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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY OF BOOPHILUS MICROPLUS

Location: Tick and Biting Fly Research

2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Ticks can cause direct economic impact because of a general loss of host condition due to irritation, anemia, secondary infection, paralysis, and toxicoses. However, their major impact upon animal production is their ability to vector a wide spectrum of pathogenic microorganisms, including protozoa, rickettsiae, spirochetes, and viruses. Disease transmitted by ticks is a major factor in limiting animal production in many subtropical and tropical regions of the world. In North America, the tick vectors of Babesia bovis and B. bigemina, the protozoan agents of Cattle Fever, are the southern cattle tick, Boophilus microplus, and the cattle tick, Boophilus annulatus. These tick species were declared eradicated from the U.S. in 1943. A quarantine zone was created on the Texas-Mexico border, and an import-dipping program has been maintained to prevent reentry of the tick vector into the U.S. Reentry of the tick and babesiosis into susceptible U.S. cattle herds would present serious economic consequences for U.S. cattle producers. Effective control of ticks is paramount to the control of animal and human tick-borne diseases. Ticks have been controlled primarily with pesticides. Development of pesticide resistance and a lack of new classes of pesticides in development have stimulated interest in novel means of tick control that are both environmentally safe and sustainable in the U.S. and in other regions of the world. Genomic and proteomic information for Boophilus sp. is quite limited. Information obtained from this project will allow for the elucidation of the complex interactions between the tick-host-pathogen. This information should aid in the definition of novel non-chemical control alternatives.

The long-term goal of this research is to develop the biological and molecular databases that will provide for the rational design of effective control technologies for the important animal disease vector, Boophilus microplus. The specific objectives for the 5 years of this project are:

1. Develop genomic resources that would facilitate the initiation of a Boophilus microplus genome project. 2.Develop techniques of gene silencing with B. microplus as a tool for use in gene function studies. 3. Investigate components of the B. microplus proteome that are involved in the various events associated with successful tick feeding and the facilitation of pathogen transmission. This would specifically include expressed proteins of the salivary gland and midgut, and lifestage-related differences in protein expression.

It is anticipated that this integrated long-term approach to the problem of tick control based upon the development of molecular genomic and proteomic databases will provide important physiological targets for the rational design of control technologies for the cattle fever tick, B. microplus. The research to be undertaken falls under NP 104 - Veterinary, Medical and Urban Entomology and addresses goal: 3.1 Genomics and Host-Pathogen Interaction. Goal 3.1.1 specifies the use of ultrastructural methods, as well as expressed sequence tag (EST) and bacterial artificial chromosome (BAC) libraries, to investigate the basis of tick digestion, salivation, egg production, and susceptibility to protozoan parasites of livestock. This goal includes identifying and cloning genes crucial to these processes and designing agents that will inhibit or disrupt their actions. Goal 3.1.2 specifies the organization of and participation in an international consortium to sequence the entire genome of the tick B. microplus and manage the bioinformatics to allow comparison of homologous genes in different genera.

Achieving these objectives will provide benefits to producers, regulators of animal diseases, and other scientists. The public will benefit from a continued flow of safe and affordable food products. The producers will realize the economic benefits of avoiding costly quarantine and control efforts. In addition to the importance of a safe and nutritious food supply, is the importance of developing environmentally safe and sustainable control technologies for ticks and the diseases they transmit. Knowledge of the tick genome and gene products will provide the database from which to develop those new control technologies.


