Location: Tick and Biting Fly Research2011 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.
3. Progress Report
Regarding Objective 1 "Develop and test anti-tick vaccines for immunization of deer", we have made significant progress on evaluating vaccine candidates using genomic data to select 8 novel candidate antigens expressed as recombinant proteins in yeast for evaluation in cattle trials. Three antigens have been evaluated in one cattle trial, and invention disclosure was filed. We completed the cloning, expression, and purification of Bm86, the antigen from the only existing anti-cattle tick vaccine, from cattle tick outbreak populations in Texas. Cattle trials expected to begin in FY12. We have completed repeated tick infestations of white-tailed deer under conditions preventing grooming. Blood and skin biopsies at tick attachment sites were collected at relevant timepoints, and these are being analyzed by flow cytometry, quantitative real-time PCR, histology, and immunohistochemistry to describe the white-tailed deer immune response to repeated tick infestation. We have isolated, cultured, and in vitro differentiated bovine and deer macrophages from blood to determine immunoregulatory effects of R. microplus salivary gland extract on host immune responses. Changes in cytokine expression and cell marker regulation were measured by real-time PCR and ELISA at time points after cell activation. The effect of repeated infestation on tick fecundity parameters were also assessed, including tick engorgement weights, egg mass production and weight, and estimated larval hatch. 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 identification of G protein-coupled receptor (GPCR) genes in the R. microplus genome. We have identified 60 candidate GPCRs using amino acid-based similarity analyses and more rigorous structural bioinformatic approaches. These GPCRs have been prioritized for expression studies in later years of the project based upon putative identity, complexity of expression and purification, and anticipated expression patterns in tick tissues. We have completed gene expression experiments to identify R. microplus genes whose expression is activated or repressed by blood-feeding. Feeding-responsive expression has been quantified by next-generation sequencing on the Illumina platform, and bioinformatics is underway to determine identities of feeding-responsive genes. The octopamine receptor is a GPCR involved in regulating responses to internal and external stimuli. We conducted gene silencing experiments to evaluate the receptor as a potential anti-tick vaccine candidate, and this methodology can be used to test involvement of this GPCR in resistance to the acaricide amitraz. Gene silencing of the octopamine receptor resulted in 83% reduction in tick survival and production of progeny.
1. Anti-tick vaccine evaluated for use against cattle ticks, Rhipicephalus annulatus. The occurrence of widespread outbreaks of cattle fever ticks within South Texas has demonstrated the need for new approaches to maintain the cattle tick eradication status of the U.S. that work within the normal eradication procedures of the USDA Cattle Fever Tick Eradication Program (CFTEP). An anti-tick vaccine based on the tick molecule Bm86 that is commercially available outside of the U.S. was evaluated using cattle held in barns and artificially infested with ticks. The vaccine was 99.6% effective immediately after the initial vaccination series and 82% effective 5 months after the initial vaccination series. Computer modeling of the habitat known to harbor this tick indicated that only 45% control is needed to maintain eradication. This is the first time an anti-tick vaccine was tested at the USDA, and the results indicate it has great potential to be used on a wide scale in the CFTEP. The vaccine is safe, non-toxic, and non-polluting, and provides a highly effective control approach for CFTEP to eradicate outbreaks of Rhipicephalus annulatus.
Rothwell, J.T., Morgan, J., James, P., Brown, G., Guerrero, F., Jorgensen, W. 2011. Mechanism of resistance to synthetic pyrethroids in buffalo flies in south-east Queensland. Australian Veterinary Journal. 89(3):70-72.