd T cells, and antibody dependent platelet activation.
Goal 1A2: Identify the functional antibody profile that best predicts protection from disease. Following challenge with A. marginale, nearly all animals immunized with OMPs are protected from severe disease, however, the degree of protection among individual animals tends to be variable. We will leverage this variation to identify the functional antibody profiles that best predict protective immunity.
Goal 1B: Use the functional antibody profile predictive of protective immunity to select vaccine candidates for immunization and challenge trials. We will have identified the Fc mediated effector functions that correlate with protective immunity to bovine anaplasmosis. We will then use these correlates of immunity to identify individual proteins against which the antibodies that mediate these protective immune functions are directed. This will allow us to prioritize the existing vaccine candidates for testing in immunization and challenge trials. The vaccine candidates will be expressed as recombinant protein and used as antigen in the functional antibody assays. We will identify the candidates that elicit an antigen-specific antibody profile that mirrors the profile predictive of protection.
Hypothesis 2A1: Immunization of cattle with A. marginale Omps expressed in C. burnetii phase II induce antibodies that recognize the corresponding native A. marginale proteins. As a proof of principle, we will immunize animals with proteins expressed in C. burnetii phase II and determine if the resulting antibodies bind native A. marginale proteins.
Hypothesis 2A2: The vaccine candidates identified in Sub-objective 1B, when expressed in C. burnetii phase II, induce protection against A. marginale challenge. We will then express the vaccine candidates prioritized in Sub-objective 1B in C. burnetii phase II and determine if they induce protective immunity.
Goal 2B: Develop an axenic growth medium for A. marginale. An efficient method to culture A. marginale in the absence of animals or host cells will allow for the use of OMPs in a vaccine and circumvent the need to identify a subset of proteins and the appropriate formulation to produce a recombinant vaccine. Using a step-wise approach we will identify the nutrients and other components required for A. marginale metabolism as measured first by protein synthesis and then by replication. Once axenic replication is achieved, we will verify expression of a full array of outer membrane proteins.
Progress Report
This report documents the FY 2025 progress of project 2090-32000-043-000D, titled, “Identifying Effective Immune Responses and Vaccine Development for Bovine Anaplasmosis”, which began in October 2021.
In support of Objective 1, to characterize the immune response that correlates with protection from infection and/or disease, ARS researchers in Pullman, Washington, have developed assays and measured antibody dependent responses mediated by complement and gamma delta T cells directed against Anaplasma marginale top vaccine candidates including Msp5, Msp1a, Msp1b, OmpA, and the variable and conserved regions of Omp7, 8, and 9. The functional responses were measured using immune serum collected at the time of control of infection. To date, antibodies directed against Msp5 that activate monocytes and neutrophils correlate with less severe disease. Additionally, antibodies directed against Msp1b that activate neutrophils correlate with reduced disease severity. Antibodies directed against OmpA that activate neutrophils and gamma delta T cells correlate with decreased disease severity. Ongoing work includes measurement of IgG, IgG1 and IgG2 specific for the different proteins and data analysis. This work will inform vaccine development by prioritizing vaccine candidates and identifying the protective components of the anti-A. marginale immune response.
In support of Objective 2, to develop a vaccine platform for A. marginale antigen expression, ARS researchers are working to develop medium that supports A. marginale replication in the absence of host cells. ARS researchers have verified pyruvate as a carbon source for A. marginale. A. marginale produces a broad array of proteins for up to 24 hours in the cell free system. Additionally, there is no additional response of A. marginale to vitamin stocks, cholesterol, other carbon sources including all forms of glucose, beef heart infusion, peptone, or yeast extract. However, protein synthesis is increased with 5% carbon dioxide (CO2), but this effect is variable. Current efforts are focused on developing a method to further purify bacteria to ensure the added nutrients are not being degraded by residual tick cell enzymes. Additionally, the effect of pyruvate on A. marginale protein synthesis will be verified using bacteria isolated from bovine red blood cells (RBC), rather than tick cell culture. This work will allow for production of native A. marginale proteins needed for vaccine development.