2.List by year the currently approved milestones (indicators of research progress)
Year 1 (2005)

Cot Analysis BAC Library Synthesis BAC Library End Sequencing BAC Clone Screening EST Sequencing EST Annotation and Gene Index Develop a List of Tick Researchers With Genomic Interest Draft a White Paper Proposing a Boophilus microplus Genome Project Submit a White Paper to Funding Agencies Construction of dsRNAs

Year 2 (2006)

BAC Clone Sequencing EST Annotation and Gene Index Introduction of dsRNAs in vivo RNAi silencing of additional targets Proteome Analysis: Eggs, Salivary Gland, Midgut Identification of antigens; larval Production of stage-specific Antisera Identification of antigens; nymphal Preparation of stage-specific antigens Purification of anticoagulants adult female Isolation/Characterization of anti-thrombin gene Immunogenicity of anti-thrombin in the bovine Inhibition of anti-thrombin by specific antibody elicited by natural exposure

Year 3 (2007)

EST Annotation and Gene Index Quantitation of target mRNAs and Proteins RNAi silencing of additional targets Proteome Analysis: Unfed Larvae, Unfed Nymphs, Salivary Gland, Midgut Salivary Gland Protein Polymorphisms Identification of antigens; adult male Purification of anticoagulants nymphal Evaluation of anti-thrombin gene polymorphisms and tissue expression Inhibition of anti-thrombin by specific antibody elicited by vaccination

Year 4 (2008)

EST Annotation and Gene Index RNAi silencing of additional targets Proteome Analysis: Unfed Adult Male and Female Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Identification of antigens; adult female Purification of anticoagulants larval Evaluation of anti-thrombin gene polymorphisms and tissue expression

Year 5 (2009)

EST Annotation and Gene Index RNAi silencing of additional targets Proteome Analysis: Salivary Gland Proteome Analysis: Midgut Purification of anticoagulants adult male Vaccination with anti-thrombin and Challenge


4a.List the single most significant research accomplishment during FY 2006.
Evaluation of the genetic relatedness of B. microplus ticks collected from separate outbreak sites – The Cattle Fever Tick Eradication Program (CFTEP) is a joint federal-state program designed to prevent B. microplus from reentering the U.S. In support of this program, and specifically Dr. Roberta Duhaime DVM, epidemiologist, with APHIS-VS, we have provided technologies that will aid in elucidating the genetic relatedness of ticks collected from independent outbreaks of B. microplus ticks within and outside of the quarantine zone. This works lays the foundation for surveying genetic relationships of ticks collected throughout Mexico. The group fully sequenced the glucose-6-phosphate gene of B. microplus and evaluated nucleotide sequence variation of one of the introns in approximately 500 individual tick specimens collected from 15 outbreak sites within the quarantine zone. Nine intron sequence variations have been identified, the frequencies of which distinguish collections from independent outbreaks. This accomplishment is aligned with NP-104 and addresses Component 2, Detection and Surviellance, specifically performance measure 2.2.4.


4b.List other significant research accomplishment(s), if any.
Identification of serologically defined B. microplus larval antigens – Life-stage-specific B. microplus proteins (6) that putatively elicit antibodies in cattle as a result of natural exposure were identified. A serologically defined 19.1 kDa protein was used to develop a diagnostic ELISA for cattle exposure to B. microplus. The 19.1 kDa protein has been partially amino acid sequenced, and we are attempting to isolate the gene. The information offers the potential for serological diagnosis of B. microplus exposure to cattle in support of the CFTEP program of APHIS-VS. In addition, it adds to a pool of candidate antigens for vaccine development. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of National Program 104 Action Plan. In addition the accomplishment addresses performance measure 3.2.1 in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health.

Identification of potential targets for the development of novel control strategies for B. microplus - Characterization of molecules that are essential for tick feeding is one approach to the identification of targets for alternative novel control, specifically molecules produced in the salivary gland tissue of fed adult females that are potentially introduced to the bovine host via tick saliva. We have identified transcripts encoding five zinc metalloproteases and four kunitz-like proteins from salivary gland tissue of adult, female ticks that had fed on the host for either 1-2 days or 4-5 days. In other ticks, the zinc metalloproteases are believed to have a fibrinolytic role and inhibit host angiogenesis (prevent wound healing) while kunitz-like proteins have been implicated in the inhibition of the host blood coagulation system. Functional characterization of these molecules with respect to their role in successful tick feeding of B. microplus will lay the foundation for their use as potential targets for alternative control technologies. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of National Program 104 Action Plan. In addition the accomplishment addresses performance measure 3.2.1.

