2010 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.
We have made significant progress on immunohistological detection of serotonin and octopamine in the tick nervous system and localizing their receptors. This has provided a foundation for choosing agonist and antagonist compounds that can be used in later experiments. Capillary feeding experiments on the cattle tick will be completed by September. We have set up an in vitro tick membrane feeding system at Moore Field and are currently optimizing the system. Progress was made toward identification of G protein-coupled receptor (GPCR) genes in the R. microplus gene database and agonist/antagonists affecting function. The octopamine receptor is a GPCR involved in regulating metabolic and physiological responses to internal and external stimuli. Gene silencing experiments were performed on the octopamine receptor, and this methodology can be used to test involvement of this GPCR in resistance to amitraz. With bioinformatic approaches based on DNA sequence similarities to known GPCRs, we have identified 30 putative GPCRs in our R. microplus gene database. These serve as a first set of candidates for novel targets for chemical-based control technologies. We have also made progress toward a more rigorous analysis based on comparison of protein coding regions from our R. microplus gene database to known GPCR structural features. We have made progress on our goal of a better understanding of the white-tailed deer immune system through studies of ungulate immune system responses to tick salivary gland extract. Real-time PCR probes have been designed and tested to measure bovine cytokine response and maturation and activation of immune cells. We have based this year's work on cattle immune system responses, as there are many similarities between the bovine and deer immune system. Several anti-tick vaccine candidates have been identified by bioinformatic, functional genomic, and proteomic approaches. Three candidates have been evaluated in cattle trials as DNA vaccines, and the positive results led to filing an invention disclosure on two antigens. Four of these candidates have been produced as recombinant proteins and are scheduled for evaluation in cattle trials in conjunction with EMBRAPA Brazil.
Facilities for working with white-tailed deer were designed and constructed at our lab. These facilities include pens to contain individual deer during tick infestation studies and a lift chute for blood and tissue sampling. We are using this facility to maintain deer obtained through collaborations with Kerr Wildlife Management area and Texas A&M Kingsville. Tame, bottle-raised does are required for the duration of the proposed research due to the frequent manipulations and sampling involved. Four bottle-fed fawns were raised at our lab from 2 days of age, and these does are being used in tick infestation studies that commenced in July 2010.
Multiple acetylcholinesterases of R. microplus. Resistance to organophosphate (OP) acaricides presents a major problem in controlling the cattle fever tick, Rhipicephalus microplus. The toxicity of OPs is due to their ability to inactivate the nerve protein acetylcholinesterase (AChE). ARS researchers at Kerrville, Texas, demonstrated that the genetics of AChE is more complex in cattle ticks than expected and that R. microplus utilizes at least three biochemically active AChEs in its central nervous system. At least two of these AChEs can exist in mutant forms that are resistant to the inactivation by OPs. This new information explains previous failures to find simple mechanisms for OP resistance that has been identified in field populations of this tick.
Identification of effective anti-cattle tick vaccine candidates. Cattle fever ticks are arthropods of veterinary and medical importance because of blood loss and physical damage associated with their feeding and because they transmit the causative agents of bovine babesiosis and anaplasmosis. ARS scientists in Kerrville, Texas, collaborating with scientists from EMBRAPA Brazil, identified two anti-cattle tick vaccine candidates in cattle trials. These candidates had been prioritized in a prior ARS project through bioinformatic and molecular biological approaches. In the cattle trials conducted in Brazil, the candidates outperformed the recombinant Bm86 Campo Grande antigen, which is an antigen similar to that used in the only current commercially available anti-tick vaccine. An invention disclosure was filed and cattle trials are scheduled to evaluate various parameters in the vaccination protocol to optimize efficacy. This accomplishment is of interest to the global animal health industry and cattle producers worldwide.
5.Significant Activities that Support Special Target Populations
A specific cooperative agreement entitled "Sequencing of BAC ends from a Rhipicephalus microplus BAC library" was created with University of Houston-Clear Lake, a Hispanic-Serving Institution. Through this agreement, a laboratory-centered university course was established and funded with tick BAC clone sequencing as the primary objective in the course. Sequence assembly and bioinformatic analysis results will be provided to ARS, and this will contribute to progress toward project objectives.
Temeyer, K.B., Pruett Jr, J.H., Olafson, P.U. 2010. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus. Veterinary Parasitology. 172:114-121.
Guerrero, F., Moolhuijzen, P., Peterson, D.G., Bidwell, S., Caler, E., Appels, R., Bellgard, M., Nene, V.M., Djikeng, A. 2010. Reassociation kinetics analysis-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus. Biomed Central (BMC) Genomics. 11:Article 374).
Temeyer, K.B., Olafson, P.U., Miller, R. 2009. Genotyping mutation in BmAChE3: A survey of laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus that are resistant or susceptible to coumaphos. Journal of Medical Entomology. 46(6):1355-1360.
Bastos, R., Ueti, M.W., Guerrero, F., Knowles Jr, D.P., Scoles, G.A. 2009. Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus) microplus increases the infection rate of Babesia bovis in larval progeny. BioMed Central (BMC) Vectors and Parasites. 2(1):57.
Kurscheid, S., Lew-Tabor, A.E., Rodriguez Valle, M., Bruyeres, A.G., Doogan, V.J., Munderloh, U.G., Guerrero, F., Barrero, R.A., Bellard, M.I. 2009. Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila. BioMed Central (BMC) Molecular Biology. 10:Article 26.
Lew-Tabor, A.E., Moolhuijzen, P.M., Jones, M.E., Kurscheid, S., Rodriguez Valle, M., Jarrett, S., Minchin, C.M., Jackson, L.A., Jonnson, N.N., Guerrero, F. 2010. Suppressive subtractive hybridization analysis of Rhipicephalus (Boophilus) microplus larval and adult transcript expression during attachment and feeding. Veterinary Parasitology. 167:304-320.
Rodriguez, V.M., Lew-Tabor, A., Gondro, C., Moolhuijzen, P., Vance, M., Guerrero, F., Bellgard, M., Jorgensen, W. 2010. Comparative microarray analysis of Rhipicephalus (Boophilus) microplus expression profiles of larvae pre-attachment and feeding adult female stages on Bos indicus and B. taurus cattle. Biomed Central (BMC) Genomics. 11:Article 437.