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
As we near completion of the 4th year (2008) of this project we have made significant contributions to our defined milestones. Considerable Boophilus microplus databases for genomics and proteomics have been established, and the databases continue to grow, providing us and the research community with resources for the design of novel ways to control this important pest of livestock. The interaction between our parent projects and subordinate projects has been very beneficial to the progress and success we have experienced. It is planned that in the final year of the project we will begin to evaluate defined candidate vaccine antigens in dose-challenge studies. The information that has been developed, and will be accumulated in the final year of this project, is both timely and enabling regarding two aspects:.
1)the immediate need to address the role of the deer in B. microplus movement and population expansion, and the rapid design of vaccine technology to suppress that role; and.
2)providing our research team with insight regarding the design of our next project along a continuum of research to develop novel control technologies. (NP104; Component 3; Goal 3.1.1)

4.Accomplishments
1. Antigenicity of Boophilus microplus female and male salivary proteins in cattle and deer: Immunological resistance is primarily manifested as a reduction in engorgement weight of the female tick. Thus, feeding of the female tick is negatively affected by the host response. Our goal is to identify tick salivary proteins that can be used for vaccination. Cattle respond to tick proteins that are introduced into the host with tick feeding. We have evaluated the bovine response to salivary proteins of female B. microplus (1-2 day fed and 4-5 day fed), male B. microplus, and feeding females of Amblyomma americanum, a related ixodid tick. Many of the defined proteins are immunogenic in cattle, and of particular interest is a low molecular weight protein (13.8 to 14.0 kDa) that is present in 1-2 day fed B. microplus salivary gland extracts, but is not present in 4-5 day fed B. microplus salivary gland extracts and salivary gland extracts of feeding A. americanum females. The protein has potential as a diagnostic antigen for distinguishing the difference between B. microplus and A. americanum host exposure. Additionally, antibodies elicited to either an infestation with B. microplus or A. americanum elicit antibodies that cross react with proteins of each genera. These data suggest similar molecular homology between the salivary proteins of related species. Deer are emerging as an alternative host that may be responsible for B. microplus movement beyond the quarantine zone for B. microplus. Thus, we have expanded our investigations to include the deer response to these salivary proteins. Deer immunologically recognize and respond to B. microplus salivary proteins much like cattle, to include antibody production to the 13.8 kDa protein. These data suggest that defined vaccine candidate antigens can potentially be protective for both the cow and deer host. (NP104; Component 3; Goal 3.1.1)

2. Salivary proteins of female and male adult Boophilus microplus ticks: Many of the proteins expressed in the salivary glands of female and male ticks are known to fulfill important roles, enabling both male and female ticks to obtain sufficient nutrient resources (blood) for reproduction. Therefore, in order to define vaccine candidate antigens for vaccination it is important to identify and purify specific salivary gland proteins. We have analyzed with SDS-PAGE electrophoresis soluble protein salivary gland extracts of female B. microplus (1-2 day fed and 4-5 day fed), male B. microplus, and feeding females of Amblyomma americanum, a related ixodid tick. The total number of abundantly expressed proteins for all extracts was 34. Twelve of these proteins were common between female and male B. microplus ticks, and 12 were shared with the related ixodid tick A. americanum. The total number of proteins visualized was greater for 4-5 day fed B. microplus, but the 1-2 day fed banding pattern reflected an abundance of low molecular weight proteins, presumably associated with enzyme inhibitors that are known to be involved in anticoagulation. The results suggest considerable molecular homology and perhaps conservation of these important salivary proteins between different genera of the ixodidea. This information will enable us to look for vaccine candidate antigens that may provide for cross protection between different ixodid species. (NP104; Component 3; Goal 3.1.1)

3. Purification of larval and adult female anticoagulants: The expression pattern of the KU-domain transcripts, should they serve a role in anti-coagulation, would provide support for their role in successful tick feeding. The penthaplus transcript is detectable at the larval stage, and in unfed females and salivary glands of fed females with a marked increase in expression that is coincident with feeding. The transcript is not expressed in adult males (unfed or fed). The Bmi-KU1 transcript is only detectable in salivary glands of fed females. The Bmi-KU2 and KU3 transcripts are detectable in larvae, unfed adult females and males, and salivary glands of fed adult females and males. Bmi-KU2 expression is elevated in fed females relative to unfed females, while Bmi-KU3 is elevated in fed males relative to unfed males. These results support that these proteins are important in tick feeding, and if their function can be inhibited they may represent viable candidate antigens for vaccination. (NP104; Component 3; Goal 3.1.1)

4. Evaluation of anti-thrombin gene polymorphism and tissue expression: In an effort to study key proteins involved in successful tick feeding, we have identified and characterized four kunitz-like proteins (enzyme and coagulation inhibitors) from the salivary gland EST libraries, i.e., penthaplus (5 kunitz domains) and Bmi-KU1, KU2, and KU3 (single kunitz domains). Evaluation of their tissue expression by RT-PCR revealed that all four transcripts are differentially expressed throughout the lifecycle (larvae versus unfed adult males and females vs. fed adult males and females). In addition, all four have been expressed as His-fusion proteins in E. coli. Mice were immunized with the penthaplus recombinant protein, and antibody production was verified using a plate ELISA assay. The mouse polyclonal antibodies will be used to characterize penthaplus protein expression throughout development. (NP-104; Component 3; Goal 3.1.1)

