Location: Animal Disease Research
Project Number: 2090-32000-038-00-D
Project Type: In-House Appropriated
Start Date: Oct 1, 2016
End Date: Sep 30, 2021
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.
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.