1a.Objectives (from AD-416)
Develop genomic tools for the initiation of a Boophilus microplus genome project, and assemblage of an international team to seek additional funding for a joint genomic project; develop techniques of gene silencing to allow for evaluation of the biological function and significance of identified genes; and develop a proteome database, that is gender and stage specific, from which the biological significance of identified proteins can be evaluated.

1b.Approach (from AD-416)
Basic information on the structure of the B. microplus genome will be obtained from information derived from a normalized cDNA library approach and the creation of a BAC library. Gene silencing through use of RNA interference will be used to reduce expression of targeted genes, and assist in the elucidation of their metabolic, biochemical, or structural roles. By focusing on the components of the tick proteome associated with events associated with tick feeding and the facilitation of pathogen transmission, proteins involved in tick feeding and life-stage related differences in protein expression will be identified and related to function.

3.Progress Report
This report documents research conducted under in-house project 6205-32000-026-00D, Molecular Biology of Ripicelphalus (Boophilus) microplus. The research focus of the project is to develop biological and molecular databases that will provide for the rational design of effective control technologies for the Southern cattle tick, R. microplus, an important animal disease vector. Key areas of research include genomic resources, development of gene silencing techniques for the study of functional genomics, and an investigation of the proteome for components associated with successful tick feeding and the facilitation of pathogen transmission. This report marks the end of the third year of the five-year project. Proposed genomics work is essentially complete, with the continued development of the EST database and the gene index. Considerable genomic resources have been developed, and a white paper has been published. What now waits is adequate funding for the conduct of a R. microplus sequencing project. The proteome database continues to grow to include expressed proteins of the ovaries, gut, and salivary gland, with those accomplishments addressed in question 4 of this report. Several proteins of larval, nymph, and adult salivary gland tissues have been shown to be immunogenic in the bovine host. During the next two years the databases will continue to expand, but emphasis will be given toward the identity of expressed proteins, their function, and evaluation of candidates as targets for tick control.

4.Accomplishments
Identification of R. microplus life-stage specific proteins that elicit an immune response in cattle: Identification and isolation of R. microplus life-stage proteins that elicit an antibody response in the host should lead to the selection of potential vaccine antigens for tick control. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, the list of immunogenic proteins (immunome) has been increased by 15 proteins from the 1-2 day fed adult female salivary gland and 6 proteins from fed nymphs. Continued development of this database will allow for the rational design of protective vaccines for R. microplus control. (NP104; Component 3, Biology and Physiology; Goal 3.1.4)

Identification of bovine host genes that associate with the tick-resistant phenotype: It is essential to identify and define host genes that are involved in natural tick innate and acquired resistance. DRB3 is a class IIa gene of the bovine leukocyte antigen complex that is being studied. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, analysis of a region of the DRB3 gene from the phenotyped calves in our genomic DNA database herd revealed a significant association between the DRB3*4401 allele and the tick-resistant phenotype. Identification of resistance associated genes will allow for selective markers and enhanced vaccine efficacy. (NP-104; Component 3, Biology and Physiology; Goal 3.1.4)

Analysis of host-pathogen interaction between Babesia bovis and R. microplus: In order to better understand the mechanisms of pathogen transmission it is necessary to compare and contrast gene expression in ovarial and gut tissues of B. bovis infected and uninfected R. microplus. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, the ovarial and gut proteomes of R. microplus adult females infected with R. bovis have been analyzed by 2-D PAGE and specific proteins that are differentially expressed in response to infection have been identified by mass spectrometry and bioinformatic analysis. Differential gene expression in response to R. bovis infection in these tissues has also been analyzed by microarray analysis and subtraction library screening, allowing examination of this critical interaction at the gene transcript and protein level. This information allows for the study of vector competence regarding the transmission of the pathogen, B. bovis, by the tick, R. microplus, to cattle. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1)

Variation within Glucose 6-phosphate dehydrogenase (G6PDH), a tick enzyme in R. microplus: Developing molecular epidemiological techniques to determine genetic relatedness of outbreak tick strains is necessary to provide better support of the APHIS-VS, Cattle Fever Tick Eradication Program. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, four different transcripts for this enzyme have been identified. In addition, on non-denaturing polyacrylamide gels, more than one protein band with G6PDH activity was observed, suggesting the presence of multiple forms of this enzyme expressed by the tick. We postulate that gender and life-stage differential expression of these transcripts of G6PDH may play a role in tolerance of oxidative stress that is induced upon feeding, and that the transcript abundance in fed females is a function of bloodmeal volume and the time adult females spend on the host relative to adult males. Both male and female ticks require an adequate blood meal to insure successful reproduction. Physiological events that protect ticks while feeding represent targets for control intervention. Identification of those targets is essential to the development of novel control technologies. Molecular tools that can provide information as to the genetic relationships between ticks collected from outbreak sites provide epidemiologists with valuable information for the management and understanding of outbreaks. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1, and NP-104 Component 2, Detection and Surviellance Technology; Goal 2.1.1)

Development of an internal standard for quantitative-PCR (Q-PCR) and quantitative reverse transcriptase-PCR (QRT-PCR) assays in R. microplus: Quantitation of mRNA in different tissues or under different conditions requires an internal invariant reference to use as a normalization standard to allow comparison of expression of the test mRNA. "Housekeeping" genes are generally used for this purpose, but previous studies in other organisms has shown that multiple reference genes are needed, as none have been found to be truly invariant. To date, the only published report of quantitative PCR in R. microplus utilized beta-actin as an internal reference for normalization between samples. Our studies have indicated that there are multiple transcripts or isoforms of beta-actin in R. microplus, making it a poor choice as an internal reference standard. We have developed 18S DNA/RNA as an internal reference standard, chosen because it is a major component of ribosomes, expressed at high levels in most tissues, and is generally known to be under tight transcriptional control. This represents a versatile and useful internal standard for relative quantization within essentially any tissue for genes that are highly expressed, or those expressed at relatively low levels, providing functional genomic researchers with a valuable tool. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1)

6.Technology Transfer

Number of new CRADAs and MTAs	3
Number of active CRADAs and MTAs	10
Number of non-peer reviewed presentations and proceedings	5

Review Publications
Olafson, P.U., Pruett Jr, J.H., Steelman, C.D. 2007. Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star Tick, Amblyomma americanum. Veterinary Parasitology. 145(1-2):190-195.