Analysis of the B. microplus ovarial proteome - The ovarial proteome of B. microplus has been analyzed by 2-D PAGE, focusing on Babesia bovis infection-induced differences. Specific proteins that are differentially expressed have been identified, extracted and submitted for initial mass spec analysis at the Protein Chemistry Laboratory, University of Texas Health Science Center in San Antonio, TX. Functional characterization of these molecules with respect to their role in pathogen transmission will lay the foundation for their use as potentials targets for alternative control technologies of the vector and the pathogen. This accomplishment addresses component 3 biology and physiology, and performance measure 3.1.1 genomics and host-pathogen interaction.

Baculovirus expression of a B. microplus acetylcholinesterase – This is the first report of the cloning and successful expression of a B. microplus acetylcholinesterase. Knowledge of the wild-type molecule allows the search for mutations that may account for resistance to organophosphates. An organophosphate acaricide is used in the import dipping vats maintained by the Cattle Fever Tick Eradication Program on the border with Mexico; as a result concerns regarding the development of resistance to organophosphates necessitates the need to understand the specific mechanisms of acetylcholinesterase target-site insensitivity. This work is in direct support of a related project at the Knipling-Bushland U.S. Livestock Insects Research Laboratory entitled, "Developing and using molecular and biochemical methods for the diagnosis of acaricide resistance in B. microplus," as well as APHIS-VS. The accomplishment addresses component 2, detection and diagnostics 2.1 of the National Program 104 Action Plan.

Biochemical diagnosis of B. microplus acetylcholinesterase organophosphate target-site insensitivity - The principal mechanism of resistance to organophosphate acaricides in B. microplus ticks is acetylcholinesterase (target-site) insensitivity to inhibition. This work describes a method of neural acetylcholinesterase extraction that allows for the diagnosis of insensitivity and the genotyping of the individual tick. This technology will find utility in the CFTEP conducted by APHIS-VS, as it allows for diagnosis as well as the screening of additional organophosphate compounds. In addition, the genotype frequency can be monitored within the tick population. The accomplishment addresses component 2, detection and diagnostics 2.1 of the National Program 104 Action Plan.

Association of bovine immune system markers with acquired resistance to Amblyomma americanum tick infestation - In an effort to evaluate the role that the host immune system plays in acquired resistance to tick infestation, three bovine genetic markers located within the bovine leukocyte antigen (BoLA) complex were evaluated using a genomic DNA database of calves phenotyped for tick-resistance/susceptibility to the Lone Star tick, A. americanum. Statistically significant associations were identified between two of the genetic markers and the tick-resistant phenotype, suggesting that genes in these regions may play a role in the manifestation of acquired resistance to tick infestation. The region analyzed is of interest, as it encodes genes responsible for the presentation of pathogen-derived antigens to the bovine immune system. Characterization of these gene sequences will enable the identification of genetic markers that can be utilized in a genetic marker-assisted selective breeding program to either include calves resistant to tick infestation or exclude calves susceptible to tick infestation. In addition, knowledge of these bovine immune response genes allows us to evaluate more specifically the bovine immune response to selected B. microplus candidate vaccine antigens. The accomplishment addresses component 3, biology and physiology, and specifically performance measure 3.2.4, identify food animal genotypes naturally resistant to blood feeding arthropods, characterize the mechanisms, and isolate the responsible genes.


4c.List significant activities that support special target populations.
None.


4d.Progress report.
BAC Clone screening – A 125 kb BAC clone that contains the gene encoding CzEST9 has been completely sequenced. Screening of the BAC library for clones containing the coding region for a CYP41-like organophosphate resistance-associated cytochrome P450 gene has begun. Screening of SAGE libraries found this P450 gene was overexpressed in the San Roman strain upon exposure to moderate levels of coumaphos and is more abundant in untreated San Roman compared to the Munoz organophosphate susceptible strain.

