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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPING AND USING MOLECULAR AND BIOCHEMICAL METHODS FOR THE DIAGNOSIS OF ACARICIDE RESISTANCE IN 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?
The U.S. livestock industry generates about 50% of the annual agricultural income of the United States. Together, horn flies and ticks account for an estimated annual potential loss to the U.S. cattle industry of several billion dollars. This potential loss is presently being averted, in part, by control of these ectoparasites with good animal husbandry practices and chemical pesticides. In addition to the control of ectoparasites, chemical pesticides are critical in maintaining an import barrier to Boophilus microplus, a vector of cattle fever (babesiosis). Boophilus ticks were eradicated from the U.S. following a 50-year program of dipping cattle and vacating tick-infested pastures. Boophilus ticks and babesiosis are still widespread in Mexico, and a quarantine zone along the U.S. border with Mexico is maintained to prevent their reintroduction into the U.S. Loss of the import barrier and reintroduction of Boophilus ticks and their associated diseases would cause great losses to the U.S. cattle industry. In Mexico, populations of B. microplus that are resistant to organophosphate (OP) and pyrethroid acaricides are widespread, and the emergence of resistance to the formamidine pesticide amitraz has been observed within the last few years.

Pesticide (acaricide) resistant populations of B. microplus, the southern cattle tick, in Mexico will compromise the Cattle Fever Tick Eradication Program of USDA, APHIS, Veterinary Services, if technology is not available to diagnose resistance in outbreaks of ticks from Mexico and then enable the choice of an acaricide to eradicate them. Infestations of southern cattle ticks from Mexico occur in south Texas along the Rio Grande River as a result of tick-infested livestock or wildlife that cross the river into the U.S. and disseminate ticks. Rapid, accurate methods are needed to diagnose acaricide resistance in a population of ticks. These methods could provide the basis for a rapid response with an acaricide to which the ticks are vulnerable.

This research project seeks to elucidate the mechanisms of pesticide resistance to pyrethroid OP and amitraz resistance in the southern cattle tick, B. microplus. Rapid and sensitive molecular tests will be developed to allow identification and monitoring of resistance to these pesticides after the elucidation of the resistance mechanisms. Mechanisms of emerging resistance to newer pesticides, such as ivermectin, will also be pursued.

The project has four specific goals: .
1)Identify and characterize mechanisms involved in tick resistance to different acaricides;.
2)Develop rapid, accurate and sensitive diagnostic protocols employing molecular probes or best available technology based upon information on resistance mechanisms to detect resistant genotypes and determine resistance status in tick populations;.
3) Determine the reliability, sensitivity, and utility of molecular and biochemical methods for the diagnosis of acaricide resistance in populations of B. microplus to multiple chemical groups of acaricides; and.
4)Use molecular and biochemical assays for resistance diagnosis in assessments of the fitness and mode of inheritance in organophosphate (OP), pyrethroid (P), and formamidine (F) resistance in B. microplus. The research falls under NP 104, Action Plan Goal 2.1 Detection and Diagnostics. The research addresses Goal 3, Objective 3.2 of the ARS Strategic Plan, Develop and Deliver Science-Based Information and Technologies to Reduce the Number and Severity of Agricultural Pest, Insect, Weed, and Disease Outbreaks.

The research provides assays for resistance diagnosis that will facilitate the selection and use of efficacious pesticides that will be used to protect dairy and beef producers from losses caused by pesticide-resistant blood-feeding flies and ticks, and tick-borne disease agents.


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

Analysis of adenylyl cyclase involved in the regulation of cellular function that might play a role in resistance to acaricides.

Cloning of octopamine receptor that is suspected to be responsible for acaricide resistance.

Development of probes for mutations in octopamine receptor associated with acaricide resistance.

Year 2 (FY 2006)

Electrophysiological studies on nervous and muscles.

Cloning of acetylcholinesterase, an enzyme critical for proper nerve functions and suspected to be responsible for resistance to certain pesticides.

Cloning of esterases, enzymes that break down pesticides, suspected to have increased activity resulting in resistance.

Identification of genes responsible for pesticide resistance.

Correlation of molecular diagnosis of resistance with bioassay.

Selection and study of genetics of pesticide resistant strains.

Year 3 (FY 2007)

Further electrophysiological studies.

Cloning of two families of enzymes (cytochrome P450s and glutathione-S-transferases) responsible for the breakdown of pesticides.

Further cloning of esterases.

Continued identification of genes associated with pesticide resistance.

Analysis of function of octopamine receptor.

Development of molecular probes for enzymes that break down pesticides.

Investigation on the inheritance of pesticide resistance.

Year 4 (FY 2008)

Cloning of ligand-gated ion channels.

Further identification of genes responsible for pesticide resistance.

Further development of molecular probes for enzymes that break down pesticides.

Evaluation of resistance of field-collected ticks and correlate with results obtained by molecular assays.

Study on whether mutations that confer pesticide resistance on the tick would make them less fit under natural selection.

Investigation on inheritance of pesticide resistance.

Year 5 (FY 2009)

Further cloning of ligand-gated ion channels.

Development of diagnostic probes for mutations in ligand-gated ion channels that lead to pesticide resistance.

Further identification of genes responsible for pesticide resistance.

Further development of molecular probes for enzymes that break down pesticides.

Continued analysis of functions of octopamine receptor.

