Location: Animal Disease Research2018 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Substantial progress was made on both Objectives. For Objective 1, various combinations of a phage display library that express all outer membrane proteins of Anaplasma marginale were used in selection (biopanning) assays to identify the proteins that mediate adhesion and entry into tick cells. Initially small libraries consisting of equal numbers of three different phage were hybridized with tick cells to optimize the assays and phage recovery conditions. The entire library consisting of 100 members was used in subsequent biopanning experiments (Sub-objective 1A). Each experiment consisted of hybridizing the library a minimum of three sequential times with whole tick cells. Next all members of the phage display library will be quantified using digital polymerase chain reaction (PCR) before and after each passage in order to identify the member of the library that are selected during each passage, thus identifying the A. marginale outer membrane proteins that mediate adhesion to tick cells. In work related to Objective 1, a method to deplete tick cells of iron was developed. The presence and sequence of ferritin 1 (fer1) was identified in Dermacentor andersoni ticks. This gene encodes a protein involved in iron regulation and thus serves as a marker for alteration in iron levels within the tick. An iron chelator was used to deplete tick cells of iron. The efficacy of the iron chelator was demonstrated by the reduction in fer1 transcript levels as well as a reduction in protein levels. Next, the effect of iron depletion in the tick cells on A. marginale replication will be determined. Iron is an essential nutrient for most bacterial pathogens as well as their hosts. Nothing is known regarding iron acquisition and regulation in ticks and how tick borne bacterial pathogens acquire iron from the tick. Thus, the ability to starve stick cells of iron will allow for the identification of the mechanisms and molecules ticks and tick- borne pathogens use to acquire this essential nutrient. For Objective 2, a putative adhesion and vaccine candidate has been sequenced from A. marginale-infected cattle (Sub-objective 2A). This is a collaborative effort with researchers at the University of Ghana. The data indicate this gene is highly conserved in relation to other outer membrane proteins and vaccine candidates. The magnitude of the antibody response as measured by titers to the North American, St. Maries strain is similar between North American and Ghanaian cattle, indicating this protein could be used to induce broadly protective immunity to many strains of A. marginale. Based on predicted B cell epitopes, it is likely that the functional domain of this protein is poorly immunogenic. To empirically determine if this is the case, the antibody response to the functional domain of this protein is currently being measured. Construction of recombinant proteins and various adjuvants will be tested for their ability to enhance the immune response to this domain.
1. Defining the microbiome in different populations of ticks. The composition of the tick microbiome strongly affects the ability of Anaplasma marginale, the cause of bovine anaplasmosis, to colonize the tick. ARS researchers in Pullman, Washington, in collaboration with investigators at Washington State University, determined that the microbiome of Dermacentor andersoni ticks, one of the primary vectors of A. marginale in the U.S., is distinct in different geographic populations, and not necessarily representative of laboratory reared populations. This is an important discovery as it helps identify the underlying factors that lead to the currently unpredictable outbreaks of bovine anaplasmosis. Additionally, manipulation of the microbiome may lead to ways to prevent transmission of A. marginale and other tick-borne pathogens.
2. Validation of a test to diagnose ovine anaplasmosis in sheep. A. ovis infects small ruminants worldwide, resulting in economic losses due to weight loss, anemia and decreased milk production. Few diagnostic tests are available to diagnose this disease. However, a competitive enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine anaplasmosis is easy to use, inexpensive and commercially available in the U.S. ARS researchers in Pullman, Washington, and Dubois, Idaho, in collaboration with investigators at Washington State University, determined that the competitive ELISA for bovine anaplasmosis has a high sensitivity and specificity when used for the diagnosis of ovine anaplasmosis. This allows for more accurate and less costly diagnosis of diseases of sheep.
3. Distinct phylogenetic split between Dermacentor variabilis tick populations discovered. In the U.S., Dermacentor variabilis is a major vector of bovine anaplasmosis, which is often characterized by sporadic and unpredictable outbreaks of severe disease. The many factors that lead to these outbreaks are unknown, though in part are likely due to vector population genetics and the capacity of particular populations to efficiently transmit the pathogen. In collaboration with researchers at Northern Arizona University, ARS researchers in Pullman, Washington, determined that geographic isolation played a role in shaping the populations of this tick in the U.S. However, at a regional level, there are high levels of genetic mixture, indicating these ticks commonly move between locations. These findings help advance our ability to understand how tick populations spread and define genetically distinct tick populations. This allows for differentiating vector populations that are highly efficient and poorly efficient in terms of their ability to transmit pathogens.
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