EST Sequencing - This is ongoing, as 6,000 ESTs from Babesia-infected larvae, midgut and ovary subtracted libraries have been newly sequenced. Organophosphate-responsive gene expression in R. microplus has been analyzed using Serial Analysis of Gene Expression (SAGE) tag libraries and the Gene Index Version 2 to identify specific genes which respond to coumaphos exposure in both organophosphate-resistant and -susceptible strains of R. microplus.

EST Annotation and Gene Index - Bioinformatic analysis of these ESTs is ongoing, including a gene ontology analysis of the updated R. microplus Gene Index Version 2 with plans to focus upon R. microplus putative G-protein coupled receptors.


5.Describe the major accomplishments to date and their predicted or actual impact.
Genomics Objective:

Submission of a Genomic White Paper - ARS scientists at the Knipling-Bushland U. S. Livestock Insects Research Laboratory (KBUSLIRL) and the Cattle Tick Fever Research Laboratory (CFTRL) in collaboration with scientists at The Institute for Genomic Research (TIGR) and the Center for Disease Control (CDC) were able to complete genomic objectives 1 through 9 of the project plan, FY 2005 milestones. This newly acquired knowledge and compilation of genomic resources has led to the completion of a white paper entitled "Proposal for the sequencing of a new target genome: the southern cattle tick, Boophilus microplus," F. D. Guerrero, V. M. Nene, J. E. George, S. C. Barker, and P. Willadsen, describing the resources, rationale, needs and scientific support for a B. microplus genome sequencing project. The completed white paper was submitted to funding agencies (NHGRI and NIAID-NIH) in FY 2005. Submission of the white paper represents a major accomplishment in the first year of this research project, and it represents the initial and essential event in securing adequate funding for the conduct of a sequencing project. Accomplishment of these goals has led to International Research collaboration with Dr. Ala Lew, Department of Primary Industries and Fisheries, Brisbane, QLD, Australia. The project is funded at $1,394,000.00 by the Smart State Innovation Projects Fund, National and International Research Alliances Program, and entitled "A Cattle Tick Vaccine for Tropical and Subtropical Beef Industries." Dr. F. G. Guerrero is a Co-PI, and Dr. J. H. Pruett is a collaborator on the project. The ever-increasing genomic and proteomic databases that are originating from this research project, and are being made available to investigators worldwide, should substantially impact the rational design of future strategies for the control of ticks and the pathogens they transmit. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of the National Program 104 Action Plan. In addition, the accomplishment addresses performance measure 3.2.1 in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health.

Publication of Gene Index 1 – ARS scientists at the KBUSLIRL in collaboration with scientists at TIGR, in an effort to develop genomic resources that would facilitate the initiation of a B. microplus genome project, have assembled a genomic database entitled Gene Index version 1 that has been published and posted on TIGR website. The database includes 45,000 EST sequences that have been assembled into a gene index of approximately 13,000 coding regions covering an estimated 35-60% of B. microplus genes. This database is available to investigators worldwide as a resource for their research projects. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of the National Program 104 Action Plan. In addition, the accomplishment addresses performance measure 3.2.1 in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health.

FY 2006 EST Sequencing – 6000 EST's from Babesia-infected B. microplus larvae, midgut, and ovary substracted libraries have been newly sequenced. Organophosphate-responsive gene expression in B. microplus has been analyzed using Serial Analysis of Gene Expression (SAGE) tag libraries and the Gene Index Version 2 to identify specific genes that respond to coumaphos exposure in both organophosphate-resistant and –susceptible strains of B. microplus. Bioinformatic analysis of these EST's is ongoing, including gene ontology analysis of the updated B. microplus Gene Index Version 2 with plans to focus upon B. microplus putative G-protein coupled receptors. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of the National Program 104 Action Plan. In addition, the accomplishment addresses performance measure 3.2.1 in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health.

