Location: Animal Disease Research
2020 Annual Report
Objectives
Anaplasma marginale is a tick-transmitted, obligate intracellular bacterial pathogen of ruminants. Survival and ongoing transmission of this pathogen requires entry and replication in specific cells types in the ruminant host and tick vector, both of which serve as potentially effective sites of intervention. First, in the ruminant host, induction of an immune response that blocks A. marginale adhesion and entry into erythrocytes, the primary host cell, would result in protection from challenge. Second, delivery of an immune response to the tick midgut targeting A. marginale adhesins and the corresponding midgut receptors could prevent colonization of the tick midgut, thus preventing or limiting ongoing transmission.
However, little is known about the molecules and mechanisms required for entry either into bovine erythrocytes or the tick midgut, which is the first barrier to tick colonization. We propose to use a phage display library as an unbiased screen to identify A. marginale surface proteins that mediate adhesion to either bovine erythrocytes or Dermacentor andersoni tick midguts. The ability of the identified adhesin candidates to bind their respective host cells will then be confirmed.
The tick midgut is biologically unusual and thus likely has unique surface receptors that could additionally be targeted by the bovine immune response. Consequently, candidate receptors for the A. marginale midgut adhesins will be identified using pull-down assays. Specific binding between each A. marginale adhesin and its corresponding midgut receptor candidate will be confirmed. Because antibody is the most likely effector molecule, the ability of bovine antibody to block binding of the adhesin to its target cell will be tested in vitro. If successful, immunization and challenge experiments will be done to determine the in vivo efficacy of these two approaches.
Objective 1: Identify the determinants of tick colonization of A. marginale in the host with the long-term goal of blocking transmission.
• Subobjective 1A: Identify the A. marginale proteins that mediate adhesion to the D. andersoni midgut.
• Subobjective 1B: Identify D. andersoni midgut receptors that serve as the binding partners for the A. marginale adhesins.
• Subobjective 1C: Determine if bovine antibody directed against A. marginale adhesins and D. andersoni midgut binding partners will reduce A. marginale midgut colonization.
• Subobjective 1D: Determine the efficacy of immunization against A. marginale adhesins and tick midgut receptors in blocking D. andersoni colonization.
• Subobjective 1E: Determine the amount of heterogeneity among the A. marginale adhesins and midgut receptors proteins in A. marginale and North American populations of Dermacentor spp, respectively.
Objective 2: Develop a safe and efficacious vaccine for Anaplasma marginale using novel platforms and techniques.
• Subobjective 2A: Identify A. marginale adhesins for bovine erythrocytes.
• Subobjective 2B: Determine if immunization against adhesins will confer protective immunity to challenge with A. marginale.
Approach
In Objective 1 we will identify the determinants of tick colonization of A. marginale in the host with the long-term goal of blocking transmission. Due to the unusual nature of the A. marginale outer membrane, algorithms to identify outer membrane proteins are often inaccurate and identification of functionally relevant vaccine targets is difficult due to the obligate intracellular nature of A. marginale. Thus, we will use a phage display library to perform an unbiased screen of the A. marginale proteome to identify midgut adhesins. The functional significance of the adhesin candidates will then be determined using a combination of adhesion assays, immunofluorescence and competitive inhibition of A. marginale invasion of tick cells. It is possible the initial phage display library will be of low diversity. If this is the case, an alternative phage display systems will be used.
Next we will identify D. andersoni midgut receptors that serve as the binding partners for the A. marginale adhesins because both bacterial ligands and their receptors on the midgut epithelial cells could serve as targets of the bovine immune system to disrupt A. marginale colonization of the tick midgut. To identify the midgut receptors, we will use pull-down assays. Once the putative midgut receptors are identified, siRNA will be used to knock-down gene expression of the midgut receptor candidates and A. marginale infection rate and levels will be measured in ticks. Finally, we will determine if bovine antibody directed against the A. marginale adhesins and D. andersoni midgut binding partners will reduce A. marginale midgut colonization in vivo and in vitro. It is possible that individual anti-ligand or anti-receptor antibodies will fail to significantly block A. marginale colonization of DAE cells. If this is the case, mapping of the specific binding domains will be done and the immune response will be directed specifically against the binding domains of each ligand. The focus of Objective 2 is to develop a safe and efficacious vaccine to prevent anaplasmosis. Toward this end, we will identify A. marginale outer membrane proteins that serve as adhesins for bovine erythrocytes using the phage display library developed in Objective 1. Next we will conduct an immunization and challenge trial to determine if the identified adhesins provide protection from challenge.
