Research Project: Identification of Tick Colonization Mechanisms and Vaccine Development for Anaplasmosis

Location: Animal Disease Research

2019 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
We have identified a number of candidate A. marginale proteins that could serve as adhesins to the midgut (Objective 1) and bovine erythrocytes (Objective 2). Together, these proteins constitute a robust set of vaccine candidates. In support of Sub-objective 1A, a number of A. marginale surface proteins have been identified that may serve as midgut adhesions. Most notably, OmpA, experimentally demonstrated to be an adhesion, was identified which helps validate our experimental system. Other candidates we identified include a number of the major surface proteins (Msp1a-like proteins 2-4, several copies of Msp1b and Msp 3,4, and 5). Among this group, the Msp1a-like proteins and Msp1b are of particular interest because in vivo msp1a and msp1b form a complex and there is some evidence that this complex also serve as adhesion for tick midguts. Interestingly, there is no published information regarding the msp1a-like proteins, which the exception of the initial description of the genes in the A. marginale genome paper. We also identified three of the components of the type 4 secretion system (T4SS), including VirB3, VirB7, and VirB8. These proteins are excellent vaccine candidates because the T4SS is responsible for injecting proteins into the host cells, which alter the host cell, thus allowing for pathogen colonization and replication. The T4SS in A. marginale is made up of approximately 20 different proteins and the surface exposed components that interact directly with the host cell have not been identified. This work helps us to identify the relevant T4SS proteins in the context of vaccine development. Finally, we identified a number of other outer membrane proteins and hypothetical proteins about which little is known. In support of Sub-objective 2A, we have identified a similar set of potential red blood cell adhesins as compared to the midgut adhesins, including OmpA and members of the Msp1 superfamily, but fewer members of the T4SS, which may be beneficial in the development of a vaccine for Anaplasma marginale.

Accomplishments
1. Domestic goats can serve as a reservoir for Anaplasma marginale. Bovine anaplasmosis, caused by the tick-borne bacterial pathogen A. marginale, is a production-limiting disease of cattle with a worldwide distribution and an estimated cost of $10-30 million annually in the U.S. alone. Knowing the reservoirs of A. marginale is essential because methods to control this disease are limited and often rely heavily on herd management. ARS researchers in Pullman, Washington, in collaboration with researchers in Brazil, have determined that goats can serve as reservoir species for A. marginale. Previously it was unknown that domestic small ruminants could harbor A. marginale. Consequently, this finding will guide the development of individualized management plans for the control of bovine anaplasmosis.

2. Experimental tools to understand the molecular determinants of vector competence. Tick-borne diseases are a growing burden affecting human and animal health. Our ability to control these diseases is limited due to a lack of experimental tools that can be used to understand the factors that determine if a tick species can efficiently transmit a particular pathogen. ARS researchers in Pullman, Washington, in collaboration with colleagues at Washington State University in Pullman, Washington, determined that cultured tick cells from different tick species differ in their permissiveness to invasion and replication by a tick-borne pathogen. Importantly, the response of the tick cells to infection mimics the biology of the tick vector. This experimental system serves as a platform for identifying the fundamental factors that determine if and how a pathogen enters and replicates in a particular tick species.

3. Environmental temperature can affect tick-borne disease. Tick borne-diseases are a growing threat to animal and human health. Environmental factors that affect the risk of tick transmission and severity of disease are poorly understood. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University, have determined that the ambient temperature affects the longevity of host cells and bacterial levels of infected tick cells, suggesting that at higher temperatures, the pathogen will develop more rapidly in ticks thus increasing the potential for pathogen transmission. These findings help us understand the epidemiology of tick-borne disease and improve our ability to identify the factors that lead to disease outbreaks.

Review Publications
Awada, L., Tizzani, P., Noh, S.M., Ducrot, C., Ntsama, F., Caceres, P., Chalvet-Monfray, K. 2018. Global dynamics of highly pathogenic avian influenza outbreaks between 2005 and 2016 - Focus on distance and rate of spread. Transboundary and Emerging Diseases. 65(6):2006-2016. https://doi.org/10.1111/tbed.12986.
Reif, K.E., Ujczo, J.K., Alperin, D.C., Noh, S.M. 2018. Differences in francisella tularensis subsp. novicida infection competence in cell lines from a natural vector and non-vector tick species. Scientific Reports. 8(1):12685. https://doi.org/10.1038/s41598-018-30419-4.
Dasilva, N.B., Ueti, M.W., Johnson, W.C., Mira, A., Schnittger, L., Valente, J.D., Vidotto, O., Masterson, H.E., Taus, N.S., Vieira, T.S. 2018. First report of Anaplasma marginale infection in goats, Brazi. PLoS One. 13(8):e0202140. https://doi.org/10.1371/journal.pone.0202140.