Evaluation of the genetic relatedness of B. microplus ticks collected from separate outbreak sites – The Cattle Fever Tick Eradication Program (CFTEP) is a joint federal-state program designed to prevent B. microplus from reentering the U.S. In support of this program and specifically Dr. Roberta Duhaime DVM, epidemiologist, with APHIS-VS, we have provided technologies that will aid in elucidating the genetic relatedness of ticks collected from independent outbreaks of B. microplus ticks within and outside of the quarantine zone. This works lays the foundation for surveying genetic relationships of ticks collected throughout Mexico. The group fully sequenced the glucose-6-phosphate gene of B. microplus and evaluated nucleotide sequence variation of one of the introns in approximately 500 individual tick specimens collected from 15 outbreak sites within the quarantine zone. Nine intron sequence variations have been identified, the frequencies of which distinguish collections from independent outbreaks. This accomplishment is aligned with NP-104 and addresses Component 2, Detection and Surviellance, specifically performance measure 2.2.4.

Baculovirus expression of a B. microplus acetylcholinesterase – This is the first report of the cloning and successful expression of a B. microplus acetylcholinesterase. An organophosphate acaricide is used in the import dipping vats maintained by the CFTEP on the border with Mexico; as a result concerns’ regarding the development of resistance to organophosphates necessitates the need to understand the specific mechanisms of target-site insensitivity. This work is in direct support of a related project at the Knipling-Bushland U.S. Livestock Insects Research Laboratory entitled, "Developing and using molecular and biochemical methods for the diagnosis of acaricide resistance in B. microplus" as well as APHIS-VS. The accomplishment addresses component 2, detection and diagnostics 2.1 of the National Program 104 Action Plan.

Association of bovine immune system markers with acquired resistance to Amblyomma americanum tick infestation - In an effort to evaluate the role that the host immune system plays in acquired resistance to tick infestation, three bovine genetic markers located within the bovine leukocyte antigen (BoLA) complex were evaluated using a genomic DNA database of calves phenotyped for tick-resistance/susceptibility to the Lone Star tick, A. americanum. Statistically significant associations were identified between two of the genetic markers and the tick-resistant phenotype, suggesting that genes in these regions may play a role in the manifestation of acquired resistance to tick infestation. The region analyzed is of interest, as it encodes genes responsible for the presentation of pathogen-derived antigens to the bovine immune system. Characterization of these gene sequences will enable the identification of genetic markers that can be utilized in a genetic marker-assisted selective breeding program to either include calves resistant to tick infestation or exclude calves susceptible to tick infestation. In addition, knowledge of these bovine immune response genes allows us to evaluate more specifically the bovine immune response to selected B. microplus candidate vaccine antigens. This work has led to a collaborative submission (June 2006) of a NRI grant entitled "Identification of genes associated with tick resistance or susceptibility in cattle" with Dr. Patricia Holman and Dr. James Womack of the School of Veterinary Medicine, Texas A&M University, College Station. Dr. J. H. Pruett and Dr. P. M. Untalan of the Knipling-Bushland U.S. Livestock Insects Research Laboratory USDA-ARS are Co-PI's on the project. The accomplishment addresses component 3, biology and physiology, and specifically performance measure 3.2.4, identify food animal genotypes naturally resistant to blood feeding arthropods, characterize the mechanisms, and isolate the responsible genes.

Proteomics Objective:

Identification of serologically defined B. microplus larval antigens – Life-stage specific B. microplus proteins (6) that putatively elicit antibodies in cattle as a result of natural exposure were identified. A serologically defined 19.1 kDa protein was used to develop a diagnostic ELISA for cattle exposure to B. microplus. The 19.1 kDa protein has been partially amino acid sequenced and we are attempting to isolate the gene. The information offers the potential of serological diagnosis of B. microplus exposure to cattle in support of the CFTEP program of APHIS-VS. In addition, it adds to a pool of candidate antigens for vaccine development. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of National Program 104 Action Plan. In addition the accomplishment addresses performance measure 3.2.1 in that it provides scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health.

