2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Develop and test anti-tick vaccines for immunization of deer.
Sub-objective 1A. Refine understanding of white-tailed deer immune system.
Sub-objective 1B. Define quantitative and qualitative gene and protein responses in R. microplus during feeding on B. bovis-infected deer.
Sub-objective 1C. Evaluation of candidate vaccine antigens.
Objective 2: Identify sensory, physiological, and biological targets for development of novel acaricides and drugs for use as chemical control technology.
Sub-objective 2A. Identify neuroregulatory processes in tick pharyngeal muscles and salivary glands.
Sub-objective 2B. Identify inhibitors of pharyngeal pump function and tick feeding.
Sub-objective 2C. Identify GPCRs and agonists/antagonists as candidates for novel acaricide development.
1b.Approach (from AD-416):
To design effective vaccines and vaccination protocols for this project, we must first better understand the nature of the immune system of the white-tailed deer, specifically the suitability of deer as hosts for R. microplus, the deer immune response upon exposure to tick antigens, and the ability of deer to serve as reservoirs for the transmission of Babesia to cattle. This will help define the role of deer in tick distribution and population maintenance. Infestation of the deer with R. microplus and B. bovis induces responses in the deer but also in the tick at the gene, protein and immunochemical level. We will determine these Babesia-induced responses in R. microplus using functional genomics and proteomics. Differentially expressed genes/proteins will be prioritized as candidates for vaccine development.
To help identify these genes and proteins, we will use an established in vitro feeding system adapted for use with R. microplus females. Quantitative gene expression associated with ingestion of Babesia-infected blood will be analyzed using available R. microplus microarrays probed with RNA isolated from dissected tick tissues. Tick proteins will also be purified from the dissected tissues and SDS polyacrylamide gel electrophoresis used for comparisons between infected and uninfected samples. Candidate vaccine antigens will be evaluated for their effectiveness in controlling R. microplus infestations on deer and cattle and their capacity to block transmission of B. bovis between individual animals.
Neurotransmitters and neuromodulators, including dopamine, GABA, and acetylcholine, play key roles in many tick physiological processes. We will identify these neuroregulators in tick synganglia and neurons innervating pharyngeal muscles and salivary glands. Literature-derived protocols will also be applied to study the neuromuscular organization of the pharyngeal pump.
We will test effects and determine the mechanisms of action of various pharmacological agents, peptides, and vaccine candidates on pharyngeal pump function and tick feeding. Functional studies, including gene silencing studies, will confirm target identity and target validation. This information would facilitate development of novel acaricides that target genes that are critical to feeding success. It is necessary to identify muscular components involved in blood sucking and salivation to understand the physiology of feeding and test the pharmacology of molecules that regulate the tick pharyngeal pump. We will use the electrical pharyngeal graph to record muscle contractions associated with blood ingestion and/or salivation and to test effects of neuroactive compounds. Additionally, we will identify feeding-induced changes in R. microplus gene expression with a functional genomics approach.
We will identify candidate tick-specific G-protein coupled receptor genes in our R. microplus expressed gene database and agonist/antagonists that affect the function of these GPCRs. Our database of sequenced expressed genes, BACs, and genomic DNA will serve as the foundation for bioinformatic and analytical approaches aimed at finding genes encoding R. microplus GPCRs.
Regarding Objective 1 "Develop and test anti-tick vaccines for immunization of deer," significant progress was made toward evaluating vaccine candidates selected from genomic and transcriptomic data generated by this project. A patent application covering two novel vaccine antigens discovered in this project has been filed. Sufficient vaccine antigen for 200-head field trials for three antigens was prepared, including the two covered by the patent application. A vaccine trial to evaluate the immune response of white-tailed deer vaccinated with two vaccine candidate antigens, including one of the antigens covered in the patent application, is in its final stages. Three additional vaccine candidate antigens have been successfully produced and are available for small group cattle trials. A quantitative study of feeding-induced gene expression in adult R. microplus has been completed, and bioinformatic analysis has identified several feeding-induced genes that are candidates for use as a tick vaccine antigen. Repeated infestation of white-tailed deer with a one-host tick resulted in development of a response representing classic acquired immunological resistance, as evidenced by tick biological parameters. All tick-infested deer displayed an increased CD4:CD8 ratio in peripheral blood during the course of the infestation relative to a control animal, supporting a role for T-cells in the immunological response of deer. H&E staining of skin sections suggested cellular recruitment at the tick attachment site, and analysis of cytokine gene expression profiles by quantitative real-time PCR revealed the localized skin response was characterized by an upregulation of Th2 cytokines over the course of the study.