Objective 3, is to devise functional genomics strategies, including gene editing, to develop traits in ruminant livestock that enhance disease resistance. One main focus of this objective is to identify the receptors used by A. marginale to invade bovine red blood cells. The long-term goal of this work is to edit those receptors to maintain function but prevent pathogen entry. This will allow for breeding cattle resistant to bovine anaplasmosis. A. marginale surface proteins Msp1a and Msp1b form a heterodimer and mediate A. marginale entry into red blood cells. To identify the red blood receptor, ARS researchers have used Msp1b in pull down assays with red blood cell membranes. The RBC cell proteins that bound Msp1b are currently being identified using mass spectrometry. Once identified, the candidates will be verified using reciprocal pull-down assays and site directed mutagenesis.
Accomplishments
1. Stimulation of the innate immune system provides protection against bovine anaplasmosis in high transmission settings. Bovine anaplasmosis, a tick-borne disease of cattle, has a major negative impact on cattle production worldwide. Current prevention methods, which include the heavy use of acaricides and antibiotics, are expensive and inadequate for disease prevention. Animals that survive initial infection develop immunity and typically benefit from life-long protection from disease. ARS researchers in Pullman, Washington, in collaboration with researchers at the University of Ghana determined that stimulation of the innate immune system with a Toll-like receptor against protected highly susceptible cross-bred animals from bovine anaplasmosis in natural field conditions in Ghana. This foundational knowledge may lead to the development of new methods to control bovine anaplasmosis, which will directly support cattle producers worldwide.
2. Tick transmission of bovine anaplasmosis may be enhanced at warmer temperatures. Bovine anaplasmosis, caused by Anaplasma marginale, is a high impact, production limiting, tick-borne disease of cattle common throughout the U.S. There are large gaps in our understanding of the factors that lead to high tick transmission and thus disease outbreaks. The pathogen is first acquired by the tick during feeding. For successful transmission, the pathogen must replicate in the tick midgut and salivary glands to high levels, and the tick must then feed on a new host. Consequently, the tick may spend a significant amount of time in the environment waiting for a second host. ARS researchers in Pullman, Washington, determined that replication of the pathogen in the tick vector while it is off the host is enhanced at temperatures between 32-37 degrees Celsius. Understanding how temperature impacts ticks and the pathogens they transmit is a critical step towards assessing the risk of transmission under various conditions, which will help cattle producers implement management strategies to help control bovine anaplasmosis.
3. Improved understanding of the living components of the environment that affect tick numbers, predisposing to disease outbreaks. Bovine anaplasmosis, a major production limiting disease of cattle in the U.S., is transmitted by Dermacentor ticks. Bovine anaplasmosis often occurs as high mortality outbreaks. The unpredictable nature of these outbreaks complicates control efforts. The number of adult ticks in the environment is one major risk factor for transmission of bovine anaplasmosis. However, the environmental factors conducive to large Dermacentor populations are largely unknown. The immature life stages feed primarily on rabbits and rodents. The adult ticks, responsible for transmission of the pathogen, feed on large mammals, including cattle. ARS researchers in Pullman, Washington, in collaboration with investigators at Washington State University, determined that immature tick stages infesting hosts that had been previously parasitized were less likely to feed and ingested less blood compared to ticks feeding on naïve hosts. This reduced feeding success is estimated to reduce tick population growth by 68%. This information is useful for understanding the risk factors and thus developing control strategies to prevent bovine anaplasmosis.
Review Publications
Futse, J.E., Zumor-Baligi, S., Ashiagbor, C.N., Noh, S.M., Fox, C.B., Palmer, G.H. 2024. An adjuvant formulation containing Toll-like Receptor 7 agonist stimulates protection against morbidity and mortality due to Anaplasma marginale in a highly endemic region of west Africa. PLOS ONE. 19(8). Article e0306092. https://doi.org/10.1371/journal.pone.0306092.
Devnath, P., Noh, S.M., Jarvis, S.M., Earls, K., Oyen, K.J. 2025. The replication rate of Anaplasma marginale is temperature-mediated in ticks. microPublication Biology. https://doi.org/10.17912/micropub.biology.001442.
Owen, J.P., Gibbs, A., Jones, C., Brunner, J., Mason, K.L., Noh, S.M., Scoles, G.A. 2025. Linked empirical studies reveal the cumulative impact of acquired tick resistance across the tick life cycle. Ticks and Tick Borne Diseases. 16(3). Article 102460. https://doi.org/10.1016/j.ttbdis.2025.102460.