Progress Report
In support of Objective 1, two approaches were used to verify and narrow the list of candidate adhesins identified in the previous fiscal year. The first approach did not work due to technical limitations. The second approach involved expressing protein from all candidates and testing the ability of the recombinant protein to compete with A. marginale binding to cells and thus reduce A. marginale adhesion and entry. Sixteen of seventen candidates have been cloned and three shown to express protein. Also, in support of Objective 1, we have developed a method to reduce iron in tick cells, identified a comprehensive set of tick genes involved in iron metabolism and determined that iron is required for A. marginale survival and replication in tick cells. This work will lead to the identification of both tick and pathogen genes that can be targeted to disrupt the tick lifecycle and target the pathogen within the tick.
In support of Sub-objective 1B, all fucosyltransferase genes in Dermacentor andersoni have been cloned and sequenced. These genes are responsible for adding fucose to surface exposed proteins in the tick midgut. In general, fucose moieties serve as adhesins for pathogen binding. The next step is to determine if the fucose moieties play an essential role in the ability of A. marginale to colonize the tick.
In support of Objective 2, and in collaboration with researchers at Washington State University, we have determined that A. marginale outer membrane proteins are readily expressed by the non-virulent Coxiella burnetii Nine Mile phase II grown in media, rather than host cells. This is important progress in A. marginale vaccine development for two reasons. First, A. marginale vaccine candidates made by C. burnetii are expected to produce a stronger immune response than those same candidates produced by E. coli, though this remains to be tested. Second, the ability to grow C. burnetii in media allows for large- scale protein production.
Accomplishments
1. In vitro tick feeding system designed to facilitate the development of methods to reduce tick burdens and prevent tick borne diseases in cattle. Ticks and tick-borne diseases cause heavy losses to the U.S. cattle industry. Methods to control ticks and prevent transmission of tick-borne diseases are limited to management practices and use of acaricides, which are expensive and partially effective. Experimental systems for testing anti-tick and pathogen-blocking interventions are limited because of the requirement of ticks to feed on cattle for consistent delivery of the blood meal and candidate interventions. ARS researchers in Pullman, Washington, in collaboration with colleagues at Washington State University in Pullman, Washington, developed a system to feed ticks on an artificial membrane and determined that ticks can be reliably infected with the bovine pathogen, Anaplasma marginale using this system. This technology, which is published, will be a valuable tool for the controlled delivery of anti-tick and pathogen-blocking molecules to ticks, which will facilitate the development of new methods to control ticks and prevent tick-borne diseases.
2. Anaplasma marginale outer membrane protein vaccine candidates, OmpA Omp7, Omp8, Omp9, Am779, have high sequence and antigenic conservation in geographically distinct cattle populations. Bovine anaplasmosis, caused by the tick-borne pathogen A. marginale, is a production-limiting disease of cattle with a worldwide distribution and an estimated cost of $10 to 30 million annually in the United States alone. Vaccine development is hampered by variation between strains and the need for a cross-protective vaccine. OmpA, Omp7, Omp8, Omp9, Am779 are all high priority vaccine candidates. ARS researchers in Pullman, Washington, in collaboration with colleagues at the University of Ghana and the University of Pretoria in South Africa have determined: 1) That all of these proteins are highly conserved in A. marginale strains from west and south Africa; and 2) Antibody in animals protectively immunized with a North American A. marginale strain readily recognize the corresponding proteins in African strains. These findings indicate that a single multi-valent vaccine has the potential to protect against multiple strains of A. marginale.
Review Publications
Vimonish, R., Johnson, W.C., Mousel, M.R., Brayton, K.A., Scoles, G.A., Noh, S.M., Ueti, M.W. 2020. Quantitative analysis of Anaplasma marginale acquisition and transmission by Dermacentor andersoni fed in vitro. Scientific Reports. 10:740. https://doi.org/10.1038/s41598-019-57390-y.
Futse, J.E., Buami, G., Kayang, B.B., Koku, R., Palmer, G.H., Graca, T., Noh, S.M. 2019. Sequence and immunologic conservation of Anaplasma marginale OmpA within strains from Ghana as compared to the predominant OmpA variant. PLoS One. 14(7):e0217661. https://doi.org/10.1371/journal.pone.0217661.
Hove, P., Brayton, K.A., Lienbenberg, J., Pretorius, A., Oosthuizen, M.C., Noh, S.M., Collins, N.E. 2020. Anaplasma marginale outer membrane protein vaccine candidates are conserved in North American and South African strains. Ticks and Tick Borne Diseases. 11(4). https://doi.org/10.1016/j.ttbdis.2020.101444.