5. Identification of expressed acetylcholinesterases in the synganglion of Boophilus microplus: Acetylcholinesterase is the target enzyme for organophosphate acaricides. One mechanism of organophosphate resistance is mutation within acetylcholinesterase. We found that genes encoding acetylcholinesterases BmAChE1, BmAChE2, and BmAChE3 are all expressed in adult tick synganglion, and that more than 2 transcripts are expressed for each of the 3 AChEs within an individual tick. In addition, preliminary evidence indicates a copy number of about 4 for each of the BmAChEs, suggesting that these genes have been duplicated in the tick genome, potentially allowing multiple different alleles to be expressed in a single individual, as well as potentially increasing AChE expression due to gene amplification. Although the data is incomplete, it is clear that the acetylcholinesterases of B. microplus and their involvement in resistance to organophosphate are vastly more complex than previously believed. To date, neural targets for acaricides have been the most effective at tick control, consistent with their key role in physiological integration, responses to environmental stimuli, and developmental processes. (NP104; Component 3; Goal 3.1.1)

6. Development of genomic resources for Boophilus microplus: In conjunction with the subordinate project with J. Craig Venter Institute, the "high Cot" fraction of tick genomic DNA was used for 6 plates of 454 pyrosequencing for the continued development of genomic tools. This fraction of genomic DNA contains the gene regulatory and coding regions of the genome and is largely void of the highly repetitive genomic DNA regions which are very difficult to sequence and assemble. The 454 sequencing protocol returned 2.7 million sequencing reads (average read length of 237 bp) from the "high Cot" DNA and was assembled into 15,221 sequence contigs with average length of 643 bp. A total of 11,856 of these contigs matched protein coding regions in gene coding region sequence databases. This new sequence data is expected to add significantly to the number of known gene coding regions from B. microplus and should produce many novel candidates to screen for their potential as anti-tick vaccine antigens. (NP104; Component 3; Goal 3.1.1)

7. An analysis of expressed proteins in the ovary of Boophilus microplus: Boophilus microplus microarrays developed under the subordinate project with Washington State University were used in an effort to investigate ovarian proteins that are differentially expressed as a result of Babesia bovis infection. Differentially expressed proteins were observed, suggesting a role for those proteins in successful infection by Babesia bovis. As a result a collaboration with the Animal Disease Research Unit, Pullman, WA, is underway to investigate the potential for exploitation of these differentially expressed genes for either an anti-tick or anti-Babesia vaccine. (NP104; Component 3; Goal 3.1.1)

8. An analysis of expressed proteins in the tick midgut following infection with Babesia bovis: Boophilus microplus microarrays developed under the subordinate project with Washington State University were used in an effort to investigate expressed proteins in midgut tissues. We identified midgut-expressed genes that are differentially expressed following infection with Babesia bovis. As a result, a collaboration with the Animal Disease Researh Unit, Pullman, WA, is underway to investigate the exploitation of these differentially expressed genes for either an anti-tick or anti-Babesia vaccine. (NP104; Component 3; Goal 3.1.1)

9. Progress associated with BAC library end-sequencing: To further our effort in BAC library end-sequencing, in conjunction with the subordinate project with JCVI and Purdue University, we have sequenced 4 BACs to completion. In conjunction with the CRADA with the Australians, we have processed 11,520 BAC ends sequence reads from the Boophilus microplus BAC library. Additionally, 5 BACs were selected and sequenced to completion. BACS selected for sequencing were determined by identity to putative anti-B. microplus vaccine candidate sequences, which had been identified through in silico bioinformatic approaches. Sequence information should provide insight into function, and development of recombinant constructs to allow recombinant protein expression for candidate vaccines. (NP104; Component 3; Goal 3.1.1)

10. Continued expansion of the project EST annotation and gene index: To continue the building of genomic and proteomic resources, in conjunction with the JCVI subordinate project, over 15,000 sequence contigs have been produced recently through 454 sequencing of "Cot-enriched" genomic DNA. Bioinformatic analysis is underway to identify contigs that contain protein coding regions. The new protein coding regions will be annotated and added to the Boophilus microplus gene index that is currently available to the scientific community at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus. The continued development of the EST annotation and gene index transfers to the research community additional genomic information about Boophilus microplus. (NP104; Component 3; Goal 3.1.1)

6.Technology Transfer

Number of Active CRADAs	1
Number of New Commercial Licenses Executed	4

Review Publications
Rachinsky, A., Guerrero, F.D., Scoles, G.A. 2007. Differential protein expression in ovaries of uninfected and Babesia-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus. Insect Biochemistry and Molecular Biology. 37:1291-1308.