2013 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," a vaccine trial to evaluate the immune response of white-tailed deer vaccinated with an ARS-patented antigen has been completed. Vaccination elicited a specific antibody response to the ARS-patented antigen, and a manuscript describing the immune response of the deer in this trial has been drafted. Serum from these vaccinated deer was used to probe synthetic peptide microarrays, and specific linear epitopes on the ARS-patented antigen were identified. Hybrid antigens that combine these validated epitopes with predicted epitopes from other anti-tick vaccine candidate antigens have been synthesized. The vaccinated deer serum was also used in an in vitro feeding study on the brown dog tick, Rhipicephalus sanguineus. A provisional patent application regarding a vaccine for companion animals has been filed on the resultant discovery that the ARS-patented vaccine antigen is efficacious against the brown dog tick. As supporting evidence for the final patent document, a vaccination trial to evaluate this antigen's effectiveness in dogs has been initiated in Brazil. A vaccination trial on pastured cattle is underway to evaluate the ARS-patented antigen as an anti-tick vaccine for cattle in the southern region of Brazil. We have completed and published two quantitative genomics studies of cattle ticks as they respond to ingestion of blood infected with Babesia bovis. We identified specific genes in the adult cattle tick gut and ovary that respond to the infection process, and interference with these genes is expected to impact the tick's feeding or reproductive success.
Regarding Objective 2 "Identify sensory, physiological, and biological targets for development of novel acaricides and drugs for use as chemical control technology," the genome sequence of the cattle tick has been sequenced by 3rd-generation sequencing methodology to a 10-fold coverage, suitable for assembly, and publication of a draft level genome sequence. The assembly of this large and complex genome is underway, and the bioinformatic annotation is expected to be completed, published, and transferred to the scientific community via the genome sequencing project's website, CattleTickBase, within the next year. We have begun analysis of this genome dataset for G-protein coupled receptor sequences, proteins that are promising targets for development of novel chemical control technologies. Additional data on the number and role of acetylcholinesterases in ticks has added to our understanding of this complex, but key system integrating physiological process of the tick and manipulating host responses to tick and tick feeding.
Dual luciferase reporter system for evaluating gene silencing constructs in tick cell culture. Scientists utilize gene silencing for functional genomics to elucidate gene function in vivo, and for target validation in vaccine development. Previously available gene silencing technology for ticks required microinjection of RNAi constructs in live ticks, with subsequent tissue collection and evaluation of gene silencing efficiency. This procedure is laborious, very slow, costly, and requires extensive travel to the USDA-ARS tick quarantine facility. ARS scientists at Kerrville, Texas, have discovered tick gene promoters and utilized them to construct a dual luciferase reporter system that provides high throughput capability for evaluation of RNAi constructs for tick gene silencing, with high efficiency and greatly reduced time and cost. This accomplishment is expected to significantly accelerate gene silencing procedures for functional genomics and target validation within USDA and the scientific community.
Sequencing the genome of the cattle fever tick. Outbreaks of cattle fever ticks within South Texas are threatening the cattle tick eradication status of the U.S. Knowing the sequence of the tick's genome would accelerate the development of vaccines and novel pesticides for use in the USDA Cattle Fever Tick Eradication Program (CFTEP). ARS scientists at Kerrville, Texas, in collaboration with researchers at Murdoch University, Perth, Australia, and National Center for Genome Resources, have sequenced the genome of the cattle tick. Data from the genome sequence has resulted in the identification of anti-tick vaccine candidates that should provide an effective tool for integration into the CFTEP. Additionally, with this sequence, targets for development of novel pesticides can be more rapidly identified and evaluated. This is expected to accelerate acaricide research and development within USDA and the global animal health industry.
Temeyer, K.B., Chen, A.C., Davey, R.B., Guerrero, F., Freeman, J.M., Kammlah, D.M., Li, A.Y., Lohmeyer, K.H., Olafson, P.U., Perez De Leon, A.A., Phillips, P.L., Pound, J.M., Welch, J.B. 2012. Novel approaches for control of Rhipicephalus (Boophilus) microplus. J Tecnica Pecuaria En Mexico. 3(Suppl. 1):25-40.
Temeyer, K.B., Tuckow, A.P., Brake, D.K., Li, A.Y., Perez De Leon, A.A. 2013. Acetylcholinesterases of blood-feeding flies and ticks. Chemico Biological Interactions. 203:319-322.
Heekin, A.M., Guerrero, F., Bendele, K.G., Saldivar, L., Scoles, G.A., Dowd, S.E., Gondro, C., Nene, V., Djikeng, A., Brayton, K.A. 2013. Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host. International Journal for Parasitology. 112:3075–3090.
Temeyer, K.B., Olafson, P.U., Brake, D.K., Tuckow, A.P., Li, A.Y., Perez De Leon, A.A. 2013. Acetylcholinesterase of Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi: Gene identification, expression, and biochemical properties of recombinant proteins. Pesticide Biochemistry and Physiology. 106:118-123.
Palavesam, A., Guerrero, F., Perez De Leon, A.A., Heekin, A.M., Wang, J., Dowd, S., Sun, Y., Foil, L.D. 2012. Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans. PLoS One. 7:44390.
Andreotti, R., Casquero Cunha, R., Aparecida Soares, M., Guerrero, F., Leivas Leite, F.P., Perez De Leon, A.A. 2012. Protective immunity against tick infestation in cattle vaccinated with recombinant trypsin inhibitor of Rhipicephalus microplus. Vaccine. 30:6678-6685.
Lovis, L., Guerrero, F., Miller, R., Bodine, D.L., Betschart, B., Sager, H. 2012. Distribution Pptterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia. International Journal for Parasitology: Drug and Drug Resistance. 2:216-224.