Adaptation of molecular diagnostic tools developed in the laboratory for field application.

Further investigation on whether mutations that confer pesticide resistance on the tick would make them less fit in natural selection.

Further investigation on inheritance of pesticide resistance.


4a.List the single most significant research accomplishment during FY 2006.
Elucidation of the molecular mechanism of organic phosphate (OP) resistance in the southern cattle tick. Coumaphos is currently the only registered acaricide in the U.S. for treating the southern cattle tick on cattle imported from Mexico and for tick outbreaks in the U.S. Resistance to coumaphos is wide spread in Mexico and resistance in the outbreaks in the U.S. was discovered for the first time by ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory last year. In order to combat the resistance problem it is critical to understand the molecular mechanism of resistance. We have now cloned an acetylcholinesterase (AChE) gene, an enzyme crucial in the proper function of the nervous system in Arthropods and the suspected target of OP pesticides, from the southern cattle tick and expressed it in the laboratory. Mutations in the enzyme were found in B. microplus strains resistant to OP, and expression of the AChE gene containing the mutations resulted in production of an altered AChE that was resistant to inactivation by OP. This accomplishment addresses Milestone 1.2 in the Project Plan and the ARS Action Plan Component 2, Detection and Surveillance Technology, Goal 2.1, Detection and Diagnostics.


4b.List other significant research accomplishment(s), if any.
First report of pyrethroid-resistant ticks found within the USA. ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory produced the first report of pyrethroid-resistant ticks found inside the USA. Ticks were collected in Hidalgo County and tested for the presence of resistance to pyrethroids. The discovery of permethrin-resistant ticks keeps the APHIS, VS, Cattle Fever Tick Eradication Program informed regarding the risk to the success of this program and should initiate the establishment of contingency plans developed for this situation. This accomplishment addresses the ARS Action Plan Component 2, Detection and Surveillance Technology, Goal 2.1, Detection and Diagnostics.

Resistant ticks found in Coahuila, Mexico. ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory produced a report of permethrin–resistant ticks found close to the USA in the state of Coahuila, Mexico. Ticks collected in Mexico were tested for the presence of resistance to 4 different acaricides and found to be highly resistant to pyrethroids. This information helps the APHIS, VS, Cattle Fever Tick Eradication Program assess the risk to the continued success of the program and make any proactive adjustments necessary. This accomplishment addresses the ARS Action Plan Component 2, Detection and Surveillance Technology, Goal 2.1, Detection and Diagnostics.


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


5.Describe the major accomplishments to date and their predicted or actual impact.
Discovery of the first case of coumaphos-resistant ticks found inside the USA. USDA, APHIS-VS, Cattle Fever Tick Eradication Program (CFTEP) uses the results of the research to develop effective eradication strategies for the southern cattle tick. Of the 29 outbreak populations in Texas submitted by the APHIS-VS-CFTEP, one strain was determined to be resistant to organophosphate acaricides and another strain to pyrethroid acaricides. Coumaphos is currently the only pesticide that is registered for the control of southern cattle tick in the U.S. In addition, these results were important to personnel of the CFTEP because they showed that acaricide-resistant ticks were likely to occur within the U.S. at any time. Discovery of coumaphos-resistant ticks in south Texas should help APHIS-VS in the development of eradication strategies when outbreaks of organophosphate resistant ticks occur in the U.S. This accomplishment addresses Milestone 4.1 in the Project Plan and the ARS Action Plan Component 2, Detection and Surveillance Technology, Goal 2.1, Detection and Diagnostics. The accomplishment contributes to Performance Measure 3.2.1 in the ARS Strategic 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?
None.


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).
Flores, A. 2006. The continuing fight against cattle ticks. Agricultural Research, June 2006. p. 8-9.


Review Publications
Foil, L.D., Guerrero, F., Alison, M.W., Kimball, M. 2005. Association of the kdr and superkdr sodium channel mutations with resistance to pyrethroids in Louisiana populations of the horn fly, Haematobia irritans irritans (L.). Veterinary Parasitology. 129:149-158.

Li, A.Y., Davey, R.B., Miller, R., George, J.E. 2005. Mode of inheritance of amitraz resistance in a Brazilian strain of the southern cattle tick, Boophilus microplus (Acari: Ixodidae). Experimental and Applied Acarology. 37:183-198.

Miller, R., Davey, R.B., George, J.E. 2005. First report of organophosphate-resistant Boophilus microplus (Acari: Ixodidae) within the United States of America. Journal of Medical Entomology. 45(2):912-917.

Miller, R., Wing, J., Cope, S., Davey, R.B., Kline, D.L. 2005. Comparison of carbon dioxide- and octenol-baited encephalitis virus surveillance mosquito traps at the Shoal Water Bay training area, Queensland, Australia. Journal of the American Mosquito Control Association. 21(4):497-500.

Rosario-Cruz, R., Guerrero, F., Miller, R., Rodrigues-Vivas, R.I., Dominguez-Garcia, D.I., Cornel, A.J., Hernandez-Ortiz, R., George, J.E. 2005. Roles played by esterase activity and by a sodium channel mutation involved in pyrethroid resistance in populations of Boophilus microplus (Acari: Ixodidae) collected from Yucatan, Mexico. Journal of Medical Entomology. 42(6):1020-1025.

Last Modified: 7/31/2014
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