Regarding Objective 2 "Identify sensory, physiological, and biological targets for development of novel acaricides and drugs for use as chemical control technology," progress was made toward identifying novel tick-specific G protein-coupled receptor (GPCR) genes in the R. microplus genome. This has been enabled through genomic and transcriptomic sequencing using next-generation and 3rd generation technologies. Expression of one high priority GPCR has been quantified in 10 tissues of R. microplus, and RNAi evaluations have been completed for this GPCR by in vivo microinjection of double-stranded RNA. Results demonstrated significant mortality, indicating this GPCR is essential to the cattle tick. Significant progress was achieved toward development of a novel dual luciferase reporter plasmid for use in cell culture that will enable screening of siRNA (short interfering RNA, approx. 21-23 bp in length), providing improved analysis of gene silencing efficiencies in vitro prior to followup in vivo. These advances will provide significantly better data and cost savings due to reduction in number of in vivo experiments and travel required to conduct these studies in quarantine facilities.
Evaluating novel anti-tick vaccine antigen for use against cattle tick infestations in cattle or white-tailed deer. Widespread outbreaks of cattle fever ticks within South Texas are threatening the cattle tick eradication status of the U.S., demonstrating the need for new approaches that can be integrated with standard eradication procedures of the USDA Cattle Fever Tick Eradication Program (CFTEP). ARS scientists at Kerrville, Texas, in collaboration with EMBRAPA scientists in Campo Grande, Brazil, completed analysis of gene expression and vaccine trials of a novel vaccine antigen. The vaccine was 75% effective against R. microplus infesting cattle. Vaccination of white-tailed deer with this antigen was shown to induce a robust immune response. RNAi established that silencing this gene resulted in significant lethality in ticks. This novel antigen was highly expressed in tick nerve tissues, and the results indicate that vaccination against cattle ticks has great potential for integration in the CFTEP.
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Perez De Leon, A.A., Pete, T.D., Auclair, A.N., Messenger, M.T., Guerrero, F., Schuster, G., Miller, R. 2012. Integrated strategy for sustainable cattle fever tick eradication in U.S.A. is required to mitigate the impact of global change. Frontiers in Systems Biology. 3:195.
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Guerrero, F., Miller, R., Perez De Leon, A.A. 2012. Anti-cattle tick vaccines: Many candidate antigens, but will a commercially viable product emerge? International Journal for Parasitology. 42(5):421-427.
Temeyer, K.B., Olafson, P.U., Pruett, J.H. 2012. Sequence polymorphism in acetylcholinesterase transcripts and genotyping survey of BmAChE1 in laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus. Journal of Medical Entomology. 49(3):555-562.
Brake, D.K., Perez De Leon, A.A. 2012. Immunoregulation of bovine macrophages by factors in the salivary glands of Rhipicephalus microplus. Parasites & Vectors. 14(5):Article 38.
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Miller, R., Estrada-Pena, A., Almazan, C., Yeater, K.M., Messenger, M., Ellis, D., Perez De Leon, A.A. 2012. Exploring the use of anti-tick vaccine as tool for integrated eradication of the cattle fever tick, Rhipicephalus (Boophilus) annulatus. Vaccine. 30(38):5682-5687.
Heekin, A.M., Guerrero, F., Bendele, K.G., Saldivar, L., Scoles, G.A., Gondro, C., Nene, V., Djikeng, A., Brayton, K.A. 2012. Analysis of Babesia bovis-induced gene expression changes in the cattle tick, Rhipicephalus (Boophilus) microplus. Parasites & Vectors. 5:162.