Identification of potential targets for the development of novel control strategies for B. microplus - Characterization of molecules that are essential for tick feeding is one approach to the identification of targets for alternative novel control, specifically molecules produced in the salivary gland tissue of fed adult females that are potentially introduced to the bovine host via tick saliva. We have identified transcripts encoding five zinc metalloproteases and four kunitz-like proteins from salivary gland tissue of adult, female ticks that had fed on the host for either 1-2 days or 4-5 days. In other ticks, the zinc metalloproteases are believed to have a fibrinolytic role and inhibit host angiogenesis (prevent wound healing) while kunitz-like proteins have been implicated in the inhibition of the host blood coagulation system. Functional characterization of these molecules with respect to their role in successful tick feeding of B. microplus will lay the foundation for their use as potential targets for alternative control technologies. The accomplishment addresses component 3, biology and physiology, and component 4, control technology, of National Program 104 Action Plan. In addition the accomplishment addresses performance measure 3.2.1.

Analysis of the B. microplus ovarial proteome - The ovarial proteome of B. microplus has been analyzed by 2-D PAGE, focusing on Babesia bovis infection-induced differences. Specific proteins which are differentially expressed have been extracted and submitted for initial mass spec analysis at Dr. Sue Weintraub's laboratory at the UTHSC in San Antonio, TX. Functional characterization of these molecules with respect to their role in pathogen transmission will lay the foundation for their use as potentials targets for alternative control technologies of the vector and the pathogen. This accomplishment addresses component 3 biology and physiology, and performance measure 3.1.1 genomics and host-pathogen interaction.

Biochemical diagnosis of B. microplus acetylcholinesterase organophosphate target-site insensitivity - The principal mechanism of resistance to organophosphate acaricides in B. microplus ticks is acetylcholinesterase (target-site) insensitivity to inhibition. This work describes a method of neural acetylcholinesterase extraction that allows for the diagnosis of insensitivity and the genotyping of the individual tick. This technology will find utility in the CFTEP conducted by APHIS-VS, as it allows for diagnosis as well as the screening of additional organophosphate compounds. In addition, the genotype frequency can be monitored within the tick population. The accomplishment addresses component 2, detection and diagnostics 2.1 of the National Program 104 Action Plan.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Publication of Gene Index 1 and posting on the TIGR website of 45,000 EST sequences, assembled into a gene index of approximately 13,000 coding regions covering an estimated 35-60% of B. microplus genes represents a significant advance in B. microplus genomics that is available to investigators worldwide. Gene Index Version 2 has been made available to grant collaborators through material transfer agreements.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
None.


Review Publications
Guerrero, F.D., Nene, V.M., George, J.E., Barker, S.C., Willadsen, P. 2006. Sequencing a new target genome: the Boophilus microplus (Acari: Ixodidae) genome project. Journal of Medical Entomology. 43(1):9-16.

Neupert, S., Predel, R., Russell, W.K., Davey, R.B., Pietrantonio, P.V., Nachman, R.J. 2005. Identification of tick periviscerokinin, the first neurohormone of Ixodidae: Single cell analysis by means of MALDI-TOF/TOF mass spectrometry. Biochemical and Biophysical Research Communications. 338:1860-1864.

Pruett Jr., J.H., Pound, J.M. 2006. Biochemical diagnosis of organophosphate-insensitivity with neural acetylcholinesterase extracted by sonication from the adult tick synganglion. Veterinary Parasitology. 135:355-363.

Temeyer, K.B., Pruett Jr., J.H., Untalan, P.M., Chen, A.C. 2006. Baculovirus expression of BmAChE3, a cDNA encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae). Journal of Medical Entomology. 43(4):707-712.

Pruett Jr., J.H., Untalan, P.M., Davey, R.B. 2006. Identification and partial purification of serologically defined Boophilus microplus larval antigens by natural ectoparasite exposure. Veterinary Parasitology. 140:148-157.

Last Modified: 4/16/2014
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